Crit Care Med, 2002 Jul, 30(7), 1528 - 34 Fas/Fas ligand pathway is involved in the resolution of type II pneumocyte hyperplasia after acute lung injury: evidence from a rat model; Wang HC et al.; OBJECTIVE: We used a rat model of acute lung injury to evaluate the role of apoptosis of type II pneumocytes in alveolar remodeling during the resolution phase . DESIGN: Controlled animal study . SETTING: University research laboratory . SUBJECTS: Sprague-Dawley rats . INTERVENTIONS: Sprague-Dawley rats had Escherichia coli lipopolysaccharide instilled transtracheally to induce acute lung injury . Animals were killed on various days after lipopolysaccharide instillation . Lung specimens from all animals were examined for the presence of apoptosis in type II pneumocytes by an in situ apoptosis assay and for proliferative nuclear antigen, cytokeratin-18, Fas, and Fas ligand with an immunohistochemical stain . Fas and Fas ligand expression in both lung tissue and bronchoalveolar lavage fluid was examined by Western blot analysis . MEASUREMENTS AND MAIN RESULTS: Histologic examination revealed that the lungs of rats with acute lung injury showed infiltration of numerous inflammatory cells in the intra-alveolar and/or interstitial space and hyperplasia of type II pneumocytes . Type II pneumocyte proliferation, detected by proliferative nuclear antigen staining, developed maximally around day 3 after acute lung injury . In the in situ apoptosis assay, positive signals in type II pneumocytes were obvious and were distributed diffusely in the lung parenchyma from day 1 after acute lung injury, became maximal around day 7, then declined until day 21 . DNA fragmentation analysis revealed that a DNA ladder pattern was detectable from day 3, persisted until day 10, and disappeared after day 14 . The major cell types expressing Fas ligand are macrophages and neutrophils . Western blot analysis showed that Fas ligand, both membrane-bound form and soluble form, was present from day 1 to day 21 after acute lung injury, with highest level occurring during the first week of acute lung injury . Fas expression in type II pneumocytes reached its maximum on days 3-5 and then gradually declined until day 21 . Fas and Fas ligand expression appeared to proceed type II pneumocyte apoptosis . After the acute stage, Fas and Fas ligand expression declined, and type II pneumocyte apoptosis also decreased . These findings correlate with histologic resolution of type II pneumocyte hyperplasia . CONCLUSIONS: Our results confirm that type II pneumocyte proliferation in response to acute lung injury is mainly a reparative phenomenon . During the resolution phase of acute lung injury, extensive apoptosis of type II pneumocytes is the main cellular mechanism that accounts for the disappearance of these cells, and Fas/Fas ligand is involved in the resolution of type II pneumocytes . Our model may provide a useful tool to assess the mechanisms of tissue remodeling after acute lung injury. J Biol Chem, 2002 Oct 18, 277(42), 39722 - 7 Epub 2002 Jul 18. Carbamoyl-phosphate synthetase . Creation of an escape route for ammonia; Thoden JB et al.; Carbamoyl-phosphate synthetase catalyzes the production of carbamoyl phosphate through a reaction mechanism requiring one molecule of bicarbonate, two molecules of MgATP, and one molecule of glutamine . The enzyme from Escherichia coli is composed of two polypeptide chains . The smaller of these belongs to the Class I amidotransferase superfamily and contains all of the necessary amino acid side chains required for the hydrolysis of glutamine to glutamate and ammonia . Two homologous domains from the larger subunit adopt conformations that are characteristic for members of the ATP-grasp superfamily . Each of these ATP-grasp domains contains an active site responsible for binding one molecule of MgATP . High resolution x-ray crystallographic analyses have shown that, remarkably, the three active sites in the E . coli enzyme are connected by a molecular tunnel of approximately 100 A in total length . Here we describe the high resolution x-ray crystallographic structure of the G359F (small subunit) mutant protein of carbamoyl phosphate synthetase . This residue was initially targeted for study because it resides within the interior wall of the molecular tunnel leading from the active site of the small subunit to the first active site of the large subunit . It was anticipated that a mutation to the larger residue would "clog" the ammonia tunnel and impede the delivery of ammonia from its site of production to the site of utilization . In fact, the G359F substitution resulted in a complete change in the conformation of the loop delineated by Glu-355 to Ala-364, thereby providing an "escape" route for the ammonia intermediate directly to the bulk solvent . The substitution also effected the disposition of several key catalytic amino acid side chains in the small subunit active site. J Biol Chem, 2002 Oct 25, 277(43), 40309 - 23 Epub 2002 Jul 18. Global gene expression profiling in Escherichia coli K12 . The effects of leucine-responsive regulatory protein; Hung SP et al.; Leucine-responsive regulatory protein (Lrp) is a global regulatory protein that affects the expression of multiple genes and operons in bacteria . Although the physiological purpose of Lrp-mediated gene regulation remains unclear, it has been suggested that it functions to coordinate cellular metabolism with the nutritional state of the environment . The results of gene expression profiles between otherwise isogenic lrp(+) and lrp(-) strains of Escherichia coli support this suggestion . The newly discovered Lrp-regulated genes reported here are involved either in small molecule or macromolecule synthesis or degradation, or in small molecule transport and environmental stress responses . Although many of these regulatory effects are direct, others are indirect consequences of Lrp-mediated changes in the expression levels of other global regulatory proteins . Because computational methods to analyze and interpret high dimensional DNA microarray data are still an early stage, much of the emphasis of this work is directed toward the development of methods to identify differentially expressed genes with a high level of confidence . In particular, we describe a Bayesian statistical framework for a posterior estimate of the standard deviation of gene measurements based on a limited number of replications . We also describe an algorithm to compute a posterior estimate of differential expression for each gene based on the experiment-wide global false positive and false negative level for a DNA microarray data set . This allows the experimenter to compute posterior probabilities of differential expression for each individual differential gene expression measurement. Blood, 2002 Aug 1, 100(3), 1026 - 30 Deletion of leucine 61 in glucose-6-phosphate dehydrogenase leads to chronic nonspherocytic anemia, granulocyte dysfunction, and increased susceptibility to infections; van Bruggen R et al.; In this study the blood cells of 4 male patients from 2 unrelated families with chronic nonspherocytic anemia and recurrent bacterial infections were investigated . The activity of glucose-6- phosphate dehydrogenase (G6PD) in the red blood cells and in the granulocytes of these patients was below detection level . Moreover, their granulocytes displayed a decreased respiratory burst upon activation . Sequencing of genomic DNA revealed a novel 3-base pair (TCT) deletion in the G6PD gene, predicting the deletion of a leucine at position 61 . The mutant G6PD protein was undetectable by Western blotting in the red blood cells and granulocytes of these patients . In phytohemagglutinin-stimulated lymphocytes the G6PD protein was present, but the amount of G6PD protein was strongly diminished in the patients' cells . Purified mutant protein from an Escherichia coli expression system showed decreased heat stability and decreased specific activity . Furthermore, we found that the messenger RNA of G6PD(180-182delTCT) is unstable, which may contribute to the severe G6PD deficiency observed in these patients . We propose the name "G6PD Amsterdam" for this new variant. Biophys Chem, 2002 Jul 10, 98(1-2), 127 - 48 Myoglobin as a model system for designing heme protein based blood substitutes; Dou Y et al.; The ligand binding properties and resistances to denaturation of >300 different site-directed mutants of sperm whale, pig, and human myoglobin have been examined over the past 15 years . This library of recombinant proteins has been used to derive chemical mechanisms for ligand binding and to examine the factors governing holo- and apoglobin stability . We have also examined the effects of mutagenesis on the dioxygenation of NO by MbO(2) to form NO(3)(-) and metMb . This reaction rapidly detoxifies NO and is a key physiological function of both myoglobins and hemoglobins . The mechanisms derived for O(2) binding and NO dioxygenation have been used to design safer, more efficient, and more stable heme protein-prototypes for use as O(2) delivery pharmaceuticals in transfusion therapy (i.e . blood substitutes) . An interactive database is being developed to allow rapid access to the ligand binding parameters, stability properties, and crystal structures of the entire set of recombinant myoglobins . The long-range goal is to use this library for developing general protein engineering principles and for designing individual heme proteins for specific pharmacological and industrial uses. Comp Biochem Physiol B Biochem Mol Biol, 2002 Aug, 132(4), 769 - 77 Expression of a hepatocyte growth-factor activator protein in turkey (Meleagris gallopavo) deferent duct epithelial cells; Holsberger DR et al.; In previous research, we discovered that turkey deferent duct epithelial cells express a serine protease . Our experimental objective was to identify the gene that encodes this protein . A lambda phage cDNA library from duct cell mRNA was constructed . The library was screened using monoclonal antibodies previously produced against the turkey deferent-duct serine protease . Phage containing the protease cDNA was excised and re-circularized into plasmids . E . coli were transformed with plasmids containing protease cDNA, which was then isolated for sequencing . NCBI BLAST searches within the GenBank database returned 63.5 and 61.7% identity with murine and human hepatocyte growth-factor activator (HGFA) precursor, respectively . The turkey protease cDNA was then cloned into the pQE-32 expression vector and transformed into M15 cells for HIS-tagged expression of the recombinant protein, which was then purified using nickel-chelated Sepharose spin columns . Afterwards, Western blot analysis of the purified recombinant turkey protein revealed recognition by a monoclonal antibody specific to the proteolytic subunit of the turkey deferent duct protease . Therefore, these findings indicate that the recombinant HGFA precursor isolated from the deferent duct is the turkey seminal plasma protease that is secreted from the deferent duct . HGFA, a member of the Kringle-serine proteinase superfamily, can initiate diverse mitogenic, morphogenic and motogenic effects through its substrate hepatocyte growth factor . Although the presence of hepatocyte growth factor and its c-MET receptor have been reported in male mammalian reproductive tracts, our novel findings on the secretion of HGFA precursor from turkeys may help to elucidate the regulation of activated hepatocyte growth factor . Biochem Biophys Res Commun, 2002 Jul 26, 295(4), 952 - 7 Heterologous expression and purification of SpaB involved in subtilin biosynthesis; Xie L et al.; Lantibiotic peptides contain thioether bridges termed lanthionines that are putatively generated by dehydration of Ser and Thr residues followed by Michael addition of cysteine residues within the peptide . The LanB and LanC proteins have been proposed to catalyze the dehydration and formation of the thioether rings, respectively . We report here the first heterologous overexpression in Escherichia coli of SpaB, the putative dehydratase for subtilin . Sequence analysis of spaB revealed several nucleotide differences with current gene database entries . The solubility of SpaB was increased dramatically when co-expressed with GroEL/ES, and soluble His(6)-tagged SpaB was purified . The protein is at least a dimer, and interaction between SpaB and SpaC was observed . SpaS the putative substrate for SpaB was overexpressed in E . coli as an intein fusion protein, and after cleavage, the peptide was obtained in good yield. Biochem Biophys Res Commun, 2002 Jul 26, 295(4), 818 - 25 A novel amylolytic enzyme from Thermotoga maritima, resembling cyclodextrinase and alpha-glucosidase, that liberates glucose from the reducing end of the substrates; Lee MH et al.; The gene previously designated as putative cyclodextrinase from Thermotoga maritima (TMG) was cloned and overexpressed in Escherichia coli . The recombinant TMG was partially purified and its enzymatic characteristics on various substrates were examined . The enzyme hydrolyzes various maltodextrins including maltotriose to maltoheptaose and cyclomaltodextrins (CDs) to mainly glucose and maltose . Although TMG could not degrade pullulan, it rapidly hydrolyzes acarbose, a strong amylase and glucosidase inhibitor, to acarviosine and glucose . Also, TMG initially hydrolyzes p-nitrophenyl-alpha-pentaoside to give maltopentaose and p-nitrophenol, implying that the enzyme specifically cleaves a glucose unit from the reducing end of maltooligosaccharides unlike to other glucosidases . Since its enzymatic activity is negligible if alpha-methylglucoside is present in the reducing end, the type of the residue at the reducing end of the substrate is important for the TMG activity . These results support the fact that TMG is a novel exo-acting glucosidase possessing the characteristics of both CD-/pullulan hydrolyzing enzyme and alpha-glucosidase. Biochem Biophys Res Commun, 2002 Jul 26, 295(4), 812 - 7 Role of cis prolines 112 and 126 in the functional activity of ribonucleolytic toxin restrictocin; Goyal A et al.; Restrictocin is a 149 amino acid ribonucleolytic toxin