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| 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· and H2O2 . Both cell types showed very active in vitro phagocytosis and production of both O2· 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 K12 . The expression of three structural genes for alpha-galactosidase (rafA), Raf permease (rafB) and sucrose hydrolase (rafD) is negatively controlled by the binding of RafR repressor (rafR) to two operator sites, O1 and O2, that flank the -35 sequence of the raf promoter, PA . In vitro, O1 and O2 are occupied on increasing the concentration of RafR, without detectable preference for one site or the other or any indication of cooperative binding . Nucleotide substitutions at positions 3, 4 or 5 in an operator half-site prevented repressor binding, supporting a model that postulates specific interactions of these base pairs with the recognition helix of RafR . To study the role of each operator site, we have compared by gel shift analysis the binding of purified RafR repressor to DNA fragments containing the original O1O2 configuration or mutant O1 or O2 . When either one of the two operators was inactivated by site-directed mutagenesis, both O1 and O2 exhibited the same affinity for repressor and the same sensitivity to arrest of repressor binding by the natural inducer, melibiose . However, in the native O1O2 configuration, simultaneous binding of RafR to both operators was sterically hindered, leading to a 13-fold decrease in the intrinsic affinity of an operator site for repressor, once the other site had been occupied . To assess the role of each operator in vivo, rafA was used as a reporter gene . A 1200-fold repression (100%) was exerted by RafR binding to the native O1O2 configuration, whereas O2 alone exerted 45% and O1 alone 6% repression of rafA transcription . The differential effects of O1 versus O2 on transcription (despite matching affinities of O1 and O2 for repressor) suggest that positioning of the O2-repressor complex between the -35 and -10 signals is crucial for transcription control and that repressor binding to the upstream O1 serves to enhance this effect. Mol Gen Genet, 1994 Jan, 242(1), 65 - 72 RNA editing of a conserved reading frame in plant mitochondria increases its similarity to two overlapping reading frames in Escherichia coli; Sunkel S et al.; An open reading frame (orfx) in mitochondria of the higher plants Oenothera berteriana and Arabidopsis thaliana is homologous to orf244 in the mitochondrial genome of Marchantia polymorpha . Homologous sequences are also present in carrot, potato and sugar beet . Profile analysis revealed similarity to two overlapping reading frames in the Escherichia coli genome . Potential translation initiation at conserved ATA (isoleucine) and TTG (leucine) codons is discussed . Transcripts of the open reading frame are altered by RNA editing in Arabidopsis and Oenothera downstream of these codons, suggesting this to be the functionally important region. Mol Gen Genet, 1994 Jan, 242(1), 23 - 32 A pleîotropic acid phosphatase-deficient mutant of Escherichia coli shows premature termination in the dsbA gene . Use of dsbA::phoA fusions to localize a structurally important domain in DsbA; Belin P et al.; A one-step mutant of Escherichia coli K-12 lacking both glucose-1-phosphatase (Agp) and pH 2.5 acid phosphatase (AppA) activities in the periplasmic space was isolated . The mutation which mapped close to chlB, at 87 min on the E . coli linkage map, also caused the loss of alkaline phosphatase (PhoA) activity, even when this activity was expressed from TnphoA fusions to genes encoding periplasmic or membrane proteins . A DNA fragment that complements the mutation was cloned and shown to carry the dsbA gene, which encodes a periplasmic disulphide bond-forming factor . The mutant had an ochre triplet in dsbA, truncating the protein at amino acid 70 . Introduction of TnphoA fusions into a plasmid-borne dsbA gene resulted in DsbA-PhoA hybrid proteins that were all exported to the periplasmic space in both dsbA+ and dsbA strains . They belong to three different classes, depending on the length of the DsbA fragment fused to PhoA . When PhoA was fused to an amino-terminal DsbA heptapeptide, the protein was only seen in the periplasm of a dsbA+ strain, as in the case of wild-type PhoA . Hybrid proteins missing up to 29 amino acids at the carboxy-terminus of DsbA were stable and retained both the DsbA and PhoA activities . Those with shorter DsbA fragments that still carried the -Cys-Pro-His-Cys- motif were rapidly degraded (no DsbA activity) . The presence is discussed of a structural domain lying around amino acid 170 of DsbA and which is probably essential for its folding into a proteolytic-resistant and enzymatically active form. Mol Gen Genet, 1994 Jan, 242(1), 111 - 5 Comparative amino acid sequence analysis of Thermotoga maritima beta-glucosidase (BglA) deduced from the nucleotide sequence of the gene indicates distant relationship between beta-glucosidases of the BGA family and other families of beta-1,4-glycosyl hydrolases; Liebl W et al.; The primary structure of the bglA gene region encoding a beta-glucosidase of Thermotoga maritima strain MSB8 was determined . The bglA gene has the potential to code for a polypeptide of 446 amino acids with a predicted molecular mass of 51,545 Da . The T . maritima beta-glucosidase (BglA) was overexpressed in E . coli at a level comprising approximately 15-20% of soluble cellular protein . Based on its amino acid sequence, as deduced from the nucleotide sequence of the gene, BglA can be classified as a broad-specificity beta-glucosidase and as a member of the beta-glucosidase family BGA, in agreement with the results of enzymatic characterization of the recombinant protein . Comparative sequence analysis revealed distant amino acid sequence similarities between BGA family beta-glucosidases, a beta-xylosidase, beta-1,4-glycanases of the enzyme family F (mostly xylanases), and other families of beta-1,4-glycosyl hydrolases . This result indicates that BGA beta-glucosidases may comprise one enzyme family within a large 'enzyme order' of retaining beta-glycosyl hydrolases, and that the members of these enzyme groups may be inter-related at the level of active site architecture and perhaps even on the level of overall three-dimensional fold. J Infect Dis, 1994 Jan, 169(1), 112 - 8 The presence of K54 capsular polysaccharide increases the pathogenicity of Escherichia coli in vivo; Russo TA et al.; Proven isogenic capsule-negative derivatives (CP9.29, CP9.108, CP9.137, CP9.171, CP9.443, and CP9.C56), generated from an O4/K54/H5 blood isolate (CP9) of Escherichia coli by IS50L::phoA (TnphoA)-mediated transposon mutagenesis, were used to assess the function of a non-K1 capsule in three animal models . Intraperitoneal injection of CP9 (K54+) into mice resulted in an LD50 at 24 h of 5.5 x 10(6) cfu compared with LD50s of 2.6 x 10(7) cfu and 3.8 x 10(7) cfu for CP9.108 (K54-) and CP9.C56 (K54-) (P < .001) . CP9 was cleared less rapidly from the bloodstream, after intravascular injection, than was CP9.108 (P < .01) . In the rat granuloma pouch model, CP9 could proliferate from starting inocula as low as 1.0 x 10(3) cfu/mL . In contrast, capsule-deficient derivatives underwent transient log kills with starting inocula as high as 1.0 x 10(6) cfu/mL . Because proven isogenic strains were evaluated, a clear contribution of the K54 capsular polysaccharide to virulence in vivo is demonstrated. Hepatology, 1994 Jan, 19(1), 217 - 28 Cytokine stimulation of nitric oxide formation and differential regulation in hepatocytes and nonparenchymal cells of endotoxemic rats; Spitzer JA; Some disease processes in which increased endotoxin and cytokine levels exist (e.g., sepsis and infantile diarrhea) are also associated with increased levels of blood nitrates, the stable end products of nitric oxide . Available evidence suggests that the effects of an endotoxic environment, with its attendant complex cytokine networks, on liver function are mediated in part by modulation of hepatic nitric oxide synthesis . This hypothesis was tested by means of studying nitric oxide formation and its regulation in liver parenchymal and nonparenchymal cells of rats that had been continuously infused with endotoxin for 30 hr . Hepatocytes of such rats responded to in vitro stimulation for 20 hr by single cytokines, tumor necrosis factor, interleukin-1 beta and interferon-gamma with enhanced nitric oxide formation . In combination, interferon-gamma and endotoxin had greater synergistic effect on hepatocytes than did tumor necrosis factor and endotoxin . Kupffer cells of these endotoxic rats responded to 20 hr of interferon-gamma stimulation with the same enhanced nitric oxide formation we documented previously for endotoxin . Potentiation of the effect, through combination of endotoxin and interferon-gamma, was not as marked as it was with hepatocytes . Challenge of Kupffer cells with tumor necrosis factor or interleukin-1 beta evoked no response . Hepatocytes and Kupffer cells of time-matched, saline solution-treated rats were unresponsive to endotoxin or cytokine stimulation . Small quantities of nitric oxide were produced by endothelial cells spontaneously; this production was somewhat enhanced in cells of the endotoxin-infused rats by a 20-hr in vitro endotoxin challenge . Studies with inhibitors suggest that enhanced nitric oxide formation by endotoxic hepatocytes and Kupffer cells in response to in vitro endotoxin stimulation is differentially regulated . Our findings indicate modulation of nitric oxide generation by cytokines in vitro in various liver cell types of endotoxic rats . A similar scenario may exist in vivo because of the prevailing inflammatory response to endotoxin administration. Endocrinology, 1994 Jan, 134(1), 27 - 34 Threonine for alanine substitution at position 109 of transthyretin differentially alters human transthyretin's affinity for iodothyronines; Rosen HN et al.; The heterozygous substitution of threonine for alanine at amino acid 109 of human transthyretin (TTR) increases its affinity for T4 . We compared the affinity of recombinant wild-type (WT) and Thr109-TTRs for various iodothyronines in an attempt to elucidate how this mutation alters the T4-binding site . Homozygous WT and Thr109-TTRs were expressed recombinantly in Escherichia coli, and heterozygous Thr109-TTR was purified from plasma . The affinities of the iodothyronines for TTR were determined by measuring {125I}T4 bound by TTR in the presence of increasing concentrations of unlabeled iodothyronines . Homozygous Thr109-TTR bound T4 with an affinity slightly, but not significantly, greater than that of heterozygous Thr109-TTR . The affinity of Thr109-TTR for all iodothyronines was higher than that of WT TTR . However, the Thr109 mutation increased TTR's affinity for T4, Triac (triiodothyroacetic acid), and T3 to a greater extent than it did for Tetrac (tetraiodothyroacetic acid), EMD21388 (3',5'-dibromo-4',6'-dihydroxy-3-methylflavone), and dextro-T4 . These data demonstrate that a subtle change in the structure of the T4-binding channel in TTR differentially alters the affinity of binding of various iodothyronines and suggests that site-directed mutagenesis of residues within the binding channel might clarify the relative importance of specific domains of this binding channel. Infect Immun, 1994 Jan, 62(1), 99 - 105 Similar cytokine but different coagulation responses to lipopolysaccharide injection in D-galactosamine-sensitized versus nonsensitized rats; Bahrami S et al.; To compare cytokine release and coagulation disturbances induced by administration of high versus low doses of endotoxin (lipopolysaccharide {LPS}), we used two endotoxin test systems similar in mortality but different in the degree of endotoxemia . One group of rats (n = 11) randomly received endotoxin (15.0 mg/kg of body weight intraperitoneally {i.p.}) and 1 ml of Ringer's solution (nonsensitized animals) . The second group (n = 11) received 1 ml of D-galactosamine (500 mg/kg i.p.) and endotoxin (100 micrograms/kg i.p.) simultaneously (sensitized animals) . Endotoxin levels in the plasma of nonsensitized rats were 1,000-fold higher than those in the plasma of sensitized rats (69.33 x 10(3) +/- 22.42 x 10(3) versus 75.8 +/- 27.08 ng of LPS per ml), leading to a mortality of 91% in nonsensitized rats versus 82% in the sensitized-rat model within 48 h postendotoxemia . Serum transaminase activity increased up to 100-fold in sensitized rats as a sign of hepatocyte damage . Despite the large difference in LPS levels in plasma, the time courses of the plasma tumor necrosis factor (TNF) increase were similar in the two groups, with a peak at 2 h (54 +/- 12 ng/ml in nonsensitized rats versus 43 +/- 12 ng/ml in sensitized rats), and also similar to that of a group of nonsensitized rats (n = 5) that received a low dose of LPS (100 micrograms/kg) only (52 +/- 21 ng/ml), while D-galactosamine alone did not induce TNF release . Despite similar TNF levels, a more pronounced coagulation disorder was observed at 4 h in nonsensitized rats (with the high LPS dose) as measured by platelet counts, plasma fibrinogen levels, and activated partial thromboplastin time prolongation (191 x 10(3) +/- 107 x 10(3) cells per microliter, 40 +/- 24 mg/dl, and 53 +/- 15 s, respectively) than in rats with the low LPS dose either sensitized (495 x 10(3) +/- 153 x 10(3), 95 +/- 49, and 38 +/- 16, respectively) or nonsensitized (439 x 10(3) +/- 62 x 10(3), 170 +/- 18, and 35 +/- 11, respectively).(ABSTRACT TRUNCATED AT 400 WORDS) Infect Immun, 1994 Jan, 62(1), 244 - 51 Cloning and sequencing of the genes encoding Escherichia coli cytolethal distending toxin; Scott DA et al.; Escherichia coli strains expressing cytolethal distending toxin (CDT) cause elongation of CHO cells at 24 h, followed by progressive cellular distention and death for up to 120 h . Similar distention and cytotoxicity are seen in HeLa, HEp-2, and, to a lesser extent, Vero cells . The initial elongation in CHO cells is indistinguishable from that caused by E . coli heat-labile toxin (LT) . In contrast to those from LT strains, supernatants from these strains have no effect on Y-1 adrenal cells . TnphoA was introduced into CDT-positive E . coli E6468/62 (O86:H34), isolated from a child with diarrhea, and 13 CDT-negative transconjugants were identified . DNA probes constructed from DNA flanking the TnphoA insertion sites of CDT-negative mutants were used to identify a CDT-positive clone from an E6468/62 genomic library with a 5.5-kb insert . Exonuclease deletions were created and assayed in CHO cells . In this manner, a 2.3-kb CDT-active region was defined, and the nucleotide sequence was determined . Sequence analysis identified three open reading frames (ORFs), designated cdtA, cdtB, and cdtC . These contain 711, 819, and 570 bp, respectively, and encode polypeptides with predicted molecular masses of 25.5, 29.8, and 20.3 kDa, respectively . Each ORF has a putative signal sequence, and there are 4-bp overlaps between cdtA and cdtB and between cdtB and cdtC . The nucleotide and predicted amino acid sequences have no significant homology with those of any previously reported genes or proteins . By in vitro transcription-translation and an anti-alkaline phosphatase immunoblot, native proteins and/or fusion proteins corresponding to each ORF were identified. Virology, 1994 Jan, 198(1), 275 - 81 Proteinase 3C of hepatitis A virus (HAV) cleaves the HAV polyprotein P2-P3 at all sites including VP1/2A and 2A/2B; Schultheiss T et al.; Thus far, the only virus-encoded proteinase of hepatitis A virus (HAV) detected is 3C, which was shown to catalyze proteolysis of most of the suggested cleavage sites within the HAV precursor polyprotein . To elucidate whether or not HAV proteinase 3C and its precursors are involved in processing of the yet unidentified sites in the polyprotein P2-P3, the genomic region of 3C including flanking sequences were expressed in a bacterial system and by cell-free translation . In both systems 2A-reactive proteins of 10 (2A) and 16 kDa (delta VP1-2A) were processing products of a polyprotein representing delta VP1-P2-P3* (delta and * denote N- or C-terminally truncated proteins, respectively), thus providing evidence for cleavage at sites VP1/2A and 2A/2B by proteinase 3C . In the cell-free expression system, processing at the P2/P3 junction was rapid and complete, whereas sites 3A/3B, 3B/3C, and 3C/3D were inefficiently cleaved, as evidenced by the accumulation of the stable precursor polypeptides P3* and 3ABC . In contrast to the eukaryotic system, mature 3C was produced in Escherichia coli . Intermolecular cleavage by recombinant 3C occurred at all putative sites within the proteolytically inactive polyprotein P2-P3* mu . The results of this study indicate that proteinase 3C mediates the primary as well as the secondary cleavages of the HAV polyprotein and thus shows an activity profile broader than that of 3C proteinases of other picornaviruses. Virology, 1994 Jan, 198(1), 257 - 64 Antibody response in cats to the envelope proteins of feline immunodeficiency virus: identification of an immunodominant neutralization domain; de Ronde A et al.; Overlapping fragments of the envelope protein of feline immunodeficiency virus (FIV) have been expressed in Escherichia coli . Screening of cat sera for antibodies to these fragments revealed that the immunodominant domain of the FIV envelope is localized within the transmembrane protein (amino acids 687-741) and that both the variable region 3 (SU3, aa 385-417) and the COOH-terminus (aa 599-611) of the surface protein (SU) are highly immunogenic . Of all rabbit sera raised to the envelope protein fragments only the serum directed to SU3 was neutralizing . Both FIV-infected and SU3-immunized cats elicited neutralizing antibodies to SU3 . Neutralizing antibodies in sera of infected cats could be absorbed by SU3, showing that SU3 is a major neutralization domain of FIV. Virology, 1994 Jan, 198(1), 118 - 28 A poxvirus protein with a RING zinc finger motif is of crucial importance for virulence; Senkevich TG et al.; The DNA sequence of a 2060-bp fragment from the left-hand end of the ectromelia (mousepox) virus genome was determined . Two genes were identified coding for 28 kDa (p28) and 16 kDa (p16) proteins, both of which are disrupted in vaccinia virus but conserved in variola (smallpox) virus . p16 contains an N-terminal hydrophobic region and may be a membrane or secreted protein . p28 contains a C3HC4 (RING) zinc finger motif that has been found in a large family of proteins involved mostly in transcription regulation . p28 was expressed in bacteria and shown to bind Zn in vitro . Disruption of the p28 gene had no appreciable effect on the multiplication of the virus in cell culture but abolished its lethality for susceptible mice . The p28- mutant virus replicated to significantly lower titers than the wild-type virus in different organs of infected mice . It is proposed that the p28 gene is an important determinant of orthopoxvirus pathogenicity, and its product may positively or negatively regulate expression of host or viral gene(s) involved in virus-host interaction. J Virol, 1994 Jan, 68(1), 338 - 45 Capsid assembly and involved function analysis of twelve core protein mutants of duck hepatitis B virus; Yang W et al.; The roles of different regions of the duck hepatitis B virus (DHBV) core protein on viral capsid assembly and related functions were examined . Twelve deletion and insertion mutations which covered 80% of the DHBV C open reading frame were constructed and expressed in Escherichia coli . The N-terminal region (amino acids 3 to 66) of DHBV core protein was important for its tertiary structure and function in E . coli . The expressed core mutants without this region apparently inhibited E . coli growth . The results of transmission electron microscopy of E . coli thin sections, capsid agarose gel, and sucrose gradient sedimentation demonstrated that a few DHBV core mutants with insertion in the N terminus and deletion in the C terminus retained the ability to form core-like particles in E . coli . However, other mutations in most of N-terminal and central regions strongly inhibited the self-assembly ability of DHBV core protein in E . coli . In addition, the mutant with a C-terminal region deletion (amino acids 181 to 228) lost most of the nucleic acid-binding activity of the DHBV core protein. J Virol, 1994 Jan, 68(1), 240 - 50 Proteolytic activity of novel human immunodeficiency virus type 1 proteinase proteins from a precursor with a blocking mutation at the N terminus of the PR domain; Zybarth G et al.; The mature human immunodeficiency virus type 1 proteinase (PR; 11 kDa) can cleave all interdomain junctions in the Gag and Gag-Pol polyprotein precursors . To determine the activity of the enzyme in its precursor form, we blocked release of mature PR from a truncated Gag-Pol polyprotein by introducing mutations into the N-terminal Phe-Pro cleavage site of the PR domain . The mutant precursor autoprocessed efficiently upon expression in Escherichia coli . No detectable mature PR was released; however, several PR-related products ranging in size from approximately 14 to 18 kDa accumulated . Products of the same size were generated when mutant precursors were digested with wild-type PR . Thus, PR can utilize cleavage sites in the region upstream of the PR domain, resulting in the formation of extended PR species . On the basis of active-site titration, the PR species generated from mutated precursor exhibited wild-type activity on peptide substrates . However, the proteolytic activity of these extended enzymes on polyprotein substrates provided exogenously was low when equimolar amounts of extended and wild-type PR proteins were compared . Mammalian cells expressing the mutated precursor produced predominantly precursor and considerably reduced amounts of mature products . Released particles consisted mostly of uncleaved or partially cleaved polyproteins . Our results suggest that precursor forms of PR can autoprocess but are less efficient in processing of the Gag precursor for formation of mature virus particles. J Immunol, 1994 Jan 1, 152(1), 96 - 105 IL-2-induced expression of TTK, a serine, threonine, tyrosine kinase, correlates with cell cycle progression; Schmandt R et al.; We have recently isolated the cDNA for a unique human 97-kDa kinase, TTK, by expression screening of a cDNA expression library using anti-phosphotyrosine antibodies . When expressed in Escherichia coli, TTK can phosphorylate serine, threonine, and tyrosine residues . Thus TTK appears to belong to a newly described family of kinases able to phosphorylate all three hydroxy amino acids . This family of multispecific kinases includes several other kinases involved in cell cycle progression . In support of a possible role in regulating cell cycle progression, TTK message is readily detected in rapidly proliferating tissues in vivo including testes, thymus, bone marrow, and many malignant tumors, but not in benign tissues with a low proliferative rate in vivo . To determine the effect of cell activation and cell cycle progression on TTK expression, we measured TTK mRNA and protein levels as well as kinase activity in freshly isolated T cells or IL-2-expanded T cell blasts activated to proliferate by the addition of a variety of mitogens . TTK mRNA levels, protein levels, and kinase activity were greatly enhanced when either freshly isolated PBL or T cell blasts were activated by cross-linking the TCR complex by mitogenic lectins or by bypassing the TCR with phorbol esters and cation ionophores . Incubation with IL-2 increased TTK expression in PBL blasts, which proliferate in response to IL-2, but not in fresh PBL, which do not proliferate in response to IL-2 . TTK expression was blocked by either cyclosporin A or FK520, which inhibit IL-2 production and could be recovered by the addition of exogenous IL-2 . Furthermore, TTK expression was prevented by incubation of the cells with rapamycin, which blocks IL-2 signaling . Thus, TTK expression in T cells appears to be a consequence of IL2-induced cell proliferation . Agonist-induced TTK expression was a delayed event occurring 12 to 24 h after activation of PBL blasts and 48 to 72 h after activation of fresh PBL . TTK protein and mRNA expression increased in both fresh PBL and T cell blasts concurrently with passage of cells through S phase as indicated by {3H}TdR incorporation and cell cycle analysis of propidium iodide-stained cells . TTK mRNA and protein levels reached a maximum as cells entered the G2 phase of the cell cycle . These results were confirmed by cell cycle blockade studies with aphidicolin and nocodazole wherein TTK protein levels are not detected in cells in G1 and are readily detectable in cells in the S and G2 phases of the cell cycle.