J Bacteriol, 1992 Dec, 174(23), 7620 - 8 Expression of the repA1 gene of IncFII plasmid NR1 is translationally coupled to expression of an overlapping leader peptide; Wu R et al.; Examination of a group of mutants of plasmid NR1 that had lost the expression of IncFII plasmid incompatibility (Inc-) revealed a group that had also lost replication proficiency (Rep-) . These mutants were obtained from plasmids in which the NR1 replication control region was present in a cointegrate with plasmid pBR322 . Whereas the wild-type parental cointegrate plasmid was capable of replicating in a polA host owing to the PolA independence of NR1 replication, the mutants were not able to transform a polA host . Losses of both expression of IncFII plasmid incompatibility and replication proficiency were found to result from the same single base-pair substitution in four independently isolated Inc- Rep- mutants . The mutation inactivates promoter PE for the transcription of RNA-E, a trans-acting repressor of translation of the essential RepA1 replication initiation protein of NR1 . Although the loss of RNA-E synthesis had been expected to increase the expression of repA1, the efficiency of translation of repA1 mRNA from these mutants was at least 100-fold lower than that from the wild type, as revealed by repA1-lacZ translational fusions . The PE mutation introduced a stop codon into a 24-amino-acid reading frame that precedes the repA1 gene and terminates just 2 bp downstream from the repA1 start codon . This putative leader peptide was also expressed in a lacZ translational fusion, and its expression was reduced by a factor of 10(4) by the PE mutation . The expression of the leader peptide and the expression of repA1 were regulated by RNA-E . These results suggest that the expression of repA1 is coupled to the translation of the leader peptide and that the repression of repA1 translation by RNA-E may occur via inhibition of the translation of the leader peptide. J Bacteriol, 1992 Dec, 174(23), 7533 - 41 Expression and regulation of the RepA protein of the RepFIB replicon from plasmid P307; Spiers AJ et al.; The control of RepFIB replication appears to rely on the interaction between an initiator protein (RepA) and two sets of DNA repeat elements located on either side of the repA gene . Limited N-terminal sequence information obtained from a RepA:beta-galactosidase fusion protein indicates that although the first residue of RepA is methionine, the initiation of translation of RepA occurs from a CTG codon rather than from the predicted GTG codon located further downstream . Overexpressed RepA in trans is capable of repressing a repA:lacZ fusion plasmid in which the expression of the fusion protein is under the control of the repA promoter . The repA promoter has been located functionally by testing a series of repA:lacZ fusion plasmids . Both in vivo genetic tests and in vitro DNA-binding studies indicate that repA autoregulation can be achieved by RepA binding to one or more repeat elements which overlap the repA promoter sequence. J Bacteriol, 1992 Dec, 174(23), 7517 - 26 Identification and characterization of the smbA gene, a suppressor of the mukB null mutant of Escherichia coli; Yamanaka K et al.; The mukB gene encodes a protein involved in chromosome partitioning in Escherichia coli . To study the function of this protein, we isolated from the temperature-sensitive mukB null mutant and characterized 56 suppressor mutants which could grow at 42 degrees C . Ten of the mutants also showed cold-sensitive growth at 22 degrees C . Using one of the cold-sensitive mutants as host, the wild type of the suppressor gene was cloned . The cloned suppressor gene complemented all of the 56 suppressor mutations . DNA sequencing revealed the presence of an open reading frame of 723 bp which could encode a protein of 25,953 Da . The gene product was indeed detected . The previously undiscovered gene, named smbA (suppressor of mukB), is located at 4 min on the E . coli chromosome, between the tsf and frr genes . The smbA gene is essential for cell proliferation in the range from 22 to 42 degrees C . Cells which lacked the SmbA protein ceased macromolecular synthesis . The smbA mutants are sensitive to a detergent, sodium dodecyl sulfate, and they show a novel morphological phenotype under nonpermissive conditions, suggesting a defect in specific membrane sites. Endocrinology, 1992 Dec, 131(6), 3120 - 2 Nuclear localization and hepatic zonation of rat "spot 14" protein: immunohistochemical investigation employing anti-fusion protein antibodies; Kinlaw WB et al.; S14 protein and mRNA levels are rapidly regulated by hormones and diet . We have purified a 45-Kd fusion protein from lysates of transformed E . coli that includes the entire S14 polypeptide . Affinity-purified rabbit anti-fusion protein antibodies were used in immunohistochemistry to determine the distribution of S14 protein across the hepatic lobule, and to reassess its intracellular location . In hyperthyroid liver, S14 protein clustered near the central venous zone, and was not detectable in the periportal area of the acinus . The signal in perivenous hepatocytes was primarily nuclear in location, in stark contrast to previous subcellular fractionation studies . Visualization of identical hepatic distribution and subcellular localization employing anti-synthetic peptide antiserum provided evidence for the specificity of the immunostaining, as did attenuation of the signal by preincubation of the antibody with its antigen . No staining was observed in sections of heart or hypothyroid liver, as expected from the low levels of S14 protein in those tissues . The data indicate that induction of S14 protein expression by T3 occurs through enhanced expression by perivenous hepatocytes, rather than by recruitment of cells in more peripheral zones of the lobule . Nuclear localization of the S14 protein by immunohistochemistry suggests that it is lost from nuclei during standard fractionation procedures, and prompts consideration of a role for S14 in regulation of nuclear structure and/or function. Chest, 1992 Dec, 102(6), 1730 - 6 Reduction of pulmonary surfactant in patients with human immunodeficiency virus infection and Pneumocystis carinii pneumonia; Hoffman AG et al.; We assessed qualitative and quantitative differences in surfactant lipid composition of bronchoalveolar lavage (BAL) fluid in patients with acquired immune deficiency syndrome (AIDS) and Pneumocystis carinii (PC) pneumonia . Five normal volunteers and 27 patients with human immunodeficiency virus (HIV) infection underwent BAL for evaluation of possible pulmonary infection . Bronchoalveolar lavage studies in eight patients were negative for PC organisms, and 19 were positive . Pneumocystis carinii pneumonia was graded (mild vs moderate to severe) by initial alveolar-arterial oxygen gradient . Bronchoalveolar lavage fluid was centrifuged, the lipids were extracted from the supernatant, and total lipid profiles of dephosphorylated glycerolipids were analyzed as trimethylsilylether derivatives by high temperature gas-liquid chromatography . Phospholipase A2 levels were determined using a radiolabeled E coli membrane method . Compared to the normal volunteers (109 +/- 13 micrograms/5 ml) and the PC negative group (107 +/- 13 micrograms/5 ml), total BAL lipid was reduced for both the mild PC pneumonia group (73 +/- 10 micrograms/5 ml) and the moderate to severe PC pneumonia group (46 +/- 4 micrograms/5 ml) . There was a parallel reduction of diacylglycerol lipids: normal volunteers, 52 +/- 7 micrograms/5 ml; PC negative, 52 +/- 9 micrograms/5 ml; mild PC pneumonia, 35 +/- 7 micrograms/5 ml; and moderate to severe PC pneumonia, 15 +/- 2 micrograms/5 ml . Phospholipase A2 activity in moderate to severe PC pneumonia was twice that of the PC negative patients, and 30 times that for normals . The data demonstrate a marked diminution in surfactant glycerophospholipid in patients with AIDS and PC pneumonia and suggest a potential role for surfactant abnormality in the pathophysiology of this disease. Arch Biochem Biophys, 1992 Dec, 299(2), 295 - 301 Effects of mutations at residue 309 of the large subunit of ribulosebisphosphate carboxylase from Synechococcus PCC 6301; Morell MK et al.; Previous studies {G . S . Hudson et al . (1989) J . Biol . Chem . 265, 808-814} showed that the faster turnover rates and lower affinities for CO2 of ribulosebisphosphate carboxylase/oxygenases from C4 plants, compared to C3 and C3/C4 plants, were specified by the chloroplast-encoded large subunits . In pairs of closely related C3 and C4 species from three genera, these kinetic changes were accompanied by only three to six amino acid residue substitutions, depending on the genus . None of these substitutions occurred near the active site and only one, 309Met (C3) to Ile (C4), was common to all three genera . Unlike the plant carboxylases, the highly homologous enzyme from the cyanobacterium Synechococcus PCC 6301 folds and assembles properly when its rbcL and rbcS genes are coexpressed in Escherichia coli . Furthermore, the cyanobacterial enzyme has Ile at position 309 of the large subunit, a high turnover number, and a poor affinity for CO2 . 309Ile was replaced with Met and several other residues by site-directed mutagenesis of the cyanobacterial rbcL . Met and Leu were tolerated at this position with no alteration in the kinetic or structural properties of the assembled holoenzyme . However, substitution with Val, Gly, Trp, or Arg prevented the assembly of the subunits . The indifference to Met or Ile at this position, as well as the tolerance for Leu which is not observed with any natural ribulosebisphosphate carboxylase, leads to the conclusion that either the 309Met/Ile substitution has no effect on the kinetic properties of the plant enzyme, despite the correlation apparent in previous studies, or the cyanobacterial enzyme is sufficiently different from the plant enzyme in other respects that the influence of residue 309 is masked. J Virol, 1992 Dec, 66(12), 7469 - 80 Human immunodeficiency virus type 1 Rev activation can be achieved without Rev-responsive element RNA if Rev is directed to the target as a Rev/MS2 fusion protein which tethers the MS2 operator RNA; Venkatesan S et al.; The posttranscriptional trans activation of unspliced or partially spliced human immunodeficiency virus RNAs by the Rev regulatory protein is crucial for virus replication and is dependent on sequence-specific RNA binding by Rev . The cognate RNA target of Rev is contained within a highly structured, 244-nucleotide Rev-responsive element (RRE) RNA in the viral env gene . Here, we show that specific interaction with the RRE is not an absolute requirement for Rev function . When the RRE is replaced by a heterologous MS2 phage operator sequence, Rev will facilitate the cytoplasmic expression of human immunodeficiency virus mRNAs containing this sequence if directed to the MS2 operator via the RNA binding motif of the MS2 phage coat protein (MS-C) as a Rev/MS-C fusion protein . Rev/MS-C efficiently activated both RRE and MS2 targets . A mutation in the MS2 operator that abolished the coat protein binding in vitro rendered the mutant RNA nonresponsive to the fusion protein in vivo . Notwithstanding that Rev can be tethered to the viral RNAs via another RNA binding motif, the structural integrity of the N terminus of Rev was still required for optimal trans activation. J Virol, 1992 Dec, 66(12), 7293 - 302 Identification of the domains required for direct interaction of the helicase-like and polymerase-like RNA replication proteins of brome mosaic virus; Kao CC et al.; Brome mosaic virus is a positive-strand RNA virus whose RNA replication requires viral protein 1a, which has putative helicase and capping functions, and 2a, which has putative polymerase function . Since domains of related sequence are conserved in a wide range of plus-strand RNA viruses, analysis of 1a and 2a function should have applicability to many other viruses . We have recently demonstrated that 1a and 2a form a complex in vivo and in vitro . Using immune coprecipitation and mutant polypeptides made in reticulocyte lysates, we have now mapped both the 1a and 2a domains necessary for complex formation . The sequences needed to bind 2a map to the carboxy-terminal helicase-like domain of 1a . Truncated polypeptides containing this domain were able to bind to 2a, while several small insertions in the helicase-like domain disrupted binding . The sequence required for binding 1a lies within a 115-residue subset of the 2a N-terminal segment preceding the polymerase-like domain . Truncations or fusion polypeptides containing this segment can bind 1a . We also determined that highly purified 2a protein made in insect cells can form a complex with highly purified 1a helicase-like domain made in Escherichia coli, suggesting that no other factor is required to mediate 1a-2a interaction . Previous genetic analyses of 1a and 2a are consistent with this mapping and show that the newly defined 1a and 2a binding regions are required for RNA synthesis . The locations of these interacting regions are discussed with regard to models of viral replication and the evolution of positive-strand RNA virus genomes. EMBO J, 1992 Dec, 11(12), 4481 - 7 Mapping of the in vivo start site for leading strand DNA synthesis in plasmid R1; Bernander R et al.; We have previously constructed Escherichia coli strains in which an R1 plasmid is integrated into the origin of chromosome replication, oriC . In such intR1 strains, oriC is inactive and initiation of chromosome replication instead takes place at the integrated R1 origin . Due to the large size of the chromosome, replication intermediates generated at the R1 origin in these strains are considerably more long-lived than those in unintegrated R1 plasmids . We have taken advantage of this and performed primer extensions on total DNA isolated from intR1 strains, and mapped the free 5' DNA ends that were generated as replication intermediates during R1 replication in vivo . The sensitivity of the mapping was considerably improved by the use of a repeated primer extension method (RPE) . The free DNA ends were assumed to represent normal in vivo start sites for leading strand DNA synthesis in plasmid R1 . The ends were mapped to a short region approximately 380 bp away from the R1 minimal origin, and the positions agreed well with previous in vitro mappings . The same start positions were also utilized in the absence of the DnaA protein, indicating that DnaA is not required for determination of the position at which DNA synthesis starts during initiation of replication at the R1 origin. EMBO J, 1992 Dec, 11(12), 4451 - 7 Epimutation of repeated genes in Ascobolus immersus; Rhounim L et al.; Ascobolus immersus artificial gene repeats were shown previously to be subject premeiotically to both cytosine methylation and inactivation . We studied sexual progenies of strains harbouring two wild type copies of the endogenous met2 gene lying either in tandem array or at ectopic unlinked positions, by (i) investigating the methylation status, (ii) searching for mutations and (iii) analysing the inheritance of inactivation both in mitotic and sexual offspring . 