produced by the fungus Aspergillus, which specifically cleaves a single phosphodiester bond within 28S rRNA resulting in a potent inhibition of protein synthesis in eukaryotic cells . Restrictocin has 12 prolines out of which three at positions 48, 112, and 126 are cis . Prolines at position 112, 118, and 126 were individually mutated to alanine to investigate their role in the catalytic and membrane interaction activity of restrictocin . All mutants were expressed in Escherichia coli, and recombinant proteins purified to homogeneity . Mutation of P112 resulted in a remarkable 50- and 100-fold reduction, respectively, in the ribonucleolytic and cytotoxic activities of restrictocin, whereas the interaction of P112A with phospholipid membranes increased . Mutants P118A and P126A exhibited 3-5-fold decreased ribonucleolytic and cytotoxic activities, however, their membrane interaction activity was marginally reduced compared to restrictocin . The study demonstrates that P112 is absolutely essential to maintain the functionally active conformation of restrictocin . Also, prolines 112, 118, and 126 do not appear to be directly involved in the membrane interaction activity of restrictocin. Curr Opin Struct Biol, 2002 Jun, 12(3), 374 - 82 Pathway evolution, structurally speaking; Rison SC et al.; Small-molecule metabolism forms the core of the metabolic processes of all living organisms . As early as 1945, possible mechanisms for the evolution of such a complex metabolic system were considered . The problem is to explain the appearance and development of a highly regulated complex network of interacting proteins and substrates from a limited structural and functional repertoire . By permitting the co-analysis of phylogeny and metabolism, the combined exploitation of pathway and structural databases, as well as the use of multiple-sequence alignment search algorithms, sheds light on this problem . Much of the current research suggests a chemistry-driven 'patchwork' model of pathway evolution, but other mechanisms may play a role . In the future, as metabolic structure and sequence space are further explored, it should become easier to trace the finer details of pathway development and understand how complexity has evolved. Curr Opin Struct Biol, 2002 Jun, 12(3), 311 - 9 Protein surface salt bridges and paths for DNA wrapping; Saecker RM et al.; The organization of large regions of DNA on the surface of proteins is critical to many DNA 'transactions', including replication, transcription, recombination and repair, as well as the packaging of chromosomal DNA . Recent thermodynamic and structural studies of DNA binding by integration host factor indicate that the disruption of protein surface salt bridges (dehydrated ion pairs) dominates the observed thermodynamics of integration host factor binding and, more generally, allows the wrapping of DNA on protein surfaces . The proposed thermodynamic signature of wrapping with coupled salt bridge disruption includes large negative salt-concentration-dependent enthalpy, entropy and heat capacity changes and smaller than expected magnitudes of the observed binding constant and its power dependence on salt concentration . Examination of the free structures of proteins recently shown to wrap DNA leads us to hypothesize that a pattern of surface salt bridges interspersed with cationic sidechains provides a structural signature for wrapping and that the number and organization of salt bridges and cationic groups dictate the thermodynamics and topology of DNA wrapping, which in turn are critical to function. Vet Immunol Immunopathol, 2002 Sep 25, 88(3-4), 209 - 16 Antigen dose modulates the immunoglobulin isotype responses of pigs against intramuscularly administered F4-fimbriae; Van der Stede Y et al.; Parenteral immunisation normally induces a systemic antibody response characterised by high IgG and low IgA responses . In the present study, the effect of different doses of F4-fimbriae on the isotype-specific antibody response after intramuscular immunisation was studied in pigs . Pigs were injected twice with a 9 weeks interval with either 1, 0.1 or 0.01 mg of F4-ETEC fimbriae . The dose of 1mg F4 induced significantly lower primary F4-specific IgG and IgM responses than the doses of 0.1 and 0.01 mg F4, but primed for an enhanced F4-specific IgM serum antibody response after the booster immunisation . Furthermore, the dose of 0.1mg induced the highest F4-specific IgA serum response which was significantly higher than after injection with 0.01 and 1mg F4 . Moreover, both lower doses (0.1 and 0.01 mg) showed a higher number of F4-specific IgA and IgG antibody secreting cells (ASC) in the local draining lymph nodes of the pigs . This study demonstrated that low doses of purified F4-ETEC fimbriae, especially the 0.1mg dose, are optimal for inducing F4-specific IgA responses after IM immunisation. Biologicals, 2002 Jun, 30(2), 85 - 92 Development of a highly sensitive in vitro assay method for biological activity of endotoxin contamination in biological products; Yamamoto A et al.; The pyrogen test in rabbits has been replaced by the bacterial endotoxin test . The endotoxin test, however, showed a considerable discrepancy with pyrogenicity and was, therefore, assumed to have an efficacy limitation in directly predicting harmful biological effects of endotoxin . We developed a sensitive in vitro assay method by making use of tumour necrosis factor alpha (TNF-alpha) induction in RAW264.7 cells, which showed a fine correlation with pyrogenicity in rabbits . RAW264.7 cells maintained by serial subculture under an endotoxin-free condition have gained the similar level of sensitivity as the endotoxin test to allow extensive dilutions of a drug for eliminating adverse effects on the cells . The in vitro TNF-alpha induction assay was shown to be capable to detect quantitatively a synergistic effect of a drug and endotoxin . The synergy is assumed necessary to be taken into consideration to define the limit value for the endotoxin test for guaranteeing the similar level of safety as by the pyrogen test . Semin Cell Dev Biol, 2002 Apr, 13(2), 109 - 19 Say when: reversible control of gene expression in the mouse by lac; Scrable H; In bacteria, coordinate expression of genes involved in lactose metabolism is regulated by the lac repressor and its DNA binding sequence, the lac operator . The lac operator-repressor complex can also be used to regulate gene expression in the laboratory mouse . In this review, I discuss the current state of murine trans-operons, and suggest ways this lac-based system might be used to build more advanced models of human diseases in the mouse. Arch Biochem Biophys, 2002 Aug 1, 404(1), 25 - 37 Role of cysteine 306 in the catalytic mechanism of Ascaris suum phosphoenolpyruvate carboxykinase; Rios SE et al.; Biochemical and metabolic data lead to the conclusion that the enzyme phosphoenolpyruvate carboxykinase (PEPCK) contributes to a critical point of divergence in energy conservation pathways between mammals and nematodes . The Ascaris suum PEPCK shares considerable homology with PEPCK from avian liver and is a good candidate for mutagenesis studies . The Cys306 substitution by Ser and Ala produced active enzymes and the two mutants are kinetically indistinguishable from each other . This substitution affects the catalytic affinity for the formation of the specific enzyme-nucleotide complex (k(cat)/K(m)) in the forward and reverse reactions . Studies with the substrate analogs 2(')dGDP and 2(')dGTP indicate that Cys306 in A . suum PEPCK is one of the residues important in nucleotide binding and may interact with the 2(')OH group in the ribose ring . Alternatively, mutation of this residue could cause protein changes that interfere with the proper conformation of the nucleotides for optimal catalysis to take place. Vaccine, 2002 Jul 26, 20(23-24), 2995 - 3004 Different kinetic of antibody responses following infection of newly weaned pigs with an F4 enterotoxigenic Escherichia coli strain or an F18 verotoxigenic Escherichia coli strain; Verdonck F et al.; To develop a vaccine against Escherichia coli-induced post-weaning diarrhea and edema disease, insights in the induction of the protective immune response following infection with these pathogenic E . coli is needed . Therefore, the fimbriae-specific antibody response of newly weaned pigs following infection with the Shiga-like toxin type II variant (SLT-IIv) producing F18(+) verotoxigenic E . coli (VTEC) (strain 107/86) was compared with the response following an infection with LT producing F4(+) enterotoxigenic E . coli (ETEC) (strain GIS 26) . F4(+) ETEC were able to colonize the gut very soon after infection, as peak excretion of F4(+) E . coli bacteria was seen 2 days post-infection (dpi), but had already disappeared 7dpi . On the other hand, F18(+) VTEC infection resulted in a slower colonization of the gut as the peak excretion of F18(+) E . coli was observed between 3 and 5dpi, but this colonization remained longer as F18(+) E . coli were detected till 9dpi in feces . Furthermore, this fast colonization pattern of F4(+) ETEC is accompanied with the presence of F4-specific antibodies in mucosal tissues and serum from 4dpi onward, with maximal amounts of F4-specific IgA in the jejunal lamina propria and serum 7dpi . In contrast, F18-specific IgA was only readily detected in the jejunal lamina propria 15dpi and showed a maximum serum titer 21dpi . Besides this faster induction and higher antibody response, the switch from IgM to IgA and IgG was also earlier following the F4(+) ETEC infection. Phytochemistry, 2002 Jul, 60(6), 589 - 93 In vitro properties of a recombinant flavonol synthase from Arabidopsis thaliana; Prescott AG et al.; cDNA corresponding to a flavonol synthase gene from Arabidopsis thaliana was cloned and expressed in Escherichia coli . The recombinant protein was purified to near-homogeneity and the catalytic properties of the enzyme were studied in vitro . Together with kaempferol and apigenin the recombinant protein synthesised the (2R,3S)-cis- and (2S,3S)-trans-isomers of dihydrokaempferol from the (2S)- and (2R)-isomers of naringenin, respectively . Flavanones and dihydroflavanols differing in degree of A- or B-ring hydroxylation were also accepted as substrates. J Mol Biol, 2002 Jul 26, 320(5), 1135 - 45 The natively helical chain segment 169-188 of Escherichia coli adenylate kinase is formed in the latest phase of the refolding transition; Ratner V et al.; The refolding transition of Escherichia coli adenylate kinase (AK) was investigated by monitoring the refolding kinetics of a selected 20 residue helical segment in the CORE domain of the protein . Residues 169 and 188 were labeled by 1-acetamido-methyl-pyrene, and by bimane, respectively . The experiment combines double-jump stopped-flow fast mixing initiation of refolding and time-resolved Forster energy transfer spectroscopy for monitoring the conformational transitions (double-kinetics experiment) . Two kinetic phases were found in the denaturant-induced unfolding of AK . In the first phase, the fluorescence quantum yields of both probes decreased . The distribution of the distances between them transformed from the native state's narrow distribution with the mean distance corresponding to the distance in the crystal structure, to a distribution compatible with an unordered structure . In the second, slow step of denaturation, neither the fluorescence parameters of the probes nor the distance distribution between them changed . This step appeared to be a transformation of the fast-folding species formed in the first phase, to the slow-folding species . Refolding of the fast-folding species of the denatured state of AK was also a two-phase process . During the first fast phase, within less than 5ms, the fluorescence emission of both probes increased, but the distance distribution between the labeled sites was unchanged . Only during the second slow refolding step did the intramolecular distance distribution change from the characteristic of the denatured state to the narrow distribution of the native state . This experiment shows that for the case of the CORE domain of AK, the large helical segment of residues 169-188 was not formed in the first compaction step of refolding . The helical conformation of this segment is established only in the second, much slower, refolding phase, simultaneously with the completion of the native structure. J Mol Biol, 2002 Jul 26, 320(5), 1119 - 33 Folding mechanism of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus: a test of the conservation of folding mechanisms hypothesis in (beta(alpha))(8) barrels; Forsyth WR et al.; As a test of the hypothesis that folding mechanisms are better conserved than sequences in TIM barrels, the equilibrium and kinetic folding mechanisms of indole-3-glycerol phosphate synthase (sIGPS) from the thermoacidophilic archaebacterium Sulfolobus solfataricus were compared to the well-characterized models of the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli . A multifaceted approach combining urea denaturation and far-UV circular dichroism, tyrosine fluorescence total intensity, and tyrosine fluorescence anisotropy was employed . Despite a sequence identity of only 13%, a stable intermediate (I) in sIGPS was found to be similar to a stable intermediate in alphaTS in terms of its thermodynamic properties and secondary structure . Kinetic experiments revealed that the fastest detectable folding event for sIGPS involves a burst-phase (<5ms) reaction that leads directly to the stable intermediate . The slower of two subsequent phases reflects the formation/disruption of an off-pathway dimeric form of I . The faster phase reflects the conversion of I to the native state and is limited by folding under marginally stable conditions and by isomerization or rearrangement under strongly folding conditions . By contrast, alphaTS is thought to fold via an off-pathway burst-phase intermediate whose unfolding controls access to a set of four on-pathway intermediates that comprise the stable equilibrium intermediate . At least three proline isomerization reactions are known to limit their interconversions and lead to a parallel channel mechanism . The simple sequential mechanism deduced for sIGPS reflects the dominance of the on-pathway burst-phase intermediate and the absence of prolyl residues that partition the stable intermediate into kinetically distinguishable species . Comparison of the results for sIGPS and alphaTS demonstrates that the thermodynamic properties and the final steps of the folding reaction are better conserved than the early events . The initial events in folding appear to be more sensitive to the sequence differences between the two TIM barrel proteins. J Mol Biol, 2002 Jul 26, 320(5), 979 - 89 Functional studies of the 900 tetraloop capping helix 27 of 16S ribosomal RNA; Belanger F et al.; The 900 tetraloop (positions 898-901) of Escherichia coli 16S rRNA caps helix 27, which is involved in a conformational switch crucial for the decoding function of the ribosome . This tetraloop forms a GNRA motif involved in intramolecular RNA-RNA interactions with its receptor in helix 24 of 16S rRNA . It is involved also in an intersubunit bridge, via an interaction with helix 67 in domain IV of 23S rRNA . Using a specialized ribosome system and an instant-evolution procedure, the four nucleotides of this loop were randomized and 15 functional mutants were selected in vivo . Positions 899 and 900, responsible for most of the tetraloop/receptor interactions, were found to be the most critical for ribosome activity . Functional studies showed that mutations in the 900 tetraloop impair subunit association and decrease translational fidelity . Computer modeling of the mutations allows correlation of the effect of mutations with perturbations of the tetraloop/receptor interactions. BMC Biotechnol . 