Nat Struct Mol Biol, 2004 Sep, 11(9), 894 - 900 Epub 2004 Aug 01. A novel tunnel in mycobacterial type III polyketide synthase reveals the structural basis for generating diverse metabolites; Sankaranarayanan R et al.; The superfamily of plant and bacterial type III polyketide synthases (PKSs) produces diverse metabolites with distinct biological functions . PKS18, a type III PKS from Mycobacterium tuberculosis, displays an unusual broad specificity for aliphatic long-chain acyl-coenzyme A (acyl-CoA) starter units (C(6)-C(20)) to produce tri- and tetraketide pyrones . The crystal structure of PKS18 reveals a 20 A substrate binding tunnel, hitherto unidentified in this superfamily of enzymes . This remarkable tunnel extends from the active site to the surface of the protein and is primarily generated by subtle changes of backbone dihedral angles in the core of the protein . Mutagenic studies combined with structure determination provide molecular insights into the structural elements that contribute to the chain length specificity of the enzyme . This first bacterial type III PKS structure underlines a fascinating example of the way in which subtle changes in protein architecture can generate metabolite diversity in nature. Crit Care Med, 2004 Aug, 32(8), 1740 - 6 Escherichia coli pneumonia enhances granulopoiesis and the mobilization of myeloid progenitor cells into the systemic circulation; Shahbazian LM et al.; OBJECTIVE: The process by which hematopoietic tissues respond to a pulmonary infection remains poorly understood . This study investigated the potential role of lung-derived granulopoietic cytokines in facilitating this response . DESIGN: Laboratory investigation . SETTING: University laboratory . SUBJECTS: Male Balb/c mice . INTERVENTIONS: Mice were challenged with intratracheal Escherichia coli or granulocyte colony-stimulating factor (G-CSF) . Bone marrow cells were isolated from normal mice and treated in vitro with G-CSF . MEASUREMENTS AND MAIN RESULTS: Bronchoalveolar lavage fluid concentrations of G-CSF, macrophage inflammatory protein-2, and keratinocyte-derived chemokine were elevated 3 and 6 hrs after intratracheal E . coli . The increases in intrapulmonary G-CSF and keratinocyte-derived chemokine were associated with increases of their concentrations in the plasma . The numbers of granulocyte-macrophage colony forming units in bone marrow, spleen, and blood were increased 48 hrs after intratracheal E . coli or G-CSF . In addition, plasma G-CSF and the number of progenitor cells (lin-ckit+Sca-1(-)) in the blood were increased at 30 mins and 48 hrs, respectively, following intratracheal G-CSF . Signal transducer and activator of transcription-3 in bone marrow cells was activated following intratracheal E . coli or G-CSF in addition to activation by in vitro G-CSF stimulation . CONCLUSIONS: During pulmonary infection, locally produced cytokines enter the circulation and may play an important role in initiating a granulopoietic response. Crit Care Med, 2004 Aug, 32(8), 1715 - 21 Expression and secretion of procalcitonin and calcitonin gene-related peptide by adherent monocytes and by macrophage-activated adipocytes; Linscheid P et al.; OBJECTIVE: To explore the roles of peripheral blood mononuclear cells (PBMCs) and PBMC-derived macrophages in sepsis-related increased procalcitonin and calcitonin gene-related peptide (CGRP) I production . DESIGN: Prospective, in vitro primary human cell culture study and human tissue samples gene expression analysis . SETTING: University hospital research laboratories . PATIENTS: Cells from healthy donors and septic patients . INTERVENTIONS: PBMCs were obtained from healthy donors . Isolation of pure monocyte cultures was performed by magnetic depletion of nonmonocyte cells from PBMCs . Adipose tissue biopsies and circulating leukocytes were collected from septic patients . Expressions of calcitonin messenger RNA and CGRP I messenger RNA were analyzed using reverse transcriptase-polymerase chain reaction and quantitative real-time polymerase chain reaction . Supernatant procalcitonin and CGRP protein content were determined by ultrasensitive chemiluminometric and radioimmunoassays, respectively . MEASUREMENTS AND MAIN RESULTS: PBMCs expressed and secreted procalcitonin and CGRP within 3-5 hrs after adherence to endothelial cells or plastic surfaces . This induction was transient, as it was not detectable after 18 hrs . No calcitonin or CGRP I messenger RNA was observed in leukocytes obtained from septic patients with markedly increased serum procalcitonin concentrations . Stimulation with cytokines, endotoxin, or Escherichia coli did not induce expression of calcitonin and CGRP I messenger RNA in PBMC-derived macrophages . However, inflammatory factors released from activated macrophages induced a marked expression of procalcitonin and CGRP in co-cultured human adipocytes . CONCLUSIONS: The adhesion-induced, transient expression and secretion of procalcitonin and CGRP in vitro may play an important role during monocyte adhesion and migration in vivo . PBMC-derived macrophages may contribute to the marked increase in circulating procalcitonin by recruiting parenchymal cells within the infected tissue, as exemplified with adipocytes. J Biosci, 2004 Jun, 29(2), 153 - 61 A novel potassium deficiency-induced stimulon in Anabaena torulosa; Alahari A et al.; Potassium deficiency enhanced the synthesis of fifteen proteins in the nitrogen-fixing cyanobacterium Anabaena torulosa and of nine proteins in Escherichia coli . These were termed potassium deficiency-induced proteins or PDPs and constitute hitherto unknown potassium deficiency-induced stimulons . Potassium deficiency also enhanced the synthesis of certain osmotic stress-induced proteins . Addition of K+ repressed the synthesis of a majority of the osmotic stress-induced proteins and of PDPs in these bacteria . These proteins contrast with the dinitrogenase reductase of A . torulosa and the glycine betaine-binding protein of E . coli, both of which were osmo-induced to a higher level in potassium-supplemented conditions . The data demonstrate the occurrence of novel potassium deficiency-induced stimulons and a wider role of K+ in regulation of gene expression and stress responses in bacteria Science, 2004 Jul 30, 305(5684), 673 - 6 Synthetic mammalian prions; Legname G et al.; Recombinant mouse prion protein (recMoPrP) produced in Escherichia coli was polymerized into amyloid fibrils that represent a subset of beta sheet-rich structures . Fibrils consisting of recMoPrP(89-230) were inoculated intracerebrally into transgenic (Tg) mice expressing MoPrP(89-231) . The mice developed neurologic dysfunction between 380 and 660 days after inoculation . Brain extracts showed protease-resistant PrP by Western blotting; these extracts transmitted disease to wild-type FVB mice and Tg mice overexpressing PrP, with incubation times of 150 and 90 days, respectively . Neuropathological findings suggest that a novel prion strain was created . Our results provide compelling evidence that prions are infectious proteins. Mol Pharmacol, 2004 Nov, 66(5), 1147 - 59 Epub 2004 Jul 30. Specific inhibition of nuclear factor-kappaB-dependent inflammatory responses by cell type-specific mechanisms upon A2A adenosine receptor gene transfer; Sands WA et al.; Adenosine is a potent inhibitor of inflammatory processes, and the A(2A) adenosine receptor (A(2A)AR) plays a key nonredundant role as a suppresser of inflammatory responses in vivo . In this study, we demonstrate that increasing A(2A)AR gene expression suppressed multiple inflammatory responses in both human umbilical vein endothelial cells (HUVECs) and rat C6 glioma cells in vitro . In particular, the induction of the adhesion molecule E-selectin by either tumor necrosis factor alpha (TNFalpha) or Escherichia coli lipopolysaccharide (LPS) was reduced by more than 70% in HUVECs, whereas inducible nitric-oxide synthase (iNOS) induction was abolished in C6 cells after exposure to interferon-gamma in combination with LPS and TNFalpha, suggesting that the receptor inhibited a common step in the induction of each of these pro-inflammatory genes . Consistent with this hypothesis, A(2A)AR expression inhibited the activation of NF-kappaB, a key transcription factor whose proper function was essential for optimal iNOS and E-selectin induction . However, although NF-kappaB binding to target DNA was severely compromised in both cell types, the mechanisms by which this occurred were distinct . In C6 cells, A(2A)AR expression blocked IkappaBalpha degradation by inhibiting stimulus-induced phosphorylation, whereas in HUVECs, A(2A)AR expression inhibited NF-kappaB translocation to the nucleus independently of any effect on IkappaBalpha degradation . Together, these observations suggest that A(2A)AR-mediated inhibition NF-kappaB activation is a critical aspect of its anti-inflammatory signaling properties and that the molecular basis of this inhibition varies in a cell type-specific manner. J Biol Chem, 2004 Sep 24, 279(39), 40358 - 61 Epub 2004 Jul 30. The Epstein-Barr virus polymerase accessory factor BMRF1 adopts a ring-shaped structure as visualized by electron microscopy; Makhov AM et al.; Epstein-Barr virus (EBV) encodes a set of core replication factors used during lytic infection in human cells that parallels the factors used in many other systems . These include a DNA polymerase and its accessory factor, a helicase/primase, and a single strand binding protein . The EBV polymerase accessory factor has been identified as the product of the BMRF1 gene and has been shown by functional assays to increase the activity and processivity of the polymerase . Unlike other members of this class of factors, BMRF1 is also a transcription factor regulating certain EBV genes . Although several polymerase accessory factors, including eukaryotic proliferating cell nuclear antigen, Escherichia coli beta protein, and T4 gene 45 protein have been shown to form oligomeric rings termed sliding clamps, nothing is known about the oligomeric state of BMRF1 or whether it forms a ring . In this work, BMRF1 was purified directly from human cells infected with an adenovirus vector expressing the BMRF1 gene product . The protein was purified to near homogeneity, and examination by negative staining electron microscopy revealed large, flat, ring-shaped molecules with a diameter of 15.5 +/- 0.8 nm and a distinct 5.3-nm diameter hole in the center . The size of these rings is consistent with an oligomer of 6 monomers, nearly twice as large as the trimeric proliferating cell nuclear antigen ring . Unlike the herpes simplex virus UL42 homologue, BMRF1 was found to self-associate in solution . These findings extend the theme of polymerase accessory factors adopting ring-shaped structures and provide an example in which the ring is significantly larger than any previously described sliding clamp . Inorg Chem, 2004 Aug 9, 43(16), 4907 - 10 Resonance Raman detection of the Fe2+-C-N modes in heme-copper oxidases: a probe of the active site; Pinakoulaki E et al.; Resonance Raman spectroscopy has been employed to investigate the reduced cyano complexes of cytochrome aa(3) from bovine heart and Rhodobacter sphaeroides and of cytochrome bo(3) from E . coli . In the aa(3)-type oxidases, the frequency of the Fe-CN stretching mode is located at 468 cm(-1), and the bending Fe-C-N vibration, at 500 cm(-1) . The fully reduced cytochrome bo(3)-CN complex gives rise to a stretching vibration at 468 cm(-1), a bending vibration at 491 cm(-1), and a stretching C-N vibration at 2037 cm(-1) . The observed differences between aa(3) and bo(3) oxidases in the frequencies of the Fe-C-N group suggest a quantitative difference in the structure of the His-heme a(3)(2+)/Cu(B)(1+) and His-heme o(3)(2+)/Cu(B)(1+) binuclear pockets upon CN- binding. World J Gastroenterol, 2004 Aug 15, 10(16), 2352 - 6 Structure prediction and activity analysis of human heme oxygenase-1 and its mutant; Xia ZW et al.; AIM: To predict wild human heme oxygenase-1 (whHO-1) and hHO-1 His25Ala mutant (delta hHO-1) structures, to clone and express them and analyze their activities . METHODS: Swiss-PdbViewer and Antheprot 5.0 were used for the prediction of structure diversity and physical-chemical changes between wild and mutant hHO-1 . hHO-1 His25Ala mutant cDNA was constructed by site-directed mutagenesis in two plasmids of E . coli DH5alpha . Expression products were purified by ammonium sulphate precipitation and Q-Sepharose Fast Flow column chromatography, and their activities were measured . RESULTS: rHO-1 had the structure of a helical fold with the heme sandwiched between heme-heme oxygenase-1 helices . Bond angle, dihedral angle and chemical bond in the active pocket changed after Ala25 was replaced by His25, but Ala25 was still contacting the surface and the electrostatic potential of the active pocket was negative . The mutated enzyme kept binding activity to heme . Two vectors pBHO-1 and pBHO-1(M) were constructed and expressed . Ammonium sulphate precipitation and column chromatography yielded 3.6-fold and 30-fold higher purities of whHO-1, respectively . The activity of delta hHO-1 was reduced 91.21% after mutation compared with whHO-1 . CONCLUSION: Proximal His25 ligand is crucial for normal hHO-1 catalytic activity . delta hHO-1 is deactivated by mutation but keeps the same binding site as whHO-1 . delta hHO-1 might be a potential inhibitor of whHO-1 for preventing neonatal hyperbilirubinemia. World J Gastroenterol, 2004 Aug 15, 10(16), 2340 - 3 Antigen epitope of Helicobacter pylori vacuolating cytotoxin A; Liu XL et al.; AIM: To construct and select antigen epitopes of vacuolating cytotoxin A (VacA) for nontoxic VacA vaccine against Helicobacter pylori (H pylori ) infection . METHODS: Eleven VacA epitopes were predicted according to VacA antigenic bioinformatics . Three candidates of VacA epitope were constructed through different combined epitopes . The candidate was linked with E . coli heat-labile enterotoxin B (LTB) by a linker of 7 amino acids, and cloned into plasmid pQE-60 in which fusion LTB-VacA epitope was efficiently expressed . To test the antigencity of the candidate, 6 BALB/c mice were treated with the fusion LTB-VacA epitope through intraperitoneal injection . To explore the ability of inhibiting the toxicity of VacA,cantiserum against the candidate was used to counteract VacA that induced HeLa cells to produce cell vacuoles in vitro . RESULTS: Serum IgG against the candidate was induced in the BALB/c mice . In vitro, the three antisera against the candidate efficiently counteracted the toxicity of VacA, and decreased the number of cell vacuoles by 14.17%, 20.20% and 30.41% respectively . CONCLUSION: Two of the three candidates, LZ-VacA1and LZ-VacA2, can be used to further study the mechanism of vacuolating toxicity of VacA, and to construct nontoxic VacA vaccine against H pylori infection. J Biol Chem, 2004 Oct 22, 279(43), 44258 - 69 Epub 2004 Jul 28. Deamidation affects structural and functional properties of human alphaA-crystallin and its oligomerization with alphaB-crystallin; Gupta R et al.; To determine the effects of deamidation on structural and functional properties of alphaA-crystallin, three mutants (N101D, N123D, and N101D/N123D) were generated . Deamidated alphaB-crystallin mutants (N78D, N146D, and N78D/N146D), characterized in a previous study (Gupta, R., and Srivastava, O . P . (2004) Invest . Ophthalmol . Vis . Sci . 