Zh Mikrobiol Epidemiol Immunobiol, 2003 Jul-Aug, (4), 110 - 7 {Development of mucosal influenza inactivated vaccines}; Gendon IuZ; In this review recent data on the development of mucosal influenza inactivated vaccines with the use of mucosoadhesive adjuvants are presented . After the intranasal administration of such vaccines the formation of not only systemic, but also local immunity (IgA antibodies), as well as cross protection against the variants of influenza virus within its serotype, can be observed . Some mucosal influenza vaccines have passed clinical tests . Certain drawbacks of Escherichia thermolabile enterotoxin, used as mucosoadhesive adjuvant, and difficulties which may arise in mass immunization with mucosal vaccines due to the necessity of introducing them in two or three intranasal administrations are indicated. Biotechnol Bioeng, 2003 Oct 20, 84(2), 129 - 44 Analysis of Escherichia coli anaplerotic metabolism and its regulation mechanisms from the metabolic responses to altered dilution rates and phosphoenolpyruvate carboxykinase knockout; Yang C et al.; The gluconeogenic phosphoenolpyruvate (PEP) carboxykinase is active in Escherichia coli during its growth on glucose . The present study investigated the influence of growth rates and PEP carboxykinase knockout on the anaplerotic fluxes in E . coli . The intracellular fluxes were determined using the complementary methods of flux ratio analysis and metabolic flux analysis based on {U-(13)C(6)}glucose labeling experiments and 2D nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids and glycerol . Significant activity of PEP carboxykinase was identified in wild-type E . coli, and the ATP dissipation for the futile cycling via this reaction accounted for up to 8.2% of the total energy flux . Flux analysis of pck deletion mutant revealed that abolishment of PEP carboxykinase activity resulted in a remarkably reduced flux through the anaplerotic PEP carboxylase and the activation of the glyoxylate shunt, with 23% of isocitrate found being channeled in the glyoxylate shunt . The changes in intracellular metabolite concentrations and specific enzyme activities associated with different growth rates and pck deletion, were also determined . Combining the measurement data of in vivo fluxes, metabolite concentrations and enzyme activities, the in vivo regulations of PEP carboxykinase flux, PEP carboxylation, and glyoxylate shunt in E . coli are discussed . Microbiol Mol Biol Rev, 2003 Sep, 67(3), 454 - 72, table of contents Transmembrane movement of exogenous long-chain fatty acids: proteins, enzymes, and vectorial esterification; Black PN et al.; The processes that govern the regulated transport of long-chain fatty acids across the plasma membrane are quite distinct compared to counterparts involved in the transport of hydrophilic solutes such as sugars and amino acids . These differences stem from the unique physical and chemical properties of long-chain fatty acids . To date, several distinct classes of proteins have been shown to participate in the transport of exogenous long-chain fatty acids across the membrane . More recent work is consistent with the hypothesis that in addition to the role played by proteins in this process, there is a diffusional component which must also be considered . Central to the development of this hypothesis are the appropriate experimental systems, which can be manipulated using the tools of molecular genetics . Escherichia coli and Saccharomyces cerevisiae are ideally suited as model systems to study this process in that both (i) exhibit saturable long-chain fatty acid transport at low ligand concentrations, (ii) have specific membrane-bound and membrane-associated proteins that are components of the transport apparatus, and (iii) can be easily manipulated using the tools of molecular genetics . In both systems, central players in the process of fatty acid transport are fatty acid transport proteins (FadL or Fat1p) and fatty acyl coenzyme A (CoA) synthetase (FACS; fatty acid CoA ligase {AMP forming} {EC 6.2.1.3}) . FACS appears to function in concert with FadL (bacteria) or Fat1p (yeast) in the conversion of the free fatty acid to CoA thioesters concomitant with transport, thereby rendering this process unidirectional . This process of trapping transported fatty acids represents one fundamental mechanism operational in the transport of exogenous fatty acids. JAMA, 2003 Sep 10, 290(10), 1337 - 44 Effect of an oral Shiga toxin-binding agent on diarrhea-associated hemolytic uremic syndrome in children: a randomized controlled trial; Trachtman H et al.; CONTEXT: Diarrhea-associated hemolytic uremic syndrome (HUS) is the most common cause of acute renal failure in children . Most cases are caused by an intestinal infection with Shiga toxin-producing strains of Escherichia coli . OBJECTIVE: To determine if administration of an oral agent that binds Shiga toxin could diminish the severity of diarrhea-associated HUS in pediatric patients . DESIGN, SETTING, AND PATIENTS: Multicenter, randomized, double-blind, placebo-controlled clinical trial of 145 children (96 experimental and 49 placebo) aged 6 months to 18 years with diarrhea-associated HUS conducted between July 27, 1997, and April 14, 2001, at 26 tertiary care pediatric nephrology centers in the United States and Canada . Trial included 2 phases, the hospital course for treatment of the acute illness and a 60-day outpatient follow-up period after discharge from the hospital . INTERVENTION: Patients were assigned to receive the binding agent, 500 mg/kg daily, or cornmeal placebo orally for 7 days in a 2:1 randomization scheme . MAIN OUTCOME MEASURES: Combined frequency of death or serious extrarenal events and need for dialysis in the experimental vs placebo group . RESULTS: A total of 62 patients (43%) were male and 123 (85%) were white . The median age of the patients was 4.2 years . Most patients (59%) were transferred from other hospitals to participating sites . The severity of disease at the time of randomization was comparable in the 2 groups . The prevalence of death or serious extrarenal events was 18% and 20% in the experimental and placebo groups, respectively (P =.82) . Dialysis was required in 42% of experimental and 39% of placebo groups (P =.86) . CONCLUSIONS: Oral therapy with a Shiga toxin-binding agent failed to diminish the severity of disease in pediatric patients with diarrhea-associated HUS. J Biol Chem, 2003 Nov 21, 278(47), 46424 - 31 Epub 2003 Sep 08. Biochemical characterization of a CDC6-like protein from the crenarchaeon Sulfolobus solfataricus; De Felice M et al.; Cdc6 proteins play an essential role in the initiation of chromosomal DNA replication in Eukarya . Genes coding for putative homologs of Cdc6 have been also identified in the genomic sequence of Archaea, but the properties of the corresponding proteins have been poorly investigated so far . Herein, we report the biochemical characterization of one of the three putative Cdc6-like factors from the hyperthermophilic crenarchaeon Sulfolobus solfataricus (SsoCdc6-1) . SsoCdc6-1 was overproduced in Escherichia coli as a His-tagged protein and purified to homogeneity . Gel filtration and glycerol gradient ultracentrifugation experiments indicated that this protein behaves as a monomer in solution (molecular mass of about 45 kDa) . We demonstrated that SsoCdc6-1 binds single- and double-stranded DNA molecules by electrophoretic mobility shift assays . SsoCdc6-1 undergoes autophosphorylation in vitro and possesses a weak ATPase activity, whereas the protein with a mutation in the Walker A motif (Lys-59 --> Ala) is completely unable to hydrolyze ATP and does not autophosphorylate . We found that SsoCdc6-1 strongly inhibits the ATPase and DNA helicase activity of the S . solfataricus MCM protein . These findings provide the first in vitro biochemical evidence of a functional interaction between a MCM complex and a Cdc6 factor and have important implications for the understanding of the Cdc6 biological function. J Biol Chem, 2003 Nov 21, 278(47), 46994 - 7001 Epub 2003 Sep 08. Mutations in the alpha8 loop of human APE1 alter binding and cleavage of DNA containing an abasic site; Shen JC et al.; Recent crystallographic studies reveal loops in human AP endonuclease 1 (APE1) that interact with the major and minor grooves of DNA containing apurinic/apyrimidinic (AP) sites . These loops are postulated to stabilize the DNA helix and the flipped out AP residue . The loop alpha8 interacts with the major groove on the 3' side of the AP site . To determine the essentiality of the amino acids that constitute the alpha8 loop, we created a mutant library containing random nucleotides at codons 222-229 that, in wild-type APE1, specify the sequence NPKGNKKN . Upon expression of the library (2 x 10(6) different clones) in Escherichia coli and multiple rounds of selection with the alkylating agent methyl-methane sulfonate (MMS), we obtained approximately 2 x 10(5) active mutants that complemented the MMS sensitivity of AP endonuclease-deficient E . coli . DNA sequencing showed that active mutants tolerated amino acid substitutions at all eight randomized positions . Basic and uncharged polar amino acids together comprised the majority of substitutions, reflecting the positively charged, polar character of the wild-type loop . Asn-222, Asn-226, and Asn-229 exhibited the least mutability, consistent with x-ray data showing that each asparagine contacts a DNA phosphate . Substitutions at residues 226-229, located nearer to the AP site, that reduced basicity or hydrogen bonding potential, increased Km 2- to 6-fold and decreased AP site binding; substitutions at residues 222-225 exhibited lesser effects . This initial mutational analysis of the alpha8 loop supports and extends the conclusion of crystallographic studies that the loop is important for binding of AP.DNA and AP site incision. J Biochem (Tokyo), 2003 Aug, 134(2), 277 - 85 Reaction of aspartate aminotransferase with C5-dicarboxylic acids: comparison with the reaction with C4-dicarboxylic acids; Islam MM et al.; The reaction of Escherichia coli aspartate aminotransferase (AspAT) with glutamate and other C5-dicarboxylates was analyzed in order to compare its mechanism of action toward C5 substrates with that toward C4 substrates, which had been extensively characterized . The association of the amino-group protonated and unprotonated forms of glutamate (SH(+) and S, respectively) with the Schiff-base protonated and unprotonated forms of the enzyme (E(L)H(+) and E(L), respectively) yields at least three forms of the Michaelis complex, whereas in the case of aspartate, only two species of this complex exist, E(L).SH(+) and E(L)H(+).S . The reaction of AspAT with 2-methylglutamate can be explained only when we consider all the protonation states of the Michaelis complex . Based on the previous crystallographic studies {Miyahara et al . (1994) J . Biochem . 116, 1001-1012}, we consider that glutamate binds to the open form of AspAT and takes an extended conformation in the Michaelis complex, with the alpha-amino group of glutamate oriented in the opposite direction to the Schiff base . This is in contrast to the Michaelis complex of aspartate, in which a strong interaction of the alpha-amino group of aspartate and the Schiff base excludes the presence of the species E(L)H(+).SH(+) . It is concluded that AspAT recognizes the two types of dicarboxylates with different chain lengths by changing the gross conformation of the enzyme protein. J Biochem (Tokyo), 2003 Aug, 134(2), 251 - 7 Identification and biochemical characterization of plant acylamino acid-releasing enzyme; Yamauchi Y et al.; Plant acylamino acid-releasing enzyme (AARE) catalyzing the N-terminal hydrolysis of N(alpha)-acylpeptides to release N(alpha)-acylated amino acids, was biochemically characterized using recombinant and native AAREs . A cDNA encoding a deduced Arabidopsis thaliana AARE (AtAARE) was cloned and sequenced . The deduced amino acid sequence encoded a 764 amino acid protein of 83.9 kDa, which was 31.8% identical with that of rat AARE . In particular, the proposed catalytic residues (Ser, Asp, and His) of AARE, called the "catalytic triad residues, " were completely conserved . Recombinant AtAARE was expressed in Escherichia coli and confirmed to be a functional AARE . Native AAREs were prepared from A . thaliana and cucumber (Cucumis sativus, L.) plants . Both native AAREs were tetrameric proteins of 350 kDa comprising four subunits of 82 kDa, and showed typical enzymological properties of other AAREs, i.e . sensitivity to diisopropyl fluorophosphate, an optimum pH of around 7.0, and an optimum temperature of 37 degrees C . Both the native and recombinant AAREs were immunochemically homologous . Intracelluar fractionation analysis showed that the AARE was mainly present in the stroma of chloroplasts . Native AARE degraded the glycated ribulose-1,5-bisphoshate carboxylase/oxygenase protein but not the native protein . Thus, plant AARE might be involved in not only catalysis of the N-terminal hydrolysis of N(alpha)-acylpeptides but also the elimination of glycated proteins. J Biochem (Tokyo), 2003 Aug, 134(2), 211 - 7 A HEAT-repeats containing protein, IaiH, stabilizes the iron-sulfur cluster bound to the cyanobacterial IscA homologue, IscA2; Morimoto K et al.; IscA homologues are involved in iron-sulfur cluster biosynthesis . In the non-nitrogen-fixing cyanobacterium Synechocystis PCC 6803, there are two IscA homologues, SLR1417 and SLR1565 (designated IscA1 and IscA2), of which only IscA2 exists as a protein complex with the HEAT-repeat-containing protein, SLR1098 (IaiH) . We observed that the absorption spectrum of the recombinant IscA2/IaiH complex resembles that of IscA2 alone, although it is sharper . In the presence of dithiothreitol, the {2Fe-2S} cluster of IscA2 alone, but not of the IscA2/IaiH complex, became reductively labile upon the addition of sodium dithionite . This implies that the IscA2 moiety of the {2Fe-2S} cluster is stabilized by the presence of IaiH . The {2Fe-2S} cluster of the IscA2/IaiH complex was destabilized by sodium dithionite in the absence of dithiothreitol, suggesting