Biosci Biotechnol Biochem, 1995 Jun, 59(6), 1099 - 106 Cloning and nucleotide sequence of the gene responsible for chlorination of tetracycline; Dairi T et al.; Two cosmid clones containing distinct types of self-defense gene of a 6-demethylchlortetracycline producer, Streptomyces aureofaciens NRRL3203, were isolated . The gene responsible for chlorination of tetracycline (chl gene) was subcloned from one of the cosmid clones by complementation of a chlorination-deficient mutant, using a gene cloning system for strain NRRL3203 developed in this study . The nucleotide sequence analysis of a 4.4-kb SacI-BamHI fragment containing the chl gene showed that the predicted product of the chl gene is a polypeptide of 452 amino acids, and that the chl gene was preceded by two open reading frames, which could endode polypeptides of 50 kDa and 32 kDa, respectively . A search for sequences homologous to these ORFs found that the former product strongly resembles that of the 6-hydroxylation enzyme for oxytetracycline biosynthesis, and that the latter product has a weak but significant similarity to the hydroxyindole O-methyltransferase of bovine pineal gland . By Northern blot analysis, these three genes were suggested to be polycistronically transcribed. Biosci Biotechnol Biochem, 1995 Jun, 59(6), 1095 - 8 Culture conditions for improvement of L-threonine production using a genetically self-cloned L-threonine hyperproducing strain of Escherichia coli K-12; Shimizu E et al.; We had constructed an L-threonine-hyperproducing strain of E . coli K-12 by recombinant DNA techniques . In this paper, culture conditions for the practical production of L-threonine were investigated using this strain . Cultivation temperature, concentration of required amino acids, and dissolved oxygen greatly influenced the yield of L-threonine . High production of L-threonine was obtained at a high level of dissolved oxygen for the recombinant strain, but not for the parent . This improved production was accompanied by a high copy number of recombinant plasmids and high activity of aspartokinase . Initial addition of L-threonine together with required amino acids greatly reduced the net production of L-threonine . To remove the reductive effect, methods for the addition of the required amino acids were tested . Lowering the required amino acids at a later stage of cultivation seemed to be effective to avoid the reductive effect of the accumulated L-threonine . By using the optimal conditions, the highest level of L-threonine production, 65 g/l, 48% yield, was achieved. Biochim Biophys Acta, 1995 Jun 1, 1230(1-2), 62 - 8 Arrangement of the epsilon subunit in the Escherichia coli ATP synthase from the reactivity of cysteine residues introduced at different positions in this subunit; Aggeler R et al.; ECF1F0 has been purified from three mutants in which a Cys has been incorporated by site-directed mutagenesis in the epsilon subunit: these mutants are epsilon S10C, epsilon H38C and epsilon S108C, respectively . ECF1F0 from the mutant epsilon S10C had a 2-fold higher activity than wild-type enzyme, due to altered association of the epsilon subunit with the rest of the complex, and yet showed normal proton pumping function . The other two mutants had ATPase activities similar to wild-type enzyme . The introduced Cys was exposed for reaction with maleimides in epsilon S10C and epsilon S108C . In epsilon H38C, the introduced Cys reacted readily with N-ethylmaleimide in isolated ECF1, but was unavailable for reaction with this or other maleimides in ECF1F0 . When this Cys at position 38 in the epsilon subunit was reacted with various maleimides in isolated ECF1 and then the ECF1 bound back to F0, the interaction between the two parts was perturbed . While ECF1F0 reconstituted with unmodified ECF1 functioned normally, enzyme with maleimide-reacted Cys-38 showed much reduced proton pumping, had only around 50% of the DCCD inhibition of unmodified or wild-type enzyme, and had a much higher LDAO activation (as much as 8.3-fold, c.f . 