Escherichia coli

Nucleic Acids Res, 1993 Jun 25, 21(12), 2899 - 905
Cleavage and binding of a DNA fragment containing a single 8-oxoguanine by wild type and mutant FPG proteins; Castaing B et al.; A 34-mer oligonucleotide containing a single 7,8-dihydro-8-oxoguanine (8-OxoG) residue was used to study the enzymatic and DNA binding properties of the Fpg protein from E . coli . The highest rates of incision of the 8-OxoG containing strand by the Fpg protein were observed for duplexes where 8-OxoG was opposite C (*G/C) or T (*G/T) . In contrast, the rates of incision of duplexes containing 8-OxoG opposite G (*G/G) and A (*G/A) were 5-fold and 200-fold slower . Gel retardation studies showed that the Fpg protein had a strong affinity for duplexes where the 8-OxoG was opposite pyrimidines and less affinity for duplexes where the 8-OxoG was opposite purines . KDapp values were 0.6 nM (*G/C), 1.0 nM (*G/T), 6.0 nM (*G/G) and 16.0 nM (*G/A) . The Fpg protein also binds to unmodified (G/C) duplex and a KDapp of 90 nM was measured . The cleavage and binding of the (*G/C) duplex were also studied using bacterial crude lysates . Wild type E . coli crude extract incised the 8-OxoG containing strand and formed a specific retardation complex with the (*G/C) duplex . These two reactions were mediated by the Fpg protein, since they were not observed with a crude extract from a bacterial strain whose fpg gene was inactivated . Furthermore, we have studied the properties of 6 mutant Fpg proteins with Cys-->Gly mutations . The results showed that the 2 Fpg proteins with Cys-->Gly mutations outside the zinc finger sequence cleaved the 8-OxoG containing strand, formed complexes with the (*G/C) duplex and suppressed the mutator phenotype of the fpg-1 mutant . In contrast, the 4 Fpg proteins with Cys-->Gly mutations within the zinc finger motif neither cleave nor bind the (*G/C) duplex, nor do these proteins suppress the fpg-1 mutator phenotype.

Biochim Biophys Acta, 1993 Jun 25, 1173(3), 289 - 93
Synthesis of mammalian prolylendopeptidase in Escherichia coli and analysis of the recombinant protein; Sommer J; Prolylendopeptidase is a cytoplasmic serine proteinase which hydrolyses peptide bonds at the C-terminal side of prolines . Here, the expression in Escherichia coli of the cDNA coding for the enzyme from porcine brain is reported . Furthermore, the purification of active prolylendopeptidase from the extract of recombinant bacterial cells is described . The large amounts of protein obtained were used to gain insight in the substrate specificity of recombinant prolylendopeptidase, by using internally quenched fluorescent substrates.

J Biol Chem, 1993 Jun 25, 268(18), 13253 - 60
Selective decay of Escherichia coli dnaG messenger RNA is initiated by RNase E; Yajnik V et al.; The rpsU-dnaG-rpoD operon messenger RNA that encodes S21, primase, and sigma-70 is cleaved under normal physiological conditions . The dnaG coding portion of the mRNA is then rapidly degraded . An endonuclease activity has been isolated from wild type Escherichia coli cells that cleaves dnaG mRNA . This activity has been identified as RNase E, and the identity confirmed by the accumulation of the unprocessed operon polycistronic mRNA in RNase E mutants, rne-3071 and ams-1, when incubated at nonpermissive temperatures . Extracts prepared from RNase E mutant strains failed to cleave dnaG mRNA in vitro . The dnaG mRNA RNase E cleavage site (5'-AAGUGAUUUA-3') is only 50% homologous to the ribosomal RNA RNase E cleavage site . By using computer programs predicting secondary structure, the dnaG RNase E cleavage site appears to be in a single stranded RNA loop.

Nature, 1993 Jun 24, 363(6431), 744 - 7
Functional dissection of TFIIB domains required for TFIIB-TFIID-promoter complex formation and basal transcription activity; Hisatake K et al.; The protein TFIIB is a general transcription initiation factor that interacts with a promoter complex (D.DNA) containing the TATA-binding subunit (TFIID tau, or TBP) of TFIID to facilitate subsequent interaction with RNA polymerase II (ref . 2) through the associated TFIIF (ref . 3) . The potential bridging function of TFIIB raises the possibility of two structural domains and emphasizes the importance of TFIIB structure-function studies for a further understanding of preinitiation complex assembly and function . Here we show that human TFIIB (refs 5,6) is comprised of functionally distinct N- and C-terminal domains . The C-terminal domain, containing the direct repeats and associated basic regions, is necessary and sufficient for interaction with the D.DNA complex . By contrast, the N-terminal domain that is dispensable for formation of the TFIID tau-TFIIB-promoter (D.B.DNA) complex is required for subsequent events leading to basal transcription initiation . On the basis of these results, we discuss structural and functional similarities between TFIIB and TFIID tau, which have similar structural organization and motifs.

Nature, 1993 Jun 24, 363(6431), 722 - 4
Characterization and localization of the FMR-1 gene product associated with fragile X syndrome; Verheij C et al.; The fragile X syndrome is the most frequent form of inherited mental retardation after Down's syndrome, having an incidence of one in 1,250 males . The fragile X syndrome results from amplification of the CGG repeat found in the FMR-1 gene . This CGG repeat shows length variation in normal individuals and is increased significantly in both carriers and patients; it is located 250 base pairs distal to a CpG island which is hypermethylated in fragile X patients . The methylation probably results in downregulation of FMR-1 gene expression . No information can be deduced about the function of the FMR-1 protein from its predicted sequence . Here we investigate the nature and function of the protein encoded by the FMR-1 gene using polyclonal antibodies raised against the predicted amino-acid sequences . Four different protein products, possibly resulting from alternative splicing, have been identified by immunoblotting in lymphoblastoid cell lines of healthy individuals . All these proteins were missing in cell lines from patients not expressing FMR-1 messenger RNA . The intracellular localization of the FMR-1 gene products was investigated by transient expression in COS-1 cells and found to be cytoplasmic . Localization was also predominantly cytoplasmic in the epithelium of the oesophagus, but in some cells was obviously nuclear.

Biochim Biophys Acta, 1993 Jun 24, 1164(1), 36 - 46
Improved synthetic methods for the selective deuteration of aromatic amino acids: applications of selective protonation towards the identification of protein folding intermediates through nuclear magnetic resonance; Wishart DS et al.; In this report we describe several novel methods for the preparation of selectively deuterated aromatic amino acids . New syntheses for {2,3,5,6-2H4}phenylalanine and {2,4,6,7-2H4}tryptophan, as well as improved catalytic exchange methods for {2,3,5,6-2H4}tyrosine and {2,3,4,5,6-2H5}phenylalanine are presented . Isotopic substitution levels for all compounds are generally found to be greater than 95% . Biosynthetic incorporation of these amino acids is also shown to be possible with little or no evidence of isotopic scrambling . The products from these new syntheses, in combination with other selectively deuterated aromatic amino acids, are found to permit group-specific 'single-proton' labelling of proteins . This highly-efficient and very cost-effective method of selective protonation is shown to produce greatly simplified 1H-NMR spectra of the aromatic region of proteins . The utility of this approach to isotopic editing is demonstrated with the identification of a transient folding intermediate of Escherichia coli thioredoxin which is undetectable by standard 2-D NMR techniques.

Biochemistry, 1993 Jun 22, 32(24), 6206 - 13
Environmental influences on the in vivo level of intramolecular triplex DNA in Escherichia coli; Ussery DW et al.; We describe an assay for detecting intramolecular triple-stranded DNA in living cells . The assay involves quantitative analysis of the differential photobinding of 4,5',8-trimethylpsoralen to 5'TA and 5'AT dinucleotides in a region of plasmid DNA containing the triplex-forming sequence (GA)7TA(GA)7 . Psoralen photobinds to the central 5'TA within the (GA)7TA(GA)7 sequence in duplex DNA but not when the sequence exists as an intramolecular triplex structure . The reactivity of photobinding sites in regions flanking the intramolecular triplex-forming sequence, those that comprise the duplex-triplex junctions, either increase or decrease upon formation of the intramolecular triplex structure from duplex DNA . The pattern of trimethylpsoralen reactivity provides an indication of the conformation of the (GA)7TA(GA)7 sequence in plasmid DNA . The formation of the intramolecular triplex structure is dependent on both pH and negative superhelical density in vitro . The fraction of the (GA)7TA(GA)7 sequence that existed as an intramolecular triplex structure in vivo was dependent on the level of DNA supercoiling in vivo and changes in growth conditions which influence the intracellular pH . The Hy3 isomer was detected in living Escherichia coli cells.

Biochemistry, 1993 Jun 22, 32(24), 6134 - 40
Promoter recognition by Escherichia coli RNA polymerase . Effects of single base pair deletions and insertions in the spacer DNA separating the -10 and -35 regions are dependent on spacer DNA sequence; Warne SE et al.; Escherichia coli RNA polymerase contacts promoter DNA at two upstream regions separated by a spacer DNA . We had previously studied the effects of substitutions of simple DNA sequences in a stretch of the spacer DNA devoid of any known specific contacts with RNA polymerase . It was found that substitution of nine consecutive nonalternating dG-dC base pairs, but not nine alternating dG-dC base pairs, impaired promoter function . We proposed that this effect was due to the fact that the oligo(dG)-oligo(dC) sequence adopted a conformation (possibly A-helical) resulting in a reduction in its length and twist as compared with the B-form DNA of the alternating sequence . Here we test this hypothesis by combining the substitutions with single base pair insertions and deletions in the spacer DNA, which affect the length and the twist in known ways . Deletion and substitutions equally affect the activities of promoters with the presumed B-DNA substitutions . However, for promoters bearing the oligo(dG)-oligo(dC) substitution, a deletion in the spacer DNA impairs promoter activity to a much greater extent than the insertion of a base pair . This asymmetry is consistent with our hypothesis that the deleterious effects of the substitution are due to its having the reduced twist and/or length characteristic of A-DNA . Additionally, we present data that concern the sequence requirements for adoption of this structure that leads to reduced promoter function.

Biochemistry, 1993 Jun 22, 32(24), 6171 - 8
Stabilization of Escherichia coli ribonuclease HI by cavity-filling mutations within a hydrophobic core; Ishikawa K et al.; The crystal structure of Escherichia coli ribonuclease HI has a cavity near Val-74 within the protein core . In order to fill the cavity space, we constructed two mutant proteins, V74L and V74I, in which Val-74 was replaced with either Leu or Ile, respectively . The mutant proteins are stabilized, as revealed by a 2.1-3.7 degrees C increase in the Tm values, as compared to the wild-type protein at pH values of 3.0 and 5.5 . The mutant protein V74A, in which Val-74 is replaced with Ala, was also constructed to analyze the reverse effect . The stability of V74A decreases by 7.6 degrees C at pH 3.0 and 12.7 degrees C at pH 5.5 in Tm as compared to those values for the wild-type protein . None of the three mutations significantly affect the enzymatic activity . The crystal structures of V74L and V74I, determined at 1.8-A resolution, are almost identical to that of the wild-type protein, except for the mutation site . In the two mutant proteins, calculation by the Voronoi procedure shows that the cavity volumes around the individual mutation sites are remarkably reduced as compared to that in the wild-type protein . These results indicate that the introduction of a methylene group into the cavity, without causing steric clash, contributes to an increase in the hydrophobic interaction within the protein core and thereby enhances protein stability . We also discuss the role of the Leu side chain, which can assume many different local conformations on a helix without sacrificing thermostability.

FEBS Lett, 1993 Jun 21, 324(3), 361 - 6
The KH domain occurs in a diverse set of RNA-binding proteins that include the antiterminator NusA and is probably involved in binding to nucleic acid; Gibson TJ et al.; New findings are presented for the approximately 50 residue KH motif, a domain recently discovered in RNA-binding proteins . The conserved sequence is approximately 10 residues larger than previously reported . Profile searches have revealed new members of this family, including two, E . coli NusA and human GAP-associated p62 phosphoprotein, for which RNA-binding data exists . A nusA homolog was detected in the RNA polymerase gene complex of six archaebacterial species and may encode an antiterminator . All KH-containing proteins are linked with RNA and the KH motif most probably functions as a nucleic acid binding domain.

FEBS Lett, 1993 Jun 21, 324(3), 247 - 52
cDNA cloning and expression of cysteine synthase B localized in chloroplasts of Spinacia oleracea; Saito K et al.; The cDNA clones for cysteine synthase B, which is localized in chloroplasts of Spinacia oleracea L., were isolated by screening a library with synthetic oligonucleotides encoding a partial peptide sequence of the purified protein . Nucleotide sequence analysis revealed an open reading frame encoding a polypeptide of 383 amino acids containing a putative transit peptide of 52 amino acids . A bacterial expression vector of the cDNA clone could genetically complement an Escherichia coli auxotroph lacking cysteine synthase and could produce the functionally active and immuno-reactive cysteine synthase in E . coli . RNA blot hybridization suggested that the transcripts were primarily accumulated in leaves of spinach.

Ann N Y Acad Sci, 1993 Jun 21, 681, 219 - 37
Approaches for studying the pathogenic T cells in autoimmune patients; Willcox N et al.; Our provisional conclusions from this work are as follows . (1) For screening responses of established lines, native human AChR is not prohibitively scarce, especially if it is concentrated onto beads, and class II-transfected TE671 cells may be useful too; both may give vital evidence of AChR-specificity, but it is still crucial to confirm that with synthetic peptides . (2) For mapping epitopes, panels of full-length and shorter recombinant human polypeptides, and of synthetic peptides, are invaluable complementary material: longer peptides tend to stimulate particularly strongly . (3) Initial selection with pooled synthetic peptides can easily generate interesting lines from both patients and controls, but they may depend on the artificial processing sites that are an inevitable consequence of arbitrarily chosen start and stop points . Of course, these might conceivably be employed in unusual antigen-presenting cells (such as thymic myoid cells), so we cannot totally dismiss such "cryptic" epitopes . This system can sometimes select T cells responding to "natural" epitopes too, as now reported for tetanus toxin . Nevertheless, for these and other reasons, at present, we strongly favor using the longest human recombinant material possible, because it is apparently processed more naturally . This must be combined with rigorous screening for reactivity to E . coli-derived contaminants plus concomitant mapping of epitopes as above . Use of intact AChR for initiating lines may yet become feasible . (4) The T cells thus isolated and characterized so far are proving to be heterogeneous in the epitopes and presenting class II molecules they recognize, and in their T-cell receptor gene usage . It is premature to claim key myasthenogenic epitopes or clonotypes, but HLA-DR3 and the linked -DQw2 do not appear to monopolize presentation . (5) Assessing the disease-relevance of these T cells is a separate problem, highlighted by their apparent similarity in healthy controls . In the meantime, to test their potential pathogenicity, we are assaying their cytokine profiles and ability to help specific antibody production in vitro . In the hope that they do prove to be relevant, we are also using some of them to test possible therapeutic strategies that might prove applicable in the patients.

J Mol Biol, 1993 Jun 20, 231(4), 950 - 9
Factors influencing the repair of the mutagenic lesion O6-methylguanine in DNA by human O6-methylguanine-DNA methyltransferase; Liem LK et al.; Oligodeoxynucleotides of various chain lengths (p(Bp)nB, n < or = 9) and the eight possible dinucleotide phosphates (pm6GpB and pBpm6G), each containing a single O6-methylguanine residue (m6G), were used to study the repair kinetics of this lesion by the cloned DNA repair proteins; human 21 kDa O6-methylguanine-DNA methyltransferase (MGMT), human 43 kDa glutathione-S-transferase fused MGMT (GSTMGMT) and the Escherichia coli 39 kDa ada protein . The observed second-order repair rate constants are dependent upon both the chain length of the oligonucleotide substrates for all three proteins and in the case assuming O6-methylguanine is similar to B) . The differences observed in the ratios of the rate constants for the substrates with five and four base residues; 125 for the E . coli 39 kDa ada protein, 640 for the human MGMT and 27,800 for the human fusion protein GSTMGMT, suggest that the pentanucleotide phosphate containing this lesion is the "optimal" substrate for the proteins . Surprisingly, the human GSTMGMT is shown to be more effective in the repair of longer substrates with the second-order repair rate constants for TATA-Cm6GTATA being 6.16 x 10(6) for GSTMGMT, 2.00 x 10(6) for MGMT and 0.27 x 10(6) M-1 s-1 for the E . coli 39 kDa ada protein . Thus, the presence of an additional protein domain at the N terminus of human MGMT can alter its selectivity towards certain substrates . Although a number of peptide domains are conserved between the E . coli 39 kDa ada protein and phosphates can also be used to explain the observed sequence specific repair of this lesion within certain DNA sequences.

J Mol Biol, 1993 Jun 20, 231(4), 1122 - 5
Crystallization and preliminary crystallographic analysis of Escherichia coli DNA photolyase; Park HW et al.; DNA photolyase from Escherichia coli (M(r) 54,000) consists of a polypeptide chain of 471 amino acids and the non-covalently bound cofactors methenyltetrahydrofolate (MTHF) and flavin adenine dinucleotide (FADH2) . Two crystal forms of the enzyme were obtained; both have symmetry of space group P1 . Form I has the unit cell dimensions a = 89.4 A, b = 97.3 A, c = 62.1 A, alpha = 108.3 degrees, beta = 97.4 degrees and gamma = 90.0 degrees . Diffraction from this form extends beyond 3 A resolution, but the crystals are radiation-sensitive and difficult to reproduce . Form II has the unit cell dimensions a = 62.6 A, b = 72.2 A, c = 58.5 A, alpha = 99.1 degrees, beta = 101.5 degrees and gamma = 72.0 degrees; most likely, the unit cell contains two molecules . High diffraction quality and reproducibility make form II suitable for structure analysis.

J Mol Biol, 1993 Jun 20, 231(4), 1078 - 89
Methionyl-tRNA synthetase zinc binding domain . Three-dimensional structure and homology with rubredoxin and gag retroviral proteins; Fourmy D et al.; Methionyl-tRNA synthetase from Escherichia coli contains one tightly bound zinc atom per subunit . The region encompassing residues 138 to 163 of this enzyme is responsible for the metal binding . A 28-mer peptide corresponding to these residues was expressed in vivo and shown to contain approximately 1 mol of tightly bound Zn/mol of peptide . In this study, the three-dimensional solution structure of this peptide was solved by means of two-dimensional proton NMR spectroscopy . A total of 133 nuclear Overhauser effect distance constraints and 22 dihedral angle restraints were used for the calculations, using a hybrid distance-geometry-simulated annealing strategy . Excluding the first four residues, the resulting structure is well-defined (r.m.s.d . 0.71 A for backbone atoms) and composed of a series of four tight turns . The second and the fourth turns are composed of CXXC sequences which are structurally homologous to the NH-S turns found in the metal binding sites of gag retroviral proteins and rubredoxin . The solution structure of the zinc binding peptide shows significant discrepancies with the crystal structure of methionyl-tRNA synthetase.

J Mol Biol, 1993 Jun 20, 231(4), 1068 - 77
Mapping of the zinc binding domain of Escherichia coli methionyl-tRNA synthetase; Fourmy D et al.; Cys/His motifs, found in several nucleic acid binding proteins, generally correspond to sites for the binding of metal atoms . Such a motif, comprising four Cys residues, occurs in the subunits of Escherichia coli methionyl-tRNA synthetase, a dimeric enzyme known to bind two zinc atoms . In this study, each of the four cysteines in the cysteine cluster (region 145 to 161) of E . coli methionyl-tRNA synthetase were successively changed into an alanine . Either substitution is sufficient to destabilize the tight binding of the zinc ion . Moreover, a peptide having a sequence corresponding to that of the 138 to 163 region of methionyl-tRNA synthetase has been prepared . It strongly binds one zinc atom, even in the presence of ethylene diamine tetraacetate . These data establish that, in methionyl-tRNA synthetase, the Cys motif of region 145 to 161 is actually the binding site for zinc . In addition, the mutation of each cysteine modifies the parameters of the methionine activation reaction, and appears to change the structure of the enzyme, as probed by an increased sensitivity of the mutant enzymes to trypsin attack . The possible role of the zinc atom and of its chelating residues in the folding of the active centre of methionyl-tRNA synthetase is discussed.

Cas Lek Cesk, 1993 Jun 20, 132(14), 434 - 6
{Thrombosis of the inferior vena cava--an unusual cause of a chronic septic condition in a female patient with type 1 diabetes mellitus}; Klein D et al.; The authors describe the accidental detection of a clinically not very marked thrombosis of the vena cava inferior in a 29-year-old female diabetic which was the cause of permanent bacteriaemia and the source of embolization of the lungs . The patient was delivered of an infant six years previously by Caesarean section, subsequently complicated by a paranephritic abscess on the right which called for surgical treatment . Since then the patient suffered from frequent dermal infections and was dyspnoic . Because of deterioration of polyneuropathic complaints of the lower extremities resulting from diabetes the patient was admitted to the authors' clinic . Sonographic examination of the abdominal cavity aroused urgent suspicion of a thrombus in the vena cava inferior, as confirmed by subsequent cavography and computed tomography . The patient was treated for prolonged periods with anticoagulants and antibiotics . During the subsequent check-up examination after four months marked diminution of the thrombus was recorded and improvement of the patient's general condition.

Cell, 1993 Jun 18, 73(6), 1165 - 73
LexA and lambda Cl repressors as enzymes: specific cleavage in an intermolecular reaction; Kim B et al.; During the SOS response, LexA repressor is inactivated by specific cleavage . Although cleavage requires RecA protein in vivo, RecA acts indirectly as a coprotease by stimulating an inherent self-cleavage activity of LexA . In lambda lysogens, cleavage of lambda Cl repressor in a similar but far slower reaction results in prophage induction . We describe an intermolecular cleavage reaction in which the C-terminal fragment of LexA acted as an enzyme to cleave other molecules of LexA . The C-terminal fragment of lambda repressor cleaved the LexA substrates about as efficiently as did the LexA enzyme, suggesting that the slow rate of Cl self-cleavage results from a weak interaction between its cleavage site and the active site.

Science, 1993 Jun 18, 260(5115), 1773 - 7
From RNA to DNA, why so many ribonucleotide reductases?
Reichard P.
It is generally accepted that DNA appeared after RNA during the chemical evolution of life . To synthesize DNA, deoxyribonucleotides are required as building blocks . At present, these are formed from the corresponding ribonucleotides through the enzymatic action of ribonucleotide reductases . Three classes of enzymes are present in various organisms . There is little sequence similarity among the three classes of reductases . However, enzymic mechanisms and the allosteric behavior of the enzymes from various organisms are strongly conserved, suggesting that the enzymes might have evolved from a common ancestor, with the class III anaerobic Escherichia coli reductase as its closest relative.

Biochim Biophys Acta, 1993 Jun 18, 1149(1), 29 - 39
Interaction of immunologically-active lipopeptides with membranes; Metzger JW et al.; Synthetic tripalmitoyl-S-glycerylcysteinyl (Pam3Cys) peptides are derived from the N-terminal part of bacterial lipoprotein and constitute polyclonal B-lymphocyte and macrophage activators . In order to elucidate the primary events of leukocyte activation, we investigated the biophysical interaction of lipopeptides containing spin labels or fluorescent markers with phosphatidylcholine vesicles or immune cells . Utilizing fluorescence microscopy and FACS analysis we found, that the surface of cells, after incubation with a fluorescein-labelled lipopeptide, was highly fluorescent . In addition, capping and patching was observed . Furthermore, fluorescence quenching experiments and electron paramagnetic resonance studies using vesicles incubated with lipopeptides suggested, that the peptide moiety and other more polar molecules linked to the lipo-amino acid are exposed to the hydrophilic compartment . These results show that in lipopeptide conjugates the Pam3Cys moiety acts as an efficient membrane anchor for molecules covalently coupled to it . The sequestering of the fatty-acid chains of the lipopeptide within the membrane is an early step of interaction, which might induce the uptake of the lipopeptide into the cell and the stimulation of immunocompetent cells.

Biochim Biophys Acta, 1993 Jun 18, 1149(1), 151 - 65
Antibody epitope mapping of the gastric H+/K(+)-ATPase; Mercier F et al.; Several antibodies against the gastric H+/K(+)-ATPase were analysed for the topological and sequence location of their epitopes . Topological mapping was done by comparing indirect immunofluorescent staining in intact and permeabilised rat parietal cells . Epitope definition was by Western analysis of intact and of trypsin or V8-proteinase-fragmented hog gastric ATPase combined with N-terminal sequencing of the fragments; by Western analysis of fragments of rabbit alpha subunit expressed in Escherichia coli; by analysis of rabbit alpha and beta subunits expressed in baculovirus-transfected SF 9 cells and by ELISA assay of synthetic octamers of one region of the hog alpha subunit . It was confirmed that the monoclonal antibody, mAb 95-111, recognised a cytoplasmic region between M4 and M5, close to the ATP-binding domain . The major epitope for monoclonal antibody mAb 12-18 was also in this region, but a second epitope was confirmed to be present in the M7/M8 region . The monoclonal antibody, mAb 146-14, was shown to recognise an extracytoplasmic epitope dependent on intact disulfide bonds, present in the rat and the rabbit, but absent in the hog beta subunit, due to non-conservative amino-acid substitutions . This antibody also recognised an epitope present in the alpha subunit of the H+/K(+)-ATPase at the M7 extracytoplasmic interface, perhaps indicating structural association of these two regions . The polyclonal antibody, pAb39, raised against the C-terminal portion of the enzyme, reacted only with the cytoplasmic surface of the H+/K(+)-ATPase, showing that the alpha subunit of the enzyme has an even number of membrane spanning segments.

Nature, 1993 Jun 17, 363(6430), 648 - 52
New eukaryotic transcriptional repressors; Saha S et al.; Transcriptional activating sequences have been described that are encoded by parts of the genome of Escherichia coli . These acidic peptides, fused to a DNA-binding fragment of the yeast transcriptional activator GAL4, activate transcription of a gene in a wide array of eukaryotes, provided that gene bears GAL4-binding sites nearby . Here we describe an E . coli-encoded sequence that, when attached to the same DNA-binding fragment (GAL4(1-147)), converts that fragment into a repressor . Thus, as assayed in yeast or in vitro in yeast extracts, this molecule represses transcription when bound upstream of a variety of different activators . Two additional repressing regions that work when tethered upstream, a multiple mutant derivative of the original isolate and a synthetic peptide are, like the original isolate, highly basic . At least one activator can be inhibited by the mutant but not by the parental repressing region . These and other findings suggest that these repressing regions interact with and inhibit the activity of activating regions bound nearby on DNA.

Nature, 1993 Jun 17, 363(6430), 640 - 4
Fusion of CHOP to a novel RNA-binding protein in human myxoid liposarcoma; Crozat A et al.; Human myxoid liposarcomas contain a characteristic chromosomal translocation, t(12;16)(q13;p11), that is associated with a structural rearrangement of the gene encoding CHOP, a growth arrest and DNA-damage inducible member of the C/EBP family of transcription factors residing on 12q13.1 . Using a CHOP-specific complementary probe and antiserum we report here the presence of an abnormal CHOP transcript and protein in these tumours . Cloning of the translocation-associated CHOP gene product revealed a fusion between CHOP and a gene provisionally named TLS (translocated in liposarcoma) . TLS is a novel nuclear RNA-binding protein with extensive sequence similarity to EWS, the product of a gene commonly translocated in Ewing's sarcoma . In TLS-CHOP the RNA-binding domain of TLS is replaced by the DNA-binding and leucine zipper dimerization domain of CHOP . Targeting of a conserved effector domain of RNA-binding proteins to DNA may play a role in tumour formation.

Proc Natl Acad Sci U S A, 1993 Jun 15, 90(12), 5863 - 7
Spectroscopic evidence for a heme-heme binuclear center in the cytochrome bd ubiquinol oxidase from Escherichia coli; Hill JJ et al.; The cytochrome bd complex is a ubiquinol oxidase, which is part of the aerobic respiratory chain of Escherichia coli . This enzyme is structurally unrelated to the heme-Cu oxidases such as cytochrome c oxidase . While the cytochrome bd complex contains no copper, it does have three heme prosthetic groups: heme b558, heme b595, and heme d (a chlorin) . Heme b558 appears to be involved in the oxidation of quinol, and heme d is known to be the site where oxygen binds and is reduced to water . The role of heme b595, which is high spin, is not known . In this paper, CO is used to probe the oxygen-binding site by use of Fourier transform infrared spectroscopy to monitor the stretching frequency of CO bound to the enzyme . Photodissociation at low temperature (e.g., 20 K) of the CO-heme d adduct results in CO associated with the protein within the heme binding pocket . This photodissociated CO can subsequently relax to form a kinetically trapped CO-heme b595 adduct . The data clearly show that heme d and heme b595 must reside within a common binding pocket in the enzyme . The catalytic active site where oxygen is reduced to water is, thus, properly considered to be a heme d-heme b595 binuclear center . This is analogous to the heme alpha 3-Cu(B) binuclear center in the heme-Cu oxidases . Heme b595 may play roles analogous to those proposed for the Cu(B) component of cytochrome c oxidase.

Proc Natl Acad Sci U S A, 1993 Jun 15, 90(12), 5796 - 800
A stationary-phase protein of Escherichia coli that affects the mode of association between the trp repressor protein and operator-bearing DNA; Yang W et al.; Highly purified preparations of trp repressor (TrpR) protein derived from Escherichia coli strains that were engineered to overexpress this material were found to contain another protein, of 21 kDa . The second protein, designated WrbA {for tryptophan (W) repressor-binding protein} remained associated with its namesake through several sequential protein fractionation steps . The N-terminal amino acid sequence of the WrbA protein guided the design of two degenerate oligonucleotides that were used as probes in the cloning of the wrbA gene (198 codons) . The WrbA protein, in purified form, was found by several criteria to enhance the formation and/or stability of noncovalent complexes between TrpR holorepressor and its primary operator targets . The formation of an operator-holorepressor-WrbA ternary complex was demonstrated by gel mobility-shift analysis . The WrbA protein alone does not interact with the trp operator . During the stationary phase, cells deficient in the WrbA protein were less efficient than wild type in their ability to repress the trp promoter . It is proposed that the WrbA protein functions as an accessory element in blocking TrpR-specific transcriptional processes that might be physiologically disadvantageous in the stationary phase of the bacterial life cycle.

Proc Natl Acad Sci U S A, 1993 Jun 15, 90(12), 5791 - 5
Molecular characterization of the 128-kDa immunodominant antigen of Helicobacter pylori associated with cytotoxicity and duodenal ulcer; Covacci A et al.; Helicobacter pylori has been associated with gastritis, peptic ulcer, and gastric adenocarcinoma . We report the nucleotide sequence and expression of an immunodominant antigen of H . pylori and the immune response to the antigen during disease . The antigen, named CagA (cytotoxin-associated gene A), is a hydrophilic, surface-exposed protein of 128 kDa produced by most clinical isolates . The size of the cagA gene and its protein varies in different strains by a mechanism that involves duplication of regions within the gene . Clinical isolates that do not produce the antigen do not have the gene and are unable to produce an active vacuolating cytotoxin . An ELISA to detect the immune response against a recombinant fragment of this protein detects 75.3% of patients with gastroduodenal diseases and 100% of patients with duodenal ulcer (P < 0.0005), suggesting that only bacteria harboring this protein are associated with disease.

Proc Natl Acad Sci U S A, 1993 Jun 15, 90(12), 5638 - 42
Functional domains of the AraC protein; Bustos SA et al.; The AraC protein, which regulates the L-arabinose operons in Escherichia coli, was dissected into two domains that function in chimeric proteins . One provides a dimerization capability and binds the ligand arabinose, and the other provides a site-specific DNA-binding capability and activates transcription . In vivo and in vitro experiments showed that a fusion protein consisting of the N-terminal half of the AraC protein and the DNA-binding domain of the LexA repressor dimerizes, binds well to a LexA operator, and represses expression of a LexA operator-beta-galactosidase fusion gene in an arabinose-responsive manner . In vivo and in vitro experiments also showed that a fusion protein consisting of the C-terminal half of the AraC protein and the leucine zipper dimerization domain from the C/EBP transcriptional activator binds to araI and activates transcription from a PBAD promoter-beta-galactosidase fusion gene . Dimerization was necessary for occupancy and activation of the wild-type AraC binding site.

Proc Natl Acad Sci U S A, 1993 Jun 15, 90(12), 5598 - 602
Essential role of the HMG domain in the function of yeast mitochondrial histone HM: functional complementation of HM by the nuclear nonhistone protein NHP6A; Kao LR et al.; The yeast mitochondrial histone protein HM is required for maintenance of the mitochondrial genome, and disruption of the gene encoding HM (HIM1/ABF2) results in formation of a respiration-deficient petite mutant phenotype . HM contains two homologous regions, which share sequence similarity with the eukaryotic nuclear nonhistone protein, HMG-1 . Experiments with various deletion mutants of HM show that a single HMG domain of HM is functional and can restore respiration competency to cells that lack HM protein (him1 mutant cells) . The gene encoding the putative yeast nuclear HMG-1 homolog, the NHP6A protein, can functionally complement the him1 mutation . These results suggest that the HMG domain is the basic unit for the function of HM in mitochondria and that the function of HMG-1 proteins in the nucleus and HM in the mitochondrion may be equivalent.

Proc Natl Acad Sci U S A, 1993 Jun 15, 90(12), 5469 - 73
On the directional specificity of ribosome frameshifting at a "hungry" codon; Lindsley D et al.; Limitation for aminoacyl-tRNA promotes ribosome frameshifting at certain sites . We have previously demonstrated ribosome frameshifting to the right (3') at an AAG site in one context, and to the left (5') at an AAG site in a different context . Here, we demonstrate that the "rightwing" context is largely specific for frameshifting to the right, and the "leftwing" context is largely specific for frameshifting to the left . Analysis of these context rules, and the conversion of a sequence that promotes leftward frameshifting to one that promotes rightward frameshifting, demonstrated here, permits us to define a minimal heptanucleotide sequence sufficient for shiftiness in each direction at an AAG codon whose lysyl-tRNA is in short supply.

Proc Natl Acad Sci U S A, 1993 Jun 15, 90(12), 5433 - 7
Yeast DNA-repair gene RAD14 encodes a zinc metalloprotein with affinity for ultraviolet-damaged DNA; Guzder SN et al.; Xeroderma pigmentosum (XP) patients suffer from a high incidence of skin cancers due to a defect in excision repair of UV light-damaged DNA . Of the seven XP complementation groups, A-G, group A represents a severe and frequent form of the disease . The Saccharomyces cerevisiae RAD14 gene is a homolog of the XP-A correcting (XPAC) gene . Like XP-A cells, rad14-null mutants are defective in the incision step of excision repair of UV-damaged DNA . We have purified RAD14 protein to homogeneity from extract of a yeast strain genetically tailored to overexpress RAD14 . As determined by atomic emission spectroscopy, RAD14 contains one zinc atom . We also show in vitro that RAD14 binds zinc but does not bind other divalent metal ions . In DNA mobility-shift assays, RAD14 binds specifically to UV-damaged DNA . Removal of cyclobutane pyrimidine dimers from damaged DNA by enzymatic photoreactivation has no effect on binding, strongly suggesting that RAD14 recognizes pyrimidine(6-4)pyrimidone photoproduct sites . These findings indicate that RAD14 functions in damage recognition during excision repair.

Proc Natl Acad Sci U S A, 1993 Jun 15, 90(12), 5428 - 32
A double-filter method for nitrocellulose-filter binding: application to protein-nucleic acid interactions; Wong I et al.; Nitrocellulose-filter binding is a powerful technique commonly used to study protein-nucleic acid interactions; however, its utility in quantitative studies is often compromised by its lack of precision . To improve precision and accuracy, we have introduced two modifications to the traditional technique: the use of a 96-well dot-blot apparatus and the addition of a DEAE membrane beneath the nitrocellulose membrane . Using the dot-blot apparatus, an entire triplicate set of data spanning 20-24 titrant concentrations can be collected on a single 4.5 x 5 inch sheet of nitrocellulose, obviating the need to manipulate separate filters for each titration point . The entire titration can then be quantitated simultaneously with direct two-dimensional beta-emission imaging technology . The DEAE second membrane traps all DNA that does not bind to the nitrocellulose, enabling a direct determination of the total amount of DNA filtered . This measurement improves precision by allowing the amount of DNA retained by the nitrocellulose to be normalized against the total amount of DNA filtered . The DEAE membrane also permits a more accurate quantitation of filter-retention efficiency and nonspecific background retention based on free DNA rather than total DNA filtered . The general approach and methods of analysis to obtain equilibrium binding isotherms are discussed, using as examples our studies of the Escherichia coli Rep protein, a helicase, and its interactions with short oligodeoxynucleotides.

Proc Natl Acad Sci U S A, 1993 Jun 15, 90(12), 5418 - 22
Phosphorylation of the regulatory subunit of type II beta cAMP-dependent protein kinase by cyclin B/p34cdc2 kinase impairs its binding to microtubule-associated protein 2; Keryer G et al.; Subcellular localization of type II cAMP-dependent protein kinase is determined by the interactions of the regulatory subunit (RII) with specific RII-anchoring proteins . By using truncated NH2-terminal RII beta fusion proteins expressed in Escherichia coli and the mitotic protein kinase p34cdc2 isolated from HeLa cells or starfish oocytes, we investigated the in vitro phosphorylation of RII beta by these kinases . The putative site for phosphorylation by the mitotic kinases is Thr-69 in the NH2-terminal domain of RII beta . This phosphorylation site matches the consensus sequence X(T/S)PX(K/R) for p34cdc2 recognition and belongs to a well-conserved sequence found in all RII beta sequences known to date . In contrast to phosphorylation by casein kinase II or the cAMP-dependent protein kinase catalytic subunit, phosphorylation of RII beta by mitotic kinases impaired its interaction with a well-known RII-anchoring protein, the neuronal microtubule-associated protein 2 . The potential regulatory significance of the phosphorylation of this site on the interaction with microtubule-associated protein 2 and other RII-anchoring proteins and the physiological relevance of this cyclin B/p34cdc2 kinase-catalyzed modification of RII beta (or phosphorylation by other proline-directed protein kinases) are discussed.

Biochem Biophys Res Commun, 1993 Jun 15, 193(2), 779 - 86
Cloning and expression of a novel human DNA binding protein, PO-GA; Lu Y et al.; We have cloned a novel human DNA-binding protein from a HeLa cDNA expression library using the cognate DNA binding site of a transcription factor, PO-B . Further hybridization screening with the initial clone produced contiguous cDNA sequence of 4508 bp and a complete open reading frame encoding a 128 kDa protein, PO-GA . Northern analysis revealed a wide distribution of PO-GA mRNA in most human tissue . However, PO-GA mRNA levels were lower in lung and kidney and undetectable in placental tissue . A DNA-binding fragment of PO-GA expressed in E . Coli bound selectively to the PO-B element and other GA-rich double-stranded DNA sequences and to certain single-stranded DNA sequences . PO-GA has regions of homology to E . coli and yeast DNA ligases, and to proteins involved in DNA repair . Thus, in addition to a potential role in transcription, PO-GA may also be involved in DNA repair or replication.

