Microbiology Reader
Equipment to run microbiology work automatically

Growth Curves of any strain.
Microbiological calculations.

Microbiology Home
Microbioloy Reader
Growth Curves
Photo Album
Microorganisms
Software
Download
Purchasing
Contact Us



Biochemistry, 1994 Jun 14, 33(23), 7278 - 87
Pseudosubstrate inhibition of CDPK, a protein kinase with a calmodulin-like domain; Harmon AC et al.; Between the catalytic and regulatory domains of calmodulin-like domain protein kinase, CDPK, is a junction domain which has some identity to the autoinhibitory domain of calmodulin-dependent protein kinase type II (Harper, J . F., Sussman, M . R., Schaller, G . E., Putnam-Evans, C., Charbonneau, H., & Harmon, A . C . (1991) Science 252, 951-954) . To investigate whether CDPK's junction domain also functions as an autoinhibitory domain, we determined the effect of synthetic peptides, corresponding to sequences within the junction domain, on the activity of native soybean CDPK . Three peptides, corresponding to residues 310-332, 318-332, 302-317, were competitive inhibitors with respect to syntide-2 and had Ki values of 5, 25, and 85 microM, respectively . These peptides were uncompetitive inhibitors with respect to ATP and had Ki values of 24, 220, and 510 microM, respectively . A fourth peptide, CDPK alpha 302-332, inhibited activity by a mixed mechanism with respect to both syntide-2 (Ki = 1.9 microM; K'i = 5.0 microM) and ATP (Ki = 15 microM; K'i = 4.5 microM) . Three of the peptides, CDPK alpha 302-332, 310-332, and 318-332, formed complexes with soybean calmodulin during electrophoresis in native polyacrylamide gels and were able to inhibit calmodulin-dependent protein kinases . Given the similarity between CDPK's calmodulin-like domain and calmodulin (40% sequence identity), it was possible that these peptides could inhibit activity through interaction with the calmodulin-like domain rather than the catalytic domain . To address this possibility, a cDNA encoding the first 312 residues of soybean CDPK alpha was constructed and expressed in Escherichia coli . This enzyme, which is missing most of the junction domain and all of the calmodulin-like domain, was active in the presence and absence of calcium . Peptide CDPK alpha 310-332 inhibited this truncated enzyme competitively with respect to syntide-2 (Ki = 4 microM) . These results show that the junction domain is capable of functioning as an autoinhibitory domain, possibly through a pseudosubstrate site located between residues 310 and 332.

Biochemistry, 1994 Jun 14, 33(23), 7267 - 77
Genetic identification of an autoinhibitor in CDPK, a protein kinase with a calmodulin-like domain; Harper JF et al.; CDPKs are a family of calcium (Ca2+)-dependent protein kinases which are defined by a carboxyl-terminal calmodulin-like domain . Mutational analysis indicates that the junction domain, which joins the kinase and calmodulin-like domains, contains an autoinhibitor . CDPK isoform AK1 from Arabidopsis was expressed in Escherichia coli as a fusion protein sandwiched between glutathione S-transferase and six consecutive histidines at the N- and C-terminal ends, respectively . This fusion, called AK1-6H, was purified and displayed kinase activity which was stimulated up to 127-fold by Ca2+, with a typical specific activity of 2000 nmol min-1 mg-1, using syntide-2 as peptide substrate . A truncation which deletes the calmodulin-like domain, as in mutant delta C-6H, disrupts Ca2+ activation and leaves the enzyme with a basal level of activity . Delta C-6H could be activated 87-fold by preincubation with a purified polyclonal IgG which was raised against a junction domain fusion . A further deletion of the junction domain, as in mutant delta JC, results in a constitutively active enzyme . This indicates that the junction domain in delta C-6H can function as an autoinhibitor . Its function as an autoinhibitor in a full-length enzyme was confirmed by site-specific mutagenesis, as shown by mutant KJM23-6H, which had a six-residue substitution in the junction domain between A422 and A432 . Both delta JC and KJM23-6H encoded Ca(2+)-independent enzymes which had specific activities greater than 70% that of a fully active AK1-6H and displayed equivalent Km values for ATP and syntide-2 . Inhibition studies on delta JC, using peptides based on the autoinhibitory domains of Ca2+/calmodulin-dependent protein kinases, are consistent with a model where the junction domain contains a similar pseudosubstrate-type autoinhibitor.

Biochemistry, 1994 Jun 14, 33(23), 7174 - 83
Mannose transporter of Escherichia coli . Backbone assignments and secondary structure of the IIA domain of the IIABMan subunit; Seip S et al.; The mannose transporter of Escherichia coli consists of two transmembrane and one peripheral protein subunit . The complex acts by a mechanism which couples translocation of the substrate with substrate phosphorylation . The peripheral IIABMan is a homodimer . The IIABMan monomer itself contains two domains which are linked by an Ala-Pro-rich hinge and which are both transiently phosphorylated at histidyl residues . The IIA and IIB domains can be separated by limited proteolysis . The IIA domain has a dimer molecular mass of 2 x 14 kDa . Almost complete 1H, 13C, and 15N NMR assignments of the backbone resonances of IIAMan have been achieved using 3D and 4D double-and triple-resonance techniques . Secondary structure elements were derived from NOE data . The IIA domain consists of a central beta-sheet of four parallel and one antiparallel strand (strand order 5 4 3 1 2) with helices on both sides of the sheet . The active-site His-10 is located in a loop at the C-terminus of beta-strand 1 . This loop and the loop after strand 3 are at the topological switch point of the sheet.

Biochemistry, 1994 Jun 14, 33(23), 7127 - 33
Replication of DNA templates containing the alpha-anomer of deoxyadenosine, a major adenine lesion produced by hydroxyl radicals; Ide H et al.; The alpha-anomer of deoxyadenosine (alpha-dA) is a major adenine lesion produced by hydroxyl radicals in DNA . To assess its biochemical effects on DNA replication, alpha-dA was site-specifically incorporated into oligodeoxyribonucleotide templates using phosphoramidite chemistry . alpha-dA in the template constituted a transient block to DNA synthesis catalyzed by Escherichia coli DNA polymerase I Klenow fragment (polI), but translesional synthesis occurred after prolonged incubation . Primer extension assays and Maxam-Gilbert sequencing of newly synthesized products revealed that alpha-dA directed not only incorporation of the correct nucleotide, dTMP, opposite the lesion but also misincorporation of dAMP and dCMP . dGMP was barely incorporated under these conditions . The order of the incorporation frequency at the alpha-dA site was affected by the nearest neighbor base pair 3' to the lesion . T7 and Taq DNA polymerases, as well as RAV-2 reverse transcriptase, showed a selectivity similar to that of PolI with respect to the nucleotide incorporation opposite alpha-dA, suggesting that the discrimination of nucleotides associated with alpha-dA is independent of the origin of DNA polymerases and is an intrinsic feature of the lesion . The mutational spectrum predicted for alpha-dA (i.e., A-->G transitions and A-->T transversions) is significantly different from those reported for other hydroxyl radical induced DNA lesions such as abasic sites or 7,8-dihydro-8-oxoguanine, both primarily directing misincorporation of A . Possible biological consequences and the mechanism of dNTP discrimination associated with alpha-dA are discussed.

Biochemistry, 1994 Jun 14, 33(23), 7120 - 6
Sequence-specific DNA displaces 6-p-toluidino-2-naphthalenesulfonate bound to a hydrophobic site on the DNA-binding domain of Drosophila c-myb; Madan A et al.; The N-terminal DNA-binding domain of c-myb oncoprotein binds to DNA in a sequence-specific manner . The domain, consisting of three imperfect tandem repeats, has tryptophan residues at very regular intervals and this is believed to be of some significance in the DNA-binding activity of the protein . We have found that the hydrophobic-site-specific probe 6-p-toluidino-2-naphthalenesulfonate (TNS) binds to the bacterially expressed DNA-binding domain of Drosophila c-myb protein (R123) . TNS has a single binding site on this protein with an apparent dissociation constant in the range of (5-8) x 10(-7) M . When the TNS-protein complex was treated with an oligomeric DNA duplex having a cognate myb-binding site, the TNS was displaced from the complex . Nonspecific DNA duplex oligomers were ineffective, indicating that TNS displacement was a sequence-specific process . We examined further some features of the TNS-binding site on the protein, taking advantage of the fluorescence properties of the protein and the bound TNS . Our data indicate that the TNS binding occurs in a peripheral site on the protein in a manner that allows the bound TNS to be solvent accessible . Furthermore, there are indications that tyrosine(s) and tryptophans of the protein mediate resonance energy transfer to the bound TNS . From fluorescence-quenching data of the protein and protein-TNS complex, we could assess that both solvent-accessible and internal tryptophans are in the vicinity of the bound TNS . (ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1994 Jun 14, 33(23), 7107 - 12
Substrate specificity is determined by amino acid binding pocket size in Escherichia coli phenylalanyl-tRNA synthetase; Ibba M et al.; Alanine at position 294 (Ala294) within the motif 3 consensus of Escherichia coli phenylalanyl-tRNA synthetase alpha subunit has previously been implicated as a determinant of amino acid specificity . To characterize the role of Ala294, the catalytic effects of amino acid replacements at this position were tested with purified wild-type and mutant phenylalanyl-tRNA synthetases . We show that Ala294 is involved in amino acid binding and that it influences specificity as a determinant of binding pocket size . Replacement of Ala294 by either glycine or serine, thereby increasing or decreasing the size of the binding pocket, respectively, reduces affinity for phenylalanine . The Gly294 mutant shows a relaxed specificity toward synthetic para-halogenated phenylalanine analogues, the apparent dissociation constant Km increasing in direct relation to an increase of the van der Waals radius of the para group, thus confirming the role of position 294 in determining amino acid binding pocket size . For the substrate analogue p-chlorophenylalanine, attachment to tRNA and in vivo incorporation into cellular protein by the Gly294 mutant were demonstrated . Tyrosine activation was also improved with this mutant, but the resulting enzyme-Tyr-adenylate complex was rapidly hydrolyzed, indicating the presence of a proofreading mechanism in E . coli phenylalanyl-tRNA synthetase.

Biochemistry, 1994 Jun 14, 33(23), 7062 - 8
Site-directed mutagenesis and NMR studies of histidine-385 mutants of 5-enolpyruvylshikimate-3-phosphate synthase; Shuttleworth WA et al.; The site-directed mutagenesis of His-385 of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase is reported . The steady-state kinetics for two mutants, H385Q and H385A, are compared with that of the wild-type enzyme . H385Q EPSP synthase was found to have 25% wild-type enzyme activity, whereas H385A EPSP synthase retained 1% activity . The KM values for Pi and shikimate 3-phosphate were unaffected, whereas the KM for phosphoenolpyruvate (PEP) was increased 10 times for H385Q EPSP synthase . The KM for EPSP was unaffected in H385Q but raised by a factor of 10 in H385A EPSP synthase . The binding of glyphosate was studied by fluorescence spectroscopy and by 31P NMR spectroscopy . Direct observation of the enzyme-intermediate complexes by 13C NMR spectroscopy with {2,3-13C}phosphoenolpyruvate was studied for the mutant enzymes and compared with the wild type . Under equilibrium conditions, H385A EPSP synthase does not accumulate enzyme-bound EPSP . These results suggest that, while critically located in the PEP binding site, His-385 is not the residue responsible for initiating catalysis through the protonation of PEP.

Biochemistry, 1994 Jun 14, 33(23), 7041 - 6
Serine 90 is required for enzymic activity by tRNA-guanine transglycosylase from Escherichia coli; Reuter K et al.; An Escherichia coli mutant described by Noguchi et al . {Noguchi, S., et al . (1982) J . Biol . Chem . 275, 6544-6550} contains tRNA lacking the hypermodified wobble nucleoside queuosine (Q) due to an inactive tRNA-guanine transglycosylase (TGT) . TGT catalyzes the posttranscriptional base exchange of the Q precursor preQ1 with the genetically encoded guanine in tRNA(Asp,Asn,His,Tyr) . The mutant tgt gene was cloned and sequenced; it contained a single point mutation resulting in the change of serine 90 to phenylalanine . Overexpression of the mutant gene yielded TGT(S90F) that showed a reduced solubility and did not purify in the same fashion as the wild-type enzyme . TGT(S90F) has no detectable enzymic activity . To determine whether serine 90 performs a catalytic role in the TGT reaction or whether the loss of activity was caused solely by a conformational change of the enzyme, we used site-specific mutagenesis to construct serine-to-alanine (S90A) and serine-to-cysteine (S90C) mutants . Both S90A and S90C mutants were purified in a manner identical to that used for the wild-type enzyme . SDS-PAGE of dimethyl suberimidate-cross-linked mutants showed a pattern identical to that of the wild-type TGT, indicative of a trimeric quaternary structure . Native PAGE of wild-type and mutant TGTs in the absence and presence of substrate tRNA exhibited band shifts indicating that both mutants retain the ability to bind tRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1994 Jun 14, 33(23), 7021 - 6
Determination by Raman spectroscopy of the pKa of N5 of dihydrofolate bound to dihydrofolate reductase: mechanistic implications; Chen YQ et al.; Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate (H2folate) to tetrahydrofolate by NADPH, and this requires that the pteridine ring be protonated at N5 . A long-standing puzzle has been how, at physiological pH, the enzyme can protonate N5 in view of its solution pKa of 2.6 and the fact that the only proton-donating group in the pterdine binding site, Asp-27, hydrogen bonds not to N5 but to the 2-amino group and N3 of the pterin ring . We have determined the pKa of N5 of dihydrofolate in the Escherichia coli DHFR/NADP+/H2folate ternary complex by Raman difference spectroscopy and found that the value is 6.5 . In contrast, the pKa of N5 is less than 4.0 in either the binary complex, the ternary complex with an analogue of NADPH (H2NADPH), or the Asp27 to serine mutant DHFR (D27S) ternary complex with NADP+ . Thus, one need not invoke proton donation from Asp-27 to N5 via a series of bound water molecules and/or pteridine-ring substituents . We propose instead that the N5 protonated form of H2folate is stabilized directly at the active site in the DHFR/NADPH/H2folate complex by specific interactions that form only in the ternary complex, involving perhaps a bound water molecule, the carboxamide moiety of the coenzyme, and/or the local electrostatic field of the enzyme molecule, to which an important contribution may be made by Asp-27.

FEBS Lett, 1994 Jun 13, 346(2-3), 263 - 7
Distinct kinetics of subunit autolysis in mammalian m-calpain activation; Saido TC et al.; Subunit autolysis of mammalian m-calpain upon activation was examined in kinetic terms using a set of antibodies recognizing different portions of the protease . Activation of m-calpain by calcium resulted in no apparent autolysis in the large catalytic subunit, whereas the small regulatory subunit underwent immediate autolysis followed by substrate proteolysis . This profile of subunit autolysis is distinct from that of the other ubiquitous isozyme, mu-calpain, in which autolysis of the large subunit and then of the small subunit precedes substrate proteolysis under the normal conditions . The activation state of m-calpain thus is not reflected by the large subunit autolysis . The mode and role of autolysis may vary among calpain isozymes.

FEBS Lett, 1994 Jun 13, 346(2-3), 217 - 20
The immobilized movement proteins of two tobamoviruses form stable ribonucleoprotein complexes with full-length viral genomic RNA; Ivanov KI et al.; The movement proteins of two tobamoviruses (tobacco mosaic virus, TMV, common strain U1 and cruciferous TMV strain) containing amino-terminal hexahistidine affinity tags were overexpressed in Escherichia coli and purified by metal chelate affinity chromatography . Purified recombinant proteins were immobilized to a Ni(2+)-chelate adsorbent and their ability to interact with full-length genomic TMV RNA was tested . Here we report that binding of viral RNA to hexahistidine fusion movement proteins results in the formation of stable ribonucleoprotein complexes.

FEBS Lett, 1994 Jun 13, 346(2-3), 175 - 80
In vitro mutation analysis of Arabidopsis thaliana small GTP-binding proteins and detection of GAP-like activities in plant cells; Anai T et al.; Previously, we have reported the molecular cloning of ara genes encoding a small GTP-binding protein from Arabidopsis thaliana . The criterion based on amino acid sequences suggest that such an ara gene family can be classified to be of the YPT/rab type . To examine the biochemical properties of ARA proteins, several deletions and point mutations were introduced into ara cDNAs . Mutant proteins were expressed in E . coli as GST-chimeric molecules and analyzed in terms of their GTP-binding or GTP-hydrolysing ability in vitro . The results indicate that four conserved amino acid sequence regions of ARA proteins are necessary for GTP-binding . A point mutation of Asn at position 72 for ARA-2, or 71 for ARA-4, to Ile decreased GTP-binding and a point mutation of Gln at position 126 for ARA-2, or 125 for ARA-4, to Leu suppressed GTP-hydrolysis activity . Furthermore, certain factors associated with the membrane fraction accelerated GTPase activities of ARA proteins, suggesting the presence of GTPase activating protein(s) (GAP(s)) in the vesicular transport system of higher plant cells.

FEBS Lett, 1994 Jun 13, 346(2-3), 151 - 5
Cloning and expression of a human pro(tea)some beta-subunit cDNA: a homologue of the yeast PRE4-subunit essential for peptidylglutamyl-peptide hydrolase activity; Gerards WL et al.; The cDNA encoding a human prosome beta-subunit (HSBpros26) was isolated from a lymphoma library using the cDNA of the Xenopus homologue as a probe . The cDNA contains an open reading frame encoding a protein of 233 amino acids and a calculated molecular weight of 25,909 . Comparison with interspecies homologues of HSBpros26 from Xenopus (XLB), rat (RN3) and yeast (PRE4) reveals a high degree of identity between the beta-subunits except for the N-terminal end, which is probably cleaved post-translationally . The complete coding sequence of HSBpros26 has been expressed in E . coli . The produced protein of about 27 kDa reacts with the prosomal monoclonal antibody MCP205, kindly provided by Dr . K . Hendil . The molecular weight of the native protein is about 28 kDa indicating that the protein is present as monomers . Finally partially purified HSBpros26 preparations do not contain any proteolytical activity.

Brain Res, 1994 Jun 13, 648(1), 171 - 5
Adenoviral vectors as functional retrograde neuronal tracers; Ridoux V et al.; Adenoviruses have been recently recognized as a highly efficient system for gene delivery to various tissues . The ability of replication-defective recombinant adenovirus to transfer the lacZ reporter gene encoding beta-galactosidase to nerve cells in various brain structures has been demonstrated . Here, on the continuation of these studies, we present evidence that the adenovirus can be transported in a retrograde manner to nerve cell bodies from axonal terminals . This method may be of great value for infecting selected subsets of specific neurons for either anatomo-functional studies or even therapeutic purposes.

Biochim Biophys Acta, 1994 Jun 12, 1206(2), 197 - 202
Characterization and use of biotinylated Escherichia coli K99 lectin; Berger S et al.; K99 lectin from Escherichia coli was purified and biotinylated via the amino groups of lysine residues using N-biotinyl-6-amino-caproic acid N-hydroxysuccinimide ester (BcapNHS) . Biotin was detected on Lys-47 and Lys-87 . It was previously demonstrated (Jacobs, A.A.C., Van den Berg, P.A., Bak, H.J . and De Graaf, F.K . (1986) Biochim . Biophys . Acta 872, 92-97) that modification of lysine residues 132 and 133 with 4-chloro-3,5-dinitrobenzoate (CDNB) resulted in the loss of the binding capacity of K99 fimbriae . Due to the higher size of the biotin derivative compared to CDNB, Lys-132 or Lys-133, essential for the biological activity, were not modified . The biotinylation did not cause the loss of the haemagglutinating activity but was sufficient to permit detection of the lectin by streptavidin . A flow cytometric analysis was used for the detection of the receptors on the surface of erythrocytes.

Nucleic Acids Res, 1994 Jun 11, 22(11), 2065 - 70
Phage P4 DNA replication in vitro; Diaz Orejas R et al.; Phage P4 DNA is replicated in cell-free extracts of Escherichia coli in the presence of partially purified P4 alpha protein {Krevolin and Calendar (1985), J . Mol . Biol . 182, 507-517} . Using a modified in vitro replication assay, we have further characterized this process . Analysis by agarose gel electrophoresis and autoradiography of in vitro replicated molecules demonstrates that the system yields supercoiled monomeric DNA as the main product . Electron microscopic analysis of in vitro generated intermediates indicates that DNA synthesis initiates in vitro mainly at ori, the origin of replication used in vivo . Replication proceeds from this origin bidirectionally, resulting in theta-type molecules . In contrast to the in vivo situation, no extensive single-stranded regions were found in these intermediates . The initiation proteins of the host, DnaB and DnaG, and the chaperones DnaJ and DnaK are not required for P4 replication, because polyclonal antibodies against those polypeptides do not inhibit the process . The reaction is inhibited by antibodies against the SSB protein, and by ara-CTP, a specific inhibitor of DNA polymerase III holoenzyme . Consistent with previous reports, P4 in vitro replication is independent of transcription by host RNA polymerase . Novobiocin, a DNA gyrase inhibitor, strongly inhibits P4 DNA synthesis, indicating that form I DNA is the required substrate.

Nucleic Acids Res, 1994 Jun 11, 22(11), 2028 - 35
The heterodimeric subunit SRP9/14 of the signal recognition particle functions as permuted single polypeptide chain; Bovia F et al.; The targeting of nascent polypeptide chains to the endoplasmic reticulum is mediated by a cytoplasmic ribonucleoprotein, the signal recognition particle (SRP) . The 9 kD (SRP9) and the 14 kD (SRP14) subunits of SRP are required to confer elongation arrest activity to the particle . SRP9 and SRP14 form a heterodimer which specifically binds to SRP RNA . We have constructed cDNAs that encode single polypeptide chains comprising SRP9 and SRP14 sequences in the two possible permutations linked by a 17 amino acid peptide . We found that both fusion proteins specifically bound to SRP RNA as monomeric molecules folded into a heterodimer-like structure . Our results corroborate the previous hypothesis that the authentic heterodimer binds to SRP RNA in equimolar ratio . In addition, both fusion proteins conferred elongation arrest activity to SRP(-9/14), which lacks this function, and one fusion protein could functionally replace the heterodimer in the translocation assay . Thus, the normal N-and C-termini of both proteins have no essential role in folding, RNA-binding and in mediating the biological activities . The possibility to express the heterodimeric complex as a single polypeptide chain facilitates the analysis of its functions and its structure in vivo and in vitro.

Nucleic Acids Res, 1994 Jun 11, 22(11), 1933 - 47
Molecular evolution of SRP cycle components: functional implications; Althoff S et al.; Signal recognition particle (SRP) is a cytoplasmic ribonucleoprotein that targets a subset of nascent presecretory proteins to the endoplasmic reticulum membrane . We have considered the SRP cycle from the perspective of molecular evolution, using recently determined sequences of genes or cDNAs encoding homologs of SRP (7SL) RNA, the Srp54 protein (Srp54p), and the alpha subunit of the SRP receptor (SR alpha) from a broad spectrum of organisms, together with the remaining five polypeptides of mammalian SRP . Our analysis provides insight into the significance of structural variation in SRP RNA and identifies novel conserved motifs in protein components of this pathway . The lack of congruence between an established phylogenetic tree and size variation in 7SL homologs implies the occurrence of several independent events that eliminated more than half the sequence content of this RNA during bacterial evolution . The apparently non-essential structures are domain I, a tRNA-like element that is constant in archaea, varies in size among eucaryotes, and is generally missing in bacteria, and domain III, a tightly base-paired hairpin that is present in all eucaryotic and archeal SRP RNAs but is invariably absent in bacteria . Based on both structural and functional considerations, we propose that the conserved core of SRP consists minimally of the 54 kDa signal sequence-binding protein complexed with the loosely base-paired domain IV helix of SRP RNA, and is also likely to contain a homolog of the Srp68 protein . Comparative sequence analysis of the methionine-rich M domains from a diverse array of Srp54p homologs reveals an extended region of amino acid identity that resembles a recently identified RNA recognition motif . Multiple sequence alignment of the G domains of Srp54p and SR alpha homologs indicates that these two polypeptides exhibit significant similarity even outside the four GTPase consensus motifs, including a block of nine contiguous amino acids in a location analogous to the binding site of the guanine nucleotide dissociation stimulator (GDS) for E . coli EF-Tu . The conservation of this sequence, in combination with the results of earlier genetic and biochemical studies of the SRP cycle, leads us to hypothesize that a component of the Srp68/72p heterodimer serves as the GDS for both Srp54p and SR alpha . Using an iterative alignment procedure, we demonstrate similarity between Srp68p and sequence motifs conserved among GDS proteins for small Ras-related GTPases . The conservation of SRP cycle components in organisms from all three major branches of the phylogenetic tree suggests that this pathway for protein export is of ancient evolutionary origin.

J Biol Chem, 1994 Jun 10, 269(23), 16502 - 7
Drosophila kinesin motor domain extending to amino acid position 392 is dimeric when expressed in Escherichia coli; Huang TG et al.; A truncated domain of the alpha-subunit of Drosophila kinesin was obtained by expression in Escherichia coli and purified to homogeneity in the presence of MgATP . This domain (designated DKH392) extends to amino acid 392 and contains the complete N-terminal region of kinesin which is highly conserved between species . The DKH392 construct includes an additional 52 amino acids beyond the minimal motor domain of 340 amino acid residues which has been previously characterized as DKH340 (Huang, T.-G., and Hackney, D . D . (1994) J . Biol . Chem . 269, 16493-16501) . The s20,w values for DKH340 and DKH392 are 3.3 and 5.2 S and the D20,w values are 7.7 x 10(-7) and 4.9 x 10(-7) cm3 s-1, respectively . These results indicate that DKH340 is a monomer in solution, but DKH392 is a dimer . In the presence of adenosine 5-(beta,gamma-imido)triphosphate, DKH392 binds to microtubules with a stoichiometry of two head domains (one DKH392 dimer) per tubulin heterodimer in contrast to the tight binding of one DKH340 per tubulin heterodimer . Electron microscopy indicates that both DKH340 monomers and DKH392 dimers decorate microtubules with a periodicity of 8 nm.

J Biol Chem, 1994 Jun 10, 269(23), 16493 - 501
Drosophila kinesin minimal motor domain expressed in Escherichia coli . Purification and kinetic characterization; Huang TG et al.; A truncated motor domain of the alpha subunit of Drosophila kinesin was obtained by expression in Escherichia coli and purified to homogeneity in the presence of MgATP . This domain (designated DKH340) extends from the N terminus to amino acid 340 . The isolated protein contains a stoichiometric level of tightly bound ADP and has a low basal rate of ATP hydrolysis of 0.029 +/- 0.002 s-1 in the absence of microtubules . The rate of release of bound ADP is 0.026 +/- 0.003 s-1 . The approximate equality of the ADP release rate and the steady state ATPase rate indicates that ADP release is the rate-limiting step in ATP hydrolysis in the absence of microtubules . The rate of ATP hydrolysis is stimulated 3000 fold-by addition of microtubules (MT) (kcat = 80 s-1; KMT0.5,ATPase = 160 nM for half-saturation of the ATPase rate by microtubules at saturating ATP levels; KMT0.5ATPase = 43 microns for half-saturation of the ATPase rate by ATP at saturating microtubule levels) . Binding of DKH340 to MTs is biphasic in the presence of adenosine 5-(beta-gamma-imido)t-riphosphate . One DKH340 binds tightly per tubulin heterodimer, but greater than one DKH340/tubulin heterodimer can be bound at higher ratios of DKH340/microtubules . In the presence of MgATP, KMT0.5,Binding for physical binding of DKH340 to microtubules is weaker than KMT0.5,ATPase for stimulation of hydrolysis . These results are consistent with a model in which DKH340 cycles on and off the microtubule during hydrolysis of each ATP molecule . For this model, the kcat/KMT0.5,ATPase ratio of 5 x 10(8) M-1 s-1 is at least as large as the bimolecular rate constant for association with microtubules, and this value approaches the diffusion controlled limit . Nucleotide-free DKH340 can be produced by gel filtration in the absence of Mg2+, but it reforms tightly bound ADP slowly in the presence of MgATP (t1/2 > or = 10 min), and thus it is likely to be in a conformational state which is not produced during steady state ATP hydrolysis.

J Biol Chem, 1994 Jun 10, 269(23), 16449 - 54
Identification of volatile forms of methyl groups released by Halobacterium salinarium; Nordmann B et al.; halobacterium salinarium (formerly H . halobium) is a chemotactic and phototactic archaeon from which volatile methyl groups are released continually, a phenomenon related to its sensory system . We found that released methyl groups comprised two different chemical species, methanol and methanethiol, the sulfur analog of methanol . Radiolabeling experiments showed that the methyl groups of both compounds, as well as the sulfur of methanethiol, were derived from methionine but were donated to cellular components and subsequently cleaved to produce the respective volatile compounds . Previous work had shown that chemostimuli and photostimuli result in transient increases in the rate of release of volatile methyl groups . We found that these increases reflected increased release of methanol but not of methanethiol . Thus, the methyl group chemistry of the H . salinarium sensory system is analogous to the well-studied chemotactic system of Escherichia coli . The reactions that result in methanethiol release are of unknown function and have unusual features . They may involve a methionine-gamma-lyase activity we detected in H . salinarium . Sulfur derived from methionine was found attached to specific proteins in reduction-sensitive disulfide linkages.

J Biol Chem, 1994 Jun 10, 269(23), 16371 - 5
Topoisomerase IV can support oriC DNA replication in vitro; Hiasa H et al.; Escherichia coli has two type II topoisomerases, DNA gyrase and topoisomerase IV (Topo IV) . Topo IV is required for the decatenation of the linked daughter chromosomes at the terminal stages of DNA replication, whereas gyrase, because of its ability to convert to negative supercoils the positive supercoils generated by replication fork progression in a circular chromosome, is required to support nascent chain elongation . Using an oriC DNA replication system in vitro, we show that Topo IV, which can relax positive supercoils, can also support replication fork progression . This activity is only observed at substoichiometric ratios of Topo IV to template, at higher ratios, the template becomes relaxed and initiation of DNA replication cannot occur . Topo IV was capable of supporting bidirectional DNA replication from oriC, although, unlike the case with gyrase, some templates apparently replicated unidirectionally . This suggests that either gyrase itself or a certain minimum superhelical density is required for proper initiation of DNA replication from oriC.

J Biol Chem, 1994 Jun 10, 269(23), 16364 - 70
Plasmodium falciparum S-adenosylhomocysteine hydrolase . cDNA identification, predicted protein sequence, and expression in Escherichia coli; Creedon KA et al.; Compounds that specifically inhibit S-adenosylhomocysteine hydrolase (SAHH; EC 3.3.1.1) interfere with the proliferation of Plasmodium malarial parasites, but efforts to identify the enzyme directly in parasite extracts have been unsuccessful . Here we report genetic and biochemical evidence for the presence of a gene encoding P . falciparum SAHH . The gene is transcribed as a 2.8-kilobase mRNA in erythrocytic stage parasites . Analysis of the open reading frame predicts a 53.9-kDa protein having conserved regions thought to be involved in NAD binding . The cDNA sequence has been incorporated into an Escherichia coli expression construct to confirm the function of the sahh product . Transformed E . coli cells produce a protein with a relative molecular weight of 56,000 which possesses SAHH activity as evidenced by the conversion of 3-deazaadenosine to S-3-deazaadenosylhomocysteine . Several amino acid residues that have been suggested to be at the SAHH active site in other organisms show nonconserved replacements in P . falciparum, suggesting that some current proposals for the enzyme mechanism may need to be revised . The structural differences between the P . falciparum and mammalian SAHH enzymes may foster innovative strategies for drug development against malaria.

J Biol Chem, 1994 Jun 10, 269(23), 16333 - 9
rheb, a growth factor- and synaptic activity-regulated gene, encodes a novel Ras-related protein; Yamagata K et al.; Neuronal activity results in long term cellular changes that underlie normal brain development and synaptic plasticity . To examine the molecular basis of activity-dependent plasticity, we have used differential cloning techniques to identify genes that are rapidly induced in brain neurons by synaptic activity . Here we describe an inducible novel member of the Ras family of small GTP-binding proteins we have termed Rheb . rheb mRNA is rapidly and transiently induced in hippocampal granule cells by seizures and by NMDA-dependent synaptic activity in the long term potentiation paradigm . The predicted amino acid sequence of Rheb is most closely homologous to yeast Ras1 and human Rap2 . The putative GTP binding regions are highly conserved . A bacterial fusion protein of Rheb binds GTP and exhibits intrinsic GTPase activity . Like Ha-Ras, the carboxylterminal sequence encodes a CAAX box that is predicted to signal post-translational farnesylation and to target Rheb to specific membranes . rheb mRNA is expressed at comparatively high levels in normal adult cortex as well as a number of peripheral tissues, including lung and intestine . In the developing brain, rheb mRNA is expressed at relatively high levels in embryonic day 19 cortical plate, and expression remains at stable levels throughout the remainder of prenatal and postnatal development . Its close homology with ras and its rapid inducibility by receptor-dependent synaptic activity suggest that rheb may play an important role in long term activity-dependent neuronal responses.

J Biol Chem, 1994 Jun 10, 269(23), 16305 - 10
Signal peptide cleavage regions . Functional limits on length and topological implications; Jain RG et al.; As a first step toward understanding the topology of the signal peptide with respect to the membrane during the protein export process, we have examined the constraints on the length of the cleavage region needed to achieve signal peptidase recognition and cleavage . Using the signal peptide of Escherichia coli alkaline phosphatase, a series of cleavage region mutants has been constructed . Variations in length were brought about by replacing the wild type cleavage region of the signal peptide with polymers of increasingly more residues . In each case, alanine residues are used exclusively in the -1 and -3 positions to provide only one viable cleavage site . Glutamine residues are used in all other positions in order to vary the length from 3 to 13 total residues . Analysis of these mutants revealed that cleavage regions ranging from 3 to 9 residues are completely and efficiently processed . The extent of processing drops substantially thereafter, with no processing observed for signal peptides with 13-residue long cleavage regions . A second mutant with a 13-residue long cleavage region was designed and analyzed to ensure that the lack of processing reflected a cleavage problem and not a translocation defect . The results are consistent with the notion that the signal peptidase active site is in close proximity to the periplasmic surface of the inner membrane and that interaction of the cleavage region with the signal peptidase probably depends on, and is constrained by, other interactions involving the signal peptide.

J Biol Chem, 1994 Jun 10, 269(23), 16260 - 8
Purification and characterization of a novel deoxyinosine-specific enzyme, deoxyinosine 3' endonuclease, from Escherichia coli; Yao M et al.; We have purified a novel endonuclease from Escherichia coli that recognizes deoxyinosine, a deamination product of deoxyadenosine in DNA . This activity, which we named deoxyinosine 3' endonuclease, is different from the known hypoxanthine DNA N-glycosylases which have been partially characterized in E . coli and other organisms . The enzyme was purified 24,800-fold to apparent homogeneity . SDS- and activity PAGE analyses indicate that the enzyme has an apparent molecular mass of 25 kDa . Deoxyinosine 3' endonuclease recognized deoxyinosine in both single- and double-stranded DNA but exhibited a 4-fold preference for double stranded over single-stranded DNA . In addition to deoxyinosine, the enzyme recognized urea residues and AP sites . Deoxyinosine 3' endonuclease has an obligatory requirement for Mg2+, but other cations such as Co2+ and Mn2+ could partially replace Mg2+ . The optimal pH for deoxyinosine 3' endonuclease was around 7.5 . In contrast to most of the known repair enzymes, deoxyinosine 3' endonuclease makes an incision at the second phosphodiester bond 3' to a deoxyinosine or AP site, leaving behind the intact lesion on the nicked DNA . Therefore, deoxyinosine 3' endonuclease recognizes, but does not remove, the lesion from the DNA molecule . The biological significance of this novel activity is discussed with reference to other repair activities in E . coli.

J Biol Chem, 1994 Jun 10, 269(23), 16236 - 41
Effects of guanosine 3',5'-bisdiphosphate (ppGpp) on rate of transcription elongation in isoleucine-starved Escherichia coli; Vogel U et al.; We measured the transcription elongation rate on two mRNA genes, i.e . infB and lacZ, and on a part of the rrnB gene under conditions when wild type (rel+) Escherichia coli and relaxed (relA) mutants were exposed to isoleucine starvation . The RNA chain growth rates were calculated from the time lag between induction of transcription and the appearance of specific hybridization to probes complementary to the 3' ends of the genes, i.e . from the transcription time . The rate of mRNA chain elongation responded differently in rel+ and relA strains exposed to isoleucine starvation as it decreased (approximately 50%) in rel+ strains that accumulated high concentrations of guanosine 3',5'-bisdiphosphate (ppGpp) and increased (approximately 15%) the relA mutant whose ppGpp pool decayed during starvation . These results show that ppGpp inhibits mRNA chain elongation in vivo . However, stable RNA chain elongation appeared unaffected by ppGpp pool size and was twice as fast as mRNA chain elongation in exponentially growing cells.

J Biol Chem, 1994 Jun 10, 269(23), 16223 - 8
Effect of selenium deficiency on type I 5'-deiodinase; DePalo D et al.; The type I iodothyronine 5'-deiodinase (5'-DI) present in rat liver and kidney has recently been demonstrated to be a selenoprotein . The goal of the present study was to examine in detail the effect of selenium (Se) deficiency on 5'-DI at the protein and mRNA levels . In weanling rats fed a selenium-deficient (Se(-)) diet for 6 weeks, 5'-DI activity was decreased 91 and 69% relative to control activities in liver and kidney, respectively . Administration of 3,5,3'-triiodothyronine resulted in a 2-fold increase in 5'-DI activity in control animals, but had little or no effect on 5'-DI activity in Se(-) animals . Western analysis using a specific antiserum directed against a bacterial fusion protein containing the carboxyl-terminal half of the 5'-DI protein demonstrated that this decrease in 5'-DI activity in Se(-) animals was explained by a marked decrease in 5'-DI protein . Administration of Se to Se(-) animals resulted in parallel increases in 5'-DI protein and activity over a 72-h time period . It was also shown that selenium deficiency was accompanied by a 40% decrease in 5'-DI mRNA levels in the kidney, but not in the liver . In both tissues, the administration of 3,5,3'-triiodothyronine resulted in increased 5'-DI mRNA levels which were not altered by selenium status . These studies indicate that selenium deficiency decreases 5'-DI activity by decreasing the amount of 5'-DI protein . The mechanism of this impairment in enzyme synthesis appears to be a defect in translation, presumably due to a block in the UGA-directed selenocysteine incorporation in selenium deficiency.

J Biol Chem, 1994 Jun 10, 269(23), 16163 - 9
The importance of conserved nucleotides of 23 S ribosomal RNA and transfer RNA in ribosome catalyzed peptide bond formation; Lieberman KR et al.; We have constructed the double mutant G2252C/G2253C in Escherichia coli 23 S rRNA by site-directed mutagenesis . These phylogenetically conserved residues are protected from chemical modification by the 3' CCA terminus of the peptidyl-tRNA site (P site)-bound tRNA . Expression of C2252/C2253 23 S rRNA in E . coli severely compromises cell growth . Mutant rRNA is assembled into 50 S subunits and 70 S ribosomes but is discriminated against in polysomes . Mutant ribosomes function at lower rates in peptidyltransferase assays than wild type ribosomes . To test whether this defect derives from disruption of base pairing with the 2 cytidines of the invariant 3' CCA terminus of tRNA, a mutant E . coli tRNAPhe gene was constructed, with the CCA sequence changed to GGA . As deacylated species, mutant and wild type tRNAPhe inhibit peptidyl transfer identically . Mutant tRNAPhe was aminoacylated in vitro but failed to react as a P site substrate, with either mutant or wild type ribosomes . These results support a role for G2252 and G2253 of 23 S rRNA in peptidyltransferase function and a role for the 3' residues of peptidyl-tRNA in catalytically productive P site interaction; but they fail to provide evidence supporting canonical base pairing between these 23 S residues and the 3' end of peptidyl-tRNA.

J Biol Chem, 1994 Jun 10, 269(23), 16091 - 100
Identification of the gene encoding lipoate-protein ligase A of Escherichia coli . Molecular cloning and characterization of the lplA gene and gene product; Morris TW et al.; R(+)-Lipoic acid is a cofactor required for function of the alpha-keto acid dehydrogenase and glycine cleavage enzyme complexes . The naturally occurring form of lipoate is attached by amide linkage to the epsilon-amino group of a specific lysine residue within conserved lipoate-accepting protein domains . Lipoate-protein ligase(s) catalyze the formation of this amide bond between lipoyl groups and specific apoproteins . We report the isolation of the lplA gene which encodes an Escherichia coli lipoate-protein ligase . Strains with lplA null mutations transport lipoic acid normally but have severe defects in the incorporation and utilization of exogenously supplied lipoic acid and lipoic acid analogs . These strains are also highly resistant to selenolipoate (a growth-inhibiting lipoate analog) and contain no detectable lipoate-protein ligase activity in cell extracts . The lplA gene has been cloned, sequenced, and physically mapped to min 99.6 (4657 kilobases) of the E . coli chromosome . Upon overexpression, the 38-kDa lplA gene product was purified to homogeneity and shown to have a mass, N-terminal sequence and amino acid composition consistent with the deduced 337 residue primary sequence . Enzyme assays show that purified LplA catalyzes the ATP-dependent attachment of {35S}lipoic acid to apoprotein, thus confirming that lplA encodes lipoate-protein ligase A . Analysis of lplA null mutants also indicates the existence of a second (lplA-independent) lipoyl-ligase enzyme in E . coli . This is the first identification of a lipoate ligase gene and the first analysis of a purified lipoate ligase enzyme.

