Biochem J, 1994 Jul 1, 301 ( Pt 1), 17 - 20 Assisted refolding of recombinant prochymosin with the aid of protein disulphide isomerase; Tang B et al.; Protein disulphide isomerase (PDI) was shown to be able to accelerate the refolding of unfolded recombinant prochymosin and to enhance the overall yield of active protein . Unlike previous reports in this study PDI was found to be active at pH values as high as 11 . The coincidence of the similar apparent optimum pH values of uncatalysed and PDI-catalysed reactions suggests that conditions favourable to spontaneous refolding of proteins may help PDI to catalyse thiol/disulphide interchange . Under the conditions described here no exogenously added dithiothreitol was required for PDI-catalysed renaturation, implying that the disulphide form of PDI was reduced to its active form by the free thiol groups in prochymosin molecules. J Pharmacol Exp Ther, 1994 Jul, 270(1), 356 - 61 Enhanced elimination of biotinylated antibodies by avidin-based hemoperfusion in rats; Burkhart KK et al.; We designed a hemoperfusion system using immobilized avidin for selective removal of biotinylated therapeutic antibodies from rats . Two prototype therapeutic antibodies, ovine antidigoxin Fab fragment and murine monoclonal 8A1 against Escherichia coli J5 lipopolysaccharide endotoxin, were biotinylated using biotin-long-chain-N-hydroxysuccinimide ester . Biotinylated antibodies were administered i.v . to anesthetized rats which were instrumented for measurement of cardiovascular parameters and connected to avidin-hemoperfusion devices . By using several different protocols of antibody administration and hemoperfusion, we found that the half-lives and areas under the time vs . concentration curves of biotinylated antibodies were reduced significantly after passage over immobilized avidin . Avidin hemoperfusion was not associated with adverse changes in blood pressure, heart rate or excess hemolysis . These data suggest that avidin hemoperfusion affords a means for selective removal of biotinylated ligands from serum, and that other therapeutic and potentially toxic compounds could be removed in this manner. Eur J Biochem, 1994 Jul 1, 223(1), 69 - 77 Expression of phytochrome apoprotein from Avena sativa in Escherichia coli and formation of photoactive chromoproteins by assembly with phycocyanobilin; Hill C et al.; Phytochrome DNAs from oat (Avena sativa L.) encoding the full-length 124-kDa polypeptide, a 118-kDa fragment lacking the first 65 amino acids, and two N-terminal fragments of 65 kDa and 45 kDa were subcloned and expressed in Escherichia coli . Reducing the temperature to 25 degrees C during cell growth and the coexpression of chaperones improved the folding into a functional conformation for most of the polypeptides, and in one case the yield of polypeptides was also enhanced . A maximum yield of reconstitutable apoprotein was obtained by expressing the 65-kDa fragment consisting of 595 amino acids . The apoproteins could be assembled in the dark with phycocyanobilin into photoreversible chromoproteins . The yield of photoreversible pigment could be further increased by far-red/red irradiation cycles, indicating that the presence of the chromophore promotes the correct folding of the binding site . The chromoproteins with an intact N-terminal domain exhibit Pr and Pfr absorption bands, which are blue-shifted relative to the corresponding bands of native phytochrome due to the particular phycocyanobilin structure . The 118-kDa fragment, only lacking the 6-kDa N-terminus, exhibits a strong Pr band, but only a weak Pfr absorbance . This indicates an essential role of the front 6-kDa region of the protein in the formation of the far-red absorbing chromophore-protein complex . Otherwise, the C-terminal region seems to be less important for photoreversibility as indicated by the function of the shorter fragments. Eur J Biochem, 1994 Jul 1, 223(1), 285 - 92 Isolation and refolding of a mutant methionine-free interleukin-2-receptor alpha chain synthesized as a fusion protein in Escherichia coli; Seipelt I et al.; A soluble domain of the interleukin(IL)-2 receptor, the alpha chain synthesized in Escherichia coli, was employed to study expression and refolding of the protein . The results showed that it is possible to obtain biologically active synthetic methionine-free IL-2 receptor alpha chain (synIL-2R alpha) after BrCN cleavage and renaturation of the crude cleavage material, although the alpha chain is expressed as a deglycosylated, methionine-free protein . The soluble receptor comprises amino acids 1-219 and forms 5 disulfide bonds in its biologically active state . Biological activity has been analysed by affinity chromatography and ELISA with mutant {Ala125}IL-2 and monoclonal antibodies as ligands . Renaturation yield is limited mainly by the high aggregation rate of incorrectly folded protein . Aggregation could be limited by varying the oxidation conditions . The deletion of a non-bridging cysteine at position 192 in the synIL-2R alpha did not affect the renaturation yield of the receptor protein . Additionally a cysteine-free and methionine-free beta-galactosidase derivative was fused to the soluble synIL-2R alpha derivatives to prevent reoxidation of incorrect disulfide bonds in the crude BrCN-cleavage material . It is suggested that cysteine impurities from cyanogen-bromide-cleaved peptides might interfere seriously with the refolding process of the synthetic IL-2 receptor alpha-subunit. Eur J Biochem, 1994 Jul 1, 223(1), 275 - 83 Purification of ATP synthase from Acetobacterium woodii and identification as a Na(+)-translocating F1F0-type enzyme; Reidlinger J et al.; The ATPase of Acetobacterium woodii was purified after solubilization of membranes with Triton X-100 by poly(ethylene glycol) precipitation and gel filtration . The enzyme consists of at least six subunits of apparent molecular masses of 57, 52, 35, 19, 15 and 4.8 kDa, as determined by SDS/PAGE . The 52-kDa band is immunologically related to the F1F0-ATPase beta subunit of Escherichia coli . The enzyme is not inhibited by vanadate but is inhibited by nitrate, azide and N,N'-dicyclohexylcarbodiimide; the 4.8-kDa subunit specifically reacts with N,N'-dicyclohexyl{14C}carbodiimide, indicating that the enzyme is of the F1F0 type . The enzyme activity is dependent on MgATP (Km = 0.4), has a pH optimum of pH 7-9 and is stimulated by sulfite . ATP hydrolysis is strictly dependent on sodium ions with a Km for Na+ of 0.4 mM . The purified enzyme was reconstituted into liposomes . Upon addition of ATP, primary and electrogenic 22Na+ transport into the lumen of the proteoliposomes was determined . These experiments demonstrate that the ATPase of Acetobacterium woodii is a Na(+)-translocating F1F0-type ATPase. Carcinogenesis, 1994 Jul, 15(7), 1371 - 5 Synthesis of a 25 base oligonucleotide containing a styrene oxide modification at the O6 position of 2'-deoxyguanosine at a defined site and incorporation studies of the similarly modified 2'-deoxyguanosine-5'-triphosphate; Pongracz K et al.; A diastereomeric mixture of the regioisomers O6-(2-hydroxy-2-phenylethyl)-2'-deoxyguanosine (st6G, beta-isomer) and O6-(2-hydroxy-1-phenylethyl)-2'-deoxyguanosine (alpha-isomer) was site-specifically placed in a 25 base oligonucleotide template 5'-CCGCTAst6GCGGGTACCGAGCTCGAAT-3' using CED phosphoramidite chemistry . Using 32P-post-labeling we found the oligonucleotide to contain 95% of the beta-isomer and 5% of the alpha-isomer of st6G . st6G as the 3'-phosphate was found to be considerably more acid labile than O6-methyl-2'-deoxyguanosine-3'-phosphate, leading to dealkylation during oligonucleotide synthesis . The diastereomeric mixture of O6-(2-hydroxy-2-phenylethyl)-2'-deoxy-guanosine-5'-triphosphate (st6dGTP) was chemically synthesized and used as a substrate for the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I . This study demonstrated that st6dGTP could be incorporated opposite deoxycytidine and did not completely block replication. Arch Biochem Biophys, 1994 Jul, 312(1), 59 - 66 Expression of modified human cytochrome P450 2E1 in Escherichia coli, purification, and spectral and catalytic properties; Gillam EM et al.; Human cytochrome P450 (P450) 2E1 is of interest because of its role in the oxidation of numerous drugs and carcinogens . The purification of the protein from human liver is difficult, and we report the development of a system for relatively high-level expression in Escherichia coli . A cDNA was prepared from liver cDNA by polymerase chain reaction methods and several variants with modified 5'-termini were constructed . Analysis of seven of these indicated that the highest levels of expression were found when the first 21 codons of the native sequence were deleted and the Trp immediately following the resulting N-terminal Met was changed to Ala (GCT) . Levels of 40-nmol membrane-bound P450 2E1 (liter culture)-1 were routinely recovered . The recombinant P450 2E1 was purified to electrophoretic homogeneity from the bacterial membranes in two ion-exchange steps in > 80% yield . Ferric P450 2E1 was isolated in a mixed spin state . The enzyme was active in chlorzoxazone 6-hydroxylation; the addition of human liver cytochrome b5 lowered the Km for the substrate and increased Vmax . N-Terminal amino acid sequence analysis yielded the expected first 21 residues . The expression system should facilitate the availability of human P450 2E1 and antibodies for studies of the enzyme. Arch Biochem Biophys, 1994 Jul, 312(1), 210 - 8 High substrate specificity factor ribulose bisphosphate carboxylase/oxygenase from eukaryotic marine algae and properties of recombinant cyanobacterial RubiSCO containing "algal" residue modifications; Read BA et al.; Marine algae play an important role in removing carbon dioxide from the atmosphere . In this investigation, we have determined the substrate specificity factor of ribulose 1,5-bisphosphate carboxylase/oxygenase from several marine chromophytic and rhodophytic algae . The enzymes were purified to homogeneity and all possessed significantly higher substrate specificity factors than the enzymes from terrestrial plants, green algae, or bacteria . There are substantial differences in the sequence in a helix 6 of the large subunit of these enzymes, which is intriguing since residues of this region had been previously shown to influence the ability of ribulose bisphosphate carboxylase to discriminate between CO2 and O2, presumably by influencing the adjacent flexible loop 6 region . Sequence divergence at this and other key regions might contribute to the substantial differences in the substrate specificity factor of the chromophyte/rhodophyte enzyme . Initial studies on probing the basis for the high substrate specificity factor employed single amino acid substitutions in the recombinant cyanobacterial ribulose bisphosphate carboxylase . Residues in the vicinity of loop 6 were changed to reflect the corresponding residues in the chromophyte/rhodophyte large subunit . Some changes in the substrate specificity factor were noted, as were alterations in other important kinetic parameters . Since marine algae show little evidence of photorespiratory metabolism, the high substrate specificity of ribulose bisphosphate carboxylase is consistent with the physiology of these organisms . The results of this study provide further evidence that the properties of this enzyme may evolve or change according to the environment in which the host organism is found. Arch Biochem Biophys, 1994 Jul, 312(1), 121 - 4 Identification of the N-terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system produced by proteolytic digestion; Lee BR et al.; The phosphoenolpyruvate:sugar phosphotransferase system of bacteria plays an important role in the concomitant uptake and phosphorylation of numerous sugars . The first protein in the pathway of phosphotransfer of the phosphoenolpyruvate:sugar phosphotransferase system is Enzyme I . It has been shown that a stable N-terminal domain can be produced by treatment of the purified protein with various proteolytic enzymes . We show here that the region from glutamate-252 to leucine-264 is accessible to proteolysis resulting in N-terminal cores ranging from M(r) 27521 to 28799. Am J Obstet Gynecol, 1994 Jul, 171(1), 223 - 30 Phosphorylation of 17 beta-hydroxysteroid dehydrogenase in BeWo choriocarcinoma cells; Barbieri RL et al.; OBJECTIVE: The purpose of this study was to test whether 17 beta-hydroxysteroid dehydrogenase might exist in a phosphorylated form . STUDY DESIGN: Phosphorylation of 17 beta-hydroxysteroid dehydrogenase was evaluated in BeWo choriocarcinoma cells . The phosphorylation of 17 beta-hydroxysteroid dehydrogenase expressed in Escherichia coli as a glutathione transferase fusion protein was also studied . RESULTS: Human BeWo choriocarcinoma cells were metabolically labeled with phosphorus 32 orthophosphate . Immunoprecipitates were prepared with rabbit anti-17 beta-hydroxysteroid dehydrogenase antiserum from the labeled cells and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . A phosphorylated protein with a molecular size of 35 kd was obtained from anti-17 beta-hydroxysteroid dehydrogenase immunoprecipitates, which suggested that 17 beta-hydroxysteroid dehydrogenase was phosphorylated in BeWo cells . The predominant phosphoamino acid was phosphoserine . 