J Biol Chem, 1993 Jun 15, 268(17), 12958 - 63
NADPH inhibits transcription of the Escherichia coli manganese superoxide dismutase gene (sodA) in vitro; Gardner PR et al.; We have previously reported that the thiols glutathione, dithiothreitol, and beta-mercaptoethanol suppress transcription of the Escherichia coli manganese-containing superoxide dismutase gene (sodA) in an in vitro coupled transcription plus translation system (Gardner, P . R., and Fridovich, I . (1987) J . Biol . Chem . 262, 17591-17595) . We now report that NADPH, but not NADH, selectively decreases transcription of sodA in vitro and that an NADPH generating system utilizing glucose 6-phosphate and the corresponding dehydrogenase markedly augments this suppressive effect . A redox buffer containing various ratios of oxidized and reduced glutathione also modulated transcription of sodA thus demonstrating the existence of a redox-sensitive mechanism controlling sodA transcription . Fusion of a 120-base pair fragment, containing 90 base pairs of DNA upstream of the sodA transcription initiation site, to a promoterless galactokinase gene (galK) conferred redox-sensitivity to GalK synthesis . We propose that these redox effects act through a redox-sensitive regulator of sodA and that the anabolic reduction charge, {NADPH}/({NADPH}+{NADP+}), is one cellular signal controlling sodA transcription.

J Biol Chem, 1993 Jun 15, 268(17), 12901 - 11
Messenger RNAs expressed in intestine of adult but not baby rabbits . Isolation of cognate cDNAs and characterization of a novel brush border protein with esterase and phospholipase activity; Boll W et al.; Using a subtractive hybridization method, we have cloned cDNAs corresponding to 10 different mRNAs which share the property of being expressed in the intestine of adult but not baby rabbits . Four could be identified as coding for previously known gene products (sucrase-isomaltase, a glutathione S-transferase, a cytochrome P450, and a long form of ferritin mRNA), while six code for previously unknown proteins . One clone, AdRab-B, codes for a protein of 1458 amino acids, including (i) a putative signal sequence at the NH2 terminus, (ii) four internal repeats, 308-346 amino acids in length, (iii) a hydrophobic stretch near the COOH terminus, which represents a potential membrane anchor, and (iv) a short hydrophilic stretch at the very COOH terminus . The corresponding protein was studied with the aid of antibodies prepared against polypeptides expressed from segments of the cDNA in Escherichia coli . The protein was shown to be proteolytically processed in the intestine (but not when expressed in COS cells) and to be targeted to the brush border membrane of the enterocytes . Finally, the protein was found to have esterase and phospholipase A/lysophospholipase activity.

J Biol Chem, 1993 Jun 15, 268(17), 12812 - 7
Topological analysis of quinoprotein glucose dehydrogenase in Escherichia coli and its ubiquinone-binding site; Yamada M et al.; Topological structure of quinoprotein glucose dehydrogenase in the inner membrane of Escherichia coli was determined by constructing protein fusions with alkaline phosphatase or beta-galactosidase . Analysis of the fusions revealed that the dehydrogenase possesses five membrane-spanning segments, and the N-terminal and C-terminal portions resided at the cytoplasmic and periplasmic side of the membrane, respectively . These results agreed with the hydropathy profile based on its primary structure . The topological structure suggests that the predicted binding site of the prosthetic group pyrroloquinoline quinone is located at the periplasmic side and that the amino acid residues corresponding to those that were presumed to interact with ubiquinone in one subunit of mitochondrial NADH dehydrogenase also occur at the periplasmic side . When the purified glucose dehydrogenase and cytochrome o ubiquinol oxidase were reconstituted together with ubiquinone into liposomes, a membrane potential could be generated by the electron transfer at the site of the ubiquinol oxidase but not of the dehydrogenase . These results suggest that glucose dehydrogenase has a ubiquinone reacting site close to the periplasmic side of the membrane, and thus its electron transfer to ubiquinone appears to be incapable of forming a proton electrochemical gradient across the inner membrane of E . coli.

J Biol Chem, 1993 Jun 15, 268(17), 12787 - 95
Carbohydrate regulation of the rat L-type pyruvate kinase gene requires two nuclear factors: LF-A1 and a member of the c-myc family; Liu Z et al.; Transcription of the L-type pyruvate kinase (L-PK) gene is induced in response to increased carbohydrate metabolism in the liver . We have demonstrated previously that a segment of the 5'-flanking region of the L-PK gene between -183 and -96 is necessary and sufficient for the glucose response in primary hepatocytes . To explore the protein factors that are involved in carbohydrate regulation, we have performed mutational analyses and in vitro binding studies of this segment . Sequences critical for the glucose response were mapped from -171 to -124 . This segment contains the consensus binding sites for two nuclear transcription factors: LF-A1 and MLTF . Both factors are capable of binding to the corresponding L-PK sites in vitro . Mutational and functional analyses indicated that LF-A1 is indeed involved in glucose induction of the L-PK gene . The PK MLTF-like site consists of two imperfect CACGTG motifs, the core binding site for a family of transcription factors related to c-myc . Unexpectedly, mutations in either motif that resulted in defective glucose stimulation retained in vitro binding to MLTF . Furthermore, an authentic MLTF binding site from the adenovirus major late promoter was not functionally interchangeable with the natural sequence . These data indicate that binding of MLTF, in presence of LF-A1, is not capable of supporting the glucose response . Conversion of either imperfect motif to CACGTG within the context of the mutations disrupting the opposite site restored the response to elevated glucose . Thus, the factor that recognizes the PK MLTF-like site and participates in mediating the carbohydrate response of the L-PK gene appears to be a member of the c-myc family distinct from MLTF.

J Biol Chem, 1993 Jun 15, 268(17), 12744 - 8
Production and characterization of recombinant heteropolymers of human ferritin H and L chains; Santambrogio P et al.; Vertebrate ferritins are iron storage proteins composed by 24 subunits of one or more types . The recombinant homopolymers of human ferritin H- and L-type chains differ in iron uptake and in physical stability, but the properties of heteropolymers with various proportions of H- and L-type chains cannot be predicted . Present study shows that unfolded human ferritin H- and L- type chains renature under similar conditions to form homopolymers indistinguishable from the native ones and that, when mixed, the unfolded H and L chains renature to form heteropolymers with restricted heterogeneity and with the expected H:L ratios . Seven of these ferritins with different H:L ratios were analyzed; electrophoretic mobility, immunological reactivity, and stability to guanidine denaturation varied as predicted, based on the homopolymers . In contrast, the rate of iron uptake, monitored by the variation of absorbance at 310 nm, increased in the ferritins that ranged in H chain content from 0 to 35%; further increments in H chains had no additional effect . This finding indicates that, under the present conditions, only a limited number of H chains are needed for the maximum rate of ferritin iron uptake . Variations of L- and H-type chains in vivo may thus have biological relevance.

J Biol Chem, 1993 Jun 15, 268(17), 12730 - 5
Peptide-dependent stimulation of the ATPase activity of the molecular chaperone BiP is the result of conversion of oligomers to active monomers; Blond-Elguindi S et al.; The molecular chaperone BiP purified from bovine liver (bBiP) exhibits a low basal level of ATPase activity that can be stimulated 3-6-fold by synthetic peptides (Flynn, G . C., Chappell, T . G., and Rothman, J . E . (1989) Science 245, 385-390) . By contrast, recombinant murine BiP (rBiP) purified to homogeneity following expression in Escherichia coli exhibits a higher basal level of ATPase activity and is much less stimulated by synthetic peptides . Nondenaturing gel electrophoresis showed that rBiP is predominantly monomeric, while bBiP exists in multiple forms probably corresponding to differentially modified monomeric, dimeric, and higher oligomeric species . Some, but not all, synthetic peptides cause conversion of the oligomeric and modified species of bBiP to a monomeric form . We propose that the peptide-dependent ATPase stimulation observed for BiP reflects the conversion of inactive oligomeric and/or modified species into active monomers.

J Biol Chem, 1993 Jun 15, 268(17), 12504 - 11
The influence of effectors and subunit interactions on Escherichia coli carbamoyl-phosphate synthetase studied by differential scanning calorimetry; Cervera J et al.; Differential scanning calorimetry of Escherichia coli carbamoyl-phosphate synthetase and its isolated large and small subunits reveals in each case an irreversible, kinetically controlled transition, at a temperature 14 degrees C higher for the holoenzyme than for the subunits, indicating dramatic stabilization of the subunits in the heterodimer . The deletion of the COOH-terminal 171 (mutant CarB'2373) or 385 (mutant CarB2177) residues of the large subunit results in more asymmetric transitions at a temperature 7 degrees C lower than for the wild type . The allosteric effectors IMP, UMP, and ornithine induce small reversible transitions at low temperature in the endotherm for the wild-type enzyme, but not for CarB'2373, as expected if the effectors bind in the 171-residue, COOH-terminal region . In contrast, two ligands that bind outside the deleted region, Ap5A (a ligand of both ATP sites) and glycine (an analog of glutamine) decrease and increase, respectively, the stability of the two mutants and of the wild type . The stabilization by glycine requires that the subunits are associated . The results support the implication of the 20-kDa COOH-terminal domain of the large subunit in the allosteric modulation by all the effectors and are consistent with the folding of the large subunit as a pseudohomodimer of its two homologous halves.

J Biol Chem, 1993 Jun 15, 268(17), 12498 - 503
Rabbit liver microsomal endopeptidase with substrate specificity for processing proproteins is structurally related to rat testes metalloendopeptidase 24.15; Kawabata S et al.; The detergent extract of rabbit liver microsomes contains an endopeptidase (MEP) with substrate specificity for peptides containing Arg residues at the P1 and P4 positions in the cleavage site (Kawabata, S., and Davie, E . W . (1992) J . Biol . Chem . 267, 10331-10336) . These sequences occur in many proproteins such as the vitamin K-dependent proproteins and prohormones . A cDNA coding for MEP has been obtained from three overlapping clones isolated from two rabbit liver lambda gt10 cDNA libraries . The longest open reading frame of the 3507-base pair cDNA codes for a protein of 704 amino acids, of which 406 residues were confirmed by amino acid sequence analysis . MEP contains a putative active site of -His-Glu-X-X-His-, which is typical of mammalian zinc metallopeptidases . Based on a hydropathy plot, MEP is a hydrophilic protein with no transmembrane domain and no NH2-terminal signal sequence . Amino acid sequence analysis identified Asn at the three potential N-glycosylation sites in the enzyme, indicating that MEP contains no N-linked sugar . MEP is homologous with rat testes metalloendopeptidase 24.15 (60% identity), rat mitochondrial intermediate peptidase (24% identity), Escherichia coli dipeptidyl carboxypeptidase (25% identity), and the open reading frame YCL57w present in yeast chromosome III (35% identity).

J Biol Chem, 1993 Jun 15, 268(17), 12325 - 33
The involvement of the arginine 17 residue in the active site of the histidine-containing protein, HPr, of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli; Anderson JW et al.; Histidine-containing protein, HPr, of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system has an active site His-15 that is phosphorylated to form N delta 1-P-histidine . The nearby conserved residue, Arg-17, has been replaced by: lysine, histidine, glutamate, glycine, serine, and cysteine . All mutations resulted in impairment of the phosphoacceptor function of HPr with enzyme I: kcat/Km values between 6% (Ser-17) and 0.1% (Glu-17), relative to wild type . Several sugar-specific enzymes II had different responses . Both the Vmax and Km of enzyme IIN-acetylglucosamine were altered, while for enzyme IImannose only Km was affected, except for R17E . For both enzymes, kcat/Km values were between 0.5 and 3%, with R17E being 10-fold lower . Except for R17E, minimal effects were observed for enzyme IImannitol . These results suggest that there are different rate-limiting steps in the enzymes II . Phosphohydrolysis properties and the pKa values for His-15 and phosphorylated His-15 determined by NMR for both wild type and mutant HPrs suggest that Arg-17 is partly responsible for the instability of P-His-15 and the depressed pKa values in wild type HPr . Other feature(s) of the tertiary structure influence the protonation of His-15 and the phosphohydrolysis properties of phosphorylated His-15.

J Biol Chem, 1993 Jun 15, 268(17), 12282 - 8
Structural analysis of the purine repressor, an Escherichia coli DNA-binding protein; Schumacher MA et al.; The purine repressor protein, PurR, is a member of the lac repressor, LacI, family of Escherichia coli DNA-binding proteins that bind DNA via a highly conserved N-terminal helix-turn-helix motif . Additionally, the members of this family display strong sequence homologies between their larger C-terminal effector binding/oligomerization domains . Analysis of the PurR primary and secondary structures reveals that its C-terminal corepressor binding domain is highly homologous to another group of E . coli-binding proteins, the periplasmic binding proteins, particularly to the ribose-binding protein (RBP) . The high resolution x-ray structure of RBP allows this protein to serve as a template with which to model the predicted secondary structure of the corepressor binding domain of PurR . Similarly, the N-terminal DNA binding domain of PurR can be modeled using the NMR-determined structure of the corresponding region (residues 1-56) from LacI as a template . Combining the two, results in a description of the likely secondary structure topology of PurR and implicates residues important for corepressor binding and dimerization . CD spectroscopic studies on PurR, its corepressor binding domain and RBP result in secondary structure estimates nearly identical with those obtained by sequence analyses, thereby providing further corroborating physical evidence for this topological assignment.

J Biol Chem, 1993 Jun 15, 268(17), 12250 - 2
The aleu207-->arg mutation in F1F0-ATP synthase from Escherichia coli . A model for human mitochondrial disease; Hartzog PE et al.; The mitochondrial ATPase 6 gene encodes a subunit of F1F0 adenosine triphosphate (ATP) synthase . A mutation in the ATPase 6 gene has been genetically linked to two maternally inherited genetic diseases: neurological muscle weakness, ataxia, and retinitis pigmentosa (NARP) and certain cases of subacute necrotizing encephalopathy (SNE) . Although the severity of both NARP and SNE disease were correlated with the quantity of the ATPase 6leu156-->arg mutation in each patient, the mutation could not be shown to alter F1F0-ATP synthase activity . To investigate the biochemical effects of the ATPase 6leu156-->arg mutation on F1F0-ATP synthase, the aleu207-->arg mutation was constructed in the F1F0-ATP synthase from Escherichia coli to serve as a model for the disease mutation . Characterization of the model bacterial enzyme revealed that the mutation abolishes detectable ATP synthesis via oxidative phosphorylation . The aleu207-->arg mutation results in a structural perturbation blocking proton translocation through F1F0-ATP synthase . The results suggest that a structural defect in human F1F0-ATP synthase is the biochemical basis for NARP and SNE.

Gene, 1993 Jun 15, 128(1), 135 - 40
Functional display of human plasminogen-activator inhibitor 1 (PAI-1) on phages: novel perspectives for structure-function analysis by error-prone DNA synthesis; Pannekoek H et al.; The synthesis of the human plasminogen-activator inhibitor 1 (PAI-1) protein in the cytoplasm of transformed Escherichia coli cells results in inactive protein preparations that can be activated by denaturation and renaturation . We have used the phagemid pComb3, designed for combinatorial immunoglobulin repertoire cloning, for routing of PAI-1 to the periplasm and subsequent exposure on the surface of filamentous phages . Phage-displayed PAI-1 specifically binds to immobilized polyclonal and monoclonal anti-human PAI-1 antibodies . In addition, PAI-1 retains its capacity to form equimolar complexes with its target serine protease tissue-type plasminogen activator (t-PA), as well as its ability to inhibit t-PA activity . Finally, we have explored and manipulated the error-prone property of TaqI DNA polymerase during PCR amplification of the full-length PAI-1 cDNA to generate a large library of predominantly single, random PAI-1 mutants . In addition, a computer simulation program has been devised that converts the number of mutations per codogenic region (in this case PAI-1) into actual mutant proteins . The PAI-1-phage mutant library is composed of 46% single and 34% double mutants and 20% wild-type PAI-1 and can be employed to isolate mutants defective in interactions of PAI-1 with other components . The method described here is applicable to other studies on the structure-function analysis of eukaryotic proteins.

Gene, 1993 Jun 15, 128(1), 13 - 7
Biochemical diversity in a phage display library of random decapeptides; DeGraaf ME et al.; Detailed knowledge and understanding of the structure and function of biologically important macromolecules is frequently insufficient to permit rational, de novo design of recognition molecules and therapeutics . Traditional drug discovery has, thus, focused on screening and identifying native molecules as drugs or as templates for genetic engineering or organic synthesis . The number and novelty of lead compounds for drug discovery might be expanded significantly, however, by the ability to express and screen large libraries of peptide structures with phage display technologies {Scott and Smith, Science 249 (1990) 386-390} . The significance of such libraries as sources of novel biological ligands will depend in part on the depth and degree of biochemical diversity they comprise . We have prepared a phage display library of greater than 2 x 10(6) individual decapeptides produced as N-terminal fusions to the pIII surface protein of fd filamentous phage . The decapeptides were expressed from a degenerate DNA insert sequence that was chemically synthesized with an equal mixture of all four nucleotide bases at the three positions in each of the ten codons . Fifty-two clones were picked randomly and without prior selection from the population and the sequences of their peptide inserts were determined . Our results confirm and document the broad representation at the primary amino acid sequence level that is expected in a library expressed from random DNA inserts . More significantly, biochemical characterization shows these insert sequences correspond to structures comprising a wide range and combination of isoelectric, hydropathic, and biochemical properties necessary in drug discovery to access a significant percentage of the repertoire of possible peptide structures by affinity or activity screening.

Biochemistry, 1993 Jun 15, 32(23), 6011 - 8
Molecular characterization of a conserved, guanine nucleotide-dependent ADP-ribosylation factor in Drosophila melanogaster; Murtagh JJ Jr et al.; ADP-Ribosylation factors (ARFs) are ubiquitous approximately 20-kDa guanine nucleotide-binding proteins that stimulate cholera toxin-catalyzed ADP-ribosylation in vitro . Because the functional role(s) of ARF in mammalian systems is (are) elusive, we looked for ARF in Drosophila melanogaster, and report the partial purification and molecular cloning of an ARF from Drosophila . We cloned the Drosophila ARF 1 gene without library screening by a combination of 5 polymerase chain reactions (PCRs), yielding a 546-base open reading frame encoding 182 amino acids, which are > 93% identical to those of mammalian class I ARFs . This ARF gene maps to 79F3-6 in the proximal region of the left arm of Drosophila chromosome 3 . The Drosophila ARF1 gene structure, including placement of introns, is highly conserved relative to mammalian class 1 ARF genes . A single ARF mRNA species of 1.8 kb was abundant in all Drosophila body segments . Recombinant Drosophila ARF 1 synthesized in Escherichia coli had biochemical and immunochemical activities similar to those of mammalian ARF . The similarities of sequence and biochemical properties between Drosophila and mammalian ARFs contrast with their differences from Drosophila arl (ARF-like protein), which does not stimulate cholera toxin-catalyzed ADP-ribosylation, and is only approximately 52-56% identical in amino acid sequence to mammalian ARFs.

FEMS Microbiol Lett, 1993 Jun 15, 110(2), 185 - 9
Evidence for protein kinase C stimulation in rat enterocytes pretreated with heat stable enterotoxin of Escherichia coli; Chaudhuri AG et al.; Rat intestinal epithelial cells were isolated and the activity of the calcium- and phospholipid-dependent protein kinase C (PKC) was investigated . The stimulation of activity by Escherichia coli heat stable enterotoxin (STa) was about 5-fold compared to control activity (16.91 +/- 1.69 vs 93.56 +/- 10.40 nmol/mg protein/min) and was dose dependent . Maximum enzyme activity was observed after incubation for 1 min with 6 ng of purified STa . The synergistic effects of calcium, phosphatidylserine and diolein on the enzyme activity were noted both in control and STa-treated cells . Staurosporine, a potent PKC inhibitor, significantly reduced the enzyme activity . Autoradiographic analysis of polyacrylamide gel electrophoresis revealed that pretreatment of the cells with STa also resulted in the phosphorylation of specific membrane proteins each with a molecular mass of 37 kDa, 100 kDa and 140 kDa . However, STa had no direct role on the enzyme activity . Our results, therefore, provide evidence for the involvement of PKC in STa-induced signal transduction in rat enterocytes.

Proc Natl Acad Sci U S A, 1993 Jun 15, 90(12), 5833 - 7
Cloning, heterologous expression, and chromosomal localization of human inositol polyphosphate 1-phosphatase; York JD et al.; Inositol polyphosphate 1-phosphatase, an enzyme in the phosphatidylinositol signaling pathway, catalyzes the hydrolysis of the 1 position phosphate from inositol 1,3,4-trisphosphate and inositol 1,4-bisphosphate . We used a cDNA that encodes bovine inositol polyphosphate 1-phosphatase as a probe to isolate the human counterpart by low-stringency hybridization . The 1.74-kb human cDNA has 341 bp of 5' untranslated region, 180 bp of 3' untranslated region, poly(A)32, and predicts a protein of 399 amino acids . Human and bovine inositol polyphosphate 1-phosphatases show 84% amino acid sequence identity . Northern blot analysis from a variety of human tissues demonstrates that a 1.9-kb mRNA is ubiquitously expressed with highest levels in pancreas and kidney . Several higher molecular weight mRNAs also are expressed in brain, muscle, heart, and liver . We have confirmed the functional identity of the human cDNA by heterologous expression in NIH 3T3 fibroblasts, COS-7 cells and Escherichia coli . Polymerase chain reaction assay of a panel of human-rodent somatic cell hybrid DNA using human inositol polyphosphate 1-phosphatase-specific DNA primers resulted in amplification of a specific product using chromosome 2 DNA as template . Fluorescence in situ hybridization of metaphase chromosomes localizes the gene to chromosome 2 band q32 . The identification of the human inositol polyphosphate 1-phosphatase gene locus provides a target for linkage analysis to identify defects in patients with inherited psychiatric disorders that respond to lithium ions, an inhibitor of the enzyme.

Biochemistry, 1993 Jun 15, 32(23), 6065 - 72
Structure of the heme-copper binuclear center of the cytochrome bo complex of Escherichia coli: EPR and Fourier transform infrared spectroscopic studies; Tsubaki M et al.; The cytochrome bo complex is a terminal quinol oxidase in the aerobic respiratory chain of Escherichia coli and functions as a redox-coupled proton pump . To clarify the structural differences of the binuclear reaction center between the cytochrome bo complex and the mitochondrial cytochrome c oxidase, a combined study using EPR and Fourier transform infrared spectroscopies was carried out . The EPR spectrum of the highly purified cytochrome bo complex in the air-oxidized state showed a broad EPR signal (peak g* = 3.7) from an integer spin system . This confirms the existence of the spin-spin exchange-coupled binuclear site, in which the Feo3+ and CuB2+ centers were bridged by an unknown ligand (X) . Binding of azide at the binuclear site as an ionic modulator weakened the strength of the spin-spin exchange coupling and thus caused a narrowing of the broad EPR signal . Binding of another modulator, formate, at the binuclear site caused the formation of EPR signals at g' = 12 and 2.7, which are very similar to those observed for cytochrome c oxidase . Cyanide replaced the bridging ligand (X) to form an Feo(3+)-C-N-CuB2+ structure in which strong spin-spin exchange coupling is expected, leading to a complete EPR-invisible state . Infrared evidence (a 2146 cm-1 C-N stretching band for the cyanide complex and a 2041 cm-1 azide antisymmetric stretching band for the azide complex) supported the theory that these ligands form bridging structures at the binuclear center, as previously observed for cytochrome c oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)

FEMS Microbiol Lett, 1993 Jun 15, 110(2), 243 - 8
Distribution in the genus Streptomyces of a homolog to nusG, a gene encoding a transcriptional antiterminator; Puttikhunt C et al.; The presence of the vbrA gene encoding the transcriptional antiterminator NusG equivalent protein of Streptomyces virginiae was tested for in 73 Streptomyces species by Southern hybridization . Fifty-five strains (75%) including S . griseus, S . lividans TK-21 and S . coelicolor A3(2) showed clear hybridization signals, indicating wide distribution of vbrA or vbrA homologs in Streptomyces species . With hybridization patterns against 3 different probes, i.e., probes covering vbrA alone, the downstream gene rplK alone, and both vbrA-rplK, the 55 strains were classified into 4 groups . In the groups I, II and III (total 50 strains) vbrA was found to be adjacent to rplK, indicating that the gene arrangement vbrA-rplK is common in Streptomyces and that these genes may constitute a part of gene cluster encoding several components of the transcription and translation apparatus, as in Escherichia coli.

FEMS Microbiol Lett, 1993 Jun 15, 110(2), 223 - 9
Eimeria refractile body proteins contain two potentially functional characteristics: transhydrogenase and carbohydrate transport; Vermeulen AN et al.; cDNA encoding an immunogenic protein from partially sporulated oocysts of Eimeria acervulina was cloned and used to search for the homologous counterpart in Eimeria tenella . Monospecific antibodies were raised against the recombinant expression product . Using these antibodies, the parasite proteins were found to be localised in the refractile bodies . The derived amino-acid sequences were compared by computer using the SWISSPROT protein database . In addition to high homology between the Eimeria species, extensive similarity was found with pyridine nucleotide transhydrogenase from Escherichia coli . Comparison with the sugar signature database also resulted in a possible sugar binding domain present only in the Eimeria proteins . It is possible that the corresponding parasite proteins play a role in the recently discovered mannitol cycle of Eimeria.

Eur J Biochem, 1993 Jun 15, 214(3), 787 - 94
Expression and mutation of soybean beta-amylase in Escherichia coli; Totsuka A et al.; The cDNA clones corresponding to soybean beta-amylase mRNA were isolated and sequenced . The cDNA contained an open-reading frame composed of 496 amino acids . The comparison of the amino acid sequence deduced from the cDNA with the N-terminal peptide sequence from mature enzyme proved that beta-amylase had no leader sequence . Employing the cDNA, the beta-amylase was directly synthesized in Escherichia coli by the expression vector pKK233-2 controlled by the tac promoter . The enzyme activity detected in E . coli lysate drastically increased with a lower cultivation temperature, and the total activity and specific activity of the enzyme in E . coli lysate cultured at 13 degrees C was 130-fold and 280-fold, respectively, the value at 37 degrees C . The enzyme produced in E . coli was purified by the affinity column chromatography of cyclomaltohexaose-immobilized Sepharose 6B . Employing the established expression and purification system of the enzyme, the functional ionizable groups in the active site were searched . His93, involving an imidazole, and Asp348, involving a carboxylate, in the highly conserved regions within the beta-amylases were replaced by Arg (H93R) and Ash (D348N) by site-directed mutagenesis, respectively . All beta-amylases, including the non-mutant and mutant beta-amylases, produced in E . coli exhibited lower Vmax values than that of beta-amylase isolated conventionally from soybean seeds . Especially the Vmax value of {H93R}beta-amylase was reduced drastically compared to that of the non-mutant; however, none of them lost their enzyme activities completely . Therefore, neither His93 nor Asp348 may participate in the catalytic reaction directly.

Eur J Biochem, 1993 Jun 15, 214(3), 695 - 701
Chemical structure of the lipid A of Escherichia coli J-5; Holst O et al.; The lipopolysaccharide, and particularly its lipid A moiety, of the J-5 mutant of Escherichia coli O111 plays a central role in studies on potential induction of cross-reactive and cross-protective antibodies, however, its chemical and antigenic structure was hitherto unknown . Here, the chemical structure of the J-5 lipid A is reported . It is composed of the bisphosphorylated disaccharide beta-D-GlcpN-4-P-(1-6)-alpha-D-GlcpN-1-P which carries four residues of 3-hydroxytetradecanoic acid, one each at positions 2, 3, 2', and 3' . The hydroxyl groups of the acyl residues at 2' and 3' are esterified with dodecanoic and tetradecanoic acid, respectively . The hydroxyl group at C-6' functions in the lipopolysaccharide as the attachment site of the core oligosaccharide . Furthermore, a new method to isolate the hydrophilic backbone, i.e . the 1,4'-bisphosphorylated glucosamine disaccharide, and its structural analysis by 1H-, 13C-, and 31P-NMR spectroscopy, are described, leading to a new and easier strategy in structural analysis of lipid A from bacterial lipopolysaccharides.

Eur J Biochem, 1993 Jun 15, 214(3), 635 - 9
Characterization of the receptor and translocator domains of colicin N; el Kouhen R et al.; Intact colicin N and various colicin derivatives, including a natural fragment lacking the first 36 amino-acid residues, a chymotryptic fragment lacking the first 66 amino acids and a thermolytic fragment comprising residues 183-387, were used to locate the regions involved in colicin-N uptake by sensitive Escherichia coli cells . Two separate domains of the molecule participate in colicin-N entry . Specific binding to OmpF receptor site requires a region located between residues 67-182 . A N-terminal domain, located between residues 17-66, is involved during the translocation step after binding to receptor . Two sub-regions, residues 17-36 and residues 37-36, can be defined in this domain . The location and interactions between these domains are discussed in comparison to other colicins which use similar cell components for their uptake.

Biochem J, 1993 Jun 15, 292 ( Pt 3), 833 - 8
Characterization of phosphorylation sites in the cytoplasmic domain of the 300 kDa mannose-6-phosphate receptor; Rosorius O et al.; The human 300 kDa mannose-6-phosphate receptor (MPR 300) is phosphorylated in vivo at serine residues of its cytoplasmic domain . Two-dimensional separation can resolve tryptic phosphopeptides into four major species . To identify the kinases involved in MPR 300 phosphorylation and the phosphorylation sites the entire coding sequence of the cytoplasmic tail was expressed in Escherichia coli . The isolated cytoplasmic domain was used as a substrate for four purified serine/threonine kinases {casein kinase II (CK II), protein kinase A (PKA), protein kinase C and Ca2+/calmodulin kinase} . All kinases phosphorylate the cytoplasmic tail exclusively on serine residues . Inhibition studies using synthetic peptides, partial sequencing of isolated tryptic phosphopeptides and co-migration with tryptic phosphopeptides from MPR 300 labelled in vivo showed that (i) PKA phosphorylates the cytoplasmic MPR 300 domain at Ser20 and at a non-identified site, neither of which are phosphorylated in vivo, and that (ii) the two sites phosphorylated by CK II in vivo and in vitro are Ser82 and Ser157 . The results indicate that the human MPR 300 is a physiological substrate of either CK II or a related kinase which may play a role in the transport function of MPR 300 and/or interaction with other proteins.

Biochem J, 1993 Jun 15, 292 ( Pt 3), 825 - 32
Expression and characterization of rat kallikrein-binding protein in Escherichia coli; Ma JX et al.; Rat kallikrein-binding protein is a novel serine-proteinase inhibitor that forms a covalent complex with tissue kallikrein . We have purified rat kallikrein-binding protein and cloned the cDNA and the gene encoding rat kallikrein-binding protein {Chao, Chai, Chen, Xiong, Chao, Woodley-Miller, Wang, Lu and Chao (1990) J . Biol . Chem . 265, 16394-16401; Chai, Ma, Murray, Chao and Chao (1991) J . Biol . Chem . 266, 16029-16036} . In the present study, we have expressed rat kallikrein-binding protein in Escherichia coli with a T7-polymerase/promoter expression system . A high level of expression was detected by an e.l.i.s.a . with an average of 24.2 mg of recombinant rat kallikrein-binding protein per 1 of culture . The recombinant protein appeared as a major protein in a crude extract of Escherichia coli on SDS/PAGE . It showed a molecular mass of 43 kDa and was recognized by polyclonal antibody to the native rat kallikrein-binding protein in Western-blot analysis . The recombinant rat kallikrein-binding protein has been purified to apparent homogeneity by DEAE-Sepharose CL-6B, hydroxyapatite Bio-Gel HPHT and Mono P 5/5 column chromatography . The purified recombinant rat kallikrein-binding protein showed immunological identity with the native rat kallikrein-binding protein purified from rat serum, in a specific e.l.i.s.a . To confirm the fidelity of the expression, the N-terminal ten amino acids of the recombinant rat kallikrein-binding protein were sequenced and were shown to match perfectly with those of the native rat kallikrein-binding protein . The purified recombinant rat kallikrein-binding protein formed SDS- and heat-stable complexes with rat tissue kallikrein (rK1) and T-kininogenase (rK10) in vitro, but not with other enzymes in the rat kallikrein gene family, such as tonin (rK2) and S3 protein (rK9), which indicates enzyme-specific binding . The properties of the recombinant rat kallikrein-binding protein including its size, charge, complex formation with target enzymes and immunological characteristics were compared with those of the native protein . This expression system provides a simple way to obtain a large amount of the biologically active recombinant protein, to study structure-function relationships of the rat kallikrein-binding protein and its interaction with its target enzymes.

J Biol Chem, 1993 Jun 15, 268(17), 12837 - 42
Recombinant vaccinia virus K3L gene product prevents activation of double-stranded RNA-dependent, initiation factor 2 alpha-specific protein kinase; Carroll K et al.; Deletion of the vaccinia virus K3L gene, a homologue of the alpha subunit of protein synthesis initiation factor 2, has been reported to reduce the ability of the virus to grow in interferon-treated cells (Beattie, E., Tattaglia, J., and Paoletti, E . (1991) Virology 183, 419-422) . Purified recombinant K3L gene product, pK3r, has potent effects on activation of double-stranded (ds) RNA-dependent, initiation factor-2 alpha (eIF-2 alpha)-specific protein kinase (PKR) in in vitro reactions . Recombinant pK3 prevents the inhibition of protein synthesis by dsRNA in a cell-free translation system from rabbit reticulocytes at levels equal to, or lower than, the level of endogenous eIF-2 alpha . In the cell-free translation system, pK3r exerts its effects at all dsRNA concentrations tested, by preventing phosphorylation of eIF-2 alpha . In addition, pK3r reduces the autophosphorylation of immunopurified PKR, as well as its ability to phosphorylate the alpha subunit of purified eIF-2 . At 400 mM NaCl, in vitro translated {35S}methionine-radiolabeled pK3 can be co-immunoprecipitated with human PKR, using a monoclonal antibody to PKR . This tight binding is consistent with a role for pK3 as a pseudosubstrate for the kinase, and identifies the amino-terminal 30% of eIF-2 alpha as the domain recognized by the eIF-2 alpha-specific protein kinases . In addition, the tight binding opens up the possibility of using binding assays to identify functional domains within the kinase and pK3 . Recombinant pK3 also prevents activation of the heme-sensitive eIF-2 alpha-specific protein kinase, eIF-2 alpha-PKh, in both cell-free translation systems as well as in partially purified preparations . This suggests some similarity between the eIF-2 alpha binding domains of the two eIF-2 alpha specific protein kinases.

J Biol Chem, 1993 Jun 15, 268(17), 12724 - 9
Cysteine to serine replacements in 6-hydroxy-D-nicotine oxidase . Consequences for enzyme activity, cofactor incorporation, and formation of high molecular weight protein complexes with molecular chaperones (GroEL); Brandsch R et al.; 6-Hydroxy-D-nicotine oxidase, an enzyme with FAD covalently attached to the protein, contains 6 cysteine residues in positions 45 (Cys1), 59 (Cys2), 136 (Cys3), 173 (Cys4), 260 (Cys5) and 433 (Cys6) . Cys2, 3, 5, and 6 were replaced with serine by site-directed mutagenesis . The effects of these exchanges on enzyme activity, the autocatalytic incorporation of the cofactor, and the interaction of the mutant proteins with molecular chaperones were analyzed . The flavinylation of 6-hydroxy-D-nicotine oxidase is dependent on the presence of allosteric effectors, e.g . glycerol 3-phosphate or other phosphorylated tricarbon compounds . Replacement of Cys2 or Cys5 abolished this dependence . Covalent incorporation of FAD was reduced to an undetectable level in the Cys3 and Cys5 mutants . Replacement of Cys6 by Ser had no significant effect on enzyme activity and cofactor attachment . Deletion of two amino acids, Phe and Arg, situated 12 and 11 amino acid residues, respectively, from the carboxyl terminus of the protein, resulted in an inactive enzyme with no covalently bound FAD . This result indicates that almost the entire protein chain has to be synthesized before the cofactor can be incorporated, making a cotranslational flavinylation step rather unlikely . The distribution of the 6-hydroxy-D-nicotine oxidase polypeptide between the high molecular weight complexes and the free soluble form was analyzed by gel filtration on Sephacryl S-200 . The wild-type holoenzyme as well as the wild-type apoenzyme were recovered in the eluent fraction of the column while the mutant proteins were retained in high molecular weight complexes, predominantly in those associated with GroEL, as revealed by immunoprecipitation . The extent of complex formation with this molecular chaperone depended on the position of the mutated Cys residue within the protein . Complex formation was highest with protein from the mutants Cys2 and Cys3, less with the Cys5, and absent with the Cys6 mutant protein . Thus, alterations in the amino-terminal part of the 6-hydroxy-D-nicotine oxidase appear more important for the interaction with molecular chaperones than alterations situated in the carboxyl-terminal part of the protein.

Eur J Biochem, 1993 Jun 15, 214(3), 703 - 10
Structural analysis of two oligosaccharide bisphosphates isolated from the lipopolysaccharide of a recombinant strain of Escherichia coli F515 (Re chemotype) expressing the genus-specific epitope of Chlamydia lipopolysaccharide; Holst O et al.; The lipopolysaccharide of the recombinant strain Escherichia coli F515-207, expressing the genus-specific epitope of Chlamydia lipopolysaccharide, was sequentially de-O- and de-N-acylated by mild hydrazinolysis and treatment with 4 M KOH, respectively, yielding two oligosaccharide bisphosphates which were isolated by high-performance anion-exchange chromatography and gel-permeation chromatography . Their structures were determined by chemical analysis, NMR spectroscopy, and mass spectrometry as alpha-Kdo-(2-4)-alpha-Kdo-(2-6)-beta-D-GlcN-(1-6)-alpha-D-GlcN 1,4'-P2 (tetrasaccharide bisphosphate) and alpha-Kdo-(2-8)-alpha-Kdo-(2-4)-alpha-Kdo-(2-6)-beta-D-GlcN-(1-6)-alpha- D-GlcN 1,4'-P2 (pentasaccharide bisphosphate).

J Immunol, 1993 Jun 15, 150(12), 5391 - 9
Cloning and expression of immunologically active recombinant Amb a V allergen of short ragweed (Ambrosia artemisiifolia) pollen; Ghosh B et al.; We have cloned and sequenced Amb a V, an Ambrosia artemisiifolia (short ragweed) pollen allergen that has proved to be particularly useful in the genetic analysis of human immune responsiveness . The amino acid sequence deduced from the cloned cDNA sequence corresponds to the published sequence of the protein, except that the cDNA sequence encodes an extra 10 amino acids at the C-terminus . The expressed 55-residue protein is then presumably cleaved enzymatically at the C-terminal lysine found in the 45-residue protein isolated from the pollen . The cloning and sequencing of Amb a V genomic DNA confirmed the cDNA sequence and showed that the Amb a V gene has no introns . Recombinant Amb a V allergen, expressed in Escherichia coli, bound to IgG and IgE antibodies in all Amb a V-allergic individuals tested and inhibition studies demonstrated that the recombinant protein contains a subset of the antigenic epitopes found on native Amb a V . In addition, recombinant Amb a V released histamine efficiently from basophils from Amb a V-allergic patients . The recombinant Amb a V allergen and mutants of Amb a V should, therefore, be useful in studies of allergen epitopes in humans, as well as providing a diagnostic tool.