J Biol Chem, 1994 Jun 10, 269(23), 16082 - 90
Direct binding of myosin II to phospholipid vesicles via tail regions and phosphorylation of the heavy chains by protein kinase C; Murakami N et al.; Recent cloning and sequencing studies suggest that heavy chains of all non-muscle myosins II have a protein kinase C (PKC) phosphorylation site within their tail regions . A fragment of human macrophage myosin heavy chain, encompassing its COOH-terminal 396 amino acids (MIIAF46), was expressed in Escherichia coli to provide a model system for study of PKC-mediated phosphorylation . PKC phosphorylated this fragment when phosphatidylserine (PS) liposomes were present, but not when liposomes made from PS/phosphatidylcholine (PC) were used . The reaction required Ca2+, but not other activators such as diacylglycerol (DG) or phosphatidylinositol 4,5-bisphosphate . Phosphorylation of MIIAF46 was not observed in the presence of micelles of PS or PS/DG . Similar results were obtained using native myosin II purified from bovine brain and chicken intestine brush border . Phosphorylation of light chains, in contrast, occurred even with PS/PC liposomes if DG was present . Addition of the PS and PS/DG liposomes significantly increased the turbidities at 340 nm of MIIAF46 and native myosin II, and the extent of increase depended upon the type of myosin used . Also, PS and PS/DG liposomes shifted the gel filtration elution positions of MIIAF46 and myosin II . In contrast, liposomes of PS/PC and PS/PC/DG gave only a slight increase in turbidity with all myosins and fragments and did not noticeably shift their gel filtration elution positions . These results suggest that myosins II bind to PS liposomes via the COOH-terminal regions of their heavy chains with affinities specific to each myosin isoform, that the binding is dependent upon the PS composition, and that PKC phosphorylates the PS-bound heavy chains.

J Biol Chem, 1994 Jun 10, 269(23), 16020 - 8
Model of subunit composition of gamma-aminobutyric acid A receptor subtypes expressed in rat cerebellum with respect to their alpha and gamma/delta subunits; Quirk K et al.; Antibodies specific for subunits of the gamma-aminobutyric acid A (GABAA) receptor have been used to immunoprecipitate {3H}muscimol, {3H}Ro 15-4513, and {3H}Ro 15-1788 binding sites from deoxycholate-solubilized preparations of rat cerebellum . Of the antisera raised against alpha subunits, those specific for alpha 6 immunoprecipitated the largest proportion of receptors . Two populations of alpha 6-containing GABAA receptors were identified . The first was labeled with {3H}Ro 15-4513 and exhibited a pharmacological profile consistent with that observed for alpha 6 beta 2 gamma 2 in transfected cells (Luddens, H., Pritchett, D . B., Kohler, M., Killisch, I., Keinanen, K., Monyer, H., Sprengel, R., and Seeberg, P . H . (1990) Nature 346, 648-651) . The second population was labeled only with {3H}muscimol and was deduced, from quantitative immunoprecipitation studies using combinations of antibodies, to contain both alpha 6 and delta subunits . The alpha 6 subunit was not observed to be present in combination with other alpha subunits or the gamma 1 subunit . Each of the other alpha subunits was found to be present in only one population of receptors in the cerebellum . Some subunits (alpha 4, alpha 5, and gamma 3) were not detectable . By combining information from quantitative immunoprecipitation experiments and Western blot analysis, a model describing the composition of all GABAA receptors in the cerebellum was constructed that defined the following alpha and gamma/delta combinations (percentage of cerebellar GABAA receptors): alpha 6 gamma 2 (36%), alpha 6 delta (23%), alpha 1 gamma 2 (28%), alpha 2 gamma 1 (8%), and alpha 3 gamma 2 (5%).

Gene, 1994 Jun 10, 143(2), 239 - 43
Secretory production of chicken ovomucoid domain 3 by Escherichia coli and alteration of inhibitory specificity toward proteases by substitution of the P1 site residue; Kojima S et al.; Ovomucoids are commonly present in bird egg white and exhibit inhibitory activity toward various serine proteases . To investigate the structure-function relationship of ovomucoid domain 3, we established a secretory expression system for the chicken ovomucoid domain 3 (OMCHI3)-encoding gene in Escherichia coli by ligating it downstream from the tac promoter and signal peptide of E . coli alkaline phosphatase . E . coli JM105 was transformed with the resulting plasmid and induced with 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) . The mature OMCHI3 was detected in the culture supernatant, and was purified to homogeneity by three-step chromatography . Amino-acid sequence analysis showed that processing by the signal peptidase was carried out exactly at the expected site . Measurements of circular dichroism spectra and inhibitory activity indicated that OMCHI3 was produced in the properly folded form . Furthermore, site-specific replacement of the Ala residue at the P1 site with Met or Lys resulted in acquisition of inhibitory activity toward chymotrypsin or trypsin, respectively, indicating that the P1 site is the predominant determinant for inhibitory specificity.

Gene, 1994 Jun 10, 143(2), 201 - 9
Translational regulation of a recombinant operon containing human platelet-derived growth factor (PDGF)-encoding genes in Escherichia coli: genetic titration of the peptide chains of the heterodimer AB; Schneppe B et al.; A new strategy is described for the production of recombinant heteromultimeric proteins using Escherichia coli as host . A recombinant operon was constructed containing modified cDNA sequences encoding the mature A and B chains of human platelet-derived growth factor (PDGF) . The relative expression rates of the PDGF genes were varied over a range equivalent to A:B ratios from 0.8 to 3.7 by means of translational regulation . This was achieved using two different translational initiation sequences (TIS) upstream from the respective coding regions, one derived from the E . coli atpE translational initiation region, and the other containing a sequence with extended complementarity to the 3' end of the 16S rRNA . The generation of mature PDGF A and B chains in different relative amounts in E . coli provided the basis for developing a novel procedure for the production of the biologically active PDGF heterodimer AB in large quantities . The general strategy is applicable to the preparation of a wide range of heteromultimeric complexes . Moreover, the described PDGF operon constitutes a compact and versatile model system for studies of the posttranscriptional regulation of gene expression.

J Mol Biol, 1994 Jun 10, 239(3), 433 - 5
Nucleotide sequence of the Escherichia coli K12 histidine operon revisited; Jovanovic G et al.; We report on a significant difference between the published nucleotide sequence of the Escherichia coli K12 histidine operon and our sequencing results repeatedly obtained from a number of different E . coli K12 strains . The discrepancies include 39 base-pair changes and one addition located predominantly in the proximal portion of the operon . Our data also suggest that neutral and near-neutral mutations do not accumulate to a significant extent in the histidine operon of E . coli strains harbouring strong mutator alleles.

Biochemistry, 1994 Jun 7, 33(22), 6998 - 7004
Effects of autophosphorylation on casein kinase II activity: evidence from mutations in the beta subunit; Lin WJ et al.; Casein kinase II is a heterotetramer composed of two catalytic (alpha) and two regulatory (beta) subunits . To examine the effects of autophosphorylation of the beta subunit on enzyme activity, two mutants of the beta subunit from Drosophila were constructed in which either Ser4 or Ser2-4 was changed to alanine residues by oligonucleotide-directed mutagenesis and the proteins were expressed in Escherichia coli . The wild-type alpha and individual beta subunits present in inclusion bodies were renatured, and the biochemical properties of the reconstituted holoenzymes were examined . Analysis of autophosphorylation revealed that phosphate incorporation was about 0.8 mol/mol of beta subunit for the wild type and Ala4 mutant; Ser2 and Ser3 were the major sites of autophosphorylation with some phosphate in Ser4 as shown by Edman degradation . No autophosphorylation was observed with the Ala2-4 mutant . Substitution of alanine for serine residues at positions 4 or 2-4 of the beta subunits did not influence the reassociation of the alpha and beta subunits to form holoenzyme, or the function of the beta subunit in stimulating catalytic activity or in responding to basic compounds . To measure the effects of autophosphorylation on casein kinase II activity, the wild-type and mutant holoenzymes were preincubated in the presence and absence of ATP, and the rate of phosphorylation was measured with various substrates . In the absence of autophosphorylation, the wild-type, Ala4, and Ala2-4 forms of the holoenzyme displayed similar rates of phosphorylation of glycogen synthase . After preincubation with ATP, the rate of phosphorylation of glycogen synthase by the wild-type and Ala4 enzymes was inhibited by 30%.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1994 Jun 7, 33(22), 6812 - 21
Time-resolved solid-state NMR spectroscopy of 5-enolpyruvylshikimate-3-phosphate synthase; Appleyard RJ et al.; The novel technique of time-resolved solid-state NMR spectroscopy has been used to characterize the enzyme, 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, in both the forward and reverse directions over time periods ranging from 5 to 300 ms . The wealth of data currently available for EPSP synthase, in particular the pre-steady-state kinetics performed using chemical quench-flow experiments {Anderson, K . S., Sikorski, J . A., & Johnson, K . A . (1988) Biochemistry 27, 7395-7406}, has made the enzyme an obvious choice as a proving ground for this new technique . Pre-steady-state 13C TOSS CP-MAS spectra have been obtained with a much improved signal-to-noise ratio, and corrections have been made to some previously reported assignments {Evans, J.N.S., Appleyard, R.J., & Shuttleworth, W.A . (1993) J . Am . Chem . Soc . 115, 1588-1590} . Peak fitting has allowed the extrapolation of NMR integral intensities of species involved in the reaction . These show a good correlation with concentrations calculated by simulations using the kinetic parameters obtained from the chemical quench-flow experiments . It is proposed that careful optimization of the contact time used will be necessary to obtain accurate, relative concentrations that will enable an independent kinetic simulation by time-resolved solid-state NMR . The technique shows much promise due to its nondestructive quenching procedure, which allows the direct observation of enzyme intermediates on a reaction pathway . However, its requirement of significantly larger amounts of enzyme does limit the technique to those proteins which naturally occur in high abundance or have been hyperexpressed.

Proc Natl Acad Sci U S A, 1994 Jun 7, 91(12), 5508 - 12
Stable DNA transformation in the obligate intracellular parasite Toxoplasma gondii by complementation of tryptophan auxotrophy; Sibley LD et al.; The protozoan parasite Toxoplasma gondii infects a wide range of vertebrate hosts and is an important opportunistic pathogen in immunocompromised humans . Although Toxoplasma is amenable to both biochemical and cellular experimental approaches, the molecular basis of its success as an intracellular parasite is poorly understood . To provide a system for molecular genetic analyses, we have developed a stable DNA transformation system for Toxoplasma based on complementation of its naturally occurring tryptophan auxotrophy . Complementation was accomplished by expressing the Escherichia coli trpB gene, encoding the beta subunit of tryptophan synthase (EC 4.2.1.20), the enzyme that catalyzes the formation of tryptophan from indole plus serine . Transformants were obtained by electroporation of a plasmid, called SAG1/trpB, containing the trpB gene flanked by Toxoplasma surface antigen 1 (SAG1) gene sequences and selection for growth on indole . Transformants were obtained with circular forms of the SAG1/trpB plasmid with efficiencies of 10(-4) per cell . Transformation with either circular or linear SAG1/trpB resulted in integration into the genome at distinct, nonhomologous sites . Trp+ transformants typically contained tandemly repeated copies of the SAG1/trpB plasmid and were stable in the absence of continued selection . The Trp phenotype provides a dominant selectable marker that should allow expression of foreign or altered genes in Toxoplasma and facilitate molecular analyses of genes important for intracellular survival.

Proc Natl Acad Sci U S A, 1994 Jun 7, 91(12), 5446 - 50
A role for destabilizing amino acid replacements in light-chain amyloidosis; Hurle MR et al.; Light-chain (L-chain) amyloidosis is characterized by deposition of fibrillar aggregates composed of the N-terminal L-chain variable region (VL) domain of an immunoglobulin, generally in individuals overproducing a monoclonal L chain . In addition to proteolytic fragmentation and high protein concentration, particular amino acid substitutions may also contribute to the tendency of an L chain to aggregate in L-chain amyloidosis, although evidence in support of this has been limited and difficult to interpret . In this paper we identify particular amino acid replacements at specific positions in the VL domain that are occupied at frequencies significantly higher in those L chains associated with amyloidosis . Analysis of the structural model for the VL domain of the Bence-Jones protein REI suggests that these positions play important roles in maintaining domain structure and stability . Using an Escherichia coli expression system, we prepared single-point mutants of REI VL incorporating amyloid-associated amino acid replacements that are both rare and located at structurally important positions . These mutants support ordered aggregate formation in an in vitro L-chain fibril formation model in which wild-type REI VL remains soluble . Moreover, the ability of these sequences to aggregate in vitro correlates well with the extent to which domain stability is decreased in denaturant-induced unfolding . The results are consistent with a mechanism for the disease process in which the VL domain, either before or after proteolytic cleavage from the L-chain constant region domain, unfolds by virtue of one or more destabilizing amino acid replacements to generate an aggregation-prone nonnative state.

Proc Natl Acad Sci U S A, 1994 Jun 7, 91(12), 5421 - 5
Properties of permease dimer, a fusion protein containing two lactose permease molecules from Escherichia coli; Sahin-Toth M et al.; An engineered fusion protein containing two tandem lactose permease molecules (permease dimer) exhibits high transport activity and is used to test the phenomenon of negative dominance . Introduction of the mutation Glu-325-->Cys into either the first or the second half of the dimer results in a 50% decrease in activity, whereas introduction of the mutation into both halves of the dimer abolishes transport . Lactose transport by permease dimer is completely inactivated by N-ethylmaleimide; however, 40-45% activity is retained after N-ethylmaleimide treatment when either the first or the second half of the dimer is replaced with a mutant devoid of cysteine residues . The observations demonstrate that both halves of the fusion protein are equally active and suggest that each half may function independently . To test the possibility that oligomerization between dimers might account for the findings, a permease dimer was constructed that contains two different deletion mutants that complement functionally when expressed as untethered molecules . Because this construct does not catalyze lactose transport to any extent whatsoever, it is unlikely that the two halves of the dimer interact or that there is an oligomeric interaction between dimers . The approach is consistent with the contention that the functional unit of lactose permease is a monomer.

Proc Natl Acad Sci U S A, 1994 Jun 7, 91(12), 5242 - 6
The cooperativity and allosteric inhibition of Escherichia coli phosphofructokinase depend on the interaction between threonine-125 and ATP; Auzat I et al.; During the reaction catalyzed by the phosphofructokinase (EC 2.7.1.11) from Escherichia coli, the phosphoryl group transferred from ATP interacts with Thr-125 {Shirakihara, Y . & Evans, P . R . (1988) J . Mol . Biol . 204, 973-994} . The replacement of Thr-125 by serine changes the saturation by fructose 6-phosphate from cooperative to hyperbolic and abolishes the allosteric inhibition by phosphoenolpyruvate . The same changes, a saturation by fructose 6-phosphate that is no longer cooperative and an activity that is no longer inhibited by phosphoenolpyruvate, are observed with wild-type phosphofructokinase when adenosine 5'-{gamma-thio}triphosphate is used instead of ATP as the phosphoryl donor . These two perturbations of the ATP-Thr-125 interaction lead to the suppression of both the allosteric inhibition by phosphoenolpyruvate and the cooperativity of fructose-6-phosphate saturation, as if replacing the neutral oxygen of ATP by sulfur or removing the methyl group of Thr-125 had "locked" phosphofructokinase in its active conformation . The geometry of this ATP-Thr-125 interaction and/or the presence of the methyl group on the beta-carbon of Thr-125 are crucial for the regulatory properties of phosphofructokinase . This interaction could be a hydrogen bond between the neutral oxygen of the gamma-phosphate of ATP and the hydroxyl group of Thr-125.

Biochemistry, 1994 Jun 7, 33(22), 7014 - 20
A single mutation in the recombinant light chain of tetanus toxin abolishes its proteolytic activity and removes the toxicity seen after reconstitution with native heavy chain; Li Y et al.; Specific proteolysis by the tetanus toxin light chain of a vesicle-associated membrane protein (VAMP) involved in exocytosis is thought to underlie its intracellular blockade of neurotransmitter release . To substantiate this mechanism, recombinant light chain was expressed as a maltose binding protein-light chain fusion product in Escherichia coli . After purification of affinity chromatography and cleavage with factor Xa, the resultant light chain was isolated and its identity confirmed by Western blotting and N-terminal sequencing . It exhibited activity similar to that of the native light chain in proteolyzing its target in isolated bovine small synaptic vesicles and in hydrolyzing a 62-residue synthetic polypeptide spanning the cleavage site of the substrate . The importance of Glu234 in the catalytic activity of the light chain, possibly analogous to Glu143 of thermolysin, was examined using site-directed mutagenesis . Changing Glu234 to Ala abolished the protease activity of the light chain, but its ability to bind the polypeptide substrate was retained . Each recombinant light chain could be reconstituted with the heavy chain of tetanus toxin, yielding the same level of disulfide-linked species as the two native chains . Whereas the toxin formed with wild-type light chain exhibited appreciable neuromuscular paralysis activity and mouse lethality, the equivalent dichain material containing the Ala234 mutant lacked neurotoxicity in both the in vitro and in vivo assays . Thus, these results demonstrate directly, for the first time, that the lethality of tetanus toxin and its inhibition of exocytosis in intact neurons are attributable largely, if not exclusively, to endoprotease activity.

FEBS Lett, 1994 Jun 6, 346(1), 65 - 8
Biochemical characterization of the presecretory protein translocation machinery of Escherichia coli; Tokuda H; The protein translocation apparatus in Escherichia coli has been studied both genetically and biochemically . In vitro protein translocation systems involving everted membrane vesicles or reconstituted proteoliposomes have significantly contributed to biochemical clarification of the structure, mechanism and energetics of the apparatus . It is established that SecA, SecY and SecE are essential components, and play fundamental roles in the translocation reaction, and that both ATP and a proton motive force are required for the translocation . A new membrane factor, SecG, was found to participate in the formation of the apparatus, causing significant enhancement of the activity . SecD was found to play a role in the release of translocated proteins from the outer surface of the cytoplasmic membrane.

FEBS Lett, 1994 Jun 6, 346(1), 55 - 8
Maltose transport system of Escherichia coli: an ABC-type transporter; Nikaido H; The maltose transport system of E . coli is composed of a periplasmic maltose-binding protein (MBP), the presumed transmembrane channel made up of MalF and MalG proteins, and two copies of the ATPase subunit, MalK . The membrane-associated transporter complex was purified in a functional form both from the wild-type strain and from mutants that do not require MBP for transport, and was reconstituted into proteoliposomes . A major function of MBP is to send a transmembrane signal, in the presence of ligands, to the ATPase subunits on the inner side of the membrane . In addition, MBP performs a special function in the translocation of the larger ligands, maltodextrins, perhaps by aligning them for entry into the channel.

FEBS Lett, 1994 Jun 6, 346(1), 48 - 54
Mitochondrial carrier proteins; Palmieri F; Ten mitochondrial carriers have been purified from animal mitochondria . They are small proteins with a molecular mass ranging from 28 to 34 kDa on SDS-PAGE . So far, five of these proteins have been sequenced . Their polypeptide chain consists of three tandemly related sequences of about 100 amino acids . The repeats of the different proteins are related and probably fold into two transmembrane alpha-helices linked by an extra-membrane loop . The features of this family are also present in several proteins of unknown function characterized by DNA sequencing . Isoforms of some carriers have been found . All mitochondrial carriers investigated in proteoliposomes function according to a simultaneous (sequential) mechanism of transport . The only exception is the carnitine carrier that proceeds via a ping-pong mechanism . Three mitochondrial carriers have been expressed in yeast and two overexpressed in E . coli and refolded in active form.

Mol Gen Genet, 1994 Jun 3, 243(5), 584 - 92
Different UmuC requirements for generation of different kinds of UV-induced mutations in Escherichia coli; Nowicka A et al.; An Escherichia coli strain bearing the dnaQ49 mutation, which results in a defective epsilon subunit of DNA polymerase III, and carrying the lexA71 mutation, which causes derepression of the SOS regulon, is totally unable to maintain high-copy-number plasmids containing the umuDC operon . The strain is also unable to maintain the pAN4 plasmid containing a partial deletion of the umuD gene but retaining the wild-type umuC gene . These results suggest that a high cellular level of UmuC is exceptionally harmful to the defective DNA polymerase III of the dnaQ49 mutant . We have used this finding as a basis for selection of new plasmid umuC mutants . The properties of two such mutants, bearing the umuC61 or umuC95 mutation, are described in detail . In the umuC122::Tn5 strain harbouring the mutant plasmids, UV-induced mutagenesis is severely decreased compared to that observed with the parental umuDC+ plasmid . Interestingly, while the frequency of UV-induced GC-->AT transitions is greatly reduced, the frequency of AT-->TA transversions is not affected . Both mutant plasmids bear frameshift mutations within the same run of seven A residues present in umuC+; in umuC61 the run is shortened to six A whereas in umuC95 is lengthened to eight A . We have found in both umuC61 and umuC95 that translation is partially restored to the proper reading frame . We propose that under conditions of limiting amounts of UmuC, the protein preferentially facilitates processing of only some kinds of UV-induced lesions.

Mol Gen Genet, 1994 Jun 3, 243(5), 525 - 31
Structure of the 5' upstream region and the regulation of the rpoS gene of Escherichia coli; Takayanagi Y et al.; The nucleotide sequence of the 5' upstream region of the Escherichia coli rpoS gene was determined and analyzed . At least four promoters responsible for rpoS transcription were identified, and designated P1, P2, P3 and P4, P1 being furthest from the upstream . Using lacZ fusion genes, the P2 promoter was found to be the strongest of the four . All of these promoters are regulated similarly, and their activity is enhanced 2 to 3-fold in stationary phase . P1 and P2 transcription start sites were determined by primer extension analyses . The P2 promoter region shows similarity to the consensus sigma 70-type promoter sequence, and was recognized by both E sigma 70 and E sigma 38 holoenzymes in vitro . The mRNA transcribed from the most distal promoter, P1, appears to include another open reading frame (orf-281), indicating that the two open reading frames comprise an operon . The rpoS gene product (sigma 38) was rapidly degraded after addition of chloramphenicol to cultures in the exponential, but not the stationary phase . This strongly suggests that posttranslational regulation is involved in the control of rpoS expression.

Mol Gen Genet, 1994 Jun 3, 243(5), 500 - 5
Hydrogen peroxide induces G:C to T:A and G:C to C:G transversions in the supF gene of Escherichia coli; Akasaka S et al.; A vector plasmid, pZ189, carrying an Escherichia coli supF gene as a target for mutations, was treated with a combination of hydrogen peroxide and Fe3+/EDTA complex and propagated in E . coli host cells that had been induced for SOS functions by ultraviolet irradiation . The mutations frequency increased by up to 30-fold over spontaneous background levels with increasing concentrations of hydrogen peroxide . The increase in mutation frequency correlated with an increase in the formation of 8-hydroxydeoxyguanosine in the pZ189 DNA . Sequence analysis of 82 independent supF mutant plasmids revealed that 70 mutants contained base substitutions, with 63 of the 70 involving a G:C base pair, and with G:C-->C:G (28 cases) and G:C-->T:A (26 cases) transversions predominating . Investigation of the influence of the local DNA sequence on the transversions revealed that the guanine at the center of the triplet 5'-PuGA-3' was five times more likely to mutate after treatment with hydrogen peroxide than that at the center of 5'PyGN3' . G:C-->T:A transversions presumably resulted from mispairing of an altered G (probably 8-hydroxydeoxyguanosine) with deoxyadenosine . The origin of the G:C-->C:G transversions may be an as yet unidentified lesion generated by hydrogen peroxide . Mutagenic hotspots for base substitutions were found at positions 133, 160 and 168 . Mutation spectra and the positions of mutagenic hotspots, when compared with a previously determined spontaneous mutagenesis spectrum, also provide information on the mechanism of spontaneous mutagenesis.

J Mol Biol, 1994 Jun 3, 239(2), 285 - 305
Crystal structures of Escherichia coli aspartate aminotransferase in two conformations . Comparison of an unliganded open and two liganded closed forms; Jager J et al.; Three crystal structures of wild type E . coli aspartate aminotransferase (E.C.2.6.1.1) in space group P2(1) have been determined at resolution limits between 2.6 and 2.35 A . The unliganded enzyme and its complexes with the substrate analogues maleate and 2-methylaspartate resulted in different conformations . The unit cell parameters of the unliganded and the inhibited enzyme are a = 87.2, b = 79.9, c = 89.8 A and beta = 119.1 degrees, and a = 85.4, b = 79.8, c = 89.5 A and beta = 118.6 degrees, respectively . The crystallographic symmetry is pseudo-C222(1) . The liganded enzyme structures were solved by difference Fourier techniques from that of a Val39-->Leu mutant partially refined to an R-factor of 0.22 at 2.85 A . They have a "closed" conformation like the chicken mAATase:maleate complex . The models were refined to R-factors of 0.19 (maleate complex) and 0.18 (2-methylaspartate complex) by molecular dynamics and restrained least squares methods . The unliganded crystal form was solved by molecular replacement and refined to an R-factor of 0.19 at 2.5 A resolution . The structure is in a "half-open" conformation, with the small domain rotated about 6 degrees from the closed conformation . The cofactor pyridoxal phosphate has a more relaxed conformation than in mAATase . Both maleate and 2-methylaspartate are hydrogen-bonded to the active site as in mAATase . The C alpha-CH3 bond of 2-methylaspartate is oriented at right angles to the cofactor pyridine ring, the most productive orientation for alpha-deprotonation of the substrate L-aspartate . Comparisons with earlier determined eAATase structures in space group C222(1) revealed differences that can probably be attributed to the somewhat lower resolution of the orthorhombic structures and/or mutations in the eAATases used in those studies . The present P2(1) structures confirm the justification of extrapolating properties of active site point mutants to the vertebrate isozymes . They will serve as reference in the interpretation of the properties of further site-directed mutants in continued studies of structure-function relationships of this enzyme.

J Biol Chem, 1994 Jun 3, 269(22), 15838 - 45
Molecular cloning of a chiral-specific 3 alpha-hydroxysteroid sulfotransferase; Lee YC et al.; A novel stereoselective hydroxysteroid sulfotransferase (HST) that acts on neutral steroids having the 3-hydroxyl group in the alpha orientation but not on steroids where the 3-hydroxyl group is oriented in the beta position has been cloned and expressed . Primary screening of a guinea pig adrenal cDNA library was performed by colony hybridization using an oligonucleotide probe based on a highly homologous region among known steroid sulfotransferases . Selected clones consisted of overlapping sequences but were incomplete . The rapid amplification of cDNA ends procedure was used to construct a full-length guinea pig HST cDNA . The guinea pig HST cDNA transiently transfected into Chinese hamster ovary K1 cells expressed a protein identical in size to that of purified guinea pig HST specific for 3 alpha-hydroxylated neutral steroids that was recently reported (Driscoll, W . J., Martin, B . M., Chen, H.-C., and Strott, C . A . (1993) J . Biol . Chem . 268, 23496-23503) . The expressed HST likewise exhibited sulfotransferase activity that was directed specifically toward steroid substrates containing a 3-hydroxyl group in the alpha orientation; on the other hand, steroids with a 3 beta-hydroxyl group were not sulfonated by the expressed HST . Thus, the cloned HST cDNA clearly coded for a steroid sulfotransferase with chiral specificity for 3 alpha-hydroxylated neutral steroids and was, therefore, given the designation of guinea pig 3 alpha-hydroxysteroid sulfotransferase (gp3 alpha-HST) . The full-length gp3 alpha-HST cDNA consisted of 1182 base pairs and coded for a protein containing 287 amino acids . The deduced amino acid sequence of the protein shares 65/79, 65/80, and 62/76% identity/similarity with rat, mouse, and human HST, respectively . Northern blot analysis of guinea pig tissues revealed no apparent gender differences in either mRNA species or distribution; a single 1.4-kilobase HST mRNA species was present in adrenal and liver tissue . Interestingly, the adrenal mRNA content was considerably more abundant than that found in the liver . Evidence for 3 alpha-HST mRNA was not detected in kidney, heart, lung, muscle, spleen, or uterus.

J Biol Chem, 1994 Jun 3, 269(22), 15808 - 13
Recombinant pulmonary surfactant protein D . Post-translational modification and molecular assembly; Crouch E et al.; Pulmonary surfactant protein D (SP-D) is a member of a family of collagenous C-type lectins that includes the serum mannose binding proteins and surfactant protein A . Recent studies have shown that rat SP-D (rSP-D) molecules are assembled as tetramers of trimeric subunits (12 mers) and that dodecamers can participate in higher orders of molecular assembly involving interactions of the amino-terminal peptide domains . In order to further study the assembly of SP-D in vitro, Chinese hamster ovary K1 cells were transfected with a full-length rat SP-D cDNA, and stable transfectants with high levels of SP-D production (approximately 6 x 10(6) dodecamers/cell/24 h) were obtained using a glutamine synthetase selection system . The secreted molecules (RrSP-D), which were purified by affinity chromatography on maltosyl-agarose, comigrated with rSP-D on SDS-polyacrylamide gel electrophoresis in the presence and absence of reduction, and coeluted with rSP-D dodecamers from 4% agarose . The major bacterial collagenase-resistant peptide showed a decreased mobility on reduction consistent with the formation of intrachain disulfide bonds . A 17-kDa pepsin-resistant fragment was isolated following overnight digestion with pepsin at 27 degrees C, confirming the formation of a triple helical domain comparable in size and thermal stability to that of natural SP-D . The expressed protein contained sialylated endoglycosidase F-sensitive carbohydrate; amino acid analysis of acid and alkaline hydrolysates demonstrated essentially normal levels of hydroxyproline, hydroxylysine, and hydroxylysine-glycosides . Electron microscopic studies showed a molecular structure indistinguishable from lung SP-D, with a similar small subpopulation of molecules showing higher orders of multimerization . Solid-phase neoglycoprotein binding assays gave the same saccharide inhibition profile as natural rat SP-D, and both proteins showed efficient saccharide-dependent agglutination of Escherichia coli . These studies demonstrate that a single genetically distinct chain type can account for the various and complex molecular assemblies of SP-D, and further verify the potential physiologic significance of the disulfide-bonded multimers and higher aggregates isolated from rat, bovine, and human lung lavage.

J Biol Chem, 1994 Jun 3, 269(22), 15795 - 802
High level expression in Escherichia coli and characterization of the EF-hand calcium-binding protein caltractin; Weber C et al.; Caltractin is a member of the calmodulin superfamily of Ca(2+)-binding proteins that was originally cloned at the DNA level from the unicellular green alga Chlamydomonas reinhardtii . Human and mouse homologs to algal caltractin have been recently characterized . In the studies reported here, recombinant Chlamydomonas caltractin was expressed at high levels in Escherichia coli and purified to homogeneity . The use of the ompT-host BL21 proved critical for obtaining high yields of homogeneous full-length protein . Growth and purification protocols were optimized to allow reproducible and efficient production of tens of milligrams of pure protein from 1-liter cultures . Caltractin has a distinct UV spectrum which is largely dominated by the fine structure due to the 9 Phe residues . Unlike other members of the same protein family, the UV and the CD spectra do not change upon addition of Ca2+ to the apoprotein . However, the 1H NMR spectrum shows distinct changes upon Ca2+ binding, which are indicative of structural and/or dynamic changes largely reminiscent of other members of the calmodulin superfamily . Ca2+ binding measurements demonstrated the binding of four Ca2+ ions to caltractin with two higher affinity (Kd = 1.2 x 10(-6) M) and two lower affinity (Kd = 1.6 x 10(-4) M) sites . Caltractin is highly stable in both the apo- and the Ca(2+)-loaded states . The unusual stability of apocaltractin makes this protein highly suited for structural studies by multidimensional NMR aimed at understanding the structural and dynamic consequences of Ca2+ binding, and the molecular basis of Ca2+ signal transduction.

J Biol Chem, 1994 Jun 3, 269(22), 15697 - 702
Purification and characterization of a novel organometallic receptor protein regulating the expression of the broad spectrum mercury-resistant operon of plasmid pDU1358; Yu H et al.; The narrow spectrum mercury-resistant (mer) operons of transposons Tn21 and Tn501 are inducible by inorganic mercury salts . The major regulatory gene merR is transcribed divergently from the other mer genes, which are cotranscribed . The MerR protein represses its own expression, as well as the expression of the other mer genes in the absence of the inducers . The synthesis of the polycistronic mer message is stimulated by MerR in the presence of the inducers . The MerRBS protein encoded by the broad spectrum mer operon of plasmid pDU1358 was characterized as a novel organomercurial receptor, distinguishing it from the narrow spectrum MerRNS proteins, described above . Several organomercurial compounds directly effected cellular activation of the mer operon transcription via the receptor protein MerRBS, but not by MerRNS . The merR gene from pDU1358 was cloned under the tac promoter, and the overexpressed MerRBS protein was soluble in buffer solutions containing 0.5 M NaCl at pH 7.5, but precipitated when NaCl concentration was reduced to 0.1 M (MerRBS concentrations at or above 0.1 mg/ml) . MerRBS was purified to near homogeneity by selective precipitation and solubilization by varying the salt concentration in buffer solutions, followed by Sephadex G-75 column chromatography . Both MerRBS and Tn21-encoded MerRNS bound with DNA fragments containing the pDU1358 mer operator sequence with comparable affinities . In vitro run-off transcription studies revealed that MerRBS activated mer operon expression in the presence of Hg2+ or phenylmercuric acetate . Phenylmercuric acetate did not induce mer operon expression when the MerRNS was used in the assay.

J Biol Chem, 1994 Jun 3, 269(22), 15583 - 7
Isolation of recombinant ADP-ribosylation factor 6, an approximately 20-kDa guanine nucleotide-binding protein, in an activated GTP-bound state; Welsh CF et al.; ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins, which, like other members of the ras superfamily, are activated by exchanging bound GDP for GTP and inactivated through hydrolysis of the gamma-phosphate of bound GTP to form GDP in a highly regulated cycle . ARF 6, a class III ARF, was expressed in Escherichia coli with its amino terminus fused to maltose-binding protein . Following release from maltose-binding protein, recombinant ARF 6 (rARF 6) exhibited maximal activity with or without GTP . Such constitutive activation was due to the predominance of ARF-GTP over ARF-GDP, as demonstrated by nucleotide analysis . rARF 6 expressed in E . coli without amino-terminal extension was bound primarily to GDP and exhibited typical GTP-dependent activity . After release from maltose-binding protein, rARF 6-GTP was stable; only a fraction of the nucleotide was removed using EDTA, whereas urea denaturation restored complete GTP dependence . {alpha-32P}GTP bound to rARF 6 was in part protected from hydrolysis by alkaline phosphatase and resulted in the formation of {alpha-32P}GTP, -GDP, and -GMP, whereas unbound nucleotide was completely hydrolyzed to guanosine . Thus, amino-terminal extension of rARF 6, by maltose-binding protein, promoted the formation of a constitutively activated GTP-bound species . By analysis of this species, we confirmed that rARF 6 lacks the intrinsic ability to hydrolyze bound GTP and speculate that maltose-binding protein may inhibit hydrolysis by extrinsic factors.

J Biol Chem, 1994 Jun 3, 269(22), 15577 - 82
Biochemical mechanism of HIV-I Vpr function . Specific interaction with a cellular protein; Zhao LJ et al.; vpr is an accessory gene of human immunodeficiency virus I (HIV-I) . Although unnecessary for viral replication in T cell lines, growing evidence suggests that it is essential for virus replication in monocytes/macrophages and for replication in vivo . We expressed HIV-I vpr in Escherichia coli and purified Vpr by affinity chromatography . In a coprecipitation assay, the purified Vpr interacted specifically with a cellular protein designated as Vpr-interacting protein, or RIP . Mutational analysis suggested that this interaction required a domain rich in leucine/isoleucine residues and highly conserved between HIV-I and SIVmac Vprs . During transient expression in mammalian cells, HIV-I Vpr was localized in the nucleus . However, mutational analysis failed to identify in Vpr a typical nuclear localization signal rich in basic amino acid residues . Instead, Vpr nuclear localization seemed to correlate with Vpr interaction with RIP . Mutations in the C-terminal 20-amino acid region containing a cryptic nuclear localization signal did not abolish Vpr nuclear localization or interaction with RIP, whereas point mutations in the leucine/isoleucine-rich domain abolished Vpr interaction with RIP and rendered Vpr unstable during transient expression . These results suggest that RIP may be involved in Vpr function.

J Biol Chem, 1994 Jun 3, 269(22), 15546 - 52
The effect of Met-->Leu mutations on calmodulin's ability to activate cyclic nucleotide phosphodiesterase; Zhang M et al.; Calmodulin (CaM) has two hydrophobic surface patches that are particularly rich in Met residues, and these are the major contact areas where CaM interacts with its target enzymes . The amino acid Leu has been introduced by site-directed mutagenesis to replace all the Met residues in CaM . All nine individual Met-->Leu mutants of CaM as well as some double and quadruple mutants were expressed in Escherichia coli . All mutants could be purified by calcium-dependent hydrophobic affinity chromatography, indicating that they still expose their hydrophobic surfaces upon binding calcium . Each single Met-->Leu mutation in the C-terminal domain of the protein had little effect on its ability to activate phosphodiesterase (PDE), while a quadruple mutant with four C-terminal Leu residues instead of Met has a significantly lower affinity for PDE . The M36L mutant is a poor activator compared with the other three N-terminal single Met-->Leu mutants, which have a slightly lower affinity for PDE than wild-type CaM . The introduction of a positively charged Arg for Met-145 resulted in an almost complete loss of CaM's ability to activate PDE . Nuclear magnetic resonance spectroscopy was used to show that most CaM mutants retain their overall three-dimensional structure . Thus, the altered activation properties appear to arise from differences in the flexibility and polarizability of the Met and Leu sidechains, rather than from structural perturbations.

J Biol Chem, 1994 Jun 3, 269(22), 15498 - 504
Mammalian topoisomerase I has base mismatch nicking activity; Yeh YC et al.; The all-type nicking enzyme (ATE) from human HeLa cells or calf thymus can nick DNA at the first phosphodiester bond 5' to all 8 possible mismatched bases . The strand disparity of this nicking is influenced by the neighboring nucleotide sequences . After nicking, the ATE covalently binds to the 3' end of the DNA product to form a cleavable complex, whose formation is insensitive to camptothecin, a specific inhibitor of eukaryotic topoisomerase I (Topo-I) . During the purification of ATE from calf thymus, a Mg(2+)-independent relaxation activity, characteristic of eukaryotic Topo-I, copurifies with the mismatch-nicking activity . The ATE from calf thymus may be a breakdown product of Topo-I . N-terminal amino acid analysis indicates that one of the polypeptides with ATE activity contains the C-terminal portion of Topo-I . Moreover, active human Topo-I, expressed as a fusion protein in Escherichia coli, is also capable of nicking all 8 base mispairs in the absence of Mg2+ . This mismatch-specific nicking activity may be a novel property of the mammalian Topo-I.

J Biol Chem, 1994 Jun 3, 269(22), 15427 - 30
Rab-GDI presents functional Rab9 to the intracellular transport machinery and contributes selectivity to Rab9 membrane recruitment; Dirac-Svejstrup AB et al.; Rab proteins occur in the cytosol bound to Rab-GDP dissociation inhibitor (GDI) . We demonstrate here that cytosolic complexes of Rab9 bound to GDI represent a functional pool of Rab9 protein that can be utilized for transport from late endosomes to the trans Golgi network in vitro . Immunodepletion of GDI and Rab proteins bound to GDI led to the loss of cytosol activity; readdition of pure Rab9-GDI complexes fully restored cytosol activity . Delipidated serum albumin could solubilize prenylated Rab9 protein, but unlike Rab9-GDI complexes, Rab9-serum albumin complexes led to indiscriminate membrane association of Rab9 protein . Rab9 delivered to membranes by serum albumin was functional, but GDI increased the efficiency of Rab9 utilization, presumably because it suppressed Rab9 protein mistargeting . Finally, GDI inhibited transport of proteins from late endosomes to the trans Golgi network, likely because of its capacity to inhibit the membrane recruitment of cytosolic Rab9 . These experiments show that GDI contributes to the selectivity of Rab9 membrane recruitment and presents functional Rab9 to the endosome-trans Golgi network transport machinery.

J Chromatogr B Biomed Appl, 1994 Jun 3, 656(1), 127 - 33
Production and simple purification of a protein encoded by part of the gag gene of HIV-1 in the Escherichia coli HB101F+ expression system inducible by lactose and isopropyl-beta-D-thiogalactopyranoside; Liska V et al.; The development of the Escherichia coli expression system, which was prepared by transferring the F' episome from strain 71/18 to a highly to a transformable F- strain HB101, is described . These new HB101 (F+) cells, which produced high levels of lac repressor, were capable of taking up lactose and grew under strict selection conditions . A relatively simple two-step purification of part of a protein (M(r) 27,000) encoded by the gag gene of HIV-1 in this expression system is described . The supernatant prepared by removal of cell debris was precipitated by 30% saturation of ammonium sulphate . The protein spectrum was characterized by gel electrophoresis, immunoblotting and ion-exchange titration curves . Optimum separation was achieved using a strong anion exchanger (Mono Q) at pH 8.0 . The purified protein did not cross-react with antibodies to E . coli.

