Escherichia coli

J Biol Chem, 1993 Feb 25, 268(6), 4259 - 66
Isolation and characterization of a novel member of the gene family encoding the cAMP response element-binding protein CRE-BP1; Nomura N et al.; Among multiple CRE (cyclic AMP response element)-binding proteins, CRE-BP1 (also designated ATF-2) has two unique characteristics: it mediates the adenovirus E1A-induced trans-activation and forms a heterodimer with c-Jun . Two structures, a putative metal finger and a leucine zipper, in CRE-BP1 are responsible for these capacities . As a new member of a CRE-BP1 family that has similar metal finger and leucine zipper structures, we have isolated cDNA clones of CRE-BPa by cross-hybridization with CRE-BP1 cDNA . CRE-BPa protein consists of 508 amino acids and has a molecular weight of 56,840 . CRE-BPa protein is highly homologous with CRE-BP1 in four regions: two of them are the regions containing the putative metal finger or the DNA-binding domain consisting of the basic amino acid cluster and the leucine zipper . Like CRE-BP1, CRE-BPa binds to CRE with higher affinity than to the 12-O-tetradecanoylphorbol-13-acetate response element as a homodimer or a CRE-BPa/c-Jun or CRE-BPa/CRE-BP1 heterodimer . However, using the c-Myb-CRE-BPa fusion protein, it was show that CRE-BPa could not mediate the E1A-induced trans-activation . Expression of CRE-BPa mRNA was found in a limited number of cell lines, and multiple sizes of CRE-BPa mRNA species were detected in some cell lines and tissues . CRE-BPa will be useful to clarify the mechanism of CRE-mediated transcriptional activation by E1A or c-Jun.

J Biol Chem, 1993 Feb 25, 268(6), 3925 - 37
Protein folding in a cell-free translation system . The fate of the precursor to mitochondrial aspartate aminotransferase; Mattingly JR Jr et al.; The precursor to rat mitochondrial aspartate aminotransferase (pmAspAT) can be expressed in and purified from Escherichia coli as a fully active enzyme with remarkable trypsin resistance . Only two sites within the presequence are readily hydrolyzed (Martinez-Carrion, M., Altieri, F., Iriarte, A., Mattingly, J . R., Youssef, J., and Wu, T . (1990) Ann . N.Y . Acad . Sci . 585, 346-356) . In contrast, pmAspAT freshly synthesized in rabbit reticulocyte lysate is significantly less resistant to proteolysis and is completely digested by trypsin . Extended incubation of the pmAspAT translation product slowly converts it to a species with qualitatively the same trypsin resistance as the purified pmAspAT . In addition, this species binds pyridoxal 5'-phosphate, exhibits catalytic activity, and loses its ability to be imported into mitochondria . This process appears to reflect protein folding . The rate of folding is unaffected by the addition of cofactor or the depletion of endogenous cofactor and is not significantly affected by the concentration of translation product in the reaction . Agents that decrease the availability of ATP partially inhibit the folding, whereas the sulfhydryl alkylating reagent N-ethylmaleimide and the detergent Triton X-100 completely prevent the conversion . Although the folding of pmAspAT in reticulocyte lysate is slow, folding is rapid once the translation product is sequestered within the mitochondria as the mature form of the enzyme . These results are presented as a model for the in vivo folding of pyridoxal-dependent, oligomeric mitochondrial precursors in the presence of cytoplasmic components and for the fate of true mitochondrial precursor proteins when not imported.

J Biol Chem, 1993 Feb 25, 268(6), 3911 - 9
Molecular cloning, DNA sequencing, and biochemical analyses of Escherichia coli glyoxylate carboligase . An enzyme of the acetohydroxy acid synthase-pyruvate oxidase family; Chang YY et al.; Glyoxylate carboligase (Gcl) (EC 4.1.1.47) of Escherichia coli catalyzes the condensation of two molecules of glyoxylate to give tartronic semialdehyde, a key intermediate in glyoxylate catabolism . We report the cloning, genomic location, and DNA sequence of the gene (called gcl) encoding E . coli Gcl and isolation of mutants lacking the enzyme . Gcl is a protein of 593 amino acid residues (64,738 Da) that has a high level (30%) of sequence similarity to the acetohydroxy acid synthases (AHAS) of branched chain amino acid synthetic pathway . Significant sequence identity (26%) was also observed with E . coli pyruvate oxidase, a redox flavoprotein, previously shown to be related to the AHAS enzymes (Chang, Y.-Y., and Cronan, J . E., Jr . (1988) J . Bacteriol . 170, 3937-3945) . Consistent with a grouping of Gcl with the AHAS and pyruvate oxidase enzymes . Gcl contains a quinone binding site as well as binding site for thiamine pyrophosphate and FAD . We also found that a gene (orf258) immediately downstream of the gcl gene encoded a protein (Orf258) of 258 residues . Although the gene organization of gcl and orf258 is analogous to that of the ilv gene operons which encode the E . coli AHAS isozyme large and small subunits, Orf258 does not function as a Gcl subunit . Moreover, disruption of the chromosomal copy of orf258 did not affect growth on glyoxylate or glycolate.

J Biol Chem, 1993 Feb 25, 268(6), 3897 - 902
Characterization of the DNA-melting function of the Rickettsia prowazekii RNA polymerase; Ding HF et al.; In vitro specific transcription by the Rickettsia prowazekii RNA polymerase was investigated . The purified rickettsial RNA polymerase, in striking contrast to that of Escherichia coli, could specifically transcribe two R . prowazekii genes (ATP/ADP translocase and citrate synthase genes) and one E . coli gene (RNA-I) on negative supercoiled plasmids but not the same genes on linear plasmids . Following the specific binding of the rickettsial RNA polymerase to the translocase gene promoter on a linear plasmid, there was no detectable open complex formation . Both the E . coli and the R . prowazekii RNA polymerases worked well when poly(dA-dT).poly(dA-dT) or poly(dI-dC).poly-(dI-dC) was used as template for generalized transcription . However, the rickettsial RNA polymerase, in contrast to the E . coli enzyme, had little activity on poly(dG-dC).poly(dG-dC), a template with a larger number of hydrogen bonds . These data indicate that the rickettsial RNA polymerase is weak, at least relative to E . coli, in the function required for the opening of DNA duplex . It appears that this operation in R . prowazekii is aided by the negative supercoiling and the high 72% AT composition of the rickettsial genome.

J Biol Chem, 1993 Feb 25, 268(6), 3845 - 9
Reduction of mutant phage T4 glutaredoxins by Escherichia coli thioredoxin reductase; Nikkola M et al.; Fifteen mutant T4 glutaredoxins (previously T4 thioredoxin) have been assayed for activity with Escherichia coli thioredoxin reductase . The mutations include substitutions in the region of the active site, in the 2 cysteines, and in the 2 residues between the cysteines forming the active-site disulfide bridge . Mutant thioredoxins where substitutions have been made in charged residues around the active site show the biggest differences in activity . The positive residues Lys-13 and Lys-21 were found to be important for efficient binding to thioredoxin reductase . Substitution of the aspartic acid at position 80 with a serine produced a glutaredoxin with superior activity . This mutant glutaredoxin has earlier been shown to be more efficient than the wild type in thiol transferase activity (Nikkola, M., Gleason, F . K., Saarinen, M., Joelson, T., Bjornberg, O., and Eklund, H . (1991) J . Biol . Chem . 266, 16105-16112) . Even the glutaredoxin P66A, where the active-site cis-proline has been substituted, could be efficiently reduced by thioredoxin reductase . Glutaredoxins lacking one or both cysteines were not active.

J Biol Chem, 1993 Feb 25, 268(6), 4441 - 6
Structural requirement of CRK SH2 region for binding to phosphotyrosine-containing proteins . Evidence from reactivity to monoclonal antibodies; Matsuda M et al.; The SH2 region of signal-transducing proteins mediates binding to phosphotyrosine-containing proteins . We analyzed the structure-function relationship of the SH2 region of the human CRK protein by using a series of monoclonal antibodies (mAbs) . Seventeen mAbs against the CRK SH2 region were classified into 5 groups according to the reactivity with mutant CRK proteins expressed in COS7 cells and in Escherichia coli and by epitope scanning with synthetic nonapeptides . Two groups of mAbs (groups A and B) were reactive only with intact SH2 . Mutation(s) in either the amino-terminal B box or the carboxyl-terminal C box, which are the two subdomains of SH2, abolished the reactivity of the CRK mutants to the mAbs of groups A and B . Group A mAbs competed the binding of the CRK SH2 region to the phosphotyrosine-containing proteins . Moreover, the spectrum of the CRK mutants which were recognized by group A mAbs coincided with that of the CRK mutants which could bind phosphotyrosine-containing proteins, suggesting that group A mAbs were directed against the site of binding to phosphotyrosine-containing proteins . Group C mAbs, directed against the region between the B and C boxes, were reactive with both wild-type and mutant CRK proteins and did not affect the capacity of SH2 to bind phosphotyrosine-containing proteins . Contrary to group A and B mAbs, mAbs belonging to groups D and E, which were mapped onto the C box, did not bind well to the native CRK proteins, but bound to CRK mutants with mutation(s) in either the B or the C box . These results suggest that the B and C boxes, which are separated by a hinge region, coordinately form the functional SH2 domain that binds to the phosphotyrosine-containing proteins and to the group A mAbs.

J Chromatogr, 1993 Feb 24, 633(1-2), 273 - 80
Evaluation of several affinity chromatographic supports for the purification of maltose-binding protein from Escherichia coli; Kroviarski Y et al.; To obtain affinity adsorbents with good mechanical resistance, suitable for the purification of maltose-binding protein (MBP) from Escherichia coli and genetically engineered proteins fused to MBP, a series of supports were prepared by grafting amylose on to agarose by different chemistries . Their capacities for MBP and their abilities to be used at relatively high flow-rates were examined . Efficient supports were most conveniently prepared by coupling amylose to epoxy-activated agarose in an aqueous-organic mixture.

Biochim Biophys Acta, 1993 Feb 24, 1166(2-3), 301 - 4
Lecithin-cholesterol acyltransferase: effects of mutagenesis at N-linked oligosaccharide attachment sites on acyl acceptor specificity; Francone OL et al.; Site-directed mutagenesis was used to generate lecithin-cholesterol acyltransferase (LCAT) species in which individual attachment sites for N-linked oligosaccharide residues were replaced with residues that prevent the attachment of carbohydrate . Mutants at three of four sites retained significant acyltransferase activity, and phospholipase activity in the absence of cholesterol . Mutation at one site (asn272) converted LCAT to a phospholipase generating fatty acids not cholesteryl esters.

Biochemistry, 1993 Feb 23, 32(7), 1810 - 5
A hydrogen-bonding network modulating enzyme function: asparagine-194 and tyrosine-225 of Escherichia coli aspartate aminotransferase; Yano T et al.; The electron distribution within the coenzyme or coenzyme-substrate conjugate needs to be properly regulated during the catalytic process of aspartate aminotransferase (AspAT) . Asn194 and Tyr225 may function in regulating the electron distribution through hydrogen-bonding to O(3') of the coenzyme, pyridoxal 5'-phosphate (PLP) or pyridoxamine 5'-phosphate (PMP) . The roles of Tyr225 have already been explored by site-directed mutagenesis (Inoue et al., 1991; Goldberg et al., 1991) . In the present studies, the mutant enzymes Asn194-->Ala and Asn194-->Ala + Tyr225-->Phe were analyzed kinetically and spectroscopically and were compared with the wild-type and Tyr225-->Phe enzymes . The kinetic studies showed that Asn194 is not essential for AspAT catalysis, although the Kd values for the substrates were increased by 10- to 50-fold upon the replacement of Asn194 . The measurements of the absorption and fluorescence excitation spectra revealed that the ratio of an enolimine to a ketoenamine form was considerably increased as a tautomeric form of the protonated PLP in the active site of the double mutant enzyme . The pH-pKd relationship for the binding of maleate to AspAT could be explained by a simple thermodynamic cycle where only one ionizing group (the imine nitrogen of the internal aldimine bond) affects the binding of maleate . The analyses of the pH-pKd curves for the wild-type and mutant enzymes showed that (i) the hydrogen bond between O(3') of PLP and Asn194 is weakened by the binding of maleate to AspAT, while the hydrogen bond between O(3') and Tyr225 is not changed, and that (ii) the replacement of Asn194 causes some effect hampering the binding of maleate.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 Feb 23, 32(7), 1759 - 69
Mutagenesis by the (+)-anti-diol epoxide of benzo{a}pyrene: what controls mutagenic specificity?
Rodriguez H, Loechler EL.
Mutagenesis by (+)-anti-benzo{a}pyrene-7,8-dihydrodiol-9,10-epoxide {(+)-anti-B{a}PDE}, an important mutagenic/carcinogenic metabolite of benzo{a}pyrene (B{a}P), is being studied in order to understand the factors that influence mutagenesis both quantitatively and qualitatively . A new mutational system, which permits the selection of supF- mutations in an Escherichia coli plasmid, pUB3, was used . The work described herein is an extension of previous work, which involved plasmid adduction and then immediate transformation (Rodriguez & Loechler, 1993), and began with the observation that mutation frequency (MF) decreased approximately 2-fold when the (+)-anti-B{a}PDE-adducted plasmid pUB3 is either (1) frozen and then thawed prior to transformation or (2) heated at 80 degrees C for 10 min prior to transformation . Several results suggest that this decrease is not due to the loss of labile adducts . To begin to understand this phenomenon, the mutagenic spectra are compared for (+)-anti-B{a}PDE in supF for the unheated (187 mutants), the freeze/thawed (134 mutants), and the heated (254 mutants) samples . In general, freeze/thawing and heating cause a decrease in all classes of mutations . Considering substitution mutations at G.C base pairs, which predominate, the mutagenic specificity for the combined data sets is GC-->TA (57%), GC-->AT (23%), and GC-->CG (20%) . This raises the question, how does (+)-anti-B{a}PDE generate this complex mutagenic specificity, which contrasts with the situation for, e.g., simple methylating agents? One factor is that mutagenic specificity at a particular guanine residue can be influenced by the base on its immediate 5'-side, most notably where mutations are virtually exclusively restricted to GC-->TA in 5'-TG-3' sequence contexts . One unexpected finding may provide additional insight . G115 in supF, which is the major hot spot for base-pairing mutagenesis, is the only site where the qualitative pattern of mutagenesis is significantly affected by heating the (+)-anti-B{a}PDE-adducted plasmid prior to transformation . Without heating, G115-->T mutations predominate, but following heating there is a statistically significant increase in the fraction of G115-->A and G115-->C mutations . The most likely model to explain this and other results is (1) a particular DNA adduct can adopt multiple conformations, (2) the conformation adopted by an adduct can be influenced by various factors, including DNA sequence context, as well as heating and freeze/thawing, and (3) each of these conformations can cause a different pattern of mutation.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochemistry, 1993 Feb 23, 32(7), 1689 - 94
A pre-transition-state mimic of an enzyme: X-ray structure of adenosine deaminase with bound 1-deazaadenosine and zinc-activated water; Wilson DK et al.; The refined 2.4-A structure of adenosine deaminase, recently discovered to be a zinc metalloenzyme {Wilson, D . K., Rudolph, F . B., & Quiocho, F . A . (1991) Science 252, 1278-1284}, complexed with the ground-state analog 1-deazaadenosine shows the mode of binding of the analog and, unexpectedly, a zinc-activated water (hydroxide) . This structure of a pre-transition-state mimic, combined with that previously determined for the complex with 6(R)-hydroxy-1,6-dihydropurine ribonucleoside, a nearly ideal transition-state analog, sheds new understanding of the precise stereospecificity and hydrolytic catalysis of an important and well-characterized member of a large group of zinc metalloenzymes . As both of these excellent mimics were generated in the active site, they demonstrate a powerful means of dissecting the course of an enzymatic reaction by direct crystallographic analysis.

Biochemistry, 1993 Feb 23, 32(7), 1770 - 3
Photo-cross-linking of CRP to nonspecific DNA in the absence of cAMP . DNA interacts with both the N- and C-terminal parts of the protein; Katouzian-Safadi M et al.; Adenosine cyclic 3',5'-phosphate receptor protein (CRP or CAP) is a regulatory protein involved in the transcription of several operons in Escherichia coli . cAMP-independent, nonspecific complexes of CRP and DNA were investigated by photochemical cross-linking of the protein to nonspecific DNA, whose thymines are substituted by 5-bromouracil (BrUra) . The cross-linked protein was completely digested by trypsin, and the covalently bound peptides were sequenced . We identified two regions of the protein in close contact with DNA: one in the C-terminal part, overlapping the canonical helix-turn-helix motif, and the other one in the N-terminal part, which is usually not considered to belong to the DNA-interacting domain of CRP . This result lead us to propose models for nonspecific interaction, where the DNA is in contact with both the N- and C-terminal parts of the protein.

FEBS Lett, 1993 Feb 22, 318(1), 11 - 6
Identification of zinc-binding ligands in the class II fructose-1,6-bisphosphate aldolase of Escherichia coli; Berry A et al.; An expression and mutagenesis system for the E . coli Class II fructose-1,6-bisphosphate aldolase has been created by modification of the vector pKfda (Biochem . J . 257 (1989) 529-534) . Large amounts of Class II aldolase (about 1 g/l in crude extracts), with properties consistent with those previously reported for the naturally occurring enzyme (Biochem . J . 169 (1978) 633-641) are obtained . The enzyme contains 2 zinc ions per enzyme dimer . We have investigated the nature of the zinc-binding site of the enzyme by site-directed mutagenesis . His-108, His-111, Cys-112 and His-142 were identified as possible zinc-binding ligands by sequence alignments and comparisons with other known zinc-containing enzymes . Mutation of these residues identified His-108 and His-111 as two of the ligands directly responsible for the tight binding of zinc . Mutation of the other two residues results in only a small effect on the amount of zinc bound per monomer and a corresponding change in specific activity . These residues are, therefore, unlikely to be directly involved in zinc binding, but may be indirectly involved in some manner in the zinc-binding environment.

J Mol Biol, 1993 Feb 20, 229(4), 833 - 48
Significant dispersed recurrent DNA sequences in the Escherichia coli genome . Several new groups; Blaisdell BE et al.; New computer and statistical methods were used to determine significant direct and inverted repeats in the Escherichia coli contig sequence collection of aggregate 1.6 x 10(6) base-pairs . Eight groups of mostly new structural repeat identities were uncovered . Apart from the high statistical significance of these repeat sequences, there are suggestive relationships of the group matches in terms of neighboring genes, of genomic distributions, of their texts, and of their potentials for secondary structure . Four of these groups are relatively numerous, 11 to 26 members, one is in coding sequences and three are in non-coding . The coding group consists of the ATP-activated transmembrane component of a typical high-affinity protein-binding transport system . One of the non-coding groups consists of a special rho-independent transcription termination signal closely following an operon . The gene neighbors of this group often appear to be involved in some way in processing RNA or DNA . A second non-coding group has, for one or both neighboring genes, a component of a system responding to stress or starvation for some nutrient.

J Mol Biol, 1993 Feb 20, 229(4), 1159 - 62
Formation in vivo, purification and crystallization of a complex of the gamma and epsilon subunits of the F0F1-ATPase of Escherichia coli; Cox GB et al.; A complex comprising the epsilon subunit of Escherichia coli F1-ATPase (ECF1-ATPase) and a glutathione-S-transferase gamma subunit (of ECF1-ATPase) fusion protein was formed in vivo and purified from cell extracts by binding to glutathione-agarose beads . The glutathione-S-transferase was released from the complex by digestion with thrombin and the gamma/epsilon complex purified by cation-exchange chromatography . Crystals of the complex were grown by vapour diffusion using PEG8000 as precipitant . The crystals are orthorhombic, space-group P2(1)2(1)2 with a = 161.9 A, b = 44.1 A and c = 63.4 A . The volume of the asymmetric unit is consistent with the presence of a complex of one gamma subunit and one epsilon subunit.

J Mol Biol, 1993 Feb 20, 229(4), 1157 - 8
Crystallization and preliminary X-ray crystallographic analysis of the protease inhibitor ecotin; Shin DH et al.; Ecotin, a novel serine protease inhibitor isolated from Escherichia coli, has been crystallized using polyethylene glycol 1500 as the precipitating agent . The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell parameters of a = 39.22 A, b = 84.86 A, and c = 98.74 A . The asymmetric unit contains one dimeric molecule of ecotin, with a crystal volume per protein mass (Vm) of 2.55 A3/Da and a solvent content of 51.8% by volume . The crystals diffract to at least 2.2 A using a conventional X-ray source, and X-ray data have been collected to 2.7 A Bragg spacing from a native crystal.

J Mol Biol, 1993 Feb 20, 229(4), 1150 - 2
Crystallization and preliminary X-ray diffraction studies of recombinant human interleukin-5; Hassell AM et al.; Recombinant human interleukin-5 (rhIL-5) has been crystallized by the hanging drop vapor diffusion method using 0.1 M-Tris.HCl buffer (pH 8.5) containing 0.2 to 0.25 M-sodium acetate and 26 to 30% PEG 4000 at 22 degrees C . The parallel-piped crystals belong to the space group C2 with unit cell dimensions of a = 122.1 A, b = 36.11 A, c = 56.42 A, beta = 98.59 degrees . They diffract to at least 2.0 A resolution on a rotating anode X-ray source . The molecular mass weight of the protein and the volume of the unit cell suggest that the asymmetric unit contains one intermolecular disulfide-bonded homodimer.

J Mol Biol, 1993 Feb 20, 229(4), 1083 - 100
Three-dimensional structure of the glutathione synthetase from Escherichia coli B at 2.0 A resolution; Yamaguchi H et al.; Glutathione synthetase (gamma-L-glutamyl-L-cysteine: glycine ligase (ADP-forming) EC 6.3.2.3: GSHase) catalyzes the synthesis of glutathione from gamma-L-glutamyl-L-cysteine and Gly in the presence of ATP . The enzyme from Escherichia coli is a tetramer with four identical subunits of 316 amino acid residues . The crystal structure of the enzyme has been determined by isomorphous replacement and refined to a 2.0 A resolution . Two regions, Gly164 to Gly167 and Ile226 to Arg241, are invisible on the electron density map . The refined model of the subunit includes 296 amino acid residues and 107 solvent molecules . The crystallographic R-factor is 18.6% for 17.914 reflections with F > 3 sigma between 6.0 A and 2.0 A . The structure consists of three domains: the N-terminal, central, and C-terminal domains . In the tetrameric molecule, two subunits that are in close contact form a tight dimer, two tight dimers forming a tetramer with two solvent regions . The ATP molecule is located in the cleft between the central and C-terminal domains . The ATP binding site is surrounded by two sets of the structural motif that belong to those respective domains . Each motif consists of an anti-parallel beta-sheet and a glycine-rich loop.

Biochim Biophys Acta, 1993 Feb 20, 1172(1-2), 12 - 6
Identification and characterization of a yeast gene encoding an adenylate kinase homolog; Konrad M; Screening for genes homologous to adenylate kinase in the yeast Saccharomyces cerevisiae resulted in the isolation of a homolog of the previously characterized ADK1 . The derived protein sequence is most closely related to mammalian GTP:AMP phosphotransferase (adenylate kinase isozyme 3; AK3); this novel gene is therefore named ADK3 . Its deletion from the yeast genome does not lead to an observable change in cellular phenotype . A strain defective for both ADK1 and ADK3 is viable . When introduced on a multicopy plasmid into an ADK1-deficient yeast strain, which shows a reduced proliferation rate, ADK3 did not rescue this growth defect . The protein was also highly overexpressed in E . coli cells . However, no change in enzymatic activity was detected in cellular extracts of yeast or bacteria.

J Mol Biol, 1993 Feb 20, 229(4), 1007 - 21
X-ray analysis and spectroscopic characterization of M121Q azurin . A copper site model for stellacyanin; Romero A et al.; The dependence of the properties of the azurin blue copper site on the nature of the axial ligand at position 121 was tested by site-directed mutagenesis . This residue was substituted for a glutamine, the purported fourth copper ligand in the related protein stellacyanin . M121Q azurin was isolated and purified from Escherichia coli and characterized by spectroscopic methods . The mutant copper site has the ultra-violet-vis and electron paramagnetic resonance (EPR) characteristics of a type I site, but the spectroscopic details differ significantly from wild-type (wt) azurin . The X and S-band EPR spectra of M121Q azurin can be well stimulated with the parameters for stellacyanin, indicating that the copper sites of both proteins in the oxidized state are similar . The midpoint potential of M121Q is 263 mV, 25 mV lower than for wt azurin . The reactivity of the mutant was probed by measuring the electron self exchange rate by nuclear magnetic resonance spectroscopy . The rate was 8 x 10(3) mol-1 s-1, almost two orders of magnitude lower than the value for wt azurin (5 x 10(5) mol-1 s-1) . Detailed structural information on the M121Q Cu(II)-site was obtained by X-ray analysis of M121Q azurin crystals at 1.9 A resolution . The histidine and cysteine copper ligand distances and angles in the equatorial plane around the copper are very similar to the wt protein . Gln121 is co-ordinated in a monodentate fashion via its side-chain oxygen atom at a distance of 2.26 A . The distance between copper and the carbonyl group of Gly45 is increased from 3.13 A (wt) to 3.37 A resulting in a distorted tetrahedral N2SO copper co-ordination . The possible significance of these results for the structure of the copper site of stellacyanin, the only small blue copper protein lacking a methionine ligand, is discussed . Conformational changes with respect to the wt azurin are seen in some of the connecting loops between secondary structure elements, in the mutation site and in the beta-strand 2a . The side-chains involved in the hydrophobic patch surrounding His117 are subject to large changes in their conformations . In contrast to wt azurin, the copper site in M121Q azurin undergoes significant structural changes on reduction.(ABSTRACT TRUNCATED AT 400 WORDS)

J Mol Biol, 1993 Feb 20, 229(4), 860 - 72
Quantitative model of ColE1 plasmid copy number control; Brendel V et al.; Initiation of replication of the Escherichia coli plasmid ColE1 is inhibited by formation of a complex between a small plasmid RNA (RNA I) and the pre-primer for DNA synthesis (RNA II) . Complex formation (and inhibition of replication) is enhanced by the plasmid-encoded Rom protein . The in vitro kinetics of complex formation were previously studied both experimentally and theoretically . The in vivo concentrations and half-lives of RNA I, RNA II and Rom protein have been measured recently . We present a dynamic model for the in vivo replication control mechanism that accounts for the measured concentration values . From the model we deduce a simple formula for the steady-state plasmid concentration . Our results agree with a previous simple steady-state analysis done by Brenner and Tomizawa, in that plasmid copy number is most strongly dependent on the per plasmid rate of RNA I synthesis . However, our model predicts other parameter dependencies that are not evident from or at variance with the previous analysis . Accordingly, we predict that plasmid copy number is greatly influenced by changes in the rate constant describing the formation of an initial unstable RNA I-RNA II complex, but is only slightly influenced by changes in the dissociation rate of this complex . Plasmid copy number per average cell volume is predicted to increase linearly with increases in the RNA II synthesis rate and with increases in the generation time of the host culture . Rom protein, which promotes conversion of the unstable RNA I-RNA II complex to a stable complex, serves to decrease copy number; however, its presence or absence does not seem to qualitatively alter the copy number control mechanism . Our model predicts the quantitative increase of plasmid copy number in rom- mutants . Several experiments are suggested to investigate the predictions of the model.

Biochim Biophys Acta, 1993 Feb 20, 1172(1-2), 161 - 6
Characterization of a cDNA encoding a human kidney, cytochrome P-450 4A fatty acid omega-hydroxylase and the cognate enzyme expressed in Escherichia coli; Palmer CN et al.; A cDNA encoding a cytochrome P-450 4A (CYP4AII) was cloned from a human kidney cDNA library . Northern blot analysis and RNase protection assays indicate that related mRNAs occur in kidney and liver with the highest abundance found in kidney . The enzyme was expressed from its cDNA in Escherichia coli . A solubilized preparation of the enzyme reconstituted with cytochrome P-450 reductase catalyzed the omega-hydroxylation of lauric acid, palmitic acid, and arachidonic acid with turnover numbers of 9.8, 2.2 and 0.55 min-1, respectively . Little or no activity was detected toward prostaglandins A1 and E1.

Science, 1993 Feb 19, 259(5098), 1154 - 7
The nonhelical structure of antifreeze protein type III; Sonnichsen FD et al.; Antifreeze proteins (AFPs) are present in the blood of some marine fishes and inhibit the growth of ice crystals at subzero temperatures by adsorption to the ice lattice . The solution structure of a Type III AFP was determined by two-dimensional nuclear magnetic resonance spectroscopy . These measurements indicate that this 66-residue protein has an unusual fold in which eight beta strands form two sheets of three antiparallel strands and one sheet of two antiparallel strands, and the triple-stranded sheets are packed orthogonally into a beta sandwich . This structure is completely different from the amphipathic, helical structure observed for Type I AFPs.

Biochemistry, 1993 Feb 16, 32(6), 1601 - 9
Magnesium in the active site of Escherichia coli alkaline phosphatase is important for both structural stabilization and catalysis; Janeway CM et al.; Site-specific mutagenesis was used to explore the roles of the side chains of residues Lys-328 and Asp-153 in Escherichia coli alkaline phosphatase . The D153H enzyme exhibits a 3.5-fold decrease in activity at pH 8.0 compared to that of the wild-type enzyme, while a double mutant D153H/K328H exhibits a 16-fold decrease in activity under these conditions . However, the Km values for both enzymes, employing the substrate p-nitrophenyl phosphate, are lower than the value for the wild-type enzyme . The Ki for phosphate, which is pH- and Mg(2+)-dependent, is decreased for the D153H enzyme and increased for the D153H/K328H enzyme . Relative to the wild-type enzyme, both mutant enzymes bind Mg2+ more weakly and undergo a time-dependent activation induced by Mg2+ . The half-time of the activation process is independent of the Mg2+ concentration, indicating that the activation most probably involves a conformational change . The pH versus activity profiles of both enzymes are altered relative to that of the wild-type enzyme and exhibit greatly enhanced activity, relative to that of the wild-type enzyme, at high pH values . The pre-steady-state kinetics for the D153H and D153H/K328H enzymes exhibit a transient burst of product formation at pH 8.0, under conditions at which the wild-type enzyme exhibits no transient burst, indicating that at pH 8.0 the hydrolysis of the covalent enzyme-phosphate complex is rate-determining and not the release of phosphate from the noncovalent enzyme-phosphate complex as is observed for the wild-type enzyme . Therefore, these mutations are directly influencing catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 Feb 16, 32(6), 1548 - 54
Use of adenosine (5')polyphospho(5')pyridoxals to study the substrate-binding region of glutathione synthetase from Escherichia coli B; Hibi T et al.; Adenosine(5')polyphospho(5')pyridoxals (APn-PLs, n = 2, 3, 4) were examined for affinity labeling of glutathione synthetase (EC 6.3.2.3) from Escherichia coli B . When the enzyme was incubated with an APn-PL or pyridoxal phosphate in the presence of Mg2+ and then reduced with sodium borohydride, it was most rapidly inactivated by AP4-PL . AP4-PL had a high affinity to the enzyme . The dissociation constant of AP4-PL in the inactivation process was 23 microM . The enzyme was almost completely protected from inactivation by addition of either ATP or gamma-glutamylcysteine . Complete inactivation corresponded to the incorporation of 1 mol of AP4-PL/mol of subunit of the tetrameric enzyme . Proteolytic digestion and sequence analysis of the AP4-PL-labeled enzyme revealed that only Lys-18 was modified . In contrast, the less efficient AP3-PL was found attached to Lys-17, Lys-18, Lys-144, and Lys-148 . In the three-dimensional structure of the enzyme, Lys-18 is located close to the putative gamma-glutamylcysteine-binding site, but Lys-17, Lys-144, and Lys-148 are in the mouth of the inner-solvent region, at the bottom of which is the active-site cleft . Furthermore, difference Fourier analysis with the AP4-PL-soaked crystal of the enzyme showed that the adenosine moiety of the bound AP4-PL was in the crevice, which is the ATP-binding site of the enzyme . These results demonstrate the bivalent binding of AP4-PL lying across the gamma-glutamylcysteine- and ATP-binding sites.

Biochemistry, 1993 Feb 16, 32(6), 1541 - 7
Human nucleotide excision nuclease incises synthetic double-stranded DNA containing a pyrimidine dimer at the fourth phosphodiester linkage 3' to the pyrimidine dimer; Tateishi S et al.; Linear 75mer double-stranded DNA containing a single pyrimidine dimer at a unique site was used to investigate pyrimidine dimer-dependent endonuclease activities from human cells . HeLaS3 cell extract incised the target DNA at the fourth phosphodiester linkage 3' to the pyrimidine dimer . However, incision of the DNA at 5' side of the pyrimidine dimer was not detected . The incision was also detected in cell extracts prepared from other excision repair-proficient cell lines . Incision was detected only on the DNA strand containing a pyrimidine dimer in the presence of poly(dI-dC)-poly(dI- dC) double strand . The reaction required Mg2+ but not ATP . The extract prepared from excision repair-deficient xeroderma pigmentosum (XP) cells belonging to the complementation group A was unable to incise the DNA . Extracts from the complementation groups C, D, and G incised the DNA very weakly at the third phosphodiester linkage 3' to the pyrimidine dimer, a site different from that incised by normal human cell extract . These results suggest that the observed incision reaction is associated with excision repair in human cells.

Biochemistry, 1993 Feb 16, 32(6), 1510 - 8
Engineering the zinc binding site of human carbonic anhydrase II: structure of the His-94-->Cys apoenzyme in a new crystalline form; Alexander RS et al.; The structure of the His-94-->Cys variant of human carbonic anhydrase II (CAII) has been determined by X-ray crystallographic methods to a resolution of 2.3 A with a final crystallographic R factor of 0.155 . This variant of CAII crystallizes in orthorhombic space group P2(1)2(1)2(1) which is the first example of a new crystal form for this important zinc hydrase (the wild-type enzyme crystallizes in monoclinic space group P21 under similar crystallization conditions) . Although the overall structure of the enzyme in the orthorhombic crystal form is similar to that of the wild-type protein in the monoclinic crystal form, the rms deviation of C alpha atoms between the two structures is 0.5 A . Larger structural deviations occur in regions of the protein molecule involved in crystal lattice contacts, and significant structural changes are found in the polypeptide strand containing Cys-94 . Surprisingly, no electron density corresponding to a zinc ion is found in the active site of crystalline His-94-->Cys CAII, even though the stoichiometry of zinc binding to this variant in solution is confirmed by atomic absorption spectroscopy . However, the KD for zinc dissociation from the variant is increased 10(4)-fold compared with wild-type enzyme; furthermore, under the crystallization conditions of high ionic strength (1.75-2.5 M ammonium sulfate), the observed KD is increased further, which leads to zinc dissociation . Spectroscopic analysis of Co(2+)-substituted His-94-->Cys CAII indicates that the metal binds in a tetrahedral geometry with a new thiolate bond.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 Feb 16, 32(6), 1454 - 65
Further characterization of the psbH locus of Synechocystis sp . PCC 6803: inactivation of psbH impairs QA to QB electron transport in photosystem 2; Mayers SR et al.; The psbH gene encodes a small protein which copurifies with photosystem 2 . In the cyanobacterium Synechocystis sp . PCC 6803, psbH is located upstream of the cytochrome b6-f complex genes petC and petA . In striking contrast, in the genomes of plant chloroplasts, psbH is cotranscribed with petB and petD, encoding the other two major subunits of the cytochrome b6-f complex . We report that in Synechocystis sp . PCC 6803 monocistronic psbH and dicistronic petCA transcripts are probably initiated separately, each from DNA regions bearing some similarity to Escherichia coli sigma 70 promoters . Synechocystis sp . PCC 6803 psbH null mutants were generated by cartridge mutagenesis . Studies using a rapid screening procedure involving in situ complementation showed that the PsbH protein is not absolutely required for the assembly of a functionally active photosystem 2 complex since psbH insertion and deletion strains were able to grow photoautotrophically . The rate of photoautotrophic growth was, however, slower than the wild type, and studies of oxygen evolution, chlorophyll fluorescence, and thermoluminescence indicated that this reduction in growth rate is probably due mainly to an impairment in electron flow from QA to QB . We conclude, therefore, that the PsbH protein is not an absolute requirement for photosystem 2 activity but that it functions to optimize electron flow between the two secondary plastoquinone acceptors by interacting with the QB site on the D1 protein.

Biochemistry, 1993 Feb 16, 32(6), 1396 - 400
Mannitol-specific enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli: physical size of enzyme IImtl and its domains IIBA and IIC in the active state; Lolkema JS et al.; The size of enzyme IImtl solubilized in the active state has been determined by size-exclusion chromatography under conditions that favor the association of the enzyme . The contribution of the detergent bound to the enzyme was determined by solubilizing the enzyme and running the TSK250 column in a number of detergents with decreasing micellar sizes . The size, expressed as the equivalent molecular mass of a globular protein, decreased from 315 kDa in decylPEG, to 275 kDa in octylPEG and octyl glucoside, and then to 245 kDa in cholate . Enzyme IImtl is not active in the latter three detergents when at concentrations above their cmc values but still binds mannitol with high affinity without significant loss of sites . This, together with the full reversibility of the inactivation, is taken as evidence that the enzyme does not unfold or dissociate in these detergents . The sizes of the separated domains IIBA and IIC of enzyme IImtl were 38 and 175 kDa, respectively . The cytoplasmic domain, IIBA, was monomeric at high concentration, whereas the membrane-bound domain, IIC, was associated at much lower concentration . Apparently, the sites that interact to keep enzyme IImtl in the associated state are exclusively located in the membrane-bound domain.

Biochemistry, 1993 Feb 16, 32(6), 1519 - 27
Cloning, overexpression, and characterization of the functional dihydroorotase domain of the mammalian multifunctional protein CAD; Zimmermann BH et al.; Mammalian dihydroorotase (DHOase) is part of a large multidomain protein called CAD, which initiates the first three steps in the de novo pyrimidine biosynthetic pathway . DHOase activity is carried out by a 44-kDa structural domain which could be isolated in active form from elastase digests . A core domain the same size as monofunctional dihydroorotases was defined, although the domain borders were uncertain . Two recombinants were overexpressed in Escherichia coli . The first encoded the core domain with 55 and 13 residues added to the amino and carboxyl ends, respectively, and was expressed in insoluble form . The recombinant protein was refolded from urea into a soluble form which was resistant to protease digestion but was catalytically inactive . In contrast, the proteolytic fragment from CAD could be unfolded and refolded with recovery of 40-100% catalytic activity . The second construct, which approximated the proteolytic fragment, had 21 residues on the amino end and 65 residues on the carboxyl end of the core domain . A 46-kDa soluble protein was expressed at 4% of the total soluble protein . The recombinant protein was catalytically active and had the expected amino-terminal sequence . The protein was purified to homogeneity . Dihydroorotase saturation curves gave a Km = 41.9 +/- 3.5 microM and a kcat = 2.79 +/- 0.06 s-1, parameters that were similar to those obtained for the proteolytic fragment . The Km was 6-fold higher and the kcat 2-fold lower than the values obtained for the parent protein, which suggests that interdomain interactions stabilize the active conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

FEMS Microbiol Lett, 1993 Feb 15, 107(1), 71 - 6
Temperature-sensitive amber suppression of ompF'-'lacZ fused gene expression in a supE mutant of Escherichia coli K12; Kuriki Y; In an ompF'-'lacZ fusion system carried by the open reading frame vector pORF1 in a supE mutant of Escherichia coli K12, read-through of an amber codon was decreased at temperatures higher than 40 degrees C . This effect of temperature was dependent on the nucleotide sequence surrounding the amber codon, which was inserted into a site between the ompF and lacZ cistrons . Upon a temperature shift-up from 30 to 42 degrees C, beta-galactosidase synthesis directed by this fusion showed a transient arrest.