that the in vivo stability of the iron-sulfur cluster in the IscA2/IaiH complex is influenced by the redox state of cellular thiols . When any one of three conserved cysteine residues in IscA2, potential ligands for the {2Fe-2S} cluster, was replaced with serine, the amount of assembled {2Fe-2S} cluster and protein complex was significantly reduced in E . coli cells . The cysteine mutated IscA2/IaiH complexes that were present all contained a {2Fe-2S}-like cluster suggesting that the assembly of a stable iron-sulfur cluster bound to IscA2 is required for efficient and stable complex formation . Truncated IaiH proteins were analyzed using the yeast two-hybrid assay to identify the essential domain of IaiH that interacts physically with IscA2 . At least 2 of the 5 N-terminal HEAT repeats of IaiH were found to be required for interaction with IscA2. FEBS Lett, 2003 Sep 11, 551(1-3), 63 - 70 Two mammalian glucosamine-6-phosphate deaminases: a structural and genetic study; Arreola R et al.; Glucosamine-6-phosphate deaminase (EC 3.5.99.6) is an allosteric enzyme that catalyzes the reversible conversion of D-glucosamine-6-phosphate into D-fructose-6-phosphate and ammonium . Here we describe the existence of two mammalian glucosamine-6-phosphate deaminase enzymes . We present the crystallographic structure of one of them, the long human glucosamine-6-phosphate deaminase, at 1.75 A resolution . Crystals belong to the space group P2(1)2(1)2(1) and present a whole hexamer in the asymmetric unit . The active-site lid (residues 162-182) presented significant structural differences among monomers . Interestingly the region with the largest differences, when compared with the Escherichia coli homologue, was found to be close to the active site . These structural differences can be related to the kinetic and allosteric properties of both mammalian enzymes. FEBS Lett, 2003 Sep 11, 551(1-3), 53 - 7 PmAV, a novel gene involved in virus resistance of shrimp Penaeus monodon; Luo T et al.; Diseases caused by viruses especially by white spot syndrome virus (WSSV) are the greatest challenge to worldwide shrimp aquaculture . The innate immunity of shrimp has attracted extensive attention, but no factor involved in the virus resistance has been reported . Here we report for the first time the identification of an antiviral gene from shrimp Penaeus monodon . A differential cDNA (designated as PmAV) cloned from virus-resistant shrimp P . monodon by differential display (DD) was found to have an open reading frame (ORF) encoding a 170 amino acid peptide with a C-type lectin-like domain (CTLD) . The PmAV gene was expressed in Escherichia coli and the protein was purified . Recombinant PmAV protein displayed a strong antiviral activity in inhibiting virus-induced cytopathic effect in fish cell in vitro . Moreover, native PmAV protein was isolated from shrimp hemolymph by immuno-affinity chromatography and confirmed by Western blot . No agglutination activity was observed both in recombinant and native PmAV protein . Immunohistological study showed that PmAV protein was located mainly in the cytoplasm, and not bound to the shrimp WSSV . It implies that the antiviral mechanism of PmAV protein is not by inhibiting the attachment of virus to target host cell . The discovery of PmAV gene might provide a clue to elucidate the innate immunity of marine invertebrates and would be helpful to shrimp viral disease control. Anal Chem, 2003 Jul 1, 75(13), 3010 - 8 Quantitative determination of noncovalent binding interactions using automated nanoelectrospray mass spectrometry; Zhang S et al.; Electrospray ionization mass spectrometry (ESI-MS) has proven to be an extremely powerful tool for studying biomolecular structures and noncovalent interactions . Here we report a method using a fully automated, chip-based nanoESI-MS system to determine the dissociation constants (Kd) for the complexes of two different proteins with their ligands . The automated nanoelectrospray system, consisting of the NanoMate and ESI chip, serves functionally as a combination of autosampler and nanoelectrospray ionization source . This system provides all the advantages of conventional nanoelectrospray plus automated, high-throughput analyses without carryover . The automated nanoESI system was used to investigate quantitative noncovalent interactions between ribonuclease A (RNase A) and cytidylic acid ligands (2'-CMP, CTP), a well-characterized model protein-ligand complex, and between an inactive endocellulase mutant (Thermobifida fusca Cel6A D117Acd) and four oligosaccharide ligands (cellotriose, cellotetraose, cellopentaose, cellohexaose) . Both titration and competitive binding approaches were performed prior to automated nanoESI-MS analysis with a Q-TOF mass spectrometer . Dissociation constants for each complex were calculated from the sum of ion peak areas of free and complexed proteins during the titration and competition experiments . The measured Kd values for the RNase A-CMP and Cel6A D117Acd-G3 complexes were found to be in excellent agreement with the available published values obtained by standard spectroscopic titration techniques . To our knowledge, this is the first report of using an ESI-MS approach to study the interactions between a cellulase and oligosaccharides . The results provide new insights for understanding the nature of cellulase-cellulose interactions. Mol Imaging, 2003 Apr, 2(2), 93 - 112 Development of a new reporter gene system--dsRed/xanthine phosphoribosyltransferase-xanthine for molecular imaging of processes behind the intact blood-brain barrier; Doubrovin M et al.; We report the development of a novel dual-modality fusion reporter gene system consisting of Escherichia coli xanthine phosphoribosyltransferase (XPRT) for nuclear imaging with radiolabeled xanthine and Discosoma red fluorescent protein for optical fluorescent imaging applications . The dsRed/XPRT fusion gene was successfully created and stably transduced into RG2 glioma cells, and both reporters were shown to be functional . The level of dsRed fluorescence directly correlated with XPRT enzymatic activity as measured by ribophosphorylation of {14C}-xanthine was in vitro (Ki = 0.124 +/- 0.008 vs . 0.00031 +/- 0.00005 mL/min/g in parental cell line), and {*}-xanthine octanol/water partition coefficient was 0.20 at pH = 7.4 (logP = -0.69), meeting requirements for the blood-brain barrier (BBB) penetrating tracer . In the in vivo experiment, the concentration of {14C}-xanthine in the normal brain varied from 0.20 to 0.16 + 0.05% dose/g under 0.87 + 0.24% dose/g plasma radiotracer concentration . The accumulation in vivo in the transfected flank tumor was to 2.4 +/- 0.3% dose/g, compared to 0.78 +/- 0.02% dose/g and 0.64 +/- 0.05% dose/g in the control flank tumors and intact muscle, respectively . {14C}-Xanthine appeared to be capable of specific accumulation in the transfected infiltrative brain tumor (RG2-dsRed/XPRT), which corresponded to the 585 nm fluorescent signal obtained from the adjacent cryosections . The images of endogenous gene expression with the "sensory system" have to be normalized for the transfection efficiency based on the "beacon system" image data . Such an approach requires two different "reporter genes" and two different "reporter substrates." Therefore, the novel dsRed/XPRT fusion gene can be used as a multimodality reporter system in the biological applications requiring two independent reporter genes, including the cells located behind the BBB. Adv Appl Microbiol, 2003, 52, 167 - 86 Acid resistance in Escherichia coli; Richard HT et al.; To colonize and cause disease, enteric pathogens must overcome environmental challenges that include acid stress in the host's stomach as well as short-chain fatty acid stress in the intestine of the host and reservoir . Three known inducible systems have evolved for stationary phase acid resistance in E . coli . These systems each provide a different level of protection with different requirements and induction conditions . Acid resistance system 1 (AR1) is acid induced in stationary phase, requires the presence of RpoS, and provides the least level of protection at pH 2.5 . Acid resistance system 2 (AR2) is glutamate dependent and stationary phase induced, requires the presence of glutamate decarboxylase and a putative glutamate:GABA antiporter, and provides the highest level of protection . Acid resistance system 3 (AR3) is arginine dependent and acid induced under anaerobic conditions, requires the presence of arginine decarboxylase (AdiA), and provides only a modest level of protection . These three systems along with log phase acid tolerance protect cells from the acid stresses in both the reservoir and host, which can range from pH 2 to 4.5 . They also protect against acid stress involved in food processing and facilitate the low infectious dose characteristic of E . coli, significantly contributing to the pathogenesis of this organism. J Basic Microbiol, 2003, 43(5), 399 - 406 Mutation analysis of the feedback inhibition site of phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase of Escherichia coli; Hu C et al.; In Escherichia coli, the phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS) AroG catalyzes the first committed step in the biosynthesis of aromatic compounds . To investigate the feedback inhibition site of AroG, mutated enzymes prepared with sequence-overlap extension PCR were expressed and purified . The enzymatic activity assay showed that the amino acid replacements at Phe144, Leu175, Leu179, Phe209, Trp215Ala and Val221 completely or partially relieved feedback inhibition of AroG addressed by the phenylalanine . Ile10Ala and Delta(1-15) desensitized feedback inhibition and caused a 70 approximately 90% loss of the specific catalytic activities . These results strongly suggest an involvement of the interior region and the N-terminus of the polypeptide chain of AroG in the formation of the feedback inhibition site of DAHPS. Chembiochem, 2003 Sep 5, 4(9), 870 - 7 NMR chemical shift perturbation study of the N-terminal domain of Hsp90 upon binding of ADP, AMP-PNP, geldanamycin, and radicicol; Dehner A et al.; Hsp90 is one of the most abundant chaperone proteins in the cytosol . In an ATP-dependent manner it plays an essential role in the folding and activation of a range of client proteins involved in signal transduction and cell cycle regulation . We used NMR shift perturbation experiments to obtain information on the structural implications of the binding of AMP-PNP (adenylyl-imidodiphosphate-a non-hydrolysable ATP analogue), ADP and the inhibitors radicicol and geldanamycin . Analysis of (1)H,(15)N correlation spectra showed a specific pattern of chemical shift perturbations at N210 (ATP binding domain of Hsp90, residues 1-210) upon ligand binding . This can be interpreted qualitatively either as a consequence of direct ligand interactions or of ligand-induced conformational changes within the protein . All ligands show specific interactions in the binding site, which is known from the crystal structure of the N-terminal domain of Hsp90 . For AMP-PNP and ADP, additional shift perturbations of residues outside the binding pocket were observed and can be regarded as a result of conformational rearrangement upon binding . According to the crystal structures, these regions are the first alpha-helix and the "ATP-lid" ranging from amino acids 85 to 110 . The N-terminal domain is therefore not a passive nucleotide-binding site, as suggested by X-ray crystallography, but responds to the binding of ATP in a dynamic way with specific structural changes required for the progression of the ATPase cycle. Chembiochem, 2003 Sep 5, 4(9), 863 - 9 The structural plasticity of the C terminus of p21Cip1 is a determinant for target protein recognition; Esteve V et al.; The cyclin-dependent kinase inhibitory protein p21(Cip1) might play multiple roles in cell-cycle regulation through interaction of its C-terminal domain with a defined set of cellular proteins such as proliferating cell nuclear antigen (PCNA), calmodulin (CaM), and the oncoprotein SET . p21(Cip1) could be described as an intrinsically unstructured protein in solution although the C-terminal domain adopts a well-defined extended conformation when bound to PCNA . However, the molecular mechanism of the interaction with CaM and the oncoprotein SET is not well understood, partly because of the lack of structural information . In this work, a peptide derived from the C-terminal domain of p21(Cip1) that covers the binding domain of the three above-mentioned proteins was used to demonstrate that the C-terminal domain of p21 recognizes multiple ligands through its ability to adopt multiple conformations . The conformation is dictated by tertiary contacts rather than by the primary sequence of the protein . Our results suggest that the C-terminal domain of p21(Cip1) adopts an extended structure when bound to PCNA and probably when bound to the oncoprotein SET, but an alpha helix when bound to CaM. J Infect Dis, 2003 Sep 15, 188(6), 938 - 43 Epub 2003 Aug 28. Heterozygous toll-like receptor 4 polymorphism does not influence lipopolysaccharide-induced cytokine release in human whole blood; von Aulock S et al.; The heterozygous Asp299Gly mutation of the toll-like receptor (TLR) 4, the key receptor for lipopolysaccharide (LPS), has been associated with attenuated inflammatory responses . When 160 healthy volunteers (9% heterozygous and 0.6% homozygous) were genotyped and their LPS-inducible cytokine release was assessed in an ex vivo whole blood test, the responses of heterozygotes did not differ significantly from those of wild-type carriers for any of the cytokines (tumor necrosis factor-alpha, interleukin {IL}-1beta, IL-6, interferon-gamma, and granulocyte colony-stimulating factor) or eicosanoids measured or for serum cytokines and C-reactive protein . Ten heterozygous subjects and 12 wild-type control subjects responded similarly to a graded series of LPS and Escherichia coli concentrations, excluding the possibility that allele-specific differences are evident only at low stimulus concentrations or in response to whole pathogens . These data demonstrate that the