Escherichia coli

Appl Environ Microbiol, 2002 Sep, 68(9), 4546 - 53
Enzymatic properties and intracellular localization of the novel Trichoderma reesei beta-glucosidase BGLII (cel1A); Saloheimo M et al.; This paper describes the characterization of an intracellular beta-glucosidase enzyme BGLII (Cel1a) and its gene (bgl2) from the cellulolytic fungus Trichoderma reesei (Hypocrea jecorina) . The expression pattern of bgl2 is similar to that of other cellulase genes known from this fungus, and the gene would appear to be under the control of carbon catabolite repression mediated by the cre1 gene . The BGLII protein was produced in Escherichia coli, and its enzymatic properties were analyzed . It was shown to be a specific beta-glucosidase, having no beta-galactosidase side activity . It hydrolyzed both cellotriose and cellotetraose . BGLII exhibited transglycosylation activity, producing mainly cellotriose from cellobiose and sophorose and cellobiose from glucose . Antibodies raised against BGLII showed the presence of the enzyme in T . reesei cell lysates but not in the culture supernatant . Activity measurements and Western blot analysis of T . reesei strains expressing bgl2 from a constitutive promoter further confirmed the intracellular localization of this beta-glucosidase.

Appl Environ Microbiol, 2002 Sep, 68(9), 4407 - 15
Characterization of the 5-carboxyvanillate decarboxylase gene and its role in lignin-related biphenyl catabolism in Sphingomonas paucimobilis SYK-6; Peng X et al.; Sphingomonas paucimobilis SYK-6 degrades a lignin-related biphenyl compound, 5,5'-dehydrodivanillate (DDVA), to 5-carboxyvanillate (5CVA) by the enzyme reactions catalyzed by the DDVA O-demethylase (LigX), the ring cleavage oxygenase (LigZ), and the meta-cleavage compound hydrolase (LigY) . In this study we examined the degradation step of 5CVA . 5CVA was transformed to vanillate, O-demethylated, and further degraded via the protocatechuate 4,5-cleavage pathway by this strain . A cosmid clone which conferred the 5CVA degradation activity to a host strain was isolated . In the 7.0-kb EcoRI fragment of the cosmid we found a 1,002-bp open reading frame responsible for the conversion of 5CVA to vanillate, and we designated it ligW . The gene product of ligW (LigW) catalyzed the decarboxylation of 5CVA to produce vanillate along with the specific incorporation of deuterium from deuterium oxide, indicating that LigW is a nonoxidative decarboxylase of 5CVA . LigW did not require any metal ions or cofactors for its activity . The decarboxylase activity was specific to 5CVA . Inhibition experiments with 5CVA analogs suggested that two carboxyl groups oriented meta to each other in 5CVA are important to the substrate recognition by LigW . Gene walking analysis indicated that the ligW gene was located on the 18-kb DNA region with other DDVA catabolic genes, including ligZ, ligY, and ligX.

Biochem Biophys Res Commun, 2002 Aug 30, 296(4), 991 - 9
Physical association of the APIS complex and general transcription factors; Sun L et al.; It has recently been demonstrated that a fragment of the proteasome, called the APIS complex, plays an important role in RNA polymerase II-mediated transcription . Here, it is shown that the APIS complex is physically associated with many general transcription factors, including components of yeast FACT (Cdc68/Pob3), TFIID, TFIIH, and the RNA polymerase II holoenzyme . Depletion of this APIS transcription factor complex from a yeast whole cell extract resulted in reduced transcription, indicating that it is functionally relevant . The APIS/transcription factor complex does not include detectable levels of the 20S proteolytic sub-unit of the proteasome . Furthermore, immunopurified 26S proteasome contains little or no transcription factors, suggesting that transcription factors and the 20S bind competitively to the APIS complex . These data add to the growing body of evidence that the APIS complex has a role in transcription, independent of its role in proteolysis and, furthermore, argues that it functions in association with the general transcription complex.

Biochem Biophys Res Commun, 2002 Aug 30, 296(4), 988 - 90
A designed apoplastocyanin variant that shows reversible folding; Datta D et al.; Plastocyanin, like many other metalloproteins, does not undergo reversible folding, which is thought to be due to an irreversible conformational change in the copper-binding site . Moreover, apoplastocyanin's ability to adopt a native tertiary structure is highly salt-dependent, and even in high salt, it has an irreversible thermal denaturation . Here, we report a designed apoplastocyanin variant, PCV, that is well folded and has reversible folding in both high and low salt conditions . This variant provides a tractable model for understanding and designing protein beta-sheets.

Biochem Biophys Res Commun, 2002 Aug 30, 296(4), 983 - 7
RecA-promoted sliding of base pairs within DNA repeats: quantitative analysis by a slippage assay; Navadgi VM et al.; RecA that catalyses efficient homology search and exchange of DNA bases has to effect major transitions in the structure as well as the dynamics of bases within RecA-DNA filament . RecA induces slippage of paired strands in poly(dA)-poly(dT) duplex using the energy of ATP hydrolysis . Here, we have adopted the targeted ligation assay and quantified the strand slippage within a short central cassette of (dA)(4)-(dT)(4) duplex . The design offers a stringent test case for scoring a cross-talk between A residues with those of T that are diagonally placed on the opposite strand at either -3, -2, -1, +1, +2, or +3 pairing frames . As expected, the cross-talk levels in RecA mediated as well as thermally annealed duplexes were maximal in non-diagonal pairing frame (i.e., 0-frame), the levels of which fell off gradually as the frames became more diagonal, i.e., -3<-2<-1 or +3<+2<+1 . Interestingly, the level of cross-talk in naked duplexes was intrinsically less efficient in minus frames than their plus frame counterparts . The asymmetry created in naked duplexes by such a disparity between minus versus plus frames was partially obviated by RecA . Moreover, RecA promoted a significantly higher level of cross-talk selectively in -2 and -1 frames, as compared to that in naked DNA, which suggests a model that the elevated cross-talk in RecA filament may be limited to base pairs housed within the same rather than adjacent RecA monomers.

Biochem Biophys Res Commun, 2002 Aug 30, 296(4), 847 - 56
Redox regulation of pro-inflammatory cytokines and IkappaB-alpha/NF-kappaB nuclear translocation and activation; Haddad JJ; Reduction-oxidation (redox) state constitutes such a potential signaling mechanism for the regulation of an inflammatory signal associated with oxidative stress . Exposure of alveolar epithelial cells to ascending DeltapO(2) regimen+/-reactive oxygen species (ROS)-generating systems induced a dose-dependent release of interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha . Similarly, the Escherichia coli-derived lipopolysaccharide-endotoxin (LPS) up-regulated cytokine biosynthesis in a dose- and time-dependent manner . Irreversible inhibition of gamma-glutamylcysteine synthetase, the rate-limiting enzyme in the biosynthesis of glutathione (GSH), by L-buthionine-(S,R)-sulfoximine (BSO), induced the accumulation of ROS and augmented DeltapO(2) and LPS-mediated release of cytokines . Analysis of the molecular mechanism implicated revealed an inhibitory-kappaB (IkappaB-alpha)/nuclear factor-kappaB (NF-kappaB)-independent pathway in mediating redox-dependent regulation of inflammatory cytokines . BSO stabilized cytosolic IkappaB-alpha and down-regulated its phosphorylation, thereby blockading NF-kappaB activation, yet it augmented cytokine secretion . Glutathione depletion is associated with the augmentation of oxidative stress-mediated inflammatory state in a ROS-dependent mechanism and the IkappaB-alpha/NF-kappaB pathway is redox-sensitive but differentially involved in regulating redox-dependent regulation of cytokines.

Eur J Biochem, 2002 Sep, 269(17), 4267 - 76
Medium-chain dehydrogenases/reductases (MDR) . Family characterizations including genome comparisons and active site modeling; Nordling E et al.; Completed eukaryotic genomes were screened for medium-chain dehydrogenases/reductases (MDR) . In the human genome, 23 MDR forms were found, a number that probably will increase, because the genome is not yet fully interpreted . Partial sequences already indicate that at least three further members exist . Within the MDR superfamily, at least eight families were distinguished . Three families are formed by dimeric alcohol dehydrogenases (ADH; originally detected in animals/plants), cinnamyl alcohol dehydrogenases (originally detected in plants) and tetrameric alcohol dehydrogenases (originally detected in yeast) . Three further families are centred around forms initially detected as mitochondrial respiratory function proteins, acetyl-CoA reductases of fatty acid synthases, and leukotriene B4 dehydrogenases . The two remaining families with polyol dehydrogenases (originally detected as sorbitol dehydrogenase) and quinone reductases (originally detected as zeta-crystallin) are also distinct but with variable sequences . The most abundant families in the human genome are the dimeric ADH forms and the quinone oxidoreductases . The eukaryotic patterns are different from those of Escherichia coli . The different families were further evaluated by molecular modelling of their active sites as to geometry, hydrophobicity and volume of substrate-binding pockets . Finally, sequence patterns were derived that are diagnostic for the different families and can be used in genome annotations.

Eur J Biochem, 2002 Sep, 269(17), 4212 - 8
Structure and function of N-acetylglucosamine kinase . Identification of two active site cysteines; Berger M et al.; N-Acetylglucosamine is a major component of complex carbohydrates . The mammalian salvage pathway of N-acetylglucosamine recruitment from glycoconjugate degradation or nutritional sources starts with phosphorylation by N-acetylglucosamine kinase . In this study we describe the identification of two active site cysteines of the sugar kinase by site-directed mutagenesis and computer-based structure prediction . Murine N-acetylglucosamine kinase contains six cysteine residues, all of which were mutated to serine residues . The strongest reduction of enzyme activity was found for the mutant C131S, followed by C143S . Determination of the kinetic properties of the cysteine mutants showed that the decreased enzyme activities were due to a strongly decreased affinity to either N-acetylglucosamine for C131S, or ATP for C143S . A secondary structure prediction of N-acetylglucosamine kinase showed a high homology to glucokinase . A model of the three-dimensional structure of N-acetylglucosamine kinase based on the known structure of glucokinase was therefore generated . This model confirmed that both cysteines are located in the active site of N-acetylglucosamine kinase with a potential role in the binding of the transferred gamma-phosphate group of ATP within the catalytic mechanism.

Eur J Biochem, 2002 Sep, 269(17), 4194 - 201
A structural basis for the pH-dependence of cofilin . F-actin interactions; Blondin L et al.; A marked pH-dependent interaction with F-actin is an important property of typical members of the actin depolymerizing factor (ADF)/cofilin family of abundant actin-binding proteins . ADF/cofilins tend to bind to F-actin with a ratio of 1 : 1 at pH values around 6.5, and to G-actin at pH 8.0 . We have investigated the mechanism for the pH-sensitivity . We found no evidence for pH-dependent changes in the structure of cofilin itself, nor for the interaction of cofilin with G-actin . None of the actin-derived, cofilin-binding peptides that we had previously identified {Renoult, C., Ternent, D., Maciver, S.K., Fattoum, A., Astier, C., Benyamin, Y . & Roustan, C . (1999) J . Biol . Chem . 274, 28893-28899} bound cofilin in a pH-sensitive manner . However, we have detected a conformational change in region 75-105 in the actin subdomain 1 by the use of a peptide-directed antibody . A pH-dependent conformational change has also been detected spectroscopically in a similar peptide (84-103) on binding to cofilin . These results are consistent with a model in which pH-dependent motion of subdomain 1 relative to subdomain 2 (through region 75-105) of actin reveals a second cofilin binding site on actin (centered around region 112-125) that allows ADF/cofilin association with the actin filament . This motion requires salt in addition to low pH.

Eur J Biochem, 2002 Sep, 269(17), 4176 - 84
De-regulation of D-3-phosphoglycerate dehydrogenase by domain removal; Bell JK et al.; Escherichia coli 3-phosphoglycerate dehydrogenase (PGDH) catalyzes the first step in serine biosynthesis, and is allosterically inhibited by serine . Structural studies revealed a homotetramer in which the quaternary arrangement of subunits formed an elongated ellipsoid . Each subunit consisted of three domains: nucleotide, substrate and regulatory . In PGDH, extensive interactions are formed between nucleotide binding domains . A second subunit-subunit interaction occurs between regulatory domains creating an extended beta sheet . The serine-binding sites overlap this interface . In these studies, the nucleotide and substrate domains (NSDs) were subcloned to identify changes in both catalytic and physical properties upon removal of a subunit-subunit interface . The NSDs did not vary significantly from PGDH with respect to kinetic parameters with the exception that serine no longer had an effect on catalysis . Temperature dependent dynamic light scattering (DLS) revealed the NSDs aggregated > 5 degrees C before PGDH, indicating decreased stability . DLS and gel filtration studies showed that the truncated enzyme formed a tetramer . This result negated the hypothesis that the removal of the regulatory domain would create an enzyme mimic of the unregulated, closely related dimeric enzymes . Expression of the regulatory domain, to study conformational changes induced by serine binding, yielded a product that by CD spectra contained stable secondary structure . DLS and pulsed field gradient NMR studies of the regulatory domain showed the presence of higher oligomers instead of the predicted dimer . We have concluded that the removal of the regulatory domain is sufficient to eliminate serine inhibition but does not have the expected effect on the quaternary structure.

Acta Virol, 2002, 46(1), 11 - 7
Selection and expression of peptides which can change the conformation of p20 protein of rice stripe virus; Zhang HW et al.; Phages with high affinity to the P20 protein of rice stripe virus (RSV) were enriched from phage-displayed random 12-mer peptide library after three rounds of phage display screening . Nine different peptides from the enriched library were selected by enzyme-linked immunosorbent assay (ELISA) . The P20 protein from raw extracts of rice leaves infected with RSV could be detected by those 9 peptides displayed on the phage, which suggested that a peptide could be an effective tool for diagnosis of RSV in rice and planthopper . Circular dichroism (CD) spectra of P20 fusion proteins with the binding phages and non-binding phages showed that the conformation of P20 protein was changed after binding to each of the 9 selected 12-mer peptides, which suggested that these peptides might disrupt the function of the P20 protein . Thereafter, those peptides might be used to develop plant resistance and disrupt virus transmission . Three of the 12-mer peptide genes were fused with the glutathione-S-transferase (GST) gene in the vector pGEX 3X . The fusion proteins were obtained from an Escherichia coli expression system and purified . The fusion proteins might have a potential to develop a plant peptide-based resistance to its pathogens and virus diagnosis . It also provided a tool (i) to confirm the inhibition of the function of P20 protein by the fusion peptides in vivo, and (ii) to detect the function of P20 protein and the interaction between the virus and its vector.

Hepatology, 2002 Sep, 36(3), 623 - 30
Demonstration of direct lineage between hepatocytes and hepatocellular carcinoma in diethylnitrosamine-treated rats; Bralet MP et al.; The question whether hepatocellular carcinoma (HCC) arises from dedifferentiation of mature hepatocytes or from proliferation of liver stem cells is still debated . In the present study, we used retroviral-mediated genetic labeling to investigate the fate of mature hepatocytes in rats after administration of diethylnitrosamine (DEN) . Mature hepatocytes were genetically labeled by intravenous injection of retroviral vectors containing the Escherichia coli beta-galactosidase gene coupled to a nuclear localization signal (nls-LacZ) 1 day after partial hepatectomy . Liver biopsies performed after completion of hepatic regeneration showed that 18.3% of hepatocytes expressed the nls-LacZ transgene . Rats were then treated with DEN in drinking water for 12 weeks and sacrificed between 98 and 151 days after the onset of DEN administration . Clones of beta-galactosidase positive cells were observed, half of which (53%) also expressed the placental form of glutathione-S-transferase (GSTp), a marker of preneoplastic cells . HCCs of various sizes expressing GSTp were present in all animals . Careful examination of 90 HCCs revealed that 16 (17.7%) also expressed nls-LacZ . This figure precisely matched the proportion of labeled hepatocytes before DEN treatment (18.3%) . In conclusion, a random clonal origin of HCC from mature hepatocytes is seen in the DEN model of hepatocarcinogenesis.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 Sep, 34(5), 583 - 8
{Redox properties and conformational changes of DsbA protein from Escherichia coli periplasm}; Li Q et al.; DsbA protein, a disulfide bond enzyme in Escherichia coli periplasm, catalyzes mainly disulfide bond formation of proteins . Site-directed mutagenes is combined with Trp-analog labeling technique was used to investigate the redox properties and conformational changes of DsbA . The results show that: (1) DsbA, as an oxidase, can catalyze disulfide bond formation efficiently . The reduced form is more stable than the oxidized form, suggesting that the strong oxidizing force of DsbA comes from the tense conformation of the oxidized form . (2) The alteration of local environment around Trp(76) between redox forms is responsible for the special fluorescence phenomenon of DsbA . (3) The result from (19)F-NMR study provides further evidence that the local conformation of Trp(76) is dramatically affected during the transformation of redox forms, but that of Trp(126) remains unaffected.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 Sep, 34(5), 571 - 5
{Restin expressed in vivo suppresses the growth of tumors in nude mice}; Xu R et al.; Restin, a homologous protein of endostatin (62% homology), is the NC domain of collagen XV at C-terminal . The recombinant restin expressed in E . coli had the ability to suppress the proliferation of bovin aortic endothelial cells and cause apoptosis . In this report, mouse restin gene was fused with a sequence of human plasminogen signal peptide by PCR and cloned into eukaryotic expression vector pCDNA3 . The plasmid containing restin gene was named pCDNAXV and was transfected into human hepatoma cell line Bel7404 . Stable transfected clones were screened and expression of restin was confirmed by RT-PCR and Western blot . The proliferated cells were injected subcutaneusly into nude mice . The growth of tumors formed by cells transfected with restin gene was much slower than that of control group . These results indicated that the expressed restin in vivo could suppress the growth of tumor, and this suppression might be achieved by restraining angiogenesis since the restin had no effect on the proliferation of tumor cells . At the same time, this report provided a new method to investigate the effect of anti-angiogenetic proteins on the tumor growth.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 Sep, 34(5), 565 - 70
{Self-aggregation of the structural protein encoded by rice ragged stunt Oryzavirus genome segment 8}; Lu HJ et al.; Rice ragged stunt oryzavirus (RRSV) is a member of the genus oryzavirus within the family Reoviridae . Its genome consists of ten segments of dsRNA . The functions of all products encoded by these viral genome segments, except one encoded by S9, have not yet been elucidated . In the present study, the ORF of S 8 of RRSV-Philippines isolate was sequenced and expressed in E . coli . The 67 kD product of S8 could be self-cleaved into two fragments with molecular weights of 43 kD and 26 kD . Western blotting indicated that both 67 kD and 43 kD products were major structural proteins of the virus . It was also found that the 67 kD protein could self aggregate into aggregates with higher sedimentation rate in sucrose gradients during centrifugation . Moreover, the self-aggregation process could be accelerated by the complex of S6 product and genome dsRNAs of RRSV . These results suggest that the S8 products, 67 kD or 43 kD, may be the structural components of the viral inner-capsid.

Alcohol Clin Exp Res, 2002 Aug, 26(8 Suppl), 70S - 74S
Oral contraceptives worsen endotoxin-induced liver injury in rats; Konno A et al.; BACKGROUND: Oral contraceptives are widely used; however, these drugs occasionally cause liver injury . Recently, it was reported that estriol worsens alcoholic liver injury by the mechanism involving activation of Kupffer cells as a result of gut-derived endotoxin . However, the relationship between oral contraceptives and endotoxin-induced liver injury has not been elucidated . Here we show that oral contraceptives sensitize Kupffer cells via a mechanism dependent on increased gut permeability to endotoxin . METHODS: Female Wistar rats (200-250 g) were given intraperitoneally a combination of estradiol (35 ng/kg of 17 alpha-Ethynylestradiol) and progesterone (2 microg/kg of Norethindrone), each dose being similar to that contained in oral contraceptives (EP treatment) . After 24 hr, a sublethal dose of lipopolysaccharide (LPS; 5 mg/kg) was injected via the tail vein . In some experiments, antibiotics (150 mg/kg/day of polymyxin B and 450 mg/kg/day of neomycin) were administered orally for 4 days before EP treatment . Gut permeability was measured in isolated segments of ileum by translocation of horseradish peroxidase . Kupffer cells were isolated and cultured in RPMI 1640 + 10% fetal bovine serum for 24 hr . After addition of LPS (100 ng/ml) to the culture medium, intracellular calcium concentration ({Ca2+}(i) ) was measured with fura-2 . RESULTS: Liver histology in rats given EP treatment intraperitoneally followed by an injection of LPS (5 mg/kg) 24 hr later revealed pronounced liver damage with massive necrosis . Whereas mean values of alanine aminotransferase (ALT) in the control, nontreated rats were 30 +/- 6 IU/liter, ALT increased to 75 +/- 21 IU/liter 24 hr after LPS injection . This increase was aggravated 6-fold (483 +/- 118 IU/liter; p< 0.05) by EP treatment . The EP treatment-induced increase in ALT was completely blocked by antibiotics (82 +/- 26 IU/liter; p< 0.05) . Gut permeability was increased approximately 10-fold with EP treatment . This increase in gut permeability was not altered by treatment with nonabsorbable antibiotics . In isolated Kupffer cells, LPS increased {Ca2+}(i) of Kupffer cells in control rats from basal levels (36 +/- 8 nmol/liter) to 100 +/- 8 nmol/liter . In contrast, the LPS-induced {Ca2+}(i) elevation was approximately 3-fold greater in the group given EP treatment before 24 hr (305 +/- 17 nmol/liter; p< 0.05) . This increase was also blocked completely by treatment with antibiotics (128 +/- 13 nmol/liter) . Similar results were obtained for LPS-induced tumor necrosis factor-alpha production by Kupffer cells from either control or EP treatment group . The increased tumor necrosis factor-alpha production as a result of EP treatment was blocked completely by antibiotics . CONCLUSIONS: These results indicate that EP treatment in vivo sensitizes Kupffer cells to LPS via a mechanism dependent on the portal increase of gut-derived endotoxin . This event suggests that oral contraceptives exacerbate alcoholic liver injury.

Acta Crystallogr D Biol Crystallogr, 2002 Sep, 58(Pt 9), 1504 - 6 Epub 2002 Aug 23.
Cloning, purification, crystallization and preliminary X-ray analysis of DOS heme domain, a new heme oxygen sensor in Escherichia coli; Park H et al.; The heme-containing PAS domain of the direct oxygen-sensor protein (DOS(H)), a bona fide oxygen-sensor protein, has been cloned from Escherichia coli strain K12 and successfully purified . The oxidized form of this protein was crystallized by the hanging-drop method with a PEG 8000-based precipitant . Preliminary X-ray diffraction studies of the PAS-domain crystal show that it belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 46.1, b = 68.1, c = 82.6 A . A complete diffraction data set was collected to 1.9 A for MAD phasing . The electron-density map shows two molecules in an asymmetric unit and a unique six-coordination of the heme iron.

Acta Crystallogr D Biol Crystallogr, 2002 Sep, 58(Pt 9), 1501 - 3 Epub 2002 Aug 23.
Crystallization and preliminary X-ray data of a bifunctional peroxiredoxin from poplar; Echalier A et al.; Two variants (wild type and V152C mutant) of a bifunctional poplar peroxiredoxin have been overexpressed in Escherichia coli cells . The two recombinant enzymes were purified and crystallized using the hanging-drop vapour-diffusion technique . Data sets were collected to 1.62 and 2.48 A resolution using X-ray synchrotron-source radiation from two crystal forms of wild-type peroxiredoxin which belonged to the monoclinic space group P2(1) (with unit-cell parameters a = 59.26, b = 68.80, c = 75.71 A, beta = 93.45 degrees ) and to the orthorhombic space group P2(1)2(1)2 (with unit-cell parameters a = 64.70, b = 130.73, c = 35.59 A), respectively . Data were also collected to 2.17 A resolution using a home X-ray source from a V152C peroxiredoxin crystal which belongs to the triclinic space group (P1), with unit-cell parameters a = 36.65, b = 41.53, c = 58.06 A, alpha = 70.52, beta = 93.45, gamma = 64.31 degrees . Phases have been obtained using molecular replacement with the structure of human peroxiredoxin V (PDB code 1hd2) as a search model . Refinement of the structures is in progress.

Plant Cell Physiol, 2002 Aug, 43(8), 903 - 10
Expression of mangrove allene oxide cyclase enhances salt tolerance in Escherichia coli, yeast, and tobacco cells; Yamada A et al.; To analyze the mechanisms of salt tolerance in the mangrove plant, Bruguiera sexangula, functional screening for cDNAs encoding proteins essential for salt tolerance was performed using Escherichia coli as the host organism . A transformant expressing a protein homologous to Lycopersicon (tomato) allene oxide cyclase (AOC) displayed enhanced salt tolerance . However, this unusual trait is not conferred by Lycopersicon AOC or its Arabidopsis homolog . Analysis of the functional region revealed a sequence of only 70 amino acids, which contains an unusual sequence that is essential for the salt-tolerant phenotype . On the basis of its unusual function, the mangrove AOC homolog is designated "mangrin" . Furthermore, expression of mangrin driven by the GAL1 promoter and the 35S cauliflower mosaic virus (CaMV) promoter in Saccharomyces cerevisiae and tobacco cell lines, respectively, also gave rise to enhanced salt tolerance . Mangrin transcripts increased in cultured B . sexangula cells in response to salt stress . We propose that mangrin plays an important role in the salt-tolerance mechanism of B . sexangula, and that the biosynthesis of mangrin might be an effective means of enhancing salt tolerance in higher plants.

Proc Natl Acad Sci U S A, 2002 Sep 17, 99(19), 12108 - 13 Epub 2002 Aug 27.
Biosynthesis of terpenes: studies on 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase; Adam P et al.; Earlier in vivo studies showed the involvement of IspH protein in the conversion of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate into isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) . We have demonstrated now that cell extract of an Escherichia coli strain engineered for hyperexpression of the ispH (lytB) gene catalyzes the in vitro conversion of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate into IPP and DMAPP . The reaction requires NADH, FAD, divalent cations (preferably Co2+), and probably one or more as-yet-unidentified proteins . The low intrinsic catalytic activities of wild-type E . coli cell extract and isolated chromoplasts of red pepper (Capsicum annuum) are enhanced by the addition of purified recombinant IspH protein.

J Biol Chem, 2002 Nov 8, 277(45), 43512 - 8 Epub 2002 Aug 26.
Aminoacylation of the N-terminal cysteine is essential for Lol-dependent release of lipoproteins from membranes but does not depend on lipoprotein sorting signals; Fukuda A et al.; Lipoproteins are present in a wide variety of bacteria and are anchored to membranes through lipids attached to the N-terminal cysteine . The Lol system of Escherichia coli mediates the membrane-specific localization of lipoproteins . Aspartate at position 2 functions as a Lol avoidance signal and causes the retention of lipoproteins in the inner membrane, whereas lipoproteins having residues other than aspartate at position 2 are released from the inner membrane and localized to the outer membrane by the Lol system . Phospholipid:apolipoprotein transacylase, Lnt, catalyzes the last step of lipoprotein modification, converting apolipoprotein into mature lipoprotein . To reveal the importance of this aminoacylation for the Lol-dependent membrane localization, apolipoproteins were prepared by inhibiting lipoprotein maturation . Lnt was also purified and used to convert apolipoprotein into mature lipoprotein in vitro . The release of these lipoproteins was examined in proteoliposomes . We show here that the aminoacylation is essential for the Lol-dependent release of lipoproteins from membranes . Furthermore, lipoproteins with aspartate at position 2 were found to be aminoacylated both in vivo and in vitro, indicating that the lipoprotein-sorting signal does not affect lipid modification.

Clin Exp Immunol, 2002 Sep, 129(3), 453 - 63
Generation of a recombinant Fab antibody reactive with the Alzheimer's disease-related Abeta peptide; Tammer AH et al.; A recombinant Fab antibody, designated 1E8-4b, which reacts with the Alzheimer's disease (AD)-related Abeta peptides, Abeta{1-40}, Abeta{1-42} and Abeta{1-43} has been developed . The 1E8-4b Fab was constructed by cloning the V(H)C(H1) and V(L)C(L) domains from the parent hybridoma 1E8 antibody, reported previously to recognize these Abeta peptides . Briefly, a C-terminal Flag tag sequence was incorporated into this construct, which was ligated into the vector pHFA2 and expressed in Escherichia coli . Following purification on an M2 anti-Flag affinity column, the 1E8-4b recombinant Fab antibody was shown to bind plaques within sections of brain tissue from CERAD-defined AD patients by immunohistochemistry . ELISA, epitope mapping and immunoblotting confirmed the recognition of the Abeta1-40/42/43} peptides by the 1E8-4b Fab . The 1E8-4b Fab did not recognize APP695 or APP770 which contain the Abeta sequence . The Abeta specificity of the recombinant 1E8-4b Fab antibody was identical to the parent 1E8 monoclonal antibody.

Arch Gynecol Obstet, 2002 Jul, 266(3), 178 - 80
Small cell carcinoma of the endometrium with concomitant pelvic inflammatory disease; Chuang J et al.; BACKGROUND: Small cell carcinoma of the endometrium is a rare disease entity characterized by bulkiness and predisposition to necrosis . Clinical presentations include postmenopausal bleeding, lower abdominal mass, chronic abdominal pain and menorrhagia . We present a case of small cell carcinoma of the endometrium with concomitant pelvic inflammatory disease . The literature is also reviewed . CASE: A 64 year old female presented was admitted with the principal complaints of fever, lower abdominal pain and malodorous vaginal discharge . Bimanual examination revealed cervical motion tenderness with a WBC of 9400 cells/microL and increased levels of neutrophils, band cells and C-reactive protein . Sonography revealed an adnexal echocomplex compatible with tubo-ovarian abscess . Culture of the vaginal discharge revealed the presence of E . coli . Symptoms persisted despite three days of antibiotics administration so a laparotomy was performed with a friable hemorrhagic uterus revealed and an area of necrosis evident in the left adnexa . Malignancy was confirmed from frozen section . Total abdominal hysterectomy, with bilateral salpingooophorectomy and optimal debulking, was performed . The final pathology report confirmed small cell carcinoma of the endometrium . CONCLUSION: Malignancy and pelvic inflammatory disease have overlapping clinical characteristics . Once pelvic inflammatory disease is suspected in a postmenopausal patient, malignancy should also be suspected, and a thorough examination and a tumor-marker analysis performed.

J Parasitol, 2002 Aug, 88(4), 804 - 7
Development and evaluation of an enzyme-linked immunosorbent assay with recombinant SAG2 for diagnosis of Toxoplasma gondii infection in cats; Huang X et al.; Cats are pivotal in the transmission of Toxoplasma gondii . To develop a sensitive and specific serodiagnostic method for feline toxoplasmosis, surface antigen 2 (SAG2) of T . gondii was expressed in Escherichia coli and its diagnostic potential evaluated in an enzyme-linked immunosorbent assay (ELISA) . The ELISA with recombinant SAG2 (rSAG2) was able to differentiate very clearly between sera from cats experimentally infected with T . gondii and sera from normal cats . Serum samples collected from domestic cats in Japan were investigated by the ELISA, and the results were compared with those of a commercially available latex agglutination test (LAT) kit . Of the 192 samples screened, 42 (21.9%) were positive by ELISA . Among the 42 ELISA-positive samples, 39 were positive by LAT . There was a significant correlation between ELISA and LAT titers . All the 150 ELISA-negative samples were negative by LAT . These results indicate that the ELISA with rSAG2 expressed in E . coli should be a useful method for detection of T . gondii infection in cats.

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2002 Jun, 16(2), 150 - 3
{Expression and antigenicity analysis of hepatitis G virus NS5 gene}; Cong Y et al.; BACKGROUND: To determine the antigenicity of HGV NS5 recombinant proteins expressed in E.coli . METHODS: HGV NS5a,NS5b and core/NS5b fusion genes were cloned into pThioC vector . Three expression plasmids were transformed into JM109(DE3) competent cells then expressed with induction by IPTG . Western blot and ELISA were used to determine the antigenicity after the three recombinant proteins were purified . RESULTS: After identification by restriction enzyme and sequencing, it was confirmed that the expressed was target proteins espected . Purified expression proteins were found strongly immunoreactive among anti HGV positive sera by Western blot and ELISA . Compared with mixed recombinant antigen (including core, NS5a synthetic peptide and NS3 recombinant proteins), in the 22 positive sera detected with mixed antigen, 68%(15/22), 90%(20/22) and 73%(16/22) were positive by P5a,P5b and Pc?5b antigens; In the 70 negative samples with mixed antigen, 7%(5/70), 1%(1/70) and 6%(4/70) were positive by P5a, P5b and Pc?5b antigens . The positive alone was found among RTPCR positive specimen using these recombinant antigens . CONCLUSIONS: NS5 gene expressed in E.coli?which couldn't be covered with other regions of antigens was one of the essential epitopes to HGV immunologic diagnosis.

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2002 Jun, 16(2), 132 - 5
{Mutation of LoopAB in HuIFN 1c/86D and enhancement of antiviral activity}; Hu R et al.; BACKGROUND: Based on the earlier mapping of the epitope recognized by neutralizing antibody, the authors directly replaced binding domain of IFN in AB-loop for enhancement of biological activity . METHODS: Two unique restriction sites (EcoR? and BsE?) were created into region flanking LoopAB . Casette mutagesis, restriction enzyme digestion, DNA sequencing, antiviral activity assay and antiproliferative activity assay have been used in the project . RESULTS: The mutated residues M31?D,D32?P of LoopAB in parent IFN were produced . The recombinant phagemid pCANTAB5E/3132IFN 1c/86D and expression plasmid PBV322-132IFN 1c/86D were constructed respectively by replacing the corresponding LoopAB with DNA fragment mutated in the residues M31?D,D32?P, which have been confirmed . The recombinant protein has been expressed in E.coli JM103 . The crude 3132IFN 1c/86D has been assayed on human WISH cells challenged with VSV and on HeLa cells by colorimetric MTT . 3132IFN 1c/86D showed 8-old antiviral activity compared to that of parent IFN 1c/86D, while IFN?induced growth inhibition of both types had no difference . CONCLUSIONS: The authors concluded that a mutant IFN with enhanced antiviral activity can be obtained via a targeted replacement of receptor binding domain in AB-loop.

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2002 Jun, 16(2), 119 - 23
{HIV-1 integrase enzyme linked immunosorbent assay and inhibitors}; Guo Z et al.; BACKGROUND: To establish an ELISA method for detecting the activity of HIV-1 integrase and screening of inhibitors . METHODS: HIV recombinant plasmid F185K/C280S IN1-288 was transformed into the E.coli strain BL21(DE3) integrase with a histag was induced by adding isopropyl-?-D?thiogalactopyranoside (IPTG)and purified by nickel affinity chromatography . The synthesized donor substrate oligonucleotide representing the HIV-1 U5LTR was immobilized onto covalink polystyrene microtiter plates, and a synthesized biotinlated 20 bp oligonucleotide was used as the target substrate . The products were detected and quantified using a colorimetic avidin?linked alkaline phosphatase reporter system,and identified by 32? autoradiography . Some natural products and chemically synthesized compounds were screened for HIV-1 integrase inhibitors . RESULTS: The purified integrase was identified by SDS?PAGE and showed integration activity by ELISA and?32P radioisotopic assay.?Coefficients of variation (CV)of ELISA in a lot and among the lots were 4.63% and 5.89% respectively, the mean of P/N was 2.836 0.161 under the optimal experimental condition . Some plant extracts were found as potent integrase inhibitors . The IC50s for CEH and CEHL were (20.41 5.68)?g/ml and (7.56 1.86)?g/ml respectively . CONCLUSIONS: The authors have established a simple and rapid ELISA method with stable repeatability for detecting integrase activity, which can be used for screening and studying of specific inhibitors of HIV-1 integrase.

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 2002 Jun, 16(2), 109 - 13
{Expression of the main structural antigen VP6 of human rotavirus by recombinant adenovirus and immune responses induced in vivo}; He J et al.; BACKGROUND: Constructing replication defective recombinant adenovirus vector expressing the group specific antigen VP6 of human rotavirus and studying the immune responses induced in vivo . METHODS: The cDNA of full length VP6 was inserted into the adenovirus vector pShuttle-CMV, and recombinant adenovirus genome DNA was obtained through homological recombination in E.coli,then the recombinant adenovirus was gained after transfecting 293 cell line with the genome DNA . Gene integration of VP6 in resultant adenovirus was confirmed by PCR and Southern blot, respectively gene expression was confirmed in 293 cells by Western blot . BALB/c mice were immunized intranasally(inl)and orally(ora), respectively, to test the immunization effects of the adenovirus . RESULTS: Recombinant adenovirus named rvAd-VP6 was obtained . The cDNA of VP6 was integrated in the adenovirus and was able to be expressed in 293 cells stably . The systemic immune responses to rotavirus VP6 could be induced effectively in both oral and intranasal group, the titer of serum IgG antibody in the two group of mice were 1?1 000 and 1?10 000-1?100 000, respectively . In addition to IgG, the serum IgA specific to VP6 could also be detected at a titer of 1?10-1?100.Secretory IgA(sIgA) was detected in both lung lavage fluid and intestinal homogenate when administered intranasally to BALB/c mice, whereas only found in intestinal homogenate in the oral group . The results indicated that the immunization efficacy of intranasal inoculation was superior to that of oral inoculation . CONCLUSIONS: The recombinant adenovirus vector expressing human rotavirus VP6 was successfully constructed, its ability to induce immune responses has laid a solid foundation for the development of rotavirus genetically engineering vaccine against rotavirus infection.

