Microbiology Reader
Equipment to run microbiology work automatically

Growth Curves of any strain.
Microbiological calculations.

Microbiology Home
Microbioloy Reader
Growth Curves
Photo Album
Microorganisms
Software
Download
Purchasing
Contact Us



Proc Natl Acad Sci U S A, 1998 Jun 23, 95(13), 7357 - 62
The yeast halotolerance determinant Hal3p is an inhibitory subunit of the Ppz1p Ser/Thr protein phosphatase; de Nadal E et al.; Components of cellular stress responses can be identified by correlating changes in stress tolerance with gain or loss of function of defined genes . Previous work has shown that yeast cells deficient in Ppz1 protein phosphatase or overexpressing Hal3p, a novel regulatory protein of unknown function, exhibit increased resistance to sodium and lithium, whereas cells lacking Hal3p display increased sensitivity . These effects are largely a result of changes in expression of ENA1, encoding the major cation extrusion pump of yeast cells . Disruption or overexpression of HAL3 (also known as SIS2) has no effect on salt tolerance in the absence of PPZ1, suggesting that Hal3p might function upstream of Ppz1p in a novel signal transduction pathway . Hal3p is recovered from crude yeast homogenates by using immobilized, bacterially expressed Ppz1p fused to glutathione S-transferase, and it also copurifies with affinity-purified glutathione S-transferase-Ppz1p from yeast extracts . In both cases, the interaction is stronger when only the carboxyl-terminal catalytic phosphatase domain of Ppz1p is expressed . In vitro experiments reveal that the protein phosphatase activity of Ppz1p is inhibited by Hal3p . Overexpression of Hal3p suppresses the reduced growth rate because of the overexpression of Ppz1p and aggravates the lytic phenotype of a slt2/mpk1 mitogen-activated protein kinase mutant (thus mimicking the deletion of PPZ1) . Therefore, Hal3p might modulate diverse physiological functions of the Ppz1 phosphatase, such as salt stress tolerance and cell cycle progression, by acting as a inhibitory subunit.

Biochemistry, 1998 Jun 23, 37(25), 9193 - 202
Strong selective pressure to use G:U to mark an RNA acceptor stem for alanine; Chihade JW et al.; The identity of alanine tRNAs is dependent on a G:U base pair at the 3:70 position of the acceptor helix . This system of molecular recognition is widely distributed from bacteria to human-cell cytoplasm . In contrast, some mitochondrial alanine acceptor helices are markedly different and contain nucleotides known to block aminoacylation by a nonmitochondrial enzyme . Thus, acceptor helix recognition may differ in these systems and may not depend on G:U . Here we report an example of a Caenorhabditis elegans mitochondrial system where the G:U pair is preserved but where proximal nucleotides known to block charging by a nonmitochondrial enzyme are also present . We show that, as expected, the mitochondrial substrate is not charged by the bacterial enzyme . In contrast, the cloned mitochondrial enzyme charged both mitochondrial and bacterial microhelices . Strikingly, charging of each required the G:U pair . Thus, G:U recognition persists even with an acceptor helix context that inactivates nonmitochondrial systems . The results suggest strong selective pressure to use G:U in a variety of contexts to mark an acceptor stem for alanine . Separate experiments also demonstrate that, at least for the mitochondrial enzyme, helix instability or irregularity is not important for recognition of G:U.

Biochemistry, 1998 Jun 23, 37(25), 9052 - 7
Thermodynamics of a transition state analogue inhibitor binding to Escherichia coli chorismate mutase: probing the charge state of an active site residue and its role in inhibitor binding and catalysis; Lee AY et al.; Electrostatic interactions play important roles in the catalysis of chorismate to prephenate by chorismate mutase . Mutation of Gln88 to glutamate in the monofunctional chorismate mutase from Escherichia coli results in an enzyme with a pH profile of activity significantly different from that of the wild type protein . To investigate whether the mutation alters the substrate binding process or the catalysis, we have directly determined the thermodynamic parameters of a transition state analogue inhibitor binding to the wild-type chorismate mutase and its Q88E mutant using isothermal titration calorimetry . The results demonstrate that solvent reorganization and hydrophobic interactions contribute the predominant free energy to inhibitor binding . The charge state of Glu88 in the Q88E mutant was experimentally determined and was shown to be protonated at pH 4.5 and ionized at pH 7.8, consistent with earlier hypotheses . Most surprisingly, inhibitor binding energetics do not exhibit significant pH dependency for both enzymes . Our findings indicate that the charge state of Glu88 has a small impact on inhibitor binding but plays an important role in the catalytic process.

Biochemistry, 1998 Jun 23, 37(25), 9033 - 7
Glutamate-459 is important for Escherichia coli branching enzyme activity; Binderup K et al.; The branching enzyme belongs to the amylolytic family, a group of enzymes that cleave and/or transfer chains of glucan . The amylolytic enzymes are homologous and all contain four conserved regions, proposed to contain the active site . By primary structure analysis, a conserved position unique to branching enzymes has been identified . This residue, which is either Asp or Glu, depending on the species, is located immediately after the putative catalytic Glu-458 (Escherichia coli numbering) . Branching enzymes differ from other amylolytic enzymes in having this acid pair, and we asked if this motif could be essential for branching enzyme action.We used site-directed mutagenesis of the Glu-459 residue in the E . coli branching enzyme in order to determine the significance of the conserved Asp/Glu in branching enzymes . A substitution of Glu-459 to Asp resulted in increased specific activity compared to wild-type, suggesting that the mutation had created a more efficient enzyme . Changing Glu-459 to Ala, Lys, or Gln lowered the specific activities and altered the preferred substrate from amylose to amylopectin.

Biochemistry, 1998 Jun 23, 37(25), 8832 - 8
Autocatalytic formation of a hydroxy group at C beta of trp171 in lignin peroxidase; Blodig W et al.; In the high-resolution crystal structures of two lignin peroxidase isozymes from the white rot fungus Phanerochaete chrysosporium a significant electron density at single bond distance from the C beta of Trp171 was observed and interpreted as a hydroxy group . To further clarify the nature of this feature, we carried out tryptic digestion of the enzyme and isolated the Trp171 containing peptide . Under ambient conditions, this peptide shows an absorbance spectrum typical of tryptophan . At elevated temperature, however, the formation of an unusual absorbance spectrum with lambda max = 333 nm can be followed that is identical to that of N-acetyl-alpha, beta-didehydrotryptophanamide, resulting upon water elimination from beta-hydroxy tryptophan . The Trp171 containing tryptic peptide isolated from the recombinant and refolded lignin peroxidase produced from Escherichia coli does not contain the characteristic 333 nm absorbance band at any temperature . However, treatment with 3 equiv of H2O2 leads to complete hydroxylation of Trp171 . Reducing substrates compete with this process, e.g., in the presence of 0.5 mM veratryl alcohol, about 7 equiv of H2O2 is necessary for complete modification . We conclude that the hydroxylation at the C beta of Trp171 is an autocatalytic reaction which occurs readily under conditions of natural turnover, e.g., in the ligninolytic cultures of P . chrysosporium, which are known to contain an oxidase-based H2O2-generating system . No dependence on dioxygen was found for this oxidative process . Chemical modification of fungal lignin peroxidase with the tryptophan-specific agent N-bromo succinimide leads to a drastically reduced activity with respect to the substrate veratryl alcohol . This suggests that Trp171 is involved in catalysis and that electron transfer from this surface residue to the oxidized heme cofactor is possible under steady-state conditions.

Biochemistry, 1998 Jun 23, 37(25), 8825 - 31
Carbamoyl phosphate synthetase: caught in the act of glutamine hydrolysis; Thoden JB et al.; Carbamoyl phosphate synthetase from Escherichia coli catalyzes the production of carbamoyl phosphate from two molecules of Mg2+ATP, one molecule of bicarbonate, and one molecule of glutamine . The enzyme consists of two polypeptide chains referred to as the large and small subunits . While the large subunit provides the active sites responsible for the binding of nucleotides and other effector ligands, the small subunit contains those amino acid residues that catalyze the hydrolysis of glutamine to glutamate and ammonia . From both amino acid sequence analyses and structural studies it is now known that the small subunit belongs to the class I amidotransferase family of enzymes . Numerous biochemical studies have suggested that the reaction mechanism of the small subunit proceeds through the formation of the glutamyl thioester intermediate and that both Cys 269 and His 353 are critical for catalysis . Here we describe the X-ray crystallographic structure of carbamoyl phosphate synthetase from E . coli in which His 353 has been replaced with an asparagine residue . Crystals employed in the investigation were grown in the presence of glutamine, and the model has been refined to a crystallographic R-factor of 19.1% for all measured X-ray data from 30 to 1.8 A resolution . The active site of the small subunit clearly contains a covalently bound thioester intermediate at Cys 269, and indeed, this investigation provides the first direct structural observation of an enzyme intermediate in the amidotransferase family.

Biochemistry, 1998 Jun 23, 37(25), 8817 - 24
Solution structure of the transmembrane H+-transporting subunit c of the F1F0 ATP synthase; Girvin ME et al.; Subunit c is the H+-translocating component of the F1F0 ATP synthase complex . H+ transport is coupled to conformational changes that ultimately lead to ATP synthesis by the enzyme . The properties of the monomeric subunit in a single-phase solution of chloroform-methanol-water (4:4:1) have been shown to mimic those of the protein in the native complex . Triple resonance NMR experiments were used to determine the complete structure of monomeric subunit c in this solvent mixture . The structure of the protein was defined by >2000 interproton distances, 64 (3)JN alpha, and 43 hydrogen-bonding NMR-derived restraints . The root mean squared deviation for the backbone atoms of the two transmembrane helices was 0.63 A . The protein folds as a hairpin of two antiparallel helical segments, connected by a short structured loop . The conserved Arg41-Gln42-Pro43 form the top of this loop . The essential H+-transporting Asp61 residue is located at a slight break in the middle of the C-terminal helix, just prior to Pro64 . The C-terminal helix changes direction by 30 +/- 5 degrees at the conserved Pro64 . In its protonated form, the Asp61 lies in a cavity created by the absence of side chains at Gly23 and Gly27 in the N-terminal helix . The shape and charge distribution of the molecular surface of the monomeric protein suggest a packing arrangement for the oligomeric protein in the F0 complex, with the front face of one monomer packing favorably against the back face of a second monomer . The packing suggests that the proton (cation) binding site lies between packed pairs of adjacent subunit c.

Biochemistry, 1998 Jun 16, 37(24), 8803 - 7
Mutagenic specificity of model estrogen-DNA adducts in mammalian cells; Terashima I et al.; Site-specifically modified oligodeoxynucleotides were used to explore the mutagenic properties of the model estrogen-DNA adducts N2-{3-methoxyestra-1,3,5(10)-trien-6(alpha, beta)-yl}-2'-deoxyguanosine (dG-N2-3MeE) and N6-{3-methoxyestra-1,3, 5(10)-trien-6(alpha,beta)-yl}-2'-deoxyadenosine (dA-N6-3MeE) in simian kidney (COS-7) cells . Oligodeoxynucleotides (5'TCCTCCTCXCCTCTC; X = dG, dA, dG-N2-3MeE, or dA-N6-3MeE) containing an unmodified or model estrogen lesion were inserted into single-stranded (ss) phagemid vectors . These ss vectors were transfected into COS-7 cells . The progeny plasmid obtained were used to transform Escherichia coli DH10B . The transformants were analyzed by oligodeoxynucleotide hybridization and sequencing to determine the mutation frequency and spectrum . Preferential incorporation of dCMP, the correct base, was observed opposite the dG-N2-3MeE lesion . Targeted mutations showing G --> T transversions were detected, along with a small number of G --> C transversions . When a dA-N6-3MeE-modified oligodeoxynucleotide was used, preferential incorporation of dTMP, the correct base, was also observed . Targeted mutations representing A --> T transversions were detected, accompanied by a small amount of A --> G transitions . The frequency of mutation observed opposite dA-N6-3MeE (17.5%) was 2.3 times higher than that observed opposite dG-N2-3MeE (7.5%) . These results indicate that estrogen DNA adducts have mutagenic potential in mammalian cells.

Biochemistry, 1998 Jun 16, 37(24), 8776 - 82
Evaluation of the kinetic mechanism of Escherichia coli glycinamide ribonucleotide transformylase; Shim JH et al.; A kinetic scheme is presented for Escherichia coli glycinamide ribonucleotide transformylase (GAR transformylase, EC 2.1.2.2) based on a steady-state and pre-steady-state kinetic analysis of the reaction in both directions employing stopped-flow absorbance and fluorescence spectroscopy . Steady-state parameters showed that kcat for the reverse direction is about 10 times lower than that for the forward direction although the Km values for formyl dideazafolate and dideazafolate or for glycinamide ribonucleotide and formyl glycinamide ribonucleotide are similar . No pre-steady-state transient was observed in either direction, and the single-turnover rate constant under saturating levels of substrates in each direction was found to be very close to the respective steady-state kcat value . This indicates that steps involving ternary complexes are rate-determining for steady-state turnover in each direction . By conducting the single-turnover reactions under various preincubation and mixing conditions, a random sequential kinetic mechanism was implicated in which the enzyme binds glycinamide ribonucleotide or formyl dideazafolate productively in no obligatory order . The collective data provided a quantitative kinetic scheme to serve as a basis for the analysis of mutations.

Biochemistry, 1998 Jun 16, 37(24), 8735 - 42
Resistance to HIV protease inhibitors: a comparison of enzyme inhibition and antiviral potency; Klabe RM et al.; Resistance of HIV-1 to protease inhibitors has been associated with changes at residues Val82 and Ile84 of HIV-1 protease (HIV PR) . Using both an enzyme assay with a peptide substrate and a cell-based infectivity assay, we examined the correlation between the inhibition constants for enzyme activity (Ki values) and viral replication (IC90 values) for 5 active site mutants and 19 protease inhibitors . Four of the five mutations studied (V82F, V82A, I84V, and V82F/I84V) had been identified as conferring resistance during in vitro selection using a protease inhibitor . The mutant protease genes were expressed in Escherichia coli for preparation of enzyme, and inserted into the HXB2 strain of HIV for test of antiviral activity . The inhibitors included saquinavir, indinavir, nelfinavir, 141W94, ritonavir (all in clinical use), and 14 cyclic ureas with a constant core structure and varying P2, P2' and P3, P3' groups . The single mutations V82F and I84V caused changes with various inhibitors ranging from 0.3- to 86-fold in Ki and from 0.1- to 11-fold in IC90 . Much larger changes compared to wild type were observed for the double mutation V82F/I84V both for Ki (10-2000-fold) and for IC90 (0.7-377-fold) . However, there were low correlations (r2 = 0.017-0.53) between the mutant/wild-type ratio of Ki values (enzyme resistance) and the mutant/wild-type ratio of viral IC90 values (antiviral resistance) for each of the HIV proteases and the viruses containing the identical enzyme . Assessing enzyme resistance by "vitality values", which adjust the Ki values with the catalytic efficiencies (kcat/Km), caused no significant improvement in the correlation with antiviral resistance . Therefore, our data suggest that measurements of enzyme inhibition with mutant proteases may be poorly predictive of the antiviral effect in resistant viruses even when mutations are restricted to the protease gene.

Biochemistry, 1998 Jun 16, 37(24), 8653 - 8
The influence of the regulatory chain amino acids Glu-62 and IIe-12 on the heterotropic properties of Escherichia coli aspartate transcarbamoylase; Dutta M et al.; In the structure of Escherichia coli aspartate transcarbamoylase with CTP bound {Kosman, R . P., Gouaux, J . E., and Lipscomb, W . N . (1993) Proteins, Struct . Funct . Genet . 15, 147-177} Lys-6 and Glu-62 form a salt-link between two regulatory chains . However, recent X-ray structural studies suggest that side chain and backbone interactions existed between Glu-62 and Ile-12 . Thus the interaction between Glu-62 and Ile-12 may help to establish the correct conformation of the nucleotide binding site . The present study of two single-site mutant enzymes, Glu-62r-->Ala and Ile-12r-->Ala, was undertaken to investigate whether the role of Glu-62 is to maintain the stability of the interface between the regulatory chains in the dimer, or interacts with the side chain of Ile-12r to define the nucleotide binding site . For both the mutant enzymes, the maximal velocity, the aspartate saturation at half the maximal velocity, and Hill coefficient were close to wild-type values . The Glu-62r-->Ala enzyme showed enhanced regulatory effects with ATP, CTP, and UTP . As a result of this mutation the enzyme losses its ability to discriminate between CTP and UTP . For the Ile-12r-->Ala enzyme, the heterotropic properties were reduced or eliminated . The enhanced regulatory effects observed with the Glu-62r-->Ala enzyme do not seem to be consistent with the presence of a salt-link between Glu-62r and Lys-6r . However, based upon kinetic data of the unique but completely opposite heterotropic properties of the two mutant enzymes, it is suggested that the side chain interaction between Glu-62r and Ile-12r helps to define the conformation of the effector binding pocket . In this study, we report the properties of both the Glu-62r-->Ala and Ile-12r-->Ala enzymes and their importance for the heterotropic activation and inhibition of aspartate transcarbamoylase.

Biochim Biophys Acta, 1998 May 20, 1392(1), 119 - 26
Molecular cloning, expression in Escherichia coli, and characterization of a novel L-3-hydroxyacyl coenzyme A dehydrogenase from pig liver; He XY et al.; A novel L-3-hydroxyacyl-CoA dehydrogenase from pig liver has been cloned, expressed, purified, and characterized . This enzyme is a homodimer with a molecular mass of 65.6 kDa, and is distinguished from the dehydrogenase of pig heart by its structural features and catalytic properties . Its subunit, consisting of 302 amino acid residues, has two additional residues in a key region of the active center while it lacks a sequence of seven residues in the NAD+-binding domain, when compared with the subunit of pig heart enzyme . In addition, there are substitutions of four single residues . The catalytic efficiency of pig liver dehydrogenase was significantly greater than that of the heart enzyme for short-chain substrate, but its catalytic rates declined with an increase in substrate chain-lengths . The distinction between pig liver and heart dehydrogenases cannot be attributed to a species difference, and thus it is concluded that there exist different isoforms of monofunctional L-3-hydroxyacyl-CoA dehydrogenases in pig . High level expression of mitochondrial L-3-hydroxyacyl-CoA dehydrogenase in Escherichia coli has provided a very convenient way to purify this important beta-oxidation enzyme . There is substantial homology between pig liver dehydrogenase and various multifunctional beta-oxidation enzymes in the active center of these enzymes; a consensus sequence, HX3PX1-3MXLXE, is proposed as the signature sequence of l-3-hydroxyacyl-CoA dehydrogenases .

Brain Res Mol Brain Res, 1998 Apr, 55(2), 181 - 97
Identification of a novel serine protease-like molecule in human brain; Meckelein B et al.; Proteolysis of the amyloid beta protein precursor (APP) is a key event in the development of Alzheimer's disease . In our search for proteases that can cleave APP and liberate the amino terminus of the amyloidogenic beta protein, we characterized a calcium-dependent serine protease (CASP) which is present in reactive astrocytes and cross-reacts with anti-cathepsin G antibodies . We wanted to take advantage of this cross-reactivity to clone the cDNA of CASP and eventually evaluate its tissue distribution . Screening of two human fetal brain cDNA libraries with anti-cathepsin G antibodies led to the identification of a cDNA coding for a novel protein whose only homology to known proteins is to the active site of trypsin-type serine proteases . We called this protein the novel serine protease (NSP) . NSP exists in at least three differentially spliced forms, one of which is expressed predominantly in brain and testis . Immunohistochemistry and immunoprecipitation with antibodies generated against NSP show that it is expressed and secreted by a variety of cells and that, in brain, it is found primarily in cerebrovascular smooth muscle cells and reactive astrocytes .

Biophys J, 1998 Jun, 74(6), 2889 - 902
Electromechanical coupling model of gating the large mechanosensitive ion channel (MscL) of Escherichia coli by mechanical force; Gu L et al.; We have developed a theoretical electromechanical coupling (EMC) model of gating of the large-conductance mechanosensitive ion channel (MscL) . The model presents the first attempt to explain the pressure-dependent transitions between the closed and open channel conformations on a molecular level by assuming 1) a homohexameric structural model of the channel, 2) electrostatic interactions between various domains of the homohexamer, 3) structural flexibility of the N-terminal portion of the monomer, and 4) mechanically and electrostatically induced displacement of the N-terminal domain relative to other structural domains of the protein . In the EMC model, 12 membrane-spanning alpha-helices (six each of the M1 and M2 transmembrane domains of the MscL monomer), are envisaged to line the channel pore with a diameter of 40 A, whereas the N- and C-termini are oriented toward each other inside the pore when the channel is closed . The model proposes that stretching the membrane bilayer by mechanical force causes the monomers to be pulled away from and slightly tilted toward each other . This relative movement of alpha-helices could serve as a trigger to initiate a "swing-like" motion of the N-terminus around the glycine residue G14 that may act as a pivot . The analysis of the attractive and repulsive coulomb forces between all domains of the channel homohexamer suggested that an inclination angle of approximately 3.0 degrees - 4.1 degrees between the oppositely oriented channel monomers should suffice for the N-terminus to turn away from other domains causing the channel to open . According to the EMC model the minimal free energy change, deltaG, that could initiate the opening of the channel was 2 kT . Also, the model predicted that the negative pressure required for channel open probability, Po = 0.5, should be between 50 and 80 mmHg . These values were in a good agreement with the experimentally estimated pressures of 60-70 mmHg obtained with the MscL reconstituted in liposomes . Furthermore, consistent with a notion that the N-terminus may present a mechanosensitive structural element providing a mechanism to open the MscL by mechanical force, the model provides a simple explanation for the variations in pressure sensitivity observed with several MscL mutants having either deletions or substitutions in N- or C-terminus, or site-directed mutations in the S2-S3 loop.

Biophys J, 1998 Jun, 74(6), 2786 - 801
A molecular dynamics study of the pores formed by Escherichia coli OmpF porin in a fully hydrated palmitoyloleoylphosphatidylcholine bilayer; Tieleman DP et al.; In this paper we study the properties of pores formed by OmpF porin from Escherichia coli, based on a molecular dynamics simulation of the OmpF trimer, 318 palmitoyl-oleoyl-phosphatidylethanolamine lipids, 27 Na+ ions, and 12,992 water molecules . After equilibration and a nanosecond production run, the OmpF trimer exhibits a C-alpha root mean square deviation from the crystal structure of 0.23 nm and a stable secondary structure . No evidence is found for large-scale motions of the L3 loop . We investigate the pore dimensions, conductance, and the properties of water inside the pore . This water forms a complicated pattern, even when averaged over 1 ns of simulation time . Around the pore constriction zone the water dipoles are highly structured in the plane of the membrane, oriented by the strong transversal electric field . In addition, there is a net orientation along the pore axis pointing from the extracellular to the intracellular side of the bilayer . The diffusion coefficients of water inside the pore are greatly reduced compared to bulk . We compare our results to results from model pores (Breed et al., 1996 . Biophys . J . 70:1 643-1 661; Sansom et al . 1997 . Biophys . J . 73:2404-241 5) and discuss implications for further theoretical work.

Trends Genet, 1998 Jun, 14(6), 236 - 43
Combinatorial control in ubiquitin-dependent proteolysis: don't Skp the F-box hypothesis; Patton EE et al.; The ubiquitin-dependent proteolytic pathway targets many key regulatory proteins for rapid intracellular degradation . Specificity in protein ubiquitination derives from E3 ubiquitin protein ligases, which recognize substrate proteins . Recently, analysis of the E3s that regulate cell division has revealed common themes in structure and function . One particularly versatile class of E3s, referred to as Skp1p-Cdc53p-F-box protein (SCF) complexes, utilizes substrate-specific adaptor subunits called F-box proteins to recruit various substrates to a core ubiquitination complex . A vast array of F-box proteins have been revealed by genome sequencing projects, and the early returns from genetic analysis in several organisms promise that F-box proteins will participate in the regulation of many processes, including cell division, transcription, signal transduction and development.

Biochem Mol Biol Int, 1998 Jun, 45(1), 155 - 61
A novel method for selection of chymotrypsin inhibitors from a phage peptide library; Liu Y et al.; A novel screening strategy has been developed for the identification of alpha-chymotrypsin inhibitors from a phage peptide library . In this strategy, the standard affinity selection protocol was modified by adding a proteolytic cleavage period to avoid recovery of alpha-chymotrypsin substrates . After four cycles of selection and further activity assay, a group of related peptides were identified by DNA sequencing . These peptides share a consensus sequence motif as (S/T)RVPR(R/H) . Then, a corresponding short peptide (Ac-ASRVPRRG-NH2) was synthesized chemically and proved to be an inhibitor of alpha-chymotrypsin . The present work provides a useful way for searching proteinase inhibitors without detailed knowledge of the molecular structure.

Biotechnology (N Y), 1995 Aug, 13(8), 801 - 4
Improved mimicry of a foot-and-mouth disease virus antigenic site by a viral peptide displayed on beta-galactosidase surface; Benito A et al.; A major antigenic site (site A) of foot-and-mouth disease virus includes multiple overlapping epitopes located within the flexible G-H loop of capsid protein VP1 . We have studied the antigenicity of several recombinant E . coli beta-galactosidases displaying the site A from a serotype C virus in different surface regions of the bacterial enzyme . In each one of the explored insertion sites, the recombinant peptide shows different specificity with a set of anti-virus monoclonal antibodies directed to site A . In some of them, the inserted stretch mimics better than free or haemocyanin-coupled peptide the antigenicity of site A in the intact virus . In particular, an insertion within an exposed loop involved in the activating interface of beta-galactosidase (amino acids 272 to 287) led to a significant improvement of the overall reactivity . Since insertions at this site renders proteins enzymatically active, the activating interface could be an adequate place for the presentation of foreign antigens in correctly assembled beta-galactosidase tetramers . These results also suggest that anti-virus antibodies directed against the major antigenic site of FMDV recognize different conformations of the G-H loop, which are better reproduced in some of the recombinant proteins because of the dissimilar restrictions imposed by each particular insertion site.

Biotechnology (N Y), 1995 May, 13(5), 504 - 6
Improved N-terminal processing of recombinant proteins synthesized in Escherichia coli; Sandman K et al.; Preparations of rHMfA (recombinant histone A from Methanothermus fervidus) synthesized in E . coli by the heterologous expression of the hmfA gene were found to contain a mixture of rHMfA molecules, approximately 40% that retained the N-terminal formyl-methionyl residue (f-met-rHMfA), approximately 50% that lacked the formyl moiety but retained the methionyl residue (met-rHMfA), and only approximately 10% that had lost both components of the protein synthesis initiating amino acid residue and therefore had the same N-terminal sequence as native HMfA molecules synthesized in Mt . fervidus . Expression of the hmfA gene in E . coli cells grown in the presence of trimethoprim and thymidine, coupled with the concurrent over-expression of a methionine aminopeptidase-encoding map gene, has been shown to overcome this N-terminal heterogeneity problem and to result in rHMfA preparations in which > 85% of the molecules have the fully processed, native N-terminal sequence . This procedure should be generally useful for ensuring N-terminal processing of recombinant proteins synthesized in E . coli.

Biotechnology (N Y), 1995 May, 13(5), 475 - 9
Antibody VH domains as small recognition units; Davies J et al.; To develop immunoglobulin based recognition units of minimum size, a human heavy chain variable domain (VH) was designed for selection of phage displayed VH . Non-specific binding of the VH through its interface for the light chain variable domain (VL) was prevented through three mutations (G44E, L45R and W47G) in this interface . These mutations were introduced to mimic camelid antibody heavy chains naturally devoid of light chain partners . The third hypervariable loop of the modified VH was then randomised to yield a repertoire of 2 x 10(8) independent clones, which was displayed on phage and selected through antigen binding . VH clones specific for hapten and protein antigens were isolated . Soluble VH was expressed with an isoleucine residue at position 47 to improve expression and stability compared to VH containing a glycine residue at this position, which however was preferable for phage selection . Affinities of soluble VH for hapten were between 100 nM and 400 nM . The VH domains were highly specific, stable and well expressed in Escherichia coli . These positive biophysical properties and their small size make them attractive for biotechnological applications.

Biotechnology (N Y), 1995 Apr, 13(4), 373 - 7
Calmodulin as a versatile tag for antibody fragments; Neri D et al.; Calmodulin is a highly acidic protein (net charge -24 at pH 8.0 in the absence of calcium) that binds to peptide and organic ligands with high affinity (Ka > 10(9) M-1) in a calcium-dependent manner . We have exploited these properties to develop calmodulin as a versatile tag for antibody fragments . Fusions of calmodulin with single chain Fv fragments (scFv) could be expressed by secretion from bacteria in good yield (5-15 mg/l in shaker flasks), and purified from periplasmic lysates or broth to homogeneity in a single step, either by binding to anion-exchange resin (DEAE-Sephadex), or to an organic ligand of calmodulin (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide-agarose) . The antibody fusions could be detected by binding of fluorescently labeled peptide ligands, as illustrated by their use in confocal microscopy, fluorescent activated cell sorting and "band shift" gel electrophoresis . Moreover, the interaction between calmodulin and peptide ligands could provide a means of heterodimerization of proteins, as illustrated by the assembly of an antibody-calmodulin fusion with maltose binding protein tagged with a peptide ligand of calmodulin.

Biotechnology (N Y), 1995 Apr, 13(4), 366 - 72
Expression of thioredoxin random peptide libraries on the Escherichia coli cell surface as functional fusions to flagellin: a system designed for exploring protein-protein interactions; Lu Z et al.; We have developed a system for probing protein/protein interactions which makes use of the bacterial flagellum to display random peptide libraries on the surface of E . coli . In developing the system the entire coding sequence of E . coli thioredoxin (trxA) was inserted into a dispensable region of the gene for flagellin (fliC), the major structural component of the E . coli flagellum . The resulting fusion protein (FLITRX) was efficiently exported and assembled into partially functional flagella on the bacterial cell surface . A diverse library of random dodecapeptides were displayed in FLITRX on the exterior of E . coli as conformationally constrained insertions into the thioredoxin active-site loop, a location known to be a highly permissive site for the insertion of exogenous peptide sequences into native thioredoxin . To demonstrate that members of this library could be bound and selected via specific protein/protein interactions to a target protein, a method was devised to enable efficient isolation of those bacteria displaying peptides with affinity to immobilized antibodies . We have unambiguously mapped three different antibody epitopes using this method . Peptides selected as FLITRX active-site fusions retain their binding specificity when made as native thioredoxin active-site loop fusions . This will facilitate future structural characterizations and broaden the general utility of the system for exploring other classes of protein-protein interactions.

Biotechnology (N Y), 1995 Mar, 13(3), 276 - 8
A computer program to determine a protein sequence from an amino acid analysis; Cordier-Ochsenbein F et al.; We have developed a computational method for analyzing the proteolytic products of a protein, knowing its sequence and the amino acid percentages of its products . For all fragments, amino acid percentages are calculated and compared to the experimental results (calculating the error within the experiment) . The program keeps the best fitted fragment using a least squared method . This program was written to determine the sequence of the proteolytic products that appeared during the purification of annexin I domain 2 . The reliability of the method was verified in this case . However the latter depends on the length and on the amino acid composition of the entire protein and of its fragments . This program may be suitable for analyzing the sequence of the products in any protease digestion, whether designed or accidental.

Biotechnology (N Y), 1995 Feb, 13(2), 175 - 9
Tight transcriptional control mechanism ensures stable high-level expression from T7 promoter-based expression plasmids; Mertens N et al.; One of the more efficient systems for high-level expression of cloned genes in Escherichia coli makes use of a phage T7 late promoter whose activity depends on a regulatable transcription unit supplying the specific T7 RNA polymerase . Using various T7 RNA polymerase/T7 promoter-based vector host systems with differential control on expression of the T7 RNA polymerase, we document that leaky expression of the latter is responsible for the frequently observed loss of the culture's ability to express genes of interest . We further show that the inability to achieve detectable expression levels can be overcome by using a tightly repressed expression system . We describe a novel and efficient control system in which basal level expression of T7 RNA polymerase is attenuated by a series of tandemly arranged transcription terminators . The plasmids also incorporate the phage lambda-derived nutL/N protein antitermination function, allowing conditional reversion of attenuation upon induction . The applicability of the system is illustrated by the strictly regulatable, high-level production of several cytokines of human and murine origin.

Biotechnology (N Y), 1995 Feb, 13(2), 151 - 4
Red-shifted excitation mutants of the green fluorescent protein; Delagrave S et al.; Using optimized combinatorial mutagenesis techniques and Digital Imaging Spectroscopy (DIS), we have isolated mutants of the cloned Aequorea victoria green fluorescent protein (GFP) that show red-shifted excitation spectra similar to that of Renilla reniformis GFP . Selective excitation of wild-type versus Red-Shifted GFP (RSGFP) enables spectral separation of these proteins . Six contiguous codons spanning the tyrosine chromophore region were randomized and sequence analysis of the mutants revealed a tyrosineglycine consensus . These mutants will enable the simultaneous analysis of two promoters or proteins per cell or organism . In consideration of the multitude of applications which are developing for GFP alone, we envisage that spectrally shifted fluorescent proteins will be of value to a diversity of research programs, including developmental and cell biology, drug-screening, and diagnostic assays.

Biotechnology (N Y), 1995 Jan, 13(1), 53 - 7
Malarial epitopes expressed on the surface of recombinant tobacco mosaic virus; Turpen TH et al.; Using malaria as a model disease, we engineered the surface of tobacco mosaic tobamovirus (TMV) for presentation of selected epitopes to the mammalian immune system . The TMV coat protein is a well-characterized and abundant self-assembling polymer previously shown to be a highly immunogenic carrier . Selected B-cell epitopes were either inserted into the surface loop region of the TMV coat protein or fused to the C terminus using the leaky stop signal derived from the replicase protein reading frame . Tobacco plants systemically infected with each of these constructs contained high titers of genetically stable recombinant virus, enabling purification of the chimeric particles in high yield . Symptoms induced in tobacco ranged from a normal mosaic pattern similar to that induced by the parental U1 strain to a unique bright yellow mosaic . As measured by quantitative ELISA against synthetic peptide standards, wild type TMV coat protein and fusion protein synthesized by the leaky stop mechanism coassembled into virus particles at the predicted ratio of approximately 20:1 . Recombinant plant viruses have the potential to meet the need for scalable and cost effective production of subunit vaccines that can be easily stored and administered.

Q Rev Biophys, 1997 Nov, 30(4), 333 - 64
From membrane to molecule to the third amino acid from the left with a membrane transport protein; Kaback HR et al.; The lac permease of E . coli is a paradigm for secondary active transporter proteins that transduce the free energy stored in electrochemical ion gradients into work in the form of a concentration gradient . This hydrophobic, polytopic, cytoplasmic membrane protein catalyses the coupled, stoichiometric translocation of beta-galactosides and H+, and it has been solubilized, purified, reconstituted into artificial phospholipid vesicles and shown to be solely responsible responsible for beta-galactoside transport as a monomer . The lacY gene which encodes the permease has been cloned and sequenced, and all available evidence indicates that the protein has 12 transmembrane domains in alpha-helical configuration that traverse the membrane in zigzag fashion connected by hydrophilic loops with the N and C termini on the cytoplasmic face of the membrane . Extensive use of site-directed and Cys-scanning mutagenesis indicates that very few residues in the permease are directly involved in the transport mechanism, but the permease appears to be a highly flexible protein that undergoes widespread conformational changes during turnover . Based on a variety of site-directed approaches which include second-site suppressor analysis and site-directed mutagenesis, excimer fluorescence, engineered divalent metal binding sites, chemical cleavage, EPR, thiol crosslinking and identification of discontinuous mAb epitopes, a helix packing model has been formulated.A mechanism for the coupled translocate ion of substrate and H+ by the lac permease of E . coli is proposed . Four residues are irreplaceable with respect to coupling, and the residues are paired in the tertiary structure--Arg-302 (helix IX) with Glu-325 (helix 10) and His-322 (helix 10) with Glu-269 (helix VIII) . In an adjacent region of the molecule at the interface between helices VIII and V is the substrate translocation pathway in which Glu-126 and Arg-144 appear to play key roles . Because of this arrangement, interfacial changes between helices VIII and V are transmitted to the interface between helices IX and X and vice versa . Upon ligand binding, a structural change at the interface between helices V and VIII disrupts the interaction between Glu-269 and His-322, Glu-269 displaces Glu-325 from Ag-302 and Glu-325 is protonated.Simultaneously, protonated Glu-325 becomes inaccessible to water which drastically increases its pKa . In this configuration, the permease undergoes a freely reversible conformational change that corresponds to translocation of the ternary complex . In order to return to ground state after release of substrate, the Arg-302-Glu-325 interaction must be reestablished which necessitates loss of H+ from Glu-325 . The H+ is released into a water-filled crevice between helices IX and X which becomes transiently accessible to both sides of the membrane due to a change in helix tilt, where it is acted upon equally by either the membrane potential or the pH gradient across the membrane . Remarkably few amino-acid residues appear to be critically involved in the transport mechanism of lac permease, suggesting that relatively simple chemistry drives the mechanism . On the other hand, widespread, cooperative conformational changes appear to be involved in turnover . As a whole the data suggest that the 12 helices which comprise the permease are loosely packed with a considerable amount of water in the interstices and that surface contours are important for sliding or tilting motions that occur during turnover . This surmise coupled with the indication that few residues are essential to the mechanism is encouraging in that it suggest that the possibility that a relatively low resolution structure (i.e . helix packing) plus localization of the critical residues and the translocation pathway can provide important insights into the mechanism . (ABSTRACT TRUNCATED)

Plant Cell, 1998 Jun, 10(6), 981 - 93
Pollen profilin function depends on interaction with proline-rich motifs; Gibbon BC et al.; The actin binding protein profilin has dramatic effects on actin polymerization in vitro and in living cells . Plants have large multigene families encoding profilins, and many cells or tissues can express multiple profilin isoforms . Recently, we characterized several profilin isoforms from maize pollen for their ability to alter cytoarchitecture when microinjected into living plant cells and for their association with poly-L-proline and monomeric actin from maize pollen . In this study, we characterize a new profilin isoform from maize, which has been designated ZmPRO4, that is expressed predominantly in endosperm but is also found at low levels in all tissues examined, including mature and germinated pollen . The affinity of ZmPRO4 for monomeric actin, which was measured by two independent methods, is similar to that of the three profilin isoforms previously identified in pollen . In contrast, the affinity of ZmPRO4 for poly-L-proline is nearly twofold higher than that of native pollen profilin and the other recombinant profilin isoforms . When ZmPRO4 was microinjected into plant cells, the effect on actin-dependent nuclear position was significantly more rapid than that of another pollen profilin isoform, ZmPRO1 . A gain-of-function mutant (ZmPRO1-Y6F) was created and found to enhance poly-L-proline binding activity and to disrupt cytoarchitecture as effectively as ZmPRO4 . In this study, we demonstrate that profilin isoforms expressed in a single cell can have different effects on actin in living cells and that the poly-L-proline binding function of profilin may have important consequences for the regulation of actin cytoskeletal dynamics in plant cells.

Plant Cell, 1998 Jun, 10(6), 905 - 15
Srchi13, a novel early nodulin from Sesbania rostrata, is related to acidic class III chitinases; Goormachtig S et al.; On the tropical legume Sesbania rostrata, stem-borne nodules develop after inoculation of adventitious root primordia with the microsymbiont Azorhizobium caulinodans . A cDNA clone, Srchi13, with homology to acidic class III chitinase genes, corresponds to an early nodulin gene with transiently induced expression during nodule ontogeny . Srchi13 transcripts accumulated strongly 2 days after inoculation, decreased from day 7 onward, and disappeared in mature nodules . Induction was dependent on Nod factor-producing bacteria . Srchi13 was expressed around infection pockets, in infection centra, around the developing nodule and its vascular bundles, and in uninfected cells of the central tissue . The specific and transient transcript accumulation together with the lipochitooligosaccharide degradation activity of the recombinant protein hint at a role of Srchi13 in normal nodule ontogeny by limiting the action of Nod factors.

J Biol Chem, 1998 Jun 19, 273(25), 15860 - 5
Biochemical properties of two protein kinases involved in disease resistance signaling in tomato; Sessa G et al.; In tomato plants, resistance to bacterial speck disease is mediated by a phosphorylation cascade, which is triggered by the specific recognition between the plant serine/threonine protein kinase Pto and the bacterial AvrPto protein . In the present study, we investigated in vitro biochemical properties of Pto, which appears to function as an intracellular receptor for the AvrPto signal molecule . Pto and its downstream effector Pti1, which is also a serine/threonine protein kinase, were expressed in Escherichia coli as maltose-binding protein and glutathione S-transferase fusion proteins, respectively . The two kinases each autophosphorylated at multiple sites as determined by phosphopeptide mapping . In addition, Pto and Pti1 autophosphorylation occurred via an intramolecular mechanism, as their specific activity was not affected by their molar concentration in the assay . Moreover, an active glutathione S-transferase-Pto fusion failed to phosphorylate an inactive maltose-binding protein-Pto(K69Q) fusion excluding an intermolecular mechanism of phosphorylation for Pto . Pti1 phosphorylation by Pto was also characterized and found to occur with a Km of 4.1 microM at sites similar to those autophosphorylated by Pti1 . Pto and the product of the recessive allele pto phosphorylated Pti1 at similar sites, as observed by phosphopeptide mapping . This suggests that the inability of the kinase pto to confer resistance to bacterial speck disease in tomato is not caused by altered recognition specificity for Pti1 phosphorylation sites.

J Biol Chem, 1998 Jun 19, 273(25), 15675 - 81
A revised model for the oligomeric state of the N-ethylmaleimide-sensitive fusion protein, NSF; Fleming KG et al.; The N-ethylmaleimide-sensitive fusion protein (NSF) is an ATPase that plays an essential role in intracellular membrane trafficking . Previous reports have concluded that NSF forms either a tetramer or a trimer in solution, and that assembly of the oligomer is essential for efficient activity in membrane transport reactions . However, in recent electron microscopic analyses NSF appears as a hexagonal cylinder similar in size to related ATPases known to be hexamers . We have therefore reevaluated NSF's oligomeric state using a variety of quantitative biophysical techniques . Sedimentation equilibrium and sedimentation velocity analytical ultracentrifugation, transmission electron microscopy with rotational image analysis, scanning transmission electron microscopy, and multiangle light scattering all demonstrate that, in the presence of nucleotide, NSF is predominantly a hexamer . Sedimentation equilibrium results further suggest that the NSF hexamer is held together by oligomerization of its D2 domains . The sedimentation coefficient, s20,w0, of 13.4 (+/-0 . 1) S indicates that NSF has unusual hydrodynamic characteristics that cannot be solely explained by its shape . The demonstration that NSF is a hexameric oligomer highlights structural similarities between it and several related ATPases which act by switching the conformational states of their protein substrates in order to activate them for subsequent reactions.

J Biol Chem, 1998 Jun 19, 273(25), 15546 - 52
The ATPase activity of Hsp104, effects of environmental conditions and mutations; Schirmer EC et al.; Hsp104 is crucial for stress tolerance in Saccharomyces cerevisiae, and both of its nucleotide-binding domains (NBD1 and NBD2) are required . Here, we characterize the ATPase activity and oligomerization properties of wild-type (WT) Hsp104 and of NBD mutants . In physiological ionic strength buffers (pH 7.5, 37 degreesC) WT Hsp104 exhibits Michaelis-Menten kinetics between 0.5 and 25 mM ATP (Km approximately 5 mM, Vmax approximately 2 nmol min-1 microg-1) . ATPase activity is strongly influenced by factors that vary with cell stress (e.g . temperature, pH, and ADP) . Mutations in the P-loop of NBD1 (G217V or K218T) severely reduce ATP hydrolysis but have little effect on oligomerization . Analogous mutations in NBD2 (G619V or K620T) have smaller effects on ATPase activity but impair oligomerization . The opposite relationship was reported for another member of the HSP100 protein family, the Escherichia coli ClpA protein, in studies employing lower ionic strength buffers . In such buffers, the Km of WT Hsp104 for ATP hydrolysis decreased 10-fold and its stability under stress conditions increased, but the effects of the NBD mutations on ATPase activity and oligomerization remained opposite to those of ClpA . Either the functions of the two NBDs in ClpA and Hsp104 have been reversed or both contribute to ATP hydrolysis and oligomerization in a complex manner that can be idiosyncratically affected by such mutations.