17 beta-Hydroxysteroid dehydrogenase expressed in E . coli as a glutathione transferase fusion protein was a substrate of protein kinase A in vitro . Protein kinase A phosphorylated the recombinant 17 beta-hydroxysteroid dehydrogenase exclusively on serine . Incubation of BeWo cell lysates with bacterial alkaline phosphatase led to a decrease in the oxidative activity of 17 beta-hydroxysteroid dehydrogenase . Incubation of the alkaline phosphatase inhibitor levamisole with BeWo cell lysates resulted in a higher estradiol-to-estrone conversion rate, compared with cell lysates without any treatment . CONCLUSION: Our data suggest that 17 beta-hydroxysteroid dehydrogenase may exist in phosphorylated forms and that phosphorylation may regulate the activity of 17 beta-hydroxysteroid dehydrogenase in vivo. J Trauma, 1994 Jul, 37(1), 82 - 9; discussion 89-90 Pre-exposure to hypoxia or septic stimuli differentially regulates endotoxin release of tumor necrosis factor, interleukin-6, interleukin-1, prostaglandin E2, nitric oxide, and superoxide by macrophages; West MA et al.; Shock states with resulting inadequate cellular oxygen delivery may contribute to macrophage (M phi) activation or dysregulation . In this study we compared the effects of transient anoxia and endotoxin pretreatment (LPS1) on M phi mediator release with a second endotoxin stimulus (LPS2) . METHODS: In vitro cultures of murine peritoneal exudate M phi were exposed to 2 hours of hypoxic or normoxic conditions, then incubated 22 hours under identical normoxic conditions +/- 10 ng/mL of LPS1 pretreatment . During the final 24 hours all M phis were exposed to a range of LPS2 concentrations . The M phi supernatants were assayed for tumor necrosis factor (TNF), interleukin 1 (IL-1), interleukin 6 (IL-6), prostaglandin E2 (PGE2), nitric oxide (NO), and superoxide release . RESULTS: LPS1 markedly inhibited M phi TNF release by LPS2, but hypoxia had no effect on LPS2-triggered TNF release . Hypoxia increased M phi IL-6 production in the absence of LPS1, but inhibited the LPS1 augmentation seen under normoxic conditions . Pretreatment with LPS1 increased NO production from LPS2 under normoxic conditions, but hypoxia inhibited this effect . Superoxide production increased by LPS1 under normoxic conditions, but hypoxia significantly inhibited superoxide release . CONCLUSIONS: The effects of transient anoxic exposure on LPS2-triggered M phi function are markedly different from the effects of pretreatment with septic stimuli (LPS1). J Invest Dermatol, 1994 Jul, 103(1), 7 - 12 A potential laboratory test for dysplastic nevus syndrome: ultraviolet hypermutability of a shuttle vector plasmid; Moriwaki S et al.; The diagnosis of the melanoma-prone disorder dysplastic nevus syndrome (DNS) is based currently on a combination of clinical and histopathologic examinations of patients . To develop a potential laboratory test for DNS, we utilized the observation that an ultraviolet light (UV)-treated mutagenesis plasmid shuttle vector has an abnormally increased frequency of mutations after transfection into lymphoblastoid cells from a patient with familial DNS . pSP189 (containing the bacterial suppressor tRNA gene supF as a marker for mutations and a gene for ampicillin resistance for selection) was treated with UV and transfected into familial DNS, xeroderma pigmentosum complementation group A (XP-A), and normal lymphoblastoid cells by electroporation or diethylaminoethyl (DEAE) dextran . Untreated plasmid pZ189K (containing a gene for kanamycin resistance) was co-transfected as an internal standard to reduce the variability of plasmid survival measurements . After 2 d, plasmids were extracted, used to transform an indicator strain of Escherichia coli, and assayed on plates containing ampicillin or kanamycin . Counting light blue or white colonies (containing mutated supF in the plasmid) and blue colonies (with wild type supF) permitted measurement of the plasmid survival and mutation frequency . Transfection by electroporation or DEAE dextran resulted in abnormally reduced survival of UV-treated plasmid after passage through the XP-A but normal survival in the three DNS lines . Transfection of UV-treated plasmid by DEAE dextran yielded a greater hypermutability with the familial DNS lines than by electroporation . These results suggest that pSP189 UV hypermutability with normal UV survival using DEAE dextran transfection may form the basis of a potential laboratory assay for familial DNS. J Allergy Clin Immunol, 1994 Jul, 94(1), 88 - 94 IgE-binding capacity of recombinant timothy grass (Phleum pratense) pollen allergens; Laffer S et al.; A panel of 60 cDNA clones coding for IgE-binding proteins from timothy grass pollen was immunocharacterized with sera from 30 patients allergic to grass pollen and antibodies raised against natural grass pollen allergens . In the cases of five representative patients in whom the IgE reactivity pattern with the recombinant allergens had been determined, IgE immunoadsorption experiments were performed . Recombinant Phl p I, Phl p V, and Phl p II and recombinant timothy grass profilin were used for immunoadsorption of the sera, and the percentage of remaining grass pollen-specific IgE was estimated . Although most of the patients showed IgE reactivity to a number of different natural and recombinant timothy grass pollen allergens, up to 66% of IgE directed against blotted total natural grass pollen allergens could be immunoadsorbed from the sera with recombinant Phl p V and Phl p I . The data point to the usefulness of recombinant allergens not only to determine IgE specificities of allergic patients but also to estimate the percentage of specific IgE that individuals produce against certain allergens . The fact that only a limited number of recombinant timothy grass pollen allergens account for a high percentage of grass pollen-specific IgE points to the possible usefulness of recombinant allergens not only for in vitro diagnosis but probably also for specific immunotherapy. Crit Care Med, 1994 Jul, 22(7), S3 - 7 Biology of proinflammatory cytokines and their antagonists; Moldawer LL; OBJECTIVES: To review the role of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) in the proinflammatory cascade, with particular emphasis on the association between increased concentrations of these cytokines and the sepsis-associated shock syndrome . Data that support the role of these cytokines as proinflammatory mediators provide a rationale for specific anticytokine therapies . DATA SOURCES: Information presented at the 22nd Educational and Scientific Symposium of the Society of Critical Care Medicine on June 9-13, 1993 in New York City was reviewed, along with supportive documentation from the English language literature . STUDY SELECTION: Controlled animal studies that elucidate the relationship between TNF-alpha and IL-1, and endotoxin-induced shock were selected for review . DATA EXTRACTION: This review focused on those data that described the roles of IL-1 and TNF-alpha in the induction of the inflammatory cascade . DATA SYNTHESIS: Information concerning the many aspects of attenuating the systemic inflammatory response was integrated into a description of emerging therapies for septic shock . CONCLUSIONS: Induction of inflammation during sepsis is a complex biological cascade that may be effectively attenuated by novel anticytokine biotherapies. J Toxicol Environ Health, 1994 Jul, 42(3), 275 - 88 Measurement of environmental formylmethionyl-peptides; Siegel PD et al.; Formylmethionyl-peptides are naturally occurring, biologically active ligands produced by bacteria . They produce a variety of biological effects including neutrophil chemotaxis, cellular degranulation, oxygen-free radical production, and smooth muscle contraction . Our studies have demonstrated that oxidized and reduced forms of formylmethionyl-leucyl-phenylalanine (fMLP) can be detected in bulk environmental organic dust samples . Organic dust fMLP content may not reflect total formylmethionyl-peptide content and pathological sequelae . Attempts to develop a total formylmethionyl-peptide assay that would reflect its pathological potential have thus far been unsuccessful . Information has been derived concerning the biology of formylmethionyl-peptides from these studies . Chromatographic, radioenzymatic, and radioreceptor-ligand binding studies were performed . High-performance liquid chromatography (HPLC) analysis of synthetic and environmental fMLP demonstrated that fMLP is labile, forming three oxidation products . HPLC is limited by inadequate sensitivity for air sample analysis and the probability of the presence of multiple formylmethionyl-peptides . Deformylases were isolated from Escherichia coli, but their usefulness in a competitive assay to detect formylmethionyl-peptides was limited by specificity differences from that for biological receptors . Receptor binding studies were conducted in an attempt to replace the deformylase with a biological receptor . The receptor binding patterns noted were consistent with the existence of three distinct formylmethionyl-peptide receptor subsets in neutrophils and alveolar macrophages . The plurality of fMLP receptor subtypes interfered with formylmethionyl-peptide measurement in a competitive assay . Formylmethionyl-peptides may contribute to organic dust-induced disease, but better techniques for the assessment of exposure to these agents are needed to properly assess their health impact. J Mol Biol, 1994 Jul 1, 240(1), 87 - 91 Crystals of glutamine-binding protein in various conformational states; Hsiao CD et al.; Crystals of glutamine-binding protein (GlnBP) in various conformational states have been obtained . Crystals of the ligand-free "open" state (denoted form B) have unit cell dimensions a = 86.3 A, b = 86.3 A, c = 81.5 A, alpha = beta = gamma = 90 degrees and diffract to about 2.3 A resolution . An analysis of the intensity data using an Requiv plot indicates that the crystal system is orthorhombic, space group P2(1)2(1)2(1) . Crystals of the ligand-bound "open" state (form B*) are obtained by soaking form B crystals with glutamine (Gln) and diffract to about 1.9 A . Crystals of the GlnBP-Gln complex in a ligand-bound "closed" state (form C) belong to space group P2(1)2(1)2(1) with a = 62.0 A, b = 65.7 A and c = 121.8 A and diffract to about 2.3 A . Crystals of a selenomethionyl GlnBP (form B') are isomorphous to form B crystals and diffract to about 2.1 A resolution. J Mol Biol, 1994 Jul 1, 240(1), 78 - 86 Structural model of the 50 S subunit of Escherichia coli ribosomes from solution scattering . II . Neutron scattering study; Svergun DI et al.; The X-ray and neutron contrast variation data of the 50 S ribosomal subunit of Escherichia coli in solution are interpreted in the frame of a two-phase model described by the shapes of the 50 S subunit and its RNA-rich core taking into account density fluctuations inside the RNA and the protein moiety . The shape of the envelope of the 50 S subunit and of the RNA-rich core are evaluated with a resolution of about 4 nm . The shape of the envelope is in good agreement with the models of the 50 S subunit obtained from electron microscopy on isolated particles . The shape of the RNA-rich core correlates well with the model of the entire particle determined by the image reconstruction from ordered sheets indicating that the latter model which is based on the subjective contouring of density maps is heavily biased towards the RNA. J Mol Biol, 1994 Jul 1, 240(1), 66 - 77 Structural model of the 50 S subunit of Escherichia coli ribosomes from solution scattering . I . X-ray synchrotron radiation study; Svergun DI et al.; The application of new methods of small-angle scattering data interpretation to a contrast variation study of the 50 S ribosomal subunit of Escherichia coli in solution is described . The experimental X-ray data from contrast variation with sucrose are analysed in terms of the basic functions in real space and the scattering curves from the volume inaccessible to sucrose and from the regions inside this volume occupied mainly by RNA and by proteins are obtained . From these curves models of the shape of the 50 S subunit and its RNA-rich core are evaluated . These two shapes are positioned so that their difference, which approximates the volume occupied by the proteins, produces a scattering curve which is in good agreement with the scattering from the protein moiety. J Mol Biol, 1994 Jul 1, 240(1), 52 - 65 Genetic evidence for IS1 transposition regulated by InsA and the delta InsA-B'-InsB species, which is generated by translation from two alternative internal initiation sites and frameshifting; Matsutani S; Insertion sequence IS1 contains two reading frames, insA and B'-insB, which are responsible for its transposition, and was previously shown to express two proteins . The first, InsA, is the product of insA . The second, InsA-B'-InsB is a fusion of InsA with the product of B'-insB . Synthesis of this protein occurs by a -1 frameshift from the 3' region of the insA frame to the open reading frame B', extending from the 5' end of the insB frame . Here, I have shown genetically that IS1 encodes the third species delta InsA-B'-InsB: delta InsA-B'-InsB uses two alternative initiation codons in the middle of the insA frame, and is produced by a frameshift mechanism similar to that used in InsA-B'-InsB expression . Deletion of the small region preceding these initiation codons resulted in decreased expression of delta InsA-B'-InsB, suggesting that the small region play some role in the translation initiation . Surprisingly, it was found that delta InsA-B'-InsB has a transposase-like function and InsA can stimulate the transposition promoted by delta InsA-B'-InsB, while delta InsA-B'-InsB seemed to bind to the left terminal inverted repeat (IRL) of IS1 and inhibit transposition when it was present in excess, as well as InsA represses transposition . It is likely that IS1 transposition activity depends on the ratio of InsA to delta InsA-B'-InsB . A double missense mutation of the internal initiation codons resulted in decreased cointegration activity, showing that delta InsA-B'-InsB is responsible for transposition but InsA-B'-InsB is probably not . Some IS elements, which also contain two tandem, out-of-phase, overlapping genes, appear to express deleted fusion proteins like delta InsA-B'-InsB, but the functions are unknown . The complex phenomena of transposition and its control found in IS1 may be more general in the other mobile DNAs. J Mol Biol, 1994 Jul 1, 240(1), 1 - 7 Suppression of yeast RNA polymerase III mutations by the URP2 gene encoding a protein homologous to the mammalian ribosomal protein S20; Hermann-Le Denmat S et al.; URP2 was cloned as a multicopy suppressor of several