45, 206-214) were also used . The biophysical and chaperone properties were determined in (a) homoaggregates of alphaA mutants (N101D, N123D, and N101D/N123D) and (b) reconstituted heteroaggregates of alpha-crystallin containing (i) wild type alphaA (WT-alphaA): WT-alphaB crystallins, (ii) individual alphaA-deamidated mutants:WT-alphaB crystallins, and (iii) WT-alphaA:individual alphaB-deamidated mutant crystallins . Compared with the WT-alphaA, the three alphaA-deamidated mutants showed reduced levels of chaperone activity, alterations in secondary and tertiary structures, and larger aggregates . These altered properties were relatively more pronounced in the mutant N101D compared with the mutant N123D . Further, compared with heteroaggregates of WT-alphaA and WT-alphaB, the heteroaggregates containing deamidated subunits of either alphaA- or alphaB-crystallins and their counterpart WT proteins showed higher molecular mass, altered tertiary structures, lower exposed hydrophobic surfaces, and reduced chaperone activity . However, the heteroaggregate containing WT-alphaA and deamidated alphaB subunit showed lower chaperone activity, smaller oligomers, and 3-fold lower subunit exchange rate than heteroaggregate containing deamidated alphaA- and WT-alphaB subunits . Together, the results suggested that (a) both Asn residues (Asn-101 and Asn-123) are required for the structural integrity and chaperone function of alphaA-crystallin and (b) the presence of WT-alphaB in the alpha-crystallin heteroaggregate leads to packing-induced structural changes which influences the oligomerization and modulate chaperone activity. Trends Biotechnol, 2004 Aug, 22(8), 381 - 3 Extracting novel information from gene expression data; Li Z et al.; Data from high throughput technologies, such as DNA microarrays, necessitated the development of new computational methodologies for analyzing the high dimensional information contained within the gene expression data . Liao's group suggested the use of network component analysis to predict transcription factor activities by integrating gene expression data from Escherichia coli with known connectivity information between their genes and transcription factors . This introduces an approach for obtaining novel information from gene expression data. Biochem J, 2004 Nov 15, 384(Pt 1), 129 - 37 A novel member of the GCN5-related N-acetyltransferase superfamily from Caenorhabditis elegans preferentially catalyses the N-acetylation of thialysine {S-(2-aminoethyl)-L-cysteine}; Abo-Dalo B et al.; The putative diamine N-acetyltransferase D2023.4 has been cloned from the model nematode Caenorhabditis elegans . The 483 bp open reading frame of the cDNA encodes a deduced polypeptide of 18.6 kDa . Accordingly, the recombinantly expressed His6-tagged protein forms an enzymically active homodimer with a molecular mass of approx . 44000 Da . The protein belongs to the GNAT (GCN5-related N-acetyltransferase) superfamily, and its amino acid sequence exhibits considerable similarity to mammalian spermidine/spermine-N1-acetyltransferases . However, neither the polyamines spermidine and spermine nor the diamines putrescine and cadaverine were efficiently acetylated by the protein . The smaller diamines diaminopropane and ethylenediamine, as well as L-lysine, represent better substrates, but, surprisingly, the enzyme most efficiently catalyses the N-acetylation of amino acids analogous with L-lysine . As determined by the k(cat)/K(m) values, the C . elegans N-acetyltransferase prefers thialysine {S-(2-aminoethyl)-L-cysteine}, followed by O-(2-aminoethyl)-L-serine and S-(2-aminoethyl)-D,L-homocysteine . Reversed-phase HPLC and mass spectrometric analyses revealed that N-acetylation of L-lysine and L-thialysine occurs exclusively at the amino moiety of the side chain . Remarkably, heterologous expression of C . elegans N-acetyltransferase D2023.4 in Escherichia coli, which does not possess a homologous gene, results in a pronounced resistance against the anti-metabolite thialysine . Furthermore, C . elegans N-acetyltransferase D2023.4 exhibits the highest homology with a number of GNATs found in numerous genomes from bacteria to mammals that have not been biochemically characterized so far, suggesting a novel group of GNAT enzymes closely related to spermidine/spermine-N1-acetyltransferase, but with a distinct substrate specificity . Taken together, we propose to name the enzyme 'thialysine N(epsilon)-acetyltransferase'. Ann Ital Chir, 2004 Jan-Feb, 75(1), 97 - 106; discussion 106 {Fournier's gangrene: case report and review of recent literature}; Geraci G et al.; OBJECTIVE: The authors report their experience in diagnosis and treatment of one case of Fournier's gangrene; recent international literature review . EXPERIMENTAL DESIGN: Complete clinical report . Diagnostic, clinical and prognostic indication, evaluation of effectiveness of surgical treatment (debridement and necrosectomy) and follow-up; comparison between indications and multidisciplinary approach proposed by international literature . SETTING: Operative Unit of General and Thoracic Surgery . University "Paolo Giaccone" of Palermo . INTERVENTION: Repeated surgical treatment previous multimodal approach, according to international guide-lines . RESULTS: Complete recovery with "restitutio ad integrum" . No relapse were recorded at follow up . CONCLUSIONS: Fournier's gangrene is an uncommon and aggressive synergistic fasciitis of the perineum and genital organs, which may bring the patient to death; it is a true surgical emergency . The disease can no longer be considered to be idiopathic; in most cases a urologic, colorectal or cutaneous source can be identified . Despite antibiotics and aggressive debridement, the mortality rate remains high, particularly in the elderly, in patients with renal failure, and in patients with extensive disease . The presentation is highly variable, necessitating a high index of suspicion . High risk patients include diabetics, alcoholics and debilitated and immunosuppressed individuals . As the AIDS population increases, the incidence of Fournier's gangrene may increase as well . In questionable cases, imaging modalities should be performed to allow early diagnosis and to reduce misses diagnosis . Broad spectrum antibiotics (while waiting for the results of culture and antibiogram effectuated on tissue specimens obtained during necrosectomy) and aggressive debridement remain the hallmarks of treatment . Hyperbaric oxygen therapy and improved local wound care may decrease the extent of tissue destruction . The surgical operation has to be performed in emergency to avoid a rapid spread of tissue necrosis and a possible development towards septic shock . Reconstructive techniques afford better cosmetic results . With early recognition, prompt treatment, improved wound care and reconstructive efforts, the mortality rates and cosmetic results should continue to improve. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 2004 Feb 28, 22(1), 46 - 9 {Expression of adenylate kinase of Schistosoma japonicum and evaluation on the immunoreactivity of the recombinant protein}; Peng HJ et al.; OBJECTIVE: To express and purify the Schistosoma japonicum adenylate kinase (AK) protein of which the cDNA sequence was subcloned into the pET32a(+) soluble expression plasmid, and to evaluate its immunoreactivity . METHODS: The recombinant plasmid was transformed into E . coli BL21, and induced with IPTG for expression . The bacterial lysis was conducted by ultrasonication and the supernatant was analyzed by SDS-PAGE after boiling and centrifugation . The target protein was purified with the Ni-NTA agarose . The proteins on the gel were transferred to the PVDF film and then the immunoreactivity was tested by Western blotting using anti-6-His antibody, sera from rabbits 6 weeks after infected with cercariae or UV-attenuated cercariae . The purified protein was coated for ELISA to test the cercariae infected rabbit serum and the normal rabbit serum . RESULTS: The molecular weight of the target fusion protein was Mr 40 000 after being induced with IPTG . The fused protein showed a single band when reacted with anti-6-His antibody, the cercariae infected rabbit serum and attenuated cercariae infected rabbit serum . CONCLUSION: The AK protein is expressed as a fused protein with thioredoxin and its molecular weight is about Mr 40000 . This protein has a positive immunoreactivity with sera of rabbits infected with cercariae and UV-attenuated cercariae. Plant Physiol Biochem, 2004 Feb, 42(2), 143 - 8 Copper deficiency induced expression of Fe-superoxide dismutase gene in Matteuccia struthiopteris; Murao K et al.; Iron-superoxide dismutase (Fe-SOD) activity was not detected in extracts from the leaves of ferns, Equisetum arvense and Matteuccia struthiopteris . To know why ferns lack Fe-SOD activity, the Fe-SOD like gene (MsFeSOD1) was isolated from M . struthiopteris and its expression was investigated with a focus on the metals Fe and Cu using the prothalli of the fern . The expression of MsFeSOD1 mRNA was induced by a deficiency of Cu, but Fe-SOD activity was not detected . The recombinant protein of MsFeSOD1 produced in E . coli showed Fe-SOD activity . These findings suggest that the fern Fe-SOD like gene was transcriptionally regulated by Cu but an additional mechanism is involved in the formation of an active enzyme. Biomaterials, 2005 Feb, 26(6), 679 - 86 Enhanced gene delivery to PC12 cells by a cationic polypeptide; Zeng J et al.; Targeted gene delivery to diseased subtypes of neurons will be beneficial to the success of gene therapy of neurological disorders . We designed a recombinant cationic polypeptide to facilitate gene delivery to neuronal-like PC12 cells that express the nerve growth factor (NGF) receptors . The recombinant polypeptide was composed of a targeting moiety derived from loop 4-containing hairpin motif of NGF and a DNA-binding moiety of 10-lysine sequence and expressed in Escherichia coli . It activated NGF receptor, TrkA and its downstream signaling pathways in PC12 and promoted the survival of neuronally differentiated PC12 cells deprived of serum . The polypeptide could also bind plasmid DNA and enhance polycation-mediated gene delivery in NGF receptor-expressing PC12 cells, but not in COS7 cells lacking NGF receptors . The enhancement of gene transfer in PC12 was inhibited by pretreatment of free, unbound polypeptides, suggesting a NGF-receptor-specific effect of the polypeptide . These observations demonstrated the concept of using receptor-mediated mechanism for targeted gene delivery to neurons. Microb Cell Fact . 2004 Jul 28;3(1):9. Characterisation of the Escherichia coli membrane structure and function during fedbatch cultivation; Shokri A et al.; BACKGROUND: Important parameters during recombinant protein production in Escherichia coli, such as productivity and protein activity, are affected by the growth rate . This includes the translocation of protein over the membrane to gain better folding capacity or reduced proteolysis . To vary the growth rate two techniques are available: fedbatch and continuous cultivation, both controlled by the ingoing feed rate . RESULTS: During fedbatch cultivation, E . coli contains phosphatidylethanolamine, phosphatidylglycerol, cardiolipin and saturated fatty acids in amounts which are stable with growth rate . However, the levels of cardiolipin are very high compared to continuous cultivation . The reason for fedbatch triggering of this metabolism is not known but hypothesised to result from an additional need for carbon and energy . The reason could be the dynamic and sometimes rapid changes in growth rate to which the fedbatch cell has at all times to adjust . The membrane flexibility, essential for translocation of various components, is however to some degree sustained by production of increased amounts of unsaturated fatty acids in phosphatidylglycerol . The result is a functionally stiff membrane which generally promotes low cell lysis and is constant with respect to protein leakage to the medium . At comparatively high growth rates, when the further stabilising effect of cyclic fatty acids is gone, the high level of unsaturated fatty acids results in a pronounced effect upon sonication . This is very much in contrast to the membrane function in continuous cultivation which shows very specific characteristics as a function of growth rate . CONCLUSIONS: The stiff and unchanging fedbatch membrane should promote a stable behaviour during downstream processing and is less dependent on the time of harvest . However, optimisation of protein leakage can only be achieved in the continuously cultivated cell where leakage is twice as high compared to the constant leakage level in fedbatch . If leakage is undesired, continuous cultivation is also preferred since it can be designed to lead to the lowest values detected . Induction at low growth rate (<0.2 h-1) should be avoided with respect to productivity, in any system, since the specific and total protein production shows their lowest values at this point. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 2004 Apr 30, 22(2), 65 - 9 {Screening and expression of recombinant human monoclonal antibody Fab fragments specific to Entamoeba histolytica}; Shu Q et al.; OBJECTIVE: To prepare recombinant human monoclonal antibody Fab fragments specific to the surface antigen of Entamoeba histolytica . METHODS: Total RNA was isolated from lymphocytes which were separated from an asymptomatic E . histolytica cyst carrier . The genes of IgG light chain and Fd region of heavy chain were amplified by a reverse transcriptase PCR and ligated with a plasmid vector . After the genes were introduced into Escherichia coli, the clones expressing Fab fragments specific to the surface antigen of E . histolytica were screened and the product was purified . RESULTS: Thirty thousand clones were screened and one of them was proved positive to the surface antigen of E . histolytica . CONCLUSION: This study demonstrated that the bacterial system can be used to produce recombinant human monoclonal antibody Fab fragments specific to the surface antigen of E . histolytica and they may be applicable for the future diagnosis and treatment of the infection. Biotechnol Bioeng, 2004 Aug 5, 87(3), 293 - 302 Impact of engineering flow conditions on plasmid DNA yield and purity in chemical cell lysis operations; Meacle FJ et al.; Chemical lysis of bacterial cells using an alkaline solution containing a detergent may provide an efficient scalable means for selectively removing covalently closed circular plasmid DNA from high-molecular-weight contaminating cellular components including chromosomal DNA . In this article we assess the chemical lysis of E . coli cells by SDS in a NaOH solution and determine the impact of pH environment and shear on the supercoiled plasmid and chromosomal DNA obtained . Experiments using a range of plasmids from 6 kb to 113 kb determined that in an unfavorable alkaline environment, where the NaOH concentration during lysis is greater than 0.15 +/- 0.03 M (pH 12.9 +/- 0.2), irreversible denaturation of the supercoiled plasmid DNA occurs . The extent of denaturation is shown to increase with time of exposure and NaOH concentration . Experiments using stirred vessels show that, depending on NaOH concentration, moderate to high mixing rates are necessary to maximize plasmid yield . While NaOH concentration does not significantly affect chromosomal DNA contamination, a high NaOH concentration is necessary to ensure complete conversion of chromosomal DNA to single-stranded form . In a mechanically agitated lysis reactor the correct mixing strategy must balance the need for sufficient mixing to eliminate potential regions of high NaOH concentrations and the need to avoid excessive breakage of the shear sensitive chromosomal DNA . The effect of shear on chromosomal DNA is examined over a wide range of shear rates (10(1)-10(5) s(-1)) demonstrating that, while increasing shear leads to fragmentation of chromosomal DNA to smaller sizes, it does not lead to significantly increased chromosomal DNA contamination except at very high shear rates (about 10(4)-10(5) s(-1)) . The consequences of these effects on the choice of lysis reactor and scale-up are discussed. Antonie Van Leeuwenhoek, 2004 Aug, 86(2), 189 - 99 Relationships between Escherichia coli cells and the surrounding medium during survival processes; Arana I et al.; In Escherichia coli, during survival under adverse conditions, namely starvation and luminous radiation, two things occur . On the one hand organic substances are released into the surrounding medium and on the other there is a transition from the culturable state to viable but non-culturable (VBNC) . An analysis of organic molecules released into the surrounding medium showed the presence of proteins, dissolved free amino acids, and dissolved monomeric carbohydrates . The concentration of these substances in the medium changed with exposure time, type of stress and type of molecule . The proteins accumulated in the medium and in some cases their identification revealed the presence of components of the outer membrane . Variations in the concentration of amino acids and carbohydrates point to a twofold process of excretion and uptake . Indeed, cell free supernatants supported the growth of several generations of a population of 10(4) cells ml(-1) . The survival of E . coli in supernatants previously colonized by cells in the VBNC state was greater than that observed in the control experiments, with a short delay in the loss of culturability . It was thus clear that organic molecules released into the medium play a role in the transition from culturable to VBNC state. J Virol, 2004 Aug, 78(16), 8935 - 41 The gene encoding the nucleocapsid protein of Gill-associated nidovirus of Penaeus monodon prawns is located upstream of the glycoprotein gene; Cowley JA et al.; The ORF2 gene of Gill-associated virus (GAV) of Penaeus monodon prawns resides 93 nucleotides downstream of the ORF1a-ORF1b gene and encodes a 144-amino-acid hydrophilic polypeptide (15,998 Da; pI, 9.75) containing 20 basic (14%) and 13 acidic (9%) residues and 19 prolines (13%) . Antiserum to a synthetic ORF2 peptide or an Escherichia coli-expressed glutathione S-transferase-ORF2 fusion protein detected a 20-kDa protein in infected lymphoid organ and gill tissues in Western blots . The GAV ORF2 fusion protein antiserum also cross-reacted with the p20 nucleoprotein in virions of the closely related Yellow head virus . By immuno-gold electron microscopy, it was observed that the ORF2 peptide antibody localized to tubular GAV nucleocapsids, often at the ends or at lateral cross sections . As GAV appears to contain only two structural protein genes (ORF2 and ORF3), these data indicate that GAV differs from vertebrate nidoviruses in that the gene encoding the nucleocapsid protein is located upstream of the gene encoding the virion glycoproteins. J Virol, 2004 Aug, 78(16), 8630 - 40 Characterization of nucleocapsid binding by the measles virus and mumps virus phosphoproteins; Kingston RL et al.; We report an analysis of the interaction between the P protein and the RNA-associated N protein (N-RNA) for both measles and mumps viruses with proteins produced in a bacterial expression system . During this study, we verified that the C-terminal tail of the N protein is not required for nucleocapsid formation . For both measles and mumps virus N, truncated proteins encompassing amino acids 1 to 375 assemble into nucleocapsid-like particles within the bacterial cell . For measles virus N, the binding site for the P protein maps to residues 477 to 505 within the tail of the molecule, a sequence relatively conserved among the morbilliviruses . For mumps virus N, a binding site for the P protein maps to the assembly domain of N (residues 1 to 398), while no strong binding of the P protein to the tail of N was detected . These results suggest that the site of attachment for the polymerase varies among the paramyxoviruses . Pulldown experiments demonstrate that the last 50 amino acids of both measles virus and mumps virus P (measles virus P, 457 to 507; mumps virus P, 343 to 391) by themselves constitute the nucleocapsid-binding domain (NBD) . Spectroscopic studies show that the NBD is predominantly alpha-helical in both viruses . However, only in measles virus P is the NBD stable and folded, having a lesser degree of tertiary organization in mumps virus P . With isothermal titration calorimetry, we demonstrate that the measles virus P NBD binds to residues 477 to 505 of measles virus N with 1:1 stoichiometry . The dissociation constant (K(d)) was determined to be 13 microM at 20 degrees C and 35 microM at 37 degrees C . Our data are consistent with a model in which an alpha-helical nucleocapsid binding domain, located at the C terminus of P, is responsible for tethering the viral polymerase to its template yet also suggest that, in detail, polymerase binding in morbilliviruses and rubulaviruses differs significantly. J Biol Chem, 2004 Oct 1, 279(40), 41333 - 9 Epub 2004 Jul 27. Purification and spectropotentiometric characterization of Escherichia coli NrfB, a decaheme homodimer that transfers electrons to the decaheme periplasmic nitrite reductase complex; Clarke TA et al.; Escherichia coli can reduce nitrite to ammonium via a 120-kDa decaheme homodimeric periplasmic nitrite reductase (NrfA) complex . Recent structure-based spectropotentiometric studies are shedding light on the catalytic mechanism of NrfA; however, electron input into the enzyme has not been addressed biochemically . This study reports the first purification of NrfB, a novel 20-kDa pentaheme c-type cytochrome encoded by the nrfB gene that follows the nrfA gene in many bacterial nrf operons . Analyses by gel filtration demonstrated that NrfB purifies as a decaheme homodimer . Analysis of NrfB by UV-visible and magnetic circular dichroism spectroscopy demonstrates that all five NrfB ferric heme irons are low spin and are most likely coordinated by two axial histidine ligands . Spectropotentiometry revealed that the midpoint redox potentials of five ferric hemes were in the low potential range of 0 to -400 mV . Analysis by low temperature EPR spectroscopy revealed signals that arise from two classes of bis-His ligated low spin hemes, namely a rhombic trio at g(1,2,3) = 2.99, 2.27, and 1.5 that arises from two hemes in which the planes of histidine imidazole rings are near-parallel and a large g(max) signal at g = 3.57 that arises from three hemes in which the planes of the histidine imidazole rings are near-perpendicular . NrfB was also overexpressed as a recombinant protein, which had similar spectropotentiometric properties as the native protein . Reconstitution experiments demonstrated that the reduced decaheme NrfB dimer could serve as a direct electron donor to the oxidized decaheme NrfA dimer, thus forming a transient 20-heme {NrfB}(2){NrfA}(2) electron transfer complex. J Biol Chem, 2004 Sep 24, 279(39), 40351 - 7 Epub 2004 Jul 26. Essential role of B-helix calcium binding sites in annexin V-membrane binding; Jin M et al.; Crystal structures of annexin V have shown up to 10 bound calcium ions in three different types of binding sites, but previous work concluded that only one of these sites accounted for nearly all of the membrane binding affinity of the molecule . In this study we mutated residues contributing to potential calcium binding sites in the AB and B helices in each of the four domains (eight sites in total) and in DE helices in the first, second, and third domains (three sites in total) . We measured the affinity of each protein for phospholipid vesicles and cell membranes by quantitative calcium titration under low occupancy conditions (< 1% saturation of available membrane binding sites) . Affinity was calculated from the midpoint and slope of the calcium titration curve and the concentration of membrane binding sites . The results showed that all four AB sites were essential for high affinity binding, as were three of the four B sites (in domains 1, 2, and 3); the DE site in the first domain made a slight contribution to affinity . Multisite mutants showed that each domain contributed additively and independently to binding affinity; in contrast, AB and B sites within the same domain were interdependent . The number of functionally important sites identified was consistent with the Hill coefficient observed in calcium titrations . This study shows an essential and previously unappreciated role for B-helix calcium binding sites in the membrane binding of annexins and indicates that all four domains of the molecule are required for maximum membrane binding affinity . J Biol Chem, 2004 Sep 24, 279(39), 40622 - 8 Epub 2004 Jul 26. Neuronal apoptosis-inhibitory protein does not interact with Smac and requires ATP to bind caspase-9; Davoodi J et al.; The neuronal apoptosis-inhibitory protein (NAIP) is the founding member of the mammalian family of inhibitor of apoptosis (IAP) proteins (also known as BIRC proteins) and has been shown to be antiapoptotic both in vivo and in vitro . The 160-kDa NAIP contains three distinct regions: an amino-terminal cluster of three baculoviral inhibitory repeat (BIR) domains, a central nucleotide binding oligomerization domain (NOD), and a carboxyl-terminal leucine-rich repeat (LRR) domain . The presence of the NOD and LRR domains renders NAIP unique among the IAPs and suggests that NAIP activity is regulated in a manner distinct from that of other members of the family . In this report, we examined the interaction of various regions of NAIP with caspase-9 and Smac . Recombinant NAIPs with truncations of the carboxyl-terminal LRR or NOD-LRR regions bound to caspase-9 . In contrast, the full-length protein did not, suggesting some form of structural autoregulation . However, the association of the wild type full-length protein with caspase-9 was observed when interaction analysis was performed in the presence of ATP . Furthermore, mutation of the NAIP ATP binding pocket allowed full-length protein to interact with caspase-9 . Thus, we conclude that NAIP binds to caspase-9 with a structural requirement for ATP and that in the absence of ATP the LRR domain negatively regulates the caspase-9-inhibiting activity of the BIR domains . Interestingly, and in contrast to the X-chromosome-linked inhibitor of apoptosis protein (XIAP), NAIP-mediated inhibition of caspase-9 was not countered by a peptide containing an amino-terminal IAP binding motif (IBM) . Consistent with this observation was the failure of Smac protein to interact with the NAIP BIR domains . These results demonstrate that NAIP is distinct from the other IAPs, both in demonstrating a ligand-dependent caspase-9 interaction and in demonstrating a distinct mechanism of inhibition . J Biol Chem, 2004 Oct 8, 279(41), 43198 - 206 Epub 2004 Jul 26. Crystal structure and mutagenesis of a protein phosphatase-1:calcineurin hybrid elucidate the role of the beta12-beta13 loop in inhibitor binding; Maynes JT et al.; Protein phosphatase-1 and protein