(ABSTRACT TRUNCATED AT 250 WORDS) J Immunol, 1994 Jan 1, 152(1), 201 - 12 Human epidermal Langerhans cells secrete a soluble receptor for IgG (Fc gamma RII/CD32) that inhibits the binding of immune complexes to Fc gamma R+ cells; Astier A et al.; Langerhans cells (LC) express Fc gamma RII on their cell surface . In this paper, we demonstrate that these cells also release soluble Fc gamma RII (sFc gamma RII) molecules . LC express transcripts encoding a membrane-associated receptor and a transmembrane-deleted Fc gamma RIIA . The latter form was identified in LC culture supernatants using specific antibodies . CHO cells, transfected with LC-derived cDNA encoding the transmembrane-deleted Fc gamma RIIA, secrete sFc gamma RIIA that include the intracellular domain and exhibit the same backbone as the protein identified in LC supernatants . Secreted sFc gamma RIIA exhibits the same pattern of binding to human and mouse IgG subclasses as do membrane Fc gamma RII and inhibits the binding of immune complexes to Fc gamma RII+ cells . In addition, CHO cells expressing the membrane-associated Fc gamma RIIA release truncated and unstable Fc gamma RIIA molecules that lack the intracellular domain . Thus, sFc gamma RII can result from shedding of membrane molecules and/or from secretion of soluble receptors lacking the transmembrane domain. Acta Haematol Pol, 1994, 25(1), 19 - 29 {Biological properties and clinical applications of interferon gamma (IFN-gamma)}; Robak T; Interferon-gamma (IFN-gamma) is produced by activated T cells and probably by NK cells . Its production can be induced by mitogens, antigens and other molecules . IFN-gamma interacts with cells by binding to specific membrane receptors . IFN-gamma--1b is an Escherichia coli--derived recombinant DNA product, which has biological activity identical to natural human IFN-gamma . This IFN type is a more potent immunomodulator than IFN-alpha and IFN-beta . Long term treatment with a therapeutic dosage of IFN-gamma--1b produces a significant reduction in the incidence of serious infections in patients with chronic granulomatous disease . This cytokine can be also useful in the treatment of patients with visceral leishmaniasis, Epstein-Barr virus infections, lepromatous leprosy and other infectious diseases . Phase I and II studies have demonstrated it to be capable of producing antitumor effects, especially in metastatic renal cell carcinoma and some hematologic malignancies . Clinical trials have suggested efficacy of IFN-gamma in the treatment of severe atopic dermatitis and rheumatoid arthritis . The most common adverse reactions are fever, headaches and erythema at the injection site. Virus Genes, 1994 Jan, 8(1), 7 - 13 Detection of a 45 kD protein derived from the N terminus of the pea seedborne mosaic potyvirus polyprotein in vivo and in vitro; Albrechtsen M et al.; A 45 kD protein (Pro1) derived from the N terminus of the pea seedborne mosaic potyvirus (PSbMV) polyprotein has been detected in extracts of infected pea plants and among in vitro translation products of PSbMV genomic RNA . The genomic region coding for the first 231 amino acids of the PSbMV polyprotein was cloned and expressed in Escherichia coli as a fusion protein with beta-galactosidase . A rabbit antiserum raised against the fusion protein recognized an approximately 45 kD protein in immunoblots of extracts of PSbMV-infected pea leaves that was not present in extracts of healthy leaves . The highest concentration of the 45 kD protein was found in extracts of young leaves, suggesting the protein may be rapidly degraded in vivo . After in vitro translation of PSbMV genomic RNA in a wheat germ extract, the antiserum immunoprecipitated a 45 kD polypeptide as well as some lower molecular weight translation products . On the other hand, an approximately 90 kD polypeptide was immunoprecipitated from in vitro translation products of genomic RNA in a rabbit reticulocyte lysate, corresponding to the combined molecular weights of Pro1 and the helper component predicted from genomic sequence data. Microbios, 1994, 77(313), 231 - 7 Changes in translational accuracy of Escherichia coli when folate metabolism is perturbed; Basso J et al.; Translational accuracy was monitored in Escherichia coli mutants which contain abnormal folate pools . A decrease in translational accuracy was indicated by the increased production of a T4 phage mutant containing a UGA mutation in a tail fibre gene . An E . coli folC mutant suppressed the phage mutant under conditions where it presumably accumulated methyl-tetrahydrofolate (methyl-THF) . A UGA suppressor strain with the mutation affecting the primary structure of the tRNA(trp) normally suppressed the phage mutant . When the strain was made thymine-requiring it no longer suppressed . The accumulation of methyl-THF which permits suppression by thymine-requiring strains may act to interfere with suppression by the tRNA suppressor, possibly by changing the modification pattern of the tRNA. Mutagenesis, 1994 Jan, 9(1), 73 - 7 Similarity in the molecular profile of mutations induced by UV light in shuttle vector plasmids propagated in mouse and human cells; Yagi T et al.; Shuttle vector plasmids pYZ289 were irradiated with UV and transfected to mouse cells to permit repair of damage, mutation and replication in the cells . The frequency and types of mutations were compared with those of UV-irradiated shuttle vector plasmids pZ189 which were propagated in normal human and xeroderma pigmentosum (XP) patient cells defective in DNA repair . Both shuttle vector plasmids contain a bacterial suppressor tRNA gene supF as a common mutation target . pYZ289 propagated in the mouse cells showed survival and mutation frequency similar to pZ189 propagated in the normal human cells . All single base substitution mutations were induced in dipyrimidine sequences and G:C to A:T transition was most frequently observed (47%) in the mouse cells; however, the frequency was significantly lower than that in the XP cells . The frequency of the base substitution mutations at A:T base pairs was significantly higher in the mouse cells (29%) than in the normal human (12%) and the XP cells (6%) . These results show that similar types of mutations are induced by UV in mouse and normal human cells, and that the A:T base pair is relatively more mutable in mouse than in normal human and XP cells. Mutagenesis, 1994 Jan, 9(1), 67 - 72 Analysis of 4-nitroquinoline-1-oxide induced mutations at the hprt locus in mammalian cells: possible involvement of preferential DNA repair; Inga A et al.; Mutation spectra induced by 4-nitroquinoline 1-oxide (4NQO) at the hprt locus for both normal (AA8) and 4NQO-sensitive (UV5) Chinese hamster ovary cells were determined to investigate the effect of DNA repair on the nature of induced mutations . The UV5 cell line is three times more sensitive to 4NQO than the AA8 parental cell line . In UV5 cells, the dGuo-N2-AQO adduct, which is considered to be the most toxic and mutagenic adduct in Escherichia coli, is poorly repaired . The molecular nature of 30 hprt mutants isolated from AA8 and 20 isolated from UV5 cells was determined by sequence analysis of in vitro amplified hprt cDNA . Both similarities and differences emerged . In both cell lines we found that (i) 4NQO is basically a base substitution mutagen acting almost exclusively at G residues and (ii) G transversions are prevalent over G transitions in both cell lines, independently from the ability to repair dGuo-N2-AQO . A high proportion (13/25) of splice mutations was observed in AA8 cells, statistically different (P < 0.04, Fisher's exact test) from the incidence of splice mutants in UV5 cells (4/20) . In AA8 mutants, all but two of the point mutations were due to lesions localized on the non-transcribed strand, suggesting preferential repair of the transcribed strand . Compared with AA8, the proportion of mutants due to lesions present on the transcribed strand was higher in UV5 cells, as expected if a preferential repair mechanism was impaired in the sensitive cell line . Our data are consistent with the molecular defect in DNA repair recently characterized in UV5. J Ocul Pharmacol, 1994 Spring, 10(1), 329 - 34 Steroidal and nonsteroidal drugs in endotoxin-induced uveitis; Kulkarni PS; Various classes of anti-inflammatory compounds like steroids (dexamethasone), cyclooxygenase inhibitors (indomethacin and flurbiprofen), 5-lipoxygenase inhibitors (BWA 218C and BWA 4C), immunosuppressive drugs (cyclosporin and rapamycin) and cod liver oil were tested for their antiinflammatory activities in endotoxin-induced uveitis model in rabbits . Intraocular inflammation was assessed in terms of two inflammatory responses i.e . breakdown of blood-aqueous barrier (BAB) and leukocyte infiltration into aqueous humor and iris ciliary body (ICB) . Prostaglandin (PG) E2 and leukotriene (LT) B4 release into aqueous humor was also measured . Indomethacin significantly inhibited PGE2 release without affecting leukocyte or BAB response . Flurbiprofen prevented leukocyte, PGE2 and LTB4 release into aqueous humor but not ICB chemotaxis . BWA 218C and BWA 4C also significantly inhibited leukocyte and LTB4 release but not BAB responses . Dexamethasone (2mg/kg, i.m.) and cyclosporin A (25 mg/kg i.m.) significantly inhibited leukocyte infiltration into aqueous humor and ICB, and PGE2 release but they failed to inhibit breakdown of BAB and LTB4 release . On the other hand, rapamycin (10mg/kg i.m.) and cod liver oil (1 ml daily i.m . up to 15 days) significantly prevented leukocyte and BAB response . Cod liver oil also significantly inhibited PGE2 and LTB4 release but rapamycin affected only PGE2 release into aqueous humor . It is concluded that arachidonic acid metabolites may not play a vital role in this uveitis model and additional proinflammatory mediators like cytokines may be involved. Arch Virol, 1994, 135(1-2), 131 - 42 Comparison of three different recombinant hepatitis B virus core particles expressed in Escherichia coli; Maassen A et al.; The properties of three different recombinant hepatitis B virus core proteins expressed in Escherichia coli were compared: an N-terminal fusion protein, a C-terminally truncated protein and a sequence-authentic protein . All three proteins assembled into capsid-like particles with typical HBc-antigenicity, sedimentation behavior and distinctive electron microscopical images . Apart from this, however, variant HBc proteins displayed properties different from sequence-authentic HBc protein p21.4 . Unlike p21.4, the particles of the N-terminal fusion protein p22.2 were sensitive to proteolytic attack by trypsin at variable sites within its arginine-rich C-terminus but not in its extended N-terminus . We therefore conclude that the C-terminal region is located on the surface of the p22.2 particle . These particles also showed increased HBe-antigenicity, as did the C-terminally truncated core particles p17.6, and to an even greater extent p18* particles which were derived from p22.2 by tryptic digestion . This might be interpreted as evidence for an--albeit minor--structural change . All variant core particles were less stable and contained less RNA . Electron microscopic indication for DNA binding of C-terminal deleted p17.6 particles was obtained using an aqueous spreading technique. Life Sci, 1994, 54(20), 1523 - 9 Effect of interferon-gamma on nitric oxide hemoglobin production in endotoxin-treated rats and its synergism with interleukin 1 or tumor necrosis factor; Kosaka H et al.; We studied the in vivo effect of interferon-gamma (IFN-gamma) on nitric oxide (NO) generation . ESR spectra of nitric oxide hemoglobin (HbNO) appeared after a lag time of 2h in the blood of rats treated with Escherichia coli lipopolysaccharide (LPS) . IFN-gamma enhanced LPS-induced HbNO formation in rats without modifying the time lag, although IFN-gamma alone did not induce HbNO formation . The plasma nitrate concentration was approximately one order of magnitude higher than the HbNO concentration . On treatment with LPS alone, the amount of tumor necrosis factor (TNF) released decreased after 2 h . Simultaneous addition of IFN-gamma and LPS increased TNF release for at least 8 h . Interleukin 1 (IL-1) release was detected only at 2 h in both groups . We also investigated the in vivo interactions of these cytokines . TNF plus IL-1 induced the greatest HbNO generation, followed by TNF plus IFN-gamma, and then IL-1 plus IFN-gamma . These results suggest that increase of TNF release by IFN-gamma plays a key role in NO generation in LPS-treated rats. J Biochem (Tokyo), 1994 Jan, 115(1), 66 - 75 Expression of octopus rhodopsin in Escherichia coli; Harada Y et al.; Apoproteins, having various molecular weights, of octopus rhodopsin (oRh, 455 amino acids), which is a typical transmembrane protein, were expressed in Escherichia coli with an inducible expression system using promoter phi 10 of the T7 phage . Fifteen synthetic genes (212-1,365 bp) for oRh (1) were cloned downstream from gene 10 of the T7 phage (846 bp), under the control of promoter phi 10 . An expression vector for mature oRh containing no extra peptide resulting from gene 10 was also constructed . Protein productivities were mainly evaluated by ELISA using monoclonal antibodies . The expression level in E . coli of the fused oRh genes varied between 1 and 200 mg/liter of the culture medium (from approximately 0.25 to 50% of the total cell protein, respectively), depending on the fused oRh genes . The amount of mature oRh protein expressed in E . coli was approximately 0.1 to 1 mg/liter . Hydropathy index analysis of gene products showed a significant negative correlation (rho = - 0.63) between expression level of oRh gene products in E . coli and their hydrophobic characteristics . Wavelength shifting of the absorption maximum by exogenous addition of retinal to apoprotein similar to that of authentic oRh was demonstrated in the membrane fraction of E . coli expressing mature opsin. J Biochem (Tokyo), 1994 Jan, 115(1), 156 - 61 Protonation state of the active-site Schiff base of aromatic amino acid aminotransferase: modulation by binding of ligands and implications for its role in catalysis; Iwasaki M et al.; The Schiff base formed between Lys258 of Escherichia coli aromatic amino acid aminotransferase (ArAT) and the coenzyme pyridoxal 5'-phosphate (PLP) has a pKa value of 6.65 . The pH dependency of the kinetic parameters was consistent with a mechanism by which the enzymatic form with the nonprotonated Schiff base productively binds aspartate, phenylalanine, and tryptophan . The Schiff base pKa value rose by 1.7-2.1 unit on binding of substrate analogs, and this strongly suggested protonation of the Schiff base upon formation of the Michaelis complex with substrates . The protonated "internal" Schiff base in the Michaelis complex is supposed to be attacked by the deprotonated substrate amino group, and this explains excellently the mechanism of transaldimination to form the PLP-substrate Schiff base . Phenylpropionate and indolepropionate caused similar increases in the pKa value to maleate . {Arg292-->Ala} ArAT showed the same pKa value as the wild-type enzyme . Therefore, neutralization of Arg292 by omega-carboxylate of dicarboxylic ligands, which had been well documented in aspartate aminotransferase to increase the Schiff base pKa, has little effect on the protonation of the Schiff base in ArAT . Thus the structure of ArAT is deliberately organized so that the Schiff base pKa is effectively modulated by substrates having only one carboxylate group. J Biochem (Tokyo), 1994 Jan, 115(1), 108 - 12 Replacement of active-site lysine-239 of thermostable aspartate aminotransferase by S-(2-aminoethyl)cysteine: properties of the mutant enzyme; Matsushima Y et al.; The active-site lysine residue of thermostable aspartate aminotransferase, Lys-239, to which the cofactor, pyridoxal 5'-phosphate (PLP), is bound, has been converted to Cys by site-directed mutagenesis . The thiol group of Cys-239 was chemically aminoethylated with ethylenimine . Amino acid analysis of the modified enzyme showed that it contained about 1 mol of S-(2-aminoethyl)cysteine (SAEC) per mol subunit . The activity of the mutant enzyme (K239SAEC) was about 14% of that of the wild-type enzyme . No significant difference in thermostability was found between the wild-type and K239SAEC enzymes . The UV-visible spectrum of K239SAEC showed a peak (lambda max 380 nm), due to absorption by the cofactor, at a 20 nm longer wavelength than that of the wild-type enzyme . The circular dichroism band due to the bound cofactor of K239SAEC also shifted toward a 20 nm longer wavelength . We determined kinetic parameters (rate constants, kmax, and dissociation constants, Kd, for the substrates) for each half transamination catalyzed by the wild-type and K239SAEC mutant enzymes by the stopped-flow method . The kmax values for the mutant enzyme reactions were 2.6-24 times lower than those for the wild-type enzyme ones . The two enzymes showed similar Kd values for the same substrates except glutamate; the mutant enzyme showed higher affinity for glutamate than the wild-type enzyme. Genetika, 1994 Jan, 30(1), 37 - 44 {Dynamics of evolutionary rates of proteins from small ribosomal subunits}; Morozov PS; Phylogenetic analysis of small ribosomal subunit proteins was performed . Plausible taxon-specific acceleration of evolutionary rates ("leading") of base substitution of some ribosomal proteins and 16S rRNA sites in different phyletic lines were revealed . The limited number of synchronously changing proteins is discussed in terms of relay-race model of coevolution of ribosome . The analogy with dilemma of Haldane is drawn . Plausible differences in the evolutionary rates of regions with different secondary structure were revealed . Changes in correlation of evolutionary rates of regions with different secondary structure during leading were determined . It is anticipated that S14 with S19 ribosomal proteins and S7 with corresponding site on 16S rRNA change co-evolutionary. Folia Microbiol (Praha), 1994, 39(1), 3 - 6 Effect of ethanol on Escherichia coli cells . Enhancement of DNA synthesis due to ethanol treatment; Basu T et al.; Increased ethanol concentration in the nutrient medium gradually slowed down the growth of Escherichia coli cells . However, during growth in the presence of 5% ethanol, DNA synthesis per cell increased about 2.5-fold compared to control cells . There was a 40-45% increase in plasmid copy number in the ethanol-treated cells. Int J Prosthodont, 1994 Jan-Feb, 7(1), 22 - 9 Endotoxin adherence to and elution from two casting alloys; Knoernschild KL et al.; The purpose of this study was to compare the relative affinity of bacterial lipopolysaccharide for two casting alloys with varied surface finishes by measuring lipopolysaccharide adherence and elution . Air borne particle abraded and polished Rexillium III and Harmony Hard alloy discs were exposed to 600,000 endotoxin units/mL E . coli lipopolysaccharide in water for 24 hours, whereas control discs were placed in 37 degrees C, lipopolysaccharide-free water . Lipopolysaccharide-exposed and control discs were then transferred every 24 hours to fresh, lipopolysaccharide-free water for up to 96 hours, and previously incubated eluates were tested for the presence of lipopolysaccharide . Initial lipopolysaccharide adherence to abraded Rexillium III discs was significantly greater than lipopolysaccharide adherence to the other groups (P < .05) . Polished and abraded Rexillium III also exhibited significantly greater lipopolysaccharide elution (P = .0001) . However, after 96 hours, more than 99% of the initially adhering lipopolysaccharide remained on both Rexillium III and Harmony Hard discs, regardless of surface treatment. Arch Toxicol, 1994, 68(2), 129 - 33 Metabolic activation of aromatic and heterocyclic N-hydroxyarylamines by wild-type and mutant recombinant human NAT1 and NAT2 acetyltransferases; Hein DW et al.; Recombinant human NAT1 and polymorphic NAT2 wild-type and mutant N-acetyltransferases (encoded by NAT2 alleles with mutations at 282/857, 191, 282/590, 341/803, 341/481/803, and 341/481) were expressed in Escherichia coli strains XA90 and/or JM105, and tested for their capacity to catalyze the metabolic activation (via O-acetylation) of the N-hydroxy (N-OH) derivatives of 2-aminofluorene (AF), and the heterocyclic arylamine mutagens 2-amino-3-methylimidazo {4,5-f}quinoline (IQ), 2-amino-3,4-dimethyl-imidazo{4,5-f}quinoxaline (MeIQx), and 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine (PhIP) . Both NAT1 and NAT2 (including all mutant human NAT2s tested) catalyzed the metabolic activation of each of the N-hydroxyarylamines to products that bound to DNA . Metabolic activation of N-OH-AF was greater than that of the heterocyclic N-hydroxyarylamines . The relative capacity of NAT1 versus NAT2 to catalyze activation varied with N-hydroxyarylamine substrate . N-OH-MeIQx and N-OH-PhIP exhibited a relative specificity for NAT2 . These results provide mechanistic support for a role of the genetic acetylation polymorphism in the metabolic activation of heterocyclic amine mutagens and carcinogens. J Virol Methods, 1994 Jan, 46(1), 95 - 105 A novel intergenic site for integration and expression of foreign genes in the genome of pseudorabies virus; Fuchs W et al.; Restriction enzyme analysis of DNA of a number of Pseudorabies virus (PRV) single plaque isolates revealed in several cases the existence of a unique EcoRI cleavage site, which has not been observed in PRV DNA before . This EcoRI site was mapped to the right end of the unique long region of the PRV genome, in BamHI-fragment 6 . Sequence analysis of this region demonstrated the presence of an 11 bp tandem repeat in variable copy numbers in different PRV strains, suggesting the creation of the EcoRI recognition site by a recombinational event . The occurrence of variable reiterations and Northern blot analysis indicated an intergenic region . We therefore, used this site for integration and expression of heterologous DNA (the multiple cloning site of phage M13 and the E . coli lacZ gene) . Viable PRV recombinants could be obtained which showed no detectable differences in virus growth in vitro compared to wild-type PRV . The novel insertion site can be used for the construction of PRV recombinants expressing foreign genes without apparent impairment of PRV genes. J Cell Sci, 1994 Jan, 107 ( Pt 1), 29 - 37 Up-regulation of the connexin43+ gap junction network in haemopoietic tissue before the growth of stem cells; Rosendaal M et al.; The early developmental stages of haemopoiesis are thought to be regulated by paracrine growth factors and by the haemopoietic environment . Are gap junctions involved here? Gap junctions are structures in cell membranes allowing the direct transfer of ions and small molecules between adjacent cells and are known to be involved in development . We have found that although connexin43 gap junctions are rare (0.00016 +/- 0.0002/microns2 tissue) in normal adult mouse marrow their expression is 80-fold higher (0.0292 +/- 0.0147/microns2) in neonatal marrow . One difference between neonatal and adult haemopoietic tissue is that in the latter more haemopoietic cells are dividing . To test if more gap junctions were due to increased division we altered adult blood-formation by mobilizing or destroying end cells--granulocytes and red cells--or by forcing stem cells to divide by making them regenerate an ablated blood-forming system . Mobilizing end cells had no effect on the number or distribution of gap junctions in marrow but forced stem cell division caused a 100-fold increase in gap junction expression and did so before any recognizable haemopoietic cells formed . There were greater than normal numbers of gap junctions in radio-protected adult mouse marrow . The cells coupled by gap junctions are TE-7+ mesodermally derived fibroblasts, STRO-1+ stromal cells, and CD45+ and CD34+ haemopoietic cells . We propose that there is a latent network of Cx43+ gap junctions in normal quiescent marrow . In response to events that call for active division of stem cells this network is amplified and coupled to haemopoietic stem cells, perhaps enabling them to divide. J Appl Physiol, 1994 Jan, 76(1), 361 - 9 Loss of endothelium-dependent vasodilation in the pulmonary vessels of sheep after prolonged endotoxin; Spath JA Jr et al.; We examined the hemodynamic response of awake sheep to prolonged endotoxin infusion (10 ng.kg-1 x min-1 for 12 h) and the in vitro endothelium-dependent relaxation of pulmonary arterial vessels excised 12 h after the end of endotoxin infusion to determine whether the development of pulmonary hypertension after endotoxin is associated with loss of endothelium-dependent relaxation . In seven of nine sheep, there was a maintained increase (4-68% of baseline) in pulmonary arterial pressure 24 h after the beginning of endotoxin infusion . The greater the increase in pulmonary arterial pressure in vivo, the greater was the in vitro deficit in endothelium-dependent relaxation of the pulmonary vessels . The maximum in vitro vessel dilation was 59% for pulmonary artery rings isolated from sheep without a sustained increase in pulmonary arterial pressure 24 h after endotoxin . Prolonged endotoxin infusion did not alter the in vitro response of pulmonary arterial vessels to KCl or 10(-5) M norepinephrine . Force development, response to 10(-5) M norepinephrine, and vasodilation in response to acetylcholine were also not altered in pulmonary vessels taken from control sheep and exposed in vitro to tumor necrosis factor-alpha (400 U/ml) . Our results suggest that loss of endothelium-dependent relaxation in pulmonary vessels supports the sustained pulmonary hypertension that develops after prolonged exposure to endotoxin. Plasmid, 1994 Jan, 31(1), 31 - 9 Characterization of plasmid pVT745 isolated from Actinobacillus actinomycetemcomitans; Novak KF et al.; A 25.1-kb plasmid, pVT745, was isolated from Actinobacillus actinomycetemcomitans strain VT745 . This plasmid hybridized under stringent conditions to chromosomal DNA from 15 A . actinomycetemcomitans isolates obtained from geographically diverse regions of the United States . Southern blot analyses of HincII-digested A . actinomycetemcomitans genomic DNA revealed five strain-specific patterns of hybridization, which clearly indicated that intact pVT745 was not inserted into any of the genomes . Plasmid pVT745 was digested with BamHI and PstI into three, non-overlapping fragments of approximately 7.0, 8.0, and 10.0 kb . The fragments were cloned in Escherichia coli on the low copy number vector pGB2 . Although these three fragments exhibited no cross-hybridization, genomic DNA from isolates representing each of the strain-specific patterns hybridized with two or three of the cloned fragments . The results suggest that two or more unique sequences within pVT745 also may be present in genomic DNA obtained from various strains of A . actinomycetemcomitans. Plasmid, 1994 Jan, 31(1), 1 - 11 Nucleotide sequence analysis of IS1533 from Leptospira borgpetersenii: identification and expression of two IS-encoded proteins; Zuerner RL; The nucleotide sequence of IS1533, an insertion sequence-like element cloned from the spirochete Leptospira borgpetersenii, was determined . IS1533 contains imperfect terminal inverted repeats (IVR) of 31 bp flanking a 1402-bp internal sequence . A putative target sequence was identified, and insertion may result in duplication of 2 bp . The internal sequence has a single open reading frame (ORF) . IS1533 encodes two proteins (43.5 and 41 kDa) initiating alternatively at either the first or the second AUG codons of the ORF . These proteins are related to a recently recognized family of IS-encoded transposases and bacterial recombinases, all which share a region of homology with the active site of the HIV reverse transcriptase . The IS1533-encoded proteins were expressed in Escherichia coli . Both the 43.5- and 41-kDa proteins bound IS1533 DNA probes in a Southwestern blot assay . These data suggest that one or both proteins function during transposition of IS1533. Mol Microbiol, 1994 Jan, 11(2), 391 - 402 Escherichia coli periplasmic chaperone FaeE is a homodimer and the chaperone-K88 subunit complex is a heterotrimer; Mol O et al.; The interaction of FaeE, a periplasmic chaperone involved in K88 biosynthesis, and the major fimbrial subunit FaeG was investigated . The genes encoding the two proteins were subcloned together in the expression vector pINIIIA1 . Cells expressing the subcloned genes accumulated in their periplasm a complex of FaeE and FaeG . This complex was purified by isoelectric focusing and anion-exchange fast-protein liquid chromatography . SDS-PAGE, native gel electrophoresis, immunoblotting and determination of the N-terminal amino acid sequences and the molar ratio of the N-terminal amino acid residues revealed that the complex is a heterotrimer consisting of two molecules of FaeE and one molecule of FaeG . The periplasmic chaperone FaeE was purified from the periplasm of cells expressing only the subcloned faeE gene . Gel filtration, protein cross-linking analysis and a biophysical approach in which the rotation diffusion coefficient of the purified FaeE was determined led to the conclusion that the native FaeE chaperone is a homodimer. Mol Microbiol, 1994 Jan, 11(2), 383 - 90 Location and orientation of an activating region in the Escherichia coli transcription factor, FNR; Bell A et al.; We have characterized a number of mutations in fnr that interfere with FNR-dependent transcription activation at two promoters where the FNR-binding site is centred around 41 1/2 bp upstream from the transcription start site . The