Nucleic Acids Res, 1994 Jan 11, 22(1), 72 - 8 Major oxidative products of cytosine, 5-hydroxycytosine and 5-hydroxyuracil, exhibit sequence context-dependent mispairing in vitro; Purmal AA et al.; Two major stable oxidation products of 2'-deoxycytidine are 2'-deoxy-5-hydroxycytidine (5-OHdC) and 2'-deoxy-5-hydroxyuridine (5-OHdU) . In order to study the in vitro incorporation of 5-OHdC and 5-OHdU into DNA by DNA polymerase, and to check the base pairing specificity of these modified bases, 5-OHdCTP and 5-OHdUTP were synthesized . Incorporation studies showed that 5-OHdCTP can replace dCTP, and to a much lesser extent dTTP, as a substrate for Escherichia coli DNA polymerase I Klenow fragment (exonuclease free) . However, 5-OHdUTP can only be incorporated into DNA in place of dTTP . To study the specificity of nucleotide incorporation opposite 5-hydroxypyrimidines in template DNA, 18- and 45-member oligodeoxyribonucleotides, containing an internal 5-OHdC or 5-OHdU in two different sequence contexts, were used . Translesion synthesis past 5-OHdC and 5-OHdU in both oligonucleotides occurred, but pauses both opposite, and one nucleotide prior to, the modified base in the template were observed . The specificity of nucleotide incorporation opposite 5-OHdC and 5-OHdU in the template was sequence context dependent . In one sequence context, dG was the predominant nucleotide incorporated opposite 5-OHdC with dA incorporation also observed; in this sequence context, dA was the principal nucleotide incorporated opposite 5-OHdU . However in a second sequence context, dC was the predominant base incorporated opposite 5-OHdC . In that same sequence context, dC was also the predominant nucleotide incorporated opposite 5-OHdU . These data suggest that the 5-hydroxypyrimidines have the potential to be premutagenic lesions leading to C-->T transitions and C-->G transversions. Biochemistry, 1994 Jan 11, 33(1), 90 - 7 Significant improvement to the catalytic properties of aspartate aminotransferase: role of hydrophobic and charged residues in the substrate binding pocket; Kohler E et al.; The substrate specificity of tyrosine aminotransferase (eTAT) from Escherichia coli has been tested by transferring the critically different residues Leu39, Glu141, and Arg293 into equivalent positions of aspartate aminotransferase (eAAT) . These residues are not directly involved in the catalytic process . The single mutant eAAT V39L possesses greater values of kcat/KM not only for tyrosine but also for aspartate and glutamate . In contrast, the double mutant eAAT P141E,A293R and also the triple mutant eAAT V39L,P141E,A293R exhibit smaller changes of kcat/KM . The converse mutants of tyrosine aminotransferase, in which critical residues of eAAT (Val39) and of mitochondrial AAT (Ala39, Val37) were transferred into equivalent positions of eTAT, exhibited generally decreased values of kcat/KM for both dicarboxylic and aromatic substrates . On the basis of the known structures of eAAT and eAAT V39L as well as of a refined model of eTAT, these results indicate that the different substrate specificities of eAAT and eTAT are due to multiple side chain differences and minor rearrangements of the backbone . The generally improved catalytic efficiency of the mutant eAAT V39L appears to be due to an indirect effect, namely, the facilitated closure of the active site upon substrate binding. Biochemistry, 1994 Jan 11, 33(1), 340 - 7 RNA displacement pathways during transcription from synthetic RNA-DNA bubble duplexes; Daube SS et al.; Previously {Daube, S.S., & von Hippel, P.H . (1992) Science 258, 1320} we have shown that functional transcription elongation complexes can be formed by adding ribonucleotide triphosphates, Mg2+, and either Escherichia coli or T7 RNA polymerase to synthetic RNA-DNA bubble-duplex constructs . Here these observations are extended to show that the RNA transcripts synthesized from these bubble-duplex constructs are properly displaced from the DNA template during transcription . Some details of the displacement process differ between the polymerases tested . Thus the transcript is fully and processively displaced in the course of T7 polymerase-catalyzed synthesis from the bubble-duplex constructs, while the presence of a large excess of an RNA (or DNA) oligomer complementary to the DNA template sequence within the "permanent" DNA bubble is required to attain complete displacement of the nascent RNA from the construct during synthesis with the core E . coli enzyme . In addition, a correlation is shown between proper RNA displacement and the achievement of full-length transcript synthesis . We conclude that both the T7 polymerase and the E . coli core enzyme actively displace the nascent transcript during elongation and that the requirement for an RNA trap with the E . coli enzyme reflects its slower rate of synthesis . This suggests that these experiments may provide insight into the relative rates of transcript elongation and secondary structure formation within the nascent RNA in elongation and termination . By use of the RNA oligomer trap methodology, multiple rounds of transcript synthesis should be achievable on these bubble-duplex constructs with any polymerase. FEBS Lett, 1994 Jan 10, 337(2), 189 - 94 Ribosome-messenger recognition in the absence of the Shine-Dalgarno interactions; Tzareva NV et al.; In an attempt to understand how Escherichia coli ribosomes recognize the initiator codon on mRNAs lacking the Shine-Dalgarno (SD) sequence, we have studied 30S initiation complex formation in extension inhibition (toeprinting) experiments using (-SD)mRNAs which are known to be reliably translated in E . coli: the plant viral messenger A1MV RNA 4 and two chimaeric mRNAs coding for beta-glucuronidase (GUS) and bearing the 5'-untranslated sequence of TMV RNA (omega) or the omega-derived sequence (CAA)n as 5'-leaders . Ribosomal protein S1 and IF3 have been found to be indispensable for translational initiation . Protein S1 appears to be a key recognition element . S1 binds to sequences within the leaders of (-SD)mRNAs thus providing their affinity to E . coli ribosomes. FEBS Lett, 1994 Jan 10, 337(2), 135 - 8 Assembly of Alzheimer-like filaments from full-length tau protein; Crowther RA et al.; The principal fibrous component of neurofibrillary pathology in Alzheimer's disease, the paired helical filament, is formed from hyperphosphorylated microtubule-associated protein tau . Here we show that recombinant tau protein either in a non-phosphorylated state or following phosphorylation with brain extract can be assembled in vitro into filaments resembling those seen in Alzheimer's disease. J Mol Biol, 1994 Jan 7, 235(1), 389 - 95 Evidence that GHN phase bias does not constitute a framing code; Curran JF et al.; It has been suggested that a triplet repeated pattern found in coding sequences, the G-nonG-N or GHN phase bias, serves a framing function during protein synthesis . To test this idea, the framing characteristics of a highly GHN biased sequence are examined . No effects on reading frame maintenance are observed despite the use of sensitive frameshift assays . Specifically, first the GHN phase is not more accurate than the alternative overlapping phases (i.e., HNG and NGH) . Second, ribosomes do not exhibit any significant tendency to slip from the alternative frames into the GHN pahse . In addition, examination of Escherichia coli programmed frameshift sites does not support roles for GHN phase bias in programmed frameshifting . Framing functions for GHN phase bias, if they occur at all, must be extremely limited. J Mol Biol, 1994 Jan 7, 235(1), 33 - 41 Oxygen radical induced mutagenesis is DNA polymerase specific; Feig DI et al.; Oxygen free radicals are produced in large amounts by normal cellular processes . Damage to DNA by these reactive species has been implicated in mutagenesis and may be important in the etiology of a variety of human diseases . In this study we investigate the types of mutations produced in vitro as a result of DNA damage by oxygen free radicals . We used a lacZ alpha forward mutation assay in which M13 viral DNA is damaged in vitro, replicated with purified DNA polymerase alpha or beta, transfected into E . coli, and screened for mutations by reduced alpha-complementation of beta-galactosidase activity . By determining the effects of damaged templates on the fidelity of individual DNA polymerases involved in replication and repair, we address the role of specific DNA polymerases in mutagenesis induced by reactive oxygen species . Aerobic incubation of DNA with 100 microM CuCl, 10 microM H2O2 and 100 microM ascorbic acid results in a 3.3-fold and a 3.6-fold elevation in mutation frequency for polymerases alpha and beta, respectively . The specificity and location of the induced mutations, however, are entirely different . For polymerase alpha, A to C, and C to A transversions and deletions of C are each elevated more than 10-fold over their frequencies on undamaged template . For polymerase beta, A to T, C to T, C to A, G to C, and G to T substitutions, and deletions of G are elevated by damage . The frequency of mutants containing two or more closely spaced substitutions is also markedly increased by template damage although the types of mutations and their positions are again specific to each DNA polymerase . We conclude that, for oxidative lesions, the frequency and the types of mutations are determined in part by the DNA polymerase that encounters the site of damage. J Mol Biol, 1994 Jan 7, 235(1), 221 - 30 Explanation for different types of regulation of arginine biosynthesis in Escherichia coli B and Escherichia coli K12 caused by a difference between their arginine repressors; Tian G et al.; In Escherichia coli K12, formation of the enzymes of arginine biosynthesis are controlled by arginine, with complete repression during growth with added arginine, severe repression (about 95%) during growth without added arginine and complete derepression during arginine-limited growth . In E . coli B, the degree of repression is not correlated with arginine concentrations . Under all conditions of growth enzyme formation is repressed, with repression being somewhat less in a medium with arginine than in a medium without arginine . These differences in repressibility between the two strains have been shown previously to be due to the presence of different alleles of argR, the gene for the arginine repressor . Here we have compared the binding of the two repressors to the operator sites of argF (ARG boxes) . In DNase I footprinting and gel retardation experiments with argF ARG boxes we have shown that the arginine repressor of E . coli K12 bound to arginine (ArgRK-arg) has a greater affinity than the arginine repressor of E . coli B bound to arginine (ArgRB-arg), whereas free ArgRB (ArgRBf) has a much stronger affinity than free ArgRK (ArgRKf) . The stronger binding of ArgRBf can explain the repression seen in E . coli B during arginine-limited growth and indicates that ArgRBf, but not ArgRKf, is able to repress enzyme synthesis under physiological conditions . The weaker repression of E . coli B than of E . coli K12 seen in the presence of arginine can be explained by the lower affinity of ArgRB-arg for operator sites as compared to ArgRK-arg . Another contributing cause for the weaker repression is the reduction of ArgRBf concentration due to autoregulation of the gene for the repressor . Thus the combined effects of repression by ArgRBf, but not ArgRKf, with the weaker repression by ArgRB-arg as compared to ArgRK-arg, convert the arginine dependent regulation in E . coli K12 to arginine independent regulation in E . coli B. J Mol Biol, 1994 Jan 7, 235(1), 13 - 26 Redefining the goals of protein secondary structure prediction; Rost B et al.; Secondary structure prediction recently has surpassed the 70% level of average accuracy, evaluated on the single residue states helix, strand and loop (Q3) . But the ultimate goal is reliable prediction of tertiary (three-dimensional, 3D) structure, not 100% single residue accuracy for secondary structure . A comparison of pairs of structurally homologous proteins with divergent sequences reveals that considerable variation in the position and length of secondary structure segments can be accommodated within the same 3D fold . It is therefore sufficient to predict the approximate location of helix, strand, turn and loop segments, provided they are compatible with the formation of 3D structure . Accordingly, we define here a measure of segment overlap (Sov) that is somewhat insensitive to small variations in secondary structure assignments . The new segment overlap measure ranges from an ignorance level of 37% (random protein pairs) via a current level of 72% for a prediction method based on sequence profile input to neural networks (PHD) to an average 90% level for homologous protein pairs . We conclude that the highest scores one can reasonably expect for secondary structure prediction are a single residue accuracy of Q3 > 85% and a fractional segment overlap of Sov > 90%. J Mol Biol, 1994 Jan 7, 235(1), 125 - 39 Post-transcriptional regulation of the str operon in Escherichia coli . Structural and mutational analysis of the target site for translational repressor S7; Saito K et al.; In the Escherichia coli str operon, translation of the S12 and S7 genes is largely coupled, and the translational repressor S7 inhibits S7 translation, which is coupled to that of S12, but does not inhibit independent translation of S7 by free ribosomes in the intracellular pool . We have studied the S12-S7 intercistronic region of mRNA by analyzing RNA synthesized in vitro using structure-specific nucleases and a chemical probe, dimethyl sulfate . Based on the results obtained, we have deduced a secondary structure model of the S12-S7 intercistronic region and identified nucleotide residues "protected" by S7 . We then carried out site-directed mutagenesis to identify nucleotide residues important for S7 translation as well as for repression by S7 . The results showed that two distinct regions are important for S7-mediated repression; one