J Biol Chem, 1998 Jun 19, 273(25), 15487 - 93
Defining the interleukin-8-binding domain of heparan sulfate; Spillmann D et al.; Interleukin-8, a member of the CXC chemokine family, has been shown to bind to glycosaminoglycans . It has been suggested that heparan sulfate on cell surfaces could provide specific ligand sites on endothelial cells to retain the highly diffusible inflammatory chemokine for presentation to leukocytes . By using selectively modified heparin and heparan sulfate fragments in a nitrocellulose filter trapping system, we have analyzed sequence requirements for interleukin-8 binding to heparin/heparan sulfate . We demonstrate that the affinity of a monomeric interleukin-8 molecule for heparin/heparan sulfate is too weak to allow binding at physiological ionic strength, whereas the dimeric form of the protein mediates binding to two sulfated domains of heparan sulfate . These domains, each an N-sulfated block of approximately 6 monosaccharide units, are contained within an approximately 22-24-mer sequence and are separated by a region of </=14 monosaccharide residues that may be fully N-acetylated . Binding to interleukin-8 correlates with the occurrence of the di-O-sulfated disaccharide unit -IdceA(2-OSO3)-GlcNSO3(6-OSO3)- . We suggest that the heparan sulfate sequence binds in horseshoe fashion over two antiparallel-oriented helical regions on the dimeric protein.

J Biol Chem, 1998 Jun 19, 273(25), 15479 - 86
RNA-dependent RNA polymerase activity of the soluble recombinant hepatitis C virus NS5B protein truncated at the C-terminal region; Yamashita T et al.; The hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRP), which is the central catalytic enzyme of HCV replicase . We established a new method to purify soluble HCV NS5B in the glutathione S-transferase-fused form NS5Bt from Escherichia coli which lacks the C-terminal 21 amino acid residues encompassing a putative anchoring domain (anino acids 2990-3010) . The recombinant soluble protein exhibited RdRP activity in vitro which was dependent upon the template and primer, but it did not exhibit the terminal transferase activity that has been reported to be associated with the recombinant NS5B protein from insect cells . The RdRP activity of purified glutathione S-transferase-NS5Bt and thrombin-cleavaged non-fused NS5Bt shares most of the properties . Substitution mutations of NS5Bt at the GDD motif, which is highly conserved among viral RdRPs, and at the clustered basic residues (amino acids 2919-2924 and 2693-2699) abolished the RdRP activity . The C-terminal region of NS5B, which is dispensable for the RdRP activity, dramatically affected the subcellular localization of NS5B retaining it in perinuclear sites in transiently overexpressed mammalian cells . These results may provide some clues to dissecting the molecular mechanism of the HCV replication and also act as a basis for developing new anti-viral drugs.

Biochem Pharmacol, 1998 May 15, 55(10), 1673 - 81
Metabolism and metabolic actions of 6-methylpurine and 2-fluoroadenine in human cells; Parker WB et al.; Activation of purine nucleoside analogs by Escherichia coli purine nucleoside phosphorylase (PNP) is being evaluated as a suicide gene therapy strategy for the treatment of cancer . Because the mechanisms of action of two toxic purine bases, 6-methylpurine (MeP) and 2-fluoroadenine (F-Ade), that are generated by this approach are poorly understood, mechanistic studies were initiated to learn how these compounds differ from agents that are being used currently . The concentration of F-Ade, MeP, or 5-fluorouracil required to inhibit CEM cell growth by 50% after a 4-hr incubation was 0.15, 9, or 120 microM, respectively . F-Ade and MeP were also toxic to quiescent MRC-5, CEM, and Balb 3T3 cells . Treatment of CEM, MRC-5, or Balb 3T3 cells with either F-Ade or MeP resulted in the inhibition of protein, RNA, and DNA syntheses . CEM cells converted F-Ade and MeP to F-ATP and MeP-ribonucleoside triphosphate (MeP-R-TP), respectively . The half-life for disappearance of HeP-ribonucleoside triphosphate from CEM cells was approximately 48 hr, whereas the half-lives of F-ATP and ATP were approximately 5 hr . Both MeP and F-Ade were incorporated into the RNA and DNA of CEM cells . These studies indicated that the mechanisms of action of F-Ade and MeP were quite different from those of other anticancer agents, and suggested that the generation of these agents in tumor cells by E . coli PNP could result in significant advantages over those generated by either herpes simplex virus thymidine kinase or E . coli cytosine deaminase . These advantages include a novel mechanism of action resulting in toxicity to nonproliferating and proliferating tumor cells and the high potency of these agents during short-term treatment.

Arch Biochem Biophys, 1998 Jun 1, 354(1), 158 - 64
Cross-linking and disulfide bond formation of introduced cysteine residues suggest a modified model for the tertiary structure of URF13 in the pore-forming oligomers; Rhoads DM et al.; URF13 is a mitochondrially encoded protein in the inner mitochondrial membrane of maize (Zea mays L.) carrying the cms-T cytoplasm . This protein is responsible for Texas-type cytoplasmic sterility and is a ligand-gated, pore-forming receptor for the pathotoxins of fungal pathogens Bipolaris maydis race T and Phyllosticta maydis . URF13 contains three transmembrane alpha-helices, with amphipathic helices II and III likely involved in pore formation, and is present as oligomers in cms-T maize mitochondria and when expressed in Escherichia coli cells . To study tertiary and quaternary structures of URF13 oligomers, we employed combinations of site-directed mutagenesis and chemical cross-linking . We introduced Cys residues individually into consecutive positions 78-82, believed to be in helix III . We expressed these proteins in E . coli cells and tested for cross-linking through disulfide bond formation or by using Cys-Cys cross-linkers . URF13-R79C, URF13-R81C, and URF13-T82C were cross-linked using Cys-Cys-specific cross-linkers, as were double mutants URF13-C27R/R79C, URF13-C27R/R81C, and URF13-C27R/T82C, indicating that the cross-linking was between introduced Cys residues on adjacent URF13 molecules . Disulfide bond formation, induced by diamide, was seen only in URF13-T82C and URF13-C27R/T82C, indicating that Cys residues introduced into position 82 are closely juxtaposed in the oligomers . Based on these observations, we modified the models for the secondary structure of URF13 and the tertiary structure of the URF13 oligomers . Sequential cross-linking of URF13-R81C oligomers with bismaleimidohexane (Cys-Cys cross-linker) and N,N'-dicyclohexylcarbodiimide (Lys-Asp/Glu cross-linker) suggests that URF13 oligomers consist of an even number of monomers.

J Biol Chem, 1998 Jun 26, 273(26), 16339 - 45
The mechanism of action of steroidogenic acute regulatory protein (StAR) . StAR acts on the outside of mitochondria to stimulate steroidogenesis; Arakane F et al.; Steroidogenic acute regulatory protein (StAR) plays an essential role in steroidogenesis, facilitating delivery of cholesterol to cytochrome P450scc on the inner mitochondrial membrane . StAR is synthesized in the cytoplasm and is subsequently imported by mitochondria and processed to a mature form by cleavage of the NH2-terminal mitochondrial targeting sequence . To explore the mechanism of StAR action, we produced 6-histidine-tagged N-62 StAR (His-tag StAR) constructs lacking the NH2-terminal 62 amino acids that encode the mitochondrial targeting sequence and examined their steroidogenic activity in intact cells and on isolated mitochondria . His-tag StAR proteins stimulated pregnenolone synthesis to the same extent as wild-type StAR when expressed in COS-1 cells transfected with the cholesterol side-chain cleavage system . His-tag StAR was diffusely distributed in the cytoplasm of transfected COS-1 cells whereas wild-type StAR was localized to mitochondria . There was no evidence at the light or electron microscope levels for selective localization of His-tag StAR protein to mitochondrial membranes . In vitro import assays demonstrated that wild-type StAR preprotein was imported and processed to mature protein that was protected from subsequent trypsin treatment . In contrast, His-tag StAR was not imported and protein associated with mitochondria was sensitive to trypsin . Using metabolically labeled COS-1 cells transfected with wild-type or His-tag StAR constructs, we confirmed that wild-type StAR preprotein was imported and processed by mitochondria, whereas His-tag StAR remained largely cytosolic and unprocessed . To determine whether cytosolic factors are required for StAR action, we developed an assay system using washed mitochondria isolated from bovine corpora lutea and purified recombinant His-tag StAR proteins expressed in Escherichia coli . Recombinant His-tag StAR stimulated pregnenolone production in a dose- and time-dependent manner, functioning at nanomolar concentrations . A point mutant of StAR (A218V) that causes lipoid congenital adrenal hyperplasia was incorporated into the His-tag protein . This mutant was steroidogenically inactive in COS-1 cells and on isolated mitochondria . Our observations conclusively document that StAR acts on the outside of mitochondria, independent of mitochondrial import, and in the absence of cytosol . The ability to produce bioactive recombinant StAR protein paves the way for refined structural studies of StAR and StAR mutants.

Immunogenetics, 1998 Jul, 48(2), 108 - 15
High RAD51 mRNA levels in young rabbit appendix . A role in B-cell gene conversion?
Schiaffella E, Fuschiotti P, Bensinger SJ, Mage RG.
The rabbit has a limited number of VH genes that rearrange . As in the chicken, the 3'-most VH1 gene is rearranged in most B lymphocytes . This laboratory reported that by 6 weeks after birth, diversification of rearranged VH genes occurs, at least in part, by gene conversion-like events in the appendix, suggesting that this organ is a homologue of the avian bursa of Fabricius . Rad51 contributes to the repair of double-strand breaks in DNA during somatic and meiotic recombination . The gene was first identified in lower eukaryotes, and later in vertebrates including chicken, as encoding an Escherichia coli RecA-like protein . We report the cloning and sequencing of RAD51 from the rabbit . Because the chicken bursa was shown to express high levels of RAD51 message, we investigated the expression of RAD51 in the rabbit appendix and other tissues . Using a quantitative polymerase chain reaction mimic assay and conventional northern analyses, we found high RAD51 expression in young rabbit appendix comparable to levels in testis where there is an abundance of meiotic recombination . RAD51 levels were three times higher in appendix B lymphocytes compared with T lymphocytes and were lower in adult appendix, as well as in spleen and Peyer's patches of young rabbits . We measured the levels of message in several appendix cell sub-populations obtained by fluorescence-activated cell sorting and found that sub-populations of B lymphocytes corresponding to different stages of B-cell development as well as B cells undergoing isotype switch did not have significantly different mRNA levels.

Sex Transm Infect, 1998 Feb, 74(1), 11 - 9
Influence of ovarian hormones on urogenital infection; Sonnex C; Numerous studies have examined the influence of hormones on infectious diseases and there is now a wealth of data relating to the more specific effect of the sex hormones, oestrogen and progesterone, on urogenital infections . The interaction between these hormones and the immune system is complex and the variation of hormonal effect between species further complicates the true picture as related to humans . Although it is difficult therefore to draw general conclusions regarding predominant effects of specific hormones, there is the suggestion that oestrogen enhances the pathogenicity of many urogenital micro-organisms . Our understanding of the influential role played by sex hormones in disease pathogenesis is at an early stage and illustrates well the importance of drawing together and interpreting as a whole both epidemiological and molecular studies.

J Gen Virol, 1998 Jun, 79 ( Pt 6), 1531 - 7
Citrus psorosis virus: nucleotide sequencing of the coat protein gene and detection by hybridization and RT-PCR; Barthe GA et al.; Citrus psorosis virus (CPV) is a multicomponent ssRNA virus with a coat protein of approximately 48 kDa . The viral genome is encapsidated in short and long particles that are readily separated by sucrose density-gradient centrifugation . CPV particles are spiral filaments that are referred to as spiroviruses (SV) . A cDNA library of purified short particles from isolate CPV-4 was prepared in a Lambda vector and screened for expression of the coat protein gene (CPG) with a monoclonal antibody to the coat protein . Sequencing of immunopositive clones indicated a single ORF encoding a 49 kDa protein . This ORF, when expressed in E . coli, gave a protein identical in size and immunoreactivity to the CPV coat protein . A full-length clone of the CPG was transcribed and used in Northern hybridization assays to establish that short particle RNA of CPV is negative sense and contains the CPG . Moreover, the CPG was not found on RNA extracted from long particles or on the sedimentable dsRNA from CPV infected tissue . RT-PCR assays were developed for the amplification of a 600 bp fragment of CPG and for the complete CPG (1317 bp) . The 600 bp fragment from a biologically and serologically different isolate, CPV-6, was cloned, sequenced and found to share 86% (nucleotide) and 96% (amino acid) identity with CPV-4 . BLAST analysis of sequences from CPV-4 and CPV-6 detected no significant nucleic acid or protein similarity with any known viral sequences.

J Gen Virol, 1998 Jun, 79 ( Pt 6), 1509 - 17
Use of modified plum pox virus coat protein genes developed to limit heteroencapsidation-associated risks in transgenic plants; Jacquet C et al.; Aphid transmission of a non-aphid-transmissible strain of zucchini yellow mosaic virus (ZYMV-NAT) occurs in transgenic plants expressing the plum pox potyvirus (PPV) coat protein (CP) gene . Heteroencapsidation has been shown to be responsible for this modification in the epidemiological characteristics of the infecting virus . In order to prevent this biological risk, several modified PPV CP constructs were produced that were designed to interfere with heteroencapsidation itself or to block aphid transmission of heteroencapsidated virions . These constructs were first expressed in Escherichia coli in order to check for the accumulation of pseudo-particles by electron microscopy . Virus-like particles (VLPs) were found with the full-length CP and with a PPV CP lacking the DAG amino acid triplet involved in aphid transmission . However, no VLPs were observed with CP lacking R220, Q221 or D264, amino acids known to be essential for the assembly of other potyvirus CPs . Transgenic Nicotiana benthamiana lines expressing the different PPV CP constructs were infected with ZYMV-NAT . Aphid transmission assays performed with these plants demonstrated that the strategies developed here provide an effective means of minimizing the biological risks associated with heteroencapsidation.

J Gen Virol, 1998 Jun, 79 ( Pt 6), 1487 - 94
Detection and assignment of proteins encoded by rice black streaked dwarf fijivirus S7, S8, S9 and S10; Isogai M et al.; The proteins encoded by rice black streaked dwarf fijivirus (RBSDV) genomic segments 7-10 (S7-S10) were characterized . Open reading frames (ORFs) from these segments were expressed as fusion proteins in Escherichia coli . Antibodies raised against the expressed products were used as probes to determine whether the viral ORFs encode structural proteins . In Western blots, antibodies to the expressed S8 and S10 products reacted with a core capsid (65 kDa) and a major outer capsid (56 kDa) protein, respectively, while none of the antibodies to S7 and S9 products reacted with structural proteins . Antisera to RBSDV S7 ORF1 and S9 ORF1 each detected a single protein of the predicted size in total protein extracts from infected rice plants and viruliferous Laodelphax striatellus . Immunoelectron microscopy revealed that antibodies to RBSDV S7 ORF1 and RBSDV S9 ORF1 reacted with tubular structures and viroplasm, respectively, in sections of both infected maize plants and viruliferous L . striatellus . Antisera to ORF2 of S7 and S9 failed to detect any proteins in the infected tissue using either Western blotting or immuno-electron microscopic techniques.

Circ Res, 1998 Jun 15, 82(11), 1173 - 88
Increased protein kinase C activity in myotrophin-induced myocyte growth; Sil P et al.; Myotrophin, a novel protein that has been shown to stimulate myocyte growth, has been isolated, purified, and sequenced from the hearts of spontaneously hypertensive rats and dilated cardiomyopathic human tissue . Recently, the cDNA clones encoding myotrophin have been isolated and expressed in Escherichia coli, and the recombinant myotrophin was found to be as biologically and immunologically active as natural myotrophin . The mechanism by which myotrophin stimulates protein synthesis and initiates myocardial hypertrophy is not known . To evaluate the involvement of protein kinase C (PKC) in myotrophin-induced hypertrophy, PKC activity and its distribution in the subcellular fraction were determined in cultured neonatal and adult myocytes . PKC activity was determined by measuring the incorporation of 32P into histone type III-S and PKCepsilon substrate peptide (epsilon(pep)) from {gamma-32P}ATP in neonatal myocytes . Myotrophin significantly stimulated PKC activity in neonatal myocytes and was associated with a significant increase in protein synthesis . The effect of myotrophin on the stimulation of PKC activity and {3H}leucine incorporation was abolished by pretreatment with either staurosporine or H-7, two selective, pharmacological PKC inhibitors . Pretreatment of myocytes with staurosporine also reduced the myotrophin-induced mRNA levels of c-fos and beta-myosin heavy chain . To evaluate the subcellular events whose occurrence was due to myotrophin and translocation of PKC, we studied the effect of genistein, a tyrosine kinase inhibitor, on myotrophin-induced neonatal myocyte growth . Genistein attenuated the {3H}leucine incorporation induced by myotrophin . To define the specificity of the PKC isoform(s) involved in myotrophin-stimulated myocyte growth, both neonatal and adult myocytes were treated with myotrophin, and Western blot analyses were performed by using the antibodies of different PKC isoforms . Results showed that both PKCalpha and PKCepsilon isoforms participated in the myotrophin-induced neonatal myocyte growth, whereas only the PKCepsilon isoform was involved in myotrophin-induced adult myocyte hypertrophy . PKCdelta and PKCzeta do not seem to participate in either neonatal or adult myocyte growth induced by myotrophin . Treatment with antisense oligonucleotides specific for PKCalpha and PKCepsilon isoforms further supported this result . PKCalpha is the major PKC isoform in neonatal myocytes and needs Ca2+ and phospholipids for its activation, and PKCepsilon (the Ca2+-independent PKC isoform) is present in both neonatal and adult myocytes; the 15-mer antisense oligodeoxynucleotides of each were used for this study . Treatment of neonatal myocytes with the PKCalpha and PKCepsilon antisense oligodeoxynucleotides for 5 days significantly reduced Ca2+-dependent and Ca2+-independent PKC activity, respectively, as well as the {3H}leucine incorporation induced by myotrophin . Furthermore, myotrophin-induced PKC activity was primarily located in the particulate fraction and did not result in a concomitant decrease in the cytosolic fraction . Myotrophin does not change PKC isoform expression (both Ca2+ dependent and independent PKC isoforms used in this study) in rat neonatal cardiac fibroblasts . Our data suggest that myotrophin exerts its action on protein synthesis, possibly through a tyrosine kinase-coupled pathway and translocation of PKC from the cytosol to the cell membrane.

J Appl Microbiol, 1998 Apr, 84(4), 585 - 92
Development and use of a multiplex PCR system for the rapid screening of heat labile toxin I, heat stable toxin II and shiga-like toxin I and II genes of Escherichia coli in water; Tsen HY et al.; Enterotoxigenic Escherichia coli (ETEC) may produce heat-labile toxin (LT) I and LTII and heat-stable toxin (ST) I and STII, while shiga toxin producing E . coli (STEC) strains, including enterohaemorrhagic E . coli (EHEC), may produce shiga-like toxin (SLT) I and/or SLTII . Both ETEC and STEC are pathogenic to humans, pigs and cattle . As contamination of environmental water by any of these pathogenic E . coli cells is possible, a multiplex polymerase chain reaction (PCR) system for the rapid screening of LTI, STII, and SLTI and SLTII genes of E . coli was developed . The PCR primers used were the SLTI and SLTII genes specific primers developed by the present authors and the LTI and STII genes specific primers reported by other laboratories . The detection specificity of this multiplex PCR system was confirmed by PCR assay of ETEC, STEC and other E . coli cells as well as non-E . coli bacteria . Its detection limit was 10(2)-10(3) cfu each of the target cells per assay . When this multiplex PCR system was used for the rapid screening of LTI, STII ETEC and STEC in water samples such as tap, underground and lake waters, it was found that after the enrichment step, as few as 10(0) cells 100 ml-1 of the water sample could be detected . Therefore, this PCR system could be used for the rapid monitoring of ETEC and/or STEC cells contaminating water samples.

Radiats Biol Radioecol, 1998 Mar-Apr, 38(2), 232 - 7
{Features of modifications of cytotoxic consequences of microwave and thermal heating}; Morozov II et al.; The incubation of bacteria Escherichia coli B/r and Escherichia coli Bs-1, preliminarily heated by microwave (frequency 7 GHz) or equal usual heat in water isotonic (0.9%) or hypertonic (10%) solution of sodium chloride resulted in decrease or increase of the cell lethality independently from heating manner . Hypertonic solutions of this compound, as was established, protected the cells against heat damages during the microwave heating less effectively than during thermal heating.

Arch Biochem Biophys, 1998 Jun 1, 354(1), 24 - 30
The fibronectin-like domain is required for the type V and XI collagenolytic activity of gelatinase B; O'Farrell TJ et al.; Gelatinase B (matrix metalloproteinase-9) is able to degrade several extracellular matrix proteins, including gelatin, elastin, and collagen types IV, V, XI, and XIV . This enzyme contains a "fibronectin-like" domain which is composed of three tandem copies of a fibronectin type 2 homology unit inserted into its catalytic domain . We have studied the involvement of this domain in the substrate specificity of gelatinase B by expressing a mutant of the enzyme, in Escherichia coli, in which this domain has been deleted . This mutant enzyme retained its ability to cleave the peptide substrate Mca-PLGL(Dpa)AR-NH2, possessing K(m) and kcat values similar to those of the wild-type enzyme . In addition, the NH2-terminal, 14-kDa, inhibitory domain of recombinant tissue inhibitor of metalloproteinase-2 was able to inhibit the mutant and the wild-type enzymes with the same potency . The mutant's gelatinolytic activity was also retained but reduced in comparison to that of the wild-type enzyme . However, contrary to the wild-type enzyme, the mutant was not able to digest or bind fibrillar collagen types V and XI . These data indicate that the fibronectin-like domain of gelatinase B is an important determinant of the enzyme's fibrillar collagen substrate specificity . It allows the enzyme to bind to and cleave collagen types V and XI, events which are thought to be involved in several normal physiological and pathological processes such as metastasis and arthritis.

Exp Cell Res, 1998 May 25, 241(1), 23 - 35
Myristoyl-coA:protein N-myristoyltransferase from bovine cardiac muscle: molecular cloning, kinetic analysis, and in vitro proteolytic cleavage by m-calpain; Raju RV et al.; Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes the attachment of myristate onto the amino terminal glycine residue of select polypeptides . Cardiac tissue expresses high levels of cAMP-dependent protein kinase whose catalytic subunit is myristoylated; however, cardiac muscle extracts were found to contain low NMT activities . Northern blot analysis of bovine heart poly(A)+ RNA probed with bovine spleen NMT cDNA revealed a 1.7-kb mRNA . Western blot analysis of cardiac muscle extracts with human NMT antibody indicated a prominent immunoreactive band with a molecular mass of 50 kDa . The expression of mRNA and protein levels in cardiac muscle is not correlated with NMT activities, suggesting the presence of regulators of the enzyme activity . We have isolated the cDNA encoding bovine cardiac muscle NMT (cNMT) by reverse transcription polymerase chain reaction . The single long open reading frame of 1248 bp of bovine cNMT specifies a protein of 416 amino acids with a predicted mass of 46,686 Da . The cDNA clone expressed in Escherichia coli resulted in the production of functionally active 50-kDa NMT . Ultrastructural and immunolocalization of NMT utilizing the immunogold labeling technique demonstrated cytoplasmic distribution with occasional mitochondrial and myofilaments localization of the NMT antibody . Cardiac muscle NMT has a higher affinity for myristoyl-CoA than toward palmitoyl-CoA . Substrate specificity indicated that cNMT has a higher affinity toward pp60src and M2 gene segment of reovirus type 3-derived peptide substrates than toward cAMP-dependent protein kinase-derived peptide . Primary translational product of cNMT sequence contained several regions rich in proline, glutamic acid, serine, and threonine, which are known as "PEST" regions . PEST-FIND analysis of the amino acid sequences indicated eight PEST regions were present in the cNMT . These PEST regions are suggested to be recognized by specific proteases, particularly Ca(2+)-dependent neutral proteases, calpains, which are responsible for the degradation of PEST-containing proteins . We have demonstrated the abolishment of NMT activity and NMT protein degradation in vitro by m-calpain . The proteolysis of cNMT by m-calpain and the abolishment of NMT activity was prevented by the calpain inhibitor, calpastatin . These observations indicate that calpains may regulate NMT activity.

Lett Appl Microbiol, 1998 Apr, 26(4), 275 - 8
The production of D-acetoin by a transgenic Escherichia coli; Ui S et al.; A 6 kbp SphI fragment encoding genes for the enzymes alpha-acetolactate synthase (ALS), alpha-acetolactate decarboxylase (ALDC) and meso-2,3-butanediol dehydrogenase (meso-BDH), involved in the formation of meso-2,3-butanediol (meso-BD) from pyruvic acid, was cloned into plasmid pUC118 . When derivatives of this plasmid were introduced into Escherichia coli JM109, the transformants were able to produce D-acetoin (D-AC) without contamination with L-acetoin (L-AC).

Biochemistry (Mosc), 1998 May, 63(5), 579 - 83
Bioluminescence of free and poly(vinyl alcohol) cryogel-entrapped recombinant Escherichia coli cells expressing firefly luciferase; Romanova NA et al.; Bioluminescence of free and poly(vinyl alcohol) cryogel-entrapped recombinant E . coli cells expressing firefly luciferase was investigated . It was shown that bioluminescence intensity and time-course of the bioluminescent signal changed upon immobilization and depended on intracellular ATP concentration and permeability of the cell membrane.

J Biol Chem, 1998 Jun 26, 273(26), 16615 - 20
TorD, a cytoplasmic chaperone that interacts with the unfolded trimethylamine N-oxide reductase enzyme (TorA) in Escherichia coli; Pommier J et al.; Reduction of trimethylamine N-oxide (TMAO) in Escherichia coli involves the terminal molybdoreductase TorA, located in the periplasm, and the membrane anchored c type cytochrome TorC . In this study, the role of the TorD protein, encoded by the third gene of torCAD operon, is investigated . Construction of a mutant, in which the torD gene is interrupted, showed that the absence of TorD protein leads to a two times decrease of the final amount of TorA enzyme . However, specific activity and biochemical properties of TorA enzyme were similar to those of the enzyme produced in the wild type . Excess of TorD protein restores the normal level of TorA enzyme, and also, leads to the appearance of a new cytoplasmic form of TorA on SDS-polyacrylamide gel electrophoresis using gentle conditions . This probably indicates a new folding state of the cytoplasmic TorA protein when TorD is overexpressed . BIAcore techniques demonstrated direct specific interaction between the TorA and TorD proteins . This interaction was enhanced when TorA was previously unfolded by heating . Finally, as TorA is a molybdoenzyme, we demonstrated that TorD can interact with TorA before the molybdenum cofactor has been inserted . As TorD homologue encoding genes are found in various TMAO reductase loci, we propose that TorD is a chaperone protein specific for the TorA enzyme . It belongs to a family of TorD-like chaperones present in several bacteria, and, probably, involved in TMAO reductase folding.

J Biol Chem, 1998 Jun 26, 273(26), 16555 - 60
Thiamin biosynthesis in Escherichia coli . Identification of this thiocarboxylate as the immediate sulfur donor in the thiazole formation; Taylor SV et al.; ThiFSGH and ThiI are required for the biosynthesis of the thiazole moiety of thiamin in Escherichia coli . The overproduction, purification, and characterization of ThiFS and the identification of two of the early steps in the biosynthesis of the thiazole moiety of thiamin are described here . ThiS isolated from E . coli thiI+ is post-translationally modified by converting the carboxylic acid group of the carboxyl-terminal glycine into a thiocarboxylate . The thiI gene plays an essential role in the formation of the thiocarboxylate because ThiS isolated from a thiI- strain does not contain this modification . ThiF catalyzes the adenylation by ATP of the carboxyl-terminal glycine of ThiS . This reaction is likely to be involved in the activation of ThiS for sulfur transfer from cysteine or from a cysteine-derived sulfur donor.

J Biol Chem, 1998 Jun 26, 273(26), 16544 - 50
Critical role for the 310 helix region of p57(Kip2) in cyclin-dependent kinase 2 inhibition and growth suppression; Hashimoto Y et al.; Although crystal structural analysis of cyclin A/cyclin-dependent kinase 2 (Cdk2)/p27 (Russo, A . A., Jeffrey, P . D., Pattern, A . K., Massague, J., and Pavletich, N . P . (1996) Nature 382, 325-331) has suggested that the 310 helix region in Cdk inhibitors of the Cip/Kip family may be involved in the inhibition of cyclin/Cdk activities, there is no biochemical evidence supporting this hypothesis . In the present study, we demonstrated that cyclin and Cdk binding domains of p57 were necessary but were not sufficient in themselves for the inhibition of cyclin A/Cdk2 and cyclin E/Cdk2, and that the 3(10) helix region of this protein is indispensable for the inhibition of these complexes . In contrast, the 3(10) helix regions of p21 and p27 were not required, and cyclin- and Cdk-binding domains alone were sufficient for the inhibition of all cyclin/Cdk complexes examined . Site-directed mutagenesis identified phenylalanine 79 and tyrosine 80 within the 3(10) helix region of p57 as crucial residues for kinase inhibition, supporting the structural evidence that the 3(10) helix binds deep inside the catalytic cleft of Cdk2, mimicking ATP . Mutations within the 3(10) helix region of the p57 molecule completely abolished the ability to arrest the cell cycle at G1 in vivo . These results indicate that this region is specifically utilized by p57 in selectively inhibiting cyclin A or E/Cdk2+ activities . Thus the 3(10) helix motif may confer a specific regulatory mechanism by which p57 differentially regulates Cdk2 and Cdk4 activities.

J Biol Chem, 1998 Jun 26, 273(26), 16476 - 86
The reduced levels of chi recognition exhibited by the RecBC1004D enzyme reflect its recombination defect in vivo; Arnold DA et al.; Homologous recombination in Escherichia coli is initiated by the RecBCD enzyme and is stimulated by an 8-nucleotide element known as Chi (chi) . We present a detailed biochemical characterization of a mutant RecBCD enzyme, designated RecBC1004D, that displays a reduced level of chi site recognition . Initially characterized genetically as unable to respond to the chi sequence, we provide evidence to indicate that the ability of this mutant enzyme to respond to chi is reduced rather than lost; the mutant displays about 20-fold lower chi recognition than wild-type RecBCD enzyme . Although this enzyme exhibits wild-type levels of double-stranded DNA exonuclease, helicase, and ATPase activity, its ability to degrade single-stranded DNA is enhanced 2-3-fold . The data presented here suggest that the reduced recombination proficiency of the recBC1004D strain observed in vivo results from a basal level of modification of the RecBC1004D enzyme at both chi-specific, as well as nonspecific, DNA sequences.

J Biol Chem, 1998 Jun 26, 273(26), 16400 - 8
Leukocystatin, a new Class II cystatin expressed selectively by hematopoietic cells; Halfon S et al.; We describe a new cystatin in both mice and humans, which we termed leukocystatin . This protein has all the features of a Class II secreted inhibitory cystatin but contains lysine residues in the normally hydrophobic binding regions . As determined by cDNA library Southern blots, this cystatin is expressed selectively in hematopoietic cells, although fine details of the distribution among these cell types differ between the human and mouse mRNAs . In addition, we have determined the genomic organization of mouse leukocystatin, and we found that in contrast to most cystatins, the leukocystatin gene contains three introns . The recombinant proteins corresponding to these cystatins were expressed in Escherichia coli as N-terminal glutathione S-transferase or FLAGTM fusions, and studies showed that they inhibited papain and cathepsin L but with affinities lower than other cystatins . The unique features of leukocystatin suggests that this cystatin plays a role in immune regulation through inhibition of a unique target in the hematopoietic system.

J Biol Chem, 1998 Jun 26, 273(26), 16358 - 65
Synthesis of polyribonucleotide chains from the 3'-hydroxyl terminus of oligodeoxynucleotides by Escherichia coli primase; Sun W et al.; Escherichia coli primase synthesizes RNA primers on DNA templates for the initiation of DNA replication . The sole known activity of primase is to catalyze synthesis of short RNA chains de novo . We now report a novel activity of primase, namely that it can synthesize RNA from the 3'-hydroxyl terminus of a pre-existing oligodeoxynucleotide . The oligonucleotide-primed synthesis of RNA by primase occurs in both of the G4oric-specific priming system and the dnaB protein associated general priming system . This priming reaction of primase is verified by a number of biochemical methods, including inhibition by modified 3'-phosphate of oligonucleotides and deoxyribonuclease I and ribonuclease H cleavages . We also show that the primed RNA is an effective primer for the synthesis of DNA chain by E . coli DNA polymerase III holoenzyme . The significance of this finding to primases generating multimeric length RNA is discussed.

J Biol Chem, 1998 Jun 26, 273(26), 16273 - 80
Role of electrostatic interactions on the affinity of thioredoxin for target proteins . Recognition of chloroplast fructose-1, 6-bisphosphatase by mutant Escherichia coli thioredoxins; Mora-Garcia S et al.; Chloroplast thioredoxin-f functions efficiently in the light-dependent activation of chloroplast fructose-1, 6-bisphosphatase by reducing a specific disulfide bond located at the negatively charged domain of the enzyme . Around the nucleophile cysteine of the active site (-W-C-G-P-C-), chloroplast thioredoxin-f shows lower density of negative charges than the inefficient modulator Escherichia coli thioredoxin . To examine the contribution of long range electrostatic interactions to the thiol/disulfide exchange between protein-disulfide oxidoreductases and target proteins, we constructed three variants of E . coli thioredoxin in which an acidic (Glu-30) and a neutral residue (Leu-94) were replaced by lysines . After purification to homogeneity, the reduction of the unique disulfide bond by NADPH via NADP-thioredoxin reductase proceeded at similar rates for all variants . However, the conversion of cysteine residues back to cystine depended on the target protein . Insulin and difluoresceinthiocarbamyl-insulin oxidized the sulfhydryl groups of E30K and E30K/L94K mutants more effectively than those of wild type and L94K counterparts . Moreover, the affinity of E30K, L94K, and E30K/L94K E . coli thioredoxin for chloroplast fructose-1,6-bisphosphatase (A0.5 = 9, 7, and 3 microM, respectively) increased with the number of positive charges, and was higher than wild type thioredoxin (A0.5 = 33 microM), though still lower than that of thioredoxin-f (A0.5 = 0.9 microM) . We also demonstrated that shielding of electrostatic interactions with high salt concentrations not only brings the A0.5 for all bacterial variants to a limiting value of approximately 9 microM but also increases the A0.5 of chloroplast thioredoxin-f . While negatively charged chloroplast fructose-1,6-bisphosphatase (pI = 4.9) readily interacted with mutant thioredoxins, the reduction rate of rapeseed napin (pI = 11.2) diminished with the number of novel lysine residues . These findings suggest that the electrostatic interactions between thioredoxin and (some of) its target proteins controls the formation of the binary noncovalent complex needed for the subsequent thiol/disulfide exchange.

J Biol Chem, 1998 Jun 26, 273(26), 16241 - 7
Transmembrane topography of subunit a in the Escherichia coli F1F0 ATP synthase; Valiyaveetil FI et al.; Subunit a is the least understood of the three subunits that compose the F0 sector in the Escherichia coli F0F1 ATP synthase . In this study, we have substituted Cys into predicted extramembranous loops of the protein and used chemical modification to obtain topographical information on the folding of subunit a . The extent of labeling of the substituted Cys residues by fluorescein-5'-maleimide was determined . The localization of reactive Cys residues was inferred from differences in the extent of labeling in inside out and right side out membrane vesicles . The NH2-terminal segment of subunit a was localized to the outside (periplasmic) surface and the COOH terminus to the cytoplasmic surface by these procedures . Loop residues in two periplasmic extramembranous loops and in two cytoplasmic extramembranous loops were also localized . The localization of two cytoplasmic Cys residues was confirmed by using 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid to block fluorescein-5'-maleimide labeling . From the localization of the Cys residues, a model for the topography is proposed that consists of five transmembrane segments with the NH2 terminus periplasmic and the COOH terminus cytoplasmic . The positions of second site suppressors, including several isolated here to the nonfunctional E219C and H245C substitutions, provide support for the topographical model proposed.

J Biol Chem, 1998 Jun 26, 273(26), 16235 - 40
Membrane topology of subunit a of the F1F0 ATP synthase as determined by labeling of unique cysteine residues; Long JC et al.; The membrane topology of the a subunit of the F1F0 ATP synthase from Escherichia coli has been probed by surface labeling using 3-(N-maleimidylpropionyl) biocytin . Subunit a has no naturally occurring cysteine residues, allowing unique cysteines to be introduced at the following positions: 8, 24, 27, 69, 89, 128, 131, 172, 176, 196, 238, 241, and 277 (following the COOH-terminal 271 and a hexahistidine tag) . None of the single mutations affected the function of the enzyme, as judged by growth on succinate minimal medium . Membrane vesicles with an exposed cytoplasmic surface were prepared using a French pressure cell . Before labeling, the membranes were incubated with or without a highly charged sulfhydryl reagent, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid . After labeling with the less polar biotin maleimide, the samples were solubilized with octyl glucoside/cholate and the subunit a was purified via the oligohistidine at its COOH terminus using immobilized nickel chromatography . The purified samples were electrophoresed and transferred to nitrocellulose for detection by avidin conjugated to alkaline phosphatase . Results indicated cytoplasmic accessibility for residues 69, 172, 176, and 277 and periplasmic accessibility for residues 8, 24, 27, and 131 . On the basis of these and earlier results, a transmembrane topology for the subunit a is proposed.

J Biol Chem, 1998 Jun 26, 273(26), 16229 - 34
Insertion scanning mutagenesis of subunit a of the F1F0 ATP synthase near His245 and implications on gating of the proton channel; Vik SB et al.; Subunit a of the E . coli F1F0 ATP synthase was probed by insertion scanning mutagenesis in a region between residues Glu219 and His245 . A series of single amino acid insertions, of both alanine and aspartic acid, were constructed after the following residues: 225, 229, 233, 238, 243, and 245 . The mutants were tested for growth yield, binding of F1 to membranes, dicyclohexylcarbodiimide sensitivity of ATPase activity, ATP-driven proton translocation, and passive proton permeability of membranes stripped of F1 . Significant loss of function was seen only with insertions after positions 238 and 243 . In contrast, both insertions after residue 225 and the alanine insertion after residue 245 were nearly identical in function to the wild type . The other insertions showed an intermediate loss of function . Missense mutations of His245 to serine and cysteine were nonfunctional, while the W241C mutant showed nearly normal ATPase function . Replacement of Leu162 by histidine failed to suppress the 245 mutants, but chemical rescue of H245S was partially successful using acetate . An interaction between Trp241 and His245 may be involved in gating a "half-channel" from the periplasmic surface of F0 to Asp61 of subunit a.

J Biol Chem, 1998 Jun 26, 273(26), 16098 - 103
TIMP-2 promotes activation of progelatinase A by membrane-type 1 matrix metalloproteinase immobilized on agarose beads; Kinoshita T et al.; Membrane-type 1 matrix metalloproteinase (MT1-MMP)/MMP-14 is the activator of progelatinase A (proGelA)/proMMP-2 on the cell surface . However, it was a paradox that a tissue inhibitor of metalloproteinase-2 (TIMP-2), which is an inhibitor of MT1-MMP, is required for proGelA activation by the cells expressing MT1-MMP . In this study, a truncated MT1-MMP having a FLAG-tag sequence at the C terminus (MT1-F) was immobilized onto agarose beads (MT1-F/B) and used to analyze the role of TIMP-2 . The proteolytic activity of MT1-F/B against a synthetic peptide substrate was inhibited by TIMP-2 in a dose-dependent manner . In contrast, TIMP-2 promoted the processing of proGelA by MT1-F/B at low concentrations and inhibited it at higher concentrations . TIMP-2 promoted the binding of proGelA to the MT1-F on the beads by forming a trimolecular complex, which was followed by processing of proGelA . A stimulatory effect of TIMP-2 was observed under conditions in which unoccupied MT1-F was still available . Thus, the ternary complex is thought to act as a means to concentrate the substrate to the bead surface and to present it to the neighboring free MT1-F.

J Biol Chem, 1998 Jun 26, 273(26), 16067 - 74
Novel recombinant analogues of bovine placental lactogen . G133K and G133R provide a tool to understand the difference between the action of prolactin and growth hormone receptors; Helman D et al.; Two new analogues of bovine placental lactogen (bPL), bPL(G133K) and bPL(G133R), were expressed in Escherichia coli, refolded, and purified to a native form . Binding experiments, which are likely to represent the binding to site 1 only, to intact FDC-P1 cells transfected with rabbit (rb) growth hormone receptor (GHR) or with human (h) GHR, to Nb2 rat lymphoma cells, or to rabbit mammary gland membranes prolactin receptor (PRLR), revealed only small or no reduction in binding capacity . The complex formation between these analogues and receptor extracellular domains (R-ECD) of various hormones was determined by gel filtration . Wild type bPL yielded 1:2 complex with hGHR-ECD, rat PRLR-ECD, and rbPRLR-ECD, whereas both analogues formed only 1:1 complexes with all R-ECDs tested . Real time kinetics experiments demonstrated that the ability of the analogues to form homodimeric complexes was compromised in both PRLR- and GHR-ECDs . The biological activity transduced through lactogenic receptors in in vitro bioassays in rabbit mammary gland acini culture and in Nb2 cells was almost fully retained, whereas the activity transduced through somatogenic receptors in FDC-P1 cells transfected with rbGHRs or with hGHRs was abolished . Both analogues exhibited antagonistic activity in the latter cells . To explain the discrepancy between the effect of the mutation on the signal transduced by PLR versus GHRs we suggest that: 1) the mutation impairs the ability of site 2 of bPL to form a stable homodimeric complex with both lactogenic and somatogenic receptors by a drastic shortening of the half-life of 2:1 complex; 2) the transient existence of the homodimeric complex is still sufficient to initiate the signal transduced through lactogenic receptors but not through somatogenic receptors; and 3) one possible reason for this difference is that JAK2, which serves as a mediator of both receptors, is already associated with lactogenic receptors prior to hormone binding-induced receptor dimerization, whereas in somatogenic receptors the JAK2 receptor association occurs subsequently to receptor dimerization.

J Biol Chem, 1998 Jun 26, 273(26), 16000 - 4
Ambiguities in mapping the active site of a conformationally dynamic enzyme by directed mutation . Role of dynamics in structure-function correlations in Escherichia coli adenylosuccinate synthetase; Wang W et al.; On the basis of ligated crystal structures, Asn21, Asn38, Thr42, and Arg419 are not involved in the chemical mechanism of adenylosuccinate synthetase from Escherichia coli, yet these residues are well conserved across species . Purified mutants (Asp21 --> Ala, Asn38 --> Ala, Asn38 --> Asp, Asn38 --> Glu, Thr42 --> Ala, and Arg419 --> Leu) were studied by kinetics, circular dichroism spectroscopy, and equilibrium ultracentrifugation . Asp21 and Arg419 are not part of the active site, yet mutations at positions 21 and 419 lower kcat 20- and 10-fold, respectively . Thr42 interacts only through its backbone amide with the guanine nucleotide, yet its mutation to alanine significantly increases Km for all substrates . Asn38 hydrogen-bonds directly to the 5'-phosphoryl group of IMP, yet its mutation to alanine and glutamate has no effect on Km values, but reduces kcat by 100-fold . The mutation Asn38 --> Asp causes 10-57-fold increases in Km for all substrates along with a 30-fold decrease in kcat . At pH 5.6, however, the Asn38 --> Asp mutant is more active, yet binds IMP 100-fold more weakly, than the wild-type enzyme . Proposed mechanisms of ligand-induced conformational change and subunit aggregation can account for the properties of mutant enzymes reported here . The results underscore the difficulty of using directed mutations alone as a means of mapping the active site of an enzyme.

