Escherichia coli

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 addition of KCl during ATPase activation by reduced thioredoxin increases then decreases this process . We proposed that three basic amino acids corresponding to arginine 73 and lysines 82 and 96 in Escherichia coli thioredoxin play an important role in the anchorage of the thioredoxin to the negatively charged surface of the CF1 and are involved in the dual effect of KCl . The variations in the screening effect of the negative charges of the CF1 surface by K+ ions can indeed explain the changes in the anchorage of these 3 basic amino acids with concomitant variation in ATPase activity . Human thioredoxin must be 10 times more concentrated than Escherichia coli or spinach chloroplast thioredoxin to exhibit the same activation effect on the ATPase . This fact was related to the properties of a sequence equivalent to the part from amino acid 59 to 72 in Escherichia coli thioredoxin . This part which joins the two lobes of the thioredoxin is more hydrophilic and more negatively charged in human thioredoxin than in Escherichia coli or spinach chloroplast thioredoxin . Although ATPase activation was obtained at a very low concentration of the reduced spinach chloroplast thioredoxin, the thioredoxin formed only a loose complex with CF1.

Free Radic Biol Med, 1990, 8(2), 117 - 9
Quinolinate phosphoribosyl transferase is not the oxygen-sensitive site of nicotinamide adenine dinucleotide biosynthesis; Gardner PR et al.; Quinolinate phosphoribosyl transferase (QPT) activity was not affected when Escherichia coli were treated with hyperbaric oxygen . This result is not in accord with a previous report (Biochem . Biophys . Res . Comm . 91:982-990; 1979) in which the enzyme was shown to be rapidly inactivated in E . Coli exposed to 4.2 atmospheres of oxygen . Our data rule out QPT as a site of oxygen toxicity and suggest other mechanisms for the inhibitory effects of the hyperbaric oxygen on pyridine nucleotide biosynthesis.

Free Radic Biol Med, 1990, 8(2), 113 - 6
Paraquat toxicity and pyridine nucleotide coenzyme synthesis: a data correction; Brown OR et al.; The decrease in pyridine nucleotide coenzymes which occurs during poisoning of Escherichia coli by hyperbaric oxygen or paraquat is not due to impairment of nicotinatemononucleotide pyrophosphorylase (carboxylating) {EC 2.4.2.19} as was previously proposed (Brown, O.R . et al . Biochem . Biophys . Res . Commun . 91:982-990; 1979) . This was shown directly using extracts of E . coli, prepared after exposure to 1 mM paraquat or 4.2 atmospheres of oxygen . The enzyme also was not impaired in Neurospora crassa by 1 mM paraquat . A naturally-occurring, non-dialyzable inhibitor of the enzyme was found in E . coli extracts . The inhibitor caused the erroneous, low nicotinatemononucleotide pyrophosphorylase (carboxylating) activities previously reported in extracts of E . coli poisoned by paraquat.

Proteins, 1990, 7(2), 185 - 97
Substrate recognition by the EcoRI endonuclease; Heitman J et al.; The EcoRI restriction endonuclease is one of the most widely used tools for recombinant DNA manipulations . Because the EcoRI enzyme has been extremely well characterized biochemically and its structure is known at 3 A resolution as an enzyme-DNA complex, EcoRI also serves as a paradigm for other restriction enzymes and as an important model of DNA-protein interactions . To facilitate a genetic analysis of the EcoRI enzyme, we devised an in vivo DNA scission assay based on our finding that DNA double-strand breaks induce the Escherichia coli SOS response and thereby increase beta-galactosidase expression from SOS::lacZ gene fusions . By site-directed mutagenesis, 50 of 60 possible point mutations were generated at three amino acids (E144, R145, and R200) implicated in substrate recognition by the crystal structure . Although several of these mutant enzymes retain partial endonuclease activity, none are altered in substrate specificity in vivo or in vitro . These findings argue that, in addition to the hydrogen bond interactions revealed by the crystal structure, the EcoRI enzyme must make additional contacts to recognize its substrate.

FEMS Microbiol Lett, 1990 Jan 1, 54(1-3), 129 - 33
Correlation between the effects of fosfomycin and chloramphenicol on Escherichia coli; Mengin-Lecreulx D et al.; It was speculated that the increase of the UDP-GlcNAc pool observed with chloramphenicol can modulate the residual PEP:UDP-GlcNAc-enolpyruvate activity of fosfomycin-treated cells . This provided an explanation on how chloramphenicol can insure the formation of enough UDP-MurNAc-pentapeptide to sustain peptidoglycan synthesis at a rate that will antagonize fosfomycin-induced lysis.

Curr Genet, 1990 Jan, 17(1), 65 - 71
The divergently transcribed rbcL and atpB genes of tobacco plastid DNA are separated by nineteen base pairs; Orozco EM Jr et al.; The in vivo transcripts of the tobacco chloroplast gene for the beta subunit of the ATPase (atpB) were examined . In tobacco, like spinach, the atpB gene encodes multiple transcripts . Six tobacco atpB transcripts are present in vivo, with 5' ends at positions "-90", "-255", "-290", "-490", "-500" and "-610" relative to the translation initiation site . The 5' end of the atpB gene ("-610" position) is 20 base pairs from the 5' end of the rbcL gene, coded for on the complementary strand . The "-255", "-490" and "-610" regions are recognized as promoters in vitro by spinach chloroplast and E . coli RNA polymerases.

J Acquir Immune Defic Syndr, 1990, 3(2), 115 - 22
Antibody response to viral proteins U (vpu) and R (vpr) in HIV-1-infected individuals; Reiss P et al.; Antibodies to E . coli-produced HIV-1 vpr and vpu were determined by enzyme immunoassay in serial sets of sera from 72 men seroconverting for antibodies to HIV-1 structural proteins, and from 196 initially symptom-free men who were positive for such antibodies at study entry . First detection of vpr- and vpu-specific antibodies always was within 12 months of seroconversion for antibodies to structural proteins . In the combined cohort of 268 men, vpr- and vpu-specific antibodies were found persistently in 26 and 43% of men, respectively . Vpr- and vpu-specific antibodies were transiently detected in 3 and 7%, respectively, and intermittently detected in 18 and 13% of men, respectively . No association was found between the patterns of vpr- or vpu-specific antibody response and clinical outcome . In subjects with different patterns of vpr- and vpu-specific antibody response, no clear temporal relationship existed between the appearance or disappearance of antibodies and the onset of HIV-1-related disease.

Braz J Med Biol Res, 1990, 23(12), 1263 - 6
Near-ultraviolet illumination inhibits the liquid holding recovery in Escherichia coli B; Aragao BR et al.; Liquid holding recovery (LHR) consists of an increase in the survival of UV-irradiated cells when they are held under non-nutrient conditions before plating . In this study we investigated in E . coli B cells the effect of the growth inhibition induced by near UV (365 nm) illumination (growth delay, GD) before irradiation with UV-254 nm on the amplitude of LHR and the induction of an SOS function (filamentation) during the liquid holding period . Our results demonstrated that pre-illumination with near-UV inhibits LHR and the induction of filamentation when the cells are incubated in nutrient medium . Moreover, this inhibition is due to GD, an effect caused by a photoproduct in the E . coli t-RNA, the 8-13 link.

Pulm Pharmacol, 1990, 3(2), 79 - 87
Recombinant human C5a-induced bronchoconstriction in the guinea-pig: a histamine independent mechanism; Regal JF et al.; Recombinant human C5a (rHuC5a), produced by a synthetic gene expressed in Escherichia coli, causes a decrease in dynamic lung compliance and an increase in pulmonary resistance when injected intravenously in anesthetized mechanically ventilated guinea pigs over a dose range of 5-20 micrograms/kg . Intravenous injection of rHuC5a also caused an immediate decrease in mean arterial blood pressure followed by a transient increase . The purpose of this study was to determine the mediators responsible for these effects . To assess the role of histamine, plasma levels of histamine were monitored and the effects of the H1 antagonist pyrilamine were assessed . rHuC5a caused a significant increase in plasma histamine . However, the H1 antagonist did not alter the maximum or the time course of the bronchoconstrictor response indicating that histamine did not play a major role . The LTD4 antagonist L-649,923 did not inhibit the rHuC5a-induced bronchoconstriction whereas the cyclo-oxygenase inhibitor indomethacin did . Thus, to assess the role of cyclo-oxygenase products, plasma levels of thromboxane (TX) B2, prostaglandin (PG) D2 and PGF2 alpha were monitored after injection of rHuC5a . In addition, guinea pigs were treated with either the TX synthetase inhibitor U-63557A or with the TX receptor antagonist SQ 29,548 . rHuC5a challenge caused an increase in plasma concentrations of TXB2, PGD2 and PGF2 alpha which peaked before the maximum of the bronchoconstriction . SQ 29,548 significantly inhibited the maximum of the bronchoconstrictor response, whereas U-63557A did not inhibit the maximum but did inhibit the time course of the response.(ABSTRACT TRUNCATED AT 250 WORDS)

Histol Histopathol, 1990 Jan, 5(1), 43 - 8
Morphological studies of cytotoxic lesions in reversible endotoxic shock; Garcia R et al.; Reversible endotoxic shock was induced in adult rats by intravenous injection of E . coli 0111:B4 lipopolysaccharide (LPS) and the progression of metabolic and morphological alterations was evaluated . Serum samples and biopsies from adrenal gland, liver and lung were studied at different times after LPS injection . Histological changes in these tissues were observed after endotoxin administration, coinciding with both the acute-phase and the recovery-phase of shock (24-72h after LPS injection) . Signs of tissue regeneration can be correlated with the regression of some serum parameters to their normal values . All these results indicate that in this experimental model of endotoxic shock, a reversible status was established, which will allow further studies of the endotoxic pathophysiological mechanisms in vivo, avoiding the complexity of the non-reversible process.

DNA Seq, 1990, 1(2), 147 - 50
Completed sequence of pKG1800, a vector for determination of transcription terminators; Bernardi F et al.; We have determined the complete sequence of the galactose promoter as well as the amino terminal part of the epimerase gene from Escherichia coli and localized the regulatory elements of the galactose promoter . In this way the sequence of pKG1800, a vector constructed for detection of transcription terminator is now completed.

DNA Seq, 1990, 1(2), 141 - 5
Nucleotide sequence and expression of the gutQ gene within the glucitol operon of Escherichia coli; Yamada M et al.; The glucitol operon in Escherichia coli is known to consist of five structural genes with the order: gut O P A B D M R . We have sequenced downstream from gutR and have identified an open reading frame encoding a water-soluble protein (223 amino acids; molecular weight = 23,562) with a putative ATP binding site . Expression of this protein in a maxicell system has been demonstrated . A repetitive extragenic palindromic (REP) sequence capable of forming stem-loop structures follows gutQ in the downstream, presumptive intercistronic region . The function of the Gut Q protein is not known.

DNA Seq, 1990, 1(1), 73 - 8
Semiautomated preparation of DNA templates for large-scale sequencing projects; Smith V et al.; The rate limiting step in a large-scale sequencing project is the generation of single-stranded DNA templates . We describe a fast, semiautomated procedure, using 96-well microtitre plates, in which 192 templates can be readily prepared in 1 day . The technique can be carried out manually or can be semiautomated using a robot pipetting device . We also provide evidence for the reliability and applicability of this method to a large-scale sequencing project.

Biomed Sci, 1990, 1(6), 597 - 604
Expression of the hAng gene in Escherichia coli; isolation and characterization of human recombinant Ser-(-1) angiogenin; Lapidus AL et al.; Recombinant human angiogenin has been synthesized in Escherichia coli with the aid of a human angiogenin gene (hAng) cloned by Neznanov et al (1990) from a human complementary DNA (cDNA) library . The gene has been expressed by use of a new type of expression vector called a 'TGATG vector' (plasmid pPR-TGATG-1; Mashko et al 1990a) . The highest level of accumulation of the recombinant angiogenin (6%-8% of the total cell protein) was observed in E . coli strain BL21 carrying a temperature-amplifiable version of the plasmid . The synthesized polypeptide carries an additional serine residue at its N terminus in comparison with natural angiogenin . Furthermore, the initiator methionine residue of the recombinant protein is removed with high efficiency by E . coli terminal aminopeptidase . Simple procedures for purification of the recombinant angiogenin from the insoluble fraction of cell protein, and for refolding the protein allowed the isolation of almost 5 mg recombinant angiogenin g-1 wet bacterial biomass . The recombinant Ser-(-1) angiogenin displayed the same biological properties (specific RNAase activity and the ability to induce blood vessel growth on the sclera of experimental animals) as its natural counterpart isolated from human blood.

Pol Arch Weter, 1990, 30(3-4), 113 - 24
{Immunomodulating effects of low doses of nitrogen mustard derivatives}; Debowy J et al.; Chlormethine (Nitrogen mustard) in small doses proved to have immunopotentiating and anti-inflammatory activities . The influence of two nitrogen mustard derivatives : chlorambucil (1 or 10 micrograms/kg p.o.) and cyclophosphamide (0.03 or 0.3 mg/kg i.v.) as well as busulphan (0.5 or 5 micrograms/kg p.o.)--the agent of ++alkylating cytostatic group were investigated in rabbits . Whole blood count, the number of T and B lymphocytes, serum IgG level, phagocytic and microbicidal activities of neutrophils and the plasma level of free glucocorticoids were estimated . The drugs were used in the doses 10-100 times lower than cytostatic ones . Moreover, the ability of alkylating drugs to enhance or to suppress the changes evoked by lipopolysaccharide of E . coli in examined parameters was assessed . The results were compared with chlormethine data obtained previously . None of two nitrogen mustard derivatives (chlorambucil or cyclophosphamide) in the doses many times lower than cytostatic ones, exhibited an immunostimulating and adjuvant properties characteristic of chlormethine . Such properties did not demonstrate small doses of busulphan, another compound of alkylating drugs.

Chin J Biotechnol, 1990, 6(4), 269 - 77
Sequence analysis of D-xylose isomerase gene from Escherichia coli; Hou YM et al.; Gene coding for E . coli D-xylose isomerase was cloned into the plasmid pWR13 from the plasmid pXO100 containing D-xylose operon . Several subclones harboring DNA fragments of different size obtained by step- by-step shortening with Bal31 nuclease were directly sequenced . The complete nucleotide sequence of the 1.6kb DNA fragment carrying D-xylose isomerase gene was determined . The structural gene of 1320 nucleotides codes for a protein of 440 amino acids . The additional 209 nucleotides at 5' end and 82 nucleotides at 3' end include Shine-Dalgarno ribosome-binding site and the transcriptional termination codon, respectively.

Chin J Biotechnol, 1990, 6(4), 251 - 7
Cloning and expression of bovine prolactin cDNA in Escherichia coli; Shu YM et al.; Total mRNA isolated from bovine pituitary was used as a template to synthesize double-stranded cDNA with reverse transcriptase and E . coli DNA polymerase . Recombination was performed using pBR322 as the cloning vector and the oligo dG-tailed and oligo dC-tailed method . The recombinant plasmid was then introduced into E . coli to construct the cDNA library of bovine pituitary mRNA . The labelled synthetic bovine prolactin (bPrl) gene fragment was used as hybridization probe to screen the positive clones, which were then subjected to enzymatic mapping and DNA sequence analysis . The results demonstrate that the positive clones contain a full length bPrl cDNA sequence . The clones obtained were subsequently trimmed, linked to a tac promoter, introduced into E . coli JM103, and expressed under the induction of IPTG . The SDS-PAGE indicates the existence of expression product, and the result of ELISA shows that the product has the same immune activity as native bPrl.

Chin J Biotechnol, 1990, 6(4), 243 - 50
Chemical synthesis and cloning of the human growth hormone releasing factor gene; Xiong KY et al.; Human growth hormone releasing factor (hGRF) gene has been synthesized and cloned . The sequence of the synthetic hGRF gene, consisting of preferred codons for expression in E . coli, was designed with the aid of computer programs . Six segments with lengths ranging from 39 to 51 nucleotides were synthesized by solid-phase phosphoramidite method . The entire gene of 141 base pairs was constructed by enzymatic ligation of all synthetic segments and then cloned into plasmid pUC-19 . The positive colonies were confirmed by the screening of ampicillin resistance, inactive beta-galactosidase, and analyzing by use of restriction enzymes and dot-blot hybridization . The cloned gene was sequenced by M13 dideoxynucleotide chain termination method and proven correct.

Chin J Biotechnol, 1990, 6(4), 229 - 36
A study on an enhancer-like element in Escherichia coli; Pan W et al.; An enhancer-like element of 1.0kb (JM103-M) from E . coli JM103 chromosome was isolated using an enhancer-probing vector . The JM103-M fragment was shown to stimulate the nearby CAT or beta-galactosidase gene expression in E . coli in both directions at a stimulation rate of 3-6 . The same fragment was also demonstrated to have promoter function when a promoter-probing vector was tested . Results obtained from sequencing data showed that a sequence TGACTAA homologous to the AP-1 binding DNA motif of SV40 and two GC boxes were present.

Chin J Biotechnol, 1990, 6(1), 35 - 44
Molecular cloning and restriction mapping of human lymphotoxin gene; Li LH et al.; In order to clone the human lymphotoxin (HuLT) gene, we practiced a concise and time-saving method: homologous recombination in vivo (1) . By using the mouse lymphotoxin (MuLT) cDNA (1.3 kb) as a probe, we isolated the HuLT gene from a human genomic library which was constructed with cosmid pcos2EMBL as a vector . After linearization, the recombinant cosmid was partially digested with BamHI, EcoRI, PstI, and PvuII respectively, and either cos end was labelled by hybridization with radioactive oligos complementary to the cohesive end sequence (2) . The physical map of HuLT gene was made by this method.

Physiol Chem Phys Med NMR, 1990, 22(4), 241 - 5
Aspartokinase III repression and lysine analogs utilization for protein synthesis; Di Girolamo M et al.; The extents of thialysine and selenalysine incorporation into cell proteins were compared in E . coli KL16 and in a mutant able to grow equally well in the presence or in the absence of both lysine analogs . The mutant differs from the parental strain in the repression of aspartokinase III (AKIII), the first enzyme of the lysine biosynthetic pathway . No analog incorporation into proteins was observed in mutant cells grown in the presence of either analog, whereas a marked analog incorporation was observed in the parental strain, where up to 17% and 12% of protein lysine can be substituted by thialysine and selenalysine respectively . In the parental strain grown in media containing either analog at different concentration the extent of analog incorporation into proteins is related to the extent of AKIII repression.

Z Kardiol, 1990, 79 Suppl 3, 167 - 70
Properties of a novel plasminogen activator (BM 06.022) produced in Escherichia coli; Martin U et al.; Since currently available thrombolytics still show disadvantages, such as administration by infusion, occurrence of intracranial hemorrhage, major hemorrhagic complications, allergic reactions, and high price, a novel tissue plasminogen activator has been developed . BM 06.022 is a t-PA mutant produced in Escherichia coli by DNA technology . It has no oligosaccharide side-chains and comprises the kringle 2- and protease domains of t-PA . Like t-PA, the enzymatic activity of BM 06.022 can be stimulated by fibrin . However, BM 06.022 binds to neither endothelial cells nor fibrin . Despite this, BM 06.022 demonstrates the same fibrin selectivity in vivo as t-PA . Investigation of the pharmacokinetic properties in rats, rabbits, dogs, and primates reveals, depending on species, a 4.5- - 10.4-fold longer dominant half-life and 3- - 8.4-fold slower plasma clearance than t-PA (alteplase) . In spite of its lower specific activity in vitro, BM 06.022 has a thrombolytic potency which is 4.6 - 11.5 times higher in vivo than that of alteplase in the rabbit model of venous thrombosis and the canine model of coronary artery thrombosis . BM 06.022 achieves reperfusion significantly more rapid than long-acting anistreplase . Therefore, because of its improved pharmacokinetic properties, BM 06.022 might be administered to infarct patients by i.v . injection in the pre-hospital phase to achieve rapid lysis . Due to its lack of fibrin- and endothelial-cell-binding, combined with retained fibrin selectivity, BM 06.022 further promises to induce fewer hemorrhagic complications than either t-PA or streptokinase.