J Chromatogr B Biomed Appl, 1994 Jun 3, 656(1), 123 - 6
Procedure for refolding and purification of recombinant proteins from Escherichia coli inclusion bodies using a strong anion exchanger; Suttnar J et al.; Using Escherichia coli system expressing papilloma virus HPV16 E7MS2 fusion protein as a model system, a novel procedure was applied to solubilize, purify and refold recombinant proteins from E . coli inclusion bodies . The necessity to reactivate proteins at low protein concentrations (owing to their tendency to aggregate at high concentrations) was overcome by solubilization of inclusion bodies in alkaline solution and immobilization of proteins on a strong and resistant anion exchanger . This procedure has an inherent advantage of combining refolding and purification procedures in one step . The solubilization of the fusion protein in an alkaline reagent with the use of an anion exchanger resulted in considerable purification of the recombinant protein at a fairly high concentration . The protein was soluble under mild conditions and reacted with antibodies against the "native" papilloma virus.

J Mol Biol, 1994 Jun 3, 239(2), 339 - 41
Crystallization and preliminary X-ray diffraction study of a bacterially produced T-cell antigen receptor V alpha domain; Fields BA et al.; A recombinant form of the variable domain of the alpha chain of a murine T-cell receptor specific for the N-terminal nonapeptide of myelin basin protein in association with the major histocompatibility complex class II I-Au molecule has been crystallized in a form suitable for X-ray diffraction analysis . This protein was secreted into the periplasmic space of Escherichia coli cells and affinity-purified using a nickel chelate adsorbent . The crystals are orthorhombic, space group P2(1)2(1)2, with unit cell dimensions a = 97.7 A, b = 79.6 A, c = 30.4 A and diffract to beyond 2.2 A resolution . The ability to crystallize a T-cell receptor domain produced in bacteria strongly suggests that the periplasmic space can provide a suitable environment for the correct in vivo folding of this class of antigen recognition molecules.

J Neurosci, 1994 Jun, 14(6), 3548 - 64
Spinal cord neuroblasts proliferate in response to basic fibroblast growth factor; Ray J et al.; Trophic factors may function as one of the epigenic signals responsible for the proliferation, growth, migration, and differentiation of neurons and glia during embryogenesis . The present study reports that basic fibroblast growth factor (bFGF) at high concentrations (10-100 ng/ml) is a mitogen for embryonic spinal cord cells that have already committed to a neuronal pathway and are expressing neuronal phenotypes (neuroblasts) . Neuroblasts proliferate with a doubling time of 2.5 d . To characterize the nature of cells proliferating in response to bFGF, we have established long-term cultures of neuroblasts that can be passaged, freeze thawed, and recultured . In cultures the proportion of astrocytes remained the same, indicating limited survival and proliferation of these cells in response to bFGF . These results indicate that bFGF has mitogenic effects preferably on neuroblasts . The morphological and biochemical characterizations of the neuronal populations present in the long-term neuroblast cultures are presented here . The presence of cholinergic and GABAergic neurons in the cultures was established by immunocytochemical analysis . The cultures contain a small number of motoneurons as judged by their immunostaining with ChAT, low-affinity NGF receptor (LNGFR), and large size . Among all other growth factors tested for their mitogenic effects on embryonic spinal cord cells in culture, only epidermal growth factor (EGF) showed such effects, but to a lesser degree . The proliferative nature of neuroblasts has made it possible to transduce the Escherichia coli beta-galactosidase (LacZ) gene stably into these cells in vitro using a retroviral vector . The transfected cells expressing the foreign gene can be passaged, freeze thawed, and recultured without the loss of transgenes . The ability to transduce foreign genes stably into these cells permits implantation of these cells in the spinal cord to study cellular and biochemical behaviors and gene expression in defined neuronal populations in in vivo environments.

J Bacteriol, 1994 Jun, 176(12), 3785 - 9
The RNA polymerase of Chlamydia trachomatis has a flexible sequence requirement at the -10 and -35 boxes of its promoters; Mathews SA et al.; Mutated variants of the predicted promoter of the countertranscript of the Chlamydia trachomatis plasmid were tested by in vitro transcription with chlamydial extract . A 3-bp deletion within the -10 region of the putative promoter caused the RNA polymerase to initiate transcription 3 bases downstream . Many single mutations in the -10 and -35 regions did not alter promoter function . However, some multiple mutations in both hexamers rendered the promoter inefficient or ineffective . Taken together, these results indicate that (i) the sequence requirement for chlamydial promoters differs from that for Escherichia coli and (ii) chlamydial RNA polymerase can tolerate considerably more variation at the -10 and -35 regions . These results are paradoxical considering the homology between C . trachomatis sigma 66 and E . coli sigma 70.

J Bacteriol, 1994 Jun, 176(12), 3775 - 84
The gene coding for polynucleotide phosphorylase in Photorhabdus sp . strain K122 is induced at low temperatures; Clarke DJ et al.; Photorhabdus sp . strain K122 was found to produce higher levels of the protein CAP87K when cultured at 9 degrees C than when cultured at 28 degrees C . NH2-terminal sequencing of this protein revealed homology with the NH2 terminus of Escherichia coli polynucleotide phosphorylase . A 4.5-kb DNA fragment from strain K122 was cloned and sequenced and found to have 75% identity to the E . coli rpsO-pnp operon coding for ribosomal protein S15 and polynucleotide phosphorylase, respectively . Predicted proteins encoded by this sequence were found to have 86% identity with ribosomal protein S15 and polynucleotide phosphorylase from E . coli, and the genes were called rpsO and pnp, respectively . Quantitation of rpsO and pnp mRNA transcripts from K122 revealed that there was a 2.4-fold increase in the level of pnp mRNA and a 1.9-fold decrease in the level of rpsO mRNA at 9 degrees C relative to 28 degrees C . Primer extension analysis revealed the positions of possible promoters controlling the expression of rpsO and pnp in K122, suggesting that the genes are expressed independently . The increase in the level of pnp mRNA at 9 degrees C was not due to any relative increase in its stability compared with that of the rpsO transcript . However, there was evidence to suggest that it may be a result of a cold-inducible promoter, P2, in the intergenic region between rpsO and pnp . Several features of P2 support the suggestion that it may be cold inducible.

J Bacteriol, 1994 Jun, 176(12), 3738 - 48
Growth phase variation of integration host factor level in Escherichia coli; Ditto MD et al.; We have measured the intracellular abundance of integration host factor (IHF), a site-specific, heterodimeric DNA-binding protein, in exponential- and stationary-phase cultures of Escherichia coli K-12 . Western immunoblot analysis showed that cultures that had been growing exponentially for several generations contained 0.5 to 1.0 ng of IHF subunits per microgram of total protein and that this increased to 5 to 6 ng/microgram in late-stationary-phase cultures . IHF is about one-third to one-half as abundant in exponentially growing cells as HU, a structurally related protein that binds DNA with little or no site specificity . Wild-type IHF is metabolically stable, but deletion mutations that eliminated one subunit reduced the abundance of the other when cells enter stationary phase . We attribute this reduction to the loss of stabilizing interactions between subunits . A mutation that inactivates IHF function but not subunit interaction increased IHF abundance, consistent with results of previous work showing that IHF synthesis is negatively autoregulated . We estimate that steady-state exponential-phase cultures contain about 8,500 to 17,000 IHF dimers per cell, a surprisingly large number for a site-specific DNA-binding protein with a limited number of specific sites . Nevertheless, small reductions in IHF abundance had significant effects on several IHF-dependent functions, suggesting that the wild-type exponential phase level is not in large excess of the minimum required for occupancy of physiologically important IHF-binding sites.

J Bacteriol, 1994 Jun, 176(12), 3661 - 72
RecOR suppression of recF mutant phenotypes in Escherichia coli K-12; Sandler SJ et al.; The recF, recO, and recR genes form the recFOR epistasis group for DNA repair . recF mutants are sensitive to UV irradiation and fail to properly induce the SOS response . Using plasmid derivatives that overexpress combinations of the recO+ and recR+ genes, we tested the hypothesis that high-level expression of recO+ and recR+ (recOR) in vivo will indirectly suppress the recF mutant phenotypes mentioned above . We found that overexpression of just recR+ from the plasmid will partially suppress both phenotypes . Expression of the chromosomal recO+ gene is essential for the recR+ suppression . Hence we call this RecOR suppression of recF mutant phenotypes . RecOR suppression of SOS induction is more efficient with recO+ expression from a plasmid than with recO+ expression from the chromosome . This is not true for RecOR suppression of UV sensitivity (the two are equal) . Comparison of RecOR suppression with the suppression caused by recA801 and recA803 shows that RecOR suppression of UV sensitivity is more effective than recA803 suppression and that RecOR suppression of UV sensitivity, like recA801 suppression, requires recJ+ . We present a model that explains the data and proposes a function for the recFOR epistasis group in the induction of the SOS response and recombinational DNA repair.

J Bacteriol, 1994 Jun, 176(12), 3638 - 45
The Escherichia coli proU promoter element and its contribution to osmotically signaled transcription activation; Mellies J et al.; The proU operon of Escherichia coli encodes a high-affinity glycine betaine transport system which is osmotically inducible and enables the organism to recover from the deleterious effects of hyperosmotic shock . Regulation occurs at the transcriptional level . KMnO4 footprinting showed that the preponderance of transcription initiated at a single primary promoter region and that proU transcription activation did not occur differentially at alternate promoters in response to various levels of salt shock . Mutational analysis confirmed the location of the primary promoter and identified an extended -10 region required for promoter activity . Specific nucleotides within the spacer, between position -10 and position -35, were important for maximal expression, but every mutant which retained transcriptional activity remained responsive to osmotic signals . A chromosomal 90-bp minimal promoter fragment fused to lacZ was not significantly osmotically inducible . However, transcription from this fragment was resistant to inhibition by salt shock . A mutation in osmZ, which encodes the DNA-binding protein H-NS, derepressed wild-type proU expression by sevenfold but did not alter expression from the minimal promoter . The current data support a model in which the role of the proU promoter is to function efficiently at high ionic strength while other cis-acting elements receive and respond to the osmotic signal.

J Bacteriol, 1994 Jun, 176(12), 3606 - 13
Comparative analysis of functional and structural features in the primase-dependent priming signals, G sites, from phages and plasmids; Tanaka K et al.; The primase-dependent priming signals, G sites, are directly recognized by the Escherichia coli primase (dnaG gene product) and conduct the synthesis of primer RNAs . In nucleotide sequence and secondary structure, there is no striking resemblance between the phage- and plasmid-derived G sites, except for the limited sequence homology near the start position of primer RNA synthesis . In this study, we analyzed the structure and function of a G site of plasmid R100, G site (R100), and discovered the necessity of the coexistence of two domains (domains I and III), which contains blocks A, B, and C, which are nucleotide sequences highly conserved among the plasmid-derived G sites . However, neither the internal region, domain II, between domains I and III nor the potential secondary structure proposed by Bahk et al . (J . D . Bahk, N . Kioka, H . Sakai, and T . Komano, Plasmid 20:266-270, 1988) is essential for single-stranded DNA initiation activity . Furthermore, chimeric G sites constructed between a G site of phage G4, G site(G4), and G site(R100) maintained significant single-stranded DNA initiation activities . These results strongly suggest that phage- and plasmid-derived G sites have functionally equivalent domains . The primase-dependent priming mechanisms of phage- and plasmid-derived G sites are discussed.

J Bacteriol, 1994 Jun, 176(12), 3559 - 67
Characterization of genetic determinants for R body synthesis and assembly in Caedibacter taeniospiralis 47 and 116; Heruth DP et al.; Caedibacter taeniospiralis, an obligate bacterial endosymbiont of Paramecium tetraurelia, confers a killing trait upon its host paramecium . Type 51 R bodies (refractile inclusion bodies) are synthesized by these endosymbionts and are required for expression of the killing trait . The nucleotide sequence of the genetic determinants for type 51 R body synthesis and assembly was determined for C . taeniospiralis 47 and 116 . Three independently transcribed genes (rebA, rebB, and rebC) were characterized . To date these are the only genes from C . taeniospiralis to be sequenced and characterized . DNA regulatory regions are recognized by Escherichia coli, and codon usage appears similar to that in E . coli . A fourth open reading frame with appropriate regulatory sequences was found within the reb locus, but no evidence was obtained to suggest that this putative gene is expressed in E . coli . The R body-encoding sequences from both strains are identical . Two-dimensional gel electrophoresis of deletion derivatives shows that two polymerization events are involved in R body assembly . One polymerization event requires only RebB and RebC; the other requires all three proteins . Expression of RebC is necessary for the posttranslational modification of RebA and RebB into species with three and two different molecular weights, respectively . In the presence of RebC, each species of RebB with a different molecular weight has six different isoelectric points.

J Bacteriol, 1994 Jun, 176(12), 3466 - 73
Isolation and characterization of the Methylophilus sp . strain DM11 gene encoding dichloromethane dehalogenase/glutathione S-transferase; Bader R et al.; The restricted facultative methylotroph Methylophilus sp . strain DM11 utilizes dichloromethane as the sole carbon and energy source . It differs from other dichloromethane-utilizing methylotrophs by faster growth on this substrate and by possession of a group B dichloromethane dehalogenase catalyzing dechlorination at a fivefold-higher rate than the group A enzymes of slow-growing strains . We isolated dcmA, the structural gene of the strain DM11 dichloromethane dehalogenase, to elucidate its relationship to the previously characterized dcmA gene of Methylobacterium sp . strain DM4, which encodes a group A enzyme . Nucleotide sequence determination of dcmA from strain DM11 predicts a protein of 267 amino acids, corresponding to a molecular mass of 31,197 Da . The 5' terminus of in vivo dcmA transcripts was determined by primer extension to be 70 bp upstream of the translation initiation codon . It was preceded by a putative promoter sequence with high resemblance to the Escherichia coli sigma 70 consensus promoter sequence . dcmA and 130 bp of its upstream sequence were brought under control of the tac promoter and expressed in E . coli to approximately 20% of the total cellular protein by induction with isopropylthiogalactopyranoside (IPTG) and growth at 25 degrees C . Expression at 37 degrees C led to massive formation of inclusion bodies . Comparison of the strain DM11 and strain DM4 dichloromethane dehalogenase sequences revealed 59% identity at the DNA level and 56% identity at the protein level, thus indicating an ancient divergence of the two enzymes . Both dehalogenases are more closely related to eukaryotic class theta glutathione S-transferases than to a number of bacterial glutathione S-transferases.

Arch Biochem Biophys, 1994 Jun, 311(2), 509 - 16
Initial kinetic and mechanistic characterization of Escherichia coli fumarase A; Flint DH; The protein encoded by the fumA gene in Escherichia coli is shown herein to be a highly efficient and specific catalyst of the fumarase reaction . In an investigation of 21 substrate analogs, this protein only had substantial activity as a hydro-lyase on fumarate, malate, acetylene dicarboxylate, fluorofumarate, and 2(S),3(S)-tartrate . The kcat and kcat/Km for the hydration of fumarate by this protein are 3100 s-1 and 5 x 10(6) mol-1 s-1, respectively . It is likely that one physiological role of this protein is a catalyst of the fumarase reaction; therefore, it is appropriate to name it fumarase A . Fumarase A specifically removes the 3-pro-R in the dehydration of (2S)-malate . The product of the action of fumarase A on acetylene dicarboxylate, fluorofumarate and 2(S),3(S)-tartrate is oxalacetate . The nitronate form of 2-hydroxy-3-nitro-propionate is a potent inhibitor of fumarase A, implying that the enzyme forms an intermediate with an anion at C-3 . No kinetic isotope effect was found with (2S,3R)-3-{2H}malate . The effects of pH on the kcat and kcat/Km for fumarate as a substrate show that the pKas of the groups involved in catalysis differ markedly from porcine fumarase . The possible roles of the proteins encoded by the three fumarase genes in E . coli are briefly discussed.

Arch Biochem Biophys, 1994 Jun, 311(2), 487 - 95
A comparison of the enzymatic and physicochemical properties of human glutathione transferase M4-4 and three other human Mu class enzymes; Comstock KE et al.; The multigene family of cytosolic glutathione S-transferases (GSTs) consists of four classes (Alpha, Mu, Pi, and Theta), all involved in the detoxication of reactive electrophiles . The human Mu class GSTs consist of at least four expressed isozyme subunits, GST M1, GST M2, GST M3, and GST M4, which have 70-90% amino acid sequence identity . The gene and cDNA sequences for GST M4 have been determined recently (K . E . Comstock, K . J . Johnson, D . Rifenbery, and W . D . Henner, J . Biol . Chem . (1993) 268, 16958-16965) . Cloning of GST M4 cDNA into an Escherichia coli expression system permitted the production of the corresponding protein . The enzyme was purified and shown to have a relatively low specific activity with the standard GST substrate 1-chloro-2,4-dinitrobenzene (1.4 +/- 0.2 mumol min-1 mg-1 protein), but an activity equivalent to other Mu class enzymes with other tested substrates . The protein forms functional dimers composed of subunits with a M(r) of approximately 26,400 . A detailed comparison of the activity with various substrates and inhibitors was performed between GST M4-4 and other human Mu class GSTs, GST M1a-1a, GST M2-2, and GST M3-3, produced in bacterial expression systems . Despite the high level of amino acid sequence identity, the enzymatic properties of these enzymes were quite different . Comparisons with the crystallographic structure of a homologous rat GST, GST 3-3, indicate that a number of the nonconserved amino acid residues can be assigned to the putative active site of GST M4-4 . This suggests that diversification in the evolution of these genes has occurred primarily in the substrate binding regions to cope with an increasing variety of foreign compounds.

Arch Biochem Biophys, 1994 Jun, 311(2), 418 - 24
Interaction of the catalytic and the membrane subunits of an oxyanion-translocating ATPase; Dey S et al.; Resistance to arsenical and antimonial compounds in Escherichia coli is due to active extrusion of these compounds from cells expressing the ars operon . The arsenical pump is an ion-translocating ATPase which consists of two polypeptide components, the ArsA and ArsB proteins . The ArsB protein, the inner membrane component of the pump, has been shown to function as the membrane anchor for the catalytic subunit, the ArsA protein . The properties and nature of interaction between these two components of the pump were investigated using an in vitro binding assay . Purified ArsA protein bound to the membrane in a saturable manner . In the absence of arsenite or antimonite an apparent positive cooperativity in the binding of the ArsA protein to membrane vesicles containing the ArsB protein was observed . In the presence of arsenite or antimonite binding became hyperbolic, with a 10-fold decrease in the concentration of ArsA protein required for half-maximal binding, without any change in the stoichiometry of the complex . Addition of ATP had little affect on membrane binding of the ArsA ATPase subunit . In the presence or absence of the anionic substrates binding was maximal in a pH range 7.5-8.5.

Arch Biochem Biophys, 1994 Jun, 311(2), 369 - 77
Prochiral sulfoxidation as a probe for multiple forms of the microsomal flavin-containing monooxygenase: studies with rabbit FMO1, FMO2, FMO3, and FMO5 expressed in Escherichia coli; Rettie AE et al.; Multiple forms of the microsomal flavin-containing monooxygenase (FMO) exist in rabbit tissues . In order to better understand the catalytic properties of these isoforms, we have expressed rabbit FMO1, FMO2, FMO3, and FMO5 in Escherichia coli and examined their kinetic parameters and prochiral selectivities for the sulfoxidation of methyl-, ethyl-, n-propyl-, and n-butyl-substituted p-tolyl sulfides . FMO1 and FMO2 exhibited high affinities for these substrates (Km < 10 microM), in contrast to the low-affinity FMO3 form for which Km values ranged between 100 and 280 microM . FMO5 did not form quantifiable levels of sulfoxide metabolites at the concentrations used . The individual stereochemical metabolite profiles generated by FMO1, FMO2, and FMO3 were unique and served to distinguish among these three cDNA-expressed isoforms . To investigate the relationship between the kinetic parameters for the cDNA-expressed enzymes and the native microsomal enzymes, we examined the kinetics and stereoselectivity of metabolism of methyl p-tolyl sulfide by detergent-solubilized rabbit liver microsomes . We analyzed these data with respect to FMO1 and FMO3, the two predominant hepatic isoforms . Sulfoxidation of methyl p-tolyl sulfide by FMO1 and FMO3 solubilized from E . coli microsomes proceeded with apparent Kms of 18 and 270 microM, respectively . FMO1 was essentially stereospecific for formation of (R)-methyl p-tolyl sulfoxide, whereas FMO3 generated this metabolite with little prochiral selectivity . Sulfoxidation of methyl p-tolyl sulfide by detergent-solubilized rabbit liver microsomes was best described by a two-enzyme model, with apparent Km values of 11 and 340 microM . The enantiomeric purity of the (R)-methyl p-tolyl sulfoxide metabolite, generated by detergent-solubilized rabbit liver microsomes, decreased progressively with increasing substrate concentration, from a high of 96% enantiomeric excess at a substrate concentration of 5 microM to a low of 63% enantiomeric excess at a substrate concentration of 2 mM . The kinetic and stereochemical properties of the high-affinity and low-affinity components of detergent-solubilized rabbit liver microsomes were similar to those exhibited by cDNA-expressed FMO1 and FMO3, respectively . Therefore, methyl p-tolyl sulfide, used at the appropriate substrate concentrations, is useful for discriminating between FMO1- and FMO3-mediated catalysis in rabbit liver microsomal preparations.

Clin Pediatr (Phila), 1994 Jun, 33(6), 325 - 8
The effect of nutritional additives on anti-infective factors in human milk; Quan R et al.; It has become a common practice to supplement human milk with a variety of additives to improve the nutritive content of the feeding for the premature infant . Twenty-two freshly frozen human milk samples were measured for lysozyme activity, total IgA, and specific IgA to Escherichia coli serotypes 01, 04, and 06 . One mL aliquots were mixed with the following: 1 mL of Similac, Similac Special Care, Enfamil, Enfamil Premature Formula, and sterile water; 33 mL of Poly-Vi-Sol, 33 mg of Moducal, and 38 mg of breast-milk fortifier, and then reanalyzed . Significant decreases (41% to 74%) in lysozyme activity were seen with the addition of all formulas; breast-milk fortifier reduced activity by 19%, while no differences were seen with Moducal, sterile water, or Poly-Vi-Sol . No differences were seen in total IgA content, but some decreases were seen in specific IgA to E . coli serotypes 04 and 06 . E . coli growth was determined after 3 1/2 hours of incubation at 37 degrees C after mixing . All cow-milk formulas enhanced E . coli growth; soy formulas and other additives preserved inhibition of bacterial growth . Nutritional additives can impair anti-infective properties of human milk, and such interplay should be considered in the decision on the feeding regimen of premature infants.

Can J Surg, 1994 Jun, 37(3), 240 - 2
Lost gallstones during laparoscopic cholecystectomy: are they really benign?
Leslie KA, Rankin RN, Duff JH.
The long-term effect of stones spilled into the peritoneal cavity during laparoscopic cholecystectomy is unknown . The course of a 58-year-old man who had recurrent right subphrenic abscesses and a right empyema secondary to spilled gallstones during laparoscopic cholecystectomy is described . The authors outline techniques for minimizing the spillage of stones during laparoscopic cholecystectomy: the application of hemoclips, endoloops and sutures, and placement of the necrotic, friable gallbladder in an endoscopic bag immediately upon completion of the dissection, before extraction of the gallbladder . They conclude that spillage of stones during laparoscopic cholecystectomy may lead to serious infection and should be recorded in the operative notes so that stones may be suspected when a patient presents with abdominal infection after laparoscopic cholecystectomy.

Br J Cancer, 1994 Jun, 69(6), 1038 - 42
Anti-tumour activity of low-toxicity lipopolysaccharide of Bordetella pertussis; Ohnishi M et al.; A lipopolysaccharide (BP-LPS) isolated from killed Bordetella pertussis (Tohama strain) was determined to have low toxicity based on the mortality and decrease in body weight of BP-LPS-injected mice . BP-LPS, administered intradermally or intraperitoneally, clearly inhibited the growth of an MM46 murine mammary carcinoma . When compared with a toxic Escherichia coli-derived LPS, BP-LPS displayed excellent anti-tumour activity against MH134 hepatoma and Meth A fibrosarcoma . As part of a combined chemotherapy/immunotherapy regimen, BP-LPS also seemed to prolong the lifespan of mice inoculated with Lewis lung carcinoma . BP-LPS thus appears to have valuable characteristics as an anti-tumour agent.

Mol Cell Biol, 1994 Jun, 14(6), 4311 - 23
Mutations that alter ligand-induced switches and dimerization activities in the retinoid X receptor; Zhang XK et al.; The retinoid X receptor (RXR) heterodimerizes with a variety of nuclear receptors . In addition, RXR forms homodimers in the presence of its ligand, 9-cis-retinoic acid . From deletion and point mutation analysis we present evidence that a short region (amino acids 413 to 443) in the carboxy terminus of RXR alpha is critical for both homo- and heterodimeric interactions as well as for diverse functional activities . In addition, we present evidence that homo- and heterodimer functions can be separated . The deletion of 19 amino acids from the C-terminal end of RXR dramatically reduced the transcriptional activation function of RXR . The removal of 10 additional amino acids resulted in a receptor (delta RXR3) that had completely lost its ligand-dependent homodimer function but retained its heterodimer activities . Heterodimer function was abolished by the deletion of an additional 20 amino acids . Single amino acid substitutions in the region generated receptors with altered RXR homodimer DNA binding, while simultaneous mutation of three Leu residues (Leu-418, -419 and -422) completely abolished both RXR homodimer and heterodimer DNA binding activities . Mutation of Leu-430 to Phe (L430-F) resulted in a receptor that bound to DNA strongly as homodimers in a ligand-independent manner, while another single amino acid exchange (L422-Q) led to a mutant that behaved in a manner exactly opposite to that of wild-type RXR in that the homodimerization of the mutant occurred in the absence of ligand and was inhibited by 9-cis-retinoic acid . In transfection assays, both L422-Q and L430-F failed to act as homodimers but retained their heterodimer function . Our studies demonstrate the unique properties of the RXR ligand binding domain and point to specific residues that mediate homo- and heterodimer activities and ligand-induced conformational switches.

Mol Cell Biol, 1994 Jun, 14(6), 4173 - 82
Homologous recombination of monkey alpha-satellite repeats in an in vitro simian virus 40 replication system: possible association of recombination with DNA replication; Kawasaki I et al.; To study homologous recombination between repeated sequences in an in vitro simian virus 40 (SV40) replication system, we constructed a series of substrate DNAs that contain two identical fragments of monkey alpha-satellite repeats . Together with the SV40-pBR322 composite vector encoding Apr and Kmr, the DNAs also contain the Escherichia coli galactokinase gene (galK) positioned between two alpha-satellite fragments . The alpha-satellite sequence used consists of multiple units of tandem 172-bp sequences which differ by microheterogeneity . The substrate DNAs were incubated in an in vitro SV40 DNA replication system and used to transform the E . coli galK strain DH10B after digestion with DpnI . The number of E . coli galK Apr Kmr colonies which contain recombinant DNAs were determined, and their structures were analyzed . Products of equal and unequal crossovers between identical 172-bp sequences and between similar but not identical (homeologous) 172-bp sequences, respectively, were detected, although those of the equal crossover were predominant among all of the galK mutant recombinants . Similar products were also observed in the in vivo experiments with COS1 cells . The in vitro experiments showed that these recombinations were dependent on the presence of both the SV40 origin of DNA replication and SV40 large T antigen . Most of the recombinant DNAs were generated from newly synthesized DpnI-resistant DNAs . These results suggest that the homologous recombination observed in this SV40 system is associated with DNA replication and is suppressed by mismatches in heteroduplexes formed between similar but not identical sequences.

Mol Cell Biol, 1994 Jun, 14(6), 3791 - 9
The yeast GAL11 protein is involved in regulation of the structure and the position effect of telomeres; Suzuki Y et al.; GAL11 is an auxiliary transcription factor that functions either positively or negatively, depending on the structure of the target promoters and the combination of DNA-bound activators . In this report, we demonstrate that a gal11 delta mutation caused a decrease in the length of the telomere C1-3A tract, a derepression of URA3 when it is placed next to telomere, and an increase in accessibility of the telomeric region to dam methylase, indicating that GAL11 is involved in the regulation of the structure and the position effect of telomeres . The defective position effect in a gal11 delta strain was suppressed by overproduction of SIR3, whereas overexpression of GAL11 failed to restore the telomere position effect in a sir3 delta strain . Hyperproduced GAL11 could partially suppress the defect in silencing at HMR in a sir1 delta mutant but not that in a sir3 delta mutant, suggesting that GAL11 can replace SIR1 function partly in the silencing of HMR . Overproduced SIR3 also could restore silencing at HMR in sir1 delta cells . In contrast, SIR1 in a multicopy plasmid relieved the telomere position effect, especially in a gal11 delta mutant . Since chromatin structure is thought to play a major role in the silencing at both the HM loci and telomeres, GAL11 is likely to participate in the regional regulation of transcription by the HM loci and telomeres, GAL11 is likely to participate in the regional regulation of transcription by modulating the chromatin structure.

J Exp Med, 1994 Jun 1, 179(6), 1809 - 21
Isolation, cDNA cloning, and overexpression of a 33-kD cell surface glycoprotein that binds to the globular "heads" of C1q; Ghebrehiwet B et al.; This work describes the functional characterization, cDNA cloning, and expression of a novel cell surface protein . This protein designated gC1q-R, was first isolated from Raji cells and was found to bind to the globular "heads" of C1q molecules, at physiological ionic strength, and also to inhibit complement-mediated lysis of sheep erythrocytes by human serum . The NH2-terminal amino acid sequence of the first 24 residues of the C1q-binding protein was determined and this information allowed the synthesis of two degenerate polymerase chain reaction primers for use in the preparation of a probe in the screening of a B cell cDNA library . The cDNA isolated, using this probe, was found to encode a pre-pro protein of 282 residues . The NH2 terminus of the protein isolated from Raji cells started at residue 74 of the predicted pre-pro sequence . The cDNA sequence shows that the purified protein has three potential N-glycosylation residues and is a highly charged, acidic molecule . Hence, its binding to C1q may be primarily but not exclusively due to ionic interactions . The "mature" protein, corresponding to amino acid residues 74-282 of the predicted pre-pro sequence, was overexpressed in Escherichia coli and was purified to homogeneity . This recombinant protein was also able to inhibit the complement-mediated lysis of sheep erythrocytes by human serum and was shown to be a tetramer by gel filtration in nondissociating conditions . Northern blot and RT-PCR studies showed that the C1q-binding protein is expressed at high levels in Raji and Daudi cell lines, at moderate levels in U937, Molt-4, and HepG2 cell lines, and at a very low level in the HL60 cell line . However, it is not expressed in the K562 cell line . Comparison of gC1q-R NH2-terminal sequence with that of the receptor for the collagen-like domain of C1q (cC1q-R) showed no similarity . Furthermore, antibodies to gC1q-R or an 18-amino acid residue-long NH2-terminal synthetic gC1q-R peptide did not cross-react with antibodies to cC1q-R . Anti-gC1q-R immunoblotted a 33-kD Raji cell membrane protein, whereas anti cC1q-R recognized a molecule of approximately 60 kD . The NH2-terminal sequence of gC1g-R appears to be displayed extracellularly since anti-gC1g-R peptide reacted with surface molecules on lymphocytes, polymorphonuclear leukocytes, and platelets, as assessed by flow cytometric and confocal laser scanning microscopic analyses.(ABSTRACT TRUNCATED AT 400 WORDS)

J Bacteriol, 1994 Jun, 176(11), 3438 - 41
Analysis of nonmethylated GATC sites in the Escherichia coli chromosome and identification of sites that are differentially methylated in response to environmental stimuli; Hale WB et al.; Seven GATC sites that are nonmethylated in logarithmic growth phase cells using glycerol as a carbon source were isolated from the Escherichia coli chromosome . Three of these GATC sites are located upstream of the operons gut, mtl, and ppiA, whereas DNA sequences adjacent to three other nonmethylated GATC sites are not homologous to previously identified genes . The seventh nonmethylated GATC site is located downstream of uspA . The protection of this site from DNA methylation requires leucine-responsive regulatory protein and is leucine responsive . The carbon source and the growth phase influenced the protection of the GATC site 5' of the ppiA gene . The other five sites were protected under all the environmental conditions examined.

J Bacteriol, 1994 Jun, 176(11), 3397 - 9
Thirty-three amino acids of the mature moiety of an unprocessed maltose-binding protein are sufficient for export in Escherichia coli; Barkocy-Gallagher GA et al.; Maltose-binding protein (MBP) is translocated across the cytoplasmic membrane of Escherichia coli; successful export depends on information in both the signal peptide and the mature moiety of the protein . To determine the shortest portion of the mature region that would maintain detectable entry of MBP into the export pathway, we took advantage of the properties of an MBP species with proline substituted in the +1 position relative to the cleavage site (MBP27-P) . This protein efficiently crosses the cytoplasmic membrane but is not processed and acts as a competitive inhibitor of signal peptidase I (leader peptidase) . Export of MBP27-P is measured by the inhibition of processing of other proteins, such as ribose-binding protein (RBP) . A series of truncated derivatives of MBP27-P were tested for the ability to inhibit processing of RBP . An MBP27-P species with only 33 amino acids of the mature moiety inhibited processing of RBP, indicating that this truncated polypeptide was probably exported and interacted with signal peptidase I.

J Bacteriol, 1994 Jun, 176(11), 3389 - 92
Primary structures of the wild-type and mutant alleles encoding the phosphatidylglycerophosphate synthase of Escherichia coli; Usui M et al.; The nucleotide sequence of the Escherichia coli pgsA gene, encoding phosphatidylglycerophosphate synthase, is revised to code for an enzyme of 182 amino acid residues, instead of the 216 of a previous work (A . S . Gopalakrishnan, Y.-C . Chen, M . Temkin, and W . Dowhan, J . Biol . Chem . 261:1329-1338, 1986) . The revised structure now explains the properties of the enzyme . Three pgsA mutants of different phenotypes were also analyzed: pgsA3, pgsA36, and pgsA10 have single-base replacements in codons 60 (Thr-->Pro), 1 (ATG-->ATA), and 92 (Thr-->Ile), respectively.

J Bacteriol, 1994 Jun, 176(11), 3383 - 5
The DNA damage-inducible dinD gene of Escherichia coli is equivalent to orfY upstream of pyrE; Lundegaard C et al.; The DNA damage-inducible gene dinD, originally identified by Kenyon and Walker (C . J . Kenyon and G . C . Walker, Proc . Natl . Acad . Sci . USA 77:2819-2823, 1980) by selection of the dinD::MudI (Ap lac) fusion, is shown here to be equivalent to the open reading frame orfY near pyrE . The evidence for identity between the two genes includes results from P1 transduction, Southern hybridization, and cloning and sequencing of the dinD fusion . No data were obtained that reveal any hints about the function of the dinD gene.

J Bacteriol, 1994 Jun, 176(11), 3278 - 85
Roles of LysP and CadC in mediating the lysine requirement for acid induction of the Escherichia coli cad operon; Neely MN et al.; Expression of the Escherichia coli cadBA operon, encoding functions required for the conversion of lysine to cadaverine and for cadaverine excretion, requires at least two extracellular signals: low pH and a high concentration of lysine . To better understand the nature of the lysine-dependent signal, mutants were isolated which expressed a cadA-lacZ transcription fusion in the absence of lysine while retaining pH regulation . The responsible mutation in one of these isolates (EP310) was in cadC, a gene encoding a function necessary for transcriptional activation of cadBA . This mutation (cadC310) is in a part of the gene encoding the periplasmic domain of CadC and results in an Arg-to-Cys change at position 265, indicating that this part of the protein is involved in responding to the presence of lysine . Three other mutants had mutations mapping in or near lysP (cadR), a gene encoding a lysine transport protein that has previously been shown to regulate cadA expression . One of these mutations is an insertion in the lysP coding region . Thus, in the absence of exogenous lysine, LysP is a negative regulator of cadBA expression . Negative regulation by LysP was further demonstrated by showing that lysP expression from a high-copy-number plasmid rendered cadA-lacZ uninducible . Expression of cadA-lacZ in a strain carrying the cadC310 allele, however, was not affected by the plasmid-expressed lysP . Cadaverine was shown to inhibit expression of the cadA-lacZ fusion in cadC+ cells but not in a cadC310 background.

J Bacteriol, 1994 Jun, 176(11), 3224 - 30
DNA alkylation repair limits spontaneous base substitution mutations in Escherichia coli; Mackay WJ et al.; The Escherichia coli Ada and Ogt DNA methyltransferases (MTases) are known to transfer simple alkyl groups from O6-alkylguanine and O4-alkylthymine, directly restoring these alkylated DNA lesions to guanine and thymine . In addition to being exquisitely sensitive to the mutagenic effects of methylating agents, E . coli ada ogt null mutants display a higher spontaneous mutation rate than the wild type . Here, we determined which base substitution mutations are elevated in the MTase-deficient cells by monitoring the reversion of six mutated lacZ alleles that revert via each of the six possible base substitution mutations . During exponential growth, the spontaneous rate of G:C to A:T transitions and G:C to C:G transversions was elevated about fourfold in ada ogt double mutant versus wild-type E . coli . Furthermore, compared with the wild type, stationary populations of the MTase-deficient E . coli (under lactose selection) displayed increased G:C to A:T and A:T to G:C transitions (10- and 3-fold, respectively) and increased G:C to C:G, A:T to C:G, and A:T to T:A transversions (10-, 2.5-, and 1.7-fold, respectively) . ada and ogt single mutants did not suffer elevated spontaneous mutation rates for any base substitution event, and the cloned ada and ogt genes each restored wild-type spontaneous mutation rates to the ada ogt MTase-deficient strains . We infer that both the Ada MTase and the Ogt MTase can repair the endogenously produced DNA lesions responsible for each of the five base substitution events that are elevated in MTase-deficient cells . Simple methylating and ethylating agents induced G:C to A:T and A:T to G:C transitions in these strains but did not significantly induce G:C to C:G, A:T to C:G, and A:T to T:A transversions . We deduce that S-adenosylmethionine (known to e a weak methylating agent) is not the only metabolite responsible for endogenous DNA alkylation and that at least some of the endogenous metabolites that cause O-alkyl DNA damage in E . coli are not simple methylating or ethylating agents.

J Bacteriol, 1994 Jun, 176(11), 3210 - 7
Conjugation-independent, site-specific recombination at the oriT of the IncW plasmid R388 mediated by TrwC; Llosa M et al.; Plasmids containing a direct repeat of plasmid R388 oriT are capable of site-specific recombination, which results in deletion of the intervening DNA . This reaction occurs in the presence, but not in the absence, of the region of R388 implicated in DNA processing during conjugation . This region contains three genes, trwA, trwB, and trwC . By using mutants of each of the three genes, it was demonstrated that only trwC is required for the oriT-specific recombination . Further analysis showed that the N-terminal 272 amino acids of the protein are sufficient to catalyze recombination . TrwC is also capable of promoting intermolecular recombination between two plasmids containing oriT, suggesting that double-strand breaks in both plasmid DNAs are involved in the process . Additionally, intramolecular recombination between R388 oriT and R46 oriT did not occur in the presence of both nickases . This suggests that the half-reactions at each oriT are not productive if they occur separately; therefore, an interaction between the recombination complexes formed at each recombining site is required . This is the first report in which a nicking-closing enzyme involved in conjugal DNA transfer promotes oriT-specific recombination of double-stranded DNA in the absence of conjugation.

J Bacteriol, 1994 Jun, 176(11), 3188 - 95
Plasmid pSC101 harbors a recombination site, psi, which is able to resolve plasmid multimers and to substitute for the analogous chromosomal Escherichia coli site dif; Cornet F et al.; Plasmid pSC101 harbors a 28-bp sequence which is homologous to dif, the target site of the XerC/XerD-dependent recombination system in Escherichia coli . Using a technique which allows very sensitive detection of plasmid loss, we show that recombination at this site, termed psi for pSC101 stabilized inheritance, causes a moderate increase in pSC101 stability . The role of the psi sequence in site-specific recombination has been explored in two other contexts . It was cloned in a derivative of plasmid p15A and inserted into the chromosome in place of dif . In the first situation, psi activity requires accessory sequences and results in multimer resolution; in the second situation, it suppresses the effects of the dif deletion and can promote intermolecular exchanges . Thus, psi is a site whose recombinational activity depends on the context, the first in the cer/dif family known to exhibit such flexibility.