J Biol Chem, 2002 Nov 1, 277(44), 41379 - 89 Epub 2002 Aug 23.
Weak strand displacement activity enables human DNA polymerase beta to expand CAG/CTG triplet repeats at strand breaks; Hartenstine MJ et al.; Using synthetic DNA constructs in vitro, we find that human DNA polymerase beta effectively catalyzes CAG/CTG triplet repeat expansions by slippage initiated at nicks or 1-base gaps within short (14 triplet) repeat tracts in DNA duplexes under physiological conditions . In the same constructs, Escherichia coli DNA polymerase I Klenow Fragment exo(-) is much less effective in expanding repeats, because its much stronger strand displacement activity inhibits slippage by enabling rapid extension through two downstream repeats into flanking non-repeat sequence . Polymerase beta expansions of CAG/CTG repeats, observed over a 32-min period at rates of approximately 1 triplet added per min, reveal significant effects of break type (nick versus gap), strand composition (CTG versus CAG), and dNTP substrate concentration, on repeat expansions at strand breaks . At physiological substrate concentrations (1-10 microm of each dNTP), polymerase beta expands triplet repeats with the help of weak strand displacement limited to the two downstream triplet repeats in our constructs . Such weak strand displacement activity in DNA repair at strand breaks may enable short tracts of repeats to be converted into longer, increasingly mutable ones associated with neurological diseases.

J Biol Chem, 2002 Nov 8, 277(45), 43175 - 84 Epub 2002 Aug 23.
Pag, a putative tumor suppressor, interacts with the Myc Box II domain of c-Myc and selectively alters its biological function and target gene expression; Mu ZM et al.; The highly conserved Myc Box II (MBII) domain of c-Myc is critically important for transformation and transcriptional regulation . A yeast two-hybrid screen identified Pag as a MBII-interacting protein . Pag, a member of the peroxiredoxin family, has been reported previously to bind to and inhibit the cytostatic properties of the c-Abl oncoprotein . We now show that Pag promotes increased cell size and confers a proapoptotic phenotype, two hallmark features of ectopic c-Myc overexpression . Pag and c-Myc also confer resistance to oxidative stress, a previously unrecognized property of the latter protein . In contrast, Pag inhibits tumorigenesis by c-Myc-overexpressing fibroblasts and causes a broad but selective loss of c-Myc target gene regulation . Pag is therefore an MBII-interacting protein that can either mimic or enhance some of the c-Myc properties while at the same inhibiting others . These features, along with the previously identified interaction with c-Abl, provide support for the idea that Pag functions as a tumor suppressor.

J Biol Chem, 2002 Nov 1, 277(44), 42164 - 70 Epub 2002 Aug 23.
The X-ray crystallographic structure of Escherichia coli branching enzyme; Abad MC et al.; Branching enzyme catalyzes the formation of alpha-1,6 branch points in either glycogen or starch . We report the 2.3-A crystal structure of glycogen branching enzyme from Escherichia coli . The enzyme consists of three major domains, an NH(2)-terminal seven-stranded beta-sandwich domain, a COOH-terminal domain, and a central alpha/beta-barrel domain containing the enzyme active site . While the central domain is similar to that of all the other amylase family enzymes, branching enzyme shares the structure of all three domains only with isoamylase . Oligosaccharide binding was modeled for branching enzyme using the enzyme-oligosaccharide complex structures of various alpha-amylases and cyclodextrin glucanotransferase and residues were implicated in oligosaccharide binding . While most of the oligosaccharides modeled well in the branching enzyme structure, an approximate 50 degrees rotation between two of the glucose units was required to avoid steric clashes with Trp(298) of branching enzyme . A similar rotation was observed in the mammalian alpha-amylase structure caused by an equivalent tryptophan residue in this structure . It appears that there are two binding modes for oligosaccharides in these structures depending on the identity and location of this aromatic residue.

J Biol Chem, 2002 Nov 8, 277(45), 42549 - 56 Epub 2002 Aug 23.
Active site mapping and substrate channeling in the sterol methyltransferase pathway; Nes WD et al.; Sterol methyltransferase (SMT) from Saccharomyces cerevisiae was purified from Escherichia coli BL21(DE3) and labeled with the mechanism-based irreversible inhibitor {3-3H}26,27-dehydrozymosterol (26,27-DHZ) . A 5-kDa tryptic digest peptide fragment containing six acidic residues at positions Glu-64, Asp-65, Glu-68, Asp-79, Glu-82, and Glu-98 was determined to contain the substrate analog covalently attached to Glu-68 by Edman sequencing and radioanalysis using C18 reverse phase high performance liquid chromatography . Site-directed mutagenesis of the six acidic residues to leucine followed by activity assay of the purified mutants confirmed Glu-68 as the only residue to participate in affinity labeling . Equilibration studies indicated that zymosterol and 26,27-DHZ were bound to native and the E68L mutant with similar affinity, whereas S-adenosylmethionine was bound only to the native SMT, K(d) of about 2 microm . Analysis of the incubation products of the wild-type and six leucine mutants by GC-MS demonstrated that zymosterol was converted to fecosterol, 26,27-DHZ was converted to 26-homo-cholesta-8(9),23(24)E,26(26')-trienol as well as 26-homocholesta-8(9),26(26')-3beta,24beta-dienol, and in the case of D79L and E82L mutants, zymosterol was also converted to a new product, 24-methylzymosta-8,25(27)-dienol . The structures of the methylenecyclopropane ring-opened olefins were determined unambiguously by a combination of (1)H and (13)C NMR techniques . A K(m) of 15 microm, K(cat) of 8 x 10(-4) s(-1), and partition ratio of 0.03 was established for 26,27-DHZ, suggesting that the methylenecyclopropane can serve as a lead structure for a new class of antifungal agents . Taken together, partitioning that leads to loss of enzyme function is the result of 26,27-DHZ catalysis forming a highly reactive cationic intermediate that interacts with the enzyme in a region normally not occupied by the zymosterol high energy intermediate as a consequence of less than perfect control . Alternatively, the gain in enzyme function resulting from the production of a delta(25(27))-olefin originates with the leucine substitution directing substrate channeling along different reaction channels in a similar region at the active site.

Genetics, 2002 Aug, 161(4), 1363 - 71
Saturation of DNA mismatch repair and error catastrophe by a base analogue in Escherichia coli; Negishi K et al.; Deoxyribosyl-dihydropyrimido{4,5-c}{1,2}oxazin-7-one (dP) is a potent mutagenic deoxycytidine-derived base analogue capable of pairing with both A and G, thereby causing G . C --> A . T and A . T --> G . C transition mutations . We have found that the Escherichia coli DNA mismatch-repair system can protect cells against this mutagenic action . At a low dose, dP is much more mutagenic in mismatch-repair-defective mutH, mutL, and mutS strains than in a wild-type strain . At higher doses, the difference between the wild-type and the mutator strains becomes small, indicative of saturation of mismatch repair . Introduction of a plasmid containing the E . coli mutL(+) gene significantly reduces dP-induced mutagenesis . Together, the results indicate that the mismatch-repair system can remove dP-induced replication errors, but that its capacity to remove dP-containing mismatches can readily be saturated . When cells are cultured at high dP concentration, mutant frequencies reach exceptionally high levels and viable cell counts are reduced . The observations are consistent with a hypothesis in which dP-induced cell killing and growth impairment result from excess mutations (error catastrophe), as previously observed spontaneously in proofreading-deficient mutD (dnaQ) strains.

Biochem J, 2002 Dec 1, 368(Pt 2), 589 - 95
Molecular cloning, expression and characterization of a novel class glutathione S-transferase from the fungus Cunninghamella elegans; Cha CJ et al.; The structural gene for glutathione S-transferase (CeGST1-1) in the fungus Cunninghamella elegans was cloned by screening a cDNA library using a degenerate oligonucleotide probe based on the N-terminal sequence of the purified protein . Open reading frame analysis indicated that the cegst1 gene encodes a protein of 210 amino acid residues . The deduced amino acid sequence showed 25% sequence identity with the sequence of the Pi-class GST from Danio rerio (zebrafish) . Similarity was also shown with the Alpha-class GST from Fasciola hepatica (liver fluke; 23% identity), the Mu class from Mus musculus (22%) and the Sigma class from Ommastrephes sloani (squid; 21%) . Further screening of a cDNA library with the cegst1 gene probe revealed the presence of another GST isoenzyme (CeGST2-2) in this fungus, which shows 84% sequence identity with CeGST1-1 at the amino acid level . Reverse transcription PCR revealed that cegst2 was also expressed at the mRNA level in the fungus C . elegans . Both cegst genes were overexpressed in Escherichia coli using the expression vector pQE51, displaying specific activities with 1-chloro-2,4-dinitrobenzene of 2.04 and 0.75 micromol/min per mg of protein respectively . Both enzymes exhibited a similar substrate specificity and inhibition profile, indicating that CeGST1-1 and CeGST2-2 belong to the same GST class . Mutagenesis analysis revealed that Tyr(10) in the N-terminal region is essential for catalysis of CeGST1-1 . We propose from these results that the CeGSTs are novel Gamma-class GSTs and designated as GSTG1-1 and GSTG2-2 respectively.

Biochem Soc Trans, 2002 Aug, 30(4), 708 - 10
Bipartite gating in the outer membrane protein FecA; van der Helm D et al.; The recent structure determination of FecA, with and without ligand, shows the existence of two gates . These are the extracellular loops closing over the binding site and the plug located inside the barrel . It indicates a process which is described as bipartite gating and allows for a rational distinction between the binding event and the transport process.

Biochem Soc Trans, 2002 Aug, 30(4), 674 - 80
Metal insertion into NiFe-hydrogenases; Blokesch M et al.; The synthesis and the insertion of the metallocentre of NiFe-hydrogenases is a complex process, in which seven maturation enzymes plus ATP, GTP and carbamoyl phosphate are involved . The review summarizes what is known about the properties and activities of these auxiliary proteins, and postulates a pathway along which maturation may take place.

Biochem Soc Trans, 2002 Aug, 30(4), 646 - 8
Cobalamin (vitamin B(12)) biosynthesis in Rhodobacter capsulatus; McGoldrick H et al.; In Rhodobacter capsulatus, cobalamin biosynthesis has been shown to occur when the bacteria are grown either aerobically or anaerobically . However, a comparison of the main cobalamin biosynthetic operon found within R . capsulatus would suggest that the encoded proteins belong to the oxygen-dependent pathway for cobalamin biosynthesis, although, significantly, no homologue of the essential mono-oxygenase CobG has yet been detected . Nonetheless, within this main cob operon is found a large open reading frame termed orf663 that is not found in any other cobalamin biosynthetic operon . When overproduced in Escherichia coli, orf663 was found to encode a 90 kDa integral membrane protein . Some of this protein is cleaved within E . coli to give a soluble N-terminal region that can easily be purified and yields a 50 kDa flavoprotein . When expressed in harness with the genes for precorrin-3a synthesis, ORF663 appears to mediate the transformation of precorrin-3a into a new chromophoric compound . Another open reading frame in close proximity to orf663 is termed orf647, and was found to encode a 2Fe-2S ferredoxin-like protein . We suggest that these two proteins may provide an alternative oxygen-independent mechanism for ring contraction within R . capsulatus.

Biochem Soc Trans, 2002 Aug, 30(4), 633 - 8
Cytochrome c maturation: a complex pathway for a simple task?
Thony-Meyer L.
Post-translational maturation of c-type cytochromes involves covalent attachment of haem to the apocytochrome polypeptide by two thioether bonds . In bacteria, haem attachment occurs in the periplasm, after the separate translocation of haem and the polypeptide across the cytoplasmic membrane . In Escherichia coli, delivery and attachment of the cofactor requires eight or nine specific proteins, which are believed to be organized in a membrane protein complex . After transport across the membrane, haem is attached covalently to the haem chaperone CcmE in an unusual way at a single histidine residue . However, haem binding to CcmE is transient and is succeeded by a further transfer to apocytochrome c . Both haem binding to and release from CcmE involve integral membrane proteins, CcmC and CcmF respectively, which carry a conserved tryptophan-rich motif in a periplasmic domain . Apocytochrome c polypeptides are synthesized as precursors and reach the periplasm by sec-dependent translocation . There they are prepared for haem binding by reduction of the cysteine residues in the motif Cys-Xaa-Xaa-Cys-His, which is characteristic of such proteins . This reduction is achieved in a thio-reduction pathway, whereby electrons are passed from cytoplasmic thioredoxin to the transmembrane protein DsbD, across the membrane, and on to the specific reductases CcmG/CcmH . The merging of the haem delivery and the thio-reduction pathways leads to the stereospecific insertion of haem into various type c cytochromes.

Biochem Soc Trans, 2002 Aug, 30(4), 601 - 4
Site-directed mutagenesis of Tyr-189 and Lys-193 in NADPH: protochlorophyllide oxidoreductase from Synechocystis; Heyes DJ et al.; NADPH:protochlorophyllide oxidoreductase (POR) catalyses the light-dependent reduction of protochlorophyllide to chlorophyllide, a key regulatory reaction in the chlorophyll biosynthetic pathway . Sequence comparisons have revealed that POR is a member of the short-chain alcohol dehydrogenase family of enzymes . A tyrosine and a lysine residue are conserved throughout all members of this family, and are proposed to be within the active site . This present study describes how site-directed mutagenesis has been used to change Tyr-189 to Phe and Lys-193 to Arg in the Synechocystis POR enzyme . The mutant enzymes were produced with a His tag in Escherichia coli and subsequently purified on a Ni(2+)-affinity column . The two mutations resulted in inactive enzymes, indicating that both residues are crucial for activity . The K(d) value for NADPH binding to the K193R mutant was significantly higher than for the wild-type enzyme, suggesting that the affinity for NADPH has also been reduced.

Biochem Soc Trans, 2002 Aug, 30(4), 584 - 90
5-Aminolaevulinic acid dehydratase: metals, mutants and mechanism; Shoolingin-Jordan PM et al.; 5-Aminolaevulinic acid dehydratase catalyses the formation of porphobilinogen from two molecules of 5-aminolaevulinic acid . The studies described highlight the importance of a bivalent metal ion and two active-site lysine residues for the functioning of 5-aminolaevulinic acid dehydratase . Dehydratases fall into two main categories: zinc-dependent enzymes and magnesium-dependent enzymes . Mutations that introduced zinc-binding ligands into a magnesium-dependent enzyme conferred an absolute requirement for zinc . Mutagenesis of lysine residues 247 and 195 in the Escherichia coli enzyme lead to dramatic effects on enzyme activity, with lysine 247 being absolutely essential . Mutation of either lysine 247 or 195 to cysteine, and treatment of the mutant enzyme with 2-bromethylamine, resulted in the recovery of substantial enzyme activity . The effects of the site-directed alkylating inhibitor, 5-chlorolaevulinic acid, and 4,7-dioxosebacic acid, a putative intermediate analogue, were investigated by X-ray crystallography . These inhibitors reacted with both active-site lysine residues . The role of these two lysine residues in the enzyme mechanism is discussed.

Thromb Haemost, 2002 Aug, 88(2), 242 - 52
Adverse effect of heparin on antithrombin action during endotoxemia: microhemodynamic and cellular mechanisms; Hoffmann JN et al.; A recent clinical sepsis trial reported a significant reduction in 90-day mortality by antithrombin (AT) exclusively in the subgroup of patients without simultaneous heparin prophylaxis . Patients additionally receiving heparin did not benefit from AT treatment . Herein, we studied the microhemodynamic and cellular mechanisms of this adverse effect of heparin on AT actions by the use of intravital microscopy and granulocyte culturing . In Syrian golden hamsters normotensive endotoxemia was induced by 2 mg/kg endotoxin (LPS, E . coli) i.v . In a first group of animals, AT (AT, 250 IU/kg i.v., n = 6) was given 5 min before LPS administration . A second group of animals (Heparin + AT, n = 5) received AT (250 IU/kg i.v.) combined with unfractionated heparin (sodium heparin, 100 IU/kg/24 h, i.v.) . Additional animals (LMWH + AT, n = 5) received AT (250 IU/kg i.v.) combined with LMWH (nadroparin 47.5 IU anti-Xa/kg, s.c., 2 h before LPS) . LPS-treated animals, which received only saline, served as controls (control, n = 6) . Using dorsal skinfold fold preparations, LPS-induced microvascular leukocyte-endothelial cell interaction (LE) and alteration of functional capillary density (FCD) were studied by intravital video fluorescence microscopy . In controls, LPS induced a massive increase in LE with a maximum at 8 h and an impressive decrease in FCD over a 24-hour period . Both LPS effects were effectively prevented by AT treatment (p < 0.01), whereas Heparin + AT and LMWH + AT animals showed microcirculatory alterations comparable to that in controls . In additional in vitro chemotaxis assays . AT blocked neutrophil chemotaxis, an effect reversed by both unfractionated heparin and LMWH . Thus, our study elucidates a relevant in vivo and in vitro unfractionated heparin and LMWH adverse effect in the microcirculatory actions of AT during endotoxemia . These results indicate that heparin should be avoided to permit AT to modulate LPS-induced inflammatory responses.

Thromb Haemost, 2002 Aug, 88(2), 221 - 9
Von Willebrand factor modulates factor VIII immunogenicity: comparative study of different factor VIII concentrates in a haemophilia A mouse model; Behrmann M et al.; The development of an immune response towards factor VIII (FVIII) remains the major complication of haemophilia A replacement therapy . Product-related risk factors have recently been identified on the basis of epidemiological studies, but the mechanism is not understood . To this end, various commercially available FVIII concentrates were administered by the IV route to FVIII-knockout mice and the resulting immune response was characterised . Significantly higher inhibitor titres (Bethesda assay) were observed for one recombinant FVIII and one plasma-derived FVIII product depleted in von Willebrand factor (VWF) . Inhibitor titres were reduced upon pre-incubation of FVIII with purified VWF . Epitope specificity of anti-FVIII IgG was characterised using FVIII-fragments produced in E . coli . Concentrates with no or reduced VWF-level elicited antibodies recognising predominantly the acidic a1 and a3 regions . Addition of VWF prior to injection also modified the epitope specificity . FVIII concentrates, therefore, show qualitative and quantitative variations in immunogenicity, which are at least partly modulated by VWF.

J Infect Dis, 2002 Sep 1, 186(5), 644 - 51 Epub 2002 Aug 09.
Serologic responses to epitopes of the major surface glycoprotein of Pneumocystis jiroveci differ in human immunodeficiency virus-infected and uninfected persons; Daly KR et al.; The major surface glycoprotein (Msg) of Pneumocystis jiroveci (P . jiroveci) is important in the immunopathogenesis of Pneumocystis pneumonia (PcP), but is difficult to study in humans . We generated 3 overlapping recombinant Msg fragments (MsgA, MsgB and MsgC), and analyzed their reactivity with serum samples from 95 healthy blood donors and 94 human immunodeficiency virus (HIV)-infected persons . Reactivity to the Msg fragments varied with HIV infection and prior episodes of PcP but not with geographic origin . Recognition of MsgA was lower-and recognition of MsgB was significantly lower-in HIV(+) serum compared with donor serum . Serum samples from HIV-positive patients with prior PcP recognized MsgC more frequently than did serum samples from those without PcP . None of the serum samples drawn from 9 patients before they had developed PcP recognized MsgC . These data suggest that these novel recombinant proteins are useful for the analysis of antibody responses to Msg.

Dev Cell, 2002 Aug, 3(2), 271 - 82
Escrt-III: an endosome-associated heterooligomeric protein complex required for mvb sorting; Babst M et al.; The sorting of transmembrane proteins (e.g., cell surface receptors) into the multivesicular body (MVB) pathway to the lysosomal/vacuolar lumen requires the function of the ESCRT protein complexes . The soluble coiled-coil-containing proteins Vps2, Vps20, Vps24, and Snf7 are recruited from the cytoplasm to endosomal membranes where they oligomerize into a protein complex, ESCRT-III . ESCRT-III contains two functionally distinct subcomplexes . The Vps20-Snf7 subcomplex binds to the endosomal membrane, in part via the myristoyl group of Vps20 . The Vps2-Vps24 subcomplex binds to the Vps20-Snf7 complex and thereby serves to recruit additional cofactors to this site of protein sorting . We provide evidence for a role for ESCRT-III in sorting and/or concentration of MVB cargoes.

Int J Radiat Biol, 2002 Aug, 78(8), 733 - 41
Modification of ionizing radiation clustered damage: estimate of the migration distance of holes through DNA via guanyl radicals under physiological conditions; Milligan JR et al.; PURPOSE: Guanyl radicals are produced in DNA when it is subjected to oxidation or ionizing radiation . The sites at which stable products can be identified can be located dozens of base pairs away from the initial site of the electron loss . This migration will modify the spatial distribution of damage and tends to mitigate the clustering of initial damage generally associated with ionizing radiation . The migration distance is presumably a function of the lifetime of the intermediate guanyl radical, and we wished to quantify the relationship between them . MATERIALS AND METHODS: Aqueous solutions containing plasmid DNA and thiocyanate ions were treated with gamma-irradiation . These conditions result in the very efficient production of guanyl radicals in the plasmid . We quantified the formation of stable guanine oxidation products in the plasmid as strand breaks by using the E . coli base excision repair endonuclease formamidopyrimidine-DNA N-glycosylase (FPG) . The effect of two additives on the yield of guanine oxidation, nitrite ions and the DNA binding ligand doxorubicin (adriamycin), were examined . RESULTS: The presence during irradiation of the DNA-binding ligand doxorubicin attenuated the yields of stable oxidized guanine products formed . The additional presence of nitrite decreased this effect of doxorubicin . CONCLUSION: Because doxorubicin binds strongly to DNA, its ability to attenuate guanine oxidation can be interpreted in terms of the migration distance of the intermediate guanyl radical . Because nitrite repairs these intermediate guanyl radicals by electron transfer, its presence during irradiation decreases their lifetime . Therefore, we derived an estimate of the migration distance of guanyl radicals as a function of their lifetime . The presence in cells of antioxidants such as glutathione sets an upper limit to the likely lifetime and, therefore, the migration distance of guanyl radicals . It was concluded that the migration of guanyl radicals may not decrease the clustering of DNA damage in vivo to a great extent.

Ann Trop Med Parasitol, 2002 Jul, 96(5), 469 - 76
The identification, cloning and functional expression of the gene encoding orotidine 5'-monophosphate (OMP) decarboxylase from Plasmodium falciparum; Menz RI et al.; The coding region of a putative orotidine 5'-monophosphate decarboxylase gene from Plasmodium falciparum was identified in genomic data from the Malarial Genome Sequencing Project . The gene encodes a protein of 323 amino acids with a predicted molecular weight of 37.8 kDa . The gene was cloned into a bacterial expression vector and over-expressed in Escherichia coli . The recombinant protein was purified and shown to have orotidine 5'-monophosphate decarboxylase activity, confirming the identity of the gene.

Radiat Prot Dosimetry, 2002, 99(1-4), 109 - 12
Simulation of 125I induced DNA strand breaks in a CAP-DNA complex; Li W et al.; The E . coli catabolite gene activator protein (CAP)-DNA complex with 125I located at the position of the H5 atom of the cytosine near the centre was incorporated into the PARTRAC track structure code . DNA strand breaks due to irradiation were calculated by track structure and radical attack simulations; strand breaks due to neutralisation of the highly charged 125Te ion were derived from a semi-empirical distribution . According to the calculations, the neutralisation effect dominates the strand breakage frequency at 2 bases away from the 125I decay site on both strands . The first breakage distribution counted from a 32P labelled end on the strand with 125I agreed well with experimental data, but on the opposite strand, the calculated distribution is more concentrated around the decay site and its yield is about 20% larger than the measured data.

Am J Obstet Gynecol, 2002 Aug, 187(2), 469 - 74
Inefficient transduction of sheep in utero after intra-amniotic injection of retroviral producer cells; Galan HL et al.; OBJECTIVE: Our purpose was to test the hypothesis that the intra-amniotic injection of a retroviral vector producer cell line into pregnant sheep will result in retrovirus-mediated transduction and stable gene transfer to the ovine fetus . STUDY DESIGN: Thirteen pregnant ewes at various gestational ages underwent amniocentesis and injection of cells producing the retrovirus vector LIRESgeo, which is derived from Maloney murine leukemia virus and encodes Escherichia coli LacZ (beta-galactosidase) as a marker gene . Pregnant ewes and fetuses were killed, and amniotic fluid, placenta, and fetal tissues were collected and assayed for transgene expression 7 to 77 days after intraamniotic injection . In addition, serum was collected and analyzed for evidence of specific immune responses against the producer cells, and amniotic fluid was collected and analyzed for deleterious effects on producer cell viability, vector production, and vector transduction . RESULTS: Only 1 of 10 fetuses exposed to the retroviral producer cells demonstrated beta-galactosidase activity that correlated with positive immunohistochemistry for LacZ in lung, trachea, pancreas, and small intestine . However, the presence of the LacZ gene could not be confirmed by polymerase chain reaction . Thus, we could not confirm transduction after any of the injections . The retroviral producer cells survived well in amniotic fluid and continued to produce high levels of retroviral vector after intra-amniotic injection, although amniotic fluid inhibited the transducing activity of the vector in a manner dependent on gestational age . CONCLUSIONS: Intra-amniotic retroviral gene transfer with the use of these amphotropic producer cells does not result in reproducible gene transfer in the ovine fetus although amniotic fluid sustains producer cell viability and vector production . Possible reasons for the inefficient transduction are discussed.

J Immunol, 2002 Sep 1, 169(5), 2368 - 73
Distinct response of human B cell subpopulations in recognition of an innate immune signal, CpG DNA; Jung J et al.; Innate immunity has recently gained renewed interest in its ability to regulate adaptive immunity . Among the innate immune signals, CpG DNA has revealed its potential as a vaccine adjuvant . However, the cellular mechanism for the effect of CpG DNA on the humoral immune response is not well understood . Here, we investigated the effects of CpG DNA on human B cell differentiation using highly purified B cell subsets: naive, germinal center (GC), and memory B cells . In the in vitro culture system that mimics the primary or secondary immune response in vivo, CpG DNA markedly augmented the proliferation and generation of plasma cells from naive and memory B cells . CpG DNA dramatically increased plasma cell generation from GC B cells . However, CpG DNA did not have effect on memory B cell generation from GC B cells . These results suggest that CpG DNA potentiates the B cell adaptive immune response by enhancing terminal differentiation, but does not affect the generation of memory B cells.

Pediatr Res, 2002 Sep, 52(3), 356 - 62
Postnatal lung inflammation increased by ventilation of preterm lambs exposed antenatally to Escherichia coli endotoxin; Ikegami M et al.; Chorioamnionitis resulting in exposure of the fetal lung to inflammation is frequent before preterm delivery . The initiation of mechanical ventilation in the preterm recruits granulocytes to the lungs and increases proinflammatory cytokine expression in the lungs . We hypothesized that when the prematurely born newborn with chorioamnionitis was ventilated, inflammation would increase . Therefore, we asked whether inflammatory exposure to the fetal lung caused by intra-amniotic endotoxin (10 mg, Escherichia coli 055:beta 5) given at 100 d gestation would alter the inflammatory responses to the mechanical ventilation in surfactant-treated preterm lambs delivered at 130 d gestation . Cells in alveolar washes, proinflammatory cytokine expression, and surfactant protein mRNA expression were not different for saline and endotoxin exposed lambs that were not ventilated . The endotoxin- and saline-exposed control animals had similar lung function for 6 h of ventilation . Bronchoalveolar lavage fluid from the ventilated and antenatal endotoxin-exposed animals contained 5.7 times more monocytes, 12 times more lymphocytes, and a nonsignificant increase in neutrophils . Cells from the bronchoalveolar lavage fluid expressed 3-fold more IL-6 and IL-8 mRNA than did cells from the saline exposed comparison animals . An antenatal exposure of the fetal lung to endotoxin enhanced the subsequent inflammatory response of the ventilated preterm lung.

Proc Natl Acad Sci U S A, 2002 Sep 3, 99(18), 11682 - 7 Epub 2002 Aug 22.
Using mechanical force to probe the mechanism of pausing and arrest during continuous elongation by Escherichia coli RNA polymerase; Forde NR et al.; Escherichia coli RNA polymerase translocates along the DNA discontinuously during the elongation phase of transcription, spending proportionally more time at some template positions, known as pause and arrest sites, than at others . Current models of elongation suggest that the enzyme backtracks at these locations, but the dynamics are unresolved . Here, we study the role of lateral displacement in pausing and arrest by applying force to individually transcribing molecules . We find that an assisting mechanical force does not alter the translocation rate of the enzyme, but does reduce the efficiency of both pausing and arrest . Moreover, arrested molecules cannot be rescued by force, suggesting that arrest occurs by a bipartite mechanism: the enzyme backtracks along the DNA followed by a conformational change of the ternary complex (RNA polymerase, DNA and transcript), which cannot be reversed mechanically.

J Bacteriol, 2002 Sep, 184(18), 5170 - 3
ExbB and ExbD do not function independently in TonB-dependent energy transduction; Held KG et al.; ExbB and ExbD proteins are part of the TonB-dependent energy transduction system and are encoded by the exb operon in Escherichia coli . TonB, the energy transducer, appears to go through a cycle during energy transduction, with the absence of both ExbB and ExbD creating blocks at two points: (i) in the inability of TonB to respond to the cytoplasmic membrane proton motive force and (ii) in the conversion of TonB from a high-affinity outer membrane association to a high-affinity cytoplasmic membrane association . The recent observation that ExbB exists in 3.5-fold molar excess relative to the molarity of ExbD in E . coli suggests the possibility of two types of complexes, those containing both ExbB and ExbD and those containing only ExbB . Such distinct complexes might individually manifest one of the two activities described above . In the present study this hypothesis was tested and rejected . Specifically, both ExbB and ExbD were found to be required for TonB to conformationally respond to proton motive force . Both ExbB and ExbD were also required for association of TonB with the cytoplasmic membrane . Together, these results support an alternative model where all of the ExbB in the cell occurs in complex with all of the ExbD in the cell . Based on recently determined cellular ratios of TonB system proteins, these results suggest the existence of a cytoplasmic membrane complex that may be as large as 520 kDa.

J Bacteriol, 2002 Sep, 184(18), 5096 - 103
Differential expression and localization of Mn and Fe superoxide dismutases in the heterocystous cyanobacterium Anabaena sp . strain PCC 7120; Li T et al.; Superoxide dismutases (Sods) play very important roles in preventing oxidative damages in aerobic organisms . The nitrogen-fixing heterocystous cyanobacterium Anabaena sp . strain PCC 7120 has two Sod-encoding genes: a sodB, encoding a soluble iron-containing Sod (FeSod), and a sodA, encoding a manganese-containing Sod (MnSod) . The FeSod was purified and characterized . A recombinant FeSod was also obtained by overproduction in Escherichia coli . Immunoblot study of the FeSod during induction of heterocyst differentiation showed that the cells produced six- to eightfold more FeSod 8 h after a shift from a nitrogen-replete condition to a nitrogen-depleted condition . However, the amount of FeSod protein in filaments with mature heterocysts was the same as that in filaments grown with combined nitrogen . Superoxide anion-generating chemicals such as methyl viologen did not induce upregulation of the sodB gene expression . The predicted preprotein of the sodA gene has a leader peptide and a motif for membrane attachment at the N terminus of the mature protein . Activity staining after gel electrophoresis of the purified thylakoid membranes showed that most of the MnSod in Anabaena sp . strain PCC 7120 was located on thylakoids toward the lumenal side . Expression of the sodA gene in E . coli shows that the leader peptide was required for its activity and the membrane localization of the MnSod . Northern hybridization detected one 0.82-kb transcript of sodA . The sodA gene was upregulated by methyl viologen, whereas its amount was unchanged during heterocyst differentiation . Immunoblotting and activity staining showed that isolated heterocysts contained a lower but still significant amount of FeSod, suggesting that its function is required in heterocysts . No MnSod was observed in isolated heterocysts . These results show that the two different Sod proteins have differentiated roles in Anabaena sp . strain PCC 7120.

J Bacteriol, 2002 Sep, 184(18), 5077 - 87
Role of ppGpp in rpoS stationary-phase regulation in Escherichia coli; Hirsch M et al.; The bacterial sigma factor RpoS is strongly induced under a variety of stress conditions and during growth into stationary phase . Here, we used rpoS-lac fusions in Escherichia coli to investigate control acting at the level of RpoS synthesis, which is especially evident when cells approach stationary phase in rich medium . Previous work has shown that the small molecule ppGpp is required for normal levels of RpoS in stationary phase . Despite the attraction of a model in which the ppGpp level controls stationary-phase induction of RpoS, careful measurement of rpoS-lac expression in a mutant lacking ppGpp showed similar effects during both exponential growth and stationary phase; the main effect of ppGpp was on basal expression . In addition, a modest regulatory defect was associated with the mutant lacking ppGpp, delaying the time at which full expression was achieved by 2 to 3 h . Deletion analysis showed that the defect in basal expression was distributed over several sequence elements, while the regulatory defect mapped to the region upstream of the rpoS ribosome-binding site (RBS) that contains a cis-acting antisense element . A number of other genes that have been suggested as regulators of rpoS were tested, including dksA, dsrA, barA, ppkx, and hfq . With the exception of the dksA mutant, which had a modest defect in Luria-Bertani medium, none of these mutants was defective for rpoS stationary-phase induction . Even a short rpoS segment starting at 24 nucleotides upstream of the AUG initiation codon was sufficient to confer substantial stationary-phase regulation, which was mainly posttranscriptional . The effect of RBS-proximal sequence was independent of all known trans-acting factors, including ppGpp.

J Bacteriol, 2002 Sep, 184(18), 5058 - 66
Temperature- and H-NS-dependent regulation of a plasmid-encoded virulence operon expressing Escherichia coli hemolysin; Madrid C et al.; Proteins H-NS and Hha form a nucleoprotein complex that modulates expression of the thermoregulated hly operon of Escherichia coli . We have been able to identify two H-NS binding sites in the hly regulatory region . One of them partially overlaps the promoter region (site II), and the other is located about 2 kbp upstream (site I) . In contrast, Hha protein did not show any preference for specific sequences . In vitro, temperature influences the affinity of H-NS for a DNA fragment containing both binding sites and H-NS-mediated repression of hly operon transcription . Deletion analysis of the hly regulatory region confirms the relevance of site I for thermoregulation of this operon . We present a model to explain the temperature-modulated repression of the hly operon, based on the experiments reported here and other, preexisting data.

J Bacteriol, 2002 Sep, 184(18), 5052 - 7
Mutational eidence for a functional connection between two domains of 23S rRNA in translation termination; Arkov AL et al.; Nucleotide 1093 in domain II of Escherichia coli 23S rRNA is part of a highly conserved structure historically referred to as the GTPase center . The mutation G1093A was previously shown to cause readthrough of nonsense codons and high temperature-conditional lethality . Defects in translation termination caused by this mutation have also been demonstrated in vitro . To identify sites in 23S rRNA that may be functionally associated with the G1093 region during termination, we selected for secondary mutations in 23S rRNA that would compensate for the temperature-conditional lethality caused by G1093A . Here we report the isolation and characterization of such a secondary mutation . The mutation is a deletion of two consecutive nucleotides from helix 73 in domain V, close to the peptidyltransferase center . The deletion results in a shortening of the CGCG sequence between positions 2045 and 2048 by two nucleotides to CG . In addition to restoring viability in the presence of G1093A, this deletion dramatically decreased readthrough of UGA nonsense mutations caused by G1093A . An analysis of the amount of mutant rRNA in polysomes revealed that this decrease cannot be explained by an inability of G1093A-containing rRNA to be incorporated into polysomes . Furthermore, the deletion was found to cause UGA readthrough on its own, thereby implicating helix 73 in termination for the first time . These results also indicate the existence of a functional connection between the G1093 region and helix 73 during translation termination.