temperature-sensitive mutations defective in RNA polymerase III-dependent transcription, but without effect on mutations affecting RNA polymerase I or II . This single-copy gene encodes a hydrophilic polypeptide of 121 amino acid residues with a predicted molecular mass of 13.9 kDa and a basic isoelectric point of 9.7 . URP2 is a highly expressed gene, judging from its abundant messenger RNA and strong codon bias . The Urp2p protein is essential for cell growth, as shown by the lethal phenotype of the urp2::HIS3 null allele . Given its striking similarity to the S20 ribosomal polypeptide of rat (55% identical residues), Urp2p is in all likelihood the yeast form of this polypeptide . Both proteins are significantly related to S10, a component of the small ribosomal subunit of Escherichia coli that is known to operate as a transcriptional elongation factor . The latter observation suggests that the suppressor effect of URP2 may be due to a direct involvement of Urp2p in RNA polymerase III-dependent transcription . Alternatively, the overexpression of Urp2p could bypass a partial preribosomal RNA processing defect associated with RNA polymerase III mutants . URP2 was assigned to the left arm of chromosome VIII, and maps between DUR3 and YLF1 . The latter gene product has homology to the E . coli gtp1 gene product, and may define a new family of putative GTP-binding proteins. J Gen Virol, 1994 Jul, 75 ( Pt 7), 1525 - 33 Partial characterization of the lettuce infectious yellows virus genomic RNAs, identification of the coat protein gene and comparison of its amino acid sequence with those of other filamentous RNA plant viruses; Klaassen VA et al.; Purified virions of lettuce infectious yellows virus (LIYV), a tentative member of the closterovirus group, contained two RNAs of approximately 8500 and 7300 nucleotides (RNAs 1 and 2 respectively) and a single coat protein species with M(r) of approximately 28,000 . LIYV-infected plants contained multiple dsRNAs . The two largest were the correct size for the replicative forms of LIYV virion RNAs 1 and 2 . To assess the relationships between LIYV RNAs 1 and 2, cDNAs corresponding to the virion RNAs were cloned . Northern blot hybridization analysis showed no detectable sequence homology between these RNAs . A partial amino acid sequence obtained from purified LIYV coat protein was found to align in the most upstream of four complete open reading frames (ORFs) identified in a LIYV RNA 2 cDNA clone . The identity of this ORF was confirmed as the LIYV coat protein gene by immunological analysis of the gene product expressed in vitro and in Escherichia coli . Computer analysis of the LIYV coat protein amino acid sequence indicated that it belongs to a large family of proteins forming filamentous capsids of RNA plant viruses . The LIYV coat protein appears to be most closely related to the coat proteins of two closteroviruses, beet yellows virus and citrus tristeza virus. J Biol Chem, 1994 Jul 1, 269(26), 17723 - 9 The N-terminal alpha-helix of fragment B of diphtheria toxin promotes translocation of fragment A into the cytoplasm of eukaryotic cells; Madshus IH; Diphtheria toxin consists of two parts, fragments A and B . Fragment A has enzymatic activity inhibiting protein synthesis . Fragment B binds to cellular receptors and, upon exposure to low pH, inserts into the membrane, forms cation-selective channels, and facilitates translocation of fragment A . Previous data have suggested that the N-terminal part of fragment B, including the amphipathic alpha-helix TH1, plays an active role during translocation of fragment A (Madshus, I . H., Wiedlocha, A., and Sandvig, K . (1994) J . Biol . Chem . 269, 4648-4652) . When replacing charged residues in TH1 with uncharged amino acids, translocation of fragment A was strongly inhibited, virtually without affecting binding of the toxin or channel activity . These data suggest that TH1 may act as a targeting/anchoring sequence . In a mutant with eight positive charges and one negative charge in TH1, increased specific binding was observed, even if TH1 was outside the toxin's binding domain . This suggests that TH1 could be important in binding to parts of the translocation machinery . Fragment A associated with this mutant fragment B was translocated 10-fold more efficiently than wild-type toxin . The fact that this mutant TH1 efficiently promoted translocation, while, a hydrophobic TH1 did not, suggests that TH1 does not interact with the hydrophobic part of the membrane phospholipids. J Biol Chem, 1994 Jul 1, 269(26), 17663 - 9 DNA-binding domain and recognition sequence of the yeast BAS1 protein, a divergent member of the Myb family of transcription factors; Hovring I et al.; The yeast BAS1 protein is a transcriptional activator with an amino-terminal domain homologous to the DNA-binding domain of the oncoprotein Myb containing three imperfect tryptophan-rich repeats . In contrast to Myb-related transcription factors from higher eukaryotes, where the second and third repeat constitutes a minimal independent DNA-binding domain, all three repeats of BAS1 were found to be necessary for sequence-specific DNA binding . Moreover, an active DNA-binding subdomain was obtained only if the first repeat was enlarged in the amino-terminal direction to include 3 tryptophans and a 23-amino acid insertion and if 55 amino acids carboxyl-terminal to the third repeat were included . The BAS1 DNA-binding site was analyzed in detail and found to cover 8-9 base pairs with no similarity to the Myb recognition element . The binding site included a conserved hexameric TGACTC motif, the methylation of which abolished BAS1 binding, as well as a 3-base pair extension that seemed to have a modulatory effect on BAS1 affinity and where binding was less affected by methylation. J Biol Chem, 1994 Jul 1, 269(26), 17642 - 8 Breakpoint cluster region gene product-related domain of n-chimaerin . Discrimination between Rac-binding and GTPase-activating residues by mutational analysis; Ahmed S et al.; The breakpoint cluster region gene product (Bcr) is a GTPase-activating protein (GAP) for members of the Rho family, Cdc42Hs, and Rac1, as is the brain protein n-chimaerin . At least 15 proteins have sequence identity to the GAP domain (150 amino acid residues) of Bcr . The widespread occurrence of proteins that possess sequence identity to the Bcr-related GAP domain makes it especially important to understand its structure/function relationships . Amino acid sequence alignment of these proteins reveals three blocks of conservation in the GAP domain . Here, we present a mutational analysis of this domain using n-chimaerin sequences . Ten mutations were constructed (at least two in each of the blocks of conservation), expressed as glutathione S-transferase fusion proteins in Escherichia coli, and purified . Seven of the mutants, including deletions, still possessed GAP activity for Rac1 . Three of the mutants had no Rac1-GAP activity but were still able to bind Rac1 . IC50 values obtained from competition experiments suggest that n-chimaerin and the mutants with no GAP activity bound Rac1 with similar apparent binding constants . Thus, this mutant analysis allows discrimination between Rac1-binding and Rac1 GTPase- activating residues. J Biol Chem, 1994 Jul 1, 269(26), 17626 - 34 Purification, cloning, and expression of a murine phosphoprotein that binds the kappa B motif in vitro identifies it as the homolog of the human heterogeneous nuclear ribonucleoprotein K protein . Description of a novel DNA-dependent phosphorylation process; Ostrowski J et al.; The kappa B enhancer element regulates expression of many genes involved in immune responses and other processes . kappa B motif binds a number of proteins, some but not all, are related to the NF-kappa B family of transcription factors . We have previously identified a 65-kDa phosphoprotein that is specifically recognized by the kappa B motif (Ostrowski, J., Sims, J . E., Sibley, C . H., Valentine, M . A., Dower, S . K., Meier, K . E., and Bomsztyk, K . (1991) J . Biol . Chem . 266, 12722-12733) . This protein is closely associated with a serine/threonine kinase that is responsive to treatment of cells with interleukin-1 and other agents . We report here purification, cloning, and expression of this kappa B motif-binding phosphoprotein . The primary structure deduced from the isolated murine cDNA, identifies the protein as the homolog of the human heterogeneous nuclear ribonucleoprotein K protein . Antipeptide antibodies and expression of the cloned cDNA in Escherichia coli, demonstrated that the K protein is the authentic phosphoprotein that binds the kappa B motif in vitro . We also demonstrate that the in vitro phosphorylation of the natural and the recombinant K proteins by the associated kinase is stimulated by the kappa B motif. J Biol Chem, 1994 Jul 1, 269(26), 17464 - 8 The 86-kDa subunit of autoantigen Ku is a somatostatin receptor regulating protein phosphatase-2A activity; Le Romancer M et al.; We previously reported the immunopurification of a somatostatin receptor from the human tumoral gastric cell HGT1 using the monoclonal antibody 30F3 (Reyl-Desmars, F., Le Roux, S., Linard, C., Benkouka, F., and Lewin, M . J . M . (1989) J . Biol . Chem . 264, 18789-18795) . Screening of a lambda gt11 HGT1-cDNA library with 30F3 led us to isolate a cDNA encoding an 86-kDa polypeptide displaying 100% structural identity with the 86-kDa subunit (p86-Ku) of the Ku autoantigen . Recombinant p86 expressed in Escherichia coli cross-reacted with 30F3 and specifically bound {125I-Tyr11}somatstatin-14 . Binding was totally displaced by somatostatin-14, somatostatin-28, and SMS 201-995, with IC50 values of 0.7, 1.0, and 1.2 nM, respectively . In a search for a biological effect associated with binding, we purified a 36-kDa, okadaic acid-sensitive phosphatase (protein phosphatase-2A (PP2A)) from rat gastric cytosol . PP2A catalyzed 32P release from p34cdc2-phosphorylated histone H1 . However, PP2A-induced 32P release was concentration dependently inhibited by recombinant p86-Ku, with a decrease in maximal velocity without a change in Km . Steric exclusion high pressure chromatography indicated that the inhibition resulted from direct interaction of the enzyme with p86-Ku . Furthermore, it was antagonized by increased concentrations of somatostatin-14 and prevented by preincubating p86-Ku with 30F3 . Given the key role played by PP2A in cell cycle regulation, the current findings suggest that p86-Ku could be a physiological target of somatostatin antiproliferative action. J Biol Chem, 1994 Jul 1, 269(26), 17454 - 7 Expression in Escherichia coli of the two subunits of the isozyme form of wheat germ protein synthesis initiation factor 4F . Purification of the subunits and formation of an enzymatically active complex; van Heerden A et al.; The subunits (p28 and p86) of the isoenzyme form of eukaryotic initiation factor 4F (eIF-(iso)4F) from wheat were expressed separately in Escherichia coli . The subunits were purified by affinity chromatography (p28) and ion-exchange chromatography (p86) . The purified subunits alone did not support polypeptide synthesis in an eIF-(iso)4F and eIF-4F-deficient translation system from wheat germ . However, when the two subunits were mixed together, activity equal to that of the native form of eIF-(iso)4F was obtained . These results show that subunits expressed separately are able to associate and form an enzymatically active complex. J Biol Chem, 1994 Jul 1, 269(26), 17394 - 6 Ribonuclease A can be transformed into a dimeric ribonuclease with antitumor activity; Di Donato A et al.; A cDNA coding for bovine pancreatic RNase A was mutagenized to insert a proline, a leucine, and 2 cysteine residues, i.e . the residues present at corresponding positions in the subunit of seminal RNase, the only dimeric RNase of the pancreatic-type superfamily . The mutant, expressed in Escherichia coli, eventually aggregated into catalytically active dimers . Like naturally dimeric seminal RNase, at equilibrium the mutant dimeric RNase A adopted two quaternary structures (one with an exchange of the N-terminal segments between partner subunits, the other with no exchange) and displayed a selective toxicity for malignant cells, absent in the monomeric, parent protein. J Bacteriol, 1994 Jul, 176(14), 4455 - 8 Requirements for mobilization of plasmids RSF1010 and ColE1 by the IncW plasmid R388: trwB and RP4 traG are interchangeable; Cabezon E et al.; Mobilization of plasmid RSF1010 by the IncW plasmid R388 requires the genes involved in W pilus synthesis plus trwB . traG of the IncP plasmid RP4 can substitute for trwB in RSF1010 mobilization by R388 but not in self-transfer of R388 . This result suggests a dual specificity of TrwB-like proteins in conjugation . The same genetic requirements were found for R388 to mobilize the unrelated plasmid ColE1. J Bacteriol, 1994 Jul, 176(14), 4444 - 7 ssaD1, a suppressor of secA51(Ts) that renders growth of Escherichia coli cold sensitive, is an early amber mutation in the transcription factor gene nusB; Rajapandi T et al.; Complementation analysis of the ssaD1 mutation, isolated as a suppressor of the secA51(Ts) mutation that renders growth of Escherichia coli cold sensitive, was used to show that ssaD corresponds to nusB, a gene known to be important in transcription antitermination . DNA sequence analysis of the ssaD1 allele showed that it creates an amber mutation in the 15th codon of nusB . Analysis of the effect of different levels of NusB protein on secA transcription and translation suggested that NusB plays little or no role in the control of secA expression . Accordingly, mechanisms by which nusB inactivation can lead to suppression of secA51(Ts) and secY24(Ts) mutations without affecting secA expression need to be considered. J Bacteriol, 1994 Jul, 176(14), 4424 - 9 Genetic determination of the meso-diaminopimelate biosynthetic pathway of mycobacteria; Cirillo JD et al.; The increasing incidence of multiple-drug-resistant mycobacterial infections indicates that the development of new methods for