J Biol Chem, 1998 Jun 26, 273(26), 15980 - 4
Regulation of casein kinase I epsilon and casein kinase I delta by an in vivo futile phosphorylation cycle; Rivers A et al.; Casein kinase I delta (CKIdelta) and casein kinase I epsilon (CKIepsilon) have been implicated in the response to DNA damage, but the understanding of how these kinases are regulated remains incomplete . In vitro, these kinases rapidly autophosphorylate, predominantly on their carboxyl-terminal extensions, and this autophosphorylation markedly inhibits kinase activity (Cegielska, A., Gietzen, K . F., Rivers, A., and Virshup, D . M . (1998) J . Biol . Chem . 273, 1357-1364) . However, we now report that while these kinases are able to autophosphorylate in vivo, they are actively maintained in the dephosphorylated, active state by cellular protein phosphatases . Treatment of cells with the cell-permeable serine/threonine phosphatase inhibitors okadaic acid or calyculin A leads to rapid increases in kinase intramolecular autophosphorylation . Since CKI autophosphorylation decreases kinase activity, this dynamic autophosphorylation/dephosphorylation cycle provides a mechanism for kinase regulation in vivo.

J Biol Chem, 1998 Jun 26, 273(26), 15940 - 5
Unisite catalysis without rotation of the gamma-epsilon domain in Escherichia coli F1-ATPase; Garcia JJ et al.; Unisite {gamma-32P}ATP hydrolysis was studied in ECF1 from the mutant betaE381C after generating a single disulfide bond between beta and gamma subunits to prevent the rotation of the gamma/epsilon domain . The single beta-gamma cross-link was obtained by removal of the delta subunit from F1 and then treating with CuCl2 as described previously (Aggeler, R., Haughton, M . A., and Capaldi, R . A . (1996) J . Biol . Chem . 270, 9185-9191) . The mutant enzyme, betaE381C, had an increased overall rate of unisite hydrolysis of {gamma-32P}ATP compared with the wild type ECF1 due to increases in the rate of ATP binding (k+1), Pi release (k+3), and ADP release (k+4) . Release of bound substrate ({gamma-32P}ATP) was also increased in the betaE381C mutant . Cross-linking between Cys-381 and the intrinsic Cys-87 of gamma caused a further increase in the rate of unisite catalysis, mainly by additional effects on nucleotide binding in the high affinity catalytic site (k+1 and k+4) . In delta-subunit-free ECF1 from wild type or betaE381C F1, addition of an excess of ATP accelerated unisite catalysis . After cross-linking, unisite catalysis of betaE381C was not enhanced by the cold chase . The covalent linkage of gamma to beta increased the rate of unisite catalysis to that obtained by cold chase of ATP of the noncross-linked enzyme . It is concluded that the conversion of Glu-381 of beta to Cys induces an activated conformation of the high affinity catalytic site with low affinity for substrate and products . This state is stabilized by cross-linking the Cys at beta381 to Cys-87 of gamma . We infer from the data that rotation of the gamma/epsilon rotor in ECF1 is not linked to unisite hydrolysis of ATP at the high affinity catalytic site but to ATP binding to a second or third catalytic site on the enzyme.

J Magn Reson, 1998 Jun, 132(2), 191 - 6
13C-NOESY-HSQC with Split Carbon Evolution for Increased Resolution with Uniformly Labeled Proteins; Baur M et al.; Two new pulse sequences are presented for the recording of 2D 13C-HSQC and 3D 13C-NOESY-HSQC experiments, containing two consecutive carbon evolution periods . The two periods are separated by a z-filter which creates a clean CxHz-quantum state for evolution in the second period . Each period is incremented (in a non-constant-time fashion) only to the extent that the defocusing of carbon inphase magnetization through J-coupling with neighboring carbons remains insignificant . Therefore, 13C homonuclear J-couplings are rendered ineffective, reducing the loss of signal and peak splitting commonly associated with long 13C evolution times . The two periods are incremented according to a special acquisition protocol employing a 13C-13C gradient echo to yield a data set analogous to one obtained by evolution over the added duration of both periods . The spectra recorded with the new technique on uniformly 13C-labeled proteins at twice the evolution time of the standard 13C-HSQC experiment display a nearly twofold enhancement of resolution in the carbon domain, while maintaining a good sensitivity even in the case of large proteins . Applied to the IIAMan protein of E . coli (31 kDa), the 13C-HSQC experiment recorded with a carbon evolution time of 2 x 8 ms showed a 36% decrease in linewidths compared to the standard 13C-HSQC experiment, and the S/N ratio of representative cross-peaks was reduced to 40% . This reduction reflects mostly the typical loss of intensity observed when recording with an increased resolution . The 13C-NOESY-HSQC experiment derived from the 13C-HSQC experiment yielded additional NOE restraints between resonances which previously had been unresolved .

Microb Pathog, 1998 Jun, 24(6), 361 - 72
Cloning and molecular analysis of genes encoding two immunodominant antigens of Ehrlichia risticii; Vemulapalli R et al.; Ehrlichia risticii, the causative agent of Potomac horse fever, is an obligate intracellular rickettsial organism . To understand the role of 55 and 51 kilodalton immunodominant antigens of E . risticii in strain variation, their genes from the 25-D and 90-12 strains were cloned, sequenced, and expressed in E . coli . Sequence analysis revealed that the gene for the 55 kDa antigen was present in a heat shock operon along with the gene for a ;10 kDa protein . Homology searches indicated that the 55 kDa antigen and the 10 kDa protein were homologues of E . coli GroEL and GroES proteins, respectively . There was no nucleotide sequence difference between the genes of the 55 kDa antigen, nor between the entire operons, from both strains of E . risticii . The sequence-based estimation of the sizes of the putative mature 51 kDa antigens of the 90-12 and 25-D strains were 52.7 kDa and 52.9 kDa, respectively . The 51 kDa antigens from the 90-12 and 25-D strains shared a 98% identity in their deduced amino acid sequences . The difference in some of the amino acids may be responsible for variation in their mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, where the 51 kDa antigen of the 25-D strain migrates towards a ;2 kDa lower molecular weight region . In Western blots, a 155 kDa protein that appeared to be a trimer product of the 51 kDa antigen was identified . The 55 and 51 kDa antigens were overexpressed in E . coli using a commercial expression system, pRSET A,B,C (Invitrogen Inc., San Diego, CA, U.S . A.) . The purified recombinant proteins cross-reacted with antisera to E . canis and E . sennetsu .

Biochemistry, 1998 May 26, 37(21), 7885 - 91
Delineation of the Cdc42/Rac-binding domain of p21-activated kinase; Thompson G et al.; p21-activated kinases (PAKs) serve as effector proteins for the GTP-binding proteins Cdc42 and Rac . They are serine/threonine kinases containing the Cdc42/Rac interactive binding (CRIB) motif . The main aim of this study was to define the minimal domain of alphaPAK required for Cdc42/Rac binding . Eight stable PAK fragments of varying lengths, each containing the CRIB motif (residues 75-88), were expressed in Escherichia coli, and their ability to interact with Cdc42 and Rac was assessed using scintillation proximity assays, isothermal titration calorimetry, and fluorescence techniques . The shortest fragments examined (residues 70-94 and 75-94) bound only weakly to either Cdc42 or Rac . A longer fragment starting at residue 75 and ending at residue 105 showed binding to Q61L Rac.GTP with Kd = 1.9 microM . Highest affinity binding (Kd approximately 0.05 microM) was seen with longer fragments ending at residue 118 or 132 . A small increase in affinity was seen with those fragments starting at residue 70 rather than residue 75 . PAK fragments bound with approximately 3-10-fold higher affinity to Cdc42 than to Rac and bound Q61L variants with 5-10-fold higher affinity than wild type . The dissociation rates of Q61L Rac.mant-GTP and of Q61L Cdc42 . mant-GTP from PAK fragment residues 70-132 were measured to be 0.66 and 0.25 min-1, respectively, which are 100-fold lower than dissociation rates for Ras:Ras-effector domains, although their affinities are similar . Calorimetric measurements revealed that binding was associated with a relatively slow heat change . It is suggested that these PAK fragments (in the absence of Cdc42 or Rac) might exist predominantly in an inactive conformation that slowly interconverts with an active conformation and/or a slow conformational change may occur upon binding to Cdc42/Rac . In conclusion, the PAK CRIB motif itself is insufficient for high-affinity binding to Cdc42/Rac, but a 30 amino acid region of PAK (residues 75-105), containing this motif, is sufficient.

Biochemistry, 1998 May 26, 37(21), 7850 - 8
Single-tryptophan mutants of monomeric tryptophan repressor: optical spectroscopy reveals nonnative structure in a model for an early folding intermediate; Shao X et al.; A monomeric version of the dimeric tryptophan repressor from Escherichia coli, L39E TR, has previously been shown to resemble a transient intermediate that appears in the first few milliseconds of folding {Shao, X., Hensley, P., and Matthews, C . R . (1997) Biochemistry 36, 9941-9949} . In the present study, the optical properties of the two intrinsic tryptophans were used to compare the structure and dynamics of the monomeric form with those of the native, dimeric form . The urea-induced unfolding equilibria of Trp19/L39E TR (Trp99 replaced with Phe) and Trp99/L39E TR (Trp19 replaced with Phe) mutants were monitored by circular dichroism and fluorescence spectroscopies at pH 7.6 and 25 degrees C . Coincident normalized transitions show that the urea denaturation process for each single-tryptophan mutant follows a two-state model involving monomeric native and unfolded forms . The free energies at standard state in the absence of denaturant for Trp19/L39E TR and Trp99/L39E TR are less than that for L39E TR, indicating that both tryptophans are involved in stabilizing the monomer . Fluorescence and near-UV circular dichroism spectroscopies indicate that the tryptophan side chains in monomeric Trp19/L39E TR and Trp99/L39E TR occupy hydrophobic, well-structured environments that are distinctively different from those found in their dimeric counterparts . Acrylamide quenching experiments show that both Trp19 and Trp99 are partially exposed to solvent in the native state, with Trp99 having a slightly greater degree of exposure . Measurements of the steady-state anisotropies of Trp19/L39E and Trp99/L39E TR demonstrate that the motions of both tryptophan side chains are restricted in the folded conformation . On the basis of these data, it can be concluded that this monomeric form of the tryptophan repressor adopts a well-folded, stable conformation with nonnative tertiary structure . When combined with previous results, the current findings demonstrate that the development of higher order structure during the folding of this intertwined dimer does not follow a simple hierarchical model.

Biochemistry, 1998 May 26, 37(21), 7844 - 9
Tyr254 hydroxyl group acts as a two-way switch mechanism in the coupling of heterotropic and homotropic effects in Escherichia coli glucosamine-6-phosphate deaminase; Montero-Moran GM et al.; The involvement of tyrosine residues in the allosteric function of the enzyme glucosamine 6-phosphate deaminase from Escherichia coli was first proposed on the basis of a theoretical analysis of the sequence and demonstrated by spectrophotometric experiments . Two tyrosine residues, Tyr121 and Tyr254, were indicated as involved in the mechanism of cooperativity and in the allosteric regulation of the enzyme {Altamirano et al . (1994) Eur . J . Biochem . 220, 409-413} . Tyr121 replacement by threonine or tryptophan altered the symmetric character of the T --> R transition {Altamirano et al . (1995) Biochemistry 34, 6074-6082} . From crystallographic data of the R allosteric conformer, Tyr254 has been shown to be part of the allosteric pocket {Oliva et al . (1995) Structure 3, 1323-1332} . Although it is not directly involved in binding the allosteric activator, N-acetylglucosamine 6-phosphate, Tyr 254 is hydrogen bonded through its phenolic hydroxyl to the backbone carbonyl from residue 161 in the neighboring polypeptide chain . Kinetic and binding experiments with the mutant form Tyr254-Phe of the enzyme reveal that this replacement caused an uncoupling of the homotropic and heterotropic effects . Homotropic cooperativity diminished and the allosteric activation pattern changed from one of the K-type in the wild-type deaminase to a mixed K-V pattern . On the other hand, Tyr254-Trp deaminase is kinetically closer to a K-type enzyme and it has a higher catalytic efficiency than the wild-type protein . These results show that the interactions of Tyr254 are fundamental in coupling binding in the active site to events occurring in the allosteric pocket of E . coli glucosamine 6-P deaminase.

Biochemistry, 1998 May 26, 37(21), 7778 - 86
Molecular properties of ClpAP protease of Escherichia coli: ATP-dependent association of ClpA and clpP; Maurizi MR et al.; The ClpAP protease from Escherichia coli consists of the ATP-binding regulatory component, ClpA (subunit Mr 84 165), and the proteolytic component, ClpP (subunit Mr 21 563) . Our hydrodynamic studies demonstrate that the predominant forms of these proteins in solution correspond to those observed by electron microscopy . ClpP and proClpP(SA), which in electron micrographs appear to have subunits arranged in rings of seven subunits, were found by ultracentrifugation to have s20,w values of 12.2 and 13.2 S and molecular weights of 300 000 and 324 000 +/- 3000, respectively, indicating that the native form of each consists of two such rings . The two intact rings of ClpP were separated in the presence of >/= 0.1 M sulfate at low temperatures, suggesting that ring-ring contacts are polar in nature and more easily disrupted than subunit contacts within individual rings . Sedimentation equilibrium analysis indicated that ClpA purified without nucleotide exists as an equilibrium mixture of monomers and dimers with Ka = (1.0 +/- 0.2) x 10(5) M-1 and that, upon addition of MgATP or adenosine 5'-O-(3-thiotriphosphate), ClpA subunits associated to a form with Mr 505 000 +/- 5000, consistent with the hexameric structure seen by electron microscopy . Sedimentation velocity and gel-filtration analysis showed that the nucleotide-promoted hexamer of ClpA (s20,w = 17.2 S) binds tightly to ClpP producing species with s20,w values of 21 and 27 S (f/f0 = 1.5 and 1.8, respectively), consistent with electron micrographs of ClpAP that show a single tetradecamer of ClpP associated with either one or two ClpA hexamers {Kessel et al . (1995) J . Mol . Biol . 250, 587-594} . Under assay conditions in the presence of ATP and Mg2+, the apparent dissociation constant of hexameric ClpA and tetradecameric ClpP was approximately 4 +/- 2 nM . By the method of continuous variation, the optimal ratio of ClpA to ClpP in the active complex was 2:1 . The specific activities of limiting ClpA and ClpP determined in the presence of an excess of the other component indicated that the second molecule of ClpA provides very little additional activation of ClpP.

Biochemistry, 1998 May 26, 37(21), 7757 - 63
The ring fragmentation product of thymidine C5-hydrate when present in DNA is repaired by the Escherichia coli Fpg and Nth proteins; Jurado J et al.; Various forms of oxidative stress, including gamma-radiolysis and UV irradiation, result in the formation of damaged bases . (5R)-Thymidine C5-hydrate is one of several modified nucleosides produced from thymidine under these conditions . N-(2-Deoxy-beta-D-erythro-pentofuranosyl)-N-3-{(2R)-hydroxyisobutyric acid}urea or alphaRT is the respective fragmentation product formed from (5R)-thymidine C5-hydrate upon hydrolysis . This modified nucleoside has potential mutagenic or lethal properties . No enzymatic activity responsible for the removal of alphaRT has been identified . We report here that when present in DNA, alphaRT is a substrate for two purified enzymes from Escherichia coli involved in the repair of oxidized bases: the Nth and the Fpg proteins . The Fpg protein removes the alphaRT lesion more efficiently than the Nth protein . This is the first example of efficient excision of a ring-opened form of a pyrimidine by the Fpg protein . The high efficacy of the Fpg protein suggests that it is likely to be involved in vivo in the excision of alphaRT . The kinetics of the reaction of the Fpg protein with DNA containing alphaRT suggest substrate inhibition . Duplex oligodeoxynucleotides containing alphaRT positioned opposite T, dG, dC, and dA were cleaved efficiently by both enzymes, although the profiles of activity of the two enzymes were different . The Nth enzyme preferentially excises alphaRT when opposite a dG, followed by alphaRT.dA, alphaRT . T, and alphaRT.dC . For the Fpg protein, the order is alphaRT.dC >/= alphaRT.dG approximately alphaRT.T > alphaRT.dA . Moreover, we show that human cell extract exhibits an activity that excises alphaRT from an oligonucleotide, suggesting that human homologues of the Nth and/or Fpg proteins could be involved in repair of this lesion in human cells.

Biochemistry, 1998 May 26, 37(21), 7741 - 8
Absence of pyridoxine-5'-phosphate oxidase (PNPO) activity in neoplastic cells: isolation, characterization, and expression of PNPO cDNA; Ngo EO et al.; Major differences in the metabolism of vitamin B6 in various cancers compared to their normal cellular counterparts have been documented . In particular, pyridoxine- 5'-phosphate oxidase (PNPO), the rate-limiting enzyme in pyridoxal 5'-phosphate (PLP) biosynthesis, is absent in liver and neurally-derived tumors . We show that the expression of PNPO is developmentally regulated not only in liver but also in brain . Specifically, PNPO activity in fetal brain tissue is 7.5-fold lower than that found in adult brain tissue . Furthermore, the isolation and characterization of a PNPO cDNA are described . The isolated cDNA was verified to be the authentic PNPO cDNA on the basis of two criteria . First, the translated product from the PNPO cDNA is immunologically reactive to a polyclonal PNPO antibody . Second, PNPO negative hepatoma cell lines stably transfected with the PNPO cDNA express enzymatically active PNPO protein . The availability of these biological reagents will not only facilitate in depth investigations of the reasons for the absence of PNPO in liver and brain malignancies but also aid in an understanding of the biochemical regulation of B6 metabolism in development.

Biochemistry, 1998 May 26, 37(21), 7670 - 5
Mapping the promoter DNA sites proximal to conserved regions of sigma 70 in an Escherichia coli RNA polymerase-lacUV5 open promoter complex; Owens JT et al.; Base-specific interactions between promoter DNA and Escherichia coli RNA polymerase are regulated by a sigma (sigma) protein during transcription initiation . To map spatial relations between evolutionarily conserved regions of the primary sigma (sigma 70) and each DNA strand along the lacUV5 promoter in the transcriptionally active "open" complex, we have used a cysteine-tethered cutting reagent to cleave DNA strands . The chemical nuclease FeBABE {iron (S)-1-(p-bromoacetamidobenzyl)ethylenediaminetetraacetate} was conjugated to single-cysteine mutants of sigma 70 at sites 132C, 376C, 396C, 422C, 496C, 517C, or 581C . After formation of open promoter complexes between lacUV5 DNA and RNA polymerase holoenzymes carrying conjugated sigma 70 subunits, we observed promoter DNA cleavage spanning at least 60 bases, between positions -48 and +12 . The results show that sigma 70 region 2.1, otherwise implicated in core enzyme binding, is proximal to the nontemplate strand of lacUV5 DNA between the -10 promoter element and positions as far downstream of the transcription start site as +12 . Conserved region 3.2 of sigma 70 is proximal to the template strand near the +1 transcription start site, and region 3.1 is positioned between the lacUV5-10 and -35 promoter elements . We propose a model for the orientation of sigma 70 and DNA in the open complex.

J Mol Biol, 1998 May 22, 278(5), 935 - 48
Simultaneous expression of leukocyte-type 12-lipoxygenase and reticulocyte-type 15-lipoxygenase in rabbits; Berger M et al.; In rabbit reticulocytes an arachidonic acid 15-lipoxygenase (15-LOX) is expressed at high yield . Rescreening a rabbit reticulocyte cDNA library for alternative 15-LOX transcripts, a full length cDNA which encodes a novel lipoxygenase was isolated . The predicted amino acid sequence of this enzyme shared a high degree (99%) of identity with the reticulocyte-type 15-lipoxygenase . Among the six amino acid residues different in both enzymes a Phe-Leu exchange was detected at position 353 . Recently, site-directed mutagenesis studies have revealed that this amino acid exchange converts a 15-lipoxygenase to a 12-lipoxygenase . In fact, when the novel enzyme was expressed in Escherichia coli, mainly 12-lipoxygenation of arachidonic acid was observed . The recombinant enzyme exhibited a rather broad substrate specificity . Various C-18 and C-20 polyenoic fatty acids and even complex substrates such as biomembranes were effectively oxygenated . Thus, the novel enzyme may be classified as leukocyte-type 12-lipoxygenase . Genomic polymerase chain reaction of the 3' region of the leukocyte-type 12-lipoxygenase gene indicated that introns 10 to 13 differed to about 10% from the corresponding sequences of the 15-lipoxygenase gene although their size and the intron-exon organization were very similar . In the 3'-untranslated region of the novel mRNA a C+U-rich, 20-fold repetitive element was found which appears to be highly related to the differentiation control element of the 15-lipoxygenase mRNA . Activity assays with a variety of cells and tissues prepared from normal rabbits suggested that only peripheral monocytes abundantly express the enzyme, suggesting a tissue-specific regulation of gene expression . These data indicate for the first time the co-expression of two separate genes for a reticulocyte-type 15-lipoxygenase and for a leukocyte-type 12-lipoxygenase in one species . This is of importance for the implication of both enzymes in red blood cell development and atherogenesis .

J Mol Biol, 1998 May 8, 278(3), 549 - 58
Operator search by mutant Lac repressors; Barker A et al.; The Escherichia coli Lac and Gal repressors are two members of a large family of bacterial repressor proteins that share significant sequence and structural homology . Efficient repression by all family members requires specific binding to a site or sites close to the transcriptional start of the genes regulated . Both LacR and GalR have to bind to at least two sites for efficient repression, yet they differ in one important respect: LacR is a homotetramer whereas GalR is a homodimer . In an attempt to understand this difference, we studied the operator binding activity of a LacR variant that has the DNA-binding specificity of GalR (LacR-V17A18) . A tetrameric version of this protein shows a 30-fold decrease in association rate to operator located on a long (lambda) DNA molecule, in comparison to wild-type LacR, while a dimeric version of this protein shows an unaltered association rate in comparison to dimeric LacR . This reduction in association rate correlates with a broadened DNA-binding specificity for base-pairs 4 and 5 of the operator: examination of an additional LacR variant with an even broader DNA-binding specificity indicates that a tetrameric version also shows a 30-fold decrease in association rate in comparison to wild-type LacR, while a dimeric version again shows an unaltered association rate in comparison to dimeric LacR . This difference in association rate in vitro correlates with whether a tetrameric or dimeric variant of LacR of a given DNA-binding specificity will repress lacZ under control of a single operator more efficiently in vivo . We therefore propose that the formation of stable homotetramers becomes a distinct disadvantage unless a high degree of DNA-binding specificity is also present, and demonstrate that this in indeed the case for GalR-mediated repression of the gal operon . This functional constraint seems to have influenced the evolution of the LacI-GalR family of repressors, most of which have a relatively broad specificity of DNA-binding and most of which form only stable homodimers .

J Mol Biol, 1998 May 8, 278(3), 539 - 48
Arm-domain interactions in AraC; Saviola B et al.; N-terminal deletions extending beyond the sixth amino acid of the Escherichia coli regulator of the l-arabinose operon, AraC, were found to generate constitutive regulatory behavior of the promoter pBAD . Mutagenesis of the DNA coding for the first 20 amino acids of the protein and screening for constitutives yielded mutants across the region whereas screening for mutants that cannot induce pBAD, even in the presence of arabinose, yielded none . These results indicate that the N-terminal arm is not essential for transcription activation, but that it plays an important and active role in holding the system in a non-activating state . Despite the fact that arabinose binds to the N-terminal domain of AraC, mutations were found in the C-terminal domain that weaken the binding of arabinose to the protein . The effects of the mutations could be suppressed by specific mutation in the N-terminal arm or by deletion of the arm . These results, in conjunction with the crystal structures of the N-terminal domain determined in the presence and absence of arabinose, indicate that in the absence of arabinose, the N-terminal arms of the protein bind to the C-terminal DNA binding domains to hold them in a state where the protein prefers to loop . When arabinose is added, the arms are pulled off the C-terminal domains, thereby releasing them to bind to adjacently located DNA half-sites and activate transcription .

J Mol Biol, 1998 May 8, 278(3), 529 - 38
Apo-AraC actively seeks to loop; Seabold RR et al.; In the absence of arabinose and interactions with other proteins, AraC, the activator-repressor that regulates the araBAD operon in Escherichia coli, was found to prefer participating in DNA looping interactions between the two well-separated DNA half-sites, araI1 and araO2 at their normal separation of 211 base-pairs rather than binding to these same two half-sites when they are adjacent to one another . On the addition of arabinose, AraC preferred to bind to the adjacently located half-sites . Inverting the distally located araO2 half-site eliminated the looping preference . These results demonstrate that apo-AraC possesses an intrinsic looping preference that is eliminated by the presence of arabinose . We developed a method for the accurate determination of the relative affinities of AraC for the DNA half-sites araI1, araI2, and araO2 and non-specific DNA . These affinities allowed accurate calculation of basal level and induced levels of expression from pBAD under a wide variety of natural and mutant conditions . The calculations independently predicted the looping preference of apo-AraC .

Chem Res Toxicol, 1998 May, 11(5), 550 - 6
Mutations induced in the supF gene of pSP189 by hydroxyl radical and singlet oxygen: relevance to peroxynitrite mutagenesis; Jeong JK et al.; We previously showed that the oxidant peroxynitrite (ONOO-) was strongly mutagenic in the supF shuttle vector pSP189 replicated in bacteria or human cells . Qualitative characteristics of the mutational spectra induced by ONOO- differed significantly from those reportedly caused by hydroxyl radical (OH.) in other experimental systems but showed similarities to spectra reportedly produced by singlet oxygen (1O2) . The molecular mechanisms of ONOO--mediated DNA damage are unknown . The objective of the present set of experiments was to characterize mutational effects induced in the supF gene of pSP189 by OH* and 1O2 to permit direct comparison with mutational spectra induced by ONOO- in this system . Base substitutions were the major form of mutation induced in plasmids replicated in human (AD293) cells by ONOO- (84%) and 1O2 (71%), whereas OH* induced fewer of them (49%) . In plasmids replicated in bacteria (Escherichia coli MBL50), frequencies of base substitutions induced by the three treatments were similar . G:C-to-T:A transversions were the most common form of base substitution induced by ONOO- (75% and 67%, respectively, in AD293- and MBL50-replicated plasmids) and 1O2 (68% and 71%); they were induced at lower frequencies by OH . (51% and 47%) . G:C-to-C:G transversions or G:C-to-A:T transitions were induced at almost equal frequencies by both ONOO- and 1O2, whereas OH* induced these mutations at different frequencies in the AD293 system . Collectively, our results confirm that in several important respects mutational spectra induced by ONOO- have greater similarity to spectra induced by 1O2 than to those induced by OH* and suggest that genotoxic derivatives of ONOO- are likely to include species that have DNA-damaging properties resembling those of 1O2 in selectivity for guanine but not identical in sequence specificity.

Chem Res Toxicol, 1998 May, 11(5), 420 - 7
Comparison of the formation of 8-hydroxy-2'-deoxyguanosine and single- and double-strand breaks in DNA mediated by fenton reactions; Lloyd DR et al.; The formation of 8-hydroxydeoxyguanosine (8-OHdG) and both single- and double-strand breaks in DNA by Fenton-type reactions has been investigated . Salmon sperm DNA was exposed to hydrogen peroxide (50 mM) and one of nine different transition-metal ions (25 microM-1 mM) . Modified DNA was isolated and subjected to analysis by liquid chromatography coupled to an electrochemical detection system (LC-ECD), to evaluate the formation of 8-OHdG . The highest yield of 8-OHdG was obtained following treatment of DNA with the chromium(III) Fenton reaction (a maximum of 19 400/10(6) nucleotides), followed by iron(II) (13 600), vanadium(III) (5800), and copper(II) (5200) . The chromium(VI) Fenton reaction generated a moderate yield of 8-OHdG (3600/10(6) nucleotides), while the yield obtained in DNA treated with cobalt(II), nickel(II), cadmium(II), and zinc Fenton reactions was not significantly higher than in control incubations of DNA with hydrogen peroxide alone . Similar treatment of the double-stranded plasmid pBluescript K+ with hydrogen peroxide (1 mM) and each transition-metal ion (1-100 microM) followed by quantitative agarose gel electrophoresis demonstrated that open-circle DNA, resulting from single-strand breaks, was generated in Fenton reactions involving all nine metal ions . In contrast, linear DNA was only formed in Fenton reactions involving chromium(III), copper(II), iron(II), and vanadium(III) ions . Formation of linear DNA, under conditions that generated relatively few single-strand breaks, suggests that these four transition-metal ions partake in Fenton reactions to generate true double-strand breaks . Furthermore, the generation of 8-OHdG exhibits a good correlation with the formation of double-strand breaks, suggesting that they arise by a similar mechanism.

Mol Cell Biol, 1998 Jun, 18(6), 3563 - 71
XRCC1 is specifically associated with poly(ADP-ribose) polymerase and negatively regulates its activity following DNA damage; Masson M et al.; Poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30) is a zinc-finger DNA-binding protein that detects and signals DNA strand breaks generated directly or indirectly by genotoxic agents . In response to these breaks, the immediate poly(ADP-ribosyl)ation of nuclear proteins involved in chromatin architecture and DNA metabolism converts DNA damage into intracellular signals that can activate DNA repair programs or cell death options . To have greater insight into the physiological function of this enzyme, we have used the two-hybrid system to find genes encoding proteins putatively interacting with PARP . We have identified a physical association between PARP and the base excision repair (BER) protein XRCC1 (X-ray repair cross-complementing 1) in the Saccharomyces cerevisiae system, which was further confirmed to exist in mammalian cells . XRCC1 interacts with PARP by its central region (amino acids 301 to 402), which contains a BRCT (BRCA1 C terminus) module, a widespread motif in DNA repair and DNA damage-responsive cell cycle checkpoint proteins . Overexpression of XRCC1 in Cos-7 or HeLa cells dramatically decreases PARP activity in vivo, reinforcing the potential protective function of PARP at DNA breaks . Given that XRCC1 is also associated with DNA ligase III via a second BRCT module and with DNA polymerase beta, our results provide strong evidence that PARP is a member of a BER multiprotein complex involved in the detection of DNA interruptions and possibly in the recruitment of XRCC1 and its partners for efficient processing of these breaks in a coordinated manner . The modular organizations of these interactors, associated with small conserved domains, may contribute to increasing the efficiency of the overall pathway.

Mol Cell Biol, 1998 Jun, 18(6), 3475 - 82
Concurrent replication and methylation at mammalian origins of replication; Araujo FD et al.; Observations made with Escherichia coli have suggested that a lag between replication and methylation regulates initiation of replication . To address the question of whether a similar mechanism operates in mammalian cells, we have determined the temporal relationship between initiation of replication and methylation in mammalian cells both at a comprehensive level and at specific sites . First, newly synthesized DNA containing origins of replication was isolated from primate-transformed and primary cell lines (HeLa cells, primary human fibroblasts, African green monkey kidney fibroblasts {CV-1}, and primary African green monkey kidney cells) by the nascent-strand extrusion method followed by sucrose gradient sedimentation . By a modified nearest-neighbor analysis, the levels of cytosine methylation residing in all four possible dinucleotide sequences of both nascent and genomic DNAs were determined . The levels of cytosine methylation observed in the nascent and genomic DNAs were equivalent, suggesting that DNA replication and methylation are concomitant events . Okazaki fragments were also demonstrated to be methylated, suggesting that the rapid kinetics of methylation is a feature of both the leading and the lagging strands of nascent DNA . However, in contrast to previous observations, neither nascent nor genomic DNA contained detectable levels of methylated cytosines at dinucleotide contexts other than CpG (i.e., CpA, CpC, and CpT are not methylated) . The nearest-neighbor analysis also shows that cancer cell lines are hypermethylated in both nascent and genomic DNAs relative to the primary cell lines . The extent of methylation in nascent and genomic DNAs at specific sites was determined as well by bisulfite mapping of CpG sites at the lamin B2, c-myc, and beta-globin origins of replication . The methylation patterns of genomic and nascent clones are the same, confirming the hypothesis that methylation occurs concurrently with replication . Interestingly, the c-myc origin was found to be unmethylated in all clones tested . These results show that, like genes, different origins of replication exhibit different patterns of methylation . In summary, our results demonstrate tight coordination of DNA methylation and replication, which is consistent with recent observations showing that DNA methyltransferase is associated with proliferating cell nuclear antigen in the replication fork.

Mol Cell Biol, 1998 Jun, 18(6), 3455 - 65
RNA-induced changes in the activity of the endonuclease encoded by the R2 retrotransposable element; Yang J et al.; R2 is a non-long terminal repeat retrotransposable element that inserts itself site specifically in the 28S rRNA genes of arthropods . The 120-kDa protein encoded by R2 has been shown to cleave one strand of the 28S gene at the target site and to use the 3' hydroxyl group generated from this nick to prime reverse transcription of its own RNA . This reaction has been termed target-primed reverse transcription (TPRT) . Cleavage of the second DNA strand can occur in the presence or absence of reverse transcription but requires RNA . In this study, more sensitive in vitro assays have enabled further characterization of these reactions . R2 protein is capable of only a single round of TPRT because, once bound to the target DNA, it does not dissociate at physiological ionic strengths . Analysis of the role of RNA in the DNA cleavage reaction has revealed that the binding of RNA induces the R2 protein to form a multimeric complex . While larger complexes may form, the active component appears to be a dimer based on sedimentation studies and the change in stoichiometry of the cleavage reaction from a 1:1 ratio of protein subunit to target DNA in the absence of RNA to a 2:1 ratio of subunit to DNA target in the presence of RNA . Nonspecific RNA can also induce formation of this RNA-protein (RNP) complex, but the association of the protein with R2 RNA is stronger as revealed by its stability in 0.4 M NaCl . Finally, formation of the RNP complex gives rise to a 150-fold increase in the ability of the R2 endonuclease to find the target site . The specificity of this RNP complex is sufficiently great that it can find the 28S gene target site and conduct the TPRT reaction with total genomic DNA.

J Biol Chem, 1998 May 15, 273(20), 12536 - 42
Cloning and expression of cDNA encoding rat liver 60-kDa lysophospholipase containing an asparaginase-like region and ankyrin repeat; Sugimoto H et al.; Mammalian tissues contain small form and large form lysophospholipases . Here we report the cloning, sequence, and expression of cDNA encoding the latter form of lysophospholipase using antibody raised against the enzyme purified from rat liver supernatant (Sugimoto, H., and Yamashita, S . (1994) J . Biol . Chem . 269, 6252-6258) . The 2,539-base pair cDNA encoded 564 amino acid residues with a calculated Mr of 60,794 . The amino-terminal two-thirds of the deduced amino acid sequence significantly resembled Escherichia coli asparaginase I with the putative asparaginase catalytic triad Thr-Asp-Lys and was followed by leucine zipper motif . The carboxyl-terminal region carried ankyrin repeat . When the cDNA was transfected into HEK293 cells, not only lysophospholipase activity but also asparaginase and platelet-activating factor acetylhydrolase activities were expressed . Reverse transcription-polymerase chain reaction revealed that the transcript occurred at high levels in liver and kidney but was hardly detectable in lung and heart from which large form lysophospholipases had been purified, suggesting the presence of multiple forms of large form lysophospholipase in mammalian tissues.

J Biol Chem, 1998 May 15, 273(20), 12509 - 14
Complementation analysis of mutants of 1-aminocyclopropane- 1-carboxylate synthase reveals the enzyme is a dimer with shared active sites; Tarun AS et al.; The pyridoxal phosphate-dependent enzyme 1-aminocyclopropane-1-carboxylate synthase (ACS, EC 4.4.1.14) catalyzes the rate-limiting step in the ethylene biosynthetic pathway . ACS shares the conservation of 11 invariant residues with a family of aminotransferases that includes aspartate aminotransferase . Site-directed mutagenesis on two of these residues, Tyr-92 and Lys-278, in the tomato isoenzyme Le-ACS2 greatly reduces enzymatic activity, indicating their importance in catalysis . These mutants have been used in complementation experiments either in vivo in Escherichia coli or in an in vitro transcription/translation assay to study whether the enzyme functions as a dimer . When the Y92L mutant is coexpressed with the K278A mutant protein, there is partial restoration of enzyme activity, suggesting that the mutant proteins can dimerize and form active heterodimers . Coexpressing a double mutant with the wild-type protein reduces wild-type activity, indicating that inactive heterodimers are formed between the wild-type and the double mutant protein subunits . Furthermore, hybrid complementation shows that another tomato isoenzyme, Le-ACS4, can dimerize and that Le-ACS2 and Le-ACS4 have limited capacity for heterodimerization . The data suggest that ACS functions as a dimer with shared active sites.

J Biol Chem, 1998 May 15, 273(20), 12476 - 81
Enzymatic and structural similarities between the Escherichia coli ATP-dependent proteases, ClpXP and ClpAP; Grimaud R et al.; Escherichia coli ClpX, a member of the Clp family of ATPases, has ATP-dependent chaperone activity and is required for specific ATP-dependent proteolytic activities expressed by ClpP . Gel filtration and electron microscopy showed that ClpX subunits (Mr 46, 000) associate to form a six-membered ring (Mr approximately 280, 000) that is stabilized by binding of ATP or nonhydrolyzable analogs of ATP . ClpP, which is composed of two seven-membered rings stacked face-to-face, interacts with the nucleotide-stabilized hexamer of ClpX to form a complex that could be isolated by gel filtration . Electron micrographs of negatively stained ClpXP preparations showed side views of 1:1 and 2:1 ClpXP complexes in which ClpP was flanked on either one or both sides by a ring of ClpX . Thus, as was seen for ClpAP, a symmetry mismatch exists in the bonding interactions between the seven-membered rings of ClpP and the six-membered rings of ClpX . Competition studies showed that ClpA may have a slightly higher affinity (approximately 2-fold) for binding to ClpP . Mixed complexes of ClpA, ClpX, and ClpP with the two ATPases bound simultaneously to opposite faces of a single ClpP molecule were seen by electron microscopy . In the presence of ATP or nonhydrolyzable analogs of ATP, ClpXP had nearly the same activity as ClpAP against oligopeptide substrates (>10,000 min-1/tetradecamer of ClpP) . Thus, ClpX and ClpA interactions with ClpP result in structurally analogous complexes and induce similar conformational changes that affect the accessibility and the catalytic efficiency of ClpP active sites.

J Biol Chem, 1998 May 15, 273(20), 12451 - 6
A mutation in the Escherichia coli secY gene that produces distinct effects on inner membrane protein insertion and protein export; Newitt JA et al.; E . coli strains that contain the secY40 mutation are cold-sensitive, but protein export defects have not been observed even at the nonpermissive temperature . Here we describe experiments designed to explain the conditional phenotype associated with this allele . We found that combining the secY40 mutation with defects in the signal recognition particle targeting pathway led to synthetic lethality . Since the signal recognition particle is required for the insertion of inner membrane proteins (IMPs) into the cytoplasmic membrane but not for protein export, this observation prompted us to examine the effect of the secY40 mutation on IMP biogenesis . The membrane insertion of all IMPs that we tested was impaired at both permissive and nonpermissive temperatures in secY40 cells grown in either rich or minimal medium . The magnitude of the insertion defects was greatest in cells grown at low temperature in rich medium, conditions in which the growth defect was most pronounced . Consistent with previous reports, we could not detect protein export defects in secY40 cells grown in minimal medium . Upon growth in rich medium, only slight protein export defects were observed . Taken together, these results suggest that the impairment of IMP insertion causes the cold sensitivity of secY40 strains . Furthermore, these results provide the first evidence that the protein export and membrane protein insertion functions of the translocon are genetically separable.

J Biol Chem, 1998 May 15, 273(20), 12427 - 35
High level expression and dimer characterization of the S100 EF-hand proteins, migration inhibitory factor-related proteins 8 and 14; Hunter MJ et al.; The phenotypical and functional heterogeneity of different macrophage subpopulations are defined by discrete changes in the expression of two S100 calcium-binding proteins, migration inhibitory factor-related proteins (MRPs) 8 and 14 . To further our understanding of MRP8 and MRP14 in the developmental stages of inflammatory responses, overexpression of the MRPs was obtained through a combination of a T7-based expression vector and the Escherichia coli BL21 (DE3) cell line . An efficient, two-step chromatographic protocol was then developed for rapid, facile purification . Extensive biophysical characterization and chemical cross-linking experiments show that MRP8 and MRP14 form oligomers with a strong preference to associate as a heterodimer . Heteronuclear NMR experiments indicate that a specific well packed dimer is formed only in equimolar mixtures of the two proteins . Our results suggest that there is a unique complementarity in the interface of the MRP8/MRP14 complex that cannot be fully reproduced in the MRP8 and MRP14 homodimers.

J Biol Chem, 1998 May 15, 273(20), 12274 - 80
DNA strand invasion promoted by Escherichia coli RecT protein; Noirot P et al.; The RecT protein of Escherichia coli is a DNA-pairing protein required for the RecA-independent recombination events promoted by the RecE pathway . The RecT protein was found to bind to both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) in the absence of Mg2+ . In the presence of Mg2+, RecT binding to dsDNA was inhibited drastically, whereas binding to ssDNA was inhibited only to a small extent . RecT promoted the transfer of a single-stranded oligonucleotide into a supercoiled homologous duplex to form a D (displacement)-loop . D-loop formation occurred in the absence of Mg2+ and at 1 mM Mg2+ but was inhibited by increasing concentrations of Mg2+ and did not require a high energy cofactor . Strand transfer was mediated by a RecT-ssDNA nucleoprotein complex reacting with a naked duplex DNA and was prevented by the formation of RecT-dsDNA nucleoprotein complexes . Finally, RecT mediated the formation of joint molecules between a supercoiled DNA and a linear dsDNA substrate with homologous 3'-single-stranded tails . Together these results indicate that RecT is not a helix-destabilizing protein promoting a reannealing reaction but rather is a novel type of pairing protein capable of promoting recombination by a DNA strand invasion mechanism . These results are consistent with the observation that RecE (exonuclease VIII) and RecT can promote RecA-independent double-strand break repair in E . coli.

J Biol Chem, 1998 May 15, 273(20), 12259 - 66
Expression and characterization of the catalytic core of tryptophan hydroxylase; Moran GR et al.; Wild type rabbit tryptophan hydroxylase (TRH) and two truncated mutant proteins have been expressed in Escherichia coli . The wild type protein was only expressed at low levels, whereas the mutant protein lacking the 101 amino-terminal regulatory domain was predominantly found in inclusion bodies . The protein that also lacked the carboxyl-terminal 28 amino acids, TRH102-416, was expressed as 30% of total cell protein . Analytical ultracentrifugation showed that TRH102-416 was predominantly a monomer in solution . The enzyme exhibited an absolute requirement for iron (ferrous or ferric) for activity and did not turn over in the presence of cobalt or copper . With either phenylalanine or tryptophan as substrate, stoichiometric formation of the 4a-hydroxypterin was found . Steady state kinetic parameters were determined with both of these amino acids using both tetrahydrobiopterin and 6-methyltetrahydropterin.

J Biol Chem, 1998 May 15, 273(20), 12239 - 43
The glucose transporter of the Escherichia coli phosphotransferase system . Mutant analysis of the invariant arginines, histidines, and domain linker; Lanz R et al.; The glucose transporter of the bacterial phosphotransferase system (PTS) consists of a hydrophilic (IIAGlc) and a transmembrane subunit (IICBGlc) . IICBGlc has two domains (C and B), which are linked by a highly invariant sequence . Transport of glucose by IIC and phosphorylation by IIB are tightly coupled processes . Three motifs that are strongly conserved in 12 homologous PTS transporters, namely two invariant arginines (Arg-424 and Arg-426) adjacent to the phosphorylation site (Cys-421), the invariant interdomain sequence KTPGRED, and two conserved histidines (His-211 and His-212) in the IIC domain were mutated and the mutant proteins characterized in vivo and in vitro for transport and phosphorylation activity . Replacement of the strongly beta-turn favoring residues Thr and Gly of the linker by alpha-helix favoring Ala results in strong reduction of activity, whereas the substitutions of the other residues have only minor effects . The R424K and R426K mutants can be phosphorylated by IIAGlc but can no longer donate the phosphoryl group to glucose . The H211Q and H212Q mutants continue to phosphorylate glucose at a reduced rate but H212Q can no longer transport glucose . Mixtures of purified R424K/H212Q and R426K/H212Q have 10% of wild-type phosphorylation activity and when coexpressed in Escherichia coli support glucose transport.