heterozygous Asp299Gly polymorphism does not exhibit a functional defect in cytokine release after the stimulation of blood monocytes. J Biol Chem, 2003 Nov 21, 278(47), 46219 - 29 Epub 2003 Sep 08. Regulation of the Escherichia coli antiterminator protein BglG by phosphorylation at multiple sites and evidence for transfer of phosphoryl groups between monomers; Gorke B; Activity of antiterminator protein BglG regulating the beta-glucoside operon in Escherichia coli is controlled by the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) in a dual manner . It requires HPr phosphorylation to be active, whereas phosphorylation by the beta-glucoside-specific transport protein EIIBgl inhibits its activity . BglG and its relatives carry two PTS regulation domains (PRD1 and PRD2), each containing two conserved histidines . For BglG, histidine 208 in PRD2 was reported to be the negative phosphorylation site . In contrast, other antiterminators of this family are negatively regulated by phosphorylation of the first histidine in PRD1, and presumably activated by phosphorylation of the histidines in PRD2 . In this work, a screen for mutant BglG proteins that escape repression by EIIBgl yielded exchanges of nine residues within PRD1, including conserved histidines His-101 and His-160, and C-terminally truncated proteins . Genetic and phosphorylation analyses indicate that His-101 in PRD1 is phosphorylated by EIIBgl and that His-160 contributes to negative regulation . His-208 in PRD2 is essential for BglG activity, suggesting that it is phosphorylated by HPr . Surprisingly, phosphorylation by HPr is not fully abolished by exchanges of His-208 . However, phosphorylation by HPr is inhibited by exchanges in PRD1 and the phosphorylation of these mutants is restored in the presence of wild-type BglG . These results suggest that the activating phosphoryl group is transiently donated from HPr to PRD1 and subsequently transferred to His-208 of a second BglG monomer . The active His-208-phosphorylated BglG dimer can subsequently be inhibited in its activity by EIIBgl-catalyzed phosphorylation at His-101. J Mol Biol, 2003 Sep 19, 332(3), 741 - 56 Repacking the Core of T4 lysozyme by automated design; Mooers BH et al.; Automated protein redesign, as implemented in the program ORBIT, was used to redesign the core of phage T4 lysozyme . A total of 26 buried or partially buried sites in the C-terminal domain were allowed to vary both their sequence and side-chain conformation while the backbone and non-selected side-chains remained fixed . A variant with seven substitutions ("Core-7") was identified as having the most favorable energy . The redesign experiment was repeated with a penalty for the presence of methionine residues . In this case the redesigned protein ("Core-10") had ten amino acid changes . The two designed proteins, as well as the constituent single mutants, and several single-site revertants were over-expressed in Escherichia coli, purified, and subjected to crystallographic and thermal analyses . The thermodynamic and structural data show that some repacking was achieved although neither redesigned protein was more stable than the wild-type protein . The use of the methionine penalty was shown to be effective . Several of the side-chain rotamers in the predicted structure of Core-10 differ from those observed . Rather than changing to new rotamers predicted by the design process, side-chains tend to maintain conformations similar to those seen in the native molecule . In contrast, parts of the backbone change by up to 2.8A relative to both the designed structure and wild-type.Water molecules that are present within the lysozyme molecule were removed during the design process . In the redesigned protein the resultant cavities were, to some degree, re-occupied by side-chain atoms . In the observed structure, however, water molecules were still bound at or near their original sites . This suggests that it may be preferable to leave such water molecules in place during the design procedure . The results emphasize the specificity of the packing that occurs within the core of a typical protein . While point substitutions within the core are tolerated they almost always result in a loss of stability . Likewise, combinations of substitutions may also be tolerated but usually destabilize the protein . Experience with T4 lysozyme suggests that a general core repacking methodology with retention or enhancement of stability may be difficult to achieve without provision for shifts in the backbone. J Mol Biol, 2003 Sep 19, 332(3), 701 - 13 Effects of confinement in chaperonin assisted protein folding: rate enhancement by decreasing the roughness of the folding energy landscape; Baumketner A et al.; Chaperonins, such as the GroE complex of the bacteria Escherichia coli, assist the folding of proteins under non-permissive folding conditions by providing a cavity in which the newly translated or translocated protein can be encapsulated . Whether the chaperonin cage plays a passive role in protecting the protein from aggregation, or an active role in accelerating folding rates, remains a matter of debate . Here, we investigate the role of confinement in chaperonin mediated folding through molecular dynamics simulations . We designed a substrate protein with an alpha/beta sandwich fold, a common structural motif found in GroE substrate proteins and confined it to a spherical hydrophilic cage which mimicked the interior of the GroEL/ES cavity . The thermodynamics and kinetics of folding were studied over a wide range of temperature and cage radii . Confinement was seen to significantly raise the collapse temperature, T(c), as a result of the associated entropy loss of the unfolded state . The folding temperature, T(f), on the other hand, remained unaffected by encapsulation, a consequence of the folding mechanism of this protein that involves an initial collapse to a compact misfolded state prior to rearranging to the native state . Folding rates were observed to be either accelerated or retarded compared to bulk folding rates, depending on the temperature of the simulation . Rate enhancements due to confinement were observed only at temperatures above the temperature T(m), which corresponds to the temperature at which the protein folds fastest . For this protein, T(m) lies above the folding temperature, T(f), implying that encapsulation alone will not lead to a rate enhancement under conditions where the native state is stable (T<T(f)) . For confinement to positively impact folding rates under physiological conditions, it is hence necessary for the protein to exhibit a folding transition above the temperature at which it exhibits its fastest folding rate (T(m)<T(f)) . We designed a protein with this property by reducing the energetic frustration in the original alpha/beta sandwich substrate protein . The modified protein exhibited a twofold acceleration in folding rates upon encapsulation . This rate enhancement is due to a mechanistic change in folding involving the elimination, upon encapsulation, of accessible local energy minima corresponding to structures with large radii of gyration . For this protein, confinement hence plays more than the role of a passive cage, but rather adopts an active role, accelerating folding rates by decreasing the roughness of the energy landscape of the protein. J Mol Biol, 2003 Sep 19, 332(3), 689 - 99 Essential role of histidine 84 in elongation factor Tu for the chemical step of GTP hydrolysis on the ribosome; Daviter T et al.; Elongation factor Tu (EF-Tu) is a GTP-binding protein that delivers aminoacyl-tRNA to the A site of the ribosome during protein synthesis . The mechanism of GTP hydrolysis in EF-Tu on the ribosome is poorly understood . It is known that mutations of a conserved histidine residue in the switch II region of the factor, His84 in Escherichia coli EF-Tu, impair GTP hydrolysis . However, the partial reaction which is directly affected by mutations of His84 was not identified and the effect on GTP hydrolysis was not quantified . Here, we show that the replacement of His84 with Ala reduces the rate constant of GTP hydrolysis more than 10(6)-fold, whereas the preceding steps of ternary complex binding to the ribosome, codon recognition and, most importantly, the GTPase activation step are affected only slightly . These results show that His84 plays a key role in the chemical step of GTP hydrolysis . Rate constants of GTP hydrolysis by wild-type EF-Tu, measured using the slowly hydrolyzable GTP analog, GTPgammaS, showed no dependence on pH, indicating that His84 does not act as a general base . We propose that the catalytic role of His84 is to stabilize the transition state of GTP hydrolysis by hydrogen bonding to the attacking water molecule or, possibly, the gamma-phosphate group of GTP. J Mol Biol, 2003 Sep 19, 332(3), 657 - 74 Dynamical properties of the MscL of Escherichia coli: a normal mode analysis; Valadie H et al.; The mechanosensitive channel (MscL) is an integral membrane protein which gates in response to membrane tension . Physiological data have shown that the gating transition involves a very large change in the conformation, and that the open state of the channel forms a large non-specific pore with a high conductance . The Escherichia coli channel structure was first modeled by homology modeling, starting with the X-ray structure of the homologous from Mycobacterium tuberculosis . Then, the dynamical and conformational properties of the channel were explored, using normal mode analysis . Such an analysis was also performed with the different structures proposed recently by Sukharev and co-workers . Similar dynamical behaviors are observed, which are characteristic of the channel architecture, subtle differences being due to the different relative positioning of the structural elements . The ability of particular regions of the channel to deform is discussed with respect to the functional and structural properties, implied in the gating process . Our results show that the first step of the gating mechanism can be described with three low-frequency modes only . The movement associated to these modes is clearly an iris-like movement involving both tilt and twist rotation. J Mol Biol, 2003 Sep 19, 332(3), 575 - 84 The role of RbfA in 16S rRNA processing and cell growth at low temperature in Escherichia coli; Xia B et al.; RbfA, a 30S ribosome-binding factor, is a multicopy suppressor of a cold-sensitive C23U mutation of the 16S rRNA and is required for efficient processing of the 16S rRNA . At 37 degrees C, DeltarbfA cells show accumulation of ribosomal subunits and 16S rRNA precursor with a significantly reduced polysome profile in comparison with wild-type cells . RbfA is also a cold-shock protein essential for Escherichia coli cells to adapt to low temperature . In this study, we examined its association with the ribosome and its role in 16S rRNA processing and ribosome profiles at low temperature . In wild-type cells, following cold shock at 15 degrees C, the amount of free RbfA remained largely stable, while that of its 30S subunit-associated form became several times greater than that at 37 degrees C and a larger fraction of total 30S subunits was detected to be RbfA-containing . In DeltarbfA cells, the pre-16S rRNA amount increased after cold shock with a concomitant reduction of the mature 16S rRNA amount and the formation of polysomes was further reduced . A closer examination revealed that 30S ribosomal subunits of DeltarbfA cells at low temperature contained primarily pre-16S rRNA and little mature 16S rRNA . Our results indicate that the cold sensitivity of DeltarbfA cells is directly related to their lack of translation initiation-capable 30S subunits containing mature 16S rRNA at low temperature . Importantly, when the C-terminal 25 residue sequence was deleted, the resulting RbfADelta25 lost the abilities to stably associate with the 30S subunit and to suppress the dominant-negative, cold-sensitive phenotype of the C23U mutation in 16S rRNA but was able to suppress the 16S rRNA processing defect and the cold-sensitive phenotype of the DeltarbfA cells, suggesting that RbfA may interact with the 30S ribosome at more than one site or function in more than one fashion in assisting the 16S rRNA maturation at low temperature. J Mol Biol, 2003 Sep 19, 332(3), 529 - 36 Allosteric switching by mutually exclusive folding of protein domains; Radley TL et al.; Many proteins are built from structurally and functionally distinct domains . A major goal is to understand how conformational change transmits information between domains in order to achieve biological activity . A two-domain, bi-functional fusion protein has been designed so that the mechanical stress imposed by the folded structure of one subunit causes the other subunit to unfold, and vice versa . The construct consists of ubiquitin inserted into a surface loop of barnase . The distance between the amino and carboxyl ends of ubiquitin is much greater than the distance between the termini of the barnase loop . This topological constraint causes the two domains to engage in a thermodynamic tug-of-war in which only one can exist in its folded state at any given time . This conformational equilibrium, which is cooperative, reversible, and controllable by ligand binding, serves as a model for the coupled binding and folding mechanism widely used to mediate protein-protein interactions and cellular signaling processes . The position of the equilibrium can be adjusted by temperature or ligand binding and is monitored in vivo by cell death . This design forms the basis for a new class of cytotoxic proteins that can be activated by cell-specific effector molecules, and can thus target particular cell types for destruction. Protein Expr Purif, 2003 Sep, 31(1), 161 - 5 Purification of recombinant human apometallothionein-3 and reconstitution with zinc; Eriste E et al.; Metallothioneins (MT) are small cysteine-rich proteins, expressed in many life forms . They are involved primarily in the metabolism of zinc and copper, and in metal detoxification processes . Metallothionein-3 is a mammalian brain-specific MT, which is down-regulated in