substituted residues in all but one of these FNR mutants are clustered around a presumed surface-exposed beta-turn containing G85 which, we suggest, forms an activating region that contacts RNA polymerase at these promoters . Using the 'oriented heterodimers' method described elsewhere, we show that this activating region on the promoter-proximal subunit of the FNR dimer is sufficient to activate transcription initiation . In contrast, this region is not essential for activation of a third FNR-dependent promoter where the FNR-binding site is centred at 61 1/2 bp upstream from the transcription start site . However, a substitution at S73 interferes with FNR-dependent activation at both this promoter and promoters in which the FNR site is located at 41 1/2 bp from the transcript start, suggesting that FNR may contain a second activating region. Mol Microbiol, 1994 Jan, 11(2), 303 - 13 The function of a ribosomal frameshifting signal from human immunodeficiency virus-1 in Escherichia coli; Yelverton E et al.; A 15-17 nucleotide sequence from the gag-pol ribosome frameshift site of HIV-1 directs analogous ribosomal frameshifting in Escherichia coli . Limitation for leucine, which is encoded precisely at the frameshift site, dramatically increased the frequency of leftward frameshifting . Limitation for phenylalanine or arginine, which are encoded just before and just after the frameshift, did not significantly affect frameshifting . Protein sequence analysis demonstrated the occurrence of two closely related frameshift mechanisms . In the first, ribosomes appear to bind leucyl-tRNA at the frameshift site and then slip leftward . This is the 'simultaneous slippage' mechanism . In the second, ribosomes appear to slip before binding aminoacyl-tRNA, and then bind phenylalanyl-tRNA, which is encoded in the left-shifted reading frame . This mechanism is identical to the 'overlapping reading' we have demonstrated at other bacterial frameshift sites . The HIV-1 sequence is prone to frame-shifting by both mechanisms in E . coli. Mol Microbiol, 1994 Jan, 11(2), 293 - 302 In vivo study of engineered G-domain mutants of Escherichia coli translation initiation factor IF2; Laalami S et al.; During the IF2-catalysed formation of the 30S initiation complex, the GTP requirement and its subsequent hydrolysis during 70S complex formation are considered to be essential for translation initiation in Escherichia coli . In order to clarify the role of certain amino acid residues believed to be crucial for the GTP hydrolytic activity of E . coli IF2, we have introduced seven single amino acid substitutions into its GTP-binding site (Gly for Val-400; Thr for Pro-446; Gly, Glu, Gln for His-448; and Asn, Glu for Asp-501) . These mutated IF2 proteins were expressed in vivo in physiological quantities and tested for their ability to maintain the growth of an E . coli strain from which the functional chromosomal copy of the infB gene has been deleted . Only one of the mutated proteins (Asp-501 to Glu) was able to sustain cell viability and several displayed a dominant negative effect . These results emphasize that the amino acid residues we substituted are essential for the IF2 functions and demonstrate the importance of GTP hydrolysis in translation initiation . These findings are discussed in relation to a previously proposed theoretical model for the IF2 G-domain. Mol Microbiol, 1994 Jan, 11(2), 273 - 80 Characterization of a 3-dehydroquinase gene from Actinobacillus pleuropneumoniae with homology to the eukaryotic genes qa-2 and QUTE; Lalonde G et al.; A gene was cloned from Actinobacillus pleuropneumoniae strain 4074 by complementation of an aroD strain of Escherichia coli . The E . coli gene aroD codes for a 3-dehydroquinase enzyme of type I, active in the aromatic biosynthesis pathway . The A . pleuropneumoniae gene, termed aroQ, displays no base or amino acid sequence homology to aroD of E . coli . It is instead homologous to the QUTE and qa-2 genes, respectively of Aspergillus nidulans and Neurospora crassa . These genes code for 3-dehydroquinase enzymes of type II, involved in the catabolism of quinic acid . The 1.8 kb fragment, which includes aroQ, carries four overlapping or adjacent open reading frames: a dapD gene; aroQ; one without homology to sequences in GenBank; and one with homology to the C-terminal 40% of chIN of E . coli. Mol Microbiol, 1994 Jan, 11(2), 249 - 60 The homologous operons for P1 and P7 plasmid partition are autoregulated from dissimilar operator sites; Hayes F et al.; The plasmid-partition regions of the P1 and P7 plasmid prophages in Escherichia coli are homologues which each encode two partition proteins, ParA and ParB . The equivalent P1 and P7 proteins are closely related . In each case, the proteins are encoded by an operon that is autoregulated by the ParA and ParB proteins in concert . This regulation is species-specific, as the P1 proteins are unable to repress the P7 par operon and vice versa . The homologous ParA proteins are primarily responsible for repression and bind to regions that overlap the operon promoter in both cases . The DNA-binding domain of the P7 autorepressor lies in the amino-terminal end of the P7 ParA protein . This region includes a helix-turn-helix motif that has a clear counterpart in the P1 ParA sequence . However, despite the common regulatory mechanism and the similarity of the proteins involved in repression, the promoter-operator sequences of these two operons are very different in sequence and organization . The operator is located downstream of the promoter in P1 and upstream of it in P7, and the two regions show little, if any, homology . How these differences may have arisen from a common ancestral form is discussed. Virus Res, 1994 Jan, 31(1), 123 - 37 Genetic instability of vaccinia virus containing artificially duplicated genome regions; Kriajevska MV et al.; A double recombinant of vaccinia virus (W-lacZ/J-tk/F) was obtained, which contains two inverted copies of the virus tk gene, separated by 45 kb: (i) the native copy located in the HindIII J fragment of the virus genome was inactivated due to insertion of E . coli lacZ gene; (ii) the second active copy was artificially inserted into the HindIII F fragment . The virus expressing both thymidine kinase and beta-galactosidase (tk+lac+ phenotype) was cloned . Due to the presence of duplicated inverted sequences of the tk gene in the virus genome extensive recombination was observed leading to genetic heterogeneity of the virus population . The population consisted mainly of the virions with the tk+lac- (77%) and tk+lac+ (23%) phenotypes . Passages in the presence of BUdR revealed minor fractions of the tk-lac+ and tk-lac- phenotypes . Structural analysis of DNA isolated from virions confirmed the genetic heterogeneity of the virus population . Nine different HindIII fragments were detected containing HindIII F, J and (or) lacZ sequences . The structure of these fragments indicates that predominantly two types of recombination events occur in the population: (i) translocation of the lacZ gene between duplicated sequences of the tk gene or displacement of lacZ by tk via intergenome and intragenome double crossing over; (ii) inversion of a 45 kb sequence in the conserved region of the genome between duplicated sequences of the tk gene due to a intragenome single crossing over. Indian J Med Res, 1994 Jan, 99, 18 - 20 Comparative evaluation of labelling intensity for detection of enterotoxigenic Escherichia coli; Lahiri SS et al.; Twenty four Esch . coli isolates obtained from patients of diarrhoea were tested by DNA hybridization for presence of enterotoxigenic Esch . coli (ETEC) . The probe generated for this study was labelled by two different ways using the large Klenow fragment of DNA polymerase-I . It was observed that labelling by sequential harnessing of the exonuclease and polymerase activity of the enzyme was superior to extension of random hexanucleotide primers . This method besides being economic, dispenses with the critical step involved in the thermodynamics of oligoannealing and initiation of DNA synthesis. Environ Mol Mutagen, 1994, 23 Suppl 24, 78 - 85 Evolution of concepts in DNA repair; Hanawalt PC; A short personalized history of the development of the field of DNA excision repair is presented, beginning with the early insights of radiation biologists and extending to the present-day convergence of the fields of DNA repair and transcription . It is becoming increasingly clear that excision repair is not merely an extraordinary scheme to help UV-exposed cells survive but rather one that operates upon a wide range of structural defects in DNA, some of which are due to environmental chemicals and others are a consequence of normal metabolic activities . It is an important challenge to researchers and risk assessors to determine the relative contributions to biological endpoints from endogenous events and the intrinsic instability of DNA as compared to exogenous environmental exposures . This should be one of the goals of the Environmental Mutagen Society as it embarks upon its second quarter-century. Environ Mol Mutagen, 1994, 23 Suppl 24, 59 - 66 Induction of genetic recombination: consequences and model systems; Hoffmann GR; Radiation and many chemicals have been found to induce homologous genetic recombination . Experimental systems that allow the detection and characterization of recombinagens exist in organisms as diverse as bacteria, fungi, plants, insects, and mammals . Recombination plays an important role in many biological processes, and studies of recombinagens can provide insight into underlying mechanisms . Studies of recombinagens are also of applied interest in genetic toxicology, because recombinational events in somatic cells can contribute to human disease . Clear connections have been established between mitotic recombination and the etiology of some cancers . This article briefly reviews two aspects of the induction of genetic recombination by radiation and chemicals--the health implications of recombinagenic effects and assays for detecting recombinagens. Cell Transplant, 1994 Jan-Feb, 3(1), 55 - 60 In vitro inhibition of insulin release by blood mononuclear cells from insulin-dependent diabetic and healthy subjects: synergistic action of IL-1 and TNF; Ablamunits V et al.; Previous studies have demonstrated that peripheral blood mononuclear cells (BMC) from type 1 (insulin-dependent) diabetic patients inhibit insulin release (IR) from rat or mouse islet cells in vitro . This phenomenon is of great interest as a model for islet graft rejection . We found that lipopolysaccharide (LPS)-stimulated BMC of healthy donors and type 1 diabetic patients suppress both basal and stimulated insulin secretion . To study whether this inhibition was due to soluble mediators we added supernatants of LPS-stimulated BMC or recombinant human interleukin-1 beta (IL-1) and tumor necrosis factor-alpha (TNF) at concentrations comparable to those found in the supernatants to rat islet cells . The inhibitory effect of BMC on islet cells could be transferred by supernatants of LPS-stimulated BMC . We found that neither IL-1 nor TNF alone inhibit IR from dispersed adult rat islet cells . However, the combination of IL-1 and TNF was highly effective . Ultrafiltration of supernatants of LPS-stimulated BMC through a PM-10 membrane (10 kDa cutoff) deprived the supernatants of the inhibitory activity indicating that only intact IL-1 and TNF (m.w . about 17 kDa), but not smaller IL-1 and TNF fragments, were responsible for the effects on islet cells . These data suggest that activation of BMC and cytokine release at islet graft site may result in an early loss of graft function . Islet transplantation using microcapsules not permeable for molecules with m.w . > 10 kDa would be preferable. Arch Microbiol, 1994, 161(3), 199 - 206 Colicins: structures, modes of action, transfer through membranes, and evolution; Braun V et al.; This article intends to inform a broader audience on a fascinating class of protein toxins (bacteriocins) which usually kill only cells of the same species . Those who gained a deeper interest in bacteriocins can find a comprehensive description of the field in a recent book based on a conference (James et al . 1992), and in more specialized review articles dealing with certain aspects (Pugsley 1984a, b), or certain colicins (De Graaf and Oudega 1986; Harkness and Olschlager 1991; Lazdunski et al . 1988) . The older literature has been reviewed by Brandis and Smarda (1971), Reeves (1972), Hardy (1975) and Konisky (1982). Vopr Virusol, 1994 Jan-Feb, 39(1), 6 - 10 {The antiviral action of recombinant receptorotoxins based on the diphtheria toxin and the human T-lymphocyte CD4-receptor}; Sidorov AV et al.; A number of vectors expressing in E . coli hybrid proteins (receptorotoxins) composed of diphtheria toxin lacking the C-terminal region and CD4-receptor fragment including N-terminal region of the natural protein have been constructed . The receptorotoxins consisting of the CD4 fragment in their N-terminal region were more stable . These recombinant receptorotoxins were cultured with HIV-infected Hut-78 cells and were shown to block HIV infection in vitro. Mol Gen Genet, 1994 Jan, 242(2), 237 - 40 Deletion of the prc (tsp) gene provides evidence for additional tail-specific proteolytic activity in Escherichia coli K-12; Silber KR et al.; The Escherichia coli protease Prc (Tsp) exhibits specificity in vitro for proteins with nonpolar carboxyl termini . To determine whether Prc is responsible for the selective degradation in vivo of proteins with nonpolar carboxyl termini, we constructed a prc (tsp) deletion strain . Deletion of the prc gene did not prevent the rapid intracellular degradation of a variant of the amino-terminal domain of lambda repressor with a nonpolar carboxyl terminus, even though this protein is a substrate for Prc in vitro . Our results indicate that at least one additional carboxy-terminal-specific proteolytic system must exist in E . coli. Mol Gen Genet, 1994 Jan, 242(2), 163 - 8 Cloning, sequencing and expression of the gene encoding 2-phosphoglycerate kinase from Methanothermus fervidus; Lehmacher A et al.; The gene encoding 2-phosphoglycerate kinase (2PGK), which catalyses the first step in the biosynthesis of cyclic 2,3-diphosphoglycerate in methanogens, was cloned and sequenced from the hyperthermophilic Methanothermus fervidus . The 2pgk gene codes for 304 amino acids, corresponding to a relative molecular mass of 35040 . The 2pgk mRNA was estimated to be 1600 nucleotides in size . Putative transcription signals and the ribosome-binding site of 2pgk are discussed . Production of 2PGK from M . fervidus in Es-cherichia coli reveals the same apparent molecular weights for the native enzyme and its denatured subunit as those shown by the 2PGK purified from M . fervidus . Also the kinetic parameters of 2PKG produced in E . coli correspond well with those from the enzyme isolated from the natural host M . fervidus. Mol Gen Genet, 1994 Jan, 242(2), 130 - 6 Replication of the Streptomyces plasmid pSN22 through single-stranded intermediates; Kataoka M et al.; The replication of the 11 kb conjugative multicopy Streptomyces plasmid pSN22 was analyzed . Mutation and complementation analyses indicated that the minimal region essential for plasmid replication was located on a 1.9 kb fragment of pSN22, containing a transacting element encoding a replication protein and a cis-acting sequence acting as a replication origin . Southern hybridization showed that minimal replicon plasmids accumulated much more single-stranded plasmid molecules than did wild-type pSN22 . Only one strand was accumulated . A 500 bp fragment from the pSN22 transfer region was identified which reduced the relative amount of single-stranded DNA, when added in the native orientation to minimal replicon plasmids . This 500 bp DNA sequence may be an origin for second-strand synthesis . It had no effect on the efficiency of co-transformation, plasmid incompatibility, or stability . The results indicate that pSN22 replicates via single-stranded intermediates by a rolling circle mechanism. FEMS Immunol Med Microbiol, 1994 Jan, 8(1), 13 - 26 The significance of the hydrophilic backbone and the hydrophobic fatty acid regions of lipid A for macrophage binding and cytokine induction; Kirikae T et al.; Natural partial structures of lipopolysaccharide (LPS) as well as synthetic analogues and derivatives of lipid A were compared with respect to inhibit the binding of 125I-labelled Re-chemotype LPS to mouse macrophage-like J774.1 cells and to induce cytokine-release in J774.1 cells . LPS, synthetic Escherichia coli-type lipid A (compound 506) and tetraacyl precursor Ia (compound 406) inhibited the binding of 125I-LPS to macrophage-like J774.1 cells and induced the release of tumor necrosis factor alpha (TNF alpha) and interleukin 6 (IL-6) . Deacylated R-chemotype LPS preparations were completely inactive in inhibiting binding and in inducing cytokine-release . Among tetraacyl compounds, the inhibition-capacity of LPS-binding was in decreasing order: PE-4 (alpha-phosphonooxyethyl analogue of 406) > 406 >> 404 (4'-monophosphoryl partial structure of 406) > 405 (1-monophosphoryl partial structure of 406) . In the case of hexaacyl preparations, compounds 506, PE-1 (alpha-phosphonooxyethyl analogue of 506) and PE-2 (differing from PE-1 in having 14:0 at positions 2 and 3 of the reducing GlcN) inhibited LPS-binding and induced cytokine release equally well, whereas preparation PE-3 (differing from PE-2 in containing a beta-phosphonooxyethyl group) showed a substantially lower capacity in binding-inhibition and cytokine-induction . The conclusion is that chemical changes in the hydrophilic lipid A backbone reduce the capacity of lipid A to bind to cells, whereas the number of fatty acids determines the capacity of lipid A to activate cells . These results indicate that the bisphosphorylated hexosamine backbone of lipid A is essential for specific binding of LPS to macrophages and that the acylation pattern plays a critical role for LPS-promoted cell activation, i.e . cytokine induction. Bioelectromagnetics, 1994, 15(1), 77 - 83 Magnetic fields after translation in Escherichia coli; Goodman EM et al.; Quantitative two-dimensional gel electrophoresis of proteins in E . coli exposed for 60 min to weak, pulsed magnetic fields (1.5 mT peak) show that numerous proteins are both increased and decreased by a factor of 2 or more . An increase in the levels of two proteins, the a subunit of DNA-dependent RNA polymerase and NusA, was confirmed by Western blot analysis. Parasite Immunol, 1994 Jan, 16(1), 27 - 34 Studies on the immunogenicity of a recombinant ookinete surface antigen Pbs21 from Plasmodium berghei expressed in Escherichia coli; Matsuoka H et al.; Plasmodium berghei ookinete surface antigen (Pbs21), was produced as a fusion product with maltose binding protein (MBP) in Escherichia coli and used to induce transmission-blocking immunity in mice . Specificity of induced antibody was confirmed by Western blotting with native ookinete Pbs21, and by the indirect immunofluorescent antibody test on ookinete bloodfilms . Immunized mice were infected with P . berghei and transmission to Anopheles stephensi mosquitoes determined by both the intensity and prevalence of oocyst infections . Compared with a control group immunized with MBP alone the maximum blockade of oocyst intensity was 66% in the mice immunized with recombinant MBP-Pbs21 . Over nine experiments blockade averaged only 33% . By comparison with native Pbs21 protein, which usually induces > or = 90% blockade, our data suggests the recombinant protein produced in this bacterial system is a less effective immunogen despite expressing epitopes recognized by known transmission-blocking monoclonal antibodies. Mol Endocrinol, 1994 Jan, 8(1), 116 - 25 Stress-induced regulation of a human proenkephalin-beta-galactosidase fusion gene in the hypothalamus of transgenic mice; Borsook D et al.; Transgenic mice expressing an Escherichia coli beta-galactosidase reporter gene under the control of 3 kilobases of human proenkephalin gene 5'-flanking sequence and 1.2 kilobases of 3'-flanking sequence exhibited an anatomically correct pattern of basal and stress-regulated transgene expression within the hypothalamus . Acute osmotic stress and hypovolemia induced transgene expression in neurons within both the paraventricular and supraoptic nuclei . Chronic osmotic stress resulted in dramatic induction of transgene expression in both nuclei . These results demonstrate that the information required for correct hypothalamic expression and stress regulation of the proenkephalin gene is contained within our fusion construct. Life Sci, 1994, 54(15), 1059 - 71 Tissue distribution of histamine N-methyltransferase-like immunoreactivity in rodents; Takemura M et al.; The rat kidney histamine N-methyltransferase was purified to homogeneity from Escherichia coli transfected with its recombinant cDNA . An antiserum to the enzyme was raised in rabbit by immunization with the purified protein . Western blot analysis of rat tissues with the antiserum revealed a band with identical mobility to that of purified enzyme in the extracts of kidney, jejunum, and brain, where the enzyme activity was detected . The antiserum cross-reacted with a 32K protein in mouse liver, brain, stomach, kidney and lung, and a 33K protein in guinea pig brain, stomach jejunum, spleen, lung, and kidney . The intensity of the staining in western blotting correlated well with the enzyme activity in all the tissues in these three species, suggesting that our antiserum is useful for quantifying histamine N-methyltransferase protein in rodent tissues. J Photochem Photobiol B, 1994 Jan, 22(1), 9 - 15 Photobiological activity of sulphur and selenium analogues of psoralen; Jakobs A et al.; Eight psoralen analogues, in which sulphur or selenium replaces one or both intracyclic oxygen atoms, were synthesized . Photoreaction with M13mp19 RF DNA in the presence and absence of oxygen (wavelength, greater than 320 nm) was studied . The damaged viral DNA was transfected into Escherichia coli and scored for infectivity towards Ca-treated wild-type E . coli . This allowed a comparative evaluation to be made of the heteropsoralens in terms of the photoreaction with DNA and the photodynamic effect . Most of the seleno- and thio-psoralens show very high photoactivity towards DNA compared with psoralen and 8-methoxypsoralen (8-MOP) . Their photoreactivity is due mainly to a {2 + 2} photoreaction, since only a minor influence of molecular oxygen could be detected . Some of the studied seleno- and thio-psoralens are very efficient DNA photoinactivating agents and show great promise in photochemotherapy. Circ Shock, 1994 Jan, 42(1), 44 - 52 Pulmonary vascular effects of endotoxin in canine lobes pretreated with dapsone; Mayers I et al.; Endotoxin results in a granulocyte mediated loss of hypoxic pulmonary vasoconstriction (HPV) . Dapsone blocks the granulocyte respiratory burst and might, therefore, preserve HPV following endotoxin . Isolated-perfused canine lobes (n = 6) were pretreated with 18 mg/kg dapsone (dapsone group), and compared to six lobes which did not receive dapsone (control group) . Total pulmonary vascular resistance (Rtot) and arterial, middle (Rm), and venous segmental resistances were calculated by a vascular occlusion technique . We then administered endotoxin (2 mg/kg) and repeated measurements at 5, 30, and 90 min . The increase in Rm during 3% O2 compared to 35% O2 ventilation was used to define the presence of HPV . In the control group, following endotoxin, values of Rm did not change (P > 0.05) during 3% O2 ventilation (0.011 +/- 0.006 cm H2O/ml/min) compared with 35% O2 ventilation (0.014 +/- 0.005 cm H2O/ml/min) . In the dapsone group, following endotoxin, values of Rm increased (P < 0.05) during 3% O2 ventilation (0.06 +/- 0.026 cm H2O/ml/min) compared with 35% O2 ventilation (0.03 +/- 0.015 cm H2O/ml/min) . Changes in 6-keto PGF1 alpha or thromboxane B2 do not explain these observations . We conclude that in this experimental preparation, pretreatment with dapsone prevents the loss of HPV associated with endotoxin. Circ Shock, 1994 Jan, 42(1), 14 - 9 Platelet activating factor impairs pressor responses to noradrenaline in the anaesthetized rat but does not mediate endotoxin-induced hyporeactivity; Bouvier C et al.; A nonhypotensive dose of endotoxin (Escherichia coli lipopolysaccharide, 250 micrograms kg-1 h-1) impaired both the pressor responsiveness to noradrenaline and its effects in reducing renal and hindquarter blood flow, measured using ultrasound Doppler flow probes . Platelet activating factor (PAF, 50 ng kg-1 h-1) similarly impaired pressor responsiveness to noradrenaline, although this effect was accompanied by marked hypotension . These actions of PAF were prevented by pretreatment with the PAF antagonists WEB 2086 (20 mg kg-1) or BN 50739 (10 mg kg-1) 15 min before commencing the infusion . However, neither antagonist modified the effect of endotoxin in impairing vascular responsiveness to noradrenaline . Thus, these results do not support a role for PAF in mediating endotoxin-induced vascular hyporeactivity, at least in the early stages of endotoxaemia. Mol Biol (Mosk), 1994 Jan-Feb, 28(1), 150 - 7 {Study of the biogenesis and secretion of alkaline phosphatase and its mutant forms in Escherichia coli . I . Introduction of directed mutations into the alkaline phosphatase gene}; Karamyshev AL et al.; Various mutations in E . coli alkaline phosphatase gene were obtained by oligonucleotide-directed mutagenesis . They result in amino acid substitutions in the signal peptide cleavage site {Val for Ala(-1)} and in the N terminus of mature polypeptide chain: Ala for Arg(+1) and Gln for Glu(+4); Gln for Glu(+4) . Enzyme activity was observed in all E . coli strains transformed by plasmids with cloned mutant genes . In addition, an amber mutation was introduced into the Arg(+1) position, and the synthesis of mutant alkaline phosphatase was shown in E . coli strains containing suppressor tRNAs specific for Ser, Gln, Tyr, Leu, Ala, Glu, Phe, Gly, His, Pro, and Cys. Mol Microbiol, 1994 Jan, 11(1), 99 - 109 Evidence that residues -15 to -46 of the haemolysin secretion signal are involved in early steps in secretion, leading to recognition of the translocator; Kenny B et al.; We previously identified three well-dispersed mutations, E978-K, F989-L and D1009-R within the haemolysin A signal region, located at positions -46, -35 and -15, with respect to the C-terminus, respectively . Each mutation reduces the efficiency of secretion two- to threefold leaving 30-45% of the wild-type activity . We have constructed by in vitro manipulations double mutants of HlyA carrying all combinations of these mutations and a triple mutant carrying all three mutations . The effects on secretion were determined and the results, including residual levels of secretion with the triple mutant of only 0.6%, compared with the wild type, indicated that these residues may interact to form a single function in the wild-type signal . To test this further, we developed a secretion competition assay in order to classify signal mutations . We demonstrated that a CIZ-HlyA fusion protein, containing the