Plant Mol Biol, 1993 Oct, 23(2), 387 - 96 Functional expression and molecular characterization of AtUBC2-1, a novel ubiquitin-conjugating enzyme (E2) from Arabidopsis thaliana; Bartling D et al.; The first member of a novel subfamily of ubiquitin-conjugating E2-proteins was cloned from a cDNA library of Arabidopsis thaliana . Genomic blots indicate that this gene family (AtUBC2) consists of two members and is distinct from AtUBC1, the only other E2 enzyme known from this species to date (M.L . Sullivan and R.D . Vierstra, Proc . Natl . Acad . Sci . USA 86 (1989) 9861-9865) . The cDNA sequence of AtUBC2-1 extends over 794 bp which would encode a protein of 161 amino acids and a calculated molecular mass of 18.25 kDa . The protein encoded by AtUBC2-1 is shown to accept 125I-ubiquitin from wheat E1 enzymes, when expressed from Escherichia coli hosts as fusion protein carrying N-terminal extensions . It is deubiquitinated in the presence of lysine and, by these criteria, is considered a functional E2 enzyme. Plant Mol Biol, 1993 Oct, 23(2), 287 - 95 The plant mitochondrial open reading frame orf221 encodes a membrane-bound protein; Prioli LM et al.; We have shown that the open reading frame orf221 is an active mitochondrial gene which encodes a novel mitochondrial polypeptide . The orf221 sequence is common to higher plants but absent in animal and fungal mitochondria . A mitochondrial polypeptide with an apparent molecular weight of 21,000 was detected with a polyclonal antibody raised against an ORF221 fusion protein . In organello translation followed by immunoprecipitation with the anti-ORF221 antibody demonstrated that this polypeptide is encoded by the orf221 gene in plant mitochondria . The ORF221 was found to be a mitochondrial membrane protein in normal (N), cms-T, and cms-C cytoplasms of several inbred lines of maize (Zea mays L.) and in other plant species. Am Rev Respir Dis, 1993 Oct, 148(4 Pt 1), 878 - 81 Changes of pulmonary glucocorticoid receptor and phospholipase A2 in sheep with acute lung injury after high dose endotoxin infusion; Liu LY et al.; In a sheep model of acute lung injury induced by an Escherichia coli endotoxin (5 micrograms/kg) with chronic lung lymph fistula (n = 15), we measured the changes in glucocorticoid receptor (GCR) binding capacity in lung tissue by means of radioligand binding assay . The content of cortisol and the activity of phospholipase A2 (PLA2) were also measured . The results showed that the maximal binding capacity (Bmax) of GCR in lung cytoplasma decreased continuously 2 h (113 +/- 3 versus 66 +/- 2 fmol/mg protein, p < 0.01), 4 h (105 +/- 6 versus 52 +/- 3 fmol/mg protein, p < 0.01), and 6 h (105 +/- 5 versus 37 +/- 2 fmol/mg protein, p < 0.01) after endotoxin infusion . Its affinity decreased markedly (p < 0.05) at 6 h after the infusion . The contents of cortisol in plasma elevated at 0.5 h and remained at a high level until 4 h after the infusion . PLA2 activity rose from 97 +/- 25 to 188 +/- 12 U (p < 0.05), 99 +/- 13 to 285 +/- 25 U (p < 0.01), and 106 +/- 14 to 354 +/- 32 U (p < 0.01) at 2, 4, and 6 h after endotoxin infusion, respectively . There was a negative correlation between the Bmax of GCR and PLA2 activity (r = -0.87, p < 0.01) . The findings indicate that there was a secondary GCR abnormality and a higher PLA2 activity during endotoxin-induced lung injury . The glucocorticoid hypofunction caused by reduced GCR binding capacity may accelerate the pathologic response of acute lung injury.(ABSTRACT TRUNCATED AT 250 WORDS) Mol Mar Biol Biotechnol, 1993 Oct, 2(5), 280 - 90 Kinetic and physical properties of a recombinant RuBisCO from a chemoautotrophic endosymbiont; Stein JL et al.; Ribulose 1,5 bisphosphate carboxylase/oxygenase (RuBisCO), which catalyzes the key step in autotrophic CO2 fixation, is present at high activity in the symbiont-containing tissues of many hydrothermal vent invertebrates . The genes encoding RuBisCO from a gill endosymbiont of the hydrothermal vent gastropod Alviniconcha hessleri have been cloned, sequenced, and functionally expressed in Escherichia coli under control of the lac promoter in the vector pBlueScript . The purified protein is a hexadecamer (L8S8) with an apparent molecular weight of 554 kDa and a specific carboxylase activity of 2.9 mumol/min/mg protein . Unlike previously characterized RuBisCOs, which display sharp temperature optima, the symbiont RuBisCO maintains a high level of activity over a broad temperature span, although it is not thermally stable after extended exposure to temperatures above 50 degrees C . This funding suggests an adaptation to the thermal transients measured at vent openings where the host snail resides . The enzyme also maintains 90% of its 1 atm activity at 370 atm pressure, whereas spinach RuBisCO retains 28% activity under similar conditions . At 1 atm pressure and 20 degrees C, the Km(CO2) and the relative substrate specificity (Srel) of the symbiont enzyme were 80 and 32.5 mumol/L, respectively, which are similar to values reported for RuBisCOs from cyanobacteria and the purple photosynthetic bacteria . The relatively low specificity of the enzyme for substrate CO2 indicates that the intracellular environment of the endosymbionts may be microaerophilic for RuBisCO to maintain net carboxylation. Wei Sheng Wu Xue Bao, 1993 Oct, 33(5), 331 - 8 {Oligonucleotide directed in vitro mutagenesis}; Su G; In this study, a method for oligonucleotide directed in vitro mutagenesis was described . It includes the following steps: clone of the gene to be mutated into the phagemid pGCI; preparation of single strand template; design and synthesis of mutated oligonucleotides; synthesis of double strand DNA . Using this method, a new BamHI site was originated at the N terminal of Stx-B and BglII site at the C terminal of Stx-B . It is ready to fuse the Stx-B gene to LamB . This method is a very useful tool for genetic engineering. Int Surg, 1993 Oct-Dec, 78(4), 354 - 6 Neutrophil dynamics in abdominal cavity of peritonitic rats treated with antiseptics; Celdran A et al.; Peritonitis was induced in Wistar rats by intraperitoneal inoculation of pure Escherichia coli . Rats in which 2 ml of 1% povidone iodine had been injected intraperitoneally 5 min after the bacterial challenge, showed a decrease in the neutrophil percentage of the peritoneal cell population during the first hours of peritonitis compared to the control group . When the same experiment was performed with 2 ml of 0.05% chlorhexidine the percentage of neutrophils was superior to the control group in the first hours after bacterial challenge . These results concur with a previous study in which the deleterious effect of povidone iodine and the beneficial effect of chlorhexidine were demonstrated. Indian J Biochem Biophys, 1993 Oct, 30(5), 252 - 6 Denatured supercoiled DNA--structural and biological activity; Santra CR et al.; Supercoiled DNA on treatment with NaOH followed by neutralization produces a condensed structure (form Id) . This structure does not split into topoisomers when run on long gel in presence of intercalating agents and the migration of this form does not change appreciably in presence or absence of ethidium bromide . Relaxation of form Id by topoisomerase I from pea chloroplast is facilitated more than form I . Single-stranded binding (SSB) protein binds more to form Id as evidenced from gel retardation study . Hydroxyl radical nicking is facilitated in this form . Compared to form I, this form produces half the number of transformants, but adsorption and penetration remain almost same in both the forms . Post-transformational growth using 32P labelled form I and form Id showed greater amount of degradation in form Id. J Protein Chem, 1993 Oct, 12(5), 571 - 7 Reactivity of cysteinyl, arginyl, and lysyl residues of Escherichia coli phosphoenolpyruvate carboxykinase against group-specific chemical reagents; Bazaes S et al.; Calcium-activated phosphoenolpyruvate carboxykinase from Escherichia coli is not inactivated by a number of sulfhydryl-directed reagents {5,5'-dithiobis(2-nitrobenzoate), iodoacetate, N-ethylmaleimide, N-(1-pyrenyl)maleimide or N-(iodoacetyl)-N'-(5-sulfo-1-naphthylethylenediamine)}, unlike phosphoenolpyruvate carboxykinase from other organisms . On the other hand, the enzyme is rapidly inactivated by the arginyl-directed reagents 2,3-butanedione and 1-pyrenylglyoxal . The substrates, ADP plus PEP in the presence of Mn2+, protect the enzyme against inactivation by the diones . Quantitation of pyrenylglyoxal incorporation indicates that complete inactivation correlates with the binding of one inactivator molecule per mole of enzyme . Chemical modification by pyridoxal 5'-phosphate also produces inactivation of the enzyme, and the labeled protein shows a difference spectrum with a peak at 325 nm, characteristic of a pyridoxyl derivative of lysine . The inactivation by this reagent is also prevented by the substrates . Binding stoichiometries of 1.25 and 0.30 mol of reagent incorporated per mole of enzyme were found in the absence and presence of substrates, respectively . The results suggest the presence of functional arginyl and lysyl residues in or near the active site of the enzyme, and indicate lack of reactive functional sulfhydryl groups. Avian Dis, 1993 Oct-Dec, 37(4), 1092 - 6 Association of K-1 capsule, smooth lipopolysaccharides, traT gene, and Colicin V production with complement resistance and virulence of avian Escherichia coli; Wooley RE et al.; A group of complement-resistant, virulent avian Escherichia coli isolates were compared with a group of complement-sensitive, avirulent avian isolates for the presence of K-1 capsule, smooth lipopolysaccharides (LPS), the traT gene, and Colicin V (ColV) production . These parameters were selected because of their reported association with complement resistance and virulence in E . coli . Lethality in chicken embryos has also been shown to be correlated with virulence of avian E . coli for chickens . The complement-resistant, virulent E . coli isolates did not possess a K-1 capsule . Production of ColV and the presence of smooth LPS were significantly correlated with embryo lethality . There was no correlation between the presence of traT and embryo lethality . These results suggest that complement resistance and virulence in avian E . coli are associated with ColV production and smooth LPS but not with K-1 antigen or traT. J Bioenerg Biomembr, 1993 Oct, 25(5), 483 - 92 Phosphate transport in mitochondria: past accomplishments, present problems, and future challenges; Ferreira GC et al.; The requirement of inorganic phosphate (Pi) for oxidative phosphorylation in eukaryotic cells is fulfilled through specific Pi transport systems . The mitochondrial proton/phosphate symporter (Pic) is a membrane-embedded protein which translocates Pi from the cytosol into the mitochondrial matrix . Pic is responsible for the very rapid transport of most of the Pi used in ATP synthesis . During the past five years there have been advances on several fronts . Genomic and cDNA clones for yeast, bovine, rat, and human Pic have been isolated and sequenced . Functional expression of yeast Pic in yeast strains deficient in Pi transport and expression in Escherichia coli of a chimera protein involving Pic and ATP synthase alpha subunit have been accomplished . Pic, in contrast to other members of the family of transporters involved in energy metabolism, was demonstrated to have a presequence, which optimizes the import of the precursor protein into mitochondria . Six transmembrane segments appear to be a structural feature shared between Pic and other mitochondrial anion carriers, and recent-site directed mutagenesis studies implicate structure-functional relationships to bacteriorhodopsin . These recent advances on Pic will be assessed in light of a more global interpretation of transport mechanism across the inner mitochondrial membrane. J Bioenerg Biomembr, 1993 Oct, 25(5), 435 - 46 The mitochondrial transport protein superfamily; Walker JE et al.; The ADP/ATP, phosphate, and oxoglutarate/malate carrier proteins found in the inner membranes of mitochondria, and the uncoupling protein from mitochondria in mammalian brown adipose tissue, belong to the same protein superfamily . Established members of this superfamily have polypeptide chains approximately 300 amino acids long that consist of three tandem related sequences of about 100 amino acids . The tandem repeats from the different proteins are interrelated, and probably have similar secondary structures . The common features of this superfamily are also present in nine proteins of unknown functions characterized by DNA sequencing in various species, most notably in Caenorhabditis elegans and Saccharomyces cerevisiae . The high level expression in Escherichia coli of the bovine oxoglutarate/malate carrier, and the reconstitution of active carrier from the expressed protein, offers encouragement that the identity of superfamily members of known sequence but unknown function may be uncovered by a similar route. Thromb Res, 1993 Oct 1, 72(1), 71 - 82 Fibrin-specific lysis of microthrombosis in endotoxemic rats by saruplase; Schneider J; The dissolution by the fibrinolytic agent saruplase of microthrombi due to disseminated intravascular coagulation (DIC) has been studied in anesthetized rats . The intravenous infusion of E . coli lipopolysaccharide (endotoxin) for 4 hours (total dose: 25 mg/kg) induced marked thrombocytopenia and hypofibrinogenemia . DIC-related microthrombosis, detected as increased deposition of 125I-labelled human fibrin, was found in the liver and the kidneys, but not in the lungs, the heart, the mesenterium, the spleen and the M . rectus abdominis of endotoxemic rats . Treatment with 1-20 micrograms/kg.min saruplase, that was infused concomitantly with endotoxin, dose-dependently and significantly reduced endotoxin-induced microthrombosis in the liver and the kidneys by 85 resp . 