4-fold for wild type) . Nucleotide-dependent conformational changes have been observed previously, in studies of ECF1 from the mutants epsilon S10C and epsilon S108C . Identical nucleotide-dependent structural changes were observed in cross-linking experiments with tetrafluorophenylazide maleimides when the intact ECF1F0 from these mutants was examined . Taken together, the Cys reactivity data and cross-linking results provide the orientation of the epsilon subunit in the enzyme complex. Am J Physiol, 1995 Jun, 268(6 Pt 2), H2252 - 9 Differential influence of arachidonic vs . eicosapentaenoic acid on experimental pulmonary hypertension; Grimminger F et al.; The impact of the 2- and 3-series prostanoid precursors arachidonic acid (AA) and eicosapentaenoic acid (EPA) on experimental pulmonary hypertension was investigated . The model of buffer-perfused rabbit lungs was stimulated by infusion of Escherichia coli hemolysin (HlyA), which is known to provoke sustained thromboxane (Tx)-mediated pulmonary hypertension . Release of di- and trienoic Tx into the recirculating perfusate was quantified by a post-high-performance liquid chromatography enzyme-linked immunosorbent assay technique . HlyA at 0.08 hemolytic unit/ml caused a sustained rise in pulmonary arterial pressure (PAP; maximum increase 14 +/- 2 mmHg) accompanied by progressive TxB2 liberation (maximum perfusate concn 33 +/- 4 pg/ml, baseline < 2 pg/ml) . Between 5 and 30 nM, AA provoked a transient monophasic rise in PAP (maximum pressor response 1.5-15 mmHg) and concomitant TxB2 release (peak concn 2-30 pg/ml) . Simultaneous administration of HlyA and AA exhibited additive effects with regard to mediator release and pressor responses . EPA at 200-2,000 nM caused a transient rise in PAP similar to that provoked by 5-30 nM AA (maximum pressor response 3-18 mmHg) . This was accompanied by liberation of TxB2 (peak concn 16 +/- 5 and 28 +/- 4 pg/ml after 1,000 and 2,000 nM EPA) and TxB3 (peak concn 9 +/- 4 and 30 +/- 3 pg/ml) . Combined application of HlyA and EPA resulted in approximate addition of the TxB2 release reaction to each single compound, and TxB3 liberation more than doubled (maximum concn 59 +/- 12 pg/ml) . The pressor responses to HlyA-EPA (200-2,000 nM) did not, however, surpass those to HlyA-AA (5-30 nM).(ABSTRACT TRUNCATED AT 250 WORDS) Plant Physiol, 1995 Jun, 108(2), 551 - 61 Molecular and biochemical analysis of calmodulin interactions with the calmodulin-binding domain of plant glutamate decarboxylase; Arazi T et al.; We previously provided what to our knowledge is the first evidence that plant glutamate decarboxylase (GAD) is a calmodulin (CaM)-binding protein . Here, we studied the GAD CaM-binding domain in detail . A synthetic peptide of 26 amino acids corresponding to this domain forms a stable complex with Ca2+/CaM with a 1:1 stoichiometry, and amino acid substitutions suggest that tryptophan-485 has an indispensable role in CaM binding . Chemical cross-linking revealed specific CaM/GAD interactions even in the absence of Ca2+ . However, increasing KCI concentrations or deletion of two carboxy-terminal lysines abolished these interactions but had a mild effect on CaM/GAD interactions in the presence of Ca2+ . We conclude that in the presence of Ca(2+)-hydrophobic interactions involving tryptophan-485 and electrostatic interactions involving the carboxy-terminal lysines mediate CaM/GAD complex formation . By contrast, in the absence of Ca2+, CaM/GAD interactions are essentially electrostatic and involve the carboxy-terminal lysines . In addition, a tryptophan residue and carboxy-terminal lysines are present in the CaM-binding domain of an Arabidopsis GAD . Finally, we demonstrate that petunia GAD activity is stimulated in vitro by Ca2+/CaM . Our study provides a molecular basis for Ca(2+)-dependent CaM/GAD interactions and suggests the possible occurrence of Ca(2+)-independent CaM/GAD interactions. Eur J Biochem, 1995 Jun 1, 230(2), 788 - 96 High-level biosynthetic substitution of methionine in proteins by its analogs 2-aminohexanoic acid, selenomethionine, telluromethionine and ethionine in Escherichia coli; Budisa N et al.; We have utilized a T7 polymerase/promoter system for the high-level incorporation of methionine analogs with