treatment of mycobacterial diseases should be a high priority . meso-Diaminopimelic acid (DAP), a key component of a highly immunogenic subunit of the mycobacterial peptidoglycan layer, has been implicated as a potential virulence factor . The mycobacterial DAP biosynthetic pathway could serve as a target for design of new antimycobacterial agents as well as the construction of in vivo selection systems . We have isolated the asd, dapA, dapB, dapD, and dapE genes involved in the DAP biosynthetic pathway of Mycobacterium bovis BCG . These genes were isolated by complementation of Escherichia coli mutations with an expression library of BCG DNA . Our analysis of these genes suggests that BCG may use more than one pathway for biosynthesis of DAP . The nucleotide sequence of the BCG dapB gene was determined . The activity of the product of this gene in Escherichia coli provided evidence that the gene may encode a novel bifunctional dihydrodipicolinate reductase and DAP dehydrogenase. J Bacteriol, 1994 Jul, 176(14), 4416 - 23 Cloning, sequencing, and mutational analysis of the hyb operon encoding Escherichia coli hydrogenase 2; Menon NK et al.; The genes encoding the two structural subunits of Escherichia coli hydrogenase 2 (HYD2) have been cloned and sequenced . They occur in an operon (hyb) which contains seven open reading frames . An hyb deletion mutant (strain AP3) failed to grown on dihydrogen-fumarate medium and also produced very low levels of HYD1 . All seven open reading frames are required for restoration of wild-type levels of active HYD2 in AP3 . The hyb operon was mapped at 65 min on the E . coli chromosome. J Bacteriol, 1994 Jul, 176(14), 4321 - 7 Replacement of diaminopimelic acid by cystathionine or lanthionine in the peptidoglycan of Escherichia coli; Mengin-Lecreulx D et al.; In Escherichia coli, auxotrophy for diaminopimelic acid (A2pm) can be suppressed by growth with exogenous cystathionine or lanthionine . The incorporation of cystathionine into peptidoglycan metabolism was examined with a dapA metC mutant, whereas for lanthionine, a dapA metA mutant strain was used . Analysis of peptidoglycan precursors and sacculi isolated from cells grown with epimeric cystathionine or lanthionine showed that meso-A2pm was totally replaced in the same position by either sulfur-containing amino acid . Moreover, mainly L-allo-cystathionine (95%) or meso-lanthionine (93%) was incorporated into the precursors and sacculi . For this purpose, a new, efficient high-pressure liquid chromatography (HPLC) technique for analysis of the cystathionine isomers was developed . The formation of the UDP-MurNAc tripeptide appeared to be a critical step, since the MurE synthetase accepted meso-lanthionine or D-allo- or L-allo-cystathionine in vitro as good substrates, although with higher Km values . Presumably, the 10-fold-higher UDP-MurNAc-L-Ala-D-Glu pool of cells grown with cystathionine or lanthionine ensured a normal rate of synthesis . The kinetic parameters of the MurF synthetase catalyzing the addition of D-alanyl-D-alanine were very similar for the meso-A2pm-,L-allo-cystathionine-, and meso-lanthionine-containing UDP-MurNAc tripeptides . HPLC analysis of the soluble fragments resulting from 95% digestion by Chalaropsis N-acetylmuramidase of the peptidoglycan material in isolated sacculi revealed that the proportion of the main dimer was far lower in cystathionine and lanthionine sacculi. J Bacteriol, 1994 Jul, 176(14), 4311 - 5 Physiological role of the chaA gene in sodium and calcium circulations at a high pH in Escherichia coli; Ohyama T et al.; Ohyama et al . previously isolated Escherichia coli mutant RS1, which had a negligible activity for sodium ion extrusion at alkaline pH (T . Ohyama, R . Imaizumi, K . Igarashi, and H . Kobayashi, J . Bacteriol . 174:7743-7749, 1992) . Our present study showed that the mutation of RS1 was compensated for by a cloned chaA gene . It has been proposed that sodium ion extrusion by ChaA is prevented under physiological conditions (D . M . Ivey, A . A . Guffanti, J . Zemsky, E . Pinner, R . Karpel, E . Padan, S . Schuldiner, and T . A . Krulwich, J . Biol . Chem . 268:11296-11303, 1993) . In order to clarify the physiological role of chaA in sodium ion circulation at alkaline pH, we constructed a delta chaA mutant . The resultant mutant, TO112, deficient in both nhaA and chaA, was unable to grow at pH 8.5 in medium containing 0.1 M sodium chloride and had negligible sodium ion extrusion activity . However, TO112 grew at pH 7.0 in medium containing 0.4 M sodium chloride . Sodium ions were extruded from TO112 cells at neutral pH . The extrusion activity at pH 7.5 was greatly reduced by the deletion of nhaB . These data demonstrate that the activity of nhaB is low at high pH and that ChaA extrudes sodium ions at alkaline pH . The uptake of calcium ions by everted membrane vesicles prepared from the delta chaA mutant TO110 was 60% of the activity observed in the vesicles of the wild-type strain at pH 8.5, but the activity at neutral pH was not reduced by the deletion of chaA . Therefore, it was also suggested that ChaA plays a role in calcium ion circulation at alkaline pH. J Bacteriol, 1994 Jul, 176(14), 4306 - 10 A statistical analysis of the formation of plasmid-free cells in populations of Escherichia coli; Tolker-Nielsen T et al.; By methods analogous to those used in the classical statistical analysis of bacterial mutation, we have analyzed the formation of plasmid-free cells in populations of Escherichia coli harboring pBR322-derived plasmids . Application of fluctuation tests and papilla analysis suggested that there is a high variance in the probability that a plasmid-containing cell will produce a plasmid-free daughter cell . Apparently a subpopulation of plasmid-containing cells gives rise to progeny that produces plasmid-free cells with a high and unpredictable rate . This finding raises the question of whether plasmid maintenance can be adequately described by the conventional mathematical models. J Bacteriol, 1994 Jul, 176(14), 4296 - 305 Membrane insertion defects caused by positive charges in the early mature region of protein pIII of filamentous phage fd can be corrected by prlA suppressors; Peters EA et al.; The filamentous phage coat protein pIII has been used to display a variety of peptides and proteins to allow easy screening for desirable binding properties . We have examined the biological constraints that restrict the expression of short peptides located in the early mature region of pIII, adjacent to the signal sequence cleavage site . Many functionally defective pIII fusion proteins contained several positively charged amino acids in this region . These residues appear to inhibit proper insertion of pIII into the Escherichia coli inner membrane, blocking the assembly and extrusion of phage particles . Suppressor mutations in the prlA (secY) component of the protein export apparatus dramatically alleviate the phage growth defect caused by the positively charged residues . We conclude that insertion of pIII fusion proteins into the inner membrane can occur by a sec gene-dependent mechanism . The suppressor strains should be useful for increasing the diversity of peptides displayed on pIII in phage libraries. J Bacteriol, 1994 Jul, 176(14), 4285 - 95 Essential motifs of relaxase (TraI) and TraG proteins involved in conjugative transfer of plasmid RP4; Balzer D et al.; Two essential transfer genes of the conjugative plasmid RP4 were altered by site-directed mutagenesis: traG of the primase operon and traI of the relaxase operon . To evaluate effects on the transfer phenotype of the point mutations, we have reconstituted the RP4 transfer system by fusion of the transfer regions Tra1 and Tra2 to the small multicopy replicon ColD . Deletions in traG or traI served to determine the Tra phenotype of mutant plasmids by trans complementation . Two motifs of TraG which are highly conserved among TraG-like proteins in several other conjugative DNA transfer systems were found to be essential for TraG function . One of the motifs resembles that of a nucleotide binding fold of type B . The relaxase (TraI) catalyzes the specific cleaving-joining reaction at the transfer origin needed to initiate and terminate conjugative DNA transfer (W . Pansegrau, W . Schroder, and E . Lanka, Proc . Natl . Acad . Sci . USA 90:2925-2929, 1993) . Phenotypes of mutations in three motifs that belong to the active center of the relaxase confirmed previously obtained biochemical evidence for the contributions of the motifs to the catalytic activity of TraI . Expression of the relaxase operon is greatly increased in the absence of an intact TraI protein . This finding suggests that the relaxosome which assembles only in the presence of the TraI in addition to its enzymatic activity plays a role in gene regulation. J Bacteriol, 1994 Jul, 176(14), 4197 - 203 In vivo studies of the role of SecA during protein export in Escherichia coli; Chun SY et al.; SecA is found in Escherichia coli both tightly associated with the cytoplasmic membrane where it functions as a translocation ATPase during protein export and free in the cytosol (R . J . Cabelli, K . M . Dolan, L . Qian, and D . B . Oliver, J . Biol . Chem . 266:24420-24427, 1991; D . B . Oliver and J . Beckwith, Cell 30:311-319, 1982; W . Wickner, A . J . M . Driessen, and F.-U . Hartl, Annu . Rev . Biochem . 60:101-124, 1991) . Here we show that SecA can be immunoprecipitated from the cytosol in complex with both fully elongated and nascent species of the precursor of maltose-binding protein, an exported, periplasmic protein . In addition, under conditions in which the distribution of SecA between the cytosolic and membrane-bound states changes from that normally observed, the distribution of precursor maltose-binding protein changes in parallel . These results support the idea that cytosolic SecA plays a role in export . With the aim of determining the roles of the multiple binding sites for ATP on SecA, we compared the export defect in a culture of E . coli expressing a temperature-sensitive allele of secA with the defect in a culture treated with sodium azide . The results indicate that the mutational change and treatment with sodium azide inhibit export by affecting different steps in the cycle of ATP binding and hydrolysis by SecA. J Bacteriol, 1994 Jul, 176(13), 4177 - 81 Purification and characterization of the cytochrome bd complex from Azotobacter vinelandii: comparison to the complex from Escherichia coli; Kolonay JF Jr et al.; Partial purification of a cytochrome bd complex from Azotobacter vinelandii grown under high aeration was achieved by isolating respiratory particles enriched in this hemoprotein via differential centrifugation and detergent extraction . The cytochrome bd complex was subsequently solubilized from the inner membrane with dodecyl maltoside and purified to near homogeneity via DEAE-Sepharose chromatography . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the complex consisted of two subunits, with sizes in good agreement with those predicted from the cloned cyd locus (59.7 and 42 kDa) . Spectral analysis of the purified complex indicated that the heme components present were cytochromes b560, b595, and d; CO difference spectral studies identified cytochrome d as a CO-reactive component . The complex had a Km for ubiquinol-1 approximately seven times larger than that for the analogous bd complex from Escherichia coli, and O2 consumption curves revealed a Km value for O2 three times greater than that which we determined for the E . coli bd complex. J Bacteriol, 1994 Jul, 176(13), 4165 - 7 Flanking sequences affect replication arrest at the Escherichia coli terminator TerB in vivo; Bierne H et al.; We have analyzed the effect of flanking sequences on Tus-induced replication arrest . pBR322 plasmid derivatives which carry the Escherichia coli replication terminator TerB at different locations were used . Efficiency of the replication arrest was estimated from the plasmid copy number and transformation frequency of tus+ cells . We found that flanking sequences do affect replication arrest efficiency, a weak arrest being correlated with the presence of an AT-rich region which is replicated just before TerB . Some sequences located after the replication terminator can also affect replication termination . We propose that the AT-rich regions might impair binding of the Tus protein to the TerB sequence or facilitate helicase-induced unwinding of DNA and Tus displacement from the TerB site. J Bacteriol, 1994 Jul, 176(13), 4111 - 6 SecF stabilizes SecD and SecY, components of the protein translocation machinery of the Escherichia coli cytoplasmic membrane; Sagara K et al.; The effect of the overproduction of SecF encoded by the tac-secF gene on a plasmid on the synthesis of other Sec proteins was studied in Escherichia coli . SecF overproduction resulted in the simultaneous overproduction of SecD encoded by the tac-secD gene on a plasmid . Deletion of the orf6 gene, located downstream of the secF gene, had no effect on SecD overproduction . A pulse-chase experiment revealed that the overproduction was due to stabilization of SecD with SecF . SecF overproduction also resulted in the overproduction of SecY encoded by the tac-secY gene on a plasmid as well . SecF overproduction also enhanced the level of SecY expressed by the chromosomal secY gene . This SecF effect was not due to its effect on SecD or SecE, since SecF overproduction did not affect the levels of SecD and SecE expressed by the chromosomal secD and secE genes, respectively . SecE-dependent overproduction of SecY has already been demonstrated . It is suggested that SecF interacts with both SecD and SecY . SecE-SecY interaction has been demonstrated . It is likely, therefore, that all Sec proteins in the cytoplasmic membrane interact with each other. J Bacteriol, 1994 Jul, 176(13), 4081 - 91 Autoregulation of hip, an operon that affects lethality due to inhibition of peptidoglycan or DNA synthesis; Black DS et al.; The hip locus of Escherichia coli affects the frequency of persistence