Alzheimer's disease brains . In this report, we describe a new procedure for purification of recombinant human apo-MT-3 by three steps, size exclusion at neutral pH, followed by cation-exchange and reverse-phase HPLC, both at low pH . Purified apo-MT-3 was reconstituted with seven Zn(2+) ions and reconstitution products were analyzed with electrospray ionization mass spectrometry . The mass spectrum of reconstituted ZnMT-3 was identical with that of native ZnMT-3 isolated by size exclusion chromatography proving the efficiency of the reconstitution process . It showed that ZnMT-3 exists in solution as a dynamic mixture of several metalloforms, where the main metalloform is Zn(7)MT-3 and minor forms are Zn(6)MT-3 and Zn(8)MT-3. Protein Expr Purif, 2003 Sep, 31(1), 149 - 54 Dialysis strategies for protein refolding: preparative streptavidin production; Sorensen HP et al.; We have investigated different dialysis strategies for the refolding of recombinant streptavidin, and present a novel dialysis setup featuring gradual dilution dialysis and continuous protein feeding into a dialysis sack . A denaturing dialysis buffer is exchanged gradually by dilution with refolding buffer and it is demonstrated that the refolding yield can be increased from 45 to 75% by lowering the dilution rate . In addition, continuous feeding of protein to the dialysis sack increases the yield by 5 to 10% . The principle of gradual dilution dialysis is amenable to stringent regulation and we suggest it to be applied for other insoluble protein targets. Protein Expr Purif, 2003 Sep, 31(1), 133 - 9 Expression, purification, and characterization of human enteropeptidase catalytic subunit in Escherichia coli; Gasparian ME et al.; Enteropeptidase (synonym:enterokinase, EC 3.4.21.9) is a heterodimeric serine protease of the intestinal brush border that activates trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the sequence (Asp)(4)-Lys . The DNA sequence encoding the light chain (catalytic subunit) of human enteropeptidase (GenBank Accession No . U09860) was synthesized from 26 oligonucleotides by polymerase chain reaction and cloned into plasmid pET-32a downstream to the gene of fusion partner thioredoxin immediately after the DNA sequence encoding enteropeptidase recognition site . The fusion protein thioredoxin/human enteropeptidase light chain was expressed in Escherichia coli BL21(DE3) strain in both soluble and insoluble forms . The soluble recombinant fusion protein failed to undergo autocatalytic cleavage and activation; however, autocatalytic cleavage and activation of recombinant human enteropeptidase light chain (L-HEP) were achieved by solubilization and renaturation of the fusion protein from inclusion bodies and the active L-HEP was purified on agarose-linked soybean trypsin inhibitor . The purified L-HEP cleaved the synthetic peptide substrate Gly-Asp-Asp-Asp-Asp-Lys-beta-naphthylamide with kinetic parameters K(m)=0.16 mM and k(cat)=115 s(-1) and small ester Z-Lys-SBzl with K(m)=140 microM, k(cat)=133 s(-1) . L-HEP associated with soybean trypsin inhibitor slowly and small ester Z-Lys-SBzl cleavage was inhibited with K(i)(*)=2.3 nM . L-HEP digested thioredoxin/human epidermal growth factor fusion protein five times faster than equal activity units of bovine recombinant light chain (EKMax, Invitrogen) at the same conditions. Protein Expr Purif, 2003 Sep, 31(1), 79 - 87 Refolding from denatured inclusion bodies, purification to homogeneity and simplified assay of MGDG synthases from land plants; Nishiyama Y et al.; In plant cells, the synthesis of monogalactosyldiacylglycerol (MGDG) is catalyzed within plastid envelope membranes by MGD proteins . MGDG synthesis was also reported in apicomplexan parasites, a phylum of protists harbouring a plastid that proved essential for the parasite survival . MGD activity is therefore a potent target for herbicidal and anti-parasitic molecules . In this study, we describe a detailed in vitro refolding protocol for denatured recombinant MGD accumulated in inclusion bodies from transformed Escherichia coli . The refolding process was dependent on CHAPS detergent and lipids, such as diacylglycerol and phosphatidylglycerol, as well as bivalent metals . Owing to this refolding procedure, the recombinant MGD protein from spinach was purified to homogeneity, allowing a definite characterization of its non-processivity and an investigation of its dimerization using cross-linking reagents . Additionally, using the portion of recombinant enzyme that accumulates in an active form in bacterial membranes, we developed a miniature assay for high-throughput screening for inhibitors. Protein Expr Purif, 2003 Sep, 31(1), 72 - 8 Cloning, expression, and purification of the His6-tagged hyper-thermostable dUTPase from Pyrococcus woesei in Escherichia coli: application in PCR; Dabrowski S et al.; The gene encoding dUTPase from Pyrococcus woesei was cloned into Escherichia coli expression system . It shows 100% gene identity to homologous gene in Pyrococcus furiosus . The expression of N-terminal His(6)-tagged Pwo dUTPase was performed in E . coli BL21(DE3)pLysS and E . coli Rosetta(DE3)pLysS strain that contains plasmid encoding additional copies of rare E . coli tRNAs . E . coli Rosetta(pLysS) strain was found with two times higher expression yield of His(6)-tagged Pwo dUTPase than E . coli BL21(DE3)pLysS . The His(6)-tagged Pwo dUTPase was purified on Ni(2+)-IDA-Sepharose, dialyzed, and the enzyme activity was investigated . We found that His(6)-tag domain has no influence on dUTP hydrolytic activity . dUTP is generated during PCR from dCTP, which inhibits the polymerization of DNA catalyzed by DNA polymerase with 3(')-5(') exonuclease activity . We observed that the thermostable His(6)-tagged Pwo dUTPase used for the polymerase chain reaction with P . woesei DNA polymerase improves the efficiency of PCR and it allows for amplification of longer targets. Protein Expr Purif, 2003 Sep, 31(1), 64 - 71 On-column refolding and characterization of soluble human interleukin-15 receptor alpha-chain produced in Escherichia coli; Matsumoto M et al.; Interleukin-15 receptor alpha-chain (IL-15Ralpha) is a member of the new cytokine receptor family, which possesses the sushi domain . To investigate the biochemical and biophysical characteristics of soluble human IL-15Ralpha (shIL-15Ralpha), shIL-15Ralpha was recombinantly expressed in Escherichia coli . The shIL-15Ralpha containing a six histidine-tag was expressed as inclusion bodies, which were solubilized with urea, immobilized on a Ni-nitrilotriacetic acid column, and refolded by a decreasing gradient of urea concentration . The refolded shIL-15Ralpha exhibited a highly flexible structure, neutralized human interleukin-15-induced cell proliferation effectively, and bound to its ligand with the same affinity as human IL-15Ralpha on the cell surface, as demonstrated by circular dichroism, a cell proliferation assay, and surface plasmon resonance, respectively . Thus, we succeeded in refolding shIL-15Ralpha to an active form on an affinity column. Protein Expr Purif, 2003 Sep, 31(1), 47 - 55 Expression and purification of receptor for activated C-kinase 1 (RACK1); Bjorndal B et al.; Receptor for activated C-kinase (RACK1) binds to protein kinase C and functions as an anchor for several other cellular components . Most in vitro studies of RACK1 have been carried out with RACK1 fused to a soluble fusion protein partner, such as GST or MBP . Here, we show that fusion complexes may exist as large soluble aggregates and thereby lead to false conclusions about the biological activity of RACK1 . We developed a purification procedure that gave soluble monodisperse molecules of the protein . The RACK1 gene was cloned and expressed in a pMAL vector . After purification of the resulting MBP-RACK1 fusion protein, RACK1 was excised from MBP by thrombin, rendering RACK1 in a soluble monodisperse form as monitored by fluorimetric static light scattering, gel filtration, and ultracentrifugation . Circular dichroism analysis revealed that RACK1 was properly folded with a T(m) of approximately 62 degrees C and contained the predicted portions of secondary structures . The biological activity of the purified protein was verified by binding to activated protein kinase C . The production of soluble, high-purity RACK1 will allow structural studies and functional in vitro studies to identify interacting partners to this important scaffold protein. Protein Expr Purif, 2003 Sep, 31(1), 12 - 8 Abrus pulchellus type-2 RIP, pulchellin: heterologous expression and refolding of the sugar-binding B chain; Goto LS et al.; Abrus pulchellus type-2 RIP, or pulchellin, is a heterodimeric glycoprotein found in A . pulchellus seeds . These chimerolectins, like all type-2 RIPs, are characterized as highly toxic proteins with enzymatic and lectin properties performed by two separate polypeptide subunits . Intending to obtain pure and homogeneous protein for structural and biological studies, the A . pulchellus type-2 RIP lectin subunit or pulchellin binding chain encoding gene fragment (PBC) was cloned . Oligonucleotides based on the sequence homologies between other RIPs like abrin and ricin were synthesized and used to amplify the complete PBC from A . pulchellus genomic DNA . The amplification product was inserted into plasmid pET28a to express the recombinant PBC (rPBC) in Escherichia coli BL21(DE3) . The rPBC was expressed as inclusion bodies that were recovered and denatured in a buffer containing urea . Repeated dialysis rounds against the oxidation buffer, which presented the redox pair cysteine-cystine, D-galactose, and decreasing urea concentrations, conducted the protein refolding . The refolding process of rPBC was successfully confirmed by biological assays and circular dichroism. Biochem Biophys Res Commun, 2003 Sep 26, 309(3), 672 - 8 Blocking the dengue virus 2 infections on BHK-21 cells with purified recombinant dengue virus 2 E protein expressed in Escherichia coli; Chiu MW et al.; Dengue viruses (DVs) are mosquito-borne infectious pathogens . They have become an expanding public health problem in the tropics and subtropics . The dengue envelope (E) protein is one of the viral structure proteins responsible mainly for the virus attachment and entry onto host cells . It is also the major immunogen for virus neutralization . In this study, we have constructed a recombinant plasmid expressing a truncated E protein of DV-2 virus PL046 strain . The C-terminal hydrophobic domain of the E protein was removed and replaced with the sequence of S peptide to facilitate expression and purification . When expressed in Escherichia coli, the recombinant E proteins were found to be in the form of aggregated state . Through denaturation and dialysis processes, the receptor-interacting function of the purified recombinant E proteins was maintained, which was demonstrated by its ability to inhibit the DV-2 plaque-forming efficiency on mammalian BHK-21 host cells. Toxicol Lett, 2003 Nov 1, 145(1), 36 - 45 N-Nitrosodiethylamine mutagenicity at low concentrations; Aiub CA et al.; N-Nitrosodiethylamine (NDEA) requires metabolic activation by cytochrome P450 enzymes, leading to electrophile species that react in DNA . Although, carcinogenicity is not an end point in genotoxicity assays, NDEA has been considered a weak carcinogen . In this study, we carried out an analysis of the mutagenicity at low concentrations of NDEA . Using SOS chromotest in the presence of metabolic activation, we detected positive mutagenicity response for NDEA doses between 0.75 and 36.46 microg/ml . In Ames test, using more sensitive strains in the presence of S9 metabolic activation mixture (S9 mix), positive results were also detected for NDEA doses between 1.01 x 10(-3) and 50.64 x 10(-3 microg per plate . Our results indicate that NDEA mutagenicity can be detected at low concentrations when more sensitive conditions are used. Biochemistry, 2003 Sep 16, 42(36), 10843 - 52 Coupled kinetics of ATP and peptide hydrolysis by Escherichia coli FtsH protease; Bruckner RC et al.; FtsH from Escherichia coli is an ATP- and Zn(2+)-dependent integral membrane protease that is involved in the degradation of regulatory proteins such as sigma(32) and uncomplexed subunits of membrane protein complexes such as secY of the protein translocase . We describe a protocol for solubilizing the recombinant enzyme from inclusion bodies and its subsequent refolding and purification to near homogeneity . This is a high-yield protocol and produces in excess of 20 mg of purified FtsH per liter of E . coli culture . We found that refolded FtsH has biochemical properties similar to detergent extracted overexpressed protein described previously . FtsH forms a large complex with an apparent mass of 1200 kDa as determined by gel filtration . Both ATPase and protease activities are coincident with this large complex; smaller forms of FtsH do not exhibit either activity . While FtsH-catalyzed hydrolysis of ATP can occur in the absence of protein substrate (k(c) = 22 min(-1); K(m) = 23 microM), proteolysis shows an absolute dependence on nucleoside-5'-triphosphates, including ATP, CTP, and various analogues . In the presence of 5 mM ATP, FtsH catalyzes the hydrolysis of sigma(32) with the following observed kinetic parameters: k(c) = 0.18 min(-1) and K(m) = 8.5 microM . Significantly, this reaction is processive and generates no intermediate species, but rather, approximately 10 peptide products, all of MW <3 kDa . FtsH protease also efficiently hydrolyzes the peptide Phe-Gly-His-(NO)2Phe-Phe-Ala-Phe-OMe . Hydrolysis occurs exclusively at the (NO)2Phe-Phe bond (k(c) = 2.1 min(-1); K(m) = 12 microM), and like proteolysis, shows an absolute dependence on NTPs . We propose a mechanism for the coupled hydrolytic activities of FtsH toward ATP and peptide substrates that is consistent with a recently proposed structural model for FtsH. Biochemistry, 2003 Sep 16, 42(36), 10809 - 21 Electron transfer in flavocytochrome P450 BM3: kinetics of flavin reduction and oxidation, the role of cysteine 999, and relationships with mammalian cytochrome P450 reductase; Roitel O et al.; Cys-999 is one component