J Biol Chem, 1998 May 15, 273(20), 12120 - 7
The role of negative superhelicity and length of homology in the formation of paranemic joints promoted by RecA protein; Wong BC et al.; Escherichia coli RecA protein pairs homologous DNA molecules to form paranemic joints when there is an absence of a free end in the region of homologous contact . Paranemic joints are a key intermediate in homologous recombination and are important in understanding the mechanism for a search of homology . The efficiency of paranemic joint formation depended on the length of homology and the topological forms of the duplex DNA . The presence of negative superhelicity increased the pairing efficiency and reduced the minimal length of homology required for paranemic joint formation . Negative superhelicity stimulated joint formation by favoring the initial unwinding of duplex DNA that occurred during the homology search and was not essential in the maintenance of the paired structure . Regardless of length of homology, formation of paranemic joints using circular duplex DNA required the presence of more than six negative supercoils . Above six negative turns, an increasing degree of negative superhelicity resulted in a linear increase in the pairing efficiency . These results support a model of two distinct kinds of DNA unwinding occurring in paranemic joint formation: an initial unwinding caused by heterologous contacts during synapsis and a later one during pairing of the homologous molecules.

J Biol Chem, 1998 May 15, 273(20), 11999 - 2002
D-peptide ligands for the co-chaperone DnaJ; Feifel B et al.; The molecular chaperone DnaK, the Hsp70 homolog of Escherichia coli, binds hydrophobic polypeptide segments in extended conformation . The co-chaperone DnaJ (Hsp40) has been reported to bind native and denatured proteins as well as peptides . We tested pseudo-peptides of D-amino acids as ligands for both chaperones . In comparison to the parent all-L peptide, these mimetics had either enantiomorphic side chain positions combined with retained main chain direction (normal all-D peptide) or unchanged side chain topology together with reverse direction of the peptide backbone (retro all-D peptide) . The peptides were labeled with acrylodan (a), and their binding to DnaK and DnaJ was monitored by the accompanying increase in fluorescence intensity . The parent all-L peptide a-CALLLSAARR bound to both DnaK (Kd = 0.1 microM) and DnaJ (Kd = 9.2 microM) . In contrast, the normal all-D and retro all-D peptides did not bind to DnaK; they bound, however, to DnaJ with Kd values of 6.8 microM and 0.9 microM, respectively . The emission spectra of the DnaJ-bound peptides suggests that DnaJ bound both D-peptides with the same main chain direction as L-peptides . Binding of the normal all-D and all-L peptides inhibited the DnaJ-induced stimulation of DnaK ATPase . However, binding of the retro all-D analog to DnaJ did not impair the stimulation, indicating the existence of separate binding sites for peptides and DnaK.

Blood, 1998 May 15, 91(10), 3980 - 5
Examination of ferrochelatase mutations that cause erythropoietic protoporphyria; Sellers VM et al.; Ferrochelatase (E.C . 4.99.1.1), the enzyme that catalyzes the terminal step in the heme biosynthetic pathway, is the site of defect in the human inherited disease erythropoietic protoporphyria (EPP) . Previously it has been demonstrated that patients with EPP may have missense mutations leading to amino acid substitutions, early chain termination, or exon deletions . While it has been clearly demonstrated that two missense mutations result in lowered enzyme activity, it has never been shown what effect specific exon deletions may have . In the current work, recombinant human ferrochelatase has been engineered to have individual exon deletions corresponding to exons 3 through 11 . When expressed in Escherichia coli, none of these possesses significant enzyme activity and all lack the {2Fe-2S} cluster . One of the human missense mutations, F417S, and a series of amino acid replacements at this site (ie, F417W, F417Y, and F417L) were examined . With the exception of F417L, all lacked enzyme activity and did not contain the {2Fe-2S} cluster in vivo or as isolated in vitro.

Blood, 1998 May 15, 91(10), 3927 - 34
Secondary mutation maintains the transformed state in BaF3 cells with inducible BCR/ABL expression; Klucher KM et al.; The BCR/ABL gene product of the Philadelphia (Ph) chromosome induces chronic myelogenous leukemia (CML) . We generated a hematopoietic cell line, TonB210.1, with tetracycline-dependent BCR/ABL expression to investigate the pathways by which BCR/ABL transforms cells . TonB210.1 demonstrates conditional growth factor independence in tissue culture and rapidly forms tumors in mice fed the tetracycline analog doxycycline . The tumors regress completely upon doxycycline withdrawal, but ultimately reform in all animals . After a long latency, tumors also develop in animals never exposed to doxycycline . Subclones of TonB210.1 established from doxycycline-independent tumors demonstrate distinct mechanisms of transformation . Most subclones manifest increased basal levels of BCR/ABL expression; some have lost the capacity to augment expression upon induction, whereas others remain inducible . More interestingly, some subclones maintain tight conditional expression of BCR/ABL and are therefore transformed by secondary mechanisms that no longer require BCR/ABL expression . These subclones show constitutive phosphorylation of the STAT5 protein, suggesting that activating mutations have occurred upstream in the signaling pathway to STAT5 . The tight conditional expression of BCR/ABL in the TonB210.1 cell line affords the opportunity to study several interesting aspects of the biology of BCR/ABL, including activation of critical signaling pathways and transcriptional programs, and its potential role in genomic instability.

EMBO J, 1998 May 1, 17(9), 2677 - 86
hMSH2 and hMSH6 play distinct roles in mismatch binding and contribute differently to the ATPase activity of hMutSalpha; Iaccarino I et al.; In extracts of human cells, base-base mismatches and small insertion/deletion loops are bound primarily by hMutSalpha, a heterodimer of hMSH2 and hMSH6 (also known as GTBP or p160) . Recombinant hMutSalpha bound a G/T mismatch-containing oligonucleotide with an apparent dissociation constant Kd = 2.6 nM, while its affinity for a homoduplex substrate was >20-fold lower . In the presence of ATP, hMutSalpha dissociated from mismatched oligonucleotide substrates, and this reaction was attenuated by mutating the conserved lysine in the ATP-binding domains of hMSH6, hMSH2 or both to arginine . Surprisingly, this reaction required only ATP binding, not hydrolysis . The ATPase activity of hMutSalpha variants carrying the Lys-->Arg mutation in hMSH2 or in hMSH6 was severely affected, but these mutants were still proficient in mismatch binding and were able to complement, albeit to different extents, mismatch repair-deficient cell extracts . The mismatch binding-proficient, ATPase-deficient double mutant was inactive in the complementation assay and its presence in repair-proficient extracts was inhibitory . We conclude that although the ATPase activity of hMutSalpha is dispensible for mismatch binding, it is required for mismatch correction.

EMBO J, 1998 May 1, 17(9), 2629 - 36
The redox-regulated SoxR protein acts from a single DNA site as a repressor and an allosteric activator; Hidalgo E et al.; The SoxR protein of Escherichia coli responds to redox signals by activating the transcription of soxS, which encodes another transcription activator that directly stimulates oxidative stress genes . We show here that transcription of the soxR gene, which is positioned head-to-head with soxS in the chromosome, initiates in the intergenic region and is itself repressed by SoxR protein in in vitro transcription experiments . Analysis of single-copy operon fusions to soxR, combined with the results of Northern blotting experiments, verified this regulation and the transcription start site in vivo . The structure of the overlapping promoters is such that the single SoxR-binding site is located in the -10/-35 spacer of the soxS promoter, but just downstream of the -10 element of the soxR promoter . Activated and non-activated SoxR bind this site equally well, exerting nearly constant repression of soxR; activated SoxR simultaneously stimulates the soxS promoter >/=30-fold . The functional soxR promoter depresses soxS transcription when SoxR is not activated and enhances soxS transcription when SoxR is activated, as shown by comparing the expression of soxS'::lacZ fusions with and without the soxR -35 element (induction ratio only approximately 7-fold) . SoxR thus represents a highly polar, redox-regulated transcriptional switch that maximizes the change in expression of soxS.

EMBO J, 1998 May 1, 17(9), 2504 - 12
The Escherichia coli SRP and SecB targeting pathways converge at the translocon; Valent QA et al.; Two distinct protein targeting pathways can direct proteins to the Escherichia coli inner membrane . The Sec pathway involves the cytosolic chaperone SecB that binds to the mature region of pre-proteins . SecB targets the pre-protein to SecA that mediates pre-protein translocation through the SecYEG translocon . The SRP pathway is probably used primarily for the targeting and assembly of inner membrane proteins . It involves the signal recognition particle (SRP) that interacts with the hydrophobic targeting signal of nascent proteins . By using a protein cross-linking approach, we demonstrate here that the SRP pathway delivers nascent inner membrane proteins at the membrane . The SRP receptor FtsY, GTP and inner membranes are required for release of the nascent proteins from the SRP . Upon release of the SRP at the membrane, the targeted nascent proteins insert into a translocon that contains at least SecA, SecY and SecG . Hence, as appears to be the case for several other translocation systems, multiple targeting mechanisms deliver a variety of precursor proteins to a common membrane translocation complex of the E.coli inner membrane.

Nucleic Acids Res, 1998 May 1, 26(9), 2156 - 60
The importance of base pairing in the penultimate stem of Escherichia coli 16S rRNA for ribosomal subunit association; Firpo MA et al.; The influence of base pairing in the penultimate stem of Escherichia coli 16S rRNA (defined as nt 1409-1491) on ribosome function has been addressed by the construction of mutations in this region of rRNA . Two sets of mutations were made on either side of a structurally conserved region in the penultimate stem that disrupted base pairing, while a third set of mutations replaced the wild-type sequence with other base pair combinations . The effects of these mutations were analyzed in vivo and in vitro . The mutations that disrupted base pairing caused significant increases in cell doubling times as well as a severe subunit association defect and a modest increase in frame shifting and stop codon read-through . Restoration of base pairing restored wild-type growth rates, decoding and subunit association, indicating that base pairing in this region is essential for proper ribosome function.

Nucleic Acids Res, 1998 May 1, 26(9), 2125 - 31
A stimulatory RNA associated with RecBCD enzyme; Amundsen SK et al.; RecBCD enzyme acts in the major pathway of homologous recombination of linear DNA in Escherichia coli . The enzyme unwinds DNA and is an ATP-dependent double-strand and single-strand exonuclease and a single-strand endonuclease; it acts at Chi recombination hotspots (5'-GCTGGTGG-3') to produce a recombinogenic single-stranded DNA 3'-end . We found that a small RNA with a unique sequence of approximately 24 nt was tightly bound to RecBCD enzyme and co-purified with it . When added to native enzyme this RNA, but not four others, increased DNA unwinding and Chi nicking activities of the enzyme . In seven similarly active enzyme preparations the molar ratio of RNA molecules to RecBCD enzyme molecules ranged from 0.2 to <0.008 . These results suggest that, although this unique RNA is not an essential enzyme subunit, it has a biological role in stimulating RecBCD enzyme activity.

Nucleic Acids Res, 1998 May 1, 26(9), 2075 - 81
Transcription activation by Escherichia coli FNR protein: similarities to, and differences from, the CRP paradigm; Li B et al.; During transcription activation at FNR-dependent promoters where the DNA site for FNR overlaps the -35 element, a surface-exposed activating region in the upstream subunit of the FNR dimer interacts with the C-terminal domain of the RNA polymerase alpha subunit . Starting with a cloned fnr gene encoding a defective FNR derivative carrying substitutions in this activating region, we screened a library of random mutations to identify substitutions that restored FNR activity . Activity can be restored by substitutions at residues T118, E47 and K60 . The locations of these residues identify three separate surface-exposed regions of FNR that can play a role in transcription activation . These three regions appear to be analogues of Activating Region 1, Activating Region 2 and Activating Region 3 of the cyclic AMP receptor protein, CRP: our results underscore the similarities between FNR and CRP.

Mol Microbiol, 1998 May, 28(3), 641 - 53
Effects of the Escherichia coli DNA-binding protein H-NS on rRNA synthesis in vivo; Afflerbach H et al.; The Escherichia coli DNA-binding protein H-NS is known to interact specifically with the upstream region of ribosomal RNA transcription units, where it causes transcriptional repression in vitro . Here, we present results demonstrating the effect of H-NS on rRNA transcription in vivo . rRNA synthesis rates were compared in cells that differ in the expression of functional H-NS or FIS molecules . We could show that in the absence of H-NS derepression of rRNA synthesis occurs at low growth rates . During the cell cycle H-NS is responsible for the rapid shut-off of rRNA synthesis at the end of the exponential phase . As it is known for FIS-dependent activation, the inhibitory function of H-NS is specific for P1, the first of the tandem rRNA promoters . The effect of H-NS on rRNA synthesis was further assessed under stress conditions . While under osmotic upshift the reduction in rRNA synthesis is clearly H-NS-dependent, no such influence could be detected at cold shock . Determination of the cellular ppGpp concentrations revealed that H-NS does not mediate its function via alterations in the synthesis of the global effector ppGpp.

Mol Microbiol, 1998 May, 28(3), 629 - 40
Genetic uncoupling of the dsRNA-binding and RNA cleavage activities of the Escherichia coli endoribonuclease RNase III--the effect of dsRNA binding on gene expression; Dasgupta S et al.; RNase III, a double-stranded RNA-specific endonuclease, is proposed to be one of Escherichia coli's global regulators because of its ability to affect the expression of a large number of unrelated genes by influencing post-transcriptional control of mRNA stability or mRNA translational efficiency . Here, we describe the phenotypes of bacteria carrying point mutations in rnc, the gene encoding RNase III . The substrate recognition and RNA-processing properties of mutant proteins were analysed in vivo by measuring expression from known RNase III-modulated genes and in vitro from the proteins' binding and cleavage activities on known double-stranded RNA substrates . Our results show that although the point mutation rnc70 exhibited all the usual rnc null-like phenotypes, unlike other mutations, it was dominant over the wild-type allele . Multicopy expression of rnc70 could suppress a lethal phenotype of the wild-type rnc allele in a certain genetic background; it could also inhibit the RNase III-mediated activation of lambdaN gene translation by competing for the RNA-binding site of the wild-type endonuclease . The mutant protein failed to cleave the standard RNase III substrates in vitro but exhibited an affinity for double-stranded RNA when passed through poly(rI):poly(rC) columns . Filter binding and gel-shift assays with purified Rnc70 showed that the mutant protein binds to known RNase III mRNA substrates in a site-specific manner . In vitro processing reactions with purified enzyme and labelled RNA showed that the in vivo dominant effect of the mutant enzyme over the wild-type was not necessarily caused by formation of mixed dimers . Thus, the rnc70 mutation generates a mutant RNase III with impaired endonucleolytic activity but without blocking its ability to recognize and bind double-stranded RNA substrates.

Mol Microbiol, 1998 May, 28(3), 603 - 13
The control of Azorhizobium caulinodans nifA expression by oxygen, ammonia and by the HF-I-like protein, NrfA; Kaminski PA et al.; The control of Azorhizobium caulinodans nifA expression in response to oxygen and ammonia involves FixLJ, FixK, NtrBC, NtrXY and the HF-I-like protein NrfA . The regulation is thus complex and possibly involves post-transcriptional regulation by NrfA . The coding region of nifA was determined using a translational lacZ fusion and by site-directed mutagenesis to identify which of four in frame AUG codons was used . The major NifA protein is translated from the second AUG codon and is predicted to consist of 613 amino acids . Primer extension analysis showed a major transcript starting 34 bp downstream from the anaerobox in wild-type, nifA, rpoN, ntrC and nrfA strains, but not in a fixK mutant . FixK- and oxygen-dependent transcription of nifA was confirmed by the analysis of four transcriptional nifA-lacZ fusions with fusion junctions at positions +1, +47, +110 and +181 with respect to the start site . Regulation by ammonia was independent of FixK and RpoN, NtrC being only partially required . Thus, there may be another type of nitrogen control that does not involve NtrC in A . caulinodans . NrfA is not required for the initiation of nifA transcription but, most probably, has an effect on nifA mRNA stability and/or translation . NrfA also restores the defect in rpoS translation to an Escherichia coli hfq mutant, indicating that HF-I and NrfA have similar activities in both A . caulinodans and E . coli.

Mol Microbiol, 1998 May, 28(3), 583 - 92
Functional and structural conservation in the mechanosensitive channel MscL implicates elements crucial for mechanosensation; Moe PC et al.; mscL encodes a channel in Escherichia coli that is opened by membrane stretch force, probably serving as an osmotic gauge . Sequences more or less similar to mscL are found in other bacteria, but the degree of conserved function has been unclear . We subcloned and expressed these putative homologues in E . coli and examined their products under patch clamp . Here, we show that each indeed encodes a conserved mechanosensitive channel activity, consistent with the interpretation that this is an important and primary function of the protein in a wide range of bacteria . Although similar, channels of different bacteria differ in kinetics and their degree of mechanosensitivity . Comparison of the primary sequence of these proteins reveals two highly conserved regions, corresponding to domains previously shown to be important for the function of the wild-type E . coli channel, and a C-terminal region that is not conserved in all species . This structural conservation is providing insight into regions of this molecule that are vital to its role as a mechanosensitive channel and may have broader implications for the understanding of other mechanosensitive systems.

Mol Microbiol, 1998 May, 28(3), 571 - 82
Conjugal transfer of chromosomal DNA in Mycobacterium smegmatis; Parsons LM et al.; The genus Mycobacterium includes the major human pathogens Mycobacterium tuberculosis and Mycobacterium leprae . The development of rational drug treatments for the diseases caused by these and other mycobacteria requires the establishment of basic molecular techniques to determine the genetic basis of pathogenesis and drug resistance . To date, the ability to manipulate and move DNA between mycobacterial strains has relied on the processes of transformation and transduction . Here, we describe a naturally occurring conjugation system present in Mycobacterium smegmatis, which we anticipate will further facilitate the ability to manipulate the mycobacterial genome . Our data rule out transduction and transformation as possible mechanisms of gene transfer in this system and are most consistent with conjugal transfer . We show that recombinants are not the result of cell fusion and that transfer occurs from a distinct donor to a recipient . One of the donor strains is mc(2)155, a highly transformable derivative that is considered the prototype laboratory strain for mycobacterial genetics; the demonstration that it is conjugative should increase its genetic manipulability dramatically . During conjugation, extensive regions of chromosomal DNA are transferred into the recipient and then integrated into the recipient chromosome by multiple recombination events . We propose that DNA transfer is occurring by a mechanism similar to Hfr conjugation in Escherichia coli.

Mol Microbiol, 1998 May, 28(3), 521 - 30
The ArcA/ArcB two-component regulatory system of Escherichia coli is essential for Xer site-specific recombination at psi; Colloms SD et al.; Two recombinases, XerC and XerD, act at the recombination sites psi and cer in plasmids pSC101 and Co1E1 respectively . Recombination at these sites maintains the plasmids in a monomeric state and helps to promote stable plasmid inheritance . The accessory protein PepA acts at both psi and cer to ensure that only intramolecular recombination takes place . An additional accessory protein, ArgR, is required for recombination at cer but not at psi . Here, we demonstrate that the ArcA/ArcB two-component regulatory system of Escherichia coli, which mediates adaptation to anaerobic growth conditions, is required for efficient recombination in vivo at psi . Phosphorylated ArcA binds to psi in vitro and increases the efficiency of recombination at this site . Binding of ArcA to psi may contribute to the formation of a higher order synaptic complex between a pair of psi sites, thus helping to ensure that recombination is intramolecular.

Mol Microbiol, 1998 May, 28(3), 435 - 47
NarJ is a specific chaperone required for molybdenum cofactor assembly in nitrate reductase A of Escherichia coli; Blasco F et al.; The formation of active membrane-bound nitrate reductase A in Escherichia coli requires the presence of three subunits, NarG, NarH and NarI, as well as a fourth protein, NarJ, that is not part of the active nitrate reductase . In narJ strains, both NarG and NarH subunits are associated in an unstable and inactive NarGH complex . A significant activation of this complex was observed in vitro after adding purified NarJ-6His polypeptide to the cell supernatant of a narJ strain . Once the apo-enzyme NarGHI of a narJ mutant has become anchored to the membrane via the NarI subunit, it cannot be reactivated by NarJ in vitro . NarJ protein specifically recognizes the catalytic NarG subunit . Fluorescence, electron paramagnetic resonance (EPR) spectroscopy and molybdenum quantification based on inductively coupled plasma emission spectroscopy (ICPES) clearly indicate that, in the absence of NarJ, no molybdenum cofactor is present in the NarGH complex . We propose that NarJ is a specific chaperone that binds to NarG and may thus keep it in an appropriate competent-open conformation for the molybdenum cofactor insertion to occur, resulting in a catalytically active enzyme . Upon insertion of the molybdenum cofactor into the apo-nitrate reductase, NarJ is then dissociated from the activated enzyme.

Oncogene, 1998 May, 16(20), 2657 - 70
Complex formation between EphB2 and Src requires phosphorylation of tyrosine 611 in the EphB2 juxtamembrane region; Zisch AH et al.; The cellular components of the neuronal signaling pathways of Eph receptor tyrosine kinases are only beginning to be elucidated . Here we show that in vivo tyrosine phosphorylation sites of the Eph receptors EphA3, EphA4, and EphB2 in embryonic retina serve as binding sites for the Src-homology 2 (SH2) domain of Src kinase . Furthermore, tyrosine-phosphorylated EphB2 was detected in Src immunoprecipitates from transfected Cos cells, indicating that EphB2 and Src can physically associate . Interestingly, a form of Src with reduced electrophoretic mobility and increased tyrosine phosphorylation was detected in Cos cells expressing tyrosine-phosphorylated EphB2, suggesting a functional interaction between EphB2 and Src . Yeast two-hybrid analysis in conjunction with site-directed mutagenesis demonstrated that phosphorylated tyrosine 611 in the juxtamembrane region of EphB2 is crucial for the interaction with the SH2 domain of Src . In contrast, binding of the carboxy-terminal SH2 domain of phospholipase Cgamma was not abolished upon mutation of tyrosine 611 in EphB2 . Phosphopeptide mapping of autophosphorylated full-length EphB2, and wild-type and tyrosine to phenylalanine mutants of the EphB2 cytoplasmic domain fused to LexA, showed tyrosine 611 in the sequence motif YEDP as a major site of autophosphorylation in EphB2 . Our mutational analysis also indicated that tyrosines 605 and 611 are important for EphB2 kinase activity . We propose Src kinase as a downstream effector that mediates the neuron's response to Eph receptor activation.

Oncogene, 1998 May, 16(20), 2597 - 607
Activation of Ras and its downstream extracellular signal-regulated protein kinases by the CDC25 homology domain of mouse Son-of-sevenless 1 (mSos1); Kim JH et al.; A fragment consisting of residues 584-1071 of the mouse Son-of-sevenless 1 (mSos1) protein was found to be sufficient for stimulation of the guanine nucleotide exchange of Ras in vitro, which defines the CDC25 homology (CDC25H) domain of mSos1 . Furthermore, we found that the CDC25H-domain fragment activated the extracellular signal-regulated protein kinases (ERKs), and was mainly membrane localized, when expressed in unstimulated human embryonic kidney 293 cells . Then, we examined the roles of other mSos1 domains in autoinhibition of the CDC25H-domain functions in unstimulated cellular environments . First, longer fragments that have the CDC25H domain and the following proline-rich Grb2-binding domain exhibited negligible membrane localization, and accordingly much lower ERK-activation activities, under serum-starved conditions . On the other hand, the preceding Pleckstrin-homology (PH) domain affects neither the ERK-activation activity nor the membrane-localization activity of the CDC25H domain . By contrast, the cells expressing a fragment containing the Dbl homology (DH) domain in addition to the PH and CDC25H domains exhibited remarkably low ERK activities under serum-starved conditions . This autoinhibitory effect of the DH domain on the CDC25H-domain function was shown to be relieved when cells were stimulated with epidermal growth factor . The DH-domain extension affected neither the in vitro guanine nucleotide exchange activity nor the membrane-localization activity of the CDC25H domain . Therefore, one of the roles of the DH domain is to exert an autoinhibition over the CDC25H-domain function on the cell membrane, in the absence, but not in the presence, of extracellular stimuli.

J Rheumatol, 1998 Jun, 25(6), 1218 - 20
Pancytopenia secondary to hemophagocytic syndrome in rheumatoid arthritis treated with methotrexate and sulfasalazine; Sibilia J et al.; Hemophagocytic syndrome is an exceptional cause of pancytopenia . Its etiologies are most commonly viral or bacterial infections, lymphoproliferative syndromes, acquired or congenital immunodeficiencies, systemic diseases, or immunomodulatory treatment . We describe a patient with rheumatoid arthritis (RA) treated with methotrexate (MTX), sulfasalazine, and low dose corticosteroids, whose case was seriously complicated by the occurrence of acute febrile pancytopenia . The pancytopenia appeared secondary to hemophagocytic syndrome triggered by Escherichia coli septicemia . The evolution was marked by severe aggravation of RA, probably due to release of cytokines from macrophages (tumor necrosis factor-alpha, interleukin 6) . Reintroduction of MTX (without sulfasalazine) resulted in partial remission and there was no reappearance of new hematological anomalies after 16 month followup . A knowledge of this syndrome is particularly important, since it mimics drug toxicity and other complications such as lymphoproliferative diseases.

FEMS Microbiol Lett, 1998 Jun 1, 163(1), 85 - 9
Molecular cloning of a light-inducible gene (lipA) encoding a novel pilin from Arthrobacter photogonimos; Yang HS et al.; Development of pili on cells of Arthrobacter photogonimos is induced by photo-oxidative conditions . The nucleotide sequence was determined of a light-inducible gene (lipA) that encodes the precursor of a light-inducible pilin (designated LIP), a polypeptide of 212 amino acids . The N-terminal leader peptide includes a typical signal sequence with a consensus cleavage site for signal peptidase 1 after residue 28, which should generate N-terminal arginine . However, the next amino acid, alanine, is the N-terminal residue of the mature protein . The abundance of charged amino acids (27% of total), a calculated pI of 9.98, and recovery of mostly monomers when cells were washed with 1 M NaCl suggest that electrostatic interactions play a dominant role in association of LIP, a novel mechanism for assembly of pili.

FEMS Microbiol Lett, 1998 Jun 1, 163(1), 37 - 42
Thermostable alpha-galactosidase from Thermotoga neapolitana: cloning, sequencing and expression; King MR et al.; A gene encoding a thermostable alpha-galactosidase from the hyperthermophile Thermotoga neapolitana was cloned and sequenced . Sequence analysis showed that the 552-amino acid protein is similar to Escherichia coli Raf type alpha-galactosidase and belongs to Family 36 of the glycosyl hydrolases . Recombinant alpha-galactosidase expressed in E . coli has a molecular mass of ca . 61 kDa, and an optimum activity at 93 degrees C at pH 7.0 . The enzyme is highly thermostable and retains 75% of activity after heating to 80 degrees C for 4 h . The potential application of the enzyme to high temperature processing of soy molasses has been demonstrated.

Vet Microbiol, 1998 Mar 31, 61(3), 229 - 35
Serotypes of Escherichia coli isolated from septicaemic chickens in Galicia (northwest Spain); Blanco JE et al.; The purpose of this study was to establish the serogroups of Escherichia coli that cause avian colibacillosis in Spain . The serogroups of 625 avian E . coli isolated between 1992 and 1993 were determined . The 458 E . coli from chickens with septicaemia belonged to 62 different O serogroups; however, 59% were of one of 18 serogroups (O1, O2, O5, O8, O12, O14, O15, O18, O20, O53, O78, O81, O83, O102, O103, O115, O116 and O132) . These 18 serogroups were also determined as an important percentage (29%) of control isolates from faeces of healthy birds . Nevertheless, a significant difference (59% versus 29%; P < 0.001) was observed . Furthermore, the serogroups O12, O14, O18, O53, O78, O81, O102, O115, O116 and O132 were almost exclusively identified among septicaemic E . coli (31% versus 3%; P < 0.001) . The high prevalence of O18, O81, O115, O116 and O132 isolates was not expected and may indicate the emergence of five new serogroups associated with avian colibacillosis not yet reported.

Vet Microbiol, 1998 Mar 31, 61(3), 191 - 7
Attachment of different Escherichia coli strains to cultured rumen epithelial cells; Galfi P et al.; The attachment to fully characterized primary rumen epithelial cell cultures of Escherichia coli strains isolated from different animal species and expressing F1-F4 or F17 fimbriae was examined . As the cell cultures contained stratified (keratinized) and non-stratified (non-keratinized) cells which grew either confluently or non-confluently, the strength of attachment of the different bacterial strains was assessed in relation to the differentiation state of the cells . Thus, strains having F1 fimbriae attached to all types of cultured cells, while strains with F2 and F3 fimbriae did not bind at all . E . coli strains having F4 or F17 fimbriae attached only to non-keratinized cells, particularly to confluent areas . As membrane glycosylation is known to change with differentiation (keratinization), our results suggest that the attachment of fimbriated E . coli strains which were capable of binding to rumen cells was more likely to be dependent on differentiation than the host specificity of the bacteria.

Protein Expr Purif, 1998 Jun, 13(1), 120 - 6
Purification and biochemical properties of soluble recombinant human Bax; Lewis S et al.; Bax is a member of the Bcl-2 protein family with proapoptotic properties . The proteins of this family contain three highly conserved regions termed BH1, BH2, and BH3 as well as a hydrophobic COOH-terminal domain, which is responsible for the membrane attachment of the proteins . We have expressed human Bax truncated of the 20 amino acid COOH-terminal hydrophobic domain to obtain large amounts of soluble protein suitable for biochemical and structural studies . The truncated protein was expressed as a glutathione S-transferase (GST) fusion protein in Escherichia coli . The GST-Bax fusion protein was bound to glutathione-Sepharose, and Bax was released by thrombin cleavage and further purified by sequential chromatography on heparin-Sepharose and DEAE-Sepharose . The purified protein was present in solution as a heptamer and multimers of the heptamer complex . Limited tryptic digestion cleaved the protein in the region preceding the BH3 domain and produced a specific stable protein fragment of 15 kDa . Phosphorylation has been proposed as a possible regulatory mechanism of the bcl-2 proteins . The Bax protein was an in vitro substrate for specific serine/threonine protein kinases.

Protein Expr Purif, 1998 Jun, 13(1), 97 - 103
Expression of sunflower homeodomain containing proteins in Escherichia coli: purification and functional studies; Palena CM et al.; Complementary DNA sequences encoding different portions of two sunflower homeodomain proteins were cloned in-frame in the expression vectors pRSET and pGEX-3X . When introduced into competent Escherichia coli cells and induced, the resulting plasmids directed the expression of large amounts (5-10% of total cellular protein) of the encoded polypeptides . As a rule, fusions in pRSET rendered insoluble proteins, while fusions in pGEX were soluble and could be purified in a single step by selective absorption onto glutathione-agarose beads, followed by elution with free glutathione . The purified proteins showed both glutathione S-transferase and DNA-binding activity, indicating that they retain their native conformation . The expression-purification protocol that was employed allowed the isolation of up to 0.7 mg of protein per gram of transformed cells . One of the fusion proteins, RH11 (which is a fusion of the homeodomain protein HAHR1 in pRSET), though insoluble, was able to bind DNA when spotted onto a nitrocellulose filter . This protein could also be simply purified in large amounts by electroelution from sodium dodecyl sulfate-polyacrylamide gels and used to elicit antibodies which recognized both the transgenic fusion and the native protein from sunflower nuclei . Our results clearly show that vector choice is a critical parameter for obtaining large amounts of a desired protein for particular purposes.

Protein Expr Purif, 1998 Jun, 13(1), 90 - 6
Overproduction, in Escherichia coli, of soluble taxadiene synthase, a key enzyme in the Taxol biosynthetic pathway; Huang KX et al.; Taxadiene synthase catalyzes the conversion of the universal precursor of diterpenoids, geranylgeranyl diphosphate, to taxadiene, a key intermediate in Taxol (paclitaxel) biosynthesis . The gene encoding taxadiene synthase was cloned recently . Here we report a method for the heterologous overexpression of cDNA encoding taxadiene synthase in Escherichia coli using a thioredoxin fusion expression system, which increases the solubility of expressed protein . Taxadiene synthase cDNA was amplified by polymerase chain reaction and then subcloned into pET3d and pET32a(+) to form pET3dTX and pET32TX, respectively . The expressed taxadiene synthase from E . coli BL21(DE3)/pET3dTX was present completely as inclusion bodies . The transformant E . coli BL21(DE3)/pET32TX produced a thioredoxin fusion taxadiene synthase (15-20% of total soluble protein) when induced with isopropyl beta-D-thiogalactopyranoside at low temperature (20 degrees C) . The recombinant enzyme was purified by a single step with a His-binding metal affinity column . The maximal production attained was 13 mg of purified, active fusion protein per 500 ml culture of E . coli BL21(DE3)/pET32TX . The purified recombinant taxadiene synthase fusion protein was similar to native protein in steady-state kinetic parameters and mobility on sodium sulfate-polyacrylamide gel electrophoresis . The protein purified from E . coli BL21(DE3)/pET3dTX had the expected N-terminal (AQLSFNA) sequence.

Protein Expr Purif, 1998 Jun, 13(1), 67 - 72
Presence of an N-terminal polyhistidine tag facilitates stable expression of an otherwise unstable N-terminal domain of mouse tissue inhibitor of metalloproteinase-1 in Escherichia coli; Rajan SS et al.; The active N-terminal domain of the mouse tissue inhibitor of metalloproteinases-1 is a 14.1-kDa polypeptide with three disulfide bonds . When expressed using a T7 system in Escherichia coli, this truncated protein, in contrast to the WT protein, was found only in trace amounts in the cell . However, when the coding sequence was placed downstream of a 60-bp sequence that encoded an in-frame histidine-rich "tag," the fusion product (NF.TIMP*His) was expressed in considerably increased abundance . WT.TIMP-1 was expressed in abundance with or without the tag . The mRNAs encoding the various forms of TIMP were present in similar amounts in all four cases . NF.TIMP*His, renatured and purified on a nickel affinity column, was found to be about 10-fold less effective than native human TIMP-2 at inhibiting cleavage of collagen type I by human fibroblast collagenase . A thrombin cleavage site in the tag was susceptible to cleavage by low levels of a contaminating proteinase.

Protein Expr Purif, 1998 Jun, 13(1), 61 - 6
Addition of a poly-(6X) His tag to Milk Bundle-1 and purification using immobilized metal-affinity chromatography; Grundy JE et al.; Milk Bundle 1 (MB-1) is a de novo designed protein enriched in M, T, K, and L . Its future application is as a high-quality dietary protein source for ruminants . The protein is currently expressed in Escherichia coli and is being characterized to solve its folded conformation . MB-1 has marginal stability at room temperature, which has hindered our attempts at characterization . To increase the stability of the protein at room temperature, the purification procedure was examined and changed to hopefully increase its effectiveness . We describe here the production and purification of a new MB-1 with six His residues at the C-terminal end . This allows the new mutant (MB-1-His) to bind metal ions and to be purified with immobilized metal-affinity chromatography (IMAC) . MB-1-His obtained using IMAC was purer on SDS-PAGE than both MB-1 or MB-1-His isolated using the current protocol . The IMAC protocol is more economical and more efficient; preliminary results show that the protein purified by this method is also quite stable at room temperature.

Protein Expr Purif, 1998 Jun, 13(1), 23 - 9
Large-scale preparation, purification, and crystallization of UDP-N-acetylmuramoyl-L-alanine: D-glutamate ligase from Escherichia coli; Auger G et al.; The UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase from Escherichia coli, an enzyme involved in the biosynthesis of the bacterial peptidoglycan monomer unit, was overproduced and purified to homogeneity on a large scale, yielding 4 mg of protein per liter of bacterial culture . Crystals of the complex with the substrate UDP-MurNAc-L-Ala were grown by the hanging drop method using ammonium sulfate as the precipitant . They are tetragonal with cell dimensions a = b = 65.5 A and c = 134.59 A, space group P4(1) or P4(3), and contain one monomer of 46,842 Da in the asymmetric unit . In order to use the multiple-wavelength anomalous diffraction method for phasing, a selenomethionine derivative of the protein has also been overproduced, purified, and crystallized.

Protein Expr Purif, 1998 Jun, 13(1), 1 - 8
Expression of maize gamma zein C-terminus in Escherichia coli; Ems-McClung SC et al.; Prolamins containing a highly conserved cysteine-rich C-terminal domain have been poorly expressed as soluble protein in model systems such as Escherichia coli . Possible reasons have included a combination of the reducing environment of the bacterial cytoplasm and protein secondary structure . Using a bacterial thioredoxin fusion expression system, full-length native gamma zein, native gamma zein C-terminus, and modified gamma zein C-terminus, containing 13 amino acid changes, were found to accumulate up to 58, 50, and 42% of the total cellular protein, respectively . The native gamma zein C-terminus fusion protein was six times more soluble (70%) than the full-length fusion protein (12%), four times more soluble than the N-terminus (19%), and eight times more soluble than the modified C-terminus (9%) . The modified C-terminal domain contained amino acid changes that improved the lysine, isoleucine, and tryptophan content, while removing two evolutionarily conserved cysteines and one nonconserved cysteine . Expression of the native C-terminal domain without thioredoxin resulted in decreased solubility (13%) and decreased expression (8%) . In contrast, coexpression with thioredoxin resulted in a sevenfold increase in solubility (86%) . These results suggest that insolubility of full-length gamma zein results from structural interactions of the N-terminus and that solubility of the C-terminal domain is dependent on proper disulfide bond formation . The ability to express the C-terminal domain of gamma zein as soluble protein should allow future identification of important structural elements in gamma zein and similar proteins.

Chin J Biotechnol, 1997, 13(4), 239 - 46
Fusion expression of green fluorescent protein and HCV capsid antigene in Escherichia coli cells; Yue L et al.; A chimeric gene of the green fluorescent protein (GFP) and hepatitis C virus (HCV) core antigene were constructed and expressed in E . coli cells . The expressed fusion protein was examined by Dot-ELISA and Western blot and the three antigenic determinants were detected . The GFP-Core fusion protein showed not only the striking green fluorescence under natural light but also the HCV antigenic activity . A new method of immunological diagnosis is greatly anticipated in the light of this fusion protein which can be seen as the HCV antigen tagged with the green fluorescent protein.

Chin J Biotechnol, 1997, 13(4), 233 - 8
High-level expression and purification of human pro-UK cDNA in Escherichia coli; Xue Y et al.; A chemically synthesized human pro-urokinase (pro-UK) CDNA was cloned into the expression vector PET-11d and expressed in E . coli BL21(DE3) pLysS under the control of the T7 promoter . Using 0.1 mmol/L IPTG induction, the expression level of the recombinant pro-UK attained up to 15% of the total bacterial proteins and existed as inclusion bodies . After denaturation and renaturation in vitro, the expressed pro-UK was purified to be identified by Zn2+ selective precipitation, immuno-affinity chromatography, and benzamidine affinity adsorption . The specific activity of the purified human pro-UK was about 110,000 IU/mg.

Cell Biochem Biophys, 1998, 29(1-2), 89 - 111
Expression and characterization of deletion recombinants of two cGMP-inhibited cyclic nucleotide phosphodiesterases (PDE-3); He R et al.; cDNAs encoding two PDE-3 or cyclic GMP-inhibited (cGI) cyclic nucleotide phosphodiesterase (PDE) isoforms, RPDE-3B (RcGIP1) and HPDE-3A (HcGIP2), were cloned from rat (R) adipose tissue and human (H) heart cDNA libraries . Deletion and N- and C-terminal truncation mutants were expressed in Escherichia coli in order to define their catalytic core . Active mutants of both RPDE-3B and HPDE-3A included the domain conserved among all PDEs plus additional upstream and downstream sequences . An RPDE-3B mutant consisting of the conserved domain alone and one from which the RPDE-3B 44-amino acid insertion was deleted exhibited little or no activity . All active recombinants exhibited a high affinity (< 1 microM) for cyclic AMP (cAMP) and cyclic GMP (cGMP), were inhibited by cAMP, cGMP, and cilostamide, but not by rolipram, and were photolabeled with {32P}-cGMP . The IC50 values for cGMP inhibition of cAMP hydrolysis were lower for HPDE-3A than for RPDE-3B recombinants . The deduced amino acid sequences of HPDE-3A and RPDE-3B catalytic domains are very similar except for the 44-amino acid insertion not found in other PDEs . It is possible that this insertion may not only distinguish PDE-3 catalytic domains from other PDEs and identify catalytic domains of PDE-3 subfamilies or conserved members of the PDE-3 gene family, but may also be involved in the regulation of sensitivity of PDE-3s to cGMP.

Arch Oral Biol, 1998 Mar, 43(3), 173 - 82
Production of rat salivary cystatin S variant polypeptides in Escherichia coli; Bedi GS et al.; Cystatins are protein inhibitors of papain and related cysteine proteinases . A series of continuous synthetic peptides corresponding to the entire sequence of rat salivary cystatin was used to localize the binding domains of the cystatin to papain . Several synthetic peptides, one from the aminoterminal sequence (peptide 1-24) and others from the carboxylterminal (peptides 66-79, 66-90, 79-90, 79-114), showed binding to papain, but none of the peptides showed inhibition of papain activity . Three recombinant rat salivary cystatin variants (N-terminal truncated protein lacking amino acid residues 1-9; variant 49-53, in which amino acid residues QVVAG of rat salivary cystatin had been replaced with amino acid residues LVL in mutant protein; and variant 65-78, in which amino acid residues 65-78 had been replaced with amino acids PG in mutant protein) were produced using the Escherichia coli expression system pGex-4T . To generate N-terminal truncated protein the desired coding region of the cystatin gene was amplified by polymerase chain reaction (PCR) . To produce the variants 49-53 and 65-78, a PCR-based approach of gene splicing by overlap extension was used . Recombinant cystatin proteins were produced as insoluble inclusion bodies as fusion proteins with a glutathione S-transferase (GST) carrier . After solubilization with urea the GST carrier was cleaved from the fusion protein with thrombin and cystatin variants purified by fast liquid chromatography on a MonoQ column . The purified proteins reacted with antibodies to rat salivary cystatin . The N-terminal truncated and variant 49-53 exhibited very little inhibitory activity towards papain, whereas variant 65-78 exhibited papain-inhibitory activity similar to the full-length recombinant cystatin.

AIDS, 1998 May 28, 12(8), 873 - 8
Pneumocystis carinii mutations associated with sulfa and sulfone prophylaxis failures in AIDS patients; Kazanjian P et al.; BACKGROUND: Failures of prophylaxis against Pneumocystis carinii pneumonia (PCP) in AIDS patients do occur, but no evidence for drug resistance has yet been presented . OBJECTIVE: To determine whether mutations in the sulfa and sulfone drug target are associated with failure of prophylaxis using a sulfa-containing agent . METHODS: Portions of the gene for P . carinii dihydropteroate synthase (DHPS), the sulfa and sulfone target, from 27 patients (20 of whom had AIDS) diagnosed with PCP between 1976 and 1997 were amplified using polymerase chain reaction and sequenced . Seven of the 27 patients (all of whom had AIDS) were receiving sulfa or sulfone drugs as prophylaxis for PCP . RESULTS: Mutations were found at only two amino-acid positions and were significantly more common in patients who received sulfa/sulfone prophylaxis . Mutations were observed in five (71%) out of seven isolates from AIDS patients receiving sulfa/sulfone as prophylaxis compared with only two (15%) out of 13 specimens from AIDS patients who did not (P = 0.022) . No mutations were seen in isolates from seven non-HIV-infected patients, none of whom were on prophylaxis . Mutations were only observed in specimens obtained in 1995-1997 . CONCLUSIONS: Mutations in two amino-acid positions were significantly more common in AIDS patients with PCP who failed sulfa/sulfone prophylaxis . These amino acids appeared to be directly involved in both substrate and sulfa binding, based on homology to the Escherichia coli DHPS crystal structure . Thus, the results were consistent with the possibility that mutations in the P . carinii DHPS are responsible for some of the failures of sulfa/sulfone prophylaxis in AIDS patients.