is the S7 binding region identified by the protection analysis and the second is the stem structure that sequesters the Shine-Dalgarno sequence for the S7 gene . Some of the base alterations in the first region abolished S7 binding and, as a consequence, abolished S7-mediated repression, without affecting the efficiency of S7 translation . Other mutations disrupting the stem structure in the second region abolished S7-mediated repression without significantly affecting the S7-mRNA interaction . We also found that certain mutations drastically decrease S7 translation achieved by translational coupling without affecting S7 translation achieved by independent initiation . These mutations are in base-paired regions and evidence was obtained to suggest that these base-paired structures are important for translational coupling . We suggest that some specific RNA structures in the intercistronic region play an active role in achieving translational coupling in this system, and that repression of S7 translation by S7 protein is due to disruption of such structures induced by binding of S7 protein to the target site, rendering translational coupling very inefficient, but leaving independent translation initiation unaffected. J Med Chem, 1994 Jan 7, 37(1), 206 - 9 A recombinant human stromelysin catalytic domain identifying tryptophan derivatives as human stromelysin inhibitors; Ye QZ et al.; The human stromelysin catalytic domain (SCD) has been expressed in Escherichia coli and purified to homogeneity (Ye et al . Biochemistry 1992, 31, 11231) . We have used this recombinant SCD for inhibitor screening and identified tryptophan derivatives as competitive inhibitors of SCD . Both Cbz-L-Trp-OH (1, IC50 2.5 microM, Ki 2.1 microM) and Boc-L-Trp-OH (3, IC50 10 microM, Ki 8 microM) showed good inhibitory activity . Modification at the indole nitrogen with formyl or mesitylene-2-sulfonyl group (16, IC50 34 microM, Ki 28 microM; 17, IC50 63 microM, Ki 52 microM) showed reduced activity . The amide Cbz-L-Trp-NH2 (13) was not active, but esters Cbz-L-Trp-OSu (14, IC50 13 microM, Ki 11 microM) and Boc-L-Trp-OSu (15, IC50 102 microM, Ki 84 microM) showed activity . Aromatic amino acid derivatives Cbz-L-Tyr-OH (18, IC50 24 microM, Ki 20 microM) and Cbz-L-Phe-OH (26, IC50 40 microM, Ki 33 microM) were also active, but other amino acid derivatives had no activity . Although Cbz-D-Trp-OH (2, IC50 86 microM, Ki 71 microM) was active, the L-configuration is consistently preferred for inhibitory activity . Some of the SCD inhibitors were tested on full-length human stromelysin purified from cultured human cells, and they showed the same potency rank order . These results demonstrate the usefulness of recombinant DNA technology in generating the authentic human protein with improved properties for drug discovery. J Biol Chem, 1994 Jan 7, 269(1), 755 - 9 Characterization and crystallization of recombinant human neurotrophin-4; Fandl JP et al.; Neurotrophin-4 (NT-4) is the most recently discovered member of the neurotrophin family . We have expressed, refolded, and purified recombinant human NT-4 from Escherichia coli and compared it with recombinant human NT-4 secreted into the culture medium of baculovirus-infected insect cells . Both preparations were characterized and determined to be indistinguishable according to several biochemical criteria . Recombinant NT-4 from E . coli was crystallized in a form suitable for x-ray analysis, and characterization of these crystals indicated that NT-4 was present as a dimer within the asymmetric unit . NT-4 was active in promoting the survival of rat TrkB receptor-expressing fibroblasts, but was inactive on embryonic chicken sensory neurons, unlike the other members of the neurotrophin family and in contrast to the reported activities of partially purified NT-4. J Biol Chem, 1994 Jan 7, 269(1), 747 - 54 The REC1 gene of Ustilago maydis involved in the cellular response to DNA damage encodes an exonuclease; Thelen MP et al.; Mutation in the REC1 gene of Ustilago maydis is known to lead to a complex phenotype with alterations in DNA repair, recombination, mutagenesis, meiosis, and cell division . The predicted product of the REC1 gene is a polypeptide of 522 amino acid residues with a molecular mass of 56,866 daltons, with no overall sequence homology to any other known protein . The open reading frame of the REC1 gene placed by itself in a U . maydis expression vector was found to be sufficient to complement the rec1 mutant . Overexpression of REC1 in Escherichia coli gave rise to the anticipated 57-kDa product together with a 3'-->5' exonuclease activity . This activity was only present in cells overexpressing REC1 and its characteristics were distinguishable from the major bacterial nucleases, but it had certain enzymatic features in common with epsilon, the proofreading exonuclease subunit of E . coli DNA polymerase III holoenzyme . To facilitate isolation of the protein product from bacteria, the REC1 gene was overexpressed from a vector that fused a hexa-histidine-leader sequence onto the amino terminus, enabling the isolation of the HisREC1 product on an immobilized metal ion affinity column . The His-REC1 protein co-eluted with the novel exonuclease activity . Alignment of the amino acid sequence of the REC1 gene product with the conserved proofreading exonuclease motifs of DNA polymerases indicated significant homology. J Biol Chem, 1994 Jan 7, 269(1), 66 - 70 Properties of purified recombinant poliovirus protein 3aB as substrate for viral proteinases and as co-factor for RNA polymerase 3Dpol; Lama J et al.; The poliovirus-specific polypeptide 3AB (B = VPg) was expressed in Escherichia coli and purified to near homogeneity . Corresponding to its known association with membranes in poliovirus-infected HeLa cells, 3AB expressed in E . coli was also membrane-associated, and it could be solubilized only in detergent-containing buffers . In soluble form, 3AB was resistant to digestion with the virus-specific proteinases 3Cpro and 3CDpro . However, it was cleaved by these enzymes to 3A and VPg when bound to the bacterial membranes, an observation suggesting that 3AB may deliver the genome-linked protein VPg to the membrane-associated poliovirus replication complex . The specific activity of 3CDpro in processing 3AB was significantly higher than that of 3Cpro . Soluble 3AB was found to stimulate nearly 100-fold poly (A)-dependent, primer-dependent poly(U) synthesis, catalyzed by purified poliovirus RNA polymerase 3Dpol . We propose that 3AB has a dual function in poliovirus genome replication: as a precursor for VPg, and as a co-factor for 3Dpol. J Biol Chem, 1994 Jan 7, 269(1), 573 - 8 Mutations of vaccinia virus DNA topoisomerase I that stabilize the cleavage complex; Gupta M et al.; Two mutations in vaccinia virus topoisomerase I, K167D and G226N, have been characterized . SOS induction was observed in Escherichia coli expressing vaccinia topoisomerase I with either one of these mutations . The mutant enzymes were purified to homogeneity and compared with the wild type enzyme for relaxation activity and the partial activities of substrate binding, site-specific DNA cleavage and DNA religation to determine the mechanism of SOS induction . The K167D mutant enzyme had reduced binding affinity for the DNA substrate with a Kapp that was 10-fold higher than wild type . Nevertheless, in reactions with high enzyme concentration, its substrate cleavage activity was 90% that of wild type . The G226N mutant enzyme had virtually wild type binding and cleavage activities . However, intermolecular religation by these two mutants were observed to be significantly reduced . The cleavage complexes formed with the K167D and G226N mutants were more stable to high salt than the wild type cleavable complex . We propose that these mutants in vivo induce the SOS response in E . coli due to the shift of topoisomerase cleavage-religation equilibrium towards cleavage and increased stability of the cleavage complex . The mutation thus has a similar effect as the topoisomerase-targeting inhibitors that turn topoisomerases into DNA damaging agents. J Biol Chem, 1994 Jan 7, 269(1), 485 - 92 Sequence-specific interactions of UvrABC endonuclease with psoralen interstrand cross-links; Ramaswamy M et al.; The nature of the Uvr protein-DNA complexes formed on psoralen-DNA interstrand cross-links was analyzed by DNase I footprinting and correlated with the incision efficiency of the UvrABC endonuclease on the cross-links of different DNA sequences . Our results indicate that the repair specificity is dependent on the DNA sequence and the psoralen orientation in the cross-link . On the strand that will be cut, a 30-nucleotide long UvrAB footprint with a DNase I hypersensitive site at the 11th nucleotide 5' to the lesion was observed and subsequently rearranged to a 22-nucleotide long UvrB-lesion footprint . On the strand that will not be cut, the UvrAB-lesion footprint had no 5' DNase I hypersensitive site and did not form the UvrB-lesion footprint . Although UvrABC incision requires the formation of UvrB-lesion complex on the strand which will be cut, the affinities of these complexes do not correlate with the incision efficiencies, suggesting that the overall reaction can be driven forward by a favorable next step such as UvrC incision . A study of the time-dependent interconversion of UvrAB-lesion complex to UvrB-lesion complex on a cross-link revealed a secondary recognition of the UvrB-lesion complex by UvrA2(B) proteins in vitro. J Biol Chem, 1994 Jan 7, 269(1), 470 - 7 Photoaffinity-labeling peptide substrates for farnesyl-protein transferase and the intersubunit location of the active site; Ying W et al.; --CAAX motif peptides, which are substrates for isoprenylation, were synthetically derivatized with the light-sensitive benzophenone (Bz) group in order to determine their potential use as catalytic site-directed covalent photocross-linking ligands for one of the enzymes catalyzing protein isoprenylation, farnesyl-protein transferase (FPTase) . Bz-peptides could be synthesized with {3H}benzophenone and possessed eiter one or two benzophenone groups located at or near the peptide's NH2 terminus (e.g . the mono-Bz probes Bz-ACVIM and Bz-LPCVVM, and the di-Bz derivatized probe Bz-GY-(Bz)PCVVM, referred to as Bz2-GYPCVVM) . Each type of derivatized peptide behaved as a substrate for farnesylation in vitro without irradiation, while under 366-nm irradiation each demonstrated covalent cross-linking ability as a catalytic site-directed photoaffinity ligand with tissue-purified or enriched but impure fractions from rat and bovine brain FPTase, as well as with a recombinant human FPTase variant, FPTase (beta alpha t) expressed in Escherichia coli . Without photoactivation, Bz-ACVIM yielded a Kd of 37 nM for the cloned variant of human FPTase . Pseudo first-order photolytic inhibition of FPTase preparations with Bz-peptides, as well as protection from photoinactivation by unmodified -CAAX motif peptides, supported the capacity of these Bz-peptides to serve as co-substrates and their specificity for seeking the catalytic site of the enzyme . SDS-polyacrylamide gel electrophoresis analysis subsequent to photolysis indicated that the mono-Bz-derivatized peptides (e.g . {3H}Bz-LPCVVM or 3H}Bz-ACVIM) became covalently cross-linked preferentially to the approximately 49-kDa beta subunit of the alpha beta dimeric FPTase . The farnesyl-PP cosubstrate bound equally well to unmodified and Bz-ACVIM-labeled enzyme . The di-Bz derivative, {3H}Bz2-GYPCVVM, in contrast, revealed exclusive photocovalent cross-linking with a species of molecular mass approximately 95-97 kDa, indicating that both FPTase subunits were tethered together covalently by the di-Bz probe . Similar differential SDS-polyacrylamide gel electrophoresis cross-linking patterns were obtained with homogeneous FPTases as well as with partially purified rat or bovine brain enzyme preparations . The absence of nonspecific photolabeling of any proteins in the partially purified rat or bovine brain enzyme preparations other than FPTase independently attested to the high efficiency of photocross-linking of the FTPase, and the selective catalytic site-seeking ability of these Bz-derivatized peptide substrates, verifying their potential as structural probes for the active site domain on the enzyme.