J Biol Chem, 2004 Aug 6, 279(32), 33290 - 7 Epub 2004 Jun 10. Evidence for multiple pathways in the assembly of the Escherichia coli maltose transport complex; Kennedy KA et al.; We used the maltose transport complex MalFGK2 of the Escherichia coli cytoplasmic membrane as a model for the study of the assembly of hetero-oligomeric membrane protein complexes . Analysis of other membrane protein complexes has led to a general model in which a unique, ordered pathway is followed from subunit monomers to a final oligomeric structure . In contrast, the studies reported here point to a fundamentally different mode for assembly of this transporter . Using co-immunoprecipitation and quantification of interacting partners, we found that all subunits of the maltose transport complex efficiently form heteromeric complexes in vivo . The pairwise complexes were stable over time, suggesting that they all represent assembly intermediates for the final MalFGK2 transporter . These results indicate that several paths can lead to assembly of this oligomer . We also characterized MalF and MalG mutants that caused reduced association between some or all of the subunits of the complex with this assay . The mutant analysis highlights some important motifs for subunit contacts and suggests that the promiscuous interactions between these Mal proteins contribute to the efficiency of complex assembly . The behaviors of the wild type and mutant proteins in the co-immunoprecipitations support a model of multiple assembly pathways for this complex. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2004 Mar, 20(2), 243 - 7 {Construction of human phage-displayed scFv library and selection of the ScFv against rabies virus}; Zhao XL et al.; AIM: To construct a human phage displayed single chain Fv antibody (scFv) library and screen specific scFv against rabies virus . METHODS: Using a phage-display technique, IgVH and IgVL genes were cloned from peripheral blood lymphocytes from three donors immunized with the WISTAR PM strain vaccine . Genes encoding scFv fragments were constructed by random linkage of VH gene and VL gene by SOE PCR, and then the constructed scFv genes were cloned into phagemid vector and transfected into E . coli TG1 . The recombinant phages were screened by four rounds of panning with rabies virus vaccine . The positive recombinant phages were sequenced and antigen-binding activity of recombinant scFv was detected by competition ELISA . RESULTS: A human phage-displayed scFv library with about 7 x 10(8) sink size was constructed . A new human scFv was selected, which bound specifically to rabies virus and had higher affinity . CONCLUSION: The results lay a solid foundation for preparation of human engineering antibody to rabies virus reported herein with higher affinity. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2004 Mar, 20(2), 195 - 8 {Cloning and expression of human IL-1R II gene}; Hu YQ et al.; AIM: To clone human IL-1R II cDNA and construct its recombinant retrovirus vector so as to explore its role in IL-1R II related diseases . METHODS: Human IL-1R II cDNA was amplified by RT-PCR from peripheral blood mononuclear cells (PBMCs) and inserted into the vector PET22b to construct recombinant vector PET22b-IL-R II . The recombinant was transfected into E . coli BL21 and expressed under IPTG induction . Expressed products were detected by Western blot . In addition, human IL-1R-II cDNA was subcloned into retrovirus vector LZRSPBMN and transfected into 293 cells by calcium phosphate precipitation . IL-1R II expression was detected by immunohistochemical staining . RESULTS: IL-1R II cDNA with 1,203 bp was amplified by RT-PCR from human PBMCs . The recombinant of this cDNA could be expressed in E . coli,which was confirmed by Western blot results . Immunohistochemistry detection showed IL-1R II protein was expressed in 293 cells . CONCLUSION: Human IL-1R II gene was cloned successfully . PET22b-IL-1R II and LZR-IL-1R II were constructed and the recombinant protein IL-1R II was expressed in E.coli BL21 . The results reported herein lay the foundation for further research on the role of IL-1R II in certain diseases. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2004 Mar, 20(2), 183 - 5 {Preparation and characterization of anti-human IL-15 monoclonal antibody}; Wang P et al.; AIM: To prepare monoclonal antibody (mAb) against human interleukin-15 (hIL-15) and identify its characterization . METHODS: The GST-IL-15 was extracted from the gene-engineering bacteria E . coli and identified by SDS-PAGE . The gel strip containing GST-IL-15 was cut off to immunize BALB/c mice . The splenocytes of immunized mice were fused with Sp2/0 myeloma cells by a routine method and the hybridomas were selected in HAT medium . The hybridoma cells secreting specific antibody were detected by ELISA and cloned by limiting dilution . The stability of the obtained hybridoma cells and the specificity of anti-hIL-15 mAb the hybridoma cells secreted were identified . In addition, the New Zealand rabbits were immunized with the rhIL-15 inclusion body protein (rhIL-15IBP) to prepare the polyclonal antibody (pAb) against hIL-15 . A sandwich ELISA was established with the anti-IL-15 mAb and pAb as coating and sandwich antibodies, respectively, to detect hIL-15 . RESULTS: One hybridoma cell line which could stably secrete specific mAb was obtained . A sandwich ELISA for detecting rhIL-15 protein was established and its sensitivity was as low as 10 microg/L . CONCLUSION: The anti-hIL-15 mAb was prepared successfully . A sandwich ELISA for the detection of hIL-15 was established. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2004 Mar, 20(2), 174 - 7 {Preparation and characterization of rabbit antibody against gastric cancer-related protein GCRG213}; Wu YQ et al.; AIM: To prepare the rabbit antibody against gastric cancer-related protein GCRG213 . METHODS: The thioredoxin/GCRG213 fusion protein was expressed in E . coli . The rabbit antibody against GCRG213 was obtained by immunizing a rabbit with the purified GCRG213 protein . The titer and specificity of the antibody was determined by ELISA and Western-blot, respectively . RESULTS: The thioredoxin/GCRG213 fusion protein with relative molecular mass (Mr) of 29,400 was overexpressed in E . coli . The purity of expressed product directly purified from a denaturing polyacrylamide gel was about 100% . The rabbit antibody against GCRG213 was obtained . The ELISA titer of antiserum against GCRG213 was about 1:256,000 . Western blot analysis showed that the antiserum could bind to the expressed fusion protein specifically . CONCLUSION: The rabbit antibody against GCRG213 has been successfully prepared, which lays the foundation for further studying the biological function and the possible role of the GCRG213 in the development of gastric carcinoma. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2004 Mar, 20(2), 171 - 3 {The expression of truncated YggG protein and preparation of its polyclonal antibody}; Huang Y et al.; AIM: To express truncated YggG protein (TYP) in Escherichia coli and to prepare anti-TYPE antibody . METHODS: Truncated yggg gene was amplified by PCR from the plasmid containing full length yggg gene and was cloned into the expression vector pDH2.The expression of TYP was achieved by thermal induction . Antiserum was prepared by immunizing rabbit with TYP,The titer and specificity of polyclonal antibody were detected by Western blot . RESULTS: Thin-layer scan analysis showed that TYP accounted for about 37.1% of total bacterial protein.The antiserum was about 1:2,000 in titier and highly specific . Full-length rggG protein could also be recognized by the antiserum . CONCLUSION: The TYP was highly expressed, and specific anti-TYP antiserum was prepared successfully. Crit Care Med, 2004 May, 32(5), 1136 - 40 Effect of interleukin-6 blockade on tissue factor-induced coagulation in human endotoxemia; Derhaschnig U et al.; OBJECTIVE: Clinical trials show that interleukin (IL)-6 represents a predictive marker in human sepsis . Furthermore, IL-6 has been proposed as a candidate mediator for endotoxin (lipopolysaccharide)-induced coagulation activation: In a primate model, an (alphaIL-6 antibody (alphaIL-6 Ab) almost abolished lipopolysaccharide-induced coagulation activation . Therefore, we wished to determine if an alphaIL-6 Ab (B-E8) may also attenuate lipopolysaccharide-induced activation of coagulation in humans . DESIGN: The study was a randomized, double blind, placebo-controlled parallel group trial (n = 12 per group) . SETTING: University medical center . PATIENTS: Healthy volunteers . INTERVENTIONS: Healthy volunteers were randomized to receive either 80 mg of a monoclonal anti-IL-6 Ab (B-E8) or placebo intravenously before bolus infusion of 2 ng/kg lipopolysaccharide . MEASUREMENTS AND MAIN RESULTS: B-E8 effectively decreased IL-6 bioactivity as measured by a Bg-bioassay in vitro and concentrations of C reactive protein . However, B-E8 did not decrease lipopolysaccharide-induced tissue factor-messenger RNA transcription or plasma concentrations of downstream coagulation variables (prothrombin fragment 1 + 2, thrombin-antithrombin III complexes, and D-dimer concentrations) . Similarly, tumor necrosis factor-alpha concentrations, fibrinolytic activity (plasmin-antiplasmin complexes), endothelial activation (soluble E-selectin), and IL-10 were unaffected . CONCLUSION: IL-6 does not appear to mediate early-phase lipopolysaccharide-induced coagulation activation in humans. Free Radic Res, 2004 Apr, 38(4), 413 - 20 Localization of alpha-tocopherol transfer protein in trophoblast, fetal capillaries' endothelium and amnion epithelium of human term placenta; Muller-Schmehl K et al.; Vitamin E has been linked to fertility since its discovery in 1922 . However, the exact mechanism by which alpha-tocopherol allows pregnancy to continue until term has remained puzzling over the last 80 years . Alpha-tocopherol transfer protein (TTPA) is expressed in liver and in Purkinje cells of the cerebellum . TTPA is suggested to be responsible for the transfer of alpha-tocopherol across barrier membranes . Ttpa-knockout mice are infertile and show symptoms similar to those observed in severe vitamin E deficiency . We thus investigated TTPA expression in human placenta and whether clues from its localization in different parts of the placenta might be of functional significance . TTPA-mRNA transcripts were quantified with a fluorescent 5'-nuclease assay (TaqMan) in five different tissues . Placental expression ranged second behind that of liver . Immunohistochemistry identified TTPA in the cytosol but also in nuclei of the trophoblast and in the endothelium of the fetal capillaries . Expression in trophoblast and fetal capillaries' endothelium indicates a role of TTPA in the stereoselective transport of RRR-alpha-tocopherol from the maternal to the fetal plasma . In amnion epithelial cells, however, TTPA was predominantly located in the nuclei . Nuclear localization of the protein may represent a novel function of TTPA. Nucleic Acids Res, 2004 Jun 09, 32(10), 3169 - 79 Print 2004. Altered DNA binding specificity of Arnt by selection of partner bHLH-PAS proteins; Kinoshita K et al.; The Ah receptor (AhR) and HLF are transcription factors involved in xenobiotic metabolism and hypoxic response, respectively . AhR and HLF heterodimerize with Arnt as the common partner, and bind to asymmetric E-boxes termed XRE and HRE, respectively . In order to investigate nucleotide preference of the heterodimers, reporter plasmids with oligonucleotides for XREs or HREs with systematic mutations were constructed and their activity was determined . Comparison of the activity revealed that DNA length and nucleotide preference recognized by Arnt subunit in the two heterodimers were largely different between XRE and HRE . We expressed AhR-Arnt and HLF-Arnt in Escherichia coli and used them for DNA binding . The dissociation constant of HLF-Arnt-HRE was 10.4 +/- 1.6 nM . Competition activity of mutated XREs or HREs with wild type was consistent with their transcription activity . Bending of XRE and HRE induced by binding of the relevant heterodimers was observed with stronger bending of XRE than of HRE . By deletional and mutational analyses, an alanine and three arginine (Ala 8, Arg 9, Arg 11 and Arg 12) residues in the basic sequence of HLF were found to be indispensable for the transcriptional activity. Biophys J, 2004 Jun, 86(6), 3905 - 13 Competing hydrophobic and screened-coulomb interactions in hepatitis B virus capsid assembly; Kegel WK et al.; Recent experiments show that, in the range from approximately 15 to 45 degrees C, an increase in the temperature promotes the spontaneous assembly into capsids of the Escherichia coli-expressed coat proteins of hepatitis B virus . Within that temperature interval, an increase in ionic strength up to five times that of standard physiological conditions also acts to promote capsid assembly . To explain both observations we propose an interaction of mean force between the protein subunits that is the sum of an attractive hydrophobic interaction, driving the self-assembly, and a repulsive electrostatic interaction, opposing the self-assembly . We find that the binding strength of the capsid subunits increases with temperature virtually independently of the ionic strength, and that, at fixed temperature, the binding strength increases with the square root of ionic strength . Both predictions are in quantitative agreement with experiment . We point out the similarities of capsid assembly in general and the micellization of surfactants . Finally we make plausible that electrostatic repulsion between the native core subunits of a large class of virus suppresses the formation in vivo of empty virus capsids, that is, without the presence of the charge-neutralizing nucleic acid. Biophys J, 2004 Jun, 86(6), 3882 - 92 Unusual heme iron-lipid acyl chain coordination in Escherichia coli flavohemoglobin; D'Angelo P et al.; Escherichia coli flavohemoglobin is endowed with the notable property of binding specifically unsaturated and/or cyclopropanated fatty acids both as free acids or incorporated into a phospholipid molecule . Unsaturated or cyclopropanated fatty acid binding to the ferric heme results in a spectral change observed in the visible absorption, resonance Raman, extended x-ray absorption fine spectroscopy (EXAFS), and x-ray absorption near edge spectroscopy (XANES) spectra . Resonance Raman spectra, measured on the flavohemoglobin heme domain, demonstrate that the lipid (linoleic acid or total lipid extracts)-induced spectral signals correspond to a transition from a five-coordinated (typical of the ligand-free protein) to a hexacoordinated, high spin heme iron . EXAFS and XANES measurements have been carried out both on the lipid-free and on the lipid-bound protein to assign the nature of ligand in the sixth coordination position of the ferric heme iron . EXAFS data analysis is consistent with the presence of a couple of atoms in the sixth coordination position at 2.7 A in the lipid-bound derivative (bonding interaction), whereas a contribution at 3.54 A (nonbonding interaction) can be singled out in the lipid-free protein . This last contribution is assigned to the CD1 carbon atoms of the distal LeuE11, in full agreement with crystallographic data on the lipid-free protein at 1.6 A resolution obtained in the present work . Thus, the contributions at 2.7 A distance from the heme iron are assigned to a couple of carbon atoms of the lipid acyl chain, possibly corresponding to the unsaturated carbons of the linoleic acid. Biophys J, 2004 Jun, 86(6), 3335 - 48 A structural model of EmrE, a multi-drug transporter from Escherichia coli; Gottschalk KE et al.; Using a recently reported computational method, we describe an approach to model the structure of EmrE, a proton coupled multi-drug transporter of Escherichia coli . EmrE is the smallest ion-coupled transporter known; it functions as an oligomer and each monomer comprises four transmembrane segments . Because of its size, EmrE provides a unique experimental paradigm . The computational method does not afford a unique solution for the monomer . The experimental constraints available were used to select the most likely structure and to dock two monomers together to yield a dimer . The model is further validated by modeling of Hsmr, an EmrE homolog with a remarkable amino acid composition with over 40% of Ala and Val . The Hsmr model is similar to that of EmrE, with the majority of the Ala or Val residues facing the lipid . In addition, the model of EmrE features a putative substrate-binding site very similar to that observed in BmrR, a transcription activator of multi-drug transporters, with a similar substrate profile . The two crucial residues that couple proton fluxes with substrate binding in the homo-dimer of EmrE, Glu-14, have a spatial arrangement that agrees with proposed molecular mechanisms of transport. BMC Immunol . 2004 Jun 09;5(1):10. NF-kappaB p50 facilitates neutrophil accumulation during LPS-induced pulmonary inflammation; Mizgerd JP et al.; BACKGROUND: Transcription factors have distinct functions in regulating immune responses . During Escherichia coli pneumonia, deficiency of NF-kappaB p50 increases gene expression and neutrophil recruitment, suggesting that p50 normally limits these innate immune responses . p50-deficient mice were used to determine how p50 regulates responses to a simpler, non-viable bacterial stimulus in the lungs, E . coli lipopolysaccharide (LPS) . RESULTS: In contrast to previous results with living E . coli, neutrophil accumulation elicited by E . coli LPS in the lungs was decreased by p50 deficiency, to approximately 30% of wild type levels . Heat-killed E . coli induced neutrophil accumulation which was not decreased by p50 deficiency, demonstrating that bacterial growth and metabolism were not responsible for the different responses to bacteria and LPS . p50 deficiency increased the LPS-induced expression of kappaB-regulated genes essential to neutrophil recruitment, including KC, MIP-2, ICAM-1, and TNF-alpha suggesting that p50 normally limited this gene expression and that decreased neutrophil recruitment did not result from insufficient expression of these genes . Neutrophils were responsive to the chemokine KC in the peripheral blood of p50-deficient mice with or without LPS-induced pulmonary inflammation . Interleukin-6 (IL-6), previously demonstrated to decrease LPS-induced neutrophil recruitment in the lungs, was increased by p50 deficiency, but LPS-induced neutrophil recruitment was decreased by p50 deficiency even in IL-6 deficient mice . CONCLUSION: p50 makes essential contributions to neutrophil accumulation elicited by LPS in the lungs . This p50-dependent pathway for neutrophil accumulation can be overcome by bacterial products other than LPS and does not require IL-6. Genes Cells, 2004 Jun, 9(6), 509 - 22 Molecular mechanism of DNA replication-coupled inactivation of the initiator protein in Escherichia coli: interaction of DnaA with the sliding clamp-loaded DNA and the sliding clamp-Hda complex; Su'etsugu M et al.; In Escherichia coli, the ATP-DnaA protein initiates chromosomal replication . After the DNA polymerase III holoenzyme is loaded on to DNA, DnaA-bound ATP is hydrolysed in a manner depending on Hda protein and the DNA-loaded form of the DNA polymerase III sliding clamp subunit, which yields ADP-DnaA, an inactivated form for initiation . This regulatory DnaA-inactivation represses extra initiation events . In this study, in vitro replication intermediates and structured DNA mimicking replicational intermediates were first used to identify structural prerequisites in the process of DnaA-ATP hydrolysis . Unlike duplex DNA loaded with sliding clamps, primer RNA-DNA heteroduplexes loaded with clamps were not associated with DnaA-ATP hydrolysis, and duplex DNA provided in trans did not rescue this defect . At least 40-bp duplex DNA is competent for the DnaA-ATP hydrolysis when a single clamp was loaded . The DnaA-ATP hydrolysis was inhibited when ATP-DnaA was tightly bound to a DnaA box-bearing oligonucleotide . These results imply that the DnaA-ATP hydrolysis involves the direct interaction of ATP-DnaA with duplex DNA flanking the sliding clamp . Furthermore, Hda protein formed a stable complex with the sliding clamp . Based on these, we suggest a mechanical basis in the DnaA-inactivation that ATP-DnaA interacts with the Hda-clamp complex with the aid of DNA binding . Copyright Blackwell Publishing Limited Annu Rev Biochem, 2004, 73, 539 - 57 Structural insights into the signal recognition particle; Doudna JA et al.; The signal recognition particle (SRP) directs integral membrane and secretory proteins to the cellular protein translocation machinery during translation . The SRP is an evolutionarily conserved RNA-protein complex whose activities are regulated by GTP hydrolysis . Recent structural investigations of SRP functional domains and interactions provide new insights into the mechanisms of SRP activity in all cells, leading toward a comprehensive understanding of protein trafficking by this elegant pathway. Chaos, 2004 Jun, 14(2), 279 - 92 Dynamics and bistability in a reduced model of the lac operon; Yildirim N et al.; It is known that the lac operon regulatory pathway is capable of showing bistable behavior . This is an important complex feature, arising from the nonlinearity of the involved mechanisms, which is essential to understand the dynamic behavior of this molecular regulatory system . To find which of the mechanisms involved in the regulation of the lac operon is the origin of bistability, we take a previously published model which accounts for the dynamics of mRNA, lactose, allolactose, permease and beta-galactosidase involvement and simplify it by ignoring permease dynamics (assuming a constant permease concentration) . To test the behavior of the reduced model, three existing sets of data on beta-galactosidase levels as a function of time are simulated and we obtain a reasonable agreement between the data and the model predictions . The steady states of the reduced model were numerically and analytically analyzed and it was shown that it may indeed display bistability, depending on the extracellular lactose concentration and growth rate . (c) 2004 American Institute of Physics Reprod Nutr Dev, 2004 Jan-Feb, 44(1), 37 - 48 Effect of intrauterine infusion of Escherichia coli on hormonal patterns in gilts during the oestrous cycle; Jana B et al.; The aim of this study was to determine the effect of intrauterine Escherichia coli infusion on the patterns of plasma LH, prolactin, progesterone, androstenedione, testosterone, oestrone, oestradiol-17beta, cortisol and 13,14-dihydro-15-keto-prostaglandin F2alpha (PGFM) in gilts during the oestrous cycle . On day 4 of the oestrous cycle (day 0), 25 mL of saline or 25 mL of Escherichia coli suspension, containing 10(7) colony forming units x mL(-1), was infused once into the each uterine horn in group I or II respectively . The control gilts developed a new oestrous cycle at the expected time but not bacteria-treated . Endometritis and vaginal discharge developed in all gilts after Escherichia coli infusion . The administration of Escherichia coli resulted in a reduction of plasma levels of LH, prolactin, oestrone and oestradiol-17beta (P < 0.05-0.001), mainly on days 15-18 after treatment (expected perioestrous period) . During this time, the plasma androstenedione level was elevated (P < 0.05-0.001) after bacteria infusion . In the gilts receiving bacteria, progesterone concentration decreased from day 8 after treatment and was low until the end of the study (P < 0.05-0.001) . On days 8-12 after bacteria administration, the level of PGFM was higher (P < 0.001) than that found in the control group . These results suggest that the developing inflammatory process of the endometrium in gilts following Escherichia coli infusion significantly affects the pituitary-ovarian axis function as well as prostaglandin production leading to anoestrus. J Vet Intern Med, 2004 May-Jun, 18(3), 295 - 300 Hepatic abscesses in cats: 14 cases (1985-2002); Sergeeff JS et al.; In this retrospective study, we describe 14 cats diagnosed with hepatic abscesses . The objective of the study was to report the clinical signs, physical examination findings, clinicopathologic findings, and outcomes in affected cats . These findings were then compared with those previously reported in dogs and humans . Clinical signs were vague and included anorexia, lethargy, and weight loss . Only 23% of cats had fever, whereas 31% were hypothermic . Increases in serum activities of alanine aminotransferase and alkaline phosphatase were found in 45 and 18%, respectively, of the 11 cats that had laboratory work performed . Abdominal ultrasound examinations were performed in 7 cats, and abnormalities were found in 71% of them . Four cats had solitary abscesses, all of which were located in the right liver lobes . The other 10 cats had multifocal small abscesses or microabscesses, and all of these cats had clinical signs suggestive of sepsis . Cytologic evaluation of samples obtained by abdominocentesis indicated septic inflammation in 67% of cats in which peritoneal fluid was analyzed . Hepatic abscess cultures yielded polymicrobial growth in 66% of the cats: Escherichia coli was the most commonly cultured organism . Overall mortality rate was 79% . All survivors underwent exploratory laparotomy for partial hepatectomy to resect the abscess followed by medical management . Hepatic abscesses should be considered in cats with signs consistent with sepsis . More routine use of ultrasonography may aid in earlier diagnosis of hepatic abscesses, potentially improving prognosis and outcome. World J Gastroenterol, 2004 Jun 15, 10(12), 1755 - 8 Effects of lactose as an inducer on expression of Helicobacter pylori rUreB and rHpaA, and Escherichia coli rLTKA63 and rLTB; Yan J et al.; AIM: To demonstrate the effect of lactose as an inducer on expression of the recombinant proteins encoded by Helicobacter pylori ureB and hpaA, and Escherichia coli LTB and LTKA63 genes and to determine the optimal expression parameters . METHODS: By using SDS-PAGE and BIO-RAD gel image analysis system, the outputs of the target recombinant proteins expressed by pET32a-ureB-E.coliBL21, pET32a-hpaA-E.coliBL21, pET32a-LTKA63-E.coliBL21 and pET32a-LTB-E.coliBL21 were measured when using lactose as inducer at different dosages, original bacterial concentrations, various inducing temperatures and times . The results of the target protein expression induced by lactose were compared to those by isopropyl-beta-D-thiogalactoside (IPTG) . The proteins were expressed in E.coli . RESULTS: Lactose showed higher efficiency of inducing the expression of rHpaA, rUreB, rLTB and rLTKA63 than IPTG . The expression outputs of the target recombinant proteins induced at 37 degrees were remarkably higher than those at 28 degrees . Other optimal expression parameters for the original bacterial concentrations, dosages of lactose and inducing time were 0.8, 50 g/L and 4 h for rHpaA; 0.8, 100 g/L and 4 h for rLTKA63; 1.2, 100 g/L and 5 h for both rUreB and rLTB, respectively . CONCLUSION: Lactose, a sugar with non-toxicity and low cost, is able to induce the recombinant genes to express the target proteins with higher efficiency than IPTG . The results in this study establish a beneficial foundation for industrial production of H pylori genetic engineering vaccine. World J Gastroenterol, 2004 Jun 15, 10(12), 1746 - 9 Transactivating effect of hepatitis C virus core protein: a suppression subtractive hybridization study; Liu M et al.; AIM: To investigate the transactivating effect of hepatitis C virus (HCV) core protein and to screen genes transactivated by HCV core protein . METHODS: pcDNA3.1(-)-core containing full-length HCV core gene was constructed by insertion of HCV core gene into EcoRI/BamHI site . HepG2 cells were cotransfected with pcDNA3.1(-)-core and pSV-lacZ . After 48 h, cells were collected and detected for the expression of beta-gal by an enzyme-linked immunosorbent assay (ELISA) kit . HepG2 cells were transiently transfected with pcDNA3.1(-)-core using Lipofectamine reagent . Cells were collected and total mRNA was isolated . A subtracted cDNA library was generated and