J Biol Chem, 1993 Jun 15, 268(17), 12983 - 9
Primary structure of human deoxycytidylate deaminase and overexpression of its functional protein in Escherichia coli; Weiner KX et al.; The cDNA encoding human dCMP deaminase was isolated from a lambda ZAPII expression library using an antibody generated against highly purified HeLa cell dCMP deaminase . The cloned cDNA consists of 1856 base pairs and encodes a protein of 178 amino acids with a calculated molecular mass of 19,985 daltons . The sequence of several cyanogen bromide-cleaved peptides derived from HeLa cell dCMP deaminase are all contained within the deduced amino acid sequence . A zinc binding region is present in the enzyme, similar to that reported for cytidine deaminase (Yang, E . C., Carlow, D., Wolfenden, R., and Short, S . A . (1992) Biochemistry 31, 4168-4174) . Northern blot analysis revealed a predominant messenger RNA species of 1.9 kilobases . Expression of the active protein to about 10% of Escherichia coli's total protein was achieved by subcloning the open reading frame into a high expression system using the polymerase chain reaction . Polyacrylamide gel electrophoresis revealed a prominent protein band which comigrated with affinity purified HeLa dCMP deaminase, while Western blot analysis yielded an immunoreactive band which comigrated with the single immunoreactive affinity column purified dCMP deaminase band . The enzyme which possesses a kcat of 1.02 x 10(3) s-1 was purified to homogeneity in over 60% yield . The overexpression of dCMP deaminase should permit more exacting studies on the regulation of this important allosteric enzyme which provides substrate for DNA synthesis.

J Biol Chem, 1993 Jun 15, 268(17), 12655 - 62
The synapse-specific phosphoprotein F1-20 is identical to the clathrin assembly protein AP-3; Zhou S et al.; F1-20 and AP-3 are independently described, synapse-associated, developmentally regulated phosphoproteins with similar apparent molecular masses on SDS-polyacrylamide gel electrophoresis (PAGE) . F1-20 was cloned and characterized because of its synapse specificity . AP-3 was purified and studied biochemically because of its function as a clathrin assembly protein . Here we present evidence that establishes the identity of F1-20 and AP-3 . Monoclonal antibodies against F1-20 and AP-3 both specifically recognize a single protein from mouse brain with an apparent molecular mass of 190 kDa on SDS-PAGE . These monoclonal antibodies also specifically recognize the cloned F1-20 protein expressed in Escherichia coli . The anti-F1-20 monoclonal antibody (mAb) stains a bovine protein with an apparent molecular mass on SDS-PAGE of 190 kDa that copurifies with brain clathrin-coated vesicles (CCVs) and that can be extracted from the brain CCVs under conditions that extract AP-3 . The anti-F1-20 and anti-AP-3 mAbs specifically recognize the same spot on a two-dimensional gel run on a bovine brain clathrin-coated vesicle extract . AP-3 purified from bovine brain CCVs is recognized by both the anti-F1-20 and anti-AP-3 mAbs . Purified preparations of bovine AP-3 and bacterially expressed mouse F1-20 give identical patterns of protease digestion with bromelain and subtilisin . Sequence analyses reveal that F1-20 has an essentially neutral 30-kDa NH2-terminal domain with an amino acid composition typical of a globular structure and an acidic COOH-terminal domain rich in proline, serine, threonine, and alanine . This is consistent with proteolysis experiments that suggested that AP-3 could be divided into a 30-kDa globular uncharged clathrin-binding domain and an acidic, anomalously migrating domain.

Gene, 1993 Jun 15, 128(1), 97 - 101
A family of vectors for surface display and production of antibodies; Dubel S et al.; Expression vectors for surface display and production of single-chain (Fv) antibodies (scAb) have been constructed based on the phagemid pSEX, which expresses DNA encoding a scAb fused to the gene III product of filamentous phage {Breitling et al., Gene 104 (1991) 147-153} . A smaller version of this phagemid, pSEX20, was made by removing an unnecessary cat . To produce a vector for the surface display of other proteins and peptides, the scAb of pSEX20 was substituted by a polycloning site (MCS) to give pSEX40 . For the presentation of Ab on the surface of Escherichia coli, phagemid pAP10 was derived from pSEX20 by substituting gene III with a gene encoding the peptidoglycan-associated lipoprotein (PAL) . Vectors for producing scAb that can be purified by antibody and metal affinity chromatography were constructed by substituting gene III in the vector pSEX20 with DNA encoding a peptide with a C-terminal epitope recognised by a monoclonal antibody (phagemid pOPE40) or with five C-terminal histidines (pOPE 90).

Gene, 1993 Jun 15, 128(1), 43 - 9
Identification of a peptide which binds to the carbohydrate-specific monoclonal antibody B3; Hoess R et al.; The monoclonal antibody (mAb) B3 recognizes an antigen found on the surface of many adenocarcinoma cells . While the structure of the cellular antigen is unknown, epitope mapping using neoglycoproteins with known carbohydrate moieties indicates that the mAb B3 reacts with the LewisY (LeY) antigen {Pastan et al., Cancer Res . 51 (1991) 3781-3787} . We have used mAb B3 to select for peptides that mimic the carbohydrate structure using libraries of filamentous phage displaying random peptides on their surface . Phage that were selected coded for the sequence APWLYGPA . The corresponding peptide was synthesized and tested for its ability to bind to mAb B3 . The peptide was found to inhibit specifically the binding of 111In-labeled mAb B3 to A431 adenocarcinoma cells, as well as to inhibit killing of these cells by a B3 immunotoxin . In addition, the LeY carbohydrate, lactodifucotetraose, was able to compete with the phage displaying this peptide for binding to mAb B3 . Alanine-scanning mutagenesis of the sequence coding for this peptide indicates that four residues, PWLY, were critical for binding to the mAb . The sequence is similar to other sequences known to mimic carbohydrate structures.

Blood, 1993 Jun 15, 81(12), 3313 - 7
Activation of the kallikrein-kinin system after endotoxin administration to normal human volunteers; DeLa Cadena RA et al.; The objective of this study was to determine the role of the kallikrein-kinin system in healthy humans after intravenous administration of either Escherichia coli endotoxin or saline . We studied a total of 18 healthy nonsmoking volunteers, 23 to 38 years old, in an open-label study at the Critical Care Medicine Department, Clinical Center, National Institutes of Health (Bethesda, MD) in which some of the patients served as their own controls . After baseline data collection, the subjects received intravenously either E coli endotoxin (n = 15, 4 ng/kg of body weight) or saline (n = 8, controls) . Signs, symptoms, systemic blood pressure, factor XII, plasma prekallikrein (PK), factor XI (FXI), antithrombin III (AT-III), high molecular weight kininogen (HK), and alpha 2-macroglobulin-kallikrein complexes were measured at baseline and 1, 2, 3, 5, and 24 hours after injection of either saline or endotoxin . After infusion of endotoxin, we found the functional plasma levels of FXI decreased at 2 hours (P < .05) and at 5 hours (P < .05) . Functional PK was significantly depressed by 2 hours (P < .05), at 5 hours (P < .05), and at 24 hours (P < .01), whereas the PK antigen was only low at 5 hours (P < .05) . These changes were accompanied by a significant increase in circulating alpha 2-macroglobulin-kallikrein complexes at 3 hours (P < .05) and 5 hours (P < .01) . No significant changes occurred in the plasma levels of factor XII or HK . We concluded that clinical response to intravenous endotoxin in healthy human volunteers is associated with activation of the kallikrein-kinin systems . Further investigation is needed with specific inhibitors of the kallikrein-kinin system to define its primary or secondary role in the endotoxin-mediated reactions.

Blood, 1993 Jun 15, 81(12), 3226 - 33
Recombinant human leukemia inhibitory factor induces acute phase proteins and raises the blood platelet counts in nonhuman primates; Mayer P et al.; Recombinant human leukemia inhibitory factor (rhLIF) produced by Escherichia coli was administered subcutaneously (sc) to rhesus monkeys at doses of 2, 10, and 50 micrograms/kg body weight/d for 14 days to assess its biologic activities in vivo . Serum levels of positively regulated acute phase proteins (APP) (C-reactive protein, alpha 1-antitrypsin, haptoglobin, and ceruloplasmin) were increased, whereas the negatively regulated APP prealbumin decreased in response to rhLIF treatment . During the second week of treatment, blood platelet counts began to increase, resulting in a maximum of a twofold increase above normal levels a week after termination of the rhLIF treatment . No changes were seen in total and differential white blood cell counts in blood progenitor levels and in red blood cell numbers . The low- and medium-dose rhLIF treatments were tolerated without significant side effects . The animals treated with the high dose showed a reduction in body weight of approximately 10% . In conclusion, rhLIF was shown to stimulate APP and to increase the number of platelets in circulation in nonhuman primates.

FEBS Lett, 1993 Jun 14, 324(2), 167 - 70
Crystallization of the seryl-tRNA synthetase:tRNAS(ser) complex of Escherichia coli; Price S et al.; Crystals of the complex between seryl-tRNA synthetase and tRNA(2ser) from Escherichia coli have been obtained from ammonium sulphate solutions . The crystals are of the 1:2 enzyme:tRNA complex, belong to the space group C222(1), have cell dimensions of a = 128.9 A, b = 164.9 A, c = 127.3 A and diffract anisotropically from 3.5 to 4.5 A . An X-ray diffraction data set to 4 A has been collected . The combination of molecular replacement using the refined structure of the catalytic domain of the native enzyme, data from a heavy atom derivative and solvent flattening was used to produce a map at 4 A resolution . This shows that a tRNA molecule binds across the dimer, the anticodon stem and loop do not contact the protein and the helical arm of the enzyme contacts the T psi C loop and the long extra arm of the tRNA.

FEBS Lett, 1993 Jun 14, 324(2), 131 - 5
The in vivo assembly and function of the N- and C-terminal halves of the Tn10-encoded TetA protein in Escherichia coli; Yamaguchi A et al.; The tetA gene was cut into its N- and C-terminal halves at the central EcoRI site and the two halves were subcloned individually or together under a separate lac promoter/operator . The expression of the C-terminal half was detected with a C-terminal-specific antibody . The amount of the N-terminal half in the cytoplasmic membrane was not affected by the presence of the C-terminal half . In contrast, the amount of the C-terminal half in the membrane was increased in the presence of the N-terminal half, indicating that the N-terminal half helps the stable folding of the C-terminal half in the membrane . Each half individually showed no tetracycline transport activity, however, when both halves were expressed together, the resultant complex showed about 40% of the tetracycline transport activity of the wild-type per number of the C-terminals of TetA protein in the membrane.

FEBS Lett, 1993 Jun 14, 324(2), 162 - 6
In vivo overexpression and purification of Escherichia coli tRNA(ser); Borel F et al.; DNA fragments corresponding to the sequences of Escherichia coli tRNA(2ser) and amber suppressor tRNA(ser), were synthesized from overlapping oligonucleotides . These were interposed between a strong promotor and a synthetic transcriptional terminator to ensure the production of a transcript of the correct size . The genes of promotor, fragment and terminator were cloned into a conditional runaway replication plasmid . At temperatures below 37 degrees C this vector has a low copy number but, following a temperature shift to 42 degrees C, the copy number is no longer regulated . Using these constructs an overexpression of tRNA(ser) of about 20 times the level of the wild-type pool could be obtained (corresponding e.g . to 200 times the expression tRNA(2ser)) . From these systems 10 mg quantities of tRNA(ser)s could be isolated with a serine acceptance of 1,100 pmol/A280 unit.

Science, 1993 Jun 11, 260(5114), 1646 - 9
Evidence of DNA bending in transcription complexes imaged by scanning force microscopy; Rees WA et al.; Complexes of Escherichia coli RNA polymerase with DNA containing the lambda PL promoter have been deposited on mica and imaged in air with a scanning force microscope . The topographic images reveal the gross spatial relations of the polymerase relative to the DNA template . The DNA appears bent in open promoter complexes containing RNA polymerase bound to the promoter and appears more severely bent in elongation complexes in which RNA polymerase has synthesized a 15-nucleotide transcript . This difference could be related to the conformational changes that accompany the maturation of open promoter complexes into elongation complexes and suggests that formation of the elongation complex involves a considerable modification of the spatial relations between the polymerase and the DNA template.

Nucleic Acids Res, 1993 Jun 11, 21(11), 2709 - 14
Elimination of band compression in sequencing gels by the use of N4-methyl-2'-deoxycytidine 5'-triphosphate; Li S et al.; Taq DNA polymerase, Sequenase, and the large fragment of E.coli polymerase I effectively utilize N4-methyl-2'-deoxycytidine 5'-triphosphate (N4-methyl-dCTP) in the place of dCTP in dideoxynucleotide terminator sequencing reactions on single-stranded templates . When the resulting fragment mixtures are resolved on sequencing gels, they are found to be free of band compressions even in cases where such compressions remain unresolved by the substitution of 7-deaza-dGTP for dGTP . Sequencing reactions using N4-methyl-dCTP instead of dCTP are somewhat more prone to false stops than are sequencing reactions using 7-deaza-dGTP instead of dGTP; this difference is more pronounced when sequencing with Sequenase at 37 degrees C than when sequencing with Taq DNA polymerase at 72 degrees C . For the three polymerases investigated, replacement of dCTP by N4-methyl-dCTP does not fundamentally change the characteristic variations in band intensities seen in the C-lane . N4-methyl-dCTP can also be used for sequencing double-stranded DNA and for DNA amplification by the polymerase chain reaction.

Nucleic Acids Res, 1993 Jun 11, 21(11), 2627 - 31
A PCR-based method for high stringency screening of DNA libraries; Israel DI; A rapid method for cloning genomic DNA utilizing a PCR-based screening protocol is described . A murine genomic library in lambda phage was subdivided into 64 wells, each containing 1000 clones, and propagated in bacteria . Amplified phage from each of 8 wells across columns, and each of 8 wells down rows, were pooled . The pooled phage were screened for the presence of murine M-CSF DNA by PCR using specific oligonucleotide primers . A single well that contained an M-CSF genomic clone was identified by the synthesis of a PCR product of the correct size that hybridized to an internal M-CSF oligonucleotide probe . This well was subdivided into 64 wells, each containing approximately 30 individual phage, reamplified, and rescreened utilizing the same protocol . A positive well was then subdivided and amplified a third time starting with an average of 2 phage per well, and rescreened for M-CSF DNA by PCR . Phage from a PCR-positive well, now highly enriched for M-CSF DNA, were grown as individual plaques . PCR-screening of randomly picked plaques demonstrated that the majority contained an M-CSF genomic insert . This method obviates the more labor and time intensive method of plaque hybridization screening of DNA libraries, and is more stringent since three oligonucleotides (the two PCR primers, and the hybridization probe) are required to give a true positive signal . Similar methodology has also been used to clone a cDNA gene contained within a plasmid library.

Nucleic Acids Res, 1993 Jun 11, 21(11), 2579 - 84
Nuclear and mitochondrial forms of human uracil-DNA glycosylase are encoded by the same gene; Slupphaug G et al.; Recent cloning of a cDNA (UNG15) encoding human uracil-DNA glycosylase (UDG), indicated that the gene product of M(r) = 33,800 contains an N-terminal sequence of 77 amino acids not present in the presumed mature form of M(r) = 25,800 . This led to the hypothesis that the N-terminal sequence might be involved in intracellular targeting . To examine this hypothesis, we analysed UDG from nuclei, mitochondria and cytosol by western blotting and high resolution gel filtration . An antibody that recognises a sequence in the mature form of the UNG protein detected all three forms, indicating that they are products of the same gene . The nuclear and mitochondrial form had an apparent M(r) = 27,500 and the cytosolic form an apparent M(r) = 38,000 by western blotting . Gel filtration gave essentially similar estimates . An antibody with specificity towards the presequence recognised the cytosolic form of M(r) = 38,000 only, indicating that the difference in size is due to the presequence . Immunofluorescence studies of HeLa cells clearly demonstrated that the major part of the UDG activity was localised in the nuclei . Transfection experiments with plasmids carrying full-length UNG15 cDNA or a truncated form of UNG15 encoding the presumed mature UNG protein demonstrated that the UNG presequence mediated sorting to the mitochondria, whereas UNG lacking the presequence was translocated to the nuclei . We conclude that the same gene encodes nuclear and mitochondrial uracil-DNA glycosylase and that the signals for mitochondrial translocation resides in the presequence, whereas signals for nuclear import are within the mature protein.

Nucleic Acids Res, 1993 Jun 11, 21(11), 2747 - 54
Synthetic gene for the hepatitis C virus nucleocapsid protein; Khudyakov YE et al.; A synthetic gene encoding the hepatitis C virus (HCV) nucleocapsid protein was constructed and expressed in E . coli . To synthesize this gene, we developed a new method that results in the enzymatic synthesis of long polydeoxyribonucleotides from oligodeoxyribonucleotides . The method, designated as the 'Exchangeable Template Reaction' (ETR), uses oligonucleotides as templates for DNA polymerase . A special mechanism was designed to exchange the templates during the polymerase reaction . The mechanism relies on the formation of a single-stranded 3'-protrusion at the 'growing point' of the elongating DNA such that it can be subsequently annealed, in a sequence-specific manner, with the next synthetic oligonucleotide . When annealed to the 3'-protrusion, the added oligonucleotide becomes a template for DNA polymerase, and the protruding 3'-end of the double-stranded DNA is used as the primer . The HCV nucleocapsid gene was assembled with DNA ligase from three fragments synthesized by ETR . The data verify that this method is efficient . The main advantage of ETR is the ability to combine more than two oligonucleotides in one tube together with polymerase and an enzymatic activity that produces a 3'-protrusion (e.g., BstXI) rather than the sequential addition of each component . The data demonstrate that as many as five oligonucleotides can be used simultaneously, resulting in a synthesized DNA fragment of designed sequence . The synthetic gene expressed in E . coli produced a 27 kDa protein that specifically interacted with antibodies from sera obtained from HCV-infected individuals.

Biochim Biophys Acta, 1993 Jun 10, 1143(1), 62 - 6
ATP-driven potassium transport in right-side-out membrane vesicles via the Kdp system of Escherichia coli; Kollmann R et al.; The ATP-generating system described by Hugenholtz, J., Hong, J.-S . and Kaback, H.R . ((1981) Proc . Natl . Acad . Sci . USA 78, 3446-3449) has been used to synthesize ATP up to 1.8 mM in right-side-out membrane vesicles from Escherichia coli . This ATP level was sufficient to drive uptake of potassium ions via the Kdp-ATPase . In the kdp wild type strain about 110 nmoles K+/mg membrane protein were accumulated . This process was still partially sensitive to the well-known inhibitors of P-type ATPases, orthovanadate and bafilomycin B1.

Biochim Biophys Acta, 1993 Jun 10, 1143(1), 109 - 11
PCR cloning, sequence analysis and expression of the cybC genes encoding soluble cytochrome b-562 from Escherichia coli B strain OP7 and K strain NM522; Trower MK; The cybC genes encoding cytochrome b-562 from B and K strains of Escherichia coli were shown to differ in size following their isolation by in vitro amplification using the polymerase chain reaction . Nucleotide sequencing of the genes and flanking regions revealed the discrepancy was due to a 26 bp deletion at the 5' end of the K strain sequence that included the initiation codon . Expression studies confirmed that the K strain gene was untranslated . These data indicate that the cybC gene product is non-essential in E . coli.

Mol Cell Biochem, 1993 Jun 9-23, 123(1-2), 39 - 44
Identification of high affinity membrane-bound fatty acid-binding proteins using a photoreactive fatty acid; Gerber GE et al.; A photoaffinity labeling method was developed to identify and characterize high affinity fatty acid-binding proteins in membranes . The specific labeling of these sites requires the use of low concentrations (nanomolar) of the photoreactive fatty acid 11-m-diazirinophenoxy-{11-3H}undecanoate . It was delivered as a bovine serum albumin (BSA) complex which serves as a reservoir for fatty acid and thus allows precise control of unbound fatty acid concentrations . The fadL protein of E . coli, which is required for fatty acid permeation of its outer membrane, was labeled by the photoreactive fatty acid neither specifically nor saturably when the probe was added in the absence of BSA; however when a nanomolar concentration of the uncomplexed probe was maintained in the presence of BSA, the labeling of the fadL protein was highly specific and saturable . This photoaffinity labeling method was also used to characterize a 22 kDa, high affinity fatty acid-binding protein which we have recently identified in the plasma membrane of 3T3-L1 adipocytes . This protein bound the probe with a Kd of 216 nM . The approach described is easily capable of identifying membrane-bound fatty acid-binding proteins and can distinguish between those of high and low affinities for fatty acids . It represents a general method for the identification and characterization of fatty acid-binding proteins.

Mol Cell Biochem, 1993 Jun 9-23, 123(1-2), 3 - 13
High resolution X-ray studies of mammalian intestinal and muscle fatty acid-binding proteins provide an opportunity for defining the chemical nature of fatty acid: protein interactions; Scapin G et al.; The structure of E . coli-derived rat intestinal fatty acid-binding protein has recently been refined to 1.2 A without bound fatty acid and to 2.0 A and 1.75 A with bound hexadecanoate (palmitate) and 9Z-octadecenoate (oleate), respectively . The structure of E . coli-derived human muscle fatty acid-binding protein has also been solved to 2.1 A with a C16 bacterial fatty acid . Both proteins contain 10 anti-parallel beta-strands in a +1, +1, +1.. . motif . The strands are arranged in two beta-pleated sheets that are orthogonally oriented . In each case, the fatty acid is enclosed by the beta-sheets and is bound to the proteins by feeble forces . These feeble forces consist of (i) a hydrogen bonding network between the fatty acid's carboxylate group, ordered solvent, and side chains of polar/ionizable amino acid residues; (ii) van der Waals contacts between the methylene chain of the fatty acid and the side chain atoms of hydrophobic and aromatic residues; (iii) van der Waals interactions between the omega-terminal methyl and the component methenyls of the phenyl side chain of a Phe which serves as an adjustable terminal sensor situated over a surface opening or portal connecting interior and exterior solvent; and (iv) van der Waals contacts between methylenes of the alkyl chain and oxygens of ordered waters that have been located inside the binding cavity . These waters are positioned over one face of the ligand and are held in place by hydrogen bonding with one another and with the side chains of protein's polar and ionizable residues . Binding of the fatty acid ligand is associated with minimal adjustments of the positions of main chain or side chain atoms . However, acquisition of ligand is associated with removal of ordered interior solvent suggesting that the free energy of dehydration of the binding site may be as important for the energy of the binding reaction as the free energy of stabilization of the fatty acid: protein complex.

Biochemistry, 1993 Jun 8, 32(22), 5913 - 6
Phosphoryl transfer between phosphorylated histidine-containing protein and histidine-containing protein is not autocatalytic; Anderson JW et al.; Histidine-containing protein, HPr, is a phosphocarrier protein that is part of the bacterial phosphoenolpyruvate:sugar phosphotransferase system . HPr is phosphorylated by enzyme I, and P-HPr transfers the phosphoryl group to the IIA domain of a number of sugar-specific enzyme II complexes . Autocatalytic phosphoryl transfer between P-HPr and HPr has recently been reported {van Dijk, A . A., Eisermann, R., Hengstenberg, W., & Robillard, G . T . (1991) Biochemistry 30, 2876-2882} . Our results show that this phosphoryl transfer is due to an unidentified contaminant of HPr preparations . The phosphoryl transfer activity is not present in all HPr preparations . When present, the phosphoryl transfer activity can be removed by further purification or destroyed over time by resuspension of HPr preparations in water . There is no autocatalytic phosphoryl transfer between P-HPr and HPr.

Biochemistry, 1993 Jun 8, 32(22), 5888 - 900
Negative cooperativity in the binding of nucleotides to Escherichia coli replicative helicase DnaB protein . Interactions with fluorescent nucleotide analogs; Bujalowski W et al.; The interactions of nucleotides with Escherichia coli replicative helicase DnaB protein have been systematically studied using fluorescent nucleotide analogs, 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP), 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP), 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-monophosphate (TNP-AMP), 3'-O-(N-methylantraniloyl) 5'-diphosphate (MANT-ADP), and 1,N6-ethenoadenosine diphosphate (epsilon ADP) . The binding of the analogs is accompanied by strong quenching of the protein fluorescence; 0.76 +/- 0.05, 0.76 +/- 0.05, 0.58 +/- 0.05, and 0.53 +/- 0.5 for TNP-ATP, TNP-ADP, MANT-ADP, and epsilon ADP, respectively . A thermodynamically rigorous method has been applied to obtain all binding parameters from fluorescence titration curves independent of the assumption of strict proportionality between the observed quenching of the protein fluorescence and the degree of nucleotide binding . An exact representation of the observed fluorescence quenching, as a function of the nucleotide binding, is introduced through an empirical function which enables analysis of single binding isotherms without the necessity of determining all quenching constants for different binding sites . Using this method, we determined that, at saturation, the DnaB hexamer binds six molecules of TNP-ATP, TNP-ADP, MANT-ADP, and epsilon ADP, and that there is strong heterogeneity among nucleotide binding sites . The binding isotherms are biphasic . Three molecules of nucleotide are bound in the first high-affinity binding phase, and the subsequent three molecules are bound in the second low-affinity binding phase . The separation of the two binding steps is even more pronounced at higher temperatures . The change of the monitored fluorescence is sequential . The binding of the first nucleotide causes the largest quenching of the protein fluorescence with subsequent nucleotide binding inducing progressively less quenching . The simplest explanation of this behavior is that there is a negative cooperativity among nucleotide binding sites on a DnaB hexamer . The negative cooperativity is an intrinsic property of the DnaB helicase, since it is observed in the binding of nucleotide analogs which are different in type and location of the modifying group . A statistical thermodynamic model is proposed, the hexagon, which provides an excellent description of the binding process using only two interaction parameters, intrinsic binding constant K and cooperativity parameter sigma . The data suggest an important role of the phosphate groups in binding and in recognition of nucleotides by the DnaB helicase.

Biochemistry, 1993 Jun 8, 32(22), 5848 - 54
Binding of the substrate analogue perseitol to phosphorylated and unphosphorylated enzyme IImtl of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli; Lolkema JS et al.; Enzyme IImtl catalyzes the concomitant transport and phosphorylation of the hexitol mannitol . Here we demonstrate that the heptitol perseitol is not phosphorylated and not transported by the enzyme . However, the enzyme binds perseitol with an affinity comparable to the affinity for mannitol . Apparent affinities of the phosphorylated enzyme for perseitol were inferred from the inhibition by perseitol of mannitol phosphorylation and uptake . Apparent affinities of the unphosphorylated enzyme follow from the inhibition of mannitol binding to the enzyme . Mechanistic interpretations of the apparent inhibition constants are discussed, and it is concluded that phosphorylation of the cytoplasmic domain of enzyme IImtl has little effect on the affinity of the membrane-bound domain of the enzyme for perseitol.

Biochemistry, 1993 Jun 8, 32(22), 5855 - 61
Topological characterization of Escherichia coli DMSO reductase by electron paramagnetic resonance spectroscopy of an engineered {3Fe-4S} cluster; Rothery RA et al.; We have applied the technique of distance estimations using the exogenous paramagnetic probe dysprosium (III) complexed with EDTA (DyEDTA) to study the topology of Escherichia coli dimethyl sulfoxide reductase (DmsABC) in situ in cytoplasmic membrane and whole cell preparations . The electron transfer subunit (DmsB) of this enzyme contains four {4Fe-4S} clusters and has complex EPR properties making it unsuitable for studies utilizing exogenous paramagnetic probes . We have utilized a mutant of DmsABC in which one of the {4Fe-4S} clusters of DmsB has been changed to a {3Fe-4S} cluster {Rothery, R . A., & Weiner, J . H . (1991) Biochemistry 30, 8296-8305} . This mutant (DmsB-C102S) has a single magnetically isolated EPR visible {3Fe-4S} cluster in its fully oxidized state, making it suitable for studies using DyEDTA as paramagnetic probe . We have studied the effect of DyEDTA on the microwave power saturation properties of the EPR signal of the DmsB-C102S mutant . DyEDTA enhances the spin relaxation of the {3Fe-4S} signal in everted membrane vesicles . It has a smaller effect on the spin relaxation of the {3Fe-4S} signal in whole cell preparations . We conclude that the {3Fe-4S} cluster of the DmsB-C102S mutant is located on the inside of the cytoplasmic membrane . We have estimated the distance of this center from the surface of the DmsAB dimer to be approximately 21 A, close to the cytoplasmic-side membrane surface level, by calibrating the DyEDTA effect using a myoglobin nitroxide standard.

Biochemistry, 1993 Jun 8, 32(22), 5800 - 8
Determination of the pKa values of active-center cysteines, cysteines-32 and -35, in Escherichia coli thioredoxin by Raman spectroscopy; Li H et al.; We have determined the pKa values for thiol-thiolate equilibria of Escherichia coli thioredoxin and compared structural properties of reduced and oxidized forms of the protein in solution by Raman spectroscopy . Ionization and hydrogen-bonding states of the two cysteine sulfhydryls (Cys32 and Cys35) in reduced thioredoxin were determined by monitoring the complex Raman SH stretching band (Li & Thomas, 1991) as a function of pH in the range 4.0 < pH < 12.2 . The Raman SH markers indicate the following: (i) Both sulfhydryls of the native protein are relatively robust hydrogen-bond donors, but one is a stronger hydrogen-bond donor than the other . (ii) The sulfhydryl which donates the weaker hydrogen bond, assigned tentatively to Cys32, is preferentially titrated to the thiolate ion (S-) as the solution pH is increased from 4 to 7 . Water or a hydroxyl oxygen is the probable acceptor for the Cys32 S-H donor . (iii) The sulfhydryl which donates the stronger hydrogen bond, Cys35, resists substantial ionization until roughly 50% of the more acidic sulfhydryl has been titrated . A carbonyl oxygen is proposed as the likely acceptor of the Cys35 S-H donor . (iv) The Raman titration data indicate pK1 = 7.1 +/- 0.2 and pK2 = 7.9 +/- 0.2 for the two thiol-thiolate equilibria . The lower pKa, which is the more strongly perturbed, is assigned tentatively to Cys32.(ABSTRACT TRUNCATED AT 250 WORDS)

FEBS Lett, 1993 Jun 7, 324(1), 87 - 92
1H and 15N assignments and secondary structure of the Src SH3 domain; Yu H et al.; The 1H and 15N sequential assignments of the Src SH3 domain have been determined through a combination of 2D and 3D Nuclear Magnetic Resonance (NMR) methods . The secondary structure of the protein has been identified based on long-range NOE patterns . The SH3 domain of Src consists largely of six beta-strands that form two anti-parallel beta-sheets.

FEBS Lett, 1993 Jun 7, 324(1), 67 - 70
Mutational analysis of the specific priming signal essential for DNA replication of the broad host-range plasmid RSF1010; Honda Y et al.; To analyze the RSF1010-specific priming mechanism, a library of randomly mutagenized ssiA sequences was constructed by chemical synthesis using mixed nucleotide phosphoramidites . Synthetic ssiA sequences with the single base-substitutions were assayed for the SSI activity in E . coli JM109 expressing RepB' primase . It was demonstrated that the activity of ssiA was damaged markedly by single base-substitutions within the possible stem-loop structure and its 3'-flanking region . It is conceivable that these domains are critical in recognition and primer synthesis by RepB' primase.

FEBS Lett, 1993 Jun 7, 324(1), 27 - 32
Efficient transient expression system based on square pulse electroporation and in vivo luciferase assay of fertilized fish eggs; Muller F et al.; Electroporation mediated DNA transfer into fish eggs has been improved by using a train of square pulses . Fertilized eggs of African catfish (Clarias gariepinus), zebrafish (Brachydanio rerio) and rosy barb (Barbus conchonius) were dechorionated enzymatically followed by application of pulses . Efficiency of plasmid DNA delivery was significantly increased by applying multiple pulses on dechorionated eggs . Optimization of physical parameters such as field strength, pulse width and pulse numbers resulted in reproducible transient expression in 25-50% of embryos and larvae by using the firefly luciferase and the E . coli beta-galactosidase (lacZ) genes both driven by CMV IE1 promoter . Temporal luciferase expression was assayed using both qualitative (sheet film) and quantitative (scintillation counting) methods in developing embryos and fry in vivo . Spatial expression of lacZ was assayed by histochemical staining . A number of embryos revealed foreign gene product also localised in the vegetal pole of the embryo.

FEBS Lett, 1993 Jun 7, 324(1), 113 - 6
Phosphatidylglycerol dependent protein translocation across the Escherichia coli inner membrane is inhibited by the anti-cancer drug doxorubicin . Evidence for an electrostatic interaction between the signal sequence and phosphatidylglycerol; Phoenix DA et al.; OmpF-Lpp, a model secretory protein, requires both a positively charged signal sequence and phosphatidylglycerol (PG) for efficient translocation across the E . coli inner membrane . Modification of the signal sequence can, however, remove both these prerequisites for translocation providing OmpF-Lpp mutants which undergo either PG and charge dependent or PG and charge independent translocation . Here we show that positively charged membrane interactive compounds (polylysine & doxorubicin) are able to inhibit PG dependent translocation of the OmpF-Lpp signal sequence mutants but not PG independent translocation . Doxorubicin is also shown to bind more efficiently to liposomes containing increased levels of anionic lipid indicating that in these assays it may be inhibiting translocation by preventing electrostatic interaction between the anionic lipid head group and the positively charged signal sequences.

FEBS Lett, 1993 Jun 7, 324(1), 109 - 12
The glucose transporter of Escherichia coli . Purification and characterization by Ni+ chelate affinity chromatography of the IIBCGlc subunit; Waeber U et al.; The IIBCGlc transmembrane subunit of the glucose transporter of E . coli containing a carboxy-terminal affinity tag consisting of six adjacent histidines was purified by nickel chelate affinity chromatography . The protein was constitutively overexpressed from a high copy number plasmid . 1.5 mg of 95% pure protein was obtained from 5 g (wet weight) cells . 70% of the phosphotransferase activity present in cell membranes was recovered . Adsorption to the nickel resin allows delipidation as well as rapid detergent exchange . The procedure was used to demonstrate exchange of subunits in the IIBCGlc dimer and it holds promise for the investigation of other protein-protein interactions.

FEBS Lett, 1993 Jun 7, 324(1), 41 - 4
Stabilization of a compact conformation of monomeric GroEL at low temperature by adenine nucleotides; Lissin NM et al.; E . coli GroEL chaperonin monomers, isolated after urea-induced dissociation of GroEL14, undergo cold denaturation below 5 degrees C . Above 5 degrees C, these monomers undergo MgATP-dependent self-assembly . We have demonstrated a conformational transition at 0 degree C induced by interaction of monomeric GroEL with adenine nucleotides . This conformation has a dramatically decreased Stokes radius and enhanced resistance to trypsin but it is slightly less compact than the conformation of monomers at 23 degrees C in the absence of MgATP and it is not capable of spontaneous self-assembly . A second, temperature-dependent conformational change with a transition at about 5 degrees C is required for GroEL to undergo oligomerization.

J Mol Biol, 1993 Jun 5, 231(3), 689 - 97
Protein-protein interactions during filamentous phage assembly; Russel M; Filamentous phage proteins pI and pIV are morphogenetic proteins required for phage assembly but not part of the virion . Neither pI nor pIV from the related phages f1 and IKe can substitute for its equivalent in the other phage . When the two proteins are supplied as pairs, however, partial restoration of heterologous phage assembly occurs . This observation strongly suggests that the two proteins interact . A selection for revertants of a temperature sensitive mutant of f1 gene IV resulted in the isolation of a suppressor mutation in gene I . This suppressor is allele specific, and thus supports the hypothesis that pI and pIV interact . A selection for IKe phage that can efficiently utilize paired pI and pIV from from f1 led to the isolation of a phage with a mutation in gene VIII, which encodes the major coat protein of the virus . Analysis of the system suggests that it is pI that interacts with both pIV and pVIII . Thus the process by which filamentous phage are concomitantly assembled and secreted across the cell membranes is likely to involve a series of protein-protein interactions that are accessible to genetic analysis.

J Mol Biol, 1993 Jun 5, 231(3), 646 - 57
Genetic and biochemical analysis of the integration host factor of Escherichia coli; Mengeritsky G et al.; Integration host factor (IHF) is a small, heterodimeric DNA-binding protein of Escherichia coli composed of two subunits, alpha and beta, encoded by the himA and hip genes, respectively . IHF binds to the minor groove at a consensus sequence and bends DNA . We mutagenized the hip gene and studied the activity of the mutant IHF proteins in vivo and in vitro . Substitutions at the C-terminal alpha-helix (alpha-helix 3) reduced IHF activity and relaxed the specificity to DNA without abolishing the ability of IHF to bend DNA . These results indicate that the C-terminal region of Hip participates in determining IHF specificity . Alanine substitutions in beta-strands 2 and 3 generally had no effect on IHF activity in vivo suggesting that individually, many of these residues make only small contributions to the binding of IHF to DNA . Replacing the single amino acid of Hip that differs from HU in a highly conserved region of the arm did not affect IHF activity . This finding led us to conclude that this region of Hip does not contribute to specific DNA recognition by IHF . The binding of IHF to DNA is probably not restricted to one domain, but requires the co-operative participation of a number of regions of the protein.

J Mol Biol, 1993 Jun 5, 231(3), 549 - 53
The spatial structure of the class II L-fuculose-1-phosphate aldolase from Escherichia coli; Dreyer MK et al.; The three-dimensional structure of L-fuculose-1-phosphate aldolase (FucA) from Escherichia coli was determined by X-ray crystallography at a resolution of 2.13 A . The enzyme is a homotetramer with an M(r) of 23,775 per subunit . Since its activity depends on the presence of metal ions (Zn2+) the enzyme belongs to the class II aldolases . As expected from amino acid sequence comparisons, this first structure of a class II aldolase shows no similarity to the known structures of class I aldolases . It has some unusual features concerning the overall chain fold, the quaternary structure, and the co-ordination of the catalytically active zinc ion . A sequence comparison with the data bank indicated that the middle domain of the enzyme L-ribulose-5-phosphate-4-epimerase is homologous to FucA and may contain an active-center metal ion.

J Biol Chem, 1993 Jun 5, 268(16), 12185 - 92
Analysis of structure-function relationships in the platelet membrane glycoprotein Ib-binding domain of von Willebrand's factor by expression of deletion mutants; Sugimoto M et al.; We have used a series of Escherichia coli-expressed deletion mutants of the glycoprotein (GP) Ib-binding domain of von Willebrand factor (vWF) to study the structural basis of its function . In addition to the prototypic molecule (rvWF441-733), we constructed 11 mutants; seven had deletions of sequence on the amino- and/or carboxyl-terminal side of the Cys509-Cys695 intrachain disulfide loop, and four had limited deletions inside the loop . Other cysteine residues in addition to 509 and 695, when present in the corresponding native sequence, were mutated to glycine; all molecules were purified in the oxidized as well as reduced and alkylated state . The smallest species retaining the ability to interact with GP Ib in the absence of modulators was the oxidized rvWF508-696; the latter, as well as rvWF441-696, became inactive after reduction and alkylation . In contrast, all the other fragments with deletions outside of the loop, but extending at least to residue 700, showed better binding to platelets after reduction and alkylation than when the Cys509-Cys695 disulfide bond was oxidized . Any limited deletion of sequence inside the loop caused complete loss of GP Ib-binding function both in the absence or in the presence of botrocetin, and this persisted even after reduction and alkylation . In contrast, all mutants with intact sequence between residues 509 and 695 bound to GP Ib in the presence of botrocetin, regardless of whether the 2 cysteine residues were oxidized or reduced and alkylated . Ristocetin, unlike botrocetin, appeared to have no effect in modulating the binding of any of the expressed fragments to platelets . Our findings suggest that the GP Ib-binding domain of vWF contains multiple interaction sites, but integrity of the sequence 509-695 is important for function.

