Int Arch Allergy Appl Immunol, 1990, 91(2), 124 - 9 Expression of Dermatophagoides pteronyssinus allergen, Der p II, in Escherichia coli and the binding studies with human IgE; Chua KY et al.; Lambda gt11 clones expressing the major house dust mite allergen, Der p II, have been reported to react with IgE in the serum of a high proportion of allergic patients . The clones described, however, only produced small quantities of protein which was not fused to the beta-galactosidase of the vector . A construct of the Der p II is described which produces a fusion of Der p II, minus its leader sequence, with the glutathione-S transferase in the pGEX vector . This could be readily isolated and was shown to react with IgE in 22 of 24 patient sera showing reactivity to native Der p II, the sera not reacting having low reactivity to native protein . Absorption analysis showed that the recombinant material removed most of the IgE reactivity of patients to native Der p II . The construct described, therefore, should be valuable for quantitative studies of a pure mite allergen. Chem Pharm Bull (Tokyo), 1990 Jan, 38(1), 153 - 5 An enzymatic method for the kinetic measurement of L-asparaginase activity and L-asparagine with an ammonia gas-sensing electrode; Tagami S et al.; A simple kinetic method to assay L-asparaginase and L-asparagine with an ammonia gas-sensing electrode is described . The method is based upon the de-amination of L-asparagine by L-asparaginase from Escherichia coli, resulting in the production of ammonia . The initial rate (mV/min) of ammonia release is proportional to the activity of L-asparaginase and also to the concentration of L-asparagine in the presence of a large amount of the enzyme . Optimal temperature, buffer composition and pH for the assays are specified . L-Asparaginase was determined in the range of 0.4-1.6 U in a 0.1 ml sample; the recovery was 98.1-103.8% for 16 determinations and sigma n was 1.59 . L-Asparagine was determined in the concentration range of 1 x 10(-4)--1 x 10(-3) M with sigma n-1 1.92 . The method was applied to the determination of 1-5 x 10(-4) M asparagine added to human serum with sigma n-1 1.96 for 5 determinations. Minerva Dietol Gastroenterol, 1990 Jan-Mar, 36(1), 51 - 4 {Infectious complications of LeVeen peritoneovenous shunt . Description of a case}; Franciosini MF et al.; A lethal case of E . coli induced meningitis and sepsis in patient with Le Veen peritoneo-venous shunt (PVS) for refractory ascites during alcohol induced cirrhosis of the liver is reported in confirmation of the high number of infectious complications that affects the cirrhotic, especially if he has been subjected to PVS . This report owes its interest to the unusual site of the infection which is however fairly frequent in these particular patients. Klin Med (Mosk), 1990 Jan, 68(1), 76 - 8 {Acute abscesses and gangrene of the lungs in patients with diabetes mellitus}; Bykov VP et al.; Decompensation of diabetes mellitus is known to promote acute pulmonary suppuration taking a complicated course in 46% of the cases . New effective methods of the prevention and treatment of purulent diseases in diabetics are awaited. Biull Eksp Biol Med, 1990 Jan, 109(1), 78 - 82 {Ultrastructure of the epithelial cells of the duodenum at the early stages of experimental Escherichia infection}; Parkhomenko IuG et al.; Ultrastructural changes of duodenal epitheliocytes were studied at the period from 15 minutes to 24 hours after inoculation using the model of experimental esherichiosis . The results obtained allow to determine the succession of ultrastructural changes and dynamics of adenylate cyclase activity of epithelial cells, involvement of endocrine cells in the pathological process . Combination of the certain morphological and cytochemical reactions and their dynamics allowed to make conclusions about typical ultrastructural changes in epitheliocytes at the early stages of experimental esherichiosis. Microb Pathog, 1990 Jan, 8(1), 47 - 60 Cloning and nucleotide sequencing of Vero toxin 2 variant genes from Escherichia coli O91:H21 isolated from a patient with the hemolytic uremic syndrome; Ito H et al.; Cellular DNA extracted from Escherichia coli strain B2F1 (O91:H21) was found to contain two separate DNA sequences that hybridized with a Vero toxin 2 (VT2)-specific gene probe under stringent conditions . These two sequences were cloned and both were shown to encode a variant of Vero toxin 2 (VT2vh) . The nucleotide sequences of the operons encoding VT2vh, designated as vtx2ha and vtx2hb, were determined . The two operons were nearly identical (99% overall DNA homology) and both encoded A subunits of 319 amino acid residues and B subunits of 89 amino acid residues, the A and B subunit genes being separated by a stretch of 14 bp . The A and B subunit genes of the vtx2ha operon exhibited 98.6% and 95.5% DNA homology, respectively, with those of the slt-II operon encoding Shiga-like toxin II (or VT2) cloned from a strain from a patient with hemorrhagic colitis, while the A and B subunit genes of the vtx2ha operon showed 94.5% and 82.8% DNA homology, respectively, with those of the slt-IIv operon encoding a SLT-II variant cloned from a strain isolated from a pig with edema disease . The nucleotide sequences of the presumed promoters and presumptive ribosome binding sites in the vtx2ha, vtx2hb, and slt-II, and slt-IIv operons were identical . These results indicate that nucleotide sequences encoding a family of VT2-related toxins are present in various strains of E . coli and that the sequences of the genes for A subunits are better conserved than those of the B subunit genes. J Biochem (Tokyo), 1990 Jan, 107(1), 73 - 6 Complete primary structure of the human estrogen-responsive gene (pS2) product; Mori K et al.; pS2 is a human gene whose transcription is directly triggered by estrogen in human breast cancer cells (MCF-7) . We described here the complete primary structure of the pS2 gene product . The pS2 protein purified from conditioned medium of MCF-7 cells was S-pyridylethylated and digested with TPCK-trypsin . Five major fragments were obtained by reverse-phase HPLC . Amino acid sequence analysis of these tryptic peptides established that the pS2 protein comprises a 60-amino acid polypeptide . The sequence of the pS2 protein was completely identical to that deduced from the nucleotide sequence of the pS2 gene, if the signal polypeptide is excluded . Furthermore, two cDNA clones encoding an 84-amino acid precursor pS2 protein were isolated from a cDNA library which was constructed with RNA from MCF-7 cells cultured in the presence of estrogen . The nucleotide sequence of one clone (pS2B1) was identical to that of pS2 cDNA previously reported except for one nucleotide in the 3' untranslated region . The other clone (pS2B2) was longer by 73 nucleotides, at the 5' end, than pS2B1 . The additional 73 nucleotides are located just upstream of the sequence of pS2B1 in the structure of the pS2 gene, indicating that the pS2 gene has two start sites for transcription . However, a mRNA molecule corresponding to pS2B1 but not to pS2B2 was detected in the cells on RNA blot hybridization analysis, indicating that one transcriptional start site is mainly used. J Biochem (Tokyo), 1990 Jan, 107(1), 21 - 6 Nucleotide sequence and characteristics of the gene for L-lactate dehydrogenase of Thermus aquaticus YT-1 and the deduced amino acid sequence of the enzyme; Ono M et al.; The gene for L-lactate dehydrogenase (LDH) from Thermus aquaticus YT-1 was cloned in Escherichia coli, using the Thermus caldophilus LDH gene as a hybridization probe, and its complete nucleotide sequence was determined . The LDH gene comprised 930 base pairs, starting with a GTG initiation codon . Its sequence had high homology (85.8% identity) with the LDH gene of T . caldophilus . The G + C content of the T . aquaticus gene was 70.9%, higher than that of the chromosomal DNA (67.4%) . In particular, that in the third position of the codons used was 91.0%, similar to the T . caldophilus gene . The primary structure of T . aquaticus LDH was deduced from the nucleotide sequence of the LDH gene . It comprises 310 amino acid residues, as does T . caldophilus LDH, and its molecular mass was calculated to be 33,210 daltons . The amino acid sequence of the T . aquaticus LDH had 87.1% identity with that of the T . caldophilus LDH . At 23 positions, the respective residues differed in charge and polarity . These differences must be related to the differences in kinetic properties between the two enzymes . The constructed plasmid overproduced the T . aquaticus LDH in E . coli. Proteins, 1990, 7(1), 41 - 51 An expectation maximization (EM) algorithm for the identification and characterization of common sites in unaligned biopolymer sequences; Lawrence CE et al.; Statistical methodology for the identification and characterization of protein binding sites in a set of unaligned DNA fragments is presented . Each sequence must contain at least one common site . No alignment of the sites is required . Instead, the uncertainty in the location of the sites is handled by employing the missing information principle to develop an "expectation maximization" (EM) algorithm . This approach allows for the simultaneous identification of the sites and characterization of the binding motifs . The reliability of the algorithm increases with the number of fragments, but the computations increase only linearly . The method is illustrated with an example, using known cyclic adenosine monophosphate receptor protein (CRP) binding sites . The final motif is utilized in a search for undiscovered CRP binding sites. Protein Eng, 1990 Jan, 3(3), 205 - 13 Metallothioneins with interdomain hinges expanded by insertion mutagenesis; Rhee IK et al.; Specific peptides of varying lengths were inserted between the two metal cluster domains of metallothionein (MT), which normally are spanned by only three amino acids, Lys-Lys-Ser . These interdomain expansions were made to test if such structural alterations would affect MT function . These constructs were engineered by inserting defined oligonucleotides of up to four tandem repeats of dodecanucleotides and hexanucleotides into an Alu-1 endonuclease cleavage site, which separates the two exonic regions of an MT-coding sequence from Chinese hamster ovary cells, MT-2 . The native and altered sequences were cloned into a high expression Escherichia coli-yeast shuttle vector and used to transform yeast cells whose endogenous MT genes had been previously deleted . Using metal resistance as a biological marker, all constructs were shown to be functional in rendering the host cells resistant to either copper or cadmium . As the inserts, by nature of their amino acid sequence, could add flexibility to the otherwise compact molecule, the two domains apparently are active independently . The level of activity, however, diminished with the length of the insert . Determinations for copy number of the chimeric plasmids and MT mRNAs in the transformed cells showed that the replicational and transcriptional capacity of the long and short constructs were equivalent. Protein Eng, 1990 Jan, 3(3), 173 - 80 Crystallization and structure solution at 4 A resolution of the recombinant synthase domain of N-(5'-phosphoribosyl)anthranilate isomerase:indole-3-glycerol-phosphate synthase from Escherichia coli complexed to a substrate analogue; Wilmanns M et al.; The recombinant synthase domain of the bifunctional enzyme N-(5'-phosphoribosyl)anthranilate isomerase:indole-3-glycerol-phosphate synthase from Escherichia coli has been crystallized, and the structure has been solved at 4 A resolution . Two closely related crystal forms grown from ammonium sulphate diffract to 2 A resolution . One form (space group R32, a = 163 A, alpha = 29.5 degrees) contains the unliganded synthase domain; the second crystal form (space group P6(3)22, a = 144 A, c = 158 A) is co-crystallized with the substrate analogue N-(5'-phosphoribit-1-yl)anthranilate . The structure of the synthase-inhibitor complex has been solved by the molecular replacement method . This achievement represents the first successful use of a (beta alpha)8-barrel monomer as a trial model . The recombinant synthase domain associates as a trimer in the crystal, the molecules being related by a pseudo-crystallographic triad . The interface contacts between the three domains are mediated by those residues that are also involved in the domain interface of the bifunctional enzyme . This system provides a model for an interface which is used in both intermolecular and intramolecular domain contacts. Mutagenesis, 1990 Jan, 5(1), 35 - 8 Mutagenic DNA repair in Escherichia coli . XVIII . Involvement of DNA polymerase III alpha-subunit (DnaE protein) in mutagenesis after exposure to UV light; Bridges BA et al.; UV light was unable to induce rifampicin-resistant mutations at 43 degrees C in Escherichia coli ER11 dnaE486 . Although DnaE486 gene product is inactive at 43 degrees C, these bacteria contain the pcbA1 mutation which allows DNA replication provided DNA polymerase I is functional . The experiments were carried out under conditions where full expression of rifampicin-resistant mutations could occur so that the lack of induced mutations cannot be ascribed to an effect of incubation at 43 degrees C on mutation expression . UV-mutability at 43 degrees C was restored by the presence of the dnaE+ allele on a plasmid . It is concluded that functional DnaE protein is essential for UV mutagenesis . The dnaE486 mutation also blocked the induction at 43 degrees C of mutations induced by UV plus delayed photoreversal, a procedure that has been postulated to reflect an early misincorporation step in the UV mutagenic process. Int J Immunopharmacol, 1990, 12(3), 297 - 305 Production of interleukin 1 from human monocytes stimulated by synthetic lipid A subunit analogues; Saiki I et al.; We have investigated that synthetic lipid A subunit analogues (GLA compounds) as well as E . coli type lipopolysaccharide (LPS) and synthetic lipid A (compound 506) are able to stimulate human monocytes to release IL-1 in vitro . Of monosaccharide-type GLA compounds, GLA-60 was found to be more active for the induction of IL-1 production than GLA-59 and GLA-27, and similar to that of LPS or compound 506 . GLA-60 could induce not only the secretion of IL-1 into culture supernatant but also the expression of membrane-associated form of IL-1 in human monocytes . Furthermore, no detectable IL-2 activity was observed in the culture supernatant . These results show that synthetic lipid A analogues of low toxicity, in particular GLA-60, are active in inducing IL-1 production in human monocytes. Biopolymers, 1990 Jan, 29(1), 139 - 47 A comparative analysis of single- and multiple-residue substitutions in the alkaline phosphatase signal peptide; Kendall DA et al.; The alkaline phosphatase signal peptide participates in transport of the enzyme to the periplasmic space of Escherichia coli . The signal sequence, like that of other signal peptides, is composed of a polar amino-terminal segment, a central region rich in hydrophobic residues and a carboxy-terminal region recognized by signal peptidase . We have previously shown that an alkaline phosphatase signal peptide mutant containing a polyleucine core region functions efficiently in transport of the enzyme {D . A . Kendall, S . C . Bock, and E . T . Kaiser (1986) Nature 321, 706-708} . In this study, some of the amino acid changes involved in the polyleucine sequence are examined individually . A Phe to Leu substitution as the sole change results in impaired transport properties in contrast to when it is combined with three other amino acid changes in the polyleucine-containing sequence . A mutant with a Pro to Leu substitution in the hydrophobic core region is comparable to wild type while the same type of substitution (Pro to Leu) in the carboxy-terminal segment results in substantial accumulation of the mutant precursor . Finally, introduction of a basic residue into the hydrophobic segment (Leu to Arg substitution) results in a complete export block . These results exemplify the spectrum of properties produced by individual residue changes and suggest there is some interplay between hydrophobicity and conformation for signal peptide function. Biochem Int, 1990, 20(1), 191 - 9 Overproduction of superoxide dismutase does not protect Escherichia coli from stringency-induced growth inhibition by 1mM paraquat; Siwecki G et al.; Two plasmid-containing Escherichia coli strains which overproduce manganese superoxide dismutase by 4- to 5-fold and iron superoxide dismutase by about 7-fold were not more resistant than parent strains to 1 mM paraquat (a known generator of superoxide) as measured by effects on growth, survival and induction of stringency . These results indicate that overproduction of superoxide dismutase does not mitigate the growth-inhibitory effects of 1 mM paraquat, including those which are expressed through induction of the stringency mechanism. Basic Life Sci, 1990, 52, 351 - 4 Proteolytic activation of UmuD and MucA proteins for SOS mutagenesis; Shiba T et al.; SOS mutagenesis in Escherichia coli requires the functions of the umuD, C genes, or their functional analogues mucA, B derived from a plasmid pKM101, and the recA gene . However, mere derepression of these SOS genes does not increase the ability of the cell to perform mutagenesis . Activation of RecA protein to a form (RecA*) that mediates cleavage of the LexA repressor is required for mutagenesis . We present evidence that UmuD and MucA are proteolytically processed by RecA* and that the processed products are the active forms involved in mutagenesis. Basic Life Sci, 1990, 52, 277 - 87 Position of a single acetylaminofluorene adduct within a mutational hot spot is critical for the related mutagenic event; Burnouf D et al.; 2-Acetylaminofluorene, a potent rat liver carcinogen, which binds primarily to C8 of guanines, has been shown to induce mainly frameshift mutations in the bacteria Escherichia coli . Mutations occur at specific sequences, known as mutation hot spots, of which two types may be considered . First, repetitive sequences, where deletions of a single unit occur (GGGGG----GGGG) . Second, the so-called NarI site, 5'GGCGCC3', where only -2-bp deletions are observed (G1G2CG3CC----GGCC) . Mutagenesis within repetitive sequences is dependent on the UmuCD+ gene functions, whereas mutagenesis in the NarI site is not . These differences in the genetic requirements of mutagenesis at these hot spots suggest that two different pathways operate . In order to precisely determine the actual involvement of each of the three premutagenic lesions that may form in the NarI site in the course of the mutational process, we designed a single adduct mutagenesis experiment, and found that AAF binding to the G3 induced only a -2 frameshift mutation event . This result will be discussed in terms of local DNA conformation. Antimicrob Agents Chemother, 1990 Jan, 34(1), 164 - 6 Decay of the ampicillin-induced lysis process in amino acid-deprived Escherichia coli; Kusser W et al.; The ability to induce the ampicillin-mediated lysis of amino acid-deprived Escherichia coli by relaxing the stringent response decreased progressively during the course of amino acid deprivation, apparently because of a time-dependent decay in a key lysis event . The decay of this labile activity was not apparent when ampicillin treatment was initiated early and maintained continuously throughout the amino acid starvation period. Adv Exp Med Biol, 1990, 256, 141 - 5 Cloning and analysis of rfb gene synthesizing the mannan 0 side chain of Escherichia coli 09 lipopolysaccharide; Kido N et al.; The rfb gene encoding the proteins responsible for the synthesis of the repeating units (O side-chain) of Escherichia coli 09 lipopolysaccharide was cloned into a conjugative plasmid RP4::miniMu and was expressed in E . coli K-12. Proteins, 1990, 7(2), 99 - 111 Understanding structural relationships in proteins of unsolved three-dimensional structure; Burbaum JJ et al.; The locations of functionally important sequences and general structural motifs have been assigned to Ile-tRNA synthetase . However, a function has not been established for some segments of the protein (e.g., CP1) . The method of structural modeling described here cannot establish the details of a 3 A crystal structure, and, in contrast to a crystal structure, the precision of the model varies according to the extent of a sequence similarity or the functional importance of a region . In Ile-tRNA synthetase, the signature sequence and the flanking regions are likely to be similar in structure to the proteins on which the model is based . For other regions, it may be possible to build a three-dimensional model by connecting well defined regions and refining the positions of the connecting elements by energy minimization . Structural modelling of this kind must be done cautiously, because the order and orientation of the elements of a structural motif can change in subtle ways . In the case of Tyr-tRNA synthetase, the beta-strand nearest the N-terminus is the outermost strand of the nucleotide binding fold; in Met-tRNA synthetase, the same strand is innermost . Furthermore, the orientation of this strand may be antiparallel (Tyr-tRNA synthetase) or parallel (Met-tRNA synthetase) . Because multiple structures that differ in their orientations of structural elements are possible, the structural analogies between proteins should not be naively extrapolated without independent experimental support . As described above, some regions of proteins tolerate internal deletions and insertions . This provides further experimental support for the practice of allowing for gaps in computer-generated sequence alignments . Nevertheless, because some regions are more tolerant of insertions and deletions than others, the structural and functional significance of a region of broken alignment must be assessed carefully . All gaps in sequence alignments cannot be treated equally, and each must be evaluated within its own context . In the synthetases of known structure, structural analogy can be used to identify important functional elements . For example, the amino acid binding site of Met-tRNA synthetase might be formed, at least in part, by a peptide that encompasses Ala50; this amino acid aligns with Gly94 of the Ile-tRNA synthetase . This is an example in which results on a protein of unknown structure (Ile-tRNA synthetases) can lead to identification of a potential substrate binding site in a protein of known structure (Met-tRNA synthetase). Mol Gen Genet, 1990 Jan, 220(2), 325 - 8 Nucleotide sequence of the region encompassing the int gene of a cryptic prophage and the dna Y gene flanked by a curved DNA sequence of Escherichia coli K12; Muramatsu S et al.; The nucleotide sequence of a 2.5 kb region encompassing a curved DNA segment (BENT-9) randomly cloned from the total Escherichia coli chromosome was determined . This region was found to contain the dna Y gene encoding a transfer RNA . The curved DNA structure was demonstrated to be located just upstream of the dna Y promoter . The results of sequencing further revealed that the int gene of a cryptic prophage, qsr', which has been shown to be present in the E . coli genome, is located next to the dna Y gene. Mol Gen Genet, 1990 Jan, 220(2), 317 - 9 Genetic mapping of pheV, an Escherichia coli gene for tRNA(Phe); Caillet J; We report the physical and genetic mapping of pheV, an Escherichia coli gene for phenylalanine tRNA, to 64 min on the chromosomal map in the near vicinity of speC coding for ornithine decarboxylase. Mol Gen Genet, 1990 Jan, 220(2), 277 - 82 Roles of Escherichia coli heat shock proteins DnaK, DnaJ and GrpE in mini-F plasmid replication; Kawasaki Y et al.; A subset of Escherichia coli heat shock proteins, DnaK, DnaJ and GrpE were shown to be required for replication of mini-F plasmid . Strains of E . coli K12 carrying a missense mutation or deletion in the dnaK, dnaJ, or grpE gene were virtually unable to be transformed by mini-F DNA at the temperature (30 degrees C) that permits cell growth . When excess amounts of the replication initiator protein (repE gene product) of mini-F were provided by means of a multicopy plasmid carrying repE, these mutant bacteria became capable of supporting mini-F replication under the same conditions . However, the copy number of a high copy number mini-F plasmid was reduced in these mutant bacteria as compared with the wild type in the presence of excess RepE protein . Furthermore, mini-F plasmid mutants that produce altered initiator protein and exhibit a very high copy number were able to replicate in strains deficient in any of the above heat shock proteins . These results indicate that the subset of heat shock proteins (DnaK, DnaJ and GrpE) play essential roles that help the functioning of the RepE initiator protein in mini-F DNA replication. Mol Gen Genet, 1990 Jan, 220(2), 197 - 203 Maintenance of plasmids in HU and IHF mutants of Escherichia coli; Ogura T et al.; Complementation and sequencing analyses revealed that the hopD mutants, which could not support stable maintenance of mini-F plasmids (Niki et al . 1988), had mutations in the hupB gene, and that the hopD410 mutation was an ochre mutation at the 5th Gln position of HU-1 . Maintenance and stability of various plasmids, mini-P1 plasmids, mini-F plasmids, and oriC plasmids, were studied in the hupA and hupB mutants (HU mutants), and himA and hip mutants (IHF mutants) . Mini-P1 plasmids and mini-F plasmids could not be introduced into the delta hupA-delta hupB double deletion mutant . Replication of mini-F plasmids was partially inhibited in the hupB mutants, including the delta hupB and hopD(hupB) mutants, whereas replication of oriC plasmids was not significantly affected even in the delta hupA-delta hupB double deletion mutant . The mini-P1 plasmid was slightly unstable in the himA-hip mutant, whereas the mini-F plasmid was stable. Mol Gen Genet, 1990 Jan, 220(2), 191 - 6 Physical and genetic analysis of the phosphoenolpyruvate carboxykinase (pckA) locus from Escherichia coli K12; Goldie H et al.; An 8 kb BamHI fragment of the Escherichia coli K12 chromosome has been cloned which complemented the pheotype of CRM+ pckA mutants with inactive phosphoenolpyruvate (PEP) carboxykinase . The pckA+ clones expressed levels of enzyme activity elevated up to 30-fold and produced a Mr 55,000 product in maxicells, which co-electrophoresed with purified PEP carboxykinase . The cloned fragment expressed the pckA, ompR and envZ gene products in maxicells . The order of genes on the chromosome inferred from restriction mapping, was (74 min)...pckA envZ ompR...