suitable labels for structural research (X-ray and NMR studies) on recombinant annexin V produced in Escherichia coli . Here, we describe, to our knowledge, the first biosynthetic high-level substitution of methionine by 2-aminohexanoic acid (norleucine), ethionine and telluromethionine in a protein . The replacement has been confirmed by electrospray mass spectroscopy, amino acid analysis and X-ray structural analysis . Conditions for expression were optimized concerning the frequency of appearance of revertants, high-level replacement and maximal protein yield . For the incorporation of norleucine and ethionine, E . coli B834 (DE3)(hsd metB), which is auxotrophic for methionine, was grown under methionine-limited conditions with an excess of the analog in the culture medium, and the expression of protein under the control of the T7 promoter was induced after the methionine supply had been exhausted . The factor limiting the high-level incorporation of telluromethionine into protein is its sensitivity towards oxidation . To overcome this problem, bacteria were grown with a limited amount of methionine, harvested after its exhaustion and resuspended in fresh media without methionine; telluromethionine was added and protein synthesis induced . Under these conditions, significant amounts of protein can be expressed before telluromethionine has been completely degraded (within hours) . Biosynthetic incorporation of heavy atoms such as tellurium into recombinant proteins can accelerate the process of obtaining heavy-atom derivatives suitable for X-ray structural analysis, supplementing the traditional trial-and-error preparation of heavy-atom derivatives for the method of multiple isomorphous replacement . Furthermore, the successful high-level incorporation of amino acid analogs can provide single-atom mutations for the detailed study of the structure and function of proteins. Eur J Biochem, 1995 Jun 1, 230(2), 741 - 51 cDNA cloning and tissue-specific regulation of expression of rat calcium-binding protein 65/67 . Identification as a homologue of annexin VI; Fan H et al.; We isolated a cDNA encoding the rat membrane-associated 65/67-kDa calcium-binding protein, CBP 65/67, from a lambda ZAP II cDNA-expression library of rat liver by immunoscreening using monospecific polyclonal anti-(CBP 65/67) antibodies and monoclonal anti-(CBP 65/67) IgG . The product of this cDNA expressed in Escherichia coli was confirmed as CBP 65/67 both by immunostaining and by comparison of the molecular mass with the CBP 65/67 isolated from rat liver by SDS/PAGE . The cDNA sequence and the deduced amino acid sequence of CBP 65/67 both show a high degree of identity to human p68 and human calelectrin, which belong to a family of calcium-dependent, membrane-associated, phospholipid-binding proteins, called annexins . This means that CBP 65/67 is a homolog of the two human proteins just mentioned above . We are not aware that a rat annexin VI has previously been isolated and sequenced . The mRNA expression of CBP 65/67 in different rat organs during development was investigated by Northern blot analysis . In adult tissues, high mRNA levels of CBP 65/67 were found in lung, heart, muscle, spleen and especially in thymus and pancreas, whereas in liver, kidney, intestine, stomach and brain only low levels of CBP 65/67 mRNA could be detected . The amount of mRNA during tissue development in kidney, stomach and muscle showed only slight changes . In contrast, a significant increase of CBP 65/67 expression was observed in liver, lung, heart and brain . In most of the organs investigated, the level of mRNA correlated closely with the level of protein expression, indicating that the expression of CBP 65/67 in most organs is controlled primarily at the transcriptional level. Eur J Biochem, 1995 Jun 1, 230(2), 538 - 48 Isolation and characterization of the proton-translocating NADH: ubiquinone oxidoreductase from Escherichia coli; Leif H et al.; The proton-translocating NADH:ubiquinone oxidoreductase (complex I) was isolated from Escherichia coli by chromatographic steps performed in the presence of an alkylglucoside detergent at pH 6.0 . The