Biochem Biophys Res Commun, 1997 Dec 8, 241(1), 122 - 6 Control of expression of PLCbeta1 by LAC repressor system: relationship between nuclear PLCbeta1 overexpression and growth factor stimulation; Billi AM et al.; Swiss 3T3 cells have a nuclear phosphoinositide signalling system which is under the control of insulin-like growth factor I (IGF-I) and acts separately from that at the plasma membrane . By using the Lac repressor system we were able both to obtain the inducible overexpression of phospholipase C beta1 (PLC beta1) and to determine its subcellular localisation and partitioning . Moreover, by comparing the level of expression at the nucleus and the percentage of cells actively incorporating bromodeoxyuridine (BrdU) in S phase it has strengthened the issue of the importance of this PLC in the onset of DNA synthesis mediated by IGF-I . In addition, this system appears to be a very powerful tool for further analysis of the downstream events following the activation of nuclear PLC beta1 . Biochem Biophys Res Commun, 1997 Dec 8, 241(1), 79 - 85 Evidence for CYP2D6 expression in human lung; Guidice JM et al.; The cytochrome P450 CYP2D6 gene (CYP2D6) expression was examined in samples from human bronchial mucosa and lung parenchyma using reverse transcription-polymerase chain reaction (RT-PCR) and immunochemistry . Except specimen from a patient previously genotyped as homozygous for a complete deletion of the gene, all tissue samples were positive . When compared to that in the liver, the mean level of CYP2D6 mRNA was 3-fold lower in bronchial mucosa and 6-fold lower in lung parenchyma . To our knowledge, these data demonstrate for the first time the presence of CYP2D6 protein in human lung . They also indicate that the gene is nonuniformly distributed within this organ . The possibility that CYP2D6 has a role in lung carcinogenesis by locally activating inhaled chemicals should therefore be considered . Methods, 1997 Oct, 13(2), 123 - 33 Development of molecular genetics for Neospora caninum: A complementary system to Toxoplasma gondii; Howe DK et al.; The development of molecular genetics has greatly enhanced the study of Toxoplasma gondii, and investigations into the biology and pathology associated with neosporosis will be similarly benefited by the development of molecular tools for Neospora caninum . We have demonstrated the feasibility of using the existing DNA vectors developed for T . gondii to transfect and transform the Nc-1 strain of Neospora . We have also shown that T . gondii proteins are faithfully expressed and targeted in N . caninum, indicating the suitability of using Neospora as a heterologous expression system for studying T . gondii . These studies provide the basis for initiating molecular genetic studies on N . caninum and will allow for a number of molecular comparisons of these two closely related, though phenotypically distinct, parasites . Here we describe the methods and reagents used to perform genetic manipulations of N . caninum, and we present some of the principles and potential utilities of these molecular studies, including the use of N . caninum as a heterologous system for the study of T . gondii proteins . J Pharmacol Exp Ther, 1997 Dec, 283(3), 1425 - 32 Isolation, heterologous expression and functional characterization of a novel cytochrome P450 3A enzyme from a canine liver cDNA library; Fraser DJ et al.; A cDNA encoding a new member of the cytochrome P450 3A subfamily, P450 3A26, has been isolated from phenobarbital-induced canine liver . The sequence encodes a protein of 503 amino acids with 33 nucleotide differences conferring 22 amino acid substitutions when compared with the previously identified canine CYP3A12 enzyme . Nine of the amino acid differences are within the substrate recognition sites (SRSs) identified for P450 family 2, with five residue substitutions clustered within SRS-6 . To facilitate heterologous expression in Escherichia coli, the N-terminus of 3A26 was modified . The expressed protein comigrated with a 3A-immunoreactive protein in dog liver microsomes with a slightly greater electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than 3A12, which suggests that 3A26 corresponds to a previously noted but never characterized 3A enzyme in dogs . Functional characterization of 3A26 was undertaken with use of progesterone, testosterone and androstenedione as substrates . Assays of expressed 3A26 and 3A12 demonstrated that 3A26 displays low steroid hydroxylase activity . Identification of an additional canine 3A enzyme should increase our understanding of xenobiotic metabolism in this important animal model . These findings also suggest that 3A26 and 3A12 may be an interesting model system for the investigation of structure-function relationships involved in steroid metabolism catalyzed by members of the cytochrome P450 3A subfamily. J Mol Biol, 1997 Nov 28, 274(2), 268 - 75 Folding of barnase in the presence of the molecular chaperone SecB; Stenberg G et al.; SecB is a molecular chaperone dedicated to interact exclusively with proteins destined for translocation across membranes . We find that SecB interacts with barnase during its folding in a similar manner to its interaction with GroEL . On mixing acid-denatured barnase with SecB in a stopped-flow spectrofluorimeter under conditions that favour refolding, we observe a series of fluorescence changes, corresponding to the binding of the denatured protein and the subsequent refolding of multiply and singly bound forms . The different phases were assigned using a combination of kinetics and mutant proteins . The refolding of barnase when bound to SecB is strongly retarded but never blocked . Multiply bound barnase is less tightly bound and refolds with a higher rate constant than singly bound barnase . Up to 4 mol of denatured barnase bind to 1 mol of tetrameric SecB . J Mol Biol, 1997 Nov 28, 274(2), 145 - 51 Clarification of the dimerization domain and its functional significance for the Escherichia coli nucleoid protein H-NS; Ueguchi C et al.; The Escherichia coli nucleoid protein, H-NS, functions as a global regulator for expression of a wide variety of genes . We recently analyzed the structure-function relationship of H-NS with special reference to the domains responsible for transcriptional repression and DNA-binding, respectively . However, identification of the presumed dimerization domain of H-NS and its functional significance was elusive . To address this particular issue, we first examined a set of N-terminally or C-terminally truncated forms of H-NS, in terms of their so-called dominant-negative effect on the in vivo function of the wild-type H-NS . The results showed that certain truncated forms exhibit such a dominant-negative effect, but others did not . As judged by the results of the dominant-negative effect, it was assumed that a relatively central portion of H-NS extending from residues 21 to 63 is involved in dimerization . This was confirmed by an in vitro chemical cross-linking analysis and a gel filtration analysis with these truncated forms of H-NS . Furthermore, the use of the dominant-negative phenotype, caused by a truncated form of H-NS (named N91), allowed us to isolate a missense mutant, which was expected to be specifically defective in dimerization . This mutant had an amino acid substitution at position 30 (Leu30 to Pro) in N91 consisting of the N-terminal 91 amino acids of H-NS . This mutant was indeed defective in the in vitro ability to form a heterodimer with the wild-type H-NS . When this particular single amino acid substitution was introduced into the full-length H-NS, the resultant H-NS mutant had lost the ability to form dimers in vitro and to function as a transcriptional repressor . These findings collectively provided us with evidence that the ability of H-NS to form a dimer is crucial for H-NS to function as a transcriptional repressor . J Biol Chem, 1997 Dec 12, 272(50), 31712 - 8 Specific interaction of eukaryotic translation initiation factor 5 (eIF5) with the beta-subunit of eIF2; Das S et al.; Eukaryotic translation initiation factor 5 (eIF5) interacts with the 40 S initiation complex (40 S.mRNA . eIF3.Met-tRNAf.eIF2.GTP) and mediates hydrolysis of the bound GTP . To characterize the molecular interactions involved in eIF5 function, we have used 32P-labeled recombinant rat eIF5 as a probe in filter overlay assay to identify eIF5-interacting proteins in crude initiation factor preparations . We observed that eIF5 specifically interacted with the beta subunit of initiation factor eIF2 . No other initiation factors including the gamma subunit of eIF2 tested positive in this assay . Furthermore, both yeast and mammalian eIF5 bind to the beta subunit of either mammalian or yeast eIF2 . Binding analysis with human eIF2beta deletion mutants expressed in Escherichia coli identified a 22-amino acid domain, between amino acids 68 and 89, as the primary eIF5-binding region of eIF2beta . These results along with our earlier observations that (a) eIF5 neither binds nor hydrolyzes free GTP or GTP bound as Met-tRNAf.eIF2.GTP ternary complex, and (b) eIF5 forms a specific complex with eIF2 suggests that the specific interaction between eIF5 and the beta subunit of eIF2 may be critical for the hydrolysis of GTP during translation initiation. J Biol Chem, 1997 Dec 12, 272(50), 31625 - 9 Cloning, expression, and properties of the regulatory subunit of bovine pyruvate dehydrogenase phosphatase; Lawson JE et al.; cDNA encoding the regulatory subunit of bovine mitochondrial pyruvate dehydrogenase phosphatase (PDPr) has been cloned . Overlapping cDNA fragments were generated by the polymerase chain reaction from bovine genomic DNA and from cDNA synthesized from bovine poly(A)+ RNA and total RNA . The complete cDNA (2885 base pairs) contains an open reading frame of 2634 nucleotides encoding a putative presequence of 31 amino acid residues and a mature protein of 847 residues with a calculated Mr of 95,656 . This value is in agreement with the molecular mass of native PDPr (95,800 +/- 200 Da) determined by matrix-assisted laser desorption-ionization mass spectrometry . The mature form of PDPr was expressed in Escherichia coli as a maltose-binding protein fusion, and the recombinant protein was purified to near homogeneity . It exhibited properties characteristic of the native PDPr, including recognition by antibodies against native bovine PDPr, ability to decrease the sensitivity of the catalytic subunit to Mg2+, and reversal of this inhibitory effect by the polyamine spermine . A BLAST search of protein data bases revealed that PDPr is distantly related to the mitochondrial flavoprotein dimethylglycine dehydrogenase, which functions in choline degradation. J Biol Chem, 1997 Dec 12, 272(50), 31533 - 41 A new mechanism-based radical intermediate in a mutant R1 protein affecting the catalytically essential Glu441 in Escherichia coli ribonucleotide reductase; Persson AL et al.; The invariant active site residue Glu441 in protein R1 of ribonucleotide reductase from Escherichia coli has been engineered to alanine, aspartic acid, and glutamic acid . Each mutant protein was structurally and enzymatically characterized . Glu441 contributes to substrate binding, and a carboxylate side chain at position 441 is essential for catalysis . The most intriguing results are the suicidal mechanism-based reaction intermediates observed when R1 E441Q is incubated with protein R2 and natural substrates (CDP and GDP) . In a consecutive reaction sequence, we observe at least three clearly discernible steps: (i) a rapid decay (k1 >/= 1.2 s-1) of the catalytically essential tyrosyl radical of protein R2 concomitant with formation of an early transient radical intermediate species, (ii) a slower decay (k2 = 0.03 s-1) of the early intermediate concomitant with formation of another intermediate with a triplet EPR signal, and (iii) decay (k3 = 0.004 s-1) of the latter concomitant with formation of a characteristic substrate degradation product . The characteristics of the triplet EPR signal are compatible with a substrate radical intermediate (most likely localized at the 3'-position of the ribose moiety of the substrate nucleotide) postulated to occur in the wild type reaction mechanism as well. J Biol Chem, 1997 Dec 12, 272(50), 31258 - 64 Molecular and biochemical characterization of an endo-beta-1,3- glucanase of the hyperthermophilic archaeon Pyrococcus furiosus; Gueguen Y et al.; We report here the first molecular characterization of an endo-beta-1,3-glucanase from an archaeon . Pyrococcus furiosus is a hyperthermophilic archaeon that is capable of saccharolytic growth . The isolated lamA gene encodes an extracellular enzyme that shares homology with both endo-beta-1,3- and endo-beta-1,3-1,4-glucanases of the glycosyl hydrolase family 16 . After deletion of the N-terminal leader sequence, a lamA fragment encoding an active endo-beta-1,3-glucanase was overexpressed in Escherichia coli using the T7-expression system . The purified P . furiosus endoglucanase has highest hydrolytic activity on the beta-1,3-glucose polymer laminarin and has some hydrolytic activity on the beta-1,3-1,4 glucose polymers lichenan and barley beta-glucan . The enzyme is the most thermostable endo-beta-1,3-glucanase described up to now; it has optimal activity at 100-105 degrees C . In the predicted active site of glycosyl hydrolases of family 16 that show predominantly endo-beta-1,3-glucanase activity, an additional methionine residue is present . Deletion of this methionine did not change the substrate specificity of the endoglucanase, but it did cause a severe reduction in its catalytic activity, suggesting a structural role of this residue in constituting the active site . High performance liquid chromatography analysis showed in vitro hydrolysis of laminarin by the endo-beta-1,3-glucanase proceeds more efficiently in combination with an exo-beta-glycosidase from P . furiosus (CelB) . This most probably reflects the physiological role of these enzymes: cooperation during growth of P . furiosus on beta-glucans. Proc Natl Acad Sci U S A, 1997 Dec 9, 