constructed into a pGEM-Teasy vector . The library was amplified with E . coli strain JM109 . The cDNAs were sequenced and analyzed in GenBank with BLAST search after polymerase chain reaction (PCR) . RESULTS: The core mRNA and protein could be detected in HepG2 cell lysate which was transfected by the pcDNA3.1(-)-core . The activity of beta-galactosidase in HepG2 cells transfected by the pcDNA3.1(-)-core was 5.4 times higher than that of HepG2 cells transfected by control plasmid . The subtractive library of genes transactivated by HCV core protein was constructed successfully . The amplified library contained 233 positive clones . Colony PCR showed that 213 clones contained 100-1 000 bp inserts . Sequence analysis was performed in 63 clones . Six of the sequences were unknown genes . The full length sequences were obtained with bioinformatics method, accepted by GenBank . It was suggested that six novel cDNA sequences might be target genes transactivated by HCV core protein . CONCLUSION: The core protein of HCV has transactivating effects on SV40 early promoter/enhancer . A total of 63 clones from cDNA library were randomly chosen and sequenced . Using the BLAST program at the National Center for Biotechnology Information, six of the sequences were unknown genes . The other 57 sequences were highly similar to known genes. Arch Microbiol, 2004 Jun, 181(6), 418 - 27 Epub 2004 May 12. Use of Tn KPK2 for sequencing a 10.6-kb PstI DNA fragment of Bradyrhizobium japonicum and for the construction of aspA and ndvA mutants; Wiedemann G et al.; Transposon Tn KPK2 was used to saturate a randomly cloned Bradyrhizobium japonicum PstI fragment and the insertions were used as starting points for the sequence determination . The first gene of the 10.6-kb DNA insert encodes a homologue to ndvA, the product of which is known to be involved in the formation of periplasmic cyclic glucans . Selected Tn KPK2 insertions were introduced into the B . japonicum wild-type strain . The resulting mutants were subsequently tested for their symbiotic interactions with soybeans . As in Sinorhizobium meliloti, a B . japonicum ndvA mutant was affected in salt-stress tolerance and exhibited symbiotic defects in that it induced the formation of ineffective soybean nodules . The central nodule tissue was infected by bacteroids, but within the infected cells the mutant was not properly maintained . Another gene was found to be highly similar to bacterial aspartases and thus was named aspA . The putative function of the product of this gene was confirmed by genetic complementation of aspartase-less Escherichia coli strain TK237 . The symbiotic phenotype of a B . japonicum aspA:Tn KPK2 mutant consisted of enlarged symbiosomes that made the system ineffective . In general, Tn KPK2 is a suitable means for fast sequencing . In combination with pJQ200SK, the resulting recombinant plasmids can be directly used to create genetically defined mutants. Acta Biochim Biophys Sin (Shanghai), 2004 Jun, 36(6), 437 - 42 Fusion protein of interleukin 4 and diphtherial toxin with high cytotoxicity to cancer cells; Zhang YJ et al.; Receptor of human interleukin 4 (hIL4R) has been found to be present on many types of cancer, so it may be a good target for cancer therapy . Here, fusion toxin gene DT4H has been constructed by fusing DNA sequence encoding the first 389 amino acids of diphtherial toxin (DT), which can not bind its own receptor, to human interleukin 4 (hIL4) gene . In order to improve the affinity of fusion toxin for hIL4R, a circularly permuted form of hIL4 (cpIL4) was used . The fusion gene was expressed in Escherichia coli where the fusion toxin DT4H was highly expressed . Purified DT4H was very cytotoxic to cancer cell line U251 cells, and moderate cytotoxic to HepG2 and MCF-7 cells . SGC-7901 cells were insensitive to it . The cytotoxic action of DT4H was specific because it was blocked by excess hIL4 . These results suggest that DT4H may be a useful agent in the treatment of certain malignancies. Acta Biochim Biophys Sin (Shanghai), 2004 Jun, 36(6), 425 - 9 Cloning and characterization of Adinbitor, a novel disintegrin from the snake venom of Agkistrodon halys brevicaudus stejneger; Wang JH et al.; Adinbitor was cloned from Agkistrodon halys brevicaudus stejneger and characterized as a novel disintegrin . In this study, total RNA was extracted from venom gland and used in RT-PCR to generate a cDNA which is 219 bp long . The sequence encoded a polypeptide composed of 73 amino acids, including 12 cysteines, an RGD motif, and the signature motif of disintergrin . Recombinant Adinbitor (rAdinbitor) was expressed in E . coli and purified by using the His.Bind affinity chromatography . The IC(50) for inhibiting human platelet aggregation and bFGF-induced proliferation of ECV304 cells was 6 microM and 0.89 microM respectively . Furthermore, Adinbitor significantly inhibited angiogenesis both in vivo and in vitro . Taken together, these results suggested that Adinbitor had typical functions of disintegrins. Acta Biochim Biophys Sin (Shanghai), 2004 Jun, 36(6), 397 - 404 Expression of overlapping PreS1 fragment recombinant proteins for the determination of immunogenic domains in HBsAg PreS1 region; Hu WG et al.; In this study, eight preS1 fragments overlapped in preS1 (21-119) region of HBV adr subtype, i.e . preS1 (21-47), preS1 (34-59), preS1 (48-70), preS1 (60-85), preS1 (71-94), preS1 (86-109), preS1 (95-119) and preS1 (21-119), were cloned by PCR, and expressed as GST fusion proteins . These GSTpreS1 fusion proteins were highly expressed in soluble form in E . coli, and about 50 to 90 mg soluble fusion proteins were purified from 1 L culture . Using these fusion proteins, the immunogenic domains in preS1 (21-119) region were identified by Western blot analysis and competitive ELISA . The results showed that the immunogenic domains mainly existed in preS1 (21-59) in N-terminus and preS1 (95-109) in C-terminus, and more importantly, a major immunogenic domain preS1 (34-59), which has much stronger immunogenicity, was identified . It was also supported by the predictions of secondary structure and immunological property in the preS1 (21-119) region . The results here would be helpful for the design of new vaccines against HBV. Crit Care Med, 2004 Jun, 32(6), 1322 - 6 Effects of dexmedetomidine on mortality rate and inflammatory responses to endotoxin-induced shock in rats; Taniguchi T et al.; OBJECTIVES: The aim of this study was to document the effects of a new sedative agent, dexmedetomidine, on the mortality rate and inflammatory responses to endotoxin-induced shock in rats . DESIGN: Randomized laboratory study . SETTING: University experimental laboratory . SUBJECTS: Fifty-seven male rats . INTERVENTIONS: The animals were randomly assigned to one of four groups . The endotoxemic group (n = 16) received intravenous Escherichia coli endotoxin (15 mg/kg over 2 mins) . The saline control group (n = 10) was given saline alone . The dexmedetomidine alone group (n = 15) was treated identically to the control group but also received dexmedetomidine (infusion at 5 microg.kg(-1).hr(-1)) immediately after the injection of 0.9% saline . The dexmedetomidine-endotoxin group (n = 16) was treated identically to the endotoxemic group with the additional administration of dexmedetomidine (infusion at 5 microg.kg(-1).hr(-1)) immediately after endotoxin injection . MEASUREMENTS AND MAIN RESULTS: Hemodynamics and arterial blood gases were recorded and plasma cytokine concentrations measured during the observation . The mortality rate was assessed up to 8 hrs after endotoxin or saline injection . In addition, microscopic findings of lung tissue for each group were obtained at necropsy . Mortality rates 8 hrs after endotoxin injection were 94%, 10%, 0%, and 44% for the endotoxemic, saline control, dexmedetomidine alone, and dexmedetomidine-endotoxin groups, respectively . Hypotension and increases in plasma cytokine (tumor necrosis factor-alpha and interleukin-6) concentrations and infiltration of neutrophils in the airspace or vessel walls of the lungs were less in the dexmedetomidine-endotoxin group than in the endotoxemic group . CONCLUSIONS: Dexmedetomidine reduced mortality rate and had an inhibitory effect on inflammatory response during endotoxemia . These findings suggest that dexmedetomidine administration may inhibit the inflammatory response. Eur J Mass Spectrom (Chichester, Eng), 2004, 10(3), 429 - 36 Complexes between recombinant intracellular carriers of vitamin A and their specific ligands investigated by electrospray-mass spectrometry; Careri M et al.; The intracellular carriers of vitamin A, cellular retinol-binding protein type I, cellular retinol-binding protein type II and cellular retinoic acid-binding protein type I are members of the intracellular lipid-binding proteins family, in which the ligand-binding cavity is located in the interior of a barrel-like structure . The dissociation constants of the specific complexes in water solutions around neutrality are very low (in the 0.1 to 10 nM range) . Because of their high stability, they represent ideal systems to verify the adequacy of electrospray ionization-mass spectrometry in the analysis of non-covalent protein-ligand complexes . The electrospray interface parameters were varied to detect the presence of species not present in solution but generated as artefacts during transfer of complexes from the condensed state to the gas-phase . The results clearly indicate that mass-spectrometry data reflect the situation present in solution only if the electrospray conditions are carefully selected . In particular, the values of cone voltage and temperature compatible with persistence of the complexes in the gas phase were determined for each vitamin A carrier . Lack of correlation between complex stability in solution and in the gas phase is attributable to the specific and differential effects of the two environments on protein conformation and ligand-protein interactions. J Biol Chem, 2004 Aug 27, 279(35), 36268 - 76 Epub 2004 Jun 08. HsAtg4B/HsApg4B/autophagin-1 cleaves the carboxyl termini of three human Atg8 homologues and delipidates microtubule-associated protein light chain 3- and GABAA receptor-associated protein-phospholipid conjugates; Tanida I et al.; In yeast, Atg4/Apg4 is a unique cysteine protease responsible for the cleavage of the carboxyl terminus of Atg8/Apg8/Aut7, a reaction essential for its lipidation during the formation of autophagosomes . However, it is still unclear whether four human Atg4 homologues cleave the carboxyl termini of the three human Atg8 homologues, microtubule-associated protein light chain 3 (LC3), GABARAP, and GATE-16 . Using a cell-free system, we found that HsAtg4B, one of the human Atg4 homologues, cleaves the carboxyl termini of these three Atg8 homologues . In contrast, the mutant HsAtg4B(C74A), in which a predicted active site Cys(74) was changed to Ala, lacked proteolytic activity, indicating that Cys(74) is essential for the cleavage activity of cysteine protease . Using phospholipase D, we showed that the modified forms of endogenous LC3 and GABARAP are lipidated and therefore were designated LC3-PL and GABARAP-PL . When purified glutathione S-transferase-tagged HsAtg4B was incubated in vitro with a membrane fraction enriched with endogenous LC3-PL and GABARAP-PL, the mobility of LC3-PL and GABARAP-PL was changed to those of the unmodified proteins . These mobility shifts were not seen when Cys(74) of HsAtg4B was changed to Ala . Overexpression of wild-type HsAtg4B decreased the amount of LC3-PL and GABARAP-PL and increased the amount of unmodified endogenous LC3 and GABARAP in HeLa cells . Expression of CFP-tagged HsAtg4B (CFP-HsAtg4B) and YFP-tagged LC3 in HeLa cells under starvation conditions resulted in a significant decrease in the punctate pattern of distribution of YFP-tagged LC3 and an increase in its cytoplasmic distribution . RNA interference of HsAtg4B increased the amount of LC3-PL in HEK293 cells . Taken together, these results suggest that HsAtg4B negatively regulates the localization of LC3 to a membrane compartment by delipidation. J Biol Chem, 2004 Aug 13, 279(33), 34406 - 10 Epub 2004 Jun 08. Effects of inorganic polyphosphate on the proteolytic and DNA-binding activities of Lon in Escherichia coli; Nomura K et al.; Lon belongs to a unique group of proteases that bind to DNA and is involved in the regulation of several important cellular functions, including adaptation to nutritional downshift . Previously, we revealed that inorganic polyphosphate (polyP) increases in Escherichia coli in response to amino acid starvation and that it stimulates the degradation of free ribosomal proteins by Lon . In this work, we examined the effects of polyP on the proteolytic and DNA-binding activities of Lon . An order-of-addition experiment suggested that polyP first binds to Lon, which stimulates Lon-mediated degradation of ribosomal proteins . A polyP-binding assay using Lon deletion mutants showed that the polyP-binding site of Lon is localized in the ATPase domain . Because the same ATPase domain also contains the DNA-binding site, polyP can compete with DNA for binding to Lon . In fact, an equimolar amount of polyP almost completely inhibited DNA-Lon complex formation, suggesting that Lon binds to polyP with a higher affinity than it binds to DNA . Collectively, our results showed that polyP may control the cellular activity of Lon not only as a protease but also as a DNA-binding protein. J Biol Chem, 2004 Aug 13, 279(33), 35106 - 12 Epub 2004 Jun 08. Membrane topology of the H+-pyrophosphatase of Streptomyces coelicolor determined by cysteine-scanning mutagenesis; Mimura H et al.; The H+-translocating pyrophosphatase (H+-PPase) is a proton pump that is found in a wide variety of organisms . It consists of a single polypeptide chain that is thought to possess between 14 and 17 transmembrane domains . To determine the topological arrangement of its conserved motifs and transmembrane domains, we carried out a cysteine-scanning analysis by determining the membrane topology of cysteine substitution mutants of Streptomyces coelicolor H+-PPase expressed in Escherichia coli using chemical