100% of the 'tandem' progeny and 64% of the 'ectopic' progeny had methylated repeats and displayed gene inactivation . Similar methylation patterns involving all or most of the cytosine residues within the repeats were observed in both arrangements . The inactivated met2 copies were totally devoid of mutation, as deduced from: (i) extensive restriction site analysis and DNA sequencing; (ii) the finding that all the Met- derivatives tested reverted to prototrophy in selective conditions; and (iii) the finding that an inactivated copy of met2 stripped of its methylation through amplification in Escherichia coli regained activity when reintroduced in A.immersus . In the absence of selection, gene silencing and methylation were faithfully maintained through mitotic divisions and through five successive sexual cycles . Altogether, these data show the epimutational nature of this methylation induced premeiotically (MIP) process. EMBO J, 1992 Dec, 11(12), 4445 - 50 High plasticity of multispecific DNA methyltransferases in the region carrying DNA target recognizing enzyme modules; Walter J et al.; Multispecific cytosine C5 DNA methyltransferases (MTases) methylate more than one specific DNA target . This is due to the presence of several target recognizing domains (TRDs) in these enzymes . Such TRDs form part of a variable centre in the MTase primary sequence, which separates conserved enzyme core sequences responsible for general steps in the methylation reaction . By deleting, rearranging and exchanging several TRDs of multispecific MTases, we demonstrate their modular character; they mediate target recognition independent of a particular TRD or core sequence context . We show also that multispecific MTases can accommodate inert material of non-MTase origin within their variable region without losing their activity . The remarkable plasticity with respect to the material that can be integrated into this region suggests that the enzyme core sequences preceding or following it form separable functional domains . In spite of the documented flexibility multispecific MTases could not be endowed with novel specificities by integration of putative TRDs of monospecific MTases, pointing to differences between multi- and monospecific MTases in the way their core and TRD sequences interact. Circulation, 1992 Dec, 86(6 Suppl), III26 - 9 von Willebrand factor as a target for antithrombotic intervention; Ruggeri ZM; The adhesive protein von Willebrand factor is essential for the formation of platelet thrombi under flow conditions characterized by high shear stress . This function requires the interaction with two distinct platelet receptors, the glycoprotein complexes Ib-IX-V and IIb-IIIa . Interaction with the former results in platelet activation, a necessary step for binding to the latter and supporting stable aggregation . The inhibition of von Willebrand factor binding to glycoprotein Ib can be achieved with small recombinant fragments containing the specific functional domain of the molecule that interacts with this platelet receptor . Such fragments may provide a new selective approach to antithrombotic therapy. Circ Res, 1992 Dec, 71(6), 1508 - 17 Optimization of retroviral vector-mediated gene transfer into endothelial cells in vitro; Kahn ML et al.; Retroviral vector-mediated gene transfer into endothelial cells is relatively inefficient with transduction rates as low as 1-2% in vitro and even lower in vivo . To increase the efficiency of gene transfer into endothelial cells, we used retroviral vectors expressing beta-galactosidase and urokinase and measured endothelial cell transduction efficiencies with quantitative assays for beta-galactosidase and urokinase protein . We evaluated several techniques reported to improve the efficiency of retroviral transduction in vitro, including 1) extended periods of exposure to vector, 2) repeated exposures to vector, 3) maximization of the ratio of vector particles to endothelial cells by increasing the volume and concentration of vector particles or by decreasing the number of endothelial cells exposed, 4) cocultivation of endothelial cells with vector-producing cells, and 5) variation of the type and concentration of polycation used with the retroviral vector . Only the use of more concentrated (higher titer) vector-containing supernatant and the use of the polycation DEAE-dextran improved the efficiency of gene transfer into endothelial cells in vitro . In an optimized transduction protocol, a 60-second exposure to 1 mg/ml DEAE-dextran followed by a single 6-hour exposure to supernatant of a titer of 10(5)-10(6) colony-forming units/ml resulted in transduction efficiencies of 50-90% with both vectors . Decreasing the time of the supernatant exposure to 15 minutes permitted transduction efficiencies of 15-20% while significantly minimizing the duration of the transduction . Therefore, the optimized protocol allows high efficiency in vitro gene transfer into endothelial cells within several hours . The briefer protocol may prove useful for in vivo gene transfer in which the time of exposure to the supernatant is limited. Cancer Res, 1992 Dec 1, 52(23), 6423 - 30 Intracellular localization of human DNA repair enzyme methylguanine-DNA methyltransferase by antibodies and its importance; Ayi TC et al.; The human DNA repair enzyme, methylguanine-DNA methyltransferase (MGMT, M(r) 21,000), which protects cells against the mutagenic effect of alkylating carcinogens, was found to be localized in the cell nucleus (except the nucleolus) by immunofluorescence staining using polyclonal and monoclonal antibodies . The supporting experiments came from differential staining of the MGMT-deficient (mer-) and -proficient (mer+) cells, Western blotting analysis, and specific antibody depletion studies with the immobilized fusion protein, GSTMGMT-glutathione-Sepharose . Its localization in the nucleus agrees with its biological function and possibly explains the ineffective protection of mammalian cells (mer-) transfected with the Escherichia coli MGMT genes from bifunctional alkylating agents. Biotechnology (N Y), 1992 Dec, 10(12), 1557 - 61 An algorithmically optimized combinatorial library screened by digital imaging spectroscopy; Goldman ER et al.; Combinatorial cassettes based on a phylogenetic "target set" were used to simultaneously mutagenize seven amino acid residues on one face of a transmembrane alpha helix comprising a bacteriochlorophyll binding site in the light harvesting II antenna of Rhodobacter capsulatus . This pigmented protein provides a model system for developing complex mutagenesis schemes, because simple absorption spectroscopy can be used to assay protein expression, structure, and function . Colony screening by Digital Imaging Spectroscopy showed that 6% of the optimized library bound bacteriochlorophyll in two distinct spectroscopic classes . This is approximately 200 times the throughput (ca . 0.03%) of conventional combinatorial cassette mutagenesis using {NN(G/C)} . "Doping" algorithms evaluated in this model system are generally applicable and should enable simultaneous mutagenesis at more positions in a protein than currently possible, or alternatively, decrease the screening size of combinatorial libraries. Biotechnology (N Y), 1992 Dec, 10(12), 1550 - 6 Recombinant protein expression in high cell density fed-batch cultures of Escherichia coli; Yee L et al.; Whereas cell concentrations of 5-10 grams dry cell weight per liter (gDCW/l) are typical of batch cultures, fed-batch techniques can be used to achieve concentrations greater than 50 gDCW/l . Feeding strategies for fed-batch cultures include feed-back control as well as pre-determined feeding profiles . The volumetric yield of recombinant products can be improved by controlling the specific growth rate and the substrate concentration . Furthermore, inhibitory by-product formation can be minimized in fed-batch cultures . This review focuses on the use of fed-batch techniques to produce recombinant products in Escherichia coli . The modes of nutrient feeding that have been employed are discussed, and the factors important in attaining high cell concentrations as well as high specific yields of recombinant product are described. J Formos Med Assoc, 1992 Dec, 91(12), 1133 - 7 Blood levels of platelet-activating factor in endotoxin-sensitive and endotoxin-resistant mice during endotoxemia; Chang TW et al.; Platelet-activating factor (PAF) is a phosphoglyceride secreted by a variety of cells and has been implicated in endotoxin toxicities . To further confirm its role in endotoxin-induced tissue injuries and death, we conducted an experiment on endotoxin-resistant (C3H/HeJ strain) and endotoxin-sensitive (C3H/HeN strain) mice . The experiment consisted of three parts: 1) the LD50 of endotoxin from E . coli 0127:B8 cells was quantitated in C3H/HeN mice; 2) the lethality of PAF in C3H/HeJ mice at a dose lethal to C3H/HeN mice was determined; and 3) the blood levels of PAF in C3H/HeJ and C3H/HeN mice were measured after a dose of endotoxin lethal to the C3H/HeN strain was injected . PAF contained in the blood samples was extracted by a solid phase procedure and assayed by a radioimmunoassay method . The results showed that endotoxin-resistant and endotoxin-sensitive mice were equally susceptible to death induced by the same lethal dose of PAF . After injection with endotoxin, the blood PAF levels in C3H/HeN mice increased significantly (p < 0.01) at 60 minutes and 90 minutes, with a peak level three times that of the control group . The blood PAF levels in C3H/HeJ mice, however, remained unelevated throughout the experiment . The timing of the occurrence of the peak blood PAF level in the C3H/HeN mice corresponded with the emergence of their illness from the endotoxin injection . These findings shed new light on our understanding of the resistant mechanisms of C3H/HeJ mice to bacterial endotoxin and affirm the possible role of PAF in mediating endotoxin toxicities. Mol Biol Cell, 1992 Dec, 3(12), 1437 - 42 The role of Gln61 and Glu63 of Ras GTPases in their activation by NF1 and Ras GAP; Nur-E-Kamal MS et al.; Two distinct GAPs of 120 and 235 kDa called GAP1 and NF1 serve as attenuators of Ras, a member of GTP-dependent signal transducers, by stimulating its intrinsic guanosine triphosphatase (GTPase) activity . The GAP1 (also called Ras GAP) is highly specific for Ras and does not stimulate the intrinsic GTPase activity of Rap1 or Rho . Using GAP1C, the C-terminal GTPase activating domain (residues 720-1044) of bovine GAP1, we have shown previously that the GAP1 specificity is determined by the Ras domain (residues 61-65) where Gln61 plays the primary role . The corresponding domain (residues 1175-1531) of human NF1 (called NF1C), which shares only 26% sequence identity with the GAP1C, also activates Ras GTPases . In this article, we demonstrate that the NF1C, like the GAP1C, is highly specific for Ras and does not activate either Rap1 or Rho GTPases . Furthermore, using a series of chimeric Ras/Rap1 and mutated Ras GTPases, we show that Gln at position 61 of the GTPases primarily determines that NF1C as well as GAP1C activates Ras GTPases, but not Rap1 GTPases, and Glu at position 63 of the GTPases is required for maximizing the sensitivity of Ras GTPases to both NF1C and GAP1C . Interestingly, replacement of Glu63 of c-HaRas by Lys reduces its intrinsic GTPase activity and abolishes the GTPase activation by both NF1C and GAP1C . Thus, the potentiation of oncogenicity by Lys63 mutation of c-HaRas appears primarily to be due to the loss of its sensitivity to the two major Ras signal attenuators (NF1 and GAP1). FEMS Microbiol Lett, 1992 Dec 1, 78(2-3), 125 - 30 Thermal regulation of fimA, the Escherichia coli gene coding for the type 1 fimbrial subunit protein; Dorman CJ et al.; The effect of temperature on expression of fimA, the gene coding for the phase-variable type 1 fimbrial subunit protein of Escherichia coli K-12 was investigated . In a genetic background in which the orientation of the DNA fragment carrying the fimA promoter was determined by the activity of the 'orientationally unbiased' FimB recombinase, fimA transcription was consistently higher at 30 degrees C than at 37 degrees C . This apparent increase in fimA expression was found to be due to the fact that the fimA site-specific recombination system had become directionally biased at the lower temperature such that the bacterial population contained more cells in the ON phase than at 37 degrees C . When expression of fimA was studied in a strain genetically incapable of switching the fimA promoter to the OFF phase (i.e . all cells in the culture were phase-locked ON), fimA transcription was found to be higher at 37 degrees C than at 30 degrees C . Thus, transcription from the fimA promoter was subject to temperature control and the site-specific recombination system determining the orientation of the promoter DNA fragment was temperature-modulated . Furthermore, it was found that the thermosensitive fimA promoter was subject to transcriptional silencing