complex is obtained in a monodisperse state with a molecular mass of approximately 550,000 Da and is composed of 14 subunits . The subunits were assigned to the 14 genes of the nuo operon, partly based on their N-terminal sequences and partly on their apparent molecular masses . The preparation contains one noncovalently bound FMN/molecule . At least two binuclear (N1b and N1c) and three tetranuclear (N2, N3 and N4) iron-sulfur clusters were detected by EPR in the preparation when reduced with NADH . Their EPR characteristics remained mostly unaltered during the isolation process . After reconstitution in phospholipid membranes, the preparation catalyses piericidin-A-sensitive electron transfer from NADH to ubiquinone-2 with Km values similar to those of complex I in cytoplasmic membranes but with only 10% of the Vmax value . The isolated complex I was cleaved into three fragments when the pH was raised from 6.0 to 7.5 and the detergent exchanged to Triton X-100 . One of these fragments is a water-soluble NADH dehydrogenase fragment which is composed of three subunits bearing at least four iron-sulfur clusters (N1b, N1c, N3 and N4) that can be reduced with NADH, one of them bearing FMN . The second, amphipathic, fragment, which is presumed to connect the NADH dehydrogenase fragment with the membrane, contains four subunits and at least one EPR-detectable iron-sulfur cluster whose spectral properties are reminiscent of the eucaryotic cluster N2 . The third membrane fragment is composed of seven homologues of the mitochondrially encoded subunits of the eucaryotic complex I . This subunit arrangement coincidences to some extent with the order of the genes on the nuo operon . A topological model of the E . coli complex I is proposed. Eur J Biochem, 1995 Jun 1, 230(2), 533 - 7 cDNA cloning, overexpression in Escherichia coli, purification and characterization of sheep liver cytosolic serine hydroxymethyltransferase; Jagath-Reddy J et al.; A sheep liver cDNA clone for the cytosolic serine hydroxymethyltransferase (SHMT) was isolated and its nucleotide sequence determined . The full-length cDNA of SHMT was placed under the control of T7 promoter in pET-3C plasmid and expressed in Escherichia coli . The overexpressed enzyme, present predominantly in the soluble fraction, was catalytically active . The recombinant SHMT was purified to homogeneity with a yield of 10 mg/l bacterial culture . The recombinant enzyme was capable of carrying out tetrahydrofolate-dependent and tetrahydrofolate-independent reactions as effectively as the native enzyme . The Km values for serine (1 mM) and tetrahydrofolate (0.82 mM) were similar to those of the native enzyme . The recombinant enzyme had a characteristic visible spectrum indicative of the presence of pyridoxal 5'-phosphate as an internal aldimine . The apoenzyme obtained upon removal of the cofactor was inactive and could be reconstituted by the addition of pyridoxal 5'-phosphate demonstrating that the recombinant SHMT was functionally very similar to the native SHMT . This overexpression of eukaryotic tetrameric SHMT in E . coli and the purification and characterization of the recombinant enzyme should thus allow studies on the role of specific amino acids and domains in the activity of the enzyme. Eur J Biochem, 1995 Jun 1, 230(2), 525 - 32 Transketolase A of Escherichia coli K12 . Purification and properties of the enzyme from recombinant strains; Sprenger GA et al.; Transketolase A was purified to apparent homogeneity from recombinant Escherichia coli K12 cells carrying the homologous cloned tktA gene on a pUC19-derived plasmid . These recombinant cells exhibited a transketolase activity in crude extracts of up to 9.7 U/mg compared to < or = 0.1 U/mg in wild-type cells . Transketolase A was purified from crude extracts of a recombinant strain by successive ammonium sulfate precipitations and two anion-exchange chromatography steps (Q-Sepharose FF, Fractogel EMD-DEAE column) and afforded an apparently homogeneous protein band on SDS/PAGE . The enzyme, both in its active and apoform, had a molecular mass of 145,000 Da (+/- 10,000 Da), judged by gel-filtration