reagents . First, we prepared a synthetic DNA that encoded the enzyme and constructed a functional cysteine-less mutant by substituting the four cysteine residues . We then introduced cysteine residues individually into 42 sites in its hydrophilic regions and N- and C-terminal segments . Thirty-six of the mutant enzymes retained both pyrophosphatase and H+-translocating activities . Analysis of 29 of these mutant forms using membrane-permeable and -impermeable sulfhydryl reagents revealed that S . coelicolor H+-PPase contains 17 transmembrane domains and that several conserved segments, such as the substrate-binding domains, are exposed to the cytoplasm . Four essential serine residues that were located on the cytoplasmic side were also identified . A marked characteristic of the S . coelicolor enzyme is a long additional sequence that includes a transmembrane domain at the C terminus . We propose that the basic structure of H+-PPases has 16 transmembrane domains with several large cytoplasmic loops containing functional motifs. Cell, 2004 Jun 11, 117(6), 713 - 20 Using a quantitative blueprint to reprogram the dynamics of the flagella gene network; Kalir S et al.; Detailed understanding and control of biological networks will require a level of description similar to that of electronic engineering blueprints . Currently, however, even the best-studied systems are usually described using qualitative arrow diagrams . A quantitative blueprint requires in vivo measurements of (1) the relative strength of the interactions (numbers on the arrows) and (2) the functions that integrate multiple inputs . Here, we address this using a well-studied system, the flagella biosynthesis transcription network in Escherichia coli . We use theory and high-resolution experiments to obtain a quantitative blueprint with (1) numbers on the arrows, finding different hierarchies of activation coefficients for the two regulators, FlhDC and FliA; and (2) cis-regulatory input functions, which summate the input from the two regulators (SUM gates) . We then demonstrate experimentally how this blueprint can be used to reprogram temporal expression patterns in this system, using controlled expression of the regulators or point mutations in their binding sites . The present approach can be used to define blueprints of other gene networks and to quantitatively reprogram their dynamics. Cell, 2004 Jun 11, 117(6), 689 - 90 Flagellar biosynthesis in silico: building quantitative models of regulatory networks; Herrgard MJ et al.; In this issue of Cell, describe the construction of an in silico model for the regulatory network responsible for the control of flagellar biosynthesis in E . coli based on quantitative gene expression data . They show how the model can be used as a quantitative blueprint to design genetic modifications with predictable influence on the dynamics of the system . The work by provides a general approach for building detailed models of transcriptional regulatory networks. Mol Microbiol, 2004 Jun, 52(6), 1813 - 26 HrpA, a DEAH-box RNA helicase, is involved in mRNA processing of a fimbrial operon in Escherichia coli; Koo JT et al.; Endonucleolytic cleavage of mRNA in the daa operon of Escherichia coli is responsible for co-ordinate regulation of genes involved in F1845 fimbrial biogenesis . Cleavage occurs by an unidentified endoribonuclease, is translation dependent and involves a unique recognition mechanism . Here, we present the results of a genetic strategy used to identify factors involved in daa mRNA processing . We used a reporter construct consisting of the daa mRNA processing region fused to the gene encoding green fluorescent protein (GFP) . A mutant defective in daa mRNA processing and expressing high levels of GFP was isolated by flow cytometry . To determine the location of mutations, two different genetic approaches, Hfr crosses and P1 transductions, were used . The mutation responsible for the processing defect was subsequently mapped to the 32 min region of the E . coli chromosome . A putative DEAH-box RNA helicase-encoding gene at this position, hrpA, was able to restore the ability of the mutant to cleave daa mRNA . Site-directed mutagenesis of the hrpA regions predicted to encode nucleotide triphosphate binding and hydrolysis functions abolished the ability of the gene to restore the processing defect in the mutant . We propose that HrpA is a novel enzyme involved in mRNA processing in E . coli. Mol Microbiol, 2004 Jun, 52(6), 1769 - 79 Effects of RNA polymerase modifications on transcription-induced negative supercoiling and associated R-loop formation; Broccoli S et al.; Transcription in the absence of topoisomerase I, but in the presence of DNA gyrase, can result in the formation of hypernegatively supercoiled DNA and associated R-loops . In this paper, we have used several strategies to study the effects of elongation/termination properties of RNA polymerase on such transcription-induced supercoiling . Effects on R-loop formation were exacerbated when cells were exposed to translation inhibitors, a condition that stimulated the accumulation of R-loop-dependent hypernegative supercoiling . Translation inhibitors were not acting by decreasing (p)ppGpp levels as the absence of (p)ppGpp in spoT relA mutant strains had little effect on hypernegative supercoiling . However, an rpoB mutation leading to the accumulation of truncated RNAs considerably reduced R-loop-dependent hypernegative supercoiling . Transcription of an rrnB fragment preceded by a mutated and inactive boxA sequence to abolish the rrnB antitermination system also considerably reduced R-loop-dependent supercoiling . Taken together, our results indicate that RNA polymerase elongation/termination properties can have a major impact on R-loop-dependent supercoiling . We discuss different possibilities by which RNA polymerase directly or indirectly participates in R-loop formation in Escherichia coli . Finally, our results also indicate that what determines the steady-state level of hypernegatively supercoiled DNA in topA null mutants is likely to be complex and involves a multitude of factors, including the status of RNA polymerase, transcription-translation coupling, the cellular level of RNase HI, the status of DNA gyrase and the rate of relaxation of supercoiled DNA. Mol Microbiol, 2004 Jun, 52(6), 1691 - 702 The Mycobacterium tuberculosis protein serine/threonine kinase PknG is linked to cellular glutamate/glutamine levels and is important for growth in vivo; Cowley S et al.; The function of the Mycobacterium tuberculosis eukaryotic-like protein serine/threonine kinase PknG was investigated by gene knock-out and by expression and biochemical analysis . The pknG gene (Rv0410c), when cloned and expressed in Escherichia coli, encodes a functional kinase . An in vitro kinase assay of the recombinant protein demonstrated that PknG can autophosphorylate its kinase domain as well as its 30 kDa C-terminal portion, which contains a tetratricopeptide (TPR) structural signalling motif . Western analysis revealed that PknG is located in the cytosol as well as in mycobacterial membrane . The pknG gene was inactivated by allelic exchange in M . tuberculosis . The resulting mutant strain causes delayed mortality in SCID mice and displays decreased viability both in vitro and upon infection of BALB/c mice . The reduced growth of the mutant was more pronounced in the stationary phase of the mycobacterial growth cycle and when grown in nutrient-depleted media . The PknG-deficient mutant accumulates glutamate and glutamine . The cellular levels of these two amino acids reached approximately threefold of their parental strain levels . Higher cellular levels of the amine sugar-containing molecules, GlcN-Ins and mycothiol, which are derived from glutamate, were detected in the DeltapknG mutant . De novo glutamine synthesis was shown to be reduced by 50% . This is consistent with current knowledge suggesting that glutamine synthesis is regulated by glutamate and glutamine levels . These data support our hypothesis that PknG mediates the transfer of signals sensing nutritional stress in M . tuberculosis and translates them into metabolic adaptation. Mol Microbiol, 2004 Jun, 52(6), 1613 - 25 SepL, a protein required for enteropathogenic Escherichia coli type III translocation, interacts with secretion component SepD; O'Connell CB et al.; Enteropathogenic Escherichia coli (EPEC), an important cause of infantile diarrhoea in the developing world, disrupts host cell microvilli, causes actin rearrangements and attaches intimately to the host cell surface . This characteristic phenotype, referred to as the attaching and effacing (A/E) effect, is encoded on a 36 kb pathogenicity island called the locus of enterocyte effacement (LEE) . The LEE includes genes involved in type III secretion and translocation, the eae gene encoding an outer membrane adhesin known as intimin, the tir gene for the translocated intimin receptor, a regulator and various genes of unknown function . Among this last group is sepL . To determine the role of SepL in EPEC pathogenesis, we constructed and tested a non-polar sepL mutant . We found that this sepL mutant is deficient for A/E and that it secretes markedly reduced quantities of those proteins involved in translocation (EspA, EspB and EspD), but normal levels of those proteins presumed to be effectors (Tir, EspF and EspG) . Despite normal levels of secretion, the mutant strain was unable to translocate EspF and Tir into host cells and formed no EspA filaments . Fractionation studies revealed that SepL is a soluble cytoplasmic protein . Yeast two-hybrid and affinity purification studies indicated that SepL interacts with the LEE-encoded protein SepD . In contrast to SepL, we found that SepD is required for type III secretion of both translocation and effector proteins . Together, these results demonstrate that SepL has a unique role in type III secretion as a functional component of the translocation system that interacts with an essential element of the secretion machinery. Mol Microbiol, 2004 Jun, 52(6), 1597 - 612 Replication fork and SeqA focus distributions in Escherichia coli suggest a replication hyperstructure dependent on nucleotide metabolism; Molina F et al.; Replication from the origin of Escherichia coli has traditionally been visualized as two replisomes moving away from each other, each containing a leading and a lagging strand polymerase . Fluorescence microscopy studies of tagged polymerases or forks have, however, indicated that the polymerases may be confined to a single location (or a few locations in cells with overlapping replication cycles) . Here, we have analysed the exact replication patterns of cells growing with four different growth and replication rates, and compared these with the distributions of SeqA foci . The SeqA foci represent replication forks because the SeqA protein binds to the newly formed hemimethylated DNA immediately following the forks . The results show that pairs of forks originating from the same origin stay coupled for most of the cell cycle and thus support the replication factory model . They also suggest that the factories consisting of four polymerases are, at the time immediately after initiation, organized into higher order structures consisting of eight or 12 polymerases . The organization into replication factories was lost when replication forks experienced a limitation in the supply of nucleotides or when the thymidylate synthetase gene was mutated . These results support the idea that the nucleotide synthesis apparatus co-localizes with the replisomes forming a 'hyperstructure' and further suggest that the integrity of the replication factories and hyperstructures is dependent on nucleotide metabolism. J Agric Food Chem, 2004 Jun 16, 52(12), 3982 - 7 Constitution of stable artificial oil bodies with triacylglycerol, phospholipid, and caleosin; Chen MC et al.; Seed oil bodies are lipid storage organelles of 0.5-2 microm in diameter and comprise a triacylglycerol matrix shielded by a monolayer of phospholipids and proteins . These proteins include abundant structural proteins, oleosins, and at least two minor proteins termed caleosin and steroleosin . This study examined if artificial oil bodies (AOBs) composed of triacylglycerol and phospholipid could be stabilized by oleosin, caleosin, or steroleosin . Our results showed that stabilization effects could be realized by oleosin or caleosin but not by steroleosin . The sizes of the AOBs constituted with oleosin (0.5-2 microm) or caleosin (50-200 nm) were similar to or 10 times smaller than those of the native oil bodies . Recombinant caleosin expressed in Escherichia coli also encapsulated AOBs with a size, topology, and stability comparable to those encapsulated with native caleosin . A proteinase K digestion indicated that caleosin anchored the AOBs via its central hydrophobic domain of approximately 4 kDa . Isoelectrofocusing revealed that the isoelectric point of the caleosin-stabilized AOBs was pH 4.0 . Aggregation of AOBs was observed at a pH lower than 4.5; thus, their stability and integrity were presumably contributed by surface caleosin via electronegative repulsion and steric hindrance . The caleosin-stabilized AOBs were thermostable up to 70 degrees C and potentially useful for biotechnological applications. J Struct Funct Genomics, 2003, 4(4), 235 - 43 Solution structure of a putative ribosome binding protein from Mycoplasma pneumoniae and comparison to a distant homolog; Rubin SM et al.; The solution structure of MPN156, a ribosome-binding factor A (RBFA) protein family member from Mycoplasma pneumoniae, is presented . The structure, solved by nuclear magnetic resonance, has a type II KH fold typical of RNA binding proteins . Despite only approximately 20% sequence identity between MPN156 and another family member from Escherichia coli, the two proteins have high structural similarity . The comparison demonstrates that many of the conserved residues correspond to conserved elements in the structures . Compared to a structure based alignment, standard alignment methods based on sequence alone mispair a majority of amino acids in the two proteins . Implications