Escherichia coli

J Bacteriol, 2002 Nov, 184(21), 5898 - 902
Hydroxylamine reductase activity of the hybrid cluster protein from Escherichia coli; Wolfe MT et al.; The hybrid cluster protein (HCP; formerly termed the prismane protein) has been extensively studied due to its unique spectroscopic properties . Although the structural and spectroscopic characteristics are well defined, its enzymatic function, up to this point, has remained unidentified . While it was proposed that HCP acts in some step of nitrogen metabolism, a specific role for this enzyme remained unknown . Recent studies of HCP purified from Escherichia coli have identified a novel hydroxylamine reductase activity . These data reveal the ability of HCP to reduce hydroxylamine in vitro to form NH(3) and H(2)O . Further biochemical analyses were completed in order to determine the effects of various electron donors, different pH levels, and the presence of CN(-) on in vitro hydroxylamine reduction.

J Bacteriol, 2002 Nov, 184(21), 5871 - 9
Truncation analysis of TatA and TatB defines the minimal functional units required for protein translocation; Lee PA et al.; The TatA and TatB proteins are essential components of the twin arginine protein translocation pathway in Escherichia coli . C-terminal truncation analysis of the TatA protein revealed that a plasmid-expressed TatA protein shortened by 40 amino acids is still fully competent to support protein translocation . Similar truncation analysis of TatB indicated that the final 30 residues of TatB are dispensable for function . Further deletion experiments with TatB indicated that removal of even 70 residues from its C terminus still allowed significant transport . These results imply that the transmembrane and amphipathic helical regions of TatA and TatB are critical for their function but that the C-terminal domains are not essential for Tat transport activity . A chimeric protein comprising the N-terminal region of TatA fused to the amphipathic and C-terminal domains of TatB supports a low level of Tat activity in a strain in which the wild-type copy of either tatA or tatB (but not both) is deleted.

J Bacteriol, 2002 Nov, 184(21), 5862 - 70
Molecular characterization of the growth phase-dependent expression of the lsrA gene, encoding levansucrase of Rahnella aquatilis; Seo JW et al.; Expression of the lsrA gene from Rahnella aquatilis, encoding levansucrase, is tightly regulated by the growth phase of the host cell; low-level expression was observed in the early phase of cell growth, but expression was significantly stimulated in the late phase . Northern blot analysis revealed that regulation occurred at the level of transcription . The promoter region was identified by primer extension analysis . Two opposite genetic elements that participate in the regulation of lsrA expression were identified upstream of the lsrA gene: the lsrS gene and the lsrR region . The lsrS gene encodes a protein consisting of 70 amino acid residues (M(r), 8,075), which positively activated lsrA expression approximately 20-fold in a growth phase-dependent fashion . The cis-acting lsrR region, which repressed lsrA expression about 10-fold, was further narrowed to two DNA regions by deletion analysis . The concerted action of two opposite regulatory functions resulted in the growth phase-dependent activation of gene expression in Escherichia coli independent of the stationary sigma factor sigma(S).

J Bacteriol, 2002 Nov, 184(21), 5855 - 61
Transcription activation by FNR: evidence for a functional activating region 2; Blake T et al.; The FNR protein of Escherichia coli controls the transcription of target genes in response to anoxia via the assembly-disassembly of oxygen-labile iron-sulfur clusters . Previous work identified patches of surface-exposed amino acids (designated activating regions 1 and 3 {AR1 and AR3, respectively}) of FNR which allow it to communicate with RNA polymerase (RNAP) and thereby activate transcription . Previously it was thought that FNR lacks a functional activating region 2 (AR2), although selecting for mutations that compensate for defective AR1 or a miscoordinated iron-sulfur cluster can reactivate AR2 . Here we show that the substitution of two surface-exposed lysine residues (Lys49 and Lys50) of FNR impaired transcription from class II (FNR box centered at -41.5) but not class I (FNR box centered at -71.5) FNR-dependent promoters . The degree of impairment was greater when a negatively charged residue (Glu) replaced either Lys49 or Lys50 than when uncharged amino acid Ala was substituted . Oriented heterodimers were used to show that only the downstream subunit of the FNR dimer was affected by the Lys-->Ala substitutions at a class II promoter . Site-directed mutagenesis of a negatively charged patch ((162)EEDE(165)) within the N-terminal domain of the RNAP alpha subunit that interacts with the positively charged AR2 of the cyclic AMP receptor protein suggested that Lys49 and Lys50 of FNR interact with this region of the alpha subunit of RNAP . Thus, it was suggested that Lys49 and Lys50 form part of a functional AR2 in FNR.

J Bacteriol, 2002 Nov, 184(21), 5842 - 7
Study of second-site suppression in the pheP gene for the phenylalanine transporter of Escherichia coli; Pi J et al.; Site-directed mutagenesis was used to investigate a region of the PheP protein corresponding to the postulated consensus amphipathic region (CAR) in the GabP protein . Whereas some critical residues are conserved in both proteins, there are major differences between the two proteins which may reflect different functions for this region . Replacement of R317, Y313, or P341 by a number of other amino acids destroyed the PheP function . An R317E-E234R double mutant exhibited low levels of PheP transport activity, indicating that there is a possible interaction between these two residues in the wild-type protein . E234 is highly conserved in members of the superfamily of amino acid-polyamine-organocation transporters and also is critical for PheP function in the wild-type protein . Second-site suppressors were isolated for mutants with mutations in E234, Y313, R317, and P341 . Most suppressor mutations were found to cluster towards the extracellular face of spans III, IX, and X . Some mutations, such as changes at M116, were able to suppress each of the primary changes at positions E234, Y313, R317, and P341 but were unable to restore function to a number of other primary mutants . The possible implications of these results for the tertiary structure of the protein are discussed.

J Bacteriol, 2002 Nov, 184(21), 5833 - 41
Escherichia coli insertion sequence IS150: transposition via circular and linear intermediates; Haas M et al.; IS150, a member of the widespread IS3 family, contains two consecutive out-of-phase open reading frames, orfA and orfB, that partially overlap . These open reading frames encode three proteins, InsA, InsB, and the InsAB protein, which is jointly encoded by both open reading frames by means of programmed translational frameshifting . We demonstrate that the InsAB protein represents the IS150 element's transposase . In vivo, the wild-type IS150 element generates circular excision products and linear IS150 molecules . Circular and linear species have previously been detected with mutant derivatives of other members of the IS3 family . Our finding supports the assumption that these products represent true transposition intermediates of members of this family . Analysis of the molecular nature of these two species suggested that the circular forms are precursors of the linear molecules . Elimination of InsA synthesis within the otherwise intact element led to accumulation of large amounts of the linear species, indicating that the primary role of InsA may be to prevent abortive production of the linear species and to couple generation of these species to productive insertion events.

J Biol Chem, 2002 Dec 20, 277(51), 49935 - 44 Epub 2002 Oct 08.
Phosphoinositide binding by the pleckstrin homology domains of Ipl and Tih1; Saxena A et al.; The Ipl protein consists of a single pleckstrin homology (PH) domain with short N- and C-terminal extensions . This protein is highly conserved among vertebrates, and it acts to limit placental growth in mice . However, its biochemical function is unknown . The closest paralogue of Ipl is Tih1, another small PH domain protein . By sequence comparisons, Ipl and Tih1 define an outlying branch of the PH domain superfamily . Here we describe phosphatidylinositol phosphate (PIP) binding by these proteins . Ipl and Tih1 bind to immobilized PIPs with moderate affinity, but this binding is weaker and more promiscuous than that of prototypical PH domains from the general receptor for phosphoinositides (GRP1), phospholipase C delta1, and dual adaptor for phosphoinositides and phosphotyrosine 1 . In COS7 cells exposed to epidermal growth factor, green fluorescent protein (GFP)-Ipl and GFP-Tih1 accumulate at membrane ruffles without clearing from the cytoplasm, whereas control GFP-GRP1 translocates rapidly to the plasma membrane and clears from the cytoplasm . Ras*-Ipl and Ras*-Tih1 fusion proteins both rescue cdc25ts Saccharomyces cerevisiae, but Ras*-Ipl rescues more efficiently in the presence of phosphatidylinositol 3-kinase (PI3K), whereas PI3K-independent rescue is more efficient with Ras*-Tih1 . Site-directed mutagenesis defines amino acids in the beta1-loop1-beta2 regions of Ipl and Tih1 as essential for growth rescue in this assay . Thus, Ipl and Tih1 are bona fide PH domain proteins, with broad specificity and moderate affinity for PIPs.

J Biol Chem, 2002 Dec 13, 277(50), 48241 - 7 Epub 2002 Oct 08.
The Aes protein and the monomeric alpha-galactosidase from Escherichia coli form a non-covalent complex . Implications for the regulation of carbohydrate metabolism; Mandrich L et al.; Aes, a 36-kDa acetylesterase from Escherichia coli, belongs to the hormone-sensitive lipase family, and it is involved in the regulation of MalT, the transcriptional activator of the maltose regulon . The activity of MalT is depressed through a direct protein-protein interaction with Aes . Although the effect is clear-cut, the meaning of this interaction and the conditions that trigger it still remain elusive . To perform a comparative thermodynamic study between the mesophilic Aes protein and two homologous thermostable enzymes, Aes was overexpressed in E . coli and purified . At the last step of the purification procedure the enzyme was eluted from a Mono Q HR 5/5 column as a major form migrating, anomalously, at 56 kDa on a calibrated Superdex 75 column . A minor peak that contains the Aes protein and a polypeptide of 50 kDa was also detected . By a combined analysis of size-exclusion chromatography and surface-enhanced laser desorption ionization-time of flight mass spectrometry, it was possible to demonstrate the presence in this peak of a stable 87-kDa complex, containing the Aes protein itself and the 50-kDa polypeptide in a 1:1 ratio . The homodimeric molecular species of Aes and of the 50-kDa polypeptide were also detected . The esterase activity associated with the 87-kDa complex, when assayed with p-nitrophenyl butanoate as substrate, proved 6-fold higher than the activity of the major Aes form of 56 kDa . Amino-terminal sequencing highlighted that the 50-kDa partner of Aes in the complex was the alpha-galactosidase from E . coli . The E . coli cells harboring plasmid pT7-SCII-aes and, therefore, expressing Aes were hampered in their growth on a minimal medium containing raffinose as a sole carbon source . Because alpha-galactosidase is involved in the metabolism of raffinose, the above findings suggest a potential role of Aes in the regulation of carbohydrate metabolism in E . coli.

J Biol Chem, 2002 Dec 13, 277(50), 48276 - 81 Epub 2002 Oct 08.
Purification, characterization, cloning, and expression of a novel xyloglucan-specific glycosidase, oligoxyloglucan reducing end-specific cellobiohydrolase; Yaoi K et al.; A novel oligoxyloglucan-specific glycosidase, oligoxyloglucan reducing end-specific cellobiohydrolase (OXG-RCBH), with a molecular mass of 97 kDa and a pI of 6.1, was isolated from the fungus Geotrichum sp . M128 . Analysis of substrate specificity using various xyloglucan oligosaccharide structures revealed that OXG-RCBH had exoglucanase activity . It recognized the reducing end of oligoxyloglucan and released two glucosyl residue segments from the main chain . The full-length cDNA encoding OXG-RCBH was cloned and sequenced, and it had a 2436-bp open reading frame encoding an 812amino acid protein . The deduced protein showed approximately 35% identity to members of glycoside hydrolase family 74 . The cDNA encoding OXG-RCBH was then expressed in Escherichia coli . Although the recombinant protein was expressed as an inclusion body, renaturation was successful, and enzymatically active recombinant OXG-RCBH was obtained.

EMBO J, 2002 Oct 15, 21(20), 5577 - 85
Holliday junction resolution in human cells: two junction endonucleases with distinct substrate specificities; Constantinou A et al.; Enzymatic activities that cleave Holliday junctions are required for the resolution of recombination intermediates and for the restart of stalled replication forks . Here we show that human cell-free extracts possess two distinct endonucleases that can cleave Holliday junctions . The first cleaves Holliday junctions in a structure- and sequence-specific manner, and associates with an ATP-dependent branch migration activity . Together, these activities promote branch migration/resolution reactions similar to those catalysed by the Escherichia coli RuvABC resolvasome . Like RuvC-mediated resolution, the products can be religated . The second, containing Mus81 protein, cuts Holliday junctions but the products are mostly non-ligatable . Each nuclease has a defined substrate specificity: the branch migration-associated resolvase is highly specific for Holliday junctions, whereas the Mus81-associated endonuclease is one order of magnitude more active upon replication fork and 3'-flap structures . Thus, both nucleases are capable of cutting Holliday junctions formed during recombination or through the regression of stalled replication forks . However, the Mus81-associated endonuclease may play a more direct role in replication fork collapse by catalysing the cleavage of stalled fork structures.

EMBO J, 2002 Oct 15, 21(20), 5323 - 30
Ionic regulation of MscK, a mechanosensitive channel from Escherichia coli; Li Y et al.; Three gene products that form independent mechanosensitive channel activities have been identified in Escherichia coli . Two of these, MscL and MscS, play a vital role in allowing the cell to survive acute hypotonic stress . Much less is known of the third protein, MscK (KefA) . Here, we characterize the MscK channel activity and compare it with the activity of its structural and functional homologue, MscS . While both show a slight anionic preference, MscK appears to be more sensitive to membrane tension . In addition, MscK, but not MscS activity appears to be regulated by external ionic environment, requiring not only membrane tension but also high concentrations of external K(+), NH(4)(+), Rb(+) or Cs(+) to gate; no activity is observed with Na(+), Li(+) or N-methyl-D-glucamine (NMDG) . An MscK gain-of-function mutant gates spontaneously in the presence of K(+) or similar ions, and will gate in the presence of Na(+), Li(+) and NMDG, but only when stimulated by membrane tension . Increased sensitivity and the highly regulated nature of MscK suggest a more specialized physiological role than other bacterial mechanosensitive channels.

Electrophoresis, 2002 Sep, 23(19), 3289 - 99
Escherichia coli single-stranded DNA-binding protein, a molecular tool for improved sequence quality in pyrosequencing; Ehn M et al.; Pyrosequencing is a four-enzyme bioluminometric DNA sequencing technique based on a DNA sequencing by synthesis principle . Currently, the technique is limited to analysis of short DNA sequences exemplified by single-nucleotide polymorphism analysis . In order to expand the field for pyrosequencing, the read length needs to be improved and efforts have been made to purify reaction components as well as add single-stranded DNA-binding protein (SSB) to the pyrosequencing reaction . In this study, we have performed a systematic effort to analyze the effects of SSB by comparing the pyrosequencing result of 103 independent complementary DNA (cDNA) clones . More detailed information about the cause of low quality sequences on templates with different characteristics was achieved by thorough analysis of the pyrograms . Also, real-time biosensor analysis was performed on individual cDNA clones for investigation of primer annealing and SSB binding on these templates . Results from these studies indicate that templates with high performance in pyrosequencing without SSB possess efficient primer annealing and low SSB affinity . Alternative strategies to improve the performance in pyrosequencing by increasing the primer-annealing efficiency have also been evaluated.

Biol Neonate, 2002, 82(3), 188 - 96
Detrimental effects of nicotine and endotoxin in the newborn piglet brain during severe hypoxemia; Froen JF et al.; Hypoxia-ischemia is a major cause of perinatal brain damage, but evidence shows that brain injury also is associated with intrauterine infections and maternal smoking . The mechanisms are not known, and we therefore explored the effects of experimental inflammation or nicotine on perinatal brain metabolism and injury during severe hypoxemia . Twenty-eight 1-week-old piglets were anesthetized and instrumented with microdialysis probes in the striatum and brainstem . We studied three pretreatment groups: (1) 20 microg/kg i.v . nicotine (n = 9); (2) 1 microg/kg i.v . endotoxin from Escherichia coli (n = 11), or (3) control (n = 8) . The piglets were subsequently exposed to 30 min of hypoxemia (6% O(2)) . In order to minimize any ischemic component and increase survival, this was abrupted for 1 min if blood pressure fell to 30 mm Hg . During hypoxemia, both the pretreatment with endotoxin and nicotine induced higher levels of extracellular lactate and peak lactate/pyruvate ratio compared with controls (54.7 +/- 9.6 (p < 0.01) and 65.2 +/- 13.1 (p < 0.02) vs . 15.9 +/- 7.4, respectively), reflecting a deterioration of the metabolic status in these groups . The two pretreated groups reached significantly higher peak levels of extracellular glycerol (30.9 +/- 4.1 vs . 77.9 +/- 12.7 and 89.4 +/- 14.2 micromol/l, respectively, p = 0.01), indicating a higher level of cellular membrane disintegration or leakage . In addition, 3 endotoxin piglets and 4 nicotine piglets died during reoxygenation, while all controls survived (p = 0.13 and p < 0.04, respectively) . Mortality was associated with a rise in extracellular glutamate at the end of hypoxemia/start reoxygenation (p = 0.02) . These findings contribute in explaining how nicotine and inflammatory response to bacterial toxins could act as cofactors for hypoxic-ischemic neurologic injury in the immature brain .

J Biomed Sci, 2002, 9(6 Pt 1), 542 - 8
Dysfunction of the mitochondrial respiratory chain in the rostral ventrolateral medulla during experimental endotoxemia in the rat; Chuang YC et al.; We investigated the functional changes in the mitochondrial respiratory chain at the rostral ventrolateral medulla (RVLM), the medullary origin of sympathetic vasomotor tone, in an experimental model of endotoxemia that mimics systemic inflammatory response syndrome . In Sprague-Dawley rats maintained under propofol anesthesia, intravenous administration of Escherichia coli lipopolysaccharide (LPS; 30 mg/kg) induced a reduction (Phase I), followed by an augmentation (Phase II) and a secondary decrease (Phase III) in the power density of vasomotor components (0-0.8 Hz) in systemic arterial pressure signals . LPS also elicited progressive hypotension, and death ensued within 4 h . Enzyme assay revealed significant depression of the activity of nicotinamide adenine dinucleotide cytochrome c reductase (Complexes I + III) and cytochrome c oxidase (Complex IV) in the RVLM during all three phases of endotoxemia . On the other hand, the activity of succinate cytochrome c reductase (Complexes II + III) remained unaltered . We conclude that selective dysfunction of respiratory enzyme Complexes I and IV in the mitochondrial respiratory chain at the RVLM, whose neuronal activity is intimately related to the death process, is closely associated with fatal endotoxemia in the rat .

J Biol Chem, 2002 Dec 6, 277(49), 47878 - 84 Epub 2002 Oct 07.
Phosphorylation of the catalytic subunit of protein kinase A . Autophosphorylation versus phosphorylation by phosphoinositide-dependent kinase-1; Moore MJ et al.; The identification of phosphoinositide-dependent kinase-1 (PDK-1) as an activating kinase for members of the AGC family of kinases has led to its implication as the activating kinase for cAMP-dependent protein kinase . It has been established in vitro that PDK-1 can phosphorylate the catalytic (C) subunit (), but the Escherichia coli-expressed C-subunit undergoes autophosphorylation . To assess which of these mechanisms occurs in mammalian cells, a set of mutations was engineered flanking the site of PDK-1 phosphorylation, Thr-197, on the activation segment of the C-subunit . Two distinct requirements appeared for autophosphorylation and phosphorylation by PDK-1 . Autophosphorylation was disrupted by mutations that compromised activity (Thr-201 and Gly-200) or altered substrate recognition (Arg-194) . Conversely, only residues peripheral to Thr-197 altered PDK-1 phosphorylation, including a potential hydrophobic PDK-1 binding site at the C terminus . To address the in vivo requirements for phosphorylation, select mutant proteins were transfected into COS-7 cells, and their phosphorylation state was assessed with phospho-specific antibodies . The phosphorylation pattern of these mutant proteins indicates that autophosphorylation is not the maturation mechanism in the eukaryotic cell; instead, a heterologous kinase with properties resembling the in vitro characteristics of PDK-1 is responsible for in vivo phosphorylation of PKA.

J Biol Chem, 2002 Dec 13, 277(50), 48199 - 204 Epub 2002 Oct 07.
In vivo interactions between gene products involved in the final stages of molybdenum cofactor biosynthesis in Escherichia coli; Magalon A et al.; The final stages of bacterial molybdenum cofactor (Moco) biosynthesis correspond to molybdenum chelation and nucleotide attachment onto an unique and ubiquitous structure, the molybdopterin . Using a bacterial two-hybrid approach, here we report on the in vivo interactions between MogA, MoeA, MobA, and MobB implicated in several distinct although linked steps in Escherichia coli . Numerous interactions among these proteins have been identified . Somewhat surprisingly, MobB, a GTPase with a yet unclear function, interacts with MogA, MoeA, and MobA . Probing the effects of various mo . mutations on the interaction map allowed us (i) to distinguish Moco-sensitive interactants from insensitive ones involving MobB and (ii) to demonstrate that molybdopterin is a key molecule triggering or facilitating MogA-MoeA and MoeA-MobA interactions . These results suggest that, in vivo, molybdenum cofactor biosynthesis occurs on protein complexes rather than by the separate action of molybdenum cofactor biosynthetic proteins.

J Biol Chem, 2002 Dec 6, 277(49), 47885 - 90 Epub 2002 Oct 07.
Zinc mediates assembly of the T1 domain of the voltage-gated K channel 4.2; Jahng AW et al.; An intermolecular Zn(2+)-binding site was identified in the structure of the T1 domain of the Shaw-type potassium channels (aKv3.1) . T1 is a BTB/POZ-type domain responsible for the ordered assembly of voltage-gated potassium channels and interactions with other macromolecules . In this structure, a Zn(2+) ion was found to be coordinated between each of the four assembly interfaces of the T1 tetramer by three Cys and one His encoded in the sequence motif (HX(5)CX(20)CC) of the T1 domain . This sequence motif is conserved among all non-Shaker-type voltage-dependent potassium (Kv) channels, but not in Shaker-type channels . The presence of this conserved Zn(2+)-binding site is a primary molecular determinant that distinguishes the tetrameric assembly of non-Shaker Kv channel subunits from that of Shaker channels . We report here that tetramerization of the Shal (rKv4.2) T1 in solution requires the presence of Zn(2+), and the addition/removal of Zn(2+) reversibly switches the protein between a stable tetrameric or monomeric state . We further show that the conversion from tetramers to monomers is profoundly pH-dependent: as the solution pH gets lower, the dissociation rate increases significantly . The unfolding energy of the T1 tetramer as a measure of the conformational stability of the structure is also pH-dependent . Surprisingly, at a lower pH we observe a distinctly altered conformational state of the T1 tetramer trapped during the process of unfolding of the T1 tetramer in the presence of Zn(2+) . The conformational alteration may be responsible for increased rate of dissociation at lower pH by allowing Zn(2+) to be removed more effectively by EDTA . The ability of the T1 domain to adopt stable alternative conformations may be essential to its function as a protein-protein interaction/signaling domain to modulate the ion conduction properties of intact full-length Kv channels.

FEBS Lett, 2002 Oct 9, 529(2-3), 332 - 6
Recombinant Escherichia coli biotin synthase is a {2Fe-2S}(2+) protein in whole cells; Cosper MM et al.; EPR and Mossbauer spectroscopies have been used to determine the type and properties of the iron-sulfur clusters present in homologously expressed recombinant Escherichia coli BioB in whole cells prior to purification . Difference EPR spectra of samples of whole cells from a strain over-expressing E . coli BioB and a strain containing the same plasmid but without the bioB insertion showed an axial S=1/2 resonance that was attributed to the {2Fe-2S}(+) cluster of the E . coli iron-sulfur cluster assembly 2Fe ferredoxin, based on principal g-values, linewidths and relaxation behavior . Comparison of the Mossbauer spectra of whole cells with and without the bioB insertion revealed that the E . coli cells with over-expressed BioB contain an additional species that exhibits a spectrum identical to that of the {2Fe-2S}(2+) cluster in purified recombinant BioB . The concentration of this {2Fe-2S}(2+) species in the whole cell sample was quantified using a Mossbauer standard and found to be approximately 260 microM, which was comparable to the BioB protein concentration estimated for the cell paste . The results demonstrate that the {2Fe-2S}(2+) cluster found in purified samples of recombinant BioB is not an artifact of the protein purification procedure, and indicate that recombinant BioB is over-expressed in an inactive form during aerobic growth.

FEBS Lett, 2002 Oct 9, 529(2-3), 237 - 42
Thermal inactivation of reduced ferredoxin (flavodoxin):NADP+ oxidoreductase from Escherichia coli; Jarrett JT et al.; Ferredoxin (flavodoxin):NADP+ oxidoreductase (FNR) is an essential enzyme that supplies electrons from NADPH to support flavodoxin-dependent enzyme radical generation and enzyme activation . FNR is a monomeric enzyme that contains a non-covalently bound FAD cofactor . We report that reduced FNR from Escherichia coli is subject to inactivation due to unfolding of the protein and dissociation of the FADH(2) cofactor at 37 degrees C . The inactivation rate is temperature-dependent in a manner that parallels the thermal unfolding of the protein and is slowed by binding of ferredoxin or flavodoxin . Understanding factors that minimize inactivation is critical for utilizing FNR as an accessory protein for S-adenosyl-L-methionine-dependent radical enzymes and manipulating FNR as an electron source for biotechnology applications.

FEBS Lett, 2002 Oct 9, 529(2-3), 225 - 31
3'-end processing of precursor M1 RNA by the N-terminal half of RNase E; Sim S et al.; M1 RNA, the catalytic component of Escherichia coli RNase P, is derived from the 3'-end processing of precursor M1 RNA, a major transcript of the rnpB gene . In this study, we investigated the mechanism of 3'-end processing of M1 RNA using the recombinant N-terminal half RNase E . The cleavage site preference of RNase E differed from that of the 40% ammonium sulfate precipitate (ASP-40), a partially purified cell extract containing processing activity . However, the addition of a trace amount of ASP-40 changed the cleavage site preference of RNase E to that of ASP-40 suggesting the involvement of a soluble factor in cleavage site preference.

FEBS Lett, 2002 Oct 9, 529(2-3), 151 - 6
HU: promoting or counteracting DNA compaction?
Dame RT, Goosen N.
The role of HU in Escherichia coli as both a protein involved in DNA compaction and as a protein with regulatory function seems to be firmly established . However, a critical look at the available data reveals that this is not true for each of the proposed roles of this protein . The role of HU as a regulatory or accessory protein in a number of systems has been thoroughly investigated and in many cases has been largely elucidated . However, almost 30 years after its discovery, convincing evidence for the proposed role of HU in DNA compaction is still lacking . Here we present an extensive literature survey of the available data which, in combination with novel microscopic insights, suggests that the role of HU could be the opposite as well . The protein is likely to play an architectural role, but instead of being responsible for DNA compaction it could be involved in antagonising compaction by other proteins such as H-NS.

Biochem Biophys Res Commun, 2002 Oct 11, 297(5), 1096 - 101
Change of product specificity of hexaprenyl diphosphate synthase from Sulfolobus solfataricus by introducing mimetic mutations; Hemmi H et al.; The introduction of several sets of amino acid substitutions into the region around a substrate-binding site of a medium-chain (all-E) prenyl diphosphate synthase, hexaprenyl diphosphate synthase from a thermoacidophilic archaeon Sulfolobus solfataricus, to mimic the product determination mechanisms of various kinds of short-chain enzymes revealed that the structure around the region of the medium-chain enzyme resembles those of eukaryotic farnesyl diphosphate synthases but not those of the other short-chain enzymes, reflecting the evolutional relationships among these enzymes.

J Hosp Infect, 2002 Sep, 52(1), 43 - 51
Genetic relationship between Escherichia coli strains isolated from the intestinal flora and those responsible for infectious diseases among patients hospitalized in intensive care units; Mereghetti L et al.; The exact origin of strains of Escherichia coli responsible for infectious diseases in intensive care units (ICUs) remains partly unknown . Our aim was to determine the nature of the link between strains from the intestinal flora of hospital staff, strains from the intestinal flora of patients hospitalized in ICUs and strains isolated from ICU patients with invasive diseases . For this purpose, 77 strains of E . coli were genetically characterized by exploring their entire genomes by random amplified polymorphism of DNA (RAPD), and by determining their phylogenetic position in ECOR (E . coli reference) groups, the virulence factors harboured (pap, sfa, afa, hly, aer and cnf) and their ability to mutate . The strains isolated from the intestinal flora of hospital staff were found to constitute a genetically heterogeneous population compared with the strains isolated from ICU carriers, which were highly clustered . The latter strains harboured numerous virulence factors, and 80% belonged to the group ECOR B2 . The strains isolated from infected patients harboured fewer virulence factors than those from the ICU carriers, and only half belonged to ECOR B2 . Moreover, these strains were more genetically related to strains from hospital staff than to strains from ICU carriers . Thus, the exogenous origin of the E . coli strains is probably almost as important as translocation from intestinal flora in ICUs . Moreover, a strong mutator phenotype had a minor, or no, role in the rapid adaptation to modifications in the ecological environment.

Cell, 2002 Oct 4, 111(1), 129 - 40
Orientation of ribosome recycling factor in the ribosome from directed hydroxyl radical probing; Lancaster L et al.; Ribosome recycling factor (RRF) disassembles posttermination complexes in conjunction with elongation factor EF-G, liberating ribosomes for further rounds of translation . The striking resemblance of its L-shaped structure to that of tRNA has suggested that the mode of action of RRF may be based on mimicry of tRNA . Directed hydroxyl radical probing of 16S and 23S rRNA from Fe(II) tethered to ten positions on the surface of E . coli RRF constrains it to a well-defined location in the subunit interface cavity . Surprisingly, the orientation of RRF in the ribosome differs markedly from any of those previously observed for tRNA, suggesting that structural mimicry does not necessarily reflect functional mimicry.

Cell, 2002 Oct 4, 111(1), 105 - 15
Crystal structure and functional analysis of the histone methyltransferase SET7/9; Wilson JR et al.; Methylation of lysine residues in the N-terminal tails of histones is thought to represent an important component of the mechanism that regulates chromatin structure . The evolutionarily conserved SET domain occurs in most proteins known to possess histone lysine methyltransferase activity . We present here the crystal structure of a large fragment of human SET7/9 that contains a N-terminal beta-sheet domain as well as the conserved SET domain . Mutagenesis identifies two residues in the C terminus of the protein that appear essential for catalytic activity toward lysine-4 of histone H3 . Furthermore, we show how the cofactor AdoMet binds to this domain and present biochemical data supporting the role of invariant residues in catalysis, binding of AdoMet, and interactions with the peptide substrate.

J Am Chem Soc, 2002 Oct 16, 124(41), 12144 - 53
Solution NMR techniques for large molecular and supramolecular structures; Riek R et al.; Transverse relaxation-optimized spectroscopy (TROSY) or generation of heteronuclear multiple quantum coherences during the frequency labeling period and TROSY during the acquisition period have been combined either with cross-correlated relaxation-induced polarization transfer (CRIPT) or cross-correlated relaxation-enhanced polarization transfer (CRINEPT) to obtain two-dimensional (2D) solution NMR correlation spectra of (15)N,(2)H-labeled homo-oligomeric macromolecules with molecular weights from 110 to 800 kDa . With the experimental conditions used, the line widths of the TROSY-components of the (1)H- and (15)N-signals were of the order of 60 Hz at 400 kDa, whereas, for structures of size 800 kDa, the line widths were about 75 Hz for (15)N and 110 Hz for (1)H . This paper describes the experimental schemes used and details of their setup for individual measurements . The performance of NMR experiments with large structures depends critically on the choice of the polarization transfer times, the relaxation delays between subsequent recordings, and the water-handling routines . Optimal transfer times for 2D {(15)N,(1)H}-CRIPT-TROSY experiments in H(2)O solutions were found to be 6 ms for a molecular weight of approximately 200 kDa, 2.8 ms for 400 kDa, and 1.4 ms for 800 kDa . These data validate theoretical predictions of inverse proportionality between optimal transfer time and size of the structure . The proton longitudinal relaxation times in H(2)O solution were found to be of the order of 0.8 s for structure sizes around 200 kDa, 0.4 s at 400 kDa, and 0.3 s at 800 kDa, which enabled the use of recycle times below 1 s . Since improper water handling results in severe signal loss, the water resonance was kept along the z-axis during the entire duration of the experiments by adjusting each water flip-back pulse individually.

Oncogene, 2002 Oct 10, 21(46), 7126 - 30
Elevated mutant frequencies and predominance of G:C to A:T transition mutations in Msh6(-/-) small intestinal epithelium; Mark SC et al.; The DNA mismatch repair (MMR) system is primarily responsible for purging newly synthesized DNA of errors incurred during semi-conservative replication . Lesion recognition is initially carried out by one of two heterodimeric protein complexes, MutS(alpha) or MutS(beta) . While the former, comprised of MSH2 and MSH6, recognizes mispairs as well as short (1-2 nucleotide) insertions/deletions (IDLs), the latter, made up of MSH2 and MSH3, is primarily responsible for recognizing 2-6 nucleotide IDLs . As most of the functional information on these heterodimers is derived from in vitro studies, it was of interest to study the in vivo consequences of a lack of MutS(alpha) . To this end, Big Blue( trade mark ) mice, that carry a lacI(+) transgenic lambda shuttle-phage mutational reporter, were crossed with Msh6(-/-) mice to evaluate the specific contribution of MutS(alpha) to genome integrity . Consistent with the importance of MutS(alpha) in lesion surveillance, small intestine epithelial cell DNA derived from lacI(+) Msh6(-/-) mice exhibited striking increases (average of 41-fold) in spontaneous mutant frequencies . Furthermore, the lacI gene mutation spectrum was dominated by G:C to A:T transitions, highlighting the critical importance of the MutS(alpha) complex in suppressing this frequently observed type of spontaneous mutation.

Proc Natl Acad Sci U S A, 2002 Oct 15, 99(21), 13538 - 43 Epub 2002 Oct 07.
Single molecule analysis of RNA polymerase elongation reveals uniform kinetic behavior; Adelman K et al.; By using single-molecule measurements, we demonstrate that the elongation kinetics of individual Escherichia coli RNA polymerase molecules are remarkably homogeneous . We find no evidence of distinct elongation states among RNA polymerases . Instead, the observed heterogeneity in transcription rates results from statistical variation in the frequency and duration of pausing . When transcribing a gene without strong pause sites, RNA polymerase molecules display transient pauses that are distributed randomly in both time and distance . Transitions between the active elongation mode and the paused state are instantaneous within the resolution of our measurements (<1 s) . This elongation behavior is compared with that of a mutant RNA polymerase that pauses more frequently and elongates more slowly than wild type.

Proc Natl Acad Sci U S A, 2002 Oct 15, 99(21), 13533 - 7 Epub 2002 Oct 07.
Lipid-protein interactions in DHPC micelles containing the integral membrane protein OmpX investigated by NMR spectroscopy; Fernandez C et al.; Intermolecular nuclear Overhauser effects (NOEs) between the integral outer membrane protein OmpX from Escherichia coli and dihexanoylphosphatidylcholine (DHPC) provided a detailed description of protein-detergent interactions . The NOEs were measured in 3D (15)N- and (13)C-resolved {(1)H,(1)H}-NOESY spectra recorded with selectively methyl-protonated and otherwise uniformly (2)H,(13)C,(15)N-labeled OmpX in micelles of DHPC at natural isotope abundance . In these mixed micelles the NMR structure of OmpX consists of an eight-stranded antiparallel beta-barrel . The OmpX surface area covered with intermolecular NOEs to the DHPC hydrophobic tails forms a continuous cylinder jacket of approximately 28 A in height, which is centered about the middle of the long axis through the beta-barrel . In addition, some intermolecular NOEs with methyl groups of the DHPC polar head were identified along both boundaries of this cylinder jacket . The experimental data suggest that the hydrophobic surface areas of OmpX are covered with a monolayer of DHPC molecules, which appears to mimic quite faithfully the embedding of the beta-barrel in a double-layer lipid membrane.

Mol Cell Biol, 2002 Nov, 22(21), 7701 - 11
A human mitochondrial GTP binding protein related to tRNA modification may modulate phenotypic expression of the deafness-associated mitochondrial 12S rRNA mutation; Li X et al.; Human mitochondrial 12S rRNA A1555G mutation has been found to be associated with deafness . However, putative nuclear modifier gene(s) has been proposed to regulate the phenotypic expression of this mutation . In yeast cells, mutant alleles of MSS1, encoding a mitochondrial GTP-binding protein, manifest a respiratory-deficient phenotype only when coupled with mitochondrial 15S rRNA P(R)(454) mutation corresponding to human A1555G mutation . This suggests that an MSS1-like modifier gene may influence the phenotypic expression of the A1555G mutation . We report here the identification and characterization of human MSS1 homolog, GTPBP3, the first identified vertebrate gene related to mitochondrial tRNA modification . The Gtpbp3 is the mitochondrial GTPase evolutionarily conserved from bacteria to mammals . Functional conservation of this protein is supported by the observation that isolated human GTPBP3 cDNA can complement the respiratory-deficient phenotype of yeast mss1 cells carrying P(R)(454) mutation . GTPBP3 is ubiquitously expressed in various tissues as multiple transcripts, but with a markedly elevated expression in tissues of high metabolic rates . We showed that Gtpbp3 localizes in mitochondrion . These observations suggest that the human GTPBP3 is a structural and functional homolog of yeast MSS1 . Thus, allelic variants in GTPBP3 could, if they exist, modulate the phenotypic manifestation of human mitochondrial A1555G mutation.

Mol Cell Biol, 2002 Nov, 22(21), 7524 - 34
High-resolution mapping of changes in histone-DNA contacts of nucleosomes remodeled by ISW2; Kassabov SR et al.; The imitation switch (ISWI) complex from yeast containing the Isw2 and Itc1 proteins was shown to preferentially slide mononucleosomes with as little as 23 bp of linker DNA from the end to the center of DNA . The contacts of unique residues in the histone fold regions of H4, H2B, and H2A with DNA were determined with base pair resolution before and after chromatin remodeling by a site-specific photochemical cross-linking approach . The path of DNA and the conformation of the histone octamer in the nucleosome remodeled or slid by ISW2 were not altered, because after adjustment for the new translational position, the DNA contacts at specific sites in the histone octamer had not been changed . Maintenance of the canonical nucleosome structure after sliding was also demonstrated by DNA photoaffinity labeling of histone proteins at specific sites within the DNA template . In addition, nucleosomal DNA does not become more accessible during ISW2 remodeling, as assayed by restriction endonuclease cutting . ISW2 was also shown to have the novel capability of counteracting transcriptional activators by sliding nucleosomes through Gal4-VP16 bound initially to linker DNA and displacing the activator from DNA.

J Biol Chem, 2002 Dec 13, 277(50), 48657 - 63 Epub 2002 Oct 04.
Escherichia coli glutamyl-tRNA reductase . Trapping the thioester intermediate; Schauer S et al.; In the first step of tetrapyrrole biosynthesis in Escherichia coli, glutamyl-tRNA reductase (GluTR, encoded by hemA) catalyzes the NADPH-dependent reduction of glutamyl-tRNA to glutamate-1-semialdehyde . Soluble homodimeric E . coli GluTR was made by co-expressing the hemA gene and the chaperone genes dnaJK and grpE . During Mg(2+)-stimulated catalysis, the reactive sulfhydryl group of Cys-50 in the E . coli enzyme attacks the alpha-carbonyl group of the tRNA-bound glutamate . The resulting thioester intermediate was trapped and detected by autoradiography . In the presence of NADPH, the end product, glutamate-1-semialdehyde, is formed . In the absence of NADPH, E . coli GluTR exhibited substrate esterase activity . The in vitro synthesized unmodified glutamyl-tRNA was an acceptable substrate for E . coli GluTR . Eight 5-aminolevulinic acid auxotrophic E . coli hemA mutants were genetically selected, and the corresponding mutations were determined . Most of the recombinant purified mutant GluTR enzymes lacked detectable activity . Based on the Methanopyrus kandleri GluTR structure, the positions of the amino acid exchanges are close to the catalytic domain (G7D, E114K, R314C, S22L/S164F, G44C/S105N/A326T, G106N, S145F) . Only GluTR G191D (affected in NADPH binding) revealed esterase but no reductase activity.

Protein Pept Lett, 2002 Oct, 9(5), 419 - 26
Cloning and high-level expression of scorpion toxin BmKITa1 in Escherichia coli and insect cells; Liu Z et al.; BmK ITa1 cDNA was cloned and highly expressed in E . coli and insect cell . SDS-PAGE and western blot analysis revealed that subunit molecular weight of expression products is about 40 kDa and 10 kDa respectively . The expression product purified by a Ni(2+)-IDA-sepharose 6B column was toxic for insect, which indicated that it was biologically activity . Furthermore, Quantitative estimation show that the biological activity of recombinant BmK ITa1 from Tn cells was more powerful than from E . coli.