of a triad (Cys-999, Ser-830, and Asp-1044) located in the FAD domain of flavocytochrome P450 BM3 that is almost entirely conserved throughout the diflavin reductase family of enzymes . The role of Cys-999 has been studied by steady-state kinetics, stopped-flow spectroscopy, and potentiometry . The C999A mutants of BM3 reductase (containing both FAD and FMN cofactors) and the isolated FAD domain are substantially compromised in their capacity to reduce artificial electron acceptors in steady-state turnover with either NADPH or NADH as electron donors . Stopped-flow studies indicate that this is due primarily to a substantially slower rate of hydride transfer from nicotinamide coenzyme to FAD cofactor in the C999A enzymes . The compromised rates of hydride transfer are not attributable to altered thermodynamic properties of the flavins . A reduced enzyme-NADP(+) charge-transfer species is populated following hydride transfer in the wild-type FAD domain, consistent with the slow release of NADP(+) from the 2-electron-reduced enzyme . This intermediate does not accumulate in the C999A FAD domain or wild-type and C999A BM3 reductases, suggesting more rapid release of NADP(+) from these enzyme forms . Rapid internal electron transfer from FAD to FMN in wild-type BM3 reductase releases NADP(+) from the nicotinamide-binding site, thus preventing the inhibition of enzyme activity through the accumulation of a stable FADH(2)-NADP(+) charge-transfer complex . Hydride transfer is reversible, and the observed rate of oxidation of the 2-electron-reduced C999A BM3 reductase and FAD domain is hyperbolically dependent on NADP(+) concentration . With the wild-type BM3 reductase and FAD domain, the rate of flavin oxidation displays an unusual dependence on NADP(+) concentration, consistent with a two-site binding model in which two coenzyme molecules bind to catalytic and regulatory regions (or sites) within a bipartite coenzyme binding site . A kinetic model is proposed in which binding of coenzyme to the regulatory site hinders sterically the release of NADPH from the catalytic site . The results are discussed in the light of kinetic and structural studies on mammalian cytochrome P450 reductase. Biochemistry, 2003 Sep 16, 42(36), 10800 - 8 The naphthoquinol oxidizing cytochrome bc1 complex of the hyperthermophilic knallgasbacterium Aquifex aeolicus: properties and phylogenetic relationships; Schutz M et al.; Phylogenetic analysis of constituent proteins of Rieske/cytochrome b complexes {Schutz et al . (2000) J . Mol . Biol . 300, 663-675} indicated that the respective enzyme from the hyperthermophile Aquifex (A.) aeolicus is closely related to proteobacterial counterparts, in disagreement with positioning of its parent species on small subunit rRNA trees . An assessment of the details and possible reasons for this discrepancy necessitates a thorough understanding of the biochemical and biophysical properties of the enzyme in addition to the bioinformatic data . The cytochrome bc(1) complex from A . aeolicus, which is part of the "Knallgasreaction" pathway, was therefore studied in membranes and in detergent-solubilized, isolated complex . Hemes b(L) (E(m,7) = -190 mV; g(z)= 3.7), b(H) (E(m,7) = -60 mV; g(z )= 3.45), and c(1) (E(m,7) = +160 mV; g(z )= 3.55) were identified by EPR and optical spectroscopy in combination with electrochemical methods . Two electrochemically distinct (E(m,7) = +95 mV; E(m,7) = +210 mV) Rieske centers were detected in membranes, and the +210 mV species was shown to correspond to the Rieske center of the cyt bc(1) complex . The gene coding for this latter Rieske protein was heterologously expressed in Escherichia coli, and the resulting protein was characterized in detail . The pool quinone of A . aeolicus was determined to be naphthoquinone . The redox poises of the individual electron-transfer steps are compared to those of other Rieske/cyt b complexes . The Aquifex enzyme was found to represent the only extant naphthoquinol oxidizing true cyt bc(1) complex described so far . An improved scenario for the phylogenetic positioning of the Aquifex cyt bc(1) complex is proposed. Biochemistry, 2003 Sep 16, 42(36), 10790 - 9 Pronounced conversion of the metal-specific activity of superoxide dismutase from Porphyromonas gingivalis by the mutation of a single amino acid (Gly155Thr) located apart from the active site; Yamakura F et al.; Glycine 155, which is located approximately 10 A from the active metal sites, is mostly conserved in aligned amino acid sequences of manganese-specific superoxide dismutases (Mn-SODs) and cambialistic SOD (showing the same activity with Fe and Mn) from Porphyromonas gingivalis, but is substituted for threonine in most Fe-SODs . Since Thr155 is located between Trp123 and Trp125, and Trp123 is one member of the metal-surrounding aromatic amino acids, there is a possibility that the conversion of this amino acid may cause a conversion of the metal-specific activity of cambialistic P . gingivalis SOD . To clarify this possibility, we have prepared a mutant of the P . gingivalis SOD with conversion of Gly155 to Thr . The ratios of the specific activities of Fe- to Mn-reconstituted enzyme, which are measured by the xanthine oxidase/cytochrome c method, increased from 0.6 in the wild-type to 11.2 in the mutant SODs, indicating the conversion of the metal-specific activity of the enzyme from a cambialistic type to an Fe-specific type . The visible absorption spectra of the Fe- and Mn-reconstituted mutant SODs closely resembled those of Fe-specific SOD . Furthermore, the EPR spectra of the Fe- and Mn-reconstituted mutant SODs also closely resembled those of Fe-specific SOD . Three-dimensional structures of the Fe-reconstituted wild-type SOD and Mn-reconstituted mutant SOD have been determined at 1.6 A resolution . Both structures have identical conformations, orientations of residues involved in metal binding, and hydrogen bond networks, while the side chain of Trp123 is moved further toward the metal-binding site than in wild-type SOD . A possible contribution of the structural differences to the conversion of the metal-specific activity through rearrangement of the hydrogen bond network among Trp123, Gln70, Tyr35, and the metal-coordinated solvent is discussed. Biochemistry, 2003 Sep 16, 42(36), 10718 - 25 Effect of varying the supercoiling of DNA on transcription and its regulation; Lim HM et al.; The effect of superhelicity of DNA templates on transcription is well documented in several cases . However, the amount of supercoiling that is needed to bring about any changes and the steps at which such effects are exerted were not systematically studied . We investigated the effect of DNA supercoiling on transcription from a set of promoters present on a plasmid by using a series of topoisomers with different superhelical densities ranging from totally relaxed to more than physiological . In vitro transcription assays with these topoisomers in the absence and presence of gene regulatory proteins showed that the effect of negative supercoiling on intrinsic transcription varies from promoter to promoter . Some of those promoters, in which DNA superhelicity stimulated transcription, displayed specific optima of superhelical density while others did not . The results also showed that the amounts of abortive RNA synthesis from two of the promoters decreased and full-length RNA increased with increasing supercoiling, indicating for the first time an inverse relationship between full-length and abortive RNA synthesis and supporting a role of DNA superhelicity in promoter clearance . DNA supercoiling might also influence the point of RNA chain termination . Furthermore, the effect of varying the amount of supercoiling on the action of gene regulatory proteins suggested the mode of action, which is consistent with previous results . Our results underscore the importance of DNA supercoiling in fine-tuning promoter activities, which should be relevant in cell physiology given that local changes in chromosomal supercoiling must occur in different environments. Biochemistry, 2003 Sep 16, 42(36), 10674 - 82 Phosphorylation by cAMP-dependent protein kinase modulates the structural coupling between the transmembrane and cytosolic domains of phospholamban; Li J et al.; We have used frequency-domain fluorescence spectroscopy to investigate the structural linkage between the transmembrane and cytosolic domains of the regulatory protein phospholamban (PLB) . Using an engineered PLB having a single cysteine (Cys(24)) derivatized with the fluorophore N-(1-pyrenyl)maleimide (PMal), we have used fluorescence resonance energy transfer (FRET) to measure the average spatial separation and conformational heterogeneity between PMal bound to Cys(24) in the transmembrane domain and Tyr(6) in the cytosolic domain near the amino terminus of PLB . In these measurements, PMal serves as a FRET donor, and Tyr(6) serves as a FRET acceptor following its nitration by tetranitromethane . The native structure of PLB is retained following site-directed mutagenesis and chemical modification, as indicated by the ability of the derivatized PLB to fully regulate the Ca-ATPase following their co-reconstitution . To assess how phosphorylation modulates the structure of PLB itself, FRET measurements were made following reconstitution of PLB in membrane vesicles made from extracted sarcoplasmic reticulum membrane lipids . We find that the cytosolic domain of PLB assumes a wide range of conformations relative to the transmembrane sequence, consistent with other structural data indicating the presence of a flexible hinge region between the transmembrane and cytosolic domains of PLB . Phosphorylation of Ser(16) by PKA results in a 3 A decrease in the spatial separation between PMal at Cys(24) and nitroTyr(6) and an almost 2-fold decrease in conformational heterogeneity, suggesting a stabilization of the hinge region of PLB possibly through an electrostatic linkage between phosphoSer(16) and Arg(13) that promotes a coil-to-helix transition . This structural transition has the potential to function as a conformational switch, since inhibition of the Ca-ATPase requires disruption of the secondary structure of PLB in the vicinity of the hinge element to permit association with the nucleotide binding domain at a site located approximately 50 A above the membrane surface . Following phosphorylation, the stabilization of the helical content in the hinge domain will disrupt this inhibitory interaction by reducing the maximal dimension of the cytosolic domain of PLB . Thus, stabilization of the structure of PLB following phosphorylation of Ser(16) is part of a switching mechanism, which functions to alter binding interactions between PLB and the nucleotide binding domain of the Ca-ATPase that modulates enzyme inhibition. Biochemistry, 2003 Sep 16, 42(36), 10667 - 73 Solution structure and dynamics of a heat shock protein assembly probed by hydrogen exchange and mass spectrometry; Wintrode PL et al.; The solution conformation and dynamics of the 16.9 kDa small heat shock protein from wheat have been studied using a combination of hydrogen/deuterium exchange, proteolytic digestion, and mass spectrometry . At room temperature, HSP16.9 exists as a dodecameric assembly . Regions of HSP16.9 that form extensive and essential intersubunit contacts in the assembly, including residues 1-40 and 131-151, show little or no protection against hydrogen/deuterium exchange after incubation in D(2)O for 5 s . The high levels of hydrogen/deuterium exchange indicate that these regions have experienced large conformational fluctuations in solution, breaking intersubunit contacts and exposing buried amide hydrogens to solvent . When HSP16.9 is pulse labeled for 10 ms, residues 1-40 and 131-151 are substantially more protected than they are after 5 s . Thus, the breaking of intersubunit contacts occurs on a time scale between 10 milliseconds and 5 s . At 42 degrees C, HSP16.9 exists in a suboligomeric form . When the intrinsic temperature dependence of hydrogen/deuterium exchange is taken into account, exchange patterns at 25 and 42 degrees C are identical within experimental error, suggesting that the conformation of individual HSP16.9 subunits is the same in both the dodecameric and subdodecameric forms . Significant protection is seen in regions that form the dimeric interface, suggesting that the stable suboligomeric form is a dimer . Taken together, these results suggest that heat activation of HSP16.9 occurs by shifting the dodecamer <--> dimer equilibrium in favor of free dimers . The conformation of the dimers themselves does not appear to be altered with an increase in temperature. Biochemistry, 2003 Sep 16, 42(36), 10609 - 18 Molecular cloning and expression of a functional snake venom serine proteinase, with platelet aggregating activity, from the Cerastes cerastes viper; Dekhil H et al.; The venoms of Viperidae snakes contain numerous serine proteinases that have been recognized to possess one or more of the essential activities of thrombin on fibrinogen and platelets . Among them, a platelet proaggregant protein, cerastocytin, has been isolated from the venom of the Tunisian viper Cerastes cerastes . Using the RACE-PCR technique, we isolated and identified the complete nucleotide sequence of a cDNA serine proteinase precursor . The recombinant protein was designated rCC-PPP (for C . cerastes platelet proaggregant protein), since its deduced amino acid sequence is more than 96% identical to the partial polypeptide sequences that have been determined for natural cerastocytin . The structure of the rCC-PPP cDNA is similar to that of snake venom serine proteinases . The expression of rCC-PPP in Escherichia coli system allowed, for the first time, the preparation and purification of an active protein from snake venom with platelet proaggregant and fibrinogenolytic activities . Purified rCC-PPP efficiently activates blood platelets at nanomolar (8 nM) concentrations, as do natural cerastocytin (5 nM) and thrombin (1 nM) . It is able to clot purified fibrinogen and to hydrolyze alpha-chains . Thus, rCC-PPP could be therefore considered a cerastocytin isoform . By comparison