FEMS Microbiol Lett, 1993 Feb 15, 107(1), 25 - 30
A yeast gene for trehalose-6-phosphate synthase and its complementation of an Escherichia coli otsA mutant; McDougall J et al.; A Saccharomyces cerevisiae gene for trehalose-6-phosphate synthase (TPS1) was sequenced . The gene appeared to code for a protein of 495 amino acid residues, giving the protein a molecular mass of 56 kDa . The TPS1 gene was able to restore both osmotolerance and trehalose accumulation during salt stress in an Escherichia coli strain mutated in the otsA gene encoding trehalose-6-phosphate synthase . Complementation studies with E . coli galU mutants showed that the TPS1-encoded trehalose-6-phosphate synthase is UDP-glucose-dependent . Sequence analysis and data base searches showed that TPS1 is allelic to GGS1, byp1, cif1 and fdp1 . A possible gene for trehalose-6-phosphate synthase in Methanobacterium thermoautotrophicum was identified.

FEMS Microbiol Lett, 1993 Feb 15, 107(1), 111 - 4
Purification of a fibroblast-inhibitory factor from Actinobacillus actinomycetemcomitans Y4; Kataoka M et al.; A factor showing inhibitory activity against human gingival fibroblasts was extracted from the cytosol fraction of Actinobacillus actinomycetemocimitans Y4 . The activity markedly inhibited the proliferation of human gingival fibroblasts, but had no effect on cell viability or gross morphology . No such activity was found in cytosol fractions from either Porphyromonas gingivalis 381 or Escherichia coli HB101 . The extract from A . actinomycetemocomitans Y4 was then purified by anion-exchange chromatography, hydroxyapatite chromatography and gel-filtration chromatography to give a single band on SDS-PAGE with an apparent molecular mass of 65 kDa . The purification ratio was 183-fold with a recovery rate of 5% compared with the crude extract (starting material) when the activity was assessed by direct cell counts.

FEMS Microbiol Lett, 1993 Feb 15, 107(1), 101 - 5
Non-specific hole formation in the Escherichia coli inner membrane by lambda S proteins in independent of cellular secY and secA functions and of the proportion of membrane acidic phospholipids; Rietsch A et al.; Formation of the lesion in the Escherichia coli inner membrane caused by lambda lysis protein S was examined by electron microscopy . We also show that macromolecules exceeding the size of the lambda R transglycosylase can pass through the S-dependent hole and that assembly of the S-dependent hole is independent of the proportion of acidic phospholipids in the inner membrane and of components of the cellular transport machinery.

Anal Biochem, 1993 Feb 15, 209(1), 1 - 5
Enzymatic synthesis of 6R-{U-14C}tetrahydrobiopterin from {U-14C}GTP; Hatakeyama K et al.; An enzymatic method for preparing 6R-{U-14C}-tetrahydrobiopterin from {U-14C}GTP is presented . This method utilizes purified preparations of three biosynthetic enzymes for 6R-tetrahydrobiopterin, i.e., Escherichia coli GTP cyclohydrolase I, rat 6-pyruvoyl-tetrahydropterin synthase, and rat sepiapterin reductase . Based on the catalytic properties of these enzymes, the reaction conditions were optimized to complete the entire conversion reaction from GTP to 6R-tetrahydrobiopterin in a single reaction solution without the need to isolate unstable intermediates after each enzymatic reaction . The reaction conditions thereby established yielded {U-14C}biopterin in an amount equivalent to 75%, on a molar basis, of the initial amount of {U-14C}GTP . The product was subsequently isolated by high-performance liquid chromatography . The method produced labeled 6R-tetrahydrobiopterin with a specific activity of 450 Ci/mol and an overall yield of more than 60%.

Eur J Biochem, 1993 Feb 15, 212(1), 27 - 34
Isolation of a ripening and wound-induced cDNA from Cucumis melo L . encoding a protein with homology to the ethylene-forming enzyme; Balague C et al.; A cDNA clone (pMEL1) was isolated from a climacteric melon fruit cDNA library using the tomato ethylene-forming-enzyme (EFE) cDNA, pTOM13, as a probe . Northern analysis of RNA isolated from wounded leaf and fruit tissue and from preclimacteric and climacteric pericarp tissue was used to determine the pattern of pMEL1 RNA expression . pMEL1 hybridized to a 1.3-kb transcript in climacteric fruit and wounded leaf tissue but was undetectable in preclimacteric fruit and unwounded leaves . Maximal expression of pMEL1 RNA occurred in wounded ripe fruit . pMEL1 contained a 1230-bp insert containing a predicted open reading frame of 318 amino acids and molecular mass of 35.3 kDa . The predicted amino acid sequence of pMEL1 was 73-81% identical to the deduced amino acid sequences of tomato (pTOM13) EFE and EFE-related genes isolated from tomato and avocado fruit and senescent carnation petals . Genomic Southern analysis indicated that pMEL1 hybridized to a number of genomic fragments consistent with the presence of more than one pMEL1-related gene in melon . On Western analysis of total protein extracts from ripe tomato and melon fruit, using an antibody raised against tomato EFE (pTOM13) expressed in Escherichia coli, a single 35.5-kDa protein was detected . A 35-kDa product translated from in-vitro transcribed pMEL1 and immunoadsorbed by anti-EFE serum was very similar in size to the predicted 35.3-kDa pMEL1 cDNA protein product . These results indicate that pMEL1 may encode an EFE gene involved in ethylene biosynthesis during fruit ripening and may be identical to or share extensive sequence similarity with an EFE expressed in response to tissue wounding.

Eur J Biochem, 1993 Feb 15, 212(1), 201 - 10
Thymidine phosphorylase activity of platelet-derived endothelial cell growth factor is responsible for endothelial cell mitogenicity; Finnis C et al.; Recombinant human platelet-derived endothelial cell growth factor, expressed in the yeast Saccharomyces cerevisiae was purified to greater than 98% purity by anion-exchange and hydroxyapatite chromatography . It was shown to possess thymidine phosphorolytic activity in vitro (pH optimum, pH 5.3; Km, 0.11 mM; Vmax, 12.5 mmol min-1 mg-1; turnover number, 9.4 s-1) . Covalent modification simultaneously inhibited the enzymatic and mitogenic properties of the protein, while interaction with a cell-surface receptor was not required to stimulate mitogenesis . Purified Escherichia coli thymidine phosphorylase was also mitogenic toward endothelial cells . It is proposed that platelet-derived endothelial cell growth factor is human thymidine phosphorylase which promotes endothelial cell proliferation by reducing thymidine levels that would otherwise be inhibitory to endothelial cell growth.

Arch Biochem Biophys, 1993 Feb 15, 301(1), 64 - 70
Regulation of the mitochondrial ATP synthase/ATPase complex: cDNA cloning, sequence, overexpression, and secondary structural characterization of a functional protein inhibitor; Lebowitz MS et al.; The ATPase inhibitor protein of the rat liver mitochondrial ATP synthase/ATPase complex has been cloned from a rat liver cDNA library, and its nucleotide sequence determined . The sequence is highly homologous to both the bovine heart (approximately 70%) and the yeast inhibitor proteins (approximately 40%) . The deduced protein sequence is 107 amino acids in length, and based on homology to the bovine heart protein, the first 25 N-terminal amino acids encode a putative mitochondrial targeting sequence . The "mature" protein (without the targeting sequence) fused to the maltose binding protein has been overexpressed in Escherichia coli . The maltose binding protein was used as a handle for the development of a rapid one-step purification of the fusion protein by affinity chromatography on an amylose resin . The purified fusion protein was cleaved with Factor Xa protease at the fusion junction, and the resulting ATPase inhibitor protein was purified to > 90% purity . The purified, overexpressed inhibitor protein displays normal inhibitor activity . The protein inhibits ATP hydrolysis catalyzed by the ATP synthase/ATPase complex in submitochondrial particles in a manner kinetically indistinguishable from the same protein purified from rat liver mitochondria, and exhibits a specific activity of approximately 10,000 units/mg . The secondary structure of the inhibitor protein was determined by circular dichroism spectropolarimetry . The experimentally determined structure shows a high content of alpha-helix and is in good agreement with sequence-based structural predictions . As the function of the inhibitor protein is known to exhibit a high dependence on pH, a study of the pH dependence of inhibitor secondary structure was performed . It is shown that as pH is lowered, conditions which activate inhibitory capacity, the protein loses significant alpha-helical structure . This is the first report of the overexpression in E . coli of a functional ATPase inhibitor protein . Secondary structural analysis of this protein indicates that conversion from its active to its inactive form involves a significant conformational change.

Arch Biochem Biophys, 1993 Feb 15, 301(1), 58 - 63
Production and characterization of polyclonal antibodies in rabbits to 4S-limonene synthase from spearmint (Mentha spicata); Alonso WR et al.; Limonene synthase, a monoterpene cyclase from the oil glands of spearmint (Mentha spicata) leaves that catalyzes the conversion of geranyl pyrophosphate to (-)-4S-limonene, was purified, and polyclonal antibodies were generated in rabbits against the sodium dodecyl sulfate-denatured protein . Immunoblotting analysis revealed that the antibodies were very specific for denatured limonene synthase from all Mentha species tested . However, no immunological cross-reactivity was observed with denatured limonene synthases from Valencia oranges (Citrus sinensis, Rutaceae) or wormseed (Chenopodium ambrosioides, Chenopodiaceae) . Furthermore, the antibody preparation did not detectably cross-react with other monoterpene cyclases from related angiosperm species of the Lamiaceae, Asteraceae, and Umbellifereae, or from conifer species, and no cross-reactivity was demonstrated toward several sesquiterpene cyclases of higher plant and fungal origin . Although the antibody preparation was highly selective for denatured limonene cyclase from Mentha, the antibodies did not recognize the native protein in several different types of experiments . Nevertheless, specificity for the target enzyme was unambiguously demonstrated when the antibody preparation was shown to cross-react with the cyclase protein expressed in Escherichia coli that harbored the corresponding limonene synthase cDNA gene from M . spicata.

Biochem Biophys Res Commun, 1993 Feb 15, 190(3), 794 - 800
Membrane association of leucyl-tRNA synthetase during leucine starvation in Escherichia coli; Williamson RM; The influence of leucine starvation on the subcellular location of leucyl-tRNA synthetase in Escherichia coli was examined in a leucine auxotrophic strain by sucrose density sedimentation analysis . Analysis of the subcellular distribution of leucyl-tRNA synthetase activity revealed that during unrestricted growth, the leucine synthetase enzyme activity was found to be localized in the soluble protein fraction of the gradient . However, during restricted growth on low levels of leucine, leucyl-tRNA synthetase activity was localized in the cytoplasmic membrane fraction of the gradient . The transition from soluble to membrane-association of the enzyme also occurred following inhibition of protein synthesis by treatment of cells with chloramphenicol . These results collectively suggest that leucyl-tRNA synthetase may be recruited to the cytoplasmic membrane in response to shortages of leucine or perturbation of protein synthesis.

Biochem Biophys Res Commun, 1993 Feb 15, 190(3), 1066 - 72
pH-dependent decarboxylation of 2-amino-3-ketobutyrate, the unstable intermediate in the threonine dehydrogenase-initiated pathway for threonine utilization; Marcus JP et al.; 2-Amino-3-ketobutyrate can be readily formed enzymatically by the action of L-threonine dehydrogenase . A convenient assay for determining the half-life of this beta-keto acid is afforded by its rapid and quantitative conversion to glycine (+ acetyl CoA), as catalyzed by 2-amino-3-ketobutyrate CoA lyase . Using this system, we have found the half-life of 2-amino-3-ketobutyrate varies with pH from 8.6 minutes at pH 5.9 to 140 minutes at pH 11.1 yielding a theoretical titration curve that predicts a pKa value of 8.15 for the alpha-amino group of this intermediate . These data are considered relevant to discussions pertaining to a threonine dehydrogenase/2-amino-3-ketobutyrate CoA lyase enzyme complex in the threonine utilization pathway and to mechanistic aspects of the 5-aminolevulinate synthase-catalyzed reaction where 2-amino-3-ketoadipate is involved.

Biochem J, 1993 Feb 15, 290 ( Pt 1), 279 - 87
Purification and characterization of 5-aminolaevulinic acid dehydratase from Escherichia coli and a study of the reactive thiols at the metal-binding domain; Spencer P et al.; 5-Aminolaevulinic acid dehydratase (ALAD) from a recombinant strain of Escherichia coli was purified to homogeneity . The enzyme is a homo-octamer of subunit M(r) 36554 +/- 17 . Enzyme activity was dependent on the presence of Zn2+ ions and an exogenous thiol . Two molar equivalents of Zn2+ are bound/mol of subunit under reducing conditions . On exposure to the metal chelator EDTA, the two Zn2+ ions are removed, giving an inactive metal-depleted apo-ALAD . On oxidation of holo-ALAD, two disulphide bonds are formed with the loss of 1 mol of Zn2+/mol of subunit . The formation of the first disulphide led to the loss of catalytic activity . Replacement of the two bound Zn2+ ions with Co2+ resulted in the formation of a green protein with a spectrum indicative of the presence of charge-transfer bands from one or more cysteine-Co2+ ligands . While Mg2+ could not activate apo-ALAD alone, it was able to substitute for the second molar equivalent of bound Zn2+, leading to a further 4-fold stimulation in activity . The four cysteine residues involved in the formation of the two disulphide bonds were identified by protein-chemistry studies and were all located in a region of the protein extending from amino acid residues 120-134 . Protein sequence data obtained in the present study has permitted the resolution of several differences between the published gene-derived protein sequences for ALAD from E . coli.

Biochem J, 1993 Feb 15, 290 ( Pt 1), 15 - 9
The pKa of the catalytic histidine residue of chloramphenicol acetyltransferase; Lewendon A et al.; A catalytically essential histidine residue (His-195) of chloramphenicol acetyltransferase (CAT) acts as a general base in catalysis, abstracting a proton from the primary hydroxy group of chloramphenicol . The pKa of His-195 has been determined from the pH-dependence of chemical modification . Both methyl 4-nitrobenzenesulphonate and iodoacetamide inactivate CAT by irreversible modification of His-195 . The kinetics of inactivation by methyl 4-nitrobenzenesulphonate are pseudo-first-order, and the pH-dependence of inactivation yields a pKa value of 6.60 . Iodoacetamide inactivation proceeds with second-order kinetics and a pKa value of 6.80 . An alternative site of modification at the active site of CAT is the thiol group of Cys-31, a residue which has no catalytic role . On replacement of Cys-31 with alanine (Ala-31 CAT), the pH-dependence of iodoacetamide inactivation gives a pKa value of 6.66 . The pKa values derived from chemical-modification experiments directed at His-195 are in agreement with the pKa values of 6.62 and 6.61 determined for wild-type and Ala-31 CAT respectively from the pH-dependence of kcat/Km.

Proc Natl Acad Sci U S A, 1993 Feb 15, 90(4), 1576 - 9
In vitro selection of optimal DNA substrates for T4 RNA ligase; Harada K et al.; We have used in vitro selection techniques to characterize DNA sequences that are ligated efficiently by T4 RNA ligase . We find that the ensemble of selected sequences ligated about 10 times as efficiently as the random mixture of sequences used as the input for selection . Surprisingly, the majority of the selected sequences approximated a well-defined consensus sequence.

Proc Natl Acad Sci U S A, 1993 Feb 15, 90(4), 1556 - 60
5' sequences are important positive and negative determinants of the longevity of Chlamydomonas chloroplast gene transcripts; Salvador ML et al.; We have found that sequences in the 5' leader of the Chlamydomonas chloroplast rbcL gene, when fused 5' to foreign genes, destabilize transcripts of these chimeric genes in the chloroplast of transgenic Chlamydomonas but that 5' sequences of the rbcL structural gene prevent this destabilization . Transcripts of the chloroplast rbcL gene are about equally abundant at all times in Chlamydomonas reinhardtii growing on an alternating 12-h light/12-h dark cycle . However, Chlamydomonas chloroplast transformants, harboring chimeric genes containing the same rbcL promoter with 63 or 92 bp of the rbcL 5' leader sequence fused upstream of the Escherichia coli uidA (beta-glucuronidase, GUS) gene, accumulated GUS transcripts only in the dark . Transcripts disappeared rapidly upon illumination of the cells . The same phenomenon was exhibited by transcripts of chimeric genes in which the GUS gene coding sequence was replaced by other unrelated genes . The precipitous light-induced drop in GUS transcript abundance was found to be due to an approximately 16-fold increase in the rate of degradation of GUS transcripts in light rather than to a decrease in the rate of transcription of the GUS gene . Transcripts of a chimeric rbcL-GUS construct in which the leader sequence of the rbcL gene was replaced by 103 bp of the leader sequence of the atpB gene were stable in illuminated cells . The destabilizing effect of the rbcL 5' leader sequence was reversed by adding 257 bp of the 5' coding region of the rbcL gene . The results show that chloroplast transcript levels in illuminated Chlamydomonas cells--and perhaps in other cases--can be determined, at least to some extent, by sequences and interactions of sequences transcribed from the 5' ends of genes.

Proc Natl Acad Sci U S A, 1993 Feb 15, 90(4), 1536 - 40
A Drosophila photoreceptor cell-specific protein, calphotin, binds calcium and contains a leucine zipper; Ballinger DG et al.; The calphotin protein, encoded by the calphotin (cap) gene, is expressed in the soma and axons of all Drosophila photoreceptor cells . It is expressed early in photo-receptor cell development, at the time when cell-type decisions are being made . Expression of calphotin is not altered by the glass mutation, which blocks photoreceptor cell development . The calphotin protein binds calcium and contains a long C-terminal leucine zipper . Potential implications of these properties are discussed.

Proc Natl Acad Sci U S A, 1993 Feb 15, 90(4), 1526 - 30
Pigment epithelium-derived factor: neurotrophic activity and identification as a member of the serine protease inhibitor gene family; Steele FR et al.; Cultured pigment epithelial cells of the fetal human retina secrete a protein, pigment epithelium-derived factor (PEDF), that induces a neuronal phenotype in cultured human retinoblastoma cells . Morphological changes include the induction of an extensive neurite meshwork and the establishment of corona-like cellular aggregates surrounding a central lumen . The differentiated cells also show increases in the expression of neuron-specific enolase and the 200-kDa neurofilament subunit . Amino acid and DNA sequence data demonstrate that PEDF belongs to the serine protease inhibitor (serpin) family . The PEDF gene contains a typical signal-peptide sequence, initiator methionine codon, and polyadenylylation signal and matches the size of other members of the serpin superfamily (e.g., alpha 1-antitrypsin) . It lacks homology, however, at the putative serpin reactive center . Thus, PEDF could exert a paracrine effect in the embryonic retina, influencing neuronal differentiation by a mechanism that does not involve classic inhibition of serine protease activity.

Proc Natl Acad Sci U S A, 1993 Feb 15, 90(4), 1518 - 22
The short form of the CheA protein restores kinase activity and chemotactic ability to kinase-deficient mutants; Wolfe AJ et al.; Escherichia coli expresses two forms of the chemotaxis-associated CheA protein, CheAL and CheAS, as the result of translational initiation at two distinct, in-frame initiation sites in the gene cheA . The long form, CheAL, plays a crucial role in the chemotactic signal transduction mechanism by phosphorylating two other chemotaxis proteins: CheY and CheB . CheAL must first autophosphorylate at amino acid His-48 before transferring its phosphono group to these other signal transduction proteins . The short form, CheAS, lacks the N-terminal 97 amino acids of CheAL and, therefore, does not possess the site of autophosphorylation . Here we demonstrate that although it lacks the ability to autophosphorylate, CheAS can mediate phosphorylation of kinase-deficient variants of CheAL each of which retains a functional autophosphorylation site . This transphosphorylation enables these kinase-deficient CheAL variants to phosphorylate CheY . Because it mediates this activity, CheAS can restore to kinase-deficient E . coli cells the ability to tumble and, thus, to perform chemotaxis in swarm plate assays.

Proc Natl Acad Sci U S A, 1993 Feb 15, 90(4), 1474 - 8
Crystal structure of Escherichia coli L-asparaginase, an enzyme used in cancer therapy; Swain AL et al.; The crystal structure of Escherichia coli asparaginase II (EC 3.5.1.1), a drug (Elspar) used for the treatment of acute lymphoblastic leukemia, has been determined at 2.3 A resolution by using data from a single heavy atom derivative in combination with molecular replacement . The atomic model was refined to an R factor of 0.143 . This enzyme, active as a homotetramer with 222 symmetry, belongs to the class of alpha/beta proteins . Each subunit has two domains with unique topological features . On the basis of present structural evidence consistent with previous biochemical studies, we propose locations for the active sites between the N- and C-terminal domains belonging to different subunits and postulate a catalytic role for Thr-89.

Proc Natl Acad Sci U S A, 1993 Feb 15, 90(4), 1470 - 3
Identification of C-terminal amino acid residues of cauliflower mosaic virus open reading frame III protein responsible for its DNA binding activity; Mougeot JL et al.; We cloned in Escherichia coli truncated versions of the protein p15 encoded by open reading frame III of cauliflower mosaic virus . We then compared the ability of the wild-type p15 (129 amino acids) and the deleted p15 to bind viral double-stranded DNA genome . Deletions of > 11 amino acids in the C-terminal proline-rich region resulted in loss of DNA binding activity of wild-type p15 . Moreover, a point mutation of the proline at position 118 sharply reduced the interaction between the viral protein and DNA . These results suggest that cauliflower mosaic virus p15 belongs to the family of DNA binding proteins having a proline-rich motif involved in interaction with double-stranded DNA.

Proc Natl Acad Sci U S A, 1993 Feb 15, 90(4), 1397 - 401
Sequence conversion during postreplicative adenovirus overlap recombination; Bennett KL et al.; Sequence conversion efficiently transfers genetic information in high yield during postreplicative adenovirus overlap recombination . This process is intrinsically nonreciprocal, depends on adenovirus-specific strand-displacement replication by both partner molecules, and requires that complementary sequences on displaced strands must exceed a minimal length to form a heteroduplex intermediate.

Proc Natl Acad Sci U S A, 1993 Feb 15, 90(4), 1364 - 8
Evidence for functional interaction between elongation factor Tu and 16S ribosomal RNA; Powers T et al.; Translation of the genetic code requires the accurate selection of elongation factor (EF)-Tu.GTP.tRNA ternary complexes at the ribosomal acceptor site, or A site . Several independent lines of evidence have implicated the universally conserved 530 loop of 16S rRNA in this process; yet its precise role has not been identified . Using an allele-specific chemical probing strategy, we have examined the functional defect caused by a dominant lethal G-->A substitution at position 530 . We find that mutant ribosomes are impaired in EF-Tu-dependent binding of aminoacyl-tRNA in vitro; in contrast, nonenzymatic binding of tRNA to the A and P sites is unaffected, indicating that the defect involves an EF-Tu-related function rather than tRNA-ribosome interactions per se . In vivo, the mutant ribosomes are found in polysomes at low levels and contain reduced amounts of A-site-bound tRNA, but normal levels of P-site tRNA, in agreement with the in vitro results; thus the dominant lethal phenotype of mutations at G530 can be explained by impaired interaction of mutant ribosomes with ternary complex . These results provide evidence for a newly defined function of 16S rRNA--namely, modulation of EF-Tu activity during translation.

Proc Natl Acad Sci U S A, 1993 Feb 15, 90(4), 1315 - 9
RuvA and RuvB proteins of Escherichia coli exhibit DNA helicase activity in vitro; Tsaneva IR et al.; The SOS-inducible ruvA and ruvB gene products of Escherichia coli are required for normal levels of genetic recombination and DNA repair . In vitro, RuvA protein interacts specifically with Holliday junctions and, together with RuvB (an ATPase), promotes their movement along DNA . This process, known as branch migration, is important for the formation of heteroduplex DNA . In this paper, we show that the RuvA and RuvB proteins promote the unwinding of partially duplex DNA . Using single-stranded circular DNA substrates with annealed fragments (52-558 nucleotides in length), we show that RuvA and RuvB promote strand displacement with a 5'-->3' polarity . The reaction is ATP-dependent and its efficiency is inversely related to the length of the duplex DNA . These results show that the ruvA and ruvB genes encode a DNA helicase that specifically recognizes Holliday junctions and promotes branch migration.

Proc Natl Acad Sci U S A, 1993 Feb 15, 90(4), 1166 - 71
Two light-transducing membrane proteins: bacteriorhodopsin and the mammalian rhodopsin; Khorana HG; Site-directed mutagenesis has provided insight into the mechanisms of action of bacteriorhodopsin and rhodopsin . These studies are summarized here.

Proc Natl Acad Sci U S A, 1993 Feb 15, 90(4), 1146 - 53
Molecular recognition analyzed by docking simulations: the aspartate receptor and isocitrate dehydrogenase from Escherichia coli; Stoddard BL et al.; Protein docking protocols are used for the prediction of both small molecule binding to DNA and protein macromolecules and of complexes between macromolecules . These protocols are becoming increasingly automated and powerful tools for computer-aided drug design . We review the basic methodologies and strategies used for analyzing molecular recognition by computer docking algorithms and discuss recent experiments in which (i) substrate and substrate analogues are docked to the active site of isocitrate dehydrogenase and (ii) maltose binding protein is docked to the extracellular domain of the receptor, which signals maltose chemotaxis.

J Biol Chem, 1993 Feb 15, 268(5), 3715 - 9
The regulatory domain of protein kinase C beta 1 contains phosphatidylserine- and phorbol ester-dependent calcium binding activity; Luo JH et al.; Certain isoforms of protein kinase C (PKC) require both Ca2+ and phospholipid for optimum activity . However, little is known about the nature of the interaction between PKC and Ca2+ . The present study demonstrates that the isolated regulatory domain of PKC beta 1, when synthesized as a fusion protein in Escherichia coli, binds 45Ca2+ with high affinity, but only in the presence of phosphatidylserine or 12-O-tetradecanoyl-phorbol-13-acetate . This binding is highly selective for Ca2+ since it is preferentially inhibited by excess non-radioactive Ca2+ when compared with the cations Mg2+, Mn2+, Na+, or K+ . It appears, therefore, that the binding of Ca2+ to PKC requires a complex tertiary structure in the regulatory domain.

J Biol Chem, 1993 Feb 15, 268(5), 3616 - 24
The P1 plasmid partition complex at parS . II . Analysis of ParB protein binding activity and specificity; Funnell BE et al.; The P1 plasmid prophage is partitioned by a very high affinity protein complex at its partition site, parS, that contains the P1 ParB protein and Escherichia coli integration host factor (IHF) . ParB binds to regions of parS that flank the IHF binding site . In this report, we have examined the sequences to which ParB binds, the spatial relationship between them, and the effect of IHF on ParB binding patterns . Methylation protection and interference experiments were performed on supercoiled plasmids . Mutations that interfered with the action of both proteins in vivo were identified following random mutagenesis of parS . These studies revealed that ParB binds to a complicated, nonsymmetrical region in the right side of parS . ParB recognizes a partial copy of this sequence, TCGCCA, in the left side of parS with much lower affinity . The presence of IHF greatly facilitates the interaction of ParB with parS such that both sides bind with an equal affinity that is much greater than to either side alone . The stimulation by IHF is strongly influenced by helical phasing . These observations support the proposal that ParB is directed, by the bend created by IHF, to bind simultaneously to properly placed sequences flanking the IHF site.

J Biol Chem, 1993 Feb 15, 268(5), 3586 - 93
Translocation of conjugated presecretory proteins possessing an internal non-peptide domain into everted membrane vesicles in Escherichia coli; Kato M et al.; Polypeptides comprising 20 amino acid residues (Y2) were covalently bound to the carboxyl terminus of a truncated proOmpA (proOmpA-D72C) through N,N'-bis(3-maleimidopropionyl)-2-hydroxy-1,3-propanediamine (X) . The length of the inverted linker domain was 2.8 nm . proOmpA-D72C-X-Y2 thus synthesized was subjected to in vitro translocation into everted membrane vesicles of Escherichia coli . The conjugated protein was translocation-competent in terms of both proteinase K resistance and signal peptide cleavage, when a proton motive force (delta microH+) was imposed . The translocation was ATP-dependent . The proteinase K-treatment resulted in the digestion of SecA, SecE, and SecY in the membrane, suggesting that the proteinase K resistance of the Y2 domain was not due to its interaction with these Sec proteins in the secretory machinery . In the absence of delta microH+, the translocation ceased at the linker domain . Upon the imposition of delta microH+, the linker-Y2 domain underwent translocation, which did not require ATP hydrolysis as in the case of the translocation of the latter portion of usual secretory proteins . The translocation was prevented by anti-Y2 IgG even when delta microH+ was imposed . Another conjugated protein, which possesses a polypeptide comprising 61 amino acid residues after the linker (proOmpA-D72C-X-Lpp'), was synthesized . This compound was also translocated into everted membrane vesicles with cleavage of the signal peptide . These results suggest that substances to be translocated through the secretory machinery need not necessarily be solely held together by polypeptide bonds.

J Biol Chem, 1993 Feb 15, 268(5), 3507 - 13
Characterization of a DNA mismatch-binding activity in yeast extracts; Miret JJ et al.; An activity present in nuclear extracts of the yeast Saccharomyces cerevisiae binds specifically to oligonucleotides containing DNA mismatches, as judged by a band shift assay . The specificity of this activity for mismatched DNA was confirmed by competition experiments; binding to radiolabeled heteroduplexes was abolished in the presence of excess unlabeled heteroduplex but not when excess unlabeled homoduplex was added . Both T/G and T/- (single base deletion) mispairs were recognized in each of two sequence contexts . Binding was also observed with G/G, G/A, A/C, and T/C mismatches, but recognition of a C/C mispair was very weak . Competition studies with the various mismatches were consistent with the idea that a single activity recognizes all mispairs tested . Extracts from strains mutant in either or both of two putative mismatch recognition functions, MSH2 and MSH3, were also tested . Mismatch-binding activity was present in extracts of msh3- strains but completely absent in msh2- strains . The molecular weight of the major binding protein was estimated by UV cross-linking experiments to be approximately 110 kDa, in good agreement with the size predicted for Msh2 protein (Reenan, R . A . and Kolodner, R . D . (1992) Genetics 132, 963-973).

J Biol Chem, 1993 Feb 15, 268(5), 3414 - 9
Functional organization of microtubule-associated protein tau . Identification of regions which affect microtubule growth, nucleation, and bundle formation in vitro; Brandt R et al.; Tau protein is a microtubule-associated protein that is almost exclusively expressed in the brain and is enriched in the axon . Determination of tau's sequence has revealed three to four tandem repeats that have been shown to constitute the microtubule binding site . In order to study the functional organization of tau, we prepared a series of truncated tau fragments using an Escherichia coli expression system . We assayed each fragment's activity in promoting growth of microtubules and in nucleating free microtubules . We found that tau's ability to nucleate microtubules requires the presence of additional sequence amino-terminal to that required for growth . We demonstrate that tau's carboxyl and amino termini differentially affect microtubule growth and nucleation . Finally, we show that in vitro microtubule bundle formation occurs when tubulin is assembled in the presence of an amino- and carboxyl-terminally truncated tau protein, whereas almost no bundling is observed in the presence of full-length tau or tau fragments that contain the amino terminus in addition to the repeat domain . We conclude that although the presence of the repeat domain promotes the growth of microtubules, the structural requirements for nucleation activity are more stringent . The differentiation between the growth promoting and nucleation activities on the structural level makes it possible for the two activities to be differentially regulated in vivo.

J Biol Chem, 1993 Feb 15, 268(5), 3268 - 71
Expression and characterization of the heme-binding domain of Chlorella nitrate reductase; Cannons AC et al.; A recombinant protein corresponding to the putative heme-binding domain of assimilatory NADH:nitrate reductase from Chlorella vulgaris has been expressed and purified from transformed Escherichia coli BL21 cells . The recombinant protein, exhibited a subunit molecular mass of approximately 10 kDa with a N-terminal sequence beginning with the residues PAGA in agreement with that predicted by cDNA analysis . The UV-visible spectrum of the protein confirmed the incorporation of heme with maxima at 413 nm and 423, 528, and 557 nm for the oxidized and reduced forms, respectively . Circular dichroism spectra indicated the environment of the heme chromophore was very similar to that of the native enzyme . Potentiometric titrations of the recombinant heme domain yielded a midpoint potential of +16 mV (n = 1, pH 7), substantially higher than the values of -160 mV obtained for the native enzyme and -28 mV obtained for a previously expressed recombinant heme domain that contained part of the Mo-pterin domain . These results indicate that portions of the amino acid sequence that are involved in the formation of the Mo-pterin domain of Chlorella nitrate reductase influence the redox potential of the heme prosthetic group.

J Biol Chem, 1993 Feb 15, 268(5), 3222 - 5
Only one of the charged amino acids located in the transmembrane alpha-helices of the gamma-aminobutyric acid transporter (subtype A) is essential for its activity; Pantanowitz S et al.; The gamma-aminobutyric acid (GABA) transporter (subtype A) is located in nerve terminals and catalyses coupled electrogenic uptake of the neurotransmitter with two or three sodium and one chloride ions . It contains 599 amino acids and 12 putative membrane spanning alpha-helices and is the first described member of a neurotransmitter transporter superfamily . The membrane domain contains 5 charged amino acids which are basically conserved . Using site-directed mutagenesis, we show that only one of them, arginine 69, is absolutely essential for activity . It is located in a highly conserved region encompassing parts of helices 1 and 2 . The three other positively charged amino acids and the only negative charged one, glutamate 467, are not critical . These results suggest that the translocation pathway of the sodium ions through the membrane does not involve charged amino acid residues and underline the importance of the highly conserved stretch between amino acids 66 and 86.

J Biol Chem, 1993 Feb 15, 268(5), 3216 - 21
Mutagenesis of acidic residues in putative membrane-spanning segments of the melibiose permease of Escherichia coli . II . Effect on cationic selectivity and coupling properties; Zani ML et al.; Individual substitution of Cys or Asn for Asp-31, Asp-51, Asp-55, or Asp-120, distributed in different membrane spanning segments of the NH2-terminal domain of melibiose (mel) permease partially or completely inactivates Na(+)-linked sugar transport and stimulation of sugar binding on mel permease by Na+ ions (Pourcher, T., Zani, M.-L., and Leblanc, G . (1993) J . Biol . Chem . 268, 3209-3215) . To investigate further the effect of these substitutions on the cationic selectivity and coupling properties of mel permease, H(+)-melibiose coupled transport, coupling between H+ and melibiose movements, sugar counterflow, and zero-trans sugar efflux by the mutant permeases were analyzed . The results provide additional evidence indicating that manipulation of some of these Asp in the membrane-spanning segments of mel permease alters its cationic selectivity properties . The results also indicate that the individual mutations diversely affect mel permease-coupling properties . For example, only permease with Asn in place of Asp-31 or Cys in place of Asp-51 retains the capacity to actively transport melibiose . On the other hand, replacing Asp-55 by Cys produces uncoupling of cosubstrate flows by the carrier but does not hamper sugar translocation . These and other features of the mutant permeases are used to discuss the relative participation of Asp-31, Asp-51, Asp-55, or Asp-120 to the mel symport mechanism and to its ionic selectivity and also the existence of a possible gating mechanism that may contribute the obligatory coupling of cosubstrate flows by the symporter.

J Biol Chem, 1993 Feb 15, 268(5), 3209 - 15
Mutagenesis of acidic residues in putative membrane-spanning segments of the melibiose permease of Escherichia coli . I . Effect on Na(+)-dependent transport and binding properties; Pourcher T et al.; Four aspartic acids, distributed in different putative membrane-spanning segments of the NH2-terminal domain of melibiose (mel) permease (D31 in helix I, D51 and D55 in helix II, and D120 in helix IV) were individually replaced by either Asn or Cys using site-directed mutagenesis . mel permease with either neutral residues at position 51, 55, or 120 or permease with a Cys in place of D31 does not catalyze significant Na(+)-linked methyl-1-thio-beta-D-galactopyranoside (TMG) accumulation . Binding studies of a high affinity ligand (p-nitrophenyl-alpha-D-galactopyranoside (NPG)) on de-energized membrane vesicles indicate that these modified transporters (i) retain the ability to bind the alpha-galactosides NPG or melibiose and the beta-galactoside TMG and (ii) exhibit a Na(+)-independent sugar-binding phenotype . In contrast, mel permease with an Asn residue at position 31 mediates Na(+)-coupled TMG transport and displays a Na(+)-dependent sugar binding phenotype, but requires a higher concentration of sodium than wild-type permease to produce maximal stimulation of sugar binding . The observation that individual mutation of the Asp residue at position 31, 51, 55, or 120 systematically and selectively modifies the contribution of the coupling ion to the early step of the transport reaction, i.e . cosubstrate binding, raises the possibility that (i) these 4 aspartic residues are at or near the cationic binding site of mel permease, (ii) the NH2-terminal domain of mel permease in which they are distributed accommodates or is part of the cationic binding site, and (iii) the oxygen atoms of these Asp side chains contribute to coordination of the coupling ion.

J Biol Chem, 1993 Feb 15, 268(5), 3156 - 60
Domains near ATP gamma phosphate in the catalytic site of H+-ATPase . Model proposed from mutagenesis and inhibitor studies; Iwamoto A et al.; The beta Gly-149 residue is in a glycine-rich sequence (Gly-Gly-Ala-Gly-Val-Gly-Lys-Thr; residues 149-156) of the Escherichia coli H(+)-ATPase (ATP synthase) beta subunit . Substitution of beta Gly-149 by Ser suppressed the effect of the beta Ser-174-->Phe mutation (Iwamoto, A., Omote, H., Hanada, H., Tomioka, N., Itai, A., Maeda, M., and Futai, M . (1991) J . Biol . Chem . 266, 16350-16355), suggesting that beta Gly-149 is located near beta Ser-174 . In this study, we introduced different residues at position 149 and found that a single mutant beta Cys-149 was defective . The effect of beta Cys-149 mutation was suppressed by beta Gly-172-->Glu, beta Ser-174-->Phe, beta Glu-192-->Val, or beta Val-198-->Ala replacement . These results suggest that beta Gly-149, beta Gly-172, beta Ser-174, beta Glu-192, and beta Val-198 residues are located close together in the catalytic site . From these findings we propose a model of the catalytic site of the enzyme near the gamma phosphate moiety of ATP . F1 enzymes with the double mutations beta Cys-149/beta Glu-172, beta Cys-149/beta Phe-174, beta Cys-149/beta Val-192, and beta Cys-149/beta Ala-198 were less sensitive than wild-type F1 to dicyclohexylcarbodiimide and adenosine triphosphopyridoxal (an affinity analogue of ATP forming a Schiff base with the epsilon-amino group of beta Lys-155 or beta Lys-201), and became sensitive to N-ethylmaleimide in an ATP-protected manner . These results of inhibitor studies are consistent with the proposed model.