phosphatase-2B (calcineurin) are eukaryotic serine/threonine phosphatases that share 40% sequence identity in their catalytic subunits . Despite the similarities in sequence, these phosphatases are widely divergent when it comes to inhibition by natural product toxins, such as microcystin-LR and okadaic acid . The most prominent region of non-conserved sequence between these phosphatases corresponds to the beta12-beta13 loop of protein phosphatase-1, and the L7 loop of toxin-resistant calcineurin . In the present study, mutagenesis of residues 273-277 of the beta12-beta13 loop of the protein phosphatase-1 catalytic subunit (PP-1c) to the corresponding residues in calcineurin (312-316), resulted in a chimeric mutant that showed a decrease in sensitivity to microcystin-LR, okadaic acid, and the endogenous PP-1c inhibitor protein inhibitor-2 . A crystal structure of the chimeric mutant in complex with okadaic acid was determined to 2.0-A resolution . The beta12-beta13 loop region of the mutant superimposes closely with that of wild-type PP-1c bound to okadaic acid . Systematic mutation of each residue in the beta12-beta13 loop of PP-1c showed that a single amino acid change (C273L) was the most influential in mediating sensitivity of PP-1c to toxins . Taken together, these data indicate that it is an individual amino acid residue substitution and not a change in the overall beta12-beta13 loop conformation of protein phosphatase-1 that contributes to disrupting important interactions with inhibitors such as microcystin-LR and okadaic acid. FEBS Lett, 2004 Jul 30, 571(1-3), 192 - 6 Identification of the catalytic residues in the double-zinc aminopeptidase from Streptomyces griseus; Fundoiano-Hershcovitz Y et al.; The aminopeptidase from Streptomyces griseus (SGAP) has been cloned and expressed in Escherichia coli . By growing the cells in the presence of 1 M sorbitol at 18 degrees C, the protein was obtained in a soluble and active form . The amino acid sequence of the recombinant SGAP contained four amino acids differing from the previously published sequence . Re-sequencing of the native protein indicated that asparagines 70 and 184 are in fact aspartic acids as in the recombinant protein . Based on the crystal structure of SGAP, Glu131 and Tyr246 were proposed to be the catalytic residues . Replacements of Glu131 resulted in loss of activity of 4-5 orders of magnitude, consistent with Glu131 acting as the general base residue . Mutations in Tyr246 resulted in about 100-fold reduction of activity, suggesting that this residue is involved in the stabilization of the transition state intermediate. FEBS Lett, 2004 Jul 30, 571(1-3), 182 - 6 Stemar-13-ene synthase, a diterpene cyclase involved in the biosynthesis of the phytoalexin oryzalexin S in rice; Nemoto T et al.; In suspension-cultured rice cells, diterpenoid phytoalexins are produced in response to exogenously applied elicitors . We isolated a cDNA encoding a diterpene cyclase, OsDTC2, from suspension-cultured rice cells treated with a chitin elicitor . The OsDTC2 cDNA was overexpressed in Escherichia coli as a fusion protein with glutathione S-transferase, and the recombinant OsDTC2 was indicated to function as stemar-13-ene synthase that converted syn-copalyl diphosphate to stemar-13-ene, a putative diterpene hydrocarbon precursor of the phytoalexin oryzalexin S . The level of OsDTC2 mRNA in suspension-cultured rice cells began to increase 3 h after addition of the elicitor and reached the maximum after 8 h . The expression of OsDTC2 was also induced in UV-irradiated rice leaves . In addition, we indicated that stemar-13-ene accumulated in the chitin-elicited suspension-cultured rice cells and the UV-irradiated rice leaves. FEBS Lett, 2004 Jul 30, 571(1-3), 141 - 6 Crystal structure and reactivity of YbdL from Escherichia coli identify a methionine aminotransferase function; Dolzan M et al.; The ybdL gene of Escherichia coli codes for a protein of unknown function . Sequence analysis showed moderate homology to several vitamin B(6) dependent enzymes, suggesting that it may bind pyridoxal-5'-phosphate . The structure analysis of YbdL to 2.35 A resolution by protein crystallography verifies that it is a PLP dependent enzyme of fold type I, the typical aspartate aminotransferase fold . The active site contains a bound pyridoxal-5'-phosphate, covalently attached to the conserved active site lysine residue Lys236 . The pattern of conserved amino acids in the putative substrate binding pocket of the enzyme reveals that it is most closely related to a hyperthermophilic aromatic residue aminotransferase from the archeon Pyrococcus horikoshii . Activity tests with 10 amino acids as amino-donors reveal, however, a preference for Met, followed by His and Phe, results which can be rationalized by modelization studies. FEBS Lett, 2004 Jul 30, 571(1-3), 50 - 4 Inhibitory action of novel aromatic diamine compound on lipopolysaccharide-induced nuclear translocation of NF-kappaB without affecting IkappaB degradation; Shin HM et al.; 4-Methyl-N1-(3-phenyl-propyl)-benzene-1,2-diamine (JSH-23) is a novel chemically synthetic compound . The aromatic diamine JSH-23 compound exhibited inhibitory effect with an IC(50) value of 7.1 microM on nuclear factor (NF)-kappaB transcriptional activity in lipopolysaccharide (LPS)-stimulated macrophages RAW 264.7, and interfered LPS-induced nuclear translocation of NF-kappaB without affecting IkappaB degradation . This mechanism of action is very rare for controlling NF-kappaB activation . Furthermore, the compound inhibited not only LPS-induced expressions of tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6 and inducible nitric oxide synthase and cyclooxygenase-2 but also LPS-induced apoptosis of the RAW 264.7 cells. Mol Biochem Parasitol, 2004 Sep, 137(1), 65 - 74 Translation initiation factor eIF-5A from Plasmodium falciparum; Molitor IM et al.; Eukaryotic translation initiation factor (eIF-5A) is a highly conserved and essential protein that contains the unique amino acid hypusine . The first step in the post-translational biosynthesis of hypusine, the transfer of an aminobutyl moiety from the polyamine substrate spermidine to the -amino group of a specific lysine residue in the eIF-5A precursor, is catalyzed by the enzyme deoxyhypusine synthase . A cDNA encoding a protein homologous to eIF-5A was isolated by plaque hybridization from a cDNA library of Plasmodium falciparum . The cloned cDNA contains an open reading frame encoding a protein of 161 amino acids, which shares a high sequence identity with other eukaryotic eIF-5A sequences . A phylogenetic tree constructed with eIF-5A from P . falciparum and 16 other eIF-5A sequences of eukaryotic and archaeal origin reveals that plasmodial eIF-5A together with other apicomplexan eIF-5A show a higher degree of homology to plant proteins than to animal and fungal sequences . The plasmodial eIF-5A gene was expressed as a six-histidine tagged fusion protein in Escherichia coli . Radioactive incorporation studies with {1,8-3H} spermidine indicated that this protein can serve as a substrate for human deoxyhypusine synthase . Results of quantitative real-time PCR studies with synchronized erythrocytic stages of P . falciparum revealed no significant induction or downregulation but only some variation in the expression level of plasmodial eIF-5A in ring, trophozoite and schizont stage. Mol Biochem Parasitol, 2004 Sep, 137(1), 43 - 53 Molecular characterization of bifunctional hydroxymethyldihydropterin pyrophosphokinase-dihydropteroate synthase from Plasmodium falciparum; Kasekarn W et al.; A 2118-base pair gene encoding the bifunctional hydroxymethyldihydropterin pyrophosphokinase-dihydropteroate syntheses of Plasmodium falciparum (pfPPPK-DHPS) was expressed under the control of the T5 promoter in a DHPS-deficient Escherichia coli strain . The enzyme was purified to near homogeneity using nickel affinity chromatography followed by gel filtration and migrates as an intense band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with apparent mass of approximately 83 kDa . Gel filtration suggested that the native pfPPPK-DHPS might exist as a tetramer of identical subunits . The enzyme was found to be Mg2+ - and ATP-dependent and had optimal temperature ranging from 37 to 45 degrees C with peak activity at pH 10 . Sodium chloride and potassium chloride at 0.2 and 0.4 M, respectively, activated the activity of the enzyme but higher salt concentrations were inhibitory . Guanidine-HCl and urea inhibited the enzyme activity by 50% at 0.25 and 0.9 M, respectively . Kinetic properties of the recombinant pfPPPK-DHPS were investigated . Sulfathiazole and dapsone were potent inhibitors of pfPPPK-DHPS, whilst sulfadoxine, sulfanilamide, sulfacetamide and p-aminosalicylic acid were less inhibitory . Our construct provides an abundant source of recombinant pfPPPK-DHPS for crystallization and drug screening. J Microbiol Methods, 2004 Sep, 58(3), 313 - 20 Binding interactions of Escherichia coli with globotetraosylceramide (globoside) using a surface plasmon resonance biosensor; Pourshafie MR et al.; Surface plasmon resonance with an alkane L1 chip was used to investigate the binding of uropathogenic Escherichia coli, carrying adhesion receptors, to globotetraosylceramide (globoside; GbO4) . The immobilization of globoside was reproducible and resulted in a stable globoside layer on the L1 chip . The data indicated that the globoside-immobilized L1 chip could be used for studying interactions with live or chemically fixed E . coli . The results indicated that the dissociation time was significantly reduced in glutaraldehyde-fixed E . coli as compared to living cells . Overall, the report demonstrates the significance of the L1 chip in terms of sensitivity, specificity, handling, and speed when studying globoside/E . coli interactions . This model may assist in screening for compounds that can inhibit the binding of uropathogenic E . coli to glycolipid ligands on target cells. Viral Immunol, 2004, 17(2), 315 - 21 Detection of Puumala hantavirus antibody with ELISA using a recombinant truncated nucleocapsid protein expressed in Escherichia coli; Maes P et al.; A truncated recombinant nucleocapsid protein (rNp118) consisting of the first 118 amino-terminal amino acids (AA) of the Puumala hantavirus (PUUV) nucleocapsid protein expressed in Escherichia coli, was evaluated for its antigenicity and reliability as serodiagnostic antigen in an enzyme-linked immunosorbent assay (ELISA) for detection of PUUV antibodies in human sera . The PUUV nucleocapsid protein has been shown to contain several B-cell epitopes, mapped within the first 118 amino-terminal AA . This finding makes the rNp118 an interesting recombinant protein to use as serodiagnostic antigen . The sensitivity of this new PUUV-rNp118 ELISA, was compared with those of a commercially available PUUV ELISA assay and an home-made ELISA based on a recombinant whole nucleocapsid protein of PUUV . Eighty-six human serum samples clinically suspected for PUUV-induced nephropathia epidemica, and previously screened with the reference assays, were tested . The sensitivity of the new assay was compared with that of the reference assays and an excellent correlation between the assays was found . Sera found to be negative by other methods were also negative in our assay . The ELISA based on rNp118 represents an alternative and valid test for detection of antibodies to PUUV in human sera . Copyright Mary Ann Liebert, Inc. Environ Mol Mutagen, 2004, 44(2), 128 - 50 In vivo mutation in gene A of splenic lymphocytes from phiX174 transgenic mice; Valentine CR et al.; Single-burst analysis was applied to a forward assay for gene A mutation in splenic lymphocytes of phiX174 transgenic mice for the purpose of optimizing analytical parameters for identifying in vivo mutations . The effect of varying the cutoff value for an in vivo burst on induced mutant frequency, fold increase, and the significance of the difference between control and N-ethyl-N-nitrosourea (ENU)-treated mice was calculated by two different methods . The plating density was reduced to an average of less than 10 background mutant plaques per aliquot in order to separate in vitro bursts . The spectrum of mutations contributing < 60 plaques per aliquot from control animals was not significantly different from the control spectra from E . coli or transgenic phiX174 cells in culture . The mutant spectra from ENU-treated animals was highly different between mutant bursts of > 80 plaques per aliquot compared to mutations contributing < 60 plaques per aliquot (P < 0.000001), the former fitting the spectrum expected for ENU-induced mutations . The latter spectrum was also different from control animals and E . coli (P < 0.000001), suggesting the difference was caused by ex vivo mutation . With the mutations found in this study, the total number of reported target sites for gene A is now 33 . The results support the interpretation that, in contrast to results for the lacI transgene, 100% of mutants isolated in gene A from control animals and cells were fixed in E . coli . We attribute the difference between the two genes to hot-spot sites for mutation in gene A and to a testable hypothesis that the mosaic plaque assay for the lacI transgene underestimates the frequency of ex vivo mutants . Planta, 2004 Nov, 220(1), 129 - 39 Epub 2004 Nov. Cloning, characterization and expression of OsGLN2, a rice endo-1,3-beta-glucanase gene regulated developmentally in flowers and hormonally in germinating seeds; Akiyama T et al.; We report here the isolation and characterization of a new endo-1,3-beta-glucanase (1,3-beta-GLU) cDNA, OsGLN2, that is expressed both in flowers and in germinating seeds of rice (Oryza sativa L.) . The isolated OsGLN2 gene encoded a protein which displayed 72%, 93% and 92% identity at the amino acid level with those encoded by barley GII, rice Gns4 and glu1 1,3-beta-GLU genes, respectively . A GST-OsGLN2 recombinant protein expressed in Escherichia coli preferentially hydrolyzed Laminaria digitata 1,3;1,6-beta-glucan and liberated only oligosaccharides, suggesting that the enzyme can be classified as a 1,3-beta-GLU . Northern analysis with a 