Adv Exp Med Biol, 1990, 279, 219 - 30
Inhibition of human phospholipases A2 by cis-unsaturated fatty acids and oligomers of prostaglandin B1; Franson R et al.; Inhibition of human phospholipases A2 by cis-unsaturated fatty acids and their oxidative metabolites and/or polymers was studied using partially purified human phospholipases A2 and {1-14C}oleate labelled, autoclaved E . coli as substrate . As previously reported for other phospholipases A2, oleic and arachidonic acids inhibited human synovial fluid phospholipase A2 with IC50s of 15 and 30 microM respectively . Air oxidation of arachidonic acid or hydroxylation of oleic acid (12-hydroxy-oleate) substantially relieved that inhibition . Similarly, the enzymatically oxidatized metabolite of arachidonate, prostaglandin B1 (PGB1), did not inhibit enzymatic activity . However, prostaglandin Bx (PGBx), an oligomer (n = 6) of PGB1, was a potent inhibitor of Ca(++)-dependent, neutral-active phospholipase A2 activities . Enzymatic activity in acid extracts from human neutrophils, platelets, sperm, plasma, synovial fluid, endometrium, degenerative disc, and snake venom was inhibited by PGBx with IC50s ranging from 0.5-7.0 microM . Inhibition was independent of substrate phospholipid concentration over a 24-fold range (5-120 microM) and PGBx quenched the tryptophan fluorescence of snake venom phospholipase A2 in a dose-dependent manner . Agonist-induced (A23187) release of arachidonic acid from prelabelled human neutrophils and cultured human endothelial cells was also inhibited by PGBx with IC50s of 3 and 20 microM, respectively . These results illustrate that oxidative reactions of cis-unsaturated fatty acids relieve their natural inhibitory activity, and polymerization of an inactive fatty acid metabolite yields a potent inhibitor of in vitro and in situ phospholipase A2 activity.

Bull World Health Organ, 1990, 68 Suppl, 138 - 44
The role of cytokines in malaria infection; Maheshwari RK; We have tested the prophylactic effect of Escherichia coli-derived recombinant human interferon gamma (rHuIFN-(gamma} against sporozoite- or trophozoite-induced Plasmodium cynomolgi B malaria infection in rhesus monkeys . Data show that treatment with only five doses of rHuIFN-(gamma) (0.1 mg/kg body weight) given on days -2, 0, and +2 after infection protected the monkeys against sporozoite-induced P . cynomolgi infection . Animals initially protected by rHuIFN-(gamma) treatment remained susceptible to reinfection . No inhibitory effect of rHuIFN-(gamma) was seen against trophozoite-induced infection . We have also tested the effect of recombinant human tumour necrosis factor (rHuTNF) in rhesus monkeys . No significant activity of TNF was seen against trophozoite-induced P . cynomolgi B infection . rHuIFN-(gamma) inhibited schizogony in functional human hepatocytes infected with P . falciparum sporozoites . These results suggest that the inhibitory effect of IFN is limited to the exoerythrocytic stage of parasite development . Interleukin-1 (IL-1) also inhibited hepatic development of P . falciparum sporozoites; however, IL-1 treatment was effective only when applied before sporozoite inoculation . IL-2 and TNF were effective in higher doses.

J Basic Microbiol, 1990, 30(10), 729 - 35
The Streptomyces aureofaciens plasmid pIMB R8 and its use for shuttle vector construction; Godany A et al.; The cryptic low copy plasmid designated as pIMB R8 was isolated from an industrial strain of the chlortetracycline producer Streptomyces aureofaciens R8/26 . Using restriction endonucleases the pIMB R8 plasmid was characterized to have 15 kb . Subcloning of the Bam HI fragments of pIMB R8 into replication probe vector resulted in the identification of the replication part . The 0.7 kb Bc/I--Bg/I fragment is sufficient for normal replication, but produces about fourty times high plasmid copy numbers than the original pIMB R8 . Using the replication part of pIMB R8 was constructed several shuttle cloning vectors for the E . coli-streptomycete system.

Microbios, 1990, 64(260-261), 185 - 95
Influence of caffeine on the induction of SOS functions recA and umuC by mitomycin C in Escherichia coli; Kim J et al.; Caffeine alone induced the SOS functions recA and umuC in wild type recA-lacZ and umuC-lacZ fusion systems in Escherichia coli strains . Caffeine was also able to induce umuC in a umuC-lacZ fusion strain defective in excision repair . Caffeine however was unable to significantly induce recA in a recA-lacZ fusion strain defective in excision repair . Mitomycin C (MMC) at a concentration of 0.5 microgram/ml functioned as a stronger inducer of recA and umuC than caffeine at 2 mg/ml . Cell growth in the presence of both caffeine (2 mg/ml) and MMC (0.05, 0.1 and 0.5 microgram/ml) resulted in primarily an additive induction of recA and umuC.

Microbios, 1990, 64(260-261), 181 - 4
Tetralysine endopeptidase activity in a cell-free extract of Escherichia coli AJ005; Ota A; The activity of tetralysine endopeptidase was examined with cell-free extracts of Escherichia coli AJ005 . The peptidase activity of cell-free extracts increased as the concentration became higher, and reached its maximum at about 30 mg protein/ml . The peptidase activity was stable and maintained most of its activity throughout a 5 h period of observation during incubation at 35 degrees C.

Biol Met, 1990, 3(3-4), 237 - 41
Novel effect of aromatic compounds on the iron-dependent expression of the Escherichia coli K12 manganese superoxide dismutase (sodA) gene; Niederhoffer EC et al.; In Escherichia coli, the superoxide dismutase genes (sodA and sodB) sense the availability of Fe through the action of the fur locus {E . C . Niederhoffer, C . M . Naranjo, K . L . Bradley, J . A . Fee (1990) Control of Escherichia coli superoxide dismutases (sodA and sodB) genes by the ferric uptake regulation (fur) locus, J . Bacteriol . 172, 1930-1938} . Previous work from other laboratories has shown that a variety of metal chelators and of redox-active aromatic compounds can dramatically induce expression of sodA . Here we show that non-redox-active, non-metal-chelating aromatic compounds also enhance expression of a chromosomal sodA gene fusion and that these effects are strongly modulated by the Fur phenotype (Fur +/-) and by the availability of iron in the culture medium . The compounds studied were ethidium bromide, hemin, 2,2'-bipyridine, 1,10-phenantroline, 4,7-phenantroline, rhodamine B1, rhodamine 6G, and, for comparison to previous studies, Paraquat.

J Basic Microbiol, 1990, 30(6), 435 - 42
The effect of non-ionic surfactants on the SOS-inducing potency of 4-nitroquinoline-1-oxide in Escherichia coli PQ37; Raabe F et al.; Various non-ionic surfactants affect the SOS-inducing potency (SOSIP) of the model genotoxin, 4-nitroquinoline-1-oxide, in Escherichia coli PQ37 to varying degrees, as measured by an automated version of the SOS chromotest . While there is little effect on the SOSIP value and other test parameters from Tween 20 and 80 and, with some reservation, Triton X305 and Tyloxapol, over the critical micelle concentration range, the SOSIP value increases in the presence of comparable concentrations of Triton X15, 45 and 100 . A possible dependence of the tester strain's beta-galactosidase production and its growth inhibition on the HLB of the non-ionic surfactants added is discussed.

Life Sci, 1990, 47(24), 2241 - 9
Impairment of histone H1 DNA binding by adduct formation with acetaldehyde; Niemela O et al.; Incubation of histone H1 with pharmacologically relevant concentrations of acetaldehyde resulted in the formation of spontaneously stable acetaldehyde-protein linkages . The reaction of acetaldehyde and H1 purified from rat liver either by a DNA recognition site affinity chromatography or by perchloric acid extraction occurred primarily at the lysine residues in the carboxyterminal tail of H1, which is crucial for its function as a eukaryotic repressor . It was further shown using an H1-lacZ fusion protein produced in E . coli and the protein isolated from rat liver that the formation of acetaldehyde adducts with H1 impair its DNA binding properties . We propose that such a reaction may occur in vivo and lead to an inability to repress genes in the liver upon excessive alcohol consumption . This mechanism may play a role in acetaldehyde-induced collagen synthesis in alcoholics.

Prep Biochem, 1990, 20(1), 75 - 85
Purine nucleoside phosphorylase: purification using an ether-linked formycin B/sepharose 6B resin with unusual properties; Hall WW et al.; Formycin B {9-deazainosine} was reacted with epoxy-activated Sepharose 6B to form an affinity resin for purine nucleoside phosphorylase (PNPase) . This resin had a large capacity (7,600 units/ml) for the enzyme from Escherichia coli . Enzyme retention was dependent on high ionic strength . Although this property is reminiscent of hydrophobic interaction chromatography, analogous resins prepared with pseudouridine or monoethanolamine instead of with formycin B, did not retain the enzyme even at high ionic strength . Furthermore, hypoxanthine facilitatted elution of the enzyme from the resin . It appeared, therefore, that the enzyme was not bound simply by hydrophobic interactions . A simple two-step purification procedure for PNPase from Escherichia coli was devised using this resin . Overall recovery was 50%, and purity of the final preparation was greater than 95% . This resin was also useful in the purification of PNPase from human erythrocytes . The ether linkage between formycin B and Sepharose 6B, together with the carbon-to-carbon linkage between the pentose and heterocyclic moieties of formycin B, provided stability to both chemical and enzymatic degradation . After 5 years of use and exposure to a variety of biological preparations, the resin showed no detectable decrease in its ability to bind PNPase.

Appl Biochem Biotechnol, 1990 Spring-Summer, 24-25, 151 - 60
Characterization of cryptic plasmids from marine cyanobacteria and construction of a hybrid plasmid potentially capable of transformation of marine cyanobacterium, Synechococcus sp., and its transformation; Matsunaga T et al.; Among forty strains of marine cyanobacteria isolated in our laboratory, five strains had 1-3 different plasmids . The unicellular marine cyanobacterium, Synechococcus sp . NKBG 042902, contains at least three plasmids (pSY09, pSY10, and pSY11) . However, these plasmids are cryptic . Therefore, a hybrid plasmid pUSY02 containing the 1.4-kb HindIII fragment of pSY11 and Escherichia coli plasmid pUC18 was constructed . The plasmid pUSY02 transformed both marine Synechococcus sp . NKBG042902-YG1116, which is a cured strain, and fresh water Anacystis nidulans R2 by dark incubation or Ca2+ treatment . However, the plasmid pSG111 constructed from the plasmid DNA of A . nidulans R2 failed to transform marine Synechococcus sp . Electroporation was also applicable to transformation of marine Synechococcus sp . and fresh water A . nidulans R2 . The plasmid pUSY02 was rapidly introduced into marine Synechococcus sp.

Chem Biol Interact, 1990, 74(3), 343 - 52
Detection of DNA strand breaks in Escherichia coli treated with platinum(IV) antitumor compounds; Defais M et al.; DNA strand breaks were observed in bacteria treated with Pt(IV) but not Pt(II) antitumor compounds by two methods . First, compounds which cause DNA strand breaks produced an SOS induction signal which was detected by a rapid bacterial assay . In addition, the capacity of these compounds to cut DNA in vivo was directly measured by agarose gel electrophoresis of pBR322 DNA extracted from bacteria treated with these drugs . cis-Diamminetetrachloroplatinum(IV) (cis-DTP) and cis-dichloro-trans-dihydroxo-cis-bis(isopropylamine)-platinum(IV) (iproplatin) produced strand breaks in both assays while cis-diamminedichloroplatinum(II) (cisplatin) did not . These results indicate that Pt(IV) antitumor complexes may cause DNA damage in vivo which is not produced by Pt(II) compounds.

Probl Tuberk, 1990, (2), 49 - 51
{Production and characteristics of monoclonal antibodies reacting with human-type M . tuberculosis}; Chernousova LN et al.; Monoclonal antibodies to M . tuberculosis were produced by hybrid technology . They were described via enzyme immunoassay and immunoblotting . It was shown that these antibodies should be included into IgG class . Besides, they are oriented towards and antigen with a molecular weight of 20 kDa, react with H37Rv at a concentration of 12 ng/ml and with BCG at a concentration of 50 micrograms/ml and fail to react with M . Intracellulare, M . Scrofulaceum and E . coli.

Ann Rech Vet, 1990, 21(1), 49 - 55
{The in vitro activity of veterinary antiseptics}; Maris P; The in vitro activity of 28 veterinary antiseptic drugs was studied on 4 bacterial strains either with or without calf serum . Eight preparations (VT Dose, Dulcidrine, Collyre Clement, Cronic, Detecaine, Lotion Maudor, Tano-Patt, Aurikan), half of which were eye-lotions, were found to be non antiseptic according to AFNOR standards . In the presence of calf serum, 4 other preparations (Albacetine, Nybocaine, Pediplasme, Pedifort) did not meet the standard NF T 72.171 criteria.

Biochimie, 1990 Jan, 72(1), 33 - 40
Enzymatic properties, renaturation and metabolic role of mannitol-1-phosphate dehydrogenase from Escherichia coli; Teschner W et al.; Enzymatic properties, renaturation and metabolic role of mannitol-1-phosphate dehydrogenase from Escherichia coli . D-mannitol-1-phosphate dehydrogenase was purified to homogeneity from Escherichia coli, and its physicochemical and enzymatic properties were investigated . The molecular weight of the polypeptide chain is 45,000 as determined by polyacrylamide gel electrophoresis in denaturing conditions . High performance size exclusion chromatography gives an apparent molecular weight of 47,000 for the native enzyme, showing that D-mannitol-1-phosphate dehydrogenase is a monomeric NAD-dependent dehydrogenase . D-mannitol-1-phosphate dehydrogenase is rapidly denatured by 6 M guanidine hydrochloride . Non-superimposable transition curves for the loss of activity and the changes in fluorescence suggest the existence of a partially folded inactive intermediate . The protein can be fully renatured after complete unfolding, and the regain of both native fluorescence and activity occurs rapidly within a few seconds at pH 7.5 and 20 degrees C . Such a high rate of reactivation is unusual for a protein of this size . D-mannitol-1-phosphate dehydrogenase is specific for mannitol-1-phosphate (or fructose-6-phosphate) as a substrate and NAD+ (or NADH) as a cofactor . Zinc is not required for the activity . The affinity of D-mannitol-1-phosphate dehydrogenase for the reduced or oxidized form of its substrate or cofactor remains constant with pH . The affinity for NADH is 20-fold higher than for NAD+ . The forward and reverse catalytic rate constants of the reaction: mannitol-1-phosphate + NAD+ in equilibrium fructose-6-phosphate + NADH have different pH dependences . The oxidation of mannitol-1-phosphate has an optimum pH of 9.5, while the reduction of fructose-6-phosphate has its maximum rate at pH 7.0 . At pH values around neutrality the maximum rate of reduction of fructose-6-phosphate is much higher than that of oxidation of mannitol-1-phosphate . The enzymatic properties of isolated D-mannitol-1-phosphate dehydrogenase are discussed in relation to the role of this enzyme in the intracellular metabolism.

J Acquir Immune Defic Syndr, 1990, 3(6), 584 - 90
Use of IFN-gamma in patients with AIDS; Heagy W et al.; The tolerance and toxicity of interferon-gamma (IFN-gamma) was assessed in a phase I/II study of 21 patients with acquired immune deficiency syndrome (AIDS) . A highly purified preparation of human recombinant E . coli-produced IFN-gamma was given i.v . twice weekly for an 8 week period . Patients were enrolled in the study in groups of four or five; the initial group received an IFN-gamma dose of 0.03 mg/m2 and subsequent groups received higher IFN-gamma doses of 0.3, 1, or 3 mg/m2 . Toxicity resulting from IFN-gamma was minimal and the therapy was well tolerated even at the maximum dose (3 mg/m2) . No patients developed antibodies that neutralized IFN-gamma . Clinical responses were observed in 3 of 17 patients with Kaposi's sarcoma (KS) . A complete clinical response was achieved in one individual and a partial, temporary regression of KS lesions was observed in two other patients . HIV p24 antigen was decreased in plasma samples obtained from six of nine patients with initially detectable HIV protein . These data suggest that IFN-gamma should be considered as a therapeutic agent, possibly with other antivirals, in the treatment of patients with AIDS.

JPEN J Parenter Enteral Nutr, 1990 Jan-Feb, 14(1), 56 - 9
Evaluation of the endotoxin retention capabilities of inline intravenous filters; Horibe K et al.; The capabilities of inline filters to retain bacteria and endotoxin were examined during simulated extended infusions for up to 168 hr . The tested inline filters were the ELD96 (Pall Biomedical Corp) and the IVEX 2 (Millipore Corp) . Approximately 1 x 10(8) total cells of Escherichia coli B . were challenged to the upstream site of the filter . The test solution of 5% dextrose in water, 0.9% saline, Paremental A (A basic solution for total parenteral nutrition (TPN), a TPN solution in use in our clinic were infused continuously up to 168 hr and flow rate was maintained at 83 ml/hr . The effluents were analyzed using the Limulus Amebocyte Lysate (LAL) test to detect endotoxin and also passage of the challenged bacteria was tested at 24-hr intervals over 168 hr . The results were as follows: (1) The viability control culture showed the presence of viable bacteria throughout the 168-hr period of the experiment . (2) During the experiments, all filters produced sterile effluents . (3) LAL assay indicated that only the effluents from the ELD96 contained no detectable endotoxin for 168 hr.

FEMS Microbiol Lett, 1990 Jan 1, 54(1-3), 169 - 73
Differences between the mechanisms of action of heat-stable and heat-labile enterotoxins of Escherichia coli; Goyal J et al.; The mucosal-to-serosal and serosal-to-mucosal fluxes of Na+ and Cl- were measured in control mice and mice treated with heat-stable (ST) and heat-labile (LT) enterotoxins in the presence or absence of: Ca2(+)-ionophore A23187, an activator of Ca2(+)-calmodulin; or phorbol-12-myristate-13-acetate (PMA), an activator of protein kinase C(PKC); or 1-(5-isoquinolinyl sulphonyl)-2-methyl piperazine (H-7), an inhibitor of PKC . There was net secretion of Na+ and CL- in both experimental groups in contrast to net absorption in the control group . The addition of ionophore or PMA or ionophore + PMA resulted in net secretion of Na+ and Cl- in the control group and the effect of ionophore and pMA was found to be additive . The addition of ionophore did not cause any change in electrolyte fluxes in the ST toxin treated group, however, it increased the net secretion of Na+ and Cl- in the LT toxin treated group . PMA increased the net secretion of Na+ and Cl- in the St toxin treated group, however, it did not cause any change in Na+ and Cl- fluxes in the LT toxin treated group . H-7 did not reverse the effect of ST toxin, however, it reversed the effect of LT toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

FEMS Microbiol Lett, 1990 Jan 1, 54(1-3), 141 - 5
Organization and nucleotide sequence of the genes for ribosomal protein S2 and elongation factor Ts in Spirulina platensis; Sanangelantoni AM et al.; A 6.5 kb region from the genome of the cyanobacterium Spirulina platensis was cloned using as a probe the Escherichia coli gene for ribosomal protein S2 . Sequence analysis revealed, in this region, the presence of the gene for ribosomal protein S2 and part of the gene for the elongation factor Ts (EF-Ts) . The arrangement rpsB-spacer-tsf resembles that reported for E . coli . The deduced amino acid sequences of the platensis S2 and EF-Ts show significant homology with the E . coli counterparts.

Anal Biochem, 1990 Jan, 184(1), 48 - 54
Heterogeneity among membrane vesicles of Escherichia coli: effects of production and fractionation techniques; Jacoby GH et al.; We have previously shown that Escherichia coli membrane proteins are associated with subpopulations of membrane vesicles beyond the well-defined inner versus outer membrane classification . To separate these vesicles we used a shallow sucrose gradient which differed in many respects from established procedures . Here we compare this revised technique to the classical sucrose density centrifugation procedure . We found that certain manipulations common to the latter obscured heterogeneity among membrane vesicles . The following treatments degraded vesicle separation: growth in rich medium addition of EDTA to buffers, spheroplasting, sonication, pelleting of membranes prior to sucrose gradient centrifugation, overloading the gradient, and long centrifugation times . Some treatments, such as EDTA addition, affected only selected vesicles . When determining protein localization within bacterial membranes experiments should be designed to avoid or at least minimize these manipulations.