by the HNS nucleoid protein, in a manner analogous to that described for other thermoregulated adhesins. Mol Biochem Parasitol, 1992 Dec, 56(2), 311 - 21 Glycosyl phosphatidylinositol-specific phospholipase C of Trypanosoma brucei: expression in Escherichia coli; Mensa-Wilmot K et al.; Glycosyl phosphatidylinositol-specific phospholipase C (GPI-PLC) from Trypanosoma brucei cleaves the glycosyl phosphatidylinositol (GPI) anchor of the trypanosome variant surface glycoprotein (VSG) and other GPI structures . We have expressed this enzyme in Escherichia coli, using a protocol designed to produce the native enzyme rather than a fusion protein . We have purified large amounts of GPI-PLC from E . coli membranes, using a single step immunoaffinity technique . The expressed enzyme is identical to its trypanosome counterpart in enzymatic specificity, mobility on SDS-PAGE, and isoelectric point . Recombinant GPI-PLC is a membrane enzyme; it associates with E . coli membranes and, like the T . brucei GPI-PLC, partitions into the detergent phase in Triton X-114 phase separation experiments . The Michaelis constants for the two enzymes are similar (400 nM, with VSG as substrate) . The turnover number (kcat, 72 min-1) of the recombinant enzyme (expressed from a . T . brucei rhodesiense WRATat 1.1 cDNA) is about one-tenth that of GPI-PLC from T . brucei brucei (ILTat 1.3). Am J Vet Res, 1992 Dec, 53(12), 2251 - 8 Effect of age on activation of porcine intestinal guanylate cyclase and binding of Escherichia coli heat-stable enterotoxin (STa) to porcine intestinal cells and brush border membranes; Jaso-Friedmann L et al.; Development of age-dependent resistance to enterotoxigenic Escherichia coli was studied, using isolated enterocytes and brush border membranes (BBM) from 7-day-old and 7-week-old pigs . Binding of 125I-labeled heat-stable (125I-STa) enterotoxin to enterocytes and BBM was specific, temperature- and time-dependent, saturable, and partially reversible . Scatchard analysis revealed a single class of receptors . Mean +/- SD avidity of binding (apparent affinity constant, Ka) of 125I-STa to enterocytes from 7-day-old and 7-week-old pigs was 2.14 +/- 0.29 x 10(8) and 2.72 +/- 0.25 x 10(8) L/mol, respectively . Numbers of STa receptors were calculated to be 64,903 +/- 2,900/enterocyte for 7-day-old pigs and 53,029 +/- 3,117/enterocyte for 7-week-old pigs . Numbers of STa receptors expressed per milligram of BBM protein from 7-day-old pigs were 2.66 x 10(11), compared with 2.29 x 10(11) for BBM from 7-week-old pigs . By 5 minutes after addition of STa to reaction mixtures, intracellular cyclic guanosine monophosphate concentration increased 13.9-fold in enterocytes from 7-day-old pigs and 8.7-fold in enterocytes from 7-week-old pigs . The particulate guanylate cyclase activity associated with BBM from 7-week-old pigs was slightly more sensitive to low amounts of STa, compared with BBM from 7-day-old pigs; however, differences were not observed at intermediate and high amounts . These data indicate that lack of a secretory response to STa by older pigs is not attributable either to decreased numbers of STa receptors or to decreased signal response between the STa receptor and membrane-bound guanylate cyclase.(ABSTRACT TRUNCATED AT 250 WORDS) J Pharm Pharmacol, 1992 Dec, 44(12), 1036 - 7 Decrease by psychotropic drugs and local anaesthetics of membrane fluidity measured by fluorescence anisotropy in Escherichia coli; Tanji K et al.; The effects of psychotropic drugs and local anaesthetics on the fluidity of Escherichia coli cell membranes were examined . Chlorpromazine was shown to increase 1,6-diphenyl-1,3,5-hexatriene fluorescence anisotropy, indicating that it decreased the membrane fluidity . This increase was significant at a temperature of more than 24 degrees C . Dibucaine, lignocaine, imipramine, tetracaine and procaine also increased the fluorescence anisotropy. EMBO J, 1992 Dec, 11(13), 4757 - 65 Chaperonin-mediated protein folding: GroES binds to one end of the GroEL cylinder, which accommodates the protein substrate within its central cavity; Langer T et al.; The mechanism of GroEL (chaperonin)-mediated protein folding is only partially understood . We have analysed structural and functional properties of the interaction between GroEL and the co-chaperonin GroES . The stoichiometry of the GroEL 14mer and the GroES 7mer in the functional holo-chaperonin is 1:1 . GroES protects half of the GroEL subunits from proteolytic truncation of the approximately 50 C-terminal residues . Removal of this region results in an inhibition of the GroEL ATPase, mimicking the effect of GroES on full-length GroEL . Image analysis of electron micrographs revealed that GroES binding triggers conspicuous conformational changes both in the GroES adjacent end and at the opposite end of the GroEL cylinder . This apparently prohibits the association of a second GroES oligomer . Addition of denatured polypeptide leads to the appearance of irregularly shaped, stain-excluding masses within the GroEL double-ring, which are larger with bound alcohol oxidase (75 kDa) than with rhodanese (35 kDa) . We conclude that the functional complex of GroEL and GroES is characterized by asymmetrical binding of GroES to one end of the GroEL cylinder and suggest that binding of the substrate protein occurs within the central cavity of GroEL. Infect Immun, 1992 Dec, 60(12), 5004 - 12 Regulation of intestinal guanylate cyclase by the heat-stable enterotoxin of Escherichia coli (STa) and protein kinase C; Crane JK et al.; The heat-stable enterotoxin of Escherichia coli (STa) stimulates membrane-bound guanylate cyclase in intestinal epithelium and induces fluid and ion secretion . Using the T84 human colon carcinoma cell line as a model, we observed that phorbol esters markedly enhanced STa-stimulated cyclic GMP accumulation in T84 cells (C . S . Weikel, C . L . Spann, C . P . Chambers, J . K . Crane, J . Linden, and E . L . Hewlett, Infect . Immun . 58:1402-1407, 1990) . In this study we document that the phorbol ester treatment increases 125I-STa-binding sites as well as membrane-bound guanylate cyclase activity in T84 cells and provide evidence that both effects are mediated by phosphorylation . Guanylate cyclase activity was increased approximately 50% in membranes prepared from intact T84 cells treated with phorbol-12,13-dibutyrate (beta-PDB) and after treatment of homogenates with beta-PDB in a manner dependent on ATP, MgCl2, and cytosol . Similarly, treatment of membranes with purified bovine brain protein kinase C in the presence of appropriate cofactors and beta-PDB resulted in an increase in STa-stimulated guanylate cyclase activity of about 70% . Likewise, the number of 125I-STa-binding sites was increased by about 25 to 40% in membranes prepared from intact cells or homogenates treated with beta-PDB; no effect on binding affinity (Kd = 0.15 nM) was noted . These experiments suggest that protein kinase C may phosphorylate the STa receptor-guanylate cyclase or a closely related protein and increase guanylate cyclase activity . The stimulatory effects of protein kinase C on STa-sensitive guanylate cyclase are opposite in direction to the profound inhibitory effects of the kinase on atrial natriuretic peptide-stimulated guanylate cyclase, demonstrating differential regulation by protein kinases within the guanylate cyclase-receptor family. J Infect Dis, 1992 Dec, 166(6), 1295 - 310 Localized, aggregative, and diffuse adherence to HeLa cells, plastic, and human small intestines by Escherichia coli isolated from patients with diarrhea; Yamamoto T et al.; Adherence of diarrhea-associated Escherichia coli was studied by scanning electron microscopy . Enteropathogenic E . coli (EPEC) adherence factor-positive (EAF+) E . coli of EPEC serotypes (class I EPEC) adhered to plastic and human jejunal and ileal mucosa, similar to case and HeLa cells . Localized adherence, elongation of cell microvilli, and "locking" of the bacterial aggregates by the elongated microvilli were evident after incubation for 20 min . EAF+ E . coli adhered strikingly to mucus but rarely to M cells in Peyer's patch-associated epithelium . Most enteroaggregative E . coli (EAggEC) strains adhered to plastic, similar to HeLa cells . Some diffuse-adhering E . coli (DAEC) strains displayed no adherence to plastic but formed "dimples" on HeLa cells . Both EAggEC and DAEC adhered at lower levels to human small intestines (except M cells) than did EAF+ E . coli . In all cases of EAF+ E . coli, EAggEC, and DAEC, strains were found with atypical characteristics . The data demonstrate the unique adherence characteristics of EAF+ E . coli, EAggEC, and DAEC. Genes Dev, 1992 Dec, 6(12B), 2646 - 54 A novel DNA-binding protein with regulatory and protective roles in starved Escherichia coli; Almiron M et al.; A starvation-inducible DNA-binding protein was discovered as a result of the analysis of proteins synthesized in 3-day-old cultures of Escherichia coli . This 19-kD protein, designated Dps, is abundant in starved cells . In vitro, Dps forms extremely stable complexes with DNA, without apparent sequence specificity . When complexed with Dps, DNA is rendered DNase resistant . Mutant cells lacking Dps show dramatic changes in the pattern of proteins synthesized during starvation . The mutants also fail to develop starvation-induced resistance to hydrogen peroxide, an agent that can cause oxidative damage to DNA in vivo . These results have prompted us to postulate that Dps plays an important role both in gene expression and DNA protection during stationary phase . The existence of similar proteins, heretofore with no known function, in bacterial species distantly related to Escherichia coli suggests that Dps may define a novel class of widely conserved DNA-binding proteins. Genes Dev, 1992 Dec, 6(12B), 2569 - 79 The Drosophila RNA-binding protein RBP1 is localized to transcriptionally active sites of chromosomes and shows a functional similarity to human splicing factor ASF/SF2; Kim YJ et al.; An RNA-binding protein gene (rbp1) from Drosophila melanogaster, encoding an RNA recognition motif and an Arg-Ser rich (RS) domain, has been characterized . The predicted amino acid sequence of rbp1 is similar to those of the human splicing factor ASF/SF2, the Drosophila nuclear phosphoprotein SRp55, and the Drosophila puff-associated protein B52 . Northern and immunohistochemical analyses showed that rbp1 is expressed at all stages in all tissues and that the RBP1 protein is localized to the nucleus . Consistent with a role in mRNA metabolism, indirect immunofluorescence reveals that the RBP1 protein colocalizes with RNA polymerase II on larval salivary gland polytene chromosomes . RBP1 protein made in Escherichia coli was tested for splicing activity using human cell extracts in which ASF has been shown previously both to activate splicing and to affect the choice of splice sites in alternatively spliced pre-mRNAs . In these assays, RBP1 protein, like ASF, is capable of both activating splicing and switching splice site selection . However, in each case, clear differences in the behavior of the two proteins were detected, suggesting that they have related but not identical functions . The general nuclear expression pattern, colocalization on chromosomes with RNA polymerase II, the similarity to ASF/SF2, SRp55, and B52, along with the effect on alternative splicing shown in vitro, suggest that rbp1 is involved in the processing of precursor mRNAs. Zhonghua Wai Ke Za Zhi, 1992 Dec, 30(12), 710 - 2, 777 {Interleukin I activity and prostaglandin E2 content in peripheral blood monocytes in patients with colorectal cancer}; Luo YQ; Immunoregulatory function of peripheral blood monocytes was studied in 20 patients with colorectal cancer, by assaying interleukin 1 (IL-1) and prostaglandin E2 (PGE2) in the culture supernatant of lipopolysaccharide-stimulated monocytes . The results showed that IL-1 activity of the monocyte culture supernatant was decreased in the preoperative patients, compared with that of controls or postoperative groups; the PGE2 content of the culture supernatant of monocytes from the preoperative cases was increased, compared to normal controls or to the postoperative groups . Again, we found that there was significant negative correlation between IL-1 activity and the PGE2 content, with the activity of IL-1 the lowest and the content of PGE2 the highest in advanced colonic cancer patients. Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1992 Dec, 14(6), 441 - 6 {Construction of an EB virus based shuttle vector plasmid pZH32}; Zhou X; The shuttle vector can replicate in both bacteria and eukaryotic cells . We have constructed a shuttle vector pZH32 based on the EB virus . Three component parts of the plasmid come from pSV2gpt, pS189 and pMCi5 . An E . coli tyrosine suppressor tRNA gene, supF, was used as a mutagenic target in this plasmid, and a gpt gene was used as a selectable marker gene for transformed cells . This pZH32 based on EBV replicates autonomously in the nuclei of human cells . It has a low background mutation frequency . The plasmid can be used to examine the mutagenic mechanism of potential mutagens and carcinogens in mammalian cells. Zhonghua Bing Li Xue Za Zhi, 1992 Dec, 21(6), 343 - 5 {Immunohistochemical observation of angiotensin converting enzyme in acute lung injury}; Yu XD et al.; The morphological changes of acute lung injury of rabbits, particularly the ACE content in the vascular endothelium of lung were studied with immunohistochemical methods and Q-970 image analysor . The activity of ACE in serum collected from the arteries was also estimated . The results showed that during the process of acute lung injury, the lung vascular endothelium was severely damaged with an obvious decrease of ACE content and activity . It indicates that decrease of ACE content and activity may play an important role in the pathogenesis of acute lung injury and may also be the sensitive criteria in expressing damage of the vascular endothelium in the lungs. Biol Chem Hoppe Seyler, 1992 Dec, 373(12), 1223 - 5 Fast quantitative assay of sequence-specific endonuclease activity based on DNA sequencer technology; Glasner W et al.; A quantitative assay of the sequence-specific endonuclease activity of Vsr DNA mismatch endonuclease is described . The procedure rests on fluorescently labelled oligonucleotide substrates and an automated DNA sequencer to determine amounts of both educt and product of the reaction; thus each individual measurement is internally standardized . The assay achieves high sample throughput by parallel measurement of multiple samples . Because of its capacity to produce and process large sets of experimental data, the system is particularly well suited for the determination of reaction kinetics . The procedure lends itself to further simplification by implementing software additions for direct peak integration . Obviously, the principle of the assay can be extended to the study of other enzymes, such as restriction endonucleases or sequence-specific proteases. Vet Immunol Immunopathol, 1992 Dec, 35(1-2), 143 - 53 A recombinant feline immunodeficiency virus envelope fusion protein stimulates peripheral blood lymphocytes from naive cats to proliferate in vitro; Beatty JA et al.; A region of feline immunodeficiency virus (FIV)/Glasgow-8 external envelope glycoprotein (env) incorporating the third and fourth variable regions (V3/V4) was cloned, inserted into the pGEX vector and expressed in Escherichia coli to yield milligram quantities of the recombinant polypeptide as a fusion protein with glutathione S-transferase . The fusion protein V3/V4GST was used in lymphocyte proliferation assays, where it consistently caused peripheral blood lymphocytes from naive cats to proliferate in a dose-dependent manner . Other FIV fusion proteins produced under identical conditions (V5GST and p24GST) and glutathione S-transferase alone did not cause proliferation in this system . The monoclonal antibody vpg15, which has been shown to block infection of susceptible cells in vitro, did not decrease the response to V3/V4GST . Human peripheral blood lymphocytes did not proliferate in response to V3/V4GST. Vet Immunol Immunopathol, 1992 Dec, 35(1-2), 133 - 41 A recombinant-based feline immunodeficiency virus antibody enzyme-linked immunosorbent assay; Mermer B et al.; We have developed an antibody detection enzyme-linked immunosorbent assay (ELISA) for the identification of animals infected by feline immunodeficiency virus (FIV) . The ELISA solid-phase antigen consists of recombinant FIV gag proteins expressed in bacteria . The proteins are purified from bacterial lysates as insoluble inclusion bodies . In the case of bacterially expressed p24gag, it is shown that all of the linear, sequential epitopes presented by viral p24 during infection are retained . Purified preparations can be substituted for solid-phase whole virus in the IDEXX PetChektm immunoassay . The antibody ELISA duplicates the sensitivity and specificity of the whole virus based PetChek plate assay. Mol Biol Cell, 1992 Dec, 3(12), 1373 - 88 Chromosome condensation caused by loss of RCC1 function requires the cdc25C protein that is located in the cytoplasm; Seki T et al.; We cloned the hamster cdc25C cDNA by using the human cdc25C cDNA as a probe and prepared an antibody to Escherichia coli-produced hamster cdc25C protein that is specific to the human cdc25C protein . The microinjected antibody inhibited a chromosome condensation induced by tsBN2 mutation, indicating that the cdc25C protein is required for an activation of p34cdc2 kinase caused by loss of RCC1 function . The hamster cdc25C protein located in the cytoplasm, prominently in a periphery of the nuclei of cells arrested with hydroxyurea, and seemed to move into the nuclei by loss of RCC1 function . Also, we found a molecular shift of the cdc25C protein in cells showing premature chromosome condensation (PCC), in addition to normal mitotic cells . This molecular-shift appeared depending on an activation of p34cdc2 kinase. Mol Endocrinol, 1992 Dec, 6(12), 2079 - 89 Activating transcription factor-2 DNA-binding activity is stimulated by phosphorylation catalyzed by p42 and p54 microtubule-associated protein kinases; Abdel-Hafiz HA et al.; Recent studies have detailed the ability of activating transcription factor-2 (ATF-2) to mediate adenoviral E1a stimulation of gene expression; however, an endogenous regulator for the transcriptional activity of this protein has not been described . To characterize the regulation of ATF-2 activity, we have expressed full-length and truncated peptides corresponding to various regions of the ATF-2 protein in bacteria and the baculovirus insect cell system . Bacterially expressed truncated (350-505) but not full-length ATF-2, was able to bind a consensus cAMP response element-containing oligonucleotide, suggesting the N-terminal moiety may serve as a negative regulator of DNA-binding activity . In contrast, the full-length ATF-2 protein expressed in Spodoptera frugiperda (Sf9) cells using a recombinant baculovirus was fully competent to bind DNA . Protein phosphatase 2A reversed the DNA-binding activity by dephosphorylating the ATF-2 polypeptide . Microtubule-associated protein kinase catalyzed the phosphorylation and stimulated the DNA-binding activity of bacterially expressed full-length ATF-2 . Phosphopeptide mapping of phosphorylated ATF-2 proteins identified a single peptide in the N-terminal moiety of ATF-2 phosphorylated by p42 or p54 microtubule-associated protein kinase . Therefore, we propose that phosphorylation of this regulatory site is sufficient to induce an allosteric structural change in the ATF-2 protein, which allows dimerization and subsequent DNA binding. Protein Expr Purif, 1992 Dec, 3(6), 508 - 11 Isolation and characterization of pro-barley lectin expressed in Escherichia coli; Schroeder MR et al.; Lectins are a class of proteins with specific carbohydrate-binding properties found in a wide variety of plants and animals . Gramineae lectins are presumably defense-related proteins in plants that exert their effect by binding to N-acetylglucosamine . Barley lectin is a vacuolar protein synthesized with an amino-terminal signal sequence for entering the secretory pathway and a carboxyl-terminal propeptide necessary for proper targeting to the vacuole . To analyze the three-dimensional structure of barley lectin with the carboxyl-terminal extension and to investigate whether the conversion of the prolectin into the mature molecule leads to a conformational change, the precursor and the mature forms of barley lectin were expressed in Escherichia coli . Both proteins accumulated in denatured form in inclusion bodies were solubilized in 8 M urea and renatured in a redox buffer system . Active pro- and mature barley lectins were purified to homogeneity by affinity chromatography. Jpn J Cancer Res, 1992 Dec, 83(12), 1244 - 7 Retrovirus-mediated gene transfer targeted to malignant glioma cells in murine brain; Yamada M et al.; A murine model for meningeal metastasis of malignant glioma was developed to study selective gene transfer into tumor cells and to establish a reliable means of determining the rate of tumor cell infection . A murine ecotropic retroviral vector was created in which the Escherichia coli beta-galactosidase gene served as a marker for gene expression from the integrated retrovirus . This retrovirus exhibited a high rate of infectivity in RSV-M mouse glioma cells in vitro . The recombinant retrovirus was injected directly into the cisterna magna of the mice . Staining of beta-galactosidase showed that the rate of gene integration was high in the disseminated glioma cells . These results suggest the possibility of retrovirus-mediated gene therapy for meningeal dissemination of malignant glioma. Appl Environ Microbiol, 1992 Dec, 58(12), 3826 - 9 The Entner-Doudoroff pathway in Escherichia coli is induced for oxidative glucose metabolism via pyrroloquinoline quinone-dependent glucose dehydrogenase; Fliege R et al.; The Entner-Doudoroff pathway was shown to be induced for oxidative glucose metabolism when Escherichia coli was provided with the periplasmic glucose dehydrogenase cofactor pyrroloquinoline quinone (PQQ) . Induction of the Entner-Doudoroff pathway by glucose plus PQQ was established both genetically and biochemically and was shown to occur in glucose transport mutants, as well as in wild-type E . coli . These data complete the body of evidence that proves the existence of a pathway for oxidative glucose metabolism in E . coli . PQQ-dependent oxidative glucose metabolism provides a metabolic branch point in the periplasm; the choices are either oxidation to gluconate followed by induction of the Entner-Doudoroff pathway or phosphotransferase-mediated transport . The oxidative glucose pathway might be important for survival of enteric bacteria in aerobic, low-phosphate, aquatic environments. Biochem J, 1992 Dec 1, 288 ( Pt 2), 649 - 55 Spectroscopic studies on an oxygen-binding haemoglobin-like flavohaemoprotein from Escherichia coli; Ioannidis N et al.; The Escherichia coli haemoglobin-like flavohaemoprotein (Hmp) has been purified to near homogeneity using two chromatographic steps . The prosthetic groups are identified as FAD and protohaem IX . SDS/PAGE has indicated a molecular mass of 44 kDa for the monomeric protein consistent with the amino-acid sequence deduced from the hmp+ gene . The protein, as isolated, is in the Fe(III) state, exhibiting absorbance maxima at 403.5, 540 (shoulder) and 627 nm . The ferrous and carbonmonoxyferrous states resemble those of haemoglobin, showing maxima at 431.5 and 558 nm, and 421, 542 and 566 nm respectively . Upon aerobic addition of NAD(P)H, the ferric state is reduced to the oxygenated Fe(II) state, characterized by maxima at 413, 544 and 580 nm . This oxy form is not stable and slowly decays to the ferric state . Addition of dithionite and nitrite to the ferric protein results in the formation of a nitrosyl complex, whose e.p.r . characteristics indicate that the b-type haem is attached to the protein through a nitrogenous ligand, probably originating from a histidine residue. Biochem J, 1992 Dec 1, 288 ( Pt 2), 503 - 9 Domains of the catalytically self-sufficient cytochrome P-450 BM-3 . Genetic construction, overexpression, purification and spectroscopic characterization; Miles JS et al.; 1 . The gene CYP102 encoding cytochrome P-450 BM-3 and subgenes encoding the cytochrome P-450 and cytochrome P-450 reductase domains have been cloned in Escherichia coli . 2 . The protein products of these genes have been overexpressed and purified to homogeneity . 3 . The cytochrome P-450 domain is purified in the ferric low-spin state, but is readily converted into the high-spin state by addition of the substrate palmitate (Ks = 1 microM) . The cytochrome P-450 reductase domain readily reduces cytochrome c . Mixing the two domains reconstitutes only about one-thousandth of the fatty acid hydroxylase activity associated with the intact cytochrome P-450 BM-3 . 4 . The X-band e.p.r . spectra of both the cytochrome P-450 domain and intact cytochrome P-450 BM-3 give g-values indicating low-spin ferric haem . The spectra are virtually identical with those of the equivalent form of cytochrome P-450 cam indicating that the haem ligation in cytochrome P-450 BM-3 is identical with that of cytochrome P-450 cam . 5 . Resonance Raman spectra of the substrate-free and substrate-bound forms of the cytochrome P-450 domain are given . Spectral differences in comparison with cytochrome P-450 cam may reflect subtle electronic differences between the respective haem environments. J Bacteriol, 1992 Dec, 174(24), 8094 - 101 A mer-lux transcriptional fusion for real-time examination of in vivo gene expression kinetics and promoter response to altered superhelicity; Condee CW et al.; We constructed mercury resistance operon-luciferase (mer-lux) transcriptional fusion plasmids to evaluate in vivo gene expression rates of the mer structural gene promoter (PTPCAD) of transposon Tn21 . In vivo gene expression kinetics corresponded well with those previously determined in vitro, yielding an apparent K0.5 for Hg(II)-stimulated induction by MerR of 9.3 x 10(-8) M with the same ultrasensitive threshold effect seen in vitro . We also used the mer-lux fusions to elucidate subtle variations in promoter activity brought about by altered superhelicity . Binding of inducer {Hg(II)} to the transcriptional activator MerR is known to result in DNA distortion and transcriptional activation of the mer operon; it has recently been demonstrated that this distortion is a consequence of MerR-Hg(II)-induced local DNA unwinding to facilitate RNA polymerase open complex formation at PTPCAD . Since negative supercoiling results in DNA unwinding similar to this MerR activation, we hypothesized that a global increase in plasmid supercoiling would facilitate MerR-mediated activation and compromise MerR-mediated repression, while removal of plasmid supercoils would compromise MerR's ability to induce transcription and facilitate its ability to repress transcription . Indeed, we found that increased negative supercoiling results in increased gene expression rates and decreased supercoiling results in reduced gene expression rates for the induced, repressed, and derepressed conditions of PTPCAD . Thus, luciferase transcriptional fusions can detect subtle variations in initial rates of gene expression in a real-time, nondestructive assay. J Bacteriol, 1992 Dec, 174(24), 8030 - 5 Escherichia coli cyclic AMP receptor protein mutants provide evidence for ligand contacts important in activation; Moore J et al.; The three-dimensional model of the Escherichia coli cyclic AMP (cAMP) receptor protein (CRP) shows that several amino acids are involved as chemical contacts for binding cAMP . We have constructed and characterized mutants at four of these positions, E72, R82, S83, and R123 . The mutations were made in wild-type crp as well as a cAMP-independent crp, crp* . The activities of the mutant proteins were characterized in vivo for their ability to activate the lac operon . These results provide genetic evidence to support that E72 and R82 are essential and S83 and R123 are important in the activation