94(25), 13967 - 72 Two forms of replication initiator protein: positive and negative controls; Wu J et al.; The pir gene of plasmid R6K encodes the protein, pi, a replication and transcription factor . Two translational options for the pir gene give rise to two forms of pi protein: a 35.0-kDa form (pi35.0) and a shortened 30.5-kDa form (pi30.5) . Although both proteins bind to a series of 22-bp direct repeats essential for plasmid R6K replication, only pi35.0 can bind to a site in the (A.T)-rich segment of its gamma ori and activate the gamma ori in vivo and in vitro . However, unlike pi35.0, pi30.5can inhibit in vivo and in vitro replication (activated by pi35.0) . We propose that the two forms of pi might have distinct functions in replication . We show that although both forms of pi produce dimers, the nature of these dimers is not identical . The N-terminal 37 amino acid residues appear to control the formation of the more stable pi35.0 dimers, whereas another, apparently weaker interface holds together dimers of pi30.5 . We speculate that the leucine zipper-like motif, absent in pi30.5, controls very specific functions of pi protein. Proc Natl Acad Sci U S A, 1997 Dec 9, 94(25), 13944 - 9 Mutations in dihydropteroate synthase are responsible for sulfone and sulfonamide resistance in Plasmodium falciparum; Triglia T et al.; Plasmodium falciparum causes the most severe form of malaria in humans . An important class of drugs in malaria treatment is the sulfone/sulfonamide group, of which sulfadoxine is the most commonly used . The target of sulfadoxine is the enzyme dihydropteroate synthase (DHPS), and sequencing of the DHPS gene has identified amino acid differences that may be involved in the mechanism of resistance to this drug . In this study we have sequenced the DHPS gene in 10 isolates from Thailand and identified a new allele of DHPS that has a previously unidentified amino acid difference . We have expressed eight alleles of P . falciparum PPPK-DHPS in Escherichia coli and purified the functional enzymes to homogeneity . Strikingly, the Ki for sulfadoxine varies by almost three orders of magnitude from 0.14 microM for the DHPS allele from sensitive isolates to 112 microM for an enzyme expressed in a highly resistant isolate . Comparison of the Ki of different sulfonamides and the sulfone dapsone has suggested that the amino acid differences in DHPS would confer cross-resistance to these compounds . These results show that the amino acid differences in the DHPS enzyme of sulfadoxine-resistant isolates of P . falciparum are central to the mechanism of resistance to sulfones and sulfonamides. Proc Natl Acad Sci U S A, 1997 Dec 9, 94(25), 13909 - 14 How Escherichia coli can bias the results of molecular cloning: preferential selection of defective genomes of hepatitis C virus during the cloning procedure; Forns X et al.; Cloned PCR products containing hepatitis C virus (HCV) genomic fragments have been used for analyses of HCV genomic heterogeneity and protein expression . These studies assume that the clones derived are representative of the entire virus population and that subsets are not inadvertently selected . The aim of the present study was to express HCV structural proteins . However, we found that there was a strong cloning selection for defective genomes and that most clones generated initially were incapable of expressing the HCV proteins . The HCV structural region (C-E1-E2-p7) was directly amplified by long reverse transcription-PCR from the plasma of an HCV-infected patient or from a control plasmid containing a viable full-length cDNA of HCV derived from the same patient but cloned in a different vector . The PCR products were cloned into a mammalian expression vector, amplified in Escherichia coli, and tested for their ability to produce HCV structural proteins . Twenty randomly picked clones derived from the HCV-infected patient all contained nucleotide mutations leading to absence or truncation of the expected HCV products . Of 25 clones derived from the control plasmid, only 8% were fully functional for polyprotein synthesis . The insertion of extra nucleotides in the region just upstream of the start codon of the HCV insert led to a statistically significant increase in the number of fully functional clones derived from the patient (42%) and from the control plasmid (72-92%) . Nonrandom selection of clones during the cloning procedure has enormous implications for the study of viral heterogeneity, because it can produce a false spectrum of genomic diversity . It can also be an impediment to the construction of infectious viral clones. Proc Natl Acad Sci U S A, 1997 Dec 9, 94(25), 13792 - 7 Multiple pathways for SOS-induced mutagenesis in Escherichia coli: an overexpression of dinB/dinP results in strongly enhancing mutagenesis in the absence of any exogenous treatment to damage DNA; Kim SR et al.; dinP is an Escherichia coli gene recently identified at 5.5 min of the genetic map, whose product shows a similarity in amino acid sequence to the E . coli UmuC protein involved in DNA damage-induced mutagenesis . In this paper we show that the gene is identical to dinB, an SOS gene previously localized near the lac locus at 8 min, the function of which was shown to be required for mutagenesis of nonirradiated lambda phage infecting UV-preirradiated bacterial cells (termed lambdaUTM for lambda untargeted mutagenesis) . A newly constructed dinP null mutant exhibited the same defect for lambdaUTM as observed previously with a dinB::Mu mutant, and the defect was complemented by plasmids carrying dinP as the only intact bacterial gene . Furthermore, merely increasing the dinP gene expression, without UV irradiation or any other DNA-damaging treatment, resulted in a strong enhancement of mutagenesis in F'lac plasmids; at most, 800-fold increase in the G6-to-G5 change . The enhanced mutagenesis did not depend on recA, uvrA, or umuDC . Thus, our results establish that E . coli has at least two distinct pathways for SOS-induced mutagenesis: one dependent on umuDC and the other on dinB/P. Proc Natl Acad Sci U S A, 1997 Dec 9, 94(25), 13548 - 53 Escherichia coli RNA polymerase terminates transcription efficiently at rho-independent terminators on single-stranded DNA templates; Uptain SM et al.; Several models have been proposed for the mechanism of transcript termination by Escherichia coli RNA polymerase at rho-independent terminators . Yager and von Hippel (Yager, T . D . & von Hippel, P . H . (1991) Biochemistry 30, 1097-118) postulated that the transcription complex is stabilized by enzyme-nucleic acid interactions and the favorable free energy of a 12-bp RNA-DNA hybrid but is destabilized by the free energy required to maintain an extended transcription bubble . Termination, by their model, is viewed simply as displacement of the RNA transcript from the hybrid helix by reformation of the DNA helix . We have proposed an alternative model where the RNA transcript is stably bound to RNA polymerase primarily through interactions with two single-strand specific RNA-binding sites; termination is triggered by formation of an RNA hairpin that reduces binding of the RNA to one RNA-binding site and, ultimately, leads to its ejection from the complex . To distinguish between these models, we have tested whether E . coli RNA polymerase can terminate transcription at rho-independent terminators on single-stranded DNA . RNA polymerase cannot form a transcription bubble on these templates; thus, the Yager-von Hippel model predicts that intrinsic termination will not occur . We find that transcript elongation on single-stranded DNA templates is hindered somewhat by DNA secondary structure . However, E . coli RNA polymerase efficiently terminates and releases transcripts at several rho-independent terminators on such templates at the same positions as termination occurs on duplex DNAs . Therefore, neither the nontranscribed DNA strand nor the transcription bubble is essential for rho-independent termination by E . coli RNA polymerase. Proc Natl Acad Sci U S A, 1997 Dec 9, 94(25), 13515 - 9 Topology of allosteric regulation of lactose permease; Seok YJ et al.; Sugar transport by some permeases in Escherichia coli is allosterically regulated by the phosphorylation state of the intracellular regulatory protein, enzyme IIAglc of the phosphoenolpyruvate:sugar phosphotransferase system . A sensitive radiochemical assay for the interaction of enzyme IIAglc with membrane-associated lactose permease was used to characterize the binding reaction . The binding is stimulated by transportable substrates such as lactose, melibiose, and raffinose, but not by sugars that are not transported (maltose and sucrose) . Treatment of lactose permease with N-ethylmaleimide, which blocks ligand binding and transport by alkylating Cys-148, also blocks enzyme IIAglc binding . Preincubation with the substrate analog beta-D-galactopyranosyl 1-thio-beta-D-galactopyranoside protects both lactose transport and enzyme IIAglc binding against inhibition by N-ethylmaleimide . A collection of lactose permease replacement mutants at Cys-148 showed, with the exception of C148V, a good correlation of relative transport activity and enzyme IIAglc binding . The nature of the interaction of enzyme IIAglc with the cytoplasmic face of lactose permease was explored . The N- and C-termini, as well as five hydrophilic loops in the permease, are exposed on the cytoplasmic surface of the membrane and it has been proposed that the central cytoplasmic loop of lactose permease is the major determinant for interaction with enzyme IIAglc . Lactose permease mutants with polyhistidine insertions in cytoplasmic loops IV/V and VI/VII and periplasmic loop VII/VIII retain transport activity and therefore substrate binding, but do not bind enzyme IIAglc, indicating that these regions of lactose permease may be involved in recognition of enzyme IIAglc . Taken together, these results suggest that interaction of lactose permease with substrate promotes a conformational change that brings several cytoplasmic loops into an arrangement optimal for interaction with the regulatory protein, enzyme IIAglc . A topological map of the proposed interaction is presented. Proc Natl Acad Sci U S A, 1997 Dec 9, 94(25), 13481 - 6 A mutant RNA polymerase that forms unusual open promoter complexes; Severinov K et al.; We describe a mutant Escherichia coli RNA polymerase (RNAP) that forms stable open promoter complexes even at -20 degrees C but with a shortened melted region that extends downstream to only position -7 . In the presence of initiating transcription substrates, the mutant RNAP undergoes a temperature-dependent isomerization, resulting in a promoter complex that is indistinguishable from the wild-type RNAP-promoter complex, with the melted region extended downstream to position +4 . We propose that the open complex formed by the mutant RNAP represents an intermediate on the normal promoter-opening pathway and that our results support earlier findings that initial promoter opening occurs in the upstream region of the -10 promoter consensus element and subsequently extends downstream to encompass the transcription start site. Proc Natl Acad Sci U S A, 1997 Dec 9, 94(25), 13452 - 7 Structure of the recombinant full-length hamster prion protein PrP(29-231): the N terminus is highly flexible; Donne DG et al.; The prion diseases seem to be caused by a conformational change of the prion protein (PrP) from the benign cellular form PrPC to the infectious scrapie form PrPSc; thus, detailed information about PrP structure may provide essential insights into the mechanism by which these diseases develop . In this study, the secondary structure of the recombinant Syrian hamster PrP of residues 29-231 {PrP(29-231)} is investigated by multidimensional heteronuclear NMR . Chemical shift index analysis and nuclear Overhauser effect data show that PrP(29-231) contains three helices and possibly one short beta-strand . Most striking is the random-coil nature of chemical shifts for residues 30-124 in the full-length PrP . Although the secondary structure elements are similar to those found in mouse PrP fragment PrP(121-231), the secondary structure boundaries of PrP(29-231) are different from those in mouse PrP(121-231) but similar to those found in the structure of Syrian hamster PrP(90-231) . Comparison of resonance assignments of PrP(29-231) and PrP(90-231) indicates that there may be transient interactions between the additional residues and the structured core . Backbone dynamics studies done by using the heteronuclear {1H}-15N nuclear Overhauser effect indicate that almost half of PrP(29-231), residues 29-124, is highly flexible . This plastic region could feature in the conversion of PrPC to PrPSc by template-assisted formation of beta-structure. Proc Natl Acad Sci U S A, 1997 Dec 9, 94(25), 13437 - 41 Regulation of ribonuclease III processing by double-helical sequence antideterminants; Zhang K et al.; The double helix is a ubiquitous feature of RNA molecules and provides a target for nucleases involved in RNA maturation and decay . Escherichia coli ribonuclease III participates in maturation and decay pathways by site-specifically cleaving double-helical structures in cellular and viral RNAs . The site of cleavage can determine RNA functional activity and half-life and is specified in part by local tertiary structure elements such as internal loops . The involvement of base pair sequence in determining cleavage sites is unclear, because RNase III can efficiently degrade polymeric double-stranded RNAs of low sequence complexity . An alignment of RNase III substrates revealed an exclusion of specific Watson-Crick bp sequences at defined positions relative to the cleavage site . Inclusion of these "disfavored" sequences in a model substrate strongly inhibited cleavage in vitro by interfering with RNase III binding . Substrate cleavage also was inhibited by a 3-bp sequence from the selenocysteine-accepting tRNASec, which acts as an antideterminant of EF-Tu binding to tRNASec . The inhibitory bp sequences, together with local tertiary structure, can confer site specificity to cleavage of cellular and viral substrates without constraining the degradative action of RNase III on polymeric double-stranded RNA . Base pair antideterminants also may protect double-helical elements in other RNA molecules with essential functions. Proc Natl Acad Sci U S A, 1997 Dec 9, 94(25), 13431 - 6 Overexpression of a glutamate receptor (GluR2) ligand binding domain in Escherichia coli: application of a novel protein folding screen; Chen GQ et al.; Expression of the S1S2 ligand binding domain {Kuusinen, A., Arvola, M . & Keinanen, K . (1995) EMBO J . 