J Bacteriol, 2002 Sep, 184(18), 5045 - 51
Isoprenoid biosynthesis in Synechocystis sp . strain PCC6803 is stimulated by compounds of the pentose phosphate cycle but not by pyruvate or deoxyxylulose-5-phosphate; Ershov YV et al.; The photosynthetic cyanobacterium Synechocystis sp . strain PCC6803 possesses homologs of known genes of the non-mevalonate 2-C-methyl-D-erythritol 2-phosphate (MEP) pathway for synthesis of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) . Isoprenoid biosynthesis in extracts of this cyanobacterium, measured by incorporation of radiolabeled IPP, was not stimulated by pyruvate, an initial substrate of the MEP pathway in Escherichia coli, or by deoxyxylulose-5-phosphate, the first pathway intermediate in E . coli . However, high rates of IPP incorporation were obtained with addition of dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GA3P), as well as a variety of pentose phosphate cycle compounds . Fosmidomycin (at 1 micro M and 1 mM), an inhibitor of deoxyxylulose-5-phosphate reductoisomerase, did not significantly inhibit phototrophic growth of the cyanobacterium, nor did it affect {(14)C}IPP incorporation stimulated by DHAP plus GA3P . To date, it has not been possible to unequivocally demonstrate IPP isomerase activity in this cyanobacterium . The combined results suggest that the MEP pathway, as described for E . coli, is not the primary path by which isoprenoids are synthesized under photosynthetic conditions in Synechocystis sp . strain PCC6803 . Our data support alternative routes of entry of pentose phosphate cycle substrates derived from photosynthesis.

J Bacteriol, 2002 Sep, 184(18), 5027 - 35
A novel histidine-rich CPx-ATPase from the filamentous cyanobacterium Oscillatoria brevis related to multiple-heavy-metal cotolerance; Tong L et al.; A novel gene related to heavy-metal transport was cloned and identified from the filamentous cyanobacterium Oscillatoria brevis . Sequence analysis of the gene (the Bxa1 gene) showed that its product possessed high homology with heavy-metal transport CPx-ATPases . The CPC motif, which is proposed to form putative cation transduction channel, was found in the sixth transmembrane helix . However, instead of the CXXC motif that is present in the N termini of most metal transport CPx-ATPases, Bxa1 contains a unique Cys-Cys (CC) sequence element and histidine-rich motifs as a putative metal binding site . Northern blotting and real-time quantitative reverse transcription-PCR showed that expression of Bxa1 mRNA was induced in vivo by both monovalent (Cu(+) and Ag(+)) and divalent (Zn(2+) and Cd(2+)) heavy-metal ions at similar levels . Experiments on heavy-metal tolerance in Escherichia coli with recombinant Bxa1 demonstrated that Bxa1 conferred resistance to both monovalent and divalent heavy metals . This is the first report of a CPx-ATPase responsive to both monovalent and divalent heavy metals.

J Bacteriol, 2002 Sep, 184(18), 5018 - 26
Carbonic anhydrase is essential for growth of Ralstonia eutropha at ambient CO(2) concentrations; Kusian B et al.; Mutant strain 25-1 of the facultative chemoautotroph Ralstonia eutropha H16 had previously been shown to exhibit an obligately high-CO(2)-requiring (HCR) phenotype . Although the requirement varied with the carbon and energy sources utilized, none of these conditions allowed growth at the air concentration of CO(2) . In the present study, a gene designated can and encoding a beta-carbonic anhydrase (CA) was identified as the site altered in strain 25-1 . The mutation caused a replacement of the highly conserved glycine residue 98 by aspartate in Can . A can deletion introduced into wild-type strain H16 generated mutant HB1, which showed the same HCR phenotype as mutant 25-1 . Overexpression of can in Escherichia coli and mass spectrometric determination of CA activity demonstrated that can encodes a functional CA . The enzyme is inhibited by ethoxyzolamide and requires 40 mM MgSO(4) for maximal activity . Low but significant CA activities were detected in wild-type H16 but not in mutant HB1, strongly suggesting that the CA activity of Can is essential for growth of the wild type in the presence of low CO(2) concentrations . The HCR phenotype of HB1 was overcome by complementation with heterologous CA genes, indicating that growth of the organism at low CO(2) concentrations requires sufficient CA activity rather than the specific function of Can . The metabolic function(s) depending on CA activity remains to be identified.

J Biol Chem, 2002 Nov 1, 277(44), 41448 - 54 Epub 2002 Aug 21.
Importance of glutamate 279 for the coenzyme binding of human glutamate dehydrogenase; Yoon HY et al.; Although the structure of glutamate dehydrogenase (GDH) has been reported from various sources including mammalian GDH, there are conflicting views regarding the location and mechanism of actions of the coenzyme binding . We have expanded these speculations by photoaffinity labeling and cassette mutagenesis . Photoaffinity labeling with a specific probe, {(32)P}nicotinamide 2-azidoadenosine dinucleotide, was used to identify the NAD(+) binding site within human GDH encoded by the synthetic human GDH gene and expressed in Escherichia coli as a soluble protein . Photolabel-containing peptides generated with trypsin were isolated by immobilized boronate affinity chromatography . Photolabeling of these peptides was most effectively prevented by the presence of NAD(+) during photolysis, demonstrating a selectivity of the photoprobe for the NAD(+) binding site . Amino acid sequencing and compositional analysis identified Glu(279) as the site of photoinsertion into human GDH, suggesting that Glu(279) is located at or near the NAD(+) binding site . The importance of the Glu(279) residue in the binding of NAD(+) was further examined by cassette mutagenesis with mutant enzymes containing Arg, Gly, Leu, Met, or Tyr at position 279 . The mutagenesis at Glu(279) has no effects on the expression or stability of the different mutants . The K(m) values for NAD(+) were 10-14-fold greater for the mutant GDHs than for wild-type GDH, whereas the V(max) values were similar for wild-type and mutant GDHs . The efficiency (k(cat)/K(m)) of the mutant GDH was reduced up to 18-fold . The decreased efficiency of the mutants results from the increase in K(m) values for NAD(+) . In contrast to the K(m) values for NAD(+), wild-type and mutant GDHs show similar K(m) values for glutamate, indicating that substitution at position 279 had no appreciable effect on the affinity of enzyme for glutamate . There were no differences in sensitivities to ADP activation and GTP inhibition between wild-type and mutant GDH, suggesting that Glu(279) is not directly involved in allosteric regulation . The results with photoaffinity labeling and cassette mutagenesis studies suggest that Glu(279) plays an important role for efficient binding of NAD(+) to human GDH.

J Biol Chem, 2002 Nov 8, 277(45), 43050 - 7 Epub 2002 Aug 21.
Randomization of the entire active-site helix alpha 1 of the thiol-disulfide oxidoreductase DsbA from Escherichia coli; Philipps B et al.; DsbA from Escherichia coli is the most oxidizing member of the thiol-disulfide oxidoreductase family (E(o)' = -122 mV) and is required for efficient disulfide bond formation in the periplasm . The reactivity of the catalytic disulfide bond (Cys(30)-Pro(31)-His(32)-Cys(33)) is primarily due to an extremely low pK(a) value (3.4) of Cys(30), which is stabilized by the partial positive dipole charge of the active-site helix alpha1 (residues 30-37) . We have randomized all non-cysteine residues of helix alpha1 (residues 31, 32, and 34-37) and found that two-thirds of the resulting variants complement DsbA deficiency in a dsbA deletion strain . Sequencing of 98 variants revealed a large number of non-conservative replacements in active variants, even at well conserved positions . This indicates that tertiary structure context strongly determines alpha-helical secondary structure formation of the randomized sequence . A subset of active and inactive variants was further characterized . All these variants were more reducing than wild type DsbA, but the redox potentials of active variants did not drop below -210 mV . All inactive variants had redox potentials lower than -210 mV, although some of the inactive proteins were still re-oxidized by DsbB . This demonstrates that efficient oxidation of substrate polypeptides is the crucial property of DsbA in vivo.

Biol Reprod, 2002 Sep, 67(3), 953 - 60
Identification of genes encoding mouse oocyte secretory and transmembrane proteins by a signal sequence trap; Taft RA et al.; The oocyte plays a key role in follicular development . At all stages of follicular development, oocytes interact with surrounding granulosa cells and promote their differentiation into the types of cells that support further oocyte growth and developmental competence . These interactions suggest the existence of an oocyte-granulosa cell regulatory loop that includes both secreted proteins and cell surface receptors on both cell types . Factors involved in the regulatory loop will therefore contain a signal sequence, which can be used to identify them through a signal sequence trap (SST) . A screen of an oocyte SST library identified three classes of oocyte-expressed sequences: known mouse genes, sequences homologous to known mammalian genes, and novel sequences of unknown function . Many of the recovered genes may have roles in the oocyte-granulosa cell regulatory loop . For several of the known mouse genes, new roles in follicular development are implied by identification of their expression, for the first time, in the oocyte . The future characterization of novel sequences may lead to the identification of novel proteins participating in the regulatory loop.

BMC Immunol . 2002 Aug 22;3(1):10.
The kappa B transcriptional enhancer motif and signal sequences of V(D)J recombination are targets for the zinc finger protein HIVEP3/KRC: a site selection amplification binding study; Allen CE et al.; BACKGROUND: The ZAS family is composed of proteins that regulate transcription via specific gene regulatory elements . The amino-DNA binding domain (ZAS-N) and the carboxyl-DNA binding domain (ZAS-C) of a representative family member, named kappaB DNA binding and recognition component (KRC), were expressed as fusion proteins and their target DNA sequences were elucidated by site selection amplification binding assays, followed by cloning and DNA sequencing . The fusion proteins-selected DNA sequences were analyzed by the MEME and MAST computer programs to obtain consensus motifs and DNA elements bound by the ZAS domains . RESULTS: Both fusion proteins selected sequences that were similar to the kappaB motif or the canonical elements of the V(D)J recombination signal sequences (RSS) from a pool of degenerate oligonucleotides . Specifically, the ZAS-N domain selected sequences similar to the canonical RSS nonamer, while ZAS-C domain selected sequences similar to the canonical RSS heptamer . In addition, both KRC fusion proteins selected oligonucleoties with sequences identical to heptamer and nonamer sequences within endogenous RSS . CONCLUSIONS: The RSS are cis-acting DNA motifs which are essential for V(D)J recombination of antigen receptor genes . Due to its specific binding affinity for RSS and kappaB-like transcription enhancer motifs, we hypothesize that KRC may be involved in the regulation of V(D)J recombination.

Scand J Immunol, 2002 Sep, 56(3), 260 - 9
Endomorphins 1 and 2 inhibit IL-10 and IL-12 production and innate immune functions, and potentiate NF-kappaB DNA binding in THP-1 differentiated to macrophage-like cells; Azuma Y et al.; We evaluated immunological effects of opioid peptides endomorphins 1 and 2 on the production of interleukin-10 (IL-10) and IL-12 cytokines, functions related to innate immunity and NF-kappaB DNA binding in human cell line THP-1 . Endomorphins 1 and 2 inhibited lipopolysaccharide (LPS)-stimulated IL-10 and IL-12 production in THP-1 differentiated to macrophage-like cells by phorbol 12-myristate 13-acetate (PMA) . Similarly, they suppressed LPS-stimulated IL-10 and IL-12 production in THP-1 matured to monocytes by 1alpha,25-dihydroxyvitamin D3 . In addition, endomorphins 1 and 2 led to marked potentiation of NF-kappaB binding in THP-1 differentiated to macrophage-like cells . Furthermore, these endomorphins further potentiated LPS-induced NF-kappaB binding . Moreover, they inhibited chemotaxis, phagocytosis of Escherichia coli and PMA-stimulated production of hydrogen peroxide in THP-1 differentiated to macrophage-like cells . These results suggest that endomorphins 1 and 2 may inhibit THP-1 functions, such as cytokine production and functions related to innate immune, and potentiate NF-kappaB DNA binding in THP-1.

J Nat Prod, 2002 Aug, 65(8), 1190 - 3
New and biologically active imidazole alkaloids from two sponges of the genus Leucetta; Gross H et al.; Chemical investigation of two sponges, Leucetta chagosensis and Leucetta cf chagosensis, collected from the Great Barrier Reef and the Fiji Islands, respectively, has led to the isolation of three new imidazole alkaloids (1-3), along with the known compounds isonaamine B (4) and naamine A (5) . The structures of the new compounds (1-3) were elucidated by employing spectroscopic techniques (NMR, MS, UV, and IR) . The structures of the known compounds 4 and 5 were determined by comparison of their (1)H and (13)C NMR spectroscopic data with published values . Compounds 1 and 2 were found to be cytotoxic toward several tumor cell lines (GI(50) values ranged from 1.3 to 7.0 microg/mL).

Sheng Wu Gong Cheng Xue Bao, 2002 May, 18(3), 339 - 42
{Site-directed mutation of PoIFN-alpha and its expression in Escherichia coli}; Chen T et al.; By using huge primer PCR Cys86 (TGC) of PoIFN-alpha was mutated to Tyr(TAC), and the first code TGT was simultaneously changed to TGC, which is a bias code of E . coli . The expression plasmid pGEX-IFN was constructed successfully . Recombinant porcine IFN alpha, which is expressed as inclusion bodies, was about 20% of the total proteins . The inclusion body was dissolved in 8 mol/L urea and subsequently renatured by dilution in refolding buffer . In order to obtain pure protein, the renatured IFN alpha was purified by FPLC, and the cytokine activity (5200 IU/mg) was verified by inhibiting the cytopathic effect.

Sheng Wu Gong Cheng Xue Bao, 2002 May, 18(3), 304 - 7
{Overexpression and detection of the mutated glucose isomerase GIG138P and GIG138P-G247D in Streptomyces lividans}; Zhu GP et al.; The shuttle expression vectors pHZGI1 and pHZGI2 were successfully constructed by inserting structural genes of GI containing single mutated site G138P and double mutated site G138P-G247D into E . coli-Streptomyces shuttle vector pHZ-1272, respectively . Then they were transformed into S . lividans TK54 strain by protoplast transformation . SDS-PAGE indicated that two shuttle vectors in TK54 strain expressed obviously specific bands at 42.5 kD after inducted by 2 micrograms/mL thiostrepton . Optical densitometric scan showed that the content of the mutant enzymes GIG138P and GIG138P-G247D were about 19% and 22% of dissoluble proteins, respectively . Western blotting farther proved that GIG138P and GIG138P-G247D were expressed in S . lividans TK54.

Sheng Wu Gong Cheng Xue Bao, 2002 May, 18(3), 291 - 4
{Refolding and purification of the huGM-CSF(9-127)-IL-6(29-184) fusion protein}; Sun QM et al.; The huGM-CSF(9-127)-IL-6(29-184) fusion protein was precipitated on column when being purified by Q Sepharose H.P . ion exchange chromatography after renaturation by dilution . To solve this problem, a novel purification and refolding strategy was adopted . Inclusion bodies was first purified by Q Sepharose H.P . ion exchange in 8 mol/L urea, followed by in situ refolding on column by Sephacryl S-200 . Renatured fusion protein was obtained in a purity of more than 95% . It was showed that the method of refolding on gel filtration column is efficient, with relative refolding rate at 80% . By the whole procedure, refolding and purification of recombinant protein can be performed within one day . This strategy is also promising to be applied in large scale purification and refolding of recombinant protein from inclusion bodies in E . coli.

Sheng Wu Gong Cheng Xue Bao, 2002 May, 18(3), 261 - 6
{Escherichia coli disulfide-forming related proteins: structures, functions and their application in gene engineering for expressing heterologous proteins in Escherichia coli}; Zhang Z et al.; The formation of disulfide bonds in secreted proteins of E . coli is a synergetic process depending on a series of Dsb proteins containing DsbA, DsbB, DsbC, DsbD, DsbE and DsbG . DsbA functions as an oxidant to form a disulfide bond between two -SH- in vivo and DsbB reactivates DsbA by reoxidizing it . Both DsbC and DsbG, two periplasmic proteins with isomerase activity, can correct mis-paired disulfide bonds introduced by DsbA although they recognize different substrates . DsbD, an inner membrane protein, plays a role in reducing DsbC and DsbG in vivo . It is regarded that DsbE has the similar function with DsbD . All DsbA, DsbC and DsbG have chaperone activity besides involving in the formation of disulfide bonds . Furthermore, their chaperone activity can promote the formation of protein disulfide bonds . There are a few reports dealing with soluble expression of heterologous proteins containing disulfide bonds assisted by DsbA and DsbC in E . coli . So far there has been no reports about the soluble expression of heterologous proteins promoted by DsbG . Our experiments first demonstrated that both DsbC and DsbG can improve the expression of single chain antibodies as soluble and functional forms in E . coli, and DsbG has additive effects with DsbC.

Nature, 2002 Aug 22, 418(6900), 880 - 4
Multiple regulatory sites in large-conductance calcium-activated potassium channels; Xia XM et al.; Large conductance, Ca(2+)- and voltage-activated K(+) channels (BK) respond to two distinct physiological signals -- membrane voltage and cytosolic Ca(2+) (refs 1, 2) . Channel opening is regulated by changes in Ca(2+) concentration spanning 0.5 micro M to 50 mM (refs 2-5), a range of Ca(2+) sensitivity unusual among Ca(2+)-regulated proteins . Although voltage regulation arises from mechanisms shared with other voltage-gated channels, the mechanisms of Ca(2+) regulation remain largely unknown . One potential Ca(2+)-regulatory site, termed the 'Ca(2+) bowl', has been located to the large cytosolic carboxy terminus . Here we show that a second region of the C terminus, the RCK domain (regulator of conductance for K(+) (ref . 12)), contains residues that define two additional regulatory effects of divalent cations . One site, together with the Ca(2+) bowl, accounts for all physiological regulation of BK channels by Ca(2+); the other site contributes to effects of millimolar divalent cations that may mediate physiological regulation by cytosolic Mg(2+) (refs 5, 13) . Independent regulation by multiple sites explains the large concentration range over which BK channels are regulated by Ca(2+) . This allows BK channels to serve a variety of physiological roles contingent on the Ca(2+) concentration to which the channels are exposed.

Nature, 2002 Aug 22, 418(6900), 876 - 80
Mechanism of magnesium activation of calcium-activated potassium channels; Shi J et al.; Large-conductance (BK type) Ca(2+)-dependent K(+) channels are essential for modulating muscle contraction and neuronal activities such as synaptic transmission and hearing . BK channels are activated by membrane depolarization and intracellular Ca(2+) and Mg(2+) (refs 6-10) . The energy provided by voltage, Ca(2+) and Mg(2+) binding are additive in activating the channel, suggesting that these signals open the activation gate through independent pathways . Here we report a molecular investigation of a Mg(2+)-dependent activation mechanism . Using a combined site-directed mutagenesis and structural analysis, we demonstrate that a structurally new Mg(2+)-binding site in the RCK/Rossman fold domain -- an intracellular structural motif that immediately follows the activation gate S6 helix -- is responsible for Mg(2+)-dependent activation . Mutations that impair or abolish Mg(2+) sensitivity do not affect Ca(2+) sensitivity, and vice versa . These results indicate distinct structural pathways for Mg(2+)- and Ca(2+)-dependent activation and suggest a possible mechanism for the coupling between Mg(2+) binding and channel opening.

Protein Sci, 2002 Sep, 11(9), 2267 - 72
Characterization of herpes simplex virus type 1 thymidine kinase mutants engineered for improved ganciclovir or acyclovir activity; Kokoris MS et al.; Herpes Simplex Virus type 1 (HSV-1) thymidine kinase (TK) is currently the most widely used suicide agent for gene therapy of cancer . Tumor cells that express HSV-1 thymidine kinase are rendered sensitive to prodrugs due to preferential phosphorylation by this enzyme . Although ganciclovir (GCV) is the prodrug of choice for use with TK, this approach is limited in part by the toxicity of this prodrug . From a random mutagenesis library, seven thymidine kinase variants containing multiple amino acid substitutions were identified on the basis of activity towards ganciclovir and acyclovir based on negative selection in Escherichia coli . Using a novel affinity chromatography column, three mutant enzymes and the wild-type TK were purified to homogeneity and their kinetic parameters for thymidine, ganciclovir, and acyclovir determined . With ganciclovir as the substrate, one mutant (mutant SR39) demonstrated a 14-fold decrease in K(m) compared to the wild-type enzyme . The most dramatic change is displayed by mutant SR26, with a 124-fold decrease in K(m) with acyclovir as the substrate . Such new "prodrug kinases" could provide benefit to ablative gene therapy by now making it feasible to use the relatively nontoxic acyclovir at nanomolar concentrations or ganciclovir at lower, less immunosuppressive doses.

Protein Sci, 2002 Sep, 11(9), 2148 - 57
Characterization and multiple molecular forms of TorD from Shewanella massilia, the putative chaperone of the molybdoenzyme TorA; Tranier S et al.; Several bacteria use trimethylamine N-oxyde (TMAO) as an exogenous electron acceptor for anaerobic respiration . This metabolic pathway involves expression of the tor operon that codes for a periplasmic molybdopterin-containing reductase of the DMSO/TMAO family, a pentahemic c-type cytochrome, and the TorD cytoplasmic chaperone, possibly required for acquisition of the molybdenum cofactor and translocation of the reductase by the twin-arginine translocation system . In this report, we show that the TorD chaperone from Shewanella massilia forms multiple and stable oligomeric species . The monomeric, dimeric, and trimeric forms were purified to homogeneity and characterized by analytical ultracentrifugation . Small-angle X-ray scattering (SAXS) and preliminary diffraction data indicated that the TorD dimer is made of identical protein modules of similar size to the monomeric species . Interconversion of the native oligomeric forms occurred at acidic pH value . In this condition, ANS fluorescence indicates a non-native conformation of the polypeptide chain in which, according to the circular dichroism spectra, the alpha-helical content is similar to that of the native species . Surface plasmon resonance showed that both the monomeric and dimeric species bind the mature TorA enzyme, but that the dimer binds its target protein more efficiently . The possible biologic significance of these oligomers is discussed in relation to the chaperone activity of TorD, and to the ability of another member of the TorD family to bind the Twin Arginine leader sequences of the precursor of DMSO/TMAO reductases.

J Biol Chem, 2002 Nov 8, 277(45), 42540 - 8 Epub 2002 Aug 20.
Yeast flavohemoglobin from Candida norvegensis . Its structural, spectral, and stability properties; Kobayashi G et al.; Flavohemoglobin was isolated directly from the yeast Candida norvegensis and studied on its structural, spectral, and stability properties . In Candida flavohemoglobin, the 155 N-terminal residues make a heme-containing domain, while the remaining 234 C-terminal residues serve as a FAD-containing reductase domain . A pair of His-95 and Gln-63 was assigned to the proximal and distal residues, respectively . In purification procedure FAD was partially dissociated on a Butyl-Toyopearl column, so that FAD-lacking flavohemoglobin was also obtainable . In this ferric species, the Soret and charge-transfer bands were all characteristic of a penta-coordinate form . Compared with the recombinant heme domain expressed in Escherichia coli, we have measured the autoxidation rate over a wide pH range . The resulting pH dependence curves were then analyzed in terms of a nucleophilic displacement mechanism . As a result, the heme domain was found to be extremely susceptible to autoxidation, its rate being more than 100 times higher than that of sperm whale MbO2 . However, this inherently high oxidation rate was dramatically suppressed in Candida flavohemoglobin to an extent almost comparable to the stability of mammalian myoglobins . These new findings lead us to conclude that Candida flavohemoglobin, differently from bacterial flavohemoglobins, can serve as an oxygen storage protein in aerobic conditions.

J Biol Chem, 2002 Nov 8, 277(45), 43126 - 36 Epub 2002 Aug 20.
Mechanistic characterization of Toxoplasma gondii thymidylate synthase (TS-DHFR)-dihydrofolate reductase . Evidence for a TS intermediate and TS half-sites reactivity; Johnson EF et al.; This study describes the use of rapid transient kinetic methods to characterize the bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) enzyme from Toxoplasma gondii . In addition to elucidating the detailed kinetic scheme for this enzyme, this work provides the first direct kinetic evidence for the formation of a TS intermediate and for half-sites TS reactivity in human and Escherichia coli monofunctional TS and in T . gondii and Leishmania major bifunctional TS-DHFR . Comparison of the T . gondii TS-DHFR catalytic mechanism to that of the L . major enzyme reveals the mechanistic differences to be predominantly in DHFR activity . Specifically, TS ligand induced domain-domain communication involving DHFR activation is observed only in the L . major enzyme and, whereas both DHFR activities involve a rate-limiting conformational change, the change occurs at different positions along the kinetic pathway.

J Biol Chem, 2002 Nov 1, 277(44), 41352 - 60 Epub 2002 Aug 20.
Functional characterization of choline monooxygenase, an enzyme for betaine synthesis in plants; Hibino T et al.; In plants, the first step in betaine synthesis was shown to be catalyzed by a novel Rieske-type iron-sulfur enzyme, choline monooxygenase (CMO) . Although CMO so far has been found only in Chenopodiaceae and Amaranthaceae, the recent genome sequence suggests the presence of a CMO-like gene in Arabidopsis, a betaine non-accumulating plant . Here, we examined the functional properties of CMO expressed in Escherichia coli, cyanobacterium, and Arabidopsis thaliana . We found that E . coli cells in which choline dehydrogenase (CDH) was replaced with spinach CMO accumulate betaine and complement the salt-sensitive phenotype of the CDH-deleted E . coli mutant . Changes of Cys-181 in spinach CMO to Ser, Thr, and Ala and His-287 to Gly, Val, and Ala abolished the accumulation of betaine . The Arabidopsis CMO-like gene was transcribed in Arabidopsis, but its protein was not detected . When the Arabidopsis CMO-like gene was expressed in E . coli, the protein was detected but was found not to promote betaine sysnthesis . Overexpression of spinach CMO in E . coli, Synechococcus sp . PCC7942, and Arabidopsis conferred resistance to abiotic stress . These facts clearly indicate that CMO, but not the CMO-like protein, could oxidize choline and that Cys-181 and His-287 are involved in the binding of Fe-S cluster and Fe, respectively.

Biochim Biophys Acta, 2002 Sep 2, 1592(1), 63 - 77
Mitochondrial processing peptidases; Gakh O et al.; Three peptidases are responsible for the proteolytic processing of both nuclearly and mitochondrially encoded precursor polypeptides targeted to the various subcompartments of the mitochondria . Mitochondrial processing peptidase (MPP) cleaves the vast majority of mitochondrial proteins, while inner membrane peptidase (IMP) and mitochondrial intermediate peptidase (MIP) process specific subsets of precursor polypeptides . All three enzymes are structurally and functionally conserved across species, and their human homologues begin to be recognized as potential players in mitochondrial disease.

Mol Cell, 2002 Aug, 10(2), 339 - 46
Important contribution to catalysis of peptide bond formation by a single ionizing group within the ribosome; Katunin VI et al.; The catalytic mechanism of peptide bond formation on the ribosome is not known . The crystal structure of 50S ribosomal subunits shows that the catalytic center consists of RNA only and suggests potential catalytic residues . Here we report rapid kinetics of the peptidyl transferase reaction with puromycin at rates up to 50 s(-1) . The rate-pH profile of the reaction reveals that protonation of a single ribosomal residue (pK(a) = 7.5), in addition to protonation of the nucleophilic amino group, strongly inhibits the reaction (>100-fold) . The A2451U mutation within the peptidyl transferase center has about the same inhibitory effect . These results suggest a contribution to overall catalysis of general acid-base and/or conformational catalysis involving an ionizing group at the active site.

Clin Exp Allergy, 2002 Aug, 32(8), 1203 - 10
Identification of shared and unique immunoglobulin E epitopes of the highly conserved tropomyosins in Blomia tropicalis and Dermatophagoides pteronyssinus; Yi FC et al.; BACKGROUND: Tropomyosin belongs to a class of highly conserved proteins in invertebrates and vertebrates . The invertebrate tropomyosins are allergenic in man with high IgE cross-reactivity and have been therefore referred to as pan-allergens . OBJECTIVES: This study aimed to clone and identify the IgE epitopes of tropomyosin from Blomia tropicalis (Blo t 10) mite . Cross-reactivity between the IgE epitopes of Blo t 10 and Der p 10 was also evaluated . METHODS: Blo t 10 was isolated using mouse anti-Der p 10 antibodies . Allergenicity of the cloned Blo t 10 was confirmed by skin prick test (SPT) and enzyme-linked immunosorbent assay (ELISA) . Dose-dependent inhibition assay was performed to determine the degree of IgE cross-reactivity between Blo t 10 and Der p 10 . Overlapping polymerase chain reaction-derived cDNA were generated and expressed as glutathione-S-transferase (GST) recombinant proteins in Escherichia coli and used to identify shared and unique IgE epitopes of Blo t 10 and Der p 10 . RESULTS: The cloned Blo t 10 shared up to 96% amino acid identity to tropomyosin of other mites . SPT and ELISA IgE-immunoassay showed recombinant Blo t 10 sensitization rates of between 20% and 29% in atopic subjects . Results of SPT and dose-dependent inhibition assays showed that some allergic individuals had unique IgE epitopes for Blo t 10 . IgE epitope mapping of Blo t 10 revealed that the epitopes were mainly located at N- and C-termini of the molecule . The results of ELISA inhibition assays of overlapping recombinant fragments indicated that the unique IgE epitopes of Blo t 10 were located at the C-terminal . CONCLUSION: Although Blo t 10 and Der p 10 are highly conserved (shared 95% amino acids identity) and significantly cross-reactive, unique IgE epitopes do exist . The results suggest the potential deficiency of using only one of these highly conserved allergens as diagnostic or therapeutic reagents.

Arzneimittelforschung, 2002, 52(7), 515 - 23
Local treatment of hemorrhoidal disease and perianal eczema . Meta-analysis of the efficacy and safety of an Escherichia coli culture suspension alone or in combination with hydrocortisone; Wienert V et al.; The objective of this paper was to assess the available clinical data on the efficacy and safety of ointments containing either a bacterial culture suspension (BCS) from Escherichia coli or a combination of BCS with hydrocortisone (CAS 50-23-7) (BCS: Posterisan, and BCS + HC: Posterisan forte) . The BCS is assumed to act by immunomodulation in hemorrhoidal disease and perianal eczema . Six randomized, double-blind trials are reported: three of them using BCS ointment and one using BCS + HC, against ointment base, and two trials using BCS + HC against hydrocortisone ointment alone . Patients with hemorrhoids and/or perianal eczema were included and treated over 2 weeks with weekly assessments . Efficacy parameters included score changes for burning, itching, redness and soiling as well as the investigators' overall efficacy rating . Safety was assessed from adverse drug reactions and an overall safety rating . Out of 1,070 patients (mean age 50 years), 273 received BCS and 229 BCS + HC; 568 patients were given the various controls . In the overall efficacy rating, BCS ointment was significantly superior to the ointment base in all three studies (p = 0.028, p = 0.016, and p = 0.045) . Moreover, BCS + HC was superior to the ointment base (p < 0.001) and to hydrocortisone alone (p = 0.156 and p = 0.021), confirming the distinct effect of the E . coli suspension . Satisfactory results were achieved in 83% of patients after the BCS + HC combination, 77% after BCS-containing ointment, 75% after hydrocortisone ointment and 52% after ointment base . Symptom scores decreased consistently more after administration of BCS than after the ointment base (p = 0.095, p = 0.006, and p = 0.029), and likewise, the combination of BCS + HC was significantly superior to the ointment base (p < 0.001) and to hydrocortisone alone (p = 0.036 and p = 0.019) . Adverse events were less frequent for BCS and BCS + HC than for the ointment base . It can therefore be concluded that ointments containing either only E . coli BCS or a combination of BCS and hydrocortisone provide significant relief in perianal eczema as well as in early stages of hemorrhoidal disease.

EMBO Rep, 2002 Sep, 3(9), 887 - 92 Epub 2002 Aug 16.
Rapid translocation of NTF2 through the nuclear pore of isolated nuclei and nuclear envelopes; Siebrasse JP et al.; The mechanism by which macromolecules are translocated through the nuclear pore complex (NPC) is little understood . However, recent measurements of nuclear transport in permeabilized cells showed that molecules binding to phenylalanine-glycine-rich repeats (FG repeats) in NPC proteins were translocated much faster through the NPC than molecules not interacting with FG repeats . We have studied that substrate preference of the NPC in isolated oocyte nuclei and purified nuclear envelopes by optical single transporter recording . NTF2, the transport receptor of RanGDP, was exported approximately 30 times faster than green fluorescent protein, an inert molecule of approximately the same size . The data confirm that restricted diffusion of inert molecules and facilitated transport of FG-repeat binding proteins are basic types of translocation through the NPC, demonstrating that the functional integrity of the NPC can be conserved in isolated nuclei and nuclear envelopes and thus opening new avenues to the analysis of nucleocytoplasmic transport.

J Biol Chem, 2002 Nov 1, 277(44), 41827 - 34 Epub 2002 Aug 19.
Malarial dihydroorotate dehydrogenase . Substrate and inhibitor specificity; Baldwin J et al.; The malarial parasite relies on de novo pyrimidine biosynthesis to maintain its pyrimidine pools, and unlike the human host cell it is unable to scavenge preformed pyrimidines . Dihydroorotate dehydrogenase (DHODH) catalyzes the oxidation of dihydroorotate (DHO) to produce orotate, a key step in pyrimidine biosynthesis . The enzyme is located in the outer membrane of the mitochondria of the malarial parasite . To characterize the biochemical properties of the malarial enzyme, an N-terminally truncated version of P . falciparum DHODH has been expressed as a soluble, active enzyme in E . coli . The recombinant enzyme binds 0.9 molar equivalents of the cofactor FMN and it has a pH maximum of 8.0 (k(cat) 8 s(-1), K(m)(app) DHO (40-80 microm)) . The substrate specificity of the ubiquinone cofactor (CoQ(n)) that is required for the oxidation of FMN in the second step of the reaction was also determined . The isoprenoid (n) length of CoQ(n) was a determinant of reaction efficiency; CoQ(4), CoQ(6) and decylubiquinone (CoQ(D)) were efficiently utilized in the reaction, however cofactors lacking an isoprenoid tail (CoQ(0) and vitamin K(3)) showed decreased catalytic efficiency resulting from a 4 to 7-fold increase in K(m)(app) . Five potent inhibitors of mammalian DHODH, Redoxal, dichloroallyl lawsone (DCL), and three analogs of A77 1726 were tested as inhibitors of the malarial enzyme . All five compounds were poor inhibitors of the malarial enzyme, with IC(50)'s ranging from 0.1-1.0 mm . The IC(50) values for inhibition of the malarial enzyme are 10(2)-10(4)-fold higher than the values reported for the mammalian enzyme, demonstrating that inhibitor binding to DHODH is species specific . These studies provide direct evidence that the malarial DHODH active site is different from the host enzyme, and that it is an attractive target for the development of new anti-malarial agents.

J Biol Chem, 2002 Nov 8, 277(45), 43194 - 205 Epub 2002 Aug 19.
Alanine-scanning mutagenesis of alpha-helix D segment of interleukin-13 reveals new functionally important residues of the cytokine; Madhankumar AB et al.; We documented that alpha-helices A, C, and D in human interleukin-13 (IL13) participate in interaction with its respective receptors . We hypothesized that alpha-helix D is the site II of the cytokine that binds IL13Ralpha1, a component of the normal tissue heterodimeric signaling IL13/4 receptor (IL13/4R), and that alpha-helix D independently binds a monomeric IL13Ralpha2 receptor, which is a non-signaling glioma-restricted receptor for IL13 . Therefore, we alanine-scanned mutagenized helix D of IL13 to identify the residues involved in the respective receptors interaction . Recombinant muteins of IL13 were produced in Escherichia coli, and their structural integrity and identity were verified . The alanine mutants were tested in functional cellular assays, in which IL13 interaction with IL13Ralpha2 (glioma cells) or an ability to functionally stimulate IL13/4R (TF-1 cells) were examined, and also in binding assays . We found that residues 105, 106, and 109 of the d-helix of IL13 are responsible for interacting with the glioma-associated receptor . Moreover, glutamic acids at positions 92 and 110, and leucine at position 104 was found to be important for IL13/4R stimulation . Thus, alpha-helix D of IL13 is the primary site responsible for interaction with the IL13 binding proteins . We propose a model that illustrates the binding mode of IL13 with cancer-related IL13Ralpha2 and physiological IL13/4R.

