J Biotechnol, 1998 Jul 16, 62(3), 231 - 9 The spectroscopic analysis, inhibition and binding studies demonstrate the equivalence of Erythrina caffra trypsin inhibitor and the recombinant substitution variant recSerETI; Minuth T et al.; A recombinant substitution mutant (recSerETI) of the Erythrina caffra trypsin inhibitor, with the N-terminal valine residue substituted by serine, was produced in E . coli and compared to the wildtype protein (wtETI) with respect to physicochemical and functional properties . The spectral properties, including UV absorbance, fluorescence emission and circular dichroism, were indistinguishable . Furthermore, the inhibitory activities of the two proteins regarding the inhibition of trypsin, chymotrypsin, tissue plasmininogen activator (t-PA) and reteplase (BM 06.022, t-PA deletion variant comprising the kringle 2 and the protease domains, isolated from transformed E . coli cells) and the affinity of the immobilized inhibitors for reteplase were closely similar . Five repetitive cycles of guanidinium chloride (GdmCl)-induced denaturation-renaturation yield the native mutant protein with its inhibitory activity fully restored . The only difference between the wildtype and the mutant protein refers to the intrinsic stability . Comparing the pH- and GdmCl-dependent transitions, as well as the thermal denaturation, recSerETI exhibits decreased stability compared to the wildtype protein . The pH range of stability is shifted from pH 1-9.5, for wtETI, to pH 2-9, for recSerETI; similarly the GdmCl-induced denaturation is found to occur at a GdmCl half concentration of 3.7 M instead of 4.5 M; in both cases the renaturation exhibits strong hysteresis . The mid-point of the thermal unfolding transition of the mutant protein is at approximately 65 degrees C, as compared to approximately 75 degrees C for the wildtype protein. J Biotechnol, 1998 Jul 16, 62(3), 163 - 7 A convenient method for affinity purification of maltose binding protein fusions; Srinivasan U et al.; Maltose binding protein of Escherichia coli is frequently employed as a fusion partner for the biosynthesis of polypeptides and proteins, largely because of the advantage provided by affinity purification of such a fusion . A mixture of cellulose and starch is shown to be a simple and effective alternative to the use of other, more complicated media which are often used for affinity chromatography of maltose binding protein fusions. J Biomol NMR, 1998 Jul, 12(1), 145 - 59 NMR detection of slow conformational dynamics in an endonuclease toxin; Whittaker SB et al.; The cytotoxic activity of the secreted bacterial toxin colicin E9 is due to a non-specific DNase housed in the C-terminus of the protein . Double-resonance and triple-resonance NMR studies of the 134-amino acid 15N- and 13C/15N-labelled DNase domain are presented . Extensive conformational heterogeneity was evident from the presence of far more resonances than expected based on the amino acid sequence of the DNase, and from the appearance of chemical exchange cross-peaks in TOCSY and NOESY spectra . EXSY spectra were recorded to confirm that slow chemical exchange was occurring . Unambiguous sequence-specific resonance assignments are presented for one region of the protein, Pro65-Asn72, which exists in two slowly exchanging conformers based on the identification of chemical exchange cross-peaks in 3D 1H-1H-15N EXSY-HSQC, NOESY-HSQC and TOCSY-HSQC spectra, together with C alpha and C beta chemical shifts measured in triple-resonance spectra and sequential NH NOEs . The rates of conformational exchange for backbone amide resonances in this stretch of amino acids, and for the indole NH of either Trp22 or Trp58, were determined from the intensity variation of the appropriate diagonal and chemical exchange cross-peaks recorded in 3D 1H-1H-15N NOESY-HSQC spectra . The data fitted a model in which this region of the DNase has two conformers, NA and NB, which interchange at 15 degrees C with a forward rate constant of 1.61 +/- 0.5 s-1 and a backward rate constant of 1.05 +/- 0.5 s-1 . Demonstration of this conformational equilibrium has led to a reappraisal of a previously proposed kinetic scheme describing the interaction of E9 DNase with immunity proteins {Wallis et al . (1995) Biochemistry, 34, 13743-13750 and 13751-13759} . The revised scheme is consistent with the specific inhibitor protein for the E9 DNase, Im9, associating with both the NA and NB conformers of the DNase and with binding only to the NB conformer detected because the rate of dissociation of the complex of Im9 and the NA conformer, NAI . is extremely rapid . In this model stoichiometric amounts of Im9 convert, the E9 DNase is converted wholly into the NBI form . The possibility that cis-trans isomerisation of peptide bonds preceding proline residues is the cause of the conformational heterogeneity is discussed . E9 DNase contains 10 prolines, with two bracketing the stretch of amino acids that have allowed the NA {symbol: see text} NB interconversion to be identified, Pro65 and Pro73 . The model assumes that one or both of these can exist in either the cis or trans form with strong Im9 binding possible to only one form. J Biomol NMR, 1998 Jul, 12(1), 109 - 21 A new general method for the biosynthesis of stable isotope-enriched peptides using a decahistidine-tagged ubiquitin fusion system: an application to the production of mastoparan-X uniformly enriched with 15N and 15N/13C; Kohno T et al.; A new strategy is described for the production of peptides enriched with stable isotopes . Peptides of interest are expressed in Escherichia coli (E . coli) cells as recombinant fusion proteins with Saccharomyces cerevisiae ubiquitin . This method yields as much as 30-100 mg/l of isotope-enriched fusion proteins in minimal media . A decahistidine tag attached to the N-terminus of ubiquitin enables a one-step purification of the fusion protein via Ni(2+)-chelating affinity chromatography . The ubiquitin moiety is then easily and specifically cleaved off by a protease, yeast ubiquitin hydrolase . Since this enzyme is also expressed at a high level in E . coli cells and can be purified in one step, the presented strategy has an advantage in view of costs over others that use commercially available proteases . In addition, since ubiquitin fusion proteins easily refold, the fusion protein can be expressed either in a soluble form or as inclusion bodies . This flexibility enables us to prepare peptides that are unstable in a soluble state in E . coli cells . As an example, the expression and the uniform stable isotope enrichment with 15N and/or 13C are described for mastoparan-X, a tetradecapeptide known to activate GTP-binding regulatory proteins . An amide group at the C-terminus of this peptide can also be formed by our method . The presented system is considered powerful for the stable isotope enrichment of short peptides with proton resonances that are too severely overlapped to be analyzed solely by proton NMR. Microbiol Mol Biol Rev, 1998 Sep, 62(3), 985 - 1019 Linkage map of Escherichia coli K-12, edition 10: the physical map; Rudd KE; A physical map, EcoMap10, of the now completely sequenced Escherichia coli chromosome is presented . Calculated genomic positions for the eight restriction enzymes BamHI, HindIII, EcoRI, EcoRV, BglI, KpnI, PstI, and PvuII are depicted . Both sequenced and unsequenced Kohara/Isono miniset clones are aligned to this calculated restriction map . DNA sequence searches identify the precise locations of insertion sequence elements and repetitive extragenic palindrome clusters . EcoGene10, a revised set of genes and functionally uncharacterized open reading frames (ORFs), is also depicted on EcoMap10 . The complete set of unnamed ORFs in EcoGene10 are assigned provisional names beginning with the letter "y" by using a systematic nomenclature. Clin Diagn Lab Immunol, 1998 Sep, 5(5), 627 - 31 Detection of human Toxoplasma-specific immunoglobulins A, M, and G with a recombinant Toxoplasma gondii rop2 protein; Martin V et al.; The Toxoplasma gondii rhoptry protein Rop2 was expressed in Escherichia coli as a fusion protein containing 44 kDa of the 55-kDa mature Rop2, supplied with six histidyl residues at the N-terminal end (Rop2196-561) . Humoral response during Toxoplasma infection of humans was analyzed by immunoglobulin G (IgG), IgA, and IgM enzyme-linked immunosorbent assay with Rop2196-561 as the antigen substrate . The analyzed sera were divided according to T . gondii-specific serological tests (IgG, IgA, or IgM indirect immunofluorescence and IgA or IgM immunosorbent agglutination assay) as group A (IgG+ IgA- IgM-; n = 35), group B (IgG+ IgA+ IgM+; n = 21), group C (IgG+ IgA+ IgM-; n = 5), and group D (IgG+ IgA- IgM+; n = 16) . Twenty-six T . gondii-seronegative sera from individuals with other infections were also included (group E) . Anti-Rop2 IgG antibodies were detected in 82.8% of group A sera and in 97.6% of the sera with acute-phase marker immunoglobulins (groups B, C, and D) . The percentage of IgA antibody reactivity against Rop2196-561 was 17.1% in group A, 50% in group D, and 80.8% in groups B and C . The percentage of IgM antibody reactivity was 0% in groups A and C and 62% in groups B and D . Sera from group E failed to show IgA, IgM, or IgG antibody reactivity . Since T . gondii Rop2 elicits a strong humoral response from an early stage of infection, it is suggested that recombinant Rop2196-561 would be suitable for use in diagnostic systems, in combination with other T . gondii antigens, to detect specific IgG, IgA, and IgM antibodies. Development, 1998 Oct, 125(19), 3765 - 74 Two distinct mechanisms for differential positioning of gene expression borders involving the Drosophila gap protein giant; Wu X et al.; We have combined genetic experiments and a targeted misexpression approach to examine the role of the gap gene giant (gt) in patterning anterior regions of the Drosophila embryo . Our results suggest that gt functions in the repression of three target genes, the gap genes Kruppel (Kr) and hunchback (hb), and the pair-rule gene even-skipped (eve) . The anterior border of Kr, which lies 4-5 nucleus diameters posterior to nuclei that express gt mRNA, is set by a threshold repression mechanism involving very low levels of gt protein . In contrast, gt activity is required, but not sufficient for formation of the anterior border of eve stripe 2, which lies adjacent to nuclei that express gt mRNA . We propose that gt's role in forming this border is to potentiate repressive interaction(s) mediated by other factor(s) that are also localized to anterior regions of the early embryo . Finally, gt is required for repression of zygotic hb expression in more anterior regions of the embryo . The differential responses of these target genes to gt repression are critical for the correct positioning and maintenance of segmentation stripes, and normal anterior development. Biochem J, 1998 Sep 15, 334 ( Pt 3), 677 - 84 A novel family of ubiquitin-specific proteases in chick skeletal muscle with distinct N- and C-terminal extensions; Baek SH et al.; We have recently identified a cDNA for a ubiquitin-specific protease (UBP), UBP41, that encodes the smallest functional UBP identified to date, using an Escherichia coli-based in vivo screening method . In the present study we isolated highly related cDNAs encoding a new family of UBP enzymes, named UBP46, UBP52 and UBP66 . These UBPs have virtually identical catalytic domains spanning the sequence of UBP41 between the active-site Cys and the His box (95% identity) . However, they possess distinct N- and/or C-terminal extensions . Moreover, they are more closely related to each other than to any other members of the UBP family . Thus these chick UBPs must define a novel family of de-ubiquitinating enzymes and should represent the first example among the UBP family enzymes, whose multiplicity is achieved by variation in their N- and C-terminal extensions . The chick UBPs were expressed in E . coli, and purified from the cells to apparent homogeneity using 125I-labelled ubiquitin-alphaNH-MHISPPEPESEEEEEHYC as a substrate . Each of the purified UBP46, UBP52 and UBP66 enzymes behaved as proteins of similar sizes under both denaturing and non-denaturing conditions, suggesting that all of them consist of a single polypeptide chain . The UBP enzymes cleaved the C-terminus of the ubiquitin moiety in natural and engineered fusions irrespective of their sizes and thus are active against ubiquitin-beta-galactosidase as well as a ubiquitin C-terminal extension protein of 80 amino acids . All UBPs except UBP66 released free ubiquitin from poly-His-tagged di-ubiquitin . However, the isopeptidase activity for hydrolysing polyubiquitinated lysozyme conjugates was not detected from these UBPs, which makes these UBPs distinct from UBP41 . These results suggest that the chick UBPs may play an important role in production of free ubiquitin from linear polyubiquitin chains and of certain ribosomal proteins from ubiquitin fusion proteins. Biochem J, 1998 Sep 15, 334 ( Pt 3), 659 - 67 Leishmania major parasites express cyclophilin isoforms with an unusual interaction with calcineurin; Rascher C et al.; The immunosuppressive effects of the fungal metabolite cyclosporin A (CsA) are mediated primarily by binding to cyclophilins (Cyps) . The resulting CsA-Cyp complex inhibits the Ca2+-regulated protein phosphatase calcineurin and down-regulates signal transduction events . Previously we reported that CsA is a potent inhibitor of infections transmitted by the human pathogenic protozoan parasite Leishmania major in vitro and in vivo, but does not effect the extracellular growth of L . major itself . It is unknown how L . major exerts this resistance to CsA . Here we report that a major Cyp, besides additional isoforms with the same N-terminal amino acid sequence, was expressed in L . major . The cloned and sequenced gene encodes a putative 174-residue protein called L . major Cyp 19 (LmCyp19) . The recombinant LmCyp19 exhibits peptidyl-prolyl cis/trans isomerase activity with a substrate specificity and an inhibition by CsA that are characteristic of other eukaryotic Cyps . To determine