(ABSTRACT TRUNCATED AT 400 WORDS) J Biol Chem, 1994 Jan 7, 269(1), 427 - 32 Defective energy coupling in delta-subunit mutants of Escherichia coli F1F0-ATP synthase; Hazard AL et al.; Membrane vesicles from 13 strains carrying mutations in the C-terminal region of the delta-subunit of Escherichia coli F1F0-ATP synthase were characterized in respect to ATPase activity, ATP-driven proton-pumping, dicyclohexylcarbodiimide sensitivity of ATPase, and oxidative phosphorylation . The salient finding was that energy-coupling between F1 and F0 sectors of the enzyme is impaired by several of the mutations . The delta G150N mutant appeared completely uncoupled in vitro . The data emphasize the role of the C-terminal region of delta-subunit in integration of the proton conduction machinery in F0 with the three F1 catalytic sites . It is suggested that the C-terminal region of delta-subunit, speculatively located in the central region of the alpha 3 beta 3 hexagon, acts functionally at the interface between the helical domain of the stalk and the F1 subunits to relay conformational signals which alter the affinities of the catalytic sites for substrates and products. J Biol Chem, 1994 Jan 7, 269(1), 418 - 26 Mutagenesis of subunit delta from Escherichia coli F1F0-ATP synthase; Hazard AL et al.; In Escherichia coli, the F1 sector of the F1F0-ATP synthase is connected to the membrane-embedded F0 sector by a narrow stalk, thought to be formed by subunits delta and b . Mutagenic analysis was used here to study the structure and function of subunit delta . First, random mutations in the protein were generated by bisulfite mutagenesis . Two single missense mutations causing impaired growth by oxidative phosphorylation were found, namely delta A149T and delta G150D . Both occur at the conserved C-terminal region, which has been suggested previously to be functionally important . Two techniques were applied to study the C-terminal region in greater detail . Cassette mutagenesis was used to randomly mutate the sequence from delta 145 to delta 167, and residues delta A149 and delta G150 were specifically mutated by site-directed mutagenesis to obtain multiple substitutions at each position . Fifteen of the residues between delta 145 and delta 167 were mutated . None was found to be absolutely essential for function . However, the properties of the mutants obtained, which included partial impairment of growth by oxidative phosphorylation, temperature sensitivity, and specific structural requirements at residues delta A149 and delta G150, confirmed that this region is important for enzyme function . Based on these studies, and on secondary and tertiary structure predictions, a model for subunit delta and its orientation in F1F0-ATP synthase is proposed. J Biol Chem, 1994 Jan 7, 269(1), 412 - 7 Active site mutants of Escherichia coli citrate synthase . Effects of mutations on catalytic and allosteric properties; Pereira DS et al.; We report properties of five active site mutants of Escherichia coli citrate synthase, in which histidine 264, aspartate 362, and phenylalanine 383 were replaced by alanines, and arginines 387 and 407 by leucines . All mutants have much lower turnover numbers than wild type enzyme; the strongest effect was with the arginine 387 mutant, perhaps because the substrate, oxaloacetate, binds in a different orientation . The arginine 407 mutant has lost most of its ability to distinguish alpha-ketoglutarate, a competitive inhibitor, from oxaloacetate . The mutations of histidine 264 and aspartate 362 affect steady-state kinetics as would be anticipated from current models for citrate synthase catalysis, and resemble mutations of these residues, in pig heart and E . coli enzyme, reported by others . Mutations of residues 264, 362, and 383 also affect allosteric properties . With the phenylalanine 383 mutant, acetyl-CoA saturation is strongly sigmoid, even in the presence of the activator, KCl, implying a marked shift of the allosteric equilibrium toward the T state . The histidine 264 mutant appears to be shifted toward R state and shows weaker binding of the allosteric inhibitor, NADH; thus this mutation also affects the allosteric site, 25-30 A away. J Biol Chem, 1994 Jan 7, 269(1), 390 - 5 Mammalian ferrochelatase . Expression and characterization of normal and two human protoporphyric ferrochelatases; Dailey HA et al.; Ferrochelatase (EC 4.99.1.1) catalyzes the terminal step in the heme biosynthetic pathway, the insertion of ferrous iron into protoporphyrin IX . Herein we report the expression, purification, and characterization of the mature processed form of human and mouse ferrochelatase in Escherichia coli JM109 . Metal analysis of the recombinant normal human ferrochelatase reveals that there are approximately 2 iron atoms/molecule of enzyme . This, along with the presence of spectral absorbance near 320 nm, is strongly suggestive that recombinant mammalian ferrochelatase as expressed in E . coli may contain an iron sulfur cluster . Two human protoporphyric ferrochelatases, F417S and M267I, were also expressed and characterized . The M267I mutant possesses the same Km and Vmax as the normal enzyme but exhibits increased thermolability when compared with normal human ferrochelatase . The F417S mutant has less than 2% of the normal activity . Since the Phe-->Ser substitution in this mutation is both chemically and structurally significant, three single amino acid substitutions (Lys, Tyr, and Trp) were engineered and characterized . None of these resulted in a protein with wild type activity . Additionally the carboxyl-terminal 10-amino acid segment, which contains Phe-417, from the yeast sequence was substituted, but this construct had no activity . Elimination of the carboxyl-terminal 30 amino acid residues (which include Phe-417) results in a protein the same length as the bacterial ferrochelatases, but it is an inactive enzyme. J Biol Chem, 1994 Jan 7, 269(1), 272 - 6 Molecular cloning of rat liver sulfite oxidase . Expression of a eukaryotic Mo-pterin-containing enzyme in Escherichia coli; Garrett RM et al.; The cDNA encoding sulfite oxidase has been cloned from a rat liver cDNA library . The gene contains a single open reading frame of 1464 nucleotides encoding a protein of 488 amino acids . The deduced amino acid sequence contains a 22-residue amino-terminal presequence that may serve as a mitochondrial targeting signal . The amino acid sequence deduced from the cDNA shows significant similarity to those of sulfite oxidase from chicken liver and nitrate reductases from algal, fungal, and plant sources . Two cysteine residues are conserved in all of these proteins, and it is proposed that one or both of these cysteines serve as ligands to molybdenum . The gene has been expressed in Escherichia coli to a level equivalent to that observed in rat liver . The recombinant enzyme has been found to contain the molybdopterin form of the molybdenum cofactor and is active as determined by the sulfite dependent reduction of cytochrome c. J Biol Chem, 1994 Jan 7, 269(1), 199 - 206 Construction, expression, and activity of a bivalent bispecific single-chain antibody; Mallender WD et al.; This report describes the design, construction, and expression of a bivalent bispecific single-chain antibody (SCA) protein in Escherichia coli . The bispecificity of the bivalent protein was based on two previously constructed monovalent single-chain antibody molecules possessing distinct specificities, SCA 4-4-20 (anti-fluorescein) and SCA 04-01 (anti-single-stranded DNA) . A flexible linker, modeled after a secreted fungal cellulase protein, was incorporated as the interdomain linker covalently joining the two active sites . Bivalent bispecific SCA protein that accumulated in bacteria as insoluble inclusion bodies was harvested, denatured, refolded, and affinity-purified in vitro . Affinity-purified bivalent bispecific SCA showed nearly identical ligand binding properties at each site relative to the individual monovalent single-chain antibody prototype molecules . In both solid and solution phase binding assays, the bivalent bispecific single-chain antibody simultaneously bound both ligands (fluorescein and (dT)6) . Construction of a model bivalent bispecific molecule provides a foundation for future assembly of similar molecules designed to identify parameters involved in enhanced binding of antibodies due to avidity and dual specificity. J Mol Biol, 1994 Jan 7, 235(1), 68 - 72 The role of Glu187 in the regulation of phosphofructokinase by phosphoenolpyruvate; Auzat I et al.; In bacterial phosphofructokinases, either a glutamic or an aspartic residue is present at position 187, and the mechanism of inhibition by phosphoenolpyruvate seems to be correlated to the nature of residue 187 . Upon binding phosphoenolpyruvate, only the enzymes with a Glu187 would undergo a major allosteric conformational change from an active into an inactive state, whereas the enzymes with an Asp187 would only show a simple upward shift in their pH-profile of activity . The phosphofructokinase from Spiroplasma citri, which has an Asp187, has been purified and its properties follow this pattern . The behaviour of mutants of the enzyme from Escherichia coli in which Glu187 is replaced by either aspartate or leucine confirms the importance of residue 187 . The major allosteric transition of E . coli phosphofructokinase is abolished by the substitution Glu187-->Asp, suggesting that a glutamate at position 187 is necessary (but not sufficient) for the protein to undergo the change from the active into the inactive state induced by phosphenolpyruvate . In addition, the presence of an acidic residue, aspartate or glutamate, at position 187 is required (but not sufficient) for the binding of ADP (or GDP) . This requirement of a negative charge for ADP binding could explain the striking conservation of an aspartate residue at position 187 in all the eukaryotic phosphofructokinases. J Mol Biol, 1994 Jan 7, 235(1), 47 - 52 The symmetry of Escherichia coli cpn60 (GroEL) determined by X-ray crystallography; Svensson LA et al.; The internal symmetries of the Escherichia coli molecular chaperone cpn60 oligomer, also called GroEL, have been examined by X-ray crystallography and self-rotation functions calculated at a resolution of 8.9 A . The oligomer ({cpn60}14) has one 7-fold symmetry axis and seven 2-fold axes that are all perpendicular to the 7-fold . The symmetry can be explained if oligomeric cpn60 is arranged as two heptamers stacked on top of each other, where the heptameric arrangement generates the 7-fold symmetry axis and the head-to-head assembly of two heptamers results in the seven 2-fold axes . This is an agreement with interpretations of electron microscopy data . However, the experimental determination of the symmetries reported here are made with an independent technique and at higher resolution . In addition self-rotation function calculations show that the symmetries observed are valid also for the internal parts of GroEL and not only for surface views . The orientations of the symmetry axes of the two independent cpn60 oligomers in the triclinic unit cell have been determined relative to the crystallographic axes . The planes formed by the 2-fold axes in the two oligomers deviate by about 2 degrees from the plane formed by the crystallographic a and c axes, while the 7-fold axes form angles of about 16 degrees with the b-axis . The two oligomers in the unit cell are arranged with their 7-fold axis parallel, but the second oligomer is rotated 26 degrees around the 7-fold axis relative to the first oligomer . Knowledge of the symmetry and orientation of the oligomers in the unit cell will be of great help in further crystallographic work. J Mol Biol, 1994 Jan 7, 235(1), 367 - 9 Preliminary X-ray crystallographic analysis of biotin carboxylase isolated from Escherichia coli; Waldrop G et al.; Acetyl CoA carboxylase catalyzes the first committed step in the biosynthesis of long chain fatty acids . In Escherichia coli, the enzyme consists of three subunits that are isolated separately and display distinct functional properties . Here we report the crystallization and preliminary X-ray analysis of one of these components, namely biotin carboxylase . The crystals are grown by microdialysis against 10 mM potassium phosphate (pH 7.0), 1 mM EDTA, 2 mM DTT and 1 mM NaN3 at 4 degrees C . They belong to the space group P2(1)2(1)2(1) with unit cell dimensions of a = 61.9 A, b = 96.1 A and c = 180.6 A and contain one dimer per asymmetric unit . The crystals diffract to a nominal resolution of 2.2 A . From a mechanistic standpoint, biotin carboxylase is especially interesting in that it is the smallest protein within its class and is one of only two carboxylases that can utilize free biotin as a substrate. J Biol Chem, 1994 Jan 7, 269(1), 44 - 6 Direct demonstration that ATP is in contact with Cys-137 in chaperonin GroEL; Bochkareva ES et al.; The nonhydrolyzable ATP analogue ATP gamma S (adenosine 5'-3-O-(thio)triphosphate) is affinity cross-linked to GroEL by formation of a disulfide bridge in a peroxide-promoted reaction . By replacing with serine each of 3 cysteine residues in GroEL, it is shown that ATP gamma S specifically cross-links to Cys-137 . It is thus demonstrated that the ATP bound to GroEL is in direct contact with Cys-137. J Mol Biol, 1994 Jan 7, 235(1), 111 - 24 Post-transcriptional regulation of the str operon in Escherichia coli . Ribosomal protein S7 inhibits coupled translation of S7 but not its independent translation; Saito K et al.; The str operon of Escherichia coli consists of the genes for ribosomal proteins S12 (rpsL) and S7 (rpsG) and elongation factors G (fusA) and Tu (tufA) . Previous studies have shown that S7 is a translational feedback repressor and inhibits the synthesis of itself and of elongation factor G . We have now shown that induction of S7 synthesis from the S7 gene fused to the arabinose promoter on a plasmid also leads to inhibition of the synthesis of S12 from the chromosomal S12 gene, and that this regulation takes place using the same target site as that used for distal gene regulation, i.e . S7 retroregulates S12 . We have then demonstrated that S7 synthesis is mostly translationally coupled with the translation of the preceding S12 gene . Using a rpsG'-'lacZ fusion gene as a reporter for S7 synthesis, we found that abolishing S12 translation by a mutational alteration of the AUG start codon of the S12 gene leads to about tenfold reduction of S7 synthesis without significantly affecting its rate of transcription . Deletion of the proximal portion of the S12 gene or a premature termination of S12 translation by an amber mutation at the 26th codon also led to a large reduction of S7 synthesis . Unexpectedly, we have discovered that overproduction of S7 in trans from a plasmid leads to repression of the rpsG'-'lacZ fusion gene when the fusion gene is preceded by the intact S12 gene, but not when the S12 gene carried the above-mentioned mutations that abolish S12 translation . Thus, a novel feature of this regulatory system is that translation of S7 achieved by independent initiation is not inhibited by S7 in vivo, whereas translation of S7 achieved by translational coupling is sensitive to S7 repression . These observations also suggest that the coupled S7 translation is probably achieved by the use of ribosomal subunits employed for translation of the upstream S12 gene. J Biol Chem, 1994 Jan 7, 269(1), 716 - 20 A computer-assisted analysis of conserved residues in the three-dimensional structures of the polymerase domains of Escherichia coli DNA polymerase I and HIV-1 reverse transcriptase; Yadav PN et al.; Using a computer-assisted molecular modeling protocol, we have completed the three-dimensional structures of HIV-1 reverse transcriptase and the Klenow fragment of DNA polymerase I based on the