chromatography . Subunits of 73,000 Da (+/- 2000 Da) were determined on SDS/PAGE, thus, transketolase A most likely forms a homodimer . N-terminal amino acid sequencing of the protein verified the identity with the cloned gene tktA . The specific activity of the purified enzyme, determined at 30 degrees C with the substrates xylulose 5-phosphate (donor of C2 compound) and ribose 5-phosphate (acceptor) at an optimal pH (50 mM glycylglycine, pH 8.5), was 50.4 U/mg . Km values for the substrates xylulose 5-phosphate and ribose 5-phosphate were 160 microM and 1.4 mM, respectively . Km values for the other physiological substrates of transketolase A were 90 microM for erythrose 4-phosphate (best acceptor substrate), 2.1 mM for D,L-glyceraldehyde 3-phosphate, 1.1 mM for fructose 6-phosphate, and 4 mM for sedoheptulose 7-phosphate . Hydroxypyruvate served as alternative donor (Km = 18 mM) . Unphosphorylated acceptor compounds were formaldehyde (Km = 31 mM), glycolaldehyde (14 mM), D,L-glyceraldehyde (10 mM) and D-erythrose (150 mM) . The enzyme was competitively inhibited by D-arabinose 5-phosphate (K = 6 mM at a concentration of 2.5 mM D-arabinose 5-phosphate) or by the chelating agent EDTA . The inactive apoform of transketolase A was yielded by dialysis against buffer containing 10 mM EDTA, thus removing the cofactors thiamine diphosphate and divalent cations . The reconstitution of the apoenzyme proceded faster in the presence of manganese ions (Kd = 7 microM at 10 microM thiamine diphosphate) than with other divalent cations. Eur J Biochem, 1995 Jun 1, 230(2), 447 - 53 Characterisation of two major cellular poly(rC)-binding human proteins, each containing three K-homologous (KH) domains; Leffers H et al.; We have revealed and characterised two nucleic-acid-binding proteins, termed PCBP-1 (M(r) 37,525, pI 7.07) and PCBP-2 (M(r) 38,579, pI 6.76), that together with heterogeneous ribonucleoparticle (hnRNP)-K correspond to the major cellular poly(rC)-binding proteins . mRNA for both PCBPs were detected in all the human tissues analysed . Both proteins contain three K-homologous (KH) domains which share similarity with other KH domain proteins, including the fragile-X protein FMR1, and which are positioned as in hnRNP-K and nova, i.e . with two closely spaced domains at the N-terminus and one at the C-terminus . PCBPs do not contain RGG boxes or any other known nucleic-acid-binding motifs . Expression in the vaccinia virus system showed that both proteins are post-translationally modified in vivo, a fact that was confirmed by {32P}orthophosphate labelling . Northwestern-blot analysis showed that the non-phosphorylated forms bind tenaciously to poly(rC) in vitro, while significantly less binding was observed for the phosphorylated variants . Escherichia coli expressed proteins also bound poly(rG), albeit at a lower level . In addition, PCBP-2 bound poly(rU), whereas very little binding to poly(rA) was observed for both proteins. Eur J Biochem, 1995 Jun 1, 230(2), 424 - 30 Alternative splicing of calretinin mRNA leads to different forms of calretinin; Schwaller B et al.; cDNA clones for calretinin, a member of the troponin-C family of calcium-binding proteins, were isolated from a cDNA library of the human colon carcinoma cell line WiDr . Sequence analysis revealed two forms of alternatively spliced calretinin mRNAs encoding C-terminally truncated proteins . Exon 7 was either spliced to exon 9 (delta 8) or to exon 10 (delta 8,9); both resulted in a frame shift and a translational stop at the second codon of exon 9 (delta 8), or at codon 15 of exon 10 (delta 8,9), respectively . The presence of delta 8 and delta 8,9 calretinin mRNA in WiDr cells was confirmed using reverse-transcriptase PCR and sequence analysis of the amplicon, as well as by a ribonuclease protection assay . Co115/3 and three other human colon carcinoma cell lines were found, by reverse-transcriptase PCR to also contain delta 8,9 calretinin mRNA . The truncated proteins were able to bind calcium, as evidenced by a calcium blot of the delta 8 form (calretinin-20k) and delta 8,9 form (calretinin-22k) expressed in Escherichia coli . Immunohistochemical