whether calcineurin is involved in the discrimination of the effects of CsA we also examined the presence of a parasitic calcineurin and tested the interaction with Cyps . Despite the expression of functionally active calcineurin by L . major, neither LmCyp19 nor other L . major Cyps bound to its own or mammalian calcineurin . The amino acid sequence of most Cyps includes an essential arginine residue around the calcineurin-docking side . In LmCyp19 this is replaced by an asparagine residue . This exchange and additional charged residues are apparently responsible for the lack of LmCyp19 interaction with calcineurin . These observations indicate that resistance of L . major to CsA in vitro is mediated by the lack of complex formation with calcineurin despite CsA binding by parasitic Cyp. Biochem J, 1998 Sep 15, 334 ( Pt 3), 559 - 63 Muscle-specific mRNA isoform encodes a protein composed mainly of the N-terminal 175 residues of type 2 Ins(1,4,5)P3 receptor; Futatsugi A et al.; We have found a novel isoform of the mouse type 2 Ins(1,4,5)P3 receptor {Ins(1,4,5)P3R} mRNA by reverse transcriptase-mediated PCR analysis . The novel isoform, which was expressed specifically in skeletal muscle and heart, was generated by the inclusion of a novel exon . As this exon contains a stop codon, the isoform encodes a putative protein (designated TIPR) consisting of 175 acid residues of the type 2 Ins(1,4,5)P3R and the following six residues derived from this exon . We transfected the cDNA of this isoform into COS-7 cells; these cells expressed a 24 kDa protein that was recognized by an antibody against TIPR produced in Escherichia coli . The isoform encoding TIPR was also found in human skeletal muscle and heart . The N-terminal region of Ins(1,4,5)P3R is suggested to have a role in ligand binding and to interact with the C-terminal channel domain of Ins(1,4,5)P3R itself . TIPR might regulate the Ins(1,4,5)P3 signal pathway in both muscles. Glia, 1998 Oct, 24(2), 244 - 51 Allograft-inflammatory factor-1 in rat experimental autoimmune encephalomyelitis, neuritis, and uveitis: expression by activated macrophages and microglial cells; Schluesener HJ et al.; Allograft inflammatory factor-1 (AIF-1) is a Ca2+ binding peptide expressed predominantly by activated monocytes . In order to investigate the role of AIF-1 in autoimmune lesions of the rat nervous system, we have used a synthetic gene to express AIF-1 in E . coli and have produced monoclonal antibodies against AIF-1 . AIF-1 was localized to monocytes/macrophages with rather selective staining of a minor rat monocyte subpopulation of lymphoid tissue . We then investigated expression of AIF-1 in experimental autoimmune encephalomyelitis (EAE), neuritis (EAN), and uveitis (EAU) . Within the local inflammatory lesions, infiltrating macrophages are prominently stained . In the diseased brain, AIF-1-positive microglial cells are not only found in the direct vicinity of the infiltrate, but widespread activation is seen in the parenchyma . This is the first demonstration that AIF-1 is present in autoimmune lesions . Immunostaining of microglial cells is noteworthy, as these cells are strategically placed regulatory elements of CNS immunosurveillance . Thus, AIF-1 might be a valuable marker to dissect the local monocyte heterogeneity in autoimmune disease. J Infect Dis, 1998 Sep, 178(3), 769 - 75 Mechanisms of isoniazid resistance in Mycobacterium tuberculosis: enzymatic characterization of enoyl reductase mutants identified in isoniazid-resistant clinical isolates; Basso LA et al.; Mutants in the structural gene of the inhA-encoded NADH-dependent 2-trans enoyl-acyl carrier protein reductase were identified from isoniazid-resistant clinical isolates of Mycobacterium tuberculosis . Recombinant InhA proteins with defined single amino acid replacements were expressed in Escherichia coli and purified to homogeneity . Steady-state kinetic parameters for wild type (WT) and I16T, I21V, I47T, and I95P mutants of the enoyl reductase were measured spectrophotometrically . NADH binding to WT and I16T, I21V, I47T, S94A, and I95P mutant reductases were determined by fluorescence spectroscopy and demonstrated that all mutant enzymes had reduced NADH affinity and that NADH binding to all mutants was cooperative as compared with the hyperbolic binding of NADH to the WT enzyme . Since KatG-produced electrophilic derivatives of isoniazid have been suggested to inactivate the enoyl reductase-NADH complex, the kinetics of inactivation for the WT and I21V and I95P mutants was determined . Both mutations resulted in significantly increased values for the apparent first-order rate constant of inactivation. Biochim Biophys Acta, 1998 Aug 24, 1381(3), 319 - 30 Impaired growth of an Escherichia coli rpe mutant lacking ribulose-5-phosphate epimerase activity; Lyngstadaas A et al.; We present evidence that ribulose-5-phosphate epimerase, a central metabolic enzyme acting in the non-oxidative branch of the pentose-phosphate pathway, is encoded by a gene in the dam containing operon of Escherichia coli . Enzymatic assays confirm that this gene encodes ribulose-5-phosphate epimerase activity . Disruption of the gene (rpe) causes loss of enzymatic activity and renders the rpe mutant unable to utilize single pentose sugars, indicating that rpe supplies the only ribulose-5-phosphate epimerase activity in E . coli . Growth of the rpe mutant is impaired in complex LB medium and severely impaired in minimal medium containing glycolytic carbon sources or gluconate . Enrichment with casamino acids abolishes or strongly relieves growth suppression in minimal medium . Aspartate counteracts the impaired growth in glycolytic carbon sources but not in gluconate . We suggest that the absence of the Rpe enzyme causes changes in the pentose-phosphate levels which alter the regulation of (a) metabolic enzyme(s) and thereby cause growth suppression and that the severity of growth suppression is related to the internal concentration of pentose-phosphates . Target enzymes for negative regulation may be located in the early parts of the Embden-Meyerhof-Parnas pathway and of the Entner-Doudoroff pathway and/or of carbohydrate transport systems feeding sugars into these sections of central metabolic pathways . Mutat Res, 1998 Sep 1, 417(1), 39 - 46 Detection of oxidative mutagenesis by isoniazid and other hydrazine derivatives in Escherichia coli WP2 tester strain IC203, deficient in OxyR: strong protective effects of rat liver S9; Blanco M et al.; Strain IC203, deficient in the OxyR function, was sensitive to both cytotoxic and mutagenic effects of isoniazid (INH) whereas its parent, WP2 uvrA/pKM101, was resistant to these effects . Four other hydrazine compounds, hydrazine hydrate (HZH), phenylhydrazine (PHZ), hydralazine (HLZ) and nialamide (NLD), were mutagenic in WP2 uvrA/pKM101 . Increases in mutagenicity were observed in IC203 for HZH and PHZ but not for HLZ and NLD . Growth inhibition zones by HZH, PHZ and NLD were larger in IC203 than in WP2 uvrA/pKM101 . The enhancements in the effects of INH, HZH and PHZ in IC203 with respect to its oxyR+ parent are considered to be caused by the production of reactive oxygen species . This is consistent with its inhibition in IC203 by S9 from liver of uninduced rats, probably through the action of catalase . Mutagenicities of INH, PHZ and HLZ were low in strains IC204, a derivative of WP2 uvrA carrying a deletion of the umuDC genes, and IC206, a derivative of IC204 deficient in the MutY glycosylase . In these strains, HZH and NLD induced a high level of revertants which carry suppressor mutations resulting exclusively from G:C-A:T transitions, thus suggesting a direct reaction of the two hydrazines with cytosine . Radiat Environ Biophys, 1998 Jul, 37(2), 101 - 6 Formation of plasmid DNA strand breaks induced by low-energy ion beam: indication of nuclear stopping effects; Chen Y et al.; Plasmid pGEM 3zf(+) was irradiated by nitrogen ion beam with energies between 20 and 100 keV and the fluence kept as 1x10(12)ions/cm2 . The irradiated plasmid was assayed by neutral electrophoresis and quantified by densitometry . The yields of DNA with single-strand and double-strand breaks first increased then decreased with increasing ion energy . There was a maximal yield value in the range of 20-100 keV . The relationship between DNA double-strand breaks (DSB) cross-section and linear energy transfer (LET) also showed a peak-shaped distribution . To understand the physical process during DNA strand breaks, a Monte Carlo calculation code known as TRIM (Transport of Ions in Matter) was used to simulate energy losses due to nuclear stopping and to electronic stopping . It can be assumed that nuclear stopping plays a more important role in DNA strand breaks than electronic stopping in this energy range . The physical mechanisms of DNA strand breaks induced by a low-energy ion beam are also discussed. J Infect Dis, 1998 Sep, 178(3), 887 - 90 Circulating leptin levels during acute experimental endotoxemia and antiinflammatory therapy in humans; Bornstein SR et al.; Leptin, a newly discovered adipose tissue-derived weight-reducing hormone, is increased in acute inflammation and may be involved in the anorexia and wasting syndrome associated with infection . To determine whether this hormone responds to an acute inflammatory stimulus, plasma leptin concentrations were measured in 12 healthy subjects after intravenous administration of endotoxin . These subjects were randomized to receive concurrently ibuprofen or placebo normal saline (6 in each group) . Endotoxin administration resulted in fever, leukocytosis, and an increase in plasma levels of the stress hormones adrenocorticotropic hormone (3.2 +/- 0.3 to 132.6 +/- 75.5 pmol/L, P = .001) and cortisol (431.6 +/- 44 to 796.9 +/- 99 mmol/L, P = .001) . Plasma leptin levels, however, did not change significantly from baseline values after administration of endotoxin (0 h: 6.9 +/- 3.1 ng/mL; 6 h: 6.0 +/- 2.2; 24 h: 6.5 +/- 2.8) . While ibuprofen suppressed fever and symptoms associated with endotoxemia, it had no effect on the plasma levels of leptin . In conclusion, acute experimental human endotoxinemia is not associated with acute changes in circulating leptin levels. Physiol Res, 1997, 46(1), 1 - 8 The effect of antigen stimulation on splenic transplants; Bergmann ES et al.; The present paper deals with the regeneration of splenic tissue after autologous transplantation . Control and transplanted rats (60 days after operation (10(6) cells per injection) . The effect of a primary response was studied by a single injection, long-lasting bacteraemia was imitated by 5 injections in weekly intervals . Spleens and transplants were investigated by flow-cytometry and immunohistochemistry . Additionally, the proliferation activity and the specific antibody production against Escherichia coli proteins were tested . Flow-cytometric analysis showed altered behaviour of T-helper cells and B-cells in transplants following a primary response, whereas in the multiple injection group a difference between the splenic and transplant response was restricted to macrophages and MHC II+ cells . The results of the morphometric analysis revealed that the cellular composition of unstimulated transplants was very similar to that of the spleen with some subtle alterations . Only the marginal zone showed more striking differences concerning the homing of several cell classes . Under stimulatory conditions, these subtle alterations became more drastic so that CD5+ cells, B-cells and macrophages responded in an abnormal manner in both groups . The analysis of thymidine kinase disclosed decreased activity in the spleen after weekly antigen stimulation . The stimulation index of all transplant groups was significantly lower than that of the spleen . The specific antibody (IgG) production after a single immunization was highest in the transplant group . All groups responded after the multiple challenge . In conclusion, the results demonstrate that splenic transplants differs in several, but subtle aspects from normal splenic tissue . The main reason for most of these alterations may be a slightly misguided recirculation and/or homing of cells. Cell, 1998 Aug 21, 94(4), 525 - 36 Crystal structure of the hexamerization domain of N-ethylmaleimide-sensitive fusion protein; Lenzen CU et al.; N-ethylmaleimide-sensitive fusion protein (NSF) is a cytosolic ATPase required for many intracellular vesicle fusion reactions . NSF consists of an amino-terminal region that interacts with other components of the vesicle trafficking machinery, followed by two homologous ATP-binding cassettes, designated D1 and D2, that possess essential ATPase and hexamerization activities, respectively . The crystal structure of D2 bound to Mg2+-AMPPNP has been determined at 1.75 A resolution . The structure consists of a nucleotide-binding and a helical domain, and it is unexpectedly similar to the first two domains of the clamp-loading subunit delta' of E . coli DNA polymerase III . The structure suggests several regions responsible for coupling of ATP hydrolysis to structural changes in full-length NSF. Dev Growth Differ, 1998 Aug, 40(4), 395 - 402 Mapping of cholecystokinin transcription in transgenic mouse brain using Escherichia coli beta-galactosidase reporter gene; Itoh Y et al.; Cholecystokinin (CCK), a neuro-gut peptide, occurs not only in the nervous but also in the digestive system . As a first step in elucidating whether CCK gene expression and its physiological functions co-operate in these separate organs, transgenic mice were produced using CCK promoter that directs bacterial beta-galactosidase as a reporter gene . A new transgenic vector was constructed, inserting the SV40 poly A signal 5' to the CCK promoter to impede any transcription upstream of the transgene . A 2.4 kb.p . region upstream to the transcription start site of the mouse CCK gene was used as the promoter . Transgene expression was detected first at embryonic 13.5 days in the central nervous system and increased after birth . The distribution of cells expressing beta-galactosidase transgene agreed fairly well with that of in situ hybridization . In addition, the upregulation of CCK gene expression was clearly demonstrated by expressing beta-galactosidase after injury to the brain . These results indicated that the 2.4 kb.p . of the CCK gene promoter region was sufficient to direct appropriate tissue-specific gene expression in mice. J Biol Chem, 1998 Sep 11, 273(37), 24165 - 72 DNA-based positive control mutants in the binding site sequence of 434 repressor; Xu J et al.; As detected by chemical nuclease treatments, the conformation of the 434 repressor-DNA complex depends on the sequence of the bound DNA (Bell, A . C., and Koudelka, G . B . (1993) J . Mol . Biol . 