of CRP by cAMP. Eur J Biochem, 1992 Dec 1, 210(2), 455 - 60 Purification and characterization of a mitochondrial endonuclease from Drosophila melanogaster embryos; Harosh I et al.; A mitochondrial endonuclease from Drosophila melanogaster embryos was purified to near homogeneity by successive fractionation with DEAE-cellulose and heparin--avidgel-F, followed by FPLC chromatography on mono S, Superose 12 and a second mono S column . This enzyme digests double-stranded DNA more efficiently than heat-denatured DNA . The endonuclease activity has a molecular mass of 44 kDa, as determined under native conditions using a gel-filtration Superose 12 column . The prominent peptide detected by SDS/polyacrylamide gel electrophoresis likewise has a molecular mass of 44 kDa, suggesting a monomeric protein . The enzyme has an absolute requirement for divalent cations, preferring Mg2+ over Mn2+ . No activity could be detected when these cations were replaced by Ca2+ or Zn2+ . The pH optimum for this enzyme activity is 6.5-7.4 and its isoelectric point is 4.9 . Both single-strand and double-strand breaks are introduced simultaneously into a supercoiled substrate in the presence of MgCl2 or MnCl2 . Endonuclease-treated DNA serves as a substrate for DNA polymerase I from Escherichia coli, suggesting that 3'-OH termini are generated during cleavage . The enzyme is free from any detectable DNA exonuclease activity but not from RNase activity . Partial inhibition by antibodies raised against mitochondrial endonucleases derived from bovine heart and Saccharomyces cerevisiae have revealed a potential structural homology between these nucleases. Blood, 1992 Dec 1, 80(11), 2755 - 64 Tumor necrosis factor and interleukin-1 induce expression of the verocytotoxin receptor globotriaosylceramide on human endothelial cells: implications for the pathogenesis of the hemolytic uremic syndrome; van de Kar NC et al.; The epidemic form of the hemolytic uremic syndrome (HUS), beginning with an acute gastroenteritis, has been associated with a verocytotoxin-producing Escherichia coli infection . The endothelial cell is believed to play an important role in the pathogenesis of HUS . Endothelial cell damage by verocytotoxin-1 (VT-1) in vitro is potentiated by the additional exposure of inflammatory mediators, such as tumor necrosis factor-alpha (TNF-alpha) . Preincubation of human umbilical vein endothelial cells (HUVEC) with TNF-alpha resulted in a 10- to 100-fold increase of specific binding sites for 125I-VT-1 . Furthermore, interleukin-1 (IL-1), lymphotoxin (TNF-beta), and lipopolysaccharide (LPS) also markedly increase VT-1 binding . Several hours' exposure to TNF-alpha was enough to enhance the number of VT-1 receptors on the endothelial cells for 2 days . The TNF-alpha-induced increase in VT-1 binding could be inhibited by simultaneous addition of the protein synthesis inhibitor cycloheximide . Glycolipid extracts of TNF-alpha-treated cells tested on thin-layer chromatography demonstrated an increase of globotriaosylceramide (GbOse3cer), a functional receptor for VT-1, which suggests that preincubation of human endothelial cells with TNF-alpha leads to an increase in GbOse3cer synthesis in these cells . We conclude from this study that TNF-alpha and IL-1 induce one (or more) enzyme(s) that is (are) rate-limiting in the synthesis of the glycolipid VT-1 receptor, GbOse3cer . These in vitro studies suggest that, in addition to VT-1, inflammatory mediators play an important role in the pathogenesis of HUS. Virology, 1992 Dec, 191(2), 783 - 92 Identification and functional analysis of the fowlpox virus homolog of the vaccinia virus p37K major envelope antigen gene; Calvert JG et al.; A fowlpox virus (FPV) gene with homology to the vaccinia virus p37K major envelope antigen gene was identified and sequenced . The predicted product has a molecular weight of 43,018 Da (p43K) . The FPV p43K gene has 37.5% identity with its vaccinia counterpart and higher homology with a molluscum contagiosum virus gene (42.6% identity) . Based on upstream sequences, p43K appears to be regulated as a late gene . Recombinant FPV were generated in which a large portion of p43K was replaced by the Escherichia coli lacZ gene . These recombinants failed to produce visible plaques under standard conditions . After prolonged incubation the microplaques developed into small macroscopic plaques . Plaques were purified on the basis of lacZ expression . Single-cycle growth curves comparing the p43K-deleted recombinant (designated fJd43Z) with parental FPV showed that the two viruses produce identical amounts of intracellular virions, but that fJd43Z released 20-fold fewer infectious particles into the medium . CsCl gradient centrifugation of {3H}thymidine-labeled virus was employed to examine differences in the production of physical particles . The two viruses produced equivalent levels of intracellular virions, but fJd43Z failed to produce detectable levels of released particles . FPV p43K is therefore involved in the release of virions from infected cells. J Cell Biol, 1992 Dec, 119(5), 1047 - 61 Antibodies against 70-kD heat shock cognate protein inhibit mediated nuclear import of karyophilic proteins; Imamoto N et al.; Previously, we found that anti-DDDED antibodies strongly inhibited in vivo nuclear transport of nuclear proteins and that these antibodies recognized a protein of 69 kD (p69) from rat liver nuclear envelopes that showed specific binding activities to the nuclear location sequences (NLSs) of nucleoplasmin and SV-40 large T-antigen . Here we identified this protein as the 70-kD heat shock cognate protein (hsc70) based on its mass, isoelectric point, cellular localization, and partial amino acid sequences . Competition studies indicated that the recombinant hsc70 expressed in Escherichia coli binds to transport competent SV-40 T-antigen NLS more strongly than to the point mutated transport incompetent mutant NLS . To investigate the possible involvement of hsc70 in nuclear transport, we examined the effect of anti-hsc70 rabbit antibodies on the nuclear accumulation of karyophilic proteins . When injected into the cytoplasm of tissue culture cells, anti-hsc70 strongly inhibited the nuclear import of nucleoplasmin, SV-40 T-antigen NLS bearing BSA and histone H1 . In contrast, anti-hsc70 IgG did not prevent the diffusion of lysozyme or 17.4-kD FITC-dextran into the nuclei . After injection of these antibodies, cells continued RNA synthesis and were viable . These results indicate that hsc70 interacts with NLS-containing proteins in the cytoplasm before their nuclear import. J Bacteriol, 1992 Dec, 174(23), 7716 - 28 FtsL, an essential cytoplasmic membrane protein involved in cell division in Escherichia coli; Guzman LM et al.; We have identified a gene involved in bacterial cell division, located immediately upstream of the ftsI gene in the min 2 region of the Escherichia coli chromosome . This gene, which we named ftsL, was detected through characterization of TnphoA insertions in a plasmid containing this chromosomal region . TnphoA topological analysis and fractionation of alkaline phosphatase fusion proteins indicated that the ftsL gene product is a 13.6-kDa cytoplasmic membrane protein with a cytoplasmic amino terminus, a single membrane-spanning segment, and a periplasmic carboxy terminus . The ftsL gene is essential for cell growth and division . A null mutation in ftsL resulted in inhibition of cell division, formation of long, nonseptate filaments, ultimate cessation of growth, and lysis . Under certain growth conditions, depletion of FtsL or expression of the largest ftsL-phoA fusion produced a variety of cell morphologies, including Y-shaped bacteria, indicating a possible general weakening of the cell wall . The FtsL protein is estimated to be present at about 20 to 40 copies per cell . The periplasmic domain of the protein displays a sequence with features characteristic of leucine zippers, which are involved in protein dimerization. J Bacteriol, 1992 Dec, 174(23), 7689 - 96 Purification and characterization of a mutant DnaB protein specifically defective in ATP hydrolysis; Shrimankar P et al.; The dnaB gene of Escherichia coli encodes an essential DNA replication enzyme . Fueled by the energy derived from the hydrolysis of ATP to ADP+P(i), this enzyme unwinds double-stranded DNA in advance of the DNA polymerase . While doing so, it intermittently stimulates primase to synthesize an RNA primer for an Okazaki fragment . To better understand the structural basis of these and other aspects of DnaB function, we have initiated a study of mutant DnaB proteins . Here, we report the purification and characterization of a mutant DnaB protein (RC231) containing cysteine in place of arginine at residue 231 . The mutant protein attains a stable, properly folded structure that allows association of six promoters to form a hexamer, as is also true for wild-type DnaB . Further, the mutant protein interacts with ATP, the nonhydrolyzable ATP analog adenosine-5'-O-(3-thiotriphosphate) (ATP gamma S), ADP, and poly(dT), and it stimulates primase action . It is, however, profoundly deficient in ATP hydrolysis, helicase activity, and replication activity at the chromosomal origin of replication . In addition, while general priming reactions with wild-type DnaB and ATP elicited the synthesis of short primers, reactions with DnaB and ATP gamma S or with RC231 and either ATP or ATP gamma S stimulated the synthesis of significantly longer primers . On the basis of these observations, we suggest that primase interacts directly with DnaB throughout primer synthesis during general priming, until dissociation of DnaB from DNA or ATP hydrolysis by DnaB disrupts the interaction and leads to primer termination. J Bacteriol, 1992 Dec, 174(23), 7629 - 34 Autoregulation of the stability operon of IncFII plasmid NR1; Tabuchi A et al.; The stb locus of IncFII plasmid NR1, which mediates stable inheritance of the plasmid, is composed of an essential cis-acting DNA site located upstream from two tandem genes that encode essential stability proteins . The two tandem genes, stbA and stbB, are transcribed as an operon from promoter PAB . Using PAB-lacZ gene fusions, it was found that the stb operon is autoregulated . A low-copy-number stb+ plasmid introduced into the same cell with the PAB-lacZ fusion plasmid repressed beta-galactosidase activity about 5-fold, whereas a high-copy-number stb+ plasmid repressed beta-galactosidase about 15-fold . The details of autoregulation were analyzed by varying the concentrations of StbA and StbB to examine their effects on expression from the PAB-lacZ fusion plasmid . StbB protein by itself had autorepressor activity . Although StbA protein by itself had no detectable repressor activity, plasmids that encoded both stbA and stbB repressed more effectively than did those that encoded stbB alone . Plasmids with a mutation in stbA had reduced repressor activity . One mutation in stbB that inactivated the stability function also reduced, but did not eliminate, repressor activity . Repressor activity of the mutant StbB protein was effectively enhanced by stbA . These results indicate that StbB serves two functions, one for stable inheritance and one for autoregulation of the stb operon, both of which may be influenced by StbA protein. J Bacteriol, 1992 Dec, 174(23), 7606 - 12 Regulation of virulence-associated plasmid genes in enteroinvasive Escherichia coli; Dagberg B et al.; The transposon TnphoA was used for construction of gene fusions and for studies of gene regulation in an enteroinvasive strain of Escherichia coli . Several plasmid-encoded virulence genes (e.g., the ipaB and virG operons) of such enteroinvasive strains are subject to coordinated thermoregulation involving both operon-specific (the VirB and VirF activators) and global regulators . The nucleoid-associated E . coli protein H-NS was shown to be a negative regulator as judged by studies using H-NS gene deletion mutations and by increasing the level of H-NS protein in the cells . An increased gene dosage of H-NS led to enhanced repression of the ipa and virG operons, particularly at low (30 degrees C) growth temperature . The cyclic AMP receptor protein complex, which is another global transcriptional regulator in E . coli, was not required for the regulation of ipa and virG expression . The virG operon was expressed in an activator-independent manner in cells lacking H-NS protein . We suggest that the role of the VirF activator is to counteract the silencing effect of H-NS. J Bacteriol, 1992 Dec, 174(23), 7527 - 32 Stimulation of glucose catabolism in Escherichia coli by a potential futile cycle; Patnaik R et al.; Fifteen-fold overexpression of phosphoenolpyruvate synthase (Pps) (EC 2.7.9.2) in Escherichia coli stimulated oxygen consumption in glucose minimal medium . A further increase in Pps overexpression to 30-fold stimulated glucose consumption by approximately 2-fold and resulted in an increased excretion of pyruvate and acetate . Insertion of two codons at the PvuII site in the pps gene abolished the enzymatic activity and eliminated the above-described effects . Both the active and the inactive proteins were detected at the predicted molecular weight by polyacrylamide gel electrophoresis . Therefore, the observed physiological changes were due to the activity of Pps . The higher specific rates of consumption of oxygen and glucose indicate a potential futile cycle between phosphoenolpyruvate (PEP) and pyruvate . A model for the stimulation of glucose uptake is presented; it involves an increased PEP/pyruvate ratio caused by the overexpressed Pps activity, leading to a stimulation of the PEP:sugar phosphotransferase system. J Bacteriol, 1992 Dec, 174(23), 7509 - 16 Levels of epsilon, an essential replication subunit of Escherichia coli DNA polymerase III, are controlled by heat shock proteins; Foster PL et al.; In Escherichia coli, epsilon, the proofreading subunit of DNA polymerase III, is encoded by dnaQ . A random search for mutants that affect the expression of dnaQ revealed that mutations in the genes encoding the heat shock proteins (HSPs) DnaK, DnaJ, and GrpE result