Pflugers Arch, 1990 Jan, 415(4), 440 - 3
Fever response induced by intravenous and intracerebroventricular injection of pyrogen in thyroidectomised and protein-calorie malnourished rabbits; Macari M et al.; The development of a fever in response to intravenous (IV, 1.5 micrograms/kg body mass) and intracerebroventricular (ICV, 1.5 micrograms/animal) injections of Escherichia coli lipopolysaccharide (LPS) was studied in control, thyroidectomised and protein-calorie malnourished rabbits (New Zealand Whites, n = 55) . ICV injection of LPS in control rabbits produced a fever response, the characteristics of which differed from those obtained after IV pyrogen injection . Thyroid deficiency caused an attenuated fever response, irrespective of whether LPS had been administered by IV or ICV injection . Protein-calorie malnourished rabbits showed a smaller fever response after IV or ICV pyrogen injections . Malnourished rabbits, refed over a period of 15 days, showed a typical biphasic fever response, but with lower magnitude than controls . The results of these experiments suggest that ICV injection of LPS is not an appropriate model for the study of fever mechanisms in disease states, and that the attenuated fever response observed in protein-calorie malnourished rabbits may be related, at least in part, to a decreased ability to produce the endogenous pyrogen interleukin-1.

J Mol Evol, 1990 Jan, 30(1), 16 - 25
The nucleotide sequence of five ribosomal protein genes from the cyanelles of Cyanophora paradoxa: implications concerning the phylogenetic relationship between cyanelles and chloroplasts; Evrard JL et al.; The nucleotide sequences of the ribosomal protein genes rps18, rps19, rpl2, rpl33, and partial sequence of rpl22 from cyanelles, the photosynthetic organelles of the protist Cyanophora paradoxa, have been determined . These genes form two clusters oriented in opposite and divergent directions . One cluster contains the rpl33 and rps18 genes; the other contains the rpl2, rps19, and rpl22 genes, in that order . Phylogenetic trees were constructed from both the DNA sequences and the deduced protein sequences of cyanelles, Euglena gracilis and land plant chloroplasts, and Escherichia coli, using parsimony or maximum likelihood methods . In addition, a phylogenetic tree was built from a distance matrix comparing the number of nucleotide substitutions per site . The phylogeny inferred from all these methods suggests that cyanelles fall within the chloroplast line of evolution and that the evolutionary distances between cyanelles and land plant chloroplasts are shorter than between E . gracilis chloroplasts and land plant chloroplasts.

Arch Biochem Biophys, 1990 Jan, 276(1), 294 - 8
Expression of the yeast LEU4 gene is subject to four different modes of control; Peters MH et al.; A translational fusion of yeast LEU4 and Escherichia coli lacZ which contains 679 bp of the LEU4 5'-flanking region and the first two codons of LEU4 was used to study LEU4 expression . Eight recipient strains with different genetic backgrounds, transformed with a plasmid containing the fusion, were grown under a variety of conditions, and beta-galactosidase activity was measured . Evidence was obtained for at least four modes of expression of LEU4: general amino acid control, leucine-specific control, basal level expression, and branched-chain amino acid-mediated repression . Determination of steady-state levels of LEU4 mRNA suggested that LEU4 expression is regulated transcriptionally.

Proc Natl Acad Sci U S A, 1990 Jan, 87(1), 458 - 62
Neurons and glia arise from a common progenitor in chicken optic tectum: demonstration with two retroviruses and cell type-specific antibodies; Galileo DS et al.; We used a recombinant retrovirus to study cell lineage in the chicken optic tectum . The virus inserts the Escherichia coli lacZ (beta-galactosidase) gene into the genome of an infected cell; a histochemical stain marks the progeny of infected cells with a blue precipitate . We had previously shown that individual clones frequently contain diverse neuronal types . Now we asked whether individual clones contain glia as well as neurons . To this end, we constructed a virus in which lacZ is fused to a nuclear localization signal sequence from the simian virus 40 large tumor antigen . Cells infected with this virus are marked with blue nuclei instead of blue somata . In embryos injected with a mixture of the two retroviruses, individual clusters contained cells with only one label type (nuclear or cytoplasmic), thus verifying that clusters of cells were clones . Furthermore, it was possible to immunostain the somata of cells that had blue nuclei, whereas the blue cytoplasmic precipitate hampered immunostaining . Together, these methods allowed us to show that some clones contained neurons (neurofilament-positive) and two types of glia (glutamine synthetase-positive and glial fibrillary acidic protein-positive) . This result demonstrates the existence of a common progenitor for neurons and glia in optic tectum.

J Exp Med, 1990 Jan 1, 171(1), 339 - 44
A mycobacterial 65-kD heat shock protein induces antigen-specific suppression of adjuvant arthritis, but is not itself arthritogenic; Billingham ME et al.; A recombinant (r)65-kD protein from Mycobacterium leprae, at levels far in excess of those present in whole mycobacteria, was unable to induce arthritis . Even when combined with a synthetic adjuvant, CP20961, to mimic the peptidoglycan adjuvant component of the mycobacterial cell wall, the r65-kD protein failed to induce arthritis . Pretreatment with as little as 1 microgram r65-kD protein protected rats against arthritis induced by M . tuberculosis, but this r65-kD protein was markedly less able to protect against arthritis induced by the synthetic adjuvant, CP20961, or type II collagen . The r65-kD protein appears, therefore, to produce an antigen-specific protection against arthritis induced by bacterial cell walls containing the 65-kD protein . Such protection can be overcome, however, by arthritogenic T lymphocytes, suggesting that protection occurs by preventing clonal proliferation of autoreactive T lymphocytes that are induced by the adjuvant properties of mycobacterial cell walls . How the r65-kD protein abrogates this particular adjuvant activity, and the nature of the arthritogenic self antigen(s), remain to be elucidated.

J Clin Invest, 1990 Jan, 85(1), 185 - 91
Interferon gamma drastically modifies the regulation of interleukin 1 genes by endotoxin in U937 cells; Ucla C et al.; IL-1 alpha, IL-1 beta, and tumor necrosis factor alpha (TNF-alpha) gene expression is induced by LPS (endotoxin) in monocytes/macrophages and in some monocytic cell lines . IFN gamma and 1 alpha,25-dihydroxyvitamin D3 (1,25{OH}2D3) are important macrophage-activating factors . They induce changes in the human monocyte cell line U937 that reflect cellular differentiation . We have studied the effect of IFN-gamma and of 1,25(OH)2D3 on the expression of IL-1 and TNF-alpha messenger RNA in response to LPS . The induction of these genes by LPS is immediate and transient, with a maximum in 3 h . Preincubation of the cells with IFN-gamma or with 1,25(OH)2D3 increases these mRNA responses to LPS about fourfold . More importantly, cells exposed to IFN-gamma for 72 h exhibit a drastically different and unexpected pattern of IL-1 alpha and IL-1 beta gene response to LPS . Instead of the normal transient response, one then observes a sustained increase in IL-1 alpha and IL-1 beta gene expression over at least 16 h after LPS stimulation . This was measured both at the level of mRNA and by direct transcription assays (run-off) . This striking effect of IFN-gamma on the kinetics of IL-1 gene response does not apply to the TNF-alpha gene . Interestingly, 1,25(OH)2D3, which shares with IFN-gamma a number of important effects on monocytes/macrophages, does not affect the kinetics of IL-1 gene response to LPS . In view of the biological relevance of endotoxin as a macrophage activator, the potential clinical implication of this prolonged induction of IL-1 gene expression is discussed.

J Bacteriol, 1990 Jan, 172(1), 377 - 82
Role of threonine residue 154 in ligand recognition of the tar chemoreceptor in Escherichia coli; Lee L et al.; The Tar chemoreceptor of Escherichia coli mediates attractant responses to aspartate, maltose, and phenol, repellent responses to Ni2+ and Co2+, and thermoresponses . To understand the role of threonine residue 154, which is located in the ligand-binding domain of Tar, we replaced the residue with serine, isoleucine, and proline by site-directed mutagenesis . The replacements caused reductions in aspartate sensing but had only a small effect on maltose sensing and almost no effect on phenol sensing, repellent sensing, and thermosensing . These results indicate that Thr-154 of Tar is rather specifically involved in aspartate sensing . The reductions in the response threshold for aspartate by the replacements with serine, isoleucine, and proline were less than 1, about 2, and more than 5 orders of magnitude, respectively . When the corresponding threonine residue in the Tsr chemoreceptor was replaced with the same amino acids, roughly similar reductions in the response threshold for serine resulted . Thus, these threonine residues seem to have a common role in detecting the aspartate and serine attractant families . A mechanism by which these chemoreceptors detect the amino acid attractants is discussed.

J Bacteriol, 1990 Jan, 172(1), 266 - 72
Agmenellum quadruplicatum M.AquI, a novel modification methylase; Karreman C et al.; The complete type II modification methylase of Agmenellum quadruplicatum was cloned in Escherichia coli as an R.Sau3A fragment of approximately 4.5 kilobases . The coding sequence was contained in a stretch of 1,156 base pairs which was organized into two parallel, partly overlapping open reading frames of 248 and 139 codons . In vivo complementation experiments showed that the synthesis of both predicted peptides was required for full methylase activity . The amino acid sequences were considerably similar to regions of other deoxycytidylate methylases.

Chin J Biotechnol, 1990, 6(3), 165 - 72
Molecular cloning of a DNA segment essential for replication from Streptomyces hygroscopicus strain 10-22 and localization of its minimal functional region; Deng ZX et al.; The 13 kb DNA segment responsible for replication has been isolated from Streptomyces hygroscopicus subsp . yingchengensis strain 10-22 using a pBR322-derived Streptomyces replication-probe vector pIJ2703 . The restriction map of the hybrid plasmid, pHZ54, has been constructed . Following a series of deletion and subcloning experiments, the minimal functional region for replication was located within a 2.86 kb DNA fragment . Subsequently, this region was further shortened into a 2 kb region by exonuclease III deletion . A few small bifunctional plasmids able to replicate in E . coli as well as in Streptomyces, were obtained during the above process . Some plasmids could be used as shuttle vectors.

Int J Tissue React, 1990, 12(6), 363 - 8
Alpha 1-antitrypsin variants produced by recombinant DNA: differences in elastase inhibitory activity and resistance to oxidant agents; Luisetti M et al.; Inherited or "acquired" deficiency of alpha 1-antitrypsin (believed to be the cause of pulmonary emphysema) will probably be treated in the future by replacement with alpha 1-antitrypsin purified from human plasma or produced by recombinant DNA, which seems promising because it permits site-specific mutagenesis in the oxidizable active site of the normal human alpha 1-antitrypsin . The aim of this in-vitro study was to investigate the elastase inhibitory activity and the resistance to oxidizing agents of normal human alpha 1-antitrypsin, a recombinant yeast-produced variant (VAL 358) and a recombinant E . coli-produced variant (LEU 358) . The inhibitors were exposed to chemical oxidants (NCS, H2O2, xanthine/xanthine oxidase, chloramine-T) and to PMA-activated neutrophils . The elastase inhibitory activity was assayed on porcine pancreatic elastase and neutrophil elastase . Normal alpha 1-antitrypsin and VAL 358 variant were good inhibitors of both elastases . LEU 358 variant was the best inhibitor for neutrophil elastase, but it poorly inhibited the porcine pancreatic elastase . Normal alpha 1-antitrypsin was affected by all oxidants; both variants were almost totally resistant to chemical oxidants and to activated neutrophils . We conclude that recombinant alpha 1-antitrypsin variants differ in their elastase inhibitory activity and offer increased resistance to oxidant agents.

Nucleic Acids Symp Ser, 1990, (22), 119 - 20
Recognition of tRNA identity determinants by aminoacyl-tRNA synthetases; Muramatsu T et al.; By analyzing aminoacylation activities of variants of isoleucine tRNAs and glutamic acid tRNA from Escherichia coli, it was found that the anticodons are the major determinants for "identities" of these tRNAs . It was also shown that the post-transcriptional modifications are essential to aminoacylation of these tRNAs . Interactions between the tRNAs and the aminoacyl-tRNA synthetases were analyzed by NMR spectroscopy.

Indian J Public Health, 1990 Jan-Mar, 34(1), 66 - 7
A profile of diarrhoea in an urban slum area; Mandal AK et al.; PIP: Researchers followed 90 households (445 people) in Sunderpur slum in Varanasi in Upper Pradesh, India for 1 year and collected stool samples when people were ill with diarrhea to determine diarrhea incidence and causes of diarrheal disease . The water supply consisted of a well, public tap, or house tap with 30 households in each group . They noted 106 diarrheal episodes for an incidence of around 23% . Incidence decreased significantly with age (p.001) . For example, it was 62.9% for children 5 years old, 34% in the school age population, and 8.7% in people =or 15 years old . Improved resistance to infection and/or improved personal hygiene could have accounted for this difference . Diarrheal incidence was considerably lower in the autumn (9.3%) and winter months (11.1%) than the spring (49.1%) and summer months {rainy season} (30.5%) (p.001) . Researchers found at least 1 parasite in the stool sample of 81.5% of cases . The leading causative agents included Ascaris lumbricoides (42.1%), Entamoeba histolytica (35.2%), hookworm (7.9%), and Escherichia coli (5.7%) . Diarrhea incidence was much higher in persons whose water supply was a well (35.8%) compared to 23.2% for those with a public tap, and 12.8% for those with a private tap . These results concerning the water supply corroborated those of the Planning Research and Action Institute's (Upper Pradesh) pilot piped water supply program in the areas of Banki, Parendra, and Mokhampur in which incidence was highest in Banki where the water supply was an open well . The next highest and the lowest incidences were among those whose water supply consisted of public taps and private taps respectively .

Arch Exp Veterinarmed, 1990, 44(5), 765 - 79
{Experimental udder infections with Escherichia coli . 4 . Discussion of the pathogenesis of acute mastitis}; Sterba P et al.; The correlations among findings obtained from experimental Escherichia coli udder infection and reported in the three previous communications are discussed in this paper . The defence capabilities elucidated included specific and unspecific activities of non-epithelial cells, primarily neutrophilic granulocytes, macrophages, lymphocytes, plasma cells, and mast cells, as well as the capability of epithelial cells proper of absorbing pathogenic material from lumens of lactiferous ducts and milk cisterns and of storing such material within intracytoplasmic vacuoles . Hence, alterations in the course of pathogenesis of acute coli mastitis were found to be of complex nature and could be properly followed up by their morphological patterns.

Arch Exp Veterinarmed, 1990, 44(5), 757 - 64
{Experimental udder infection with Escherichia coli . 3 . Morphological changes directly to subepithelially situated structures in the area of the glands of the milk cistern and the large milk ducts}; Sterba P et al.; Alterations were recorded from capillaries and connective tissue by means of optical light and electron microscopy in investigations of subepithelial structures, following experimental Escherichia coli infection . These alterations led to an impairment of the blood-udder barrier and thus to increased extravasation of cells (especially neutrophilic granulocytes) . They were found to accumulate in subepithelial connective tissue and, eventually, to penetrate in high quantity the epithelium proper, and, consequently, to be considerably involved in alterations to the latter.

Arch Exp Veterinarmed, 1990, 44(5), 727 - 39
{Experimental udder infection with Escherichia coli . 1 . The behavior of selected clinical, hematological and other parameters concerned with milk composition}; Sterba P et al.; Four cows with clinically intact udders in high lactation were intracisternally infected by a pathogenic field strain of Escherichia coli . Infections were induced by quarters and with time intervals . Hence, when the animals were slaughtered six hours from beginning of the experiment, epithelial samples could be collected from cisternal walls and lactiferous ducts which, by the time of sampling, had been in contact with pathogens for one, four, and six hours . Highly acute mastitis began to develop two hours from experimental infection and exhibited typical clinical symptoms, including general disorders, unambiguous alterations to the white blood count, and changes in milk composition . These changes were typical of acute toxin action on the udder quarters . Concomitant reactions of non-infected control quarters, detectable by clinical manifestations or by laboratory diagnosis, were not observed.

Proteins, 1990, 8(4), 377 - 85
Refinement of the NMR structures for acyl carrier protein with scalar coupling data; Kim Y et al.; Structure determination of small proteins using NMR data is most commonly pursued by combining NOE derived distance constraints with inherent constraints based on chemical bonding . Ideally, one would make use of a variety of experimental observations, not just distance constraints . Here, coupling constant constraints have been added to molecular mechanics and molecular dynamics protocols for structure determination in the form of a psuedoenergy function that is minimized in a search for an optimum molecular conformation . Application is made to refinement of a structure for a 77 amino acid protein involved in fatty acid synthesis, Escherichia coli acyl carrier protein (ACP) . 54 3JHN alpha coupling constants, 12 coupling constants for stereospecifically assigned side chain protons, and 450 NOE distance constraints were used to calculate the 3-D structure of ACP . A three-step protocol for a molecular dynamics calculation is described, in analogy to the protocol previously used in molecular mechanics calculations . The structures calculated with the molecular mechanics approach and the molecular dynamics approach using a rigid model for the protein show similar molecular energies and similar agreement with experimental distance and coupling constant constraints . The molecular dynamics approach shows some advantage in overcoming local minimum problems, but only when a two-state averaging model for the protein was used, did molecular energies drop significantly.

Proteins, 1990, 8(4), 305 - 8
Preliminary crystallographic analysis of class 3 rat liver aldehyde dehydrogenase; Rose JP et al.; NAD-linked aldehyde dehydrogenases (A1DH) (EC 1.2.1.3) catalyze the irreversible oxidation of a wide variety of aldehydes to their respective carboxylic acids . Crystals of a class 3 AIDH (from an Escherichia coli expression system) suitable for X-ray analysis have been obtained . These crystals, which can be grown to a size of 0.8 x 0.3 x 0.2 mm, diffract to 2.5 A resolution . Analysis of the diffraction pattern indicates that the crystals belong to the monoclinic space group P21, with cell parameters a = 65.11 A, b = 170.67 A, c = 47.15 A, and beta = 110.5 degrees . Assuming one dimer per asymmetric unit, the value Vm is calculated to be 2.45 and the solvent content of the crystal is estimated to be 50% . A self-rotation function study produced significant rotation peaks (58% of the origin) on the kappa = 180 section at psi = 90 degrees and phi = 71 degrees and 341 degrees, indicating that the pseudo-dimer axis is (or is very nearly) perpendicular to the b-axis.

J Basic Microbiol, 1990, 30(10), 769 - 84
Cellular role of DNA polymerase I; Savic DJ et al.; Escherichia coli possesses three well-established DNA polymerases, I, II, and III . DNA polymerase I (Pol I) is the main repair polymerase in E . coli and also has a minor but important role in chromosomal replication . A major advantage of Pol I as an experimental system is its simplicity; unlike other replication enzymes, it is active as a single subunit . To a large extent, mutagenesis appears to be the result of (dis)functions of the DNA replication machinery . It is the purpose of this review to provide an integrated view of this relationship with particular emphasis on the role of Pol I in mutagenic events.

Immunol Ser, 1990, 49, 329 - 57
Large-scale production of hematopoietic growth factors; Thatcher DR; Genetic engineering has given us the means to produce large quantities of hematopoietic growth factors for clinical evaluation as therapeutic agents . The production technology for exploiting advances in genetic engineering is itself a new and rapidly evolving field of endeavor that has added another layer of choice for production . Commercial manufacturers of growth factors are now faced with an ever-expanding variety of alternatives in primary production systems, from E . coli to transgenic cows and a variety of choices in separation technologies for the isolation of these factors in a pharmaceutically acceptable form . Overlaying this plethora of options is the possibility of using site-specific mutagenesis to change the natural structure of growth factors to produce new chemical entities with novel pharmaceutical properties . For example, soluble forms of M-CSF have been created by specific deletion of hydrophobic transmembrane regions of the molecule and improved forms of G-CSF have been claimed to have been synthesized by directed amino acid substitution . The utility of these approaches will ultimately be decided by the relative efficacy of each product in the clinic and to a lesser extent process economics in the commercial marketplace.