to the lethal consequences of selective inhibition of either DNA or peptidoglycan synthesis . Regulation of the hip operon, which consists of a regulatory region and two genes, hipB and hipA, was examined with strains containing a hip-lac transcriptional fusion placed in single copy at the lambda att site . Disruption of the hip locus increased activity from the fusion 16-fold . Repression was restored by supplying HipB in trans . HipB was overexpressed and purified . On the basis of gel filtration and cross-linking studies, HipB is a dimer in solution . Sequence analysis revealed that HipB is a Cro-like DNA-binding protein . The interaction of HipB with the hip regulatory region was examined by gel retardation, DNase I protection, and methylation protection studies . HipB binds with a Kapp (K apparent) of 40 pM to four operator sites with the conserved sequence TATCCN8GGATA (N represents any nucleotide) . Binding to the operators is nearly simultaneous and appears to be cooperative . Analysis of the role of HipA in the regulation of the hip operon is complicated by the toxicity of HipA in the absence of HipB . Strains disrupted in hipB but not in hipA could not be recovered . Moreover, hipA-containing plasmids cannot be replicated in strains defective in or lacking hipB . HipA is found exclusively in a tight complex with HipB . Although disruption of hipA slightly increased expression from the hip-lac fusion, in vitro studies suggest that HipA does not bind to the hip regulatory region directly but indirectly via HipB. J Bacteriol, 1994 Jul, 176(13), 4011 - 6 Mutational analysis of sequences in the recF gene of Escherichia coli K-12 that affect expression; Sandler SJ et al.; The level of translation of recF-lacZ fusions is reduced 20-fold by nucleotides 49 to 146 of recF . In this region of recF, we found a previously described ribosome-interactive sequence called epsilon and a hexapyrimidine tract located just upstream of the epsilon sequence . Mutational studies indicate that the hexapyrimidine sequence is involved in at least some of the reduced translation . When the hexapyrimidine sequence is mutant, mutating epsilon increases the level of translation maximally . We ruled out the possibility that ribosome frameshifting explains most of the effect of these two sequences on expression and suspect that multiple mechanisms may be responsible . In a separate report, we show that mutations in the hexapyrimidine tract and epsilon increase expression of the full-sized recF gene. J Bacteriol, 1994 Jul, 176(13), 3944 - 55 Promoter and operator determinants for fur-mediated iron regulation in the bidirectional fepA-fes control region of the Escherichia coli enterobactin gene system; Hunt MD et al.; The fepA-entD and fes-entF operons in the enterobactin synthesis and transport system are divergently transcribed from overlapping promoters, and both are inhibited by the Fur repressor protein under iron-replete conditions . A plasmid harboring divergent fepA'-phoA and fes-entF'-'lacZ fusions, both under the control of this bidirectional regulatory region, was constructed for the purpose of monitoring changes in expression of the two operons simultaneously . Deletion analysis, site-directed mutagenesis, and primer extension were employed to define both a single promoter governing the expression of fes-entF and two tandemly arranged promoters giving rise to the opposing fepA-entD transcript . A single Fur-binding site that coordinately regulates the expression of all transcripts emanating from this control region was identified by in vitro protection from DNase I nicking . The substitution of one base pair in the Fur recognition sequence relieved Fur repression but did not change the in vitro affinity of Fur for its binding site . Additional mutations in a limited region outside of the promoter determinants for either transcript inhibited expression of both fes and fepA . These observations suggest a mechanism of Fur-mediated regulation in this compact control region that may involve other regulatory components. J Bacteriol, 1994 Jul, 176(13), 3928 - 35 Characterization of the sigma 38-dependent expression of a core Escherichia coli starvation gene, pexB; Lomovskaya OL et al.; A reverse genetics approach was used to clone a pex starvation gene that codes for an 18-kDa polypeptide, designated PexB . Single-copy pexB-lacZ operon fusions were constructed to study transcriptional regulation and the promoter region of this gene . The induction by carbon starvation or osmotic stress was transcriptional and controlled by sigma 38 but was independent of this sigma factor by the oxidative stress; presumably, it was sigma 70 mediated under the latter stress . During nitrogen starvation, the induction was controlled at the posttranscriptional level . The pexB upstream region contained 245 nucleotides within which sequences approximating the consensus for cyclic AMP receptor protein and integration host factor binding sites were discernible . Deletion of 164 bp of the upstream region, which included these consensus sequences, did not affect starvation-or osmotic stress-mediated induction of pexB but abolished its induction by oxidative stress . The same start site was used in transcription during carbon starvation, osmotic stress, or oxidative stress, suggesting that the pexB promoter can be recognized in vivo by both sigma 38 and sigma 70, depending, presumably, on the presence of appropriate transcriptional factors . The -10 and -35 regions of pexB resembled those of some but not all genes known to be controlled by sigma 38. Exp Cell Res, 1994 Jul, 213(1), 253 - 65 Adhesion activity in fibronectin's alternatively spliced domain EDa (EIIIA) and its neighboring type III repeats: oncogene-dependent regulation; Xia P et al.; EDa (EIIIA) is one of two alternatively spliced type III repeats in cellular fibronectin (cFN) lacking in plasma fibronectin (pFN) . Previous studies using proteolytic fragments of cFN suggested that EDa may harbor adhesion activity for various Balb/c 3T3 cell derivatives . This putative adhesion activity has now been analyzed more directly . EDa and neighboring type III repeats III11 and III12 from human cFN cDNA were subcloned in various permutations and recombinant minigenes expressed in Escherichia coli . Purified recombinant polypeptide corresponding to EDa type III repeat alone is capable of promoting 3T3 cell attachment and limited cytoplasmic spreading, as are neighboring repeats III11 and III12 when tested as single repeats . While EDa alone exhibited 40-60% of the attachment activity of human pFN depending upon cell type, EDa with both neighboring repeats displayed 70-90% of pFN activity; furthermore, cell spreading was more extensive with the three-repeat molecule . Two experimental approaches indicated that cell surface proteoglycans do not participate in these adhesion processes . Finally, the effects of various oncogenes upon transformation of Balb/c 3T3 cells were investigated . Adhesion activity to all three repeats is completely abrogated by two different ras oncogenes, unaffected by the sis oncogene, and elevated by the src oncogene . Furthermore, ras- revertants of ras-transformed cells had reacquired adhesion activity for EDa and its neighboring repeats . Comparison of individual repeats confirmed oncogene-dependent regulation of receptor activity to these sequences--for 3T3 cells, EDa > III11 = III12, but for src-transformed cells III12 >> EDa > III11 . These studies reveal a new adhesion-promoting activity in alternatively spliced EDa and in neighboring repeats III11 and III12; this receptor activity is regulated either positively or negatively subsequent to transformation by specific oncogenes. Exp Cell Res, 1994 Jul, 213(1), 128 - 42 Structural elements of the amino-terminal head domain of vimentin essential for intermediate filament formation in vivo and in vitro; Beuttenmuller M et al.; The biological functions of the non-alpha-helical, N- and C-terminal head and tail domains of intermediate filament (IF) proteins are still ill-defined . Previously, it has been shown that the basic, N-terminal head piece of the type III IF protein vimentin is essential for regular IF assembly and that arginine residues within the N-terminus may be involved . In order to identify particular regions within this domain essential for filament formation and stabilization, N-terminally truncated and arginine substitution forms of vimentin were constructed via site-directed in vitro mutagenesis of murine vimentin cDNA . The de novo filament assembly properties of these modified forms were compared with those of wild-type vimentin after transient expression in vimentin-free, cultured cells . In order to investigate their filament assembly competence in vitro, they were also produced in an E . coli expression system . It could be demonstrated that deletion of the first 10, 13, 17, and 32 amino acid residues, respectively, from the N-terminus of vimentin has an increasingly deleterious effect on filament assembly in vitro and network formation in vivo and that, thus, the highly conserved sequence motif, SSYRRXFGG, located in the N-terminus of various IF proteins and partially or totally removed by the above deletions plays a particularly important role in both activities . These results were confirmed and extended by arginine point mutations in the N-terminal head region, which showed that only one of the two adjacent arginine residues located within the conserved sequence motif is essential for filament assembly and stability in vitro as well as network formation in vivo . The neighboring arginine residues could be replaced by lysine residues without severe effects on the assembly properties of the respective mutant proteins . Distinction between the assembly-promoting potentials of the two arginine residues of the N-terminal doublet was considerably facilitated by a Val389-->Asp substitution toward the carboxy-end of the 2B segment of the vimentin rod domain . The synergistic effect of point mutations in this and the N-terminal region of the vimentin molecule implies the interaction of both protein domains in the process of filament assembly . Mutant vimentin proteins that were characterized by distinct incompetence to assemble into IFs caused a massive collapse of the endogenous vimentin filament system when expressed in mouse skin fibroblasts. Exp Cell Res, 1994 Jul, 213(1), 107 - 12 Minisatellite DNA-binding proteins in mouse brain, liver, and kidney; Shinder GA et al.; Minisatellites are tandemly repeated DNA sequences that are found in most higher eukaryotes . They are genetically unstable and often gain or lose repeat units . Minisatellite repeats contain a "core" sequence which is highly conserved among a family of minisatellites . The core sequence resembles the chi sequence of Escherichia coli which is a binding site for recombination proteins . It has therefore been suggested that minisatellite core sequences may also be binding sites for proteins involved in recombination . In this paper, we report several proteins in mouse brain, liver, and kidney nuclear extracts which bind to various minisatellite sequences . We have found several proteins which have not been previously reported and, in addition, have noted that brain has a different profile of minisatellite-binding proteins than liver and kidney . Moreover, we have also observed probe-specificity in the binding of some of these proteins, suggesting that different "families" of minisatellites may have qualitatively different functions. Cell, 1994 Jul 1, 77(7), 1093 - 100 Differential evolution of substrates for an RNA enzyme in the presence and absence of its protein cofactor; Liu F et al.; Selection of substrates for an RNA enzyme, the catalytic subunit of RNAase P from E . coli, has been carried out by simulation of evolution in vitro in the presence and absence of the protein cofactor of the enzyme . In the presence of the protein, substrates resembling precursor tRNAs, which were readily cleaved by the catalytic RNA, were selected in addition to others, with different sequences and structures (one of which resembled the precursor to 4.5S RNA) that were not readily cleaved by the catalytic RNA alone . The ribonucleoprotein enzyme is more versatile than the RNA enzyme, and our results suggest that it and 4.5S RNA may have evolved after ancestral tRNAs. Proc Soc Exp Biol Med, 1994 Jul, 206(3), 273 - 9 Interaction of lactogenic hormones with the soluble extracellular domain of prolactin receptors; Gertler A et al.; Two variants of rabbit prolactin receptor extracellular domain (rbPRLR-ECD) were prepared using insect/baculovirus (amino acids 1-198) and E . coli (amino acids 4-210) expression systems . Bovine PRLR-ECD (bPRPL-ECD amino acids 1-210) and human growth hormone receptor ECD (hGHR-ECD amino acids 1-246) were also prepared using E . coli expression system . All four proteins were purified as monomers with > 95% homogeneity . Their affinity for various lactogenic and somatogenic hormones was determined by binding assays . The stoichiometry of complex formation with these hormones was studied by gel filtration on a Superdex 75 column, and bioactivity was determined by in vitro bioassays . The results summarized in this paper indicate that, in contrast to hGHR-ECD, in which the ability to form a 2:1 complex with hGH is indicative of the biological activity of the hormone, the ability or inability of prolactin and placental lactogen to form 2:1 complexes with rb or bPRLR-ECD cannot predict their biological activity . This conclusion does not preclude however, hormone- or antibody-induced dimerization of the membrane-embedded receptor. J Histochem Cytochem, 1994 Jul, 42(7), 953 - 6 Development of endogenous beta-galactosidase and autofluorescence in rat brain microvessels: implications for cell tracking and gene transfer studies; Lal B et al.; Cell transplantation is commonly used in studies of CNS development, tumor biology, and gene therapy . Fluorescent dyes and the E . coli lacZ reporter gene are used to identify transplanted cells in host tissues . The