J Biol Chem, 1993 Jun 5, 268(16), 12077 - 82
Structure/function analysis of the transmembrane domain of DAB389-interleukin-2, an interleukin-2 receptor-targeted fusion toxin . The amphipathic helical region of the transmembrane domain is essential for the efficient delivery of the catalytic domain to the cytosol of target cells; vanderSpek JC et al.; Cassette and deletion mutagenesis were used to analyze the function of the amphipathic alpha-helices in the transmembrane domain of DAB389-interleukin-2 (IL-2), a fusion protein which is targeted to the interleukin-2 receptor . We demonstrate that the in-frame deletion of 60 amino acids, from Asn204 to Glu263 in DAB389-IL-2, results in complete loss of cytotoxic activity, whereas when the amphipathic regions from Asp208 to Ser220 and Ala244 to His258 are replaced with idealized amphipathic helices composed of repeating Glu, Lys, and Leu residues, the mutant fusion toxin has low but detectable activity . DAB389-IL-2 and both variants form channels in artificial phospholipid bilayers with conductances identical to those formed by diphtheria toxin . Both mutant fusion toxins bind to the high affinity IL-2 receptor with affinities similar to that of DAB389-IL-2 . The fact that these mutants have markedly reduced or absent cytotoxic activity, but possess "wild type" catalytic activity, binding affinities, and channel conductances, suggests the existence of a step in the intoxication pathway, defective in the mutants, which occurs after DAB389-IL-2 binds to the IL-2 receptor . It is unknown whether this step occurs prior or subsequent to channel formation, but it is essential for the efficient delivery of the ADP-ribosyltransferase from DAB389-IL-2 to the cytosol of target cells.

J Biol Chem, 1993 Jun 5, 268(16), 11939 - 45
Regulation of autoproteolysis of the HIV-1 and HIV-2 proteases with engineered amino acid substitutions; Rose JR et al.; Autoproteolysis of the retroviral aspartyl proteases is a major obstacle to purification and analysis of these enzymes . A mutagenic approach to rendering autolytic cleavage sites less labile was applied to the primary cleavage site between Leu5 and Trp6 in human immunodeficiency virus-1 (HIV-1) protease . From predictions based on known substrates it was concluded that amino acids Lys or Ser in place of Gln at position 7 would prevent cleavage at the Leu5-Trp6 peptide bond, therefore stabilizing the protein . Autoproteolytic stability was enhanced at least 100-fold by these mutations . At longer time points the protease was degraded at secondary sites which contained adequate substrate sequences but were conformationally restricted . Conversely, a mutation in HIV-2 protease which changed Lys7 to Gln rendered the protein 3-fold less stable and shifted the position of the initial autoproteolytic cleavage from Phe3-Ser4 to Leu5-Trp6 . The effects of these mutations demonstrate that small changes in protein sequence can have a major impact on their autoproteolytic stability . The work described here suggests a general method for stabilizing proteases and perhaps other recombinantly produced proteins to autolysis.

J Biol Chem, 1993 Jun 5, 268(16), 11910 - 6
Cloning of human cDNAs encoding mitochondrial and cytosolic serine hydroxymethyltransferases and chromosomal localization; Garrow TA et al.; Human cDNAs for cytosolic and mitochondrial serine hydroxymethyltransferase (SHMT) were cloned by functional complementation of an Escherichia coli glyA mutant with a human cDNA library . The cDNA for the cytosolic enzyme encodes a 483-residue protein of M(r) 53,020 . The cDNA for the mitochondrial enzyme encodes a mature protein of 474 residues of M(r) 52,400 . The deduced protein sequences share a high degree of sequence identity to each other (63%), and the individual isozymes are highly homologous to the analogous rabbit liver cytosolic (92% identity) and mitochondrial (97% identity) SHMT isozymes (Martini, F., Angelaccio, S., Pascarella, S., Barra, D., Bossa, F., and Schirch, V . (1987) J . Biol . Chem . 262, 5499-5509; Martini, F., Maras, B., Tanci, P., Angelaccio, S., Pascarella, S., Barra, D., Bossa, F., and Schirch, V . (1989) J . Biol . Chem . 264, 8509-8519) . SHMT is a highly conserved protein with the human isozymes retaining about 43% sequence identity with the E . coli protein . The human cytosolic and mitochondrial SHMT genes were localized to chromosome regions 17p11.2 and 12q13, respectively . The high degree of nucleotide sequence identity between the two isozymes, and the presence of keratin genes in both chromosomal regions, is consistent with these regions of chromosome 12 and 17 arising by a duplication event.

J Biol Chem, 1993 Jun 5, 268(16), 11830 - 7
Bidirectional excision in methyl-directed mismatch repair; Grilley M et al.; Using electron microscopy and indirect end-labeling methods, we have examined excision tracts produced by the Escherichia coli methyl-directed mismatch repair system on a closed circular G-T heteroduplex that contains a single d(GATC) site . Despite differing polarities of the unmodified strand in the two hemimethylated derivatives of the heteroduplex, that portion of the unmethylated strand spanning the shorter path between the d(GATC) site and mismatch is targeted for excision in both cases . Mismatch-provoked excision occurring on both hemimethylated DNAs requires DNA helicase II, but exonuclease requirements for the reaction depend on heteroduplex orientation . When the d(GATC) sequence on the unmodified strand resides 3' to the mismatch as viewed along the shorter path, excision requires exonuclease I . Excision occurring on the alternate hemimethylated heteroduplex depends on the 5'--> 3' hydrolytic activity of exonuclease VII . Coupled with the previous demonstration that repair initiates via the mismatch-provoked, MutHLS-dependent incision of the unmethylated strand at a d(GATC) sequence (Au, K.G., Welsh, K., and Modrich, P . (1992) J . Biol . Chem . 267, 12142-12148), these findings indicate an excision mechanism in which helicase II displacement renders the incised strand sensitive to the appropriate single-strand exonuclease . Our data imply that hydrolysis commences at the d(GATC) site, proceeds to a point beyond the mismatch, and terminates at a number of discrete sites within a 100-nucleotide region just beyond this site . The extent of excision is therefore controlled by one or more components of the repair system.

J Biol Chem, 1993 Jun 5, 268(16), 11792 - 7
Characterization of yeast plasma membrane H(+)-ATPase mutant pma1-A135V and its revertants; Na S et al.; An A135V substitution in the first transmembrane segment of the yeast plasma membrane H(+)-ATPase (PMA1) confers cellular resistance to hygromycin B, exhibits growth sensitivity to low external pH, and results in a defective enzyme that hydrolyzes ATP at 33% of wild type level . The importance of the A135 residue was probed genetically by analysis involving both site-directed mutagenesis and randomly generated second-site intragenic suppressor mutations . No other amino acid at position 135 gave either the wild type phenotype or the normal enzyme activity of A135 . Substitutions with the bulkier amino acid residues A135L, A135I, and A135F produced more severe cellular phenotypes than the original A135V mutation . The substitution of the smaller side chain residue Gly was also a mutant, although not as severe as the A135V mutant . The introduction of a bulky Trp or a polar Ser residue produced dominant lethality, while charged amino acids produced recessive lethality . Reduced rates of proton transport measured by acidification of the medium by whole cells correlate closely with the severity of cellular phenotype . Some of the mutant enzymes exhibit an apparent instability in vitro . Thus, the localized structure around A135 is highly constrained . The cellular sensitivity to low external pH of the A135V mutant was used to select intragenic revertants . Most full revertants (low pHR, HygS) restored A135, but second-site mutations in putative transmembrane segments 2 (V146I and V157F) and 4 (L327V) were also observed . Two partial revertants (low pHR, HygR) have secondary mutations at S660C or a double change at F611L-S660F in the putative ATP binding domain . These results provide additional evidence for functional coupling between the cytoplasmic domain catalyzing ATP hydrolysis and transmembrane helices 1 and 2.

J Biol Chem, 1993 Jun 5, 268(16), 11785 - 91
DNA polymerase III accessory proteins . V . Theta encoded by holE; Studwell-Vaughan PS et al.; The polIII core subassembly of DNA polymerase III holoenzyme is composed of the alpha (DNA polymerase), epsilon (editing exonuclease), and theta subunits . We have identified holE encoding theta (8.6 kDa) at 40.4 min, expressed and purified 300 mg of theta, and have studied its function by constituting the polIII core from pure alpha, epsilon, and theta subunits . The theta subunit binds the epsilon proofreader tightly, but it does not form a detectable complex with alpha . The epsilon subunit also binds to alpha (Maki, H., and Kornberg, A . (1987) Proc . Natl . Acad . Sci . U . S . A . 84, 4389-4392) . Hence, the subunit arrangement of the polIII core is linear, alpha epsilon theta . Interaction of theta with epsilon slightly stimulated epsilon in excision of a 3' terminal mismatched nucleotide, suggesting a possible role for theta in fidelity.

J Biol Chem, 1993 Jun 5, 268(16), 11779 - 84
DNA polymerase III accessory proteins . IV . Characterization of chi and psi; Xiao H et al.; The gamma complex (gamma delta delta' chi psi) subassembly of the Escherichia coli replicase initiates processive replication upon assembling the ring-shaped beta subunit around DNA . The beta ring acts as a clamp to hold the replicase down to DNA for highly processive synthesis . In this report we characterize the chi and psi subunits of the gamma complex . Both chi and psi are monomeric, and they associate to form a 1:1 complex . The chi subunit does not form a complex with gamma, but psi binds gamma tightly thereby acting as a bridge to assimilate chi into the gamma complex structure . The psi subunit stimulated the ATPase and replication activities of gamma . The chi subunit only stimulated the activities of gamma when the psi subunit was also present thus reflecting the structure where psi bridges the interaction of chi with gamma.

J Biol Chem, 1993 Jun 5, 268(16), 11758 - 65
DNA polymerase III accessory proteins . I . holA and holB encoding delta and delta'; Dong Z et al.; The genes encoding the delta and delta' subunits of the 10-subunit Escherichia coli replicase, DNA polymerase III holoenzyme, have been identified and sequenced . The holA gene encoding delta is located downstream of rlpB at 15.2 min and predicts a 38.7 kda protein . The holB gene encoding delta' is located at 24.3 min and predicts a 36.9-kDa protein . Hence the delta and delta' subunits are unrelated proteins encoded by separate genes . The genes have been used to express and purify delta and delta' in quantity . The predicted amino acid sequence of delta' is homologous to the sequences of the tau and gamma subunits revealing a large amount of structural redundancy within the holoenzyme.

J Biol Chem, 1993 Jun 5, 268(16), 11703 - 10
On the mechanism of frameshift (deletion) mutagenesis in vitro; Shibutani S et al.; An experimental system has been developed to quantify frameshift deletions and base substitutions formed during DNA synthesis in vitro . Oligodeoxynucleotides, modified site-specifically with acetylaminofluorene or other adducts and lesions, were used as templates in primer extension reactions catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I . The influence of DNA sequence context on frameshift mutagenesis was determined by modifying systematically the bases flanking the lesion . Frequencies of nucleotide insertion opposite the lesion and chain extension from the 3'-primer terminus were established by steady state kinetic analysis . The ability of a damaged nucleotide to generate one-base and two-base frameshift deletions was determined primarily by two parameters: the nature of the base inserted opposite the adduct with respect to the sequence context in which the lesion is embedded and the overall rate of translesional DNA synthesis . Frameshift deletions generated during DNA synthesis were greatly enhanced in the absence of proofreading exonuclease . Misinsertion of bases opposite the lesion precedes misalignment of the template-primer . Extending on earlier studies (Kunkel, T . A . (1990) Biochemistry 29, 8003-8011), a model has been proposed and used in various sequence contexts to predict the propensity of aminofluorene adducts, exocyclic DNA adducts, 8-oxopurines, and synthetic abasic sites to generate frameshift deletions in vitro.

J Biol Chem, 1993 Jun 5, 268(16), 11604 - 9
Cysteine phosphorylation of the glucose transporter of Escherichia coli; Meins M et al.; The glucose transporter (IIBCGlc/IIAGlc complex) of the bacterial phosphotransferase system couples vectorial translocation to phosphorylation of the transported sugar . The IIAGlc subunit transfers the phosphoryl group from the phosphoryl carrier protein P-HPr to the IIBCGlc subunit . IIBCGlc translocates and phosphorylates glucose . The site of IIBCGlc phosphorylation is cysteine 421 as shown by mass spectrometric and biochemical analyses of phosphorylated peptides . Site-directed mutagenesis of Cys421 (C421S) afforded a stable but completely inactive protein (Nuoffer, C., Zanolari, B., and Erni, B . (1988) J . Biol . Chem . 263, 6647-6655) . Cys421 is located in the C-terminal cytoplasmic domain of the IIBCGlc subunit in a sequence context (LDACITRL) which is well conserved in other transporters of the bacterial phosphotransferase system . Phosphocysteine has been shown previously to be the catalytic intermediate of the mannitol transporter (Pas, H . H., Meyer, G . H., Kruizinga, W . H., Tamminga, K . S., van Weeghel, R . P., and Robillard, G . T.

J Biol Chem, 1993 Jun 5, 268(16), 11599 - 603
Membrane topology of the glucose transporter of Escherichia coli; Buhr A et al.; The glucose transporter of the bacterial phosphotransferase system couples translocation with phosphorylation of the substrate in a 1:1 stoichiometry . It consists of a transmembrane subunit (IIBCGlc) and a hydrophilic subunit (IIAGlc) . Both subunits are transiently phosphorylated . The IIBCGlc subunit is 477 residues long and consists of two domains . The amino-terminal hydrophobic domain is involved in glucose binding and translocation, the carboxyl-terminal domain contains the phosphorylation site (Cys421) . Protein fusions between IIBCGlc and beta-galactosidase (LacZ) as well as alkaline phosphatase (PhoA) were analyzed to determine the membrane topology of the IIBCGlc subunit . The protein fusions were generated by progressively deleting ptsG from its 3' end and ligating the truncated gene to lacZ and 'phoA lacking promoter and leader sequences . LacZ fusions of high activity (32 out of 54) occur at the amino and carboxyl termini and three internal clusters, and 41 active PhoA fusions occur in four internal clusters . Accordingly the hydrophobic domain of IICGlc (residues 19-336) is suggested to contains eight membrane-spanning segments, with the amino terminus and the COOH-terminal hydrophilic domain (IIBGlc) located on the cytoplasmic face of the membrane . A sequence comparison of IIBCGlc with three related proteins indicates that the periplasmic loops differ in size and sequence while the cytoplasmic loops are better conserved.

J Mol Biol, 1993 Jun 5, 231(3), 621 - 33
The mutant recBCD enzyme, recB2109CD enzyme, has helicase activity but does not promote efficient joint molecule formation in vitro; Eggleston AK et al.; The Escherichia coli recB2109CD enzyme displays a defect in homologous recombination . In vitro, it possesses significant levels of non-specific nuclease activity but is deficient in chi-dependent nicking activity . To determine whether an alteration in helicase activity contributes further to its in vivo defect, the ability of recB2109CD enzyme to unwind dsDNA was examined . The mutant enzyme is able to unwind DNA but has a kcat which is one-third that of the wild-type enzyme . While the Km for DNA ends of the wild-type and mutant enzymes at low NaCl concentrations are essentially equivalent, the Km for ATP of recB2109CD enzyme is nearly six times greater . The processivity of unwinding (i.e . the average length of DNA unwound before recB2109CD enzyme dissociates from the DNA substrate) at 1 mM-Mg2+ ion and 1 mM-ATP is approximately 13 kb/end, whereas that of wild-type recBCD enzyme is 30 kb/end . In an assay which requires the co-ordinate actions of the recBCD, recA, and SSB proteins, joint molecule formation in the presence of recB2109CD enzyme is up to sixfold slower and proceeds to a lower extent than that mediated by the wild-type enzyme . We conclude that although the reduced helicase activity of the mutant recBCD enzyme may contribute to its recombination deficiency, its defect in the chi-dependent attenuation of non-specific nuclease activity is primarily responsible for the recombination-deficiency of E . coli strains bearing the recB2109 mutation.

J Mol Biol, 1993 Jun 5, 231(3), 605 - 20
Biochemical characterization of a mutant recBCD enzyme, the recB2109CD enzyme, which lacks chi-specific, but not non-specific, nuclease activity; Eggleston AK et al.; RecBCD enzyme of Escherichia coli is a DNA helicase which also possesses ATP-dependent nuclease activities . We have purified a mutant recBCD enzyme, designated recB2109CD enzyme, and have examined the nuclease activities of this protein in vitro to determine whether any alteration in these activities is responsible for the recombination-deficient phenotype of the recB2109 strain . The recB2109CD enzyme possesses all of the non-specific nuclease activities (dsDNA exonuclease and ssDNA exo- and endonuclease) associated with wild-type recBCD enzyme although they are reduced approximately 2 to 3-fold relative to the wild-type enzyme . The ATP-dependent dsDNA exonuclease activity of recB2109CD enzyme requires significantly higher ATP concentrations for optimal activity when compared to the wild-type enzyme . The ATP-independent ssDNA endonuclease activity of the two enzymes is similar, but the ATP-stimulated ssDNA endonuclease and ATP-dependent ssDNA exonuclease activities of the mutant enzyme are reduced relative to those of wild-type recBCD enzyme . Despite its ability to degrade linear dsDNA non-specifically, recB2109CD enzyme lacks sequence-specific nicking activity at chi sites, which are hotspots for genetic recombination . Since this interaction with chi significantly attenuates the non-specific dsDNA exonuclease activity of wild-type recBCD enzyme, these results suggest that the non-specific dsDNA exonuclease activity of the mutant enzyme cannot be attenuated, with the consequence that a DNA substrate which is suitable for recombination is not produced.

J Biol Chem, 1993 Jun 5, 268(16), 11823 - 9
Methyl-directed mismatch repair is bidirectional; Cooper DL et al.; Methyl-directed mismatch repair is initiated by the mismatch-provoked, MutHLS-dependent cleavage of the unmodified strand at a hemimethylated d(GATC) sequence . This reaction is independent of the polarity of the unmodified strand and can occur either 3' or 5' to the mismatch on the unmethylated strand (Au, K . G., Welsh, K., and Modrich, P . (1992) J . Biol . Chem . 267, 12142-12148) . The overall repair reaction also occurs without regard to polarity of the unmethylated strand . Both hemimethylated configurations of a linear heteroduplex containing a single d(GATC) sequence are subject to methyl-directed correction in Escherichia coli extracts and in a purified repair system . Repair of both heteroduplex orientations requires MutH, MutL, MutS, DNA helicase II, SSB, and DNA polymerase III holoenzyme, but the two substrates differ with respect to exonuclease requirements for correction . When the unmethylated d(GATC) sequence that directs repair is located 5' to the mismatch on the unmodified strand, mismatch correction requires the 5'--> 3' hydrolytic activity of exonuclease VII or RecJ exonuclease . Repair directed by an unmodified d(GATC) sequence situated 3' to the mismatch depends on the 3'--> 5' activity of exonuclease I . Specific requirements for these activities are evident with circular heteroduplexes containing a single asymmetrically placed d(GATC) sequence, with the requirement for a 5'--> 3' or 3'--> 5' hydrolytic activity being determined by the orientation of the unmethylated strand along the shorter path joining the two sites in the DNA circle . This observation suggests that the methyl-directed repair system utilizes the proximal d(GATC) sequence to direct correction . To our knowledge, these experiments represent the first instance in which exonuclease I, exonuclease VII, and RecJ have been implicated in a particular DNA metabolic pathway.

J Biol Chem, 1993 Jun 5, 268(16), 11773 - 8
DNA polymerase III accessory proteins . III . holC and holD encoding chi and psi; Xiao H et al.; Genes encoding the chi and psi accessory proteins of the DNA polymerase III holoenzyme replicase of Escherichia coli have been identified, sequenced, and used to express and purify both chi and psi in quantity . The holC gene encoding chi is located between the xerB and valS genes at 96.5 min on the chromosome; it encodes a 147-amino acid protein of 16.6 kDa . holD encoding psi lies upstream of rimI at 99.3 min and encodes a 137-amino acid protein of 15.2 kDa . The genes have been cloned into expression vectors, and both chi and psi have been purified in quantity . The accompanying report characterizes the function and physical interactions of chi and psi with other holenzyme subunits (Xiao, H., Dong, Z., and O'Donnell, M . (1993) J . Biol . Chem . 268, 11779-11784).

J Biol Chem, 1993 Jun 5, 268(16), 12129 - 35
Avian 3-hydroxy-3-methylglutaryl-CoA synthase . Characterization of a recombinant cholesterogenic isozyme and demonstration of the requirement for a sulfhydryl functionality in formation of the acetyl-enzyme reaction intermediate; Misra I et al.; cDNA encoding avian liver hydroxymethylglutaryl-CoA synthase has been cloned into a pET vector, and the resulting expression plasmid has been used to transform Escherichia coli BL21 (DE3) . Heterologous expression of hydroxymethylglutaryl-CoA synthase occurs upon growth of this bacterial strain in the presence of isopropyl-1-thio-beta-D-galactopyranoside, with the target enzyme representing over 20% of total cellular protein . Recombinant enzyme is soluble and stable in crude E . coli extracts, facilitating its isolation in homogeneous form . With respect to specific activity, acylation stoichiometry, Km,Ac-CoA, and binding of a spin-labeled substrate analog, the recombinant enzyme is equivalent to avian enzyme, suggesting its utility for mechanistic and structural studies . Our earlier prediction that this avian cDNA encodes the cholesterogenic cytosolic isozyme is supported by a series of experimental observations . Upon SDS-polyacrylamide gel electrophoresis, the recombinant synthase exhibits mobility in agreement with the 57.6-kDa deduced molecular mass, which exceeds the 53-kDa estimate and experimental observation for the ketogenic mitochondrial isozyme . Activity of the recombinant synthase is stimulated by Mg2+, as predicted for the cholesterogenic cytosolic isozyme and in contrast to the inhibition observed for the mitochondrial isozyme . Although antibody prepared against avian mitochondrial synthase effectively detects both avian mitochondrial and recombinant synthases on Western blots, antibody prepared against rodent cytosolic synthase discriminates between the two proteins, sensitively detecting recombinant enzyme while reacting poorly with authentic mitochondrial enzyme . Directed mutagenesis of the recombinant synthase has been performed to produce a C129S variant, in which the sulfhydryl previously implicated in formation of the acetyl-S-enzyme reaction intermediate is replaced by a hydroxyl group . EPR measurements on the binary C129S-spin-labeled acyl-CoA complex demonstrate that the mutant's substrate binding site is unperturbed in comparison with wild-type protein . These data illustrate the utility of spin-labeled substrate analogs as tools to stringently evaluate the structural integrity of engineered proteins . C129S is catalytically inactive (10(5)-fold decrease in kcat) despite retaining the ability to form noncovalent complexes with acetyl-CoA or a spin-labeled acetyl-CoA analog . The demonstrated failure of C129S to form a covalent acyl-O-enzyme species accounts for these observations; data derived from experiments performed with a C129G mutant confirm this conclusion . These results distinguish hydroxymethylglutaryl-CoA synthase from beta-ketoacyl thiolase.(ABSTRACT TRUNCATED AT 400 WORDS)

J Mol Biol, 1993 Jun 5, 231(3), 678 - 88
Synthesis of proteins in Escherichia coli is limited by the concentration of free ribosomes . Expression from reporter genes does not always reflect functional mRNA levels; Vind J et al.; Induction of beta-galactosidase from high copy-number plasmids was found to reduce the synthesis of other cellular proteins in Escherichia coli . The reduction depends on the protein in question and on the induction level of the beta-galactosidase . It could be observed transiently within one minute after induction and in some cases also during steady-state induction . Our interpretation is that the concentration of the free ribosomal subunits decreases after induction, leading to an increased competition among the individual ribosome binding sites for ribosomes . The immediate reduction in the synthesis individual proteins after induction of beta-galactosidase was used as an assay to measure in vivo the efficiency of a ribosome binding site . These efficiencies were compared to the calculated affinities between the ribosome binding site of specific mRNA species and the 3' end of 16 S RNA . For several mRNAs with similar Shine-Dalgarno sequences, the sensitivity to competition differed twofold . Our results show, that both transiently during induction of lacZ and also at very high steady-state expression levels, the expression from reporter genes, including the lacZ gene itself, does not reflect the levels of the mRNAs in a simple way.

J Mol Biol, 1993 Jun 5, 231(3), 594 - 604
Identification of a region within M1 RNA of Escherichia coli RNase P important for the location of the cleavage site on a wild-type tRNA precursor; Kirsebom LA et al.; To investigate the function of the catalytic subunit of Escherichia coli RNase P, M1 RNA, we studied cleavage by different M1 RNA mutants of wild-type precursors to tRNA(TyrSu3), tRNA(His) and tRNA(SerSu1) . We showed that deletion or substitution of the conserved nucleotides G291, G292, U294 and A295 in M1 RNA resulted in a shift of the cleavage site for the tRNA(SerSu1) precursor, whereas the other two tRNA precursors were cleaved at the normal position . By using chimeric tRNA precursors in which the acceptor-stem of the tRNA(TyrSu3) precursor was replaced by the acceptor-stem derived from the tRNA(SerSu1) precursor, we showed that the aberrant cleavage by M1 RNA mutants could be reversed by substituting the nucleotide at position -2 in one of the chimeric precursors . These results suggest, in support of our previous findings, that different tRNA precursors are processed differently and that the primary structure of the amino acid acceptor-stem of a tRNA precursor plays a significant role in the RNase P cleavage reaction . Furthermore, in agreement with a previous report, a truncated tRNA(TyrSu3) precursor was cleaved aberrantly by a mutant M1 RNA in which the nucleotide at position 92 had been deleted . In contrast, a corresponding truncated tRNA(SerSu1) precursor was cleaved at the same position both by the wild-type and by this mutant M1 RNA . We conclude that not only the primary structures of the acceptor-stems of tRNA precursors, but also the primary structures in different regions of M1 RNA determine the location of the cleavage site on various tRNA precursors . Here we have identified the region G291 to A295 to be important for the selection of the cleavage site on the tRNA(SerSu1) precursor . We discuss the possibility that the conformation of M1 RNA in the enzyme-substrate complex is dependent on the identity of the substrate.

J Mol Biol, 1993 Jun 5, 231(3), 569 - 80
Transcriptional slippage during the transcription initiation process at a mutant lac promoter in vivo; Xiong XF et al.; A C.G to A.T transversion at position +10 of the lac promoter activates a nascent sigma 70-dependent promoter (the +10A promoter) . The lac +10A promoter has two unusual properties; it programs a large family of transcripts with multiple 5' ends, and its sequence bears little resemblance to other sigma 70-dependent promoters . The 5' end of the +10A in vivo mRNA was determined to contain oligo(U) sequences of varying lengths suggesting that the true start site was at a run of three T.A base-pairs located 20 to 22 bp downstream of the lac wild-type promoter start site, and that the transcription initiation process involved a transcriptional slippage event (which resulted in multiple rU incorporation) . Only mutations at or near the start site and those deletions that changed the location of the start site abolished this transcriptional slippage property of the transcription initiation process . This transcriptional slippage was also found to be promoter independent because changing the lac UV5 start site to a run of five T.A base-pairs (-1 to +4) resulted in similar transcriptional slippage . Saturated mutagenesis of the +10A promoter identified a potential -10-like region and indicated that sequences immediately upstream of the -10 region contributed to the promoter's activity . Decreasing the weak -35 region homology did not change promoter strength; however, introduction of the consensus -35 hexamer TTGACA increased expression tenfold . RNA polymerase bound to the +10A promoter partially protects a 20 base-pair sequence from DNase I digestion upstream of the start site . These results suggest that RNA polymerase interacts with the +10A promoter in a different manner from that for the majority of sigma 70 promoters.

J Immunol Methods, 1993 Jun 4, 162(1), 1 - 7
A rapid and simple microfluorometric phagocytosis assay; Wan CP et al.; A microfluorometric method for phagocytosis study has been developed using fluorescein conjugated Escherichia coli K-12 particles . This technique is based on the uptake of fluorescent particles and quenching of extracellular fluorescence at the end of the assay . A murine macrophage cell line, J774, was used as a phagocyte model . The cells were harvested from tissue culture flasks and adjusted to 1 x 10(6) cells/ml . They were then dispensed into a 96-well tissue culture plate, 100 microliters/well, and incubated at 37 degrees C in 5% CO2 for 1 h to allow cells to adhere to the bottom of the wells . The culture medium was aspirated and 100 microliters of fluorescent E . coli particles suspended in Hanks' buffer were added . The plates were further incubated for various time periods . Buffer solution in the wells was removed by aspiration . Extracellular fluorescence was then quenched by adding 100 microliters of trypan blue (250 micrograms/ml, pH 4.4) . The dye was removed after 1 min . The intensity of fluorescence associated with intracellular fluorescent particles was measured directly in the wells using a computerized microplate fluorometer at 485 nm excitation and 530 nm emission . This assay provided a rapid and objective measurement of phagocytosis activity . Using a cultured cell line and a 96-well microtiter plate format, this assay can facilitate the screening of a large number of various biological and pharmacological substances for their modulating effects on phagocytosis.

J Chromatogr, 1993 Jun 4, 639(1), 67 - 74
Increased yield of homogeneous HIV-1 reverse transcriptase (p66/p51) using a slow purification approach; Bhikhabhai R et al.; A chromatographic procedure to purify recombinant reverse transcriptase (RT) from human immunodeficiency virus-1 is reported . A bacterial system which expressed large amounts of p66 RT polypeptide was used . The purification scheme was optimized for high-yield production of homogeneous p66/p51 RT using a combination of chromatographic matrices in the following order: Q-Sepharose, heparin-Sepharose, phenyl-Sepharose, S-Sepharose, Poly(A)-Sepharose and Q-Sepharose . The p66 polypeptide remained intact after the first chromatographic step on Q-Sepharose, where it was recovered in the non-adsorbed fraction . A high yield of p66/p51 RT was obtained when the time from application to elution of heparin-Sepharose in the second chromatographic step was prolonged . Phenyl-Sepharose was used in the next chromatographic step to separate the heterodimeric forms of RT from p66 RT on the basis of hydrophobicity . The chromatography on S-Sepharose resolved the major heterodimeric form, p66/p51, from other heterodimeric variants . Further purification was done by affinity chromatography on Poly(A)-Sepharose followed by anion-exchange chromatography on Q-Sepharose . Amounts of 25-35 mg of the pure heterodimer p66/p51 RT were recovered from 50 g of bacterial cells.

Epidemiol Infect, 1993 Jun, 110(3), 575 - 81
Genetic analysis of Escherichia coli from porcine postweaning diarrhoea; Hampson DJ et al.; A total of 79 Australian isolates of beta-haemolytic Escherichia coli from cases of porcine postweaning diarrhoea (PWD), and 18 isolates of serotype O 149:K91:K88 (F4) from unweaned pigs from Australia, Indonesia and Denmark, were examined by multilocus enzyme electrophoresis . These were divided into 57 electrophoretic types (ETs), with an overall mean genetic diversity per enzyme locus of 0.466 . This value closely resembled that previously recorded for the whole species . Not only was the collection diverse, but there was considerable genetic heterogeneity amongst PWD isolates of the same serogroup . Isolates from serogroups O 8 and O 138 were most varied, whilst many from serogroups O 141 and O 149 were more closely related . In contrast, the isolates from the unweaned pigs all belonged to only one ET.

Epidemiol Infect, 1993 Jun, 110(3), 469 - 75
Phage typing of Vero cytotoxin-producing Escherichia coli O 157 isolated in the United Kingdom: 1989-91; Frost JA et al.; Between 1989 and 1991 a total of 1092 Vero cytotoxin-producing Escherichia coli O 157 isolated in the United Kingdom were phage typed in the Laboratory of Enteric Pathogens (LEP) . Twenty-three phage types was identified, the most frequent being types 2 (36.1%), 49 (29.6%), 1 (10.3%) and 4 (8.9%) . Although isolations of O 157 VTEC have increased each year from 1 in 1982 to 532 in 1991, the predominant phage types have remained unchanged although the proportion of strains belonging to types 2 and 49 has increased . O 157 VTEC from 17 outbreaks were phage typed during this period with phage type 49 predominating (7 of 17 outbreaks).

Protein Expr Purif, 1993 Jun, 4(3), 215 - 22
Chemical synthesis, molecular cloning, overexpression, and site-directed mutagenesis of the gene coding for pumpkin (Curcubita maxima) trypsin inhibitor CMTI-V; Wen L et al.; The gene encoding for a pumpkin (Curcubita maxima) trypsin inhibitor CMTI-V was synthesized chemically . The synthetic gene was prepared from four overlapping oligonucleotides by overlapping extension . The synthetic gene was amplified by polymerase chain reaction, cloned into a T7 expression vector and expressed in Escherichia coli as a fusion protein . The clone, namely 70-1, encoded a fusion protein containing 7 amino acid residues of the N-terminus of the bacterial protein rho 10 and the entire 68 residues of CMTI-V . The wild-type fusion protein constituted approximately 15% of the total bacterial protein mass and was purified to homogeneity in a single step by antibody affinity chromatography . The wild-type fusion protein possesses inhibitory activity toward trypsin and beta-Factor XIIa, but to a lesser extent when compared to the natural CMTI-V . A mutant, T43A, in which threonine at position 43 (P2 position) was replaced by alanine, was constructed . This mutant showed considerably lower specific inhibitory activity toward both trypsin and beta-Factor XIIa.

DNA Cell Biol, 1993 Jun, 12(5), 371 - 9
Construction and function of fusion enzymes of the human cytochrome P450scc system; Harikrishna JA et al.; Type I cytochrome P450 enzyme systems are found in mitochondria and consist of three components, a flavoprotein (adrenodoxin reductase, AdRed), an iron-sulfur protein (adrenodoxin, Adx), and the cytochrome P450; Type II P450 enzymes in the endoplasmic reticulum consist of only two components, P450 reductase and the P450 . Genetically engineered fusion proteins of Type II cytochromes P450 (such as steroid 17 alpha- and 21-hydroxylases) produce enzymes with increased activity . To test the consequences of constructing fusions of Type I enzymes, we built fusion proteins based on the cholesterol side-chain cleavage enzyme, P450scc . We constructed expression vectors for three fusion proteins: NH2-P450scc-AdRed-COOH, P450-AdRed-Adx, and P450scc-Adx-AdRed . The various components were assembled from cassette-like cDNA fragments modified and amplified by polymerase chain reaction (PCR), subcloned into a specially tailored vector, and linked by DNA segments encoding hydrophilic linker peptides . The final vectors were transfected into COS-1 cells, incubated with 22R-hydroxycholesterol, and assayed by the secretion of pregnenolone into the culture medium . Triple transfection of three individual vectors expressing P450scc, AdRed, and Adx yielded more pregnenolone than did transfection with P450scc alone . The P450scc-AdRed and P450scc-Adx-AdRed fusion proteins produced levels of pregnenolone similar to the control triple transfection . However, the P450scc-AdRed-Adx fusion produced substantially more pregnenolone, having an apparent Vmax of 9.1 ng of pregnenolone produced per milliliter of medium per 24 hr, compared to a Vmax of 1.7 ng/ml per day for the triple transfection.(ABSTRACT TRUNCATED AT 250 WORDS)

J Clin Invest, 1993 Jun, 91(6), 2754 - 60
Endotoxin-induced pulmonary dysfunction is prevented by C1-esterase inhibitor; Guerrero R et al.; In septic shock, hypotension, disseminated intravascular coagulation, and neutrophil activation are related to the activation of the blood coagulation contact system . This study evaluates in dogs the effect of the C1-esterase inhibitor (C1-INH), a main inhibitor of the blood coagulation contact system, on the cardiovascular and respiratory dysfunction associated with endotoxic shock . Two groups were included: controls, which received Escherichia coli endotoxin, and a C1-INH group in which C1-INH was infused before E . coli endotoxin administration . In both groups, endotoxin produced hypodynamic shock; however, the decrease in the systolic index and the ventricular systolic work indexes were greater in controls than the C1-INH group . In controls, the arterial O2 partial pressure decreased by 30% and the alveolo-arterial O2 difference increased by 625%, these parameters remained unchanged in the C1-INH group . Hypoxemia was associated with increased intrapulmonary shunt, decreased blood coagulation contact factors, and decreased C3c . In contrast, C1-INH administration prevented endotoxin-induced hypoxemia, the increase in intrapulmonary shunt, and the decrease in blood coagulation contact factors . This study shows that, in dogs with endotoxic shock, pulmonary dysfunction is associated with an activation of the blood coagulation contact phase system . An inhibition of this system by C1-INH prevented the hypoxemia induced by endotoxic shock.

Hepatology, 1993 Jun, 17(6), 1062 - 5
The role of 16,16-dimethyl prostaglandin E2 on the intrahepatic biliary branches in dogs; Ohta T et al.; We studied the effects of the oral administration of a stable prostaglandin E2 analog, 16,16-dimethyl prostaglandin E2, on the intrahepatic biliary branches in a canine model . Obstructive cholestasis with a bacterial infection was induced surgically in two liver lobes in healthy mongrel dogs, and 16,16-dimethyl prostaglandin E2 was administered orally . We examined the morphological changes in the intrahepatic biliary branches and quantitatively estimated density of mucus-producing glandular elements in the ductal wall by counting these glands per unit area . Dogs treated with 16,16-dimethyl prostaglandin E2 (group 1) demonstrated fibrous thickening of the ductal wall, moderate infiltration by inflammatory cells and severe adenomatous hyperplasia of the bile duct epithelium, including striking proliferation of the mucous glands . The mean number of these mucous glands per unit area (4 mm2) was 43.0 +/- 9.0 (mean +/- S.D.; range = 36 to 56) . In contrast, in a control group whose members did not receive 16,16-dimethyl prostaglandin E2 (group 2), the mean number of mucous glands per unit area was 19.4 +/- 8.0 (range = 10 to 29), significantly lower than that in group 1, although histological examination revealed chronic inflammation in the region of the large bile duct similar to that in group 1 . These findings suggest that the increase in the number of mucous glands that typically occur in the setting of bile stasis and biliary infection is enhanced by 16,16-dimethyl prostaglandin E2.

Eur J Biochem, 1993 Jun 1, 214(2), 451 - 7
Oligomerization of the Nef protein from human immunodeficiency virus (HIV) type 1; Kienzle N et al.; The nef genes, derived from two different human immunodeficiency-virus-type-1 (HIV-1) strains, were expressed in procaryotic cells (Escherichia coli) and in eucaryotic cells (insect cells infected with nef-containing baculovirus) . The oligomerization of recombinant Nef protein was studied by NMR spectroscopy and immunoblotting under various experimental conditions . 1H-NMR spectroscopy shows that native folded protein has the tendency to polymerize under low-salt conditions . These oligomers become covalently linked by disulfide bonds after decreasing the reduction potential, a process which is fully reversible . Cross-linking studies with bis(sulfo-succinimidyl)suberate and alkylation with iodoacetic acid under non-reducing and reducing conditions document for the first time that Nef can also form homomeric structures including monomers, dimers, trimers and tetramers in cell lysates and intact cells . We found disulfide-linked as well as non-covalently associated oligomers . Since the Nef molecules are not exclusively found in the cytoplasm of HIV infected cells and since the reduced glutathione concentration in lymphocytes of virus infected persons is known to be unusually low, it might be possible that these Nef oligomers have a biological function in vivo as well.