Arch Oral Biol, 1990, 35 Suppl, 85S - 91S
Molecular cloning and expression of antigens from Actinobacillus actinomycetemcomitans in Escherichia coli; Sunday GJ et al.; In order to study the role of membrane proteins in mucosal colonization by Actinobacillus actinomycetemcomitans, a plasmid library was constructed by ligating EcoRI-digested genomic DNA from A . actinomycetemcomitans strain Y4 into EcoRI-digested and dephosphorylated pUC13 DNA . A second library was constructed by ligating Sau3A-digested and size-fractionated A . actinomycetemcomitans strain Y4 DNA into BamHI-cleaved and dephosphorylated pUC13 DNA . The DNA was transformed into Escherichia coli strain MC1022 and plated onto Luria-Bertani agar containing 100 micrograms/ml ampicillin and X-gal . Recombinant colonies were examined for the expression of A . actinomycetemcomitans antigens by colony immunoscreening using rabbit antiserum to strain Y4 . Nine positive clones were isolated . Three of these clones produced proteins that reacted with rabbit antiserum to strain Y4 on a Western transfer, and seven of these clones reacted with rabbit antiserum to strain Y4 as measured by indirect immunofluorescence . One reactive clone contained an 8.4 kb plasmid, which directed the synthesis of a peptide with an Mr of 20,000 as measured by SDS-PAGE . Partial DNA sequence analysis revealed the presence of a signal-peptide leader sequence at the amino terminus suggesting that the native protein is a membrane component of A . actinomycetemcomitans . Thus A . actinomycetemcomitans antigens can be expressed in E . coli, and some of these proteins may be expressed on the E . coli cell surface.

Acta Biochim Pol, 1990, 37(1), 21 - 30
Preliminary characterization of the repeated DNA sequence from Vicia sativa; Lehmann P et al.; We have identified a family of interspersed repeated sequences present in about 40,000 copies in the genomes of Vicia sativa and its near relative Vicia faba . The element vif is at least 6 kb in length, and members of the repeat family display a degree of heterogeneity in restriction pattern . Homologous elements in V . faba are much more heterogeneous than their V . sativa counterparts; however, analysis of five different V . faba lines revealed substantially the same patterns, suggesting that this family was reamplified prior to the divergence of the lines studied.

Adv Biophys, 1990, 26, 33 - 49
Regulation in repressor inactivation by RecA protein; Ogawa H et al.; Treatments that damage DNA or inhibit DNA synthesis in E . coli induce the expression of a set of functions called SOS functions that are involved in DNA repair, mutagenesis, arrest of cell division and prophage induction . Induction of SOS functions is triggered by inactivation of the LexA repressor or a phage repressor . Inactivation of these repressors results from their cleavage by the E . coli RecA protein in the presence of single-stranded DNA and a nucleoside triphosphate . We found that these cleavage reactions are controlled by two mechanisms in vitro: one is through the structural change of the RecA protein in the ternary complex, RecA-ssDNA-ATP-gamma-S . The active ternary complex is formed by binding of ATP-gamma-S to a complex of RecA protein and ssDNA . On the other hand, when the RecA protein binds to ATP-gamma-S prior to its binding to ssDNA, the resulting complex has no or only very weak cleavage activity toward the repressor . This structural change is negatively controlled by its C-terminal part . The loss of the 25 amino acid residues from the C-terminal leads the RecA protein to stable binding to dsDNA as well as ssDNA, and the protein takes the activated form for the repressor cleavage constitutively . The other mechanism is through the structural change of the repressor . The cleavage reaction of a phi 80cI repressor is greatly stimulated by the presence of d(G-G), and d(G-G) stimulates the cleavage by binding to the C-terminal half of the phi 80cI repressor . Moreover, the C-terminal fragment of the cleaved products of the 80cI repressor was able to cleave a phi 80cI-lambda chimeric repressor . These results strongly suggested that the active site of the repressor cleavage was located in the C-terminal domain of the repressor and that the C-terminal fragment produced by the cleavage could cleave the repressor.

Acta Neurochir (Wien), 1990, 107(3-4), 140 - 6
Otogenic intracranial abscesses; Kulai A et al.; The commonest cause of the intracranial abscesses collected prospectively during the last two years was chronic middle ear infection (73%) . The diagnosis was based on the clinical history, otological investigations, contrast enhanced computerized tomography and surgical findings . The clinical presentation was characterized by chronic otitis with an exacerbation of otorrhea, otalgia or pain in the temporal region or headache with high fever, vomiting and nausea . A review of our 14 patients with otogenic intracranial abscesses is reported to highlight that prompt diagnosis, appropriate therapy and careful monitoring can provide vastly improved results.

Microbiol Immunol, 1990, 34(10), 879 - 83
Cloning of genes for a hemolytic factor of Leptospira interrogans serovar autumnalis strain Congo 21-543; Fukunaga M et al.; A DNA fragment encoding a hemolytic factor was cloned from the parasitic spirochete Leptospira interrogans serovar autumnalis strain Congo 21-543 . Initial clones were isolated by screening a genomic library in pBR322 in Escherichia coli for hemolytic activity . Hemolytic activity was coded by a 4.5 kilobase BamHI-HindIII fragment . Southern hybridization with DNAs from other strains of Leptospira using this gene as a probe showed that DNAs from non-parasitic strains failed to hybridize with the probe, whereas those from all parasitic strains tested had the sequence which hybridize to the probe.

Chin J Biotechnol, 1990, 6(1), 27 - 34
In vivo cloning of proline genes and its expression in Escherichia coli; Wang AQ et al.; The wild type proA+, B+ genes of E . coli were cloned in vivo using a plasmid containing a mini-Mu replicon, pEG5005 . The cloning frequency was about 1.46 x 10(-3)/Kanr transductant . Genetic and biochemical analysis of these clones indicated that the proA+, B+ genes are on the plasmid pEG5005 . The secretion of proline were assayed for 500 Pro+ clones . However, no proline accumulation was detected . A Pro+ clone pPR3 was mutagenized in vivo by NTG and the mutants resistant to D-proline were obtained . One of the Dpr mutants pPR7 was found to produce 0.35 mg/ml proline in a proA B deletion strain . When pPR7 was transferred into a proline producing strain, the yield of proline increased up to 2.5 mg/ml, which is 7 and 2.5 times higher than that of the donor and recipient respectively . The physical maps of pEG5005 and pPR3 were roughly established.

Comp Biochem Physiol B, 1990, 96(3), 491 - 5
Chicken growth hormone: cDNA-synthesis and base sequence; Baum D et al.; 1 . Growth hormone (GH)-cDNA was synthesized from poly A(+)-mRNA extracts of chicken pituitary glands . 2 . Chicken-cDNA library was cloned into E . coli . 3 . Base sequence analysis of chicken GH-cDNA revealed only 70% similarity compared with duck GH-cDNA, and 97% similarity with a previously published chicken GH-cDNA sequence . 4 . Dissimilarities in base sequences are primarily observed in the 3'-non-coding region of GH-cDNAs (chicken and duck) . 5 . Comparisons of amino acid sequences of chicken and duck GH exhibit only three substitutions, while the amino acid sequences of GHs of chicken are identical.

Mol Gen Genet, 1990 Jan, 220(2), 334 - 8
The fimD gene required for cell surface localization of Escherichia coli type 1 fimbriae; Klemm P et al.; The fimD gene of Escherichia coli K12 was shown to be necessary for surface localization of type 1 fimbriae, since deletion of the gene resulted in a virtually bald phenotype . The FimD protein was found to be located in the outer membrane . Expressed alone, this protein had a very deleterious effect on cell growth . The DNA sequence of the fimD gene was determined; the corresponding amino acid sequence of the FimD protein was compared with those of the PapC and FaeD proteins . A deletion derivative of FimD gave clues as to which parts of the protein were necessary for outer membrane integration.

FEMS Microbiol Lett, 1990 Jan 1, 54(1-3), 55 - 60
A new fimbrial type (PCFO9) on enterotoxigenic Escherichia coli 09:H- LT+ isolated from a case of infant diarrhea in central Australia; Heuzenroeder MW et al.; The genes determining the biosynthesis of a new putative colonization factor, designated PCF09 have been cloned from an LT+ enterotoxigenic Escherichia coli 09:H- isolated during an outbreak of infant diarrhea in Central Australia . Electron microscopy has shown it to be of the fibrillar type . Purification of the major pilin subunit showed it to have a size of approximately 27 kDa . NH2-terminal analysis of the major subunit has shown the PCFO9 determinant to be distinct from other fimbriae although there is some conservation of certain residues . A synthetic oligodeoxynucleotide probe based on the NH2-terminal amino acid sequence of the purified protein has been used in Southern hybridization analyses to define the region on pPM1320 encoding the structural gene for the major pilin subunit.

FEMS Microbiol Lett, 1990 Jan 1, 54(1-3), 107 - 12
The binding of colonization factor antigens of enterotoxigenic Escherichia coli to intestinal cell membrane proteins; Wenneras C et al.; We examined the binding of colonization factor antigens (CFAs) of enterotoxigenic Escherichia coli to electrophoretically separated membrane components of rabbit intestinal brush borders or human intestinal (and non-intestinal) cell lines using an immunoblotting technique . Both CFA/I and CFA/II bound to distinct membrane components which seemed to be identical in rabbit brush borders and in a human intestinal cell line; these binding structures were mainly missing in membranes from epithelial cell lines of non-intestinal origin . Both shared and specific binding components were identified for CFA/I and the different subcomponents of CFA/II (CS1, CS2 and CS3), respectively . Chloroform-methanol extraction of lipids from the cell membranes did not change the binding pattern for either CFA/I or CFA/II suggesting that the binding occurred to (glyco)proteins rather than to (glyco)lipids.

Mol Microbiol, 1990 Jan, 4(1), 39 - 47
Engineering upstream transcriptional and translational signals of Bordetella pertussis serotype 2 fimbrial subunit protein for efficient expression in Escherichia coli: in vitro autoassembly of the expressed product into filamentous structures; Walker MJ et al.; Escherichia coli containing a cloned gene encoding the Bordetella pertussis serotype 2 fimbrial subunit failed to produce detectable levels of the gene product in whole-cell extracts . To engineer plasmids capable of directing the expression in E . coli of high levels of this product, both as a pre-protein and as a methionylated mature form the upstream signals of the fimbrial subunit gene were replaced by the lambda P(L) and P(R) promoters and the E . coli atpE translational initiation region . These constructs did not result in the expression of fimbrial subunit at detectable levels in several E . coli strains including DH5 . However, they did in E . coli CAG629, which is lon protease and heat shock protein deficient . Both pre-protein and methionylated mature protein had molecular weights of 25.0 kD, which indicated that correct processing of the leader sequence had occurred and thus that it was transposed across the inner membrane . Electron microscopic investigation of the cell surface of E . coli cells expressing either form of the fimbrial gene failed to detect the presence of filamentous structures . The methionylated mature form of the recombinant fimbrial subunit was purified to apparent homogeneity . After dialysis in appropriate conditions it was seen to autoassemble into protein polymers . Antibodies raised against polymerized recombinant subunit reacted weakly with whole B . pertussis serotype 2 fimbriae in immunodot blot assays . However, such antibodies reacted in Western blots equally well with the recombinant and wild-type form of the fimbrial subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Microbiol, 1990 Jan, 4(1), 101 - 6
Functional analysis of the fsoC gene product of the F7(1) (fso) fimbrial gene cluster; Riegman N et al.; Contrary to what would be expected from data in the literature, mutations in the fsoC gene of the F7(1) (fso) P-fimbrial gene cluster do not completely block fimbrial biogenesis . fsoC mutants still express small amounts of fimbriae of normal length, which carry the non-adhesive minor subunit protein, FsoE, but lack the adhesin, FsoG . The FsoC protein operates at the same stage in fimbrial biogenesis as the FsoF and FsoG proteins . The data suggest that FsoC, FsoF and FsoG interact to form an initiation complex for fimbrial biogenesis.

Scand J Rheumatol, 1990, 19(1), 11 - 6
Salicylazosulfapyridine (Salazopyrin) effect on endotoxin-induced production of interleukin-1-like factor from human monocytes in vitro; Remvig L et al.; Monocytes (M phi) were simultaneously preincubated with salicylazosulfapyridine (Salazopyrin) (SAZ), 0.78-12.5 mM, and lipopolysaccharide from E . coli, 1 X 10(-9) g/ml . Presence of SAZ resulted in a dose-dependent decrease in the co-stimulatory activity in M phi culture supernatants, corresponding to a 50% reduction by SAZ, 2.0 mM . Co-stimulatory activity was estimated by the mitogen-induced thymocyte proliferation assay (THY assay) . The results indicate an inhibitory effect of SAZ in vitro on the production of IL-1 and other possible co-stimulatory factors . This inhibitory effect was not due to decreased M phi viability, production of suppressive substances, or to drug interference with the THY assay . Equimolar preincubations with sulfapyridine, 5-aminosalicylic acid and N-acety-1-5-aminosalicylic acid were without effect on the production of co-stimulatory factors.

Proc Natl Acad Sci U S A, 1990 Jan, 87(2), 696 - 700
Searching sequence space by definably random mutagenesis: improving the catalytic potency of an enzyme; Hermes JD et al.; How easy is it to improve the catalytic power of an enzyme? To address this question, the gene encoding a sluggish mutant triose-phosphate isomerase (D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1) has been subjected to random mutagenesis over its whole length by using "spiked" oligonucleotide primers . Transformation of an isomerase-minus strain of Escherichia coli was followed by selection of those colonies harboring an enzyme of higher catalytic potency . Six amino acid changes in the Glu-165----Asp mutant of triosephosphate isomerase improve the specific catalytic activity of this enzyme (from 1.3-fold to 19-fold) . The suppressor sites are scattered across the sequence (at positions 10, 96, 97, 167, and 233), but each of them is very close to the active site . These experiments show both that there are relatively few single amino acid changes that increase the catalytic potency of this enzyme and that all of these improvements derive from alterations that are in, or very close to, the active site.

Infect Immun, 1990 Jan, 58(1), 279 - 81
The Dr hemagglutinin, afimbrial adhesins AFA-I and AFA-III, and F1845 fimbriae of uropathogenic and diarrhea-associated Escherichia coli belong to a family of hemagglutinins with Dr receptor recognition; Nowicki B et al.; The receptor specificities of four Escherichia coli cloned hemagglutinins, AFA-I, AFA-III, F1845 fimbriae, and the Dr hemagglutinin were studied . Evidence is provided that all four hemagglutinins recognize as their receptor the Dr blood group antigen . However, results of experiments using enzyme-treated erythrocytes and monoclonal antibodies indicate that the four adhesins recognize different epitopes on the Dr antigen and thus constitute a family of Dr receptor-recognizing bacterial adhesins . Furthermore, the same results suggest that the Dr antigen itself may be divided into subcomponents on the basis of bacterial adhesins.

Infect Immun, 1990 Jan, 58(1), 149 - 56
The 987P fimbrial gene cluster of enterotoxigenic Escherichia coli is plasmid encoded; Schifferli DM et al.; A clone containing the 987P fimbrial gene cluster was selected from a cosmid library of total DNA of the prototype Escherichia coli strain 987 by using 987P-specific antiserum . A subclone of 12 kilobases containing all of the genes required for fimbrial expression on a nonfimbriated K-12 strain of E . coli and a DNA fragment internal to the fimbrial subunit gene were used to probe the prototype strain and various isolates of 987P-fimbriated enterotoxigenic E . coli . All strains had several plasmids, as shown by agarose gel electrophoresis, and each of five strains which expressed 987P fimbriae showed a plasmid of 35 to 40 megadaltons (MDa) hybridizing to both 987P-specific probes . Hybridization to restricted DNA of strain 987 supported a plasmid origin for the cloned 987P gene cluster . Moreover, an isogenic strain which had lost its 35-MDa plasmid was no longer capable of synthesizing fimbrial subunits, but regained fimbrial expression after reintroduction of the TnphoA (Tn5 IS50L::phoA)-tagged 35-MDa plasmid . Absence of fimbrial subunit synthesis in K-12 strains transformed with the 35-MDa plasmid alone suggested the requirement of regulatory elements existing in strain 987 but missing in K-12 strains . A probe for the heat-stable enterotoxin STIa hybridized in each of the 987P-fimbriated strains to the plasmid containing the 987P genes and in most of these strains to an additional plasmid which contained the gene for the heat-stable enterotoxin STII . Occurrence of the 987P and STIa genes on the same replicon correlates with epidemiological observations, STIa being the most prevalent toxin produced by 987P-fimbriated E . coli.

Acta Biochim Biophys Hung, 1990, 25(1-2), 101 - 9
Rec mutants of Escherichia coli deficient in subunits of rec BC (D) complex; Tenke E et al.; The inactivation of rec BC (D) DNase upon chromatography on DEAE-cellulose was observed . Simultaneously DNA-stimulated ATPases (I and II) and DNase activities on single- and double-stranded DNA substrates were measured in Escherichia coli rec+ and rec- cell extracts . Normal levels of ATPase I and II were detected in rec+ cells . Rec A- cells were lacking DNA dependent ATPase I, while rec B single and rec BC double mutants were defective in DNA dependent ATPase II, the second major enzyme of this type . Rec B and C mutations did not change DNase activities . Rec A mutation significantly increased DNase activity on linear single-stranded substrate.

Arch Exp Veterinarmed, 1990, 44(6), 925 - 30
New ELISA test for detection of bovine leukemia virus infections in cattle, using bacterially synthesized p24; Siakkou H et al.; A new ELISA test is described for the detection of antibodies to bovine leukemia virus protein p24 . This test employs a bacterially synthesized p24 antigen which represents a hybrid protein consisting of beta-galactosidase and about 70% of the mature viral p24 . The antigen preparation was enriched from Escherichia coli cells to 95% purity and was used for the detection of antibodies in cattle . In a selected set of 100 positive field sera, 97 could be verified by the new test.

Arch Exp Veterinarmed, 1990, 44(6), 909 - 16
Synthesis of bovine leukemia virus antigens in Escherichia coli; Ulrich R et al.; Plasmids were constructed by the use of pEX vectors that encode and express different parts of the bovine leukemia virus (BLV): main core protein p24, nucleic acid-binding protein p12, transmembrane protein gp30, and different segments of envelope protein gp51 . Expression of fusion proteins with molecular weights higher than 117 kD for all recombinant plasmids was shown in Coomassie-blue stained gels and by Western blot analysis with rabbit anti-BLV sera . Coupling of a gp51-encoding with a p24-encoding DNA fragment in pEX vectors led to synthesis of a fusion protein that was recognized by monoclonal antibodies directed against gp51 and p24 epitopes . Using another vector, a gp51-encoding DNA fragment of BLV was expressed as a fusion protein with 100 amino acids of the MS2 polymerase . The fusion protein was recognized by monoclonal antibodies directed against gp51.

Pharmacol Ther, 1990, 48(2), 259 - 80
Ribonucleases H of retroviral and cellular origin; Wintersberger U; Ribonucleases H (RNases H) are enzymes which catalyse the hydrolysis of the RNA-strand of an RNA-DNA hybrid . Retroviral reverse transcriptases possess RNase H activity in addition to their RNA- as well as DNA-dependent DNA-polymerizing activity . These enzymes transcribe the viral single stranded RNA-genome into double stranded DNA, which then can be handled by the host cell like one of its own genes . Various, sometimes highly repeated, sequences related to retroviruses and like these encompassing two separate domains, one of which potentially codes for a DNA polymerizing, the other for an RNase H activity, are found in genomes of uninfected cells . In addition proteins coded for by cellular genes (e.g . from E . coli and from yeast) are known, which exhibit RNase H activity, the biological function of which is not fully understood . In the light of these facts the question of whether retroviral RNases H could be promising targets for antiviral drugs is discussed.

Adv Exp Med Biol, 1990, 278, 231 - 42
Prevalence of specific antibodies to herpes simplex virus type 2 as revealed by an enzyme-linked immunoassay and western blot analysis; Spiezia KV et al.; A solid-phase ELISA for the detection of antibodies to gG-2 was developed . The assay utilizes a recombinant DNA-derived gG-2 as a solid-phase "capture" reagent and goat anti-human IgG (gamma) conjugate to horseradish peroxidase as a probe (detector) reagent . A total of 229 serum samples collected from various populations were tested by ELISA and western blot analysis . On comparison with confirmed HSV-2 infection, the sensitivity and specificity of the ELISA were 92.9% and 98.7%, respectively . Western blot had a sensitivity of 83.9% and a specificity similar to the ELISA . The ELISA is fast and easy to perform and may be used to diagnose previous exposure to genital herpes and to monitor human response to future HSV-2 vaccines.