Curr Protein Pept Sci, 2002 Aug, 3(4), 451 - 60
Structures and interactions of proteins involved in the coupling function of the protonmotive F(o)F(1)-ATP synthase; Gaballo A et al.; The mitochondrial F(1)F(o) ATP synthase complex has a key role in cellular energy metabolism . The general architecture of the enzyme is conserved among species and consists of a globular catalytic moiety F(1), protruding out of the inner side of the membrane, a membrane integral proton translocating moiety F(o), and a stalk connecting F(1) to F(o) . The X-ray crystallographic analysis of the structure of the bovine mitochondrial F(1) ATPase has provided a structural basis for the binding-change rotary mechanism of the catalytic process in F(1), in which the gamma subunit rotates in the central cavity of the F(1) alpha3/beta3 hexamer . Rotation of gamma and eta subunits in the E . coli enzyme and of, gamma and delta subunits in the mitochondrial enzyme, is driven, during ATP synthesis, by proton motive rotation of an oligomer of c subunits (10-12 copies) within the F(o) base piece . Average analysis of electron microscopy images and cross-linking results have revealed that, in addition to a central stalk, contributed by gamma and delta/eta subunits, there is a second lateral one connecting the peripheries of F(o) and F(1) . To gain deeper insight into the mechanism of coupling between proton translocation and catalytic activity (ATP synthesis and hydrolysis), studies have been undertaken on the role of F(1) and F(o) subunits which contribute to the structural and functional connection between the catalytic sector F(1) and the proton translocating moiety F(o) . These studies, which employed limited proteolysis, chemical cross-linking and functional analysis of the native and reconstituted F(1)F(o) complex, as well as isolated F(1), have shown that the N-terminus of alpha subunits, located at the top of the F(1) hexamer is essential for energy coupling in the F(1)F(o) complex . The alpha N-terminus domain appears to be connected to F(o) by OSCP (F(o) subunit conferring sensitivity of the complex to oligomycin) . In turn, OSCP contacts F(o)I-PVP(b) and d subunits, with which it constitutes a structure surrounding the central gamma and delta rotary shaft . Cross-linking of F(o)I-PVP(b) and gamma subunits causes a dramatic enhancement of downhill proton translocation decoupled from ATP synthesis but is without effect on ATP driven uphill proton transport . This would indicate the existence of different rate-limiting steps in the two directions of proton translocation through F(o) . In mitochondria, futile ATP hydrolysis by the F(1)F(o) complex is inhibited by the ATPase inhibitor protein (IF(1)), which reversibly binds at one side of the F(1)F(o) connection . The trans-membrane deltapH component of the respiratory deltap displaces IF(1) from the complex; in particular the matrix pH is the critical factor for IF(1)association and its related inhibitory activity . The 42L-58K segment of the IF(1) has been shown to be the most active segment of the protein; it interacts with the surface of one alpha/beta pairs of F(1), thus inhibiting, with the same pH dependence as the natural IF(1), the conformational interconversions of the catalytic sites involved in ATP hydrolysis . IF(1) has a relevant physiopathological role for the conservation of the cellular ATP pool in ischemic tissues . Under these conditions IF(1), which appears to be over expressed, prevents dissipation of the glycolytic ATP.

Mini Rev Med Chem, 2001 Sep, 1(3), 293 - 306
Aerobic nitroreduction by flavoproteins: enzyme structure, mechanisms and role in cancer chemotherapy; Skelly JV et al.; NQO1 (DT-diaphorase) and its truncated isoenzyme, the metalloenzyme NQO2, can reduce quinone substrates by two-electron transfer . While NQO1 is a known detoxification enzyme, the function of NQO2 is less well understood . Both rat NQO1 and human NQO2 reductively bioactivate the dinitroarene CB 1954 to a cytotoxic product that behaves as a difunctional DNA-crosslinking species with potent anti-tumour activity, although human NQO1 is much less effective . A FMN-dependent nitroreductase from E . coli B also reduces quinones and reductively bioactivates CB 1954 . However, this enzyme reduces CB 1954 to the 2- and 4-hydroxylamines in equivalent yield, whereas NQO1 and NQO2 generate only the 4-isomer . The reduction profile is a key factor in the development of anti-tumour prodrugs, where distinct delivery strategies are being evaluated: prodrug therapy, antibody-, macromolecule and gene-directed enzyme prodrug therapy (ADEPT, MDEPT or GDEPT) . The flavoprotein enzymes are explored in terms of structure and bioreduction mechanism, particularly for use in the design of novel prodrugs with potential application as chemotherapeutic agents.

Curr Protein Pept Sci, 2001 Sep, 2(3), 227 - 44
Chaperone-assisted protein folding in the cell cytoplasm; Houry WA; Folding of polypeptides in the cell typically requires the assistance of a set of proteins termed molecular chaperones . Chaperones are an essential group of proteins necessary for cell viability under both normal and stress conditions . There are several chaperone systems which carry out a multitude of functions all aimed towards insuring the proper folding of target proteins . Chaperones can assist in the efficient folding of newly-translated proteins as these proteins are being synthesized on the ribosome and can maintain pre-existing proteins in a stable conformation . Chaperones can also promote the disaggregation of preformed protein aggregates . Many of the identified chaperones are also heat shock proteins . The general mechanism by which chaperones carry out their function usually involves multiple rounds of regulated binding and release of an unstable conformer of target polypeptides . The four main chaperone systems in the Escherichia coli cytoplasm are as follows . (1) Ribosome-associated trigger factor that assists in the folding of newly-synthesized nascent chains . (2) The Hsp 70 system consisting of DnaK (Hsp 70), its cofactor DnaJ (Hsp 40), and the nucleotide exchange factor GrpE . This system recognizes polypeptide chains in an extended conformation . (3) The Hsp 60 system, consisting of GroEL (Hsp 60) and its cofactor GroES (Hsp 10), which assists in the folding of compact folding intermediates that expose hydrophobic surfaces . (4) The Clp ATPases which are typically members of the Hsp 100 family of heat shock proteins . These ATPases can unfold proteins and disaggregate preformed protein aggregates to target them for degradation . Several advances have recently been made in characterizing the structure and function of all of these chaperone systems . These advances have provided us with a better understanding of the protein folding process in the cell.

Biochemistry, 2002 Oct 15, 41(41), 12520 - 8
GTPase activation of elongation factors Tu and G on the ribosome; Mohr D et al.; The GTPase activity of elongation factors Tu and G is stimulated by the ribosome . The factor binding site is located on the 50S ribosomal subunit and comprises proteins L7/12, L10, L11, the L11-binding region of 23S rRNA, and the sarcin-ricin loop of 23S rRNA . The role of these ribosomal elements in factor binding, GTPase activation, or functions in tRNA binding and translocation, and their relative contributions, is not known . By comparing ribosomes depleted of L7/12 and reconstituted ribosomes, we show that, for both factors, interactions with L7/12 and with other ribosomal residues contribute about equally and additively to GTPase activation, resulting in an overall 10(7)-fold stimulation . Removal of L7/12 has little effect on factor binding to the ribosome . Effects on other factor-dependent functions, i.e., A-site binding of aminoacyl-tRNA and translocation, are fully explained by the inhibition of GTP hydrolysis . Based on these results, we propose that L7/12 stimulates the GTPase activity of both factors by inducing the catalytically active conformation of the G domain . This effect appears to be augmented by interactions of other structural elements of the large ribosomal subunit with the switch regions of the factors.

Biochemistry, 2002 Oct 15, 41(41), 12421 - 6
A positive charge preservation at position 116 of alpha A-crystallin is critical for its structural and functional integrity; Bera S et al.; An autosomal dominant congenital cataract associated with a missense mutation, Arg-116 to Cys (R116C), in the coding sequence of human alphaA-crystallin has been reported . Subsequent study of this mutant, generated by site-directed mutagenesis, showed significant changes in secondary and tertiary structures, partial loss of chaperone activity, and substantially increased oligomeric size . The study presented here aims to show whether these changes are due to the loss of a positive charge at this position or due to the presence of an extra Cys . To show this, Arg-116 in alphaA-crystallin was mutated to Lys (R116K), Cys (R116C), Gly (R116G), and Asp (R116D) and expressed in Escherichia coli cells . The wild-type (alphaA-wt) and mutant proteins were purified by size exclusion chromatography and characterized by measurements of circular dichroism, intrinsic tryptophan fluorescence, and TNS fluorescence and by determination of molecular masses and chaperone function which was assessed as the ability to suppress target protein aggregation or enhance target protein refolding . Mutation of Arg-116 to a Cys or Gly showed very similar changes in structure, oligomerization, and chaperone function which suggest that the presence of this Cys per se is not the cause of the changes . The R116K mutant, on the other hand, had nearly the same structure, oligomeric size, and chaperone function as alphaA-wt, whereas the mutant with an acidic amino acid in this position, R116D, showed drastic changes in protein structure . Thus, a positive charge must be preserved at this position for the structural and functional integrity of alphaA-crystallin.

Biochemistry, 2002 Oct 15, 41(41), 12284 - 96
NMR solution structure of ATTp, an Arabidopsis thaliana trypsin inhibitor; Zhao Q et al.; The three-dimensional structure of the precursor form of the Arabidopsis thaliana trypsin inhibitor (ATT(p), GenBank entry Z46816), a 68-residue (approximately 7.5 kDa) rapeseed class proteinase inhibitor, has been determined in solution at pH 5.0 and 25 degrees C by multinuclear magnetic resonance spectroscopy . The protein contains one alpha-helix and two strands of antiparallel beta-sheet, with a type IV beta-turn connecting the two strands . The alpha-helix and the inhibitory loop are connected to the beta-sheet through three disulfide bridges; a fourth disulfide bridge connects the N- and C-termini . The overall structural topology of ATT(p) is similar to those of the sweet tasting protein brazzein (rmsd of 3.0 A) and the antifungal protein Rs-Afp1 {a knottin protein from radish (Raphanus sativus), rmsd of 2.7 A} . The precursor segment in ATT(p) is disordered, as visualized by the final 20-conformer ensemble and as confirmed by (15)N heteronuclear NOE analysis . The overall fold of ATT(p) is distinct from those of other classes of serine proteinase inhibitors except in the inhibitor loop; therefore, it represents a new inhibitor fold.

Plant Mol Biol, 2002 Oct, 50(3), 511 - 21
Cloning and functional expression of two plant thiol methyltransferases: a new class of enzymes involved in the biosynthesis of sulfur volatiles; Attieh J et al.; Glucosinolates are defensive compounds found in several plant families . We recently described five distinct isoforms of a novel plant enzyme, thiol methyltransferase (TMT), which methylate the hydrolysis products of glucosinolates to volatile sulfur compounds that have putative anti-insect and anti-pathogen roles . In the work presented here, two cDNAs encoding these enzymes (cTMT1 and cTMT2) were isolated by screening a cabbage cDNA library with an Arabidopsis EST showing high sequence homology to one TMT isoform . The genomic clone of cTMT1 was subsequently amplified by PCR . Both cDNAs encoded polypeptides of identical lengths (227 amino acids) and similar predicted masses (ca . 25 kDa), but differing in 13 residues . The cDNAs contained the typical methyltransferase signatures, but were otherwise distinct from conventionally known N-, O- or S-methyltransferases . A chloride methyl transferase was the only gene with an assigned function that shared significant similarity with the TMT cDNAs . Southern analysis indicated single copy for each TMT gene . The two cDNAs were expressed in Escherichia coli . The substrate range, kinetic properties and molecular sizes of the purified recombinant proteins were comparable to those of the native enzyme . These data, together with the detection of the sequenced amino acid motif of one native TMT peptide in the cDNAs, confirmed that the latter were authentic TMTs . The expression pattern of the TMTs in various cabbage tissues was consistent with their association with glucosinolates . The cloning of this new class of plant genes furnishes crucial molecular tools to understand the role of this metabolic sector in plant defenses against biotic stress.

Plant Mol Biol, 2002 Oct, 50(3), 393 - 403
Molecular analysis of de novo pyrimidine synthesis in solanaceous species; Giermann N et al.; The de novo synthesis of pyrimidine nucleotides in plants has been analysed on a molecular level with special focus on cDNA cloning and structure analysis of all genes involved and their expression pattern during development . The exhaustive cloning of all cDNAs resulted from screening with heterologous cDNAs or by using complementation strategies with Escherichia coli mutants and subsequent enzyme activity measurements . Southern hybridization and comparison with the Arabidopsis genome reveals plant specific aspects and a simple genomic organization of pyrimidine synthesis in plants, which is superimposed by the postulated, complex subcellular compartmentalization . Northern hybridization evinces coordinated expression of all genes under developmental control during tobacco leaf growth.

Nat Struct Biol, 2002 Nov, 9(11), 862 - 9
The catalytic mechanism of the ESA1 histone acetyltransferase involves a self-acetylated intermediate; Yan Y et al.; Yeast ESA1 is a member of the MYST subfamily of histone acetyltransferases (HATs), which use acetyl-coenzyme A (CoA) to acetylate specific Lys residues within histones to regulate gene expression . The structure of an ESA1-CoA complex reveals structural similarity to the catalytic core of the GCN5/PCAF subfamily of HAT proteins . Here we report additional structural and functional studies on ESA1 that demonstrate that histone acetylation proceeds through an acetyl-cysteine enzyme intermediate . This Cys residue is strictly conserved within the MYST members, suggesting a common mode of catalysis by this HAT subfamily . However, this mode of catalysis differs dramatically from the GCN5/PCAF subfamily, which mediate direct nucleophilic attack of the acetyl-CoA cofactor by the enzyme-deprotonated substrate lysine of the histone . These results demonstrate that different HAT subfamilies can use distinct catalytic mechanisms, which have implications for their distinct biological roles and for the development of HAT-specific inhibitors.

Nat Biotechnol, 2002 Nov, 20(11), 1140 - 5 Epub 2002 Oct 07.
Engineering tolerance and hyperaccumulation of arsenic in plants by combining arsenate reductase and gamma-glutamylcysteine synthetase expression; Dhankher OP et al.; We have developed a genetics-based phytoremediation strategy for arsenic in which the oxyanion arsenate is transported aboveground, reduced to arsenite, and sequestered in thiol-peptide complexes . The Escherichia coli arsC gene encodes arsenate reductase (ArsC), which catalyzes the glutathione (GSH)-coupled electrochemical reduction of arsenate to the more toxic arsenite . Arabidopsis thaliana plants transformed with the arsC gene expressed from a light-induced soybean rubisco promoter (SRS1p) strongly express ArsC protein in leaves, but not roots, and were consequently hypersensitive to arsenate . Arabidopsis plants expressing the E . coli gene encoding gamma-glutamylcysteine synthetase (gamma-ECS) from a strong constitutive actin promoter (ACT2p) were moderately tolerant to arsenic compared with wild type . However, plants expressing SRS1p/ArsC and ACT2p/gamma-ECS together showed substantially greater arsenic tolerance than gamma-ECS or wild-type plants . When grown on arsenic, these plants accumulated 4- to 17-fold greater fresh shoot weight and accumulated 2- to 3-fold more arsenic per gram of tissue than wild type or plants expressing gamma-ECS or ArsC alone . This arsenic remediation strategy should be applicable to a wide variety of plant species.

Plant Cell, 2002 Oct, 14(10), 2325 - 38
Rose scent: genomics approach to discovering novel floral fragrance-related genes; Guterman I et al.; For centuries, rose has been the most important crop in the floriculture industry; its economic importance also lies in the use of its petals as a source of natural fragrances . Here, we used genomics approaches to identify novel scent-related genes, using rose flowers from tetraploid scented and nonscented cultivars . An annotated petal EST database of approximately 2100 unique genes from both cultivars was created, and DNA chips were prepared and used for expression analyses of selected clones . Detailed chemical analysis of volatile composition in the two cultivars, together with the identification of secondary metabolism-related genes whose expression coincides with scent production, led to the discovery of several novel flower scent-related candidate genes . The function of some of these genes, including a germacrene D synthase, was biochemically determined using an Escherichia coli expression system . This work demonstrates the advantages of using the high-throughput approaches of genomics to detail traits of interest expressed in a cultivar-specific manner in nonmodel plants . EST sequences were submitted to the GenBank database (accession numbers BQ 103855 to BQ 106728).

Microbiology, 2002 Oct, 148(Pt 10), 3265 - 75
Intragenic suppressors of a mutation in the aspartate chemoreceptor gene that abolishes binding of the receptor to methyltransferase; Shiomi D et al.; In the chemotaxis of Escherichia coli, receptor methylation is the key process of adaptation . The methyltransferase CheR binds to the carboxy-terminal NWETF sequence of major chemoreceptors . The substitution of Ala for Trp of this sequence (W550A) of the aspartate chemoreceptor (Tar) abolishes its CheR-binding ability . In this study, six independent intragenic suppressors of the mutation were isolated . They were divided into two classes . Tar carrying the class I suppressors (G278A-L488M, T334A, G278A, G278C and A398T) showed signal biases toward tumbling, corresponding to increased activities of the receptor-associated histidine kinase CheA . These suppressors further reduced the unstimulated methylation level of Tar-W550A, but allowed slight but significant stimulation of methylation by aspartate . Some other CheA-activating mutations were also found to serve as class I suppressors . These results suggest that the class I suppressors compensate for the signal bias of Tar-W550A caused by its low methylation level and that the NWETF sequence is required primarily to maintain an appropriate level of methylation by increasing the local concentration of CheR around the receptor . The class II suppressor was a mutation in the termination codon (Op554W) resulting in the addition of 11 residues containing an xWxxF motif . This revertant Tar supported chemotaxis and was methylated almost as effectively as wild-type Tar . This effect was reversed by introducing a mutation in the xWxxF motif . These results reinforce the importance of the xWxxF motif and suggest that the motif does not have to be located at the extreme carboxy terminus.

Microbiology, 2002 Oct, 148(Pt 10), 3213 - 22
Effects of the Min system on nucleoid segregation in Escherichia coli; Akerlund T et al.; The Min system of Escherichia coli directs cell division to the mid-cell by a mechanism that involves the dynamic localization of all of its three constituent proteins, MinC, MinD and MinE . Both the Min system and the nucleoid regulate cell division negatively and strains of E . coli lacking a functional Min system can divide at nucleoid-free cell poles in addition to the nucleoid-free region between newly segregated nucleoids . Interestingly, E . coli strains with a defective Min system have disturbed nucleoid segregation and the cause for this disturbance is not known . It is reported here that growth conditions promoting a higher frequency of polar divisions also lead to a more pronounced disturbance in nucleoid segregation . In strains with an intact Min system, expression of MinE, but not of MinD, from an inducible promoter was followed by impaired nucleoid segregation . These results suggest that the disturbed nucleoid segregation in min mutants is not caused by polar divisions per se, nor by impaired resolution of chromosome dimers in min mutants, leaving open the possibility that the Min system has a direct effect on nucleoid segregation . It is also shown how the disturbed nucleoid segregation can explain in part the unexpected finding that the clear majority of cells in min mutant populations contain 2(n) (n=0, 1, 2.) origins of replication.

J Virol, 2002 Nov, 76(21), 10598 - 607
Nuclear localization of human immunodeficiency virus type 1 preintegration complexes (PICs): V165A and R166A are pleiotropic integrase mutants primarily defective for integration, not PIC nuclear import; Limon A et al.; Retroviral replication requires the integration of reverse-transcribed viral cDNA into a cell chromosome . A key barrier to forming the integrated provirus is the nuclear envelope, and numerous regions in human immunodeficiency virus type 1 (HIV-1) have been shown to aid the nuclear localization of viral preintegration complexes (PICs) in infected cells . One region in integrase (IN), composed of Val-165 and Arg-166, was reportedly essential for HIV-1 replication and nuclear localization in all cell types . In this study we confirmed that HIV-1(V165A) and HIV-1(R166A) were replication defective and that less mutant viral cDNA localized to infected cell nuclei . However, we present three lines of evidence that argue against a specific role for Val-165 and Arg-166 in PIC nuclear import . First, results of transient transfections revealed that V165A FLAG-tagged IN and green fluorescent protein-IN fusions carrying either V165A or R166A predominantly localized to cell nuclei . Second, two different strains of previously described class II IN mutant viruses displayed similar nuclear entry profiles to those observed for HIV-1(V165A) and HIV-1(R166A), suggesting that defective nuclear import may be a common phenotype of replication-defective IN mutant viruses . Third, V165A and R166A mutants were defective for in vitro integration activity, when assayed both as PICs isolated from infected T-cells and as recombinant IN proteins purified from Escherichia coli . Based on these results, we conclude that HIV-1(V165A) and HIV-1(R166A) are pleiotropic mutants primarily defective for IN catalysis and that Val-165 and Arg-166 do not play a specific role in the nuclear localization of HIV-1 PICs in infected cells.

J Biol Chem, 2002 Dec 6, 277(49), 47770 - 8 Epub 2002 Oct 03.
Non-canonical transit peptide for import into the chloroplast; Miras S et al.; The large majority of plastid proteins are nuclear-encoded and, thus, must be imported within these organelles . Unlike most of the outer envelope proteins, targeting of proteins to all other plastid compartments (inner envelope membrane, stroma, and thylakoid) is strictly dependent on the presence of a cleavable transit sequence in the precursor N-terminal region . In this paper, we describe the identification of a new envelope protein component (ceQORH) and demonstrate that its subcellular localization is limited to the inner membrane of the chloroplast envelope . Immunopurification, microsequencing of the natural envelope protein and cloning of the corresponding full-length cDNA demonstrated that this protein is not processed in the N-terminal region during its targeting to the inner envelope membrane . Transient expression experiments in plant cells were performed with truncated forms of the ceQORH protein fused to the green fluorescent protein . These experiments suggest that neither the N-terminal nor the C-terminal are essential for chloroplastic localization of the ceQORH protein . These observations are discussed in the frame of the endosymbiotic theory of chloroplast evolution and suggest that a domain of the ceQORH bacterial ancestor may have evolved so as to exclude the general requirement of an N-terminal plastid transit sequence.

Genome Res, 2002 Oct, 12(10), 1523 - 32
Factors influencing the identification of transcription factor binding sites by cross-species comparison; McCue LA et al.; As the number of sequenced genomes has grown, the questions of which species are most useful and how many genomes are sufficient for comparison have become increasingly important for comparative genomics studies . We have systematically addressed these questions with respect to phylogenetic footprinting of transcription factor (TF) binding sites in the gamma-proteobacteria, and have evaluated the statistical significance of our motif predictions . We used a study set of 166 Escherichia coli genes that have experimentally identified TF binding sites upstream of the gene, with orthologous data from nine additional gamma-proteobacteria for phylogenetic footprinting . Just three species were sufficient for approximately 74.0% of the motif predictions to correspond to the experimentally reported E . coli sites, and important characteristics to consider when choosing species were phylogenetic distance, genome size, and natural habitat . We also performed simulations using randomized data to determine the critical maximum a posteriori probability (MAP) values for statistical significance of our motif predictions (P = 0.05) . Approximately 60% of motif predictions containing sites from just three species had average MAP values above these critical MAP values . The inclusion of a species very closely related to E . coli increased the number of statistically significant motif predictions, despite substantially increasing the critical MAP value.

Am J Pathol, 2002 Oct, 161(4), 1475 - 84
Lipopolysaccharide induces overexpression of MUC2 and MUC5AC in cultured biliary epithelial cells: possible key phenomenon of hepatolithiasis; Zen Y et al.; Bacterial infection, bile stasis, mucin hypersecretion, and an alteration of the mucin profile such as an aberrant expression of gel-forming apomucin (MUC2 and MUC5AC) in the intrahepatic biliary tree are thought to be important in the lithogenesis of hepatolithiasis . So far, there have been no detailed studies linking bacterial infection to altered mucus secretion of biliary epithelium . In this study, the influence of lipopolysaccharide (LPS), a bacterial component, on apomucin expression in cultured murine biliary epithelial cells was examined with emphasis on the participation of tumor necrosis factor (TNF)-alpha . It was found that LPS up-regulated the expression of MUC2 and MUC5AC in cultured murine biliary epithelial cells . LPS also induced the expression of TNF-alpha in biliary epithelial cells and its secretion into the culture medium . The up-regulation of these apomucins was inhibited by pretreatment with TNF-alpha antibody . TNF-alpha alone also induced the overexpression of MUC2 and MUC5AC in cultured biliary epithelial cells . This overexpression was inhibited by pretreatment with calphostin C, an inhibitor of protein kinase C . These findings suggest that LPS can induce overexpression of MUC2 and MUC5AC in biliary epithelial cells via synthesis of TNF-alpha and activation of protein kinase C . This mechanism might be involved in the lithogenesis of hepatolithiasis.

Toxicon, 2002 Oct, 40(10), 1383 - 7
Bordetella pertussis adenylate cyclase toxin: a versatile screening tool; Dautin N et al.; The calmodulin-activated adenylate cyclase (AC) toxin is an essential virulence factor of Bordetella pertussis, the causative agent of whooping cough . This toxin has been exploited to devise screening techniques for investigating diverse biological processes . This mini-review describes several such applications . First, AC has been utilized as a selective reporter for protein translocation from bacteria to eukaryotic cells, in particular to study protein targeting by type III secretion machinery . More recently, AC has been used as a signal transducer in Escherichia coli to elaborate genetic screens for protein-protein interactions ("bacterial two-hybrid system") or site-specific proteolytic activities .

Trends Biochem Sci, 2002 Oct, 27(10), 489 - 92
Nanotransducers in cellular redox signaling: modification of thiols by reactive oxygen and nitrogen species; Cooper CE et al.; The control of signal transduction involves post-translational modification of proteins at key amino acids . Cysteine residues are important in the control of 'redox' cell-signaling pathways, as thiol chemistry offers the possibility of modification by structurally diverse species, including those derived from oxidized lipids, peroxides or nitric oxide . An important and provocative study of the modification of thiols in the transcription factor OxyR recently extended this hypothesis . The findings offer the enticing possibility that the cell can distinguish between different degrees of oxidant and nitrosative exposure by modification at a single site on a signaling molecule.

J Virol Methods, 2002 Oct, 106(1), 141 - 51
Generation of recombinant fowlpox virus using the non-essential F11L orthologue as insertion site and a rapid transient selection strategy; Boulanger D et al.; Avipoxviruses show an abortive replication phenotype in mammalian cells and are under evaluation as safe vectors for vaccination . Non-essential gene sequences located in highly conserved regions of virus genomes are considered particularly useful to integrate heterologous DNA . Fowlpox virus F11L orthologue is described in this paper as a suitable locus for insertion into fowlpox virus genome . Disruption of the F11L coding sequence by integration of an expression cassette for the Escherichia coli lacZ and guanine phosphoribosyltransferase marker genes resulted in the isolation of replication competent knockout viruses . Growth of F11L-knockout viruses in primary chicken embryo fibroblasts was unimpaired in comparison to wild type-virus . To test the generation of vector viruses, an insertion plasmid was constructed that contains F11L-specific sequences for homologous recombination, the E . coli lacZ and gpt genes as transient selectable marker, and the vaccinia virus early/late promoter P7.5 for transcriptional control of target gene expression . The coding sequence of the melanoma-associated antigen tyrosinase was chosen as model recombinant gene . Isolation of tyrosinase-recombinant viruses, which produced stably the insert, demonstrated the usefulness of the F11L-insertion site for the generation of fowlpox vectors . Rapid isolation of those recombinants was achieved by using a double selective system and linearising the vector plasmid before transfection.

Antiviral Res, 2002 Nov, 56(2), 99 - 114
Biochemical characterization of rhinovirus RNA-dependent RNA polymerase; Hung M et al.; Human rhinoviruses (HRV) represent the single most important causative agent of the common cold . The HRV genome encodes an RNA-dependent RNA polymerase (RdRp) designated 3D polymerase that is required for replication of the HRV RNA genome . We have expressed and purified recombinant HRV-16 3D polymerase to near homogeneity from Escherichia coli transformed with an expression plasmid containing the full-length 460 amino acid HRV-16 3D sequence with a methionine at the N-terminus and a glycine-serine linker followed by a 6-histidine affinity tag at the C-terminus . The purified recombinant protein has rifampicin-resistant activity in a poly(A)-dependent poly(U) polymerase assay while corresponding fractions similarly purified from E . coli transformed with an expression plasmid without the HRV-16 3D sequence showed no activity . The optimal conditions for temperature, pH, divalent cations Mg(2+) and Mn(2+), and KCl were determined . The recombinant protein has RNA polymerase activity on homopolymeric templates poly(A) and poly(C) and heteropolymeric RNA templates primed with either RNA or DNA oligonucleotide primers or self-primed by a copy-back mechanism . A unique, secondary structureless heteropolymeric RNA template that is an efficient substrate was developed to facilitate kinetic characterizations of the enzyme . In the presence of Mg(2+), the enzyme displayed strong base and sugar specificity . However, when Mg(2+) was replaced by Mn(2+) specificity for ribonucleotides was lost, utilization of deoxynucleotides became possible and primer-independent activity was observed on the poly(C) template . Zn(2+) was found to inhibit HRV-16 3D polymerase with an IC(50) as low as 0.6 microM by a mechanism distinct from the magnesium ion stimulation . The activity of this 6His-tagged HRV-16 3D polymerase was compared with that of a recombinant HRV-16 3D polymerase expressed without the 6His-tag and was found to be identical . The availability of recombinant rhinovirus RdRp in a purified form will facilitate the structure-function analysis of this enzyme as well as the identification of specific inhibitors to the rhinovirus 3D polymerase that have therapeutic value in the treatment of the common cold .

J Mol Biol, 2002 Oct 4, 322(5), 1135 - 46
Oligomeric properties and signal peptide binding by Escherichia coli Tat protein transport complexes; de Leeuw E et al.; The Escherichia coli Tat apparatus is a protein translocation system that serves to export folded proteins across the inner membrane . The integral membrane proteins TatA, TatB and TatC are essential components of this pathway . Substrate proteins are directed to the Tat apparatus by specialized N-terminal signal peptides bearing a consensus twin-arginine sequence motif . Here we have systematically examined the Tat complexes that can be purified from overproducing strains . Our data suggest that the TatA, TatB and TatC proteins are found in at least two major types of high molecular mass complex in detergent solution, one consisting predominantly of TatA but with a small quantity of TatB, and the other based on a TatBC unit but also containing some TatA protein . The latter complex is shown to be capable of binding a Tat signal peptide . Using an alternative purification strategy we show that it is possible to isolate a TatABC complex containing a high molar excess of the TatA component.

J Mol Biol, 2002 Oct 4, 322(5), 1039 - 52
Quantitative assessment of peptide sequence diversity in M13 combinatorial peptide phage display libraries; Rodi DJ et al.; Novel statistical methods have been developed and used to quantitate and annotate the sequence diversity within combinatorial peptide libraries on the basis of small numbers (1-200) of sequences selected at random from commercially available M13 p3-based phage display libraries . These libraries behave statistically as though they correspond to populations containing roughly 4.0+/-1.6% of the random dodecapeptides and 7.9+/-2.6% of the random constrained heptapeptides that are theoretically possible within the phage populations . Analysis of amino acid residue occurrence patterns shows no demonstrable influence on sequence censorship by Escherichia coli tRNA isoacceptor profiles or either overall codon or Class II codon usage patterns, suggesting no metabolic constraints on recombinant p3 synthesis . There is an overall depression in the occurrence of cysteine, arginine and glycine residues and an overabundance of proline, threonine and histidine residues . The majority of position-dependent amino acid sequence bias is clustered at three positions within the inserted peptides of the dodecapeptide library, +1, +3 and +12 downstream from the signal peptidase cleavage site . Conformational tendency measures of the peptides indicate a significant preference for inserts favoring a beta-turn conformation . The observed protein sequence limitations can primarily be attributed to genetic codon degeneracy and signal peptidase cleavage preferences . These data suggest that for applications in which maximal sequence diversity is essential, such as epitope mapping or novel receptor identification, combinatorial peptide libraries should be constructed using codon-corrected trinucleotide cassettes within vector-host systems designed to minimize morphogenesis-related censorship.

J Mol Biol, 2002 Oct 4, 322(5), 983 - 95
Fur-DNA interactions at the bidirectional fepDGC-entS promoter region in Escherichia coli; Lavrrar JL et al.; The transcriptional repressor Fur binds to a 19-bp consensus sequence, 5'-GATAATGATAATCATTATC-3', under high iron conditions . The fepDGC-entS promoter of Escherichia coli contains two Fur-binding sites (FBS) offset by 6bp . Genetic studies of this promoter region revealed two mutations that exhibited a loss of iron regulation in vivo . One mutation altered the upstream portion of FBS 1, whereas the other, originally created to improve entS promoter strength, inadvertently altered the downstream portion of FBS 2 . In both cases, there remains a 19-bp sequence that by current models should be sufficient for Fur binding . The effect of these mutations on Fur binding was examined using in vitro gel retardation assays and DNase I footprinting experiments . Though Fur bound wild-type DNA with high affinity, its affinity for the mutants was reduced, suggesting that both sites are required . In addition, gel shift studies demonstrated that the Fur-promoter complexes exhibit a unique hierarchy of binding, with distinct species forming at increasing concentrations of Fur . The DNA sequences bound in each gel-shifted species were determined using a coupled gel shift/footprint technique . The data presented here, with previously published data, suggest a new model for Fur-DNA interactions similar to that seen with the transcriptional repressor, DtxR . The model predicts that the 19-bp consensus Fur operator is configured as overlapping 13-mer sequences, and that two Fur dimers interact with these sequences from opposite faces of the helix.

Mol Microbiol, 2002 Oct, 46(1), 245 - 56
Acidic phospholipids inhibit the DNA-binding activity of DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli; Makise M et al.; In order to initiate chromosomal DNA replication in Escherichia coli, the DnaA protein must bind to both ATP and the origin of replication (oriC) . Acidic phospholipids are known to inhibit DnaA binding to ATP, and here we examine the effects of various phospholipids on DnaA binding to oriC . Among the phospholipids in E . coli membrane, cardiolipin showed the strongest inhibition of DnaA binding to oriC . Synthetic phosphatidylglycerol containing unsaturated fatty acids inhibited binding more potently than did synthetic phosphatidylglycerol containing saturated fatty acids, suggesting that membrane fluidity is important . Thus, acidic phospholipids seem to inhibit DnaA binding to both oriC and adenine nucleotides in the same manner . Adenine nucleotides bound to DnaA did not affect the inhibitory effect of cardiolipin on DnaA binding to oriC . A mobility-shift assay re-vealed that acidic phospholipids inhibited formation of a DnaA-oriC complex containing several DnaA molecules . DNase I footprinting of DnaA binding to oriC showed that two DnaA binding sites (R2 and R3) were more sensitive to cardiolipin than other DnaA binding sites . Based on these in vitro data, the physiological relevance of this inhibitory effect of acidic phospholipids on DnaA binding to oriC is discussed.

Mol Microbiol, 2002 Oct, 46(1), 215 - 22
Sequence-specific interaction of nascent antiterminator RNA with the zinc-finger motif of Escherichia coli RNA polymerase; Sen R et al.; The N-terminal Zn-finger motif of the beta' subunit of RNA polymerase contains two pairs of invariant cysteines flanking a moderately well-conserved segment of 13 amino acids that is rich in basic residues . Previous work showed that replacement of certain Zn-finger residues prevented transcription antitermination in response to phage HK022 put sites . Nascent put RNA binds to and modifies transcribing polymerase, so that it becomes resistant to termination . To characterize the Zn finger further, we replaced each of the basic residues with alanine and determined the effects of the substitutions on termination, antitermination and cell viability . All the mutants were defective in put-mediated antitermination . The severity of the defect depended on the mutant and on the sequence of the upstream stem-loop of put RNA . Some, but not all, mutants distinguished between put variants that differed in this region . This suggests that the Zn-finger motif interacts directly and specifically with put RNA . All the mutants in the basic residues complemented a temperature-sensitive beta' mutant for cell growth at a non-permissive temperature, and those mutant enzymes that were tested transcribed and terminated normally in vitro on a template that lacked a put site.

Mol Microbiol, 2002 Oct, 46(1), 203 - 14
Physiological role of the GlnK signal transduction protein of Escherichia coli: survival of nitrogen starvation; Blauwkamp TA et al.; Escherichia coli contains two PII-like signal trans-duction proteins, PII and GlnK, involved in nitrogen assimilation . We examined the roles of PII and GlnK in controlling expression of glnALG, glnK and nac during the transition from growth on ammonia to nitrogen starvation and vice versa . The PII protein exclusively controlled glnALG expression in cells adapted to growth on ammonia, but was unable to limit nac and glnK expression under conditions of nitrogen starvation . Conversely, GlnK was unable to limit glnALG expression in cells adapted to growth on ammonia, but was required to limit expression of the glnK and nac promoters during nitrogen starvation . In the absence of GlnK, very high expression of the glnK and nac promoters occurred in nitrogen-starved cells, and the cells did not reduce glnK and nac expression when given ammonia . Thus, one specific role of GlnK is to regulate the expression of Ntr genes during nitrogen starvation . GlnK also had a dramatic effect on the ability of cells to survive nitrogen starvation and resume rapid growth when fed ammonia . After being nitrogen starved for as little as 10 h, cells lacking GlnK were unable to resume rapid growth when given ammonia . In contrast, wild-type cells that were starved immediately resumed rapid growth when fed ammonia . Cells lacking GlnK also showed faster loss of viability during extended nitrogen starvation relative to wild-type cells . This complex phenotype resulted partly from the requirement for GlnK to regulate nac expression; deletion of nac restored wild-type growth rates after ammonia starvation and refeeding to cells lacking GlnK, but did not improve viability during nitrogen starvation . The specific roles of GlnK during nitrogen starvation were not the result of a distinct function of the protein, as expression of PII from the glnK promoter in cells lacking GlnK restored the wild-type phenotypes.

Mol Microbiol, 2002 Oct, 46(1), 113 - 24
IHF and HU stimulate assembly of pre-replication complexes at Escherichia coli oriC by two different mechanisms; Ryan VT et al.; Pre-replication complexes (pre-RC) assemble on replication origins and unwind DNA in the presence of chromatin proteins . As components of Escherichia coli pre-RC, two histone-like proteins HU and IHF (integration host factor), stimulate initiator DnaA-catalysed unwinding of the chromosomal replication origin, oriC . Using in vivo footprint analysis just before DNA synthesis initiates, we detect IHF binding coincident with a shift of DnaA to weaker central oriC sites . Integration host factor redistributed pre-bound DnaA to identical sites in vitro . HU did not redistribute DnaA, but suppressed binding specifically at I3 . These results suggest that different pathways mediated by bacterial chromatin proteins exist to regulate pre-RC assembly and unwind oriC.

Mol Microbiol, 2002 Oct, 46(1), 63 - 74
The P1 plasmid is segregated to daughter cells by a 'capture and ejection' mechanism coordinated with Escherichia coli cell division; Li Y et al.; The fate of the P1 plasmid of Escherichia coli was followed by time-lapse photomicroscopy . A GFP-ParB fusion marked the plasmid during partition (segregation) to daughter cells at slow growth rate . The process differs from that previously inferred from statistical analysis of fixed cells . A focus of plasmid copies is captured at the cell centre . Immediately before cell division, the copies eject bidirectionally along the long axis of the cell . Cell division traps one or more plasmid copies in each daughter . They are not directed to a prescribed position but are free to move, associate and disassociate . Later, they are captured to the new cell centre to restart the cycle . A null P1 par mutant associates to form a focus, but it is neither captured nor ejected . A dominant negative ParB protein forms a plasmid focus that attaches to the cell centre but never ejects . It remains captive at the centre and blocks host cell division . The cells elongate . Eventually the intact focus is pushed to one side and the cells divide simultaneously in several places at the same time . This suggests that the wild-type plasmid imposes a regulatory node on the host cell cycle, preventing cell division until its own segregation is completed.

Plant J, 2002 Oct, 32(1), 93 - 103
Two long-chain acyl-CoA synthetases from Arabidopsis thaliana involved in peroxisomal fatty acid beta-oxidation; Fulda M et al.; Post-germinative growth of oilseeds is dependent on the breakdown of the stored lipid reserves . Long-chain acyl-CoA synthetase activities (LACS) are critically involved in this process by activating the released free fatty acids and thus feeding the beta-oxidation cycle in glyoxysomes . Here we report on the identification of two LACS genes, AtLACS6 and AtLACS7 from Arabidopsis thaliana coding for peroxisomal LACS proteins . The subcellular localization was verified by co-expression studies of spectral variants of the green fluorescent protein (GFP) . While AtLACS6 is targeted by a type 2 (PTS2) peroxisomal targeting sequence, for AtLACS7 a functional PTS1 as well as a PTS2 could be demonstrated . Possible explanations for this potentially redundant targeting information will be discussed . Expression studies of both genes revealed a strong induction 1 day after germination resembling the expression pattern of other genes involved in beta-oxidation . Analysis of the substrate specificities of the two LACS proteins demonstrated enzymatic activity for both enzymes with the whole spectrum of fatty acids found in stored lipid reserves . These results suggest that both LACS proteins might have overlapping functions and are able to initiate beta-oxidation in plant peroxisomes.