Br J Cancer, 1993 Jun, 67(6), 1196 - 202
Expression in mammalian cells of the Escherichia coli O6 alkylguanine-DNA-alkyltransferase gene ogt reduces the toxicity of alkylnitrosoureas; Harris LC et al.; V79 Chinese hamster cells expressing either the O6-alkylguanine-DNA-alkyltransferase (ATase) encoded by the E . coli ogt gene or a truncated version of the E . coli ada gene have been exposed to various alkylnitrosoureas to investigate the contribution of ATase repairable lesions to the toxicity of these compounds . Both ATases are able to repair O6-alkylguanine (O6-AlkG) and O4-alkylthymine (O4-AlkT) but the ogt ATase is more efficient in the repair of O4-methylthymine (O4-MeT) and higher alkyl derivatives of O6-AlkG than is the ada ATase . Expression of the ogt ATase provided greater protection against the toxic effects of the alkylating agents then the ada ATase particularly with N-ethyl-N-nitrosourea (ENU) and N-butyl-N-nitrosourea (BNU) to which the ada ATase expressing cells were as sensitive as parent vector transfected cells . Although ogt was expressed at slightly higher levels than the truncated ada in the transfected cells, this could not account for the differential protection observed . For-N-methyl-N-nitrosourea (MNU) the increased protection in ogt-transfected cells is consistent with O4-MeT acting as a toxic lesion . For the longer chain alkylating agents and chloroethylating agents, the protection afforded by the ogt protein may be a consequence of the more efficient repair of O6-AlkG, O4-AlkT or both of these lesions in comparison with the ada-encoded ATase.

Arch Biochem Biophys, 1993 Jun, 303(2), 274 - 80
Escherichia coli leader peptidase: production of an active form lacking a requirement for detergent and development of peptide substrates; Kuo DW et al.; The report by Zimmermann et al . (J . Biol . Chem . 257, 1982, 6529-6536) that the active site of Escherichia coli leader peptidase (LPase) is located in the periplasm led us to explore the possibility that soluble, active short forms of LPase could be produced . Detergent-free delta 2-75 mutant protein (LPase-sf) lacking the two N-terminal transmembrane spanning and the cytoplasmic domains was produced in high yield . The mass of the protein determined by electrospray ionization mass spectrometry was 27,952 amu . The increase of 42 amu over that predicted by the expected amino acid sequence indicates that the N-terminus of LPase-sf is acetylated . This is consistent with the inability to obtain an N-terminal sequence . LPase-sf lacks the site of autolysis present in LPase, thus circumventing problems associated with enzyme autocatalytic instability . LPase-sf and wild type LPase displayed comparable activity versus two peptide substrates . The peptides, based upon the cleavage sites of procoat and the precursor of maltose binding protein, were processed at the expected sites . In addition, the activity of both LPase's was not inhibited by classical inhibitors of the four classes of proteases . LPase-sf displayed similar activity in the presence and absence of detergent . Wild type LPase displayed specificity for alanine in the P1 subsite of the peptide WSASALX*KI and did not hydrolyze peptides with glycine, valine, or serine in that position . The availability of a detergent-free active form of LPase should facilitate mechanistic and structural studies.

J Gen Virol, 1993 Jun, 74 ( Pt 6), 1157 - 62
The N-terminal protein of the polyprotein encoded by the potyvirus tobacco vein mottling virus is an RNA-binding protein; Brantley JD et al.; The first predicted polypeptide encoded by the potyvirus tobacco vein mottling virus (TVMV) is a highly positively charged protein of predicted M(r) 29K that functions as a protease to perform the first predicted cleavage in the potyvirus polyprotein . We expressed this protein (P1pro) fused with glutathione S-transferase (GST) and purified the fusion protein from engineered Escherichia coli . We found that the intact fusion protein, as well as samples in which the P1pro portion was liberated from GST by pretreatment with thrombin, was able to bind RNA . Binding activity was optimal at relatively high KCl concentrations, suggesting an interaction dependent on a specific protein structure and not just on the binding of the negatively charged phosphate backbone by the positively charged P1pro polypeptide . The TVMV P1pro preferred ssRNA over DNA or dsRNA, and showed a possible preference for sequences containing oligo(G) tracts . Like other potyvirus-encoded proteins, the TVMV P1pro therefore possesses more than one demonstrable biochemical activity and probably plays multiple roles in the TVMV life cycle.

J Bacteriol, 1993 Jun, 175(12), 3900 - 4
Cloning and sequencing of a gene encoding carminomycin 4-O-methyltransferase from Streptomyces peucetius and its expression in Escherichia coli; Madduri K et al.; Sequence analysis of a portion of the Streptomyces peucetius daunorubicin biosynthetic gene cluster revealed a complete open reading frame (dnrK) that showed DNA and protein sequence homology to several O-methyltransferases . Expression of dnrK in Streptomyces lividans and Escherichia coli was done to show that this gene codes for carminomycin 4-O-methyltransferase . The deduced carminomycin 4-O-methyltransferase protein shows a conserved nucleotide binding site for its S-adenosyl-L-methionine cofactor.

J Bacteriol, 1993 Jun, 175(12), 3876 - 86
Nucleotide sequences and heterologous expression of tcmG and tcmP, biosynthetic genes for tetracenomycin C in Streptomyces glaucescens; Decker H et al.; The nucleotide sequence of the tcmIII, tcmIc, and tcmVII region of the tetracenomycin (TCM) C gene cluster of Streptomyces glaucescens ETH 22794 (GLA.0) revealed the presence of two genes, tcmP and tcmG . The deduced product of tcmG resembles flavoprotein hydroxylases found in several other bacteria, whereas the predicted amino acid sequence of tcmP is not significantly similar to those of any known proteins in the available data bases . Southern blot hybridization revealed an approximately 180-bp deletion in a tcmIII (tcmG) mutant and a 1,800-bp insertion in a tcmVII (tcmP) mutant . Heterologous expression of tcmG and tcmP in Streptomyces lividans and tcmP in Escherichia coli established that tcmP encodes an O-methyltransferase, catalyzing the methylation of the C-9 carboxy group of TCM E to yield TCM A2, and that tcmG is responsible for the hydroxylation of TCM A2 at positions C-4, C-4a, and C-12a to give TCM C . These are the final two steps of TCM C biosynthesis.

J Bacteriol, 1993 Jun, 175(12), 3790 - 7
Cloning and characterization of ftsN, an essential cell division gene in Escherichia coli isolated as a multicopy suppressor of ftsA12(Ts); Dai K et al.; A new cell division gene, ftsN, was identified in Escherichia coli as a multicopy suppressor of the ftsA12(Ts) mutation . Remarkably, multicopy ftsN suppressed ftsI23(Ts) and to a lesser extent ftsQ1(Ts); however, no suppression of the ftsZ84(Ts) mutation was observed . The suppression of ftsA12(Ts), ftsI23(Ts), and ftsQ1(Ts) suggests that FtsN may interact with these gene products during cell division . The ftsN gene was located at 88.5 min on the E . coli genetic map just downstream of the cytR gene . ftsN was essential for cell division, since expression of a conditional null allele led to filamentation and cell death . DNA sequence analysis of the ftsN gene revealed an open reading frame of 319 codons which would encode a protein of 35,725 Da . The predicted gene product had a hydrophobic sequence near its amino terminus similar to the noncleavable signal sequences found in several other Fts proteins . The presumed extracellular domain was unusual in that it was rich in glutamine residues . A 36-kDa protein that was localized to the membrane fraction was detected in minicells containing plasmids with the ftsN gene, confirming that FtsN was a membrane protein.

EMBO J, 1993 Jun, 12(6), 2503 - 12
DNA looping and the helical repeat in vitro and in vivo: effect of HU protein and enhancer location on Hin invertasome assembly; Haykinson MJ et al.; Site-specific DNA inversion by the Hin recombinase requires the formation of a multicomponent nucleo-protein structure called an invertasome . In this structure, the two recombination sites bound by Hin are assembled together at the Fis-bound recombinational enhancer with the requisite looping of the intervening DNA segments . We have analyzed the role of the HU protein in invertasome assembly when the enhancer is located at variable positions close to one of the recombination sites . In the absence of HU in vitro and in hupA hupB mutant cells in vivo, invertasome assembly is very inefficient when there is < 104 bp of DNA between the enhancer and recombination site . Invertasome assembly in the presence of HU in vitro or in vivo displayed a periodicity beginning with 60 bp of intervening DNA that reflected its helical repeat . The average helical repeat for this DNA region was calculated by autocorrelation and Fourier transformation to be 11.2 bp per turn for supercoiled DNA both in the presence of HU in vitro and in hup+ cells in vivo . HU is the only protein in Escherichia coli that can promote invertasome formation with short DNA lengths between the enhancer and recombination sites . However, the presence of certain polyamines and a protein activity present in HeLa nuclear extracts can efficiently substitute for HU in invertasome assembly . These data support a model in which HU binds non-specifically to the DNA between the enhancer and recombination site to facilitate DNA looping.

EMBO J, 1993 Jun, 12(6), 2495 - 501
Lrp, a major regulatory protein in Escherichia coli, bends DNA and can organize the assembly of a higher-order nucleoprotein structure; Wang Q et al.; Lrp (Leucine-responsive regulatory protein) is a global regulatory protein that controls the expression of many operons in Escherichia coli . One of those operons, ilvIH, contains six Lrp binding sites located within a several hundred base pair region upstream of the promoter region . Analysis of the binding of Lrp to a set of circularly permuted DNA fragments from this region indicates that Lrp induces DNA bending . The results of DNase I footprinting experiments suggest that Lrp binding to this region facilitates the formation of a higher-order nucleoprotein structure . To define more precisely the degree of bending associated with Lrp binding, one or two binding sites were separately cloned into a pBend vector and analyzed . Lrp induced a bend of approximately 52 degrees upon binding to a single binding site, and the angle of bending is increased to at least 135 degrees when Lrp binds to two adjacent sites . Lrp-induced DNA bending, and a natural sequence-directed bend that exists within ilvIH DNA, may be architectural elements that facilitate the assembly of a nucleoprotein complex.

EMBO J, 1993 Jun, 12(6), 2449 - 57
Translational repression of brlA expression prevents premature development in Aspergillus; Han S et al.; The Aspergillus nidulans brlA developmental regulatory locus consists of two overlapping transcription units, brlA alpha and brlA beta, which encode functionally related polypeptides . We used translational fusions between each of the predicted brlA reading frames and the Escherichia coli lacZ gene to test the hypothesis that developmental regulation of brlA alpha and brlA beta expression occurs through different mechanisms . brlA alpha is transcriptionally controlled and a large portion of brlA alpha-directed beta-galactosidase activity is regulated in a brlA-dependent manner . In contrast, brlA beta mRNA is constitutively transcribed but translation of the brlA polypeptide is prevented by the presence of a short open reading frame (microORF) present in the 5' end of brlA beta mRNA . Removing the microORF initiation codon leads to deregulated brlA expression, resulting in an inappropriate activation of development . We propose that one mechanism for developmental induction in A.nidulans involves translational control.

EMBO J, 1993 Jun, 12(6), 2241 - 7
The vacuolar membrane protein gamma-TIP creates water specific channels in Xenopus oocytes; Maurel C et al.; The vacuolar membrane (tonoplast) of higher plant cells contains an abundant 27 kDa protein called TIP (tonoplast intrinsic protein) that occurs in different isoforms and belongs to a large family of homologous channel-like proteins found in bacteria, plants and animals . In the present study, we identified and characterized the function of gamma-TIP from Arabidopsis thaliana by expression of the protein in Xenopus oocytes . gamma-TIP increased the osmotic water permeability of oocytes 6- to 8-fold, to values in the range 1-1.5 x 10(-2) cm/s . Similar results were obtained with the homologous human erythrocyte protein CHIP28, recently identified as the erythrocyte water channel . The bacterial homolog GlpF did not affect the osmotic water permeability of oocytes, but facilitated glycerol uptake, in accordance with its known function . By contrast, gamma-TIP did not promote glycerol permeability . Voltage clamp experiments provided evidence showing that gamma-TIP induced no electrogenic ion transport in oocytes, especially during osmotic challenge that resulted in massive transport of water . These results allow us to conclude that the various protein members of the MIP family have unique and specific transport functions and that the plant protein gamma-TIP likely functions as a water specific channel in the vacuolar membrane.

Circ Shock, 1993 Jun, 40(2), 132 - 8
Oxygen extraction and vascular dilation are dependently increased in skeletal muscle during canine endotoxemia; Hershey JC et al.; The principal aim of these experiments was to evaluate the ability of a skeletal muscle to extract oxygen during endotoxemia, and determine if the decompensatory decrease in skeletal muscle vascular resistance that occurs after exposure to endotoxin is related to muscle oxygen uptake (VO2) . A vascularly isolated denervated canine gracilis muscle was perfused in situ at a constant flow (5-7 mL/min/100 g) . Endotoxemia was induced by a 30-min intravenous infusion of Escherichia coli endotoxin (2 mg/kg) . Perfusion pressure and arteriovenous oxygen difference (a-v O2) were continuously measured, and muscle O2 extraction was calculated (VO2 = flow x a-v O2) . These studies found that gracilis muscle oxygen uptake increased from a resting value of 0.30 mL O2/min/100 g to 0.63 mL O2/min/100 g (111% increase) by 90 min post-endotoxin . The arterial conductance (i.e., arterial dilation) increased 58% during this time . The amount of oxygen the muscle extracts was found to be directly related to the degree of vasodilation (r = .97), and inversely correlated to mean arterial pressure (r = .97) . Pre-dilating the muscle with sodium nitroprusside did not alter oxygen extraction . However, after the introduction of endotoxin, a pre-dilated muscle increased VO2 94% by 90 min . These observations support the concept that endotoxin causes an increase in VO2 without producing a defect in the ability of muscle to extract oxygen . The vasodilation typically observed in skeletal muscle during endotoxemia is not the cause of the increased oxygen uptake . It seems likely that mediators released by endotoxin metabolically stimulate skeletal muscle cells, which increases oxygen demand, thus promoting vasodilation.

Am J Surg, 1993 Jun, 165(6), 681 - 5
Small bowel myoelectric activity in peritonitis; Frantzides CT et al.; Peritonitis is associated clinically with paralytic ileus, but the physiologic mechanisms of the effects of peritonitis on bowel myoelectric activity have not been explored . Bipolar electrodes were inserted into the rats, and myoelectric control recordings were obtained for 4 h/d for 5 consecutive days . Peritonitis was then induced, and myoelectric recordings were again obtained . Each animal served as its own control . Prior to induction of peritonitis (control), phase I, II, and III myoelectric activity was present in all recordings . The cycle duration of the migrating myoelectric complex was 17.17 +/- 0.39 minutes, and the migration velocity of phase III was 0.61 +/- 0.02 cm/min . The most striking feature during peritonitis was the complete inhibition of phase II activity . Phase III activity, however, was present with a cycle duration of 16.69 +/- 0.42 minutes . This study shows that some features of intestinal myoelectric activity (phase III) are preserved during episodes of peritonitis, and others are changed (phase I) or lost (phase II) . Disappearance of phase II activity in this type of ileus emphasizes its importance in normal small bowel motility.

Proc Natl Acad Sci U S A, 1993 Jun 1, 90(11), 5355 - 8
The beta subunit of the mitochondrial processing peptidase from rat liver: cloning and sequencing of a cDNA and comparison with a proposed family of metallopeptidases; Paces V et al.; Most nuclearly encoded mitochondrial proteins are synthesized with amino-terminal leader peptides that are removed by the mitochondrial processing peptidase (MPP) after translocation . Earlier we reported cloning and sequencing of a cDNA for the larger subunit (MPP alpha subunit) of this enzyme from rat liver mitochondria . We have now completed the cloning and sequencing of a cDNA encoding the smaller subunit of the enzyme (MPP beta subunit) from the same source . The cDNA consists of 1570 bp: 17 bp of 5'-untranslated sequence, 1467 bp of coding sequence, and 86 bp of 3'-untranslated sequence . The predicted protein consists of 489 amino acid residues, including a 45-amino acid leader peptide at the amino terminus and a 444-amino acid mature protein . The amino acid sequences of four tryptic peptides derived from purified MPP beta subunit precisely match those predicted by the cDNA sequence, as does the predicted mature amino terminus . The amino-terminal sequence is typical of a mitochondrial leader peptide, with eight positively charged arginine residues and a single negatively charged aspartate residue . When the amino acid sequence of rat MPP beta subunit is compared with sequences in the protein data bases, significant homology is found with the protease-enhancing protein of Neurospora crassa, the smaller subunit of MPP from Saccharomyces cerevisiae, and the core I protein of bovine ubiquinol:cytochrome c reductase . Lower homology is found with other members of a recently proposed class of endoproteases, which includes human insulinase and protease III from Escherichia coli.

Proc Natl Acad Sci U S A, 1993 Jun 1, 90(11), 5307 - 11
Probability of DNA knotting and the effective diameter of the DNA double helix; Rybenkov VV et al.; During the random cyclization of long polymer chains, knots of different types are formed . We investigated experimentally the distribution of knot types produced by random cyclization of phage P4 DNA via its long cohesive ends . The simplest knots (trefoils) predominated, but more complex knots were also detected . The fraction of knots greatly diminished with decreasing solution Na+ concentration . By comparing these experimental results with computer simulations of knotting probability, we calculated the effective diameter of the DNA double helix . This important excluded-volume parameter is a measure of the electrostatic repulsion between segments of DNA molecules . The calculated effective DNA diameter is a sensitive function of electrolyte concentration and is several times larger than the geometric diameter in solutions of low monovalent cation concentration.

Proc Natl Acad Sci U S A, 1993 Jun 1, 90(11), 5011 - 5
Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases; Henzel WJ et al.; A rapid method for the identification of known proteins separated by two-dimensional gel electrophoresis is described in which molecular masses of peptide fragments are used to search a protein sequence database . The peptides are generated by in situ reduction, alkylation, and tryptic digestion of proteins electroblotted from two-dimensional gels . Masses are determined at the subpicomole level by matrix-assisted laser desorption/ionization mass spectrometry of the unfractionated digest . A computer program has been developed that searches the protein sequence database for multiple peptides of individual proteins that match the measured masses . To ensure that the most recent database updates are included, a theoretical digest of the entire database is generated each time the program is executed . This method facilitates simultaneous processing of a large number of two-dimensional gel spots . The method was applied to a two-dimensional gel of a crude Escherichia coli extract that was electroblotted onto poly(vinylidene difluoride) membrane . Ten randomly chosen spots were analyzed . With as few as three peptide masses, each protein was uniquely identified from over 91,000 protein sequences . All identifications were verified by concurrent N-terminal sequencing of identical spots from a second blot . One of the spots contained an N-terminally blocked protein that required enzymatic cleavage, peptide separation, and Edman degradation for confirmation of its identity.

Biochemistry, 1993 Jun 1, 32(21), 5692 - 7
Cloning and functional expression of dendrotoxin K from black mamba, a K+ channel blocker; Smith LA et al.; Mamba dendrotoxins, 7K M(r) polypeptides with three disulfide bonds, selectively inhibit certain fast-activating, voltage-sensitive neuronal K+ channels and have been instrumental in their identification, localization, and purification . However, derivatives with more refined specificity are essential to define the structural and functional properties of the multiple subtypes known to reside in the nervous system . Hence, utilizing a constructed cDNA library from the venom glands of the black mamba (Dendroaspis polylepis), the gene encoding dendrotoxin K was isolated, amplified, and expressed as a maltose-binding fusion protein in the periplasmic space of Escherichia coli . After cleavage of the chaperone from the affinity-purified product, a recombinant protein was isolated and shown to be identical to native dendrotoxin K in its N-terminal sequence, chromatographic behavior, convulsive-inducing activity, and binding to voltage-activated K+ channels in bovine synaptic membranes . This successful expression of refolded active toxin, in adequate yield, makes possible for the first time the preparation of mutants with specificity tailored for each K+ channel subtype, based both on the recently derived three-dimensional structure of alpha-dendrotoxin and the identified binding site on cloned K+ channels.

Biochemistry, 1993 Jun 1, 32(21), 5526 - 38
Laser-induced protein-DNA cross-links via psoralen furanside monoadducts; Sastry SS et al.; We have developed a technique for cross-linking DNA binding proteins to DNA using psoralen furanside monoadducts as photoaffinity probes and a continuous-wave argon ion laser (366 nm) as a light source . Several DNA binding proteins (T7 RNA polymerase, UvrB, single-stranded DNA binding protein of Escherichia coli, T4 gp32, and RecA of E . coli) are shown to cross-link to single-stranded psoralen monoadducted DNA oligos differing in length and sequence . Increasing fluences of laser light on a fixed ratio of DNA/protein resulted in an increase in the yield of cross-links . Titration experiments were carried out to measure the apparent cross-linking constant (KappXL) for T7 RNA polymerase or UvrB to a monoadducted 24 mer DNA . The estimated values for the apparent cross-linking constant were in the range of (2-3) x 10(-7) M for both T7 RNA polymerase and UvrB . The efficiency of cross-linking was investigated as a function of the length of adducted DNA and also as a fraction of the total noncovalent binding of proteins of psoralenated DNAs . The results showed that in the cases of T7 RNA polymerase and UvrB cross-linking was more efficient with short oligos (8 and 19 mers) as compared to longer oligos (50 mer) . A tryptic peptide of T7 RNA polymerase that was conjugated to a psoralen furanside monoadducted 12 mer DNA was isolated by high-performance liquid chromatography . Mass spectrometry and amino acid composition of this peptide revealed that it originated from a region between residues 558 and 608 of the primary structure of T7 RNA polymerase . Two other peptides cross-linked to oligos were also purified . Repeated attempts to perform Edman sequencing of the peptide-DNA conjugates failed . Overall evidence indicates that photo-cross-linking of furanside monoadducts occurred at multiple sites on the proteins . We have shown that T7 RNA polymerase molecules in a ternary complex arrested at the furanside monoadduct can be cross-linked to the DNA templates with laser light . Evidence suggests that the arrested polymerase molecules existed in multiple conformations on the DNA template . This method of transcriptional cross-linking offers a new method for preparing highly stable elongation complexes for further studies.

Am Rev Respir Dis, 1993 Jun, 147(6 Pt 1), 1371 - 9
Neutrophil elastase inhibitors, SC-37698 and SC-39026, reduce endotoxin-induced lung dysfunction in awake sheep; Gossage JR et al.; Neutrophils have been implicated as important cellular mediators of the pulmonary dysfunction observed following endotoxemia in chronically instrumented awake sheep . Several areas of research suggest that neutrophil-derived proteases may be mediators of this dysfunction . We hypothesized that neutrophil elastase inhibitors would attenuate the effects of endotoxemia in sheep . To test this hypothesis, we studied the effects of two putative neutrophil elastase inhibitors, SC-37698 and SC-39026 (Searle, Skokie, IL), on endotoxin-induced lung dysfunction in awake sheep . Sheep were given intravenous neutrophil elastase inhibitor alone (20 mg/kg/h for 6 h), intravenous endotoxin (E . coli endotoxin, 0.5 microgram/kg over 20 min) 1 h after beginning the 6-h infusion of elastase inhibitor, or endotoxin 1 h after beginning a 6-h infusion of elastase inhibitor vehicle . SC-37698 attenuated the increase in lung lymph flow and lung lymph protein clearance, the alterations in lung mechanics, and the fall in white blood count . Qualitatively similar effects were seen with SC-39026 . These data suggest the need for further research examining the role of protease-antiprotease interactions and the potential utility of neutrophil elastase inhibitors in acute lung injury like that observed in the adult respiratory distress syndrome (ARDS) in the human.

Proc Soc Exp Biol Med, 1993 Jun, 203(2), 179 - 92
Sleep as a prognostic indicator during infectious disease in rabbits; Toth LA et al.; Infectious disease alters sleep patterns in rabbits, but the recuperative value of enhanced sleep during infectious disease has not been experimentally verified . To evaluate the relationship between specific sleep patterns and the clinical response to infectious disease, we classified sleep patterns in rabbits inoculated with E . coli, S . aureus, or C . albicans on the basis of the duration of the period of enhanced sleep . Patterns characterized by a long period of enhanced sleep were associated with a more favorable prognosis and less severe clinical signs than were patterns characterized by relatively short periods of enhanced sleep followed by prolonged sleep suppression . A contrasting analysis of these data indicated that animals that eventually died demonstrated reduced sleep compared to rabbits that survived the infection . These observations are consistent with the hypothesis that dynamic changes in sleep over the course of an infectious disease aid in recuperation.

Oncogene, 1993 Jun, 8(6), 1559 - 66
Human ERG-2 protein is a phosphorylated DNA-binding protein--a distinct member of the ets family; Murakami K et al.; We describe the identification of the ERG-2 gene products using an antibody raised against recombinant human ERG-2 protein . ERG-2 is a nuclear phosphoprotein and binds to purine-rich sequences (C/G)(C/a)GG-AA(G/a)T . ERG-2 protein, with a half-life of 21 h, is considerably more stable than the short-lived ETS-1 or ETS-2 proteins . Its phosphorylation is stimulated by phorbol myristate acetate (PMA), but not by Ca2+ ionophore treatment . ETS-1 protein is phosphorylated by Ca(2+)-dependent events, whereas ERG-2 protein is phosphorylated by activation of protein kinase C, suggesting their involvement in distinct signal transduction mechanisms . The expression of ERG-2 protein is restricted to few cell types and is high in early myeloid cells, indicating that it may function at an early stage of hematopoietic lineage determination . The DNA-binding sequence for ERG-2 protein is identified by using a random oligonucleotide selection procedure . The selected sequence is very similar to the binding sequence determined for human ETS-1 using the same method . Like other ets proteins, ERG-2 is a sequence-specific DNA-binding protein and is expressed at higher levels in early myeloid cells than in mature lymphoid cells . These results suggest that it may act as a regulator of genes required for maintenance and/or differentiation of early hematopoietic cells.

J Infect Dis, 1993 Jun, 167(6), 1464 - 6
Interleukin-8 release in baboon septicemia is partially dependent on tumor necrosis factor; Redl H et al.; The role of tumor necrosis factor (TNF) in interleukin (IL)-8 release in septicemia in the baboon (2-h infusion of live Escherichia coli, 5 x 10(8) cfu/kg) was investigated . Four experiments were done: one control (n = 7) and three with pretreatment to reduce TNF plasma levels . Pretreatment was with anti-TNF antibody (anti-TNF) 15 mg/kg, which neutralized circulating TNF (n = 4); 0.5 mg/kg of anti-TNF, which reduced peak TNF from 6.2 ng/mL (controls) to 0.6 ng/mL (n = 4); and a xanthine derivate (HWA138), which reduced TNF to 1 ng/mL (n = 5) . With TNF levels < 1 ng/mL, a significant reduction of circulating IL-8 from 10.4 ng/mL (peak) in controls to 1 ng/mL (peak) in anti-TNF-treated animals was found, but with HWA138 only some decrease in IL-8 was seen . Despite high endotoxin levels (10-30 ng/mL peak), neutralization of TNF resulted in diminished release of IL-8 and significantly lower levels of granulocyte elastase.

J Bacteriol, 1993 Jun, 175(11), 3689 - 91
Physical mapping of the scattered methionine genes on the Escherichia coli chromosome; Old IG et al.; Methionine is an important amino acid which acts not only as a substrate for protein elongation but also as the initiator of protein synthesis . The genes of the met regulon, which consists of 10 biosynthetic genes (metA, metB, metC, metE, metF, metH, metK, metL, metQ, and metX), two regulatory genes (metJ and metR), and the methionyl tRNA synthetase gene (metG), are scattered throughout the chromosome . The only linked genes are metK and metX at 63.6 min, metE and metR at 86.3 min, and the metJBLF gene cluster at 89 min . metBL form the only met operon.

J Bacteriol, 1993 Jun, 175(11), 3591 - 7
Evidence for a novel glycinamide ribonucleotide transformylase in Escherichia coli; Nygaard P et al.; We demonstrate here that Escherichia coli synthesizes two different glycinamide ribonucleotide (GAR) transformylases, both catalyzing the third step in the purine biosynthetic pathway . One is coded for by the previously described purN gene (GAR transformylase N), and a second, hitherto unknown, enzyme is encoded by the purT gene (GAR transformylase T) . Mutants defective in the synthesis of the purN- and the purT-encoded enzymes were isolated . Only strains defective in both genes require an exogenous purine source for growth . Our results suggest that both enzymes may function to ensure normal purine biosynthesis . Determination of GAR transformylase T activity in vitro required formate as the C1 donor . Growth of purN mutants was inhibited by glycine . Under these conditions GAR accumulated . Addition of purine compounds or formate prevented growth inhibition . The regulation of the level of GAR transformylase T is controlled by the PurR protein and hypoxanthine.

J Bacteriol, 1993 Jun, 175(11), 3563 - 9
Functional characterization of a replication initiator protein; Gammie AE et al.; Functional domains in the RepI replication initiator protein have been identified by classical and site-directed mutagenesis techniques . Mutations conferring an increase in plasmid copy number contained alterations in a key position of a putative helix-turn-helix DNA binding motif . The mutations did not appear to affect autorepressing functions . Regions of RepI important for autorepression were localized as well . Two classes of mutations resulting in diminished autorepression functions were identified . One class was distinguished by an elevated copy number, while the other class remained at the wild-type copy number level . Analysis of the various mutations leading to changes in copy number or autorepressing functions suggest that in some cases the autorepression and initiating functions of the RepI protein are separable . Finally, analysis with deletion clones suggests that the trans-acting autorepressing functions of RepI might depend on intermolecular coupling control.

J Bacteriol, 1993 Jun, 175(11), 3546 - 55
Heat shock proteins DnaJ, DnaK, and GrpE stimulate P1 plasmid replication by promoting initiator binding to the origin; Sozhamannan S et al.; Binding of the P1-encoded protein RepA to the origin of P1 plasmid replication is essential for initiation of DNA replication and for autoregulatory repression of the repA promoter . Previous studies have shown defects in both initiation and repression in hosts lacking heat shock proteins DnaJ, DnaK, and GrpE and have suggested that these proteins play a role in the RepA-DNA binding required for initiation and repression . In this study, using in vivo dimethyl sulfate footprinting, we have confirmed the roles of the three heat shock proteins in promoting RepA binding to the origin . The defects in both activities could be suppressed by increasing the concentration of wild-type RepA over the physiological level . We also isolated RepA mutants that were effective initiators and repressors without requiring the heat shock proteins . These data suggest that the heat shock proteins facilitate both repression and initiation by promoting only the DNA-binding activity of RepA . In a similar plasmid, F, initiator mutants that confer heat shock protein independence for replication were also found, but they were defective for repression . We propose that the initiator binding involved in repression and the initiator binding involved in initiation are similar in P1 but different in F.

J Bacteriol, 1993 Jun, 175(11), 3335 - 42
A new putative sigma factor of Myxococcus xanthus; Apelian D et al.; A third putative sigma factor gene, sigC, has been isolated from Myxococcus xanthus by using the sigA gene (formerly rpoD of M . xanthus) as a probe . The nucleotide sequence of sigC has been determined, and an open reading frame of 295 residues (M(r) = 33,430) has been identified . The deduced amino acid sequence of sigC exhibits the features which are characteristic of other bacterial sigma factors . The characterization of a sigC-lacZ strain has demonstrated that sigC expression is induced immediately after cells enter into the developmental cycle and is dramatically reduced at the onset of sporulation . A deletion mutant of sigC grows normally in vegetative culture and is able to develop normally . However, in contrast to the wild-type cells, the sigC deletion mutant cells became capable of forming fruiting bodies and myxospores on semirich agar plates . This suggests that sigC may play a role in expression of genes involved in negatively regulating the initiation of fruiting body formation.

J Bacteriol, 1993 Jun, 175(11), 3327 - 34
Signal sequence processing is required for the assembly of LamB trimers in the outer membrane of Escherichia coli; Carlson JH et al.; Proteins destined for either the periplasm or the outer membrane of Escherichia coli are translocated from the cytoplasm by a common mechanism . It is generally assumed that outer membrane proteins, such as LamB (maltoporin or lambda receptor), which are rich in beta-structure, contain additional targeting information that directs proper membrane insertion . During transit to the outer membrane, these proteins may pass, in soluble form, through the periplasm or remain membrane associated and reach their final destination via sites of inner membrane-outer membrane contact (zones of adhesion) . We report lamB mutations that slow signal sequence cleavage, delay release of the protein from the inner membrane, and interfere with maltoporin biogenesis . This result is most easily explained by proposing a soluble, periplasmic LamB assembly intermediate . Additionally, we found that such lamB mutations confer several novel phenotypes consistent with an abortive attempt by the cell to target these tethered LamB molecules . These phenotypes may allow isolation of mutants in which the process of outer membrane protein targeting is altered.

J Bacteriol, 1993 Jun, 175(11), 3259 - 68
Dual response regulators (NarL and NarP) interact with dual sensors (NarX and NarQ) to control nitrate- and nitrite-regulated gene expression in Escherichia coli K-12; Rabin RS et al.; Two sensor proteins, NarX and NarQ, mediate nitrate regulation of anaerobic respiratory gene expression . Either of these sensors is sufficient to signal the presence of nitrate to the response regulator protein, NarL, a transcriptional activator and repressor . Two observations suggested the existence of a second response regulator that is also involved in nitrate regulation . First, narL null mutants retain residual nitrate induction of fdnG operon expression; this residual induction is absent in narX narQ double-null strains . Second, nitrate induction of aeg-46.5 operon expression is substantially enhanced in narL null strains (M.H . Choe and W.S . Reznikoff, J . Bacteriol . 173:6139-6146, 1991) . We found that this nitrate induction requires either the NarX or the NarQ protein, consistent with the existence of a second response regulator . We designate this second regulator NarP . We isolated insertion mutants that are defective in aeg-46.5 operon expression . These insertions are in the narP gene, which encodes a response regulator that is 44% identical to the NarL protein . Null alleles of narP abolished aeg-46.5 induction and also eliminated the residual NarL-independent nitrate induction of fdnG operon expression . Both the NarX and NarQ proteins communicate with both the NarP and NarL proteins . We found that the primary signal for NarP-dependent aeg-46.5 operon induction is nitrite rather than nitrate . By contrast, nitrite is a relatively weak signal for NarL-dependent induction . In narX null strains, nitrate was an efficient signal for NarL-dependent induction, and this induction required the NarQ protein . We conclude that, in wild-type strains, the NarQ protein communicates the presence of nitrite to both the NarP and NarL proteins and that the NarX protein inhibits this communication with the NarL protein.

Infect Immun, 1993 Jun, 61(6), 2708 - 12
Expression of contact-dependent cytolytic activity by Mycobacterium tuberculosis and isolation of the genomic locus that encodes the activity; King CH et al.; We investigated the presence of cytolytic activity in the virulent H37Rv and attenuated H37Ra strains of Mycobacterium tuberculosis and in the vaccine strain Mycobacterium bovis BCG . The virulent strain H37Rv expressed > 3-fold more contact-dependent cytolytic activity than the attenuated strain H37Ra, and the vaccine strain M . bovis BCG did not produce cytolytic activity . We also isolated an approximately 3.2-kbp fragment of the M . tuberculosis H37Rv genome that was capable of inducing this contact-dependent hemolytic activity in a nonhemolytic strain of Escherichia coli.

FEBS Lett, 1993 Jun 1, 323(3), 257 - 60
Prediction of a novel topology in the N-terminal, 14 kDa fragment of Ada protein; Swindells MB; Previously determined protein structures have been analysed, in order to find folding motifs similar to that proposed by NMR spectroscopy, for the N-terminal, 14 kDa fragment of the Ada protein . The analyses reveal only limited similarities with the NMR-derived structural data and strongly suggest that this region of the Ada protein adopts a previously unobserved topology . Characteristic structural features, which arise from the inferred chain connectivity, are examined through comparisons with other structures . Using this information, the topology of the Ada protein 14 kDa fragment has been predicted in order to provide structural data not yet attainable from NMR experiments.

FEBS Lett, 1993 Jun 1, 323(3), 252 - 6
Folding topology and DNA binding of the N-terminal fragment of Ada protein; Sakashita H et al.; Three amino terminal fragments of Escherichia coli Ada protein (39 kDa) with different molecular masses (14 kDa, 16 kDa and 20 kDa) were prepared in large quantities from an E . coli strain harboring plasmids constructed for the overproduction of the truncated proteins . The three fragments can be methylated to an extent similar to that of the intact molecule . The methylated 16 kDa fragment specifically binds to the ada box on a DNA duplex . NMR analyses revealed that the 14 kDa fragment comprises two alpha-helices and a beta-sheet with parallel and anti-parallel mixed strands . A comparison of the 15N-1H HMQC spectra of the fragments has led to the conclusion that this tertiary structure within the 14 kDa fragment is retained in the larger 16 kDa and 20 kDa fragments.

FEBS Lett, 1993 Jun 1, 323(3), 243 - 6
Mobility in pyruvate dehydrogenase complexes with multiple lipoyl domains; Machado RS et al.; High-field NMR studies were carried out with genetically-reconstructed pyruvate dehydrogenase (PDH) complexes of Escherichia coli containing from zero to nine lipoyl domains per lipoate acetyltransferase (E2p) subunit . The only significant differences between the NMR spectra were the increasing intensities of the signals derived from the lipoyl domains and their associated linkers, and the much enhanced signal from the E3-binding domain and its linker in complexes that are devoid of lipoyl domains . The results suggest an explanation for the presence of three lipoyl domains per E2p subunit in the wild-type PDH complex, based on its greater inherent mobility, and potentially more efficient active-site coupling, than any of the other complexes.

FEBS Lett, 1993 Jun 1, 323(3), 218 - 22
Production of biologically active light chain of tetanus toxin in Escherichia coli . Evidence for the importance of the C-terminal 16 amino acids for full biological activity; Fairweather NF et al.; The activity of the light (L) chain of tetanus toxin, and of mutants constructed by site-directed mutagenesis, was studied by expression and purification of the proteins from E . coli . Wild-type recombinant L chain (pTet87) was active in the inhibition of exocytosis from cultured bovine adrenal chromaffin cells, although at a level 5-15% of that of L chain purified from tetanus toxin . L chain mutants which terminated at Leu-438 (pTet89), or which contained a Cys-to-Ser mutation at residue 439 (pTet88) were equally as active as the full-length recombinant protein . The reduced activity of pTet87 L chain correlated with C-terminal proteolysis of the protein upon purification . A tryptic fragment derived from native light chain and which terminated at Leu-434 also showed reduced activity in the exocytosis assay, consistent with a requirement of the C-terminal region of the L chain for maximal activity . pTet87 L chain, but neither of the mutants, could be associated with purified H (heavy) chain to form a covalent dimer which induced the symptoms of tetanus in mice . The ability to form biologically active toxin using recombinant L chain will be of great value in structure-function studies of tetanus toxin.

Clin Immunol Immunopathol, 1993 Jun, 67(3 Pt 1), 249 - 56
Cloning of a human IgM autoantibody bearing a cross-reactive idiotype in a lambda expression vector: a new approach to studying autoantibodies; Dang H et al.; The monoclonal antibody 4B4 is a human IgM,kappa which expresses a cross-reactive lupus-associated idiotype and has anti-Sm binding activity . We find that the VL nucleotide sequence of 4B4, like the 4B4 VH region, is encoded by unmutated germline genes . The 4B4 VH and VL were cloned into the ImmunoZap lambda expression vector to produce three recombinant immunoglobulin polypeptides . These recombinant polypeptides expressed, respectively, either the 4B4 VH or VL alone or a VH/VL heterodimer . ELISA showed that the VH/VL heterodimer retained anti-Sm antibody activity . The 4B4 idiotype was found predominantly on the VH . This report describes: (i) a method for producing recombinant immunoglobulin molecules from an IgM-secreting B cell line and (ii) the ability of recombinant antibody fragments expressed in Escherichia coli to retain the structural and antigenic properties of the native molecule.