J Biol Chem, 1993 Feb 15, 268(5), 3107 - 13
Inactivation of the recA protein by mutation of histidine 97 or lysine 248 at the subunit interface; Nguyen TT et al.; We have used site-directed mutagenesis to prepare two new mutant recA proteins, one in which histidine 97 has been replaced by alanine, and another in which lysine 248 has been replaced by alanine . Although these mutant proteins were originally designed from different considerations, they turned out to have remarkably similar properties . Both the {H97A}recA protein and the {K248A}recA protein bind poorly to single-stranded DNA, have no single-stranded DNA-dependent ATP hydrolysis activity, and do not promote renaturation of complementary single-stranded DNA molecules or the ATP-dependent three-strand exchange reaction . Furthermore, both mutant proteins are defective in Mg(2+)-induced helical filament formation . To account for these results, we propose that the mutation of either histidine 97 or lysine 248 alters subunit interactions between recA monomers and that this leads to the loss of cooperative single-stranded DNA binding and DNA pairing activities . This proposal is consistent with the recently determined x-ray structure of the recA protein, which shows that although histidine 97 and lysine 248 are distant from one another in the monomer structure, these two residues are on the opposing complementary faces of the recA subunit and pack against each other at the interface between adjacent recA monomers in the helical filament (Story, R . M., Weber, I . T., and Steitz, T . A . (1992) Nature 355, 318-325).

J Biol Chem, 1993 Feb 15, 268(5), 3099 - 106
Glutamate synthase genes of the diazotroph Azospirillum brasilense . Cloning, sequencing, and analysis of functional domains; Pelanda R et al.; A 10-kilobase EcoRI fragment of Azospirillum brasilense genomic DNA was cloned in Escherichia coli . Two open reading frames of 4548 and 1446 base pairs (bp) were identified within the fragment as the structural genes for the alpha and beta subunits (gltB and gltD, respectively) of A . brasilense GltS . The organization of the gltBD region of A . brasilense differs from that of the corresponding region in E . coli: in A . brasilense, gltD is upstream relative to gltB, and its stop codon is separated by 141 bp from the first ATG of gltB . The deduced amino acid sequences reveal a high similarity with GltS from E . coli and with the ferredoxin-dependent GltS from maize . Binding domains for flavin cofactors and NADPH, a domain for glutamine binding and activation, and cysteine clusters for iron-sulfur centers formation were tentatively identified on the basis of sequence comparison with flavoproteins, pyridine nucleotide-dependent enzymes, amidotransferases, and iron-sulfur proteins.

FEBS Lett, 1993 Feb 15, 317(3), 267 - 70
ATP-driven Na+ transport and Na(+)-dependent ATP synthesis in Escherichia coli grown at low delta mu H+; Avetisyan AV et al.; In inverted subcellular vesicles of Escherichia coli grown at high delta mu H+ (neutral pH, no protonophorous uncoupler), ATP-driven Na+ transport and oxidative phosphorylation are completely inhibited by the protonophore CCCP . If E . coli was grown at low delta mu H+, i.e . at high pH or in the presence of uncoupler, some oxidative phosphorylation was observed in the vesicles even in CCCP-containing medium, and Na+ transport was actually stimulated by CCCP . The CCCP-resistant transport and phosphorylation were absent from the unc mutant lacking F0F1 ATPase . Both processes proved to be sensitive to (i) the Na+/H+ antiporter monensin, (ii) the Na+ uniporter ETH 157, (iii) the F0 inhibitors DCCD and venturicidin, and (iv) the F1 inhibitor aurovertin . The CCCP-resistant oxidative phosphorylation was stimulated by Na+ and arrested by oppositely directed delta pNa . These data are consistent with the assumption that, under appropriate growth conditions, the F0F1-type ATPase of E . coli becomes competent in transporting Na+ ions.

FEBS Lett, 1993 Feb 15, 317(3), 237 - 40
Synthesis of homocysteine thiolactone by methionyl-tRNA synthetase in cultured mammalian cells; Jakubowski H et al.; Homocysteine thiolactone is a product of an error-editing reaction, catalyzed by Escherichia coli and Saccharomyces cerevisiae methionyl-tRNA synthetases, which prevents incorporation of homocysteine into tRNA and protein both in vitro and in vivo . Here, homocysteine thiolactone is also shown to be synthesized by cultured mammalian cells such as human cervical carcinoma (HeLa), mouse renal adenocarcinoma (RAG), and Chinese hamster ovary (CHO) cells labeled with {35S}methionine, but not by normal human and mouse (Balb/c 3T3) fibroblasts . A temperature-sensitive methionyl-tRNA synthetase mutant of CHO cells, Met-1, does not make the thiolactone at the non-permissive temperature . The data indicate that methionyl-tRNA synthase is involved in synthesis of homocysteine thiolactone in CHO cells, thereby extending this important proofreading mechanism to mammalian cells.

Arch Biochem Biophys, 1993 Feb 15, 301(1), 98 - 102
Effect of glutathione on aconitase in Escherichia coli; Gardner PR et al.; The effect of glutathione (GSH) on the superoxide-sensitive {4Fe-4S}-containing aconitase of Escherichia coli was explored . A mutant deficient in GSH biosynthesis, designated gshA, grew slower in a defined medium than did the parental strain and this effect was more pronounced when succinate was supplied as the carbon source in place of glucose . This suggested that the citric acid cycle was compromised in the gshA strain . Aconitase activity was approximately 25% lower in GSH-deficient cells growing on either glucose or succinate, and was lower still in strains producing less superoxide dismutase . Addition of GSH to the medium stimulated growth of the gshA strain on succinate . It also elevated the aconitase activity in the presence of chloramphenicol, which was added to block protein synthesis . Dithiothreitol and 2-mercaptoethanol were much less effective in this regard than was GSH . Exposure of cultures to 4.2 atm O2 caused a rapid decline in aconitase activity and this was the case in both GSH-proficient and GSH-deficient E . coli; however, the reactivation which was seen when the hyperoxic exposure was terminated was significantly impaired in the gshA strain . There is a dynamic balance between inactivation of aconitase by superoxide and reactivation by Fe(II) and this balance is altered in GSH-deficient E . coli . GSH may facilitate reactivation of aconitase, and of other {4Fe-4S}-containing dehydratases, by increasing the rate of transfer of Fe(II) to the {3Fe-4S} site.

Proc Natl Acad Sci U S A, 1993 Feb 15, 90(4), 1212 - 6
Histidine-226 is part of the pH sensor of NhaA, a Na+/H+ antiporter in Escherichia coli; Gerchman Y et al.; The nhaA gene of Escherichia coli, which encodes a pH-activated Na+/H+ antiporter, has been modified; six of its eight histidine codons were mutated to arginine codons by site-directed mutagenesis, yielding the mutations H254R-H257R (a double mutant), H226R, H39R, H244R, and H319R . In addition a deletion (delta nhaA1-14) lacking the remaining two histidines, His-3 and His-5, has been constructed . By comparing the phenotypes conferred by plasmids bearing the various mutations to the phenotype of the wild type upon transformation of strains NM81 (delta nhaA) or EP432 (delta nhaA, delta nhaB) we found that none of the NhaA histidines are essential for the Na+/H+ antiporter activity of the NhaA protein . However, the replacement of His-226 by Arg markedly changes the pH dependence of the antiporter . All mutants except H226R confer to NM81 and EP432 Na+ resistance up to pH 8.5 as well as Li+ resistance . Cells bearing H226R are resistant to Li+ and to Na+ at neutral pH, but they become sensitive to Na+ above pH 7.5 . Analysis of the Na+/H+ antiporter activity of membrane vesicles derived from H226R cells shows that the mutated protein is activated by pH to the same extent as the wild type . However, whereas the activation of the wild-type NhaA occurs between pH 7 and pH 8, that of H226R antiporter occurs between pH 6.5 and pH 7.5 . Furthermore, while the wild-type antiporter remains almost fully active at least up to pH 8.5, H226R is reversibly inactivated above pH 7.5, reaching 10-20% of the maximal activity at pH 8.5 . We suggest that His-226 is part of a pH-sensitive site that regulates the activity of NhaA.

Proc Natl Acad Sci U S A, 1993 Feb 15, 90(4), 1455 - 9
mRNA processing independent of RNase III and RNase E in the expression of the F1845 fimbrial adhesin of Escherichia coli; Bilge SS et al.; F1845, the fimbrial adhesin of a diarrhea-associated Escherichia coli, confers upon the bacteria the ability to adhere to cultured epithelial cells in a diffuse pattern . The fimbrial subunit gene, daaE, is encoded on a polycistronic mRNA which is processed endoribonucleolytically to produce a stable message encoding only daaE . The processing event occurs in bacterial strains with mutations in RNase III or RNase E, the only endoribonucleases which have been implicated in the processing of E . coli mRNA . Sequences encoding a stem-loop structure downstream of daaE play an essential role in determining the stability of the daaE mRNA . Rapid degradation of the sequences upstream of the cleavage site occurs upon processing, suggesting that processing of the F1845 polycistronic mRNA results in differential expression of genes involved in the biogenesis of fimbriae.

Proc Natl Acad Sci U S A, 1993 Feb 15, 90(4), 1160 - 5
A genetic approach to the generation of antibodies with enhanced catalytic activities; Lesley SA et al.; A hydrolytic catalytic antibody, generated against a nitrophenyl phosphonate transition state analogue, has been cloned and expressed in Escherichia coli for use as a model system to demonstrate the feasibility of using genetic selections to enhance catalytic activity . Conditions were found that permit the secretion of active recombinant antibody into the periplasm of a strain of E . coli deficient in the biotin biosynthetic genes (delta bio-gal) . A number of substrates were synthesized that, upon hydrolysis by the antibody, yield free biotin, which is required for cell growth . The substrates and selections can be used to identify mutants of the antibody with altered activities . This approach should be generalizable to a wide number of hydrolytic reactions including the selective cleavage of peptide, polysaccharide, phosphodiester, and ester bonds.

Arch Biochem Biophys, 1993 Feb 15, 301(1), 77 - 84
Topological switching of the COOH-terminal domain of peptidylglycine alpha-amidating monooxygenase by alternative RNA splicing; Yun HY et al.; Proteins encompassing the two catalytic domains (monooxygenase and lyase) and the COOH-terminal domain of rat peptidylglycine alpha-amidating monooxygenase (rPAM)3 were purified from recombinant Escherichia coli overexpressing each domain and used to raise domain-specific polyclonal antibodies . Four alternatively spliced forms of PAM RNA (PAM-1, -2, -3, and -4) were transcribed in vitro and used to synthesize PAM proteins in a cell-free translation system . The orientation of the proteins in microsomal membrane vesicles was analyzed using trypsin protection assays and immunoprecipitation with the domain-specific antibodies . Only one of the two potential N-glycosylation sites (Asn765-Phe-Ser) in PAM-1 was efficiently utilized by microsomal membranes . PAM-1 and PAM-2 were shown to be type Ia membrane proteins with their two catalytic domains residing within microsomal vesicles and their COOH-terminal domains exposed to the cytosol . In contrast, PAM-3 and PAM-4 were shown to be soluble proteins contained entirely within vesicles . Thus, the COOH-terminal domain underwent topological switching between the cytosolic (PAM-1 and -2) and luminal (PAM-3) compartments as a function of alternative splicing of exons Ba/Bb . Computer analyses of the PAM protein sequence correlated the exons encoding PAM-1 with a model for the structural and functional domains of the PAM protein . The dual topologies of the PAM proteins confer an important means of functional regulation to this secretory granule associated neuropeptide processing enzyme.

Proc Natl Acad Sci U S A, 1993 Feb 15, 90(4), 1345 - 9
Vitamin D and adaptation to dietary calcium and phosphate deficiencies increase intestinal plasma membrane calcium pump gene expression; Cai Q et al.; The effect of vitamin D and other variables on the synthesis of the chicken intestinal plasma membrane calcium pump (PMCA) mRNA was assessed . The DNA probe for Northern analysis was obtained by reverse transcription and PCR with intestinal poly(A)+ RNA, using two 20-mer oligonucleotide primers homologous to the 3' coding region of the human teratoma PMCA . An EcoRI restriction fragment of the PCR product was cloned into the pBluescript II KS(-) phagemid vector, and the chimeric plasmid was used to transform Escherichia coli . The amino acid sequence deduced from the nucleotide DNA sequence of the PCR product and the cloned DNA were 96% homologous with the teratoma sequence . Northern blots of intestinal poly(A)+ RNA with 32P-labeled DNA showed the presence of three major species of chicken PMCA mRNAs at about 6.6, 5.4, and 4.5 kb . Northern analysis with the chicken PMCA DNA indicated that repletion of vitamin D-deficient chickens with vitamin D increased PMCA mRNAs in the duodenum, jejunum, ileum, and colon . After injection of 1,25-dihydroxyvitamin D3 intravenously into vitamin D-deficient chickens, duodenal PMCA mRNA tended to increase by 2 hr, reached a maximum at about 16 hr, and returned to baseline levels at 48 hr . Adaptation of chickens to either a calcium- or phosphorus-deficient diet resulted in a 2- to 3-fold increase in duodenal PMCA mRNA . These results indicate that vitamin D and specific variables that affect calcium absorption through the vitamin D-endocrine system increase intestinal PMCA gene expression.

J Inorg Biochem, 1993 Feb 15, 49(3), 171 - 5
Metallobiochemistry of RNA . Co(NH3)6(3+) as a probe for Mg2+(aq) binding sites; Cowan JA; Co(NH3)6(3+) may serve as a probe of outer sphere complexation by Mg(H2O)6(2+) with RNA . The number of metal binding sites may be quantitatively assessed by optical spectroscopy (55-130, depending on background {K+}) . Novel coordination-induced features have been observed in circular dichroism spectra.

J Biol Chem, 1993 Feb 15, 268(5), 3670 - 6
Molecular cloning of chicken myosin-binding protein (MyBP) H (86-kDa protein) reveals extensive homology with MyBP-C (C-protein) with conserved immunoglobulin C2 and fibronectin type III motifs; Vaughan KT et al.; The complete nucleotide sequence of a cDNA clone encoding the chicken skeletal muscle myosin-binding protein H (MyBP-H), formerly termed 86-kDa protein, has been established and the predicted amino acid sequence compared with other proteins entered into the GenBank data base . The full-length cDNA of 2066 base pairs contains a single open reading frame of 1611 base pairs encoding a muscle-specific protein of 58,487 Da . The predicted molecular weight differs significantly from the relative mobility of 86-kDa protein in reducing sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) . The full-length protein expressed in Escherichia coli also exhibits an anomalously slow mobility in SDS-PAGE; this gel retardation is a property of the N-terminal 24 kDa of the protein which contain two extended motifs of alternating alanine and proline residues, resembling the N terminus of skeletal muscle myosin light chain 1 (Nabeshima, Y . I., Fujii-Kuriyama, Y., Muramatsu, M., and Ogata, K . (1984) Nature 308, 333-338) . The C-terminal 40 kDa share 49.6% sequence identity and 17% conservative substitutions with chicken skeletal muscle MyBP-C (C-protein) (Einheber, S., and Fischman, D . A . (1990) Proc . Natl . Acad . Sci . U.S.A . 87, 2157-2161) . The protein contains four internal repeats of approximately 100 amino acids each, two of which bear significant resemblance to the C2 set of the immunoglobulin superfamily, and the other two are related to the type III fibronectin repeat . The arrangement of these repeats, -III-C2-III-C2-, is identical to that seen in the C-terminal 40-kDa section of MyBP-C . This repeat structure is implicated in myosin binding for the MyBP family . Finally, genomic Southern blots indicate that a single gene encodes fast skeletal muscle MyBP-H.

J Biol Chem, 1993 Feb 15, 268(5), 3625 - 31
Mechanism of platelet-derived growth factor (PDGF) AA, AB, and BB binding to alpha and beta PDGF receptor; Fretto LJ et al.; The biological effects of platelet-derived growth factor (PDGF) are mediated by cell surface alpha and beta PDGF receptors, which, as a result of ligand binding, undergo dimerization in a manner consistent with PDGF being bivalent . In order to directly demonstrate PDGF bivalency and to define the binding of PDGF AB to isolated beta receptor, we developed solid-phase binding assays using purified recombinant extracellular domain of human PDGF receptors . PDGF AA, AB, and BB were prepared from the monomeric chains expressed in Escherichia coli, and each was purified to homogeneity; PDGF AB contained < 0.5% of either homodimer . The interactions of these isoforms with immobilized PDGF receptors were examined by several approaches . Scatchard analysis revealed high affinity binding (Kd = 0.5-1.0 nM) of radiolabeled PDGF AA and AB to alpha receptor and of PDGF BB to both receptor subtypes . Contrary to previous reports, PDGF AB also bound beta receptor with high affinity (Kd = 0.9 nM) . When a B-chain-specific monoclonal antibody that recognizes the putative binding domain of PDGF BB was used for ligand detection, we found that PDGF AB binding to beta receptor occurred exclusively through the B-chain subunit, whereas binding to alpha receptor occurred through either subunit . In addition, site-directed mutagenesis was used to specifically inactivate the B chain of PDGF AB, which eliminated binding to the beta receptor without affecting alpha receptor binding . These results establish that PDGF is bivalent and that monovalent ligand retains high affinity receptor binding.

J Biol Chem, 1993 Feb 15, 268(5), 3282 - 8
Properties and sequence of a female-specific, juvenile hormone-induced protein from locust hemolymph; Zhang J et al.; In the fat body of Locusta migratoria, an RNA transcript of about 800 nucleotides has been detected that is specific to the adult female and dependent on induction by juvenile hormone (JH) or an analog . The corresponding cDNA has been cloned (lambda 21) and a 718-base pair sequence determined . It encodes a 196-amino acid polypeptide, including a signal peptide . An NH2-terminal sequence has 24 out of 28 amino acids identical with those of a previously described 19K locust hemolymph protein, but the remainder of the sequence shows no similarity . From adult female hemolymph, a 21-kDa protein, designated 21K protein, has been purified, with an NH2-terminal sequence exactly matching that deduced from clone lambda 21 . This 21K protein is found only in the adult female, is dependent on induction by JH, and is assumed to represent the product of the lambda 21 gene . It shows no immunochemical cross-reaction with locust 19K protein, apolipophorin III, nor with vitellogenin (Vg) . Its isoelectric point is pH 5.4; it contains some carbohydrate . 21K protein is synthesized in adult female fat body, accumulates in hemolymph, and is taken up into the developing oocytes in parallel with Vg . In locusts deprived of JH with precocene, production of 21K protein and of lambda 21-hybridizing transcripts is induced by the JH analog, methoprene, in parallel with Vg and its mRNA . Because of its sex-, stage-, and JH-dependent regulation, coordinate with Vg, the 21K protein will be valuable for analysis of gene expression.

Blood, 1993 Feb 15, 81(4), 973 - 9
Endotoxin-induced tissue factor messenger RNA in human monocytes is negatively regulated by a cyclic AMP-dependent mechanism; Ollivier V et al.; Tissue factor (TF) is a transmembrane receptor that serves as the major cofactor for factor VIIa-catalyzed proteolytic activation of factors IX and X . In response to bacterial lipopolysaccharide (LPS), monocytes transcribe, synthesize, and express TF on their surface, thereby conveying to activated monocytes the ability to initiate the blood coagulation protease cascades . Agents that elevate cellular cyclic AMP (cAMP) inhibit the functional expression of TF by LPS-stimulated monocytes . In this study, we investigated the mechanism of this suppression . Northern blot analysis of total RNA from LPS-stimulated monocytes showed a concentration-dependent decrease in TF messenger RNA (mRNA) levels in response to dibutyryl-cAMP (dBt-cAMP) . TF mRNA and procoagulant activity were inhibited as early as 1 hour after the addition of dBt-cAMP and the inhibition persisted through 4 hours . Suppression of specific mRNA abundance was also observed with agents, including forskolin and iso-butyl-methyl-xanthine (IBMX), that increase cAMP levels by independent mechanisms . Flow immunocytometric analysis confirmed that cell-surface TF protein levels declined in parallel with TF functional activity . The rate of decay of TF mRNA after the arrest of transcription by actinomycin D was not altered by the addition of dBt-cAMP, IBMX, or forskolin, thus excluding effects on TF mRNA stability . We conclude that elevated cAMP levels suppress TF mRNA by reducing the rate of TF gene transcription.

Gene, 1993 Feb 14, 124(1), 87 - 92
Physical and functional characterization of the ColS8 plasmid; Garcia ME et al.; The restriction map of the 5.1-kb colicinogenic plasmid ColS8 is reported . Transposon-insertion mutagenesis has been carried out to investigate the location of the various functional regions of this plasmid . Twenty-six independent ColS8::Tn1 insertions and six different deletion mutant plasmids were isolated, and the locations of the insertions and deletions were determined . The mapping of the transposon-insertion sites, together with characterization of the phenotypes of these mutants, permitted the localization of the regions of DNA involved in colicin production, colicin S8 immunity and mobilization by other plasmids . There is a polar effect in some mutants in which single insertions result in the non-expression of all three functions . In minicell preparations, the ColS8 plasmid directed the synthesis of colicin S8 (60 kDa) and a 14-kDa immunity protein . One deletion mutant with a colicin-less phenotype can synthesize colicin S8 in minicells, which placed the DNA region involved in colicin release between the colicin production and immunity regions . Both the 60-kDa and 14-kDa proteins are expressed from pColS8 in maxicell preparations.

Gene, 1993 Feb 14, 124(1), 83 - 5
The pRSET family of T7 promoter expression vectors for Escherichia coli; Schoepfer R; A family of eight T7 promoter-based expression plasmids is presented . These are high-copy-number vectors featuring translational start and stop elements and a multiple cloning site (polylinker) with eleven unique restriction sites in all six reading frames . Depending on the cloning strategy used, recombinant proteins may contain either short vector-encoded fusion fragments or no fusion fragments at all . Following promoter induction, proteins are usually produced at a high level.

Gene, 1993 Feb 14, 124(1), 57 - 65
Cloning, sequencing, and heterologous expression of a cellulase-encoding cDNA (cbh1) from Penicillium janthinellum; Koch A et al.; From a Penicillium janthinellum cDNA library, two clones with 1.8- and 1.9-kb inserts were isolated by hybridization to a Trichoderma reesei cellulase-encoding gene probe (egl1) . Both cDNAs have identical 5' ends and coding sequences, but different polyadenylation start points in their 3' untranslated regions . In the nucleotide (nt) sequence, one open reading frame of 537 amino acids was detected which shows 56% homology with endoglucanase I of T . reesei and 70% homology with cellobiohydrolase I of T . reesei, Phanerochaete chrysosporium, and Humicola grisea . Expression of the 1.9-kb cDNA in the Escherichia coli T7 system led to the detection of a 57-kDa protein, in agreement with the theoretical value . Fusion to the promoter of the yeast phosphoglycerokinase-encoding gene led to efficient expression and partial secretion of the cDNA-encoded cellulase with cellobiohydrolase I activity in Saccharomyces cerevisiae.

Gene, 1993 Feb 14, 124(1), 29 - 35
Escherichia coli host strains SURE and SRB fail to preserve a palindrome cloned in lambda phage: improved alternate host strains; Doherty JP et al.; We have attempted to produce Escherichia coli strains with the optimal combination of host mutations required for the construction of genomic libraries in lambda and cosmid vectors . For lambda vectors, we defined this as a strain that combined high efficiency of phage plating with optimal tolerance to DNA methylation and the ability to propagate recombinants containing regions of potential secondary structure . To optimize this latter property, we have tested a series of strains for the ability to propagate a lambda phage containing a palindromic sequence . These included an mcr- derivative of a strain shown by Ishiura et al . {J . Bacteriol . 171 (1989) 1068-1074} to allow optimal stability of inserts in cosmid clones . All the sbcC strains allowed plaque formation of the palindrome-containing lambda phage . However, while the palindrome-containing phage plated with reasonable efficiency on SURE (recB sbcC recJ umuC uvrC) and SRB (sbcC recJ umuC uvrC), the majority of phage recovered from these strains no longer required an sbcC host for subsequent plating . These two strains also gave poorer titres with a low-yielding phage clone from the human Prader-Willi chromosome region . Optimal phage hosts appear to be those that are mcrA delta(mcrBC-hsd-mrr) combined with mutations in sbcC plus recBC or recD and without mutations in additional recombination functions such as recJ or recJ umuC uvrC (all of our E . coli strains are available on request).

Gene, 1993 Feb 14, 124(1), 115 - 20
Isolation of a yeast essential gene, COF1, that encodes a homologue of mammalian cofilin, a low-M(r) actin-binding and depolymerizing protein; Iida K et al.; We have cloned a Saccharomyces cerevisiae gene (COF1) encoding a low-M(r) actin-binding protein of 143 amino acid (aa) residues (yeast cofilin; Cof); its aa sequence is 35% identical to porcine Cof . The yeast recombinant Cof produced in Escherichia coli exhibited in vitro activities on actin filaments similar to those of mammalian and avian Cof . Gene disruption and tetrad analysis showed that gene COF1 is essential for yeast cell growth . Expression of the cDNA of porcine Cof or destrin (Des), the latter a Cof-related protein, complemented the cof1 null allele in yeast cells.

Gene, 1993 Feb 14, 124(1), 105 - 9
Cloning of the lysA gene from Mycobacterium tuberculosis; Andersen AB et al.; The lysA and proC genes of Mycobacterium tuberculosis were cloned by screening of a recombinant lambda gt11 M . tuberculosis DNA library for phages able to complement lysA and proC Escherichia coli mutants . The lysA gene encodes diaminopimelic acid decarboxylase which catalyzes the conversion of diaminopimelic acid (DAP) to lysine . The lysA gene from M . tuberculosis encodes a 44-kDa protein, as determined by maxicell experiments . The nucleotide sequence of the structural gene was established . The deduced amino acid sequence was found to exhibit significant homology (from 55% to 73% similarity, and from 27% to 53% identity) to DAP decarboxylase sequences from other bacterial species.

Gene, 1993 Feb 14, 124(1), 93 - 8
Lentivirus envelope sequences and proviral genomes are stabilized in Escherichia coli when cloned in low-copy-number plasmid vectors; Cunningham TP et al.; A promoter-selection vector (pKK232-8) was used to identify sequences with strong Escherichia coli promoter activity positioned near the start of the envelope-encoding genes (env) of two lentiviruses, simian immunodeficiency virus (SIV) and equine infectious anemia virus (EIAV) . For EIAV, cloning the cryptic promoter sequences together with downstream sequences encoding the envelope glycoprotein (gp90) in moderate- to high-copy-number (hcn) plasmid vectors, such as pBR322 or pUC, resulted in rearrangements and point mutations in env when propagated in E . coli . To alleviate this problem, low-copy-number (lcn) cloning vectors, pLG338-30 and pLG339-SPORT, were constructed . The plasmids carry resistance markers for ampicillin (ApR) or kanamycin (KmR), the pSC101 origin of replication (ori) from plasmid pLG338 {Stoker et al., Gene 18 (1982) 335-341}, and a multiple cloning site (MCS) from plasmids pIBI30 or pSPORT . Full-length env and genomic proviral sequences of EIAV were genetically stable when subcloned into these lcn vectors . Proviral sequences of an SIV clone (pBK28-SIV) that are genetically unstable in the hcn vector pUC18 were also stabilized and remained fully infectious when subcloned into the lcn vector pLG339-SPORT . These lcn vectors appear to be generally useful in stabilizing lentivirus genomic sequences for subcloning, propagation, and manipulation in E . coli, possibly as a result of reducing the level of toxic gene expression from cryptic promoter sequences.

Gene, 1993 Feb 14, 124(1), 37 - 44
Modified-cytosine restriction-system-induced recombinant cloning artefacts in Escherichia coli; Williamson MR et al.; We have tested whether, and to what extent, recombinant clones from DNA segments with 5-methylation of cytosines recovered in methylation-restrictive (mcr+) hosts contain mutations . We constructed a model system in which the tetracycline-resistance-encoding gene (tet) from pBR322 was cloned into the plasmid pGEM3Zf+ . The central region of tet was removed from the construct, methylated in vitro and then religated back into the unmethylated remainder of the construct . The central region of tet was either (1) methylated with a combination of four bacterial methyltransferases (M.AluI, M.HaeIII, M.HpaII plus M.HhaI) or (2) methylated with M.SssI which methylates at all CpG dinucleotides . These two protocols generated theoretical levels of DNA methylation in the central fragment of 10.5% and 33%, respectively . The construct was transformed into a series of isogenic (recA+) bacterial strains that were mcrA+ mcrB+C+, mcrA+ mcrB-C+, mcrA- mcrB+C+, mcrA- mcrB-C+ or mcrA- delta mcrBC, and also into a set of isogenic recA- derivatives of these strains . With the two methylation protocols, there was an average 48- and 141-fold reduction, respectively, in the number of transformants recovered from the recA+ mcr+ hosts compared with a methylation-tolerant host (mcr-) . Of the clones recovered in recA+mcr+ hosts, > 20% of clones had an inactivating mutation in tet . The majority of such mutant clones contained deletions that frequently extended into the unmethylated portion of tet and even into the plasmid sequences beyond the end of the polylinker . With the recA- mcr+ hosts, effective restriction was much more stringent, rendering the plasmid containing the methylated segment effectively unclonable.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene, 1993 Feb 14, 124(1), 135 - 6
A double-cos-site vector containing a multiple cloning site flanked by T7 and T3 RNA polymerase promoters; Reilly JD et al.; A double-cos-site cosmid vector, c2XMCS, contains a multiple cloning site (MCS) with fifteen restriction sites flanked by phage T7 and T3 RNA polymerase promoters . The two cos sites in the cosmid vector enable the construction of cosmid libraries from unfractionated partial digests of genomic DNA . This simple construct combines the efficiency of a double-cos-site cosmid with the versatility provided by the MCS from pBluescript.

Gene, 1993 Feb 14, 124(1), 133 - 4
Derivatives of pUC18 that have BglII sites flanking a modified multiple cloning site and that retain the ability to identify recombinant clones by visual screening of Escherichia coli colonies; Janssen GR et al.; We report the construction of a series of derivatives of pUC18 in which a modified multiple cloning site is flanked by one or two BglII sites . The reading frame of the lacZ alpha fragment was retained in each construct, permitting the recognition of transformants containing plasmids with inserts by visual screening for loss of blue coloration.

Biochim Biophys Acta, 1993 Feb 13, 1161(2-3), 272 - 8
Differential scanning calorimetric study of 5-enolpyruvoyl shikimate-3-phosphate synthase and its complexes with shikimate-3-phosphate and glyphosate: irreversible thermal transitions; Merabet EK et al.; The thermal denaturation of native Escherichia coli 5-enolpyruvoyl shikimate-3-phosphate (EPSP) synthase, its binary complex with shikimate-3-phosphate (S3P) and its ternary complex with S3P and glyphosate have been studied using highly-sensitive differential scanning calorimetry (DSC) . All observed transitions are strongly scanning-rate-dependent and irreversible . Consistent with these observations, the data were better fit by a simple irreversible model than by the controversial reversible model more commonly employed . The results obtained provide additional support for the application of irreversible models to the thermal denaturation of proteins . The calculated parameters, activation energy (Ea), enthalpy of denaturation (delta H) and transition temperature (Tm), obtained from fitting to an irreversible model agree well with values obtained from approximation techniques . Further, the results show that the formation of the ternary complex greatly enhances the thermal stability of the enzyme (delta Tm = 10.6 degrees C), while the binding of S3P alone increases the transition temperature only slightly (delta Tm = 3 degrees C) . The heat of binding calculated at the transition temperature also demonstrates the greater stability of the ternary complex (delta H = -70 kcal/mol) versus the binary complex (delta H = -10 kcal/mol).

Biochim Biophys Acta, 1993 Feb 13, 1161(2-3), 244 - 8
Physical and conformational properties of staphylokinase in solution; Damaschun G et al.; The structure of staphylokinase has been analyzed by solution X-ray scattering, dynamic light scattering, ultracentrifugation and ultraviolet circular dichroism spectroscopy . Staphylokinase has a radius of gyration of 2.3 nm, a Stokes radius of 2.12 nm and a maximum dimension of 10 nm . The sedimentation coefficient is 1.71 S . These physical parameters indicate that the shape of staphylokinase is very elongated . The protein molecule consists of two folded domains of similar size . The mean distance of the centres of gravity of the domains is 3.7 nm . The mutual positions of the two domains are variable in solution . Thus, the molecule is shaped like a flexible dumbbell . About 18% of the amino acids of staphylokinase are organized in helical structures, 30% are incorporated in beta-sheets and 20% form turns.

Biochim Biophys Acta, 1993 Feb 13, 1161(2-3), 187 - 93
Antibodies to recombinant fragment 212-276 of protein C specifically recognize the intact human molecule; Fijalkowska I et al.; The peptide fragment Pro212-Ile276 of human protein C was produced as a part of a fusion protein in Escherichia coli . The identity of the peptide was confirmed by immunoblotting experiments using specific antibodies to intact protein C . The peptide Pro212-Ile276 was isolated from the fusion protein after mild hydrolysis with formic acid by gel filtration and reverse-phase HPLC . This peptide fragment was used to produce antibodies specific for the heavy chain of protein C which recognized native protein C present in blood plasma . Antibodies to intact protein C reacted also with the Pro212-Ile276 peptide fragment, indicating that this region is immunogenic in intact protein C and may represent a native epitope.

Biochim Biophys Acta, 1993 Feb 13, 1161(2-3), 216 - 22
The role of Cys-17 in the pyridoxal 5'-phosphate inhibition of the bovine liver low M(r) phosphotyrosine protein phosphatase; Cirri P et al.; Mammalian tissues contain two low M(r) phosphotyrosine protein phosphatase isoforms (type-1 and type-2) that differ in the 40-73 amino-acid sequence . Only one isoform (type-2) is strongly inhibited by pyridoxal 5'-phosphate, whereas the other is poorly inhibited by this compound . The mechanism of pyridoxal 5'-phosphate inhibition of the bovine liver enzyme (a type-2 isoform) has been studied by kinetic methods using a series of pyridoxal 5'-phosphate analogues . These studies indicate that pyridoxal 5'-phosphate interacts with the enzyme in both the phosphate and aldehyde groups . Active site-directed mutagenesis has been used to investigate the sites of pyridoxal 5'-phosphate binding . Our results indicate that Cys-17, essential for enzyme activity, interacts with the phosphate moiety of pyridoxal 5'-phosphate . On the other hand, Cys-12, which is also involved in the catalytic mechanism, does not participate in pyridoxal 5'-phosphate binding.

J Chromatogr, 1993 Feb 12, 631(1-2), 277 - 80
Separation of interleukins by a preparative chromatofocusing-like method; Monkarsh SP et al.; A chromatofocusing-like method used in the large-scale separation of deamidated from amidated recombinant human interleukin-1 alpha (amino acids 117-271), derived from Escherichia coli, is described . Two major protein species having isoelectric points (pI) of approximately 5.3 and 5.1 were separated by high-performance liquid chromatography using a sulfopropyl strong cation-exchange column . Unlike standard chromatofocusing technique, this method does not use carrier ampholytes during gradient separation of proteins, nor does it employ increased ionic strength for protein elution, the usual method for performing standard ion-exchange chromatography . N-Terminal sequence analysis of the protein with a pI of 5.3 revealed an Asn residue at position 32 as predicted by the cDNA sequence . The pI 5.1 species showed an Asp residue at the same position as a result of deamidation of Asn . This method was also used in the large-scale separation of N-Met from des-Met recombinant human interleukin-1 beta.

J Chromatogr, 1993 Feb 12, 631(1-2), 269 - 75
Immunoaffinity chromatography of recombinant Amb a I in the presence of a denaturing agent; Keating KM et al.; Recombinant proteins expressed in E . coli are often sequestered into inclusion bodies and require the use of denaturing agents in order to solubilize them . The recombinant form of Amb a I, the major allergen from short ragweed pollen, is one such protein . In some cases solubility can be maintained after the removal of the denaturing agent, particularly if the protein can be folded into its native conformation . However, not all proteins refold readily and after the removal of the denaturing agent the proteins will reaggregate and/or precipitate . In the case of Amb a I, the recombinant protein stays in solution at low concentrations but aggregates with itself and other proteins . The recombinant Amb a I is not expressed at high levels and may be toxic to E . coli . Therefore, isolation from a complex mixture of E . coli proteins was necessary . Monoclonal antibodies which recognize the denatured form of Amb a I were available, allowing for immunoaffinity purification . However, because the protein was not monomeric, this chromatographic technique did not provide an improvement in the purity level when run in normal buffer solutions . Analysis of one monoclonal antibody's stability to urea indicated it could tolerate the presence of 2 M urea and recover full activity . Use of this antibody as an immunoaffinity reagent in a column run in 2 M urea, which minimized aggregation of the E . coli produced proteins, gave a high degree of purification of recombinant Amb a I in one step . This illustrates the potential for the use of denaturing and other solubilizing agents in immunoaffinity chromatography of recombinant proteins.

Nucleic Acids Res, 1993 Feb 11, 21(3), 733 - 9
Quantitative sequence-activity models (QSAM)--tools for sequence design; Jonsson J et al.; Models have been developed that allow the biological activity of a DNA segment to be altered in a desired direction . Partial least squares projections to latent structures (PLS) was used to establish a quantitative model between a numerical description of 68 bp fragments of 25 E.coli promoters and their corresponding quantitative measure of in vivo strength . This quantitative sequence-activity model (QSAM) was used to generate two 68 bp fragments predicted to be more potent promoters than any of those on which the model originally was based . The optimized structures were experimentally verified to be strong promoters in vivo.

Nucleic Acids Res, 1993 Feb 11, 21(3), 523 - 9
Oncogenic conversion of Ets affects redox regulation in-vivo and in-vitro; Wasylyk C et al.; The avian acute leukemia virus E26 encodes a fusion protein between viral Gag and the cellular transcription factors cMyb and cEts1(p68) . vEts on its own transforms more mature erythroid cells . We have compared the properties of vEts and cEts1(p68) . vEts interacts preferentially with an antibody that recognizes the active conformation of the DNA-binding domain . The DNA-binding activity of vEts is particularly sensitive to incubation conditions for band-shift assays, phosphorylation and modification by sulphydryl-specific reagents . Increased sensitivity is due to loss of a protective function of cEts1 C-terminal sequences . cEts2 has a related C-terminal sequence with a similar role . These results suggest that the vEts DNA-binding domain is more accessible to protein-protein interactions and to regulatory mechanisms . Indeed, vEts DNA binding is preferentially inactivated by oxidizing conditions in-vivo . We suggest that the 'open' conformation of the vEts DNA-binding domain favours interactions with other proteins or DNA and facilitates transformation.