2002 Jul 18;2(1):13. Signal and noise in bridging PCR; Liu S et al.; BACKGROUND: In a variant of the standard PCR reaction termed bridging, or jumping, PCR the primer-bound sequences are originally on separate template molecules . Bridging can occur if, and only if, the templates contain a region of sequence similarity . A 3' end of synthesis in one round of synthesis that terminates in this region of similarity can prime on the other . In principle, Bridging PCR (BPCR) can detect a subpopulation of one template that terminates synthesis in the region of sequence shared by the other template . This study considers the sensitivity and noise of BPCR as a quantitative assay for backbone interruptions . Bridging synthesis is also important to some methods for computing with DNA . RESULTS: In this study, BPCR was tested over a 328 base pair segment of the E . coli lac operon and a signal to noise ratio (S/N) of approximately 10 was obtained under normal PCR conditions with Taq polymerase . With special precautions in the case of Taq or by using the Stoffel fragment the S/N was improved to 100, i.e . 1 part of cut input DNA yielded the same output as 100 parts of intact input DNA . CONCLUSIONS: In the E . coli lac operator region studied here, depending on details of protocol, between 3 and 30% per kilobase of final PCR product resulted from bridging . Other systems are expected to differ in the proportion of product that is bridged consequent to PCR protocol and the sequence analyzed . In many cases physical bridging during PCR will have no informational consequence because the bridged templates are of identical sequence, but in a number of special cases bridging creates, or, destroys, information. J Org Chem, 2002 Jul 26, 67(15), 5402 - 4 Kottamides A-D: novel bioactive imidazolone-containing alkaloids from the New Zealand ascidian Pycnoclavella kottae; Appleton DR et al.; Kottamides A-D (1-4), novel 2,2,5-trisubstituted imidazolone-containing alkaloids, were isolated from the New Zealand endemic ascidian Pycnoclavella kottae and structurally characterized using 15N natural abundance 2-D NMR in addition to standard spectroscopic methods . The kottamides exhibited anti-inflammatory and anti-metabolic activity as well as cytotoxicity toward tumor cell lines. J Mol Microbiol Biotechnol, 2002 Jul, 4(4), 417 - 26 Increasing the efficiency of heterologous promoters in actinomycetes; Wilkinson CJ et al.; An Escherichia coli-actinomycete shuttle vector, pCJW93, was constructed which places cloned genes under the control of the thiostrepton-inducible tip promoter from Streptomyces lividans . We also constructed expression vectors bearing the actII-ORF4/PactI activator-promoter system of the actinorhodin biosynthetic pathway of Streptomyces coelicolor . With both types of vector, levels of expression varied widely in different actinomycete strains, indicating different levels of the host factors needed for optimal expression . Deletion of the actII-ORF4 activator gene from one such plasmid in Saccharopolyspora erythraea drastically reduced expression from the cognate actI promoter, showing that host factors are required for optimal production of the activator protein itself . However, a low copy number expression vector pWIZ1 for the polyketide synthase DEBS1-TE, in which the promoter for the activator gene was replaced by the strong heterologous ermE* promoter of S . erythraea directed highly efficient production of polyketide synthase protein in Streptomyces cinnamonensis; and the levels of triketide lactone product found were up to 100-fold greater than were produced by the same plasmid in which actII-ORF4 was expressed from its own promoter . Ensuring appropriate expression of a specific activator protein should enable more convenient and consistent heterologous expression of genes in a broad range of actinomycete hosts. J Mol Microbiol Biotechnol, 2002 Jul, 4(4), 379 - 88 Specific growth inhibition by acetate of an Escherichia coli strain expressing Era-dE, a dominant negative Era mutant; Inoue K et al.; Escherichia coli Era is a GTP binding protein and essential for cell growth . We have previously reported that an Era mutant, designated Era-dE, causes a dominant negative effect on the growth and the loss of the ability to utilize TCA cycle metabolites as carbon source when overproduced . To investigate the role of Era, the gene expression in the cells overproducing Era-dE was examined by DNA microarray analysis . The expression of lipA and nadAB, which are involved in lipoic acid synthesis and NAD synthesis, respectively, was found to be reduced in the cells overproducing Era-dE . Lipoic acid and NAD are essential cofactors for the activities of pyruvate dehydrogenase complex, 2-oxoglutarate dehydrogenase complex and glycine cleavage enzyme complex . The expression of numerous genes involved in dissimilatory carbon metabolism and carbon source transport was increased . This set of genes partially overlaps with the set of genes controlled by cAMP-CAP in E coli . Moreover, the growth defect of Era-dE overproduction was specifically enhanced by acetate but not by TCA cycle metabolites both in rich and synthetic media . Intracellular serine pool in Era-dE overproducing cells was found to be increased significantly compared to that of the cells overproducing wild-type Era . It was further found that even the wild-type E . coli cells not overproducing Era-dE became sensitive to acetate in the presence of serine in a medium . We propose that when Era-dE is overproduced, carbon fluxes to the TCA cycle and to C1 units become impaired, resulting in a higher cellular serine concentration . We demonstrated that such cells with a high serine concentration became sensitive to acetate, however the reason for this acetate sensitivity is not known at the present. Arch Insect Biochem Physiol, 2002 Aug, 50(4), 191 - 206 Cloning, partial purification and in vivo developmental profile of expression of the juvenile hormone epoxide hydrolase of Ctenocephalides felis; Keiser KC et al.; cDNAs encoding two different epoxide hydrolases (nCfEH1 and nCfEH2) were cloned from a cDNA library prepared from the wandering larval stage of the cat flea, Ctenocephalides felis . Predicted translations of the open reading frames indicated the clones encoded proteins of 464 (CfEH1) and 465 (CfEH2) amino acids . These proteins have a predicted molecular weight of 53 kDa and a putative 22 amino acid N-terminal hydrophobic membrane anchor . The amino acid sequences are 77% identical, and both are homologous to previously isolated epoxide hydrolases from Manduca sexta, Trichoplusia ni, and Rattus norvegicus . Purification of native juvenile hormone epoxide hydrolase (JHEH) from unfed adult cat fleas generated a partially pure protein that hydrolyzed juvenile hormone III to juvenile hormone III-diol . The amino terminal sequence of this;50-kDa protein is identical to the deduced amino terminus of the protein encoded by the nCfEH1 clone . Affinity-purified rabbit polyclonal antibodies raised against Escherichia coli-expressed HisCfEH1 recognized a approximately 50-kDa protein present in the partially purified fraction containing JHEH activity . Immunohistochemistry experiments using the same affinity-purified rabbit polyclonal antibodies localized the epoxide hydrolase in developing oocytes, fat body, and midgut epithelium of the adult flea . The presence of JHEH in various flea life stages and tissues was assessed by Northern blot and enzymatic activity assays . JHEH mRNA expression remained relatively constant throughout the different flea larval stages and was slightly elevated in the unfed adult flea . JHEH enzymatic activity was highest in the late larval, pupal, and adult stages . In all stages and tissues examined, JHEH activity was significantly lower than juvenile hormone esterase (JHE) activity, the other enzyme responsible for JH catalysis . Biopolymers, 2002 Sep, 64(6), 283 - 91 Solution structure of a pentasaccharide representing the repeating unit of the O-antigen polysaccharide from Escherichia coli O142: NMR spectroscopy and molecular simulation studies; Landersjo C et al.; Conformational studies have been performed of a pentasaccharide derived from the O-polysaccharide from Escherichia coli O142 . The polymer was selectively degraded by anhydrous hydrogen fluoride and reduced to yield an oligosaccharide model of its repeating unit, which in the branching region consists of four aminosugars . A comparison of (1)H and (13)C chemical shifts between the pentasaccharide and the polymer showed only minor differences, except where the cleavage had taken place, indicating that the oligomer is a good model of the repeating unit . Langevin dynamics and molecular dynamics simulations with explicit water molecules were carried out to sample the conformational space of the pentasaccharide . For the glycosidic linkages between the hexopyranoside residues, small but significant changes were observed between the simulation techniques . One-dimensional (1D) (1)H,(1)H double pulsed field gradient spin echo (DPFGSE) transverse rotating-frame Overhauser effect spectroscopy (T-ROESY) experiments were performed, and homonuclear cross-relaxation rates were obtained . Subsequently, a comparison of interproton distances from NMR experiment and the two simulation approaches showed that in all cases the use of explicit water in the simulations resulted in better agreement . Hydrogen-bond analysis of the trajectories from the molecular dynamics simulation revealed interresidue interactions to be important as a cluster of different hydrogen bonds and as a distinct highly populated hydrogen bond . NMR data are consistent with the presence of hydrogen bonding within the model of the repeating unit . J Biol Chem, 2002 Oct 4, 277(40), 37064 - 9 Epub 2002 Jul 17. Iron detoxification properties of Escherichia coli bacterioferritin . Attenuation of oxyradical chemistry; Bou-Abdallah F et al.; Bacterioferritin (EcBFR) of Escherichia coli is an iron-mineralizing hemoprotein composed of 24 identical subunits, each containing a dinuclear metal-binding site known as the "ferroxidase center." The chemistry of Fe(II) binding and oxidation and Fe(III) hydrolysis using H(2)O(2) as oxidant was studied by electrode oximetry, pH-stat, UV-visible spectrophotometry, and electron paramagnetic resonance spin trapping experiments . Absorption spectroscopy data demonstrate the oxidation of two Fe(II) per H(2)O(2) at the ferroxidase center, thus avoiding hydroxyl radical production via Fenton chemistry . The oxidation reaction with H(2)O(2) corresponds to {Fe(II)(2)-P}(Z) + H(2)O(2) --> {Fe(III)(2)O-P}(Z) + H(2)O, where {Fe(II)(2)-P}(Z) represents a diferrous ferroxidase center complex of the protein P with net charge Z and {Fe(III)(2)O-P}(Z) a micro-oxo-bridged diferric ferroxidase complex . The mineralization reaction is given by 2Fe(2+) + H(2)O(2) + 2H(2)O --> 2FeOOH((core)) + 4H(+), where two Fe(II) are again oxidized by one H(2)O(2) . Hydrogen peroxide is shown to be an intermediate product of dioxygen reduction when O(2) is used as the oxidant in both the ferroxidation and mineralization reactions . Most of the H(2)O(2) produced from O(2) is rapidly consumed in a subsequent ferroxidase reaction with Fe(II) to produce H(2)O . EPR spin trapping experiments show that the presence of EcBFR greatly attenuates the production of hydroxyl radical during Fe(II) oxidation by H(2)O(2), consistent with the ability of the bacterioferritin to facilitate the pairwise oxidation of Fe(II) by H(2)O(2), thus avoiding odd electron reduction products of oxygen and therefore oxidative damage to the protein and cellular components through oxygen radical chemistry. J Biol Chem, 2002 Sep 27, 277(39), 36345 - 50 Epub 2002 Jul 17. Distinct sites on G protein beta gamma subunits regulate different effector functions; Mirshahi T et al.; G proteins interact with effectors at multiple sites and regulate their activity . The functional significance of multiple contact points is not well understood . We previously identified three residues on distinct surfaces of Gbetagamma that are crucial for G protein-coupled inward rectifier K(+) (GIRK) channel activation . Here we show that mutations at these sites, S67K, S98T, and T128F, abolished or reduced direct GIRK current activation in inside-out patches, but, surprisingly, all mutants synergized with sodium in activating K(+) currents . Each of the three Gbeta(1) mutants bound