C alpha crystal coordinates of the individual enzymes . The two model-built structures were then used to compare the electrostatic potential contours and analyze the spatial positions of residues conserved in the catalytic domains of the two enzymes . In spite of rather weak sequence similarity and different folding patterns between the DNA-dependent DNA polymerase (pol I) and the RNA-dependent DNA polymerases (RT), we have noted the occurrence of identical or similar residues at common spatial positions in pol I and RT in a three-dimensional context . The homologous residues present at equivalent spatial position in the Klenow fragment and the p66 subunit of HIV-1 RT may therefore imply their functional similarity . Furthermore, these conserved residues may represent a similar structure-function feature in all polymerases. J Biol Chem, 1994 Jan 7, 269(1), 51 - 4 Activation of the cystic fibrosis transmembrane conductance regulator by cGMP in the human colonic cancer cell line, Caco-2; Tien XY et al.; Intestinal chloride (Cl-) secretion can be induced by the heat-stable enterotoxin (STa) from Escherichia coli via generation of cGMP . We investigated the regulatory pathway responsible for cGMP-mediated Cl- secretion in the human colonic carcinoma cell line Caco-2 using whole-cell voltage clamp techniques . Cyclic GMP or cAMP induced a 5-fold increase in Cl- conductance (gCl) in the presence of intracellular ATP and 3-isobutyl-1-methylxanthine . Current activation by cGMP persisted in the presence of the type I cGMP-dependent protein kinase (PKG) inhibitor, KT5823, but was inhibited by the specific peptide inhibitor of the cAMP-dependent protein kinase A (PKA), PKI5-24 . The stimulatory effects of cGMP and cAMP on gCl were not additive . The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel that is regulated by intracellular ATP and by cAMP-dependent phosphorylation . In order to determine whether CFTR was involved in the cGMP-dependent increase in gCl, we tested the effect of intracellularly injected anti-CFTR505-511 antibodies previously shown to inhibit CFTR function . Antibodies introduced into individual cells via the patch pipette completely inhibited cGMP-dependent current activation . Cyclic GMP also failed to activate gCl in cystic fibrosis cells . Taken together, these studies demonstrate that activation of the CFTR via PKA-dependent phosphorylation accounts for the cGMP-mediated increase in Cl- secretion in Caco-2 cells. J Biol Chem, 1994 Jan 7, 269(1), 161 - 8 The mechanism of human immunodeficiency virus reverse transcriptase-catalyzed strand transfer from internal regions of heteropolymeric RNA templates; DeStefano JJ et al.; The mechanism of human immunodeficiency virus reverse transcriptase-catalyzed strand transfer synthesis (i.e . switching of the primer to a new template) from internal regions of natural sequence RNA was investigated . The system consisted of a 142-nucleotide RNA template (donor) primed with a specific 20-nucleotide DNA oligonucleotide used to initiate synthesis . DNA oligonucleotides with homology to internal regions of the donor were used as acceptor templates . In reactions performed in the absence of acceptor template, a prominent DNA synthesis product 75 nucleotides in length resulting from pausing DNA synthesis within the homology zone was observed . Prominent donor RNA degradation products of 47 or 54 nucleotides were also observed, in reactions with 80 or 150 mM KCl, respectively . The lengths indicated a potential 13- or 20-nucleotide long, respectively, complementary region between the DNA and RNAs . The 54-, but not the 47-, nucleotide RNA was susceptible to Escherichia coli RNase H, indicating that the DNA was annealed only to the 54-mer . When acceptor was added, a portion of the 75-nucleotide DNA was chased into transfer product at both salt concentrations, and a portion of the 54-mer RNA became resistant to E . coli RNase H . Evidently, this donor RNA was annealed to the 75-nucleotide long DNA but could be actively displaced by the acceptor . Overall, these observations support two mechanisms for transfer . In one, the pause site-specific DNA dissociates from the donor template before transferring . In the other, the acceptor actively displaces the DNA from the donor. Biochim Biophys Acta, 1994 Jan 4, 1183(3), 547 - 9 Molecular cloning, sequencing and expression of cDNA encoding human coproporphyrinogen oxidase; Taketani S et al.; A complete cDNA clone encoding human coproporphyrinogen (coprogen) oxidase, the sixth enzyme in the heme biosynthetic pathway, has been isolated from a human placenta cDNA library . The cDNA had an open reading frame of 1062 base pairs encoding a protein of 354 amino acid residues (M(r) 40,291) . Amino acid sequencing showed that the mature enzyme consists of 323 amino acid residues (M(r) 36,842) with a putative leader peptide of 31 amino acid residues . The human enzyme showed an 86% identity to the mouse enzyme . In addition, the recombinant enzyme which did not contain leader peptide was actively expressed in Escherichia coli . The isolation and expression of cDNA for human coprogen oxidase should facilitate studies of the structure of the gene as well as characterization of molecular lesions causing hereditary coproporphyria. Biochim Biophys Acta, 1994 Jan 4, 1183(3), 521 - 32 Flash photolysis of the carbon monoxide compounds of wild-type and mutant variants of cytochrome bo from Escherichia coli; Brown S et al.; The carbon monoxide compounds of the fully reduced and mixed valence forms of cytochrome bo from Escherichia coli were laser photolysed under anaerobic conditions at room temperature . The carbon monoxide recombined with characteristic rate constants of 50 s-1 or 35 s-1 in the fully reduced and mixed valence forms, respectively . Rates of CO recombination with the fully reduced enzyme were examined in a variety of mutant forms of cytochrome bo, produced by site-directed mutagenesis . A method was developed to deconvolute cytochromes bo and bd, leading to some reassessment of histidine ligands to the metals . Significant changes in the rate constant of recombination of carbon monoxide occurred in many of these mutants and these results could be rationalised generally in terms of our current working model of the folding structure of subunit I . In the mixed valence form of the enzyme the transient photolysis spectra in the visible region are consistent with a rapid electron redistribution from the binuclear centre to the low-spin haem . This electron transfer is biphasic, with rate constants of around 10(5) and 8000 s-1 . The process was also examined in the His-333-Leu mutant, in which a putative histidine ligand to CuB is replaced by leucine, and which results in the loss of the CuB . It appeared that rapid haem-haem electron transfer could still occur . The observation that CuB is apparently not required for rapid haem-haem electron transfer is consistent with the recently proposed model in which the two haems are positioned on opposite sides of transmembrane helix X in subunit I of the oxidase. Biochim Biophys Acta, 1994 Jan 4, 1183(3), 499 - 503 Effects of deletions in the uncA-uncG intergenic regions on expression of uncG, the gene for the gamma subunit of the Escherichia coli F1Fo-ATPase; Angov E et al.; The gamma subunit of the E . coli F1Fo-ATPase is coded for by uncG . This gene is poorly expressed compared to uncA (alpha subunit), which precedes uncG in the unc operon . The genes are separated by a 50-nucleotide intergenic region . We examined the effects of a set of deletions in this region on the relative expression of uncA'-'lacZ and uncG'-'lacZ translational fusion genes located either in the chromosomal unc operon or at the chromosomal lambda att site . The gene for the alpha subunit was expressed 3-6-times better than the gene for the gamma subunit, depending upon chromosomal location . Deletion analysis revealed that the uncA-uncG intergenic region significantly affects expression of uncG, but the Shine-Dalgarno region is not absolutely required for expression of uncG . Different deletions resulted in either increased or decreased expression of uncG. Proc Natl Acad Sci U S A, 1994 Jan 4, 91(1), 306 - 10 Osmoprotective compounds in the Plumbaginaceae: a natural experiment in metabolic engineering of stress tolerance; Hanson AD et al.; In common with other zwitterionic quarternary ammonium compounds (QACs), glycine betaine acts as an osmoprotectant in plants, bacteria, and animals, with its accumulation in the cytoplasm reducing adverse effects of salinity and drought . For this reason, the glycine betaine biosynthesis pathway has become a target for genetic engineering of stress tolerance in crop plants . Besides glycine betaine, several other QAC osmoprotectants have been reported to accumulate among flowering plants, although little is known about their distribution, evolution, or adaptive value . We show here that various taxa of the highly stress-tolerant family Plumbaginaceae have evolved four QACs, which supplement or replace glycine betaine-namely, choline O-sulfate and the betaines of beta-alanine, proline, and hydroxyproline . Evidence from bacterial bioassays demonstrates that these QACs function no better than glycine betaine as osmoprotectants . However, the distribution of QACs among diverse members of the Plumbaginaceae adapted to different types of habitat indicates that different QACs could have selective advantages in particular stress environments . Specifically, choline O-sulfate can function in sulfate detoxification as well as in osmoprotection, beta-alanine betaine may be superior to glycine betaine in hypoxic saline conditions, and proline-derived betaines may be beneficial in chronically dry environments . We conclude that the evolution of osmoprotectant diversity within the Plumbaginaceae suggests additional possibilities to explore in the metabolic engineering of stress tolerance in crops. Proc Natl Acad Sci U S A, 1994 Jan 4, 91(1), 316 - 20 Cysteine-rich LIM domains of LIM-homeodomain and LIM-only proteins contain zinc but not iron; Archer VE et al.; The structure of LIM domains has major implications for transcription because proteins such as Is1-1 contain two LIM domains associated with a homeodomain, and RBTN1/Ttg-1 and RBTN2/Ttg-2 contain two LIM domains but no homeodomain . Conserved cysteine and histidine residues in the LIM domains suggest a metal-binding role . RBTN and Is1-1 LIM proteins have been made in Escherichia coli and insect cell expression systems and their metal content has been determined using atomic absorption spectroscopy and electron paramagnetic resonance spectroscopy . LIM proteins expressed in soluble form contain zinc atoms, whereas bacterial inclusion bodies invariably also have Fe-S clusters . The latter are identified as linear {Fe3S4}+ clusters and appear to result from incorrect metal coordination by E . coli . These studies show that RBTN1, RBTN2, and Is1-1 are metalloproteins that contain zinc but not iron and, therefore, that the LIM domain represents a zinc-binding domain. Proc Natl Acad Sci U S A, 1994 Jan 4, 91(1), 291 - 5 Functional communication in the recognition of tRNA by Escherichia coli glutaminyl-tRNA synthetase; Rogers MJ et al.; Wild-type Escherichia coli glutaminyl-tRNA synthetase (GlnRS; EC 6.1.1.18) poorly aminoacylates opal suppressors (GLN) derived from tRNA(Gln) . Mutations in glnS (the gene encoding GlnRS) that compensate for impaired aminoacylation were isolated by genetic selection . Two glnS mutants were obtained by using opal suppressors differing in the nucleotides composing the base pair at 3.70: glnS113 with an Asp-235-->Asn change selected with GLNA3U70 (GLN carrying G3-->A and C70-->U changes), and glnS114 with a Gln-318-->Arg change selected with GLNU70 (GLN carrying a C70-->U change) . The Asp-235-->Asn change was identified previously by genetic selection . Additional mutants were isolated by site-directed mutagenesis followed by genetic selection; the mutant enzymes have single amino acid changes (Lys-317-->Arg and Gln-318-->Lys) . A number of mutants with no phenotype also were obtained randomly . In vitro aminoacylation of a tRNA(Gln) transcript by GlnRS enzymes with Lys-317-->Arg, Gln-318-->Lys, or Gln-318-->Arg changes shows that the enzyme's kinetic parameters are not greatly affected by the mutations . However, aminoacylation of a tRNA(Gln) transcript with an opal (UCA) anticodon shows that the specificity constants (kcat/Km) for the mutant enzymes were 5-10 times above that of the wild-type GlnRS . Interactions between Lys-317 and Gln-318 with the inside of the L-shaped tRNA and with the side chain of Gln-234 provide a connection between the acceptor end-binding and anticodon-binding domains of GlnRS . The GlnRS mutants isolated suggest that perturbation of the interactions with the inside of the tRNA L shape results in relaxed anticodon recognition. Proc Natl Acad Sci U S A, 1994 Jan 4, 91(1), 271 - 5 Attenuation of the induction of nitric oxide synthase by endogenous glucocorticoids accounts for endotoxin tolerance in vivo; Szabo C et al.; An enhanced formation of nitric oxide (NO) due to induction of a calcium-independent (inducible) NO synthase (iNOS) contributes importantly to the cardiovascular failure caused by bacterial endotoxin . Repeated challenges with small doses of endotoxin result in tolerance to both peripheral vascular failure and death caused by subsequent injection of a higher dose of endotoxin . Here we investigate whether tolerance to endotoxin is associated with a lack of induction of iNOS in vivo and whether endogenous glucocorticoids play a role in the development of endotoxin tolerance . In anesthetized rats, i.v . administration of Escherichia coli endotoxin {lipopolysaccharide (LPS); 2 mg.kg-1} resulted in a prolonged decrease in mean arterial blood pressure (MAP) and hyporeactivity to the contractile responses elicited by norepinephrine (NE; 10 nM) in aortic rings ex vivo . Hyporeactivity to NE was partially reversed by NG-nitro-L-arginine methyl ester (0.3 mM) in vitro, suggesting that an enhanced formation of NO contributes to this hyporeactivity . There was a substantial increase in the activity of iNOS in the lung 3 h after i.v . injection of LPS (0.2 +/- 0.1 to 6.6 +/- 0.6 pmol.mg-1.min-1; n = 5; P < 0.01) . Rats injected i.p . with LPS (0.5 mg.kg-1) for 4 consecutive days became tolerant to an i.v . injection of LPS (2 mg.kg-1) in that both hypotension and vascular hyporeactivity to NE were significantly attenuated . Moreover, in these endotoxin-tolerant rats, the induction of iNOS by LPS in the lung was attenuated by 63% +/- 6% . Injection of LPS caused a 9-fold increase in plasma corticosterone (CCS) levels within 2 h and CCS levels remained significantly elevated 6 and 24 h after LPS . Animals rendered tolerant to endotoxin by administration of a low dose of LPS (0.5 mg.kg-1, i.p.) for 4 days still had a 6-fold increase in plasma CCS levels 24 h after the last injection of LPS . When endotoxin-tolerant rats were treated with the glucocorticoid receptor antagonist RU 486 (50 mg.kg-1, p.o . 