3'-UTR gene-specific probe revealed that OsGLN2 is expressed exclusively in the paleae and lemmas during flowering, and no expression of OsGLN2 was detected in other tissues such as leaf blades, leaf sheaths, stems, nodes and roots in mature rice plants . The OsGLN2 gene is also expressed in germinating seeds, where its expression is predominant in endosperms rather than embryos . In de-embryonated rice half-seeds, addition of gibberellin A3 (GA) greatly enhanced expression of the OsGLN2 gene, while the GA-induced gene expression was suppressed strongly by abscisic acid (ABA) . This is the first report, to our knowledge, that OsGLN2 encodes a 1,3-beta-GLU and is expressed specifically in paleae and lemmas during flowering and in germinating seeds, where its expression is enhanced by GA and suppressed by ABA. Arch Microbiol, 2004 Sep, 182(1), 60 - 6 Epub 2004 Jul 20. Ribosome modulation factor protects Escherichia coli during heat stress, but this may not be dependent on ribosome dimerisation; Niven GW; The role of ribosome modulation factor (RMF) in protecting heat-stressed Escherichia coli cells was identified by the observation that cultures of a mutant strain lacking functional RMF (HMY15) were highly heat sensitive in stationary phase compared to those of the parent strain (W3110) . No difference in heat sensitivity was observed between these strains in exponential phase, during which RMF is not synthesised . Studies by differential scanning calorimetry demonstrated that the ribosomes of stationary-phase cultures of the mutant strain had lower thermal stability than those of the parent strain in stationary phase, or exponential-phase ribosomes . More rapid breakdown of ribosomes in the mutant strain during heating was confirmed by rRNA analysis and sucrose density gradient centrifugation . Analyses of ribosome composition showed that the 100S dimers dissociated more rapidly during heating than 70S particles . While ribosome dimerisation is a consequence of the conformational changes caused by RMF binding, it may not therefore be essential for RMF-mediated ribosome stabilisation. Biosci Biotechnol Biochem, 2004 Jul, 68(7), 1565 - 8 Expression of a single-chain antibody against indole-3-acetic acid in Escherichia coli; Mitani N et al.; A hybridoma cell line that produces a monoclonal antibody specific for indole-3-acetic acid (IAA) was prepared . The DNA fragments coding the variable regions of the light and the heavy chains of the antibody were prepared by PCR using the cDNA of the antibody as a template . A chimera DNA for a single chain variable fragment (scFv) was constructed, and expressed in Escherichia coli . The scFv antibody expressed in E . coli as well as the original monoclonal antibody showed a specific binding to IAA. Biosci Biotechnol Biochem, 2004 Jul, 68(7), 1557 - 64 Cloning of the Ruminococcus albus cel5D and cel9A genes encoding dockerin module-containing endoglucanases and expression of cel5D in Escherichia coli; Taguchi H et al.; An EcoRI chromosomal DNA fragment of Ruminococcus albus F-40 that conferred endoglucanase activity on Escherichia coli was cloned . An open reading frame (ORF1) and another incomplete reading frame (ORF2) were found in the EcoRI fragment . The ORF2 was completed using inverse PCR genome walking technique . ORF1 and ORF2, which confront each other, encoded cellulases belonging to families 5 and 9 of the glycoside hydrolases and were designated cel5D and cel9A respectively . The cel5D gene encodes 753 amino acids with a deduced molecular weight of 83,409 . Cel5D consists of a signal peptide of 24 amino acids, a family-5 catalytic module, a dockerin module, and two family-4 carbohydrate-binding modules (CBMs) . The cel9A gene encodes 936 amino acids with a deduced molecular weight of 104,174, consisting of a signal peptide, a family-9 catalytic module, a family-3 CBM, and a dockerin module . The catalytic module polypeptide (rCel5DCat) derived from Cel5D was constructed, expressed, and purified from a recombinant E . coli . The truncated enzyme hydrolyzed cellohexaose, cellopentaose, and cellotetraose to yield mainly cellotriose and cellobiose with glucose as a minor product, but the enzyme was less active toward cellotriose and not active toward cellobiose, suggesting that this enzyme is a typical endoglucanase . rCel5DCat had a Km of 3.9 mg/ml and a Vmax of 37.2 micromol/min/mg for carboxymethycellulose. Biosci Biotechnol Biochem, 2004 Jul, 68(7), 1481 - 8 Cloning and overexpression of the Exiguobacterium sp . F42 gene encoding a new short chain dehydrogenase, which catalyzes the stereoselective reduction of ethyl 3-oxo-3-(2-thienyl)propanoate to ethyl (S)-3-hydroxy-3-(2-thienyl)propanoate; Wada M et al.; Exiguobacterium sp . F42 was screened as a producer of an enzyme catalyzing the NADPH-dependent stereoselective reduction of ethyl 3-oxo-3-(2-thienyl)propanoate (KEES) to ethyl (S)-3-hydroxy-3-(2-thienyl)propanoate ((S)-HEES) . (S)-HEES is a key intermediate for the synthesis of (S)-duloxetine, a potent inhibitor of the serotonin and norepinephrine uptake carriers . The responsible enzyme (KEES reductase) was partially purified, and the gene encoding KEES reductase was cloned and sequenced via an inverse PCR approach . Sequence analysis of the gene for KEES reductase revealed that the enzyme was a member of the short chain dehydrogenase/reductase family . The probable NADPH-interacting site and 3 catalytic residues (Ser-Tyr-Lys) were fully conserved . The gene was highly expressed in Escherichia coli, and the gene product was purified to homogeneity from the recombinant E . coli by simpler procedures than from the original host . The molecular mass of the purified enzyme was 27,500 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 55,000 as determined by gel filtration chromatography . Our results show that this enzyme can be used for the practical production of (S)-HEES. Proc Natl Acad Sci U S A, 2004 Aug 3, 101(31), 11239 - 44 Epub 2004 Jul 26. The ion channel of F-ATP synthase is the target of toxic organotin compounds; von Ballmoos C et al.; ATP is the universal energy currency of living cells, and the majority of it is synthesized by the F1F0 ATP synthase . Inhibitors of this enzyme are therefore potentially detrimental for all life forms . Tributyltin chloride (TBT-Cl) inhibits ATP hydrolysis by the Na(+)-translocating ATP synthase of Ilyobacter tartaricus or the H(+)-translocating counterpart of Escherichia coli with apparent Ki of 200 nM . To target the site of this inhibition, we synthesized a tritium-labeled derivative of TBT-Cl in which one of the butyl groups was replaced by a photoactivatable aryldiazirine residue . Upon illumination, subunit a of the ATP synthase becomes specifically modified, and this labeling is suppressed in the presence of the original inhibitor . In case of the Na+ ATP synthase, labeling is also suppressed in the presence of Na+ ions, suggesting an interference in Na+ or TBT-Cl binding to subunit a . This interference is corroborated by the protection of ATP hydrolysis from TBT-Cl inhibition by 105 mM Na+ . TBT-Cl strongly inhibits Na+ exchange by the reconstituted I . tartaricus ATP synthase . Taken together these results indicate that the subunit a ion channel is the target site for ATPase inhibition by toxic organotin compounds . An inhibitor interacting specifically with this site has not been reported previously. Proc Natl Acad Sci U S A, 2004 Aug 3, 101(31), 11471 - 6 Epub 2004 Jul 26. Cardioprotective effects of thioredoxin in myocardial ischemia and reperfusion: role of S-nitrosation {corrected}; Tao L et al.; Apoptosis contributes to myocardial ischemia/reperfusion (MI/R) injury, and both thioredoxin (Trx) and nitric oxide have been shown to exert antiapoptotic effects in vitro . Recent evidence suggests that this particular action of Trx requires S-nitrosation at Cys-69 . The present study sought to investigate whether or not exogenously applied Trx reduces MI/R injury in vivo and to which extent this effect depends on S-nitrosation . Adult mice were subjected to 30 min of MI and treated with either vehicle or human Trx (hTrx, 2 mg/kg, i.p.) 10 min before reperfusion . Native hTrx was incorporated into myocardial tissue as shown by immunostaining, and reduced MI/R injury as evidenced by decreased terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining, DNA fragmentation, caspase-3 activity, and infarct size . When hTrx was partially S-nitrosated by preincubation with S-nitrosoglutathione, its cardioprotective effect was markedly enhanced . Treatment with hTrx significantly reduced p38 mitogen-activated protein kinase (MAPK) activity, and this effect was also potentiated by S-nitrosation . To further address the role of S-nitrosation for the overall antiapoptotic effect to Trx, the action of Escherichia coli Trx (eTrx) was investigated in the same model . Whereas eTrx inhibited MI/R-induced apoptosis to a degree similar to hTrx, S-nitrosation of this protein, which lacks Cys-69, failed to further enhance its antiapoptotic action . Collectively, our results demonstrate that systemically applied Trx is taken up by the myocardium to exert potent cardioprotective effects in vivo, offering interesting therapeutic avenues . In the case of hTrx, these effects are further potentiated by S-nitrosation, but this posttranslational modification is not essential for protection. J Biol Chem, 2004 Sep 24, 279(39), 40584 - 92 Epub 2004 Jul 23. In vivo and in vitro reconstitution of atg8 conjugation essential for autophagy; Ichimura Y et al.; In an analogous manner to protein ubiquitination, The C terminus of Atg8p, a yeast protein essential for autophagy, conjugates to a head group of phosphatidylethanolamine via an amide bond . Though physiological role of this reaction is assigned to membrane organization during autophagy, its molecular details are still unknown . Here, we show that Escherichia coli cells coexpressed Atg8p, Atg7p (E1), and Atg3p (E2) allowed to form conjugate of Atg8p with endogenous PE . Further, we established an in vitro Atg8p-PE reconstitution system using purified Atg8pG116, Atg7p, Atg3p, and PE-containing liposomes, demonstrating that the Atg7p and the Atg3p are minimal catalysts for Atg8p-PE conjugate reaction . Efficiency of this lipidation reaction depends on the state of the substrate, PE (phospholipid bilayer and its lipid composition) . It is also suggested that the lipidation induces a conformational change in the N-terminal region of Atg8p . In vitro system developed here will provide a powerful system for further understanding the precise role of lipidation and interaction of two ubiquitin-like systems essential for autophagy . Invest Ophthalmol Vis Sci, 2004 Aug, 45(8), 2497 - 502 Effects of rolipram, a selective inhibitor of type 4 phosphodiesterase, on lipopolysaccharide-induced uveitis in rats; Chi ZL et al.; PURPOSE: To investigate effects of rolipram, an inhibitor of type 4 phosphodiesterase, on lipopolysaccharide (LPS)-induced uveitis in Wistar rats . METHODS: A total of 100 microg LPS was injected into the rat footpad . Rolipram (Wako Pure Chemical, Osaka, Japan) was injected intraperitoneally 30 minutes before administration of LPS . Levels of intracameral protein, cells, E-selectin, tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and nitrite were determined . E-selectin, TNF-alpha, IL-6, and inducible nitric oxide synthase (iNOS) mRNAs and immunohistochemical reactivity of nuclear factor (NF)-kappa B and TNF-alpha were also examined in the iris-ciliary body . RESULTS: After LPS injection, intracameral protein and cells increased from 18 to 30 hours later . Rolipram, however, inhibited elevation of protein and cells . After LPS injection, mRNA levels of E-selectin, TNF-alpha, and IL-6 in the iris-ciliary body increased 3 hours later, and iNOS mRNA increased 6 hours later . Elevation of mRNA levels for E-selectin, TNF-alpha, and IL-6 was inhibited by rolipram . After LPS injection, intracameral TNF-alpha and IL-6 levels increased 4 to 6 hours later, and nitrite levels increased 14 to 20 hours later . Elevation of TNF-alpha and IL-6 levels was decreased by rolipram . Rolipram did not affect iNOS mRNA and nitrite levels . Immunoreactivity of NF-kappa B was strong 1 hour after LPS injection, and was decreased by rolipram . Immunoreactivity of TNF-alpha was strong 4 hours after LPS injection and was decreased by rolipram . CONCLUSIONS: NF-kappa B translocation and expression of E-selectin, TNF-alpha, and IL-6 are involved in the pathogenesis of LPS-induced uveitis and are inhibited by rolipram . The inhibitory effect of rolipram in uveitis may be independent of iNOS synthesis. Avian Pathol, 2004, 33(2), 117 - 125 Colibacillosis in caged layer hens: characteristics of the disease and the aetiological agent; Vandekerchove D et al.; In Europe, outbreaks of acute mortality in layer flocks due to colisepticaemia have frequently been observed since the mid-1990s . The aims of this study were to describe the disease, to identify the serotypes of the avian pathogenic Escherichia coli (APEC) present in these outbreaks, and to detect the presence of F11 fimbriae and flagella in the isolates . For this purpose, 20 flocks with APEC-associated increased mortality and 20 control flocks matched for age were examined . Weekly mortality rates in the colibacillosis-affected flocks reached 1.71%, versus 0.30% in the control flocks . The maximum cumulative mortality over an entire colibacillosis outbreak reached 9.19% . The disease was often flock and hen house associated, with recurrent outbreaks within one round and in successive rounds in the same house . Disease was usually acute without clinical symptoms . Peritonitis with yolk material deposited in the peritoneal cavity and polyserositis were the main lesions at necropsy . O78 strains were isolated in 15 of the 20 colibacillosis flocks, and in only one of the control flocks . The majority of strains from the control flocks could not be serotyped by the 28 O-antisera used . In general, F11 fimbriae and flagella were present in the majority of the strains . F11 fimbriae were significantly more often found in O78 isolates than in the other serotypes, and are thus more often present