Clin Exp Pharmacol Physiol, 2002 Nov, 29(11), 990 - 5
Nitric oxide inhibits renal cytochrome P450-dependent epoxygenases in the rat; Oyekan A; 1 . Nitric oxide (NO), or peroxynitrite, is known to inhibit haemoproteins, including cytochrome P450 mono-oxygenases . The present study explores the functional correlates of the inhibition by NO of renal epoxygenase on the vascular responses to arachidonic acid (AA) in the perfused kidney . 2 . Control kidneys produce measurable amounts of epoxyeicosatrienoic acids (epoxides), which were increased from 0.6 +/- 0.2 to 1.8 +/- 0.9 ng/min (P < 0.05) following the addition of AA 5 micro g . Sodium nitroprusside (SNP; 100 micro mol/L), an NO donor, blunted the basal and AA-stimulated efflux of epoxides . 3 . Sodium nitroprusside at 10 and 100 micro mol/L inhibited renal microsomal conversion of {14C}-AA to epoxides and its hydration products dihydroxyeicosatrienoic acid (diols) . Microsomes harvested from rats 3 h after treatment with Escherichia coli endotoxin (lipopolysaccharide; LPS) also inhibited renal epoxygenase activity (81 +/- 8%; P < 0.05) . 4 . In the phenylephrine-preconstricted and indomethacin (2.8 micro mol/L)-treated kidney, AA at 5, 10 and 25 micro g elicited vasodilation that was blunted by miconazole (2 micro mol/L), 80 mmol/L KCl, tetraethylammonium (10 mmol/L), a K+ channel blocker, or SNP (100 micro mol/L) . 5 . Vasodilation induced by AA, but not 5,6-epoxide, was reduced in rats treated with LPS, an effect that was abolished by Nomega-nitro-l-arginine (100 mg/kg in drinking water for 10 days) . 6 . These data suggest that NO inhibits renal epoxygenase activity and inhibits epoxide-mediated AA-induced vasodilation in the rat kidney.

Phys Rev E Stat Nonlin Soft Matter Phys . 2002 Sep;66(3 Pt 1):031910 . Epub 2002 Sep 24.
Recognition of an organism from fragments of its complete genome; Anh VV et al.; This paper considers the problem of matching a fragment to an organism using its complete genome . Our method is based on the probability measure representation of a genome . We first demonstrate that these probability measures can be modeled as recurrent iterated function systems (RIFS) consisting of four contractive similarities . Our hypothesis is that the multifractal characteristics of the probability measure of a complete genome, as captured by the RIFS, is preserved in its reasonably long fragments . We compute the RIFS of fragments of various lengths and random starting points, and compare with that of the original sequence for recognition using the Euclidean distance . A demonstration on five randomly selected organisms supports the above hypothesis.

Pharmacology, 2002 Oct, 66(2), 57 - 60
In vitro effect of fluticasone propionate on interleukin 8 production by monocytes obtained from patients affected by moderate-severe allergic asthma; Gangemi S et al.; BACKGROUND: Interleukin-8 (IL-8), a potent chemotactic and activating factor for neutrophils and eosinophils, may be a crucial factor in severe asthma . The aim of this study was to evaluate the effect of fluticasone propionate (FP), a corticosteroid with potent anti-inflammatory activity, on the in vitro release of IL-8 by monocytes obtained from asthmatic patients . METHODS: Monocytes from 15 non-atopic healthy donors and from 15 patients affected by moderate-severe allergic asthma were isolated and incubated (37 degrees C, 5% CO(2)) for 24 h with varying combinations of lipopolysaccharide (LPS) from Escherichia coli (1 microg/ml) and FP (100 nmol/l) . IL-8 concentration in the culture supernatant was measured by an immuno-enzymatic method (ELISA) . RESULTS: A highly significant inverse correlation between FEV1 (forced expiratory volume) values before withdrawal and in vitro IL-8 production by unstimulated monocytes from asthmatic patients was observed (Rho = -0.787; p = 0.0032) . IL-8 production by either LPS-stimulated or unstimulated monocytes from asthmatic subjects was statistically increased compared to monocytes from healthy donors (p < 0.05) . FP addition reduced IL-8 production by monocytes from asthmatic patients and also from healthy donors (p < 0.05) . CONCLUSIONS: The partial IL-8 inhibition by FP could be closely related to the anti-inflammatory activity of this corticosteroid . Based on these results, we propose that the clinical improvement of asthma, observed following FP administration, may depend, at least in part, on the ability of this drug to modulate cytokine production by monocytes .

Nucleic Acids Res, 2002 Oct 1, 30(19), 4250 - 63
RNA canonical and non-canonical base pairing types: a recognition method and complete repertoire; Lemieux S et al.; The problem of systematic and objective identification of canonical and non-canonical base pairs in RNA three-dimensional (3D) structures was studied . A probabilistic approach was applied, and an algorithm and its implementation in a computer program that detects and analyzes all the base pairs contained in RNA 3D structures were developed . The algorithm objectively distinguishes among canonical and non-canonical base pairing types formed by three, two and one hydrogen bonds (H-bonds), as well as those containing bifurcated and C-H.X...H-bonds . The nodes of a bipartite graph are used to encode the donor and acceptor atoms of a 3D structure . The capacities of the edges correspond to probabilities computed from the geometry of the donor and acceptor groups to form H-bonds . The maximum flow from donors to acceptors directly identifies base pairs and their types . A complete repertoire of base pairing types was built from the detected H-bonds of all X-ray crystal structures of a resolution of 3.0 A or better, including the large and small ribosomal subunits . The base pairing types are labeled using an extension of the nomenclature recently introduced by Leontis and Westhof . The probabilistic method was implemented in MC-Annotate, an RNA structure analysis computer program used to determine the base pairing parameters of the 3D modeling system MC-Sym.

Nucleic Acids Res, 2002 Oct 1, 30(19), 4158 - 65
Investigating the endonuclease activity of four Pyrococcus abyssi inteins; Saves I et al.; Among the 14 inteins of the Pyrococcus abyssi genome, 10 harbour the LAGLIDADG motifs of dodecapeptide endonucleases . Four of these were cloned, expressed in Escherichia coli and purified to assay their potential endonuclease activity . PabRIR1-2 and PabRIR1-3 are specific endonucleases, named PI-PabI and PI-PabII, respectively, cleaving the sequence spanning their homing site . This is consistent with their size and with the relative positions and sequences of their endonuclease motifs . However, PI-PabI is 10-fold more active than PI-PabII and a discrepancy of the DNA recognition and cleavage mechanisms was observed between the two inteins . In particular, analysis of the DNA cleavage reactions by MALDI-TOF highlighted that while the cleavage of DNA by PI-PabI consists of two steps corresponding to the cleavage of each DNA strand, PI-PabII processes the two DNA strands simultaneously . Furthermore, the two inteins interact differently with DNA . In addition, we did not detect any endonuclease activity for PabLon and PabRIR1-1 . Deletions in the intein sequences and mutations in the putative endonuclease motifs probably abolish this activity . Hence, inteins from the same archaebacteria, even if contained in the same host protein, did not evolve uniformly and are presumably at different stages of the invasion cycle.

Protein Eng, 2002 Aug, 15(8), 635 - 42
A major IgE epitope-containing grass pollen allergen domain from Phl p 5 folds as a four-helix bundle; Maglio O et al.; Phl p 5, a 29 kDa major allergen from timothy grass pollen, is one of the most reactive members of group 5 allergens . Its sequence comprises two repeats of a novel alanine-rich motif (AR) whose structure and allergenic response are still mostly unknown . We report here a structural characterization of an immunodominant fragment of Phl p 5, Phl p 5(56-165) which comprises the first AR repeat . Recombinant (r)Phl p 5(56-165) was expressed in Escherichia coli, purified to homogeneity and shown to be sufficient to react with serum IgE from 90% of grass pollen allergic patients . Using NMR spectroscopy, we show conclusively that the fragment forms a compact globular domain which is, however, prone to degradation with time . The rPhl p 5(56-165) fold consists of a four-helix bundle held together by hydrophobic interactions between the aromatic rings and aliphatic side chains . This evidence gives clear indications about the structure of the full-length Phl p 5 and provides a rational basis for finding ways to stabilize the fold and designing therapeutic vaccines against grass pollen allergy.

Protein Eng, 2002 Aug, 15(8), 627 - 33
Structural consequences of replacement of an alpha-helical Pro residue in Escherichia coli thioredoxin; Rudresh et al.; While it is well known that introduction of Pro residues into the interior of protein alpha-helices is destabilizing, there have been few studies that have examined the structural and thermodynamic effects of the replacement of a Pro residue in the interior of a protein alpha-helix . We have previously reported an increase in stability in the P40S mutant of Escherichia coli thioredoxin of 1-1.5 kcal/mol in the temperature range 280-330 K . This paper describes the structure of the P40S mutant at a resolution of 1.8 A . In wild-type thioredoxin, P40 is located in the interior of helix two, a long alpha-helix that extends from residues 32 to 49 with a kink at residue 40 . Structural differences between the wild-type and P40S are largely localized to the above helix . In the P40S mutant, there is an expected additional hydrogen bond formed between the amide of S40 and the carbonyl of residue K36 and also additional hydrogen bonds between the side chain of S40 and the carbonyl of K36 . The helix remains kinked . In the wild-type, main chain hydrogen bonds exist between the amide of 44 and carbonyl of 40 and between the amide of 43 and carbonyl of 39 . However, these are absent in P40S . Instead, these main chain atoms are hydrogen bonded to water molecules . The increased stability of P40S is likely to be due to the net increase in the number of hydrogen bonds in helix two of E.coli thioredoxin.

Circ Res, 2002 Oct 4, 91(7), 618 - 25
Adrenomedullin reduces endothelial hyperpermeability; Hippenstiel S et al.; Endothelial hyperpermeability induced by inflammatory mediators is a hallmark of sepsis and adult respiratory distress syndrome . Increased levels of the regulatory peptide adrenomedullin (ADM) have been found in patients with systemic inflammatory response . We analyzed the effect of ADM on the permeability of cultured human umbilical vein endothelial cell (HUVEC) and porcine pulmonary artery endothelial cell monolayers . ADM dose-dependently reduced endothelial hyperpermeability induced by hydrogen peroxide (H2O2), thrombin, and Escherichia coli hemolysin . Moreover, ADM pretreatment blocked H2O2-related edema formation in isolated perfused rabbit lungs and increased cAMP levels in lung perfusate . ADM bound specifically to HUVECs and porcine pulmonary artery endothelial cells and increased cellular cAMP levels . Simultaneous inhibition of cAMP-degrading phosphodiesterase isoenzymes 3 and 4 potentiated ADM-dependent cAMP accumulation and synergistically enhanced ADM-dependent reduction of thrombin-induced hyperpermeability . However, ADM showed no effect on endothelial cGMP content, basal intracellular Ca2+ levels, or the H2O2-stimulated, thrombin-stimulated, or Escherichia coli hemolysin-stimulated Ca2+ increase . ADM diminished thrombin- and H2O2-related myosin light chain phosphorylation as well as stimulus-dependent stress fiber formation and gap formation in HUVECs, suggesting that ADM may stabilize the barrier function by cAMP-dependent relaxation of the microfilament system . These findings identify a new function of ADM and point to ADM as a potential interventional agent for the reduction of vascular leakage in sepsis and adult respiratory distress syndrome.

J Biol Chem, 2002 Dec 20, 277(51), 50155 - 9 Epub 2002 Oct 02.
Mechanism of action of RNase T . I . Identification of residues required for catalysis, substrate binding, and dimerization; Zuo Y et al.; Escherichia coli RNase T, an RNA-processing enzyme and a member of the DEDD exonuclease superfamily, was examined using sequence analysis and site-directed mutagenesis . Like other DEDD exonucleases, RNase T was found to contain three conserved Exo motifs that included four invariant acidic residues . Mutagenesis of these motifs revealed that they are essential for RNase T activity, indicating that they probably form the RNase T catalytic center in a manner similar to that found in other DEDD exonucleases . We also identified by sequence analysis three short, but highly conserved, sequence segments rich in positively charged residues . Site-directed mutagenesis of these regions indicated that they are involved in substrate binding . Additional analysis revealed that residues within the C-terminal region of RNase T are essential for RNase T dimerization and, consequently, for RNase T activity . These data define the domains necessary for RNase T action, and together with information in the accompanying article, have led to the formulation of a detailed model for the structure and mechanism of action of RNase T.

J Biol Chem, 2002 Dec 20, 277(51), 50160 - 4 Epub 2002 Oct 02.
Mechanism of action of RNase T . II . A structural and functional model of the enzyme; Zuo Y et al.; A detailed structural and functional model of E . coli RNase T was generated based on sequence analysis, homology modeling, and experimental observation . In the accompanying article, three short sequence segments (nucleic acid binding sequences (NBS)) important for RNase T substrate binding were identified . In the model, these segments cluster to form a positively charged surface patch . However, this patch is on the face of the RNase T monomer opposite the DEDD catalytic center . We propose that by dimerization, the NBS patch from one subunit is brought to the vicinity of the DEDD center of the second monomer to form a fully functional RNase T active site . In support of this model, mutagenetic studies show that one NBS1 residue, Arg(13), sits at the catalytic center despite being on the opposite side of the monomer . Second, the complementarity of the RNase T subunits through the formation of homodimers was demonstrated by reconstitution of partial RNase T activity from monomers derived from two inactive mutant proteins, one defective in catalysis and one in substrate binding . These data explain why RNase T must dimerize to function . The model provides a detailed framework on which to explain the mechanism of action of RNase T.

Biotechnol Prog, 2002 Sep-Oct, 18(5), 1054 - 9
Protein purification via aqueous two-phase extraction (ATPE) and immobilized metal affinity chromatography . Effectiveness of salt addition to enhance selectivity and yield of GFPuv; Li Y et al.; This study illustrates the compatibility and complementary nature of aqueous two-phase extraction (ATPE) and immobilized metal affinity chromatography (IMAC) in a general recovery scheme . The purification of green fluorescent protein (GFPuv) from extracts of Eschericia coli was investigated using a combination of these two techniques . High molarity of sodium chloride was found effective in increasing selectivity, with the promotion of hydrophobic interaction the probable mechanism that drove the target protein to a particular phase in ATPE, as well as that which enhanced GFPuv adsorption in IMAC . Moreover, the similar salt condition allows the direct application of the GFPuv-containing phase to the IMAC column without additional adjustment step . A simple screen of conditions was therefore performed to generate a favorable two-step purification scheme for GFP leading to an overall high purity.

J Comput Aided Mol Des, 2002 Mar, 16(3), 167 - 79
Investigation of the metal binding site in methionine aminopeptidase by density functional theory; Jorgensen AT et al.; All methionine aminopeptidases exhibit the same conserved metal binding site . The structure of this site with either Co2+ ions or Zn2+ ions was investigated using density functional theory . The calculations showed that the structure of the site was not influenced by the identity of the metal ions . This was the case for both of the systems studied; one based on the X-ray structure of the human methionine aminopeptidase type 2 (hMetAP-2) and the other based on the X-ray structure of the E . coli methionine aminopeptidase type I (eMetAP- 1) . Another important structural issue is the identity of the bridging oxygen, which is part of either a water molecule or a hydroxide ion . Within the site of hMetAP-2 the results strongly indicate that a hydroxide ion bridges the metal ions . By contrast, the nature of the oxygen bridging the metal ions within the metal binding site of eMetAP-1 cannot be determined based on the results here, due to the similar structural results obtained with a bridging water molecule and a bridging hydroxide ion.

Oral Dis, 2002 Sep, 8(5), 261 - 7
Effect of high-density lipoprotein on lipopolysaccharide-induced alveolar bone resorption in rats; Jonarta AL et al.; OBJECTIVE: To determine whether treatments with high-density lipoprotein (HDL) may alter the lipopolysaccharide (LPS)-induced alveolar bone resorption in rats . MATERIALS AND METHODS: Rats were injected with 500 microg of LPS from Escherichia coli at the alveolar mucosa of lower right first molar once every 2 days for 8 days . The negative and positive control were injected with phosphate buffered saline (PBS) and LPS alone, respectively . In HDL-treated animals various concentration of HDL were injected immediately before, after the third or the final LPS injection . The bone sections were stained with tartrate-resistant acid phosphatase (TRAP) and the numbers of both osteoclasts and preosteoclasts and the levels of alveolar bone resorption were assessed . RESULTS: The numbers of both osteoclasts and preosteoclasts and the levels of alveolar bone resorption in animals treated with HDL before or during LPS injections were lower than those in the positive control, but higher than those in the negative control, regardless of HDL doses . Similar results were also observed in animals treated with 250 and 500 microg of HDL after the final LPS injection . Only treatments with 1000 microg of HDL after LPS injections completely reduced the number of both osteoclasts and preosteoclasts, but only partially decreased the alveolar bone resorption . CONCLUSION: HDL treatments partially reduced the LPS-induced alveolar bone resorption in vivo in rats, suggesting that HDL may neutralize the ability of LPS to induce alveolar bone resorption.

Microb Drug Resist, 2002 Fall, 8(3), 179 - 85
Inactivation of the acrA gene is partially responsible for chloramphenicol sensitivity of Escherichia coli CM2555 strain expressing the chloramphenicol acetyltransferase gene; Potrykus J et al.; An Escherichia coli CM2555 strain, sensitive to chloramphenicol when expressing the cat gene and producing active chloramphenicol acetyltransferase (CAT), was described recently . It was proposed that this sensitivity is due to decreased levels of acetyl coenzyme A (Acetyl CoA) in cat-expressing CM2555 cells in the presence of chloramphenicol . CAT catalyzes transfer of the acetyl moiety from Acetyl CoA to a chloramphenicol molecule . Thus, a very efficient acetylation of chloramphenicol may cause deprivation of Acetyl CoA and cell death . A specific mutation causing the chloramphenicol sensitivity phenotype of CM2555 was not reported to date . Therefore, we aimed to identify a genetic defect causing this phenotype . Here, we found that overexpression of the acrEF genes, encoding a transmembrane pump, or the acrE gene alone, results in restoration of chloramphenicol-resistance of cat-expressing CM2555 strain . Although no mutation exists in the CM2555 acrE locus, a nonsense mutation in the 67th codon of the acrA gene, which encodes a component of another transmembrane pump, has been found . Although introduction of the deltaacrAB allele into CM732, a parental strain of CM2555, and into some other commonly used E . coli strains led to their chloramphenicol sensitivity in the presence of CAT, the same genetic manipulation did not result in such a phenotype in other genetic backgrounds, including "wild-type" E . coli MG1655 . These results suggest that the acrA dysfunction is one of more mutations responsible for chloramphenicol sensitivity of cat-expressing CM2555 strain.

Acta Biochim Pol, 2002, 49(2), 509 - 13
A comparison between the crystal and solution structures of Escherichia coli asparaginase II; Kozak M et al.; The small angle X-ray scattering (SAXS) pattern of the homotetrameric asparaginase II from Escherichia coli was measured in solution in conditions resembling those in which its crystal form was obtained and compared with that calculated from the crystallographic model . The radius of gyration measured by SAXS is about 5% larger and the maximum dimension in the distance distribution function about 12% larger than the corresponding value calculated from the crystal structure . A comparison of the experimental and calculated distance distribution functions suggests that the overall quaternary structure in the crystal and in solution are similar but that the homotetramer is less compact in solution than in the crystal.

Proteomics, 2002 Sep, 2(9), 1247 - 53
Rapid analysis of protein interactions: On-chip micropurification of recombinant protein expressed in Esherichia coli; Natsume T et al.; We describe a rapid analysis of interactions between antibodies and a recombinant protein present in total cell lysates . Using a surface plasmon resonance biosensor, a low concentration of glutathione-S-transferase (GST) fused protein expressed in small scale Esherichia coli culture was purified on an anti-GST antibody immobilized sensor chip . The 'on-chip purification' was verified using matrix-assisted laser desorption/ionization-time of flight mass spectrometry by measuring the molecular masses of recombinant proteins purified on the sensor chip . The specific binding of monoclonal antibodies for the on-chip micropurified recombinant proteins can then be monitored, thus enabling kinetic analysis and epitope mapping of the bound antibodies . This approach reduced time, resources and sample consumption by avoiding conventional steps related to concentration and purification.

Proteomics, 2002 Sep, 2(9), 1220 - 8
Proteomic analysis of lipopolysaccharide-induced apoptosis in PC12 cells; Huang YH et al.; We employed rat pheochromocytoma PC12 cells as our model system to identify cellular proteins that accompany Escherichia coli lipopolysaccharide (LPS)-induced apoptosis, based on a proteomic approach . Cell viability tests revealed that naive PC12 cells underwent cell death in a dose-dependent manner after treatment with LPS . Flow cytometric analysis confirmed that apoptosis was primarily responsible for the observed cell death . Two-dimensional electrophoresis in conjunction with N-terminal sequencing, immunoblot, matrix-assisted laser desorption/ionization-time of flight analysis or computer matching with protein databases further revealed that the LPS-induced apoptosis is accompanied by an augmented level of calreticulin, calcium binding protein 50, endoplasmic reticulum protein 60 (ERP60), heat shock protein 60 (HSP60) or HSP90, and a reduced level of amphoterin, cytochrome c oxidase polypeptide VIa-liver or ERP29 . These proteins are associated with endoplasmic reticulum, mitochondria or cell membrane, and are with known or potential roles in apoptosis . Their identification therefore provides an impetus for further delineation of the cellular and molecular basis of apoptotic cell death and sepsis based on proteomic profiling of PC12 cells.

Proc Natl Acad Sci U S A, 2002 Oct 15, 99(21), 13549 - 53 Epub 2002 Oct 02.
Combinatorial mutagenesis to restrict amino acid usage in an enzyme to a reduced set; Akanuma S et al.; We developed an effective strategy to restrict the amino acid usage in a relatively large protein to a reduced set with conservation of its in vivo function . The 213-residue Escherichia coli orotate phosphoribosyltransferase was subjected to 22 cycles of segment-wise combinatorial mutagenesis followed by 6 cycles of site-directed random mutagenesis, both coupled with a growth-related phenotype selection . The enzyme eventually tolerated 73 amino acid substitutions: In the final variant, 9 amino acid types (A, D, G, L, P, R, T, V, and Y) occupied 188 positions (88%), and none of 7 amino acid types (C, H, I, M, N, Q, and W) appeared . Therefore, the catalytic function associated with a relatively large protein may be achieved with a subset of the 20 amino acid . The converged sequence also implies simpler constituents for proteins in the early stage of evolution.

J Biol Chem, 2002 Dec 20, 277(51), 49134 - 42 Epub 2002 Oct 01.
Protein kinase C delta associates with the interleukin-6 receptor subunit glycoprotein (gp) 130 via Stat3 and enhances Stat3-gp130 interaction; Novotny-Diermayr V et al.; The transcriptional regulation of Stat proteins is controlled through their C-terminal domains, which harbor both a tyrosine phosphorylation site, required for dimerization and subsequent nuclear translocation, and a serine phosphorylation site, required for maximum transcriptional activity . Previously, we reported that protein kinase Cdelta (PKCdelta) phosphorylates and interacts with Stat3 in an interleukin (IL)-6-dependent manner . In this study, we further characterized this interaction, and investigated the potential role of such an interaction . We show here that the catalytic domain of PKCdelta interacts with the Src homology 2 domain and part of the adjacent C-terminal transactivation domain of Stat3 . This interaction, which does not seem to involve a classical phosphotyrosine SH2-mediated binding, however, significantly enhances the interaction of Stat3 and the IL-6 receptor subunit glycoprotein (gp) 130, which is the initial step for Stat3 activation by IL-6 . Expression of a dominant negative PKCdelta or depletion of the endogenous PKCdelta by phorbol 12-myristate 3-acetate treatment abrogates the association of Stat3 with gp130 . At the same time, PKCdelta is recruited to gp130 via association with Stat3, which may facilitate its phosphorylation on the gp130 receptor . Finally, we identified Thr-890, a putative PKC phosphorylation site on gp130, to be critical for the effect of PKCdelta . Our data indicate that PKCdelta plays important regulatory roles in IL-6 signaling.

J Biol Chem, 2002 Dec 20, 277(51), 49863 - 9 Epub 2002 Oct 01.
Crystal structure of SEDL and its implications for a genetic disease spondyloepiphyseal dysplasia tarda; Jang SB et al.; SEDL is an evolutionarily highly conserved protein in eukaryotic organisms . Deletions or point mutations in the SEDL gene are responsible for the genetic disease spondyloepiphyseal dysplasia tarda (SEDT), an X-linked skeletal disorder . SEDL has been identified as a component of the transport protein particle (TRAPP), critically involved in endoplasmic reticulum-to-Golgi vesicle transport . Herein, we report the 2.4 A resolution structure of SEDL, which reveals an unexpected similarity to the structures of the N-terminal regulatory domain of two SNAREs, Ykt6p and Sec22b, despite no sequence homology to these proteins . The similarity and the presence of unusually many solvent-exposed apolar residues of SEDL suggest that it serves regulatory and/or adaptor functions through multiple protein-protein interactions . Of the four known missense mutations responsible for SEDT, three mutations (S73L, F83S, V130D) map to the protein interior, where the mutations would disrupt the structure, and the fourth (D47Y) on a surface at which the mutation may abrogate functional interactions with a partner protein.

Free Radic Biol Med, 2002 Oct 1, 33(7), 886 - 93
Repair of deaminated bases in DNA; Kow YW; Deamination of DNA bases can occur spontaneously, generating highly mutagenic lesions such as uracil, hypoxanthine, and xanthine . When cells are under oxidative stress that is induced either by oxidizing agents or by mitochondrial dysfunction, additional deamination products such as 5-hydroxymethyluracil (5-HMU) and 5-hydroxyuracil (5-OH-Ura) are formed . The cellular level of these highly mutagenic lesions is increased substantially when cells are exposed to DNA damaging agent, such as ionizing radiation, redox reagents, nitric oxide, and others . The cellular repair of deamination products is predominantly through the base excision repair (BER) pathway, a major cellular repair pathway that is initiated by lesion specific DNA glycosylases . In BER, the lesions are removed by the combined action of a DNA glycosylase and an AP endonuclease, leaving behind a one-base gap . The gapped product is then further repaired by the sequential action of DNA polymerase and DNA ligase . DNA glycosylases that recognize uracil, 5-OH-Ura, 5-HMU (derived from 5-methylcytosine) and a T/G mismatch (derived from a 5-methylcytosine/G pair) are present in most cells . Many of these glycosylases have been cloned and well characterized . In yeast and mammalian cells, hypoxanthine is efficiently removed by methylpurine N-glycosylase, and it is thought that BER might be an important pathway for the repair of hypoxanthine . In contrast, no glycosylase that can recognize xanthine has been identified in either yeast or mammalian cells . In Escherichia coli, the major enzyme activity that initiates the repair of hypoxanthine and xanthine is endonuclease V . Endonuclease V is an endonuclease that hydrolyzes the second phosphodiester bond 3' to the lesion . It is hypothesized that the cleaved DNA is further repaired through an alternative excision repair (AER) pathway that requires the participation of either a 5' endonuclease or a 3'-5' exonuclease to remove the damaged base . The repair process is then completed by the sequential actions of DNA polymerase and DNA ligase . Endonuclease V sequence homologs are present in all kingdoms, and it is conceivable that endonuclease V might also be a major enzyme that initiates the repair of hypoxanthine and xanthine in mammalian cells.

J Clin Virol, 2002 Aug, 25 Suppl 2, S37 - 49
Animal models of congenital cytomegalovirus infection: an overview of progress in the characterization of guinea pig cytomegalovirus (GPCMV); Schleiss MR; BACKGROUND: The strict species-specificity of cytomegalovirus (CMV) precludes preclinical evaluation of human CMV (HCMV) vaccines in animal models and necessitates the study of nonhuman CMVs . Among the CMVs of small mammals, the guinea pig cytomegalovirus (GPCMV) has unique advantages, due to its ability to cross the placenta, causing infection in utero . OBJECTIVE AND STUDY DESIGNS: Progress in GPCMV studies has been hampered by a lack of detailed molecular characterization of the viral genome . Therefore, recent efforts have been undertaken to characterize the GPCMV genome, and apply this information to in vivo subunit vaccine studies . RESULTS: Progress in the sequencing of the GPCMV genome has revealed the presence of both highly conserved as well as novel open reading frames (ORFs) . Cloning of GPCMV vaccine candidates, such as the glycoprotein B (gB) and UL83 proteins, has facilitated subunit vaccine evaluation . Protein vaccines and DNA vaccines have shown evidence of protection in pregnancy/challenge experiments . In addition, the GPCMV genome has proved amenable to cloning as a bacterial artificial chromosome (BAC) in Escherichia coli, and BAC-derived recombinants retain the ability to replicate in vivo . CONCLUSIONS: Progress has been made in molecular characterization of GPCMV . Insights from these studies should prove germane to the understanding of the correlates of protective immunity for the fetus in vaccine studies, and should assist in prioritization of vaccine strategies in HCMV vaccine trials.

Arch Biochem Biophys, 2002 Oct 15, 406(2), 289 - 95
Ligand-dependent structural changes and limited proteolysis of Escherichia coli phosphofructokinase-2; Cabrera R et al.; Binding of MgATP to the allosteric site of phosphofructokinase-2 promotes a dimer to tetramer conversion . In the presence of Fru-6-P the enzyme remains as a dimer . Limited proteolysis in the presence of MgATP completely protects the enzyme against inactivation and cleavage, while Fru-6-P provides a partial protection . A 28-kDa proteolytic fragment containing the N-terminus of the protein is inactive, but retains the ability to bind Fru-6-P and the allosteric effector MgATP . The fragment remains as a dimer but does not form a tetramer in the presence of MgATP . The results suggest major conformational changes of the enzyme upon ligand binding that confer a higher degree of compactness to the monomers in the dimer and in the tetramer, demonstrate the presence of the active and allosteric sites in this N-terminus fragment, and stress the importance of the C-terminus region of the protein for catalytic activity and ligand-induced oligomerization.

Arch Biochem Biophys, 2002 Oct 15, 406(2), 261 - 70
Novel S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase, an enzyme responsible for biosynthesis of methyl salicylate and methyl benzoate, is not involved in floral scent production in snapdragon flowers; Negre F et al.; Using a functional genomic approach we have isolated and characterized a cDNA that encodes a salicylic acid carboxyl methyltransferase (SAMT) from Antirrhinum majus . The sequence of the protein encoded by SAMT has higher amino acid identity to Clarkia breweri SAMT than to snapdragon benzoic acid carboxyl methyltransferase (BAMT) (55 and 40% amino acid identity, respectively) . Escherichia coli-expressed SAMT protein catalyzes the formation of the volatile ester methyl salicylate from salicylic acid with a K(m) value of 83 microM . It can also methylate benzoic acid to form methyl benzoate, but its K(m) value for benzoic acid is 1.72 mM . Snapdragon flowers do not emit methyl salicylate . The potential involvement of SAMT in production and emission of methyl benzoate in snapdragon flowers was analyzed by RNA gel blot analysis . SAMT mRNA was not detected in floral tissues by RNA blot hybridization, but low levels of SAMT gene expression were detected after real-time RT-PCR in the presence of SAMT-specific primers, indicating that this gene does not contribute significantly, if at all, in methyl benzoate production and emission in snapdragon flowers . Expression of SAMT in petal tissue was found to be induced by salicylic and jasmonic acid treatments.

Water Sci Technol, 2002, 46(4-5), 427 - 34
A novel approach to pathogen reduction in biosolids: the enzymic hydrolyser; Mayhew ME et al.; Revision of the Sludge (Use in Agriculture) Regulations in the UK has resulted in the requirement of a final product standard in terms of E . coli per gram of dry solids . Conventional mesophilic digestion including 14-day secondary storage should normally provide adequate treatment to meet the Treated Sludge Standard . Any process capable of greater pathogen reduction would ensure more process security and compliance comfort . Such a process would be a welcome alternative to extra secondary storage where an existing works does not have sufficient capacity, particularly if the differences in costs between the options are small . Enzymic hydrolysis was found to be up to ten-fold more effective in E . coli reduction than conventional secondary digestion . A two-stage digestion process based on this technique has been developed by United Utilities and Montgomery Watson Harza (termed the enzymic hydrolyser, patent pending) . Studies showed that the mean numbers of E . coli were significantly lower in the enzymic hydrolyser systems (P > 0.05; t = 13.19) compared to conventional digesters . Increased stability was a secondary benefit of the system (foam was eliminated or greatly reduced in the enzymic hydrolyser units) . Another benefit of the system for retrofit to existing assets is the decreased tankage volumes required compared to secondary digestion to achieve more than twice the log kill of pathogens.

Rev Med Chil, 2002 Aug, 130(8), 841 - 9
{Antibodies against recombinant Ro 60 Kd, Ro 52 and La 48 Kd proteins in primary Sjogren's Syndrome . Utility of analytic methods combination to detect antibodies anti Ro and La}; Aguilera S et al.; BACKGROUND: The use of new recombinant antigens may increase the sensitivity and specificity of the detection of anti Ro and anti La antibodies in Sjogren's syndrome . AIM: To determine the immune reactivity of sera from patients with Sjogren's syndrome, against fusion recombinant proteins (prf) Ro60 Kd, Ro52 Kd and La48 Kd expressed in E coli and recombinant protein Ro52 Kd, expressed in baculovirus (prb) . MATERIAL AND METHODS: Serum samples from 46 patients with a diagnosis of Sjogren's syndrome, according to the European criteria of 1997, were studied . Using conventional ELISA assays, 32 patients had positive anti Ro antibodies (group A) and 16 patients had negative anti Ro and anti La antibodies, but had positive antinuclear antibodies or rheumatoid factors (group B) . Antibodies against recombinant proteins were measured by ELISA or Western Blot . RESULTS: Reactivity against prf Ro60 was present in 69% of samples from group A patients and in 36% of samples from group B . Reactivity against prf Ro52 was present in 94% of samples from group A and 50% of samples from group B . Reactivity against prb Ro52 was present in 75% of samples from group A and 40% of samples from group B . Reactivity against prf La was present in 78% of samples by ELISA and 97% of samples by Western Blot . In 10 of 14 serum samples from group B patients, there was reactivity against at least one recombinant protein . CONCLUSIONS: A high prevalence of reactivity against recombinant Ro and La proteins was detected in serum samples from patients with Sjogren syndrome.

Nat Cell Biol, 2002 Oct, 4(10), 821 - 5
One-Eyed Pinhead and Spadetail are essential for heart and somite formation; Griffin KJ et al.; Mutant analysis in the zebrafish Danio rerio has demonstrated distinct developmental roles for the T-box transcription factor Spadetail (Spt) and the Nodal-receptor cofactor One-Eyed Pinhead (Oep) in the formation of mesoderm and endoderm . Here, we show that spt and oep genetically interact and are together essential for the formation of cardiac and somitic mesoderm . These two mesodermal defects are dependent on different effectors of Nodal signalling; cardiac mesoderm formation involves the mix-like transcription factor Bonnie and Clyde (Bon), whereas somitogenesis is dependent on a different pathway . Analysis of the somite defect in Zoep;spt embryos has provided insights into the control of somitic mesoderm formation by Spt, which was previously implicated in the regulation of cell adhesion and motility . We show that the failure to form somites in Zoep;spt embryos is independent of this and that Spt must have an additional function . We propose that the major role of Spt in somitogenesis is to promote the differentiation of presomitic mesoderm from tailbud progenitors by antagonizing progenitor-type gene expression and behaviour.

Pharmacogenetics, 2002 Oct, 12(7), 517 - 28
Characterization of human soluble high and low activity catechol-O-methyltransferase catalyzed catechol estrogen methylation; Goodman JE et al.; The major detoxification pathway of the carcinogenic catechol estrogens is methylation by catechol- -methyltransferase (COMT) . It has been hypothesized that the enzyme encoded by the low-activity allele (COMT(L) ) has a lower catalytic activity for catechol estrogen methylation than that encoded by the high activity allele (COMT(H) ) . We expressed and purified human soluble (S)-COMT(H) and S-COMT(L) in and characterized the methylation of 2- and 4-hydroxyestradiol (2- and 4-OH-E2) . There were no differences between the kinetic parameters for COMT(H) and COMT(L) . The kinetic parameters for S-adenosylmethionine (SAM), the methyl donor in these reactions, also did not differ for COMT(H) and COMT(L) . S-adenosylhomocysteine, the demethylated SAM metabolite, inhibited methylation of the catechol estrogens in a non-competitive manner similarly for COMT(H) and COMT(L) . Each COMT substrate tested inhibited the methylation of other substrates in a mixed competitive and non-competitive fashion similarly for COMT(H) and COMT(L) . Furthermore, in cytosolic fractions of COMT(HH)(MCF-10A and ZR-75-1) and COMT(LL)(MCF-7 and T47D) human breast epithelial cell lines, no differences were detected between the kinetic parameters of COMT with respect to 2- and 4-OH-E2 methylation; nor were COMT protein levels associated with the COMT genotype . These data suggest that the decreased COMT enzymatic activity that has been detected in human tissue in association with the COMT(L) allele is not reflected by differences in the affinity or capacity of COMT(H) and COMT(L) for catechol estrogen methylation . These results raise the question of what accounts for the difference in COMT activity associated with the COMT(HH) and COMT(LL) genotypes in human tissue.

Proc Natl Acad Sci U S A, 2002 Oct 15, 99(21), 13481 - 6 Epub 2002 Oct 01.
Interaction of dihydrofolate reductase with methotrexate: ensemble and single-molecule kinetics; Rajagopalan PT et al.; The thermodynamics and kinetics of the interaction of dihydrofolate reductase (DHFR) with methotrexate have been studied by using fluorescence, stopped-flow, and single-molecule methods . DHFR was modified to permit the covalent addition of a fluorescent molecule, Alexa 488, and a biotin at the N terminus of the molecule . The fluorescent molecule was placed on a protein loop that closes over methotrexate when binding occurs, thus causing a quenching of the fluorescence . The biotin was used to attach the enzyme in an active form to a glass surface for single-molecule studies . The equilibrium dissociation constant for the binding of methotrexate to the enzyme is 9.5 nM . The stopped-flow studies revealed that methotrexate binds to two different conformations of the enzyme, and the association and dissociation rate constants were determined . The single-molecule investigation revealed a conformational change in the enzyme-methotrexate complex that was not observed in the stopped-flow studies . The ensemble averaged rate constants for this conformation change in both directions is about 2-4 s(-1) and is attributed to the opening and closing of the enzyme loop over the bound methotrexate . Thus the mechanism of methotrexate binding to DHFR involves multiple steps and protein conformational changes.

J Biol Chem, 2002 Dec 13, 277(50), 48535 - 49 Epub 2002 Sep 30.
Interfacial kinetic and binding properties of the complete set of human and mouse groups I, II, V, X, and XII secreted phospholipases A2; Singer AG et al.; Expression of the full set of human and mouse groups I, II, V, X, and XII secreted phospholipases A(2) (sPLA(2)s) in Escherichia coli and insect cells has provided pure recombinant enzymes for detailed comparative interfacial kinetic and binding studies . The set of mammalian sPLA(2)s display dramatically different sensitivity to dithiothreitol . The specific activity for the hydrolysis of vesicles of differing phospholipid composition by these enzymes varies by up to 4 orders of magnitude, and yet all enzymes display similar catalytic site specificity toward phospholipids with different polar head groups . Discrimination between sn-2 polyunsaturated versus saturated fatty acyl chains is <6-fold . These enzymes display apparent dissociation constants for activation by calcium in the 1-225 microm range, depending on the phospholipid substrate . Analysis of the inhibition by a set of 12 active site-directed, competitive inhibitors reveals a large variation in the potency among the mammalian sPLA(2)s, with Me-Indoxam being the most generally potent sPLA(2) inhibitor . A dramatic correlation exists between the ability of the sPLA(2)s to hydrolyze phosphatidylcholine-rich vesicles efficiently in vitro and the ability to release arachidonic acid when added exogenously to mammalian cells; the group V and X sPLA(2)s are uniquely efficient in this regard.

J Biol Chem, 2002 Dec 6, 277(49), 47581 - 7 Epub 2002 Sep 30.
Phosphorylation of isolated human phosphodiesterase-5 regulatory domain induces an apparent conformational change and increases cGMP binding affinity; Francis SH et al.; Substrate binding to the phosphodiesterase-5 (PDE5) catalytic site increases cGMP binding to the regulatory domain (R domain) . The latter promotes PDE5 phosphorylation by cyclic nucleotide-dependent protein kinases, which activates catalysis, enhances allosteric cGMP binding, and causes PDE5A1 to apparently elongate . A human PDE5A1 R domain fragment (Val(46)-Glu(539)) containing the phosphorylation site (Ser(102)) and allosteric cGMP-binding sites was studied . The rate, cGMP dependence, and stoichiometry of phosphorylation of the PDE5 R domain by the catalytic subunit of cAMP-dependent protein kinase are comparable with that of the holoenzyme . Migration in native polyacrylamide gels suggests that either cGMP binding or phosphorylation produces distinct conformers of the R domain . Phosphorylation of the R domain increases affinity for cGMP approximately 10-fold (K(D) values 97.8 +/- 17 and 10.0 +/- 0.5 nm for unphospho- and phospho-R domains, respectively) . {(3)H}cGMP dissociates from the phospho-R domain with a single rate (t(12) = 339 +/- 30 min) compared with the biphasic pattern of the unphospho-R domain (t(12) = 39.0 +/- 4.8 and 265 +/- 28 min, for the fast and slow components, respectively) . Thus, cGMP-directed regulation of PDE5 phosphorylation and the resulting increase in cGMP binding affinity occur largely within the R domain . Conformational change(s) elicited by phosphorylation of the R domain within the PDE5 holoenzyme may also cause or participate in stimulating catalysis.