3 h prior to LPS), there was a restoration of the effects of LPS (2 mg.kg-1, i.v.) in causing hypotension, vascular hyporeactivity to NE, and iNOS induction in the lung . However, in control rats RU 486 enhanced neither the decrease in MAP nor the induction of iNOS in response to LPS (2 mg.kg-1, i.v.) . Thus, cardiovascular tolerance to endotoxin is accompanied and explained by reduced induction of iNOS in vivo due to the elevation of endogenous glucocorticoid levels. FEBS Lett, 1994 Jan 3, 337(1), 66 - 70 The transglycosylation reaction of cyclodextrin glucanotransferase is operated by a Ping-Pong mechanism; Nakamura A et al.; A new photometric assay of the disproportionation activity of cyclodextrin glucanotransferase (CGTase) using 3-ketobutylidene-beta-2-chloro-4-nitrophenyl-maltopentaoside as the donor, proved that the transglycosylation reaction of CGTase was operated by a Ping-Pong Bi Bi mechanism . The values of the kcat/Km(acceptor) proved that the same configurations of free hydroxyl groups with those of D-glucopyranose at C2, C3 and C4 positions were required for the acceptors used by CGTase . The structure around C6 on acceptors was not essential for acceptor function, but it was recognized by CGTase, since the values of kcat/Km for D-xylose were smaller than that for D-glucose . The value of kcat/Km for maltose was about 20-times larger than that for D-glucose, indicating that at least two glucopyranosyl rings are recognized by the acceptor binding sites. FEBS Lett, 1994 Jan 3, 337(1), 52 - 5 A novel plant glutathione peroxidase-like protein provides tolerance to oxygen radicals generated by paraquat in Escherichia coli; Holland D et al.; Citrus salt-stress associated protein (Cit-SAP) reveals significant sequence homology to mammalian glutathione peroxidase (GP) . In an attempt to assign biological function to this protein, transformed E . coli cells expressing Cit-SAP were examined for their ability to tolerate free radicals formed by paraquat, an O2- radical forming agent . In the presence of paraquat, the survival rate of the transformed bacteria expressing Cit-SAP was much higher as compared to the wild-type bacteria . The results support the assumption that Cit-SAP is a plant GP-like protein which participate in the enzymatic system aimed at scavenging oxygen free-radicals in plants. FEBS Lett, 1994 Jan 3, 337(1), 114 - 8 The human HIP gene, overexpressed in primary liver cancer encodes for a C-type carbohydrate binding protein with lactose binding activity; Christa L et al.; HIP was originally identified as a gene expression in primary liver cancers, and in normal tissues such as pancreas and small intestine . Based on gene data base homologies, the HIP protein should consist of a signal peptide linked to a single carbohydrate recognition domain . To test this hypothesis HIP and the putative carbohydrate recognition domain encoded by the last 138 C-terminal amino acids, were expressed as glutathione-S-transferase proteins (GST-HIP and GST-HIP-142, respectively) . Both recombinant proteins were purified by a single affinity purification step from bacterial lysates and their ability to bind saccharides coupled to trisacryl GF 2000M were tested . Our results show that HIP and HIP-142 proteins bind to lactose, moreover the binding requires divalent cations . Thus the HIP protein is a lactose-binding lectin with the characteristics of a C-type carbohydrate recognition domain of 138 amino acids in the C-terminal region. Neurosci Lett, 1994 Jan 3, 165(1-2), 153 - 6 Cultured neurons infected with an HSV-1-derived vector remain electrically excitable and responsive to neurotransmitter; Farkas RH et al.; Vectors derived from herpes simplex virus type 1 (HSV-1) have allowed foreign genes to be expressed in differentiated postmitotic neurons, but the usefulness of these infected neurons for electrophysiological studies has not been examined . Using a latent, non-pathogenic recombinant HSV-1 virus, we expressed E . coli beta-galactosidase in identified rat cholinergic and dopaminergic neurons in culture . The electrophysiological properties of the infected cholinergic neurons were examined . The neurons remained electrically excitable and responsive to neurotransmitter. J Exp Biol, 1994 Feb, 187(1), 143 - 58 CHARACTERIZATION OF MACROPHAGES AND NEUTROPHILIC GRANULOCYTES FROM THE PRONEPHROS OF CARP (CYPRINUS CARPIO) Kemenade B, Groeneveld A, Rens B, Rombout J. To analyse the functional activity of different leucocyte types, carp pronephros cells were separated on Percoll density gradients and by use of fluorescence-activated cell sorting . Cell populations were characterised by light and electron microscopy and by flow cytometry . Fractions enriched in macrophages and neutrophilic granulocytes were subsequently analysed for phagocytic activity in vitro by quantification of the uptake of Escherichia coli bacteria or yeast cells, and for respiratory burst response by measurement of the production of the reactive oxygen intermediates O2&middot; and H2O2 . Both cell types showed very active in vitro phagocytosis and production of both O2&middot; and H2O2 . When activated with phorbol myristate acetate or bacteria, it was evident that the neutrophilic granulocytes were significantly more active than the macrophages . Analysis of single-cell respiratory burst activity in fish phagocytes was investigated after preloading of cells with dihydrorhodamine123 . Cells were subsequently separated and analysed for fluorescence using flow cytometry . Both the macrophage-enriched fraction and the granulocyte-enriched fraction appeared to consist of active and inactive subpopulations . In comparison with the inactive populations, active populations had characteristic high forward/sideward scatter profiles. Eksp Med Morfol, 1994, 32(3-4), 27 - 39 {The ultrastructural changes in type-2 pneumocytes in experimental heat and endotoxic shock}; Angelov A; The early ultrastructural changes in type 2 pneumocytes (PN2) as a result of burn and endotoxic shock in rats and rabbits were studied . Stereotype morphological damages in PN2 were established . Differences between experimental models were quantitative and depend mainly from the severity of shock and animals species . Morphofunctional heterogenity of PN2 population leads to the appearance of three groups of PN2: 1) PN2 with degenerative changes; 2) PN2 with "stress" hyperactivity and exocytosis of lamelar bodies; and 3) PN2 with increased number of lamelar bodies . Disturbances of lamelar bodies morphogenesis (conglomerates, giant lamelar bodies) as well as lamelar bodies formation by alternative way - directly from rough endoplasmic reticulum were observed . The predominant part of the described PN2 lesins were adaptive in character. J Comput Biol, 1994 Spring, 1(1), 39 - 50 Combined use of sequence similarity and codon bias for coding region identification; States DJ et al.; A computer program called BLASTX was previously shown to be effective in identifying and assigning putative function to likely protein coding regions by detecting significant similarity between a conceptually translated nucleotide query sequence and members of a protein sequence database . We present and assess the sensitivity of a new option to this software tool, herein called BLASTC, which employs information obtained from biases in codon utilization, along with the information obtained from sequence similarity . A rationale for combining these diverse information sources was derived, and analyses of the information available from codon utilization in several species were performed, with wide variation seen . Codon bias information was found on average to improve the sensitivity of detection of short coding regions of human origin by about a factor of 5 . The implications of combining information sources on the interpretation of positive findings are discussed. Acta Cient Venez, 1994, 45(2), 96 - 101 A mutation affecting gluconate catabolism in Escherichia coli: the locus for the main high affinity transport; De Rekarte UD et al.; The bioH-malA region of the E . coli chromosome (min 75.5) includes the gntT gene which encodes a high affinity transport for gluconate . Other gnt loci have not been characterized in this region; nevertheless, because lesions in it affect severely the utilization of gluconate, it has been suggested as being more complex . This region was investigated with respect to gluconate catabolism through the characterization of suitable E . coli strains lysogenized with a specialized transducing phage carrying the bioH-malA region of the bacterial chromosome (lambda cI857st68h80d2bioH-malA) . It was found that the region transduced by this phage while includes the gntT gene lacks other gnt loci that might code additional activities for transport of gluconate or its phosphorylation . Moreover, the pleiotropic lesion gntM2, previously mapped into this region and suggested as altering gntT or a presumptive regulator gene that might be involved in this catabolism, resulted recessive in lysogens (partial diploids) containing the defective prophage . The results obtained supported the idea that gntM2 is an allele of gntT; consequently those results suggested the precise position of this gene on the cromosomic map and the central role that its product might have in the initial incorporation of gluconate in E . coli. Arch Immunol Ther Exp (Warsz), 1994, 42(5-6), 425 - 31 Seasonal modulation of interferon response in spotted sousliks (Spermophilus suslicus); Kandefer-Szerszen M et al.; Interferon production in spotted sousliks in different physiological states: in summer activity, summer torpor, winter hibernation and awaked from winter hibernation was studied . Three different interferon inducers: poly rI:rC complexed with DEAE-dextran, lipopolysaccharide from E . coli (LPS) and tilorone hydrochloride were used . In comparison with sousliks active in summer, the interferon levels in serum and different organs of sousliks in winter hibernation after induction with tilorone hydrochloride, LPS and poly rI:rC were significantly lower . Decreased interferon response was also observed in sousliks in summer torpor and awaked from winter hibernation . In contrast to hibernation, artificial acidosis induced by placing sousliks in the atmosphere with a high concentration of CO2 enhanced interferon production. Folia Microbiol (Praha), 1994, 39(6), 452 - 8 Overproduction of the Hsd subunits leads to the loss of temperature-sensitive restriction and modification phenotype; Weiserova M et al.; The genes hsdM and hsdS for M . EcoKI modification methyltransferase and the complete set of hsdR, hsdM and hsdS genes coding for R . EcoKI restriction endonuclease, both with and without a temperature-sensitive (ts) mutation in hsdS gene, were cloned in pBR322 plasmid and introduced into E . coli C (a strain without a natural restriction-modification (R-M) system) . The strains producing only the methyltransferase, or together with the endonuclease, were thus obtained . The hsdSts-1 mutation, mapped previously in the distal variable region of the hsdS gene with C1 245-T transition has no effect on the R-M phenotype expressed from cloned genes in bacteria grown at 42 degrees C . In clones transformed with the whole hsd region an alleviation of R-M functions was observed immediately after the transformation, but after subculture the transformants expressed the wild-type R-M phenotype irrespective of whether the wild-type or the mutant hsdS allele was present in the hybrid plasmid . Simultaneous overproduction of HsdS and HsdM subunits impairs the ts effect of the hsdSts-1 mutation on restriction and modification. Arch Biochem Biophys, 1994 Jan, 308(1), 24 - 30 Determination of the relative percentage deuteration of the ribosomal protein S4 by electrospray ionization mass spectrometry; Gulcicek EE et al.; Electrospray ionization mass spectrometry provides a fast, economical, and accurate new method for determining levels of nonexchangeable deuterium incorporation in proteins derived from organisms grown on deuterated media . Results are shown for determining the percentage deuteration of ribosomal protein S4 from Escherichia coli. Surg Neurol, 1994 Jan, 41(1), 34 - 9 Rapid bony destruction with pyogenic vertebral osteomyelitis; Heary RF et al.; We report the case of a previously healthy woman who had an extremely rapid progression of pyogenic vertebral osteomyelitis . The plain film radiographs from 1 month before admission, from the time of admission, and from her 1 year follow-up are presented . Although it is a well known fact that pyogenic vertebral osteomyelitis can evolve rapidly and that the radiographic images often lag behind the clinical symptoms, it is rare to find a case with such clear radiographic documentation . A film that was interpreted as having mild arthritic changes, 1 month before admission, progressed to one that demonstrates severe bony destruction with a kyphotic angulation of 90 degrees . The current methods of diagnosis and treatment of pyogenic vertebral osteomyelitis are reviewed. Gut, 1994 Jan, 35(1), 40 - 5 Effects of endotoxin and dexamethasone on group