Redox Rep, 2002, 7(2), 79 - 84
Yeast thioredoxin peroxidase expression enhances the resistance of Escherichia coli to oxidative stress induced by singlet oxygen; 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 . A soluble protein from Saccharomyces cerevisiae specifically provides protection against a thiol-containing metal-catalyzed oxidation system (thiol/Fe(3+)/O(2)) but not against an oxidation system without thiol . This 25 kDa protein acts as a peroxidase but requires the NADPH-dependent thioredoxin system or a thiol-containing intermediate, and was named thioredoxin peroxidase (TPx) . The role of TPx in the cellular defense against oxidative stress induced by singlet oxygen was investigated in Escherichia coli containing an expression vector with a yeast genomic DNA fragment that encodes TPx and mutant in which the catalytically essential amino acid cysteine (Cys-47) has been replaced with alanine by a site-directed mutagenesis . Upon exposure to methylene blue and visible light, which generates singlet oxygen, there was a distinct difference between the two strains in regard to growth kinetics, viability, the accumulation of oxidized proteins and lipids, and modulation of activities of superoxide dismutase and catalase . The results suggest that TPx may play an important protective role in a singlet oxygen-mediated cellular damage.

Curr Cancer Drug Targets, 2001 Aug, 1(2), 109 - 19
Selective destruction of tumor cells through specific inhibition of products resulting from chromosomal translocations; Rodriguez-Garcia A et al.; A key problem in the effective treatment of patients with cancer (both leukemia and solid tumors) is to distinguish between tumor and normal cells . This problem is the main reason why current treatments for cancer are often ineffective . There have been remarkable advances in our understanding of the molecular biology of cancer that provides new selective tumor destruction mechanisms . The molecular characterization of the tumor-specific chromosomal abnormalities has revealed that fusion proteins are the consequence in the majority of cancers . These fusion proteins result from chimeric genes created by the translocations, which form chimeric mRNA species that contain exons from the genes involved in the translocation . Obviously, these chimeric molecules are attractive therapeutic targets since they are unique to the disease (they only exist in the tumor cells but not in the normal cells of the patient), allowing the design of specific anti-tumor drugs . Inhibition of chimeric gene expression by anti-tumor agents specifically kills leukemic cells without affecting normal cells . As therapeutic agents targeting chimeric genes, zinc-finger proteins, antisense RNAs or hammerhead-based ribozymes have been used . All of these agents have some limitations, indicating that new therapeutic tools are required as gene inactivating agents that should be able to inhibit any chimeric fusion gene product . Recently, we have used the catalytic RNA subunit of RNase P from Escherichia coli, which can be specifically directed to cut any mRNA sequence, to specifically destroy tumor-specific fusion genes created as a result of chromosomal translocations . In this chapter, we will review the advances made to selectively destroy tumor cells through specific inhibition of products resulting from chromosomal translocations.

J Am Chem Soc, 2002 Aug 28, 124(34), 10025 - 35
Four-dimensional NMR spectroscopy of a 723-residue protein: chemical shift assignments and secondary structure of malate synthase g; Tugarinov V et al.; A four-dimensional (4-D) NMR study of Escherichia coli malate synthase G (MSG), a 723-residue monomeric enzyme (81.4 kDa), is described . Virtually complete backbone (1)HN, (15)N, (13)C, and (13)C(beta) chemical shift assignments of this largely alpha-helical protein are reported . The assignment strategy follows from our previously described approach based on TROSY triple resonance 4-D NMR spectroscopy {Yang, D.; Kay, L . E . J . Am . Chem . Soc . 1999, 121, 2571-2575 . Konrat, R; Yang, D; Kay, L . E . J . Biomol . NMR 1999, 15, 309-313} with a number of modifications necessitated by the large size of the protein . A protocol for refolding deuterated MSG in vitro was developed to protonate the amides deeply buried in the protein core . Of interest, during the course of the assignment, an isoaspartyl linkage in the protein sequence was unambiguously identified . Chemical shift assignments of this system are a first step in the study of how the domains of the protein change in response to ligand binding and for characterizing the dynamical properties of the enzyme that are likely important for function.

Water Sci Technol, 2002, 45(10), 237 - 42
Coliform and helminth eggs removal in a combined UASB reactor-baffled pond system in Brazil: performance evaluation and mathematical modelling; von Sperling M et al.; The paper presents the monitoring results of a pilot UASB reactor followed by a baffled polishing pond treating domestic sewage in Brazil . Longitudinal profiles of E coli and helminth eggs along the baffled pond have been undertaken . The experimental results have been compared with von Sperling's model for coliform removal and Ayres' model for helminth eggs removal, and the fitting was considered satisfactory in both cases . The distribution of the helminth species along the system is also presented.

Biotechniques, 2002 Aug, 33(2), 430 - 4
Robust and sensitive nylon hybridization membrane suitable for high-throughput robotic arraying applications; Green MT et al.; An important aspect of automated macroarraying is the suitability of the nylon membrane selected on which samples are to be arrayed . PerForma is a positively charged nylon membrane that has been developed specificallyfor automated macroarraying . Tests usingfluorescent hybridization detection methods have shown that immobilized DNA amounts as low as 0.25 pg can be detected and that positive signals are obtainable after 21 stripping cycles . This report describes the improved colony growth, improved handling characteristics, increased hybridization detection sensitivity, and increased stripping and reprobing capability obtained using PerForma.

Biotechniques, 2002 Aug, 33(2), 424 - 9
Nonradioactive detection of retroviral-associated RNase H activity in a microplate-based, high-throughput format; McLellan N et al.; None of the available antiretroviral drugs that are currently used in the clinic to treat infection with HIV-1 is directed against the RNase H active site of the reverse transcriptase . Here we developed a nonradioactive, 96-well plate assay designed to be used for high-throughput screening of compounds capable of inhibiting the RNase H activity of HIV-1 reverse transcriptase . We employed a tRNA as substrate that was labeled with digoxygenin-modified reporter residues . The labeled tRNA was prehybridized with a DNA oligonucleotide that contained a single biotinylated residue at its 5'-terminus to ensure its attachment to streptavidin-coated microplates . The uncleaved, immobilized DNA/tRNA substrate was detected through the use of established ELISA protocols . Incubation with purified HIV-1 reverse transcriptase initiated RNase H degradation and caused a signal reduction to negligible background levels . In contrast, the signal intensity remained unaffected when using an RNase H deficient mutant enzyme . The assay was validated using the hydrazone derivative BBNH that was previously shown to inhibit RNase H degradation below concentrations of 10 microM.

Biotechniques, 2002 Aug, 33(2), 310, 312, 314 - 5
Rapid generation of nested deletions by differential restriction digestion; Dennis JJ et al.; A method was devised for generating nested deletions in DNA that exploits the difference in frequency of restriction sites recognized by compatible restriction endonucleases . A cloning vector was constructed that contains no common blunt-end or RsaI restriction sites and two 8-bp blunt-end restriction sites flanking a commodious multiple cloning site . DNA fragments are cloned into the multiple cloning site using blue-white selection, and nested deletions are generated by digesting the resulting plasmid with either SwaI or PmeI and partially digesting the insert DNA with RsaI . The DNAs are ligated and transformed, producing afamily of plasmids with different-sized deletions . The DNA sequence of these inserts can be rapidly determined, and the overlapping sequences can be assembled in silico to produce a large DNA contig . Nested deletions generated in this manner can also be used for the structure-function analysis of proteins.

Nippon Yakurigaku Zasshi, 2002 Aug, 120(2), 115 - 22
{Pharmacological and clinical properties of didanosine (VIDEX), a nucleoside reverse transcriptase inhibitor}; Okiyama M et al.; An active metabolite, ddATP, of didanosine that is an analogue of purine-nucleoside (a component of nucleic acid) was known to inhibit the activity of DNA polymerase for E . coli . In 1985, Dr . Michiya et al . of NCI reported that didanosine and ddA inhibited replication of the human immunodeficiency virus (HIV) . This discovery led to the clinical application of both the compounds . Didanosine, after being uptaken into a cell, becomes an active metabolite, ddATP, to inhibit a reverse transcriptase of HIV . Compared with zidovudine, didanosine has weak cytotoxicity both in vitro and in vivo . Didanosine, which is recommended as a first-line therapy drug in the Japanese Guideline on an anti-HIV Infection Therapy, was approved as twice-daily Videx Tablet and Dry Syrup formulations for launch in June 1992 . In March 2001, a once-daily Videx EC Capsule formulation was approved and launched, having expected adherence improvements in HIV/AIDS patients.

Curr Eye Res, 2002 Feb, 24(2), 92 - 8
Characterization of T lymphocyte subtypes in endotoxin-induced uveitis and effect of pentoxifylline treatment; Avunduk AM et al.; PURPOSE: The aims of the study were twofold: 1) to investigate the role of T lymphocyte subtypes in the pathogenesis of endotoxin-induced uveitis (EIU) and 2) to study the possible beneficial effect of pentoxifylline, an inhibitor of neutrophil motility, and Tumor Necrosis Factor-alpha on this disease . METHODS: Forty-two inbred male Lewis rats were divided into seven equal groups . 200 microg of Escherichia coli 055: B55 lipopolysaccharide (LPS) was injected in one hind footpad of the Group 2, 3, 4, 5, 6, and 7 rats . Group 5, 6, and 7 rats also received concomitant intraperitoneal pentoxifylline (PTX) during food pad injection of LPS . Group 1 rats were used as controls with intra-peritoneal normal saline injection . Eight, 24, and 48 hours after treatment, the rats were euthanized . Neutrophil leukocyte, mononuclear cells, and CD4+, CD8+, and CD45RA+ cell infiltration in the anterior uveal tissue were determined either by hematoxylin-eosin or monoclonal antibody staining . Tumor Necrosis Factor-alpha (TNF-alpha) levels were also measured in the aqueous and blood samples . We compared the numbers of infiltrating cells in the different groups . RESULTS: We found that peak infiltration of lymphocyte, neutrophils, and CD4+ cells occurred at 24 hours . However, CD8+ and CD45RA+ cell number reached their highest levels at 48 hours . There was no inflammatory cell infiltration in the control rats . Concomitant pentoxifylline treatment did not affect any of these parameters, although it effectively reduced TNF-alpha concentrations in the anterior chamber and the serum . CONCLUSION: We conclude that, 1) T lymphocytes might be involved in the pathogenesis of endotoxin-induced uveitis . 2) The potential role of pentoxifylline in the treatment of human uveitis is questionable . However, these are initial findings and need confirmation by additional studies.

J Urol, 2002 Sep, 168(3), 1182 - 7
Adenovirus mediated gelsolin gene therapy for orthotopic human bladder cancer in nude mice; Sazawa A et al.; PURPOSE: Gelsolin is an actin regulatory protein that is undetectable or reduced in human bladder tumors compared with normal epithelial cells . Whether the over expression of gelsolin could inhibit tumor growth was investigated in an orthotopic bladder cancer nude mouse model using recombinant adenovirus encoding wild-type gelsolin (Ad-GSN) . MATERIALS AND METHODS: The 2 human bladder cancer cell lines KU-7 and UMUC-2 were transduced with Ad-GSN in vitro . Flow cytometric analysis was done to examine the cell cycle after transducing the adenovirus . Cell growth was compared with control groups of these cells transduced with adenovirus containing the Escherichia coli beta-galactosidase gene Ad-betagal . In vivo KU-7 cells were introduced into the bladder of nude mice (day 0), followed by 3 injections into the urethra (days 2 to 4) with Ad-GSN or Ad-betagal (1 x 10 pfu) . At 8 days after initial adenovirus exposure (day 10) each bladder was sectioned and stained, and the mass of the tumor was digitally determined . RESULTS: Bladder cancer cell growth (KU-7 and UMUC-2) was inhibited after these cells were transduced with Ad-GSN in vitro . Based on flow cytometric analysis over expression of gelsolin may cause these cells to arrest or delay at the G2/M phase of the cell cycle . In the orthotopic bladder cancer model the mass of the tumor was approximately 90% less in Ad-GSN treated animals than in controls . CONCLUSIONS: Ad-GSN provides a significant tumor suppressive effect on human bladder cancer cells in this orthotopic nude mouse model . Adenovirus mediated over expression of gelsolin may be useful therapy for human bladder cancer.

J Virol, 2002 Sep, 76(18), 9551 - 5
The human cytomegalovirus ribonucleotide reductase homolog UL45 is dispensable for growth in endothelial cells, as determined by a BAC-cloned clinical isolate of human cytomegalovirus with preserved wild-type characteristics; Hahn G et al.; An endothelial cell-tropic and leukotropic human cytomegalovirus (HCMV) clinical isolate was cloned as a fusion-inducing factor X-bacterial artificial chromosome in Escherichia coli, and the ribonucleotide reductase homolog UL45 was deleted . Reconstituted virus RVFIX and RV Delta UL45 grew equally well in human fibroblasts and human endothelial cells . Thus, UL45 is dispensable for growth of HCMV in both cell types.

J Biol Chem, 2002 Nov 1, 277(44), 41517 - 24 Epub 2002 Aug 16.
New roles for conserved regions within a sigma 54-dependent enhancer-binding protein; Lew CM et al.; 23 amino acid substitutions were made in the C7 and C3 regions of pspFDeltaHTH, a protein required to convert varsigma(54) closed promoter complexes to open complexes . These mutants were assayed for transcriptional competence, for the ability to hydrolyze ATP, for their multimerization state, and for their ability to interact with varsigma(54) and its holoenzyme . C7 region mutants caused the protein to assume a compact form . This property could be mimicked by the addition of ATP, implying that compaction via C7 and ATP is part of the activation process . A number of C3 mutants were important for energy coupling, as indicated previously for several members of this activator family (, ) . However, a patch within C3 influenced oligomerization . The C3 region was especially important in interacting with varsigma(54) during the transition state but not important in inducing varsigma(54) holoenzyme to engage the nontemplate strand of the promoter . It is proposed that both regions contain deterrent functions that prevent premature activation . Overall, the results imply unexpected roles for the C7 and C3 regions of this protein family during promoter activation.

J Biol Chem, 2002 Nov 15, 277(46), 44121 - 30 Epub 2002 Aug 16.
A new type 2 copper cysteinate azurin . Involvement of an engineered exposed cysteine in copper binding through internal rearrangement; van Amsterdam IM et al.; The double mutant H117G/N42C azurin exhibits tetragonal type 2 copper site characteristics with Cys(42) as one of the copper ligands as concluded from spectroscopic evidence (UV-visible, EPR, and resonance Raman) . Analysis of the kinetics of copper uptake by the apoprotein by means of stopped flow spectroscopy suggests that the solvent-exposed Cys(42) assists in binding the metal ion and carrying it over to the active site where it becomes coordinated by, among others, a second cysteine, Cys(112) . A structure is proposed in which the loop from residue 36 to 47 has rearranged to form a tetragonal type 2 copper site with Cys(42) as one of the ligands . The process of copper uptake as observed for the double mutant may be relevant for a better understanding of the way copper chaperones accept and transfer metal ions in the living cell.

J Cell Biol, 2002 Aug 19, 158(4), 801 - 15 Epub 2002 Aug 19.
Conditional control of selectin ligand expression and global fucosylation events in mice with a targeted mutation at the FX locus; Smith PL et al.; Glycoprotein fucosylation enables fringe-dependent modulation of signal transduction by Notch transmembrane receptors, contributes to selectin-dependent leukocyte trafficking, and is faulty in leukocyte adhesion deficiency (LAD) type II, also known as congenital disorder of glycosylation (CDG)-IIc, a rare human disorder characterized by psychomotor defects, developmental abnormalities, and leukocyte adhesion defects . We report here that mice with an induced null mutation in the FX locus, which encodes an enzyme in the de novo pathway for GDP-fucose synthesis, exhibit a virtually complete deficiency of cellular fucosylation, and variable frequency of intrauterine demise determined by parental FX genotype . Live-born FX(-/-) mice exhibit postnatal failure to thrive that is suppressed with a fucose-supplemented diet . FX(-/-) adults suffer from an extreme neutrophilia, myeloproliferation, and absence of leukocyte selectin ligand expression reminiscent of LAD-II/CDG-IIc . Contingent restoration of leukocyte and endothelial selectin ligand expression, general cellular fucosylation, and normal postnatal physiology is achieved by modulating dietary fucose to supply a salvage pathway for GDP-fucose synthesis . Conditional control of fucosylation in FX(-/-) mice identifies cellular fucosylation events as essential concomitants to fertility, early growth and development, and leukocyte adhesion.

J Exp Med, 2002 Aug 19, 196(4), 417 - 30
Accumulation of immature Langerhans cells in human lymph nodes draining chronically inflamed skin; Geissmann F et al.; The coordinated migration and maturation of dendritic cells (DCs) such as intraepithelial Langerhans cells (LCs) is considered critical for T cell priming in response to inflammation in the periphery . However, little is known about the role of inflammatory mediators for LC maturation and recruitment to lymph nodes in vivo . Here we show in human dermatopathic lymphadenitis (DL), which features an expanded population of LCs in one draining lymph node associated with inflammatory lesions in its tributary skin area, that the Langerin/CD207(+) LCs constitute a predominant population of immature DCs, which express CD1a, and CD68, but not CD83, CD86, and DC-lysosomal-associated membrane protein (LAMP)/CD208 . Using LC-type cells generated in vitro in the presence of transforming growth factor (TGF)-beta1, we further found that tumor necrosis factor (TNF)-alpha, as a prototype proinflammatory factor, and a variety of inflammatory stimuli and bacterial products, increase Langerin expression and Langerin dependent Birbeck granules formation in cell which nevertheless lack costimulatory molecules, DC-LAMP/CD208 and potent T cell stimulatory activity but express CCR7 and respond to the lymph node homing chemokines CCL19 and CCL21 . This indicates that LC migration and maturation can be independently regulated events . We suggest that during DL, inflammatory stimuli in the skin increase the migration of LCs to the lymph node but without associated maturation . Immature LCs might regulate immune responses during chronic inflammation.

J Biochem Mol Biol Biophys, 2002 Feb, 6(1), 17 - 21
Mitochondrial factors modulate the activity of endonuclease G, the major nuclease of Mammalian mitochondria; Ikeda S et al.; Endonuclease G (Endo G), a sugar-nonspecific nuclease preferring single-stranded DNA (ssDNA), is responsible for major nuclease activity in mitochondria . If the enzyme provides an important nicking function for mitochondrial DNA (mtDNA) in vivo, then mitochondrial factors likely exist which modulate the enzyme's activity and prevent cleavage at single-stranded moieties of mtDNA . In the present paper, we report that specific membrane phospholipids, polyamines and single-stranded DNA-binding protein (SSB) appear to exert such effects in vitro . Phosphatidylcholine and phosphatidylethanolamine, the major constituents of the mitochondrial inner membrane, stimulated purified Endo G activity 5- to 10-fold . Spermine at 5-100 microM also stimulated activity about 4-fold . However, at more than 500 microM, the spermine largely inhibited the degradation of ssDNA and duplex DNA . Escherichia coli SSB, which has physicochemical properties analogous to those of mitochondrial SSB (mtSSB), markedly inhibited the degradation of phiX174 ssDNA by Endo G, indicating the possible involvement of mtSSB in vivo in protection of single-stranded regions of mtDNA from nucleolytic attacks.

J Biochem Mol Biol Biophys, 2002 Apr, 6(2), 123 - 8
Molecular characterization and expression of a putative ATPase domain from Eimeria tenella; Chong SP et al.; VCP (Valosin-Containing Protein), a member of the AAA (ATPases Associated to a variety of cellular Activities) family of proteins, possesses a duplicated highly conserved ATPase domain . An expressed sequence tag (EST), representing a clone from the Eimeria tenella merozoite cDNA library, was found to have high similarity to VCP genes from other organisms . A complete sequence derived from the corresponding clone (designated eth060) shows amino acid identity of 42-62% with other members of the VCP subfamily . Sequence analysis identified a putative ATPase domain in the eth060 sequence . This domain was PCR-amplified using gene-specific primers and cloned into a pBAD/Thio-TOPO expression vector . Expression in Escherichia coli demonstrated that the putative ATPase domain, which consists of 414 amino acid residues, produced a fusion protein of approximately 60 kDa in size.

J Biochem Mol Biol Biophys, 2002 Aug, 6(4), 243 - 8
Glu 103 Gln site-directed mutation causes an alteration in physical properties of spinach betaine aldehyde dehydrogenase; Incharoensakdi A et al.; Site-directed mutant of spinach betaine aldehyde dehydrogenase (BADH) was obtained by replacing Glu 103 with Gln 103 and the resulting E 103 Q mutant was expressed in Escherichia coli . The mutant BADH as compared to the wild-type BADH was slightly more sensitive to inhibition by NaCl but less sensitive to inhibition by (NH(4))(2)SO(4) . Glycinebetaine (GB) activated the wild-type enzyme but not the mutant enzyme . Stronger inhibition by choline was observed in the mutant enzyme than in the wild-type enzyme whereas the reverse was observed for the inhibition by isovaleraldehyde . The mutant enzyme exhibited a broader temperature optimum than the wild-type enzyme, however, the mutant enzyme appeared to be more heat labile . Both mutant and wild-type enzymes could be protected by NAD(+) against thermal inactivation in a similar manner . However, neither GB nor NaCl could afford protection against thermal inactivation in the mutant enzyme whereas some protection was observed in the wild-type enzyme . Similar pH activity profile was obtained for both mutant and wild-type enzymes . The mutant enzyme was less stable than the wild-type enzyme under the pH range of 5-11 . Overall results suggest that the negative charge of Glu 103 at the surface of the spinach BADH plays some roles in the maintenance of the structural integrity of the enzyme.

Genome Biol . 2002 Jul 25;3(8):RESEARCH0040 . Epub 2002 Jul 25.
The dominance of the population by a selected few: power-law behaviour applies to a wide variety of genomic properties; Luscombe NM et al.; BACKGROUND: The sequencing of genomes provides us with an inventory of the 'molecular parts' in nature, such as protein families and folds, and their functions in living organisms . Through the analysis of such inventories, it has been shown that different genomes have very different usage of parts; for example, the common folds in the worm are very different from those in Escherichia coli . RESULTS: Despite these differences, we find that the genomic occurrence of generalized parts follows a well-known mathematical framework called the power law, with a few parts occurring many times and most occurring only a few times . This observation is true in a wide variety of genomic contexts . Earlier studies found power laws in a few specific cases, such as the occurrence of protein families . Here, we find many further cases of power-law behavior, for example in the occurrence of pseudogenes and in levels of gene expression . We show comprehensively that this behavior applies across many different genomes, for many different types of parts (DNA words, InterPro families, protein superfamilies and folds, pseudogene families and pseudomotifs), and for the many disparate attributes associated with these parts (their functions, interactions and expression levels) . CONCLUSIONS: Power-law behavior provides a concise mathematical description of an important biological feature: the sheer dominance of a few members over the overall population . We present this behavior in a unified framework and propose that all these observations are connected to an underlying DNA duplication process as genomes evolved to their current state.

Biochemistry, 2002 Aug 27, 41(34), 10700 - 9
Peptidyl aldehydes as reversible covalent inhibitors of protein tyrosine phosphatases; Fu H et al.; Protein tyrosine phosphatases (PTPs) are a large family of enzymes that catalyze the hydrolytic removal of the phosphoryl group from phosphotyrosyl (pY) proteins . PTP inhibitors provide potential treatment of human diseases/conditions such as diabetes and obesity as well as useful tools for studying the function of PTPs in signaling pathways . In this work, we have shown that certain aryl-substituted aldehydes act as reversible, slow-binding inhibitors of modest potency against PTP1B, SHP-1, and a dual-specificity phosphatase, VHR . Attachment of the tripeptide Gly-Glu-Glu to the para position of cinnamaldehyde resulted in an inhibitor (Cinn-GEE) of substantially increased potency against all three enzymes (e.g., K(I) = 5.4 microM against PTP1B) . The mechanism of inhibition was investigated using Cinn-GEE specifically labeled with (13)C at the aldehyde carbon and (1)H-(13)C heteronuclear single-quantum coherence spectroscopy . While Cinn-GEE alone showed a single cross-peak at delta 9.64 ((1)H) and delta 201 ((13)C), the PTP1B/Cinn-GEE complex showed three distinct cross-peaks at delta 7.6-7.8 ((1)H) and 130-137 ((13)C) . Mutation of the catalytic cysteine (Cys-215 in PTP1B) into alanine had no effect on the cross-peaks, whereas mutation of a conserved active-site arginine (Arg-221 in PTP1B) to alanine abolished all three cross-peaks . Similar experiments with Cinn-GEE that had been labeled with (13)C at the benzylic position revealed a change in the hybridization state (from sp(2) to sp(3)) for the benzylic carbon as a result of binding to PTP1B . These results rule out the possibility of a free aldehyde, aldehyde hydrate, or hemithioacetal as the enzyme-bound inhibitor form . Instead, the data are consistent with the formation of an enamine between the aldehyde group of the inhibitor and the guanidine group of Arg-221 in the PTP1B active site . These aldehydes may provide a general core structure that can be further developed into highly potent and specific PTP inhibitors.

Biochemistry, 2002 Aug 27, 41(34), 10675 - 9
Identification of the residues involved in stabilization of the semiquinone radical in the high-affinity ubiquinone binding site in cytochrome bo(3) from Escherichia coli by site-directed mutagenesis and EPR spectroscopy; Hellwig P et al.; During turnover of cytochrome bo(3) from Escherichia coli, a semiquinone radical is stabilized in a high-affinity binding site . To identify binding partners of this radical, site-directed mutants have been designed on the basis of a recently modeled quinone binding site (Abramson et al., 2000) . The R71H, H98F, D75H, and I102W mutant enzymes were found to show very little or no quinol oxidase activity . The thermodynamic and EPR spectroscopic properties of semiquinone radicals in these mutants were characterized . For the H98F and the R71H mutants, no EPR signal of the semiquinone radical was observed in the redox potential range from -100 to 250 mV . During potentiometric titration of the D75H mutant enzyme, a semiquinone signal was detected in the same potential range as that of the wild-type enzyme . However, the EPR spectrum of the D75H mutant lacks the characteristic hyperfine structure of the semiquinone radical signal observed in the wild-type oxidase, indicating that D75 or the introduced His, interacts with the semiquinone radical . For the I102W mutant, a free radical signal was observed with a redox midpoint potential downshifted by about 200 mV . On the basis of these observations, it is suggested that R71, D75, and H98 residues are involved in the stabilization of the semiquinone state in the high-affinity binding site . Details of the possible binding motif and mechanistic implications are discussed.

Biochemistry, 2002 Aug 27, 41(34), 10635 - 45
Attenuation of the editing activity of the Escherichia coli leucyl-tRNA synthetase allows incorporation of novel amino acids into proteins in vivo; Tang Y et al.; The fidelity of translation is dependent on the specificity of the aminoacyl-tRNA synthetases (aaRSs) . The aaRSs that activate the hydrophobic amino acids leucine, isoleucine, and valine employ a proofreading mechanism that hydrolyzes noncognate aminoacyl adenylates and misaminoacylated tRNAs . Discrimination between structurally similar amino acids by these AARSs is believed to operate by a double-sieve principle, wherein a separate editing domain governs hydrolysis on the basis of the size and hydrophilicity of the amino acid side chain . Leucyl-tRNA synthetase (LeuRS) relies on its editing function to correct misaminoacylation of tRNA(Leu) by isoleucine and methionine . Thr252 of Escherichia coli LeuRS has been shown previously to be important in defining the size of the editing cavity . Here we report the isolation and characterization of three LeuRS mutants with point mutations at this position (T252Y, T252L, and T252F) . The proofreading activity of the synthetase is significantly impaired when an amino acid bulkier than threonine is introduced . The rate of misaminoacylation of tRNA(Leu) by isoleucine and valine increases with the increasing size of the amino acid substituent at position 252, and the noncognate amino acids norvaline and norleucine are inserted efficiently at the leucine sites of recombinant proteins under conditions of constitutive overexpression of the T252Y mutant in E . coli . In addition, the unsaturated amino acids allylglycine, homoallylglycine, homopropargylglycine, and 2-butynylalanine all support protein synthesis in E . coli hosts harboring the mutant synthetase . These results demonstrate that programmed manipulation of the editing cavity can allow in vivo incorporation of novel protein building blocks.

Biochemistry, 2002 Aug 27, 41(34), 10623 - 8
Discrimination of tRNA(Leu) isoacceptors by the mutants of Escherichia coli leucyl-tRNA synthetase in editing; Du X et al.; Leucyl-tRNA synthetase (LeuRS), one of the class Ia aminoacyl-tRNA synthetases, joins Leu to tRNA(Leu) and excludes noncognate amino acids in protein synthesis . In this study, Escherichia coli LeuRS mutants at amino acid E292, which was located in the connective polypeptide 1 insertion region, were synthesized . Although mutated LeuRS showed little change in structure compared with wild-type LeuRS, the mutants were impaired in activity to varying extents . It was also showed that mutations did not affect the adenylation reaction . However, mutated LeuRS can mischarge tRNA(Leu) isoacceptors tRN or tRN with isoleucine to different extents . Isoleucylation of tRN was more than that of tRN . The mutant LeuRS-E292S, which was picked out as an example for the investigation of the relationship between tRNA(Leu) isoacceptors and editing function, can discriminate the Watson-Crick base pair of the first base pair of tRNA(Leu) from the wobble base pair . The tRNA(Leu) with the Watson-Crick base pair may result in more isoleucylated product than that with the wobble base pair . The same phenomenon happened to another mutant, LeuRS-A293D . It seems that the flexibility of the first base pair affects the editing reaction of LeuRS . The results indicate that the flexibility of the first base pair of tRNA(Leu) may probably affect the mischarged 3'-end of tRNA(Leu) shuttling from synthetic site to editing site and that the transferred acceptor arm of tRNA(Leu) may interact with LeuRS in the region around E292.

Biochemistry, 2002 Aug 27, 41(34), 10585 - 92
The different energetic state of the intra A-chain/domain disulfide of insulin and insulin-like growth factor 1 is mainly controlled by their B-chain/domain; Guo ZY et al.; Insulin and insulin-like growth factor 1 (IGF-1) share homologous sequence, similar three-dimensional structure, and weakly overlapping biological activity, but different folding information is stored in their homologous sequences: the sequence of insulin encodes one unique thermodynamically stable three-dimensional structure while that of IGF-1 encodes two disulfide isomers with different three-dimensional structure but similar thermodynamic stability . Their different folding behavior probably resulted from the different energetic state of the intra A-chain/domain disulfide: the intra A-chain disulfide of insulin is a stable bond while that of IGF-1 is a strained bond with high energy . To find out the sequence determinant of the different energetic state of their intra A-chain/domain disulfide, the following experiments were carried out . First, a local chimeric single-chain insulin (PIP) with the A8-A10 residues replaced by the corresponding residues of IGF-1 was prepared . Second, the disulfide stability of two global hybrids of insulin and IGF-1, Ins(A)/IGF-1(B) and Ins(B)/IGF-1(A), was investigated . The local segment swap had no effect on the fidelity of disulfide pairing and the disulfide stability of PIP molecule although the swapped segment is close to the intra A-chain/domain disulfide . In redox buffer which favors the disulfide formation for most proteins, Ins(A)/IGF-1(B) cannot form and maintain its native disulfides just like that of IGF-1, while the disulfides of Ins(B)/IGF-1(A) are stable in the same condition . One major equilibrium intermediate with two disulfides of Ins(A)/IGF-1(B) was purified and characterized . V8 endoproteinase cleavage and circular dichroism analysis suggested that the intra A-chain/domain disulfide was reduced in the intermediate . Our present results suggested that the energetic state of the intra A-chain/domain disulfide of insulin and IGF-1 was not controlled by the A-chain/domain sequence close to this disulfide but was mainly controlled by the sequence of the B-chain/domain.

Biol Pharm Bull, 2002 Aug, 25(8), 995 - 9
Platonin, a photosensitizing dye, improves circulatory failure and mortality in rat models of endotoxemia; Hsiao G et al.; Platonin, a cyanine photosensitizing dye, is a potent macrophage-activating agent and an immunomodulator . In this study, we compare the inhibitory effects of platonin with those of the three clinical drugs minocycline, clindamycin, and cyclosporin, on hypotension, tachycardia, and nitric oxide (NO) formation in a rat model of circulatory shock induced by Escherichia coli lipopolysaccharide (LPS) . We also evaluate the effect of drugs on the 6 h survival rate in LPS-treated rats . Administration of LPS (15 mg/kg) caused a rapid drop in mean arterial blood pressure (MAP) . Minocycline (10 mg/kg, i.v.) significantly prevented the fall of MAP at 3 h, and platonin (100 microg/kg, i.v.) markedly prevented the fall of MAP within the 0-3 h period after LPS administration . However, neither clindamycin (10 mg/kg, i.v.) nor cyclosporin (15 mg/kg, i.v.) had any effects in this study . On the other hand, an inducible NO synthase inhibitor, NG-nitro-L-arginine ester (L-NAME), caused a significantly increase in MAP and a moderate bradycardia after LPS administration . In addition, an increase in plasma nitrate formation elicited by endotoxemia was significantly reduced by pretreatment with either minocycline (10 mg/kg) or platonin (100 microg/kg) . However, only platonin (100 microg/kg) markedly reduced the mortality and prolonged the mean survival time in LPS-treated rats . Minocycline, clindamycin, and cyclosporin had no effects under the same conditions . Further studies using an electron spin resonance (ESR) method were conducted on the scavenging activity of platonin on the free radicals formed . Platonin (10 microm) greatly reduced the ESR signal intensity of superoxide anion, hydroxyl radical, and methyl radical formation . In conclusion, platonin has beneficial effects on ameliorating endotoxaemia . This protective effect of platonin may be mediated, at least partly, by the reduced drop in MAP and the inhibition of NO and free radical formation in rat models of endotoxemia.

J Gen Virol, 2002 Sep, 83(Pt 9), 2269 - 78
The terminal repeats and latency-associated nuclear antigen of herpesvirus saimiri are essential for episomal persistence of the viral genome; Collins CM et al.; The simian herpesvirus saimiri (HVS) induces malignant T cell lymphomas and is closely related to Kaposi's sarcoma-associated herpesvirus (KSHV or HHV-8) . Both belong to the gamma-2 herpesvirus subgroup . The viral genome of HVS consists of a unique region (L-DNA) that contains all of the viral genes flanked by non-coding terminal repeats (H-DNA) . Here we describe the cloning of a 113 kb restriction fragment containing the L-DNA of an oncogenic HVS strain in an F' replicon-based E . coli vector . Cloned DNA was infectious and the ends of the progeny viral genome consisted of amplified tandem alternating repeats of vector and a single H-DNA unit . T cells infected with these viruses contained the linear DNA typically found a few weeks after infection, but were unable to form episomal circular viral DNA, which is the latent form of the viral genome . Recombinant viruses with reconstructed H-DNA were generated and T cells infected with these rescued viruses contained high copy numbers of episomal DNA . Plasmids expressing the latency-associated nuclear antigen (LANA) and containing various numbers of H-DNA repeats stably replicated as episomes, but constructs containing three repeat units produced the highest copy numbers . These data show that intact and multiple terminal repeats are essential components for episomal replication in latently infected T cells . Moreover, LANA and terminal repeats are sufficient for stable plasmid persistence . Cloned HVS can also be utilized for mutagenesis of HVS and for the expression of foreign genes through efficient manipulation of plasmids in E . coli.

J Gen Virol, 2002 Sep, 83(Pt 9), 2211 - 4
Intramolecular disulfide bonding is essential for betanodavirus coat protein conformation; Krondiris JV et al.; Here we report on the conformational changes that are responsible for the appearance of the Dicentrarchus labrax encephalitis virus (DlEV) coat protein as a doublet in SDS-PAGE . Wild-type and mutated forms of the coat protein cDNA were expressed in E . coli . The study of the resulting recombinant molecules excluded the possibility of the involvement of a precursor autocatalysis mechanism or a ribosomal frameshifting event in the doublet formation . The appearance of the coat protein doublet was found to be beta-mercaptoethanol sensitive . Based on this observation, we carried out substitution of all cysteine residues . The obtained results demonstrated the importance of intramolecular disulfide bonding between cysteines 187 and 201 on coat protein conformational changes.