in dramatic decreases in the cellular levels of epsilon . dnaQ is arranged in an overlapping divergent transcriptional unit with rnhA, which encodes RNase H1, and mutations in the same HSPs also reduced the apparent levels of RNase H1 . The HSPs had only small effects on transcriptional fusions to these genes; thus, it is likely that they operate primarily at the protein level . Since survival and mutagenesis after DNA damage are affected by epsilon and RNase H1, HSPs may have a broad influence on various aspects of DNA replication and repair. Biochemistry, 1992 Dec 1, 31(47), 11785 - 92 Study of calmodulin binding to the alternatively spliced C-terminal domain of the plasma membrane Ca2+ pump; Kessler F et al.; The C-terminal regions of the four human plasma membrane Ca2+ pump isoforms 1a-d generated from alternatively spliced RNA have been expressed in Escherichia coli, and the recombinant proteins have been purified to a very high degree . The C-termini of isoforms 1a, 1c, and 1d contain an insert encoded by an alternatively spliced exon which is homologous to the calmodulin binding domain of isoform 1b . In isoforms 1c and 1d (29 and 38 amino acid insertions, respectively), subdomain A of the original calmodulin binding site of isoform 1b is followed by the spliced-in domain, which is then followed by subdomain B of the original calmodulin binding site . The positive charges of histidine residues at positions 27, 28, and 38 of the alternatively spliced sequence are likely to be responsible for the observed pH-dependent calmodulin binding to the novel "duplicated" binding site . The affinity of calmodulin for the C-terminal domains of isoforms 1a, 1c, and 1d, which contain the histidine-rich inserts, is much higher at pH 5.9 than at pH 7.2 . A synthetic peptide (I31) containing 31 amino acids of the alternatively spliced sequence (from residue 9 to 40) also binds calmodulin with strong pH dependency . Alternative splicing in the C-terminal domain is proposed to confer pH dependence to the regulation of the activity of Ca2+ pump isoforms. Biochemistry, 1992 Dec 1, 31(47), 11698 - 706 The cationic locus on the recombinant kringle 2 domain of tissue-type plasminogen activator that stabilizes its interaction with omega-amino acids; De Serrano VS et al.; The properties of the cationic locus within the recombinant (r) kringle 2 domain (residues 180-261) of tissue-type plasminogen activator ({K2tPA}) that are responsible for stabilization of its interaction with the carboxylate moiety of omega-amino acid ligands have been assessed by determination of the binding constants of several such ligands to a variety of r-{K2tPA} mutants obtained by oligonucleotide-directed mutagenesis . We have generated, expressed in Escherichia coli, and purified alanyl mutants of individual histidyl,lysyl, and arginyl residues of r-{K2tPA} and determined the dissociation constants of several omega-amino acids, viz., 6-aminohexanoic acid (6-AHxA), 7-aminoheptanoic acid (7-AHpA), L-lysine (L-Lys), and trans-(aminomethyl)cyclohexane-1-carboxylic acid (AMCHA), to each of the r-{K2tPA} variants . We find that K33 plays the most significant role as a cationic partner of the complementary carboxylate group of these ligands . When K33 is altered to a variety of other amino acids, the K33R mutant best stabilizes binding of all of these ligands . However, the r-K33L and r-K33F variants selectively interact with 7-AHpA almost as strongly (ca . 2-fold reduction in binding strength) as wild-type r-{K2tPA} . Increased polarity (K33Q) or a negative charge (K33E) at this sequence position significantly destabilizes binding of omega-amino acids to the muteins . We also found that the r-K33E mutant and, to a lesser extent, the r-K33Q variant selectively interact with a new ligand, 1,6-diaminohexane . These observations show that the omega-amino acid binding site of wtr-{K2tPA} could be redesigned to provide a new binding specificity.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1992 Dec 1, 31(47), 11684 - 8 Characterization of the phosphorylated enzyme intermediate formed in the adenosine 5'-phosphosulfate kinase reaction; Satishchandran C et al.; Adenosine 5'-phosphosulfate (APS) kinase (ATP:APS 3'-phosphotransferase) catalyzes the ultimate step in the biosynthesis of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), the primary biological sulfuryl donor . APS kinase from Escherichia coli is phosphorylated upon incubation with ATP, yielding a protein that can complete the overall reaction through phosphorylation of APS . Rapid-quench kinetic experiments show that, in the absence of APS, ATP phosphorylates the enzyme with a rate constant of 46 s-1, which is equivalent to the Vmax for the overall APS kinase reaction . Similar pre-steady-state kinetic measurements show that the rate constant for transfer of the phosphoryl group from E-P to APS is 91 s-1 . Thus, the phosphorylated enzyme is kinetically competent to be on the reaction path . In order to elucidate which amino acid residue is phosphorylated, and thus to define the active site region of APS kinase, we have determined the complete sequence of cysC, the structural gene for this enzyme in E . coli . The coding region contains 603 nucleotides and encodes a protein of 22,321 Da . Near the amino terminus is the sequence 35GLSGSGKS, which exemplifies a motif known to interact with the beta-phosphoryl group of purine nucleotides . The residue that is phosphorylated upon incubation with ATP has been identified as serine-109 on the basis of the amino acid composition of a radiolabeled peptide purified from a proteolytic digest of 32P-labeled enzyme . We have identified a sequence beginning at residue 147 which may reflect a PAPS binding site . This sequence was identified in the carboxy terminal region of 10 reported sequences of proteins of PAPS metabolism. J Virol, 1992 Dec, 66(12), 7511 - 6 An autophosphorylating but not transphosphorylating activity is associated with the unique N terminus of the herpes simplex virus type 1 ribonucleotide reductase large subunit; Conner J et al.; We report on a protein kinase function encoded by the unique N terminus of the herpes simplex virus type 1 (HSV-1) ribonucleotide reductase large subunit (R1) . R1 expressed in Escherichia coli exhibited autophosphorylation activity in a reaction which depended on the presence of the unique N terminus . When the N terminus was separately expressed in E . coli and partially purified, a similar autophosphorylation reaction was observed . Importantly, transphosphorylation of histones and of proteins in HSV-1-infected cell extracts was also observed with purified R1 and with truncated R1 mutants in which most of the N terminus was deleted . Ion-exchange chromatography was used to separate the autophosphorylating activity of the N terminus from the transphosphorylating activity of an E . coli contaminant protein kinase . We propose a putative function for this activity of the HSV-1 R1 N terminus during the immediate-early phase of virus replication. J Virol, 1992 Dec, 66(12), 7481 - 9 Purification and characterization of poliovirus polypeptide 3CD, a proteinase and a precursor for RNA polymerase; Harris KS et al.; A cDNA clone encoding the 3CD proteinase (3CDpro) of poliovirus type 2 (Sabin), the precursor to proteinase 3Cpro and RNA polymerase 3Dpol, was expressed in bacteria by using a T7 expression system . Site-specific mutagenesis of the 3C/3D cleavage site was performed to generate active proteolytic precursors impaired in their ability to process themselves to 3Cpro and 3Dpol . Of these mutations, the exchange of the Thr residue at the P4 position of the 3C/3D cleavage site for a Lys residue (3CDpro T181K) resulted in a mutant polypeptide exhibiting the smallest amount of autoprocessing . This mutant was purified to 86% homogeneity and used for subsequent proteolytic studies . Purified 3CDproM (M designates the cleavage site mutant 3CDpro T181K) was capable of cleaving the P1 capsid precursor, a peptide representing the 2BC cleavage site, and the 2BC precursor polypeptide . Purified 3CDproM demonstrated the same detergent sensitivity in processing experiments with the capsid precursor as was observed by using P1 and crude extracts of poliovirus-infected HeLa cell lysates . Purified 3CDproM did not have any detectable RNA polymerase activity, whereas 3Dpol, separated from 3CDproM by gel filtration in the last step of purification, did . We conclude that 3CDproM can process both structural and nonstructural precursors of the poliovirus polyprotein and that it is active against a synthetic peptide substrate . Moreover, cleavage of 3CD to 3Dpol is needed to activate the 3D RNA polymerase. J Virol, 1992 Dec, 66(12), 7362 - 7 Herpes simplex virus type 1 protease expressed in Escherichia coli exhibits autoprocessing and specific cleavage of the ICP35 assembly protein; Deckman IC et al.; The UL26 gene of herpes simplex virus type 1 (HSV-1) encodes a protease which is responsible for the C-terminal cleavage of the nucleocapsid-associated proteins, ICP35 c and d, to their posttranslationally modified counterparts, ICP35 e and f . To further characterize the HSV-1 protease, the UL26 gene product was expressed in Escherichia coli . The expressed protease underwent autoproteolytic processing at two independent sites . The first site is shared with ICP35 and results in removal of 25 amino acids from the C terminus of the protease . The second unique site gives rise to protein species consistent with deletion of a 28-kDa fragment at the N terminus . A mutant protease, which showed no activity in a mammalian cell cotransfection assay (F . Liu and B . Roizman, Proc . Natl . Acad . Sci . USA 89:2076-2080, 1992), failed to exhibit autoproteolytic processing at either site when expressed in bacteria . The inactive mutant was able to serve as a substrate in a trans assay in which the substrate and protease were coexpressed in bacteria . This experiment demonstrated that the unique N-terminal processing was mediated exclusively by the HSV-1 protease . ICP35 c,d also served as a substrate in this assay and was correctly processed by HSV-1 protease in E . coli . This trans-cleavage assay will aid in the characterization of HSV-1 protease and assist in investigation of the role of proteolytic processing in the virus. J Virol, 1992 Dec, 66(12), 7245 - 52 Analytical study of avian reticuloendotheliosis virus dimeric RNA generated in vivo and in vitro; Darlix JL et al.; The retroviral genome consists of two identical RNA molecules associated at their 5' ends by a stable structure called the dimer linkage structure . The dimer linkage structure, while maintaining the dimer state of the retroviral genome, might also be involved in packaging and reverse transcription, as well as recombination during proviral DNA synthesis . To study the dimer structure of the retroviral genome and the mechanism of dimerization, we analyzed features of the dimeric genome of reticuloendotheliosis virus (REV) type A and identified elements required for its dimerization . Here we report that the REV dimeric genome extracted from virions and infected cells, as well as that synthesized in vitro, is more resistant to heat denaturation than avian sarcoma and leukemia virus, murine leukemia virus, or human immunodeficiency virus type 1 dimeric RNA . The minimal domain required to form a stable REV RNA dimer in vitro was found to map between positions 268 and 452 (KpnI and SalI sites), thus corresponding to the E encapsidation sequence (J . E . Embretson and H . M . Temin, J . Virol . 61:2675-2683, 1987) . In addition, both the 5' and 3' halves of E are necessary in cis for RNA dimerization and the extent of RNA dimerization is influenced by viral sequences flanking E . Rapid and efficient dimerization of REV RNA containing gag sequences in addition to the E sequences and annealing of replication primer tRNA(Pro) to the primer-binding site necessitate the nucleocapsid protein. J Virol, 1992 Dec, 66(12), 7040 - 8 The 5' end of the equine arteritis virus replicase gene encodes a papainlike cysteine protease; Snijder EJ et al.; The presence of a papainlike cysteine protease (PCP) domain in the N-terminal region of the equine arteritis virus (EAV) replicase, which had been postulated on the basis of limited sequence similarities with cellular and viral thiol proteases, was confirmed by in vitro translation and mutagenesis studies . The EAV protease was found to direct an autoproteolytic cleavage at its C terminus which leads to the production of an approximately 30-kDa N-terminal replicase product (nsp1) containing the PCP domain . Amino acid residues Cys-164 and His-230 of the EAV replicase polyprotein were identified as the most likely candidates for the role of PCP catalytic residues . By means of N-terminal sequence analysis of a PCP cleavage product, derived from a bacterial expression system, it was shown that cleavage occurs between Gly-260 and Gly-261 . No evidence for PCP-directed cleavages at other positions in the EAV replicase was obtained . In cotranslational and posttranslational trans-cleavage assays, neither EAV nsp1 nor its precursor was able to process the PCP cleavage site in trans. J Virol, 1992 Dec, 66(12), 6868 - 77 Immunological characterization of the gag gene products of bovine immunodeficiency virus; Battles JK et al.; The bovine immunodeficiency virus (BIV) gag gene encodes a 53-kDa precursor (Pr53gag) that is involved in virus particle assembly and is further processed into the putative matrix (MA), capsid (CA), and nucleocapsid (NC) functional domains in the mature virus . Gag determinants are also found in the Gag-Pol polyprotein precursor . To immunologically identify the major precursors and processed products of the BIV gag gene, monospecific rabbit sera to recombinant BIV MA protein and Pr53gag and peptides predicted to correspond to the CA and NC proteins and the MA-CA cleavage site were developed and used in immunoprecipitations and immunoblots of BIV antigens . Monospecific antisera to native and recombinant human immunodeficiency virus type 1 proteins were also used to identify analogous BIV Gag proteins and to determine whether cross-reactive epitopes were present in the BIV Gag precursors or processed products . The BIV MA, CA, and NC Gag proteins were identified as p16, p26, and p13, respectively . In addition to BIV Pr53gag, the major Gag precursor, two other Gag-related precursors of 170 and 49 kDa were identified that have been designated pPr170gag-pol and Pr49gag, respectively; pPr170gag-pol is the Gag-Pol polyprotein precursor, and Pr49gag is the transframe Gag precursor present in pPr170gag-pol . Several alternative Gag cleavage products were also observed, including p23, which contains CA and NC determinants, and p10, which contains a peptide sequence conserved in the CA proteins of most lentiviruses . The monospecific antisera to human immunodeficiency virus type 1 CA (p24) and NC (p7) proteins showed cross-reactivity to and aided in the identification of analogous BIV proteins . Based on the present data, a scheme for the processing of BIV Gag precursors is proposed. Protein Sci, 1992 Dec, 1(12), 1661 - 5 A kinetic model for binding protein-mediated arabinose transport; Kehres DG; A kinetic model is presented based on the simplest plausible mechanism for bacterial binding protein-dependent transport . The transport phenotypes of the 18 variant arabinose-binding proteins analyzed by Kehres and Hogg (1992, Protein Sci . 