Nucleic Acids Res, 1993 Feb 11, 21(3), 505 - 12
Atomic force microscopy of DNA in aqueous solutions; Hansma HG et al.; DNA on mica can be imaged in the atomic force microscope (AFM) in water or in some buffers if the sample has first been dehydrated thoroughly with propanol or by baking in vacuum and if the sample is imaged with a tip that has been deposited in the scanning electron microscope (SEM) . Without adequate dehydration or with an unmodified tip, the DNA is scraped off the substrate by AFM-imaging in aqueous solutions . The measured heights and widths of DNA are larger in aqueous solutions than in propanol . The measured lengths of DNA molecules are the same in propanol and in aqueous solutions and correspond to the base spacing for B-DNA, the hydrated form of DNA; when the DNA is again imaged in propanol after buffer, however, it shortens to the length expected for dehydrated A-DNA . Other results include the imaging of E . coli RNA polymerase bound to DNA in a propanol-water mixture and the observation that washing samples in the AFM is an effective way of disaggregating salt-DNA complexes . The ability to image DNA in aqueous solutions has potential applications for observing processes involving DNA in the AFM.

Nucleic Acids Res, 1993 Feb 11, 21(3), 373 - 9
Cloning, production and characterisation of wild type and mutant forms of the R.EcoK endonucleases; Weiserova M et al.; The hsdR, hsdM and hsdS genes coding for R.EcoK restriction endonuclease, both with and without a temperature sensitive mutation (ts-1) in the hsdS gene, were cloned in pBR322 plasmid and introduced into E.coli C3-6 . The presence of the hsdSts-1 mutation has no effect on the R-M phenotype of this construct in bacteria grown at 42 degrees C . However, DNA sequencing indicates that the mutation is still present on the pBR322-hsdts-1 operon . The putative temperature-sensitive endonuclease was purified from bacteria carrying this plasmid and the ability to cleave and methylate plasmid DNA was investigated . The mutant endonuclease was found to show temperature-sensitivity for restriction . Modification was dramatically reduced at both the permissive and non-permissive temperatures . The wild type enzyme was found to cleave circular DNA in a manner which strongly suggests that only one endonuclease molecule is required per cleavage event . Circular and linear DNA appear to be cleaved using different mechanisms, and cleavage of linear DNA may require a second endonuclease molecule . The subunit composition of the purified endonucleases was investigated and compared to the level of subunit production in minicells . There is no evidence that HsdR is prevented from assembling with HsdM and HsdSts-1 to produce the mutant endonuclease . The data also suggests that the level of HsdR subunit may be limiting within the cell . We suggest that an excess of HsdM and HsdS may produce the methylase in vivo and that assembly of the endonuclease may be dependent upon the prior production of this methylase.

Nucleic Acids Res, 1993 Feb 11, 21(3), 531 - 5
Replacement of RNA hairpins by in vitro selected tetranucleotides; Dichtl B et al.; An in vitro selection method based on the autolytic cleavage of yeast tRNA(Phe) by Pb2+ was applied to obtain tRNA derivatives with the anticodon hairpin replaced by four single-stranded nucleotides . Based on the rates of the site-specific cleavage by Pb2+ and the presence of a specific UV-induced crosslink, certain tetranucleotide sequences allow proper folding of the rest of the tRNA molecule, whereas others do not . One such successful tetramer sequence was also used to replace the acceptor stem of yeast tRNA(Phe) and the anticodon hairpin of E.coli tRNA(Phe) without disrupting folding . These experiments suggest that certain tetramers may be able to replace structurally nonessential hairpins in any RNA.

Nucleic Acids Res, 1993 Feb 11, 21(3), 427 - 34
Determinants of Escherichia coli RNase P cleavage site selection: a detailed in vitro and in vivo analysis; Svard SG et al.; The location of the Escherichia coli RNase P cleavage site was studied both in vitro and in vivo . We show that selection of the cleavage site is dependent on the nucleotide at the cleavage site and the length of the acceptor-stem . Within the acceptor-stem the number of nucleotides on the 5'-half of the acceptor-stem appears to be the important determinant, rather than the number of base pairs in the acceptor-stem . We also demonstrate that the length of the T-stem and a G to C substitution at position 57 in the tRNA(Tyr)Su3 precursor influence the location of the cleavage site under certain conditions . With respect to the function of the subunits of RNase P our data suggest that the nucleotide at position 333 in M1 RNA, and the C5 protein, are important for the identification of the cleavage site.

Biochemistry, 1993 Feb 9, 32(5), 1354 - 63
Assembly of the Arc repressor-operator complex: cooperative interactions between DNA-bound dimers; Brown BM et al.; Arc repressor, a member of the beta-ribbon family of DNA binding proteins, binds to its 21-base-pair operator as a tetramer . Here, the Arc dimer is shown to bind specifically to DNA fragments containing operator half-sites, and the equilibrium and kinetic constants for these reactions are determined . DNA-bound dimers are also shown to be transient intermediates in association experiments, indicating that assembly of the Arc tetramer-operator complex occurs by sequential addition of dimers to operator half-sites . When the left or right operator half-site is occupied by an Arc dimer, cooperative interactions increase the affinity of the second dimer by approximately 5900-fold {delta delta G = -5.1 (+/- 0.5) kcal/mol} . This increase in affinity is largely caused by an increase in the half-life of the complex; "non-cooperatively" bound dimers dissociate with a half-life of a few seconds while "cooperatively" bound dimers have half-lives of more than 1 h.

Biochemistry, 1993 Feb 9, 32(5), 1332 - 7
Bacteriorhodopsin D85N: three spectroscopic species in equilibrium; Turner GJ et al.; Ground-state absorbance measurements show that BR from Halobacterium halobium containing asparagine at residue 85 (D85N) exists as three distinct chromophoric states in equilibrium . In the pH range 6-12 the absorbance spectra of the three states are demonstrated to be similar to flash-induced spectral intermediates which comprise the latter portion of the wild-type BR photocycle . One of the states absorbs maximally at 405 nm, has a deprotonated Schiff base, and contains predominantly the 13-cis form of retinal, identifying it as a close homologue of the M intermediate in the BR photocycle . The other species possess absorbance maxima with correspondence to those of the wild-type N (570 nm) and O (615 nm) photointermediates . The retinal composition of the O-like form was found to be dominated by all-trans isomer . The pH dependence of the concentrations of the equilibrium species corresponds closely with the pH dependence of the M, N, and O photointermediates . These data support kinetic models which emphasize the role of back-reactions during the photocycle of bacteriorhodopsin . Energetic and spectral characterization of the D85N ground-state equilibrium supports its use as a model for elucidating molecular transitions comprising the latter portion of the BR photocycle.

Biochemistry, 1993 Feb 9, 32(5), 1251 - 6
Titration of protein transport activity by incremental changes in signal peptide hydrophobicity; Doud SK et al.; A systematic series of mutants has been generated which provides a means for titrating the dependence of protein transport activity on signal peptide hydrophobicity . These mutants involve replacement of the hydrophobic core segment of the Escherichia coli alkaline phosphatase signal peptide while maintaining the natural amino- and carboxyl-terminal segments and the overall length . The new core regions vary in composition from 10:0 to 0:10 in the ratio of alanine to leucine residues . Thus, a nonfunctional polyalanine-containing signal peptide is titrated with the more hydrophobic residue, leucine . Using precursor processing to quantify transport activity, we observe a clear, nonlinear dependence on hydrophobicity . At ratios of alanine to leucine of less than or equal to 8:2, the signal peptide is essentially nonfunctional; at ratios greater than or equal to 3:7, the signal peptide functions efficiently . The midpoint is between alanine to leucine ratios of 6:4 and 5:5 . Signal peptides with hydrophobicity just below the midpoint show substantial, additional precursor processing over time while the others do not . The data are consistent with a simple model involving a two-state equilibrium between the untransported and transported species and a change in the delta G of -0.85 kcal/mol for every alanine to leucine conversion.

Biochemistry, 1993 Feb 9, 32(5), 1235 - 42
Recombinant human hemoglobin: modification of the polarity of the beta-heme pocket by a valine67(E11)-->threonine mutation; Fronticelli C et al.; Using the mutagenesis and a gene expression system previously described {Fronticelli et al . (1991) J . Protein Chem . 10, 495-501}, we have replaced Val67E11 in the distal heme pocket of the beta-chains of hemoglobin with Thr . The valine to threonine substitution is isosteric and only modifies the polarity of the beta-heme environment . The absorption and CD spectra of the resultant mutant hemoglobin were essentially the same as that of wild-type protein, indicating that the mutation did not cause any large conformational changes and that a water molecule was not coordinated to the ferrous iron atom . Equilibrium measurements of oxygen binding to the mutant indicate a 2-fold decrease in overall affinity relative to native or wild-type human hemoglobin . Thermodynamic analyses of O2 binding curves, based either on the sequential Adair model or on the MWC two-state model, indicated that the overall decrease of O2 affinity in the system was due to a lower association equilibrium constant for the intermediates of oxygenation, particularly those involved at the third ligation step . The functional characteristics of the mutant hemoglobin in either the T- or R-state were not modified greatly by the mutation; however, the Bohr effect and sensitivity to C1- were increased, suggesting a role of the intermediates of oxygenation in the modulation of these parameters.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem Pharmacol, 1993 Feb 9, 45(3), 783 - 6
Inhibition of phospholipase A2 purified from human herniated disc; Carabaza A et al.; The effect on human herniated intervertebral disc phospholipase A2 (HD-PLA2) of a number of retinoids, antirheumatic drugs and reported PLA2 inhibitors was evaluated using autoclaved {1-14C}-oleate-labeled Escherichia coli membranes as the substrate . Dexamethasone, non-steroidal antiinflammatory drugs, aristolochic acid and retinol were inactive, whereas a marked inhibition was found for manoalide, retinal, nordihydroguaiaretic acid and p-bromophenacyl bromide after preincubation with the enzyme (IC50 values 0.25, 4, 5 and 5 microM, respectively) . The results are parallel to those obtained with the PLA2 purified from human synovial fluid.

Biochemistry, 1993 Feb 9, 32(5), 1278 - 84
Substitution of Glu841 by lysine in the carbamate domain of carbamyl phosphate synthetase alters the catalytic properties of the glutaminase subunit; Lusty CJ et al.; In previous studies a Glu841-->Lys replacement in the carbamate phosphorylating domain located in the COOH half of the synthetase subunit of Escherichia coli carbamyl phosphate synthetase was shown to reduce overall synthesis of carbamyl phosphate by 4 orders of magnitude with either glutamine or NH3 as nitrogen donor (Guillou et al., 1992) . In the present study, the mutant enzyme has been further analyzed for its glutamine hydrolytic activity . The glutaminase activity of the mutant enzyme has the following properties . (1) In the absence of other substrates the turnover number is only marginally different from that of the wild-type complex . (2) The Km for glutamine is 60 times higher than in wild-type complex and three times higher than in the separated glutaminase subunit . (3) In the present study wild-type carbamyl phosphate synthetase has been shown to catalyze glutamine hydrolysis by a mechanism involving an enzyme-bound acyl ester intermediate (gamma-glutamyl thioester) . This intermediate is formed and is hydrolyzed with rates consistent with overall glutamine hydrolysis . At physiological concentrations of glutamine (1.2 mM), the steady-state concentration of gamma-glutamyl thioester is 0.3 mol/mol of wild-type enzyme . Under the same conditions, only 0.02 mol of thioester is measured in the mutant enzyme . Maximal accumulation of this covalent intermediate by the mutant enzyme required 10 times higher concentrations of free glutamine . (4) The rate of reaction with 2-amino-4-oxo-5-chloropentanoate, a glutamine analog known to specifically alkylate an active site cysteine residue, is 2 orders of magnitude slower in the mutant.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochim Biophys Acta, 1993 Feb 8, 1141(1), 95 - 104
Reactions of the membrane-bound cytochrome bo terminal oxidase of Escherichia coli with carbon monoxide and oxygen; Bolgiano B et al.; The cytochrome 'bo' quinol oxidase of Escherichia coli contains 2 mol of haem, one or both of which are 'haem O' . One of the haems forms, with the single copper present, a binuclear site for ligand binding and oxygen reduction . Cytoplasmic membranes from a strain of E . coli lacking the alternative cytochrome bd quinol oxidase, and having amplified levels of cytochrome bo, were used to study oxygen and carbon monoxide reactivity with this oxidase . The high-spin ligand-binding haem was identified from its contribution to the Soret region and the shift in midpoint potential from +211 to +477 mV in the presence of CO . Oxidative titration of a CO-liganded sample was accompanied by a decrease in the contribution from a photodissociable CO-binding haem . The photodissociation spectrum was typical of a high-spin haem . Photolysis of CO-liganded, reduced membranes in the presence of O2 at sub-zero temperatures revealed O2 binding and cytochrome oxidation characterized by differential absorbance changes in the alpha-spectral region . Monitoring by epr spectroscopy of the same reaction sequence at -80 degrees C revealed a slight increase in g = 6 signal intensity immediately after photolysis attributable to cytochrome o oxidation prior to Cu oxidation . Subsequent decline in the g = 6 signal and appearance of a g = 3 signal indicated sequential electron flow from low-spin to high-spin haems and copper oxidation, suggesting that a second haem carries electrons from ubiquinol to the binuclear centre.

J Mol Biol, 1993 Feb 5, 229(3), 794 - 6
Crystallization and preliminary X-ray analysis of the periplasmic fragment of CyoA-a subunit of the Escherichia coli cytochrome o complex; van der Oost J et al.; CyoA, an integral membrane protein, is a subunit of the Escherichia coli cytochrome o quinol oxidase complex . The C-terminal periplasmic domain of CyoA has been expressed in E . coli, purified and crystallized . Crystals were grown using ammonium sulphate as a precipitant . They have space group I222 or I2(1)2(1)2(1) and diffract X-rays to 2.3 A resolution.

J Mol Biol, 1993 Feb 5, 229(3), 735 - 46
Refined solution structures of the Escherichia coli trp holo- and aporepressor; Zhao D et al.; The solution structures of the trp-repressor from Escherichia coli in both the liganded (holo-) and unliganded (apo-) form, have been refined by restrained molecular dynamics with simulated annealing using the program XPLOR and additional experimental constraints . The ensemble of refined holorepressor structures have a root-mean-square deviation (r.m.s.d.) of 0.8 A relative to the average structure for the backbone of the dimer core (helices A, B, C, A', B', C') and 2.5 A for the helix-turn-helix DNA-binding domain (helices D and E) . The corresponding values for the aporepressor are 0.9 A for the backbone of the ABC-dimer core and 3.2 A for the DE helix-turn-helix . The r.m.s.d . of the average structures from the corresponding crystal structures are 2.3 A for the holorepressor ABC core and 4.2 A for its DE region; 2.3 A for the aporepressor core and 5.5 A for its DE region . The relative disorder of the DNA-binding domain is reflected in a number of experimental parameters including substantially more rapid backbone proton exchange rates, exchange-limited relaxation times and crystallographic B-factors . The stabilizing effect of the L-Trp ligand is evident in these measurements, as it is in the higher precision of the holorepressor structure.

Science, 1993 Feb 5, 259(5096), 806 - 9
Probing the structure and mechanism of Ras protein with an expanded genetic code; Chung HH et al.; Mutations in Ras protein at positions Gly12 and Gly13 (phosphate-binding loop L1) and at positions Ala59, Gly60, and Gln61 (loop L4) are commonly associated with oncogenic activation . The structural and catalytic roles of these residues were probed with a series of unnatural amino acids that have unusual main chain conformations, hydrogen bonding abilities, and steric features . The properties of wild-type and transforming Ras proteins previously thought to be uniquely associated with the structure of a single amino acid at these positions were retained by mutants that contained a variety of unnatural amino acids . This expanded set of functional mutants provides new insight into the role of loop L4 residues in switch function and suggests that loop L1 may participate in the activation of Ras protein by effector molecules.

Science, 1993 Feb 5, 259(5096), 796 - 8
Formation of an Fe(III)-tyrosinate complex during biomineralization of H-subunit ferritin; Waldo GS et al.; An iron(III)-tyrosinate complex was identified in ferritin by ultraviolet-visible and resonance Raman spectroscopies . Previously, a specific amino acid side chain coordinated to iron in ferritin was not known . Ferritin protein was overexpressed in Escherichia coli from complementary DNA sequences of bullfrog red cell ferritin . The purple iron(III)-tyrosinate intermediate that formed during the first stages of iron uptake was replaced by the amber multinuclear iron(III)-oxo complexes of fully mineralized ferritin . Only the H subunit formed detectable amounts of the iron(III)-tyrosinate complex, which may explain the faster rates of iron biomineralization in H- compared to L-type ferritin.

J Biol Chem, 1993 Feb 5, 268(4), 2662 - 6
Domain structure of an N-ethylmaleimide-sensitive fusion protein involved in vesicular transport; Tagaya M et al.; N-Ethylmaleimide-sensitive fusion protein (NSF) is an essential component for protein transport between Golgi cisternae . Sequence analysis and proteolytic dissection reveal that NSF contains two tandem "ATP domains," each containing the consensus sequence for the binding of nucleotide . When Escherichia coli-produced Chinese hamster ovary NSF is purified, it exhibits a low, but significant, ATPase activity . The ATPase activity of NSF is sensitive to N-ethylmaleimide and influenced by monoclonal antibodies against recombinant NSF.

J Biol Chem, 1993 Feb 5, 268(4), 2535 - 41
Iron content of human 5-lipoxygenase, effects of mutations regarding conserved histidine residues; Zhang YY et al.; Recombinant human 5-lipoxygenase was expressed in Escherichia coli and purified to more than 95% homogeneity by ammonium sulfate precipitation and agarose-ATP column chromatography . The specific activity of the purified enzyme was 21-28 mumol/mg, as assessed by the generation of 5-hydro(pero)xyeicosatetraenoic acid . The iron content was analyzed by graphite furnace atomic absorption spectrophotometry for six preparations of the enzyme . The average value of the iron content was 0.86 mol/mol (iron/protein) with a range of 0.74-1.15 mol/mol . All lipoxygenases that have been sequenced contain 6 conserved histidine residues . Mutants of 5-lipoxygenase, with substitutions of these 6 conserved histidines, were purified and analyzed . Mutants H372Q and H550Q had no detectable enzyme activity and were also practically devoid of iron . Three mutants regarding His367 (H367Q, H367N, and H367S) were all inactive but had partial iron contents (0.5, 0.2, and 0.5 mol/mol, respectively) . Finally, the mutated proteins H362Q, H390Q, and H399Q displayed reduced enzyme activity but contained similar amounts of iron as non-mutated 5-lipoxygenase . We conclude that histidines 372 and 550 constitute two of the iron ligands in 5-lipoxygenase . Also His367 is necessary for the enzyme activity, but this residue is not crucial for binding of iron.

J Biol Chem, 1993 Feb 5, 268(4), 2486 - 92
Deletion mutagenesis of high molecular weight kininogen light chain . Identification of two anionic surface binding subdomains; Kunapuli SP et al.; The light chain (LC) of cleaved high molecular weight kininogen (HK) binds to anionic surfaces as well as the zymogens prekallikrein and factor XI and thus accelerates activation of the kallikrein-kinin, fibrinolytic, and coagulation pathways . The binding sites on HK LC for factor XI (amino acid residues 574-631) and prekallikrein (residues 583-613) have been localized to domain 6 . Domain 5 (residues 438-520) has been postulated to contain the anionic surface binding subdomain . In order to define this subdomain we have expressed HK LC (residues Lys438-Ser644) as a fusion protein with glutathione-S-transferase (GST) in Escherichia coli and generated various HK LC deletion mutants . The recombinant HK LC (rHK LC) and various HK LC fragments were purified as GST fusion proteins by glutathione-Sepharose affinity chromatography from bacterial cell extracts . The rHK LC and recombinant fragments His459-Ser644, Glu466-Ser644, Leu483-Ser644, His493-Ser644, Lys438-Asp492, Lys438-Ser531, and His493-Lys520 inhibited 125I-HKa binding to kaolin, a model anionic surface used in the contact system, in a concentration-dependent manner . Deletion mutant proteins lacking domain 5, Thr521-Ser644 and Ser583-Ser644, did not inhibit the radiolabeled HKa binding to kaolin . The rHK LC and recombinant fragments Lys438-Asp492, Lys438-Ser531, His493-Ser644, His493-Lys520, Thr521-Ser644, and Ser583-Ser644 were radiolabeled with 125I and were then tested for their ability to bind to kaolin in the presence of fibrinogen and albumin . Except for the Thr521-Ser644 and Ser583-Ser644 fragments, all other radiolabeled HK LC deletion mutant proteins and rHK LC bound to kaolin in a concentration-dependent manner . This binding to kaolin was specific since it was inhibited by the addition of excess unlabeled HKa . The rHK LC, His493-Ser644 and delta 493-520 HK LC have coagulant activity, while other deletion mutant proteins did not exhibit coagulant properties . We conclude that there are at least two anionic surface binding subdomains, one in the histidine-glycine-rich region (Lys438-Asp492) and the other in the histidine-glycine-lysine-rich region (His493-Lys520), in the domain 5 of HK LC . Either subdomain, in the presence of the zymogen binding domain 6, is sufficient to impart coagulant activity to HK LC, while the presence of both did not increase the coagulant activity of HK LC additively.

J Biol Chem, 1993 Feb 5, 268(4), 2307 - 11
Guanosine tetraphosphate inhibits protein synthesis in vivo . A possible protective mechanism for starvation stress in Escherichia coli; Svitil AL et al.; Guanosine 3',5'-bispyrophosphate (ppGpp) accumulates in bacteria in response to either amino acid or energy source starvation . Here we demonstrate that levels of ppGpp similar to those induced by amino acid starvation inhibit the rate of protein synthesis by 84-91% . The intracellular concentration of ppGpp is manipulated in our studies by increasing the expression of a truncated relA gene encoding a smaller but catalytically active peptide with ppGpp synthetase activity . We find that the intracellular activity of the truncated RelA peptide is insensitive to chloramphenicol, unlike the product of the wild-type relA gene, ppGpp synthetase I . Previously, this same ppGpp expression system was used (Schreiber, G., Metzger, S., Aizenman, E., Roza, S., Cashel, M., and Glaser, G . (1991) J . Biol . Chem . 226, 3760-3767) to demonstrate that increasing the ppGpp concentration inhibits growth and ribosomal RNA transcription, and they found suggestive evidence for ppGpp inhibition of protein synthesis . We further investigated the effect of ppGpp on protein synthesis and find that ppGpp is a potent inhibitor of protein synthesis as well as glycerol accumulation but has no effect on transport of methionine, the amino acid used in measuring protein synthesis rates, or on uptake of alpha-methylglucoside, a non-metabolizable analogue of glucose.

J Med Chem, 1993 Feb 5, 36(3), 378 - 84
Novel 6-alkoxypurine 2',3'-dideoxynucleosides as inhibitors of the cytopathic effect of the human immunodeficiency virus; Burns CL et al.; Twenty-one 6-alkoxypurine 2',3'-dideoxynucleosides were enzymatically synthesized with nucleoside phosphorylases purified from E . coli . Eighteen analogs exhibited anti-HIV-1 activity in MT4 cells . Two analogs, 6-(hexyloxy)-(17) and 6-(heptyloxy)-(18) purine 2',3'-dideoxynucleoside, were as potent as 2',3'-dideoxyinosine (ddI, didanosine, Videx) . Although the antiviral activities of 17 and 18 were equivalent, 18 was more cytotoxic . Analogs containing less than four carbons in the 6-alkoxypurine substituent exhibited weak anti-HIV-1 activity . Analogs containing more than seven carbons in the 6-alkoxypurine substituent were too cytotoxic to be effectively evaluated for antiviral activity . Several 6-alkoxypurine 2',3'-dideoxynucleosides were evaluated for substrate activity with calf intestinal adenosine deaminase (ADA) . Increasing the carbon chain length of the 6-alkoxypurine substituent decreased the rate of dealkoxylation . The best substrate in this series was 6-methoxypurine 2',3'-dideoxynucleaside (1); however, the rate of dealkoxylation of 100 microM 1 was 0.17% of the rate of deamination of 100 microM 2',3'-dideoxyadenosine . Compound 17, the most potent anti-HIV-1 analog, was not a substrate for ADA . EHNA (erthro-9-(2-hydroxy-3-nonyl)adenine), a potent inhibitor of ADA, had little effect on the antiviral activities of 17 and ddI . In contrast, coformycin, a potent inhibitor of both ADA and AMP deaminase, dramatically decreased the antiviral activity of 17, but not the antiviral activity of ddI . Thus, AMP deaminase appeared to be involved in the anabolism of 17 . The pharmacokinetic profile of 17, the most promising analog in this series, was determined in the rat . At least seventeen metabolites of 17, including ddI, were detected in plasma samples . This analog also had poor oral bioavailability.

J Med Chem, 1993 Feb 5, 36(3), 370 - 7
Inhibition of the tetracycline efflux antiport protein by 13-thio-substituted 5-hydroxy-6-deoxytetracyclines; Nelson ML et al.; A series of 13-(alkylthio) and 13-(arylthio) derivatives of 5-hydroxy-6-deoxytetracycline (tetracycline, Tc) were synthesized and compared to the clinically used antibiotics tetracycline, methacycline, and minocycline for their ability to inhibit tetracycline efflux in an everted membrane vesicle assay of bacterial resistance to tetracyclines . The assay screened for the ability of tetracycline analogues to inhibit {3H}tetracycline uptake into everted membrane vesicles, thereby evaluating the molecular prerequisites for inhibition of an efflux-dependent resistant bacterial system . Thiol adducts attached at the exocyclic C13 carbon of methacycline led to an increase in inhibitor potency as compared to the reference antibiotics . The most potent inhibitors of {3H}tetracycline uptake into everted vesicles among these analogues, particularly members of the alkyl series, revealed important structure-activity relationships between inhibitor potency, steric parameters, and lipophilicity at the C13 sulfur position.

J Biol Chem, 1993 Feb 5, 268(4), 2888 - 92
Alanine-scanning mutagenesis of the epidermal growth factor-like domains of human thrombomodulin identifies critical residues for its cofactor activity; Nagashima M et al.; Thrombomodulin (TM) is an endothelial cell surface-bound cofactor in thrombin-dependent formation of activated protein C, a potent anticoagulant . Cofactor activity has been localized to the carboxyl-terminal half of the six epidermal growth factor-like (EGF) domains of TM (TME) . To identify residues in TME that are critical for activity, 77 alanine point mutants were made between Cys-333 and Cys-462 by site-directed mutagenesis (all residues except Ala, Cys, Gly, and Pro) . Mutants were expressed in Escherichia coli and cofactor activity measured directly in periplasmic extracts obtained by osmotic shock . Critical residues were defined as those which when mutated had less than 25% cofactor activity of a reference TME . Western blots of non-reduced samples confirmed that alanine substitutions did not significantly decrease expression levels or result in the formation of multimers . In EGF4, which is essential for protein C activation by the thrombin-TM complex, critical residues were: Asp-349, Glu-357, Tyr-358, and Phe-376 . In EGF5-EGF6, critical residues within a proposed acidic thrombin-binding region were: Glu-408, Tyr-413, Ile-414, Leu-415, Asp-416, Asp-417, Asp-423, Ile-424, Asp-425, and Glu-426 . A potential Ca(2+)-binding site, which is comprised of residues Asp-423, Asp-425, Glu-426, Asn-439, Leu-440, and Phe-444, was also identified and overlaps the thrombin-binding region . Asp-461, in the C-loop of EGF6 previously shown to be critical for thrombin binding, was also critical . Asp-398, Asp-400, Asn-402, and Asn-429 in EGF5 were also critical . Thus, rapid alanine-scanning mutagenesis of TME has identified 22 critical residues in the region comprising EGF4-6, which is essential for thrombin binding and protein C activation by the thrombin-TM complex.

J Biol Chem, 1993 Feb 5, 268(4), 2296 - 9
An iron-sulfur center and a free radical in the active anaerobic ribonucleotide reductase of Escherichia coli; Mulliez E et al.; Anaerobically grown Escherichia coli contain an oxygen-sensitive ribonucleotide reductase . The enzyme requires anaerobic activation by two E . coli fractions with S-adenosylmethionine, NADPH, dithiothreitol, and KCl . We now find that photochemically reduced deazaflavin can substitute for these two fractions and NADPH . The reductase contained roughly equimolar amounts of iron and sulfide, suggesting the presence of an Fe-S complex . The cluster is characterized by a charge transfer band at 420 nm and a low temperature EPR signal centered at g = 2.01 that is difficult to saturate at 14 K, suggested to be a (3Fe-4S)+ cluster . In five different preparations of essentially protein-pure reductase containing widely different amounts of iron, the catalytic activity correlated well with the iron content . The iron signal disappeared during reductive anaerobic activation, with the appearance of a new EPR signal at g = 2.0033 showing a temperature behavior and microwave power saturability consistent with an organic free radical . The signal disappeared after exposure of the activated enzyme to air . We suggest that activation involves generation of a specific amino acid free radical that is dependent on the reduced Fe-S cluster and S-adenosylmethionine . From other work it appears likely that the free radical is localized on glycine 681 of the polypeptide chain.

J Mol Biol, 1993 Feb 5, 229(3), 579 - 84
Two-dimensional crystals of the molecular chaperone GroEL reveal structural plasticity; Zahn R et al.; For two-dimensional (2-D) crystallization we have purified the molecular chaperone GroEL from Escherichia coli to homogeneity . The final and important step for crystallization in the purification procedure was an ATP-agarose column, on which the spacer between ATP and agarose was attached to C8 of adenine . Using the mica spreading "negative staining-carbon film" procedure and polyethylene glycol as a precipitant, we obtained four different 2-D periodic arrays . Two of them turned out to be true crystals . One crystal has P2 symmetry and lattice constants of a = 24.3 nm and b = 16.9 nm, the other has essentially P4 symmetry and shows smoothly varying local changes in the lattice parameters (a = b = 23 (+/- 1.3) nm) . Very striking in the P4 crystal is the departure within each individual GroEL particle from the GroEL-typical seven-fold symmetry, which seems to be required for GroEL to accommodate to a crystal symmetry.

J Biol Chem, 1993 Feb 5, 268(4), 3009 - 15
Neutrophil activation by nascent FimH subunits of type 1 fimbriae purified from the periplasm of Escherichia coli; Tewari R et al.; Previous studies of type 1 fimbriae of Escherichia coli have implicated FimH, a minor subunit, as the determinant of its mannose binding property . Structure-function analysis of FimH has not been possible because of the difficulty in obtaining adequate amounts of the subunit from type 1 fimbriae . We have obtained nascent FimH that has not been incorporated into the fimbrial structure from the periplasm of an E . coli strain expressing the cloned fimH gene . Nascently translocated FimH was initially degraded in the periplasm; however, when co-expressed with FimC, a putative fimbrial chaperone, the FimH molecules were stabilized and readily isolated from the periplasmic extract by fractionation on a sodium dodecyl sulfate-polyacrylamide gel followed by electroelution of the FimH band from the gel . The eluted protein was purified to homogeneity by affinity chromatography on a mannose-Sepharose column . Purified FimH displayed the same mannose-inhibitable binding to human neutrophils as type 1 fimbriated bacteria, including triggering an oxidative burst with concomitant release of reactive oxygen metabolites . In addition, inert microspheres coated with FimH, but not those coated with bovine serum albumin, were phagocytosed by neutrophils . These data provide direct evidence that FimH is the determinant on type 1 fimbriae which is responsible for mediating mannose-specific adherence and that isolated FimH is a potent activator of human neutrophils.

J Biol Chem, 1993 Feb 5, 268(4), 2767 - 72
Interactions of phage P22 tailspike protein with GroE molecular chaperones during refolding in vitro; Brunschier R et al.; Because efficient folding in vivo and reconstitution in vitro of phage P22 tailspike protein is temperature-sensitive, and because a chaperone function of the GroE proteins for tailspike folding in vivo has been suggested by genetic observations, the interactions of purified Escherichia coli GroE proteins with phage P22 tailspikes during refolding in vitro were investigated . At elevated temperature (> 30 degrees C), in the absence of ATP, GroEL effectively trapped refolding tailspike protein and prevented reconstitution . Tailspike protein was released from GroEL by addition of ATP around 35 degrees C or without added ATP upon cooling to 25 degrees C, and native tailspike trimers were formed . In accordance with the cold release, tailspike reconstitution at < or = 25 degrees C was unaffected by GroE . No formation of native tailspike trimers was observed, when refolding was initiated at 42 degrees C in the presence of the GroE proteins and ATP or when tailspike protein was dissociated from a preformed complex with the chaperone by addition of ATP at 42 degrees C . In contrast to other GroE ligands, the tailspike polypeptide was bound by and released from GroE in similar states of folding, and the presence of GroES in addition to GroEL had no effect on reconstitution yields at any temperature . Thus, the GroE proteins may exhibit widely differing interactions even with proteins showing similarly temperature-sensitive yields of folding.

J Mol Biol, 1993 Feb 5, 229(3), 609 - 22
Ribosome initiation complex formation with the pseudoknotted alpha operon messenger RNA; Spedding G et al.; The Escherichia coli alpha mRNA has a complex pseudoknot secondary structure that forms the recognition site for a translational repressor, ribosomal protein S4, and also encompasses the regulated ribosome binding site . To find out whether the pseudoknot is a stable structure under the conditions of ribosome initiation complex formation, thermal denaturation of the RNA was monitored by calorimetry and ultraviolet light hyperchromicity . The secondary structure formed by the coding region melts in a single transition and has a stability of -7.4 kcal/mol at 37 degrees C (5 mM-Mg2+, 100 mM-Na+, pH 7.0) . A broad transition with tm approximately 38 degrees C may be a rearrangement of pseudoknot secondary or tertiary structure . Using reverse transcriptase primer extension assays ("toeprints") to measure the kinetics of ternary 30 S subunit-tRNAf(met)-alpha mRNA translational initiation complex formation, we find a fast and a slow phase in the reaction . The fraction reacting rapidly is sensitive to temperature and mutations in the mRNA . We interpret these results in terms of "active" and "inactive" mRNA conformations that are trapped by 30 S subunits and react rapidly or slowly with tRNAf(met), respectively; the active form is predominant above 37 degrees C . The binary 30 S-mRNA complex in the inactive form stops MMLV reverse transcriptase near the 3' edge of the pseudoknot structure, apparently by stabilizing the pseudoknot . We propose the following mechanism for translational initiation with the alpha mRNA . The intact pseudoknot stimulates 30 S subunit binding, at low temperatures, but prevents proper binding of tRNAf(met) . The inactive to active transition of the pseudoknot, which may be related to the 38 degrees C transition seen in melting experiments, is required for tRNAf(met) to pair with the anticodon and is rate-limiting for initiation complex formation at lower temperatures . A novel feature of this proposal is that the mRNA structure affects a kinetic step in initiation complex formation, as well as ribosome binding affinity.

J Biol Chem, 1993 Feb 5, 268(4), 2542 - 53
Cell- and heparin-binding domains of the hexabrachion arm identified by tenascin expression proteins; Aukhil I et al.; We have produced a set of bacterial expression proteins corresponding to 10 segments of tenascin and two of fibronectin and tested them for heparin binding and cell adhesion . We used polymerase chain reaction cloning to terminate the segments precisely at domain boundaries . Heparin binding activity was mapped to two different tenascin segments: one comprising the fourth and fifth fibronectin type III domains, and to TNfbg, the fibrinogen-like terminal knob . TNfbg, but none of the other tanascin segments, also supported adhesion of primary rat embryo skin fibroblasts . The fibroblasts did not spread on TNfbg but remained rounded . Cell binding to TNfbg occurred in the presence or absence of divalent cations and was not inhibited by RGD peptides, suggesting that integrins are not involved . Fibroblast binding to TNfbg was strongly inhibited by soluble heparin, by treating the cells with heparitinase, or by culture conditions that cause undersulfation of proteoglycans . These observations suggest that cell attachment to TNfbg is mediated by cell surface proteoglycans . We have also made full-length cDNA constructs for the largest and smallest splice variants of human tenascin, as well as one truncated after the 14th epidermal growth factor-like domain, in the pNUT mammalian cell expression vector . Stably transfected baby hamster kidney cell lines secreted large quantities of tenascin, and this was assembled into normal hexabrachions, the arm length corresponding to the construct.

Nature, 1993 Feb 4, 361(6411), 464 - 7
A mammalian guanine-nucleotide-releasing protein enhances function of yeast secretory protein Sec4; Burton J et al.; Small GTP-binding proteins of the ras superfamily are important for exocytosis from eukaryotic cells . GTP-binding proteins can exist in two different conformations depending on whether they are bound to GDP or GTP, and are thought to function as molecular switches that regulate a variety of cellular processes . The GTP-GDP cycle is controlled by accessory proteins that promote the exchange of bound GDP or the hydrolysis of GTP . The protein Sec4, a member of the Sec4/Ypt1/Rab branch of the Ras superfamily, is involved in a late stage of the secretory pathway in yeast . Here we report the isolation of a mammalian complementary DNA, mss4, encoding a GDP-releasing protein that enhances Sec4 function . The Mss4 protein also stimulates GDP release from Ypt1 and from the mammalian protein Rab3a, but not from Ras2 . Mss4 shows sequence similarity to Dss4, a yeast protein with similar biochemical properties.

Zentralbl Bakteriol, 1993 Feb, 278(1), 73 - 82
Binding of Escherichia coli to Penrose rubber drains--an in vitro study; Guo W et al.; Ten different isolates of Escherichia coli were used to compare bacterial attachment to Penrose rubber drains at different temperatures and to investigate a possible relation with cell surface hydrophobicity and charges, as well as the capacity of autoaggregation . Penrose rubber drain pieces of 1 cm2 were incubated with 4.8 x 10(6) E . coli cells for 1 h at 22, 37 and 42 degrees C, respectively . After rinsing with phosphate buffered saline (PBS), the number of adhering bacteria on the drain pieces was calculated by measuring cellular ATP (adenosine triphosphate) bioluminescence . Bacterial cell surface properties at different temperatures were determined by two-phase partitioning and autoaggregation was determined by arbitrary scoring under the dissecting microscope . For most strains of E . coli, the number of adherence to Penrose rubber drain was higher at 22 degrees C than at 37 degrees C and 42 degrees C . Bacterial cell-surface properties and autoaggregation capacity were influenced by the growth temperature, but without correlation to bacterial ability to attach to rubber drains . Thus, the present study demonstrates that attachment of E . coli to Penrose rubber drains was significantly influenced by temperature, but bacterial cell-surface hydrophobicity and charge as well as autoaggregation capacity had no influence on bacterial attachment ability.