C-terminal 81 kDa of HlyA fused to virtually the whole LacZ protein, strongly inhibits the secretion of the wild-type HlyA co-expressed in the same cell . The properties of the fusion indicate that it blocks the translocator . The three mutations singly and in combinations were recombined in vitro into the 3'-end of the hybrid gene . In every case, the presence of a mutation in the secretion signal of the hybrid protein alleviated the inhibition of secretion of the co-expressed HlyA . All the mutations are therefore essentially recessive and we propose that they all affect an early function, probably recognition of the translocator, rather than a subsequent step involved in translocation or final release of the toxin to the medium . This would indicate that residues involved in recognition (or steps leading to recognition) extend from at least -15 to -46 with respect to the HlyA C-terminus. Mol Microbiol, 1994 Jan, 11(1), 3 - 8 Regulatory implications of translational frameshifting in cellular gene expression; Engelberg-Kulka H et al.; The genetic code, once thought to be rigid, has been found to be quite flexible, permitting several different reading alternatives . One of these is translational frameshifting, a process programmed in the mRNA sequence and which enables a +1 or -1 shift from the reading frame of the initiation codon . So far, the involvement of translational frameshifting in gene expression has been described mainly in viruses (particularly retroviruses), retrotransposons, and bacterial insertion elements . In this MicroReview, we present a survey of the cellular genes, mostly in Escherichia coli, which have been found to be expressed through a translational frameshifting process, as well as a discussion of the regulatory implications of this process. Invest Radiol, 1994 Jan, 29(1), 68 - 71 Effect of radiographic contrast media on granulocyte phagocytosis of Escherichia coli in a whole blood flow cytometric assay; Lillevang ST et al.; RATIONALE AND OBJECTIVES . Earlier studies have demonstrated an adverse effect of radiographic contrast media (CM) on granulocyte phagocytosis . Most studies in the past have depended on granulocyte separative procedures that may themselves affect granulocyte functions . This study was performed to evaluate the effect of CM on phagocytosis using a flow cytometric assay allowing more physiological assay conditions . METHODS . Twenty consecutive patients were blindly randomized to receive the nonionic ratio 3.0 CM iohexol or the ionic ratio 3.0 CM ioxaglate for intravenous urography . Granulocyte phagocytic potential was measured before and at 1, 5, and 20 minutes after CM administration with a flow cytometric whole blood method evaluating the ingestion of complement- and immunoglobulin G (IgG)-opsonized fluorescent Escherichia Coli bacteria . RESULTS . The ability of granulocytes to phagocytize opsonized E . Coli was adversely affected by both CM used . Compared with baseline values, significantly decreased phagocytic activity was observed for iohexol at 1, 5, and 20 minutes and for ioxaglate at 1 and 5 minutes . The largest decrease with ioxaglate was from 85.3 +/- 10.5 to 69.3 +/- 16.3 (5 minutes), and the largest change with iohexol was from 87.1 +/- 8.5 to 74.5 +/- 15.9 (5 minutes) . CONCLUSION . These results confirm earlier reports that ionic and nonionic CM adversely affect the phagocytic ability of granulocytes after intravenous administration. Environ Mol Mutagen, 1994, 23(2), 110 - 5 Assay by inhibition of repair to measure O6-methylguanine in DNA; Mironov NM et al.; A procedure for measuring the level of O6-methylguanine (O6-meG) in DNA is described . Repair of 32P-oligodeoxynucleotides containing O6-meG adducts by O6-alkylguanine alkyltransferase (AGT) was performed in the presence of different quantities of DNA containing unknown concentrations of O6-meG . Each methylated DNA sample inhibited the repair of oligodeoxynucleotide substrate to an extent dependent upon O6-meG concentration . Each DNA sample tested at different concentrations in the assay therefore had a characteristic inhibition curve and could be compared to the curves generated using reference DNA samples of known O6-meG concentration . We report the method of calculation of the O6-meG level in a given DNA sample by comparison of its inhibition curve with that of reference DNAs . This method of calculation does not require a knowledge of the exact quantity of the labelled substrate or AGT used . The method requires only 0.1-10 micrograms of DNA, with a limit of detection of 0.8 fmol of O6-meG per microgram of DNA. Can J Vet Res, 1994 Jan, 58(1), 67 - 70 Effect of repeated administration of tirilazad mesylate on healthy and endotoxemic calves: a pilot study; Semrad SD et al.; Tirilazad mesylate (TM:U74006F), a nonglucocorticoid 21-aminosteroid (lazaroid), is beneficial in the treatment of experimentally-induced ischemic injury following brain and spinal cord trauma, subarachnoid hemorrhage, hypovolemic shock and endotoxemia . This study investigated the effects of TM following repeated administration in sixteen healthy and endotoxemic calves . Group A calves received TM 3 mg/kg IV; group B calves received Escherichia coli endotoxin in increasing doses (0.1 to 20 micrograms/kg IV); group C calves received TM and endotoxin and group D calves received sterile saline (10 mL) . Endotoxin, TM and saline were given every eight hours for five days . Mild, transient tachypnea was observed following TM administration . The drug suppressed clinical signs of endotoxemia until larger doses of endotoxin were given . At necropsy no substantial lesions were observed in groups A and D . Groups B and C had lesions consistent with endotoxemia but only group C calves had evidence of abomasal and ruminal ulceration . Although TM may be of benefit in the treatment of endotoxemia, further studies are needed to determine the optimal dosage and potential side effects in the endotoxic bovine neonate. Protein Sci, 1994 Jan, 3(1), 70 - 81 Solution structure of villin 14T, a domain conserved among actin-severing proteins; Markus MA et al.; The solution structure of the N-terminal domain of the actin-severing protein villin has been determined by multidimensional heteronuclear resonance spectroscopy . Villin is a member of a family of actin-severing proteins that regulate the organization of actin in the eukaryotic cytoskeleton . Members of this family are built from 3 or 6 homologous repeats of a structural domain of approximately 130 amino acids that is unrelated to any previously known structure . The N-terminal domain of villin (14T) contains a central beta-sheet with 4 antiparallel strands and a fifth parallel strand at one edge . This sheet is sandwiched between 2 helices on one side and a 2-stranded parallel beta-sheet with another helix on the other side . The strongly conserved sequence characteristic of the protein family corresponds to internal hydrophobic residues . Calcium titration experiments suggest that there are 2 binding sites for Ca2+, a stronger site near the N-terminal end of the longest helix, with a Kd of 1.8 +/- 0.4 mM, and a weaker site near the C-terminal end of the same helix, with a Kd of 11 +/- 2 mM . Mutational and biochemical studies of this domain in several members of the family suggest that the actin monomer binding site is near the parallel strand at the edge of the central beta-sheet. Protein Sci, 1994 Jan, 3(1), 22 - 9 Hydrogen bond interactions of G proteins with the guanine ring moiety of guanine nucleotides; Weng G et al.; We have utilized Raman difference spectroscopy to investigate hydrogen bonding interactions of the guanine moiety in guanine nucleotides with the binding site of two G proteins, EF-Tu (elongation factor Tu from Escherichia coli) and the c-Harvey ras protein, p21 (the gene product of the human c-H-ras proto-oncogene) . Raman spectra of proteins complexed with GDP (guanosine 5' diphosphate), IDP (inosine 5' diphosphate), 6-thio-GDP, and 6-18O-GDP were measured, and the various difference spectra were determined . These were compared to the difference spectra obtained in solution, revealing vibrational features of the nucleotide that are altered upon binding . Specifically, we observed significant frequency shifts in the vibrational modes associated with the 6-keto and 2-amino positions of the guanine group of GDP and IDP that result from hydrogen bonding interactions between these groups and the two proteins . These shifts are interpreted as being proportional to the local energy of interaction (delta H) between the two groups and protein residues at the nucleotide binding site . Consistent with the tight binding between the nucleotides and the two proteins, the shifts indicate that the enthalpic interactions are stronger between these two polar groups and protein than with water . In general, the spectral shifts provide a rationale for the stronger binding of GDP and IDP with p21 compared to EF-Tu . Despite the structural similarity of the binding sites of EF-Tu and p21, the strengths of the observed hydrogen bonds at the 6-keto and 2-amino positions vary substantially, by up to a factor of 2.(ABSTRACT TRUNCATED AT 250 WORDS) Protein Sci, 1994 Jan, 3(1), 132 - 8 Construction of an overproduction vector containing the novel srp (sterically repressed) promoter; Ezaz-Nikpay K et al.; We report the design, synthesis, and evaluation of a novel Escherichia coli promoter intended for use in overproduction of proteins that are deleterious to the host . In this sterically repressed promoter (srp), the lac operator site is positioned between the -10 and -35 elements, where it can interfere sterically with RNA polymerase and thereby prevent assembly of a poised transcriptional complex . An srp-containing phagemid, pKEN1, and a tac-containing phagemid, pHN1, which has been widely used in protein overproduction but is often unstable, are compared with respect to levels of uninduced and induced protein expression . The level of uninduced protein synthesis by the srp promoter in vivo is approximately 50% of that observed with tac, whereas the levels of induced protein synthesis with the 2 vectors are approximately equal . A remarkable increase in stability of overproduction and growth was observed when the toxic Ada protein was overproduced in pKEN1, demonstrating the potential utility of this vector in overproducing toxic proteins. Protein Sci, 1994 Jan, 3(1), 109 - 17 On the rate of proton exchange with solvent of the catalytic histidine in flavocytochrome b2 (yeast L-lactate dehydrogenase); Balme A et al.; The family of FMN-dependent, alpha-hydroxy acid-oxidizing enzymes catalyzes substrate dehydrogenation by a mechanism the first step of which is abstraction of the substrate alpha-proton (so-called carbanion mechanism) . For flavocytochrome b2 and lactate oxidase, it was shown that once on the enzyme this proton is lost only slowly to the solvent (Lederer F, 1984, In: Bray RC, Engel PC, Mayhew SG, eds, Flavins & flavoproteins, Berlin: Walter de Gruyter & Co., pp 513-526; Urban P, Lederer F, 1985, J Biol Chem 260:11115-11122) . This suggested the occurrence of a pKa increase of the catalytic histidine upon enzyme reduction by substrate . For flavocytochrome b2, the crystal structure indicated 2 possible origins for the stabilization of the imidazolium form of His 373: either a network of hydrogen bonds involving His 373, Tyr 254, flavin N5 and O4, a heme propionate, and solvent molecules, and/or electrostatic interactions with Asp 282 and with the reduced cofactor N1 anion . In this work, we probe the effect of the hydrogen bond network at the active site by studying proton exchange with solvent for 2 mutants: Y254F and the recombinant flavodehydrogenase domain, in which this network should be disrupted . The rate of proton exchange, as determined by intermolecular hydrogen transfer experiments, appears identical in the flavodehydrogenase domain and the wild-type enzyme, whereas it is about 3-fold faster in the Y254F mutant . It thus appears that specific hydrogen bonds to the solvent do not play a major role in stabilizing the acid form of His 373 in reduced flavocytochrome b2 . Removal of the Y254 phenol group induces a pKa drop of about half a pH unit for His 373 in the reduced enzyme . Even then, the rate of exchange of the imidazolium proton with solvent is still lower by several orders of magnitude than that of a normally ionizing histidine . Other factors must then also contribute to the pKa increase, such as the electrostatic interactions with D282 and the anionic reduced cofactor, as suggested by the crystal structure. Protein Sci, 1994 Jan, 3(1), 103 - 8 Kinetics and thermodynamics of thermal denaturation in acyl carrier protein; Horvath LA et al.; The denaturation of Escherichia coli acyl carrier protein (ACP) in buffers containing both monovalent and divalent cations was followed by variable-temperature NMR and differential scanning calorimetry . Both high concentrations of monovalent salts (Na+) and moderate concentrations of divalent salts (Ca2+) raise the denaturation temperature, but calorimetry indicates that a significant increase in the enthalpy of denaturation is obtained only with the addition of a divalent salt . NMR experiments in both low ionic strength monovalent buffers and low ionic strength monovalent buffers containing calcium ions show exchange between native and denatured forms to be slow on the NMR time scale . However, in high ionic strength monovalent buffers, where the temperature of denaturation is elevated as it is in the presence of Ca2+, the transition is fast on the NMR time scale . These results suggest that monovalent and divalent cations may act to stabilize ACP in different ways . Monovalent ions may nonspecifically balance the intrinsic negative charge of this protein in a way that is similar for native, denatured, and intermediate forms . Divalent cations provide stability by binding to specific sites present only in the native state. Bioessays, 1994 Jan, 16(1), 13 - 22 DNA helicases: enzymes with essential roles in all aspects of DNA metabolism; Matson SW et al.; DNA helicases catalyze the disruption of the hydrogen bonds that hold the two strands of double-stranded DNA together . This energy-requiring unwinding reaction results in the formation of the single-stranded DNA required as a template or reaction intermediate in DNA replication, repair and recombination . A combination of biochemical and genetic studies have been used to probe and define the roles of the multiple DNA helicases found in E . coli . This work and similar efforts in eukaryotic cells, although far from complete, have established that DNA helicases are essential components of the machinery that interacts with the DNA molecule. Acta Anaesthesiol Scand, 1994 Jan, 38(1), 33 - 9 Amelioration of respiratory and circulatory changes in established endotoxic shock by ketoprofen; Sigurdsson GH et al.; In an intensive-care setting we studied the effects of ketoprofen, a dual inhibitor of cyclooxygenase and lipoxygenase, on circulatory and respiratory changes during established endotoxic shock in sheep . Two groups (n = 7 in each) were exposed to E . coli endotoxin, which caused a sharp increase in pulmonary artery pressure (200%; PAP), intrapulmonary shunt fraction (300%; QS/QT%), and oxygen extraction ratio (50%; VO2/DO2%) . There was also a significant decrease in mean arterial pressure (25%; MAP), respiratory compliance (60%; CT), arterial oxygen tension (65%; PaO2), and oxygen delivery index (15%; DO2) in both groups . After 30 min of endotoxin infusion, group K received ketoprofen, 2.5 mg/kg b.w . i.v., while group E served as shock controls . After 4 h there had been a significant improvement in MAP, PaO2, DO2, QS/QT%, and CT in the ketoprofen-treated group compared with the controls (P < 0.01) . In addition, the oxygen extraction ratio normalised in group K, but remained 70-100% increased in group E (P < 0.01) . The wet-to-dry weight ratios of the lungs and the liver were significantly lower in the ketoprofen-treated group compared with the controls (P < 0.05) . It was concluded that ketoprofen significantly ameliorated the respiratory and circulatory effects of established endotoxic shock in sheep. Urol Int, 1994, 52(1), 45 - 7 Seminal vesicle abscess: two case reports and a review of the literature; Gutierrez R et al.; Two new cases of seminal vesicle abscess diagnosed by computerized tomography and treated with percutaneous drainage are reported . A review of the 8 cases published so far is also presented. Trends Biochem Sci, 1994 Jan, 19(1), 38 - 42 Repression versus activation in the control of gene transcription; Cowell IG; Studies on the regulation of transcription often focus on mechanisms of transcriptional activation . However, transcriptional repression is also an important factor in the regulation of many genes . Transcription of specific genes can be downregulated in various ways, and examination of a number of different systems has revealed that most or all steps required for transcriptional activation can be interfered with by transcriptional repressors. Protein Eng, 1994 Jan, 7(1), 83 - 9 Herpes thymidine kinase mutants with altered catalytic efficiencies obtained by random sequence selection; Munir KM et al.; We have obtained 190 active Herpes simplex virus type 1 thymidine kinase mutants by substituting a 33 nucleotide sequence with 20% degeneracy for a portion of the nucleotide sequence that encodes the putative thymidine binding site {K.M . Munir, D.C . French, D.K . Dube and L.A . Loeb (1992) J . Biol . Chem., 167, 6584-6589} . In order to classify these mutants with respect to thymidine kinase activity we determined the ability of Escherichia coli harboring these mutants to form colonies in the presence of varying concentrations of thymidine . Escherichia coli harboring one of the mutant enzymes was able to form colonies at a concentration of thymidine lower than did the wild type . It was able to phosphorylate thymidine more rapidly than the wild type both in vivo and in vitro . The increased thymidine kinase activity was manifested by (i) a 42% enhanced uptake of {methyl-3H}thymidine into E . coli, (ii) a 2.4 times higher rate of {methyl-3H}thymidine incorporation into acid-insoluble material and (iii) a 5-fold increase in the kcat of the purified enzyme compared to the wild type . Herpes thymidine kinase purified from other mutants that formed colonies at higher thymidine concentrations than that of the wild type exhibited a decrease in kcat . The kcat of one of these mutant thymidine kinases was 10(-4) of that of the wild type enzyme . This study demonstrates that a spectrum of mutant enzymes with different catalytic properties can be obtained by selection from a plasmid with random sequence substitutions and this can be done in the absence of rational protein design. Protein Eng, 1994 Jan, 7(1), 125 - 30 Cloning, expression and purification of a sarcoplasmic calcium-binding protein from the sandworm Nereis diversicolor via a fusion product with chloramphenicol acetyltransferase; Dekeyzer N et al.; A gene coding for the Nereis sarcoplasmic calcium-binding protein (NSCP) was synthesized and expressed in Escherichia coli . The sequence of the gene was derived from the protein sequence by reverse translation . It possesses a number of unique, regularly spaced, restriction endonuclease cleavage sites to facilitate future site-directed mutagenesis . For the cloning strategy the gene sequence was divided into four parts . Three parts were cloned by ligation of hybridized oligomers and one part by inverse PCR . The protein was expressed as a fusion protein with the bacterial chloramphenicol acetyl-transferase (CAT), which could be easily purified by affinity chromatography . At the junction of the CAT and NSCP moieties a recognition site for the proteolytic enzyme factor Xa was built in . However, the distance between the moieties appeared to be crucial to warrant cleavage . A kinetic analysis showed that NSCP prepared from the sandworm and the one expressed by E . coli behaved in the same way . This system provides a basis for site-specific mutagenesis studies, in order to elucidate the molecular mechanism of cation binding and concomitant conformational changes. Protein Eng, 1994 Jan, 7(1), 109 - 15 A calmodulin-target peptide hybrid molecule with unique calcium-binding properties; Porumb T et al.; This paper describes the production and properties of a hybrid protein comprising the full length of the Xenopus laevis calmodulin (CaM) sequence, followed, through a glycylglycine linker, by the 26-residue CaM-binding region of myosin light-chain kinase (M13) . This hybrid molecule appears to have high thermal stability (Tm > 75 degrees C in the presence of Ca2+) as well as unusual Ca(2+)-binding properties: (i) a wide-range biphasic Ca(2+)-binding response (extending over pCa 4.8-7.4) and (ii) a high apparent binding constant (pCa50% = 6.3, a 10-fold increase from that of wild-type CaM) . NMR and CD data indicate that the CaM-M13 hybrid molecule exists in equilibrium in an approximate 1:1 ratio between two major conformations, one of which is similar to the compact globular structure of the CaM-M13 complex {M.Ikura, G.M . Clore, A.M . Gronenborn, G . Zhu, C.B . Klee and A . Bax (1992) Science, 256, 632-638} and the other to the dumb-bell-like structure of the wild type CaM {Y.S . Babu, C.E . Bugg and W.J . Cook (1988) J . Mol . Biol., 204, 191-204} . The biphasic Ca(2+)-binding curve can be interpreted using a linear combination of two Hill binding curves with significantly different dissociation constants (2 x 10(-9) M and 8 x 10(-8) M), which can be attributed to the two conformations in equilibrium . The present study has opened an avenue to engineer proteins with higher Ca(2+)-binding affinities using the known CaM structures as a template. Genetics, 1994 Jan, 136(1), 403 - 16 Statistical approaches for analyzing mutational spectra: some recommendations for categorical data; Piegorsch WW et al.; In studies examining the patterns or spectra of mutational damage, the primary variables of interest are expressed typically as discrete counts within defined categories of damage . Various statistical methods can be applied to test for heterogeneity among the observed spectra of different classes, treatment groups and/or doses of a mutagen . These are described and compared via computer simulations to determine which are most appropriate for practical use in the evaluation of spectral data . Our results suggest that selected, simple modifications of the usual Pearson X2 statistic for contingency tables provide stable false positive error rates near the usual alpha = 0.05 level and also acceptable sensitivity to detect differences among spectra . Extensions to the problem of identifying individual differences within and among mutant spectra are noted. Eur Urol, 1994, 25(2), 171 - 3 Secondary psoas abscess twenty-seven years after nephrectomy; Guillaume MP et al.; Abscesses of the psoas muscle are due to a hematogenous dissemination, to the spread of infection from adjacent intestinal structures, to osteomyelitis of the spine or to tuberculous infection of a disc space . In contrast, psoas abscesses related to the urological tract have only been described on exception . The present report focuses on a right psoas abscess which developed 27 years after a nephrectomy . The infectious process resulted from the spread of an acute vesical infection through the residual ureter . Analysis of 4 other cases reported in the literature allows us to delineate the clinical features of psoas abscesses of urological origin. J Steroid Biochem Mol Biol, 1994 Jan, 48(1), 61 - 8 Immunolocalization of retinoic acid receptors in rat, mouse and human ovary and uterus; Zhuang YH et al.; We raised an antibody against a synthetic peptide corresponding to amino acids 155-174 of human retinoic acid receptor alpha (RAR-alpha) . The sequence is highly homologous in all RARs and their isoforms . When mouse and human RARs (alpha, beta and gamma) expressed in Cos cell were analysed with immunoblot, all receptors gave a specific 51 K signal . Mouse RAR-gamma gave an additional signal corresponding to 58 K . In human teratocarcinoma cells (F9) both 51 and 58K molecule sizes were detected . The RAR expression in F9 cells was slightly down-regulated in charcoal-stripped culture medium and returned to normal level after retinoic acid treatment . The 51 K protein was found in all ovarian and uterine samples, but the quantity of the 58 K protein varied in different species and organs, being highest in the mouse uterus and the rat and human ovary . Using immunohistochemistry the RARs were found in the nuclear compartment . In the rat uterus, positive immunoreaction was found mainly in the nuclei of epithelial, uterine glandular and stromal cells . In the rat ovary, positive reaction was found in the nuclei of germinal epithelial, follicular and stromal cells. Arzneimittelforschung, 1994 Jan, 44(1), 102 - 8 Escherichia coli as a biologically active ingredient of suppositories . Solid phase extraction and quantification in a double antibody ELISA; Barthold E et al.; Techniques for isolation and quantification of an active ingredient of biological origin from a pharmaceutical product (Posterisan suppositories) were developed . By means of ELISA (enzyme-linked immunosorbent assay) in connection with a computerized evaluation, the antigenic material, a bacterial culture suspension (BCS) of Escherichia coli as a raw material was shown to be specifically and reproducibly detectable and quantifiable . As a limit of detection, a bacteria concentration of 6 x 10(4) cells/ml was determined, corresponding to 0.02% of the concentration in the product . After treating of the suppositories with organic solvents, the E . coli antigens were extracted with silica columns . The complete validations of both methods, the ELISA itself and the extraction procedure of the antigens from the matrix, in accordance with pharmaceutically accepted principles are presented . The eventual application of the new technique to the analysis of other pharmaceuticals with similar ingredients as well as the possibility of substituting conventional methods like total cell counting is discussed. Anal Biochem, 1994 Jan, 216(1), 67 - 76 A study of protein secondary structure by Fourier transform infrared/photoacoustic spectroscopy and its application for recombinant proteins; Luo S et al.; FT-IR/PAS (Fourier transform infrared/photoacoustic spectroscopy) was used to evaluate the secondary structure of proteins . Four well-studied proteins, concanavalin A, hemoglobin, lysozyme, and trypsin, which have different distributions of secondary structures, were used for assignments of the infrared bands and evaluating the accuracy of FT-IR/PAS methods . Secondary structure contents estimated from FT-IR/PAS and other physical methods (e.g., X-ray diffraction, CD, and traditional FT-IR) show good agreement . In addition, the secondary structure can be evaluated with as little as 0.5 micrograms of protein (concanavalin A), suggesting that FT-IR/PAS is a sensitive and useful technique that could be applied to studies of the folding of recombinant and mutant proteins where only small amounts of material are available . Recombinant phosphorylase kinase gamma 1-300 subunit expressed