Mol Cell Biol, 1993 Jun, 13(6), 3782 - 91
Fos is a preferential target of glucocorticoid receptor inhibition of AP-1 activity in vitro; Kerppola TK et al.; Several regulatory interactions between the AP-1 and the nuclear hormone receptor families of transcription factors have been reported . However, the molecular mechanisms that underlie these interactions remain unknown, and models derived from transient-transfection experiments are contradictory . We have investigated the effect of the purified glucocorticoid receptor (GR) DNA-binding domain (GR residues 440 to 533 {GR440-533}) on DNA binding and transcription activation by Fos-Jun heterodimers and Jun homodimers . GR440-533 differentially inhibited DNA binding and transcription activation by Fos-Jun heterodimers . Inhibition of Jun homodimers required a 10-fold-higher concentration of GR440-533 . An excess of Fos monomers protected Fos-Jun heterodimers from inhibition by GR440-533 . Surprisingly, regions outside the leucine zipper and basic region were required for GR inhibition of Fos and Jun DNA binding . The region of GR440-533 required for inhibition of Fos-Jun DNA binding was localized to the zinc finger DNA-binding domain . However, inhibition of Fos-Jun DNA binding was independent of DNA binding by GR440-533 . GR440-533 also differentially inhibited Fos-Jun heterodimer binding to the proliferin plfG element . Differential inhibition of DNA binding by different AP-1 family complexes provides a potential mechanism for the diverse interactions between nuclear hormone receptors and AP-1 family proteins at different promoters and in different cell types.

Mol Cell Biol, 1993 Jun, 13(6), 3530 - 40
The octamer/mu E4 region of the immunoglobulin heavy-chain enhancer mediates gene repression in myeloma x T-lymphoma hybrids; Shen L et al.; We have shown previously that the immunoglobulin heavy-chain enhancer acts as a repressor of gene transcription in hybrids between immunoglobulin-producing myelomas and a T-lymphoma line . We have now mapped this repressive activity to a 51-bp enhancer subfragment which contains the octamer and mu E4 protein-binding motifs . Even a single copy of this subfragment will repress gene expression in hybrid cells . Mutational analyses of the repressor fragment suggest that in non-B cells, a strong transcriptional repressor(s) functions through the same motifs important for gene activation in B cells . Changes in chromatin structure that accompany reporter gene repression suggest a general mechanism for prohibiting immunoglobulin heavy-chain locus activation in inappropriate cell types.

Mol Cell Biol, 1993 Jun, 13(6), 3494 - 504
Hel-N1: an autoimmune RNA-binding protein with specificity for 3' uridylate-rich untranslated regions of growth factor mRNAs; Levine TD et al.; We have investigated the RNA binding specificity of Hel-N1, a human neuron-specific RNA-binding protein, which contains three RNA recognition motifs . Hel-N1 is a human homolog of Drosophila melanogaster elav, which plays a vital role in the development of neurons . A random RNA selection procedure revealed that Hel-N1 prefers to bind RNAs containing short stretches of uridylates similar to those found in the 3' untranslated regions (3' UTRs) of oncoprotein and cytokine mRNAs such as c-myc, c-fos, and granulocyte macrophage colony-stimulating factor . Direct binding studies demonstrated that Hel-N1 bound and formed multimers with c-myc 3' UTR mRNA and required, as a minimum, a specific 29-nucleotide stretch containing AUUUG, AUUUA, and GUUUUU . Deletion analysis demonstrated that a fragment of Hel-N1 containing 87 amino acids, encompassing the third RNA recognition motif, forms an RNA binding domain for the c-myc 3' UTR . In addition, Hel-N1 was shown to be reactive with autoantibodies from patients with paraneoplastic encephalomyelitis both before and after binding to c-myc mRNA.

J Virol, 1993 Jun, 67(6), 3142 - 50
Human papillomavirus type 18 E7 protein requires intact Cys-X-X-Cys motifs for zinc binding, dimerization, and transformation but not for Rb binding; McIntyre MC et al.; Human papillomavirus type 18 (HPV-18) E7 proteins bind zinc through Cys-X-X-Cys repeats located at the C terminus of the protein . In order to examine the role of these cysteine motifs in E7 function, we expressed the HPV-18 E7 protein in bacteria and found that purified E7 forms a dimer through interactions with zinc . Mutants with single mutations within the Cys-X-X-Cys motifs bound a reduced level of zinc in a zinc blot assay, while a double mutant lost all zinc-binding activity . When expressed in vivo, none of the mutants cooperated with an activated ras oncogene to transform primary rat embryo fibroblasts, but all mutants retained nearly wild-type Rb-binding activity . The results indicate that the cysteine motifs play an important role in transformation by HPV-18 E7 but do not contribute to Rb binding.

J Exp Med, 1993 Jun 1, 177(6), 1745 - 53
Alteration of the glycolipid binding specificity of the pig edema toxin from globotetraosyl to globotriaosyl ceramide alters in vivo tissue targetting and results in a verotoxin 1-like disease in pigs; Boyd B et al.; All members of the verotoxin (VT) family specifically recognize globo-series glycolipids on the surface of susceptible cells . Those toxins that are associated with human disease, VT1, VT2, and VT2c, bind to globotriaosyl ceramide (Gb3) while VT2e, which is associated with edema disease of swine, binds preferentially to globotetraosyl ceramide (Gb4) . We were recently able to identify, using site-directed mutagenesis, amino acids in the binding subunit of these toxins that are important in defining their glycosphingolipid (GSL) binding specificity (Tyrrell, G . J., K . Ramotar, B . Boyd, B . W . Toye, C . A . Lingwood, and J . L . Brunton . 1992 . Proc . Natl . Acad . Sci . USA . 89:524) . The concomitant mutation of Gln64 and Lys66 in the VT2e binding subunit to the corresponding residues (Glu and Gln, respectively) found in VT2 effectively converted the GSL binding specificity of the mutant toxin from Gb4 to Gb3 in vitro . We now report that the altered carbohydrate recognition of the mutant toxin (termed GT3) has biological significance, resulting in a unique disease after intravascular injection into pigs as compared with classical VT2e-induced edema disease . The tissue localization of radiolabeled GT3 after intravascular injection was elevated in neural tissues compared with VT2e accumulation, while localization of GT3 to the gastrointestinal tract was relatively reduced . Accordingly, the pathological lesions after challenge with GT3 involved gross edema of the cerebrum, cerebellum, and brain stem, while purified VT2e caused hemorrhage and edema of the cerebellum, and submucosa of the stomach and large intestine . In addition, both radiolabeled toxins bound extensively to tissues not directly involved in the pathology of disease . VT2e, unlike GT3 or VT1, bound extensively to red cells, which have high levels of Gb4 . The overall tissue distribution of VT2e was thus found to be influenced by regional blood flow to each organ and not solely by the Gb4 levels of these tissues . Conversely, the distribution of GT3 (and VT1), which cleared more rapidly from the circulation, correlated with respective tissue Gb3 levels rather than blood flow . These studies indicate the primary role of carbohydrate binding specificity in determining systemic pathology, suggest that the red cells act as a toxin carrier in edema disease, and indicate that red cell binding does not protect against the pathology of systemic verotoxemia.

J Surg Res, 1993 Jun, 54(6), 584 - 91
Intestinal production of interleukin-1 alpha during endotoxemia in the mouse; Mester M et al.; Interleukin-1 (IL-1) may be involved in gut permeability to macromolecules and gut glutamine metabolism during endotoxemia . We developed a sensitive radioimmunoassay specific for mouse IL-1 alpha (detection limit of 100 pg/ml, or 5 pM) and measured intestinal levels of IL-1 alpha in response to endotoxin . CD-1 mice (N = 190) were randomized to intraperitoneal (ip) or intravenous (i.v.) lipopolysaccharide (LPS) infusion (15 micrograms/g or 1.5 micrograms/g Escherichia coli 0111:B4 LPS) or saline . Mice were sacrificed at Time 0, 30 min, 1 hr, 2.5 hr, 4 hr, 6 hr, 12 hr, and 24 hr (3 mice/group/time point) . Small bowel (SB) and large bowel (LB) were harvested and compared to liver . Duodenum, upper jejunum, midjejunum, terminal ileum, cecum, ascending colon, and sigmoid were analyzed in separate experiments . Tissues were frozen, weighed, and homogenized, the homogenates were centrifuged, and the supernates were assayed for immunoreactive IL-1 alpha . IL-1 alpha was expressed as pg/g wt +/- SEM (lowest detectable amount = 1000 pg/g wet tissue (WT)) . SB but not LB from normal controls had constitutively elevated levels of IL-1 alpha (6177 +/- 1640 pg/g WT) . LPS ip or i.v . produced lethargy, diarrhea, and a dramatic elevation of IL-1 alpha levels in both SB and LB . In SB, IL-1 alpha was elevated compared to baseline at 1 hr (19201 +/- 626 pg/g WT) and reached a fivefold maximal increase at 2.5 hr (31775 +/- 503 pg/g WT) following 15 micrograms/g ip.(ABSTRACT TRUNCATED AT 250 WORDS)

J Diarrhoeal Dis Res, 1993 Jun, 11(2), 101 - 4
Epithelial cell invasiveness of non-enteropathogenic serotypes of Escherichia coli; Albert MJ et al.; Current evidence suggests that enteropathogenic Escherichia coli (EPEC) of traditional serotypes possess a three-stage pathogenesis: localised adherence (LA), to, attachment-effacement (AE) of, and penetration of, enterocytes, all of which can be reproduced in tissue culture models in vitro . Three E . coli isolates of non-traditional serotypes (02:H2, 02:H25 and 015:H2) isolated from children with diarrhoea were previously shown to be positive for LA and AE activities in laboratory models . In the present study, they were, in addition, shown to be positive for invasion of a HEp-2 cell monolayer . These findings further establish the pathogenicity of non-traditional serotypes of E . coli and their role in the causation of diarrhoea.

Rev Sci Tech, 1993 Jun, 12(2), 425 - 33
Advances in the diagnosis of some parasitic diseases by monoclonal antibody-based enzyme-linked immunosorbent assays; Knowles DP Jr et al.; Advances in diagnostic assays for parasitic diseases include the use of monoclonal antibodies (MAbs) in antigen capture and competitive inhibition enzyme-linked immunosorbent assays (C-ELISA) . Antigen capture ELISAs for Anaplasma marginale and Cryptosporidium parvum provide direct detection of these parasites during clinical disease, and the C-ELISA format has been adapted for detection of anti-Babesia equi, anti-A . marginale and anti-bluetongue virus antibodies . False-positive results may occur when antigen preparations in other ELISA formats are contaminated with Escherichia coli, erythrocyte or cell-culture antigens . The C-ELISA format overcomes problems of antigen purity, since the specificity of the C-ELISA depends solely on the MAb used . For this reason, the C-ELISA format is highly suited for use with recombinant antigens . Also, the use of recombinant protein in diagnostic assays precludes the need to infect animals for antigen production when the antigen cannot be produced in cell culture.

J Protein Chem, 1993 Jun, 12(3), 323 - 7
Large scale purification and refolding of HIV-1 protease from Escherichia coli inclusion bodies; Hui JO et al.; The protease encoded by the human immunodeficiency virus type 1 (HIV-1) was engineered in Escherichia coli as a construct in which the natural 99-residue polypeptide was preceded by an NH2-terminal methionine initiator . Inclusion bodies harboring the recombinant HIV-1 protease were dissolved in 50% acetic acid and the solution was subjected to gel filtration on a column of Sephadex G-75 . The protein, eluted in the second of two peaks, migrated in SDS-PAGE as a single sharp band of M(r) approximately 10,000 . The purified HIV-1 protease was refolded into an active enzyme by diluting a solution of the protein in 50% acetic acid with 25 volumes of buffer at pH 5.5 . This method of purification, which has also been applied to the purification of HIV-2 protease, provides a single-step procedure to produce 100 mg quantities of fully active enzyme.

J Biochem (Tokyo), 1993 Jun, 113(6), 769 - 75
Dual roles of 90-kDa heat shock protein in the function of the mineralocorticoid receptor; Nemoto T et al.; Association of the 90-kDa heat shock protein (HSP90) is required for the high-affinity ligand-binding of the glucocorticoid receptor (GR), but not for that of the androgen receptor {Ohara-Nemoto, Y., Nemoto, T., & Ota, M . (1991) J . Biochem . 109, 113-119} . In the present study, we investigated the ligand- and HSP90-binding characteristics of the mineralocorticoid receptor (MR), which shares to some degree the ligand-binding specificity of the GR . A truncated human MR (designated MR351) starting from Gly-351 was translated in vitro with rabbit reticulocyte lysate . Scatchard analysis revealed the presence of a single class of high-affinity binding sites for {3H}aldosterone (Kd = 0.35 +/- 0.2 nM), comparable to those of the native receptor in target tissues . Glycerol gradient centrifugation and immunoadsorption analyses showed that MR351 associated with rabbit HSP90 . Exposure to 0.4 M NaCl induced the dissociation of HSP90 from MR351 and simultaneously enhanced binding of MR351 to DNA-cellulose . Moreover, when measured at 10 nM, HSP90-free MR351 showed only 9% of the {3H}aldosterone-binding found in the presence of HSP90 . On the other hand, when MR351 was expressed in Escherichia coli as a protein tagged with a histidine hexamer at the N-terminus (designated H6MR351), specific binding of {3H}aldosterone was detected . The binding affinity (Kd = 338 +/- 45 nM) was, however, 1,000-fold lower than that of MR351 translated in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biochem (Tokyo), 1993 Jun, 113(6), 705 - 9
Dephosphorylation of microtubule-binding sites at the neurofilament-H tail domain by alkaline, acid, and protein phosphatases; Hisanaga S et al.; The dephosphorylation-induced interaction of neurofilaments (NFs) with microtubules (MTs) was investigated by using several phosphatases . Escherichia coli alkaline and wheat germ acid phosphatases increased the electrophoretic mobility of NF-H and NF-M by dephosphorylation, and induced the binding of NF-H to MTs . The binding of NFs to MTs was observed only after the electrophoretic mobility of NF-H approached the exhaustively dephosphorylated level when alkaline phosphatase was used . The number of phosphate remaining when NF-H began to bind to MTs was estimated by measuring phosphate bound to NF-H . NF-H did not bind to MTs even when about 40 phosphates from the total of 51 had been removed by alkaline phosphatase . The removal of 6 further phosphates finally resulted in the association of NF-H with MTs . A similar finding, that the restricted phosphorylation sites in the NF-H tail domain, but not the total amount of phosphates, were important for binding to MTs, was also obtained with acid phosphatases . In contrast to alkaline and acid phosphatases, four classes of protein phosphatases (protein phosphatases 1, 2A, 2B, and 2C) were ineffective for shifting the electrophoretic mobility of NF proteins and for inducing the association of NFs to MTs.

Virus Genes, 1993 Jun, 7(2), 171 - 86
In vitro characterization of a herpes simplex virus type 1 ICP22 deletion mutant; Poffenberger KL et al.; We report the construction of a deletion mutant (del22Z) that is unable to synthesize any detectable messenger RNA or protein products from the herpes simplex virus type 1 (HSV-1) immediate early ICP22 gene upon infection . The del22Z deletion mutant lacks all but 18 nucleotides of the ICP22 coding sequence and carries the bacterial lacZ gene at the site of the deletion . No other known open reading frames or flanking sequences were disrupted . Del22Z was able to infect Vero cells productively but was severely restricted in human and rodent cells that were permissive for the parental HSV-1(F) . The yield of del22Z was not enhanced significantly, either by increasing the multiplicity of infection or by increasing the duration of the infection . There was a prolonged expression of some early gene products and a delayed appearance of some late gene products in both permissive and restrictive cells . This phenotype of cell-line restricted growth and alteration of the normal gene expression cascade maps specifically to the ICP22 coding region.

J Appl Bacteriol, 1993 Jun, 74(6), 652 - 61
PhoE porin of Escherichia coli and phosphate reversal of acid damage and killing and of acid induction of the CadA gene product; Rowbury RJ et al.; The lethal effects of inorganic acid on phoE+ Escherichia coli strains, grown at neutral pHo, were enhanced by chloramphenicol, apparently because some organisms acquire acid tolerance (habituate) during challenge and chloramphenicol stops this . Phosphate (and/or polyphosphate) present during challenge prevented killing and damage by acid to outer membranes, DNA and cellular enzymes but did not prevent acid pHo enhancing novobiocin activity . To reverse acid effects, phosphate must interact with or cross the outer membrane but need not enter the cytoplasm; it is probable that it competes with H+ (or protonated anions) for passage through the PhoE pore . Phosphate also prevented induction of beta-galactosidase in a strain with the cadA promoter fused to lacZ . Four unc mutants showed essentially normal acid sensitivity and habituation; the same was true for strains with lesions in fur, oxyR, katF, phoP, cadA and hycB . In contrast, deletion of rpoH led to slightly increased acid sensitivity for cells grown at pHo 7.0, although habituation was relatively normal.

Chem Biol Interact, 1993 Jun, 87(1-3), 269 - 78
Stereoselectivity of soman detoxication by organophosphorus acid anhydrases from Escherichia coli; Hoskin FC et al.; Three organophosphorus acid anhydrases have been isolated from E . coli by gel filtration and ion exchange column procedures, and further identified by gel electrophoresis . All three have molecular weights in the 120,000-140,000 range . Two of them hydrolyze racemic 1,2,2-trimethylpropylmethylphosphonofluoridate (soman) to completion at a single rate and, in parallel with this, detoxify soman at a comparable rate . The third enzyme appears to show stereoselectivity with respect to the two pairs of isomers of soman in that it hydrolyzes the racemic mixture at a fast and a slow rate, the latter approaching the non-enzymatic rate, and detoxifies soman only at the slower rate . In the past, organophosphorus acid anhydrases from bacterial and mammalian sources have been assayed either as crude sonicates or homogenates, or as cold ethanol precipitated fractions . Major discrepancies among laboratories have probably been due either to the assay of mixtures of varying proportions of these three enzymes depending on the various organs or organisms used as the source, or to the purification of one of the enzymes at the expense of the others . For E . coli, a fourth organophosphorus acid anhydrase is also present but at a considerably lower activity.

Free Radic Biol Med, 1993 Jun, 14(6), 609 - 13
Near ultraviolet light inactivation of dihydroxyacid dehydratase in Escherichia coli; Smyk-Randall E et al.; The effects of near ultraviolet (NUV) light on a NUV chromophore-containing oxidant-sensitive enzyme, dihydroxyacid dehydratase (DHAD), were measured in seven strains of Escherichia coli . The strains differed in production of the oxidant-defense enzymes, superoxide dismutases (Fe-SOD and Mn-SOD), and catalases HPI and HPII . With the stress of aerobic growth but without NUV exposure, the strains lacking either Fe or Mn SOD or both SODs had 57%, 25%, and 12%, respectively, of the DHAD-specific activity of the parent (K12) strain . Under the same conditions, the catalase strains that were wild type, overproducing, and deficient had comparable DHAD-specific activities . When aerobic cultures were exposed for 30 min to NUV with a fluence of 216 J/m2/s at 310-400 nm, the percentage decreases in DHAD-specific activities were similar (ranging from 75% to 89%) in strains with none, either, or both SODs missing, and in the catalase-overproducing strain . However, the decreases were only 58% and 52% in the strain with catalase missing and in its parent, respectively . The NUV-induced loss of DHAD enzyme activity was not accompanied by any detectable loss of the DHAD protein as measured by polyclonal antibody to DHAD.

Mol Reprod Dev, 1993 Jun, 35(2), 105 - 13
Gene transfer in bovine blastocysts using replication-defective retroviral vectors packaged with Gibbon ape leukemia virus envelopes; Kim T et al.; With this work we demonstrate that murine leukemia virus (MLV)-based replication-defective retroviral vectors encapsidated with Gibbon ape leukemia virus (GaLV) envelopes are significantly more infectious to bovine embryonic trachea (EBTr) cells than vectors encapsidated with murine xenotropic envelope proteins . In a test of internal promoter activity in an MLV retroviral vector, the rat beta-actin promoter was shown to be better than the herpes simplex virus type 1 thymidine kinase (TK) and human cytomegalovirus (CMV) immediate early promoters for the expression of an E . coli beta-galactosidase marker gene in bovine target cells . By co-culture of bovine blastocysts and virus-producing cells, or by culture of embryos in the medium harvested from virus-producing cells, we transferred the E . coli beta-galactosidase gene into trophoblasts and also into inner cell mass (ICM) cells of a bovine embryo through the infection of the MLV-based replication-defective retroviruses encapsidated with GaLV envelope proteins . The infection was confirmed by the expression of the E . coli beta-galactosidase gene under a beta-actin internal promoter . In addition, co-culture of ICM cells with virus-producing cells resulted in differentiation of ICM cells into embryoid bodies expressing the marker genes.

Mol Gen Genet, 1993 Jun, 239(3), 371 - 7
The wild-type allele of tonB in Escherichia coli is dominant over the tonB1 allele, encoding TonBQ160K, which suppresses the btuB451 mutation; Anton M et al.; The entire coding sequence of the tonB gene, except for nine codons at the 3' end, was deleted from the chromosome of Escherichia coli . Introduction of the btuB451 suppressor mutant tonB1 into the chromosome of such a tonB deletion strain showed that the tonB1 allele was active as a suppressor in a single copy at 37 degrees C and 42 degrees C but not at 28 degrees C . No temperature dependence was seen when FepA- or FhuA-dependent activities of the tonB1 gene product (TonBQ160K) were tested . The btuB451 suppressor activity of tonB1 was inhibited by the simultaneous presence within the cells of the tonB+ allele on a multicopy plasmid . This represents the first case of dominance among different tonB alleles . Inhibition of suppression was abolished by overexpression of the btuB451-encoded receptor protein . Competition for binding of TonB+ and TonBQ150K to ExbB was excluded as the cause of dominance . Based on our data we conclude that competition for binding of TonB+ and TonBQ160K to the btuB451 gene product is the reason for the observed dominance . The implications of these findings for the mechanism of btuB451 suppression by tonB1 are discussed.

Protein Expr Purif, 1993 Jun, 4(3), 256 - 64
Renaturation of casein kinase II from recombinant subunits produced in Escherichia coli: purification and characterization of the reconstituted holoenzyme; Lin WJ et al.; Casein kinase II is a heterotetrameric protein kinase with an alpha 2 beta 2 composition; the subunits can be separated only under harsh denaturing conditions . In this study, the optimal conditions for renaturation of denatured casein kinase II have been established . Purified casein kinase II from rabbit reticulocytes was denatured with 8 M urea and 0.1 M dithiothreitol at 25 degrees C . Various parameters, including arginine, oxidized glutathione/dithiothreitol, substrate, and temperature were optimized for renaturation . Under optimal conditions, the denatured protein kinase was successfully renatured with a recovery of 75% activity and eluted around 160,000 Da upon gel filtration, indicating the tetrameric structure . When the alpha (catalytic) and beta (regulatory) subunits of casein kinase II from Drosophila were cloned and overexpressed with the pET3a vector in Escherichia coli, the majority of the subunits were in an insoluble form . The optimal conditions for denaturation/renaturation of the recombinant casein kinase II from Drosophila were identical to those developed for the holoenzyme, except for the redox state and temperature . When the alpha subunit was solubilized and renatured in an approximate 1:1 ratio with the beta subunit, the catalytic activity was stimulated fourfold over that of the alpha subunit alone . The reconstituted enzyme was purified to apparent homogeneity in one step by chromatography on heparin--TSK . Gel filtration indicated the formation of an alpha 2 beta 2 tetramer . The reconstituted recombinant casein kinase II exhibited characteristics of the native holoenzyme in subunit composition, inhibition by heparin, stimulation by basic compounds, and the KCl concentration required for optimal activity.(ABSTRACT TRUNCATED AT 250 WORDS)

J Clin Invest, 1993 Jun, 91(6), 2423 - 8
Guanylin stimulation of Cl- secretion in human intestinal T84 cells via cyclic guanosine monophosphate; Forte LR et al.; Intestinal salt and fluid secretion is stimulated by Escherichia coli heat-stable enterotoxins (ST) through activation of a membrane guanylate cyclase found in the intestine . Guanylin is an endogenous intestinal peptide that has structural similarity to the bacterial peptides . Synthetic preparations of guanylin or E . coli ST 5-17 stimulated Cl- secretion in T84 cells cultured on semipermeable membranes as measured by increases in short circuit current (Isc) . The guanylin/ST receptors appeared to be on the apical surface of T84 cells, since addition of guanylin to the apical, but not basolateral, reservoir stimulated Isc . Bumetanide added to the basolateral side effectively inhibited the Isc responses of T84 cells to either guanylin or ST 5-17 . Guanylin appeared to be about one-tenth as potent as ST in stimulating transepithelial Cl- secretion . Guanylin and E . coli ST 5-17 both caused massive (> 1,000-fold) increases in cGMP levels in T84 cells, but guanylin was less potent than ST . Both peptides fully inhibited the binding of 125I-ST to receptor sites on intact T84 cells . The radioligand binding data obtained with guanylin or ST 5-17 best fit a model predicting two receptors with different affinity for these ligands . The Ki values for guanylin were 19 +/- 5 nM and 1.3 +/- 0.5 microM, whereas the Ki values for ST 5-17 were 78 +/- 38 pM and 4.9 +/- 1.4 nM . We conclude that guanylin stimulated Cl- secretion via the second messenger, cGMP, in T84 human colon cells . At least two guanylin receptors with different affinities for these ligands may exist in the cultured T84 cells . It may be postulated that guanylin is an endogenous hormone that controls intestinal Cl- secretion by a paracrine mechanism via cGMP and that E . coli ST stimulates Cl- secretion by virtue of an opportunistic mechanism through activation of guanylin receptors.

Eur J Biochem, 1993 Jun 1, 214(2), 497 - 501
Interaction of calmodulin with a putative calmodulin-binding domain of inositol 1,4,5-triphosphate 3-kinase . Effects of synthetic peptides and site-directed mutagenesis of Trp165; Erneux C et al.; Recombinant rat brain inositol 1,4,5-triphosphate {Ins(1,4,5)P3} 3-kinase was expressed in Escherichia coli as a beta-galactosidase fusion product . It could be adsorbed onto calmodulin-Sepharose and eluted in Ca(2+)-free medium as a 48-kDa protein . Purification could be achieved in a single step . Molecular evidence for a calmodulin-binding domain on Ins(1,4,5)P3 3-kinase can be shown by the following approaches . (a) Inhibition of Ca2+/calmodulin stimulation by a synthetic peptide based on a candidate calmodulin-binding domain . The inhibition was mimicked by a well-characterized peptide derived from the sequence of smooth muscle myosin light-chain kinase calmodulin-binding site . (b) The construction of two mutants by site-directed mutagenesis of Trp165 to Gly or Arg . Both mutants displayed kinase activity but were no longer Ca2+/calmodulin sensitive, supporting, therefore, the role of Trp165 in calmodulin binding.

Arch Biochem Biophys, 1993 Jun, 303(2), 402 - 6
Expression and characterization of rat protein phosphatases-1 alpha, -1 gamma 1, -1 gamma 2, and -1 delta; Zhang Z et al.; Four distinct cDNAs for rat protein phosphatase-1 have been isolated from rat tissues (Sasaki et al., Jpn . J . Cancer Res . 81, 1272-1280, 1990) . These cDNAs encode proteins of highly similar sequence, the major differences being located at their N and C termini . In order to demonstrate that these cDNAs encode functional proteins and to investigate their enzymatic properties, it would be desirable to obtain purified preparations of these proteins . Using a system that was developed for the expression of rabbit muscle protein phosphatase-1 (Zhang et al., J . Biol . Chem . 267, 1484-1490, 1992) we have expressed these isoforms in Escherichia coli . The four recombinant isoforms were purified to near homogeneity and their properties were examined in terms of substrate specificity and sensitivity to okadaic acid and inhibitor-2.

J Gen Virol, 1993 Jun, 74 ( Pt 6), 1011 - 6
Kinetic studies of the predicted substrate-binding site of varicella-zoster virus thymidine kinase; Suzutani T et al.; To investigate the mechanism of kinetic action and substrate recognition of varicella-zoster virus (VZV) thymidine kinase (TK), we designed and isolated a site-directed mutant VZV TK which has double amino acid substitutions, 136threonine to leucine and 137isoleucine to leucine (SDM TK) . This mutant was designed to alter the substrate-binding site of the VZV TK to duplicate that of the herpes simplex virus type 2 enzyme . Kinetic studies of the activity of wild-type TK indicated that the binding order of ATP and thymidine is random and that wild-type VZV TK possessed high thymidylate kinase (TM-K) activity . The sensitivity of VZV TK to bisubstrate analogues, dinucleotides of adenosine and thymidine, showed that the optimum distance between the ATP- and substrate-binding sites is two phosphoryl groups greater than with the natural substrate for TK activity . SDM TK lost deoxycytidine kinase activity and had reduced TK and TM-K activities . Inhibition studies on both WT and SDM TK by 5-halogenovinyluridine analogues and their 5' monophosphate derivatives revealed that amino acids at positions 136 and 137 are involved in substrate binding, probably through a role in the formation of the binding pocket for bulky substrates.

Proc Natl Acad Sci U S A, 1993 Jun 1, 90(11), 5229 - 33
Functional substitution of the signal recognition particle 54-kDa subunit by its Escherichia coli homolog; Bernstein HD et al.; The 54-kDa subunit of the mammalian signal recognition particle (SRP54) binds to the signal sequences of nascent secretory and transmembrane proteins and facilitates their cotranslational targeting to the membrane translocation apparatus in the endoplasmic reticulum (ER) . A 48-kDa Escherichia coli protein that shares extensive sequence similarity with SRP54 was identified in homology searches . Recent genetic experiments by Phillips and Silhavy {Phillips, G . J . & Silhavy, T . J . (1992) Nature (London) 359, 744-746} have shown that depletion of this protein, designated Ffh (fifty-four homolog), leads to a significant secretory defect in vivo . We demonstrate here that Ffh is structurally and functionally related to SRP54 by virtue of its ability to mimic closely its mammalian counterpart in several established biochemical assays, thereby suggesting that it plays a direct role in protein export . Ffh assembled efficiently with mammalian SRP components into a chimeric ribonucleoprotein {"SRP(Ffh)"} and bound at the site normally occupied by SRP54 . Like SRP54, the Ffh moiety of the chimeric particle specifically recognized the signal sequence of preprolactin in a photocrosslinking assay . Moreover, Ffh could also act in concert with other SRP components to arrest elongation of preprolactin upon recognition of the signal sequence . In all of these assays, Ffh had approximately the same specific activity as SRP54 . In contrast, SRP(Ffh) did not promote the translocation of preprolactin across the membrane of microsomal vesicles, suggesting that Ffh cannot mediate an interaction with a membrane component that is required for the translocation of nascent chains.

Biochemistry, 1993 Jun 1, 32(21), 5698 - 704
Role of the conserved quartets of residues located in the N- and C-terminal halves of the transposon Tn10-encoded metal-tetracycline/H+ antiporter of Escherichia coli; Yamaguchi A et al.; In the putative secondary structure of the transposon Tn10-encoded metal-tetracycline/H+ antiporter {Yamaguchi, et al . (1992) J . Biol . Chem . 267, 7490-7498}, Tyr50-XXX-Gln54 in transmembrane helix 2 and Gly80-XXX-Asp84 in helix 3 are thought to face each other in the N-terminal region . At the corresponding positions in the C-terminal region, a similar quartet of residues, His257-XXX-Gln261 in helix 8 and Gly281-XXX-Asp285 in helix 9, is also located . The quartets involve the residues Asp84 and Asp285, which have been revealed to be essential for the tetracycline transport function . When Gln54 and Gln261 were replaced with Ala by site-directed mutagenesis, the active tetracycline transport activity decreased to about 10% and 40% of the wild-type level, respectively . The Km values of the Q54A and Q261A mutants for tetracycline were 140 and 160 microM, respectively, which are about 8-fold higher than that of the wild type . Thus, the two Gln residues may contribute to the substrate recognition . On the other hand, the replacement of Gly80 and Gly281 with Leu caused a complete loss of the transport activity, whereas the G80A and G281A mutants retained about 10% and 30% of the activity of the wild-type, respectively, suggesting that a bulky side chain at positions 80 and 281 causes steric hindrance of the transport . The mutation of Tyr50 to Ala or His caused a decrease in activity by a factor of about 3 without a significant change in the Km value, whereas the Y50C mutant showed no transport activity at all.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1993 Jun 1, 32(21), 5656 - 69
Complete assignments of magnetic resonances of ribonuclease H from Escherichia coli by double- and triple-resonance 2D and 3D NMR spectroscopies; Yamazaki T et al.; Assignments of 1H, 15N, and 13C magnetic resonances for ribonuclease H from Escherichia coli have been completed using double- and triple-resonance 2D and 3D NMR experiments . These assignments include all types of 1H, 15N, and 13C nuclei detectable by NMR . The enzyme used, which cleaves the RNA moiety of an RNA-DNA duplex, consists of 155 amino acid residues and has 1962 nuclei (227 nitrogen, 762 carbons, and 973 protons) observable independently by NMR . Among those, 1868 nuclei (95%) have been assigned . Two methods, 3D HCH and 13C-13C-1H heteroSQC/homoSQC, were newly devised to complete the side chain assignments . These methods were used to elucidate the -CH2- and -C-CH-substructures . Triple-resonance experiments to detect other types of substructures, (e.g., -N-CH- and -C-NH-) were also applied . In total, 10 kinds of 3D NMR experiments were used to complete the assignments . The chemical shifts obtained through the assignments were analyzed in terms of the tertiary structure of the protein molecule . Among the 13C chemical shifts, larger secondary shifts (deviations from shifts at the random coil state) were observed for the C alpha, C beta, and C' nuclei, which reflect the local structures on the backbone, that is, the alpha-helix, beta-sheet, and left-handed helix, respectively.

Am Rev Respir Dis, 1993 Jun, 147(6 Pt 1), 1380 - 5
Lung protein leakage in feline septic shock; Schutzer KM et al.; The aim of the present study was to explore lung microvascular leakage of protein and water in a feline model of septic shock, using a double isotope technique with external gamma camera detection and gravimetric lung water measurements . The experiments were performed on artificially ventilated cats . One group of cats (n = 8) was given an infusion of live Escherichia coli bacteria, and another group (n = 5) served as a control group receiving saline . Plasma transferrin was radiolabeled in vivo with indium-113m-chloride, and erythrocytes were labeled with technetium-99m . The distribution of these isotopes in the lungs was continuously measured with a gamma camera . A normalized slope index (NSI) was calculated, indicative of the transferrin accumulation corrected for changes in local blood volume that reflect protein leakage . In the septic group there was a protein leakage after bacterial infusion, with a NSI of 39 x 10(-4) +/- 5 x 10(-4) min-1 (mean +/- SEM), and the PaO2 diminished from 21 +/- 1 to 9.5 +/- 1 kPa . In control cats a slight protein leakage with a NSI of 9 +/- 10(-4) +/- 2 x 10(-4) min-1 was detected, probably caused by the operative procedure, but PaO2 did not change . Wet-to-dry-weight ratios of postmortem lungs were not significantly different between the groups . It was concluded that an intravenous infusion of live E . coli bacteria induces a lung capillary protein leakage without increased lung water and a concomitantly disturbed gas exchange.(ABSTRACT TRUNCATED AT 250 WORDS)

Virology, 1993 Jun, 194(2), 807 - 14
The RER-localized rotavirus intracellular receptor: a truncated purified soluble form is multivalent and binds virus particles; Taylor JA et al.; A budding event transfers the immature, single-shelled rotavirus particle (SSP) across the RER membrane prior to assembly of mature virions in the ER lumen . Budding is triggered by the interaction of the SSP with a viral receptor glycoprotein (NS28) which is located in the RER membrane . We have expressed the cytoplasmic domain of the NS28 receptor as a glutathione S-transferase fusion protein to generate a soluble polypeptide that in turn can be cleaved to yield a carboxy-terminal receptor domain . The soluble terminal domain (delta 1-85 NS28) has been purified to homogeneity and retains SSP-binding activity when immobilized on a solid matrix . Integral membrane status therefore is not an essential prerequisite for ligand binding . The Kd for the interaction between immobilized delta 1-85 NS28 and purified particles is 4.6 x 10(-11) M, a value indistinguishable from the value obtained for the full-length and membrane-anchored receptor . Cross-linking with the bifunctional reagent dimethylsuberimidate indicates that delta 1-85 NS28 is a tetramer . When delta 1-85 NS28 is added to a monodisperse suspension of purified virus, the particles aggregate, indicating that the receptor is multivalent . The rotavirus intracellular receptor therefore provides a model for the detailed analysis of the early events that trigger the budding of cytoplasmically located particles across cell membranes.

Virology, 1993 Jun, 194(2), 647 - 53
The C-terminal third of UL42, a HSV-1 DNA replication protein, is dispensable for viral growth; Gao M et al.; UL42 is the herpes simplex virus type 1 DNA polymerase (Pol) accessory protein and is required for viral DNA replication and growth . Previous results from this laboratory demonstrated that the N-terminal two thirds of the protein contains all of the biochemical activities of the protein which can be measured in vitro . These activities include dsDNA-binding, association with DNA polymerase, and stimulation of polymerase activity . To better understand the functions of UL42 in infected cells, we have isolated and characterized two viral recombinants, UL42lacZ and n338 . In the mutant virus UL42lacZ, the UL42 gene was disrupted by insertion of the Escherichia coli lacZ gene, while in the mutant virus n338, a termination codon was introduced after amino acid position 338 . Analysis of the mutant phenotypes suggest that (1) the first 338 residues of UL42 retain all the functions necessary for viral DNA replication and growth in lytic infection, (2) localization of UL42 to the cell nucleus is independent of Pol, and (3) localization of ICP8 (ssDNA-binding protein) to prereplication sites is independent of functional UL42.

J Infect Dis, 1993 Jun, 167(6), 1344 - 50
Detection of tumor necrosis factor soluble receptor p55 in blood samples from healthy and endotoxemic humans; Shapiro L et al.; The tumor necrosis factor (TNF) soluble receptor derived from the cell surface p55 TNF receptor (TNFsRp55) is a naturally occurring substance generated during infection and inflammation . TNFsRp55 inhibits biologic effects of TNF . An RIA was developed to quantitate TNFsRp55 in human blood . Recovery of TNFsRp55 from blood anticoagulated with EDTA was optimal compared with recovery from serum or heparinized plasma . TNF did not interfere with the assay . With the RIA based on radiolabeled nonglycosylated Escherichia coli-derived recombinant TNFsRp55, a mean concentration of 198 +/- 15 pg/mL was found in 14 volunteers . When glycosylated CHO cell-derived TNFsRp55 was used, the mean level was 1656 +/- 95 pg/mL . Infusion of endotoxin into volunteers induced TNFsRp55, which peaked at 517 +/- 99 pg/mL for the E . coli-based RIA and 7300 +/- 1810 pg/mL for the CHO cell-based RIA . These findings demonstrate that blood collected in EDTA is optimal for measuring circulating TNFsRp55 and that this soluble receptor is present in health but elevated during endotoxemia.

J Bacteriol, 1993 Jun, 175(11), 3598 - 606
Regulation of Escherichia coli glnB, prsA, and speA by the purine repressor; He B et al.; A strategy was devised to identify Escherichia coli genes subject to coregulation by purR . From a data base search, similarities to the pur regulon cis-acting control site were found in 26 E . coli genes . Of five genes examined in which the putative pur operator is upstream of the coding sequence, glnB, prsA, and speA bound purified purine repressor in vitro . Binding of the repressor to a pur operator in these genes was dependent upon a corepressor . The pur operator in glnB is located between two major transcription start sites that were located by primer extension mapping . The effect of purR on expression of glnB, prsA, and speA was examined by using a lacZ reporter . The results indicated two- to threefold repression of these genes by purR . Coregulation by purR provides evidence that expands the pur regulon to include glnB, prsA, and speA . These genes have functions related to nucleotide metabolism.