Biomed Sci, 1990 Jan, 1(1), 68 - 72
Latex-agglutination analysis of human recombinant interleukin-2 with monoclonal antibodies; Lunev VE et al.; Hybridomas producing monoclonal antibodies (Mabs) to human interleukin-2 have been obtained . The antibodies have been characterised in terms of binding constant, subclass, and cross-reaction with proteins from Escherichia coli and human blood serum . The epitope for the monoclonal antibody LNKB-2, namely the 66-72 fragment of the interleukin-2 molecule, has been located . From the antibodies obtained, two capable of reacting simultaneously with the monomeric form of interleukin-2 have been selected and a rapid simple Mab-based latex-agglutination assay which can detect as little as 1.5 ng ml-1 interleukin-2 has been developed.

Chin J Biotechnol, 1990, 6(3), 179 - 87
Synthesis of a recombinant DNA-derived HBV e antigen and its application in diagnosis; Wu GH et al.; The gene coding for HBV e antigen (HBeAg) and core antigen (HBeAg) are located in the C region of the HBV genome . In this paper 1.8 kb BamHI-EcoRI DNA fragment carrying HBc gene from adw2 HBV DNA was digested with Bal31 and HapII, a series of fragment with different length were produced and inserted into pUR222 and pUC18 vectors to construct various recombinant plasmids . Two new recombinants able to direct high level synthesis of HBeAg in E . coli were obtained . Recombinant HBeAg was then purified by DEAE chromatography and affinity chromatography . The preparation obtained were identified by electrophoresis and immunochemical analysis . Molecular weight of the recombinant HBeAg determined by SDS-PAGE was about 38 kd . It is considered to be a dimer of two fused antigen molecules in size of 19 kd . Bacterial extracts prepared from cells harboring one of the constructed plasmids have been used successfully as a diagnostic reagent for the detection of HBeAg and anti-HBe in human sera by ELISA.

Autoimmunity, 1990, 6(4), 257 - 68
Mapping of autoantigenic epitopes on recombinant thyroid peroxidase fragments using the polymerase chain reaction; Banga JP et al.; Cloned cDNA templates of thyroid peroxidase (TPO) have been used in conjunction with the polymerase chain reaction (PCR) to express selected segments of the thyroid microsomal/peroxidase antigen (TMA/TPO) as recombinant protein in E . coli . Six small, different recombinant fragments averaging 120 amino acid residues and one large fragment (269 amino acids) of TPO which together encompass 80% of the extracellular region of the molecule have been produced and autoantibody (aAb) binding sites analysed by immunoblotting . A minimum of six independent, sequential antigenic determinants have been localized on the recombinant proteins and these map to the amino terminal, the central core region and the carboxyl terminal of the TPO molecule . More accurately, the six antigenic sites reside on overlapping recombinant TPO preparations termed R1a + R1b (residues 1 to 160) R1c (residues 145 to 250), R2b (residues 457 to 589), R3a (residues 577-677), R3b (residues 657-767) and R3c (residues 737-845) . The large fragment of TPO termed R3 (residues 577-845) encompassing R3a, R3b and R3c also reacts with the aAbs . Different sera from patients with autoimmune thyroid disease contain antibodies to TMA/TPO which differ in their fine specificity . The use of recombinant molecular biological techniques together with PCR to prepare small segments of a large autoantigen as recombinant protein will now allow studies to progress on autoepitope mapping of the precise amino acid sequences of the TPO molecule with the use of synthetic peptides.

Nucleic Acids Symp Ser, 1990, (22), 87 - 8
Reinvestigation of phosphorylation of tRNA in Escherichia coli; Mizutani T et al.; This report shows the results of the reinvestigation of tRNA phosphorylation in E . coli . The phosphorylation did not occur on suppressor seryl-tRNA but occurred on other tRNA species . The activity of tRNA phosphorylation was found in E . coli extracts and partially purified . On DEAE-Sephadex A50 and PAGE gel, the phosphorylated-tRNA showed a pattern different from that the natural suppressor serine tRNA.

Nucleic Acids Symp Ser, 1990, (22), 75 - 6
Stabilization of mRNA in the cell-free translation system from Escherichia coli; Hirao I et al.; In the RNA directed cell-free protein synthesizing system from E . coli, there is a problem of contaminating 3'-exonucleases which attack the mRNAs . Thus, we tried the following two methods to stabilize mRNAs against nucleases: (A) To use mRNAs having hairpin structures at their termini and (B) To hybridize mRNAs with small DNA fragments to the 3'-termini of mRNAs . It was found that degradation of a mRNA was inhibited by the method B rather than the method A in the translation system.

Nucleic Acids Symp Ser, 1990, (22), 39 - 40
Possible evolutionary origin of primitive protein-encoding mRNAs as a virusoid-like ribo-organism; Ohnishi K; E . coli rnpA and rpmH genes encoding the protein portion of ribonuclease (RNase) P and L34 ribosomal protein were found to be homologous to the entire sequence of M1 RNA and virusoids . The resulting alignment strongly suggests that most primitive mRNAs must have emerged from virusoid-like ribo-organism.

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 1990, 8(4), 249 - 52
{Construction of a cDNA library of Schistosoma japonicum}; Yan WY et al.; The template mRNA was extracted from Schistosoma japonicum . The first strand of cDNA was synthesized by AMV-reverse transcriptase . The second strand cDNA was first digested by RNase H to remove mRNA and was then synthesized by AMV-reverse transcriptase, T4-DNA polymerase . Sizing of cDNA was applied on a NACS column to remove small fragments of less than 1 kb . Homopolymeric tailing of vector (pUC18) was done with dGTP and DNA terminal transferase and tailing of the cDNA with dCTP was carried out under the same conditions . After annealing, the plasmids with cDNA were transformed into E . coli MC1061 . The efficiency of cloning was about 10(4)/micrograms mRNA with 30% of the transformants having the inserts of cDNA (Figs . 1-2).

Proteins, 1990, 8(4), 309 - 14
Hormone phage: an enrichment method for variant proteins with altered binding properties; Bass S et al.; Human growth hormone (hGH), a 191 residue protein containing two disulfide bonds, was fused to the carboxyl-terminal domain of the gene III protein, a minor coat protein exposed at one end of the filamentous phage M13 . The gene fusion was cloned into a plasmid containing origins of replication for Escherichia coli and filamentous phage and was packaged into phagemid particles upon infection by an M13KO7 helper phage . Transcription of the hGH-gene III fusion was controlled so that usually no more than one copy of the fusion protein was displayed along with the four copies of the wild-type gene III protein . The hGH-gene III fusion protein was properly folded, as judged by reactivity with six hGH monoclonal antibodies whose epitopes are sensitive to the folded conformation of hGH . Moreover, the hGH-gene III phagemid particles were enriched over 5000-fold from non-hGH phage, and 8-fold from a mutant hGH phagemid following a single hGH-specific elution step from hGH receptor-coated beads . The hGH phagemid should be useful for isolating new receptor binding mutants of hGH . More generally, this expression system may allow other large proteins with discontinuous binding epitopes to be displayed, and binding selections applied to their mutated gene III fusions on filamentous phage.

Arch Oral Biol, 1990, 35 Suppl, 69S - 78S
Molecular approaches to leucotoxin as a virulence component in Actinobacillus actinomycetemcomitans; Ebersole JL et al.; A strategy has been developed to examine the hypothesis that leucotoxin is a critical virulence factor of Actinobacillus actinomycetemcomitans in a non-human primate (Macaca fascicularis) . Firstly the leucotoxin gene from A . actinomycetemcomitans was cloned and sequenced . This DNA contained a functional leucotoxin gene, as protein extracts of Escherichia coli with the cloned sequences lysed appropriate human cell lines . The protein encoded by lktA shared at least 42% identity with P . haemolytica leucotoxin and with the alpha-haemolysins from E . coli and A . pleuropneumoniae . The lktA gene of A . actinomycetemcomitans was linked to another gene, lktC, which is thought to be related to the LktC proteins from these other bacteria and with which it shared at least 49% amino acid identity . Despite the overall homology to the other leucotoxins/haemolysins, the LktA from A . actinomycetemcomitans has several unique properties including a very basic pI of 9.7, as compared to pIs approx . 6.2 for lktA proteins in other bacteria . Using the cloned genes as probes produced evidence that a TOX- strain contains the leucotoxin gene but fails to transcribe it at high levels . The second avenue of investigation was to develop methods for examining the humoral immune responses in the monkey to bacterial toxins such as lktA . A . actinomycetemcomitans was detected in subgingival plaque samples from approx . 40% of the animals . A . actinomycetemcomitans comprised less than 1% to 9% of the flora . Most A . actinomycetemcomitans isolates were serotype b and each of the monkeys had serum IgG antibody to A . actinomycetemcomitans serotype b (generally considered to be lktA-producing strains) . An ELISA was developed to examine the isotype/subclass distribution, level and avidity of serum antibody in the monkey following parenteral immunization with a prototype bacterial exotoxin (tetanus toxoid) . IgG1 and IgG3 antibody predominated over IgG2 and IgG4 after primary immunization . Secondary immunization elicited enriched IgG1 and IgG4 responses . Primary immunization increased avidity indices of IgG to tetanus toxoid from approx . 0.9 (baseline) to a mean of 1.72 and secondary immunization significantly increased the avidity index to 2.56.

Microbios, 1990, 64(260-261), 135 - 51
Mechanisms of hyperbaric-oxygen inhibition of growth and net biosynthesis of RNA, DNA, protein and lipids in Escherichia coli; Brown OR; The effects of hyperbaric oxygen at 4.2 atmospheres on growth, induction of stringency and net synthesis of RNA, DNA, protein and lipid by Escherichia coli were evaluated by measuring culture absorbance and incorporation of specific 14C-labelled substrates . Combinations of different bacterial strains, various nutritional conditions and chloramphenicol were used to suppress or allow induction of stringency and to mitigate or enhance other consequences of reported sites of oxygen toxicity . In minimal medium with glucose as the sole organic nutrient, there was almost instantaneous inhibition of net biosynthesis of four major macromolecular constituents: lipid, RNA, DNA and protein . Net lipid biosynthesis primarily failed indirectly due to induction of stringency . Protein net synthesis declined due to induction of stringency and lack of amino acids . RNA net biosynthesis stopped from a direct effect and indirectly via induction of stringency . Net DNA biosynthesis was indirectly primarily impaired.

Eur J Nucl Med, 1990, 17(1-2), 28 - 33
Leucocyte sequestration in endotoxemia and the effect of low-molecular-weight dextran; Christenson JT et al.; Leucocyte sequestration in various organs during endotoxin-induced shock in sheep was studied using leucocytes labelled with indium 111 oxine . A moderate dose of Escherichia coli endotoxin (10 micrograms/kg body weight) was slowly infused intravenously in 16 sheep, 9 of which subsequently received a continuous i.v . infusion of low-molecular-weight dextran (LMWD) given at an infusion rate of 15 ml/h over 4 h, starting 30 min after administration of the endotoxin . By that time, signs of acute lung injury had developed, thus mimicking a clinical situation . The remaining animals were untreated and served as controls . A marked increase in lung, liver and kidney leucocyte sequestration, together with a sharp, corresponding drop in splenic activity and leucocyte count in peripheral blood, occurred shortly after the endotoxin infusion in both groups . However, after 90 min there was a significantly lower leucocyte activity in the lungs, liver and kidneys of LMWD-treated animals as compared with controls . Less marked hemodynamic and respiratory alterations were also observed in animals treated with LMWD . The present study confirms previous reports that significant leucocyte sequestration in the lungs occurs early during endotoxemia . Furthermore, we found that leucocyte sequestration also occurs in the liver and kidneys, which could explain the development of multi-organ failure, frequently described in clinical sepsis . Even after injury to organs, LMWD infusion seems to be beneficial by significantly lowering leucocyte sequestration and could therefore be justified as an addition to the arsenal of interventions used in the treatment of endotoxemia.

Acta Biochim Pol, 1990, 37(2), 299 - 307
Inhibition of RNA synthesis in vitro by 9-aminoacridine carboxamide antitumor agents . Effects on overall RNA synthesis and synthesis of the initiating dinucleotide; Piestrzeniewicz MK et al.; A series of 9-aminoacridine carboxamide derivatives of systematically varied structure was assayed in an RNA synthesis in vitro system . Escherichia coli DNA-dependent RNA polymerase and DNA derived from phage T7 or calf thymus were used to measure the effect of the drugs on overall RNA and the initiating dinucleotide (pppApU) syntheses . By means of multiple linear regression analysis it was shown that the inhibition of these reactions depends both on the drug equilibrium binding constant and kinetic parameters of dissociation of drug-DNA complexes.

J Ocul Pharmacol, 1990 Fall, 6(3), 219 - 26
Immunosuppression by gramicidin S of experimental autoimmune uveoretinitis, pinealitis and autoimmune encephalomyelitis; Matsushima S et al.; Using an in vitro lymphocyte proliferation assay we screened several cyclic peptide antibiotics (bacitracin, oleandomycin, capreomycin, colistin, virginiamycin, and gramicidin S) for their immunosuppressive activity . Gramicidin S (GrS) was found to inhibit {3H}-thymidine incorporation into concanavalin A-stimulated and E coli lipopolysaccharide-stimulated lymphocytes . In vivo studies, experimental autoimmune uveoretinitis (EAU) and pinealitis were induced in female Lewis rats by immunization with bovine S-antigen and experimental autoimmune encephalomyelitis (EAE) were induced by immunization of rats with rat brain homogenates . GrS suppressed the onset of these inflammatory diseases at nontoxic concentrations . Evidence was obtained that GrS inhibits {3H}-thymidine incorporation into lymphocytes by preventing transport of the compound across the membrane . Since GrS binds to various cell membranes, GrS would suppress the proliferation of not only lymphocytes but also of other immune cells by modifying cell membrane properties . The present study indicates that a search for compounds which cause proper cell membrane modification should be a worthwhile strategy for development of immunosuppressive drugs.

Microbiol Immunol, 1990, 34(7), 607 - 15
Two kinds of P-fimbrial variants of uropathogenic Escherichia coli recognizing forssman glycosphingolipid; Orino K et al.; Various types of fimbriae on pathogenic Escherichia coli strains have been classified by their antigenicities and recognition specificities for receptors . However, the antigenicity of fimbrial proteins does not always correlate with the fimbrial recognition specificity . In this communication, the exact carbohydrate structures recognized by the fimbriae of two human uropathogenic E . coli strains, KS71 (O4) and IH11024 (O6), that have P-fimbrial antigen, were examined . Strain KS71 showed mannose-resistant (MR) hemagglutination (HA) of human blood group OP1 phenotype erythrocytes, and its HA was inhibited by blood group Pk antigen, Gal(alpha,1-4)Gal(beta,1-4)Glc-ceramide and P antigen, GalNAc(beta,1-3)Gal (alpha,1-4)Gal(beta,1-4)Glc-ceramide but not by Forssman antigen, GalNAc(alpha,1-3)GalNAc(beta,1-3)Gal(alpha,1-4)Gal (beta,1-4)Glc-ceramide, as previously described in many papers . The cells also showed MR HA of sheep erythrocytes, which was potently inhibited by Forssman, and weakly by P and Pk antigens . These phenomena could not be explained by the above P adhesin specificity . This adhesin was called Forssman-like adhesin . Strain IH11024 also caused MR HA of sheep erythrocytes but not of human erythrocytes . The HA was inhibited specifically by Forssman but neither by Pk nor P antigen . This adhesin was completely different from P adhesin and Forssman-like adhesin in recognition of the carbohydrate epitope . This adhesin, until now called a pseudotype of P fimbriae, was renamed Forssman adhesin.

Haemostasis, 1990, 20(4), 229 - 32
Human monocytes release plasma serine protease inhibitors in vitro; Kloczko J et al.; The ability of human peripheral blood monocytes to secrete plasma serine protease inhibitors was studied . Monocytes from blood obtained from healthy young adult volunteers were cultured for up to 36 h with and without lipopolysaccharide from Escherichia coli . The concentrations of plasma serine protease inhibitors in monocyte culture supernatants were measured by using rocket immunoelectrophoresis . The study showed that human monocytes stimulated with lipopolysaccharide in vitro release antithrombin III, C1 esterase inhibitor, alpha 2-antiplasmin, and alpha 2-macroglobulin.

Microbiol Immunol, 1990, 34(6), 509 - 21
Overproduction of human immunodeficiency virus type I reverse transcriptase in Escherichia coli and purification of the enzyme; Saitoh A et al.; Overexpression of the reverse transcriptase was designed in E . coli . For a high level of expression, HIV protein was expressed as a protein fusion with beta-galactosidase . When the proviral DNA fragment covering the 3' half of the gag gene and the entire pol gene was ligated to the 3' end of the lacZ gene to fuse the truncated gag to lacZ in frame, a small quantity of reverse transcriptase was produced, indicating that frameshifting and post-translational processing have occurred . Much more reverse transcriptase was produced when the entire pol region was directly fused to the lacZ gene . From a one liter culture of bacteria, 1 mg of highly purified reverse transcriptase consisting of approximately equimolar amounts of two species (p64 and p51) was obtained . These proteins had identical N-termini consistent with the deduced amino acid sequence and therefore, might be correctly processed from the fusion protein in E . coli by the protease encoded by the pol region . The purified reverse transcriptase was enzymatically as active as the enzyme purified from the virus particles, and immunoreactive to the sera of HIV carriers with high sensitivity and specificity.

Biomed Biochim Acta, 1990, 49(4), 161 - 6
Synthesis of oligonucleotide probes containing 2'-deoxy-2'-fluoronucleosides for cleavage of RNA by RNase H; Krug A et al.; Modified oligodeoxyribonucleotides containing 3'-terminal 2'-deoxy-2'-fluorouridine (UF) or 2'-fluorothymidine (TF) were successfully applied for specific RNA hydrolysis by RNase H from E . coli . The nonanucleotides d(CACCGCGCTF) and d(CACCGCGCUF) were synthesized using the phosphoramidite solid support method . The modified nucleosides were immobilized on the CPG support and provided the starting nucleoside residues . Model experiments were carried out using the 5S RNA from E . coli ribosomes and its 1-41 fragment . It was found that the use of this type of modified probes did not decrease neither the efficiency nor the specificity of the RNase H reaction.

Folia Haematol Int Mag Klin Morphol Blutforsch, 1990, 117(2), 331 - 8
Investigation of prostaglandins in the culture supernatant of mononuclear cells and its influence on platelet aggregation; Giedrojc J et al.; The prostaglandins can be synthesized by many cells types . Cells of the immune system also metabolise arachidonic acid to prostaglandins . However, the specific class of immunocompetent cells that synthesize prostaglandins as well as the spectrum of arachidonic acid metabolides produced by these cells is not firmly established . The aim of our study was to investigate the behaviour of prostaglandins in the culture supernatant of mononuclear cells and the influence of this supernatant on platelet aggregation . Blood cells were separated from peripheral blood according to a modification of the procedure of Boyum . The level of prostaglandins was determined by means of radioimmunoassay kits . The PGF2 alpha concentrations were significantly higher in the culture supernatant (F) in comparison with supernatant (K) and (O) . The supernatant of lymphocytes culture does not influence platelet aggregation . The highest concentrations of PGE1 and PGF2 alpha were noted at the 12 and 24 hours of the monocyte cultivation, however the lowest at the 36 and 48 hours . The supernatant obtained from monocyte cultivation at the 36 hours exert an independent effect on platelets aggregation, whereas at 24 and 48 hours it plays a role in platelet aggregation . The presented results may indicate the influence of monocytes on AA metabolism and platelet function.