Proc Natl Acad Sci U S A, 1993 Feb 1, 90(3), 1043 - 7
Mutants in disulfide bond formation that disrupt flagellar assembly in Escherichia coli; Dailey FE et al.; We report the isolation and characterization of Escherichia coli mutants (dsbB) that fail to assemble functional flagella unless cystine is present . Flagellar basal bodies obtained from these mutants are missing the L and P rings . This defect in assembly appears to result from an inability to form a disulfide bond in the P-ring protein (FlgI) . Cystine suppresses this defect in dsbB strains . We also show that dsbA strains {Bardwell, J . C . A., McGovern, K . & Beckwith, J . (1991) Cell 67, 581-589} fail to assemble P rings, apparently from a similar failure in disulfide bond formation . However, cystine does not completely suppress this defect in dsbA strains . Thus, disulfide bond formation in FlgI is essential for assembly . DsbA likely puts in that bond directly, whereas the DsbB product(s) play a role in oxidizing DsbA, so that it can be active.

Microb Pathog, 1993 Feb, 14(2), 85 - 94
Exchange of phospholipids between Escherichia coli cells and environment; Galdiero F et al.; We studied the exchange of phospholipids between Escherichia coli K-12 cells and the suspension medium containing inactivated guinea-pig serum . In this medium, the release of 3H-labelled phospholipids was proportional both to the quantity of serum and to the temperature of incubation . No phospholipids were released when no guinea-pig serum was added to the medium, or the incubation temperature was 4 degrees C . The release of phospholipids into the medium was accompanied by an uptake of serum phospholipids by the cells, as demonstrated by incorporation of labelled phospholipids from the suspension medium . We conclude that an exchange occurs between the cellular phospholipids and those of the medium . Control tests with 3H-thymidine showed that cellular lysis was not involved.

Indian J Exp Biol, 1993 Feb, 31(2), 136 - 41
Induction of prophage lambda by nitrofurantoin and its modulation by butylated hydroxytoluene, sodium arsenite and alpha tocopherol; Rahman MS et al.; Nitrofurantoin induced prophage-lambda in E . coli K12 strain GY5027(lambda) in a dose dependent manner, the maximum induction being 10-fold the spontaneous induction level and the maximum efficiency of induction 74% . The lever extract used as a metabolizing mixture enhanced the induction level significantly . Chloramphenicol at a concentration of 20 micrograms/ml inhibited the prophage induction by nitrofurantoin, indicating that the induction required concomitant protein synthesis . Butylated hydroxytoluene(BHT) and sodium arsenite enhanced the nitrofurantoin induced prophage-lambda induction in E . coli GY 5027(lambda) cells in a dose dependent manner . The maximum modulations in induction level (I/Io) were achieved with 100 micrograms/ml BHT and 250 micrograms/ml sodium arsenite corresponding to a nitrofurantoin concentration of 15 micrograms/ml and were found significant on statistical analysis . alpha-tocopherol, however, did not produce any effect on the prophage-lambda induction by nitrofurantoin.

Hum Mol Genet, 1993 Feb, 2(2), 107 - 14
Isolation of the human Xp21 glycerol kinase gene by positional cloning; Walker AP et al.; The gene for human glycerol kinase deficiency (GK) maps in Xp21.3 in a critical region of about 50-250 kb located distal to the Duchenne muscular dystrophy gene (DMD) by analysis of patient deletions and YAC contigs . We have used a genomic exon amplification strategy to isolate potential exons from two cosmids which mapped to this interval . The genomic exons were used to isolate six overlapping cDNA clones from human fetal liver which encode the X-linked glycerol kinase gene . The cDNA clones map to cosmids, YAC clones and deletions in patients which define the GK critical region and also hybridize to several autosomal fragments and one Xq fragment in genomic DNA . The GK gene is expressed most in human liver with three transcript sizes of 1.85, 2.7, and 3.7 kb . Sequence analysis of 1.5 kb of several overlapping liver cDNA clones predicted a protein with approximately 63% similarity to the E . coli and B . subtilis glycerol kinase genes . The liver cDNA clones have sequence identity with four genomic exons and the 3' untranslated region from an Xp21.3 cosmid thus indicating that this is the expressed GK gene which when deleted in patients gives rises to GK deficiency.

Anim Genet, 1993 Feb, 24(1), 1 - 7
Construction of a library of bovine genomic fragments enriched in CpG islands; De Rubertis F et al.; A procedure is described to isolate DNA probes from the bovine genome that are enriched in sites for the so-called rare cutter restriction endonucleases . A collection of SacII (CvCGCGG)-Hin-dIII fragments from bovine sperm was established in the plasmid Bluescript . 180 clones were picked at random and analysed for the presence of inserts with sites for the following rare cutters: EagI, BsshII, NarI, MluI, NruI, NaeI: 70% of the clones contained at least 1 site and 5% contained four different such sites . 22.8% had multiple sites for one or more of the rare cutters tested . Sequence analysis for 16 clones confirmed the cloning of DNA with a G+C content and a proportion of CpG vs GpCs indicative of CpG islands.

Circ Shock, 1993 Feb, 39(2), 83 - 8
Complement and leukocyte activation in septic baboons; Bengtsson A et al.; The effect of Escherichia coli infusion on complement and leukocytes was evaluated in a baboon model . During 8 hr, different amounts of live E . coli (5 x 10(8), 2.5 x 10(9) and 10(10) live bacteria kg body weight) were infused . Twenty-one baboons were investigated . Activation of complement (terminal C5b-9 complement complex; TCC) and activation of leukocytes (PMN elastase) and plasma concentrations of endotoxin (lipopolysaccharide; LPS) were determined before the start of bacteria infusion and 2, 4, 6, and 8 hr after the start of infusion . In baboons receiving 2.5 x 10(9) and 10(10) live E . coli per kilogram body weight, increasing plasma levels of TCC were found (P < 0.05) . No significant alterations of TCC were observed when animals were infused with 5 x 10(8) live E . coli per kilogram body weight during an 8 hr period . Plasma levels of PMN elastase increased significantly in baboons receiving 5 x 10(8), 2.5 x 10(9), and 10(10) live bacteria per kilogram body weight . High levels of LPS were detected in animals receiving 10(10) live E . coli bacteria per kilogram body weight and in animals receiving 5 x 10(8) or 2.5 x 10(9) live E . coli bacteria per kilogram body weight . There was a positive correlation between the formation of TCC and the plasma levels of PMN elastase and LPS and between plasma levels of PMN elastase and of LPS . Activation of complement and leukocytes may contribute to the development of organ dysfunction seen in baboons infused with high amounts of live E . coli.

J Med Virol, 1993 Feb, 39(2), 101 - 8
Competitive EIA for anti-HIV-2 detection in The Gambia: use as a screening assay and to identify possible dual infections; Berry N et al.; The performance of a competitive EIA for the detection of HIV-2-specific antibody utilising a viral lysate antigen was assessed over a 3 year period in The Gambia, West Africa, and compared with a commercially available assay, ELAVIA-2, using three panels of sera . An immunodominant region of the transmembrane glycoprotein of an HIV-2 isolate (ANT 53) was also cloned and expressed in E . coli as a beta-galactosidase fusion protein and the resulting recombinant protein substituted in place of the existing viral lysate antigen . Competitive EIAs were found to be both a specific and sensitive means of reliably determining the HIV-2 status of an individual with a high predictive value, particularly when a strategy of concordant positive results in the two EIAs was used . When either anti-HIV-2 competitive EIAs were used in conjunction with a competitive EIA for anti-HIV-1 detection it was possible in the vast majority of cases to identify the virus-type infecting an individual and speciate HIV-1 and HIV-2 infections . A few sera which showed similar regression profiles when diluted over a serial tenfold dilution steps were identified as possible dual infections.

Biokhimiia, 1993 Feb, 58(2), 224 - 33
{Features of interaction of Escherichia coli DNA polymerase I and its Klenow fragment with dTTP gamma-p-azidoanilide}; Kudriashova NV et al.; gamma-p-Azidoanilidate of dTTP was used to study the photoaffinity modification of DNA polymerase I and Klenow fragment . The analog was found to be a mixed-type inhibitor with respect to dTTP of the polymerization reaction catalyzed by DNA polymerase I and Klenow fragment . In the absence of the reagent both UV-irradiated enzymes were rapidly inactivated . Substrates (dNTP and template-primer) protected the enzymes from inactivation by UV-light with different efficiency . In the presence of the template-primer UV-irradiation induced activation of DNA polymerase I . The effect of the analog on both enzyme forms under irradiation is different . At concentration of 10(-5)M gamma-p-anilidate of dTTP accelerated the activation of DNA polymerase I initiated by UV-irradiation and at 10(-4)M concentration it inactivated the enzyme by 20-25% . Under such conditions one enzyme molecule covalently bound two molecules of the analog . While the template-complementary substrate (dTTP) protected DNA polymerase I both from inactivation and modification, the non-complementary one (dCTP) worked only against modification . In contrast to DNA polymerase I Klenow fragment was not inactivated when exposed to UV-irradiation and gamma-p-anilidate of dTTP neither modified the protein nor exerted any significant effect on its polymerization activity . The data accumulated suggest the presence on the DNA polymerase I molecule of a regulatory region providing additional dNTP binding sites.

Enferm Infecc Microbiol Clin, 1993 Feb, 11(2), 80 - 3
{Application of PCR for the detection of mutations in the genes coding for beta-lactamases}; Gallego L et al.; BACKGROUND: The purpose of this study was to develop a satisfactory technique to detect punctual mutations in blaTEM genes . METHODS: The strains {E . coli HB 101 pBR322 (TEM-1), E . coli J62 RP4 (TEM-2)} were submitted to PCR with primers PL1 and PL2 which amplify the genetic region susceptible of punctual mutations . Then, we developed Southern blot and hybridization with oligonucleotide probes (GIn 37, Lys 37 y Thr 261), corresponding to first and last mutations . RESULTS: A region of 841 bp was amplified using the primers previously described . Hybridization experiments with the Thr 261 probe gave positive signal with both strains (both carry the mutation); GIn 37 only hybridized with TEM-1 and Lys 37 only with TEM-2 . CONCLUSIONS: The use of primers which amplify all the region susceptible of mutations in blaTEM and oligonucleotide probes allows the specific detection of point modifications in the original genes by the use of digoxigenin-labeled oligonucleotide probes.

Protein Eng, 1993 Feb, 6(2), 195 - 200
Characterization of the DNA binding domain of the mouse IRF-2 protein; Uegaki K et al.; The DNA binding domain of the interferon regulatory factor-2 protein (IRF-2) has been produced and characterized . alpha-chymotrypsin digestion of the purified IRF-2 protein bound to a synthetic binding site yields a peptide fragment of 14 K in molecular weight . N-terminal analysis of this peptide fragment showed that its sequence is the same as that of the intact IRF-2 . A peptide fragment of approximately 14 K, IRF-2(113), which corresponds to the N-terminal 113 amino acids of the intact IRF-2 protein, has been expressed in a functional form in Escherichia coli . The first methionine was processed during the expression and the purified IRF-2(113) thus contains 112 amino acids . DNase I footprinting and gel retardation assaying showed that IRF-2(113) binds to a synthetic DNA having the consensus binding site and to the upstream regulatory sequence of the IFN-beta gene as intact IRF-2 does . These results showed that this peptide fragment, IRF-2(113), may be a good material for investigation of the DNA binding domain of IRF-2 and of the DNA-protein interaction.

J Rheumatol, 1993 Feb, 20(2), 325 - 30
Synovial type (group II) phospholipase A2 in cartilage; Nevalainen TJ et al.; Phospholipase A2 (PLA2) was produced in E . coli by a recombinant technique using a synthetic gene coding for PLA2 found in synovial fluid (SF) of patients suffering from rheumatoid arthritis (RA) . Polyclonal antibodies were produced against the recombinant synovial type PLA2 (syn-PLA2) in a rabbit . The IgG fraction of the antiserum was isolated and the specificity was tested by immunoblotting . The antiserum detected a protein with an apparent molecular weight of 15 kDa in extracts of cartilage, but did not react with PLA2 isolated from human pancreas or ascitic fluid . In immunohistochemistry, the anti-syn-PLA2 antibody decorated chondrocytes and matrix of articular, laryngeal and auricular cartilage, but did not decorate synovial tissue or its contained inflammatory cells in RA or other human tissues tested (pancreas, liver, thyroid, tonsil, lung, nasal polyp, skin, placenta, myometrium, bone) . The results show that syn-PLA2 is present both in articular and extraarticular cartilage and support the view that PLA2 found in SF might originate from chondrocytes, and not from synovial lining or inflammatory cells.

Mol Cell Endocrinol, 1993 Feb, 91(1-2), 35 - 41
High expression of the hormone binding active extracellular domain (1-294) of rat lutropin receptor in Escherichia coli; Chen W et al.; Using the polymerase chain reaction (PCR) technique, the cDNA fragment corresponding to the receptor coding region for residues 1-294 was prepared from rat lutropin receptor (LHR) cDNA and subsequently subcloned into Escherichia coli expression vector pT7-7 . This truncated receptor was efficiently expressed in E . coli as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining . The recombinant protein present in the inclusion bodies was solubilized in 6 M guanidine-HCl and purified in two successive steps of fast performance liquid chromatography (FPLC) using Superose-12 and Mono Q columns . Refolding of the purified recombinant protein was achieved in 1.5 M guanidine-HCl in the presence of an equimolar proportion of cysteine and cystine . The refolded soluble truncated LHR(1-294) had apparent molecular weights of 33 kDa and 140 kDa under reducing and nonreducing conditions, respectively . The multimeric nature of the extracellular domain of the receptor is believed to be due to its self-association by intermolecular disulfide bond formation since the 1-294 amino acid segment has nine cysteine residues and thus has one or possibly more free sulfhydryl groups . The purified truncated receptor had high binding affinity for human choriogonadotropin (hCG) as indicated by ligand blotting on SDS-PAGE and radioligand receptor assays . The amount of hCG required for 50% inhibition of binding of 125I-hCG to the soluble truncated receptor was 2.7 x 10(-10) M (or 12.25 ng) . Similarly, the amount of soluble truncated receptor required for 50% inhibition of 125I-hCG binding to the rat ovarian receptor in the radioligand receptor assay was 58 ng.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biochem (Tokyo), 1993 Feb, 113(2), 208 - 13
Inhibition of aspartate chemotaxis of Escherichia coli by site-directed sulfhydryl modification of the receptor; Gomi S et al.; Thr-154 of the chemoreceptor Tar in Escherichia coli is important for aspartate sensing . Taking advantage of the fact that Tar has no Cys residues, we have further investigated the role of Thr-154 by replacing it with Cys in order to subject it to SH modification . Tar-T154C retained the abilities of aspartate sensing and repellent sensing . However, when cells with Tar-T154C were treated with an SH-modifying reagent, 5,5'-dithiobis-2-nitrobenzoic acid (DTNB), they specifically lost the ability to sense aspartate; the ability was restored by the reducing reagent, 1,4-dithiothreitol . DTNB showed no detectable effect on the function of wild-type Tar or serine-replaced Tar, Tar-T154S . Thus, DTNB modifies Cys-154 of Tar-T154C in intact cells and causes a specific defect in the aspartate-sensing ability of Tar . The addition of 1 mM or higher concentrations of aspartate resulted in protection of Cys-154 from the modification; serine had no effect in this regard . These results that not only is Thr-154 important for aspartate sensing but also, it may be located at the actual aspartate-binding site.

Int J Androl, 1993 Feb, 16(1), 47 - 52
Immuno-competent cells in the murine epididymis following infection with Escherichia coli; Nashan D et al.; Epididymitis was induced by retrograde injection of Escherichia coli into the vas deferens of 28 mice . A group of 28 saline-injected animals served as controls . On Days 1, 3, 7 and 28, groups of seven animals were killed . Bacterial culture was performed . Leucocyte numbers and distribution were determined in epididymides . In infected mice, E . coli were isolated from all epididymides on Days 1 and 3, but only from five of seven epididymides on Days 7 and 28 . One week after infection, the total number of macrophages rose from about 10 to 28% . Significantly increased macrophage percentages were also found in animals killed 28 days after infection . A simultaneous increase in MHC class II positive cells was seen on Day 7 . A total of 20% of the cells expressed MHC class II in infected epididymides (normal = 6%) . A similar increase was found on Day 28 after infection . Most of the macrophages and MHC class II positive cells were located in the interstitium, fewer in the peritubular layer and nearly none in the epithelium . The main increase in these cells occurred in the interstitium and, to a lesser but significant extent, in the peritubular area . T-helper and T-suppressor/cytotoxic lymphocytes reached peak values on Day 28 . The increase in T-lymphocytes and simultaneous appearance of plasma cells followed the increase in numbers of macrophages and MHC class II positive cells . They were located mainly in the interstitium . A sequential increase in leucocyte subsets and negative culture results for E . coli were observed on Days 7 and 28 (2/7 on each day) . The inflammatory process was restricted to the interstitium.(ABSTRACT TRUNCATED AT 250 WORDS)

Genet Res, 1993 Feb, 61(1), 1 - 8
Expression of the Escherichia coli ftsZ gene: trials and tribulations of gene fusion studies; Robin A et al.; The ftsZ gene of Escherichia coli, which codes for an essential cell division protein, is subjected to multiple regulation, as shown in part with studies using an ftsZ::lacZ operon fusion located on phage lambda JFL100 . Using this same fusion, we sought to isolate regulatory mutants overexpressing ftsZ by selecting mutants able to grow on lactose . One Lac+ mutant was obtained which overexpressed the ftsZ::lacZ fusion 70-fold . The mutation responsible for the overexpression lies in a new gene, cot, located near 56 min on the E . coli genetic map . The cot mutation probably affects the transcription of a chromosomal open reading frame, ORF1, lying downstream of the bioA gene and adjacent to the ftzZ::lacZ fusion of the lambda JFL100 prophage integrated at att lambda . Using an ftsZ84(Ts) strain, in which there was a double selection for overexpression of both ftsZ::lacZ and ftsZ+, no Lac+Tr mutants were obtained from 3.6 x 10(10) bacteria; the introduction of a mutL allele, increasing spontaneous base substitution mutation rates 75-fold, did not permit us to isolate such a mutant . We conclude that Lac+ ftsZ-constitutive mutations cannot be obtained in lambda JFL100 lysogens by a single base substitution.

Exp Parasitol, 1993 Feb, 76(1), 1 - 12
Trypanosoma cruzi: characterization of two recombinant antigens with potential application in the diagnosis of Chagas' disease; Gruber A et al.; A genomic library of Trypanosoma cruzi, constructed in the vector lambda gt11, was screened with a hyperimmune rabbit antiserum against tissue culture trypomastigotes . Two clones, B12 and B13, containing inserts of 350 and 600 bp, respectively, were isolated . Sequencing data indicated that both clones present a pattern of tandemly repeated nucleotide units of 60 bp for B12 and 36 bp for B13 . Southern blot analysis suggests that both corresponding genes exist as a single copy . The inserts of both recombinants were subcloned in the vector pMSgt11, in phase with the lacZ gene . Recombinant proteins were affinity purified on pAPTG-agarose columns and employed to immunize rabbits, as well as to immunoselect human chagasic antibodies . By Western blot, antibodies to B12 reacted with bands of 230 kDa in trypomastigotes and 200 kDa in epimastigotes, while those to B13 identified bands of 140 and 116 kDa in trypomastigotes and epimastigotes . Immunoprecipitation of radioiodinated parasites revealed that the 140-kDa antigen recognized by antibodies to B13 is located on the membrane of trypomastigotes but not epimastigotes . The potential application of either recombinant antigen in the serological diagnosis of Chagas' disease was evaluated initially by RIA . It was observed that B13 presents a very good performance with sensitivity of 97% . For B12, the corresponding value was 82% . The reactivity to B13 was also evaluated by ELISA tests run in parallel with conventional serological reactions for Chagas' disease . Analysis of 209 serum samples indicates that B13 presents similar or even better performance in relation to the use of total epimastigote antigens, making it a promising candidate for the diagnosis of Chagas' disease.

Eur J Cell Biol, 1993 Feb, 60(1), 185 - 95
Sequential O-glycosylation of nuclear pore complex protein gp62 in vitro; Cordes VC et al.; Gp62 is a nuclear pore complex glycoprotein of vertebrates containing multiple O-linked N-acetylglucosamine monosaccharides . We have recently shown (Cordes et al., Eur . J . Cell Biol . 55, 31-47 (1991)) that gp62 of mouse and Xenopus laevis is predominantly glycosylated in the amino-terminal half of the molecule . Here we describe in vitro glycosylation in rabbit reticulocyte lysate of gp62 and of individual segments of this protein expressed as recombinant polypeptides in E . coli . Competition experiments between purified polypeptides revealed that gp62 exhibits in vitro at least two types of binding sites for a cytosolic N-acetylglucosaminyltransferase resulting in a sequential glycosylation of the protein . High affinity binding sites for this glycosyltransferase were located in the amino-terminal domain between amino acids 248-341 of mouse gp62, whereas low affinity binding sites were scattered in the entire amino-terminal half . The segment containing amino acids 248-341 was able to compete with in vitro O-glycosylation of gp62 subdomains containing low affinity binding sites . This gp62 segment was also able to compete with O-glycosylation of other unrelated cellular proteins in vitro.

J Vet Med Sci, 1993 Feb, 55(1), 173 - 5
A cryptic DNA sequence, isolated from Actinobacillus pleuropneumoniae, confers a hemolytic activity upon Escherichia coli K12 strains; Ito H et al.; A hemolytic recombinant clone on Escherichia coli was obtained from a genomic library of Actinobacillus pleuropneumoniae . The clone possessed a recombinant plasmid, pHLY1, carrying an insert DNA of 1 kilobases . A 21 kilodaltons protein, which is assumed to be a fusion protein with a tetracycline resistant protein of pBR322, was encoded by pHLY1 . The nucleotide sequence and its deduced amino acid sequence were different from those of the previously reported hemolysin genes of A . pleuropneumoniae . Furthermore, the amino acid sequence did not show a significant homology with other published sequences in the SWISS-PROT Protein Sequence Data Bank.

Mol Microbiol, 1993 Feb, 7(4), 523 - 36
The RpoS sigma factor relieves H-NS-mediated transcriptional repression of csgA, the subunit gene of fibronectin-binding curli in Escherichia coli; Olsen A et al.; Curli encoded by the curlin subunit gene, csgA, are fibronectin- and laminin-binding fibres expressed by many natural Escherichia coli and E . coli K-12 strains in response to low temperature, low osmolarity and stationary-phase growth conditions . Curli expression is dependent on RpoS, a sigma factor that controls many stationary phase-inducible genes . Many commonly used K-12 strains carry an amber mutation in rpoS . Strains able to form curli carry an amber suppressor whereas curli-negative E . coli K-12 strains, in general, are sup0 . Introduction of SupD, SupE, or supF suppressors into sup0 strains resulted in expression of temperature-regulated curli . In curli-deficient, RpoS- E . coli K-12 strains, csgA is transcriptionally activated by mutations in hns, which encodes the histone-like protein H-NS . Curli expression, fibronectin binding, and csgA transcription remain temperature- and osmoregulated in such double mutants . Our data suggest that RpoS+ strains, and hence curli-proficient strains of E . coli K-12, are relieved for the transcriptional repression mediated by the H-NS protein upon accumulating RpoS as cells reach stationary phase.

Mol Microbiol, 1993 Feb, 7(3), 359 - 69
Isolation of dnaJ, dnaK, and grpE homologues from Borrelia burgdorferi and complementation of Escherichia coli mutants; Tilly K et al.; The heat-shock proteins DnaJ, DnaK, and GrpE are involved in the replication of various species of DNA in Escherichia coli, in addition to their roles in other processes, including protein disaggregation and export . We have cloned the Borrelia burgdorferi homologues of these genes . DNA sequence analysis revealed an open reading frame encoding a protein that is 62% identical to the E . coli DnaK protein . Genes homologous to the E . coli grpE and dnaJ genes, encoding products 28% and 39% identical to their homologues, are located up- and downstream, respectively, of the B . burgdorferi dnaK gene . No obvious promoters were detected in the sequenced DNA, although a potential transcription terminator was found downstream of the dnaJ gene, so these three genes may form an operon, perhaps with a fourth gene located upstream of the grpE gene . The grpE homologue complemented an E . coli grpE mutant and the dnaJ homologue complemented an E . coli dnaJ mutant, whereas the B . burgdorferi dnaK gene did not complement dnaK mutants.

Biophys J, 1993 Feb, 64(2), 426 - 34
Mechanical properties of vesicles . I . Coordinated analysis of osmotic swelling and lysis; Ertel A et al.; To determine how transmembrane osmotic gradients perturb the structure and dynamics of biological membranes, we examined the effects of medium dilution on the structures of osmolyte-loaded lipid vesicles . Our preparations were characterized by dynamic light scattering (DLS) and nuclear magnetic resonance (NMR) spectroscopies . Populations of Escherichia coli phosphatidylethanolamine (PE) or dioleoylphosphatidylglycerol (DOPG) vesicles prepared by the pH jump technique were variable and polymodal in size distribution . Complex and variable structural changes occurred when PE vesicles were diluted with hypotonic buffer . Such vesicles could not be used as model systems for the analysis of membrane mechanical properties . NaCl-loaded, DOPG vesicles prepared by extrusion through 100 nm (diameter) pores were reproducible and monomodal in size distribution and unilamellar, whereas those prepared by extrusion through 200-, 400-, or 600-nm pores were variable and polymodal in size distribution and/or multilamellar . Time and pressure regimes associated with osmotic lysis of extruded vesicles were defined by monitoring release of carboxyfluorescein, a self-quenching fluorescent dye . Corresponding effects of medium dilution on vesicle structure were assessed by DLS spectroscopy . These experiments and the accompanying analysis (Hallett, F.R., J . Marsh, B.G . Nickel, and J.M . Wood . 1993 . Biophys . J . 64:000-000) revealed conditions under which vesicles are expected to reside in a consistently strained state.

AIDS Res Hum Retroviruses, 1993 Feb, 9(2), 175 - 81
The galactosyl ceramide/sulfatide receptor binding region of HIV-1 gp120 maps to amino acids 206-275; Bhat S et al.; Our recent studies have indicated that galactosyl ceramide (GalCer) or sulfatide (sul) may serve as an alternate receptor for human immunodeficiency virus (HIV) in neural cells . In this paper, we describe the mapping of GalCer/sul binding region of HIV env glycoprotein gp120 . Deglycosylated gp120 binds to GalCer, suggesting that the amino acids of glycoprotein gp120 and not the carbohydrates are responsible for the observed binding . Specific regions of gp120 responsible for the binding were analyzed by using varying-length truncations of gp120 expressed in Escherichia coli and vaccinia virus . Purified recombinant gp120 containing amino acids 200-295 of gp120 bind to GalCer/sul, whereas recombinant env proteins that deleted this region did not bind . These recombinant proteins also bind to SK-N-MC-derived neuroblastoma cells, the binding of which is inhibited by anti-GalCer . In addition, 125I-labeled gp120 binding to GalCer is inhibited by these proteins . Studies using lysates containing truncated gp120 expressed in vaccinia virus also gave similar results . By eliminating the overlapping regions that do not bind, we conclude that the amino acids responsible for GalCer/sul binding reside between amino acids 206 and 275 . The significance of this mapping is discussed in relation to the neurotropism of HIV.

Mol Gen Genet, 1993 Feb, 237(1-2), 81 - 8
Autoregulation by cooperative binding of the PemI and PemK proteins to the promoter region of the pem operon; Tsuchimoto S et al.; The low copy number plasmid R100 carries the pem region, consisting of two genes, pemI and pemK, which are required for stable maintenance of the plasmid . Here, to understand the regulation of the expression of the pem region, we constructed plasmids carrying either the pemI or the pemK gene, whose initiation codons were fused in frame with the lacZ gene, and examined their expression by assaying beta-galactosidase (LacZ) activity . The synthesis of both PemI and PemK proteins was found to be repressed coordinately in the presence of a plasmid carrying the entire pem region . This indicates that pemK and pemI cistrons form an operon, and that the expression of the operon is negatively regulated by its own products . We then conducted a gel retardation assay in vitro and found that the two pem products, each of which was obtained as a tripartite protein (PemI-collagen-LacZ and PemK-collagen-LacZ), bound cooperatively to a specific fragment containing the proximal region of the pem operon . The binding region, determined by DNase I footprinting analysis, included the promoter for the pem operon . This indicates that both PemI and PemK proteins bind to the promoter region to autoregulate their synthesis.

Mol Gen Genet, 1993 Feb, 237(1-2), 129 - 33
DNA specificity of Escherichia coli deoP1 operator-DeoR repressor recognition; Hammer K et al.; We have studied the importance of the specific DNA sequence of the deo operator site for DeoR repressor binding by introducing symmetrical, single basepair substitutions at all positions in the deo operator and tested the ability of these variants to titrate DeoR in vivo . Our results show that a 16 bp palindromic sequence constitutes the deo operator . Positions outside this palindrome (positions +/- 9, +/- 10) can be changed without any major effect on DeoR binding . Most of the central 6-8 bp of the palindrome (positions +/- 1, +/- 2, +/- 3) can be substituted with other nucleotides with no or only minor effects on DeoR binding, while changes at position +/- 4 and +/- 5 give a more heterogeneous response . Finally, changes at positions +/- 6, +/- 7 and +/- 8 severely disrupt DeoR binding.

Mol Gen Genet, 1993 Feb, 237(1-2), 113 - 22
Function of the Escherichia coli nucleoid protein, H-NS: molecular analysis of a subset of proteins whose expression is enhanced in a hns deletion mutant; Yoshida T et al.; The expression of numerous Escherichia coli cellular proteins was previously demonstrated to be greatly enhanced in a hns deletion background, relative to the levels in wild-type cells . In this study, a subset of such proteins, expression of which is affected by H-NS, was partially purified, and the genes coding for some of the proteins were identified and characterized . Two of the proteins thus characterized, 19K and 17K, were found to be encoded by previously predicted genes that are located adjacent to, and downstream of, the trpABCDE operon (27.6 min on the E . coli genetic map) . The genes coding for the other two proteins, 10K-L and 10K-S, are located at 77.5 min on the genetic map . Their nucleotide sequences were determined and revealed that they may constitute an operon . To characterize the putative promoters for these genes, a set of promoter-lacZ transcriptional fusion genes was constructed on the E . coli chromosome . The results of such promoter-probe analyses indicated that H-NS represses the expression of these genes at the transcriptional level . Furthermore, H-NS appeared to exhibit relatively strong affinity for the putative promoter sequences in vitro . These results are compatible with the hypothesis that H-NS functions as a transcriptional repressor.

Curr Opin Genet Dev, 1993 Feb, 3(1), 91 - 6
Geminiviruses: plant viral vectors; Stanley J; Geminiviruses are being used as convenient autonomously replicating vectors for foreign gene amplification in plants . Using tissue culture techniques, they have been adapted for the analysis of the regulation of gene expression in a wide range of hosts, including both mono- and dicotyledonous species . In monocotyledonous plants that are particularly recalcitrant to transformation, geminivirus symptom-induction has been used as a sensitive marker for DNA uptake.

Anal Biochem, 1993 Feb 1, 208(2), 300 - 5
Bioluminescent immunoassay with a protein A-luciferase fusion protein; Kobatake E et al.; Protein A and firefly luciferase were genetically fused and the resulting fusion protein was applied to a bioluminescent immunoassay . The gene fusion plasmid, pMALU2, was constructed by inserting the structural gene of luciferase into a protein A expression vector, and was expressed in Escherichia coli . The resulting fusion protein of molecular weight 91 kDa retained not only the enzymatic activity of luciferase but also the binding capability of protein A to the Fc region of immunoglobulin G (IgG) . The bioluminescent immunoassay was performed with the fusion protein and human IgG was determined in the concentration range from 10(-3) to 10(-7) g/ml.

Vet Microbiol, 1993 Feb, 34(2), 123 - 30
Characteristics of Escherichia coli isolated from septic foals; Hirsh DC et al.; Fifteen Escherichia coli isolates from the blood and tissue of foals with septicemia were compared with 15 from the feces of clinically normal horses . Comparisons were made with respect to survival in normal equine serum, production of aerobactin, and production of hemolysin . Isolates from the blood and tissues of septic foals were more likely to be resistant to equine serum than were isolates from feces of clinically normal horses . There were minimal differences between the isolates with respect to aerobactin and hemolysin production, almost all being nonhemolytic and aerobactin negative . Serum resistance is probably a virulence determinant for invasive E . coli in horses.

Photochem Photobiol, 1993 Feb, 57(2), 352 - 5
Gene expression of the B875 light-harvesting prepolypeptides from Rhodospirillum rubrum in Escherichia coli; Ghosh R et al.; The gene coding for the prepolypeptides of alpha and beta, obtained as a 429 bp fragment from chromosomal DNA of Rhodospirillum rubrum S1 by polymerase chain reaction amplification, were cloned in tandem into the high-level expression vector pOTSNco 12 for expression in Escherichia coli . The vector pOTSNco12 is a derivative of the pAS vector system, which contains the strong lambda PL promotor and is under tight control by the cI857 repressor encoded by the expression strain AR58 . Induction of transcription from the lambda PL promotor is achieved by shifting the growth temperature from 32 to 42 degrees C . Expression of the gene products was monitored by sodium dodecylsulfate polyacrylamide gel electrophoresis and western blotting . The expressed B875 light-harvesting prepolypeptides were located in the E . coli inner membrane and could not be removed by washing with high salt . The amount of expressed B875 light-harvesting prepolypeptides was estimated to be about 0.1% of the total soluble protein.

Genomics, 1993 Feb, 15(2), 418 - 20
Two members of the S-lac lectin gene family, LGALS1 and LGALS2, reside in close proximity on human chromosome 22q12-q13; Mehrabian M et al.; S-lac lectins are a family of soluble lactose-binding proteins thought to function in the control of cell growth . We now report the chromosomal mapping of two members of the family, termed L-14-I and L-14-II, to the q12-q13 region of human chromosome 22, suggesting the possibility of a cluster of genes for lactose-binding proteins.

Clin Nephrol, 1993 Feb, 39(2), 75 - 80
In vivo exposure to the currently available peritoneal dialysis fluids decreases the function of peritoneal macrophages in CAPD; de Fijter CW et al.; Previous in vitro studies have revealed that the currently available peritoneal dialysis fluids (PDF) inhibit several functions of phagocytic cells . To investigate the clinical relevance of those in vitro findings, we compared the in vivo effect of PDF pH on peritoneal macrophage (PMO) function in chronic peritoneal dialysis patients . In a randomized crossover setting, each of eight patients used exclusively PDF at pH five (D5) or pH seven (D7) on day one . The next day the patients who used D5 were switched to D7 and vice versa . Likewise the effect of glucose-mediated hypertonicity was studied in eight other patients, using PDF with 1.36% glucose (D136) or 3.86% glucose (D386) . PMO were isolated from the effluents and studied for their phagocytic and killing capacity, and their ability to mount a respiratory burst (chemiluminescence response) . PMO obtained after the intraperitoneal instillation of D7 were significantly better able to phagocytize both S . epidermidis (65 +/- 9 vs 37 +/- 8% uptake, p < 0.005) and E . coli (43 +/- 8 vs 25 +/- 4% uptake, p < 0.005) . In addition, PMO harvested from D7 effluents revealed a significantly higher killing capacity than PMO derived from D5 effluents for S . epidermidis (60 +/- 5 vs 38 +/- 6%, p < 0.005) as well as for E . coli (51 +/- 10 vs 25 +/- 9%, p < 0.025) . Moreover, PMO derived from D7 effluents mounted a significantly higher respiratory burst as compared to PMO in vivo exposed to D5 for the same time.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucl Med Biol, 1993 Feb, 20(2), 225 - 30
Abscess scintigraphy with 99mTc-human immunoglobulin (IgG) using a one-step labeling method; Oster ZH et al.; It was shown earlier that non-specific human gamma globulin (IgG) labeled with 111In can be used as an agent for abscess localization . We describe experimental results with 99mTc-IgG in animals bearing abscesses and tumors using a one-step labeling method with 99mTc . We studied this compound in several animal models: mice bearing turpentine abscesses and subcutaneously transplanted sarcomas, in rats with turpentine or E . coli abscesses and intracerebrally implanted gliomas and in rabbits with E . coli or turpentine abscesses . Blood clearance was studied in dogs . It was found that the absolute concentration of 111In-IgG in abscess and tumor was higher than that of 99mTc-IgG . However, the abscess-to-tumor ratio was higher for 99mTc-IgG . The 99mTc-IgG images were of high quality and abscesses could be detected as early as 30 min post-injection (p.i.) . It appears that 99mTc-IgG has many potential advantages over 111In-IgG because of better physical properties of 99mTc, simpler preparation, lower cost and greater availability and the possibility of using higher 99mTc doses.

Am J Physiol, 1993 Feb, 264(2 Pt 2), R456 - 9
Cortical spreading depression blocks prostaglandin E1 and endotoxin fever in rats; Monda M et al.; We have tested the hypothesis that the cortex may play a role in the development of fever . Male Sprague-Dawley rats equipped with AM transmitters for telemetric measurement of body temperature were given intracerebroventricular prostaglandin E1 (PGE1), corticotropin-releasing hormone (CRH), or intravenous E . coli endotoxin . Application of cotton pellets soaked with 3.3 M KCl to the frontal cortex (to induce spreading depression) significantly reduced fevers to PGE1 and endotoxin when compared with fever magnitude with 0.9% NaCl application to the cortex . Neither CRH-induced hyperthermia nor normal body temperatures were altered by the spreading depression . Our results reveal a novel action of spreading depression on thermoregulatory function and indicate cortical involvement in the development of fever.