J Bacteriol, 1993 Jun, 175(11), 3430 - 42
Mutational analysis of an Escherichia coli fourteen-gene operon for phosphonate degradation, using TnphoA' elements; Metcalf WW et al.; All genes for phosphonate (Pn) utilization in Escherichia coli are in a large cluster of 14 genes named, in alphabetical order, phnC to phnP . Plasmids carrying these genes were mutagenized by using TnphoA'-1, and 43 mutants containing simple insertions were studied in detail . Their insertion sites were defined by restriction mapping and by DNA sequencing . One or more mutations in each phn gene was identified . In 23 mutants, expression of the TnphoA'-1 lacZ gene was phosphate starvation inducible . These mutants had TnphoA'-1 oriented in line behind the phnC promoter, i.e., in the + orientation . In 20 mutants, the TnphoA'-1 lacZ gene was expressed at a low basal level . These mutants had insertions in the opposite orientation . All 43 phn::TnphoA'-1 insertions were recombined onto the chromosome to test for mutational effects, and their structures on the chromosome were verified by DNA hybridization . Those in the + orientation were switched to TnphoA'-9, which has an outward promoter for expression of downstream genes . These insertions were tested for polar effects by measuring beta-glucuronidase synthesis from a uidA gene transcriptionally fused to the 3' end of the phnP gene . The results indicate the following: (i) the phnC-to-phnP gene cluster is an operon of 14 genes, and the phnC promoter is the sole psi promoter; (ii) three gene products (PhnC, PhnD, and PhnE) probably constitute a binding protein-dependent Pn transporter; (iii) seven gene products (PhnG, PhnH, PhnI, PhnJ, PhnK, PhnL, and PhnM) are required for catalysis and are likely to constitute a membrane-associated carbon-phosphorus (C-P) lyase; (iv) two gene products (PhnN and PhnP) are not absolutely required and may therefore be accessory proteins for the C-P lyase; and (v) two gene products (PhnF and PhnO) are not required for Pn use and may have a regulatory role because they have sequence similarities to regulatory proteins . The mechanism for breaking the C-P bond by a lyase is discussed in light of these results.

FEBS Lett, 1993 Jun 1, 323(3), 261 - 6
An EPR investigation of non-haem iron sites in Escherichia coli bacterioferritin and their interaction with phosphate . A study using nitric oxide as a spin probe; Le Brun NE et al.; EPR studies of bacterioferritin (BFR), an iron-storage protein of Escherichia coli {1993, Biochem . J . 292, 47-56}, have revealed the presence of non-haem iron (III) (NHI) sites within the protein coat which may be involved in iron uptake and release . When nitric oxide was used as an EPR spin probe of the Fe(II) state of the NHI sites, two distinct mononuclear NHI species were found . Under certain conditions, an iron dimer was also observed . The reaction of phosphate with NHI species has been investigated . Results point to a function for this anion in core nucleation.

Mol Cell Biol, 1993 Jun, 13(6), 3811 - 20
Analysis of a protein-binding domain of p53; Ruppert JM et al.; The tumor suppressor protein p53 was first isolated as a simian virus 40 large T antigen-associated protein and subsequently was found to function in cell proliferation control . Tumor-derived mutations in p53 occur predominantly in four evolutionarily conserved regions spanning approximately 50% of the polypeptide . Previously, three of these regions were identified as essential for T-antigen binding . We have examined the interaction between p53 and T antigen by using Escherichia coli-expressed human p53 . By a combination of deletion analysis and antibody inhibition studies, a region of p53 that is both necessary and sufficient for binding to T antigen has been localized . This function is contained within residues 94 to 293, which include the four conserved regions affected by mutation in tumors . Residues 94 to 293 of p53 were expressed in both wild-type and mutant forms . T-antigen binding was unaffected by tumor-derived mutations which have been associated with the wild-type conformation of p53 but was greatly reduced by mutations which were previously shown to alter p53 conformation . Our results show that, like T-antigen binding to the Rb tumor suppressor protein, T antigen appears to interact with the domain of p53 that is commonly mutated in human tumors.

J Virol, 1993 Jun, 67(6), 3470 - 80
Herpes simplex virus type 1 DNA cleavage and encapsidation require the product of the UL28 gene: isolation and characterization of two UL28 deletion mutants; Tengelsen LA et al.; The herpes simplex virus type 1 UL28 gene contains a 785-amino-acid open reading frame that codes for an essential protein . Studies with temperature-sensitive mutants which map to the UL28 gene indicate that the UL28 gene product (ICP18.5) is required for packaging of viral DNA and for expression of viral glycoproteins on the surface of infected cells (C . Addison, F . J . Rixon, and V . G . Preston, J . Gen . Virol . 71:2377-2384, 1990; B . A . Pancake, D . P . Aschman, and P . A . Schaffer, J . Virol . 47:568-585, 1983) . In this study, we describe the isolation of two UL28 deletion mutants that were constructed and propagated in Vero cells transformed with the UL28 gene . The mutants, gCB and gC delta 7B, contained deletions of 1,881 and 537 bp, respectively, in the UL28 gene . Although the mutants synthesize viral DNA, they fail to form plaques or produce infectious virus in cells that do not express the UL28 gene . Transmission electron microscopy and Southern blot analysis demonstrated that both mutants are defective in cleavage and encapsidation of viral DNA . Analysis by cell surface immunofluorescence showed that the UL28 gene is not required for expression of viral glycoproteins on the surface of infected cells . A rabbit polyclonal antiserum was made against an Escherichia coli-expressed Cro-UL28 fusion protein . This antibody reacted with an infected-cell protein having an apparent molecular mass of 87 kDa . The 87-kDa protein was first detected at 6 h postinfection and was expressed as late as 24 h postinfection . No detectable UL28 protein was synthesized in gCB- or gC delta 7B-infected Vero cells.

J Comput Aided Mol Des, 1993 Jun, 7(3), 305 - 23
An approximate but efficient method to calculate free energy trends by computer simulation: application to dihydrofolate reductase-inhibitor complexes; Gerber PR et al.; Derivatives of free energy differences have been calculated by molecular dynamics techniques . The systems under study were ternary complexes of Trimethoprim (TMP) with dihydrofolate reductases of E . coli and chicken liver, containing the cofactor NADPH . Derivatives are taken with respect to modification of TMP, with emphasis on altering the 3-, 4- and 5-substituents of the phenyl ring . A linear approximation allows the encompassing of a whole set of modifications in a single simulation, as opposed to a full perturbation calculation, which requires a separate simulation for each modification . In the case considered here, the proposed technique requires a factor of 1000 less computing effort than a full free energy perturbation calculation . For the linear approximation to yield a significant result, one has to find ways of choosing the perturbation evolution, such that the initial trend mirrors the full calculation . The generation of new atoms requires a careful treatment of the singular terms in the non-bonded interaction . The result can be represented by maps of the changed molecule, which indicate whether complex formation is favoured under movement of partial charges and change in atom polarizabilities . Comparison with experimental measurements of inhibition constants reveals fair agreement in the range of values covered . However, detailed comparison fails to show a significant correlation . Possible reasons for the most pronounced deviations are given.

Cancer Metastasis Rev, 1993 Jun, 12(2), 195 - 212
The bioactivation of CB 1954 and its use as a prodrug in antibody-directed enzyme prodrug therapy (ADEPT); Knox RJ et al.; Walker cells in vivo or in vitro are exceptionally sensitive to the monofunctional alkylating agent CB 1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) . The basis of the sensitivity is that CB 1954 forms DNA interstrand crosslinks in Walker cells but not in insensitive cells . Crosslink formation is due to the aerobic reduction of CB 1954 to form 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide by the enzyme DT diaphorase . The 4-hydroxylamine can not crosslink DNA directly but requires further activation by a non-enzymatic reaction with a thioester (such as acetyl coenzyme A) . As predicted from their measured DT diaphorase activities, a number of rat hepatoma and hepatocyte cell lines are also sensitive to CB 1954 . However, no CB 1954-sensitive tumours or cell lines of human origin have been found . This is because the rate of reduction of CB 1954 by the human form of DT diaphorase is much lower than that of the Walker enzyme (ratio of kcat = 6.4) . To overcome this intrinsic resistance of human cells towards CB 1954 a number of strategies have been developed . First, analogues have been developed that are more rapidly reduced by the human form of CB 1954 . Second, the cytotoxicity of CB 1954 can be potentiated by reduced pyridinium compounds . Third, a CB 1954 activating enzyme can be targeted to human tumours by conjugating it to an antibody (ADEPT) . A nitroreductase enzyme has been isolated from E . coli that can bioactivate CB 1954 much more rapidly than Walker DT diaphorase and is very suitable for ADEPT . Thus CB 1954 may have a role in the therapy of human tumours.

Childs Nerv Syst, 1993 Jun, 9(3), 163 - 5
A concerted effort to prevent shunt infection; Kestle JR et al.; In an attempt to reduce the rate of shunt infection a new protocol for shunt surgery was introduced on July 1, 1988 at The Hospital for Sick Children in Toronto . The operations were done at the beginning of the day, operating room personnel were kept to a minimum, no visitors were allowed in the room, a staff neurosurgeon or neurosurgical fellow attended all operations and two doses of perioperative cloxacillin 50 mg/kg were given intravenously . From July 1, 1988 to June 30, 1989 there were 576 shunt procedures on the Neurosurgical Service and 22 (3.8%) of these resulted in a shunt infection . During the preceding year (July 1, 1987 to June 30, 1988) 581 shunt operations were performed, 75 (12.9%) of which resulted in an infection (chi 2 = 29.9, P < 0.001) . It appears that the introduction of this protocol for shunt surgery has helped to reduce the risk of shunt infection from 12.9% to 3.8% (a reduction of 70.5%) . The rate of infection occurring after shunt revisions was not significantly different from that occurring after new shunt insertions . When the individual factors in the protocol were analyzed, the use of antibiotics and a shorter duration of surgery appeared to be related to a lower shunt infection rate.

Biull Eksp Biol Med, 1993 Jun, 115(6), 599 - 600
{The effect of the mast cells on interleukin-1 production by exudate and bone marrow macrophages in inflammation}; Klimenko NA et al.; It was shown on the model of acute infectious peritonitis in mice that the inflammation induced in the absence of mast cells was characterized by an earlier interleukin-1 (IL-1) production by bone marrow macrophages and by the lack of relapsed IL-1 production by macrophages of both exudate and bone marrow . This correlated with the previous data of the authors on the earlier haematopoiesis activation but weaker bone marrow hyperplasia expression in inflammation induced in the absence of mast cell . The results testify to essential role of mast cells in regulation of IL-1 production by macrophages of exudate and bone marrow . The previously established modulatory effect of mast cells on haematopoiesis in inflammation may be mediated by their effect on IL-1 production by macrophages of the inflammatory focus and haematopoiesis-induced microenvironment.

J Biochem (Tokyo), 1993 Jun, 113(6), 734 - 7
Recombinant human synovial fluid phospholipase A2 and N-terminal variant: kinetic parameters and response to inhibitors; Marki F et al.; To further explore the role of the N-terminus of group II phospholipase A2 (PLA2) for enzymatic activity, kinetics of recombinant human synovial fluid PLA2 (rPLA2) and a variant with an extra N-terminal methionine (Met-rPLA2) were analyzed with the substrates {1-14C}oleate-labeled Escherichia coli, vesicular phosphatidylethanolamine, and micellar phosphatidylcholine . While N-terminal variation did not affect assay parameters such as pH-optimum, or optimal concentration of calcium and E . coli substrate, it drastically reduced maximum velocities with all three substrates, without any decrease of substrate affinities, suggesting a role of the N-terminus in the catalytic step(s) subsequent to binding of the substrate . Eleven compounds from various structural classes were found to potently inhibit both enzymes . Several of these compounds were equipotent on rPLA2 and Met-rPLA2; others had significantly lower potency on Met-rPLA2 than on rPLA2 . This suggests participation of the N-terminal region of the enzyme in the mechanism of inhibition by the latter compounds . The study provides new evidence for the role of the N-terminus of group II PLA2 for catalytic activity.

Clin Exp Allergy, 1993 Jun, 23(6), 518 - 23
The effect of zardaverine, an inhibitor of phosphodiesterase isoenzymes III and IV, on endotoxin-induced airway changes in rats; Kips JC et al.; Zardaverine is a novel phosphodiesterase III/IV inhibitor, developed as a potential therapeutic agent for asthma . In this study we evaluated the effect of zardaverine in an in vivo animal model of airway inflammation and hyperresponsiveness . Endotoxin exposure in rats causes a transient increase in airway responsiveness and a neutrophilic inflammation of the bronchi, which are both at least partly mediated through the secondary release of tumour necrosis factor alpha (TNF alpha) . Groups of 10 animals each were pretreated with placebo or zardaverine (1, 10, 30 mumol/kg) i.p., 30 min prior to exposure to aerosolized endotoxin (LPS) or saline . Ninety minutes later, airway responsiveness to 5-HT was assessed and bronchoalveolar lavage (BAL) performed . Zardaverine did not influence baseline lung resistance (RL), but inhibited dose dependently the 5-HT induced increase in RL in control animals . In placebo pretreated animals LPS exposure caused a significant decrease in PC50RL5-HT (provocative concentration of 5-HT causing a 50% increase in RL), compared to the saline exposed control group (1.1 +/- 0.1 vs 2.7 +/- 0.4 micrograms/kg) (P < 0.01) . This decrease in PC50RL5-HT was significantly inhibited by zardaverine 30 mumol/kg (5.4 +/- 1.8 vs 1.1 +/- 0.1 micrograms/kg) (P < 0.05) . Compared to placebo pre-treated, LPS exposed animals, zardaverine 30 mumol/kg also significantly inhibited to LPS induced neutrophil increase (193.0 +/- 50.0 vs 915.6 +/- 181.3 x 10(3)) (P < 0.01), increase in elastase activity (23 +/- 11 vs 54 +/- 9 nmol substrate/h/ml) (P < 0.05) and TNF alpha release in BAL fluid (93.1 +/- 19.5 vs 229.5 +/- 24.8 U/ml BAL fluid) (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Acta Med Port, 1993 Jun, 6(6), 271 - 4
{Emphysematous pyelonephritis}; Rosado J et al.; A case of emphysematous pyelonephritis to E . coli, in a 63-year-old female diabetic patient is presented herein . Imaging techniques (plain abdomen X-Ray, ultra-sonography and computed tomography) allowed the authors to make the diagnosis . Percutaneous nephrostomy, combined with medical therapeutics, contributed to a favourable clinical evolution of the patient.

Photochem Photobiol, 1993 Jun, 57(6), 1027 - 31
Effects of mutagenetic substitution of prolines on the rate of deprotonation and reprotonation of the Schiff base during the photocycle of bacteriorhodopsin; Zhang YN et al.; Membrane-buried proline residues are found in many transport proteins . To study their roles in the structure and function of bacteriorhodopsin (bR), effects of the individual substitutions of Pro-50, Pro-91 and Pro-186 on the deprotonation and reprotonation kinetics of the Schiff base (SB) were determined by flash photolysis . The obtained rate constants and the amplitudes of the slow and fast components were compared with those of ebR (wild-type bR, the native protein that is expressed in Escherichia coli) . The deprotonation rates of PSB were found to be 10 times faster than that of ebR for P50A, P91A and P91G mutants, and 4 times faster for the P50G mutant . These mutations also increased the initial reprotonation rate of the SB, although the overall change in the reprotonation rate is not as significant as that in the deprotonation rate . Our results indicate that Pro-50 and Pro-91, as well as Pro-186, are important for the proton-pumping function of bR.

Photochem Photobiol, 1993 Jun, 57(6), 1011 - 7
Cu(II) sensitizes pBR322 plasmid DNA to inactivation by UV-B (280-315 nm); Lloyd RE et al.; Copper(II), in the presence of UV-B radiation (280-315 nm), can generate single-strand breaks in the sugar-phosphate backbone of pBR322 plasmid DNA . A low level of single-strand backbone breaks occurs in the presence of Cu(II) alone, but UV-B irradiation increases the rate by the more than 100-fold . Concomitant with the damage to the DNA backbone is a loss of transforming activity . Oxygen is required for generation of the single-strand breaks but not for the loss of transforming activity . A DNA glycosylase (Fpg), which participates in the repair of certain DNA nitrogenous base damage, does not repair plasmid DNA damaged by Cu(II) . The hydroxyl radical scavenging compound DMSO is only somewhat effective at protecting the physical and biological properties of the DNA . These results with Cu(II) are compared to those obtained previously with pBR322 plasmid DNA in the presence of Fe(III) and UV-A.

Genet Res, 1993 Jun, 61(3), 195 - 204
Evolution of the mitochondrial ATPase 6 gene in Drosophila: unusually high level of polymorphism in D . melanogaster; Kaneko M et al.; We have determined 1990 bp mitochondrial DNA sequence which extends from 3' end of the cytochrome oxidase subunit I (COI) gene to 5' end of the COIII gene from two sibling species of Drosophila, D . simulans and D . mauritiana . Analyses of the sequences and part of the NADH dehydrogenase subunit 2 gene and the COI gene together with those from D . melanogaster and D . yakuba revealed that amino-acid substitution rate of the ATPase 6 gene seems to be higher in some strains of D . melanogaster than in the other species . High level of amino-acid polymorphism in this gene was observed in D . melanogaster . Synonymous substitution rate is relatively constant in all the genes examined, suggesting that mutation rate is not higher in the ATPase 6 gene of D . melanogaster . The amino-acid substitutions found specifically in D . melanogaster are at the sites which are not conserved among mammals, yeast and E . coli . These sites of the ATPase 6 gene might lose the selective constraint in D . melanogaster, and the amino-acid substitutions can be explained by neutral mutations and random genetic drift.

Biol Pharm Bull, 1993 Jun, 16(6), 552 - 7
A trypsin inhibitor trans-4-guanidinomethylcyclohexanecarboxylic acid 4-tert-butylphenyl ester suppresses the onset of DNA synthesis in Escherichia coli cells synchronized by phosphate starvation; Kato M et al.; trans-4-Guanidinomethylcyclohexanecarboxylic acid 4-tert-butylphenyl ester (GMCHA-OPh'Bu), a trypsin inhibitor, dose-dependently inhibited the growth of Escherichia coli K-12 IAM1264 . Growth inhibition was preceded by dose- and time-dependent inhibition of DNA synthesis . These results strongly suggested participation of a trypsin-like proteinase in DNA synthesis . To clarify this suggestion, the effects of GMCHA-OPh'Bu on the doubling time and on the uptake of {methyl-3H}thymidine into DNA were examined with E . coli K-12 IAM1264 synchronized by a modified version of phosphate starvation . The synchrony lasted for two or three cycles with a doubling time of 55 min and a cell division period of 15 min . The cell cycle of E . coli was divided into three periods, cell division period (P), the period between cell division and initiation of chromosome replication (Q) and the period between initiation of chromosome replication and cell division (R) . The R period was subdivided into two periods, R1 in which the rate of thymidine uptake into DNA was increasing, and R2 in which it was constant . The addition of GMCHA-OPh'Bu at the R1 period did not affect the already-initiated round of cell division, however, it retarded the next round . The addition at P, Q or R2 retarded the cell division in the same round, causing prolongation of the R1 period . A sharp and momentary appearance of trypsin-like proteinase activity peaked at the Q/R1 boundary in one cell cycle, and inhibition of the activity prolonged the R1 period.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem Mol Biol Int, 1993 Jun, 30(2), 377 - 83
Changes in intracellular potassium and thiol levels in Escherichia coli K12 under various stresses; Oktyabrsky ON et al.; The levels of intracellular K+ and thiols were measured synchronously in E . coli cells under starvation stress and osmotic upshock,alkaline shift and cytoplasm acidification . The level of nonprotein thiols increased in response to osmotic upshock and alkaline shift but decreased under cytoplasm acidification . A positive correlation between the changes in the K+ pool and the level of nonprotein thiols was found under osmotic shock and alkaline shift . Inverse correlation between these parameters was observed after the acidification of cytoplasm . It is supposed that there is a tight relationship between the levels of K+, nonprotein thiols and intracellular pH.

Biokhimiia, 1993 Jun, 58(6), 908 - 12
{A new method of isolation and purification of SsoII restriction endonuclease}; Kariagina AS et al.; A new method for isolation and purification of restriction endonuclease SsoII which results in a homogeneous preparation suitable for all types of fine physico-chemical assays has been elaborated . The procedure includes four chromatographic steps: fractionation on butyl-Toyopearl, combined chromatography on SP-Toyopearl and phosphocellulose PII, and chromatography on DEAE-Toyopearl and on QAE-Toyopearl . The use of fast flow sorbents (Toyopearl) makes it possible to reduce the time needed for the separation of proteins and to optimize the fractionation conditions, thus avoiding the dialysis between the chromatographic steps which significantly decreased the enzyme activity yields in previous purification schemes . The isolation of restriction endonuclease SsoII by the new method usually takes four days.

AIDS, 1993 Jun, 7(6), 807 - 12
Augmentation of HIV-specific lymphoproliferation in HIV-infected individuals by TraT: a novel T-cell immunopotentiating agent; Bell SJ et al.; OBJECTIVE: To evaluate the potential of TraT to restore HIV-specific cell-mediated immunity . DESIGN: CD4+ T cell-associated antiviral and recall antigen-specific lymphoproliferative responses are generally impaired or absent in HIV-infected individuals . METHODS: Using peripheral blood mononuclear cells (PBMC) from a group of asymptomatic and symptomatic HIV-infected individuals, we compared the immunomodulatory effects of exogenous interleukin-2 (IL-2) with the effects elicited by the bacterial integral membrane protein, TraT . RESULTS: Exogenous IL-2 enhanced lymphoproliferation induced by an immunodominant synthetic HIV gp41 analogue, gp41{8} (amino acids 593-604), in four out of 10 asymptomatics and six out of 19 symptomatics . In contrast, TraT acted synergistically with gp41{8} to augment HIV-specific proliferation with higher frequency and greater magnitude than exogenous IL-2 . Moreover, this TraT-mediated enhancement of HIV-specific lymphoproliferation occurred in the majority of HIV-infected individuals, irrespective of CD4+ T-cell count in peripheral blood or disease status, and thus appears not to be major histocompatibility complex-restricted . TraT also augmented lymphoproliferation induced by well-known recall antigens and other less immunodominant HIV analogues . CONCLUSIONS: These findings suggest that TraT, in combination with HIV-derived peptides, could be used to maintain or restore cell-mediated immune functions of HIV-infected individuals, as well as cellular immune functions in individuals suffering from other immunodeficiency disorders.

Infect Immun, 1993 Jun, 61(6), 2462 - 7
A recombinant 15-kilodalton carboxyl-terminal fragment of Plasmodium yoelii yoelii 17XL merozoite surface protein 1 induces a protective immune response in mice; Daly TM et al.; Since the developmental stages of malarial parasites which replicate within erythrocytes are responsible for the morbidity and mortality associated with this disease, antigens produced by these stages have been proposed as candidates for a vaccine . One surface protein of merozoites (MSP-1) has been shown to immunize both rodents and primates against virulent challenge infection in experimental systems . However, little is known of relevant epitopes on the molecule, and attempts to obtain recombinant MSP-1 polypeptides in a native configuration have proven difficult . We have found that the cysteine-rich, carboxyl-terminal region of the MSP-1 protein from the rodent malarial parasite Plasmodium yoelii yoelii can be expressed in a native configuration as a fusion protein in Escherichia coli . This recombinant polypeptide containing 15 kDa of the predicted 197-kDa protein elicits antibodies in mice which recognize the native parasite MSP-1 . Most significantly, both inbred and outbred mice immunized with the fusion protein in Ribi adjuvant are partially and in some cases completely protected against challenge infection with an otherwise lethal parasite strain . This is the first observation of such significant protection obtained with a small portion of the MSP-1 produced in recombinant systems.

Mol Microbiol, 1993 Jun, 8(6), 1145 - 53
Identification of an operon involved in the assimilatory nitrate-reducing system of Azotobacter vinelandii; Ramos F et al.; A number of mutants lacking nitrate reductase (Nas-) or nitrite reductase (Nis-) activities have been isolated and characterized . An operon including two new genes (nasA and nasB) has been defined and cloned from an Azotobacter vinelandii gene bank . nasA encodes for nitrite reductase apoenzyme, whereas nasB is specific for nitrate reductase activity . Nitrate reductase exerts a regulatory effect on nasAB.

Mol Microbiol, 1993 Jun, 8(6), 1071 - 81
Molecular genetic analysis of the moa operon of Escherichia coli K-12 required for molybdenum cofactor biosynthesis; Rivers SL et al.; A 3.2 kb chromosomal DNA fragment which complements the defects in a series of twelve moa::Mucts insertion mutants has been sequenced . Five open reading frames (ORFs) were identified and these are arranged in a manner consistent with their forming an operon . The encoded proteins (MoaA-MoaE) have predicted molecular weights of 37,346, 18,665, 17,234, 8843 and 16,981 respectively . Examination of subclones of the whole locus in an expression system demonstrated the predicted products . N-terminal amino acid sequences for the moaA, B, C and E products confirmed the translational starts . Genetic analysis distinguished four classes of moa mutants corresponding to genes moaA, C, D and E . Potential promoter sequences upstream of moaA and a possible transcription termination signal have been identified . Genetic analysis of the chlA1 and chlM mutants, which have been biochemically characterized as defective in molybdopterin biosynthesis, indicates that these carry lesions in moaA and moaD respectively . The moa locus is orientated clockwise at 17.7 minutes in the chromosome.

Mol Microbiol, 1993 Jun, 8(6), 1031 - 8
Multicopy plasmid instability: the dimer catastrophe hypothesis; Summers DK et al.; Multimer formation reduces plasmid copy number and is an established cause of segregational instability . Nevertheless, it is difficult to rationalize observations that low levels of dimers can cause severe instability, if we assume they are distributed evenly in cell populations . We report here that dimer distribution is in fact heterogeneous in recombination-proficient strains . Most cells in the population contain only monomers; dimers are confined to a small subpopulation from which plasmid-free daughters arise at high frequency . In a rec+ culture where 4% of pBR322 molecules are dimers, more than half are in dimer-only cells . We show that this situation is inevitable because dimers replicate at twice the rate of monomers . Runaway multimerization is avoided because dimer-containing cells grow more slowly than their monomer-containing counterparts . A computer simulation is used to show how dimers proliferate after formation by homologous recombination . The equilibrium concentration of dimers is proportional to the inter-plasmid recombination rate and is essentially independent of the rate at which homologous recombination converts dimers to monomers.

J Gen Microbiol, 1993 Jun, 139 ( Pt 6), 1235 - 43
Genetic organization, sequence and biochemical characterization of recombinant beta-xylosidase from Thermoanaerobacterium saccharolyticum strain B6A-RI; Lee YE et al.; Endoxylanase (xynA) and beta-xylosidase (xynB) genes from Thermoanerobacterium saccharolyticum were subcloned from a cosmid clone (pXDM1) to generate pXPH3 . The nucleotide sequence of a PstI-HindIII fragment in pXPH3 that contained xynB revealed an open reading frame (ORF) of 1500 bp encoding a 55 kDa protein . Another open reading frame (ORF1) of unknown function was found 21 bp downstream from the first stop codon of xynB . xynB, ORF1 and xynA had the same direction of transcription . xynB from T . saccharolyticum strain B6A-RI exhibited 45% amino acid similarity, with 18% amino acid identity to xynA of T . saccharolyticum strain B6A-RI, and 61% similarity and 37% identity with the beta-xylosidase gene from Caldocellum saccharolyticum . Recombinant beta-xylosidase was purified from E . coli (pXPH3) cells . The enzyme was a monomer with a molecular mass of 55 kDa . The specific activity and pH and temperature optima for hydrolysis of p-nitrophenyl-beta-D-xylopyranoside (pNPX) were 5.53 U mg-1, 5.5 and 70 degrees C, respectively . The beta-xylosidase was stable at 65 degrees C, but lost activity at 85 degrees C . The purified enzyme had hydrolytic activity towards xylopentose, xylotriose, xylobiose and pNPX, but had no activity toward xylan.

Hum Mol Genet, 1993 Jun, 2(6), 673 - 6
A gene from chromosome 4p16.3 with similarity to a superfamily of transporter proteins; Duyao MP et al.; We have previously used exon amplification to identify the ADD1 gene in cosmid Y24 from the Huntington's disease (HD) region of 4p16.3 . The same technique has now yielded a second gene from this cosmid . This gene appears to encode a novel member of a superfamily of transporter proteins that includes active and passive transporters in a number of species . The predicted protein of 455 amino acids displays sequence similarity with the E . coli tetracycline resistance efflux protein encoded by cloning vector pBR322, and with a number of related transporters . This gene should open a route to isolating additional mammalian members of this growing superfamily.

Res Commun Chem Pathol Pharmacol, 1993 Jun, 80(3), 367 - 70
J5 prevents endotoxin shock in suckling rats; Yoshioka T et al.; A sublethal dose of endotoxin (lipopolysaccharide: LPS) can induce endotoxin tolerance . It is not well known whether LPS feeding causes endotoxemia and endotoxin tolerance in the newborn . Since Rc mutant Escherichia coli LPS (J5) does not have toxic or lethal effects, J5 is used to induce endotoxin tolerance in this study . This study showed that both an intraperitoneal (ip) injection and feeding of Rc mutant Escherichia coli LPS (J5) induced endotoxin tolerance in suckling rats.

J Mol Evol, 1993 Jun, 36(6), 586 - 95
Development of artificial neural filters for pattern recognition in protein sequences; Schneider G et al.; Four different artificial neural network architectures have been tested for their suitability to extract and predict sequence features . For optimization of the network weights an evolutionary computing method has been applied . The networks have feedforward architecture and provide adaptive neural filter systems for pattern recognition in primary structures and sequence classification . The recognition and prediction of signal peptidase cleavage sites of E . coli periplasmic protein precursors serves as an example for filter development . The primary structures are represented by seven physicochemical residue properties . This amino acid description provides the feature space for network optimization . The properties hydrophobicity, hydrophilicity, side-chain volume, and polarity allowed an accurate classification of the data . A three-layer network architecture reached a learning success of 100%; the highest prediction accuracy in an independent test set of sequences was 97% . This network architecture appears to be most suited for the analysis of E . coli signal peptidase cleavage sites . Further suggestions about the design and future applications of artificial neural networks for protein sequence analysis are made.

Int Immunol, 1993 Jun, 5(6), 681 - 90
Expression of recombinant rabbit IL-8 in Escherichia coli and establishment of the essential involvement of IL-8 in recruiting neutrophils into lipopolysaccharide-induced inflammatory site of rabbit skin; Harada A et al.; In order to establish the pathophysiological roles of IL-8, rabbit IL-8 was expressed in Escherichia coli and purified to homogeneity by sequential chromatography on heparin agarose, CM-HPLC, and RP-HPLC . The purified recombinant rabbit IL-8 was homogeneous on SDS-PAGE and the ED50 of neutrophil chemotactic activity for rabbit peritoneal neutrophils was 2 ng/ml . The binding of 125I-labeled rabbit IL-8 to rabbit neutrophils was inhibited by unlabeled human IL-8 as well as rabbit IL-8 but not by another leucocyte chemotactic cytokine (chemokine), monocyte chemotactic and activating factor . Scatchard plot analysis of the binding of 125I-labeled rabbit IL-8 to rabbit peritoneal neutrophils revealed that the rabbit neutrophils have two affinity classes of receptors for IL-8 (Kd = 2.3 nM, 4.1 x 10(4) sites/cell; Kd = 18.0 nM, 11.4 x 10(4) sites/cell) . It was found that a previously generated mouse anti-human IL-8 mAb, WS-4, inhibited the binding of 125I-labeled rabbit IL-8 to rabbit neutrophils, and blocked neutrophil chemotaxis in vitro in a specific and dose-dependent manner . An ELISA system for rabbit IL-8 was established using this mAb and guinea pig polyclonal antibodies to recombinant rabbit IL-8 to measure the levels of IL-8 in rabbit plasma . Intravenous administration of lipopolysaccharide (LPS) (100 micrograms) in rabbits caused the highest level of IL-8 in blood at around 2 h . Intravenous administration of WS-4 (10 mg) inhibited neutrophil infiltration at the site of LPS injection into the rabbit skin, suggesting that IL-8 is essential in the recruitment of neutrophils at sites of acute inflammation in vivo.

Virus Res, 1993 Jun, 28(3), 273 - 83
The gene encoding the late nonstructural 36K protein of vaccinia virus is essential for virus reproduction; Shchelkunov SN et al.; Two genetic markers--the thymidine kinase gene of herpes simplex virus, and the beta-galactosidase gene of Escherichia coli--were incorporated into the 36K protein gene (IL1 gene according to the nomenclature of the Copenhagen strain of vaccinia virus; Goebel et al., 1990) from the HindIII-P DNA fragment of the LIVP strain (variant of Lister strain) of vaccinia virus (VV) . After recombination of the obtained integration plasmid pVZ64-TK with the VV genome (tk-), it was found that the resultant TK+ viruses were unstable with respect to the Lac+ phenotype . On the basis of hybridization of DNA fragments of selected clones, a scheme for the formation of hybrid viruses is proposed, and an approach to a simple phenotypical discrimination between essential and non-essential genes for VV viability is described.

J Crit Care, 1993 Jun, 8(2), 109 - 16
Effect of anesthesia and surgery on plasma cytokine levels; Shimada M et al.; Cytokines released in response to stress may have a profound impact on circulatory stability . There is no information on the effect of general anesthesia alone on plasma cytokine levels and little information on cytokine release following surgery . Plasma cytokine levels and hemodynamic parameters were measured during anesthesia and abdominal surgery under sterile and nonpyrogenic conditions in seven pigs anesthetized with ketamine and pentobarbital . Tumor necrosis factor (TNF) was measured by bioassay . Bioassays of low and high sensitivity were used to measure interleukin 6 (IL-6) . Measurements were made sequentially during: (1) 4 hours observation with anesthesia alone; (2) 2 hours following laparotomy and traumatic intestinal manipulation (IM) sufficient to produce shock; and (3) after an intravenous bolus of 1 microgram/kg endotoxin as a positive control . Arterial blood pressure decreased following IM from 91.5 +/- 5.8 to 48.6 +/- 3.2 mm Hg, (mean +/- SE, P < .05), with no further change following endotoxin . Heart rate was unchanged during the experiment, and central venous pressure decreased after endotoxin (P < .05) . There were no increases in TNF or IL-6 (using a low sensitivity assay) with anesthesia alone or following IM with shock, but both increased after endotoxin administration (P < .05); using a high sensitivity assay, IL-6 did not change during anesthesia alone but did increase fivefold following IM with shock (P < .05) and 50-fold following endotoxin administration (P < .05) . We conclude that in a porcine model under sterile and nonpyrogenic conditions, prolonged anesthesia does not increase plasma cytokine levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Arch Esp Urol, 1993 Jun, 46(5), 419 - 21
{The infected solitary renal cyst . A case review and review of the literature}; Lopez Caballero I et al.; A case of solitary infected renal cyst is presented . The clinical features, diagnosis and treatment of this disease entity are discussed and the literature reviewed . Less than thirty cases have been reported until the last decade.

Mol Biochem Parasitol, 1993 Jun, 59(2), 327 - 9
Rapid isolation of DNA from trypanosomatid protozoa using a simple 'mini-prep' procedure; Medina-Acosta E et al.; Although several methods for isolating genomic DNA from trypanosomatid protozoa exist, all are time-consuming and cumbersome . Faster, simpler and efficient protocols for preparation of DNA from these protozoa are needed to ease the screening of mutants and transfectants . We describe the use of a bacterial lysis method to isolate chromosomal DNA from a wide range of trypanosomatids . The method is based on the finding reported by He et al., who noticed that phenol/chloroform treatment of Escherichia coli cells in the presence of LiCl and Triton X-100 solubilizes plasmid DNA, while precipitating unwanted chromosomal DNA and denatured cellular proteins . In applying this lysis method to the isolation of episomal DNA from transfected trypanosomatids, we found that, unlike bacterial genomic DNA, chromosomal DNA of trypanosomatids was soluble in the phenol/chloroform/Triton/LiCl mixture . This observation prompted us to use the bacterial lysis method as a routine protocol for extraction of DNA from trypanosomatids.

Mol Biochem Parasitol, 1993 Jun, 59(2), 223 - 34
Molecular cloning and localization of an abundant novel protein of Plasmodium berghei; Uparanukraw P et al.; Screening of Plasmodium berghei genomic libraries using DNA insert corresponding to the 3' half of P . falciparum 70-kDa heat shock protein gene identified several abundant clones which represent a novel gene in the parasite . The complete sequence was obtained using an approach based on inverse polymerase chain reaction . Analysis of the deduced amino acid sequence revealed the presence of 19 imperfect repeats of the sequence Gly-Gly-Met-Pro toward the carboxy terminus . Except for the similar sequence repeated seven times in the malarial 70-kDa heat shock protein, the sequence of the cloned gene product is very different . Moreover, the sequence also revealed acidic and basic domains in the protein which are more than 60% similar in sequence to functional domains present in numerous DNA binding transcription factors . A 56-kDa protein was identified by immunoprecipitation from labeled P . berghei extract using antisera raised in mice against gene products expressed in Escherichia coli . The protein is present in all the different life cycle stages of the parasites as revealed by immunoelectron microscopy.

Mol Biochem Parasitol, 1993 Jun, 59(2), 191 - 200
A mitochondrial heat shock protein from Crithidia fasciculata; Effron PN et al.; MCP72 is a mitochondrial hsp70 protein from the trypanosomatid Crithidia fasciculata . An MCP72 cDNA clone was isolated from a C . fasciculata cDNA library by screening with antiserum specific for the homologous protein of Trypanosoma cruzi {9} . The MCP72 cDNA encodes a polypeptide of 663 amino acids which is 84% identical to the Trypanosoma cruzi protein and 56% identical to the Escherichia coli hsp70 protein DnaK . MCP72 is less similar to other hsp70 proteins . Native MCP72 was purified to homogeneity by ATP-agarose affinity chromatography . Comparison of its N-terminal amino acid sequence with that deduced from the cDNA sequence shows that 20 amino acid residues had been cleaved from the N-terminus; this sequence probably represents a mitochondrial import signal which is cleaved during translocation into the mitochondrion . Fluorescence microscopy, using antibodies specific for MCP72, indicates that the protein is concentrated in a region of the mitochondrial matrix which surrounds the kinetoplast.

Mol Biochem Parasitol, 1993 Jun, 59(2), 181 - 9
Cloning and characterisation of cDNA clones encoding two Babesia bovis proteins with homologous amino- and carboxy-terminal domains; Dalrymple BP et al.; A dextran sulphate protein (DSP) fraction derived from Babesia bovis has previously been shown to induce a protective immune response in cattle . A B . bovis cDNA library was screened with both the complete anti-DSP serum and a subfraction of the anti-DSP serum affinity purified on a native B . bovis protein of approx . 80 kDa . cDNA clones encoding two different B . bovis proteins were identified . The product of one gene, Bv80, has a single divergent copy of a sequence of 149 amino acids (approx . 30% amino acid identity) in both the amino- and carboxy-terminal domains . These domains are separated by an array of short variant repeat sequences rich in proline and glutamic acid . The product of the other gene, BvVAl (homologous to the previously described 225-kDa B . bovis protein){19}, is predicted to have a single divergent copy of a sequence of 170-171 amino acids (approx . 35% amino acid identity) in both the amino- and carboxy-terminal domains . These domains are also separated by an array of repeats . The 73-amino acid repeat unit of this array is composed of a number of variant derivatives of shorter repeat units . Detailed analysis of genomic clones flanking two alleles of the gene encoding BvVAl/225 kDa identified further members of a multi-gene family . This region of the genome of B . bovis has been subject to a large number of amplification processes.