1, 1652-1660) (wild type and 17 mutants) are interpreted to mean that in wild-type arabinose uptake the forward transport rate (k(for)) greatly exceeds the dissociation rate (kund) of a binding protein docked with the AraG:AraH membrane complex, and that k(for) dominance is preserved in all of the binding protein surface mutants . The assumptions and predictions of the model are consistent with existing data from other periplasmic transport systems. Protein Sci, 1992 Dec, 1(12), 1652 - 60 Escherichia coli K12 arabinose-binding protein mutants with altered transport properties; Kehres DG et al.; The arabinose-binding protein (ABP) of Escherichia coli binds L-arabinose in the periplasm and delivers it to a cytoplasmic membrane complex consisting of the AraG and AraH proteins, for uptake into the cell . To study the interaction between the soluble and membrane components of this periplasmic transport system, regions of the ABP surface containing the opening of the arabinose-binding cleft were subjected to site-directed mutagenesis . Thirty-eight ABP variants containing one to three amino acid substitutions were recovered . ABP variants were expressed with wild-type AraG and AraH from a plasmid, in a strain lacking the chromosomal araFGH operon, and the whole cell uptake parameters, Ven (maximum initial velocity of arabinose entry) and K(en) (concentration of arabinose yielding half-maximal entry) were determined . Twenty-four mutants had normal Ven values, 3 mutants had Ven and K(en) values twice wild type, and 11 mutants had Ven and K(en) values 20-50% of wild type . Binding proteins that had altered uptake properties were each expressed, processed, and localized to the periplasm at levels equivalent to wild type . The mutant binding proteins behaved the same as wild type during purification, and each had a Kd (dissociation constant for bound arabinose) comparable to that of wild-type ABP . Mutations that resulted in altered uptake identified nine amino acids surrounding the arabinose-binding cleft, all of which are charged in the wild-type protein, and all of whose side chains project outward from the cleft . The evidence suggests that this surface of the binding protein and these nine charged loci play a major role in ABP interactions with the membrane complex. Protein Sci, 1992 Dec, 1(12), 1642 - 51 Functional mapping of the surface of Escherichia coli ribose-binding protein: mutations that affect chemotaxis and transport; Binnie RA et al.; Ribose-binding protein is a bifunctional soluble receptor found in the periplasm of Escherichia coli . Interaction of liganded binding protein with the ribose high affinity transport complex results in the transfer of ribose across the cytoplasmic membrane . Alternatively, interaction of liganded binding protein with a chemotactic signal transducer, Trg, initiates taxis toward ribose . We have generated a functional map of the surface of ribose-binding protein by creating and analyzing directed mutations of exposed residues . Residues in an area on the cleft side of the molecule including both domains have effects on transport . A portion of the area involved in transport is also essential to chemotactic function . On the opposite face of the protein, mutations in residues near the hinge are shown to affect chemotaxis specifically. Protein Sci, 1992 Dec, 1(12), 1604 - 12 Effects of calcium on recombinant bovine chromogranin A; Angeletti RH et al.; Bovine chromogranin A, the acidic calcium-binding protein characteristic of endocrine secretory vesicles, has been expressed in Escherichia coli using the pET3a vector system under T7 polymerase control . The expressed protein is located in the bacterial cytosol and can be purified from bacterial proteins by a heat treatment step, followed by gel filtration, anion-exchange, and reversed-phase chromatography . The purified recombinant chromogranin A has an apparent M(r) of ca . 72,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in spite of its 432-amino acid polypeptide chain, consistent with observations on natural chromogranin A . The primary structure has been confirmed by mass spectral analysis of tryptic peptides, by Edman degradation of the intact protein, and by immunoreactivity with sequence-specific antibodies . Analysis by circular dichroism spectroscopy shows pH- and concentration-dependent spectra . The spectra are Ca2(+)-dependent from 5 to 40 microM. Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1992 Dec, 14(6), 429 - 32 {Effect of lipopolysaccharide on protein kinase C activity in human erythrocytes}; Wei X; Membrane-associated protein kinase C activity was significantly increased after normal human erythrocytes were incubated with lipopolysaccharide . The effect was dose and time dependent . The results indicate that protein kinase C is activated by lipopolysaccharide in intact human erythrocytes. Plant Mol Biol, 1992 Dec, 20(5), 763 - 80 Induction, purification and characterisation of acyl-ACP thioesterase from developing seeds of oil seed rape (Brassica napus); Hellyer A et al.; The level of two thioesterases, acyl-CoA thioesterase and acyl-ACP thioesterase was determined during seed maturation in oil seed rape . Both thioesterase activities rose markedly prior to the onset of lipid accumulation, but the induction kinetics suggest that the activities reside on distinct polypeptides . Acyl-ACP thioesterase (EC 3.1.2.14) was purified 2000-fold using a combination of ion exchange, ACP-affinity chromatography, chromatofocusing and gel filtration . Using native gel electrophoresis, and assays for enzymic activity, two polypeptides were identified on SDS-PAGE as associated with the activity . Cleveland mapping of these polypeptides, of 38 kDa component and 33 kDa respectively, demonstrated that they are related . An antibody was prepared against the 38 kDa component, and this also recognises the 33 kDa polypeptide in highly purified preparations . Western blotting of a crude extract identifies one band at 38 kDa consistent with the 33 kDa component being a degradation product generated during purification . The native molecule has a M(r) of 70 kDa indicating a dimeric structure . The enzyme has a pH optimum of 9.5 and shows strong preference for oleoyl-ACP as substrate . The intact enzyme has an N-terminus blocked to protein sequencing . We also found that two other polypeptides co-purify with acyl-ACP thioesterase under native conditions . The N-terminal amino-acid sequence of these polypeptides is shown and their possible identity is discussed. Chin Med J (Engl), 1992 Dec, 105(12), 998 - 1003 Isolation and sequencing of the cDNA encoding the 75-kD human sperm protein related to infertility; Zhang ML et al.; Serum was obtained from an infertile woman (IS) inducing head-to-head agglutination of human sperm and was used to screen a human testis lambda gt11 cDNA library . A plaque producing the interacting antigen was located . The recombinant lambda gt11 was isolated and cut with EcoRI releasing a 0.7-kb cDNA . Using the 0.7-kb cDNA as a probe, a larger cDNA of 2.4 kb was isolated and its nucleotide sequence determined . It was composed of 2 427 nucleotides with an open reading frame of 1584 nucleotides encoding 528 amino acid residues . The specific antisperm antibody was isolated from IS by epitope selection, using positive plaques of E . coli Y1090 . The epitope-selected antibodies interacted with a 75-kD human sperm protein and with a polypeptide in the form of a beta-galactosidase fusion protein in the recombinant lysate of E . coli Y1089, determined by immunoblot . The fusion protein was purified by affinity chromatography on an anti-beta-galactosidase-Sepharose column . It is proposed that production of anti-75-kD antibodies may be the underlying cause of the infertility. Biochem Cell Biol, 1992 Dec, 70(12), 1332 - 8 Inhibition of mammalian ribonucleotide reductase by cis-diamminedichloroplatinum(II); Chiu CS et al.; Ribonucleotide reductase is a highly regulated, rate-limiting activity in the synthesis of DNA . A previous study has shown that the Escherichia coli enzyme is inhibited by the clinically important antitumor agent cis-diamminedichloroplatinum(II) (DDP), and this has led to the hypothesis that ribonucleotide reductase is an important site of action for this chemotherapeutic agent . This hypothesis has been directly tested in this investigation . We observed that DDP inhibits the mammalian ribonucleotide reductase, with 50% inhibition occurring at 0.3 mM . Unlike the E . coli enzyme where only one of the two protein components is targeted by DDP, we observed that both of the mammalian proteins (R1 and R2) were sites for the inhibitory activity of the drug . Colony-forming experiments, enzyme activity studies, and analyses of R1 and R2 message levels in mutant cell lines containing either high levels of ribonucleotide reductase activity or exhibiting resistance to the cytotoxic effects of DDP were used to further investigate the potential role of ribonucleotide reductase in DDP cytotoxic action and drug resistance . These studies did not support a hypothesis formulated in the earlier investigation that inhibition of ribonucleotide reductase is an important component of DDP cytotoxic activity or that it is a major participant in DDP resistance mechanisms . From a biological point of view, DDP is a very active drug, and in addition to its cytotoxic effects it is capable of inducing a variety of cellular changes . Whether or not the inhibition of mammalian ribonucleotide reductase activity that we have described in this study plays a role in mediating any of these other effects remains to be determined. J Korean Med Sci, 1992 Dec, 7(4), 307 - 13 Polymorphonuclear leukocyte functions enhanced by chemotaxis; Hong YS; Human polymorphonuclear leukocytes (PMN) migrate into tissues in response to chemoattractants, yet it is not known whether this process alters the functional capabilities of the PMN . Using recombinant human interleukin-8 (rHIL-8, 100 ng/ml) as a stimulus, we compared a population of PMN that migrated through a polyvinylpyrrolidone-coated polycarbonate filter containing 8.0 microns diameter pores with PMN stimulated in suspension . PMN were analyzed by flow cytometry according to functional and phenotypic criteria . CD11b/CD16 expression was unaltered by chemotaxis . In contrast, chemotaxis enhanced phagocytosis of E . coli, independent of opsonization with IgG . Similarly, chemotaxis increased baseline hydrogen peroxide production . We conclude that the chemotactic motion of PMN "primes" the cell for increased oxidative burst activity and augments the ability of PMN to ingest bacteria . This increased functional capability is distinct from rHIL-8 stimulation and appears to be independent of complement-and Fc-receptor expression. J Diarrhoeal Dis Res, 1992 Dec, 10(4), 201 - 4 Effect of berberine on enterotoxin-induced intestinal fluid accumulation in rats; Khin-Maung-U et al.; We have determined the effects of berberine (Berberis aristita) on intestinal fluid accumulation due to enterotoxigenic E . coli (ETEC) heat-stable (ST) toxin in suckling (24-days old) Wistar rats . Intestinal fluid accumulation occurred in suckling Wistar rats by administration of culture filtrate containing ST-producing ETEC in serial dilutions up to 1/8 dilution by oral or intragastric route . When berberine (0.1 mg) and 1/8 dilution of ST-toxin were given together by oral or intragastric injection, a significant (p < 0.01) reduction in fluid accumulation was observed . Neither treatment with berberine orally before intragastric injection of ST-toxin nor intragastric administration of berberine after ST-toxin reduced the fluid accumulation due to ST-toxin. J Biochem (Tokyo), 1992 Dec, 112(6), 725 - 8 Site-directed mutagenesis of amino acid residues involved in the glutathione binding of human glutathione S-transferase P1-1; Kong KH et al.; The four residues of human glutathione S-transferase P1-1 whose counterparts were indicated by X-ray crystallography to reside in the GSH-binding site of pig glutathione S-transferase P1-1 were individually replaced with threonine or alanine by site-directed mutagenesis to obtain mutants R13T, K44T, Q51A, and Q64A . The kinetic parameters, susceptibilities to an inhibitor, S-hexyl-GSH, and affinities for GSH-Sepharose of the latter were compared with those of the wild-type enzyme, and pKa of the thiol group of GSH bound in R13T was shown to be equivalent to that in the wild type . From the results, Lys44, Gln51, and Gln64 were deduced to contribute to the binding of GSH . On the other hand, Arg13 seems to be essential for the enzymatic activity as mainly involved in the construction of a proper structure of the active site. Cell Struct Funct, 1992 Dec, 17(6), 433 - 42 Inhibition of expression of a mouse alpha-globin gene by plasmids that include