14, 6327-6332} of the rat alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid-selective glutamate receptor GluR2 in Escherichia coli under control of a T7 promoter leads to production of >100 mg/liter of histidine-tagged S1S2 protein (HS1S2) in the form of inclusion bodies . Using a novel fractional factorial folding screen and a rational, step-by-step approach, multiple conditions were determined for the folding of the HS1S2 alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid binding domain . Characterization of the HS1S2 ligand binding domain showed that it is water-soluble, monomeric, has significant secondary structure, and is sensitive to trypsinolysis at sites close to the beginning of the putative transmembrane regions . Application of a fractional factorial folding screen to other proteins may provide a useful means to evaluate E . coli as an economical and convenient expression host. Proc Natl Acad Sci U S A, 1997 Dec 9, 94(25), 13420 - 5 Identification of a second aryl phosphate-binding site in protein-tyrosine phosphatase 1B: a paradigm for inhibitor design; Puius YA et al.; The structure of the catalytically inactive mutant (C215S) of the human protein-tyrosine phosphatase 1B (PTP1B) has been solved to high resolution in two complexes . In the first, crystals were grown in the presence of bis-(para-phosphophenyl) methane (BPPM), a synthetic high-affinity low-molecular weight nonpeptidic substrate (Km = 16 microM), and the structure was refined to an R-factor of 18 . 2% at 1.9 A resolution . In the second, crystals were grown in a saturating concentration of phosphotyrosine (pTyr), and the structure was refined to an R-factor of 18.1% at 1.85 A . Difference Fourier maps showed that BPPM binds PTP1B in two mutually exclusive modes, one in which it occupies the canonical pTyr-binding site (the active site), and another in which a phosphophenyl moiety interacts with a set of residues not previously observed to bind aryl phosphates . The identification of a second pTyr molecule at the same site in the PTP1B/C215S-pTyr complex confirms that these residues constitute a low-affinity noncatalytic aryl phosphate-binding site . Identification of a second aryl phosphate binding site adjacent to the active site provides a paradigm for the design of tight-binding, highly specific PTP1B inhibitors that can span both the active site and the adjacent noncatalytic site . This design can be achieved by tethering together two small ligands that are individually targeted to the active site and the proximal noncatalytic site. J Clin Invest, 1997 Dec 1, 100(11), 2766 - 76 Therapeutic effects of interleukin-4 gene transfer in experimental inflammatory bowel disease; Hogaboam CM et al.; Inflammatory bowel disease (IBD) is characterized by altered immunoregulation and augmented intestinal synthesis of nitric oxide . The purpose of this study was to determine the effects of exogenous IL-4, introduced by a recombinant human type 5 adenovirus (Ad5) vector, on the tissue injury associated with an experimental model of colonic immune activation and inflammation . Colitis was induced in rats by the intrarectal administration of trinitrobenzene sulfonic acid (TNB) dissolved in 50% ethanol, and control rats received saline via the same route . 1 h later, all rats were randomized into two groups . The first group was injected intraperitoneally (ip) with 3.0 x 10(6) plaque forming units (PFUs) of Ad5 transfected with murine interleukin-4 (Ad5IL-4) and the second group was injected ip with the same amount of Ad5 expressing the Escherichia coli Lac Z gene (Ad5LacZ) . One-half of the colitic and control rats were injected again with 3.0 x 10(6) PFUs of Ad5IL-4 or Ad5LacZ on day 3 of the 6-d study . When introduced once or twice via the peritoneal route into control rats, Ad5LacZ was localized to the serosal lining of the peritoneal cavity, the diaphragm and the liver on day 6 . One or two injections of Ad5IL-4 into rats also produced measurable levels of circulating IL-4 . TNB-colitis in both Ad5LacZ-treated groups was associated with pronounced elevations in serum IFN-gamma, and mucosal ulceration of the distal colon . Myeloperoxidase and inducible nitric oxide synthase II (NOS II) synthetic activity were also increased by 30- and fivefold, respectively, above control levels in the distal colon . However, two injections of Ad5IL-4 into colitic rats caused the overexpression of IL-4, and significantly inhibited tissue damage, serum and colon IFN-gamma levels and myeloperoxidase activity in the distal colon . In addition, NOS II gene expression and NOS II nitric oxide synthesis was significantly inhibited . No therapeutic effect was observed in rats injected once with Ad5IL-4 . Thus, IL-4, introduced by Ad5, is therapeutic during acute inflammation in the rat colon . The therapeutic effect of IL-4 was associated with an inhibition of inducible nitric oxide expression and a reduction in nitric oxide synthesis. Protein Sci, 1997 Nov, 6(11), 2474 - 6 Purification, characterization, and crystallization of Escherichia coli ribokinase; Sigrell JA et al.; Ribokinase phosphorylates ribose to form ribose-5-phosphate in the presence of ATP and magnesium . The phosphorylated sugar can enter the pentose phosphate pathway or be used for the synthesis of nucleotides, histidine, and tryptophan . Ribokinase belongs to the PfkB family of carbohydrate kinases, for which no three-dimensional structure is currently known . We describe an improved purification protocol for Escherichia coli ribokinase and give evidence from light-scattering and gel filtration studies that the protein forms a dimer in solution . Several types of crystals are also described that have been obtained of apo ribokinase, ribokinase in the presence of ATP, and in a ternary complex with an ATP-analogue and ribose . The latter crystals give the best X-ray diffraction . A complete data set has been collected at the synchrotron source in Hamburg, to 2.6 A resolution using a frozen crystal . The crystals belong to space group P6(1)22 or P6(5)22 with cell parameters a = b = 95 A and c = 155 A. Protein Sci, 1997 Nov, 6(11), 2426 - 35 Covalent methionylation of Escherichia coli methionyl-tRNA synthethase: identification of the labeled amino acid residues by matrix-assisted laser desorption-ionization mass spectrometry; Gillet S et al.; Methionyl-adenylate, the mixed carboxylic-phosphoric acid anhydride synthesized by methionyl-tRNA synthetase (MetRS) is capable of reacting with this synthetase or other proteins, by forming an isopeptide bond with the epsilon-NH2 group of lysyl residues . It is proposed that the mechanism for the in vitro methionylation of MetRS might be accounted for by the in situ covalent reaction of methionyl-adenylate with lysine side chains surrounding the active center of the enzyme, as well as by exchange of the label between donor and acceptor proteins . Following the incorporation of 7.0 +/- 0.5 mol of methionine per mol of a monomeric truncated methionyl-tRNA synthetase species, the enzymic activities of {32P}PPi-ATP isotopic exchange and tRNA(Met) aminoacylation were lowered by 75% and more than 90%, respectively . The addition of tRNA(Met) protected the enzyme against inactivation and methionine incorporation . Matrix-assisted laser desorption-ionization mass spectrometry designated lysines-114, -132, -142 (or -147), -270, -282, -335, -362, -402, -439, -465, and -547 of truncated methionyl-tRNA synthetase as the target residues for covalent binding of methionine . These lysyl residues are distributed at the surface of the enzyme between three regions {114-150}, {270-362}, and {402-465}, all of which were previously shown to be involved in catalysis or to be located in the binding sites of the three substrates, methionine, ATP, and tRNA. Protein Sci, 1997 Nov, 6(11), 2375 - 84 1H, 13C, and 15N resonance assignments of Fusarium solani pisi cutinase and preliminary features of the structure in solution; Prompers JJ et al.; Essentially complete (96%) sequence-specific assignments were made for the backbone and side-chain 1H, 13C, and 15N resonances of Fusarium solani pisi cutinase, produced as a 214-residue heterologous protein in Escherichia coli, using heteronuclear NMR techniques . Three structural features were noticed during the assignment . (1) The secondary structure in solution corresponds mostly with the structure from X-ray diffraction, suggesting that both structures are globally similar . (2) The HN of Ala32 has a strongly upfield-shifted resonance at 3.97 ppm, indicative of an amide-aromatic hydrogen bond to the indole ring of Trp69 that stabilizes the N-terminal side of the parallel beta-sheet . (3) The NMR data suggest that the residues constituting the oxyanion hole are quite mobile in the free enzyme in solution, in contrast to the existence of a preformed oxyanion hole as observed in the crystal structure . Apparently, cutinase forms its oxyanion hole upon binding of the substrate like true lipases. Biotechniques, 1997 Nov, 23(5), 920 - 6 Ribonuclease H renaturation gel assay using a fluorescent-labeled substrate; Han LY et al.; Ribonucleases H (RNases H) are enzymes that specifically degrade the RNA of RNA-DNA hybrids . These enzymes are involved in DNA replication, reverse transcription (RT) and antisense oligodeoxyribonucleotide-mediated arrest of translation . One of the most valuable tools for assaying RNase H activity is the renaturation gel assay with which such activities can be detected using purified protein preparations or crude extracts . Radioactive substrates {32P labeled poly(rA)-poly(dT) hybrid} are commonly used with exposure of the gel to X-ray film; this is possible at any time without disturbing the renaturation-degradation process . Here, we describe a method using fluorescent-labeled substrates . RNA-DNA substrates are synthesized by first transcribing DNA with T7 RNA polymerase using Bodipy-TR-14-UTP and the four normal nucleoside triphosphates . The run-off transcript is annealed to a short oligomeric DNA complementary to the 3'-end of the transcript, and the DNA portion of the hybrid is formed by RT . This RNA-DNA is added to the polyacrylamide mixture before polymerization, and SDS-PAGE is performed as usual . After various periods of renaturation, the gel is scanned to detect fluorescent substrate using the red-excited laser of a fluorescence scanner . This fluorescence method has all of the advantages of using radio-labeled substrates and none of its disadvantages, and the sensitivities of the two methods are comparable . In addition, we show that the sensitivity of this procedure can be increased if damaging chemicals remaining in the gel after polymerization are eliminated by simultaneous electrophoresis of the RNase H and a protein with higher mobility. Biotechniques, 1997 Nov, 23(5), 892 - 6 Photoimmunodetection: a nonradioactive labeling and detection method for DNA; Marvik OJ et al.; We describe a simple detection system for DNA based on antibody detection of UV-induced photoproducts . It includes a convenient and inexpensive labeling procedure, which is completed in 5-10 min . The only equipment required is a UV source such as an ordinary transilluminator or a DNA crosslinker . Using a monoclonal antibody specific for thymine dimers, coupled to horseradish peroxidase, we are able to detect subpicogram amounts of UV-irradiated DNA directly, and approximately 10 pg homologues DNA indirectly by hybridization with an irradiated probe. Biotechniques, 1997 Nov, 23(5), 858 - 62, 864 Cloning and assembly of PCR products using modified primers and DNA repair enzymes; Watson DE et al.; We present a method for the creation of ligatable 3' overhangs by the incorporation of a modified base, uracil, at a specific position in the PCR primer and subsequent treatment with the DNA-modifying enzyme uracil DNA glycosylase and then either T4 endonuclease V or human apurinic/apyrimidinic endonuclease 1 . In this study, we describe the cloning of a fragment specifying the chloramphenicol-resistance gene into a SacI vector site . To further test this method, three segments of the lacZ gene were amplified by PCR, and after treatment with the DNA-modifying enzymes, the properly oriented segments were ligated into a SacI-cleaved plasmid . Using the methods described, we were able to assemble PCR products into appropriate structures. Chem Biol, 1995 Oct, 2(10), 661 - 6 Use of semi-synthetic transfer RNAs to probe molecular recognition by Escherichia coli proline-tRNA synthetase; Yap LP et al.; BACKGROUND: The attachment of specific amino acids to the 3'-end of cognate transfer of RNAs (tRNAs) is catalyzed by a class of enzymes known as aminoacyl-tRNA synthetases (aaRS) . We have previously demonstrated that Escherichia coli proline-tRNA synthetase (ProRS) can aminoacylate semi-synthetic tRNAs prepared by annealing two RNA oligonucleotides . We set out to examine the factors that are important in selective recognition of tRNAPro by ProRS, using semi-synthetic tRNAs and full-length tRNA transcripts . RESULTS: Deletion of nucleotides A58, A59, and U60 in the T psi C-loop of semi-synthetic tRNAs has no adverse effect on aminoacylation . Nucleotide deletions that extend into the T psi stem, particularly beyond C61, significantly reduce the efficiency of aminoacylation, however . Site-directed mutagenesis of full-length tRNAPro transcripts shows that, although there is no strict sequence requirement at base pair 52.62 in the T psi C stem, helix destabilizing purine-purine mismatches at