of these discrepancies for sequence based structural modeling are discussed. Amyloid, 2004 Mar, 11(1), 14 - 20 Non-glycosylphosphatidylinositol (GPI)-anchored recombinant prion protein with dominant-negative mutation inhibits PrPSc replication in vitro; Kishida H et al.; Dominant-negative mouse prion protein (PrP) with a lysine mutation at codon 218 (Q218K) is known to inhibit prion replication . In order to gain further mechanistic insight into such dominant negative inhibition, non-glycosylphosphatidylinositol (GPI)-anchored recombinant PrP with Q218K (rPrP-Q218K) was investigated . When applied into scrapie-infected mouse neuroblastoma (ScN2a) cells, rPrP-Q218K but not wild-type rPrP (rPrP-WT) exclusively inhibited abnormal protease-resistant pathogenic isoform (PrPSc) replication without reducing the viability of the cells . It was even more efficient than quinacrine, which has already been prescribed for sporadic Creutzfeldt-Jakob disease (CJD) patients; 50% effective concentration (EC50) = 0.20 microM, 99% effective concentration (EC99) = 0.86 microM vs . EC50 = 0.45 microM, EC99 = 1.5 microM . Besides, no apparent cell damage was observed at the concentration of up to 4.3 microM (100 micrograms/ml) . In combination treatment with 0.43 microM (10 micrograms/ml) of rPrP-Q218K, EC99 of quinacrine was decreased from 1.5 microM to 0.5 microM, and the cell viability was recovered from 50% to over 90% as inversely proportional to the concentration of quinacrine . Such combination could alleviate the side effects of quinacrine by reducing its effective concentration without changing or even acceleration the inhibition efficacy . Since homogeneous, high-quality rPrPs could be easily prepared from Escherichia coli in large quantities, rPrP-Q218K is a good candidate for a prion replication antagonist. Chembiochem, 2004 Apr 2, 5(4), 527 - 33 Selective inhibition of HIV-1 reverse transcriptase (HIV-1 RT) RNase H by small RNA hairpins and dumbbells; Hannoush RN et al.; We present here the design of a novel class of RNA inhibitors of the RNase H domain of HIV-1 RT, a ribonuclease activity that is essential for viral replication in vivo . Specifically, we show that small RNA hairpins and dumbbells can selectively inhibit the RNase H activity of HIV-1 RT without affecting other cellular RNases H (e.g., E . coli and human RNase H) . These results suggest that the inhibitors do not interact with the nucleic acid binding site of RT RNase H, as this region should be well conserved among the various enzymes . The most potent inhibitors displayed IC50 values in the 3-8 microM range . Remarkably, the DNA polymerase activity, an intrinsic property of HIV RT, was not inhibited by the hairpin and dumbbell aptamers, a property not previously observed for any nucleic acid aptamer directed against RT RNase H . The results described here suggest a noncompetitive binding mechanism, as outlined in the differential inhibitory characteristics of each of the nucleic acid aptamers against the bacterial, human, and viral RNase H homologues. Chembiochem, 2004 Apr 2, 5(4), 467 - 73 Membrane protein-lipid interactions in mixed micelles studied by NMR spectroscopy with the use of paramagnetic reagents; Hilty C et al.; For solution NMR studies of the structure and function of membrane proteins, these macromolecules have to be reconstituted and solubilized in detergent micelles . Detailed characterization of the mixed detergent/protein micelles is then of key importance to validate the results from such studies, and to evaluate how faithfully the natural environment of the protein in the biological membrane is mimicked by the micelle . In this paper, a selection of paramagnetic probes with different physicochemical properties are used to characterize the 60 kDa mixed micelles consisting of about 90 molecules of the detergent dihexanoylphosphatidylcholine (DHPC) and one molecule of the Escherichia coli outer-membrane protein X (OmpX), which had previously been extensively studied by solution NMR techniques . The observation of highly selective relaxation effects on the NMR spectra of OmpX and DHPC from a water-soluble relaxation agent and from nitroxide spin labels attached to lipophilic molecules, confirmed data obtained previously with more complex NMR studies of the diamagnetic OmpX/DHPC system, and yielded additional novel insights into the protein-detergent interactions in the mixed micelles . The application of paramagnetic probes to the well-characterized OmpX/DHPC system indicates that such probes should be widely applicable as an efficient support of NMR studies of the topology of mixed membrane protein-detergent micelles. Pediatr Nephrol, 2004 Aug, 19(8), 915 - 6 Epub 2004 Jun 04. Isolated abducens nerve palsy in hemolytic uremic syndrome; Durkan A et al.; Isolated abducens nerve (VI cranial nerve) palsies are reported in a dialyzed child with Escherichia coli 0157:H7-associated hemolytic uremic syndrome (HUS) . There were no other neurological manifestations and he made a complete recovery, suggesting that isolated abducens nerve palsy in HUS may represent a minor neurological complication. Proc Natl Acad Sci U S A, 2004 Jun 15, 101(24), 8876 - 81 Epub 2004 Jun 07. Crystal structures of the DsbG disulfide isomerase reveal an unstable disulfide; Heras B et al.; Dsb proteins control the formation and rearrangement of disulfide bonds during the folding of secreted and membrane proteins in bacteria . DsbG, a member of this family, has disulfide bond isomerase and chaperone activity . Here, we present two crystal structures of DsbG at 1.7and 2.0-A resolution that are meant to represent the reduced and oxidized forms, respectively . The oxidized structure, however, reveals a mixture of both redox forms, suggesting that oxidized DsbG is less stable than the reduced form . This trait would contribute to DsbG isomerase activity, which requires that the active-site Cys residues are kept reduced, regardless of the highly oxidative environment of the periplasm . We propose that a Thr residue that is conserved in the cis-Pro loop of DsbG and DsbC but not found in other Dsb proteins could play a role in this process . Also, the structure of DsbG reveals an unanticipated and surprising feature that may help define its specific role in oxidative protein folding . Thus, the dimensions and surface features of DsbG show a very large and charged binding surface that is consistent with interaction with globular protein substrates having charged surfaces . This finding suggests that, rather than catalyzing disulfide rearrangement in unfolded substrates, DsbG may preferentially act later in the folding process to catalyze disulfide rearrangement in folded or partially folded proteins. Proc Natl Acad Sci U S A, 2004 Jun 15, 101(24), 9143 - 8 Epub 2004 Jun 07. AtNAP7 is a plastidic SufC-like ATP-binding cassette/ATPase essential for Arabidopsis embryogenesis; Xu XM et al.; In bacteria, yeast, and mammals, iron-sulfur (Fe-S) cluster-containing proteins are involved in numerous processes including electron transfer, metabolic reactions, sensing, signaling, and regulation of gene expression . In humans, iron-storage diseases such as X-linked sideroblastic anemia and ataxia are caused by defects in Fe-S cluster availability . The biogenesis of Fe-S clusters involves several pathways, and in bacteria, the SufABCDSE operon has been shown to play a vital role in Fe-S biogenesis and repair during oxidative stress . Although Fe-S proteins play vital roles in plants, Fe-S cluster biogenesis and maintenance and physiological consequences of dysfunctional Fe-S cluster assembly remains obscure . Here we report that Arabidopsis plants deficient for the SufC homolog AtNAP7 show lethality at the globular stage of embryogenesis . AtNAP7 is expressed in developing embryos and in apical, root, and floral meristems and encodes an ATP-binding cassette/ATPase that can partially rescue growth defects in an Escherichia coli SufC mutant during oxidative stress . AtNAP7 is plastid-localized, and mutant embryos contain abnormal developing plastids with disorganized thylakoid structures . We found that AtNAP7 can interact with AtNAP6, a plastidic Arabidopsis SufD homolog, and because Arabidopsis plastids also harbor SufA, SufB, SufS, and SufE homologs, plastids probably contain a complete SUF system . Our results imply that AtNAP7 represents a conserved SufC protein involved in the biogenesis and/or repair of oxidatively damaged Fe-S clusters and suggest an important role for plastidic Fe-S cluster maintenance and repair during Arabidopsis embryogenesis. Proc Natl Acad Sci U S A, 2004 Jun 15, 101(24), 8864 - 9 Epub 2004 Jun 07. Human RAD9 checkpoint control/proapoptotic protein can activate transcription of p21; Yin Y et al.; When human cells incur DNA damage, two fundamental responses can follow, cell cycle arrest or apoptosis . Human RAD9 (hRAD9) and p53 function in both processes, but the mechanistic relationship between their activities is unknown . p53 mediates checkpoint control at G(1) by transcriptional regulation of p21 . In this report, we show that hRAD9, like p53, can also regulate p21 at the transcriptional level . We demonstrate that overexpression of hRAD9 leads to increased p21 RNA and encoded protein levels . The promoter region of p21 fused to a luciferase reporter can be transactivated by either hRAD9 or p53, indicating that hRAD9 regulates the p21 promoter for transcriptional control of expression . Using an electrophoretic mobility-shift assay, we show that hRAD9 specifically binds to a p53-consensus DNA-binding sequence in the p21 promoter . Microarray screening coupled with Northern analysis reveals that hRAD9 regulates the abundance of other messages in addition to p21 . Our data reveal a previously undescribed mechanism for regulation of p21 and demonstrate that hRAD9 can control gene transcription . We suggest that hRAD9 and p53 co-regulate p21 to direct cell cycle progression by similar molecular mechanisms . Furthermore, hRAD9 might regulate other cellular processes as well by modulating transcription of multiple down-stream target genes. Microbiology, 2004 Jun, 150(Pt 6), 1797 - 808 Evolution of compatible replicons of the related IncQ-like plasmids, pTC-F14 and pTF-FC2; Gardner MN et al.; Two closely related but compatible plasmids of the IncQ-2alpha and IncQ-2beta groups, pTF-FC2 and pTC-F14, were discovered in two acidiphilic chemolithotrophic bacteria . Cross-complementation and cross-regulation experiments by the replication proteins were carried out to discover what changes were necessary when the plasmids evolved to produce two incompatibility groups . The requirement of a pTC-F14 oriV for a RepC DNA-binding protein was plasmid specific, whereas the requirement for the RepA helicase and RepB primase was less specific and could be complemented by the IncQ-2alpha plasmid pTC-FC2, and the IncQ-1beta plasmid pIE1108 . None of the IncQ-1alpha plasmid replication proteins could complement the pTC-F14 oriV, and pTC-F14 and RSF1010 were incompatible . This incompatibility was associated with the RepC replication protein and was not due to iteron incompatibility . Replication of pTC-F14 took place from a 5.7 kb transcript that originated upstream of the mobB gene located within the region required for mobilization . A pTC-F14 mobB-lacZ fusion was regulated by the pTC-F14 repB gene product and was plasmid specific, as it was not regulated by the RepB proteins of pTF-FC2 or the IncQ-1alpha and IncQ-1beta plasmids . Plasmid pTC-F14 appears to have evolved independently functioning iterons and a plasmid-specific RepC-binding protein; it also has a major replication transcript that is independently regulated from that of pTF-FC2 . However, the RepA and RepB proteins have the ability to function with either replicon. Microbiology, 2004 Jun, 150(Pt 6), 1637 - 48 Specific growth rate and not cell density controls the general stress response in Escherichia coli; Ihssen J et al.; In batch cultures of Escherichia coli, the intracellular concentration of the general stress response sigma factor RpoS typically increases during the transition from the exponential to the stationary growth phase . However, because this transition is accompanied by complex physico-chemical and biological changes, which signals predominantly elicit this induction is still the subject of debate . Careful design of the growth environment in chemostat and batch cultures allowed the separate study of individual factors affecting RpoS . Specific growth rate, and not cell density or the nature of the growth-limiting nutrient, controlled RpoS expression and RpoS-dependent hydroperoxidase activity . Furthermore, it was demonstrated that the standard E . coli minimal medium A (MMA) is not suitable for high-cell-density cultivation because it lacks trace elements . Previously reported cell-density effects in chemostat cultures of E . coli can be explained by a hidden, secondary nutrient limitation, which points to the importance of medium design and appropriate experimental set-up for studying cell-density effects. J Clin Microbiol, 2004 Jun, 42(6), 2675 - 81 Characterization of novel coding sequences specific to Mycobacterium avium subsp . paratuberculosis: implications for diagnosis of Johne's Disease; Paustian ML et al.; Mycobacterium avium subsp . paratuberculosis is the causative agent of Johne's Disease, an economically important intestinal ailment of ruminants . Due to the considerable genetic and serologic cross-reactivity with closely related and ubiquitous members of the M . avium complex, a species-specific method for the serological diagnosis of Johne's disease is unavailable . Computational and PCR-based analysis of the complete genome sequence of M . avium subsp . paratuberculosis led to the identification of 13 open reading frames with no identifiable homologs . One of these sequences is a putative insertion element present in six copies on the M . avium subsp . paratuberculosis genome . These novel M . avium subsp . paratuberculosis genes were cloned into Escherichia coli expression vectors, and nine were successfully expressed as recombinant fusion proteins . Five of these proteins were purified in sufficient amounts to