J Gen Virol, 2002 Sep, 83(Pt 9), 2201 - 9
The putative capsid protein of the newly identified avian hepatitis E virus shares antigenic epitopes with that of swine and human hepatitis E viruses and chicken big liver and spleen disease virus; Haqshenas G et al.; We recently identified a novel virus, designated avian hepatitis E virus (avian HEV), from chickens with hepatitis-splenomegaly (HS) syndrome in the USA . We showed that avian HEV is genetically related to swine and human HEVs . Here we report the antigenic cross-reactivity of the putative open reading frame 2 (ORF2) capsid protein of avian HEV with those of swine and human HEVs and the Australian chicken big liver and spleen disease virus (BLSV) . The region encoding the C-terminal 268 amino acid residues of avian HEV ORF2 was cloned into expression vector pRSET-C . The truncated ORF2 protein was expressed in E . coli as a fusion protein and purified by affinity chromatography . Western blot analysis revealed that the avian HEV ORF2 protein reacted with antisera against the Sar-55 strain of human HEV and with convalescent antisera against swine HEV and the US2 strain of human HEV, as well as with antiserum against BLSV . Convalescent sera from specific-pathogen-free chickens experimentally infected with avian HEV also reacted with the recombinant capsid proteins of swine HEV and Sar-55 human HEV . Antisera against the US2 human HEV also reacted with recombinant ORF2 proteins of both swine HEV and Sar-55 human HEV . The antigenic cross-reactivity of the avian HEV putative capsid protein with those of swine and human HEVs was further confirmed, for the most part, by ELISA assays . The data indicate that avian HEV shares certain antigenic epitopes in its putative capsid protein with swine and human HEVs, as well as with BLSV . The results have implications for HEV diagnosis and taxonomy.

J Gen Virol, 2002 Sep, 83(Pt 9), 2161 - 8
Quasispecies in the 5' untranslated genomic region of bovine viral diarrhoea virus from a single individual; Jones LR et al.; The variability of the 5' untranslated genomic region (5'UTR) of bovine viral diarrhoea virus (BVDV) RNA obtained from a single individual was analysed . Lung, kidney and spleen tissues from a naturally infected foetus were used as the source of viral RNA . A fragment of 288 bases of the internal ribosome entry site from the BVDV 5'UTR was amplified by RT-PCR using a proofreading DNA polymerase . PCR products were cloned into pGem and, subsequently, transformed into Escherichia coli . The single-strand conformational polymorphisms of 158 lung-derived clones were analysed; a total of 11 banding patterns was observed . DNAs corresponding to all patterns were sequenced . Of the randomly selected clones, 11 and 10 clones derived from the kidney and spleen, respectively, were also sequenced . All sequences presented differences ranging from 1 to 6 nt substitutions . Analysis of the secondary structure of the variant sequences and comparisons to variant nucleotide sites from the 5'UTR of several BVDV isolates showed that the observed changes were almost free of randomness . Clustering and phylogenetic analyses suggested the existence of low-kinetic variants . BVDV quasispecies may be involved in establishing persistent infections by means of eluding maternal antibodies . The methods described here may be adapted easily both to analyse large numbers of samples from other genomic regions and for the study of BVDV quasispecies evolution in other systems.

Proc Natl Acad Sci U S A, 2002 Sep 3, 99(18), 11646 - 51 Epub 2002 Aug 15.
A far-red fluorescent protein with fast maturation and reduced oligomerization tendency from Entacmaea quadricolor (Anthozoa, Actinaria); Wiedenmann J et al.; We performed the biochemical and biophysical characterization of a red fluorescent protein, eqFP611, from the sea anemone Entacmaea quadricolor cloned in Escherichia coli . With an excitation maximum at 559 nm and an emission maximum at 611 nm, the recombinant protein shows the most red-shifted emission and the largest Stokes shift of all nonmodified proteins in the green fluorescent protein family . The protein fluoresces with a high quantum yield of 0.45, although it resembles the nonfluorescent members of this protein class, as inferred from the absence of the key amino acid serine at position 143 . Fluorescence is constant within the range pH 4-10 . Red fluorophore maturation reaches a level of 90% after approximately 12 h by passing through a green intermediate . After complete maturation, only a small fraction of the green species (less than 1%) persists . The protein has a reduced tendency to oligomerize, as shown by its monomeric appearance in SDS/PAGE analysis and single-molecule experiments . However, it forms tetramers at higher concentrations in the absence of detergent . Fluorescence correlation spectroscopy reveals light-driven transitions between bright and dark states on submillisecond and millisecond time scales . Applicability of eqFP611 for in vivo labeling in eukaryotic systems was shown by expression in a mammalian cell culture.

J Biol Chem, 2002 Nov 1, 277(44), 41318 - 25 Epub 2002 Aug 15.
PH-induced collapse of the extracellular loops closes Escherichia coli maltoporin and allows the study of asymmetric sugar binding; Andersen C et al.; LamB (maltoporin) is essential for the uptake of maltose and malto-oligosaccharides across the outer membrane of Escherichia coli . Purified LamB was reconstituted in artificial lipid bilayer membranes forming channels in the permanently open configuration at neutral pH . Almost complete channel closure was observed when the pH on both sides of the membrane was lowered to pH 4 . When LamB was added to only one side of the membrane, the cis-side, and the pH was lowered at either side of the membrane, the cis- or the trans-side, the response to pH was asymmetric, suggesting preferential orientation of maltoporin channels and pH- dependent closure of only one side of the channel . In experiments with LamB mutants in which major external loops L4, L6, and/or L9 were deleted, we identified the surface-exposed loops L4 and L6 as the cause of pH-mediated closure . The pH dependence of the LamB channel is consistent with the assumption that it inserts in a preferential orientation into the lipid bilayer . About 70-80% of the reconstituted channels are oriented with the extracellular entrance toward the side to which the protein was added (the cis-side) and with the periplasmic opening on the opposite side (the trans-side) . The possibility of closing the channels, which are oriented in the reverse direction by low pH at the trans-side, allowed the deduction of channel asymmetry with respect to carbohydrate binding kinetics . Whereas maltose binding was found to be almost symmetric with respect to the channel orientation, the sucrose and trehalose binding to LamB was asymmetric . The results are discussed in respect to possible physiological function of the pH-dependent closure of maltoporin.

J Aerosol Med, 2002 Summer, 15(2), 221 - 8
Intranasal immunization against influenza; Gluck R; Nasalflu is a novel influenza subunit vaccine, which is administered by the intranasal route using a spray device . Nasalflu is based on the virosomal concept which is registered in the EU as Epaxal Berna, a vaccine against Hepatitis A, and Inflexal Berna V, a subunit influenza vaccine . The virosome is a carrier system which delivers antigens to cells and is able to induce both B- and T-cell immunity . When virosomal vaccines are given parenterally, an immune response is elicited fast and sufficiently.

Chem Res Toxicol, 2002 Aug, 15(8), 1001 - 9
Substrate recognition by a family of uracil-DNA glycosylases: UNG, MUG, and TDG; Liu P et al.; In response to continuous hydrolytic and oxidative DNA damage, cells of all organisms have a complex network of repair systems that recognize, remove, and rebuild the injured sites . Damaged pyrimidines are generally removed by glycosylases that must scan the entire genome to locate lesions with sufficient fidelity to selectively remove the damage without inadvertent removal of normal bases . We report here studies conducted with a series of base analogues designed to test mechanisms of base recognition suggested by structural studies of glycosylase complexes . The oligonucleotide series examined here includes 5-halouracils with increasing substituent size and purine analogues placed opposite the target uracil with hydrogen, amino, and keto substituents in the 2- and 6-positions . The glycosylases studied here include Escherichia coli uracil-DNA glycosylase (UNG), E . coli mismatch uracil-DNA glycosylase (MUG), and the Methanobacterium thermoautotrophicum mismatch thymine-DNA glycosylase (TDG) . The results of this study suggest that these glycosylases utilize several strategies for base identification, including (1) steric limitations on the size of the 5-substituent, (2) electronic-inductive properties of the 5-substituent, (3) reduced thermal stability of mispairs, and (4) specific functional groups on the purine base in the opposing strand . Contrary to predictions based upon the crystal structure, the preference of MUG for mispaired uracil over thymine is not based upon steric exclusion . Furthermore, the preference for mispaired uracil over uracil paired with adenine is more likely due to reduced thermal stability as opposed to specific recognition of the mispaired guanine . On the other hand, TDG, which exhibits modest discrimination among various pyrimidines, shows strong interactions with functional groups present on the purine opposite the target pyrimidine . These results provide new insights into the mechanisms of base selection by DNA repair glycosylases.

J Pediatr, 2002 Aug, 141(2), 253 - 8
Idiopathic thrombocytopenic purpura diagnosed during the second decade of life; Lowe EJ et al.; OBJECTIVE: To retrospectively review our institutional experience of adolescents with idiopathic thrombocytopenic purpura (ITP) . STUDY DESIGN: Medical record review of all patients diagnosed with ITP between the ages of 10 and 18 years seen at our center from January 1976 to March 2000 . RESULTS: Data were collected from 126 patients . Of the evaluable 110 cases, 63 (57%) satisfied the criteria for chronic ITP, 30 (27%) for acute ITP, and 17 (15%) were uncertain . Sex distribution and mean ages were similar in all 3 groups . Platelet count at presentation was higher in patients with chronic ITP . Splenectomy was performed in 24 patients, with 17 (77%) of 22 having normal platelet counts at last follow-up . Outcome for the nonsplenectomized patients with chronic ITP included normalization of platelet count (n = 4), minimal or no bleeding without treatment (n = 29), treatment for ongoing symptoms (n = 5), and unknown (n = 1) . Two patients died, 1 from intracranial hemorrhage and 1 from Escherichia coli sepsis and pulmonary hemorrhage . CONCLUSIONS: Patients 10 to 18 years of age with ITP are more likely than younger children to have chronic disease . Many patients with ITP recover without drug therapy or need for splenectomy . ITP in adolescents shares features of both childhood and adult ITP.

Science, 2002 Aug 16, 297(5584), 1183 - 6
Stochastic gene expression in a single cell; Elowitz MB et al.; Clonal populations of cells exhibit substantial phenotypic variation . Such heterogeneity can be essential for many biological processes and is conjectured to arise from stochasticity, or noise, in gene expression . We constructed strains of Escherichia coli that enable detection of noise and discrimination between the two mechanisms by which it is generated . Both stochasticity inherent in the biochemical process of gene expression (intrinsic noise) and fluctuations in other cellular components (extrinsic noise) contribute substantially to overall variation . Transcription rate, regulatory dynamics, and genetic factors control the amplitude of noise . These results establish a quantitative foundation for modeling noise in genetic networks and reveal how low intracellular copy numbers of molecules can fundamentally limit the precision of gene regulation.

Infect Immun, 2002 Sep, 70(9), 5316 - 8
Shiga toxin 2 induces macrophage-granulocyte colonies from human bone marrow and cord blood stem cells; Chiyoda S et al.; Addition of Shiga toxin 2 to human bone marrow or cord blood cell culture induced macrophage-granulocyte colonies . Although Shiga toxin 2 alone induced colonies mainly composed of macrophages, it induced colonies mainly consisting of granulocytes when combined with physiological doses of interleukin-1beta, granulocyte colony-stimulating factor, or stem cell factor with interleukin-3.

J Biol Chem, 2002 Oct 18, 277(42), 39548 - 53 Epub 2002 Aug 14.
Cofactor binding to Escherichia coli D-3-phosphoglycerate dehydrogenase induces multiple conformations which alter effector binding; Grant GA et al.; The inhibition of Escherichia coli d-3-phosphoglycerate dehydrogenase by l-serine is positively cooperative with a Hill coefficient of approximately 2, whereas the binding of the inhibitor, l-serine, to the apoenzyme displays positive cooperativity in the binding of the first two serine molecules and negative cooperativity in the binding of the last two serine molecules . An earlier report demonstrated that the presence of phosphate appeared to lessen the degree of both the positive and negative cooperativity, but the cause of this effect was unknown . This study demonstrates that the presence of intrinsically bound NADH was responsible to a substantial degree for this effect . In addition, this study also provides evidence for negative cooperativity in NADH binding and for at least two NADH-induced conformational forms of the enzyme that bind the inhibitor in the physiological range . Successive binding of NADH to the enzyme resulted in an increase in the affinity for the first inhibitor ligand bound and a lessening of both the positive and negative cooperativity of inhibitor binding as compared with that seen in the absence of NADH . This effect was specific for NADH and was not observed in the presence of NAD+ or the substrate alpha-ketoglutarate . Conversely, the binding of l-serine did not have a significant effect on the stoichiometry of NADH binding, consistent with it being a V-type allosteric system . Thus, cofactor-related conditions were found in equilibrium binding experiments that significantly altered the cooperativity of inhibitor binding . Since the result of inhibitor binding is a reduction in the catalytic activity, the binding of inhibitor to these NADH-induced conformers must also induce additional conformations that lead to differential inhibition of catalytic activity.

J Biol Chem, 2002 Oct 25, 277(43), 41060 - 9 Epub 2002 Aug 14.
Structure-function analysis of HscC, the Escherichia coli member of a novel subfamily of specialized Hsp70 chaperones; Kluck CJ et al.; Hsp70 chaperones assist protein folding processes through nucleotide-controlled cycles of substrate binding and release . In our effort to understand the structure-function relationship within the Hsp70 family of proteins, we characterized the Escherichia coli member of a novel Hsp70 subfamily, HscC, and identified considerable differences to the well studied E . coli homologue, DnaK, which together suggest that HscC is a specialized chaperone . The basal ATPase cycle of HscC had k(cat) and K(m) values that were 8- and 10,000-fold higher than for DnaK . The HscC ATPase was not affected by the nucleotide exchange factor of DnaK GrpE and stimulated 8-fold by DjlC, a DnaJ protein with a putative transmembrane domain, but not by other DnaJ proteins tested . Substrate binding dynamics and substrate specificity differed significantly between HscC and DnaK . These differences are explicable by distinct structural variations . HscC does not have general chaperone activity because it did not assist refolding of a denatured model substrate . In vivo, HscC failed to complement temperature sensitivity of DeltadnaK cells . Deletion of hscC caused a slow growth phenotype that was suppressed after several generations . Triple knock-outs of all E . coli genes encoding Hsp70 proteins (DeltadnaK DeltahscA DeltahscC) were viable, indicating that Hsp70 proteins are not strictly essential for viability . An extensive search for DeltahscC phenotypes revealed a hypersensitivity to Cd(2+) ions and UV irradiation, suggesting roles of HscC in the cellular response to these stress treatments . Together our data show that the Hsp70 structure exhibits an astonishing degree of adaptive variations to accommodate requirements of a specialized function.

Genes Dev, 2002 Aug 15, 16(16), 2147 - 55
YaeL (EcfE) activates the sigma(E) pathway of stress response through a site-2 cleavage of anti-sigma(E), RseA; Kanehara K et al.; Escherichia coli YaeL (EcfE) is a homolog of human site-2 protease (S2P), a membrane-bound zinc metalloprotease involved in regulated intramembrane proteolysis . We have shown previously that YaeL, having essential metalloprotease active site motifs in the cytoplasmic domain, is indispensable for viability . Here, we obtained rpoE, encoding an extracytoplasmic stress response sigma factor (sigma(E)), as a multicopy suppressor against the yaeL disruption . Whereas sigma(E) is thought to be activated by regulated cleavage of RseA on the periplasmic side by the DegS protease, we found that a degradation intermediate of RseA consisting of the transmembrane and the cytoplasmic domains accumulated in the YaeL-depleted cells . This intermediate was degraded on expression of YaeL but not of its metalloprotease motif mutants . Cells depleted of YaeL were incapable of activating a sigma(E)-dependent promoter in response to an envelope stress . It is suggested that sigma(E) activation involves two successive proteolytic cleavages: first, at a periplasmic site by DegS; second, at a cytoplasmic or intramembrane site by YaeL . Thus, YaeL is positively required for the sigma(E) extracytoplasmic stress response.

Antimicrob Agents Chemother, 2002 Sep, 46(9), 2772 - 8
Heterologous expression of epothilone biosynthetic genes in Myxococcus xanthus; Julien B et al.; Epothilones are potential anticancer drugs that stabilize microtubules in a manner similar to paclitaxel (Taxol) . Epothilones are produced from the myxobacterium Sorangium cellulosum, which has a 16-h doubling time and produces only milligram-per-liter amounts of epothilone A and epothilone B . Furthermore, genetic manipulation of S . cellulosum is difficult . To produce epothilones in a more genetically amenable and rapidly growing host, we chose the closely related and best-characterized myxobacteria Myxococcus xanthus . We inserted 65.4 kb of S . cellulosum DNA that encompassed the entire epothilone gene cluster into the chromosome of M . xanthus by a series of homologous recombination events . The resulting strain produced epothilones A and B . Construction of a strain that contained a mutation in epoK, the P450 epoxidase, resulted in production of epothilones C and D.

Biochim Biophys Acta, 2002 Aug 19, 1591(1-3), 109 - 118
Therapeutic efficacy of anti-ErbB2 immunoliposomes targeted by a phage antibody selected for cellular endocytosis; Nielsen UB et al.; Many targeted cancer therapies require endocytosis of the targeting molecule and delivery of the therapeutic agent to the interior of the tumor cell . To generate single chain Fv (scFv) antibodies capable of triggering receptor-mediated endocytosis, we previously developed a method to directly select phage antibodies for internalization by recovering infectious phage from the cytoplasm of the target cell . Using this methodology, we reported the selection of a panel of scFv that were internalized into breast cancer cells from a nonimmune phage library . For this work, an immunotherapeutic was generated from one of these scFv (F5), which bound to ErbB2 (HER2/neu) . The F5 scFv was reengineered with a C-terminal cysteine, expressed at high levels in Escherichia coli, and coupled to sterically stabilized liposomes . F5 anti-ErbB2 immunoliposomes were immunoreactive as determined by surface plasmon resonance (SPR) and were avidly internalized by ErbB2-expressing tumor cell lines in proportion to the levels of ErbB2 expression . F5-scFv targeted liposomes containing doxorubicin had antitumor activity and produced significant reduction in tumor size in xenografted mice compared to nontargeted liposomes containing doxorubicin . This strategy should be applicable to generate immunotherapeutics for other malignancies by selecting phage antibodies for internalization into other tumor types and using the scFv to target liposomes or other nanoparticles.

Biochim Biophys Acta, 2002 Aug 19, 1591(1-3), 1 - 10
Identification of methylated proteins by protein arginine N-methyltransferase 1, PRMT1, with a new expression cloning strategy; Wada K et al.; Methylation at arginines has recently come to attention as a posttranslational modification of proteins, which is implicated in processes from signaling, transcriptional activation, to mRNA processing . Here we report that several proteins extracted from HeLa cells were methylated by PRMT1 (protein arginine N-methyltransferease 1) even on a nitrocellulose membrane, while proteins from Escherichia coli are not methylated with this protein . Screening PRMT1 substrates from a lambdagt11-HeLa cDNA library, we found that more than half of the 48 cDNA clones obtained encode putative RNA-binding proteins that have RGG (arginine-glycine-glycine) motifs, such as hnRNP R (heterogeneous nuclear ribonucleoprotein R) and hnRNP K . We cloned two novel arginine methylation substrates, ZF5 (zinc finger 5) and p137GPI (GPI-anchor protein p137), which do not possess typical RGG motifs . We also cloned a novel protein that has RGG motifs, but does not have any other RNA-binding motifs . We tentatively termed this clone SAMT1 (substrate of arginine methyl transferase 1) . A(63-)VLD(-65) to AAA mutation of PRMT1 suppressed the methylation of recombinant SAMT1 and other RGG proteins in the HeLa extracts . This systematic screening of substrate proteins with the solid phase methylation reaction will contribute to identify new roles of PRMT family.

Bioorg Med Chem Lett, 2002 Sep 16, 12(18), 2651 - 4
Oligonucleotides comprised of alternating 2'-deoxy-2'-fluoro-beta-D-arabinonucleosides and D-2'-deoxyribonucleosides (2'F-ANA/DNA 'altimers') induce efficient RNA cleavage mediated by RNase H; Min KL et al.; Chimeric oligonucleotides comprised of alternating residues of 2'-deoxy-2'-fluoro-D-arabinonucleic acid (2'F-ANA) and DNA were synthesized and evaluated for an important antisense property-the ability to elicit ribonuclease H (RNase H) degradation of complementary RNA . Experiments used both human RNase HII and Escherichia coli RNase HI . Mixed backbone oligomers comprising alternating three-nucleotide segments of 2'F-ANA and three-nucleotide segments of DNA were the most efficient at eliciting RNase H degradation of target RNA, and were significantly better than oligonucleotides entirely composed of DNA, suggesting that these mixed backbone oligonucleotides may be potent antisense agents.

Protein Expr Purif, 2002 Aug, 25(3), 547 - 57
Heterologous expression, purification, and enzymatic activity of Mycobacterium tuberculosis NAD(+) synthetase; Bellinzoni M et al.; The enzyme NAD(+) synthetase (NadE) catalyzes the last step of NAD biosynthesis . Given NAD vital role in cell metabolism, the enzyme represents a valid target for the development of new antimycobacterial agents . In the present study we expressed and purified two putative forms of Mycobacterium tuberculosis NAD(+) synthetase, differing in the polypeptide chain length (NadE-738 and NadE-679) . Furthermore, we evaluated several systems for the heterologous expression and large scale purification of the enzyme . In particular, we compared the efficiency of production, the yield of purification, and the catalytic activity of recombinant enzyme in different hosts, ranging from Escherichia coli strains to cultured High Five (Trichoplusia ni BTI-TN-5B1-4) insect cells . Among the systems assayed, we found that the expression of a thioredoxin-NadE fusion protein in E . coli Origami(DE3) is the best system in obtaining highly pure, active NAD(+) synthetase . The recombinant enzyme maintained its activity even after proteolytic cleavage of thioredoxin moiety . Biochemical evidence suggests that the shorter form (NadE-679) may be the real M . tuberculosis NAD(+) synthetase . These results enable us to obtain a purified product for structure-function analysis and high throughput assays for rapid screening of compounds which inhibit enzymatic activity.

Protein Expr Purif, 2002 Aug, 25(3), 533 - 40
Expression, purification, and biochemical characterization of Mycobacterium tuberculosis aspartate decarboxylase, PanD; Chopra S et al.; Like all bacteria, Mycobacterium tuberculosis (Mtb) possesses the genes necessary for coenzyme A biosynthesis and metabolism . In the present work, the Mtb panD gene was PCR amplified, overexpressed, and purified by metal affinity chromatography . The recombinant Mtb panD was found to exist as a tetramer in solution . Incubation of Mtb panD at 37 degrees C for several hours resulted in a complete cleavage of the inactive (pi) form into the two subunits (alpha and beta) . The cleavage was confirmed by Western blot analysis as well as by N-terminal sequencing . Cleaved Mtb panD was assayed for decarboxylase activity with L-aspartate as substrate . The kinetic parameters K(m) and k(cat) were found to be 219 microM and 0.65s(-1), respectively . These results provide the means for further studies based on the identification of the Mtb panD as well as other components of pantothenate metabolism as potential drug targets.

Protein Expr Purif, 2002 Aug, 25(3), 508 - 18
Overexpression, purification, and characterization of the periplasmic space thiamin-binding protein of the thiamin traffic ATPase in Escherichia coli; Hollenbach AD et al.; Thiamin (Vitamin B(1)) transport in Escherichia coli occurs by the superfamily of traffic ATPases in which the initial receptor is the periplasmic binding protein . We have cloned the periplasmic thiamin-binding protein (TBP) of the E . coli periplasmic thiamin transport system and purified the overexpressed protein to apparent homogeneity . A subsequent biochemical characterization demonstrates that TBP is a 34.205kDa monomer . TBP also contains one tightly bound thiamin species {thiamin, thiamin monophosphate (TMP), or thiamin diphosphate (TDP)} per monomer (K(D)=0.8 microM) when isolated under conditions that would remove any loosely bound ligands . We also demonstrate that thiamin is readily exchangeable in the presence of exogenous thiamin with a k(off)=0.12s(-1) . The biochemical characteristics of the overexpressed, plasmid-derived TBP are indistinguishable from those determined for endogenous TBP purified from E . coli . The overexpression and purification of TBP that we present here allows the rapid isolation of large amounts of pure protein that are required for further mechanistic and structural studies and demonstrates a vast improvement over previously reported purifications.

Protein Expr Purif, 2002 Aug, 25(3), 503 - 7
Removal of DnaK contamination during fusion protein purifications; Rial DV et al.; The use of fusion proteins for recombinant protein expression in Escherichia coli has become popular because the carrier increases protein solubility, standardizes expression levels, and facilitates purification of the fusion products . However, we have observed that the peptide regions that fuse the carrier to the protein of interest bind E . coli Hsp70 molecular chaperones (DnaK) depending on their amino acid composition, resulting in an unwanted contamination during protein purification . Here we describe an approach that helps to circumvent this unwanted contamination . First, the appropriate amino acids surrounding and comprising the cloning site are chosen by using a software based on an algorithm already developed to decrease to a minimum the propensity of the fusion protein to bind DnaK . Second, DnaK contamination is significantly reduced by washing the fusion protein bound to the purification resin with MgATP plus soluble denatured E . coli proteins before elution . The approach can also be applied to eliminate other molecular chaperones.

Protein Expr Purif, 2002 Aug, 25(3), 448 - 55
Large-scale production of functional human adrenomedullin: expression, cleavage, amidation, and purification; Mitsuda Y et al.; Human adrenomedullin (hAM) is a 52-amino-acid regulatory peptide containing a six-membered ring structure and an amidated C-terminus, features that are essential for its biological activity . Here, we describe a simple and effective protocol for producing large quantities of highly pure, functional recombinant hAM . A peptide precursor (hAM-Gly) was expressed in Escherichia coli as a fusion protein with thioredoxin and collected as inclusion bodies . The fusion protein was then digested with BLase, a glutamate-specific endopeptidase, to prepare hAM-Gly . The essential ring structure formed spontaneously, while the terminal amide was generated by conversion of the added glycine residue using peptidylglycine alpha-amidating enzyme . The low solubility of hAM-Gly enabled the use of a selective precipitation/extraction method to generate a product that was 80-90% pure, which was sufficient to proceed with the alpha-amidating enzyme reaction . The resultant hAM was then purified further by column chromatography . The final yield was about 82 mg/L of bacterial culture, and the purity, determined by reverse phase HPLC, was >99.5% . The recombinant hAM was biologically active, eliciting concentration-dependent increases in cAMP in CHO-K1 cells expressing a specific hAM receptor and hypotensive responses when intravenously injected into rats . This new approach to the synthesis of hAM is simpler and more cost-effective for large-scale production than chemical synthesis . It therefore represents a new powerful tool that has the potential to facilitate analysis of the structure and function of hAM, as well as the development of new therapeutic protocols for the treatment of ailments such as hypertension.

Protein Expr Purif, 2002 Aug, 25(3), 363 - 71
Expression and purification of recombinant full-length NS3 protease-helicase from a new variant of Hepatitis C virus; Poliakov A et al.; Viral mRNA extracted from the serum of a patient infected with HCV strain 1a was used for cloning, expression, and purification of full-length Hepatitis C NS3 protein . Sequencing of the protease gene identified the virus to be a new variant closely related to strain H77, differing in 15 out of 631 amino acids in the NS3 protein, none of which were predicted to be directly involved in catalysis, binding of substrate, or cofactor . A pBAD expression system was used to express the enzyme with an N-terminal tag in Escherichia coli . Purification from the soluble cellular fraction was achieved by Ni(2+)-IMAC and PolyU Sepharose affinity chromatography . The dependence of the proteolytic activity of the full-length NS3 protein on ionic strength, glycerol concentration, and a peptide corresponding to the activating region of NS4A was analyzed and used to design an activity assay that is suitable for inhibition studies . The kinetic constants (k(cat) and K(M)) for catalysis and the inhibitory potencies (IC(50) and K(i)) of five product-based hexapeptide inhibitors were comparable to those reported for the truncated NS3 protein . Detailed kinetic and inhibition studies using this variant of full-length NS3 can increase the understanding of the enzymatic characteristics of NS3, reveal the importance of the substituted amino acids and the significance of the genetic variability for design of effective inhibitors of the virus, and is thus of relevance for drug discovery.

J Food Prot, 2002 Aug, 65(8), 1253 - 8
Inactivation of Escherichia coli by a combination of nisin, pulsed electric fields, and water activity reduction by sodium chloride; Terebiznik M et al.; The effect of nisin combined with pulsed electric fields (PEF) and water activity reduction by sodium chloride (NaCl) on the inactivation of E . coli in simulated milk ultrafiltrate media was studied with a Doehlert design and a response surface method . The reduction of water activity from 0.99 to 0.95 by the addition of NaCl (without any other hurdle) did not affect E . coli viability of approximately 10(8) CFU/ml . A reduction in PEF effectiveness occurred when the NaCl concentration was increased because of an increase in conductance, which reduced the pulse decay time . In cells submitted to PEF nisin activity was decreased, probably as a consequence of the nonspecific binding of nisin to cellular debris or the emergence of new binding sites in or from cells . However, the lethal effect due to nisin was reestablished and further improved when water activity was reduced to 0.95 . A synergistic effect was evidenced when low-intensity PEF were applied . Decreasing water activity to 0.95 and applying PEF at 5 kV/cm (a nonlethal intensity when no other hurdle is used) with the further addition of nisin (1,200 IU/ml) resulted in a 5-log cycle reduction of the bacterial population.

Yi Chuan Xue Bao, 2002, 29(3), 189 - 95
A method for constructing reshaping single-domain antibody; Cheng JL et al.; The aim of this research was to demonstrate a novel and practical method for constructing reshaping Single-domain antibodies . Different from other methods, our method does not need to model the configuration of antibodies with specific sequences to determine the sequences of human acceptor FRs and then determine which amino acid residues in human acceptor FRs should be substituted . Most importantly, reshaping and enhancing the antigen binding affinity shared one procedure at the same time . Using this method, the reshaping anti-CD28 single-domain antibodies were constructed . According to the amino acid sequence of a mouse anti-human CD28 monoclonal antibody VH, two most homologous sequences of human antibodies were selected from GenBank and one of them was used as a main framework region for constructing the reshaping antibody . Before the original mouse antibody CDRs were inserted into the human acceptor FRs, some amino acid residues which were different from those of the original mouse antibody in the corresponding positions of the human acceptor FRs were determined or alternatively mutated by their conservative properties in Kabat classification . When the synthesized nucleotide fragments in different length were spliced by overlap PCR into the entire reshaping genes, Taq DNA polymerase and high Mg2+ concentration were used to introduce more mutation in FRs and CDRs randomly . A phage library was constructed using these PCR products and several reshaping Single-domain antibodies with high antigen binding affinity were selected after three rounds of panning . Two of them were expressed in E . coli BL21 (DE3) . The antigen-binding affinity of refolded proteins was still in a high level measured by ELISA . These results suggested that this method was feasible and efficient for constructing reshaping Single-domain antibodies.

J Biol Chem, 2002 Oct 18, 277(42), 39477 - 84 Epub 2002 Aug 13.
The role of apolipoprotein A-I helix 10 in apolipoprotein-mediated cholesterol efflux via the ATP-binding cassette transporter ABCA1; Panagotopulos SE et al.; Recent studies of Tangier disease have shown that the ATP-binding cassette transporter A1 (ABCA1)/apolipoprotein A-I (apoA-I) interaction is critical for high density lipoprotein particle formation, apoA-I integrity, and proper reverse cholesterol transport . However, the specifics of this interaction are unknown . It has been suggested that amphipathic helices of apoA-I bind to a lipid domain created by the ABCA1 transporter . Alternatively, apoA-I may bind directly to ABCA1 itself . To better understand this interaction, we created several truncation mutants of apoA-I and then followed up with more specific point mutants and helix translocation mutants to identify and characterize the locations of apoA-I required for ABCA1-mediated cholesterol efflux . We found that deletion of residues 221-243 (helix 10) abolished ABCA1-mediated cholesterol efflux from cultured RAW mouse macrophages treated with 8-bromo-cAMP . Point mutations in helix 10 that affected the helical charge distribution reduced ABCA1-mediated cholesterol efflux versus the wild type . We noted a strong positive correlation between cholesterol efflux and the lipid binding characteristics of apoA-I when mutations were made in helix 10 . However, there was no such correlation for helix translocations in other areas of the protein as long as helix 10 remained intact at the C terminus . From these observations, we propose an alternative model for apolipoprotein-mediated efflux.

J Biol Chem, 2002 Oct 25, 277(43), 41157 - 62 Epub 2002 Aug 13.
DEAD box RhlB RNA helicase physically associates with exoribonuclease PNPase to degrade double-stranded RNA independent of the degradosome-assembling region of RNase E; Liou GG et al.; The Escherichia coli RNA degradosome is a multicomponent ribonucleolytic complex consisting of three major proteins that assemble on a scaffold provided by the C-terminal region of the endonuclease, RNase E . Using an E . coli two-hybrid system, together with BIAcore apparatus, we investigated the ability of three proteins, polynucleotide phosphorylase (PNPase), RhlB RNA helicase, and enolase, a glycolytic protein, to interact physically and functionally independently of RNase E . Here we report that Rh1B can physically bind to PNPase, both in vitro and in vivo, and can also form homodimers with itself . However, binding of RhlB or PNPase to enolase was not detected under the same conditions . BIAcore analysis revealed real-time, direct binding for bimolecular interactions between Rh1B units and for the RhlB interaction with PNPase . Furthermore, in the absence of RNase E, purified RhlB can carry out ATP-dependent unwinding of double-stranded RNA and consequently modulate degradation of double-stranded RNA together with the exonuclease activity of PNPase . These results provide evidence for the first time that both functional and physical interactions of individual degradosome protein components can occur in the absence of RNase E and raise the prospect that the RNase E-independent complexes of RhlB RNA helicase and PNPase, detected in vivo, may constitute mini-machines that assist in the degradation of duplex RNA in structures physically distinct from multicomponent RNA degradosomes.

Eur J Biochem, 2002 Aug, 269(16), 4134 - 42
Functional expression and mutational analysis of flavonol synthase from Citrus unshiu; Wellmann F et al.; Flavonols are produced by the desaturation of flavanols catalyzed by flavonol synthase . The enzyme belongs to the class of intermolecular dioxygenases which depend on molecular oxygen and FeII/2-oxoglutarate for activity, and have been in focus of structural studies recently . Flavonol synthase cDNAs were cloned from six plant species, but none of the enzymes had been studied in detail . Therefore, a cDNA from Citrus unshiu (Satsuma mandarin) designated as flavonol synthase was expressed in Escherichia coli, and the purified recombinant enzyme was subjected to kinetic and mutational chacterizations . The integrity of the recombinant synthase was revealed by a molecular ion from MALDI-TOF mass spectrometry at m/z 37888 +/- 40 (as compared to 37899 Da calculated for the translated polypeptide), and by partial N-terminal sequencing . Maximal flavonol synthase activity was observed in the range of pH 5-6 with dihydroquercetin as substrate and a temperature optimum at about 37 degrees C . Km values of 272, 11 and 36 micro m were determined for dihydroquercetin, FeII and 2-oxoglutarate, respectively, with a sixfold higher affinity to dihydrokaempferol (Km 45 micro m) . Flavonol synthase polypeptides share an overall sequence similarity of 85% (47% identity), whereas only 30-60% similarity were apparent with other dioxygenases . Like the other dioxygenases of this class, Citrus flavonol synthase cDNA encodes eight strictly conserved amino-acid residues which include two histidines (His221, His277) and one acidic amino acid (Asp223) residue for FeII-coordination, an arginine (Arg287) proposed to bind 2-oxoglutarate, and four amino acids (Gly68, His75, Gly261, Pro207) with no obvious functionality . Replacements of Gly68 and Gly261 by alanine reduced the catalytic activity by 95%, while the exchange of these Gly residues for proline completely abolished the enzyme activity . Alternatively, the substitution of Pro207 by glycine hardly affected the activity . The data suggest that Gly68 and Gly261, at least, are required for proper folding of the flavonol synthase polypeptide.

Eur J Biochem, 2002 Aug, 269(16), 3998 - 4008
A sucrose-binding protein homologue from soybean exhibits GTP-binding activity that functions independently of sucrose transport activity; Pirovani CP et al.; The sucrose binding protein (SBP) has been implicated as an important component of the sucrose uptake system in plants . SBP-mediated sucrose transport displays unique kinetic features and the protein is not similar to other transport proteins . Here, we report the characterization of a member of the SBP family from soybean {Glycine max (L) Merrill} designated S64 or SBP2 . Subcellular fractionation and precipitation by GTP-agarose demonstrated that S64/SBP2 is a membrane-associated protein that exhibits GTP binding activity . Purified recombinant S64/SBP2 protein, expressed as a histidine-tagged protein in Escherichia coli, exhibited nucleotide-binding specificity to guanine nucleotides . The GTP binding site was mapped to an imperfect Walker A type-sequence, Ala279-Leu-Ala-Pro-Thr-Lys-Lys-Ser286, by site-directed mutagenesis . Escherichia coli-produced wild-type protein and a truncated version of the protein containing the putative binding-sequence-bound GTP, although not with the same efficiency . In contrast, replacement of Thr283 and Lys284 residues to Leu and Glu residues prevented GTP binding . The site directed mutant failed to bind GTP but retained the ability to undergo oligomerization andto promote growth of the susy7 yeast strain, deficient inutilizing extracellular sucrose, on medium containing sucrose as the sole carbon source . Our results indicate that GTP binding and sucrose transport by SBP are separable and function independently . The implications of our findings with respect to the function and membrane topology of SBP are discussed.