in Escherichia coli was found in the inclusion bodies . We found that renatured phosphorylase kinase gamma 1-300 subunit has two kinase forms: one has a 10-fold higher activity than the other one . Both fractions, however, are the same as judged from sodium dodecylsulfate-polyacrylamide gel electrophoresis . Differences in conformation were demonstrated by using the FT-IR/PAS method, which showed that the low-activity form has more beta-sheet structure than the form with high activity . Analysis of these kinase forms by CD confirms the interpretation made by the FT-IR/PAS method. Anal Biochem, 1994 Jan, 216(1), 47 - 51 Separation of inner and outer membrane vesicles from Escherichia coli in self-generating Percoll gradients; Morein S et al.; A rapid and simple method for separating and isolating the inner and outer membranes of Escherichia coli is described . Membrane vesicles were prepared either by passing the bacteria through a French press or by conversion of the cells to spheroplasts by the lysozyme-EDTA treatment and disruption of the spheroplasts by sonication . The membrane vesicles were collected by ultracentrifugation and suspended in a Percoll-containing buffer . The membranes were separated by centrifugation of the membrane-Percoll mixture in a fixed angle rotor at 27,000gmax for 30 min in a preparative centrifuge . One low-density and one high-density band was obtained, corresponding to the inner and outer membranes, respectively . For the membranes prepared by French pressing 69 and 3.3% of the total activity in the gradient of the inner membrane marker NADH-oxidase was found in the low-density and the high-density bands, respectively . For the outer membrane marker 2-keto-3-deoxyoctonate (KDO), 69 and 7.3% of the total amount of KDO in the gradient was found in the high-density and the low-density bands, respectively . For the membranes prepared by sonication of spheroplasts the same figures were 39 and 6.5% for NADH-oxidase and 52 and 9.0% for KDO . The total time of preparation of membrane vesicles, from harvesting the bacteria to the separation of the inner and outer membrane vesicles, is about 6 h . A good separation of the inner and outer membranes was still obtained when samples corresponding to about 10 mg of membrane protein were added to a 33-ml gradient. Plant J, 1994 Jan, 5(1), 111 - 22 Characterization of two cDNAs that encode MAP kinase homologues in Arabidopsis thaliana and analysis of the possible role of auxin in activating such kinase activities in cultured cells; Mizoguchi T et al.; Two cDNA clones, cATMPK1 and cATMPK2, encoding MAP kinases (mitogen-activated protein kinases) have been cloned from Arabidopsis thaliana and their nucleotide sequences have been determined . Putative proteins encoded by ATMPK1 and ATMPK2 genes, designated ATMPK1 and ATMPK2, contain 370 and 376 amino acid residues, respectively, and are 88.7% identical at the amino acid sequence level . ATMPK1 and ATMPK2 exhibit significant similarity to rat ERK2 (49%) and Xenopus MAP kinase (50%) . The amino acid residues corresponding to the sites of phosphorylation (Thr-Glu-Tyr) that are involved in the activation of MAP kinases are conserved in ATMPK1 and ATMPK2 . Northern blot analysis indicates that the ATMPK1 and ATMPK2 mRNAs are significantly present in all the organs except seeds . Genomic Southern blot analysis suggests that there are a few additional genes that are related to ATMPK1 and ATMPK2 in the Arabidopsis genome . Purified Xenopus MAP kinase kinase (MAPK kinase) phosphorylates ATMPK1 and ATMPK2 proteins that have been expressed in Escherichia coli, activating these enzymes . A rapid and transient activation of 46-kDa protein kinase activity that phosphorylated myelin basic protein (MBP) was detected when auxin-starved tobacco BY-2 cells were treated with synthetic auxin, 2,4-dichlorophenoxyacetic acid (2,4-D) . Protein kinase activities which phosphorylated the recombinant ATMPK2 protein also increased rapidly after auxin treatment in the auxin-starved BY-2 cells . These results suggest that auxin may function as an activator of plant MAP kinase homologues, as do various mitogens in animal systems. J Biomol NMR, 1994 Jan, 4(1), 35 - 46 Application of neural networks to automated assignment of NMR spectra of proteins; Hare BJ et al.; Simulated neural networks are described which aid the assignment of protein NMR spectra . A network trained to recognize amino acid type from TOCSY data was trained on 148 assigned spin systems from E . coli acyl carrier proteins (ACPs) and tested on spin systems from spinach ACP, which has a 37% sequence homology with E . coli ACP and a similar secondary structure . The output unit corresponding to the correct amino acid is one of the four most activated units in 83% of the spin systems tested . The utility of this information is illustrated by a second network which uses a constraint satisfaction algorithm to find the best fit of the spin systems to the amino acid sequence . Application to a stretch of 20 amino acids in spinach ACP results in 75% correct sequential assignment . Since the output of the amino acid type identification network can be coupled with a variety of sequential assignment strategies, the approach offers substantial potential for expediting assignment of protein NMR spectra. Arch Virol, 1994, 134(3-4), 345 - 56 Detection of antibodies to caprine arthritis-encephalitis virus using recombinant gag proteins; Rimstad E et al.; The coding sequences of the core proteins p17 and p28 of caprine arthritis-encephalitis virus (CAEV) were amplified using the polymerase chain reaction and cloned into the plasmid expression vector p-GEX-2T . Both p17 and p28 were expressed as fusion proteins with glutathione S-transferase . The recombinant proteins were affinity purified from induced bacterial lysates using glutathione-agarose beads . The purified proteins were used in an enzyme-linked immunosorbent assay (ELISA) to detect antibodies against CAEV in goat sera and milk samples . Three different ELISA tests were developed based on p17, p28 or the combination of these two recombinant proteins (p17 + p28) . A comparison was made to an ELISA based on purified whole virus particles and to agar immunodiffusion test (AGID) . Sera with conflicting results in the different ELISA tests were examined by Western blotting . There was a high correlation between the ELISA tests based on p17 + p28 recombinant proteins and whole virus ELISA, with an estimated kappa value of 0.92 . Only 72-75% of the sera that tested positive in these two ELISA tests were positive in AGID . Antibodies to CAEV were detected in significantly more animals when serum samples were tested compared to milk samples . Based on the time and materials required to prepare the reagents, the recombinant based ELISA test was less expensive than the whole virus ELISA. Environ Mol Mutagen, 1994, 23(1), 17 - 31 Sources of variability in data from a lacI transgenic mouse mutation assay; Piegorsch WW et al.; Experimental features of a transgenic mouse mutation assay based on a lacI target transgene from Escherichia coli are considered in detail . Sources of variability in the experimental protocol that can affect the statistical nature of the observations are examined with the goal of identifying sources of excess variation in the observed mutant fractions . The sources include plate-to-plate (within packages), package-to-package (within animals), and animal-to-animal (within study) variability . Data from two laboratories are evaluated, using various statistical methods to identify excess variability . Results suggest only scattered patterns of excess variability, except possibly in those cases where genomic DNA from test animals is stored for extended periods (e.g., > 90 days) after isolation from tissues . Further study is encouraged to examine the validity and implications of this time/storage-related effect. Cell Growth Differ, 1994 Jan, 5(1), 87 - 93 Human immunodeficiency virus 1 Tat stimulates transcription of the transforming growth factor alpha gene in an epidermal growth factor-dependent manner; Nabell LM et al.; The human immunodeficiency virus 1 (HIV-1) tat gene encodes a protein of critical importance for viral transcription . In addition, Tat has been shown capable of entering cells, stimulating cell proliferation, and altering host cell gene expression . We examined the effect of Tat on the expression of the transforming growth factor alpha (TGF-alpha) gene in MDA468 human breast carcinoma cells . We showed that these cells were capable of supporting the activation of the HIV-1 long terminal repeat by Tat . Then, in cotransfection assays, in which the TGF-alpha promoter was linked to a luciferase reporter gene and the tat gene was expressed under the control of the SV40 early promoter, we showed that tat gene expression increased TGF-alpha-luciferase reporter function but only in cells stimulated with epidermal growth factor (EGF) . The effects of tat and EGF were dose dependent . To confirm these cotransfection data, Tat was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase (GST) and purified on glutathione-agarose . GST-Tat was introduced into the MDA468 cells either in the presence of chloroquine or by scrape loading . The biological activity of GST-Tat was tested on cells that had been stablely transfected with the HIV-1 long terminal repeat linked to luciferase as a reporter . GST-Tat was then introduced into the cells, and the level of TGF-alpha mRNA was determined.(ABSTRACT TRUNCATED AT 250 WORDS) Hum Mutat, 1994, 3(1), 19 - 24 Enzymatic amplification of synthetic oligodeoxyribonucleotides: implications for triplet repeat expansions in the human genome; Behn-Krappa A et al.; The triplet repeat sequences (CGG)n, (GCT)n, and (CAG)n, which naturally occur in the human genome, can be autonomously expanded in human DNA by an as yet unknown mechanism . These in part excessive expansions have been causally related to human genetic diseases, the fragile X (Martin-Bell) syndrome, to myotonic dystrophy (Curschmann-Steinert), to spinal and bulbar muscular atrophy (Kennedy disease), and recently to Huntington disease . A GCC trinucleotide repeat was found to be expanded and methylated in the fragile site FRAXE on the human X chromosome . These findings were associated with mental retardation (Knight et al., 1993) . In spinocerebellar ataxia type 1 (SCA1), a polymorphic CAG repeat was found to be unstable and expanded in individuals with that disease (Orr et al., 1993) . We have demonstrated in in vitro experiments that the synthetic oligodeoxyribonucleotides (CGG)17, (CGG)12, (GCC)17, (CG)25, (CTG)17, or (CAG)17 plus (GTC)17, in the absence of added natural DNA, can be expanded with Taq polymerase in the polymerase chain reaction (PCR) . Some expansion can already be detected after 4 PCR cycles . The E . coli Klenow DNA polymerase also functions in a similar amplification and expansion reaction performed at 37 degrees C without cycling . Other oligodeoxyribonucleotides, like, (CGG)7, (CGGT)13, or (TAA)17, are devoid of this property or have very low activity . The cytidine-methylated polymers (GCC)17 or (CG)25 yield expansion products of considerably reduced chain lengths . The expansion of the polymer (CGG)17 is affected by cytidine methylation to a lesser degree . A specific sequence and/or secondary structure and high CG content appear to be requirements for this expansion reaction by a possible slippage mechanism.(ABSTRACT TRUNCATED AT 250 WORDS) Biokhimiia, 1994 Jan, 59(1), 113 - 7 {Kinetics of luciferase gene expression from fireflies Photinus pyralis and Luciola mingrelica in Escherichia coli cells . II . The role of pH in the biosynthesis of proteins and their transformation into active conformers}; Leont'eva OV et al.; The Photinus pyralis and Luciola mingrelica luciferases genes expression kinetics has been studied, repectively, on the pME61 and pJG lambda plasmids with a thermoinducible lambda PR promoter in the E . coli strain CA under conditions of a pH shift . An increase in the enzyme activity after a pH shift has been revealed . The maximal amount of the firefly Ph . pyralis luciferase reaches 5.3%, while the maximal amount of the L . mingrelica enzyme reaches 4.9% of the total amount of cellular proteins in case of pHout shift to 9.0 according to the SDS gel electrophoresis data; that in case of a pHout shift to 8.5 is 4.9% and 4.5%, respectively . The enzyme activation correlates well with the dynamics of pHout and pHin. Biokhimiia, 1994 Jan, 59(1), 102 - 12 {Kinetics of luciferase gene expression from fireflies Photinus pyralis and Luciola mingrelica in Escherichia coli cells . I . Kinetics of the transformation of recombinant luciferase into enzymatically-active conformers}; Kutuzova GD et al.; The Photinus pyralis and Luciola mingrelica luciferases genes expression has been studied on the pME61 or pJG lambda plasmids with a thermoinducible lambda Pr promoter in the E . coli strain CA using three independent methods: SDS gel electrophoresis to quantify the synthesized luciferase protein, EIA to quantify the enzyme native conformer and activity measurements . The cultures were incubated by the temperature schemes 28 degrees-42 degrees-21 degrees C and 28 degrees-21 degrees C . The maximal amount of the Ph . pyralis protein reached 4.5%, while that of L . mingrelica luciferase was 4.1% of the total amount of the cellular proteins after 10-hr incubation . The amount of the native conformer and the luciferase activity began to grow after a long induction period, reaching the maximal level by hour 50-60 after the thermoinduction . The increase in the enzyme activity correlated well with the increase in the ATP content in the cell . The observed "low-temperature induction" of the enzyme activity is interpreted as a protein transition to an active conformation . This transformation is considerably behind the protein biosynthesis process . Intracellular metabolic reactions have been shown to play an active part in these conformational changes. Am J Ind Med, 1994 Jan, 25(1), 103 - 4 Lipopolysaccharide (LPS) inhalation in healthy subjects causes bronchoalveolar neutrophilia, lymphocytosis, and fibronectin increase; Sandstrom T et al.; Eight healthy volunteers were exposed to purified lipopolysaccharide (LPS) by controlled inhalation . Bronchoalveolar lavage (BAL) 3 hours after exposure revealed a pronounced neutrophilia, increase in lymphocytes, fibronectin concentration, and decrease in alveolar macrophage phagocytosis, as compared to a reference BAL. J Gen Virol, 1994 Jan, 75 ( Pt 1), 239 - 42 Unusual amino-terminal sequence repeat characterizes the capsid protein of dasheen mosaic potyvirus; Pappu SS et al.; The 3'-terminal region of a Florida isolate of dasheen mosaic potyvirus (DMV-LA) genome including the coat protein (CP) gene was cloned and sequenced . Protease digestion was predicted to occur between the glutamine and alanine residues at positions 79 and 80 of the 408 residue long polypeptide to produce a CP of 329 amino acids with an estimated M(r) of 36,229 . Following the putative protease recognition site is a DAG sequence, which is conserved among aphid-transmitted potyviral CPs . There is an unusual and unique stretch of 52 amino acids after the DAG that is repetitive and rich in threonine and asparagine . A sequence of 10 residues (GNNTNTNTSNT) was repeated three times in tandem within this stretch and was followed by six proline residues . Several potential glycosylation sites were found clustered within this region . Expression in Escherichia coli and Western blotting of the CP confirmed its size and serological identity . Sequence comparisons and phylogenetic reconstructions indicated that DMV is a distinct potyvirus within the passionfruit woodiness virus subgroup cluster. Am J Respir Crit Care Med, 1994 Jan, 149(1), 128 - 33 Effects of capsaicin on the airway responses to inhaled endotoxin in the guinea pig; Jarreau PH et al.; Inhalation of lipopolysaccharide (LPS) has been associated with increased airway responsiveness and inflammation both in humans and in animals . To investigate the contribution of capsaicin-sensitive nerves to these changes, we compared airway responsiveness and inflammation after intratracheal administration of 10 micrograms/kg LPS (Escherichia coli O55:B5 lipopolysaccharide) or saline in guinea pigs treated 10 days previously with 50 mg/kg capsaicin and in those pretreated with the capsaicin vehicle . Four hours after LPS, airway responsiveness and cell counts in the bronchoalveolar lavage were assessed . To determine airway responsiveness, guinea pigs were anesthetized, tracheotomized, and mechanically ventilated before exposure to increasing concentrations of aerosolized histamine (10(-4) to 10(-3) M) . Capsaicin pretreatment prevented the LPS-induced increase in airway responsiveness in response to aerosolized histamine . It significantly reduced total cell recovery in the bronchoalveolar lavage after LPS (1,167 +/- 167 10(3) cells/ml in capsaicin-treated guinea pigs versus 2,171 +/- 184 10(3) in vehicle-treated guinea pigs) by reducing the LPS-induced influx of neutrophils and macrophages . Additional experiments demonstrated that the activity of neutral endopeptidase (NEP) in the tracheal epithelium was not significantly different in guinea pigs injected with LPS from that in the saline-treated control animals, and that the pretreatment with the NEP inhibitor phosphoramidon did not increase the LPS-induced influx of neutrophils into the bronchoalveolar lavage . These results demonstrate that in the guinea pig, capsaicin-sensitive nerves are involved in LPS-induced airway hyperresponsiveness and inflammation. Int Arch Allergy Immunol, 1994, 103(3), 307 - 10 Inhibition of enteropathogenic Escherichia coli (EPEC) adhesion to HeLa cells by human colostrum: detection of specific sIgA related to EPEC outer-membrane proteins; Camara LM et al.; Human colostrum and a high molecular weight colostrum fraction (HMWF; > 14,000 D) prevented the adhesion of localized adherent (LA+) O111:H-enteropathogenic Escherichia coli (EPEC) to HeLa cells . This effect was abolished after absorption with an O111:H-LA + EPEC strain, but absorption with a LA- strain of same serotype had no effect on the process . A low molecular weight fraction (< 14,000 D), absorbed or not with LA+ or LA- bacterial strains, did not inhibit the adherence of E . coli to HeLa cells . IgA-depleted colostrum had no inhibitory effect on bacterial adhesion, demonstrating the critical role of this protein in the phenomenon . Heat inactivation of whole colostrum did not significantly modify the inhibition adherence levels . Immunoblots of O111:H-LA+ strain outer-membrane complex reacted with colostrum and HMWF showing that IgA antibodies were predominantly reactive with a 94-kD protein . These data confirm and extend observations about colostrum sIgA participation in adhesion inhibition of EPEC to HeLa cells and its response to a 94-kD outer-membrane protein. Plant Mol Biol, 1994 Jan, 24(2), 381 - 8 Control of gene expression in plant cells using a 434:VP16 chimeric protein; Wilde RJ et al.; The herpes simplex virus type 1 VP16 polypeptide is a potent trans-activator of viral gene expression . We have tested the ability of the VP16 activation domain to activate gene expression in plant cells . A plasmid encoding a translational fusion between the full-length 434 repressor and the C-terminal 80 amino acids of VP16, was constructed . When expressed in Escherichia coli, the chimeric protein binds efficiently to 434-binding motifs (operators) . For expression in plant cells, the chimeric activator gene was placed between the cauliflower mosaic virus (CaMV) 35S promoter and nos terminator sequences in a pUC-based plasmid . The 434 operators were placed upstream of a minimal CaMV 35S promoter linked to the E . coli gus reporter gene . This reporter-expression cassette was then incorporated into the same plasmid as the 434 cI/VP16 activator-expression cassette . Two control plasmids were also constructed, one encoding the 434 protein with no activator domain and the second a chimeric activator with no DNA-binding domain . The chimeric activator was tested for its ability to activate gene expression in a tobacco protoplast transient assay system . Results are presented to show that we can obtain in plant cells significant activation of gene expression that is dependent on both DNA-binding and the presence of the activator domain. Plant Mol Biol, 1994 Jan, 24(2), 369 - 79 A novel carotenoid biosynthesis gene coding for zeta-carotene desaturase: functional expression, sequence and phylogenetic origin; Linden H et al.; A DNA fragment which has been isolated previously from an Anabaena DNA expression library was subcloned . The corresponding protein was overexpressed in Escherichia coli . The recombinant enzyme was fully active in converting zeta-carotene into lycopene in vitro with neurosporene as an intermediate . A smaller fragment which still contained the active enzyme was sequenced . An open reading frame of 1497 bp was found coding for a protein consisting of 499 amino acids with the calculated molecular weight of 56,740 . In a computer search of nucleotide sequences contained in the EMBL nucleotide sequence library, all the best-fitting comparisons were carotenoid desaturases . The highest similarity was found with the crtI phytoene desaturase genes of bacteria and the al-1 gene from Neurospora crassa . A much lower similarity was found with the pds genes coding for phytoene desaturase from cyanobacteria and higher plants . It is shown in protein similarity plots that the amino acid similarity of zeta-carotene desaturase to the latter is mainly limited to the N terminus of the polypeptides . In contrast, the protein similarity plots and a comparison of a conserved region clearly demonstrate that there is a strong relationship between zeta-carotene desaturase and the phytoene desaturases from various bacteria and fungi . Therefore we propose that the zeta-carotene desaturase gene is homologous to the crt I phytoene desaturase genes of bacteria and fungi. Plant Mol Biol, 1994 Jan, 24(1), 253 - 7 The gene for ribosomal protein L27 is located on the plastid rather than the nuclear genome of the chlorophyll c-containing alga Pleurochrysis carterae; Fujiwara S et al.; The gene for ribosomal protein L27 (rpl27) has not been found in plastid genomes . We report here that the rpl27 gene is located in the plastid genome of the prymnesiophyte Pleurochrysis carterae . The deduced amino acid sequence showed 59% identity with E . coli L27 . 1.0 kb transcript of the gene was detected by Northern blot analysis . Nucleotide sequence analysis of PCR products suggested that rpl27 is widespread in the genomes of Prymesiophyta and Rhodophyta . In all species of Prymnesiophyta examined in this study, the gene is located at the 3' downstream region of Rubisco operon. Nephron, 1994, 66(1), 21 - 8 Verotoxin-binding in human renal sections; Lingwood CA; Gastrointestinal infection with verotoxin-producing Escherichia coli (VTEC) has been strongly implicated in the etiology of the hemolytic uremic syndrome (HUS), the leading cause of pediatric acute renal failure . The binding of fluorescein-conjugated VT1 overlaid on to frozen human renal sections has been examined . Sections from biopsies of infants aged < 2 years were compared with those from adult autopsies . VT primarily stained distal convoluted tubules, particularly those adjacent to glomeruli, and collecting ducts . VT-binding was detected within the infant glomerulus but not the adult . Binding of the toxin was removed when the section was pretreated with alpha-galactosidase, confirming the receptor-binding specificity for globotriaosyl ceramide (gal alpha 1-4gal beta 1-4 glucosylceramide), the glycolipid receptor for VT . These studies may suggest that differential localization of this glycolipid in the pediatric renal glomerulus is a risk factor for the development of HUS following infection with VTEC. J Comp Neurol, 1994 Jan 1, 339(1), 3 - 11 Comparative efficacy of expression of genes delivered to mouse sensory neurons with herpes virus vectors; Davar G et al.; To achieve gene delivery to sensory neurons of the trigeminal ganglion, thymidine kinase-negative (TK-) herpes simplex viruses (HSV) containing the reporter gene lacZ (the gene for E . coli beta-galactosidase) downstream of viral (in vectors RH116 and tkLTRZ1) or mammalian (in vector NSE-lacZ-tk) promoters were inoculated onto mouse cornea and snout . Trigeminal ganglia were removed 4, 14, 30, and 60 days after inoculation with vectors and histochemically processed with 5-bromo-4-chloro-3 indolyl-beta-galactoside (X-Gal) . With vector tkLTRZ1, large numbers of labeled neurons were observed in rostromedial and central trigeminal ganglion at 4 days after inoculation . A gradual decline in the number of labeled neurons was observed with this vector at subsequent time points . With vectors RH116 and NSE-lacZ-tk, smaller numbers of labeled neurons were seen at 4 days following inoculation than were observed with vector tkLTRZ1 . No labeled neurons could be observed at 14 days after inoculation with vectors RH116 and NSE-lacZ-tk . Immunocytochemistry for E . coli beta-galactosidase and in situ hybridization to HSV latency-associated transcripts revealed labeled neurons in regions of the trigeminal ganglion similar to that observed with X-Gal staining . A comparable distribution of labeled neurons in trigeminal ganglion was also observed after application of the retrograde tracer Fluoro-Gold to mouse cornea and snout . These data provide evidence that retrogradely transported tk- herpes virus vectors can be used to deliver a functional gene to sensory neurons in vivo in an anatomically predictable fashion. Otolaryngol Pol, 1994, 48(3), 275 - 8 {Unasyn in perioperative prophylaxis in patients with laryngeal cancer}; Szmeja Z et al.; In comparative examination of patients with larynx cancer, the authors evaluated the effectiveness of postoperative prophylactic treatment with Unasyn, which was administered to 25 patients who underwent total laryngectomy . Despite the very varied bacterial spectrum the usefulness and effectiveness of this preparation in preventing inflammatory complications of general and local character. Biol Cell, 1994, 80(2-3), 175 - 80 Scanning transmission electron microscopy of biological structures; Colliex C et al.; The design of the scanning transmission electron microscope (STEM) has been conceived to optimize its detection efficiency of the different elastic and inelastic signals resulting from the interaction of the high energy primary electrons with the specimen . Its potential use to visualize and measure biological objects was recognized from the first studies by Crewe and coworkers in the seventies . Later the real applications have not followed the initial hopes . The purpose of the present paper is to describe how the instrument has practically evolved and recently begun to demonstrate all its potentialities for quantitative electron microscopy of a wide range of biological specimens, from freeze-dried isolated macromolecules to unstained cryosections . Emphasis will be put on the mass-mapping, multi-signal and elemental mapping modes which are unique features of the STEM instruments. Life Sci, 1994, 55(13), 1045 - 52 