the channel indicating that the defects reflected mainly functional impairments . We tested these mutants for functional interactions with effectors other than K(+) channels . With N-type calcium channels, Gbetagamma wild type and mutants all inhibited basal currents . A depolarizing pre-pulse relieved Gbetagamma inhibition of Ca(2+) currents by the wild type and the S98T and T128F mutants but not the S67K mutant . Both wild type and mutant Gbetagamma subunits activated phospholipase C beta(2) with similar potencies; however, the S67K mutant showed reduced maximal activity . These data establish a pattern where mutations can alter the Gbetagamma regulation of a specific effector function without affecting other Gbetagamma-mediated functions . Moreover, Ser-67 showed this pattern in all three effectors tested, suggesting that this residue participates in a common functional domain on Gbeta(1) that regulates several effectors . These data show that distinct domains within Gbetagamma subserve specific functional roles. J Biol Chem, 2002 Oct 4, 277(40), 37582 - 9 Epub 2002 Jul 17. Membrane-protein interactions contribute to efficient 27-hydroxylation of cholesterol by mitochondrial cytochrome P450 27A1; Murtazina D et al.; Mitochondrial cytochrome P450 27A1 (P450 27A1) catalyzes 27-hydroxylation of cholesterol, the first step in the alternative bile acid biosynthetic pathway . Although several crystal structures of P450s are known, no structural information is available for the mammalian, membrane-bound enzymes involved in the removal of cholesterol from the body . We prepared a three-dimensional model of P450 27A1 based on the structure of P450 BM-3 . Conservative and non-conservative mutations were introduced at hydrophobic and positively charged residues in the putative F-G loop and the adjacent helix G (positions 219-237) . Subcellular distribution of the mutant P450s expressed in Escherichia coli was used as a measure of membrane-protein interactions . Conservative substitutions of residues located on the surface, according to our model, L219V, L219I, Y220F, F223Y, L224I, R229K, V231L, F234Y, K236R, and R237K, weakened the association of the mutant P450s with the membrane and led to the appearance of up to 21% of P450 27A1 in the bacterial cytosol . It is likely that the mutated side chains are involved in binding to membrane phospholipids . Substitutions in the F-G loop did not significantly affect the K(m) value for cholesterol hydroxylation . However, non-conservative mutants, L219N, Y220A, Y220S, F223A, K226R, and R229A, had significantly impaired catalytic properties, indicating strict requirements for the size and polarity of the side chains at these positions for the catalysis . The results provide insight into the membrane topology of mitochondrial P450s and indicate the importance of membrane-protein interactions in the efficiency of reactions catalyzed by P450 27A1. Drug Metab Dispos, 2002 Aug, 30(8), 869 - 74 Metabolism of tamoxifen by recombinant human cytochrome P450 enzymes: formation of the 4-hydroxy, 4'-hydroxy and N-desmethyl metabolites and isomerization of trans-4-hydroxytamoxifen; Crewe HK et al.; The cytochrome P450 (P450)-mediated biotransformation of tamoxifen is important in determining both the clearance of the drug and its conversion to the active metabolite, trans-4-hydroxytamoxifen . Biotransformation by P450 forms expressed extrahepatically, such as in the breast and endometrium, may be particularly important in determining tissue-specific effects of tamoxifen . Moreover, tamoxifen may serve as a useful probe drug to examine the regioselectivity of different forms . Tamoxifen metabolism was investigated in vitro using recombinant human P450s . Forms CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7 were coexpressed in Escherichia coli with recombinant human NADPH-cytochrome P450 reductase . Bacterial membranes were harvested and incubated with tamoxifen or trans-4-hydroxytamoxifen under conditions supporting P450-mediated catalysis . CYP2D6 was the major catalyst of 4-hydroxylation at low tamoxifen concentrations (170 +/- 20 pmol/40 min/0.2 nmol P450 using 18 microM tamoxifen), but CYP2B6 showed significant activity at high substrate concentrations (28.1 +/- 0.8 and 3.1 +/- 0.5 nmol/120 min/0.2 nmol P450 for CYP2D6 and CYP2B6, respectively, using 250 microM tamoxifen) . These two forms also catalyzed 4'-hydroxylation (13.0 +/- 1.9 and 1.4 +/- 0.1 nmol/120 min/0.2 nmol P450, respectively, for CYP2B6 and CYP2D6 at 250 microM tamoxifen; 0.51 +/- 0.08 pmol/40 min/0.2 nmol P450 for CYP2B6 at 18 microM tamoxifen) . Tamoxifen N-demethylation was mediated by CYP2D6, 1A1, 1A2, and 3A4, at low substrate concentrations, with contributions by CYP1B1, 2C9, 2C19 and 3A5 at high concentrations . CYP1B1 was the principal catalyst of 4-hydroxytamoxifen trans-cis isomerization but CYP2B6 and CYP2C19 also contributed. Biophys J, 2002 Aug, 83(2), 1050 - 73 Polarized fluorescence depletion reports orientation distribution and rotational dynamics of muscle cross-bridges; Bell MG et al.; The method of polarized fluorescence depletion (PFD) has been applied to enhance the resolution of orientational distributions and dynamics obtained from fluorescence polarization (FP) experiments on ordered systems, particularly in muscle fibers . Previous FP data from single fluorescent probes were limited to the 2(nd)- and 4(th)-rank order parameters, <P(2)(cos beta)> and <P(4)(cos beta)>, of the probe angular distribution (beta) relative to the fiber axis and <P(2d)>, a coefficient describing the extent of rapid probe motions . We applied intense 12-micros polarized photoselection pulses to transiently populate the triplet state of rhodamine probes and measured the polarization of the ground-state depletion using a weak interrogation beam . PFD provides dynamic information describing the extent of motions on the time scale between the fluorescence lifetime (e.g., 4 ns) and the duration of the photoselection pulse and it potentially supplies information about the probe angular distribution corresponding to order parameters above rank 4 . Gizzard myosin regulatory light chain (RLC) was labeled with the 6-isomer of iodoacetamidotetramethylrhodamine and exchanged into rabbit psoas muscle fibers . In active contraction, dynamic motions of the RLC on the PFD time scale were intermediate between those observed in relaxation and rigor . The results indicate that previously observed disorder of the light chain region in contraction can be ascribed principally to dynamic motions on the microsecond time scale. FEBS Lett, 2002 Jul 17, 523(1-3), 234 - 8 Escherichia coli 6-pyruvoyltetrahydropterin synthase ortholog encoded by ygcM has a new catalytic activity for conversion of sepiapterin to 7,8-dihydropterin; Woo HJ et al.; The putative gene (ygcM) of Escherichia coli was verified in vitro to encode the ortholog of 6-pyruvoyltetrahydropterin synthase (PTPS) . Unexpectedly, the enzyme was found to convert sepiapterin to 7,8-dihydropterin without any cofactors . The enzymatic product 7,8-dihydropterin was identified by HPLC and mass spectrometry analyses, suggesting a novel activity of the enzyme to cleave the C6 side chain of sepiapterin . The optimal activity occurred at pH 6.5-7.0 . The reaction rate increased up to 3.2-fold at 60-80 degrees C, reflecting the thermal stability of the enzyme . The reaction required no metal ion and was activated slightly by low concentrations (1-5 mM) of EDTA . The apparent K(m) value for sepiapterin was determined as 0.92 mM and the V(max) value was 151.3 nmol/min/mg . The new catalytic function of E . coli PTPS does not imply any physiological role, because sepiapterin is not an endogenous substrate of the organism . The same activity, however, was also detected in a PTPS ortholog of Synechocystis sp . PCC 6803 but not significant in Drosophila and human enzymes, suggesting that the activity may be prevalent in bacterial PTPS orthologs. Anal Biochem, 2002 Jul 15, 306(2), 188 - 96 Electrochemical detection of nucleic base mismatches with ferrocenyl naphthalene diimide; Yamashita K et al.; Electrochemical detection of nucleic base mismatches was attempted successfully with ferrocenyl naphthalene diimide (FND) in a model system with 20-meric double-stranded oligonucleotides with or without a mismatch(es) . Thus, dA(20) or a 20-meric sequence of the lac Z gene was immobilized on a gold electrode and complementary oligonucleotides with different numbers of mismatches were allowed to hybridize in the presence of FND to give rise to an electrochemical signal . The signal intensity varied depending on the number of unpaired bases on the DNA duplex . From experiments with a quartz crystal microbalance, eight molecules of FND were found to bind to the 20-meric double-stranded oligos and this number decreased as the number of mismatches increased . These findings were further supported by matrix-assisted laser desorption ionization time-of-flight mass spectroscopy . This novel method will be useful for the analysis of single-nucleotide polymorphisms present on human genes. Neuron, 2002 Jul 3, 35(1), 39 - 50 Drosophila homeodomain protein dHb9 directs neuronal fate via crossrepressive and cell-nonautonomous mechanisms; Broihier HT et al.; Here we present the identification and characterization of dHb9, the Drosophila homolog of vertebrate Hb9, which encodes a factor central to motorneuron (MN) development . We show that dHb9 regulates neuronal fate by restricting expression of Lim3 and Even-skipped (Eve), two homeodomain (HD) proteins required for development of distinct neuronal classes . Also, dHb9 and Lim3 are activated independently of each other in a virtually identical population of ventrally and laterally projecting MNs . Surprisingly, dHb9 represses Lim3 cell nonautonomously in a subset of dorsally projecting MNs, revealing a novel role for intercellular signaling in the establishment of neuronal fate in Drosophila . Lastly, we provide evidence that dHb9 and Eve regulate each other's expression through Groucho-dependent crossrepression . This mutually antagonistic relationship bears similarity to the crossrepressive relationships between pairs of HD proteins that pattern the vertebrate neural tube. BMC Plant Biol . 2002 Jul 18;2(1):6. High frequency of phenotypic deviations in Physcomitrella patens plants transformed with a gene-disruption library; Egener T et al.; BACKGROUND: The moss Physcomitrella patens is an attractive model system for plant biology and functional genome analysis . It shares many biological features with higher plants but has the unique advantage of an efficient homologous recombination system for its nuclear DNA . This allows precise genetic manipulations and targeted knockouts to study gene function, an approach that due to the very low frequency of targeted recombination events is not routinely possible in any higher plant . RESULTS: As an important prerequisite for a large-scale gene/function correlation study in this plant, we are establishing a collection of Physcomitrella patens transformants with insertion mutations in most expressed genes . A low-redundancy moss cDNA library was mutagenised in E . coli using a derivative of the transposon Tn1000 . The resulting gene-disruption library was then used to transform Physcomitrella . Homologous recombination of the mutagenised cDNA with genomic coding sequences is expected to target insertion events preferentially to expressed genes . An immediate phenotypic analysis of transformants is made possible by the predominance of the haploid gametophytic state in the life cycle of the moss . Among the first 16,203 transformants analysed so far, we observed 2636 plants (= 16.2%) that differed from the wild-type in a variety of developmental, morphological and physiological characteristics . CONCLUSIONS: The high proportion of phenotypic deviations and the wide range of abnormalities observed among the transformants suggests that mutagenesis by gene-disruption library transformation is a useful strategy to establish a highly diverse population of Physcomitrella patens mutants for functional genome analysis. Mol Microbiol, 2002 Jul, 45(2), 521 - 32 LrhA as a new transcriptional key regulator of flagella, motility and chemotaxis genes in Escherichia coli; Lehnen D et al.; The function of the LysR-type regulator LrhA of Escherichia coli was defined by comparing whole-genome mRNA profiles from wild-type E . coli and an isogenic lrhA mutant on a DNA microarray . In the lrhA mutant, a large number (48) of genes involved in flagellation, motility and chemotaxis showed relative mRNA abundances increased by factors between 3 and 80 . When a representative set of five flagellar, motility and chemotaxis genes was tested in lacZ reporter gene fusions, similar factors for derepression were found in the lrhA mutant . In gel retardation experiments, the LrhA protein bound specifically to flhD and lrhA promoter DNA (apparent K(D) approximately 20 nM), whereas the promoters of fliC, fliA and trg were not bound by LrhA . The expression of flhDC (encoding FlhD(2)C(2)) was derepressed by a factor of 3.5 in the lrhA mutant . FlhD(2)C(2) is known as the master regulator for the expression of flagellar and chemotaxis genes . By DNase I footprinting, LrhA binding sites at the flhDC and lrhA promoters were identified . The lrhA gene was under positive autoregulation by LrhA as shown by gel retardation and lrhA expression studies . It is suggested that LrhA is a key regulator controlling the transcription of flagellar, motility and chemotaxis genes by regulating the synthesis and concentration of FlhD(2)C(2). Mol Microbiol, 2002 Jul, 45(2), 501 - 10 Rapid induction and reversal of a bacteriostatic condition by controlled expression of toxins and antitoxins; Pedersen K et al.; RelE and ChpAK (MazF) toxins of Escherichia coli have previously been described as proteins that mediate efficient cell killing . We show here that induction of relE or chpAK transcription does not confer cell killing but, instead, induces a static condition in which the cells are still viable but unable to proliferate . Later induction of transcription of the antitoxin genes relB or chpAI