Eur J Biochem, 2002 Aug, 269(16), 3978 - 89
Leishmania donovani phosphofructokinase . Gene characterization, biochemical properties and structure-modeling studies; Lopez C et al.; The characterization of the gene encoding Leishmania donovani phosphofructokinase (PFK) and the biochemical properties of the expressed enzyme are reported . L . donovani has a single PFK gene copy per haploid genome that encodes a polypeptide with a deduced molecular mass of 53 988 and a pI of 9.26 . The predicted amino acid sequence contains a C-terminal tripeptide that conforms to an established signal for glycosome targeting . L . donovani PFK showed most sequence similarity to inorganic pyrophosphate (PPi)-dependent PFKs, despite being ATP-dependent . It thereby resembles PFKs from other Kinetoplastida such as Trypanosoma brucei, Trypanoplasma borreli (characterized in this study), and a PFK found in Entamoeba histolytica . It exhibited hyperbolic kinetics with respect to ATP whereas the binding of the other substrate, fructose 6-phosphate, showed slight positive cooperativity . PPi, even at high concentrations, did not have any effect . AMP acted as an activator of PFK, shifting its kinetics for fructose 6-phosphate from slightly sigmoid to hyperbolic, and increasing considerably the affinity for this substrate, whereas GDP did not have any effect . Modelling studies and site-directed mutagenesis were employed to shed light on the structural basis for the AMP effector specificity and on ATP/PPi specificity among PFKs.

Mol Microbiol, 2002 Aug, 45(4), 1107 - 17
A Rho-dependent phase-variable transcription terminator controls expression of the FimE recombinase in Escherichia coli; Joyce SA et al.; The fim switch is a 314 bp segment of invertible chromosomal DNA that is responsible for phase-variable expression of type 1 fimbriae in Escherichia coli . The switch harbours the promoter of the fimA gene . This codes for the type 1 fimbrial subunit protein and, when the promoter is directed towards fimA (phase ON), the bacteria are fimbriate and, when it is directed away, the cells are afimbriate . The switch lies immediately downstream from the fimE gene, coding for a tyrosine site-specific recombinase that catalyses inversion of the switch from the ON to the OFF phase . It has been suggested previously that, because the fim switch lies immediately downstream from the fimE gene, expression of FimE could be subject to control by antisense RNA in phase OFF bacteria in which the promoter harboured within the fim switch is oriented against the direction of transcription of the fimE gene . In this study, no effect of inducible fimE antisense RNA, expressed in cis or in trans, on FimE expression was detected . In phase ON cells, fimE mRNA extends across the switch into fimA, whereas in phase OFF cells, it terminates within the switch . This termination is Rho dependent and is abolished in a rho mutant . The extended fimE found in phase ON cells is more stable and results in an approximately fivefold increase in FimE protein compared with phase OFF bacteria . In the absence of Rho factor, fimE mRNA is equally stable in phase ON and phase OFF cells, and the levels of FimE recombinase are also equal.

Mol Microbiol, 2002 Aug, 45(4), 933 - 41
Characterization of a fungal protein kinase from Cryphonectria parasitica and its transcriptional upregulation by hypovirus; Kim MJ et al.; The chestnut blight fungus Cryphonectria parasitica and its hypovirus comprise useful model system to study the mechanisms of hypoviral infection . We used degenerate primers based on fungal protein kinases to isolate a gene, cppk1, which encodes a novel Ser/Thr protein kinase of C . parasitica . The gene showed highest homology to ptk1, a Ser/Thr protein kinase from Trichoderma reesei . The encoded protein had a predicted mass of 70.5 kDa and a pI of 7.45 . Northern blot analyses revealed that the cppk1 transcript was expressed from the beginning of culture, with a slight increase by 5 days of culture . However, its expression was specifically affected by the presence of virus, and it was transcriptionally upregulated in the fungal strain infected with the hypovirus . A kinase assay using Escherichia coli-derived CpPK1 revealed CpPK1-specific phosphorylated proteins with estimated masses of 50 kDa and 44 kDa . In addition, the phosphorylation of both proteins was higher in a cell-free extract from the hypovirulent strain . The increased expression of cppk1 by the introduction of an additional copy results in a subset of viral symptoms of reduced pigmentation and conidiation in a virus-free isolate . cppk1 overexpression also causes the downregulation of mating factor genes Mf2/1 and Mf2/2, resulting in female sterility . The present study suggests that the hypovirus disturbs fungal signalling by transcriptional upregulation of cppk1, which results in reduced pigmentation and conidiation and female sterility.

Transpl Immunol, 2002 May, 9(2-4), 323 - 9
Efficient in vitro transduction of epithelial cells and keratinocytes with improved adenoviral gene transfer for the application in skin tissue engineering; Doebis C et al.; The adenovirus-mediated transfer of therapeutic genes into keratinocytes may be a useful approach to treat several skin diseases or to improve the graft take of in vitro generated skin equivalents used for wound coverage . However, in contrast to many other tissues, keratinocytes are relatively difficult to transduce by adenoviral vectors . To achieve high efficiency of adenoviral transduction into epithelial cells we investigated the effects of the polycation polybrene on the infection process . The human (HaCaT, A549) and rat (NBT II, MHICI) epithelial cell lines, as well as human and rat primary keratinocytes, were transduced with recombinant Ad(beta)-gal adenovirus, encoding for the reporter gene E . coli beta-galactosidase, in the presence of various polybrene concentrations . We determined the amount of beta-gal positive cells by X-gal staining and the beta-gal expression by ONPG-assay after 24 h . In all tested human and rat epithelial cell lines, as well as in human and rat primary keratinocytes, the addition of polybrene during adenoviral transduction of Ad(beta)-gal resulted in a marked increase of beta-gal positive cells and beta-gal protein expression . The efficacy of polybrene showed a clear dose dependency . The improvement of adenoviral gene transfer into various types of human and rat epithelial cells by polybrene allows us to reduce the amount of recombinant virus particles resulting in a decreased inflammation induced by this therapeutic agent . In addition, the efficient transduction and expression with enhanced adenoviral transfer of therapeutic genes into primary keratinocytes provides a powerful tool for analysing the functions and the regulation of a gene of interest in vitro.

Int Microbiol, 2002 Jun, 5(2), 91 - 4
Identification and partial purification of K88ab Escherichia coli receptor proteins in porcine brush border membranes; Caloca MJ et al.; Six receptor proteins, with molecular masses ranging from 94 to 27 kDa, that bind to Escherichia coli K88ab fimbriae were recovered from brush border membranes and were detected after solubilization with Triton X-114 . The recovery of these receptor proteins in the aqueous phase suggests their peripheral localization . The 63-, 60- and 33-kDa K88ab binding proteins were recovered using gel-filtration chromatography of the aqueous phase.

Pharm Res, 2002 Jul, 19(7), 960 - 7
The lower-generation polypropylenimine dendrimers are effective gene-transfer agents; Zinselmeyer BH et al.; OBJECTIVE: To evaluate polypropylenimine dendrimers (generations 1-5: DAB 4, DAB 8, DAB 16, DAB 32, and DAB 64) as gene delivery systems . METHODS: DNA binding was evaluated by measuring the reduced fluorescence of ethidium bromide, and molecular modelling of dendrimer-DNA complexes also was performed . Cell cytotoxicity was evaluated against the A431 cell line using the MTT assay . In vitro transfection was evaluated against the A431 cell line using the beta-galactosidase reporter gene and N-{1-(2,3-dioleoyloxy)propyl}-N,N,N-trimethylammonium methylsulphate (DOTAP) served as a positive control . RESULTS: Molecular modeling and experimental data revealed that DNA binding increased with dendrimer generation . Cell cytotoxicity was largely generation dependent, and cytotoxicity followed the trend DAB 64 > DAB 32 > DAB 16 > DOTAP > DAB 4 > DAB 8, whereas transfection efficacy followed the trend DAB 8 = DOTAP = DAB 16 > DAB 4 > DAB 32 = DAB 64 . CONCLUSION: The generation 2 polypropylenimine dendrimer combines a sufficient level of DNA binding with a low level of cell cytoxicity to give it optimum in vitro gene transfer activity.

Life Sci Space Res, 1975, 13, 187 - 93
Peculiarities of biological action of hadrons of space radiation; Akoev IG et al.; Biological investigations in space enable one to make a significant contribution on high-energy hadrons to biological effects under the influence of factors of space flights . Physical and molecular principles of the action of high-energy hadrons are analysed . Genetic and somatic hadron effects produced by the secondary radiation from 70 GeV protons have been studied experimentally . The high biological effectiveness of hadrons, great variability in biological effects, and specifically of their action, are associated with strong interactions of high-energy hadrons . These are the probability of nuclear interaction with any atom nucleus, generation of a great number of secondary particles (among them, probably, highly effective multicharged and heavy nuclei, antiprotons, pi(-)-mesons), and the spatial distribution of secondary particles as a narrow cone with extremely high density of particles in its first part . The secondary radiation generated by high- and superhigh-energy hadrons upon their interaction with the spaceship is likely to be the greatest hazard of radiation to the crew during space flights.

Izv Akad Nauk Ser Biol, 2002 Jul-Aug, (4), 393 - 401
{Hemopoiesis on stromal sublayers formed by constant lines of fibroblasts}; Michurina TV et al.; We studied the formation of hemopoietic colonies on an artificial sublayer in the peritoneal cavity of mice under the influence of stromal sublayers consisting of fibroblasts of six constant cell limes . All studied lines of stromal cells supported the formation of granulocytic foci and some of them supported the formation of erythroid foci as well . It was shown that hemopoiesis was preserved or, in some cases, enhanced on sublayers of fixed (metabolically inactive) cells . The treatment of fibroblasts by E . coli lipopolysaccharide did not lead, as a rule, to significant stimulation of hemopoiesis.

Br J Cancer, 2002 Aug 12, 87(4), 405 - 13
Production and characterisation of a recombinant scFv reactive with human gastrointestinal carcinomas; Kim DJ et al.; SC142-reactive antigen are highly glycosylated glycoproteins expressed on tissues of gastric and colon cancers but not on normal tissues . Murine SC142 antibody specific for the SC142-reactive antigen has been produced by immunisation with SNU16 stomach cancer cells . However, SC142 antibody has several potential problems such as high immunogenicity and poor tumour penetration owing to their large size . To improve tumour penetration potential in vivo, recombinant single-chain fragments have been produced using the original hybridoma cells as a source of variable heavy- and variable light-chain-encoding antibody genes . The use of the polymerase chain reaction, expression cloning technology and gene expression systems in E . coli has led to the production of SC142 single-chain fragments, which was similar in activity to the SC142 parent antibody confirmed by immunohistochemistry . Analysis by DNA sequencing, SDS-PAGE and Western blotting has demonstrated the integrity of the single-chain fragments . Competitive ELISA showed that SC142 single-chain fragments originated from parent SC142 antibody . BIAcore biosensor binding experiments showed that the SC142 single-chain fragments had an ideal dissociation rate constant as a tumour imaging reagent . These results illustrate the potential application of these novel products as an immunodiagnostic and further immunotherapeutic reagent.

Plant Physiol, 2002 Aug, 129(4), 1892 - 8
Inhibition of phospholipase D alpha by N-acylethanolamines; Austin-Brown SL et al.; N-Acylethanolamines (NAEs) are endogenous lipids in plants produced from the phospholipid precursor, N-acylphosphatidylethanolamine, by phospholipase D (PLD) . Here, we show that seven types of plant NAEs differing in acyl chain length and degree of unsaturation were potent inhibitors of the well-characterized, plant-specific isoform of PLD-PLD alpha . It is notable that PLD alpha, unlike other PLD isoforms, has been shown not to catalyze the formation of NAEs from N-acylphosphatidylethanolamine . In general, inhibition of PLD alpha activity by NAEs increased with decreasing acyl chain length and decreasing degree of unsaturation, such that N-lauroylethanolamine and N-myristoylethanolamine were most potent with IC(50)s at submicromolar concentrations for the recombinant castor bean (Ricinus communis) PLD alpha expressed in Escherichia coli and for partially purified cabbage (Brassica oleracea) PLD alpha . NAEs did not inhibit PLD from Streptomyces chromofuscus, and exhibited only moderate, mixed effects for two other recombinant plant PLD isoforms . Consistent with the inhibitory biochemical effects on PLD alpha in vitro, N-lauroylethanolamine, but not lauric acid, selectively inhibited abscisic acid-induced closure of stomata in epidermal peels of tobacco (Nicotiana tabacum cv Xanthi) and Commelina communis at low micromolar concentrations . Together, these results provide a new class of biochemical inhibitors to assist in the evaluation of PLD alpha physiological function(s), and they suggest a novel, lipid mediator role for endogenously produced NAEs in plant cells.

Plant Physiol, 2002 Aug, 129(4), 1866 - 71
Redox regulation of Arabidopsis 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase; Entus R et al.; The cDNA for 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase of Arabidopsis encodes a polypeptide with an amino-terminal signal sequence for plastid import . A cDNA fragment encoding the processed form of the enzyme was expressed in Escherichia coli . The resulting protein was purified to electrophoretic homogeneity . The enzyme requires Mn(2+) and reduced thioredoxin (TRX) for activity . Spinach (Spinacia oleracea) TRX f has an apparent dissociation constant for the enzyme of about 0.2 microM . The corresponding constant for TRX m is orders of magnitude higher . In the absence of TRX, dithiothreitol partially activates the enzyme . Upon alkylation of the enzyme with iodoacetamide, the dependence on a reducing agent is lost . These results indicate that the first enzyme in the shikimate pathway of Arabidopsis appears to be regulated by the ferredoxin/TRX redox control of the chloroplast.

Plant Physiol, 2002 Aug, 129(4), 1710 - 22
Arabidopsis contains nine long-chain acyl-coenzyme a synthetase genes that participate in fatty acid and glycerolipid metabolism; Shockey JM et al.; Long-chain acyl-coenzyme A (CoA) synthetases (LACSs) activate free fatty acids to acyl-CoA thioesters and as such play critical roles in fatty acid metabolism . This important class of enzymes factors prominently in several fatty acid-derived metabolic pathways, including phospholipid, triacylglycerol, and jasmonate biosynthesis and fatty acid beta-oxidation . In an effort to better understand the factors that control fatty acid metabolism in oilseeds, we have sought to identify and characterize genes that encode LACSs in Arabidopsis . Nine cDNAs were identified, cloned, and tested for their ability to complement a LACS-deficient strain of yeast (Saccharomyces cerevisiae) . Seven of the nine successfully restored growth, whereas two cDNAs encoding putative peroxisomal isoforms did not . Lysates from yeast cells overexpressing each of the nine cDNAs were active in LACS enzyme assays using oleic acid as a substrate . The substrate specificities of the enzymes were determined after overexpression in LACS-deficient Escherichia coli . Most of the LACS enzymes displayed highest levels of activity with the fatty acids that make up the common structural and storage lipids in Arabidopsis tissues . Analysis of the tissue-specific expression profiles for these genes revealed one flower-specific isoform, whereas all others were expressed in various tissues throughout the plant . These nine cDNAs are thought to constitute the entire LACS family in Arabidopsis, and as such, will serve as powerful tools in the study of acyl-CoA metabolism in oilseeds.

Proc Natl Acad Sci U S A, 2002 Aug 20, 99(17), 11061 - 6 Epub 2002 Aug 12.
Two distinct modes of RecA action are required for DNA polymerase V-catalyzed translesion synthesis; Pham P et al.; SOS mutagenesis in Escherichia coli requires DNA polymerase V (pol V) and RecA protein to copy damaged DNA templates . Here we show that two distinct biochemical modes for RecA protein are necessary for pol V-catalyzed translesion synthesis . One RecA mode is characterized by a strong stimulation in nucleotide incorporation either directly opposite a lesion or at undamaged template sites, but by the absence of lesion bypass . A separate RecA mode is necessary for translesion synthesis . The RecA1730 mutant protein, which was identified on the basis of its inability to promote pol V (UmuD'(2)C)-dependent UV-mutagenesis, appears proficient for the first mode of RecA action but is deficient in the second mode . Data are presented suggesting that the two RecA modes are "nonfilamentous" . That is, contrary to current models for SOS mutagenesis, formation of a RecA nucleoprotein filament may not be required for copying damaged DNA templates . Instead, SOS mutagenesis occurs when pol V interacts with two RecA molecules, first at a 3' primer end, upstream of a template lesion, where RecA mode 1 stimulates pol V activity, and subsequently at a site immediately downstream of the lesion, where RecA mode 2 cocatalyzes lesion bypass . We posit that in vivo assembly of a RecA nucleoprotein filament may be required principally to target pol V to a site of DNA damage and to stabilize the pol V-RecA interaction at the lesion . However, it is only a RecA molecule located at the 3' filament tip, proximal to a damaged template base, that is directly responsible for translesion synthesis.

Neurology, 2002 Aug 13, 59(3), 451 - 4
Distal myopathy with rimmed vacuoles: novel mutations in the GNE gene; Tomimitsu H et al.; The authors present three novel missense mutations in the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) gene, the causative gene for hereditary inclusion body myopathy, in Japanese patients with distal myopathy with rimmed vacuoles . Seven out of nine patients had homozygous V572L mutation, one was a compound heterozygote with C303V and V572L mutations, and the remaining patient bore homozygous A631V mutation.

Microbiology, 2002 Aug, 148(Pt 8), 2573 - 8
Silencing of the Escherichia coli bgl operon by RpoS requires Crl; Schnetz K; Silencing of the Escherichia coli bgl operon is mediated by histone-like protein H-NS and affected by other pleiotropic regulators, including sigma factor RpoS . Silencing is relieved and the bgl operon is activated in hns mutants and by mutations that map in the vicinity of the bgl promoter . However, the expression level of activated bgl operon derivatives varies with the strain background . Here it is shown that the repression of the bgl operon by RpoS requires Crl . Crl is a protein that is necessary for the RpoS-dependent expression of the csgBA operon and that enhances the expression of other RpoS-dependent genes . In a Crl-negative strain RpoS had no effect on the bgl operon . The crl gene maps close to the proBA locus in the lac operon region and is deleted in many commonly used E . coli strains . Crl may therefore account for some of the observed strain-dependent variations of bgl operon expression levels and effects of pleiotropic regulators on bgl operon regulation.

Microbiology, 2002 Aug, 148(Pt 8), 2507 - 18
BfpU, a soluble protein essential for type IV pilus biogenesis in enteropathogenic Escherichia coli; Schreiber W et al.; A cluster of 14 genes located on the large plasmid of enteropathogenic Escherichia coli (EPEC) strains is sufficient to direct the biogenesis of the type IV bundle-forming pilus (BFP) in a recombinant E . coli host . The fifth gene in the cluster, bfpU, encodes a protein that is predicted to be localized to the periplasmic space . To determine whether BfpU is necessary for pilus biogenesis, the authors constructed a non-polar bfpU mutant EPEC strain by allelic exchange . The mutant strain was unable to perform localized adherence and auto-aggregation, two phenotypes associated with BFP expression, and it failed to make BFP . These phenotypes were restored to the bfpU mutant by a plasmid containing bfpU . There was no difference between the wild-type and bfpU mutant strains in their expression or processing of the pre-pilin protein or in their localization of the pilin protein in the inner and outer membranes . Fractionation studies revealed that BfpU is completely soluble and is detected in both the periplasm and the cytoplasm . Thus, BfpU represents a novel protein required for type IV pilus assembly.

Microbiology, 2002 Aug, 148(Pt 8), 2413 - 26
Regulation of phasin expression and polyhydroxyalkanoate (PHA) granule formation in Ralstonia eutropha H16; Potter M et al.; Regulation of expression of the phasin PhaP, which is the major protein at the surface of polyhydroxyalkanoate (PHA) granules in Ralstonia eutropha H16, was studied and analysed at the molecular level . The regulation of PhaP expression is achieved by an autoregulated repressor, which is encoded by phaR in R . eutropha . The occurrence of PhaR homologues and the organization of phaR genes was analysed in detail in 29 different bacteria . Three kinds of molecule to which PhaR binds were identified in cells of R . eutropha, as revealed by gel-mobility-shift assays, DNaseI footprinting, cell fractionation, immunoelectron microscopy studies employing anti-PhaR antibodies raised against purified N-terminal hexahistidine-tagged PhaR and in vitro binding studies employing artificial PHA granules . PhaR binds upstream of phaP at two sites comprising the transcriptional start site plus the -10 region and a region immediately upstream of the -35 region of the sigma(70) promoter of phaP, where two imperfect 12 bp repeat sequences (GCAMMAAWTMMD) were identified on the sense and anti-sense strands . PhaR also binds 86 bp upstream of the phaR translational start codon, where the sigma(54)-dependent promoter was identified . PhaR also binds to the surface of PHA granules . In the cytoplasm of a phaROmegaKm mutant of R . eutropha H16, increased quantities of PhaP were detected and the cells formed by this strain were much smaller and had many more PHA granules present than the wild-type . These data support the following model for the regulation of phaP expression . Under cultivation conditions not permissive for PHA biosynthesis or in mutants defective in PHA biosynthesis, PhaR binds to the phaP promoter region and represses transcription of this gene . After the onset of PHA biosynthesis, under conditions that are permissive for the formation of nascent granules, PhaR binds to PHA granules and phaP is transcribed . At the later stages of PHA accumulation, PhaR no longer binds to the granules and the transcription of phaP is again repressed . In addition to this, phaR expression is subject to autoregulation . Excess PhaR that has not bound to the phaP upstream region or to PHA granules binds to the phaR upstream region, thereby repressing its own transcription.

Microbiology, 2002 Aug, 148(Pt 8), 2393 - 403
Permeability of Coxiella burnetii to ribonucleosides; Miller JD et al.; Knowledge about transport in Coxiella burnetii, an obligate phagolysosomal parasite, is incomplete . The authors investigated the capability of isolated, intact, host-free Coxiella to transport ribonucleosides while incubated at a pH value typical of lysosomes . Because of the low activities and limitations of obtaining experimental quantities of isolated, purified Coxiella, incorporation of substrate into nucleic acid was used as a trap for determination of uptake abilities . Virulent wild-type (phase I) organisms possessed uptake capability for all ribonucleosides . Both phase I and phase II (avirulent) organisms incorporated the purine nucleosides guanosine, adenosine and inosine, and showed a more limited uptake of thymidine and uridine . Both phases were poorly active in cytidine uptake . Neither phase of the organism was capable of transport and incorporation of NTPs, CMP, cytosine or uracil . Water space experiments confirmed that the uptake process concentrated the purine nucleosides within the cytoplasm of both wild-type and phase II Coxiella via a low-pH-dependent mechanism . Comparison of uptake rates in Escherichia coli versus Coxiella verified that the incorporation of ribonucleosides by Coxiella is a slow process . It is concluded that Coxiella possesses some transport pathways consistent with utilization of pools of nucleosides found within its host cell lysosomal pathway.

Microbiology, 2002 Aug, 148(Pt 8), 2283 - 91
Prospecting for novel lipase genes using PCR; Bell PJ et al.; A PCR method suitable for the isolation of lipase genes directly from environmental DNA is described . The problems associated with the low levels of similarity between lipase genes were overcome by extensive analysis of conserved regions and careful primer design . Using this method, a lipase gene (oli-lipase) was isolated directly from environmental DNA . This lipase showed less than 20% similarity with other known lipases at the amino acid level . The study also revealed that distantly related members of the alpha/beta hydrolase superfamily share similar conserved motifs with the lipases, thus making these genes targets for gene prospecting by PCR.

Nucleic Acids Res . 2002 Aug 15;30(16):e88.
Removal of impurities from transcription factor preparations that alter their DNA-binding properties; Sun L et al.; Biochemical studies of transcriptional activators are important for understanding their detailed mechanism of action . Such experiments generally employ chimeric constructs comprised of fused DNA- binding and activation domains that are expressed in, and purified from, Escherichia coli, since full-length activators are usually difficult to express . We report here that such preparations contain chaperone impurities that affect the DNA-binding properties of the activator, for example sharply reducing the half-life of the protein-DNA complex . A simple method to remove these troublesome contaminants is described.

Nucleic Acids Res, 2002 Aug 15, 30(16), 3558 - 65
C.EcoO109I, a regulatory protein for production of EcoO109I restriction endonuclease, specifically binds to and bends DNA upstream of its translational start site; Kita K et al.; The EcoO109I restriction-modification system, which recognizes 5'-(A/G)GGNCC(C/T)-3', has been cloned, and contains convergently transcribed endonuclease and methylase . The role and action mechanism of the gene product, C.EcoO109I, of a small open reading frame located upstream of ecoO109IR were investigated in vivo and in vitro . The results of deletion analysis suggested that C.EcoO109I acts as a positive regulator of ecoO109IR expression but has little effect on ecoO109IM expression . Assaying of promoter activity showed that the expression of ecoO109IC was regulated by its own gene product, C.EcoO109I . C.EcoO109I was overproduced as a His-tag fusion protein in recombinant Escherichia coli HB101 and purified to homogeneity . C.EcoO109I exists as a homodimer, and recognizes and binds to the DNA sequence 5'-CTAAG(N)(5)CTTAG-3' upstream of the ecoO109IC translational start site . It was also shown that C.EcoO109I bent the target DNA by 54 +/- 4 degrees.

J Biol Chem, 2002 Oct 18, 277(42), 39456 - 62 Epub 2002 Aug 12.
A novel antioxidant mechanism of ebselen involving ebselen diselenide, a substrate of mammalian thioredoxin and thioredoxin reductase; Zhao R et al.; The antioxidant mechanism of ebselen involves recently discovered reductions by mammalian thioredoxin reductase (TrxR) and thioredoxin (Trx) forming ebselen selenol . Here we describe a previously unknown reaction; ebselen reacts with its selenol forming an ebselen diselenide with a rate constant of 372 m(-1)s(-1) . The diselenide also was a substrate of TrxR forming the selenol with K(m) of 40 microm and k(cat) of 79 min(-1) (k(cat)/K(m) of 3.3 x 10(4) m(-1)s(-1)) . Trx increased the reduction because of its fast reaction with diselenide (rate constant 1.7 x 10(3) m(-1)s(-1)) . Diselenide stimulated the H2O2 reductase activity of TrxR, even more efficiently with Trx present . Because the mechanism of ebselen as an antioxidant has been assumed to involve glutathione peroxidase-like activity, we compared the H2O2 reductase activity of ebselen with the GSH and Trx systems . TrxR at 50 nm, far below the estimated physiological level, gave 8-fold higher activity compared with 1 mm GSH; addition of 5 microm Trx increased this difference to 13-fold . The rate constant of ebselen selenol reacting with H2O2 was estimated to be faster than 350 m(-1)s(-1) . We propose novel mechanisms for ebselen antioxidant action involving ebselen selenol and diselenide formation, with the thioredoxin system rather than glutathione as the predominant effector and target.

J Biol Chem, 2002 Oct 18, 277(42), 39450 - 5 Epub 2002 Aug 12.
FAD is a preferred substrate and an inhibitor of Escherichia coli general NAD(P)H:flavin oxidoreductase; Louie TM et al.; Escherichia coli general NAD(P)H:flavin oxidoreductase (Fre) does not have a bound flavin cofactor; its flavin substrates (riboflavin, FMN, and FAD) are believed to bind to it mainly through the isoalloxazine ring . This interaction was real for riboflavin and FMN, but not for FAD, which bound to Fre much tighter than FMN or riboflavin . Computer simulations of Fre.FAD and Fre.FMN complexes showed that FAD adopted an unusual bent conformation, allowing its ribityl side chain and ADP moiety to form an additional 3.28 H-bonds on average with amino acid residues located in the loop connecting Fbeta5 and Falpha1 of the flavin-binding domain and at the proposed NAD(P)H-binding site . Experimental data supported the overlapping binding sites of FAD and NAD(P)H . AMP, a known competitive inhibitor with respect to NAD(P)H, decreased the affinity of Fre for FAD . FAD behaved as a mixed-type inhibitor with respect to NADPH . The overlapped binding offers a plausible explanation for the large K(m) values of Fre for NADH and NADPH when FAD is the electron acceptor . Although Fre reduces FMN faster than it reduces FAD, it preferentially reduces FAD when both FMN and FAD are present . Our data suggest that FAD is a preferred substrate and an inhibitor, suppressing the activities of Fre at low NADH concentrations.

J Biol Chem, 2002 Oct 18, 277(42), 39443 - 9 Epub 2002 Aug 12.
PDZ domains facilitate binding of high temperature requirement protease A (HtrA) and tail-specific protease (Tsp) to heterologous substrates through recognition of the small stable RNA A (ssrA)-encoded peptide; Spiers A et al.; The Escherichia coli protease HtrA has two PDZ domains, and sequence alignments predict that the E . coli protease Tsp has a single PDZ domain . PDZ domains are composed of short sequences (80-100 amino acids) that have been implicated in a range of protein:protein interactions . The PDZ-like domain of Tsp may be involved in binding to the extreme COOH-terminal sequence of its substrate, whereas the HtrA PDZ domains are involved in subunit assembly and are predicted to be responsible for substrate binding and subsequent translocation into the active site . E . coli has a system of protein quality control surveillance mediated by the ssrA-encoded peptide tagging system . This system tags misfolded proteins or protein fragments with an 11-amino acid peptide that is recognized by a battery of cytoplasmic and periplasmic proteases as a degradation signal . Here we show that both HtrA and Tsp are able to recognize the ssrA-encoded peptide tag with apparent K(D) values of approximately 5 and 390 nm, respectively, and that their PDZ-like domains mediate this recognition . Fusion of the ssrA-encoded peptide tag to the COOH terminus of a heterologous protein (glutathione S-transferase) renders it sensitive to digestion by Tsp but not HtrA . These observations support the prediction that the HtrA PDZ domains facilitate substrate binding and the differential proteolytic responses of HtrA and Tsp to SsrA-tagged glutathione S-transferase are interpreted in terms of the structure of HtrA.

J Biol Chem, 2002 Oct 18, 277(42), 39989 - 98 Epub 2002 Aug 09.
Network of interactions of a novel plant-specific Arg/Ser-rich protein, atRSZ33, with atSC35-like splicing factors; Lopato S et al.; Arg/Ser-rich (RS) proteins play a crucial role in splicing and are implicated in splice site selection in metazoa . In plants, intron recognition seems to differ from the one in animals due to specific factor requirements . Here we describe a new plant-specific RS-rich protein, atRSZ33, with a unique domain structure consisting of an RNA recognition motif (RRM), two zinc knuckles embedded in a basic RS region, and an acidic C-terminal domain . atRSZ33 was found to be a phosphoprotein that concentrates in nuclear speckles and is predominantly present in roots and flowers . In a yeast two-hybrid screen, atRSZ33 interacted with splicing factors atSRp34/SR1, an Arabidopsis ortholog of human SF2/ASF; atRSZp21 and atRSZp22, which are similar to the human 9G8; and three novel SC35-like splicing factors termed atSCL28, atSCL30, and atSCL33/SR33 . Two further members of the SCL family, namely SCL30a and the ortholog of mammalian SC35, atSC35, were also found to interact with atRSZ33 . These interactions were verified by in vitro binding assays; furthermore, the transcriptional activity of atRSZ33 was found to overlap with the ones of its interacting partners . These specific interactions coupled with the many similarities of atRSZ33 to SR proteins suggest that its main activity is in spliceosome assembly . Mapping of regions necessary for protein-protein interaction between atRSZ33 and atSCL33/SR33 revealed that both zinc knuckles together with a small part of the RS and the RRM domain are required for efficient binding . However, the interacting domain is relatively small, allowing binding of additional proteins, a feature that is consistent with the proposed role of atRSZ33 in spliceosome assembly.

Genome Res, 2002 Aug, 12(8), 1246 - 56
CpG methylation modifies the genetic stability of cloned repeat sequences; Nichol K et al.; The genetic stability of tandemly repeated DNAs is affected by repeat sequence, tract length, tract purity, and replication direction . Alterations in DNA methylation status are thought to influence many processes of mutagenesis . By use of bacterial and primate cell systems, we have determined the effect of CpG methylation on the genetic stability of cloned di-, tri-, penta- and minisatellite repeated DNA sequences . Depending on the repeat sequence, methylation can significantly enhance or reduce its genetic stability . This effect was evident when repeat tracts were replicated from either direction . Unexpectedly, methylation of adjacent sequences altered the stability of contiguous repeat sequences void of methylatable sites . Of the seven repeat sequences investigated, methylation stabilized five, destabilized one, and had no effect on another . Thus, although methylation generally stabilized repeat tracts, its influence depended on the sequence of the repeat . The current results lend support to the notion that the biological consequences of CpG methylation may be affected through local alterations of DNA structure as well as through direct protein-DNA interactions . In vivo CpG methylation in bacteria may have technical applications for the isolation and stable propagation of DNA sequences that have been recalcitrant to isolation and/or analyses because of their extreme instability.

Eur J Med Res, 2002 Jul 24, 7(7), 304 - 8
Intrapulmonary neutrophil apoptosis in a pig model of pneumonia; Bauer TT et al.; BACKGROUND: Apoptosis and necrosis are two distinct cell death modalities . Polymorphonuclear leukocytes (PMNL) play an important role in pneumonia and little information is available on whether these cells are cleared by necrosis or apoptosis . We therefore compared the proportion of apoptotic PMNL in lung tissues with and without pneumonia . In addition, the association of PMNL apoptosis and mechanical ventilation (MV) was investigated . METHODS: Samples obtained from a pig model developed to tracheobronchial stenting were analysed . Pneumonia was induced with that model in 10 animals (white-Landrace piglets, Pittman-Moore or Gottingen minipigs) . Animals were sacrificed when clinical parameters of pneumonia became evident and 17 lung samples could be analysed for histologic evidence of pneumonia, growth of micro-organisms, and the apoptotic index (proportion of apoptotic PMNL over all PMNL in a 400-power field) . In addition, lung samples (n = 7) from two pigs without stent implantation on mechanically ventilated for more than 48 h were studied . RESULTS: A total of 9/17 samples (53%) showed histologic evidence of pneumonia, 11/17 (65%) had significant bacterial growth (> 10(3) cfu/g), and 7/17 (41%) fulfilled both criteria . The apoptotic index was not significantly different in samples with and without histologic evidence of pneumonia (pneumonia: 25.3 +/- 6.1% vs . No pneumonia: 25.2 +/- 11.9%; 95%CI: -9.7 - 9.5, p = 0.989) or in samples with and without significant bacterial growth (significant bacterial growth: 23.5+/-9.3% vs . No significant bacterial growth: 28.5+/-8.2%; 95%CI: -4.6 - 16.7; p = 0.284) . However, the apoptotic index was higher in samples of pigs, mechanically ventilated for more than 48 h, compared to the stent group (MV: 36.9+/-10.7% vs . Stent: 25.2 +/- 9.0%; 95% CI: -20.5 - -2.9; p = 0.012) . CONCLUSION: In an animal model, the proportion of apoptotic PMNL was not different in lung samples with and without histologic or bacterial pneumonia . However, MV for more than 48 h was associated with a higher proportion of apoptotic PMNL in lung tissue . This study may be helpful for the understanding of the evolution of lung tissue damage induced by bacterial pneumonia and MV.

J Virol Methods, 2002 Aug, 105(1), 57 - 65
Use of heat labile UNG in an RT-PCR assay for enterovirus detection; Taggart EW et al.; A reverse transcription-polymerase chain reaction (RT-PCR) assay was developed to replace the Roche AMPLICOR Enterovirus Test used in our laboratory from 1996 to 1999 . The new assay design was optimized to match or exceed the performance of the Roche AMPLICOR Enterovirus test kit with respect to analytical sensitivity and specificity, contamination control, ease of use and availability of reagents . This new assay uses a heat labile form of the enzyme uracil DNA glycosylase (UNG) for amplicon contamination control and an RT-PCR enzyme mixture, enabling a one tube/one step amplification . RNA preparation was undertaken using a commercial extraction kit . End detection was accomplished using a probe-capture enzyme immuno assay (EIA) plate format . This EV RT-PCR assay exceeds the performance of the Roche AMPLICOR Enterovirus assay in a direct comparison . The combined enzymological approach has potential application to a wide variety of assays requiring sensitive RNA detection and stringent contamination control, including those utilizing real time detection methods.