234, 542-553) . We show here that these DNA sequence-dependent conformational changes alter the efficiency with which the repressor activates transcription from 434 PRM . Several lines of evidence suggest that binding site sequence affects the repressor's ability to activate transcription by altering the accessibility of the activation surface on the repressor to RNA polymerase . The results presented here show that in addition to affecting transcription by altering the overall binding affinity of protein for DNA, DNA sequence may also modulate the activity of the DNA-bound protein. J Biol Chem, 1998 Sep 11, 273(37), 24083 - 7 Functional characterization of a plant importin alpha homologue . Nuclear localization signal (NLS)-selective binding and mediation of nuclear import of nls proteins in vitro; Jiang CJ et al.; Nuclear import of most nuclear proteins is initiated by recognition of the nuclear localization signal (NLS) by importin alpha . We recently isolated an importin alpha homologue from rice (rice importin alpha1) and demonstrated that transcription of the gene is down-regulated by light in rice leaves . To address the function of rice importin alpha1 in the process of nuclear import of proteins, we performed in vitro binding and nuclear import assays . The rice importin alpha1 showed specific binding to fusion proteins containing either monopartite or bipartite NLSs, but not to a fusion protein containing a Matalpha-2-type NLS, suggesting that there exists selective binding of rice importin alpha1 to different plant NLSs . The rice importin alpha1 is also capable of forming a complex with mouse importin beta and NLS protein in vitro . An in vitro nuclear import assay using permeabilized HeLa cells revealed that rice importin alpha1, in conjunction with other vertebrate transport factors, mediates the nuclear envelope docking of NLS proteins and their subsequent translocation into the nucleus . These data provide strong, direct evidence suggesting that rice importin alpha1 functions as a component of the NLS receptor in plant cells. J Biol Chem, 1998 Sep 11, 273(37), 24030 - 6 Enhanced binding of altered H-NS protein to flagellar rotor protein FliG causes increased flagellar rotational speed and hypermotility in Escherichia coli; Donato GM et al.; H-NS is an Escherichia coli nucleoid protein known only to function as a modulator of gene expression . In this study, we found that specific single amino acid substitutions in H-NS caused an approximately 50% increase in flagellum rotational speed . In fluorescence anisotropy and chemical cross-linking assays, H-NS interacted with the flagellar torque-generating rotor protein FliG to form a complex with a Kd of 2.15 microM . Furthermore, one of the altered H-NS proteins that exhibited high speed flagellum rotation bound FliG 50% tighter than wild-type H-NS . These results demonstrate the first non-regulatory role for H-NS and provide a direct correlation between H-NS-FliG binding affinities, flagellar rotation, and motor torque generation. J Biol Chem, 1998 Sep 11, 273(37), 23993 - 9 Flagellar motor-switch binding face of CheY and the biochemical basis of suppression by CheY mutants that compensate for motor-switch defects in Escherichia coli; Shukla D et al.; CheY is a response regulator protein of Escherichia coli that interacts with the flagellar motor-switch complex to modulate flagellar rotation during chemotaxis . The switch complex is composed of three proteins, FliG, FliM, and FliN . Recent biochemical data suggest a direct interaction of CheY with FliM . In order to determine the FliM binding face of CheY, we isolated dominant suppressors of fliM mutations in cheY with limited allele specificity . The protein products of suppressor cheY alleles were purified and assayed for FliM binding . Six out of nine CheY mutants were defective in FliM binding . Suppressor amino acid substitutions were mapped on the crystal structure of CheY showing clustering of reduced binding mutations on a solvent-accessible face of CheY, thus revealing a FliM binding face of CheY . To examine the basis of genetic suppression, we cloned, purified, and tested FliM mutants for CheY binding . Like the wild-type FliM, the mutants were also defective in binding to various CheY suppressor mutants . This was not expected if CheY suppressors were compensatory conformational suppressors . Furthermore, a comparison of flagellar rotation patterns indicated that the cheY suppressors had readjusted the clockwise bias of the fliM strains . However, a chemotaxis assay revealed that the readjustment of the clockwise bias was not sufficient to make cells chemotactic . Although the suppressors did not restore chemotaxis, they did increase swarming on motility plates by a process called "pseudotaxis." Therefore, our genetic selection scheme generated suppressors of pseudotaxis or switch bias adjustment . The binding results suggest that the mechanism for this adjustment is the reduction in binding affinity of activated CheY . Therefore, these suppressors identified the switch-binding surface of CheY by loss-of-function defects rather than gain-of-function compensatory conformational changes. J Biol Chem, 1998 Sep 11, 273(37), 23976 - 83 A novel mutation in the switch 3 region of Gsalpha in a patient with Albright hereditary osteodystrophy impairs GDP binding and receptor activation; Warner DR et al.; Albright hereditary osteodystrophy (AHO), a disorder characterized by skeletal abnormalities and obesity, is associated with heterozygous inactivating mutations in the gene for Gsalpha . A novel Gsalpha mutation encoding the substitution of tryptophan for a nonconserved arginine within the switch 3 region (Gsalpha R258W) was identified in an AHO patient . Although reverse transcription-polymerase chain reaction studies demonstrated that mRNA expression from wild type and mutant alleles was similar, Gsalpha expression in erythrocyte membranes from the affected patient was reduced by 50% . A Gsalpha R258W cDNA, as well as one with arginine replaced by alanine (Gsalpha R258A), was generated, and the biochemical properties of in vitro transcription/translation products were examined . When reconstituted with cyc- membranes, both mutant proteins were able to stimulate adenylyl cyclase normally in the presence of guanosine- 5'-O-(3-thiotriphosphate) (GTPgammaS) but had decreased ability in the presence of isoproterenol or AlF4- (a mixture of 10 microM AlCl3 and 10 mM NaF) . The ability of each mutant to bind and be activated by GTPgammaS or AlF4- was assessed by trypsin protection assays . Both mutants were protected normally by GTPgammaS but showed reduced protection in the presence of AlF4- . The addition of excess GDP (2 mM) was able to rescue the ability of AlF4- to protect the mutants, suggesting that they might have reduced affinity for GDP . A Gsalpha R258A mutant purified from Escherichia coli had decreased affinity for GDP and an apparent rate of GDP release that was 10-fold greater than that of wild type Gsalpha . Sucrose density gradient analysis demonstrated that both Gsalpha R258W and Gsalpha R258A were thermolabile at higher temperatures and that denaturation of both mutants was prevented by the presence of 0.1 mM GTPgammaS or 2 mM GDP . The crystal structure of Gsalpha demonstrates that Arg258 interacts with a conserved residue in the helical domain (Gln170) . Arg258 substitutions would be predicted to open the cleft between the GTPase and helical domains, allowing for increased GDP release in the inactive state, resulting in enhanced thermolability and reduced AlF4--induced adenylyl cyclase stimulation and trypsin protection, since activation by AlF4- requires bound GDP. J Biol Chem, 1998 Sep 11, 273(37), 23866 - 76 Probing the role of cysteine residues in the EcoP15I DNA methyltransferase; Reddy YV et al.; Chemical modification using thiol-directed agents and site-directed mutagenesis has been used to investigate the role of cysteine residues of EcoP15I DNA methyltransferase . Irreversible inhibition of enzymatic activity was provoked by chemical modification of the enzyme by N-ethylmaleimide and iodoacetamide . 5, 5'-Dithiobis(2-nitrobenzoic acid) titration of the enzyme under nondenaturing and denaturing conditions confirmed the presence of six cysteine residues without any disulfides in the protein . Aware that relatively bulky reagents inactivate the methyltransferase by directly occluding the substrate-binding site or by locking the methyltransferase in an inactive conformation, we used site-directed mutagenesis to sequentially replace each of the six cysteines in the protein at positions 30, 213, 344, 434, 553, and 577 . All the resultant mutant methylases except for the C344S and C344A enzymes retained significant activity as assessed by in vivo and in vitro assays . The effects of the substitutions on the function of EcoP15I DNA methyltransferase were investigated by substrate binding assays, activity measurements, and steady-state kinetic analysis of catalysis . Our results clearly indicate that the cysteines at positions other than 344 are not essential for activity . In contrast, the C344A enzyme showed a marked loss of enzymatic activity . More importantly, whereas the inactive C344A mutant enzyme bound S-adenosyl-L-methionine, it failed to bind to DNA . Furthermore, in double and triple mutants where two or three cysteine residues were replaced by serine, all such mutants in which the cysteine at position 344 was changed, were inactive . Taken together, these results convincingly demonstrate that the Cys-344 is necessary for enzyme activity and indicate an essential role for it in DNA binding. J Biol Chem, 1998 Sep 11, 273(37), 23625 - 8 The copper chaperone CCS directly interacts with copper/zinc superoxide dismutase; Casareno RL et al.; Dominantly inherited mutations in the gene encoding copper/zinc superoxide dismutase (SOD1) result in the fatal motor neuron disease familial amyotrophic lateral sclerosis (FALS) . These mutations confer a gain-of-function to SOD1 with neuronal degeneration resulting from enhanced free radical generating activity of the copper present in the mutant enzyme . The delivery of copper to SOD1 is mediated through a soluble factor identified as the copper chaperone for SOD1 (CCS) . Amino acid sequence alignment of SOD1 and CCS reveals a striking homology with conservation of the amino acids essential for mediating SOD1 homodimerization . Here we demonstrate that CCS and SOD1 directly interact in vitro and in vivo and that this interaction is mediated via the homologous domains in each protein . Importantly, CCS interacts not only with wild-type SOD1 but also with SOD1 containing the common missense mutations resulting in FALS . Our findings therefore reveal a common mechanism whereby different SOD1 FALS mutants may result in neuronal injury and suggest a novel therapeutic approach in patients affected by this fatal disease. J Toxicol Environ Health A, 1998 Aug 21, 54(8), 647 - 62 Thermoregulation in rats exposed perinatally to dioxin: core temperature stability to altered ambient temperature, behavioral thermoregulation, and febrile response to lipopolysaccharide; Gordon CJ et al.; Recent studies have shown that perinatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) alters thermoregulatory function in adult rats and hamsters, indicated by a reduced body temperature during the animal's nocturnal phase . The present study was designed to assess the behavioral thermoregulation, ability to develop a fever, and thermoregulatory stability as a function of ambient temperature (Ta) in rats exposed perinatally to TCDD . Pregnant Long-Evans rats were exposed on gestational day (GD) 15 to 1 microg TCDD/kg (po) . The male offspring were implanted with transmitters to monitor core temperature (Tc) and motor activity (MA) . The 24-h pattern of core temperature was affected by TCDD exposure, characterized by a reduced nocturnal Tc . At some ages, the diurnal Tc of the TCDD group was elevated . This dysfunction in temperature regulation was most apparent at 7 and 11 mo of age . The 24-h pattern of MA was also altered by TCDD . The hypothermic effects of TCDD were most pronounced at cooler Ta values of 10 to 22 degrees C . In contrast, behavioral thermoregulation, assessed by measuring the selected Ta and Tc of rats in a temperature gradient, was unaffected by TCDD . The ability to develop a fever following administration of lipopolysaccharide (LPS) endotoxin (Escherichia coli; 50 microg/kg) was accentuated in the TCDD-treated animals . The data confirm a nocturnal hypothermia in rats prenatally exposed to TCDD . However, the normal behavioral regulation of Tc suggests that hypothalamic thermoregulatory centers are not permanently altered . The accentuated fever in TCDD animals shows possible functional alterations in the neuroimmune and/or thermoregulatory axes involved in fever. Chest, 1998 Aug, 114(2), 569 - 76 Intrathoracic and extrathoracic sources of exhaled nitric oxide in porcine endotoxemic shock; Fujii Y et al.; OBJECTIVES: Nitric oxide (NO), a highly reactive species produced by the activity of NO synthases (NOS), is normally present in the exhaled air of humans and animals . Exhaled NO concentration increases significantly in humans with sepsis and animals, but neither the source nor NOS isoforms responsible for this rise in pulmonary NO production are known . The main objective of this study is to determine the sites and the mechanisms of enhanced NO production in the exhaled air of endotoxemic pigs . DESIGN: Randomized, controlled, animal study . SETTING: University-based animal research facility . SUBJECTS: Thirteen pathogen-free adult female pigs (22 to 27 kg) . INTERVENTIONS: Anesthetized pigs were divided into two groups: control and lipopolysaccharides (LPS) (septic) groups . In both groups, extrathoracic (upper airways, nasal, and paranasal) and intrathoracic (bronchi, bronchioles, and alveoli) compartments were ventilated equally with two separate ventilators connected to two tracheal tubes . The LPS group received slow infusion (over 2 h) of Escherichia coli endotoxin (10 microg/kg/h), whereas saline solution was infused into the control group . Expired air of the two compartments was collected throughout the 2-h observation period . The animals were then killed and the lungs were quickly excised and frozen . MEASUREMENTS: Hemodynamic variables were measured in both groups . NO concentration in the exhaled air of both compartments was measured with a chemiluminescence analyser . Pulmonary NOS activity was evaluated by measuring the conversion of L-{2,3H}-arginine to L-{2,3H}-citrulline, and pulmonary expression of NOS was evaluated by immunoblotting . RESULTS: Baseline NO concentration in both groups was significantly higher in the extrathoracic vs intrathoracic compartment (average of 5.2 vs 3.4 parts per billion) . Endotoxin infusion elicited a significant and early (after 45 min) rise in exhaled NO concentration in the extrathoracic compartment . Exhaled NO in the intrathoracic compartment also rose significantly but after 90 min of endotoxin infusion . Measurement of lung NOS activity showed a substantial rise in Ca++/calmodulin-dependent activity in the LPS group with no rise in Ca++/calmodulin-independent activity . Immunoblotting of lung tissue samples indicated the absence of the inducible isoform in both groups of animals . Moreover, LPS injection elicited no significant alterations in the pulmonary expression of the endothelial and the neuronal isoforms . CONCLUSIONS: Both extrathoracic and intrathoracic compartments contribute to the rise in exhaled NO production in experimental septic shock . The rise in exhaled NO production is due to increased activity of constitutive NOS isoforms as a result of increased cofactor availability and/or downregulation of the endogenous inhibitors of NOS. DNA Cell Biol, 1998 Aug, 17(8), 727 - 34 Cloning and functional analysis of C . elegans 7B2; Lindberg I et al.; The neuroendocrine protein 7B2 is a binding protein for the prohormone convertase 2 (PC2) and is required for the intracellular conversion of proPC2 to active PC2 . Both full-length 7B2 and its carboxy-terminal 31-residue peptide (CT peptide) are capable of potent inhibition of PC2; the 7B2 protein thus regulates both the biosynthesis and the activity of PC2 . Vertebrate 7B2s are highly conserved (92%-97% homology), and thus, species comparison has not been informative in assessing the crucial protein domains responsible for bioactivity . We here report the cloning of the Caenorhabditis elegans 7B2 protein . Although weakly conserved with the vertebrate sequences (23% similarity with mouse 7B2), C . elegans 7B2 contains the signature PPNPCP motif as well as a highly conserved heptapeptide within the CT peptide . In in vitro assays, C . elegans 7B2 possessed significant inhibitory activity against recombinant vertebrate PC2 (IC50 130 nM), and in two functional tests, the amino-terminal domain of C . elegans 7B2 facilitated the activation of proPC2 . We conclude that despite low amino acid conservation overall, both functional domains within 7B2 have been conserved between the C . elegans and the vertebrate proteins. Mutat Res, 1998 Jul 17, 403(1-2), 199 - 214 Comparison of mutant frequencies and types of mutations induced by thiotepa in the endogenous Hprt gene and transgenic lacI gene of Big Blue rats; Chen T et al.; The utility of the lacI transgene of Big Blue rats as a reporter of in vivo mutation was evaluated by comparing the frequency and types of mutations induced by thiotepa in the transgene and the endogenous Hprt gene . Transgenic rats were given i.p . injections of 1.4 mg/kg of thiotepa three times per week over a period of 4 weeks (a total dose of 16.8 mg/kg); 1 week after the last injection, mutation assays were performed on spleen lymphocytes isolated from the animals . Thiotepa treatment increased the lacI mutant frequency from 34.8 +/- 4.1 x 10(-6) in control animals to 140.9 +/- 64.8 x 10(-6) (p = 0.0020) and the Hprt mutant frequency from 3.5 +/- 1.5 x 10(-6) to 41.1 +/- 23.2 x 10(-6) (p = 0.0028) . Sequence analysis of lacI mutant DNA and Hprt mutant cDNA produced similar overall mutation patterns: G:C-->T:A transversion was the most common base pair substitution (32% of independent mutations in the lacI gene and 28% of Hprt mutations), and deletions and insertions accounted for 34% of mutations in the lacI gene and 28% in the Hprt gene . The majority of thiotepa-induced base pair substitutions in the Hprt gene occurred with the mutated purine on the non-transcribed DNA strand, while no strand-related bias was found for mutations in the lacI gene . Substitutions at G:C base pairs in the lacI gene, but not in the Hprt gene, were found disproportionately in CpG sites . In addition, multiplex polymerase chain reaction analysis of genomic DNA from the Hprt mutants indicated that 34% had relatively large deletions; none of these deletions was detected by the cDNA analysis . The results indicate that the frequency of thiotepa-induced mutants in Big Blue rats was 2.8-fold greater in the lacI gene than in the Hprt gene . Although the Hprt gene recovered a fraction of large deletions not found among the lacI mutants, the effects of transcription-coupled DNA repair in the Hprt gene and the targeting of base pair substitutions to G:C base pairs in CpG sites may have contributed to the higher mutant frequencies induced by thiotepa in the lacI transgene compared with the Hprt gene. Mutat Res, 1998 Jul 17, 403(1-2), 137 - 47 Mutation spectrum in the lacI gene, induced by gamma-radiation in aqueous solution under oxic conditions; Wijker CA et al.; Irradiation of DNA in a cellular environment leads to many types of DNA damage, resulting from various effects of gamma-radiation . One of these effects is the formation of water-derived radicals (e.g., .OH radicals), which are formed in the vicinity of DNA (indirect effect) . To study the influence of the indirect effect on gamma-radiation-induced mutations, a newly constructed plasmid, containing the lacI gene as a target gene, was irradiated with 60Co gamma-radiation in aqueous solution, in the presence of oxygen . Under these circumstances, only .OH radicals will be responsible for the induced mutations . Sequence analysis of the gamma-radiation-induced mutations showed that 96% of all mutations were base pair substitutions, 87% of which occurred in the lacI gene, the others are formed in the lac operator part . All gamma-radiation-induced mutations in the lacI gene occurred exclusively on G:C base pairs, and no mutations at A:T base pairs could be detected . In the spontaneous mutation spectrum, 83% of all mutations were base pair substitutions, 35% of which occurred in the lacI gene and 48% in the lac operator part . Base pair substitutions on G:C base pairs were very similar in the gamma-radiation-induced and in the spontaneous mutation spectrum, implying a high contribution of .OH radicals to spontaneous mutagenesis . A:T to G:C transitions accounted for 10% of all spontaneous base pair substitutions in the lacI gene and are probably the result of effects, other than just .OH radicals . It can be concluded that .OH radicals are an important source for mutations at G:C base pairs . In this paper, the extracellular gamma-radiation-induced mutation spectrum is also compared to the previously obtained, intracellular gamma-radiation-induced mutation spectrum of the lacI gene . Comparison shows some differences, such as relative high amounts of mutations at A:T base pairs, G:C to T:A transversions and frameshift mutations in the intracellular gamma-radiation-induced mutation spectrum, as compared to the extracellular gamma-radiation-induced mutation spectrum . Since the extracellular gamma-radiation-induced mutation spectrum shows that .OH radicals are mainly responsible for base pair substitutions on G:C base pairs, mutations at A:T base pairs in the intracellular gamma-radiation-induced mutation spectrum are apparently the result of additional or other factors. Mutat Res, 1998 Jul 17, 403(1-2), 21 - 8 Effect of endogenous carotenoids and defective RpoS sigma factor on spontaneous mutation under starvation conditions in Escherichia coli: evidence for the possible involvement of singlet oxygen; Bridges BA et al.; Under starvation conditions, a variety of stationary phase genes are up-regulated under the control of the stationary phase sigma factor RpoS including at least two peroxidases and a protective DNA binding protein Dps . Previous work suggested that the reversion to prototrophy of certain amino acid auxotrophs of Escherichia coli that occurs when the bacteria are starved of a required amino acid results from the accumulation of oxidative damage to guanine residues in DNA . We report here that three strains lacking RpoS are indistinguishable from wild type in their ability to undergo this starvation-associated mutation, suggesting that basal levels of catalase activity are more than adequate in these strains, and that the induction of catalases and other proteins controlled by rpoS does not contribute to the protection of the DNA, at least in cells starved in early stationary phase . In comparison, the introduction of a plasmid specifying the production of singlet oxygen scavengers (carotenoids) in stationary phase cells led to a roughly twofold reduction in mutant yield . The results suggest that singlet oxygen may be an important endogenously produced mutagen in resting cells. Mutat Res, 1998 Aug 7, 416(1-2), 37 - 57 Application of the comet assay for monitoring DNA damage in workers exposed to chronic low-dose irradiation . II . Base damage; Kruszewski M et al.; In the preceding paper {M . Wojewodzka, M . Kruszewski, T . Iwanenko, A.R . Collins, I . Szumiel, Application of the comet assay for monitoring DNA damage in workers exposed to chronic low dose irradiation . I . Strand breakage., Mutat . Res . 416 (1998) 21-35}, we reported the results of DNA damage examination carried out for a group of people (49 individuals) professionally at risk of exposure to low doses of ionizing radiation as measured by the alkaline comet assay . Here, we used the method in combination with oxidative base damage-specific endonucleases to estimate base damage in the same individuals . These were endonuclease III (endoIII) and formamidopyrimidine glycosylase (FPG) . In contrast to the previous investigations, we found no statistically significant difference in base damage between the control and hazard groups . Interestingly, the hazard group exhibited lower level of enzyme-sensitive sites than the control; however, this different was not significant . No correlation of base damage with age was found, similarly as in the case of DNA damage measured by the alkaline comet assay . Interindividual variability of base damage precluded exposure estimation for single individuals, since several members of the control group exhibited high comet parameters. Int J Cancer, 1998 Sep 25, 78(1), 106 - 11 Targeting tumor cells via EGF receptors: selective toxicity of an HBEGF-toxin fusion protein; Chandler LA et al.; Over-expression of the epidermal growth factor receptor (EGFR) is a hallmark of numerous solid tumors, thus providing a means of selectively targeting therapeutic agents . Heparin-binding epidermal growth factor (HBEGF) binds to EGFRs with high affinity and to heparan sulfate proteoglycans, resulting in increased mitogenic potential compared to other EGF family members . We have investigated the feasibility of using HBEGF to selectively deliver a cytotoxic protein into EGFR-expressing tumor cells . Recombinant fusion proteins consisting of mature human HBEGF fused to the plant ribosome-inactivating protein saporin (SAP) were expressed in Escherichia coli . Purified HBEGF-SAP chimeras inhibited protein synthesis in a cell-free assay and competed with EGF for binding to receptors on intact cells . A construct with a 22-amino-acid flexible linker (L22) between the HBEGF and SAP moieties exhibited an affinity for the EGFR that was comparable to that of HBEGF . The sensitivity to HBEGF-L22-SAP was determined for a variety of human tumor cell lines, including the 60 cell lines comprising the National Cancer Institute Anticancer Drug Screen . HBEGF-L22-SAP was cytotoxic in vitro to a variety of EGFR-bearing cell lines and inhibited growth of EGFR-over-expressing human breast carcinoma cells in vivo . In contrast, the fusion protein had no effect on small-cell lung carcinoma cells, which are EGFR-deficient . Our results demonstrate that fusion proteins composed of HBEGF and SAP exhibit targeting specificity and cytotoxicity that may be of therapeutic value in treating a variety of EGFR-bearing malignancies. J Biochem (Tokyo), 1998 Sep, 124(3), 634 - 41 Roles of the Ser146, Tyr159, and Lys163 residues in the catalytic action of 7alpha-hydroxysteroid dehydrogenase from Escherichia coli; Tanabe T et al.; The Escherichia coli 7alpha-hydroxysteroid dehydrogenase (7alpha-HSDH; EC 1.1.1.159) has been the subject of our studies, including the cloning of its gene, and determination of the crystal structures of its binary and ternary complexes {J . Bacteriol . 173, 2173-2179 (1991); Biochemistry 35, 7715-7730 (1996)} . Through these studies, the Ser146, Tyr159, and Lys163 residues were found to be involved in its catalytic action . In order to clarify the roles of these residues, we constructed six single mutants of 7alpha-HSDH, Tyr159-Phe (Y159F), Tyr159-His (Y159H), Lys163-Arg (K163R), Lys163-Ile (K163I), Ser146-Ala (S146A), and Ser146-His (S146H), by site-directed mutagenesis . These mutants were overexpressed in E . coli WSD, which is a 7alpha-HSDH null strain, and the expressed enzymes were purified to homogeneity . The kinetic constants of the mutant enzymes were determined, and the structures of the Y159F, Y159H, and K163R mutants were analyzed by X-ray crystallography . The Y159F mutant showed no activity, while the Y159H mutant exhibited 13.3% of the wild-type enzyme activity . No remarkable conformational change between the Y159F (or Y159H) and wild-type proteins was detected on X-ray crystallography . On the other hand, the K163I mutant showed just 5.3% of the native enzyme activity, with a 8 . 