J Biol Chem, 2002 Dec 6, 277(49), 47300 - 4 Epub 2002 Sep 30.
Stabilization of the R allosteric structure of Escherichia coli aspartate transcarbamoylase by disulfide bond formation; West JM et al.; Here we report the first use of disulfide bond formation to stabilize the R allosteric structure of Escherichia coli aspartate transcarbamoylase . In the R allosteric state, residues in the 240s loop from two catalytic chains of different subunits are close together, whereas in the T allosteric state they are far apart . By substitution of Ala-241 in the 240s loop of the catalytic chain with cysteine, a disulfide bond was formed between two catalytic chains of different subunits . The cross-linked enzyme did not exhibit cooperativity for aspartate . The maximal velocity was increased, and the concentration of aspartate required to obtain one-half the maximal velocity, {Asp}(0.5), was reduced substantially . Furthermore, the allosteric effectors ATP and CTP did not alter the activity of the cross-linked enzyme . When the disulfide bonds were reduced by the addition of 1,4-dithio-dl-threitol the resulting enzyme had kinetic parameters very similar to those observed for the wild-type enzyme and regained the ability to be activated by ATP and inhibited by CTP . Small-angle x-ray scattering was used to verify that the cross-linked enzyme was structurally locked in the R state and that this enzyme after reduction with 1,4-dithio-dl-threitol could undergo an allosteric transition similar to that of the wild-type enzyme . The complete abolition of homotropic and heterotropic regulation from stabilizing the 240s loop in its closed position in the R state, which forms the catalytically competent active site, demonstrates the significance that the quaternary structural change and closure of the 240s loop has in the functional mechanism of aspartate transcarbamoylase.

Virology, 2002 Sep 15, 301(1), 165 - 75
Ornithine decarboxylase encoded by chlorella virus PBCV-1; Morehead TA et al.; Sequence analysis of the 330-kb genome of chlorella virus PBCV-1 revealed an open reading frame, A207R, which encodes a protein with 37-41% amino acid identity to ornithine decarboxylase (ODC) from many eukaryotic organisms . The a207r gene was cloned and the protein was expressed as a His-A207R fusion protein in Escherichia coli . The recombinant protein catalyzes pyridoxal 5'-phosphate-dependent decarboxylation of ornithine to putrescine, the first step in the polyamine biosynthetic pathway . The enzyme has a pH optimum of 9.0 and a temperature optimum of 42 degrees C, and it requires dithiothreitol for maximal activity . The enzyme has a K(m) for ornithine of 0.78 mM and a specific activity of 100 micromol/min/mg protein . PBCV-1 ODC is quite sensitive to the competitive inhibitor L-arginine and the irreversible inhibitor difluoromethylarginine but it is less sensitive to the irreversible inhibitor difluoromethylornithine . The a207r gene is expressed both early and late in PBCV-1 infection and is highly conserved among the chlorella viruses . The 42-kDa PBCV-1 ODC (372 amino acids) is the smallest ODC in the databases and, to our knowledge, is the first virus-encoded ODC.

Biochim Biophys Acta, 2002 Sep 27, 1577(3), 445 - 51
Cloning, characterisation, and expression of a novel gene encoding chlorite dismutase from Ideonella dechloratans; Thorell HD et al.; The gene for chlorite dismutase was isolated from a lambda ZAP genomic library of Ideonella dechloratans using peptide sequences obtained from the purified protein . A nucleotide sequence of 1200 bp was determined . The chlorite dismutase gene codes for a 285-residue polypeptide, with the experimentally determined N-terminus of the mature protein at residue 38 . The N-terminal part of the encoded peptide has the characteristics of a signal sequence for periplasmic proteins . The nucleotide sequence and deduced protein showed no homology to sequences found in the databases . The transcriptional start point of the chlorite dismutase gene was determined with a new primer extension technique using capillary electrophoresis . Expression of recombinant chlorite dismutase in E . coli resulted in an active enzyme with similar spectral properties as the native enzyme.

Biochem Biophys Res Commun, 2002 Oct 4, 297(4), 756 - 9
Gammaherpesviruses encode functional dihydrofolate reductase activity; Gaspar G et al.; We overexpressed and purified from Escherichia coli the dihydrofolate reductase (DHFR) of the gammaherpesviruses human herpesvirus 8 (HHV-8), herpesvirus saimiri (HVS), and rhesus rhadinovirus (RRV) . All three enzymes proved catalytically active . The K(m) value of HHV-8 DHFR for dihydrofolate (DHF) was 2.02+/-0.44 microM, that of HVS DHFR was 4.31+/-0.56 microM, and that of RRV DHFR is 7.09+/-0.11 microM . These values are approximately 5-15-fold higher than the K(m) value reported for the human DHFR . The K(m) value of HHV-8 DHFR for NADPH was 1.31+/-0.23 microM, that of HVS DHFR was 3.78+/-0.61 microM, and that of RRV DHFR was 7.47+/-0.59 microM . These values are similar or slightly higher than the corresponding K(m) value of the human enzyme . Methotrexate, aminopterin, trimethoprim, pyrimethamine, and N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine (PT523), all well-known folate antagonists, inhibited the DHFR activity of the three gammaherpesviruses competitively with respect to DHF but proved markedly less inhibitory to the viral than towards the human enzyme.

Mol Genet Metab, 2002 Sep-Oct, 77(1-2), 68 - 79
Identification of isobutyryl-CoA dehydrogenase and its deficiency in humans; Nguyen TV et al.; The acyl-CoA dehydrogenases (ACDs) are a family of related enzymes that catalyze the alpha,beta-dehydrogenation of acyl-CoA esters . Two homologues active in branched chain amino acid metabolism have previously been identified . We have used expression in Escherichia coli to produce a previously uncharacterized ACD-like sequence (ACAD8) and define its substrate specificity . Purified recombinant enzyme had a k(cat)/K(m) of 0.8, 0.23, and 0.04 (microM(-1)s(-1)) with isobutyryl-CoA, (S) 2-methylbutyryl-CoA, and n-propionyl-CoA, respectively, as substrates . Thus, this enzyme is an isobutyryl-CoA dehydrogenase . A single patient has previously been described whose fibroblasts exhibit a specific deficit in the oxidation of valine . Amplified ACAD8 cDNA made from patient fibroblast mRNA was homozygous for a single nucleotide change (905G>A) in the ACAD8 coding region compared to the sequence from control cells . This encodes an Arg302Gln substitution in the full-length protein (position 280 in the mature protein), a position predicted by molecular modeling to be important in subunit interactions . The mutant enzyme was stable but inactive when expressed in E . coli . It was also stable and appropriately targeted to mitochondria, but inactive when expressed in mammalian cells . These data confirm further the presence of a separated ACD in humans specific to valine catabolism (isobutyryl-CoA dehydrogenase, IBDH), along with the first enzymatic and molecular confirmation of a deficiency of this enzyme in a patient.

J Biochem (Tokyo), 2002 Oct, 132(4), 629 - 34
Membrane topology inversion of SecG detected by labeling with a membrane-impermeable sulfhydryl reagent that causes a close association of SecG with SecA; Nagamori S et al.; SecG stimulates protein translocation in Escherichia coli by facilitating the membrane insertion-deinsertion cycle of SecA . SecG was previously shown to undergo membrane topology inversion, since SecA-dependent protein translocation renders the membrane-protected region of SecG sensitive to external proteases . To examine this topology inversion in more detail without protease-treatment, SecG derivatives with a single cysteine residue at various positions were labeled in the presence and absence of protein translocation with a membrane impermeable SH reagent, 4-acetamido-4'-maleimidylstilbene-2-2'-disulfonic acid (AMS) . Treatment of spheroplasts with AMS revealed that a cysteine residue in the cytoplasmic region of SecG could be labeled from the periplasm side only in the presence of protein translocation, whereas a cytoplasmic protein, elongation factor, Tu, remained unlabeled . Treatment of inverted membrane vesicles with AMS also revealed that cysteine residues in the periplasmic region were labeled from the cytoplasmic side of membranes only when protein translocation was in progress . This labeling required ATP, SecA and a precursor protein, and became more efficient as the position of the cysteine residue became closer to the C-terminus . Crosslinking analyses revealed that the interaction between SecG and SecA in membranes markedly increases when SecA and SecG undergo membrane-insertion and topology inversion, respectively . Thus, the two most dynamic components of the translocation machinery were found for the first time to interact with each other when both undergo conformational changes.

J Biochem (Tokyo), 2002 Oct, 132(4), 551 - 5
Mutagenicity of 5-formylcytosine, an oxidation product of 5-methylcytosine, in DNA in mammalian cells; Kamiya H et al.; To examine the mutagenicity of 5-formylcytosine (5-fC), an oxidation product of 5-methylcytosine (5-mC), 5-fC was incorporated into predetermined sites of double-stranded shuttle vectors . The nucleotide sequences in which the modified base was incorporated were 5'-AFGCGT-3' and 5'-ACGFGT-3' (F represents 5-fC), the recognition site for the restriction enzyme MluI (5'-ACGCGT-3') . 5-fC was incorporated into the template strand of either the leading or lagging strand of DNA replication . The modified DNAs were transfected into simian COS-7 cells, and the DNAs replicated in the cells were recovered and analyzed after a second transfection into Escherichia coli . 5-fC weakly blocked DNA replication in mammalian cells . The 5-fC residues were mutagenic, with mutation frequencies in double-stranded vectors of 0.03-0.28% . The mutation spectrum of 5-fC was broad, and included targeted (5-fC-->G, 5-fC-->A, and 5-fC-->T) and untargeted mutations . These results suggest that the oxidation of 5-mC results in mutations at and around the modified sites.

Allergy, 2002 Nov, 57(11), 1053 - 8
Severe IgE-mediated anaphylaxis following consumption of fried frog legs: definition of alpha-parvalbumin as the allergen in cause; Hilger C et al.; BACKGROUND: IgE-mediated allergic reactions to bullfrog and edible frog have been reported . The implicated allergens have not been defined so far . The frog material and the patient's serum from a case of severe food-induced anaphylaxis were used to define the implicated allergen at the protein and DNA level . METHODS: Immunoblotting techniques and N-terminal protein microsequencing were used to define the allergen recognized by the patient's serum . Back translation from the identified protein sequence was used to design degenerated primers to amplify the allergen's cDNA by polymerase chain reaction (PCR) . We defined the nucleotide sequence of the allergen from the frog of Indonesian origin that was consumed by the patient, and the homologous cDNA from Rana esculenta . RESULTS: Protein microsequencing revealed that the implicated frog allergen belonged to the parvalbumin family . cDNAs coding for alpha- and beta-parvalbumin of R . esculenta and Rana species were cloned . Recombinant proteins were expressed in Escherichia coli . The patient's serum IgE antibodies recognized parvalbumin prepared from frog muscle and recombinant alpha-parvalbumin from R . species but not from R . esculenta . Recombinant beta-parvalbumin was not recognized by the IgE antibodies . CONCLUSION: This work defines at the protein and DNA levels alpha-parvalbumin as the allergen implicated in a case of IgE-mediated anaphylaxis to frog muscle . It also shows that a protein belonging to the parvalbumin family is implicated in type I allergies outside the fish species.

Biochem J, 2002 Dec 15, 368(Pt 3), 789 - 97
Functional interaction between nuclear inhibitor of protein phosphatase type 1 (NIPP1) and protein phosphatase type 1 (PP1) in Drosophila: consequences of over-expression of NIPP1 in flies and suppression by co-expression of PP1; Parker L et al.; The catalytic subunit of type 1 Ser/Thr protein phosphatases (PP1c) forms complexes with many proteins that target it to particular subcellular locations and regulate its activity towards specific substrates . We report the identification of a Drosophila orthologue of nuclear inhibitor of PP1 (NIPP1Dm) through interaction with PP1c in the yeast two-hybrid system . NIPP1Dm shares many properties with mammalian NIPP1 including inhibition of PP1c in vitro, binding to RNA and PP1c, and localization to nuclear speckles . However, the mechanism controlling interaction of PP1c with NIPP1 is not conserved in Drosophila . NIPP1 can function independently of PP1c as a splicing factor, but the relative importance of this function is unknown . Over-expression of NIPP1Dm in Drosophila is cell-lethal in a range of tissues and developmental stages . The effects of ectopic NIPP1Dm are suppressed by co-expression of PP1c, indicating that the only effect of ectopic NIPP1Dm is to affect PP1c function . Co-expression of NIPP1Dm and PP1c does not have any detectable physiological effect in vivo, suggesting that the NIPP1Dm-PP1c holoenzyme is not normally limiting in Drosophila . These data show that NIPP1Dm and PP1c interact in vivo and suggest that NIPP1's role as a phosphatase regulator is conserved in Drosophila.

RNA, 2002 Sep, 8(9), 1137 - 47
Protein S1 counteracts the inhibitory effect of the extended Shine-Dalgarno sequence on translation; Komarova AV et al.; There are two major components of Escherichia coli ribosomes directly involved in selection and binding of mRNA during initiation of protein synthesis-the highly conserved 3' end of 16S rRNA (aSD) complementary to the Shine-Dalgarno (SD) domain of mRNA, and the ribosomal protein S1 . A contribution of the SD-aSD and S1-mRNA interactions to translation yield in vivo has been evaluated in a genetic system developed to compare efficiencies of various ribosome-binding sites (RBS) in driving beta-galactosidase synthesis from the single-copy (chromosomal) lacZ gene . The in vivo experiments have been supplemented by in vitro toeprinting and gel-mobility shift assays . A shortening of a potential SD-aSD duplex from 10 to 8 and to 6 bp increased the beta-galactosidase yield (four- and sixfold, respectively) suggesting that an extended SD-aSD duplex adversely affects translation, most likely due to its redundant stability causing ribosome stalling at the initiation step . Translation yields were significantly increased upon insertion of the A/U-rich S1 binding targets upstream of the SD region, but the longest SD remained relatively less efficient . In contrast to complete 30S ribosomes, the S1-depleted 30S particles have been able to form an extended SD-aSD duplex, but not the true ternary initiation complex . Taken together, the in vivo and in vitro data allow us to conclude that S1 plays two roles in translation initiation: It forms an essential part of the mRNA-binding track even when mRNA bears a long SD sequence, and through the binding to the 5' untranslated region, it can ensure a substantial enhancing effect on translation.

J Med Microbiol, 2002 Sep, 51(9), 731 - 9
Recombinant OspC from Borrelia burgdorferi sensu stricto, B . afzelii and B . garinii in the serodiagnosis of Lyme borreliosis; Panelius J et al.; Genes for the outer-surface protein C (OspC) from three north European human isolates of Borrelia burgdorferi sensu stricto, B . afzelii and B . garinii were cloned and sequenced . Polyhistidine-tagged recombinant OspC (rOspC) proteins were produced in Escherichia coli and used, after biotinylation, as antigens on streptavidin-coated plates in enzyme-linked immunosorbent assays (ELISA) . In IgM ELISA, 30% (5/17) and 35% (6/17) of patients with erythema migrans (EM) in the acute or convalescent phase, respectively, reacted with one to three rOspCs . Of the patients, 53% (8/15) with neuroborreliosis (NB) and 53% (8/15) with Lyme arthritis (LA) had IgM antibodies to OspC . The immunoreactivity was stronger against rOspC from B . afzelii and B . garinii than against rOspC from B . burgdorferi sensu stricto . In early Lyme borreliosis (LB), rOspC and flagella performed equally well in detecting IgM antibodies . Cross-reactive antibodies to rOspC were observed in serum samples from patients with rheumatoid factor positivity and with syphilis or Epstein-Barr virus (EBV) infection . In IgM ELISA, thiocyanate in the serum dilution buffer reduced EBV-associated non-specific positive reactions . Of the patient sera examined in IgG ELISA, 30% (5/17) with EM in the acute phase, 35% (6/17) with EM in the convalescent phase, 33% (5/15) with NB and 60% (9/15) with LA were positive . Because of the heterogeneity of OspC, a polyvalent antigen with several OspC variants from at least B . afzelii and B . garinii is needed to improve the sensitivity of OspC ELISA in the serodiagnosis of LB in Europe.

Proc Natl Acad Sci U S A, 2002 Oct 15, 99(21), 13448 - 52 Epub 2002 Sep 30.
Subunit rotation of ATP synthase embedded in membranes: a or beta subunit rotation relative to the c subunit ring; Nishio K et al.; ATP synthase F(o)F(1) (alpha(3)beta(3)gammadelta epsilon ab(2)c(10-14)) couples an electrochemical proton gradient and a chemical reaction through the rotation of its subunit assembly . In this study, we engineered F(o)F(1) to examine the rotation of the catalytic F(1) beta or membrane sector F(o) a subunit when the F(o) c subunit ring was immobilized; a biotin-tag was introduced onto the beta or a subunit, and a His-tag onto the c subunit ring . Membrane fragments were obtained from Escherichia coli cells carrying the recombinant plasmid for the engineered F(o)F(1) and were immobilized on a glass surface . An actin filament connected to the beta or a subunit rotated counterclockwise on the addition of ATP, and generated essentially the same torque as one connected to the c ring of F(o)F(1) immobilized through a His-tag linked to the alpha or beta subunit . These results established that the gamma epsilon c(10-14) and alpha(3)beta(3)deltaab(2) complexes are mechanical units of the membrane-embedded enzyme involved in rotational catalysis.

Invest Ophthalmol Vis Sci, 2002 Oct, 43(10), 3165 - 73
Relaxation of beta-structure in tear lipocalin and enhancement of retinoid binding; Gasymov OK et al.; PURPOSE: To study binding of retinoids to human tear lipocalin (TL) to assess factors influencing ligand affinity and delivery . Mechanistic features of retinoid interactions with TL were investigated, including the influence of the retinoid functional group on ligand affinity, the relative affinity of retinol versus fatty acids, the influence of relaxation of secondary structure in TL on ligand binding, the role of specific conserved hydrophobic residues in maintaining the rigidity of the secondary structure, and the potential release of retinol in a low-pH environment that promotes structural relaxation at lipid interfaces . METHODS: The binding and displacement of retinoids were monitored by quenching of protein fluorescence . Circular dichroic spectra were used to evaluate structural and conformational changes in TL-retinoid complexes . Site-directed mutagenesis was performed to determine the influence of the residues Trp17, Ile98, Gly15, and Leu19 in retinoid binding to TL and to correlate these effects with changes in secondary structure . RESULTS: Retinal and retinol bound TL with similar affinity . Fatty acids competed with retinoids for the same binding site on TL . Optical activity associated with retinal binding to TL was reduced in the presence of palmitic acid . In comparison with TL, the mutants W17C and I98C displayed relaxation of secondary structure, manifested as diminution of beta-sheet content in conjunction with a destabilization in urea, reduced aromatic asymmetry, and greater binding affinity for retinoids . Unlike fatty acids, retinol is not released from TL at low pH . CONCLUSIONS: The unique spectral properties of retinoids permit the simultaneous study of structural changes in TL and ligand binding . Retinoid binding is enhanced by specific mutations that induce relaxation of TL structure but is altered minimally by the functional group in retinoids . Two key hydrophobic residues, Trp17 (A strand) and Ile98 (G strand), contribute to backbone rigidity and influence retinoid binding through their participation in an internal hydrophobic cluster and external hydrophobic patch, respectively . The contributions of these sites to ligand binding may explain their conserved nature in the lipocalin family . Information regarding the binding and release of retinoids compared with fatty acids favors a role for TL in the delivery of lipids other than retinol to the tear film interfaces.

J Biol Chem, 2002 Dec 6, 277(49), 47619 - 25 Epub 2002 Sep 28.
A single amino acid difference between human and monkey interleukin (IL)-1beta dictates effective binding to soluble type II IL-1 receptor; Smith DE et al.; Soluble type II interleukin (IL)-1 receptor (sIL1R-II) binds human IL-1beta with high affinity and neutralizes its activity . Recombinant sIL1R-II is considered a potentially useful anti-IL-1 therapeutic, and preclinical studies have been undertaken with this molecule in primates . To better understand the cytokine-receptor interactions occurring in this nonhuman context, monkey IL-1 and IL1R-II were cloned, and their binding abilities were examined in vitro . IL-1beta from cynomolgus monkey was capable of binding and activating the human type I IL-1 receptor . However, unlike human IL-1beta, it was unable to effectively bind and become neutralized by sIL1R-II . Human and cynomolgus IL-1beta proteins are 96% identical, differing by only six amino acids . Structural and mutational analysis revealed that the unique sIL1R-II binding ability of human IL-1beta is due to a single amino acid difference compared with monkey IL-1beta.

EMBO J, 2002 Oct 1, 21(19), 5281 - 91
Monitoring intermediate folding states of the td group I intron in vivo; Waldsich C et al.; Group I introns consist of two major structural domains, the P4-P6 and P3-P9 domains, which assemble through interactions with peripheral extensions to fold into an active ribozyme . To assess group I intron folding in vivo, we probed the structure of td wild-type and mutant introns using dimethyl sulfate . The results suggest that the majority of the intron population is in the native state in accordance with the current structural model, which was refined to include two novel tertiary contacts . The importance of the loop E motif in the P7.1-P7.2 extension in assisting ribozyme folding was deduced from modeling and mutational analyses . Destabilization of stem P6 results in a deficiency in tertiary structure formation in both major domains, while weakening of stem P7 only interferes with folding of the P3-P9 domain . The different impact of mutations on the tertiary structure suggests that they interfere with folding at different stages . These results provide a first insight into the structure of folding intermediates and suggest a putative order of events in a hierarchical folding pathway in vivo.

Am J Respir Cell Mol Biol, 2002 Oct, 27(4), 446 - 54
Decreased distribution of lung epithelial junction proteins after intratracheal antigen or lipopolysaccharide challenge: correlation with neutrophil influx and levels of BALF sE-cadherin; Evans SM et al.; Distribution of airway junctional complex proteins after antigen or lipopolysaccharide challenge in sensitized or naive mice, respectively, was investigated . E-cadherin immunoreactivity was detected continuously along neighboring epithelial cell borders and between adjacent alveolar epithelial cells in naive and saline-challenged mice . Occludin and ZO-1 immunoreactivity were observed in the tight junction areas . Both challenges induced changes in epithelial morphology and phenotype, accompanied initially by focal loss of epithelial E-cadherin that increased in size with time and number of allergen challenges . Allergen challenge also led to focal loss of occludin and ZO-1 . Western blot analysis revealed increased levels of sE-cadherin in lavage fluid after either challenge, and this increase correlated with lavage neutrophil numbers (P = 0.002) . Immunocytochemistry of lavage cells 6 h after either challenge revealed E-cadherin epitopes within cytoplasmic vacuoles of neutrophils, the major cell type . In contrast, peripheral blood neutrophils or tissue neutrophils before epithelial transmigration were negative, suggesting that in airway inflammation, E-cadherin extracellular domain is cleaved by neutrophils during epithelial penetration, instigating the destabilization of adherens and tight junctions . This junctional deterioration could lead to a progressive decrease in epithelial integrity and induce alterations in epithelial morphology, with consequent enhanced paracellular transit of antigens and pathogens.

Protein Expr Purif, 2002 Oct, 26(1), 171 - 8
A fusion protein system for the recombinant production of short disulfide-containing peptides; Fairlie WD et al.; A recombinant fusion protein system for the production, oxidation, and purification of short peptides containing a single disulfide bond is described . The peptides are initially expressed in Escherichia coli as a fusion to an engineered mutant of the N-terminal SH2 domain of the intracellular phosphatase, SHP-2 . This small protein domain confers several important properties which facilitate the production of disulfide-containing peptides: (i) it is expressed at high levels in E . coli; (ii) it can be purified via a hexahistidine tag and reverse-phase HPLC; (iii) it contains no endogenous cysteine residues, allowing the formation of an intrapeptide disulfide bond while still attached to the fusion partner; (iv) it is highly soluble in native buffers, facilitating the production of very hydrophobic peptides and the direct use of fusion products in biochemical assays; (v) it contains a unique methionine residue at the junction of the peptide and fusion partner to facilitate peptide cleavage by treatment with cyanogen bromide (CNBr) . This method is useful for producing peptides, which are otherwise difficult to prepare through traditional chemical synthesis approaches, and this has been demonstrated by preparing a number of hydrophobic disulfide-containing peptides derived from phage-display libraries.

Protein Expr Purif, 2002 Oct, 26(1), 89 - 95
Expression of Torpedo californica creatine kinase in Escherichia coli and purification from inclusion bodies; Wang PF et al.; The pET17 expression vector was used to express creatine kinase from the electric organ of Torpedo californica as inclusion bodies in Escherichia coli BL21(DE3) cells . The insoluble aggregate was dissolved in 8M urea and, following extraction with Triton X-100, the enzyme was refolded by dialysis against Tris buffer (pH 8.0) containing 0.2M NaCl . After two buffer changes, chromatography on Blue Sepharose was used as a final step in the purification procedure . Approximately 54mg active protein was recovered from a 1L culture and the refolded enzyme had a specific activity of 75U/mg . The molecular mass of the purified protein was consistent with that predicted from the amino acid sequence and the CD spectrum of the refolded enzyme was essentially identical to that of creatine kinase from human muscle (HMCK) . The K(m) values of ATP and ADP were also similar to those of HMCK, while the K(m) values for both phosphocreatine and creatine were approximately 5-10-fold higher . The purification described here is in marked contrast with earlier attempts at purification of this isozyme where, in a process yielding less than 1mg/L culture, enzyme with a specific activity of ca . 5U/mg was obtained.

Protein Expr Purif, 2002 Oct, 26(1), 71 - 81
Identification, characterization of levoglucosan kinase, and cloning and expression of levoglucosan kinase cDNA from Aspergillus niger CBX-209 in Escherichia coli; Zhuang X et al.; The first enzyme responsible for assimilating levoglucosan in Aspergillus niger CBX-209 was corroborated to be levoglucosan kinase that catalyzes the transfer of a phosphate group from ATP to levoglucosan to yield a glucose 6-phosphate in the presence of magnesium ion and ATP by FAB-mass spectrometric method combined with previous observations from HPLC and enzymological experiments . Levoglucosan kinase was purified to apparent homogeneity by using a combination of seven purification steps . SDS-PAGE revealed a single protein band of 56 KDa . It is a monomeric enzyme and maximal enzyme activity was measured at pH 9.3 and 30 degrees C . This kinase is stable below 20 degrees C at a quite broad pHs ranging from 6 to 10 and levoglucosan could protect the enzyme from thermal inactivation . Exclusive substrate specificity for levoglucosan suggested that not only the structure of the intramolecular glucosidic linkage but also the configuration of the pyranose frame would be specific for recognition by levoglucosan kinase . The K(m) values of this enzyme were 71.2mM for levoglucosan and 0.25 mM for ATP, determined by double reciprocal plottings and ADP inhibited on the enzyme activity competitively with a Ki value of 0.20mM . A cDNA library from A . niger was constructed in Escherichia coli DH5alpha . The library was screened for levoglucosan kinase gene on NCE selective medium and three positive recombinants were selected after a five day culture . Detection of activities of levoglucosan kinase in the cell extracts indicated that levoglucosan kinase gene (lgk) was expressed by the recombinant strain of E . coli DH5alpha.

Protein Expr Purif, 2002 Oct, 26(1), 35 - 41
Cloning, expression, and purification of the functional Delta(3)-Delta(2)-enoyl-CoA isomerase fusion protein; Li D et al.; Delta(3)-Delta(2)-Enoyl-CoA isomerase (EC 5.3.3.8) is a key enzyme for the beta-oxidation of unsaturated fatty acids . The cDNA of the full-length rat liver Delta(3)-Delta(2)-enoyl-CoA isomerase was previously cloned as pAG847 . PCR methodologies were used to subclone the gene encoding the functional Delta(3)-Delta(2)-enoyl-CoA isomerase from pAG847 with primers that were designed to add six continuous histidine codon to the 5(') primer . The PCR product was inserted into a pLM1 expression vector and overexpressed in Escherichia coli . The soluble expressed protein was purified with a nickel HiTrap chelating metal affinity column to apparent homogeneity based on Coomassie blue-stained SDS-PAGE and the molecular weight of the protein subunit was 30 kDa . The purified protein had a dimeric structure composed of identical subunits, and the molecular weight of the enzyme determined by gel chromatography was 60 kDa . Kinetic studies have been carried out and K(M) of 81 microM and V(max) of 292 micromol/min/mg were determined . The specific activity of the protein is 201 U/mg, which is significantly higher than that reported before for the same protein isolated from a natural source . The one-step purification of the highly active Delta(3)-Delta(2)-enoyl-CoA isomerase will greatly facilitate the further investigation of this enzyme through site-directed mutagenesis and enzyme catalyzed reactions with substrate analogues.

Biochemistry, 2002 Oct 8, 41(40), 12224 - 35
Kinetic analysis of interdomain coupling in a lidless variant of the molecular chaperone DnaK: DnaK's lid inhibits transition to the low affinity state; Slepenkov SV et al.; DnaK, the Escherichia coli Hsp70, possesses two functional domains, the N- and C-terminal ATPase and peptide-binding domains, respectively . Elucidation of the mechanism of allosteric coupling between the two domains is key to understanding how Hsp70 chaperones interact with their substrates . We previously reported that ATP reacts with wild-type DnaK-peptide complexes according to the two-step reaction, ATP + DnaK-P if ATP-DnaK-P if ATP-DnaK + P, where ATP binds in the first step, and a conformational change that quenches DnaK's tryptophan fluorescence (denoted by the asterisk) and expels bound peptide occurs in the second step . Here we report that DnaK(2-517), a lidless variant, also reacts with ATP and peptide by this two-step mechanism . Compared to wild-type DnaK, we found that, depending on the sequence of the bound peptide and the temperature, deletion of the lid produces a 27- to 66-fold increase in the rate constant (k(2)) for the ATP-triggered conformational change (ATP-DnaK-P --> ATP-DnaK+P) but only a approximately 2-fold increase in the rate constant (k(-)(2)) for the reverse reaction (ATP-DnaK+P --> ATP-DnaK-P) . A model is proposed in which the lid regulates the rate of interdomain communication by retarding motions within the beta-sandwich that occur as a consequence of ATP binding . New evidence in support of the reversible, two-step conformational switch mechanism is also presented.

Biochemistry, 2002 Oct 8, 41(40), 12062 - 75
Isolation and characterization of a family of stable RNA tetraloops with the motif YNMG that participate in tertiary interactions; Proctor DJ et al.; RNA is known to fold into a variety of structural elements, many of which have sufficient sequence complexity to make the thermodynamic study of each possible variant impractical . We previously reported a method for isolating stable and unstable RNA sequences from combinatorial libraries using temperature gradient gel electrophoresis (TGGE) . This method was used herein to analyze a six-nucleotide RNA hairpin loop library . Three rounds of in vitro selection were performed using TGGE, and unusually stable RNAs were identified by cloning and sequencing . Known stable tetraloops were found, including sequences belonging to the UNCG motif closed by a CG base pair, and the CUUG motif closed by a GC base pair . In addition, unknown tetraloops were found that were nearly as stable as cUNCGg, including sequences related through substitution of the U with a C (Y), the C with an A (M), or both . These substitutions allow hydrogen bonding and stacking interactions in the UNCG loop to be maintained . Thermodynamic analysis of YNMG and variant loops confirmed optimal stability with Y at position 1 and M at position 3 . Similarity in structure and stability among YNMG loops was further supported by deoxyribose substitution, CD, and NMR experiments . A conserved tertiary interaction in 16S rRNA exists between a YAMG loop at position 343 and two adenines in the loop at position 159 (Escherichia coli numbering) . NMR and functional group substitution experiments suggest that YNAG loops in particular have enhanced flexibility, which allows the tertiary interaction to be maintained with diverse loop sequences at position 159 . Taken together, these results support the existence of an extended family of UNCG-like tetraloops with the motif cYNMGg that are thermodynamically stable and structurally similar and can engage in tertiary interactions in large RNA molecules.

Biochemistry, 2002 Oct 8, 41(40), 12032 - 42
Two (betaalpha)(8)-barrel enzymes of histidine and tryptophan biosynthesis have similar reaction mechanisms and common strategies for protecting their labile substrates; Henn-Sax M et al.; The enzymes N'-{(5'-phosphoribosyl)formimino}-5-aminoimidazole-4-carboxamide ribonucleotide isomerase (HisA) and phosphoribosylanthranilate isomerase (TrpF) are sugar isomerases that are involved in histidine and tryptophan biosynthesis, respectively . Both enzymes have the (betaalpha)(8)-barrel fold and catalyze Amadori rearrangements of a thermolabile aminoaldose into the corresponding aminoketose . To identify those amino acids that are essential for catalysis, conserved residues at the active sites of both HisA and TrpF from the hyperthermophile Thermotoga maritima were replaced by site-directed mutagenesis, and the purified variants were investigated by steady-state enzyme kinetics . Aspartate 8, aspartate 127, and threonine 164 appeared to be important for the HisA reaction, whereas cysteine 7 and aspartate 126 appeared to be important for the TrpF reaction . On the basis of these results and the X-ray structure of a complex between TrpF and a bound product analogue, a reaction mechanism involving general acid-base catalysis and a Schiff base intermediate is proposed for both enzymes . A comparison of the HisA and TrpF enzymes from T . maritima and Escherichia coli showed that, at the physiological temperatures of 80 and 37 degrees C, respectively, the enzymes from the hyperthermophile have significantly higher catalytic efficiencies than the corresponding enzymes from mesophiles . These results suggest that HisA and TrpF have similar chemical reaction mechanisms and use the same strategy to prevent the loss of their thermolabile substrates.

Biochemistry, 2002 Oct 8, 41(40), 12010 - 24
The crystal structure of YdcE, a 4-oxalocrotonate tautomerase homologue from Escherichia coli, confirms the structural basis for oligomer diversity; Almrud JJ et al.; The tautomerase superfamily consists of three major families represented by 4-oxalocrotonate tautomerase (4-OT), 5-(carboxymethyl)-2-hydroxymuconate isomerase (CHMI), and macrophage migration inhibitory factor (MIF) . The members of this superfamily are structurally homologous proteins constructed from a simple beta-alpha-beta fold that share a key mechanistic feature; they use an amino-terminal proline, which has an unusually low pK(a), as the general base in a keto-enol tautomerization . Several new members of the 4-OT family have now been identified using PSI-BLAST and categorized into five subfamilies on the basis of multiple-sequence alignments and the conservation of key catalytic and structural residues . The members of subfamily 5, which includes a hypothetical protein designated YdcE from Escherichia coli, are predicted not to form hexamers . The crystal structure of YdcE has been determined to 1.35 A resolution and confirms that it is a dimer . In addition, YdcE complexed with (E)-2-fluoro-p-hydroxycinnamate, identified as a potent competitive inhibitor of this enzyme, as well as N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) and benzoate are also presented . These latter crystal structures reveal the location of the active site and suggest a mechanism for the observed YdcE-catalyzed tautomerization reaction . The dimeric arrangement of YdcE represents a new structure in the 4-OT family and demonstrates structural diversity within the 4-OT family not previously reported.

Biochemistry, 2002 Oct 8, 41(40), 11954 - 62
Three-dimensional solution NMR structure of Apo-L75F-TrpR, a temperature-sensitive mutant of the tryptophan repressor protein; Tyler R et al.; L75F-TrpR is a temperature-sensitive mutant of the tryptophan repressor protein of Escherichia coli in which surface-exposed residue leucine 75 in the DNA binding domain is replaced with phenylalanine . Biochemical and biophysical studies had suggested global alterations in dynamics for L75F-TrpR, although the structure was apparently similar to that of wild-type TrpR . Herein, we report the three-dimensional solution structure of apo-L75F-TrpR determined by multidimensional ((1)H, (15)N, and (13)C) solution NMR spectroscopy . An ensemble of structures was generated from 769 unique NOE-based distance restraints, 68 dihedral angle restraints, and 62 hydrogen bond distance restraints . Apo-L75F-TrpR exhibits a three-dimensional (3D) fold very similar to that of apo-WT-TrpR, with a dimeric core of four alpha-helices (A-C and F) from each subunit, and less well-defined D and E helical regions of the DNA binding domains . Despite their many similarities, wild-type and mutant proteins display significant chemical shift differences, one cluster of which is in the B-C turn, too distant to be ascribed solely to ring current effects from Phe75 . Differences in NOE patterns and amide proton exchange rates are also observed in the B-C turn region . The data provide evidence that this point mutation exerts local effects on structure and stability in the DNA binding domain, and propagates long-range effects through the tertiary structure.

Biotechnol Bioeng, 2002 Dec 5, 80(5), 544 - 51
Modeling of the controlled expression of a harmful protein by a three-plasmid harboring system; Unzaga TV et al.; A genetically structured mathematical model was developed and used to evaluate the influence of molecular parameters involved in the expression of a harmful recombinant protein (SPA::EcoRI) . The system consists of the controlled expression of the endonuclease EcoRI cloned in the plasmid pMTC48 . The control is exerted by the lambda CI repressor expressed from the plasmid pRK248cIts . The deleterious effect of the activity of the enzyme EcoRI on the host DNA is prevented by the action of the EcoRI methylase that is expressed constitutively from a third plasmid, pEcoR4 . The model includes molecular mechanisms involved in the regulation of the expression of these genes and is used to determine cultural conditions that maximize the production of the recombinant protein .

Biotechnol Bioeng, 2002 Dec 5, 80(5), 516 - 24
Production of cytidine 5'-monophosphate N-acetylneuraminic acid using recombinant Escherichia coli as a biocatalyst; Lee SG et al.; An Escherichia coli strain expressing three recombinant enzymes, i.e., cytidine 5'-monophosphate (CMP) kinase, sialic acid aldolase and cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-NeuAc) synthetase, was utilized as a biocatalyst for the production of CMP-NeuAc . Both recombinant E . coli extract and whole cells catalyzed the production of CMP-NeuAc from CMP (20 mM), N-acetylmannosamine (40 mM), pyruvate (60 mM), ATP (1 mM), and acetylphosphate (60 mM), resulting in 90% conversion yield based on initial CMP concentration used . It was confirmed that endogenous acetate kinase can catalyze not only the ATP regeneration in the conversion of CMP to CDP but also the conversion of CDP to CTP . On the other hand, endogenous pyruvate kinase and polyphosphate kinase could not regenerate ATP efficiently . The addition of exogenous acetate kinase to the reaction mixture containing the cell extract increased the conversion rate of CMP to CMP-NeuAc by about 1.5-fold, but the addition of exogenous inorganic pyrophosphatase had no influence on the reaction . This E . coli strain could also be employed as an enzyme source for in situ regeneration of CMP-NeuAc in a sialyltransferase catalyzed reaction . About 90% conversion yield of alpha2,3-sialyl-N-acetyllactosamine was obtained from N-acetyllactosamine (20 mM), CMP (2 mM), N-acetylmannosamine (40 mM), pyruvate (60 mM), ATP (1 mM), and acetyl phosphate (80 mM) using the recombinant E . coli extract and alpha2,3-sialyltransferase .

J Comp Physiol A Neuroethol Sens Neural Behav Physiol, 2002 Sep, 188(8), 589 - 94 Epub 2002 Aug 03.
Neuronal involvement in the effect of an antisecretory factor-derived peptide on induced secretion in the porcine small intestine; Grondahl ML et al.; The antisecretory factor is a protein inhibiting enterotoxin-induced intestinal inflammation and hypersecretion . We studied the signaling pathway of three antisecretory factor-derived peptides (A1, A3 and A4) in the proximal and distal porcine small intestine . In vivo (ligated loops), only A3 significantly reduced the cholera toxin-induced fluid accumulation and only in proximal loops . A3 and A4 reduced Escherichia coli heat-labile enterotoxin-induced fluid accumulation in the proximal segment, whereas A1 and A3 reduced the Escherichia coli heat-labile enterotoxin-induced fluid accumulation in the distal segment . The secretory response to intraluminally added 5-hydroxytryptamine was not significantly inhibited by the peptides . The amount of intraluminal 5-hydroxytryptamine accumulated in cholera toxin-stimulated loops from the proximal segment was significantly reduced by A3 . In vitro,the effect of A3 on secretagogue-induced increases in short-circuit current was recorded from proximal small intestine by the Ussing chamber technique . A3 decreased the tetrodotoxin sensitive effect of substance P . The in vivo results suggest that the antisecretory effect may involve inhibition of the local release of 5-hydroxytryptamine induced by cholera toxin, but not inhibition of secretory reflexes induced by 5-hydroxytryptamine . The in vitro results suggest that the effect of A3 lies beyond the surface epithelium, and involves mucosal neuronal structures.