I and II phospholipase A2 in rat ileum and stomach; Lilja I et al.; Phospholipase A2 (EC 3.1.1.4) is a key enzyme in inflammation and is thought to play an important part in inflammatory diseases of the gastrointestinal tract . To investigate the nature and regulation of phospholipase A2 activity in the gastrointestinal mucosa, the distribution of messenger ribonucleic acid (mRNA) for group II phospholipase A2 in various parts of the rat gastrointestinal tract was studied, as well as the influence of endotoxin or dexamethasone, or both, on the group I and II phospholipase A2 mRNA expression and activity in the rat glandular stomach and distal ileum . The results show that (a) group II phospholipase A2 is present along the whole gastrointestinal tract, but in particularly large amounts in the distal ileum, (b) endotoxin increases group II, but not group I, phospholipase A2 mRNA expression in the glandular stomach and distal ileum, and (c) dexamethasone reduces the endotoxin induced increases in group II phospholipase mRNA expression and activity in the gastrointestinal mucosa . These findings suggest that phospholipase A2 of type II is a mediator of endotoxin effects in the gastrointestinal mucosa and that its expression at the mRNA level can be inhibited by corticosteroids. Gut, 1994 Jan, 35(1), 34 - 9 Lipid peroxidation and electrogenic ion transport in the jejunum of the vitamin E deficient rat; Lindley KJ et al.; Increased concentrations of reactive oxygen species in children with depleted antioxidant defences have been implicated in a cycle of malnutrition, malabsorption, and infection leading to protracted diarrhoea . A rat model of chronic vitamin E deficiency has been used to study the effects of antioxidant depletion on jejunal structure and function in vitro . Basal intestinal short circuit current (Isc), a measure of net electrogenic ion movement across the intestinal epithelium, was greater in chronically vitamin E deficient jejuna than controls, as was the electrogenic secretory response to aminophylline and Escherichia coli STa but not to bethanechol . The galactose stimulated current was also greater in vitamin E deficient jejuna . Indices of lipid peroxidation (concentrations of thiobarbituric acid reactive substances and malondialdehyde) were increased in the vitamin E deficient small bowel . Small intestinal brush border membranes from vitamin E deficient animals displayed changes in both static and dynamic components of membrane fluidity measured by steady state fluorescence polarography . The results of these studies support the hypothesis that oxidative stress in subjects with compromised antioxidant defences results in small intestinal hypersecretion, which could predispose to or perpetuate protracted diarrhoea. EMBO J, 1994 Jan 1, 13(1), 34 - 41 The N-terminal domain of a rab protein is involved in membrane-membrane recognition and/or fusion; Steele-Mortimer O et al.; Proteins of the YPT1/SEC4/rab family are well documented to be involved in the regulation of membrane transport . We have previously reported that rab5 regulates endosome-endosome recognition and/or fusion in vitro . Here, we show that this process depends on the rab5 N-terminal domain . Treatment of early endosomal membranes at a low trypsin concentration essentially abolished fusion and cleaved rab5 to a 1 kDa smaller polypeptide . Two-dimensional gel analysis suggested that rab5 is one of the few, if not the only, polypeptides cleaved by trypsin under these conditions . Whereas endosome fusion could be stimulated by cytosol prepared from cells overexpressing rab5 (and thus containing high amounts of the protein), this stimulation was abolished by trypsin-treatment of the cytosol . Trypsin-treated cytosol prepared from mock-transfected cells, which contains very low amounts of rab5, showed no inhibitory activity indicating that rab5 is the target of trypsin in these experiments . Purified rab5 prepared after expression in Escherichia coli was treated with trypsin, which cleaved the protein at the N-terminus . A synthetic peptide of rab5 N-terminal domain inhibited endosome fusion in our cell-free assay . A version of the same peptide truncated at the N-terminus or a peptide of rab3 N-terminal domain were without effects . Altogether, these observations suggest that the N-terminal domain of rab5 is involved in the process of early endosome recognition and/or fusion, presumably because it interacts with another component of the transport machinery. EMBO J, 1994 Jan 1, 13(1), 249 - 57 The second to last amino acid in the nascent peptide as a codon context determinant; Mottagui-Tabar S et al.; Forty-two different sense codons, coding for all 20 amino acids, were placed at the ribosomal E site location, two codons upstream of a UGA or UAG codon . The influence of these variable codons on readthrough of the stop codons was measured in Escherichia coli . A 30-fold difference in readthrough of the UGA codon was observed . Readthrough is not related to any property of the upstream codon, its cognate tRNA or the nature of its codon-anticodon interaction . Instead, it is the amino acid corresponding to the second upstream codon, in particular the acidic/basic property of this amino acid, which seems to be a major determinant . This amino acid effect is influenced by the identity of the A site stop codon and the efficiency of its decoding tRNA, which suggests a correlation with ribosomal pausing . The magnitude of the amino acid effect is in some cases different when UGA is decoded by a wildtype form of tRNA(Trp) as compared with a suppressor form of the same tRNA . This indicates that the structure of the A site decoding tRNA is also a determinant for the amino acid effect. EMBO J, 1994 Jan 1, 13(1), 232 - 40 Editing of the mitochondrial small subunit rRNA in Physarum polycephalum; Mahendran R et al.; Post-transcriptional insertion, substitution or deletion of nucleotides in RNA (RNA editing) has been observed in RNAs from a number of organisms but always in messenger RNA or transfer RNA . We report here that the 17S rRNA of the mitochondrial ribosome of Physarum polycephalum is edited at 40 sites with single cytidine insertions . The locations of the editing sites are fairly evenly distributed throughout the RNA and do not correspond to any obvious feature of the primary sequence or secondary structure . In addition to these cytidine editing sites are editing sites in which a nucleotide other than cytidine is inserted . At two sites a uridine is inserted and at two sites adenosine residues are inserted . This is the first report of mixed nucleotide insertional editing . These results imply that the editing mechanism in Physarum may be different from those proposed for the kinetoplastid protozoa. EMBO J, 1994 Jan 1, 13(1), 138 - 46 An iron-sulfur center essential for transcriptional activation by the redox-sensing SoxR protein; Hidalgo E et al.; The soxRS oxidative stress regulon of Escherichia coli is triggered by superoxide (O2.-) generating agents or by nitric oxide through two consecutive steps of gene activation . SoxR protein has been proposed as the redox sensing gene activator that triggers this cascade of gene expression . We have now characterized two forms of SoxR: Fe-SoxR contained non-heme iron (up to 1.6 atoms per monomer); apo-SoxR was devoid of Fe or other metals . The spectroscopic properties of Fe-SoxR indicated that it contains a redox active iron-sulfur (FeS) cluster that is oxidized upon extraction from E . coli . Fe-SoxR and apo-SoxR bound the in vivo target, the soxS promoter, with equal affinities and protected the same region from DNase I in vitro . However, only Fe-SoxR stimulated transcription initiation at soxS in vitro > 100-fold, similar to the activation of soxS expression in vivo . This stimulation occurred at a step after the binding of RNAP and indicates a conformational effect of oxidized Fe-SoxR on the soxS promoter . The variable redox state of the SoxR FeS cluster may thus be employed in vivo to modulate the transcriptional activity of this protein in response to specific types of oxidative stress. Am J Physiol, 1994 Jan, 266(1 Pt 2), R9 - 14 Hypothermia to endotoxin involves the cytokine tumor necrosis factor and the neuropeptide vasopressin in rats; Derijk RH et al.; Previously, we have reported that intravenous administration of bacterial endotoxin (lipopolysaccharide, LPS) in rats kept at a subthermoneutral ambient temperature of 24 degrees C results in a fall in colonic temperature that involved the release of antipyretic products by peripheral macrophages . Here, we demonstrate that treatment of rats with a biologically active antiserum to tumor necrosis factor (TNF) markedly attenuates the hypothermia in response to administration of LPS (0.5 mg/kg) . Moreover, this hypothermia was prevented by central injection of a selective antagonist of V1 vasopressin receptors, dPTyr(Me) arginine vasopressin (AVP; 2 micrograms icv) . AVP is thought to act as an antipyretic in the ventral septal area (VSA) of the brain . Because the AVP content of this area has been shown to be eliminated after long-term castration, we have tested the hypothesis that castration would attenuate the hypothermia in response to administration of LPS . Castrated rats displayed a markedly less hypothermic response than age-matched controls in response to administration of LPS . We conclude that hypothermia in response to intravenous injection of LPS involves the release of TNF from peripheral macrophages . Moreover, our results are consistent with the possibility that androgen-dependent vasopressinergic neurons in the VSA are mediating the hypothermia in response to intravenous administration of LPS. Am J Physiol, 1994 Jan, 266(1 Pt 2), R1 - 8 Hypothermia to endotoxin involves reduced thermogenesis, macrophage-dependent mechanisms, and prostaglandins; Derijk RH et al.; At a subthermoneutral ambient temperature of 24 degrees C, intravenous administration of bacterial endotoxin (lipopolysaccharide, LPS) to rats resulted in hypothermia associated with a fall in oxygen consumption followed by fever . At the thermoneutral ambient temperature of 30 degrees C, animals only responded to LPS with fever . The hypothermia and reduction in oxygen consumption were attenuated in rats with eliminated peripheral macrophages . By contrast, macrophage elimination did not affect the febrile response to LPS . Both the hypothermia and the febrile response to LPS were prevented by peripheral administration of the cyclooxygenase inhibitor indomethacin . We conclude that hypothermia in response to LPS is caused by reduced thermogenesis, involves antipyretic products released from peripheral macrophages, and is mediated by prostaglandins . In addition, the febrile response likewise involves prostaglandins, but in contrast to the hypothermia appears to be independent of pyrogens released from peripheral macrophages . Previously, we reported the induction of the pyrogen interleukin-1 in the brain during the time course of the febrile response to LPS (34) . The latter observations support the hypothesis that the second phase of biphasic fever is mediated by synthesis and action of pyrogens inside the blood-brain barrier. Oncogene, 1994 Jan, 9(1), 121 - 32 Comparison of the expression and function of the transcription factor PU.1 (Spi-1 proto-oncogene) between murine macrophages and B lymphocytes; Ross IL et al.; The expression of mRNA encoding the DNA-binding protein PU.1 (Spi-1) is restricted to B lymphocytes and macrophages . The role of PU.1 in tissue-specific transcriptional regulation in the two cell types was examined by co-transfection of a PU.1 expression plasmid with vectors containing B cell (IgH enhancer) or macrophage-specific (c-fms) transcription control elements . Cotransfection of the PU.1 expression plasmid in MOPC31C B cells trans-repressed the IgH enhancer but trans-activated the c-fms promoter . The latter was insufficient to overcome a block to transcription elongation that determines macrophage-specific c-fms gene expression . In the macrophage line RAW264, PU.1 had no effect on the c-fms promoter, but trans-repressed the activity of a c-fms reporter plasmid containing the transcription attenuator . The effects of PU.1 in both cell types were distinct from those of c-ets-2, a related factor, which trans-activated the c-fms promoter in both B cells and macrophages but also repressed the IgH enhancer . PU.1 was shown to be one of several nuclear proteins that bound a critical cis-acting element of the IgH enhancer, microB, but analysis of nuclear extracts of a wide range of B cell and macrophage lines demonstrated a strong correlation between macrophage phenotype and nuclear PU.1 expression . The data suggest that differences in nuclear PU.1 expression and function between macrophages and B cells may play a role in lineage divergence. J Pharmacol Exp Ther, 1994 Jan, 268(1), 238 - 47 Modulation of superoxide generation in in vivo lipopolysaccharide-primed Kupffer cells by staurosporine, okadaic acid, manoalide, arachidonic acid, genistein and sodium orthovanadate; Mayer AM et al.; Continuous infusion of a nonlethal dose of Escherichia coli lipopolysaccharide (LPS) into rats induced extravasation of mononuclear phagocytes into the liver and the priming of Kupffer cells for in vitro phorbol myristate acetate (PMA)-stimulated superoxide anion (O2-) release . The purpose of this investigation was to determine the role of protein kinase C (PKC), protein serine-threonine phosphatase(s) 1 and 2a, protein tyrosine kinase(s) and phosphatase(s), phospholipase A2 (PLA2), arachidonic acid (AA) and its cyclooxygenase (CO) and 5-lipoxygenase (5-LO) metabolites in the modulation of PMA-stimulated O2-generation in in vivo LPS-primed rat Kupffer cells . The following inhibitors blocked PMA-stimulated O2- generation in the absence (-AA) or presence of AA (+AA) (50 microM): 1) staurosporine, a putative PKC inhibitor (150 nM, 95% inhibition without AA, 88% inhibition with AA); 2) okadaic acid, a protein serine-threonine phosphatase inhibitor (2 microM, 65% inhibition with or without AA); 3) the