this position result in decreased aminoacylation activity . Moreover, aminoacylation is severely affected when a DNA-RNA hybrid helix is incorporated into the acceptor-T psi C stem domain . CONCLUSIONS: At least three nucleotides in the T psi C-loop are dispensable for aminoacylation of E . coli tRNAPro . These results, combined with previous data, demonstrate that four out of five of the so-called 'variable pocket' nucleotides are not important for recognition of tRNAPro by E . coli ProRS . ProRS is also sensitive to changes that are likely to alter the helical conformation in the T psi C stem. Chem Biol, 1995 Feb, 2(2), 83 - 9 A direct comparison of the properties of natural and designed zinc-finger proteins; Shi Y et al.; BACKGROUND: Zinc-finger proteins of the Cys2His2 type constitute an important family of DNA-binding proteins . Each zinc-finger domain has three residues that are thought to be important in determining DNA binding site specificity . Proteins have been designed previously by combining zinc-finger domains with a fixed sequence framework with different DNA-contacting residues . RESULTS: We compared the DNA-binding properties of the DNA-binding domain from the human transcription factor Sp1, which contains three zinc fingers, with designed proteins in which the sequences of the structural framework were greatly modified but the presumed DNA-contacting residues were retained . Frameworks based on a zinc-finger consensus sequence and on a minimalist sequence consisting largely of alanine residues were studied . The preference for binding to the target sequence, 5'-(G,T)GG G(C,A)G GG(G,T)-3', was retained in all cases tested . The consensus framework-based protein was found to be superior to the natural one in terms of overall DNA-binding affinity, the degree of sequence discrimination, and the resistance to inactivation by chelating agents . CONCLUSIONS: Our observations provide direct evidence that the residues previously observed to interact with the DNA bases are indeed the most important residues for determining DNA-binding specificity . We have also shown that these domains can tolerate considerable sequence variation while retaining function as well as three-dimensional structure . Finally, they show that framework modification can be used to generate proteins that have normal or enhanced DNA-binding activity but have different metal-binding properties. Chem Biol, 1995 Feb, 2(2), 71 - 5 Evolution in action; Scanlan TS et al.; The alpha/beta barrel enzyme phosphotriesterase from soil bacteria appears to have evolved the ability to hydrolyze the insecticide paraoxon at the diffusion limit in only a few decades . A newly-identified open reading frame from Escherichia coli may offer a clue to its origins. Chem Biol, 1995 Jan, 2(1), 53 - 60 Grb2 SH3 binding to peptides from Sos: evaluation of a general model for SH3-ligand interactions; Simon JA et al.; BACKGROUND: Grb2 acts as an adaptor protein in the transduction of signals from receptor tyrosine kinases to Ras . It binds to phosphotyrosine on the cytoplasmic tail of cell-surface receptors via its central SH2 domain, and to its immediate downstream target, Sos, via two SH3 domains . The basis of the Grb2-Sos interaction is not fully understood . We previously proposed a model for SH3 domain binding specificity, based on two solution structures of the Src SH3 domain complexed with high-affinity ligands, in which the ligands are bound in a polyproline type II conformation in two distinct orientations, class I and class II . Here, we have used this model to predict the identity and orientation of Grb2 SH3 ligands in the human Sos protein . RESULTS: Six contiguous fragments from the carboxy-terminal portion of hSos (amino acids 1000-1333), each containing a single potential SH3 binding site, were expressed in E . coli as GST fusion proteins . Four of these proteins were predicted to associate with SH3 domains . The amino-terminal Grb2 SH3 domain was shown to bind strongly to only these four fragments . CONCLUSIONS: We have used a general model for SH3-ligand interactions to predict the nature of Grb2 SH3 interactions with the hSos protein . Comparison of the four hSos sequences that bind Grb2 revealed a preference for the PXXPXR motif, consistent with the predicted class II-type binding interaction . The interaction between Grb2 and hSos peptides is predominantly via the amino-terminal SH3 domain, although the carboxy-terminal SH3 domain may increase the overall stability of the Grb2-hSos complex. Chem Biol, 1994 Oct, 1(2), 119 - 24 Genetically engineered synthesis of precorrin-6x and the complete corrinoid, hydrogenobyrinic acid, an advanced precursor of vitamin B12; Roessner CA et al.; BACKGROUND: Genetically engineered synthesis, in which the gene products, cofactors, and substrates of a complete pathway are combined in vitro in a single flask to give the target, can be a viable alternative to conventional chemical construction of molecules of complex structure and stereochemistry . We chose to attempt to synthesize the metal-free corrinoid hydrogenobyrinic acid, an advanced precursor of vitamin B12 . RESULTS: Cloning and overexpression of the genes necessary for the S-adenosyl methionine dependent conversion of 5-aminolevulinic acid (ALA) to precorrin-3 and those required for the synthesis of hydrogenobyrinic acid from precorrin-3 completed the repertoire of the 12 biosynthetic enzymes involved in corrin synthesis . Using these enzymes and the necessary cofactors, the multi-enzyme synthesis of hydrogenobyrinic acid from ALA can be achieved in 20% overall yield in a single reaction vessel, corresponding to an average of at least 90% conversion for each of the 17 steps involved . CONCLUSIONS: By replacing the cell wall with glass, and by mixing the soluble biosynthetic enzymes and necessary cofactors, the major segment of the physiological synthesis of vitamin B12 has been accomplished . Since only those enzymes necessary for the synthesis of hydrogenobyrinic acid from ALA are supplied, none of the intermediates is deflected from the direct pathway . This results in an efficiency which in fact surpasses that of nature. Chem Biol, 1994 Oct, 1(2), 91 - 7 Metal-coordination sphere in the methylated Ada protein-DNA co-complex; Myers LC et al.; BACKGROUND: The Ada protein of Escherichia coli repairs methyl phosphotriesters in DNA by direct, irreversible methyl transfer to one of its own cysteine residues . This residue, Cys69, is ligated to a tightly bound zinc ion in the protein . After methyl transfer, Ada can bind DNA sequence-specifically, inducing the transcription of genes that confer resistance to the toxic effects of methylating agents . Coordination of zinc via a thioether-S is exceedingly rare . We therefore investigated whether methylation causes ligand exchange of Cys69, replacing the thioether with a new zinc ligand with higher affinity for the metal . RESULTS: We added a 13C-labeled methyl group to Cys69 of Ada and used isotope-edited NMR to observe the behavior of its protons . Comparison of the spectra for the Zn- and 112Cd-bound forms of the methylated protein with that of the 113Cd-bound form provided clear evidence that S-Me-Cys69 is coordinated to the metal in Ada when Ada is bound specifically to DNA . CONCLUSIONS: The transcriptionally competent form of Ada, in which Cys69 is methylated and the protein is bound to DNA, maintains the coordination of S-Me-Cys69 to the metal ion . Thus, ligand exchange is not responsible for switching Ada from a DNA-repair protein to a transcriptional activator . We propose that the lability of the thioether-zinc coordinate bond may provide a mechanism for down-regulation of the adaptive response by inactivation of the Ada DNA-binding domain. Mol Microbiol, 1997 Oct, 26(2), 387 - 98 Polyphosphate kinase is a component of the Escherichia coli RNA degradosome; Blum E et al.; The Escherichia coli degradosome is a multienzyme complex with four major protein components: the endoribonuclease RNase E, the exoribonuclease PNPase, the RNA helicase RhlB and enolase . The first three of these proteins are known to have important functions in mRNA processing and degradation . In this work, we identify an additional component of the degradosome, polyphosphate kinase (PPK), which catalyses the reversible polymerization of the gamma-phosphate of ATP into polyphosphate (poly(P)) . An E . coli strain deleted for the ppk gene showed increased stability of the ompA mRNA . Purified His-tagged PPK was shown to bind RNA, and RNA binding was prevented by hydrolysable ATP . Chemical modification of RNA by PPK, for example the addition or removal of 3' or 5' terminal phosphates, could not be detected . However, polyphosphate was found to inhibit RNA degradation by the degradosome in vitro . This inhibition was overcome by the addition of ADP, required for the degradation of polyphosphate and for the regeneration of ATP by PPK in the degradosome . Thus, PPK in the degradosome appears to maintain an appropriate microenvironment, removing inhibitory polyphosphate and NDPs and regenerating ATP. Mol Microbiol, 1997 Oct, 26(2), 337 - 46 Proline 21, a residue within the alpha-helical domain of phiX174 lysis protein E, is required for its function in Escherichia coli; Witte A et al.; PhiX174 lysis protein E-mediated lysis of Escherichia coli is characterized by a protein E-specific fusion of the inner and outer membrane and formation of a transmembrane tunnel structure . In order to understand the fusion process, the topology of protein E within the envelope complex of E . coli was investigated . Proteinase K protection studies showed that, during the time course of protein E-mediated lysis process, more of the fusion protein E-FXa-streptavidin gradually became accessible to the protease at the cell surface . These observations postulate a conformational change in protein E during induction of the lysis process by movement of the C-terminal end of the protein throughout the envelope complex from the inner side to the outer side spanning the entire pore and fusing the inner and outer membranes at distinct areas . The initiation mechanism for such a conformational change could be the cis-trans isomerization of proline residues within alpha-helical membrane-spanning segments . Conversion of proline 21, presumed to be in the membrane-embedded alpha-helix of protein E, to alanine, glycine, serine and valine, respectively, resulted in lysis-negative E mutant proteins . Proteinase K accessibility studies using streptavidin as a reporter fused to the P21G mutant protein showed that the C-terminal part of the fusion protein is not translocated to the outer side of the membrane, suggesting that this proline residue is essential for the correct folding of protein E within the cell wall complex of E . coli . Oligomerization of protein P21G-StrpA was not disturbed. Mol Microbiol, 1997 Oct, 26(2), 321 - 35 Deletion analysis of cspA of Escherichia coli: requirement of the AT-rich UP element for cspA transcription and the downstream box in the coding region for its cold shock induction; Mitta M et al.; In order to analyse the mechanism of cold shock induction of CspA, a major cold shock protein of Escherichia coli, deletion analysis of the cspA gene was carried out . It was found that (i) the AT-rich sequence (-47 to -38) upstream of the cspA -35 region may act as the UP element playing a crucial role in cspA transcription at both 37 degrees C and 15 degrees C; (ii) the unusually long 5'-UTR of the cspA mRNA has negative effects on cspA expression at 37 degrees C; and (iii) in contrast, the 5'-UTR exerts a positive effect on mRNA stabilization at low temperature . Furthermore, it was demonstrated that the 14 base downstream box (DB) locating 12 bases downstream of the initiation codon of the cspA mRNA and complementary to a region near the decoding region of 16S rRNA was essential for the mRNA translation during the growth lag acclimation phase immediately after cold shock . During this phase, translation of non-cold shock gene mRNAs is blocked, since they require cold shock-specific ribosomal factors for the formation of the translation initiation complex . It is proposed that DB in cold shock mRNAs allows the formation of a stable initiation complex at low temperature in the absence of the cold shock ribosomal factors. Mol Microbiol, 1997 Oct, 26(2), 311 - 20 Sok antisense RNA from plasmid R1 is functionally inactivated by RNase E and polyadenylated by poly(A) polymerase I; Dam Mikkelsen N et al.; The hok/sok system of plasmid R1, which mediates plasmid stabilization by the killing of plasmid-free cells, codes for two RNA species, Sok antisense RNA and hok mRNA . Sok RNA, which is unstable, inhibits translation of the stable hok mRNA . The 64nt Sok RNA folds into a single stem-loop domain with an 11 nt unstructured 5' domain . The initial recognition reaction between Sok RNA and hok mRNA takes place between the 5' domain and the complementary region in hok mRNA . In this communication we examine the metabolism of Sok antisense RNA . We find that RNase E cleaves the RNA 6nt from its 5' end and that this cleavage initiates Sok RNA decay . The RNase E cleavage occurs in the part of Sok RNA that is responsible for the initial recognition of the target loop in hok mRNA and thus leads to functional inactivation of the antisense . The major RNase E cleavage product (denoted pSok-6) is rapidly degraded by polynucleotide phosphorylase (PNPase) . Thus, the RNase E cleavage tags pSok-6 for further rapid degradation by PNPase from its 3' end . We also show that Sok RNA is polyadenylated by poly(A) polymerase I (PAP I), and that the poly(A)-tailing is prerequisite for the rapid 3'-exonucleolytic degradation by PNPase. Mol Microbiol, 1997 Oct, 26(2), 261 - 75 Role of architectural elements in combinatorial regulation of initiation of DNA replication in Escherichia coli; Polaczek P et al.; Bending of DNA is a prerequisite of site-specific recombination and gene expression in many regulatory systems involving the assembly of specific nucleoprotein complexes . We have investigated how the uniquely