Methods Enzymol, 1990, 184, 328 - 40
Z-DNA affinity chromatography; Fishel R et al.; In this chapter we have detailed a method that can be generalized to link virtually any DNA substrate to a chromatography matrix at its ends via an avidin-biotin linkage . We have used this technique to construct a left-handed Z-DNA column for the purpose of identification and purification of Z-DNA-binding proteins . This technique for the linkage of DNA to a column matrix by avidin-biotin technology can be modified, however, to produce linked multimeric sequences specific for regulatory or other DNA-binding proteins.

Arch Virol, 1990, 113(1-2), 1 - 16
Cloning and characterization of major antigenic determinants of human cytomegalovirus Ad169 seen by the human immune system; Lindenmaier W et al.; Starting from a cosmid library of HCMV Ad169-DNA random fragments of DNA were generated . Fragments about 200 to 600 bp in length were selected and cloned into open reading frame (ORF) expression vectors to create ORF-libraries that represent either the entire viral genome or defined subregions . About 120,000 clones were isolated and screened immunologically for the synthesis of fusion proteins consisting of an antigenic peptide encoded by the CMV sequence coupled to a truncated E . coli beta-galactosidase molecule . Anti-CMV sera raised in animals as well as human hyperimmune globulin were used for colony screening . Distinct sets of antigenic fusion proteins were recognized by different antisera . Ten of the clones giving strong reactions with human immune sera were mapped on the CMV genome and the sequences of the CMV inserts determined . Antibodies against fusion proteins were raised in mice or rabbits to identify the corresponding CMV proteins . Antigenic fusion proteins described here were recognized by most individual human CMV immune sera tested . They allow determination of the humoral immune response to defined determinants and may therefore be particularly useful in diagnosis and vaccine development.

J Acquir Immune Defic Syndr, 1990, 3(9), 859 - 72
Export and one-step purification from Escherichia coli of a MalE-CD4 hybrid protein that neutralizes HIV in vitro; Szmelcman S et al.; The 177 N-terminal amino acids of CD4, the receptor of the human immunodeficiency virus (HIV), have been expressed in Escherichia coli as genetic fusions to the periplasmic maltose-binding protein (MalE) from this organism . A large fraction of the hybrid proteins can be released from the periplasm by osmotic shock and purified in one step on a cross-linked amylose column eluted with maltose under mild conditions . One hybrid protein binds HIV envelope protein gp160 and neutralizes the virus in vitro . This provides the first example of the production and one-step purification of an active form of an eukaryotic protein by fusion to MalE . The use of this system for mass screening of CD4 mutants, high-scale production of the hybrid protein for structural studies on CD4, testing antiviral compounds, and therapeutic assays is discussed.

Intervirology, 1990, 31(2-4), 116 - 21
Localization of antigenic sites in the primary sequence of the Sendai virus NP protein; Fisher LE; Monoclonal antibodies were used to localize five antigenic sites in the sequence of the Sendai virus NP gene . NP-specific peptides, expressed in Escherichia coli from the cloned gene served as antigens for blot analyses to determine the specificity of antibody binding . The antigenic sites were associated primarily with the C-terminal half of the protein.

Nauchnye Doki Vyss Shkoly Biol Nauki, 1990, (2), 22 - 30
{The 5S rRNA-protein complex of Escherichia coli studied by carbodiimide modification}; Dontsova OA et al.; 5S rRNA-protein complex has been reconstituted from 5S rRNA and total protein of large (L) ribosomal subunit of Escherichia coli . The complex consists of 5S rRNA and 3 proteins only: L5, L18, L25 . A water-soluble carbodiimide {N-cyclohexyl-N'-(2-morpholinoethyl)-carbodiimide-methyl-p-toluolsulp honate} cross-links L18 to 5S rRNA at pH 7.2 and L25 to 5S rRNA at pH 7.7 . This pH-dependence of cross-linked proteins is a consequence of the difference in stability of the initial complex: the complex has all three proteins at pH 7.7 but L18 mainly at pH 7.2 . It has been shown that L18 stimulates the chemical modification of U87 and U89 residues of 5S rRNA by carbodiimide . A model of L18-5S rRNA complex has been proposed.

Mikrobiol Zh, 1990 Jan-Feb, 52(1), 91 - 5
{A simple method for counting Rickettsia cells}; Emel'ianov VV; A simple modification of the method for counting Rickettsiae is described . The Escherichia coli cells (ECC) which served as reference particles were stained in suspension with methylene blue mixed with Rickettsia prowazekii (RP) and quickly sprayed over the glass slide . After fixation the samples were stained according to the technique of Gimenez and examined in the light microscope under oil immersion . Through a grid in the eye-piece it was not so difficult to count red-coloured RP and dark-blue ECC against a background formed by impurities . To calculate RP concentration, the reference particles' concentration was multiplied by the dilution factor of RP suspension by the ratio of RP to ECC enumerated . The statistical approach has shown that the wash of the slides during staining procedure does not change this ratio . Differential staining of Rickettsiae with fuchsin is the main clue of this new method to count them even in the crude preparations of infected yolk sacs.

Biochem Cell Biol, 1990 Jan, 68(1), 169 - 79
Study of the function of Escherichia coli ribosomal RNA through site-directed mutagenesis; Leclerc D et al.; Various approaches have been developed to study how mutations in Escherichia coli ribosomal RNA affect the function of the ribosome . Most of them are in vivo approaches, where mutations are introduced in a specialized plasmid harboring the ribosomal RNA genes . The mutated plasmids are then expressed in an appropriate host, where they can confer resistance to antibiotics whose target is the ribosome . Conditions can be used where the host ribosomal RNA genes or the host ribosomes are selectively inactivated, and the effect of the mutations on ribosome assembly and function can be studied . Another approach, which has been developed mainly with 16S ribosomal RNA, can be used entirely in vitro . In this approach, a plasmid has been constructed which contains the 16S ribosomal RNA gene under control of a T7 promoter . Mutations can be introduced in the 16S ribosomal RNA sequence and the mutated 16S ribosomal RNAs are produced by in vitro transcription . It is then possible to investigate how the mutations affect the assembly of the 16S ribosomal RNA into 30S subunits and the activity of the reconstituted 30S subunits in cell-free protein synthesis assays . Although these approaches are recent, they have already provided a large body of interesting information, relating specific RNA sequences to interactions with ribosomal proteins, to ribosome function, and to its response to antibiotics.

Int J Biochem, 1990, 22(3), 247 - 51
The effects of anthranilic acid on gene expression; Stevens SM et al.; 1 . A Drosophila pseudoobscura amylase gene cloned in Escherichia coli is expressed at high levels . The expression of this gene is repressed when glucose (0.5% final concentration) is added to a starch minimal medium culture of E . coli cells containing the amylase plasmid . 2 . Addition of anthranilic acid (5 and 7 mM final exogenous concentration) to catabolite repressed cells mimics the action of adenosine 3'5' cyclic monophosphate (cAMP) by depressing the expression of the amylase . 3 . The results suggest that anthranilic acid acts either indirectly, possibly through the glucose transport system, or directly, by way of an intercalative model of initiation, to alter the levels of transcription.

Proteins, 1990, 7(1), 16 - 31
A genetic screen to identify variants of bovine pancreatic trypsin inhibitor with altered folding energetics; Coplen LJ et al.; A genetic screening procedure has been developed to identify mutant forms of bovine pancreatic trypsin inhibitor (BPTI) that can fold to an active conformation but are inactivated more rapidly than the wild-type protein . Small cultures of Escherichia coli containing plasmids with mutagenized BPTI genes were grown in microtiter plates, lysed, and treated with dithiothreitol (DTT) . Under these conditions, unfolding and inactivation of the wild-type protein has a half-time of about 10 hours . Variants of BPTI that are inactivated within 1 hour were identified by adding trypsin and a chromogenic substrate . Approximately 11,000 mutagenized clones were screened in this way and 75 clones that produce proteins that can fold but are inactivated by DTT were isolated . The genes coding for 68 "DTT-sensitive" mutant proteins were sequenced, and 25 different single amino acid substitutions at 15 of the 58 residues of the protein were identified . Most of the altered residues are largely buried in the core of the native wild-type structure and are highly conserved among proteins homologous to BPTI . These results indicate that a large fraction of the sequence of the protein contributes to the kinetic stability of the active conformation, but it also appears that substitutions can be tolerated at most sites without completely preventing folding . Because this genetic screen is based on changes in folding energetics, further studies of the isolated mutants are expected to provide information about the roles of the altered residues in folding and unfolding.

Protein Eng, 1990 Jan, 3(3), 221 - 6
A method for construction of long randomized open reading frames and polypeptides; Mandecki W; A method is presented for construction of randomized open reading frame sequences (ORFs) and gene libraries containing them . The building blocks for the ORFs were 75 bp long DNA fragments generated by cloning sequences from a single synthetic oligonucleotide preparation by bridge mutagenesis . The fragments had the property that, regardless of their orientation in the ligated product, the ORF of the construct was maintained . The heterogeneity of the ORFs resulted from the random ligation of 2000 different DNA fragments . The randomized ORFs were cloned downstream from the lac promoter in a multicopy plasmid in Escherichia coli . To test the method, a library of 10(6) clones was constructed.

Microbios, 1990, 61(248-249), 135 - 43
Small, heat-stable, DNA-binding proteins from Caulobacter crescentus; Paterczyk B et al.; From a heterogenous cell population of Caulobacter crescentus a deoxyribonucleoprotein fraction (DNP) was obtained by the modified technique of Sjastad et al . (1982) . Under the electron microscope DNP had a smooth fibrillar structure . The chemical properties of the proteins associated with the DNA were similar to those of other bacteria . An abundant, heat-stable, basic and DNA-binding protein, termed HCc, which has a molecular weight of 13.4 kD may be an analogue of the HU-histone-like protein from Escherichia coli.

Mol Gen Genet, 1990 Jan, 220(2), 341 - 4
A possible role for the pcnB gene product of Escherichia coli in modulating RNA: RNA interactions; Masters M et al.; The sequence of the PcnB protein of Escherichia coli, a protein required for copy number maintenance of ColE1-related plasmids, was compared with the PIR sequence database . Strong local similarities to the sequence of the E . coli protein tRNA nucleotidyltransferase were found . Since a substrate of the latter protein, tRNA, structurally resembles the RNAs that control ColE1 copy number we believe that we may have identified a region in PcnB that interacts with these RNAs . Consistent with this idea is our observation that PcnB is required for the replication of R1, a plasmid whose replication is also regulated by a small RNA.

Mol Gen Genet, 1990 Jan, 220(2), 301 - 6
Escherichia coli glyA mRNA decay: the role of 3' secondary structure and the effects of the pnp and rnb mutations; Plamann MD et al.; The Escherichia coli glyA structural gene is followed by two REP sequences and a rho-independent transcription terminator . These sequences are essential for maintaining glyA mRNA stability and gene expression by blocking the 3' to 5' exonucleolytic activities of polynucleotide phosphorylase and ribonuclease II . The results support the model of cooperative endonucleolytic and 3' to 5' exonucleolytic activities in mRNA decay.

J Appl Bacteriol, 1990 Jan, 68(1), 69 - 74
Effect of short-chain organic acids on macromolecular synthesis in Escherichia coli; Cherrington CA et al.; Incubating cultures of Escherichia coli with propionic acid (5 mmol/l) or formic acid (10 mmol/l) at pH 5.0 produced bacteriostasis lasting 30 and 120 min respectively . During this time rates of RNA, DNA, protein, lipid and cell wall synthesis were reduced . Growth resumed after continued incubation in the presence of acid, but cells from acid-treated cultures were larger than controls . DNA synthesis was particularly sensitive to the presence of the propionic or formic acid.

Proc Natl Acad Sci U S A, 1990 Jan, 87(2), 743 - 7
In vitro trimerization of OmpF porin secreted by spheroplasts of Escherichia coli; Sen K et al.; It is not yet clear how bacterial outer membrane proteins reach their correct destination after they are secreted across the cytoplasmic membrane . We show here that porin OmpF is secreted into the medium as a water-soluble monomeric protein by spheroplasts of Escherichia coli . Furthermore, this monomeric porin is taken up by cell envelope preparations or purified lipopolysaccharides in the presence of 0.03% Triton X-100 and is converted correctly into the mature trimeric conformation . These results appear to reproduce a part of the physiological export and targeting steps of this protein.

EMBO J, 1990 Jan, 9(1), 295 - 304
Mutations affecting primer RNA interaction with the replication repressor RNA I in plasmid CoIE1: potential RNA folding pathway mutants; Polisky B et al.; The control of plasmid ColE1 copy number is mediated by the kinetics of interaction of two complementary plasmid-encoded RNAs . One RNA is the primer precursor and the other is a small counter-transcript called RNA I . The interaction of these highly structured RNAs results in inhibition of formation of mature primer RNA necessary for replication initiation . We have studied several plasmid copy number mutants which have single base changes in the primer which render the primer resistant to inhibition by RNA I despite the fact that the mutations are located outside the overlap between primer and RNA I . We propose a model to account for the resistance of the mutant primers which is based on the differential folding of the nascent primer transcripts during transcription . We propose that the mutant primers diverge in structure from their wild-type counterparts during a discrete period during transcription . During this brief divergence, they are proposed to interact kinetically more slowly with RNA I than wild-type primer because a particular domain (the anti-tail) required for efficient interaction with RNA I is buried in a stem-loop structure while this same domain is predicted to be single-stranded in the wild-type . Despite substantial sequence divergence from ColE1, the primer precursors of the related plasmids CloDF13, RSF1030 and p15A also have retained the potential to expose their anti-tail in a similar manner to ColE1, suggesting that the folding pathway has been conserved in evolution.(ABSTRACT TRUNCATED AT 250 WORDS)

Carcinogenesis, 1990 Jan, 11(1), 117 - 21
Increased uracil-DNA glycosylase, AP-DNA binding protein and deoxyribonuclease activities in tumor and SV40-transformed cell lines of human origin; Kuhnlein U et al.; The activities of three human DNA metabolizing enzymes--uracil-DNA glycosylase, apurinic/apyrimidinic(AP)-DNA binding protein (an AP-DNA endonuclease) and the major cellular deoxyribonuclease (presumably DNase III and/or DNase IV)--were measured in logarithmic growing (diploid non-established) fibroblast strains, tumor-derived cell lines and SV40-transformed cell lines . The levels of activity of uracil-DNA glycosylase and DNase were increased, on average, 5- to 6-fold in tumor cell lines and 10-fold in SV40-transformed cell lines compared to those observed in normal fibroblast strains . AP-DNA binding activity was only 2- to 3-fold higher in both tumor-derived and SV40-transformed cell lines . Measurements in serum-deprived (and hence growth-retarded) SV40-transformed cells indicated that the observed increase in enzyme activity was only partially due to a higher proportion of S-phase cells in the rapidly growing transformed lines . Cell extract mixing experiments indicated that the relatively low levels of activity of the three enzymes in normal fibroblasts could not be ascribed to the presence of an inhibitory factor(s) in the crude extract.

J Bacteriol, 1990 Jan, 172(1), 281 - 6
Identification and sequence of the drpA gene from Escherichia coli; Zhou Z et al.; The drpA gene of Escherichia coli encodes a factor that is involved in global RNA synthesis . We establish that the drpA gene has been successfully cloned and describe the fine-structure map of three drpA-(Ts) mutations as well as the complete nucleotide sequence of the drpA gene . We identified a major sigma-70 promoter for the drpA gene on the bases of (i) its similarity to the consensus sequence and (ii) S1 protection and primer extension mapping data . In addition, the nucleotide sequence revealed a pair of dnaA boxes and a factor-independent terminator at the 5' end and 3' end of the gene, respectively . The deduced amino acid sequence of the DrpA protein showed a nucleotide-binding pocket found in some ATPases.

FEBS Lett, 1990 Jan 1, 259(2), 318 - 20
Antigenic determinants synthesized in a library of randomly cloned fragments of the HIV-1 genome; Mikhailov MV et al.; A DNA expression library of randomly selected fragments of the HIV-1 genome was constructed and used to search for antigenic determinants . A large segment of the HIV-1 provirus was sonicated, and 150-250 bp DNA fragments were cloned in a system of expression vectors developed to obtain high yields of recombinant proteins in Escherichia coli . The expressed library was immunoscreened with sera of AIDS patients . Eleven identified immunoreactive clones were found to correspond to already known and new antigenic regions of HIV-1 proteins gp41, p24, and reverse transcriptase.

Acta Physiol Pol, 1990, 41(1-3), 45 - 52
Antipyretic activity of prazosin; Szreder Z; Thermal responses to prazosin (0.75 mg/kg, i.v . as a bolus injection or 3 h infusion) were investigated in febrile rabbits (treated with E . coli lipopolysaccharide, PLPS) at 3 ambient temperatures (Ta) of 5, 19, 28 degrees C . The drug produced antipyresis which increased with the simultaneous drop of Ta . This antipyretic activity was accompanied by an inhibition of heat production or enhanced elimination of heat, depending on Ta . It is suggested that antipyresis produced by prazosin is mainly due to the effector part of the thermoregulatory system.

SAAS Bull Biochem Biotechnol, 1990 Jan, 3, 129 - 33
Fat-body-specific expression of the Drosophila Lsp-2 gene; Benes H et al.; The larval serum protein-2 gene (Lsp-2) of Drosophila melanogaster is expressed at a very high level in the fat body of third-instar larvae . Here we report that Lsp-2 transcription in adult flies produces a unique mRNA localized in the adult adipose tissue of the head in both sexes . To identify regulatory regions of this Drosophila gene, Lsp-2 5'-flanking DNA sequences were fused to the E . coli beta-galactosidase gene (lacZ) . Transient expression of the hybrid gene in third-instar larvae indicates that 230 bp just upstream from the 'TATA box' of the Lsp-2 gene are sufficient for larval fat body-specific expression.

Bioseparation, 1990, 1(3-4), 305 - 10
Combined use of extraction and genetic engineering for protein purification: recovery of beta-galactosidase fused proteins; Enfors SO et al.; Partitioning of beta-galactosidase in aqueous two-phase systems of poly(ethylene glycol) and potassium phosphate is reviewed . The affinity of Escherichia coli beta-galactosidase for the PEG-rich phase dominates also in beta-galactosidase fusion proteins and the concept of using beta-galactosidase as an affinity handle for extraction of other proteins, after fusion, is discussed . A hypothesis is presented, assuming that tryptophan residues at the surface of beta-galactosidase is responsible for its partitioning to the PEG rich phase, and the concept of poly-tryptophan handles fused to the target protein for extraction is introduced.

Bioseparation, 1990, 1(1), 9 - 21
Optimization and simulation of continuous affinity-recycle extraction (care); Gordon NF et al.; Simulation and optimization of continuous affinity recycle extraction (CARE), a protein purification unit operation based on protein adsorption to solid phase adsorbents, is described in this paper . Rather than packing conventional adsorbent particles in a fixed bed (column), solid/liquid contact is carried out in well-mixed reactors . Continuous operation is achieved by recirculation of the adsorbent particles between two or more contactors . The feasibility of this purification scheme was established with the recovery and isolation of the enzyme beta-galactosidase from E.coli, using the affinity support PABTG/Agarose . A mathematical model describing system performance was developed . The mathematical model was used to optimize several facets of the system design and operation . The base two-stage contractor design was modified by the addition of an intermediate wash stage as well as the incorporation of multiple adsorption stages . These design modifications serve to increase purification, concentration and recovery while utilizing the same amount of adsorbent . The methodology for defining and optimizing objective functions was developed and experimentally validated . Finally, optimum system start-up protocols, minimizing the time required to reach steady-state operation, were developed and experimentally validated . The impact of early introduction of adsorptive purification in a downstream processing sequence, with CARE, was evaluated and is described . Through the early introduction of a highly specific adsorptive step, significant purification is achieved simultaneously with clarification and concentration . In addition, purification performance in CARE was contrasted with that achievable in conventional column chromatography.