Arch Biochem Biophys, 2002 Sep 1, 405(1), 130 - 6
Geranyl diphosphate synthase from Abies grandis: cDNA isolation, functional expression, and characterization; Burke C et al.; Geranyl diphosphate synthase catalyzes the condensation of dimethylallyl diphosphate and isopentenyl diphosphate to generate geranyl diphosphate, the essential precursor of monoterpene biosynthesis . Using geranylgeranyl diphosphate synthase from Taxus canadensis as a hybridization probe, four full length cDNA clones, sharing high sequence identity to each other (>69%) and to the Taxus geranylgeranyl diphosphate synthase (>66%), were isolated from a grand fir (Abies grandis) cDNA library . When expressed in Escherichia coli, three of the recombinant enzymes produced geranyl diphosphate and one produced geranylgeranyl diphosphate as the dominant product when supplied with isopentenyl diphosphate and dimethylallyl diphosphate as cosubstrates . One enzyme (AgGPPS2) was confirmed as a specific geranyl diphosphate synthase, in that it accepted only dimethylallyl diphosphate as the allylic cosubstrate and it produced exclusively geranyl diphosphate as product, with a k(cat) of 1.8s(-1) . Gel filtration experiments performed on the recombinant geranyl diphosphate synthases, in which the plastidial targeting sequences had been deleted, revealed that these enzymes are homodimers similar to other short-chain prenyltransferases but different from the heterotetrameric geranyl diphosphate synthase of mint.

Arch Biochem Biophys, 2002 Sep 1, 405(1), 122 - 9
Recognition of diverse RNAs by a single protein structural framework; Spingola M et al.; The coat proteins of different single-strand RNA phages utilize a common structural framework to recognize different RNA targets, making them suitable models for studies of RNA-protein recognition generally, especially for the class of proteins that bind RNA on a beta-sheet surface . Here we show that structurally distinct molecules are capable of satisfying the requirements for binding to Qbeta coat protein . Although the predicted secondary structures of the RNAs differ markedly, we contend that they are approximately equivalent structurally in their complexes with coat protein . Based on our prior observations that the RNA-binding specificities of Qbeta and MS2 coat proteins can be interconverted with as few as one amino acid substitution each, and taking into account details of the structures of complexes of MS2 coat protein with wild-type and aptamer RNAs, we propose a model for the Qbeta coat protein-RNA complex.

Arch Biochem Biophys, 2002 Sep 1, 405(1), 112 - 21
Molecular cloning and characterization of a new linalool synthase; Crowell AL et al.; Mentha citrata Ehrh . (bergamot mint; Lamiaceae) produces an essential oil containing only the acyclic monoterpenol (-)-3R-linalool and its acetate ester . A cloning strategy based upon the assumption that the responsible monoterpene synthase would resemble, in sequence, monoterpene cyclases from this plant family yielded a cDNA encoding the (--)-3R-linalool synthase . The nucleotide sequence of this monoterpene synthase is similar to those of several monoterpene cyclases from the mint (Lamiaceae) family (62-72% identity), but differs substantially from that of 3S-linalool synthase from Clarkia (41% identity; this composite gene appears to be of recent origin) and from that of 3R-linalool synthase from Artemisia (52% identity; the functional role of this gene is uncertain) . Heterologous expression in Escherichia coli of a truncated version of the cDNA (in which the plastidial transit peptide was deleted) allowed purification and characterization of the enzyme, which was shown to possess most properties similar to other known monoterpene cyclases, but with a K(m) value for the natural substrate, geranyl diphosphate, of 56 microM with k(cat) of 0.83 s(-1) . These kinetic constants for this 3R-linalool synthase are higher than those of any defined monoterpene cyclase, but the kinetic efficiency does not approach that reported for the 3S-linalool synthase from Clarkia . Although linalyl diphosphate is an enzyme-bound intermediate of monoterpene cyclase reactions, this tertiary allylic isomer of the geranyl substrate is not an efficient precursor of linalool with the M . citrata synthase . Modeling of the active site of this linalool synthase from Mentha and comparison to the modeled active sites of phylogenetically related monoterpene cyclases revealed structural differences in the binding of the diphosphate moiety which initiates the ionization step of the electrophilic reaction sequence and in the access of water to the active site to permit stereoselective quenching of the initially formed carbocationic intermediate to produce 3R-linalool.

Arch Biochem Biophys, 2002 Sep 1, 405(1), 87 - 94
Evidence for Cu(I)-thiolate ligation and prediction of a putative copper-binding site in the Escherichia coli NADH dehydrogenase-2; Rapisarda VA et al.; NADH dehydrogenase-2 (NDH-2) from Escherichia coli is a membrane-bound flavoprotein linked to the respiratory chain . We have previously shown that this enzyme has cupric reductase activity that is involved in hydroperoxide-induced oxidative stress . In this paper we present spectroscopic evidence that NDH-2 contains thiolate-bound Cu(I) with luminescence properties . Purified NDH-2 exhibits an emission band at 670nm with excitation wavelengths of 280 and 580nm . This emission is quenched by the specific Cu(I) chelator bathocuproine disulfonate, but not by EDTA . The luminescence intensity is sensitive to the enzyme substrates and, thus, the Cu(I)-thiolate chromophore reflects the redox and/or conformational states of the protein . There is one copper atom per polypeptide chain of the purified NDH-2, as determined by atomic absorption spectroscopy . Bioinformatics allowed us to recognize a putative copper-binding site and to predict four structural/functional domains in NDH-2: (I) the FAD-binding domain, (II) the NAD(H)-binding domain, (III) the copper-binding domain, and (IV) the domain of anchorage to the membrane containing two transmembrane helices, at the C-terminus . A NDH-2 topology model, based on the secondary structure prediction, is proposed . This is the first description of a copper-containing NADH dehydrogenase . Comparative sequence analysis allowed us to identify a branch of homologous dehydrogenases that bear a similar metal-binding motif.

Biochem Biophys Res Commun, 2002 Aug 23, 296(3), 749 - 54
Specificity of elongation factor EF-TU for hydrophobic peptides; Malki A et al.; The elongation factor EF-Tu carries aminoacyl-tRNAs to the A-site of the ribosome during the elongation process of protein biosynthesis . We, and others, have recently reported that the Escherichia coli EF-Tu interacts with unfolded and denatured proteins and behaves like a chaperone in protein folding and protection against protein thermal denaturation . In this study, we have identified EF-Tu binding sites in protein substrates by screening cellulose-bound peptides scanning the sequences of several proteins . The binding motifs recognized by EF-Tu in protein substrates are also recognized by the chaperone DnaK and mainly consist of hydrophobic clusters . EF-Tu interacts as efficiently as DnaK with the membrane spanning sequence of the membrane protein phospholemman and with the signal sequence of alkaline phosphatase . It interacts less efficiently with several other hydrophobic clusters of lysozyme and alkaline phosphatase, which are also DnaK substrates and fails to bind to several DnaK binding sites . Our results suggest that EF-Tu, like DnaK, interacts albeit more weakly with the hydrophobic regions of substrate protein and are consistent with the hypothesis that it possesses chaperone properties.

J Inorg Biochem, 2002 Aug 30, 91(3), 463 - 9
Effects of chaotropic anions on the distribution of conformational substates of amicyanin, wild type and Cys3Ala/Cys26Ala azurin mutant; Stirpe A et al.; The effect of azide and thiocyanate on the structure and dynamics of wild type and disulfide bond depleted azurin and of amicyanin has been investigated by electron paramagnetic resonance (EPR) spectroscopy at low temperature . The analysis of the EPR spectra, which can be described in terms of Gaussian distributions of the components of the axial symmetric <--> g and <--> A tensors of the spin-Hamiltonian, has shown that the two small exogenous ligands, known as chaotropic agents, are effective in reducing the structural heterogeneity of the proteins . Such a reduction, quantified by the standard deviations sigma(g axially) and sigma(A axially) and obtained by simulation of the experimental EPR spectra, depends on azide and thiocyanate concentration in solution . In particular, the comparison of the sigma(g axially) and sigma(A axially) values found for the protein samples investigated points out that the lower the protein to anion molar ratios (1:50; 1:100) are, the more marked the reduction in structural heterogeneity is . The thiocyanate effect is stronger than the azide one . Furthermore, the reduction in structural heterogeneity is more marked in the azurins than in amicyanin and the Cys3Ala/Cys26Ala azurin mutant is less flexible compared to the wild-type protein . The effect observed upon N(-)(3) and SCN(-) addition in solution is very similar to that observed when glycerol is added to the solution, suggesting that such perturbing agents behave like cryoprotectors, affecting the protein-solvent interactions in such a way as to suppress the large amplitude motions.

Biochim Biophys Acta, 2002 Aug 31, 1564(2), 421 - 8
Thiamine transport in Escherichia coli: the mechanism of inhibition by the sulfhydryl-specific modifier N-ethylmaleimide; Hollenbach AD et al.; Active transport of thiamin (vitamin B(1)) into Escherichia coli occurs through a member of the superfamily of transporters known as ATP-binding cassette (ABC) transporters . Although it was demonstrated that the sulfhydryl-specific modifier N-ethylmaleimide (NEM) inhibited thiamin transport, the exact mechanism of this inhibition is unknown . Therefore, we have carried out a kinetic analysis of thiamin transport to determine the mechanism of inhibition by NEM . Thiamin transport in vivo exhibits Michaelis-Menten kinetics with K(M)=15 nM and V(max)=46 U mg(-1) . Treatment of intact E . coli KG33 with saturating NEM exhibited apparent noncompetitive inhibition, decreasing V(max) by approximately 50% without effecting K(M) or the apparent first-order rate constant (k(obsd)) . Apparent noncompetitive inhibition is consistent with an irreversible covalent modification of a cysteine(s) that is critical for the transport process . A primary amino acid analysis of the subunits of the thiamin permease combined with our kinetic analysis suggests that inhibition of thiamin transport by NEM is different from other ABC transporters and occurs at the level of protein-protein interactions between the membrane-bound carrier protein and the ATPase subunit.

Mutat Res, 2002 Aug 29, 505(1-2), 13 - 25
Efficient repair of bulky anti-BPDE DNA adducts from non-transcribed DNA strand requires functional p53 but not p21(waf1/cip1) and pRb; Wani MA et al.; Wild-type p53 protein is known to regulate the global genomic repair (GGR), removing bulky chemical DNA adducts as well as cyclobutane pyrimidine dimers from the genome overall and from non-transcribed strands (NTS) in DNA . To investigate the role of cellular factor(s) relevant to p53 regulated DNA repair processes, we examined the repair kinetics of chemical carcinogen, anti-benzo{a}pyrene-diol epoxide (anti-BPDE), induced bulky DNA adducts in normal human mammary epithelial cells (HMECs) and HMEC transformed by human papillomavirus (HPV)-16E6 or -16E7 oncoproteins, which, respectively targets p53 or pRb proteins for degradation . The results show that the removal of anti-BPDE DNA adducts from the genome overall and NTS by GGR was significantly reduced in HPV-16E6 protein expressing cells as compared to that in normal and HPV-16E7 protein expressing cells, indicating the role of p53 and not pRb in nucleotide excision repair (NER) . We further determined the potential effects of the p53-regulated p21(waf1/cip1) gene product in NER in human colon carcinoma, HCT116 cells expressing wild-type p53 but different p21(waf1/cip1) genotypes (p21+/+, p21+/-, p21-/-) . The results donot show a discernible difference in the removal of anti-BPDE DNA adducts from the genome overall and the transcribed strand (TS) and NTS irrespective of the presence or absence of p21(waf1/cip1) expression . Based on these results, we suggest that: (i) the wild-type p53 function but not p21(waf1/cip1) expression is necessary for GGR of chemical induced bulky DNA adducts; (ii) the Rb gene product does not play a significant role in NER; and (iii) the modulation of NER by p53 may be independent of its function in the regulation of cell cycle arrest upon chemically induced DNA damage.

Mech Dev, 2002 Jun, 114(1-2), 177 - 80
A novel noncollagenous protein encoded by an alternative transcript of the chick type III collagen gene is expressed in cartilage, bone and muscle; Cohen AJ et al.; We have identified a noncollagenous protein, Col3alt, encoded by an alternative transcript of the chick type III collagen gene; its amino acid sequence is out of frame with the collagen coding sequence . This 178-amino-acid protein is unique and has no recognizable motifs other than a hydrophobic domain . Col3alt is found in embryonic cartilage, muscle and bone and in the proliferative and prehypertrophic zones of juvenile chicken growth plates . The protein is intracellular in immature chondrocytes and myoblasts, but is extracellular in well-differentiated cartilage, muscle and bone, despite the lack of a conventional signal peptide . These results demonstrate an unexpected economy of genome utilization in which a single gene, using alternative promoters, gives rise to two unrelated proteins, type III collagen and Col3alt .

Nippon Hinyokika Gakkai Zasshi, 2002 Jul, 93(5), 648 - 51
{A case report of Fournier's gangrene in a diabetic patient induced by transrectal prostate biopsy (TRPB)}; Kumagai A et al.; A 70-year-old man with poorly controlled diabetes mellitus, and an elevated serum prostatic specific antigen, underwent transrectal prostate biopsy . He received one dose of cefotium before, and three doses of cefotium (1.0 gram every 12 hours intravenously) after prostatic biopsy . He was doing well until postbiopsy day 1, when he developed high fever, dysuria and lower abdominal pain . His perineal area exhibited black-purpish discoloration . On postbiopsy day 3, laboratory data showed leukopenia and DIC . Operative findings during laparotomy on the same day, included malodorous cloudy fluid and tissue edema involving the perivesical space . Intraoperative tissue cultures as well as postoperative cultures of blood and drainage revealed Escherichia coli, serotype O-6 . Despite maximal supportive therapy, the patient developed multiorgan failure and died on the tenth postbiopsy day . This patient's history and hospitalization course suggests that transrectal prostatic biopsy induced Fournier's gangrene.

World J Gastroenterol, 2002 Aug, 8(4), 619 - 23
Identification of tumor associated single-chain Fv by panning and screening antibody phage library using tumor cells; Nie YZ et al.; AIM:To study the feasibility of panning and screening phage-displaying recombinant single-chain variable fragment (ScFv) of anti-tumor monoclonal antibodies for fixed whole cells as the carriers of mAb-binding antigens . METHODS: The recombinant phage displaying libraries for anti-colorectal tumor mAb MC3Ab, MC5Ab and anti-gastric tumor mAb MGD1 was constructed . Panning and screening were carried out by means of modified fixation of colorectal and gastric tumor cells expressed the mAb-binding antigens . Concordance of binding specificity to tumor cells between phage clones and parent antibodies was analyzed . The phage of positive clones was identified with competitive ELISA, and infected by E.coli HB2151 to express soluble ScFv . RESULTS:The ratio of positive clones to MC3-ScF-MC5-ScFv and MGD1-ScFv were 60 %, 24 % and 30 % . MC3-ScFv had M(r) 32 000 confirmed by Western blot . The specificity to antigen had no difference between 4 positive recombinant phage antibodies and MC3Ab . CONCLUSION:The modified process of fixing whole tumor cells is efficient, convenient and feasible to pan and screen the phage-displaying ScFv of anti-tumor monoclonal antibodies.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1998, 30(1), 63 - 69
Phosphorylation of PHO4 Protein by the PHO85-PAP1 Kinase Complex; Wu JS et al.; Homology comparison of the novel PAP1 protein indicated that PAP1 protein is highly homologous to PHO85-associated protein PHO80, PCL1 and PCL2 . We constructed the fused HA-PAP1 gene in frame for immunoprecipitation with anti-HA monoclonal antibody . Coimmunoprecipitation of fused HA-PAP1 and PHO85 protein, which were translated in vitro, verified the association of PAP1 with PHO85 protein . Simultaneously, we purified PHO4 protein expressed in E . coli and constructed a PHO85::TRP1 strain . After arranging the fused gene under the control of yeast ADH1 promoter, we transformed the resulting plasmids into yeast YPH499 and PHO85::TRP1 respectively, then prepared the yeast lysates for immunoprecipitation with anti-HA . We found that the immunoprecipitant complex of PAP1 can phosphorylate PHO4 protein in vitro, and the kinase activity is PHO85-associated, but is not related to CDC28 protein kinase . These data suggest that PAP1 is a putative cyclin and can form cyclin-CDK complex with PHO85 protein . Northern bolt with PAP1 gene as probe indicated there was no obvious difference during the different phase of the cell-cycle in the transcription of PAP1 gene . We constructed the PAP1::TRP1 strain, and the rAPase activity analysis showed PAP1 had no function in the PHO system.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1998, 30(1), 1 - 8
Use of Carcinoembryonic Antigen Gene Promoter in Colorectal Carcinoma-specific Suicidal Gene Therapy; Jiang Q et al.; The possibility of tumor-specific suicidal gene therapy of colorectalcarcinoma was investigated using carcinoembryonic antigen (CEA) -positive human colorectal carcinoma cell line LoVo and CEA-negative HeLa cell line as a model . After confirming cellular specificity of cea promoter by CAT assay, eukaryotic expression plasmid pCEACD was constructed in which the expresstion of E . coli cytosine deaminase (CD) gene was under the control of cea promoter . The expression of CD gene increased the sensitivity of LoVo cells to 5-fluorocytosine (5FC) by 750 fold, while the sensitivity of HeLa cells to 5FC was increased by only 7.5 fold . These results suggest that the expression of CD gene driven by cea promoter specifically killed CEA-positive colorectal carcinoma cells . Transmission electron microscopy and DNA fragmentation assay demonstrate that CD/5FC system induced apoptosis in LoVo cells.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1998, 30(2), 203 - 206
Cloning of Schistosoma japonicum Chinese Strain Fatty Acid Binding Protein (Sj-FABPc) Gene and Its Overproduction in Escherichia coli; Cai XZ et al.; A 600 bp DNA fragment was amplified by PCR, from an adult Schistosoma japonicum cDNA library . Sequence analysis revealed that this fragment containedthe S . japonicum Chinese Mainland strain fatty acid binding protein (Sj-14FABPc) gene . This gene was then cloned into the expression vector pGEX-2T, and subsequently expressed in Escherichia coli . The recombinant GST-fusion protein could be purified by glutathione agarose affinity chromatography . Its molecular weight was about 41 kD . The yield of expression was around 25 mg/L E . coli culture . The immunological test suggested that the recombinant protein had good antigenicity, and could be developed into a new vaccine molecule of S . japonicum.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1998, 30(2), 198 - 202
Nuclear Factors Enhance the Binding of Estrogen Receptor to Estrogen Response Element; Zhu GZ et al.; We obtained the full length of human estrogen receptor (hER) through the in vitro translation . It was shown that the translated product could bind to the estrogen response element (ERE) . The nuclear extract prepared from the rat uterus after ovariectomy could enhance the binding of hER-ERE in an estrogen dependent manner . However, the enhancing effect was sharply decreased when the nuclear extract was pre-incubated at 50 degrees for 15 minutes before being used for the binding reaction . These results indicated the presence in the rat uterus extracts after ovariectomy of a heat labile factor that can enhance the binding of hER-ERE in an estrogen-dependent manner . The DNA binding domain of estrogen receptor (ER DBD) fused to the Scistosoma japonicam glutathione S-transferase (GST) was expressed in the E . coli . The expression product also could bind the ERE . However, the binding was not affected by the uterine extract, indicating that the heat-sensitive nuclear factors may interact with hER outside the DBD to enhance the binding of ER to ERE.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1998, 30(2), 174 - 178
C-terminal His-tag Fusion Expression and Purification of Truncated cAMP-dependent Protein Kinase; Xu ZP et al.; The truncated mCalpha lacking 3'-coding sequence of 96 base paires was fused with a His-tag (mCalpha4H) and a C-terminal fusion expression plasmid pZP mCalpha4H was constructed . With the induction of IPTG, the Expressed mCalpha4H was up to about 20% of the total bacterial proteins in E . coli BL21 (DE3) . Using immobilized metal (Ni(2+)) chelation affinity chromatography, the target protein mCalpha4H was purified from crude lysates and inclusion bodies respectively . The results of in vitro and in vivo myristoylation assay showed that the purifed mCalpha4H is a substrate of NMT as the mCalpha.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1998, 30(2), 164 - 168
The Construction of an Adenovirus Vector Containing Cytosine Deaminase Gene and Its Application; Xu DH et al.; A replication-defective recombinant adenovirus vector containing CMV promoter and Escherichia coli cytosine deaminase (cd) gene (AdCMVCD) was constructed . It was shown by Southern blotting and RT-PCR that cd gene had been inserted into AdCMVCD and was expressed in the infected cells . The recombinant virus was purified by gradient centrifugation in CsCl and its titer was 1x10(15) pfu/L . HeLa and C6 cells infected with AdCMVCD(m.o.i=100) became sensitive to the prodrug 5FC, and the number of viable cells decreased to less than 20% of the control after treatment with 100 &mgr;mol/L 5FC . Significant bystander effect was also observed . When cells infected with AdCMVCD were mixed with wild type cells at a ratio of 3.3 : 96.7, more than 60% of the cells could be killed in the presence of 50 &mgr;mol/L 5FC . These results suggest that the AdCMVCD/5FC system may provide a new approach for gene therapy of cancers.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1998, 30(3), 236 - 240
Study on the Overexpression of the Gene Encoding Arginyl-tRNA Synthetase under Induction; Wu JF et al.; Arginyl-tRNA synthetase (ArgRS) from E . coli was overproduced from transformant containing the gene encoding this enzyme (argS) by 550 fold higher than that from host cell . By site-directed mutagenesis the cleavage site for NcoI restriction endonuclease was introduced into the start codon of argS . The mutant gene was recombinated with plasmid pTrc99B under IPTG control . In the transformant containing the recombinated plasmid, argS can overexpressed 2 000 times higher than that in host cell . Through one step DEAE-Sepharose column chromatography, ArgRS was purified to be one band by SDS-PAGE and its specific activity was 15 000 u/mg, similar to reported values . The mutant ArgRS2ND which had the second residue asparagine replaced by aspartic acid showed no change of activity, kinetic constants nor the thermal and denaturational stabilities.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1998, 30(3), 220 - 224
Cloning and Expression of the cDNA Encoding Human Tissue Inhibitor of Metalloproteinase-3 and Its Inhibition on Angiogenesis; Sun JX et al.; We have isolated the cDNA encoding the mature tissue inhibitor of metalloproteinase-3 (TIMP-3) by using RT-PCR method with total RNA extracted from human fresh placenta . The result of sequencing indicated that the mature TIMP-3 polypeptide contained 188 aa residues, including 12 conserved cysteinyl residues in the TIMP family . The expression plasmid pET-TIMP3 was constructed by inserting TIMP-3 cDNA into plasmid pET-24 (a(+)) containing T7 promoter and transformed into E . coli BL21 (DE3) . An expression strain BLTIMP3 was selected . SDS-PAGE analysis revealed that the human TIMP-3 protein was highly expressed and accumulated up to above 30% of the total bacterial proteins in the form of inclusion body after induced by 1 mmol/L IPTG for 3--5 h . The partially purified and renatured TIMP-3 can significantly inhibit angiogenesis as shown by CAM assay.

Cell Microbiol, 2002 Aug, 4(8), 503 - 14
Enhanced Escherichia coli invasion of human brain microvascular endothelial cells is associated with alternations in cytoskeleton induced by nicotine; Chen YH et al.; Although epidemiological studies have shown that exposure to tobacco smoking significantly increases the risk of bacterial meningitis, heretofore the pathogenic effects of smoking on this disease have been poorly understood . In order to dissect this issue, we have investigated the effects of nicotine, the major component of tobacco, on E . coli invasion of human brain microvascular endothelial cells (HBMEC) . Our studies showed that E . coli invasion of HBMEC was significantly enhanced by nicotine in a dose-dependent manner . The nicotine-mediated enhancement was associated with actin cytoskeleton rearrangement and morphological changes in the eukaryotic host cell that are essential for bacterial entry . The recombinant IbeA protein and alpha-bungarotoxin (a nicotinic acetylcholine receptor antagonist) were able to efficiently block the nicotine-mediated cellular effects, suggesting the involvement of the IbeA and nicotinic receptors . Blocking of phosphatidylinositol 3-kinase (PI3K) by LY294002 abolished the entry of E . coli in HBMECs treated with nicotine in a dose-dependent manner . Inhibition of PI3K was associated with decreased phosphorylation of Akt and actin cytoskeletal rearrangement . In contrast to PI3K, blockage of Rho kinase (ROCK) by Y27632 upregulated both nicotine- and E . coli-mediated cellular responses . Thus, this study provides experimental evidence for the first time that the major component of tobacco, nicotine, enhances meningitic E . coli invasion of HBMEC through modulation of cytoskeleton.

Biochemistry, 2002 Aug 20, 41(33), 10540 - 53
Membrane charge and curvature determine interaction with acyl-CoA binding protein (ACBP) and fatty acyl-CoA targeting; Chao H et al.; Although acyl-CoA binding protein (ACBP) stimulates utilization of long-chain fatty acyl-CoA by a variety of membrane-bound enzymes, it is not known whether ACBP directly interacts with membranes . To test this hypothesis, mouse recombinant (mr) ACBP was engineered to contain the native mouse ACBP amino acid sequence expressed as a fusion protein at high levels (>150 mg/L) in Escherichia coli . Purification and cleavage of the fusion tag resulted in mrACBP identical to native ACBP as shown by mass (10000.5 Da) and amino acid sequence (peptide mapping after proteolysis) determined by matrix-assisted laser desorption time of flight (MALDI-TOF) mass spectroscopy . The mrACBP was functionally active as shown by binding of cis-parinaroyl-CoA with high affinity, K(d) = 12 +/- 2 nM, at a single binding site, stimulating oleoyl-CoA utilization by microsomal glycerol-3-phosphate acyltransferase 3.2-fold and protecting oleoyl-CoA from microsomal acyl-CoA hydrolase . Direct interaction of mrACBP with membranes was demonstrated by two independent methods: (i) Circular dichroism showed an 8% increase in alpha-helix content of mrACBP in the presence of anionic phospholipid-rich, but not neutral, small unilamellar vesicles (SUV) . (ii) Membrane filtration confirmed that mrACBP bound to anionic phospholipid-rich SUV but only weakly interacted with neutral SUV or large unilamellar vesicles (LUV), regardless of charge . (iii) The mrACBP-oleoyl-CoA complex transferred 2-3-fold more oleoyl-CoA to anionic phospholipid-rich SUV than to anionic phospholipid-rich LUV and neutral SUV or LUV . Conversely, mrACBP extracted less oleoyl-CoA from anionic phospholipid-rich SUV . Taken together, these data indicated for the first time that mrACBP interacted preferentially with anionic phospholipid-rich, highly curved membranes to facilitate transfer of ACBP-bound ligands.

Biochemistry, 2002 Aug 20, 41(33), 10472 - 81
Structural mobility of the extracellular ligand-binding core of an ionotropic glutamate receptor . Analysis of NMR relaxation dynamics; McFeeters RL et al.; Ionotropic glutamate receptors play important roles in a variety of neuronal processes and have been implicated in multiple neurodegenerative diseases . The extracellular ligand-binding (S1S2) core of the GluR2 subtype can be expressed in bacteria as a soluble, monomeric protein with binding properties essentially identical to those of the intact receptor . The crystal structure of this protein has been determined in the presence and absence of various agonists and antagonists {Armstrong, N., Sun, Y., Chen, G . Q., and Gouaux, E . (1998) Nature 395, 913-917; Armstrong, N., and Gouaux, E . (2000) Neuron 28, 165-181} . The protein consists of two lobes, with the S1 segment composing the majority of lobe 1 and the S2 segment composing most of lobe 2 . A domain closure upon ligand binding has been postulated, but details of intradomain motions have not been investigated . In this paper, the backbone motions of the ligand-binding core of GluR2 bound to glutamate were studied using (15)N longitudinal (T1) and transverse (T2) relaxation measurements as well as {1H}-15N nuclear Overhauser effects at 500 and 600 MHz . Residues in the agonist-binding pocket exhibited two main classes of motion . Those contacting the alpha-substituents of the ligand glutamate exhibited minimal internal motion, while those contacting the gamma-constituents exhibited exchange dynamics, indicating two dynamically distinct portions of the binding pocket . Also, two residues in transdomain linkers between lobes 1 and 2 show exchange, lending new insight into the previously proposed domain closure hypothesis . Finally, concerted motion of helix F suggests a pathway for ligand dissociation without the necessity of domain reopening.

Gene Expr, 2002, 10(4), 193 - 200
Novel binding of GTP to the phosphoprotein (P) of vesicular stomatitis virus; Mathur M et al.; The phosphoprotein (P) of vesicular stomatitis virus (VSV) is a subunit of the RNA polymerase (L) that transcribes the negative strand genome RNA into mRNAs both in vitro and in vivo . We have previously shown that the P protein of VSV, expressed in E . coli, is biologically inactive unless phosphorylated at specific serine residues by cellular casein kinase II (CKII) . In the present study we present evidence that the P protein, in addition to being phosphorylated, binds covalently to GTP only when it is phosphorylated . Competition experiments show that ATP, ADP, GTP, and GDP can compete for the binding site(s) of GTP but not AMP, GMP, CTP, or UTP . Interestingly, once GTP is bound to P protein it cannot be displaced by unlabeled GTP . The GTP binding site has been mapped within the domain where the phosphorylation of P protein by CKII occurs . Finally, we show that phosphorylation negative P mutants P3A (P60A, P62A, P64A), P3E (P60E, P62E, P64E), and P3R (P60R, P62R, P64R) failed to bind to GTP, indicating that phosphorylation of P is indeed essential for binding to GTP . Although the precise role of binding of GTP to P is unclear, it appears that phosphorylation of P may initiate a structural change within the P protein allowing GTP to bind, thus manifesting biological function to the transcription factor.

Mol Biol (Mosk), 2002 Jul-Aug, 36(4), 682 - 8
{Distribution and functional significance of A/T tracts in promotor sequences of Escherichia coli}; Chasov VV et al.; Distribution of the A/T tracts described in earlier publications in the region extending from nucleotide -250 to +150 relative to the transcription initiation site of gene transcribed regions adjacent to promoter was studied . Upstream of the -35 region a succession of A/T tracts was discovered distributed at a shorter distance one from another than in analogous elements of the transcribed region (1 and 1.5 helix turns, respectively) . Such a positional dependence suggests different functional manifestation of A/T tracts at different transcription steps . Single initiation using the T7D promoter mutant derivatives devoid of A/T tracts in two critical positions, +41 and , yielded shortened products of the corresponding length . One might speculate that such elements adjacent to promoter region play a significant role in transcription complex functioning.

Mol Biol (Mosk), 2002 Jul-Aug, 36(4), 605 - 9
{Construction of an artificial digenic network with epigenetic properties}; Tropynina TS et al.; A model of the simplest epigene was constructed to have two alternative states, which proved to be stable and transmittable through Escherichia coli cell generations . A switch from one to another state was induced by thermal and chemical external signals . The results gave further insight into the dynamic mechanism of preservation, coding, and transmission of the genetic information.

Exp Parasitol, 2002 Mar, 100(3), 143 - 9
Expression and characterization of Ov-47, a dominant antigen of Onchocerca volvulus; Ghogomu SM et al.; The expression and characterization of a recombinant antigen termed Ov-47 are described . Ov-47 was identified and isolated from a lambda gt-11 cDNA expression library derived from adult female Onchocerca volvulus mRNA using rabbit antiserum raised against the surface proteins of O . volvulus female worms . The antiserum was earlier found to mediate, in vitro, cytoadherence and cytotoxicity reactions to microfilariae in the presence of heat-labile serum factors . The deduced amino acid sequence of the gene was assigned the EMBL GenBank Accession No . Y15993 . The open reading frame (1077 bp) of the gene was then subcloned into pQE-60 and expressed in Escherichia coli JM109 cells . The gene encodes a protein with an apparent molecular weight of 47,000 Da as revealed by SDS-PAGE . Up to 100 micrograms/ml pure Ov-47 recombinant protein could be isolated from E . coli cultures by Ni-agarose affinity chromatography . The 47-kDa protein was recognized by sera from both infected and endemic normal subjects . The parent protein was found to have a molecular weight of 60 kDa . IgG3 subclass responses to Ov-47 were significantly higher in endemic normals than in infected subjects (P < 0.05) . In contrast, IgG4 responses were higher in infected subjects than in endemic normals (P < 0.05) . IgG2 response exhibited marked age dependency with lower responses in younger patients, which rose to higher levels in elderly patients . IgG1, IgG3, and IgG4 responses did not show any age dependency . This study clearly shows that Ov-47 is a dominant antigen of O . volvulus adult worms with an important role in the host-parasite-interplay.

Clin Immunol, 2002 Jun, 103(3 Pt 1), 324 - 33
IgE binding conformational epitopes of Asp f 3, a major allergen of Aspergillus fumigatus; Ramachandran H et al.; sp f 3 has been identified as one of the major allergens of Aspergillus fumigatus associated with the sensitization and immune responses in allergic bronchopulmonary aspergillosis (ABPA) . In order to understand the structure/function relationship of Asp f 3, we studied synthetic peptides and constructed mutants deleted of specific IgE binding regions . The mutated allergens were obtained by expressing the genes and studied by ELISA for their reactivity with IgE from patients with ABPA . Seven linear IgE binding regions spanning the whole Asp f 3 molecule were demonstrated . The results demonstrated strong binding of IgE from ABPA patients with Asp f 3 and one mutant, Asp f 3(1-150), but not with other mutant constructs . The results identified 12 amino acids at the N-terminal end and 8 amino acids (143-150) at the C-terminal end as significant in the conformational constraints for IgE binding . The Fourier transfer spectra showed comparable beta-sheet structure of Asp f 3(1-150) and Asp f 3, indicating the role of secondary structure in IgE binding . The primary and secondary structures may help understanding of the functional role the allergens play in the disease and may have implications in immunodiagnosis and probably immunotherapy.

Curr Genet, 2002 Jul, 41(4), 268 - 74 Epub 2002 Jul 05.
A novel method used to delete a new Aspergillus fumigatus ABC transporter-encoding gene; Langfelder K et al.; Aspergillus fumigatus is an important opportunistic human pathogenic fungus . In severely immunocompromised patients, the fungus causes life-threatening diseases, such as pneumonia and invasive aspergillosis . In order to obtain a better understanding of the key elements involved in A . fumigatus virulence and for identifying possible drug targets, it is essential to be able to generate gene-deletion strains . Until recently, the molecular techniques available did not provide a rapid method for gene deletion . A novel method described for A . nidulans was adapted for A . fumigatus . This method is quick and produces an increased homologous recombination efficiency . By using an Escherichia coli strain expressing the lambda red operon, it is possible to induce an in vivo recombination of a PCR fragment flanked by >50-bp regions with a cosmid containing the gene of interest . This produces cosmids in which the gene of interest has been replaced by a bi-functional marker . Such cosmids have large flanking regions surrounding the selectable marker pyrG of A . fumigatus used here, which result in high recombination efficiencies in A . fumigatus . Here, we identified a new ABC transporter-encoding gene in A . fumigatus, designated abcA . By using this method, an A . fumigatus knock-out mutant was generated, providing evidence that this method of generating gene deletions can also be used in A . fumigatus and significantly broadens our repertoire of molecular techniques to study A . fumigatus.

Planta, 2002 Aug, 215(4), 630 - 8 Epub 2002 May 21.
Immunocharacterization of Vitis vinifera L . ferredoxin-dependent glutamate synthase, and its spatial and temporal changes during leaf development; Loulakakis KA et al.; The grapevine (Vitis vinifera L.) partial fragment of cDNA clone pGOGAT1 {Loulakakis and Roubelakis-Angelakis (1997) Physiol Plant 101:220-228}, encoding the ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1), was overexpressed in Escherichia coli cells . A hybrid between the Fd-GOGAT fragment and maltose-binding protein was purified and used to raise a polyclonal antibody in a rabbit . The prepared antibody appeared to be specific towards Fd-GOGAT; it recognized a protein band of approximately 160 kDa on nitrocellulose blots after SDS-PAGE of total proteins from leaves, internodes, roots and calluses, and precipitated most of the enzyme activity present in grapevine protein extracts . The quantity of Fd-GOGAT protein was substantially higher in leaves than in other grapevine tissues tested, coincident with a similar distribution of the enzyme specific activity . Intracellular localization studies revealed that both the enzyme activity and the 160-kDa immunoreactive protein were associated with the chloroplastic fraction . Furthermore, the accumulation of Fd-GOGAT, glutamine synthetase (GS) and glutamate dehydrogenase (GDH), at the activity and protein levels, was monitored during leaf development of field-grown plants, from the stage of the newly expanding leaf to the senescing old leaf . Both the specific activity and quantity of the 160-kDa polypeptide of Fd-GOGAT were higher in the mature, full sized leaves and substantially lower in young and senescing leaves . GS specific activity and immunoreactive protein followed the same trend as Fd-GOGAT, while GDH showed opposite developmental patterns of accumulation . The biological significance of the presence of Fd-GOGAT in the various grapevine tissues and its physiological role during early development and natural senescence of the leaves are discussed.