88% . When saruplase (20 micrograms/kg.min) was administered only during the last two hours of endotoxin infusion, liver microthrombosis was still significantly dissolved by 69%, whereas renal microthrombosis was insignificantly reduced by 34% . The inhibition of endotoxin-induced microthrombosis took place in the same dosage range as the shortening of the euglobulin clot lysis time in normal rats by saruplase as a measure of its fibrinolytic activity . Saruplase did not modify thrombocytopenia and hypofibrinogenemia in endotoxemic rats . Saruplase per se did not affect plasma fibrinogen levels . Thus, in a fibrin-selective dose range saruplase is able to dissolve microthrombosis associated with DIC in endotoxemic rats. Mol Biol Rep, 1993 Oct, 18(3), 209 - 15 Comparison of the homologous carboxy-terminal domain and tail of alpha-crystallin and small heat shock protein; Merck KB et al.; The C-terminal domain and tail, which is the most conserved region of the alpha-crystallin/small heat shock protein (HSP) family, was obtained from rat alpha A-crystallin, bovine alpha B-crystallin and mouse HSP25 . All three domains have primarily beta-sheet conformation and less than 10% of alpha-helix, like the proteins from which they are derived . Whereas the C-terminal part of alpha A-crystallin forms dimers or tetramers, the corresponding regions of alpha B-crystallin and HSP25 form larger aggregates . The heat-protective activity, recently described for the alpha-crystallin/small HSP family, is not retained in the C-terminal domain and tail . In the course of this study some differences with the previously published sequence of HSP25 were observed, and a revision is proposed. Mol Biol Rep, 1993 Oct, 18(3), 183 - 7 Polypyrimidine/polypurine sequence in plasmid DNA enhances formation of dimer molecules in Escherichia coli . Dimerization of plasmid DNA in Escherichia coli; Kato M; Formation of dimer molecules of a recombinant plasmid, pTIR10, which carries a pyrimidine/purine-biased stretch occurs about 6-fold more efficiently than for the control plasmid pUC19 in Escherichia coli strain JM107 . Since pyrimidine/purine-biased sequences have a potential to form unusual DNA structures, this observation suggests that the inserted sequence affects the replication process of plasmid DNA, probably by forming a triple helix under physiological conditions. Proc Natl Acad Sci U S A, 1993 Oct 1, 90(19), 9120 - 4 Neurotrophic activity of the Antennapedia homeodomain depends on its specific DNA-binding properties; Le Roux I et al.; In previous reports we have demonstrated that the 60-aa peptide corresponding to the homeodomain of the Drosophila protein Antennapedia (pAntp) translocates through the membrane of neurons in culture, accumulates in neuronal nuclei, and promotes neurite growth . To analyze the importance of specific pAntp DNA-binding properties in this phenomenon we have constructed three mutant versions of pAntp that differ in their ability to translocate through the membrane and to bind specifically the cognate sequence for homeodomains present in the promoter of HoxA5 . We demonstrate that removing two hydrophobic residues of the third helix inhibits pAntp internalization and suppresses its neurotrophic activity . We also show that pAntp neurotrophic activity is lost when mutations are introduced in positions preserving its penetration and nuclear accumulation but abolishing its capacity to bind specifically the cognate DNA-binding motif for homeoproteins . Our results strongly suggest that pAntp neurotrophicity requires both its internalization and its specific binding to homeobox cognate sequences . We propose that homeoproteins might regulate important events in the morphological differentiation of the postmitotic neuron. Poult Sci, 1993 Oct, 72(10), 1823 - 31 Major histocompatibility complex class IV restriction fragment length polymorphism markers in replicated meat-type chicken lines divergently selected for high or low early immune response; Uni Z et al.; Information on MHC may improve the efficiency of selection for immunological traits via the application of marker assisted selection or by selecting directly for a specific restriction fragment length polymorphism (RFLP) band or MHC haplotype . An experimental procedure is presented here for identifying MHC genes that are related to early immune response . A Class IV cDNA clone was used to probe Southern blots of erythrocyte genomic DNA from chickens . Chickens were taken from the second (S2) and third (S3) generations of replicated lines divergently selected for high antibody response (HC1, HC2) or low antibody response (LC1, LC2) to Escherichia coli vaccination at 10 days of age . These selection criteria have been found to be associated with other immunological parameters . The hypothesis that these selected lines differ in their MHC loci was evaluated by comparing the frequencies of MHC RFLP markers (single RFLP bands) and haplotypes (patterns of RFLP bands) . The significant differences between LC and HC in the frequency of many MHC RFLP bands and of five MHC haplotypes indicate that early antibody production is influenced by MHC genes . The reliability of the association between the selection and frequency differences was tested and proven in most cases by analysis of the replicated lines . These differences in RFLP markers represent a change in allelic frequencies in MHC genes, probably due to selection . The results imply a connection between the Class IV genes and early antibody production, and they show the potential of prospective breeding not only by immunological phenotype but also by genotype (i.e., using RFLP markers of the MHC). J Bacteriol, 1993 Oct, 175(19), 6186 - 93 Environmental regulation of the fim switch controlling type 1 fimbrial phase variation in Escherichia coli K-12: effects of temperature and media; Gally DL et al.; Expression of type 1 fimbriae in Escherichia coli K-12 is phase variable and associated with the inversion of a short DNA element (switch) . The fim switch requires either fimB (on-to-off or off-to-on switching) or fimE (on-to-off switching only) and is affected by the global regulators leucine-responsive regulatory protein (Lrp), integration host factor (IHF), and H-NS . Here it is shown that switching frequencies are regulated by both temperature and media and that these effects appear to be independent . fimE-promoted on-to-off switching occurs far more rapidly than previously estimated (0.3 per cell per generation in defined rich medium at 37 degrees C) and faster at lower than at higher temperatures . In direct contrast, fimB-promoted switching increases with temperature, with optima between 37 and 41 degrees C . Switching promoted by both fimB and fimE is stimulated by aliphatic amino acids (alanine, isoleucine, leucine, and valine), and this stimulation requires lrp . Furthermore, lrp appears to differentially regulate fimB- and fimE-promoted switching in different media. Infect Immun, 1993 Oct, 61(10), 4518 - 22 Multivalent binding of K99 fimbriae to the N-glycolyl-GM3 ganglioside receptor; Willemsen PT et al.; The ganglioside N-glycolyl-GM3 binds laterally at numerous positions to K99 fimbriae, as shown by electron microscopic detection and erythrocyte-binding activity . The data demonstrate the multivalent nature of K99 fimbriae with respect to their receptor-binding sites. Infect Immun, 1993 Oct, 61(10), 4480 - 4 Sialyloligosaccharide chains of laminin as an extracellular matrix target for S fimbriae of Escherichia coli; Virkola R et al.; S fimbriae purified from recombinant Escherichia coli HB101(pANN801-13) bound strongly to extracellular matrices of cultured endothelial and epithelial cells; only poor binding was seen with the fimbriae purified from the sfaS mutant strain HB101(pANN801-1321) . E . coli HB101(pANN801-13) adhered strongly to laminin immobilized on glass; no adhesion was seen to type I, III, IV, or V collagen . Strain HB101(pANN801-1321) failed to adhere to any of the target proteins . Adhesion to laminin of strain HB101(pANN801-13) was inhibited by sialyl-alpha-2,3-lactose as well as by periodate oxidation and neuraminidase treatment of laminin . In Western blotting, the purified S fimbriae recognized more strongly the A chain than the B chains of laminin. J Infect Dis, 1993 Oct, 168(4), 1037 - 41 Distribution of the bundle-forming pilus structural gene (bfpA) among enteropathogenic Escherichia coli; Giron JA et al.; Enteropathogenic Escherichia coli (EPEC) express an inducible bundle-forming pilus (BFP) associated with the presence of the EPEC adherence factor (EAF) plasmid and localized adherence (LA) on HEp-2 cells . The cloned structural gene (bfpA) encoding BFP was found to be specific for EPEC, as homologous sequences were found only in EPEC and not in other enteropathogens . The bfpA probe was slightly more sensitive than the EAF probe; among EPEC strains with LA, the bfpA and EAF probes hybridized with 99% and 96% of the strains, respectively . Immunoblotting of whole cell lysates of BFP-positive, EAF-positive or -negative, and LA-positive or -negative EPEC strains revealed variations in the size (18,500-21,000) of the expressed structural subunit of BFP, suggesting differences in processing that may account for discrepancies between the bfpA, EAF, and LA . Because the bfpA probe consists primarily of sequences encoding an important EPEC virulence factor, in contrast to the unknown function of sequences contained in the EAF probe, the bfpA probe may be useful in epidemiologic studies to detect EPEC. Rev Latinoam Microbiol, 1993 Oct-Dec, 35(4), 433 - 41 Mutagenic assessment of 1,N6-ethenodeoxyadenosine in DNA; Maldonado-Rodriguez R et al.; The mutagenic role of 1-N6-Ethenodeoxydenosine (epsilon A) was assessed by a genetic assay of mutations in the 5' coding region of the lacl gene of Escherichia coli . 1-N6-Etheno-2-deoxydadenosine 5'-triphosphate (epsilon dATP) was substituted for dATP during in vitro DNA synthesis on M13 recombinant uracil single stranded DNA bearing the lacl gene, catalyzed by the large fragment of E . coli DNA polymerase I . DNA products were transfected into a strain of E . coli lacking a chromosomal copy of the lacl gene, and i- phenotypic mutants were seen as blue plaques in the absence of inducer . Mutant progeny were characterized by dideoxy sequencing in the N-terminal region of the lacl gene where epsilon A had originally replaced A, and were found to have T-->C transitions . The frequency of base substitution mutation was different in each of three target sites tested . Taking into account the sequence changes and the coding properties at target sites, we conclude that in general, epsilon A increases the mutation frequency when compared with the control (transfection with unsubstituted DNA) . This increase was similar to that produced by in vitro primer elongation in absence of dATP . The combined results of the electrophoretic assay of primer elongation, measurements of mutation frequency, and sequence analysis of mutant phage progeny support the proposal that epsilon A in template DNA can be mutagenic through epsilon AC mispairing during in vivo replication. Mol Microbiol, 1993 Oct, 10(1), 181 - 91 The response of the picoplanktonic marine cyanobacterium Synechococcus species WH7803 to phosphate starvation involves a protein homologous to the periplasmic phosphate-binding protein of Escherichia coli; Scanlan DJ et al.; During phosphate-limited growth the marine phycoerythrin-containing picoplanktonic cyanobacterium Synechococcus sp . WH7803 synthesizes novel polypeptides, including two abundant species of 100 kDa and 32 kDa . The 32 kDa polypeptide was localized to the cell wall, although in a related strain, Synechococcus sp . WH8103, it could be detected in both the cell wall fraction and the periplasm . The gene (designated pstS) encoding this polypeptide was cloned and shown to be present in a single copy . The deduced amino acid sequence indicated a polypeptide consisting of 326 amino acids with a calculated M(r) of 33,763 . Comparison of this sequence with that obtained by microsequencing the N-terminus of the 32 kDa polypeptide showed that the mature protein was synthesized as a precursor, the first 24 amino acid residues being cleaved between two alanine residues at positions 24 and 25 . The amino acid sequence of the mature polypeptide showed 35% identity and 52% similarity to the periplasmic phosphate-binding protein (PstS) from Escherichia coli, including three regions of much stronger homology which, by comparison with E . coli PstS, are directly involved in phosphate binding . Northern blot analysis revealed a pstS transcript of 1.2 kb in RNA extracted from cells grown in Pi-replete conditions and one of 1.4 kb in considerably increased abundance under Pi-depleted conditions . Homologues of the pstS gene were detected in other marine phycoerythrin-containing Synechococcus strains, but not in freshwater or halotolerant species. Mol Microbiol, 1993 Oct, 10(1), 171 - 9 Proteolytic cleavage at arginine residues within the hydrophilic disulphide loop of the Escherichia coli Shiga-like toxin I A subunit is not essential for cytotoxicity; Burgess BJ et al.; Escherichia coli Shiga-like toxin I is a type II ribosome-inactivating protein composed of an A subunit with RNA-specific N-glycosidase activity, non-covalently associated with a pentamer of B subunits possessing affinity for galabiose-containing glycolipids . The A subunit contains a single intrachain disulphide bond encompassing a hydrophilic sequence containing two trypsin-sensitive arginine residues . By analogy with other bacterial toxins it has been proposed that proteolytic nicking, deemed essential for a cytotoxic effect, occurs within this disulphide-bonded loop to generate the A1 and A2 fragments . Reduced A1 is then believed to translocate an internal membrane to inactivate protein synthesis in the cytosol . In this report, the disulphide-loop arginines of the SLT I A subunit were mutated to block the specific proteolysis presumed to occur . However, the mutant generated remained an effective toxin having similar catalytic activity to wild-type toxin and only a marginally reduced cytotoxicity towards cultured cells . We conclude that the disulphide-loop arginine