fully reversed the static condition induced by RelE and ChpAK respectively . We also provide a mechanistic explanation for these findings . Thus, induction of relE transcription severely inhibited translation, whereas induction of chpAK transcription inhibited both translation and replication . Hence, most likely, lack of colony formation is due to inhibition of translation in the case of relE and inhibition of translation and/or replication in the case of chpAK . Consistent with this proposal, later induction of transcription of the cognate antitoxin genes simultaneously reversed cell stasis and the inhibitory effects of RelE and ChpAK on macromolecular syntheses . These results preclude that RelE and ChpAK mediate cell killing during the conditions used here . In vivo and in vitro analyses of a mutant RelE protein supported that inhibition of colony formation was due to inhibition of translation. Mol Microbiol, 2002 Jul, 45(2), 439 - 52 Identification of amino acids in the Dr adhesin required for binding to decay-accelerating factor; Van Loy CP et al.; Members of the Dr family of adhesins of Escherichia coli recognize as a receptor the Dr(a) blood-group antigen present on the complement regulatory and signalling molecule, decay-accelerating factor (DAF) . One member of this family, the Dr haemagglutinin, also binds to a second receptor, type IV collagen . Structure/function information regarding these adhesins has been limited and domains directly involved in the interaction with DAF have not been determined . We devised a strategy to identify amino acids in the Dr haemagglutinin that are specifically involved in the interaction with DAF . The gene encoding the adhesive subunit, draE, was subjected to random mutagenesis and used to complement a strain defective for its expression . The resulting mutants were enriched and screened to obtain those that do not bind to DAF, but retain binding to type IV collagen . Individual amino acid changes at positions 10, 63, 65, 75, 77, 79 and 131 of the mature DraE sequence significantly reduced the ability of the DraE adhesin to bind DAF, but not collagen . Over half of the mutants obtained had substitutions within amino acids 63-81 . Analysis of predicted structures of DraE suggest that these proximal residues may cluster to form a binding domain for DAF. Mol Microbiol, 2002 Jul, 45(2), 423 - 38 Novel mode of transcription regulation of divergently overlapping promoters by PhoP, the regulator of two-component system sensing external magnesium availability; Yamamoto K et al.; PhoP is a response regulator of the PhoQ-PhoP two-component system controlling a set of the Mg(II)-response genes in Escherichia coli . Here we demonstrate the mode of transcription regulation by phosphorylated PhoP of divergently transcribed mgtA and treR genes, each encoding a putative Mg(II) transporter and a repressor for the trehalose utilization operon respectively . Under Mg(II)-limiting conditions in vivo, two promoters, the upstream constitutive P2 and the downstream inducible P1, were detected for the mgtA gene . Gel-shift analysis in vitro using purified PhoP indicates its binding to a single DNA target, centred between -43 and -24 of the mgtAP1 promoter . This region includes the PhoP box, which consists of a direct repeat of the heptanucleotide sequence (T)G(T)TT(AA) . Site-directed mutagenesis studies indicate the critical roles for T (position 3), T (position 4) and A (position 6) for PhoP-dependent transcription from mgtAP1 . DNase I footprinting assays reveal weak binding of PhoP to this PhoP box, but the binding becomes stronger in the simultaneous presence of RNA polymerase . Likewise the RNA polymerase binding to the P1 promoter becomes stronger in the presence of PhoP . For the PhoP-assisted formation of open complex at the mgtAP1 promoter, however, the carboxy-terminal domain of alpha subunit (alpha CTD) is not needed . For transcription in vivo of the treR gene, four promoters were identified . The most upstream promoter treRP4 divergently overlaps with the mgtAP1 promoter, sharing the same sequence as the respective -10 signal in the opposite direction . In vitro transcription using mutant promoters support this prediction . In the presence of PhoP, transcription from the promoter treRP3 was repressed with concomitant activation of mgtAP1 transcription . The PhoP box is located between -46 and -30 with respect to treRP3, and the alpha CTD is needed for this repression. Mol Microbiol, 2002 Jul, 45(2), 351 - 63 Nac-mediated repression of the serA promoter of Escherichia coli; Blauwkamp TA et al.; Escherichia coli and related bacteria contain two paralogous PII-like proteins involved in nitrogen regulation, the glnB product, PII, and the glnK product, GlnK . Previous studies have shown that cells lacking both PII and GlnK have a severe growth defect on minimal media, resulting from elevated expression of the Ntr regulon . Here, we show that this growth defect is caused by activity of the nac product, Nac, a LysR-type transcription factor that is part of the Ntr regulon . Cells with elevated Ntr expression that also contain a null mutation in nac displayed growth rates on minimal medium similar to the wild type . When expressed from high-copy plasmids, Nac imparts a growth defect to wild-type cells in an expression level-dependent manner . Neither expression of Nac nor lack thereof significantly affected Ntr gene expression, suggesting that the activity of Nac at one or more promoters outside the Ntr regulon was responsible for its effects . The growth defect of cells lacking both PII and GlnK was also eliminated upon supplementation of minimal medium with serine or glycine for solid medium or with serine or glycine and glutamine for liquid medium . These observations suggest that high Nac expression results in a reduction in serine biosynthesis . beta-Galactosidase activity expressed from a Mu d1 insertion in serA was reduced approximately 10-fold in cells with high Nac expression . We hypothesize that one role of Nac is to limit serine biosynthesis as part of a cellular mechanism to reduce metabolism in a co-ordinated manner when cells become starved for nitrogen. Mol Microbiol, 2002 Jul, 45(2), 289 - 306 Gene expression profiling of Escherichia coli growth transitions: an expanded stringent response model; Chang DE et al.; When conditions cause bacterial growth to stop, extensive reprogramming of physiology and gene expression allows for the cell's survival . We used whole-genome DNA arrays to determine the system response in Escherichia coli cells experiencing transient growth arrest caused by glucose-lactose diauxie and H2O2 treatment, and also entry into stationary phase . The results show that growth-arrested cells induce stringent control of several gene systems . The vast majority of genes encoding the transcription and translation apparatus immediately downregulate, followed by a global return to steady state when growth resumes . Approximately one-half of the amino acid biosynthesis genes downregulate during growth arrest, with the notable exception of the his operon, which transiently upregulates in the diauxie experiment . Nucleotide biosynthesis downregulates, a result that is again consistent with the stringent response . Likewise, aerobic metabolism downregulates during growth arrest, and the results led us to suggest a model for stringent control of the ArcA regulon . The stationary phase stress response fully induces during growth arrest, whether transient or permanent, in a manner consistent with known mechanisms related to stringent control . Cells similarly induce the addiction module anti-toxin and toxin genes during growth arrest; the latter are known to inhibit translation and DNA replication . The results indicate that in all aspects of the response cells do not distinguish between transient and potentially permanent growth arrest (stationary phase) . We introduce an expanded model for the stringent response that integrates induction of stationary phase survival genes and inhibition of transcription, translation and DNA replication . Central to the model is the reprogramming of transcription by guanosine tetraphosphate (ppGpp), which provides for the cell's rapid response to growth arrest and, by virtue of its brief half-life, the ability to quickly resume growth as changing conditions allow. Glycobiology, 2002 Jul, 12(7), 435 - 42 Molecular cloning, gene organization, and expression of mouse Mpi encoding phosphomannose isomerase; Davis JA et al.; Phosphomannose isomerase (PMI) interconverts fructose-6-P (Fru-6-P) and mannose-6-P (Man-6-P), linking energy metabolism to protein glycosylation . We have cloned the mouse Mpi cDNA, analyzed its genomic organization, and studied the expression in different tissues . The Mpi gene has eight exons covering 7.2 kb . The structure and intron-exon boundaries are essentially the same as its human ortholog with 85% amino acid identity . Mpi is alternatively spliced at the 3' end, resulting in three messages with different 3'-untranslated regions . Mpi expression is regulated at both the transcription and translation levels, with the highest expression level in testis . Rabbit antibodies prepared against mouse PMI expressed in E . coli recognize a single 47-kDa band . Immunohistochemistry of mouse tissues shows general cytosolic staining in all cells . In testis, staining is intense in round spermatids and residual bodies, moderate in pachytene spermatocytes, and weak in spermatogonia and spermatozoa . In contrast, northern blot analysis shows comparable transcripts of 1.8 and 1.6 kb in pachytene spermatocytes and round spermatids, suggesting delayed translation of PMI . The stage-specific expression of PMI in testis may be important for KDN synthesis, which requires Man-6-P, or it may be needed to ensure sufficient glycosylation precursors in cells that do not utilize glucose and instead rely on lactate and pyruvate. J Biol Chem, 2002 Sep 27, 277(39), 35853 - 61 Epub 2002 Jul 16. A single enzyme catalyses formation of Trypanothione from glutathione and spermidine in Trypanosoma cruzi; Oza SL et al.; Protozoa of the order Kinetoplastida differ from other organisms in their ability to conjugate glutathione (l-gamma-glutamyl-cysteinyl-glycine) and spermidine to form trypanothione {N(1),N(8)-bis(glutathionyl)spermidine}, a metabolite involved in defense against chemical and oxidant stress and other biosynthetic functions . In Crithidia fasciculata, trypanothione is synthesized from GSH and spermidine via the intermediate glutathionylspermidine in two distinct ATP-dependent reactions catalyzed by glutathionylspermidine synthetase (GspS; EC ) and trypanothione synthetase (TryS; EC ), respectively . Here we have cloned a single copy gene (TcTryS) from Trypanosoma cruzi encoding a protein with 61% sequence identity with CfTryS but only 31% with CfGspS . Saccharomyces cerevisiae transformed with TcTryS were able to synthesize glutathionylspermidine and trypanothione, suggesting that this enzyme is able to catalyze both biosynthetic steps, unlike CfTryS . When cultures were supplemented with aminopropylcadaverine, yeast transformants contained glutathionylaminopropylcadaverine and homotrypanothione {N(1),N(9)-bis(glutathionyl)aminopropylcadaverine}, metabolites that have been previously identified in T . cruzi, but not in C . fasciculata . Kinetic studies on recombinant TcTryS purified from Escherichia coli revealed that the enzyme displays high-substrate inhibition with glutathione (K(m) and K(i) of 0.57 and 1.2 mm, respectively, and k(cat) of 3.4 s(-1)), but obeys Michaelis-Menten kinetics with spermidine, aminopropylcadaverine, glutathionylspermidine, and MgATP as variable substrate . The recombinant enzyme possesses weak amidase activity and can hydrolyze trypanothione, homotrypanothione, or glutathionylspermidine to glutathione and the corresponding polyamine. J Inorg Biochem, 2002 Jul 25, 91(1), 59 - 69 Reactivity of recombinant and mutant vanadium bromoperoxidase from the red alga Corallina officinalis; Carter JN et al.; Vanadium bromoperoxidase (VBPO) from the marine red alga Corallina officinalis has been cloned and heterologously expressed in Esherichia coli . The sequence for the full-length cDNA of VBPO from C . officinalis is reported . Steady state kinetic analyses of monochlorodimedone bromination reveals the recombinant enzyme behaves similarly to native VBPO from the alga . The kinetic parameters (K(m)(Br-)=1.2 mM, K(m)(H(2)O(2))=17.0 microM) at the optimal pH 6.5 for recombinant VBPO are similar to reported values for enzyme purified from the alga . The first site-directed mutagenesis experiment on VBPO is reported . Mutation of a conserved active site histidine residue to alanine (H480A) results in the loss of the ability to efficiently oxidize bromide, but retains the ability to oxidize iodide . Kinetic parameters (K(m)(I-)=33 mM, K(m)(H(2)O(2))=200 microM) for iodoperoxidase activity were determined for mutant H480A . The presence of conserved consensus sequences for the active sites of VBPO from marine sources shows its usefulness in obtaining recombinant forms of VBPO . Furthermore, mutagenesis of the conserved extra-histidine residue shows the importance of this residue in the oxidation of halides by hydrogen peroxide. Prog Lipid Res, 2002 Sep, 41(5), 407 - 35 Multi-subunit acetyl-CoA carboxylases; Cronan JE Jr et al.; Acetyl-CoA carboxylase (ACC) catalyses the first committed step of fatty acid synthesis, the carboxylation of acetyl-CoA to malonyl-CoA . Two physically distinct types of enzymes are found in nature . Bacterial and most plant chloroplasts contain a multi-subunit ACC (MS-ACC) enzyme that is readily dissociated into its component proteins . Mammals, fungi, and plant cytosols contain the second type of ACC, a single large multifunctional polypeptide . This review will focus on the structures, regulation, and enzymatic mechanisms of the bacterial and plant MS-ACCs. J Surg Res, 2002 Jun 15, 105(2), 189 - 94 Cold storage sensitizes rat femoral artery to an endotoxin-induced decrease in endothelium-dependent relaxation; Piepot HA et al.; Cold-stored arteries, tissues or organs are transferred in