Gastroenterology, 1994 Jun, 106(6), 1638 - 44
Adenovirus-mediated transfer of human lipase complementary DNA to the gallbladder; Maeda H et al.; BACKGROUND/AIMS: Despite many improvements in current therapy, exocrine pancreatic insufficiency remains a significant problem in cystic fibrosis . To establish a new therapy for exocrine pancreatic insufficiency, the feasibility of transferring the human pancreatic lipase complementary DNA to the gallbladder as a possible target using a recombinant adenovirus vector was evaluated . METHODS: The adenovirus vector AdCMV.Lip was constructed using the cytomegalovirus immediate early promoter to drive the human pancreatic lipase complementary DNA . In vitro infection of the human gallbladder epithelial cell line HS-181 and ex vivo infection of the sheep gallbladder with AdRSV . beta-gal (an adenovirus vector containing the Escherichia coli lacZ {(beta-galactosidase} gene) or AdCMV.Lip were evaluated . RESULTS: The supernatant from AdCMV.Lip-infected HS-181 showed the secretion of active lipase for at least 2 weeks in vitro . The epithelium of gallbladder infected with AdRSV.beta-gal ex vivo showed the expression of the beta-galactosidase . The fluid from the gallbladder lumen infected with AdCMV.Lip showed the increased lipase activity . CONCLUSIONS: These observations show that an adenovirus vector can transfer a human pancreatic enzyme in vitro and ex vivo, suggesting the feasibility of in vivo gene therapy for exocrine pancreatic insufficiency in cystic fibrosis.

Endocrinology, 1994 Jun, 134(6), 2581 - 8
Tumor necrosis factor increases the rate of lipolysis in primary cultures of adipocytes without altering levels of hormone-sensitive lipase; Green A et al.; To investigate the effects of cytokines on adipocyte lipolysis, a macrophage cell line (RAW 264.7) was treated with Escherichia coli lipopolysaccharide (1 microgram/ml) for 18 h to induce cytokine release . Conditioned medium (5%, vol/vol) from these cells was added to rat epididymal adipocytes isolated and incubated under sterile conditions . After incubation, the adipocytes were washed, and the rate of lipolysis (glycerol release) was determined after a further 1-h incubation . The conditioned medium caused an approximately 2.7-fold increase in lipolysis, detectable after 6-12 h, maximal by 24 h, and reversible by 48 h after washing the cells . The effect of conditioned medium was reversed by a neutralizing antibody to mouse tumor necrosis factor-alpha (TNF alpha), and the direct addition of recombinant human TNF alpha (0.1-50 ng/ml) reproduced the effect, with a half-maximally effective concentration of approximately 3 ng/ml . The effect of TNF on the expression of hormone-sensitive lipase (HSL; the rate-limiting enzyme for lipolysis) was investigated by Western immunoblots using an antibody raised to a bacterially expressed 96-amino acid portion of the HSL enzyme . TNF treatment did not alter the concentration of immunoreactive HSL . From these data we conclude that 1) macrophages release a cytokine(s) in response to lipopolysaccharide that stimulates lipolysis in freshly isolated adipocytes; 2) TNF alpha can account for most, or perhaps all, of this effect; 3) TNF alpha increases the rate of lipolysis by a mechanism that does not involve increased expression of HSL . Based on the time-dependent aspects of TNF alpha stimulation and the lack of change in immunoreactive HSL, the findings suggest a TNF-induced posttranslational modification of the enzyme.

Blood, 1994 Jun 1, 83(11), 3113 - 9
Direct interaction of proteoglycan macrophage colony-stimulating factor and basic fibroblast growth factor; Suzu S et al.; The proteoglycan form of macrophage colony-stimulating factor (PG-M-CSF), but not M-CSF with a molecular weight of 85 kD (85-kD M-CSF), bound to immobilized basic fibroblast growth factor (bFGF), and, conversely, bFGF bound to immobilized PG-M-CSF, but not to the 85-kD M-CSF . PG-M-CSF has an additional amino acid sequence at its carboxyl terminus (part of a precursor sequence that is removed in 85-kD M-CSF by proteolytic processing) and it has one or two chondroitin sulfate glycosaminoglycan chains at the carboxyl terminus . Enzymatic removal of the chondroitin sulfate chain from PG-M-CSF had no effect on the binding between PG-M-CSF and bFGF . Ligand blotting analysis with radioiodinated bFGF showed that bFGF specifically bound to the polypeptide that corresponded to the carboxyl terminus of PG-M-CSF and was produced in Escherichia coli transfected with its gene . The exogeneous addition of heparan sulfate, which has strong affinity for bFGF, efficiently inhibited the binding between PG-M-CSF and bFGF . These results show that PG-M-CSF binds bFGF through its carboxyl terminal peptide and that the binding sites for PG-M-CSF and heparan sulfate on bFGF are located close together . PG-M-CSF also significantly reduced the mitogenic action of bFGF on Balb/c 3T3 mouse fibroblastic cells . Therefore, we conclude that PG-M-CSF not only binds bFGF, but also neutralizes the activity of the growth factor.

J Virol, 1994 Jun, 68(6), 3982 - 9
Interactions among the major and minor coat proteins of polyomavirus; Barouch DH et al.; Murine polyomavirus contains two related minor coat proteins, VP2 and VP3, in addition to the major coat protein, VP1 . The sequence of VP3 is identical to that of the carboxy-terminal two-thirds of VP2 . VP2 may serve a role in uncoating of the virus, and both minor coat proteins may be important for viral assembly . In this study, we show that VP3 and a series of deletion mutants of VP3 can be expressed in Escherichia coli as fusion proteins to glutathione S-transferase and partially solubilized with a mild detergent . Using an in vitro binding assay, we demonstrate that a 42-amino-acid fragment near the carboxy terminus of VP3 (residues 140 to 181) is sufficient for binding to purified VP1 pentamers . This binding interaction is rapid, saturable, and specific for the common carboxy terminus of VP2 and VP3 . The VP1-VP3 complex can be coimmunoprecipitated with an antibody specific to VP1, and a purified VP3 fragment can selectively extract VP1 from a crude cell lysate . The stoichiometry of the binding reaction suggests that each VP1 pentamer in the virus binds either one VP2 or one VP3, with the VP1-VP2/3 complex stabilized by hydrophobic interactions . These results, taken together with studies from other laboratories on the expression of polyomavirus capsid proteins in mouse and insect cells (S . E . Delos, L . Montross, R . B . Moreland, and R . L . Garcea, Virology, 194:393-398, 1993; J . Forstova, N . Krauzewicz, S . Wallace, A . J . Street, S . M . Dilworth, S . Beard, and B . E . Griffin, J . Virol . 67:1405-1413, 1993), support the idea that a VP1-VP2/3 complex forms in the cytoplasm and, after translocation into the nucleus, acts as the unit for viral assembly.

J Virol, 1994 Jun, 68(6), 3971 - 81
Central nervous system-derived cells express a kappa B-binding activity that enhances human immunodeficiency virus type 1 transcription in vitro and facilitates TAR-independent transactivation by Tat; Taylor JP et al.; The Tat protein of human immunodeficiency virus type 1 (HIV-1) is a potent activator of long terminal repeat-directed transcription . While in most cell types, activation requires interaction of Tat with the unusual transcription element TAR, astrocytic glial cells support TAR-independent transactivation of HIV-1 transcription by Tat . This alternative pathway of Tat activation is mediated by the viral enhancer, a kappa B domain capable of binding the prototypical form of the transcription factor nuclear factor kappa B (NF-kappa B) present in many cell types, including T lymphocytes . Tat transactivation mediated by the kappa B domain is sufficient to allow replication of TAR-deleted mutant HIV-1 in astrocytes . The present study demonstrates the existence of kappa B-specific binding factors present in human glial astrocytes that differ from prototypical NF-kappa B . The novel astrocyte-derived kappa B-binding activity is retained on an HIV-1 Tat affinity column, while prototypical NF-kappa B from Jurkat T cells is not . In vitro transcription studies demonstrate that astrocyte-derived kappa B-binding factors activate transcription of the HIV-1 long terminal repeat and that this activation is dependent on the kappa B domain . Moreover, TAR-independent transactivation of HIV-1 transcription is reproduced in vitro in an astrocyte factor-dependent manner which correlates with kappa B-binding activity . The importance of the central nervous system-enriched kappa B transcription factor in the regulation of HIV-1 expression is discussed.

J Virol, 1994 Jun, 68(6), 3943 - 54
UL69 of human cytomegalovirus, an open reading frame with homology to ICP27 of herpes simplex virus, encodes a transactivator of gene expression; Winkler M et al.; The UL69 open reading frame of human cytomegalovirus (HCMV) is homologous to the immediate-early protein ICP27 of herpes simplex virus, an essential viral regulatory protein involved in the transition from early to late gene expression . Genes with homology to ICP27 have been detected in all subclasses of herpesviruses so far . While the respective proteins in alpha- and gammaherpesviruses have been defined as trans-regulatory molecules, nothing is known about these genes in betaherpesviruses . This study was therefore undertaken in order to investigate expression from the UL69 gene locus of HCMV . Northern (RNA) blot experiments revealed a complex pattern of transcripts that changed during the time course of the HCMV replicative cycle: two transcripts of 2.7 and 3.5 kb that were regulated differentially could be detected as early as 7 h after infection . However, these transcripts could not be detected in the presence of cycloheximide . Additional, larger transcripts were present exclusively at late times after infection . To analyze protein expression from the UL69 gene region, the UL69 open reading frame was expressed as a histidine-tagged protein in Escherichia coli . A specific antiserum was generated and used to detect the UL69 protein in HCMV-infected cells which revealed its localization within the intranuclear inclusions that are characteristic for HCMV infection . In cotransfection experiments, an HCMV true late promoter could not be activated by UL69, whereas an early promoter and several heterologous promoters were stimulated about 10-fold . Complementation studies showed that the UL69 protein cannot substitute for ICP27 in the context of the HSV infection, suggesting functional differences between these two proteins . In summary, these experiments define a novel regulatory protein encoded by HCMV that is expressed as an early-late gene and appears to exert a broad stimulatory effect on gene expression.

J Virol, 1994 Jun, 68(6), 3809 - 20
Localization of a 34-amino-acid segment implicated in dimerization of the herpes simplex virus type 1 ICP4 polypeptide by a dimerization trap; Gallinari P et al.; The herpes simplex virus type 1 immediate-early protein ICP4 plays an essential role in the regulation of the expression of all viral genes . It is the major trans activator of early and late genes and also has a negative regulatory effect on immediate-early gene transcription . ICP4 is a sequence-specific DNA-binding protein and has always been purified in a dimeric form . The part of the protein that consists of the entire highly conserved region 2 and of the distal portion of region 1 retains the ability to specifically associate with DNA and to form homodimers in solution . In an attempt to map the dimerization domain of ICP4, we used a dimerization trap assay, in which we screened deletion fragments of this 217-amino-acid stretch for sequences that could confer dimerization properties on a heterologous cellular transcription factor (LFB1), which binds to its cognate DNA sequence only as a dimer . The analysis of these chimeric proteins expressed in vitro ultimately identified a stretch of 34 amino acids (343 to 376) that could still confer DNA-binding activity on the LFB1 reporter protein and thus apparently contained the ICP4 dimerization motif . Consistent with this result, a truncated ICP4 protein containing amino acids 343 to 490, in spite of the complete loss of DNA-binding activity, appeared to retain the capacity to form a heterodimer with a longer ICP4 peptide after coexpression in an in vitro translation system.

J Virol, 1994 Jun, 68(6), 3742 - 52
Proteolytic activity of human cytomegalovirus UL80 protease cleavage site mutants; Jones TR et al.; The human cytomegalovirus UL80 open reading frame encodes protease and assembly protein from its N- and C-terminal regions, respectively . We reported previously that a 30-kDa protease is derived by autoproteolytic processing of a polyprotein which is the translation product of the entire UL80 open reading frame (E . Z . Baum, G . A . Bebernitz, J . D . Hulmes, V . P . Muzithras, T . R . Jones, and Y . Gluzman, J . Virol . 67:497-506, 1993) . Three autoproteolytic cleavage sites within the UL80 polyprotein were characterized; site 143 is within the protease domain and inactivates the protease . In this article, we report (i) expression analyses of UL80 in infected cells, including the processing kinetics of the UL80 polyprotein; (ii) the existence of an additional cleavage site (site 209) within the protease domain of the UL80 polyprotein; and (iii) the effect of mutagenesis at each of the cleavage sites upon proteolytic activity and steady-state levels of the UL80 processing products . During the course of infection, UL80 polyprotein processing begins at cleavage site 643 and follows at sites 256 and 143 . Cleavage at site 643 and/or 256 within the polyprotein is not a prerequisite for efficient protease activity, since all three proteases (85-, 80-, and 30-kDa proteins) were equally active in cleaving the assembly protein precursor to its mature form . Inhibition of cleavage at site 143 resulted in a three- to sixfold increase in the steady-state level of the 30-kDa protease, supporting the hypothesis that cleavage at this site may represent a mechanism by which cytomegalovirus regulates the level of active protease.

J Virol, 1994 Jun, 68(6), 3558 - 69
In vitro activities of purified visna virus integrase; Katzman M et al.; Although integration generally is considered a critical step in the retrovirus life cycle, it has been reported that visna virus, which causes degenerative neurologic disease in sheep, can productively infect sheep choroid plexus cells without detectable integration . To ascertain whether the integrase (IN) of visna virus is an inherently defective enzyme and to create tools for further study of integration of the phylogenetically related human immunodeficiency virus type 1 (HIV-1), we purified visna virus IN by using a bacterial expression system and applied various in vitro oligonucleotide-based assays to studying this protein . We found that visna virus IN demonstrates the full repertoire of in vitro functions characteristic of retroviral integrases . In particular, visna virus IN exhibits site-specific endonuclease activity following the invariant CA found two nucleotides from the 3' ends of viral DNA (processing activity), joins processed oligonucleotides to various sites on other oligonucleotides (strand transfer or integration activity), and reverses the integration reaction by resolving a complex that mimics one end of viral DNA integrated into host DNA (disintegration activity) . In addition, although it has been reported that purified HIV-1 IN cannot specifically nick visna virus DNA ends, purified visna virus IN does specifically process and integrate HIV-1 DNA ends.

J Immunol, 1994 Jun 1, 152(11), 5368 - 74
Efficient tumor cell lysis mediated by a bispecific single chain antibody expressed in Escherichia coli; Gruber M et al.; Recent advances in the expression of Abs in Escherichia coli have raised the possibility that virtually any specificity can be obtained by either cloning Ab genes from characterized hybridomas or by de novo selection using Ab gene libraries . Bispecific Abs have been more difficult to engineer because of problems inherent in the proper folding and association of VH and VL domains . In this report, a model system for expressing and testing the activity of a single chain bispecific Ab was used . The Ab contained the VH and VL genes from the anti-TCR Ab 1B2 joined by a 25 amino acid residue linker to the VH and VL genes from the anti-fluorescein Ab 4420 . The 57-kDa single chain bispecific Ab (scFv2) was purified in a single step by affinity chromatography through a fluorescein column at a yield of 1 mg/L of bacterial culture . Despite the presence of 1B2 V regions at the NH2-terminus and a 10-residue c-myc peptide at the COOH-terminus, the refolded protein had an affinity for fluorescein that was nearly identical with the monospecific single chain Ab . The scFv2 also bound the TCR of the mouse CTL clone 2C and redirected the lysis of human tumor cells that had fluorescein covalently linked to their surface . Lysis was mediated at scFv2 concentrations that were 100-fold lower than the concentrations of Ab that inhibited normal recognition by CTL 2C . These results show that single chain bispecific Abs can mediate CTL lysis of target cells without the immunosuppressive side effects associated with the use of anti-TCR Abs.

Infect Immun, 1994 Jun, 62(6), 2508 - 14
Recombinant Mycobacterium bovis BCG secreting functional interleukin-2 enhances gamma interferon production by splenocytes; O'Donnell MA et al.; Mycobacterium bovis BCG was genetically engineered to express and secrete mouse interleukin-2 (IL-2) and rat IL-2 . Genes encoding IL-2 were inserted into an Escherichia coli-BCG shuttle plasmid under the control of the BCG HSP60 promoter . To facilitate study of proteins produced in this system, the IL-2 gene product was expressed (i) alone, (ii) with the mycobacterial alpha-antigen secretion signal sequence at the amino terminus, (iii) with an influenza virus hemagglutinin epitope tag at the amino terminus, and (iv) with both the secretion signal sequence and the epitope tag . When expressed with the alpha-antigen signal sequence, biologically active IL-2 was secreted into the extracellular medium . Western blot (immunoblot) analysis of the intracellular IL-2 and extracellular IL-2 revealed that the secretion signal was appropriately cleaved from the recombinant lymphokine upon secretion . To assess the ability of recombinant BCG to stimulate cytokine production in a splenocyte population, mouse splenocytes were cultured together with wild-type or IL-2-producing BCG . IL-2-secreting BCG clones stimulated substantial increases in gamma interferon production, which could be reproduced by the addition of exogenous IL-2 to BCG . Levels of IL-6, IL-10, tumor necrosis factor alpha, and granulocyte-macrophage colony-stimulating factor were not significantly changed, while IL-4 and IL-5 remained undetectable (less than 50 pg/ml) . The enhanced production of gamma interferon in response to IL-2-secreting BCG was strain independent . Recombinant BCG expressing mammalian cytokines provides a novel means to deliver cytokines and may augment the immunostimulatory properties of BCG in immunization and cancer therapy.

Infect Immun, 1994 Jun, 62(6), 2295 - 301
Effect of alterations of basic amino acid residues of Escherichia coli heat-stable enterotoxin II on enterotoxicity; Fujii Y et al.; Escherichia coli heat-stable enterotoxin II (STII) is composed of 48 amino acid residues . Among these, one histidine, two arginine, and six lysine residues are basic . Isoelectric focusing showed that the isoelectric point of STII is 9.7, indicating that the side chains of some of these basic amino acid residues project outside the molecule . To understand the role that these basic amino acid residues play in toxicity, STII was chemically modified with ethoxyformic anhydride, maleic anhydride, and phenylglyoxal, which alter the side chains of basic amino acid residues in proteins . Maleic anhydride, which modifies the epsilon amino group, caused a significant loss of enterotoxic activity, but the other two modifiers did not . This indicated that lysine residues play an important role in the expression of the enterotoxic activity of STII and that the contribution of the other basic amino acid residues to the toxicity is relatively low . To confirm this hypothesis, we substituted these nine basic amino acid residues by oligonucleotide-directed site-specific mutagenesis and examined the enterotoxicity of these purified mutant STIIs . The enterotoxic activity was reduced when the lysine residues at positions 18, 22, 23, and 46 were substituted . In particular, the substitution at positions 22 and 23 induced a remarkable reduction . These results demonstrate that the lysine residues at positions 22 and 23 are very important in the expression of the enterotoxic activity of STII.

Cancer Res, 1994 Jun 1, 54(11), 2943 - 51
Yeast topoisomerase II mutants resistant to anti-topoisomerase agents: identification and characterization of new yeast topoisomerase II mutants selected for resistance to etoposide; Liu YX et al.; We describe a system that allows us to easily isolate and characterize mutants in yeast topoisomerase II that are resistant to antitumor agents that target this enzyme . The system uses yeast strains that are sensitive to those agents and that carry temperature-sensitive top2 mutations . The temperature-sensitive mutation allows the isolation of recessive drug-resistant mutations . The mutagenized TOP2 gene we have used is under the control of the yeast DED1 promoter; this overexpression of TOP2 is designed to avoid isolating mutants that are drug resistant solely because the mutated topoisomerase II has low enzymatic activity . We describe three mutants that we isolated using this system . Two of the three mutants show resistance to etoposide and amsacrine, while the third mutant is partially resistant to etoposide and fluoroquinolones but not to amsacrine . DNA sequence changes have been identified in all of these mutant TOP2 genes . The mutant with partial resistance to etoposide and fluoroquinolones has an amino acid change at position 738 of TOP2, which is three amino acids from the site homologous to Ser83 of E . coli gyrA, an amino acid which had previously been shown to be an important target for resistance to quinolones in bacteria . One of the alleles that confers resistance to both etoposide and amsacrine, top2-103, has changes in amino acid 824 and amino acid 1186 of TOP2 . Reconstruction of the mutations by oligonucleotide-directed mutagenesis demonstrates that the change at amino acid 824 is responsible for the drug resistance of this allele.

Proteins, 1994 Jun, 19(2), 150 - 7
Crystallization and characterization of colicin E1 channel-forming polypeptides; Elkins PA et al.; Crystals of the channel-forming domain of colicin E1 from E . coli were grown by vapor diffusion at pH 6.4 and higher pH values . Cleavage of the colicin molecule with trypsin or thermolysin produced two of the pore-forming polypeptides used in these experiments . The third polypeptide was purified from a constructed plasmid that overexpresses only the C-terminal domain of colicin E1 . Polypeptide crystals are tetragonal with space group I4, have one monomer in the asymmetric unit, and diffract to 2.2-2.4 A . Unit cell parameters for the tryptic and thermolytic polypeptides are a = 102.9 A and c = 35.6 A . Crystals of the overexpressed polypeptide have unit cell parameters of a = 87.2 A and c = 59.1 A . The crystals were characterized by precession photography, and native data sets of each channel-forming fragment were collected on a Siemens-Nicolet area detector . The crystallization and characterization of these polypeptides are the first steps in the structure determination of the channel-forming domain of colicin E1.

Cell Growth Differ, 1994 Jun, 5(6), 563 - 73
The transcription activation domains of v-Myc and VP16 interact with common factors required for cellular transformation and proliferation; Min S et al.; The amino terminus of the avian myelocytomatosis virus MC29 v-Myc oncoprotein contains sequences that are essential for cellular transformation (S . Farina, et al . J . Virol., 66: 2698-2708, 1992; S . Min and E . J . Taparowsky . Oncogene, 7:1531-1540, 1992) and for the ability to activate gene transcription (S . Min and E . J . Taparowsky . Oncogene, 7:1531-1540, 1992) . To investigate the molecular interactions that mediate these v-Myc-associated activities, we performed competition assays in which various regions of the v-Myc amino terminal transcription activation domain (TAD) were examined for their ability to inhibit transcription activation by v-Myc, VP16, and the myogenic regulatory factor MyoD . Overexpression of these transcriptional activators also was used to investigate whether Myc-interacting proteins were required for cellular transformation and cell proliferation events . Our results demonstrate that at least two distinct cellular activities interact with the v-Myc TAD and that it is the synergism between these activities that is required for v-Myc to function fully as a transcriptional activator . In addition, v-Myc activators squelch VP16- and MyoD-dependent transcription activation, suggesting that the v-Myc TAD interacts with a component of the general transcription machinery . In support of this observation, we found that overexpression of the v-Myc TAD inhibits ras-mediated cellular transformation as well as cell proliferation, underscoring the critical role these amino terminal Myc-interacting factors play in regulating the physiology of both normal and transformed cells.

J Nihon Univ Sch Dent, 1994 Jun, 36(2), 139 - 44
The inhibitory effect of endotoxins on growth of human cell lines; Yoshinuma N et al.; A study was conducted to examine the effect of endotoxin present in periodontal pockets on the proliferation and attachment of human cell lines on the culture plates (Ca9-22 and gingival fibroblasts) . The endotoxin was collected from periodontal pockets of anterior teeth in patients with periodontal disease by subgingival irrigation with sterilized distilled water . The solutions obtained were then subjected to hot phenol-water extraction . The collected endotoxin from periodontal pocket and four other kinds of endotoxin obtained commercially as positive controls were added to cell cultures and the numbers of viable cells on the culture plates were counted . Among the commercially available endotoxins used in this study, only 500 micrograms/ml of endotoxin derived from Escherichia coli 0111:B4 significantly decreased the number of attachment cells of Ca9-22 and gingival fibroblasts on the culture plates . Endotoxin from periodontal pockets at 5 micrograms/ml also significantly decreased the numbers of attachment cells of both cell lines.

Curr Genet, 1994 Jun, 25(6), 567 - 70
Transformation of Trichoderma reesei based on hygromycin B resistance using homologous expression signals; Mach RL et al.; Trichoderma reesei was transformed to hygromycin B resistance using a novel vector, which contains the E . coli hygromycin B phosphotransferase gene (hph) fused between promoter and terminator elements of the homologous Trichoderma pki1 (coding for pyruvate kinase) and cbh2 (coding for cellobiohydrolase II) genes, respectively . Transformation frequencies of over 1,800--2,500 transformants/micrograms DNA were obtained, which is a 15--20-fold increase over that with pAN7-1, which contains hph between A . nidulans expression signals . Mitotically-stable transformants contained the hph gene and the regulatory sequences of the pki1 promoter and the cbh2 terminator integrated into the genome . Evidence for preferentially ectopic integration is given.

Curr Genet, 1994 Jun, 25(6), 524 - 30
Cloning and characterization of two 3-phosphoglycerate kinase genes of Rhizopus niveus and heterologous gene expression using their promoters; Takaya N et al.; Two 3-phosphoglycerate kinase genes (pgk1 and pgk2) were cloned from Rhizopus niveus . It was deduced that both pgk genes have two introns . They have open reading frames of 1,355 bp and 1,356 bp, and code for proteins of 417 and 416 amino acids, respectively . The first introns of both genes are located at similar positions as those of pgk genes from other fungi based on the deduced amino-acid sequences of PGK proteins . The position of their second introns was similar to that of the seventh intron of the human pgk gene . The deduced amino-acid sequences of PGK proteins show high identity (64.8-72.2%) to those of PGKs of other filamentous fungi . When the promoters of each of the pgk genes were fused to the E . coli beta-glucuronidase (GUS) gene and introduced into R . niveus, significant GUS activities were detected in the cell lysates of the transformants, suggesting that GUS protein was expressed under the control of both pgk gene promoters in R . niveus . GUS activity was induced by glucose but not by glycerol, indicating that expression of R . niveus pgk genes was regulated by the carbon source.

Microbiology, 1994 Jun, 140 ( Pt 6), 1319 - 26
In vivo complementation and site-specific mutagenesis of the tellurite resistance determinant kilAtelAB from IncP alpha plasmid RK2Ter; Turner RJ et al.; The IncP alpha plasmid RK2 carries a cryptic tellurite resistance (Ter) determinant . This determinant from RK2Ter has been previously cloned into a pUC8 plasmid (pDT1558) . The Ter determinant identified as the kilA locus comprises an operon of three genes: kilA, telA and telB {also referred to as klaA, klaB and klaC on RK2(Tes)} . Each of the genes was subcloned into the expression vector pJF118EH behind an inducible tac-promotor using PCR . The PCR primers were used to engineer an efficient ribosome-binding site and adjacent sequence to improve protein expression . Expression plasmids were modified by inclusion of different resistance markers for selection during complementation . The tellurite-resistance phenotype was studied with the overexpressing plasmids . The study provides further evidence that all three genes within the kilAtelAB operon are required for cells to show resistance to potassium tellurite . Additionally, site-directed mutagenesis was carried out on the two cysteine residues in TelB . Changing either Cys125 or Cys132 to a Ser or Ala residue decreased the resistance mediated by the operon . These mutants demonstrate the requirement of cysteine residues within TelB for expression of tellurite resistance.

Jpn J Genet, 1994 Jun, 69(3), 269 - 85
Identification of the region that determines the specificity of binding of the transposases encoded by Tn3 and gamma delta to the terminal inverted repeat sequences; Maekawa T et al.; To analyze the region that determines the specificity of binding of the Tn3 transposase to the terminal inverted repeat sequences (IR), we first determined the nucleotide sequence of a Tn3-family transposon, gamma delta, which is supposed to encode a transposase similar to that of Tn3 . gamma delta was 5981 bp in length and contained three coding frames: Two were the genes, tnpA and tnpR, encoding transposase (1002 amino acids) and resolvase/repressor (183 amino acids), respectively, and the third, named tnpX, encoding a protein (698 amino acids) of unknown function but containing two NTP-binding motifs . Utilizing the tnpA sequence, we then constructed a series of Tn3-gamma delta hybrid genes encoding chimeric proteins in the N-terminal segments of the transposases (amino acid position 1 to 242 of Tn3 or 1' to 243' of gamma delta), which has been previously shown to be responsible for specific binding of transposase to IR sequences in Tn3 . Examination of their DNA-binding activities revealed that the subsegment of the N-terminus from amino acid position 1 to 109 determines the specificity of binding to the IR sequences . The third coding frame found in gamma delta, tnpX, is located downstream of tnpR and is expressed from the tnpR promoter in the absence of the tnpR gene product, resolvase/repressor, to produce a protein that inhibits the growth of the host cells . Possible roles of this protein are discussed.

Bioessays, 1994 Jun, 16(6), 419 - 24
A good eye for arthropod evolution; Osorio D et al.; Insect and crustacean lineages diverged over 500 Myr ago, and there are continuing uncertainties about whether they evolved from a common arthropod ancestor or, alternatively, they evolved independently from annelid worms . Despite the diversity of their limbs and lifestyles, the nervous systems of insects and crustaceans share many common features both in development and in function . Cellular and molecular embryology techniques reveal good evidence for homologies in the developing segmental ganglia . In the visual system, this seemingly common programme of insect and crustacean CNS development culminates in common adult neural function . Comparisons of the cellular anatomy and physiology of animals as diverse as flies and crayfishes indicate that the neural circuits in the lamina of their optic lobe have been inherited largely unchanged from a common ancestor with good compound eyes.

Anal Biochem, 1994 Jun, 219(2), 297 - 304
Optimization of an HP Scanjet for quantification of protein electrophoresis gels; Kendrick NC et al.; An inexpensive desktop scanner, the Hewlett-Packard Scanjet IIp (HP), has been optimized for analysis of protein electrophoresis gels by comparison with a calibrated laser densitometer (Laser) . Images from both densitometers were transferred to a personal computer and analyzed with QGEL software . Without correction the HP response was often in poor agreement with the Laser . However, when the HP response to Coomassie blue stained gels and x-ray films was linearized using a HP software option called Emphasis, the HP results agreed with results from the Laser . For 2D gels scanned with appropriate Emphasis applied, spot integrated density values were a constant multiple of 1.8 +/- 0.3 times the corresponding Laser value for x-ray films (CV = 17%) and 2.1 +/- 0.5 for Coomassie blue stained gels (CV = 24%) . The highest error was observed for density extremes . For proteins quantified relative to standards using sodium dodecyl sulfate-slab gel electrophoresis, the HP values were within 15% of the Laser values . Data is shown concerning linearity and reproducibility of response, optical density range (about 0 to 1.8 OD units), variability of the imaging field, and resolution of the HP.

Anal Biochem, 1994 Jun, 219(2), 195 - 200
Analysis of uracil in DNA by gas chromatography-mass spectrometry; Blount BC et al.; A sensitive method using gas chromatography and negative chemical ionization mass spectrometry for the detection of uracil in DNA is described . Uracil DNA glycosylase is used to specifically cleave uracil from DNA . Once removed, uracil is derivatized with 3,5-bis(trifluoromethyl)benzyl bromide and the sample components are separated with gas chromatography . Negative chemical ionization mass spectrometry is used to quantitatively detect as little as 1 pg of uracil per 100 micrograms DNA . This assay is 10 times more sensitive than previously described quantitative methods for the detection of uracil in DNA . We apply this method as part of a project to determine why folic acid deficiency causes DNA breaks . We demonstrate that inhibition of folic acid metabolism induces an accumulation of uracil in DNA.

J Neurosci Res, 1994 Jun 1, 38(2), 127 - 33
Preparation and affinity purification of a novel, biologically active, CNTF fusion protein; Rabinovsky ED et al.; A biologically active fusion protein comprising a short hydrophilic leader peptide fused to the N-terminus of rat CNTF was generated using commercially available materials . The coding region for rat CNTF was sub-cloned into the pFLAG-1 vector and transfected into the JM 109 strain of E . coli . The transfected cells expressed high levels of the fusion protein (FLAG-CNTF) following induction by isopropyl beta-D-thiogalactoside (IPTG) . FLAG-CNTF is expressed as insoluble material that was resolubilized by extraction with guanidine hydrochloride and purified by immuno-affinity chromatography . Analysis of the purified material by reverse phase HPLC and Western blot analysis indicated that FLAG-CNTF was composed of two closely related species that are greater than 99% pure after affinity chromatography . The purified FLAG-CNTF migrated as a 27 KD doublet on SDS polyacrylamide gel electrophoresis . Both bands of the doublet were shown to contain the FLAG peptide and CNTF by Western blot analysis, and amino acid sequence analysis demonstrated a single amino acid sequence corresponding to FLAG peptide and the N-terminus of CNTF . The purified fusion protein was tested for biological activity using the IMR-32 human neuroblastoma cell line . Treatment of IMR-32 cells with FLAG-CNTF increased the level of choline acetyltransferase (ChAT) in these cells 2-3-fold over that of control cells in a dose dependent manner . A direct comparison of the effects of FLAG-CNTF and recombinant human CNTF on IMR-32 ChAT activity showed that both factors exhibited similar potencies.(ABSTRACT TRUNCATED AT 250 WORDS)

J Pediatr Surg, 1994 Jun, 29(6), 805 - 7
The effect of endotoxin on neonatal erythrocyte intracellular calcium concentration; Todd JC 3rd et al.; Erythrocyte membrane deformability is dependent on the maintenance of "normal" intracellular calcium (Ca) levels . Red cell flexibility is known to be altered in the septic neonate . In turn, this adversely affects viscosity and compromises flow in the microcirculation . It has been suggested that this may play a role in the mesenteric hypoperfusion associated with necrotizing enterocolitis . This study was designed to determine the effect of endotoxin on erythrocyte Ca homeostasis in the neonate . Paired specimens were obtained from the umbilical cord of 10 healthy neonates . The samples were incubated with either buffered saline (control) or 2 micrograms/mL of Escherichia coli endotoxin (LPS) . Erythrocytes were then isolated, washed, and loaded with the fluorescent Ca chelator, FURA-2 . The free cytosolic Ca concentration was determined by spectroscopic analysis of the ratio of fluorescent intensities at 340 nm and 380 nm . Results were obtained every 1.8 seconds for 1 minute, and the mean value was used for analysis . In 10 additional neonates, the white blood cells were removed before incubation in saline and LPS, and the erythrocytes were evaluated as described above . Differences were analyzed statistically by the paired t test . In the presence of white blood cells, endotoxin resulted in a significant increase in free cytosolic Ca concentration (LPS, 42.602 +/- 5.166 nmol; control, 31.661 +/- 4.002 nmol; P < .02) . However, no significant difference were detected when cells were incubated in the absence of white blood cells (LPS, 32.374 +/- 2.479 nmol; control, 34.021 +/- 2.549 nmol) . Endotoxin induces a significant increase in neonatal free cytosolic Ca concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

J Clin Microbiol, 1994 Jun, 32(6), 1457 - 63
Enzyme-linked immunosorbent assay for detection of immunoglobulin G antibodies to Escherichia coli Vero cytotoxin 1; Karmali MA et al.; The frequency of Vero cytotoxin 1 (VT1)-neutralizing antibody (NAb) in serum specimens from 790 age-stratified (0 to 70 years) control individuals from Toronto was 61 of 790 (7.7%), with a peak of 19% in the 20- to 30-year-old age group and a second peak of 16.7% in the 60- to 70-year-old age group . A total of 568 serum specimens, including 538 from the 790 Toronto control subjects, 21 from patients from three outbreaks of VT-producing Escherichia coli (VTEC) infection, and 9 known VT1-NAb-positive serum specimens from patients with hemolytic-uremic syndrome (HUS), were then tested for the presence of anti-VT1 immunoglobulin G (IgG) by an enzyme-linked immunosorbent assay (ELISA) . The mean ELISA values of 522 VT1-NAb-negative serum specimens and 46 VT1-NAb-positive serum specimens were 0.09 +/- 0.06 (range, 0 to 0.56) and 0.78 +/- 0.66 (range, 0.16 to 2.91), respectively (P < 0.001; Student's t test) . With a breakpoint of 0.21 (mean ELISA value of the VT1-NAb-negative sera + 2 standard deviations), the sensitivity, specificity, positive predictive value, and negative predictive value of the VT1 IgG ELISA compared with those of the VT1-NAb assay were, respectively, 95.7, 98.7, 86.3, and 99.6% . There were nine discrepant serum specimens, of which seven were anti-VT1 IgG positive and VT1-NAb negative and two were anti-VT1 IgG negative and VT1-NAb positive . The ELISA was also used for testing 238 control serum specimens from The Netherlands, Japan, and India and acute- and convalescent-phase serum specimens from 42 Toronto patients with HUS . The frequencies of anti-VT1 IgG (with VT1-NAb frequencies in parantheses) in control sera from the Netherlands, Japan, and India were 6% (3%), 1.1% (0%), and 12% (10%), respectively, with no age clustering . The frequencies of anti-VT1 IgG seropositivity in HUS patients were 5 of 14 (35.7%) in patients with unknown toxin exposure, 2 of 22 (9.1%) in individuals with known exposure to VT1 plus VT2 or VT1 alone, and 0 of 6 (0%) in patients exposed to only VT2 . Development of serum anti-VT1 IgG response appears to be the exception rather than the rule in sporadic HUS patients infected with VTEC expressing VT1 . However, in two family outbreaks associated with VTEC strains expressing VT1 alone and VT1 plus VT2, respectively, the presence of anti-VT1 IgG in virtually all exposed individuals who remained symptom free suggests that the presence of antibody was associated with protection.

Biophys J, 1994 Jun, 66(6), 2111 - 26
13C NMR and fluorescence analysis of tryptophan dynamics in wild-type and two single-Trp variants of Escherichia coli thioredoxin; Kemple MD et al.; The rotational motion of tryptophan side chains in oxidized and reduced wild-type (WT) Escherichia coli thioredoxin and in two single-tryptophan variants of E . coli thioredoxin was studied in solution in the temperature range 20-50 degrees C from 13C-NMR relaxation rate measurements at 75.4 and 125.7 MHz and at 20 degrees C from steady-state and time-resolved trp fluorescence anisotropy measurements . Tryptophan enriched with 13C at the delta 1 and epsilon 3 sites of the indole ring was incorporated into WT thioredoxin and into two single-trp mutants, W31F and W28F, in which trp-28 or trp-31 of WT thioredoxin was replaced, respectively, with phenylalanine . The NMR relaxation data were interpreted using the Lipari and Szabo "model-free" approach (G . Lipari and A . Szabo . 1982 . J . Amer . Chem . Soc . 104:4546-4559) with trp steady-state anisotropy data included for the variants at 20 degrees C . Values for the correlation time for the overall rotational motion (tau m) from NMR of oxidized and reduced WT thioredoxin at 35 degrees C agree well with those given by Stone et al . (Stone, M . J., K . Chandrasekhar, A . Holmgren, P . E . Wright, and H . J . Dyson . 1993 . Biochemistry . 32:426-435) from 15N NMR relaxation rates, and the dependence of tau m on viscosity and temperature was in accord with the Stokes-Einstein relationship . Order parameters (S2) near 1 were obtained for the trp side chains in the WT proteins even at 50 degrees C . A slight increase in the amplitude of motion (decrease in S2) of trp-31, which is near the protein surface, but not of trp-28, which is partially buried in the protein matrix, was observed in reduced relative to oxidized WT thioredoxin . For trp-28 in W31F, order parameters near 1 (S2 > or = 0.8) at 20 degrees C were found, whereas trp-31 in W28F yielded the smallest order parameters (S2 approximately 0.6) of any of the cases . Analysis of time-resolved anisotropy decays in W28F and W31F yielded S2 values in good agreement with NMR, but gave tau m values about 60% smaller . Generally, values of tau e, the effective correlation time for the internal motion, were < or = 60 ps from NMR, whereas somewhat longer times were obtained from fluorescence . The ability of NMR and fluorescence techniques to detect subnanosecond motions in proteins reliably is examined.

Biophys J, 1994 Jun, 66(6), 1959 - 68
Reorganization of lipid domain structure in membranes by a transmembrane peptide: an ESR spin label study on the effect of the Escherichia coli outer membrane protein A signal peptide on the fluid lipid domain connectivity in binary mixtures of dimyristoyl phosphatidylcholine and distearoyl phosphatidylcholine; Sankaram MB et al.; The effect of a transmembrane peptide on the domain structure of a two-component, two-phase lipid bilayer composed of dimyristoyl phosphatidylcholine (DMPC) and distearoyl phosphatidylcholine (DSPC) was examined by spin label electron spin resonance (ESR) spectroscopy . The peptide, pOmpA, is the hydrophobic, 25-residue signal sequence of the outer membrane protein A from Escherichia coli . Nitroxide derivatives of the phospholipid DSPC, 16-DSPCSL, and of the pOmpA signal peptide, pOmpA-IASL, were used as probes . The first-derivative lineshapes of the ESR spectra were analyzed using a normalized intensity ratio, R, that gives information on the average sizes of the disconnected fluid domains and their point of connectivity (Sankaram, M.B., D . Marsh, and T.E . Thompson . 1992 . Biophys . J . 63:340-349) . In the absence of the peptide, the number of fluid lipid domains does not vary with the fraction of lipid that is in the fluid phase, and phase conversion is accomplished solely by changes in the domain size . The phase boundaries of the lipid mixture remain largely unchanged by the presence of the peptide at mole fractions up to 0.02, but both the size and number of the fluid domains is changed, and the point at which they become connected is shifted to lower fractions of the fluid phase . In addition, the number of domains in the presence of the peptide no longer remains constant but increases from a domain density at low fractions of the fluid phase that is much lower than that in the absence of peptide to one that is comparable to the natural state in the absence of peptide at the point of domain connectivity . A simple model is presented for the process of domain fission, where the latter is determined by a balance between the effects of peptide concentration in the fluid domains, the line tension at the domain boundaries, and the distributional entropy of the domains.