Am J Physiol, 1993 Feb, 264(2 Pt 2), H357 - 63
Myocardial responses to isoproterenol are altered by chronic alcoholism and infection; McDonough KH et al.; Alcohol, consumed as 36% of the caloric intake for 8-10 wk, causes a potentiation of cardiac dysfunction induced by a second insult, sepsis . Because chronic alcoholism may attenuate the responsiveness of the myocardium to catecholamine stimulation, and because catecholamine support seems to be essential for the myocardium to generate an adequate cardiac output in sepsis, we hypothesized that the heart from the alcoholic septic rat would show a compromised inotropic responsiveness to catecholamines compared with the heart from the nonalcoholic septic rat . To test this hypothesis, rats were fed an ethanol-containing or control liquid diet for 8-10 wk and were then made septic with live Escherichia coli (10(10) E . coli) through a dorsal subcutaneous catheter . The next day, hearts were removed and perfused at a constant hydrostatic pressure, and a compliant balloon was placed in the left ventricule for measurement of pressure (LVP) . Hearts were paced at 350-360 beats/min . Hearts were allowed to stabilize for 15 min, and then the response to a submaximal dose of isoproterenol (Iso) was measured . Hearts recovered for 30 min, at which time the response to a maximum dose of Iso was recorded . Basal (pre-Iso) LVP was lower in the control septic and alcoholic septic groups than in the control and alcohol groups . However, the maximum increase in LVP in response to Iso was greater in the two septic groups than in the two nonseptic groups . The peak LVP in response to Iso was similar in the control, septic, and alcoholic septic groups, and was significantly greater than in the alcohol group.(ABSTRACT TRUNCATED AT 250 WORDS)

Prostaglandins Leukot Essent Fatty Acids, 1993 Feb, 48(2), 193 - 200
Protective effect of diclofenac sodium against endotoxic shock in anaesthetized pigs; Mozes T et al.; The effect of diclofenac sodium was investigated on haemodynamics, haematologic and blood glucose values as well as the release of eicosanoids, tumor necrosis factor (TNF) and platelet activating factor (PAF) in anaesthetized pigs receiving 5 micrograms.kg-1 Escherichia coli lipopolysaccharide (LPS) over 60 min into the superior mesenteric artery . The animals were observed for an additional period of 2 h after the termination of LPS infusion . 15 of the 31 animals infused with LPS and not treated with diclofenac sodium died within 30 min after the commencement of LPS infusion (non-survivors), while the other 16 survived the experimental period of 3-h, though in a shock state (survivors) . No alterations were observed in plasma concentrations of PAF or eicosanoids (TXB2, 6-keto PGF1 alpha and LTB4), but a marked increase was detected in TNF release in the non-survivors . A significant, though transient, increase in concentrations of PAF, TNF and eicosanoids studied characterized the survivors . Another group of 7 LPS-infused pigs was treated with diclofenac sodium (2 mg, kg-1, i.v . bolus 60 min before the start of LPS infusion, followed by a continuous infusion of 1 mg kg-1 h-1) 1 mg/kg-1/h-1 . This treatment prevented death and shock despite the high concentrations of TNF and PAF . Concentrations of both cyclooxygenase and 5-lipoxygenase enzymes products were reduced . These data indicated that the beneficial effect of diclofenac sodium in LPS induced shock may be related to the reduced production of eicosanoids.

J Leukoc Biol, 1993 Feb, 53(2), 126 - 32
Characterization of interleukin-1 and interleukin-6 production by hepatic endothelial cells and macrophages; Feder LS et al.; Interleukin-1 (IL-1) and interleukin-6 (IL-6) derived from Kupffer cells are major inducers of hepatic inflammation and the acute phase response . The present studies demonstrate that liver endothelial cells also produce significant quantities of IL-1 and IL-6, suggesting that these cells also participate in these processes . Endothelial cells and macrophages were isolated from female Sprague-Dawley rats by combined collagenase and pronase perfusion of the liver followed by centrifugal elutriation . In the absence of stimulation, endothelial cells were found to spontaneously produce IL-1 and IL-6 in a time-dependent manner, reaching maximal levels after 10 h in culture for IL-1 and 6-8 h for IL-6 . The amount and kinetics of cytokine production by hepatic endothelial cells were similar to those observed with Kupffer cells . In further studies, the effects of lipopolysaccharide (LPS), a potent liver macrophage activator and inflammatory agent, on cytokine release were analyzed . Treatment of rats with LPS resulted in a decrease in IL-1 release by both cell types compared to cells from untreated animals . In contrast, LPS treatment had no major effect on IL-6 release . We also found that both macrophages and endothelial cells could be induced to produce additional IL-1 and IL-6 by treatment with LPS in vitro, but only if they were preincubated for at least 24 h prior to stimulation with LPS and analyzed for cytokine release . These data demonstrate that liver endothelial cells, like Kupffer cells, have the capacity to produce immunoregulatory and proinflammatory cytokines.

J Dairy Sci, 1993 Feb, 76(2), 414 - 20
Endotoxemia in dairy cattle: role of eicosanoids in reticulorumen stasis; Eades SC; The role of arachidonic acid metabolites in the forestomach stasis induced by Escherichia coli endotoxin was evaluated . Six adult Holstein cows received saline solution; endotoxin at 1, 10, and 100 ng/kg of body weight; flunixin meglumine at 1.1 mg/kg of body weight; and flunixin meglumine at 1.1 mg/kg plus endotoxin at 100 ng/kg . The frequency of reticulorumen contractions, mental attitude, body temperature, respiratory rate, heart rate, and plasma concentration of prostaglandin E2, prostacyclin, and thromboxane were evaluated . Administration of saline solution and endotoxin at 1 ng/kg had no significant effects . Administration of endotoxin at 10 ng/kg did not cause significant clinical effects or alter reticulorumen contractions but enhanced synthesis of thromboxane . Administration of endotoxin at 100 ng/kg caused mild clinical signs of stasis, reduced the frequency of reticulorumen contractions, and enhanced synthesis of thromboxane and prostacyclin . Reticulorumen stasis was not accompanied by an increase in the plasma concentration of prostaglandin E2 . Flunixin meglumine abolished endotoxin-induced reticulorumen stasis, tachycardia, and synthesis of arachidonic acid metabolites . Reticulorumen stasis during bovine endotoxemia is caused either by enhanced synthesis of an arachidonic acid metabolite other than prostaglandin E2 or by local synthesis of prostaglandin E2.

J Dairy Sci, 1993 Feb, 76(2), 408 - 13
Serum concentrations of copper, iron, and zinc during Escherichia coli-induced mastitis; Erskine RJ et al.; Six Holstein cows were intracisternally challenged with 50 cfu of Escherichia coli to induce acute mastitis . Clinical status, milk concentrations of bacteria, and serum albumin concentration were determined to monitor the progress and severity of infection for 72 h after bacterial challenge . Blood samples were also collected throughout infection to determine serum concentrations of Zn, Fe, and Cu . Experimental E . coli mastitis resulted in mean serum concentrations of Zn, Fe, and Cu of 28, 35, and 52% of prechallenge concentrations . These decreases first occurred 4 to 12 h after peak bacterial concentration in milk . Changes in serum trace elements may occur too late in the pathogenesis of infection to decrease peak bacterial numbers in milk . However, mediation of infection and inflammation may occur in later stages of the infection process.

J Dairy Sci, 1993 Feb, 76(2), 401 - 7
Vitamin E as an adjuvant in an Escherichia coli J5 vaccine; Hogan JS et al.; Vitamin E was tested as an adjuvant in an Escherichia coli (O111:B4) J5 vaccine . Twenty cows were assigned to five groups of 4 cows . Cows in four groups were vaccinated with an E . coli J5 bacterin containing 5 ml of 10(9) boiled cells/ml . Vaccinations were at drying off, 30 d after drying off, and within 48 h after calving . Vaccine adjuvants differed among groups . The four treatment adjuvants were 5 ml of Freund's incomplete adjuvant, 5 ml of vitamin E, 2.5 ml of Freund's plus 2.5 ml of vitamin E, and 5 ml of PBS . Cows in the fifth group were unimmunized controls . A front mammary quarter of each cow was challenged by infusion of 10 micrograms of E . coli J5 lipopolysaccharide approximately 4 wk into lactation . Vitamin E alone enhanced serum IgM titers but had no effect on milk IgM or serum and milk IgG titers . The mixture of Freund's plus vitamin E resulted in peak IgG titers in serum and milk comparable with that of Freund's alone . Persistency of IgG titers in cows immunized with the Freund's plus vitamin E mixture was greater than the persistency of titers for cows immunized with the vaccine containing Freund's alone as the adjuvant . The mixture of Freund's plus vitamin E had a synergistic effect in reducing severity of systemic clinical signs.

Int J Biochem, 1993 Feb, 25(2), 201 - 7
Construction of cDNA libraries from cultures of Armillaria mellea; O'Connell J et al.; 1 . Conditions were established for growth of mycelial cultures of Armillaria mellea such that the production of its lysine-specific proteinase was maximized . Proteinase synthesis was confirmed by immunoprecipitation . 2 . Mycelia grown under these same conditions were used as a source of RNA and this RNA was translatable in a wheat germ translation system to produce proteins with M(r) in the range < 10,000- > 90,000 . 3 . Double-stranded cDNA was prepared and was inserted into the EcoR1 site of lambda gt10 and lambda gt11 using an adaptor ligation strategy . Packaging of these materials yielded large cDNA libraries . The form lambda gt10 contained 2.9 x 10(6) pfu/ml with 70% recombinants whereas that from lambda gt11 contained 2.2 x 10(6) pfu/ml with 60% recombinants.

Protein Sci, 1993 Feb, 2(2), 264 - 76
Exploration of subsite binding specificity of human cathepsin D through kinetics and rule-based molecular modeling; Scarborough PE et al.; The family of aspartic proteinases includes several human enzymes that may play roles in both physiological and pathophysiological processes . The human lysosomal aspartic proteinase cathepsin D is thought to function in the normal degradation of intracellular and endocytosed proteins but has also emerged as a prognostic indicator of breast tumor invasiveness . Presented here are results from a continuing effort to elucidate the factors that contribute to specificity of ligand binding at individual subsites within the cathepsin D active site . The synthetic peptide Lys-Pro-Ile-Glu-Phe*Nph-Arg-Leu has proven to be an excellent chromogenic substrate for cathepsin D yielding a value of kcat/Km = 0.92 x 10(-6) s-1 M-1 for enzyme isolated from human placenta . In contrast, the peptide Lys-Pro-Ala-Lys-Phe*Nph-Arg-Leu and all derivatives with Ala-Lys in the P3-P2 positions are either not cleaved at all or cleaved with extremely poor efficiency . To explore the binding requirements of the S3 and S2 subsites of cathepsin D, a series of synthetic peptides was prepared with systematic replacements at the P2 position fixing either Ile or Ala in P3 . Kinetic parameters were determined using both human placenta cathepsin D and recombinant human fibroblast cathepsin D expressed in Escherichia coli . A rule-based structural model of human cathepsin D, constructed on the basis of known three-dimensional structures of other aspartic proteinases, was utilized in an effort to rationalize the observed substrate selectivity.

Protein Sci, 1993 Feb, 2(2), 244 - 54
A study of intermediates involved in the folding pathway for recombinant human macrophage colony-stimulating factor (M-CSF): evidence for two distinct folding pathways; Wilkins JA et al.; The folding pathway for a 150-amino acid recombinant form of the dimeric cytokine human macrophage colony-stimulating factor (M-CSF) has been studied . All 14 cysteine residues in the biologically active homodimer are involved in disulfide linkages . The structural characteristics of folding intermediates blocked with iodoacetamide reveal a rapid formation of a small amount of a non-native dimeric intermediate species followed by a slow progression via both monomeric and dimeric intermediates to the native dimer . The transition from monomer to fully folded dimer is complete within 25 h at room temperature at pH 9.0 . The blocked intermediates are stable under conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and thus represent various dimeric and folded monomeric species of the protein with different numbers of disulfide bridges . Peptide mapping and electrospray ionization mass spectrometry revealed that a folded monomeric species of M-CSF contained three of the four native disulfide bridges, and this folded monomer also showed some biological activity in a cell-based assay . The results presented here strongly suggest that M-CSF can fold via two different pathways, one involving monomeric intermediates and another involving only dimeric intermediates.

Protein Sci, 1993 Feb, 2(2), 223 - 30
Identification of a nucleic acid-binding region within the largest subunit of Drosophila melanogaster RNA polymerase II; Kontermann RE et al.; The largest and the second-largest subunit of the multisubunit eukaryotic RNA polymerases are involved in interaction with the DNA template and the nascent RNA chain . Using Southwestern DNA-binding techniques and nitrocellulose filter binding assays of bacterially expressed fusion proteins, we have identified a region of the largest, 215-kDa, subunit of Drosophila RNA polymerase II that has the potential to bind nucleic acids nonspecifically . This nucleic acid-binding region is located between amino acid residues 309-384 and is highly conserved within the largest subunits of eukaryotic and bacterial RNA polymerases . A homology to a region of the DNA-binding cleft of Escherichia coli DNA polymerase I involved in binding of the newly synthesized DNA duplex provides indirect evidence that the nucleic acid-binding region of the largest subunit participates in interaction with double-stranded nucleic acids during transcription . The nonspecific DNA-binding behavior of the region is similar to that observed for the native enzyme in nitrocellulose filter binding assays and that of the separated largest subunit in Southwestern assays . A high content of basic amino acid residues is consistent with the electrostatic nature of nonspecific DNA binding by RNA polymerases.

PCR Methods Appl, 1993 Feb, 2(3), 210 - 7
Mutagenic oligonucleotide-directed PCR amplification (Mod-PCR): an efficient method for generating random base substitution mutations in a DNA sequence element; Chiang LW et al.; Saturation mutagenesis is one approach for determining the contributions of individual base pairs to the structure and function of defined DNA sequence elements . In this paper, we describe a novel method for saturation mutagenesis involving PCR amplification with degenerate synthetic oligonucleotides as primers . The degeneracy is confined to a specific target within the primer by mixing a low percentage of the three non-wild type (non-WT) nucleotide precursors with WT at specific positions during primer synthesis . PCR amplification of WT template DNA with the degenerate primer and an opposing WT primer, followed by subsequent cloning using restriction sites designed into the primers, results in recovery of a population of randomly mutated products . Since primers with multiple mutations hybridize less efficiently to WT template DNA during PCR amplification, the recovery of mutants with multiple base changes is greatly reduced . The efficient generation of random point mutations with this method allows the construction of separate mutant populations, each mutagenized over a different portion of the DNA sequence element . If a phenotypic assay is available, these populations can be screened directly to define those regions within the element that are important for activity . Only those populations containing mutations in the important regions require further characterization by DNA sequence analysis.

Plant Mol Biol, 1993 Feb, 21(3), 503 - 13
Sigma-like transcription factors from mustard (Sinapis alba L.) etioplast are similar in size to, but functionally distinct from, their chloroplast counterparts; Tiller K et al.; Three proteins resembling bacterial sigma factors were previously isolated from mustard chloroplasts (K . Tiller, A . Eisermann and G . Link, Eur J Biochem 198: 93-99, 1991) . These sigma-like factors (SLFs) confer DNA-binding and transcription specificity to a system consisting of Escherichia coli core RNA polymerase and cloned DNA regions that carry a chloroplast promoter . Sigma-like activity was now isolated also from etioplasts and could be assigned to three polypeptides of M(r) 67,000 (SLF67), 52,000 (SLF52) and 29,000 (SLF29), i.e . the same sizes as for the chloroplast SLFs . The purification scheme for the factors from either plastid type included an initial heparin-Sepharose and a final gel filtration step . For the etioplast factors, however, an additional phosphocellulose step was required to release these polypeptides from the RNA polymerase . The etioplast SLFs have similar, but not identical, salt requirements for DNA binding as compared to their chloroplast counterparts . Under conditions of maximum binding activity there is overall preference of etioplast SLFs for the psbA promoter over the trnQ and rps16 promoters.

Proteins, 1993 Feb, 15(2), 147 - 76
Crystal structure of CTP-ligated T state aspartate transcarbamoylase at 2.5 A resolution: implications for ATCase mutants and the mechanism of negative cooperativity; Kosman RP et al.; The X-ray crystal structure of CTP-ligated T state aspartate transcarbamoylase has been refined to an R factor of 0.182 at 2.5 A resolution using the computer program X-PLOR . The structure contains 81 sites for solvent and has rms deviations from ideality in bond lengths and bond angles of 0.018 A and 3.722 degrees, respectively . The cytosine base of CTP interacts with the main chain carbonyl oxygens of rTyr-89 and rIle-12, the main chain NH of rIle-12, and the amino group of rLys-60 . The ribose hydroxyls form polar contacts with the amino group of rLys-60, a carboxylate oxygen of rAsp-19, and the main chain carbonyl oxygen of rVal-9 . The phosphate oxygens of CTP interact with the amino group of rLys-94, the hydroxyl of rThr-82, and an imidazole nitrogen of rHis-20 . Recent mutagenesis experiments evaluated in parallel with the structure reported here indicate that alterations in the hydrogen bonding environment of the side chain of rAsn-111 may be responsible for the homotropic behavior of the pAR5 mutant of ATCase . The location of the first seven residues of the regulatory chain has been identified for the first time in a refined ATCase crystal structure, and the proximity of this portion of the regulatory chain to the allosteric site suggests a potential role for these residues in nucleotide binding to the enzyme . Finally, a series of amino acid side chain rearrangements leading from the R1 CTP allosteric to the R6 CTP allosteric site has been identified which may constitute the molecular mechanism of distinct CTP binding sites on ATCase.

Lipids, 1993 Feb, 28(2), 81 - 8
Overexpression of a Rhizopus delemar lipase gene in Escherichia coli; Joerger RD et al.; A cloned complementary deoxyribonucleic acid encoding the precursor polypeptide of an extracellular lipase from the fungus Rhizopus delemar was altered by site-directed mutagenesis to generate deoxyribonucleic acid fragments that specifically code for the polypeptides of the proenzyme and the mature form of the lipase . Attempts to produce these polypeptides in enzymatically active form in Escherichia coli revealed toxic effects toward the host . Therefore the polypeptides were expressed as inactive and insoluble forms in the cytoplasm of E . coli BL21 (DE3) cells using plasmid vector pET11-d . With this tightly regulated high-level expression system, lipase and prolipase polypeptides were produced to estimated levels of up to 21% and 15%, respectively, of total cellular protein . The insoluble polypeptides were solubilized in 8 M urea . Refolding into active forms was achieved by treatment with the redox system cystine/cysteine and dilution . Refolded mature lipase was purified to homogeneity by affinity and ion exchange chromatography . The enzyme had a specific activity comparable to that of lipase from the fungal culture . The quantities of pure enzyme obtained from a 1-L culture of E . coli exceeded those obtained from the fungal culture by a factor of at least 100 . Refolded recombinant prolipase was purified essentially to homogeneity and had a specific activity similar to that of the mature enzyme . Its pH optimum was 7.5, rather than the pH 8 determined for recombinant mature lipase and for the enzyme purified from the fungal culture . Recombinant prolipase retained activity after 15 min incubation at 65 degrees C, while mature lipase retained activity only up to 45 degrees C.

FASEB J, 1993 Feb 1, 7(2), 290 - 8
Aminopeptidases: structure and function; Taylor A; Aminopeptidases catalyze the cleavage of amino acids from the amino terminus of protein or peptide substrates . They are widely distributed throughout the animal and plant kingdoms and are found in many subcellular organelles, in cytoplasm, and as membrane components . Several aminopeptidases perform essential cellular functions . Many, but not all, of these peptidases are zinc metalloenzymes and are inhibited by the transition-state analog bestatin . Some are monomeric, and others are assemblies of relatively high mass (50 kDa) subunits . cDNA sequences are available for several aminopeptidases, and a 3-dimensional structure is available for the bovine lens enzyme . Crystallographic, electron micrographic, NMR, and photoaffinity labeling studies indicate that lens leucine aminopeptidase protomers are bilobal and that bestatin and substrates are bound in an active site, which is found in the larger lobe on each protomer . Zn2+ is involved in substrate liganding in most aminopeptidases . There is no evidence of an acyl-enzyme intermediate in hydrolysis . Amino acid sequences determined directly or deduced from cDNAs indicate some amino acid sequence homologies in organisms as diverse as Escherichia coli and mammals, particularly in catalytically important residues or in residues involved in metal ion binding.

EMBO J, 1993 Feb, 12(2), 677 - 82
Rab9 functions in transport between late endosomes and the trans Golgi network; Lombardi D et al.; Rab proteins represent a large family of ras-like GTPases that regulate distinct vesicular transport events at the level of membrane targeting and/or fusion . We report here the primary sequence, subcellular localization and functional activity of a new member of the rab protein family, rab9 . The majority of rab9 appears to be located on the surface of late endosomes . Rab9, purified from Escherichia coli strains expressing this protein, could be prenylated in vitro in the presence of cytosolic proteins and geranylgeranyl diphosphate . In vitro-prenylated rab9 protein, but not C-terminally truncated rab9, stimulated the transport of mannose 6-phosphate receptors from late endosomes to the trans Golgi network in a cell-free system that reconstitutes this transport step . Rab7, a related rab protein that is also localized to late endosomes, was inactive in the in vitro transport assay, despite its efficient prenylation and capacity to bind and hydrolyze GTP . These results strongly suggest that rab9 functions in the transport of mannose 6-phosphate receptors between late endosomes and the trans Golgi network . Moreover, our results confirm the observation that a given organelle may bear multiple rab proteins with different biological functions.

EMBO J, 1993 Feb, 12(2), 625 - 30
Regulation of the Escherichia coli rmf gene encoding the ribosome modulation factor: growth phase- and growth rate-dependent control; Yamagishi M et al.; Ribosome modulation factor (RMF) is a protein specifically associated with 100S ribosome dimers which start to accumulate in Escherichia coli cells upon growth transition from exponential to stationary phase . The structural gene, rmf, encoding the 55 amino acid residues RMF protein has been cloned from the 21.8 min region of the E . coli genome and sequenced . While rmf was silent in rapidly growing exponential phase cells, a high level of transcription took place concomitantly with the growth transition to stationary phase . Under slow growth conditions, rmf was expressed even in exponential phase and there was an inverse relationship between the expression of rmf and the cell growth rate . Thus, the expression profile of rmf is contrary to those of genes for ribosomal components and ribosome-associated proteins constituting the translational apparatus . The katF gene product, a stationary phase-specific sigma factor, was not required for the expression of rmf . Disruption of rmf resulted in loss of ribosome dimers and reduction of cell viability during stationary phase.

EMBO J, 1993 Feb, 12(2), 617 - 23
Topography of the E site on the Escherichia coli ribosome; Wower J et al.; Three photoreactive tRNA probes have been utilized in order to identify ribosomal components that are in contact with the aminoacyl acceptor end and the anticodon loop of tRNA bound to the E site of Escherichia coli ribosomes . Two of the probes were derivatives of E . coli tRNA(Phe) in which adenosines at positions 73 and 76 were replaced by 2-azidoadenosine . The third probe was derived from yeast tRNA(Phe) by substituting wyosine at position 37 with 2-azidoadenosine . Despite the modifications, all of the photoreactive tRNA species were able to bind to the E site of E . coli ribosomes programmed with poly(A) and, upon irradiation, formed covalent adducts with the ribosomal subunits . The tRNA(Phe) probes modified at or near the 3' terminus exclusively labeled protein L33 in the 50S subunit . The tRNA(Phe) derivative containing 2-azidoadenosine within the anticodon loop became cross-linked to protein S11 as well as to a segment of the 16S rRNA encompassing the 3'-terminal 30 nucleotides . We have located the two extremities of the E site-bound tRNA on the ribosomal subunits according to the positions of L33, S11 and the 3' end of 16S rRNA defined by immune electron microscopy . Our results demonstrate conclusively that the E site is topographically distinct from either the P site or the A site, and that it is located alongside the P site as expected for the tRNA exit site.

J Dairy Res, 1993 Feb, 60(1), 19 - 29
Classification of newly calved cows into moderate and severe responders to experimentally induced Escherichia coli mastitis; Vandeputte-Van Messom G et al.; In the present study newly calved cows were tentatively classified as moderate and severe responders to experimentally induced Escherichia coli mastitis based upon the reactive oxygen species (ROS)-generating capacity of their blood neutrophils before infection . The groups differed in blood and milk composition prior to infection . This initial classification was supported by the corresponding variation in clinical symptoms and in the changes in milk production and composition measured during mastitis . Responses of newly calved cows to Esch . coli challenge varied from mild to severe symptoms of inflammation in infected glands and differed in the intensity of systemic disturbances and general illness . Losses in milk yield and compositional changes were most pronounced in inflamed glands and in severe responders . In inflamed glands milk yield and composition did not return to preinfection level in either moderate or severe responders . The yields of lactose, alpha-lactalbumin, casein and fat followed the same pattern as milk yield . It is concluded that the severe and long lasting systemic disturbances observed in severe responders can be ascribed to absorption of endotoxin from infected glands into circulation, indicating the important role of endotoxin in the pathology of coliform mastitis in periparturient cows . Evaluation of the ROS-generating capacity of blood neutrophils and blood and milk composition before infection might help to predict the cow's sensitivity to Esch . coli mastitis.

Eur J Immunol, 1993 Feb, 23(2), 552 - 7
Involvement of beta 1 integrins in the binding and entry of Trypanosoma cruzi into human macrophages; Fernandez MA et al.; We have investigated the role of integrin molecules in the binding and entry of Trypanosoma cruzi into human macrophages, with the help of monoclonal antibodies (mAb) . Addition of Lia 1/2 mAb and in lesser extent of Lia 1/5 mAb, which are both specific for the beta 1 subunit of the VLA integrin family, to human macrophages blocked T . cruzi uptake and subsequent replication inside the macrophages . This inhibition correlated with their respective ability to block Fibronectin (Fn) binding to macrophages . Furthermore, another anti-beta 1 mAb, Alex 1/4, which binds to a different epitope on the beta 1 molecule and was unable to block Fn binding, did not affect T . cruzi invasion . The inhibition by Lia 1/2 and Lia 1/5 was dose dependent and clearly observable with doses as low as 1 microgram/ml . Moreover, this inhibition was T . cruzi specific since the Lia 1/2 and Lia 1/5 were unable to block uptake of Leishmania pifanoi or Escherichia coli by human macrophages . In contrast, the TS 1/18 mAb, which blocks ligand binding to beta 2 integrin, inhibited entry of L . pifanoi but not of T . cruzi . Finally, mAb specific for the alpha 4 and alpha 5 subunits of the two major Fn binding molecules of macrophages (VLA-4 and VLA-5, respectively), either alone or in combination, were poor inhibitors of T . cruzi uptake, suggesting that several members of the VLA family, including VLA-4 and VLA-5, are involved in binding and entry of T . cruzi into macrophages.

Eur J Biochem, 1993 Feb 1, 211(3), 829 - 34
Cholesterol metabolism in rat adrenal gland during reversible endotoxic shock; Abarca S et al.; The adrenal glands have a crucial role for survival during endotoxic shock . Cholesterol is the obligatory intermediary in corticosteroid biosynthesis; thus any alteration in either the availability of cholesterol or in the ability of the adrenal gland to use cholesterol would have a profound effect on corticosteroid production . We have studied the effect of Escherichia coli endotoxin on cholesterol metabolism, injecting lipopolysaccharide (1.6 mg/100 g body) from E . coli 0111:B4 into the tail vein of male Wistar rat . Previous studies from this laboratory have shown that this dose of lipopolysaccharide induces a reversible endotoxic shock . During reversible endotoxic shock there is an alteration in plasma cholesterol; plasma total-cholesterol levels increase mainly at 6-24 h post-lipopolysaccharide injection, whereas cholesterol in high-density lipoproteins shows no significant variations, except a slight but significant decrease at 24 h . The cholesterol content in adrenal gland is diminished in endotoxemic rat, this decrease is more important at 6-24 h after endotoxin injection . We have also measured the acyl-CoA:cholesterol O-acyltransferase (ACAT) and cholesterol-esterase (CEH) activity during endotoxic shock . ACAT activity decreases after lipopolysaccharide injection . ACAT activity in endotoxemic rats is approximately 35-40% of the activity in control rats . This decrease is due to a defect in the functional capacity of the enzyme, since with exogenous cholesterol there is no significant variation in the ACAT activity . CEH activity, in contrast, increases during endotoxic shock; it shows a maximum (twofold the activity seen in control rats) at 6 h after lipopolysaccharide injection . These results show that lipopolysaccharide injection modifies cholesterol metabolism in plasma and in the adrenal gland, either directly or by mediators.

Eur J Biochem, 1993 Feb 1, 211(3), 749 - 55
Involvement of the Arg179 in the active site of human IL-6; Fontaine V et al.; Three internal-amino acid deletions of amino acids 171-179 of human interleukin 6 (IL-6) were introduced at the cDNA level . While all deletion proteins were biologically inactive, immunoprecipitations with a set of conformation-specific anti-(IL-6) monoclonal antibodies showed that only mutant delta 177-179 does not present major alterations in folding . This finding, together with the observation that delta 177-179 is not able to compete with IL-6 for binding to the soluble human IL-6 receptor, suggested that some or all of these three residues participate to the composition of the receptor-binding site of human IL-6 . A large number of single-amino-acid-substitution mutants were generated in residues 177, 178 and 179 . Their detailed analysis revealed that Arg179 is crucial for activity in mouse cells, because all amino acid substitutions in this position cause a dramatic drop of biological activity on murine hybridoma cells without affecting the overall protein folding . The only substitution which preserved some residual activity was the conservative Arg to Lys change . This demonstrates the absolute requirement for a positive charge in position 179 for the interaction of human IL-6 with its receptor.

Eur J Biochem, 1993 Feb 1, 211(3), 731 - 42
Localization of the tightly bound divalent-cation-dependent and nucleotide-dependent conformation changes in G-actin using limited proteolytic digestion; Strzelecka-Golaszewska H et al.; Using proteolytic susceptibility as a probe, we have identified four regions of the actin polypeptide chain where structural rearrangements, dependent on the nature of the tightly bound metal ion and/or nucleotide, take place . Replacement of the tightly bound Ca2+ by Mg2+ in ATP-actin strongly affected the regions around Arg26 and Lys68, as judged from nearly complete inhibition of tryptic cleavages of the polypeptide chain at these residues . It also significantly diminished the rates of splitting by trypsin of the peptide bonds involving carbonyl groups of Arg372 and of Lys373 in the C-terminal segment . Conversion of ATP-actin to ADP-actin (with Mg2+ as the tightly bound cation) abolished the protective effect of Mg2+ on specific tryptic cleavage and, in contrast, largely inhibited proteolysis at specific sites for subtilisin and for a novel protease from Escherichia coli A2 strain within a surface loop of residues 39-51 . We also examined the effect of proteolytic cleavage or chemical modification at certain sites on the kinetics of proteolysis at other sites of the molecule . These experiments demonstrated structural relationships between loop 39-51 and regions involving Lys61 and Lys68 . It is suggested that the conformational transitions reflected in the observed changes in proteolytic susceptibility may underlie the known influence of the nature of the tightly bound cation and nucleotide on the kinetics of actin polymerization and stability of the polymer.

Eur J Biochem, 1993 Feb 1, 211(3), 703 - 10
Overexpression of trypanosomal triosephosphate isomerase in Escherichia coli and characterisation of a dimer-interface mutant; Borchert TV et al.; In this paper, the successful expression of trypanosomal triosephosphate isomerase (TIM) from Trypanosoma brucei brucei to high yield in Escherichia coli, using a T7-polymerase-based expression system, is described . Overexpressed trypanosomal TIM is fully active . The measured physicochemical properties of this recombinant TIM and TIM purified from trypanosomes are indistinguishable . Crystals of recombinant TIM have been grown in the presence of 2.4 M ammonium sulphate under the same conditions as for trypanosomally expressed TIM . The recombinant TIM crystal structure has been refined at 0.23 nm resolution; no differences were detected between this structure and the original crystal structure . A TIM mutant was made in which a unique dimer-interface histidine residue (His47) was changed into an asparagine . This variant ({H47N}TIM) could be expressed and purified to homogeneity by a procedure which was somewhat different from the purification of recombinant wild-type TIM . It is shown that the {H47N}TIM dimer is considerably less stable than wild-type trypanosomal TIM . The catalytic activity of {H47N}TIM is concentration dependent . The dilution-dependent inactivation is reversible . His47 is involved in a water-mediated hydrogen bond with Asp385 of the other subunit . The lower stability of the {H47N}TIM dimer implies that this water-mediated hydrogen bond is important for the stability of the TIM dimer.

Eur J Biochem, 1993 Feb 1, 211(3), 615 - 24
Purification, characterization, crystallisation and X-ray analysis of selenomethionine-labelled hydroxymethylbilane synthase from Escherichia coli; Hadener A et al.; Hydroxymethylbilane synthase (HMBS) catalyses the conversion of porphobilinogen into hydroxymethylbilane, a linear tetrapyrrolic intermediate in the biosynthesis of haems, chlorophylls, vitamin B12 and related macrocycles . In the course of an investigation of the crystal structure of this enzyme, we intended to follow a new strategy to obtain the X-ray phase information, i.e . the collection of multiwavelength anomalous diffraction data from a crystal of a seleno-L-methionine (SeMet)-labelled variant of the protein . We have expressed and purified HMBS from Escherichia coli (34268 Da) in which all (six) methionine (Met) residues are replaced by SeMet . Complete replacement, as shown by amino acid composition analysis and by electrospray mass spectrometry, was achieved by growing the Met-requiring mutant E . coli PO1562 carrying the plasmid pPA410 in a medium containing 50 mg/l SeMet as the sole source of Met . {SeMet}HMBS exhibits full enzyme activity, as reflected by unchanged steady-state kinetic parameters relative to native enzyme . Rhombohedral crystals of {SeMet}HMBS could be grown at the pH optimum (7.4) of the enzyme (solutions containing 30 mg/ml protein, 0.4 mM EDTA, 20 mM dithiothreitol, 3 M NaCl and 15 mM Bristris-propane buffer were equilibrated by vapour diffusion at 20 degrees C against reservoirs of saturated NaCl) . However, being very thin plates, these crystals were not suitable for X-ray analysis . Alternatively, rectangular crystals were obtained at pH 5.3 using conditions based on those reported for wild-type HMBS {sitting drops of 50 microliters containing 6-7 mg/ml protein, 0.3 mM EDTA, 15 mM dithiothreitol, 10% (mass/vol.) poly(ethylene glycol) 6000 and 0.01% NaN3 in 0.1 M sodium acetate were equilibrated by vapour diffusion at 20 degrees C against a reservoir of 10-20 mg solid dithiothreitol} . X-ray diffraction data of the crystals were complete to 93.8% at 0.21 nm resolution and showed that {SeMet}HMBS and native HMBS crystallise isomorphously . A difference Fourier map using FSeMet-Fnative and phases derived from the native structure, which has recently been determined independently by multiple isomorphous replacement, showed positive difference peaks centered at or close to where the sulphur atoms of the Met side chains appear in the native structure . In addition, paired positive/negative peaks in the difference map near the cofactor of HMBS indicate conformational differences in the active site, probably due to differences in the state of oxidation of the cofactor in the two crystalline samples.

Eur J Biochem, 1993 Feb 1, 211(3), 609 - 14
Facile sulfitolysis of the disulfide bonds in oxidized thioredoxin and glutaredoxin; Wurfel M et al.; Thioredoxins and glutaredoxins, in their oxidized form, possess a single disulfide bridge located on an edge of the small compact molecules . In contrast to most other disulfide-containing proteins, this S-S bridge is cleaved by millimolar concentrations of sulfite in the absence of protein denaturing agents at pH 7-8 and ambient temperature; however, the reaction is not quantitative . Sulfitolysis of Escherichia coli thioredoxin was found to be associated with an increase in fluorescence at 345 nm . A comparative study of sulfitolysis in 12 different thioredoxins and glutaredoxins of bacterial and plant origin has been made . Although they are all thought to be highly conserved in three-dimensional structure, their reactivities towards sulfite and the effects of 6 M guanidinium chloride (not affecting, or enhancing sulfitolysis) vary strongly in the series, with E . coli thioredoxin being less reactive and plant thioredoxins and E . coli glutaredoxin being more susceptible molecules . Contrary to expectation, reaction with sulfite is not generally correlated with the presence of negatively or positively charged amino acid residues near the disulfide loop but is determined by individual sequence and surface features in every single protein . These results confirm our hypothesis that thioredoxin sulfitolysis and inactivation {Wurfel, M., Haberlein, I., Follmann, H . (1990) FEBS Lett . 268, 146-148} can occur in plant cells under physiological conditions and provide a biochemical rationale for the phytotoxicity of SO2.

Eur J Biochem, 1993 Feb 1, 211(3), 591 - 9
Structure/function relationships in the pyruvate dehydrogenase complex from Azotobacter vinelandii . Role of the linker region between the binding and catalytic domain of the dihydrolipoyl transacetylase component; Schulze E et al.; The role of the hinge region between the binding domain and the catalytic domain in dihydrolipoyl transacetylase (E2p) from Azotobacter vinelandii was addressed by deletion mutagenesis . Mutated dihydrolipoyl transacetylase proteins were constructed with a deletion of 11 amino acids in the hinge region between the binding domain and the N-terminal part of the catalytic domain of E2p {E2p(pAPE1)} and with a further deletion of 9 amino acids into the N-terminal sequence protruding from the globular structure of the catalytic domain {E2p(pAPE2)} and found to take part in the intratrimer interaction . Both proteins behaved as wild-type E2p with respect to catalytic activity and quaternary structure . The interaction of the peripheral components pyruvate dehydrogenase (E1p) and lipoamide dehydrogenase (E3) with the mutated E2p proteins was studied . E2p(pAPE1) assembles to a trimeric pyruvate dehydrogenase complex (PDC) with 15% decreased complex activity . No difference in affinity towards the peripheral components was detected . Upon binding of E3, E2p(pAPE2) dissociates into trimers and monomers . At saturation, two dimers of E3 were bound/E2p monomer instead of one dimer/E2p chain in trimeric wild-type E2p or E2p (pAPE1) . The monomeric E2p species was catalytically inactive . Upon binding of excess E1p, some monomer formation of the E2p mutant took place . E1p however can prevent monomerization by E3 . It is concluded that E1p is bound between two different E2p chains in the trimer . The substrates CoA and acetyl-CoA also prevent monomerization because they are bound by amino acid residues of two different E2p chains . In the presence of CoA no difference in affinity with respect to E1p and E3 binding was observed . CoA (and acetylCoA) also prevent dissociation of the 24-subunit core structure of wild-type E2p when added before addition of E1p or E3 . Therefore, it seems likely that in vivo A . vinelandii PDC is based on a 24-subunit E2p core, like Escherichia coli PDC . A functional difference between complexes based on a trimer or a 24-subunit core has not been observed . A role of the hinge region as a spacer to allow binding of E1p or E3 seems unlikely . The results are discussed on the basis of the three-dimensional structure of the catalytic domain.

Eur J Biochem, 1993 Feb 1, 211(3), 583 - 90
Interleukin-1 beta-specific partial agonists defined by site-directed mutagenesis studies; Guinet F et al.; Monocyte-derived interleukin 1 (IL-1) mediates a wide range of biological effects including destruction of the cartilage matrix in articular diseases such as rheumatoid and osteoarthritis . To elucidate further the relationships between protein structure and biological activities, we have analyzed the sequence of several IL-1 polypeptides using the algorithm of Parker, the hydrophobic cluster analysis method and published structural data . This led us to identify several residues that seemed to be strictly topologically conserved, with respect to identifiable secondary structures features, although this was not readily apparent from sequence alignments . We performed site-directed mutagenesis on some of these conserved residues, as well as on those predicted to occur in external loops of the polypeptide . Human IL-1 beta mutant polypeptides were expressed in Escherichia coli in soluble form and purified to homogeneity by anion-exchange and gel-filtration chromatography . Their biological effects (binding to EL4-6.1 murine thymocytes, Raji human B cells and rabbit chondrocytes cells, lymphocyte activation, neutral protease induction, proteoglycan degradation and synthesis) have been determined . Among the 20 IL-1 beta mutant polypeptides we present here, four showed a markedly reduced activity in cartilage matrix assays without any significant change in their binding to the cartilage matrix cells (chondrocytes) . Furthermore, some of these mutants were specific partial agonists of the effects of IL-1 on connective tissue since they have a low affinity for thymocytes.