(75 min) . Transcription of the pckA gene cloned on multicopy plasmids increased in stationary phase and was also regulated by catabolite repression . The transcriptional control region has been located by genetic fusions to the chloramphenicol acetyltransferase (cat) gene and pckA was transcribed in the direction of envZ (clockwise direction on the chromosome). Microbiol Immunol, 1990, 34(1), 11 - 24 Transcriptional control plays an important role for the production of heat-labile enterotoxin in enterotoxigenic Escherichia coli of human origin; Katayama S et al.; The production of heat-labile enterotoxin (LT) in 76 strains of human enterotoxigenic Escherichia coli (ETEC) varied by a factor of 100 . Three ETEC strains that differ in the levels of LT production were chosen for the cloning of LT genes (toxAB) into plasmid pBR322, and the gene structure and expression were compared in E . coli HB101 . The recombinant of the low LT-producing strain produced LT at the same level as that of the moderate LT-producing strain, but that of the high-level producer continued to produce at a level 14-21 times higher than the others . The restriction maps of the coding regions of the cloned LT genes (toxAB) were identical, but the flanking regions were dissimilar . The content of LT mRNA per cell, examined by Northern blot analysis, was higher in the high producer than the others by 6 times . The promoter strengths of the recombinants were all alike . LT mRNA of the high producer was more stable than that of the moderate one by 1.3 times, but the difference was not large enough to explain the difference of the content of LT mRNA . It was shown that LT production can be controlled at a transcriptional step, and DNA structure of the flanking regions may be involved in the control of the LT gene expression. Int J Pept Protein Res, 1990 Jan, 35(1), 17 - 24 Properties of a cleaved two-chain form of recombinant human growth hormone; Canova-Davis E et al.; Escherichia coli cells transformed with plasmids engineered for the expression of recombinant human growth hormone as a secreted product also produced a proteolytically cleaved form of rhGH . This variant is isolated at a high resolution anion exchange chromatography stage during the manufacturing process . The higher isoelectric point of this form is demonstrated by isoelectric focusing and chromatofocusing and the two-chain nature by tryptic mapping, N- and C-terminal sequence analyses, and sodium dodecyl sulfate polyacrylamide gel electrophoresis . These data indicate that the single site of cleavage is between Thr-142 and Tyr-143, in contrast to the two-chain variant isolated from human pituitary glands, which has a clip after residue Phe-139 . The recombinant two-chain form was further characterized by reversed-phase high performance liquid chromatography at both acidic and basic pHs . The assay utilizing bicarbonate-containing mobile phases was determined to be the most efficient and sensitive method . The bioactivity of this two-chain form was measured by the in vivo rat weight gain assay and by the in vitro Nb2 cell bioassay . Its immunological similarity to intact one-chain rhGH was demonstrated with an enzyme-linked immunosorbent assay. FEMS Microbiol Lett, 1990 Jan 1, 54(1-3), 45 - 50 Binding and hemagglutinating properties of the B subunit(s) of heat-labile enterotoxin isolated from human enterotoxigenic Escherichia coli; Sugii S et al.; The binding and hemagglutinating activities of the B subunit(s) of the heat-labile enterotoxin (LTh-B) isolated from human enterotoxigenic Escherichia coli were investigated . The binding of 125I-labeled LTh-B to neuraminidase-treated human type B erythrocytes was most effectively inhibited by ganglioside GM1 . A number of mono-, di- and polysaccharides, as well as several glycoproteins were at least 500 times less potent inhibitors . However, hemagglutination was effectively inhibited by galactose, melibiose and hog A + H but not by ganglioside GM1 . Preincubation of the LTh-B with ganglioside GM1 gave much stronger hemagglutination than LTh-B alone . These results suggest that the predominant binding substance for LTh-B on neuraminidase-treated human type B erythrocytes is ganglioside GM1, but indicate that the interaction of LTh-B with ganglioside GM1 is different in hemagglutination. FEMS Microbiol Lett, 1990 Jan 1, 54(1-3), 291 - 3 Cloning of the cellulase gene from Penicillium funiculosum and its expression in Escherichia coli; Sahasrabudhe NA et al.; A gene of Penicillium funiculosum encoding an endoglucanase was cloned and expressed in Escherichia coli using the lacZ promoter of vector pUC 18 . The gene product hydrolyzed carboxymethyl cellulose and showed strong cross reactivity with P . funiculosum anticellulases. FEMS Microbiol Lett, 1990 Jan 1, 54(1-3), 231 - 4 Cloning and expression of the putative gene coding for GTP cyclohydrolase I from Escherichia coli; Katzenmeier G et al.; The putative gene coding for GTP cyclohydrolase I of Escherichia coli was isolated from a lambda gt11 expression vector library by using antibodies as a probe and has been subcloned on a 3.8 kb Bam HI fragment in the plasmid vector pUC13 . E . coli cells carrying the recombinant plasmid designated pCYH express 100-fold increased levels of the enzyme . The protein formed under the control of the plasmid appears electrophoretically and immunochemically identical with the wild type enzyme. FEMS Microbiol Lett, 1990 Jan 1, 54(1-3), 227 - 30 Molecular cloning and expression of the genes encoding the Escherichia coli K4 capsular polysaccharide, a fructose-substituted chondroitin; Drake CR et al.; The majority of capsular polysaccharides (K antigens) are linear molecules and their genes have a common functional organisation encoding common steps in capsule biogenesis . However, the K4 antigen is a substituted polymer composed of a chondroitin backbone with a fructose side chain . In order to determine whether K4 biosynthesis uses these common mechanisms the K4 antigen genes were cloned . DNA probes taken from the two conserved regions of the K1 genes were used to isolate one plasmid, pRD1, homologous to both probes . Immunological analysis was used to show that pRD1 directs the production of the substituted K4 antigen on the cell surface . Southern hybridisation was used to show that the cloned genes are organised in the same way as other K antigen gene clusters . We conclude that the branched K4 antigen is handled by the same post-polymerisation mechanisms as other linear K antigens. Biol Chem Hoppe Seyler, 1990 Jan, 371(1), 23 - 30 The effect of N-terminal extension on the structure and function of human interleukin-1 beta; Hejnaes KR et al.; Interleukin-1 beta (IL-1 beta) and N-terminally extended Met-Glu-Ala-Glu-IL-1 beta (MEAE-IL-1 beta) were cloned and expressed in E . coli . Extension of the chain results in a limited conformational change reflected by the CD spectrum in the far ultraviolet, while the aromatic side chains responsible for the CD in the near ultraviolet are not affected . No difference in immunoreactivity between IL-1 beta and MEAE-IL-1 beta is observed in the IL-1 beta ELISA . Like IL-1 beta, MEAE-IL-1 beta exhibits biological activity tested in the costimulatory mouse thymocyte (LAF) assay . The specific biological activity of IL-1 beta is 3 x 10(8) U/mg and that of MEAE-IL-1 beta 3 x 10(6) U/mg . Like IL-1 beta, MEAE-IL-1 beta displaces {125I}IL-1 beta from mouse thymocytes and the binding affinities of the two forms differ by a factor of 10(2) . Finally the inhibitory effect of the two IL-1 beta forms on in vitro insulin secretion from isolated rat islets of Langerhans was measured . Again MEAE-IL-1 beta is 10(2) times less potent than IL-1 beta . The structure-activity relationship for IL-1 beta and MEAE-IL-1 beta is discussed. Avian Dis, 1990 Jan-Mar, 34(1), 58 - 62 An enzyme-linked immunosorbent assay for detection of antibodies against Escherichia coli: association between indirect hemagglutination test and survival; Leitner G et al.; An enzyme-linked immunosorbent assay (ELISA) was modified for detection of antibodies against the two main pathogenic serotypes of Escherichia coli: serotypes O78:K80 and O2:K1 . The ELISA was a more sensitive and repeatable test than the indirect hemagglutination test (IHT), which is a common method for detecting antibodies against E . coli . Cross-reactivity between the two strains was measured by reacting antisera of each serotype against homologous and heterologous antigens . The results suggest that aside from similar determinants expressed by the two serotypes, serotype O2:K1 expresses more strain-specific determinants than does O78:K80 . Comparison of mean antibody titers of immunized chicks by IHT and ELISA along the primary response revealed that during the first 15 days after immunization with inactivated E . coli, the titers in both tests were parallel . After 15 days post-immunization, antibody titers measured by IHT decreased rapidly, whereas titers measured by ELISA decreased only slightly . In addition, a higher correlation was found between titers detected by ELISA and survival through challenge with E . coli than between titers detected with IHT and survival through challenge . The results suggest that the ELISA is a better test for detection of antibody in flocks suspected of being infected with E . coli. Scand J Infect Dis, 1990, 22(1), 1 - 4 Enterohaemorrhagic Escherichia coli; Cryan B; In North America enterohaemorrhagic Escherichia coli have emerged as important enteric pathogens since their initial description in 1982 . They have been associated with the idiopathic haemolytic uraemic syndrome and in outbreaks, mortality rates of up to 31% have been recorded . In this paper the recent literature pertaining to the pathogenesis, laboratory identification, epidemiology and therapy of infections by these organisms is reviewed. Mol Biochem Parasitol, 1990 Jan 1, 38(1), 49 - 55 Expression, partial purification and immunogenicity of fragments of the knob protein of Plasmodium falciparum; Rashid MA et al.; Three structural domains of the histidine-rich knob protein (KP) of Plasmodium falciparum were expressed in Escherichia coli . A single-step purification scheme was devised to obtain great enrichment of expressed polypeptides for use in subsequent experiments . Immune human sera from Africa, South-East Asia and South America were tested for reactivity with each of the expressed fragments . While the two fragments which represented the central and C-terminal regions of KP showed a strong reactivity with all the antisera which were tested, the N-terminal fragment which contains the repetitive histidine-rich sequences showed almost no reactivity. Mol Biochem Parasitol, 1990 Jan 1, 38(1), 25 - 32 The major surface glycoprotein (GP63) is present in both life stages of Leishmania; Frommel TO et al.; Leishmania exist as extracellular promastigotes which multiply in the gut of the sandfly insect vector and as intracellular amastigotes which divide in the phagolysosome of mononuclear phagocytic cells of the mammalian host . Promastigotes express a major surface glycoprotein of 63 kDa, referred to as GP63 . The expression of GP63 in both Leishmania life stages was studied using rabbit antibodies against native GP63 as well as rabbit antibodies against recombinant GP63 that was synthesized in an Escherichia coli expression system . Immunofluorescence staining detected GP63 in intracellular amastigotes contained within a macrophage cell line and within freshly isolated lesion amastigotes . Western blot analysis using anti-recombinant GP63 antibodies also demonstrated that amastigotes synthesize GP63 which may undergo differential post-translational processing as compared to promastigote GP63. Mol Microbiol, 1990 Jan, 4(1), 21 - 7 Isolation of intact FNR protein (Mr 30,000) of Escherichia coli; Trageser M et al.; FNR, the activator of anaerobic respiratory genes of Escherichia coli, has previously only been isolated as a protein of Mr 29,000, which lacks nine N-terminal amino acid residues . The underlying proteolytic events have been studied with the aim of isolating intact FNR and determining whether cleavage is the result of a physiologically significant intracellular processing mechanism or proteolytic degradation during isolation . The FNR protein was present in aerobically and anaerobically grown bacteria as the intact protein (Mr 30,000) . Proteolysis only occurred during and shortly after disruption of the bacteria . The production of FNR (Mr 29,000) must therefore be regarded as an isolation artefact . The proteolysis was caused by a protease which is located outside the cytoplasmic membrane or activated upon disruption of the membrane . Protease inhibitors directed against serine, cysteine or metalloproteases failed to prevent cleavage of FNR . In E . coli strain CAG627, proteolysis was greatly reduced making it possible to isolate FNR of Mr 30,000 . The N-terminal sequence of FNR (Mr 30,000) was identical to that predicted from the fnr gene starting with the initiating methionine residue and including a four-cysteine cluster (16)Cys-X3-Cys-X2-Cys-X5-Cys(29). Mol Microbiol, 1990 Jan, 4(1), 143 - 50 Lipid involvement in protein translocation in Escherichia coli; de Vrije GJ et al.; Signal peptides play an essential role in protein translocation . This review summarizes the current knowledge of the structure of signal peptides and signal peptide-lipid interactions and addresses the possibility that signal peptide-lipid interactions initiate membrane translocation of precursor proteins . A new model for protein translocation in Escherichia coli is proposed, which includes as central features conformational changes of the signal peptide and signal-peptide-induced local changes in membrane organization (non-bilayer lipids). Mol Microbiol, 1990 Jan, 4(1), 13 - 20 Characterization of divergent NtrA-dependent promoters in the anaerobically expressed gene cluster coding for hydrogenase 3 components of Escherichia coli; Lutz S et al.; The regulatory region of two divergently oriented transcriptional units involved in the formation of the gas-evolving hydrogenase (isoenzyme 3) of Escherichia coli was investigated . DNA sequence analysis revealed the existence of a 210 bp non-coding region containing two sequences showing homology to -24/-12 NtrA-dependent promoters . These sequences were arranged in a divergent orientation entirely consistent with their being involved in transcribing the divergent operons . Through S1 protection experiments it could be shown that transcription of both promoters was NtrA-dependent and that it was regulated in an identical manner: oxygen repressed expression, as did anaerobic growth in the presence of nitrate; transcription was induced in cells grown anaerobically in the absence of exogenous electron acceptors and formate was found to be obligately required for this anaerobic induction . Lying at an approximately equal distance between both promoters was a short stretch of DNA which showed similarity to the sequence previously identified (Birkmann and Bock, 1989a) as being necessary for formate induction