Biophys J, 1994 Jun, 66(6), 1815 - 22
Modeling of the three-dimensional structure of polypeptides in solution using potential-scaled/hot-solute molecular dynamics; Tsujishita H et al.; We present here an efficient and accurate procedure for modeling of the three-dimensional structures of polypeptides in the explicit solvent water based on molecular dynamics calculations . Using the toxic domain analog of heat-stable enterotoxin as a model peptide, we examined the utilities of two molecular dynamics techniques with the system containing the explicit solvent . One is the potential-scaled molecular dynamics that had been designed for effective conformational analyses of biomolecules with the explicit solvent water by partially scaling down the potential energies involved in the solute molecules . The other is the variation of Berendsen's weak coupling method (referred to as "hot-solute" method), in which only the solute of the system is heated to a high temperature while the solvent is kept at a normal temperature . Each method successfully increased the rate of folding of the peptides, and the most effective was a combination of the two methods . Moreover, the final structure obtained via cooling process successfully reproduced the experimentally known structure from the extended amino acid sequence using only the distance restraints representing three disulfide bonds in the peptide . Additional distance restraints derived from some of the NOE cross peaks accelerated the folding of the peptide, but gave almost the same structure as in the case without these additional restraints . Because a similar calculation without the explicit solvent could not reproduce the known structure, it is suggested that the explicit solvent water could play an important role in the modeling . The methods presented here have the potential for accurate modeling even when less experimental information was available.

Blood Coagul Fibrinolysis, 1994 Jun, 5(3), 341 - 8
Haemophilia B Leyden: the effect of mutations at position +13 on the liver-specific transcription of the factor IX gene; Reijnen MJ et al.; Haemophilia B Leyden is characterized by low plasma levels (< or = 1-13% of normal) of blood coagulation factor IX during childhood . After the onset of puberty, plasma factor IX levels gradually rise, probably under the influence of androgens . Single point mutations have been detected in the factor IX promoter at -21, -20, -6, -5, +6, +8 and +13 . This paper examines how two of these mutations (a deletion of an A, and an A-->G substitution at +13) interfere with normal factor IX gene transcription . It is shown that both mutations do impair factor IX promoter activity in transiently transfected HepG2 cells . The mutations at +13 lie in a region (+1 to +18) that is considered to contain a binding site for the CCAAT/enhancer binding protein . Transactivation by the CCAAT/enhancer binding protein alpha of the wild-type and mutated factor IX promoter (-192 to +38) resulted in an approximately four-fold and approximately two-fold, respectively, increase of CAT activity . Gel mobility shift assays revealed that the binding of the CCAAT/enhancer binding protein alpha is disrupted by both the deletion of an A, and the A-->G substitution at +13 . The role of two additional bZIP factors, D-site binding protein and liver-enriched transcriptional activator protein, in the binding and activation of the +13 factor IX promoter region was examined . The D-site binding protein binds to the factor IX promoter region (+1 to +18) in gel mobility shift assays . The deletion of an A at +13 does not interfere with the binding of the D-site binding protein.(ABSTRACT TRUNCATED AT 250 WORDS)

AIDS Res Hum Retroviruses, 1994 Jun, 10(6), 665 - 74
Expression and purification of nonglycosylated SIV proteins, and their use in induction and detection of SIV-specific immune responses; Hanke T et al.; Two commercially available expression vectors were modified to generate plasmids pGEXcPk and pQ9cPk . Proteins expressed from pGEXcPk and pQ9cPk had a short oligopeptide tag termed Pk at their carboxy termini and either glutathione S-transferase (GST) or a small histidine (His) tag, respectively, at their N termini . GST fusion proteins can be purified on immobilized glutathione and proteins coupled to the His tag selectively bind to Ni(2+)-NTA columns . The Pk tag is recognized by monoclonal antibody (MAb) SV5-P-k, previously produced in our laboratory . Thus proteins expressed from the pGEXcPk and pQ9cPk vectors can be purified in a two-step procedure, first via the N-terminal tag and second via the C-terminal tag . The combination of two affinity purification steps significantly improves the antigen purity and selects for full-size proteins . Moreover, by using the MAbSV5-P-k in the second purification step, Pk-linked antigens can be assembled directly into solid matrix-antibody-antigen (SMAA) complexes for use as vaccines . The genes for nef, endonuclease, p15, p17, p27, protease, Rev, reverse transcriptase (rt), tat, vif, vpr, and vpx of simian immunodeficiency virus (SIV mac 251) were cloned and expressed as both GST-SIV-Pk and His-SIV-Pk proteins . Multivalent SMAA complexes were made that contained His-p17-Pk, His-p27-Pk, His-rt-Pk, His-vpx-Pk, and His-vpr-Pk . Following two immunizations of mice with this mixture, antibodies could be detected to all five SIV antigens . When compared to single-protein immunizations, the immunogenicity of some of the proteins in this cocktail was either enhanced or decreased . Mice were also immunized with His-p17-Pk or His-p17-Pk-antibody complexes in the presence or absence of alum . The antibody-antigen complexes induced two- to four-fold higher antibody levels than antigen alone but did not appear to be more immunogenic in inducing lymphoproliferative responses . Sera from SIV-infected macaques were tested for the presence of antibodies reacting with the recombinant proteins by Western blot analysis . Antibodies to endonuclease, p15, p17, p27, rt, and vif were readily detected, antibodies against protease and vpx were present at much lower levels, but no antibodies were detected to nef, rev, tat, or vpr . Thus, we have developed a comprehensive range of reagents (available on request) that can be used to examine immune responses to SIV in both mice and monkeys.

Biotechniques, 1994 Jun, 16(6), 1060 - 4
Lambda/plasmid vector construction by in vivo cre/lox-mediated recombination; Brunelli JP et al.; Lambda/plasmid hybrid vectors have been previously constructed in which the plasmid sequences are separated from flanking lambda arms by lox sites . The lox sequence is the substrate of Cre-mediated site-specific recombination, allowing easy excision of plasmid sequences (automatic subcloning) . We have developed a simple procedure to construct other such lambda hybrid vectors using in vivo cre/lox-mediated recombination to exchange new plasmids for plasmids previously incorporated into lambda/plasmid hybrids . Because hybrid vectors both with and without lacZ alpha plasmid sequences are available, producing either blue or clear plaques, respectively, the new lambda hybrid vectors can be distinguished from the parental hybrids by blue/clear plaque screening . This procedure has been successfully used to construct ten hybrid vectors . It generates new lambda/plasmid hybrid vectors, without ligation or lambda packaging, which retain the property of automatic subcloning.

Free Radic Biol Med, 1994 Jun, 16(6), 869 - 70
Coinduction of nitric oxide synthesis and intracellular nonheme iron-nitrosyl complexes in murine cytokine-treated fibroblasts; Lancaster JR Jr et al.; Murine fibroblasts treated with interferon-gamma plus tumor necrosis factor-alpha plus lipopolysaccharide produce nitrite and EPR-observable intracellular nonheme iron-thiol-dinitrosyl species . Inhibition of .NO synthesis or de novo tetrahydrobiopterin (BH4) synthesis decreases nitrite and the EPR signal . The effects of BH4 synthesis inhibition are prevented by sepapterin, which increases BH4 through the salvage pathway.

Genetics, 1994 Jun, 137(2), 343 - 52
Intragenic suppression of integration-defective IS10 transposase mutants; Junop MS et al.; IS10 transposase mediates excision and integration reactions in Tn10/IS10 transposition . Mutations in IS10 transposase that specifically block integration have previously been identified; however, the mechanism by which these mutations block integration has not been established . One approach to defining the basis of this block is to identify ways in which the original defect can be corrected . The approach we have taken toward this end has been to isolate and characterize intragenic second site suppressors to two different integration-defective mutants . Of the second site suppressors identified, one, CY134, is of particular interest for two reasons . First, it suppresses at least seven different mutations that confer an integration-defective phenotype . Interestingly, these mutations map in two separate segments of transposase, designated patch I and patch II . Second, CY134 on its own has previously been shown to relax the target DNA sequence requirements for Tn10 integration . We provide evidence that suppression by CY134 is not simply a consequence of this mutation conferring a general "transposition up" phenotype, but rather is due to correcting the original defect . Possible mechanisms of suppression for both CY134 and other second site suppressors are considered.

Protein Sci, 1994 Jun, 3(6), 967 - 74
A 70-amino acid zinc-binding polypeptide fragment from the regulatory chain of aspartate transcarbamoylase causes marked changes in the kinetic mechanism of the catalytic trimer; Zhou BB et al.; Interaction between a 70-amino acid and zinc-binding polypeptide from the regulatory chain and the catalytic (C) trimer of aspartate transcarbamoylase (ATCase) leads to dramatic changes in enzyme activity and affinity for active site ligands . The hypothesis that the complex between a C trimer and 3 polypeptide fragments (zinc domain) is an analog of R state ATCase has been examined by steady-state kinetics, heavy-atom isotope effects, and isotope trapping experiments . Inhibition by the bisubstrate ligand, N-(phosphonacetyl)-L-aspartate (PALA), or the substrate analog, succinate, at varying concentrations of substrates, aspartate, or carbamoyl phosphate indicated a compulsory ordered kinetic mechanism with carbamoyl phosphate binding prior to aspartate . In contrast, inhibition studies on C trimer were consistent with a preferred order mechanism . Similarly, 13C kinetic isotope effects in carbamoyl phosphate at infinite aspartate indicated a partially random kinetic mechanism for C trimer, whereas results for the complex of C trimer and zinc domain were consistent with a compulsory ordered mechanism of substrate binding . The dependence of isotope effect on aspartate concentration observed for the Zn domain-C trimer complex was similar to that obtained earlier for intact ATCase . Isotope trapping experiments showed that the compulsory ordered mechanism for the complex was attributable to increased "stickiness" of carbamoyl phosphate to the Zn domain-C trimer complex as compared to C trimer alone . The rate of dissociation of carbamoyl phosphate from the Zn domain-C trimer complex was about 10(-2) that from C trimer.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein Sci, 1994 Jun, 3(6), 960 - 6
Association of the catalytic subunit of aspartate transcarbamoylase with a zinc-containing polypeptide fragment of the regulatory chain leads to increases in thermal stability; Peterson CB et al.; The regulatory enzyme aspartate transcarbamoylase (ATCase), comprising 2 catalytic (C) trimers and 3 regulatory (R) dimers, owes its stability to the manifold interchain interactions among the 12 polypeptide chains . With the availability of a recombinant 70-amino acid zinc-containing polypeptide fragment of the regulatory chain of ATCase, it has become possible to analyze directly the interaction between catalytic and regulatory chains in a complex of simpler structure independent of other interactions such as those between the 2 C trimers, which also contribute to the stability of the holoenzyme . Also, the effect of the interaction between the polypeptide, termed the zinc domain, and the C trimer on the thermal stability and other properties can be measured directly . Differential scanning microcalorimetry experiments demonstrated that the binding of the zinc domain to the C trimer leads to a complex of markedly increased thermal stability . This was shown with a series of mutant forms of the C trimer, which themselves varied greatly in their temperature of denaturation due to single amino acid replacements . With some C trimers, for which tm varied over a range of 30 degrees C due to diverse amino acid substitutions, the elevation of tm resulting from the interaction with the zinc domain was as large as 18 degrees C . The values of tm for a variety of complexes of mutant C trimers and the wild-type zinc domain were similar to those observed when the holoenzymes containing the mutant C trimers were subjected to heat denaturation.(ABSTRACT TRUNCATED AT 250 WORDS)

Plant Physiol, 1994 Jun, 105(2), 579 - 83
Isolation and characterization of cDNAs encoding imidazoleglycerolphosphate dehydratase from Arabidopsis thaliana; Tada S et al.; cDNA clones encoding imidazoleglycerolphosphate dehydratase (IGPD; EC 4.2.1.19) from Arabidopsis thaliana were isolated by complementation of a bacterial auxotroph . The predicted primary translation product shared significant identity with the corresponding sequences from bacteria and fungi . As in yeast, the plant enzyme is monofunctional, lacking the histidinol phosphatase activity present in the Escherichia coli protein . IGPD mRNA was present in major organs at all developmental stages assayed . The Arabidopsis genome appears to contain two genes encoding this enzyme, based on DNA gel blot and polymerase chain reaction analysis.

Cell Mol Biol (Noisy-le-grand), 1994 Jun, 40(4), 489 - 93
DNA binding properties of recombinant human mitochondrial transcription factor 1; Ikeda S et al.; Recombinant mitochondrial transcription factor 1 (r-mtTF1) was overproduced in Escherichia coli, and purified to near homogeneity by DNA affinity chromatography . Binding affinities of r-mtTF1 to the light strand promoter (LSP) and heavy strand promoter (HSP) of human mitochondrial DNA and a non-promoter fragment derived from pUC vector were quantitatively measured by electrophoretic mobility shift assay . The order of the affinities for these DNAs estimated by competition experiments was LSP > HSP = pUC, which was essentially the same as that of transcriptional activity from each promoter in vitro . Recombinant mtTF1 bound more tightly to supercoiled or relaxed DNA than to linear DNA, moreover to single-stranded DNA with the same affinity for linear DNA . The results suggest that the mechanism of activation of HSP by mtTF1 is different from that of LSP, and that multiple binding of mtTF1 to mitochondrial DNA, in a non-specific manner, may significantly participate in activation of transcription from HSP and replication of mitochondrial DNA.

Plant Cell, 1994 Jun, 6(6), 885 - 92
pCyP B: a chloroplast-localized, heat shock-responsive cyclophilin from fava bean; Luan S et al.; When the immunosuppressants cyclosporin A (CsA) and FK506 bind to their intracellular receptors (immunophillins), they form complexes that bind to calcineurin and block calcineurin-dependent signaling pathways in immune cells . Previously, we reported that higher plants also express immunophilins and have a Ca(2+)-dependent signaling pathway sensitive to immunophilin-ligand complexes . Based on an N-terminal peptide sequence of a chloroplast-localized cyclophilin (pCyP B), we isolated a cDNA clone encoding the preprotein of the cyclophilin . The deduced amino acid sequence of this cDNA starts with a putative transit sequence for chloroplast targeting . The mature pCyP B protein has rotamase activity with low-substrate specificity . Enzyme activity was inhibited by CsA with an inhibition constant of 3.9 nM . Similar to other CyPs from mammalian cells, pCyP B, when complexed with CsA, inhibited the phosphatase activity of bovine calcineurin . The mRNA level of pCyP B was high in leaf tissue but was not detectable in roots . Expression of the transcript in the leaf tissues was regulated by light and induced by heat shock . These findings illustrate the conserved nature of cyclophilin proteins among all of the eukaryotes and suggest that cyclophilins have a unique mode of regulation in higher plants.

Plant Cell, 1994 Jun, 6(6), 799 - 810
Promoter elements required for developmental expression of the maize Adh1 gene in transgenic rice; Kyozuka J et al.; To define the regions of the maize alcohol dehydrogenase 1 (Adh1) promoter that confer tissue-specific expression, a series of 5' promoter deletions and substitution mutations were linked to the Escherichia coli beta-glucuronidase A (uidA) reporter gene and introduced into rice plants . A region between -140 and -99 not only conferred anaerobically inducible expression in the roots of transgenic plants but was also required for expression in the root cap, embryo, and in endosperm under aerobic conditions . GC-rich (GC-1, GC-2, and GC-3) or GT-rich (GT-1 and GT-2) sequence motifs in this region were necessary for expression in these tissues, as they were in anaerobic expression . Expression in the root cap under aerobic conditions required all the GC- and GT-rich motifs . The GT-1, GC-1, GC-2, and GC-3 motifs, and to a lesser extent the GT-2 motif, were also required for anaerobic responsiveness in rice roots . All elements except the GC-3 motif were needed for endosperm-specific expression . The GC-2 motif and perhaps the GT-1 motif appeared to be the only elements required for high-level expression in the embryos of rice seeds . Promoter regions important for shoot-, embryo-, and pollen-specific expression were proximal to -99, and nucleotides required for shoot-specific expression occurred between positions -72 and -43 . Pollen-specific expression required a sequence element outside the promoter region, between +54 and +106 of the untranslated leader, as well as a silencer element in the promoter between -72 and -43.

Gesundheitswesen, 1994 Jun, 56(6), 353 - 7
{Responsibilities of UV facilities and monitoring of UV disinfection from the viewpoint of the public health authority}; Steuer W; Extensive studies carried out for long years have shown that UV disinfection of drinking water is fundamentally a well-founded alternative to chlorination . The article states the prerequisites for installing a UV disinfection plant . Authorities responsible for checking on such plant must insists on the need for ascertaining whether the appropriate type has been selected and they must also insists on periodic controls of built-in reactors . In consequence there of Public Health authorities must shoulder advisory and controlling responsibilities . Other possible uses of UV disinfection plant are pointed out.

Ginecol Obstet Mex, 1994 Jun, 62, 153 - 6
{Chorioamnionitis as a cause of jaundice during pregnancy}; Figueroa Damian R et al.; A clinical case of a pregnant patient with chorioamniotis and E . coli sepsis during the third trimester, whose principal clinical symptom was an icteric syndrome, is presented . Jaundice during pregnancy represents many etiologic possibilities, nevertheless the most frequent causes are hepatic or biliary tract diseases, some systemic illness like infections or eclampsia can be associated to this syndrome . It is proposed that, although is not a common cause, chorioamniotis must be considered between the causes of jaundice during pregnancy.

Math Biosci, 1994 Jun, 121(2), 227 - 34
A geometric model for codon recognition logic; Halitsky D; Known types of pairings between mRNA bases and tRNA nucleosides are shown to be consistent with the notion of a translation space TS constructed such that certain wobble-pairings cannot be used in the same translation system without engendering confusion between keto-final codon twins like AAU(ASN)/AAG(LYS) and between amino-final codon twins like AAC(ASN)/AAA(LYS) . When TS is abstractly formalized using Coxeter's face-first three-dimensional projection of a four-dimensional hypercube, the resulting model suggests a specific configurational logic for codon recognition by cognate tRNAs . Although this logic will in general permit codons and anticodons to form matching configurations whose loci are six lines parallel to the axis of a cylinder, confusion of keto-final and amino-final codon twins may result from wobble-pairings whose loci are the two of these lines off the surface of the cylinder.

Parasitology, 1994 Jun, 108 ( Pt 5), 527 - 32
Characterization and localization of Schistosoma mansoni calreticulin Sm58; Khalife J et al.; Recombinant Schistosoma mansoni calreticulin (SmCaR) was expressed in Escherichia coli, using the glutathione S-transferase fusion protein, and its Ca(2+)-binding capacity was determined . Results obtained by a 45Ca2+ overlay technique showed that Ca(2+)-binding site(s) were present in the recombinant CaR indicating that proper folding of the protein was obtained using this system . An antiserum raised against the recombinant SmCaR showed that the native protein (Sm58) was expressed in all stages of the life-cycle from cercariae to the adult worm and in the egg . However, SmCaR seems to be a developmentally regulated protein whose expression can be used to study the post-transformational differentiation of the schistosomulum . Localization of SmCaR demonstrated that the majority of SmCaR was expressed in the epithelia of the digestive duct and in the genital organs . These results suggest that SmCaR, by regulating the Ca2+ concentration, may play an important role during cell proliferation . Finally the presence of SmCaR in miracidia and in the genital organs suggests that the antibody response directed against this protein could interfere in egg production.

Mol Microbiol, 1994 Jun, 12(5), 833 - 43
The glucose-starvation stimulon of Escherichia coli: induced and repressed synthesis of enzymes of central metabolic pathways and role of acetyl phosphate in gene expression and starvation survival; Nystrom T; Proteins of the glucose-starvation stimulon were identified by using two-dimensional gel electrophoresis and the gene-protein database of Escherichia coli . Members of this stimulon included enzymes of the Embden-Meyerhof-Parnas (EMP) pathway, phosphotransacetylase (Pta) and acetate kinase (AckA) of the acetyl phosphate/acetate production pathway, and formate transacetylase . The synthesis of these enzymes was found to be induced concomitantly with the decreased synthesis of enzymes of the Krebs cycle . Thus, the modulation in the synthesis of specific proteins during aerobic glucose starvation is, in part, similar to the response of cells shifted to anaerobiosis . These modulations suggest that the glucose-starved cell increases the relative flow of carbon through the Pta-AckA pathway . Indeed, the ability to synthesize acetyl phosphate, an intermediate of the pathway, appears to be indispensable for glucose-starved cells as pta and pta-ackA double mutants were found to be impaired in their ability to survive glucose starvation . The survival characteristics of ackA mutants and the wild-type parent were indistinguishable . Moreover, the pta mutant failed to induce several proteins of the glucose-starvation stimulon.

Mol Microbiol, 1994 Jun, 12(5), 707 - 16
The ompA 5' untranslated region impedes a major pathway for mRNA degradation in Escherichia coli; Hansen MJ et al.; The unusual longevity of the Escherichia coli ompA transcript is determined by its 5' untranslated region (UTR), which functions in vivo as an mRNA stabilizer . Here we show that this 5' UTR can prolong the lifetime in E . coli of a variety of heterologous mRNAs to which it is joined, either as a gene fusion or as an operon fusion . Statistical extrapolation suggests that it is quite likely that most E . coli mRNAs could be stabilized in this manner . We conclude that the ompA 5' UTR impedes a major pathway for mRNA degradation in E . coli and that stabilization by fusion to this UTR does not require translational readthrough of the heterologous mRNA segment by ribosomes that initiate translation at the ompA ribosome-binding site . Additional experiments indicate that the E . coli ribonuclease whose action is slowed by the ompA 5' UTR is not RNase III.

Mol Microbiol, 1994 Jun, 12(5), 685 - 92
Protein folding in the periplasm of Escherichia coli; Wulfing C et al.; With the discovery of molecular chaperones and the development of heterologous gene expression techniques, protein folding in bacteria has come into focus as a potentially limiting factor in expression and as a topic of interest in its own right . Many proteins of importance in biotechnology contain disulphide bonds, which form in the Escherichia coli periplasm, but most work on protein folding in the periplasm of E . coli is very recent and is often speculative . This MicroReview gives a short overview of the possible fates of a periplasmic protein from the moment it is translocated, as well as of the E . coli proteins involved in this process . After an introduction to the specific physiological situation in the periplasm of E . coli, we discuss the proteins that might help other proteins to obtain their correctly folded conformation--disulphide isomerase, rotamase, parts of the translocation apparatus and putative periplasmic chaperones--and briefly cover the guided assembly of multi-subunit structures . Finally, our MicroReview turns to the fate of misfolded proteins: degradation by periplasmic proteases and aggregation phenomena.

Plant Mol Biol, 1994 Jun, 25(3), 545 - 50
Isolation of a poplar and an Arabidopsis thaliana dihydrodipicolinate synthase cDNA clone; Vauterin M et al.; A poplar DHDPS cDNA clone has been isolated by functional rescue of the dapA-deficient AT997 mutant of Escherichia coli . By sequence comparison between the poplar and maize DHDPS cDNAs, two oligonucleotides were designed to perform polymerase chain reaction (PCR) on Arabidopsis thaliana genomic DNA . The PCR fragment was subsequently used to isolate an Arabidopsis DHDPS genomic and cDNA clone.

FEMS Microbiol Rev, 1994 Jun, 14(2), 161 - 76
Genetic approaches to study Legionella pneumophila pathogenicity; Ott M; Legionella pneumophila is an intracellular pathogen replicating in human macrophages during the course of infection of the lungs . Infection by legionellae often leads to severe pneumonia, termed Legionnaires' disease . Genetic approaches to identify the factors responsible for L . pneumophila pathogenicity started with the construction of genomic libraries in Escherichia coli . Various L . pneumophila-specific genes were cloned in E . coli K-12 by identification using functional assays, antibody screening and hybridization ('reverse genetics') . By disrupting the genes via allelic exchange, mutants have been created to assess the influence of the factors on pathogenicity . Among the cloned genes, only for the gene product of the mip gene, encoding a 24-kDa surface-associated protein (macrophage infectivity potentiator) unequivocal evidence for its contribution to pathogenicity could be provided . Two hemolytic factors that have been cloned do not seem to play a role in L . pneumophila pathogenicity . Genetic systems for transposon mutagenesis of the L . pneumophila genome (Tn5, Tn903dIIlacZ, MudphoA), including Tn phoA shuttle mutagenesis, have been established and specifically adapted to identify mutants which displayed an impaired capability to multiply inside macrophages and with a reduced in vivo virulence . Furthermore, by complementation of avirulent mutants, genetic loci could be identified which restored the virulence.

Mamm Genome, 1994 Jun, 5(6), 365 - 71
Efficient identification and regional positioning of YAC and cosmid clones to human chromosome 21 by radiation fusion hybrids; Kumlien J et al.; The use of integrated mapping strategies involving bacterial, yeast, and rodent cells as hosts simplifies the construction of maps, which combine long-range order, high resolution, and easy access to the cloned DNA . Radiation-fusion hybrids offer a specially powerful long-range mapping system for human chromosomes . We describe here techniques for establishing a radiation-fusion hybrid map of Chromosome (Chr) 21q and its integration with local information on YAC and cosmid positions.

FEMS Microbiol Lett, 1994 Jun 1, 119(1-2), 89 - 94
A reassessment of the range of c-type cytochromes synthesized by Escherichia coli K-12; Iobbi-Nivol C et al.; Five different c-type cytochromes have been detected during anaerobic growth of various Escherichia coli strains in different media . None of these cytochromes was detectable in aerobically-grown cultures . Only a single, 43 kDa cytochrome was synthesized in response to the presence of trimethylamine-N-oxide: synthesis of this cytochrome was unaffected by the presence of nitrate or nitrite, was repressed by oxygen, but was dependent upon a functional tor operon located at minute 22 (coordinate 1070 kb) on the E . coli chromosome . The other four cytochromes, masses 16, 18, 24 and 50 kDa, were induced by nitrite coordinately with formate-dependent nitrite reductase activity, but repressed by oxygen and nitrate . As only the 18 kDa and 50 kDa cytochromes are encoded by the nrf operon located at minute 92 (coordinate 4366 kb), there must be other loci, possibly essential for formate-dependent nitrite reduction, encoding the 16 kDa and 24 kDa cytochromes . No other c-type cytochrome was detected under any growth condition tested.

FEMS Microbiol Lett, 1994 Jun 1, 119(1-2), 71 - 6
Energy-dependent receptor activities of Escherichia coli K-12: mutated TonB proteins alter FhuA receptor activities to phages T5, T1, phi 80 and to colicin M; Killmann H et al.; The activity of the FhuA receptor in the outer membrane of Escherichia coli is dependent on the TonB, ExbB and ExbD proteins which are anchored to the cytoplasmic membrane . Only infection by phage T5 occurs independently of TonB, ExbB and ExbD . In this paper we describe mutated FhuA proteins which displayed either an increased or decreased FhuA activity to phage T5 when combined with mutated TonB proteins . These results suggest conformational changes in FhuA by TonB which are recognized by phage T5 . Similar results were obtained with colicin M and the phages T1 and phi 80 . It is proposed that the FhuA mutant proteins assume conformations which are either improved or impaired by the TonB derivatives . For the direct interaction of FhuA with TonB regions which are located outside the TonB box of FhuA and the region around residue 160 of TonB are important.

FEMS Microbiol Lett, 1994 Jun 1, 119(1-2), 33 - 9
The nusG gene of Streptomyces griseus: cloning of the gene and analysis of the A-factor binding properties of the gene product; Kuberski S et al.; The nusG gene of Streptomyces griseus was cloned and the nucleotide sequence determined . It encodes a protein with an identity of 76% to the reported receptor (VbrA) for VB-C, an autoregulatory factor in Streptomyces virginae . NusG protein was expressed in Escherichia coli . However, no binding activity for A-factor, an butyrolactone autoregulator in S . griseus very similar to VB-C, could be detected . The nusG gene of S . griseus does not seem to encode the A-factor-binding protein.

Genome, 1994 Jun, 37(3), 508 - 12
Construction and expression of a metallothionein-beta-glucuronidase gene fusion; Hattori J et al.; A gene fusion consisting of the Chinese hamster metallothionein II and beta-glucuronidase coding regions was constructed . The fusion protein expressed in Escherichia coli retained cadmium-binding capacity and beta-glucuronidase activity . When expressed from the constitutive 35S promoter in transgenic tobacco, the levels of 109Cd accumulation in leaves were reduced to about 70% of those in untransformed control plants . Metallothionin-beta-glucuronidase did not sequester a significant proportion of the leaf 109Cd taken up through the roots in vitro and therefore a sink for Cd was not created.

Chem Biol Interact, 1994 Jun, 92(1-3), 77 - 85
Heterologous systems for expression of mammalian sulfotransferases; Veronese ME et al.; This paper describes the use of both mammalian and bacterial expression systems as tools to study the structural and functional relationships of proteins encoded by cDNAs to both rat and human aryl sulfotransferases . In particular, we describe the use of the mammalian COS cell system for functional expression studies, and the use of Escherichia coli for the expression and purification of a sulfotransferase fusion protein suitable as an antigen for the generation of sulfotransferase antibodies.

Chem Biol Interact, 1994 Jun, 92(1-3), 25 - 31
Changes in substrate specificity of the recombinant form of phenol sulfotransferase IV (tyrosine-ester sulfotransferase); Guo WX et al.; The over-expression of mammalian enzymes in bacterial systems by means of recombinant DNA technology has provided the enzymologist with a supply of catalyst sufficiently abundant to identify suboptimal substrates . Such large quantities are particularly useful when working with the enzymes of detoxication, a family of proteins that are distinguished by their broad substrate specificity for generally lipophilic compounds, i.e., by their very low specificity for features other than the functional group {1} . We have achieved bountiful expression of a sulfotransferase active with phenols {2}, an enzyme originally purified and characterized from rat liver {3}, and classified as tyrosine-ester sulfotransferase, EC 2.8.2.9 {4,5}, but usually referred to as rat liver phenol or aryl sulfotransferase IV . Having improved the sensitivity and versatility of some of the assays for sulfotransferases, we examined the substrate spectrum of this enzyme . As presented here, the results of this examination point to the limitations of enzyme nomenclature and to the danger of equating enzymes isolated from their normal habitat with those formed by recombinant technology in a foreign cell . Our experiments also establish a greater catalytic scope for the natural rat liver enzyme than that previously described.

Chem Biol Interact, 1994 Jun, 92(1-3), 129 - 44
Major hydroxysteroid sulfotransferase STa in rat liver cytosol may consist of two microheterogeneous subunits; Ogura K et al.; The possible existence of two microheterogeneous subunits, designated ST-40P and ST-41P, of hydroxysteroid sulfotransferases in female Sprague-Dawley rat liver cytosol was demonstrated by cloning and sequencing of cDNAs, both isolated from two rat liver cDNA libraries . These subunits consisted of an equal number of amino acid residues with only one amino acid substitution . ST-40P and ST-41P expressed as homodimers from the ST-40 and ST-41 cDNAs in Escherichia coli had enzyme activities toward all of the examined 20 hydroxysteroids, 13 bile acids, and the carcinogen 5-hydroxymethylchrysene (5-HCR), with formation of the reactive metabolite 5-HCR sulfate, at rates very similar to those by STa, the major hydroxysteroid sulfotransferase in rat liver cytosol . This strongly suggested that they are essential components of STa . The present study carried out by using the recombinant enzymes provides the first direct evidence for the identity of sulfotransferases catalysing the sulfation of hydroxysteroids and bile acids and proposes that the current nomenclature system used for distinguishing hydroxysteroid sulfotransferases from bile acid sulfotransferases should be improved.

Am J Physiol, 1994 Jun, 266(6 Pt 1), G1146 - 55
Direct in vivo adenovirus-mediated gene transfer to salivary glands; Mastrangeli A et al.; Gene transfer to the salivary glands holds the potential for the therapy of salivary gland disorders and for delivery of therapeutic proteins to the mouth and upper gastrointestinal tract . Administration of the recombinant adenovirus vectors Ad.RSV beta gal {coding for the intracellular protein beta-galactosidase (beta-Gal)} and Ad alpha 1AT {coding for human alpha 1-antitrypsin (alpha 1-AT), a secreted protein} to salivary gland cell lines in vitro demonstrated exogenous gene expression . Retrograde ductal injection of the Ad.RSV beta gal vector to rat salivary glands in vivo resulted in beta-Gal expression in acinar and ductal cells . Exposure of submandibular glands in vivo to Ad alpha 1AT resulted in expression of alpha 1-AT mRNA transcripts, de novo synthesis of alpha 1-AT, and secretion in the saliva . To evaluate the feasibility of adenovirus-mediated gene transfer to human glands, human minor salivary glands were infected ex vivo with Ad.RSV beta gal, and implanted into severe combined immunodeficient mice . Evaluation of the human tissue demonstrated beta-Gal activity . These observations demonstrate that adenovirus vectors are capable of direct delivery of genes to the salivary glands, suggesting a variety of possible gene therapy applications.

Am J Physiol, 1994 Jun, 266(6 Pt 1), E986 - 92
Effect of IL-1 receptor antagonist and antiserum to TNF-alpha on LPS-induced plasma ACTH and corticosterone rise in rats; Ebisui O et al.; Using an antiserum against tumor necrosis factor (TNF)-alpha and an interleukin (IL-1) receptor antagonist, we studied putative roles of these cytokines in mediating the endotoxin-induced elevation of plasma adrenocorticotropic hormone (ACTH) and corticosterone levels in freely moving rats . Intravenous administration of Escherichia coli lipopolysaccharide (LPS) increased plasma ACTH and corticosterone levels in a dose-dependent manner . The plasma corticosterone reached to its highest level among a series of experiments after the administration of even the smallest dose (0.03 microgram/kg) tested . Plasma ACTH and corticosterone levels in these rats were completely inhibited by the intravenous administration of anti-murine TNF-alpha-rabbit antiserum (anti-TNFAS) after the administration of LPS but not by the intravenous administration of IL-1 receptor antagonist (IL-1RA) . On the other hand, both recombinant human IL-1RA and anti-TNFAS significantly inhibited plasma ACTH increase stimulated with 10 micrograms/kg LPS . These findings indicate that 1) when the plasma corticosterone increase induced by intravenous LPS remains below its maximum, the effect is exclusively mediated by TNF-alpha, and 2) when a larger amount of LPS is administered, both IL-1 beta and TNF-alpha participate at least in part in the hypothalamic-pituitary-adrenal axis activation.

Am J Physiol, 1994 Jun, 266(6 Pt 1), E863 - 9
Epinephrine-induced changes in hepatic glucose production after ethanol; Lang CH et al.; Previous studies indicate that catecholamines play an important role in mediating the glucose metabolic response to endotoxin . Because acute ethanol (EtOH) intoxication impairs this response, the present study was initiated to ascertain whether EtOH attenuates the lipopolysaccharide response by decreasing the increment in plasma catecholamines after endotoxin or by decreasing the responsiveness of rats to epinephrine . All studies were performed on chronically catheterized fasted rats infused intravenously with either EtOH or an equal volume of saline . In the first series of experiments, intravenous administration of Escherichia coli endotoxin increased, to the same extent, the plasma concentrations of epinephrine and norepinephrine in both saline- and EtOH-infused rats . In the second study, rats were infused with {3-3H}glucose to assess whole body glucose metabolism and the ability of EtOH to alter the glucose metabolic response to epinephrine . The exogenous infusion of a maximally stimulating dose of epinephrine (1 microgram.min-1.kg-1) into saline-infused control animals for 3 h produced a marked hyperglycemia that resulted from a sustained increase in the rate of hepatic glucose production and a reduction in the metabolic clearance rate for glucose . EtOH infusion did not prevent the epinephrine-induced hyperglycemia but blunted the stimulatory effect of epinephrine on glucose production . The differences in glucose metabolism between saline- and EtOH-treated rats could not be explained by changes in plasma insulin or glucagon concentrations . Furthermore, the ability of EtOH to impair the epinephrine-induced increase in glucose production was still evident in rats treated with 4-methylpyrazole, an inhibitor of alcohol dehydrogenase.(ABSTRACT TRUNCATED AT 250 WORDS)

Eur J Biochem, 1994 Jun 1, 222(2), 625 - 30
Involvement of exposed polypeptide loops in trimeric stability and membrane insertion of Escherichia coli OmpF porin; Fourel D et al.; Different ompF-ompC gene fusions were used to analyse the regions involved in the stable trimerization and membrane insertion of the Escherichia coli OmpF porin . The stability of the trimers formed from the various hybrids was analysed . Three classes of trimer instability are observed related to the presence of different exposed polypeptide loops of OmpF . In all cases, amino acids located between residue 115 and residue 144 of OmpF are necessary to promote a correct and stable trimeric conformation . However, immunogold labelling studies indicate the correct insertion of the protein in the outer membrane despite a marked instability of some hybrid porins . The location of the residues involved in trimer stability is discussed with regards to both the three-dimensional structure and the folding of OmpF.

Eur J Biochem, 1994 Jun 1, 222(2), 605 - 14
Transport of C4-dicarboxylates by anaerobically grown Escherichia coli . Energetics and mechanism of exchange, uptake and efflux; Engel P et al.; Transport activities for uptake, efflux and exchange of C4-dicarboxylates were observed in anaerobically grown Escherichia coli . All three transport modes were only present in strains containing the transcriptional activator FNR of anaerobic respiration, and were repressed by nitrate and O2 . The kinetic and energetic parameters of C4-dicarboxylate transport and the mechanism of the uptake, efflux and exchange reactions were analyzed in whole cells and in membrane vesicles . Fumarate/succinate exchange could be characterized as homologous or heterologous 1:1 counter-exchange . The external substrate was determined as divalent fumarate2- (or succinate2-) at pH 6-9, whereas monovalent H-fumarate dominated as the substrate at pH 3-4 . The exchange was not inhibited by dissipation of delta p or constituents of it (delta psi or delta pH) . We conclude that this transport mode functions as an electroneutral exchange of C4-dicarboxylates . The uptake of C4-dicarboxylates did not depend on internal counter-substrate and resulted in an accumulation of the substrate . Similar to antiport, fumarate was accepted in the divalent form at pH values greater than or equal to 6 and in the monovalent form at pH 3.5-6 . The uptake was inhibited by dissipation of delta p or delta psi . Artificially imposed delta pH, delta psi or fumarate gradients were able to drive fumarate uptake . An involvement of Na+ could not be detected . Thus the uptake is likely to operate as an electrophoretic H+/fumarate symport . Independent of the presence of an external counter-substrate, the substrates were secreted from cells or membrane vesicles loaded with succinate or fumarate . The efflux was electrogenic . Energizing the cells or membrane vesicles inhibited efflux, maximal efflux rates were obtained only after dissipation of delta p or delta psi . An imposed K(+)-diffusion potential (outside positive) inhibited succinate excretion . The efflux of succinate from de-energized membrane vesicles generated a delta psi of -70 mV . It is thus suggested that succinate efflux functions as a H+/succinate symport.

Eur J Biochem, 1994 Jun 1, 222(2), 561 - 71
Kinetics of the reduction of wild-type and mutant cytochrome c-550 by methylamine dehydrogenase and amicyanin from Thiobacillus versutus; Ubbink M et al.; To elucidate the kinetic properties of the methylamine dehydrogenase (MADH) redox chain of Thiobacillus versutus the reduction of cytochrome c-550 by MADH and amicyanin has been studied . Under steady state conditions, the rate constants of the reactions have been determined as a function of the ionic strength, both for wild type cytochrome c-550 and for mutants in which the conserved residue Lys14 has been replaced as follows: Lys14-->Gln (mutant {K14Q}cytochrome c-550) and Lys14-->Glu (mutant {K14E}cytochrome c-550) . The second-order rate constant of the reduction of cytochrome c-550 by MADH shows a biphasic ionic-strength dependence . At low ionic strength the rate constant remains unchanged (wild type) or increases ({K14Q}cytochrome c-550) with increasing ionic strength, while at high salt concentrations the rate constant decreases monotonically as the ionic strength increases . It is suggested that conformational freedom exists in the association complex and that this is favourable for electron transfer . {K14Q}cytochrome c-550 and {K14E}cytochrome c-550 are reduced at rates 20-fold and 500-fold slower than wild-type cytochrome c-550 by MADH, due to a lower association constant . It is concluded that MADH possesses a negative patch with which cytochrome c-550 associates . Lys14 plays an important role in the formation of the reaction complex . The midpoint potentials of wild-type and mutant cytochrome c-550 have been determined by using cyclic voltammetry . {K14Q}cytochrome c-550 and {K14E}cytochrome c-550 show an increase in E0 of only 2 mV and 8 mV, respectively, compared to wild-type cytochrome c-550 (241 mV at pH 8.1) . {K14Q}cytochrome c-550 and {K14E}cytochrome c-550 cytochrome c-550 are reduced by amicyanin at rates that are only slightly faster than for wild-type cytochrome c-550 . The difference is partly attributable to the change in E0 . High ionic strength results in a threefold increase in the rate in all three cases . These results indicate that charge interactions do not play a major role in the formation of the amicyanin/cytochrome c-550 reaction complex, suggesting an interaction at the hydrophobic patch of amicyanin . The reduction of cytochrome c-550 by MADH can be inhibited by Zn(2+)-substituted amicyanin . Ag(+)-amicyanin, however, has little effect on the reduction rate . These results suggest that MADH has a much higher affinity for Cu(2+)-amicyanin (substrate) than for Cu(+)-amicyanin (product) . On the basis of these findings the roles of the components of the MADH redox chain are discussed.