in isolates from colibacillosis flocks . Strains positive for F11, and for F11 and flagella, were more frequently present in heart and liver of the colibacillosis-affected flocks . Colibacillose chez les pondeuses en cage: caractEristiques de la maladie et de l'agent Etiologique En Europe, des cas aigus avec de la mortalitE chez des troupeaux de pondeuses dus a une colisepticEmie ont souvent EtE observEs depuis la mi-quatre-vingt dix . Les buts de cette Etude ont EtE de dEcrire la maladie, d'identifier les sErotypes des Escherichia coli aviaires pathogenes (APEC) et de dEtecter la prEsence des fimbriae F11 ainsi que des flagelles des souches isolEes . Pour cela, il a EtE examinE vingt troupeaux ayant prEsentE une augmentation de la mortalitE associEe aux APEC et vingt troupeaux tEmoins du meme age . Les taux de mortalitE hebdomadaire dans les troupeaux affectEs de colibacillose ont atteint 1.71% contre 0.30% chez les tEmoins . La mortalitE cumulative maximale couvrant l'Episode de colibacillose a EtE 9.19% . La maladie a souvent EtE associEe au troupeau et au batiment avec des cas rEcurrents au sein d'une bande et dans les bandes successives dans le meme batiment . La maladie Etait gEnEralement aigue sans symptome . A l'autopsie, les principales lEsions ont EtE une pEritonite avec un dEpot de jaune d'oeuf dans la cavitE pEritonEale et une polysErosite . Des souches 078 ont EtE isolEes dans 15 des 20 troupeaux atteints de colibacillose et dans seulement un des troupeaux tEmoins . La majoritE des souches isolEes des troupeaux tEmoins n'ont pas pu etre sErotypEes avec les 28 antisErums 0 utilisEs . En gEnEral, les fimbriae F11 et des flagelles ont EtE observEs pour la majoritE des souches . Les fimbriae F11 ont EtE trouvEs significativement plus souvent chez les isolats 078 que chez les autres sErotypes et ainsi ont EtE plus souvent prEsents chez les isolats des troupeaux affectEs de colibacillose . Les souches positives pour F11, et pour F11 et les flagelles ont EtE plus frEquemment isolEes du coeur et du foie des animaux des troupeaux affectEs de colibacillose . Kolibazillose in Kafiglegehennen: Charakteristika der Erkrankung und des atiologischen Agens Seit Mitte der neunziger Jahre wird in Europa das Auftreten von plotzlichen Todesfallen in Legehennenherden aufgrund von Koliseptikamie haufiger beobachtet . Ziel dieser Studie war es, das Krankheitsbild zu beschreiben, die Serotypen der aviaren pathogenen Escherichia coli-Stamme (APEC) in diesen Ausbruchen zu bestimmen und das Vorkommen von F11-Fimbrien und Flagellen in den Isolaten nachzuweisen . Aus diesem Grund wurden zwanzig Herden mit APEC-assozierter erhohter Mortalitat und zwanzig KontÉ plus frÉquemment isolÉes du cœur et du foie des animaux des troupeaux affectÉs de colibacillose . Kolibazillose in Käfiglegehennen: Charakteristika der Erkrankung und des ätiologischen Agens Seit Mitte der neunziger Jahre wird in Europa das Auftreten von plötzlichen Todesfällen in Legehennenherden aufgrund von Koliseptikämie häufiger beobachtet . Ziel dieser Studie war es, das Krankheitsbild zu beschreiben, die Serotypen der aviären pathogenen Escherichia coli-Stämme (APEC) in diesen Ausbrüchen zu bestimmen und das Vorkommen von F11-Fimbrien und Flagellen in den Isolaten nachzuweisen . Aus diesem Grund wurden zwanzig Herden mit APEC-assozierter erhöhter Mortalität und zwanzig Kontrollherden im entsprechendem Alter untersucht . Die wöchentlichen Mortalitätsraten in den von Kolibazillose betroffenen Herden erreichte 1.71% im Gegensatz zu 0.30% in den Kontrollherden . Die maximale kumulative Mortalität über die Gesamtdauer eines Kolibazillose-Ausbruchs betrug 9.19% . Die Krankheit war oft an eine Herde oder an ein Stallgebäude gebunden mit wiederkehrenden Ausbrüchen innerhalb eines Durchgangs und in nachfolgenden Durchgängen im selben Stall . Bei der Sektion waren Peritonitis verbunden mit Dottermassen in der Peritonealhöhle und Polyserositis die hauptsächlichen Veränderungen . O78-Stämme wurden in 15 der 20 Kolibazillose-Herden und nur in einem der Kontrollherden isoliert . Die Mehrzahl der Stämme aus den Kontrollherden konnten nicht mit den 28 verwendeten O-Antiseren serotypisiert werden . Im Allgemeinen waren F11-Fimbien und Flagellen bei der Mehrzahl der Stämme vorhanden . F11-Fimbrien wurden jedoch signifikant öfter bei den O78-Isolaten nachgewiesen als bei den anderen Serotypen, d.h . sie waren bei den Isolaten aus den Kolibazillose-Herden häufiger zu finden . Stämme mit F11 sowie mit F11 und Flagellen waren häufiger in Herz und Leber von Tieren aus den Kolibazillose betroffenen Herden vorhanden . Colibacilosis en gallinas de puesta en batería: características de la enfermedad y del agente etiológico En Europa, se han venido observando brotes de mortalidad súbita debido a colisepticemia en lotes de ponedoras desde mediados de los años noventa . Los objetivos de este estudio fueron: describir la enfermedad, identificar los serotipos de Escherichia coli aviar patogena (APEC) presentes en estos brotes, y detectar la presencia de fimbrias F11 y flagelos en estos aislados . Para este propósito, se examinaron veinte lotes con un incremento de mortalidad asociada a APEC y veinte lotes control de la misma edad . El porcentaje de mortalidad semanal en los lotes afectados de colibacilosis llego al 1.71%, frente a 0.30% en los lotes control . La mortalidad acumulada máxima durante todo el brote de colibacilosis llego al 9.19% . La enfermedad se asoció frecuentemente al lote y a la granja, con brotes recurrentes en una misma puesta y en puestas sucesivas en la misma granja . La enfermedad se presentaba normalmente de forma aguda sin sintomatología clínica . Peritonitis y material de la yema del huevo presente en la cavidad peritoneal y poliserositis fueron las lesiones más importantes a la necropsia . Las cepas O78 fueron aisladas en 15 de los 20 lotes con colibacilosis, y en únicamente uno de los lotes control . La mayoría de cepas de los lotes control no pudieron ser serotipadas con el antisuero frente a 28O utilizado . En general, las fimbrias F11 y los flagelos estabn presentes en la mayoría de cepas . Las fimbrias F11 se encontraron en un número significativamente más elevado en los aislados O78 que en otros serotipos, y fueron pues más frecuentes en los aislados provenientes de los lotes con colibacilosis . Las cepas positivas a F11, y a F11 y flagelos se encontraron con más frecuencia en corazón e hígado de los lotes afectados con colibacilosis. J Mol Biol, 2004 Aug 6, 341(2), 605 - 15 Iron binding and oxidation kinetics in frataxin CyaY of Escherichia coli; Bou-Abdallah F et al.; Friedreich's ataxia is associated with a deficiency in frataxin, a conserved mitochondrial protein of unknown function . Here, we investigate the iron binding and oxidation chemistry of Escherichia coli frataxin (CyaY), a homologue of human frataxin, with the aim of better understanding the functional properties of this protein . Anaerobic isothermal titration calorimetry (ITC) demonstrates that at least two ferrous ions bind specifically but relatively weakly per CyaY monomer (K(d) approximately 4 microM) . Such weak binding is consistent with the hypothesis that the protein functions as an iron chaperone . The bound Fe(II) is oxidized slowly by O(2) . However, oxidation occurs rapidly and completely with H(2)O(2) through a non-enzymatic process with a stoichiometry of two Fe(II)/H(2)O(2), indicating complete reduction of H(2)O(2) to H(2)O . In accord with this stoichiometry, electron paramagnetic resonance (EPR) spin trapping experiments indicate that iron catalyzed production of hydroxyl radical from Fenton chemistry is greatly attenuated in the presence of CyaY . The Fe(III) produced from oxidation of Fe(II) by H(2)O(2) binds to the protein with a stoichiometry of six Fe(III)/CyaY monomer as independently measured by kinetic, UV-visible, fluorescence, iron analysis and pH-stat titrations . However, as many as 25-26 Fe(III)/monomer can bind to the protein, exhibiting UV absorption properties similar to those of hydrolyzed polynuclear Fe(III) species . Analytical ultracentrifugation measurements indicate that a tetramer is formed when Fe(II) is added anaerobically to the protein; multiple protein aggregates are formed upon oxidation of the bound Fe(II) . The observed iron oxidation and binding properties of frataxin CyaY may afford the mitochondria protection against iron-induced oxidative damage. J Mol Biol, 2004 Aug 6, 341(2), 455 - 66 Crystal structure of the reaction complex of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Thermotoga maritima refines the catalytic mechanism and indicates a new mechanism of allosteric regulation; Shumilin IA et al.; 3-Deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAHPS) catalyzes the first reaction of the aromatic biosynthetic pathway in bacteria, fungi, and plants, the condensation of phosphoenolpyruvate (PEP) and d-erythrose-4-phosphate (E4P) with the formation of DAHP . Crystals of DAHPS from Thermotoga maritima (DAHPS(Tm)) were grown in the presence of PEP and metal cofactor, Cd(2+), and then soaked with E4P at 4 degrees C where the catalytic activity of the enzyme is negligible . The crystal structure of the "frozen" reaction complex was determined at 2.2A resolution . The subunit of the DAHPS(Tm) homotetramer consists of an N-terminal ferredoxin-like (FL) domain and a (beta/alpha)(8)-barrel domain . The active site located at the C-end of the barrel contains Cd(2+), PEP, and E4P, the latter bound in a non-productive conformation . The productive conformation of E4P is suggested and a catalytic mechanism of DAHPS is proposed . The active site of DAHPS(Tm) is nearly identical to the active sites of the other two known DAHPS structures from Escherichia coli (DAHPS(Ec)) and Saccharomyces cerevisiae (DAHPS(Sc)) . However, the secondary, tertiary, and quaternary structures of DAHPS(Tm) are more similar to the functionally related enzyme, 3-deoxy-d-manno-octulosonate-8-phosphate synthase (KDOPS) from E.coli and Aquiflex aeolicus, than to DAHPS(Ec) and DAHPS(Sc) . Although DAHPS(Tm) is feedback-regulated by tyrosine and phenylalanine, it lacks the extra barrel segments that are required for feedback inhibition in DAHPS(Ec) and DAHPS(Sc) . A sequence similarity search revealed that DAHPSs of phylogenetic family Ibeta possess a FL domain like DAHPS(Tm) while those of family Ialpha have extra barrel segments similar to those of DAHPS(Ec) and DAHPS(Sc) . This indicates that the mechanism of feedback regulation in DAHPS(Tm) and other family Ibeta enzymes is different from that of family Ialpha enzymes, most likely being mediated by the FL domain. J Mol Biol, 2004 Aug 6, 341(2), 443 - 54 Stopped-flow and mutational analysis of base flipping by the Escherichia coli Dam DNA-(adenine-N6)-methyltransferase; Liebert K et al.; By stopped-flow kinetics using 2-aminopurine as a probe to detect base flipping, we show here that base flipping by the Escherichia coli Dam DNA-(adenine-N6)-methyltransferase (MTase) is a biphasic process: target base flipping is very fast (k(flip)>240 s(-1)), but binding of the flipped base into the active site pocket of the enzyme is slow (k=0.1-2 s(-1)) . Whereas base flipping occurs in the absence of S-adenosyl-l-methionine (AdoMet), binding of the target base in the active site pocket requires AdoMet . Our data suggest that the tyrosine residue in the DPPY motif conserved in the active site of DNA-(adenine-N6)-MTases stacks to the flipped target base . Substitution of the aspartic acid residue of the DPPY motif by alanine abolished base flipping, suggesting that this residue contacts and stabilizes the flipped base . The exchange of Ser188 located in a loop next to the active center by alanine led to a seven- to eightfold reduction of k(flip), which was also reduced with substrates having altered GATC recognition sites and in the absence of AdoMet . These findings provide evidence that the enzyme actively initiates base flipping by stabilizing the transition state of the process . Reduced rates of base flipping in substrates containing the target base in a non-canonical sequence demonstrate that DNA recognition by the MTase starts before base flipping . DNA recognition, cofactor binding and base flipping are correlated and efficient base flipping takes place only if the enzyme has bound to a cognate target site and AdoMet is available. J Mol Biol, 2004 Aug 6, 341(2), 405 - 17 The biochemical requirements of DNA polymerase V-mediated translesion synthesis revisited; Fujii S et al.; In addition to replicative DNA polymerases, cells contain specialized DNA polymerases involved in processes such as lesion tolerance, mutagenesis and immunoglobulin diversity . In Escherichia coli, DNA polymerase V (Pol V), encoded by the umuDC locus, is involved in translesion synthesis (TLS) and mutagenesis . Genetic studies have established that mutagenesis requires both UmuC and a proteolytic product of UmuD (UmuD') . In addition, RecA protein and the replication processivity factor, the beta-clamp, were genetically found to be essential co-factors for mutagenesis . Here, we have reconstituted Pol V-mediated bypass of three common replication-blocking lesions, namely the two major UV-induced lesions and a guanine adduct formed by a chemical carcinogen (G-AAF) under conditions that fulfil these in vivo requirements . Two co-factors are essential for efficient Pol V-mediated lesion bypass: (i) a DNA substrate onto which the beta-clamp is stably loaded; and (ii) an extended single-stranded RecA/ATP filament assembled downstream from the lesion site . For efficient bypass, Pol V needs to interact simultaneously with the beta-clamp and the 3' tip of the RecA filament . Formation of an extended RecA/ATP filament and stable loading of the beta-clamp are best achieved on long single-stranded circular DNA templates . In contrast to previously published data, the single-stranded DNA-binding protein (SSB) is not absolutely required for Pol V-mediated lesion bypass provided ATP, instead of ATPgammaS, activates the RecA filament . Further discrepancies with the existing literature are explainable by the use of either inadequate DNA substrates or a UmuC fusion protein instead of native Pol