usefulness of these methods depends on host autofluorescence and beta-galactosidase (beta-Gal) activity . Our interest in the CNS vasculature led us to examine vascular autofluorescence and beta-Gal activity in postnatal and adult rat brains . Brains were perfusion-fixed (3.7% paraformaldehyde), cryoprotected, and cryostat-sectioned (12 microns) . Autofluorescent vessel profiles were quantitated in sections using rhodamine filter sets and beta-Gal-positive vessels were quantitated under bright-field after incubation of sections with X-Gal chromogenic substrate for 1-18 hr at 37 degrees C . Multifocal vessel autofluorescence appeared in postnatal Day (PND) 18 Lewis rats (0.6 +/- 0.4 vessels/field) and increased tenfold in adults (6.8 +/- 0.3/field) . The numbers of beta-Gal-positive vessels in PND 18 and adult sections incubated with X-Gal for 18 hr were 21.1 +/- 1.7 and 119 +/- 17, respectively . Host beta-Gal staining was similar to that produced by implanted endothelial cells expressing the bacterial lacZ reporter gene . Reducing incubation times in X-Gal to less than 4 hr eliminated endogenous staining and retained lacZ-specific staining . The presence of vascular autofluorescence and endogenous beta-Gal activity must be considered when either fluorescence- or lacZ-dependent cell markers are used in rat brain. Cancer Res, 1994 Jul 1, 54(13), 3511 - 5 Human monoclonal antibody identified an immunoreactive tetrapeptide sequence (Lys-Tyr-Gln-Ile) in M(r) 43,000 protein of human melanoma; Oka T et al.; The human monoclonal antibody (HuMAb) L92 reacts to an M(r) 43,000 protein associated with human melanoma . To identify the gene encoding its antigenic epitope, a complementary DNA expression library constructed from the human melanoma cell line UCLASO M14 was screened with HuMAb L92 . DNA sequence analysis of the isolated clone revealed that the immunoreactive peptide was composed of 10 amino acids (QDLT-MKYQIF) . The peptide was expressed in Escherichia coli with beta-galactosidase as a fused protein . There is no homology between the cloned sequence and other reported DNA sequences . Western blot analysis showed that the fused protein had specific binding to HuMAb L92 . An antigen-encoding peptide with 10 amino acids was synthesized and tested for its immunoreactivity in vitro . HuMAb L92 reacted specifically to the 10-amino acid peptide in both an antibody-binding inhibition to the M(r) 43,000 protein and a solid-phase enzyme-linked immunosorbent assay . Using several truncated fusion proteins, we found the minimum number of amino acids required for the antibody binding to be 4 (KYQI) . These results suggest that the identified peptide sequence encodes the antigenic epitope of the M(r) 43,000 protein. Anesth Analg, 1994 Jul, 79(1), 40 - 5 Serum but not plasma produces injury in the perfused rabbit lung; Mouchawar AM et al.; Serum contains proteins that may produce lung injury directly by affecting endothelial cells and indirectly by modulating the effects of endotoxin (lipopolysaccharide) . We studied the effects of 10% serum, 10% plasma, and 10% plasma plus 2 micrograms/mL lipopolysaccharide on pulmonary hypertension and vascular permeability in the isolated perfused rabbit lung . Control lungs perfused with Krebs-Henseleit buffer containing 3% albumin for 2 h had stable pulmonary vascular pressures and permeability (measured by the capillary filtration coefficient) . Serum produced pulmonary hypertension and increased pulmonary vascular permeability . In contrast, plasma, with and without lipopolysaccharide, did not alter pulmonary vascular pressures or permeability . Pretreatment with the cyclooxygenase inhibitor indomethacin prior to the addition of serum prevented the serum-induced increase in pulmonary vascular pressures and permeability . We conclude that the deleterious effects of serum are not due to plasma proteins per se, but instead are related to activation of the coagulation cascade during preparation of the serum . The deleterious effects of serum appear to be mediated by cyclooxygenase metabolites of arachidonic acid . Finally, endotoxin, even with the addition of plasma, does not directly produce lung injury. Mol Cell Biol, 1994 Jul, 14(7), 4958 - 74 Novel interactions between human T-cell leukemia virus type I Tax and activating transcription factor 3 at a cyclic AMP-responsive element; Low KG et al.; Human proenkephalin gene transcription is transactivated by human T-cell leukemia virus type I (HTLV-I) Tax in human Jurkat T lymphocytes . This transactivation was further enhanced in Jurkat cells treated with concanavalin A, cyclic AMP, or 12-O-tetradecanoylphorbol-13-acetate . Deletion and cis-element transfer analyses of the human proenkephalin promoter identified a cyclic AMP-responsive AP-1 element (-92 to -86) as both necessary and sufficient to confer Tax-dependent transactivation . Different AP-1 or cyclic AMP-responsive element-binding protein (CREB)/activating transcription factor (ATF) proteins which bind this element were expressed in murine teratocarcinoma F9 cells to identify those capable of mediating Tax-dependent transactivation of human proenkephalin gene transcription . Although CREB, c-Fos, c-Jun, and JunD did not have significant effects, JunB inhibited the Tax-dependent transactivation . In contrast, ATF3 dramatically induced Tax-dependent transactivation, which was further enhanced by protein kinase A . Electrophoretic mobility shift assays with recombinant fusion proteins expressed and purified from bacteria indicate that the DNA-binding activity of ATF3 is also dramatically enhanced by Tax . Chimeric fusion proteins consisting of the DNA-binding domain of the yeast transcription factor Gal4 and the amino-terminal domain (residues 1 to 66) of ATF3 were able to mediate Tax-dependent transactivation of a Gal4-responsive promoter, which suggests a direct involvement of this region of ATF3 . Recombinant fusion proteins of glutathione S-transferase with either the amino- or carboxy-terminal (residues 139 to 181) domain of ATF3 were able to specifically interact with Tax . Furthermore, specific antisera directed against Tax coimmunoprecipitated ATF3 only in the presence of Tax. Mol Cell Biol, 1994 Jul, 14(7), 4712 - 21 Repression of basal transcription by HMG2 is counteracted by TFIIH-associated factors in an ATP-dependent process; Stelzer G et al.; A basal repressor of class II gene transcription was identified, purified, and found to be identical to nonhistone chromosomal protein HMG2 . HMG2 was shown to inhibit basal transcription under conditions in which transcription templates form soluble complexes with HMG2 . Order-of-addition experiments clearly revealed that HMG2 acted after assembly of a TBP-TFIIA-promoter complex and before formation of the fourth phosphodiester bond by RNA polymerase II . Subsequently, an activity that efficiently counteracted repression of transcription by HMG2 in both TBP- and TFIID-containing transcription systems was isolated . Several lines of evidence suggested that antirepression was mediated by a TFIIH-associated factor . The antirepressor first coeluted with TFIIH, was depleted from this fraction by antibodies directed against the TFIIH subunit p62, was dependent on either ATP or dATP, and then was inhibited by the ATP analogs AMP-PNP and ATP gamma S . Relief of HMG2-mediated repression as well as basal promoter function of TFIIH may involve a helicase that coelutes with TFIIH and displays similar nucleotide specificities . Taken together, these data suggest novel consequences of chromatin-associated HMG proteins and they provide direct evidence for a role of TFIIH-associated enzymes in ATP-dependent antirepression of nonhistone chromosomal proteins. Mol Cell Biol, 1994 Jul, 14(7), 4653 - 61 Transcriptional control of the yeast PDR5 gene by the PDR3 gene product; Katzmann DJ et al.; Saccharomyces cerevisiae cells possess the ability to simultaneously acquire resistance to an array of drugs with different cytotoxic activities . The genes involved in this acquisition are referred to as pleiotropic drug resistant (PDR) genes . Several semidominant, drug resistance-encoding PDR mutations have been found that map near the centromere on chromosome II, including PDR3-1 and PDR4-1 . DNA sequencing of chromosome II identified a potential open reading frame, designated YBL03-23, that has the potential to encode a protein with strong sequence similarity to the product of the PDR1 gene, a zinc finger-containing transcription factor . Here we show that YBL03-23 is allelic with PDR3 . The presence of a functional copy of either PDR1 or PDR3 is essential for drug resistance and expression of a putative membrane transporter-encoding gene, PDR5 . Deletion mapping of the PDR5 promoter identified a region from -360 to -112 that is essential for expression of this gene . DNase I footprinting analysis using bacterially expressed Pdr3p showed specific recognition by this protein of at least one site in the -360/-112 interval in the PDR5 promoter . A high-copy-number plasmid carrying the PDR3 gene elevated resistance to both oligomycin and cycloheximide . Increasing the number of PDR3 gene copies in a delta pdr5 strain increased oligomycin resistance but was not able to correct the cycloheximide hypersensitivity that results from loss of PDR5 . These data are consistent with the notion that PDR3 acts to increase cycloheximide resistance by elevating the level of PDR5 transcription, while PDR3-mediated oligomycin resistance acts through some other target gene. Mol Cell Biol, 1994 Jul, 14(7), 4522 - 31 CCR4 is a glucose-regulated transcription factor whose leucine-rich repeat binds several proteins important for placing CCR4 in its proper promoter context; Draper MP et al.; The yeast CCR4 protein is required for the expression of a number of genes involved in nonfermentative growth, including glucose-repressible ADH2, and is the only known suppressor of mutations in the SPT6 and SPT10 genes, two genes which are believed to be involved in chromatin maintenance . We show here that although CCR4 did not bind DNA under the conditions tested, it was able to activate transcription when fused to a heterologous DNA-binding domain . The transcriptional activation ability of CCR4, in contrast to that of many other activators, was glucose regulated . Two activation domains one of which was glucose responsive and encompassed a glutamine-proline-rich region similar to that found in other eukaryotic transcriptional factors were identified . The two transactivation regions, when separated from the leucine-rich repeat and the C terminus of CCR4, were unable to complement a defective ccr4 allele, suggesting that the leucine-rich repeat and the C terminus make contacts that link the activation regions to the proper gene context . Native immunoprecipitation of CCR4 revealed that CCR4 was complexed with at least four other proteins . The leucine-rich repeat of CCR4 was both necessary and sufficient for interaction with at least two of these factors . We propose that the leucine-rich repeat links CCR4 through its associated factors to its promoter context at ADH2 and other loci where it is required for proper transcriptional regulation. J Am Coll Cardiol, 1994 Jul, 24(1), 55 - 60 Dose finding with a novel recombinant plasminogen activator (BM 06.022) in patients with acute myocardial infarction: results of the German Recombinant Plasminogen Activator Study . A study of the Arbeitsgemeinschaft Leitender Kardiologischer Krankenhausärzte (ALKK); Neuhaus KL et al.; OBJECTIVES . The aim of this study was to determine the appropriate dose of a novel recombinant tissue-type plasminogen activator (BM 06.022) for thrombolysis in patients with acute myocardial infarction . BACKGROUND . BM 06.022 is a mutant of tissue-type plasminogen activator expressed in Escherichia coli that can be given as a single bolus because of a prolonged half-life, which might obviate the need for complicated regimens . METHODS . BM 06.022 given as a single bolus was investigated in 142 patients in a multicenter sequential dose-finding study . Efficacy of the drug was assessed from infarct-related artery patency by coronary angiography . RESULTS . With the first dose of 10 MU of BM 06.022, the predefined minimal 90-min patency of 70% was not achieved, as indicated by the sequential probability ratio test after treatment of 42 patients (group A) . The second dose of 15 MU of BM 06.022 was given subsequently in the preset maximum of 100 patients (group B) . Angiography 30, 60 and 90 min after the bolus injection of BM 06.022 revealed a patent infarct-related artery (Thrombolysis in Myocardial Infarction trial {TIMI} grade 2 or 3) in 65% and 66%, 73% and 74% and 66% and 75% of patients in groups A and B, respectively . Very early reocclusion up to the 90-min angiogram occurred in 17% and 13%, late reocclusion until predischarge angiography occurred in 7% and 5%, and rescue percutaneous transluminal coronary angioplasty after the 90-min angiogram was performed in 6 and 14 patients in groups A and B, respectively . Plasma fibrinogen decreased from 2.79 g/liter (range 0.94 to 4.75) to 1.69 g/liter (range 0.0 to 3.95) in group A and from 2.54 g/liter (range 0.0 to 5.02) to 0.92 g/liter (range 0.0 to 2.68) in group B . Two bleeding complications requiring transfusion or surgical intervention and one nonfatal intracranial hemorrhage were encountered . Eight patients had a reinfarction, and five patients died, all of cardiac causes . CONCLUSIONS . With BM 06.022 given as a single bolus, a high early patency rate of the infarct-related coronary artery can be achieved . The speed of thrombolysis seems to be superior to standard thrombolytic drugs . The compound warrants further evaluation with respect to safety and efficacy by clinical end points. Infect Immun, 1994 Jul, 62(7), 3038 - 40 The eaeB gene of enteropathogenic Escherichia coli is necessary for signal transduction in epithelial cells; Foubister V et al.; An enteropathogenic Escherichia coli mutant carrying an internal deletion in the eaeB gene (UMD864) was unable to activate epithelial cell