residues are not the unique and essential processing sites previously assumed, but that processing may occur at alternative accessible sites to compensate for loss of target sites within the loop. Mol Microbiol, 1993 Oct, 10(1), 157 - 70 Analysis of the transfer region of the Streptomyces plasmid SCP2; Brolle DF et al.; pIJ903, a bifunctional derivative of the 31.4 kb low-copy-number, conjugative Streptomyces plasmid SCP2*, was mutagenized in Streptomyces lividans using Tn4560 . Mutant plasmids differing in their transfer frequencies, chromosome mobilization abilities, pock formation, and complementation properties were isolated . The mutations defined five transfer-related genes, traA, traB, traC, traD and spd, clustered in a region of 9 kb . The deduced sequences of the putative TraA and TraB proteins showed no overall similarity to known protein sequences, but the phenotype of traA mutant plasmids and sequence motifs in the putative TraA protein suggested that it might be a DNA helicase. Mol Microbiol, 1993 Oct, 10(2), 407 - 20 Identification and characterization of stationary phase-inducible genes in Escherichia coli; Weichart D et al.; During transition into stationary phase a large set of proteins is induced in Escherichia coli . Only a minority of the corresponding genes has been identified so far . Using the lambda placMu system and a plate screen for carbon starvation-induced fusion activity, a series of chromosomal lacZ fusions (csi::lacZ) was isolated . In complex medium these fusions were induced either during late exponential phase or during entry into stationary phase . csi::lacZ expression in minimal media in response to starvation for carbon, nitrogen and phosphate sources and the roles of global regulators such as the alternative sigma factor sigma s (encoded by rpoS), cAMP/CRP and the relA gene product were investigated . The results show that almost every fusion exhibits its own characteristic pattern of expression, suggesting a complex control of stationary phase-inducible genes that involves various combinations of regulatory mechanisms for different genes . All fusions were mapped to the E . coli chromosome . Using fine mapping by Southern hybridization, cloning, sequencing and/or phenotypic analysis, csi-5, csi-17, and csi-18 could be localized in osmY (encoding a periplasmic protein), glpD (aerobic glycerol-3-phosphate dehydrogenase) and glgA (glycogen synthase), respectively . The other fusions seem to specify novel genes now designated csiA through to csiF . csi-17(glpD)::lacZ was shown to produce its own glucose-starvation induction, thus illustrating the intricacies of gene-fusion technology when applied to the study of gene regulation. Mol Microbiol, 1993 Oct, 10(2), 341 - 50 A lowered concentration of cAMP receptor protein caused by glucose is an important determinant for catabolite repression in Escherichia coli; Ishizuka H et al.; A decreased intracellular concentration of cAMP is insufficient to account for catabolite repression in Escherichia coli . We show that glucose lowers the amount of cAMP receptor protein (CRP) in cells . A correlation exists between CRP and beta-galactosidase levels in cells growing under various conditions . Exogenous cAMP completely eliminates catabolite repression in CRP-overproducing cells, while it does not fully reverse the effect of glucose on beta-galactosidase expression in wild-type cells . When the CRP concentration is reduced by manipulating the crp gene, beta-galactosidase expression decreases in proportion to the concentration of CRP . These findings indicate that the lowered concentration of CRP caused by glucose is one of the major factors for catabolite repression . We propose that glucose causes catabolite repression by lowering the intracellular levels of both CRP and cAMP. Mol Microbiol, 1993 Oct, 10(2), 245 - 51 The galactose regulon of Escherichia coli; Weickert MJ et al.; Galactose transport and metabolism in Escherichia coli involves a multicomponent amphibolic pathway . Galactose transport is accomplished by two different galactose-specific transport systems . At least four of the genes and operons involved in galactose transport and metabolism have promoters containing similar regulatory sequences . These sequences are recognized by at least three regulators, Gal repressor (GalR), Gal isorepressor (GalS) and cAMP receptor protein (CRP), which modulate transcription from these promoters . The negative regulators, GalR and GalS, discriminate between utilization of the high-affinity (regulated by GalS) and low-affinity (regulated by GalR) transport systems, and modulate the expression of genes for galactose metabolism in an overlapping fashion . GalS is itself autogenously regulated and CRP dependent, while the gene for GalR is constitutive . The gal operon encoding the enzymes for galactose metabolism has two promoters regulated by CRP in opposite ways; one (P1) is stimulated and the other (P2) inhibited by CRP . Both promoters are strongly repressed by GalR but weakly by GalS . All but one of the constituent promoters of the gal regulon have two operators . The gal regulon has the potential to coordinate galactose metabolism and transport in a highly efficient manner, under a wide variety of conditions of galactose availability. Mol Microbiol, 1993 Oct, 10(2), 225 - 31 Control and function of lysyl-tRNA synthetases: diversity and co-ordination; Nakamura Y et al.; Lysyl-tRNA synthetases are synthesized from two distinct genes in Escherichia coli, lysS (constitutively) and lysU (inducibly); however, the physiological significance and the differential control mechanism of these two genes have been a long-standing puzzle . Recent studies have successfully uncovered a significant control mechanism of lysU expression, which involves the leucine-responsive regulatory protein (Lrp) and a translational enhancer element called 'downstream box' . Moreover, it is likely that there is a mechanism underlying co-ordinate expression of lysU with other genes outside the leucine-Lrp regulon under harsh conditions such as low pH and anaerobiosis . A possible mechanism of lysyl-tRNA synthetase expression and function is reviewed. Microb Pathog, 1993 Oct, 15(4), 283 - 91 Expression of a truncated guanylate cyclase (GC-C), a receptor for heat-stable enterotoxin of enterotoxigenic Escherichia coli, and its dimer formation in COS-7 cells; Hirayama T et al.; A fragment of guanylate cyclase C (GC-C) of about 1.7 k bp corresponding to amino acids 1-553 spanning the extracellular and transmembrane domains and a portion of the intracellular region was amplified using template cDNA prepared from rat intestinal cells by the polymerase chain reaction method . The cloned 1.7 k bp fragment was inserted into the mammalian expression vector pCGUT and the truncated GC-C expressed on the surface of COS-7 cells was demonstrated to bind heat-stable enterotoxin by photo affinity labeling with 125I-N-5-azidonitrobenzoyl-STh{5-19} . Analysis by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis showed that the truncated GC-C formed dimers on the surface of COS-7 cells . The intracellular region of GC-C was found not to be necessary for dimer formation by the GC-C . Comparison of the molecular weights of the truncated GC-C expressed in COS-7 cells and Escherichia coli suggested that the truncated GC-C was glycosylated in the mammalian expression system. Vet Microbiol, 1993 Oct, 37(1-2), 101 - 14 A monoclonal antibody identifies 2134P fimbriae as adhesins on enterotoxigenic Escherichia coli isolated from postweaning pigs; Dean-Nystrom EA et al.; Fimbriae (pili) of enterotoxigenic Escherichia coli (ETEC), including K88, K99, 987P, and F41, are adhesins that facilitate intestinal colonization in neonatal pigs . K88 is also associated with some ETEC isolated from weaned pigs . Many ETEC isolates from weaned pigs do not express known adhesins and are termed 4P- . A novel bacterial adhesin, 2134P, was recently identified on two 4P- ETEC isolates from weaned pigs . In this study, we identified a 2134P-specific monoclonal antibody, mAb 6C7/C1, that blocked the binding of 2134P+ bacteria to intestinal epithelial cells . Indirect immunofluorescent antibody and immunoperoxidase assays using mAb 6C7/C1 confirmed that the 2134P adhesin is expressed in vivo by adherent bacteria in pigs challenge-exposed with 2134P+ ETEC . 2134P was detected on 31% of 189 postweaning diarrhea 4P- ETEC isolates from the National Animal Disease Center's culture collection by dot blot immunoperoxidase assays using mAb 6C7/C1 . We conclude that 2134P is a bacterial adhesin and is an important virulence attribute of some ETEC that cause diarrhea in weaned pigs. Anim Genet, 1993 Oct, 24(5), 393 - 6 Evidence for linkage between K88ab, K88ac intestinal receptors to Escherichia coli and transferrin loci in pigs; Guerin G et al.; Segregation at the loci coding for the K88ab and K88ac small intestinal receptors to E . coli adhesins (K88abR, K88acR) and at the transferrin (TF) locus was studied in 38 pig families including 273 piglets . The TF locus showed a segregation deviation towards the B variant while each of the K88 receptors behaved as a single autosomal dominant gene . Recombinants between K88abR and K88acR provide evidence that they are under the control of two different loci . Thirty-two triple backcross families were selected to test linkage and estimate recombination rates (theta) . Our results demonstrate that the two K88 receptor loci are closely linked (theta = 0.02) with a maximum lod score value (Zm) of 46.0 . In addition, they are linked to the TF locus, theta = 0.14, Zm = 19.6 for the K88abR locus and theta = 0.16, Zm = 17.9 for the K88acR locus . The estimated recombination rates, smaller in males than in females, are consistent with the order TF-K88abR-K88acR . This linkage thus localizes the K88 loci, as the TF locus, on chromosome 13. Anim Genet, 1993 Oct, 24(5), 333 - 8 A linkage group on pig chromosome 4 comprising the loci for blood group L, GBA, ATP1B1 and three microsatellites; Marklund L et al.; Restriction fragment length polymorphisms (RFLPs) were described for the porcine loci for beta-glucosidase (GBA) and the beta-polypeptide 1 of the Na+,K(+)-transporting ATPase (ATP1B1) . Linkage analyses using a three-generation pedigree provided evidence for the assignment of ATP1B1, GBA and two microsatellite loci (S0001 and S0067) to a previously described linkage group comprising the loci for blood group L (EAL) and an anonymous microsatellite (S0097) . The linear order of the six markers was determined with confidence by multipoint analyses and the length of the linkage group was estimated at 88cM . This linkage group was assigned to pig chromosome 4 on the basis of a previous physical localization of the ATP1B1 gene . In situ hybridization data for S0001 presented in this study were consistent with a localization on chromosome 4 and suggested a regional localization to 4p12-p13 . The present study reveals conflicting data concerning the genetic localization of the K88 loci controlling the expression of the receptors for the E . coli pilus antigens . One group has reported data suggesting a loose linkage between K88 and EAL, now mapped to chromosome 4, whereas two other groups have found linkage between K88 and the transferrin locus (TF), mapped to chromosome 13 by in situ hybridization. J Biochem (Tokyo), 1993 Oct, 114(4), 468 - 72 Self-assembly of symbionin, a chaperonin of intracellular symbiont; Morioka M et al.; Symbionin, a homologue of Escherichia coli GroEL, which functions as a molecular chaperone in the aphid endosymbiont, exists as a double-doughnut structure, consisting of two rings of seven 63-kDa subunits . Symbionin had a more labile oligomeric structure than GroEL and completely disassembled into monomeric (1-mer) components in 3 M urea . The urea-dissociated symbionin self-assembled into 14-mer symbionin in a Mg-ATP dependent manner . When 1-mer symbionin was incubated in the presence of Mg-ATP, its reassembly proceeded hyperbolically with time . The yield of reassembled symbionin increased in response to increase in the initial concentration of 1-mer symbionin . The reassembled symbionin not only had the same molecular mass as native symbionin but also exhibited the same ATPase and phosphotransferase activity, suggesting that the correct three-dimensional structure was restored in vitro . While the self-assembly of symbionin was markedly stimulated by the presence of reassembled symbionin, it was not affected by the presence of native symbionin, which may suggest that native symbionin contains component(s) inhibitory to its chaperoning activity . As in the self-assembly of GroEL, that of symbionin was stimulated by the presence of GroES. Mol Biochem Parasitol, 1993 Oct, 61(2), 159 - 69 Cloning of the triosephosphate isomerase gene of Plasmodium falciparum and expression in Escherichia coli; Ranie J et al.; A major supply of energy in the rapidly multiplying intraerythrocytic Plasmodium falciparum is from the glycolytic pathway . We have isolated the cDNA and genomic clones of the glycolytic enzyme, triosephosphate isomerase (TPI) by polymerase chain reaction (PCR) . Degenerate oligonucleotides obtained by reverse translation of conserved polypeptide sequences derived from TPIs of other organisms, were used to prime PCR on P . falciparum DNA . The P . falciparum TPI gene is interrupted by a single intron which divides the coding region into two exons . The coding region encodes a protein of 248 amino acids which is of the same size as TPIs from other organisms and shares 42-45% homology with other known eukaryotic TPIs . On comparison with human TPI the catalytic domain was found to be highly conserved, while significant variations occurred at the other regions in the protein sequence . The P . falciparum TPI gene was cloned into the expression vector pTrc99A and hyperexpressed as an unfused protein in Escherichia coli . The 28-kDa protein was shown to be catalytically active. Enferm Infecc Microbiol Clin, 1993 Oct, 11(8), 437 - 40 {DNA probe for the detection of uropathogenic strains of