antisense oligonucleotides; Yokoyama K et al.; Plasmid-borne DNAs, corresponding to 68-base oligodeoxynucleotides, synthesized in the antisense or sense configuration and based on the nucleotide sequences of various regions of the mouse alpha-globin mRNA, were introduced with the gene for xanthine-guanine phosphoribosyl transferase from E . coli (Ecogpt) into mouse erythroleukemia (MEL) cells by protoplast fusion . Specific inhibition of the synthesis of alpha-globin was observed only in the cells transformed with the plasmids with antisense 68-mers that corresponded to the cap site as well as the site of initiation of translation of alpha-globin mRNA (Oligo-A); Other plasmids with antisense 68-mers that corresponded to the regions of the exon/intron junctions, the individual exons, or the 3' untranslated region were ineffective . This antisense RNA efficiently reduced the production of alpha-globin to 9-18% of the endogenous level after induction with hexylmethylene-bis-acetoamide (HMBA) . Moreover, most of the antisense transformants did not show any decrease in the expression of the c-myc gene at the early phases of differentiation of MEL cells . Thus, we propose a hypothesis that the early decline in levels of c-myc mRNA may be independent of and uncoupled from the program of globin synthesis during the differentiation of MEL cells. Cell Struct Funct, 1992 Dec, 17(6), 363 - 9 Small GTP-binding proteins on rat liver lysosomal membranes; Sai Y et al.; GTP-binding proteins have been identified on the membranes of highly purified dextran-filled lysosomes (dextranosomes) and Triton-filled lysosomes (tritosomes) obtained from rat liver . Autoradiography of blots of lysosomal membrane proteins incubated with {alpha-32P}GTP revealed the presence of several specific GTP-binding proteins with a relative molecular mass (M(r)) predominantly in the range of 26-30 kDa . These GTP-binding proteins migrated slower in polyacrylamide gels than purified c-Ha-ras protein expressed in E . coli, whose apparent M(r) was 23 kDa in the same blot . The relative contents of GTP-binding proteins in lysosomal membranes were comparable or greater than that of plasma membranes and of microsomes . Chemical extraction showed that lysosomal GTP-binding proteins were more tightly associated with the membranes than with microsomal GTP-binding proteins . The possible involvement of lysosomal GTP-binding proteins in cellular functions including vacuolar (lysosomal) acidification and organellar dynamics are discussed. Scanning Microsc, 1992 Dec, 6(4), 911 - 8 Atomic force microscopy of DNA on mica and chemically modified mica; Thundat T et al.; Atomic force microscopy (AFM) was used to image circular DNA adsorbed on freshly cleaved mica and mica chemically modified with Mg(II), Co(II), La(III), and Zr(IV) . Images obtained on unmodified mica show coiling of DNA due to forces involved during the drying process . The coiling or super twisting appeared to be right handed and the extent of super twisting could be controlled by the drying conditions . Images of DNA observed on chemically modified surfaces show isolated open circular DNA that is free from super twisting, presumably due to strong binding of DNA on chemically modified surfaces. Yakugaku Zasshi, 1992 Dec, 112(12), 873 - 86 {Structural studies of malaria vaccine}; Tomita K; Malaria still remains a serious health problem in large areas of the world, and in this article, recent research progress mainly made by us toward malaria vaccine development has been reviewed . 1) Peptide vaccines (antigens) of immunodominant tetrapeptide repeats (NANP and NVDP) of the circumsporozoite surface protein of the malaria parasite, Plasmodium falciparum, were genetically produced in E . coli as a fusion protein with a part of human growth hormone, which has affected on the conformations and immunogenicities of the peptide vaccines . 2) Monoclonal antibodies against the peptide antigens were produced by fusion of mouse spleen cells with myeloma cells, and the F (ab's) obtained by partial digestion of the antibodies with papain were used for the measurement of the dissociation constants of the antigen-antibody complexes . The amino acid sequence of the Hv region in F(ab) domain was also deduced from its nucleotide sequence. Farmaco, 1992 Dec, 47(12), 1529 - 41 Photobiological activity of certain new methylazapsoralens; Baccichetti F et al.; The photobiological activity of a series of psoralen isosters carrying a nitrogen atom at 8 position, new potential drugs for the photochemotherapy of hyperproliferative skin diseases, have been studied; the more active derivatives appeared to be 5,4'-dimethyl-8-azapsoralen and 3,4,4'-trimethyl-8-azapsoralen which induced a strong inhibition of DNA synthesis in Ehrlich ascites cells, very similar to that provoked by 8-methoxypsoralen, the furocoumarin at present used in photochemotherapy . Such compounds induced a small amount of inter-strand DNA cross-links and were non phototoxic when assayed on guinea-pig skin; however, both derivatives appeared to be highly mutagenic in E . coli WP2 TM6 . This strain contains the plasmid R46 and it is proficient in DNA repair, and therefore monoadducts do not should be mutagenic in such a strain . Because the first steps of excision, which remove monoadducts, and of the main cross-link repair use the same enzymes (produced by the uvrABC complex), in the presence of a great number of monofunctional lesions, it is possible that there are not sufficient enzyme molecules for removing cross-links according this pathway, which could be repaired by a second one, uvrABC independent and based on glycosilase activity, which works at reduced levels and is much less accurate. Biol Chem Hoppe Seyler, 1992 Dec, 373(12), 1187 - 91 A new ultrasensitive bioluminogenic enzyme substrate for beta-galactosidase; Geiger R et al.; A derivative of D-luciferin, D-luciferin-O-beta-galactoside, was synthesized and used as highly sensitive substrate for beta-galactosidase . The substrate was physicochemically characterized . Enzymatic cleavage of the new compound by beta-galactosidase was demonstrated and kinetic constants Km, Vmax, kcat and kcat/Km have been determined . The compound has been proved to be a highly sensitive substrate for beta-galactosidase, permitting a limit of detection of 3.7 x 10(-19) mol of enzyme per assay. Biofactors, 1992 Dec, 4(1), 33 - 6 Lipopolysaccharide-binding factors are present in bovine serum; Khemlani LS et al.; Response of vertebrates to bacterial lipopolysaccharide (LPS) may be regulated, in part, by serum factors that influence the bioavailability and cellular binding affinity of LPS . Using 3H- or 14C-labeled LPS, or a novel fluorescence-based assay for detection in CsCl isopycnic density gradients, our studies indicate the existence of factors in adult and fetal bovine serum that bind LPS . Within serum-containing gradients, labeled LPS appeared in two peaks: least-dense fractions (< or = 1.30 g/cm3) and at 1.35 g/cm3 . This profile was different from that of gradients without serum, where LPS appeared at 1.38 g/cm3 . Binding of LPS to serum component(s) at 1.35 g/cm3 was rapid (< 1 min), saturable and specific . A partial shift (50%) of LPS from a density of 1.35 g/cm3 to other serum components at < or = 1.30 g/cm3 occurred over 1 h . Flow cytometric analysis indicated that bovine serum factors influence the binding of LPS to blood monocytes, because monocyte-FITC-LPS association increased in the presence of bovine serum. Immunol Cell Biol, 1992 Dec, 70 ( Pt 6), 411 - 6 Development of human ABO blood group A antigen on Escherichia coli Y1089 and Y1090; Yang N et al.; Studies by other workers have shown that some strains of Escherichia coli have surface antigens analogous to the human blood group ABH antigens, and that these are carbohydrates associated with membrane lipopolysaccharides . This study has demonstrated that E . coli strains Y1089 and Y1090 possess the H antigen, which can be converted to the A antigen by incubation with A-transferase (N-acetyl-galactosaminyl transferase) and A-sugar (UDP-N-acetyl-galactosamine) . Such cells will then form mixed agglutinates with human A red cells and human polyclonal (but not mouse monoclonal) anti-A antibodies . E . coli Y1089 and Y1090 have endogenous enzymes that use the A-sugar (in the absence of A-transferase) to produce a variant A antigen . Cells expressing this variant antigen adsorb anti-A antibodies but do not participate in mixed agglutination with human group A red cells . It is estimated that E . coli Y1089 and Y1090 possess approximately 5000 H epitopes per cell that can be converted to A epitopes. Arzneimittelforschung, 1992 Dec, 42(12), 1512 - 5 Isolation and renaturation of bio-active proteins expressed in Escherichia coli as inclusion bodies; Fischer B et al.; Over-expression of recombinant proteins in Escherichia coli often results in the formation of insoluble and inactive material as inclusion bodies . Within the last years specific methods and strategies have been developed to produce bio-active proteins from these inclusion bodies . These methods include (i) isolation and purification of inclusion bodies, (ii) solubilization and reduction of the insoluble material, and (iii) renaturation of the proteins including formation of native disulphide bonds . Inclusion bodies are isolated from E . coli cells after desintegration of cells by mechanic forces . Due to their stability, inclusion bodies may be purified by washing with detergent solutions or low concentrations of denaturant . High concentrations of urea and guanidine hydrochloride are used in combination with reducing reagents such as 2-mercaptoethanol or dithiothreitol to reduce and solubilize proteins from inclusion bodies . After purification of solubilized materials, proteins are renatured and native disulphide bonds are formed by either air oxidation or glutathione reoxidation starting from reduced material, or by disulphide interchange starting from mixed disulphides containing peptides . Final yield of renatured proteins is raised by low concentrations of proteins and by adding low concentrations of denaturant during renaturation. Protein Eng, 1992 Dec, 5(8), 811 - 9 Expression of the phosphorylase kinase gamma subunit catalytic domain in Escherichia coli; Cox S et al.; The catalytic subunit of phosphorylase b kinase (gamma) and an engineered truncated form (gamma-trc, residues 1-297) have been expressed in Escherichia coli . The truncated protein included the entire catalytic domain as defined by sequence alignment with other protein kinases but lacked the putative calmodulin binding domain . Full-length protein was produced in insoluble aggregates . Some activity was regenerated by solubilization in urea and dilution into renaturating buffer but the activity was found to be associated with a smaller molecular weight component . Full-length protein could not be refolded successfully . The truncated gamma subunit was produced in the soluble fraction of the cell as well as in inclusion bodies . The insoluble protein was refolded by dilution from urea and purified to homogeneity, in a one step separation on DEAE-Sepharose to give a protein mol . wt 32,000 +/- 2000 with a high sp . act . of 5.3 mumol 32P incorporated into phosphorylase b(PPB)/min/nmol . Kinetic parameters gave Km for ATP 46 +/- 3 microM and Km for PPb 27 +/- 1 microM . The sp . act . and the Km values are comparable to those observed for the activated holoenzyme and indicate that the gamma-trc retains the substrate recognition and catalytic properties . The ratio of activities at pH 6.8/8.2 was 0.84 . gamma-trc was inhibited by ADP with a Ki of 52 microM and was sensitive to activation by Mg2+ and inhibition by Mn2+, properties that are characteristic of the holoenzyme and the isolated gamma subunit . Calmodulin which confers calcium sensitivity on the isolated gamma subunit had no effect on the enzymic properties of gamma-trc.(ABSTRACT TRUNCATED AT 250 WORDS) Protein Eng, 1992 Dec, 5(8), 797 - 806 Development of an optimized refolding process for recombinant Ala-Glu-IGF-1; Hejnaes KR et al.; Denatured and reduced N-terminal extended insulin-like growth factor-1 (AE-IGF-1) was purified from Escherichia coli extracts and subjected to in vitro folding . The renaturation process was shown to be a function of the redox potential of the solution . Folding by different methods had no significant effect on the renaturation . A maximal yield of 60% (w/w) was obtained . The folded AE-IGF-1 was enzymatically converted to IGF-1 . The major by-product (20% w/w) was identified as scrambled IGF-1 . Enzymatic digestion at alkaline and acidic pH suggested two possible disulphide bond arrangements; (i) Cys6-Cys47, Cys18-Cys61, Cys48-Cys52; or (ii) Cys6-Cys52, Cys18-Cys61, Cys47 and Cys48 being in their reduced forms . Energy minimization and molecular modelling suggested that the scrambled IGF-1, having reduced cysteines at positions 47 and 48, was the energetically most stable conformation of the two. Protein Eng, 1992 Dec, 5(8), 791 - 6 Construction and characterization of a single polypeptide chain containing two enzymatically active dihydrofolate reductase domains; Iwakura M et al.; A single polypeptide chain containing two dihydrofolate reductase (DHFR) sequences from Escherichia coli was constructed to determine if a repeat sequence fusion protein could be expressed in an active form . The possibility that intersequence interactions could play a significant role for this enzyme is suggested by the results of Hall and Frieden (1989, Proc . Natl Acad . Sci . USA, 86, 3060-3064) who observed a substantial decrease in the yield of active enzyme when folded in the presence of a large C-terminal fragment . The fusion protein {DHFR(Cys152Glu)--Ile--DHFR (Met1Gln)} was efficiently expressed in E . coli cells and has an activity which is twice that of the wild-type enzyme in the standard assay . The Michaelis constants of the fusion protein for the substrate, dihydrofolate and the cofactor, NADPH, are essentially unchanged from those of the wild-type protein . The urea-induced in vitro unfolding reaction of the fusion protein a