with other snake venom serine proteinases, a Gly replaces the conserved Cys(42) . This implies that rCC-PPP lacks the conserved Cys(42)-Cys(58) disulfide bridge . A structural analysis performed by molecular modeling indicated that the segment of residues Tyr(67)-Arg(80) of rCC-PPP corresponds to anion-binding exosite 1 of thrombin that is involved in its capacity to induce platelet aggregation . Furthermore, the surface of the rCC-PPP molecule is characterized by a hydrophobic pocket, comprising the 90 loop (Phe(90)-Val(99)), Tyr(172), and Trp(215) residues, which might be involved in the fibrinogen clotting activity of rCC-PPP. Biochemistry, 2003 Sep 16, 42(36), 10560 - 8 Structure and catalytic mechanism of L-rhamnulose-1-phosphate aldolase; Kroemer M et al.; The structure of L-rhamnulose-1-phosphate aldolase has been established at 1.35 A resolution in a crystal form that was obtained by a surface mutation and has one subunit of the C(4)-symmetric tetramer in the asymmetric unit . It confirms an earlier 2.7 A resolution structure which was determined in a complicated crystal form with 20 subunits per asymmetric unit . The chain fold and the active center are similar to those of L-fuculose-1-phosphate aldolase and L-ribulose-5-phosphate 4-epimerase . The active center similarity is supported by a structural comparison of all three enzymes and by the binding mode of the inhibitor phosphoglycolohydroxamate at the site of the product dihydroxyacetone phosphate for the two aldolases . The sensitivity of the catalytic rate to several mutations and a comparison with the established mechanism of the related aldolase give rise to a putative catalytic mechanism . This mechanism involves the same binding mode of the second product L-lactaldehyde in both aldolases, except for a 180 degrees flip of the aldehyde group distinguishing between the two epimers rhamnulose and fuculose . The N-terminal domain exhibits a correlated anisotropic mobility that channels the isotropic Brownian motion into a directed movement of the catalytic base and the substrate phosphate on the N-domain toward the zinc ion and the lactaldehyde on the C-terminal domain . We suggest that this movement supports the catalysis mechanically. Biochemistry, 2003 Sep 16, 42(36), 10554 - 9 Human recombinant resistin protein displays a tendency to aggregate by forming intermolecular disulfide linkages; Aruna B et al.; Resistin, a small cysteine rich protein secreted by adipocytes, has been proposed to be a link between obesity and type II diabetes by modulating the insulin signaling pathway and thus inducing insulin resistance . Resistin protein, with 11 cysteine residues, was not significantly homologous at the amino acid level to any other known cysteine rich proteins . Resistin cDNA derived from human subcutaneous adipose tissue was expressed in Escherichia coli as an N-terminal six-His-tag fusion protein . The overexpressed recombinant resistin was purified to homogeneity from inclusion bodies, after solubilization in 8 M urea, using a metal affinity column . While MALDI-TOF mass spectrometric analysis of the purified protein generated a single peak corresponding to the estimated size of 11.3 kDa, the protein exhibited a concentration-dependent oligomerization which is evident from size exclusion chromatography . The oligomeric structure was SDS-insensitive but beta-mercaptoethanol-sensitive, pointing to the importance of disulfide linkages in resistin oligomerization . Estimation of free cysteine residues using the NBD-Cl assay revealed a concentration- and time-dependent increase in the extent of formation of disulfide linkages . The presence of intermolecular disulfide bond(s), crucial in maintaining the global conformation of resistin, was further evident from fluorescence emission spectra . Circular dichroism spectra revealed that recombinant resistin has a tendency to reversibly convert from alpha-helical to beta-sheet structure as a direct function of protein concentration . Our novel observations on the biophysical and biochemical features of human resistin, particularly those shared with prion proteins, may have a bearing on its likely physiological function. Indian J Gastroenterol, 2003 Jul-Aug, 22(4), 150 - 1 Acute necrotizing gastritis; Dharap SB et al.; A 17-year-old man presented with signs of peritonitis . Laparotomy revealed gangrene of the stomach without obvious cause . The patient underwent total gastrectomy with esophago-jejunal anastomosis with formation of jejunal pouch . Bacterial culture of the peritoneal fluid grew Strept . pyogenes and E . coli . The patient was discharged on day 21 after a stormy postoperative course. Tohoku J Exp Med, 2003 Jun, 200(2), 85 - 92 Effects of ketamine and propofol on the ratio of interleukin-6 to interleukin-10 during endotoxemia in rats; Taniguchi T et al.; Our previous study reported that the change in the ratio of interleukin (IL)-6 to IL-10 influences the severity of sepsis in patients with systemic inflammatory response syndrome . We evaluated the change in the ratio of IL-6 to IL-10 after administration of ketamine or propofol in endotoxin-exposed rats in order to evaluate the relationship of pro-inflammatory and anti-inflammatory cytokines following ketamine or propofol administration during endotoxemia . We randomly assigned 40 rats to one of four equal groups: endotoxin alone, receiving Escherichia coli endotoxin (15 mg/kg, i.v.); saline control; ketamine (10 mg x kg(-1) x h(-1), i.v.) before and during exposure to endotoxin; and propofol (10 mg x kg(-1) x h(-1), i.v.) before and during exposure to endotoxin . We measured the plasma concentrations of tumor necrosis factor (TNF)-alpha, IL-6, and IL-10 and calculated the ratio of IL-6 to IL-10 in each group . The current study showed that ketamine and propofol administration attenuated the increase in TNF-alpha, IL-6, and IL-10, and ketamine attenuated the increase in the ratio of IL-6 to IL-10, but propofol increased this ratio in rats receiving a single intravenous bolus of endotoxin . While the mechanisms responsible for the inhibitory effects require further investigation, our results suggest that proper use of ketamine as an anesthetic agent may offer certain advantages in the management of patients with endotoxemia. J Protein Chem, 2003 Apr, 22(3), 285 - 93 Interaction of cAMP receptor protein from Escherichia coli with cAMP and DNA studied by differential scanning calorimetry; Blaszczyk U et al.; The cyclic AMP receptor protein (CRP) regulates the expression of many genes in Escherichia coli . The protein is a homodimer, and each monomer is folded into two distinct structural domains . In this study, we have used differential scanning calorimetry (DSC) and circular dichroism (CD) to measure the enthalpy change and melting temperature of the apo-CRP and CRP complexes with cAMP or DNA sequences lac, gal, and palindromic ICAP . DSC and CD measurements showed irreversible thermal denaturation process of CRP . Enthalpy of dissociation of the protein-DNA complex, as measured by DSC, depends on the DNA sequence . The thermal transition of the protein in CRP-DNA complexes, measured by CD, indicates that the protein stability in the complex is also DNA sequence-dependent. J Lab Clin Med, 2003 Aug, 142(2), 128 - 35 Attenuation by intravenous 2-chloroadenosine of acute lung injury induced by live escherichia coli or latex particles added to endotoxin in the neutropenic state; Sakamaki F et al.; Although neutrophil depletion can reduce the level of acute lung injury (ALI) induced by Escherichia coli endotoxin, that induced by live E coli cannot be attenuated even in neutropenia . This suggests that live E coli cause ALI by way of an mechanism independent of circulating neutrophil . Tumor necrosis factor-alpha (TNF-alpha), which is released from monocytes and macrophages, is a proinflammatory cytokine that is recognized as a central mediator of several forms of inflammation . In this controlled experimental study, we examined the effects of an adenosine-receptor agonist, 2-chloroadenosine (2CA), that has suppressive effects on various cell types and TNF-alpha, on endotoxin plus latex particles, and on ALI induced by live E coli in the neutropenic state . We studied 42 guinea pigs rendered neutropenic by means of intraperitoneal cyclophosphamide administration . Experimental groups consisted of (1) a saline-solution control group; (2) an endotoxin (0.2 mg/kg)-treated group; (3) a group treated with endotoxin plus 2CA (10 micro g/kg); (4) a group treated with latex (2 x 10(9)/kg); (5) a group exposed to endotoxin and latex; (6) a group exposed to endotoxin, latex, and 2CA; (7) a group exposed to E coli (2 x 10(9)/kg); and (8) a group exposed to E coli and 2CA . The injection of endotoxin alone in neutropenic animals did not increase the indexes of ALI (lung tissue/plasma ratio {T/P} and lung wet weight/dry weight ratio {W/D}, calculated with the use of iodine 125-labeled albumin) . In contrast, these indexes were increased in the endotoxin-and-latex groups compared with those of the control group . ALI in the endotoxin-and-latex group was attenuated by intravenous 2CA . The intravenous injection of live E coli also caused increases in T/P, W/D, and plasma TNF-alpha, but thse were limited by 2CA . In summary, ALI induced by latex particles added to endotoxin and live E coli in the neutropenic state was attenuated by 2CA, suggesting a partial contribution of various cell types or humoral mediators as a neutrophil-independent pathway in its pathogenesis. Alcohol Clin Exp Res, 2003 Aug, 27(8 Suppl), 72S - 75S The production of tumor necrosis factor-alpha by macrophages in rats with acute alcohol loading; Kitazawa T et al.; BACKGROUND: It is suggested that endotoxin, proinflammatory cytokines, and lipopolysaccharide-binding protein (LBP) play an important role in the development of alcoholic liver disease . Our previous study showed that splenic macrophages were important for endotoxin uptake and excessive production of tumor necrosis factor (TNF) in rats given large amounts of alcohol . To study the pathophysiological roles of macrophages in alcoholic liver diseases, we examined the production of TNF-alpha by rat Kupffer cells, splenic macrophages, and alveolar macrophages with acute alcohol loading in the presence or absence of LBP . METHODS: Kupffer cells, splenic macrophages, and alveolar macrophages were isolated from male Wistar rats given 5 mg/g body weight of ethanol intraperitoneally after an hour . The production of TNF-alpha by these cells incubated with endotoxin 100 ng/ml in the presence or absence of LBP (1% rat serum) was determined . RESULTS: Acute alcohol loading did not affect the production of TNF-alpha by Kupffer cells . With acute alcohol loading, splenic macrophages tended to produce more TNF-alpha . Alveolar macrophages produced more TNF-alpha than Kupffer cells, and although the production of TNF-alpha by alveolar macrophages tended to be suppressed by acute alcohol loading, the production of TNF-alpha by alveolar macrophages still remained high in the presence of rat serum . CONCLUSIONS: Splenic macrophages and alveolar macrophages may be related to excessive production of TNF-alpha in acute alcoholics with endotoxemia. Mol Biol Cell, 2003 Dec, 14(12), 4758 - 69 Epub 2003 Sep 05. Glom is a novel mitochondrial DNA packaging protein in Physarum polycephalum and causes intense chromatin condensation without suppressing DNA functions; Sasaki N et al.; Mitochondrial DNA (mtDNA) is packed into highly organized structures called mitochondrial nucleoids (mt-nucleoids) . To understand the organization of mtDNA and the overall regulation of its genetic activity within the mt-nucleoids, we identified and characterized a novel mtDNA packaging protein, termed Glom (a protein inducing agglomeration of mitochondrial chromosome), from highly condensed mt-nucleoids of the true slime mold, Physarum polycephalum . This protein could bind to the entire mtDNA and package mtDNA into a highly condensed state in vitro . Immunostaining analysis showed that Glom specifically localized throughout the mt-nucleoid . Deduced amino acid sequence revealed that Glom has a lysine-rich region with proline-rich domain in the N-terminal half and two HMG boxes in C-terminal half . Deletion analysis of Glom revealed that the lysine-rich region was sufficient for the intense mtDNA condensation in vitro . When the recombinant Glom proteins containing the lysine-rich region were expressed in Escherichia coli, the condensed nucleoid structures were observed in E . coli . Such in vivo condensation did not interfere with transcription or replication of E . coli chromosome and the proline-rich domain was essential to keep those genetic activities . The expression of Glom also complemented the E . coli mutant lacking the bacterial histone-like protein HU and the HMG-boxes region of Glom was important for the complementation . Our results suggest that Glom is a new mitochondrial histone-like protein having a property to cause intense DNA condensation without suppressing DNA functions. Proc Natl Acad Sci U S A, 2003 Sep 16, 100(19), 10706 - 11 Epub 2003 Sep 05. The conformation of neurotensin bound to its G protein-coupled receptor; Luca S et al.; G protein-coupled receptors (GPCRs) mediate the perception of smell, light, taste, and pain . They are involved in signal recognition and cell communication and are some of the most important targets for drug development . Because currently no direct structural information on high-affinity ligands bound to GPCRs is available, rational drug design is limited to computational prediction combined with mutagenesis experiments . Here, we present the conformation of a high-affinity peptide agonist (neurotensin, NT) bound to its GPCR NTS-1, determined by direct structural methods . Functional receptors were expressed in Escherichia coli, purified in milligram amounts by using optimized procedures, and subsequently reconstituted into lipid vesicles . Solid-state NMR experiments were tailored to allow for the unequivocal detection of microgram quantities of 13C,15N-labeled NT(8-13) in complex with functional NTS-1 . The NMR data are consistent with a disordered