of the fdhF gene. Cytometry, 1990, 11(2), 223 - 30 Light scattering measurement in an arc lamp-based flow cytometer; Steen HB; The epi-illumination optics employed in most arc lamp-based flow cytometers may be modified so as to produce a dark-field configuration which facilitates highly sensitive detection of both forward and large angle light scattering in an instrument with a "jet on open surface" flow chamber . Forward scattering is detected at angles upwards from about 2 degrees, while large angle scattering includes angles above 18 degrees . Theoretical considerations suggest that large angle scattering measured around 20 degrees may be as efficient as that measured at 90 degrees for the purpose of distinguishing cells on the basis of intracellular structure . This was supported by the finding that dual parameter light scattering histograms of leukocyte suspensions obtained with the arc lamp-based instrument were closely similar to such histograms recorded with a laser-based instrument with the large angle detector at 90 degrees . Different species of bacteria could be distinguished by means of the dual parameter light scattering device, as could different species of sea algae . The sensitivity of the device is sufficient to measure 0.2 microns polystyrene particles in both forward and large angle scattering. Eur Arch Otorhinolaryngol, 1990, 247(2), 89 - 92 Experimental conditions for the development of persistent otitis media with effusion; Takahashi H et al.; The purpose of this study was to investigate sufficient conditions for the development of long-lasting otitis media with effusion (OME) without any organic obstruction of the eustachian tube . Three experimental conditions were employed using 20 adult cats (27 ears) . Only tubal ventilatory dysfunction with transection of the tensor veli palatini muscle and excision of the pterygoid hamulus resulted in a small incidence of OME (7.1%), which lasted for 5 weeks . Instillation of Escherichia coli endotoxin into the middle ears formed only a transient OME in 50% of the animals . Combination of these two procedures brought a high incidence of OME (85.7%), most of which lasted for more than 8 weeks . These studies showed that tubal ventilatory dysfunction alone was not a sufficient condition for the development of OME but was important for prolongation of the pathological state of OME . The production of inflammatory exudate was considered to be a trigger for the formation of OME. DNA Cell Biol, 1990 Jan-Feb, 9(1), 71 - 6 A simple procedure for cross-linking complementary oligonucleotides; Chu BC et al.; A simple, efficient procedure for cross-linking two complementary oligonucleotides, which does not require chemical modification of either oligonucleotide, is described . One of the oligonucleotides is first converted to the 5'-phosphorothioate derivative with polynucleotide kinase . It is then incubated with its complement in the presence of 1 microM trans-platinum(II)diammine dichloride . After overnight incubation, 40-50% cross-linking is observed . DNA synthesis by the Klenow fragment of Escherichia coli DNA polymerase I is blocked at the cross-linked site, resulting in the formation of truncated products . Potassium platinous chloride (K2PtCl4) and cis-platinum(II)diammine dichloride form cross-links less efficiently than the trans isomer. DNA Cell Biol, 1990 Jan-Feb, 9(1), 1 - 9 Expression and refolding of recombinant human fibroblast procathepsin D; Conner GE et al.; Procathepsin D is a precursor of the human lysosomal protease cathepsin D . Due to its short half-life, procathepsin D is difficult to obtain in quantities sufficient to allow structural and enzymatic studies . To obtain large quantities of this precursor, procathepsin D was expressed using the T7 promoter vector pET3a in bacteria that carry a chromosomal copy of the T7 RNA polymerase gene under the control of the lac promoter . At high cell density in rich medium, basal levels of T7 RNA polymerase were sufficient to express recombinant procathepsin D without addition of an exogenous inducer of the lac promoter . The recombinant protein, constituting almost half of the total cell protein, accumulated in intracytoplasmic inclusion bodies and was isolated from the insoluble fraction of lysed cells . Antibodies prepared against the purified recombinant protein were shown to crossreact with native human placental and porcine spleen cathepsin D . Recombinant procathepsin D was solubilized in denaturants and was refolded . After extended preincubation of the denatured protein at acid pH to allow folding and activation of the zymogen, pepstatin inhibitable catalytic proteolysis was detected . These data demonstrated that the glycosylated aspartic protease, procathepsin D can be refolded and activated in an unglycosylated form and thus provides a system for the study of procathepsin D structure and function. Microbios, 1990, 61(246), 49 - 61 Cloning and expression of Thiobacillus versutus cryptic plasmid pTAV-1 DNA in Escherichia coli; Jagusztyn-Krynicka EK et al.; pTAV-1 is an approximately 100 kb Thiobacillus versutus cryptic plasmid . pTAV-1 DNA was cloned in Escherichia coli . Nine recombinant plasmids containing pTAV-1 DNA inserted into the EcoRI restriction site of pACYC184 were constructed . The origin of DNA inserts was confirmed by Southern blot hybridization . The expression of mixotrophic T . versutus plasmid genes was demonstrated in E . coli. Methods Enzymol, 1990, 183, 211 - 21 Consensus patterns in DNA; Stormo GD; Matrices can provide realistic representations of protein/DNA specificity . In many cases simple mononucleotide-based matrices are adequate representations, but more complex matrices may be needed for other cases . Unlike simple consensus sequences, matrices allow for different penalties to be assessed for different changes to a binding site, a property that is essential for accurate description of a binding site pattern . When only a collection of binding site sequences is known, the best representation for the pattern is an information content formulation, based on both thermodynamic and statistical considerations . Quantitative data on relative binding affinities may be used to determine matrices that provide a best fit to the data . Matrix representations also provide an efficient method of aligning multiple sequences to identify binding site patterns that they have in common. J Parenter Sci Technol, 1990 Jan-Feb, 44(1), 4 - 12 Dry heat inactivation of endotoxin on the surface of glass; Ludwig JD et al.; The thermal inactivation of three endotoxin preparations on the inner surface of glass capillary tubes was studied . The samples were exposed to precisely controlled dry heat conditions at study temperatures ranging from 170 degrees to 350 degrees C, and were assayed using the gel-clot method of the Limulus Amebocyte Lysate test . Plots of the log of the amount of pyrogenic material remaining versus heating time revealed apparently biphasic destruction curves . The initial slopes were linear to a minimum 3-log unit reduction, and were followed by slower destruction rates for the terminal slopes . D values were calculated from the initial slopes of the destruction data, and Z values were estimated from the D values . The D and Z values were found to vary with the initial charged amounts of endotoxin . A second-order equation was found to be an inappropriate model for the inactivation process at temperatures between 170 degrees and 250 degrees C, but was found to be suitable for temperatures between 250 degrees and 325 degrees C . The data were successfully fit to a biexponential equation for all the temperatures studied . The overall inactivation rate of the endotoxin material formulated with fillers was apparently faster than that for the pure endotoxin preparations. Immunology, 1990 Jan, 69(1), 117 - 20 Autoantibodies to c-myc nuclear protein products in autoimmune disease; Yamauchi T et al.; This report describes the identification of autoantibodies that react with c-myc proteins in the sera of some patients with autoimmune diseases . Sera from nine to 30 patients, including six with systemic lupus erythematosus, one with mixed connective tissue disease, one with dermatomyositis and one with autoimmune haemolytic anaemia, reacted in immunoblotting assays against a truncated human c-myc protein p42 produced by Escherichia coli . Furthermore, these sera exhibited double bands of 58,000 and 60,000 MW in the nuclear fraction of HL-60 cells with the amplified c-myc gene . These bands were the same as those detected by the anti-human c-myc protein monoclonal antibody (MYC-1) by immunoblotting assay . The binding activities to 58,000 and 60,000 MW were not reduced by DNase I digestion with the sera and could be absorbed by a p42-bound affinity column and recovered after elution. Curr Genet, 1990 Jan, 17(1), 21 - 4 Transformation of Aspergillus giganteus to hygromycin B resistance; Wnendt S et al.; A wild strain of A . giganteus was transformed to hygromycin B resistance using a bacterial resistance gene under the control of A . nidulans sequences . Stable transformants arose by heterogenous integration, mainly of tandem repeats of vector DNA at various sites in the host genome . Between 6 and 30 resistant colonies were obtained per microgram DNA per 3 x 10(3) viable protoplasts . Vector DNA could be recovered by transformation of Escherichia coli with undigested genomic DNA from Aspergillus giganteus transformants. Eur Arch Otorhinolaryngol, 1990, 247(1), 40 - 2 Effect of Escherichia coli endotoxin on cochlear potentials following its application to the chinchilla middle ear; Morizono T et al.; The compound action potential (CAP) of the eighth nerve and the endocochlear potential (EP) were examined in the chinchilla as an animal model when Escherichia coli endotoxin (100 micrograms) was applied to the middle ear cavity . A significant elevation of the CAP threshold at 2, 3, and 4 kHz was observed 48 h after the instillation of endotoxin, but this hearing loss was thought to be caused by a conductive component . No significant change in the CAP threshold was recognized 30 days after instillation . The EP in either period showed no significant difference . These findings indicate that the application of endotoxin at the concentration used in the present study does not cause a cochlear disturbance. Appl Environ Microbiol, 1990 Jan, 56(1), 7 - 12 Stereospecific production of the herbicide phosphinothricin (glufosinate) by transamination: cloning, characterization, and overexpression of the gene encoding a phosphinothricin-specific transaminase from Escherichia coli; Bartsch K et al.; We have cloned the gene encoding a 43-kilodalton transaminase from Escherichia coli K-12 with a specificity for L-phosphinothricin {L-homoalanine-4-yl-(methyl)phosphinic acid}, the active ingredient of the herbicide Basta (Hoechst AG) . The structural gene was isolated, together with its own promoter, and shown to be localized on a 1.6-kilobase DraI-BamHI fragment . The gene is subject to catabolite repression by glucose; however, repression could be relieved completely when 4-aminobutyrate (GABA) served as the sole nitrogen source . The regulation pattern obtained and a comparison of the restriction map of the initially cloned 15-kilobase SalI fragment with the physical map of the E . coli K-12 genome suggest that the cloned gene is identical with gabT, a locus on the gab gene cluster of E . coli K-12 which codes for the GABA:2-ketoglutartate transaminase (EC 2.6.1.19) . A number of expression plasmids carrying the isolated transaminase gene were constructed . With these constructs, the transaminase expression in transformants of E . coli could be increased up to 80-fold compared with that in a wild-type control, and the transaminase constituted up to 20% of the total soluble protein of the bacteria . Thus, the protein crude extracts of the transformants could be used, after a simple heat precipitation step, for the biotechnological production of L-phosphinothricin in an enzyme reactor. Appl Environ Microbiol, 1990 Jan, 56(1), 1 - 6 Stereospecific production of the herbicide phosphinothricin (glufosinate) by transamination: isolation and characterization of a phosphinothricin-specific transaminase from Escherichia coli; Schulz A et al.; An aminotransferase capable of transaminating 2-oxo-4-{(hydroxy)(methyl)phosphinoyl}butyric acid to L-phosphinothricin {L-homoalanine-4-yl-(methyl)phosphinic acid}, the active ingredient of the herbicide Basta (Hoechst AG), was purified to apparent homogeneity from Escherichia coli K-12 . The enzyme catalyzes the transamination of L-phosphinothricin and various analogs with 2-ketoglutarate as the amino group acceptor . The transaminase has a molecular mass of 43 kilodaltons by sodium dodecyl sulfate-gel analysis and an isoelectric point of 4.35 . The enzyme was most active in the high-pH region, with a maximum at pH 8.0 to 9.5, and had a temperature optimum of 55 degrees C . Heat stability was observed up to 70 degrees C . Substrate specificity studies suggested that the enzyme is identical with the 4-aminobutyrate:2-ketoglutarate transaminase (EC 2.6.1.19) . The first 30 amino acids of the N terminus of the protein were determined by gas phase sequencing . The transaminase was immobilized by coupling to the epoxy-activated carrier VA-Biosynth (Riedel de Haen) and used in a column reactor for the continuous production of L-phosphinothricin . The enzyme reactor was operated for 7 weeks with only a slight loss of catalytic capacity . Production rates of more than 50 g of L-phosphinothricin per liter of column per h were obtained. J Immunoassay, 1990, 11(4), 579 - 90 Human antibody response to the nucleoside triphosphate hydrolase of Toxoplasma gondii; Tenter AM et al.; An enzyme-linked immunosorbent assay (ELISA) that uses a recombinant protein of Toxoplasma gondii as antigen was used for the detection of specific antibodies in human sera . An antigenic portion