allow immunoblot analyses of their reactivity with sera from naturally infected cattle as well as mice and rabbits exposed to M . avium subsp . paratuberculosis . Fusion proteins representing MAP0862, MAP3732c, and MAP2963c were recognized by nearly all of the sera tested, including those from cattle in the clinical stages of disease . Notably, further analysis of the protein encoded by MAP0862 showed that it reacted with sera from additional infected cattle but not with sera from uninfected control animals . The fusion product of MAP0860c did not react with any of the sera tested, while the remaining four proteins were variably recognized by sera from M . avium subsp . paratuberculosis-infected cattle . Collectively, the results of this study demonstrate the utility of genomic data to identify potential diagnostic sequences. J Biol Chem, 2004 Aug 13, 279(33), 34840 - 8 Epub 2004 Jun 07. The novel gene fad158, having a transmembrane domain and leucine-rich repeat, stimulates adipocyte differentiation; Tominaga K et al.; Adipocyte differentiation is known to be regulated by a complex array of genes known as master regulators . Using a subtraction method, we previously isolated 102 genes that are expressed in the early stage of adipocyte differentiation . One of these genes named fad158 (factor for adipocyte differentiation 158) seems to be a novel gene, since there is no significantly similar gene listed in databases . Both mouse and human fad158 encode 803 amino acids and contain 4 transmembrane regions and 8 leucine-rich repeat motifs . Expression of fad158 was induced at an early stage in differentiating 3T3-L1 cells and was observed in the skeletal muscle . When the expression was knocked down with an antisense method in 3T3-L1 cells, the accumulation of oil droplets was reduced . Moreover, on overexpression of fad158 in NIH-3T3 cells, which are fibroblasts and do not usually differentiate into adipocytes, stable transformants accumulated oil droplets and showed an elevated expression of adipocyte marker genes, indicating that these cells had differentiated into mature adipocytes . fad158 has the ability to regulate adipocyte differentiation positively, especially at an early stage. J Biol Chem, 2004 Aug 13, 279(33), 35101 - 5 Epub 2004 Jun 07. Cysteine oxidation of tau and microtubule-associated protein-2 by peroxynitrite: modulation of microtubule assembly kinetics by the thioredoxin reductase system; Landino LM et al.; Alterations in the redox status of proteins have been implicated in the pathology of several neurodegenerative conditions including Alzheimer and Parkinson diseases . We report that peroxynitrite- and hydrogen peroxide-induced disulfides in the neuron-specific microtubule-associated proteins tau and microtubule-associated protein-2 are substrates for the ubiquitous thioredoxin reductase system composed of thioredoxin reductase, human or Escherichia coli thioredoxin, and NADPH . Tau and microtubule-associated protein-2 cysteine oxidation and reduction were quantitated by monitoring the incorporation of 5-iodoacetamidofluorescein, a thiol-specific labeling reagent . Cysteine oxidation of tau and microtubule-associated protein-2 to disulfides altered the ability of the proteins to promote the assembly of microtubules from purified porcine tubulin . Treatment of tau and microtubule-associated protein-2 with either the thioredoxin reductase system or small molecule reductants fully restores the ability of the MAPs to promote microtubule assembly . Thus changes in the redox state of microtubule-associated proteins may regulate microtubule polymerization in vivo. Appl Environ Microbiol, 2004 Jun, 70(6), 3566 - 74 Biotransformation of explosives by the old yellow enzyme family of flavoproteins; Williams RE et al.; Several independent studies of bacterial degradation of nitrate ester explosives have demonstrated the involvement of flavin-dependent oxidoreductases related to the old yellow enzyme (OYE) of yeast . Some of these enzymes also transform the nitroaromatic explosive 2,4,6-trinitrotoluene (TNT) . In this work, catalytic capabilities of five members of the OYE family were compared, with a view to correlating structure and function . The activity profiles of the five enzymes differed substantially; no one compound proved to be a good substrate for all five enzymes . TNT is reduced, albeit slowly, by all five enzymes . The nature of the transformation products differed, with three of the five enzymes yielding products indicative of reduction of the aromatic ring . Our findings suggest two distinct pathways of TNT transformation, with the initial reduction of TNT being the key point of difference between the enzymes . Characterization of an active site mutant of one of the enzymes suggests a structural basis for this difference. Appl Environ Microbiol, 2004 Jun, 70(6), 3352 - 9 Expression of functional recombinant mussel adhesive protein Mgfp-5 in Escherichia coli; Hwang DS et al.; Mussel adhesive proteins have been suggested as a basis for environmentally friendly adhesives for use in aqueous conditions and in medicine . However, attempts to produce functional and economical recombinant mussel adhesive proteins (mainly foot protein type 1) in several systems have failed . Here, the cDNA coding for Mytilus galloprovincialis foot protein type 5 (Mgfp-5) was isolated for the first time . Using this cDNA, we produced a recombinant Mgfp-5 fused with a hexahistidine affinity ligand, which was expressed in a soluble form in Escherichia coli and was highly purified using affinity chromatography . The adhesive properties of purified recombinant Mgfp-5 were compared with the commercial extracted mussel adhesive Cell-Tak by investigating adhesion force using atomic force microscopy, material surface coating, and quartz crystal microbalance . Even though further macroscale assays are needed, these microscale assays showed that recombinant Mgfp-5 has significant adhesive ability and may be useful as a bioadhesive in medical or underwater environments. Biochem Biophys Res Commun, 2004 Jul 2, 319(3), 929 - 35 Identification of two antigenic epitopes on SARS-CoV spike protein; Hua R et al.; The spike (S) protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) is a major virion structural protein . It plays an important role in interaction with receptor and inducing neutralizing antibodies . In the study, six tentative antigenic epitopes (S1 S2 S3 S4 S5 S6) of the spike protein of SARS-CoV were predicted by bio-informatics analysis, and a multi-epitope chimeric gene of S1-S2-S3-S4-S5-S6 was synthesized and fused to downstream GST gene in pGEX-6p-1 . The Western blotting demonstrated that SARS patient convalescent serum could recognize the recombinant fusion protein . A number of monoclonal antibodies were developed against the fusion protein . In further, the six predicted epitope genes were individually fused to GST of pGEX-6p-1 and expressed in Escherichia coli BL21, respectively . Among six fusion peptides, S5 reacted with monoclonal antibody D3C5 and S2 reacted with monoclonal antibody D3D1 against spike protein of SARS-CoV . The epitopes recognized by monoclonal antibodies D3C5 and D3D1 are linear, and correspond to 447-458 and 789-799 amino acids of spike protein of SARS-CoV, respectively . Identification of antigenic epitope of spike protein of SARS-CoV could provide the basis for the development of immunity-based prophylactic, therapeutic, and diagnostic techniques for the control of severe acute respiratory syndrome. Biochem Biophys Res Commun, 2004 Jul 2, 319(3), 787 - 94 Crystal structure of the ribonuclease P protein Ph1877p from hyperthermophilic archaeon Pyrococcus horikoshii OT3; Takagi H et al.; Ribonuclease P (RNase P) is a ribonucleoprotein complex involved in the processing of pre-tRNA . Protein Ph1877p is one of essential components of the hyperthermophilic archaeon Pyrococcus horikoshii OT3 RNase P {Biochem . Biophys . Res . Commun . 306 (2003) 666} . The crystal structure of Ph1877p was determined at 1.8A by X-ray crystallography and refined to a crystallographic R factor of 22.96% (Rfree of 26.77%) . Ph1877p forms a TIM barrel structure, consisting of ten alpha-helices and seven beta-strands, and has the closest similarity to the TIM barrel domain of Escherichia coli cytosine deaminase with a root-mean square deviation of 3.0A . The protein Ph1877p forms an oblate ellipsoid, approximate dimensions being 45Ax43Ax39A, and the electrostatic representation indicated the presence of several clusters of positively charged amino acids present on the molecular surface . We made use of site-directed mutagenesis to assess the role of twelve charged amino acids, Lys42, Arg68, Arg87, Arg90, Asp98, Arg107, His114, Lys123, Lys158, Arg176, Asp180, and Lys196 related to the RNase P activity . Individual mutations of Arg90, Arg107, Lys123, Arg176, and Lys196 by Ala resulted in reconstituted particles with reduced enzymatic activities (32-48%) as compared with that reconstituted RNase P by wild-type Ph1877p . The results presented here provide an initial step for definite understanding of how archaeal and eukaryotic RNase Ps mediate substrate recognition and process 5'-leader sequence of pre-tRNA. Biochem Biophys Res Commun, 2004 Jul 2, 319(3), 715 - 9 Soluble expression of aggregating proteins by covalent coupling to the ribosome; Sorensen HP et al.; Ribosomes are extremely soluble ribonucleoprotein complexes . Heterologous target proteins were fused to ribosomal protein L23 (rpL23) and expressed in an rpL23 deficient Escherichia coli strain . This enabled the isolation of 70S ribosomes with covalently bound target protein . Isolation of recombinant proteins from 70S ribosomes was achieved by specific proteolytic cleavage followed by efficient removal of ribosomes by centrifugation . By this procedure we isolated active green fluorescent protein, streptavidin (SA), and murine interleukin-6 (mIL-6) . Approximately 500microg of each protein was isolated per gram cellular wet weight . By pull-down assays we demonstrate that SA covalently bound to the ribosome binds d-biotin . Ribosomal coupling is therefore suggested as a method for the investigation of protein interactions . The presented strategy is in particular efficient for the expression, purification, and investigation of proteins forming inclusion bodies in the E . coli cytoplasm. FEMS Microbiol Lett, 2004 Jun 15, 235(2), 237 - 42 Epitope mapping of a single repetitive unit of the B13 Trypanosoma cruzi antigen as fusions to Escherichia coli LamB protein; Pereira CM et al.; B13, one of the immunodominant antigens of Trypanosoma cruzi, is composed of repeats of a 12-amino-acid motif . Using synthetic peptides, the sequence FGQAAAGDK was previously shown to contain the B13 immunodominant epitope recognized by chagasic patients sera . To investigate the effects of neighboring sequences in the immunodominance, we tested serum recognition of two B13 sequences fused to LamB . GDKPSPFGQAAA-LamB and FGQAAAGDKPSP-LamB were recognized, respectively, by 15% and 80% of 80 sera reactive to B13 antigen . Recognition of FGQAAAGDKPSP-LamB was inhibited by AAAGDK-containing synthetic peptides . FGQAAAGDKPSP-LamB competed with a B13 recombinant protein containing 16.6 repeats for binding to chagasic antibodies . These results strengthen previous conclusions on the immunodominant epitope of B13 and provide a comparison of two methods for epitope mapping. Cytokine, 2004 Jun 21, 26(6), 243 - 6 Heat stress decreases pulmonary MCP-1 production in endotoxemia; Urs J et al.; An exaggerated pro-inflammatory response in endotoxemia may lead to multiple organ damage including acute lung injury . Heat stress prior to endotoxemia results in attenuation of inflammation possibly by decreasing cytokine production . Monocyte chemoattractant protein (MCP)-1, a pro-inflammatory cytokine, is responsible for monocyte recruitment into the lung in acute lung injury . The objective of this study is to determine if pretreatment with heat results in decreased MCP-1 production in the lungs of endotoxemic rats at a transcriptional or post-transcriptional level . Rats were assigned to one of four groups: control, heat alone, heat with or without endotoxin . Rats were made endotoxemic by injection of Escherichia coli lipopolysaccharide . MCP-1 was measured in lavage fluid and MCP-1 mRNA in the lung tissue . Endotoxemia resulted in production of MCP-1 . Control and heat alone rats had 21+/-4 vs . 20+/-3 pg/ml, p=0.75 . MCP-1 concentration was decreased in the lavage fluid of pre-heated when compared to non-heated endotoxemic rats (37+/-28 vs . 70+/-35 pg/ml, p <0.02 ) . However, the MCP-1 mRNA was higher in the heated compared to non-heated endotoxemic rats (1.59+/-0.35 vs . 