5-fold higher Kd . However, the K163R mutant retained 64% activity, and no remarkable conformational change was detected on X-ray crystallography . In the cases of the S146A and S146H mutants, the activities fairly decreased, with 20.3 and 35.6% of kcat of the wild-type, respectively . The data presented in this paper confirm that Tyr159 acts as a basic catalyst, that Lys163 binds to NAD(H) and lowers the pKa value of Tyr159, and that Ser146 stabilizes the substrate, reaction intermediate and product in catalysis. Virus Res, 1998 Jun, 55(2), 199 - 206 Identification of the 5' terminal structure of influenza virus genome RNA by a newly developed enzymatic method; Honda A et al.; A combination of T4 polynucleotide kinase, Escherichia coli alkaline phosphatase, yeast Saccharomyces cerevisiae capping enzyme consisting of alpha (RNA guanylyltransferase) and beta (RNA 5'-triphosphatase) subunits . and its alpha subunit without RNA 5'-phosphatase activity was used to establish a simple enzymatic method for determination of RNA species with 5'-hydroxyl, 5'-monophosphate, 5'-diphosphate or 5'-triphosphate termini . Using this method, we found that viral genome RNA (vRNA) segments of both A-type and C-type influenza viruses carry tri- or diphosphates at their 5' termini . The conclusion was based on the observations that: (i) 5' phosphorylation of vRNAs by T4 polynucleotide kinase takes place only after phosphatase treatment; and (ii) capping of vRNAs can be observed with both the intact yeast capping enzyme and its alpha subunit alone devoid of RNA 5'-triphosphatase activity; but (iii) the level of capping is higher for the alphabeta holoenzyme than the alpha subunit though the relative level varies depending on RNA preparations . The results support the de novo initiation for the RNA replication although transcription of influenza vRNAs is initiated by host cell capped RNAs as primers. Protein Eng, 1998 Jun, 11(6), 457 - 65 A study of conserved in-loop and out-of-loop glycine residues in the large subunit of ribulose bisphosphate carboxylase/oxygenase by directed mutagenesis; Cheng ZQ et al.; The replacement of all 22 completely conserved glycine residues in the large subunit of ribulose bisphosphate carboxylase/oxygenase from Anacystis nidulans by directed mutagenesis is described . In each beta/alpha barrel of the large subunit there are 12 completely conserved glycines in six of eight loops at the C-termini of eight beta-strands and four in loops at N-terminal ends of the beta-strands . Two completely conserved glycines are also in each beta/alpha barrel backbone and four more are in a large N-terminal portion preceding the barrel in a given L subunit . Substitution of glycines in loops that are C-terminal to beta-strands by proline was more deleterious to carboxylase activity than that by alanine supporting the postulates that these loops contribute to catalysis and substrate binding and that in some cases the glycines may serve as hinges enabling movement of the loops . In contrast, substitution of glycines at the N-terminal ends of beta-strands in the beta/alpha barrel more often led to failure to detect L subunits or their assembly into L8S8 complex . Substitution of these and the other conserved glycines by proline was more deleterious to carboxylase activity than by alanine in enzymes that assembled. J Immunol, 1998 Sep 1, 161(5), 2642 - 7 cDNA encoding a single-chain antibody to HIV p17 with cytoplasmic or nuclear retention signals inhibits HIV-1 replication; Tewari D et al.; HIV-1 gag p17 protein is an attractive target for molecular intervention, because it is involved in the viral replication cycle at both the pre- and postintegration levels . In the present experiments, we targeted p17 by intracellularly expressing a cDNA encoding an Ab to p17 . cDNA from a hybridoma-secreting Ab to p17 was cloned, sequenced, reconstructed as a single-chain Ab fragment (scFv), and expressed in the cytoplasm or nucleus with appropriate retention signals . The expressed scFvs had no effect on T cell growth or CD4 expression and bound specifically to HIV-1 p17 . Human CD4+ Jurkat T cells that expressed scFvs and were infected with HIV-1 showed a marked reduction in virus replication compared with cells expressing vector alone . The inhibition of virus replication was more pronounced when scFvs were expressed in the cytoplasm rather than the nucleus . From these studies, we conclude that the intracellular expression of a single-chain Ab to p17 inhibits HIV replication; in addition, the degree of inhibition is related to the intracellular targeting site. Proc Natl Acad Sci U S A, 1998 Sep 1, 95(18), 10763 - 8 A cis-acting element that directs the activity of the murine methylation modifier locus Ssm1; Engler P et al.; Silencing of chromosomal domains has been described in diverse systems such as position effect variegation in insects, silencing near yeast telomeres, and mammalian X chromosome inactivation . In mammals, silencing is associated with methylation at CpG dinucleotides, but little is known about how methylation patterns are established or altered during development . We previously described a strain-specific modifier locus, Ssm1, that controls the methylation of a complex transgene . In this study we address the questions of the nature of Ssm1's targets and whether its effect extends into adjacent sequences . By examining the inheritance of methylation patterns in a series of mice harboring deletion derivatives of the original transgene, we have identified a discrete segment, derived from the gpt gene of Escherichia coli, that is a major determinant for Ssm1-mediated methylation . Methylation analysis of sequences adjacent to a transgenic target indicates that the influence of this modifier extends into the surrounding chromosome in a strain-dependent fashion . Implications for the mechanism of Ssm1 action are discussed. Proc Natl Acad Sci U S A, 1998 Sep 1, 95(18), 10751 - 6 The reductive enzyme thioredoxin 1 acts as an oxidant when it is exported to the Escherichia coli periplasm; Debarbieux L et al.; Thioredoxin 1 is a major thiol-disulfide oxidoreductase in the cytoplasm of Escherichia coli . One of its functions is presumed to be the reduction of the disulfide bond in the active site of the essential enzyme ribonucleotide reductase . Thioredoxin 1 is kept in a reduced state by thioredoxin reductase . In a thioredoxin reductase null mutant however, most of thioredoxin 1 is in the oxidized form; recent reports have suggested that this oxidized form might promote disulfide bond formation in vivo . In the Escherichia coli periplasm, the protein disulfide isomerase DsbC is maintained in the reduced and active state by the membrane protein DsbD . In a dsbD null mutant, DsbC accumulates in the oxidized form . This oxidized form is then able to promote disulfide bond formation . In both these cases, the inversion of the function of these thiol oxidoreductases appears to be due to an altered redox balance of the environment in which they find themselves . Here, we show that thioredoxin 1 attached to the alkaline phosphatase signal sequence can be exported into the E . coli periplasm . In this new environment for thioredoxin 1, we show that thioredoxin 1 can promote disulfide bond formation and, therefore, partially complement a dsbA strain defective for disulfide bond formation . Thus, we provide evidence that by changing the location of thioredoxin 1 from cytoplasm to periplasm, we change its function from a reductant to an oxidant . We conclude that the in vivo redox function of thioredoxin 1 depends on the redox environment in which it is localized. Proc Natl Acad Sci U S A, 1998 Sep 1, 95(18), 10716 - 21 Tn5/IS50 target recognition; Goryshin IY et al.; This communication reports an analysis of Tn5/IS50 target site selection by using an extensive collection of Tn5 and IS50 insertions in two relatively small regions of DNA (less than 1 kb each) . For both regions data were collected resulting from in vitro and in vivo transposition events . Since the data sets are consistent and transposase was the only protein present in vitro, this demonstrates that target selection is a property of only transposase . There appear to be two factors governing target selection . A target consensus sequence, which presumably reflects the target selection of individual pairs of Tn5/IS50 bound transposase protomers, was deduced by analyzing all insertion sites . The consensus Tn5/IS50 target site is A-GNTYWRANC-T . However, we observed that independent insertion sites tend to form groups of closely located insertions (clusters), and insertions very often were spaced in a 5-bp periodic fashion . This suggests that Tn5/IS50 target selection is facilitated by more than two transposase protomers binding to the DNA, and, thus, for a site to be a good target, the overlapping neighboring DNA should be a good target, too . Synthetic target sequences were designed and used to test and confirm this model. Proc Natl Acad Sci U S A, 1998 Sep 1, 95(18), 10547 - 52 Implications of macromolecular crowding for signal transduction and metabolite channeling; Rohwer JM et al.; The effect of different total enzyme concentrations on the flux through the bacterial phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) in vitro was determined by measuring PTS-mediated carbohydrate phosphorylation at different dilutions of cell-free extract of Escherichia coli . The dependence of the flux on the protein concentration was more than linear but less than quadratic . The combined flux-response coefficient of the four enzymes constituting the glucose PTS decreased slightly from values of approximately 1.8 with increasing protein concentrations in the assay . Addition of the macromolecular crowding agents polyethylene glycol (PEG) 6000 and PEG 35000 led to a sharper decrease in the combined flux-response coefficient, in one case to values of approximately 1 . PEG 6000 stimulated the PTS flux at lower protein concentrations and inhibited the flux at higher protein concentrations, with the transition depending on the PEG 6000 concentration . This suggests that macromolecular crowding decreases the dissociation rate constants of enzyme complexes . High concentrations of the microsolute glycerol did not affect the combined flux-response coefficient . The data could be explained with a kinetic model of macromolecular crowding in a two-enzyme group-transfer pathway . Our results suggest that, because of the crowded environment in the cell, the different PTS enzymes form complexes that live long on the time-scale of their turnover . The implications for the metabolic behavior and control properties of the PTS, and for the effect of macromolecular crowding on nonequilibrium processes, are discussed. Proc Natl Acad Sci U S A, 1998 Sep 1, 95(18), 10477 - 81 Branch migration during Rad51-promoted strand exchange proceeds in either direction; Namsaraev EA et al.; The Saccharomyces cerevisiae Rad51 protein is important for genetic recombination and repair of DNA double-strand breaks in vivo and can promote strand exchange between linear double-stranded DNA and circular single-stranded DNA in vitro . However, unlike Escherichia coli RecA, Rad51 requires an overhanging complementary 3' or 5' end to initiate strand exchange; given that fact, we previously surmised that the fully exchanged molecules resulted from branch migration in either direction depending on which type of end initiated the joint molecule . Our present experiments confirm that branch migration proceeds in either direction, the polarity depending on whether a 3' or 5' end initiates the joint molecules . Furthermore, heteroduplex DNA is formed rapidly, first at the overhanging end of the linear double-stranded DNA's complementary strand and then more slowly by progressive lengthening of the heteroduplex region until strand exchange is complete . Although joint molecule formation occurs equally efficiently when initiated with a 3' or 5' overhanging end, branch migration proceeds more rapidly when it is initiated by an overhanging 3' end, i.e., in the 5' to 3' direction with respect to the single-stranded DNA. Proc Natl Acad Sci U S A, 1998 Sep 1, 95(18), 10378 - 83 Nitric oxide dioxygenase: an enzymic function for flavohemoglobin; Gardner PR et al.; Nitric oxide (NO*) is a toxin, and various life forms appear to have evolved strategies for its detoxification . NO*-resistant mutants of Escherichia coli were isolated that rapidly consumed NO* . An NO*-converting activity was reconstituted in extracts that required NADPH, FAD, and O2, was cyanide-sensitive, and produced NO3- . This nitric oxide dioxygenase (NOD) contained 19 of 20 N-terminal amino acids identical to those of the E . coli flavohemoglobin . Furthermore, NOD activity was produced by the flavohemoglobin gene and was inducible by NO* . Flavohemoglobin/NOD-deficient mutants were also sensitive to growth inhibition by gaseous NO* . The results identify a function for the evolutionarily conserved flavohemoglobins and, moreover, suggest that NO* detoxification may be a more ancient function for the widely distributed hemoglobins, and associated methemoglobin reductases, than dioxygen transport and storage. EMBO J, 1998 Sep 1, 17(17), 5192 - 200 Mechanistic studies of initiator-initiator interaction and replication initiation; Lu YB et al.; Unlike the chromosome of Escherichia coli that needs only one replication initiator protein (origin recognition protein) called DnaA, many plasmid replicons require dual initiators: host-encoded DnaA and a plasmid-encoded origin recognition protein, which is believed to be the major determinant of replication control . Hitherto, the relative mechanistic roles of dual initiators in DNA replication