Eur J Biochem, 1993 Feb 1, 211(3), 521 - 7
Use of gene fusions of the structural gene sdaA to purify L-serine deaminase 1 from Escherichia coli K-12; Su H et al.; The purification by affinity chromatography of beta-galactosidase from strains carrying sdaA/lacZ gene fusions results in the copurification of L-serine deaminase 1 . We conclude that sdaA is the structural gene for the latter enzyme . The purified L-serine deaminase 1 obtained after collagenase treatment of an sdaA-collagen-lacZ fusion differs from the native enzyme by the addition of several amino acids at the C-terminal . Like the enzyme in crude extracts, this purified enzyme is catalytically inactive, and is activated by incubation with iron and dithiothreitol.

Eur J Biochem, 1993 Feb 1, 211(3), 501 - 7
Expression of the gamma-subunit gene of desulfoviridin-type dissimilatory sulfite reductase and of the alpha- and beta-subunit genes is not coordinately regulated; Karkhoff-Schweizer RR et al.; It has been shown {Pierik, A . J., Duyvis, M . G., van Helvoort, J . M . L . M., Wolbert, R . B . G . & Hagen, W . R . (1992) Eur . J . Biochem . 205, 111-115} that desulfoviridin, the dissimilatory sulfite reductase of sulfate-reducing bacteria of the genus Desulfovibrio, contains a third, gamma, subunit (11 kDa), in addition to the well-established alpha (50 kDa) and beta (40 kDa) subunits, and an alpha 2 beta 2 gamma 2 subunit structure has been proposed . Cloning and sequencing of the dsvC gene indicated it to encode a protein of 105 amino acids (11.9 kDa; gamma subunit) . The finding that the dsvC gene, located on a 3.5-kb SacII fragment, is transcribed in both Escherichia coli and Desulfovibrio vulgaris as an mRNA of only 400-600 nucleotides, and that both the dsvA and dsvB genes are present on a 7.2-kb SacII fragment, indicates that dsvC forms a separate transcriptional unit . The steady-state level of alpha and beta subunits expressed in D . vulgaris Hildenborough cells is rather constant, while that of the gamma subunit increased strongly in the stationary growth phase . Biochemical analysis of the purified protein, expressed in E . coli, and library comparison of its sequence, have so far failed to establish the function of gamma.

Eur J Biochem, 1993 Feb 1, 211(3), 475 - 84
Mutant aspartate aminotransferase (K258H) without pyridoxal-5'-phosphate-binding lysine residue . Structural and catalytic properties; Ziak M et al.; If the pyridoxal-phosphate-binding lysine residue 258 of aspartate aminotransferase is exchanged for a histidine residue, the enzyme retains partial catalytic competence {Ziak, M., Jaussi, R., Gehring, H . and Christen, P . (1990) Eur . J . Biochem . 187, 329-333} . The three-dimensional structures of the mutant enzymes of both chicken mitochondria and Escherichia coli were determined at high resolution . The folding patterns of the polypeptide chains proved to be identical to those of the wild-type enzymes, small conformational differences being restricted to parts of the active site . If aspartate or glutamate was added to the pyridoxal form of the mutant enzyme {lambda max 392 nm and 330 nm (weak); negative CD at 420 nm, positive CD at 370 nm and 330 nm}, the external aldimine (lambda max = 430 nm; negative CD at 360 nm and 430 nm) transiently accumulated . Upon addition of 2-oxoglutarate to the pyridoxamine form (lambda max 330 nm, positive CD), a putative ketamine intermediate could be detected; however, with oxalacetate, an equilibrium between external aldimine and the pyridoxal form, which was strongly in favour of the former, was established within seconds . The transamination cycle with glutamate and oxalacetate proceeds only three orders of magnitude more slowly than the overall reaction of the wild-type enzyme . The specific activity of the mutant enzyme is 0.1 U/mg at 25 degrees C and constant from pH 6.0 to 8.5 . Reconstitution of the mutant apoenzyme with {4'-3H}pyridoxamine 5'-phosphate resulted in rapid release of 3H with a first-order rate constant kappa' = 5 x 10(-4) s-1 similar to that of the wild-type enzyme . Apparently, in aspartate aminotransferase, histidine can to some extent substitute for the active-site lysine residue . The imidazole ring of H258, however, seems too distant from C alpha and C4' to act efficiently as proton donor/acceptor in the aldimine-ketamine tautomerization, suggesting that the prototropic shift might be mediated by an intervening water molecule . Transmination of the internal to the external aldimine apparently can be replaced by de novo formation of the latter, and by its hydrolysis in the reverse direction.

Carcinogenesis, 1993 Feb, 14(2), 311 - 3
Enhancement of binding rate of RecA protein to DNA by carcinogenic benzo{a}pyrene derivatives and selective change of adduct conformation; Kim SK et al.; The association kinetics of RecA protein from Escherichia coli to DNA is strongly enhanced if even a minor fraction of DNA bases has been modified by a carcinogenic (+)-anti metabolite of benzo{a}pyrene (BPDE) . The enhancement is much smaller with the less carcinogenic (-)-anti enantiomer of BPDE suggesting the possibility that the RecA protein binds selectively to the proto-oncogenic target . Most importantly, the binding of RecA to DNA modified with the latter enantiomer is found to give rise to a reorganization of this BPDE adduct from an intercalation site into a minor groove site . This indicates that the binding mechanism of RecA is via intercalation of some amino acid moiety, a discovery that could explain the approximately 50% contour length increase of the DNA within its fibrous complex with RecA.

Carcinogenesis, 1993 Feb, 14(2), 175 - 81
Cloning and characterization of a mouse 3-methyladenine/7-methyl-guanine/3-methylguanine DNA glycosylase cDNA whose gene maps to chromosome 11; Engelward BP et al.; In Escherichia coli, the repair of 3-methyladenine (3MeA) DNA lesions by DNA glycosylases prevents alkylation induced cell death . We described previously the isolation of a human 3MeA DNA glycosylase (AAG) cDNA that maps to chromosome 16 and hybridizes to specific genomic DNA fragments from a number of mammals, including mouse . As a first step in the generation of a 3MeA DNA glycosylase deficient mouse by homologous replacement in embryonic stem cells, we have cloned the mouse 3MeA DNA glycosylase cDNA . The cloned 1095 base pair cDNA contains a complete 333 amino acid open reading frame that predicts a 36.5 kDa protein and hybridizes to a 1.5 kb mRNA transcript . Mouse 3MeA DNA glycosylase (Aag) transcript levels vary by up to 21 fold among tissues, being highest in the testes and lowest in the heart . The Aag cDNA encodes a glycosylase able to release 3MeA, 7-methylguanine (7MeG) and 3-methylguanine (3MeG) from alkylated DNA . The expression of Aag in E . coli provides substantial resistance against killing by methylating agents, but, unlike its E . coli counterparts, the Aag glycosylase fails to protect against killing by ethylating and propylating agents . A 232 amino acid stretch of the predicted mouse protein shares extensive amino acid identity with rat (93%) and human (83%) 3MeA DNA glycosylases and we observe that all three mammalian glycosylases have a bipartite nuclear localization signal . The Aag gene maps to mouse chromosome 11, suggesting a segment of conserved synteny between mouse chromosome 11 and human chromosome 16, which bears the human 3MeA DNA glycosylase gene . Cloning the mouse 3MeA DNA glycosylase cDNA is a step toward understanding the role of this DNA repair enzyme in mammals.

Biochem J, 1993 Feb 1, 289 ( Pt 3), 771 - 5
Location of close contacts between Escherichia coli RNA polymerase and guanine residues at promoters either with or without consensus -35 region sequences; Minchin S et al.; Methylation-interference assays have been used to identify guanine residues that make important contacts with RNA polymerase during open-complex formation at two related Escherichia coli promoters . Methylation of lower-strand G-31 at a gal consensus promoter completely prevents complex formation, while modification of upper-strand G-33 has no detectable effect . At galP1, which lacks a consensus -35 region, modification of lower-strand G-33 and upper-strand G-14 reduces, but does not prevent, complex formation . G-33 is the only guanine residue in the -35 region of galP1 where modification interferes with open-complex formation . Since this guanine residue is not protected in open complexes, we conclude that its modification causes alteration of, or interference with, a transient contact during the transcription initiation pathway.

Arch Biochem Biophys, 1993 Feb 1, 300(2), 756 - 60
Quantitative in vitro assay for human immunodeficiency virus deoxyribonucleic acid integration; Carteau S et al.; An obligatory step of retroviral growth is the integration of a DNA copy of the viral RNA into the genomic DNA of the host . Recombinant human immunodeficiency virus type I (HIV-1) integrase (IN) expressed in Escherichia coli efficiently catalyzes the overall in vitro integration reaction, namely, the processing of the long terminal repeat (LTR) ends and the strand transfer reaction . Using the 3' end of synthetic oligonucleotides which match the termini of HIV-1 LTRs as substrate and supercoiled pSP65 DNA as the target, we describe an assay that is suitable for the enzymatic analysis of the integration and for testing candidate inhibitors of HIV IN protein.

Arch Biochem Biophys, 1993 Feb 1, 300(2), 629 - 34
Inhibitory effect of spermine on ribosomal peptidyltransferase; Kalpaxis DL et al.; A cell-free system derived from Escherichia coli has been used to study the kinetics of inhibition of peptide bond formation by spermine at optimal Mg2+ concentration (10 mM) . With the aid of the puromycin reaction, it was possible to show that spermine does not affect the final degree of peptide bond formation . However, spermine inhibits peptide bond formation at the kinetic phase of the reaction . A single molecule of spermine participates in the mechanism of inhibition . The type of inhibition of peptide bond formation by spermine is simple competitive, regardless of whether the ternary complex AcPhe-tRNA-poly(U)-ribosome (complex C) is formed in the presence (Ki = 190 microM) or in the absence (Ki = 84 microM) of factors washable from ribosomes . Preincubation experiments of spermine with the individual components of complex C demonstrated that the inhibitory effect of spermine is closely related with its binding to AcPhe-tRNA.

J Bacteriol, 1993 Feb, 175(4), 993 - 1000
The Escherichia coli fmt gene, encoding methionyl-tRNA(fMet) formyltransferase, escapes metabolic control; Meinnel T et al.; The genetic organization near the recently cloned fmt gene, encoding Escherichia coli methionyl-tRNA(fMet) formyltransferase (J . M . Guillon, Y . Mechulam, J . M . Schmitter, S . Blanquet, and G . Fayat, J . Bacteriol . 174:4294-4301, 1992), has been studied . The fmt gene, which starts at a GUG codon, is cotranscribed with another gene, fms, and the transcription start site of this operon has been precisely mapped . Moreover, the nucleotide sequence of a 1,379-bp fragment upstream from fmt reveals two additional open reading frames, in the opposite polarity . In the range of 0.3 to 2 doublings per h, the intracellular methionyl-tRNA(fMet) formyltransferase concentration remains constant, providing, to our knowledge, the first example of a gene component of the protein synthesis apparatus escaping metabolic control . When the gene fusion technique was used for probing, no effect on fmt expression of the concentrations of methionyl-tRNA(fMet) formyltransferase or tRNA(fMet) could be found . The possibility that the fmt gene, the product of which is present in excess to ensure full N acylation of methionyl-tRNA(fMet), could be expressed in a constitutive manner is discussed.

J Bacteriol, 1993 Feb, 175(4), 982 - 92
Structural genes for thiamine biosynthetic enzymes (thiCEFGH) in Escherichia coli K-12; Vander Horn PB et al.; Escherichia coli K-12 synthesizes thiamine pyrophosphate (vitamin B1) de novo . Two precursors {4-methyl-5-(beta-hydroxyethyl)thiazole monophosphate and 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate} are coupled to form thiamine monophosphate, which is then phosphorylated to make thiamine pyrophosphate . Previous studies have identified two classes of thi mutations, clustered at 90 min on the genetic map, which result in requirements for the thiazole or the hydroxymethylpryimidine . We report here our initial molecular genetic analysis of the thi cluster . We cloned the thi cluster genes and examined their organization, structure, and function by a combination of phenotypic testing, complementation analysis, polypeptide expression, and DNA sequencing . We found five tightly linked genes, designated thiCEFGH . The thiC gene product is required for the synthesis of the hydroxymethylpyrimidine . The thiE, thiF, thiG, and thiH gene products are required for synthesis of the thiazole . These mutants did not respond to 1-deoxy-D-threo-2-pentulose, indicating that they are blocked in the conversion of this precursor compound to the thiazole itself.

J Bacteriol, 1993 Feb, 175(4), 966 - 72
Paracrystalline inclusions of a novel ferritin containing nonheme iron, produced by the human gastric pathogen Helicobacter pylori: evidence for a third class of ferritins; Frazier BA et al.; An abundant 19.3-kDa Helicobacter pylori protein has been cloned, and the sequence is homologous with a ferritin-like protein produced by Escherichia coli K-12 . Homologies are also present with a number of eucaryotic ferritins, as well as with the heme group-containing bacterioferritins . All amino acids involved in chelation of inorganic iron by ferritins from humans and other higher species are conserved in the H . pylori protein . Consistent with the structural data indicating an iron-binding function, E . coli overexpressing the H . pylori ferritin-like protein accumulates almost 10 times more nonheme iron than vector controls, and the iron-binding activity copurifies with the 19.3-kDa protein . Immunoelectron microscopy of H . pylori, as well as of E . coli overexpressing the H . pylori gene, demonstrates that the gene product has a cytoplasmic location where it forms paracrystalline inclusions . On the basis of these structural and functional data, we propose that the H . pylori gene product (termed Pfr) forms the basis for a second class of bacterial ferritins designed to store nonheme iron.

J Bacteriol, 1993 Feb, 175(4), 952 - 8
Motility, chemokinesis, and methylation-independent chemotaxis in Azospirillum brasilense; Zhulin IB et al.; Observations of free-swimming and antibody-tethered Azospirillum brasilense cells showed that their polar flagella could rotate in both clockwise and counterclockwise directions . Rotation in a counterclockwise direction caused forward movement of free-swimming cells, whereas the occasional change in the direction of rotation to clockwise caused a brief reversal in swimming direction . The addition of a metabolizable chemoattractant, e.g., malate or proline, had two distinct effects on the swimming behavior of the bacteria: (i) a short-term decrease in reversal frequency from 0.33 to 0.17 s-1 and (ii) a long-term increase in the mean population swimming speed from 13 to 23 microns s-1 . A . brasilense therefore shows both chemotaxis and chemokinesis in response to temporal gradients of some chemoeffectors . Chemokinesis was dependent on the growth state of the cells and may depend on an increase in the electrochemical proton gradient above a saturation threshold . Analysis of behavior of a methionine auxotroph, assays of in vivo methylation, and the use of specific antibodies raised against the sensory transducer protein Tar of Escherichia coli all failed to demonstrate the methylation-dependent pathway for chemotaxis in A . brasilense . The range of chemicals to which A . brasilense shows chemotaxis and the lack of true repellents indicate an alternative chemosensory pathway probably based on metabolism of chemoeffectors.

J Bacteriol, 1993 Feb, 175(4), 921 - 5
Accumulation of the F plasmid TraJ protein in cpx mutants of Escherichia coli; Silverman PM et al.; We report here studies of the cellular control of F plasmid TraJ protein levels, focusing on the effects of chromosomal cpx mutations . The principal conclusion from our results is that the cpx mutations impair accumulation of the TraJ protein, thereby reducing tra gene expression . We measured TraJ activity in vivo by expression of a traY'-'lacZ fusion gene and TraJ protein by immuno-overlay blot . In strains with normal TraJ levels, traY expression and donor-related functions were reduced in cells carrying any of four cpxA mutations . In the strain background used to isolate cpx mutants, these reductions were especially evident in cells grown to high density, when traY expression and donor activity both increased in cpx+ cells . In each of the four cpxA mutants tested, TraJ levels were lower than in the otherwise isogenic cpxA+ strain . In cells grown to high density, the differences ranged from 4-fold in the cpxA6 strain to > 10-fold in the cpxA2, cpxA5, and cpxA9 strains . The cpxA2 mutation had little or no effect on traY expression or on donor-related functions when TraJ was present in excess of its limiting level in F' or Hfr cells or on a mutant traY promoter whose expression in vivo was independent of TraJ.

J Bacteriol, 1993 Feb, 175(4), 1206 - 10
Noninducible Tet repressor mutations map from the operator binding motif to the C terminus; Hecht B et al.; We have developed a new genetic selection system for Tet repressor mutations with a noninducible phenotype for tetracycline (TetRs) . Extensive chemical mutagenesis of tetR yielded 93 single-site Tet repressor mutations . They map from residue 23 preceding the alpha-helix-turn-alpha-helix operator binding motif to residue 196 close to the C terminus of the repressor . Thirty-three of the mutations are clustered between residues 95 and 117, and another 27 are clustered between residues 131 to 158 . Several of the mutants were characterized quantitatively in vivo for induction by tetracycline and anhydrotetracycline . While all of these are severely reduced in tetracycline-mediated induction, only some of them are affected for anhydrotetracycline-mediated induction.

J Bacteriol, 1993 Feb, 175(4), 1165 - 72
Identification of the regulatory sequence of anaerobically expressed locus aeg-46.5; Choe M et al.; A newly identified anaerobically expressed locus, aeg-46.5, which is located at min 46.5 on Escherichia coli linkage map, was cloned and analyzed . The phenotype of this gene was studied by using a lacZ operon fusion . aeg-46.5 is induced anaerobically in the presence of nitrate in wild-type and narL cells . It is repressed by the narL gene product, as it showed derepressed anaerobic expression in narL mutant cells . We postulate that aeg-46.5 is subject to multiple regulatory systems, activation as a result of anaerobiosis, narL-independent nitrate-dependent activation, and narL-mediated repression . The regulatory region of aeg-46.5 was identified . A 304-bp DNA sequence which includes the regulatory elements was obtained, and the 5' end of aeg-46.5 mRNA was identified . It was verified that the anaerobic regulation of aeg-46.5 expression is controlled on the transcriptional level . Computer analysis predicted possible control sites for the NarL and FNR proteins . The proposed NarL site was found in a perfect-symmetry element . The aeg-46.5 regulatory elements are adjacent to, but divergent from, those of the eco gene.

J Bacteriol, 1993 Feb, 175(4), 1118 - 25
Cell division inhibitors SulA and MinCD prevent formation of the FtsZ ring; Bi E et al.; Immunoelectron microscopy was used to assess the effects of inhibitors of cell division on formation of the FtsZ ring in Escherichia coli . Induction of the cell division inhibitor SulA, a component of the SOS response, or the inhibitor MinCD, a component of the min system, blocked formation of the FtsZ ring and led to filamentation . Reversal of SulA inhibition by blocking protein synthesis in SulA-induced filaments led to a resumption of FtsZ ring formation and division . These results suggested that these inhibitors block cell division by preventing FtsZ localization into the ring structure . In addition, analysis of min mutants demonstrated that FtsZ ring formation was also associated with minicell formation, indicating that all septation events in E . coli involve the FtsZ ring.

J Bacteriol, 1993 Feb, 175(4), 1110 - 7
Mutations affecting the ability of Escherichia coli Lrp to bind DNA, activate transcription, or respond to leucine; Platko JV et al.; Lrp is a regulatory protein in Escherichia coli that increases expression of some operons and decreases expression of others . Mutations in Lrp were isolated on the basis of their effects on ilvIH, one of the operons regulated positively by Lrp . The ilvIH operon encodes an enzyme involved in the biosynthesis of leucine, valine, and isoleucine, and expression of this operon is repressed when cells are grown in the presence of leucine . Three groups of mutants were isolated . Mutant strains that were resistant to the repressive effects of leucine were termed leucine response mutants . These mutants had changes in the Lrp amino acid sequence between amino acid residues 108 and 149 . Mutant strains having low expression of ilvIH in vivo were identified as colonies having reduced expression of a reporter gene . For some of these mutants, called DNA-binding mutants, binding to ilvIH DNA in vitro was markedly reduced . The mutations in these strains caused changes in Lrp between amino acids 16 and 70 . Six of ten of these mutations were within a region having a putative helix-turn-helix motif . A third group of mutants had low ilvIH expression in vivo but apparently normal DNA binding in vitro . These mutants were called activation mutants since they affected the ability of Lrp to activate expression . Lrp from these strains had changes in amino acids between residues 76 and 125 . This study suggests that Lrp has separate domains responsible for binding DNA, activating transcription, and responding to leucine.

J Bacteriol, 1993 Feb, 175(4), 1087 - 94
Glycerol kinase of Escherichia coli is activated by interaction with the glycerol facilitator; Voegele RT et al.; Glycerol transport is commonly cited as the only example of facilitated diffusion across the Escherichia coli cytoplasmic membrane . Two proteins, the glycerol facilitator and glycerol kinase, are involved in the entry of external glycerol into cellular metabolism . The glycerol facilitator is thought to act as a carrier or to form a selective pore in the cytoplasmic membrane, whereas the kinase traps the glycerol inside the cell as sn-glycerol-3-phosphate . We found that the kinetics of glycerol uptake in a facilitator-minus strain are significantly different from the kinetics of glycerol uptake in the wild type . Free glycerol was not observed inside wild-type cells transporting glycerol, and diffusion of glycerol across the cytoplasmic membrane was not the rate-limiting step for phosphorylation in facilitator-minus mutants . Therefore, the kinetics of glycerol phosphorylation are different, depending on the presence or absence of the facilitator protein . We conclude that there is an interaction between the glycerol facilitator protein and glycerol kinase that stimulates kinase activity, analogous to the hexokinase- and glycerol kinase-porin interactions in mitochondria.

Epidemiol Infect, 1993 Feb, 110(1), 9 - 16
Diarrhoea in close contacts as a risk factor for childhood haemolytic uraemic syndrome . The CPKDRC co-investigators; Rowe PC et al.; To determine whether the risk factors for childhood haemolytic uraemic syndrome (HUS) are similar to risk factors previously reported for Escherichia coli O 157 . H7 gastroenteritis, we conducted a case-control study at eight paediatric hospitals in the summer of 1990 . Thirty-four consecutive children with HUS were prospectively enrolled; all had diarrhoea and 88% had laboratory evidence of exposure to verotoxin-producing E . coli (VTEC) . The 102 controls were otherwise healthy children with minor acute injuries . Parents of all subjects responded to a questionnaire about each child's exposure to various foods, methods of food preparation, sources of water, travel, and individuals with diarrhoea . Children with HUS were significantly more likely than controls to have had close contact with an individual with diarrhoea in the 2 weeks before the onset of illness (74 v . 29%, P < 0.00001; odds ratio 7.0, 95% CI 2.7-18.5) . The onset of diarrhoea in the contacts occurred a median of 6 days (range, 1- > 14 days) before the onset of diarrhoea in the HUS patients . Exposure to undercooked ground meat was not significantly more common in the patients with HUS (15 v . 8%; P = 0.05) . These data provide evidence consistent with person-to-person transmission of VTEC in a substantial proportion of episodes of childhood HUS.

Curr Genet, 1993 Feb, 23(2), 134 - 40
Cloning and sequencing of the 3-phosphoglycerate kinase (PGK) gene from Penicillium citrinum and its application to heterologous gene expression; Nara F et al.; The gene coding for 3-phosphoglycerate kinase (PGK) in ML-236B (compactin)-producing Penicillium citrinum was isolated from the recombinant phage lambda library using the corresponding Aspergillus nidulans pgk gene as a probe . The P . citrinum pgk gene has an open reading frame of 1,254 bp, encoding a protein of 417 amino acids with a predicted molecular weight of 44,079 daltons . The position of the two introns, 59 and 60 bp respectively, was deduced from an homology comparison with the sequence of the A . nidulans pgk gene . The PGK protein of P . citrinum shows extensive high homology to the PGKs of four other fungi: P . chrysogenum (93%), A . nidulans (84%), Trichoderma reesei (78%) and Saccharomyces cerevisiae (68%) . Almost total conservation is found in P . citrinum of residues thought to be important for the structure and function of the yeast enzyme . The strong codon preference found has greater similarity to that in other filamentous fungi than in yeast . A DNA fragment encompassing the pgk gene was shown to hybridize a 1.35-kb poly(A)+RNA, sufficient to encode the PGK polypeptide . A fused gene, pgk-hpt, containing the putative pgk promoter and the open reading frame of the Escherichia coli hygromycin B phosphotransferase (hpt) gene was constructed, and was successfully used to transform P . citrinum to a hygromycin B (HmB)-resistant phenotype.

Curr Genet, 1993 Feb, 23(2), 115 - 22
Characterization of the Trichoderma reesei cbh2 promoter; Stangl H et al.; A 613-bp fragment of the 5' upstream region of the Trichoderma reesei cbh2 gene (coding for the cellulolytic enzyme cellobiohydrolase II) has been isolated and sequenced . Fusion of this fragment to the E . coli uidA gene (coding for beta-glucuronidase) leads to--albeit low--expression of beta-glucuronidase activity in the presence of cellulose and upon the addition of low molecular weight inducers (sophorose, lactose) of cellobiohydrolase II . It also governed the formation of beta-glucuronidase activity during sporulation and its transport to the conidial surface . However, despite the presence of a signal peptide in the cbh2:uidA fusion, beta-glucuronidase was not secreted in T . reesei . Defined fragments of the 613-bp promoter region were isolated and used to identify areas involved in the regulation of cbh2 expression by protein-DNA binding assays . At least two binding areas--between -443/-363 and -363/-173, respectively--were identified . In both areas, the DNA-protein complex observed was appreciably larger when cell-free extracts from sophorose-induced mycelia were used . This suggests that at least one of the proteins regulating cbh2 transcription is itself induced by cellulose.

Br J Cancer, 1993 Feb, 67(2), 304 - 10
A recombinant single chain antibody interleukin-2 fusion protein; Savage P et al.; Recombinant interleukin-2 (rIL-2) therapy has been shown to be of value in the treatment of some cases of melanoma and renal cell carcinoma . However its use can be limited by severe systemic toxicity . Targeting rIL-2 to the tumour should improve the anti-tumour immune response and decrease the systemic toxicity . With this aim we have employed recombinant DNA techniques to construct a single chain antibody interleukin-2 fusion protein (SCA-IL-2) . The protein used in this model system comprises the variable domains of the anti-lysozyme antibody D1.3 fused to human IL-2 . It has been expressed by secretion from Escherichia coli and the purified product possesses antigen binding specificity and retains the immunostimulatory activities of rIL-2 . This approach can be taken to generate SCA-IL-2 proteins that bind to appropriate cellular antigens . In vivo administration of a tumour binding SCA-IL-2 should result in a localised high concentration of IL-2 in tumour tissues, maximising the anti-tumour immune response, whilst keeping systemic side effects to a minimum.

Biotechniques, 1993 Feb, 14(2), 256 - 65
Selection of an active single chain Fv antibody from a protein linker library prepared by enzymatic inverse PCR; Stemmer WP et al.; Enzymatic inverse PCR mutagenesis was developed as a simple and reliable method for the construction of large libraries of site-directed mutants . Enzymatic inverse PCR library mutagenesis uses a single PCR fragment and is restriction-site independent . The usefulness of the technique was demonstrated by the design of a single chain linker for an antibody Fv fragment without computer modeling . The Fv fragment of an antibody specific for a metal chelate was expressed in active form in the periplasm of E . coli . The light and the heavy chains of the Fv are expressed as a bicistronic mRNA . Enzymatic inverse PCR mutagenesis was used to construct a library of 3 x 10(5) Fv mutants, in which the C-terminus of the light chain was connected to the N-terminus of the heavy chain by a 15-amino acid peptide linker of variable composition . After plating, active mutant colonies were identified by screening colony filter lifts with a radiolabeled hapten, N'-(2-hydroxyethyl)-p-thioureidobenzyl EDTA . About 0.2% of the mutants were positive, and a selected sFv clone was shown to have the same affinity as the Fv (9 x 10(9)) and was similar to the whole antibody (11 x 10(9)) . This example compares favorably with both of the other approaches to constructing sFv's; namely, molecularly modeled linkers as well as universal linkers, which have often yielded significantly lower affinities than whole antibodies or Fabs . The enzymatic inverse PCR library mutagenesis approach is simple and reliable and can be used to obtain linkers for the great majority of antibodies for which no structural data are available . More generally, it can be used to modify DNA coding for any structural protein or regulatory element.

Biotechniques, 1993 Feb, 14(2), 250 - 5
A vector for facile PCR product cloning and modification generating any desired 4-base 5' overhang: pRPM; Cease KB et al.; A pair of plasmids were developed for cloning PCR products in order to facilitate the preparation of products with 5' overhangs consisting of any desired 4-nucleotide sequence . These vectors allow DNA to be cloned into a unique restriction site by blunt-end ligation or by AT-cloning . The cloned DNA is subsequently excised using class IIS restriction enzyme sites flanking the insert yielding a fragment that is entirely free of vector sequences . These enzymes recognize sequences in the vector but "reach-over" the junction to cut within the insert thereby generating a 4-base 5' overhang sequence determined by the 5' sequences in the insert . Thus, in cases where the insert was originally generated by PCR, the overhangs are specified by the primer . More generally, these vectors offer a unique capability for the reversible cloning of any blunt DNA fragment because excision and fill-in reactions precisely regenerate the original blunt fragment regardless of its sequence . These "reach-over" product modification vectors represent general and flexible tools for the generation of fragments for use in engineering DNA constructs.

Arch Surg, 1993 Feb, 128(2), 138 - 43; discussion 143-4
Elaboration of interleukin 1-receptor antagonist is not attenuated by glucocorticoids after endotoxemia; Santos AA et al.; The body's response to infection/inflammation is initiated by the elaboration of cytokines, such as tumor necrosis factor, interleukin 1-beta (IL-1-beta), IL-6, and IL-8 . Cytokines, in turn, stimulate the pituitary-adrenal axis, and it has been suggested that the corticosteroids elaborated serve as negative feedback signals to diminish inflammatory events . To test this hypothesis, we administered hydrocortisone shortly before endotoxin administration to normal volunteers . Steroids greatly reduced the clinical response to endotoxin and attenuated the appearance of tumor necrosis factor, IL-6, and IL-8 in the circulation . In contrast, IL-1-receptor antagonist, a competitive antagonist of the IL-1 receptor, was unaffected by steroid administration . These data suggest that IL-1-receptor antagonist may act in synergism with corticosteroids to reduce inflammation . Elevation of concentrations of these two factors, corticosteroids and IL-1-receptor antagonist, in plasma appears to be the mechanism used by the body to overcome the effects of inflammatory cytokines.

Proc Natl Acad Sci U S A, 1993 Feb 1, 90(3), 970 - 4
Ribosomal RNA antitermination in vitro: requirement for Nus factors and one or more unidentified cellular components; Squires CL et al.; Using an in vitro transcription assay, we have successfully demonstrated read through of a Rho-dependent terminator by the ribosomal RNA antitermination system . The assay used a DNA template containing a promoter-antiterminator-terminator arrangement, RNA polymerase, termination factor Rho, antitermination factors NusA, NusB, NusE, and NusG, and a cellular extract depleted of NusB . Terminator read-through was highly efficient only in the presence of the extract and Nus factors, suggesting that an as yet uncharacterized cellular component is required for ribosomal antitermination . The NusB-depleted extract had no activity in the absence of NusB, confirming an absolute requirement for this protein in ribosomal RNA antitermination . The DNA template requirements were the same as those previously established in vivo; transcription of a wild-type boxA sequence is both necessary and sufficient to promote RNA polymerase modification into a terminator-resistant form.

Proc Natl Acad Sci U S A, 1993 Feb 1, 90(3), 933 - 7
Screening for in vivo protein-protein interactions; Germino FJ et al.; We describe an in vivo approach for the isolation of proteins interacting with a protein of interest . The protein of interest is "tagged" with a portion of the biotin carboxylase carrier protein (BCCP), encoded on a specially constructed plasmid, so that it becomes biotinylated in vivo . The "query" proteins (e.g., those in a cDNA library) are tagged by fusing them to the 3' end of the lacZ gene on a lambda vector in such a way that the beta-galactosidase activity is not disrupted . These phage are transfected into cells containing the plasmid encoding the BCCP-tagged protein . The infection lyses the cells and exposes the protein complexes . The BCCP-tagged protein and any associated protein(s) are "captured" by using avidin, streptavidin, or anti-biotin antibody-coated filters . The detection of bound protein is accomplished by directly assaying for beta-galactosidase activity on the filters . Positive plaques can be plaque-purified for DNA sequencing . We have tested this approach by using c-Fos and c-Jun as our model system . We show that avidin, streptavidin, or polyclonal anti-biotin (but not a monoclonal anti-biotin) antibody is capable of specifically capturing in vivo biotinylated beta-galactosidase and c-Jun and that this capture is dependent upon the presence of both avidin and the BCCP moiety . Further, complexes containing c-Jun and c-Fos can also be isolated in this manner, and the isolation of this complex is dependent on the presence of c-Fos, c-Jun, avidin, and the BCCP moiety . We discuss the possible uses and limitations of this technique for isolating proteins that interact with a known protein.

Proc Natl Acad Sci U S A, 1993 Feb 1, 90(3), 918 - 22
Recombinant human sickle hemoglobin expressed in yeast; Martin de Llano JJ et al.; Sickle hemoglobin has been expressed in the yeast Saccharomyces cerevisiae after site-directed mutagenesis of a plasmid containing normal human alpha- and beta-globin genes . Cassette mutagenesis of this plasmid was achieved by inserting a DNA fragment containing the beta-globin gene in the replicative form of M13mp18 to make a point mutation and then reconstituting the original plasmid containing the mutated beta-globin gene . Pure recombinant hemoglobin S was shown to be identical to natural sickle hemoglobin in its ultraviolet and visible absorption bands and by gel electrophoresis, isoelectric focusing, amino acid analysis, mass spectrometry, partial N-terminal sequencing, and functional properties (P50, cooperativity, and response to 2,3-bisphosphoglycerate) . In yeast and in mammalian cells, cotranslational processing yields the same N-terminal valine residues of hemoglobin alpha- and beta-chains, but in bacterial expression systems the N terminus is extended by an additional amino acid because the initiator methionine residue is retained . Since the N-terminal valine residues of both chains of hemoglobin S participate in important physiological functions, such as oxygen affinity, interaction with anions, and the Bohr coefficient, the yeast expression system is preferable to the bacterial system for recombinant DNA studies . Hence, mutagenesis employing this expression system should permit definitive assignments of the role of any amino acid side chain in hemoglobin S aggregation and could suggest additional approaches to therapeutic intervention . The engineering of this system for the synthesis of sickle hemoglobin and its purification to homogeneity in a single column procedure are described.

Proc Natl Acad Sci U S A, 1993 Feb 1, 90(3), 903 - 7
sid1, a gene initiating siderophore biosynthesis in Ustilago maydis: molecular characterization, regulation by iron, and role in phytopathogenicity; Mei B et al.; Iron uptake in Ustilago maydis is mediated by production of extracellular hydroxamate siderophores . L-Or-nithine N5-oxygenase catalyzes hydroxylation of L-ornithine, which is the first committed step of ferrichrome and ferrichrome A biosynthesis in U . maydis . We have characterized sid1, a gene coding for this enzyme, by complementation in trans, gene disruption, and DNA sequence analysis . A comparison of genomic DNA and cDNA sequences has shown that the gene is interrupted by three introns . The putative amino acid sequence revealed similarity with Escherichia coli lysine N6-hydroxylase, which catalyzes the hydroxylation of lysine, the first step in biosynthesis of aerobactin . Two transcription initiation points have been determined, both by PCR amplification of the 5' end of the mRNA and by primer extension . A 2.3-kb transcript which accumulates in cells grown under low iron conditions was detected by Northern hybridization . A less abundant 2.7-kb transcript was observed in cells grown in iron-containing medium . By contrast, constitutive accumulation of the 2.3-kb transcript was observed in a mutant carrying a disruption of urbs1, a gene involved in regulation of siderophore biosynthesis . Analysis of the pathogenicity of mutants carrying a null allele of sid1 suggests that the biosynthetic pathway of siderophores does not play an essential role in the infection of maize by U . maydis.

Proc Natl Acad Sci U S A, 1993 Feb 1, 90(3), 1122 - 6
Single-stranded shuttle phagemid for mutagenesis studies in mammalian cells: 8-oxoguanine in DNA induces targeted G.C-->T.A transversions in simian kidney cells; Moriya M; A single-stranded shuttle vector has been developed for the purpose of investigating translesional events in mammalian cells . The vector is designed to permit site-specific introduction of defined DNA lesions between a gene for neomycin resistance and its promoter . Efficiencies of translesional synthesis in simian kidney cells (COS) and Escherichia coli are established by determining the number of neomycin- and ampicillin-resistant colonies recovered, respectively, after introduction of a modified vector . Fidelity of translesional synthesis is evaluated by analyzing the nucleotide sequence of progeny phagemid DNA in the region corresponding to the lesion site . This experimental system, capable of detecting mutagenic and nonmutagenic events at and adjacent to the lesion site, was used to establish the mutagenic potential of a single 8-oxoguanine residue in DNA . This modified base, produced by attack of reactive oxygen species on cellular DNA, did not cause a decrease in the number of transformants when single-stranded DNA containing the lesion replicated in COS cells or E . coli . The predominant mutations observed (> 78%) were G-->T transversions targeted to the site of the lesion . The mutation frequencies for this event were 2.5-4.8% in COS cells and 1.8% in E . coli . It is concluded that a single-stranded shuttle vector, utilized in conjunction with a site-specific approach, can be used to investigate translesional events in mammalian cells and in bacteria.

Proc Natl Acad Sci U S A, 1993 Feb 1, 90(3), 1053 - 7
Escherichia coli cell division protein FtsZ is a guanine nucleotide binding protein; Mukherjee A et al.; FtsZ is an essential cell division protein in Escherichia coli that forms a ring structure at the division site under cell cycle control . The dynamic nature of the FtsZ ring suggests possible similarities to eukaryotic filament forming proteins such as tubulin . In this study we have determined that FtsZ is a GTP/GDP binding protein with GTPase activity . A short segment of FtsZ is homologous to a segment in tubulin believed to be involved in the interaction between tubulin and guanine nucleotides . A lethal ftsZ mutation, ftsZ3 (Rsa), that leads to an amino acid alteration in this homologous segment decreased GTP binding and hydrolysis, suggesting that interaction with GTP is essential for ftsZ function.

Proc Natl Acad Sci U S A, 1993 Feb 1, 90(3), 1038 - 42
A pathway for disulfide bond formation in vivo; Bardwell JC et al.; Protein disulfide bond formation in Escherichia coli requires the periplasmic protein DsbA . We describe here mutations in the gene for a second protein, DsbB, which is also necessary for disulfide bond formation . Evidence suggests that DsbB may act by reoxidizing DsbA, thereby regenerating its ability to donate its disulfide bond to target proteins . We propose that DsbB, an integral membrane protein, may be involved in transducing redox potential across the cytoplasmic membrane.