V. J Mol Biol, 2004 Aug 6, 341(2), 337 - 43 Scanning force microscopy of DNA translocation by the Type III restriction enzyme EcoP15I; Reich S et al.; Type III restriction enzymes are multifunctional heterooligomeric enzymes that cleave DNA at a fixed position downstream of a non-symmetric recognition site . For effective DNA cleavage these restriction enzymes need the presence of two unmethylated, inversely oriented recognition sites in the DNA molecule . DNA cleavage was proposed to result from ATP-dependent DNA translocation, which is expected to induce DNA loop formation, and collision of two enzyme-DNA complexes . We used scanning force microscopy to visualise the protein interaction with linear DNA molecules containing two EcoP15I recognition sites in inverse orientation . In the presence of the cofactors ATP and Mg(2+), EcoP15I molecules were shown to bind specifically to the recognition sites and to form DNA loop structures . One of the origins of the protein-clipped DNA loops was shown to be located at an EcoP15I recognition site, the other origin had an unspecific position in between the two EcoP15I recognition sites . The data demonstrate for the first time DNA translocation by the Type III restriction enzyme EcoP15I using scanning force microscopy . Moreover, our study revealed differences in the DNA-translocation processes mediated by Type I and Type III restriction enzymes. Phytochemistry, 2004 Jun, 65(12), 1777 - 84 Identification of barley CK2alpha targets by using the protein microarray technology; Kramer A et al.; We have successfully established a novel protein microarray-based kinase assay, which we applied to identify target proteins of the barley protein kinase CK2alpha . As a source of recombinant barley proteins we cloned cDNAs specific for filial tissues of developing barley seeds into an E . coli expression vector . By using robot technology, 21,500 library clones were arrayed in microtiter plates and gridded onto high-density filters . Protein expressing clones were detected using an anti-RGS-His6 antibody and rearrayed into a sublibrary of 4100 clones . All of these clones were sequenced from the 5'-end and the sequences were analysed by homology searches against protein databases . Based on these results we selected 768 clones expressing different barley proteins for protein purification . The purified proteins were robotically arrayed onto FAST slides . The generated protein microarrays were incubated with an expression library-derived barley CK2alpha in the presence of {gamma-33P}ATP, and signals were detected by X-ray film or phosphor imager . We were able to demonstrate the power of the protein microarray technology by identification of 21 potential targets out of 768 proteins including such well-known substrates of CK2alpha as high mobility group proteins and calreticulin. Gene, 2004 Aug 4, 337, 25 - 35 Identification, expression, modeled structure and serological characterization of Plasmodium vivax histone 2B; Rawat DS et al.; Histones play important role in DNA packaging, replication and gene expression . Here, we describe the isolation and characterization of histone 2B (PvH2B) gene from the most common but non-cultivable human malaria parasite Plasmodium vivax . The isolated cDNA clone of PvH2B was allowed to express in Escherichia coli and the recombinant protein was purified by affinity chromatography . The expressed PvH2B protein showed DNA-binding properties on the South-Western analysis and the confocal microscopy localized it in the parasite nucleus . This gene is actively expressed during blood stages of the parasite and all P . vivax patients produced antibodies against the protein . The mRNA of PvH2B was found to contain a poly(A) tail at its 3' end, unlike abundant mRNA of human H2B . The encoded polypeptide is 118 amino acid long contains a nuclear targeting site, a signature motif of H2B and showed 74% homology to its host molecule . The structure of PvH2B showed that it has certain differences from that of its host at critical functional sites (viz acetylation, methylation, trypsin cleavage, DNA-binding and inter-histone interaction) which are required for general gene expression and DNA packaging . The distinctive structural features of P . vivax H2B described here may help in designing the specific antimalarial drugs. Biochem Pharmacol, 2004 Aug 15, 68(4), 711 - 8 Enantioselectivity of ribonucleotide reductase: a first study using stereoisomers of pyrimidine 2'-azido-2'-deoxynucleosides; Roy B et al.; In this paper, the enantioselectivity of ribonucleotide reductase (RNR, EC 1.17.4.1), a pivotal enzyme involved in DNA biosynthesis, was studied using the beta-d and beta-l stereoisomers of 2'-azido-2'-deoxynucleosides of uracil and cytosine . The corresponding 5'-diphosphate derivatives in the d-configuration have been extensively studied as mechanism-based inhibitors of the enzyme . The original l-enantiomers were synthesized and evaluated in vitro . In cell culture experiments, only the cytosine derivative with a d-configuration was found cytostatic and able to deplete dNTP pools in response to RNR inhibition . In the case of the uracil enantiomeric pair, this result correlates with an inefficient intracellular monophosphorylation as demonstrated in testing their substrate properties against human uridine-cytidine kinase 1 . Regarding cytosine analogues, human deoxycytidine kinase was found to be able to phosphorylate both enantiomers with comparable efficiency but only the d-stereoisomer was active in human cell culture . The interaction of the beta-d and beta-l stereoisomers of 2'-azido-2'-deoxyuridine 5'-diphosphate with purified Escherichia coli RNR was also examined . Inactivation of the enzyme was only observed in the presence of the d-stereoisomer, demonstrating that RNR exhibits enantiospecificity with respect to the natural configuration of the sugar moiety, as far as 2'-azido-2'-deoxynucleotides are concerned. Arch Oral Biol, 2004 Sep, 49(9), 739 - 46 Immuno-localization of COX-1 and COX-2 in the rat molar periodontal tissue after topical application of lipopolysaccharide; Miyauchi M et al.; Up-regulation of prostaglandin E2 (PGE2) production in the periodontal tissue is considered to be important for periodontal tissue destruction . The purpose of the study was to demonstrate the dynamic changes of immuno-localization of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) in rat periodontal tissue after topical application of lipopolysaccharide (LPS: 5 mg/ml in physiological saline) from Escherichia coli into the rat molar gingival sulcus . In the normal periodontal tissue, small numbers of junctional epithelium (JE) cells and numerous osteocytes embedded in alveolar bone constitutively expressed COX-1 . The COX-1 expression was not effected by LPS application . JE cells, especially in the coronal portion of JE also expressed COX-2 . LPS application induced the JE cells with consequent transient expression of COX-2 with a peak at day 1 . These findings suggest that JE cells may play a critical role in first defense line against LPS challenge and PGE2 from JE cells may be responsible for the initiation of periodontal inflammation . In the deep periodontal tissue, cementoblasts and osteoblasts showed constitutive expression of COX-2, which may be induced by continuous cyclic tension force due to occlusal pressure . LPS application caused a transient up-regulation of COX-2 expression in periodontal ligament fibroblasts, cementoblasts and osteoblasts . It is suggested that the inducible production of PGE2 via COX-2 by these cells may be associated with connective tissue destruction and alveolar bone resorption. Comp Biochem Physiol A Mol Integr Physiol, 2004 Jun, 138(2), 147 - 60 DNA modification in chick heart and cerebrum; Kagawa K et al.; Heart muscle cells and cerebral neurons are known to lose the ability to proliferate and are called terminally differentiated cells . They are generated in appropriate numbers during embryogenesis and retained throughout adult life without turnover . We are interested in such a long-lived DNA . We isolated DNA from chick heart and cerebrum and compared it with DNA from other organs after incubation with DNase I . Single-strand breaks were assessed using a reaction system composed of DNA and Escherichia coli DNA polymerase . The DNA of both organs was relatively resistant to DNase I, and DNA modification occurred during embryogenesis . CIMS (chemical ionization mass spectrometry) indicated that the molecular mass of the deoxynucleoside of both DNAs was larger than that of the corresponding canonical deoxyribonucleoside by m/z 28 (or 30 for the protonated form) . The difference between these deoxynucleosides is based on a difference in sugar constituents . Cerebral deoxynucleotides were analyzed by (13)C NMR . An extra signal near 173 ppm was observed, which was assigned to the amide carbonyl . We propose a model of the deoxynucleoside where a carbonyl residue exists between the base and the 2-deoxyribose moiety of the canonical deoxyribonucleoside. Structure (Camb), 2004 Jun, 12(6), 1087 - 97 The crystal structure of the carboxy-terminal dimerization domain of htpG, the Escherichia coli Hsp90, reveals a potential substrate binding site; Harris SF et al.; Hsp90 is a ubiquitous, well-conserved molecular chaperone involved in the folding and stabilization of diverse proteins . Beyond its capacity for general protein folding, Hsp90 influences a wide array of cellular signaling pathways that underlie key biological and disease processes . It has been proposed that Hsp90 functions as a molecular clamp, dimerizing through its carboxy-terminal domain and utilizing ATP binding and hydrolysis to drive large conformational changes including transient dimerization of the amino-terminal and middle domains . We have determined the 2.6 A X-ray crystal structure of the carboxy-terminal domain of htpG, the Escherichia coli Hsp90 . This structure reveals a novel fold and that dimerization is dependent upon the formation of a four-helix bundle . Remarkably, proximal to the helical dimerization motif, each monomer projects a short helix into solvent . The location, flexibility, and amphipathic character of this helix suggests that it may play a role in substrate binding and hence chaperone activity. Structure (Camb), 2004 Jun, 12(6), 1057 - 65 RIalpha subunit of PKA: a cAMP-free structure reveals a hydrophobic capping mechanism for docking cAMP into site B; Wu J et al.; In eukaryotes the primary target for cAMP, a ubiquitous second messenger, is cAMP-dependent protein kinase (PKA) . Understanding how binding and release of cAMP changes the cAMP binding domains and then triggers long-range allosteric responses is an important challenge . This conformational switching requires structure solutions of cAMP binding domains in cAMP-bound and cAMP-free states . We describe for the first time a crystal structure of the cAMP binding domains of PKA type Ialpha regulatory subunit where site A is occupied by cGMP and site B is unoccupied . The structure reveals that the carboxyl terminus of domain B serves as a hydrophobic cap, locking the cyclic nucleotide via its adenine ring into the beta-barrel . In the absence of cAMP, the "cap" is released via an extension of the C-terminal helix . This simple hinge mechanism for binding and release of cAMP also provides a mechanism for allosteric communication between sites A and B. Structure (Camb), 2004 Jun, 12(6), 975 - 86 Crystal structure of the targeting endonuclease of the human LINE-1 retrotransposon; Weichenrieder O et al.; The human L1 endonuclease (L1-EN) is encoded by the non-LTR retrotransposon LINE-1 (L1) . L1 is responsible for more than 1.5 million retrotransposition events in the history of the human genome, contributing more than a quarter to human genomic DNA (L1 and Alu elements) . L1-EN is related to the well-understood human DNA repair endonuclease APE1, and its nicking specificity is a major determinant for retrotransposon integration site selection . The crystal structure of human L1 endonuclease is the first of a retrotransposon-encoded protein and a prototype for retrotransposon-encoded endonucleases involved in target-primed reverse transcription . Structure-based endonuclease alignments reveal a conserved threonine in addition to previously identified invariant residues and suggest that DNA recognition proceeds via the accommodation of an extrahelical nucleotide within a pocket of the enzyme . The present analysis will help to refine phylogenetic and functional relationships among metal-dependent phosphohydrolases and provides a basis for manipulating non-LTR retrotransposon integration site selection. Structure (Camb), 2004 Jun, 12(6), 927 - 35 Structure and mechanism of GDP-mannose glycosyl hydrolase, a Nudix enzyme that cleaves at carbon instead of phosphorus; Gabelli SB et al.; GDP-mannose glycosyl hydrolase (GDPMH) catalyzes the hydrolysis of GDP-mannose and GDP-glucose to GDP and sugar by substitution with inversion at C1 of the sugar . The enzyme has a modified Nudix motif and requires one divalent cation for activity . The 1.3 A X-ray structure of the GDPMH-Mg(2+)-GDP complex, together with kinetic, mutational, and NMR data, suggests a mechanism for the GDPMH reaction . Several residues and the divalent cation strongly promote the departure of the GDP leaving group, supporting a dissociative mechanism . Comparison of the GDPMH structure with that of a typical Nudix hydrolase suggests how sequence changes result in the switch of catalytic activity from P-O bond cleavage to C-O bond cleavage . Changes in the Nudix motif result in loss of binding of at least one Mg(2+) ion, and shortening of a loop by 6 residues shifts the catalytic base by approximately 10 A. Am J Reprod Immunol, 2004 Aug, 52(2), 164 - 73 Molecular cloning, expression of testicular transcript abundant in germ cells and immunobiological effects of the recombinant protein; Verma S et al.; PROBLEM: It has been well documented that antisperm antibodies can be causative factors for infertility . In this report we have identified a protein on human sperm referred as human sperm-associated protein (HSAP) using serum of an immunoinfertile