SAAS Bull Biochem Biotechnol, 1990 Jan, 3, 85 - 90
Zinc finger structure of a ribosomal gene-specific transcription factor; Hanas JS et al.; Xenopus transcription factor IIIA (TFIIIA) or TFIIIA mutants with internal deletions were expressed in E.coli, isolated from E.coli cell extracts, and identified by SDS PAGE and immunoblotting with rabbit antiserum against native TFIIIA . Specific DNA binding of intact or internally deleted TFIIIA was compared by analyzing their abilities to protect the internal control region (ICR) of the Xenopus 5S ribosomal RNA gene from DNase I digestion . Intact protein bound specifically to the entire ICR (+96 to +43) . One TFIIIA deletion mutant, expressed from cDNA lacking the coding sequence for the putative fourth zinc finger protected the ICR from DNase I digestion from nucleotide positions +96 to +78 . A second TFIIIA mutant resulting from fusion of putative zinc fingers 7 and 8 protected the 5S gene ICR from positions +96 to +63 . The regions of the protein comprising the N-terminal 3 fingers and N-terminal six fingers appear to be in contact with approximately 18 and 33 bp of DNA respectively on the 3' side of the 5S gene ICR.

Bioprocess Technol, 1990, 10, 393 - 416
Downstream processing of proteins from mammalian cells; Ogez JR et al.; Less than a decade ago, the use of continuous mammalian cell lines for the production of cloned proteins was considered strictly a research tool . At that time, few thought it possible to allay the many safety concerns associated with transformed cells . It soon became clear that mammalian expression systems had numerous advantages over bacteria for production of therapeutic proteins, initiating a multidisciplinary effort to address these concerns in a thorough and reliable manner . The success of these efforts is exemplified by the emergence of product molecules into the market . Today, there are seven recombinant human therapeutics that have received FDA approval . Almost half of them (OKT3, t-PA, and EPO) are produced in mammalian cells, with the remainder produced in bacteria (insulin, growth hormone, and alpha-interferon) or yeast (hepatitis vaccine) . At least a dozen more recombinant cell culture products are in advanced human clinical trials . With the accumulation of data and experience, continuous mammalian cell lines will no doubt be the preferred hosts for many future products of biotechnology.

Genet Eng (N Y), 1990, 12, 1 - 19
Folding of eukaryotic proteins produced in Escherichia coli; Kelley RF et al.; Although intracellular expression in E . coli may result in accumulation of the eukaryotic protein in inclusion bodies, the protein may often be recovered by first solubilizing with denaturant followed by refolding . Some general guidelines for developing a refolding procedure are apparent but the specific protocol must be empirically determined for each protein . Convenient and rapid assays for detecting native protein are critical for developing a refolding procedure . Maintaining solubility during refolding is a common feature of recovery processes . Proper folding should be assessed by a number of methods including activity, spectroscopic and stability measurements . For some proteins, properly folded protein may be obtained by secretion from E . coli; however, secretion does not ensure correct folding and protection from proteolytic degradation.

J Biotechnol, 1990 Jan, 13(1), 47 - 60
Simultaneous regulation of plasmid replication and heterologous gene expression in Escherichia coli; Chew LC et al.; An expression plasmid in which plasmid DNA replication and heterologous gene expression can be simultaneously regulated was constructed to avoid derepression prior to induction . This was achieved by placing a pBR322 origin of replication immediately downstream of an anthranilate synthase-human epidermal growth factor fusion gene (trpE-hEGF), both under the control of the promoter from the tryptophan biosynthetic operon . Regulation of plasmid copy number ensured tight repression of the trp promoter prior to induction . Upon induction, plasmid copy number increased up to six-fold and the fusion protein accumulated to approximately 12% of total cell protein . Induction experiments with a series of plasmid derivatives with sequentially lower copy numbers revealed that accumulation levels of the TrpE-hEGF fusion protein post-induction correlated well with plasmid copy number . Plasmid constructs where the native trp promoter had been replaced by derivatives deleted of the attenuator resulted in high levels of hEGF accumulation in the tryptophan-free medium prior to induction . Nevertheless, up to two-fold increase in TrpE-hEGF accumulation levels were obtained using the constructs lacking the attenuator compared to those bearing the native trp promoter.

SAAS Bull Biochem Biotechnol, 1990 Jan, 3, 1 - 7
Cooperative interactions in transcriptional regulation; Fried MG; Cooperative interactions between regulatory proteins and RNA polymerase are a common feature of transcriptional systems . We have developed a method, based on the electrophoresis mobility shift assay, for the measurement of cooperative effects in the binding of proteins to DNA restriction fragments . Using this approach we have identified a hitherto unknown interaction between the E . coli lactose repressor and CAP proteins . We suggest that this interaction plays a role in the control of the lactose operon that is not predicted by current regulatory models.

Biochem Biophys Res Commun, 1989 Dec 29, 165(3), 942 - 6
Protective effect of chlorpromazine against the lethality of interleukin 1 in adrenalectomized or actinomycin D-sensitized mice; Bertini R et al.; Interleukin 1 (IL-1) and Tumor Necrosis Factor (TNF) are thought to play a key role in septic shock and inflammation . We have tested the effect of dexamethasone (DEX) and chlorpromazine (CPZ) on the lethal effect of IL-1, TNF and endotoxin . Two different experimental models were used to sensitize mice to the lethal effect of IL-1: adrenalectomy and pretreatment with actinomycin D . CPZ (4 mg/kg) was found to protect mice against IL-1 and endotoxin toxicity in all cases, while DEX had a protective effect only in adrenalectomized mice . In contrast to its protective effect against IL-1 and endotoxin, CPZ did not protect mice against TNF . These findings might be useful in the analysis of the differences in the actions of IL-1 and TNF in vivo, and in the development of new drugs preventing their toxicity.

Biochem Biophys Res Commun, 1989 Dec 29, 165(3), 1371 - 4
A new family of 2-hydroxyacid dehydrogenases; Grant GA; The NADH-dependent hydroxypyruvate reductase from cucumber and the pdxB gene product of E . coli display significant homology to E . coli D-3-phosphoglycerate dehydrogenase . In contrast, these proteins do not display much similarity with other oxidoreductases or with other 2-hydroxyacid dehydrogenases in particular . On the basis of their relatedness and the structure of their substrates, these three enzymes constitute a new family of 2-hydroxyacid dehydrogenases distinct from malate and lactate dehydrogenase.

Biochem Biophys Res Commun, 1989 Dec 29, 165(3), 1043 - 50
Crystallizable HIV-1 protease derived from expression of the viral pol gene in Escherichia coli; Danley DE et al.; A plasmid vector was used to express the HIV-1 pol open reading frame under the regulation of the bacterial trp promoter in Escherichia coli . This expression system has been used as a source of recombinant viral protease . The self-processed active enzyme was recovered from a soluble fraction of a bacterial cell lysate and purified by a procedure involving four steps of chromatography . The protocol yielded 0.3 mg of protease for each liter of bacterial culture . The protease formed tetragonal bipyramidal crystals which have been used in high-resolution X-ray diffraction studies.

Biochem Biophys Res Commun, 1989 Dec 29, 165(3), 1422 - 7
Expression of normal and abnormal porcine kidney D-amino acid oxidase in Escherichia coli: purification and characterization of the enzymes; Watanabe F et al.; Expression plasmids for normal and abnormal porcine D-amino acid oxidases (E.C . 1.4.3.3, DAO) have been constructed from cloned cDNA that encodes the entire protein sequence of DAO, and the enzymes were expressed in Escherichia coli cells on a large scale . The expressed enzymes were purified to apparent homogeneity . The molecular weight of the normal DAO (38 kD) was identical with that of DAO purified from porcine kidney, whereas that of the abnormal DAO was 39 kD, which comprised the normal DAO with an additional decapeptide at its amino terminus . However, the specific activities of the two enzymes were comparable with that of natural DAO . The results indicate that the bulky decapeptide does not affect the structure necessary for the catalytic function of DAO in the amino-terminal region . The use of a GTG triplet in the 5'-untranslated region of DAO cDNA as the initiation codon for the synthesis of the abnormal DAO is suggested.

Biochem Biophys Res Commun, 1989 Dec 29, 165(3), 1243 - 9
Involvement of protein kinase C in activation of human granulocytes and peritoneal macrophages by type 1 fimbriated (mannose specific) Escherichia coli; Gbarah A et al.; Specific binding of bacteria to phagocytic cells mediated by antibody and complement (opsonins) or by lectin-carbohydrate interactions is required for their efficient uptake and killing by opsonophagocytosis or lectinophagocytosis, respectively (Ofek and Sharon, Infect . Immun . 56, 539, 1988) . An early step in these processes is activation of the phagocytes by the bound bacteria, as evidenced by appearance of an oxidative burst . Previous work has shown that protein kinase C (PKC) is involved in activation of human granulocytes by opsonized Escherichia coli . In the present study, we used three inhibitors of PKC to examine the possible involvement of the enzyme in activation of human granulocytes and peritoneal macrophages by type 1 fimbriated (mannose-specific) Escherichia coli in the absence of opsonins . Activation, as measured by chemiluminescence, was completely inhibited by sphingosine (50 microM) and only partially (50%) by 100 microM H-7 {1-(5-isoquinolinesulfonyl)-2-methylpiperazine}; in both cases it was fully reversible . The third inhibitor, K252a, also inhibited almost completely the activation at 1 microM . The inhibitors acted similarly on activation of the phagocytic cells by opsonized bacteria or by phorbol-12-myristate-13-acetate (0.1 microM) . Down regulation of the kinase, by pretreatment of the human granulocytes or macrophages with a high concentration (1.6 microM) of phorbol myristate acetate, abolished their ability to respond to stimulation by the bacteria . Our findings provide evidence for the involvement of PKC in the activation of phagocytic cells by type 1 fimbriated bacteria.

Biochem Biophys Res Commun, 1989 Dec 29, 165(3), 1085 - 90
Site-specific alteration of Gly-24 in streptokinase: its effect on plasminogen activation; Lee BR et al.; Oligonucleotide-directed mutagenesis was carried out to replace glycine-24 of streptokinase with histidine, glutamic acid, or alanine . Substitutions with either histidine or glutamic acid resulted in almost complete loss of streptokinase activity but streptokinase replaced with alanine retained its activity . Although streptokinases with histidine-24 or glutamic acid-24 bound normally to human plasminogen, they were not able to generate active plasmin, whereas those with alanine-24 or glycine-24 (wild-type) could generate active plasmin . The results indicate that the small, uncharged alkyl group side-chain on the 24th amino acid residue of streptokinase is indispensable for the activity of the human plasminogen-streptokinase complex.

Gene, 1989 Dec 28, 85(2), 553 - 7
Growth at sub-optimal temperatures allows the production of functional, antigen-binding Fab fragments in Escherichia coli; Cabilly S; Expression in Escherichia coli of recombinant genes coding for the kappa-chain and the Fd fragment of an antibody directed against carcinoembryonic antigen gives rise to Fab dimers . These Fab fragments possess antibody activity, as demonstrated by enzyme-linked immunosorbent assay as well as by ligand competition assay . Effective production of soluble Fab in Escherichia coli was achieved by a decrease in the growth temperature . Following a one step purification by anion exchange chromatography, the bacterially-produced Fab retains its activity at 4 degrees C for at least two months . The relatively simple methodology described in this study should be useful for the design and production of antibodies in bacteria.

Gene, 1989 Dec 28, 85(2), 453 - 9
Synthesis of glia-derived nexin in yeast; Sommer J et al.; Glia-derived nexin (GDN) is a 43-kDa glycoprotein isolated from rat glioma cell cultures . It promotes neurite extension in cultures of neuroblastoma cells and chick sympathetic neurons . Moreover, GDN is a potent serine protease inhibitor (serpin), belonging to the family of protease nexins . We report here the expression of rat GDN in the Saccharomyces cerevisiae strain GRF18 under the control of the PHO5 promoter . We describe the purification of more than 6 mg total GDN from the cellular extract of 1 liter of yeast culture . The amino acid composition and the sequence of CNBr-fragments of the recombinant protein correlate with the values deduced from the rat GDN cDNA . We provide evidence that the recombinant GDN has exactly the same properties as the glioma-derived protein with respect to its protease-inhibitory activity and its ability to promote the extension of neurites from neuroblastoma cells . The large amounts of recombinant protein obtained from this expression system will allow further biochemical and physiological analysis of GDN and of the serpins in general.

Gene, 1989 Dec 28, 85(2), 329 - 33
Cloning and expression of the gene encoding a novel proteinase from Tritirachium album limber; Samal BB et al.; We have isolated the genomic and cDNA clones encoding a novel proteinase from the fungus Tritirachium album Limber, named proteinase T, synthesis of which is induced in skim milk medium . The coding sequence for this enzyme is interrupted by two introns in the fungal genome . The amino acid sequence of proteinase T as deduced from the nucleotide sequence is about 53% identical to that of proteinase K . Four cysteines are present in the mature proteinase, probably in the form of two disulfide bonds, which might explain the thermal stability of the proteinase . We have expressed the proT cDNA in Escherichia coli . The authenticity of the product has been characterized by Western blotting and N-terminal analysis of the recombinant product.

Gene, 1989 Dec 28, 85(2), 353 - 62
Optimization and in situ detection of Escherichia coli beta-galactosidase gene expression in Dictyostelium discoideum; Dingermann T et al.; We show that a fusion gene, containing the promoter and 5'-noncoding region of a Dictyostelium discoideum actin 6 gene linked to the Escherichia coli beta-galactosidase (beta Gal) gene (lacZ), directs the production of functionally active beta Gal in D . discoideum and that the enzyme can be detected by staining in situ; a procedure which will be of great value in analyzing cell-type-specific gene expression . We illustrate this by fusing lacZ to the promoter of the prespore-specific gene, D19, and localizing expressing cells in migrating slugs . Optimal expression requires the inclusion of termination and polyadenylylation signals and we describe pDDlac, a vector containing a multiple cloning site upstream from a lacZ-Dictyostelium terminator fusion, which can be used to analyze regulated promoters.

Biochemistry, 1989 Dec 26, 28(26), 9949 - 56
Thermodynamics of protein-RNA recognition in a highly conserved region of the large-subunit ribosomal RNA; Ryan PC et al.; Ribosomal protein L11 from Escherichia coli specifically binds to a highly conserved region of 23S ribosomal RNA . The thermodynamics of forming a complex between this protein and several different rRNA fragments have been investigated, by use of a nitrocellulose filter binding assay . A 57-nucleotide region of the RNA (C1052-U1108) contains all the protein recognition features, and an RNA fragment containing this region binds L11 10(3)-10(4)-fold more tightly than tRNA . Binding constants are on the order of 10 microM-1 and are only weakly dependent on K+ concentration (delta log K/delta log {K+} = -1.4) or temperature . Binding requires multivalent cations; Mg2+ is taken up into the complex with an affinity of approximately 3 mM-1 . Other multivalent cations tested, Ca2+ and Co(NH3)63+, promote binding nearly as well . The pH dependence of binding is a bell-shaped curve with a maximum near neutral pH, but the entire curve is shifted to higher pH for the smaller of two RNA fragments tested . This result suggests that the smaller fragment favors a conformation stabilizing protonated forms of the RNA recognition site and is potentially relevant to a hypothesis that this rRNA region undergoes an ordered series of conformational changes during the ribosome cycle.

Biochemistry, 1989 Dec 26, 28(26), 10028 - 34
Substitution of amino acids in helix F of bacteriorhodopsin: effects on the photochemical cycle; Ahl PL et al.; The effects of amino acid substitutions in helix F of bacteriorhodopsin on the photocycle of this light-driven proton pump were studied . The photocycles of Ser-183----Ala and Glu-194----Gln mutants were qualitatively similar to that of wild-type bacteriorhodopsin produced in Escherichia coli and bacteriorhodopsin from Halobacterium halobium . The substitution of a Phe for either Trp-182 or Trp-189 significantly reduced the fraction of photocycling bacteriorhodopsin . The amino acid substitutions Tyr-185----Phe and Ser-193----Ala substantially increased the lifetime of the photocycle without substantially increasing the lifetime of the M photocycle intermediate . Similar results were also obtained with the Pro-186----Gly substitution . In contrast, replacing Pro-186 with the larger residue Leu inhibited the formation of the M photocycle intermediate . These results are consistent with a structural model of the retinal-binding pocket suggested by low-temperature UV/visible and Fourier transform infrared difference spectroscopies that has Trp-182, Tyr-185, Pro-186, and Trp-189 forming part of the binding pocket.

Biochemistry, 1989 Dec 26, 28(26), 9937 - 43
Function of threonine-55 in the carbamoyl phosphate binding site of Escherichia coli aspartate transcarbamoylase; Xu W et al.; Carbamoyl phosphate is held in the active site of Escherichia coli aspartate transcarbamoylase by a variety of interactions with specific side chains of the enzyme . In particular, the carbonyl group of carbamoyl phosphate interacts with Thr-55, Arg-105, and His-134 . Site-specific mutagenesis was used to create a mutant version of the enzyme in which Thr-55 was replaced by alanine in order to help define the role of this residue in the catalytic mechanism . The Thr-55----Ala holoenzyme exhibits a 4.7-fold reduction in maximal observed specific activity, no alteration in aspartate cooperativity, and a small reduction in carbamoyl phosphate cooperativity . The mutation also causes 14-fold and 35-fold increases in the carbamoyl phosphate and aspartate concentrations required for half the maximal observed specific activity, respectively . Circular dichroism spectroscopy has shown that saturating carbamoyl phosphate does not induce a conformational change in the Thr-55----Ala holoenzyme as it does for the wild-type holoenzyme . The kinetic properties of the Thr-55----Ala catalytic subunit are altered to a greater extent than the mutant holoenzyme . The mutant catalytic subunit cannot be saturated by either substrate under the experimental conditions . Furthermore, as opposed to the wild-type catalytic subunit, the Thr-55----Ala catalytic subunit shows cooperativity for aspartate and can be activated by N-(phosphonoacetyl)-L-aspartate in the presence of low concentrations of aspartate and high concentrations of carbamoyl phosphate . As deduced by circular dichroism spectroscopy, the conformation of the Thr-55----Ala catalytic subunit in the absence of active-site ligands is distinctly different from the wild-type catalytic subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1989 Dec 26, 28(26), 9932 - 7
Bleomycin-iron can degrade DNA in the presence of excess ethylenediaminetetraacetic acid in vitro; Solomon LR et al.; The antineoplastic drug bleomycin, when complexed to Fe(II), causes both single- and double-stranded lesions in DNA in vitro . EDTA is commonly used to inhibit the reaction of bleomycin-Fe with DNA, presumably by removing the metal cofactor . In this study, we utilized a simple assay involving the conversion of supercoiled plasmid DNA to the nicked or linear forms to further investigate the ability of bleomycin-Fe to degrade DNA in the presence of EDTA . We found that a 1:1 complex of bleomycin and Fe can degrade plasmid DNA even in the presence of a 10(6) molar excess of EDTA over bleomycin . Furthermore, we found that the half-life for inactivation of bleomycin-Fe by excess EDTA is about 1.5 h . Finally, we demonstrate that excess bleomycin associated with the outer plasma membranes of cells can damage DNA after the cells are lysed in buffers containing EDTA and SDS . These results suggest that EDTA may not be an efficient inhibitor of the reaction of bleomycin-Fe with DNA.

Nucleic Acids Res, 1989 Dec 25, 17(24), 10403 - 25
Purification, cloning and sequence analysis of RsrI DNA methyltransferase: lack of homology between two enzymes, RsrI and EcoRI, that methylate the same nucleotide in identical recognition sequences; Kaszubska W et al.; RsrI DNA methyltransferase (M-RsrI) from Rhodobacter sphaeroides has been purified to homogeneity, and its gene cloned and sequenced . This enzyme catalyzes methylation of the same central adenine residue in the duplex recognition sequence d(GAATTC) as does M-EcoRI . The reduced and denatured molecular weight of the RsrI methyltransferase (MTase) is 33,600 Da . A fragment of R . sphaeroides chromosomal DNA exhibited M.RsrI activity in E . coli and was used to sequence the rsrIM gene . The deduced amino acid sequence of M.RsrI shows partial homology to those of the type II adenine MTases HinfI and DpnA and N4-cytosine MTases BamHI and PvuII, and to the type III adenine MTases EcoP1 and EcoP15 . In contrast to their corresponding isoschizomeric endonucleases, the deduced amino acid sequences of the RsrI and EcoRI MTases show very little homology . Either the EcoRI and RsrI restriction-modification systems assembled independently from closely related endonuclease and more distantly related MTase genes, or the MTase genes diverged more than their partner endonuclease genes . The rsrIM gene sequence has also been determined by Stephenson and Greene (Nucl . Acids Res . (1989) 17, this issue).