of the T . gondii nucleoside triphosphate hydrolase (NTPase) was expressed in Escherichia coli as a glutathione S-transferase fusion protein with an apparent molecular mass of about 5000 . A total of 118 T . gondii positive and 63 negative sera were examined . Seven % of the T . gondii positive sera, but none of the negative sera, reacted with the recombinant NTPase . Advantages and disadvantages that are associated with the use of fusion proteins as antigens in ELISA are discussed. Int J Immunopharmacol, 1990, 12(6), 631 - 7 Elevation of cyclic adenosine monophosphate levels independently down regulates IL-1, IL-2, and IL-2 receptor (CD25) syntheses; Iwaz J et al.; In view of the central involvement of interleukin-1 (IL-1) in T-cell functions and the negative effects exerted by cyclic adenosine monophosphate (cAMP) on T-cell responses, we wondered whether these inhibitions rely on defects in IL-1 generation . We investigated the effect of a known cAMP elevating agent, cholera toxin (CT), on the generation of IL-1 from peripheral blood adherent cells as well as the role of IL-1 whenever IL-2 synthesis and IL-2 receptor (CD25 antigen) expression are inhibited . While augmenting intracellular cAMP concentration, CT inhibits from 20 to 40% the generation of IL-1 activity from E . coli lipopolysaccharide (LPS)-stimulated adherent cells . Theophylline (TH), a cAMP degradation blocking agent, induces the same decrease in IL-1 activity . The B chain of CT, devoid of cAMP activating potency, is not inhibitory . In systems where CT and TH dramatically inhibit the generation of IL-2 activity (80%), addition of exogenous IL-1 does not restore the ability of T-cells to produce or release IL-2 . Moreover, CT- and dibutyryl (db)cAMP-induced inhibition of CD25 antigen expression is not overcome by exogenous IL-1, IL-2, nor by both interleukins . It is concluded that inhibition of IL-1 and IL-2 production are independent and that inhibition of CD25 antigen expression is independent of IL-1 and IL-2 modulation . Cholera toxin and cAMP influences on interleukin synthesis are discussed. Med Microbiol Immunol (Berl), 1990, 179(4), 169 - 75 Expression of an antigenic polypeptide of the human parvovirus B19; Eiffert H et al.; The DNA fragment of the human parvovirus B19, with 715 nucleotides between nucleotide positions 3141-3856 was expressed in Escherichia coli as a beta-galactosidase fusion protein . The plasmid vector pSS20d used for this purpose permits cleavage of the viral gene product from the beta-galactosidase moiety by collagenase . After purification by p-aminophenyl-beta-D-thiogalactoside-sepharose and superose, a soluble protein with a molecular mass of 28 kDa was isolated . It represents a common part of the viral capsid proteins VP1 and VP2 . This bacterially derived parvoviral gene product can be used for detection of anti-B19 antibodies in human sera. J Recept Res, 1990, 10(1-2), 97 - 117 Different crosslinking agents identify distinctly different putative Escherichia coli heat-stable enterotoxin rat intestinal cell receptor proteins; Thompson MR et al.; The receptor for heat-stable enterotoxins (ST) produced by Escherichia coli and related organisms is located in the brush border region of intestinal villus cells . Heterobifunctional and homobifunctional crosslinkers were used to covalently couple 125I-ST to rat intestinal cell brush border membrane proteins . Experimental conditions during ligand binding and subsequent crosslinking significantly influence the efficiency of crosslinking, and the number of peptides specifically crosslinked to the 125I-ST . Multiple proteins efficiently coupled to 125I-ST with agents that can couple through the ST amino terminus . The crosslinker 1-ethyl-3-(dimethylaminopropyl)carbodiimide (EDC), which can react with the carboxy terminus of the ST, covalently crosslinked 125I-ST to a single protein with an apparent Mr of 125,000-130,000, larger than the proteins identified using longer crosslinkers . Each of the proteins identified by crosslinking migrate with the same retention time on gel filtration after solubilization, with an approximate molecular size of 150,000-200,000. Crit Rev Biochem Mol Biol, 1990, 25(4), 225 - 44 Roles of G proteins in coupling of receptors to ionic channels and other effector systems; Birnbaumer L et al.; Guanine nucleotide binding (G) proteins are heterotrimers that couple a wide range of receptors to ionic channels . The coupling may be indirect, via cytoplasmic agents, or direct, as has been shown for two K+ channels and two Ca2+ channels . One example of direct G protein gating is the atrial muscarinic K+ channel K+{ACh}, an inwardly rectifying K+ channel with a slope conductance of 40 pS in symmetrical isotonic K+ solutions and a mean open lifetime of 1.4 ms at potentials between -40 and -100 mV . Another is the clonal GH3 muscarinic or somatostatin K+ channel, also inwardly rectifying but with a slope conductance of 55 pS . A G protein, Gk, purified from human red blood cells (hRBC) activates K+ {ACh} channels at subpicomolar concentrations; its alpha subunit is equipotent . Except for being irreversible, their effects on gating precisely mimic physiological gating produced by muscarinic agonists . The alpha k effects are general and are similar in atria from adult guinea pig, neonatal rat, and chick embryo . The hydrophilic beta gamma from transducin has no effect while hydrophobic beta gamma from brain, hRBCs, or retina has effects at nanomolar concentrations which in our hands cannot be dissociated from detergent effects . An anti-alpha k monoclonal antibody blocks muscarinic activation, supporting the concept that the physiological mediator is the alpha subunit not the beta gamma dimer . The techniques of molecular biology are now being used to specify G protein gating . A "bacterial" alpha i-3 expressed in Escherichia coli using a pT7 expression system mimics the gating produced by hRBC alpha k. Rev Roum Virol, 1990 Jan-Mar, 41(1), 53 - 60 Effects of B4 Coxsackievirus infection on cell-mediated immunity, natural cytotoxicity and interleukin-1 production by spleen cells, in mice; Voiculescu C et al.; By using CBA inbred mice as responders, the influence of in vivo B4 Coxsackievirus infection (in two separate experimental procedures) on anti-virus cell-mediated immunity (CMI), natural killer (NK) cell activity and lipopolysaccharide (LPS) induced interleukin-1 (IL-1) production in spleen cells were studied . Both experimental infection schedules were able to induce a "positive" CMI response . NK cell activity was also significantly stimulated, especially by the administration of high virus amounts/inoculum, with frequent injections . On the contrary, LPS-dependent IL-1 synthesis increased particularly following application of the other experimental schedule (low virus amounts, few injections) . Some aspects concerning CMI, NK and IL-1 relationships, during several enteroviral diseases, are discussed. Med Microbiol Immunol (Berl), 1990, 179(3), 145 - 59 Escherichia coli-derived envelope protein gD but not gC antigens of herpes simplex virus protect mice against a lethal challenge with HSV-1 and HSV-2; Broker M et al.; Immunization studies with HSV-1 and HSV-2 envelope proteins expressed in Escherichia coli were performed . After active immunization of mice with a gD-1 antigen (Leu53-Ala312) expressed as a fusion protein, the animals were protected from a lethal challenge with HSV-1 and HSV-2 . In addition, antisera from rabbits immunized with the same gD-1 antigen also conferred passive immunity to mice against a challenge infection with either HSV-1 or HSV-2 . In contrast to these successful gD-1 protection experiments, various gC-1 and gC-2 fusion proteins from E . coli failed to induce protective immunity . Moreover, the mice sera from immunized animals were not able to react with the authentic, glycosylated gC-1 and gC-2 envelope proteins, whereas sera raised against authentic gC-1 and gC-2 glycoproteins do recognize the gC fusion proteins from E . coli . These results indicate, that E . coli might represent an ideal system for expressing gD antigens as a possible component of a HSV vaccine, whereas gC antigen cannot be produced in an immunocompetent form in E . coli. Biol Met, 1990, 3(1), 24 - 7 Mobilization of Escherichia coli R1 silver-resistance plasmid pJT1 by Tn5-Mob into Escherichia coli C600; Starodub ME et al.; Escherichia coli R1 is an Ag(+)-resistant strain that, as we have shown recently, harbours at least two large plasmids, pJT1 (83 kb) and pJT2 (77 kb) . Tn5-Mob was introduced into the E . coli R1 host replicon via conjugation on membrane filters . The transfer functions of plasmid RP4-4 were provided in this process and Tn5-Mob clones mated with E . coli C600 yielded Ag(+)-resistant transconjugants . This mobilization procedure allowed transfer and expression of pJT1 Ag+ resistance in E . coli C600 . Prior to use of Tn5-Mob mobilization, it was not possible to transfer Ag(+)-resistant determinant(s) into E . coli by conjugation or transformation including high-voltage electroporation . E . coli C600 containing PJT1 and PJT2 displayed decreased accumulation of Ag+ similar to E . coli R1.E . coli C600 could not tolerate 0.1 and 0.5 mM Ag+, rapidly accumulated Ag+ and became non-viable . Tn5-Mob mobilization may be useful in the study of metal resistance in bacteria, especially in strains not studied for resistance mechanisms. Cell Motil Cytoskeleton, 1990, 16(4), 229 - 38 Differential effects of gelsolins on tissue culture cells; Huckriede A et al.; Gelsolins, prepared from a number of different sources, showed similar severing activity on F-actin in vitro or on stress fibers of detergent-extracted cells but differed in their effects on actin in stress fibers of microinjected cells . When human gelsolin isolated from plasma was injected into cells in a Ca(++)-containing buffer, stress fibers were degraded, the cellular morphology was changed, and numerous actin patches appeared . These effects were particularly striking when the Ca(++)-insensitive N-terminal proteolytic fragment of this gelsolin was injected . By contrast, Ca(++)-sensitive gelsolins isolated from human platelets, pig stomach smooth muscle and pig plasma showed no comparable activity . Furthermore, the Ca(++)-independent N-terminal proteolytic fragments prepared from these gelsolins also had no effect despite their in vitro actin severing activity . Most striking was the finding that human plasma gelsolin expressed in E . coli did not degrade stress fibers, in contrast to the same protein isolated from plasma; nor was there any stress fiber disruption observed with the N-terminal half of human gelsolin expressed in Escherichia coli . The different behavior of these gelsolins in cells cannot be explained by sequence diversity between plasma and cytoplasmic forms, nor by variability in the Ca++ sensitivity of the preparations . It suggests the presence of factors, as yet unidentified, that may regulate gelsolin activity in the cytoplasm of living cells and discriminate between gelsolins of different origin . Such discrimination could be achieved as a result of post-translational modification of the gelsolin; only in this way can differences between apparently identical proteins isolated from human plasma and expressed in E . coli be reconciled. Prog Clin Biol Res, 1990, 344, 495 - 514 Gene structures of three vertebrate adenylate kinase isozymes; Nakazawa A et al.; Adenylate kinase is an ubiquitous enzyme which contributes to homeostasis of adenine nucleotide composition in the cell . In vertebrates, three isozymes (AK1, AK2, and AK3) are characterized which have distinct distribution in tissues as well as subcellular compartments . The genetic backgrounds of these adenylate kinase isozymes were analyzed . cDNA clones for AK1 were isolated from poly(A)+RNA of chicken skeletal muscle . The results of mRNA analysis in various tissues using the AK1 cDNA indicated that the AK1 gene expression is regulated both tissue-specifically and developmentally at the transcriptional level . The AK1 gene was cloned from chicken and human DNA and characterized . Both genes were split into seven exons . The intron positions in both genes coincided . cDNA clones for AK2 isolated from bovine liver poly(A)+RNA contained two types . One type (AK2A) encoded the same amino acid sequence as that reported for bovine heart AK2 . The other type (AK2B) encoded the same sequence as AK2 except for the COOH terminus . The mRNA species corresponding to the two cDNA clones were identified in bovine liver and heart . Both the cDNA sequences were found to direct the active adenylate kinase synthesis in E . coli . The AK2 gene was cloned and characterized . It consisted of seven exons and six introns . From genomic structure analysis, the two cDNA species were shown to be derived from a single gene by the alternative splicing mechanism . Three types of cDNA clones for AK3 were isolated from bovine liver poly(A)+RNA, which contained the common AK3-coding region and different 3' portions . No NH2-terminal presequence of mitochondrial targeting was identified in AK3 from the sequencing and expression analyses of the cDNA . Upon expression of the cDNA sequence in E . coli, AK3 protein was recovered in the periplasmic space of the bacteria, indicating that AK3 without presequence was exported through the inner bacterial membrane as it is imported through the mitochondrial membranes . Internal targeting signals may be responsible for the translocation process . The AK3 gene was cloned and partially characterized . It is split into at least five exons . The comparisons of amino acid sequences and genomic structure of three isozymes revealed that a segment corresponding to either exon 5 of the AK2 gene or a part of exon 3 of the AK3 gene is missing in the AK1 gene . Phylogenetic analysis suggested that AK1, a shorter molecule, would have been separated from a longer molecule very early in evolution of adenylate kinase.