were unclear . Here, we present the first evidence that DnaA communicates with the plasmid-encoded pi initiator of R6K and contacts the latter at a specific N-terminal region . Without this specific contact, productive unwinding of plasmid ori gamma and replication is abrogated . The results also show that DnaA performs different roles in host and plasmid replication as revealed by the finding that the ATP-activated form of DnaA, while indispensable for oriC replication, was not required for R6K replication . We have analyzed the accessory role of the DNA bending protein, integration host factor (IHF), in promoting initiator-origin interaction and have found that IHF significantly enhances the binding of DnaA to its cognate site . Collectively, the results further advance our understanding of replication initiation. EMBO J, 1998 Sep 1, 17(17), 5095 - 102 Transcription through a simple DNA repeat blocks replication elongation; Krasilnikova MM et al.; The influence of d(G)n.d(C)n repeats on plasmid replication in Escherichia coli cells was analyzed using electrophoretic analysis of replication intermediates . These repeats impeded the replication fork in a length- and orientation-dependent manner . Unexpectedly, the replication arrest relied primarily on the repeats' transcription . When the d(C)n sequence served as the transcriptional template, both transcription and replication were blocked . This was true for transcription driven by either bacterial or phage RNA polymerases . We hypothesize that the replication fork halts after it encounters a stalled ternary complex of the RNA polymerase, the DNA template and the r(G)n transcript . This constitutes a novel mechanism for the regulation of replication elongation . The effects of this mechanism on repeat length polymorphism and genome rearrangements are discussed. Biochemistry, 1998 Sep 1, 37(35), 12269 - 79 TNP-ATP and TNP-ADP as probes of the nucleotide binding site of CheA, the histidine protein kinase in the chemotaxis signal transduction pathway of Escherichia coli; Stewart RC et al.; The interaction of CheA with ATP has important consequences in the chemotaxis signal transduction pathway of Escherichia coli . This interaction results in autophosphorylation of CheA, a histidine protein kinase . Autophosphorylation of CheA sets in motion a chain of biochemical events that enables the chemotaxis receptor proteins to communicate with the flagellar motors . As a result of this communication, CheA allows the receptors to control the cell swimming pattern in response to gradients of attractant and repellent chemicals . To probe CheA interactions with ATP, we investigated the interaction of CheA with the fluorescent nucleotide analogues TNP-ATP {2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate} and TNP-ADP . Spectroscopic studies indicated that CheA bound TNP-ATP and TNP-ADP with high affinity (micromolar Kd values) and caused a marked enhancement of the fluorescence of the TNP moiety of these modified nucleotides . Analysis of titration experiments indicated a binding stoichiometry of two molecules of TNP-ATP (TNP-ADP) per CheA dimer and suggested that the two binding sites on the CheA dimer operate independently . Binding of TNP-ATP to CheA was inhibited by ATP, and analysis of this inhibition indicated that the CheA dimer binds 2 molecules of ATP . Competition experiments also indicated that CheA binds TNP-ATP considerably more tightly than it binds unmodified ATP . Binding of TNP-ADP to CheA was inhibited by ADP in a similar manner . TNP-ATP was not a substrate for CheA and served as a potent inhibitor of CheA autophosphorylation (Ki < 1 microM) . The glycine-rich regions (G1 and G2) of CheA and other histidine protein kinases have been presumed to play important roles in ATP binding and/or catalysis of CheA autophosphorylation, although few experimental tests of these functional assignments have been made . Here, we demonstrate that a CheA mutant protein with Gly-->Ala substitutions in G1 and G2 has a markedly reduced affinity for ATP and ADP, as measured by Hummel-Dreyer chromatography . This mutant protein also bound TNP-ATP and TNP-ADP very poorly and had no detectable autokinase activity . Surprisingly, a distinct single-site substitution in G2 (Gly470-->Lys) had no observable effect on the affinity of CheA for ATP and ADP, despite the fact that it rendered CheA completely inactive as an autokinase . This mutant protein also bound TNP-ATP and TNP-ADP with affinities and stoichiometries that were indistinguishable from those observed with wild-type CheA . These results provide some preliminary insight into the possible functional roles of G1 and G2, and they suggest that TNP-nucleotides are useful tools for exploring the effects of additional mutations on the active site of CheA. Biochemistry, 1998 Sep 1, 37(35), 12261 - 8 Translocase-bound SecA is largely shielded from the phospholipid acyl chains; van Voorst F et al.; Protein translocation in Escherichia coli is mediated by the SecA ATPase bound to the SecYEG membrane protein complex . SecA translocation ATPase activity as well as protein translocation is dependent on the presence of negatively charged lipids . By using a phospholipid with an acyl chain linked photoactivatable group, the lipid accessibility of SecA bound at the translocase was explored . SecA bound to lipid vesicles containing negatively charged lipids was found to be readily accessible for labeling by the photoactivatable phospholipid . The presence of an excess amount of SecYEG complex resulted in a remarkable reduction in the amount of lipid-accessible SecA irrespective of the nucleotide-bound form of SecA . These data demonstrate that the SecYEG-bound SecA is largely shielded from the phospholipid acyl chains and suggest the presence of two distinct pools of membrane-bound SecA that differ in the degree of lipid association. Biochemistry, 1998 Sep 1, 37(35), 12253 - 60 A recombinant monocysteine mutant (Ser to Cys-155) of fast skeletal troponin T: identification by cross-linking of a domain involved in a physiologically relevant interaction with troponins C and I; Jha PK et al.; Troponin T (TnT), a subunit of the heterotrimeric troponin (Tn) complex, is essential for the Ca2+ regulation of vertebrate striated muscle contraction both in vivo and in vitro . With the exception of bovine cardiac TnT, all known vertebrate TnT isoforms lack a thiol group, a property which makes the wild-type proteins unsuitable as cross-linking substrate . We generated a mutant human fast skeletal TnT in which Ser155 was changed to Cys (TnT-Cys155) . Mutation of this residue in TnT as well as in vitro expression in Escherichia coli and purification of the recombinant mutant protein did not affect its biological properties in terms of in vitro binding to troponin I (TnI), troponin C (TnC), actin-tropomyosin (actin-Tm), and actomyosin ATPase activity . TnT-Cys155 was labeled with 4-maleimidobenzophenone (BP-TnT155) and photo-cross-linked to TnI, TnC, Tm, and all of the thin filament proteins . BP-TnT155 did not cross-link to Tm and showed weak Ca2+/Mg2+-independent cross-linking with TnI in the binary complex and in the presence of all thin filament protein components . BP-TnT155 showed Ca2+/Mg2+-dependent cross-linking with TnC in the binary and ternary complexes and Ca2+-favored cross-linking with TnI in the ternary complex . Thus, residue 155 of TnT is within 10 A (the length of cross-linker) of TnC in the presence or absence of Ca2+ and comes within 10 A of both TnI and TnC in the presence of Ca2+ . TnT residue 155 is in close proximity to or may even partly encompass the Tm binding site . These results suggest that TnT, in association with TnI, may participate in the "information transfer" mediated by the Ca2+ binding signal from TnC to Tm and the region around TnT residue 155 probably acts as a linker between troponin and actin-Tm in this signal transmission process . Our results also suggest that TnT contains at least one Ca2+/Mg2+-dependent TnC binding region located between its Tm and TnI binding regions . A recombinant truncated fragment of TnI, TnI96-181, containing amino acid residues 96-181 and labeled with BP at Cys-133, failed to cross-link with TnT, indicating that the region around Cys-133 of TnI is not involved in binary interaction with TnT. Biochemistry, 1998 Sep 1, 37(35), 12241 - 52 Modulation of the binding of signal peptides to lipid bilayers by dipoles near the hydrocarbon-water interface; Voglino L et al.; Interactions between signal (leader) sequences and membranes are critical to protein insertion and translocation across membranes . In this paper, circular dichroism, tryptophan fluorescence, electrophoretic mobility, dipole potential, and binding measurements were used to study the interaction of the signal sequence of the Escherichia coli LamB protein with various lipid bilayers . By modifying specific chemicophysical properties of both the signal sequence and bilayer, we analyzed some of the key factors underlying peptide-lipid interactions . We synthesized three analogues of the LamB signal peptide differing in their net charge (-2 to +4) and studied their binding to bilayers containing combinations of neutral lipids {egg phosphatidylcholine (EPC), sphingomyelin, cholesterol, ketocholesterol, and nitroxide-containing phospholipid} and a charged lipid (phosphatidylserine) . All three peptides bound to EPC bilayers and underwent a random coil to alpha-helix transition upon binding . Microelectrophoresis experiments revealed that both the N and C termini were near the outer surface of the bilayer, suggesting that the peptides adopted a "hammock" configuration with both termini exposed to the aqueous phase and the core of the alpha-helix located near the hydrocarbon-water interface . The binding of these LamB peptides was not markedly dependent on the bilayer area per molecule, compressibility modulus, or dipole potential, but did depend on the charge of the peptide and bilayer interfacial region . Moreover, the binding of LamB peptides was essentially eliminated in bilayers composed of phospholipids with a nitroxide moiety at the 7 position in one of their acyl chains or in EPC bilayers containing equimolar ketocholestanol . We propose that the incorporation of nitroxide or ketone groups into the hydrocarbon region near the lipid headgroup increases the effective width of the hydrophilic interfacial region and prevents some of the hydrophobic amino acids in the alpha-helix from reaching the nonpolar hydrocarbon core, thereby diminishing the free energy of partitioning and inhibiting peptide binding . These results point to an important role for interfacial dipoles in peptide-lipid interactions. Biochemistry, 1998 Sep 1, 37(35), 12213 - 20 Truncation of limonene synthase preprotein provides a fully active 'pseudomature' form of this monoterpene cyclase and reveals the function of the amino-terminal arginine pair; Williams DC et al.; The monoterpene cyclase limonene synthase transforms geranyl diphosphate to a monocyclic olefin and constitutes the simplest model for terpenoid cyclase catalysis . (-)-4S-Limonene synthase preprotein from spearmint bears a long plastidial targeting sequence . Difficulty expressing the full-length preprotein in Escherichia coli is encountered because of host codon usage, inclusion body formation, and the tight association of bacterial chaperones with the transit peptide . The purified preprotein is also kinetically impaired relative to the mixture of N-blocked native proteins produced in vivo by proteolytic processing in plastids . Therefore, the targeting sequence, that precedes a tandem pair of arginines (R58R59) which is highly conserved in the monoterpene synthases, was removed . Expression of this truncated protein, from a vector that encodes a tRNA for two rare arginine codons (pSBET), affords a soluble, tractable 'pseudomature' form of the enzyme that is catalytically more efficient than the native species . Truncation up to and including R58, or substitution of R59, yields enzymes that are incapable of converting the natural substrate geranyl diphosphate, via the enzymatically formed tertiary allylic isomer 3S-linalyl diphosphate, to (-)-limonene . However, these enzymes are able to cyclize exogenously supplied 3S-linalyl diphosphate to the olefinic product . This result indicates a role for the tandem arginines in the unique diphosphate migration step accompanying formation of the intermediate 3S-linalyl diphosphate and preceding the final cyclization reaction catalyzed by the monoterpene synthases . The structural basis for this coupled isomerization-cyclization reaction sequence can be inferred by homology modeling of (-)-4S-limonene synthase based on the three-dimensional structure of the sesquiterpene cyclase epi-aristolochene synthase {Starks, C . M., Back, K., Chappell, J., and Noel, J . P . (1997) Science 277, 1815-1820}. Biochemistry, 1998 Sep 1, 37(35), 12206 - 12 Kinetic reaction scheme for the dihydrofolate reductase domain of the bifunctional thymidylate synthase-dihydrofolate reductase from Leishmania major; Liang PH et al.; In several species of protozoa, the catalytic activities for the enzymes dihydrofolate reductase (DHFR) and thymidylate synthase (TS) reside on a single polypeptide chain constituting a bifunctional thymidylate synthase-dihydrofolate reductase enzyme . In most other species, however, these enzymes occur as monofunctional catalytic activities on separate enzymes . In this study, the kinetic reaction scheme for the dihydrofolate reductase activity from the bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) isolated from the parasite Leishmania major is compared to that of the monofunctional DHFR purified from Escherichia coli . Examination using pre-steady-state kinetic methods reveals interesting differences between the bifunctional and monofunctional forms of the dihydrofolate reductase enzymes . The rate-limiting step in the kinetic pathway for the monofunctional E . coli enzyme is the release of product, tetrahydrofolate . In contrast, for the L . major bifunctional enzyme, the kinetic step which limits the steady-state turnover is a conformational change associated with the release of NADP+ . A complete kinetic description for the dihydrofolate reductase reaction pathway for the bifunctional enzyme is presented. Biochemistry, 1998 Sep 1, 37(35), 12189 - 94 Inhibition of the cAMP-dependent protein kinase by synthetic A-helix peptides; Gamboni S et al.; The catalytic subunit of the cAMP-dependent protein kinase from Dictyostelium discoideum, PkaC, displays the same properties as its mammalian counterpart, except for being about twice as large in size . Sequence comparisons indicated the presence of a conserved alpha-helix (A-helix) within the N-terminal region of PkaC which could potentially establish close contacts with the catalytic core {Veron, M., et al . (1993) Proc . Natl . Acad . Sci . U.S.A . 