woman; it is thus a sperm-specific protein--a candidate molecule for control of fertility . METHOD OF STUDY: An immunoinfertile woman serum showing head-head sperm agglutination and acrosomal localization, reacted with human sperm protein of apparent molecular weight of 48 kDa on Western blot . Anti-48 kDa antiserum was raised in rabbit by eluting 48 kDa protein and was used to screen the human testis cDNA expression library . A putative positive hsap cDNA clone was obtained, sequenced and subjected to tissue specificities studies by Northern blotting . The cell type-specific expression was done using in situ RNA hybridization studies . To obtain recombinant HSAP (r-HSAP), hsap cDNA was cloned in pET 22b(+) expression vector . r-HSAP was expressed as polyhistidine fusion protein in Escherichia coli and purified . Rabbits were immunized with the purified r-HSAP, which led to generation of antibodies . In order to evaluate in vitro immunocontraceptive potential, the anti-r-HSAP antibodies were characterized by agglutination assay, zona-free hamster egg penetration assay, indirect immunofluorescence (IIF) assay, and by flow cytometry analysis . RESULTS: We have cloned a human testis gene encoding a protein (HSAP) of 328 amino acids . Antibodies against the purified recombinant protein specifically recognized approximately 40 kDa r-HSAP, and a cognate 48 kDa protein band in human sperm extract in Western blot procedure . The anti-r-HSAP antibodies localized acrosomal compartment, inhibited sperm binding/attachment in zona-free hamster penetration assay and revealed surface binding with human live sperm by flow cytometry . The cDNA sequence has been submitted to EMBL and has been given the accession number Y16676 . CONCLUSION: This study has put in evidence that novel sperm-specific r-HSAP has role in sperm function and may have application in the development of a contraceptive vaccine . The availability of the recombinant protein will facilitate studies on the assessment of its potential as a contraceptive immunogen. Biochemistry, 2004 Aug 3, 43(30), 9743 - 54 Characterization of the human mitochondrial methionyl-tRNA synthetase; Spencer AC et al.; Human mitochondrial methionyl-tRNA synthetase (human mtMetRS) has been identified from the human EST database . The cDNA encodes a 593 amino acid protein with an 18 amino acid mitochondrial import signal sequence . Sequence analysis indicates that this protein contains the consensus motifs characteristic of a class I aminoacyl-tRNA synthetase but lacks the Zn(2+) binding motif and C-terminal dimerization region found in MetRSs from various organisms . The mature form of human mtMetRS has been cloned and expressed in Escherichia coli . Gel filtration experiments indicate that this protein functions as a monomer with an apparent molecular mass of 67 kDa . The kinetic parameters for activation of methionine have been determined for the purified enzyme . The K(M) and k(cat) for aminoacylation of E . coli initiator tRNA(f)(Met) are reported . The kinetics of aminoacylation of an in vitro transcript of human mitochondrial tRNA(Met) (mtRNA(Met)) have been determined . To address the effects of the modification of mtRNA on recognition of the mitochondrial tRNA by human mtMetRS, the kinetics of aminoacylation of native bovine mtRNA(Met) and of an in vitro transcript of the bovine mtRNA(Met) have also been investigated. Biochemistry, 2004 Aug 3, 43(30), 9655 - 63 Kinetic studies of Thermobifida fusca Cel9A active site mutant enzymes; Zhou W et al.; Thermobifida fusca Cel9A-90, an unusual family 9 enzyme, is a processive endoglucanase containing a catalytic domain closely linked to a family 3c cellulose binding domain (Cel9A-68) followed by a fibronectin III-like domain and a family 2 cellulose binding domain . To study its catalytic mechanism, 12 mutant genes with changes in five conserved residues of Cel9A-68 were constructed, cloned, and expressed in Escherichia coli . The purified mutant enzymes were assayed for their activities on (carboxymethyl)cellulose, phosphoric acid-swollen cellulose, bacterial microcrystalline cellulose, and 2,4-dinitrophenyl beta-D-cellobioside . They were also tested for ligand binding, enzyme processivity, and thermostability . The results clearly show that E424 functions as the catalytic acid, D55 and D58 are both required for catalytic base activity, and Y206 plays an important role in binding, catalysis, and processivity, while Y318 plays an important role in binding of crystalline cellulose substrates and is required for processivity . Several amino acids located in a loop at the end of the catalytic cleft (T245-L251) were deleted from Cel9A-68, and this enzyme showed slightly improved filter paper activity and binding to BMCC but otherwise behaved like the wild-type enzyme . The FnIII-like domain was deleted from Cel9A-90, reducing BMCC activity to 43% of the wild type. Indian J Exp Biol, 2004 Jan, 42(1), 115 - 6 Evaluation of immunomodulatory activity of Suvarnamalini vasant, a generic Ayurvedic herbomineral formulation; Sangle V et al.; Suvarnamalini vasant-a generic Ayurvedic herbomineral preparation was studied for its immunomodulatory activity by 1) evaluating it's effect on phagocytic function of polymorphonuclear white blood cells of rats and 2) it's effect in E.coli-induced peritonitis in albino mice . Pretreatment of rats with Suvarnamalini vasant improved the phagocytic function of polymorphonuclear white blood cells and also protected mice against E.coli-induced peritonitis . The results indicate the potential of Suvarnamalini vasant as an immunomodulator. Anticancer Res, 2004 May-Jun, 24(3a), 1593 - 6 Procarbacine radiolysis and experiments in vitro; Delipetar-Grudl A et al.; Steady-state radiolysis of aqueous procarbazine (PC) was studied in air-free, aerated and solutions saturated with N2O . The corresponding Gi(-PC)-values obtained at pH=7.4 were: 2.85, 5.60 and 3.45, respectively . The investigations in vitro, using E . coli (AB 1157) as a model for living systems, demonstrated that PC acts as a cytostatic in air-free as well as in aerated media . However, it shows radiation protecting ability in the presence of N2O, where OH-radicals are the predominant reactants . Similar results were observed at pH=6.2 . The experimental data contribute to a better understanding of the many-sided and frequently contradictory behavior of PC. Anticancer Res, 2004 May-Jun, 24(3a), 1393 - 9 Extracellular expression of cytosine deaminase results in increased 5-FU production for enhanced enzyme/prodrug therapy; Rehemtulla A et al.; BACKGROUND: The cytosine deaminase/5-fluorocytosine (CD/5-FC), strategy for cancer gene therapy shows considerable promise in experimental models but, because CD is a cytosolic enzyme, intracellular production of 5-fluorouracil (5-FU) causes the demise of the transduced cells before cytotoxic concentrations of' 5-FU can be achieved within the extracellular milieu . MATERIALS AND METHODS: A soluble secreted form of CD was constructed and evaluated compared to intracellular CD in vitro and in vivo . RESULTS: The secreted form of CD temporarily spared transduced cells and enhanced accumulation of extracellular 5-FU . Cytosolic CD produced rapid inhibition of thymidylate synthase and cell death before significant extracellular concentrations of 5-FU developed . Finally, tumors expressing the secreted form of CD had an improved response to 5-FC treatment compared to tumors expressing intracellular CD . CONCLUSION: Further evaluation of extracellular expression of CD for enzyme/prodrug therapy may provide improvements in this commonly studied gene therapy strategy. Biopolymers, 2004 Aug 15, 74(6), 457 - 66 Hydration of proteins: SAXS study of native and methoxy polyethyleneglycol (mPEG)-modified L-asparaginase and bovine serum albumin in mPEG solutions; Murthy NS et al.; Two mPEG-modified globular proteins {mPEG: methoxy poly(ethylene glycol)}, and their native unmodified forms, were examined by small-angle x-ray scattering to evaluate the extent of their surface hydration . The effects of free and protein-bound mPEG on the hydration shell were modeled with discrete electron density profiles . We show that an mPEG-depleted layer can account for the decrease in the measured radius of gyration R(g) from 34.1 to 31.1 A in native L-asparaginase, and from 32.4 to 31.0 A in native bovine serum albumin (BSA) in mPEG-containing solvents . For mPEG-modified proteins in mPEG-free solvents, we attribute the observed increase in the R(g) over that of the native proteins (approximately 3% in L-asparaginase, and 10% in BSA) to the presence of mPEG on the protein surface . The R(g) of the mPEG-modified proteins in mPEG solutions generally decrease with mPEG concentration . Relative to the corresponding unmodified protein, this decrease in R(g) is much larger in BSA (from 35.6 to 31.2 A) but much smaller (from 34.9 to 34.3 A) in L-asparaginase . From these studies, the thickness of the hydration layer around L-asparaginase and BSA is estimated to be approximately 15 A . Exclusion of polyols from the protein domain could be related to the presence of the hydration shell around the protein. Biopolymers, 2004 Aug 15, 74(6), 432 - 47 Protein structure preference, tRNA copy number, and mRNA stem/loop content; Luo L et al.; From statistical analyses of protein sequences for humans and Escherichia coli we found that the messenger RNA segment of m-codons (for m=2 to 6) with average high tRNA copy number (TCN) (larger than approximately 10.5 for humans or approximately 1.95 for E . coli) preferably code for the alpha helix and that with low TCN (smaller than approximately 7.5 for humans or approximately 1.7 for E . coli) preferably code for coil . Between them there is an intermediate region without correlation to structure preference . For the beta strand the preference/ avoidance tendency is not obvious . All strong preference-modes of TCN for protein secondary structures have been deduced . The mutual interaction between two factors--protein secondary structural type and codon TCN--is tested by F distribution . A phenomenological model on the relation between structure preference and translational efficiency or accuracy is proposed . It is pointed out that the structure preference of codons is related to the distribution of mRNA stem/loop content in three TCN regions. Braz J Med Biol Res, 2004 Aug, 37(8), 1103 - 9 Epub 2004 Jul 20. A high-copy T7 Escherichia coli expression vector for the production of recombinant proteins with a minimal N-terminal His-tagged fusion peptide; Ramos CR et al.; We report here the construction of a vector derived from pET3-His and pRSET plasmids for the expression and purification of recombinant proteins in Escherichia coli based on T7 phage RNA polymerase . The resulting pAE plasmid combined the advantages of both vectors: small size (pRSET), expression of a short 6XHis tag at N-terminus (pET3-His) and a high copy number of plasmid (pRSET) . The small size of the vector (2.8 kb) and the high copy number/cell (200-250 copies) facilitate the subcloning and sequencing procedures when compared to the pET system (pET3-His, 4.6 kb and 40-50 copies) and also result in high level expression of recombinant proteins (20 mg purified protein/liter of culture) . In addition, the vector pAE enables the expression of a fusion protein with a minimal amino-terminal hexa-histidine affinity tag (a tag of 9 amino acids using XhoI restriction enzyme for the 5'cloning site) as in the case of pET3-His plasmid and in contrast to proteins expressed by pRSET plasmids (a tag of 36 amino acids using BamHI restriction enzyme for the 5'cloning site) . Thus, although proteins expressed by pRSET plasmids also have a hexa-histidine tag, the fusion peptide is much longer and may represent a problem for some recombinant proteins. Ann Surg, 2004 Aug, 240(2), 313 - 20 Paradoxical effect of IL-18 therapy on the severe and mild Escherichia coli infections in burn-injured mice; Kinoshita M et al.; OBJECTIVE: To investigate the effects of IL-18 therapy on severe and mild bacterial infection after burn injury . SUMMARY BACKGROUND DATA: IL-18 therapy restores IFN-gamma production in immunosuppressive mice following burn injury and up-regulate host response to LPS and experimental bacterial peritonitis . On the other hand, the overproduction of IFN-gamma could induce an exaggerated inflammation . Therefore, in this study, we focus on the beneficial and deleterious effects of IL-18-induced IFN-gamma and investigate the behavior of IL-18 in infections . METHODS: Burn injury was induced in C57BL/6 mice and then they were i.p . injected with IL-18 (0.2 microg) on alternate days . After 1 week, severe and mild infections were made in mice by an Escherichia coli challenge (5 x 10 CFU and 1 x 10 CFU i.v., respectively) . RESULTS: IL-18 therapy decreased the mortality of burn-injured mice followed by a severe infection, whereas it unexpectedly increased the mortality of burned mice with a mild infection . The IL-18 therapy increased the number of liver mononuclear cells (MNCs), especially NK cells, and greatly up-regulated the impaired IFN-gamma production from the liver and spleen MNCs in mice with severe infection . Both the serum IFN-gamma concentrations recovered while the bacterial count in the liver decreased . In contrast, the serum IFN-gamma concentrations of the burned mice with mild infection did not decrease in comparison to the unburned mice, whereas IL-18 therapy greatly up-regulated the serum IFN-gamma levels in burned mice . However, IL-18 therapy significantly elevated the serum ALT and creatinine levels, thus suggesting that the mortality was induced by an exaggerated form of shock/multiorgan failure . These beneficial and deleterious effects of IL-18 therapy in mice with severe and mild infections, respectively, were all inhibited by anti-IFN-gamma Ab pretreatment . CONCLUSION: IL-18 therapy can be a potent therapeutic tool against severe bacterial infection in immunocompromised hosts, but careful attention should also be paid to its adverse effects. Nucleic Acids Res . 2004 Jul 25;32(13):e110. Detection of protein-DNA interaction with a DNA probe: distinction between single-strand and