marine PLA2 inhibitor manoalide (1 microM, 97.5% inhibition without AA, 75% with AA) . In addition, it was observed that exogenously added AA enhanced PMA-stimulated O2- generation in a time- and dose-dependent manner (5-50 microM) and partially reversed the inhibitory effect of manoalide . The following inhibitors did not block PMA-stimulated O2- generation in the absence or presence of AA: 1) indomethacin, a CO inhibitor (1-100 microM) and WY-50,295M tromethamine, a novel 5-LO inhibitor (1-100 microM); 2) genistein, a protein tyrosine kinase inhibitor (1-100 microM); and 3) sodium orthovanadate (1-300 microM), a protein tyrosine phosphatase inhibitor . It was concluded that, in in vivo LPS-primed Kupffer cells, PMA-stimulated O2- generation is modulated by PKC, protein serine-threonine phosphatase(s), PLA2 and AA but not by protein tyrosine kinase(s) and phosphatase(s) and CO and 5-LO products . These findings could have implications on the design of novel therapeutic approaches for the modulation of enhanced O2- release by Kupffer cells in endotoxemia. Am J Obstet Gynecol, 1994 Jan, 170(1 Pt 1), 223 - 7 Permeation of human chorioamniotic membranes by Escherichia coli in vitro; Gyr TN et al.; OBJECTIVE: Our goal was to study the permeation of Escherichia coli through human chorioamniotic membranes in vitro . STUDY DESIGN: Medium was placed in two compartments separated by chorioamniotic membranes obtained from six cesarean sections at term . The compartment faced by the chorion was inoculated with E . coli . Both compartments were sampled over 12 hours for observation of bacterial growth . Controls were performed without membranes . RESULTS: In the compartment that was inoculated, concentration of E . coli increased from 10(6) to 10(10) colony-forming units per milliliter . In the compartment faced by amnion, bacterial growth was observed after 6 hours and reached 10(3) colony-forming units per milliliter . Permeation of E . coli was confirmed histopathologically . The change of glucose and lactate was linear . In the controls the concentration of E . coli increased to 10(7) (p < 0.001) . CONCLUSIONS: E . coli organisms permeate viable chorioamniotic membranes . The membranes constitute a weak barrier against ascending infection and do not inhibit bacterial growth. Dev Biol, 1994 Jan, 161(1), 77 - 83 lacZ expression in germline transgenic zebrafish can be detected in living embryos; Lin S et al.; Use of transgenic technology in zebrafish has been limited by the inability to efficiently express transgenes in early embryos of F1 and subsequent generations and to rapidly detect transgenic fish . We generated transgenic fish by injecting fertilized eggs with the Escherichia coli lacZ gene under the control of the Xenopus elongation factor 1 alpha transcriptional regulatory element . Four of five lines of transgenic fish we obtained express the lacZ gene in early embryos . The pattern of expression was distinct for each line, with two lines showing extensive expression beginning at approximately the midblastula transition, one showing patchy expression and one showing expression almost exclusively in motor neurons . Expression patterns were stable through the F2 generation in the three lines studied to date . The availability of these lines facilitated the development of a reliable and rapid method for live-staining lacZ-expressing embryos using the substrate fluorescein-di-beta-D-galactopyranoside (FDG) . Positive embryos of the two most highly lacZ-expressing lines could be identified after 2-3 min of staining in FDG and then picked out and raised . These observations should prove useful for a variety of studies in zebrafish. J Bacteriol, 1994 Jan, 176(2), 543 - 6 Regulation of the raffinose permease of Escherichia coli by the glucose-specific enzyme IIA of the phosphoenolpyruvate:sugar phosphotransferase system; Titgemeyer F et al.; In enteric bacteria, chromosomally encoded permeases specific for lactose, maltose, and melibiose are allosterically regulated by the glucose-specific enzyme IIA of the phosphotransferase system . We here demonstrate that the plasmid-encoded raffinose permease of enteric bacteria is similarly subject to this type of inhibition. J Bacteriol, 1994 Jan, 176(2), 540 - 2 Mutations in the delta subunit influence the assembly of F1F0 ATP synthase in Escherichia coli; Stack AE et al.; Missense mutations affecting Asp-161 and Ser-163 in the delta subunit of F1F0 ATP synthase have been generated . Although most substitutions allowed substantial enzyme function, the delta Asp-161-->Pro substitution resulted in a loss of enzyme activity . The loss of activity was attributable to a structural failure altering assembly of the enzyme complex. J Bacteriol, 1994 Jan, 176(2), 535 - 9 Efficient transposition in mycobacteria: construction of Mycobacterium smegmatis insertional mutant libraries; Guilhot C et al.; The Tn611 transposon was inserted into pCG63, a temperature-sensitive plasmid isolated from an Escherichia coli-mycobacterial shuttle vector which contains the pAL5000 and pUC18 replicons . The resulting plasmid, pCG79, was used to generate a large number of insertional mutations in Mycobacterium smegmatis . These are the first mycobacterial insertional mutant libraries to be constructed by transposition directly into a mycobacterium . No highly preferential insertion sites were detected by Southern blot analysis of the chromosomal DNAs isolated from the insertion mutants . Auxotrophic mutants with various phenotypes were isolated at a frequency ranging from 0.1 to 0.4%, suggesting that the libraries are representative . The pCG79 system thus seems to be a useful tool for the study of M . smegmatis genetics and may be applicable to other mycobacteria, such as the M . tuberculosis complex. J Bacteriol, 1994 Jan, 176(2), 531 - 4 Regulation of the Escherichia coli hfq gene encoding the host factor for phage Q beta; Kajitani M et al.; The host factor (HF-I) for phage Q beta RNA replication is a small protein of 102 amino acid residues encoded by the hfq gene at 94.8 min on the Escherichia coli chromosome . The synthesis rate of HF-I at the exponential-growth phase is higher than at the stationary phase, and it increases concomitantly with the increase in cell growth rate . The intracellular level of HF-I is about 30,000 to 60,000 molecules per cell, the majority being associated with ribosomes as one of the salt wash proteins . Taken together, we suggest that HF-I is one of the growth-related proteins. J Bacteriol, 1994 Jan, 176(2), 401 - 8 Expression of Caulobacter dnaA as a function of the cell cycle; Zweiger G et al.; The initiation of DNA replication is under differential control in Caulobacter crescentus . Following cell division, only the chromosome in the progeny stalked cell is able to initiate DNA replication, while the chromosome in the progeny swarmer cell does not replicate until later in the cell cycle . We have isolated the dnaA gene in order to determine whether this essential and ubiquitous replication initiation protein also contributes to differential replication control in C . crescentus . Analysis of the cloned C . crescentus dnaA gene has shown that the deduced amino acid sequence can encode a 486-amino-acid protein that is 37% identical to the DnaA protein of Escherichia coli . The gene is located 2 kb from the origin of replication . Primer extension analysis revealed a single transcript originating from a sigma 70-type promoter . Immunoprecipitation of a DnaA'-beta-lactamase fusion protein showed that although expression occurs throughout the cell cycle, there is a doubling in the rate of expression just prior to the initiation of replication. J Bacteriol, 1994 Jan, 176(2), 378 - 87 Escherichia coli Fis and DnaA proteins bind specifically to the nrd promoter region and affect expression of an nrd-lac fusion; Augustin LB et al.; The Escherichia coli nrd operon contains the genes encoding the two subunits of ribonucleoside diphosphate reductase . The regulation of the nrd operon has been observed to be very complex . The specific binding of two proteins to the nrd regulatory region and expression of mutant nrd-lac fusions that do not bind these proteins are described . A partially purified protein from an E . coli cell extract was previously shown to bind to the promoter region and to regulate transcription of the nrd operon (C . K . Tuggle and J . A . Fuchs, J . Bacteriol . 172:1711-1718, 1990) . We have purified this protein to homogeneity by affinity chromatography and identified it as the E . coli factor for inversion stimulation (Fis) . Cu-phenanthroline footprinting experiments showed that Fis binds to a site centered 156 bp upstream of the start of nrd transcription . Mutants with deletion and site-directed mutations that do not bind Fis at this site have two- to threefold-lower expression of an nrd-lac fusion . The previously reported negative regulatory nature of this site (C . K . Tuggle and J . A . Fuchs, J . Bacteriol . 172:1711-1718, 1990) was found to be due to a change in polarity in the vectors used to construct promoter fusions . Two nine-base sequences with homology to the DnaA consensus binding sequence are located immediately upstream of the nrd putative -35 RNA polymerase binding site . Binding of DnaA to these sequences on DNA fragments containing the nrd promoter region was confirmed by in vitro Cu-phenanthroline footprinting . Footprinting experiments on fragments with each as well as both of the mutated 9-mers suggests cooperativity between the two sites in binding DnaA . Assay of in vivo expression from wild-type and DnaA box-mutated nrd promoter fragments fused to lacZ on single-copy plasmids indicates a positive effect of DnaA binding on expression of nrd. J Bacteriol, 1994 Jan, 176(2), 359 - 67 New outer membrane-associated protease of Escherichia coli K-12; Kaufmann A et al.; The gene for a new outer membrane-associated protease, designated OmpP, of Escherichia coli has been cloned and sequenced . The gene encodes a 315-residue precursor protein possessing a 23-residue signal sequence . Including conservative substitutions and omitting the signal peptides, OmpP is 87% identical to the outer membrane protease OmpT . OmpP possessed the same enzymatic activity as OmpT . Immuno-electron microscopy demonstrated the exposure of the protein at the cell surface . Digestion of intact cells with proteinase K removed 155 N-terminal residues of OmpP, while the C-terminal half remained protected . It is possible that much of this N-terminal part is cell surface exposed and carries the enzymatic activity . Synthesis of OmpP was found to be thermoregulated, as is the expression of ompT (i.e., there is a low rate of synthesis at low temperatures) and, in addition, was found to be controlled by the cyclic AMP system. J Bacteriol, 1994 Jan, 176(2), 338 - 43 Purification and properties of a membrane-bound lytic transglycosylase from Escherichia coli; Ursinus A et al.; A membrane-bound lytic transglycosylase (Mlt) has been solubilized in the presence of 2% Triton X-100 containing 0.5 M NaCl from membranes of an Escherichia coli mutant that carries a deletion in the slt gene coding for a 70-kDa soluble lytic transglycosylase (Slt70) . The enzyme was purified by a four-step procedure including anion-exchange (HiLoad SP-Sepharose and MonoS), heparin-Sepharose, and poly(U)-Sepharose 4B column chromatography . The purified protein that migrated during denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single band corresponding to an apparent molecular mass of about 38 kDa is referred to as Mlt38 . Optimal activity was found in buffers with a pH between 4.0 and 4.5 . The enzyme is stimulated by a factor of 2.5 in the presence of Mg2+ at a concentration of 10 mM and loses its activity rapidly at temperatures above 30 degrees C . Besides insoluble murein sacculi, the enzyme was able to degrade glycan strands isolated from murein by amidase treatment . The enzymatic reaction occurred with a maximal velocity of about 2.2 mg/liter/min with murein sacculi as a substrate . The amino acid sequences of four proteolytic peptides showed no identity with known sequences in the data bank . With Mlt38, the number of proteins in E . coli showing lytic transglycosylase activity rises to three. Cell Immunol, 1994 Jan, 153(1), 248 - 55 In vitro production of tumor necrosis factor-alpha by adherent human peripheral blood mononuclear cells incubated with killed coccidioidal arthroconidia and spherules; Ampel NM; We examined the in vitro production of tumor necrosis factor (TNF) by adherent human peripheral blood mononuclear cells (MNL) incubated with arthroconidia or spherules derived from the dimorphic fungus Coccidioides immitis . Using a bioassay measuring the percentage cytotoxicity of L929 cells, arthroconidia and spherules induced the production of measurable amounts of TNF by MNL . Both the arthroconidial and spherule preparations contained < 0.01 ng/ml of endotoxin, below that needed to induce cytotoxicity in the bioassay . Based on ELISA, the vast majority of TNF induced by arthroconidia or spherules was TNF-alpha, with minimal production of TNF-beta . These are the first data to show the production of TNF in human coccidioidomycosis. J Clin Invest, 1994 Jan, 93(1), 114 - 20 Inhibition of endotoxin-induced activation of coagulation and fibrinolysis by pentoxifylline or by a monoclonal anti-tissue factor antibody in chimpanzees; Levi M et al.; Knowledge of the pathogenetic mechanisms responsible for the activation of the coagulation system associated with endotoxemia is important for the development of improved modalities for prevention and treatment . We analyzed the appearance in plasma of TNF, IL-6, and indices of coagulation and fibrinolytic system activation in normal chimpanzees after intravenous infusion of endotoxin . Endotoxin infusion elicited reproducible and dose-dependent elevations in serum TNF and IL-6, as well as marked increases in thrombin generation in vivo as measured by immunoassays for prothrombin activation fragment F1 + 2, thrombin-antithrombin III complexes, and fibrinopeptide A . Activation of the fibrinolytic mechanism was monitored with assays for plasminogen activator activity and plasmin-alpha 2-antiplasmin complexes . To potentially intervene in the molecular pathways elicited by endotoxin, pentoxifylline, an agent that interrupts "immediate early" gene activation by monocytes, or a potent monoclonal antibody that neutralizes tissue factor-mediated initiation of coagulation, were infused shortly before endotoxin . Pentoxifylline markedly inhibited increases in the levels of TNF and IL-6, as well as the effects on coagulation and fibrinolysis . In contrast, the monoclonal antibody to tissue factor completely abrogated the augmentation in thrombin generation, but had no effect on cytokine levels or fibrinolysis . We conclude that the endotoxin-induced activation of coagulation appears to be mediated by the tissue factor-dependent pathway, the fibrinolytic response triggered by endotoxin is not dependent on the generation of thrombin, and that the release of cytokines may be important in mediating the activation of both the coagulation and the fibrinolytic mechanisms in vivo. J Bacteriol, 1994 Jan, 176(1), 221 - 31 Organization and functions of genes in the upstream region of tyrT of Escherichia coli: phenotypes of mutants with partial deletion of a new gene (tgs); Bosl M et al.; A delta tyrT::kan mutant from Escherichia coli K-12 (DTK-12) shows a transient growth lag that is caused by glycine starvation (U . Michelsen, M . Bosl, T . Dingermann, and H . Kersten, J . Bacteriol . 