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 re