signals, including tyrosine phosphorylation, cytoskeletal rearrangements, and the release of inositol phosphates, indicating that the eaeB locus encodes a product that is involved in stimulating signals in epithelial cells. Infect Immun, 1994 Jul, 62(7), 2917 - 29 Actin accumulation associated with clustered and localized adherence in Escherichia coli isolated from patients with diarrhea; Yamamoto T et al.; Escherichia coli D2 (serotype 07:H-) that was isolated from a child with diarrhea hybridized with an F1845 DNA probe used to detect diffuse adherence . Strain D2 adhered to tissue culture cells (HeLa and HEp-2 cells) in a clustered pattern but did not autoagglutinate on the cell surface and induced the elongation of microvilli after 3 h of incubation . After 6 h of incubation, the infected cells were positive for fluorescent-actin staining at the site of clustered adherence . When analyzed with a confocal laser scanning microscope, each D2 cell was surrounded by accumulated actin in a capsule-like formation . Capsule-like, accumulated actin was also observed with enteropathogenic E . coli (EPEC), although in this case, actin accumulation was associated with EPEC microcolonies in a localized pattern . Four other strains of F1845 DNA probe-positive, diffusely adhering E . coli were negative for actin accumulation . Strain D2 did not hybridize with EPEC attaching and effacing DNA or EPEC adherence factor DNA probes . In addition, clustered D2 cells were found inside tissue culture cells . The data suggest a novel infectious mechanism as well as genetic heterogeneity of F1845 DNA probe-positive E . coli . Capsule-like, accumulated actin may protect the bacteria from host defense mechanisms. Infect Immun, 1994 Jul, 62(7), 2653 - 61 A 9.0-kilobase-pair circular plasmid of Borrelia burgdorferi encodes an exported protein: evidence for expression only during infection; Champion CI et al.; In this study, we report the cloning, sequencing, and molecular analysis of a gene located on a 9.0-kbp circular plasmid of virulent Borrelia burgdorferi B31 designated eppA (exported plasmid protein A) . This gene encodes a precursor protein of 174 amino acids including a signal peptide of 20 amino acids and a type I signal peptidase cleavage site . The mature EppA protein of 154 amino acids has a calculated molecular weight of 17,972 . Several lines of evidence suggest that eppA is not expressed by B . burgdorferi B31 during in vitro cultivation . Immunoblot analysis using hyperimmune rabbit antiserum to recombinant EppA (rEppA) did not detect the presence of EppA in B . burgdorferi B31 cultivated in vitro . Northern blot analysis using total RNA isolated from in vitro-cultivated virulent B . burgdorferi B31 failed to detect an eppA transcript . EppA was not detected in culture supernatants of virulent B . burgdorferi B31 in a sensitive antigen-capture enzyme-linked immunosorbent assay . In contrast, evidence for expression of eppA during infection was based on the observation that patients with Lyme disease as well as rabbits experimentally infected with B . burgdorferi B31 produced antibodies that recognized rEppA . Because the cellular location of EppA in B . burgdorferi cannot be determined in vivo because of very small numbers of organisms present in vertebrate infection, we examined the cellular location of rEppA expressed in Escherichia coli . In E . coli, rEppA is targeted to the outer membrane . In addition, purified E . coli outer membranes containing rEppA treated with chaotrophic agents did not result in rEppA release . These findings are consistent with the idea that EppA is not peripherally associated with the outer membrane of E . coli but rather has an integral outer membrane association. Immunol Lett, 1994 Jul, 41(2-3), 217 - 23 Immunization of mice with Escherichia coli containing an antigen of the malaria parasite Plasmodium chabaudi chabaudi expressed in lambda gt11 induces high antibody titres; Hartz D et al.; Escherichia coli containing a recombinant malarial protein expressed in lambda gt11 have been evaluated as an antigen delivery system in vivo . They were generated by infecting non-suppressing E . coli cells with a clone from a cDNA library of Plasmodium chabaudi chabaudi in lambda gt11 . This clone (termed clone 6) expresses part of a 93 kDa blood-stage antigen of P . c . chabaudi as beta-galactosidase fusion protein . Immunization of C57B1/6 mice with these infected E . coli cells resulted in an antibody response to the malarial part of the fusion protein comparable to that obtained with purified fusion protein preparations . This method, therefore represents a rapid secondary screening of clones from lambda gt11 expression libraries for immunogenic and potentially protective components . In addition, the administration of whole infected E . coli obviates the need for an adjuvant. Immunol Lett, 1994 Jul, 41(2-3), 155 - 61 Detection of adult T-cell leukemia-derived factor/human thioredoxin in human serum; Kitaoka Y et al.; Adult T-cell leukemia (ATL)-derived factor (ADF), originally defined as an inducer of interleukin-2 receptor/alpha-chain (IL-2R/p55) of human T-lymphotropic virus type I (HTLV-I) positive T cells, is a human homologue of redox-active coenzyme thioredoxin (Trx) of Escherichia coli . In this study, an enzymatic assay system based on the dithiol-dependent insulin-reducing activity of ADF/Trx was established (insulin-reducing assay) to determine the amount of ADF/Trx in human serum using NADPH and Trx reductase purified from human placenta . Insulin-reducing activity was detected in all of the serum samples from healthy volunteers (n = 30) screened by this assay, with a mean +/- SD of 10.9 +/- 2.4 U/l . This mean value corresponds with the concentration of 223 ng recombinant ADF/Trx (rADF/Trx)/ml . Human serum is known to contain several redox-active proteins with ADF/Trx motifs . To differentiate the contribution of these proteins and ADF/Trx to the insulin-reducing activity, the anti-rADF/Trx monoclonal antibody (mAb)-conjugated affinity column-depleted sera obtained from an identical source was used for analysis . The affinity column-depleted sera demonstrated a loss of over 99% of the original activity, while control column depleted sera lost less than 4% . Furthermore, the amount of affinity-purified ADF/Trx molecules eluted from the same column almost corresponded with the amount estimated by the insulin-reducing activity.(ABSTRACT TRUNCATED AT 250 WORDS) Biol Pharm Bull, 1994 Jul, 17(7), 980 - 2 Mitogenic activity and the induction of tumor necrosis factor by lipopeptide analogs of the N-terminal part of lipoprotein in the outer membrane of Escherichia coli; Shimizu T et al.; Mitogenicity and the production of tumor necrosis factor (TNF) by a chemically synthesized lipotetrapeptide analog (KAB-8), S-{2,3-bis(palmitoyloxy)-2R-propyl}-N-{(2,2,2)-trichloro- ethoxycarbonyl: Troc group}-(R)-cysteinyl-(S)-seryl-(S)-seryl-(S)-asparagine, the amino acid sequence of which corresponds to that of the lipopeptide part of lipoprotein in Escherichia coli, and several derivatives (KAB-30-41), which possessed the altered glycerocysteine moiety, were examined . A 1-cysteinyl glycerol skeleton-type compound (KAB-8), a propane-type compound (KAB-31), a homoglycerol-type (KAB-39 and 40) and a 2-cysteinyl glycerol-type (KAB-41) exhibited mitogenic activity on splenocytes from C3H/He mice at various concentrations ranging from 0.4 to 100 micrograms/ml . However, propane-type compounds, except KAB-31, and ethane-type compounds showed lower mitogenic activity than other types of compounds . Compounds KAB-8, 31, 40 and 41 induced the production of TNF in peritoneal exudated macrophages from C3H/He mice at concentrations of 25 and 50 micrograms/ml . The results indicate that the structural differences of the glycerol moiety in the synthetic lipopeptides affect the potency of its biological activities. Bioorg Khim, 1994 Jul, 20(7), 751 - 8 {Cloning transport protein genes for two strains of tobacco mosaic virus and their expression in Escherichia coli cells}; Ivanov KI et al.; Recombinant constructs expressing fusion (His)6 movement proteins of two strains (Cruciferous and Vulgare) of tobacco mosaic virus were generated by cloning the PCR-amplified fragments into the pQE-9 vector . The movement proteins containing N-terminal (His)6 affinity tags were purified on a metal chelate adsorbent. Zh Mikrobiol Epidemiol Immunobiol, 1994 Jul-Aug, (4), 64 - 6 {Coxiella burnetii antigens--inducers of tumor necrosis factor and interleukin-1}; Tokarevich NK et al.; The cultivation of mouse peritoneal macrophages in the presence of antigenic preparations obtained from C.burnetii was accompanied by the appearance of phagocytes and considerable amounts of tumor necrosis factor and interleukin 1 in the culture medium . The production of cytokines depended on the doses of preparations used as inducers . The special treatment of C.burnetii antigen, selectively removing its phospholipid components, led to a sharp drop in its capacity for stimulating the production of cytokines. Plasmid, 1994 Jul, 32(1), 32 - 40 A set of beta-galactosidase gene fusion cassettes demonstrates usefulness in expressing HIV-1 genes in Escherichia coli; Valverde V et al.; Heterologous expression in Escherichia coli is often limited by yield and solubility of the foreign protein in the bacterial cytoplasm . In many cases, overexpression also results in growth inhibition . In order to produce retroviral proteins that are especially difficult to overexpress in E . coli, we designed a set of beta-galactosidase fusion cassettes . Fusions with beta-galactosidase increase significantly both yield and solubility of the foreign proteins, thus making purification much easier . These cassettes allow for N- or C-terminal fusions, and the retroviral proteins can be released from the fusion by automaturation in vivo for the HIV-1 protease or cleavage by thrombine for Tat . More generally, any synthetic sequence coding for a given cleavage site can be introduced 5' or 3' to the lacZ gene through a convenient set of unique restriction sites, making these fusion cassettes highly versatile. Plasmid, 1994 Jul, 32(1), 19 - 31 TrfA-dependent, inner-membrane-associated plasmid RK2 DNA synthesis in Escherichia coli maxicells; Michaels K et al.; Previous results of experiments in which plasmid-encoded proteins were selectively labeled in ultraviolet sensitive "maxicell" mutants suggested that the essential initiation proteins of RK2 (33 and 43 kDa) were bound to the inner membrane of Escherichia coli (D . Kostyal et al., 1989, Plasmid 21, 226-237) . However, in the present studies using a specific polyclonal antibody against the TrfA initiation proteins, significant levels of these proteins were also detected for the first time in the outer membrane fraction as well as the inner membrane fraction . Only in the cytosol fraction were the initiation proteins relatively absent . In order to determine whether initiation and replication were also associated with either or both of the membrane fractions, it was necessary to develop a replicating system more active than the one previously extracted from minicell membranes, which did not separate the membrane into its component parts (J . A . Kornacki and W . Firshein, 1986, J . Bacteriol . 167, 319-326) . In addition, it was also necessary to devise an extraction procedure that did not degrade the supercoil DNA template during the separation of the inner from the outer membrane fraction . Both criteria were met, first by the use of maxicells containing a miniplasmid derivative of RK2 and second by disrupting cell envelopes in the French pressure cell using low pressure . Under these conditions, not only were the two major membrane fractions separated successfully from the cytosol fraction, but supercoil DNA template was also preserved in both fractions, detergents were avoided, and replication was significantly higher than that described in the earlier experiments . TrfA-dependent initiation of DNA replication was associated primarily with the inner membrane fraction. Mol Biol (Mosk), 1994 Jul-Aug, 28(4), 926 - 31 {Study of the poly(U)-dependent interaction of tRNA(Phe) with the P-site of Escherichia coli ribosomes by chemical modification with nitrosoethylurea}; Nekhai SA et al.; The accessibility of phosphates in E . coli tRNA(Phe) bound to E . coli ribosomes and 30S subunits for attack by an alkylating agent ethylnitrosourea was studied . The experimental technique allowed us to investigate a part of the molecule between N-11 and N-72 . Being bound to the poly(U)-programmed P site of 30S subunit, tRNA(Phe) reveals protection in a discrete region including phosphates 23-44 . This region corresponds to the hairpin formed from the anticodon arm and the adjoining 3' strand of the D stem and the variable loop . On the P site of 70S-poly(U), additional protection was observed in a region between N-45 and N-63 . This region corresponds to the extra loop and T stem, and reveals a fine structure of protection: protected phosphates in positions 45-49, 51, 54-55, 58-61, and 63 alternate with unprotected ones in positions 50, 52-53, 56-57 and 62 . We conclude that ethylnitrosourea can be used for detailed study of tRNA-ribosome contacts. Mol Biochem Parasitol, 1994 Jul, 66(1), 59 - 69 The Plasmodium falciparum protein RESA interacts with the erythrocyte cytoskeleton and modifies erythrocyte thermal stability; Da Silva E et al.; The ring-infected erythrocyte surface antigen (RESA) associates with spectrin in the erythrocyte membrane (Foley, M., Tilley, L., Sawyer, W . H . and Anders, R . F . (1991) Mol . Biochem . Parasitol., 46, 137-148) . A fragment of the RESA protein, which was expressed in Escherichia coli, was found to bind to inside-out vesicles of erythrocyte membranes in an apparently saturable manner . Upon extraction of inside-out vesicles with Triton X-100, the RESA fragment remained associated with the erythrocyte cytoskeleton . Using the technique of steady-state fluorescence polarisation, we have studied the thermal denaturation of fluorescein-labelled spectrin in the presence of recombinant RESA . We found that the RESA fragment partially protected spectrin against heat-induced conformational changes . Furthermore, erythrocytes infected with a RESA (-) laboratory strain (FCR3) were shown to be more susceptible to heat-induced fragmentation than erythrocytes infected with a RESA (+) strain of the parasite . RESA does not, however, appear to play an essential role in the invasion process per se as erythrocytes resealed to contain anti-RESA antibodies were efficiently invaded. Mol Biochem Parasitol, 1994 Jul, 66(1), 111 - 8 Expression and characterization of a rac family protein kinase of Entamoeba histolytica; Que X et al.; We previously reported the isolation from Entamoeba histolytica of a novel rac family protein kinase gene, termed Ehrac1, for "related to cAMP-dependent protein kinases and protein kinase Cs" . To study the function and properties of this kinase gene further, we fused the full-length coding region and the truncated catalytic domain of the Ehrac1 gene in frame with the gene encoding glutathione S-transferase in the pGEX-KG vector and expressed the fusion in Escherichia coli . The thrombin-cleaved and uncleaved fusion proteins, GST-Ehrac1 and GST-Ehrac1-c (catalytic domain), were purified and found to exhibit similar protein kinase activities . The Ehrac1 fusion kinase was found to phosphorylate serine/threonine residues exclusively in vitro . The preferred substrate for the enzyme was histone H1 with a Km of approx . 