171:5987-5994, 1989) . The same deletion, transduced into the relA1 spoT1 mutant CA274 to construct strain DTC274, caused complete growth inhibition in glucose minimal medium . Here, we show that the tyrT 5' region contains three new open reading frames in the order ORF37-->ORF34-->ORF32-->tyrT and that the delta tyrT::kan allele used previously deletes tyrT as well as a carboxy-terminal portion of ORF32 . A plasmid encoding ORF32 totally complemented the inability of strain DTC274 to grow on glucose minimal medium as well as the transient glycine starvation phenomenon in DTK-12, and ORF32 was designated tgs . Partial deletion of tgs, cotransduced with the marker delta tyrT::kan, was responsible for the completely different phenotypes of the deletion mutants DTK-12 and DTC274 . The deduced Tgs protein sequence showed significant homology to the PurN protein of E . coli and to enzymes with glycinamide ribonucleotide transformylase activity . We discuss whether growth inhibition in strain DTC274 may be caused by synergistic effects with the preexisting mutations relA1 and spoT1 . The deduced protein sequence of ORF37 showed striking similarity to regulator response proteins and is probably a new member of this family . A spontaneous mutation in ORF37, caused by the integration of an insertion element, IS1, exhibited no phenotype. J Bacteriol, 1994 Jan, 176(1), 15 - 20 The pho regulon-dependent Ugp uptake system for glycerol-3-phosphate in Escherichia coli is trans inhibited by Pi; Brzoska P et al.; sn-Glycerol-3-phosphate (G3P) or glyceryl phosphoryl phosphodiesters, the substrates of the phoB-dependent Ugp transport system, when transported exclusively through this system, can serve as a sole source of phosphate but not as a sole source of carbon (H . Schweizer, M . Argast, and W . Boos, J . Bacteriol . 150:1154-1163, 1982) . In order to explain this phenomenon, we tested two possibilities: repression of the pho regulon by Ugp-mediated transport and feedback inhibition by internal G3P or its degradation product Pi . Using an ugp-lacZ fusion, we found that the expression of ugp does not decline upon exposure to G3P, in contrast to the repressing effect of transport of Pi via the Pst system . This indicated that the Ugp system becomes inhibited after the uptake and metabolism of G3P . Using 32P-labeled G3P, we observed that little Pi is released by cells taking up G3P via the Ugp system but large amounts of Pi are released when the cells are taking up G3P via the GlpT system . Using a glpD mutant that could not oxidize G3P but which could still phosphorylate exogenous glycerol to G3P after GlpF-mediated transport of glycerol, we could not find trans inhibition of Ugp-mediated uptake of exogenous 14C-G3P . However, when allowing uptake of Pi via Pst, we observed a time-dependent inhibition of 14C-G3P taken up by the Ugp transport system . Inhibition was half maximal after 2 min and could be elicited by Pi concentrations below 0.5 mM . Cells had to be starved for Pi in order to observe this inhibition . We conclude that the activity of the Ugp transport system is controlled by the level of internal phosphate. J Bacteriol, 1994 Jan, 176(1), 130 - 6 Mutations in ftsZ that confer resistance to SulA affect the interaction of FtsZ with GTP; Dai K et al.; Mutations in the essential cell division gene ftsZ confer resistance to SulA, a cell division inhibitor that is induced as part of the SOS response . In this study we have purified and characterized the gene products of six of these mutant ftsZ alleles, ftsZ1, ftsZ2, ftsZ3, ftsZ9, ftsZ100, and ftsZ114, and compared their properties to those of the wild-type gene product . The binding of GTP was differentially affected by these mutations . FtsZ3 exhibited no detectable GTP binding, and FtsZ9 and FtsZ100 exhibited markedly reduced GTP binding . In contrast, FtsZ1 and FtsZ2 bound GTP almost as well as the wild type, and FtsZ114 displayed increased GTP binding . Furthermore, we observed that all mutant FtsZ proteins exhibited markedly reduced intrinsic GTPase activity . It is likely that mutations in ftsZ that confer sulA resistance alter the conformation of the protein such that it assumes the active form. J Bacteriol, 1994 Jan, 176(1), 115 - 22 Determination of the growth rate-regulated steps in expression of the Escherichia coli K-12 gnd gene; Pease AJ et al.; In Escherichia coli K-12 strain W3110, the amount of 6-phosphogluconate dehydrogenase relative to that of total protein, i.e., the specific enzyme activity, increases about threefold during growth in minimal media over the range of growth rates with acetate and glucose as sole carbon sources . Previous work with gnd-lac operon and protein fusion strains indicated that two steps in the expression of the gnd gene are subject to growth rate-dependent control, with at least one step being posttranscriptional . With both Northern (RNA) and slot blot analyses, we found that the amount of gnd mRNA relative to that of total RNA was 2.5-fold higher in cells growing in glucose minimal medium than in cells grown on acetate . Therefore, since the total mRNA fraction of total RNA is essentially independent of the growth rate, the amount of gnd mRNA relative to that of total mRNA increases about 2.5-fold with increasing growth rate . This indicates that most of the growth rate-dependent increase in 6-phosphogluconate dehydrogenase can be accounted for by the growth rate-dependent increase in gnd mRNA level . We measured the decay of gnd mRNA mass in the two growth conditions after blocking transcription initiation with rifampin and found that the stability of gnd mRNA does not change with growth rate . We also used a gnd-lacZ protein fusion to measure the functional mRNA half-life and found that it too is growth rate independent . Thus, the growth rate-dependent increase in the level of gnd mRNA is due to an increase in gnd transcription, and this increase is sufficient to account for the growth rate regulation of the 6-phosphogluconate dehydrogenase level . The dilemma posed by interpretations of the properties of gnd-lac fusion strains and by direct measurement of gnd mRNA level is discussed. J Bacteriol, 1994 Jan, 176(1), 100 - 7 Molecular characterization of the promoter of osmY, an rpoS-dependent gene; Yim HH et al.; The osmY gene, which encodes a periplasmic protein with an apparent M(r) of 22,000, is induced by both osmotic and growth phase signals . We demonstrate here that osmY expression is regulated at the level of transcription and that transcription initiates 242 nucleotides upstream of the osmY open reading frame . Relative to the transcriptional start site, 5' deletions up to -36 did not inhibit osmY expression . 3' deletions that extended into the untranslated leader region affected the overall level of osmY::lacZ expression but did not affect inducibility . 5' and 3' deletions that extended past the transcriptional start region essentially abolished osmY expression, suggesting that there is a single promoter region . A putative promoter was identified, and its -10 region, TATATT, closely resembles the sigma 70 consensus -10 sequence, TATAAT . However, we show that osmY is not absolutely dependent on a functional sigma 70 for its expression . Since osmY expression does require rpoS (R . Hengge-Aronis, R . Lange, N . Henneberg, and D . Fischer, J . Bacteriol . 175:259-265, 1993), which encodes a stationary-phase sigma factor, sigma S (K . Tanaka, Y . Takayanagi, N . Fujita, A . Ishihama, and H . Takahashi, Proc . Natl . Acad . Sci . USA 90:3511-3515, 1993), E sigma S may be the form of RNA polymerase responsible for transcription of osmY. Dig Dis Sci, 1994 Jan, 39(1), 46 - 50 Effect of isolated portal hypertension on Kupffer cell function; Basista MH et al.; The increased incidence of infection in cirrhotics may in part be attributable to dysfunction of the reticuloendothelial system (RES) in removing pathogens from the circulation . The portosystemic shunting (PSS) that results from portal hypertension in cirrhotics may compromise RES function by allowing enteric pathogens to be shunted away from the Kupffer cells . A well-characterized model of portal hypertension induced by partial portal vein ligation (PVL), in which there is no hepatic parenchymal cell damage, was used . Kupffer cell function is unaltered and the effect of PSS alone on overall RES function can be evaluated . In addition to the usual immunologically inert {99mTc}sulfur colloid, an actual pathogen was also evaluated . PVL and sham-ligated rats were given either {99mTc}sulfur colloid or E . coli via the ileocolic vein . The right femurs, lungs, livers and spleens of the animals receiving 99mTc were excised and the radioactivity counted . The lungs, livers, and spleens of the animals receiving E . coli were liquefied and the bacteria were quantified . For both groups the ratios of 99mTc or E . coli in the lung, spleen, and femur to liver were calculated . PVL rats had significantly more 99mTc in the lung, spleen, and femur than the sham rats . There were also significantly more E . coli in the lungs for PVL rats but no significant difference in the spleen counts . These results imply that even in the absence of Kupffer cell dysfunction, PSS alters reticuloendothelial system function by causing a greater distribution of pathogens to the periphery . This altered distribution may contribute to an increased susceptibility to infection in cirrhotics. Dig Dis Sci, 1994 Jan, 39(1), 152 - 6 Interleukin-1 receptor antagonist does not prevent endotoxin-induced inhibition of gastric acid secretion in rats; Saperas E et al.; The underlying mechanisms involved in endotoxin-induced inhibition of gastric acid secretion were investigated in conscious rats with pylorus ligation for 2 hr . Intraperitoneal injection of endotoxin (0.1, 1, and 5 micrograms/rat) inhibited gastric acid output by 31%, 80%, and 84% respectively . Intraperitoneal endotoxin (1 microgram/rat) -induced inhibition of gastric acid secretion was not altered by pretreatment with the interleukin-1 receptor antagonist, IL-1RA, indomethacin, naloxone, or capsaicin . Treatments were injected peripherally at doses previously shown to antagonize the antisecretory effect of exogenous interleukin-1 beta, to inhibit prostaglandin synthesis in the stomach and brain, to block opiate receptors, and to alter functioning of unmyelinated afferent nerve fibers . These results indicate that the antisecretory effect of endotoxin can be expressed by factors other than interleukin-1, prostaglandins, or opioid peptides that do not require the integrity of capsaicin-sensitive afferent pathways. Biochem J, 1994 Jan 1, 297 ( Pt 1), 59 - 67 Co-variation of glutathione transferase expression and cytostatic drug resistance in HeLa cells: establishment of class Mu glutathione transferase M3-3 as the dominating isoenzyme; Hao XY et al.; Qualitative and quantitative analyses of glutathione, glutathione transferases (GSTs) and other glutathione-linked enzymes in HeLa cells have been made in order to study their significance in cellular resistance to electrophilic cytotoxic agents . The cytosolic concentrations of three GSTs, GST M1-1 (53 +/- 9 ng/mg of cytosolic protein), GST P1-1 (11 +/- 3 ng/mg) and GST A1-1 (1.1 +/- 0.4 ng/mg) were quantified by isoenzyme-specific enzyme-linked immunoassays . Electrophoretic analysis and immunoblotting demonstrated another component, GST M3-3, which was identified by amino acid sequence analysis . GST M3-3 was quantified (1550 +/- 250 ng/mg) by slot-blot immunoanalysis and was the most abundant GST in HeLa cells . An additional cytosolic 13 kDa protein with high affinity for immobilized glutathione or S-hexyglutathione was found to be identical with a macrophage migration-inhibitory factor, previously identified as a lymphokine . Cells grown in roller bottles (HR) rather than in ordinary culture flasks contain a significantly lower concentration of all the GSTs and were found to be more sensitive to the cytostatic agents doxorubicin (2.3-fold), cisplatin (1.7-fold) and melphalan (1.4-fold) . The cytosolic concentrations of glutathione reductase and glyoxalase I were also lower in HR cells, whereas the total glutathione concentration was unchanged and the glutathione peroxidase activity was increased . The results indicate that GSTs contribute to the cellular resistance phenotype. Mol Gen Genet, 1994 Jan, 242(1), 90 - 9 Role of two operators in regulating the plasmid-borne raf operon of Escherichia coli; Muiznieks I et al.; The plasmid-borne raf operon encodes functions required for the inducible uptake and utilization of raffinose in Escherichia coli K