Cent Afr J Med, 1997 Nov, 43(11), 316 - 21
Contamination of traditional drinking water sources during a period of extreme drought in the Zvimba communal lands, Zimbabwe; Moran P et al.; OBJECTIVES: Diarrhoeal disease is a significant public health concern in Zimbabwe, particularly for the population living in rural settings . The present study was undertaken to investigate the quality of water in a rural area of Zimbabwe during a period of extreme drought . DESIGN AND SETTING: A cross sectional survey study design was used . During the month of July 1995, water samples were collected from various actively used sources in the Zvimba communal lands, Zimbabwe . MAIN OUTCOME MEASURES: The level of contamination was estimated by use of the membrane filtration technique to detect the presence of Escherichia coli . RESULTS: Mean concentrations of E . coli found in boreholes and piped water were 9.3 and zero colonies per 100 ml, respectively . Using standardized criteria to define suitable drinking water quality, borehole and piped sources were determined to be more likely to provide satisfactory drinking water . CONCLUSIONS: Water samples collected from semi-protected and unprotected wells, which serve the majority of the population in the study area, were found to be unsatisfactory for drinking (range from two to 1,960 colonies of E . coli per 100 ml) . Included are suggestions on how to efficiently utilize available water.

Nat Biotechnol, 1996 Jul, 14(7), 872 - 4
In vitro hydrogen production by glucose dehydrogenase and hydrogenase; Woodward J et al.; A new in vitro enzymatic pathway for the generation of molecular hydrogen from glucose has been demonstrated . The reaction is based on the oxidation of glucose by Thermoplasma acidophilum glucose dehydrogenase with the concomitant oxidation of NADPH by Pyrococcus furiosus hydrogenase . Stoichiometric yields of hydrogen were produced from glucose with the continuous recycling of cofactor . This simple system may provide a method for the biological production of hydrogen from renewable sources . In addition, the other product of this reaction, gluconic acid, is a high-value chemical commodity.

Nat Biotechnol, 1996 Jun, 14(6), 751 - 5
Affinity-assisted in vivo folding of a secreted human peptide hormone in Escherichia coli; Samuelsson E et al.; We show that coexpression of a specific binding protein in Escherichia coli can significantly improve the relative yields of correctly folded human insulin-like growth factor I (IGF-I) . A glutathione redox buffer was used during growth to allow formation and breakage of disulfide bonds in the periplasm of the bacterial host . Both the binding protein and the peptide hormone were produced as affinity fusions, which allowed purification of the in vivo formed heterodimer by alternative affinity purification methods . The use of affinity-assisted in vivo folding has general implications for expression, folding, and purification of recombinant proteins.

Nat Biotechnol, 1996 May, 14(5), 629 - 34
Translational level is a critical factor for the secretion of heterologous proteins in Escherichia coli; Simmons LC et al.; A method for enhancing the secretion of heterologous proteins in Escherichia coli by optimizing, as opposed to simply maximizing, the translational level of a given protein is described . Random alteration of the translational initiation region (TIR) of the Heat-Stable Enterotoxin II (STII) signal sequence resulted in a library of vectors with varied translational strengths . Subsequent screening of this library using E . coli alkaline phosphatase as a reporter led to the selection of several TIR variants covering a 10-fold range of translational strength . These TIR variants, in combination with several previously generated variants, are shown to dramatically improve the secretion of a number of heterologous proteins . In fact, the heterologous proteins tested required a narrow translational range for optimal high-level secretion into the periplasm . Interestingly, the secretion of two native E . coli proteins was unaffected by TIR strength when tested over an identical range . The dependence of secretion on a narrow translational level demonstrates its critical role in the secretion of heterologous proteins in E . coli.

Nat Biotechnol, 1996 Apr, 14(4), 476 - 80
Effect of disulfide bonds on the structure, function, and stability of the trypsin/tPA inhibitor from Erythrina caffra: site-directed mutagenesis, expression, and physiochemical characterization; Lehle K et al.; Erythrina trypsin/tPA inhibitor (ETI) from the seeds of Erythrina caffra retains its native structure and inhibitory function after reducing its two disulfide bonds . In order to elucidate the specific role of these crosslinks, alanine residues were substituted for cysteines after cloning the gene in Escherichia coli . Expression of the recombinant inhibitor and the substitution mutants, C83A, CC39, 83AA, and CC132, 139AA, led to inclusion bodies . After solubilization in guanidinium-chloride (GdmCl)/dithiothreitol and oxidation in glutathione buffer, activity could be recovered at yields up to 80% . The mutant proteins exhibit full inhibitory function without detectable alterations of their native structure . However, their stability is reduced: at acid pH, where the oxidized natural inhibitor retains its native structure, the reduced wildtype protein and the mutants undergo at least partial denaturation, reflected by decreased pH ranges of stability: pH 5-7 for the reduced inhibitor, pH 2.5-8.5 for CC132, 139AA, and pH 3.5-8.5 for C83A and CC39, 83AA . Urea and GdmCl denaturation at pH 7 show hysteresis for both the oxidized inhibitor and the double mutant CC132, 139AA . In contrast, the reduced protein and the other mutants exhibit true equilibrium transitions at pH 7, with urea half-concentrations of 0.9 M and 1.9 M and GdmCl half-concentrations of 0.5 M and 1.0 M, respectively . The stability of Erythrina trypsin/tPA inhibitor follows the sequence: oxidized ETI > CC132, 139AA > CC39, 83AA and C83A > reduced ETI.

Nat Biotechnol, 1996 Mar, 14(3), 329 - 34
Improved refolding of an immobilized fusion protein; Stempfer G et al.; Fusion proteins of monomeric alpha-glucosidase from Saccharomyces cerevisiae containing N- or C-terminal hexa-arginie peptides were expressed in the cytosol of Escherichia coli in soluble form . The polycationic peptide moieties allow noncovalent binding of the denatured fusion proteins to a polyanionic solid support . Upon removal of the denaturant, refolding of the matrix-bound protein can proceed without perturbation by aggregation . However, nonspecific interactions of the denatured polypeptide, or of folding intermediates, with the matrix cause a drastic decrease in renaturation under suboptimal folding conditions . At low salt concentrations, ionic interactions of the refolding polypeptide with the matrix result in lower yields of renaturation . At higher salt concentrations, renaturation is prevented by hydrophobic interactions with the matrix . Apart from ionic strength, renaturation of the denatured matrix-bound fusion protein must be optimized with respect to pH, temperature, cosolvents, and matrix material used . Under optimum conditions, immobilized alpha-glucosidase can be renatured with a high yield at protein concentrations up to 5 mg/ml, whereas folding of the wild-type enzyme in solution is feasible only at an extremely low protein concentration (15 micrograms/ml) . Thus, folding of the immobilized alpha-glucosidase allows an extremely high yield of the renaturated model protein . The technology should be applicable to other proteins that tend to aggregate during refolding.

Nat Biotechnol, 1996 Mar, 14(3), 315 - 9
Improved green fluorescent protein by molecular evolution using DNA shuffling; Crameri A et al.; Green fluorescent protein (GFP) has rapidly become a widely used reporter of gene regulation . However, for many organisms, particularly eukaryotes, a stronger whole cell fluorescence signal is desirable . We constructed a synthetic GFP gene with improved codon usage and performed recursive cycles of DNA shuffling followed by screening for the brightest E . coli colonies . A visual screen using UV light, rather than FACS selection, was used to avoid red-shifting the excitation maximum . After 3 cycles of DNA shuffling, a mutant was obtained with a whole cell fluorescence signal that was 45-fold greater than a standard, the commercially available Clontech plasmid pGFP . The expression level in E . coli was unaltered at about 75% of total protein . The emission and excitation maxima were also unchanged . Whereas in E . coli most of the wildtype GFP ends up in inclusion bodies, unable to activate its chromophore, most of the mutant protein is soluble and active . Three amino acid mutations appear to guide the mutant protein into the native folding pathway rather than toward aggregation . Expressed in Chinese Hamster Ovary (CHO) cells, this shuffled GFP mutant showed a 42-fold improvement over wildtype GFP sequence, and is easily detected with UV light in a wide range of assays . The results demonstrate how molecular evolution can solve a complex practical problem without needing to first identify which process is limiting . DNA shuffling can be combined with screening of a moderate number of mutants . We envision that the combination of DNA shuffling and high throughput screening will be a powerful tool for the optimization of many commercially important enzymes for which selections do not exist.

Biochim Biophys Acta, 1998 Jun 11, 1385(1), 157 - 64
Conformational stability studies of the pleckstrin DEP domain: definition of the domain boundaries; Kharrat A et al.; Pleckstrin is the major substrate of protein kinase C in platelets . It contains at its N- and C-termini two pleckstrin homology (PH) domains which have been proposed to mediate protein-protein and protein-lipid interactions . A new module, called DEP, has recently been identified by sequence analysis in the central region of pleckstrin . In order to study this module, several recombinant polypeptides corresponding to the DEP module and N- and C-termini extended forms have been expressed . Using circular dichroism (CD) and nuclear magnetic resonance (NMR) techniques, the domain boundaries have been determined that yield a soluble and folded pleckstrin DEP domain . This comprises 93 amino acids with an alpha/beta fold in agreement with secondary structure predictions . Stability studies indicate that the regions surrounding the DEP domain do not contribute to its stability suggesting that the phosphorylation sites at S113, T114 and S117 are in an unstructured region . Identification of the regions of pleckstrin that are folded shall facilitate determination of its structure and function .

Mutat Res, 1998 May 11, 414(1-3), 149 - 56
4-Chloro-o-phenylenediamine: a 26-week oral (in feed) mutagenicity study in Big Blue mice; Suter W et al.; 4-Chloro-o-phenylenediamine (4-C-o-PDA) is a liver carcinogen in mice and was found to be weakly mutagenic in the liver of female Big Blue mice after short term treatment . In the present study the test compound was given subchronically in the diet for 26 weeks at doses of 0, 5000 and 10,000 ppm . The corresponding average test substance intake was 2166 mg kg-1 day-1 (males: 1794 mg kg-1 day-1; females: 2539 mg kg-1 day-1) and 4610 mg kg-1 day-1 (males: 3926 mg kg-1 day-1; females 5925 mg kg-1 day-1) at the low and high dose, respectively . After sacrifice, tissues were flash frozen in liquid nitrogen . The lacI mutant frequency in the liver was determined from three male and three female mice per dose group . The genomically integrated transgene was recovered by packaging into lambda phage using Transpack packaging extract (Stratagene, La Jolla, USA) followed by infection of Escherichia coli strain SCS-8 . Blue mutant plaques were scored against a background of clear non-mutant plaques . Food consumption decreased initially at 10,000 ppm, while no treatment related effect on food intake was observed at 5000 ppm . Body weight gain was found to be decreased in all treated animals . Absolute and relative liver weight increased in a dose-related manner, but only the latter effect was statistically significant . A clear dose dependent increase in lacI mutant frequencies was observed in the liver of both sexes . The following mutant frequencies (x10(-5)) were observed: 2.73+/-1.01 (males, untreated), 7.24+/-1.50 (females, untreated), 18.91+/-5.30 (5000 ppm, males), 24.91+/-7.58 (5000 ppm, females), 20.47+/-6.68 (10,000 ppm, males) and 36.17+/-14.98 (10,000 ppm, females) . It is therefore concluded that 4-C-o-PDA is a strong mutagen in the liver of mice treated subchronically for 26 weeks .

Gene, 1998 Jun 15, 213(1-2), 93 - 100
Control of expression by the cellulose synthase (bcsA) promoter region from Acetobacter xylinum BPR 2001; Nakai T et al.; The 5' upstream region (about 3.1kb) of the cellulose synthase operon (bcs operon) has been isolated by cloning from Acetobacter xylinum strain BPR 2001 . The expression level of the upstream region was determined using sucrose synthase cDNA as a reporter gene in the shuttle vector pSA19 . The expression occurred with the 1.1-kb upstream sequence from the ATG start codon of the bcs operon but not with the 241-bp upstream sequence in A . xylinum, although neither the 1.1-kb nor the 241-bp upstream sequence caused any expression as a promoter in Escherichia coli . The level of expression with the 1 . 1-kb upstream sequence in A . aceti was 75% of that in A . xylinum . These results suggest that the upstream region functions as a specific promoter for the Acetobacter genus . The expression was reduced by the introduction of the 241-bp upstream region between the lac promoter and the reporter gene in E . coli and was not detected in A . xylinum . This suggests that the short upstream region composed of 241bp contains the site(s) which causes a negative regulation on the transcription for bcs operon . The production of recombinant protein with the ribosome-binding site (RBS) of A . xylinum obtained from the bcs operon, was reduced to about half in E . coli, and that with the site of the lac promoter was also reduced to about half in A . xylinum . This shows that a species-specific predominance occurs during interaction between mRNA and 16S rRNA in the RBS between A . xylinum and E . coli .

Gene, 1998 Jun 15, 213(1-2), 83 - 91
Accurate insertional inactivation of lacZalpha: construction of pTrueBlue and M13TrueBlue cloning vectors; Slilaty SN et al.; Color selection plasmid and phage vectors based on insertional inactivation of the lacZalpha gene fragment have been in wide use for nearly two decades . The originals (the pUC and M13mp series) and all subsequent derivatives of these vectors contain the same mechanism for insertional inactivation of lacZalpha placing the sites for gene interruption between the initiator ATG codon and codon 7, a region that encodes a functionally nonessential part of the lacZalpha-complementation peptide . Numerous observations found in the literature and made in our laboratory suggest that the color selection process in these vectors does not function reliably and that erroneous conclusions about the success of cloning experiments are frequently drawn . We have studied the efficiency of lacZalpha inactivation by performing insertions at different intervals along the entire length of the proximal 60-amino-acid (aa)-coding region of the lacZ gene (lacZalpha) . Our results show that accurate and reliable inactivation of lacZalpha occurs only when insertions are made within the DNA region that encodes aa 11-36 . Based on these results, novel color selection plasmid and phage vectors, pTrueBlue and M13TrueBlue, have been constructed . Application of these vectors for gap-free shotgun sequencing of genomic DNA is discussed .

Gene, 1998 Jun 15, 213(1-2), 37 - 46
A shuttle mutagenesis system for tagging genes in the yeast Yarrowia lipolytica; Neuveglise C et al.; A shuttle mutagenesis system was developed for the dimorphic yeast Yarrowia lipolytica . This system combines transposon insertions generated in Escherichia coli with the transformation of yeast with the Tn-mutagenized DNA . The mini-transposon mTn-3xHA/GFP, used in Saccharomyces cerevisiae for producing stable insertions, was adapted for use in the yeast Y . lipolytica . The mTnYl1 transposon (for mini-Tn of Y . lipolytica) confers resistance to tetracycline in E . coli . It also contains the Y . lipolytica URA3 gene for selection of yeast transformants, and the coding sequence for the S65T mutant form of GFP . The rare cutter endonuclease, I-SceI, restriction site, which enables identification of the chromosomal localization of mutagenized genes, was also incorporated . mTnYl1 was first tested on the ACO1 gene, which encodes an Acyl CoA oxidase isozyme . The mutagenesis system was further validated on a Y . lipolytica genomic DNA library constructed in a pHSS6 derivative vector . Mutants with a particular morphology or defective for alkane, fatty acids and oil degradation were obtained .

Biochim Biophys Acta, 1998 Jun 11, 1385(1), 17 - 32
Antibodies specific for (6-4) DNA photoproducts: cloning, antibody modeling and construction of a single-chain Fv derivative; Morioka H et al.; We have investigated a series of four monoclonal antibodies that specifically recognize pyrimidine (6-4) pyrimidone photoproducts . One of these antibodies (64M4), bound all four possible pyrimidine-pyrimidone photoadducts with equal affinities whereas the others (64M2, 64M3 and 64M5) were selective for TC and TT sequences . In addition, 64M5 had the highest binding affinity for photodamaged DNA of the four {T . Mori et al., Photochem . Photobiol . 54 (1991) 225-232} . To help understand the differences between these antibodies, we have cloned and sequenced the variable region genes from all four . Comparing these sequences revealed that all four were highly similar to one another, although there were some differences in potential antigen-contact regions . To assess the influences of these sequence differences at the structural level, computer models were constructed for all four antibodies . Most of the sequence differences occurred in potential antigen contact regions, suggesting specific positions that might account for the observed differences in binding affinities and selectivities . A single-chain Fv derivative of 64M5 was therefore constructed and characterized to provide an experimental system in which structure-function relationships can be tested . This derivative could be isolated from Escherichia coli using two chromatographic steps and possessed the same binding specificity as the parent monoclonal antibody .

Oncol Rep, 1998 Jul-Aug, 5(4), 793 - 7
Adeno-associated virus vector mediated transduction of primary normal human breast epithelial cells; Yang J et al.; Cultured human breast epithelial cells from reduction mammoplasty specimens were transduced using an adeno-associated virus vector encoding the marker gene E . coli -galactosidase . Subconfluent, growing, breast epithelial cells were more easily transduced than confluent, quiescent, cells . Transduction of non-dividing confluent cells could be greatly increased by ultraviolet light-induced DNA damage or by prior exposure to the DNA synthesis inhibitor hydroxyurea . The effects of ultraviolet light and hydroxyurea on transduction were additive when these agents were applied together.

Biochemistry, 1998 Jun 9, 37(23), 8605 - 13
Species-specific differences in the operational RNA code for aminoacylation of tRNAPro; Stehlin C et al.; An operational RNA code relates amino acids to specific structural features located in tRNA acceptor stems . In contrast to the universal nature of the genetic code, the operational RNA code can vary in evolution due to coadaptations of the contacts between aminoacyl-tRNA synthetases and the acceptor stems of their cognate tRNA substrates . Here we demonstrate that, for class II prolyl-tRNA synthetase (ProRS), functional coadaptations have occurred in going from the bacterial to the human enzyme . Analysis of 20 ProRS sequences that cover all three taxonomic domains (bacteria, eucarya, and archaea) revealed that the sequences are divided into two evolutionarily distant groups . Aminoacylation assays showed that, while anticodon recognition has been maintained through evolution, significant changes in acceptor stem recognition have occurred . Whereas all tRNAPro sequences from bacteria strictly conserve A73 and C1.G72, all available cytoplasmic eukaryotic tRNAPro sequences have a C73 and a G1.C72 base pair . In contrast to the Escherichia coli synthetase, the human enzyme does not use these elements as major recognition determinants, since mutations at these positions have only small effects on cognate synthetase charging . Additionally, E . coli tRNAPro is a poor substrate for human ProRS, and the presence of the human anticodon-D stem biloop domain was necessary and sufficient to confer efficient aminoacylation by human ProRS on a chimeric tRNAPro containing the E . coli acceptor-TpsiC stem-loop domain . Our data suggest that the two ProRS groups may reflect coadaptations needed to accommodate changes in the operational RNA code for proline.

Biochemistry, 1998 Jun 9, 37(23), 8584 - 94
Involvement of carboxy-terminal amino acids in secretion of human lysosomal protease cathepsin L; Chauhan SS et al.; Cathepsin L, a lysosomal cysteine protease, is overexpressed and secreted by malignantly transformed cells . However, the reason for secretion of this man 6-phosphate-containing lysosomal protease into the extracellular medium is not clear . We wished to determine whether there is a region within the primary sequence of the proenzyme form of cathepsin L which affects its subcellular and extracellular localization . High-level transient expression of human procathepsin L in mouse NIH 3T3 cells results in the secretion of most of this protein into the extracellular medium . At the same time, the endogenous mouse procathepsin L in these nontransformed cells is found in its usual location in lysosomes . Mutants of human procathepsin L with carboxy-terminus deletions involving the last 11 amino acids are not secreted into the medium . Deletion of as little as two amino acids, Thr and Val, from the carboxy terminus, blocked the secretion of the protein but did not affect its enzyme activity, posttranslational processing, or subcellular distribution . Replacement of Thr-Val by two bulky amino acids Tyr-Asn allowed secretion of the procathepsin L, but the replacement of these two amino acids by nonbulky alanines prevented its secretion . Single alanine substitutions of the last six amino acids (ASYPTV) indicated that substitution by alanine of Y or T does not affect the secretion of hproCAT L, but alanine substitutions of S, P, or V completely blocked its secretion into the culture medium . We therefore conclude that the carboxy terminus of procathepsin L contains a sequence essential for its secretion.

Biochemistry, 1998 Jun 9, 37(23), 8498 - 507
In vitro disulfide-coupled folding of guanylyl cyclase-activating peptide and its precursor protein; Hidaka Y et al.; Guanylyl cyclase-activating peptide II (GCAP-II), an endogenous ligand of particulate guanylyl cyclase C (GC-C), is processed from the precursor protein and circulates in human blood . GCAP-II consists of 24 amino acid residues and contains two disulfide bridges . The correct disulfide paring of GCAP-II is an absolute requirement for its biological activity . This study shows that the folding of the peptide from the reduced form yields a peptide with the native disulfide paring as a minor product and with non-native ones as major products, regardless of the presence or absence of reduced and oxidized glutathione . The results suggest that GCAP-II does not possess sufficient information to permit the adoption of the native conformation and to effectively form the correct disulfide pairing and, as a result, that GCAP-II is correctly folded by assistance of a factor(s) such as an intra- or intermolecular chaperone . We studied whether a peptide in the pro-leader sequence of the precursor protein (proGCAP-II) contains sufficient information to facilitate the folding of GCAP-II . For this purpose, we prepared proGCAP-II in Escherichia coli by a recombinant technique and examined the disulfide-coupled folding of proGCAP-II from the reduced form . proGCAP-II was quantitatively recovered with the correctly folded structure from the reduced form both in the presence and in the absence of reduced and oxidized glutathione . The protein contains only disulfide linkages at the same positions as the mature form of proGCAP-II, GCAP-II, and the biologically active isomer of GCAP-II in the molecule . These results provide evidence that the propeptide of proGCAP-II is a critical factor in the formation of the correct disulfide paring in the folding of the protein.

Biochemistry, 1998 Jun 9, 37(23), 8417 - 25
Plant polyketide synthases: a chalcone synthase-type enzyme which performs a condensation reaction with methylmalonyl-CoA in the biosynthesis of C-methylated chalcones; Schroder J et al.; Heterologous screening of a cDNA library from Pinusstrobus seedlings identified clones for two chalcone synthase (CHS) related proteins (PStrCHS1 and PStrCHS2, 87.6% identity) . Heterologous expression in Escherichia coli showed that PStrCHS1 performed the typical CHS reaction, that it used starter CoA-esters from the phenylpropanoid pathway, and that it performed three condensation reactions with malonyl-CoA, followed by the ring closure to the chalcone . PstrCHS2 was completely inactive with these starters and also with linear CoA-esters . Activity was detected only with a diketide derivative (N-acetylcysteamine thioester of 3-oxo-5-phenylpent-4-enoic acid) that corresponded to the CHS reaction intermediate postulated after the first condensation reaction . PstrCHS2 performed only one condensation, with 6-styryl-4-hydroxy-2-pyrone derivatives as release products . The enzyme preferred methylmalonyl-CoA against malonyl-CoA, if only methylmalonyl-CoA was available . These properties and a comparison with the CHS from Pinus sylvestris suggested for PstrCHS2 a special function in the biosynthesis of secondary products . In contrast to P . sylvestris, P . strobus contains C-methylated chalcone derivatives, and the methyl group is at the position predicted from a chain extension with methylmalonyl-CoA in the second condensation of the biosynthetic reaction sequence . We propose that PstrCHS2 specifically contributes the condensing reaction with methylmalonyl-CoA to yield a methylated triketide intermediate . We discuss a model that the biosynthesis of C-methylated chalcones represents the simplest example of a modular polyketide synthase.

J Biochem (Tokyo), 1998 May, 123(5), 924 - 31
Identification of the catalytic triad residues of porcine liver acylamino acid-releasing enzyme; Mitta M et al.; Acylamino acid-releasing enzyme (AARE) {EC 3.4.19.1} is a tetrameric serine protease, which belongs to the oligopeptidase family and specifically removes acetyl amino acids from N-terminally acetylated peptides . By using diisopropyl fluorophosphate, we previously identified one of the residues comprising the catalytic triad of this enzyme as Ser587 {Miyagi, M . et al . (1995) J . Biochem . 118, 771-779} . To elucidate the other two residues forming the catalytic triad of this new serine-type protease, wild-type and four mutant AAREs, in which each candidate residue of the catalytic triad deduced from sequence alignment with other oligopeptidases was substituted by site-directed mutagenesis, were expressed in Escherichia coli as fusion proteins with short peptide chains at both N- and C-termini of a subunit of porcine liver enzyme . All of the recombinant AAREs were estimated to have similar conformational and quaternary structures to the native porcine liver enzyme from their CD spectra and behavior on gel-filtration, but the mutants in which Ala587, Asn675, or Tyr707 was substituted for Ser587, Asp675, or His707, respectively, did not show detectable hydrolytic activity toward acetyl-L-methionyl L-alanine . These facts suggest that Ser587, Asp675, and His707 are essential residues for the AARE activity and comprise the catalytic triad of the enzyme in this order . Thus, AARE has been shown to have a protease-like domain in its C-terminal region, as do other proteins classified as members of the oligopeptidase family.

J Biochem (Tokyo), 1998 May, 123(5), 891 - 8
Cloning and expression of the gene encoding flavodoxin from Desulfovibrio vulgaris (Miyazaki F); Kitamura M et al.; The gene encoding a flavodoxin of Desulfovibrio vulgaris (Miyazaki F) was cloned, and overexpressed in Escherichia coli . A 1.6-kbp DNA fragment, isolated from D . vulgaris (Miyazaki F) by double digestion with SalI and EcoRI, contained the flavodoxin gene and its regulatory region . An expression system for the flavodoxin gene under control of the T7 promoter was constructed in E . coli . The purified protein was soluble and exhibited a characteristic visible absorption spectrum . HPLC analysis of the recombinant flavodoxin revealed the presence of an identical FMN to that found in the native D . vulgaris flavodoxin, and its dissociation constant with FMN was determined to be 0.38 nM . In vitro H2 reduction analysis indicated that the recombinant flavodoxin is active, and its redox potential was determined to be E1 = -434 and E2 = -151 mV using methyl viologen and 2-hydroxy-1,4-naphthoquinone, respectively . Its redox behavior was also examined with the recombinant flavodoxin adsorbed onto a graphite electrode . The mutant, A16E, was also produced, which revealed the feature of a conserved Glu residue at the surface of the molecule.

J Biochem (Tokyo), 1998 May, 123(5), 839 - 46
Effects of point mutations at the flexible loop alanine-145 of Escherichia coli dihydrofolate reductase on its stability and function; Ohmae E et al.; To elucidate the role of a flexible loop (residues 142-149) in the stability and function of Escherichia coli dihydrofolate reductase, alanine-145 in this loop was substituted by site-directed mutagenesis with ten amino acids (Glu, Phe, Gly, His, Ile, Leu, Arg, Ser, Thr, and Val) . The amount of three mutant proteins (A145E, A145I, and A145L) in cells was too small to allow the measurement of circular dichroism (CD) spectra and urea unfolding . The CD spectra of other seven mutants were identical with those of the wild-type DHFR, indicating that the native conformation of DHFR was not affected by the mutations.The free energy change of unfolding by urea decreased with an increase in the hydrophobicity of amino acid residues introduced, A145T>A145R>A145G>=A145S>=A145H>A145V++ +>wild-type>=A145F . The steady-state kinetic parameters for the enzyme reaction, Km and ksub, were only slightly influenced by the mutations . These results suggest that site 145 in the flexible loop plays an important role in the stability but has little or no effect on the native structure and function of this enzyme . The characteristics of the mutations are discussed in comparison with those of mutations at site 67 {Ohmae et al . (1996) J . Biochem . 119, 703-710} and at site 121 {Gekko et al . (1994) J . Biochem . 116, 34-41} in two other flexible loops.

Cell, 1998 May 29, 93(5), 897 - 908
IHF modulation of Tn10 transposition: sensory transduction of supercoiling status via a proposed protein/DNA molecular spring; Chalmers R et al.; Architectural protein IHF modulates Tn10 transposition in vitro . IHF stimulates transposon excision . Also, separately, IHF forces transposon end/target DNA interactions into a constrained pathway, "channeling," that yields only unknotted intratransposon inversion circles . Negative supercoiling influences both effects, differently . We infer that IHF is an architectural catalyst: it promotes initial transpososome assembly and is then ejected from the transpososome . IHF then rebinds, altering transpososome conformation to promote channeling . We also infer that the developing transpososome is a molecular spring: DNA provides basic elasticity; a conformational change in transposase provides force; and IHF and/or supercoiling provide conformational inputs . In vivo, IHF is a sensory transducer of chromosomal supercoiling status: with supercoiling absent, IHF is "supercoiling relief factor"; with supercoiling present, stimulation and channeling comprise a homeostatic pair such that modest changes in chromosome condition strongly influence transpositional outcome.

Electrophoresis, 1998 May, 19(5), 837 - 44
Extraction of membrane proteins by differential solubilization for separation using two-dimensional gel electrophoresis; Molloy MP et al.; We describe the extraction and enrichment of membrane proteins for separation by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) after differential solubilization of an Escherichia coli cell lysate . In a simple three-step sequential solubilization protocol applicable for whole cell lysates, membrane proteins are partitioned from other cellular proteins by their insolubility in solutions conventionally used for isoelectric focusing (IEF) . As the first step, Tris-base was used to solubilize many cytosolic proteins . The resultant pellet was then subjected to conventional solubilizing solutions (urea, 3-{(3-cholamidopropyl)dimethylammonio}-1-propanesulfonate, dithiothreitol, Tris, carrier ampholytes) . Following the completion of this step, 89% of the initial E . coli sample mass was solubilized . Finally, the membrane protein rich pellet was partially solubilized using a combination of urea, thiourea, tributyl phosphine and multiple zwitterionic surfactants . Using N-terminal sequence tagging and peptide mass fingerprinting we have identified 11 membrane proteins from this pellet . Two of these outer membrane proteins (Omp), OmpW and OmpX, have previously been known only as an open reading frame in E . coli, while OmpC, OmpT and OmpTOLC have not previously been identified on a 2-D gel . The prefractionation of an entire cell lysate into multiple fractions, based on solubility, results in simplified protein patterns following 2-D PAGE using broad-range pH 3.5-10 immobilized pH gradients (IPGs) . Additional advantages of sample prefractionation are that protein identification and gel matching, for database construction, is a more manageable task, the procedure requires no specialized apparatus, and the sequential extraction is conducted in a single centrifuge tube, minimizing protein loss.

Rev Argent Microbiol, 1998 Jan-Mar, 30(1), 13 - 9
{Accumulation of actin and adhesion to HEp-2 cells of strains of Escherichia coli isolated from children with diarrhea in Mendoza, Argentina}; Ortiz A et al.; E . coli strains are the major bacterial cause of diarrhea among children under 2 years of age residing in Mendoza, Argentina . Detection of diarrheogenic E . coli is made after coproculture, by agglutination tests using O-group antisera including most enteropathogenic E . coli (EPEC) serogroups and others often isolated in diarrhea . Although there are many O serogroups and H serotypes of E . coli strongly associated with infantile diarrhea, a number of studies have shown differences in the rate of isolation of EPEC between cases and controls using DNA probes . We compared the diagnosis of EPEC infections by traditional serogrouping tests with other detection methods using cell culture, involving the screening of isolates for adherence patterns to HEp-2 cells . A total of 140 isolates from children less than 24 months old with acute, persistent and chronic diarrhea and 40 isolates from controls were recovered . Three distinct patterns of adherence, termed localised (LA), diffuse (DA) and aggregative (EAgg) adherence were found . The fluorescence actin staining assay was used as indicative of the ability of some EPEC strains that produce attaching-effacing (A/E) lesions . Positive serogrouping strains were strongly associated with adherence (P = 0.0001) . LA adherence pattern occurred in 11% of cases with acute diarrhea associated with these serogroups (P = 0.001) and children under 12 months (P = 0.0001) . The FAS test was positive in 80% of them . EAgg adherence was found only in patients (20% P = 0.0001) and DA occurred both in cases (29%) and controls (2.5% P = 0.0001) . Diagnosis of EPEC infections has traditionally been performed by identifying organisms belonging to a number of serogroups or serotypes epidemiologically linked to diarrhea . Evidence is presented in this paper to show that pathogenicity is not restricted to serogroups . Isolation of many adherent strains not belonging to traditional EPEC O serogroups, shows the need for alternative methods to be used to detect and identify E . coli.

Andrologia, 1998, 30 Suppl 1, 35 - 9
Methods for the detection of male genital tract inflammation; Wolff H; Male genital tract inflammation is reflected by increased numbers of white blood cells (WBC) in semen . An ejaculate containing more than 10(6) WBC ml-1 semen is termed leukocytospermic . Among male infertility patients, the frequency of leukocytospermia is between 10% and 20% . By conventional light microscopy or sperm staining techniques, it is not possible to reliably differentiate WBC from immature germ cells in semen . In contrast, the cytochemical peroxidase method reliably identifies granulocytes, the most prevalent WBC type in semen . The method is cheap, fast and easy to perform . The gold standard for the detection of all WBC populations in semen is immunocytology using monoclonal antibodies . However, it is expensive and time-consuming, thus remaining a research tool at present . The measurement of granulocyte elastase in semen provides information on the number of granulocytes and their inflammatory activation . However, commercial granulocyte elastase enzyme immunoassays are expensive and due to logistical reasons often delay the results for more than 1 week . Leukocyte esterase dipstick tests lack both sensitivity and specificity for the detection of inflammatory changes in semen . For clinical purposes, the peroxidase method is ideally suited to detect inflammatory changes in semen.

J Nutr, 1998 Jun, 128(6), 947 - 53
Weaning and the weanling diet influence the villous height and crypt depth in the small intestine of pigs and alter the concentrations of short-chain fatty acids in the large intestine and blood; van Beers-Schreurs HM et al.; Effects of weaning pigs to different diets have been investigated in terms of the changes in the small intestinal morphology, and in the absorption of short-chain fatty acids (SCFA) and sodium from the large intestine . One piglet from each of six litters containing nine pigs was sampled on the day of weaning; the other eight piglets were divided into four equal groups and fed different diets as follows: unweaned, weanling diet, or sow's milk at high or low level . Four and seven days after weaning, measurements of the intestinal tissue and contents were made; the plasma concentrations of SCFA, aldosterone and sodium were also measured . The villous height in the small intestine was highest in the unweaned group and greater in the high milk group than in either the weanling diet or low milk group (P < 0.001) . Apparently, villous atrophy was due more to the level of feed intake than to the composition of the diet . The concentrations of SCFA in the large intestine and portal blood were highest in the weanling diet group and lowest in the low milk group . The low milk group tended to have higher blood concentrations of aldosterone (P = 0.15), which may have compensated for the low concentrations of SCFA in maintaining a higher percentage of dry matter in the intestine . Pigs fed weanling diet may use the energy from the SCFA to maintain a body weight comparable to that of pigs fed milk at a low level.

Arch Microbiol, 1998 Jun, 169(6), 491 - 6
Exponential growth of Escherichia coli B/r during its division cycle is demonstrated by the size distribution in liquid culture; Trueba FJ et al.; The Collins and Richmond equation was used to analyze the growth of individual bacterial cells . Birth size was derived from the size of deeply constricted cells in the sample . The analysis was applied to normalized and pooled data from electron micrographs of Escherichia coli showing that cellular length, surface, and volume do not grow linearly as reported before . We present evidence that bacteria grow exponentially during the division cycle, which is consistent with previous proposals . Our results confirm previous incorporation studies that demonstrate basically exponential growth patterns for cell mass during the division cycle.

Arch Microbiol, 1998 Jun, 169(6), 483 - 90
Iron regulates transcription of the Escherichia coli ferric citrate transport genes directly and through the transcription initiation proteins; Angerer A et al.; Ferric citrate induces transcription of the ferric citrate transport genes fecABCDE in Escherichia coli by binding to the outer-membrane receptor protein FecA without entering the cell . Replete iron concentrations inhibit transcription of the fec transport system via the iron-loaded Fur repressor . Here we show that the Fur repressor activated by Mn2+ (used instead of Fe2+) binds to the promoter of the regulatory genes fecIR and to the promoter of fecABCDE . DNase I footprint analysis revealed that Mn2+-Fur (50 nM) protected 30 nucleotides of the coding strand and 24 nucleotides of the noncoding strand of the fecIR promoter . Higher amounts of Mn2+-Fur (100 nM) covered 41 nucleotides of the coding strand of the fecIR promoter and 38 nucleotides of the coding strand of the fecA promoter . The corresponding region of the noncoding strand of the fecA promoter was hypersensitive to DNase I . The results of a deletion analysis of the fecA promoter supported the previously assigned -35 and -10 regions and nucleotide position +11 for FecI-RNA polymerase interaction . Induction of fecIR transcription by iron limitation increased fecB-lacZ transcription 3.5-fold, whereas under constitutive fecIR transcription, iron limitation increased fecB-lacZ transcription twofold . The two iron-regulated sites of fec transport gene transcription suggest a fast response to sufficient intracellular iron concentrations by repression of fecABCDE transcription and a slower adaptation as the result of fecIR transcription inhibition.

Fold Des, 1998, 3(3), 161 - 71
A single dipeptide sequence modulates the redox properties of a whole enzyme family; Huber-Wunderlich M et al.; BACKGROUND: Disulfide exchange reactions are catalyzed by thiol/disulfide oxidoreductases . These enzymes possess a thioredoxin fold and contain a catalytic disulfide with the sequence Cys-X-X-Cys at the N terminus of an alpha helix . Despite these similarities, the various members differ strongly in their redox potentials (-122 mV to -270 mV) . Using the strong oxidant DsbA from Escherichia coli as a model system, we investigated whether the redox properties of these enzymes can be modulated rationally by exchange of the X-X dipeptide . RESULTS: The X-X dipeptide of DsbA (Cys30-Pro31-His32-Cys33) was exchanged by the dipeptides of eukaryotic protein disulfide isomerase (PDI; Gly-His), glutaredoxin (Pro-Tyr), and thioredoxin (Gly-Pro) from E . coli . All variants were less oxidizing than wild-type DsbA and their redox potentials were in the order of the related natural enzymes (DsbA > PDI > glutaredoxin > thioredoxin) . The equilibrium constant between glutathione and the thioredoxin-like variant increased 1200-fold compared with wild-type DsbA . The variants also showed a strong increase in the pKa of the nucleophilic cysteine (Cys30) . As for glutaredoxin and thioredoxin, the catalytic disulfide stabilized the corresponding variants while destabilizing wild-type DsbA and the PDI-like variant . CONCLUSIONS: The X-X dipeptide in the active site of thiol/disulfide oxidoreductases appears to be the main determinant of the redox properties of these enzymes . This empirical finding should be very useful for the design of new thiol/disulfide oxidoreductases with altered redox potentials and for studying the function of these enzymes in vivo.

Hybridoma, 1998 Apr, 17(2), 191 - 8
Characterization of monoclonal antibodies directed to the amino-terminus of the WT1, Wilms' tumor suppressor protein; Rauscher FJ 3rd et al.; We have produced and characterized three monoclonal antibodies (MAbs) directed to the amino terminus of the WT1 Wilms' tumor suppressor transcription factor and compared their properties to rabbit polyclonal sera raised to the same immunogen . A recombinant protein consisting of amino acids 1-181 of human WT1 was overexpressed in E . coli, purified, and used as the immunogen . Three MAbs designated 6F-H2, 6F-H7, and 6F-HC-17--all of the IgG1 subclass--were selected and further characterized . Each recognized all isoforms of the full-length WT1 protein in Western blot assays and immunoprecipitated WT1 in both physiologic buffers and under high detergent/high salt (RIPA) conditions . Preliminary epitope mapping suggests that all three MAbs recognize a region in the amino terminal 84 amino acids of WT1 and that the MAbs do not recognize the polyglycine or polyproline regions of the protein . The WT1 antibodies do not recognize the structurally and functionally related early growth response (EGR)1, EGR2, EGR3, or EGR4 proteins . All WT1 MAbs recognize the murine WT1 protein and immunohistochemical staining of murine embryonic and newborn kidney sections show strong staining of condensing metanephric mesenchyme and primitive podocytes in developing glomeruli . These WT1-specific MAbs should be useful in characterizing the biochemical and developmental roles of WT1 and in defining the emerging role of WT1 as a diagnostic and/or prognostic marker in mesothelioma, leukemias, and breast cancer.

Proteins, 1998 Jun 1, 31(4), 406 - 16
Species dependence of enzyme-substrate encounter rates for triose phosphate isomerases; Wade RC et al.; Triose phosphate isomerase (TIM) is a diffusion-controlled enzyme whose rate is limited by the diffusional encounter of the negatively charged substrate glyceraldehyde 3-phosphate (GAP) with the homodimeric enzyme's active sites . Translational and orientational steering of GAP toward the active sites by the electrostatic field of chicken muscle TIM has been observed in previous Brownian dynamics (BD) simulations . Here we report simulations of the association of GAP with TIMs from four species with net charges at pH 7 varying from -12e to +12e . Computed second-order rate constants are in good agreement with experimental data . The BD simulations and computation of average Boltzmann factors of substrate-protein interaction energies show that the protein electrostatic potential enhances the rates for all the enzymes . There is much less variation in the computed rates than might be expected on the basis of the net charges . Comparison of the electrostatic potentials by means of similarity indices shows that this is due to conservation of the local electrostatic potentials around the active sites which are the primary determinants of electrostatic steering of the substrate.

Proteins, 1998 Jun 1, 31(4), 383 - 90
Direct observation of an altered quaternary-structure transition in a mutant aspartate transcarbamoylase; Tsuruta H et al.; Time-resolved small-angle X-ray scattering (TR-SAXS) was used to monitor the structural changes that occur upon the binding of the natural substrates to a mutant version of the allosteric enzyme aspartate transcarbamoylase from Escherichia coli, in which the creation of a critical link stabilizing the R state of the enzyme is hindered . Previously, SAXS experiments at equilibrium showed that the structures of the unligated mutant enzyme and the mutant enzyme saturated with a bisubstrate analog are indistinguishable from the T and R state structures, respectively, of the wild-type enzyme (Tauc et al., Protein Sci . 3:1998-2004, 1994) . However, as opposed to the wild-type enzyme, the combination of one substrate, carbamoyl phosphate, and succinate, an analog of aspartate, did not convert the mutant enzyme into the R state . By using TR-SAXS we have been able to study the transient steady-state during catalysis using the natural substrates rather than the nonreactive substrate analogs . The steady-state in the presence of saturating amount of substrates is a mixture of 60% T and 40% R structures, which is further converted entirely to R in the additional presence of ATP . These results provide a structural explanation for the reduced cooperativity observed with the mutant enzyme as well as for the stimulation by ATP at saturating concentrations of substrates . They also illustrate the crucial role played by domain motions and quaternary-structure changes for both the homotropic and heterotropic aspects of allostery.

Exp Eye Res, 1998 Apr, 66(4), 465 - 75
Cloning, high level-expression and characterization of human lens thioltransferase; Raghavachari N et al.; Polymerase chain reaction (PCR) primers, directed against the nucleotide sequence of pig liver thioltransferase (PLTT) were used to amplify human lens thioltransferase (HLTT) from a pool of human lens cDNA . The 520 bp PCR fragment obtained was cloned unidirectionally into pCR 3.1-Uni vector and sequenced . The cDNA sequence of the lens thioltransferase had 98% and 87% homology to pig liver and human placental thioltransferases (TTase) respectively . Nhe1 and EcoR1 fragment of the recombinant PCR 3.1-Uni vector was subcloned in pET 23a Expression vector . High level expression of HLTT was accomplished in Escherichia coli and the expressed protein was characterized by immunoblot analysis with anti PLTT and N-terminal amino acid sequence analysis . The recombinant enzyme efficiently dethiolated protein thiol mixed disulfides conjugated to both cystine (PSSC) and glutathione (PSSG) and had a significant dehydroascorbate reductase activity . Human lens thioltransferase thus displayed structural and functional characteristics identical to pig liver and human placental thioltransferases .