P-fimbriated Escherichia coli}; Rodriguez E et al.; BACKGROUND: P fimbria is one of the main factors of virulence of the uropathogenic Escherichia coli strains thus developing methods for its detection is of interest . P fimbriation may manifest through associated characteristic hemagglutination patterns . Another way of directly detecting its expression is by the PF test consisting in a specific agglutination test with latex particles which have the specific receptor of the fimbria incorporated . METHODS: The two phenotypic techniques (hemagglutination pattern using human, bovine, and sheep erythrocytes, and the PF test) were compared with colony hybridization with a specific DNA probe (pap1) in 35 strains of uropathogenic E . coli . RESULTS: Eight of the 35 strains studied were positive for the PF test with 7 strains presenting mannose-resistant agglutination to human erythrocytes without agglutinating the other erythrocytes tried . However, with hybridization with the DNA probe the number of positives was higher (25/35) . CONCLUSIONS: The difference found in the number of positive strains may be due to the probe used corresponding to cluster pap, thus the use of a smaller more specific probe for fimbrial expression obtained from pap should be used given that the hybridization technique is easy to perform and is carried out in less time than phenotypic detection which requires long periods of culture prior to the test. Curr Opin Biotechnol, 1993 Oct, 4(5), 564 - 72 Baculovirus systems for the expression of human gene products; Luckow VA; Significant advances in basic and applied biology have resulted from the use of baculovirus vectors for the expression of heterologous proteins in cultured insect cells and in insect larvae . The development of improved vectors has greatly facilitated the construction of recombinant baculoviruses, both by increasing the efficiency of identifying recombinant viruses and by reducing or eliminating the tedious steps used to purify the desired recombinant virus from its non-recombinant parent virus. Curr Opin Biotechnol, 1993 Oct, 4(5), 520 - 5 Recent advances in heterologous gene expression in Escherichia coli; Olins PO et al.; Recent advances in protein expression in E . coli have focused primarily on the enhancement of protein quality . Problems in mRNA translation such as inefficient initiation, mistranslation, frame-shifting and frame-hopping can often be addressed by altering heterologous gene-coding sequences . Fusion technology can also be used to address problems in translational initiation, the authenticity of amino-terminal amino acids, in vivo protein activity and protein purification . Accessory molecules, such as chaperones, are increasingly used to enhance protein quality in vivo and in vitro . E . coli has recently gained wide use as a host both for the engineering of proteins with altered activities and for the creation of multi-functional hybrids. Biotechnology (N Y), 1993 Oct, 11(10), 1166 - 70 High level production of hybrid potyvirus-like particles carrying repetitive copies of foreign antigens in Escherichia coli; Jagadish MN et al.; Synthesis in E . coli of native coat protein of Johnsongrass mosaic virus, and hybrid protein molecules containing foreign antigens, resulted in the intracellular formation of potyvirus-like particles (PVLPs) . The foreign antigens used were an octapeptide epitope from Plasmodium falciparum and a decapeptide hormone (luteinizing hormone releasing hormone) at the N- or at both N- and C-terminal regions of the coat protein molecule, and a full length protein antigen (Sj26-glutathione S-transferase of 26 kD from Schistosoma japonicum) replacing the N-terminal 62 amino acids of the coat protein . Electron microscopy of ultrathin sections of E . coli revealed that PVLPs resulting from coat protein molecules containing peptide fusions appeared in vast arrays of parallel strands within the cytoplasm sometimes extending the length of the cell and at times the cells were strung together, with "threads" of PVLPs appearing to connect individual bacterial cells . PVLPs resulting from the fusion of the 26 kD antigen Sj26 to coat protein were shorter and wider . The physical form of the high molecular weight PVLPs enabled purification by simple size exclusion column chromatography . The Sj26-PVLPs administered to mice without adjuvant elicited antibody responses comparable to monomeric Sj26 administered with Freund's Complete Adjuvant. Biotechnology (N Y), 1993 Oct, 11(10), 1138 - 43 Use of peptide libraries to map the substrate specificity of a peptide-modifying enzyme: a 13 residue consensus peptide specifies biotinylation in Escherichia coli; Schatz PJ; I describe a technique for screening peptide libraries of over 10(9) independent clones for substrates of peptide-modifying enzymes . The peptides, linked to their genetic material by the lac repressor, are exposed to the enzyme and then screened by affinity purification on a receptor specific for the modified product . The enzyme characterized, E . coli biotin holoenzyme synthetase, normally adds biotin to a specific lysine residue in complex protein domains . The 13 residue substrate identified by this library screening approach is much smaller than the 75 amino acid required sequence of the natural substrate, and can function at either end of a fusion protein . The sequence is quite distinct at some positions from that region of the natural substrates, presumably because the peptides have to mimic the folded structure formed by the natural substrate . This technique should be useful for mapping the substrate specificity of a variety of peptide-modifying enzymes . In addition, small peptide substrates that are enzymatically biotinylated at a single site should be useful for a variety of purposes in labeling, purification, detection, and immobilization of proteins. Gut, 1993 Oct, 34(10), 1405 - 11 Electrogenic colonic ion transport in Hirschsprung's disease: reduced secretion to the neural secretagogues acetylcholine and iloprost; Hardy SP et al.; The effects of the abnormal innervation in Hirschsprung's disease on colonic ion transport were examined in vitro using Ussing chambers . The response of the mucosal/submucosal preparations to different secretagogues were investigated in aganglionic and ganglionic rectosigmoid and transverse colon from children with Hirschsprung's disease and compared with normally innervated colon from children with anorectal anomalies . Basal values were similar in aganglionic and ganglionic rectosigmoid colon . Neurally mediated secretion with iloprost (10(-6) M) and acetylcholine (900 and 9 microM) was considerably reduced in aganglionic colon compared with normally innervated ganglionic colon . The ganglionic colon proximal to the aganglionic colon also had a reduced response to acetylcholine despite a normal acetylcholinesterase staining pattern . The responses to Escherichia coli STa enterotoxin (50 MU/ml) and isobutylmethylxanthine (10(-3) M) were similar in ganglionic and aganglionic colon . The response to STa enterotoxin was not changed by the nerve blocking agent tetrodotoxin (10(-6) M) . The data show that colonocytes from aganglionic colon are capable of a normal secretory response if stimulated directly by cAMP or cGMP acting secretagogues but secretion in response to neurally mediated secretagogues is impaired . The hypertrophied acetylcholinesterase positive nerve fibres that infiltrate the aganglionic colon are likely to contribute to the reduced secretion to acetylcholine. J Lab Clin Med, 1993 Oct, 122(4), 388 - 94 Inhibitors of nitric oxide synthase attenuate human neutrophil chemotaxis in vitro; Belenky SN et al.; Products released through the L-arginine/nitric oxide biosynthetic pathway regulate soluble guanyl cyclase activity, which in turn modulates polymorphonuclear leukocyte chemotaxis . We hypothesized that inhibitors of nitric oxide synthase attenuate polymorphonuclear leukocyte chemotaxis in vitro . To test this hypothesis, unstimulated polymorphonuclear leukocytes were pretreated with buffer or the nitric oxide synthase inhibitors NG-monomethyl-L-arginine (L-NMMA), NG-nitro-L-arginine methyl ester, and L-canavanine before being exposed to three structurally unrelated chemoattractants, N-formyl-methionyl-leucyl-phenylalanine, C5a des arginine, and leukotriene B4 . Polymorphonuclear leukocyte chemotaxis was quantified with a modified blind-well chamber technique . We found that L-NMMA and L-canavanine but not NG-nitro-L-arginine significantly attenuated polymorphonuclear leukocyte chemotaxis (p < 0.05) . L-Arginine but not D-arginine, the nitric oxide donor sodium nitroprusside, and 8-bromo-cyclic guanosine monophosphate restored polymorphonuclear leukocyte chemotaxis attenuated by L-NMMA . Chemotaxis of polymorphonuclear leukocytes primed with lipopolysaccharide (Escherichia coli 0127:B8) or phorbol-13-butyrate was also significantly attenuated by pretreatment with L-NMMA and L-canavanine . Consistent with these observations, intracellular concentrations of cyclic guanosine monophosphate in polymorphonuclear leukocytes was decreased by L-NMMA during exposure to N-formyl-methionyl-leucyl-phenylalanine . These data indicate that nitric oxide synthase inhibitors attenuate chemotaxis of unstimulated and primed polymorphonuclear leukocytes in vitro . We suggest that the L-arginine/nitric oxide biosynthetic pathway plays an important role in regulating polymorphonuclear leukocyte emigration in vivo. Proc Natl Acad Sci U S A, 1993 Oct 1, 90(19), 8763 - 8 An operational RNA code for amino acids and possible relationship to genetic code; Schimmel P et al.; RNA helical oligonucleotides that recapitulate the acceptor stems of transfer RNAs, and that are devoid of the anticodon trinucleotides of the genetic code, are aminoacylated by aminoacyl tRNA synthetases . The specificity of aminoacylation is sequence dependent, and both specificity and efficiency are generally determined by only a few nucleotides proximal to the amino acid attachment site . This sequence/structure-dependent aminoacylation of RNA oligonucleotides constitutes an operational RNA code for amino acids . To a rough approximation, members of the two different classes of tRNA synthetases are, like tRNAs, organized into two major domains . The class-defining conserved domain containing the active site incorporates determinants for recognition of RNA mini-helix substrates . This domain may reflect the primordial synthetase, which was needed for expression of the operational RNA code . The second synthetase domain, which generally is less or not conserved, provides for interactions with the second domain of tRNA, which incorporates the anticodon . The emergence of the genetic from the operational RNA code could occur when the second domain of synthetases was added with the anticodon-containing domain of tRNAs. Mutat Res, 1993 Oct, 297(3), 313 - 21 Some aspects of EMS-induced mutagenesis in Escherichia coli; Grzesiuk E et al.; AB2497 and its mutS and umuDC derivatives were EMS-treated at the stationary phase and specificity of mutation measured . It was found that: (i) in mutS+ cells EMS induces predominantly GC-->AT transitions (by supB or supE(oc) formation) and in mutS- cells mainly AT-->TA transversions (by supL(NG) formation); (ii) transversions of AT-->TA are umuDC-dependent and mutational specificity is biased towards AT-->GC transitions in mutS- umuDC- strains . When mutS- umuDC- cells were transfected with plasmids bearing umuD'C or umuDC genes, mutational specificity was again biased towards AT-->TA transversions; (iii) experiments with bacteria bearing umuC::lacZ or recA::lacZ fusions suggest that processing of UmuD-->UmuD' might be poorer in EMS-treated mutS- than in mutS+ cells. Mutat Res, 1993 Oct, 294(3), 317 - 23 Analysis of mutations caused by DNA double-strand breaks produced by a restriction enzyme in shuttle vector plasmids propagated in ataxia telangiectasia cells; Tatsumi-Miyajima J et al.; Rejoining of DNA double-strand breaks (DSB) produced by a restriction endonuclease AvaI in the supF gene in a plasmid pZ189Ava, and mutations presumably due to the altered rejoinings were analyzed . After allowing the rejoining and replication of the plasmids in human cells originating from normal subjects and ataxia telangiectasia (AT) patients, the plasmids were retrieved and those containing mutated supF were screened in an indicator strain of Escherichia coli . The proportion of correctly rejoined plasmids was significantly lower in AT cells than in normal cells, suggesting that AT cells have lower fidelity in rejoining DSB . DNA sequencing of the mutated supF genes revealed that all mutations were deletions or insertions occurring exactly or closely at the rejoining site in both normal and AT cells . In AT cells, the majority of mutations were deletions, while deletions and insertions were evenly formed in normal cells . AT cells may be deficient in the mechanism to protect the broken ends of DNA strands from the exonucleolytic digestion. Mutat Res, 1993 Oct, 294(3), 263 - 74 The first zinc-binding domain of UvrA is not essential for UvrABC-mediated DNA excision repair; Visse R et al.; Specific mutations in uvrA were introduced to analyze the role of the zinc-binding domains of the protein in DNA excision repair . Zinc-coordinating cysteines were substituted into non-coordinating serine or glycine residues . Mutations leading to changes in the second zinc-binding domain had a profound effect on UV survival in vivo; however these mutant proteins could not be isolated for in vitro analyses . Amino acid substitutions in the first zinc-binding domain had very little effect on UV survival in vivo . In vitro analyses showed that although this domain no longer coordinates zinc, ATPase activity, helicase activity, DNA binding, incision of damaged DNA and DNA repair synthesis appeared to be normal . Therefore it seems that the first zinc-binding domain of UvrA is not essential for DNA excision repair. Mutat Res, 1993 Oct, 294(3), 223 - 34 mei-3, a recombination and repair gene of Neurospora crassa, encodes a