Eur J Biochem, 1994 Jun 1, 222(2), 453 - 60
Bovine inositol monophosphatase . Ligand binding to pyrene-maleimide-labelled enzyme; Greasley PJ et al.; Inositol monophosphatase can be modified at two sites by pyrene maleimide . These sites have been identified as Cys141 and Cys218 . Stoichiometric addition of pyrene maleimide allows the sole modification of Cys218 . The fluorescence of the pyrene moiety on the modified protein can be excited directly or by resonance energy transfer . The fluorescence properties of the pyrene group on Cys218 allows the interaction of ligands with the enzyme to be monitored . This feature has allowed dissociation constants for various metal ions to be determined and allowed the formation of various enzyme/ligand complexes to be observed . These studies have demonstrated that Mg2+ is required to support Pi binding and that Li+ interacts with a post-catalytic complex which is only formed in the forward reaction.

Eur J Biochem, 1994 Jun 1, 222(2), 285 - 92
Interfacial kinetic reaction of human 5-lipoxygenase; Noguchi M et al.; The kinetics of human 5-lipoxygenase were investigated in the presence of Tween 20 using a continuous spectrophotometric assay . Using the mixture at a constant molar ratio of arachidonate/Tween 20 at pH 8.0, the steady-state velocity on a varied arachidonate concentration did not follow simple Michaelis-Menten-type kinetics and double-reciprocal plot analysis gave hyperbolic curves . However, by introducing the concept of a local pH change, it was possible to analyze the kinetics as simple Michaelis-Menten type . The concept of a local pH change implies that when utilizing an acidic and amphiphilic substance as a substrate, such as arachidonate, the medium around the substrate is acidified with an increased concentration of substrate . This concept was explained rationally by two experiments . Consequently, the data were transformed according to a local pH change and analyzed according to a dual phospholipid model as has been proposed for phospholipase A2 {Hendrickson, H . S . and Dennis, E . A . (1984) Kinetic analysis of the dual phospholipid model for phosphalipase A2, J . Biol . Chem . 259, 5734-5739} . It is concluded that 5-lipoxygenase performs an interfacial reaction in the arachidonate/Tween 20 mixed micelles in the same manner as phospholipase A2 . The values of Km were almost constant (about 0.07 molar fraction), even when arachidonate molar ratios were changed in the surface of the mixed micelles . The values for Ks (the association constant of the enzyme to the micelle interface) ranged over 0.21-0.48 microM . The Vmax was 25.76 mumol.min-1.mg-1 . This concept of a local pH change could be used extensively with enzymes which utilize both amphiphilic and acidic substances as substrates.

J Surg Res, 1994 Jun, 56(6), 530 - 6
IFN-gamma decreases translocation and improves survival following transfusion and thermal injury; Gennari R et al.; The effects of recombinant murine interferon-gamma (rmIFN-gamma) on survival and host defense were studied during gut-derived sepsis that included transfusion-induced immunosuppression . Balb/c mice (n = 153) were transfused with allogeneic blood and then treated with different doses of rmIFN-gamma: 10, 100, 1000, 10,000 U, or sterile saline as control once daily for 3 days . Five days after transfusion they were gavaged with 10(10) Escherichia coli and given a 20% TBSA burn injury . Survival was significantly higher in groups receiving 10 U compared to control and the group receiving 10,000 U (P < 0.0001 and P = 0.02, respectively) . Groups receiving 100 or 1000 U also showed an improvement of survival compared to nontreated control animals (P = 0.02) . The effect of rmIFN-gamma on the degree of translocation and the host's ability to kill translocated organisms was also investigated . Mice were treated as described above, except they were gavaged with 111In oxine-labeled E . coli and then subjected to a 20% TBSA burn . Mesenteric lymph nodes (MLN), liver, and spleen were harvested aseptically . Less translocation to the liver was observed compared to the nontreated group (P = 0.002) to the MLNs and spleen of the group treated with 100 U rmIFN-gamma compared to controls and the group treated with 10 U (P < 0.005) . Animals receiving 1000 U showed fewer bacteria in the liver and spleen compared to the control group (P < 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)

EMBO J, 1994 Jun 1, 13(11), 2725 - 34
Copy-choice illegitimate DNA recombination revisited; d'Alencon E et al.; Nearly precise excision of a transposon related to Tn10 from an Escherichia coli plasmid was used as a model to study illegitimate DNA recombination between short direct repeats . The excision was stimulated 100-1000 times by induction of plasmid single-stranded DNA synthesis and did not involve transfer of DNA from the parental to the progeny molecule . We conclude that it occurred by copy-choice DNA recombination, and propose that other events of recombination between short direct repeats might be a result of the same process.

EMBO J, 1994 Jun 1, 13(11), 2508 - 15
Production of an atrial natriuretic peptide variant that is specific for type A receptor; Cunningham BC et al.; Receptor-specific variants of atrial natriuretic peptide (ANP) were selected from libraries of filamentous phage particles that displayed single copies of random ANP mutants fused to gene III protein . These ANP variants were differentially selected by binding to immobilized natriuretic peptide receptor A (NPR-A) over competing receptor C (NPR-C) in solution . This method also selected ANP variants with improved secretion expression in Escherichia coli . Several of the identified mutations were combined to produce an efficiently expressed ANP analog that was as potent as wild-type ANP in stimulating NPR-A guanylyl cyclase activity but resistant to inactivation mediated by NPR-C . Such NPR-A-selective analogs should be useful for correlating the various activities of ANP to the relevant receptor and may also be more potent therapeutics in the targeting of NPR-A.

Ann Thorac Surg, 1994 Jun, 57(6), 1395 - 401
Successful adenovirus-mediated gene transfer in an in vivo model of human malignant mesothelioma; Smythe WR et al.; Malignant mesothelioma remains a frustrating clinical problem with uniformly poor responses to current therapeutic regimens . However, the localized nature of the disease, the potential accessibility of the tumor, and the relative lack of distant metastases make it a particularly attractive candidate for somatic gene therapy . The purpose of this study was to evaluate the ability of an adenoviral vector system to transfer genetic material to human mesothelioma cells in vitro and in vivo . Using a replication-deficient recombinant adenovirus carrying the Escherichia coli lacZ marker gene, we found that human mesothelioma cell lines were susceptible to adenovirus infection . Furthermore, surprisingly effective gene transfer was accomplished within tumor implants of human mesothelioma growing within the peritoneal cavity of immunodeficient mice after intraperitoneal administration of virus . These studies demonstrate that adenoviral vectors hold promise as vehicles to deliver gene therapy in human malignant mesothelioma.

J Med Microbiol, 1994 Jun, 40(6), 428 - 34
Specific DNA probes to detect Escherichia coli strains producing cytotoxic necrotising factor type 1 or type 2; Oswald E et al.; Cytotoxic necrotising factors type 1 (CNF1) and type 2 (CNF2) are produced by many Escherichia coli strains isolated from man and animals with intestinal or extra-intestinal colibacillosis . In most laboratories, CNF-producing strains are detected by a cell cytotoxicity assay and confirmed with a neutralisation assay or a mouse footpad assay . In this study, we sought to determine whether DNA probes could detect clinical isolates of E . coli producing CNF2 or CNF1, or both, without the need for cell cultures or animal assays . Two internal fragments of the gene encoding CNF2 were used as DNA probes: a 875-bp XhoI-PstI DNA fragment and an adjacent 335-bp PstI-ClaI fragment . A positive response with both DNA probes was associated with CNF2-producing strains, whereas a positive response with only the 335-bp probe was associated with CNF1-producing strains . Results of colony hybridisation experiments with 185 clinical isolates of E . coli demonstrated that these DNA probes detected CNF2-producing strains with a sensitivity and specificity of 100% and CNF1-producing strains with a sensitivity and specificity of 99% . These two DNA probes should greatly facilitate epidemiological studies to assess the importance of CNF-producing strains as agents of diarrhoea and septicaemia.

Hum Genet, 1994 Jun, 93(6), 655 - 8
Identification of the mutations in the T-protein gene causing typical and atypical nonketotic hyperglycinemia; Nanao K et al.; We have investigated the molecular lesions of T-protein deficiency causing typical or atypical nonketotic hyperglycinemia (NKH) in two unrelated pedigrees . A patient with typical NKH was identified as being homozygous for a missense mutation in the T-protein gene, a G-to-A transition leading to a Gly-to-Asp substitution at amino acid 269 (G269D) . Sibling patients of a second family with atypical NKH had two different missense mutations in the T-protein gene (compound heterozygote), a G-to-A transition leading to a Gly-to-Arg substitution at amino acid 47 (G47R) in one allele, and a G-to-A transition leading to an Arg-to-His substitution at amino acid 320 (R320H) in the other allele . Gly 269 is conserved in T-proteins of various species, even in E . coli, whereas Gly 47 and Arg 320 are replaced by Ala and Leu, respectively, in E . coli . The mutation occurring in more conservative amino acid residues thus results in more deleterious damage to the T-protein, and gives the severe clinical phenotype, viz., typical NKH.

Biochem J, 1994 Jun 1, 300 ( Pt 2), 531 - 9
NADPH binding and control of catalase compound II formation: comparison of bovine, yeast, and Escherichia coli enzymes; Hillar A et al.; 1 . NADPH binds to bovine catalase and to yeast catalases A and T, but not to Escherichia coli catalase HPII . The association was demonstrated using chromatography and fluorimetry . Bound NADPH fluoresces in a similar way to NADPH in solution . 2 . Bound NADPH protects bovine and yeast catalases against forming inactive peroxide compound II either via endogenous reductant action or by ferrocyanide reduction during catalytic activity in the presence of slowly generated peroxide . 3 . Bound NADPH reduces neither compound I nor compound II of catalase . It apparently reacts with an intermediate formed during the decay of compound I to compound II; this postulated intermediate is an immediate precursor of stable compound II either when the latter is formed by endogenous reductants or when ferrocyanide is used . It represents therefore a new type of hydrogen donor that is not included in the original classification of Keilin and Nicholls {Keilin, D . and Nicholls, P . (1958) Biochim . Biophys . Acta 29, 302-307} 4 . A model for NADPH action is presented in which concerted reduction of the ferryl iron and of a neighbouring protein free radical is responsible for the observed NADPH effects . The roles of migrant radical species in mammalian and yeast catalases are compared with similar events in metmyoglobin and cytochrome c peroxidase reactions with peroxides.

Biochem J, 1994 Jun 1, 300 ( Pt 2), 469 - 75
Cytochrome bo from Escherichia coli: reaction of the oxidized enzyme with hydrogen peroxide; Watmough NJ et al.; Oxidized cytochrome bo reacts rapidly with micromolar concentrations of H2O2 to form a single derivative . The electronic absorption spectrum of this compound differs from that of the oxidized form of the enzyme reported by this laboratory {Watmough, Cheesman, Gennis, Greenwood and Thomson (1993) FEBS Lett . 319, 151-154} . It is characterized by a Soret maximum at 411 nm, increased absorbance at 555 nm, and reduced intensity at 624 nm . The apparent dissociation constant for this process is of the order of 4 x 10(-6) M, and the bimolecular rate constant for the formation of the new compound is (1.25-1.7) x 10(3) M-1.s-1 . Electronic absorption difference spectroscopy shows this product to be identical with the compound formed from the reaction of the mixed-valence form of the enzyme with dioxygen . Investigation of this compound by room-temperature magnetic c.d . spectroscopy shows haem o to be neither high-spin nor low-spin ferric, but to have a spectrum characteristic of an oxyferryl species . There is no evidence for oxidation of the porphyrin ring . Therefore the binuclear centre of this species must consist of an oxyferryl haem (S = 1) coupled to a Cu(II) ion (S = 1/2) to form a new paramagnetic centre . The reaction was also followed by X-band e.p.r . spectroscopy, and this showed the disappearance in parallel with the formation of the oxyferryl species, of the broad g = 3.7, signal which arises from the weakly coupled binuclear centre in the oxidized enzyme . Since no new e.p.r.-detectable paramagnetic species were observed, the Cu(II) ion is presumed to be coupled to another paramagnet, possibly an organic radical . There is no evidence in the electronic absorption spectrum to indicate further reaction of cytochrome bo with H2O2 to form a second species . We argue that the circumstances of formation of this oxyferryl species are the same as those for the P form of cytochrome c oxidase, a species often regarded as containing a bound peroxide ion . The implications of these observations for the reaction mechanism of haem-copper terminal oxidases are discussed.

Biochem J, 1994 Jun 1, 300 ( Pt 2), 413 - 8
Recombinant expression of the fdxD gene of Rhodobacter capsulatus and characterization of its product, a {2Fe-2S} ferredoxin; Armengaud J et al.; A gene called fdxD that could potentially code for a ferredoxin has recently been identified upstream of the nitrogenase structural genes in Rhodobacter capsulatus {Willison, Pierrard and Hubner (1993) Gene 133, 39-46} . In the present study, the fdxD gene product has been overproduced in Escherichia coli in a soluble form . The recombinant protein, pink in colour, was purified to homogeneity, and biochemically characterized as a new ferredoxin . It represents the fifth ferredoxin so far identified in R . capsulatus and was designated FdV . Its N-terminal sequence is identical with that of the native ferredoxin isolated from R . capsulatus . U.v-visible-absorption spectra as well as results of c.d . and e.p.r . spectroscopy demonstrated that the fdxD product contained a {2Fe-2S} cluster correctly assembled and incorporated into the polypeptide . Although similar to plant-type ferredoxins, FdV appeared poorly competent in the photo-reduction of NADP+ . On the basis of in vitro assays, FdV cannot serve as an electron donor for nitrogenase . The lack of reactivity of FdV in either of these assays may primarily be due to its relatively high mid-point redox potential (E'o = -220 mV, pH 7.5).

Biochem J, 1994 Jun 1, 300 ( Pt 2), 373 - 81
Investigation of the nature of the two metal-binding sites in 5-aminolaevulinic acid dehydratase from Escherichia coli; Spencer P et al.; Two distinct metal-binding sites, termed alpha and beta, have been characterized in 5-aminolaevulinic acid dehydratase from Escherichia coli . The alpha-site binds a Zn2+ ion that is essential for catalytic activity . This site can also utilize other metal ions able to function as a Lewis acid in the reaction mechanism, such as Mg2+ or Co2+ . The beta-site is exclusively a transition-metal-ion-binding site thought to be involved in protein conformation, although a metal bound at this site only appears to be essential for activity if Mg2+ is to be bound at the alpha-site . The alpha- and beta-sites may be distinguished from one another by their different abilities to bind divalent-metal ions at different pH values . The occupancy of the beta-site with Zn2+ results in a decrease of protein fluorescence at pH 6 . Occupancy of the alpha- and beta-sites with Co2+ results in u.v.-visible spectral changes . Spectroscopic studies with Co2+ have tentatively identified three cysteine residues at the beta-site and one at the alpha-site . Reaction with N-ethyl{14C}maleimide preferentially labels cysteine-130 at the alpha-site when Co2+ occupies the beta-site.

Biochem J, 1994 Jun 1, 300 ( Pt 2), 331 - 8
Molecular characterization of human stathmin expressed in Escherichia coli: site-directed mutagenesis of two phosphorylatable serines (Ser-25 and Ser-63); Curmi PA et al.; Stathmin, a probable relay protein possibly integrating multiple intracellular regulatory signals {reviewed in Sobel (1991) Trends Biochem . Sci . 16, 301-305}, was expressed in Escherichia coli at levels as high as 20% of total bacterial protein . Characterization of the purified recombinant protein revealed that it had biochemical properties very similar to those of the native protein . It is a good substrate for both cyclic AMP-dependent protein kinase (PKA) and p34cdc2, on the same four sites as the native eukaryotic protein . As shown by m.s., the difference in isoelectric points from the native protein is probably due to the absence of acetylation of the protein produced in bacteria . C.d . studies indicate that stathmin probably contains about 45% of its sequence in an alpha-helical conformation, as also predicted for the sequence between residues 47 and 124 by computer analysis . Replacement of Ser-63 by alanine by in vitro mutagenesis resulted in a ten times less efficient phosphorylation of stathmin by PKA which occurred solely on Ser-16, confirming that Ser-63 is the major target of this kinase . Replacement of Ser-25, the major site phosphorylated by mitogen-activated protein kinase in vitro and in vivo, by the charged amino acid glutamic acid reproduced, in conjunction with the phosphorylation of Ser-16 by PKA, the mobility shift on SDS/polyacrylamide gels induced by the phosphorylation of Ser-25 . This result strongly suggests that glutamic acid in position 25 is able to mimic the putative interactions of phosphoserine-25 with phosphoserine-16, as well as the resulting conformational changes that are probably also related to the functional regulation of stathmin.

J Commun Dis, 1994 Jun, 26(2), 103 - 8
Prevalence of intestinal parasites in rural areas of district Shahjahanpur, Uttar Pradesh; Virk KJ et al.; A stool survey was carried out in some of the villages of Dadraul and Bhawal Khera PHC's of district Shahjahanpur (Uttar Pradesh) . Out of 381 individuals examined 111 (29.2 per cent) were found positive for one or the other intestinal parasite . Ascaris lumbricoides superseded all the parasites by showing a positivity of 17.85 percent . Other parasites found were Hookworm, Hymenolepis nana, Tapeworm, Trichuris trichiura, Enterobius vermicularis, Entamoeba histolytica, E . coli and Giardia lamblia . Parasitic load was slightly higher in females (33.59 per cent) than males (28.18 per cent) . The highest positivity was encountered in the age groups between 6 to 14 years . This high prevalence of intestinal parasites may be due to the lack of awareness about personal cleanliness and hygiene and illiteracy among rural women . Majority of them had helminthic infections . It is concluded that in rural areas of district Shahjahanpur intestinal helminthic infections are more prevalent that protozoan infections.

Mil Med, 1994 Jun, 159(6), 445 - 8
Diarrheal disease aboard a U.S . Navy ship after a brief port visit to a high risk area; Haberberger RL et al.; In August 1988, a study was conducted to determine the etiology and risk factors associated with travelers' diarrhea among U.S . military personnel after a 5-day port visit to Alexandria, Egypt . Twenty-one percent of the 2,747 evaluated crew members of the USS John F . Kennedy reported an episode of acute diarrhea, which led to 155 sick-call visits and at least 110 lost man-days . The most common pathogen identified was enterotoxigenic Escherichia coli, and all isolated bacterial enteropathogens were sensitive to quinolone drugs . Independent risk factors for the development of diarrhea included: (1) consuming any meal ashore and specifically eating meats, desserts, or a buffet meal; and (2) a recent history of travelers' diarrhea . These data indicate that even brief port visits to developing countries pose a major threat to the health of U.S . shipboard personnel.

J Biochem (Tokyo), 1994 Jun, 115(6), 1185 - 9
Transcriptional attenuation and differential mRNA stability in the regulation of the Escherichia coli melibiose operon; Shimamoto T et al.; The organization of the melibiose operon of Escherichia coli is promoter-melA-melB . The amount of the product (alpha-galactosidase) of the first gene (melA) is much larger than that of the product (melibiose permease) of the second gene (melB) . Using the chloramphenicol acetyltransferase gene (cat) as reporter, we found that there was an element between melA and melB, which reduced the expression of the downstream gene, melB . This region contained a boxA-like sequence, which is known as a binding site for an attenuation factor, NusA . Northern hybridization analysis revealed that the ratio of melA mRNA and melAB mRNA was comparable with the ratio of the melA and melB products . We also found that the melA mRNA was about 3-fold more stable than the melAB mRNA . Experimental results obtained with a nusAts mutant suggested that the NusA protein is involved in the reduced expression of the melB gene . We conclude that the production ratio of alpha-galactosidase and melibiose permease is regulated at two levels: 1) transcription and 2) mRNA stability.

J Biochem (Tokyo), 1994 Jun, 115(6), 1172 - 7
Human aldolase C: characterization of the recombinant enzyme expressed in Escherichia coli; Kusakabe T et al.; To study the structure/function relationship and enzymatic properties of human aldolase C, we have constructed an Escherichia coli expression plasmid, pHAC11, for the isozyme . E . coli cells carrying this plasmid produced enzymatically active human aldolase C . The kcat and Km values for fructose-1,6-bisphosphate (Fru-1,6-P2) and fructose-1-phosphate (Fru-1-P) of the recombinant enzyme were found to be similar to those of authentic aldolase C from human brain . The Fru-1,6-P2/Fru-1-P activity ratio of the recombinant enzyme is approximately 13.5, which is comparable to that of the recombinant rat aldolase C, but is slightly higher than those of rat brain and hepatoma aldolases C . The substitution of Ser for the carboxyl-terminal Tyr (Tyr-363) of the recombinant enzyme caused a marked decrease in that of Fru-1,6-P2, with little change in that of Fru-1-P . The activity ratio changed from 13.5 for the normal enzyme to 3.8 for the engineered enzyme . Human aldolase C was found to form tetrameric hybrids with aldolase B in vivo when these enzymes were coexpressed in E . coli cells.

J Biochem (Tokyo), 1994 Jun, 115(6), 1135 - 40
Enhancement of serine-sensitivity by a gene encoding rhodanese-like protein in Escherichia coli; Hama H et al.; When cells of Escherichia coli are grown on lactate (or other carbon sources), an addition of serine to the medium causes growth inhibition . This growth inhibition is caused by inhibition by serine of homoserine dehydrogenase I, which is involved in threonine-isoleucine biosynthesis {Hama, H., Sumita, Y., Kakutani, Y., Tsuda, M., & Tsuchiya, T . (1990) Biochem . Biophys . Res . Commun . 168, 1211-1216} . We have cloned and sequenced genes which enhance the serine-sensitivity . Two open reading frames were found and designated as sseA and sseB . Introduction of either sseA or sseB gene, or both, into E . coli cells enhanced the serine-sensitivity . The sseA gene elicited stronger enhancement than sseB . The deduced amino acid sequence of SseA showed considerable similarity with that of bovine liver rhodanese, which catalyzes sulfur transfer from thiosulfate . We observed a twofold increase in rhodanese activity in E . coli cells harboring a plasmid carrying the sseA gene . The position of sseA in the genetic map is around 52' . However, sseA is different from cysM, which codes for O-acetylserine sulfhydrylase-B, an enzyme catalyzing sulfur transfer from thiosulfate to O-acetylserine, the map position of which is also around 52'.

J Biochem (Tokyo), 1994 Jun, 115(6), 1113 - 8
Autoregulation of transcription of the hupA gene in Escherichia coli: evidence for steric hindrance of the functional promoter domains induced by HU; Kohno K et al.; The molecular mechanism of autoregulation of expression of the hupA gene in Escherichia coli was examined . The promoter of the gene contains a palindromic sequence with the potential to form a cruciform DNA structure in which the -35 sequence lies at the base of the stem and the -10 sequence forms a single-stranded loop . An artificial promoter lacking the palindrome, which was constructed by replacing a 10 nucleotide repeat for the predicted cruciform arm by a sequence in the opposite orientation, was not subject to HU-repression . DNA relaxation induced by deleting HU proteins and/or inhibiting DNA gyrase in cells results in increased expression from the hupA promoter . We propose that initiation of transcription of the hupA gene is negatively regulated by steric hindrance of the functional promoter domains for formation of the cruciform configuration, which is facilitated at least in part by negative supercoiling of the hupA promoter DNA region . The promoter region of the hupB gene also contains a palindromic sequence that can assume a cruciform configuration . Negative regulation of this gene by HU proteins may occur by a mechanism similar to that operating for the hupA gene.

J Biochem (Tokyo), 1994 Jun, 115(6), 1075 - 82
Inactivation of Ca2+/calmodulin-dependent protein kinase II by Ca2+/calmodulin; Ishida A et al.; Incubation of calmodulin-dependent protein kinase II with Ca2+ and calmodulin resulted in a marked inactivation of the enzyme . Chelation of Ca2+ by EGTA or addition of calmodulin antagonists, W-7 or trifluoperazine, completely blocked this inactivation . The concentration required for the half-maximal inactivation, 127 microM, is three to four orders of magnitude higher than that for the half-maximal activation of the enzyme . The Ca2+/calmodulin-independent activity of the proteolytic fragment of the enzyme, whose calmodulin-binding site involved in the enzyme activation was deleted, was also decreased by incubation with Ca2+ and calmodulin . These results suggest that calmodulin-dependent protein kinase II possesses a second, low-affinity calmodulin-binding site, which is distinct from the calmodulin-binding site involved in the activation of the enzyme, and that the binding of calmodulin to the second binding site causes the inactivation of the enzyme . The inactivation by Ca2+/calmodulin was temperature-dependent . The addition of both 500 microM ADP and 10 mM MgCl2 markedly protected the enzyme against the inactivation, while such a marked protection was not observed after the addition of either of the two alone . The addition of 5 microM autocamtide-2, a synthetic substrate peptide containing the amino acid sequence of the autophosphorylation site (Thr286/Thr287 in alpha/beta, gamma, and delta isoforms) lying within the autoinhibitory domain, also protected the enzyme against the inactivation by Ca2+/calmodulin, while syntide-2, another synthetic substrate peptide corresponding to a phosphorylation site of glycogen synthase, did not protect it even at a concentration as high as 304 microM.

Shi Yan Sheng Wu Xue Bao, 1994 Jun, 27(2), 215 - 23
{Preparation of antibodies against zip product and the distribution of zip protein in Drosophila embryos}; Zhao DB et al.; Zip gene is required during the late neurogenesis of Drosophila . Molecular analysis of zip gene revealed that it encodes an integral membrane glycoprotein, functioning as a cellular recognition and/or adhesion molecule . It has been cloned into an expression plasmid pWR 590 . By expression of this plasmid in E . coli, a fusion protein of zip with lacZ has been purified and used to prepare polyclonal antibodies from rabbits . After being identified by West blotting, antibodies were used to react with the whole mount embryos of Drosophila . The results of this antibody labelling experiment showed that zip proteins are fundamentally synthesized after germ band shortening, supporting our previous predication that zip gene is involved in the late neurogenesis and that antibodies against zip recognize lateral neural fascicles as well as neurons in CNS, proving that zip protein expressed in CNS may play a role in the establishment and maintenance of neural fasciculation.

Wei Sheng Wu Xue Bao, 1994 Jun, 34(3), 220 - 5
{Both 987P and F41 fimbriae produced by the same enterotoxigenic Escherichia coli strain isolated from a piglet with diarrhea}; Gao S et al.; An enterotoxigenic Escherichia coli strain isolated from a piglet with diarrhea was examined for the presence of fimbriea 987P and F41 by a direct agglutination (with MAbs), an indirect immunofluorescence technique (MAbs as first antibodies), SDS-PAGE and Western blots (antisera IgG as probes) . Results of these techniques revealed that both 987P and F41 fimbrial adhesins were produced by the same strain, not by separate ones.

Wei Sheng Wu Xue Bao, 1994 Jun, 34(3), 206 - 12
{Site-directed mutagenesis of Lac Z gene in Escherichia coli and the kinetic properties of the mutated enzymes}; Chu X et al.; Glutamic acid at position of 537 of beta-D-galactosidase coded by Lac Z gene was substituted with Aspartic acid, Glutamine and Valine using synthetic oligonucleotide probes . Compared to native enzyme, the kcat values for substrate ONPG were 0.13%, 0.0006% and 0.0035% for Asp-537, Gln-537 and Val-537 mutated enzymes respectively . The Km values were of the same order of magnitude, either native or mutated enzymes . The substrate analog, IPTG was a strong inhibitor of each of the substituted enzymes, as in the case of native enzyme . The transition state analogs, 2-NH2-galactose and L-ribose were almost the same effects for the mutated enzymes as for the normal enzyme . The nucleophili, Azide, did not activate the mutated enzymes as in the case of Glu-461 substituted in beta-D-galactosidase . The effect of methanol on the mutated enzymes was less than on native enzyme . The order of the thermal stability was native enzyme > Asp-537 > Gln-537 > Val-537 enzymes . Overall, the evidence strongly supports the suggestion that Glu-537 is an essential residue of beta-D-galactosidase.

Wei Sheng Wu Xue Bao, 1994 Jun, 34(3), 184 - 90
{Isolation and sequencing of glucoamylase gene from a glucoamylase over producing strain}; Zhong L et al.; Chromosomal DNA was isolated from the mycelia of Aspergillus niger T21, a strain producing glucoamylase at a high level . Southern blot analysis indicated that the glucoamylase gene is situated on a 2.5kb EcoR I -EcoR V fragment . Chromosomal DNA was digested completely with EcoR I, EcoR V . The fragments in the range of 2.0-3.0kb were isolated through electrophoresis in agarose gel . The pooled fragments were ligated onto pBR322 vector prior to transformation into E . coli DH5 . Four glucoamylase-specific recombinants were screened by in situ hybridization from the transformants . Restriction mapping and sequencing for one of the four were performed . Data show that the glucoamylase gene from A . niger T21 is a 2.3kb fragment containing four intervening sequences in the coding region.

Mol Biochem Parasitol, 1994 Jun, 65(2), 247 - 58
Cloning and expression of the dihydrofolate reductase-thymidylate synthase gene from Trypanosoma cruzi; Reche P et al.; We have cloned, sequenced and expressed the Trypanosoma cruzi gene encoding the bifunctional protein dihydrofolate reductase-thymidylate synthase (DHFR-TS) . The strategy followed for the isolation of positive clones from a genomic library was based on the construction of a probe by the amplification of highly conserved sequences of the TS domain by the polymerase chain reaction . Translation of the open reading frame of 1563 bp yields a polypeptide of 521 amino acids with a molecular mass of 58829 Da . For heterologous expression of T . cruzi DHFR-TS in Escherichia coli, the entire coding sequence was amplified by polymerase chain reaction and cloned into the plasmid vector pKK223.3 . The presence of catalytically active DHFR-TS was demonstrated by complementation of the Thy- E . coli strain chi 2913 and the DHFR- Thy- E . coli strain PA414 . The gene is expressed as an active protein which constitutes approximately 2% of the total cell soluble protein . Recombinant bifunctional enzyme and the DHFR domain have been purified by methotrexate-Sepharose chromatography to yield 1-2 mg of active DHFR-TS per litre of culture . Southern and electrophoretic analyses using the coding sequence as probe indicated that the T . cruzi enzyme is encoded by a single copy gene which maps to two bands of approximately 990 kb and 1047 kb . It appears that T . cruzi is diploid for the DHFR-TS gene which is located on two different-sized homologous chromosomes.

Mol Biochem Parasitol, 1994 Jun, 65(2), 233 - 45
Molecular characterization and overexpression of the hypoxanthine-guanine phosphoribosyltransferase gene from Trypanosoma cruzi; Allen TE et al.; The hypoxanthine-guanine phosphoribosyltransferase (HGPRT) enzyme in Trypanosoma cruzi is a rational target for the treatment of Chagas disease . To evaluate the T . cruzi HGPRT in detail, the HGPRT gene (hgprt) was cloned from a genomic library of T . cruzi DNA and sequenced . Translation of the nucleotide sequence of the hgprt revealed an open reading frame of 663 bp that encoded a 25.5-kDa polypeptide of 221 amino acids . The T . cruzi HGPRT exhibited only 24%, 25%, and 21% amino acid sequence identity to its human, Plasmodium falciparum, and Schistosoma mansoni counterparts, respectively, but was 50% identical to the T . brucei HGPRT protein . Northern analysis of T . cruzi RNA revealed a 1.8-kb hgprt transcript, while Southern blots of genomic DNA suggested that hgprt was a single copy gene within the T . cruzi genome . The T . cruzi hgprt was inserted into the pBAce expression plasmid and transformed into Escherichia coli that are deficient in hypoxanthine and guanine phosphoribosylating activities . High levels of soluble, enzymatically active T . cruzi HGPRT were obtained, and this expression complemented the bacterial phosphoribosyltransferase deficiencies . The recombinant HGPRT was purified to apparent homogeneity by GTP-agarose affinity chromatography and recognized hypoxanthine, guanine, and allopurinol, but not adenine or xanthine, as substrates . The availability of the hgprt clone and large amounts of pure HGPRT protein provide a foundation for a structure-based drug design strategy for the treatment of Chagas disease.

J Diarrhoeal Dis Res, 1994 Jun, 12(2), 113 - 6
Evaluation of a non-radioactive chemiluminescent method for using oligonucleotide and polynucleotide probes to identify enterotoxigenic Escherichia coli; Abdul Alim A et al.; We compared the applicability of an enhanced chemiluminescent (ECL) method for using gene probes with that of radioactive probes to identify enterotoxigenic Escherichia coli (ETEC) in stools of Bangladeshi children with diarrhoea . Colony blots of E . coli isolates were hybridized using both {alpha-32P}-dCTP labelled and fluorescein-11-dUTP labelled polynucleotide and oligonucleotide gene probes for heat-labile enterotoxin (LT), and heat-stable enterotoxin (ST) . Analysis of 1,620 isolates obtained from 540 patients gave similar results by both radioactive and chemiluminescent probes . The ECL method was faster than the radioactive method . Both polynucleotide and oligonucleotide probes could be used by the ECL method . Hybridization and detection by the ECL method appeared to be a convenient alternative to radioactive probes for screening E . coli isolates for ETEC.

Hepatogastroenterology, 1994 Jun, 41(3), 217 - 21
Hepatic recovery after biliary drainage in experimental obstructive jaundice complicated by biliary infection; Kato S et al.; The effects of an association of biliary infection and bile stasis on the recovery process of the impaired structure and function of the cholestatic liver after biliary drainage were studied in experimental animals . Obstructive jaundice was induced by clamping a tube inserted into the common bile duct of rats, while at the same time Escherichia coli bacteria were introduced into the tube to induce biliary infection . After one week, the biliary obstruction was removed . After 14 days of biliary drainage, the impaired structure and function of the cholestatic liver without biliary infection were remarkably improved, having returned almost to normal, while in the cases with biliary infection, the impairments were far less improved, especially with respect to mitochondrial function . These results suggest that major hepatic surgery should not be employed in patients with obstructive jaundice in the presence of biliary infection and should be delayed until hepatic function has recovered sufficiently after biliary drainage.

Genetika, 1994 Jun, 30(6), 756 - 62
{Mutant alleles for radioresistance form the Escherichia coli strain Gam(r)444): cloning and preliminary characteristics}; Verbenko VN et al.; Mutant alleles Gamr, which are able to increase the resistance to radiation of Escherichia coli wild-type cells, were cloned from the hyperradioresistant mutant Gamr444 on a plasmid mini-Mu vector MudII4042 . The influence of recombinant plasmids on the sensitivity of wild-type and mutant (recA and htpR) cells to gamma-irradiation was studied . It was shown that the enhanced resistance of the Gamr444 strain to radiation was caused by mutations of two different classes, dominant and recessive . The cloned recessive mutation gamr12 increases resistance to radiation only after homogenization, that is, radiation-induced transfer from the plasmid to the chromosome, and it imposes constitutive expression of the heat-shock promoter htpG . Dominant mutant gamr alleles are active in the trans-position . A mutation-insertion into a chromosomal gene impaired by one of the dominant mutations, gamr18, was constructed . The insertion causes drastic cell radiosensitization on the recB sbcB background and probably disturbs the RecF pathway of recombination and repair . Dominant plasmids of the second type lead to the RecA-independent inhibition of DNA postirradiation degradation . The radioprotective action of recessive and dominant gamr mutations is additive.

Genetika, 1994 Jun, 30(6), 731 - 9
{Cloning and analysis of genetic determinants determining the production of microcin B2 and B27}; Basiuk EI et al.; Cloning of plasmid genes for the synthesis of two peptide broad-spectrum antibiotics-microcins B2 and B27- and for host cell immunity to their action was performed . Recombinant plasmids containing these genes were designated pBE108 and pVB27, respectively . Deletional derivatives of plasmid pBE108 and mutant plasmids were obtained via transposon Tn5 insertions, which did not determine production of microcin B2 and immunity to it . Phenotypic study and physical mapping of these plasmids demonstrated that a 4.2-kb DNA fragment is responsible for B2 microcin production; immunity is provided by a 1.4-kb DNA fragment . A 5-kb DNA fragment is necessary for microcin B27 synthesis and expression of immunity to its action . Homology between these fragments and with plasmid DNA for the synthesis of microcin B17 and immunity to it was found . Homology between plasmid genes determining synthesis of type B and C microcins and host cell immunity to them was not observed . The production of B27 microcin is controlled by the product of the ompR gene; B2 microcin synthesis does not depend on this product . The mutations recA and lexA increase the susceptibility of Escherichia coli cells to the action of microcins B2 and B27 but not microcin C51.

Biol Pharm Bull, 1994 Jun, 17(6), 767 - 72
Hydroxylation of phenylalanine and salicylate by stimulated polymorphonuclear leukocytes and the accelerating effect of glutathione on their hydroxylation; Fujimoto S et al.; Incubation of phorbol-myristate acetate-stimulated human polymorphonuclear leukocytes (PMNs) with phenylalanine and salicylate induced significant levels of formation of o- and m-tyrosines, and 2,3- and 2,5-dihydroxybenzoates (DHBAs), respectively, dependent on reaction time . Aromatic hydroxylation reactions were not inhibited by desferrioxamine, nor were they affected by the removal of trace ion contamination from the buffer solution used by treatment with conalbumin . Hydroxylation reactions were largely blocked by superoxide dismutase and hydroxyl radical (OH.) scavengers . The results of the present study suggest that the generation of OH . by human PMNs occurs during the respiratory burst . Hydroxylation of both phenylalanine and salicylate by stimulated human PMNs were significantly accelerated by incubation in the presence of the reduced form of glutathione (GSH) . Hydroxylation of phenylalanine by stimulated guinea pig PMNs in the presence of GSH was significantly inhibited by desferrioxamine, although the same hydroxylation in the absence of GSH was not affected . Hydroxylation of phenylalanine by the hypoxanthine (HX)-xanthine oxidase (XO) system by intact PMNs was significantly accelerated by the addition of GSH, although that in the absence of PMNs was largely inhibited . Desferrioxamine showed an inhibitory effect on hydroxylation by the HX-XO system in the presence, but not in the absence, of intact PMNs . The results suggest that the formation of OH . by stimulated PMNs is accelerated by GSH, based on the occurrence of the Harber-Weiss reaction catalyzed by transition metal ions liberated and reduced by GSH from PMNs, and by the effective accumulation of H2O2 by the GSH-induced inhibition of catalase.

Protein Expr Purif, 1994 Jun, 5(3), 291 - 5
Purification of recombinant Shiga-like toxin type I A1 fragment from Escherichia coli; Zollman TM et al.; Shiga-like toxin I A1 (Slt-IA1) is a RNA N-glycosidase which depurinates a specific adenosine of 28 S eukaryotic rRNA thus inhibiting protein synthesis and ultimately leading to cell death . We have overexpressed this protein in Escherichia coli using a high copy number plasmid and purified the enzyme to homogeneity using a three-step process . Slt-IA1 is released from the periplasm of cells using polymyxin B sulfate, precipitated with ammonium sulfate, and adsorbed to a Matrex Gel Green A dye-ligand agarose column . The enzyme is eluted from the Green A agarose as a single peak with 0.32 M NaCl . Slt-IA1 was purified approximately 1979-fold and routinely gave yields greater than 100% . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band with an apparent molecular weight of 28,000 . An isoelectric point of 5.1 was determined using analytical isoelectric focusing gels . In an in vitro protein synthesis inhibition assay, 0.02 pM of purified Slt-IA1 inhibited protein synthesis by 50%.

Protein Expr Purif, 1994 Jun, 5(3), 278 - 84
A method for the high-level expression of a parathyroid hormone analog in Escherichia coli; Oldenburg KR et al.; A recombinant parathyroid hormone analog, rPTH(1-34*), was obtained from Escherichia coli using a gene polymerization strategy . The PTH gene polymer contains up to 8 copies of the gene, each separated by a cleavable linker . The polymer was expressed at very high levels and formed inclusion bodies which could be easily isolated by low-speed centrifugation . A polyhistidine leader peptide allows rapid purification via nickel chelation chromatography of the PTH polymer solubilized from the inclusion bodies . Yields of greater than 500 mg/liter have been obtained . After isolating the polymer, monomeric rPTH(1-34*) is released from the polymer by chemical cleavage with cyanogen bromide . Following cyanogen bromide cleavage and high-performance liquid chromatography purification, highly purified, biologically active rPTH(1-34*) is obtained at a yield of approximately 300 mg/liter . This is a general strategy for the high-level production of a variety of peptides and small proteins.

Protein Expr Purif, 1994 Jun, 5(3), 252 - 8
Expression of trimeric human dUTP pyrophosphatase in Escherichia coli and purification of the enzyme; Climie S et al.; In order to rapidly purify human dUTPase, a cDNA fragment that encodes the enzyme was subcloned and expressed using the Escherichia coli plasmid vector pGEX2T . The resulting plasmid expressed high levels of a glutathione S-transferase-dUTPase fusion protein following induction with IPTG . Affinity chromatography was used to purify the fusion protein, and dUTPase was then released from the fusion protein by thrombin treatment . The purified dUTPase has two additional vector-encoded residues at the amino terminus (gly-ser), but they have no apparent effect on the activity of the enzyme since the recombinant dUTPase has catalytic properties similar to those reported for dUTPase purified from human cells (32.3 U/mg, kcat = 25 s-1, Km = 2.6 microM) . Enzyme activity was inhibited by 5-mercuri-dUTP and was shown to be sensitive to EDTA . Periodate-oxidized UTP had no effect on the activity of the enzyme, and dTTP caused only slight inhibition . The results of gel filtration experiments are consistent with a homotrimeric subunit composition for dUTPase . The ability to purify human dUTPase from E . coli should allow further characterization of the enzyme and provide material for the screening of potentially useful inhibitors.