Nucleic Acids Res, 1998 Jul 1, 26(13), 3317 - 8
A rapid, small scale method for characterization of plasmid insertions in the Dictyostelium genome; Barth C et al.; A rapid, simple method for characterization of plasmid insertions in the Dictyostelium discoideum genome was developed . It is based on the capability of linear plasmid multimers in the insertions to recircularize efficiently in Escherichia coli cells . This recombinational recircularization of plasmid multimers provides a highly sensitive and reliable tool for determining whether individual Dictyostelium transformants resulted from restriction enzyme-mediated integration (REMI) or from recombinational integration of plasmid (RIP) . The method also reveals any rearrangements in RIP insertions and provides an estimate of the vector copy number in any particular transformant.

Nucleic Acids Res, 1998 Jul 1, 26(13), 3235 - 41
Generation of circular RNAs and trans-cleaving catalytic RNAs by rolling transcription of circular DNA oligonucleotides encoding hairpin ribozymes; Diegelman AM et al.; A simple new strategy for the in vitro synthesis of circular RNAs and hairpin ribozymes is described . Circular single-strand DNA oligonucleotides 67-79 nt in length are constructed to encode both hairpin ribozyme sequences and ribozyme-cleavable sequences . In vitro transcription of these small circles by Escherichia coli RNA polymerase produces long repeating RNAs by a rolling circle mechanism . These repetitive RNAsundergo self-processing, eventually yielding unit length circular and linear RNAs as the chief products . The transcription is efficient despite the absence of promoter sequences, with RNA being produced in up to 400 times the amount of DNA circle used . It is shown that the linear monomeric hairpin ribozymes are active in cleaving RNA targets in trans , including one from HIV-1 . Several new findings are established: (i) that rolling circle transcription can be extended to the synthesis of catalytic RNAs outside the hammerhead ribozyme motif; (ii) that rolling circle transcription is potentially a very simple and useful strategy for the generation of circular RNAs in preparative amounts; and (iii) that self-processed hairpin ribozymes can be catalytically active in trans despite the presence of self-binding domains.

EMBO J, 1998 Jun 15, 17(12), 3478 - 83
EF-G-catalyzed translocation of anticodon stem-loop analogs of transfer RNA in the ribosome; Joseph S et al.; Translocation, catalyzed by elongation factor EF-G, is the precise movement of the tRNA-mRNA complex within the ribosome following peptide bond formation . Here we examine the structural requirement for A- and P-site tRNAs in EF-G-catalyzed translocation by substituting anticodon stem-loop (ASL) analogs for the respective tRNAs . Translocation of mRNA and tRNA was monitored independently; mRNA movement was assayed by toeprinting, while tRNA and ASL movement was monitored by hydroxyl radical probing by Fe(II) tethered to the ASLs and by chemical footprinting . Translocation depends on occupancy of both A and P sites by tRNA bound in a mRNA-dependent fashion . The requirement for an A-site tRNA can be satisfied by a 15 nucleotide ASL analog comprising only a 4 base pair (bp) stem and a 7 nucleotide anticodon loop . Translocation of the ASL is both EF-G- and GTP-dependent, and is inhibited by the translocational inhibitor thiostrepton . These findings show that the D, T and acceptor stem regions of A-site tRNA are not essential for EF-G-dependent translocation . In contrast, no translocation occurs if the P-site tRNA is substituted with an ASL, indicating that other elements of P-site tRNA structure are required for translocation . We also tested the effect of increasing the A-site ASL stem length from 4 to 33 bp on translocation from A to P site . Translocation efficiency decreases as the ASL stem extends beyond 22 bp, corresponding approximately to the maximum dimension of tRNA along the anticodon-D arm axis . This result suggests that a structural feature of the ribosome between the A and P sites, interferes with movement of tRNA analogs that exceed the normal dimensions of the coaxial tRNA anticodon-D arm.

EMBO J, 1998 Jun 15, 17(12), 3439 - 47
Transcription activation at Class II CRP-dependent promoters: identification of determinants in the C-terminal domain of the RNA polymerase alpha subunit; Savery NJ et al.; Many transcription factors, including the Escherichia coli cyclic AMP receptor protein (CRP), act by making direct contacts with RNA polymerase . At Class II CRP-dependent promoters, CRP activates transcription by making two such contacts: (i) an interaction with the RNA polymerase alpha subunit C-terminal domain (alphaCTD) that facilitates initial binding of RNA polymerase to promoter DNA; and (ii) an interaction with the RNA polymerase alpha subunit N-terminal domain that facilitates subsequent promoter opening . We have used random mutagenesis and alanine scanning to identify determinants within alphaCTD for transcription activation at a Class II CRP-dependent promoter . Our results indicate that Class II CRP-dependent transcription requires the side chains of residues 265, 271, 285-288 and 317 . Residues 285-288 and 317 comprise a discrete 20x10 A surface on alphaCTD, and substitutions within this determinant reduce or eliminate cooperative interactions between alpha subunits and CRP, but do not affect DNA binding by alpha subunits . We propose that, in the ternary complex of RNA polymerase, CRP and a Class II CRP-dependent promoter, this determinant in alphaCTD interacts directly with CRP, and is distinct from and on the opposite face to the proposed determinant for alphaCTD-CRP interaction in Class I CRP-dependent transcription.

EMBO J, 1998 Jun 15, 17(12), 3233 - 40
Interaction of polyomavirus internal protein VP2 with the major capsid protein VP1 and implications for participation of VP2 in viral entry; Chen XS et al.; A complex of the polyomavirus internal protein VP2/VP3 with the pentameric major capsid protein VP1 has been prepared by co-expression in Escherichia coli . A C-terminal segment of VP2/VP3 is required for tight association, and a crystal structure of this segment, complexed with a VP1 pentamer, has been determined at 2.2 A resolution . The structure shows specific contacts between a single copy of the internal protein and a pentamer of VP1 . These interactions were not detected in the previously described structure of the virion, but the location of VP2 in the recombinant complex is consistent with features in the virion electron-density map . The C-terminus of VP2/VP3 inserts in an unusual, hairpin-like manner into the axial cavity of the VP1 pentamer, where it is anchored strongly by hydrophobic interactions . The remainder of the internal protein appears to have significant flexibility . This structure restricts possible models for exposure of the internal proteins during viral entry.

Poult Sci, 1998 Jun, 77(6), 908 - 11
Serum levels of interleukin-6, alpha1-acid glycoprotein, and corticosterone in two-week-old chickens inoculated with Escherichia coli lipopolysaccharide; Nakamura K et al.; The concentration of interleukin-6 (IL-6), alpha1-acid glycoprotein (alpha1-AG), and corticosterone (CORT) was investigated chronologically (0 h to 14 d) in the sera of 2-wk-old specific-pathogen-free chicks inoculated with Escherichia coli lipopolysaccharide (LPS) . In the LPS group the IL-6 level was elevated from 1 h to 2 d and was the highest at 3 h . From 4 to 14 d the IL-6 level was low in the LPS group . In the PBS group, IL-6 was not detected except a mild increase from 1 h to 6 h . In the LPS group, the alpha1-AG level increased from 6 h to 4 d, and the peak was 2 d . In the PBS group the alpha1-AG level was always low . The CORT level in the LPS group was higher than that of PBS group at 1 h . This study suggests that E . coli LPS may elevate serum IL-6 and CORT, and that IL-6 and CORT may increase the alpha1-AG level in the chicks.

Poult Sci, 1998 Jun, 77(6), 894 - 901
Active immunization of Japanese quail hens with a recombinant chicken inhibin fusion protein enhances production performance; Moreau JD et al.; The effects of active immunization against inhibin on production performance in female Japanese quail (Coturnix coturnix japonica) were assessed in two separate trials using an MBP-cINA521 fusion protein as an immunogen . The fusion protein, MBP-cINA521, consisted of the bacterial maltose binding protein (MBP) and a truncated form of the mature alpha-subunit of chicken inhibin (cINA521) . MBP-cINA1521 was constructed by: 1) excising a 521-bp PstI fragment from a chicken inhibin alpha-subunit cDNA (cINA6; gift of P . A . Johnson), 2) cloning this fragment, which encodes all but the first 11 amino acid residues of the mature alpha-subunit, into the pMal-c2 vector of the MBP fusion expression system, and 3) expressing the fusion protein (MBP-cINA521) from the Escherichia coli and purifying it using affinity chromatography . In each trial, quail were randomly and equally assigned to one of two injection treatments as follows: 1) MBP-cINA521 in Freund's adjuvant, or 2) Freund's adjuvant (vehicular controls; CON) . All immunizations were given subcutaneously and Freund's complete and incomplete adjuvant were used for primary and booster injections, respectively . In Trial 1, birds were given a primary challenge of 0.2 mg MBP-cINA521 per bird at 25 d of age, followed by booster immunizations (0.1 mg MBP-cINA521 per bird) at 33, 40, 47, 54 and 61 d of age and every 35 d thereafter . The CON birds received vehicular immunizations at the same time intervals . In Trial 2, birds treated with MBP-cINA521 received a primary challenge of 0.2 mg MBP-cINA521 per bird at 26 d of age, followed by booster immunizations (0.1 mg MBP-cINA521 per bird) using the same schedule as that used in Trial 1, with the exception that no boosters were given after 61 d of age . The CON birds received vehicular immunizations at the same time intervals . Collection of production performance data was initiated coincident with the laying of the first egg in each trial (i.e., beginning at 41 and 44 d of age for Trials 1 and 2, respectively) and continued for 30 1-wk periods of lay . Combined data from Trials 1 and 2 indicated that the mean +/- SE age at first egg lay was markedly decreased (P < 0.005) in MBP-cINA521-treated quail (53.4 +/- 0.9 d of age) when compared to the CON (57.6 +/- 1.3 d of age) . Likewise, the mean +/- SE age at 50% egg production was reduced (P < 0.03) in quail immunized against inhibin (65.4 +/- 2.1 d of age) when compared to the CON (77.6 +/- 4.7 d of age) . Total hen-day egg production was also higher (P < 0.05, Trial 1; P < 0.01, Trial 2) in MBP-cINA521-treated quail (88.7 +/- 1.4%, Trial 1; 90.1 +/- 1.2%, Trial 2) than in the CON birds (81.9 +/- 2.9%, Trial 1; 73.6 +/- 6.5%, Trial 2) . Collectively, these findings provide evidence that inhibin immunoneutralization accelerated puberty and enhanced hen-day egg production during a 30-wk period of egg lay in Japanese quail.

Res Immunol, 1998 Feb, 149(2), 127 - 37
Does a peptide bound to a monoclonal antibody always adopt a unique conformation?
Guijarro JI, Djavadi-Ohaniance L, Baleux F, Delepierre M, Goldberg ME.
The conformation of a synthetic undecapeptide derived from the Escherichia coli tryptophan synthase beta2 subunit was studied by NMR spectroscopy when bound to a monoclonal antibody (mAb 164-2) Fab' fragment directed against the native protein . The peptide 1(H-G-R-V-G-I-Y-F-G-M-K)11, peptide 11, was recognized by the antibody and its corresponding Fab' fragments with high affinity (K(D) = 1.1+/-0.2* 10(-8) M) . Peptide 11 was labelled with 15N and its structure at the binding site of the Fab' 164-2 fragment was studied by isotope-editing techniques . 1H-15N heteronuclear spectra indicated the presence of two Fab'-peptide 11 complexes with two different conformations in slow chemical exchange on the chemical shift time scale.

Biol Chem, 1998 Apr-May, 379(4-5), 611 - 6
Dependence of McrBC cleavage on distance between recognition elements; Stewart FJ et al.; DNA cleavage by the modification-dependent restriction enzyme McrBC requires the presence of two suitably modified recognition elements appropriately spaced in the substrate . To characterize the spacing requirement in more detail, we have constructed a plasmid with a single McrBC cleavage site, in which the distance between recognition elements could be systematically varied while preserving the local sequence surrounding the recognition elements . Optimal separation between elements was 55-103 basepairs, with detectable cleavage observed at spacing of 32 bp to 2 kb; no cleavage was seen with spacing of 22 bp or less or with 3 kb between elements . Changing the spacing by 4 basepairs within the optimal range had little effect on the efficiency of cleavage, suggesting that the recognition elements need not lie on the same face of the DNA helix.

Biol Chem, 1998 Apr-May, 379(4-5), 549 - 51
Correlation between transcription and C to T mutations in the non-transcribed DNA strand; Beletskii A et al.; We recently showed that transcription can promote C to T mutations in the non-transcribed strand in E . coli . To study the relationship between the level of transcription and mutant frequency, an inactive allele of the kanamycin-resistance gene was expressed under the control of a hybrid promoter consisting of an UP element and the tac promoter . When this promoter is induced, the frequency of C to T mutations in the non-transcribed strand increases in rough proportion to the amount of mRNA . At the highest level of transcription at which cell growth is not affected, there is about a 10-fold increase in the frequency of mutations . This result is consistent with the hypothesis that transcription forces the non-transcribed strand to be in a single-stranded state and that this results in frequent C to T mutations.

Biol Chem, 1998 Apr-May, 379(4-5), 497 - 503
Protein-protein and protein-DNA interactions in the type I restriction endonuclease R.EcoR124I; Mernagh DR et al.; The type I restriction-modification system EcoR124I recognizes and binds to the split DNA recognition sequence 5'-GAAN(6)RTCG-3' . The methyltransferase, consisting of HsdM and HsdS subunits with the composition M2S, can interact with one or more subunits of the HsdR subunit to form the endonuclease . The interaction of the methyltransferase with HsdR has been investigated by surface plasmon resonance, showing that there are two non-equivalent binding sites for HsdR which differ in binding affinity by at least two orders of magnitude . DNA footprinting experiments using Exonuclease III suggest that the addition of HsdR to the methyltransferase (at a stoichiometry of either 1:1 or 2:1) increases the stability of the resulting DNA-protein complex but does not increase the size of the footprint . More extensive in situ footprinting experiments using copper-phenanthroline on the DNA-protein complexes formed by M2S, R1M2S and R2M2S also show no difference in the detailed cleavage pattern, with approximately 18 nucleotides protected on both strands in each complex . Thus the HsdR subunit(s) of the endonuclease stabilise the interaction of the M2S complex with DNA, but do not directly contribute to DNA binding . In addition, the thymidine nucleotide in the tetranucleotide recognition sequence GTCG is hyper-reactive to cleavage in each case, suggesting that the DNA structure in this region is altered in these complexes.

Biol Chem, 1998 Apr-May, 379(4-5), 459 - 65
Mutational analysis of the function of Met137 and Ile197, two amino acids implicated in sequence-specific DNA recognition by the EcoRI endonuclease; Ivanenko T et al.; The gene encoding the EcoRI endonuclease was altered by site-directed mutagenesis to introduce multiple substitutions of M137 and 1197, two amino acids which were suggested by the revised crystal structure to mediate recognition of the cytosines in the 5'-GAATTC-3' target sequence . Eight substitutions of M137 and ten substitutions of 1197 were isolated . With the exception of M137W, M137P and M137K, all mutant enzymes retained enough activity to damage cellular DNA in the absence of the EcoRI methyltransferase . All M137 replacements abolished the ability of the enzyme to restrict phage growth . Conservative replacements at 1197 (L, V) did not impair phage restriction, whereas non-conservative changes reduced (G, W) or abolished (D, P) restriction . In general, substitutions at M137 were more deleterious than substitutions at I197 . Double mutants with combinations of M137G/A and I197G/A mutations exhibited a phenotype characteristic for the respective single M137 mutant . Double mutants carrying combinations of the M137G/A replacements and substitutions at R200 were viable even in the absence of the methyltransferase, suggesting that disrupting contacts to both bases of the GC base pair inactivates the enzyme . None of the replacements resulted in relaxed recognition specificity . In summary, our findings are consistent with a role for M137 but do not support such a role for I197 in substrate recognition by the EcoRI endonuclease.

Biol Chem, 1998 Apr-May, 379(4-5), 429 - 36
Selfish restriction modification genes: resistance of a resident R/M plasmid to displacement by an incompatible plasmid mediated by host killing; Naito Y et al.; Previous work from this laboratory demonstrated that plasmids carrying a type II restriction-modification gene complex are not easily lost from their bacterial host because plasmid-free segregant cells are killed through chromosome cleavage . Here, we have followed the course of events that takes place when an Escherichia coli rec BC sbcA strain carrying a plasmid coding for the PaeR7I restriction-modification (R/M) gene complex is transformed by a plasmid with an identical origin of replication . The number of transformants that appeared was far fewer than with the restriction-minus (r-) control . Most of the transformants were very small . After prolonged incubation, the number and the size of the colonies increased, but this increase never attained the level of the r- control . Most of the transformed colonies retained the drug-resistance of the resident, r+ m+ plasmid . These results indicate that post-segregational host killing occurs when a plasmid bearing an R/M gene complex is displaced by an incompatible plasmid . Such cell killing eliminates the competitor plasmid along with the host and, thus, would allow persistence of the R/M plasmid in the neighboring, clonal host cells in nature . This phenomenon is reminiscent of mammalian apoptosis and other forms of altruistic cell death strategy against infection . This type of resistance to displacement was also studied in a wild type Escherichia coli strain that was normal for homologous recombination (rec+) . A number of differences between the recBC sbcA strain and the rec+ strain were observed and these will be discussed.

Plant J, 1998 May, 14(3), 297 - 304
Acetyl-CoA:benzylalcohol acetyltransferase--an enzyme involved in floral scent production in Clarkia breweri; Dudareva N et al.; Volatile esters impart distinct characteristics to the floral scent of many plants, and are important in attracting insect pollinators . They are also important flavor compounds in fruits . The ester benzylacetate is a major constituent of the floral scent of Clarkia breweri, an annual plant native to California . The enzyme acetyl-CoA:benzylalcohol acetyltransferase (BEAT), which catalyzes the formation of benzylacetate, has been purified from C . breweri petals, and a cDNA encoding this enzyme has been isolated and characterized . The sequence of the 433-residue BEAT protein does not show high similarity to any previously characterized protein, but a 35-residue region from position 135-163 has significant similarity (42-56% identity) to several proteins known or suspected to use an acyl-CoA substrate . E . coli cells expressing C . breweri BEAT produced enzymatically active protein, and also synthesized benzylacetate and secreted it into the medium . Of the different parts of the C . breweri flower, petals contained the majority of BEAT transcripts, and no BEAT mRNA was detected in leaves . The levels of BEAT mRNA in the petals increased as the bud matured, and peaked at anthesis, paralleling changes in BEAT activity . However, three days after anthesis, mRNA levels began a steep decline, whereas BEAT activity remained high for the next two days, suggesting that the BEAT protein is relatively stable.

Plant J, 1998 Apr, 14(2), 159 - 68
N- and C-terminal peptide sequences are essential for enzyme assembly, allosteric, and/or catalytic properties of ADP-glucose pyrophosphorylase; Laughlin MJ et al.; ADP-glucose pyrophosphorylase is a key regulatory enzyme in starch synthesis in most plant tissues . Unlike the allosteric regulatory dependent properties of the leaf enzyme, the enzymes from non-photosynthetic tissues exhibit varying levels of sensitivity to allosteric regulation, a behavior which may be an inherent property of the enzyme or a product of post-translational modification . As partial proteolysis of the holoenzyme may account for the wide variation of allosteric regulatory behavior exhibited by enzymes from non-photosynthetic tissues, small N- and C-terminal peptide deletions were made on either the potato large and small subunit and co-expressed with the counterpart wild-type subunit in Escherichia coli . Removal of the putative carboxy-terminal allosteric binding region from either subunit type results in an abolishment of enzyme formation indicating that the carboxy terminus of each subunit type is essential for proper subunit folding and/or enzyme assembly as well as its suggested role in allosteric regulation . Removal of a small 10 amino acid peptide from the N-terminus of the small subunit increased its resistance to the allosteric inhibitor Pi as well as its sensitivity to heat treatment . Likewise, removal of the corresponding peptide (17 residues) at the N-terminus of the large subunit also increased its resistance towards Pi inhibition but, in addition, increased its sensitivity to 3-PGA activation . Deletion of an additional 11 residues reversed these changes in allosteric properties but at the expense of a reduced catalytic turnover rate . Combined, these results indicate that the N- and C-terminal regions are essential for the proper catalytic and allosteric regulatory properties of the potato ADP-glucose pyrophosphorylase . The possible significance of these results on the observed insensitivity to effector molecules by ADP-glucose pyrophosphorylases from other non-photosynthetic tissues is discussed.

FEMS Microbiol Lett, 1998 May 15, 162(2), 241 - 7
Isolation and cloning of a Azospirillum lipoferum locus that complements Escherichia coli proU mutant; Tripathi AK et al.; Glycine betaine relieved sodium chloride-mediated inhibition of growth in Azospirillum lipoferum ATCC 29708 . 35S-methionine labelling of proteins after salinity up-shock revealed strong induction of a 30 kDa protein which cross-reacted with the anti-glycine betaine binding protein antibody from Escherichia coli . This suggested that A . lipoferum had a salinity-induced ProU-like high-affinity glycine betaine transport system . A genomic library of A . lipoferum ATCC 29708 was screened for the proU-like gene by complementation of a proU mutant of E . coli . Four recombinant cosmids, capable of restoring growth of the proU mutant on plates containing 600 mM NaCl and 1 mM glycine betaine were selected . Selected recombinant cosmids hybridized with a proU gene probe from E . coli . Complementation of E . coli proU mutant with the A . lipoferum genomic DNA was evident by the ability of proU mutant (containing selected recombinant cosmids) to grow on minimal medium supplemented with 600 mM NaCl and 1 mM glycine betaine.

FEMS Microbiol Lett, 1998 May 15, 162(2), 235 - 9
Association of genes encoding P fimbriae, CS31A antigen and EAST 1 toxin among CNF1-producing Escherichia coli strains from cattle with septicemia and diarrhea; Bertin Y et al.; Fifty-six CNF1-producing Escherichia coli strains isolated from cattle with diarrhea or septicemia were screened by PCR for the detection of pap, sfa, afa, clpG, or f17 adherence factor and EAST 1 toxin genes . All the isolates were pap-positive, in accordance with the close association of pap, CNF1 and alpha-hemolysin genes observed on human and porcine E . coli . Only the gene encoding the P adhesin of class III (PrsG) was detected . Genes encoding CS31A antigen (71%) and S fimbriae (34%) (but not Afa or F17) were detected among the bovine isolates . E . coli producing both CNF1 and plasmid-encoded CS31A is a new example of association between bacterial clones and plasmid-mediated virulence factors . The EAST 1 toxin-encoding gene was detected on 66% of the CNF1-producing isolates but was linked to CS31A rather than to CNF1 . These results suggest a close association between EAST 1 toxin and the adherence factor CS31A among pathogenic bovine E . coli.

Vaccine, 1998 May, 16(8), 850 - 6
Systemic immunization with urease protects mice against Helicobacter pylori infection; Guy B et al.; The ability of systemic immunization to induce protection against Helicobacter pylori infection has been evaluated in a mouse model . It was observed that if appropriate formulations and adjuvants were used such immunization elicited in outbred Swiss mice levels of protection similar or better than those induced by the oral route in the presence of cholera toxin or Escherichia coli heat labile toxin . Recombinant urease mixed with adjuvants, which induced strong Th1 and Th2 responses elicited better protection than urease mixed with adjuvants which induced a predominant Th2 type response only . These experiments demonstrate the feasibility of parenteral immunization against H . pylori and suggest that an appropriate balance between Th1 and Th2 type responses is required to achieve complete protection.

J Investig Dermatol Symp Proc, 1996 Apr, 1(1), 28 - 32
Normal levels of the vitamin D receptor and its message in psoriatic skin; Solvsten H et al.; Treatment with vitamin D3 analogs improves psoriasis . The vitamin D receptor (VDR) mediates most, if not all, the effects of vitamin D3 . The purpose of this study was to determine the levels of the VDR mRNA and VDR protein in normal and in involved and uninvolved psoriatic skin . Although VDR mRNA was not detected by Northern analysis of human skin samples, it was readily detectable by use of the more sensitive ribonuclease protection assay . The VDR mRNA levels were normal in acute guttate as well as in chronic plaque lesions . There was also no difference in VDR mRNA levels between normal and uninvolved psoriatic skin . The VDR protein was detected by Western analysis using the monoclonal 9A7 gamma anti-VDR antibody and a polyclonal rabbit anti-VDR antibody . For comparison, VDR levels were analyzed in cultures of normal human keratinocytes and the epithelial cell line MCF-7 . Studies of the extraction procedures for VDR showed that at least 60% of Escherichia coli-expressed VDR added to the skin biopsy specimens was recovered . The VDR concentration in normal human adult skin was approximately 50 pg/microgram protein, and the concentrations of VDR in involved and uninvolved psoriatic skin were of the same order of magnitude . Using the 9A7 gamma anti-VDR antibody, the VDR (M(r) 53,000) was constantly present in lower amounts than a band of M(r) 80,000 in both skin specimens and keratinocyte cultures . This high-molecular-weight band is most likely a cross-reacting protein not related to VDR, because it was absent when using the polyclonal anti-VDR antibody.

Rinsho Byori, 1998 May, 46(5), 456 - 60
{Epitope analysis of anti-serum protein antibodies and its application for amyloidosis research}; Yamada T et al.; To obtain antibodies specific to desired portions in proteins, synthetic peptides are widely used as immunogens, however, generated antibodies often react improperly with native proteins . Monoclonal antibodies (MoAb), epitopes of which are well defined, would be useful . In this review, we present epitope analysis and its application for immunohistochemical studies on four MoAbs against serum amyloid A(SAA), the serum precursor of fibrillar component in reactive amyloid deposits . Multipin-ELISA (enzyme-linked immunosorbent assay), the epitope mapping system which is based on reactivity with small amount of synthetic peptides immobilized on plastic pins, divided 13 clones of rat-mouse hybrid anti-SAA MoAbs into four . Two were determined epitopes as a portion around residue 18 and 30 of SAA while the other two could not be determined . These epitopes were analyzed using truncated recombinant SAA proteins, which were produced in E . coli, and determined as a portion around residue 90 and 100 (C-terminus), respectively . Negative immunohistochemistry of anti-SAA C-terminus MoAb for human reactive amyloid deposits suggests that SAA C-terminus is removed before or during amyloid deposition.

Rinsho Byori, 1998 May, 46(5), 450 - 5
{Recent trends in protein structural studies}; Titani K et al.; Since the 1980's, structural studies of proteins have changed remarkably . It is currently possible to predict the entire amino acid sequence of a protein by the rapid and highly sensitive analysis of the nucleotide sequence of genomic DNA or cDNA encoding the protein . In the near future, the entire sequence of a protein may be predicted from a partial sequence just by searching a variety of databases now being constructed for many biological species . The predicted protein sequence, however, is the backbone structure of the precursor protein without post-translational modifications . Therefore, the major objectives of recent structural studies of proteins are directed to 1) rapid and sensitive confirmation of the predicted sequence and identification of those modifications present in mature proteins by newly developed mass spectrometry, 2) determination of the 3D structures of intact and mutant proteins isolated or expressed in cultured E . coli, yeast or animal cells using X-ray crystallography or NMR analysis, and 3) rapid prediction of the 3D structures of proteins utilizing protein databases . The "PROTEOME" project was proposed in 1998 to bring together all the data on the structure and function of mature proteins under international cooperation . The present paper summarizes such recent trends in protein structural studies.

J Crit Care, 1998 Jun, 13(2), 81 - 90
The platelet-activating factor antagonist BB-882 does not improve tissue oxygen extraction in endotoxic shock; Spapen H et al.; PURPOSE: We investigated whether BB-882, a novel potent PAF antagonist, could influence systemic and pulmonary hemodynamics and oxygen extraction capabilities during an acute reduction in blood flow induced by cardiac tamponade after endotoxin challenge . MATERIALS AND METHODS: Twenty-one anesthetized, ventilated, and endotoxin-shocked (2 mg/kg i.v . Escherichia coli endotoxin) dogs were randomly divided in three groups . One group (N = 7) served as control . A second group (N = 7) received BB-882 as a single bolus dose of 5 mg/kg, 30 minutes before endotoxin administration . A third group (N = 7) received BB-882 as a continuous infusion of 5 mg/kg x h, started 30 minutes after endotoxin . Hemodynamic and gazometric measurements were obtained in all dogs 30 minutes after endotoxin injection and repeated 30 minutes after cardiac filling pressures were restored to baseline by generous saline infusion . Saline infusion rate was then set at 20 mL/kg x h and tamponade was induced by repeated bolus injections of warm saline into the pericardial sac . RESULTS: Compared with controls, pretreatment with BB-882 attenuated the early endotoxin-induced decrease in arterial pressure (70 +/- 17 v 51 +/- 14 mm Hg, P < .05), cardiac index (118 +/- 29 v 91 +/- 15 mL/ kg x min, P < .05), stroke index (1.0 +/- 0.2 v 0.7 +/- 0.3 mL/kg, P < .05), and left ventricular stroke work index (0.9 +/- 0.3 v 0.4 +/- 0.2 g x m/kg, P < .05), but these effects were not sustained after fluid resuscitation . In contrast, BB-882 post-treatment maintained arterial pressure and improved cardiac performance at lower filling pressures in the later phase of endotoxic shock . BB-882 did not influence pulmonary hemodynamics . Treatment with BB-882 did not influence oxygen extraction at critical oxygen delivery (51.5 +/- 9.9% and 52.8 +/- 13.9% v 46.6 +/- 9.0%, respectively BB-882 pretreatment and post-treatment v control) . CONCLUSIONS: We conclude that in this model of endotoxic shock the administration of BB-882, either before or after endotoxin challenge, has time-related beneficial hemodynamic and cardiac effects but does not improve global oxygen extraction capabilities . The potential benefit of adjunctive treatment with a platelet-activating factor antagonist in sepsis remains doubtful.

Langenbecks Arch Surg, 1998 Mar, 383(1), 95 - 8
The impact of donor organ quality on postoperative liver function after orthotopic rat liver transplantation; Knoop M et al.; The impact of donor factors for posttransplant liver function was evaluated in the model of orthotopic rearterialized liver transplantation in the rat . The effect of donor fasting, parenteral hyperalimentation, hypotension, warm ischemia and endotoxins on histology, clinical chemistry and MEGX test was analyzed in syngeneic and allogeneic recipients of livers stored for 4 hrs on ice . In syngeneic animals, 20 min of warm ischemia led to significantly elevated serum transaminase levels and degree 2 histological damage on POD 2 . Endotoxins produced a grade 1 histological damage . All groups had a lower MEGX formation rate compared to controls . In allogeneic animals, warm ischemia was the single most detrimental parameter . The strength of the rejection response on POD 8 did not depend on the type of donor pretreatment . The major finding of this non-survival study is the deleterious effect of warm ischemia and endotoxin on the functional and structural integrity of liver grafts after 4 hrs of cold ischemia.

Langenbecks Arch Surg, 1998 Mar, 383(1), 75 - 80
Failure of Kupffer cell blockade to prevent disseminated intravascular coagulation in endotoxemic rats despite improved survival; Ruttinger D et al.; OBJECTIVE: Studies were conducted to evaluate the impact of gadolinium chloride (GdCl3), an agent which blocks the phagocytosis of liver macrophages (Kupffer cells, KC), on the coagulation system and on mortality in a model of rats subjected to a lethal dose of Escherichia coli lipopolysaccharide (LPS) (10 mg/kg body weight, intravenously) . METHODS: Rats were either pretreated with GdCl3 (10 mg/kg, i.v., 48 h and 24 h prior to LPS exposure) or saline vehicle . A variety of coagulation parameters such as activated partial prothrombin time (aPTT), fibrinogen, systemic platelet count, antithrombin III (AT III), and activities of factors V, VII, and XII were monitored in the early (1 h) and late time course (16 h) following administration of E.coli LPS . RESULTS: The administration of LPS resulted in the development of disseminated intravascular coagulation (DIC) and was associated with a mortality rate of 47% within 16 h . Blockade of KC by GdCl3 completely abolished LPS-related mortality (0%) . However, despite improved survival, GdCl3 failed to prevent laboratory and clinical signs of DIC . GdCl3 per se even contributed to coagulatory and fibrinolytic disorders . CONCLUSION: These results confirm reports on the protective potential of GdCl3 pretreatment in experimental endotoxemia . However, the present study does not support the concept of DIC as a strong prognostic criterion for the outcome of sepsis and septic shock . Furthermore, the results presented suggest a minor role for KC in LPS-mediated activation of coagulation and indicate an involvement of KC in LPS-associated lethality independent of the coagulation system.

Mutat Res, 1998 Feb 26, 398(1-2), 175 - 82
Catabolite repressors are potent antimutagens in Escherichia coli plate incorporation assays: experiments with glucose, glucose-6-phosphate and methyl-alpha-D-glucopyranoside; Ambrose M et al.; Having previously found that the yields of spontaneous valine-resistant (Val(r)) Escherichia coli mutants which appeared on plates containing 40 microg/ml of valine were always much lower when glucose was present in the glycerol-containing defined medium normally used to select them, we now sought to determine whether or not the global regulatory mechanism known as catabolite repression (formerly also called glucose repression) might be involved . We therefore tested glucose (the archetypal catabolite repressor), glycerol (a non catabolite-repressing substrate), glucose-6-phosphate (G6P, an exceptionally powerful catabolite repressor) and methyl-alpha-D-glucopyranoside (alphaMG, a strongly catabolite-repressing but non-utilisable glucose analogue), as potential inhibitors of spontaneous mutagenesis in plate incorporation assays, using three distinct mutation detection systems . We found that the numbers of spontaneous Val(r) and Lac+ mutations appearing on the selective plates tended to be highest when the medium contained only a non-repressing primary carbon source (glycerol in the Val(s) --> Val(r) system, lactose in the Lac- --> Lac+ system) and lowest when it had been supplemented with a strongly catabolite-repressing compound such as alphaMG, G6P or glucose . These results would seem to establish that catabolite repression is an important factor in determining the outcome of the spontaneous mutation generation process in E . coli and hence that the numbers of spontaneous mutations which can be expected to arise in any given set of mutation assay conditions may often be dependent upon the levels of catabolite repression which prevail during the course of the assay . The implications of these results for conventional plate-incorporation mutation assays are discussed.

Mutat Res, 1998 Feb 26, 398(1-2), 143 - 9
Hepatocarcinogenesis (Z#2)/mutagenesis during initiation stage; Nichols WS et al.; Previously, we developed a model for high incidence, endogenously generated hepatocellular carcinoma (HCC), the human alpha-1-antitrypsin (alpha1AT) Z gene transgenic mouse (Z#2) . We now examine the potential utility of a model for endogenous carcinogenesis utilizing the Z#2 mouse also transgenic for the lacI gene, a convenient target for in vivo mutagenesis studies . We crossed the Z#2 line and mice transgenic for lambda/lacI shuttle vector (Big Blue), for determination of lacI mutant frequency during initiation of endogenous carcinogenesis . Five month old double transgenic mice (Z#2+/lacI+) successfully displayed: (1) the expected post-inflammatory stage of Z#2 carcinogenesis; and (2) hepatic lacI mutants measured at frequencies (10(-5)-10(-4)) useful to mutagenesis studies . In this study, hepatic lacI mutation frequencies in Z#2 transgenic mice appeared to be only slightly increased (< 2x) when compared to age matched negative controls . In the future, it may be important to reconcile possibly limited lacI mutagenesis at the time of initiation and demonstrated high incidence of hepatocarcinogenesis.

FEMS Immunol Med Microbiol, 1998 Apr, 20(4), 319 - 25
Mycobacterium tuberculosis reactive T cell clones from naturally converted PPD-positive healthy subjects: recognition of the M . tuberculosis 16-kDa antigen; Oftung F et al.; Mycobacterium tuberculosis reactive T cell clones were established from naturally converted PPD-positive healthy subjects and screened for proliferative reactivity against defined M . tuberculosis protein antigens of 16, 19, 65 (HSP65), and 71 (HSP70) kDa recombinantly expressed in Escherichia coli . Among the recombinant antigens tested, the M . tuberculosis 16-kDa protein antigen, as expressed from the lambda gt11 phage Y3155, was found to induce T cell proliferation . Crossreactivity studies showed that the epitope recognized was present in M . tuberculosis, M . africanum as well as the vaccine strain M . bovis BCG . The M . tuberculosis 16-kDa reactive T cell clone identified showed the CD4+, CD8- phenotype, secreted interferon-gamma upon antigen stimulation, and displayed major histocompatibility complex class II restricted cytotoxicity against M . tuberculosis pulsed macrophages . The results obtained suggest that the recombinant M . tuberculosis 16-kDa antigen can be recognized by human Th1 cells with potential relevance to protection.

Yale J Biol Med, 1997 Jul-Aug, 70(4), 301 - 10
Functional re-evaluation of the putative glutathione transporters, RcGshT and RsGshT; Li L et al.; Transport systems that mediate glutathione (GSH) efflux from hepatocytes into blood plasma and bile have been characterized extensively in sinusoidal and canalicular membrane vesicles, and recent reports describe two candidate GSH transport proteins: the rat sinusoidal GSH transporter (RsGshT) and rat canalicular GSH transporter (RcGshT) . However, studies in our laboratory have been unable to confirm the function of these gene products . Xenopus laevis oocytes injected with either rat liver mRNA, the cRNA for RcGshT or the cRNA for RsGshT did not transport GSH at a higher rate than water-injected oocytes, when measured either as 3H-GSH uptake or efflux, at low or high GSH concentrations, or in the presence or absence of acivicin to inhibit gamma-glutamyltransferase activity . In contrast, transport of 3H-taurocholate was markedly accelerated in oocytes injected with rat liver mRNA or the cRNA for the Na(+)-taurocholate cotransporting polypeptide (Ntcp), confirming the integrity of the mRNA and the viability of the oocytes . Northern blot analysis failed to detect an RcGshT transcript in rat liver total RNA or rat liver mRNA . Of significance, the RcGshT and RsGshT cDNA sequences are similar to those found in the Escherichia coli K-12 genome, indicating possible cloning artifacts . Further studies are needed to resolve this discrepancy, and to isolate and characterize hepatic GSH transport proteins.

Am J Pathol, 1998 Jun, 152(6), 1421 - 6
New monoclonal antibodies to the T cell antigens CD4 and CD8 . Production and characterization in formalin-fixed paraffin-embedded tissue; Williamson SL et al.; We have generated a recombinant protein representing part of the CD4 molecule and a peptide representing an epitope of predicted high antigenicity on the CD8 molecule and employed these to generate mouse monoclonal antibodies using standard hybridoma protocols . The extracellular domain of the CD4 molecule was obtained by reverse transcription of mRNA from peripheral blood lymphocytes followed by polymerase chain reaction . The amplified gene fragment was cloned into an expression vector to allow a histidine-tagged fusion protein to be produced in Escherichia coli . Purified fusion protein was used to immunize mice . The CD8 monoclonal antibody was raised against a peptide consisting of 13 amino acids within the carboxyl-terminal region of the CD8 cytoplasmic domain . The antibodies showed appropriate reactivity on Western blotting . By heat pretreatment, these antibodies have been shown to be highly effective on paraffin-embedded tissue . In normal lymphoid tissue, the expected distribution of CD4 and CD8 lymphocytes was observed . In a series of 16 T cell lymphomas and B cell lymphomas, immunostaining results were compared with those obtained using reagents effective only in frozen tissue . A high degree of correlation was observed . These results suggest that NCL-CD4 and NCL-CD8 may be of value in the characterization of T cell disorders.

Proc Natl Acad Sci U S A, 1998 Jun 9, 95(12), 6909 - 14
Heteroduplex joint formation in Escherichia coli recombination is initiated by pairing of a 3'-ending strand; Friedman-Ohana R et al.; The formation of heteroduplex joints in Escherichia coli recombination is initiated by invasion of double-stranded DNA by a single-stranded homologue . To determine the polarity of the invasive strand, linear molecules with direct terminal repeats were released by in vivo restriction of infecting chimeric phage DNA and heteroduplex products of intramolecular recombination were analyzed . With this substrate, the invasive strand is expected to be incorporated into the circular crossover product and the complementary strand is expected to be incorporated into the reciprocal linear product . Strands of both polarities were incorporated into heteroduplex structures, but only strands ending 3' at the break were incorporated into circular products . This result indicates that invasion of the 3'-ending strand initiates the heteroduplex joint formation and that the complementary 5'-ending strand is incorporated into heteroduplex structures in the process of reciprocal strand exchange . The polarity of the invasive strand was not affected by recD, recJ, or xonA mutations . However, xonA and recJ mutations increased the proportion of heteroduplexes containing 5'-ending strands . This observation suggests that RecJ exonuclease and exonuclease I may enhance recombination by degrading the displaced strands during branch migration and thereby causing strand exchange to be unidirectional.

Proc Natl Acad Sci U S A, 1998 Jun 9, 95(12), 6887 - 92
Nucleolar localization of the Werner syndrome protein in human cells; Marciniak RA et al.; Werner Syndrome (WS) is a human genetic disorder with many features of premature aging . The gene defective in WS (WRN) has been cloned and encodes a protein homologous to several helicases, including Escherichia coli RecQ, the human Bloom syndrome protein (BLM), and Saccharomyces cerevisiae Sgs1p . To better define the function of WRN protein we have determined its subcellular localization . Indirect immunofluorescence using polyclonal anti-human WRN shows a predominant nucleolar localization . Studies of WRN mutant cells lines confirmed the specificity of antibody recognition . No difference was seen in the subcellular localization of the WRN protein in a variety of normal and transformed human cell lines, including both carcinomas and sarcomas . The nucleolar localization of human WRN protein was supported by the finding that upon biochemical subcellular fractionation, WRN protein is present in an increased concentration in a subnuclear fraction enriched for nucleolar proteins . We have also determined the subcellular localization of the mouse WRN homologue (mWRN) . In contrast to human WRN protein, mWRN protein is present diffusely throughout the nucleus . Understanding the function of WRN in these organisms of vastly differing lifespan may yield new insights into the mechanisms of lifespan determination.

Proc Natl Acad Sci U S A, 1998 Jun 9, 95(12), 6803 - 8
Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins; Llopis J et al.; Many cellular events depend on a tightly compartmentalized distribution of H+ ions across membrane-bound organelles . However, measurements of organelle pH in living cells have been scarce . Several mutants of the Aequorea victoria green fluorescent protein (GFP) displayed a pH-dependent absorbance and fluorescent emission, with apparent pKa values ranging from 6.15 (mutations F64L/S65T/H231L) and 6.4 (K26R/F64L/S65T/Y66W/N146I/M153T/ V163A/N164H/H231L) to a remarkable 7.1 (S65G/S72A/T203Y/H231L) . We have targeted these GFPs to the cytosol plus nucleus, the medial/trans-Golgi by fusion with galactosyltransferase, and the mitochondrial matrix by using the targeting signal from subunit IV of cytochrome c oxidase . Cells in culture transfected with these cDNAs displayed the expected subcellular localization by light and electron microscopy and reported local pH that was calibrated in situ with ionophores . We monitored cytosolic and nuclear pH of HeLa cells, and mitochondrial matrix pH in HeLa cells and in rat neonatal cardiomyocytes . The pH of the medial/trans-Golgi was measured at steady-state (calibrated to be 6.58 in HeLa cells) and after various manipulations . These demonstrated that the Golgi membrane in intact cells is relatively permeable to H+, and that Cl- serves as a counter-ion for H+ transport and likely helps to maintain electroneutrality . The amenability to engineer GFPs to specific subcellular locations or tissue targets using gene fusion and transfer techniques should allow us to examine pH at sites previously inaccessible.