state of the ligand in the absence of receptor . Upon receptor binding, the peptide undergoes a linear rearrangement, adopting a beta-strand conformation . Our results provide a viable structural template for further pharmacological investigations. J Biol Chem, 2003 Dec 12, 278(50), 50322 - 9 Epub 2003 Sep 05. Crystal structures of the liganded and unliganded nickel-binding protein NikA from Escherichia coli; Heddle J et al.; Bacteria have evolved a number of tightly controlled import and export systems to maintain intracellular levels of the essential but potentially toxic metal nickel . Nickel homeostasis systems include the dedicated nickel uptake system nik found in Escherichia coli, a member of the ABC family of transporters, that involves a periplasmic nickel-binding protein, NikA . This is the initial nickel receptor and mediator of the chemotactic response away from nickel . We have solved the crystal structure of NikA protein in the presence and absence of nickel, showing that it behaves as a "classical" periplasmic binding protein . In contrast to other binding proteins, however, the ligand remains accessible to the solvent and is not completely enclosed . No direct bonds are formed between the metal cation and the protein . The nickel binding site is apolar, quite unlike any previously characterized protein nickel binding site . Despite relatively weak binding, NikA is specific for nickel . Using isothermal titration calorimetry, the dissociation constant for nickel was found to be approximately 10 microm and that for cobalt was approximately 20 times higher. J Biol Chem, 2003 Nov 28, 278(48), 48041 - 50 Epub 2003 Sep 05. Release of ribosome-bound ribosome recycling factor by elongation factor G; Kiel MC et al.; Elongation factor G (EF-G) and ribosome recycling factor (RRF) disassemble post-termination complexes of ribosome, mRNA, and tRNA . RRF forms stable complexes with 70 S ribosomes and 50 S ribosomal subunits . Here, we show that EF-G releases RRF from 70 S ribosomal and model post-termination complexes but not from 50 S ribosomal subunit complexes . The release of bound RRF by EF-G is stimulated by GTP analogues . The EF-G-dependent release occurs in the presence of fusidic acid and viomycin . However, thiostrepton inhibits the release . RRF was shown to bind to EF-G-ribosome complexes in the presence of GTP with much weaker affinity, suggesting that EF-G may move RRF to this position during the release of RRF . On the other hand, RRF did not bind to EF-G-ribosome complexes with fusidic acid, suggesting that EF-G stabilized by fusidic acid does not represent the natural post-termination complex . In contrast, the complexes of ribosome, EF-G and thiostrepton could bind RRF, although with lower affinity . These results suggest that thiostrepton traps an intermediate complex having RRF on a position that clashes with the P/E site bound tRNA . Mutants of EF-G that are impaired for translocation fail to disassemble post-termination complexes and exhibit lower activity in releasing RRF . We propose that the release of ribosome-bound RRF by EF-G is required for post-termination complex disassembly . Before release from the ribosome, the position of RRF on the ribosome will change from the original A/P site to a new location that clashes with tRNA on the P/E site. Endocrinology, 2003 Dec, 144(12), 5578 - 84 Epub 2003 Aug 21. In vitro and in vivo calcitonin I gene expression in parenchymal cells: a novel product of human adipose tissue; Linscheid P et al.; Circulating levels of calcitonin precursors (CTpr), including procalcitonin (ProCT), increase up to several thousand-fold in human sepsis, and immunoneutralization improves survival in two animal models of this disease . Herein, we analyzed inflammation-mediated calcitonin I gene (CALC I) expression in human adipocyte primary cultures and in adipose tissue samples from infected and noninfected patients with different levels of serum ProCT . In ex vivo differentiated adipocytes, the expression of CT mRNA increased 24-fold (P < 0.05) after the administration of Escherichia coli endotoxin (lipopolysaccharide) and 37-fold (P < 0.05) after IL-1beta administration by 6 h . ProCT protein secretion into culture supernatant increased 13.5-fold (P < 0.01) with lipopolysaccharide treatment and 15.2-fold (P < 0.01) with IL-1beta after 48 h . In coculture experiments, adipocyte CT mRNA expression was evoked by E . coli-activated macrophages in which CT mRNA was undetectable . The marked IL-1beta-mediated ProCT release was inhibited by 89% during coadministration with interferon-gamma (IFNgamma) . In patients with infection and markedly increased serum ProCT, CT mRNA was detected in adipose tissue biopsies . Hence, we demonstrate that ProCT, which is suspected to mediate deleterious effects in sepsis and inflammation, is a novel product of adipose tissue secretion . The inhibiting effect of IFNgamma on IL-1beta-induced CT mRNA expression and on ProCT secretion might explain previous observations that serum ProCT concentrations increase less in systemic viral compared with bacterial infections. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Sep, 35(9), 853 - 8 {Cloning, expression and characterization of the hypoxanthine-guanine phosphoribosyltransferase mutants from T . tengcongensis}; You DL et al.; Based on a predicted three-dimensional structure of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) from Thermoanaerobacter tengcongensis, three mutants of HGPRT were designed to modify the purine specificity of HGPRT . Site-directed mutagenesis was used to generate the three mutants, K(133)A, K(133)S, K(133)T . Wild type HGPRT and its mutants were expressed E . coli in BL21 (DE3) pLysS, and the expression products of them reached about 30% of the total protein . The molecular weight of the recombinant proteins was 22 kD . The specific activities of the enzymes were determined . Catalytic activities of mutants K(133)A, K(133)S, K(133)T retained only 4%, 1.1%, 2.7% activities, respectively, of the wild type for hypoxanthine, 1.7% 0.6% 1% activities, respectively, of wild type for guanine . However, the three mutants showed 24-fold, 7-fold, 18-fold activities, respectively, of the wild type for xanthine, and 650-fold, 210-fold, 380-fold activities, respectively, of the wild type for adenine . Comparison of kinetic data for purified recombinant mutant with wild-type HGPRT showed significant difference in the catalytic efficiency (kcat/Km) for purine, xanthine and adenine, the mutants exhibiting more than 40 to 50-fold higher kcat/Km, as a result of nearly 4 to 5-fold decrease in Km, compared with wild-type . These results demonstrate that a single amino acid substitution in HGPRT at the active site can significantly modify the specificity for binding purine. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Sep, 35(9), 834 - 40 {Baculovirus p74 gene is a species-specific gene}; Wu WW et al.; The p74 gene of Autographa californica multicasid nucleopolyhedrovirus (AcMNPV) bacmid was knockouted and substituted by the p74 gene of Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV), using RecA-mediated homologous recombination in the E . coli . No selection marker, which might influence the expression and function of p74 gene, was left in the modified p74 locus . The promoter of AcMNPV p74 gene directly controlled the expression of SpltMNPV p74 gene in the recombinant AcMNPV bacmid-polhSL74 . RT-PCR showed that the substituted p74 gene was transcribed . Bioassay showed that the recombinant virus AcMNPV bacmid-polhSL74 could not infect the Argyrogramma agnata larvae per os, and thus showing the p74 gene is species-specific. Science, 2003 Sep 5, 301(5638), 1383 - 7 Molecular basis of metal-ion selectivity and zeptomolar sensitivity by CueR; Changela A et al.; The earliest of a series of copper efflux genes in Escherichia coli are controlled by CueR, a member of the MerR family of transcriptional activators . Thermodynamic calibration of CueR reveals a zeptomolar (10(-21) molar) sensitivity to free Cu+, which is far less than one atom per cell . Atomic details of this extraordinary sensitivity and selectivity for +1transition-metal ions are revealed by comparing the crystal structures of CueR and a Zn2+-sensing homolog, ZntR . An unusual buried metal-receptor site in CueR restricts the metal to a linear, two-coordinate geometry and uses helix-dipole and hydrogen-bonding interactions to enhance metal binding . This binding mode is rare among metalloproteins but well suited for an ultrasensitive genetic switch. J Clin Microbiol, 2003 Sep, 41(9), 4480 - 2 Multiplex PCRs for identification of necrotoxigenic Escherichia coli; Van Bost S et al.; Two multiplex PCRs were developed for the detection of necrotoxigenic Escherichia coli virulence genes . M1 contained the primers for the toxins and the aerobactin, and M2 contained the primers for the adhesins . They were validated by single PCRs performed with reference E . coli strains and by multiplex PCRs with necrotoxigenic E . coli strains isolated from different animal species. Proc Natl Acad Sci U S A, 2003 Sep 16, 100(19), 10688 - 93 Epub 2003 Sep 04. Differential substrate-induced signaling through the TonB-dependent transporter BtuB; Cadieux N et al.; The BtuB transporter mediates high-affinity binding and TonB-dependent active transport of vitamin B12 {cyanocobalamin (CNCbl)} across the outer membrane of Escherichia coli . A characteristic feature of TonB-dependent transporters is the Ton box, a conserved sequence near the N terminus and exposed to the periplasm . Crosslinking to TonB and site-directed spin labeling indicated that the Ton box of BtuB undergoes a substantial conformational transition in response to CNCbl binding, but only slight movement was seen in crystal structures . An in vivo method of detecting substrate-induced changes in the Ton box environment measured reaction of a biotin maleimide derivative with cysteine substitutions through the N-terminal region of BtuB between positions 1 and 31 . The degree of maleimide labeling of different residues correlated with their accessibility in the crystal structure . Labeling of many positions was increased strongly when CNCbl was present, consistent with the undocking of this region proposed from spin-labeling analyses . The receptor-binding domain of colicin E3, which binds to BtuB competitively with CNCbl, resulted in decreased labeling . Both substrate-induced transitions occur in and beyond the Ton box and were affected by transport-uncoupling substitutions . Thus, two transport substrates that bind competitively to the extracellular face of BtuB stabilize opposite transitions of the Ton box. Appl Environ Microbiol, 2003 Sep, 69(9), 5707 - 10 Survival of F-specific RNA coliphage, feline calicivirus, and Escherichia coli in water: a comparative study; Allwood PB et al.; The relationship between the survival of enteric viral pathogens and their indicators (coliform bacteria and coliphages) is not well understood . We compared the survival rates of feline calicivirus (FCV), Escherichia coli, and a male-specific RNA coliphage MS2 at 4, 25, and 37 degrees C for up to 28 days in dechlorinated water . The survival rates of E . coli and FCV, a surrogate of noroviruses (NV), had a high degree of correlation at 4 and 25 degrees C, while MS2 phage survived significantly longer (P < 0.05) at these two temperatures . At 37 degrees C, the survival rates for all three organisms were highly correlated . Decimal reduction values indicating the number of days needed for 90% reduction in titer (D values) decreased for all three organisms as storage temperatures increased . FCV had the shortest D value among all three organisms at all temperatures investigated . These findings indicate that F-specific RNA phages may be useful indicators of NV in the environment. Appl Environ Microbiol, 2003 Sep, 69(9), 5673 - 8 Use of DNA and peptide nucleic acid molecular beacons for detection and quantification of rRNA in solution and in whole cells; Xi C et al.; DNA and peptide nucleic acid (PNA) molecular beacons were successfully used to detect rRNA in solution . In addition, PNA molecular beacon hybridizations were found to be useful for the quantification of rRNA: hybridization signals increased in a linear fashion with the 16S rRNA concentrations used in this experiment (between 0.39 and 25 nM) in the presence of 50 nM PNA MB . DNA and PNA molecular beacons were successfully used to detect whole cells in fluorescence in situ hybridization (FISH) experiments without a wash step . The FISH results with the PNA molecular beacons were superior to those with the DNA molecular beacons: the hybridization kinetics were much faster, the signal-to-noise ratio was much higher, and the specificity was much better for the PNA molecular beacons . Finally, it was demonstrated that the combination of the use of PNA molecular beacons in FISH and flow cytometry makes it possible to rapidly collect quantitative FISH data . Thus, PNA molecular beacons might provide a solution for limitations of traditional FISH methods, such as variable target site accessibility, poor sensitivity for target cells with low rRNA content, background fluorescence, and applications of FISH in microfluidic devices. Appl Environ Microbiol, 2003 Sep, 69(9), 5555 - 62 Foreshore sand as a source of Escherichia coli in nearshore water of a Lake Michigan beach; Whitman RL et al.; Swimming advisories due to excessive Escherichia coli concentrations are common at 63rd Street Beach, Chicago, Ill . An intensive study was undertaken to characterize the source and fate of E . coli in beach water and sand at the beach . From April through September 2000, water and sand samples were collected daily or twice daily at two depths on three consecutive days per week (water samples, n = 1,747; sand samples, n = 858); hydrometeorological conditions and bird and bather distributions were also recorded . E . coli concentrations in sand and water were significantly correlated, with the highest concentration being found in foreshore sand, followed by those in submerged sediment and water of increasing depth . Gull contributions to E . coli densities in sand