90, 10618-10622} . We show in this report that a synthetic peptide with the A-helix sequence inhibits PKA activity, whereas unrelated peptides display no inhibitory activity . The inhibition seems competitive with respect to the kemptide substrate rather than due to binding to a secondary site . We further show by amino acid replacements that the last lysine of the A-helix sequence is involved in this specific inhibition . A model is proposed for the possible role of the A-helix. Biochemistry, 1998 Sep 1, 37(35), 12133 - 43 Probing of DNA-binding sites of Escherichia coli RecA protein utilizing 1-anilinonaphthalene-8-sulfonic acid; Masui R et al.; RecA protein of Escherichia coli plays an essential role in homologous recombination of DNA strands . To analyze the interaction of RecA with single-stranded DNA (ssDNA), we performed a fluorescence competition assay employing 1-anilinonaphthalene-8-sulfonic acid (ANS) as an extrinsic fluorescent probe . ANS bound to RecA at three sites, leading to enhancement of ANS fluorescence . Addition of synthetic polynucleotides to the RecA-ANS complex in the absence of a nucleotide quenched the ANS fluorescence, indicating displacement of ANS molecules by ssDNA . Less effective quenching by poly(dA) suggests that the nucleoprotein filament on poly(dA) may differ from those on poly(dT) and poly(dC) . A titration experiment with poly(dT) and poly(dA) showed clear stoichiometric binding of 3.5 nucleotides per protein . The site size for poly(dC) was 7.0, which could be explained by the formation of a double helix of poly(dC) . ATP and other nucleotides also displaced the ANS . To identify ANS-binding sites, ANS was incorporated into RecA by UV irradiation, and fluorescent peptides were isolated from the proteolytic digest . Sequence analysis suggested that ANS binds to or near the ATP-binding region . These results suggest that the fluorescence quenching and photoincorporation assay using ANS may be useful for the analysis of the interaction of a protein and its ligand. Biochemistry, 1998 Sep 1, 37(35), 12042 - 50 Tryptophan substitutions surrounding the nucleotide in catalytic sites of F1-ATPase; Weber J et al.; Novel tryptophan substitutions, surrounding the nucleotide bound in catalytic sites, were introduced into Escherichia coli F1-ATPase . The mutant enzymes were purified and studied by fluorescence spectroscopy . One cluster of Trp substitutions, consisting of beta-Trp-404, beta-Trp-410, beta-Asp-158 (lining the adenine-binding pocket), and beta-Trp-153 (close to the alpha/beta-phosphates), showed the same fluorescence responses to MgADP, MgAMPPNP, and MgATP and the same nucleotide binding pattern with MgADP and MgAMPPNP, with one site of higher and two sites of lower affinity . Therefore, in absence of catalytic turnover (and of gamma-subunit rotation), sites 2 and 3 appeared similar in affinity, and the region of the catalytic site sensed by these Trp substitutions did not change conformation with different nucleotides . In contrast, alpha-Trp-291 and beta-Trp-297, both close to the gamma-phosphate, showed very different fluorescence responses to MgADP versus MgAMPPNP, and in these cases the response was due exclusively or predominantly to nucleotide binding at the first, high-affinity catalytic site, thus allowing specific detection of this site . Titration with MgATP showed that the high-affinity site was present under conditions of steady-state, Vmax MgATP hydrolysis. Am J Physiol, 1998 Sep, 275(3 Pt 1), G449 - 59 Effects of endotoxin on gastric injury from luminal irritants in rats: potential roles of nitric oxide; Mercer DW et al.; The expression and function of inducible nitric oxide synthase (iNOS) in the stomach is unclear . This study assessed the effects of endotoxin on rat gastric iNOS expression and its role in gastric injury from luminal irritants . In conscious rats, a 5-h treatment with intraperitoneal lipopolysaccharide (LPS; 1-20 mg/kg) dose dependently increased gastric mucosal iNOS immunoreactivity and increased gastric luminal nitrate and nitrite accumulation (Griess reaction) . LPS also increased gastric luminal fluid accumulation and reduced macroscopic gastric injury from orogastric acidified ethanol . Aminoguanidine (45 mg/kg) did not prevent LPS-induced gastroprotection or gastric fluid accumulation . NG-nitro-L-arginine methyl ester increased gastric luminal fluid and caused macroscopic gastric injury when given with LPS . Using an anesthetized preparation followed by removal of luminal fluid, LPS reduced gastric mucosal blood flow and exacerbated gastric injury from either acidified ethanol or acidified taurocholate, an effect that was negated by aminoguanidine . These data indicate that in conscious rats, the gastroprotective effect of endotoxin is dependent on constitutive NOS but not iNOS activity . However, the inducible isoform participates in the ability of endotoxin to exacerbate gastric injury from luminal irritants in the anesthetized rat. Mol Microbiol, 1998 Aug, 29(3), 825 - 34 On proteins of the microsporidian invasive apparatus: complete sequence of a polar tube protein of Encephalitozoon cuniculi; Delbac F et al.; The microsporidian Encephalitozoon cuniculi is an obligate intracellular parasite that can cause opportunistic infections in AIDS patients . Spore invasion of host cells involves extrusion of a polar tube . After immunocytochemical identification of several polar tube proteins (PTPs) in E . cuniculi, a major PTP was isolated from two-dimensional gels and two peptide fragments were sequenced . The complete nucleotide sequence of the corresponding gene was obtained using a combination of PCR amplification and cloning techniques . The gene exists as a single copy per haploid genome and encodes an acidic proline-rich protein, with a deduced molecular mass of 37 kDa, that contains four tandemly arranged 26-amino-acid repeats . An N-terminal region of 22 residues represents a cleaved signal peptide, probably involved in the targeting of the PTP . No similarity with known proteins has been found . The protein was expressed in Escherichia coli, purified and injected into mice . The antisera reacted specifically with the polar tube in indirect immunofluorescence assays and electron microscope immunocytochemistry . Further identification of conserved and variable PTP structural motifs should be useful for diagnostic purposes and new therapeutic strategies. Mol Microbiol, 1998 Aug, 29(3), 763 - 74 Evidence that a globular conformation is not compatible with FhaC-mediated secretion of the Bordetella pertussis filamentous haemagglutinin; Guedin S et al.; The 220 kDa Bordetella pertussis filamentous haemagglutinin (FHA) is the major extracellular protein of this organism . It is exported using a signal peptide-dependent pathway, and its secretion depends on one specific outer membrane accessory protein, FhaC . In this work, we have investigated the influence of conformation on the FhaC-mediated secretion of FHA using an 80kDa N-terminal FHA derivative, Fha44 . In contrast to many signal peptide-dependent secretory proteins, no soluble periplasmic intermediate of Fha44 could be isolated . In addition, cell-associated Fha44 synthesized in the absence of FhaC did not remain competent for extracellular secretion upon delayed expression of FhaC, indicating that the translocation steps across the cytoplasmic and the outer membrane might be coupled . A chimeric protein, in which the globular B subunit of the cholera toxin, CtxB, was fused at the C-terminus of Fha44, was not secreted in B . pertussis or in Escherichia coli expressing FhaC . The hybrid protein was only secreted when both disulphide bond-forming cysteines of CtxB were replaced by serines or when it was produced in DsbA- E . coli . The Fha44 portion of the secretion-incompetent hybrid protein was partly exposed on the cell surface . These results argue that the Fha44-CtxB hybrid protein transited through the periplasmic space, where disulphide bond formation is specifically catalysed, and that secretion across the outer membrane was initiated . The folded CtxB portion prevented extracellular release of the hybrid, in contrast to the more flexible CtxB domain devoid of cysteines . We propose a secretion model whereby Fha44 transits through the periplasmic space on its way to the cell surface and initiates its translocation through the outer membrane before being released from the cytoplasmic membrane . Coupling of Fha44 translocation across both membranes would delay the acquisition of its folded structure until the protein emerges from the outer membrane . Such a model would be consistent with the extensive intracellular proteolysis of FHA derivatives in B . pertussis. Mol Microbiol, 1998 Aug, 29(3), 731 - 40 FtsK is an essential cell division protein that is localized to the septum and induced as part of the SOS response; Wang L et al.; The role of ftsK in the growth of Escherichia coli was examined by turning off its expression . This resulted in smooth filaments without constrictions, indicating that FtsK was required at an early step in septation . Consistent with this, FtsK was found to localize to the septum in 70% of the cells, indicating that it was recruited relatively early in this process . FtsK localization required the function of FtsZ and FtsA but not FtsI and FtsQ . Consistent with this, Z rings were present in FtsK-depleted filaments . Subcellular localization of FtsK confirmed that it was a membrane protein . Only the first 202 amino acids of FtsK were essential for its role in membrane localization, cell division and viability . The expression of ftsK increased as part of the SOS response, and increased expression of ftsK conferred increased resistance to DNA damage. Mol Microbiol, 1998 Aug, 29(3), 685 - 94 The ABC maltose transporter; Ehrmann M et al.; Bacterial ATP-binding cassette (ABC) transporters and their homologues in eukaryotic cells form one of the largest superfamilies known today . They function as primary pumps that couple substrate translocation across the cytoplasmic membrane to ATP hydrolysis . Although ABC transporters have been studied for more than three decades, the structure of these multi-component systems is unknown, and the mechanism of transport is not understood . This article reviews one of the most widely studied ABC systems, the maltose transporter of Escherichia coli . A first structural model of the transport channel allows discussion of possible mechanisms of transport . In addition, recent experimental evidence suggests that regulation of gene expression and transport activity is far more complex than expected. Int Immunol, 1998 Aug, 10(8), 1211 - 6 Disease-specific recombinant allergens for the diagnosis of allergic bronchopulmonary aspergillosis; Crameri R et al.; Allergic bronchopulmonary aspergillosis (ABPA), a severe pulmonary complication caused by Aspergillus fumigatus, is considered a complex clinical syndrome with defined serological, pathological radiological and clinical features . The diagnosis of ABPA is often difficult because of several overlapping clinical and laboratory findings shared between asthma with sensitization to A . fumigatus and ABPA, but essential for treatment to prevent severe deterioration of pulmonary function . We have cloned A . fumigatus allergens from a cDNA library displayed on phage surface, sequenced the inserts and produced recombinant proteins in Escherichia coli . The single recombinant allergens were used to assess the immunological response in representative groups of A . fumigatus-sensitized asthmatics with or without ABPA and healthy controls . The allergens rAsp f 1, a 16.9 kDa ribotoxin, rAsp f 3, a 18.5 kDa peroxisomal protein, and rAsp f 6, a 23 kDa manganese superoxide dismutase, were identified as proteins with known biological function and rAsp f 4, a 30 kDa allergen, lacks sequence homology to known proteins . The secreted ribotoxin rAsp f 1 and rAsp f 3 are recognized by serum IgE of A . fumigatus-sensitized asthmatics with or without ABPA, whereas the non-secreted manganese superoxide dismutase rAsp f 6 and the rAsp f 4 allergen are exclusively recognized by serum IgE of ABPA patients . The dissection of IgE-mediated immune responses to single recombinant A . fumigatus allergens in asthmatic patients allow a discrimination between ABPA and A . fumigatus sensitization with high specificity (100%) and sensitivity (90%). J Allergy Clin Immunol, 1998 Aug, 102(2), 184 - 90 How far can we simplify in vitro diagnostics for grass pollen allergy?: A study with 17 whole pollen extracts and purified natural and recombinant major allergens; van Ree R et al.; BACKGROUND: Current diagnostics for grass pollen allergy are composed of mixtures of pollen of different grass species . Their complex composition hampers accurate standardization . OBJECTIVE: The aim of the study was to investigate whether mixtures of grass pollen extracts can be replaced by a single pollen species and whether a single pollen species can be replaced by a limited number of purified natural or recombinant major allergens . METHODS: Sera (n = 800) were selected on the basis of a general suspicion for inhalant allergy and tested in a RAST for IgE reactivity with pollen from 17 different grass species . Cross-reactivity of IgE responses was studied by means of RAST inhibition . Sera with positive test results for grass pollen were tested in a RAST for natural Lol p 1 and Lol p 5 and recombinant