Proc Natl Acad Sci U S A, 1998 Jun 9, 95(12), 6756 - 61
Terpenoid-based defenses in conifers: cDNA cloning, characterization, and functional expression of wound-inducible (E)-alpha-bisabolene synthase from grand fir (Abies grandis); Bohlmann J et al.; (E)-alpha-Bisabolene synthase is one of two wound-inducible sesquiterpene synthases of grand fir (Abies grandis), and the olefin product of this cyclization reaction is considered to be the precursor in Abies species of todomatuic acid, juvabione, and related insect juvenile hormone mimics . A cDNA encoding (E)-alpha-bisabolene synthase was isolated from a wound-induced grand fir stem library by a PCR-based strategy and was functionally expressed in Escherichia coli and shown to produce (E)-alpha-bisabolene as the sole product from farnesyl diphosphate . The expressed synthase has a deduced size of 93.8 kDa and a pI of 5 . 03, exhibits other properties typical of sesquiterpene synthases, and resembles in sequence other terpenoid synthases with the exception of a large amino-terminal insertion corresponding to Pro81-Val296 . Biosynthetically prepared (E)-alpha-{3H}bisabolene was converted to todomatuic acid in induced grand fir cells, and the time course of appearance of bisabolene synthase mRNA was shown by Northern hybridization to lag behind that of mRNAs responsible for production of induced oleoresin monoterpenes . These results suggest that induced (E)-alpha-bisabolene biosynthesis constitutes part of a defense response targeted to insect herbivores, and possibly fungal pathogens, that is distinct from induced oleoresin monoterpene production.

Proc Natl Acad Sci U S A, 1998 Jun 9, 95(12), 6722 - 7
An interplay between TATA box-binding protein and transcription factors IIE and IIA modulates DNA binding and transcription; Yokomori K et al.; The basal transcription factor IIE (TFIIE) is thought to be one of the last factors to be assembled into a preinitiation complex (PIC) at eukaryotic promoters after RNA polymerase II and TFIIF have been incorporated . It was shown that a primary function of TFIIE is to recruit and cooperate with TFIIH in promoter melting . Here, we show that the large subunit of TFIIE (E56) can directly stimulate TBP binding to the promoter in the absence of other basal factors . The zinc-finger domain of E56, required for transcriptional activity, is critical for this function . In addition, the small subunit of TFIIE (E34) directly contacts DNA and TFIIA and thus providing a second mechanism for TFIIE to help binding of a TBP/IIA complex to the promoter, the first critical step in the PIC assembly . These studies suggest an alternative PIC assembly pathway in which TFIIE affects both TBP and TFIIH functions during initiation of RNA synthesis.

Proc Natl Acad Sci U S A, 1998 Jun 9, 95(12), 6607 - 12
Interacting helical faces of subunits a and c in the F1Fo ATP synthase of Escherichia coli defined by disulfide cross-linking; Jiang W et al.; Subunits a and c of Fo are thought to cooperatively catalyze proton translocation during ATP synthesis by the Escherichia coli F1Fo ATP synthase . Optimizing mutations in subunit a at residues A217, I221, and L224 improves the partial function of the cA24D/cD61G double mutant and, on this basis, these three residues were proposed to lie on one face of a transmembrane helix of subunit a, which then interacted with the transmembrane helix of subunit c anchoring the essential aspartyl group . To test this model, in the present work Cys residues were introduced into the second transmembrane helix of subunit c and the predicted fourth transmembrane helix of subunit a . After treating the membrane vesicles of these mutants with Cu(1, 10-phenanthroline)2SO4 at 0 degrees, 10 degrees, or 20 degreesC, strong a-c dimer formation was observed at all three temperatures in membranes of 7 of the 65 double mutants constructed, i.e., in the aS207C/cI55C, aN214C/cA62C, aN214C/cM65C, aI221C/cG69C, aI223C/cL72C, aL224C/cY73C, and aI225C/cY73C double mutant proteins . The pattern of cross-linking aligns the helices in a parallel fashion over a span of 19 residues with the aN214C residue lying close to the cA62C and cM65C residues in the middle of the membrane . Lesser a-c dimer formation was observed in nine other double mutants after treatment at 20 degreesC in a pattern generally supporting that indicated by the seven landmark residues cited above . Cross-link formation was not observed between helix-1 of subunit c and helix-4 of subunit a in 19 additional combinations of doubly Cys-substituted proteins . These results provide direct chemical evidence that helix-2 of subunit c and helix-4 of subunit a pack close enough to each other in the membrane to interact during function . The proximity of helices supports the possibility of an interaction between Arg210 in helix-4 of subunit a and Asp61 in helix-2 of subunit c during proton translocation, as has been suggested previously.

J Biol Chem, 1998 Jun 12, 273(24), 15162 - 8
The b and delta subunits of the Escherichia coli ATP synthase interact via residues in their C-terminal regions; McLachlin DT et al.; An affinity resin for the F1 sector of the Escherichia coli ATP synthase was prepared by coupling the b subunit to a solid support through a unique cysteine residue in the N-terminal leader . b24-156, a form of b lacking the N-terminal transmembrane domain, was able to compete with the affinity resin for binding of F1 . Truncated forms of b24-156, in which one or four residues from the C terminus were removed, competed poorly for F1 binding, suggesting that these residues play an important role in b-F1 interactions . Sedimentation velocity analytical ultracentrifugation revealed that removal of these C-terminal residues from b24-156 resulted in a disruption of its association with the purified delta subunit of the enzyme . To determine whether these residues interact directly with delta, cysteine residues were introduced at various C-terminal positions of b and modified with the heterobifunctional cross-linker benzophenone-4-maleimide . Cross-links between b and delta were obtained when the reagent was incorporated at positions 155 and 158 (two residues beyond the normal C terminus) in both the reconstituted b24-156-F1 complex and the membrane-bound F1F0 complex . CNBr digestion followed by peptide sequencing showed the site of cross-linking within the 177-residue delta subunit to be C-terminal to residue 148, possibly at Met-158 . These results indicate that the b and delta subunits interact via their C-terminal regions and that this interaction is instrumental in the binding of the F1 sector to the b subunit of F0.

J Biol Chem, 1998 Jun 12, 273(24), 15157 - 61
Disruption of Escherichia coli hepA, an RNA polymerase-associated protein, causes UV sensitivity; Muzzin O et al.; During the development of purification procedures for Escherichia coli RNA polymerase (RNAP), we noticed the consistent co-purification of a 110-kDa polypeptide . Here, we report the identification of the 110-kDa protein as the product of the hepA gene, a member of the SNF2 family of putative helicases . We have cloned the hepA gene and overexpressed and purified the HepA protein . We show in vitro that RNAP preparations have an ATPase activity only in the presence of HepA and that HepA binds core RNAP competitively with the promoter specificity sigma70 subunit with a 1:1 stoichiometry and a dissociation constant (Kd) of 75 nM . An E . coli strain with a disruption in the hepA gene shows sensitivity to ultraviolet light.

J Biol Chem, 1998 Jun 12, 273(24), 15085 - 90
Mammalian mitochondrial methionyl-tRNA transformylase from bovine liver . Purification, characterization, and gene structure; Takeuchi N et al.; The mammalian mitochondrial methionyl-tRNA transformylase (MTFmt) was partially purified 2,200-fold from bovine liver mitochondria using column chromatography . The polypeptide responsible for MTFmt activity was excised from a sodium dodecyl sulfate-polyacrylamide gel and the amino acid sequences of several peptides were determined . The cDNA encoding bovine MTFmt was obtained and its nucleotide sequence was determined . The deduced amino acid sequence of the mature form of MTFmt consists of 357 amino acid residues . This sequence is about 30% identical to the corresponding Escherichia coli and yeast mitochondrial MTFs . Kinetic parameters governing the formylation of various tRNAs were obtained . Bovine MTFmt formylates its homologous mitochondrial methionyl-tRNA and the E . coli initiator methionyl-tRNA (Met-tRNAfMet) with essentially equal efficiency . The E . coli elongator methionyl-tRNA (Met-tRNAmMet) was also formylated although with somewhat less favorable kinetics . These results suggest that the substrate specificity of MTFmt is not as rigid as that of the E . coli MTF which clearly discriminates between the bacterial initiator and elongator Met-tRNAs . These observations are discussed in terms of the presence of a single tRNAMet gene in mammalian mitochondria.

J Biol Chem, 1998 Jun 12, 273(24), 14891 - 9
Monoterpene synthases from common sage (Salvia officinalis) . cDNA isolation, characterization, and functional expression of (+)-sabinene synthase, 1,8-cineole synthase, and (+)-bornyl diphosphate synthase; Wise ML et al.; Common sage (Salvia officinalis) produces an extremely broad range of cyclic monoterpenes bearing diverse carbon skeletons, including members of the p-menthane (1,8-cineole), pinane (alpha- and beta-pinene), thujane (isothujone), camphane (camphene), and bornane (camphor) families . An homology-based polymerase chain reaction cloning strategy was developed and used to isolate the cDNAs encoding three multiproduct monoterpene synthases from this species that were functionally expressed in Escherichia coli . The heterologously expressed synthases produce (+)-bornyl diphosphate, 1, 8-cineole, and (+)-sabinene, respectively, as their major products from geranyl diphosphate . The bornyl diphosphate synthase also produces significant amounts of (+)-alpha-pinene, (+)-camphene, and (+/-)-limonene . The 1,8-cineole synthase produces significant amounts of (+)- and (-)-alpha-pinene, (+)- and (-)-beta-pinene, myrcene and (+)-sabinene, and the (+)-sabinene synthase produces significant quantities of gamma-terpinene and terpinolene . All three enzymes appear to be translated as preproteins bearing an amino-terminal plastid targeting sequence, consistent with the plastidial origin of monoterpenes in plants . Deduced sequence analysis and size exclusion chromatography indicate that the recombinant bornyl diphosphate synthase is a homodimer, whereas the other two recombinant enzymes are monomeric, consistent with the size and subunit architecture of their native enzyme counterparts . The distribution and stereochemistry of the products generated by the recombinant (+)-bornyl diphosphate synthase suggest that this enzyme might represent both (+)-bornyl diphosphate synthase and (+)-pinene synthase which were previously assumed to be distinct enzymes.

J Biol Chem, 1998 Jun 12, 273(24), 14667 - 70
A hexameric transmembrane pore revealed by two-dimensional crystallization of the large mechanosensitive ion channel (MscL) of Escherichia coli; Saint N et al.; We have established a reconstitution method of the detergent-solubilized recombinant large mechanosensitive ion channel of Escherichia coli (MscL) that yielded two-dimensional crystals . For that purpose, we have developed a new protocol using Triton X-100 to solubilize and purify the MscL protein . This protocol not only allowed an increase in the protein yield but also made it possible to obtain a homogeneous delipidated and reproducible preparation of the purified protein . When examined by the patch-clamp method MscL channels were found to be fully functional, exhibiting characteristic conductance and activation by pressure . For electron crystallography the homogeneous Triton X-100-purified recombinant MscL was further reconstituted at low lipid-to-protein ratios using Bio-Beads SM2 to remove the detergent . Two-dimensional crystals, exhibiting a p6 plane group symmetry, have been produced and examined by negative stain electron microscopy . Image processing of selected micrographs yielded a projection map at 15-A resolution that provided the first explicit structural information about the molecular boundary and homohexameric organization of the MscL channels in the membrane bilayer.

Endocr J, 1998 Feb, 45(1), 75 - 81
Atrial natriuretic peptide has no potential to protect against endotoxin-induced acute renal failure in the absence of renal nerves; Hiki N et al.; Atrial natriuretic peptide (ANP) has been shown to have the potential to restore renal function after ischemic injury, an underlying component of endotoxin (Et)-induced acute renal failure, and is known to counteract renal sympathetic nerve activity in renal function . We have recently found that renal denervation restores the Et-induced renal dysfunction . The purpose of this study was to examine effects of ANP infusion on the Et-induced acute renal failure in the absence of renal nerves . Ten to 14 days after bilateral renal denervation (DNX), Wistar rats (250 to 300 g body wt) were used in the acute experiment . Rats with intact renal nerves (INN) served as controls . Following control clearance measurements, rats were intravenously injected with 4 mg/kg Et (Escherichia coli, 055: B5) . During endotoxemia, rats were infused with 10 microg/kg/h ANP or saline vehicle . Et injection reduced the glomerular filtration rate (GFR) significantly in saline-infused INN and DNX rats . ANP infusion restored the greatly reduced GFR to the pre-endotoxemia level in DNX rats but not in INN rats . There was significant difference between the ANP- and saline-infused DNX rats in the percentage change relative to the basal GFR value during the ANP infusion period . ANP infusion did not improve the hyponatriuresis and oliguria after Et administration, which is independent of renal nerves . In conclusion, ANP infusion has a minor reno-protective effect in rats with Et-induced acute renal failure in the absence of the renal nerves.

Hum Gene Ther, 1998 May 20, 9(8), 1217 - 21
Adenovirus-mediated in vivo gene transfer in guinea pig middle ear mucosa; Mondain M et al.; This article describes a study designed to assess the feasibility of using recombinant adenovirus for delivering therapeutic peptides in vivo in the guinea pig middle ear cleft . A recombinant adenoviral vector AdCMVsp1 LacZ containing the Escherichia coli beta-galactosidase was injected into the middle ear space . Qualitative assessment of cell middle ear transfection was performed on day 2 by light microscopy study, after injecting a multiplicity of infection (MOI) ranging from 0 to 1000 . At an MOI of 30, 30% of the promontory area epithelial cells were stained . An MOI of 50 stained 60% of the cells and an MOI of 100 or more stained more than 90% of the cells . The duration of cell transfection was studied after injecting an MOI of 50 . The percentage of stained cells was 60% on day 2, 10% on day 7, and 0% on day 14 . Middle ear mucosal inflammation, consisting of a granulocytic infiltrate, was observed when an MOI above 50 was used . Even at a high MOI (500), no staining could be found in the cochlea, in the facial nerve, in the brain, or in visceral organs . These data suggest that recombinant adenovirus vectors can be used to transfer genes in the middle ear . This method appears to be safe, and may be envisaged as a short-duration treatment to transfer genes in vivo in the treatment of middle ear diseases.

Hum Gene Ther, 1998 May 20, 9(8), 1173 - 80
Quantitation of host cell DNA contaminate in pharmaceutical-grade plasmid DNA using competitive polymerase chain reaction and enzyme-linked immunosorbent assay; Lahijani R et al.; The rising interest in gene therapy for the treatment of numerous disorders necessitates the need for the large-scale production of therapeutic biopharmaceuticals that meet stringent purity standards . Residual host cell DNA in recombinant pharmaceuticals has been identified as a potential risk factor that must be quantitated carefully both during the manufacturing process and in the final product . We describe a PCR method to quantitate contaminating levels of host cell DNA in clinical plasmid DNA preparations intended for human gene therapy . The quantitation is based on the coamplification of two similar templates, the target DNA and a synthetic competitor, and the quantitation of the resulting PCR products . The competitor is identical to the target DNA PCR product except for a 29-bp internal replacement . As a result, the two PCR products can easily be distinguished from each other . The competitive nature of the assay allows the use of the ratio of the target DNA PCR product to the competitor DNA PCR product to determine the original amount of target DNA in a sample . The primers used in this assay anneal to a conserved region of the E . coli 23S rRNA gene . One of the primers is biotinylated, allowing the PCR products to be detected colorimetrically after their capture on microtiter plates . The capture is accomplished by differential hybridization to target and competitor-specific probes covalently attached to wells of microtiter plates . The entire assay is performed in less than 2 hr postamplification . This method represents an attractive alternative to Southern blot analysis, which is the currently established method for DNA quantitation.

Hum Gene Ther, 1998 May 20, 9(8), 1143 - 56
Toxin gene-mediated growth inhibition of lung adenocarcinoma in an animal model of pleural malignancy; Hoganson DK et al.; Transduction of malignant cells with toxin genes provides a novel strategy by which to promote tumor cell destruction . Whereas the capacity of the toxin gene/prodrug combination cytosine deaminase/fluorocytosine to inhibit growth of human metastatic pulmonary adenocarcinoma cell lines in vitro is established, the in vivo efficacy of this binary system has not yet been determined . For the development of toxin gene therapy for the treatment of lung adenocarcinoma metastatic to the pleural space, a reliable, disease-specific model is required . The serosa of the rat small intestine resembles the basal lamina of the pleura and provides the basis for a more convenient model than direct injection of tumor into the pleural space . Adenocarcinoma cells are inoculated into everted denuded rat intestine configured as a sac . Immunocytochemical and histological analyses show rapid cell growth with characteristics that mimic nodular metastatic intrapleural disease . In the context of this model, systemically delivered fluorocytosine significantly inhibits the growth of cytosine deaminase-expressing human lung adenocarcinoma cells . The dosing schedule required 30 days; neither addition of an enzyme inhibitor that increases the half-life of fluorocytosine nor intralumenal drug delivery is effective in shortening (to 15 days) the protocol . We conclude that CD continues to hold promise as a toxin gene for lung adenocarcinoma gene therapy, and that prolonged prodrug administration may be required for maximum efficacy.

Antimicrob Agents Chemother, 1998 Jun, 42(6), 1495 - 8
Mode of action of sulfanilyl fluoroquinolones; Alovero F et al.; The mode of action of sulfanilyl fluoroquinolones (NSFQs) was investigated with NSFQ-104, NSFQ-105, and some structurally related compounds . Evidence arising from interactions with p-aminobenzoic acid and trimethoprim suggested that a sulfonamidelike mechanism of action makes little or no contribution to the in vitro activity of NSFQs . NSFQ-105 showed an activity that inhibits gyrase-catalyzed DNA supercoiling that is similar to the activity of other fluoroquinolones . Also, NSFQ-105 uptake was decreased by the presence of Mg2+ and increased by a lower pH . These results indicate that NSFQs having only one ionizable group could exhibit more favorable kinetics of access to the bacterial cell than zwitterionic fluoroquinolones.

Microb Pathog, 1998 May, 24(5), 277 - 88
Binding characteristics of Escherichia coli enterotoxin b (STb) to the pig jejunum and partial characterization of the molecule involved; Rousset E et al.; Escherichia coli heat-stable enterotoxin b (STb) causes severe diarrhoea in weaning piglets . STb most probably has to bind to intestinal epithelial cells in order to achieve its effect . Using biotinylated biologically active STb, we developed a semi-quantitative binding assay using indirect fluorescence microscopy . We demonstrated the attachment of the biotinylated toxin to microvilli of the pig jejunum . However, binding was abolished when biotinylated STb was either boiled or treated with 2-mercaptoethanol, treatments known to abolish biological activity . Different characteristics of STb attachment to the pig small intestine were determined . The reaction was rapid and reached maximum intensity after approximately 10 min . The binding was pH dependent showing an optimum at pH 5.8 . Incubation at either 4 degrees C, 25 degrees C or 37 degrees C did not affect the binding . No competition was observed with non-biotinylated STb . However, preincubation of biotinylated STb with streptavidin conjugated to horseradish peroxidase completely abolished the binding . Pig tissues other than jejunum demonstrated binding towards STb including duodenum, ileum, caecum, colon, liver, lung, spleen and kidney . The molecule involved was then partially characterized . Metaperiodate treatment of the jejunum sections abrogated binding but protease treatment had no effect . Enzymatic treatments of jejunal sections demonstrated that N- and O-glycosidases, and several exoglycosidases did not affect binding, whereas reduced binding was observed with ceramide glycanase and alpha-glucosidase, and was completely abolished following neuraminidase treatment . Overall, our results suggest that in vitro STb binding was rapid, pH dependent, temperature independent, not restricted to jejunum and involves a molecule that seems to be composed of a ceramide moiety, terminal neuraminic acid and/or alpha-linked terminal glucose residue(s) .

Hum Mol Genet, 1998 Jun, 7(6), 1029 - 32
Mutation detection by a two-hybrid assay; Schwartz H et al.; Yeast-based assays have been developed to detect inactivating mutations in human genes, but these assays generally rely on the human protein having a biological function in yeast . We describe a simple method to detect mutations by virtue of their ability to abolish a protein-protein interaction in the yeast two-hybrid assay . By the use of direct recombinational cloning in yeast of a reverse transcription-PCR product followed by a simple growth selection this method distinguished both homozygous and heterozygous mutations in the p53 tumor suppressor gene . This approach should be applicable to many human genes whose encoded proteins have suitable partners in the two-hybrid assay.

Hum Mol Genet, 1998 Jun, 7(6), 991 - 7
Ataxin-3 is transported into the nucleus and associates with the nuclear matrix; Tait D et al.; It has been reported that the ataxin-3 protein containing a polyglutamine sequence in the pathological range (61-84Q) is localized within the nucleus of neuronal cells, whereas ataxin-3 with a normal repeat length (12-37Q) is predominantly a cytoplasmic protein . In this study, the subcellular localization of the full-length ataxin-3 protein with a glutamine sequence in the normal range (Q3KQ22) was analysed in two mammalian cell lines . Using two affinity-purified polyclonal antibodies raised against the N- or C-terminal portion of ataxin-3, the protein was detected predominantly, but not exclusively, in the nucleus of COS-7 as well as neuroblastoma cells by immunofluorescence and confocal laser scanning microscopy (CLSM) . The distribution of the protein in these cellular compartments was confirmed by biochemical subcellular fractionations . Furthermore, CLSM revealed that the ataxin-3 protein present in the nucleus of neuroblastoma cells is associated with the inner nuclear matrix . Our results taken together with the finding of a nuclear localization signal in ataxin-3 indicate that the ataxin-3 protein per se translocates to the nucleus and that an expanded glutamine repeat is not essential for this transport.

Arch Biochem Biophys, 1998 May 1, 353(1), 181 - 9
Molecular cloning, functional expression, and characterization of pyruvate dehydrogenase kinase from anaerobic muscle of the parasitic nematode Ascaris suum; Chen W et al.; The pyruvate dehydrogenase complex (PDC) plays a key role in the anaerobic mitochondrial metabolism of the parasitic nematode Ascaris suum . A cDNA coding for an A . suum pyruvate dehydrogenase kinase (APDK) has been cloned and sequenced from poly(A)+ RNA isolated from adult A . suum muscle.2 APDK exhibited significant sequence identity to mammalian PDKs . Nucleotide sequence analysis of the APDK cDNA revealed a 22-nucleotide spliced leader, characteristic of many nematode mRNAs, a 5'-UTR of 6 nucleotides, an open reading frame of 1197 nucleotides, and a 3'-UTR of 101 nucleotides that included a putative polyadenylation signal . The open reading frame predicted a protein of 399 amino acids with a molecular weight of 45,402 that included a putative 18-aminoacid leader peptide . Recombinant APDK (rAPDK) was functionally expressed in Escherichia coli with a his tag at its N-terminus and purified to apparent homogeneity on Ni-NTA-agarose . Recombinant APDK was a dimer and was not autophosphorylated and its activity was stimulated in the presence of APDK-deficient adult A . suum muscle PDC presumably by the binding of APDK to the dihydrolipoyl transacetylase (E2) core of the complex . After binding to the core, rAPDK activity was stimulated by elevated NADH/NAD+ and acetyl CoA/CoA ratios within the same ranges as observed for the native APDK . Immunoblotting suggested that native APDK focused as a series of 43-kDa spots (pI 6.1-6.8) on two-dimensional gels of the purified adult A . suum muscle PDC .

Arch Biochem Biophys, 1998 May 1, 353(1), 152 - 9
Site-directed mutagenesis of a regulatory site of Escherichia coli ADP-glucose pyrophosphorylase: the role of residue 336 in allosteric behavior; Meyer CR et al.; Site-directed mutagenesis was used to probe the role of glycine residue 336 in the regulatory properties of Escherichia coli ADP-glucose pyrophosphorylase . This residue was previously found to be changed from glycine to aspartate in the gene of an Escherichia coli mutant strain . The mutant enzyme had altered kinetic properties, including higher activity in the absence of the activator fructose 1,6-bisphosphate (FBP), higher apparent affinity for FBP and substrates, and lower apparent affinity for the inhibitor AMP . The observed changes in activity were caused by this single mutation, because the aspartate mutant was prepared from the wild-type gene . The kinetic properties of the site-directed mutant are identical to those of the enzyme from the mutant strain . A series of mutants was prepared to explore the effects of charge, size, shape, and hydrophobicity of the amino acid at residue 336 on the enzyme regulatory properties . All of the mutants, except for the lysine and arginine enzymes, were expressed and purified for kinetic analysis . The glycine-336 residue is able to tolerate diverse substitutions without compromise of catalytic activity . A range of allosteric changes was observed, with the most dramatic effects seen with the highly active aspartate enzyme and the low-activity G336Q mutant, which exhibited lower apparent affinities for activator and substrates and higher apparent affinity for inhibitor . The altered allosteric properties of the G336D mutant enzyme were almost completely abolished by substitution of asparagine . Thus, the aspartate negative charge is essential for the altered binding of effectors .

Arch Biochem Biophys, 1998 May 1, 353(1), 131 - 40
Substrate selectivities and lipid modulation of plant phospholipase D alpha, -beta, and -gamma; Pappan K et al.; Three classes of phospholipase D (PLD), designated PLD alpha, -beta, and -gamma, have been cloned from plants, but their substrate selectivities have not been established . Using active PLDs expressed from their cDNAs in Escherichia coli, we compared the hydrolytic activities of these three PLDs toward various phospholipids and the influence of substrate composition on their substrate selectivities . When single-class phospholipid vesicles of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylinositol 4,5-bisphosphate (PIP2), N-acylphosphatidylethanolamine (NAPE), and cardiolipin (CL) were examined, PLD alpha hydrolyzed PC, PE, and PG but PLD beta and -gamma showed no activity toward any of these lipids . When PIP2 was included in mixed vesicles with the phospholipids above, PLD alpha showed the same PC-, PE-, and PG-hydrolyzing ability, whereas PLD beta and -gamma were able to hydrolyze both PE and PS . When both PE and PIP2 were included in substrate vesicles, PLD beta and PLD gamma hydrolyzed PC, PG, and NAPE, showing that both PE and PIP2 are required for PC, PG, and NAPE hydrolysis by PLD beta and -gamma . The PE activation of PLD beta and -gamma required lipid vesicles made of mostly PE, suggesting that PE may affect the substrate presentation rather than serve as a cofactor of these PLDs . Under equivalent reaction conditions, PLD beta displayed a similar preference for PC and NAPE, whereas PLD gamma preferred NAPE to PC by nearly three times . None of the three PLDs used PI, CL, or PIP2 as substrates . These results have identified PS- and NAPE-hydrolyzing PLDs and have indicated an important role for lipid composition in regulating the substrate selectivity of PLD beta and -gamma .

Arch Biochem Biophys, 1998 May 1, 353(1), 109 - 15
Expression of catalytically active human cytochrome p450scc in Escherichia coli and mutagenesis of isoleucine-462; Woods ST et al.; Cytochrome P450scc (P450scc) catalyzes the first step in steroid hormone synthesis, the conversion of cholesterol to pregnenolone . Human P450scc has been poorly studied due to the difficulty of purifying reasonable quantities of enzyme from human tissue . To provide a more convenient source of the human enzyme and to enable structure-function studies to be done using site-directed mutagenesis, we expressed the mature form of human P450scc in Escherichia coli . The expression system enabled us to produce larger quantities of active cytochrome than have previously been isolated from placental mitochondria . The expressed P450scc was purified to near homogeneity and shown to have catalytic properties comparable to the enzyme purified from the human placenta . The mature form of human adrenodoxin was also expressed in E . coli and supported cholesterol side chain cleavage activity with the same Vmax as that observed using bovine adrenodoxin but with a higher Km . Mutation of Ile-462 to Leu in human P450scc caused a decrease in the catalytic rate constant (kcat) with cholesterol as substrate, increased the Km for 22R-hydroxycholesterol, but did not affect the kinetic constants for 20 alpha-hydroxycholesterol . This suggests that Ile-462 lies close to the side chain binding site and that the side chains of cholesterol, 22R-hydroxycholesterol, and 20 alpha-hydroxycholesterol occupy slightly different positions in the active site .

Arch Biochem Biophys, 1998 May 1, 353(1), 64 - 72
Purification and characterization of maize starch synthase I and its truncated forms; Imparl-Radosevich JM et al.; Comparison of the protein sequences deduced from the cDNAs of maize granule-bound starch synthase, Escherichia coli glycogen synthase, and maize starch synthase I (SSI) reveals that maize SSI contains an N-terminal extension of 93 amino acids . In order to study the properties of maize SSI and to understand the functions of the maize SSI N-terminal extension, the gene coding for full-length SSI (SSI-1) and genes coding for N-terminally truncated SSI (SSI-2 and SSI-3) were individually expressed in E . coli . Here we describe for the first time the purification of a higher plant starch synthase to apparent homogeneity . Its kinetic properties were therefore studied in the absence of interfering amylolytic enzymes . The specific activities of the purified SSI-1, SSI-2, and SSI-3 were 22.5, 33.4, and 26.3 micromol Glc/min/mg of protein, respectively, which are eight times higher than those of partially purified SSI from developing maize endosperm . The full-length recombinant enzyme SSI-1 exhibited properties similar to those of the enzyme from maize endosperm . As observed for native maize enzyme, recombinant SSI-1 exhibited "unprimed" activity without added primer in the presence of 0.5 M citrate . Our results have clearly indicated that the catalytic center of SSI is not located in its N-terminal extension . However, N-terminal truncation decreased the enzyme affinity for amylopectin, with the Km for amylopectin of the truncated SSI-3 being about 60-90% higher than that of the full-length SSI-1 . These results suggest that the N-terminal extension in SSI may not be directly involved in enzyme catalysis, but may instead regulate the enzyme binding of alpha-glucans . Additionally, the N-terminal extension may play a role in determining the localization of SSI to specific portions of the starch granule or it may regulate its interactions with other enzymes involved in starch synthesis .

Arch Biochem Biophys, 1998 May 1, 353(1), 55 - 63
Comparative study on recombinant chloroplastic and cytosolic ascorbate peroxidase isozymes of spinach; Yoshimura K et al.; The spinach stromal, thylakoid-bound, and cytosolic ascorbate peroxidase isozymes (EC 1.11.1.11) were overexpressed in Escherichia coli, and their enzymatic properties were compared with the respective native isozymes . The purification of the recombinant stromal and cytosolic ascorbate peroxidases using the conventional column chromatography yielded 0.73 and 2.2 mg of protein/liter of bacteria culture with enzyme activities of 800 and 486 micromol min-1 mg protein-1, respectively . In every respect, the recombinant stromal, thylakoid-bound, and cytosolic ascorbate peroxidase isozymes exhibited identical enzymatic properties with each native isozyme . Specifically, the recombinant stromal and thylakoid-bound ascorbate peroxidase isozymes showed high utilization of ascorbate as an electron donor and had a very short lifetime in ascorbate-depleted medium . Polyclonal antibodies raised against both purified recombinant stromal and cytosolic ascorbate peroxidase isozymes were prepared . Both antibodies showed a cross-reaction with the recombinant and native ascorbate peroxidase isozymes .

Arch Biochem Biophys, 1998 May 1, 353(1), 16 - 28
Characterization of CYP2C19 and CYP2C9 from human liver: respective roles in microsomal tolbutamide, S-mephenytoin, and omeprazole hydroxylations; Lasker JM et al.; Individuals with drug metabolism polymorphisms involving CYP2C enzymes exhibit deficient oxidation of important therapeutic agents, including S-mephenytoin, omeprazole, warfarin, tolbutamide, and nonsteroidal anti-inflammatory drugs . While recombinant CYP2C19 and CYP2C9 proteins expressed in yeast or Escherichia coli have been shown to oxidize these agents, the capacity of the corresponding native P450s isolated from human liver to do so is ill defined . To that end, we purified CYP2C19, CYP2C9, and CYP2C8 from human liver samples using conventional chromatographic techniques and examined their capacity to oxidize S-mephenytoin, omeprazole, and tolbutamide . Upon reconstitution, CYP2C19 metabolized S-mephenytoin and omeprazole at rates that were 11- and 8-fold higher, respectively, than those of intact liver microsomes, whereas neither CYP2C9 nor CYP2C8 displayed appreciable metabolic activity with these substrates . CYP2C19 also proved an efficient catalyst of tolbutamide metabolism, exhibiting a turnover rate similar to CYP2C9 preparations (2.0-6.4 vs 2.4-4.3 nmol hydroxytolbutamide formed/min/nmol P450) . The kinetic parameters of CYP2C19-mediated tolbutamide hydroxylation (Km = 650 microM, Vmax = 3.71 min-1) somewhat resembled those of the CYP2C9-catalyzed reaction (Km = 178-407 microM, Vmax = 2.95-7.08 min-1) . Polyclonal CYP2C19 antibodies markedly decreased S-mephenytoin 4'-hydroxylation (98% inhibition) and omeprazole 5-hydroxylation (85% inhibition) by human liver microsomes . CYP2C19 antibodies also potently inhibited (>90%) microsomal tolbutamide hydroxylation, which was similar to the inhibition (>85%) observed with antibodies to CYP2C9 . Moreover, excellent correlations were found between immunoreactive CYP2C19 content, S-mephenytoin 4'-hydroxylase activity (r = 0.912; P < 0 . 001), and omeprazole 5-hydroxylase activity (r = 0.906; P < 0.001) in liver samples from 13-17 different subjects . A significant relationship was likewise observed between microsomal tolbutamide hydroxylation and CYP2C9 content (r = 0.664; P < 0.02) but not with CYP2C19 content (r = 0.393; P = 0.184) . Finally, immunoquantitation revealed that in these human liver samples, expression of CYP2C9 (88 . 5 +/- 36 nmol/mg) was 5-fold higher than that of CYP2C19 (17.8 +/- 14 nmol/mg) and nearly 8-fold higher than that of CYP2C8 (11.5 +/- 12 nmol/mg) . Our results, like those obtained with recombinant CYP2C enzymes, indicate that CYP2C19 is a primary determinant of S-mephenytoin 4'-hydroxylation and low-Km omeprazole 5-hydroxylation in human liver . Despite its tolbutamide hydroxylase activity, the low levels of hepatic CYP2C19 expression (relative to CYP2C9) may preclude an important role for this enzyme in hepatic tolbutamide metabolism and any polymorphisms thereof .

Biochemistry, 1998 May 5, 37(18), 6606 - 13
Thermal unfolding of three acclimation temperature-associated isoforms of carp light meromyosin expressed by recombinant DNAs; Kakinuma M et al.; Differential scanning calorimetry (DSC) was performed to investigate thermodynamic properties of three carp fast skeletal light meromyosin (LMM) isoforms expressed in Escherichia coli by recombinant DNAs . Three isoforms were the 10 degreesC-, intermediate-, and 30 degreesC-type LMM predominantly expressed in carp acclimated to 10, 20, and 30 degreesC . The isoforms expressed in E . coli by recombinant DNAs exhibited a typical pattern of alpha-helix in CD spectroscopy with two minima at 222 and 208 nm . Moreover, the three isoforms formed paracrystals typical of LMM, suggesting that expressed proteins retained intact structural properties . When the LMM isoforms were subjected to DSC analysis, the 10 degreesC and 30 degreesC types showed endotherms having transition temperatures (Tm) at 35.1 and 39.5 degreesC, respectively, which are responsible for thermal unfolding of alpha-helix . The intermediate type exhibited two comparable endotherms with Tm values at 34.9 and 40.6 degreesC, implying that it has intermediate thermodynamic properties between those of 10 degreesC and 30 degreesC types . However, a chimeric LMM having the 10 degreesC and 30 degreesC type as N- and C-terminal halves, respectively, showed the DSC pattern typical of the whole 30 degreesC-type molecule . On the other hand, another chimeric LMM composed of the N-terminal 30 degreesC type and C-terminal 10 degreesC type gave the pattern of the full 10 degreesC type . These results suggest that thermodynamic properties of the C-terminal half largely account for thermal unfolding of the whole molecule.

Biochemistry, 1998 May 5, 37(18), 6572 - 85
Identification of O-glycosylation sites and partial characterization of carbohydrate structure and disulfide linkages of human insulin-like growth factor binding protein 6; Neumann GM et al.; The actions of insulin-like growth factors (IGFs) are modulated by a family of high-affinity binding proteins (IGFBPs), including IGFBP-6, which preferentially binds IGF-II and is O-glycosylated . Glycosylated and nonglycosylated recombinant human IGFBP-6, expressed in Chinese hamster ovary cells and Escherichia coli, respectively, were purified using IGF-II affinity chromatography and reverse-phase medium-pressure chromatography . Electrospray ionization mass spectrometry (ESMS) of glycosylated IGFBP-6 revealed considerable heterogeneity of carbohydrate composition . Major glycoforms contained 8-16 monosaccharides, including N-acetylhexosamine, hexose, and N-acetylneuraminic acid . Glycosylation sites of IGFBP-6 were identified as Thr126, Ser144, Thr145, Thr146, and Ser152 by using a combination of ESMS and Edman sequencing of tryptic fragments separated by reverse-phase high-pressure liquid chromatography . One oligosaccharide chain contained 5-6 monosaccharides, whereas the others contained 2-4 monosaccharides . Glycosylated IGFBP-6 exhibited greater resistance to proteolysis by chymotrypsin and trypsin than nonglycosylated IGFBP-6 . Native disulfide bond positions in IGFBP-6 were localized by means of observed disulfide-linked tryptic fragments, revealing that there are two disulfide-linked subdomains within each of the N- and C-terminal regions and confirming a previous suggestion that the latter regions are not interconnected . A model of IGFBP-6 is developed in which these distinct domains are separated by a central region which is O-glycosylated.

Biochemistry, 1998 May 5, 37(18), 6485 - 90
DNA structural integrity and base composition affect ultraviolet light-induced oxidative DNA damage; Wei H et al.; We previously demonstrated that ultraviolet (UV) light (254 nm) induced the formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in DNA via a singlet oxygen mechanism . In the present paper, we provide novel findings that DNA structure and base composition significantly affect the yield of 8-OHdG by UV radiation . Unlike ionizing radiation that induces 8-OHdG both in free 2'-deoxyguanosine (dG) and in DNA, UV light induced 8-OHdG formation in intact DNA and polydG.dC, but not in dG . When thermally denatured DNA was irradiated with UV light, the yield of 8-OHdG was reduced by more than 80% compared to intact DNA . Oxygenation of the denatured DNA solution did not restore the yield of UV-induced 8-OHdG . Irradiation of DNA with different AT/GC ratios showed that the yield of UV-induced 8-OHdG varied in proportion to the AT content, suggesting that AT base pairs in DNA enhance generation of the oxidizing species and subsequent oxidation of dG . The natural antioxidants genistein, estradiol, protocatechuic acid (PCA), and oleanolic acid (OA) were investigated for their inhibition of UV-induced 8-OHdG . Genistein and estradiol, that intercalate into DNA as shown by a computer modeling, significantly quenched UV-induced 8-OHdG, whereas PCA and OA did not fit into DNA and exhibited weak or no effect . These results suggest that the intercalation of genistein and estradiol into DNA may alter the DNA structural integrity, interrupt the production of oxidizing species, and subsequently reduce the formation of 8-OHdG by UV radiation.

Biochemistry, 1998 May 5, 37(18), 6465 - 75
A substrate recognition role for the {4Fe-4S}2+ cluster of the DNA repair glycosylase MutY; Porello SL et al.; The Escherichia coli DNA repair enzyme MutY plays an important role in the recognition and repair of 7, 8-dihydro-8-oxo-2'-deoxyguanosine: 2'-deoxyadenosine (OG:A) mismatches in DNA {Michaels et al . (1992) Proc . Natl . Acad . Sci . U.S . A . 89, 7022-7025} . MutY prevents mutations due to misincorporation of A opposite OG during DNA replication by removing the adenine base . This enzyme has significant sequence homology with the {4Fe-4S}2+ cluster-containing DNA repair enzyme, endonuclease III {Michaels et al . (1990) Nucleic AcidsRes . 18, 3841-3845} . In the present study, we have investigated the importance of cluster assembly in folding of MutY . MutY was denatured and then refolded in the presence or absence of ferrous and sulfide ions . Denatured MutY can refold in the presence of ferrous and sulfide ions to provide active enzyme . This suggests the cluster can self-assemble and that this process is facile in vitro . Interestingly, CD spectra and Tm measurements of MutY refolded with and without ferrous and sulfide ions are essentially identical, implying that assembly of the cluster is not required for MutY folding . Additionally, Tm measurements indicated that the {4Fe-4S}2+ cluster does not contribute significantly to the overall thermal stability of MutY . Refolded forms of MutY which lack the cluster are unable to perform the adenine glycosylase function and bind to DNA . However, these inactive folded forms regain activity by addition of ferrous and sulfide ions . This indicates that the Fe-S cluster may have a superficial location, allowing for its assembly after folding . More importantly, these results provide evidence that the presence of the {4Fe-4S}2+ cluster is critical for the specific recognition of substrate DNA necessary for the adenine glycosylase activity of MutY.

Biochemistry, 1998 May 5, 37(18), 6419 - 26
Detection of a new substrate-derived radical during inactivation of ribonucleotide reductase from Escherichia coli by gemcitabine 5'-diphosphate; van der Donk WA et al.; Ribonucleotide reductases (RNRs) play a central role in replication and repair by catalyzing the conversion of nucleotides to deoxynucleotides . Gemcitabine 5'-diphosphate (F2CDP), the nucleoside of which was recently approved by the FDA for treatment of pancreatic cancer, is a potent mechanism-based inhibitor of class I and II RNRs . Inactivation of the Eschericia coli class I RNR is accompanied by loss of two fluorides and one cytosine . This RNR is composed of two homodimeric subunits: R1 and R2 . R1 is the site of nucleotide reduction, and R2 contains the essential diferric-tyrosyl radical cofactor . The mechanism of inactivation depends on the availability of reductant . In the presence of reductant {thioredoxin (TR)/thioredoxin reductase (TRR)/NADPH or dithiothreitol}, inhibition results from R1 inactivation . In the absence of reductant with prereduced R1 and R2, inhibition results from loss of the essential tyrosyl radical in R2 . The same result is obtained with C754S/C759S-R1 in the presence of TR/TRR/NADPH . In both cases, tyrosyl radical loss is accompanied by formation of a new stable radical (0.15-0.25 equiv/RNR) . EPR studies in 2H2O, with {U-2H}R1, and examination of the microwave power saturation of the observed signal, indicate by process of elimination that this new radical is nucleotide-based . In contrast to all previously investigated 2'-substituted nucleotide inhibitors of RNR, inactivation is not accompanied by formation of a new protein-associated chromophore under any conditions . The requirement for reductant in the R1 inactivation pathway, the lack of chromophore on the protein, the loss of two fluoride ions, and the stoichiometry of the inactivation all suggest a unique mechanism of RNR inactivation not previously observed with other 2'-substituted nucleotide inhibitors of RNR . This unique mode of inactivation is proposed to be responsible for its observed clinical efficacy.

Biochemistry, 1998 May 5, 37(18), 6387 - 93
Reversible denaturation, self-aggregation, and membrane activity of Escherichia coli alpha-hemolysin, a protein stable in 6 M urea; Soloaga A et al.; Escherichia coli alpha-hemolysin (HlyA) is an extracellular protein toxin (107 kDa) whose cell lytic activity may be preserved for months at -20 degreesC in the presence of 6 M urea, although it decays rapidly in urea-free buffers . This paper describes experiments addressed to unravel the role of urea in HlyA stabilization . Urea up to 8 M inhibits the Ca2+-binding and hemolytic activities of the protein, alters its secondary and tertiary structures, and reduces its tendency to self-aggregation . All these changes are largely reversed upon urea removal by dilution or dialysis, suggesting that they are interrelated . Furthermore, the extent of recovery of the native activities and structural features of alpha-hemolysin that follows urea removal increases with the concentration of urea during the previous phase . Thus, it seems that urea elicits the reversible transition of HlyA to a less active but more stable state whose structure differs significantly from that of the native protein . Moreover dialysis equilibration of the protein with buffers containing 3 M urea induces the formation of a molecular form of HlyA 5-10 times more active than the native protein in the absence of urea . This hyperactive intermediate appears to keep the native secondary structure of HlyA, but with a less compact tertiary structure, that increases the number of exchangeable Ca2+ ions under these conditions . Changes in the intrinsic fluorescence of HlyA also support the notion of a conformational change in the high-activity intermediate . The intermediate is only detected when assayed in the presence of Ca2+ and 3 M urea and can bind a large number of calcium ions (approximately 12 vs approximately 3 for the native protein); it shows a large tendency to self-aggregation and presumably, in the presence of membranes, a similar tendency to irreversible insertion, which may be the reason for its high lytic activity.