(ABSTRACT TRUNCATED AT 400 WORDS) Toxicon, 1990, 28(5), 493 - 500 The effect of Escherichia coli heat-stable enterotoxin on protein kinase activity; Knoop FC et al.; Isolated rat enterocytes were incubated with E . coli heat-stable enterotoxin or buffer alone and the protein kinase activity and cyclic GMP level determined on the particulate fraction or cytosol, respectively . In the control cells, particulate protein kinase activity and cyclic GMP concentration were at a maximum after 20 sec and 1 min of incubation, respectively . In heat-stable enterotoxin-treated cells the particulate protein kinase activity was significantly increased (P less than 0.05) after 20 sec of incubation, but decreased (P less than 0.05) after 30 sec, 1 min and 2 min, when compared to the control reaction . During this time period the concentration of intracellular cyclic GMP increased 10-fold . The effect of heat-stable enterotoxin on particulate protein kinase activity and cyclic GMP concentration was dose-dependent . Analysis of radioactive membrane phosphorylation products indicate a role for phosphoproteins with a mol . wt of 25,000 and 120,000 . These results suggest that the action of heat-stable enterotoxin may involve an effect on protein kinase. Biomed Biochim Acta, 1990, 49(2-3), S166 - 71 Natural and artificial mutants of the human 2,3-bisphosphoglycerate as a tool for the evaluation of structure-function relationships; Garel MC et al.; 2,3-bisphosphoglycerate mutase is a multifunctional enzyme which catalyses in red blood cells the synthesis and the degradation of 2,3-bisphosphoglycerate, the allosteric effector of hemoglobin . In order to study the structure-function relationships in BPGM, an expression vector was constructed which yielded an active protein, but with a modified electrophoretic mobility, due to a non-blocked N-terminal residue . Using site directed mutagenesis, mutants were produced with shortened chains . Results indicated the importance of residues 252-256 for the function . A natural deficient mutant with the substitution 89 Arg----Cys was described . Artificial mutant with the same substitution reproduced the same defect, as well as mutants Arg----Gly and Arg----Ser, indicating the key role of Arg 89 in the enzymatic mechanism. J Basic Microbiol, 1990, 30(4), 295 - 6 Mutagenesis of acetobacter methanolicus MB58 with the transposon Tn5; Dobrowolski P et al.; Transposon mutagenesis was applied to the isolation of mutants of the facultatively methylotrophic Acetobacter methanolicus MB 58 . The transposon Tn5 (pSU2011) was transferred from Escherichia coli SM 10 by means of conjugation to Acetobacter methanolicus MB 58 . Four out of 1850 stable Km-resistant transconjugants were identified that were formaldehyde sensitive and failed to grow on methanol. Int J Tissue React, 1990, 12(1), 47 - 52 Direct stimulation of superoxide output in neutrophils by lipid A of Escherichia coli; Tarnok I et al.; Two different results have been published in regard to the superoxide-stimulating activity of lipopolysaccharide or Lipid A in neutrophils: first, a direct stimulation after a lag time of about 30-60 sec and second, the inactivity of Lipid A if applied alone, being able only to "prime" the cells for a second challenge during a longer incubation period . In order to achieve clarity regarding these two different opinions, we asked the questions whether: (a) Lipid A is able to stimulate PMN directly, i.e . without a preincubation and a second stimulus; (b) fMLP and Lipid A show a synergistic effect; (c) a preincubation ("priming") of the PMN with Lipid A really increases the superoxide output after a second challenge . We observed (a) a direct stimulation of the chemiluminescence with Lipid A without an additional second challenge, accompanied by a seemingly unimodal kinetics of the superoxide output, i.e . mainly the second phase of the usually bimodal kinetics has been stimulated . As for question (b), a clearly detectable synergism between Lipid A and fMLP could be measured . Regarding question (c), a preincubation ("priming") with Lipid A was of no beneficial effect; the chemiluminescence count could be equally well increased without a "priming" compound. J Intern Med Suppl, 1990, 732, 125 - 32 Neuronal influence on intestinal transport; Jodal M; Reflex activation of the enteric nervous system (ENS) from the intestinal lumen and also from the serosa induces intestinal secretion . Thus mechanical distention, cholera toxin, heat-stable enterotoxin from E . coli, bile acids, mucosal inflammation and chemical peritonitis all induce an intestinal secretion that is inhibited by 60-100% by nerve-blocking agents . As a result of a large number of in vitro and in vivo studies, a picture of the organization of the secretory enteric nervous reflexes is now emerging . In secretory states with preserved intact intestinal epithelium, it is proposed that the reflex activation occurs via stimulation of receptor cells, i.e . epithelial endocrine cells such as EC and N-cells, which release peptides/amines into the interstitial space and thereby activate nerves close to the epithelium . The afferent neurones appear to transfer the reflex to the myenteric plexus, probably by using tachykinins as transmitters . This is in agreement with a superior and co-ordinating role for the myenteric plexus in the control of intestinal function by the ENS . Interneurones in turn mediate the transmission of the nerve signal to the submucosal plexus and the efferent neurones via cholinergic, nicotinic postganglionic receptors . The transmitters at the effector cells are acetylcholine and probably VIP. Arch Virol, 1990, 112(3-4), 139 - 48 Activity of a fowlpox virus late gene promoter in vaccinia and fowlpox virus recombinants; Kumar S et al.; Characterization of a late promoter of fowlpox virus (FPV) and a study of its activity in FPV and vaccinia virus (VV) was carried out . The 5'-mRNA start site of the FPV late gene mapped to a TAAAT sequence near the translation start site (ATG) . A cloned DNA fragment of FPV genome (PFL1) comprising of the 5'-end of the late gene was used to express the LacZ gene of E . coli in FPV and VV recombinants . A comparative analysis of beta-galactosidase (BG) expression from the LacZ gene under the control of the FPV promoter and a VV late promoter (PL11) was performed . Like FPV-PL11-LacZ and VV-PL11-LacZ constructs, FPV-PFL1-LacZ and VV-PFL1-LacZ virus recombinants expressed BG indicating that essential features of transcription were conserved in the two viruses . Furthermore, the LacZ transcripts originating from PFL1 in FPV and VV recombinants mapped to the expected TAAAT sequence . Time course analysis of BG expressed by VV and FPV recombinants suggested that although the transcription machinery in the two viruses was essentially conserved, subtle differences in the efficiency of transcription or translation may exist. J Gen Microbiol, 1990 Jan, 136 ( Pt 1), 65 - 7 Temperature sensitivity of transposition of class II transposons; Turner AK et al.; It has been reported that transposition of Tn3 is temperature-sensitive . The effect of temperature on the transposition of other class II bacterial transposable elements is reported here: Tn21, Tn501, Tn1721, Tn2501 and Tn3926 all also display temperature-sensitivity of transposition . The temperature at which the highest transposition frequency was observed varied between room temperature and 30 degrees C. Mol Biol (Mosk), 1990 Jan-Feb, 24(1), 220 - 30 {Expression in Escherichia coli of hybrid genes containing sequences coding the bovine adrenocorticotropic hormone}; Parsadanian ASh et al.; E . coli strains producing a hybrid protein, containing adrenocorticotropic hormone (ACTH) and protein A of S . aureus was obtained . The sequence coding for ACTH was obtained from the bovine proopiomelanocortin cDNA and, after the modification of the 5'- and 3'-terminal parts, was linked with the protein A gene and its derivatives due to synthetic adaptors . Three forms of ACTH gene, coding this hormone with differing N-terminal amino acid were used to construct the fusion gene . The hybrid proteins contain Asp-Pro or (Asp)4-Lys sequences for obtaining ACTH by acid or enterokinase treatment, respectively . It is shown that each of the constructed plasmids direct the synthesis of hybrid protein in E . coli . This protein was purified by the use of IgG-sepharose . The level of the expression of the hybrid protein is 4 mg/l of the bacterial culture . Most of the synthesized protein is secreted into the periplasmic space. Mol Biol (Mosk), 1990 Jan-Feb, 24(1), 104 - 16 {Interaction of dNTP-binding sites of human DNA polymerase alpha and The Klenow fragment of Escherichia coli DNA polymerase I with nucleotides, pyrophosphate and their analogs}; Nevinskii GA et al.; AMP and NaF each taken separately were shown to activate DNA polymerization catalyzed by Klenow fragment of DNA polymerase I by means of interaction of AMP or NaF with 3'----5'-exonuclease center of the enzyme . In the presence of NaF which is a selective inhibitor of 3'----5'-exonuclease center, AMP is an inhibitor of polymerization competitive with respect to dATP . Ki values and the pattern of inhibition with respect to dATP were determined for AMP, ADP, ATP, carboxymethylphosphonyl-5'-AMP, Pi, PPi, PPPi, methylenediphosphonic acid and its ethylated esters, phosphonoformic acid, phosphonoacetic acid and its ethylated esters as well as for some bicarbonic acids in the reactions of DNA polymerization catalyzed by Klenow fragment of DNA polymerase I (in the presence of NaF) and DNA polymerase alpha from human placenta in the presence of poly(dT) template and r(pA)10 primer . All nucleotides and their analogs were found to be capable of competing with dATP for the active center of the enzyme . Most of the analogs of PPi and phosphonoacetic acid are inhibitors of Klenow fragment competitive with respect to dATP . Nowever these analogs display a mixed-type inhibition in the case of human DNA polymerase alpha . We postulated a similar mechanism of interaction for dNTP with both DNA-polymerases . It is suggested that each phosphate group of PPi makes equal contribution to the interaction with DNA polymerases and that the distance between the phosphate groups is important for this interaction . beta-phosphate of NTP or dNTP is suggested to make negligible contribution to the efficiency of the formation of enzyme complexes with dNTP . beta-phosphate is likely to be an essential point of PPi interaction with the active center of proteins during the cleavage of the alpha-beta-phosphodiester bond of dNTP in the reaction of DNA polymerization. Genetika, 1990 Jan, 26(1), 18 - 29 {Structure and stability of cointegrating plasmids mediated by the IS1 elements in transposon delta Tn9'}; Danilevich VN et al.; In order to elucidate the structural features of the transposon Tn9', representative of the Tn9 family, which define the ability of the transposon to produce unstable cointegrates, we have obtained a derivative of this transposon carrying a deletion in its central region . The deletion in the obtained transposon delta Tn9' covers a DNA segment of about 50 bp in length, occupying the most distal position in relation to the cat gene, at its junction with the right copy of the IS1 . The structure and stability of the IS1/delta Tn9'-mediated cointegrates between the plasmids pDK57.1 (pBR322::delta Tn9') and pRP3.1, a deletion derivative of RP1, have been studied . The three types of cointegrates were found . Those of the type I are predominantly formed, due to the left copy of the IS1 which in delta Tn9' occupies proximal position to the promoter of the cat gene . These cointegrates contain three copies of IS1 and are of high stability . The cointegrates of the type II contain two entire copies of delta Tn9' (i.e . four copies of IS1) as well as the structures of the type II, representing the cointegrate equivalent of inverse transposition and also containing four copies of IS1 . Cointegrates of the type II and III dissociate efficiently in the rec+ cells but, in contrast to the cointegrates mediated by the original transposon Tn9', are unable to dissociate efficiently in the recA- cells . It was concluded that a DNA segment in the central region of Tn9' may be essential for the expression of the IS1-specific resolvase encoded by the right copy of IS1. Chem Pharm Bull (Tokyo), 1990 Jan, 38(1), 128 - 32 Toxicities of dicyanobenzofurazans with formation of superoxide in Escherichia coli; Takabatake T et al.; The toxicities of some benzofurazans (BZs), benzofurazan (1), 4,7-dimethylbenzofurazan (2), 4,7-dibromobenzofurazan (3), 4-bromo-6-cyanobenzofurazan (4), 4,7-dicyanobenzofurazan (5) and 4,5-dicyanobenzofurazan (6), were examined on Escherichia coli . Compound 5 at 4 microM and compound 6 at 7 microM completely inhibited the growth of E . coli in a simple nutritionally restricted medium (GM medium) . These compounds were more toxic in GM medium than in a nutritionally rich medium (YE medium), which contained yeast extract as an additive in GM medium . Compound 4 also inhibited the growth of E . coli at 300 microM in GM medium . The toxicities of BZs were in the order of 1 approximately 2 approximately 3 less than 4 approximately 5 approximately 6 . Compounds 4, 5 and 6 induced manganese-superoxide dismutase (Mn-SOD) and catalase activities of E . coli in YE medium . The induced SOD and catalase provide a defense against the potential cytotoxicities of O2- and H2O2 . The rate of dioxygen uptake in cyanide-resistant respiration of E . coli was dependent on the concentration of 5, and was correlated with the induction of SOD and catalase . The reduction potentials of BZs followed the order of 1 approximately 2 less than 3 less than 4 less than 5 approximately 6 . Compounds 5 and 6, which had redox potentials higher than those of the other BZs, are thought to be more readily reduced in the living system.