Parasitol Res, 2002 Sep, 88(9), 855 - 60 Epub 2002 Jun 04.
A direct sandwich ELISA to detect antibodies against the C-terminal region of merozoite surface protein 1 could be a useful diagnostic method to identify Plasmodium vivax exposed persons; Lim KJ et al.; We expressed a C-terminal 108-aa region of the Plasmodium vivax merozoite surface protein 1 (PvMSP1c) excluding the C-end transmembrane region in Escherichia coli in order to evaluate the antibody level to MSP in Korean malaria patients . We optimized a direct sandwich enzyme-linked immunosorbent assay (ELISA) method to simultaneously determine the total antibody levels, including IgG and IgM, to PvMSP1c . If the cut-off for seropositivity was determined as the mean+3SD of the antibody levels of the negative control group, the antibody levels were positive in 99.5% of the patient group (sensitivity 199/200) . The antibody levels were negative in 99.4% of the negative control group (specificity 504/507) . The positive reactions in the negative control group came from non-specific reactions, as confirmed by a competition assay . This direct sandwich ELISA for PvMSP1c antibody could prove to be a useful tool for the diagnosis of malaria patients and for blood screening in blood banks.

Mol Genet Genomics, 2002 Jul, 267(5), 684 - 94 Epub 2002 Jun 26.
The 5'-upstream cis-acting sequences of a cyanobacterial psbA gene: analysis of their roles in basal, light-dependent and circadian transcription; Shibato J et al.; Transcription of the psbA2 gene in the unicellular photosynthetic cyanobacterium Microcystis aeruginosa K-81 is modulated by light and follows a circadian rhythm . In this study, we further characterized psbA transcription using a series of 5'-upstream deletions and mutant promoters which were tested in both photosynthetic and non-photosynthetic bacteria . Specific psbA2 transcripts were obtained from a minimal promoter sequence (-38/+14) with Escherichia coli RNA polymerases (RNAPs) both in vivo and in vitro, indicating the presence of a common regulatory mechanism for basal transcription . A DNase I footprinting assay showed that the E . coli RNAP, which is structurally similar to that of cyanobacteria, specifically binds to a large segment (from -115 to +23) of the sequence upstream of psbA2 . In cyanobacteria, the -10 sequence (TAGTAT), but not the -35 motif (TTTACA), is essential for basal transcription by homologous and heterologous RNAPs that contain the major sigma factor . Each of the conserved thymidine nucleotides at positions -12 and -7 (underlined above) was essential, and both an insertion and a deletion in the spacer region of the promoter caused reductions in transcription . RNAP was able to bind to a mutant promoter lacking the -10 sequence, though this did not actually lead to transcription . Interestingly, a high level of arrhythmic circadian transcription was observed in mutants lacking the -35 region . In contrast, a mutation in the AU-box mutation, which controls the stability of the psbA2 mRNA, did not affect the circadian pattern of transcription . These findings demonstrate that light-dependent psbA2 expression is controlled at the transcriptional and post-transcriptional levels, whereas the circadian pattern of expression is regulated at the transcriptional level.

Mol Genet Genomics, 2002 Jul, 267(5), 664 - 72 Epub 2002 Jun 19.
Evolutionary relationship of Alw26I, Eco31I and Esp3I, restriction endonucleases that recognise overlapping sequences; Bitinaite J et al.; Type II restriction endonucleases (ENases) have served as models for understanding the enzyme-based site-specific cleavage of DNA . Using the knowledge gained from the available crystal structures, a number of attempts have been made to alter the specificity of ENases by mutagenesis . The negative results of these experiments argue that the three-dimensional structure of DNA-ENase complexes does not provide enough information to enable us to understand the interactions between DNA and ENases in detail . This conclusion calls for alternative approaches to the study of structure-function relationships related to the specificity of ENases . Comparative analysis of ENases that manifest divergent substrate specificities, but at the same time are evolutionarily related to each other, may be helpful in this respect . The success of such studies depends to a great extent on the availability of related ENases that recognise partially overlapping nucleotide sequences (e.g . sets of enzymes that bind to recognition sites of increasing length) . In this study we report the cloning and sequence analysis of genes for three Type IIS restriction-modification (RM) systems . The genes encoding the ENases Alw26I, Eco31I and Esp3I (whose recognition sequences are 5'-GTCTC-3', 5'-GGTCTC-3' and 5'-CGTCTC-3', respectively) and their accompanying methyltransferases (MTases) have been cloned and the deduced amino acid sequences of their products have been compared . In pairwise comparisons, the degree of sequence identity between Alw26I, Eco31I and Esp3I ENases is higher than that observed hitherto among ENases that recognise partially overlapping nucleotide sequences . The sequences of Alw26I, Eco31I and Esp3I also reveal identical mosaic patterns of sequence conservation, which supports the idea that they are evolutionarily related and suggests that they should show a high level of structural similarity . Thus these ENases represent very attractive models for the study of the molecular basis of variation in the specific recognition of DNA targets . The corresponding MTases are represented by proteins of unusual structural and functional organisation . Both M . Alw26I and M . Esp3I are represented by a single bifunctional protein, which is composed of an m(6)A-MTase domain fused to a m(5)C-MTase domain . In contrast, two separate genes encode the m(6)A-MTase and m(5)C-MTase in the Eco31I RM system . Among the known bacterial m(5)C-MTases, the m(5)C-MTases of M . Alw26I, M . Eco31I and M . Esp3I represent unique examples of the circular permutation of their putative target recognition domains together with the conserved motifs IX and X.

Mol Genet Genomics, 2002 Jul, 267(5), 587 - 92 Epub 2002 Jun 04.
Origin-specific reduction of ColE1 plasmid copy number due to mutations in a distinct region of the Escherichia coli RNA polymerase; Ederth J et al.; Mutations affecting a region of the Escherichia coli RNA polymerase have been isolated that specifically reduce the copy number of ColE1-type plasmids . The mutations, which result in a single amino acid alteration (G1161R) or a 41-amino acid deletion (Delta1149-1190) are located near the 3'-terminal region in the rpoC gene, which encodes the largest subunit (beta ') of the RNA polymerase . The rpoC deletion and the point mutation cause over 20- and 10-fold reductions, respectively, in the copy number of ColE1 . ColE1 plasmid numbers are regulated by two plasmid-encoded RNAs: RNA II, which acts as a preprimer for the DNA polymerase I to start initiation of replication, and RNA I, its antisense inhibitor . Altered expression from the RNA I and RNA II promoters in vivo was observed in the RNA polymerase mutants . The RNA I/RNA II ratio is higher in the mutants than in the wild-type strain and this is most probably the main reason for the reduction in the ColE1 copy number in the two rpoC mutants.

Cell Tissue Res, 2002 Aug, 309(2), 281 - 6 Epub 2002 Jul 05.
Transforming growth factor-beta increases Escherichia coli K1 adherence, invasion, and transcytosis in human brain microvascular endothelial cells; Zhang WG et al.; Escherichia coli K1 traversal of the human brain microvascular endothelial cells (HBMEC) that constitute the blood-brain barrier (BBB) is a complex process involving E . coli adherence to and invasion of HBMEC . In this study, we demonstrated that human transforming growth factor-beta-1 (TGF-beta1) increases E . coli K1 adherence, invasion, and transcytosis in HBMEC . In addition, TGF-beta1 increases RhoA activation and enhances actin condensation in HBMEC . We have previously shown that E . coli K1 invasion of HBMEC requires phosphatidylinositol-3 kinase (PI3K) and RhoA activation . TGF-beta1 increases E . coli K1 invasion in PI3K dominant-negative HBMEC, but not in RhoA dominant-negative HBMEC, indicating that TGF-beta1-mediated increase in E . coli K1 invasion is RhoA-dependent, but not PI3K-dependent . Our findings suggest that TGF-beta1 treatment of HBMEC increases E . coli K1 adherence, invasion, and transcytosis, which are probably dependent on RhoA.

Anal Bioanal Chem, 2002 Jul, 373(6), 501 - 7 Epub 2002 Jun 29.
Purification method for recombinant proteins based on a fusion between the target protein and the C-terminus of calmodulin; Schauer-Vukasinovic V et al.; Calmodulin (CaM) was used as an affinity tail to facilitate the purification of the green fluorescent protein (GFP), which was used as a model target protein . The protein GFP was fused to the C-terminus of CaM, and a factor Xa cleavage site was introduced between the two proteins . A CaM-GFP fusion protein was expressed in E . coli and purified on a phenothiazine-derivatized silica column . CaM binds to the phenothiazine on the column in a Ca(2+)-dependent fashion and it was, therefore, used as an affinity tail for the purification of GFP . The fusion protein bound to the affinity column was then subjected to a proteolytic digestion with factor Xa . Pure GFP was eluted with a Ca(2+)-containing buffer, while CaM was eluted later with a buffer containing the Ca(2+)-chelating agent EGTA . The purity of the isolated GFP was verified by SDS-PAGE, and the fluorescence properties of the purified GFP were characterized.

Appl Microbiol Biotechnol, 2002 Aug, 59(4-5), 462 - 6 Epub 2002 Jun 15.
Expression of two kinds of recombinant glutamate dehydrogenase from Aeropyrum pernix with different N-terminal sequence length in Escherichia coli; Helianti I et al.; Two recombinant Aeropyrum pernix glutamate dehydrogenase (GDH) enzymes with different length N-termini were cloned and expressed in Escherichia coli: sGDH begins with the amino acid sequence of the extracted native enzyme (M-Q-P-T-D-P-L-E-E), whereas lGDH begins with the sequence of the predicted ORF (M-E-V-L-A-L-Q-P-T-D) and is longer than sGDH by five amino acids (M-E-V-L-A) . Purified recombinant lGDH was more stable than sGDH, indicating that the N-terminal extension, containing mostly hydrophobic residues, affected the overall stability of recombinant lGDH . This stabilising effect of extending the N-terminal sequence on an oligomeric enzyme such as GDH is novel; factors affecting stabilisation have previously only been discussed in the context of the contribution of internal amino acids.

Nat Struct Biol, 2002 Sep, 9(9), 704 - 10
A large iris-like expansion of a mechanosensitive channel protein induced by membrane tension; Betanzos M et al.; MscL, a bacterial mechanosensitive channel of large conductance, is the first structurally characterized mechanosensor protein . Molecular models of its gating mechanisms are tested here . Disulfide crosslinking shows that M1 transmembrane alpha-helices in MscL of resting Escherichia coli are arranged similarly to those in the crystal structure of MscL from Mycobacterium tuberculosis . An expanded conformation was trapped in osmotically shocked cells by the specific bridging between Cys 20 and Cys 36 of adjacent M1 helices . These bridges stabilized the open channel . Disulfide bonds engineered between the M1 and M2 helices of adjacent subunits (Cys 32-Cys 81) do not prevent channel gating . These findings support gating models in which interactions between M1 and M2 of adjacent subunits remain unaltered while their tilts simultaneously increase . The MscL barrel, therefore, undergoes a large concerted iris-like expansion and flattening when perturbed by membrane tension.

Nat Struct Biol, 2002 Sep, 9(9), 696 - 703
Physical principles underlying the transduction of bilayer deformation forces during mechanosensitive channel gating; Perozo E et al.; In mechanosensitive (MS) channels, gating is initiated by changes in intra-bilayer pressure profiles originating from bilayer deformation . Here we evaluated two physical mechanisms as triggers of MS channel gating: the energetic cost of protein-bilayer hydrophobic mismatches and the geometric consequences of bilayer intrinsic curvature . Structural changes in the Escherichia coli large MS channel (MscL) were studied under nominally zero transbilayer pressures using both patch clamp and EPR spectroscopic approaches . Changes in membrane intrinsic curvature induced by the external addition of lysophosphatidylcholine (LPC) generated massive spectroscopic changes in the narrow constriction that forms the channel 'gate', trapping the channel in the fully open state . Hydrophobic mismatch alone was unable to open the channel, but decreasing bilayer thickness lowered MscL activation energy, stabilizing a structurally distinct closed channel intermediate . We propose that the mechanism of mechanotransduction in MS channels is defined by both local and global asymmetries in the transbilayer pressure profile at the lipid-protein interface.

Plant Cell, 2002 Aug, 14(8), 1833 - 46
The short-chain alcohol dehydrogenase ABA2 catalyzes the conversion of xanthoxin to abscisic aldehyde; Gonzalez-Guzman M et al.; Mutants able to germinate and perform early growth in medium containing a high NaCl concentration were identified during the course of two independent screenings and named salt resistant (sre) and salobreno (san) . The sre and san mutants also were able to germinate in high-osmoticum medium, indicating that they are osmotolerant in a germination assay . Complementation analyses revealed that sre1-1, sre1-2, san3-1, and san3-2 were alleles of the abscisic acid (ABA) biosynthesis ABA2 gene . A map-based cloning strategy allowed the identification of the ABA2 gene and molecular characterization of four new aba2 alleles . The ABA2 gene product belongs to the family of short-chain dehydrogenases/reductases, which are known to be NAD- or NADP-dependent oxidoreductases . Recombinant ABA2 protein produced in Escherichia coli exhibits a K(m) value for xanthoxin of 19 micro M and catalyzes in a NAD-dependent manner the conversion of xanthoxin to abscisic aldehyde, as determined by HPLC-mass spectrometry . The ABA2 mRNA is expressed constitutively in all plant organs examined and is not upregulated in response to osmotic stress . The results of this work are discussed in the context of previous genetic and biochemical evidence regarding ABA biosynthesis, confirming the xanthoxin-->abscisic aldehyde-->ABA transition as the last steps of the major ABA biosynthetic pathway.

Plant Cell, 2002 Aug, 14(8), 1767 - 85
Discrete forms of amylose are synthesized by isoforms of GBSSI in pea; Edwards A et al.; Amyloses with distinct molecular masses are found in the starch of pea embryos compared with the starch of pea leaves . In pea embryos, a granule-bound starch synthase protein (GBSSIa) is required for the synthesis of a significant portion of the amylose . However, this protein seems to be insignificant in the synthesis of amylose in pea leaves . cDNA clones encoding a second isoform of GBSSI, GBSSIb, have been isolated from pea leaves . Comparison of GBSSIa and GBSSIb activities shows them to have distinct properties . These differences have been confirmed by the expression of GBSSIa and GBSSIb in the amylose-free mutant of potato . GBSSIa and GBSSIb make distinct forms of amylose that differ in their molecular mass . These differences in product specificity, coupled with differences in the tissues in which GBSSIa and GBSSIb are most active, explain the distinct forms of amylose found in different tissues of pea . The shorter form of amylose formed by GBSSIa confers less susceptibility to the retrogradation of starch pastes than the amylose formed by GBSSIb . The product specificity of GBSSIa could provide beneficial attributes to starches for food and nonfood uses.

J Biol Chem, 2002 Oct 18, 277(42), 39953 - 9 Epub 2002 Aug 08.
Improving nucleoside diphosphate kinase for antiviral nucleotide analogs activation; Gallois-Montbrun S et al.; Antiviral nucleoside analog therapies rely on their incorporation by viral DNA polymerases/reverse transcriptase leading to chain termination . The analogs (3'-deoxy-3'-azidothymidine (AZT), 2',3'-didehydro-2',3'-dideoxythymidine (d4T), and other dideoxynucleosides) are sequentially converted into triphosphate by cellular kinases of the nucleoside salvage pathway and are often poor substrates of these enzymes . Nucleoside diphosphate (NDP) kinase phosphorylates the diphosphate derivatives of the analogs with an efficiency some 10(4) lower than for its natural substrates . Kinetic and structural studies of Dictyostelium and human NDP kinases show that the sugar 3'-OH, absent from all antiviral analogs, is required for catalysis . To improve the catalytic efficiency of NDP kinase on the analogs, we engineered several mutants with a protein OH group replacing the sugar 3'-OH . The substitution of Asn-115 in Ser and Leu-55 in His results in an NDP kinase mutant with an enhanced ability to phosphorylate antiviral derivatives . Transfection of the mutant enzyme in Escherichia coli results in an increased sensitivity to AZT . An x-ray structure at 2.15-A resolution of the Dictyostelium enzyme bearing the serine substitution in complex with the R(p)-alpha-borano-triphosphate derivative of AZT shows that the enhanced activity reflects an improved geometry of binding and a favorable interaction of the 3'-azido group with the engineered serine.

J Biol Chem, 2002 Oct 18, 277(42), 39320 - 6 Epub 2002 Aug 08.
Toll-like receptor (TLR) signaling in response to Aspergillus fumigatus; Mambula SS et al.; Aspergillus fumigatus causes life-threatening infections in patients with qualitative and quantitative defects in phagocytic function . Here, we examined the contribution of Toll-like receptor (TLR)-2, TLR4, the adapter protein MyD88, and CD14 to signaling in response to the three forms of A . fumigatus encountered during human disease: resting conidia (RC), swollen conidia (SC), and hyphae (H) . Compared with elicited peritoneal macrophages obtained from wild-type and heterozygous mice, TLR2(-/-) and MyD88(-/-) macrophages produced significantly less tumor necrosis factor-alpha (TNFalpha) following A . fumigatus stimulation . In contrast, following stimulation with RC, SC, and H, TLR4(-/-) and CD14(-/-) macrophages exhibited no defects in tumor necrosis factor-alpha release . TLR2(-/-), TLR4(-/-), MyD88(-/-), and CD14(-/-) macrophages bound similar numbers of RC and SC compared with wild-type macrophages . RC, SC, and H stimulated greater activation of a nuclear factor kappa B (NFkappaB)-dependent reporter gene and greater release of tumor necrosis factor-alpha from the human monocytic THP-1 cell line stably transfected with CD14 compared with control cells stably transfected with empty vector . A . fumigatus stimulated NFkappaB-dependent reporter gene activity in the human embryonic kidney cell line, HEK293, only if the cells were transfected with TLR2 . Moreover, activity increased when TLR2 and CD14 were co-transfected . Taken together, these data suggest that optimal signaling responses to A . fumigatus require TLR2 in both mouse and human cells . In contrast, a role for CD14 was found only in the human cells . MyD88 acts as a central adapter protein mediating signaling responses following stimulation with RC, SC, and H.

Curr Pharm Des, 2002, 8(22), 1995 - 2007
DNA topoisomerase I from mycobacteria--a potential drug target; Nagaraja V et al.; DNA topoisomerases are ubiquitous group of enzymes altering the topology of DNA by concerted breakage and rejoining of the phosphodiester backbone of DNA . The enzymes are classified based on the pattern of DNA cleavage . Type IA enzymes found in all bacteria nick the DNA and attach themselves covalently to the 5' side of the nick during the first transesterification reaction . Most of the information on this group of enzymes comes from studies with E . coli topoisomerase I and III . Members of type IA group are single subunit Zn(++) metalloenzymes recognizing single stranded DNA without high degree of sequence specificity during relaxation reaction of negatively super coiled DNA . So far no inhibitors are known for this group of enzymes inspite of their important role in maintaining homeostasis of DNA topology . Molecular characterization of DNA topoisomerase I from mycobacteria has revealed some of the important features of type IA enzymes hitherto unknown and provide scope for identifying novel inhibitors . The present review describes the recent developments in the area summarizing the distinctive features of mycobacterial topoisomerase I . The enzyme has several properties not shared by either type IA or IB enzymes with respect to DNA binding, recognition, sequence specificity and interaction pattern . The physiological basis of the unusual features is discussed . The unique properties described would aid in developing the enzyme as a target molecule in pharmaceutical design . In addition, the findings lead to address some fundamental questions on the intracellular role of topoisomerase I in the biology of mycobacteria which are one of the most formidable group of pathogenic organisms.

J Biol Chem, 2002 Oct 11, 277(41), 38339 - 44 Epub 2002 Aug 06.
Cyclobutylpyrimidine dimer base flipping by DNA photolyase; Christine KS et al.; DNA Photolyase is a flavoprotein that uses light to repair cyclobutylpyrimidine dimers in DNA . From considerations of the crystal structure of the protein, it has been hypothesized that the dimer lesion is flipped out of the DNA double helix into the substrate binding pocket . We have used a fluorescent adenine analog, 2-aminopurine (2-Ap), as a probe of local double helical structure upon binding of the substrate to the protein . Our results show that the local structure around the thymidine lesion changes dramatically upon binding to Photolyase . This is consistent with base flipping of the lesion into the protein binding cavity with concomitant destacking of the opposing complementary 2-Ap nucleotide.

J Biol Chem, 2002 Oct 18, 277(42), 39280 - 8 Epub 2002 Aug 06.
The Rad51-dependent pairing of long DNA substrates is stabilized by replication protein A; Eggler AL et al.; Rad51 protein forms nucleoprotein filaments on single-stranded DNA (ssDNA) and then pairs that DNA with the complementary strand of incoming duplex DNA . In apparent contrast with published results, we demonstrate that Rad51 protein promotes an extensive pairing of long homologous DNAs in the absence of replication protein A . This pairing exists only within the Rad51 filament; it was previously undetected because it is lost upon deproteinization . We further demonstrate that RPA has a critical postsynaptic role in DNA strand exchange, stabilizing the DNA pairing initiated by Rad51 protein . Stabilization of the Rad51-generated DNA pairing intermediates can be can occur either by binding the displaced strand with RPA or by degrading the same DNA strand using exonuclease VII . The optimal conditions for Rad51-mediated DNA strand exchange used here minimize the secondary structure in single-stranded DNA, minimizing the established presynaptic role of RPA in facilitating Rad51 filament formation . We verify that RPA has little effect on Rad51 filament formation under these conditions, assigning the dramatic stimulation of strand exchange nevertheless afforded by RPA to its postsynaptic function of removing the displaced DNA strand from Rad51 filaments.

EMBO J, 2002 Aug 15, 21(16), 4349 - 56
Operator-bound GalR dimers close DNA loops by direct interaction: tetramerization and inducer binding; Semsey S et al.; The assembly of the Gal repressosome, a higher order nucleoprotein complex that represses transcription of the gal operon in Escherichia coli, involves the formation of a DNA loop encompassing the promoter segment . GalR dimers bound to two spatially separated operators, O(E) and O(I), specifically interact with the histone-like protein HU and close the loop in supercoiled DNA . We isolated and characterized a GalR mutant containing an amino acid substitution (R282L) that can repress transcription in the absence of HU and supercoiled DNA both in vivo and in vitro . Repression involves the same DNA looping; deletion of either O(E) or O(I) makes the mutant GalR ineffective in repression . This and other results suggest that the R282L substitution increases the normal affinity between two DNA-bound GalR dimers, allowing looping . We conclude that GalR dimers interact directly and do not use HU as an adaptor in loop closure; HU and DNA supercoiling act in concert to stabilize the GalR tetramer . The stronger GalR-GalR interaction also made the gal transcription non-inducible, suggesting that the inducer binding acts by modulating tetramerization.

EMBO J, 2002 Aug 15, 21(16), 4268 - 76
Diverse recognition of non-PxxP peptide ligands by the SH3 domains from p67(phox), Grb2 and Pex13p; Kami K et al.; The basic function of the Src homology 3 (SH3) domain is considered to be binding to proline-rich sequences containing a PxxP motif . Recently, many SH3 domains, including those from Grb2 and Pex13p, were reported to bind sequences lacking a PxxP motif . We report here that the 22 residue peptide lacking a PxxP motif, derived from p47(phox), binds to the C-terminal SH3 domain from p67(phox) . We applied the NMR cross-saturation method to locate the interaction sites for the non-PxxP peptides on their cognate SH3 domains from p67(phox), Grb2 and Pex13p . The binding site of the Grb2 SH3 partially overlapped the conventional PxxP-binding site, whereas those of p67(phox) and Pex13p SH3s are located in different surface regions . The non-PxxP peptide from p47(phox) binds to the p67(phox) SH3 more tightly when it extends to the N-terminus to include a typical PxxP motif, which enabled the structure determination of the complex, to reveal that the non-PxxP peptide segment interacted with the p67(phox) SH3 in a compact helix-turn-helix structure (PDB entry 1K4U).

J Bacteriol, 2002 Sep, 184(17), 4930 - 2
The metD D-methionine transporter locus of Escherichia coli is an ABC transporter gene cluster; Gal J et al.; The metD D-methionine transporter locus of Escherichia coli was identified as the abc-yaeE-yaeC cluster (now renamed metNIQ genes) . The abc open reading frame is preceded by tandem MET boxes bracketed by the -10 and -35 boxes of a promoter . The expression driven by this promoter is controlled by the MetJ repressor and the level of methionine.

J Bacteriol, 2002 Sep, 184(17), 4906 - 11
Surface loop motion in FepA; Scott DC et al.; Using a lysine-specific cleavable cross-linking reagent ethylene glycolbis(sulfosuccimidylsuccinate) (Sulfo-EGS), we studied conformational motion in the surface loops of Escherichia coli FepA during its transport of the siderophore ferric enterobactin . Site-directed mutagenesis determined that Sulfo-EGS reacted with two lysines, K332 and K483, and at least two other unidentified Lys residues in the surface loops of the outer membrane protein . The reagent cross-linked K483 in FepA L7 to either K332 in L5, forming a product that we designated band 1, or to the major outer membrane proteins OmpF, OmpC, and OmpA, forming band 2 . Ferric enterobactin binding to FepA did not prevent modification of K483 by Sulfo-EGS but blocked its cross-linking to OmpF/C and OmpA and reduced its coupling to K332 . These data show that the loops of FepA undergo conformational changes in vivo, with an approximate magnitude of 15 A, from a ligand-free open state to a ligand-bound closed state . The coupling of FepA L7 to OmpF, OmpC, or OmpA was TonB independent and was unaffected by the uncouplers CCCP (carbonyl cyanide m-chlorophenylhydrazone) and DNP (2,4-dinitrophenol) but completely inhibited by cyanide.

J Bacteriol, 2002 Sep, 184(17), 4811 - 8
Identification by heterologous expression and gene disruption of VisA as L-lysine 2-aminotransferase essential for virginiamycin S biosynthesis in Streptomyces virginiae; Namwat W et al.; The visA gene of Streptomyces virginiae has been thought to be a part of the virginiamycin S (VS) biosynthetic gene cluster based on its location in the middle of genes that encode enzymes highly similar to those participating in the biosynthesis of streptogramin-type antibiotics . Heterologous expression of the visA gene was achieved in Escherichia coli by an N-terminal fusion with thioredoxin (TrxA), and the intact recombinant VisA protein (rVisA) was purified after cleavage with enterokinase to remove the TrxA moiety . The purified rVisA showed clear L-lysine 2-aminotransferase activity with an optimum pH of around 8.0 and an optimum temperature at 35 degrees C, with 2-oxohexanoate as the best amino acceptor, indicating that VisA converts L-lysine into Delta(1)-piperidine 2-carboxylic acid . A visA deletion mutant of S . virginiae was created by homologous recombination, and the in vivo function of the visA gene was studied by phenotypic comparison between the wild type and the visA deletion mutant . No differences in growth in liquid media or in morphological behavior on solid media were observed, indicating that visA is not involved in primary metabolism or morphological differentiation . However, the visA mutant failed to produce VS while maintaining the production of virginiamycin M(1) at a level comparable to that of the parental wild-type strain, demonstrating that visA is essential to VS biosynthesis . These results, together with the observed recovery of the defect in VS production by the external addition of 3-hydroxypicolinic acid (3-HPA), a starter molecule in VS biosynthesis, suggest that VisA is the first enzyme of the VS biosynthetic pathway and that it supplies 3-HPA from L-lysine.

J Bacteriol, 2002 Sep, 184(17), 4783 - 91
Factors affecting start site selection at the Escherichia coli fis promoter; Walker KA et al.; Transcription initiation with CTP is an uncommon feature among Escherichia coli sigma(70) promoters . The fis promoter (fis P), which is subject to growth phase-dependent regulation, is among the few that predominantly initiate transcription with CTP . Mutations in this promoter that cause a switch from utilization of CTP to either ATP or GTP as the initiation nucleotide drastically alter its growth phase regulation pattern, suggesting that the choice of the primary initiating nucleotide can significantly affect its regulation . To better understand what factors influence this choice in fis P, we made use of a series of promoter mutations that altered the nucleotide or position used for initiation . Examination of these promoters indicates that start site selection is determined by a combination of factors that include preference for a nucleotide distance from the -10 region (8 > 7 > 9 >> 6 >> 10 > 11), initiation nucleotide preference (A = G >> CTP > or = UTP), the DNA sequence surrounding the initiation region, the position of the -35 region, and changes in the intracellular nucleoside triphosphate pools . We describe the effects that each of these factors has on start site selection in the fis P and discuss the interplay between position and nucleotide preference in this important process.

J Bacteriol, 2002 Sep, 184(17), 4775 - 82
Membrane protein degradation by FtsH can be initiated from either end; Chiba S et al.; FtsH, a membrane-bound metalloprotease, with cytoplasmic metalloprotease and AAA ATPase domains, degrades both soluble and integral membrane proteins in Escherichia coli . In this paper we investigated how membrane-embedded substrates are recognized by this enzyme . We showed previously that FtsH can initiate processive proteolysis at an N-terminal cytosolic tail of a membrane protein, by recognizing its length (more than 20 amino acid residues) but not exact sequence . Subsequent proteolysis should involve dislocation of the substrates into the cytosol . We now show that this enzyme can also initiate proteolysis at a C-terminal cytosolic tail and that the initiation efficiency depends on the length of the tail . This mode of degradation also appeared to be processive, which can be aborted by a tightly folded periplasmic domain . These results indicate that FtsH can exhibit processivity against membrane-embedded substrates in either the N-to-C or C-to-N direction . Our results also suggest that some membrane proteins receive bidirectional degradation simultaneously . These results raise intriguing questions about the molecular directionality of the dislocation and proteolysis catalyzed by FtsH.

J Bacteriol, 2002 Sep, 184(17), 4733 - 8
SoxRS down-regulation of rob transcription; Michan C et al.; Rob is regarded as a constitutively expressed protein, although little is known about how rob gene is regulated . We show here by reverse transcription-PCR that the transcriptional levels of rob are strongly down-regulated in response to the superoxide-generating agent paraquat (PQ) . Repression reached a maximum of 20-fold after 10 min exposure at 10 microM PQ . The magnitude of rob repression was comparable to that of induction quantified for the most sensitive SoxS targets . beta-Galactosidase expression with the rob2::lacZ transcriptional fusion indicates that down-regulation of rob expression takes place, at least in part, at the level of transcription initiation . Moreover, ca . 50% of the rob mRNA was degraded in <1 min after the addition of rifampin to inhibit transcription . This intrinsic short half-life, which is of obvious benefit for a rapid down-regulation after transcription ceases, was unaffected by the addition of PQ . No repression was observed in a soxR-null strain, indicating that the rob transcript level might be negatively modulated by the intracellular amounts of SoxS protein . Gel retardation assays support the idea that in vivo SoxS would block rob transcription directly.

J Bacteriol, 2002 Sep, 184(17), 4715 - 21
Roles of RecJ, RecO, and RecR in RecET-mediated illegitimate recombination in Escherichia coli; Shiraishi K et al.; We analyzed effects of overexpression of RecE and RecT on illegitimate recombination during prophage induction in Escherichia coli and found that frequencies of spontaneous and UV-induced illegitimate recombination are enhanced by coexpression of RecE and RecT in the wild type, but the enhanced recombination was reduced by recJ, recO, or recR mutation . The results indicated that RecET-mediated illegitimate recombination depends on the functions of RecJ, RecO, and RecR, suggesting that the RecE and RecJ exonucleases play different roles in this recombination pathway and that the RecO and RecR proteins also play important roles in the recombination . On the other hand, the frequency of the RecET-mediated illegitimate recombination was enhanced by a recQ mutation, implying that the RecQ protein plays a role in suppression of RecET-mediated illegitimate recombination . It was also found that RecET-mediated illegitimate recombination is independent of the RecA function with UV irradiation, but it is enhanced by the recA mutation without UV irradiation . Based on these results, we propose a model for the roles of RecJOR on RecET-mediated illegitimate recombination.

J Bacteriol, 2002 Sep, 184(17), 4666 - 71
Biosynthesis of the cyanobacterial light-harvesting polypeptide phycoerythrocyanin holo-alpha subunit in a heterologous host; Tooley AJ et al.; The entire pathway for the biosynthesis of the phycobiliviolin-bearing His-tagged holo-alpha subunit of the cyanobacterial photosynthetic accessory protein phycoerythrocyanin was reconstituted in Escherichia coli . Cyanobacterial genes encoding enzymes required for the conversion of heme to 3Z-phycocyanobilin, a precursor of phycobiliviolin (namely, heme oxygenase 1 and 3Z-phycocyanobilin:ferredoxin oxidoreductase), were expressed from a plasmid under the control of the hybrid trp-lac (trc) promoter . Genes for the apo-phycoerythrocyanin alpha subunit (pecA) and the heterodimeric lyase/isomerase (pecE and pecF), which catalyzes both the covalent attachment of phycocyanobilin and its concurrent isomerization to phycobiliviolin, were expressed from the trc promoter on a second plasmid . Upon induction, recombinant E . coli used endogenous heme to produce holo-PecA with absorbance and fluorescence properties similar to those of the same protein produced in cyanobacteria . About two-thirds of the apo-PecA was converted to holo-PecA . No significant bilin addition took place in a similarly engineered E . coli strain that lacks pecE and pecF . By using immobilized metal affinity chromatography, both apo-PecA and holo-PecA were isolated as ternary complexes with PecE and PecF . The identities of all three components in the ternary complexes were established unambiguously by protein and tryptic peptide analyses performed by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Bioinformatics, 2002, 18 Suppl 1, S337 - 44
Identifying operons and untranslated regions of transcripts using Escherichia coli RNA expression analysis; Tjaden B et al.; Microarrays traditionally have been used to assay the transcript expression of coding regions of genes . Here, we use Escherichia coli oligonucleotide microarrays to assay transcript expression of both open reading frames (ORFs) and intergenic regions . We then use hidden Markov models to analyse this expression data and estimate transcription boundaries of genes . This approach allows us to identify 5' untranslated regions (5' UTRs) of transcripts as well as genes that are likely to be operon members . The operon elements we identify correspond to documented operons with 99% specificity and 63% sensitivity . Similarly we find that our 5' UTR results accurately coincide with experimentally verified promoter regions for most genes.

Bioinformatics, 2002, 18 Suppl 1, S312 - 20
Efficient multiple genome alignment; Hohl M et al.; MOTIVATION: To allow a direct comparison of the genomic DNA sequences of sufficiently similar organisms, there is an urgent need for software tools that can align more than two genomic sequences . RESULTS: We developed new algorithms and a software tool 'Multiple Genome Aligner' (MGA for short) that efficiently computes multiple genome alignments of large, closely related DNA sequences . For example, it can align 85% percent of the complete genomes of six human adenoviruses (average length 35305 bp.) in 159 seconds . An alignment of 74% of the complete genomes of three of strains of E . coli (lengths: 5528445; 5498450; 4639221 approximately bp.) is produced in 30 minutes.

Bioinformatics, 2002, 18 Suppl 1, S22 - 30
DNA sequence and structure: direct and indirect recognition in protein-DNA binding; Steffen NR et al.; MOTIVATION: Direct recognition, or direct readout, of DNA bases by a DNA-binding protein involves amino acids that interact directly with features specific to each base . Experimental evidence also shows that in many cases the protein achieves partial sequence specificity by indirect recognition, i.e., by recognizing structural properties of the DNA . (1) Could threading a DNA sequence onto a crystal structure of bound DNA help explain the indirect recognition component of sequence specificity? (2) Might the resulting pure-structure computational motif manifest itself in familiar sequence-based computational motifs? RESULTS: The starting structure motif was a crystal structure of DNA bound to the integration host factor protein (IHF) of E . coli . IHF is known to exhibit both direct and indirect recognition of its binding sites . (1) Threading DNA sequences onto the crystal structure showed statistically significant partial separation of 60 IHF binding sites from random and intragenic sequences and was positively correlated with binding affinity . (2) The crystal structure was shown to be equivalent to a linear Markov network, and so, to a joint probability distribution over sequences, computable in linear time . It was transformed algorithmically into several common pure-sequence representations, including (a) small sets of short exact strings, (b) weight matrices, (c) consensus regular patterns, (d) multiple sequence alignments, and (e) phylogenetic trees . In all cases the pure-sequence motifs retained statistically significant partial separation of the IHF binding sites from random and intragenic sequences . Most exhibited positive correlation with binding affinity . The multiple alignment showed some conserved columns, and the phylogenetic tree partially mixed low-energy sequences with IHF binding sites but separated high-energy sequences . The conclusion is that deformation energy explains part of indirect recognition, which explains part of IHF sequence-specific binding.

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