RecA-like protein; Cheng R et al.; Neurospora crassa mei-3 is a mutant which exhibits meiotic and mitotic defects and mutagen sensitivity . Its defect is believed to be in recombination and repair . We have cloned the mei-3 gene from a N . crassa cosmid library of genomic DNA . Restriction fragment length polymorphism analysis determined the location of the cloned fragment was on chromosome one in approximately the same position that was previously reported for mei-3 by classical genetic methods . Deletion analysis showed the approximate coding region of mei-3 on the cloned genomic fragment . Northern blot analysis identified a 900-bp transcript . Sequencing revealed a 798-bp open reading frame with high coding preference which could encode a protein having a molecular weight of approximately 29,000 . The predicted protein product of mei-3 has significant identity to the Rad51 and Dmc1 proteins from Saccharomyces cerevisiae . The mei-3 gene and both yeast genes have significant primary sequence homology with RecA, a recombination protein identified in Escherichia coli . The results suggest RecA-like proteins involved in DNA recombination and repair are highly conserved in eukaryotes. Mutat Res, 1993 Oct, 294(3), 215 - 22 Excision repair reduces doxorubicin-induced genotoxicity; Anderson RD et al.; LacI mutations induced by doxorubicin in a wild-type, uvr(A)BC repair-proficient E . coli strain were analyzed by DNA sequencing . These mutations were contrasted with mutations previously recovered from doxorubicin-treated uvrB- organisms in order to assess the role of excision repair in doxorubicin-induced genotoxicity . After a 30-min exposure of wild-type E . coli to 330 microM doxorubicin, survival was 34% and the overall lacI mutation frequency increased 1.8-fold to 340 x 10(-8) . The distribution of doxorubicin-induced mutants among subclasses of mutation involving the i-d and lac operator regions differed significantly between repair-proficient and -deficient strains . Distributional differences appeared to result both from a decrease in deletions involving the lac operator and an increase in base substitutions involving the i-d region in repair proficient organisms . However, elements of the doxorubicin-induced mutation spectrum in uvrB- E . coli are still discernable in wild-type organisms . These elements include the remarkable shift of 3'-deletion endpoints to palindromic sequence within the lac operator and the recovery of multiple isolates of T:A-->A:T transversions at position 96 in doxorubicin-treated cultures . These observations suggest that components of the uvr(A)BC nucleotide excision repair system function through a general mechanism prior to fixation of mutations to reduce, but not completely eliminate, the genotoxic effects of doxorubicin. Mutat Res, 1993 Oct, 292(2), 175 - 85 The Escherichia coli galK2 papillation assay: its specificity and application to seven newly isolated mutator strains; Oller AR et al.; The Escherichia coli dnaE and dnaQ genes encode, respectively, the alpha (polymerase) and epsilon (proofreading) subunits of DNA polymerase III . Mutations in these genes resulting in mutator or antimutator phenotypes provide important tools to understand the mechanisms by which mutations occur . One way to isolate such strains is the use of papillation assays . We used one such assay based on the reversion of the galK2 allele in cells grown on MacConkey-Gal plates . Here, we describe the identification of the galK2 mutation and its possible reversion pathways, and the characterization of 7 mutators isolated using this system . 1 mutator resided in dnaE and 6 in dnaQ . Sequencing of the galK2 allele revealed a G.C-->T.A transversion at base pair 571 that changed a glu codon (GAA) to a stop codon (TAA) . The analysis of 319 revertants showed that a Gal+ phenotype can be achieved by A.T-->G.C transition, A.T-->T.A transversion and A.T-->C.G transversion . We characterized the mutator phenotypes of the newly isolated mutators by determining (i) their mutation frequencies to resistance to rifampicin and nalidixic acid in both wild-type and mutL backgrounds, (ii) their temperature sensitivity and medium dependence and (iii) their mutational specificity (by analyzing the nature of galK revertants) . Based on the genomic locations of their mutations, specificity of reversion pathways and magnitude of mutator effects, the mutators can be grouped into 3 classes . These classes may represent different mutational mechanisms that include defective base insertion, defective proofreading and interference with the postreplicative mismatch-repair system. J Gen Virol, 1993 Oct, 74 ( Pt 10), 2171 - 9 Conformation-dependent recognition of baculovirus-expressed Epstein-Barr virus gp350 by a panel of monoclonal antibodies; Zhang PF et al.; The Epstein-Barr virus (EBV) major membrane protein, gp350, induces antibodies that neutralize virus infectivity in vitro and is a potential candidate for an EBV vaccine . Full-length EBV gp350 and five protein fragments, encompassing the entire protein sequence, were generated in a baculovirus expression system . The recombinant proteins were analysed using a panel of 14 monoclonal antibodies (MAbs) (13 prepared against native gp350 derived from virus-producing cells and one prepared against an Escherichia coli recombinant protein) . All 14 MAbs, including a virus-neutralizing antibody, reacted with the full-length recombinant gp350 in a dot blot immunoassay, but only four of the 14 MAbs reacted with polypeptides expressed by the five subclones, indicating that the full-length protein, but not the protein fragments, was antigenically similar to native gp350 . Treatment of the six recombinant proteins with peptide-N-glycosidase F (PNGase F) indicated that the full-length gp350 protein and the N-terminal fragment were glycosylated and that the four internally initiated polypeptides were not glycosylated . PNGase F treatment of the full-length glycosylated gp350 did not eliminate its reactivity with all of the 10 MAbs examined (including the neutralizing MAb) in a dot blot immunoassay; however, denatured glycosylated gp350 lost reactivity with all but four of the 14 MAbs when analysed by either dot blot or Western blot immunoassay . The data suggest that conformational epitopes are more important in recognition of gp350 by this panel of MAbs than glycosylation sites, and that the epitope on gp350 recognized by the neutralizing MAb is conformation- and not glycosylation-dependent. J Bacteriol, 1993 Oct, 175(20), 6663 - 70 Expression of the Escherichia coli dnaX gene; Chen KS et al.; The Escherichia coli dnaX gene encodes both the tau and gamma subunits of DNA polymerase III . This gene is located immediately downstream of the adenine salvage gene apt and upstream of orf12-recR, htpG, and adk . The last three are involved in recombination, heat shock, and nucleotide biosynthesis, respectively . apt, dnaX, and orf12-recR all have separate promoters, and the first two are expressed predominantly from those separate promoters . However, use of an RNase E temperature-sensitive mutant allowed the detection of lesser amounts of polycistronic messengers extending from both the apt and dnaX promoters through htpG . Interestingly, transcription of the weak dnaX promoter is stimulated 4- to 10-fold by a sequence contained entirely within the dnaX reading frame . This region has been localized; at least a portion of the sequence (and perhaps the entire sequence) is located within a 31-bp region downstream of the dnaX promoter. J Bacteriol, 1993 Oct, 175(19), 6293 - 8 Induction of EcoRII methyltransferase: evidence for autogenous control; Friedman S et al.; The cytosine analog 5-azacytidine kills Escherichia coli cells that carry plasmids expressing EcoRII DNA (cytosine 5)methyltransferase under control of its own promoter . We previously showed that this enzyme binds tightly to azacytidine-containing DNA in vitro and proposed that such binding is lethal in vivo . In support of this proposal, we now show that the enzyme sediments with the nucleoid of azacytidine-treated cells . Azacytidine treatment led to an increase in the amount of enzyme, and this increase required sequences in the ecoRIIM promoter region . Enzyme inducibility correlated with drug sensitivity: plasmids carrying the methyltransferase gene but lacking the wild-type promoter did not confer sensitivity . These results suggested that the ecoRIIM gene was under autogenous control . Transcriptional ecoRIIM'-lacZ fusions in E . coli were, therefore, constructed . They showed that expression from the ecoRIIM promoter was inhibited when EcoRII DNA (cytosine-5)methyltransferase was introduced into the cell in trans and inhibition was reversed by treating the cells with azacytidine . These results provide evidence that the expression of the ecoRIIM gene is under autogenous regulation and that cell death induced by azacytidine is due, in part, to the disruption of autoregulation. EMBO J, 1993 Oct, 12(10), 3739 - 45 The topology of the brown adipose tissue mitochondrial uncoupling protein determined with antibodies against its antigenic sites revealed by a library of fusion proteins; Miroux B et al.; The uncoupling protein (UCP) of brown adipose tissue mitochondria is a specialized member of the family of evolutionarily related mitochondrial membrane transporters, which also includes the ADP/ATP translocator and the phosphate carrier . We have generated a library of bacterial clones randomly expressing short subsequences of the UCP fused to the MalE periplasmic protein of Escherichia coli . Anti-UCP sera were used to select clones expressing antigenic sequences of the UCP . Ten different fusion proteins representing eight non-overlapping subsequences of the UCP were obtained . The ability of fusion proteins to select antibodies directed against a short segment of the UCP was used to study the topological organization of the UCP in the inner mitochondrial membrane . Four different fusion proteins were used to determine the orientation of the N-terminal extremities of the first, second, third and fourth predicted alpha-helices of the UCP . This topological study together with previous data on the UCP provides an experimental basis for the predicted structure of the UCP and for other homologous carrier proteins. Mutat Res, 1993 Oct, 289(2), 255 - 63 C/G to A/T transversions represent the main type of mutation induced by gamma-irradiation in double-stranded M13mp10 DNA in a nitrogen-saturated solution; Braun JE et al.; To get more insight into the possible mutagenic consequences of DNA damage induced by radiation-generated H radicals (.H), a nitrogen-saturated solution of double-stranded (ds) M13mp10 DNA in phosphate buffer was irradiated with gamma-rays . Under these conditions 55% of the DNA-damaging species consists of H radicals and 45% of OH radicals (.OH) . The mutations were investigated in a 144-bp mutational target sequence inserted into the lacZ alpha gene . A very specific mutation spectrum was obtained with respect to the type of mutations . Twenty out of the 28 radiation-induced mutations were C/G to A/T transversions; the remaining 8 mutations were 4 C/G to G/C transversions, 2 C/G to T/A transitions, one T/A to A/T transversion and only one -1 bp deletion . The mutations were rather randomly distributed along the 144-bp mutation target sequence with no clear mutational hot spots . When these results are compared with those we have obtained previously after irradiation of ds M13mp10 DNA under O2 (100% .OH) or N2O (90% .OH; 10% .H) (Hoebee et al., 1988, 1989), the data strongly suggest that H radicals may be responsible for the observed C/G to A/T transversions but not for -1 bp deletions. Mutat Res, 1993 Oct, 289(2), 205 - 14 Mutagenic and recombinagenic effects of diethylstilbestrol quinone; Korah RM et al.; Estrogens are believed to be major contributors to many cancers of the human female genital tract, but the mechanism of their carcinogenic action is not well-understood . While a tumor-promoting role for estrogens is well-supported, whether they also act as tumor initiators has remained controversial . Here, we have sought to examine the mutagenic potential of diethylstilbestrol, a synthetic estrogen that is a powerful carcinogen in hamsters, and is suspected to be a human carcinogen . Phage M13 single-stranded DNA was treated in vitro with diethylstilbestrol quinone (DES Q: 1.25 mM) and transfected into Escherichia coli cells . DES Q treatment resulted in an apparent enhancement of mutagenesis in the LacZ(alpha) gene segment . DNA sequence analysis of LacZ(alpha) mutants obtained by transfection of DES Q-treated DNA revealed that the major effect of DES Q treatment has been a 6-fold elevation of recombination between the phage-borne LacZ(alpha) sequence and the LacZ delta M15 sequence on the E . coli fertility plasmid F . To confirm whether DES Q treatment is recombinagenic, we used an experimental system that allows the detection of recombination between a defective E . coli chromosomal LacY gene and a normal counterpart borne on a plasmid . Transfection of DES Q (0.06-12 mM) treated plasmid DNA showed significant enhancement (2-100-fold) in recombination, but not in mutagenesis . These results raise the possibility that estrogen quinones may induce recombinagenic DNA damage. Mutat Res, 1993 Oct, 289(2), 181 - 6 Effect of DNA-repair enzymes on mutagenesis by oxygen free radicals; Reid TM et al.; Cytosine to thymine transitions are among the most common types of mutations produced by oxygen damage to DNA . One possible mechanism for these transitions is deamination of cytosine to uracil . Using both a forward mutation assay as well as a reversion assay specific for damage to cytosines we show that direct deamination to uracil does not play a significant role in mutagenesis induced by reactive oxygen free radicals . In contrast, lesions sensitive to repair by E . coli endonuclease III play a major role in oxidative mutagenesis as evidenced by the ability of endonuclease III to modulate the extent of mutagenesis that results from exposure of DNA to