clustered Dam methylase sites, GATCs, in the origin of Escherichia coli replication (oriC) and their methylation status modulate the geometry of oriC and its interaction with architectural proteins, such as integration host factor (IHF), factor for inversion stimulation (Fis) and DnaA initiator protein . We note that 3 of the 11 GATC sites at oriC are strategically positioned within the IHF protected region . Methylation of the GATCs enhances IHF binding and alters the IHF-induced bend at oriC . GATC motifs also contribute to intrinsic DNA curvature at oriC and the degree of bending is modulated by methylation . The IHF-induced bend at oriC is further modified by Fis protein and IHF affinity for its binding site may be impaired by protein(s) binding to GATCs within the IHF site . Thus, GATC sites at oriC affect the DNA conformation and GATCs, in conjunction with the protein-induced bends, are critical cis-acting elements in specifying proper juxtapositioning of initiation factors in the early steps of DNA replication. Mol Microbiol, 1997 Oct, 26(2), 223 - 36 Role of multiple ArcA recognition sites in anaerobic regulation of succinate dehydrogenase (sdhCDAB) gene expression in Escherichia coli; Shen J et al.; Succinate dehydrogenase (sdhCDAB) gene expression in Escherichia coli is negatively regulated by the arcA and fnr gene products during anaerobic cell growth conditions . The controlled synthesis of this sole membrane-bound enzyme of the tricarboxylic acid cycle allows optimal participation in the aerobic electron transport pathway for the generation of energy via oxidative phosphorylation reactions . To understand how ArcA participates in the anaerobic repression of sdhCDAB expression, a family of sdhC-lacZ fusions was constructed and analysed in vivo . DNase I footprint and gel shift assays using purified ArcA protein revealed the location of four distinct and independent ArcA binding sites in the sdhC promoter region . ArcA sites, designated sites 1 and 2, are centred at -205 bp and -119 bp upstream of the sdhC promoter, respectively, whereas ArcA site 3 overlaps the -35 and -10 regions of the sdhC promoter . A fourth ArcA site is centred at +257 bp downstream of the sdhC promoter . They are bound with differing affinity by ArcA and ArcA phosphate . The in vivo studies, in combination with the in vitro studies, indicate that ArcA site 3 is necessary and sufficient for the ArcA-dependent repression of sdhC gene expression, while the DNA region containing ArcA site 2 contributes to maximal gene expression . The DNA-containing ArcA sites 1 and 4 provide minor roles in the ArcA regulation of sdhC expression . Lastly, the Fnr-dependent control of sdhCDAB gene expression was shown to occur independently of the ArcA and to require DNA sequences near the start of sdhC transcription. Zh Mikrobiol Epidemiol Immunobiol, 1995 Jul-Aug, (4), 47 - 51 {An analysis of the blood sera of patients infected with HIV-1 for the presence of antibodies to the gp120 and gp41 sequences}; Sergeev OV et al.; Blood sera, originating four regions of Russia and Byelorussia and previously tested for the content of antibodies to HIV-1 proteins, were studied in the enzyme immunoassay on the basis of recombinant sequences gp160, as well as on the basis of oligopeptides corresponding to sequences V3 of six HIV-1 strains . The possibility of using sequences gp160, contained in fusion polypeptides synthesized in Escherichia coli cells, for the detection of antibodies in laboratory research was shown . Differences in the reactivity of the sera under study with respect to fragments gp160 correlated with the geographical origin of these sera: similarity between the serum samples from Elista and Rostov and their difference from serum samples collected in other regions were shown. Wiad Lek, 1997, 50(4-6), 117 - 9 {Liver abscess in the course of ulcerative colitis}; Dabrowski M et al.; The case of 19 year old female patient treated because of the ulcerative colitis . The abscess of the liver has appeared in this patient . This kind of complication has been never described in medical literature. Tsitol Genet, 1997 Jan-Feb, 31(1), 6 - 11 {The ultrastructural bases of disorders in the vascular plexuses of the lateral cerebral ventricles in rats under the action of endotoxin}; Bardakhch'ian EA et al.; Using method of electron microscopy, the ultrastructural changes in epithelium and capillaries of the vascular plexuses of the brain lateral ventricles in rats after intravenous administration of E . coli endotoxin were studied . The dynamics of ultrastructural disorders during endotoxemia and epithelial cells alterations depending on microcirculatory bed conditions were shown. Genetika, 1997 Jul, 33(7), 1020 - 4 {Use of Escherichia coli recF mutant strains for analyzing the genotoxicity of complex platinum compounds}; Ptitsyn LR et al.; The bioluminescence method of assessing SOS response in Escherichia coli cells was applied to test the genotoxicity of five complex platinum compounds: cis-diamminedichloroplatinum, cycloplatam, trans-diamminedichloroplatinum, and two trans-binuclear complexes . Strains AB1157 (pPLS-1) and JC9239 (pPLS-1), a variant of AB1157 carrying the recF143 mutation, were used in the test procedure . SOS response in JC9239 cells was shown to be induced by significantly lower concentrations of cis-DDP and cycloplatam than in AB1157 cells . A drastic increase in cis-DDP toxicity for the JC9239 strain was shown . However, the presence of the recF mutation in JC9239 cells retarded the SOS response induced by trans-platinum compounds . The results obtained indicated that the E . coli recF system plays an essential role in repair and utilization of cis-DDP-DNA adducts. J Photochem Photobiol B, 1997 Oct, 40(3), 299 - 304 RNA-protein cross-links induced by sensitization with a pyrroloquinolinone derivative, a furocoumarin analogue; Baccichetti F et al.; The capacity of 2,6-dimethyl-9-methoxy-4H-pyrrolo {3,2,1-ij} quinolin-4-one (PQ), a furocoumarin analogue, of inhibiting protein synthesis in Ehrlich cells upon UVA irradiation was investigated . Using 8-methoxypsoralen (8-MOP) as a reference, we observed that in our short-term test the block of RNA synthesis do not affect protein synthesis, which is driven by pre-synthesised molecules of m-RNA; actually 8-MOP, studied at 100 microM concentration, practically abolished RNA synthesis without affecting significantly protein synthesis . Studying PQ sensitization in HL60 cells by alkaline elution and protein precipitation, the formation of covalent RNA-protein cross-links was observed . 8-MOP, assayed in severe experimental conditions, induced only moderate amounts of such lesion . On the basis of the data obtained in experiments carried out using various scavengers or exposing cells to UVA light in a nitrogen atmosphere, this damage appeared to be due to singlet oxygen formation, which is generated by PQ to a large extent . These results are consistent with the data obtained by H . Singh and J.A . Vadasz (Singlet oxygen: a major reactive species in the furocoumarin photosensitized inactivation of E.coli ribosomes, Photochem . Photobiol., 28 (1978) 539-545) on E.coli ribosomes . The lower activity we observed with 8-MOP might be attributed to a different sensitivity of whole mammalian cells in comparison with isolated ribosomes. Biol Chem, 1997 Oct, 378(10), 1205 - 9 Site-directed mutagenesis of the Cys residues in ClpA, the ATPase component of protease Ti (ClpAP) in Escherichia coli; Seol JH et al.; The ATP-dependent casein hydrolysis by protease Ti (ClpAP) has been shown to be inhibited by sulfhydryl blocking agents, such as N-ethylmaleimide (NEM), when preincubated with ClpA but not with ClpP . To define the role of three Cys residues in ClpA, site-directed mutagenesis was performed to substitute each of them with Ser or Ala . None of the mutations showed any effect on the ATPase activity of ClpA or its ability to support the casein degradation by ClpP . However, NEM could no longer block the ability of ClpA/C47S or ClpA/C47A in supporting the ClpP-mediated proteolysis, unlike that of ClpA, ClpA/C203S, or ClpA/C243S . Furthermore, in the presence of NEM, casein could stimulate the ATPase activities of ClpA/C47S and ClpA/C47A and protect from their degradation by ClpP, but not of the other ClpA proteins . These results suggest that the inhibitory effect of NEM is due to prevention of the interaction of ClpA with casein by introduction of a bulky alkyl group to Cys47, but not linked to the catalytic function of the ATPase. Biol Chem, 1997 Oct, 378(10), 1153 - 62 A lethal mutant of the catabolite gene activator protein CAP of Escherichia coli; Lopata M et al.; The dimeric catabolite gene activator protein (CAP) of Escherichia coli uses its recognition helix to bind with each subunit the DNA sequence motif 5' G-7T-6G-5A-4 3' . It makes a direct amino acid-base contact with E181 and cytosine in position-5' on the reverse strand . While testing mutants of CAP in position 181 for specificity changes, we found that CAP E181Q is lethal in high amounts for the E . coli strains we used for cloning . We cloned this CAP mutant successfully in cya- strains, where CAP is inactive . Examination of the in vitro binding activities of CAP E181Q, and of in vivo activity when present in low, non-lethal amounts, revealed loss of specificity but not of binding capacity for its DNA targets . It binds well to CAP consensus with G or T in position-5, better to CAP consensus with A, C in position-5, quite well to lambda consensus operator with G in position-7 and rather weakly to lambda consensus. Biol Chem, 1997 Oct, 378(10), 1141 - 52 Introduction of a proline residue into position 31 of the loop of the dimeric 4-alpha-helical protein ROP causes a drastic destabilization; Peters K et al.; The exchange of an alanine with a proline residue in position 31 of the loop region of the dimeric 4-alpha-helical-bundle protein ROP causes a reduction in the alpha-helix content of 7% and a reduction in stability of about 40% compared to the wild type parameters . The Gibbs energy of unfolding by denaturants extrapolated linearly to zero denaturant concentration, delta G0D (buffer, 25 degrees C), has been determined to be 43 kJ (mol dimer)-1 . The corresponding ROPwt value is 72 kJ (mol dimer)-1 (Steif et al., 1993) . The extrapolated delta G0D values obtained from urea and GdmHCI un- and refolding studies are identical within error limits . Deconvolution of the stability values into enthalpy and entropy terms resulted in the following parameters . At T1/2 = 43 degrees C (Cprotein = 0.05 mg.ml-1) the ROP A31P mutant is characterized by delta Hv.H.0 = 272 kJ (mol dimer)-1, delta Cp = 7.2 kJ (mol dimer)-1 K-1, delta S0 = 762 J (mol dimer)-1 K-1 . These parameters are only approximately 50% as large as the corresponding values of ROPwt . We assume that the significant reduction in stability reflects the absence of at least one hydrogen bond as well as deformation of the protein structure . This interpretation is supported by the reduction in the change in heat capacity observed for the A31P mutant relative to ROPwt, by the increased aggregation tendency of the mutant and by the reduced specific CD absorption at 222 nm . All results support the view that in the case of ROP protein the loop region plays a significant role in the maintenance of native structure and conformational stability. Biol Chem, 1997 Oct, 378(10), 1125 - 30 Activation of mitochondrial 2-oxoacid dehydrogenases by thioredoxin; Bunik V et al.; The regulation of mitochondrial dehydrogenases of 2-oxoacids by thioredoxin is established . It is found that at low NAD+ and saturating concentrations of 2-oxoacids and CoA, inactivation of 2-oxoacid dehydrogenase complexes takes place, preventing NAD+ reduction under such conditions . However, addition of oxidized E . coli thioredoxin to the reaction medium without dithiothreitol allows effective NAD+ reduction at this substrate ratio . Product accumulation curves show that thioredoxin activates the complexes by protecting them from the inactivation observed in the conditions when the complex-bound dihydrolipoate is accumulated . Disappearance of the activatory effect of thioredoxin after its treatment with SH-specific reagents indicates the involvement of the redox-active cysteine couple of thioredoxin in its activation of 2-oxoacid dehydrogenase complexes . The redox-inactive thioredoxin not only shows no activation, but in fact exerts an inhibitory effect . The inhibition manifests the complex formation between SH-modified thioredoxin and dehydrogenase systems, involving amino acid residues of thioredoxin other than cysteine . High efficiency of thioredoxin from E . coli as compared to chloroplast thioredoxin f and glutathione disulfide is revealed . This indicates the importance of specific protein structure also for the influence of the redox-active thioredoxin upon the 2-oxoacid dehydrogenase complexes . The results obtained suggest that these key enzyme systems of mitochondrial metabolism represent previously unidentified targets for the action of mitochondrial thioredoxin, which is known to resemble the E . coli counterpart studies in this work. Nucleic Acids Res, 1997 Nov 15, 25(22), 4557 - 61 The yeast 8-oxoguanine DNA glycosylase (Ogg1) contains a DNA deoxyribophosphodiesterase (dRpase) activity; Sandigursky M et al.; The yeast OGG1 gene was recently cloned and shown to encode a protein that possesses N-glycosylase/AP lyase activities for the repair of oxidatively damaged DNA at sites of 7,8-dihydro-8-oxoguanine (8-oxoguanine) . Similar activities have been identified for Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) and Drosophila ribosomal protein S3 . Both Fpg and S3 also contain a deoxyribophosphodiesterase (dRpase) activity that removes 2-deoxyribose-5-phosphate at an incised 5' apurinic/apyrimidinic (AP) sites via a beta-elimination reaction . Drosophila S3 also has an additional activity that removes trans-4-hydroxy-2-pentenal-5-phosphate at a 3' incised AP site by a Mg2+-dependent hydrolytic mechanism . In view of the substrate similarities