staining using an antiserum specific for the novel C-terminus of calretinin-22k confirmed its presence in WiDr, Co115/3 and three additional colon carcinoma cell lines . The fact that alternative splicing of calretinin was found in five different cell lines suggests that alternatively spliced calretinins fulfill a physiological function. Eur J Biochem, 1995 Jun 1, 230(2), 401 - 7 Mutations at the C-terminal isoleucine and other potential iron ligands of 5-lipoxygenase; Hammarberg T et al.; The non-heme iron centre in human 5-lipoxygenase was studied . Recombinant enzyme was expressed in Escherichia coli, purified and assayed for iron content and enzyme activity . For non-mutated enzyme, the iron content was 1.01 +/- 0.19 mol/mol . Deletion of the C-terminal Ile673 resulted in an iron content of 0.03 +/- 0.07 mol/mol and undetectable lipoxygenase activity . Mutations at His367, Glu376 and Asn554 led to drastically decreased enzyme activity (< 2% of non-mutated control) but iron was still present . In addition to Glu376, eight other conserved acidic residues (Asp/Glu) in 5-lipoxygenase were replaced, none of which was crucial for enzyme activity . We conclude that Ile673 is an iron ligand in 5-lipoxygenase, while our results do not support that Glu376 or Asn554 have this function . The possible role of His367 as a replaceable iron ligand is discussed. Eur J Biochem, 1995 Jun 1, 230(2), 384 - 95 DnaA-dependent assembly of the ABC primosome at the A site, a single-stranded DNA hairpin containing a dnaA box; Masai H et al.; The ABC primosome is assembled from DnaA, DnaB and DnaC proteins at a stem-and-loop structure containing a dnaA box within its stem (A site), and catalyses primer RNA synthesis for DNA chain elongation . The DnaA protein can bind to the A site and the A-site-DnaA-protein complex can be isolated by gel-filtration chromatography in the absence of nucleotides . Mutations within the dnaA box completely abolish the binding of DnaA protein . Point mutations within the stem region outside the dnaA box also severely reduce the affinity of DnaA protein for the A site . These results indicate that not only the dnaA box but also other nucleotides and/or secondary structure features of the stem are important for proper recognition of the A site by DnaA protein . The preprimosome, which is able to synthesize RNA primers upon addition of primase, can be isolated by gel-filtration chromatography in the presence of ATP or adenosine 5'-{gamma-thio}triphosphate, a non-hydrolyzable analogue of ATP . The preprimosome can translocate along Escherichia coli single-stranded-DNA-binding protein-coated single-stranded DNA, utilizing the energy released by hydrolysis of ATP, as indicated by its helicase activity . dATP, as well as dCTP, can support the helicase activity of the preprimosome to some extent, while they are inert in helicase assays with DnaB protein in the absence of E . coli single-stranded DNA-binding protein . In keeping with this result, the isolated preprimosome, which appears to contain DnaA and DnaB proteins, is capable of hydrolyzing dATP as well as ATP and GTP . In a reconstituted replication assay, addition of excess dATP restores replication activities which have been inhibited by addition of adenosine 5'-{gamma-thio}triphosphate . The ability of dATP to support helicase and replicative activities of the ABC primosome indicates that the formation of the complex somehow modulates the structures of its component(s) so that they can utilize otherwise inert nucleotides . On the basis of these results, a scheme for the assembly of the ABC primosome at the A site is presented. Development, 1995 Jun, 121(6), 1705 - 18 Mouse chick chimera: a new model to study the in ovo developmental potentialities of mammalian somites; Fontaine-Perus J et al.; Chimeras were prepared by transplanting somites from 9-day post-coitum mouse embryos or somitic dermomyotomes from 10-day post-coitum mouse embryos into 2-day-old chick embryos at different axial levels . Mouse somitic cells then differentiated in ovo in dermis, cartilage and skeletal muscle as they normally do in the course of development