Recombinant rabbit tryptophan hydroxylase is a substrate for cAMP-dependent protein kinase; Vrana KE et al.; A full-length cDNA clone for rabbit tryptophan hydroxylase (TPH) was modified and subcloned into a bacterial expression vector . Expression of this gene in the protease-deficient strain of bacteria, BL21{DE3}, produced TPH immunoreactive protein which exhibited enzyme activity . Treatment of the recombinant enzyme (in bacterial extracts) with the purified catalytic subunit of the cAMP-dependent protein kinase and {gamma-32P}-ATP resulted in specific phosphorylation of TPH . This expression system provides a means of generating and purifying large amounts of this important enzyme . Moreover, these experiments establish that TPH will serve as an in vitro substrate for cAMP-dependent protein kinase. Crit Rev Microbiol, 1994, 20(2), 125 - 42 Homologous genetic recombination: the pieces begin to fall into place; Clark AJ et al.; One of the authors (AJC) acknowledges with gratitude the important role Fernando Bastarrachea played in the author's discovery that E . coli could carry out homologous genetic recombination by multiple pathways . This in turn led to the discovery of several genes, including recF, recO, and recR, whose role in recombination would not otherwise have been detected . Subsequent genetic and biochemical studies have led to a general formulation in which there are multiple nucleolytic ways to achieve a presynaptic intermediate bound to RecA protein . Postsynaptic events in the general formulation occur by means of multiple branch migration enzymes to form Holliday DNA structures and a specific nuclease to cleave them . The general formulation is built on synapsis catalyzed by RecA protein . A second RecA-independent synapsis catalyzed by RecT (and RecE?) protein is now under study and a third type independent of both RecA and RecT has apparently been discovered . How these will affect the general formulation remains to be seen . Some proteins, most prominently RecF, RecO, and RecR, have no role in the general formulation . The hypothesis is presented that these proteins act as a switch between replication and recombination by helping to convert replication to recombination intermediates . Universality of the general formulation is supported by the widespread occurrence of recA, recB, recC, and recD genes among bacteria . Recent discovery of recA-like genes in several eukaryotes further supports its universality . We have contributed additional support by sequencing a recA-like gene from an archaeal species, thus making it plausible that the mechanism of synapsis worked out for E . coli RecA protein will hold for all three organismal domains . The boundaries of the puzzle of homologous genetic recombination therefore seem complete and the pieces to the complex picture they encompass are falling into place. Antonie Van Leeuwenhoek, 1994, 65(1), 7 - 12 Membrane topology of the methyl-accepting chemotaxis protein DcrA from Desulfovibrio vulgaris Hildenborough; Deckers HM et al.; Alkaline phosphatase fusions were used to study the membrane topology of DcrA, a protein of 668 amino acids from Desulfovibrio vulgaris Hildenborough, which has two potentially membrane-spanning hydrophobic sequences at residues 11 to 29 and 188 to 207 . A fusion at amino acid residue 170 in the proposed periplasmic domain exhibited high alkaline phosphatase activity, while low activity was observed for a fusion at amino acid residue 284 in the proposed cytoplasmic domain . The data support a topological model for DcrA similar to that of the methyl-accepting chemotaxis proteins of the enteric bacteria. Antonie Van Leeuwenhoek, 1994, 65(1), 63 - 9 Crotonobetaine reductase from Escherichia coli--a new inducible enzyme of anaerobic metabolization of L(-)-carnitine; Roth S et al.; Crotonobetaine reductase from Escherichia coli 044 K74 is an inducible enzyme detectable only in cells grown anaerobically in the presence of L(-)-carnitine or crotonobetaine as inducers . Enzyme activity was not detected in cells cultivated in the presence of inducer plus glucose, nitrate, gamma-butyrobetaine or oxygen, respectively . Fumarate caused an additional stimulation of growth and an increased expression of crotonobetaine reductase . The reaction product, gamma-butyrobetaine, was identified by autoradiography . Crotonobetaine reductase is localized in the cytoplasm, and has been characterized with respect to pH (pH 7.8) and temperature optimum (40-45 degrees C) . The Km value for crotonobetaine was determined to be 1.1 x 10(-2M) . gamma-Butyrobetaine, D(+)-carnitine and choline are inhibitors of crotonobetaine reduction . For gamma-butyrobetaine (Ki = 3 x 10(-5M)) a competitive inhibition type was determined . Various properties suggest that crotonobetaine reductase is different from other reductases of anaerobic respiration. Virchows Arch, 1994, 424(6), 653 - 9 Skeletal muscle oedema and muscle fibre necrosis during septic shock . Observations with a porcine septic shock model; Hauptmann S et al.; In domestic pigs, intermitted application of Escherichia coli-endotoxin was used to create an animal model for a prolonged hypo- and hyperdynamic septic shock-like state and to investigate mechanisms of multiple organ failure . Here, we describe the changes in skeletal muscle after 18 h (2 animals) and 48 h (6 animals) of septic shock . Two pigs for each observation period that received physiologic saline solutions instead of endotoxin served as controls . The earliest lesions were endothelial cell damage with endomysial oedema and swelling of mitochondria in muscle fibres . With increasing degree of endothelial cell damage, pericytes showed degenerative changes with cytoplasmic fragmentation and karyolysis . After 48 h of shock, endomysial oedema was increased with fibrinogen present . Muscle fibre diameters were increased and swollen mitochondria and segmental necrosis of muscle fibres were frequently observed . However, phagocytic reaction or regenerative changes were not detected . In this respect, skeletal muscle lesions in septic shock differ from ischemic damage, which is characterized by early phagocytosis . Tumour necrosis factor alpha (TNF alpha) was increased greatly and significantly in the serum of the pigs that received endotoxin . The lesions described may be the result of both direct damage to muscle fibres by the endotoxin and/or the increased levels of TNF alpha and indirect damage because of the increased diffusion distance, due to the endomysial oedema . The loss of blood proteins into the endomysium may also play a role in generating hypoproteinemia in patients with septic shock. Biol Cell, 1994, 80(1), 1 - 10 Trypanosoma cruzi cDNA encodes a tandemly repeated domain structure characteristic of small stress proteins and glutathione S-transferases; Schoneck R et al.; The isolation, characterization, and expression of a novel cDNA encoding a Trypanosoma cruzi polypeptide (TcAc2), homologous to various small stress proteins and glutathione S-transferases, are described . The deduced amino-acid sequence revealed two domains sharing 27% identity and an additional 27% similarity to each other suggesting that the molecule may have evolved from a single domain by a process of gene duplication and fusion . The TcAc2 cDNA was subcloned into the pGEX-2T vector for expression in E coli . In vitro translation products of epimastigote mRNA, immunoprecipitated with anti-TXepi serum, showed a major radioactive band of 52 kDa . Immunoprecipitation of {35S}methionine labelled epimastigote and trypomastigote antigens after pulse chase experiments, using anti-TcAc2 fusion protein antibodies, showed that the protein is released into the culture medium . Moreover, Western blot analysis revealed a single band of 52 kDa with epimastigote, trypomastigote and amastigote antigens . Primary structure homology searches revealed that each TcAc2 domain contained within its N-terminus significant homology to Solanum tuberosum pathogenesis-related protein PR1, soybean heat shock protein 26-A, auxin regulated clone pCNT103 from Nicotiana tabacum and Drosophila melanogaster glutathione S-transferase 27 (GST27) . This finding was supported by a comparison of hydrophobicity profiles of TcAc2 and these proteins . Most of them play a central role in protection mechanisms against stress . Based on the homology between TcAc2, glutathione S-transferases (GST) and small stress proteins, it is likely that the TcAc2 gene product may play a crucial role in parasite's adaptation to its microenvironment . These molecules could be considered as members of the GST superfamily, where the T cruzi protein may take a particular place because of its internal gene duplication. Surg Today, 1994, 24(5), 441 - 8 Endotoxin-induced liver injury after extended hepatectomy and the role of Kupffer cells in the rat; Nagata Y et al.; Liver injury by endotoxin given during regeneration following a 70% hepatectomy was examined in Wistar rats . The intravenous administration of endotoxin caused an elevation of the serum GPT level, and severe damage of the remnant liver showing centrilobular necrosis with microthrombi . The highest mortality was induced by the administration of endotoxin to rats 24 h after hepatectomy . Kupffer cells in the regenerative phase of the liver showed an augmented in vitro production of both tumor necrosis factor (TNF) and interleukin-1 (IL-1) . The simultaneous administration of heparin and prostagladin E1 (PGE1), which is known to suppress the production of TNF and IL-1, reduced the magnitude of liver injury and the mortality of these rats . The absence of any direct cytotoxic effect of TNF and IL-1 against liver cells suggested that the cytokines, produced by Kupffer cells, play an important but indirect role in the remnant liver injury induced by endotoxin after hepatectomy. Surg Today, 1994, 24(2), 150 - 2 Spontaneously perforated pyometra presenting as diffuse peritonitis: report of a case; Kimura H et al.; We report herein a rare case of spontaneously perforated pyometra found in a 72-year-old woman who was admitted to our hospital with abdominal pain and vomiting . A distended abdomen with muscular rigidity, a positive Blumberg sign, and a WBC count of 11,900/mm3 indicated diffuse peritonitis, although a plain abdominal X-ray film revealed no free air in the peritoneal cavity . An emergency laparotomy was performed, which revealed a lot of pus, and perforation in the fundus of a distended uterus . The patient was therefore diagnosed as having suffered uterine perforation associating with a pyometra, and a total hysterectomy with bilateral salpingo-oophorectomy was carried out . Histological examination revealed a pyometra with inflammation and destruction of the endometrium and myometrium, and cervical occlusion with no evidence of malignancy . Postoperatively, the patient developed a subcutaneous abscess and pneumonia, but recovered and was discharged on the 74th day after her operation . Thus, although rare, spontaneously perforated pyometra should be considered when elderly women present with acute abdominal symptoms. Arch Microbiol, 1994, 161(6), 495 - 500 Expression of subunits a and c of the sodium-dependent ATPase of Propionigenium modestum in Escherichia coli; Gerike U et al.; The aim of the present study was to construct functional hybrid ATPases consisting of all Escherichia coli ATPase subunits excepts the F0 subunits a or c which were replaced by the respective subunits of the Propionigenium modestum ATPase . This would give valuable information on the subunit(s) conferring the coupling ion specificity . Plasmids were constructed that carried the gene for subunit c (uncE) or subunit a (uncB) behind a tac promoter . These plasmids were transformed into E . coli strains which differed with respect to the unc operon and the expression of the P . modestum genes was verified biochemically . Enhanced expression of the P . modestum genes led to strong growth inhibition of all E . coli strains tested . However, the expressed P . modestum proteins could not functionally complement E . coli strains that lacked the homologous subunit. Biochimie, 1994, 76(2), 187 - 91 Sites of strand breakage in DNA irradiated by fast neutrons; Isabelle V et al.; Therapeutic fast neutrons are densely ionizing particles, with a high relative biological effectiveness relative to 60Co gamma rays (RBE) and a low oxygen enhancement ratio (OER) . The molecular basis of their properties is not yet entirely understood . In a previous work, we have shown that neutrons induce a different number of DNA frank strand breaks as compared to gamma photons, and we have revealed the presence of breaks due to the direct effects of neutrons . In the present work, we searched for eventual differences in the chemical nature of the attacked sites in DNA irradiated in oxygenated diluted solution . We compare our results with neutrons to those previously reported by other authors using gamma- or X-rays . Using sequencing gel electrophoresis of short natural DNA restriction fragments, or synthetic oligonucleotides, we have shown that, in the case of neutrons, the attack occurs with almost the same probability, at each nucleotide, as reported for gamma- and X-rays . The doubling of bands in the bottom of gels shows the presence of two types of termini, the 3'-phosphate and the 3'-phosphoglycolate . Upon neutron irradiation, the 3'-phosphate end appears with a higher yield than the 3'-phosphoglycolate, whereas equal amounts were obtained with gamma- or X-rays. Biochimie, 1994, 76(2), 141 - 51 Comparison of solution structure of free and complexed lac operator by molecular modelling with NMR constraints; Gincel E et al.; The structure difference between the free operator of the lac system d(GCTCACAAT).d(ATTGTGAGC) and the same operator complexed to the headpiece of the lac repressor has been investigated by 2-D-1H NMR spectroscopy in conjunction with molecular modelling in internal coordinates (JUMNA) . The free and complexed operator adopt both a right-handed B helical conformation, but a more detailed analysis of the conformational parameters using the Curves program shows striking differences in the groove geometries, the rises, the twists and the total bending. Biochimie, 1994, 76(2), 133 - 9 Fluorescence study on the non-specific binding of cyclic-AMP receptor protein to DNA: effect of pH; Giraud-Panis MJ et al.; The binding of the cyclic-AMP receptor protein (CRP) of Escherichia coli to a non-specific DNA fragment of 46 base pairs has been studied using fluorescence spectroscopy . The equilibrium binding constant was found to be several orders of magnitude lower than in the specific binding to a DNA fragment of the same size . The salt dependence of the equilibrium binding constant indicates that the CRP makes an identical number (8) of ion pairs to this non-specific DNA fragment in the presence and absence of cAMP . This number is larger than that previously found in the specific binding process . The effect of pH on the non-specific binding was investigated . The number of ion pairs does not vary between pH 6 and 8 . From the variation of the binding constant with pH it was deduced that two histidines are involved in the binding in the absence of cAMP . These are most probably the histidines 199 of each subunit . In the presence of cAMP, only one histidine participates in the binding process, indicating an asymmetric interaction between the two subunits of the CRP and the DNA. Biochimie, 1994, 76(2), 129 - 32 Identification of the DNA-interacting sites of proteins: microsequencing of the peptides cross-linked to 5-bromouracil substituted DNA; Katouzian-Safadi M et al.; Photochemical induced cross-links between protein and nucleic acids are useful tools in the study of the protein-DNA interactions . The substitution of thymine by 5-bromouracil in DNA increases the photocross-linking yield, and reduces the direct damages to both DNA and proteins . Using the lac repressor-DNA non-specific interaction system, we have developed a procedure to identify the interaction site on the protein . Sensitive, accurate and inexpensive in time and material, this procedure is based on the possibility of sequencing peptides in the presence of a large excess of DNA . The obtained result (the implication of His 29) agrees with previous work. Arch Microbiol, 1994, 161(5), 434 - 8 Comparative aspects of utilization of sulfonate and other sulfur sources by Escherichia coli K12; Uria-Nickelsen MR et al.; Selected biochemical features of sulfonate assimilation in Escherichia coli K-12 were studied in detail . Competition between sulfonate-sulfur and sulfur sources with different oxidation states, such as cysteine, sulfite and sulfate, was examined . The ability of the enzyme sulfite reductase to attack the C-S linkage of sulfonates was directly examined . Intact cells formed sulfite from sulfonate-sulfur . In cysteine-grown cells, when cysteine was present with either cysteate or sulfate, assimilation of both of the more oxidized sulfur sources was substantially inhibited . In contrast, none of three sulfonates had a competitive effect on sulfate assimilation . In studies of competition between different sulfonates, the presence of taurine resulted in a decrease in cysteate uptake by one-half, while in the presence of isethionate, cysteate uptake was almost completely inhibited . In sulfite-grown cells, sulfonates had no competitive effect on sulfite utilization . An E . coli mutant lacking sulfite reductase and unable to utilize isethionate as the sole source of sulfur formed significant amounts of sulfite from isethionate . In cell extracts, sulfite reductase itself did not utilize sulfonate-sulfur as an electron acceptor . These findings indicate that sulfonate utilization may share some intermediates (e.g., sulfite) and regulatory features (repression by cysteine) of the assimilatory sulfate reductive pathway, but sulfonates do not exert regulatory effects on sulfate utilization . Other results suggest that unrecognized aspects of sulfonate metabolism, such as specific transport mechanisms for sulfonates and different regulatory features, may exist. Arch Microbiol, 1994, 161(5), 400 - 3 Inactivation of Escherichia coli threonine synthase by DL-Z-2-amino-5-phosphono-3-pentenoic acid; Laber B et al.; The rhizocticines and plumbemicines are two groups of di- and tripeptid antibiotics thought to interfere with threonine or threonine-related metabolism . Z-2-amino-5-phosphono-3-pentenoic acid, the common unusual amino acid constituent of the rhizocticines and plumbemicines, was found to irreversibly inhibit Escherichia coli threonine synthase in a time-dependent reaction that followed pseudo-first order and saturation kinetics . These data provide evidence that the toxicity of the rhizocticines and plumbemicines is due to the inhibition of threonine synthase by Z-2-amino-5-phosphone-3-pentenoic acid, which is liberated by peptidases after uptake into the target cell . Additionally, methods for the purification of threonine synthase from an overproducing E . coli strain and for the enzymatic synthesis of L-homoserine phosphate are described. Parasitol Res, 1994, 80(3), 223 - 9 Production of prostaglandins D2 and E2 by mouse fibroblasts and astrocytes in culture caused by Trypanosoma brucei brucei products and endotoxin; Alafiatayo RA et al.; A study was made to characterize the effects of living Trypanosoma brucei brucei and its products on prostaglandin D2 (PGD2) and PGE2 production by fibroblasts and astrocytes . Cultured fibroblasts were prepared from Microtus agrestis embryos and astrocyte cultures were prepared from neonatal rats . The cultures were maintained in low-endotoxin or defined media (i.e . endotoxin-free) . The PG production was compared with and studied in combination with a defined lipopolysaccharide (LPS) from Escherichia coli . Living T . b . brucei were without effect on PG production . Preparations of T . b . brucei prepared by freeze-thawing and sonication produced dose- and time-dependent increases in PGD2 and PGE2 synthesis by both cell types . LPS caused a similar pattern of increases . The combination of parasite products with LPS caused synergistic production to levels higher than the maximal production by each mitogen alone . The findings have important implications for several pathological features that accompany trypanosomiasis. Int Urol Nephrol, 1994, 26(2), 215 - 22 Fournier's gangrene of the scrotum following anorectal disorders; Kattan S et al.; Five cases of Fournier's gangrene of the scrotum following anorectal disorders were encountered in a period of 2 years . Perirectal abscess was the most common associated underlying condition occurring in three patients . E . coli was the predominant organism cultured in all cases . A chronic debilitating condition was encountered in 3 patients . The mortality rate was 20%, but serious life threatening complications occurred in 4 of our patients . Error in diagnosis and delay in initiating medical treatment were the main causes of the high mortality rate associated with the disease . We advocate thorough rectal examination including proctoscopy in all cases of Fournier's gangrene, especially when no obvious source is apparent. Zhonghua Yi Xue Za Zhi, 1994 Jan, 74(1), 38 - 9, 64 {The role of endotoxin in the bronchial hyperreactivity formation}; Chen JX et al.; Endotoxin was as a pathogen to investigate its effects on bronchial hyperreactivity formation . The latent periods of guinea pigs inhaled histamine and PD2 value of isoproterenol were markedly decreased 4 days after the intraperitoneal injection of endotoxin 1 mg/kg, meanwhile PD2 value of histamine and histamine levels of lung tissue and plasma were increased significantly . These results indicate that endotoxin is a very important factor in the infection induced bronchial hyperreactivity. Cancer Invest, 1994, 12(4), 390 - 4 Effects of sarcoma 180 growth on interleukin-1 and circulating immune complexes; Chasseing NA et al.; Cultured splenic mononuclear adherent cells (SMAc) from normal BALB/c mice as well as those from mice bearing 10-day sarcoma 180 (S180), exhibited a marked increase in Escherichia coli lipopolysaccharide-stimulated interleukin-1 (IL-1) production, when compared to spontaneous values . On days 20 and 30 following S180 challenge, a decrease in this effect on IL-1 production in treated and untreated SMAc was observed . Concomitantly with the alterations in the regulation of IL-1 production during tumor growth, an increase in the levels of prostaglandin E2 and serum immune complexes could be detected . These data suggest that the immunosuppression associated with later stages of tumor development may be due to direct effects on monocytes, by means of a down-regulation of IL-1 production. Arch Virol Suppl, 1994, 9, 381 - 92 Replication and translation of cowpea mosaic virus RNAs are tightly linked; Wellink J et al.; The genome of cowpea mosaic virus (CPMV) is divided among two positive strand RNA molecules . B-RNA is able to replicate independently from M-RNA in cowpea protoplasts . Replication of mutant B-transcripts could not be supported by co-inoculated wild-type B-RNA, indicating that B-RNA cannot be efficiently replicated in trans . Hence replication of a B-RNA molecule is tightly linked to its translation and/or at least one of the replicative proteins functions in cis only . Remarkably also for efficient replication of M-RNA one of its translation products was found to be required in cis . This 58K protein possibly helps in directing the B-RNA-encoded replication complex to the M-RNA . In order to identify the viral polymerase the CPMV B-RNA-specific proteins have been produced individually in cowpea protoplasts using CaMV 35S promoter based expression vectors . Only protoplasts transfected with a vector containing the 200K coding sequence were able to support replication of co-transfected M-RNA . Despite this, CPMV-specific RNA polymerase activity could not be detected in extracts of these protoplasts using a poly(A)/oligo(U) assay . These results indicate that, in contrast to the poliovirus polymerase, the CPMV polymerase is not able to accept oligo(U) as a primer and in addition support the concept that translation and replication are linked. Arch Virol Suppl, 1994, 9, 369 - 77 Nuclear targeting of Semliki Forest virus nsP2; Rikkonen M et al.; The Semliki Forest virus-specific nonstructural protein nsP2 is transported into the nuclei of both infected and transfected BHK cells . The pentapeptide sequence P 648R RRV is an essential part of the nuclear localization signal (NLS) of nsP2, the middle arginine being the most critical residue for nuclear targeting . Host DNA and RNA syntheses are rapidly inhibited in virus-infected cells, and nsP2 could be involved in these processes . It has been postulated that the inhibition of cellular replication could be due to viral NTPase activity . We have expressed and purified nsP2 in E . coli using the highly efficient T7 based expression system . Purified nsP2 was shown to have ATPase and GTPase activities, and these specific activities were increased in the presence of single-stranded RNA, a typical feature of RNA helicases . The role of nsP2 in the nucleus was studied by creating a mutant virus SFV-RDR, which contained an altered NLS (PRDRV) . The mutation affected neither the processing nor the stability of nsP2, but it did render nsP2 completely cytoplasmic . SFV-RDR was shown to be fully infectious, and no difference could be seen in the expression of viral proteins . In addition, the inhibition of host DNA synthesis was almost equally efficient in both wild-type and mutant-infected cells . The pathogenic properties of the mutant will be further studied. Biochimie, 1994, 76(1), 59 - 62 Two GTPs are hydrolysed on two molecules of EF-Tu for each elongation cycle during code translation; Scoble J et al.; A new experimental design has been used to determine the number of GTPs hydrolysed per peptide bond in EF-Tu function in a poly(U)-translation system . We find that two GTPs are consumed for every amino acid incorporated into the nascent poly(Phe)-chains, in accordance with previous findings with other techniques . These results necessitate a revision of current views concerning E coli translation; also new schemes for ribosome function are discussed. Biochimie, 1994, 76(1), 33 - 44 Modification of aminoacyl-tRNA synthetases with pyridoxal-5'-phosphate . Identification of the labeled amino acid residues; Kalogerakos T et al.; The isotopic {32P}PPi-ATP exchange activity of isoleucyl-, valyl-, histidyl-, tyrosyl- and methionyl-tRNA synthetases from Escherichia coli are lost upon incubation in the presence of pyridoxal-5'-phosphate (PLP) . When the residual activity of either isoleucyl-, valyl- or methionyl-tRNA synthetase (monomeric truncated form) was plotted as a function of the number of PLP molecules incorporated per enzyme molecule, the plots obtained appeared biphasic . Below 50% inactivation of these enzymes, PLP incorporation varied linearly with the isotopic exchange measurements, and extrapolation of the first half of the plot indicated a stoichiometry of 1.10 +/- 0.05 mol of PLP incorporated per mol of 100% inactivated synthetase . Beyond 50% inactivation, the graph deviated from its initial slope, and up to 4-5 mol of PLP were incorporated per mol