Hum Gene Ther, 1994 Jun, 5(6), 731 - 44
In vivo evaluation of the safety of adenovirus-mediated transfer of the human cystic fibrosis transmembrane conductance regulator cDNA to the lung; Yei S et al.; Cystic fibrosis (CF) is a common, fatal hereditary disease resulting from mutations of the human cystic fibrosis transmembrane conductance regulator (CFTR) gene in which epithelial cells throughout the body manifest altered regulation of apical membrane chloride secretion . Although the disease affects multiple organs throughout the body, over 90% of patients die of complications of the lung involvement . The feasibility of adenovirus-derived vectors for in vivo delivery of the human CFTR cDNA to treat the pulmonary component of CF is currently being evaluated using in vitro and in vivo approaches . Defining the therapeutic window between biological efficacy and toxicity is an important part of this work . Here we present data regarding the preclinical evaluation of the safety of in vivo delivery of the human CFTR cDNA to the cotton rat airway epithelium using the replication-deficient adenoviral vector Av1Cf2 or a similar vector, Av1LacZ4, expressing the Escherichia coli LacZ gene as a histologic marker . Gene transfer to the respiratory epithelium was efficient, as demonstrated by in situ hybridization and histochemical staining . Administration of these vectors resulted in a mild, transient, dose-dependent cellular inflammatory response similar in character to that seen with adenovirus 5 (Ad5), but far less in intensity, which was not associated with structural lung damage or mortality . Av1Cf2 DNA sequences were easily detected in the lung after pulmonary administration, but could not be demonstrated in organs other than the lung . These preclinical observations suggest that adenovirus-mediated gene transfer to the airway epithelium can be achieved efficiently, but is accompanied by a dose- and time-dependent inflammation.(ABSTRACT TRUNCATED AT 250 WORDS)

Arch Esp Urol, 1994 Jun, 47(5), 519 - 21
{Retroperitoneal abscess following extracorporeal shock-wave lithotripsy}; Canto Faubel E et al.; An additional case of perinephric abscess post-ESWL is described . The clinical and radiological features and the therapeutic approach are discussed . The literature is briefly reviewed.

Alcohol Clin Exp Res, 1994 Jun, 18(3), 602 - 7
Suppression of tumor necrosis factor production by alcohol in lipopolysaccharide-stimulated culture; Nair MP et al.; Many studies have shown that alcohol consumption is associated with alteration in immune responses and increased incidence of infection in the host . Tumor necrosis factor (TNF) is a potent soluble mediator of immunoregulation and inflammation, and plays a very important role in host's defenses against infection and tumor . We propose that one of the mechanisms of alcohol-mediated immunosuppression may be due to a defect in the synthesis and release of the TNF . To determine this, we studied the direct effect of alcohol on lipopolysaccharide (LPS)-induced TNF production by whole blood and total mononuclear cell from normal subjects . Aliquots of blood samples (1 ml) or ficoll-hypaque separated total mononuclear cells (1 x 10(6)/ml) were cultured with different concentrations of either ethanol or acetaldehyde in the presence or absence of LPS for 4 hr at 37 degrees C . Plasma samples and culture supernatants were assayed for TNF levels in a bioassay using a TNF-sensitive WEHI 164 subclone 13 cell line . LPS at 10 micrograms/ml produced a maximal level of TNF compared with lower (1 micrograms/ml) or higher concentration (50 micrograms/ml) of LPS . Kinetics studies showed that an incubation time of 4 hr with LPS produced a maximum level of TNF production by blood . Alcohol, as low as 0.1% concentration, produced significant suppression of LPS-induced TNF production by whole blood, whereas alcohol at 0.2 and 0.3% concentrations were required to produce a significant suppression of TNF production by separated mononuclear cells . Anti-TNF-alpha antibodies significantly neutralized the LPS-induced TNF that suggests that blood monocytes may be the primary source of TNF production.(ABSTRACT TRUNCATED AT 250 WORDS)

Vet Immunol Immunopathol, 1994 Jun, 41(3-4), 229 - 39
Expression and purification of recombinant ovine interleukin-1 beta from Escherichia coli; Seow HF et al.; An expression vector bearing the gene segment encoding the mature form of ovine interleukin-1 beta (OvIL-1 beta) was constructed . This vector provided a rapid method for obtaining Escherichia coli derived recombinant OvIL-1 beta (rOvIL-1 beta) using the expression plasmid pGEX-2T . The level of expression of fusion protein in the soluble fraction was approximately 20% of the total accumulated proteins . Affinity purification by glutathione-Sepharose yielded a fusion protein and subsequent thrombin cleavage of this material yielded rOvIL-1 beta . The specific activity of the purified recombinant protein was 10(3)-10(4) times higher than the fusion protein . The rOvIL-1 beta was 10-100 times more potent than human interleukin-1 beta (HuIL-1 beta) in an ovine thymocyte proliferation assay, although they were of equal potency in the NOB-1/CTLL assay . This simple purification method, which produces purified rOvIL-1 beta with a high specific activity (approximately 10(8) U mg-1), will now make it possible to evaluate the in vivo effects of IL-1 beta in sheep.

Vet Microbiol, 1994 Jun, 40(3-4), 387 - 92
ELISA to determine anti-K99 pilus antibody in the sera of normal and diarrhoeic calves; Smith P et al.; An ELISA to detect circulating antibodies against K99 pili, a major attachment factor to intestinal epithelial cells of Escherichia coli in calves, was performed . Two methods of K99 pili purification were attempted . Best results in terms of purity of the K99 antigen were achieved following the method described by Karkhanis and Bhogal (1986) . This procedure included a heat shock at 65 degrees C during 25 min to release the pili and ultracentrifugation steps to purify the antigen . SDS-PAGE showed an 18 KDa major band, identified as the K99 pilus antigen after immunoblotting against reference antisera . The purified K99 antigen was then adsorbed to the ELISA microplates . High optical density was obtained in the ELISA using a pool of sera from immunized cows . No differences in antibody levels (P > or = 0.05) could be detected between clinically healthy calves and those showing diarrhoea.

Vet Microbiol, 1994 Jun, 40(3-4), 219 - 30
Prevalence of F107 fimbriae on Escherichia coli isolated from pigs with oedema disease or postweaning diarrhoea; Imberechts H et al.; The study comprises fifty 4 to 12 weeks old pigs that died from oedema disease or severe diarrhoea . Smears were prepared from the mucosa of duodenum, jejunum and ileum, and by immunofluorescence F107 fimbrial antigens were detected . E . coli strains were isolated from the intestines and were characterised by slide agglutination (serogroup and F107 fimbriae production), by their cytotoxicity for Vero cells, and by gene amplification (genes coding for the major F107 subunit FedA, the toxin causing oedema disease SLT-IIv, and enterotoxins LTI, STIa and STII) . F107 fimbriae were demonstrated in association with E . coli of serogroups O139:K12 and O141:K85a,b but not of serogroup O149:K91:F4a,c . Expression in culture of F107 fimbriae by some isolates gave additional evidence for production of these fimbriae by ETEC strains . The genetic determinant of SLT-Ilv was found in association with F107, and could not be detected in serogroup O149:K91:F4a,c . Gene fedA was demonstrated in two isolates which were devoid of SLT-IIv . Most isolates from cases of oedema disease belonged to serogroup O139:K12 and did not contain enterotoxin genes . Isolates from pigs that suffered from diarrhoea were serotyped O141:K85a,b or O149:K91:F4a,c, and carried at least two enterotoxin genes in their genomes . In a small proportion of the cases F107 antigens were demonstrated in intestinal smears although gene fedA was not detected in the corresponding isolates . The results confirm the importance of F107 fimbriae as virulence factor in oedema disease E . coli strains, but also demonstrate that F107 fimbriae can be found in association with postweaning diarrhoea isolates . In these latter strains enterotoxins were always demonstrated, irrespective of the presence of toxin SLT-IIv.

Vet Microbiol, 1994 Jun, 40(3-4), 209 - 18
Detection of Escherichia coli strains producing cytotoxic necrotizing factor type two (CNF2) by enzyme-linked immunosorbent assay; Oswald E et al.; Sheep and rabbit antisera were produced against lysates of E . coli strain 711 (pVir) . This K-12 strain carries the Vir plasmid which codes for Cytotoxic Necrotizing Factor type 2 (CNF2) . Immunoglobulin G (IgG) fractions of both immune sera were subsequently purified by a two-step precipitation method . To increase the specificity for CNF2, the sheep IgG preparation was extensively adsorbed against both a sonicated extract of isogenic K-12 strain 711 and intact phenol-treated cells of vaccine strain 711 (pVir) . An enzyme-linked immunosorbent assay (ELISA) was developed to detect clinical isolates of E . coli producing CNF2, using the final preparations of rabbit and sheep IgG in a double sandwich technique . The results obtained with this CNF2-ELISA were compared to those obtained with the conventional HeLa cell cytotoxicity assay . The testing of 133 E . coli strains (49 CNF2 positive strains and 84 negative strains) resulted in no false-negative and no false-positive . Therefore, the CNF2-ELISA offers a good alternative to the HeLa cell culture assay for the detection of CNF2-producing strains where facilities for and experience with cell cultures is lacking.

Protein Eng, 1994 Jun, 7(6), 777 - 82
Mutational analysis of the N-capping box of the alpha-helix of chymotrypsin inhibitor 2; elMasry NF et al.; The N-terminus of the helix of the chymotrypsin inhibitor 2 from barley (CI2) has an N-capping box (Ser at the first position in the helix and Glu at position 4) as well as a frequently found Glu at position 3 . The energetic importance of this motif has been studied by determining the free energy of unfolding of the wild-type and protein mutants derived from those residues using guanidinium chloride-induced denaturation and differential scanning microcalorimetry . Mutating N-cap residue Ser31 to either Ala or Gly destabilizes CI2 by 0.8-1 kcal mol-1 . Truncation of the box in the mutants SA31EA33EA34 or SG31EA33EA34 destablizes the protein by 1.5-2 kcal mol-1 . The N-capping box is an important motif in stabilizing proteins and delineating the beginning of alpha-helices in the pathway of protein folding.

Protein Eng, 1994 Jun, 7(6), 769 - 75
Modulation of the enzymatic activity of papain by interdomain residues remote from the active site; Altschuh D et al.; The two main catalytic residues Cys25 and His159 of the monomeric cysteine protease papain are located on different walls of a cleft formed by two domains . This topology suggests a possible relationship between relative domain organization and catalytic mechanism . The effect on enzymatic parameters of structural modifications at various locations of the two-domain interface of papain was examined by individual or double replacements by Ala of pairs of interacting residues . Most modifications had no effect on enzyme activity . However, the enzyme's substrate turnover (kcat) decreased following simultaneous alteration of the two most conserved residues, forming an apolar contact located 15 A away from the active site . The pH activity profile of the double mutant was unchanged, indicating a conserved ionization state of the active site thiolate-imidazolium ion pair . This state is strongly dependent on the distance separating the two residues, thus suggesting that the active site geometry has not been significantly altered . Efficient enzymatic activity in papain requires more than a correct active site geometry and is influenced by domain packing properties in a region remote from the active site.

Pharmacol Toxicol, 1994 Jun, 74(6), 351 - 8
Pharmacological characterization of a biosynthetic trisulfide-containing hydrophobic derivative of human growth hormone: comparison with standard 22 K growth hormone; Thomsen MK et al.; Growth hormone is the classical anabolic hormone which promotes organ growth after binding to somatogenic target cell receptors, present in various target tissues . The present study elucidated the pharmacological characteristics in vitro and in vivo of human growth hormone and a recently identified by-product of a recombinant human growth hormone preparation; i.e . a trisulfide-containing (cys 182-cys 189) hydrophobic, folding derivative of growth hormone, hydrophobic derivative-growth hormone . Standard growth hormone and hydrophobic derivative-growth hormone possessed similar characteristics in vitro, both as regards binding to the somatogenic receptor on the human IM-9 cell line, and the prolactin receptor-mediated proliferation of rat Nb2 cells . This indicates that no change occurs in the binding characteristics in spite of a change in conformation of the molecule . Using an ELISA assay that detected standard and hydrophobic derivative-growth hormone equally well, the plasma pharmacokinetical profiles of the preparations following a single intravenous or subcutaneous dose were indistinguishable . Thus, following initial disposition of hydrophobic derivative-growth hormone and standard growth hormone into a volume, V1, of one to two times the plasma volume, almost 90% of either compound disappeared from plasma during the alpha-phase of the plasma decay curve . Similar half-lives of 4-5 min . were found for hydrophobic derivative-growth hormone and standard growth hormone during this phase, indicating rapid removal of drug from the circulation . Also, the AUC and Cmax values for standard and hydrophobic derivative-growth hormone did not differ following intravenous or subcutaneous administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Oral Microbiol Immunol, 1994 Jun, 9(3), 166 - 73
Stress response of Porphyromonas gingivalis; Lu B et al.; The heat shock response of Porphyromonas gingivalis was examined by 1- and 2-dimensional polyacrylamide gel electrophoresis after metabolic labeling with {14C}-amino acids . When P . gingivalis cells were shifted from 37 degrees C to 42 degrees C, elevated synthesis of 4 proteins with the apparent molecular weight of 92, 80, 74, and 62 kDa was observed, and synthesis of a 50-kDa protein decreased during heat shock . The 74- and 62-kDa proteins were identified as homologs of Escherichia coli DnaK and GroEL respectively by Western immunoblotting . On 2-dimensional Western blots, 2 forms of DnaK and 4 forms of GroEL were identified due to their slightly different isolelectric points . dnaK and groEL gene homologs were identified in several P . gingivalis strains and some other black-pigmented oral species . DnaK and GroEL homolog proteins were induced when P . gingivalis cells were challenged by ethanol . These proteins were not induced by oxidative stress or change in pH.

Mol Microbiol, 1994 Jun, 12(6), 973 - 84
Regulation of acetyl phosphate synthesis and degradation, and the control of flagellar expression in Escherichia coli; Pruss BM et al.; We investigated the relationship between Escherichia coli flagellar expression and the regulation of acetyl phosphate synthesis and degradation . Using cells either wild type for acetyl phosphate metabolism or defective for phosphotransacetylase or acetate kinase, or both, we measured flagellar expression and the intracellular concentration of acetyl phosphate relative to growth phase and temperature . Under the conditions tested, we found that elevated levels of acetyl phosphate corresponded to inhibition of flagellar synthesis . To extend these observations, we measured the intracellular concentration of acetyl-CoA, the level of expression from the pta and ackA promoters, and the activities of phosphotransacetylase and acetate kinase derived from cell lysates . Relative to increasing culture density, acetyl-CoA levels and expression from both the pta and ackA promoters decreased . Relative to increasing temperature, expression from the ackA promoter decreased and phosphotransacetylase activity increased . In contrast, temperature had little or no effect on either acetate kinase activity or expression from the pta promoter . We propose that cells regulate intracellular acetyl phosphate concentrations relative to growth phase and temperature by modulating the availability of acetyl-CoA, the expression of ackA, and the activity of phosphotransacetylase.

Mol Microbiol, 1994 Jun, 12(6), 1005 - 12
Light-regulated promoters from Synechocystis PCC6803 share a consensus motif involved in photoregulation; Marraccini P et al.; A library of Synechocystis PCC6803 (S.6803) DNA cloned in front of the promoterless cat reporter gene of the plasmid pFF11 was used to transform S.6803 to high light-dependent resistance to chloramphenicol . In five clones harbouring a stably replicating pFF11-derived plasmid, this phenotype occurred independently of the photosystem II electron transport and resulted from the correlated increase of CAT activity level and cat mRNA accumulation . The five promoter inserts contained no Escherichia coli sigma 70 promoter element, in agreement with their lack of activity in this organism, but shared two conserved motifs . Two secondary mutations, which restored light-regulated promoter activity to an inactive mutant of the smallest insert, mapped within one of the common motifs, emphasizing the probable involvement of this element in photoregulation.

J Appl Physiol, 1994 Jun, 76(6), 2840 - 5
In vivo adenovirus-mediated gene transfer to lungs via pulmonary artery; Lemarchand P et al.; On the basis of the knowledge that the pulmonary and bronchial circulations have extensive anastomoses, we hypothesized that gene transfer to the endothelium of both pulmonary and bronchial circulations might be achieved with replication-deficient recombinant adenovirus (Ad) vectors administered to the pulmonary circulation . To evaluate this concept, the right upper lobe branches of the sheep pulmonary artery and vein were temporarily occluded and a replication-deficient recombinant Ad vector containing the Escherichia coli lacZ reporter gene coding for beta-galactosidase (beta-Gal) was infused into the lumen of the occluded pulmonary artery . After 15 min, the pulmonary circulation was restored, and 1 or 4 days later the lungs were evaluated by histochemical analysis for beta-Gal activity . Gene transfer and expression were positive in 13 of 17 evaluated sheep . No beta-Gal activity was detected in any category of cells of uninfected lobes . As hypothesized, beta-Gal activity was detected in endothelial cells of the right upper lobe pulmonary and bronchial circulations . Unexpectedly, gene transfer was also observed in epithelial cells of the alveoli and the airways (bronchi and bronchioles) as well as in the epithelium of submucosal glands . These studies demonstrate that it is possible to use Ad vectors for transfer and expression of genes to lung parenchymal cells served by both the pulmonary and bronchial circulations . Furthermore, whereas administration of such vectors via the airways results in gene transfer only to the epithelium, pulmonary artery administration permits gene transfer to both endothelium and epithelium, thus expanding the target range of Ad gene transfer to the lungs.

Genes Dev, 1994 Jun 1, 8(11), 1344 - 55
Functional domains within FEN-1 and RAD2 define a family of structure-specific endonucleases: implications for nucleotide excision repair; Harrington JJ et al.; Structure-specific nucleases catalyze critical reactions in DNA replication, recombination, and repair . Recently, a structure-specific endonuclease, FEN-1, has been purified and shown to cleave DNA flap structures . Here, we describe the cloning of the murine FEN-1 gene . The nucleotide sequence of FEN-1 is highly homologous to the Saccharomyces cerevisiae genes YKL510 and RAD2 . We show that YKL510 and a truncated RAD2 protein are also structure-specific endonucleases . The substrate specificity of the truncated RAD2 protein implicates branched DNA structures as important intermediates in nucleotide excision repair . The polarity of these branched DNA structures allows us to predict the placement of DNA scissions by RAD2 and RAD1/RAD10 in this reaction.

Mol Cell Endocrinol, 1994 Jun, 102(1-2), 77 - 84
Production of antibodies to the human thyrotropin receptor and their use in characterising eukaryotically expressed functional receptor; Harfst E et al.; The structure of the human thyrotropin receptor expressed as a recombinant protein in eukaryotic cells was investigated by immunochemical and functional means using two types of polyclonal rabbit antisera: one raised against the large N-terminal extracellular region (residues 1-415) expressed in E . coli and the other raised against a synthetic peptide (residues 313-330) . Both types of antisera gave similar results, with the former being more effective . As expected from the lack of conformation of the immunogens, the antisera worked well in immunoblotting . Less predictably, the antisera also recognised the functional receptor in its native state (detected by flow cytofluorimetry and immunoprecipitation), and inhibited the binding of thyrotropin . Thus the region 313-330 is on the outside of the receptor molecule and falls within, or close to, the binding site of thyrotropin . None of the antisera stimulated cAMP production, showing that this is a very special property, largely restricted to certain human autoantibodies . The antisera were used to immunoprecipitate radioiodinated proteins from Chinese hamster ovary cell (CHO) lines expressing recombinant receptor . The most abundant and reproducible cell-surface molecule that correlated with the presence of full-length functional receptor was a glycopolypeptide of approximately 100 kDa, of which 15 kDa is attributable to carbohydrate, in good agreement with the size predicted for the polypeptide from the cDNA sequence . Three other molecular species were also variably detected at the cell surface: 55 kDa, 180 kDa and large molecular weight material at the top of the polyacrylamide gel.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Cell Endocrinol, 1994 Jun, 102(1-2), 39 - 44
A limited cytoplasmic region of the prolactin receptor critical for signal transduction; Edery M et al.; Prolactin receptors (PRL-R) are members of the cytokine receptor superfamily, which have in common, an absence of any known consensus sequence for signal transduction in their cytoplasmic domains . Four areas of high sequence homology have been identified in the cytoplasmic domains of PRL and growth hormone (GH) receptors, which may be important for signal transduction . The aim of this study was to investigate the role of these cytoplasmic regions in the functional activity of the PRL-R . Several mutant forms of PRL-R were constructed either by truncation or by deletion of the cDNA . Biological activities of these mutant receptors were assayed in CHO cells using a functional assay consisting in the co-transfection of PRL-R cDNA, along with a PRL responsive promoter fused to the coding sequence of the chloramphenicol acetyl transferase (CAT) gene . Progressive truncation of the cytoplasmic domain led to a progressive loss of ability to transactivate the CAT gene . Fully active PRL-R could be obtained when 217 of 358 aa of the cytoplasmic domain were present . Deletion of the first region of homology with the GH-R (residues 245-267) abolished the functional activity of PRL-R, whereas deletion of the second region of homology (residues 322-333) was without effect . These results indicate that a critical cytoplasmic region of 23 residues proximal to the transmembrane domain is essential for PRL signal transduction . There is strong homology within an 8-residue segment of this region with other members of the cytokine receptor superfamily, suggesting it contains a sequence necessary for signal transduction.

Mol Cell Endocrinol, 1994 Jun, 102(1-2), 111 - 7
Differential binding and activation of thyroid hormone response elements by TR alpha 1 and RXR alpha-trap heterodimers; Miyamoto T et al.; Thyroid hormone receptor (TR) forms homo- and heterodimers on various thyroid hormone response elements (TREs) . We wished to clarify the relationship of homo- and heterodimer binding to TREs and their trans-activation . We investigated binding characteristics in gel mobility shift assays using synthetic direct repeat (DR) TREs having the consensus motifs separated by different oligonucleotide gaps, and we compared binding to trans-activation mediated via the direct repeat TRE . HTR alpha 1 purified from E . coli formed a monomer and homodimer on DR-TRE +0 to +5 but binding did not closely correlate with T3-dependent trans-activation . When RXR alpha expressed in COS 1 cell was added to purified TR alpha 1 in the gel shift assays, TR/RXR heterodimers were formed, and binding of heterodimers correlated highly with the level of trans-activation . These results strongly suggest that TR/TRAP heterodimers mediate the effect of thyroid hormone on DR-TREs . We also found T3-dependent disruption of homodimer formation on DR +0 to +2 and that T3 increased heterodimer formation on these TREs.

Eur Respir J, 1994 Jun, 7(6), 1131 - 7
Leukotriene receptor antagonism prevents lung protein leakage and hypoxaemia in a septic cat model; Schutzer KM et al.; Products of the arachidonic acid cascade have been found to play an important role in the pathophysiology in experimental shock and in ARDS . The effect of cysteinyl-leukotriene (cLT) blockade on the development of respiratory failure during septic shock was examined . Ventilated cats received an infusion of Escherichia coli bacteria . Pretreatment was given with diethylcarbamazine (DEC), a leukotriene synthetase inhibitor, or a new potent cLT receptor antagonist, ICI 198,615 . With a gamma camera, the distributions of plasmatransferrin radiolabelled with indium-113m chloride (113mIn) and erythrocytes radiolabelled with technetium-99m (99mTc) were measured over the lungs . A normalized slope index (NSI) reflecting protein leakage, based on the transferrin extravasation, was calculated . In the nonseptic control group (n = 7) NSI was 4.4 x 10(-4) +/- 0.7 x 10(-4).min-1 (mean +/- SEM) . Unpretreated septic animals (n = 7) showed a protein leakage after bacterial infusion, with a NSI of 34 +/- 3.5 x 10(-4).min-1 . Pretreatment with DEC (n = 6) significantly reduced NSI to 16 +/- 1.5 x 10(-4).min-1 . In the group pretreated with ICI 198,615 (n = 8), NSI was 9 +/- 1.2 x 10(-4).min-1 . Arterial oxygen tension (PaO2) remained at baseline level of 20 +/- 1.0 kPa during the experimental period in both the nonseptic control group and the ICI 198,615 pretreated group . In the unpretreated septic group, PaO2 fell progressively from a preseptic value of 21 +/- 0.9 to 12 +/- 1.5 kPa after 3 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Comput Appl Biosci, 1994 Jun, 10(3), 295 - 9
Steady-state modelling of metabolic flux between the tricarboxylic acid cycle and the glyoxylate bypass in Escherichia coli; el-Mansi EM et al.; In this study, mathematical modelling, using the computer package MetaModel, was employed to calculate the steady-state fluxes and the concentration of various metabolites of the central pathways during growth of Escherichia coli on acetate . This package also enabled us to formulate the matrices of the elasticity coefficients and the control and response coefficients under different steady states . In this paper, we have assessed the relative contribution of the competing enzymes at the metabolic junction of isocitrate, i.e . isocitrate dehydrogenase (ICDH) and isocitrate lyase (ICL), to the overall distribution of carbon flux among the enzymes of the central pathways thus extending the pioneering work of Walsh and Koshland on the partition of carbon flux between the two metabolic cycles of the tricarboxylic acid and the glyoxylate bypass . This study revealed that ICDH is not 'rate limiting' during growth on acetate and that flux through ICL is essential not only to replenish the central pathways with biosynthetic precursors but also to sustain a high intracellular level of isocitrate . Furthermore, above certain threshold concentration of ICL, the Krebs cycle and the glyoxylate bypass work in concert and the partition of carbon flux between ICDH and ICL is no longer a problem.

Comput Appl Biosci, 1994 Jun, 10(3), 273 - 5
MC-Fit: using Monte-Carlo methods to get accurate confidence limits on enzyme parameters; Dardel F; A program is described for estimating enzymatic parameters from experimental data using Apple Macintosh computers . MC-Fit uses iterative least-square fitting and Monte-Carlo sampling to get accurate estimates of the confidence limits . This approach is more robust than the conventional covariance matrix estimation, especially in cases where experimental data is partially lacking or when the standard error on individual measurements is large . This happens quite often when analysing the properties of variant enzymes obtained by mutagenesis, as these can have severely impaired activities and reduced affinities for their substrates.

Curr Biol, 1994 Jun 1, 4(6), 477 - 87
Symmetry and asymmetry in the function of Escherichia coli integration host factor: implications for target identification by DNA-binding proteins; Werner MH et al.; BACKGROUND: Escherichia coli integration host factor (IHF) is a DNA-binding protein that participates in a wide variety of biochemical functions . In many of its activities, IHF appears to act as an architectural element, dramatically distorting the conformation of bound DNA . IHF is a dimer of non-identical subunits, each about 90 amino acids long . One dimer interacts specifically with a 30 base pair (bp) target, but well-conserved sequences are found in only half of this binding site . Thus, the IHF-DNA system has long been viewed as a paradigm of asymmetry in a protein-DNA interaction . RESULTS: We have isolated the subunits of IHF and show that either subunit is capable of specifically recognizing natural IHF-binding sites and supporting lambda site-specific recombination in vitro . Mobility shift and footprinting data indicate that the isolated subunits interact with DNA as homodimers . We also describe the design of symmetric duplexes to which heterodimeric and homodimeric IHFs can bind by recognizing specific sequences . CONCLUSIONS: Our in vitro manipulation of the IHF system demonstrates that binding and bending of target DNA can be accomplished symmetrically . The prevalence of asymmetry found for this system in nature suggests that additional selective forces may operate . We suggest that these follow from the disparity between the size of the DNA that IHF protects (30 bp) and the length of DNA that the protein can initially contact (10 bp) . This disparity implies that an IHF target is recognized in stages and may dispose the part of the protein-DNA system used for initial recognition to evolve distinctly from the remainder of the interaction surface . We suggest that a limitation in the length of DNA that can be initially contacted is a general property of DNA-binding proteins . In that case, many proteins can be expected to identify complex targets by step-wise, rather than simultaneous, contact between sequence elements and DNA-binding domains.

FEMS Immunol Med Microbiol, 1994 Jun, 9(1), 23 - 7
Molecular epidemiology of Legionella pneumophila serogroup 1 by ribotyping with a non-radioactive probe and PCR fingerprinting; Matsiota-Bernard P et al.; Hybridization with acetylaminofluorene-labelled 16 + 23 S rRNA from Escherichia coli was used to detect DNA polymorphism among Legionella pneumophila serogroup 1 isolates . Isolates from unrelated patients showed at least four different rRNA restriction patterns, whereas those from related patients showed a single pattern . Amplification of genomic regions with an arbitrary primer by polymerase chain reaction was used to further analyze the isolates . Related isolates showed closely related patterns while unrelated isolates displayed six distinct patterns . We could differentiate the majority of unrelated isolates with the combination of the patterns obtained with the ribotyping and the PCR fingerprinting, while strains from the same outbreak remained highly related . The ribotyping and the PCR fingerprinting are proposed as useful and easy to perform epidemiological markers of L . pneumophila serogroup 1 infection.

PCR Methods Appl, 1994 Jun, 3(6), 338 - 45
Cloning and analysis of PCR-generated DNA fragments; Costa GL et al.; Methods are presented for the improved yield and analysis of blunt-ended cloning of PCR-generated DNA fragments . We show that Pfu DNA polymerase polishing of Taq DNA polymerase-generated fragments increases the yield and efficiency of cloning . Using a triple primer set consisting of two outside, asymmetrically distanced primers and one fragment-specific primer, both the presence and orientation of cloned inserts can be determined . Application of these methods allows the generation and cloning of a fragment in 1 day and the analysis of putative clones the next, thereby saving a substantial amount of both time and effort.

PCR Methods Appl, 1994 Jun, 3(6), 320 - 31
Specific immunoglobulin cDNA clones produced from hybridoma cell lines and murine spleen fragment cultures by 3SR amplification; Stillman CA et al.; The isothermal 3SR amplification method has been employed to assist in cloning the VL and VH genes from cells of hybridomas and splenic fragment cultures expressing antibodies for phosphorylcholine (PC) and estradiol (E2), respectively . As a first step, pools of degenerate primer pairs were identified complementary to immunoglobulin light and heavy chain variable (V) genes and capable of amplifying immunoglobulin RNA specifically at 42 degrees C . To evaluate the functionality of the 3SR-cloned immunoglobulin genes, anti-PC VH and VL cDNAs were joined together to form a single chain (sc) antibody construct and were expressed in Escherichia coli under the regulation of the alkaline phosphatase (phoA) promoter . Similarly, the combination of a murine spleen fragment and 3SR methodologies were employed to clone a selected pool of cDNAs for cultures producing anti-estradiol antibodies . This approach of using the murine spleen fragment and 3SR isothermal amplification offers the advantages of B-cell follicle architecture for antigen-driven B-cell maturation and proliferation and RNA-specific amplification, respectively . The potential utility of these advantages for the production of monoclonal antibodies and for providing the capability of studying memory B-cell development are discussed.

Biochem Med Metab Biol, 1994 Jun, 52(1), 36 - 44
Characterization of wild-type human medium-chain acyl-CoA dehydrogenase (MCAD) and mutant enzymes present in MCAD-deficient patients by two-dimensional gel electrophoresis: evidence for post-translational modification of the enzyme; Bross P et al.; Two-dimensional gel electrophoresis was used to study and compare wild-type medium-chain acyl-CoA dehydrogenase (MCAD; EC 1.3.99.3) and mis-sense mutant enzyme found in patients with MCAD deficiency . By comparing the patterns for wild-type and mutant MCAD expressed in Escherichia coli or in eukaryotic COS-7 cells we demonstrate that variants with point mutations changing the net charge of the protein can be readily resolved from the wild-type protein . After expression of the cDNA in eukaryotic cells two spots representing mature MCAD can be distinguished, one with an isoelectric point (pI) corresponding to that obtained for the mature protein expressed in E . coli and another one shifted to lower pI . This demonstrates that MCAD protein is partially modified after transport into the mitochondria and removal of the transit peptide . The observed pI shift would be compatible with phosphorylation of one aspartic acid residue per monomer . Comparison of pulse labeling and steady-state amounts of MCAD protein in overexpressing COS-7 cells confirms that K304E MCAD is synthesized and transported into mitochondria in amounts similar to the wild-type protein, but is degraded much more readily . For wild-type MCAD, the spot representing the nonmodified form predominates after pulse labeling while that representing the modified form is relatively stronger in steady state, demonstrating that the modification occurs in mitochondria after the transit peptide has been removed . For K304E mutant MCAD, the nonmodified spot is relatively stronger both in pulse labeling and in steady state, indicating that either the efficiency of modification or the stability of the modified form is affected by the K304E mutation.(ABSTRACT TRUNCATED AT 250 WORDS)

Biologicals, 1994 Jun, 22(2), 85 - 94
The molecular biology of production cell lines; Simonsen CC et al.; The emergence of a wide variety of biological expression systems for the large-scale production of therapeutic proteins has shifted the focus from vectors to host organisms . Although expression systems now span bacteria, fungi, plants, insects, and mammalian cells, the vast majority of recombinant-derived biopharmaceuticals at the present time have been produced in Escherichia coli and in mammalian cells . This promises to change as the economic benefits of the newer systems permit the development of a new generation of proteins heretofore considered unfeasible for commercial development . Despite the impressive results which have been observed for many of the newer systems, there are many commercial considerations which suggest that E . coli and CHO cell expression systems may continue to dominate the manufacture of biopharmaceuticals for a long time to come.

Int J Biochem, 1994 Jun, 26(6), 787 - 97
Kinetic analysis of reversible closed bicyclic enzyme cascades covering the whole course of the reaction; Varon R et al.; A kinetic analysis of the closed bicyclic enzyme cascades is presented . 1 . It includes the dependence on time from the onset of the reaction, of the concentration of the modified and unmodified enzyme species involved and the time course equations of the modificational fractions of the interconvertible enzymes . 2 . The transient phase equations obtained allow the definition of new regulatory modification properties . 3 . The expressions for concentrations of the unmodified and modified forms of the interconvertible enzymes, as well as those of the fractional modifications in the steady state are derived as particular cases of the general equations . 4 . These steady state expressions coincide with those obtained by other authors . 5 . The analytical results obtained are discussed in relation to the Escherichia coli glutamine synthetase cascade.

FEMS Microbiol Lett, 1994 Jun 1, 119(1-2), 129 - 35
Heat shock protein 60 (GroEL) from Porphyromonas gingivalis: molecular cloning and sequence analysis of its gene and purification of the recombinant protein; Maeda H et al.; Porphyromonas gingivalis is associated with human periodontal disease . We cloned and sequenced the gene for heat shock protein 60 (GroEL, HSP60) from P . gingivalis FDC381 . The identified clone carried a 2.6 kb DNA fragment which contained two open reading frames (ORFs) encoding a 9.6- and a 58.4-kDa protein . The translated amino acid sequence of these ORFs showed a high degree of homology with known sequences for GroES and GroEL from several bacterial species and humans . Escherichia coli carrying this clone expressed a 65-kDa protein which was recognized by anti-Mycobacterium leprae HSP60 monoclonal antibody . We purified the 65-kDa protein by DEAE-sepharose chromatography and hydroxyapatite chromatography . This protein was immunogenic and was recognized by sera from a number of patients with periodontal disease . This immunological reactivity and the existence of molecular mimicry between the P . gingivalis GroEL and other HSP homologs may indicate an important role for this molecule in periodontal lesion.

FASEB J, 1994 Jun, 8(9), 661 - 4
Active deglycosylated mammalian gamma-glutamyl transpeptidase; Smith TK et al.; gamma-Glutamyl transpeptidase, a highly glycosylated heterodimeric enzyme that is usually attached to the external surface of cell membranes, is of major importance in the metabolism of glutathione . The enzyme, which has been isolated from many animal sources, contains a large amount of carbohydrate, which is linked to both protein subunits . Previous work has not shown whether such carbohydrate is needed for enzyme activity nor indicated its functional role . Notably, gamma-glutamyl transpeptidase isolated from Escherichia coli, which exhibits about 80% amino acid sequence homology with the rat enzyme, has only about 0.1% of its specific enzymatic activity and is not glycosylated . Here we treated the highly glycosylated gamma-glutamyl transpeptidases isolated from rat and pig kidneys with a mixture of glycosidases and then separated two completely active gamma-glutamyl transpeptidase fractions from each species . One fraction was completely devoid of carbohydrate and was fully active as compared with the respective isolated enzymes, but differed in solubility and stability . The other fraction, which contained 10-20% of the initially bound carbohydrate, exhibited a marked increase in susceptibility to proteases . The oligosaccharide chains of gamma-glutamyl transpeptidase may protect against protease action (including self-destruction by the inherent protease activity of the light subunit) during synthesis of the active enzyme from its single chain precursor, as well as after enzyme synthesis.

Infect Immun, 1994 Jun, 62(6), 2633 - 8
F17-like fimbriae from an invasive Escherichia coli strain producing cytotoxic necrotizing factor type 2 toxin; el Mazouari K et al.; The F17b fimbriae encoded by the transmissible virulence plasmid Vir, also coding for cytotoxic necrotizing factor type 2, were characterized . A 5.7-kb region of Vir mediates in vitro N-acetylglucosamine-sensitive adhesion to calf intestinal villi . Sequence analysis revealed that this region codes for a structural subunit and an adhesin closely related to the F17-A and F17-G proteins encoded by the F17 fimbrial gene cluster . The F17b-A gene presents an open reading frame of 540 bp encoding a polypeptide of 180 amino acids with a putative signal peptide of 21 residues . The mature protein shows an identity of 74% with the F17-A structural subunit . This 20-kDa protein is recognized by antiserum directed against F17 fimbriae . The F17b-G gene shows an open reading frame of 1,029 bp encoding a polypeptide of 343 amino acids with a putative signal peptide of 22 residues . The F17b-G polypeptide exhibits 95% identity with the F17-G adhesin . The functional homology of the gene products was further confirmed by demonstrating that mutants in the F17-A gene can be complemented by the F17b-A gene and vice versa . These results prove that fimbriae belonging to the F17 family are also found on pathogenic Escherichia coli strains other than enterotoxigenic isolates producing heat-labile or heat-stable enterotoxin.

Infect Immun, 1994 Jun, 62(6), 2178 - 86
Effect of salicylate, bismuth, osmolytes, and tetracycline resistance on expression of fimbriae by Escherichia coli; Kunin CM et al.; Adherence of Escherichia coli is facilitated by fimbriae and several outer membrane proteins (OMPs) . Hypertonic conditions, salicylate, and Mar mutations are known to reduce OmpF expression . We speculated that OMPs involved in export or assembly of fimbrial subunits might be similarly affected . To explore this hypothesis, E . coli expressing P, type 1, S, colonization factor antigen I (CFA/I), or CFA/II fimbriae was grown in the presence of salicylate, bismuth salts, NaCl, and nonfermented sugars . Tetracycline-resistant clones were derived from several P-fimbriated strains . The bacteria were tested for the ability to agglutinate erythrocytes, yeast cells, and alpha-D-Gal(-4)-beta-D-Gal-bonded latex (Gal-Gal) beads and were examined for fimbriae by electron microscopy . Hyperosmolar conditions decreased fimbrial expression for all strains . Expression of P fimbriae by pyelonephritic strains, all of which were OmpF+, was reversibly repressed by salicylate and bismuth salts . CFA strains were similarly affected . Tetracycline-resistant P-fimbriated strains were OmpF deficient, were unable to agglutinate erythrocytes and Gal-Gal beads, and lacked fimbriae as observed by electron microscopy . Strains with plasmid-encoded P-fimbrial genes did not demonstrate OmpF on polyacrylamide gel electrophoresis profiles and were not affected by salicylate . The type 1-fimbriated phenotype was not affected by salicylate or bismuth unless the strains also expressed P fimbriae . S-fimbriated strains were not affected . The mechanism by which salicylates, bismuth salts, and tetracycline resistance inhibit or modulate the expression of P fimbriae may be mediated through OmpF and other OMPs.

Braz J Med Biol Res, 1994 Jun, 27(6), 1291 - 7
Iron-regulated outer-membrane proteins of strains of avian septicemic Escherichia coli; Vidotto MC et al.; 1 . Outer-membrane protein patterns of Escherichia coli recovered from the peritoneal cavities of infected guinea pigs and grown in medium M9 containing 2,2'-dipyridyl were studied by polyacrylamide gel electrophoresis (SDS-PAGE) to determine whether in vivo conditions of growth affected the expression of these bacterial surface proteins . 2 . Eleven strains of septicemic E . coli studied in vitro under conditions of iron restriction expressed iron-regulated outer-membrane proteins, mainly the protein of approximately 74 kDa, whereas avirulent strains grown under similar conditions did not present the 74-kDa protein . 3 . These results show the distribution of iron-regulated outer-membrane proteins among avian E . coli and suggest that the protein of approximately 74 kDa may be important for the virulence of these strains.