Nucleic Acids Res, 1989 Dec 25, 17(24), 10163 - 70
'Sticky feet'-directed mutagenesis and its application to swapping antibody domains; Clackson T et al.; We describe a novel technique for precisely cutting and pasting two DNA sequences without using restriction sites . The method is based on site-directed mutagenesis and uses a long primer, generated by the polymerase chain reaction (PCR), to transfer large segments of DNA into a single-stranded template . The primer anneals to the template by virtue of 'sticky feet' sequences (complementary to the template) which are introduced at the ends of the primer by the PCR . Yields of desired recombinants were high (approximately 36%) and the transplanted sequences (approximately 400bp) free of errors . We have used this technique to swap CH2 domains between two mouse antibodies, and find that this domain can carry features critical for triggering complement lysis, in addition to the C1q binding motif.

J Biol Chem, 1989 Dec 25, 264(36), 21848 - 56
Enhanced recA protein binding to Z DNA represents a kinetic perturbation of a general duplex DNA binding pathway; Kim JI et al.; recA protein binding to duplex DNA is enhanced when a B form DNA substrate is replaced with a left-handed Z form helix . This represents a kinetic rather than an equilibrium effect . Binding to Z DNA is much faster than binding to B DNA . In other respects, binding to the two DNA forms is quite similar . recA protein binds to B or Z DNA with a stoichiometry of 1 monomer/4 base pairs . The final protein filament exhibits a right-handed helical structure when either B or Z form DNAs are bound . There are only two evident differences: the kcat for ATP hydrolysis is reduced 3-4-fold when Z DNA is bound, and recA binding at equilibrium is less stable on Z DNA than on B DNA . At steady state, the binding favors B DNA in competition experiments . The results indicate that Z DNA binding by recA protein follows the same pathway as for recA binding to B DNA, but that the nucleation step is faster on the Z form helix.

J Biol Chem, 1989 Dec 25, 264(36), 21842 - 7
Restoration of membrane incorporation of an Escherichia coli outer membrane protein (OmpA) defective in membrane insertion; Klose M et al.; The mechanism of sorting, to the outer membrane, of the 325-residue Escherichia coli protein OmpA has been investigated . It is thought to traverse the membrane eight times in antiparallel beta-strands, forming an amphiphilic beta-barrel which encompasses residues 1 to about 170; the COOH-terminal moiety is periplasmic . A mutant, carrying the substitutions Leu164----Pro and Val166----Asp within the last beta-strand (residues 160-170), has been described which was unable to assemble in the membrane (Klose, M., MacIntyre, S., Schwarz, H., and Henning, U . (1988) J . Biol . Chem . 263, 13297-13302) . Linkers were inserted between the codons for residues 164 and 165 of the mutant protein . Of 13 different genes recovered, five encoded proteins which had regained the ability to assemble in the membrane . The properties of the mutant proteins, together with a structure prediction method, indicate the following rules for the final beta-strand to be compatible with, or possibly initiate, membrane insertion: (i) it must be amphiphilic or hydrophobic while its primary structure as such is fairly unimportant, (ii) it must extend over at least 9 residues, and (iii) it must not contain a proline residue around its center . One of the genes recovered coded for OmpA up to residue 164 and then followed by 10 linker-encoded residues . This 174-residue polypeptide was assembled in the membrane but did not, in contrast to all other proteins, expose sites sensitive to trypsin at the inner face of the membrane . This behavior agrees perfectly well with the OmpA model.

J Biol Chem, 1989 Dec 25, 264(36), 21798 - 805
Localization of the site of cleavage of ribosomal RNA by colicin E3 . Placement on the small ribosomal subunit by electron microscopy of antibody--complementary oligodeoxynucleotide complexes; Lasater LS et al.; Colicin E3 is a ribonuclease that inactivates Escherichia coli ribosomes by cleaving the RNA of the small ribosomal subunit after nucleotide 1493 . A series of oligodeoxynucleotides that complement 16 S RNA in the region of the colicin cleavage site has been synthesized, and their ability to form complexes with 30 S ribosomal subunits has been measured using a nitrocellulose filter-binding assay . The most efficiently bound probe, complementary to residues 1485-1496, was modified with antibody-recognizable derivatives at the 5'-end, the 3'-end, or both . Antibody-oligonucleotide-subunit complexes were then generated and examined by electron microscopy . Antibody binding was seen at the tip of the platform of the 30 S subunit . The complementary oligonucleotide and thus the site at which colcin E3 cleavage occurs is therefore in the same physical region as the 3'-end of the 16 S ribosomal RNA and its message-positioning "Shine-Dal-garno" sequence.

J Biol Chem, 1989 Dec 25, 264(36), 21770 - 8
Identification of the site of phosphorylation of the chemotaxis response regulator protein, CheY; Sanders DA et al.; The protein (Escherichia coli CheY) that controls the direction of flagellar rotation during bacterial chemotaxis has been shown to be phosphorylated on the aspartate 57 residue . The residue phosphorylated is present within a conserved sequence in every member of a family of bacterial regulatory proteins . The phosphorylation is transient, with a much shorter half-life than that expected of a simple acyl phosphate intermediate, indicating that the sequence and conformation of the protein is designed to achieve a rapid hydrolysis . The CheY-phosphate linkage can be reductively cleaved by sodium borohydride . High-performance tandem mass-spectrometric analysis of proteolytic peptides derived from {3H}borohydride-reduced phosphorylated CheY protein was used to identify the position of phosphorylation . Mutants with altered aspartate 57 exhibited no chemotaxis . When aspartate 13, another conserved residue, was changed, greatly reduced chemotaxis was observed, suggesting an important role for aspartate 13 . The rate-determining step of chemotactic signaling is governed by the kinetics of formation and hydrolysis of the CheY protein phosphoaspartate bond . The CheY protein apparently functions as a protein phosphatase that possesses a transient covalent intermediate . Transient phosphorylation of an aspartate residue is an effective mechanism for producing a biochemical signal with a short concentration-independent half-life . The duration of the signal can be controlled by small structural elements within the phosphorylated protein.

J Biol Chem, 1989 Dec 25, 264(36), 21633 - 7
Signal transduction and osmoregulation in Escherichia coli . A single amino acid change in the protein kinase, EnvZ, results in loss of its phosphorylation and dephosphorylation abilities with respect to the activator protein, OmpR; Kanamaru K et al.; The EnvZ protein is a bacterial protein kinase, which specifically phosphorylates the activator protein, OmpR, involved in expression of the ompF and ompC genes in Escherichia coli . The phosphotransfer between the EnvZ and OmpR proteins was postulated to be involved in the signal transduction in response to an environmental osmotic stimulus . In this study, we isolated a novel type of envZ mutant, in which a base substitution resulted in a Tyr-to-Ser conversion at amino acid residue 351 of the EnvZ protein . This single amino acid conversion was found to dramatically affect the functions of the EnvZ protein . The mutant EnvZ protein was defective in its ability not only as to OmpR-phosphorylation but also as to OmpR-dephosphorylation . The envZ mutant, termed envZ30, was isolated as a pseudorevertant, which phenotypically suppresses an ompR3-type mutant exhibiting an OmpF- OmpC-constitutive phenotype . These results will be discussed in relation to the structure and function of the protein kinase, EnvZ.

J Biol Chem, 1989 Dec 25, 264(36), 21582 - 90
Isolation, characterization, and expression in Escherichia coli of two murine Mu class glutathione S-transferase cDNAs homologous to the rat subunits 3 (Yb1) and 4 (Yb2); Townsend AJ et al.; The cytosolic glutathione S-transferases (GSTs, EC 2.5.1.18) are a superfamily of dimeric isoenzymes which catalyze the conjugation of electrophilic substrates with glutathione . We have isolated from a murine cell line (L929) two mu class GST cDNAs, pmGT10 (1.4 kilobases (kb} and pmGT2 (1.1 kb) . Analysis of the deduced amino acid sequences revealed 93% homology between pmGT10 and the rat Yb1 (subunit 3) gene and 95% homology between pmGT2 and the rat Yb2 (subunit 4) gene . Furthermore, the 3'-untranslated regions of pmGT10 display a marked degree of homology to the 3' region of the rat Yb1 gene, while this region of pmGT2 displays marked homology to the corresponding region of the rat Yb2 gene . Using probes specific for each gene, northern blot analysis demonstrated that pmGT10 and pmGT2 hybridized to a 1.3-1.4-kb mRNA species present in L929 cells . These two murine cDNAs were subcloned into bacterial expression vectors, and enzymatically active GSTs encoded by pmGT10 (mGTmu1) or pmGT2 (mGTmu2) were purified from transformed Escherichia coli lysates . Western blot analysis of the individual GSTs produced in E . coli indicated that both genes encode GSTs which reacted with antibodies directed against mu class GST, but not with antibodies directed against alpha or pi class GSTs . The isoelectric points of these purified homodimers are 8.7 and 7.1, respectively, which are remarkably similar to those of the GST homodimers of the homologous rat subunits, 3-3 (or Yb1) and 4-4 (or Yb2), which are 8.4 and 6.9, respectively . Furthermore, the two murine subunits can form a heterodimer having a pI = 8.1 after in vitro dissociation with guanidine HCl, followed by dilution and dialysis to allow reassociation . Both homodimers of mGTmu1 and mGTmu2 and an apparent heterodimer are formed in L929 cells, as demonstrated by the isoelectric focusing profile of GST purified from this cell line . Hybridization of gene-specific probes with RNA from normal mouse tissues revealed that mGTmu1 transcripts were highly expressed in murine kidney, heart, lung, liver, and brain . Lower levels of mGTmu2 transcripts were also detected in kidney, heart, and lung . Expression of mGTmu2 RNA was not detected in murine liver or brain, which is in contrast to the regulation of the homologous rat subunit 4 (Yb2), which is expressed in liver and brain.

J Biol Chem, 1989 Dec 25, 264(36), 21498 - 503
DNA polymerase gamma from Xenopus laevis . II . A 3'----5' exonuclease is tightly associated with the DNA polymerase activity; Insdorf NF et al.; Xenopus laevis DNA polymerase gamma co-purifies with a tightly associated 3'----5' exonuclease . The purified enzyme lacks 5'----3' exonuclease and endonuclease activity . The ratio of the 3'----5' exonuclease activity to DNA polymerase gamma activity remains constant over the final three chromatographic procedures . In addition, these activities co-sediment under partially denaturing conditions in the presence of ethylene glycol . The associated 3'----5' exonuclease activity removes a terminally mismatched nucleotide more rapidly than a correctly base-paired 3'-terminal residue, as expected if this exonuclease has a proofreading function . The 3'----5' exonuclease has the ability to release a terminal phosphorothioated nucleotide, a property shared with T4 DNA polymerase, but not with Escherichia coli DNA polymerase I.

Nucleic Acids Res, 1989 Dec 25, 17(24), 10473 - 88
Hyperexpression and purification of Escherichia coli adenylate cyclase using a vector designed for expression of lethal gene products; Reddy P et al.; We describe the construction of a new generation of vectors (pRE) for the hyperexpression of lethal gene products such as adenylate cyclase in Escherichia coli . The pRE vectors are based on the lambda PL promoter and lambda cII ribosome binding site described by Shimatake and Rosenberg (Nature, 292, 128-132, 1981) . They have a unique NdeI restriction endonuclease site 3' of the lambda cII ribosome binding site that includes the ATG initiation codon, multilinker cloning sites 3' to the NdeI site, and two lambda transcription terminators 5' and 3' of the lambda PL promoter to eliminate nonspecific transcription and reduce leaky PL transcription, respectively . For hyperexpression of adenylate cyclase, tight control of transcription was necessary since elevation of cAMP levels above the physiological range is lethal to E . coli . Lethality associated with the overproduction of adenylate cyclase was shown to be mediated through the cAMP receptor protein . We used this expression system to overproduce adenylate cyclase 7500 fold, corresponding to 30% of the total cellular protein . Under these conditions the enzyme precipitated with significant loss of activity . Reducing the rate and amount of adenylate cyclase expression to 16% of the total cell protein produced one fourth of the enzyme in a soluble form with high specific activity . The soluble adenylate cyclase was purified to near homogeneity.

J Biol Chem, 1989 Dec 25, 264(36), 21885 - 90
A second type of rat phosphoinositide-specific phospholipase C containing a src-related sequence not essential for phosphoinositide-hydrolyzing activity; Emori Y et al.; A fourth type of rat phosphoinositide-specific phospholipase C (PLC IV) has been cloned for cDNA and sequenced . PLC IV is distinct from the other three types of rat PLC (PLC I, II, and III) with respect to primary structure and tissue distribution of its mRNAs . PLC IV contains two homologous regions included commonly in PLC I, II, and III and is most similar to PLC II (identity: 50.2%) . PLC IV, in common with PLC II, has a sequence homologous to the N-terminal regulatory domains of nonreceptor tyrosine kinases of the src-family of oncogenes . Using an Escherichia coli expression system, we succeeded in producing active PLC IV in E . coli crude extracts . Various truncation experiments of the PLC IV cDNA revealed that the src-related domain is not necessary for catalytic activity while both domains homologous among PLC I-IV are essential . PLC IV is expressed in various rat tissues and abundant in spleen, suggesting that PLC IV plays a fundamental role in cellular functions such as growth and secretion.

J Biol Chem, 1989 Dec 25, 264(36), 21677 - 81
Spectroscopic studies of isopenicillin N synthase . A mononuclear nonheme Fe2+ oxidase with metal coordination sites for small molecules and substrate; Chen VJ et al.; The nonheme iron oxidase isopenicillin N synthase catalyzes the formation of two new internal bonds in the tripeptide delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to form the beta-lactam and thiazolidine rings of isopenicillin N . Concomitantly, O2 is reduced to 2 H2O . The recombinant enzyme from Cephalosporium acremonium (Mr = 38,400), expressed as an apoenzyme in Escherichia coli, binds 1 g atom of Fe2+/mol of enzyme to reconstitute full activity . Mossbauer spectra of the 57Fe-enriched enzyme exhibit parameters (delta = 1.30 mm/s, delta EQ = 2.70 mm/s) which unambiguously show that the active site iron is high spin Fe2+ . Anaerobic binding of ACV causes a substantial decrease in the isomer shift parameter delta (delta = 1.10 mm/s, delta EQ = 3.40 mm/s) showing that the substrate perturbs the iron site and makes its coordination environment much more covalent . Nitric oxide (NO) binds to the EPR silent active site iron to give an EPR active species (g = 4.09, 3.95, 2.0; S = 3/2) similar to those of the nitrosyl complexes of many other mononuclear Fe2+-containing enzymes . The rhombicity of the EPR spectrum is increased (g = 4.22, 3.81, 1.99) by anaerobic addition of ACV suggesting that the substrate binds to or near the iron without displacing NO . Interestingly, the enzyme.ACV.NO complex displays an optical spectrum similar to that of ferric rubredoxin in which the iron has only thiol coordination . This suggests that the Fe2+ of the enzyme.ACV.NO complex acquires Fe3+ character and that the cysteinyl thiol moiety of ACV coordinates to the iron . Similar substrate thiol coordination to the iron of the enzyme.ACV complex is the most probable explanation for the large decrease in isomer shift observed . These results provide the first evidence for the direct involvement of iron in this unique O2-dependent reaction and suggest novel roles for iron and oxygen in biological catalysis.

Eur J Biochem, 1989 Dec 22, 186(3), 629 - 36
Lipopolysaccharide interactions with lysozyme differentially affect lipopolysaccharide immunostimulatory activity; Ohno N et al.; The effect of complex formation between lysozyme and lipopolysaccharide (LPS) on the immunostimulatory activities of LPS have been investigated in vitro . Three prototype immunostimulatory activities were examined: B-lymphocyte proliferation, B-lymphocyte differentiation and macrophage production of lymphocyte-activating factor activity . Different effects of lysozyme were noted, depending upon the structure of the LPS, even though previous studies have established that all LPS preparations readily bind lysozyme . Both Re-LPS- and lipid-A-dependent immunostimulatory activities were readily inhibited by lysozyme in a dose-dependent fashion . In contrast, S-LPS and Ra-LPS were unaffected in their immunostimulatory activities by lysozyme . These differences were not the result of quantitative differences in LPS binding of lysozyme, or effects of lysozyme on overall binding of LPS to target cells . These data suggest that the factors which dictate the initial interactions between LPS and lymphoreticular cells may not be identical for all LPS preparations and/or purified lipid A.

Eur J Biochem, 1989 Dec 22, 186(3), 621 - 7
Effects of lipopolysaccharide chemotype structure on binding and inactivation of hen egg lysozyme; Ohno N et al.; The structural features of lipopolysaccharide {LPS} which influence the binding and inactivation of lysozyme have been examined . Binding of polysaccharide-containing LPS (S-LPS) and Ra-Rc-LPS preparations was independent of temperature between 37-50 degrees C; in contrast, binding of Rd-LPS, Re-LPS and lipid A was temperature-dependent . The binding of lysozyme to Rd-LPS and Re-LPS was increased by treatment with mild alkali, which has little detectable effect on binding of lysozyme to S-LPS and Ra-Rc-LPS preparations . Competitive binding experiments using dansylated lysozyme and/or dansylated polymyxin B indicated independent binding sites on the LPS for lysozyme and polymyxin B . These results indicate significant differences between most LPS preparations and lipid A and glycolipid LPS in their interaction with proteins of mammalian origin.

Cell, 1989 Dec 22, 59(6), 1189 - 202
A gradient of nuclear localization of the dorsal protein determines dorsoventral pattern in the Drosophila embryo; Roth S et al.; The dorsoventral axis of the Drosophila embryo is determined by a morphogen gradient established by the action of 12 maternal-effect genes: the dorsal group genes and cactus . One of the dorsal group genes, dorsal (dl), encodes the putative morphogen . Although no overall asymmetry in the distribution of dorsal protein is observed, a gradient of nuclear concentration of dl protein is established during cleavage stages, with a maximum at the ventral side of the egg . At the dorsal side of the egg, the protein remains in the cytoplasm . Nuclear localization of the dl protein, and hence gradient formation, is blocked in dorsalizing alleles of all of the other dorsal group genes, while in ventralizing mutants nuclear localization extends to the dorsal side of the egg . A correlation between dl protein distribution and embryonic pattern in mutant embryos indicates that the nuclear concentration of the dl protein determines pattern along the dorsoventral axis.

Cell, 1989 Dec 22, 59(6), 1115 - 25
The v-myb oncogene product binds to and activates the promyelocyte-specific mim-1 gene; Ness SA et al.; The v-myb oncogene induces myeloid leukemias in chickens, transforms myeloid cells in vitro, and encodes a sequence-specific DNA binding protein . We used differential hybridization to screen for v-myb-regulated genes in cells transformed by a temperature-sensitive mutant of the oncogene and identified a new gene, mim-1, which encodes a specifically expressed, secretable protein contained in the granules of both normal and v-myb-transformed promyelocytes . The promoter of the mim-1 gene contains three closely spaced binding sites for v-myb protein and is strongly activated by v-myb in a cotransfection assay . Synthetic copies of the binding sites are both necessary and sufficient to confer v-myb protein-dependent activation to a heterologous promoter . We conclude that mim-1 is a cellular gene that is directly regulated by the product of the v-myb oncogene.

Science, 1989 Dec 22, 246(4937), 1595 - 7
The anticodon contains a major element of the identity of arginine transfer RNAs; Schulman LH et al.; The contribution of the anticodon to the discrimination between cognate and noncognate tRNAs by Escherichia coli Arg-tRNA synthetase has been investigated by in vitro synthesis and aminoacylation of elongator methionine tRNA (tRNA(mMet) mutants . Substitution of the Arg anticodon CCG for the Met anticodon CAU leads to a dramatic increase in Arg acceptance by tRNA(mMet) . A nucleotide (A20) previously identified by others in the dihydrouridine loop of tRNA(Arg)s makes a smaller contribution to the conversion of tRNA(mMet) identity from Met to Arg . The combined anticodon and dihydrouridine loop mutations yield a tRNA(mMet) derivative that is aminoacylated with near-normal kinetics by the Arg-tRNA synthetase.

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