(ABSTRACT TRUNCATED AT 250 WORDS) Protein Eng, 1990 Jan, 3(3), 215 - 20 The location of inhibitory specificities in human mucus proteinase inhibitor (MPI): separate expression of the COOH-terminal domain yields an active inhibitor of three different proteinases; Meckelein B et al.; Human mucus proteinase inhibitor (MPI) consists of 107 amino acids arranged in two domains showing high homology to each other . This protein is an inhibitor of different serine proteinases including trypsin, chymotrypsin, leukocyte elastase and cathepsin G . On the basis of sequence comparisons it has been suggested that the first domain inhibits trypsin, whereas the second one was thought to be active against chymotrypsin and elastase . To prove the location of the different inhibitory activities gene fragments for both domains have been cloned separately and expressed in Escherichia coli . Inhibition assays with the isolated recombinant domains showed that the second domain is active against chymotrypsin, neutrophil elastase and trypsin, whereas for the first domain only a weak activity against trypsin could be detected . These results suggest that the inhibitory activities of the native molecule towards these three proteinases are all located in the second domain. Basic Life Sci, 1990, 52, 299 - 308 Molecular mechanisms of replicational fidelity in Escherichia coli; Maki H et al.; DNA polymerase III holoenzyme is responsible for chromosomal DNA synthesis in Escherichia coli and seems to be a major determinant of the fidelity of replication of this organism . Among ten different subunits of the holoenzyme, the alpha subunit, encoded by the dnaE gene, has a polymerase activity, while the epsilon subunit, encoded by the dnaQ gene, is a proofreader with a 3'-5' exonuclease activity . Using poly(dA)/oligo(dT)20 as a template-primer, misincorporation of dGMP, dCMP, and dAMP by the alpha subunit and exonucleolytic editing of those mispairs by the epsilon subunit were investigated . When the polymerization reaction was performed with the alpha subunit, dCMP and dGMP but not dAMP were misincorporated . This would suggest that the polymerase might have a base-selecting function to avoid dA:dA mispairing . A subassembly of the DNA polymerase III consisting of alpha, epsilon, and theta subunits misincorporated only dGMP . This would imply that the proofreading function of the epsilon subunit may correct the dC:dA but not the dG:dA mispair . Addition of a protein encoded by the mutT gene, defects of which cause AT to CG transversions in vivo, diminished the misincorporation of dGMP onto poly(dA) template by the alpha subunit . A dGTPase activity was associated with the MutT protein . The significance of the dGTPase activity in the prevention of dG:dA mispairing is discussed. Mol Gen Genet, 1990 Jan, 220(2), 339 - 40 Site-directed insertion mutagenesis with cloned fragments in Escherichia coli by P1 phage transduction; Izuhara M et al.; A cloned gene with an insertion, which was made by introducing cat, was ligated to the cloning site of the phage lambda gt11 . P1 phage grown on cells lysogenized with the recombinant lambda phage could transduce the mutant gene into the original site on the Escherichia coli chromosome. FEMS Microbiol Lett, 1990 Jan 1, 54(1-3), 187 - 92 Identification of proteins encoded in Escherichia coli hydA, hydB and analysis of the hydA locus; Yamamoto T et al.; The hydB gene of Escherichia coli, which is related with the expression of hydrogenase activity, was cloned into the plasmid (pES1) . Using the maxicell protein-labeling method, the molecular weight of hydB gene product was estimated . Comparing between the gene products from the mutant strains and that of the hydB genes cloned strains, the molecular weight of the gene product was 35,000 Mr . Similarly, the molecular weight of the gene product of hydA, which had been previously cloned, was determined by maxicell analysis . The molecular weight of hydA gene product was estimated to be 80,000 Mr . Using deletion analysis and Tn1000 insertional inactivation of hydA's function, the hydA coding region was estimated between 2.2 kb and 2.8 kb in a 3.1 kb EcoRI-MluI fragment on the recombinant plasmid pEH3. Avian Dis, 1990 Jan-Mar, 34(1), 129 - 36 The use of biotin-labeled cDNA probes for the detection of infectious bursal disease viruses; Jackwood DJ et al.; A cDNA library was prepared from the double-stranded RNA genome of the infectious bursal disease virus (IBDV) strain ST-C . The cDNA molecules were annealed into the plasmid pUC9 and used to transform Escherichia coli strain JM107 . A cDNA clone that contained IBDV-specific nucleotide sequences was selected and designated STC-1 . Radiolabeled probes were prepared from STC-1 and hybridized to genome segment A of ST-C in a northern blot hybridization assay . The STC-1 cDNA was 448 base pairs in length, and its nucleotide sequence indicated that it is located near the VP-2/VP-4 junction in IBDV genome segment A . Biotin-labeled probes were prepared from STC-1 and used in a dot-blot hybridization assay to detect IBDV . Under relatively low stringency conditions of hybridization, the biotinylated probes detected four subtypes of IBDV serotype 1 and a serotype 2 IBDV isolate. Oncology, 1990, 47(2), 143 - 8 Mouse monoclonal antibody directed against hepatitis B virus X protein synthesized in Escherichia coli: detection of reactive antigen in liver cell carcinoma and chronic hepatitis; Zentgraf H et al.; A mouse monoclonal antibody directed against the protein product of the hepatitis B virus X open reading frame was prepared . This antibody was used to screen liver tissue sections from patients with chronic hepatitis (CH) and patients with liver cell carcinoma (LCC) . Reactive antigen was detected by immunohistochemistry in about 30% auf the samples from CH patients and in about 80% of the samples from LCC patients regardless of whether tumor or surrounding nontumor tissue was analyzed . A predominant localization of the antigen in the cytoplasm was observed . In liver sections of CH patients the presence of HBx or HBx-related protein appeared to correlate with the presence of the classical viral antigens HBs- and/or HBcAg . A similar correlation was not found in liver or tumor tissue samples from LCC patients . The occurrence of X-monoclonal-antibody-reactive protein (Xarp) at a low frequency in liver tissue from patients without hepatitis B virus related disease suggests that Xarp in some cases may not be identical with the putative viral X antigen. Am J Vet Res, 1990 Jan, 51(1), 40 - 5 Protection of cattle and swine against foot-and-mouth disease, using biosynthetic peptide vaccines; Morgan DO et al.; A single dose of foot-and-mouth disease (FMD) virus protein 1 (VP1) peptide, expressed in Escherichia coli as a fusion protein with 190 amino acids (AA) of the LE' protein of the tryptophan operon of E coli, elicited an immune response in steers sufficient to withstand the challenge of exposure to animals with acute FMD . The 58-micrograms dose of viral peptide, composed of a segment of the VP1 from the A12 strain (A12) of FMD virus (FMDV; A12-32dimer) in a tandem repeat configuration of AA137 through 168 and emulsified with oil adjuvant, elicited a serologic response in cattle equivalent to that obtained using conventional whole virus vaccines . Two groups of swine were vaccinated, 1 with the A12-32dimer as used in cattle and 1 with AA131 through 157 from VP1 of the A24 strain (A24) of FMDV (A24-peptide), expressed in the same system as A12-32dimer, but as a single copy per molecule . In swine, the 58-micrograms dose of the A12-32dimer repeated at 28 days was an effective immunogen; all swine were protected against A12 and, in addition, the vaccine protected 50% of the swine against A24 . The 29-micrograms dose of A24-peptide, administered according to the same schedule, elicited protection against A24 in 50% of the vaccinates and, in addition, protected 25% of those vaccinates against A12 . The serologic response elicited by A12-32dimer against A24 virus was considerably greater than the response elicited by A24-peptide against A12 virus . The evidence of multiple immunogenic epitopes between AA131 and AA168 was evaluated. Arch Biochem Biophys, 1990 Jan, 276(1), 77 - 84 Identification of different classes of nonessential sulfhydryl groups in Escherichia coli adenylosuccinate synthetase; Dong Q et al.; Reaction of Escherichia coli adenylosuccinate synthetase with the thiol reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) or N-ethylmaleimide (NEM) leads to modification of one cysteine residue per enzyme monomer without significant loss of enzyme activity . Modification of a second cysteine residue occurs under mild denaturing conditions (3.5 M urea), and derivatization of this thiol followed by dialysis results in complete loss of enzyme activity . The remaining two cysteine residues react with DTNB only after treatment with 8 M urea . The location of the various cysteine residues in the enzyme was established by using {14C}NEM followed by tryptic digestion and radiopeptide isolation . The reactive cysteine has been identified as Cys291, and the thiol exposed with 3.5 M urea is Cys344 . When Cys344 was replaced by either serine or alanine, the mutant enzymes were found to be as active as the wild-type enzyme . These findings point to the nonessential role of Cys344 in adenylosuccinate synthetase. J Leukoc Biol, 1990 Jan, 47(1), 31 - 8 Interactions of virulent and avirulent Legionella pneumophila with human monocytes; Summersgill JT et al.; Four pairs of virulent/avirulent strains of Legionella pneumophila were examined for their adherence/uptake and activation of human monocytes . Oxidative metabolic responses of monocytes were quantitated by measuring intracellular hydrogen peroxide generation using flow cytometry and by assessment of superoxide dismutase-inhibitable superoxide anion generation . All L . pneumophila strains induced less of a response than did Escherichia coli . Within each pair of isolates, virulent strains of L . pneumophila stimulated the oxidative response of monocytes less than avirulent variants . To determine effects of complement fixation by each strain on their adherence to monocytes, a phagocytic index (PI) was determined under various conditions . In autologous donor serum (AS), all L . pneumophila strains had a PI in the range of 2.1-3.1 bacteria per monocyte, with E . coli having a PI of 9.1 . No significant differences were observed between virulent L . pneumophila strains and their avirulent variants . In the presence of heat-inactivated AS, all PI fell to 0.13-0.20 for the L . pneumophila strains, and to 2.16 for E . coli . Using heat-inactivated AS reconstituted with exogenous human complement as a source of opsonization, levels of PI were indistinguishable from their respective levels in AS . This suggests that complement fixation plays an important role in the adherence of virulent and avirulent L . pneumophila to human monocytes. J Bacteriol, 1990 Jan, 172(1), 53 - 62 Inactivation, sequence, and lacZ fusion analysis of a regulatory locus required for repression of nitrogen fixation genes in Rhodobacter capsulatus; Kranz RG et al.; Transcription of the genes that code for proteins involved in nitrogen fixation in free-living diazotrophs is typically repressed by high internal oxygen concentrations or exogenous fixed nitrogen . The DNA sequence of a regulatory locus required for repression of Rhodobacter capsulatus nitrogen fixation genes was determined . It was shown that this locus, defined by Tn5 insertions and by ethyl methanesulfonate-derived mutations, is homologous to the glnB gene of other organisms . The R . capsulatus glnB gene was upstream of glnA, the gene for glutamine synthetase, in a glnBA operon . beta-Galactosidase expression from an R . capsulatus glnBA-lacZ translational fusion was increased twofold in cells induced by nitrogen limitation relative to that in cells under nitrogen-sufficient conditions . R . capsulatus nifR1, a gene that was previously shown to be homologous to ntrC and that is required for transcription of nitrogen fixation genes, was responsible for approximately 50% of the transcriptional activation of this glnBA fusion in cells induced under nitrogen-limiting conditions . R . capsulatus GLNB, NIFR1, and NIFR2 (a protein homologous to NTRB) were proposed to transduce the nitrogen status in the cell into repression or activation of other R . capsulatus nif genes . Repression of nif genes in response to oxygen was still present in R . capsulatus glnB mutants and must have occurred at a different level of control in the regulatory circuit. J Bacteriol, 1990 Jan, 172(1), 498 - 500 In vitro peptidoglycan synthesis by envelopes from Escherichia coli tolM mutants is inhibited by colicin M; Harkness RE et al.; An in vitro peptidoglycan synthesis reaction was employed to further characterize the role of the tolM product in colicin M-induced inhibition of peptidoglycan synthesis . It was found that the tolM product is not the colicin M target and that this gene product does not play a role in the interaction of the colicin with its target . Colicin M remained associated with envelopes prepared from colicin-treated tolM mutants . These findings suggested that the tolM product most likely is involved with the internalization of colicin M. J Bacteriol, 1990 Jan, 172(1), 437 - 45 mprA, an Escherichia coli gene that reduces growth-phase-dependent synthesis of microcins B17 and C7 and blocks osmoinduction of proU when cloned on a high-copy-number plasmid; del Castillo I et al.; Microcins B17 and C7 are plasmid-determined, peptide antibiotics produced by Escherichia coli when cells enter the stationary phase of growth . Microcinogenic strains are immune to the action of the microcin they synthesize . A well-characterized deficient-immunity phenotype is exhibited by microcin B17-producing cells in the absence of the immunity gene mcbG (M.C . Garrido, M . Herrero, R . Kolter, and F . Moreno, EMBO J . 