of synthetase at the highest used PLP concentrations . In the cases of homodimeric histidyl- and tyrosyl-tRNA synthetases, extrapolation of the graph at 100% inactivation indicated 2.8 +/- 0.1 and 2.4 +/- 0.1 mol of PLP incorporated per mol of enzyme, respectively . PLP-labeled peptides were obtained through trypsin digestion and RPLC purification, prior to Edman degradation analysis . PLP-labeled residues were identified as lysines 132, 332, 335 and 402 of monomeric methionyl-tRNA synthetase, lysines 332, 335, 402, 465, 596 and 640 of native dimeric methionyl-tRNA synthetase, lysines 22, 117, 601, 604 and 645 of isoleucyl-tRNA synthetase, lysines 554, 557, 559, 593 and 909 of valyl-tRNA synthetase, lysines 2, 118, 369 and 370 of histidyl-tRNA synthetase, and lysine 237 of tyrosyl-tRNA synthetase . In addition, the amino terminal residue of the polypeptide chain(s) of either isoleucyl-, valyl-, histidyl- or methionyl-tRNA synthetases was found labeled . Among these residues, lysines 332, 335 and 402 of monomeric methionyl-tRNA synthetase as well as lysines 332, 335, 402 and 596 of dimeric methionyl-tRNA synthetase, lysines 601, 604 and 645 of isoleucyl-tRNA synthetase, lysines 554, 557 and 559 of valyl-tRNA synthetase, lysines 2, 369 and 370 of histidyl-tRNA synthetase, and lysine 237 of tyrosyl-tRNA synthetase were labeled in the presence of PLP concentrations smaller than or equal to 1 mM, and are shown to be critical for the activity of the enzymes . It is concluded that these residues participate to the binding sites of the phosphates of ATP on the studied synthetases. Arch Virol, 1994, 136(3-4), 375 - 80 High level production of potyvirus-like particles in insect cells infected with recombinant baculovirus; Edwards SJ et al.; The full length gene for the coat protein (CP) of the potyvirus, Johnsongrass mosaic virus, was incorporated into recombinant baculovirus and expressed in insect cells . Western blot and Coomassie-stained polyacrylamide gel electrophoresis analysis of infected insect cells demonstrated that CP was produced in large quantity . Electron microscopic examination of these cells showed the presence of numerous potyvirus-like particles in the cytoplasm . Morphologically the particles resembled potyvirus particles assembled in vitro in the absence of viral RNA and those found in Escherichia coli expressing the recombinant CP gene. Arch Toxicol, 1994, 68(3), 167 - 73 Comparison of CYP1A1 induction and genotoxicity in vitro as indicators of potentially harmful effects of environmental samples; Kopponen P et al.; Cytochrome P450IA1 (CYP1A1) induction of Hepa-1 mouse and H4IIE rat hepatoma cell lines was compared using selected environmental samples . The results were in agreement for both cell lines: no induction was observed for the fly ash extract from peat combustion, an intermediate induction was found for the fly ash extract from biosludge combustion, and a strong induction was detected for natural peat extract . However, Hepa-1 responded to the samples more sensitively than did H4IIE: the half maximal induction (ED50) values for Hepa-1 were smaller than those for H4IIE . In a bacterial DNA repair assay without metabolic activation and in a mammalian sister chromatid exchange test in the presence of metabolic activation the samples were virtually non-genotoxic . Thus the CYP1A1-inducing potency and genotoxicity of the samples were not correlated . In light of these results, the CYP1A1 induction test might be a useful addition to conventional genotoxicity tests, which may fail to detect potentially harmful compounds/mixtures. Wien Klin Wochenschr, 1994, 106(8), 250 - 2 {Phagotest and Bursttest (Phagoburst), test kits for study of phagocyte functions}; Hirt W et al.; Phagotest and Bursttest are complete test kits for the investigation of the phagocytic function of granulocytes and monocytes in whole blood using flow cytometry . Phagotest allows the quantitative determination of leukocyte phagocytosis (uptake of bacteria) . It determines the percentage of phagocytes which ingest fluorescein-isothiocyanate (FITC) labelled opsonized E . coli bacteria and their activity (number of bacteria per cell) . Reduced phagocytosis is observed in patients with sepsis, renal failure, and recurrent bacterial infections . Bursttest allows the quantitative determination of leukocyte oxidative burst . It determines the percentage of leukocytes which oxidize the fluorogenic substrate dihydrorhodamine (DHR) 123 and their enzymatic activity (amount of rhodamine 123) . Reduced burst activity is observed in patients with chronic granulomatous disease (CGD). Biosens Bioelectron, 1994, 9(2), 111 - 7 A spectroscopically interrogated flow-through type toxicity biosensor; Bains W; A simple immobilised cell biosensor is described which uses the MUVS (Metabolic Ultraviolet Spectroscopy) response as an assay for toxic compounds in water . Bacterial cells are immobilised in an agarose membrane in a flow cell . A sample of water is passed over the flow cell . The UV absorbence of the membrane, compared to a similar membrane lacking bacterial cells, is measured at 200 nm . When the cells are stressed so that their energy metabolism is reduced, their UV spectra change, and specifically their OD200 drops within 15 seconds of exposure to the toxin . The sensor has potential for use in rapid tests, and possibly continuous monitoring, of environmental samples for toxic contamination. J Basic Microbiol, 1994, 34(2), 117 - 22 Isolation and characterization of the thermoresistant gluconokinase from Escherichia coli; Vivas EI et al.; It is known that two gluconokinases are inducibly expressed during the utilization of gluconate by E . coli . One is thermoresistant (activity stable for 3 h at 30 degrees C) and the other thermosensitive (losses 75% or more of its activity under the above conditions) . The thermoresistant gluconokinase (EC 2.7.1.12) was isolated, purified and characterized for the first time from the E . coli mutant Ca26, a K12 derivative which lacks the thermosensitive activity . The enzyme was purified 43 fold with a recovery of 11% . The M(r) of the enzyme was 100 kDa with three equal subunits of approximately 29.5 kDa . The enzyme exhibited Michaelis-Menten kinetics and the Km values for gluconate and ATP were 0.02 mM and 0.045 mM respectively. Environ Mol Mutagen, 1994, 23(4), 274 - 80 Propylene oxide mutagenesis at template cytosine residues; Snow ET et al.; Propylene oxide (PO) is a widely used industrial reagent which is mutagenic and carcinogenic . We have recently shown that a variety of aliphatic epoxides, including propylene oxide, can react with DNA to form hydroxyalkyl adducts at N-3 of cytosine which rapidly undergo hydrolytic deamination to produce uracil adducts . These 3-hydroxyalkyl uracil adducts are stable in DNA and are postulated to be an important class of potentially mutagenic lesions . Mutagenesis at cytosine residues due to PO modification of single-stranded M13mp2/C141 DNA was studied by transfection of modified DNA into SOS and non-SOS induced E . coli host cells . Mutations of the proline (CCC) codon at C141 which result in reversion of the lacZ phenotype (blue plaques) were scored . It was found that PO treatment of single-stranded DNA results in dose-dependent mutagenesis that is highly SOS dependent . The spectrum of base-substitution mutations found at this site differed when PO-modified DNA was transfected into E . coli with different DNA repair backgrounds . These results indicate that propylene oxide induced DNA adducts at template cytosine residues are mutagenic in E . coli and that this mutagenesis is greatly increased by SOS processing . They also show that these lesions may be repaired by one or more mechanisms. J Protein Chem, 1994 Jan, 13(1), 15 - 22 The chaperonin assisted and unassisted refolding of rhodanese can be modulated by its N-terminal peptide; Mendoza JA et al.; The in vitro refolding of the monomeric, mitochondrial enzyme rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1), which is assisted by the E . coli chaperonins, is modulated by the 23 amino acid peptide (VHQVLYRALVSTKWLAESVRAGK) corresponding to the amino terminal sequence (1-23) of rhodanese . In the absence of the peptide, a maximum recovery of active enzyme of about 65% is achieved after 90 min of initiation of the chaperonin assisted folding reaction . In contrast, this process is substantially inhibited in the presence of the peptide . The maximum recovery of active enzyme is peptide concentration-dependent . The peptide, however, does not prevent the interaction of rhodanese with the chaperonin 60 (cpn60), which leads to the formation of the cpn60-rhodanese complex . In addition, the peptide does not affect the rate of recovery of active enzyme, although it does affect the extent of recovery . Further, the unassisted refolding of rhodanese is also inhibited by the peptide . Thus, the peptide interferes with the folding of rhodanese in either the chaperonin assisted or the unassisted refolding of the enzyme . A 13 amino acid peptide (STKWLAESVRAGK) corresponding to the amino terminal sequence (11-23) of rhodanese does not show any significant effect on the chaperonin assisted or unassisted refolding of the enzyme . The results suggest that other sequences of rhodanese, in addition to the N-terminus, may be required for the binding of cpn60, in accord with a model in which cpn60 interacts with polypeptides through multiple binding sites. Eur J Clin Pharmacol, 1994, 46(1), 87 - 8 Concentration of meropenem in serum and in bronchial secretions in patients undergoing fibreoptic bronchoscopy; Bergogne-Berezin E et al.; The objective of the study was to evaluate the ability of meropenem to reach the bronchial lumen . 24 patients undergoing fibreoptic bronchoscopy for exploratory purposes were given a single dose of meropenem 1 g as an (i.v., infusion over 30 min . Plasma (P) sampling times were: 0, 0.5, 1, 2 and 3 h . Bronchial secretions (BS) were collected by fibreoptic bronchoscopy at the same sampling times (except for 0 and 0.5 h) in three groups of 8 patients . Meropenem was measured by bioassay using E . coli ATCC 39118 as the test-organism . The results showed that meropenem had reached a high plasma concentration at the first sampling time (59.8 mg.l-1) and then the plasma level decreased rapidly to 10.6 mg.l-1 and 2.7 mg.l-1 at 2 and 3 h respectively . The highest concentration achieved in bronchial secretion was 0.53 mg.l-1 in the third hour, ie 20% of the serum level . The data indicate significant penetration of meropenem into bronchial secretions and achievement of a local level sufficiently high to eradicate most respiratory pathogens. Biol Chem Hoppe Seyler, 1994 Jan, 375(1), 43 - 51 Asymmetry and structural changes in ECF1 examined by cryoelectronmicroscopy; Wilkens S et al.; The Escherichia coli ATPase (ECF1) has been studied by cryoelectronmicroscopy and an intrinsic asymmetry of the molecule in the hexagonal projection identified . The three beta subunits could be distinguished . One, which we have called beta 1, has a greater density in projection than the other two; the second, beta 2, is of intermediate density in projection, while the third, beta 3, is smeared out in density . These different features of the beta subunits were used to orient images, and the positions of the gamma and epsilon subunits then established . The location of the gamma subunit, as monitored by the central mass, was not fixed . This subunit could be found in positions that followed an arc from close to beta 2 to close to beta 3, a shift of around 10A, with respect to the center of the mass . The location of the epsilon subunit was monitored after reconstituting a complex of epsilon subunit-depleted ECF1 with a mutant epsilon subunit in which His at residue 38 had been replaced by Cys, and this Cys labeled with an approximately 14A gold particle . The epsilon subunit was found in positions described by an arc between an alpha subunit (alpha 1) and the neighboring beta subunit (beta 1), a shift of around 20A, with respect to the center of the gold particle . A nucleotide dependence of the position of the gamma subunit has been established by Gogol, E.P., Johnston, E., Aggeler, R . and Capaldi, R.A . (1990) Proc . Natl . Acad . Sci . USA 87, 9585-9589 . A nucleotide dependence of the position of the epsilon subunit is shown here.(ABSTRACT TRUNCATED AT 250 WORDS) Avian Dis, 1994 Jan-Mar, 38(1), 93 - 102 Effect of avian influenza virus infection on the phagocytic function of systemic phagocytes and pulmonary macrophages of turkeys; Kodihalli S et al.; The effects of avian influenza virus (AIV) infection on systemic phagocytes and pulmonary macrophages of turkeys were studied . There was a significant increase (P < 0.0001) in oxidative burst in systemic phagocytes of AIV-inoculated turkeys on 2, 4, 6, and 8 days postinoculation (PI), as measured by chemiluminescence . There was also a significant increase (P < 0.02) in oxidative burst in pulmonary macrophages on day 4 PI . The chemiluminescence response was depressed on 6, 8, and 10 days PI in AIV-inoculated turkeys compared with controls . The increase in oxidative response in both systemic phagocytes and pulmonary macrophages correlated with the peak virus titer in the lungs and trachea of AIV-inoculated inoculated turkeys . Bacterial killing by pulmonary macrophages from AIV-inoculated turkeys was reduced on days 6 and 10 PI compared with uninoculated controls . Histopathological changes in trachea were more pronounced on day 6 PI in AIV-inoculated turkeys; no significant changes were detected in the lungs . These data indicate that compromised functional capacity of pulmonary macrophages predisposes turkeys to secondary bacterial infections. Avian Dis, 1994 Jan-Mar, 38(1), 50 - 8 Bordetella avium hemagglutination and motility mutants: isolation, characterization, and pathogenicity; Moore KM et al.; Transposon mutagenesis was used to produce Bordetella avium mutants, which were screened for the lack of potential virulence factors, including a hemagglutinin, flagella, pili, and toxins . A mini-Tn10 transposon containing a kanamycin-resistance gene was introduced into the chromosomal DNA of the virulent 002/S isolate by electroporation . A hemagglutination-negative (HA-) mutant and a motility-negative mutant were obtained . Southern blot analysis showed that only the motility-negative mutant contained the transposon, whereas the HA- mutant was a spontaneous kanamycin-resistant mutant . Both mutants were stable in vitro and in vivo . Following inoculation of 2-week-old poults, the HA- mutant was determined to be less virulent than the 002/S parent, whereas the motility-negative mutant was similar in virulence to the 002/S parent . These results indicate that the hemagglutinin of B . avium is a virulence factor, but motility does not appear to contribute to virulence. Avian Dis, 1994 Jan-Mar, 38(1), 188 - 92 An outbreak of infectious laryngotracheitis in California broilers; Linares JA et al.; Infectious laryngotracheitis (ILT) was diagnosed as the cause of an outbreak of respiratory disease in broiler chickens in California . The classical form of ILT is characterized by dyspnea, gasping, coughing, and expectoration of bloody exudate . Most of the broilers submitted to the diagnostic laboratory showed a non-classical presentation of ILT, in which mucoid tracheitis and conjunctivitis were the most consistent lesions . Historically, most of the ILT cases diagnosed in our laboratory have consisted of layers with classical signs and lesions . It is not known whether this non classical presentation of ILT in broilers is due to differences in the way broilers respond to ILT infection or to the nature of the ILT virus isolate. Avian Dis, 1994 Jan-Mar, 38(1), 146 - 50 Characterization of an avirulent mutant of a virulent avian Escherichia coli isolate; Nolan LK et al.; A virulent, complement-resistant avian Escherichia coli isolate and its avirulent, complement-sensitive, transposon-insertion mutant were compared for the purpose of revealing structures associated with complement resistance . Both had a smooth lipopolysaccharide layer, contained traT, and lacked a capsule, but the mutant possessed a 16.2-kilodalton outer-membrane protein (OMP) not present in the wild-type . This protein may be the product of a coding region interrupted by transposon insertion . Such results suggest that an OMP greater than 16.2 kilodaltons in size may be responsible for the complement resistance and virulence of this wild-type E . coli. Avian Dis, 1994 Jan-Mar, 38(1), 135 - 40 Non-association of macrophage phagocytosis and oxidant stimulation with complement resistance and colicin V production by avian Escherichia coli; Bounous DI et al.; Avian isolates of Escherichia coli were classified as virulent based on their isolation from chickens with natural cases of colisepticemia, production of colicin V, and complement resistance . A second group of isolates was designated as avirulent based on their isolation from healthy chickens, their inability to produce colicin, and their classification as sensitive or intermediate to the action of complement . In vitro assays of phagocytosis and oxidant production were performed in an attempt to correlate these activities with the ability of each group of bacteria to escape the specific host defense mechanisms of phagocytosis and killing . Although oxidant production regressed with significant linearity on percent phagocytosis, neither group (virulent or avirulent) differed in ability to stimulate peritoneal macrophage phagocytic and oxidant activity when opsonized with normal chicken serum . These results differ from those in mammalian species. Avian Dis, 1994 Jan-Mar, 38(1), 127 - 34 Phenotypic expression of recombinant plasmids pKT107 and pHK11 in an avirulent avian Escherichia coli; Wooley RE et al.; An avirulent wild-type avian Escherichia coli strain (Av) was electrotransformed with plasmids coding for complement resistance (pKT107) and Colicin V (ColV) production (pHK11) in order to study the effects of complement resistance and ColV production on virulence . Transformants were also compared with the wild type for embryo lethality, uptake by macrophages, motility, growth rate, plasmid content, and hemolysis . Growth rates and complement resistance patterns of strain Av and transformant Av+pHK11 were similar, but Av+pHK11 caused a significantly greater number of deaths in embryos and acquired motility . Transformant Av+pKT107 had a lower rate of phagocytosis, a slower growth rate, and a greater sensitivity to complement, and it changed from being non-hemolytic to expressing alpha-hemolytic action . The 35-kb plasmid present in the wild type was not present in the transformants . Although some of the results demonstrate the difficulties encountered in using wild-type organisms as recipients in virulence studies, the results with Av+pHK11 indicate that ColV production plus the acquisition of motility contributes to the virulence of avian E . coli. Arch Virol, 1994, 136(1-2), 123 - 31 Immunogenicity of non-structural proteins of foot-and-mouth disease virus: differences between infected and vaccinated swine; Rodriguez A et al.; Non-structural as well as VP1 recombinant proteins of foot-and-mouth disease virus (FMDV) produced in E . coli, have been used to study the specific antibody response of infected or vaccinated swine . An analysis of sera from infected pigs, using a direct ELISA, showed that polypeptide 3ABC (spanning non-structural proteins 3A, 3B and 3C) was the most antigenic among the recombinant proteins studied and allowed specific detection of FMDV infected swine from the second week after the infection . The sensitivity of this assay was comparable to that obtained when the whole FMDV was used as ELISA antigen . Conversely, use of polypeptide 3ABC did not allow detection of significant levels of antibodies in sera from vaccinated animals . This differential pattern of ELISA reactivities offers a promising approach for the distinction of infected from vaccinated pigs . In addition, a highly specific and sensitive method of diagnosis for FMDV replication was achieved using an immunoblotting assay which detected antibodies against the 3ABC polypeptide. Int Urol Nephrol, 1994, 26(4), 389 - 93 Recurrent emphysematous pyelonephritis . A case report; Huang JJ et al.; Emphysematous pyelonephritis (EPN) is an uncommon but necrotizing renal infection due to gas-forming coliform bacteria that usually occurs in patients with diabetes mellitus and/or obstructive uropathy . Bilateral EPN is rarely seen and recurrent infection in different kidneys has never been reported in the literature . Here we present a female diabetic patient who experienced two episodes of EPN in different kidneys within 2.5 years, resulting in death eventually . We discuss the pathogenesis, incompatibility between clinical features and radiological findings of EPN, and the principle of management for this life-threatening infection. Ann Pharm Fr, 1994, 52(3), 137 - 52 {Studies of in vitro and in vivo toxicity of dyes used in affinity chromatography}; Santambien P et al.; Some reactive textile dyes are used for years as biomimetic ligands in protein purification . The reluctance to use these performant systems in large scale for therapeutically applicable proteins is related with the possible dye leakage and consequently with problems of contamination . Therefore, toxicology data are necessary to quantify the level of danger in association with sensitive assays . This study deals with a series of in vitro toxicity studies with eucaryotic cells (growth, polyploidia, ...) as well as with procaryotic cells (E . coli) for genotoxic studies . Both approaches demonstrated a total absence of toxicity for all ranges of concentrations investigated for Reactive Blue 2, Reactive Red 120 and their derivatives . Additional experiments done in vivo by the administration of dye solutions to a series of mice confirmed the non toxic character of these dyes in vitro. J Med, 1994, 25(3-4), 219 - 29 Hyperglycemia, ketoacidosis and other complications of L-asparaginase in children with acute lymphoblastic leukemia; Cetin M et al.; We show Escherichia coli derived L-asparaginase complications observed in 14 of 136 acute lymphoblastic leukemia patients during remission induction therapy according to St . Jude Children's Hospital Total XI Protocol . We observed hyperglycemia in six patients; two of them had accompanying ketoacidosis . One of the cases with ketoacidosis had peritonitis and pancreatitis . Central nervous system symptoms such as convulsions and depression with personality changes (in one case) were observed in four of these six hyperglycemic patients . Intracranial bleeding and ischemic infarction were shown in cranial computed tomographies in two cases . Hypersensitivity reactions were observed in seven patients . Patients were randomly assigned into two groups and treated with conventional dose steroids or high dose methylprednisolone . Although the frequency of hypersensitivity reactions were lower in the high dose methylprednisolone group, one patient in this group had an anaphylactic reaction . These findings once again high-light L-asparaginase complications which are not dose dependent and can be life threatening. Folia Microbiol (Praha), 1994, 39(3), 171 - 5 Construction of recombinant K88 DNA with Ptac promoter; Holoda E et al.; The gene encoding K88ab was localized on the 11.6 kb HindIII-HindIII fragment of 74 kb plasmid DNA of E . coli 7301 . The smallest recombinant DNA producing the K88ab antigen was obtained by excision of the 5.15 kb EcoRI-EcoRI fragment from recombinant DNA composed of the 11.6 kb K88ab fragment in the pBR322 vector . The size of the smallest fragment was 6.5 kb . Expression of the K88ab antigen was controlled by the P1 promoter of the pBR322 vector . Substitution of promoter Ptac for promoter P1 made it possible to achieve expression of the K88ab antigen by E . coli MT . Substitution of promoter PL for promoter P1 failed to achieve expression of the K88ab antigen in the recipient strains used. Artif Cells Blood Substit Immobil Biotechnol, 1994, 22(3), 739 - 45 Functional properties of beta(NA1)Val-deleted,(NA2)His-->Met hemoglobin synthesized in Escherichia coli; Baudin V et al.; Bovine Hb (hemoglobin) has a low oxygen affinity in the absence of chloride ions and DPG . Because of the increasing interest of this Hb as a potential blood substitute we have engineered a human Hb mutant with the aim of mimicking the functional properties of bovine Hb . This was achieved by deleting residue beta NA1 Val and substituting a methionine for histidine at the beta NA2 position as previously suggested by Perutz and Imai in 1980 . Our results show that the artificial mutant exhibits some of the characteristics of bovine Hb but does not show the low oxygen affinity which is measured in bovine blood. Artif Cells Blood Substit Immobil Biotechnol, 1994, 22(3), 733 - 8 Chimeric hemoglobin subunits: functional properties of a recombinant beta/alpha hemoglobin; Dumoulin A et al.; Our goal was to design a single hemoglobin subunit able to assemble into a stable tetrameric structure with cooperative O2 binding and low oxygen affinity . We have synthesized in E . coli a chimeric beta/alpha globin subunit composed of the first 73 residues of the beta chain and the last 73 residues of the alpha chain . Molecular building indicated that this construction could result in Hb homotetramers possessing the alpha 1 beta 2 interface, responsible for the heme-heme interaction in Hb heterotetramers . The results show that the chimeric subunits assemble into tetramers which bind oxygen reversibly without cooperativity but with an oxygen affinity slightly lower than observed for human Hb . The strong effector RSR 4 lowers the oxygen affinity . Kinetics of CO recombination in the presence of RSR 4 reveal a biphasic bimolecular rebinding . Functional studies suggest that the quaternary structure of the oligomer is intermediary between R-and T-state. Artif Cells Blood Substit Immobil Biotechnol, 1994, 22(3), 625 - 31 Denatured hemoglobin increases human blood mononuclear cell procoagulant effect; Kim SA et al.; PURPOSE: To study the effects of the common contaminants of hemoglobin solutions, red cell stroma, bacterial endotoxin, and denatured hemoglobin on the causes of the thrombotic lesions which have been reported in animal experiments after hemoglobin administration . PROTOCOL: Human blood mononuclear cells were isolated on Ficoll-Hypaque gradients and incubated with hemoglobin from the LAIR production facility, red cell stroma, bacterial endotoxin (E . coli, Wittaker Bioproducts), and hemoglobin denatured by boiling . Incubations were performed separately and in combinations . Mononuclear cells were then lysed and assayed for procoagulant activity in a recalcification time assay . RESULTS: Only bacterial endotoxin and hemoglobin denatured by boiling increased the procoagulant activity of human blood mononuclear cells . Denatured hemoglobin mixed one part in eight with undenatured hemoglobin increased mononuclear cell procoagulant activity by more than ten-fold that of the undenatured hemoglobin control . CONCLUSIONS: The study suggests that denatured but not undenatured hemoglobin causes increased blood procoagulant activity