Phl p 1 and Phl p 5 . Results: Specific IgE antibodies against one or more of the 17 pollen species were detected in 209 of 800 sera (26.1%) . The highest responses were observed against Poa pratensis followed by Festuca rubra, Phleum pratense, and Dactylis glomerata . IgE responses were clearly lower (approximately by a factor of 5) against only three species (Phragmites communis, Cynodon dactylon, and Zea mays) . With the exception of a few low-responder sera, no sera were found to have negative test results to the high responder species and positive results to any of the other species . Sera with positive test results for grass pollen (n = 154) were tested with purified Lol p 1 and Lol p 5 . IgE anti-Lol p 1 and Lol p 5 accounted for an average of 81% +/- 7% of total anti-grass pollen IgE . For 14 sera (all with low anti-grass pollen IgE titers), a RAST with purified allergens resulted in a false-negative diagnosis for grass pollen allergy . With recombinant Phl p 1 and Phl p 5, the mean IgE reactivity was 57% +/- 6% of the anti-grass pollen IgE response (n = 141), with 13 false-negative results . CONCLUSION: One grass species is sufficient for in vitro diagnosis of grass pollen allergy . With purified natural Lol p 1 and Lol p 5, greater than 90% of grass-positive sera is detected . Around 80% of the IgE response to grass pollen is directed to these major allergens . Recombinant allergens, produced in Escherichia coli, did not equal the IgE-binding capacity of their natural counterparts. Int J Biochem Cell Biol, 1998 Jul, 30(7), 773 - 82 Recombinant expression of rat histidine decarboxylase: generation of antibodies useful for western blot analysis; Dartsch C et al.; Histidine decarboxylase catalyses the formation of histamine, an important biological messenger . In spite of the essential biological functions exerted by histamine the knowledge about the mechanisms involved in the regulation of histidine decarboxylase is rather limited . This is most likely due to the limited supply of suitable tools, including highly specific antibodies . In the present study we describe the production and characterisation of specific antisera against rat histidine decarboxylase using recombinant protein synthesised in a bacterial expression system . The antisera were shown to effectively immunoprecipitate histidine decarboxylase activity in extracts of fetal rat liver as well as to detect the histidine decarboxylase protein by Western blot analysis of COS-7 cells expressing recombinant rat histidine decarboxylase . The results demonstrate the successful production of highly specific antisera to histidine decarboxylase which may become valuable tools in future studies of the structure and function of this enzyme. Arch Virol, 1998, 143(7), 1349 - 63 Serological characterization of the 3'-proximal encoded proteins of beet yellows closterovirus; He X et al.; The 3'-proximal open reading frames (ORFs) of beet yellows closterovirus, California isolate (BYV-CA), were sequenced and the expression of the corresponding proteins analyzed . The nucleotide sequence of ORF 5 (coding for p24) was most conserved compared with ORF 7 (coding for p20) and ORF 8 (coding for p21) among the isolates analyzed . Polyclonal antisera were produced to GST fusion proteins of p24, p20, and p21 . Accumulation of p24, CP, p20 and p21 was studied in infected Tetragonia expansa plants and Chenopodium quinoa protoplasts . All four proteins were expressed in all tissues (old leaves, young leaves and stems), and most abundantly in young leaves . The subcellular localization of each protein in different tissues showed that compared with p24, CP and p21, p20 accumulated less in transfected protoplasts . Immunogold labeling in sugarbeet with p24 and CP antisera demonstrated co-localization of p24 and CP in vascular petiole tissues . In infectivity neutralization tests, antisera against p24 and CP greatly reduced transmission of BYV by viruliferous aphids compared with viruliferous aphids fed on preimmune serum or antiserum to p21. J Biochem (Tokyo), 1998 Sep, 124(3), 679 - 85 A novel acyl-CoA synthetase, ACS5, expressed in intestinal epithelial cells and proliferating preadipocytes; Oikawa E et al.; We report here the identification, characterization, and expression of a novel rat acyl-CoA synthetase (ACS) designated as ACS5 . ACS5 consists of 683 amino acids and is approximately 60% identical to the previously characterized ACS1 and ACS2 . ACS5 was overproduced in Escherichia coli cells and then purified to near homogeneity . The purified enzyme utilized a wide range of saturated fatty acids similar to those utilized by ACS1 and ACS2, but differed in its preference for C16-C18 unsaturated fatty acids . Northern blot analysis revealed that ACS5 mRNA is present most abundantly in the small intestine, and to a much lesser extent in the lung, liver, adrenal gland, adipose tissue, and kidney . In situ hybridization of rat ileum revealed abundant accumulation of ACS5 transcripts in foveolar epithelial cells . The hepatic level of ACS5 mRNA was significantly increased by refeeding a fat-free high sucrose diet and reduced by fasting or refeeding a high cholesterol diet, whereas that in the small intestine was not significantly altered by various dietary conditions . In contrast to the absence of ACS1 mRNA in undifferentiated 3T3-L1 preadipocytes, ACS5 mRNA was present in proliferating 3T3-L1 preadipocytes and its level remained unaltered during differentiation, suggesting that ACS5 may provide the acyl-CoA utilized for the synthesis of cellular lipids in proliferating preadipocytes. J Biochem (Tokyo), 1998 Sep, 124(3), 615 - 21 Molecular characterization of tobacco sulfite reductase: enzyme purification, gene cloning, and gene expression analysis; Yonekura-Sakakibara K et al.; A cDNA clone, NtSiR1, that encodes the precursor of ferredoxin-dependent sulfite reductase (Fd-SiR) has been isolated from a cDNA library of tobacco (Nicotiana tabacum cv . SR1) . The identity of the cDNA was established by comparison of the purified protein and the predicted structure with the nucleotide sequence . The amino terminus of the purified enzyme was Thr62 of the precursor protein, and the mature region of NtSiR1 consisted of 632 amino acids . Tobacco Fd-SiR is 82, 77, and 48% identical with Fd-SiRs from Zea mays, Arabidopsis thaliana, and a cyanobacterium, respectively . Significant similarity was also found with Escherichia coli NADPH-SiR in the region involved in ligation of siroheme and the {4Fe-4S} cluster . On Northern blot analysis, a transcript of NtSiR1 was detected in leaves, stems, roots, and petals in similar amounts . We also isolated a genomic SiR clone named gNtSiR1 . It consists of 8 exons and 7 introns . Genomic Southern blot analysis indicated that at least two SiR genes are present in the tobacco genome. J Biochem (Tokyo), 1998 Sep, 124(3), 572 - 7 Detection of blotted antigen using a fusion protein between protein A and pepsinogen C; Aoki T et al.; Human pepsinogen (PG) A and C were fused with protein A and expressed in Escherichia coli . Although the fusion proteins (PA-PGA and PA-PGC) were not expressed at high levels and were almost totally recovered from the insoluble fraction, the renaturation and purification procedures were easy and simple . PA-PGA and PA-PGC possessed proteolytic activity equivalent to the gastric mucosal PGA and PGC, respectively . However, the activity of PA-PGC was about 3-fold higher than that of PA-PGA . Therefore, PA-PGC was applied to the subsequent immunoblotting studies . The proteolytic activity of PA-PGC was used for digesting the blocking reagent around the target antigen (in situ digestion method) or casein-clotting in the agarose plate containing skimmed milk (caseogram print method) . Although the sensitivity of these methods was lower than that of the conventional color detection, the caseogram print method was superior in that the reaction was linear over a wide range . On the other hand, the in situ digestion method possessed a unique property on Western blotting, and it was very easy to identify the relative position of the target, which could be recognized as a clear band . For PA-PGC detection, no special chemicals are required, and so the procedure is simple, rapid, and inexpensive. Nucleic Acids Res, 1998 Sep 15, 26(18), 4291 - 300 A phylogenomic study of the MutS family of proteins; Eisen JA; The MutS protein of Escherichia coli plays a key role in the recognition and repair of errors made during the replication of DNA . Homologs of MutS have been found in many species including eukaryotes, Archaea and other bacteria, and together these proteins have been grouped into the MutS family . Although many of these proteins have similar activities to the E.coli MutS, there is significant diversity of function among the MutS family members . This diversity is even seen within species; many species encode multiple MutS homologs with distinct functions . To better characterize the MutS protein family, I have used a combination of phylogenetic reconstructions and analysis of complete genome sequences . This phylogenomic analysis is used to infer the evolutionary relationships among the MutS family members and to divide the family into subfamilies of orthologs . Analysis of the distribution of these orthologs in particular species and examination of the relationships within and between subfamilies is used to identify likely evolutionary events (e.g . gene duplications, lateral transfer and gene loss) in the history of the MutS family . In particular, evidence is presented that a gene duplication early in the evolution of life resulted in two main MutS lineages, one including proteins known to function in mismatch repair and the other including proteins known to function in chromosome segregation and crossing-over . The inferred evolutionary history of the MutS family is used to make predictions about some of the uncharacterized genes and species included in the analysis . For example, since function is generally conserved within subfamilies and lineages, it is proposed that the function of uncharacterized proteins can be predicted by their position in the MutS family tree . The uses of phylogenomic approaches to the study of genes and genomes are discussed. Nucleic Acids Res, 1998 Sep 15, 26(18), 4267 - 73 The LIM domains of hic-5 protein recognize specific DNA fragments in a zinc-dependent manner in vitro; Nishiya N et al.; hic-5 protein is a member of the LIM protein family, containing four LIM domains in its C-terminal region . It is mainly localized in focal adhesions and shows striking similarity to paxillin in its LIM domains, although the function of these LIM domains has remained elusive . In the present study, we found that full-length and the C-terminal half of hic-5 protein, including four LIM domains, bound to DNA in a zinc-dependent manner in vitro . Mouse genomic fragments that specifically bound to the hic-5 protein were isolated by successive rounds of hic-5 protein-DNA complex immunoprecipitation and PCR amplification . Seven independent clones were isolated, which contained high amounts of G+A and/or a long A/T tract . A DNA binding protein blot assay revealed the specificity of the interaction between hic-5 protein and the DNA fragment . Using a series of truncated forms of the hic-5 LIM domains, each of the four LIM domains was found to contribute to DNA binding in a distinctive manner. J Biol Chem, 1998 Sep 4, 273(36), 23176 - 82 Role of MutS ATPase activity in MutS,L-dependent block of in vitro strand transfer; Worth L Jr et al.; In addition to mismatch recognition, Escherichia coli MutS has an associated ATPase activity that is fundamental to repair . Hence, we have characterized two MutS mutant gene products to define the role of ATP hydrolysis in homeologous recombination . These mutants, denoted MutS501 and MutS506, have single point mutations within the Walker A motif, and rate constants for ATP hydrolysis are down 60-100-fold as compared with wild type . Both MutS501 and MutS506 retain mismatch binding and, unlike wild type, fail to relinquish this specificity in the presence of ATP, adenosine 5'-O-(thiotriphosphate), and adenosine 5'-(beta, gamma-imino)triphosphate . Both MutS501 and MutS506 blocked the level of strand transfer between M13 and fd DNAs . The level of inhibition varied between the mutants and corresponded with the relative affinities to a G/T mispair . Neither MutS501 nor MutS506, however, would afford complete block of full-length heteroduplex in the presence of MutL . DNase I footprinting data are consistent with these results, as the region of protection by MutS501 and MutS506 was unchanged in the presence of ATP and MutL . Taken together, these studies suggest that 1) MutS impedes RecA-mediated homeologous exchange as a distinct mismatch-provoked event and 2) the role of MutL is coupled to MutS-dependent ATP hydrolysis . These observations are in good agreement with the present model for E . coli methyl-directed mismatch repair. J Bacteriol, 1998 Sep, 180(17), 4596 - 602 Overproduction of a functional fatty acid biosynthetic enzyme blocks fatty acid synthesis in Escherichia coli; Subrahmanyam S et al.; beta-Ketoacyl-acyl carrier protein (ACP) synthetase II (KAS II) is one of three Escherichia coli isozymes that catalyze the elongation of growing fatty acid chains by condensation of acyl-ACP with malonyl-ACP . Overexpression of this enzyme has been found to be extremely toxic to E . coli, much more so than overproduction of either of the other KAS isozymes, KAS I or KAS III . The immediate effect of KAS II overproduction is the cessation of phospholipid synthesis, and this inhibition is specifically due to the blockage of fatty acid synthesis . To determine the cause of this inhibition, we examined the intracellular pools of ACP, coenzyme A (CoA), and their acyl thioesters . Although no significant changes were detected in the acyl-ACP pools, the CoA pools were dramatically altered by KAS II overproduction . Malonyl-CoA increased to about 40% of the total cellular CoA pool upon KAS II overproduction from a steady-state level of around 0.5% in the absence of KAS II overproduction . This finding indicat