Plant Cell Physiol, 2002 Sep, 43(9), 1054 - 8
Salicylic acid carboxyl methyltransferase induced in hairy root cultures of Atropa belladonna after treatment with exogeneously added salicylic acid; Fukami H et al.; In Atropa belladonna hairy roots, exogeneously added salicylic acid (SA) is converted to methyl salicylate (MSA) through the reaction, which might be catalysed by S-adenosyl-L-methionine: salicylic acid carboxyl methyltransferase (SAMT) . Here we cloned a cDNA for A . belladonna SAMT (AbSAMT1), which consisted of 357 aa residues . It was expressed in E . coli, and the recombinant AbSAMT1 showed SAMT activity . When A . belladonna hairy roots were exposed to a high concentration of SA, AbSAMT1 mRNA begins to be expressed 12 h after the exposure, and steady expression continued over 144 h.

Plant Cell Physiol, 2002 Sep, 43(9), 1043 - 8
The role of plant CCTalpha in salt- and osmotic-stress tolerance; Yamada A et al.; To find key genes essential for salt tolerance in the mangrove plant, Bruguiera sexangula, functional screening was performed using Escherichia coli as the host organism . A transformant expressing a cytosolic chaperonin-containing TCP-1alpha (CCTalpha) homologue displayed enhanced salt tolerance . Analysis in E . coli of the functional region revealed that a sequence of only 218 amino acids, containing the apical domain, is necessary for osmotolerance . Furthermore, this domain shows chaperone activity in vitro . Therefore, CCTalpha facilitates the folding of proteins without ATP or the cage-like structure, and may play an important role in stress tolerance, at least in B . sexangula.

Mol Microbiol, 2002 Sep, 45(6), 1599 - 611
A third envelope stress signal transduction pathway in Escherichia coli; Raffa RG et al.; Escherichia coli uses overlapping envelope stress responses to adapt to insults to the bacterial envelope that cause protein misfolding . The sigmaE and Cpx envelope stress responses are activated by both common and distinct envelope stresses and respond by increasing the expression of the periplasmic protease DegP as well as target genes unique to each response . The sigmaE pathway is involved in outer membrane protein (OMP) folding quality control whereas the Cpx pathway plays an important role in the assembly of at least one pilus . Previously, we identified the spy gene as a new Cpx regulon member of unknown function . Interestingly, induction of spy expression by severe envelope stresses such as spheroplasting is only partially dependent on an intact Cpx signalling pathway, unlike other Cpx-regulated genes . Here we show that the BaeS sensor kinase and BaeR response regulator also control expression of spy in response to envelope stress . BaeS and BaeR do not affect expression of other known Cpx-regulated genes, however, baeR cpxR double mutants show increased sensitivity to envelope stresses relative to either single mutant alone . We propose that the Bae signal transduction pathway controls a third envelope stress response in E . coli that induces expression of a distinct set of adaptive genes.

Mol Microbiol, 2002 Sep, 45(6), 1575 - 88
Amplification of Hot DNA segments in Escherichia coli; Kodama K et al.; In Escherichia coli, a replication fork blocking event at a DNA replication terminus (Ter) enhances homologous recombination at the nearby sister chromosomal region, converting the region into a recombination hotspot, Hot, site . Using a RNaseH negative (rnhA-) mutant, we identified eight kinds of Hot DNAs (HotA-H) . Among these, enhanced recombination of three kinds of Hot DNAs (HotA-C) was dependent on fork blocking events at Ter sites . In the present study, we examined whether HotA DNAs are amplified when circular DNA (HotA plus a drug-resistance DNA) is inserted into the homologous region on the chromosome of a rnhA- mutant . The resulting HotA DNA transformants were analysed using pulsed-field gel electrophoresis, fluorescence in situ hybridization and DNA microarray technique . The following results were obtained: (i) HotA DNA is amplified by about 40-fold on average; (ii) whereas 90% of the cells contain about 6-10 copies of HotA DNA, the remaining 10% of cells have as many as several hundred HotA copies; and (iii) amplification is detected in all other Hot DNAs, among which HotB and HotG DNAs are amplified to the same level as HotA . Furthermore, HotL DNA, which is activated by blocking the clockwise oriC-starting replication fork at the artificially inserted TerL site in the fork-blocked strain with a rnhA+ background, is also amplified, but is not amplified in the non-blocked strain . From these data, we propose a model that can explain production of three distinct forms of Hot DNA molecules by the following three recombination pathways: (i) unequal intersister recombination; (ii) intrasister recombination, followed by rolling-circle replication; and (iii) intrasister recombination, producing circular DNA molecules.

Mol Microbiol, 2002 Sep, 45(6), 1557 - 73
Point mutants of EHEC intimin that diminish Tir recognition and actin pedestal formation highlight a putative Tir binding pocket; Liu H et al.; Attachment to host cells by enterohaemorrhagic Escherichia coli (EHEC) is associated with the formation of a highly organized cytoskeletal structure containing filamentous actin, termed an attaching and effacing (AE) lesion . Intimin, an outer membrane protein of EHEC, is required for the formation of AE lesions, as is Tir, a bacterial protein that is translocated into the host cell to function as a receptor for intimin . We established a yeast two-hybrid assay for intimin-Tir interaction and, after random mutagenesis, isolated 24 point mutants in intimin, which disrupted Tir recognition in this system . Analysis of 11 point mutants revealed a correlation between recognition of recombinant Tir and the ability to trigger AE lesions . Many of the mutations fell within a 50-residue region near the C-terminus of intimin . Alanine-scanning mutagenesis of this region revealed four residues (Ser890, Thr909, Asn916 and Asn927) that are critical for Tir recognition . Mapping the sequences of EHEC intimin and Tir onto the crystal structure of the intimin-Tir complex of enteropathogenic E . coli predicts that each of these four intimin residues lies at the intimin-Tir interface and contributes to a pocket that interacts with Ile298 of EHEC Tir . Thus, this genetic approach to intimin function both identified residues critical for Tir binding and demonstrated a correlation between the ability to bind Tir and the ability to trigger actin focusing.

Mol Microbiol, 2002 Sep, 45(6), 1541 - 55
High resolution contact probing of the Lrp-like DNA-binding protein Ss-Lrp from the hyperthermoacidophilic crenarchaeote Sulfolobus solfataricus P2; Enoru-Eta J et al.; Ss-Lrp, from Sulfolobus solfataricus, is an archaeal homologue of the global bacterial regulator Lrp (Leucine-responsive regulatory protein), which out of all genome-encoded proteins is most similar to Escherichia coli Lrp (E-value of 5.6 e-14) . The recombinant protein has been purified as a 68 kDa homotetramer . The specific binding of Ss-Lrp to its own control region is suggestive of negative autoregulation . A high resolution contact map of Ss-Lrp binding was established by DNase I and hydroxyl radical footprinting, small non-intercalating groove-specific ligand-binding interference, and various base-specific premodification and base removal binding interference techniques . We show that Ss-Lrp binds one face of the DNA helix and establishes the most salient contacts with two major groove segments and the intervening minor groove, in a region that overlaps the TATA-box and BRE promoter elements . Therefore, Ss-Lrp most likely exerts autoregulation by preventing promoter recognition by TBP and TFB . Moreover, the results demonstrate profound Ss-Lrp induced structural alterations of sequence stretches flanking the core contact site, and reveal that the deformability of these regions significantly contributes to binding selectivity.

Eur J Biochem, 2002 Oct, 269(19), 4753 - 61
Purification and kinetic analysis of the two recombinant arogenate dehydrogenase isoforms of Arabidopsis thaliana; Rippert P et al.; The present study reports the first purification and kinetic characterization of two plant arogenate dehydrogenases (EC 1.3.1.43), an enzyme that catalyses the oxidative decarboxylation of arogenate into tyrosine in presence of NADP . The two Arabidopsis thaliana arogenate dehydrogenases TyrAAT1 and TyrAAT2 were overproduced in Escherichia coli and purified to homogeneity . Biochemical comparison of the two forms revealed that at low substrate concentration TyrAAT1 is four times more efficient in catalyzing the arogenate dehydrogenase reaction than TyrAAT2 . Moreover, TyrAAT2 presents a weak prephenate dehydrogenase activity whereas TyrAAT1 does not . The mechanism of the dehydrogenase reaction catalyzed by these two forms has been investigated using steady-state kinetics . For both enzymes, steady-state velocity patterns are consistent with a rapid equilibrium, random mechanism in which two dead-end complexes, E-NADPH-arogenate and E-NADP-tyrosine, are formed.

Int J Cancer, 2002 Nov 1, 102(1), 51 - 9
Gene therapy for intraperitoneally disseminated pancreatic cancers by Escherichia coli uracil phosphoribosiltransferase (UPRT) gene mediated by restricted replication-competent adenoviral vectors; Oonuma M et al.; Although patients with unresectable pancreatic tumors have been treated with 5-fluorouracil (5FU)-based combination chemotherapy, the drug resistance of cancer cells presents a crucial therapeutic problem . It was reported that UPRT overcomes 5FU resistance . UPRT catalyzes the synthesis of 5-fluorouridine monophosphate (FUMP) from Uracil and phosphoribosylpyrophosphate (PRPP) . The antitumor effect of 5FU is enhanced by augmenting 5-fluorodeoxyuridine monophosphate (FdUMP) converted from FUMP, which inhibits thymidylate synthetase (TS) . We first demonstrated that injecting an E1-deficient adenoviral vector (Adv) expressing UPRT (AxCAUPRT) followed by 5-FU treatment resulted in a volume reduction of xenotransplanted human tumors . In examining the therapeutic effect of AxCAUPRT/5-FU against peritoneal dissemination, we found that non-selective gene transduction of AxCAUPRT caused severe adverse effects arising from the increase of F-dUMP in normal intestine . Because the therapeutic gene delivered by a restricted replication-competent Adv lacking 55 kDa E1B protein (AxE1AdB) is speculated to be expressed selectively in tumors, mice with established tumors were injected with AxE1AdB and E1-deleted Adv expressing the lacZ reporter gene (AxCAlacZ) . The expression of the reporter gene (lacZ) was selectively enhanced in disseminated tumors . The therapeutic advantage of restricted replication competent Adv that expresses UPRT (AxE1AdB-UPRT) was evaluated in an intraperitoneal disseminated tumor model . To study the anti-tumor effect of AxE1AdB-UPRT/5FU, mice with disseminated AsPC-1 tumors were administered the Adv, followed by the 5FU treatment . It was shown that the treatment with AxE1AdB-UPRT/5FU caused a dramatic reduction of the disseminated tumor burden without toxicity in normal tissues . Our results showed that the AxE1AdB-UPRT/5FU system is a promising tool for intraperitoneal disseminated pancreatic cancer .

Crit Care Med, 2002 Sep, 30(9), 2063 - 8
Tumor necrosis factor-alpha and interleukin-1beta mediate endothelial permeability induced by lipopolysaccharide-stimulated whole blood; Nooteboom A et al.; OBJECTIVE: To investigate the role of endotoxin-induced inflammatory mediators in blood on the permeability of endothelial monolayers . DESIGN: Whole blood of healthy volunteers was treated with bacterial lipopolysaccharide (Escherichia coli, B55:05), and the resultant plasma was added to human umbilical venular endothelial cells (HUVEC) cultured on semipermeable membrane inserts (Transwells) . SETTING: University hospital laboratory . SUBJECTS: Whole blood of healthy volunteers . INTERVENTIONS: Donor plasma was treated with excess antibodies against either tumor necrosis factor-alpha, interleukin-1beta, or both, before the incubation on HUVEC . MEASUREMENTS AND MAIN RESULTS: The permeability of HUVEC monolayers to fluorescent-labeled albumin and dextran was measured over a 6-hr period, after removal of the stimulus . The production of tumor necrosis factor-alpha and interleukin-1beta in lipopolysaccharide-treated whole blood was determined by radioimmunoassay . Individually, lipopolysaccharide (10 microg/mL), tumor necrosis factor-alpha (10 ng/mL), and interleukin-1beta (50 ng/mL) all increased endothelial permeability by about 2.5-fold . A much larger increase could be achieved by preincubation of lipopolysaccharide (10 microg/mL) in whole blood: the resultant plasma induced a ten-fold increase of the permeability . The permeability response after preincubation of lipopolysaccharide in whole blood was time- and dose-dependent . Moreover, this treatment increased the sensitivity of endothelial monolayers to lipopolysaccharide by a factor of several thousand-fold: Whereas high doses of lipopolysaccharide were required for direct stimulation of the permeability, picomolar amounts of lipopolysaccharide in whole blood induced a similar increase . Significant amounts of tumor necrosis factor-alpha and interleukin-1beta were produced in blood at similar doses of lipopolysaccharide . The addition of antibodies against tumor necrosis factor-alpha or interleukin-1beta to plasma partially but significantly abrogated the permeability increase . However, a complete inhibition could be achieved by the simultaneous addition of anti-tumor necrosis factor-alpha and anti-interleukin-1beta to plasma . CONCLUSIONS: Although lipopolysaccharide is capable of directly inducing endothelial permeability, blood-borne tumor necrosis factor-alpha and interleukin-1beta mediate lipopolysaccharide-induced endothelial permeability at low endotoxin concentrations . These findings support the idea that multifactorial inhibition of inflammatory mediators may improve survival in septic patients.

Acta Crystallogr D Biol Crystallogr, 2002 Oct, 58(Pt 10 Pt 1), 1757 - 9 Epub 2002 Sep 26.
Atomic resolution crystal structure of squid ganglion DFPase; Koepke J et al.; Diisopropylfluorophosphatases (DFP-ases) are capable of detoxifying chemical warfare agents like diisopropylfluorophosphate (DFP) by hydrolysis . The protein reported here was recombinantely expressed in E . coli . The X-ray crystal structure of this enzyme has been refined to a resolution of 0.85 A and a crystallographic R value of 9.4% . Reversible flash-cooling improved both, mosaicity and resolution of the crystals considerably . The overall structure of this protein represents a six-bladed beta-propeller with two calcium ions bound in a central water filled tunnel . 496 water, 2 glycerol, 2 MES-buffer molecules, and 18 PEG fragments of different lengths could be refined in the solvent region . The 208 most reliable residues, without disorder or reduced occupancy in their side-chains, were finally refined without restraints . A subsequent full-matrix refinement cycle for the positional parameters yielded estimated standard deviations (esds) by matrix inversion . The herewith calculated bond lengths and bond-esds were used to obtain averaged bond lengths, which have been compared to the restraints used in preceding refinement cycles.

Acta Crystallogr D Biol Crystallogr, 2002 Oct, 58(Pt 10 Pt 1), 1715 - 21 Epub 2002 Sep 26.
Scaffolds for protein crystallisation; Stura EA et al.; In the absence of a method to ensure that crystals can be obtained for any given protein, the possibility of developing scaffolds for protein crystallisation becomes attractive . Among several approaches that could yield scaffolds, two are particularly promising: the first is based on immunoglobulin Fab fragments and immunoglobulin binding proteins while the second is based on fusion proteins . In the Fab based scaffold, the protein of interest is the antigen recognised by the antibody . In the second case, it is a protein fused to one of the scaffold components . The operational difference between the two methods is the existence of a flexible covalent tether compared to a highly specific interaction . The relative merits and disadvantages of each approach are discussed here . We also describe a lattice obtained through a combinatorial approach which appears to have the required properties to be considered a scaffold . The system makes use of an Fab derived from a rheumatoid factor and an Fc-fusion protein . The Fc-fusion system is ideal for enhanced expression of glycoproteins in mammalian cells and provides a useful tag for their purification . The molecular replacement shows a mode of binding for this rheumatoid factor that is not competitive with bacterial Fc-binding proteins . Hence it may be possible to generalize the method to include bacterial expression of fusion proteins with either protein A or protein G as the fusion partner.

Acta Crystallogr D Biol Crystallogr, 2002 Oct, 58(Pt 10 Pt 1), 1664 - 9 Epub 2002 Sep 26.
Crystallization of RNA/protein complexes; Garber M et al.; Different complexes of ribosomal proteins with specific rRNA fragments have been crystallized and studied by our group during the last six years . There are several factors important for successful crystallization of RNA/protein complexes, among them: length and content of RNA fragments, homogeneity of RNA and protein preparations, stability of the complexes, conditions for mixing RNA and protein components before crystallization, effect of Se-Met on RNA/protein complex crystal quality . In this paper we describe findings and methodical details, which helped us to succeed in obtaining X-ray quality crystals of several RNA/protein complexes.

Acta Crystallogr D Biol Crystallogr, 2002 Oct, 58(Pt 10 Pt 1), 1657 - 9 Epub 2002 Sep 26.
Crystallization of biological macromolecules using agarose gel; Biertumpfel C et al.; Gellified media prevent convection and crystal sedimentation, and provide an attractive growth environment for optimising biological crystals . Agarose gels are particularly easy to use and they are compatible with most of the common crystallization methods . They also offer new possibilities like counter-diffusion techniques . This paper gives a brief overview of their general properties and presents an application of a counter-diffusion setup combining agarose gel and capillaries to the crystallization of proteins and protein / nucleic acid complexes.

Acta Crystallogr D Biol Crystallogr, 2002 Oct, 58(Pt 10 Pt 2), 1882 - 5 Epub 2002 Sep 28.
Crystallization, X-ray characterization and selenomethionine phasing of Mlc1p bound to IQ motifs from myosin V; Terrak M et al.; Mlc1p is a calmodulin-like protein from the budding yeast Saccharomyces cerevisiae, where it has been identified as a subunit of a class V myosin, Myo2p, and a binding partner of an IQGAP-like protein, Iqg1p . Through its interactions with these two proteins, Mlc1p plays a role in polarized growth and cytokinesis . Mlc1p has been crystallized in complexes with four different IQ target motifs from the neck region of Myo2p: IQ2, IQ3, IQ4 and IQ2-IQ3 (referred to as IQ2,3) . Electron-density maps for two of the complexes (Mlc1p-IQ4 and Mlc1p-IQ2,3) were obtained from multiple anomalous dispersion (MAD) experiments based on selenomethionine derivatives . The other two structures (Mlc1p-IQ2 and Mlc1p-IQ3) were determined by molecular replacement using the partially refined structure of Mlc1p-IQ2,3 as a search model.

Acta Crystallogr D Biol Crystallogr, 2002 Oct, 58(Pt 10 Pt 2), 1876 - 8 Epub 2002 Sep 28.
Purification, crystallization and preliminary X-ray diffraction analysis of yeast nucleosome-assembly factor Cia1p; Padmanabhan B et al.; Yeast Cia1p is a homologue of human CIA (CCG1-interacting factor A), which possesses nucleosome-assembly activity and interacts with the human TFIID subunit CCG1 and the C-terminal domain of histone H3 . The yeast Cia1p without the C-terminal polyanionic stretch has been expressed in Escherichia coli, purified to homogeneity and crystallized by the hanging-drop vapour-diffusion method using PEG 8000 as precipitant . The protein was crystallized in orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 106.70, b = 46.92, c = 40.60 A and one molecule in the asymmetric unit . The crystal diffracted beyond 2.95 A resolution using synchrotron radiation.

Acta Crystallogr D Biol Crystallogr, 2002 Oct, 58(Pt 10 Pt 2), 1862 - 4 Epub 2002 Sep 28.
Crystallization and preliminary X-ray diffraction studies on the DNA-binding domain of the transcriptional activator protein PhoB from Escherichia coli; Shindoh K et al.; PhoB is a transcriptional factor that activates more than 30 genes of the pho regulon in response to phosphate starvation . Crystals of its C-terminal domain (PhoBC) were obtained in two forms . The first crystal form, obtained from phosphate solution, belongs to space group P2(1), with unit-cell parameters a = 30.7, b = 105.9, c = 30.9 A, beta = 110.3 degrees . The second form, crystallized from PEG solution, belongs to the same space group, but has a smaller unit cell (a = 30.6, b = 37.5, c = 44.4 A, beta = 109.4 degrees ) . Crystals of selenomethionyl-derivatized PhoBC were obtained using the conditions for the second crystal form . Diffraction data from wild-type PhoBC (2.0 A resolution) and MAD data sets from selenomethionyl-derivative PhoBC (3.0 A resolution) have been collected at 100 K with a synchrotron-radiation source . MAD data analysis is in progress.

Acta Crystallogr D Biol Crystallogr, 2002 Oct, 58(Pt 10 Pt 2), 1848 - 50 Epub 2002 Sep 28.
Human neuroglobin: crystals and preliminary X-ray diffraction analysis; Pesce A et al.; Neuroglobin, a recently discovered member of the haemoglobin superfamily, is primarily expressed in the brain of humans and other vertebrates, where it has been proposed to enhance O(2) supply in response to hypoxia or ischaemia, protecting the neuron from hypoxic injury . Neuroglobin is the first example of a vertebrate haemoglobin in which a hexacoordinate haem geometry has been detected . A triple mutant (replacing three Cys residues) of human neuroglobin (151 amino acids) has been expressed in Escherichia coli, purified and crystallized in two crystal forms, the best of which diffracts to 1.95 A resolution using synchrotron radiation . The crystals belong to space group P2(1), with unit-cell parameters a = 39.6, b = 94.9, c = 67.5 A, beta = 94.4 degrees, and contain 2-4 protein molecules per asymmetric unit.

Acta Crystallogr D Biol Crystallogr, 2002 Oct, 58(Pt 10 Pt 2), 1841 - 2 Epub 2002 Sep 28.
Crystallization and preliminary X-ray diffraction analysis of recombinant hydrolase domain of 10-formyltetrahydrofolate dehydrogenase; Chumanevich AA et al.; 10-Formyltetrahydrofolate dehydrogenase (FDH) is an abundant enzyme in liver cytosol . It is important for the regulation of 10-formyltetrahydrofolate/tetrahydrofolate pools, for de novo purine biosynthesis and for the removal of formate in the form of CO(2) . The enzyme is a natural fusion of two unrelated genes and consists of two functional catalytic domains . Here, the crystallization of the N-terminal domain of FDH is reported . This domain binds folate and functions as a 10-formyltetrahydrofolate hydrolase . The crystals grow as either spear-shaped needles or large plates, with the largest crystals reaching dimensions of 1.2 x 0.2 x 0.05 mm . Diffraction analysis revealed the space group to be P2(1)2(1)2, with unit-cell parameters a = 100.00, b = 64.63, c = 64.59 A . Based on the estimated solvent content, there is one 34 kDa molecule in the asymmetric unit . A native data set extending to 2.3 A resolution has been collected with good merging statistics.

Acta Crystallogr D Biol Crystallogr, 2002 Oct, 58(Pt 10 Pt 2), 1830 - 2 Epub 2002 Sep 28.
Crystallization and preliminary X-ray characterization of archaeal group II chaperonin alpha-subunit from Thermococcus strain KS-1; Shomura Y et al.; The archaeal group II chaperonin from Thermococcus strain KS-1 is composed of two kinds of subunits (alpha and beta) . Each of the recombinant subunits was individually expressed in Escherichia coli and purified as homo-hexadecamers of each subunit . Both homo-oligomers facilitate the refolding of denatured proteins in vitro in an ATP-dependent manner . A mutant alpha-subunit homo-oligomer with two amino-acid substitutions, which has the ability to capture the unfolded protein but lacks the ability to refold the unfolded protein, was crystallized in two different conditions . One crystal form was obtained from a high-concentration solution of ammonium sulfate and grew to maximum dimensions of 0.15 x 0.15 x 0.4 mm . The crystals of this form belonged to the tetragonal space group P42(1)2, with unit-cell parameters a = b = 209.3, c = 156.1 A, and diffracted X-rays to 2.4 A resolution with synchrotron radiation . The other form was crystallized from a polyethylene glycol 6000 solution and belonged to the tetragonal space group, with unit-cell parameters a = b = 220.8, c = 182.4 A . This form only diffracts X-rays to 6 A resolution . Diffraction data collected from the former crystal enabled initial successful phases to be obtained by the molecular-replacement method.

Science, 2002 Oct 18, 298(5593), 580 - 4 Epub 2002 Sep 26.
Microfluidic large-scale integration; Thorsen T et al.; We developed high-density microfluidic chips that contain plumbing networks with thousands of micromechanical valves and hundreds of individually addressable chambers . These fluidic devices are analogous to electronic integrated circuits fabricated using large-scale integration . A key component of these networks is the fluidic multiplexor, which is a combinatorial array of binary valve patterns that exponentially increases the processing power of a network by allowing complex fluid manipulations with a minimal number of inputs . We used these integrated microfluidic networks to construct the microfluidic analog of a comparator array and a microfluidic memory storage device whose behavior resembles random-access memory.

J Biol Chem, 2002 Dec 6, 277(49), 46987 - 92 Epub 2002 Sep 25.
Biochemical characterization of CopA, the Escherichia coli Cu(I)-translocating P-type ATPase; Fan B et al.; Escherichia coli CopA is a copper ion-translocating P-type ATPase that confers copper resistance . CopA formed a phosphorylated intermediate with {gamma-(32)P}ATP . Phosphorylation was inhibited by vanadate and sensitive to KOH and hydroxylamine, consistent with acylphosphate formation on conserved Asp-523 . Phosphorylation required a monovalent cation, either Cu(I) or Ag(I) . Divalent cations Cu(II), Zn(II), or Co(II) could not substitute, signifying that the substrate of this copper-translocating P-type ATPase is Cu(I) and not Cu(II) . CopA purified from dodecylmaltoside-solubilized membranes similarly exhibited Cu(I)/Ag(I)-stimulated ATPase activity, with a K(m) for ATP of 0.5 mm . CopA has two N-terminal Cys(X)(2)Cys sequences, Gly-Leu-Ser-Cys(14)-Gly-His-Cys(17), and Gly-Met-Ser-Cys(110)-Ala-Ser-Cys(113), and a Cys(479)-Pro-Cys(481) motif in membrane-spanning segment six . The requirement of these cysteine residues was investigated by the effect of mutations and deletions . Mutants with substitutions of the N-terminal cysteines or deletion of the first Cys-(X)(2)-Cys motif formed acylphosphate intermediates . From the copper dependence of phosphoenzyme formation, the mutants appear to have 2-3 fold higher affinity for Cu(I) than wild type CopA . In contrast, substitutions in Cys(479) or Cys(481) resulted in loss of copper resistance, transport and phosphoenzyme formation . These results imply that the cysteine residues of the Cys-Pro-Cys motif (but not the N-terminal cysteine residues) are required for CopA function.

J Biol Chem, 2002 Dec 6, 277(49), 47420 - 7 Epub 2002 Sep 25.
Promoter use by sigma 38 (rpoS) RNA polymerase . Amino acid clusters for DNA binding and isomerization; Lee SJ et al.; Sigma(38) is a non-essential but highly homologous member of the sigma(70) family of transcription factors . In vitro mutagenesis and in vivo screening were used to identify 22 critical amino acids in the promoter interaction domain of Escherichia coli sigma(38) . Electrophoretic mobility shift assay studies showed that residues involved in duplex DNA binding largely segregated into distinct regions that coincided with those of sigma(70) . However, the majority of these amino acids were in non-conserved positions . Analysis indicates that this region of the two sigma(s) probably has a common overall organization but differs in how its amino acids are used to form functional open complexes . Placement of the mutations on the known sigma(70) holoenzyme structure shows two clusters; one appears to be used for duplex DNA recognition and the other for the subsequent isomerization events . Permanganate assays for DNA melting support this view.

J Biol Chem, 2002 Nov 29, 277(48), 46051 - 8 Epub 2002 Sep 25.
Identification of a jasmonate-regulated allene oxide synthase that metabolizes 9-hydroperoxides of linoleic and linolenic acids; Itoh A et al.; Allene oxide synthase (AOS) is a cytochrome P-450 (CYP74A) that catalyzes the first step in the conversion of 13-hydroperoxy linolenic acid to jasmonic acid and related signaling molecules in plants . Here, we report the molecular cloning and characterization of a novel AOS-encoding cDNA (LeAOS3) from Lycopersicon esculentum whose predicted amino acid sequence classifies it as a member of the CYP74C subfamily of enzymes that was hitherto not known to include AOSs . Recombinant LeAOS3 expressed in Escherichia coli showed spectral characteristics of a P-450 . The enzyme transformed 9- and 13-hydroperoxides of linoleic and linolenic acid to alpha-ketol, gamma-ketol, and cyclopentenone compounds that arise from spontaneous hydrolysis of unstable allene oxides, indicating that the enzyme is an AOS . Kinetic assays demonstrated that LeAOS3 was approximately 10-fold more active against 9-hydroperoxides than the corresponding 13-isomers . LeAOS3 transcripts accumulated in roots, but were undetectable in aerial parts of mature plants . In contrast to wild-type plants, LeAOS3 expression was undetectable in roots of a tomato mutant that is defective in jasmonic acid signaling . These findings suggest that LeAOS3 plays a role in the metabolism of 9-lipoxygenase-derived hydroperoxides in roots, and that this branch of oxylipin biosynthesis is regulated by the jasmonate signaling cascade.

J Biol Chem, 2002 Dec 13, 277(50), 48550 - 7 Epub 2002 Sep 25.
Superactive SecY variants that fulfill the essential translocation function with a reduced cellular quantity; Mori H et al.; The fifth and the sixth cytoplasmic regions (C5 and C6) of SecY are important for the SecA-driven preprotein translocation reaction . A cold-sensitive mutation, secY205 (Tyr-429 --> Asp), in C6 impairs the ATP- and precursor-dependent SecA insertion into the membrane . We now identified second site mutations that suppressed the defect . Cis-placement of these mutations proved to suppress mutations at another essential residue (Arg-357) of SecY as well . Thus, they tolerate the otherwise defective SecY alterations in the same molecule . Two alterations (Ile-195 to Ser in TM5 region and Ile-408 to Leu in TM10 region) were found to make the translocation channel more active, because it enabled cells to survive with reduced content of the SecYE complex . These mutations only very weakly suppressed a signal sequence defect of the lambda receptor protein . The mutant SecYEG translocase exhibited higher than normal activity in vitro, being accompanied by striking independence of the proton motive force as well as by stabilization of a bound and active SecA species against urea treatment . These results have been interpreted in terms of balance shifts between channel closing and channel opening alterations in the SecYEG translocase.

Mutat Res, 2002 Sep 30, 506-507, 101 - 11
Single nucleotide instability: a wide involvement in human and rat mammary carcinogenesis?
Okochi E, Watanabe N, Sugimura T, Ushijima T.
Genetic instability plays important roles in carcinogenesis . In two cell lines which we established from mammary carcinomas induced in lacI-transgenic rats by 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine (PhIP), spontaneous point mutation rates (MRs) of the endogenous hypoxanthine-guanine phosphoribosyltransferase (hprt) gene and lacI transgene were found to be increased . The two rat mammary carcinoma cell lines lacked microsatellite instability (MSI), and nuclear extracts from them were proficient in G/T mismatch binding . The increase of spontaneous point MRs was considered to be due to a mechanism(s) different from mismatch repair insufficiency, and this type of genetic instability was termed as single nucleotide instability (SNI) . SNI in the rat mammary carcinoma cell lines was characterized by the elevation of A:T to C:G transversions of the hprt and lacI genes, which were rarely observed in normal mammary epithelial cells . The elevation of A:T to C:G transversions was also present in the lacI gene of the primary carcinomas of the two cell lines, which suggested that the molecular abnormality present in the cell lines was already present in their primary carcinomas . Mth1 mutation, which is known to cause elevation of A:T to C:G transversions, was analyzed in the 2 cell lines and in 11 primary PhIP-induced mammary carcinomas, but no mutations were observed . Finally, spontaneous point MRs of the hprt gene were measured in six human breast cancer cell lines, and increase was found in five of them . These human breast cancer cell lines were proficient in G/T mismatch binding, and were reported to lack MSI . SNI was suggested to play a wide involvement in human and rat mammary carcinogenesis.

Mutat Res, 2002 Sep 30, 506-507, 55 - 63
Mutagenicity and carcinogenicity in relation to DNA adduct formation in rats fed leucomalachite green; Culp SJ et al.; Leucomalachite green is a persistent and prevalent metabolite of malachite green, a triphenylmethane dye that has been used widely as an antifungal agent in the fish industry . Concern over the use of malachite green is due to the potential for consumer exposure, evidence suggestive of tumor promotion in rodent liver, and suspicion of carcinogenicity based on structure-activity relationships . Our previous study indicated that feeding rodents malachite or leucomalachite green resulted in a dose-related increase in liver DNA adducts, and that, in general, exposure to leucomalachite green caused an increase in the number and severity of changes greater than was observed following exposure to malachite green . To characterize better the genotoxicity of leucomalachite green, female Big Blue rats were fed leucomalachite green at doses of 0, 9, 27, 91, 272, or 543 ppm for up to 32 weeks . The livers were analyzed for lacI mutations at 4, 16, and 32 weeks and DNA adducts at 4 weeks . Using a 32P-postlabeling assay, we observed a dose-related DNA adduct in the livers of rats fed 91, 272, and 543 ppm leucomalachite green . A approximately 3-fold increase in lacI mutant frequency was found in the livers of rats fed 543 ppm leucomalachite green for 16 weeks, but significant increases in mutant frequencies were not found for any of the other doses or time points assayed . We also conducted 2-year tumorigenesis bioassays in female and male F344 rats using 0, 91, 272, and 543 ppm leucomalachite green . Preliminary results indicate an increasing dose trend in lung adenomas in male rats treated with leucomalachite green, but no increase in the incidence of liver tumors in either sex of rat . These results suggest that the DNA adduct formed in the livers of rats fed leucomalachite green has little mutagenic or carcinogenic consequence.

BMC Pediatr . 2002 Sep 02;2(1):7.
The clinical and molecular spectrum of galactosemia in patients from the Cape Town region of South Africa; Henderson H et al.; BACKGROUND: The objective of this study was to document the clinical, laboratory and genetic features of galactosemia in patients from the Cape Town metropolitan region . METHODS: Diagnoses were based on thin layer chromatography for galactosuria/galactosemia and assays of erythrocyte galactose-1-phosphate uridyltransferase (GALT) and galactokinase activities . Patients were screened for the common S135L and Q188R transferase gene mutations, using PCR-based assays . Screening for the S135L mutation in black newborns was used to estimate the carrier rate for galactosemia in black South Africans . RESULTS: A positive diagnosis of galactosemia was made in 17 patients between the years 1980 to 2001 . All had very low or absent galactose-1-phosphate uridyltransferase (GALT) activity, and normal galactokinase levels . The mean age at diagnosis was 5.1 months (range 4 days to 6.5 months) . A review of 9 patients showed that hepatomegaly (9/9), and splenomegaly, failure to thrive, developmental delay, bilateral cataracts (6/9) were the most frequent features at diagnosis . Six had conjugated hyperbilirubinemia . Four experienced invasive E . coli infection before diagnosis . Ten patients were submitted to DNA analysis . All 4 black patients and 2 of mixed extraction were homozygous for the S135L allele, while all 3 white patients were homozygous for the Q188R allele . The remaining patient of mixed extraction was heterozygous for the Q188R allele . The estimated carrier frequency of the S135L mutation in 725 healthy black newborns was 1/60 . CONCLUSIONS: In the absence of newborn screening the delay in diagnosis is most often unacceptably long . Also, carrier frequency data predict a galactosemia incidence of approximately 1/14 400 for black newborns in the Cape Metropole, which is much higher than the current detection rate . It is thus likely that many patients go undetected.

Biochem J, 2002 Dec 15, 368(Pt 3), 835 - 43
The Escherichia coli cAMP receptor protein bound at a single target can activate transcription initiation at divergent promoters: a systematic study that exploits new promoter probe plasmids; El-Robh MS et al.; We report the first detailed quantitative study of divergent promoters dependent on the Escherichia coli cAMP receptor protein (CRP), a factor known to activate transcription initiation at target promoters by making direct interactions with the RNA polymerase holoenzyme . In this work, we show that CRP bound at a single target site is able to activate transcription at two divergently organized promoters . Experiments using promoter probe plasmids, designed to study divergent promoters in vivo and in vitro, show that the divergent promoters function independently . Further in vitro experiments show that two holo RNA polymerase molecules cannot be accommodated simultaneously at the divergent promoters.

Int Immunopharmacol, 2002 Jul, 2(8), 1133 - 42
Differential alteration of functions of rat peritoneal macrophages responsive to endogenous opioid peptide endomorphin-1; Inui Y et al.; Endomorphin-1 is a recently isolated endogenous opioid peptide, and potent and selective high affinity mu-opioid receptor agonist . We evaluate the role of endomorphin-1 on macrophage functions . Endomorphin-1 potentiated macrophage adhesion and the expression of adhesion molecule Mac-1 on macrophages . However, endomorphin-1 did not alter phagocytosis of Escherichia coli by macrophages . Moreover, endomorphin-1 inhibited macrophage chemotaxis and the production of superoxide anion by macrophages . On the contrary, endomorphin-1 inhibited TNF-alpha production by macrophages stimulated with both LPS and PMA, respectively . Similarly, endomorphin-1 suppressed IL-10 and IL-12 productions in response to LPS . In contrast, endomorphin-1 potentiated IL-1beta production by macrophages stimulated with PMA . These results suggest that endomorphin-1 may alter macrophage functions such as cytokine productions and functions related to natural host defense.

Mol Ther, 2002 Aug, 6(2), 287 - 97
Systemic interleukin-6 responses following administration of adenovirus gene transfer vectors to humans by different routes; Ben-Gary H et al.; Administration of adenovirus (Ad) vectors to animals induces innate immune responses, typified by elevated interleukin-6 (IL-6) . To assess innate responses to Ad vectors in humans, we evaluated serum IL-6 following administration of E1(-) E3(-) Ad vectors to different human hosts and the relationship among peak IL-6 and peak anti-Ad neutralizing antibodies . We administered: 1) Ad(GV)CFTR.10, a vector carrying the normal human CFTR cDNA (3 x 10(7) to 2 x 10(10) particle units (pu)) to airways of individuals with cystic fibrosis (CF); 2) Ad(GV)VEGF121.10, a vector carrying the normal human vascular endothelial growth factor (VEGF)121 cDNA, to the myocardium (4 x 10(8) to 4 x 10(10) pu) of individuals with coronary artery disease (CAD) and to lower extremity muscles (4 x 10(8) to 4 x 10(9.5) pu) of individuals with peripheral vascular disease (PVD); and 3) Ad(GV)CD.10, a vector carrying the Escherichia coli cytosine deaminase gene to skin (7 x 10(7) to 7 x 10(9) pu) and airways (7 x 10(8) to 7 x 10(10) pu) of normal individuals and to liver metastasis (4 x 10(8) to 4 x 10(9) pu) of individuals with colon carcinoma . IL-6 increased mildly (up to 220 pg/ml) following vector administration to skin and lung airways of normal individuals and of individuals with CF, and to muscle and liver metastasis of individuals with PVD and colon cancer, respectively . IL-6 responses were higher (up to 1100 pg/ml) following myocardial administration . Control individuals who had chest surgery and bronchoscopy, but no vector administration, had comparable IL-6 increases . Thus, both administration of Ad vectors of humans up to 10(10) pu and the procedures used to administer the vectors elicit systemic IL-6 responses . There was no correlation among peak IL-6 and peak anti-Ad antibodies . These observations indicate that the innate host responses following administration of Ad vectors to humans may result from the procedures used to administer the vector, and from the vector per se.

Health Millions, 1998 Jul-Aug, 24(4), 25 - 6
Diarrhoea deaths in Reang migrant camps; Ray DK; PIP: In May 1998, an outbreak of diarrhea epidemic was reported from the Reang migrant camps of Anandabazar, Longtaria, and Kashirampura . Due to lack of medical care, hygiene, drinking water, and most importantly, the absence of health-seeking behavior among the tribals, the initial mortality rate was very high . It was only by June 9, 1998, that the epidemic came under control . The estimated mortality rate ranged from 250-350 deaths . Experts say that the infection was bacterial (mostly E . coli) . With its limited resources, the Voluntary Health Association of Tripura helped in controlling the epidemic . It trained volunteers from the refugee camps in the prevention, control and primary treatment of diarrhea and supplied them with oral rehydration solution packets, medicines and educational materials on diarrhea control, health and hygiene . The Reang migrants are in serious struggle for survival in the camps . Urgent efforts are needed to address their needs . Recommendations include the following: 1) strengthen the infrastructural support; 2) ensure a minimum balanced diet to the camp inmates; 3) provide education to the children; 4) establish a regular health care system; and 5) improve the health-seeking behavior of the camp inmates .