oxygen free radicals. J Exp Med, 1993 Oct 1, 178(4), 1347 - 55 Lipopolysaccharides prime whole human blood and isolated neutrophils for the increased synthesis of 5-lipoxygenase products by enhancing arachidonic acid availability: involvement of the CD14 antigen; Surette ME et al.; Stimulation of heparinized blood with 1 microM formyl-methionyl-leucyl-phenylalanine (FMLP) resulted in the formation of < 30 pmol/ml plasma of 5-lipoxygenase (5-LO) products . The preincubation of blood with 1 microgram/ml of lipopolysaccharide (LPS) (Escherichia coli 0111-B4) for 30 min before stimulation with FMLP resulted in the accumulation of 250-300 pmol of 5-LO products per ml plasma . The major products detected were leukotriene B4 and (5S)-hydroxy-6,8,11,14-eicosatetraenoic acid which were produced in equivalent amounts . The priming activity was detectable with as little as 1-10 ng LPS per ml blood and was optimal using 1-10 micrograms LPS/ml blood . The priming for 5-LO product synthesis was optimal after 20-30 min of preincubation with LPS and declined at preincubation times > 30 min . The priming effect of LPS was also observed using the complement fragment C5a or interleukin 8 as agonists . Polymorphonuclear leukocytes (PMN) and peripheral blood mononuclear cells accounted for 80 and 20% of the synthesis of 5-LO products, respectively . The ability of LPS to prime isolated PMN was dependent on the presence of plasma and was inhibited by the anti-CD14 antibody IOM2, indicating a CD14-dependent priming mechanism . The priming of whole blood with tumor necrosis factor alpha (TNF-alpha) and LPS was additive and the presence of mononuclear cells did not enhance the ability of LPS to prime PMN, indicating that the priming activity of LPS is independent of LPS-induced TNF-alpha synthesis . The mechanism by which LPS enhance 5-LO product synthesis in PMN was investigated . Treatment of PMN with LPS strongly enhanced the release of arachidonic acid after stimulation with FMLP . The release of arachidonic acid was optimal 2-3 min after stimulation with FMLP, attaining levels 5-15-fold greater than those observed in unprimed cells stimulated with FMLP . These results demonstrate that LPS dramatically increases the ability of blood to generate 5-LO products, and support the putative role of leukotrienes in pathological states involving LPS. Virology, 1993 Oct, 196(2), 731 - 8 Disruption of a salt bridge between Asp 488 and Lys 465 in HIV-1 reverse transcriptase alters its proteolytic processing and polymerase activity; Goobar-Larsson L et al.; The conserved aspartic acid residue 488 in the RNase H domain of HIV-1 reverse transcriptase (RT) was mutated to alanine . RT was expressed in Escherichia coli alone or with the entire pol-gene polyprotein consisting of proteinase, RT, and integrase and processed by the HIV-1 proteinase in the bacterial cell . Expression of mutant RT together with the proteinase resulted in an overproduction of RT p51 vs p66 . The mutation also altered the conformation of the RT p66/p51 heterodimer as shown by the loss of binding of monoclonal antibodies to mutant RT in ELISA . Crystallographic data shows that a salt bridge exists between Asp 488 and Lys 465 of RNase H which stabilizes the uncleavable form of RT p66, and that substitution of Asp for Ala would prevent the formation of this salt bridge . Our results indicate that disruption of this salt bridge through mutation of Asp 488 interferes with the conformational changes that regulate the limited processing of p66 to 51 by the virus proteinase . Homology data suggest that such a bridge may be present in other lentiviruses . The mutation introduced caused a moderate decrease in both the RNase H activity and the polymerase activity of RT, indicating that the proper folding of the RNase H domain of RT is necessary to achieve full polymerase activity. Mol Microbiol, 1993 Oct, 10(1), 35 - 43 Structural and functional analyses of the FinP antisense RNA regulatory system of the F conjugative plasmid; van Biesen T et al.; The efficiency of conjugation of F-like plasmids is regulated by the FinOP fertility inhibition system . The transfer (tra) operon is under the direct control of the TraJ transcriptional activator which, in turn, is negatively regulated by FinP, an antisense RNA, and FinO, a 22 kDa protein . Recently, FinO has been shown to extend the chemical stability of FinP in vivo in the absence of traJ mRNA . The in vitro secondary structures of both the FinP and TraJ RNAs were determined by the use of single- and double-strand-specific nucleases; both RNAs were found to have double stem-loop structures that are complementary to each other and, therefore, FinP RNA and TraJ RNA have the potential to form a duplex with each other . This was verified by in vitro binding experiments . The reaction was shown to be biomolecular with an apparent rate constant (kapp) of 5 x 10(5)M-1s-1, a value that is similar to those found for other natural antisense RNA systems . Preliminary evidence for the in vivo formation of the FinP-TraJ RNA duplex was obtained by primer extension of the traJ mRNA; the presence of both FinO and FinP was required to cause a dramatic reduction in the steady-state level of traJ mRNA, perhaps as a result of RNase III degradation of the resulting RNA duplex. Plant Physiol, 1993 Oct, 103(2), 565 - 73 Identification, cDNA cloning, and gene expression of soluble starch synthase in rice (Oryza sativa L.) immature seeds; Baba T et al.; Three forms of soluble starch synthase were resolved by anion-exchange chromatography of soluble extracts from immature rice (Oryza sativa L.) seeds, and each of these forms was further purified by affinity chromatograph . The 55-, 57-, and 57-kD proteins in the three preparations were identified as candidates for soluble starch synthase by western blot analysis using an antiserum against rice granule-bound starch synthase . It is interesting that the amino-terminal amino acid sequence was identical among the three proteins, except that the 55-kD protein lacked eight amino acids at the amino terminus . Thus, these three proteins are products of the same gene . The cDNA clones coding for this protein have been isolated from an immature rice seed library in lambda gt11 using synthetic oligonucleotides as probes . The deduced amino acid sequence of this protein contains a lysine-X-glycine-glycine consensus sequence for the ADP-glucose-binding site of starch and glycogen synthases . Therefore, we conclude that this protein corresponds to a form of soluble starch synthase in immature rice seeds . The precursor of the enzyme contains 626 amino acids, including a 113-residue transit peptide at the amino terminus . The mature form of soluble starch synthase shares a significant but low sequence identity with rice granule-bound starch synthase and Escherichia coli glycogen synthase . However, several regions, including the substrate-binding site, are highly conserved among these three enzymes . Blot hybridization analysis demonstrates that the gene encoding soluble starch synthase is a single-copy gene in the rice genome and is expressed in both leaves and immature seeds . These results suggest that soluble and granule-bound starch synthases play distinct roles in starch biosynthesis of plant. J Protein Chem, 1993 Oct, 12(5), 525 - 31 Cysteine 17 of recombinant human granulocyte-colony stimulating factor is partially solvent-exposed; Arakawa T et al.; Oh-eda et al . have shown instability of granulocyte-colony stimulating factor (G-CSF) upon storage above pH 7.0 {J . Biol . Chem . (1990) 265, 11,432-11,435} . To clarify the mechanism of this instability, the accessibility of a free cysteinyl residue at position 17 for disulfide exchange reaction was examined using a sulfhydryl reagent . The results show that the cysteine is partially solvent-exposed in both glycosylated and nonglycosylated forms, suggesting that the exposure of the cysteine plays a critical role in the instability of the protein . This is supported by the facts that at low pH where the cysteine is protonated, both proteins have much greater stability and that a Cys17-->Ser analog is extremely stable at neutral pH and 37 degrees C . It was observed that the rate of sulfhydryl titration is slower for the glycosylated form than for the nonglycosylated form, suggesting that the cysteine residue is less solvent-exposed for the former protein or that the pKa is somewhat more basic . In either case, the carbohydrate appears to affect the reactivity of the sulfhydryl group through steric hindrance or alteration in local conformation . Both the glycosylated and nonglycosylated proteins showed essentially identical conformation as determined by circular dichroism, fluorescence, and infrared spectroscopy . Unfolding of these two proteins, induced either by guanidine hydrochloride or by pH, showed an identical course, indicating comparable conformational stability . Contribution of conformational changes to the observed instability at higher pH is unlikely, since little difference in fluorescence spectrum occurs between pH 6.0 and 8.0.(ABSTRACT TRUNCATED AT 250 WORDS) Avian Dis, 1993 Oct-Dec, 37(4), 1105 - 12 Cloning and partial sequence analysis of a Mycoplasma synoviae DNA fragment encoding epitopes shared with the major adhesin P1 protein of Mycoplasma pneumoniae; Morsy MA et al.; Polyclonal antibodies specific for the adhesin P1 protein of Mycoplasma pneumoniae were used to screen an expression library of M . synoviae genomic DNA constructed in the expression vector lambda gt11 . Following several cycles of immunoscreening using the anti-P1 antiserum, a lambda gt11 recombinant clone containing 3.9 kilobase pairs (kbp) of M . synoviae DNA was identified and isolated from the expression library . Expression of the recombinant clone (designated MS-1) in Escherichia coli Y1089 followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the crude E . coli lysates revealed the presence of two novel proteins . Two antibodies that recognize the adhesin polypeptide--chicken anti-M . synoviae antibodies and anti-P1 antiserum--reacted with both proteins on immunoblots . Partial sequence analysis of the M . synoviae DNA in clone MS-1 and computer comparison of the predicted amino-acid sequences with existing protein sequence files revealed homology with the adhesin P1 protein of M . pneumoniae. Vaccine, 1993 Oct, 11(13), 1341 - 6 Induction of cytolytic and antibody responses using Plasmodium falciparum repeatless circumsporozoite protein encapsulated in liposomes; White K et al.; Plasmodium circumsporozoite (CS) protein-induced antibody and T-cell responses are considered to be important in protective immunity . Since the key repeat determinant of the CS protein may actually restrict the recognition of other potential T- and B-cell sites, a modified Plasmodium falciparum CS protein lacking the central repeat region, RLF, was expressed in Escherichia coli . On purification, RLF was encapsulated into liposomes {L(RLF)} and used for the in vivo induction of cytolytic T lymphocytes (CTL) and antibodies . Immunization of B10.Br (H-2k) mice with L(RLF), but not with RLF, induced CD8+ CTL specific for the P . falciparum CS protein CTL epitope, amino acid residues 368-390 . Anti-L(RLF) serum reacted with antigens on intact sporozoites and inhibited sporozoite invasion of hepatoma cells . Antibody specificity studies in New Zealand White rabbits revealed new B-cell sites localized in amino acid residues 84-94, 91-99, 97-106 and 367-375 . Although the mechanisms by which liposomes enhance cellular and humoral immune responses remain unknown, liposome-formulated vaccines have been well tolerated in humans; hence, their use in vaccines, when efficacy depends on antibody and CTL responses, may be broadly applicable. Vaccine, 1993 Oct, 11(13), 1310 - 5 Peptide sequences with strong stimulatory activity for lymphoid cells: implications for vaccine development; Russell-Jones GJ et al.; Seven peptides derived from the bacterial major outer-membrane protein TraT were synthesized and then tested in lymphoproliferative assays using lymphoid cells from a variety of animals that had been immunized with the native TraT molecule in saline . A hierarchical pattern of responsiveness to the peptides was observed in the four animal species studied and in particular three of the peptides (T2, T4 and T6) showed very strong responses in all species . The 'universality' of the TraT-derived peptides was confirmed by studying the responsiveness of lymphoid cells obtained from the peripheral blood of twenty clinically normal human donors . Thus, following a secondary in vitro immunization with TraT-pulsed human peripheral blood mononuclear cells, responsiveness to TraT and to the TraT-derived peptides was observed in the cultures derived from all twenty donors . Taken together, our findings imply that the putative T-cell epitope peptides (T2, T4 and T6) could be employed as carriers in subunit vaccines and thereby help to overcome the unresponsiveness observed in animals and humans as a result of MHC restriction. Comput Appl Biosci, 1993 Oct, 9(5), 551 - 61 Automatic display of RNA secondary structures; Muller G et al.; A set of programs written in C language with the GL library and under UNIX has been developed for generating compact, pleasant and non-overlapping displays of secondary structures of ribonucleic acids . The first program, rnasearch, implements a new search procedure that dynamically rearranges overlapping portions of the two-dimensional drawing while preserving clear and readable displays of the two-dimensional structure . The algorithm is fast (the execution time for the command rnasearch is 38.6 s for the 16S rRNA of Escherichia coli with 1542 bases), accepts outputs from two-dimensional prediction programs and therefore allows for rapid comparison between the various two-dimensional folds generated . A second program, rnadisplay, allows the graphical display of the computed two-dimensional structures on a graphics workstation . Otherwise, it is possible to obtain a paper output of the two-dimensional structure by using