between Ogg1, Fpg and S3 at the level of base excision repair, we examined whether Ogg1 also contains dRpase activities . A glutathione S-transferase fusion protein of Ogg1 was purified and subsequently found to efficiently remove sugar-phosphate residues at incised 5' AP sites . Activity was also detected for the Mg2+-dependent removal of trans -4-hydroxy-2-pentenal-5-phosphate at 3' incised AP sites and from intact AP sites . Previous studies have shown that DNA repair proteins that possess AP lyase activity leave an inefficient DNA terminus for subsequent DNA synthesis steps associated with base excision repair . However, the results presented here suggest that in the presence of MgCl2, Ogg1 can efficiently process 8-oxoguanine so as to leave a one nucleotide gap that can be readily filled in by a DNA polymerase, and importantly, does not therefore require additional enzymes to process trans -4-hydroxy-2-pentenal-5-phosphate left at a 3' terminus created by a beta-elimination catalyst. Nucleic Acids Res, 1997 Nov 15, 25(22), 4537 - 44 High affinity binding of MEF-2C correlates with DNA bending; Meierhans D et al.; To regulate lineage-specific gene expression in many cell types, members of the myocyte enhancer factor-2 (MEF-2) family of transcription factors cooperate with basic helix-loop-helix (bHLH) proteins, which show only limited intrinsic DNA binding specificity . We investigated the DNA binding properties of MEF-2C in vitro and show that the inherent bendability of the MEF site is one of the principal structural characteristics recognized by MEF-2C . Measurements of the apparent dissociation constants of MEF-2C complexes with several DNA sequences revealed that MEF-2C bound with high affinity to DNA sequences containing a MEF site . Mutations in the MEF site which did not affect the bendability of the DNA changed the free energy of binding only marginally . However, reducing the intrinsic bendability of the DNA binding site through an AA-->GC substitution increased the half-maximal binding concentration of MEF-2C by almost one order of magnitude . Electrophoretic mobility shift assays revealed markedly reduced MEF-2C binding to DNA containing 2,6-diaminopurine . On binding to MEF-2C the maximum ellipticity at 275 nm in the CD spectrum of DNA containing a MEF site was red shifted by 4 nm and its intensity reduced significantly, while a slight blue shift of <1 nm was observed for a mutant DNA sequence with reduced bendability (AA-->GC) . Bending analysis by circular permutation assay revealed that the DNA in the cognate complex was bent by 49 degrees , while the DNA in the complex with the mutant oligonucleotide was largely unbent. Nucleic Acids Res, 1997 Nov 15, 25(22), 4493 - 9 The yeast gene YNL292w encodes a pseudouridine synthase (Pus4) catalyzing the formation of psi55 in both mitochondrial and cytoplasmic tRNAs; Becker HF et al.; The protein products of two yeast Saccharomyces cerevisiae genes (YNL292w and CBF5) display a remarkable sequence homology with Escherichia coli tRNA:pseudouridine-55 synthase (encoded by gene truB) . The gene YNL292w coding for one of these proteins was cloned in an E.coli expression vector downstream of a His6-tag . The resulting recombinant protein (Pus4) was expressed at high level and purified to homogeneity by metal affinity chromatography on Ni2+-NTA-agarose, followed by ion-exchange chromatography on MonoQ . The purified Pus4p catalyzes the formation of pseudouridine-55 in T7 in vitro transcripts of several yeast tRNA genes . In contrast to the known yeast pseudouridine synthase (Pus1) of broad specificity, no other uridines in tRNA molecules are modified by the cloned recombinant tRNA:Psi55 synthase . The disruption of the corresponding gene YNL292w in yeast, which has no significant effect on the growth of yeast cells, leads to the complete disappearance of the Psi55 formation activity in a cell-free extract . These results allow the formal identification of the protein encoded by the yeast ORF YNL292w as the only enzyme responsible for the formation of Psi55 which is almost universally conserved in tRNAs . The substrate specificity of the purified YNL292w-encoded recombinant protein was shown to be similar to that of the native protein present in yeast cell extract . Chemical mapping of pseudouridine residues in both cytoplasmic and mitochondrial tRNAs from the yeast strain carrying the disrupted gene reveals that the same gene product is responsible for Psi55 formation in tRNAs of both cellular compartments. Nucleic Acids Res, 1997 Nov 15, 25(22), 4474 - 80 The motif V of plum pox potyvirus CI RNA helicase is involved in NTP hydrolysis and is essential for virus RNA replication; Fernandez A et al.; The plum pox potyvirus (PPV) protein CI is an RNA helicase whose function in the viral life cycle is still unknown . The CI protein contains seven conserved sequence motifs typical of RNA helicases of the superfamily SF2 . We have introduced several individual point mutations into the region coding for motif V of the PPV CI protein and expressed these proteins in Escherichia coli as maltose binding protein fusions . Mutations that abolished RNA helicase activity also disturbed NTP hydrolysis . No mutations affected the RNA binding capacity of the CI protein . These mutations were also introduced in the PPV genome making use of a full-length cDNA clone . Mutant viruses carrying CI proteins with reduced RNA helicase activity replicated very poorly in protoplasts and were unable to infect whole plants without rapid pseudoreversion to wild-type . These results indicate that motif V is involved in the NTP hydrolysis step required for potyvirus RNA helicase activity, and that this activity plays an essential role in virus RNA replication inside the infected cell. Biochim Biophys Acta, 1997 Sep 12, 1353(3), 298 - 306 Heat shock-induced excessive relaxation of DNA in Escherichia coli mutants lacking the histone-like protein HU; Ogata Y et al.; Plasmid DNA in exponentially growing Escherichia coli immediately relaxes after heat shock but the relaxed DNA re-supercoils rapidly, the despite continued presence of the heat shock conditions . We have now obtained genetic evidence indicating that the histone-like protein HU of E . coli is required for this re-supercoiling of DNA . Plasmid DNA in a hupA-hupB double gene-disruption mutant relaxed excessively after heat shock, while the relaxation of DNA in a himA-himD double gene-disruption mutant and in an hns insertion mutant was transient, thereby indicating that HU protein, but not IHF or H-NS proteins, is required for the re-supercoiling of DNA . Exposure of the hupA-hupB double mutant to a temperature of 50 degrees C led to both excessive relaxation of DNA and to a decrease in viable cell number but temperatures lower than 46 degrees C did not lead to these events . Based on these results, we propose that HU protein maintains the negative supercoiling of DNA during thermal stress and contributes to cellular thermotolerance in E . coli. Biochim Biophys Acta, 1997 Sep 12, 1353(3), 277 - 86 Molecular cloning and expression of rat kallistatin gene; Chai KX et al.; We have previously purified and cloned human kallistatin and rat kallikrein-binding protein (RKBP), which are tissue kallikrein inhibitors belonging to the serine proteinase inhibitor superfamily . In this study, we have cloned and sequenced the gene encoding rat kallistatin with Phe-Phe-Ser-Ala-Gln at positions P2-P3', which is identical to the reactive center of human kallistatin . Rat kallistatin is highly similar to human kallistatin, sharing 68% and 57% sequence identity at the cDNA and the amino acid levels . The rat kallistatin gene exists in a single copy and is located on chromosome 6 . An SphI RFLP is found between SHR and WKY rats at or near the rat kallistatin gene locus . Two amino acid polymorphisms of the rat kallistatin gene between these two strains were found by sequence analysis . A candidate promoter in the 5'-flanking region (109 bp) of the rat kallistatin gene has been identified by reporter assays . The expression of rat kallistatin in the liver is growth-dependent and down-regulated during acute phase inflammation . Recombinant rat kallistatin produced in E . coli is able to bind to tissue kallikrein, and the interaction is inhibited by heparin . These characteristics define rat kallistatin as the counterpart of human kallistatin. Plant Mol Biol, 1997 Nov, 35(5), 641 - 54 Cloning and characterisation of a cytosolic glutathione reductase cDNA from pea (Pisum sativum L.) and its expression in response to stress; Stevens RG et al.; A second glutathione reductase (GR) cDNA has been cloned and sequenced from pea (Pisum sativum L . cv . Birte) . This new GR cDNA (GOR2) does not encode a pre-protein with a transit peptide and therefore is most likely to represent a cytosolic GR . It is significantly different at the DNA level from the previously cloned chloroplastidial/mitochondrial pea GR (GOR1), but retains the features characteristic of GRs from all sources and has GR activity when expressed in Escherichia coli . GOR2 maps to linkage group 6 on the pea genome map and it seems likely that this is the only locus for this gene . In contrast to GOR1, transcript levels of GOR2 increase in the recovery (post-stress) phases of both drought and chilling by about ten- and three-fold respectively . GOR2 therefore may play a role in the restoration of the post-stress redox state of the cytosolic glutathione pool. Plant Mol Biol, 1997 Nov, 35(5), 623 - 32 Heteromeric assembly of the cytosolic glutamine synthetase polypeptides of Medicago truncatula: complementation of a glnA Escherichia coli mutant with a plant domain-swapped enzyme; Carvalho H et al.; We have cloned and sequenced the cDNAs corresponding to the two cytosolic glutamine synthetase (GS) polypeptides (a and b) of Medicago truncatula . Using these two cDNAs we have prepared a construct encoding the N-terminal domain of b and the C-terminal domain of a in order to produce a domain-swapped polypeptide which should assemble to give an enzyme containing chimeric active sites . Both the native and the domain-swapped enzymes were expressed in Escherichia coli where they were catalytically and physiologically active as they were able to rescue a glnA deletion mutant . The expressed polypeptides were of the correct size and the isoenzymes behaved similarly to their native homologues on ion-exchange chromatography . We have found slight differences in the kinetic properties of the purified enzymes and in the modulation of their activities by several putative cellular effectors . In vitro dissociation of the purified a and b homo-octamers, followed by reassociation, showed that the subunits are able to self-assemble, perhaps randomly, to form heteromeric isoenzymes . Moreover, heteromeric isoenzymes occur in the plant as revealed by studies on the GS isoenzymes of nodules, roots, stems and stipules. Plant Mol Biol, 1997 Nov, 35(5), 597 - 603 UDP-glucose:sterol glucosyltransferase: cloning and functional expression in Escherichia coli; Warnecke DC et al.; Steryl glucosides are characteristic lipids of plant membranes . The biosynthesis of these lipids is catalyzed by the membrane-bound UDP-glucose:sterol glucosyltransferase (EC 2.4.1.173) . The purified enzyme (Warnecke and Heinz, Plant Physiol 105 (1994): 1067-1073) has been used for the cloning of a corresponding cDNA from oat (Avena sativa L.) . Amino acid sequences derived from the amino terminus of the purified protein and from peptides of a trypsin digestion were used to construct oligonucleotide primers for polymerase chain reaction experiments . Screening of oat and Arabidopsis cDNA libraries with amplified labeled DNA fragments resulted in the isolation of sterol glucosyltransferase-specific cDNAs with insert lengths of ca . 2.3 kb for both plants . These cDNAs encode polypeptides of 608 (oat) and 637 (Arabidopsis) amino acid residues with molecular masses of 66 kDa and 69 kDa, respectively . The first amino acid of the purified oat protein corresponds to the amino acid 133 of the deduced polypeptide . The absence of these N-terminal amino acids reduces the molecular mass to 52 kDa, which is similar to the apparent molecular mass of 56 kDa determined for the purified protein . Different fragments of these cDNAs were expressed in Escherichia coli . Enzyme assays with homogenates of the transformed cells exhibited sterol glucosyltransferase activity. Plant Mol Biol, 1997 Nov, 35(4), 471 - 81 Molecular characterization of glyoxalase II from Arabidopsis thaliana; Maiti MK et al.; Glyoxalase II is part of the glutathione-dependent glyoxalase detoxification system . In addition to its role in the detoxification of cytotoxic 2-oxo-aldehydes, specifically methylglyoxal, it has been suggested that the glyoxalase system may also play a role in controlling cell differentiation and proliferation . During the analysis of a T-DNA-tagged mutant of Arabidopsis we identified the gene for a glyoxalase II isozyme (GLY1) that appears to be mitochondrially localized . The cDNA encoding a glyoxalase II cytoplasmic isozyme (GLY2) was also isolated and characterized . Southern blot and sequence analyses indicate that glyoxalase II proteins are encoded by at least two multigene families in Arabidopsis . Escherichia coli cells expressing either GLY1 or GLY2 exhibit increased glyoxalase II activity, confirming that they do, in fact, encode glyoxalase II proteins . Northern analysis shows that the two genes are differentially expressed . Transcripts for the mitochondrial isozyme are most abundant in roots, while those for the cytoplasmic isozyme are highest in flower buds . The identification of glyoxalase II isozymes that are differentially expressed suggests that they may play different roles in the cell. Plant Mol Biol, 1997 Oct, 35(3), 377 - 81 Expression of chalcone synthase and chalcone isomerase proteins in Arabidopsis seedlings; Cain CC et al.; Antibodies have been developed against the first two enzymes of flavonoid biosynthesis in Arabidopsis thaliana . Chalcone synthase (CHS) and chalcone isomerase (CHI) were