which is thought to be a marker of macrophage activation . These findings suggest a possible mechanism of toxicity of cell-free hemoglobins and the need for sensitive measures of hemoglobin denaturation. Artif Cells Blood Substit Immobil Biotechnol, 1994, 22(3), 387 - 98 Toxicity of hemoglobin solutions: hemoglobin is a lipopolysaccharide (LPS) binding protein which enhances LPS biological activity; Roth RI et al.; Administration of alpha alpha-crosslinked stroma-free hemoglobin (SFH) as a cell-free resuscitation fluid is associated with multiple organ toxicities . Many of these toxicities are characteristic of the pathophysiological effects of bacterial endotoxins (lipopolysaccharide, LPS) . To better understand the potential role of LPS in the observed in vivo toxicities of SFH, we examined mixtures of SFH and E . coli LPS for evidence of LPS-SFH complex formation . LPS-SFH complexes were demonstrated by three techniques: ultrafiltration through 300 kDa cut-off membranes, which distinguished LPS in complexes (87-89% < 300 kDa) from LPS alone (90% > 300 kDa); density centrifugation through 5% sucrose, which distinguished denser LPS alone from LPS-SFH complexes; and precipitation by 67% ethanol, which demonstrated 2-3 fold increased precipitability of complexes compared to SFH alone . Interaction of LPS with SFH was also associated with markedly increased biological activity of LPS, as manifested by enhancement of LPS activation of Limulus amebocyte lysate (LAL), increased release of human mononuclear cell tissue factor, and enhanced production of cultured human endothelial cell tissue factor . These results demonstrated that hemoglobin can serve as an endotoxin binding protein, and that this interaction results in the alteration of several LPS physical characteristics and enhancement of LPS biological activities. Chin J Biotechnol, 1994, 10(1), 49 - 54 Synthesis and cloning of the human lysozyme gene; Qian S et al.; The human lysozyme is an enzyme with potential importance in clinical and industrial application . Owing to the limitation of its natural resource, we are making an attempt to produce the enzyme with the aid of recombinant DNA technology . Twenty-four segments with length ranging from 26 to 38 nucleotides were chemically synthesized by solid phase . The oligonucleotides were joined to form DNA duplexes by two different ligation methods . The entire gene covers a start signal ATG and a BamHI restriction site at its 5' end, and two stop signals TAA TGA and a SphI restriction site at its 3' end, besides the structural gene of human lysozyme . The synthetic gene was cloned into vector M13 . The positive colonies were confirmed by dot-blot hybridization and analysis by restriction enzymes . The DNA sequence of the cloned enzyme gene was proved to be correct by M13 dideoxynucleotide chain termination method . The study on gene expression is under way. Chin J Biotechnol, 1994, 10(1), 18 - 24 Cloning and high level expression of gene encoding ES antigen from Trichinella spiralis muscle larvae; Yan Y et al.; The partial structure gene encoding ES antigen derived from Trichinella spiralis (TSP) muscle larvae was cloned, characterized, and expressed in E . coli . The target DNA (0.7 kb) was directly obtained from the TSP total RNA by using RNA PCR technique . Based on the analysis with the RE digestion, the fragment was cloned into the fusion expression vector pEX31C . It was shown that a kind of 37kDa fusion protein was expressed in E . coli containing the recombinant plasmid by SDS-PAGE electrophoresis . The expressed protein was over 22% of the total cell protein, and it was aggregated in the form of inclusion bodies in E . coli . The purified protein could be recognized in ELISA both by sera from swine-infected with TSP and by the monoclonal antibody against TSP . These findings suggest that the recombinant protein is a potentially valuable antigen both for immunodiagnosis and vaccine development of trichinellosis. Chin J Biotechnol, 1994, 10(1), 11 - 8 Effect of 5' non-coding region on expression of LT-B gene; Ye Q et al.; The different construction of mRNA 5' non-coding region may affect gene expression . To improve gene expression levels, we constructed recombinant plasmids with different nucleotide composition of the 5' non-coding region which direct the synthesis of human toxicogenic E . coli heat-labile enterotoxin B subunit coded by LT-B gene under the control of the PRPL tandem promoter of vector pBV220 . These recombinants were expressed in E . coli HB101 and DH5 alpha, respectively . The results show that the expression levels of LT-B gene with two tandem SD sequences upstream from the initiation codon are lower than those with only one SD sequence and translation coupling can improve the expression levels; the different SD sequences can affect the expression of LT-B gene a little . The SD sequence of LT-B gene itself may be better for expression than that of the vector pBV220; the length of 5' non-coding region in the recombinants harboring only one SD sequence has no effect on the expression of LT-B gene; the expression of LT-B gene in HB101 is higher than that in DH5 alpha. C R Acad Sci III, 1994 Jan, 317(1), 5 - 10 Fast protein sequence verification by matrix assisted laser desorption mass spectrometric analysis of whole enzymatic digests; Hoang BM et al.; Matrix assisted laser desorption mass spectrometry provides a very fast and efficient way to check protein sequences by the analysis of whole proteolytic digests . This method has been applied to the sequence verification of E . coli isoleucyl tRNA synthetase, for which two different sequences are found in data banks . The result of this investigation clearly shows that no one of these sequences is correct. DNA Seq, 1994, 4(4), 259 - 65 Nucleotide sequence of the gene for peptostreptococcal protein L; Murphy JP et al.; A gene bank of Peptostreptococcus magnus DNA was established using an E . coli host-vector system . Western blot analysis identified a clone expressing protein L which bound to the light chain of human immunoglobulins . DNA sequence determination and analysis revealed an open reading frame of 992 amino acids, giving a theoretical secreted protein of 106 kD with a pl of 4.67. DNA Seq, 1994, 4(4), 253 - 7 A high throughput system for the preparation of single stranded templates grown in microculture; Kolner DE et al.; A high throughput system for the preparation of single stranded M13 sequencing templates is described . Supernatants from clones grown in 48-well plates are treated with a chaotropic agent to dissociate the phage coat protein . Using a semi-automated cell harvester, the free nucleic acid is bound to a glass fiber filter in the presence of chaotrope and then washed with ethanol by aspiration . Individual glass fiber discs are punched out on the cell harvester and dried briefly . The DNA samples are then eluted in water by centrifugation . The processing time from 96 microcultures to sequence quality templates is approximately 1 hr . Assuming the ability to sequence 400 bases per clone, a 0.5 megabase per day genome sequencing facility will require 6250 purified templates a week . Toward accomplishing this goal we have developed a procedure which is a modification of a method that uses a chaotropic agent and glass fiber filter (Kristensen et al., 1987) . By exploiting the ability of a cell harvester to uniformly aspirate and wash 96 samples, a rapid system for high quality template preparation has been developed . Other semi-automated systems for template preparation have been developed using commercially available robotic workstations like the Biomek (Mardis and Roe, 1989) . Although minimal human intervention is required, processing time is at least twice as long . Custom systems based on paramagnetic beads (Hawkins et al., 1992) produce DNA in insufficient quantity for direct sequencing and therefore require cycle sequencing . These systems require custom programing, have a fairly high initial cost and have not proven to be as fast as the method reported here. Arch Virol, 1994, 138(1-2), 149 - 60 Nucleotide sequences and expression in Escherichia coli of the coat protein genes from two strains of melon necrotic spot virus; Ohshima K et al.; The nucleotide sequences of coat protein (CP) genes of two Japanese strains of melon necrotic spot virus (MNSV-NH and MNSV-S) were determined . The size of the CP genes of both strains was 1170 nucleotides . Comparisons of the deduced amino acid sequences among MNSV strains showed more than 95% homology, and those among other carmoviruses showed 31-34% homology . cDNAs of MNSV CP genes were cloned into an Escherichia coli expression fusion vector and MNSV-NH and MNSV-S CPs were successfully produced . Furthermore, synthetic oligonucleotide primers were used for differentiating MNSV strains by reverse transcription-polymerase chain reaction method. Adv Exp Med Biol, 1994, 347, 81 - 92 Antiribosomal antibodies in SLE, infection, and following deliberate immunization; Elkon KB et al.; ARA occur in approximately 10% of randomly selected SLE patients but in up to 40% of patients with active disease . Anti-P antibodies appear to be a highly specific diagnostic marker for SLE since they are rarely detected in other multisystem autoimmune disorders . ARA are most frequently directed against the P proteins and the shared conserved C-terminus of the P proteins is immunodominant in almost all sera tested . Anti-P antibodies increase in titer in patients with active disease and have been reported to be detected more frequently in patients with severe behavioral disturbances . This may be particularly true of patients with affective disorders . The clinical utility of serological tests for anti-P in central nervous system lupus must await large, prospective studies . Other ARA antibodies have been detected in patients with SLE . These antibodies include anti-28S rRNA, anti-S10, and anti-L12 . In all cases, the frequency with which these antibodies are detected is increased in sera containing anti-P . The P proteins and the 28S rRNA epitope play essential, but as yet undefined, roles in GTPase activity on the ribosome . The L12 protein is the mammalian homologue of the E . coli and yeast proteins known to bind to the 28S rRNA epitope . These findings indicate that some SLE patients produce autoantibodies against multiple components of a functionally related domain of the ribosome . This, in turn, supports the notion that the ribosome initiates and/or maintains autoantibody production . Despite the evidence supporting an antigen driven immune response, attempts to induce anti-P antibodies by immunization with autologous ribosomes in the autoimmune strain of mouse, MRL, have been unsuccessful . It therefore seems likely that the ribosomal components must be altered in some way to break tolerance or that other abnormalities of the immune system are necessary for autoantibody production . Immunization with foreign ribosomes induce anti-P autoantibodies in mice and in apparently normal humans infected with the hemoflaggelate, T . cruzi . The ability of the P proteins to break tolerance in these situations is, most likely, explained by the provision of a T cell epitope (the foreign P protein) together with the multivalency of the P proteins on the ribosome (which activate autoreactive B cells) . We therefore propose (Fig . 5) a two-signal model for autoantibody production similar to that suggested for T-B collaboration in the normal immune response and also in the GVHD model of lupus.(ABSTRACT TRUNCATED AT 400 WORDS) Microbiol Immunol, 1994, 38(7), 535 - 41 Pig intestinal membrane-bound receptor (guanylyl cyclase) for heat-stable enterotoxin: cDNA cloning, functional expression, and characterization; Wada A et al.; A cDNA encoding the receptor protein for a heat-stable enterotoxin (STa) produced by enterotoxigenic Escherichia coli was cloned from intestinal epithelial cells of a 10-week-old pig . The cDNA had an open reading frame of 3,219 base pairs and coded for a protein with 1,073 amino acid residues . The mature protein consisted of 1,050 amino acid residues with a molecular mass of ca . 121 kDa and was 87% and 82% identical with the human and rat protein, respectively . The CHO cell line overexpressing the pig recombinant STa receptor specifically bound to a photoaffinity-labeled analog of STa and showed marked elevation of the cellular content of cGMP in response to STa. Microbiol Immunol, 1994, 38(6), 461 - 5 The non-random pattern of insertion of IS2 into the hemB gene of Escherichia coli; Lewis LA et al.; The hemB gene of Escherichia coli has been identified as a hot spot for the insertion of the transposable element IS2 . The insertional specificity of IS2 is still unclear . This study reports on the attempt to sequence a statistically significant number of insertions in hemB, in order to determine whether there might be a basis for future studies to determine a molecular basis of IS2 insertional specificity . The results indicate that IS2 inserts in a non-random manner into a 240 bp segment at the 5' end of the gene (region I) . Twenty-one of 24 insertions occurred in region I . Three insertions have been identified in the two middle 250 bp segments of the 975 bp gene, and none in the 3' terminal segment . A seventeen bp sequence showing 88.2% identity with a segment of IS2, 221 bp from the 3' terminus has been identified in region I . Four instances of repeated insertion between the same pair of nucleotides have been observed at four different sites. Microbiol Immunol, 1994, 38(6), 441 - 7 Construction of mutant genes for a non-toxic verotoxin 2 variant (VT2vp1) of Escherichia coli and characterization of purified mutant toxins; Cao C et al.; The gene encoding a Verotoxin 2 variant, VTvp1, was mutated by oligonucleotide-directed site-specific mutagenesis . Among 6 mutant toxins encoded by the mutated genes, E167Q-R170L (glutamic acid at position 167 and arginine at position 170 from N-terminus of the A subunit were replaced by glutamine and leucine, respectively) was found to have markedly decreased activities; inhibition of protein synthesis, Vero cell cytotoxicity and mouse lethality of the purified E167Q-R170L were 1/1,900, 1/125,000 and 1/2,000, respectively, of those of the purified wild-type VT2vp1 . Since the antigenic property of the E167Q-R170L was demonstrated to be similar to that of the wild-type VT2vp1 by Ouchterlony double gel diffusion test and by neutralization test of Vero cell cytotoxicity of the VT2vp1, a possibility to use the mutant VT2vp1, E167Q-R170L, as a toxoid is discussed. Microbiol Immunol, 1994, 38(6), 435 - 40 Specific detection of a verotoxin 2 variant, VT2vp1, by a bead-enzyme-linked immunosorbent assay; Cao C et al.; A bead-enzyme-linked immunosorbent assay to specifically detect a Verotoxin 2 variant, VT2vp1, was developed . The sensitivity of the bead-ELISA was 200 pg/ml of the purified VT2vp1 and it did not react with 20 ng/ml of the purified VT2 . The specificity of the bead-ELISA was examined with 107 strains of Verocytotoxin-producing Escherichia coli that include VT1-, VT2-, VT2vha-, VT2vhb- and VT2vp1-producing E . coli, and only VT2vp1-producing E . coli that were confirmed by VT2vp1-specific polymerase chain reaction gave positive results . It was noted that all 58 VT2vp1-producing E . coli strains were from pigs, but not from cows and humans. J Inherit Metab Dis, 1994, 17(4), 383 - 90 Komrower Lecture . Molecular basis of phenotype expression in homocystinuria; Kraus JP; Cystathionine beta-synthase (CBS) deficiency is the most common cause of homocystinuria in humans . The human gene maps to chromosome 21q22.3 and encodes the CBS subunit of 551 amino acid residues (63kDa) . CBS, a tetramer of these subunits, binds its two substrates, homocysteine and serine, and three additional ligands: pyridoxal 5'-phosphate, S-adenosylmethionine, and haem . Screening for mutations by expressing patient cDNA segments in E . coli permitted us to separate the parental CBS alleles, localize each mutation within one third of the cDNA, and functionally analyse the mutant protein . Using this method we identified the first 14 mutations in homocystinuria . The most common mutation in patients of predominantly 'Celtic' origin is the G919A transition which substitutes serine for glycine 307. Genetica, 1994, 92(2), 107 - 14 The microinjected Drosophila melanogaster 1731 retrotransposon is activated after the midblastula stage of the amphibian Pleurodeles waltl development; Kim MH et al.; The entire 1731 retrotransposon of Drosophila melanogaster, tagged with the E . coli lac Z gene inserted in its gag sequence, was injected into oocytes and fertilized eggs of the urodele amphibian Pleurodeles waltl . Expression of the reporter gene indicated that the 1731 promoter (its 5'LTR) is active in the embryos and not in the oocytes . It appeared that this element is regulated as amphibian genes are at the beginning of the development, i.e . that expression was detected after the mid blastula stage and maintained up to four or five days after injection . Another construction associating the modified 1731 promoter with the CAT gene is also expressed in Pleurodeles embryos during the same period of development . This indicated that the 1731 promoter issued from a Drosophila species is activated as promoting sequences of amphibian zygotic genes are, suggesting that in the case of horizontal transfer, 1731 can be expressed into vertebrate organisms. Environ Mol Mutagen, 1994, 24(3), 168 - 75 Molecular models that may account for nitrous acid mutagenesis in organisms containing double-stranded DNA; Hartman Z et al.; Nitrous acid (NA) is often presumed to cause base substitutions in organisms with double-stranded DNA as a direct consequence of oxidative deamination of adenine and of cytosine residues . Here we summarize evidence indicating that other mechanisms are involved in the case of NA-induced G/C-->A/T transition mutations . We present several models for pathways of NA mutagenesis that may account for our experimental results and overlapping data noted in the literature . One model proposes that the base substitution mutations observed are due to DNA alkylation damage mediated via nitrosation of polyamines and/or other ubiquitous cellular molecules . Other models assume that predisposing lesions, such as G-to-G cross-links, are first formed . The cross-links are pictured as leading to perturbations in DNA structure that allow subsequent opportunity for NA-induced deaminations of cytosine residues in their immediate vicinity . The deaminations preferentially result in G/C-->A/T transition mutations at sites highly dependent on adjoining base sequence context (i.e., in NA "mutational hotspots") . A final model proposes that NA-induced G/C-->A/T transition mutations arise mainly from oxidative deamination of guanosine residues and not from deamination of cytosine residues in duplex DNA. Cell Mol Biol (Noisy-le-grand), 1994, 40 Suppl 1, 37 - 44 Exploration of mucosal immunity in humans: relevance to vaccine development; Czerkinsky C et al.; Although the immune system is remarkably diverse, there is evidence that certain types of immune responses take place and are restricted to certain anatomic locations within the body . The concept of a common mucosal immune system that provides immune reactivity not only at the site of antigen deposition but also at remote mucosal sites may be explained by the utilization of organ-specific recognition molecules by circulating precursors of mucosal immunoblasts and by the production of certain maturation factors (e.g . cytokines, hormones) produced preferentially in certain organs or parts of a given organ . This notion may explain the unification of immune responses in diverse mucosal sites and the physiologic segregation of mucosal from systemic immune mechanisms . Novel methods have been developed to enable studies of antigen specific B and T cell responses in various mucosal and extramucosal tissues in primates and rodents, using cholera toxin or its B subunit as prototype immunogens and mucosal carrier-delivery systems . The tissue localization and isotype commitment of antibody-secreting cells (ASC) and the homing potential of their circulating precursors have also been examined after oral, nasal, intra-tonsillar, rectal and/or genital immunization(s) . The anatomical distribution of T- and accessory cell-derived cytokines has also been examined . These tools and approaches are being employed in studies attempting to induce optimal mucosal immune responses to several mucosal pathogens including HIV-1, in certain organs such as the lower gastrointestinal tract and the female urogenital tract.(ABSTRACT TRUNCATED AT 250 WORDS) Crit Rev Toxicol, 1994, 24(3), 255 - 80 Transgenic animal models for measuring mutations in vivo; Mirsalis JC et al.; Transgenic animal models for measuring mutations provide a powerful tool for rapidly assessing tissue-specific mutations following in vivo treatment . These models are based on the insertion into the rodent genome of the Escherichia coli lacI (lac repressor) or lacZ (beta-galactosidase) genes that serve as targets for mutations . Following in vivo treatment of animals, genomic DNA is isolated from various tissues and the target gene is packaged into lambda-phage heads; the lambda-phage are used to infect E . coli in order to produce plaques . Mutations in the target gene are then detected using colorimetric or selective procedures . In this review methods are discussed for producing these transgenic models, the target genes used, gene rescue techniques, sequencing of isolated mutants, and parameters that affect dosing regimens and design of studies . We also present a summary of data published to date with these systems and present our conclusions and proposed directions for future research. Adv Enzyme Regul, 1994, 34, 225 - 46 Human casein kinase II: structures, genes, expression and requirement in cell growth stimulation; Pyerin W; Casein kinase II (CKII) is an ubiquitous Ser/Thr protein phosphotransferase in control of a variety of crucial cellular functions including metabolism, signal transduction, transcription, translation and replication . CKII levels are consistently higher in neoplastic tissues . The human CKII is composed of subunits alpha, alpha', and beta with molecular masses of 43, 38 and 28 kDa, respectively, that form heterotetrameric holoenzymes (alpha 2 beta 2; alpha alpha' beta 2, alpha'2 beta 2) showing autophosphorylation particularly at subunit beta and hence suspected to play a regulatory role . The amino acid sequences of subunits indicate high evolutionary conservation . Employing the complete set of tissue-derived (placenta) and recombinant (expressed in E . coli) subunits and CKII holoenzymes, the catalytic function of alpha and alpha' and the several-fold stimulation by beta is shown to occur comparably in tissue-derived and recombinant CKII and the autophosphorylation of beta is shown by site-directed mutagenesis to be not decisive for the tuning of CKII activity . The human genome contains two genes encoding CKII alpha . First, there is a processed (pseudo)gene which is 99% homologous to the CKII alpha cDNA and which possesses a promoter-like region adjacently upstream with TATA and CAAT boxes so that transcription cannot be excluded . Second, there is an active gene of which we have characterized so far a 18.9 kb long central fragment which contains 8 exons comprising bases 102-824 of the CKII alpha coding region . The gene fragment contains repetitive elements, most prominently 16 Alu repeats . The genome further contains one as yet uncharacterized CKII alpha' gene and one gene encoding CKII beta . The CKII beta gene has been characterized as a 4.2 kb spanning gene composed of seven exons which possesses three transcription start sites and the translation start site in the second exon . The first intron harbors an Alu repeat also . The promoter region of the CKII beta gene contains elements such as multiple GC boxes, a CpG island, and nonstandard-positioned CAAT boxes but lacks a TATA box thus characterizing the gene as a housekeeping gene . The CKII genes are not clustered at a certain chromosome but rather are distributed over the whole human genome . Using the genomic clones as the probes for in situ hybridization, the active CKII alpha gene was mapped to chromosome 20p13, the processed CKII alpha (pseudo)gene to chromosome 11p15, and the CKII beta gene to chromosome 6p21 . (The CKII alpha' gene has been localized on chromosome 16 with a cDNA probe.).(ABSTRACT TRUNCATED AT 400 WORDS) Scand J Infect Dis, 1994, 26(3), 275 - 82 Microtiter assay for colorimetric detection of in vitro amplified Chlamydia trachomatis sequences; Lundeberg J et al.; An integrated diagnostic method for colorimetric detection of polymerase chain reaction (PCR) products in a microtitre format is described . The amplified material is labelled by using modified PCR primers introducing a biotin molecule and a lac operator handle . Positive samples are identified, after immobilization onto streptavidin-coated microtitre wells, using a reporter fusion protein consisting of the Escherichia coli lac repressor and beta-galactosidase . The analysis of the PCR products is thus, from a practical point of view, identical with the enzyme-linked immunosorbent assay (ELISA) using the enzymatic activity of beta-galactosidase . Here, we show that the assay can be used for rapid and simple detection of Chlamydia trachomatis in an automated format . The reported analysis of 90 urethral patient samples, using this microtitre assay, indicates that this technique is more sensitive and more specific than culture technique. Microbiol Immunol, 1994, 38(4), 273 - 9 Escherichia coli heat-labile enterotoxin binds to glycosylated proteins with lactose by amino carbonyl reaction; Shida K et al.; The binding of Escherichia coli heat-labile enterotoxin (LT) type I to glycosylated proteins with lactose (Gal beta 1-4Glc) by amino carbonyl reaction was studied by the Western blot assay and by the microtiter well binding assay . LT bound to a lactose-alpha-lactalbumin amino carbonyl product (Lac-LA), whereas cholera toxin did not . The binding ability of Lac-LA was abolished by beta-galactosidase treatment, indicating that the terminal galactose is essential for the binding of LT . The binding of LT to Lac-LA was inhibited by galactose and lactose, and most effectively inhibited by lactulose (Gal beta 1-4Fru), which is a structural analog of the Amadori rearrangement product of the amino carbonyl reaction between lactose and an epsilon-amino group of a lysine residue (lactuloselysine) . The results suggest that LT recognizes the portion of lactuloselysine in Lac-LA . LT also bound to a melibiose (Gal alpha 1-6Glc)-alpha-lactalbumin amino carbonyl product (Mel-LA), but the binding ability of Mel-LA was weaker than that of Lac-LA, suggesting that the beta 1-4 linked terminal galactose is dispensable but preferable for the binding . Furthermore, LT bound to the amino carbonyl products of lactose with beta-lactoglobulin, caseins, bovine serum albumin, and ovalbumin . These results indicate that LT binds to the amino carbonyl products between proteins and sugars containing the terminal galactose, such as lactose.
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