14 microM . Histone H3 and kemptide were phosphorylated at about half the rate of histone H1 . Protamine, enolase, bovine serum albumin, and poly (Glu:Tyr) were not substrates for the enzyme . The protein kinase activity was higher in the presence of Mn2+ than Mg2+ . Neither cAMP, Ca2+, nor Ca2+/calmodulin stimulated enzyme activity . The pH optimum of the enzyme was 7.5 . The Ehrac1 kinase can utilize GTP as well as ATP as a phosphate donor with an apparent Km of 80 microM . Enzyme activity was inhibited 30-40% by a crude cAMP-dependent protein kinase inhibitor from rabbit and by thiol reagents . The expression and purification of enzymatically active Ehrac1 protein kinase should allow further analysis of the regulation and signal transduction pathways of E . histolytica. Mol Microbiol, 1994 Jul, 13(2), 337 - 48 Identification and characterization of hitherto unknown Mycoplasma pneumoniae proteins; Proft T et al.; Eleven hitherto unknown Mycoplasma pneumoniae proteins were identified and characterized with respect to their size and subcellular location . This was carried out through the construction of in vitro gene fusions between a modified mouse dehydrofolate reductase (dhfr) gene and selected regions (cosmid clones) of the M . pneumoniae genome and expressing them in Escherichia coli . Positive clones were identified using antibodies against specific fractions of M . pneumoniae . The deduced protein sequences of 11 out of 30 clones did not show significant homologies to known proteins in protein data-bank searches . Monospecific antibodies against these 11 fusion proteins were used to determine the size and cellular location of the corresponding M . pneumoniae proteins by immunoscreening Western blots of SDS-acrylamide gels from M . pneumoniae cell extracts. Mol Microbiol, 1994 Jul, 13(2), 301 - 12 Cloning, sequencing, and characterization of multicopy suppressors of a mukB mutation in Escherichia coli; Yamanaka K et al.; The mukB gene codes for a 177 kDa protein, which might be a candidate for a force-generating enzyme in chromosome positioning in Escherichia coli . The mukB106 mutant produces normal-sized, anucleate cells and shows a temperature-sensitive colony formation . To identify proteins interacting with the MukB protein, we isolated three multicopy suppressors (msmA, msmB, and msmC) to the temperature-sensitive colony formation of the mukB106 mutation . The msmA gene, which could not suppress the production of anucleate cells, was found to be identical to the dksA gene . The msmB and msmC genes suppressed the production of anucleate cells as well as the temperature-sensitive colony formation . However, none of them could suppress both phenotypes in a mukB null mutation . DNA sequencing revealed that the msmB gene was identical to the cspC gene and that the msmC gene had not been described before . A homology search revealed that the amino acid sequences of both MsmB and MsmC possessed high similarity to proteins containing the cold-shock domain, such as CspA of E . coli and the Y-box binding proteins of eukaryotes; this suggests that MsmB and MsmC might be DNA-binding proteins that recognize the CCAAT sequence . Hence, the msmB and msmC genes were renamed cspC and cspE, respectively . Possible mechanisms for suppression of the mukB106 mutation are discussed. Mol Microbiol, 1994 Jul, 13(2), 265 - 72 The dps promoter is activated by OxyR during growth and by IHF and sigma S in stationary phase; Altuvia S et al.; Dps is a non-specific DNA-binding protein abundant in starved Escherichia coli cells and is important for the defence against hydrogen peroxide . We found that dps mRNA levels are controlled by rpoS-encoded sigma S, the transcriptional activator OxyR and the histone-like IHF protein . In exponentially growing cells, dps is induced by treatment with hydrogen peroxide in an OxyR-dependent manner . This OxyR-dependent induction occurs only during log phase, although the OxyR protein is present in stationary phase . In the stationary phase cells, dps is expressed in a sigma S- and IHF-dependent manner . The purified OxyR and IHF proteins are also shown to bind upstream of the dps promoter . Our results suggest that the dps promoter is recognized by both sigma 70-holoenzyme and sigma S-holoenzyme, since OxyR acts through sigma 70 and the starts of the OxyR- and sigma S-dependent transcripts are identical. Mol Microbiol, 1994 Jul, 13(2), 243 - 51 Cloning and molecular characterization of a Legionella pneumophila gene induced by intracellular infection and by various in vitro stress conditions; Abu Kwaik Y et al.; The synthesis of a global stress protein (GspA) of Legionella pneumophila is induced in the intracellular environment of the phagocytic cell and by various in vitro stress stimuli . We used techniques of reverse genetics to isolate the gspA gene from a genomic library of L . pneumophila . Sequence analysis of approximately 1700 bp of a representative clone (pBSP1) showed the presence of two open reading frames (ORFs) . ORF1 encoded for a polypeptide with an inferred molecular mass of 19 kDa and an isoelectric point of 6.1 . These predictions correlated with the migration of the GspA protein on two-dimensional SDS-polyacrylamide gels . The predicted amino acid sequence of the GspA protein was identical to 22/23 residues of the N-terminal amino acid sequence derived by Edman degradation of the purified protein . The GspA protein was 41.3% and 36.5% identical to the 16 kDa IbpA and IbpB heat-shock proteins, respectively, of Escherichia coli . Primer extension from mRNA isolated from L . pneumophila showed that transcription of the gspA gene was controlled by two overlapping promoters . One of the promoters was a sigma 70 promoter, while the other was a heat-shock promoter and was regulated by the sigma 32 transcription factor in E . coli . Northern blot analysis showed that the level of gspA mRNA was elevated 3.4-, 5.0-, and 6.7-fold after exposure of L . pneumophila to heat shock, oxidative stress and osmotic shock, respectively . The gspA gene was conserved among 13 serogroups of L . pneumophila . Our data showed that the gspA gene of L . pneumophila, which is induced by intracellular infection and by various stress stimuli, is controlled transcriptionally by two overlapping and separately regulated promoters. J Cell Sci, 1994 Jul, 107 ( Pt 7), 1949 - 57 Dephosphorylation of the largest neurofilament subunit protein influences the structure of crossbridges in reassembled neurofilaments; Gotow T et al.; Phosphorylation-dependent change in electrophoretic mobility is the most unique characteristic of NF-H, the largest molecular mass subunit of the neurofilament . We dephosphorylated NF-H using Escherichia coli alkaline phosphatase, then reassembled it into neurofilaments with NF-M and NF-L, and into NF-H filaments with NF-H alone . We compared these dephosphorylated filaments with control: projections by low-angle rotary-shadow, crossbridges by quick-freeze deep-etch, and core filament packing density by thin-section electron microscopy . Projections in the dephosphorylated filaments were basically similar in structure to those in control, although there was a tendency for them to be wider and less dense, especially in NF-H filaments . Dephosphorylated filaments were still able to form crossbridges between core filaments, but their crossbridges were significantly wider, less dense, more branched and more irregular than crossbridges in control, and core filaments were more densely packed . These structural differences may be brought about by the removal of phosphate groups from NF-H tail and consequent reduction of electrostatic repulsion between adjacent crossbridges extending from the same core filament . The results indicate that phosphorylation of NF-H is necessary for forming well developed crossbridges, straight and at constant intervals, like those of in vivo axonal neurofilaments. J Cell Sci, 1994 Jul, 107 ( Pt 7), 1935 - 48 Identification of two N-terminal non-alpha-helical domain motifs important in the assembly of glial fibrillary acidic protein; Ralton JE et al.; The non-alpha-helical N-terminal domain of intermediate filament proteins plays a key role in filament assembly . Previous studies have identified a nonapeptide motif, SSYRRIFGG, in the non-alpha-helical N-terminal domain of vimentin that is required for assembly . This motif is also found in desmin, peripherin and the type IV intermediate filament proteins . GFAP is the only type III intermediate filament protein in which this motif is not readily identified . This study has identified two motifs in the non-alpha-helical N-terminal domain of mouse GFAP that play important roles in GFAP assembly . One motif is located at the very N terminus and has the consensus sequence, MERRRITS-ARRSY . It has some characteristics in common with the vimentin nonapeptide motif, SSYRRIFGG, including its location in the non-alpha-helical N-terminal domain and a concentration of arginine residues . Unlike the vimentin motif in which even conserved sequence changes affect filament assembly, the GFAP consensus sequence, MERRRITS-ARRSY, can be replaced by a completely unrelated sequence; namely, the heptapeptide, MVRANKR, derived from the lambda cII protein . When fused to GFAP sequences with sequential deletions of the N-terminal domain, the lambda cII heptapeptide was used to help identify a second motif, termed the RP-box, which is located just upstream of the GFAP alpha-helical rod domain . This RP-box affected the efficiency of filament assembly as well as protein-protein interactions in the filament, as shown by sedimentation assays and electron microscopy . These results are supported by previous data, which showed that the dramatic reorganization of GFAP within cells was due to phosphorylation-dephosphorylation of a site located in this RP-box . The results in this study suggest the RP-box motif to be a key modulator in the mechanism of GFAP assembly, and support a role for this motif in both the nucleation and elongation phases of filament assembly . The RP-box motif in GFAP has the consensus sequence, RLSL-RM-PP . Sequences similar to the GFAP RP-box motif are also to be found in vimentin, desmin and peripherin . Like GFAP, these include phosphorylation and proteolysis sites and are adjacent to the start of the central alpha-helical rod domain, suggesting that this motif of general importance to type III intermediate filament protein assembly. Antimicrob Agents Chemother, 1994 Jul, 38(7), 1555 - 60 L-651,392, a potent leukotriene inhibitor, controls inflammatory process in Escherichia coli pyelonephritis; Tardif M et al.; In this study, the relationship between leukotrienes, peritubular cell infiltration with polymorphonuclear cells (PMNs) and renal tubular damage was investigated in a rat model of acute ascending pyelonephritis . Infection was induced by the injection of 10(5) CFU of Escherichia coli into the bladder and occlusion of the left ureter for 24 h . Treatment of infected animals was started 24 h after the induction of pyelonephritis with either hydrocortisone (25 mg/kg of body weight per day), the leukotriene inhibitor L-651,392 (10 mg/kg/day), or the vehicle of L-651,392 and was maintained for 5 days . At the end of treatment, the animals were killed, serum was collected, and both kidneys were removed for colony counts and histopathology . Renal function was evaluated by the measurement of blood urea nitrogen levels and creatinine clearance . The numbers of PMNs and mononuclear cells (MNs) in the cortex and medulla were recorded for all groups on plastic sections done from the left kidney . Infection alone (vehicle of L-651,392) resulted in intensive interstitial infiltration and a severe tubular destruction in the cortex . Treatment with hydrocortisone did not prevent PMN migration and tissue damage . By contrast, treatment with L-651,392 resulted in a significant reduction in PMNs (P < 0.001 in comparisons with all other groups) and greater preservation of the tubular structure despite identical bacterial counts than in the group receiving hydrocortisone . We conclude that L-651,392 prevents inflammatory cells from reaching the site of infection and protects the kidney from tubular damage associated with inflammation during pyelonephritis . Inhibitors of leukotrienes should be further investigated for their potential benefit as adjuvants to antibiotherapy in the treatment of pyelonephritis. Am J Vet Res, 1994 Jul, 55(7), 991 - 9 Pathogenicity of a bovine attaching effacing Escherichia coli isolate lacking Shiga-like toxins; Fischer J et al.;