vascular, reconstructive and transplantation surgery . The function of transferred vessels and tissues diminishes when infection complicates transplantation, thereby contributing to morbidity . To evaluate the mechanisms involved, the effects of cold storage on basal vascular reactivity and the sensitivity to the vascular effects of endotoxin were tested in isolated rat femoral artery segments . A crossover design was followed, so that prior to cold storage 4 vessels were incubated for 2 h at 37 degrees C with endotoxin (Escherichia coli 0127:B8, 50 microg mL(-1)) in Krebs solution and 4 with Krebs solution only, while, after cold storage, segments from the former vessels were incubated with Krebs solution only and segments from the latter with endotoxin in Krebs solution . Vascular reactivity was tested in a wire myograph by the addition of depolarizing 125 mM KCl or norepinephrine (NE) as well as the endothelium-dependent vasodilator acetylcholine (ACh) and endothelium-independent vasodilator sodium nitroprusside (SNP) . Cold storage did not affect vascular reactivity in the absence of endotoxin . Endotoxin decreased maximum response to NE prior to storage and sensitivity to SNP prior to and after cold storage . After cold storage, endotoxin decreased relaxation to ACh and increased vasoconstriction in response to KCl and NE (P < 0.05) . We conclude that cold storage does not alter endothelial and smooth muscle function but sensitizes rat femoral artery to an endotoxin-induced decrease in endothelium-dependent relaxation and thereby to an increase in vasoconstrictor responses, whereas endotoxin alone only decreases receptor-dependent vasoconstrictor responses and sensitivity to NO donors . This may explain in part the detrimental effect of infection on function of cold-stored arterial grafts and tissue/organ transfers. Bioconjug Chem, 2002 Jul-Aug, 13(4), 707 - 12 Establishment of intein-mediated protein ligation under denaturing conditions: C-terminal labeling of a single-chain antibody for biochip screening; Sydor JR et al.; Intein-mediated protein ligation is a recently developed method that enables the C-terminal labeling of proteins . This technique requires a correctly folded intein mutant that is fused to the C-terminus of a target protein to create a thioester, which allows the ligation of a peptide with an N-terminal cysteine (1, 2) . Here we describe the establishment of this method for the labeling, under denaturing conditions, of target proteins that are expressed insolubly as intein fusion proteins . A GFPuv fusion protein with the Mycobacterium xenopi gyrA intein was expressed in inclusion bodies in Escherichia coli and initially used as a model protein to verify intein cleavage activity under different refolding conditions . The intein showed activity after refolding in nondenaturing and slightly denaturing conditions . A construct of the same intein with an anti-neutravidin single-chain antibody was also expressed in an insoluble form . The intein-mediated ligation was established for this single chain antibody-intein fusion protein under denaturing conditions in 4 M urea to prevent significant precipitation of the fusion protein during the first refolding step . Under optimized conditions, the single-chain antibody was labeled with a fluorescent peptide and used for antigen screening on a biochip after final refolding . This screening procedure allowed the determination of binding characteristics of the scFv for avidin proteins in a miniaturized format. Ir J Med Sci, 2001 Jul-Sep, 170(3), 172 - 5 Inhaled nitric oxide in combination with volume resuscitation refines a porcine model of endotoxic shock; Herity NA et al.; BACKGROUND: Existing porcine models of endotoxic shock poorly represent the human situation . AIMS: To assess whether the cardiovascular profile of a porcine model could be improved by refining the protocol . METHODS: In 30 pigs, right and left heart pressures and cardiac output were measured . Lipopolysaccharide (LPS) was administered as a bolus (n=12), as a 30 minute infusion (n=6) or as a 30 minute infusion along with inhaled NO and volume resuscitation (n=6) and six sham-treated pigs received normal saline . Haemodynamic values were measured over three hours . RESULTS: LPS increased pulmonary vascular resistance (PVR) (13.3 +/- 1.4 to 37.0 +/- 3.9kPa/l per sec, p<0.05) and reduced cardiac output (6.0 +/- 0.6 to 4.8 +/- 0.41/min) . Mortality was 50% within 30 minutes . Inhaled NO and volume resuscitation controlled pulmonary vascular resistance (PVR) and preserved CO . Systemic vascular resistance (SVR) declined in the first hour (118.4 +/- 11.8 to 65.8 +/- 8.2kPa/l per sec, p<0.05) and remained low . CONCLUSIONS: Porcine models of endotoxaemia based on LPS administration are a poor model of human septic shock, but can be improved by regulating PVR and supporting CO which may contribute to future studies of septic shock Chem Commun (Camb), 2002 Jan 7, (1), 1 - 11 Expanding the genetic code; Wang L et al.; The ability to incorporate unnatural amino acids into proteins directly in living cells will provide new tools to study protein and cellular function, and may generate proteins or even organisms with enhanced properties . Due to the limited promiscuity of some synthetases, natural amino acids can be substituted with close analogs at multiple sites using auxotrophic strains . Alternatively, this can be achieved by deactivating the editing function of some synthetases . The addition of new amino acids to the genetic code, however, requires additional components of the protein biosynthetic machinery including a novel tRNA-codon pair, an aminoacyl-tRNA synthetase, and an amino acid . This new set of components functions orthogonally to the counterparts of the common 20 amino acids, i.e., the orthogonal synthetase (and only this synthetase) aminoacylates the orthogonal tRNA (and only this tRNA) with the unnatural amino acid only, and the resulting acylated tRNA inserts the unnatural amino acid only in response to the unique codon . Using this strategy, the genetic code of Escherichia coli has been expanded to incorporate unnatural amino acids with a fidelity rivaling that of natural amino acids . This methodology is being applied to other cell types and unnatural analogs with a variety of functionalities. Pancreatology, 2001, 1(5), 461 - 5 The pathobiochemistry of hereditary pancreatitis: studies on recombinant human cationic trypsinogen; Sahin-Toth M; BACKGROUND/AIMS: This study attempts to identify the biochemical alterations in human cationic trypsinogen and trypsin caused by the hereditary pancreatitis-associated mutations Arg117-->His and Asn21-->Ile . METHODS: Recombinant wild-type and mutant human cationic trypsinogens were expressed in Escherichia coli and purified to homogeneity, and trypsin autolysis and trypsinogen autoactivation were characterized . RESULTS: Both mutations significantly enhanced the autoactivation of human cationic trypsinogen . In addition, the Arg117-->His mutation inhibited autocatalytic inactivation of trypsin, while the Asn21-->Ile mutation had no such effect . CONCLUSIONS: The findings support the notion that enhanced trypsinogen activation in the pancreas is the common initiating step in hereditary pancreatitis, whereas trypsin stabilization plays a role in cases associated with the Arg117-->His mutation. Proc Natl Acad Sci U S A, 2002 Jul 23, 99(15), 9697 - 702 Epub 2002 Jul 15. Global analysis of mRNA decay and abundance in Escherichia coli at single-gene resolution using two-color fluorescent DNA microarrays; Bernstein JA et al.; Much of the information available about factors that affect mRNA decay in Escherichia coli, and by inference in other bacteria, has been gleaned from study of less than 25 of the approximately 4,300 predicted E . coli messages . To investigate these factors more broadly, we examined the half-lives and steady-state abundance of known and predicted E . coli mRNAs at single-gene resolution by using two-color fluorescent DNA microarrays . An rRNA-based strategy for normalization of microarray data was developed to permit quantitation of mRNA decay after transcriptional arrest by rifampicin . We found that globally, mRNA half-lives were similar in nutrient-rich media and defined media in which the generation time was approximately tripled . A wide range of stabilities was observed for individual mRNAs of E . coli, although approximately 80% of all mRNAs had half-lives between 3 and 8 min . Genes having biologically related metabolic functions were commonly observed to have similar stabilities . Whereas the half-lives of a limited number of mRNAs correlated positively with their abundance, we found that overall, increased mRNA stability is not predictive of increased abundance . Neither the density of putative sites of cleavage by RNase E, which is believed to initiate mRNA decay in E . coli, nor the free energy of folding of 5' or 3' untranslated region sequences was predictive of mRNA half-life . Our results identify previously unsuspected features of mRNA decay at a global level and also indicate that generalizations about decay derived from the study of individual gene transcripts may have limited applicability. Plant Cell, 2002 Jul, 14(7), 1579 - 89 Detection and localization of a chloroplast-encoded HU-like protein that organizes chloroplast nucleoids; Kobayashi T et al.; Chloroplast DNA (cpDNA) is packed into discrete structures called chloroplast nucleoids (cp-nucleoids) . The structure of cpDNA is thought to be important for its maintenance and regulation . In bacteria and mitochondria, histone-like proteins (such as HU and Abf2, respectively) are abundant and play important roles in DNA organization . However, a primary structural protein has yet to be found in cp-nucleoids . Here, we identified an abundant DNA binding protein from isolated cp-nucleoids of the primitive red alga Cyanidioschyzon merolae . The purified protein had sequence homology with the bacterial histone-like protein HU, and it complemented HU-lacking Escherichia coli mutants . The protein, called HC (histone-like protein of chloroplast), was encoded by a single gene (CmhupA) in the C . merolae chloroplast genome . Using immunofluorescence and immunoelectron microscopy, we demonstrated that HC was distributed uniformly throughout the entire cp-nucleoid . The protein was expressed constitutively throughout the cell and the chloroplast division cycle, and it was able to condense DNA . These results indicate that HC, a bacteria-derived histone-like protein, primarily organizes cpDNA into the nucleoid. Am J Respir Crit Care Med, 2002 Jul 15, 166(2), 192 - 9 Protection of human lung cells against hyperoxia using the DNA base excision repair genes hOgg1 and Fpg; Wu M et al.; Hyperoxia causes pulmonary toxicity in part by injuring alveolar epithelial cells . Previous studies have shown that toxic oxygen-derived species damage DNA and this damage is recognized and repaired by either human enzyme 8-oxoguanine DNA glycosylase (hOgg1) or Escherichia coli enzyme formamidopyrimidine DNA glycosylase (Fpg) . To determine whether these DNA repair proteins can reduce O(2)-mediated DNA damage in lung cells, A549 lung epithelial cells were transduced with either hOgg1 or Fpg using a retroviral vector containing enhanced green fluorescent protein . Expression of each gene in the transduced cells was confirmed by fluorescent microscopy, Northern blotting, Western blotting, and an enzymatic oligonucleotide cleavage assay . A549 cells expressing either hOgg1 or Fpg were protected from hyperoxia as evidenced by a decrease in DNA damage and a corresponding increase in cell survival . Further, we determined that overexpression of hOgg1 or Fpg partially mitigated the toxic effects of hydrogen peroxide in lung cells . Our data suggest that increased expression of DNA base excision repair genes might represent a new approach for protecting critical lung cells from the toxic effects of hyperoxia. Gene, 2002 Jun 12, 292(1-2), 57 - 63 Expression of long- and short-type FK506 binding proteins in hyperthermophilic archaea; Ideno A et al.; It has been reported that the hyperthermophilic archaeon, Methanococcus jannaschii, possesses two FKBP (FK506 binding protein) genes in the genome, one being 26 kDa FKBP (long-type FKBP) and the other, 18 kDa FKBP (short-type FKBP) . FKBP is a family of peptidyl-prolyl cis-trans isomerases (PPIases) . In order to clarify the difference between their roles in archaeal cells, they were expressed in Escherichia coli, and their PPIase and chaperone-like protein-folding activities were investigated . The catalytic efficiency of the PPIase activity of the long-type FKBP was significantly lower than that of short-type FKBP (less than 1/1000) which is comparable to that of human FKBP12 . Both FKBPs showed chaperone-like protein-folding activity to enhance the refolding yield of an unfolded protein (Thermoplasma citrate synthase) in vitro . The chaperone-like protein-folding activity of the short type was higher than that of the long type . While the intracellular content of long-type FKBP in M . jannaschii tended to increase, that of short-type FKBP obviously decreased at growth temperatures higher than the optimum of 85 degrees C . In Pyrococcus horikoshii, another hyperthermophilic archaeon, the intracellular content of long-type FKBP did not change with temperature (80-102 degrees C) . These results suggest that long-type FKBP functions at any temperature in the cells as a chaperone to maintain the folding states of intracellular proteins . On the other hand, short-type FKBP may be required at lower temperatures . Peptidyl-prolyl cis-trans isomerization is known to be a rate-limiting step in protein-folding and is slower at low temperature . Since the PPIase activity of short-type FKBP was much stronger than that of the long type, it may be required to accelerate the folding of intracellular proteins and for the hyperthermophilic cell to live at low growth temperatures. Biochem J, 2002 Sep 15, 366(Pt 3), 977 - 81 A non-radioactive method for the assay of many serine/threonine-specific protein kinases; Ross H et al.; The generation of drugs that modulate the activities