Eur J Pharmacol, 1993 Jun 1, 248(1), 41 - 7
Pharmacological modulation of lipopolysaccharide-induced pleural eosinophilia in the rat; a role for a newly generated protein; Bozza PT et al.; Intrathoracic injection of endotoxin lipopolysaccharide, LPS into rats induced a dose-dependent increase in the number of eosinophils recovered from the pleural cavity . The pleural eosinophil accumulation peaked within 24-48 h, and returned to basal levels within 120 h . This phenomenon was accompanied by mononuclear cell infiltration, and preceded by massive neutrophil accumulation . Pretreatment with indomethacin, BW 755C (a dual cyclo/lipoxygenase inhibitor), BW A4C (a specific lipoxygenase inhibitor) or the platelet activating factor (PAF) antagonists WEB 2086 and PCA 4248 failed to inhibit the endotoxin-induced pleural eosinophilia, whilst dexamethasone (5-10 micrograms/cavity) or cycloheximide (14-28 micrograms/cavity) abolished this phenomenon . Transfer of the cell-free pleural washing from LPS-treated donor rats to normal recipient rats led to a two-fold increase in the eosinophil counts . Treatment of donors, but not recipients, with cycloheximide or dexamethasone inhibited the eosinophil accumulation induced by the pleural washings, indicating that the generation of the eosinophilotactic activity, but not its effects, depends on protein synthesis . This eosinophilotactic activity was maintained after lyophilization and heating (100 degrees C for 30 min), but was destroyed by trypsin . This substance has a molecular weight ranging between 10 and 50 kDa . The available data suggest that the late eosinophil accumulation induced by LPS is independent of arachidonic acid metabolites and PAF, and probably depends on a newly generated heat-stable soluble protein.

Trends Genet, 1993 Jun, 9(6), 211 - 7
Nucleotide excision repair . II: From yeast to mammals; Hoeijmakers JH; An intricate network of repair systems safeguards the integrity of genetic material, by eliminating DNA lesions induced by numerous environmental and endogenous genotoxic agents . Nucleotide excision repair (NER) is one of the most versatile DNA repair systems . Deficiencies in this process give rise to the classical human DNA repair disorders xeroderma pigmentosum (XP) and Cockayne's syndrome (CS), and to a recently recognized disease called PIBIDS, a photosensitive form of the brittle hair disorder trichothiodystrophy . This is the second of a two-part review on NER . Part I (in the previous issue of TIG) concentrated on the main characteristics of the NER pathway of E . coli and yeast . Part II compares the mammalian and yeast systems, and attempts to integrate current knowledge on the eukaryotic pathway to suggest an outline for the reaction mechanism.

Am J Trop Med Hyg, 1993 Jun, 48(6), 831 - 8
Cytostatic effect of Lutzomyia longipalpis salivary gland homogenates on Leishmania parasites; Charlab R et al.; Salivary gland homogenates (SGH) of female Lutzomyia longipalpis, in concentrations as small as 0.05 pairs of glands/ml, inhibit the in vitro multiplication of promastigotes of Leishmania mexicana amazonensis . The effect seems to be cytostatic since promastigote viability 24 hr after exposure ranged from 55% to 100% in different experiments . The cells cultivated in the presence of SGH were characterized by a very slender shape, with cell bodies that were almost two times as long as controls . The promastigote growth inhibitory activity was not present in Anopheles albimanus SGH or in the gut extracts of Lu . longipalpis sand flies . Additionally, the salivary gland homogenates of Lu . longipalpis did not inhibit the growth of other cell types such as Escherichia coli or a monkey kidney cell line (LLCMK2), suggesting that the activity had a specific range of action . The SGH activity was sensitive to both trypsinization and boiling, partially resistant to heating at 56 degrees C for 30 min, and had a molecular weight of approximately 20 kD as determined by size exclusion high-performance liquid chromatography . The results suggest that vector saliva could influence the development of Leishmania parasites within the vector by inhibiting their growth and triggering them to a differentiation pathway.

Protein Eng, 1993 Jun, 6(4), 425 - 31
Mutagenesis and kinetic analysis of the active site Glu177 of ricin A-chain; Chaddock JA et al.; Ricin A-chain (RTA) is an N-glycosidase which removes a specific adenine residue from the large rRNA of eukaryotic ribosomes . As a consequence, the ribosome is inactivated and protein synthesis is inhibited leading to cell death . This report describes the effects on enzyme activity of specific mutations of the conserved active site Glu177 . The activity of mutant proteins was initially screened using an in vitro translation system . It was found that mutagenesis of Glu177 to Lys led to an apparent total inactivation of the enzyme, Glu177 to Ala had a small effect on activity, whereas the conservative Glu177 to Asp mutation had a significant effect . The properties of Glu177 to Asp were investigated more closely . Mutant protein was purified from an Escherichia coli expression system and kinetic analysis of the depurination activity assessed using salt-washed yeast ribosomes . It was shown that the Km of the mutant protein was unchanged when compared to data of wild type RTA; however, the kcat was significantly decreased (49-fold compared to wild type RTA) . This suggests that Glu177 plays a predominant role in the rate-limiting step of the enzymatic mechanism and not in substrate binding . These data are discussed in relation to other reports of ricin Glu177 substitutions.

Protein Eng, 1993 Jun, 6(4), 409 - 15
Altering substrate preference of carboxypeptidase Y by a novel strategy of mutagenesis eliminating wild type background; Olesen K et al.; To change the substrate preference of carboxypeptidase Y the putative substrate binding pocket was subjected to random mutagenesis . Based upon the three-dimensional structure of a homologous enzyme from wheat, we hypothesized that Tyr147, Leu178, Glu215, Arg216, Ile340 and Cys341 are the amino acid residues of carboxypeptidase Y that constitute S1, the binding pocket for the penultimate amino acid side chain of the substrate . We developed a new and generally applicable mutagenesis strategy to facilitate efficient screening of a large number of mutants with multiple changes in carboxypeptidase Y . The key feature is the elimination of wild type background by introducing a nonsense codon at each target site for subsequent mutagenesis by degenerate oligonucleotides . The entire hypothesized S1 binding pocket and subsets of it were subjected to saturation mutagenesis by this strategy, and screening yielded a number of mutant enzymes which have up to 150 times more activity (kcat/Km) towards CBZ-Lys-Leu-OH than the wild type enzyme . All selected mutants with increased activity have mutations at position 178 . Mutagenesis of positions 215 and 216 has virtually no effect on the activity, while mutating positions 340 and 341 generally reduces activity.

Protein Eng, 1993 Jun, 6(4), 349 - 57
X-ray structures of two single-residue mutants of DNase I: H134Q and Y76A; Weston S et al.; The structures of the single-residue mutants H134Q and Y76A of bovine pancreatic DNase I have been determined and refined including data to 2.3 and 2.4 A resolution respectively, by X-ray crystallography . H134 is an essential catalytic residue, while Y76 contributes to the binding of DNA by providing a large van der Waals contact area that stabilizes the wide minor groove seen in DNase I-DNA complexes . The mutant proteins, which show strongly reduced activities of 0.001% (H134Q) and 0.3% (Y76A), were expressed in E . coli and both crystallize in space-group C2 with almost identical unit cells . The crystal packing scheme is different from that found in wild type crystals grown under very similar conditions, presumably due to the absence of the carbohydrate moiety . In both mutants the conformation of the protein is nearly identical to that of the wild type enzyme and changes are confined to surface loops involved in packing . The disruption of the hydrogen bonds between H134, E78 and Y76 in both mutants leads to an increased mobility and positional shifts in the DNA-binding loop, mainly around residue Y76 . This in turn may further reduce DNA-binding affinity and, thus, contribute to the low activity . In contrast, symmetry contacts involving residues 97-108 lead to a stabilization of the flexible loop compared to wild type DNase I.

Cell Mol Biol (Noisy-le-grand), 1993 Jun, 39(4), 361 - 70
Aerobic responses of mouse macrophages to phagocytosis against Escherichia coli as revealed by microphotometry; Shono M et al.; Fluorescence of peritoneal macrophages, which ingested E . coli labelled with N-{7-(dimethylamino)-4-methyl-coumarinyl}-maleimide, was measured with a microfluorometer . The cellular contents of protein and formazan of a monotetrazolium produced by succinate dehydrogenase (SD) activity were assayed with a microspectrophotometer . During phagocytosis, protein increased in 30 min., but was balanced later by self-hydrolysis . SD activity was unchanged for 30 min . and then increased until 2 hrs . Upon removal of bacteria, the fluorescence and protein decreased with time, whereas SD activity increased further for 1 hr . before decrease . This indicates a delayed activation of the enzyme . The cellular ATP content was reduced during phagocytosis and restored to the normal level 3 hrs . after removal of bacteria . Both KCN-sensitive and -insensitive oxygen uptakes increased in phagocytosis . Thus, oxidative metabolism of macrophages is stimulated in phagocytosis and the stimulation can be demonstrated in situ by the microphotometric methods.

Rapid Commun Mass Spectrom, 1993 Jun, 7(6), 496 - 501
Direct observation of UV-crosslinked protein-nucleic acid complexes by matrix-assisted laser desorption ionization mass spectrometry; Jensen ON et al.; Interactions between proteins and nucleic acids are important in the fundamental cellular processes that drive replication, recombination, dynamic alteration and repair of DNA, transcription and processing of RNA, synthesis of proteins, and regulation of enzyme activities . As part of an effort to develop a general, sensitive mass spectrometric strategy for the characterization of protein-nucleic acid interactions, we have used matrix-assisted laser desorption-ionization (MALDI) time-of-flight mass spectrometry to analyze protein-nucleic acid complexes that have been covalently crosslinked by ultraviolet (UV) light . In general, the application of MALDI mass spectrometric techniques to studies of UV-induced crosslinking of nucleoprotein complexes is demonstrated to be feasible . Specifically, MALDI mass analysis was used to determine the molecular weights of the phage T4 gene 32 protein (gp32) crosslinked to the oligonucleotide (dT)20, and the Escherichia coli transcription termination factor rho, photoaffinity labeled with 4-thio-uridine-diphosphate (4sUDP) . The covalent gp32:(dT)20 complex is readily detected at a concentration of 1-2 microM in 1 microL of an unpurified solution of reactants that has been exposed to a single, 266 nm UV laser pulse . Mass spectrometric molecular weight determinations of the covalent rho:4sUDP complex add directness and specificity to the ATPase inactivation assay normally used to monitor the formation of 4sUDP photoaffinity labeled rho . It is found that successful MALDI mass spectrometry of protein-nucleic acid complexes is as critically dependent on the choice of solvents and additives as it is on the primary matrix compound.

Plant Mol Biol, 1993 Jun, 22(3), 411 - 26
Selection of Arabidopsis cDNAs that partially correct phenotypes of Escherichia coli DNA-damage-sensitive mutants and analysis of two plant cDNAs that appear to express UV-specific dark repair activities; Pang Q et al.; To resist terrestrial UV radiation, plants employ DNA-damage-repair/toleration (DRT) activities, as well as shielding mechanisms . Little is known about the structure and regulation of plant DRT genes . We isolated DRT cDNAs from Arabidopsis thaliana, by selecting for complementation of Escherichia coli mutants lacking all bacterial defenses against UV-light damage to DNA . These mutants are phenotypically deficient in recombinational and mutagenic toleration (RecA-), excision repair (Uvr-) and photoreactivation (Phr-) . Among 840 survivors of heavily UV-irradiated (10(-7) survival) mutants harboring plasmids derived from an Arabidopsis cDNA library in the vector lambda YES, we identified four unique plant cDNAs, designated DRT100, DRT101, DRT102, and DRT103 . Drt101 and Drt102 activity were specific for UV-light damage, and complemented both UvrB- and UvrC- phenotypes in the dark . Apparent Uvr- correction efficiencies were 1 to 40% for Drt101, and 0.2 to 15% for Drt102, depending on the UV fluence . Drt101 and Drt102 showed no extensive amino-acid homology with any known DNA-repair proteins . Drt100 appeared to correct RecA-, rather than Uvr-, phenotypes . Although the light dependence of Drt103 activity was consistent with its identification as a photoreactivating enzyme, its predicted amino-acid sequence did not resemble known photolyase sequences . The N-terminal coding sequence of Drt101 suggests that it is targeted to chloroplasts, as reported for Drt100 . These cDNAs afforded only modest increases in survival during the original selection procedure . The fact that they were readily isolated nevertheless suggests that selections may be made powerful enough to overcome barriers to expression and function in bacteria, at least for cDNAs of reasonable abundance.

Appl Environ Microbiol, 1993 Jun, 59(6), 1767 - 73
The GUS gene fusion system (Escherichia coli beta-D-glucuronidase gene), a useful tool in studies of root colonization by Fusarium oxysporum; Couteaudier Y et al.; The plant-pathogenic fungus Fusarium oxysporum was successfully transformed with the beta-D-glucuronidase gene from Escherichia coli (gusA) (GUS system) in combination with the gene for nitrate reductase (niaD) as the selectable marker . The frequency of cotransformation, as determined by GUS expression on plates containing medium supplemented with 5-bromo-4-chloro-3-indolyl glucuronide (GUS+), was very high (up to 75%) . Southern hybridization analyses of GUS+ transformants revealed that single or multiple copies of the gusA gene were integrated into the genomes . High levels of GUS activity are expressed in some transformants, but activity in F . oxysporum does not appear to be correlated with the copy number of the gusA gene . Since the highest activity was found in a transformant with a single copy, it can be assumed that sequence elements of F . oxysporum integrated upstream of the gene can act as a promoter or enhancer . Expression of the gusA gene was also detected during growth of the fungus in plants, indicating that the GUS system can be used as a sensitive and easy reporter gene assay in F . oxysporum.

J Trop Pediatr, 1993 Jun, 39(3), 183 - 7
Mannose-resistant haemagglutination (MRHA) and haemolysin (Hly) production of strains of Escherichia coli isolated from children with diarrhoea: effect of breastfeeding; Giugliano LG et al.; Haemolysin production (Hly) and mannose-resistant haemagglutination induction (MRHA) of human (H), bovine (B), and both (HB) erythrocytes was investigated in strains of E . coli isolated from 142 cases of children with diarrhoea aged 0-36 months . Haemolysin production was more frequent in strains isolated from children under 1 year and this characteristic was strongly associated with HB+ haemagglutination (P < 0.005) . Isolation frequency of MRHA strains was compared in 53 breastfed and 50 non-breastfed children under 1 year of age . HB+ strains were significantly more frequent in non-breastfed infants (P < 0.025) . Severity of diarrhoea evaluated by the number of watery stools per day, was significantly reduced in the breastfed group (P < 0.05) . The results suggests that breastfeeding may protect infants against the establishment of HB+ strains which might be acting either as a main pathogen or as an opportunistic strain.

J Dairy Sci, 1993 Jun, 76(6), 1568 - 74
Preinfection chemotactic response of blood polymorphonuclear leukocytes to predict severity of Escherichia coli mastitis; Kremer WD et al.; Experimental mastitis was induced by inoculating rear right quarters of 10 healthy cows with 10(3) cfu of Escherichia coli . The chemotactic responses of peripheral blood polymorphonuclear leukocytes at d-6, -5, -2, -1, and immediately prior to inoculation were measured . Chemiluminescence of polymorphonuclear leukocytes was measured immediately prior to inoculation . Severity of the experimental mastitis was assessed by bacterial growth in the inoculated quarters . Results of this study indicated that severity of the experimental mastitis may be predicted by the chemotactic response in vitro of polymorphonuclear leukocytes isolated from the peripheral blood at d 2, d 1, and immediately prior to inoculation . The number of circulating polymorphonuclear leukocytes immediately prior to inoculation also showed a negative relationship with the severity of mastitis . No relationship existed between preinfection chemiluminescence of polymorphonuclear leukocytes and the severity of the experimental mastitis.

Genomics, 1993 Jun, 16(3), 645 - 8
Ferrochelatase structural mutant (Fechm1Pas) in the house mouse; Boulechfar S et al.; The molecular basis of an inherited defect of ferrochelatase in mouse (Fechm1Pas/Fechm1Pas, described by Tutois et al., 1991, J . Clin . Invest . 88:1730-1736) was investigated . cDNA clones encoding ferrochelatase, isolated by amplification of the mRNA from the liver of a mutant mouse using the polymerase chain reaction, were sequenced by the dideoxynucleotide chain-termination method . All the clones carried a T to A transversion at nucleotide 293, leading to a methionine to lysine substitution at position 98 in the protein (mutation M98K) . Hybridization with allele-specific oligonucleotides (ASOs) confirmed the mutation at the cDNA and genomic levels . Finally, expression of the mutant ferrochelatase protein in E . coli demonstrated a marked deficiency in activity in agreement with the activity of the deficient enzyme in vivo . This Fechm1Pas/Fechm1Pas mutant mouse represents a useful model for studying the pathophysiological feature of the human disease and the first accessible model for gene therapy in the field of porphyrias.

Hepatogastroenterology, 1993 Jun, 40(3), 259 - 61
Effect of supernatants from Kupffer cells stimulated with galactosamine and endotoxin on the function of isolated rat hepatocytes; Kmiec Z et al.; Activated Kupffer cells may release substances that are involved in liver injury induced by galactosamine and endotoxin . In the present study Kupffer cells were isolated from rat livers, cultured for 24 hours and incubated with galactosamine, endotoxin (LPS) or tumor necrosis factor-alpha for 4 or 24 hours . The Kupffer cell-conditioned media were than added in separate experiments to freshly prepared isolated rat hepatocytes to determine their cytotoxic effect . No significant effects on the rate of protein synthesis, as assessed by the incorporation of 14C-leucine on lactate dehydrogenase enzyme release from hepatocytes during 1 h incubation was found as compared with conditioned media from control Kupffer cells . In further experiments, Kupffer cells incubated for 4 hours with LPS and galactosamine were shown to produce thromboxane B2 and also the potentially cytoprotective prostaglandins PGE2 and small amounts of prostacyclin measured as 6-keto-PGF1 alpha . It is concluded that under the conditions of the present experiments, factors secreted by cultured Kupffer cells have no cytotoxic effects on isolated rat hepatocytes during short-term incubation.

Genetics, 1993 Jun, 134(2), 409 - 22
The influence of primary and secondary DNA structure in deletion and duplication between direct repeats in Escherichia coli; Trinh TQ et al.; We describe a system to measure the frequency of both deletions and duplications between direct repeats . Short 17- and 18-bp palindromic and nonpalindromic DNA sequences were cloned into the EcoRI site within the chloramphenicol acetyltransferase gene of plasmids pBR325 and pJT7 . This creates an insert between direct repeated EcoRI sites and results in a chloramphenicol-sensitive phenotype . Selection for chloramphenicol resistance was utilized to select chloramphenicol resistant revertants that included those with precise deletion of the insert from plasmid pBR325 and duplication of the insert in plasmid pJT7 . The frequency of deletion or duplication varied more than 500-fold depending on the sequence of the short sequence inserted into the EcoRI site . For the nonpalindromic inserts, multiple internal direct repeats and the length of the direct repeats appear to influence the frequency of deletion . Certain palindromic DNA sequences with the potential to form DNA hairpin structures that might stabilize the misalignment of direct repeats had a high frequency of deletion . Other DNA sequences with the potential to form structures that might destabilize misalignment of direct repeats had a very low frequency of deletion . Duplication mutations occurred at the highest frequency when the DNA between the direct repeats contained no direct or inverted repeats . The presence of inverted repeats dramatically reduced the frequency of duplications . The results support the slippage-misalignment model, suggesting that misalignment occurring during DNA replication leads to deletion and duplication mutations . The results also support the idea that the formation of DNA secondary structures during DNA replication can facilitate and direct specific mutagenic events.

Comput Appl Biosci, 1993 Jun, 9(3), 315 - 24
METALGEN.DB: metabolism linked to the genome of Escherichia coli, a graphics-oriented database; Rouxel T et al.; The table of 'biochemical pathways' published by Boehringer is considered as the most exhaustive document dealing with intermediary metabolism . The present work consisted of constructing a database in which is collected everything known to date about intermediary metabolism: metabolic pathways, bioreactions, metabolites, enzymatic activities, genes and regulation . Data on genes make it possible to establish a link with existing computer structures specialized for genomic sequences . The present structure of the database presented here consists of three environments . The first environment is graphic; it is modeled upon the Boehringer table, but is more convenient to use . The second environment provides the user with all kinds of manipulations of data (entries, corrections, deletions, consistency, limiting redundancy, etc.) . These operations either are automatic or depend on a user-friendly interface (menus selected with the mouse) and so allow biologists at all levels to use the database and to develop it for their own particular needs . Finally, the third environment offers various procedures that make it possible to carry out multicriteria research on all the data in the database at once . This database can thus be substituted for the Boehringer table as a standard reference . It is also a tool that can be used to develop information systems not only in the domain of metabolism but also in the study of genomes (modeling, analysis of data, predictions, etc.).

Comput Appl Biosci, 1993 Jun, 9(3), 275 - 83
The joint distribution of patterns in random sequences with application to the RC-measure for expressivity; Kleffe J et al.; A method was previously developed for computation of pattern probabilities in random sequences under Markov chain models . We extend this method to the calculation of the joint distribution for two patterns . An application yields the distribution of the right choice measure for expressivity and how significance bounds depend on sequence length . These bounds are used to show that the choice of pyrimidine in codon position 3 of Escherichia coli genes deviates considerably from a general Markov process model for coding regions . We also derive some statistical evidence that this significant deviation is limited to codon position 3.

Am J Vet Res, 1993 Jun, 54(6), 867 - 72
Detection of passage and absorption of chicken egg yolk immunoglobulins in the gastrointestinal tract of pigs by use of enzyme-linked immunosorbent assay and fluorescent antibody testing; Yokoyama H et al.; Chicken egg yolk IgG can be absorbed and transferred as efficiently as colostral antibodies in the blood of neonatal pigs . Egg yolk IgG has a half-life of 1.85 days in newborn pig serum . This is shorter than the reported half-life (12 to 14 days) of homologous IgG in serum of pigs . Similar to colostral antibodies, egg yolk IgG absorption from intestine ceased at about 34 hours of age, after a logarithmic decrease in absorption rate from birth . Egg yolk IgG absorption inhibition time in the gastrointestinal tract took 1.73 hours to decrease by half . Egg yolk IgG was protective against experimentally induced diarrhea in pigs when it was administered at high dose, and multiple dosing was instituted . Adverse effects were not observed when chicken egg yolk IgG was administered orally to pigs.

Mol Immunol, 1993 Jun, 30(9), 833 - 40
Cloning and expression of an autoimmune DNA-binding single chain Fv . Only the heavy chain is required for binding; Barry MM et al.; Hed 10 is a murine autoimmune antibody which binds tightly to the single-stranded DNA, poly(dT) . The heavy and light chain variable region genes of Hed 10 were cloned and then joined by a 42 base-pair linker with the aid of a PCR ligation technique to produce a single chain Fv gene . After insertion into an expression vector the single chain Fv protein (scFv Hed 10) was produced in high yield and was purified by chromatography on an oligo (dT) cellulose column . The binding of scFv Hed 10 to poly (dT) was measured by fluorescence quenching and a binding constant of 3.2 x 10(6) M-1 was calculated . Previously {Lee et al . (1982) Biochemistry 21, 4940-4945} the binding constant of Fab Hed 10 to poly (dT) was found to be 12.7 x 10(6) M-1 . In addition the Vh gene of Hed 10 was expressed independently as well as another scFv which contained the Vh region of Hed 10 linked to the light chain variable region of Jel 42, an antibody specific for Hpr protein of E . coli (scFv 10 H.42 L) . Both of these proteins had binding constants for poly (dT) in the range of 6 x 10(6) M-1 . Therefore, the light chain of Hed 10 contributes little to the binding of this autoimmune antibody to DNA.

Protein Sci, 1993 Jun, 2(6), 977 - 84
Substrate specificities of catalytic fragments of protein tyrosine phosphatases (HPTP beta, LAR, and CD45) toward phosphotyrosylpeptide substrates and thiophosphotyrosylated peptides as inhibitors; Cho H et al.; The transmembrane PTPase HPTP beta differs from its related family members in having a single rather than a tandemly duplicated cytosolic catalytic domain . We have expressed the 354-amino acid, 41-kDa human PTP beta catalytic fragment in Escherichia coli, purified it, and assessed catalytic specificity with a series of pY peptides . HPTP beta shows distinctions from the related LAR PTPase and T cell CD45 PTPase domains: it recognizes phosphotyrosyl peptides of 9-11 residues from lck, src, and PLC gamma with Km values of 2, 4, and 1 microM, some 40-200-fold lower than the other two PTPases . With kcat values of 30-205 s-1, the catalytic efficiency, kcat/Km, of the HPTP beta 41-kDa catalytic domain is very high, up to 5.7 x 10(7) M-1 s-1 . The peptides corresponding to PLC gamma (766-776) and EGFR (1,167-1,177) phosphorylation sites were used for structural variation to assess pY sequence context recognition by HPTP beta catalytic domain . While exchange of the alanine residue at the +2 position of the PLC gamma (Km of 1 microM) peptide to lysine or aspartic acid showed little or no effect on substrate affinity, replacement by arginine increased the Km 35-fold . Similarly, the high Km value of the EGFR pY peptide (Km of 104 microM) derives largely from the arginine residue at the +2 position of the peptide, since arginine to alanine single mutation at the -2 position of the EGFR peptide decreased the Km value 34-fold to 3 microM . Three thiophosphotyrosyl peptides have been prepared and act as substrates and competitive inhibitors of these PTPase catalytic domains.

Protein Sci, 1993 Jun, 2(6), 927 - 35
Molecular dynamics simulations and rigid body (TLS) analysis of aspartate carbamoyltransferase: evidence for an uncoupled R state; Tanner JJ et al.; In the R form of ATCase complexed with the bisubstrate analogue, N-(phosphonacetyl)-L-aspartate, large temperature factors are reported for the allosteric domains of the regulatory chains . We studied the conformational flexibility of the holoenzyme with molecular dynamics simulations and rigid body (TLS) analysis . The results of the molecular dynamics simulations suggest that, although local atomic fluctuations account for the temperature factors of the catalytic and zinc domains, they do not account for the large temperature factors of the allosteric regions . However, the temperature factors of the allosteric domains can be satisfactorily analyzed using a rigid body model . The simulations and rigid body analysis support the idea that the allosteric regions are mechanically uncoupled from the rest of the enzyme in the PALA structure . Implications of this uncoupling for allosteric regulation are discussed.

Protein Sci, 1993 Jun, 2(6), 1024 - 33
Cysteine scanning mutagenesis of putative transmembrane helices IX and X in the lactose permease of Escherichia coli; Sahin-Toth M et al.; Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino-acid residue in putative transmembrane helices IX and X and the short intervening loop was systematically replaced with Cys (from Asn-290 to Lys-335) . Thirty-four of 46 mutants accumulate lactose to high levels (70-100% or more of C-less), and an additional 7 mutants exhibit lower but highly significant lactose accumulation . As expected (see Kaback, H.R., 1992, Int . Rev . Cytol . 137A, 97-125), Cys substitution for Arg-302, His-322, or Glu-325 results in inactive permease molecules . Although Cys replacement for Lys-319 or Phe-334 also inactivates lactose accumulation, Lys-319 is not essential for active lactose transport (Sahin-Toth, M., Dunten, R.L., Gonzalez, A., & Kaback, H.R., 1992, Proc . Natl . Acad . Sci . USA 89, 10547-10551), and replacement of Phe-334 with leucine yields permease with considerable activity . All single-Cys mutants except Gly-296 --> Cys are present in the membrane in amounts comparable to C-less permease, as judged by immunological techniques . In contrast, mutant Gly-296 --> Cys is hardly detectable when expressed at a relatively low rate from the lac promoter/operator but present in the membrane in stable form when expressed at a high rate from T7 promoter . Finally, studies with N-ethylmaleimide (NEM) show that only a few mutants are inactivated significantly . Remarkably, the rate of inactivation of Val-315 --> Cys permease is enhanced at least 10-fold in the presence of beta-galactopyranosyl 1-thio-beta-D-galactopyranoside (TDG) or an H+ electrochemical gradient (delta mu-H+) . The results demonstrate that only three residues in this region of the permease -Arg-302, His-322, and Glu-325-are essential for active lactose transport . Furthermore, the enhanced reactivity of the Val-315 --> Cys mutant toward NEM in the presence of TDG or delta mu-H+ probably reflects a conformational alteration induced by either substrate binding or delta mu-H+.

Protein Sci, 1993 Jun, 2(6), 1013 - 23
In vivo formation of active aspartate transcarbamoylase from complementing fragments of the catalytic polypeptide chains; Yang YR et al.; Despite the complexity of Escherichia coli aspartate transcarbamoylase (ATCase), composed of 12 polypeptide chains organized as two catalytic (C) trimers and three regulatory (R) dimers, it is possible to form active stable enzyme in vivo even with fragmented catalytic (c) chains . Based on the observation that chymotryptic digestion of the C trimers yields an active protein that can be dissociated into fragmented chains and then reconstituted in high yield, genetically engineered plasmids carrying the genes encoding each of the fragments were constructed . When the N-terminal peptide (residues 1-242) and the C-terminal peptide (residues 235-310) were expressed separately, each incomplete polypeptide chain was found in the insoluble fraction of the individual cell extracts . Mixing the two insoluble pellets in 6.5 M urea, followed by a 10-fold dilution in buffer, led to the formation of active C trimers composed of incomplete polypeptide chains with an 8-amino acid redundancy . When the two partial genes were linked into a single transcriptional unit separated by a 15-nucleotide untranslated region containing a sequence for ribosome binding, the cells produced high yields of active C trimers composed of the incomplete, partially overlapping chains . The resulting protein, purified as C trimers or as holoenzyme formed by the addition of R subunits, has a specific activity (Vmax) only slightly less than that of the wild-type C trimer and ATCase . However, Km for aspartate exhibited by the C trimer composed of fragmented chains is more than 10-fold larger than that of the wild-type trimer . The holoenzyme formed from the C trimer containing the coexpressed peptides is devoid of cooperativity with a Hill coefficient of 1.0, as contrasted to wild-type ATCase for which the Hill coefficient is 1.7 . Km for aspartate as well as Kd for the binding of the bisubstrate analog N-(phosphonacetyl)-L-aspartate are significantly higher than the analogous values for wild-type ATCase . Sedimentation velocity experiments indicate that the holoenzyme containing the incomplete chains has a conformation analogous to that of the R state of wild-type ATCase.

Protein Sci, 1993 Jun, 2(6), 1001 - 12
Reconstitution of active catalytic trimer of aspartate transcarbamoylase from proteolytically cleaved polypeptide chains; Powers VM et al.; Treatment of the catalytic (C) trimer of Escherichia coli aspartate transcarbamoylase (ATCase) with alpha-chymotrypsin by a procedure similar to that used by Chan and Enns (1978, Can . J . Biochem . 56, 654-658) has been shown to yield an intact, active, proteolytically cleaved trimer containing polypeptide fragments of 26,000 and 8,000 MW . Vmax of the proteolytically cleaved trimer (CPC) is 75% that of the wild-type C trimer, whereas Km for aspartate and Kd for the bisubstrate analog, N-(phosphonacetyl)-L-aspartate, are increased about 7- and 15-fold, respectively . CPC trimer is very stable to heat denaturation as shown by differential scanning microcalorimetry . Amino-terminal sequence analyses as well as results from electrospray ionization mass spectrometry indicate that the limited chymotryptic digestion involves the rupture of only a single peptide bond leading to the production of two fragments corresponding to residues 1-240 and 241-310 . This cleavage site involving the bond between Tyr 240 and Ala 241 is in a surface loop known to be involved in intersubunit contacts between the upper and lower C trimers in ATCase when it is in the T conformation . Reconstituted holoenzyme comprising two CPC trimers and three wild-type regulatory (R) dimers was shown by enzyme assays to be devoid of the homotropic and heterotropic allosteric properties characteristic of wild-type ATCase . Moreover, sedimentation velocity experiments demonstrate that the holoenzyme reconstituted from CPC trimers is in the R conformation . These results indicate that the intact flexible loop containing Tyr 240 is essential for stabilizing the T conformation of ATCase . Following denaturation of the CPC trimer in 4.7 M urea and dilution of the solution, the separate proteolytic fragments re-associate to form active trimers in about 60% yield . How this refolding of the fragments, docking, and association to form trimers are achieved is not known.

Anal Biochem, 1993 Jun, 211(2), 320 - 3
A continuous spectrophotometric assay for the aminoacylation of transfer RNA by alanyl-transfer RNA synthetase; Wu MX et al.; A continuous spectrophotometric assay is described for the aminoacylation reaction catalyzed by Escherichia coli alanyl-transfer RNA synthetase . The assay is based on coupling the alanyl-tRNA synthetase-dependent formation of AMP to the lactate dehydrogenase oxidation of NADH . Oxidation of NADH, as monitored at 340 nm, is shown to be stoichiometric with the formation of alanyl-tRNA(Ala) . This assay will facilitate the rapid accumulation and analysis of kinetic data for alanyl-tRNA synthetase and should be applicable to aminoacyl-tRNA synthetases in general.

Scand J Immunol, 1993 Jun, 37(6), 637 - 43
The structure of the variable regions of mouse monoclonal antibodies to hepatitis B virus core antigen; Skrivelis V et al.; From a panel of monoclonal antibodies (MoAbs) directed against E . coli-derived native and denatured hepatitis B virus (HBV) core antigen we have selected a set of specific MoAbs which recognize different linear antigenic determinants: MoAb C1-5--cl epitope; MoAb 14K8--less immunogenic N-terminal region; and MoAbs 13C9, 10F10 and 14E11, 14G3--the immunodominant region between amino acids 134 and 140 . We have applied the polymerase chain reaction technique to clone Ig VH and VL region genes, and appropriate full-length cDNA clones were obtained and characterized by nucleotide sequence analysis . Among the six heavy chain variable region sequences examined, three VH families were represented . Two of them belong to the 7183 (MoAb C1-5) and 3609 (14B8) families respectively and four, having only two amino acid changes in the CDR2 region, to the J558 family . These four probably are derived from a single expanded B-cell clone . The light chain sequences indicate that their VL are encoded by V kappa 21, V kappa 19 and V kappa 3 germline genes . Unlike VH genes, light chain genes are closely related to known representatives of mouse kappa light chain families and are employed also by MoAbs raised against other antigens.

Mol Pharmacol, 1993 Jun, 43(6), 854 - 7
Mechanisms of excision of 5-fluorouracil by uracil DNA glycosylase in normal human cells; Mauro DJ et al.; Recent evidence indicates that 5-fluorouracil (5-FlUra) is incorporated into DNA and is removed by the DNA repair enzyme uracil DNA glycosylase . Synthetic oligonucleotides containing either a single uracil or 5-FlUra residue were constructed to examine the mechanisms by which human cells remove 5-FlUra from DNA . The human uracil DNA glycosylase excised uracil in a manner similar to that observed for the bacterial enzyme . In contrast, a significant difference was observed in their abilities to remove 5-FlUra . In particular, both the bacterial and normal human enzymes displayed 13-17-fold increases in their apparent Km values but the apparent Vmax values remained virtually constant . These results demonstrate that normal human cells possess a defined capacity to remove 5-FlUra incorporated into DNA . However, specific kinetic differences may exist that affect their capacity to remove 5-FlUra formed in DNA after treatment with this cancer chemotherapeutic agent.

Mol Gen Genet, 1993 Jun, 239(3), 334 - 44
The positive-acting sulfur regulatory protein CYS3 of Neurospora crassa: nuclear localization, autogenous control, and regions required for transcriptional activation; Kanaan MN et al.; The positive-acting global sulfur regulatory protein, CYS3, of Neurospora crassa turns on the expression of a family of unlinked structural genes that encode enzymes of sulfur catabolism . CYS3 contains a leucine zipper and an adjacent basic region (b-zip), which together constitute a bipartite sequence-specific DNA-binding domain . Specific anti-CYS3 antibodies detected a protein of the expected size in nuclear extracts of wild-type Neurospora under conditions in which the sulfur circuit is activated . The CYS3 protein was not observed in cys-3 mutants . Nuclear extracts of wild type, but not cys-3 mutants, also showed specific DNA-binding activity identical to that obtained with a CYS3 protein expressed in Escherichia coli . A truncated CYS3 protein that contains primarily the b-zip domain binds to DNA with high specificity and affinity in vitro, yet fails to activate gene expression in vivo, and instead inhibits the function of the wild-type CYS3 protein . Amino-terminal, carboxyterminal, and internal deletions as well as alanine scanning mutagenesis were employed to identify regions of the CYS3 protein that are required for its trans-activation function . Regions of CYS3 carboxy terminal to the b-zip motif are not completely essential for function although loss of an alanine-rich region results in decreased activity . All deletions amino terminal to the b-zip motif led to a complete loss of CYS3 function . Alanine scanning mutagenesis demonstrated that an unusual prolinerich domain of CYS3 appears to be very important for function and is presumed to constitute an activation domain . It is concluded that CYS3 displays nuclear localization and positive autogenous control in Neurospora and functions as a trans-acting DNA-binding protein.

Gynecol Oncol, 1993 Jun, 49(3), 380 - 2
Malacoplakia of the vagina presenting as a pelvic mass; Fishman A et al.; Malacoplakia is an unusual type of chronic inflammation that rarely involves the female genital tract . A case of malacoplakia involving the vaginal cuff of a patient previously treated by radical hysterectomy for cervical carcinoma is presented.

Zhonghua Jie He He Hu Xi Za Zhi, 1993 Jun, 16(3), 153 - 4, 187-8
{Polydatin prevents endotoxin-induced acute lung injury in rats}; Mo GY et al.; The effects of polydatin on endotoxin-induced acute lung injury were studied in rats . The lung injury was induced by infusion of inactivated E . coli 30 min after MPS blockade with red cell membrane . It was found that pretreatment with polydatin resulted in marked reduction of lung wet-to-dry weight ratio, lung permeability index (LPI), neutrophil count in BALF and hemoconcentration . The elevation of lung tissue MDA and the decrease in plasma GSH-Px activity were greatly improved with polydatin . There was significant correlation between LPI and lung MDA level (r = 0.741, P < 0.01) . Postmortem examination revealed that polydatin attenuated the histopathological changes . We conclude that polydatin protectsral against endotoxin-induced acute lung injury.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1993 Jun, 15(3), 178 - 82
{Alterations of protein kinase C activity in rat myocardium and aorta smooth muscle during endotoxemia}; Wei X; A decline of cytosol protein kinase C activity was observed in rat myocardial cells at 4 and 8 h after endotoxin administration, and membrane-associated protein kinase C activity rose at the same time . The activity of membrane protein kinase C in aortic smooth muscle cells at 0.5 and 4 h after endotoxin injection was higher than that in control, while cytosol protein kinase C activity was lower . The results indicate that protein kinase C was activated in myocardial cells and aortic smooth muscle cells during various phases of endotoxemia.

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