Facts Infant Feed, 1993 Apr, (3), 1 - 4
Contaminated food: a major cause of diarrhoea and associated malnutrition among infants and young children; World Health Organization WHO . Food and Nutrition Programme . Food Safety Unit; PIP: An estimated 3.2 million children die annually as a result of diarrheal diseases while hundreds of millions more suffer from frequent episodes of diarrhea and impaired nutritional status . Up to 70% of the 1400 million episodes of diarrhea worldwide in children under age 5 cause this morbidity and mortality may be due to pathogens which can be transmitted through food . Breast milk is the ideal source of nutrients and protection against diarrhea and exposure to foodborne pathogens for infants in their first months of life . As foodstuffs are added to supplement childrens diets at ages 4-6 months, infants increase their potential exposure to contaminants borne in foodstuffs, especially E . coli . Food may become contaminated with nightsoil, polluted water, flies, pests, domestic animals, unclean utensils and pots, food handlers, dust, and dirt . Raw foods may also harbor pathogens or be obtained from infected animals . Making food several hours before eating and storing it at temperatures suitable for the growth of bacteria, and cooking or reheating food insufficiency to reduce or eliminate pathogens are practices which especially place food at risk for being contaminated and consumed . Cooking food thoroughly and eating it as soon as it is cool enough for consumption would therefore control the majority of contaminants in food and a significant number of foodborne episodes of diarrhea . Socioeconomic and cultural constraints, however, often impede such behavior and may be the result of food storages, beliefs or habits, inadequate supplies of safe water and lack of sanitation facilities, shortages of cooking fuel or other facilities for the safe preparation and storage of food, and/or lack of time to prepare food . Little attention has been given to teach mothers and care-givers about food safety . They need to be taught which measures to take to reduce the risk of exposure to foodborne pathogens . Moreover, an integrated approach is called for .

Chembiochem, 2002 Jul 2, 3(7), 672 - 7
Cellular internalization of enhanced green fluorescent protein ligated to a human calcitonin-based carrier peptide; Machova Z et al.; Carrier peptides offer new opportunities to overcome problems in cellular drug delivery . Their objectives are improved cellular uptake or permeation of biological membranes, which are important pharmacokinetic features for the cellular distribution of therapeutics . Previously, human calcitonin (hCT) and selected C-terminal hCT fragments have been shown to be internalized and to permeate the epithelium of the nasal mucosa . To assess the potential of hCT-derived carrier peptides for cellular internalization of a model protein we fused enhanced green fluorescent protein (EGFP) and the {C(8)}hCT8-32 fragment by using expressed protein ligation (EPL) . EGFP thioester was obtained by intein-mediated purification with an affinity chitin-binding tag (the IMPACT system, based on protein splicing) . Internalization of EGFP-{C(8)}hCT8-32 by excised bovine nasal mucosa was monitored by confocal laser scanning microscopy . This novel conjugate displayed internalization into some sectors of the mucosa, whereas EGFP itself was not capable of translocation . Thus, we demonstrate successful internalization of a model protein through ligation to an hCT-derived carrier peptide, which has potential for the delivery of therapeutics . At this point the respective mechanism of translocation is unknown.

Chembiochem, 2002 Jul 2, 3(7), 659 - 63
Picosecond time-resolved fluorescence from blue-emitting chromophore variants Y66F and Y66H of the green fluorescent protein; Kummer AD et al.; The origin of the low steady-state fluorescence quantum yield of some blue-emitting variants of the green fluorescent protein (GFP) is investigated in single-site mutants in which the tyrosine residue at position 66 has been replaced by phenylalanine or by histidine . Time-resolved fluorescence measurements reveal excited-state lifetimes of 74 ps (Y66F) and 0.9 ns (Y66H) at room temperature that increase to values close to the radiative limit as the temperature is lowered . These short lifetimes are explained by temperature-dependent internal conversion . The pronounced difference between the room-temperature lifetimes of the two mutants suggests that hydrogen bonding of the distal aromatic ring plays a more important role than tight packing in the fixation of the chromophore.

Plant Cell, 1991 Dec, 3(12), 1263 - 1274
The hy3 Long Hypocotyl Mutant of Arabidopsis Is Deficient in Phytochrome B; Somers DE et al.; The six long hypocotyl (hy) complementation groups of Arabidopsis (hy1, hy2, hy3, hy4, hy5, and hy6) share the common feature of an elongated hypocotyl when grown in white light . The varied responses of these mutants to irradiations of differing wavelengths have suggested that some of the lines may lack elements of the phytochrome signal transduction pathway . We have performed immunoblot and RNA gel blot analyses of the multiple types of phytochrome present in wild-type and mutant Arabidopsis and provide evidence that mutations at the HY3 locus cause a specific deficiency in phytochrome B . Using an Escherichia coli overexpression system, we have developed and identified monoclonal antibodies that selectively recognize phytochromes A, B, and C from Arabidopsis . In wild-type plants, phytochrome A is highly abundant in etiolated tissue, but rapidly decreases about 200-fold upon illumination . Phytochromes B and C are present at much lower levels in etiolated tissue but are unaffected by up to 24 hr of red light illumination, and together predominate in green seedlings . These data establish that phytochromes B and C are "type 2" or photostable phytochromes . Levels of phytochromes A, B, and C similar to those of the wild type are observed in strains containing mutations at the HY4 and HY5 loci . In contrast, all four hy3 mutant alleles tested here exhibit a modest (twofold to threefold) reduction in phyB transcript and a severe (20- to 50-fold) deficiency in phyB-encoded protein, relative to levels in wild-type plants . The levels of phyA- and phyC-encoded mRNA and protein, however, are indistinguishable from the wild type in these mutants . We conclude that the phenotype conferred by hy3 is due to the reduced levels of the light-stable phytochrome B.

J Biol Chem, 2002 Nov 29, 277(48), 46706 - 11 Epub 2002 Sep 24.
Three amino acids in Escherichia coli CspE surface-exposed aromatic patch are critical for nucleic acid melting activity leading to transcription antitermination and cold acclimation of cells; Phadtare S et al.; Cold-shock proteins of the CspA family of Escherichia coli help the cells to acclimate to low temperature conditions through an unknown mechanism . In vitro, these proteins bind to single-stranded nucleic acids and destabilize nucleic acid secondary structures . An unusual surface-exposed patch of 6 evolutionarily conserved aromatic amino acids is thought to be involved in RNA binding by the cold-shock proteins . Here we investigated the functional role of the aromatic patch in E . coli CspE by substituting individual aromatic residues with positively charged Arg residues . These substitutions do not affect the RNA binding activity of the CspE mutants . We show that substitutions of three centrally located aromatic patch amino acid residues, Phe(17), Phe(30), and His(32), abolish the ability of the mutant CspE to acclimatize cells to cold, antiterminate transcription and melt nucleic acids but have no effect on RNA binding . On the other hand, peripherally located Trp(10), Phe(19), and Phe(33) can be substituted with Arg without loss of any of the in vivo and in vitro CspE functions tested . The results thus indicate that these aromatic patch residues have clearly distinct functional roles and further extend the correlation between the essential function of CspA homologues in cold acclimation and their ability to antiterminate transcription.

J Biol Chem, 2002 Dec 6, 277(49), 47834 - 43 Epub 2002 Sep 24.
Lipopolysaccharide rapidly traffics to and from the Golgi apparatus with the toll-like receptor 4-MD-2-CD14 complex in a process that is distinct from the initiation of signal transduction; Latz E et al.; Mammalian responses to LPS require the expression of Toll-like receptor 4 (TLR4), CD14, and MD-2 . We expressed fluorescent TLR4 in cell lines and found that TLR4 densely localized to the surface and the Golgi . Similar distributions were observed in human monocytes . Confocal imaging revealed rapid recycling of TLR4-CD14-MD-2 complexes between the Golgi and the plasma membrane . Fluorescent LPS followed these trafficking pathways in CD14-positive cells . The TLR4- adapter protein, MyD88, translocated to the cell surface upon LPS exposure, and cross-linking of surface TLR4 with antibody induced signaling . Golgi-associated TLR4 expression was disrupted by brefeldin A, yet LPS signaling was preserved . We conclude that LPS signaling may be initiated by surface aggregation of TLR4 and is not dependent upon LPS trafficking to the Golgi.

J Biol Chem, 2002 Nov 29, 277(48), 46559 - 65 Epub 2002 Sep 24.
The RNA helicase DbpA exhibits a markedly different conformation in the ADP-bound state when compared with the ATP- or RNA-bound states; Henn A et al.; The motor enzymes that belong to the family of RNA helicases catalyze the strand separation of duplex RNA via ATP hydrolysis . Among these enzymes, Escherichia coli DbpA is a unique RNA helicase because it possesses ATPase-specific activity toward the peptidyl transferase center in 23 S ribosomal RNA . For this reason, it has been the subject of numerous biochemical and structure-function studies . The ATP-stimulated unwinding activity of DbpA toward specific and nonspecific RNA duplexes has been demonstrated . However, the underlying molecular and structural basis, which facilitates its helicase activities, is presently not known . We combined time-dependent limited proteolysis digestion, fluorescence spectroscopy, and three-dimensional structural homology modeling techniques to study the structural conformations of DbpA with respect to its binding to stoichiometric ratios of RNA and cofactors . We show that the conformational state of DbpA is markedly different in the ADP-bound state than in any other state (ATP- or RNA-bound) . These results, together with structural homology studies, suggest that a hinge region located in the core domain of DbpA mediates such conformational changes.

J Biol Chem, 2002 Dec 13, 277(50), 48558 - 64 Epub 2002 Sep 24.
RNA binding properties of the AU-rich element-binding recombinant Nup475/TIS11/tristetraprolin protein; Worthington MT et al.; Regulation of messenger RNA stability by AU-rich elements is an important means of regulating genes induced by growth factors and cytokines . Nup475 (also known as tristetraprolin, or TIS11) is the prototype for a family of zinc-binding Cys(3)His motif proteins required for proper regulation of tumor necrosis factor mRNA stability in macrophages . We developed an Escherichia coli expression system to produce soluble Nup475 protein in quantity to study its RNA binding properties . Nup475 protein bound a tumor necrosis factor AU-rich element over a broad range of pH and salt concentrations by RNA gel shift . This binding was inhibited by excess zinc metal, providing a potential mechanism for previous reports of zinc stabilization of AU-rich element (ARE) containing messenger RNAs . Immobilized Nup475 protein was used to select its optimal binding site by RNA SELEX and revealed a strong preference for the extended sequence UUAUUUAUU, rather than a simple AUUUA motif . These findings were confirmed by site-directed mutagenesis of the tumor necrosis factor ARE and RNA gel shifts on c-fos, interferon-gamma, and interferon-beta ARE fragments . A weaker binding activity toward adenine-rich sites, such as a poly(A) tail RNA fragment, can partially disrupt the Nup475-tumor necrosis factor AU-rich element complex.

J Biol Chem, 2002 Nov 22, 277(47), 45020 - 7 Epub 2002 Sep 24.
Development and fertility in Caenorhabditis elegans clk-1 mutants depend upon transport of dietary coenzyme Q8 to mitochondria; Jonassen T et al.; The Caenorhabditis elegans clk-1 mutants lack coenzyme Q(9) and instead accumulate the biosynthetic intermediate demethoxy-Q(9) (DMQ(9)) . clk-1 animals grow to reproductive adults, albeit slowly, if supplied with Q(8)-containing Escherichia coli . However, if Q is withdrawn from the diet, clk-1 animals either arrest development as young larvae or become sterile adults depending upon the stage at the time of the withdrawal . To understand this stage-dependent response to a Q-less diet, the quinone content was determined during development of wild-type animals . The quinone content varies in the different developmental stages in wild-type fed Q(8)-replete E . coli . The amounts peak at the second larval stage, which coincides with the stage of arrest of clk-1 larvae fed a Q-less diet from hatching . Levels of the endogenously synthesized DMQ(9) are high in the clk-1(qm30)-arrested larvae and sterile adults fed Q-less food . Comparison of quinones from animals fed a Q-replete or a Q-less diet establishes that the Q(8) present is assimilated from the E . coli . Furthermore, this E . coli-specific Q(8) is present in mitochondria isolated from fertile clk-1(qm30) adults fed a Q-replete diet . These results suggest that the uptake and transport of dietary Q(8) to mitochondria prevent the arrest and sterility phenotypes of clk-1 mutants and that DMQ is not functionally equivalent to Q.

Biophys J, 2002 Oct, 83(4), 2096 - 108
Phase behavior of cationic amphiphiles and their mixtures with helper lipid influences lipoplex shape, DNA translocation, and transfection efficiency; Zuhorn IS et al.; Cationic lipids are widely used for gene transfection, but their mechanism of action is still poorly understood . To improve this knowledge, a structure-function study was carried out with two pyridinium-based lipid analogs with identical headgroups but differing in alkyl chain (un)saturation, i.e., SAINT-2 (diC18:1) and SAINT-5 (diC18:0) . Although both amphiphiles display transfection activity per se, DOPE strongly promotes SAINT-2-mediated transfection, but not that of SAINT-5, despite the fact that DOPE effectively facilitates plasmid dissociation from either lipoplex . This difference appears to correlate with membrane stiffness, dictated by the cationic lipid packing in the donor liposomes, which governs the kinetics of lipid recruitment by the plasmid upon lipoplex assembly . Because of its interaction with the relatively rigid SAINT-5 membranes, the plasmid becomes inappropriately condensed, which results in formation of structurally deformed lipoplexes . This structural deformation does not affect its cellular uptake but, rather, hampers plasmid translocation across endosomal and/or nuclear membranes . This is inferred from the observation that both lipoplexes effectively translocate much smaller oligonucleotides into cells . In fact, SAINT-5/DOPE-mediated transfection is greatly improved when, before lipoplex assembly, the plasmid is stabilized by condensation with polylysine . The results emphasize a role of the structural shape of the plasmid in gaining cytosolic/nuclear access . Moreover, it has been proposed that such a translocation is promoted when the lipoplex adopts the hexagonal phase, and data are presented that demonstrate that the lamellar SAINT-5/DOPE lipoplex adopts such a phase after its interaction with acidic phospholipid-containing membranes.

Appl Environ Microbiol, 2002 Oct, 68(10), 5005 - 11
Widespread and persistent populations of a major new marine actinomycete taxon in ocean sediments; Mincer TJ et al.; A major taxon of obligate marine bacteria within the order Actinomycetales has been discovered from ocean sediments . Populations of these bacteria (designated MAR 1) are persistent and widespread, spanning at least three distinct ocean systems . In this study, 212 actinomycete isolates possessing MAR 1 morphologies were examined and all but two displayed an obligate requirement of seawater for growth . Forty-five of these isolates, representing all observed seawater-requiring morphotypes, were partially sequenced and found to share characteristic small-subunit rRNA signature nucleotides between positions 207 and 468 (Escherichia coli numbering) . Phylogenetic characterization of seven representative isolates based on almost complete sequences of genes encoding 16S rRNA (16S ribosomal DNA) yielded a monophyletic clade within the family Micromonosporaceae and suggests novelty at the genus level . This is the first evidence for the existence of widespread populations of obligate marine actinomycetes . Organic extracts from cultured members of this new group exhibit remarkable biological activity, suggesting that they represent a prolific resource for biotechnological applications.

Appl Environ Microbiol, 2002 Oct, 68(10), 4932 - 42
Phenotypic and genotypic characterization of avian Escherichia coli O86:K61 isolates possessing a gamma-like intimin; La Ragione RM et al.; Escherichia coli O86:K61 has long been associated with outbreaks of infantile diarrhea in humans and with diarrheal disease in many animal species . Studies in the late 1990s identified E . coli O86:K61 as the cause of mortality in a variety of wild birds, and in this study, 34 E . coli O86:K61 isolates were examined . All of the isolates were nonmotile, but most elaborated at least two morphologically distinct surface appendages that were confirmed to be type 1 and curli fimbriae . Thirty-three isolates were positive for the eaeA gene encoding a gamma type of intimin . No phenotypic or genotypic evidence was obtained for elaboration of Shiga-like toxins, but most isolates possessed the gene coding for the cytolethal distending toxin . Five isolates were selected for adherence assays performed with tissue explants and HEp-2 cells, and four of these strains produced attaching and effacing lesions on HEp-2 cells and invaded the cells, as determined by transmission electron microscopy . Two of the five isolates were inoculated orally into 1-day-old specific-pathogen-free chicks, and both of these isolates colonized, invaded, and persisted well in this model . Neither isolate produced attaching and effacing lesions in chicks, although some pathology was evident in the alimentary tract . No deaths were recorded in inoculated chicks . These findings are discussed in light of the possibility that wild birds are potential zoonotic reservoirs of attaching and effacing E . coli.

Antiviral Res, 2002 Oct, 56(1), 51 - 9
Recombinant antibody Fab against the hypervariable region 1 of hepatitis C virus blocks the virus adsorption to susceptible cells in vitro; Zhou YH et al.; Antibodies against hypervariable region 1 (HVR1) of hepatitis C virus (HCV) are putatively considered to be neutralizing . We previously found that monoclonal antibodies (mAbs) (30F1 and 30F3) against the HVR1 of HCV neutralize HCV in vitro . To develop potentially therapeutic molecules against HCV, we cloned cDNAs of antibody Fab fragments from the mouse hybridoma cells secreting these two mAbs . Fab fragments produced in Escherichia coli were purified by a single step of nickel-chelate affinity chromatography via a hexa-histidine tag . The specificity of the Fabs was confirmed by competition ELISA, BIAcore analysis, and N-terminal amino acid sequencing . The binding constant for the interaction with HVR1 was 1.39 nM for Fab 30F1 and 3.96 nM for Fab 30F3 . The HCV capture assay and inhibition of HCV adsorption test demonstrated that both Fabs had neutralizing activity . The data may be useful for designing immunological therapy of HCV.

Chem Biol . 2002 Sep;9(9):1043.
Genetic control by a metabolite binding mRNA; Nahvi A et al.; Messenger RNAs are typically thought of as passive carriers of genetic information that are acted upon by protein- or small RNA-regulatory factors and by ribosomes during the process of translation . We report that the 5'-untranslated sequence of the Escherichia coli btuB mRNA assumes a more proactive role in metabolic monitoring and genetic control . The mRNA serves as a metabolite-sensing genetic switch by selectively binding coenzyme B(12) without the need for proteins . This binding event establishes a distinct RNA structure that is likely to be responsible for inhibition of ribosome binding and consequent reduction in synthesis of the cobalamin transport protein BtuB . This finding, along with related observations, supports the hypothesis that metabolic monitoring through RNA-metabolite interactions is a widespread mechanism of genetic control.

Gend Action . 1998 Winter;2(1):4.
Integrating gender into natural resources management projects: USAID lessons learned; Tagless extraction-retentate chromatography: a new global protein digestion strategy for monitoring differential protein expression; Ciphergen Biosystems, Inc., Fremont, CA 94555, USA . sweinberger@ciphergen.com

A new global protein digestion and selective peptide extraction strategy for the purpose of monitoring differential protein expression, coined as tagless extraction-retentate chromatography, is introduced . Target protein populations are firstly digested under reduced and alkylated conditions, and resultant peptides selectively extracted via covalent attachment to methionine residues by bromoacetyl reactive groups tethered to the surface of glass beads packed in small reaction vessels . After conjugation, reactive beads are stringently washed to remove nonspecifically bound peptides and then later treated with beta-mercaptoethanol to release captured methionine peptides in their nascent state, without complicating affinity tags . Recovered methionine containing peptides are profiled using the surface-enhanced laser desorption/ionization (SELDI) retentate chromatography mass spectrometry (RCMS) method . Selected peptides are further studied employing ProteinChip tandem mass spectrometry (MS/MS) analysis to identify their parent proteins . This approach has been applied to an Escherichia coli lysate model system and has demonstrated facility in reducing global digest complexity, sensitivity to low protein expression levels, and significant quantitative capability . It is envisioned that tagless extraction-RCMS will evolve to be a valuable approach for both basic research and clinical proteomics endeavors.

J Crit Care, 2002 Sep, 17(3), 188 - 202
Blood pH level modulates organ metabolism of lactate in septic shock in dogs; Chrusch C et al.; OBJECTIVE: Lactic acidosis is an important complication of septic shock . Alkali treatment such as sodium bicarbonate is often used to treat the low pH level that develops in sepsis . The effect of this treatment on lactate (Lac) clearance is not clear . In the present study, the objective was to examine whether blood pH level alters Lac metabolism in sepsis . Measurements were determined in a canine model of Escherichia coli sepsis after bolus infusion (5 mmol/kg) of either lactic acid (LA) or sodium lactate (NaL) . In one preparation, Lac uptake by the splanchnic organs (SP), liver, lung, kidneys (Kid), and soft tissues of the lower extremity (SOL) was primarily determined, whereas in another preparation, Lac uptake by the head and neck region and lung was obtained . METHODS: The dogs were studied while anesthetized and ventilated . After 4 hours of sepsis, either LA or NaL was given through a catheter positioned in the abdominal aorta in respective sepsis (SepLA, SepNaL) and nonsepsis groups (ConLA, ConNaL) (n approximately equal to 6 in each preparation) . Catheters and flow probes were used to measure organ Lac uptake . Measurements were obtained at end infusion and at 15-minute intervals after infusion until 75 minutes after infusion . RESULTS: Arterial clearance of Lac in the sepsis groups was slower as compared with the nonsepsis groups . In the liver, sepsis inhibited the uptake of LA as compared with the nonseptic group . In SP, both sepsis and pH affected Lac uptake in which an increase in uptake was found only after NaL infusion in the nonseptic group . In the head and neck region, Lac uptake was pH-level dependent and was found after LA infusion in the sepsis and nonsepsis groups . In the lung, Lac was produced after either LA or NaL infusion in all groups . Neither Kid nor SOL contributed to Lac uptake in any of the groups . CONCLUSION: Lactate clearance was reduced in sepsis . Both effects of pH level and sepsis modulated the organ uptake of Lac in septic shock . Only a small amount of the total Lac infused could be accounted for by the organs measured in the present study . This suggests that additional organs may account for lactate removal in sepsis .

Biol Reprod, 2002 Oct, 67(4), 1080 - 6
Ovarian carbonyl reductase-like 20beta-hydroxysteroid dehydrogenase shows distinct surge in messenger RNA expression during natural and gonadotropin-induced meiotic maturation in nile tilapia; Senthilkumaran B et al.; Meiotic maturation in fish is accomplished by maturation-inducing hormones . 17alpha,20beta-Dihydroxy-4-pregnen-3-one (17alpha,20beta-DP) was identified as the maturation-inducing hormone of several teleosts, including Nile tilapia . A cDNA encoding 20beta-hydroxysteroid dehydrogenase (20beta-HSD), the enzyme that converts 17alpha-hydroxyprogesterone to 17alpha,20beta-DP, was cloned from the ovarian follicle of Nile tilapia . Genomic Southern analysis indicated that 20beta-HSD probably exists as a single copy in the genome . The Escherichia coli-expressed cDNA product oxidized both carbonyl and steroid compounds, including progestogens, in the presence of NADPH . Carbonyl reductase-like 20beta-HSD is broadly expressed in various tissues of tilapia, including ovary, testis, and gill . Northern blot and reverse transcription polymerase chain reaction analyses during the 14-day spawning cycle revealed that the expression of 20beta-HSD in ovarian follicles is low from Day 0 to Day 8 after spawning and is not detectable on Day 11 . Distinct expression was evident at Day 14, the day of spawning . In males, 20beta-HSD expression was observed continually in mature testes but not in immature testes of 30-day-old fish . In vitro incubation of postvitellogenic immature follicles (corresponding to Day 11 after spawning) with hCG induced the expression of 20beta-HSD mRNA transcripts within 1-2 h, followed by the final meiotic maturation of oocytes . In tissues such as gill, muscle, brain, and pituitary, however, hCG treatment did not induce any changes in the levels of mRNA transcripts . Actinomycin D blockade of hCG-induced 20beta-HSD expression and final oocyte maturation demonstrated the involvement of transcriptional factors . The carbonyl reductase-like 20beta-HSD plays an important role in the meiotic maturation of tilapia gametes.

J Biol Chem, 2002 Nov 15, 277(46), 43549 - 52 Epub 2002 Sep 23.
Neuronal leucine-rich repeat protein-3 amplifies MAPK activation by epidermal growth factor through a carboxyl-terminal region containing endocytosis motifs; Fukamachi K et al.; Neuronal leucine-rich repeat protein-3 (NLRR-3) belongs to the LRR superfamily . Expression of rat NLRR-3 gene isolated from c-Ha-ras transgenic rat tumor is regulated mainly through the Ras-MAPK signaling pathway . NLRR-3 was found to enhance phosphorylation of MAPK when COS-7 cells were transfected with NLRR-3 and stimulated with a low concentration (0.01 ng/ml) of epidermal growth factor (EGF), but the amplification of MAPK phosphorylation by NLRR-3 was no longer observed when the carboxyl-terminal 30 amino acid stretch containing clathrin-mediated endocytosis motifs was deleted . A green fluorescent protein-tagged NLRR-3 localized at the plasma membrane was efficiently internalized in COS-7 cells, but internalization of a carboxyl-terminal-deleted version (NLRRDeltaC) was less efficient . The presence of clathrin-adaptor protein complexes containing NLRR-3 in brain lysate was confirmed by immunoprecipitation and glutathione S-transferase pull-down experiments, and affinity column chromatography revealed that the carboxyl-terminal region of NLRR-3 interacts with beta-adaptin . We propose that NLRR-3 potentiates Ras-MAPK signaling by facilitating internalization of EGF in clathrin-coated vesicles.

Di Yi Jun Yi Da Xue Xue Bao, 2002 Jun, 22(6), 554 - 6
Construction of eukaryotic expression vector of thrombospondin-1 type I repeat sequence; Li XW et al.; OBJECTIVE: To construct eukaryotic expression vector of thrombospondin-1 type I repeat sequences . METHODS: Thrombospondin-1 type I repeat sequence gene was amplified from human fetal lung tissue by reverse transcriptase-PCR (RT-PCR) to construct both recombinant clone vector and expression vector through coupling reaction, followed by transforming these vectors into E.coli DH5alpha . The positive clones were selected for verification by double enzyme digestion and sequence analysis . RESULT: The expected amplification product, thrombospondin-1 type I repeat gene sequence, was acquired by RT-PCR, which had a consistency up to 99% with the sequence from the GenBank . CONCLUSION: The eukaryotic expression vector PcDNA3.1(+)/TSP-1 type I repeat sequence was successfully constructed.

Vaccine, 2002 Oct 4, 20(29-30), 3436 - 42
Influenza virosomes are an efficient delivery system for respiratory syncytial virus-F antigen inducing humoral and cell-mediated immunity; Cusi MG et al.; In the present study we investigated the efficacy of a new potential vaccine constituted of the respiratory syncytial virus (RSV)-F protein associated with influenza virosomes (RSV-F/IRIV) in combination with the mucosal adjuvant Escheriagen (Escherichia coli heat-labile toxin), administered intranasally (i.n.) to BALB/c mice . After an intramuscular "priming" with influenza virus vaccine, group A of mice was i.n . immunized with of RSV-F/IRIV+heat-labile toxin (HLT), groups B and C were inoculated i.n . with F-RSV+HLT and IRIV+HLT, respectively . The results showed that the virosomal delivery system greatly potentiate immune responses in animals . All mice immunized with the RSV-F/IRIV+HLT developed a mucosal IgA response and a high level of serum IgG . A balanced Th1/Th2 cytokine profile was observed in mice immunized with RSV-F/IRIV+HLT, while a Th2 response was observed in mice immunized with RSV-F+HLT . Histological analysis of lung tissue of RSV challenged mice did not reveal a vaccine-enhanced pulmonary eosinophilia . These results show that i.n . immunization of BALB/c mice with RSV-F/IRIV in combination with HLT can be considered a promising approach for the development of an efficacious human vaccine.

FEBS Lett, 2002 Sep 25, 528(1-3), 279 - 82
Transcriptional activator, AoXlnR, mediates cellulose-inductive expression of the xylanolytic and cellulolytic genes in Aspergillus oryzae; Marui J et al.; AoXlnR was isolated as a transcriptional activator of the major xylanase gene, xynF1, in Aspergillus oryzae . To investigate the spectrum of genes under the control of AoXlnR, expression of the xylanolytic and cellulolytic genes in an A . oryzae wild type strain, an AoxlnR disruptant and an AoXlnR overexpressed strain was analyzed by Northern blotting . AoXlnR mediated expression of at least four xylanolytic genes and four cellulolytic genes when induced by xylan and D-xylose . Moreover, AoXlnR was newly found to mediate the cellulose-inductive expression of the xylanolytic genes as well as the cellulolytic genes.

FEBS Lett, 2002 Sep 25, 528(1-3), 257 - 60
Increased stability of human growth hormone with reduced lactogenic potency; Schulga AA et al.; Human growth hormone (hGH), whose main function is the somatic growth stimulation, induces diverse effects including lactation . We examined the possibility of hGH stabilization by elimination of its lactogenic activity . Chimeric GHs were constructed by replacement of different segments of hGH with sequences derived from non-lactogenic porcine GH . As was observed in the rat Nb2-11C lymphoma cell test, lactogenic activity of some chimeric hormones was seriously destroyed . This kind of hormones displayed the substantial increase in thermal and guanidine hydrochloride stability . The more stable hGH variants were found to be more soluble in Escherichia coli cells.

FEBS Lett, 2002 Sep 25, 528(1-3), 203 - 6
NMR studies of the hydrogen bonds involving the catalytic triad of Escherichia coli thioesterase/protease I; Tyukhtenko SI et al.; Escherichia coli thioesterase/protease I (TEP-I) is a lipolytic enzyme of the serine protease superfamily with Ser(10), Asp(154) and His(157) as the catalytic triad residues . Based on comparison of the low-field (1)H nuclear magnetic resonance spectra of two mutants (S10G and S12G) and two transition state analogue complexes we have assigned the exchangeable proton resonances at 16.3 ppm, 14.3 ppm, and 12.8 ppm at pH 3.5 to His(157)-N(delta1)H, Ser(10)-O(gamma)H and His(157)-N(epsilon2)H, respectively . Thus, the presence of a strong Asp(154)-His(157) hydrogen bond in free TEP-I was observed . However, Ser(10)-O(gamma)H was shown to form a H-bond with a residue other than His(157)-N(epsilon2).

FEBS Lett, 2002 Sep 25, 528(1-3), 193 - 6
Dominant negative mutant of a lipoprotein-specific molecular chaperone, LolA, tightly associates with LolCDE; Miyamoto A et al.; Periplasmic molecular chaperone LolA and the inner membrane ATP binding cassette transporter LolCDE are essential for ATP-dependent release of outer membrane-directed lipoproteins from the inner membrane of Escherichia coli . A LolA(F47E) mutant carrying a Phe to Glu mutation at position 47 was defective in the release of lipoproteins from spheroplasts and proteoliposomes reconstituted with LolCDE . When incubated with proteoliposomes containing LolCDE, LolA remained in the supernatant whereas LolA(F47E) bound to proteoliposomes . This tight association of LolA(F47E) with LolCDE caused a dominant negative phenotype in vivo, suggesting that the LolA-LolCDE interaction is critical for lipoprotein release.

FEBS Lett, 2002 Sep 25, 528(1-3), 95 - 100
The GCM domain is a Zn-coordinating DNA-binding domain; Cohen SX et al.; Glial cells missing (GCM) proteins form a small family of transcriptional regulators involved in different developmental processes . They contain a DNA-binding domain that is highly conserved from flies to mice and humans and consists of approximately 150 residues . The GCM domain of the mouse GCM homolog a was expressed in bacteria . Extended X-ray absorption fine structure and particle-induced X-ray emission analysis techniques showed the presence of two Zn atoms with four-fold coordination and cysteine/histidine residues as ligands . Zn atoms can be removed from the GCM domain by the Zn chelator phenanthroline only under denaturating conditions . This suggests that the Zn ions are buried in the interior of the GCM domain and that their removal abolishes DNA-binding because it impairs the structure of the GCM domain . Our results define the GCM domain as a new type of Zn-coordinating, sequence-specific DNA-binding domain.

FEBS Lett, 2002 Sep 25, 528(1-3), 70 - 6
Antitumor activity of interleukin-21 prepared by novel refolding procedure from inclusion bodies expressed in Escherichia coli; Asano R et al.; Interleukin-21 (IL-21) has recently been identified as a novel 4-helix-bundle type I cytokine possessing a cytokine receptor gamma chain essential for the immune response . We report the preparation and functional characterization of Escherichia coli-expressed recombinant human IL-21 (rIL-21) . The rIL-21, expressed as insoluble inclusion bodies in E . coli, was solubilized and then refolded by using a modified dialysis method . The introduction of redox reagents during refolding led to a dramatic increase in the refolding efficiency . Circular dichroism spectrum analysis showed that the refolded rIL-21 had an alpha-helix as a secondary structure, which is a characteristic of type I cytokines . Flow cytometry confirmed previous reports that rIL-21 binds to CD3-activated T cells (T-LAK) and to cell lines Raji, HL60, and Jurkat . rIL-21 stimulated the proliferation of T-LAK but not peripheral blood mononuclear cells, and this effect seems to be identical to that of co-stimulation with anti-CD3 antibody . Growth inhibition assay indicated that enhancement of the cytotoxicity of T-LAK to the human bile duct carcinoma TFK-1 depended on the concentration of rIL-21 . Thus, refolded rIL-21 had activity identical to that of authentic IL-21 and enhanced the anti-tumor activity of T-LAK . These conclusions suggest the potential use of the refolded cytokine in adoptive immunotherapy using T-LAK cells and in the discovery of other functions of the cytokine.

Cell, 2002 Sep 20, 110(6), 701 - 11
Methylation of histone h3 at lysine 9 targets programmed DNA elimination in tetrahymena; Taverna SD et al.; Histone H3 lysine 9 methylation {Me(Lys9)H3} is an epigenetic mark for heterochromatin-dependent gene silencing, mediated by direct binding to chromodomain-containing proteins such as Heterochromatin Protein 1 . In the ciliate Tetrahymena, two chromodomain proteins, Pdd1p and Pdd3p, are involved in the massive programmed DNA elimination that accompanies macronuclear development . We report that both proteins bind H3(Lys9)Me in vitro . In vivo, H3(Lys9)Me is confined to the time period and location where DNA elimination occurs, and associates with eliminated sequences . Loss of parental Pdd1p expression drastically reduces H3(Lys9)Me . Finally, tethering Pdd1p is sufficient to promote DNA excision . These results extend the range of H3(Lys9)Me involvement in chromatin activities outside transcriptional regulation and also strengthen the link between heterochromatin formation and programmed DNA elimination.

J Biochem Mol Biol, 2002 Mar 31, 35(2), 206 - 12
In vitro determination of dengue virus type 2 NS2B-NS3 protease activity with fluorescent peptide substrates; Khumthong R et al.; The NS2B-NS3(pro) polyprotein segment from the dengue virus serotype 2 strain 16681 was purified from overexpressing E . coli by metal chelate affinity chromatography and gel filtration . Enzymatic activity of the refolded NS2B-NS3(pro) protease complex was determined in vitro with dansyl-labeled peptide substrates, based upon native dengue virus type 2 cleavage sites . The 12mer substrate peptides and the cleavage products could be separated by reversed-phase HPLC, and were identified by UV and fluorescence detection . All of the peptide substrates (representing the DEN polyprotein junction sequences at the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 sites) were cleaved by the recombinant protease NS2B-NS3(pro) . No cleavage was observed with an enzymatically inactive S135A mutant of the NS3 protein, or with a modified substrate peptide of the NS3/NS4A polyprotein site that contained a K2093A substitution . Enzymatic activity was dependent on the salt concentration . A 50% decrease of activity was observed in the presence of 0.1 M sodium chloride . Our results show that the NS3 protease activity of the refolded NS2BNS3(pro) protein can be assayed in vitro with high specificity by using cleavage-junction derived peptide substrates.

J Biochem Mol Biol, 2002 May 31, 35(3), 343 - 7
Purification and spectroscopic characterization of the human protein tyrosine kinase-6 SH3 domain; Koo BK et al.; The human protein tyrosine kinase-6 (PTK6) polypeptide that is deduced from the cDNA sequence contains a Src homology (SH) 3 domain, SH2 domain, and catalytic domain of tyrosine kinase . We initiated biochemical and NMR characterization of PTK6 SH3 domain in order to correlate the structural role of the PTK6 using circular dichroism and heteronuclear NMR techniques . The circular dichroism data suggested that the secondary structural elements of the SH3 domain are mainly composed of beta-sheet conformations . It is most stable when the pH is neutral based on the pH titration data . In addition, a number of cross peaks at the low-field area of the proton chemical shift of the NMR spectra indicated that the PTK6 SH3 domain retains a unique and folded conformation at the neutral pH condition . For other pH conditions, the SH3 domain became unstable and aggregated during NMR measurements, indicating that the structural stability is very sensitive to pH environments . Both the NMR and circular dichroism data indicate that the PTK6 SH3 domain experiences a conformational instability, even in an aqueous solution.

J Biochem Mol Biol, 2002 May 31, 35(3), 297 - 301
OxyR regulon controls lipid peroxidation-mediated oxidative stress in Escherichia coli; Yoon SJ et al.; Membrane lipid peroxidation processes yield products that may react with DNA and proteins to cause oxidative modifications . The oxyR gene product regulates the expression of enzymes and proteins that are needed for cellular protection against oxidative stress . Upon exposure to tert-butylhydroperoxide (t-BOOH) and 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH), which induce lipid peroxidation in membranes, the Escherichia coli oxyR overexpression mutant was much more resistant to lipid peroxidation-mediated cellular damage, when compared to the OxyR deletion mutant in regard to growth kinetics, viability, and DNA damage . The deletion of the OxyR gene in E . coli also resulted in increased susceptibility of superoxide dismutase to lipid peroxidation-mediated inactivation . The results indicate that the peroxidation of lipid is probably one of the important intermediary events in free radical-induced cellular damage . Also, the oxyR regulon plays an important protective role in lipid peroxidation-mediated cellular damage.

J Biochem Mol Biol, 2002 May 31, 35(3), 255 - 61
Biochemical characterization of oligomerization of Escherichia coli GTP cyclohydrolase I; Lee S et al.; GTP cyclohydrolase I (E.C . 3.5.4.16) is a homodecameric protein that catalyzes the conversion of GTP to 7,8- dihydroneopterin triphosphate (H(2)NTP), the initial step in the biosynthesis of pteridines . It was proposed that the enzyme complex could be composed of a dimer of two pentamers, or a pentamer of tightly associated dimers; then the active site of the enzyme was located at the interface of three monomers (Nar et al . 1995a, b) . Using mutant enzymes that were made by site-directed mutagenesis, we showed that a decamer of GTP cyclohydrolase I should be composed of a pentamer of five dimers, and that the active site is located between dimers, as analyzed by a series of size exclusion chromatography and the reconstitution experiment . We also show that the residues Lys 136, Arg139, and Glu152 are of particular importance for the oligomerization of the enzyme complex from five dimers to a decamer.

J Biochem Mol Biol, 2002 Jul 31, 35(4), 428 - 31
Disrupting Escherichia coli: a comparison of methods; Benov L et al.; The often-encountered problem of disrupting bacteria for the purpose of extracting soluble protein has generated various methods . Many require specialized equipment . Very often, especially during preliminary studies, investigators need a simple, fast, and inexpensive method for cell disruption that preserves biological activity . This paper compares some simple and inexpensive methods for cell disruption, such as bead-vortexing, freezing-thawing, French pressing, and sonication . It also provides some tips to increase protein yield and preserve biological activity . If performed under optimal conditions, bead-vortexing gives protein yields that are comparable to French pressing and sonication . It also preserves the activities of labile enzymes and releases periplasmic enzymes . Vortexing with glass beads appears to be the simplest method for cell disruption.

J Biochem Mol Biol, 2002 Jul 31, 35(4), 389 - 94
Dynamics of supercoiled and relaxed pTZ18U plasmids probed with a long-lifetime metal-ligand complex; Kang JS et al.; {Ru(bpy)2(dppz)}(2+) (bpy = 2,2'-bipyridine, dppz = dipyrido- {3,2-a:2',3'-c}phenazine) (RuBD), a long-lifetime metalligand complex, displays favorable photophysical properties . These include long lifetime, polarized emission, but no significant fluorescence from the complex that is not bound to DNA . To show the usefulness of this luminophore (RuBD) for probing the bending and torsional dynamics of nucleic acids, its intensity and anisotropy decays when intercalated into supercoiled and relaxed pTZ18U plasmids were examined using frequency-domain fluorometry with a blue light-emitting diode (LED) as the modulated light source . The mean lifetimes for the supercoiled plasmids (< tau > = 148 ns) were somewhat shorter than those for the relaxed plasmids (< tau > = 160 ns) . This suggests that the relaxed plasmids were shielded more efficiently from water . The anisotropy decay data also showed somewhat shorter slow rotational correlation times for supercoiled plasmids (288 ns) than for the relaxed plasmids (355 ns) . The presence of two rotational correlation times suggests that RuBD reveals both the bending and torsional motions of the plasmids . These results indicate that RuBD can be useful for studying both the bending and torsional dynamics of nucleic acids.