of particular protein kinases has become a prime focus of the pharmaceutical and biotechnology industry . Consequently, improved methods for the development of high-throughput screening formats for these enzymes is a high priority . In the present study, we have designed three generic peptide substrates that can be used to assay a diverse range of protein kinases . These peptides share a common seven-residue epitope that includes the site of phosphorylation, and against which we have generated a phospho-specific antibody . Thus a large number of serine/threonine-specific protein kinases can be screened using a simple non-radioactive format. Biochem J, 2002 Oct 15, 367(Pt 2), 321 - 7 Involvement of delta-aminolaevulinate synthase encoded by the parasite gene in de novo haem synthesis by Plasmodium falciparum; Varadharajan S et al.; The malaria parasite can synthesize haem de novo . In the present study, the expression of the parasite gene for delta-aminolaevulinate synthase (Pf ALAS ) has been studied by reverse transcriptase PCR analysis of the mRNA, protein expression using antibodies to the recombinant protein expressed in Escherichia coli and assay of ALAS enzyme activity in Plasmodium falciparum in culture . The gene is expressed through all stages of intra-erythrocytic parasite growth, with a small increase during the trophozoite stage . Antibodies to the erythrocyte ALAS do not cross-react with the parasite enzyme and vice versa . The recombinant enzyme activity is inhibited by ethanolamine and the latter inhibits haem synthesis in P . falciparum and growth in culture . The parasite ALAS is localized in the mitochondrion and its import into mitochondria in a cell-free import assay has been demonstrated . The import is blocked by haemin . On the basis of these results, the following conclusions are arrived at: PfALAS has distinct immunological identity and inhibitor specificity and is therefore a drug target . The malaria parasite synthesizes haem through the mitochondrion/cytosol partnership, and this assumes significance in light of the presence of apicoplasts in the parasite that may be capable of independent haem synthesis . The Pf ALAS gene is functional and vital for parasite haem synthesis and parasite survival. Biochemistry, 2002 Jul 23, 41(29), 9021 - 5 A critical residue in the folding pathway of an integral membrane protein; Nagy JK et al.; Although a number of common diseases are a direct consequence of membrane protein misfolding, studies of membrane protein folding and misfolding lag well behind those of soluble proteins . Here it is shown that an interfacial residue, Tyr16, of the integral membrane protein diacylglycerol kinase (DAGK) plays a critical role in the folding pathway of this protein . Properly folded Y16C exhibits kinetic parameters and stability similar to wild-type DAGK . However, when unfolded and then allowed to spontaneously fold in the presence of model membranes, Y16C exhibits dramatically lower rates and efficiencies of functional assembly compared to the wild-type protein . The Y16C mutant represents a class of mutations which may be commonly found in disease-related membrane proteins. Cell Res, 2002 Jun, 12(2), 143 - 50 Recombinant scorpion insectotoxin AaIT kills specifically insect cells but not human cells; Ji SJ et al.; The nucleotide sequence deduced from the amino acid sequence of the scorpion insectotoxin AaIT was chemically synthesized and was expressed in Escherichia coli . The authenticity of this in vitro expressed peptide was confirmed by N-terminal peptide sequencing . Two groups of bioassays, artificial diet incorporation assay and contact insecticidal effect assay, were carried out separately to verify the toxicity of this recombinant toxin . At the end of a 24 h experimental period, more than 60% of the testing diamondback moth (Plutella xylostella) larvae were killed in both groups with LC50 value of 18.4 microM and 0.70 microM respectively . Cytotoxicity assay using cultured Sf9 insect cells and MCF-7 human cells demonstrated that the toxin AaIT had specific toxicity against insect cells but not human cells . Only 0.13 microM recombinant toxin was needed to kill 50% of cultured insect cells while as much as 1.3 microM toxin had absolutely no effect on human cells . Insect cells produced obvious intrusions from their plasma membrane before broken up . We infer that toxin AaIT bind to a putative sodium channel in these insect cells and open the channel persistently, which would result in Na+ influx and finally cause destruction of insect cells. Folia Biol (Praha), 2002, 48(3), 96 - 101 Characterization of functional domains of human interferon gamma by specific monoclonal antibodies; Nacheva G et al.; For the study of functional domains of hIFNgamma, two MABs were characterized . Both MABs, named 1E11 and 5D6, were sequence-specific for hIFNgamma . According to the estimated additivity index, they recognized epitopes located at distinct non-overlapping areas of the hIFNgamma molecule . When pre-incubated with hIFNgamma, MAB 1E11 was able to neutralize the antiviral as well as the antiproliferative activity of the cytokine . This indicated that MAB 1E11 was specific either for the domain responsible for binding to the cell receptor or for a domain required for both functions . By contrast, the second MAB 5D6 did not interfere with any of the two hIFNgamma biological activities . The epitope of MAB 5D6 was located between the amino acid residues Leu 135 and Glu 143 by using different forms of C-terminally truncated hIFNgamma . These data allow the conclusion that the last nine C-terminal amino acids are not essential for the receptor binding and biological functioning of this cytokine . The possible role of hIFNgamma C-terminus in the intracellular cascade of events is discussed. Infect Immun, 2002 Aug, 70(8), 4669 - 77 Escherichia coli shiga-like toxins induce apoptosis and cleavage of poly(ADP-ribose) polymerase via in vitro activation of caspases; Ching JC et al.; Shiga-like toxin-producing Escherichia coli causes hemorrhagic colitis and hemolytic-uremic syndrome in association with the production of Shiga-like toxins, which induce cell death via either necrosis or apoptosis . However, the abilities of different Shiga-like toxins to trigger apoptosis and the sequence of intracellular signaling events mediating the death of epithelial cells have not been completely defined . Fluorescent dye staining with acridine orange and ethidium bromide showed that Shiga-like toxin 1 (Stx1) induced apoptosis of HEp-2 cells in a dose- and time-dependent manner . Stx2 also induced apoptosis in a dose-dependent manner . Apoptosis induced by Stx1 (200 ng/ml) and apoptosis induced by Stx2 (200 ng/ml) were maximal following incubation with cells for 24 h (94.3% +/- 1.8% and 81.7% +/- 5.2% of the cells, respectively) . Toxin-treated cells showed characteristic features of apoptosis, including membrane blebbing, DNA fragmentation, chromatin condensation, cell shrinkage, and the formation of apoptotic bodies, as assessed by transmission electron microscopy . Stx2c induced apoptosis weakly even at a high dose (1,000 ng/ml for 24 h; 26.7% +/- 1.3% of the cells), whereas Stx2e did not induce apoptosis of HEp-2 cells . Thin-layer chromatography confirmed that HEp-2 cells express the Stx1-Stx2-Stx2c receptor, globotriaosylceramide (Gb3), but not the Stx2e receptor, globotetraosylceramide (Gb4) . Western blot analysis of poly(ADP-ribose) polymerase (PARP), a DNA repair enzyme, demonstrated that incubation with Stx1 and Stx2 induced cleavage, whereas incubation with Stx2e did not result in cleavage of PARP . A pan-caspase inhibitor (Z-VAD-FMK) and a caspase-8-specific inhibitor (Z-IETD-FMK) eliminated, in a dose-dependent fashion, the cleavage of PARP induced by Shiga-like toxins . Caspase-8 activation was confirmed by detection of cleavage of this enzyme by immunoblotting . Cleavage of caspase-9 and the proapoptotic member of the Bcl-2 family BID was also induced by Stx1, as determined by immunoblot analyses . We conclude that different Shiga-like toxins induce different degrees of apoptosis that correlates with toxin binding to the glycolipid receptor Gb3 and that caspases play an integral role in the signal transduction cascade leading to toxin-mediated programmed cell death. Infect Immun, 2002 Aug, 70(8), 4556 - 63 Identification of Escherichia coli outer membrane protein A receptor on human brain microvascular endothelial cells; Prasadarao NV; Neonatal Escherichia coli meningitis continues to be a diagnostic and treatment challenge despite the availability of active antibiotics . Our earlier studies have shown that outer membrane protein A (OmpA) is one of the major factors responsible for Escherichia coli traversal across the blood-brain barrier that constitutes a lining of brain microvascular endothelial cells (BMEC) . In this study we showed that OmpA binds to a 95-kDa human BMEC (HBMEC) glycoprotein (Ecgp) for E . coli invasion . Ecgp was partially purified by wheat germ agglutinin and Maackia amurensis lectin (MAL) affinity chromatography . The MAL affinity-purified HBMEC proteins bound to OmpA(+) E . coli but not to OmpA(-) E . coli . In addition, the deglycosylated MAL-bound proteins still interact with OmpA(+) E . coli, indicating the role of protein backbone in mediating the OmpA binding to HBMEC . Interestingly, the MAL affinity-bound fraction showed one more protein, a 65-kDa protein that bound to OmpA(+) E . coli in addition to Ecgp . Further, the 65-kDa protein was shown to be a cleavage product of Ecgp . Immunocytochemistry of HBMEC infected with OmpA(+) E . coli by using anti-Ecgp antibody suggests that Ecgp clusters at the E . coli entry site . Anti-Ecgp antibody also reacted to microvascular endothelium on human brain tissue sections, indicating the biological relevance of Ecgp in E . coli meningitis . Partial N-terminal amino acid sequence of Ecgp suggested that it has 87% sequence homology to gp96, an endoplasmic reticulum-resident molecular chaperone that is often expressed on the cell surface . In contrast, the 65-kDa protein, which could be the internal portion of Ecgp, showed 70% sequence homology to an S-fimbria-binding sialoglycoprotein reported earlier . These results suggest that OmpA interacts with Ecgp via the carbohydrate epitope, as well as with the protein portion for invading HBMEC. Infect Immun, 2002 Aug, 70(8), 4485 - 93 Structure-function analysis of decay-accelerating factor: identification of residues important for binding of the Escherichia coli Dr adhesin and complement regulation; Hasan RJ et al.; Decay-accelerating factor (DAF), a complement regulatory protein, also serves as a receptor for Dr adhesin-bearing Escherichia coli . The repeat three of DAF was shown to be important in Dr adhesin binding and complement regulation . However, Dr adhesins do not bind to red blood cells with the rare polymorphism of DAF, designated Dr(a(-)); these cells contain a point mutation (Ser165-Leu) in DAF repeat three . In addition, monoclonal antibody IH4 specific against repeat three was shown to block both Dr adhesin binding and complement regulatory functions of DAF . Therefore, to identify residues important in binding of Dr adhesin and IH4 and in regulating complement, we mutated 11 amino acids-predominantly those in close proximity to Ser165 to alanine-and expressed these mutations in Chinese hamster ovary cells . To map the mutations, we built a homology model of repeat three based on the poxvirus complement inhibitory protein, using the EXDIS, DIAMOD, and FANTOM programs . We show that perhaps Ser155, and not Ser165, is the key amino acid that interacts with the Dr adhesin and amino acids Gly159, Tyr160, and Leu162 and also aids in binding Dr adhesin . The IH4 binding epitope contains residues Phe148, Ser155, and L171 . Residues Phe123 and Phe148 at the interface of repeat 2-3, and also Phe154 in the repeat three cavity, were important for complement regulation . Our results show that residues affecting the tested functions are located on the same loop (148 to 171), at the same surface of repeat three, and that the Dr adhesin-binding and complement regulatory epitopes of DAF appear to be distinct and are approximately 20 A apart. Infect Immun, 2002 Aug, 70(8), 4302 - 11 Identification of an aggregative adhesion fimbria (AAF) type III-encoding operon in enteroaggregative Escherichia coli as a sensitive probe for detecting the AAF-encoding operon family; Bernier C et al.; Enteroaggregative Escherichia coli (EAEC) is recognized as an emerging cause of diarrhea in children and adults worldwide, and recent studies have implicated EAEC in persistent diarrhea in patients infected with human immunodeficiency virus (HIV) . In this study, we identified aggregative adhesion fimbria type III (AAF-III) in isolate 55989, a typical EAEC strain . Analysis of the sequence of the plasmid-borne agg-3 gene cluster encoding AAF-III showed this cluster to be closely related to the agg and aaf operons and to the afa operons carried by diffusely adherent pathogenic E . coli . We investigated the adhesion properties of a collection of 25 EAEC strains isolated from HIV-infected patients presenting with persistent diarrhea . We found that a minority of strains (36%) carried sequences similar to those of the agg and aaf operons, which encode AAF-I and AAF-II, respectively . We developed PCR assays specific for the agg-3 operon . In our collection, the frequency of AAF-III strains was similar (12%) to that of AAF-I strains (16%) but higher than that of AAF-II isolates (0%) . Differences between EAEC strains in terms of the virulence factors present render detection of these strains difficult with the available DNA probes . Based on comparison of the agg, aaf, and agg-3 operons, we defined an AAF probe internal to the adhesion gene clusters and demonstrated that it was efficient for the identification of EAEC strains . We investigated 32 EAEC isolates, of which only 34.4% were detected with the classical CVD432 probe (detecting pAA virulence plasmids) whereas 65.6% were detected with the AAF probe. Infect Immun, 2002 Aug, 70(8), 4196 - 203 Evidence that the variable regions of the central