0.74+/-0.51, MCP-1/beta-actin mRNA, p <0.01) . Control and heat alone rats had undetectable mRNA MCP-1 in the lungs . Heat stress prior to endotoxemia results in decreased production of MCP-1 by a post-transcriptional mechanism. Anal Biochem, 2004 Jul 1, 330(1), 140 - 4 A rapid method for analyzing recombinant protein inclusion bodies by mass spectrometry; Grimm R et al.; The identification and characterization of a protein overexpressed in insoluble inclusion bodies in Escherichia coli are the first crucial and time-limiting steps in recombinant protein expression . Here, a straightforward approach to the analysis of recombinant proteins in inclusion bodies is presented . Inclusion bodies were dissolved in 8M urea and analyzed by matrix-assisted laser desorption ionization (MALDI)-time of flight mass spectrometry without prior desalting . Mass determination was achieved by direct spotting of the samples onto the MALDI target and serial dilution in the matrix . The masses of four different proteins, expressed in inclusion bodies, were determined with a mass accuracy better than 0.1% . Furthermore, protein modifications, such as N-terminal processing of single amino acids or artificial cyanylation caused by incubation of the inclusion bodies with urea at elevated temperatures, could be detected . Similarly, tryptic digests were directly analyzed in 2M urea to obtain peptide mass fingerprints for identification and more detailed information on the primary protein structure and secondary modifications . Due to the presence of ammonia in the urea-containing buffers, no Na(+) adducts were observed in the peptide mass fingerprint analysis . Taken together, the rapid and robust procedures presented here greatly facilitate the analysis of recombinant proteins. Anal Biochem, 2004 Jul 1, 330(1), 74 - 80 A nonradioactive 96-well plate assay for the detection of hypoxia-inducible factor prolyl hydroxylase activity; Oehme F et al.; The transcriptional activation of hypoxia-inducible genes is essential for the adaptation of mammalian tissues to oxygen deficiency . The hypoxia-inducible transcription factor (HIF) is a cellular switch for the up-regulation of these genes during hypoxia . Under normoxia, HIFs are hydroxylated on conserved prolyl residues by a recently discovered family of HIF prolyl hydroxylases (HIF-PHD1-3) . Hydroxylated HIF specifically interacts with the von Hippel-Lindau protein-elongin B-elongin C complex (VBC) which leads to ubiquitination and subsequent proteasomal degradation of HIF . We developed a nonradioactive microtiter plate assay based on the interaction of hydroxylated HIF with VBC which enabled us to detect hydroxylated HIF in the nanomolar concentration range . A biotinylated HIF peptide substrate was bound to a streptavidin-coated microtiter plate and hydroxylated with the HIF-PHD3 isoenzyme . Recombinant VBC complex with a thioredoxin (Trx) tag was purified from Escherichia coli and bound to the hydroxylated HIF peptide . The interaction between VBC and hydroxylated HIF was detected by using an anti-thioredoxin antibody. Anal Biochem, 2004 Jul 1, 330(1), 52 - 7 A cell-free biosensor for the detection of transcriptional inducers using firefly luciferase as a reporter; Pellinen T et al.; A cell-free biosensor for the detection of transcription induction by specific small-molecule ligands is presented . As model systems, tetracycline and mercury-inducible promoters were used containing firefly luciferase as reporter gene . Escherichia coli S30 extract was prepared and used for coupled transcription-translation reactions . By using purified Tet repressor and MerR regulatory proteins, we could study repressor-operator interactions for optimizing the relative concentrations of each component . Previously, detection of tetracycline and mercury using similar transcriptional regulation in whole living cells has been carried out . As compared to whole-cell biosensors, our results showed better sensitivity for the detection of tetracycline and the toxic effect of mercury was avoided in the cell-free system . Also, as the system omits cell cultivation and bacterial membranes as molecule passage inhibitors, it is possible to carry out assays in much shorter times and without the use of genetically modified organisms. Int J Biochem Cell Biol, 2004 Sep, 36(9), 1836 - 47 Proteolytic processing of an HIV-1 pol polyprotein precursor: insights into the mechanism of reverse transcriptase p66/p51 heterodimer formation; Sluis-Cremer N et al.; HIV-1 reverse transcriptase (RT) is a heterodimer comprising a 66 kDa subunit (p66) and a p66-derived 51 kDa subunit (p51) . RT is translated as part of a larger gag-pol polyprotein and subsequently processed to the p66/p51 heterodimer by HIV-1 protease (PR) during viral maturation . The processing events involved in the formation of the RT p66 and p51 subunits and the pathway(s) of RT dimer formation are poorly characterized . Attempts to study the kinetics of PR-catalyzed formation of p66/p51 HIV-1 RT in isolated HIV virions produced in the presence of HIV PR inhibitors were unsuccessful due to difficulties in removal of the tight-binding inhibitor to initiate proteolytic processing . Accordingly, an inducible bacterial expression vector encoding a 90 kDa pol polyprotein fragment was constructed . Following expression in Escherichia coli, the pol polyprotein underwent time-dependent proteolytic processing to the RT p66/p51 heterodimer . This processing was catalyzed entirely by HIV-1 PR since mutations that inactivate PR prevented RT heterodimer formation . The kinetics of RT processing follow an ordered sequential pathway in which RT p66 is first excised from the pol polyprotein, followed by formation of the p51 subunit . Processing of the p66 subunit to form p51 apparently proceeds through a p66/p66 RT homodimer intermediate since the L234A mutation in RT, a mutation that prevents RT dimerization, resulted in the formation of RT p66 only . These results provide the first experimental data defining the pathway for the HIV-1 PR catalyzed formation of the p66/p51 HIV-1 RT heterodimer . Virology, 2004 Jun 20, 324(1), 204 - 12 Isolation and characterization of the Mason-Pfizer monkey virus p12 protein; Knejzlik Z et al.; The Mason-Pfizer monkey virus (M-PMV) Gag protein, precursor to the structural proteins of the infectious virion, assembles into immature capsid-like particles when expressed at high levels in bacterial cells . Similar capsid-like particles can be obtained by in vitro assembly using a high concentration of isolated Gag . M-PMV Gag contains a p12 protein that has no corresponding analogues in most other retroviruses and has been suggested to contain an internal scaffold domain (ISD) . We have expressed and purified p12 and the N- and C-terminal halves (Np12 and Cp12) that are predicted to be structurally independent domains . The behavior of these proteins was analyzed using chemical cross-linking, CD spectroscopy, and electron microscopy . The N-terminal half of p12 is largely alpha-helical although the C-terminal portion lacks any apparent ordered structure . Both p12 and Np12 form high-order oligomers in vitro and when expressed in E . coli produce organized structures that are visible by electron microscopy . Interestingly, Cp12, as well as the whole protein, can form dimers in the presence of SDS . The data show that both domains of p12 contribute to its ability to multimerize with much of this potential residing in its N-terminal part, most probably within the leucine zipper-like (LZL) sequence. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2003 Nov, 19(6), 580 - 1 {Preparation and identification of the monoclonal antibody against human endostatin}; Feng YM et al.; AIM: To prepare monoclonal antibody against human endostatin for the in depth study on the mechanism of anti-tumor effect of endostatin . METHODS: Monoclonal antibody specific for endostatin was prepared using hybridoma technique and screened with ELISA . Ascites fluids were produced in BALB/c mice following in sequentical intraperitoneal injection of pristine and hybridoma cells . The mAb was purified by affinify chromatography with protein A sepharose CL-4B . RESULTS: One hybridoma cell line 4E7 was established, which could produce mAb against human endostatin . The titers of mAb 4E7 in culture supernatant and ascites fluid were 1:128-1:516 and 1:10(-4)-1:10(-6), respectively . The mAb 4E7 belonged to IgG1 (lambda type) . Western blot analysis showed that the mAb 4E7 could react to the human endostatin expressed by yeast and E.coli, but it had no cross reaction to other cytokines such as bFGF . CONCLUSION: The mAb 4E7 is able to react specifically with human endostatin and can be used for further investigation. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2004 Jan, 20(1), 104 - 8 {Construction, expression and biologic activities of two rhIL-6/GM-CSF fusion proteins}; Sun QM et al.; AIM: To construct and express the gene encoding hIL-6/GM-CSF fusion protein . METHODS: The genes encoding the two hIL-6/GM-CSF fusion proteins were constructed in pBV220 expression vector . Fusion proteins were expressed in E.coli BL-21 and purified through Q Sepharose HP ion exchange chromatography and Sephacryl S-200 gel filtration columns . The biologic activities of the fusion proteins were detected by proliferation of hIL-6 dependent cell line B9 and hGM-CSF dependent cell line TF1 with MTT assay . RESULTS: Both fusion proteins were expressed in E.coli BL-21 in the form of inclusion body . The expression levels were more than 25% of the total cell lysates.Both fusion proteins were obtained with high purity which had both hIL-6 and hGM-CSF biologic activities . CONCLUSION: Two hIL-6/GM-CSF fusion proteins with high purity and bilolgic activities have been acquired successfully. Zhongguo Wei Zhong Bing Ji Jiu Yi Xue, 2004 Jun, 16(6), 361 - 3 {Influence of lipopolysaccharide pretreatment on acute lung injury induced by lipopolysaccharide in rats}; Xue Y et al.; OBJECTIVE: To evaluate the effect of lipopolysaccharide pretreatment on blocking the development of lipopolysaccharide (E.Coli O(55):B(5)) induced acute lung injury . The activity of nuclear factor-kappaB (NF-kappaB) in alveolar macrophages was assessed to elucidate its mechanism . METHODS: Thirty-six Wistar rats were divided into three groups: normal saline (A), lipopolysaccharide (B), lipopolysaccharide preconditioning (C) . Rat model of acute lung injury was reproduced by administering intraperitoneally lipopolysaccharide in a dose of 6 mg/kg . Group A and B served as control . In the test group (group C) lipopolysaccharide was given intraperitoneally 0.5 mg/kg, 0.5 mg/kg and 1.0 mg/kg consecutively for 3 days before lipopolysaccharide challenge . Four hours after lipopolysaccharide/normal saline administration, the animals were killed . Blood gas was measured . And total protein of bronchoalveolar lavage fluid (BALF) was calculated by measuring the radioactivity of (99)Tc labeled serum albumin . Wet/dry ratios of the lungs of each group were determined . The nuclear protein of the alveolar macrophages was extracted from BALF, and the activity of NF-kappaB was assayed with electrophoretic mobility shift assay (EMSA) . Microscopic examination of the lung was done . RESULTS: In group C, partial pressure of oxygen in artery (PaO(2)) was significantly higher than that in group B, and total protein content of BALF was significantly lower in group A and C than that in group B . Activity of NF-kappaB in group C was higher than group A and B . CONCLUSION: Lipopolysaccharide pretreatment can reduce the severity of acute lung injury induced by lipopolysaccharide challenge . This phenomenon may be related with change in the activity of NF-kappaB of the alveolar macrophages. Eur J Biochem, 2004 Jun, 271(12), 2452 - 61 Genomic organization, tissue distribution and deletion mutation of human pyridoxine 5'-phosphate oxidase; Kang JH et al.; We used a combined computer and biochemical approach to characterize human pyridoxine 5'-phosphate oxidase (PNPO) . The human PNPO gene is composed of seven exons and six introns, and spans approximately 8 kb . All exon/intron junctions contain the gt/ag consensus splicing site . The absence of TATA-like sequences, the presence of Sp1-binding sites and more importantly, the presence of CpG islands in the regulatory region of the PNPO gene are characteristic features of housekeeping genes . Northern blot analyses showed two species of poly(A)(+) RNA of approximately 2.4 and approximately 3.4 kb at identical intensity, whereas Western blot analysis showed that no protein isoform exists in any of the tissues examined . PCR-based analysis led to the idea that two messages are transcribed from a single copy gene, and that the size difference is due to differential usage of the polyadenylation signal . The major sites of PNPO expression are liver, skeletal muscle and kidneys while a very weak signal was detected in lung . The mRNA master dot-blot for multiple human tissues provided a complete map of the tissue distribution not only for PNPO but also for pyridoxal kinase and pyridoxal phosphatase . The data indicate that mRNA expression of all three enzymes essential for vitamin B(6) metabolism is ubiquitous but is highly regulated at the level of transcription in a tissue-specific manner . In addition, human brain PNPO cDNA was expressed in Escherichia coli, and the roles of both the N- and C-terminal regions were studied by creating sequential truncation mutants . Our results showed that deletion of the N-terminal 56 residues affects neither the binding of coenzyme nor catalytic activity. Eur J Biochem, 2004 Jun, 271(12), 2400 - 7 Evidence for two different electron transfer pathways in the same enzyme, nitrate reductase A from Escherichia coli; Giordani R et al.; In order to clarify the role of cytochrome in nitrate reductase we have performed spectrophotometric and stopped-flow kinetic studies of reduction and oxidation of the cytochrome hemes with analogues of physiological quinones, using menadione as an analogue of menaquinone and duroquinone as an analogue of ubiquinone, and comparing the results with those obtained with dithionite . The spectrophotometric studies indicate that reduction of the cytochrome hemes varies according to the analogue of quinone used, and in no cases is it complete . Stopped-flow kinetics of heme oxidation by potassium nitrate indicates that there are two distinct reactions, depending on whether the hemes were previously reduced by menadiol or by duroquinol . These results, and those of spectrophotometric studies of a mutant lacking the highest-potential {Fe-S} cluster, allow us to propose a two-pathway electron transfer model for nitrate reductase A from Escherichia coli. Biochem J, 2004 Sep 1, 382(Pt 2), 751 - 7 Catalytic and structural contributions for glutathione-binding residues in a Delta class glutathione S-transferase; Winayanuwattikun P et al.; Glutathione S-transferases (GSTs) are dimeric proteins that play a major role in cellular detoxification . The GSTs in mosquito Anopheles dirus species B, an important malaria vector in South East Asia, are of interest because they can play an important role in insecticide resistance . In the present study, we characterized the Anopheles dirus (Ad)GST D3-3 which is an alternatively spliced product of the adgst1AS1 gene . The data from the crystal structure of GST D3-3 shows that Ile-52, Glu-64, Ser-65, Arg-66 and Met-101 interact directly with glutathione . To study the active-site function of these residues, alanine substitution site-directed mutagenesis was performed resulting in five mutants: I52A (Ile-52-->Ala), E64A, S65A, R66A