Mol Biotechnol, 1994 Jun, 1(3), 251 - 63
Expression of foreign genes in mammalian cells using an antibody fusion system; Forster SJ et al.; An expression system is described whereby a gene product is expressed fused to an antibody Fab fragment to form an antibody-like molecule . The antigen binding function of the original antibody is retained and the foreign gene replaces the CH2 and CH3 regions of the heavy chain . The fusion protein is secreted as if it were an antibody, and can be purified using the antigen-binding function of the Fab-like part of the molecule . In principle, any open reading frame can be expressed and it is not necessary to develop an individual purification scheme, or any analytical reagents such as antibodies, for the expressed protein, as both these functions can be performed by the Fab part of the fusion protein . In practice, the nature of the nonantibody part of the fusion influences the efficiency of expression and secretion, and detailed guidance is given on trouble-shooting and maximizing expression.

Res Microbiol, 1994 Jun-Aug, 145(5-6), 420 - 30
The role of FlbD in regulation of flagellar gene transcription in Caulobacter crescentus; Benson AK et al.; The flagellar (fla) genes in Caulobacter crescentus are organized into a regulatory hierarchy of four levels (I-IV) in which transcription of the class III and class IV genes late in the cell cycle from sigma 54-dependent promoters depends on expression of the class II genes above them . The periodicity of fla gene expression has been attributed to sequential activation and repression by specific transcription factors . We have been particularly interested in understanding the function and regulation of one such transcription factor, FlbD . FlbD belongs to the NtrC family of bacterial response regulators that catalyse the initiation of transcription by sigma 54 RNA polymerase (E sigma 54) and its function is required for transcription of the class III and IV fla genes . Here we show that purified FlbD binds to ftr elements that are required for transcription from the sigma 54-dependent class III flbG promoter (ftr1) and repression of transcription from the class II fliF promoter (ftr4) . Dimethylsulphate footprinting assays demonstrated that FlbD makes base-specific contacts at highly conserved guanine nucleotides in each half site of the ftr sequences . In a reconstituted in vitro transcription system using E . coli E sigma 54, we found that FlbD was clearly capable of driving transcriptional initiation from the flbG promoter and that this activity relied on the ftr1 binding site . Several observations suggest that phosphorylation plays a role in the regulation of FlbD activity . First, we found that a mutant form of FlbD (FlbDS140F) corresponding to the substitution found in a constitutively active NtrC protein (NtrCS160F), displayed a greater potential for activating E sigma 54-dependent transcription that the wildtype protein . We also observed that high energy-phosphate-containing molecules stimulate transcription activation by the wild type FlbD . Together, these results suggest that FlbD is responsible for mediating fla gene transcription initiation by E sigma 54 and that covalent modification is likely to play a role in governing FlbD activity during the cell cycle.

Appl Microbiol Biotechnol, 1994 Jun, 41(4), 440 - 6
Development of a transformation system for Trichoderma longibrachiatum and its use for constructing multicopy transformants for the egl1 gene; Sanchez-Torres P et al.; An efficient transformation system for the fungus Trichoderma longibrachiatum has been developed . Transformation was obtained both by electroporation and polyethyleneglycol treatment, using a plasmid carrying the Escherichia coli hygromycin B phosphotransferase gene as a dominant selectable marker . The transformation frequency was 0.5 to 5 transformants/micrograms plasmid DNA . Transformation normally occurred by tandem integration of the transforming DNA . A high percentage of the transformants were mitotically unstable . The efficiency of co-transformation was very high (around 90%), and several co-transformants containing multiple copies of the egl1 gene encoding a beta-(1,4)-endoglucanase were obtained . Some of them secrete increased levels of endoglucanase to the culture medium . In addition, the E . coli lacZ gene was expressed in an active form under control of the Aspergillus nidulans gpdA gene promoter.

Biosci Biotechnol Biochem, 1994 Jun, 58(6), 1170 - 1
Enhancement of thermostability of firefly luciferase from Luciola lateralis by a single amino acid substitution; Kajiyama N et al.; We constructed firefly luciferase mutants from Luciola lateralis in which Ala at position 217 was replaced by each of three hydrophobic amino acid residues (Ile, Leu, and Val) . These mutants were superior to the wild-type in thermostability . Especially, the purified Ala217Leu mutant still maintained over 70% of the initial activity after 60 min at 50 degrees C . This mutant is the most thermostable firefly luciferase obtained.

Biosci Biotechnol Biochem, 1994 Jun, 58(6), 1080 - 6
Expression in Escherichia coli of cDNA encoding barley beta-amylase and properties of recombinant beta-amylase; Yoshigi N et al.; To express the cloned beta-amylase cDNA in Escherichia coli under control of the tac promoter, a plasmid pBETA92 was constructed . The plasmid consisted of 6312 bp . An extract of E . coli JM109 harboring pBETA92 had beta-amylase activity that produced beta-maltose from soluble starch . The enzyme production started in the logarithmic phase, increased linearly, and reached a maximum after 12 h . The recombinant barley beta-amylase gave two major (pI 5.43 and 5.63) and four minor (pI 5.20, 5.36, 5.80, and 6.13) activity bands on isoelectric focusing, and their pIs didn't change throughout the incubation . But Western blot analysis found that one beta-amylase having a molecular weight of about 56,000 was synthesized . The recombinant beta-amylase was purified from the cells by consecutive column chromatography . The purified enzyme gave a single band of protein on SDS-PAGE but showed heterogeneity on isoelectric focusing . The N-terminal amino acid sequence showed that the recombinant beta-amylase lacked four amino acids at positions 2-5 (Glu-Val-Asn-Val) when compared with the presumed amino acid sequence of barley beta-amylase . Therefore, the recombinant beta-amylase consisted of 531 amino acids, and its molecular weight was calculated to be 59,169 . The N-terminal amino acid sequence of the recombinant beta-amylase and the nucleotide sequence of the junction position in plasmid pBETA92 indicated that GTG (Val-5 in the case of barley beta-amylase) at positions 27-29 from the SD sequence (AGGA) was the translation initiation codon . The properties of the recombinant beta-amylase were almost the same as those of barley beta-amylase except for the pI and the Km values for maltohexaose and maltoheptaose.(ABSTRACT TRUNCATED AT 250 WORDS)

Lett Appl Microbiol, 1994 Jun, 18(6), 346 - 8
Use of a commercial gene probe assay kit for rapid MPN enumeration of Escherichia coli in drinking water; Gale P et al.; A commercial gene probe assay kit for presence/absence determination of Escherichia coli in food samples has been used in the standard UK six tube format most probable number (MPN) method for enumerating E . coli in drinking water samples . Presence/absence analysis with the gene probe kit (requiring 3 h) of all MPN tubes after a 21-24 h incubation (minerals modified glutamate; 37 degrees C) enumerated confirmed E . coli in 24-27 h which offered an improvement of up to 48 h over the standard UK MPN method . MPNs determined by the gene probe method and the standard UK method agreed in nine of the 16 water samples which were analysed and for which E . coli concentrations were within the detection limits of the six tube MPN format . This was consistent with the gene probe method detecting one E . coli in a tube . For the other seven water samples, the gene probe method registered positive only 20 of the 30 tubes which the standard UK method determined to be positive . The sensitivity of the gene probe method for drinking water samples, although encouraging, needs improvement perhaps through kit quality control procedures.

Enzyme Microb Technol, 1994 Jun, 16(6), 496 - 500
A streptavidin-cellulose-binding domain fusion protein that binds biotinylated proteins to cellulose; Le KD et al.; A fusion protein, Sta-CBDCex, which comprises streptavidin with a cellulose-binding domain (CBDCex) fused to its C terminus, was produced in the cytoplasm of Escherichia coli, where it formed inclusion bodies . Renatured Sta-CBDCex, recovered from the inclusion bodies, adsorbed to Avicel, a microcrystalline cellulose . The cellulose-bound Sta-CBDCex in turn bound biotinylated alkaline phosphatase or biotinylated beta-glucosidase . The immobilized beta-glucosidase remained fully active during 2 weeks of continuous column operation at 50 degrees C.

Shock, 1994 Jun, 1(6), 419 - 24
The role of protein kinase C in lipopolysaccharide-induced myocardial depression in guinea pigs; Heard SO et al.; The effect of lipopolysaccharide (LPS) on cardiac protein kinase C (PKC) activation and cardiac depression was evaluated . Guinea pigs (n = 44) received intraperitoneal injections of saline or Escherichia coli LPS (2 mg/kg) . Left atria were harvested 16 h later and suspended in oxygenated low calcium (1 mM) (n = 24) or high calcium (5 mM) (n = 20) 30 degrees C Krebs-Henseleit buffer . Atria were treated with H-7 (n = 23), a PKC inhibitor, or vehicle (n = 21) . Contractile responses to changes in preload and stimulating frequency, in the resting and potentiated states, and to escalating doses of phenylephrine were measured . PKC activation in ventricular muscle was also determined . LPS activated ventricular PKC (p < .05) but treatment with H-7 failed to reverse LPS-induced atrial dysfunction in the low calcium buffer . Contractile function in the potentiated state indicated that LPS appears to interfere with calcium release from the sarcoplasmic reticulum (SR) . The contractile response to phenylephrine was markedly attenuated in atria harvested from endotoxic animals . These data indicate that LPS-induced cardiac depression is mediated, in part, by alterations in SR calcium release . LPS activates cardiac PKC but a causal relationship among LPS, PKC, and cardiac dysfunction remains to be established.

Shock, 1994 Jun, 1(6), 425 - 31
Early endotoxic shock results in enhanced vasodilator responses to nitroglycerin but unaltered responses to neuropeptides calcitonin gene-related peptide and substance P; Arden WA et al.; To determine the role that vasoactive neuropeptides, calcitonin gene-related peptide, and substance P play in tissue-blood flow regulation during early septic shock, we examined the responsiveness of arteries removed from pigs 3 h after administration of Escherichia coli lipopolysaccharide or saline vehicle . The carotid, cranial mesenteric, and left anterior descending coronary arteries were excised, and rings were cut from each vessel . Constrictor responses were obtained to cumulative doses of norepinephrine or potassium chloride . Rings were reconstricted and challenged with acetylcholine, substance P, calcitonin gene-related peptide, and nitroglycerin . Lipopolysaccharide significantly increased the cranial mesenteric artery's response to high concentrations of norepinephrine and the response to nitroglycerin in all vessels . This enhancement of responses to nitroglycerin suggests augmented smooth-muscle responsiveness to an exogenous source of nitric oxide, possibly associated with early depression of basal endothelial function . Depression of agonist-induced nitric oxide release may mask such enhancement with endothelial-dependent dilators and may enhance the response to adrenergic constrictors in some vascular beds.

Int J Biol Macromol, 1994 Jun, 16(3), 153 - 8
The non-enzymatic specific amino-acylation of transfer RNA at high pressure; Krzyzaniak A et al.; This paper shows that the phenylalanine-specific tRNA of Escherichia coli as well as the yellow lupin methionine initiator tRNAMet can be charged specifically with phenylalanine and methionine, respectively, in the absence of specific aminoacyl-tRNA synthetases, under high pressure of a maximum of 6 kbar (1 bar = 10(5) Pa; 1 atm = 1.01 x 10(5) Pa) . The esterification reaction takes places at the 3' end of the tRNA molecules . The yield of Phe-tRNAPhe or Met-tRNAMet at high pressure is approximately 10 times lower than that of the enzymatic aminoacylation reaction . This reaction seems to be specific, and mis-aminoacylation of tRNAPhe and tRNAMet with serine is negligible . It is well known that tRNA undergoes conformational changes during interaction with an aminoacyl-tRNA synthetase . Similarly, on the basis of circular dichroism spectra, we showed that the conformation of tRNA at high pressure differs slightly from its original A-RNA form . Therefore, it can be speculated that the chargeable conformation of tRNA induced by the aminoacyl-tRNA synthetase during enzymatic aminoacylation and the one created at high pressure are similar and are most probably formed by a dehydration mechanism . We think that the 'unique' tertiary structure of tRNA existing under high pressure creates an active centre which might itself catalyse ester bond formation . Therefore, the structure of the amino acid stem of tRNA may determine (code) the charging of the particular amino acid to specific tRNA . This code is clearly distinct from the rules of the classical genetic code.

J Appl Physiol, 1994 Jun, 76(6), 2785 - 93
Increased organ blood flow in chronic endotoxemia is reversed by nitric oxide synthase inhibition; Meyer J et al.; We evaluated regional blood flows in a hyperdynamic sepsis model and the reversal of increased flows by blockade of nitric oxide (NO) synthase . Seven awake sheep were continuously infused with Escherichia coli endotoxin {lipopolysaccharide (LPS), 10 ng.kg-1.min-1} for 48 h . The NO synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME, 25 mg/kg) was injected after 24 h . Blood flows to systemic organs were determined with the radioactive microsphere technique . LPS induced elevation of cardiac index by 36% (P < 0.05) and a fall in systemic vascular resistance index by 37% (P < 0.05) at 0 h {time of L-NAME administration, 24 h after infusion of LPS had begun} L-NAME administration normalized cardiac index {6.1 +/- 0.5 at 4 h posttreatment, 6.1 +/- 0.5 l.min-1.m-2 at -24 h (baseline)} and systemic vascular resistance index (1,333 +/- 105 at 4 h posttreatment, 1,280 +/- 163 dyn.s.cm-5.m2 at -24 h) and reduced all regional blood flows to near-baseline levels for the remainder of the study period (24 h) . O2 consumption was unaffected by treatment.

Cell Growth Differ, 1994 Jun, 5(6), 625 - 35
Cell cycle-dependent expression of Nek2, a novel human protein kinase related to the NIMA mitotic regulator of Aspergillus nidulans; Schultz SJ et al.; The serine/threonine protein kinase NIMA of Aspergillus nidulans is required for entry into mitosis and may function in parallel to the universal mitotic inducer p34cdc2 . Here, we report the isolation of complementary DNAs encoding Nek2 and Nek3, two novel human protein kinases structurally related to NIMA . Sequence comparisons revealed several unique features which may define a family of NIMA-related protein kinases . Nek2 was chosen for further study since it represents the closest known mammalian relative of NIMA . Chromosomal mapping of the nek2 gene identified two independent loci on chromosomes 1 and 14, and Northern blot analyses revealed the expression of two distinct mRNAs of approximately 2.4 and 4.7 kilobases in all human cell lines examined . In HeLa cells synchronized by both drug arrest and elutriation, a strikingly cell cycle-dependent pattern of Nek2 expression could be observed; Nek2 protein was almost undetectable during G1 but accumulated progressively throughout S, reaching maximal levels in late G2 . These observations demonstrate that Nek2 resembles Aspergillus NIMA, not only in its catalytic domain, but also in its cell cycle-dependent expression . Hence, the human Nek2 protein kinase may also function at the onset of mitosis.

Int J Exp Pathol, 1994 Jun, 75(3), 191 - 6
Endotoxin is angiogenic; Mattsby-Baltzer I et al.; The formation of new blood vessels, angiogenesis, is an important event in inflammation, wound healing and tumour growth . Mediators produced by various cells when exposed to endotoxin include cytokines (tumour necrosis factor, interleukins 1 and 6, and basic fibroblast growth factor) which, it has been suggested, stimulate angiogenesis . The angiogenic effect of endotoxin (lipopolysaccharide, LPS) was studied in rats using the quantitative mesenteric window assay . Adult rats were injected intraperitoneally with Escherichia coli LPS (5 pg/ml-20,000 ng/ml) twice daily for 4.5 consecutive days and were sacrificed 14 days after the start of this treatment . An angiogenic response was observed at concentrations of > 2 ng/ml in a dose-dependent manner . No inflammatory cellular exudate was seen in the test tissue at the time of angiogenesis analysis . Suppressed body-weight gain, a marker of the systemic effect of LPS in the rat, was significant only at the highest dose tested . The data suggest that endotoxin-mediated neovascularization could be a component of inflammation and wound healing.

Cell Adhes Commun, 1994 Jun, 2(2), 87 - 99
Activation dependent and independent VLA-4 binding sites on vascular cell adhesion molecule-1; Needham LA et al.; Vascular cell adhesion molecule-1 (VCAM) is a cytokine-inducible member of the immunoglobulin superfamily which binds to the integrin VLA-4 . VCAM is expressed predominantly on the vascular endothelium where it is involved in the recruitment of mononuclear cells and lymphocytes to sites of inflammation . Two forms of VCAM containing six and seven Ig domains (VCAM-6d; VCAM-7d) are generated by alternative splicing but the physiological significance of this is unknown . We have utilised VCAM deletion mutants, VCAM-transfected cell lines and monoclonal antibodies to assess the functional importance of the individual VCAM domains . We have identified two binding sites on VCAM-7d located in domains 1 and 4 that are involved in the adhesion of the U937 human myelomonocytic cell line . Adhesion to domain 1 is temperature-independent, inhibited by the anti-VCAM mAbs 4B2 or lE10, and insensitive to PMA activation . In contrast, adhesion to domain 4 is temperature sensitive, unaffected by mAbs 4B2 or lE10 and augmented by PMA . Adhesion to both domains can be totally inhibited by the anti-VLA-4 mAb, 2B4 . The anti-VCAM mAb 4B2 inhibits adhesion of U937 cells to stably transfected VCAM-7d-CHO cells at 4 degrees C, but, at 37 degrees C the effect of 4B2 on adhesion is modest with incubation times of less than 60 minutes duration . With longer incubation times, its effectiveness gradually increases, so that by 2 hours > 75% of the response can be blocked . Co-incubation with PMA prevents this time-dependent enhancement of 4B2 efficacy but has no significant effect on the inhibitory activity of the anti-VLA-4 mAb 2B4 . These data can be explained by postulating a two stage ligand-receptor interaction that involves activation-induced changes in the avidity of VLA-4 for domain 4 of VCAM.

Bioessays, 1994 Jun, 16(6), 437 - 44
Mutation frequency decline revisited; Witkin EM; 'Mutation frequency decline' (MFD) was discovered about forty years ago, and described as the disappearance of a particular class of ultraviolet light-induced mutations in Escherichia coli that occurred whenever protein synthesis was briefly inhibited immediately after irradiation . Later, MFD was interpreted as an excision repair anomaly uniquely affecting nonsense suppressor mutations induced in certain tRNA genes . Never fully understood, MFD has recently been linked to the newly discovered transcription-coupled rapid repair of ultraviolet damage on the template strand of active genes . This article recalls the emergence and development of the MFD story, and offers a new way to explain it and its relation to strand-specific excision repair.

AIDS Res Hum Retroviruses, 1994 Jun, 10(6), 655 - 64
Linear epitopes of HIV-1, presented as hybrids with Escherichia coli beta-galactosidase or synthetic peptides; Isaguliants MG et al.; HIV-1 B cell epitopes from gp41, the T cell epitope of p34pol, and a cluster of B and T epitopes from p17gag were selected . The epitopes were presented as synthetic peptides and as either N- or C-terminal insertions into beta-galactosidase . Hybrids were efficiently expressed in E . coli and easily purified when epitopes were inserted at the beta-galactosidase C terminus . Sera from HIV-1-infected individuals reacted in peptide- and hybrid protein-based enzyme-linked immunosorbent assays (ELISAs) mostly with the immunodominant site of gp41 . The second site of gp41 and also sites from p17 and p34 appeared to be immunorecessive . A few of the HIV-1-positive sera exhibited several immunorecessive reactivities . HIV-1-positive sera from the former Soviet Union and Cuba had reactivities similar to those of American, African, and west European sera . Some sera could not be evaluated as specifically HIV-1 seropositive because of their broad reactivities with a multitude of peptides and proteins, unrelated to HIV-1 . Extensive tests were performed to define unspecific reactivities by absorption, blocking, and sandwich ELISAs . The application of the hybrid protein assay substantially improved the specificity of the ELISA tests . Thus, hybrid protein-based ELISAs appeared to be more suitable than peptide-based ELISAs, especially for the evaluation of immunorecessive reactivities.

J Clin Pathol, 1994 Jun, 47(6), 524 - 8
Epitope analysis of antibodies recognising the cell proliferation associated nuclear antigen previously defined by the antibody Ki-67 (Ki-67 protein); Kubbutat MH et al.; AIMS--To elucidate the fine specificities of the antibodies MIB 1 and MIB 3 and of additional monoclonal antibodies which also recognise the Ki-67 protein (MIB 5, IND.64, JG-67-2a) . METHODS--Different parts of the Ki-67 protein cDNA were expressed in Escherichia coli . Bacterial lysates were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted on to nitrocellulose . Additionally different peptides were synthesised on a membrane support (SPOT-Blot) . The immunoreactivity of the antibodies with the recombinant proteins and the immobilised synthetic peptides, respectively, was analysed . A competition enzyme linked immunosorbent assay (ELISA) using a soluble synthetic peptide was also performed . RESULTS--The epitopes of all antibodies tested were contained within the same region of seven amino acids . The antibodies MIB 1 and MIB 3 required the five amino acid sequence FKELF for binding, whereas Ki-67, JG-67-2a, MIB 5 and IND.64 detected the sequence FKEL . CONCLUSIONS--It is concluded that the amino acid sequence FKELF represents an immunodominant area of the Ki-67 protein and that there is no correlation between the ability to detect the Ki-67 protein in paraffin wax sections irradiated with microwaves and the epitopes recognised by the antibodies.

Eur J Biochem, 1994 Jun 1, 222(2), 353 - 66
cDNA cloning and deduced amino acid sequence of two ferritins: soma ferritin and yolk ferritin, from the snail Lymnaea stagnalis L; von Darl M et al.; Pulmonate freshwater snails contain two different ferritin types, soma ferritin and yolk ferritin . A cDNA library was constructed from midgut gland poly(A)-rich RNA of the snail Lymnaea stagnalis L . and recombinant clones encoding both ferritin types were obtained by immunoscreening . The longest cDNA inserts had a length of 859 bp (soma ferritin) and 1548 bp (yolk ferritin) and the specificity of these inserts was confirmed by immunoprecipitation of both ferritin types translated in vitro from hybrid-selected mRNAs . The 5' untranslated region (UTR) of the soma ferritin mRNA contains a 28-bp element which shows 64% sequence identity with the iron-responsive element (IRE) of vertebrate ferritin mRNAs . The soma ferritin mRNA is strongly translated in the wheat germ system but poorly translated in rabbit reticulocyte lysate . The yolk ferritin mRNA, which contains no IRE, is equally well translated in both in vitro translation systems . The deduced amino acid sequence of the soma ferritin subunit (174 amino acid residues, M(r) 20140) shows 50-70% sequence identity with subunits of vertebrate ferritins . After removal of an 18-amino-acid-residue signal sequence the deduced protein sequence of yolk ferritin contains 221 amino acids (M(r) 25438) . Sequence identity of this chain with other eukaryotic ferritin chains is only 31-42% . Both snail ferritin sequences are more similar to the H-subunit type of vertebrate ferritins than to the L-type and both have the H-specific amino acid residues of the ferroxidase centre . The yolk ferritin sequence has a 42-amino-acid-residue insertion predicted to reside in the L loop of the subunit.

EMBO J, 1994 Jun 1, 13(11), 2677 - 85
The decoding region of 16S RNA; a cross-linking study of the ribosomal A, P and E sites using tRNA derivatized at position 32 in the anticodon loop; Doring T et al.; A photo-reactive diazirine derivative was attached to the 2-thiocytidine residue at position 32 of tRNA(Arg)I from Escherichia coli . This modified tRNA was bound under suitable conditions to the A, P or E site of E.coli ribosomes . After photo-activation of the diazirine label, the sites of cross-linking to 16S rRNA were identified by our standard procedures . Each of the three tRNA binding sites showed a characteristic pattern of cross-linking . From tRNA at the A site, a major cross-link was observed to position 1378 of the 16S RNA, and a minor one to position 936 . From the P site, there were major cross-links to positions 693 and to 957 and/or 966, as well as a minor cross-link to position 1338 . The E site bound tRNA showed major cross-links to position 693 (identical to that from the P site) and to positions 1376/1378 (similar, but not identical, to the cross-link observed from the A site) . Immunological analysis of the concomitantly cross-linked ribosomal proteins indicated that S7 was the major target of cross-linking from all three tRNA sites, with S11 as a minor product . The results are discussed in terms of the overall topography of the decoding region of the 30S ribosomal subunit.

J Thorac Cardiovasc Surg, 1994 Jun, 107(6), 1432 - 9
Experimental use of a modified fibrin glue to induce site-directed angiogenesis from the aorta to the heart; Fasol R et al.; From 10 cultures of manipulated Escherichia coli bacteria expressing the class I heparin-binding growth factor polypeptide alpha-endothelial cell growth factor, 11.2 +/- 0.7 mg alpha-endothelial cell growth factor was eluted by heparin-sepharose affinity chromatography . Analysis of molecular weight (17,000 kD) was done by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and purification of the growth factor was done by high-performance liquid chromatography . The harvested alpha-endothelial cell growth factor was proved by protein blotting . To assess the growth-promoting activity, we did an endothelial cell growth assay by comparing adult human endothelial cell control cultures, without adding growth factor to the culture medium, with adult human endothelial cell cultures with 0.02 to 20.0 ng/ml alpha-endothelial cell growth factor and 1.0 ng/ml heparin and with adult human endothelial cell cultures with alpha-endothelial cell growth factor but without heparin . Tritiated thymidine counts proved the significant growth-promoting activity of alpha-endothelial cell growth factor . In 10 experimental animals modified fibrin glue containing 1 microgram alpha-endothelial cell growth factor was implanted between the aorta and the myocardium of the left ventricle and results were compared with those in five control animals that received normal fibrin glue without growth factor . After 9 weeks of implantation, angiography and histologic investigation showed newly grown vascular structures between the aorta and the myocardium in all experimental animals, but none in the control animals . Our study proved the feasibility of initiating site-directed formation of new blood vessel structures to the heart by a modified fibrin glue implant containing angiogenic growth factor alpha-endothelial cell growth factor.

Mutat Res, 1994 Jun 1, 307(2), 533 - 40
Development of a lambda-based complementation assay for the preliminary localization of lacI mutants from the Big Blue mouse: implications for a DNA-sequencing strategy; Gu M et al.; The Big Blue transgenic mouse carrying the E . coli lacI gene as a mutational target in a lambda-based shuttle vector has been receiving increasing attention in genotoxicity testing because it offers the potential of studying mutation in a mammalian system in vivo . The system not only provides information on mutant frequency, but it also offers the potential of providing information about mutational specificity . Such data is not only important for studies of mutational mechanisms; it offers a critical advantage for determining the mutational response at levels where significant increases in mutant frequency have not been discerned . The repeated sequencing of the entire 1080-bp lacI target, however, remains a formidable task . Here we report on the adaptation of the "negative complementation" assay for the lacI-d phenotype to accommodate the lambda lacI recovered from the Big Blue transgenic animal . This assay permits the localization of mutations to an approximately 330-bp region to facilitate the production of mutational specificity data . The assay is based upon lysogenization of the lambda containing the lacI mutation into a lacI+ host . Of 107 sequenced lacI mutants recovered from Big Blue mice, 74 were identified as NC+ (lacI-d) using this assay . Of these 74, 49 occurred in the region 32-208 bp, which has traditionally been viewed as the NC+ domain . 33 of these mutations were previously identified as producing the NC+ phenotype while another 7 occurred at sites where NC+ mutants have been recovered, but involved a new base substitution . 9 mutants involved new sites . An additional 25 mutants located downstream of the presumed NC+ region were also found to be NC+ as determined by their blue colour on X-gal plates . Of these, 18 occurred in the 209-360-bp region . In parallel, 54 lacI mutants carrying unknown mutations were examined . 37 of these produced blue colonies in this assay . The sequencing of these mutants revealed that 20 (54%) of the 37 mutants were located in the 32-208-bp region . This complementation assay can potentially reduce the amount of DNA sequencing necessary to produce a mutational spectrum by optimising the choice of sequencing primers, and thus provide a significant saving of the material and time required . Furthermore, evidence indicates that the restriction of the mutational target to the NC+ region extends these savings without reducing the usefulness of the mutational specificity data.

Mutat Res, 1994 Jun 1, 307(2), 451 - 9
LacZ transgenic mouse models: their application in genetic toxicology; Gossen JA et al.; Gene mutations have been implicated in the etiology of cancer, developmental anomalies, genetic disease and aging . Many different methods for mutation detection have been developed and applied to obtain a more fundamental insight in the chain of molecular events that ultimately lead to mutations . Most of these methods, however, can only be applied to cultured cells and therefore do not allow comparative analysis of mutations in various organs and tissues in an intact organism . The main difficulty in studying mutagenesis in chromosomal DNA is to identify and isolate mutated genes with a high efficiency . Here we describe the development and application of LacZ transgenic mouse models for studying, in different organs and tissues, spontaneous or induced mutations . Such models allow study of the induction of DNA damage, repair, mutagenesis and carcinogenesis in one animal system . Accordingly, results obtained may ultimately provide greater insight into the chain of events from in vivo exposure to genotoxic agents to mutations and their ultimate physiological endpoints . In addition to their use in fundamental research, transgenic animal mutation models find a major application in the field of genetic toxicology testing, in particular with respect to organ specificity.

Infect Immun, 1994 Jun, 62(6), 2553 - 61
CS31A capsule-like antigen as an exposure vector for heterologous antigenic determinants; Bousquet F et al.; CS31A is a multimeric surface protein surrounding certain enterotoxigenic and septicemic bovine Escherichia coli strains . The possibility of using CS31A as a carrier of foreign antigenic determinants was investigated . Introduction of heterologous viral epitopes into the CS31A major subunit, ClpG, was successfully performed in the V3 region of the molecule which encodes for an immunodominant linear epitope . E . coli K-12 strains producing the hybrid CS31A molecules or the purified chimeric proteins were used for mice immunization . By using the C epitope derived from the S protein of the porcine transmissible gastroenteritis virus, significant antiviral antibody titers were elicited and seroneutralization of the virus was demonstrated, confirming that the molecular environment in V3 is favorable for antigen presentation . These results indicate that synthesis of CS31A hybrid proteins by the wild-type strain 31A could become a powerful tool for the development of oral vaccines directed against mucosal pathogens.

Biochemistry, 1994 May 31, 33(21), 6732 - 8
Purification and characterization of the recombinant human calcium-binding S100 proteins CAPL and CACY; Pedrocchi M et al.; The S100 proteins CAPL and CACY are expressed in a tissue- and cell-specific manner and have been reported to be associated with the metastatic phenotype of tumor cells . In order to study the biochemical, cation-binding, and conformational properties, we produced and purified large amounts of the recombinant human proteins in Escherichia coli . Several characteristics of native proteins are shown to correspond to those of the bacterially expressed proteins . Both are able to form homodimers in vitro, probably the biologically active species, but not heterodimers . The Ca(2+)-binding parameters were studied by flow offlysis at physiological ionic strength . Both isotherms show a maximum of two Ca2+ per protein and are insensitive to Mg2+, indicating that the sites are of the Ca(2+)-specific type . The isotherms show slight (CAPL, nH = 1.15) or pronounced (CACY, nH = 1.33) positive cooperativity with K0.5 values of 0.32 mM (CACY) and 0.15 mM (CAPL), indicating that the sites are of the low-affinity type . Conformational changes in the Tyr microenvironment of CACY indicate that Ca2+ binding induces a shift of Tyr to a less polar environment . Mg2+ does not affect the fluorescence properties nor does it induce a difference spectrum, thus suggesting that at physiological ionic conditions it does not interact with the protein . The Ca(2+)-induced difference spectra of CAPL are about 3 times smaller than those of CACY, suggesting that the additional Tyr84 in CACY is much more sensitive to Ca2+ than the two Tyr residues conserved in both proteins.

Biochemistry, 1994 May 31, 33(21), 6571 - 7
Mutagenic analysis of a receptor contact site on interleukin-2: preparation of an IL-2 analog with increased potency; Berndt WG et al.; Interleukin-2 (IL-2) is a 133 amino acid alpha-helical protein secreted by activated T-cells . Combinatorial cassette mutagenesis was used to investigate the functional role of a continuous five amino acid region of IL-2 suspected to interact with the intermediate-affinity IL-2 receptor . A limited random library of IL-2 mutants was constructed in which residues 17-21 (Leu-Leu-Leu-Asp-Leu) were simultaneously mutated . The proteins were produced in an Escherichia coli expression system and screened in a biological assay for their ability to mediate the proliferation of a murine IL-2-dependent cell line . From the over 2600 clones examined, only 42 exhibited significant activity, confirming the functional importance of this region . Selected clones were purified and further characterized by biological and receptor binding assays . Viewed in the context of the recently revised 2.5-A crystal structure for IL-2, these results suggest the following conclusions: both Asp20 and Leu21, as shown by their sensitivity to mutation, are the functionally more important residues in this region, but for different reasons . Asp20 is solvent-accessible and likely plays a direct receptor contact role as previous studies have indicated . Leu21, in contrast, is completely buried in the hydrophobic core of the protein . Substitutions at this position, even a conservative Leu-->Val substitution, were found to perturb the precise hydrophobic packing arrangements that are critical for activity, resulting in a significant loss of function . In addition, one of the analogs identified in the screen was found to be 2-3 times more potent than the wild-type protein.

Biochemistry, 1994 May 31, 33(21), 6546 - 54
Replacement of the proximal ligand of sperm whale myoglobin with free imidazole in the mutant His-93-->Gly; Barrick D; The proximal bond between the iron atom of the heme group and the N epsilon of histidine F8 in myoglobin (Mb) and hemoglobin (Hb) is presumed to be an important determinant of heme binding, protein structure, and oxygen binding . Here a system is described in which the proximal ligand is provided intermolecularly by the histidine side chain mimic imidazole . The proximal ligand of sperm whale Mb is replaced with glycine (H93G) using site-directed mutagenesis . The addition of imidazole to Escherichia coli expressing this gene reconstitutes myoglobin function . H93G Mb purified in the presence of imidazole is spectroscopically similar to wild-type Mb in combination with a wide variety of distal ligands . The crystal structure of H93G Mb, determined in the presence of imidazole, reveals that an imidazole molecule is bonded to the heme iron on the proximal side, substituting in trans for the side-chain function of the proximal histidine of wild-type Mb . Although H93G Mb is similar in spectroscopic and gross structural detail to wild-type Mb, subtle differences exist in the orientation of imidazole with respect to the heme group . trans-Complementation of proximal ligand function will allow the proximal bond in hemoproteins to be chemically substituted beyond the limits of the genetic code.

Biochemistry, 1994 May 31, 33(21), 6468 - 74
Mutations affecting transition-state stabilization by residues coordinating zinc at the active site of cytidine deaminase; Smith AA et al.; Cytidine deaminase from Escherichia coli contains 1 mol of tightly bound zinc per enzyme subunit (Yang, C., Carlow, D., Wolfenden, R., & Short, S.A . (1992) Biochemistry 31, 4168-4174) . When the metal liganding residues Cys-129 and Cys-132 were replaced by Ala, and His-102 was replaced by Ala, Asn, or Gln, deaminase activities of cell extracts containing these mutant enzymes were decreased by several orders of magnitude relative to that of the wild-type enzyme . After purification, each mutant protein was found to contain less than 0.2 mol of zinc per enzyme subunit, except mutant H102Q, which contained 1 mol of zinc per subunit . The activity of each mutant enzyme increased in the presence of added zinc but never attained wild-type activity . Mutant H102N was unique in that this protein could be purified as a stable apoenzyme, activated by added zinc, and then inhibited by EDTA . This mutant enzyme bound zinc with an apparent Kd value of 6.0 x 10(-10) M and regained maximal activity in the presence of 1 mol of zinc per subunit . Affinities of the mutant cytidine deaminases for the transition-state analogue, 5-fluoropyrimidin-2-one ribonucleoside (3,4) hydrate, were found to decrease in rough proportion to kcat/Km over a range spanning several orders of magnitude . This variation in catalytic efficiency arose mainly from effects on kcat, indicating the involvement of zinc coordination in the catalytic process rather than in substrate binding.

Biochemistry, 1994 May 31, 33(21), 6671 - 5
Effect of divalent cations on the molecular structure of the GroEL oligomer; Azem A et al.; Structural analysis, by chemical cross-linking with glutardialdehyde (GA), and by urea denaturation, was carried out for the chaperonin oligomer GroEL14 from Escherichia coli . The cross-linking reaction of GroEL14 presents two phases: a rapid intralayer cross-linking reaction, which first occurs between the monomers of individual GroEL7 heptameric rings, and a slow interlayer cross-linking reaction, which later occurs between the two stacked heptameric rings of the GroEL14 oligomer . The biphasic behavior of the cross-linking reaction indicates that the surfaces of contact between GroEL monomers within individual heptameric rings are more extensive than the surfaces of contact between the two GroEL7 rings of the oligomer . Millimolar amounts of the divalent cations Mg2+, Mn2+, Ca2+, or Zn2+, but not of monovalent ions, increase the velocity of both intra- and interlayer cross linking . Divalent cations increase the stability of the native GroEL14 oligomer in urea . In contrast, Mg2+ activates ATP hydrolysis by GroEL14, with an activation constant in the micromolar range, while Ca2+ does not significantly assist ATP hydrolysis . It is concluded that divalent cations affect the structure of GroEL14 in particular the contacts between monomers within the GroEL7 heptameric layers . The effect of divalent cations on the structure of the chaperonin molecule is quantitatively and qualitatively distinct from that of magnesium ions on the chaperonin ATPase activity.

FEBS Lett, 1994 May 30, 345(2-3), 198 - 202
Antimycin inhibition of the cytochrome bd complex from Azotobacter vinelandii indicates the presence of a branched electron transfer pathway for the oxidation of ubiquinol; Junemann S et al.; Antimycin A and UHBDT inhibit the activity of the purified cytochrome bd complex from Azotobacter vinelandii . Inhibition of activity is non-competitive and antimycin A binding induces a shift to the red in the spectrum of a b-type haem . No inhibitory effects were seen with myxothiazol . Steady-state experiments indicate that the site of inhibition for antimycin A lies on the low-potential side of haem b558 . In the presence of antimycin A at concentrations sufficient to inhibit respiration, some direct electron transfer from ubiquinol-1 to haem b595 and haem d still occurs . The results are consistent with a branched electron transfer pathway from ubiquinol to the oxygen reduction site.

FEBS Lett, 1994 May 30, 345(2-3), 143 - 6
Cleavage of supercoiled double-stranded DNA by several ribosome-inactivating proteins in vitro; Ling J et al.; Several ribosome-inactivating proteins (RIPs), such as ricin (including its A-chain), luffin, cinnamomin and camphorin, were found to express enzymatic activity to cleave supercoiled double-stranded DNA . In particular, alpha-sarcin, a RIP with a novel ribonuclease activity, was first proved to have this activity . They convert supercoiled DNA into a nicked circular conformation at low concentrations and further into a linear form at high concentrations: they have no effect on linear DNA . Although intact type II RIPs exhibited no RNA N-glucosidase activity, they were detected to cleave supercoiled DNA . Even if ricin A-chain was treated by boiling, its activity on supercoiled DNA was largely retained.

FEBS Lett, 1994 May 30, 345(2-3), 125 - 30
Refolding proteins by gel filtration chromatography; Werner MH et al.; We have developed a facile means for the refolding of milligram quantities of purified proteins that employs gel filtration chromatography . We demonstrate by electrophoretic mobility shift and NMR spectroscopy that human ETS-1 protein, bovine ribonuclease A and E . coli integration host factor can be refolded into the native conformation using this technique . We have extended this strategy to the preparation of milligram quantities of macromolecular complexes suitable for structural analysis by NMR spectroscopy or X-ray crystallography . The diverse challenges to overcome in refolding these proteins illustrates the potential of this technique as a general approach for recovery of recombinant proteins produced as insoluble inclusion bodies.






What Is Biotechnology?, What Is Anthrax?, What is Food Microbiology?, What Is Botulism?, What Is Fermentation?, i, Microorganism, i, Bacterium, a, Microbiology, s, Bacteria, i, Microorganisms, c, Escherichia coli, i, Prokaryotes, c, Pasteurella, r, Schizosaccharomyces, n, Citrobacter, i, Saccharomyces yeast, r, Escherichia coli, i, Microbial, r, S. cerevisiae, i, Escherichia coli, r, Haemophilus, c, Agrobacterium, i, S. cerevisiae, s, Enterobacteriacea, s, Corynebacter, c, Salmonella typhimurium, c, Burkholderia, o, Bacillus subtilis, s, Yeasts, o, Biodegradation, a, Campylobacter




 

   Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology
Anaerobic Microbiology
Antimicrobial Susceptibility
Artificial Atmosphere
Bioassay of Antibiotics
Biofilm Microbiology
Bioreactor Technology
Biotechnology
Cell Biology
Clinical Microbiology
Environmental Microbiology
Experiments with Yeast		 Fermentation
Food Microbiology
Functional Genomics
Gene Technology
Growth Media Development
Growth Rate and Lag Time
Industrial Microbiology
Medical/Pharmaceutical Field
Microbiological Assay
Microbiological Research
Microbiology of Cosmetics

go to a specific theme...

 Military Microbiology
Molecular Microbiology
Mutagenicity and Genotoxicity
Oral Microbiology
Patents
Postantibiotic Studies
Soil Microbiology
Spore Microbiology
Veterinary Microbiology
Waste/Wastewater Treatment
Water Microbiology
Wine Microbiology

 


 

© 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com
 

 

 

Last modified: May 25, 2005