Biochemistry, 1998 May 5, 37(18), 6351 - 60
Characterization of the ligand binding activities of vitronectin: interaction of vitronectin with lipids and identification of the binding domains for various ligands using recombinant domains; Yoneda A et al.; Vitronectin is a multifunctional plasma glycoprotein which may regulate the systems related to protease cascades such as the coagulation, fibrinolysis, and complement systems as well as cell adhesion . Solid-phase assays and affinity chromatography on immobilized glycolipids indicated that vitronectin purified under denaturing conditions bound to sulfatide (Gal(3-SO4)beta1-1ceramide), cholesterol 3-sulfate, and various phospholipids, but not gangliosides . Only the unfolded or multimeric form of vitronectin bound to sulfatide, suggesting a conformational dependency of the binding activity, while vitronectin bound to cholesterol 3-sulfate regardless of its conformational state . The recombinant domains of human vitronectin and mutants with certain domains deleted were separately expressed in E . coli as fusion proteins . Using the recombinants, sulfatide-, phosphatidylserine-, cholesterol 3-sulfate-, Type I collagen-, heparin-, and beta-endorphin-binding activities were found to be attributable to hemopexin domain 2 and hemopexin domain 1 . The possibility was suggested that the presence of a somatomedin domain and/or connecting region flanking hemopexin domain 1 inactivated its heparin binding . De-N-glycosylation of plasma vitronectin significantly affected the cholesterol sulfate- and collagen-binding activities, although its effects were opposite . These findings suggest that diverse ligand-binding activities could be attributed to pexin family motifs but that the interdomain interactions and glycosylations modulate the ligand binding activities of vitronectin.

Biochemistry, 1998 May 5, 37(18), 6336 - 42
Strength of an interloop hydrogen bond determines the kinetic pathway in catalysis by Escherichia coli dihydrofolate reductase; Miller GP et al.; On the basis of X-ray crystallographic data, Sawaya and Kraut proposed that Met20 loop conformational changes modulate ligand specificity observed in the catalytic cycle for Escherichia coli dihydrofolate reductase (DHFR) {Sawaya, M . R., and Kraut, J . (1997) Biochemistry 36, 586-603} . Interloop hydrogen bonds stabilize either a closed Met20 loop conformation observed in substrate complexes or an occluded Met20 loop conformation observed in product complexes, respectively . To test this model, we targeted a single hydrogen bond occurring exclusively in the closed Met20 loop conformation . Specifically, Asp122 in the betaF-betaG loop was independently substituted with asparagine, serine, and alanine-amino acids with decreasing abilities to hydrogen-bond . The kinetic analyses of the Asp122 mutants enabled the construction of kinetic schemes at pH 7.0 that demonstrate two striking features . First, a significant correlation exists between decreased binding of nicotinamide adenine dinucleotide phosphate, reduced (NADPH), and decreased hydride transfer rates resulting from these mutations . In other words, the interactions of Asp122 are along the reaction coordinate leading to the transition state . Second, substitutions for Asp122 alter the catalytic pathway preferred by wild-type DHFR under saturating conditions of substrate and cofactor . Overall, the steady-state rate contains contributions from the product off rates from the DHFR.5,6, 7,8-tetrahydrofolate (H4F) and DHFR.NADPH.H4F complexes and from the rate of hydride transfer . These mutational effects support the mechanistic model whereby interloop contacts regulate an equilibrium of Met20 loop conformations that, in turn, modulate ligand affinity and turnover.

Biochemistry, 1998 May 5, 37(18), 6263 - 76
Structure of reduced DsbA from Escherichia coli in solution; Schirra HJ et al.; The three-dimensional structure of reduced DsbA from Escherichia coli in aqueous solution has been determined by nuclear magnetic resonance (NMR) spectroscopy and is compared with the crystal structure of oxidized DsbA {Guddat, L . W., Bardwell, J . C . A., Zander, T., and Martin, J . L . (1997) Protein Sci . 6, 1148-1156} . DsbA is a monomeric 21 kDa protein which consists of 189 residues and is required for disulfide bond formation in the periplasm of E . coli . On the basis of sequence-specific 1H NMR assignments, 1664 nuclear Overhauser enhancement distance constraints, 118 hydrogen bond distance constraints, and 293 dihedral angle constraints were obtained as the input for the structure calculations by simulated annealing with the program X-PLOR . The enzyme is made up of two domains . The catalytic domain has a thioredoxin-like fold with a five-stranded beta-sheet and three alpha-helices, and the second domain consists of four alpha-helices and is inserted into the thioredoxin motif . The active site between Cys30 and Cys33 is located at the N terminus of the first alpha-helix in the thioredoxin-like domain . The solution structure of reduced DsbA is rather similar to the crystal structure of the oxidized enzyme but exhibits a different relative orientation of both domains . In addition, the conformations of the active site and a loop between strand beta5 and helix alpha7 are slightly different . These structural differences may reflect important functional requirements in the reaction cycle of DsbA as they appear to facilitate the release of oxidized polypeptides from reduced DsbA . The extremely low pKa value of the nucleophilic active site thiol of Cys30 in reduced DsbA is most likely caused by its interactions with the dipole of the active site helix and the side chain of His32, as no other charged residues are located next to the sulfur atom of Cys30 in the solution structure.

J Biol Chem, 1998 May 8, 273(19), 11815 - 25
An RNA pseudoknot as the molecular switch for translation of the repZ gene encoding the replication initiator of IncIalpha plasmid ColIb-P9; Asano K et al.; Translation initiation of the repZ gene encoding the replication initiator of plasmid ColIb-P9 is not only negatively regulated by the action of the antisense Inc RNA encoded in the leader region, but is also coupled to the translation and termination of a transcribed leader sequence, repY, a positive regulatory element for repZ gene expression . This translational coupling depends on base pairing between two complementary sequences, 5'-rGGCG-3' and 5'-rCGCC-3', which are located upstream of and in the middle of repY, respectively, and have the potential to form a pseudoknot with the stem-loop structure I . Another stem-loop called structure III near the 3'-end of repY sequesters both the 5'-rCGCC-3' sequence and the repZ ribosome-binding site . Here we show that the RepZ mRNA leader sequence synthesized in vitro indeed contains several stem-loop structures including structures I and III, but not the pseudoknot . However, disruption of structure III, without changing the repZ ribosome-binding site, by means of base substitution and deletion induces base pairing between the two short complementary sequences distantly separated, resulting in the formation of a pseudoknot . When the pseudoknot is allowed to form in vivo due to the same mutations, a maximum level of repZ expression is obtained comparable to one observed in the absence of Inc RNA . These results strengthen our previously proposed model that the pseudoknot induced by the translation and termination of the repY reading frame functions as the molecular switch for translational initiation of the repZ gene.

J Biol Chem, 1998 May 8, 273(19), 11752 - 7
Characterization of the glycosyltransferase enzyme from the Escherichia coli K5 capsule gene cluster and identification and characterization of the glucuronyl active site; Griffiths G et al.; Bacterial capsular polysaccharides play an important role in virulence and survival . The Escherichia coli K5 capsule consists of a repeat structure of -4)GlcA-beta(1,4)-GlcNAc alpha(1-, identical to N-acetylheparosan . A 60-kDa protein, KfiC, has been identified as a bifunctional glycosyltransferase, responsible for the alternating alpha and beta addition of each UDP-sugar to the nonreducing end of the polysaccharide chain . Using hydrophobic cluster analysis, a conserved secondary structure motif characteristic of beta-glycosyltransferases was identified along with two highly conserved aspartic acid residues at positions 301 and 352 within the KfiC protein . Site-directed mutagenesis was used to identify catalytically active amino acids within domain A of the KfiC protein . The conserved aspartic acid residues at 301 and 352 were shown to be critical for the beta addition of UDP-GlcA (uridine diphosphoglucuronic acid) to defined nonreducing end oligosaccharide acceptors, suggesting that these conserved aspartic acid residues are catalytically important for beta-glycosyltransferase activity . A deleted derivative of the kfiC gene was generated, which encoded for a truncated KfiC (kfiC') protein . This protein lacked 139 amino acids at the C terminus . This enzyme had no UDP-GlcA transferase activity but still retained UDP-GlcNAc transferase activity, indicating that two separate active sites are present within the KfiC protein.

J Biol Chem, 1998 May 8, 273(19), 11611 - 8
Isolation and characterization of thymitaq (AG337) and 5-fluoro-2-deoxyuridylate-resistant mutants of human thymidylate synthase from ethyl methanesulfonate-exposed human sarcoma HT1080 cells; Tong Y et al.; Thymidylate synthase plays an essential role in the synthesis of DNA . Recently, several new and specific thymidylate synthase inhibitors that occupy the folate binding site, including Tomudex(R), BW1843U89, and Thymitaq, have demonstrated therapeutic activity in patients with advanced cancer . In order to find drug-resistant forms of human thymidylate synthase for gene therapy applications, human sarcoma HT1080 cells were exposed to ethyl methanesulfonate and Thymitaq selection . Thymitaq-resistant clonal derived sublines were established, and analysis indicated that both gene amplification and point mutations contributed to drug resistance . Eight mutant cDNAs that were identified from Thymitaq-resistant sublines were generated by site-directed mutagenesis and transfected into thymidylate synthase-negative cells . Only K47E, D49G, or G52S mutants retain enzyme activity . Moreover, cytotoxicity studies demonstrated that D49G and G52S transfected cells, besides displaying resistance to Thymitaq with IC50 values 40- and 12-fold greater than wild-type enzyme transfected cells, respectively, also lead to fluorodeoxyuridine resistance (26- and 97-fold in IC50 values, respectively) but not to Tomudex or BW1843U89 . Characterization of the purified altered enzymes obtained from expression in Escherichia coli is consistent with the cell growth inhibition results . We postulate that the D49G or G52S mutation leads to the structural perturbation of the highly conserved Arg50 loop, decreasing the binding of thymidylate synthase to the inhibitors, Thymitaq and fluorodeoxyuridylate.

J Biol Chem, 1998 May 8, 273(19), 11605 - 10
Sequences outside recognition sets are not neutral for tRNA aminoacylation . Evidence for nonpermissive combinations of nucleotides in the acceptor stem of yeast tRNAPhe; Frugier M et al.; Phenylalanine identity of yeast tRNAPhe is governed by five nucleotides including residues A73, G20, and the three anticodon nucleotides (Sampson et al., 1989, Science 243, 1363-1366) . Analysis of in vitro transcripts derived from yeast tRNAPhe and Escherichia coli tRNAAla bearing these recognition elements shows that phenylalanyl-tRNA synthetase is sensitive to additional nucleotides within the acceptor stem . Insertion of G2-C71 has dramatic negative effects in both tRNA frameworks . These effects become compensated by a second-site mutation, the insertion of the wobble G3-U70 pair, which by itself has no effect on phenylalanylation . From a mechanistic point of view, the G2-C71/G3-U70 combination is not a "classical" recognition element since its antideterminant effect is compensated for by a second-site mutation . This enlarges our understanding of tRNA identity that appears not only to be the outcome of a combination of positive and negative signals forming the so-called recognition/identity set but that is also based on the presence of nonrandom combinations of sequences elsewhere in tRNA . These sequences, we name "permissive elements," are retained by evolution so that they do not hinder aminoacylation . Likely, no nucleotide within a tRNA is of random nature but has been selected so that a tRNA can fulfill all its functions efficiently.

J Biol Chem, 1998 May 8, 273(19), 11429 - 35
Identification and characterization of a novel human aldose reductase-like gene; Cao D et al.; We have identified a novel human protein that is highly homologous to aldose reductase (AR) . This protein, which we called ARL-1, consists of 316 amino acids, the same size as AR, and its amino acid sequence is 71% identical to that of AR . It is more closely related to the AR-like proteins such as mouse vas deferens protein, fibroblast growth factor-regulated protein, and Chinese hamster ovary reductase, with 81, 82, and 83%, respectively, of its amino acid sequence identical to the amino acid sequence of these proteins . The cDNA of ARL-1 was expressed in Escherichia coli to obtain recombinant protein for characterization of its enzymatic activities . For comparison, the cDNA of human AR was also expressed in E . coli and analyzed in parallel . These two enzymes differ in their pH optima and salt requirement, but they act on a similar spectrum of substrates . Similar to AR, ARL-1 can efficiently reduce aliphatic and aromatic aldehydes, and it is less active on hexoses . While AR mRNA is found in most tissues studied, ARL-1 is primarily expressed in the small intestines and in the colon, with a low level of its mRNA in the liver . The ability of ARL-1 to reduce various aldehydes and the locations of expression of this gene suggest that it may be responsible for detoxification of reactive aldehydes in the digested food before the nutrients are passed on to other organs . Interestingly, ARL-1 and AR are overexpressed in some liver cancers, but it is not clear if they contribute to the pathogenesis of this disease.

J Biol Chem, 1998 May 8, 273(19), 11409 - 12
Synthetic signal peptides specifically recognize SecA and stimulate ATPase activity in the absence of preprotein; Miller A et al.; Although it is known that virtually all exported proteins require a signal peptide, it is not clearly understood how the signal peptide interfaces with the translocation machinery to achieve transport . In this study we document a direct interaction between the signal peptide and SecA, a primary component of the translocase in Escherichia coli, and show that the signal peptide itself can stimulate SecA-lipid ATPase activity . Using synthetic signal peptides corresponding to the wild type alkaline phosphatase signal sequence and two model sequences, we find that the extent of stimulation of SecA ATPase activity by the different peptides parallels the hierarchy of results found for in vivo function (Izard, J . W., Doughty, M . B., and Kendall, D . A . (1995) Biochemistry 34, 9904-9912) . The peptide-induced activity requires a lipid to protein molar ratio of at least 300:1 and liposomes enriched in negatively charged phospholipids . Furthermore, specific binding of the signal peptide to SecA was demonstrated using chemical cross-linking and competition with unlabeled peptides.

Shock, 1998 Apr, 9(4), 296 - 303
Alterations in hepatic gluconeogenic amino acid uptake and gluconeogenesis in the endotoxin treated conscious dog; Meinz H et al.; We examined the effect of a 240 min intraportal infusion of a nonlethal dose of Escherichia coli endotoxin (.21 g x kg(-1) x min{-1}) on hepatic amino acid and glucose metabolism in chronically catheterized 42 h fasted conscious dogs (n = 8) . Hepatic metabolism was assessed using tracer (3-{3H}glucose {U-14C}alanine) and arteriovenous difference techniques . After endotoxin administration net hepatic glucose output increased twofold . Arterial plasma insulin levels decreased by 25%, whereas arterial plasma glucagon and cortisol levels increased 10- and 6-fold, respectively . Arterial lactate levels increased 6.4-fold, whereas net hepatic lactate uptake was not increased . Arterial alanine levels (1.6-fold) and net hepatic alanine uptake (1.3-fold) increased, whereas net hepatic alanine fractional extraction was unaltered . In contrast, the arterial levels of the other gluconeogenic amino acids (glutamine, glycine, serine, and threonine) decreased . Despite this decrease, net uptake of these amino acids by the liver did not decrease, because net hepatic amino acid fractional extraction increased . Total net hepatic gluconeogenic precursor uptake was unaltered (1.1 +/- .1 to 1.3 +/- .3 mg x kg(-1) x min(-1) expressed in glucose equivalents) . In summary, gluconeogenesis does not increase after endotoxin administration . Thus, an increase in net hepatic glycogenolysis accounts for the majority of the increase in hepatic glucose production . The lack of an increase in alanine fractional extraction, despite hyperglucagonemia and a rise in the fractional extraction of other gluconeogenic amino acids, suggests that endotoxin specifically impairs hepatic alanine entry in vivo.

Shock, 1998 Apr, 9(4), 274 - 81
Low molecular weight heparin prevents the pulmonary hemodynamic and pathomorphologic effects of endotoxin in a porcine acute lung injury model; Darien BJ et al.; Tumor necrosis factor alpha (TNF-alpha) activity, platelet and neutrophil degranulation and margination, and increased vascular permeability are central to the pathophysiology of endotoxin-mediated acute lung injury . Nonanticoagulant activities of low molecular weight heparin (LMWH) include solubilization of the TNF-alpha receptor protein, inhibition of neutrophil adhesion, and regulation of thromboxane B2 (TXB2) biosynthesis . In this study, we evaluated the ability of LMWH to modulate TNF-alpha and TXB2 activity during endotoxemia and the subsequent effects on pulmonary hemodynamics . Domestic pigs 8-10 weeks old were anesthetized and catheterized for standard cardiopulmonary measurements and the lungs harvested for cuff:vessel ratio, myeloperoxidase activity, and permeability index . Pigs were randomly assigned to one of four groups: lipopolysaccharide (LPS) (n = 6), given .5 microg/kg/h Escherichia coli LPS intravenously for 6 h; saline control (n = 5); LMWH (n = 5), given .5 mg/kg LMWH for 30 min, followed by .5 mg/kg/h; and LMWH + LPS (same dosages, n = 6) . Administration of LPS resulted in increased plasma TNF-alpha and TXB2 activity; increased pulmonary arterial pressure, pulmonary vascular resistance, and alveolar-arterial oxygen tension; decreased systemic arterial oxygen tension; and pulmonary edema . The cardiopulmonary parameters for the LMWH-treated pigs did not differ from those of the saline-treated control pigs . Pretreatment with LMWH attenuated the LPS-mediated TNF-alpha and TXB2 activity and attenuated LPS-mediated pulmonary hypertension, hypoxemia and neutrophil emigration, and edema formation . In conclusion, the data show that the protective effects of LMWH in this model of acute lung injury are associated with altered neutrophil adhesion and TNF-alpha and thromboxane activity.

Biotechnol Prog, 1998 Mar-Apr, 14(2), 343 - 6
Recovery and reuse of the molecular chaperone GroEL for in vitro protein refolding; Guise AD et al.; The chaperones GroEL and GroES from Escherichia coli are known to improve in vitro protein refolding yields . We show that, for the molecular chaperone-assisted refolding of hen egg white lysozyme, GroES is not an essential requirement and that activity is recovered with GroEL and ATP alone . The refolding yields of lysozyme in the presence of GroEL are much greater than those obtained by dilution because of a reduction in protein aggregation . On the basis of the large difference in molecular weight between the GroEL complex (MW 840 000) and lysozyme (MW 14 600), we have demonstrated that using an ultrafiltration membrane (MW 30 000) GroEL may be easily retained after refolding while lysozyme passes freely into the permeate . The chaperonin recovered from the refolding solution was then reused several times for further refolding experiments . The effectiveness of GroEL-assisted refolding was found to decrease with reuse, and this has been attributed to a reduction in the GroEL:lysozyme molar ratio.

Biotechnol Prog, 1998 Mar-Apr, 14(2), 326 - 31
Steam-sterilizable, fluorescence lifetime-based sensing film for dissolved carbon dioxide; Chang Q et al.; An autoclavable sensing film was developed for monitoring dissolved CO2 . The sensing film, based on fluorescence resonance energy transfer (FRET), consisted of a fluorescent donor, an acceptor, and a quaternary ammonium hydroxide, which were doped in a two-component silicone film . As no aqueous solution was used in the sensing film matrix, the sensing film was unaffected by osmotic pressure . Fluorescence lifetime was selected as the sensing parameter, and measured in frequency domain using phase fluorometry . Upon exposure to 20% CO2-saturated water, a 43 degrees increase in phase angle was observed at 100 MHz . The process was fully reversible when the sensing film was exposed to nitrogen-saturated water . The estimated response and recovery times for 90% signal change were 1 min (for a step change from 0 to 6.7% CO2-saturated water) and 1.5 min (for a step change from 6.7 to 3.3% CO2-saturated water) . When used for on-line monitoring of dissolved CO2 produced by a culture of Escherichia coli, the sensing film showed a similar trend to that obtained from off-line measurements using a wet chemistry analyzer.

Biotechnol Prog, 1998 Mar-Apr, 14(2), 210 - 7
Heat-induced translocation of cytoplasmic beta-galactosidase across inner membrane of Escherichia coli; Umakoshi H et al.; The behaviors of heat-induced translocation of cytoplasmic beta-galactosidase to periplasm across the inner membrane of Escherichia coli cells were investigated in order to apply such phenomena to the process for production and separation of intracellular biomolecules . The heat stress was found to induce translocation of cytoplasmic beta-galactosidase (beta-gal) together with reduction of the amounts of intracellular soluble proteins and formation of their inactive aggregates . The translocation of beta-gal was then analyzed using (a) the location factor of beta-gal (LFG), which meant enzyme location in the cells and could be determined from the kinetic analysis of enzyme release process, and (b) the percentage of beta-gal activity in periplasm after solublizing the outer membrane of E . coli cells by lysozyme/EDTA treatment . The LFG values were maximized when cells were stressed at the temperature of 42-47 degrees C . From the results on the surface properties of both beta-gal and cell membrane under the heat stress, it is suggested that (1) the conformational change of cytoplasmic oligomeric beta-gal to the partially dissociated and/or unfolded state with higher local hydrophobicity, (2) the increase in membrane fluidity of inner membrane, (3) the enhancement of hydrophobic interaction between lipid and protein, and (4) the inhibition of its translocation by GroEL restabilizing the proteins could underlie the heat-induced translocation of beta-gal across the inner membrane . The possibility to apply the heat-induced translocation of beta-gal for the enhancement of the target selectivity at the process upstream is finally presented.

Int Arch Allergy Immunol, 1998 May, 116(1), 40 - 8
Molecular cloning and expression of cynomolgus monkey interleukin-1beta cDNA; Totsuka K et al.; The cynomolgus monkey cDNA encoding interleukin-1beta (IL-1beta) was molecularly cloned by the reverse transcription polymerase chain reaction from a cDNA library of adherent splenocytes stimulated with lipopolysaccharides . The sequence analysis showed that the monkey IL-1beta cDNA encodes a protein of 268 amino acids and displays a high degree (90%) of homology with the human counterpart . Substitution of amino acids resides mainly in the leader sequences of IL-1beta when compared with those of human IL-1beta . This cloned monkey IL-1beta cDNA was used to express in Escherichia coli as a fusion protein with thioredoxin and in insect cells infected with a recombinant baculovirus after molecular modification where monkey IL-1beta signal sequences were placed prior to the mature sequences of IL-1beta for efficient secretion in insect cells . Recombinant monkey IL-1beta expressed in both systems was shown to react with rabbit antihuman IL-1beta antiserum by Western blot analysis and to have the biological activity of IL-1beta in a bioassay.

Microbiol Immunol, 1998, 42(4), 289 - 97
The 21-kDa polypeptide (VAP21) in the rabies virion is a CD99-related host cell protein; Sagara J et al.; In our monoclonal antibody (MAb) stocks prepared against the BHK-21 cell antigens, two (#11875 and 28276) recognized a 21-kDa polypeptide (referred to as VAP21) which is efficiently incorporated into the rabies virion . By using these MAbs, we isolated the cDNA clones that encoded a polypeptide of 144 amino acids from our BHK-21 cell cDNA library . Based on the following evidence, the cDNA was assumed to encode a full-length sequence of VAP21 antigen: i) expression of the cDNA in animal cells resulted in the production of a polypeptide recognized by the two MAbs, and its electrophoretic mobility was the same as that of authentic VAP21 antigen; and ii) immunization with the products from the cDNA-transformed E . coli cells raised specific antibodies in rabbits that recognized a 21-kDa polypeptide in the virion . From the deduced amino acid sequence, it is suggested that the VAP21 antigen has a molecular structure of type-I transmembrane protein containing characteristic proline-rich and glycine-rich regions in its ectodomain . Homology searches resulted in finding homologous sequences (totally about 40% homology) in the human MIC2 gene product (CD99; 32-kDa) of T lymphocytes . These results suggest that the VAP21 antigen in the rabies virion is a cellular CD99-related transmembrane protein.

J Bioenerg Biomembr, 1998 Feb, 30(1), 55 - 62
The dinuclear center of cytochrome bo3 from Escherichia coli; Watmough NJ et al.; For the study of the dinuclear center of heme-copper oxidases cytochrome bo3 from Escherichia coli offers several advantages over the extensively characterized bovine cytochrome c oxidase . The availability of strains with enhanced levels of expression allows purification of the significant amounts of enzyme required for detailed spectroscopic studies . Cytochrome bo3 is readily prepared as the fast form, with a homogeneous dinuclear center which gives rise to characteristic broad EPR signals not seen in CcO . The absence of CuA and the incorporation of protohemes allows for a detailed interpretation of the MCD spectra arising from the dinuclear center heme o3 . Careful analysis allows us to distinguish between small molecules that bind to heme o3, those which are ligands of CuB, and those which react to yield higher oxidation states of heme o3 . Here we review results from our studies of the reactions of fast cytochrome bo3 with formate, fluoride, chloride, azide, cyanide, NO, and H2O2.

Biochem Mol Biol Int, 1998 May, 44(6), 1193 - 202
Molecular cloning and expression of mouse ceramide glucosyltransferase; Ichikawa S et al.; Ceramide glucosyltransferase (EC 2.4.1.80) catalyzes the first glycosylation step of glycosphingolipid (GSL) synthesis, the transfer of glucose from UDP-Glucose to hydrophobic ceramide and generate glucosylceramide (GlcCer) . We have cloned mouse ceramide glucosyltransferase cDNA from a brain cDNA library by PCR based homology cloning . The nucleotide sequence determination revealed that mouse ceramide glucosyltransferase cDNA encodes 394 amino acids with a calculated molecular mass of 45 kDa . The amino acid sequence of mouse ceramide glucosyltransferase showed 98% identity with the human sequence . Homology searches against currently available databases identified three homologous proteins in Caenorhabditis elegans and one homologous protein in Cyanobacteria . Highly conserved sequences of ceramide glucosyltransferases and the homologs among a wide variety of organisms suggest biological significance of the lipid glucosylation system.

Biochem Mol Biol Int, 1998 May, 44(6), 1147 - 55
Amino acid residue 250 has a functional role in the assembly of rabbit liver microsomal cytochrome P450 2B4; Schulze J et al.; Cytochrome P450 2B4 lacking amino acids 2-27, CYP2B4 (delta2-27), was mutated at position 250 and expressed in E . coli fused to glutathione S-transferase . Expression of the E250S variant (holo- plus apoenzyme) proceeded to an extent comparable with that of CYP2B4 (delta2-27), while the protein level of the E250P mutant averaged 42% that of the control pigment . Comparison of these data with the corresponding reduced CO difference spectra of the various CYP2B4 (delta2-27) forms revealed that, in the control and E250S preparations, about 90% and 44%, respectively, of the total amount of hemoprotein present existed in the form of holoenzyme, whereas the E250P derivative failed to produce a reduced carbonyl complex . Thus, replacement of the negatively charged E250 with an uncharged, polar serine residue substantially hampered assembly of CYP2B4 (delta2-27); introduction of an alpha-helix-disrupting proline completely blocked the formation of holoenzyme . These phenomena suggested that the negative charge of E250, residing in the putative G helix, underwent pairing with some positively charged group, possibly H285 located in the I helix . Deletion of the negative charge obviously perturbed the active-site geometry such as to affect both the incorporation and/or retention of the heme ligand and the spectral binding of substrates such as hexobarbital.

Anat Embryol (Berl), 1998 May, 197(5), 359 - 67
Intravenous injection of guanylin induces mucus secretion from goblet cells in rat duodenal crypts; Furuya S et al.; Guanylin, structurally related to the heat-stable enterotoxin of E . coli, is a 15-amino-acid peptide isolated from rat small intestine . We investigated the morphological effects of an intravenous injection of rat and human guanylin upon the rat intestine . Various doses of rat guanylin were injected intravenously in anesthetized rats . After 5, 10 and 30 min, rats were killed by intracardiac perfusion with aldehyde fixative, and specimens of the intestine were then prepared for light and electron microscopy . Intravenously injected rat guanylin rapidly induced mucus secretion from crypt goblet cells in the duodenum . About half of the crypt goblet cells secreted mucous granules by compound exocytosis within 5 min . The villus goblet cells, in contrast, were not sensitive to guanylin . Goblet cells in the jejunum were less responsive than those in the duodenum . This secretory response was rare in the ileum and colon . Human guanylin produced similar results . The mucus secretion induced by guanylin was inhibited by a prior-injection of atropine, but not hexamethonium . Moreover, guanylin induced intense edema in the mucosa and submucosa of the small intestine 5 min after the injection, which disappeared after 30 min . A prior-injection of atropine did not block the appearance of edema . In conclusion, the intravenous injection of guanylin induces two phenomena related to water movement: (1) compound exocytosis of mucous granules from crypt goblet cells in the rat duodenum and jejunum; (2) perineural, inter-epithelial and intra-epithelial edema in the rat small intestine.

Biol Reprod, 1998 Jun, 58(6), 1463 - 8
Loss of the signature six carboxyl amino acid tail from ovine interferon-tau does not affect biological activity; Ealy AD et al.; Interferon-tau (IFN-tau) is a type I IFN that is secreted from conceptuses of Bovidae (sheep, cattle, and related ruminant ungulates) for a few days during early pregnancy . It acts to prolong the life span of the corpus luteum . All secreted forms of IFN-tau, like the related IFN-omega, are 172 amino acids in length and differ from IFN-alpha and -beta by the presence of six additional amino acids at their carboxyl termini . The aim of this study was to determine whether this carboxyl tail was important for biological activity of IFN-tau, particularly for its antiluteolytic function in ewes . Full-length ovine IFN-tau (p3) and a mutated form truncated by six amino acids at its carboxyl terminal (p3Trn6, 166 amino acids) were produced in Escherichia coli . Both proteins had similar antiviral activities (2.12 +/- 0.92 x 10(8) IU/mg for p3; 1.96 +/- 0.58 x 10(8) IU/mg for p3Trn6) when tested on Madin-Darby bovine kidney (MDBK) cells . Antiproliferative activity, as measured on human Daudi cells by determining the protein concentration required to inhibit growth by 50%, was slightly higher (p < 0.05) for p3Trn6 (7.36 +/- 0.46 pM) than for p3 (13.99 +/- 0.85 pM) . Most importantly, p3 and p3Trn6 were equally capable of prolonging the life span of the corpus luteum of nonpregnant ewes when the proteins were administered at doses of either 60 or 300 microg/day into the uterine lumen through indwelling uterine cannulae from Day 10 to Day 18 postestrus . Therefore, the carboxyl-terminal amino acid extension for IFN-tau does not appear to serve a functional role in the action of these proteins.

Xenobiotica, 1998 May, 28(5), 457 - 63
Rat CYP24 catalyses 23S-hydroxylation of 26,26,26,27,27,27-hexafluorocalcitriol in vitro; Hayashi K et al.; 1 . Kidney mitochondrial 24-hydroxylase cytochrome P450 (CYP24) catalyses sequential hydroxylation at both C-24 and C-23 positions of calcidiol and calcitriol . Here, we have investigated the in vitro metabolism of a hexafluorinated derivative of calcitriol, 26,26,26,27,27,27-hexafluorocalcitriol (ST-630), in a reconstituted system by using recombinant Escherichia coli membrane fractions containing rat CYP24 . 2 . When ST-630 was incubated with CYP24 supplemented with bovine adrenodoxin and NADPH-adrenodoxin reductase, a distinct metabolite could be observed . This metabolite was found to be 26,26,26,27,27,27-hexafluoro-23S-hydroxcalcitriol, a biologically active metabolite of ST-630, based on cochromatography on HPLC and mass spectrometric analysis . 3 . These results show the direct evidence that CYP24 plays an essential role in the metabolism of ST-630 to yield its 23S-hydroxylated metabolite, as observed in cultured cells and experimental animal studies.

Biochim Biophys Acta, 1998 May 27, 1403(1), 72 - 84
Characterization of a chloroplast isoform of serine acetyltransferase from the thermo-acidiphilic red alga Cyanidioschyzon merolae; Toda K et al.; We isolated a gene for serine acetyltransferase (SAT), a key enzyme in sulfate assimilation, from the primitive red alga Cyanidioschyzon merolae, an inhabitant of sulfurous hot springs, and designated this gene cmSAT . The N-terminal region of the cmSAT protein has characteristics of a chloroplast targeting peptide . cmSAT protein fused with a 6x histidine tag complemented a SAT deficient Escherichia coli mutant . The protein was purified with its SAT activity, which was inhibited by cysteine, using the high affinity of the histidine tag in an Ni-NTA column . The Km values for acetyl-CoA and l-serine were 0.3 and 0.1 mM, respectively . Southern blotting indicated the existence of other SAT isoforms in C . merolae . A 2.4 kb transcript was always detected when growth was synchronized under a 12-h light/dark cycle . Under these conditions, a 31-kDa protein was always detected on immunoblots, indicating processing of the cmSAT protein and constitutive expression of cmSAT . A 45-kDa protein, thought to be the unprocessed cmSAT protein, was detected in the dark period, from M phase to early G1 phase . No significant change in the level of protein expression was detected under continuous darkness or in a sulfate-deficient medium . Using immunoelectron microscopy, the cmSAT protein was primarily detected in the stroma and a few were detected in the cytoplasm, which indicate that cmSAT protein is transported to and functions in a chloroplast .

Biochim Biophys Acta, 1998 May 27, 1403(1), 1 - 4
Rise of intracellular free calcium levels with activation of inositol triphosphate in a human colonic carcinoma cell line (COLO 205) by heat-stable enterotoxin of Escherichia coli; Bhattacharya J et al.; The heat-stable enterotoxin (STa) produced by Escherichia coli has been found to increase rapidly two potential intracellular signals, inositol triphosphate and cytosolic free calcium in a human colonic cell line, COLO 205 . Addition of STa to COLO 205 cells prelabelled with myo-{2-3H}inositol resulted in a rapid rise of {3H}inositol triphosphate . Using fluorescent indicator, Fura-2AM, intracellular free Ca2+ has been found to increase 5.12-fold compared to control . Suspension of cells in calcium-free buffer demonstrated STa-induced rapid rise of cytosolic Ca2+ . The same result was found when extracellular calcium was chelated with EGTA . This effect was not observed with cells that were pretreated with dantrolene which suggest that the intracellular calcium rise might be due to mobilization from intracellular stores . This study demonstrated for the first time a change in cytosolic calcium in cultured human colonic cells by STa, which is accompanied by inositol triphosphate activation .

Biochemistry, 1998 Jun 2, 37(22), 8191 - 6
Sulfhydryl oxidation of mutants with cysteine in place of acidic residues in the lactose permease; Voss J et al.; To examine further the role of charge-pair interactions in the structure and function of lactose permease, Asp237 (helix VII), Asp240 (helix VII), Glu126 (cytoplasmic loop IV/V), Glu269 (helix VIII), and Glu325 (helix X) were replaced individually with Cys in a functional mutant devoid of Cys residues . Each mutant was then oxidized with H2O2 in order to generate a sulfinic and/or sulfonic acid at these positions . Due to the isosteric relationship between aspartate and sulfinate, in particular, and the lower pKa of the sulfinic and sulfonic acid side chains, oxidized derivatives of Cys are useful probes for examining the role of carboxylates . Asp237-->Cys or Asp240-->Cys permease is inactive, as shown previously, but H2O2 oxidation restores activity to an extent similar to that observed when a negative charge is reintroduced by other means . Glu126-->Cys, Glu269-->Cys, or Glu325-->Cys permease is inactive, but oxidation does not restore active lactose transport . The data are consistent with previous observations indicating that Asp237 and Asp240 are not critical for active lactose transport, while Glu126, Glu269, and Glu325 are irreplaceable . Although Glu269-->Cys permease does not transport lactose, the oxidized mutant exhibits significant transport of beta,D-galactosylpyranosyl 1-thio-beta,D-galactopyranoside, a property observed with Glu269-->Asp permease . The observation supports the idea that an acidic residue at position 269 is important for substrate recognition . Finally, oxidized Glu325-->Cys permease catalyzes equilibrium exchange with an apparent pKa of about 6.5, more than a pH unit lower than that observed with Glu325-->Asp permease, thereby providing strong confirmatory evidence that a negative charge at position 325 determines the rate of translocation of the ternary complex between the permease, substrate, and H+.

Biochemistry, 1998 Jun 2, 37(22), 8163 - 72
Solution structure of the ternary complex between aminoacyl-tRNA, elongation factor Tu, and guanosine triphosphate; Bilgin N et al.; Complex formation between elongation factor Tu (EF-Tu), Phe-tRNAPhe, and GTP was analyzed by small-angle neutron and X-ray scattering methods . Both techniques show that the ternary complex consists of one EF-Tu and one aminoacyl-tRNA . No shift in stoichiometry was detected when the temperature was raised from 5 to 37 degreesC, in contrast to previous observations obtained from RNase A protection experiments {Bilgin and Ehrenberg (1995) Biochemistry34, 715-719} . A small but significant increase in the radius of gyration of the complex was observed when the temperature was decreased from 37 to 5 degreesC . The X-ray solution scattering patterns were compared with those calculated from the crystal structure of the complex formed between EF-Tu from Thermus aquaticus and Phe-tRNAPhe from yeast . The comparison shows that the solution structure of the ternary complex, formed entirely from Escherichia coli components and under translationally optimal buffer conditions, is very close to the crystal structure, formed from heterologous components under very different conditions . Furthermore, for the hybrid complex in solution there is no evidence for the formation of trimers as suggested by the crystal structure.

Biochemistry, 1998 Jun 2, 37(22), 8020 - 6
Ligand-induced changes in periplasmic loops in the lactose permease of Escherichia coli; Sun J et al.; N- and C-terminal halves of lactose permease, each with a single-Cys residue in a periplasmic loop, were coexpressed, and cross-linking was studied in the presence or absence of ligand . A Cys residue at position 36 between transmembrane helices I and II (loop I/II) forms a disulfide spontaneously with a Cys residue at position 253, 254, 255, or 256 in loop VII/VIII . Moreover, in the presence of o-phenanthroline-copper, a Cys residue at position 42 in loop I/II forms a disulfide with a Cys residue at position 253, 254, 257, or 258 in loop VII/VIII . Changes in the rate of cross-linking are also observed in the presence of substrate, suggesting that ligand binding induces movement between loops I/II and VII/VIII such that positions 253-256 are brought closer to position 36 or 42, while positions 257 and 258 move away from position 42.

Biochemistry, 1998 Jun 2, 37(22), 7981 - 91
A mutational analysis of binding interactions in an antigen-antibody protein-protein complex; Dall'Acqua W et al.; Alanine scanning mutagenesis, double mutant cycles, and X-ray crystallography were used to characterize the interface between the anti-hen egg white lysozyme (HEL) antibody D1.3 and HEL . Twelve out of the 13 nonglycine contact residues on HEL, as determined by the high-resolution crystal structure of the D1.3-HEL complex, were individually truncated to alanine . Only four positions showed a DeltaDeltaG (DeltaGmutant - DeltaGwild-type) of greater than 1.0 kcal/mol, with HEL residue Gln121 proving the most critical for binding (DeltaDeltaG = 2.9 kcal/mol) . These residues form a contiguous patch at the periphery of the epitope recognized by D1.3 . To understand how potentially disruptive mutations in the antigen are accommodated in the D1.3-HEL interface, we determined the crystal structure to 1.5 A resolution of the complex between D1.3 and HEL mutant Asp18 --> Ala . This mutation results in a DeltaDeltaG of only 0.3 kcal/mol, despite the loss of a hydrogen bond and seven van der Waals contacts to the Asp18 side chain . The crystal structure reveals that three additional water molecules are stably incorporated in the antigen-antibody interface at the site of the mutation . These waters help fill the cavity created by the mutation and form part of a rearranged solvent network linking the two proteins . To further dissect the energetics of specific interactions in the D1.3-HEL interface, double mutant cycles were carried out to measure the coupling of 14 amino acid pairs, 10 of which are in direct contact in the crystal structure . The highest coupling energies, 2.7 and 2.0 kcal/mol, were measured between HEL residue Gln121 and D1.3 residues VLTrp92 and VLTyr32, respectively . The interaction between Gln121 and VLTrp92 consists of three van der Waals contacts, while the interaction of Gln121 with VLTyr32 is mediated by a hydrogen bond . Surprisingly, however, most cycles between interface residues in direct contact in the crystal structure showed no significant coupling . In particular, a number of hydrogen-bonded residue pairs were found to make no net contribution to complex stabilization . We attribute these results to accessibility of the mutation sites to water, such that the mutated residues exchange their interaction with each other to interact with water . This implies that the strength of the protein-protein hydrogen bonds in these particular cases is comparable to that of the protein-water hydrogen bonds they replace . Thus, the simple fact that two residues are in direct contact in a protein-protein interface cannot be taken as evidence that there necessarily exists a productive interaction between them . Rather, the majority of such contacts may be energetically neutral, as in the D1.3-HEL complex.

Biochemistry, 1998 Jun 2, 37(22), 7929 - 40
NMR solution structure of the 21 kDa chaperone protein DnaK substrate binding domain: a preview of chaperone-protein interaction; Wang H et al.; The solution structure of the 21 kDa substrate-binding domain of the Escherichia coli Hsp70-chaperone protein DnaK (DnaK 386-561) has been determined to a precision of 1.00 A (backbone of the beta-domain) from 1075 experimental restraints obtained from multinuclear, multidimensional NMR experiments . The domain is observed to bind to its own C-terminus and offers a preview of the interaction of this chaperone with other proteins . The bound protein region is tightly held at a single amino acid position (a leucyl residue) that is buried in a deep pocket lined with conserved hydrophobic residues . A second hydrophobic binding site was identified using paramagnetically labeled peptides . It is located in a region close to the N-terminus of the domain and may constitute the allosteric region that links substrate-binding affinity with nucleotide binding in the Hsp70 chaperones.

Development, 1998 May, 125(9), 1659 - 67
Runt determines cell fates in the Drosophila embryonic CNS; Dormand EL et al.; The segmentation gene, runt, is expressed by a subset of the 30 neuroblasts that give rise to each neuromere of the Drosophila embryo . Runt activity in the neuroblasts is necessary for expression of even-skipped in the EL neurons . runt is therefore a good candidate for a gene specifying neuroblast identities . We have ectopically expressed Runt in restricted subsets of neuroblasts and show that Runt is sufficient to activate even-skipped expression in the progeny of specific neuroblasts . Using the marker Tau-green fluorescent protein to highlight the axons, we have found that the extra Even-skipped-expressing neurons project axons along the same pathway as the EL neurons . We find that Runt is expressed in neuroblast 3-3, supporting an autonomous role for runt during neuroblast specification.






What Is Biofilter?, What Is Growth Medium?, What Is Anthrax?, What Is Bioengineering?, What Is Fermentation?, c, Microbiology, e, Bacteria, r, Microorganism, c, Microbe, o, Bacterium, n, Candida albicans, s, Microbial, i, Escherichia coli, r, Multidrug resistant, s, Acinetobacter, i, Antibiotic prophylaxis, i, Staphylococcus, e, Candida albicans, r, Biodegradation, i, Escherichia coli, a, Staphylococcus aureus, s, Culture medium, e, Streptococcal, i, Botulin, n, Culture medium, e, Vancomycin, s, Bacteria, c, Yeasts, c, Escherichia coli, s, Antimicrobials, i, Staphylococcus




 

   Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology
Anaerobic Microbiology
Antimicrobial Susceptibility
Artificial Atmosphere
Bioassay of Antibiotics
Biofilm Microbiology
Bioreactor Technology
Biotechnology
Cell Biology
Clinical Microbiology
Environmental Microbiology
Experiments with Yeast		 Fermentation
Food Microbiology
Functional Genomics
Gene Technology
Growth Media Development
Growth Rate and Lag Time
Industrial Microbiology
Medical/Pharmaceutical Field
Microbiological Assay
Microbiological Research
Microbiology of Cosmetics

go to a specific theme...

 Military Microbiology
Molecular Microbiology
Mutagenicity and Genotoxicity
Oral Microbiology
Patents
Postantibiotic Studies
Soil Microbiology
Spore Microbiology
Veterinary Microbiology
Waste/Wastewater Treatment
Water Microbiology
Wine Microbiology

 


 

© 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com
 

 

 

Last modified: May 25, 2005