7:1853-1862, 1988) . A 14.6-kilobase-pair EcoRI chromosomal fragment was isolated by its ability to suppress this phenotype when cloned into a multicopy vector . This fragment was mapped to 57.5 min on the E . coli genetic map . The position of the gene responsible for suppression, designated mprA, was determined by insertional mutagenesis and deletion analysis . mprA was shown to be transcribed clockwise on the E . coli chromosome, and its product was identified as a 19-kilodalton polypeptide . Suppression was shown to be achieved by decreasing microcin B17 production . Increased mprA gene dosage also caused a decrease in microcin C7 production and blocked the osmoinduction of the proU locus in high-osmolarity media . Our results suggest that the mprA gene product could play a regulatory role on expression of several E . coli genes, this control being exerted at the transcriptional level. J Bacteriol, 1990 Jan, 172(1), 424 - 30 Glycerol facilitator of Escherichia coli: cloning of glpF and identification of the glpF product; Sweet G et al.; The glycerol facilitator is known as the only example of a transport protein that catalyzes facilitated diffusion across the Escherichia coli inner membrane . Here we show that the gene encoding the facilitator, glpF, is the first gene in an operon with glpK, encoding glycerol kinase, at 88 min of the E . coli chromosome . The operon is transcribed counterclockwise . We cloned the glpF gene, demonstrated that it complemented a chromosomal glycerol transport-minus mutation, and identified the gene product . The GlpF protein appeared in the membrane fraction of plasmid-bearing strains and had an apparent Mr of 25,000. J Bacteriol, 1990 Jan, 172(1), 31 - 9 Positioning of replicated chromosomes in Escherichia coli; Hiraga S et al.; The positioning of replicated chromosomes at one-fourth and three-fourths of the cell length was inhibited when protein synthesis was inhibited by chloramphenicol or rifampin or by starvation for amino acids . Under these conditions, the progress of chromosome replication continued and replicated chromosomes were located close to each other as one nucleoid mass at midcell . Cells which already had two separate daughter chromosomes located at the cell quarters divided into two daughter cells under these conditions . When protein synthesis resumed, daughter chromosomes moved from midcell to the cell quarters, respectively, before any detectable increase in cell length was observed . The chromosome positioning occurred even under inhibition of the initiation of chromosome replication and under inactivation of DNA gyrase . The chromosome positioning presumably requires new synthesis of a particular protein(s) or translation itself. J Bacteriol, 1990 Jan, 172(1), 305 - 9 Autogenous control is not sufficient to ensure steady-state growth rate-dependent regulation of the S10 ribosomal protein operon of Escherichia coli; Lindahl L et al.; The regulation of the S10 ribosomal protein operon of Escherichia coli was studied by using a lambda prophage containing the beginning of the S10 operon (including the promoter, leader, and first one and one-half structural genes) fused to lacZ . The synthesis of the lacZ fusion protein encoded by the phage showed the expected inhibition during oversynthesis of ribosomal protein L4, the autogenous regulatory protein of the S10 operon . Moreover, the fusion gene responded to a nutritional shift-up in the same way that genuine ribosomal protein genes did . However, the gene did not exhibit the expected growth rate-dependent regulation during steady-state growth . Thus, the genetic information carried on the prophage is sufficient for L4-mediated autogenous control and a normal nutritional shift-up response but is not sufficient for steady-state growth rate-dependent control . These results suggest that, at least for the 11-gene S10 ribosomal protein operon, additional regulatory processes are required to coordinate the synthesis of ribosomal proteins with cell growth rate and, furthermore, that sequences downstream of the proximal one and one-half genes of the operon are involved in this control. J Bacteriol, 1990 Jan, 172(1), 24 - 30 The Myxococcus xanthus FprA protein causes increased flavin biosynthesis in Escherichia coli; Shimkets LJ; The fprA gene is immediately adjacent to the csgA gene (formerly known as spoC) of Myxococcus xanthus . Whereas the csgA gene has an essential role in cell interactions during the developmental cycle, the function of the fprA gene is unknown . Gene disruption was used to determine what affect a null mutation in this gene has on the phenotype of the cell . A csgA-fprA deletion and an fprA frameshift mutation were constructed in vitro in a cloned copy of this locus and then inserted into the M . xanthus chromosome to create a merodiploid with the wild-type and mutant alleles in tandem . The merodiploid was then allowed to segregate one of the two alleles along with the vector sequences in an effort to replace the wild-type allele with the mutant allele . All of the segregants had the wild-type allele, suggesting that a functional fprA gene is essential for vegetative growth . The fprA gene was placed under control of the lacZ transcriptional and translational signals and overexpressed in Escherichia coli, and the new host was examined for any phenotypic changes . A 27-kilodalton protein was observed in sodium dodecyl sulfate-polyacrylamide gels of total-cell protein as predicted from the DNA sequence of this gene . Overexpression of FprA caused the accumulation of a yellow pigment with spectral and redox properties similar to that of the flavins . The pigment cochromatographed with flavin mononucleotide by Silica Gel G thin-layer chromatography . Approximately two-thirds of the total cellular flavin was associated with soluble protein . The major soluble flavin-associated protein was purified on DEAE-Bio-Gel A and Phenyl-Sepharose CL-4B and by polyacrylamide gel electrophoresis . The amino acid composition of the purified protein was similar to that predicted from the DNA sequence of the FprA fusion protein . Apparently, overproduction of FprA (for flavin-associated protein A) in E . coli resulted in a large increase in flavin biosynthesis . Together, these results suggest that the fprA gene encodes a protein that is associated with flavin mononucleotide and has an essential function in M . xanthus. J Bacteriol, 1990 Jan, 172(1), 185 - 92 Increased expression of the bifunctional protein PrlF suppresses overproduction lethality associated with exported beta-galactosidase hybrid proteins in Escherichia coli; Kiino DR et al.; We have cloned and determined the nucleotide sequence of the prlF gene . An open reading frame predicting a 111-amino-acid protein (Mr 12,351) with an acidic carboxy terminus was identified . The DNA sequence preceding this open reading frame revealed a putative promoter and a ribosome-binding site . The nucleotide sequence of the prlF1 mutation revealed a 7-base-pair duplication resulting in a slightly smaller predicted gene product of Mr 12,009 that lacked the acidic carboxy terminus . Maxicell analysis of prlF and prlF1 subclones identified peptides of sizes similar to those predicted by the nucleotide sequences . The prlF sequence was shown to be expressed in vivo by both maxicell analysis and construction of a prlF-lacZ fusion . Two kanamycin resistance insertions within the prlF open reading frame were introduced into the chromosome, replacing the wild-type gene . In contrast to the prlF1 mutation, these insertions had no detectable effect on cell growth or on the beta-galactosidase activity or maltose sensitivity (two sensitive indicators of hybrid protein export) conferred by the lamB-lacZ42-1 gene fusion . Overproduction of the wild-type prlF gene product from a plasmid carrying an active hybrid promoter, however, conferred a prlF1 phenotype . In addition, both the prlF1 mutation and both kanamycin resistance insertions increased the beta-galactosidase activity of a prlF-lacZ fusion . These results suggest that prlF is autoregulated and that overproduction of the prlF gene product increases the export efficiency of beta-galactosidase hybrid proteins from the cytoplasm. J Virol, 1990 Jan, 64(1), 96 - 104 Purification and properties of Epstein-Barr virus DNase expressed in Escherichia coli; Stolzenberg MC et al.; A cDNA corresponding to the BGLF5 open reading frame of the Epstein-Barr virus (EBV) genome and coding for an early DNase was inserted into the procaryotic expression vector pKK223-3 . One bacterial clone producing the expected 52-kilodalton DNase was used as a source of EBV DNase . The 52-kilodalton Dnase was purified in the active form to near homogeneity by ammonium sulfate precipitation and successive chromatographies on phosphocellulose, DNA-cellulose, and gel filtration columns . The purified enzyme exhibited both exonuclease and endonuclease activities, an absolute requirement for divalent cations, an alkaline pH preference, and a typical residual activity in presence of 300 mM KCl . Moreover, the enzyme was specifically inhibited by human sera with high antibody titers to EBV early antigens . These properties are similar to those observed for EBV-induced DNase from lymphoblastoid cell extracts . In addition, the enzyme was recognized by both immunoglobulin G and A serum fractions from patients with nasopharyngeal carcinoma (NPC) . From these results and previous studies which demonstrated the value of antibody titers to this viral DNase as an NPC marker, it appears that EBV-encoded DNase produced in a heterologous expression system could be used in the development of a specific and early NPC diagnosis test. J Med, 1990, 21(1-2), 67 - 84 Changes in the fibrinolysin system in infantile and adult respiratory distress syndrome (ARDS), caused by trauma and/or septic shock in patients and in experimental animals; Ambrus JL et al.; Five hundred premature infants were treated on a randomized double-blind basis with human plasminogen or placebo . We found that in premature infants plasminogen levels are low; thus, defense against intra-alveolar fibrin deposition during birth trauma is reduced . A significant decrease in the incidence of respiratory distress syndrome-hyaline membrane disease and death was seen in the treated infants . Infants with established respiratory distress syndrome were treated with human plasmin or placebo . A significant decrease in death rate was found in the treated infants . Decreased plasminogen and anti-thrombin III (AT-III) levels were found in patients with adult respiratory distress syndrome and/or septic shock . These levels returned to normal within 14 days in survivors, but remained depressed in those who died . It was thought that these parameters may have diagnostic and predictive values . In experimental animals, injection of E . coli endotoxin or oleic acid produced an adult respiratory distress syndrome type phenomenon . This was also accompanied by decreases in plasminogen levels, with recovery in the survivors . It is suggested that plasminogen and anti-thrombin III should be explored as auxiliary therapeutic agents in adult respiratory distress syndrome. Annu Rev Biophys Biophys Chem, 1990, 19, 7 - 41 The proton-translocating ATPase of Escherichia coli; Senior AE; The purpose of this review is to provide an up-to-date summary of E . coli proton-translocating F1F0ATPase . From work on this enzyme, new insights have been gained in the areas of bacterial physiology and energy metabolism, mechanism of enzyme action, mechanism of ion transport through membranes, structure of membrane proteins, mechanism of energy coupling, and regulation of complex enzyme expression and assembly . An important and pressing need is for more structural information . High-resolution structural analyses of F1F0 have not progressed far, and this is likely to present a road block unless overcome . One possibility is to crystallize or apply nuclear magnetic resonance spectroscopy to isolated subunits available in native form from E . coli F1F0 . In this way, one might incrementally build a structure of the F1F0 complex . Static views, however, are unlikely to provide a complete picture of a dynamic enzyme such as this, in which long-range interactions between F0 and F1 and cooperative interactions between nucleotide-binding sites play such an important role in catalysis . Mutagenesis and reversion analysis are two powerful techniques, which, combined with direct enzymological measurements, can be exploited in the immediate future to study the intriguing dynamic aspects of F1F0 function . Many questions remain to challenge us . Regulation of enzyme activity in the cell is not understood . The role of the noncatalytic nucleotide sites is unknown . The assembly pathway and regulation of expression are not established . The mechanisms of H+ translocation and catalysis seem to be proving amenable to analysis, and further advances in these areas can be expected . Long-range conformational interaction between the H+ conduction machinery in F0 and the catalytic sites in F1 seems basic to energy coupling; a major future goal is to provide a realistic physical explanation to validate this concept. Biochimie, 1990 Jan, 72(1), 25 - 32 Induction by different thioredoxins of ATPase activity in coupling factor 1 from spinach chloroplasts; Galmiche JM et al.; ATPase activity of the coupling factor 1, CF1, isolated from spinach chloroplasts, was enhanced by reduction with dithiothreitol . Reduced thioredoxins from spinach chloroplasts, Escherichia coli and human lymphocytes replaced dithiothreitol as reductant and activator of the ATPase . CF1 must be in an oxidized activated state to be further activated by reduced thioredoxin . This state was obtained either by heating CF1 or removing the inhibitory intrinsic epsilon subunit from CF1 . Efficiency and primary structure of the different thioredoxins were compared . The progressive additi