the program print2D which builds a Postscript file . Moreover the two-dimensional drawing can be labelled for representing data coming from chemical modifications and/or enzymatic cleavages . Application to a few secondary structures such as RNaseP, 5S rRNA and 16S rRNA are given. Arch Oral Biol, 1993 Oct, 38(10), 903 - 10 The fate of genetically marked human oral keratinocytes in vitro; Garlick JA et al.; The fate of the progeny of human oral gingival keratinocytes was mapped in stratified epithelial tissues in vitro by following the expression of a marker gene in genetically related clones . Oral epithelial progenitor cells were genetically marked at high efficiency by transducing them with a retrovirus vector that carried the gene for a histochemically detectable product, Escherichia coli beta-galactosidase (beta-gal) . These cells were then grown in submerged cultures and on collagen rafts at the air-liquid interface to demonstrate the distribution of genetically marked cells in a differentiating tissue in vitro . The dynamics of transduced cells showed that clonally related cells were arranged in discrete units of labelled cells and these clusters were defined as 'clonal proliferation units' . The size and configuration of these units were related to the proliferative potential and differentiating capacity of the cell that was initially transduced . This model demonstrates the relation between clonally related cells and tissue architecture for oral keratinocytes in vitro. Bioorg Khim, 1993 Oct, 19(10), 981 - 8 {Computerized structural analysis of O-specific polysaccharides O1A, O1B, and O1C from Escherichia coli}; Nifant'ev NE et al.; A computer evaluation of 13C-NMR data for the title polysaccharides based on the monosaccharide and methylation analysis data led to the structure of the repeating unit of the O1A polysaccharide as well as to several probable structures of the O1C polysaccharide, of which the correct one was inferred by means of a single NOE experiment . The analysis of the spectrum of the O1B polysaccharide was unsuccessful, due to the presence in its structure of the fragment alpha-L-Rha-(1-->2)-alpha-D-Gal-(1-->3)-D-GlcNAc with the terminal (1-->2)-linkage, whose spectral data could not be calculated by additive schemes using only glycosylation effects . However in reevaluation of the O1B spectral data by taking into account the deviations from additivities of the chemical shifts values in spectra of the related trisaccharides, to reveal the most probable structure of the O1B's repeating unit . {formula: see text} New Microbiol, 1993 Oct, 16(4), 323 - 32 Classification of Italian isolates of Borrelia burgdorferi into three genomic groups; Cinco M et al.; In this study we investigated the genotypic characteristics of some locally isolated strains of B . burgdorferi by three different methodologies: restriction endonuclease analysis (REA), Southern blot hybridization with whole DNAs from Borrelia strains and Southern blot hybridization with rRNA 16 + 23S genes derived from E . coli . REA fingerprintings were evaluated by cluster analysis, according to the principles of numerical taxonomy . The genomas of the locally isolated strains were compared with borreliae originating from different countries of Europe, including Sweden and with the American reference strain B31 . Among the European strains, some already described by Baranton (Baranton et al., 1992) as representatives of different genomic groups Borrelia sensu stricto and Borrelia garinii were used . By the different techniques the isolates were included in three genomic groups which could correspond to the three genospecies identified by Baranton, namely B . burgdorferi sensu stricto, B . garinii and B . group VS461: in fact two strains were included in a homogeneous group, probably corresponding to the VS461 genomic group, together with other European borreliae; one isolate was included in a group consisting of B31 and some other European strains already described as belonging to Borrelia burgdorferi in sensu stricto . Finally two isolates were ascribed to a third genomic group probably corresponding to the genospecies indicated as Borrelia garinii . These findings indicate that a small number of Borrelia strains isolated from a very restricted area can be genetically heterogeneous. J Cardiovasc Pharmacol, 1993 Oct, 22(4), 550 - 6 Antishock effect of U-67,590A, methylprednisolone suleptanate, associated with restoration of lowered vascular reactivity in endotoxemic rats; Nomura S et al.; We wished to investigate the modulating effects of a glucocorticoid on mortality and sustained hypotension in endotoxemic rats in conjunction with in vitro study of responses of isolated aorta to KCl, norepinephrine (NE), and acetylcholine (ACh) . Endotoxemia was induced by intravenous (i.v.) bolus injection of 50 mg/kg Escherichia coli endotoxin in rats, resulting in high mortality . Pretreatment with U-67,590A, methylprednisolone suleptanate, at a dose of 9 mg/kg resulted in 100% survival; the survival rate of saline-treated controls was 21% . Isolated rat aorta that had been treated with endotoxin for 4 h showed decreases in contractile responses to KCl and NE and in relaxing response to ACh . Similar attenuation of contractile responsiveness was observed in endothelium-denuded preparations . Addition of endotoxin to the in vitro tissue bath did not inhibit the responses in a 4-h period . Pretreatment with U-67,590A inhibited the late gradual decrease in blood pressure (BP) but not the early hypotensive response to endotoxin . The responses to KCl or NE of aorta isolated 4 h after the endotoxin injection remained suppressed in U-67,590A-treated rats but were restored in 24 h . These results suggest that endotoxemia impairs the endothelium moderately, but this does not account for either reduced reactivity of vascular smooth muscle to vasoconstrictor agents or the sustained hypotension . The steroid inhibits endotoxemia-induced mortality and sustained hypotension. Leuk Lymphoma, 1993 Oct, 11(3-4), 249 - 62 Cytotoxicity of a recombinant diphtheria toxin-granulocyte colony-stimulating factor fusion protein on human leukemic blast cells; Chadwick DE et al.; Granulocyte colony-stimulating factor (G-CSF) is a potent stimulator of the growth of normal and malignant hematopoietic cells and synergizes with other factors such as interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) . The action of G-CSF is mediated through a specific membrane receptor, however it is not clear if all of the effects of G-CSF are direct or indirect . As a step towards addressing this problem, a recombinant diphtheria toxin (DT)-related human G-CSF fusion protein has been constructed and purified from E . coli . The 70,000 dalton chimeric protein has immunologic determinants characteristic of both DT and G-CSF . At high concentrations, DAB486-G-CSF is cytotoxic towards G-CSF-dependent OCI/AML1 cells, but not factor independent OCI/AML3 cells; colony formation by G-CSF-responsive leukemic blasts from a patient with acute myeloblastic leukemia (AML) was also inhibited . The G-CSF fusion toxin displayed ADP-ribosyltransferase activity in a cell-free system . Genetic conjugation of G-CSF to an enzymatically inactive DT mutant, CRM197, resulted in a 200-fold reduction in the ability of G-CSF to stimulate normal bone marrow colony formation . These results suggest that fusion of G-CSF to DT sequences interferes with some of the activity but not the specificity of the ligand binding domain of the molecule . Nevertheless, DAB486-G-CSF may be included with the increasing number of other toxin-hormone fusion proteins whose toxicity is directed towards specific receptor-bearing cells, and may represent a novel approach towards the study and treatment of leukemia. Plant J, 1993 Oct, 4(4), 717 - 25 DIP: a member of the MIP family of membrane proteins that is expressed in mature seeds and dark-grown seedlings of Antirrhinum majus; Culianez-Macia FA et al.; DiP, a gene from Antirrhinum majus, which encodes a protein with striking homology to other integral membrane proteins, was cloned . The gene was specifically expressed in mature seeds and during seedling germination, particularly in cotyledons of seedlings grown in the dark . The deduced product, called DiP, for dark intrinsic protein, shows strong homology with the MIP family of channel transporters which include; the bovine major intrinsic protein (MIP), the Escherichia coli glycerol facilitator (GIpF), the peribacteroid nodulin-26 (Nod26), and the tonoplast protein from kidney bean (TIP) . DiP is most similar to other plant members of this family, and in particular to the tobacco protein TobRB7 which is expressed specifically in roots . However, the expression pattern of diP suggests that its product is functionally more similar to the tonoplast intrinsic protein from kidney bean since it is most highly expressed in the cotyledons of germinating seedlings, before the cells undergo expansion growth and become photosynthetic. Protein Expr Purif, 1993 Oct, 4(5), 465 - 72 Isolation and characterization of three recombinant human granulocyte colony stimulating factor His-->Gln isoforms produced in Escherichia coli; Lu HS et al.; This report demonstrates that three variant isoforms of recombinant methionyl human granulocyte colony stimulating factor are present in small quantities in the crude preparation solubilized from Escherichia coli inclusion bodies . These isoforms were separated from the main form of the factor during purification and further isolated by a series of cationic exchange chromatographic separations . They exhibit full in vitro biological activity and have slightly lower pI's . Structural characterization of the intact proteins and their isolated peptides by sequence determination and mass spectrometric analysis revealed that they are methionyl granulocyte colony stimulating factors having a His-->Gln replacement at sequence position 53, 157, or 171, respectively . The specific His-->Gln change suggests the occurrence of mistranslation during protein synthesis . These variant forms are chromatographically separable during purification and are not detectable in the final purified form of the factor. Protein Expr Purif, 1993 Oct, 4(5), 445 - 55 Purification of an active TATA-binding protein-containing factor using a monoclonal antibody that recognizes the human TATA-binding protein; Chatterjee PK et al.; The human TATA-binding protein was expressed in Escherichia coli as a fusion with an N-terminal hexahistidine sequence, partially purified, and used to raise monoclonal antibodies . More than 50 hybridoma clones producing antibodies that reacted in immunoblot assays with HeLa cell TATA-binding protein and its bacterially synthesized derivative were identified . All antibodies examined recognized epitopes within the N-terminal 159 amino acids of the human TATA-binding protein . Further characterization of one monoclonal antibody, MTBP-6, established that it immunoprecipitates both native HeLa cell TATA-binding protein and TATA-binding protein extracted from cells in the presence of 0.5% SDS . Antibody MTBP-6 immunoprecipitates of native, human cell TATA-binding protein contained the TATA-binding protein and additional polypeptides . Immunoprecipitation of both the TATA-binding protein and several additional polypeptides was specifically blocked by bacterially synthesized, hexahistidine-tagged TATA-binding protein, suggesting that MTBP-6 can efficiently recognize the TATA-binding protein in TFIID and other complexes . Consistent with this conclusion, immunoaffinity chromatography on antibody MTBP-6 permitted purification, in active form, of a TATA-binding protein-containing factor required for transcription by RNA polymerase III . These properties suggest that MTBP-6 will be a useful reagent for the purification and characterization of the multiple TBP-containing complexes present in human cells. Nature, 1993 Sep 30, 365(6445), 464 - 8 Crystal structure of the DsbA protein required for disulphide bond formation in vivo; Martin JL et al.; Proteins that contain disulphide bonds are often slow to fold in vitro because the oxidation and correct pairing of the cysteine residues is rate limiting . The folding of such proteins is greatly accelerated in Escherichia coli by DsbA, but the mechanism of this rate enhancement is not well understood . Here we report the crystal structure of oxidized DsbA and show that it resembles closely the ubiquitous redox protein thioredoxin, despite very low sequence similarity . An important difference, however, is the presence of another domain which forms a cap over the thioredoxin-like active site of DsbA . The redox-active disulphide bond, which is responsible for the oxidation of substrates, is thus at a domain interface and is surrounded by grooves and exposed hydrophobic side chains . These features suggest that DsbA might act by binding to partially folded polypeptide chains before oxidation of cysteine residues. Nature, 1993 Sep 30, 365(6445), 454 - 6 Skeletal muscle myosin light chains are essential for physiological speeds of shortening; Lowey S et al.; In muscle each myosin head contains a regulatory light chain (LC2) that is wrapped around the head/rod junction, and an alkali light chain that is distal to LC2 (ref . 