and M101A . Interestingly, the E64A mutant was expressed in Escherichia coli in inclusion bodies, suggesting that this residue is involved with the tertiary structure or folding property of this enzyme . However, the I52A, S65A, R66A and M101A mutants were purified by glutathione affinity chromatography and the enzyme activity characterized . On the basis of steady-state kinetics, difference spectroscopy, unfolding and refolding studies, it was concluded that these residues: (1) contribute to the affinity of the GSH-binding site ('G-site') for GSH, (2) influence GSH thiol ionization, (3) participate in kcat regulation by affecting the rate-limiting step of the reaction, and in the case of Ile-52 and Arg-66, influenced structural integrity and/or folding of the enzyme . The structural perturbations from these mutants are probably transmitted to the hydrophobic-substrate-binding site ('H-site') through changes in active site topology or through effects on GSH orientation . Therefore these active site residues appear to contribute to various steps in the catalytic mechanism, as well as having an influence on the packing of the protein. Biochemistry, 2004 Jun 15, 43(23), 7628 - 36 The region adjacent to the highly immunogenic site and shielded by the middle domain is responsible for self-oligomerization/client binding of the HSP90 molecular chaperone; Nemoto TK et al.; We here investigated the mechanism of self-oligomerization of the 90-kDa heat shock protein (HSP90) molecular chaperone, because it is known that this oligomerization reflects the client-binding activity . The transition temperatures for the self-oligomerization of the full-length forms of human HSP90alpha and HtpG (bacterial HSP90), i.e., 45 and 60 degrees C, respectively, were identical to those for the dissociation of the recombinant N domain (residues 1-400 of human HSP90alpha and residues 1-336 of HtpG in our definition) from the remainder of the molecule . The N domain of human HSP90alpha expressed in Escherichia coli was oligomeric, and the oligomerization activity was localized within residues 311-350, i.e., C-terminally adjacent to the highly immunogenic site (residues 291-304) . Particularly, residues 341-350 were critical on oligomerization . On the other hand, residues 289-389 were indispensable for the interaction with the M domain (residues 401-618) of the molecule . Oligomer formation of the N domain was efficiently suppressed by its extension until Lys546, i.e., residues 401-546, which is required for the interaction with the N domain . Among highly conserved amino acids at residues 289-400, Trp297, Pro379, and Phe384 were essential for the interaction with the M domain . With these observations taken together, we propose as the activation mechanism of HSP90 molecular chaperone that heat stress induces the liberation of the oligomerization/client-binding site of residues 311-350 by disrupting the intramolecular interaction between residues 289-389 and 401-546. Biochemistry, 2004 Jun 15, 43(23), 7610 - 7 Characterization of Mycobacterium tuberculosis NAD kinase: functional analysis of the full-length enzyme by site-directed mutagenesis; Raffaelli N et al.; NAD kinase is the only known enzyme catalyzing the formation of NADP, a coenzyme implicated in most reductive biosynthetic reactions and in many antioxidant defense systems . Despite its importance, nothing is known regarding its structure or mechanism of catalysis . Mycobacterium tuberculosis NAD kinase has been overexpressed in Escherichia coli and purified to homogeneity . The molecular and kinetic properties of the enzyme resulted in significant differences from those reported by others on a proteolytically degraded form of the protein . Indeed the full-length enzyme displays an allosteric behavior and shows a strict preference for inorganic polyphosphate as the phosphate donor . It is inhibited by the reaction product NADP and by both NADH and NADPH . The mycobacterial enzyme shares with all other known NAD kinases a highly conserved region (spanning residues 189-210), particularly rich in glycines, which differs from the primary sequences of all previously identified nucleotide-binding sites . Alanine-scanning mutagenesis performed on 11 conserved residues within this domain revealed its importance in catalysis . A total of 6 of 11 mutated proteins completely lost the enzymatic activity while retaining the same oligomeric state of the wild-type protein, as demonstrated by gel-filtration analysis . Substitutions of S199 and G208 with alanine rendered enzyme versions with reduced activity . Their kinetic characterization, performed on purified proteins, revealed kinetic parameters toward ATP and polyphosphate similar to those of the wild-type enzyme . On the contrary, when the kinetic analysis was performed by using NAD as the variable substrate, significant differences were observed with respect to both the allosteric behavior and the catalytic efficiency, suggesting that the mutated region is likely involved in NAD binding. Biochemistry, 2004 Jun 15, 43(23), 7575 - 83 Contribution of the esterified amino acid to the binding of aminoacylated tRNAs to the ribosomal P- and A-sites; Fahlman RP et al.; Crystallographic studies suggest that the esterified amino acid of aminoacyl tRNA make contacts with the ribosomal A-site but not in the P-site . Biochemical evidence indicating a thermodynamic contribution of the esterified amino acid to binding aminoacyl-tRNA to either the ribosomal P- and A-sites has been inconsistent, partly because of the labile nature of the aminoacyl linkage and the long times required to reach equilibrium . Measuring the association and dissociation rates of deacylated and aminoacylated tRNAs to the A-site and P-site of E . coli ribosomes afforded an accurate estimate of the contribution of the amino acid . While esterified phenylalanine or methionine has no effect on the affinity of tRNA to the P-site, an esterified pheylalanine stabilizes binding to the A-site by 7 kJ/mol, in agreement with the contacts observed in the X-ray crystal structure . In addition, it was shown that the presence of an esterified amino acid in one ribosomal site does not affect the binding of an aa-tRNA to the other site. Biochemistry, 2004 Jun 15, 43(23), 7432 - 42 Correlation of an adenine-specific conformational change with the ATP-dependent peptidase activity of Escherichia coli Lon; Patterson J et al.; Escherichia coli Lon, also known as protease La, is a serine protease that is activated by ATP and other purine or pyrimidine triphosphates . In this study, we examined the catalytic efficiency of peptide cleavage as well as intrinsic and peptide-stimulated nucleotide hydrolysis in the presence of hydrolyzable nucleoside triphosphates ATP, CTP, UTP, and GTP . We observed that the k(cat) of peptide cleavage decreases with the reduction in the nucleotide binding affinity of Lon in the following order: ATP > CTP > GTP approximately UTP . Compared to those of the other hydrolyzable nucleotide triphosphates, the ATPase activity of Lon is also the most sensitive to peptide stimulation . Collectively, our kinetic as well as tryptic digestion data suggest that both nucleotide binding and hydrolysis contribute to the peptidase turnover of Lon . The kinetic data that were obtained were further put into the context of the structural organization of Lon protease by probing the conformational change in Lon bound to the different nucleotides . Both adenine-containing nucleotides and CTP protect a 67 kDa fragment of Lon from tryptic digestion . Since this 67 kDa fragment contains the ATP binding pocket (also known as the alpha/beta domain), the substrate sensor and discriminatory (SSD) domain (also known as the alpha-helical domain), and the protease domain of Lon, we propose that the binding of ATP induces a conformational change in Lon that facilitates the coupling of nucleotide hydrolysis with peptide substrate delivery to the peptidase active site. Biochemistry, 2004 Jun 15, 43(23), 7273 - 87 Mapping backbone dynamics in solution with site-directed spin labeling: GCN4-58 bZip free and bound to DNA; Columbus L et al.; In site-directed spin labeling, a nitroxide-containing side chain is introduced at selected sites in a protein . The EPR spectrum of the labeled protein encodes information about the motion of the nitroxide on the nanosecond time scale, which has contributions from the rotary diffusion of the protein, from internal motions in the side chain, and from backbone fluctuations . In the simplest model for the motion of noninteracting (surface) side chains, the contribution from the internal motion is sequence independent, as is that from protein rotary diffusion . Hence, differences in backbone motions should be revealed by comparing the sequence-dependent motions of nitroxides at structurally homologous sites . To examine this model, nitroxide side chains were introduced, one at a time, along the GCN4-58 bZip sequence, for which NMR (15)N relaxation experiments have identified a striking gradient of backbone mobility along the DNA-binding region {Bracken et al . (1999) J . Mol . Biol . 285, 2133} . Spectral simulation techniques and a simple line width measure were used to extract dynamical parameters from the EPR spectra, and the results reveal a mobility gradient similar to that observed in NMR relaxation, indicating that side chain motions mirror backbone motions . In addition, the sequence-dependent side chain dynamics were analyzed in the DNA/protein complex, which has not been previously investigated by NMR relaxation methods . As anticipated, the backbone motions are damped in the DNA-bound state, although a gradient of motion persists with residues at the DNA-binding site being the most highly ordered, similar to those of helices on globular proteins. Plant Physiol, 2004 Jun, 135(2), 947 - 58 Epub 2004 Jun 04. Characterization of an ultraviolet B-induced lipase in Arabidopsis; Lo M et al.; An Arabidopsis expressed sequence tag clone, 221D24, encoding a lipase has been characterized using an antisense approach . The lipase gene is expressed during normal growth and development of Arabidopsis rosette leaves but is down-regulated as the leaves senesce . When plants are exposed to sublethal levels of UV-B radiation, expression of the lipase is strongly up-regulated . The lipase protein is localized in the cell cytosol and is present in all organs of Arabidopsis plants . Recombinant lipase protein produced in Escherichia coli preferentially hydrolyzed phospholipids, indicating that the gene encodes a phospholipase . Transgenic plants in which lipase expression is suppressed showed enhanced tolerance to UV-B stress but not osmotic stress and were unable to up-regulate PR-1 expression when irradiated with UV-B . The observations collectively indicate that the lipase is capable of deesterifying membrane phospholipids and is up-regulated in response to UV-B irradiation. J Biol Chem, 2004 Aug 13, 279(33), 34269 - 76 Epub 2004 Jun 04. Stripping down the mitochondrial cholesterol hydroxylase system, a kinetics study; Schiffler B et al.; The origin of steroid hormones in mammals is cholesterol that is metabolized by the mitochondrial CYP11A1 system . The cytochrome P450 is fed with reduction equivalents via a small electron transfer chain consisting of NADPH, adrenodoxin reductase, and adrenodoxin . Though the redox behavior of the individual protein components has been studied previously, the kinetics of the system in its entirety has not yet been analyzed . In this study we combine surface plasmon resonance experiments to determine the binding constants for the different pairs of redox partners with measurements of the pre-steady-state kinetics of the different reaction steps of this system and steady-state kinetics . We could correlate the individual protein-protein interactions with the effect of distinct reduction-oxidation steps on the overall catalytic activity of the CYP11A1 system . For the first time, we were able to follow the reduction of each of the protein components of this system within one measurement when we mixed all oxidized protein components with NADPH . These measurements allowed the determination of the individual apparent rate constants for the reduction of all three proteins involved . In addition, variation of the ionic strength in these experiments revealed different optimum salt concentrations for the reduction of adrenodoxin reductase and adrenodoxin, respectively, and unraveled dramatically changing reduction rates of CYP11A1 by adrenodoxin. J Biol Chem, 2004 Aug 13, 279(33), 34123 - 9 Epub 2004 Jun 04. Redox reactions of the iron-sulfur cluster in a ribosomal RNA methyltransferase, RumA: optical and EPR studies; Agarwalla S et al.; An unprecedented {4Fe-4S} iron-sulfur cluster was found in RumA, the enzyme that methylates U1939 in Escherichia coli 23 S ribosomal RNA (Agarwalla, S., Kealey, J . T., Santi, D . V., and Stroud, R . M . (2002) J . Biol . Chem . 277, 8835-8840; Lee, T . T., Agarwalla, S., and Stroud, R . M . (2004) Structure 12, 397-407) . Methyltransferase reactions do not involve a redox step . To understand the structural and functional roles of the cluster in RumA, we have characterized redox reactions of the iron-sulfur cluster . As isolated aerobically, RumA exhibits a visible absorbance maximum at 390 nm and is EPR silent . It cannot be reduced by anaerobic additions of dithionite . Photoreduction by deazariboflavin/EDTA gives EPR spectra, the quantity (56% of S = 1/2 species) and details (g(av) approximately 1.96-1.93) of which indicate a {4Fe-4S}(1+) cluster in the reduced RumA . Oxidation of RumA by ferricyanide leads to loss of the 390-nm band and appearance of lower intensity bands at 444 and 520 nm . EPR spectra of ferricyanide-oxidized RumA show a fraction (<8%) of the FeS cluster trapped in the {3Fe-4S}(1+) form (g(av) approximately 2.011) together with unusual radical-like spectrum (g' values 2.015, 2.00, and 1.95) . RumA also reacts with nitric oxide to give EPR spectra characteristic of the protein-bound iron dinitrosyl species . Oxidation of the cluster leads to its decomposition and that could be a mechanism for regulating the activity of RumA under