domain of VlsE are antigenic during infection with lyme disease spirochetes; McDowell JV et al.; It has been postulated that the vls system of the Lyme disease spirochetes contributes to immune evasion through antigenic variation . While it is clear that vlsE undergoes sequence change within its variable regions at a high frequency during the early stages of infection, a definitive role in immune evasion has not been demonstrated . In this report we assessed the murine and human humoral immune response to recombinant (r)-VlsE variants that originally arose during infection in mice . Immunoblot analyses of r-VlsE variants were conducted by using serum samples collected from mice infected with Borrelia burgdorferi clones that carried different vlsE variants . All of the r-VlsE variants were recognized by infection sera regardless of the identity of the infecting clone or isolate . In addition, all variants were immunoreactive with a panel of human Lyme disease patient serum samples . It is evident from these analyses that the infection-induced VlsE variants share common epitopes that reside within conserved segments of these proteins . However, preabsorption experiments revealed that the variable regions of the central domain of VlsE, which undergo rapid mutation during infection, also influence the antigenic properties of the protein . A subset of the antibodies elicited against vlsE variants that differ in the sequences of their variable regions were found to be variant specific . Hence, in spite of a robust antibody response to conserved segments of VlsE, infection-induced sequence changes within the variable regions alter the antigenicity of VlsE . These results provide the first direct evidence of antigenic variation in the VlsE protein. Infect Immun, 2002 Aug, 70(8), 4106 - 11 Novel 33-kilodalton lipoprotein from Mycobacterium leprae; Maeda Y et al.; A novel Mycobacterium leprae lipoprotein LpK (accession no . ML0603) was identified from the genomic database . The 1,116-bp open reading frame encodes a 371-amino-acid precursor protein with an N-terminal signal sequence and a consensus motif for lipid conjugation . Expression of the protein, LpK, in Escherichia coli revealed a 33-kDa protein, and metabolic labeling experiments and globomycin treatment proved that the protein was lipidated . Fractionation of M . leprae demonstrated that this lipoprotein was a membrane protein of M . leprae . The purified lipoprotein was found to induce production of interleukin-12 in human peripheral blood monocytes . The studies imply that M . leprae LpK is involved in protective immunity against leprosy and may be a candidate for vaccine design. Infect Immun, 2002 Aug, 70(8), 4099 - 105 LuxS-mediated quorum sensing in Borrelia burgdorferi, the lyme disease spirochete; Stevenson B et al.; The establishment of Borrelia burgdorferi infection involves numerous interactions between the bacteria and a variety of vertebrate host and arthropod vector tissues . This complex process requires regulated synthesis of many bacterial proteins . We now demonstrate that these spirochetes utilize a LuxS/autoinducer-2 (AI-2)-based quorum-sensing mechanism to regulate protein expression, the first system of cell-cell communication to be described in a spirochete . The luxS gene of B . burgdorferi was identified and demonstrated to encode a functional enzyme by complementation of an Escherichia coli luxS mutant . Cultured B . burgdorferi responded to AI-2 by altering the expression levels of a large number of proteins, including the complement regulator factor H-binding Erp proteins . Through this mechanism, a population of Lyme disease spirochetes may synchronize production of specific proteins needed for infection processes. Infect Immun, 2002 Aug, 70(8), 4053 - 8 Proteasomal degradation of cytotoxic necrotizing factor 1-activated rac; Lerm M et al.; The cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli has been shown to activate members of the Rho family by deamidation of glutamine 63 . This amino acid is essential for hydrolysis of GTP, and any substitution results in a constitutively active Rho . Activation of Rho induces the formation of stress fibers, filopodia, and membrane ruffles due to activation of RhoA, Cdc42, and Rac, respectively . Here we show that the level of endogenous Rac decreased in CNF1-treated HEK293 and HeLa cells . The amount of mRNA remained unaffected, leaving the possibility that Rac is subject to proteolytic degradation . Treatment of cells with lactacystin, an inhibitor of the 26S proteasome, protected Rac from degradation . We have previously shown that CNF1 activates the c-Jun N-terminal kinase (JNK) only transiently in HeLa cells (M . Lerm, J . Selzer, A . Hoffmeyer, U . R . Rapp, K . Aktories, and G . Schmidt, Infect . Immun . 67:496-503, 1998) . Here we show that CNF1-induced JNK activation is stabilized in the presence of lactacystin . The data indicate that Rac is degraded by a proteasome-dependent pathway in CNF1-treated cells. Int J Parasitol, 2002 Aug, 32(9), 1107 - 15 Molecular cloning and expression of the catalytic subunit of protein kinase A from Trypanosoma cruzi; Huang H et al.; The activation of protein kinase A (cyclic adenosine monophosphate-dependent protein kinase) by cyclic adenosine monophosphate is believed to play an important role in regulating the growth and differentiation of Trypanosoma cruzi . A PCR using degenerate oligonucleotide primers against conserved motifs in the VIb and VIII subdomains of the ACG family of serine/threonine protein kinases was utilised to amplify regions corresponding to the parasite homologue of the protein kinase A catalytic subunit . This putative protein kinase A fragment was used to isolate the entire gene from T . cruzi genomic libraries . The deduced 329 amino acid sequence of this gene contained all of the signature motifs of known protein kinase A catalytic subunit proteins . The recombinant protein expressed in Escherichia coli was shown to phosphorylate Kemptide, a synthetic peptide substrate of protein kinase A, in a protein kinase inhibitor (PKI)-inhibitory manner . Immunoprecipitation with polyclonal antisera raised against recombinant protein of this gene was able to pull-down PKI-inhibitory phosphotransferase activity from epimastigote lysates . Immunoblot and Northern blot analyses, in combination with enzyme activity assays, revealed that this gene was a stage-regulated enzyme in T . cruzi with higher levels and activity being present in epimastigotes compared with amastigotes or trypomastigotes . Overall these studies indicate that the cloned gene encodes an authentic protein kinase A catalytic subunit from T . cruzi and are the first demonstration of PKI-inhibitory phosphotransferase activity in an expressed protozoan protein kinase A catalytic subunit. Cytometry, 2002 Jun 1, 48(2), 71 - 9 Measurement of nuclear factor-kappa B translocation on lipopolysaccharide-activated human dendritic cells by confocal microscopy and flow cytometry; Blaecke A et al.; BACKGROUND: Nuclear factor kappa B (NF-kappaB) is a ubiquitously expressed transcription factor that regulates cytokine and immunoglobulin (Ig) gene expression . In most cell types, the inactive p50/p65 NF-kappaB heterodimer is located in the cytoplasm, complexed to its IkappaB inhibitory unit . Stimulation of cells by various reagents such as bacterial endotoxin or cytokines leads to a dissociation of NF-kappaB from IkappaB and a rapid translocation of free NF-kappaB to the nucleus . The aim of this article is to define optimal conditions for the measurement of NF-kappaB translocation by both confocal microscopy and flow cytometry . METHODS: Four commercial anti-NF-kappaB antibodies were evaluated by confocal microscopy, after using two methods of fixation and permeabilization of the cells . These antibodies were examined further by flow cytometry on purified nuclei . RESULTS: Paraformaldehyde-methanol treatment of dendritic cells is a good combination to visualize NF-kappaB translocation by confocal microscopy . Three of the four antibodies tested gave good results on nonactivated and on lipopolysaccharide (LPS)-activated dendritic cells . The measurement of NF-kappaB translocation by flow cytometry on purified nuclei is a quick and sensitive method . Only one of the four evaluated antibodies showed a significant difference between nonactivated and activated cells . CONCLUSIONS; Microscopy and flow cytometry are quick and reproducible methods to measure NF-kappaB translocation and can be adapted to identify new molecules that activate dendritic cells . Eur J Immunol, 2002 Jun, 32(6), 1737 - 47 CD8+ T cell apoptosis induced by Escherichia coli heat-labile enterotoxin B subunit occurs via a novel pathway involving NF-kappaB-dependent caspase activation; Salmond RJ et al.; The B subunit of Escherichia coli heat-labile enterotoxin (EtxB) is a potent immunomodulatory molecule capable of treating and preventing autoimmune disease . These properties result from its ability to bind to glycolipid receptors, principally G(M1) ganglioside, and modulate immune cell function . EtxB receptor binding causes B cell activation, modulates monocyte cytokine secretion and triggers apoptosis of CD8+ T cells . These wide-ranging effects suggest that B subunit receptor interaction triggers signaling events affecting cellular differentiation . We have investigated the processes by which EtxB induces CD8+ T cell apoptosis . We show that receptor interaction by EtxB activates caspase-3 in CD8+ but not in CD4+ T cells . Inhibition of caspase-3 blocks the apoptotic process . EtxB induces the activation of NF-kappaB in both CD8+ and CD4+ T cells . The findings that (i) SN50, a peptide inhibitor of NF-kappaB nuclear translocation, prevents caspase-3 activation and subsequent apoptosis, and (ii) CD8+CD4- thymocytes from transgenic mice expressing a dominant-negative form of the IkappaBalpha protein were markedly less susceptible to EtxB-induced apoptosis than cells from wild-type mice, indicate that NF-kappaB is important in the induction of the apoptotic pathway . Further investigations revealed that while caspase-8 activity is detected concomitant to caspase-3, caspase-9 activation, following mitochondrial cytochrome c release, is detectable later on . These observations are consistent with death receptor-mediated signaling, however, experiments using lpr/lpr and p55 TNFR -/- mice rule out the involvement of Fas and the p55 TNF receptor, respectively . The data therefore indicate that EtxB-mediated apoptosis occurs via a novel pathway involving NF-kappaB. Int J Cancer, 2002 Jul 20, 100(3), 367 - 74 Pharmacokinetic and tumor-seeking properties of recombinant and nonrecombinant anti-carcinoembryonic antigen antibody fragments; Groulet A et al.; Production of recombinant antibody fragments in bacterial expression systems results in intentional or fortuitous differences compared to the original products prepared by hybridoma technology . These differences may have significant effects not only on antigen-binding properties but also on pharmacokinetic and tumor-seeking properties . Our major goal was to investigate some of these possible differences . We produced in Escherichia coli an rFab' fragment containing only 1 cysteine residue in the hinge region; the fragment was derived from a mouse MAb (F6) specific for CEA . The rFab' had a slightly lower m.w . and a higher isoelectric point relative to the corresponding nonrecombinant fragment (pFab') . This was explained by the absence of N-glycosylation on the V kappa domain of rFab' . V kappa glycosylation had no significant effect on antibody-binding affinity and kinetics . However, rFab' was eliminated from the circulation much faster than pFab', and the maximal dose accumulated in the tumor was reduced relative to pFab' . Thus, glycosylation appears to modify the targeting efficiency of antibody fragments . rF(ab')(2) fragments were obtained either spontaneously from the culture supernatant of E . coli or by chemical cross-linking {rcF(ab')(2)} . We observed improved tumor targeting with rcF(ab')(2) compared to rF(ab')(2), which could be explained by the greater stability of the thioether compared to the disulfide linkage . These results demonstrate that a single cysteine residue in the hinge region of rFab' is particularly well suited to prepare stable, chemically coupled, bivalent or bispecific antibodies, avoiding intrahinge disulfide bonding and thus achieving higher production yields . Biotechnol Bioeng, 2002 Aug 5, 79(3), 271 - 6 Affinity separation using an Fv antibody fragment-"smart" polymer conjugate; Fong RB et al.; Poly(N-isopropylacrylamide), or PNIPAAm, is considered a "smart" polymer because it sharply precipitates when heated above a critical temperature, about 32 degrees C in water, and redissolves when cooled . Conjugates made of PNIPAAm and IgG antibodies also exhibit the same critical temperature behavior . Interestingly, antigens that are complexed with these conjugates can also be phase-separated along with the conjugates . In this work, we conjugated PNIPAAm for the first time to the immunoglobulin Fv fragment, the smallest fragment of an antibody that still retains the antigenic affinity of the whole antibody . For our studies, we used an Fv fragment that strongly binds hen egg white lysozyme (HEL) . The purified Fv fragment-polymer conjugate precipitated at the same temperature as did the pure polymer . After addition of the conjugate to a mixture containing HEL and after thermal separation of the conjugate at 37 degrees C, the amount of HEL in solution was reduced by as much as 80% . We were able to demonstrate the reversibility of the separation through three cycles of precipitation and dissolution . It was also possible to recover free HEL by thermal separation of the conjugate in the presence of an eluant, 50 mM diethylamine . The conjugate can then be recycled for second use . In conclusion, immunoseparations can be performed using smart polymer conjugates made with just the variable domains of an antibody . Unlike whole antibodies, fragments of antibodies can be produced in Escherichia coli, allowing easier genetic engineering of the antibody