Mol Pharmacol, 1993 Feb, 43(2), 176 - 82
Inhibition by glucocorticoids of the formation of interleukin-1 alpha, interleukin-1 beta, and interleukin-6: mediation by decreased mRNA stability; Amano Y et al.; The mechanism by which glucocorticoids inhibit interleukin (IL)-1 and IL-6 formation in human monocytes and a promonocytic cell line activated by Escherichia coli lipopolysaccharide was analyzed . Dexamethasone (DEX) decreased levels of IL-1 alpha and IL-1 beta mRNAs in a dose-related fashion . The DEX-induced decrease in levels of IL-1 alpha and IL-1 beta mRNAs was abolished by the steroid receptor antagonist RU486 . The levels of IL-1 alpha and IL-1 beta proteins within the cells and of IL-1 beta in the culture medium were decreased by DEX to comparable extents, so that DEX had no detectable effect on cytokine secretion . DEX did not influence lipopolysaccharide-induced transcription of the IL-1 beta gene in monocytes . However, DEX markedly decreased the stability of IL-1 beta mRNA, as shown both by steady state measurements and by pulse-labeling . DEX-induced instability of IL-1 beta mRNA required protein synthesis . DEX was also found to be a potent inhibitor of IL-1-induced expression of the IL-6 gene in connective tissue-type cells from the synovium of patients with rheumatoid arthritis . Inhibition of the formation of proinflammatory cytokines, including IL-1 beta and tumor necrosis factor-alpha, is a mechanism by which glucocorticoids exert anti-inflammatory effects . Inhibition by glucocorticoids of the expression of IL-1 alpha in antigen-presenting cells could decrease the capacity of the cells to stimulate the proliferation of T lymphocytes . This activity, as well as inhibition of the production and effects of IL-1 beta, including induced formation of IL-6 and of certain lymphokines, could explain the immunosuppressive effects of glucocorticoids.

J Nucl Med, 1993 Feb, 34(2), 234 - 41
Rapid infarct imaging with a technetium-99m-labeled antimyosin recombinant single-chain Fv: evaluation in a canine model of acute myocardial infarction; Nedelman MA et al.; Studies of monoclonal antibody-based imaging agents show that blood clearance is inversely proportional to molecular size, i.e., Fab or Fab' > F(ab')2 > IgG . Indium-111-antimyosin Fab-DTPA is a highly specific and sensitive marker for myocardial necrosis . An improvement on current antibody diagnostic imaging may result from the use of smaller labeled fragments . We report the first in vivo targeting of acute myocardial infarction with a novel recombinant single-chain Fv (sFv) antimyosin protein . The sFv (MW = 27,594) is approximately one-half the size of the Fab and is comprised of the heavy and light chain variable regions from the myosin-specific murine monoclonal antibody R11D10 which were joined by a 15-amino-acid linker and expressed as a fusion protein (sFv) in E . coli . The binding affinity of the sFv for cardiac myosin was similar to the affinity observed for the Fab fragment . Technetium-99m labeling of the sFv was accomplished by the attachment of a cleavable, ester-linked bifunctional chelator (RP-1) . Comparative studies in mice showed 99mTc-sFv-RP-1 cleared significantly faster (p < 0.001) than 99mTc-Fab'-RP-1 and 111In-Fab-DTPA antimyosin fragments . Furthermore, measurement of 99mTc-sFv-RP-1 blood clearance in a canine model of acute myocardial infarction gave a mean T1/2 of 0.54 +/- 0.13 hr versus 2.80 +/- 0.57 and 2.58 +/- 0.64 hr for Fab-DTPA and Fab'-RP-1 (p < 0.05), respectively . Despite its comparatively rapid clearance, 99mTc sFv-RP-1 had similar uptake in the infarct compared to the Fab'-RP-1 . In addition, infarct visualization was more rapid with the sFv . Thus, these data demonstrate antimyosin sFv possesses characteristics necessary for rapid imaging of myocardial necrosis.

Hepatology, 1993 Feb, 17(2), 266 - 72
Hyaluronic acid uptake by the isolated, perfused rat liver: an index of hepatic sinusoidal endothelial cell function; Deaciuc IV et al.; Previous studies indicate that sinusoidal endothelial cells bind and internalize hyaluronic acid at much greater rates than do other liver cells . Thus hepatic hyaluronic acid removal rate may be indicative of sinusoidal endothelial cell function . In these studies the uptake of hyaluronic acid (molecular weight 1.3 x 10(6)) was measured in isolated perfused rat liver under a variety of conditions . Uptake was dependent on hyaluronic acid concentration . At all concentrations tested, the rate of hyaluronic acid uptake stabilized at a steady-state level 2 to 3 min after development of a high rate of apparent uptake . At saturating hyaluronic acid concentration (150 ng.ml-1), the steady-state uptake rate was 10.4 +/- 1.0 micrograms.gm-1 liver wet wt.hr-1, which is as high as or higher than the rates reported for isolated rat liver sinusoidal endothelial cells . The half-maximal rate of uptake was attained at a hyaluronic acid concentration of 80 ng.ml-1 . Hyaluronic acid uptake was inhibited by heparin (80%), a competitive ligand for the hyaluronic acid receptor on sinusoidal endothelial cells; 4 beta-phorbol 12 beta-O-myristoyl 13 alpha-acetate (25% to 50%), a tumor promoter and activator of protein kinase C; prostaglandin F2 alpha (24% to 52%), an eicosanoid secreted in the liver by Kupffer cells; A23187 (33% to 66%), a Ca2+ ionophore; and Escherichia coli lipopolysaccharide (16% to 43%) . Platelet activating factor did not affect hyaluronic acid uptake by the perfused liver . Hyaluronic acid uptake was increased by 50% after a 24-hr fast.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood, 1993 Feb 1, 81(3), 808 - 14
Analysis of mutant NADH-cytochrome b5 reductase: apparent "type III" methemoglobinemia can be explained as type I with an unstable reductase; Nagai T et al.; A patient in Kurobe, Japan, was previously reported to have a new class of hereditary methemoglobinemia, type III . In this patient, NADH cytochrome b5 reductase deficiency was observed in lymphocytes and platelets as well as in erythrocytes, but this was not associated with mental retardation . A base change was identified in the gene for NADH cytochrome b5 reductase, causing an amino acid substitution from Leu-148 to Pro . In the present study, the mutant enzyme (Leu-148-->Pro) was expressed in Escherichia coli, purified, and characterized . The mutant enzyme retained about 60% of the catalytic activity of the wild type, but was remarkably heat unstable . By incubating the mutant enzyme at 42 degrees C for 10 minutes, 80% of the enzyme activity was lost, whereas the wild-type enzyme lost < 20% activity after incubation at 50 degrees C for 30 minutes . Another mutant in which Leu-148 was replaced by Ala was prepared to establish the role of the residue . This mutant was apparently less heat stable than the wild type, implying a structural role for Leu-148 . Reinvestigation of the enzyme activity in the blood cells and fibroblasts of the type III Kurobe patient, revealed that about 40% of the normal activity was detected in these cells, in contrast to the previous report . Thus, this patient reported previously as having hereditary meth-hemoglobinemia type III was shown to have type I.

J Protein Chem, 1993 Feb, 12(1), 7 - 10
Essential sulfhydryl group of malic enzyme from Escherichia coli; Chang GG et al.; The activity of malic enzyme from Escherichia coli was unaffected by the monovalent cations Na+ or Li+ at 10 mM . At 100 mM, Li+ or Na+ inhibited the enzyme activity by 88% and 83%, respectively . However, the enzyme activity was stimulated by 40-80-fold with 10 mM K+, Rb+, Cs+, or NH4+ . Less stimulation was observed with 100 mM of these stimulating cations . The stimulatory effect was lost after the enzyme was dialyzed against Tris-Cl buffer, but was regained after incubating the dialyzed enzyme with dithiothreitol . The regenerated enzyme was inactivated by 5,5'-dithiobis(2-nitrobenzoic acid) . The resulting inactive thionitrobenzoyl enzyme could be regenerated to the active thiol-enzyme by dithiothreitol or converted to the inactive thiocyanoylated enzyme by KCN . The thiocyanoylated enzyme was insensitive to K+ stimulation, which suggested the essentiality of the sulfhydryl groups of the E . coli malic enzyme.

Leukemia, 1993 Feb, 7(2), 310 - 7
Development of a lacZ marked WEHI-3B D+ murine leukemic cell line as an in-vivo model of acute non-lymphocytic leukemia; Smith C et al.; Established leukemic cell lines have been useful models for studying the biology of leukemia . Analysis of the actions of differentiating agents on leukemic cell lines in vivo has been limited by an inability to unambiguously distinguish host hematopoietic elements from differentiated leukemic cells . In order to identify and quantify leukemic cells during in vivo studies, a derivative of the murine myelomonocytic leukemia cell line WEHI-3B D+, which stably expresses beta-galactosidase, was constructed utilizing retroviral vector gene transfer . This cell line, termed WEHI-3B D+/lacZ 2.8, demonstrated in vitro growth and differentiation properties similar to the parental cell line . WEHI-3B D+/lacZ 2.8 expressed high levels of beta-galactosidase following prolonged in vitro growth and following differentiation in suspension cultures and clonogenic assays . In vivo, WEHI-3B D+/lacZ 2.8 was leukemogenic and high level expression of beta-galactosidase was maintained . Quantification of tissue involvement with WEHI-3B D+/lacZ 2.8 leukemia was performed utilizing staining with the fluorogenic beta-galactosidase substrate fluorescein di-beta-galactoside and fluorescence-activated cell sorting analysis . In vivo differentiation efficiency following granulocyte colony-stimulating factor (G-CSF) administration was determined using a simultaneous nuclear and cytoplasmic staining procedure . Results indicate that treatment of mice inoculated with WEHI-3B D+/lacZ 2.8 cells with G-CSF administration causes detectable but limited differentiation.

Endocrinology, 1993 Feb, 132(2), 539 - 45
Regulation of proteins in the cholesterol side-chain cleavage system in JEG-3 and Y-1 cells; Black SM et al.; The conversion of cholesterol to pregnenolone, the rate-limiting step in steroid hormone synthesis, occurs on mitochondrial cytochrome P450scc, which catalyzes this reaction by receiving electrons from NADPH via a flavoprotein {adrenodoxin reductase (AdRed)} and an iron sulfur protein {adrenodoxin (Adx)} . The behavior of the genes and mRNAs encoding these proteins has been studied in several systems, but little is known about the behavior of the human proteins . Using cloned cDNAs for human P450scc and AdRed, we constructed bacterial expression vectors to make milligram quantities of the corresponding proteins . These, plus purified human Adx similarly prepared by Dr . L . Vickery, were injected into rabbits to raise antiserum to each of the proteins . Each antiserum was highly specific and did not cross-react with other mitochondrial proteins detectable by Western blotting . Human JEG-3 choriocarcinoma cells and mouse Y-1 adrenocortical carcinoma cells were then incubated for 0-24 h with 1 mM 8-bromo-cAMP (8Br-cAMP) or 30 nM phorbol 12-myristate 13-acetate (PMA; phorbol ester) plus 1 microM A23187 (calcium ionophore) to activate the protein kinase-A and -C pathways, respectively . In JEG-3 cells, 8Br-cAMP increased and PMA/A23187 slightly decreased the abundance of P450scc and Adx, but neither treatment had a detectable effect on AdRed . The production of pregnenolone by these cells increased 3-fold in response to 8Br-cAMP and fell to one third in response to PMA/A23187 . In Y-1 cells, 8Br-cAMP increased the abundance of all three proteins, while PMA/A23187 decreased the abundance of P450scc and Adx . The production of pregnenolone by these cells increased 9-fold in response to 8Br-cAMP and was unaffected by TPA/A23187 . These studies show that the three proteins of the cholesterol side-chain cleavage system behave in response to 8Br-cAMP and PMA/A23187 as predicted from the study of their genes and mRNAs, indicating that the chronic regulation of steroidogenesis in these cell systems is regulated principally at the level of mRNA abundance.

Protein Expr Purif, 1993 Feb, 4(1), 85 - 94
The nonbiotinylated form of the 1.3 s subunit of transcarboxylase binds to avidin (monomeric)-agarose: purification and separation from the biotinylated 1.3 S subunit; Shenoy BC et al.; Avidin-biotin technology is used routinely to purify biotin-containing carboxylases and also proteins that have been chemically coupled to biotin . The 1.3 S subunit of transcarboxylase (TC) studied here is the biotin-containing subunit of TC which not only acts as a carboxyl carrier between the CoA ester sites on the central 12 S subunit of TC and keto acid sites on the outer 5 S subunit of TC but also links the 12 S and 5 S subunits together to form a 26 S multisubunit TC complex . The 1.3 S subunit has been cloned, sequenced, and expressed in Escherichia coli . A method for purifying recombinant 1.3 S subunits from E . coli using avidin (monomeric)-agarose column chromatography has been developed . This affinity-purified 1.3 S was found to be homogeneous by SDS-PAGE, amino acid composition, and N-terminal sequence analysis but had a biotin content of only 28% based on moles of biotin per mole of 1.3 S . This lack of stoichiometry was found to be due to copurification of apo-1.3 S as evidenced by the holocarboxylase synthetase reaction . A procedure for separating the apo- and biotinylated 1.3 S forms using hydrophobic interaction chromatography on an Ether 5 PW column is described . The method is based on the difference in hydrophobicity between apo and biotinylated 1.3 S forms . The copurification of apo and biotinylated forms of 1.3 S on the avidin (monomeric)-agarose column was found to be due to specific interaction with avidin rather than to interaction between apo- and biotinylated 1.3 S forms as demonstrated by the fluorescence quenching studies . The results suggest that the avidin-biotin system by itself may not be sufficient to obtain homogeneous biotinyl proteins as nonbiotinyl protein can also bind avidly to such columns.

Protein Expr Purif, 1993 Feb, 4(1), 8 - 15
Expression and purification of the adenovirus proteinase polypeptide and of a synthetic proteinase substrate; Anderson CW; Simple methods are described for expressing the endoproteinase polypeptides of adenovirus serotypes 2 and 12 in Escherichia coli and for purifying these products from crude bacterial extracts using immobilized metal affinity chromatography . A plasmid for expressing an artificial adenovirus substrate that can be purified by the same method also is described . Cleavage of this substrate by the Ad2 virion proteinase confirmed that the cleavage specificity of the adenovirus proteinase is determined by the four amino acids immediately before the cleavage site . The purified recombinant Ad2 endoproteinase alone was incapable of cleaving the artificial substrate, but cleavage occurred if the artificial substrate was incubated with both recombinant endoproteinase and H2ts1 virions or heat-inactivated wild-type Ad2 virions . These results indicate that, in addition to the 23-kDa proteinase polypeptide, cofactors present in Ad2 virions are required to produce active adenovirus proteinase.

Protein Expr Purif, 1993 Feb, 4(1), 59 - 63
Improved procedure for a high-yield recovery of enzymatically active recombinant calf chymosin from Escherichia coli inclusion bodies; Tichy PJ et al.; The high-yield recovery of enzymatically active recombinant calf chymosin from Escherichia coli inclusion bodies was achieved by optimization of solubilization and renaturation conditions . The solubilization was carried out in 8 M urea at various pHs, at various temperatures, and for various periods of time . The following values were found optimal: 1 h at 31 degrees C, pH 10.4 . For successful correct refolding of solubilized prochymosin molecules it was found to be necessary to dilute the solution into an alkaline buffer (pH 10.7) in such a way that the final concentration of urea did not exceed 0.32 M and that of protein 0.275 mg/ml . Our optimized procedure gives about eight times higher yields of enzymatically active chymosin than the current published methods.

Protein Expr Purif, 1993 Feb, 4(1), 32 - 7
Expression, purification, and functional characterization of the DNA-binding domain of the herpes simplex virus type 1 UL9 protein; Martinez R et al.; The origin-binding protein (UL9 protein) encoded by the herpes simplex virus type 1 (HSV-1) UL9 gene plays a critical role in the initiation of viral DNA replication . The 317-amino acid C-terminal DNA-binding domain (UL9-COOH) of the UL9 protein was expressed as a fusion protein to glutathione S-transferase in bacteria . Milligram quantities of the fusion protein were purified by differential ammonium sulfate precipitation and affinity chromatography over reduced glutathione agarose . The UL9-COOH moiety was cleaved from the fusion protein by proteolysis with thrombin . The purified UL9-COOH displayed specific and stable binding activity toward its DNA recognition sequences within the HSV-1 origin of DNA replication.

Protein Expr Purif, 1993 Feb, 4(1), 24 - 31
Rapid purification of monomer HIV-2 Tat protein expressed in Escherichia coli; Rhim H et al.; Human immunodeficiency viruses types 1 and 2 encode transactivator proteins, named Tat-1 and Tat-2, that stimulate transcription directed by the viral long terminal repeat sequences . The Tat-1 and Tat-2 proteins are related in protein sequence and mechanism of transactivation . We expressed Tat proteins by in vitro translation and found, as expected, that both Tat-1 and Tat-2 were monomers . Unexpectedly, we found that the Tat-1 and Tat-2 proteins displayed significantly different tertiary structures . As determined by velocity sedimentation and gel filtration, Tat-1 acted as a compact structure and Tat-2 acted as an extended, asymmetric structure . Additionally, we found that the in vitro-expressed Tat-2 protein was significantly less susceptible to aggregation than the Tat-1 protein . This observation led us to overexpress Tat-2 in Escherichia coli and develop a simple and rapid method to purify monomer Tat-2 to approximately 50% purity . These results suggest that large amounts of Tat-2 protein can be purified in suitable form for biochemical and biophysical studies of Tat protein structure.

Protein Expr Purif, 1993 Feb, 4(1), 16 - 23
Refolding of recombinant Pneumocystis carinii dihydrofolate reductase and characterization of the enzyme; Delves CJ et al.; The isolation of dihydrofolate reductase (DHFR) cDNA sequences from the messenger RNA of Pneumocystis carinii using the polymerase chain reaction is described . The 206-amino acid P . carinii DHFR was expressed to high levels in Escherichia coli inclusion bodies using the T7 promoter expression system . Solubilization of the inclusion bodies in 4 M guanidine hydrochloride and refolding of the recombinant protein in the presence of 0.5% polyethylene glycol 1450 yielded correctly folded DHFR which was purified to homogeneity by methotrexate-Sepharose affinity chromatography . The refolded enzyme was readily crystallized as a ternary complex with NADPH and various inhibitors . The enzyme exhibited a sharp pH optimum with maximum activity at pH 7.0 (turnover number = 6500 min-1) . Km values for dihydrofolate (DHF) and NADPH were 2.3 and 3.0 microM, respectively, in 0.1 m imidazole buffer, pH 7 . Folate did not act as a substrate . Comparison of the kinetic properties of the refolded enzyme with soluble P . carinii DHFR expressed at low levels in the T7 expression system showed similar pH-activity profiles, Km values for DHF and NADPH, and IC50 values for several known antifolates which were tested as inhibitors of the enzyme.

Mol Cell Biol, 1993 Feb, 13(2), 1238 - 50
In vivo and in vitro analysis of transcriptional activation mediated by the human cytomegalovirus major immediate-early proteins; Klucher KM et al.; To define mechanistically how the human cytomegalovirus (HCMV) major immediate-early (IE) proteins induce early-gene transcription, the IE1 72-kDa protein, the IE2 55-kDa protein, and the IE2 86-kDa protein were analyzed for their ability to activate transcription from an HCMV early promoter in vivo and in vitro . In transient-expression assays in U373MG astrocytoma/glioblastoma and HeLa cells, only the IE2 86-kDa protein was able to activate the HCMV early promoter to high levels . In HeLa cells, the IE1 72-kDa protein was able to activate the promoter to a low but detectable level, and the level of promoter activity observed in response to the IE2 86-kDa protein was increased synergistically following cotransfection of the constructs expressing both IE proteins . To examine the interaction of the HCMV IE proteins with the RNA polymerase II transcription machinery, we assayed the ability of Escherichia coli-synthesized proteins to activate the HCMV early promoter in nuclear extracts prepared from U373MG cells, HeLa cells, and Drosophila embryos . The results of the in vitro experiments correlated well with those obtained in vivo . The basal activity of the promoter was minimal in both the HeLa and U373MG extracts but was stimulated 6- to 10-fold by the IE2 86-kDa protein . With a histone H1-deficient extract from Drosophila embryos, the HCMV early promoter was quite active and was stimulated two- to fourfold by the IE2 86-kDa protein . Addition of histone H1 at 1 molecule per 40 to 50 bp of DNA template significantly repressed basal transcription from this promoter . However, the IE2 86-kDa protein, but none of the other IE proteins, was able to counteract the H1-mediated repression and stimulate transcription at least 10- to 20-fold . The promoter specificity of the activation was demonstrated by the inability of the IE2 86-kDa protein to activate the Drosophila Kruppel promoter in either the presence or absence of histone H1 . These results suggest that one mechanism of transcription activation by the IE2 86-kDa protein involves antirepression.

J Immunol, 1993 Feb 1, 150(3), 707 - 16
A monoclonal antibody to recombinant human IFN-alpha receptor inhibits biologic activity of several species of human IFN-alpha, IFN-beta, and IFN-omega . Detection of heterogeneity of the cellular type I IFN receptor; Benoit P et al.; A cDNA encoding a 63-kDa human IFN-alpha R (hIFN-alpha R) has recently been cloned . Mouse cells transfected with this cDNA failed to show any signal transmission for IFN-beta, suggesting the involvement of additional chains in the receptor complex . We have expressed in Escherichia coli and COS 7 cells a soluble recombinant protein (sIFN-alpha R) comprising the extracellular domain of the hIFN-alpha R fused at the carboxyl terminus to a sequence of five histidine residues . The sIFN-alpha R was affinity purified and used to generate mAb . One mAb, 64G12, which recognized the cellular receptor as well as the recombinant proteins was found to neutralize the biologic activity of IFN-alpha, -beta, and -omega and natural type I IFN, but not IFN-gamma . To our knowledge, this is the first report of a neutralizing mAb against the human IFN-alpha R . Interestingly, we found that the affinity of this antibody for the cellular receptor depended on the cell line used . These results provide strong evidence that the cloned hIFN-alpha R chain is required for binding and signal transmission of all subspecies of type I IFN and suggest a structural heterogeneity of the receptor at the cell surface.

J Bacteriol, 1993 Feb, 175(3), 909 - 11
Viability of folA-null derivatives of wild-type (thyA+) Escherichia coli K-12; Krishnan BR et al.; Dihydrofolate reductase (the folA gene product) catalyzes the synthesis of tetrahydrofolate, a key methyl donor in many biosynthetic pathways . Loss of folA had been thought to be lethal to wild-type (thyA+) Escherichia coli . Viable folA-null derivatives of thyA+ E . coli were obtained, however, by recombining a folA deletion linked to a Kanr selectable marker into a lambda folA+ phage and using this phage to transduce cells to kanamycin resistance . folA-null strains were slow growing, formed small colonies, and were auxotrophic for thymidine, adenine, methionine, glycine, and pantothenate.

J Bacteriol, 1993 Feb, 175(3), 902 - 4
Positive regulation of the Escherichia coli glycine cleavage enzyme system; Wilson RL et al.; A new mutation in Escherichia coli, designated gcvA1, that results in noninducible expression of both gcv and a gcvT-lacZ gene fusion was isolated . A plasmid carrying the wild-type gcvA gene complemented the mutation and restored glycine-inducible gcv and gcvT-lacZ gene expression . These results suggest that gcvA encodes a positive-acting regulatory protein that acts in trans to increase expression of gcv.

J Bacteriol, 1993 Feb, 175(3), 858 - 65
Combinatorial mutagenesis of the lamB gene: residues 41 through 43, which are conserved in Escherichia coli outer membrane proteins, are informationally important in maltoporin structure and function; Chan WC et al.; A new strategy for combinatorial mutagenesis was developed and applied to residues 40 through 60 of LamB protein (maltoporin), with the aim of identifying amino acids important for LamB structure and function . The strategy involved a template containing a stop codon in the target sequence and a pool of random degenerate oligonucleotides covering the region . In vitro mutagenesis followed by selection for function (Dex+, ability to utilize dextrins) corrected the nonsense mutation and simultaneously forced incorporation of a random mutation(s) within the region . The relative importance of each residue within the target was indicated by the frequency and nature of neutral and deleterious mutations recovered at each position . Residues 41 through 43 in LamB accepted few neutral substitutions, whereas residues 55 through 57 were highly flexible in this regard . Consistent with this finding was that the majority of defective mutants were altered at residues 41 to 43 . Characterization of these mutants indicated that the nature of residues 41 to 43 influenced the amount of stable protein in the outer membrane . These results, as well as the conserved nature of this stretch of residues among outer membrane proteins, suggest that residues 41 to 43 of LamB play an important role in the process of outer membrane localization.

J Bacteriol, 1993 Feb, 175(3), 693 - 700
Xis and Fis proteins prevent site-specific DNA inversion in lysogens of phage HK022; Dorgai L et al.; HK022, a temperate coliphage related to lambda, forms lysogens by inserting its DNA into the bacterial chromosome through site-specific recombination . The Escherichia coli Fis and phage Xis proteins promote excision of HK022 DNA from the bacterial chromosome . These two proteins also act during lysogenization to prevent a prophage rearrangement: lysogens formed in the absence of either Fis or Xis frequently carried a prophage that had suffered a site-specific internal DNA inversion . The inversion is a product of recombination between the phage attachment site and a secondary attachment site located within the HK022 left operon . In the absence of both Fis and Xis, the majority of lysogens carried a prophage with an inversion . Inversion occurs during lysogenization at about the same time as prophage insertion but is rare during lytic phage growth . Phages carrying the inverted segment are viable but have a defect in lysogenization, and we therefore suggest that prevention of this rearrangement is an important biological role of Xis and Fis for HK022 . Although Fis and Xis are known to promote excision of lambda prophage, they had no detectable effect on lambda recombination at secondary attachment sites . HK022 cIts lysogens that were blocked in excisive recombination because of mutation in fis or xis typically produced high yields of phage after thermal induction, regardless of whether they carried an inverted prophage . The usual requirement for prophage excision was bypassed in these lysogens because they carried two or more prophages inserted in tandem at the bacterial attachment site; in such lysogens, viable phage particles can be formed by in situ packaging of unexcised chromosomes.

J Bacteriol, 1993 Feb, 175(3), 661 - 8
Transcription of the Escherichia coli rrnB P1 promoter by the heat shock RNA polymerase (E sigma 32) in vitro; Newlands JT et al.; The P1 promoters of the seven Escherichia coli rRNA operons contain recognition sequences for the RNA polymerase (RNAP) holoenzyme containing sigma 70 (E sigma 70), which has been shown to interact with and initiate transcription from rrn P1 promoters in vivo and in vitro . The rrn P1 promoters also contain putative recognition elements for E sigma 32, the RNAP holoenzyme responsible for the transcription of heat shock genes . Using in vitro transcription assays with purified RNAP holoenzyme, we show that E sigma 32 is able to transcribe from the rrnB P1 promoter . Antibodies specific to sigma 70 eliminate transcription of rrnB P1 by E sigma 70 but have no effect on E sigma 32-directed transcription . Physical characterization of the E sigma 32-rrnB P1 complex shows that there are differences in the interactions made by E sigma 70 and E sigma 32 with the promoter . E sigma 32 responds to both Fis-mediated and factor-independent upstream activation, two systems shown previously to stimulate rrnB P1 transcription by E sigma 70 . We find that E sigma 32 is not required for two major control systems known to regulate rRNA transcription initiation at normal temperatures in vivo, stringent control and growth rate-dependent control . On the basis of the well-characterized role of E sigma 32 in transcription from heat shock promoters in vivo, we suggest that E sigma 32-directed transcription of rRNA promoters might play a role in ribosome synthesis at high temperatures.

J Bacteriol, 1993 Feb, 175(3), 636 - 41
Autonomous replication of foreign DNA in Histoplasma capsulatum: role of native telomeric sequences; Woods JP et al.; Genetic transformation of the dimorphic pathogenic fungus Histoplasma capsulatum can result in chromosomal integration of the transforming DNA or the generation of multicopy linear plasmids carrying the transforming DNA . We showed previously that Escherichia coli plasmids do not replicate autonomously in H . capsulatum without significant modifications, one of which is the in vivo addition of Histoplasma telomeres at the termini of linear DNA . To address the requirements for autonomous replication in H . capsulatum, we constructed a circular E . coli plasmid containing adjacent inverted stretches of Histoplasma telomeric repeats separated by a unique restriction site . The linearized plasmid bearing telomeric termini was maintained in H . capsulatum without modification other than the addition of more telomeric sequence . We recovered the original plasmid in E . coli after removal of the telomeric termini by using engineered restriction sites . Thus, no special Histoplasma modification or sequence other than the telomeres was needed for autonomous replication in H . capsulatum . Additionally, this plasmid provides a shuttle vector that replicates autonomously in E . coli (as a circular plasmid) and in H . capsulatum (as a linear plasmid).

J Bacteriol, 1993 Feb, 175(3), 630 - 5
The product of the hypB gene, which is required for nickel incorporation into hydrogenases, is a novel guanine nucleotide-binding protein; Maier T et al.; The products of the hyp operon genes are essential for the formation of catalytically active hydrogenases in Escherichia coli . At least one of these auxiliary proteins, HYPB, appears to be involved in nickel liganding to the hydrogenase apoprotein, since mutations in hypB can be phenotypically suppressed by high nickel concentrations in the medium (R . Waugh and D . H . Boxer, Biochimie 68:157-166, 1986) . To approach the identification of the specific function of HYPB, we overexpressed the hypB gene and purified and characterized the gene product . HYPB is a homodimer of 31.6-kDa subunits, and it binds guanine nucleotides, with a Kd for GDP of 1.2 microM . The protein displays a low level of GTPase activity, with a kcat of 0.17 min-1 . The apparent Km for GTP, as measured in the GTP hydrolysis reaction, was determined to be 4 microM . A chromatography system was established to measure nickel insertion into hydrogenase 3 from E . coli and to determine the effects of lesions in hypB . Nickel appears to be associated only with the processed large subunit of hydrogenase 3 in the wild type, and hypB mutants accumulate the precursor form of this subunit, which is devoid of nickel . The results are discussed in terms of a model in which HYPB is involved in nickel donation to the hydrogenase apoprotein and in which GTP hydrolysis is thought to reverse the interaction between either HYPB or another nickel-binding protein and the hydrogenase apoprotein after the nickel has been released.

J Bacteriol, 1993 Feb, 175(3), 589 - 96
Construction and biochemical characterization of recombinant cytoplasmic forms of the IucD protein (lysine:N6-hydroxylase) encoded by the pColV-K30 aerobactin gene cluster; Thariath A et al.; The aerobactin gene cluster in pColV-K30 consists of five genes (iucABCD iutA); four of these (iucABCD) are involved in aerobactin biosynthesis, whereas the fifth one (iutA) encodes the ferriaerobactin outer membrane receptor . iucD encodes lysine:N6-hydroxylase, which catalyzes the first step in aerobactin biosynthesis . Regardless of the method used for cell rupture, we have consistently found that IucD remains membrane bound, and repeated efforts to achieve a purified and active soluble form of the enzyme have been unsuccessful . To circumvent this problem, we have constructed recombinant IucD proteins with modified amino termini by creating three in-frame gene fusions of IucD to the amino-terminal amino acids of the cytoplasmic enzyme beta-galactosidase . Two of these constructs resulted in the addition to the iucD coding region of a hydrophilic leader sequence of 13 and 30 amino acids . The other construct involved the deletion of the first 47 amino acids of the IucD amino terminus and the addition of 19 amino acids of the amino terminus of beta-galactosidase . Cells expressing any of the three recombinant IucD forms were found to produce soluble N6-hydroxylysine . One of these proteins, IucD439, was purified to homogeneity from the soluble fraction of the cell lysates, and it was capable of participating in the biosynthesis of aerobactin, as determined in vitro by a cell-free system and in vivo by a cross-feeding bioassay . A medium ionic strength of 0.25 (250 mM NaCl) or higher was required to maintain the protein in a catalytically functional, tetrameric state.(ABSTRACT TRUNCATED AT 250 WORDS)

Infect Immun, 1993 Feb, 61(2), 760 - 3
Calcium-calmodulin dependence of actin accretion and lethality in cultured HEp-2 cells infected with enteropathogenic Escherichia coli; Baldwin TJ et al.; Infection of cultured HEp-2 cells with enteropathogenic Escherichia coli causes substantial actin accretion at points of bacterial contact and cell death . Loss of viability was delayed by chelating intracellular free calcium . Actin accretion was partially inhibited by preventing elevation of free cytosolic calcium and prevented by treatment with a calmodulin inhibitor.

Infect Immun, 1993 Feb, 61(2), 565 - 72
Molecular characterization of a protective outer membrane lipoprotein (OmlA) from Actinobacillus pleuropneumoniae serotype 1; Gerlach GF et al.; An expression library was constructed from an Actinobacillus pleuropneumoniae serotype 1 clinical isolate using a plasmid vector . The library was screened with serum raised against the culture supernatant of this strain . One Escherichia coli transformant which also reacted with convalescent serum was isolated and found to express a protein with an electrophoretic mobility of approximately 50,000 . The A . pleuropneumoniae-derived DNA encoding the protein was localized and characterized by nucleotide sequence analysis and primer extension mapping . One open reading frame of 1,095 bases was detected and confirmed by TnphoA insertion mutagenesis . It encoded a protein with a calculated molecular mass of 40 kDa which was lipid modified and present in the outer membrane and in membrane blebs of A . pleuropneumoniae . This protein was designated as outer membrane lipoprotein A (OmlA), and the encoding gene as omlA . Southern blotting under low-stringency conditions revealed the presence of hybridizing sequences in all A . pleuropneumoniae type strains, and a specific serum detected a homologous protein in serotypes 2, 8, 9, 11, and 12 type strains . Pigs immunized with this recombinant protein preparation were protected from death in an aerosol challenge experiment with an A . pleuropneumoniae serotype 1 isolate.

Infect Immun, 1993 Feb, 61(2), 491 - 7
Gamma interferon-induced nitric oxide production reduces Chlamydia trachomatis infectivity in McCoy cells; Mayer J et al.; McCoy cells, murine-derived cells commonly used for propagation of chlamydiae, were found to be efficient producers of nitric oxide (NO) when primed with murine gamma interferon (IFN-gamma) and then exposed to the second signals provided by Escherichia coli lipopolysaccharide, human interleukin-1 alpha, murine tumor necrosis factor alpha, or Chlamydia trachomatis type H . Murine recombinant IFN-gamma over a range of 0 to 50 U/ml inhibited infectivity of C . trachomatis type H in a dose-dependent fashion in McCoy cells while simultaneously inducing NO production . Quantitation of infectious chlamydia progeny remaining in McCoy cells 48 or 72 h postinfection revealed that IFN-gamma-primed McCoy cells reduced chlamydial inclusion-forming units (expressed as units per milliliter) by 4 log10 units at higher IFN-gamma concentrations (50 U/ml) compared with control values . The magnitude of this antichlamydial effect was directly related to increased synthesis of NO, the production of which was IFN-gamma dose dependent . The antichlamydial effects of IFN-gamma were blocked in a dose-dependent manner by the addition of N-guanidino-monomethyl L-arginine (MLA), an inhibitor of nitric oxide synthesis . These results suggest that although IFN-gamma priming of McCoy cells is required for antichlamydial activity, nitric oxide is a necessary effector molecule involved in the mechanism(s) of IFN-gamma-induced inhibition of chlamydial proliferation in this murine cell line . The ability to block the potent antichlamydial effects of IFN-gamma by inhibition of a specific enzyme, nitric oxide synthase, may give insights into mechanisms by which IFN-gamma and perhaps other cytokines are able to control proliferation of chlamydiae and other intracellular pathogens.

Infect Immun, 1993 Feb, 61(2), 470 - 7
Cloning and sequencing of Coxiella burnetii outer membrane protein gene com1; Hendrix LR et al.; The gene for an approximately 27-kDa outer membrane-associated, immunoreactive protein was cloned from the rickettsial pathogen Coxiella burnetii . The gene, designated com1 for Coxiella outer membrane protein 1, was expressed in Escherichia coli, presumably by its own promoter . The complete nucleotide sequence of the gene was determined . The deduced amino acid sequence of 252 residues includes a putative leader sequence . The leader sequence is recognized in and removed by E . coli on the basis of the difference in the molecular mass of the protein produced in an in vitro transcription-translation system (27.6 kDa) and that of the protein immunoprecipitated from an iodinated E . coli clone (25.7 kDa) . The Com1 protein expressed in E . coli was proteinase K sensitive in nondisrupted cells and soluble in 1% Sarkosyl, suggesting a loose association with the outer membrane . While the complete predicted sequence of the Com1 protein does not show any overall similarity to those of previously described proteins, a region which includes the only two cysteines in Com1 is homologous to the catalytic site of protein disulfide oxidoreductases.

Infect Immun, 1993 Feb, 61(2), 411 - 7
Cloning, heterologous expression, and characterization of the Erysipelothrix rhusiopathiae DnaK protein; Partridge J et al.; The dnaK (hsp70) gene from the facultative intracellular pathogen Erysipelothrix rhusiopathiae was cloned by heterologous DNA hybridization of a genomic library using the Escherichia coli dnaK gene as a probe . A 3.2-kb fragment which encoded an 1,800-bp open reading frame was recovered . The deduced amino acid sequence of this open reading frame shares 56% identity with the E . coli DnaK protein . Expression of the encoded protein in E . coli by using the phage T7 promoter/polymerase system resulted in accumulation of a unique 65-kDa protein . Western blot (immunoblot) analysis of extracts from a recombinant E . coli strain using anti-E . coli DnaK polyclonal antibodies confirmed that the cloned gene encodes a DnaK homolog . The recombinant E . rhusiopathiae DnaK protein was purified to 80% homogeneity by ATP affinity chromatography . The purified material hydrolyzed ATP with a specific activity of 100 nmol min-1 mg of protein-1 . Analysis of total protein extracts from E . rhusiopathiae indicates that DnaK is a highly expressed protein in this organism.

Virology, 1993 Feb, 192(2), 643 - 50
Biological activity of cauliflower mosaic virus aphid transmission factor expressed in a heterologous system; Blanc S et al.; Aphid transmission factor (ATF) activity of cauliflower mosaic virus (CaMV) gene II product was recovered after expression of the gene by a baculovirus recombinant . The expression product, when first acquired by aphids through parafilm membrane, was able to mediate the transmission of two aphid nontransmissible isolates (CM1841, CM4-184) providing the first direct evidence that the product of the gene II is the CaMV ATF . The CaMV ATF in its active conformation has a strong tendency to aggregate and all attempts at solubilizing it resulted in the loss of the ATF activity . The CaMV ATF was also expressed in Escherichia coli, using the pGEX 3X plasmid vector, as a fusion protein to glutathione S-transferase (GST) and was purified . The fusion product (GST-P18), whether purified or not, was not able to complement the transmission of transmission-defective isolates . However, when GST-P18 was added to some extracts from a plant infected with an aphid-transmissible isolate (Cabb B-JI), the transmission was inhibited . This suggests that it could be possible to block the in vitro transmission of CaMV using a molecule analogous to the ATF.

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