overexpressed and purified from Escherichia coli as fusion proteins with glutathione S-transferase from Schistosoma japonicum . The recombinant proteins were then used to immunize chickens and the resulting IgY fraction was purified from egg yolks . Immunoblots of crude protein extracts from Arabidopsis seedlings carrying wild-type and null alleles for CHS and CHI showed that the resulting antibody preparations provide useful tools for characterizing expression of the flavonoid pathway at the protein level . An initial analysis of expression patterns in seedlings shows that CHS and CHI proteins are present at high levels during a brief period of early seedling germination that just precedes the transient accumulation of flavonoid end-products. J Bacteriol, 1998 Feb, 180(3), 705 - 13 Regulation of carAB expression in Escherichia coli occurs in part through UTP-sensitive reiterative transcription; Han X et al.; In Escherichia coli, expression of the carAB operon is subject to cumulative repression, which occurs by ArgR-mediated repression at a downstream promoter, P2, and by pyrimidine-mediated regulation at an upstream promoter, P1 . In this study, we show that pyrimidine-mediated regulation occurs in part through a mechanism involving UTP-sensitive reiterative transcription (i.e., repetitive addition of U residues to the 3' end of a nascent transcript due to transcript-template slippage) . In this case, reiterative transcription occurs at the end of a run of three T x A base pairs in the initially transcribed region of the carAB P1 promoter . The sequence of this region is 5'-GTTTGC (nontemplate strand) . In the proposed regulatory mechanism, increased intracellular levels of UTP promote reiterative transcription, which results in the synthesis of transcripts with the sequence GUUUU(n) (where n = 1 to >30) . These transcripts are not extended downstream to include structural gene sequences . In contrast, lower levels of UTP enhance normal template-directed addition of a G residue at position 5 of the nascent transcript . This addition precludes reiterative transcription and permits normal transcript elongation capable of producing translatable carAB transcripts . Thus, carAB expression, which is necessary for pyrimidine nucleotide (and arginine) biosynthesis, increases in proportion to the cellular need for UTP . The proposed mechanism appears to function independently of a second pyrimidine-mediated control mechanism that involves the regulatory proteins CarP and integration host factor. J Lipid Res, 1997 Dec, 38(12), 2492 - 501 Functional bioactive recombinant acylation stimulating protein is distinct from C3a anaphylatoxin; Murray I et al.; Acylation stimulating protein (ASP) acts upon adipose tissue to stimulate triglyceride synthesis and glucose transport . The aim of the present study was to produce recombinant ASP and to measure its bioactivity . The cDNA region of the parent complement C3 sequence coding for ASP (C3adesArg) was cloned and expressed in E . coli . Bioactivity of the purified recombinant material was tested by determining its effect on triglyceride synthesis, glucose transport, and competition binding assays . In standard assays, concentrations of 5.5 microM recombinant ASP (rASP) stimulated triglyceride synthesis comparably to plasma ASP (pASP): 228% versus 237%, respectively, in 3T3 preadipocytes and 568% versus 440% in human differentiated adipocytes . rASP also increased glucose transport in L6 myocytes (163% at 10 microm rASP) and in human differentiated adipocytes (334% rASP vs . 329% pASP at 5 microM) . rASP competitively displaced radiolabeled plasma ASP from high affinity association with the cell surface in both human differentiated adipocytes and 3T3 preadipocyte fibroblasts . Furthermore, immunoprecipitation of rASP and pASP with a specific monoclonal antibody abolished stimulation of cellular triglyceride synthesis . Lastly, we contrasted the structure:function activities of the arginated (C3a) and desarginated (ASP) proteins . The lipogenic activity and the anaphylatoxic activity result from distinct structural domains of the polypeptides . Thus rASP retains full biologic ASP activity and may provide a tool to study structure-function relationships in this physiologic system. Liver Transpl Surg, 1998 Jan, 4(1), 78 - 88 Long-term reduction of serum bilirubin levels in Gunn rats by retroviral gene transfer in vivo; Tada K et al.; Conjugation with glucuronic acid, mediated by bilirubin-uridinediphosphoglucuronate glucuronosyltransferase (bilirubin-UGT), is essential for efficient biliary excretion of bilirubin . Inherited absence of this enzyme activity results in the potentially lethal Crigler-Najjar syndrome type I in humans and lifelong hyperbilirubinemia in Gunn rats . To develop a gene therapy for bilirubin-UGT deficiency, we constructed a high-titer replication-deficient amphotropic recombinant retrovirus (MFG-S hB-UGT1) capable of transferring the gene encoding bilirubin-UGT1, the principal bilirubin-UGT isoform in human liver . To stimulate hepatocyte proliferation, Gunn rats were subjected to 66% hepatectomy . After 24 hours, the portal vein, the hepatic artery, and the inferior vena cava above and below the hepatic vein were clamped, and the portal vein and the isolated segment of the vena cava were cannulated . The liver was perfused with the MFG-S hB-UGT1 preparation through the portal vein at 5 ml/min for 10 minutes, then circulation was restored . Control rat livers were perfused with a recombinant retrovirus expressing Escherichia coli beta-galactosidase . In MFG-S hB-UGT1-perfused rats, but not in controls, expression of human bilirubin-UGT1 was shown by immunotransblotting, bilirubin-UGT assay of liver homogenates, and biliary excretion of bilirubin diglucuronide and monoglucuronide . Mean serum bilirubin levels decreased by 20% to 25% in 3 weeks and remained at that level throughout the study period (18 months) . This is the first report of long-term amelioration of inherited jaundice by retrovirus-directed gene therapy in an animal model for Crigler-Najjar syndrome. J Bacteriol, 1998 Feb, 180(3), 737 - 41 Intragenic suppressors of induction-deficient TetR mutants: localization and potential mechanism of action; Biburger M et al.; Eight Tn10 Tet repressor mutants with an induction-deficient phenotype and with primary mutations located at or close to the dimer interface were mutagenized and screened for inducibility in the presence of tetracycline . The second-site suppressors with wild-type-like operator binding activity that were obtained act, except for one, at a distance, suggesting that they contribute to conformational changes in the Tet repressor . Many of these long-range suppressors occur along the dimer interface, indicating that interactions between the monomers play an important role in Tet repressor induction. J Bacteriol, 1998 Feb, 180(3), 732 - 6 Modulation of Escherichia coli adenylyl cyclase activity by catalytic-site mutants of protein IIA(Glc) of the phosphoenolpyruvate:sugar phosphotransferase system; Reddy P et al.; It is demonstrated here that in Escherichia coli, the phosphorylated form of the glucose-specific phosphocarrier protein IIA(Glc) of the phosphoenolpyruvate:sugar phosphotransferase system is an activator of adenylyl cyclase and that unphosphorylated IIA(Glc) has no effect on the basal activity of adenylyl cyclase . To elucidate the specific role of IIA(Glc) phosphorylation in the regulation of adenylyl cyclase activity, both the phosphorylatable histidine (H90) and the interactive histidine (H75) of IIA(Glc) were mutated by site-directed mutagenesis to glutamine and glutamate . Wild-type IIA(Glc) and the H75Q mutant, in which the histidine in position 75 has been replaced by glutamine, were phosphorylated by the phosphohistidine-containing phosphocarrier protein (HPr-P) and were equally potent activators of adenylyl cyclase . Neither the H90Q nor the H90E mutant of IIA(Glc) was phosphorylated by HPr-P, and both failed to activate adenylyl cyclase . Furthermore, replacement of H75 by glutamate inhibited the appearance of a steady-state level of phosphorylation of H90 of this mutant protein by HPr-P, yet the H75E mutant of IIA(Glc) was a partial activator of adenylyl cyclase . The H75E H90A double mutant, which cannot be phosphorylated, did not activate adenylyl cyclase . This suggests that the H75E mutant was transiently phosphorylated by HPr-P but the steady-state level of the phosphorylated form of the mutant protein was decreased due to the repulsive forces of the negatively charged glutamate at position 75 in the catalytic pocket . These results are discussed in the context of the proximity of H75 and H90 in the IIA(Glc) structure and the disposition of the negative charge in the modeled glutamate mutants. J Bacteriol, 1998 Feb, 180(3), 680 - 9 Archaeal binding protein-dependent ABC transporter: molecular and biochemical analysis of the trehalose/maltose transport system of the hyperthermophilic archaeon Thermococcus litoralis; Horlacher R et al.; We report the cloning and sequencing of a gene cluster encoding a maltose/trehalose transport system of the hyperthermophilic archaeon Thermococcus litoralis that is homologous to the malEFG cluster encoding the Escherichia coli maltose transport system . The deduced amino acid sequence of the malE product, the trehalose/maltose-binding protein (TMBP), shows at its N terminus a signal sequence typical for bacterial secreted proteins containing a glyceride lipid modification at the N-terminal cysteine . The T . litoralis malE gene was expressed in E . coli under control of an inducible promoter with and without its natural signal sequence . In addition, in one construct the endogenous signal sequence was replaced by the E . coli MalE signal sequence . The secreted, soluble recombinant protein was analyzed for its binding activity towards trehalose and maltose . The protein bound both sugars at 85 degrees C with a Kd of 0.16 microM . Antibodies raised against the recombinant soluble TMBP recognized the detergent-soluble TMBP isolated from T . litoralis membranes as well as the products from all other DNA constructs expressed in E . coli . Transmembrane segments 1 and 2 as well as the N-terminal portion of the large periplasmic loop of the E . coli MalF protein are missing in the T . litoralis MalF . MalG is homologous throughout the entire sequence, including the six transmembrane segments . The conserved EAA loop is present in both proteins . The strong homology found between the components of this archaeal transport system and the bacterial systems is evidence for the evolutionary conservation of the binding protein-dependent ABC transport systems in these two phylogenetic branches. J Bacteriol, 1998 Feb, 180(3), 655 - 9 Comparison of the sensitivities of two Escherichia coli genes to in vivo variation of Lrp concentration; Chen C et al.; Transcription of the Escherichia coli genes serA and gltBDF depends on the leucine-responsive regulatory protein, Lrp, and is very much decreased in an lrp mutant . By the use of an Lrp-deficient host and the lrp gene cloned under a plasmid-borne arabinose pBAD promoter, we varied the amount of Lrp present in the cell and showed that both genes were transcribed in proportion to the amount of Lrp synthesized . The affinity of serA for Lrp was four to five times greater than the affinity of gltD . Overproduction of Lrp was lethal to the cell. J Bacteriol, 1998 Feb, 180(3), 622 - 5 Endogenous superoxide dismutase levels regulate iron-dependent hydroxyl radical formation in Escherichia coli exposed to hydrogen peroxide; McCormick ML et al.; Aerobic organisms contain antioxidant enzymes, such as superoxide dismutase (SOD) and catalase, to protect them from both direct and indirect effects of reactive oxygen species, such as O2.- and H2O2 . Previous work by others has shown that Escherichia coli mutants lacking SOD not only are more susceptible to DNA damage and killing by H2O2 but also contain larger pools of intracellular free iron . The present study investigated if SOD-deficient E . coli cells are exposed to increased levels of hydroxyl radical (.OH) as a consequence of the reaction of H2O2 with this increased iron pool . When the parental E . coli strain AB1157 was exposed to H2O2 in the presence of an alpha-(4-pyridyl-1-oxide)-N-tert-butyl-nitrone (4-POBN)-ethanol spin-trapping system, the 4-POBN-.CH(CH3)OH spin adduct was detectable by electron paramagnetic resonance (EPR) spectroscopy, indicating .OH production . When the isogenic E . coli mutant JI132, lacking both Fe- and Mn-containing SODs, was exposed to H2O2 in a similar manner, the magnitude of .OH spin trapped was significantly greater than with the control strain . Preincubation of the bacteria with the iron chelator deferoxamine markedly inhibited the magnitude of .OH spin trapped . Exogenous SOD failed to inhibit .OH formation, indicating the need for intracellular SOD . Redox-active iron, defined as EPR-detectable ascorbyl radical, was greater in the SOD-deficient strain than in the control strain . These studies (i) extend recent data from others demonstrating increased levels of iron in E . coli SOD mutants and (ii) support the hypothesis that a resulting increase in .OH formation generated by Fenton chemistry is responsible for the observed enhancement of DNA damage and the increased susceptibility to H2O2-mediated killing seen in these mutants lacking SOD. J Bacteriol, 1998 Feb, 180(3), 527 - 37 Identification and characterization of protease-resistant SecA fragments: secA has two membrane-integral forms; Chen X et al.; We have identified and characterized the protease-resistant SecA fragments (X . Chen, H . Xu, and P . C . Tai, J . Biol . Chem . 