and water were most apparent on the day following gull activity in a given area . E . coli recolonized newly placed foreshore sand within 2 weeks . Analysis of variance, correlation, cluster analyses, concentration gradients, temporal-spatial distribution, demographic patterns, and DNA fingerprinting suggest that E . coli may be able to sustain population density in temperate beach sand during summer months without external inputs . This research presents evidence that foreshore beach sand (i) plays a major role in bacterial lake water quality, (ii) is an important non-point source of E . coli to lake water rather than a net sink, (iii) may be environmentally, and perhaps hygienically, problematic, and (iv) is possibly capable of supporting an autochthonous, high density of indicator bacteria for sustained periods, independent of lake, human, or animal input. Eur Cytokine Netw, 2003 Apr-Jun, 14(2), 91 - 6 Glycosylation enhances functional stability of the chemotactic cytokine CCL2; Ruggiero P et al.; The human chemokine CCL2 gene was expressed in the yeast P.pastoris and gave rise to a mixture of differently glycosylated recombinant proteins . In comparison to non-glycosylated E.coli-derived CCL2, glycosylated yeast CCL2L was 4-20 times less active in a chemotactic assay in vitro . However, CCL2L could maintain full activity upon prolonged incubation at 37 degrees C, whereas the non-glycosylated chemokine readily lost activity . It could be hypothesized that glycosylation is a mechanism used by the organism to modulate CCL2 stability . The partial loss of specific activity due to glycosylation is balanced by the advantage of prolonging the effectiveness of chemokine . Thus, differential glycosylation allows one to obtain highly effective short-lived CCL2 or less-effective long-lived CCL2 and may thus represent a novel mechanism of adaptation to pathological versus physiological conditions. Steroids, 2003 Sep, 68(7-8), 603 - 11 Novel P450(17alpha) inhibitors: 17-(2'-oxazolyl)- and 17-(2'-thiazolyl)-androstene derivatives; Zhu N et al.; Twelve 17-(2'-oxazolyl)- and 17-(2'-thiazolyl)-androsta-5,16-diene derivatives were designed and synthesized from 3 beta-acetoxy-pregna-5,16-dien-20-one (1b) as inhibitors of 17 alpha-hydroxylase-C(17,20)-lyase (P450(17 alpha)) . Potent inhibitors of this enzyme could be of value as treatment of prostate cancer . Two substituents (methyl and phenyl) were introduced either at their 4'- or 5'-position in order to investigate their structure-activity relationship . Due to the 16,17-double bond, 17-thiazoles were generally obtained in low yield . The pharmacological results showed that the compounds containing 17-(2'-oxazolyl) (14c) and 17-(2'-thiazolyl) (8c) (41.5%) demonstrated reasonable inhibition against P450(17 alpha) . Their 3-acetate (13c and 7c) were less potent than their 3-OH counterparts . The introduction of a phenyl or methyl group generally decreased inhibitory activity . Surprisingly, 17-(5'-methyl-2'-thiazolyl) (12a) was the most potent compound in this series and was almost as potent as L-39, which has good antitumor activity. Free Radic Biol Med, 2003 Sep 15, 35(6), 595 - 602 Inhibition of 8-oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase (8-oxo-dGTPase) activity of the antimutagenic human MTH1 protein by nucleoside 5'-diphosphates; Bialkowski K et al.; The hMTH1 protein, a human homologue of E . coli MutT protein, is an enzyme converting 8-oxo-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) to 8-oxo-2'-deoxyguanosine 5'-monophosphate (8-oxo-dGMP) and inorganic pyrophosphate . It is thought to play an antimutagenic role by preventing the incorporation of promutagenic 8-oxo-dGTP into DNA . As found in our previous investigations, 8-oxo-2'-deoxyguanosine 5'-diphosphate (8-oxo-dGDP) strongly inhibited 8-oxo-dGTPase activity of MTH1 . Following this finding, in the present study we have tested the canonical ribo- and deoxyribonucleoside 5'-diphosphates (NDPs and dNDPs) for possible inhibition of 8-oxo-dGTP hydrolysis by hMTH1 extracted from CCRF-CEM cells (a human leukemia cell line) . Among them, the strongest inhibitors appeared to be dGDP (Ki=74 microM), dADP (Ki=147 microM), and GDP (Ki=502 microM) . Other dNDPs and NDPs, such as dCDP, dTDP, ADP, CDP, and UDP were much weaker inhibitors, with Ki in the millimolar range . Based on the present results and published data, we estimate that the strongest inhibitors, dGDP and dADP, at physiological concentrations not exceeding 5 microM and GDP at mean concentration of 30 microM, taken together, can decrease the cellular hMTH1 enzymatic activity vs . 8-oxo-dGTP (expected to remain below 500 pM) by up to 15% . The other five NDPs and dNDPs tested cannot markedly affect this activity. Gene, 2003 Aug 14, 313, 59 - 69 Engineering of RNase P ribozyme for gene-targeting applications; Raj SM et al.; Ribonuclease P (RNase P) is a ubiquitous ribonucleoprotein complex responsible for the biosynthesis of tRNA . This enzyme from Escherichia coli contains a catalytic RNA subunit (M1 ribozyme) and a protein subunit (C5 cofactor) . M1 ribozyme cleaves an RNA helix that resembles the acceptor stem and T-stem structure of its natural tRNA substrate . When covalently linked with a guide sequence, M1 RNA can be engineered into a sequence-specific endonuclease, M1GS ribozyme, which can cleave any target RNA sequences that base pair with the guide sequence . Recent studies indicate that M1GS ribozymes efficiently cleave the mRNAs of herpes simplex virus 1, human cytomegalovirus, and cancer causing BCR-ABL proteins in vitro and effectively inhibit the expression of these mRNAs in cultured cells . Moreover, RNase P ribozyme variants that are more active than the wild type M1 RNA can be generated using in vitro selection procedures and the selected variants are also more effective in inhibiting gene expression in cultured cells . These results demonstrate that engineered RNase P ribozymes represent a novel class of promising gene-targeting agents for applications in both basic research and clinical therapy . This review discusses the principle underlying M1GS-mediated gene inactivation and methodologies involved in effective M1GS construction, expression in vivo and emerging prospects of this technology for gene therapy. Exp Eye Res, 2003 Oct, 77(4), 505 - 14 A soluble peripherin/Rds C-terminal polypeptide promotes membrane fusion and changes conformation upon membrane association; Boesze-Battaglia K et al.; Photoreceptor rod cells contain a unique tetraspanin fusion protein known as peripherin/rds . This protein is important in membrane fusion events hypothesized to be essential to disk membrane morphogenesis and disk shedding . In vivo and in vitro fusogenic activity has been mapped to the C-terminal domain of peripherin/rds . Moreover, a fusion peptide domain localized to a 15 amino acid long region (residues 311-325) is essential for mediating lipid bilayer fusion of model membranes . To address the functional and structural properties required for peripherin/rds dependent membrane fusion, constructs of the entire C-terminal domain (residues 284-346) were generated and polypeptides expressed . A wild type-peripherin/rds C-terminal GST fusion construct that included the entire C-terminus (PERCTER) or a C-terminal truncation mutant (PERCTN) were engineered with a thrombin cleavage site . Protein expression was induced in E . coli with IPTG, expressed proteins cleaved from the GST with thrombin and purified to homogeneity on a Superdex 75 column . Purity was confirmed by SDS-PAGE and Western blot analysis . The purified wt C-terminal protein resolved as a monomer under reducing conditions on SDS-PAGE (15%) and was immunoreactive with anti peripherin/rds antibody 2B6 (gift from Dr R . Molday) . The purified polypeptide promoted the requisite steps of fusion, membrane destabilization, lipid mixing and aqueous contents mixing . Conversely, the truncation mutant lacking a portion of the fusion domain was unable to promote these steps . A common feature of most membrane fusion proteins is a change in conformation upon membrane association . Structural changes in the C-terminal polypeptide were investigated using far UV CD . The far UV CD spectra of the purified C-terminal polypeptide indicated substantial alpha-helical content in the wt peptide in isotonic aqueous buffer . An increase in intensity of 208 and 222 nm CD bands upon addition of DPC vesicles indicated an increase in alpha-helical content of the polypeptide . These results demonstrate that a purified soluble form of the C-terminus of peripherin/rds can interact with biological phospholipids; moreover, this interaction promotes a conformational change that is most consistent with an increase in alpha-helical content. J Surg Res, 2003 Aug, 113(2), 189 - 94 MAPK regulation of prostaglandin E2 production by lipopolysaccharide-stimulated macrophages is not dependent on nuclear factor kappaB; Lo CJ; BACKGROUND: Prostaglandin E2 (PGE2) is a major contributor to the production and maintenance of immunosuppression in injured and septic patients . Although the synthesis of PGE2 by various enzymes has been elucidated, the regulatory mechanism of these enzymes is not clear . The purpose of this study was to determine the role of MAPK cascades in COX-2 gene activation by lipopolysaccharide (LPS)-stimulated macrophages (Mphi) . MATERIALS AND METHODS: RAW 264.7 cells, a mouse Mphi cell line, were exposed to Escherichia coli LPS (10 microg/ml) in the presence of PD98059, a selective inhibitor of MAPKP44/P42, and SB202190, a selective inhibitor of MAPKP38 . COX-2 mRNA expression and PGE2 production were measured by Northern Blot assay and ELISA, respectively . Mphi nuclear factor (NF)kappaB and cAMP-response element (CRE) activities were determined by electrophoretic mobility shift assays . RESULTS: LPS stimulation increased Mphi COX-2 mRNA expression and PGE2 production . PD98059 or SB202190 attenuated LPS-induced COX-2 mRNA as well as PGE2 production in a dose-dependent fashion . Inhibition of MAPKP44/P42 or MAPKP38 had no effect on NFkappaB activation but reduced CRE activity induced by LPS stimulation . CONCLUSION: Our data show that MAPK cascades regulate COX-2 gene expression and PGE2 production in LPS-stimulated Mphi through NFkappaB independent pathways . The regulatory mechanism is likely to be mediated through CRE. Biochem J, 2003 Dec 15, 376(Pt 3), 633 - 44 Use of the transport specificity ratio and cysteine-scanning mutagenesis to detect multiple substrate specificity determinants in the consensus amphipathic region of the Escherichia coli GABA (gamma-aminobutyric acid) transporter encoded by gabP; King SC et al.; The Escherichia coli GABA (gamma-aminobutyric acid) permease, GabP, and other members of the APC (amine/polyamine/choline) transporter superfamily share a CAR (consensus amphipathic region) that probably contributes to solute translocation . If true, then the CAR should contain structural features that act as determinants of substrate specificity ( k (cat)/ K (m)) . In order to address this question, we have developed a novel, expression-independent TSR (transport specificity ratio) analysis, and applied it to a series of 69 cysteine-scanning (single-cysteine) variants . The results indicate that GabP has multiple specificity determinants (i.e . residues at which an amino acid substitution substantially perturbs the TSR) . Specificity determinants were found: (i) on a hydrophobic surface of the CAR (from Leu-267 to Ala-285), (ii) on a hydrophilic surface of the CAR (from Ser-299 to Arg-318), and (iii) in a cytoplasmic loop (His-233) between transmembrane segments 6 and 7 . Overall, these observations show that (i) structural features within the CAR have a role in substrate discrimination (as might be anticipated for a transport conduit) and, interestingly, (ii) the substrate discrimination task is shared among specificity determinants that appear too widely dispersed across the GabP molecule to be in simultaneous contact with the substrates . We conclude that GabP exhibits behaviour consistent with a broadly applicable specificity delocalization principle, which is demonstrated to follow naturally from the classical notion that translocation occurs synchronously with conformational transitions that change the chemical potential of the bound ligand {Tanford (1982) Proc . Natl . Acad . Sci . U.S.A . 79, 2882-2884}. Biochem J, 2003 Dec 15, 376(Pt 3), 645 - 53 Induction of substrate specificity shifts by placement of alanine insertions within the consensus amphipathic region of the Escherichia coli GABA (gamma-aminobutyric acid) transporter encoded by gabP; King SC et al.; The Escherichia coli GABA (gamma-aminobutyric acid) permease GabP is a prototypical APC (amine/polyamine/choline) super-family transporter that has a CAR (consensus amphipathic region) containing multiple specificity determinants, ostensibly organized on two helical surfaces, one hydrophobic {SHS (sensitive hydrophobic surface)} and the other hydrophilic {SPS (sensitive polar surface)} . To gauge the functional effects of placing alanine insertions at close intervals across the entire GabP CAR, 64 insertion variants were constructed . Insertions, particularly those in the SHS and the SPS, were highly detrimental to steady-state {(3)H}GABA accumulation . TSR (transport specificity ratio) analysis, employing {(3)H}nipecotic acid and {(14)C}GABA, showed that certain alanine insertions were associated with a specificity shift (i.e . a change in k (cat)/ K (m)) . An insertion (INS Ala-269) located N-terminal to the SHS increased specificity for {(3)H}nipecotic acid relative to {(14)C}GABA, whereas an insertion (INS Ala-321) located C-terminal to the SPS had the opposite effect . Overall, the results are consistent with a working hypothesis that the GabP CAR contains extensive functional surfaces that may be manipulated by insertion mutagenesis to alter the specificity ( k (cat)/ K (m)) phenotype . The thermodynamic basis of TSR analysis provides generality, suggesting that amino acid insertions could affect specificity in many other transporters, particularly those such as the E . coli phenylalanine permease PheP {Pi, Chow a