J Biochem Mol Biol, 2002 Jul 31, 35(4), 353 - 7
Control of singlet oxygen-induced oxidative damage in Escherichia coli; Kim SY et al.; Singlet oxygen ((1)O(2)) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules . The oxyR gene product regulates the expression of the enzymes and proteins that are needed for cellular protection against oxidative stress . In this study, the role of oxyR in cellular defense against a singlet oxygen was investigated using Escherichia coli oxyR mutant strains . Upon exposure to methylene blue and visible light, which generates singlet oxygen, the oxyR overexpression mutant was much more resistant to singlet oxygen-mediated cellular damage when compared to the oxyR deletion mutant in regard to growth kinetics, viability and protein oxidation . Induction and inactivation of major antioxidant enzymes, such as superoxide dismutase and catalase, were observed after their exposure to a singlet oxygen generating system in both oxyR strains . However, the oxyR overexpression mutant maintained significantly higher activities of antioxidant enzymes than did the oxyR deletion mutant . These results suggest that the oxyR regulon plays an important protective role in singlet oxygen-mediated cellular damage, presumably through the protection of antioxidant enzymes.

J Am Chem Soc, 2002 Oct 2, 124(39), 11636 - 41
Germacrene A is a product of the aristolochene synthase-mediated conversion of farnesylpyrophosphate to aristolochene; Calvert MJ et al.; The biosynthesis of several sesquiterpenes has been proposed to proceed via germacrene A . However, to date, the production of germacrene A has not been proven directly for any of the sesquiterpene synthases for which it was postulated as an intermediate . We demonstrate here for the first time that significant amounts of germacrene A (7.5% of the total amount of products) are indeed released from wild-type aristolochene synthase (AS) from Penicillium roqueforti . Germacrene A was identified through direct GC-MS comparison to an authentic sample and through production of beta-elemene in a thermal Cope rearrangement . AS also produced a small amount of valencene through deprotonation of C6 rather than C8 in the final step of the reaction . On the basis of the X-ray structure of AS, Tyr 92 was postulated to be the active-site acid responsible for protonation of germacrene A (Caruthers, J . M.; Kang, I.; Rynkiewicz, M . J.; Cane, D . E.; Christianson, D . W . J . Biol . Chem . 2000, 275, 25533-25539) . The CD spectra of a mutant protein, ASY92F, in which Tyr 92 was replaced by Phe, and of AS were very similar . ASY92F was approximately 0.1% as active as nonmutated recombinant AS . The steady-state kinetic parameters were measured as 0.138 min(-1) and 0.189 mM for k(cat) and K(M), respectively . Similar to a mutant protein of 5-epi-aristolochene (Rising, K . A.; Starks, C . M.; Noel, J . P.; Chappell, J . J . Am . Chem . Soc . 2000, 122, 1861-1866), the mutant released significant amounts of germacrene A (approximately 29%) . ASY92F also produced various amounts of a further five hydrocarbons of molecular weight 204, valencene, beta-(E)-farnesene, alpha- and beta-selinene, and selina-4,11-diene.

J Am Chem Soc, 2002 Oct 2, 124(39), 11594 - 5
Amphipols can support the activity of a membrane enzyme; Gorzelle BM et al.; Amphipathic polymers ("amphipols") were introduced several years ago (Tribet, C.; Audebert, R.; Popot, J.-L . Proc . Natl . Acad . Sci . U.S.A . 1996, 93, 15047-15050) as an alternative method for solubilizing integral membrane proteins in stable, nativelike conformations . However, direct maintenance of full membrane protein functionality in amphipol solutions has not previously been demonstrated in the absence of added lipid or detergent . In this contribution, the first zwitterionic amphipol "PMAL-B-100" is introduced . PMAL-B-100 not only maintains membrane protein structure and solubility, but also supports the full catalytic activity of an integral membrane enzyme, diacylglycerol kinase, in the complete absence of additional lipid or detergent . All of the roles which a lipid bilayer normally plays in maintaining diacylglycerol kinase's structure and in facilitating catalysis are satisfied by the environment and interactions supplied by PMAL-B-100.

Life Sci Space Res, 1979, 17, 111 - 5
A special photoproduct of UV-irradiated DNA in vacuo; Bucker H et al.; DNA isolated from E . coli B/r cells was UV-irradiated at 254 nm under high vacuum of 10(-7) torr . Compared with DNA in solution, the formation of cis-syn thymine dimers (TT) was decreased, and the formation of cytosine thymine dimers (CT) and a photoproduct of RF value 0.41 was increased . The latter photoproduct was identified as tran-syn TT which is preferentially produced in heat-denaturated DNA.

New Egypt J Med, 1992 May, 6(5), 1416 - 22
Relation between weaning and gastroenteritis; Mostafa HA et al.; PIP: More than 50% of Egyptian children who die before reaching age 2 years perish as a result of acute infantile diarrhea . Most health experts in Egypt recommend that weaning foods be given to infants beginning at age 4-6 months, the point at which breast milk alone cannot meet an infant's nutritional needs . Findings are reported from an evaluation of the relationship between the weaning period and the possible incidence of diarrheal attacks . 60 cases aged 6-24 months presented with diarrhea after the early introduction of foods during the weaning period . 100 healthy infants matched according to age, sex, and socioeconomic status served as controls . 46.6% of cases were among infants under 1 year old . Only 30% of the children were originally breast-fed . Isonatremic dehydration accounted for 75% of all dehydration among the subjects, while E . coli caused 28.3% of cases, E . histolytica 21.6%, and Giardia lamblia 16% . The presence of polymorph nuclear leucocytes in the stool was a predictive test for the bacterial etiology of diarrhea with a positive predictive value of 87% . Finally, the band/neutrophil ratio of the bacterial group was significantly different from that of the control group, although no statistically significant difference was identified when comparing the parasitic diarrheal group to the control group .

Acta Paediatr, 1992 Sep, 81 Suppl 381, 39 - 44
Persistent diarrhea in Northeast Brazil: etiologies and interactions with malnutrition; Lima AA et al.; With the improved control of acute diarrheal illness mortality with oral rehydration therapy, persistent diarrhea is now emerging as a major cause of childhood mortality in tropical developing areas like the impoverished populations in Brazil's Northeast . "Graveyard surveillance" in the rural community of Guaiuba in northeastern Brazil revealed fully half of the 70% diarrhea mortality was due to persistent diarrheal illnesses . Furthermore, 11% of 14 or more diarrheal illnesses/child/year in an urban slum in Fortaleza persisted beyond 14 days, a definition that clearly identified the high risk children for heavy diarrhea burdens . Not only did heavy diarrhea burdens ablate the key "catch-up" growth seen in severely malnourished children and in children following previous diarrheal illnesses, but malnutrition significantly predisposed children to a greater incidence and duration of diarrhea as well as a greater incidence of persistent diarrhea . Etiologic studies of 37 children presenting with persistent diarrhea to Hospital das Clinicas in Fortaleza revealed that Cryptosporidium (in 13%) and enteroadherent E . coli (36% with aggregative, 29% with diffuse, and 13% with localized adherence to HEp-2 cells) were the predominant potential pathogens found in the stool or upper small bowel . These findings suggest that persistent diarrhea is emerging as an important health problem in Brazil's Northeast, that it identifies a high risk child for heavy diarrhea burdens, that important interactions occur with malnutrition, an that Cryptosporidium and enteroadherent E . coli warrant further study as potential etiologies of this major cause of morbidity and mortality.

Acta Paediatr, 1992 Sep, 81 Suppl 381, 124 - 6
Persistent diarrhea in Vietnamese children: a preliminary report; Ngan PK et al.; The clinical and laboratory features of persistent diarrhea were investigated in 83 children under 3 years of age who were treated in the Gastroenterology Division of the Institute for the Protection of Children's Health, Hanoi, from August 1988 to August 1989 . The number of cases of diarrhea was highest in the children aged 4-5 months . The mean age of the children studies was 6.6 +or- 3.4 months . The ratio of males to females was 2.6 and mean age of 1st episode of diarrhea was 4.3 +or- 3.4 months; persistent diarrhea was more common in children under 6 months of age than in older children . Persistent diarrhea occurred in the 1st diarrheal episode in 66.5% of cases . Recent nonenteric infections were found in 30% of the study group . Of the 83 children studies, 36% had stool specimens positive for enteric pathogens; 24% had enterotoxigenic Escherichia coli isolated, 8% had enteropathogenic E . coli, 5% rotavirus, 6% Candida, and 4% Giardia lamblia . The duration of diarrhea was longer in children who received antibiotics than in those who did not (p 0.01).

Afr Health, 1992 Nov, 15(1), 10 - 1
Low-cost water supplies and their contribution to health; Watts R; PIP: The importance of low cost water supplies and sanitation facilities is heightened in drought conditions such as occurred in Zimbabwe during 1991-92 . A summary of water and toilet systems is given . Zimbabwe must recognize that it is a dry country and adopt water and sanitation services appropriate to the climate . Since 1980, 10,000 water sources have been protected . The Blair Latrine has been installed in 300,000 locations and uses little or no flush system, is odorless, free of insects, and doubles as a bathroom . The target is to install 1.4 million Blair toilets, 576 piped water supplies, and 36,000 primary water supplies between 1985-2005 . When piped water supplies are used in conjunction with the Blair toilet, a tank replaces the soil-lined pit . A Harare based company is currently manufacturing a 1-liter flush toilet instead of a 10-liter one . The Vonder Rig is another technological improvement being tested by the Blair Research Institute, which is effective in drilling through soils and rocky areas in the Epworth area which has a high water table . However, Epworth water supplies were installed in just a few homes; wells currently in use are placed too close to pit latrines and are dry due to the drought . Save the Children Fund (SCF) has been involved with rural water programs, but finds hauling water to rural areas too expensive . People must move into the city with friends and relatives . A SCF engineer has upgraded and dug 1000 wells in the past year, and finds that the Vonder Rig is suitable only where the water is near the surface and soil conditions are right . The SCF has improved existing wells by reinforcing the well lining and headworks and adding a long-lasting windlass plus an apron and cover for prevention of contamination . The SCF develops ways of obtaining water through rural participation . Africare, a US nongovernmental organization, prefers digging boreholes and has completed 800 at a cost of $10 million . However, boreholes dry out, suffer mechanical failures, and must be maintained . For communal wells, the pumps are associated with lower bacteria counts of E . coli . The Bush Pump has been found to be most successful, but cost prevents wide-scale use . Since 1991, upgrading wells has become a strategy, based on trial data . UNICEF has rated Zimbabwe highly concerning the number of people with access to safe water; hopefully, when normal rainfall returns, safe water supplies will also .

Proc Natl Acad Sci U S A, 2002 Oct 1, 99(20), 12681 - 4 Epub 2002 Sep 23.
FlgM gains structure in living cells; Dedmon MM et al.; Intrinsically disordered proteins such as FlgM play important roles in biology, but little is known about their structure in cells . We use NMR to show that FlgM gains structure inside living Escherichia coli cells and under physiologically relevant conditions in vitro, i.e., in solutions containing high concentrations (>/=400 g/liter) of glucose, BSA, or ovalbumin . Structure formation represents solute-induced changes in the equilibrium between the structured and disordered forms of FlgM . The results provide insight into how the environment of intrinsically disordered proteins could dictate their structure and, in turn, emphasize the relevance of studying proteins in living cells and in vitro under physiologically realistic conditions.

Proc Natl Acad Sci U S A, 2002 Oct 1, 99(20), 12771 - 6 Epub 2002 Sep 23.
Ultrafast ligand rebinding in the heme domain of the oxygen sensors FixL and Dos: general regulatory implications for heme-based sensors; Liebl U et al.; Heme-based oxygen sensors are part of ligand-specific two-component regulatory systems, which have both a relatively low oxygen affinity and a low oxygen-binding rate . To get insight into the dynamical aspects underlying these features and the ligand specificity of the signal transduction from the heme sensor domain, we used femtosecond spectroscopy to study ligand dynamics in the heme domains of the oxygen sensors FixL from Bradyrhizobium japonicum (FixLH) and Dos from Escherichia coli (DosH) . The heme coordination with different ligands and the corresponding ground-state heme spectra of FixLH are similar to myoglobin (Mb) . After photodissociation, the excited-state properties and ligand-rebinding kinetics are qualitatively similar for FixLH and Mb for CO and NO as ligands . In contrast to Mb, the transient spectra of FixLH after photodissociation of ligands are distorted compared with the ground-state difference spectra, indicating differences in the heme environment with respect to the unliganded state . This distortion is particularly marked for O(2) . Strikingly, heme-O(2) recombination occurs with efficiency unprecedented for heme proteins, in approximately 5 ps for approximately 90% of the dissociated O(2) . For DosH-O(2), which shows 60% sequence similarity to FixLH, but where signal detection and transmission presumably are quite different, a similarly fast recombination was found with an even higher yield . Altogether these results indicate that in these sensors the heme pocket acts as a ligand-specific trap . The general implications for the functioning of heme-based ligand sensors are discussed in the light of recent studies on heme-based NO and CO sensors.

Plant Cell, 1993 Jul, 5(7), 769 - 780
Cellular Concentrations and Uniformity of Cell-Type Accumulation of Two Lea Proteins in Cotton Embryos; Roberts JK et al.; The levels and cell-type distribution of late embryogenesis abundant (Lea) proteins D-7 and D-113 have been determined in mature cotton embryos by immunochemical methods . The two proteins were expressed in and purified from Escherichia coli and utilized for antibody production in rabbits . The antiserum to each protein was found to interact with all members of each protein family in cotton extracts by protein gel blotting . Using these antibodies in quantitative "rocket" immunoelectrophoreses, D-7 proteins were found to accumulate to ~8 x 1015 molecules per embryo, which is equivalent to ~109 molecules per "average cell." D-113 proteins accumulate to ~1016 molecules per embryo, which equates to ~1.3 x 109 molecules per average cell . These values calculate to concentrations of about 226 and 283 {mu}M, respectively, in the cell aqueous phase immediately prior to seed desiccation . In immunocytochemical studies using the fluorophor rhodamine linked to the secondary antibody, both proteins appeared to be evenly present in the cytosol of all cell types present in the embryo, including both cotyledon and axis epidermal cells . Thus, their function does not appear related to unique functions of specific cell or tissue types . The very high molar concentrations of the two proteins, coupled with their unusual predicted structure and their cytosol location, would seem to reduce the number of their conceivable functions.

J Bacteriol, 2002 Oct, 184(20), 5814 - 7
Secretion of alpha-amylase from Pseudoalteromonas haloplanktis TAB23: two different pathways in different hosts; Tutino ML et al.; Secretion of cold-adapted alpha-amylase from Pseudoalteromonas haloplanktis TAB23 was studied in three Antarctic bacteria . We demonstrated that the enzyme is specifically secreted in the psychrophilic hosts even in the absence of a protein domain that has been previously reported to be necessary for alpha-amylase secretion in Escherichia coli . The occurrence of two different secretion pathways in different hosts is proposed.

J Bacteriol, 2002 Oct, 184(20), 5789 - 99
Conserved response regulator CtrA and IHF binding sites in the alpha-proteobacteria Caulobacter crescentus and Rickettsia prowazekii chromosomal replication origins; Brassinga AK et al.; CzcR is the Rickettsia prowazekii homolog of the Caulobacter crescentus global response regulator CtrA . CzcR expression partially compensates for developmental defects in ctrA mutant C . crescentus cells, and CzcR binds to all five CtrA binding sites in the C . crescentus replication origin . Conversely, CtrA binds to five similar sites in the putative R . prowazekii replication origin (oriRp) . Also, Escherichia coli IHF protein binds over a central CtrA binding site in oriRp . Therefore, CtrA and IHF regulatory proteins have similar binding patterns in both replication origins, and we propose that CzcR is a global cell cycle regulator in R . prowazekii.

J Bacteriol, 2002 Oct, 184(20), 5781 - 8
The positive regulator, TraJ, of the Escherichia coli F plasmid is unstable in a cpxA* background; Gubbins MJ et al.; The Cpx (conjugative plasmid expression) stress response of Escherichia coli is induced in response to extracytoplasmic signals generated in the cell envelope, such as misfolded proteins in the periplasm . Detection of stress is mediated by the membrane-bound histidine kinase, CpxA . Signaling of the response regulator CpxR by activated CpxA results in the expression of several factors required for responding to cell envelope stress . CpxA was originally thought to be required for the expression of the positive regulator of the F plasmid transfer (tra) operon, TraJ . It was later determined that constitutive gain-of-function mutations in cpxA led to activation of the Cpx envelope stress response and decreased TraJ expression . In order to determine the nature of the downregulation of TraJ, the level of expression of TraJ, TraM, and TraY, the F-encoded regulatory proteins of the F tra region, was determined both in a cpxA* background and in a wild-type background in which the Cpx stress response was induced by overexpression of the outer membrane lipoprotein, NlpE . Our results suggest that TraJ downregulation is controlled by a posttranscriptional mechanism that operates in the cytoplasm in response to upregulation of the Cpx stress response by both the cpxA* gain-of-function mutation and the overexpression of NlpE.

J Bacteriol, 2002 Oct, 184(20), 5772 - 80
Pseudoknot-dependent translational coupling in repBA genes of the IncB plasmid pMU720 involves reinitiation; Praszkier J et al.; Replication of the IncB miniplasmid pMU720 requires synthesis of the replication initiator protein, RepA, whose translation is coupled to that of a leader peptide, RepB . The unusual feature of this system is that translational coupling in repBA has to be activated by the formation of a pseudoknot immediately upstream of the repA Shine-Dalgarno sequence . A small antisense RNA, RNAI, controls replication of pMU720 by interacting with repBA mRNA to inhibit expression of repA both directly, by preventing formation of the pseudoknot, and indirectly, by inhibiting translation of repB . The mechanism of translational coupling in repBA was investigated using the specialized ribosome system, which directs a subpopulation of ribosomes that carry an altered anti-Shine-Dalgarno sequence to translate mRNA molecules whose Shine-Dalgarno sequences have been altered to be complementary to the mutant anti-Shine-Dalgarno sequence . Our data indicate that translation of repA involves reinitiation by the ribosome that has terminated translation of repB . The role of the pseudoknot in this process and its effect on the control of copy number in pMU720 are discussed.

J Bacteriol, 2002 Oct, 184(20), 5696 - 705
YfcX enables medium-chain-length poly(3-hydroxyalkanoate) formation from fatty acids in recombinant Escherichia coli fadB strains; Snell KD et al.; Expression of Escherichia coli open reading frame yfcX is shown to be required for medium-chain-length polyhydroxyalkanoate (PHA(MCL)) formation from fatty acids in an E . coli fadB mutant . The open reading frame encodes a protein, YfcX, with significant similarity to the large subunit of multifunctional beta-oxidation enzymes . E . coli fadB strains modified to contain an inactivated copy of yfcX and to express a medium-chain-length synthase are unable to form PHA(MCL)s when grown in the presence of fatty acids . Plasmid-based expression of yfcX in the FadB(-) YfcX(-) PhaC(+) strain restores polymer formation . YfcX is shown to be a multifunctional enzyme that minimally encodes hydratase and dehydrogenase activities . The gene encoding YfcX is located downstream from yfcY, a gene encoding thiolase activity . Results of insertional inactivation studies and enzyme activity analyses suggest a role for yfcX in PHA monomer unit formation in recombinant E . coli fadB mutant strains . Further studies are required to determine the natural role of YfcX in the metabolism of E . coli.

J Mol Biol, 2002 Sep 27, 322(4), 891 - 901
Evaluation of structural and evolutionary contributions to deleterious mutation prediction; Saunders CT et al.; Methods for automated prediction of deleterious protein mutations have utilized both structural and evolutionary information but the relative contribution of these two factors remains unclear . To address this, we have used a variety of structural and evolutionary features to create simple deleterious mutation models that have been tested on both experimental mutagenesis and human allele data . We find that the most accurate predictions are obtained using a solvent-accessibility term, the C(beta) density, and a score derived from homologous sequences, SIFT . A classification tree using these two features has a cross-validated prediction error of 20.5% on an experimental mutagenesis test set when the prior probability for deleterious and neutral cases is equal, whereas this prediction error is 28.8% and 22.2% using either the C(beta) density or SIFT alone . The improvement imparted by structure increases when fewer homologs are available: when restricted to three homologs the prediction error improves from 26.9% using SIFT alone to 22.4% using SIFT and the C(beta) density, or 24.8% using SIFT and a noisy C(beta) density term approximating the inaccuracy of ab initio structures modeled by the Rosetta method . We conclude that methods for deleterious mutation prediction should include structural information when fewer than five to ten homologs are available, and that ab initio predicted structures may soon be useful in such cases when high-resolution structures are unavailable.

J Mol Biol, 2002 Sep 27, 322(4), 851 - 69
Ion permeation and selectivity of OmpF porin: a theoretical study based on molecular dynamics, Brownian dynamics, and continuum electrodiffusion theory; Im W et al.; Three different theoretical approaches are used and compared to refine our understanding of ion permeation through the channel formed by OmpF porin from Escherichia coli . Those approaches are all-atom molecular dynamics (MD) in which ions, solvent, and lipids are represented explicitly, Brownian dynamics (BD) in which ions are represented explicitly, while solvent and lipids are represented as featureless dielectrics, and Poisson-Nernst-Planck (PNP) electrodiffusion theory in which both solvent and local ion concentrations are represented as a continuum . First, the ability of the different theoretical approaches in reproducing the equilibrium average ion density distribution in OmpF porin bathed by a 1M KCl symmetric salt solution is examined . Under those conditions the PNP theory is equivalent to the non-linear Poisson-Boltzmann (PB) theory . Analysis shows that all the three approaches are able to capture the important electrostatic interactions between ions and the charge distribution of the channel that govern ion permeation and selectivity in OmpF . The K(+) and Cl(-) density distributions obtained from the three approaches are very consistent with one another, which suggests that a treatment on the basis of a rigid protein and continuum dielectric solvent is valid in the case of OmpF . Interestingly, both BD and continuum electrostatics reproduce the distinct left-handed twisted ion pathways for K(+) and Cl(-) extending over the length of the pore which were observed previously in MD . Equilibrium BD simulations in the grand canonical ensemble indicate that the channel is very attractive for cations, particularly at low salt concentration . On an average there is 1.55 K(+) inside the pore in 10mM KCl . Remarkably, there is still 0.17 K(+) on average inside the pore even at a concentration as low as 1microM KCl . Secondly, non-equilibrium ion flow through OmpF is calculated using BD and PNP and compared with experimental data . The channel conductance in 0.2M and 1M KCl calculated using BD is in excellent accord with the experimental data . The calculations reproduce the experimentally well-known conductance-concentration relation and also reveal an asymmetry in the channel conductance (a larger conductance is observed under a positive transmembrane potential) . Calculations of the channel conductance for three mutants (R168A, R132A, and K16A) in 1M KCl suggest that the asymmetry in the channel conductance arises mostly from the permanent charge distribution of the channel rather than the shape of the pore itself . Lastly, the calculated reversal potential in a tenfold salt gradient (0.1:1M KCl) is 27.4(+/-1.3)mV (BD) and 22.1(+/-0.6)mV (PNP), in excellent accord with the experimental value of 24.3mV . Although most of the results from PNP are qualitatively reasonable, the calculated channel conductance is about 50% higher than that calculated from BD probably because of a lack of some dynamical ion-ion correlations.

J Mol Biol, 2002 Sep 27, 322(4), 827 - 40
Chaperone-independent folding of type 1 pilus domains; Vetsch M et al.; An elementary step in the assembly of adhesive type 1 pili of Escherichia coli is the folding of structural pilus subunits in the periplasm . The previously determined X-ray structure of the complex between the type 1 pilus adhesin FimH and the periplasmic pilus assembly chaperone FimC has shown that FimH consists of a N-terminal lectin domain and a C-terminal pilin domain, and that FimC exclusively interacts with the pilin domain . The pilin domain fold, which is common to all pilus subunits, is characterized by an incomplete beta-sheet that is completed by a donor strand from FimC in the FimC-FimH complex . This, together with unsuccessful attempts to refold isolated, urea-denatured FimH in vitro had suggested that folding of pilin domains strictly depends on sequence information provided by FimC . We have now analyzed in detail the folding of FimH and its two isolated domains in vitro . We find that not only the lectin domain, but also the pilin domain can fold autonomously and independently of FimC . However, the thermodynamic stability of the pilin domain is very low (8-10kJmol(-1)) so that a significant fraction of the domain is unfolded even in the absence of denaturant . This explains the high tendency of structural pilus subunits to aggregate non-specifically in the absence of stoichiometric amounts of FimC . Thus, pilus chaperones prevent non-specific aggregation of pilus subunits by native state stabilization after subunit folding.

J Mol Biol, 2002 Sep 27, 322(4), 687 - 96
Structure of the ecto-ADP-ribosyl transferase ART2.2 from rat; Mueller-Dieckmann C et al.; The mammalian extracellular ADP-ribosyl transferases ART1 through ART5 are sequence-related to each other . Among them ART2 is involved in immuno regulation . The variant ART2.2 was expressed in the periplasm of Escherichia coli and crystallized . Its structure was determined by X-ray diffraction at 1.7A resolution in one crystal form and at slightly lower resolutions in two others . The active center was indicated by a ligated nicotinamide analogue, which also revealed a small induced-fit . The centerpiece of the chainfold of ART2.2 agrees with those of all bacterial ADP-ribosyl transferases . This correspondence and the nicotinamide position were used to model the binding structure of the whole substrate NAD(+) at ART2.2 . Two of the bacterial enzymes are structurally more closely related to ART2.2 while the others are more closely related to the eukaryotic poly(ADP-ribosyl)polymerase . This splits the ADP-ribosyl transferases into two distinct subfamilies . A special feature of ART2.2 is its long N-terminal extension and two disulfide bridges that are far away from the active center . They stabilize the protein against denaturation and presumably also against shearing forces parallel with the membrane where ART2.2 is anchored.

Mol Biochem Parasitol, 2002 Aug 28, 123(2), 115 - 23
Analysis of stage-specific gene expression in the bloodstream and the procyclic form of Trypanosoma brucei using a genomic DNA-microarray; Diehl S et al.; A microarray comprising 21,024 different PCR products spotted on glass slides was constructed for gene expression studies on Trypanosoma brucei . The arrayed fragments were generated from a T . brucei shotgun clone library, which had been prepared from randomly sheared and size-fractionated genomic DNA . For the identification of stage-specific gene activity, total RNA from in vitro cultures of the human, long slender form and the insect, procyclic form of the parasite was labelled and hybridised to the microarray . Approximately 75% of the genomic fragments produced a signal and about 2% exhibited significant differences between the transcript levels in the bloodstream and procyclic forms . A few results were confirmed by Northern blot analysis or reverse-transcription and PCR . Three hundred differentially regulated clones have been selected for sequencing . So far, of 33 clones that showed about 2-fold or more over-expression in bloodstream forms, 15 contained sequences similar to those of VSG expression sites and at least six others appeared non-protein-coding . Of 29 procyclic-specific clones, at least eight appeared not to be protein-coding . A surprisingly large proportion of known regulated genes was already identified in this small sample, and some new ones were found, illustrating the utility of genomic arrays .

Immunol Lett, 2002 Nov 1, 84(2), 153 - 7
Recombinant multi-epitope vaccine induce predefined epitope-specific antibodies against HIV-1; Li H et al.; Monoclonal antibody 2F5 recognizing ELDKWA-epitope on HIV-1 gp41 has significant neutralization potency against 90% of the investigated viruses of African, Asia, American and European strains, but antibodies responses to ELDKWA-epitope in HIV-1 infected individuals were very low . Based on the epitope-vaccine strategy suggested by us, a recombinant glutathione S-transferase (GST) fusion protein (GST-MELDKWAGELDKWAGELDKWAVDIGPGRAFYGPGRAFYGPGRAFY) as vaccine antigen containing three repeats of neutralizing epitope ELDKWA on gp41 and GPGRAFY on gp120 was designed and expressed in Escherichia coli . After vaccination course, the recombinant multi-epitope vaccine could induce high levels of predefined multi-epitope-specific antibodies in mice . These antibodies in sera could bind to both neutralizing epitopes on gp41 peptide, V3 loop peptide and recombinant soluble gp41 (aa539-684) in ELISA assay (antisera dilution: 1:1,600-25,600), while normal sera did not . Moreover, these antibodies in sera could recognize the CHO-WT cells which expressed HIV-1 envelope glycoprotein on the cell surfaces, indicating that the predefined epitope-specific antibodies could recognize natural envelope protein of HIV-1 though these antibodies were induced by recombinant multi-epitope-vaccine . These experimental results suggested a possible way to develop recombinant multi-epitope vaccine inducing multi-antiviral activities against HIV-1.

Mol Cell Probes, 2002 Aug, 16(4), 277 - 83
Chimeric RNA-DNA molecular beacon assay for ribonuclease H activity; Rizzo J et al.; Current methods to detect and assay ribonuclease H (RNase H) activity are indirect and time-consuming . Here we introduce a direct and sensitive method, based on the fluorescence quenching mechanism of molecular beacons, to assay RNA cleavage in RNA:DNA hybrids . An RNA-DNA chimeric beacon assay for RNase H enzymatic activity was developed . The substrate is a single-stranded RNA-DNA chimeric oligonucleotide labeled with a 5'-fluorescein and a 3'-DABCYL . The fluorophore (fluorescein) of the probe is held in close proximity to the quencher (DABCYL) by the RNA:DNA stem-loop structure . When the RNA sequence of the RNA:DNA hybrid stem is cleaved, the fluorophore is separated from the quencher and fluorescence can be detected as a function of time . Chimeric beacons with different stem lengths and sequences have been surveyed for this assay with E . coli RNase H . We found that the beacon kinetic parameters are in qualitative agreement with previously reported values using more cumbersome assays . This method permits real-time detection of RNase H activity and a convenient approach to RNase H kinetic and mechanistic study .

Biochem Biophys Res Commun, 2002 Sep 27, 297(3), 653 - 8
Effect of Mg(2+) on the kinetics of guanine nucleotide binding and hydrolysis by Cdc42; Zhao J et al.; The biological activities of Rho family GTPases are controlled by their guanine nucleotide binding states in cell . Mg(2+) ions play key roles in guanine nucleotide binding and in preserving the structural integrity of GTPases . We describe here the kinetics of the interaction of GTP with the Rho family small GTPase Cdc42 in the absence and presence of Mg(2+) . In contrast to the cases of Ras and Rab proteins, which require Mg(2+) for the nucleotide binding and intrinsic hydrolysis of GTP, our results show that in the absence of Mg(2+), the binding affinity of GTP to Cdc42 is in the submicromolar concentration, and the Mg(2+) cofactor has only a minor effect on the Cdc42-catalyzed intrinsic hydrolysis rate of GTP . These results suggest that the intrinsic GTPase reaction mechanism of Cdc42 may differ significantly from that of other subfamily members of the Ras superfamily.

Biochem Biophys Res Commun, 2002 Sep 27, 297(3), 463 - 7
PNUTS (phosphatase nuclear targeting subunit) inhibits retinoblastoma-directed PP1 activity; Udho E et al.; Protein phosphatase type 1 catalytic subunit (PP1c) is a serine/threonine phosphatase involved in the dephosphorylation of many proteins in eukaryotic cells . It associates with several known targeting or regulatory subunits that directly regulate PP1c activity toward specific substrates . The recently identified Phosphatase Nuclear Targeting Subunit (PNUTS) binds to PP1c and inhibits PP1 activity toward phosphorylase a . One of the substrates of PP1c has been shown to be the cell cycle regulatory protein, Retinoblastoma (pRb) . In this study, we show that PNUTS dissociates from PP1c under mildly hypoxic cell growth conditions that lead to an increase of PP1c activity toward pRb . We developed an assay that measures pRb-directed PP1c activity and show that a GST-PNUTS fusion protein inhibits phosphatase activity toward pRb when using PP1c from cell lysates, GST-PP1c, or purified PP1c . These studies suggest that PNUTS is involved in the regulation of PP1c activity toward pRb.

Biochemistry, 2002 Oct 1, 41(39), 11804 - 11
A large Ca2+-dependent channel formed by recombinant ADP/ATP carrier from Neurospora crassa resembles the mitochondrial permeability transition pore; Brustovetsky N et al.; Strong support for the central role of the ADP/ATP carrier (AAC) in the mitochondrial permeability transition (mPT) is provided by the single-channel current measurements in patch-clamp experiments with the isolated reconstituted AAC . In previous work {Brustovetsky, N., and Klingenberg, M . (1996) Biochemistry 35, 8483-8488}, this technique was applied to the AAC isolated from bovine heart mitochondria . Here we used recombinant AAC (rAAC) from Neurospora crassa expressed in E . coli, since AAC from mammalian sources cannot be expresssed in E . coli . The rAAC is free from residual mitochondrial components which might associate with the AAC in preparation from bovine heart . Ca(2+)-dependent channels with up to 600 pS are obtained, which are gated at >150 mV . The channel corresponds to a preferential matrix-outside orientation of rAAC in the patch membrane as shown with carboxyatractylate and a polar gating asymmetry . The channel is inhibited by ADP and bongkrekate, not by carboxyatractylate . Cyclophilin, isolated from Neurospora crassa, suppresses the gating, thus increasing conductivity at high positive voltage . Cyclosporin A abolishes the cyclophilin effect . ADP does not eliminate the cyclophilin effect but produces fast large-amplitude flickering of the channel without a stable decrease of the channel conductance . Also the pro-oxidant tert-butyl hydroperoxide reversibly suppresses voltage gating of the channel . The results show that the AAC can be a conducting component of the mPT pore, exhibiting similar characteristics as the mPT pore (response to Ca(2+), BKA, ADP), with a cyclophilin and pro-oxidant-sensitive gating at high voltage.

Biochemistry, 2002 Oct 1, 41(39), 11649 - 57
Multistate binding in pyridoxine 5'-phosphate synthase: 1.96 A crystal structure in complex with 1-deoxy-D-xylulose phosphate; Yeh JI et al.; We report the 1.96 A crystal structure of pyridoxine 5'-phosphate synthase (PdxJ) in complex with 1-deoxy-D-xylulose phosphate (dXP) . The octameric enzyme possesses eight distinct binding sites, and three different binding states are observed . The observation of these three states supports a mechanism in which precise conformational changes of a peptide loop and groups of active site residues modulate binding and specificity . The differences in protein conformation when one or two substrates are bound can be correlated with a condensation mechanism that leads productively to the formation of pyridoxine 5'-phosphate (PNP) . "Snapshots" of the progression from the apo form to a singly occupied "transitional binding" state and, subsequently, to a fully occupied, reactive state are revealed and indicate how the enzyme structure can be related to a plausible catalytic mechanism and, moreover, to favorable energetics of reaction.

Biochemistry, 2002 Oct 1, 41(39), 11611 - 27
Kinetic mechanism of direct transfer of Escherichia coli SSB tetramers between single-stranded DNA molecules; Kozlov AG et al.; The kinetic mechanism of transfer of the homotetrameric Escherichia coli SSB protein between ssDNA molecules was studied using stopped-flow experiments . Dissociation of SSB from the donor ssDNA was monitored after addition of a large excess of unlabeled acceptor ssDNA by using either SSB tryptophan fluorescence or the fluorescence of a ssDNA labeled with an extrinsic fluorophore {fluorescein (F) or Cy3} . The dominant pathway for SSB dissociation occurs by a "direct transfer" mechanism in which an intermediate composed of two DNA molecules bound to one SSB tetramer forms transiently prior to the release of the acceptor DNA . When an initial 1:1 SSB-ssDNA complex is formed with (dT)(70) in the fully wrapped (SSB)(65) mode so that all four SSB subunits are bound to (dT)(70), the formation of the ternary intermediate complex occurs slowly with an apparent bimolecular rate constant, k(2,app), ranging from 1.2 x 10(3) M(-1) s(-1) (0.2 M NaCl) to approximately 5.1 x 10(3) M(-1) s(-1) (0.4 M NaBr), and this rate limits the overall rate of the transfer reaction (pH 8.1, 25 degrees C) . These rate constants are approximately 7 x 10(5)- and approximately 7 x 10(4)-fold lower, respectively, than those measured for binding of the same ssDNA to an unligated SSB tetramer to form a singly ligated complex . However, when an initial SSB-ssDNA complex is formed with (dT)(35) so that only two SSB subunits interact with the DNA in an (SSB)(35) complex, the formation of the ternary intermediate occurs much faster with a k(2,app) ranging from >6.3 x 10(7) M(-1) s(-1) (0.2 M NaCl) to 2.6 x 10(7) M(-1) s(-1) (0.4 M NaBr) . For these experiments, the rate of dissociation of the donor ssDNA determines the overall rate of the transfer reaction . Hence, an SSB tetramer can be transferred from one ssDNA molecule to another without proceeding through a free protein intermediate, and the rate of transfer is determined by the availability of free DNA binding sites within the initial SSB-ssDNA donor complex . Such a mechanism may be used to recycle SSB tetramers between old and newly formed ssDNA regions during lagging strand DNA replication.

Biochemistry, 2002 Oct 1, 41(39), 11592 - 601
Crystal structures of human mitochondrial branched chain aminotransferase reaction intermediates: ketimine and pyridoxamine phosphate forms; Yennawar NH et al.; The three-dimensional structures of the isoleucine ketimine and the pyridoxamine phosphate forms of human mitochondrial branched chain aminotransferase (hBCATm) have been determined crystallographically at 1.9 A resolution . The hBCATm-catalyzed transamination can be described in molecular terms together with the earlier solved pyridoxal phosphate forms of the enzyme . The active site lysine, Lys202, undergoes large conformational changes, and the pyridine ring of the cofactor tilts by about 18 degrees during catalysis . A major determinant of the enzyme's substrate and stereospecificity for L-branched chain amino acids is a group of hydrophobic residues that form three hydrophobic surfaces and lock the side chain in place . Short-chain aliphatic amino acid side chains are unable to interact through van der Waals contacts with any of the surfaces whereas bulky aromatic side chains would result in significant steric hindrance . As shown by modeling, and in agreement with previous biochemical data, glutamate but not aspartate can form hydrogen bond interactions . The carboxylate group of the bound isoleucine is on the same side as the phosphate group of the cofactor . These active site interactions are largely retained in a model of the human cytosolic branched chain aminotransferase (hBCATc), suggesting that residues in the second tier of interactions are likely to determine the specificity of hBCATc for the drug gabapentin . Finally, the structures reveal a unique role for cysteine residues in the mammalian BCAT . Cys315 and Cys318, which immediately follow a beta-turn (residues 311-314) and are located just outside the active site, form an unusual thiol-thiolate hydrogen bond . This beta-turn positions Thr313 for its interaction with the pyridoxal phosphate oxygens and substrate alpha-carboxylate group.

Biochemistry, 2002 Oct 1, 41(39), 11552 - 65
Mechanism of sulfide-quinone reductase investigated using site-directed mutagenesis and sulfur analysis; Griesbeck C et al.; Biological sulfide oxidation is a reaction occurring in all three domains of life . One enzyme responsible for this reaction in many bacteria has been identified as sulfide:quinone oxidoreductase (SQR) . The enzyme from Rhodobacter capsulatus is a peripherally membrane-bound flavoprotein with a molecular mass of approximately 48 kDa, presumably acting as a homodimer . In this work, SQR from Rb . capsulatus has been modified with an N-terminal His tag and heterologously expressed in and purified from Escherichia coli . Three cysteine residues have been shown to be essential for the reductive half-reaction by site-directed mutagenesis . The catalytic activity has been nearly completely abolished after mutation of each of the cysteines to serine . A decrease in fluorescence on reduction by sulfide as observed for the wild-type enzyme has not been observed for any of the mutated enzymes . Mutation of a conserved valine residue to aspartate within the third flavin-binding domain led to a drastically reduced substrate affinity, for both sulfide and quinone . Two conserved histidine residues have been mutated individually to alanine . Both of the resulting enzymes exhibited a shift in the pH dependence of the SQR reaction . Polysulfide has been identified as a primary reaction product using spectroscopic and chromatographic methods . On the basis of these data, reaction mechanisms for sulfide-dependent reduction and quinone-dependent oxidation of the enzyme and for the formation of polysulfide are proposed.

Biochemistry, 2002 Oct 1, 41(39), 11543 - 51
Structure and dynamics of the beta-barrel of the membrane transporter BtuB by site-directed spin labeling; Fanucci GE et al.; Site-directed spin labeling and EPR spectroscopy were used to map two consecutive beta-strands of the putative transmembrane beta-barrel of BtuB . For these studies, a series of 29 consecutive single cysteine mutants of BtuB were produced covering residues 148-176 . The proteins were then expressed, reacted with a sulfhydryl-specific spin label, purified in octyl glucoside (OG), and reconstituted into palmitoyloleoylphosphatidylcholine (POPC) bilayers . The labeled residues spanned from the extracellular region (position 148) to the small periplasmic loop (positions 160-163) and back up to the extracellular side (position 176) of BtuB . Continuous wave power saturation in the presence of oxygen or NiAA yielded an i, i + 2 periodicity for the collision frequencies at these sites and demonstrated the presence of a beta-strand structural motif . For both strands studied, the even-numbered residues were found to be exposed to the hydrophobic phase of the bilayer, whereas the odd-numbered residues pointed toward the interior of the barrel and the core of the protein . In addition, the collision parameters yielded the position of the protein within the bilayer . The phase relationship between the oxygen and metal collision frequencies along with the corresponding membrane depth parameters, Phi, indicates that segments 151-159 and 164-172 are within the bilayer . In POPC bilayers, there is a mobility gradient for spin labels along the barrel indicating enhanced backbone flexibility toward the periplasmic surface of the barrel . In POPC/OG mixed micelles, the even-numbered residues facing the hydrocarbon show an increased mobility compared with the bilayer environment whereas the inward-facing side chains show little change in motion . The data indicate that the protein core remains folded in POPC/OG mixed micelles but that this environment increases the backbone fluctuations of the strands . A model for the beta-barrel of BtuB is presented in part on the basis of these EPR data.