271:29698-29706, 1996) through immunodetection with region-specific antibodies, chemical extraction, and sequencing analysis . The 66-, 36-, and 27-kDa proteolytic fragments in the membranes all start at Met1, whereas the 48-kDa fragment starts at Glu361 . The overlapping of the sequences of the 66- and 48-kDa fragments indicates that they are derived from different SecA molecules . These two fragments were generated differently in response to ATP hydrolysis and protein translocation . Furthermore, the presence of membrane is required for the generation of the 48-kDa fragment but not for that of the 66-kDa fragment . These data suggest that there are two different integral forms of SecA in the membrane: SecA(S) and SecA(M) . The combination of these two forms of SecA has several membrane-interacting domains . Both forms of SecA are integrated in the membrane, since both the 48- and 66-kDa fragments could be derived from urea- or Na2CO3-washed membranes . Moreover, all fragments are resistant to extraction with a high concentration of salt or with heparin, but the membrane-specific 48-kDa SecA domain is more sensitive to Na2CO3 or urea extraction . This suggests that this domain may interact with other membrane proteins in an aqueous microenvironment and therefore may form a part of the protein-conducting channel. J Bacteriol, 1998 Feb, 180(3), 505 - 13 Immunolocalization of Hsp60 in Legionella pneumophila; Garduno RA et al.; One of the most abundant proteins synthesized by Legionella pneumophila, particularly during growth in a variety of eukaryotic host cells, is Hsp60, a member of the GroEL family of molecular chaperones . The present study was initiated in response to a growing number of reports suggesting that for some bacteria, including L . pneumophila, Hsp60 may exist in extracytoplasmic locations . Immunolocalization techniques with Hsp60-specific monoclonal and polyclonal antibodies were used to define the subcellular location and distribution of Hsp60 in L . pneumophila grown in vitro, or in vivo inside of HeLa cells . For comparative purposes Escherichia coli, expressing recombinant L . pneumophila Hsp60, was employed . In contrast to E . coli, where Hsp60 was localized exclusively in the cytoplasm, in L . pneumophila Hsp60 was predominantly associated with the cell envelope, conforming to a distribution pattern typical of surface molecules that included the major outer membrane protein OmpS and lipopolysaccharide . Interestingly, heat-shocked L . pneumophila organisms exhibited decreased overall levels of cell-associated Hsp60 epitopes and increased relative levels of surface epitopes, suggesting that Hsp60 was released by stressed bacteria . Putative secretion of Hsp60 by L . pneumophila was further indicated by the accumulation of Hsp60 in the endosomal space, between replicating intracellular bacteria . These results are consistent with an extracytoplasmic location for Hsp60 in L . pneumophila and further suggest both the existence of a novel secretion mechanism (not present in E . coli) and a potential role in pathogenesis. Appl Microbiol Biotechnol, 1997 Dec, 48(6), 699 - 703 Enzymatic production of ethyl (R)-4-chloro-3-hydroxybutanoate: asymmetric reduction of ethyl 4-chloro-3-oxobutanoate by an Escherichia coli transformant expressing the aldehyde reductase gene from yeast; Kataoka M et al.; The asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (R)-4-chloro-3-hydroxybutanoate (CHBE) using Escherichia coli JM109 (pKAR) cells expressing the aldehyde reductase gene from Sporobolomyces salmonicolor AKU4429 as a catalyst was studied . The reduction required NADP+, glucose and glucose dehydrogenase for NADPH regeneration . In an aqueous system, the substrate was unstable, and inhibition of the reaction by the substrate was also observed . Efficient conversion of COBE to (R)-CHBE with a satisfactory enantiomeric excess (ee) was attained on incubation with transformant cells in an n-butyl acetate/water two-phase system containing the above NADPH-regeneration system . Under the optimized conditions, with the periodical addition of COBE, glucose and glucose dehydrogenase, the (R)-CHBE yield reached 1530 mM (255 mg/ml) in the organic phase, with a molar conversion yield of 91.1% and an optical purity of 91% ee . The calculated turnover of NADP+, based on the amounts of NADP+ added and CHBE formed, was about 5100 mol/mol. Biochemistry (Mosc), 1997 Sep, 62(9), 946 - 50 Effect of Asp-97-->Glu substitution on the pH dependence of catalysis by inorganic pyrophosphatase of Escherichia coli; Fabrichniy IP et al.; Substitution of Glu for the evolutionarily conserved Asp-97 in the active site of Escherichia coli inorganic pyrophosphatase increases the apparent pKa of the essential acidic group controlling the catalytic constant for pyrophosphate hydrolysis . In combination with previously reported data, this fact suggests that activity decreases in alkaline medium because of decreased rate of the release of the first molecule of the product phosphate from the active site. J Mol Biol, 1997 Nov 28, 274(2), 174 - 80 SelD homolog from Drosophila lacking selenide-dependent monoselenophosphate synthetase activity; Persson BC et al.; The isolation and molecular characterization of an invertebrate gene that encodes a homolog of the human selenophosphate synthetase 1 is described . This Drosophila gene, termed selD-like, is located in the cytogenetic interval 50 D/E on the right arm of chromosome 2 . It is expressed ubiquitously throughout embryogenesis and found to be highly enriched in the developing gut and in the nervous system of the embryo.The SelD-like from Drosophila was purified after expression in Escherichia coli . The purified protein does not catalyze the selenide-dependent ATP hydrolysis reaction and its gene does not complement a selD lesion in E . coli . These results and the fact that selD-like possesses an arginine residue at the position of the essential Cys17 (E . coli nomenclature) indicate that the Drosophila gene exerts a function different from that of the classical selenophosphate synthetases . Two classes of SelD proteins can therefore be differentiated . The class I proteins contain a cysteine or selenocysteine residue in the active site and display selenide-dependent selenophosphate synthetase activity . Class II proteins, including Drosophila selD-like and human selenophosphate synthetase 1 are devoid of this activity and they possess other amino acids in position 17 . Biochemistry, 1998 Jan 27, 37(4), 1124 - 30 Electron injection through a specific pathway determines the outcome of oxygen activation at the diiron cluster in the F208Y mutant of Escherichia coli ribonucleotide reductase protein R2; Parkin SE et al.; Protein R2 of ribonucleotide reductase from Escherichia coli contains a dinuclear iron cluster, which reductively activates O2 to produce the enzyme's functionally essential tyrosyl radical by one-electron oxidation of residue Y122 . A key step in this reaction is the rapid injection of a single electron from an exogenous reductant (Fe2+ or ascorbate) during formation of the radical-generating intermediate, cluster X, from the diiron(II) cluster and O2 . As this step leaves only one of the two oxidizing equivalents of the initial diiron(II)-O2 adduct, it commits the reaction to a one-electron oxidation outcome and precludes possible two-electron alternatives (as occur in the related diiron bacterial alkane hydroxylases and fatty acyl desaturases) . In the F208Y site-directed mutant of R2, Y208 is hydroxylated (a two-electron oxidation) in preference to the normal reaction {Aberg, A., Ormo, M., Nordlund, P., & Sjoberg, B . M . (1993) Biochemistry 32, 9845-9850}, implying that this substitution blocks electron injection or (more likely) introduces an endogenous reductant (Y208) that effectively competes . Here we demonstrate that O2 activation in the F208Y mutant of R2 partitions between these two-electron (Y208 hydroxylation) and one-electron (Y122 radical production) outcomes and that the latter becomes predominant under conditions which favor electron injection (namely, high concentration of the reductant ascorbate) . Moreover, we show that the sensitivity of the partition ratio to ascorbate concentration is strictly dependent on the integrity of a hydrogen-bond network involving the near surface residue W48: when this residue is substituted with F, Y208 hydroxylation predominates irrespective of ascorbate concentration . These data suggest that the hydrogen-bond network involving W48 is a specific electron-transfer pathway between the cofactor site and the protein surface. Glycobiology, 1997 Dec, 7(8), 1237 - 46 Temperature differences for trans-glycosylation and hydrolysis reaction reveal an acceptor binding site in the catalytic mechanism of Trypanosoma cruzi trans-sialidase; Ribeirao M et al.; Trypanosoma cruzi, the agent of Chagas disease, expresses on its surface a trans-sialidase that catalyzes preferentially the transference of alpha-2,3-linked sialic acid to acceptors containing terminal beta-galactosyl residues, instead of the typical hydrolysis reaction, found in most sialidases . The trans-sialidase is responsible for the acquisition of the host sialic acid by this protozoan parasite, which does not synthesize sialic acids . Here, we have studied some kinetic properties of a recombinant trans-sialidase expressed in Escherichia coli . We found that it has sequential-type kinetics for the transferase reaction, as shown for the parasite-derived enzyme . The rates of sialic acid transfer to water (hydrolysis), and to beta-galactosyl residues have a unique behavior with respect to the reaction temperature . While the hydrolysis rate of sialyllactose increases continuously up to 35 degrees C, the temperature for the maximal rate of trans-glycosylation depends on the acceptor concentration . At low acceptor concentrations the rate of trans-glycosylation is maximal at 13 degrees C and independent of the amount of sialic acid donors . With increasing acceptor concentrations, maximal rates of trans-glycosylation are shifted to higher temperatures . This finding is explained by an 8-fold increase in the Km for the acceptor from 13 degrees C to 33 degrees C . Differences in hydrolysis and transfer rates were also obtained by using 4-methylumbelliferyl-N-acetyl-neuraminic acid . However, its hydrolysis rate is much higher than the rate of transference to lactose, suggesting that a long-lived enzyme-sialosyl intermediate is not formed . In addition, lactose does not increase the rate of methyl-umbelliferone release at any temperature, indicating that the rate limiting step is the aglycon release . Based on these results we propose that transglycosylation in T . cruzi sialidase is favored by the existence of a binding site for beta-galactosyl residues, which accepts the new glycosidic bond as sialic acid is released from the donor . With increasing temperature the affinity for the acceptor decreases, resulting in a concomitant increase in the rate of transfer to water, which, in turn, can be suppressed by increasing the acceptor concentration. Clin Diagn Lab Immunol, 1998 Jan, 5(1), 41 - 4 Depressed phagocytosis and oxidative burst in polymorphonuclear leukocytes from individuals with pulmonary tuberculosis with or without human immunodeficiency virus type 1 infection; Shalekoff S et al.; Phagocytosis and oxidative burst in whole-blood granulocytes were assessed by flow cytometry with Phagotest and Bursttest kits, respectively . Seventy individuals were included in this study: 15 healthy, normal donors, 18 human immunodeficiency virus (HIV) type 1 (HIV-1)-seropositive patients, 19 patients with pulmonary tuberculosis (TB), and 18 patients co-infected with Mycobacterium tuberculosis and HIV-1 (TB-HIV) . Granulocyte phagocytosis was assessed by incubating whole blood with fluorescence-labelled Escherichia coli and measuring the proportion of granulocytes with ingested bacteria and the capacity (fluorescence intensity) of each cell to phagocytose E . coli . The percentage of granulocytes converting nonfluorescent dihydrorhodamine to fluorescent rhodamine 123 on production of reactive oxygen intermediates (ROIs) and the mean channel shift were assessed as a measure of oxidative burst . No differences in the proportion of granulocytes that were capable of phagocytosing or producing ROIs in response to E . coli were observed between any of the study groups . Phagocytosis was significantly enhanced in granulocytes from HIV-1-infected individuals . On the other hand, granulocytes from individuals infected with M . tuberculosis alone or in combination with HIV-1 had a significantly reduced capacity to phagocytose E . coli and to produce ROIs in response to E . coli as an agonist . These results provide evidence that granulocytes from individuals with pulmonary TB with or without concomitant infection with HIV-1 have an impaired ability to phagocytose and to undergo oxidative burst, possibly contributing to the enhanced susceptibility to opportunistic infections in these patients. Hum Immunol, 1997 Aug-Sep, 56(1-2), 17 - 27 Idiotypic vaccine for treatment of human B-cell lymphoma . Construction of IgG variable regions from single malignant B cells; Terness P et al.; Immunoglobulin idiotypes (Id) of malignant B cells represent highly specific markers which can be used for vaccination . PCR-amplification of immunoglobulin genes enables the rapid production of large amounts of Id vaccines . However, the separate amplification and subsequent recombination of heavy and light chains can lead to a loss of the relevant Id . To preserve the original chain pairs, we used single malignant B cells derived from an immunocytoma patient . Cytoplasm was extracted and the mRNA transcribed into cDNA . The VH and VL genes were then amplified by PCR and cloned into a vector for expression in E . coli . Id production was checked using an anti-Id mouse monoclonal Ab raised against the patient's tumor-specific IgG . One out of 3 constructs expressed the relevant Id . Analysis of the first 31 light chain residues revealed an identical sequence for the malignant B cells' IgG and the recombinant Id construct . Exchange of either the heavy or light chain with an unrelated chain resulted in loss of the Id . An unrelated sequence derived from the c-myc protein is coupled to the Id vaccine . The lymphoma patient was shown to have Abs to the c-myc sequence . This sequence therefore, increases the Id+ Ab's antigenicity . CD spect