1) . The role of these light chains in vertebrate skeletal muscle myosin has remained obscure . Here we prepare heavy chains that are free of both light chains in order to determine by a motility assay whether the light chains are necessary for movement . We find that removal of light chains from myosin reduces the velocity of actin filaments from 8.8 microns s-1 to 0.8 microns s-1 without significantly decreasing the ATPase activity . Reconstitution of myosin with LC2 or alkali light chain increases filament velocity to intermediate rates, and readdition of both classes of light chains fully restores the original sliding velocity . We conclude that even though the light chains are not essential for enzymatic activity, light-chain/heavy-chain interactions play an important part in the conversion of chemical energy into movement. Nature, 1993 Sep 30, 365(6445), 448 - 51 Generating loss-of-function phenotypes of the fushi tarazu gene with a targeted ribozyme in Drosophila; Zhao JJ et al.; The ability to isolate gene sequences and analyse their expression patterns has generated demand for mutations created to assess their biological functions . In Drosophila melanogaster this can be achieved by traditional mutagenesis, but this is time-consuming, labour-intensive and not always successful . Moreover, the functions of genes that are expressed several times during development are often obscured in the later stages because of disruptions caused by the absence of early gene function . Here we propose a new strategy to create conditional knock-out mutations using a targeted heat-inducible ribozyme . Ribozymes are catalytic RNA molecules that specifically cleave RNAs and are potentially useful for studying gene function during animal development because the expression of critical regulatory genes is usually low and their function is often dosage-dependent . The ribozyme can be delivered to a specific region or at a particular developmental stage using a region-specific or inducible promoter . The Drosophila fushi tarazu (ftz) gene is a good candidate for testing this approach . We generated transgenic flies carrying a ribozyme against the ftz gene . The two developmental phases of ftz function can be distinguished by timed induction of the ribozyme . Activation of the ribozyme in the blastoderm disrupts the ftz seven-stripe pattern and produces ftz-like pair-rule defects in larvae . The involvement of ftz in neurogenesis was verified by activation of the ribozyme during the early phase of formation of the central nervous system. Nature, 1993 Sep 30, 365(6445), 412 - 9 Association between proto-oncoprotein Rel and TATA-binding protein mediates transcriptional activation by NF-kappa B; Kerr LD et al.; The c-Rel protein is able to associate in vitro and in vivo with the TATA-binding protein (TBP) of the TFIID complex . Coexpression of TBP with c-Rel augments transactivation from the kappa B site in Drosophila Schneider cells . DNA-binding mutants of TBP not only fail to cooperate, but they repress transactivation by c-Rel . There may be a direct communication between kappa B enhancer binding proteins and basal transcription factors which leads to enhanced transcription. Gene, 1993 Sep 30, 132(1), 143 - 8 The glutamate dehydrogenase-encoding gene of the hyperthermophilic archaeon Pyrococcus furiosus: sequence, transcription and analysis of the deduced amino acid sequence; Eggen RI et al.; Glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon, Pyrococcus woesei, has been isolated, characterized and found to be very similar if not identical to the recently purified GDH from P . furiosus . Using a polymerase chain reaction, based on the N-terminal amino acid sequences of GDH, the P . furiosus gdh gene was identified, cloned into Escherichia coli and sequenced . The transcription start point of gdh has been mapped 1 nucleotide upstream from the ribosome-binding site . Using antiserum raised against purified GDH, expression of gdh was observed in E . coli . The deduced primary sequence of the P . furiosus GDH has been compared to various bacterial, archaeal and eukaryal GDHs and showed a high degree of similarity (32-52%). Biochem Biophys Res Commun, 1993 Sep 30, 195(3), 1415 - 21 Histidine residues are involved in translocation-coupled ATP hydrolysis by the Sec-A protein; Tokuda H et al.; Treatment of SecA, an essential component of the protein translocation machinery of Escherichia coli, with a histidine-specific reagent, diethylpyrocarbonate, caused significant abolition of its translocation-coupled ATPase (translocation ATPase) activity, which requires a presecretory protein and membrane vesicles, whereas its endogenous ATPase (SecA-ATPase) activity was enhanced by a factor of 2 . Diethylpyrocarbonate-treated SecA exhibited an absorption maximum at 240 nm due to the formation of N-carbethoxyhistidine . Upon the modification of about 5 of the total 22 histidine residues in the SecA molecule, both the abolition of its translocation ATPase activity and the enhancement of its SecA-ATPase activity occurred . Intact and modified SecA exhibited similar affinities for ATP, proOmpA and membranes, whereas Vmax of the translocation ATPase activity was significantly lower in the case of the modified SecA . ATP had no effect on the modification of SecA . Taken together, these results indicate that histidine residues susceptible to diethylpyrocarbonate are essential for the translocation ATPase, but not directly involved in the binding of ATP, proOmpA and membranes . A possible reason for the abolition of translocation ATPase is discussed. Biochem Biophys Res Commun, 1993 Sep 30, 195(3), 1386 - 93 cDNA cloning, expression in Escherichia coli and purification of human 6-pyruvoyl-tetrahydropterin synthase; Ashida A et al.; cDNA clones for human 6-pyruvoyl-tetrahydropterin synthase, the second enzyme in the biosynthetic pathway of tetrahydrobiopterin, were isolated from a human Molt-4 cell cDNA library by cross-hybridization with a rat cDNA . One cDNA clone contained the entire coding sequence of 435 base pairs . The cDNA was expressed in Escherichia coli using the expression vector pMAL as a fusion protein with maltose-binding protein . After affinity purification through its maltose-binding protein domain, the fusion protein was digested by factor Xa at a specific cleavage site inserted between the domains . The main product was a protein species with a native molecular mass of 90 kDa and a subunit molecular mass of 17 kDa, and the molecular masses and its kinetic properties were similar to those of the human enzyme purified from the liver. Biochem Biophys Res Commun, 1993 Sep 30, 195(3), 1237 - 44 Fluorescence properties of the three tyrosine residues in the ribose-binding protein; Kim D et al.; Three tyrosine residues but no tryptophan exist in the ribose-binding protein (RBP) of Escherichia coli . In order to assess the contribution of each tyrosine to the fluorescence properties, mutants were constructed by site-directed mutagenesis to replace tyrosines at 32, 115, and 261 by phenylalanines . The mutant proteins were functional as confirmed by in vivo tactic response and by their ability to bind to ribose . The fluorescence emission spectra of the native proteins purified from the various tyrosine mutants were measured from emission scans with a peak at 303 nm . The tyrosines, at positions 32, 115, and 261, contribute 10.0, 69.6, and 23.4%, respectively, to the total intensity of fluorescence . In completely unfolded polypeptide, these tyrosines have almost the same intensities of fluorescence, indicating that the fluorescence from tyrosines at 32 and 261 are considerably quenched in the folded, native protein. Biochem Biophys Res Commun, 1993 Sep 30, 195(3), 1211 - 7 Cloning and functional expression of a Schistosoma japonicum cDNA homologous to the enolase gene family; Waine GJ et al.; A cDNA encoding the complete open reading frame of Schistosoma japonicum enolase has been cloned . The 1494bp cDNA (C30) was isolated from a S . japonicum cDNA expression library immunoscreened with hyperimmune rabbit sera raised against soluble adult S . japonicum proteins . The ORF encodes a protein of 434 amino acids exhibiting 72% identity to human, murine, and rat enolases, and 62% identity to Saccharomyces cerevisiae enolase . The inferred molecular mass of the protein is 47,251 Daltons, similar to that reported for the enolases of other species . In vitro translation of C30 also generated a protein of 47kDa . After subcloning and expression, the recombinant protein was purified by affinity chromatography under non-denaturing conditions and shown to exhibit functional enolase enzymatic activity. Biochem Biophys Res Commun, 1993 Sep 30, 195(3), 1174 - 83 Efficient adenovirus-mediated gene transfer into human blood monocyte-derived macrophages; Haddada H et al.; The efficiency of gene transfer into human blood monocyte-derived macrophages has been evaluated using a replication-defective adenovirus vector harboring a lac Z gene of E . coli as a reporter gene . Whereas, no beta-galactosidase activity was found in freshly infected purified monocytes, 40% to 80% of infected macrophages which derived from these monocytes showed a beta-galactosidase activity, 2 to 4 days after infection and lasted for at least 3 weeks . Moreover, beta-galactosidase activity was found in infected monocyte/macrophages 7 days after their injection into a human tumor preestablished in nude mice . These data indicate that it is possible to transfer and stably express a gene of potential therapeutical function into human monocyte-derived macrophages using an adenovirus vector. Gene, 1993 Sep 30, 132(1), 89 - 94 Characterization and sequence of a 33-kDa enterohemolysin (Ehly 1)-associated protein in Escherichia coli; Stroeher UH et al.; The nucleotide sequence of the 2.1-kb EcoRI-AccI fragment of the enterohemolysin (Ehly)-associated plasmid, pEO21, has been determined . A third of this sequence encodes a 29.6-kDa protein, and coincides with the location of Tn1725 insertions which inactivate the production of Ehly . A protein of similar size (33-35 kDa) was found to be produced in large amounts by JM83{pEO21} and found to be immunologically related to the approximately 65-kDa protein made by the parental strain, C3888 . DNA sequences coding for the 29.6-kDa protein of phi C3888, now designated Ehly 1, were found only in some enterohemolytic Escherichia coli indicating the existence of multiple, genetically different Ehlys. Gene, 1993 Sep 30, 132(1), 21 - 31 Gene organization and primary structure of a ribosomal RNA gene cluster from Streptomyces griseus subsp . griseus; Kim E et al.; The Streptomyces griseus subsp . griseus KCTC 9080 genome contains six rRNA-encoding gene (rDNA) clusters . One rDNA cluster (rrnE), contained in an 8.7-kb BamHI fragment, was cloned and sequenced . The rDNA were arranged in the order 16S-23S-5S, and separated by small intergenic spacers . No tRNA-encoding sequences were found in the spacer regions . The lengths of the mature 16S, 23S and 5S rRNAs were 1528, 3120 and 120 nucleotides (nt), respectively . The G + C content of the gene cluster was lower than that of the chromosomal DNA . In general, the primary and secondary structures of the three rRNAs showed good agreement with those from other Streptomyces species . However, in comparison with Escherichia coli, two noticeable changes (mismatches and deletions) and two large insertions were identified in the 16S and 23S rRNAs, respectively . On the other hand, regions showing considerable heterogeneity, even within the genus Streptomyces, were found in both rRNAs . Putative primers and processing signals showing high sequence similarity to those from other Streptomyces species were located in the region upstream from the 5' end of the mature 16S rRNA . A potential hairpin loop structure reminiscent of a Rho-independent terminator was located just downstream from the 5S rRNA . A considerable degree of sequence conservation and variation within rDNA gene clusters was revealed in this study, both at the infra- and suprageneric levels. Biochim Biophys Acta, 1993 Sep 29, 1170(1), 62 - 71 Early catalytic steps of Euglena gracilis chloroplast type II fatty acid synthase; Worsham LM et al.; Euglena gracilis is a very ancient eukaryote whose chloroplast acquisition and evolution has been independent of higher plants . The organism in unique in possessing two de novo fatty acid synthases, a true multienzyme complex of great size in the cytosol and a plastid-localized type II fatty acid synthase composed of discrete enzymes and acyl carrier protein (ACP) . The enzymology of the early steps of fatty acid biosynthesis differed in the Euglena type II fatty acid synthase compared to those of Escherichia coli and plants . The enzymes of Euglena participating in both priming and elongation reactions to form a new carbon-carbon bond were acetyl-CoA-ACP transacylase, malonyl-CoA-ACP transacylase, and beta-ketoacyl-ACP synthase I . The effects of inhibitors on the three different enzymes were noted . All carbon-carbon bond formation was inhibited by cerulenin . Although neither fatty acid biosynthesis nor any of the isolated enzymes were sensitive to diisopropylphosphofluoridate, the three Euglena enzymes studied were sensitive to different sulfhydryl-alkylating agents . Acetyl-ACP supported fatty acid biosynthesis as effectively as did comparable amounts of ACPSH and acetyl-CoA . There was no evidence for a beta-ketoacyl-ACP synthase III for priming such as has been reported in type II fatty acid synthase of higher plants and bacteria . The roles of the acetyl-CoA-ACP transacylase and beta-ketoacyl-ACP synthase I appear to be unique in the type II fatty acid synthase of Euglena . Acetyl-CoA-ACP transacylase, malonyl-CoA-ACP transacylase, and beta-ketoacyl-ACP synthase I were separated from one another and shown to have different molecular weights. Biochemistry, 1993 Sep 28, 32(38), 9985 - 93 Human actin depolymerizing factor mediates a pH-sensitive destruction of actin filaments; Hawkins M et al.; ADF (actin depolymerizing factor) is an M(r) 19,000 actin-binding protein present in many vertebrate tissues and particularly abundant in neuronal cells . We have cloned human ADF and here show it to be identical in sequence to porcine destrin . Human ADF expressed in Escherichia coli behaves like native ADF from porcine brain . It binds to G-actin at pH 8 with a 1:1 stoichiometry and Kd approximately 0.2 microM, thereby sequestering monomers and preventing polymerization . It does not cosediment with F-actin at this pH, but severs actin filaments in a calcium-insensitive manner . The severing activity is only about 0.1% efficient . By contrast, at pH values below 7, ADF binds to actin filaments in a highly cooperative manner and at a 1:1 ratio to filament subunits . When the pH is raised to 8.0, the decorated filaments are rapidly severed