Biochemistry, 2002 Oct 1, 41(39), 11525 - 31
Weak protein-protein interactions are sufficient to drive assembly of hepatitis B virus capsids; Ceres P et al.; Hepatitis B virus (HBV) is an enveloped DNA virus with a spherical capsid (or core) . The capsid is constructed from 120 copies of the homodimeric capsid protein arranged with T = 4 icosahedral symmetry . We examined in vitro assembly of purified E . coli expressed HBV capsid protein . After equilibration, concentrations of capsid and dimer were evaluated by size exclusion chromatography . The extent of assembly increased as temperature and ionic strength increased . The concentration dependence of capsid assembly conformed to the equilibrium expression: K(capsid) = {capsid}/{dimer}(120) . Given the known geometry for HBV capsids and dimers, the per capsid assembly energy was partitioned into energy per subunit-subunit contact . We were able to make three major conclusions . (i) Weak interactions (from -2.9 kcal/mol at 21 degrees C in low salt to -4.4 kcal/mol at 37 degrees C in high salt) at each intersubunit contact result in a globally stable capsid; weak intersubunit interactions may be the basis for the phenomenon of capsid breathing . (ii) HBV assembly is characterized by positive enthalpy and entropy . The reaction is entropy-driven, consistent with the largely hydrophobic contacts found in the crystal structure . (iii) Increasing NaCl concentration increases the magnitude of free energy, enthalpy, and entropy, as if ionic strength were increasing the amount of hydrophobic surface buried by assembly . This last point leads us to suggest that salt acts by inducing a conformational change in the dimer from an assembly-inactive form to an assembly-active form . This model of conformational change linked to assembly is consistent with immunological differences between dimer and capsid.

J Obstet Gynaecol India, 1971 Dec, 21(6), 655 - 60
Septic abortion; Baxi LV et al.; PIP: 53 of 3100 abortions at Bombay hospital were septic abortions, giving an incidence of 1.7% . Various factors of possible etiological significance were analyzed, including age, parity, marital status, duration of gestation, and the mode of interference leading to sepsis . 36 of 53 patients were aged 20-30 years, but other age groups were represented . In the present study, gravidity was not relevant, for all gravidity groups, from primipara to grand multipara 5 and above, had patients suffering septic abortions . 9 patients were married and gave a history of interference; in all, 38 patients were married, 22 were unmarried, and 4 were widows . 23 patients gave a definite mode of interference, and the most common method was interference with a stick . 43% mortality occurred in patients giving a history of interference, and 36% mortality occurred in others . Vaginal and cervical cultures revealed (16 cases studied) 5 cases of CL . tetani, 1 case of E . coli, and 10 patients showing strepto-, staphylo-, pneumococcal infections . In this series, 21 of 53 patients died: 8 of tetanus, 3 or renal failure, 4 of septicemia, 2 of hemorrhagic diathesis, and 3 of endotoxic shock . 1 patient had acute bacterial endocarditis and pulmonary embolism at sutopsy . It is this article's contention that the main cause of sepsis is using an instrument to induce abortion during an unwanted pregnancy; hence, a plea is made for more liberalized abortion legislation .

Mikrobiologiia, 2002 Jul-Aug, 71(4), 452 - 4
{Effect of red and infrared radiation on the growth of Escherichia coli cells and production of recombinant barstar protein}; Trushin MV; Incoherent red and infrared low-intensity light enhanced the growth of the auxotrophic strain Escherichia coli AD494(DE3)pLysS and the production of the recombinant polypeptide barstar . Illumination also stimulated the growth of nonrecombinant E . coli cells.

Semin Thromb Hemost, 2002 Aug, 28(4), 335 - 42
Chemical derivatization as a strategy to study structure-activity relationships of glycosaminoglycans; Casu B et al.; Sulfated glycosaminoglycans (GAGs) are amenable to a number of chemical modifications that modulate their biological activity . N-sulfate groups can be exposed and N-acylated (usually N-acetylated), specific O-sulfate groups can be removed, and free hydroxyl groups (either preexisting in the original GAG or exposed by desulfation) can be sulfated . Heparin/heparan sulfate, chondroitin sulfate, and dermatan sulfate have been variously desulfated or sulfated to afford novel GAGs with protein binding and associated biological properties different from those of the original GAGs . Regiospecific sulfation of N-acetyl heparosan ( E . coli K5 polysaccharide) afforded a number of derivatives, some endowed with antithrombotic activity and others with antimetastatic properties . Most of the activities could be correlated with typical sulfation patterns along each GAG backbone . Glycol splitting of nonsulfated glucuronic residues (including a critical residue in the pentasaccharide sequence of the active site for antithrombin) leads to substantial loss of anticoagulant activity of heparin . Partial removal of sulfate groups at position 2 of iduronic acid residues followed by glycol splitting of all nonsulfated uronic acid residues afforded nonanticoagulant, antiangiogenic heparins.

Planta, 2002 Sep, 215(5), 870 - 9 Epub 2002 Jun 14.
Cloning and characterization of Arabidopsis thaliana pyridoxal kinase; Lum HK et al.; Pyridoxal kinase (PK; EC 2.7.1.35), a key enzyme in vitamin B(6) metabolism, was cloned from Arabidopsis thaliana (L.) Heynh . and characterized . The amino acid sequence of the A . thaliana PK was found to be similar to the mammalian enzyme, with a homology of more than 40% . Characterization studies showed that the kinase is a dimeric molecule consisting of two identical subunits, each subunit having a molecular mass of approximately 35 kDa . The enzyme exhibited maximal activity at pH 6.0 . Similar to the mammalian enzyme, the enzyme from A . thaliana preferred Zn(2+) instead of the commonly used Mg(2+) as the divalent cation for catalysis . Under optimal conditions, the V(max) of the enzyme was 604 nmol pyridoxal 5'-phosphate (PLP) mg(-1) min(-1), and the K(m) values for pyridoxal and ATP were 688 micro M and 98 micro M, respectively . Examination of levels of enzyme expression showed that leaves, stems, roots and flowers can generate PLP independently at similar levels . Furthermore, expression of the PK gene in A . thaliana seeds was found to start 60 h after imbibition . Results from the present study suggest that plant tissues depend on PK for the production of PLP.

Planta, 2002 Sep, 215(5), 847 - 54 Epub 2002 Jun 20.
Beta-ketoacyl-acyl carrier protein synthase IV: a key enzyme for regulation of medium-chain fatty acid synthesis in Cuphea lanceolata seeds; Schutt BS et al.; With the aim of elucidating the mechanisms involved in the biosynthesis of medium-chain fatty acids in Cuphea lanceolata Ait., a crop accumulating up to 90% decanoic acid in seed triacylglycerols, cDNA clones of a beta-ketoacyl-acyl carrier protein (ACP) synthase IV (clKAS IV, EC 2.3.1.41) were isolated from C . lanceolata seed embryos . The amino acid sequence deduced from clKAS IV cDNA showed 80% identity to other plant KAS II-type enzymes, 55% identity towards plant KAS I and over 90% towards other Cuphea KAS IV-type sequences . Recombinant clKAS IV was functionally overexpressed in Escherichia coli, and substrate specificity of purified enzyme showed strong preference for elongation of short-chain and medium-chain acyl-ACPs (C4- to C10-ACP) with nearly equal activity . Further elongation steps were catalysed with distinctly less activity . Moreover, short- and medium-chain acyl-ACPs exerted a chain-length-specific and concentration-dependent substrate inhibition of clKAS IV . Based on these findings a regulatory mechanism for medium-chain fatty acid synthesis in C . lanceolata is presented.

Planta, 2002 Sep, 215(5), 735 - 44 Epub 2002 Jul 03.
Molecular cloning and cytochemical analysis of exopolygalacturonase from carrot; Tanaka R et al.; Exopolygalacturonase (exo-PGase, EC 3.2.1.67) attacks the non-reducing terminus of the polygalacturonic acid in pectic molecules, releasing galacturonic acid . We cloned the cDNA of exo-PGase purified from cell homogenates of suspension-cultured carrot ( Daucus carota L . cv . Kintoki) cells . The nucleotide sequence of the cDNA (1.4 kb) contains an open reading frame that encodes a 391-amino-acid polypeptide . Sequence homology research showed 97.9% identity to the glycoprotein EP4 obtained from cultured carrot cells and 49.3% identity to the ENOD8 gene product of alfalfa ( Medicago sativa) . However, no significant similarity was found to known PGases . The Southern hybridization pattern indicated that this exo-PGase protein is a member of a small-sized gene family . Predominant expression of the exo-PGase gene was detected by in situ hybridization and immunohistochemistry in the root apical meristem and in the elongation region, but not in the root cap . A cross-immunoresponse with anti-exo-PGase also occurred in the root nodule meristem of alfalfa . These results suggest that this exo-PGase plays a role in the degradation of pectic molecules during root development.

Nat Biotechnol, 2002 Oct, 20(10), 1044 - 8 Epub 2002 Sep 16.
An efficient system for the evolution of aminoacyl-tRNA synthetase specificity; Santoro SW et al.; A variety of strategies to incorporate unnatural amino acids into proteins have been pursued, but all have limitations with respect to technical accessibility, scalability, applicability to in vivo studies, or site specificity of amino acid incorporation . The ability to selectively introduce unnatural functional groups into specific sites within proteins, in vivo, provides a potentially powerful approach to the study of protein function and to large-scale production of novel proteins . Here we describe a combined genetic selection and screen that allows the rapid evolution of aminoacyl-tRNA synthetase substrate specificity . Our strategy involves the use of an "orthogonal" aminoacyl-tRNA synthetase and tRNA pair that cannot interact with any of the endogenous synthetase-tRNA pairs in Escherichia coli . A chloramphenicol-resistance (Cm(r)) reporter is used to select highly active synthetase variants, and an amplifiable fluorescence reporter is used together with fluorescence-activated cell sorting (FACS) to screen for variants with the desired change in amino acid specificity . Both reporters are contained within a single genetic construct, eliminating the need for plasmid shuttling and allowing the evolution to be completed in a matter of days . Following evolution, the amplifiable fluorescence reporter allows visual and fluorimetric evaluation of synthetase activity and selectivity . Using this system to explore the evolvability of an amino acid binding pocket of a tyrosyl-tRNA synthetase, we identified three new variants that allow the selective incorporation of amino-, isopropyl-, and allyl-containing tyrosine analogs into a desired protein . The new enzymes can be used to produce milligram-per-liter quantities of unnatural amino acid-containing protein in E . coli.

Nat Struct Biol, 2002 Oct, 9(10), 740 - 4
Induced structural changes of 7SL RNA during the assembly of human signal recognition particle; Kuglstatter A et al.; The eukaryotic signal recognition particle (SRP) is a cytoplasmic ribonucleoprotein particle that targets secretory and membrane proteins to the endoplasmic reticulum . The binding of SRP54 to the S domain of 7SL RNA is highly dependent on SRP19 . Here we present the crystal structure of a human SRP ternary complex consisting of SRP19, the M domain of SRP54 and the S domain of 7SL RNA . Upon binding of the M domain of SRP54 to the 7SL RNA-SRP19 complex, the asymmetric loop of helix 8 in 7SL RNA collapses . The bases of the four nucleotides in the long strand of the asymmetric loop continuously stack and interact with the M domain, whereas the two adenines in the short strand flip out and form two A-minor motifs with helix 6 . This stabilizing interaction is only possible when helix 6 has been positioned parallel to helix 8 by the prior binding of SRP19 to the tetraloops of helices 6 and 8 . Hence, the crystal structure of the ternary complex suggests why SRP19 is necessary for the stable binding of SRP54 to the S domain RNA.

J Cell Sci, 2002 Oct 15, 115(Pt 20), 3923 - 34
Nowa, a novel protein with minicollagen Cys-rich domains, is involved in nematocyst formation in Hydra; Engel U et al.; The novel protein Nowa was identified in nematocysts, explosive organelles of Hydra, jellyfish, corals and other CNIDARIA: Biogenesis of these organelles is complex and involves assembly of proteins inside a post-Golgi vesicle to form a double-layered capsule with a long tubule . Nowa is the major component of the outer wall, which is formed very early in morphogenesis . The high molecular weight glycoprotein has a modular structure with an N-terminal sperm coating glycoprotein domain, a central C-type lectin-like domain, and an eightfold repeated cysteine-rich domain at the C-terminus . Interestingly, the cysteine-rich domains are homologous to the cysteine-rich domains of minicollagens . We have previously shown that the cysteines of these minicollagen cysteine-rich domains undergo an isomerization process from intra- to intermolecular disulfide bonds, which mediates the crosslinking of minicollagens to networks in the inner wall of the capsule . The minicollagen cysteine-rich domains present in both proteins provide a potential link between Nowa in the outer wall and minicollagens in the inner wall . We propose a model for nematocyst formation that integrates cytoskeleton rearrangements around the post-Golgi vesicle and protein assembly inside the vesicle to generate a complex structure that is stabilized by intermolecular disulfide bonds.

J Biol Chem, 2002 Nov 22, 277(47), 45466 - 72 Epub 2002 Sep 19.
Unmasking a functional allosteric domain in an allosterically nonresponsive carbamoyl-phosphate synthetase; Eroglu B et al.; Although carbamoyl-phosphate synthetases (CPSs) share sequence identity, multidomain structure, and reaction mechanism, they have varying physiological roles and allosteric effectors . Escherichia coli CPS (eCPS) provides CP for both arginine and pyrimidine nucleotide biosynthesis and is allosterically regulated by metabolites from both pathways, with inhibition by UMP and activation by IMP and ornithine . The arginine-specific CPS from Saccharomyces cerevisiae (sCPS), however, apparently responds to no allosteric effectors . We have designed and analyzed a chimeric CPS (chCPS, in which the C-terminal 136 residues of eCPS were replaced by the corresponding residues of sCPS) to define the structural basis for the allosteric nonresponsiveness of sCPS and thereby provide insight into the mechanism for allosteric selectivity and responsiveness in the other CPSs . Surprisingly, ornithine and UMP each had a significant effect on chCPS activity, and did so at concentrations that were similar to those effective for eCPS . We further found that sCPS bound both UMP and IMP and that chCPS bound IMP, although none of these interactions led to changes in enzymatic activity . These findings strongly suggest that the nonresponsive sCPS is not able to communicate occupancy of the allosteric site to the active site but does contain a latent allosteric interaction domain.

J Biol Chem, 2002 Nov 29, 277(48), 46763 - 8 Epub 2002 Sep 19.
Mapping the signal sequence-binding site on SRP reveals a significant role for the NG domain; Cleverley RM et al.; We present evidence that the signal recognition particle (SRP) recognizes signal sequences via the NG domain on the SRP54 protein subunit . Using a recently developed cross-linking method (Fancy, D . A., and Kodadek, T . (1999) Proc . Natl . Acad . Sci . U . S . A . 96, 6020-6024; Correction (1999) Proc . Natl . Acad . Sci . U . S . A . 96, 1317), we find that signal peptides cross-link to the Escherichia coli SRP protein Ffh (the homologue of the mammalian SRP54 subunit) via the NG domain . Within the NG domain, the cross-linking site maps to the ras-like C-terminal subdomain termed the G domain . This result stands in contrast to previous studies, which concluded based on nascent chain cross-linking that the signal sequence bound to the adjacent M domain . As independent evidence of a direct binding interaction between the NG domain and the signal sequence, we find that the NG domain of Ffh binds signal peptides as an isolated entity . Our results suggest that the NG domain forms a substantial part of the binding site for the signal sequence.

J Biol Chem, 2002 Nov 29, 277(48), 46456 - 62 Epub 2002 Sep 18.
Purification and properties of TrwB, a hexameric, ATP-binding integral membrane protein essential for R388 plasmid conjugation; Hormaeche I et al.; TrwB is an integral membrane protein linking the relaxosome to the DNA transport apparatus in plasmid R388 conjugation . Native TrwB has been purified in monomeric and hexameric forms, in the presence of dodecylmaltoside from overexpressing bacterial cells . A truncated protein (TrwBDeltaN70) that lacked the transmembrane domain could be purified only in the monomeric form . Electron microscopy images revealed the hexameric structure and were in fact superimposable to the previously published atomic structure for TrwBDeltaN70 . In addition, the electron micrographs showed an appendix, approximately 25 A wide, corresponding to the transmembrane region of TrwB . TrwB was located in the bacterial inner membrane in agreement with its proposed coupling role . Purified TrwB hexamers and monomers bound tightly the fluorescent ATP analogue TNP-ATP . A mutant in the Walker A motif, TrwB-K136T, was equally purified and found to bind TNP-ATP with a similar affinity to that of the wild type . However, the TNP-ATP affinity of TrwBDeltaN70 was significantly reduced in comparison with the TrwB hexamers . Competition experiments in which ATP was used to displace TNP-ATP gave an estimate of ATP binding by TrwB (K(d)((ATP)) = 0.48 mm for hexamers) . The transmembrane domain appears to be involved in TrwB protein hexamerization and also influences its nucleotide binding properties.

J Biol Chem, 2002 Dec 6, 277(49), 47844 - 53 Epub 2002 Sep 18.
HLA-B27 subtypes differentially associated with disease exhibit subtle structural alterations; Hulsmeyer M et al.; The reasons for the association of the human major histocompatibility complex protein HLA-B27 with spondyloarthropathies are unknown . To uncover the underlying molecular causes, we determined the crystal structures of the disease-associated B*2705 and the nonassociated B*2709 subtypes complexed with the same nonapeptide (GRFAAAIAK) . Both differ in only one residue (Asp(116) and His(116), respectively) in the F-pocket that accommodates the peptide C terminus . Several different effects of the Asp(116) --> His replacement are observed . The bulkier His(116) induces a movement of peptide C-terminal pLys(9), allowing the formation of a novel salt bridge to Asp(77), whereas the salt bridge between pLys(9) and Asp(116) is converted into a hydrogen bond with His(116) . His(116) but not Asp(116) adopts two alternative conformations, one of which leads to breakage of hydrogen bonds . Water molecules near residue 116 differ with regard to number, position, and contacts made . Furthermore, F-pocket atoms exhibit higher B-factors in B*2709 than in B*2705, indicating an increased flexibility of the entire region in the former subtype . These changes induce subtle peptide conformational alterations that may be responsible for the immunobiological differences between these HLA-B27 subtypes.

Vet Microbiol, 2002 Oct 22, 89(2-3), 195 - 9
Investigation of putative CDT gene in Escherichia coli isolates from pigs with diarrhea; da Silva AS et al.; In this study, 98 Escherichia coli isolates from 42 diarrheic neonatal piglets were screened for the presence of cytolethal distending toxin coding gene by polymerase chain reaction (PCR) . PCR yielded a single product which was specifically generated for E . coli cdt(+) control strain and not for other control strains . Twenty two (22.4%) of the isolates tested were cdtB positive, and 50% of the cdtB(+) isolates were also estII positive . The most prevalent pathotype was O32 cdtB(+) estII(+), which accounted for 59% of the cdtB positive strains . These results indicate an association between the presence of the cdtB gene and diarrhea, and support the need for further studies to determine the role of this toxin in diarrhea.

Avian Dis, 2002 Jul-Sep, 46(3), 749 - 53
Virulence factors of avian pathogenic Escherichia coli isolated from broilers from the south of Brazil; da Rocha AC et al.; Sixty-three Escherichia coli strains isolated from broilers with respiratory problems were examined for virulence factors, hemolysin synthesis ability, motility, hemagglutination capacity, operon pap presence, colicin production, and serum resistance . The capacity to hemagglutinate guinea pig erythrocytes was found in 53 (84.1%) of the samples, but only 30 (47.6%) agglutinated chicken erythrocytes . D-mannose-sensitive hemagglutination against guinea pig erythrocytes was found in 19 (30.2%) samples and against chicken erythrocytes, in 15 (23.8%) samples, whereas the D-mannose-resistant hemagglutination with guinea pig erythrocytes was found in 34 (54%) samples, and 13 of these (20.6%) showed this characteristic against chicken erythrocytes . Operon pap, P fimbria codifier, was detected in 26 samples in a total of 34 D-mannose-resistant samples . Colicin production was observed in 55 (87.3%) of the strains, and 41.8% presented V colicin production . Of the samples analyzed, 56 (88.9%) presented serum resistance, six (9.5%) were intermediate, and only one (1.6%) was sensitive to the action of the complement . The diversity of virulence profiles detected in the samples in this study explains in part the multifactorial characteristics of avian colibacillosis.

Avian Dis, 2002 Jul-Sep, 46(3), 721 - 4
Carriage of potentially pathogenic Escherichia coli in chickens; Kariuki S et al.; DNA-DNA hybridization, cultured cell lines, and transmission electron microscopy were used to study pathogenicity traits of 64 Escherichia coli isolated from apparently healthy chickens from 18 small-scale farms in Thika District, Kenya . A total of 39 (60.9%) isolates hybridized with the eae gene probe for enteropathogenic E . coli (EPEC) whereas another 16 (25%) hybridized with the lt and st gene probes and were categorized as enterotoxigenic E . coli . Electron microscopic examination of the eae probe-positive E . coli cultures with the HT-2919A cell line confirmed that they were able to attach intimately and produced effacement typical of EPEC . In addition, negative stain electron microscopy showed that the EPEC strains produced pili that have previously been associated with increased virulence of E . coli infections in chickens . This study has also demonstrated that apparently healthy chickens may carry enteropathogenic E . coli strains.

Avian Dis, 2002 Jul-Sep, 46(3), 668 - 78
Systemic and mucosal antibody responses to selected cell surface antigens of avian pathogenic Escherichia coli in experimentally infected chickens; Kariyawasam S et al.; The immune response to four cell surface antigens of avian pathogenic Escherichia coli (APEC) was investigated as the first step in identifying vaccine candidates . F1 pilus adhesin, P pilus adhesin, aerobactin receptor protein, and lipopolysaccharide (LPS) from an O78 E . coli (strain EC99) were used as antigens . The proteins were purified as 6xhistidine-tagged recombinant proteins and LPS was purified from a phenol/water extract . Groups of 12 broiler chickens were vaccinated intranasally with the EC99 strain and challenged with the same strain 10 days later via the intra-air sac route . The chickens that survived were euthanatized 10 days postchallenge . Scores were assigned to infected chickens on the basis of lesions and recovery of the challenge E . coli . The immunoglobulin (Ig) IgG, IgA, and IgM antibodies to the four antigens were measured in serum and air sac washings in an enzyme-linked immunosorbent assay . Among the chickens that were not vaccinated prior to challenge, two died and three of the survivors were ill, whereas, of the chickens that were vaccinated prior to challenge, one died and one of the survivors became ill . After the intranasal vaccination, high antibody activity against all four antigens was associated with each Ig isotype in serum and air sac washings . IgG was the predominant isotype of Ig in air sac washings as detected by radial immunodiffusion . Chickens that were not ill after challenge had greater IgG, IgA, and IgM antibody activity against all four antigens in serum and air sac washings than did sick chickens . Thus, all of the antigens tested appear to be suitable candidates for a vaccine to protect chickens from respiratory tract infections caused by APEC.

Avian Dis, 2002 Jul-Sep, 46(3), 660 - 7
interactions between escherichia coli and Newcastle disease virus in chickens; El Tayeb AB et al.; We investigated the interaction between Newcastle disease virus (NDV) and Escherichia coli in cell cultures, embryonated eggs, and 8-wk-old chickens . We measured the interactions on the basis of bacterial adherence and NDV hemagglutination titer in chickens, chicken embryos, and chicken embryo cell culture . Depending on the inoculation order of E . coli, a significant alteration of the growth of NDV was observed in both chickens and chicken embryos . When certain strains of E . coli were given before NDV exposure, the virus titers were lowered . In chickens, the mean virus titer was significantly (P < 0.05) lowered in the crop, the proventriculus, the gizzard, and the jejunum . However, there were no significant differences (P < 0.05) between the two groups for NDV titers in the duodenum, ileum, and cecum . In chicken embryos, when E . coli serotypes O78 and O119:B14 were inoculated before NDV exposure, the mean NDV titers were significantly (P < 0.5) lowered . However, there were no significant differences (P < 0.05) in NDV titer between the two groups when E . coli serotypes O78:K80:NM and O1ab:K NM were inoculated 24 hr before NDV exposure . When NDV was given prior to E . coli exposure, NDV titer was higher in both chickens and chicken embryos . In chickens, when NDV was given 48 hr before E . coli inoculation, NDV was detected in the proventriculus, gizzard, jejunum, ileum, and cecum, whereas no virus was detected in the control groups (NDV only) . In the crop, NDV was detected at a significantly (P < 0.05) higher titer in the E . coli-inoculated group when compared with the control group that received NDV alone . In chicken embryos, virus titer was significantly (P < 0.05) higher when NDV was given 24 hr before E . coli inoculation for all three NDV strains used (Ulster and V4 strains) . Adherence of E . coli to chicken embryo kidney (CEK) cells was significantly higher (P < 0.05) when the CEK cells were infected first with NDV and then by E . coli . The mean bacterial count per microscopic field in NDV-uninfected monolayers was eight compared with 112 for the NDV-infected monolayers . In approximately 10% of the fields in NDV-infected monolayers, the bacteria were too numerous to count.

Avian Dis, 2002 Jul-Sep, 46(3), 570 - 80
Expression of infectious laryngotracheitis virus glycoproteins in Escherichia coli and their application in enzyme-linked immunosorbent assay; Chang PC et al.; Three glycoproteins of infectious laryngotracheitis virus (ILTV), gC, gE, and gp60, were expressed in Escherichia coli as fusion proteins with a 6-histidine tag at their amino termini . The proteins expressed, designated as r-gC, r-gp60, and r-gE, all retain their antigenicity, as revealed by Western blot with chicken antiserum against ILTV . However, only r-gp60 and r-gE, but not r-gC, were found to be soluble . The soluble r-gp60 and r-gE were purified by a nickel column and then used as the enzyme-linked immunosorbent assay (ELISA) antigen for detecting ILTV-specific antibodies . The diagnostic potential of r-gE and r-gp60 ELISA was assessed with the use of sera prepared from vaccinated or unvaccinated chickens of either specific-pathogen-free (SPF) or field origins . The result shows that r-gp60 and r-gE ELISA could discriminate vaccinated SPF chickens from unvaccinated ones 2 wk postvaccination . Moreover, r-gp60 and r-gE ELISA could also discriminate vaccinated field flocks from unvaccinated ones . This result indicates that r-gp60 and r-gE might serve as an alternative ELISA antigen for detecting ILTV-specific antibodies . Moreover, r-gp60 or r-gE ELISA might play an important role in the eradication of infectious laryngotracheitis (ILT) in the future when the gp60- or gE-deleted marker vaccine of ILT is available.

Oncogene, 2002 Sep 26, 21(43), 6684 - 8
The HtrA1 serine protease is down-regulated during human melanoma progression and represses growth of metastatic melanoma cells; Baldi A et al.; Differential gene expression of cell lines derived from a malignant melanoma or its autologous lymph node metastasis using cDNA arrays indicated down-regulation of PRSS11, a gene encoding the serine protease HtrA1, a homolog of the Escherichia coli protease HtrA, in the metastatic line . Stable PRSS11 overexpression in the metastatic cell line strongly inhibited proliferation, chemoinvasion and Nm23-H1 protein expression in vitro, as well as cell growth in vivo in nu/nu mice . A polyclonal anti-HtrA1 serum demonstrated a significantly higher expression in primary melanomas when compared to unrelated metastatic lesions in a human melanoma tissue array, and down-modulation of HtrA1 expression in autologous lymph node melanoma metastases in seven out of 11 cases examined . These results suggest that down-regulation of PRSS11 and HtrA1 expression may represent an indicator of melanoma progression.

Mol Genet Genomics, 2002 Sep, 268(1), 81 - 6 Epub 2002 Jul 17.
Influence of the relA gene on ribosome frameshifting; Masucci JP et al.; We have examined the influence of genotype at the relA locus on the kinetics of leftward (or -1) frameshifting at a variety of codons calling for a limiting aminoacyl-tRNA species . We used lacZ left-frameshift reporter constructs carrying the sequenceU UUC XYZ, whereXYZ was each of three triplets coding for three different amino acids; we slowed the ribosomes at each of these by limiting for the amino acid or for the aminoacyl-tRNA . In all cases, limitation stimulated leftward frameshifting . In all cases, the stimulation was greater in relA mutant cells than in their wild-type relA(+) counterparts . In the latter genotype, the increased frameshifting was constant from the start of the limitation regime . This was also true of the relA mutant strain during limitation for lysine-tRNA or for leucine; however, during limitation for isoleucine-tRNA (or for isoleucine) the mutant showed a gradual, progressive increase in frameshifting, suggesting an indirect effect . We suggest that gradual accumulation of undermodified tRNAs, which is characteristic of the relA response, is involved . However, the specific modification involved is unknown . It is not queosine: analysis of a tgt mutant that is completely defective in queosine modification showed no increase in leftward frameshifting on the reporter which showed the larger, gradual increase during the relA response to isoleucine-tRNA limitation.

Mol Genet Genomics, 2002 Sep, 268(1), 62 - 9 Epub 2002 Aug 13.
Identification of multicopy suppressors of the pcnB plasmid copy number defect in Escherichia coli; Sarkar N et al.; Plasmids containing a ColE1 origin of replication are widely used for cloning purposes in Escherichia coli . Among the host factors that affect the copy number of ColE1 plasmids is the E . coli protein poly(A) polymerase I (PAP I), which regulates the intracellular level of RNA I, a ColE1-encoded negative regulator of plasmid replication . In strains that lack PAP I, RNA I levels are elevated, resulting in reduced levels of ColE1 plasmids in the cell . PAP I is encoded by the gene pcnB . We devised a genetic approach, based on the identification of multicopy suppressor clones, to identify trans-acting factors that can help offset the ColE1 plasmid copy number defect in a pcnB (-) genetic background . Using this strategy, we identified suppressors that mapped to two regions of the E . coli chromosome . The suppressor activity of one of the chromosomal regions was localized to the rssB gene, a response regulator gene known to be involved in the turnover of the stationary-phase sigma factor, RpoS . The second suppressor maps to min 55.4 of the E . coli chromosome, and the factor responsible for the suppressor activity appears to be a novel RNA or protein.

Genetics, 2002 Sep, 162(1), 485 - 99
Enzyme kinetics, substitutable resources and competition: from biochemistry to frequency-dependent selection in lac; Lunzer M et al.; Trade-offs in catalytic efficiency at the lac permease of Escherichia coli produce alleles with different substrate specializations that are selectively favored on different galactosides . We show that differential resource utilization during competition for mixtures of galactosides produces frequency-dependent selection at lac . However, the polymorphism is protected only in a narrow range of galactoside ratios despite intense selection on the pure galactosides . Hence, stabilizing frequency-dependent selection protecting natural allozyme polymorphisms through differential resource utilization will be sporadic and ephemeral in randomly changing environments . A comparison of predictions, based on first principles, with experimental outcomes reveals an additional, unanticipated source of weak selection.

Genetics, 2002 Sep, 162(1), 5 - 13
Escherichia coli strains (ndk) lacking nucleoside diphosphate kinase are powerful mutators for base substitutions and frameshifts in mismatch-repair-deficient strains; Miller JH et al.; Nucleoside diphosphate (NDP) kinase is one of the enzymes that maintains triphosphate pools . Escherichia coli strains (ndk) lacking this enzyme have been shown to be modest base substitution mutators, and two members of the human family of NDP kinases act as tumor suppressors . We show here that in E . coli strains lacking NDP kinase high levels of mispairs are generated, but most of these are corrected by the mismatch-repair system . Double mutants that are ndk mutS, lacking both the NDP kinase and mismatch repair, have levels of base substitutions 15-fold higher and levels of certain frameshifts up to 10-fold higher than those of the respective mutations in mutS strains that are NDP kinase proficient . A sequence analysis of the specificity of base substitution mutations generated in ndk and ndk mutS backgrounds as well as other experiments suggests that NDP kinase deficiency stimulates polymerase errors that lead to A:T --> G:C transitions and that the editing capacity of cells may be affected, leading to additional uncorrected mispairs and to A:T --> T:A transversions.

Biotechnol Appl Biochem, 2002 Oct, 36(Pt 2), 77 - 84
An analysis of target preferences of Escherichia coli outer-membrane endoprotease OmpT for use in therapeutic peptide production: efficient cleavage of substrates with basic amino acids at the P4 and P6 positions; Okuno K et al.; The Escherichia coli outer-membrane endoprotease OmpT mainly cleaves peptide bonds between consecutive basic amino acids . The effect of adjacent residues on cleavage efficiency is currently unknown, except at positions P2 and P2' . Therefore we investigated the effects of amino acid residues upstream of the cleavage site on the ability of OmpT to cleave efficiently a fusion protein carrying human glucagon-like peptide-1 (7-37) in 4 M urea . The P1-P10 residues were replaced by Ala and each substrate was subjected to OmpT digestion . The replacement of Arg residue at P1 blocked the cleavage due to the loss of the cleavage site, and the replacement of Arg residue at P4 maximally reduced the cleavage rate . Conversely, cleavage efficiency increased on replacing Glu at P6 . Substitution of the residues at P4 and P6 with several different amino acids showed that OmpT preferred basic residues at these positions, whereas acidic residues had a negative effect . This was also shown to be true with synthetic decapeptide substrates in the absence of urea . The k(cat)/ K(m) ratio increased with basic residues at P4 or P6, mainly due to a lower K(m) rather than an increase in k(cat) . On the basis of these findings, we prepared a fusion protein carrying human atrial natriuretic peptide (ANP), a drug for acute congestive heart failure . OmpT released mature ANP from the E . coli-expressed fusion protein . As expected, the introduction of an Arg residue at P4 and P6 enhanced the release of ANP.

Mol Biol Rep, 2002, 29(1-2), 79 - 82
DNA supercoiling by gyrase is linked to nucleoid compaction; Stuger R et al.; The genes of E . coli are located on a circular chromosome of 4.6 million basepairs . This 1.6 mm long molecule is compressed into a nucleoid to fit inside the 1-2 microm cell in a functional format . To examine the role of DNA supercoiling as nucleoid compaction force we modulated the activity of DNA gyrase by electronic, genetic, and chemical means . A model based on physical properties of DNA and other cell components predicts that relaxation of supercoiling expands the nucleoid . Nucleoid size did not increase after reduction of DNA gyrase activity by genetic or chemical means, but nucleoids did expand upon chemical inhibition of gyrase in chloramphenicol-treated cells, indicating that supercoiling may help to compress the genome.

Mol Biol Rep, 2002, 29(1-2), 233 - 6
Combinatorial complexity of pathway analysis in metabolic networks; Klamt S et al.; Elementary flux mode analysis is a promising approach for a pathway-oriented perspective of metabolic networks . However, in larger networks it is hampered by the combinatorial explosion of possible routes . In this work we give some estimations on the combinatorial complexity including theoretical upper bounds for the number of elementary flux modes in a network of a given size . In a case study, we computed the elementary modes in the central metabolism of Escherichia coli while utilizing four different substrates . Interestingly, although the number of modes occurring in this complex network can exceed half a million, it is still far below the upper bound . Hence, to a certain extent, pathway analysis of central catabolism is feasible to assess network properties such as flexibility and functionality.

Dis Aquat Organ, 2002 Aug 15, 51(1), 77 - 80
Production of polyclonal antiserum specific to the 27.5 kDa envelope protein of white spot syndrome virus; You Z et al.; A truncated version of the white spot syndrome virus (WSSV) 27.5 kDa envelope protein was expressed as a histidine tag fusion protein in Escherichia coli . The bacterial expression system allowed the production of up to 10 mg of purified recombinant protein per liter of bacterial culture . Antiserum from a rabbit immunized with the recombinant protein recognized the 27.5 kDa viral envelope protein of WSSV isolated from different geographic regions . The antiserum did not recognize any of the other known WSSV structural proteins . A sensitive immunodot assay for WSSV was developed using the specific rabbit polyclonal antiserum.

Chem Commun (Camb), 2001 Sep 21, (18), 1760 - 1
Identification of Tyr58 as the proton donor in the aspartate-alpha-decarboxylase reaction; Saldanha SA et al.; The decarboxylation of L-aspartate by E . coli L-aspartate-alpha-decarboxylase (ADC) is shown to occur with retention of configuration; analysis of the protein structure identifies Tyr58 as the proton donor in the decarboxylation mechanism.

Proc Natl Acad Sci U S A, 2002 Oct 1, 99(20), 12733 - 40 Epub 2002 Sep 18.
Inaugural Article: a self-replicating ligase ribozyme; Paul N et al.; A self-replicating molecule directs the covalent assembly of component molecules to form a product that is of identical composition to the parent . When the newly formed product also is able to direct the assembly of product molecules, the self-replicating system can be termed autocatalytic . A self-replicating system was developed based on a ribozyme that catalyzes the assembly of additional copies of itself through an RNA-catalyzed RNA ligation reaction . The R3C ligase ribozyme was redesigned so that it would ligate two substrates to generate an exact copy of itself, which then would behave in a similar manner . This self-replicating system depends on the catalytic nature of the RNA for the generation of copies . A linear dependence was observed between the initial rate of formation of new copies and the starting concentration of ribozyme, consistent with exponential growth . The autocatalytic rate constant was 0.011 min(-1), whereas the initial rate of reaction in the absence of pre-existing ribozyme was only 3.3 x 10(-11) M.min(-1) . Exponential growth was limited, however, because newly formed ribozyme molecules had greater difficulty forming a productive complex with the two substrates . Further optimization of the system may lead to the sustained exponential growth of ribozymes that undergo self-replication.

J Virol, 2002 Oct, 76(20), 10553 - 8
Identification and expression of immunogenic proteins of a disease-associated marine turtle herpesvirus; Coberley SS et al.; Herpesviruses are associated with several diseases of marine turtles, including lung-eye-trachea disease (LETD) and fibropapillomatosis . Two approaches were used to identify immunodominant antigens of LETV, the LETD-associated herpesvirus . The first approach targeted glycoprotein B, which is known to be immunogenic and neutralizing in other species . The second strategy identified LETV proteins recognized on Western blots by antibodies in immune green turtle plasma . A 38-kDa protein was resolved by two-dimensional gel electrophoresis, sequenced, and identified as a scaffolding protein encoded by the overlapping open reading frames of UL26 and UL26.5 . Glycoprotein B and the scaffolding protein were cloned and expressed in Escherichia coli . The expressed proteins were recognized on Western blots by antibodies in immune green turtle plasma . Phylogenetic studies based on UL26, DNA polymerase, and glycoprotein B revealed that LETV clusters with the alphaherpesviruses.

Endocrinology, 2002 Oct, 143(10), 4096 - 103
Transgenic mice harboring murine luteinizing hormone receptor promoter/beta-galactosidase fusion genes: different structural and hormonal requirements of expression in the testis, ovary, and adrenal gland; Hamalainen T et al.; In vivo regulation of the LH receptor (LHR) promoter was studied using transgenic (TG) mice harboring fusion genes containing three different lengths of the LHR promoter (7.4 kb, 2.1 kb, and 173 bp), fused with coding sequence of the Escherichia coli beta-galactosidase (beta-GAL) reporter gene . The length of the LHR promoter significantly affected the pattern of beta-GAL expression . In the testis the shortest promoter directed expression primarily of the full-length beta-GAL mRNA, but mainly truncated messages were transcribed from the longer LHR promoter/beta-GAL constructs . The case was reversed in the ovary and adrenal gland . Furthermore, we have recently detected strong LHR expression in the adrenal gland of female mice with chronically elevated serum LH . Therefore, the regulation of the adrenal LHR expression was addressed in the present study using the LHR/beta-GAL TG mice . Elevated LH levels were achieved in the LHR/beta-GAL mice either by gonadectomy or cross-breeding them with TG mice overexpressing a chimeric protein of bovine LH beta-subunit and the C-terminal fragment of human chorionic gonadotropin-beta . In both models, beta-GAL mRNA was found in the adrenal cortex when the 7.4-kb LHR promoter was applied but not in mice carrying the 173-bp LHR promoter . The 7.4-kb construct was activated also in the ovaries in the double TG LHR(beta-GAL)/bovine LH beta-subunit/C-terminal fragment of human chorionic gonadotropin-betamice in some theca-interstitial cells surrounding the follicles . Hence, the LHR promoter elements essential for directing beta-GAL expression to the adrenal gland and ovary (7.4 kb) are different from those recently shown to be essential for the testicular expression (173 bp) . In conclusion, elevated serum LH concentrations were found seminal for the LHR promoter activation in the ovaries and adrenals, and different lengths of the promoter are responsible for reporter gene expression in the testis, ovary, and adrenal gland.

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