and were able to migrate into chick host limb . To trace the behavior of somitic myogenic stem cells more closely, somites arising from mice bearing a transgene of the desmin gene linked to a reporter gene coding for Escherichia coli beta-galactosidase (lacZ) were grafted in ovo . Interestingly, the transgene was rapidly expressed in myotomal muscles derived from implants . In the limb muscle mass, positive cells were found several days after implantation . Activation of desmin nls lacZ also occurred in in vitro cultures of somite-derived cells . Our experimental method facilitates investigation of the mechanisms of mammalian development, allowing the normal fate of implanted mouse cells to be studied and providing suitable conditions for identification of descendants of genetically modified cells. Plant J, 1995 Jun, 7(6), 981 - 8 Tyrosine phosphatase signalling in a lower plant: cell-cycle and oxidative stress-regulated expression of the Chlamydomonas eugametos VH-PTP13 gene; Haring MA et al.; The first evidence for tyrosine phosphatase signalling pathways in plants is presented by characterizing a putative protein tyrosine phosphatase gene from the unicellular green alga Chlamydomonas eugametos . This cDNA, referred to as VH-PTP13, contains an open reading frame specifying a protein with a molecular weight of 30.3 kDa, that has significant homology with a distinct group of dual-specificity phosphatases . The highest homology is found with CL-100, a human stress-response gene that regulates MAPkinase activity . The purified VH-PTP13 protein expressed in E . coli had phosphatase activity and inactivated MAPkinases from alfalfa and tobacco . Nondividing C . eugametos gametes did not express the VH-PTP13 gene whereas synchronously dividing vegetative cells only expressed VH-PTP13 in the early G1-phase of the cycle, implying a function there . When vegetative cells were subjected to oxidative stress, expression of the VH-PTP13 gene was strongly induced, analogous to the human CL-100 gene . Its potential role in plant signalling pathways is discussed. Plant J, 1995 Jun, 7(6), 969 - 80 The regulatory regions of the rice tungro bacilliform virus promoter and interacting nuclear factors in rice (Oryza sativa L.); Yin Y et al.; The Rice Tungro Bacilliform Virus (RTBV) promoter confers phloem-specific gene expression in transgenic rice plants . A series of promoter deletion mutants were fused with the Escherichia coli beta-glucuronidase A (uidA) reporter gene and introduced into transgenic rice plants . The RTBV promoter confers substantially stronger expression in shoots than in roots . A fragment of the promoter comprising nucleotides -164 to +45 relative to the transcriptional start site contains sufficient information for phloem-specific gene expression . Within this region, nucleotides -164 to -43 were essential for promoter function since deletion of this fragment dramatically reduced promoter activity . Gel-retardation assays identified two groups of rice nuclear factors (RNFG1 and RNFG2) that bind to the -164 to +45 promoter fragment . Competition and DNasel footprinting experiments indicated that RNFG1 bound to nucleotides -3 to +8 (Box I) while RNFG2 bound to nucleotides -53 to -39 (Box II) . Interactions between the two groups of factors were observed . In addition, we found differences in the binding of nuclear factors from shoots versus from roots, in agreement with the different activities of the promoter in these two organs . It is proposed that binding of RNFG1 and RNFG2 between nucleotides -164 to +45 is essential for the tissue-specific expression of this promoter. Indian J Biochem Biophys, 1995 Jun, 32(3), 137 - 46 Purification and partial characterization of acyl carrier proteins from developing oil seeds of pisa (Actinodaphne hookeri) and ground nut (Arachis hypogaea); Sreenivas A et al.; Acyl carrier proteins (ACP) were purified to homogeneity in the active form from developing seeds of pisa (Actinodaphne hookeri) which synthesizes exclusively trilaurin and from ground nut (Arachis hypogaea) which synthesizes triacylglycerols containing long chain fatty acids . Two major isoforms of ACPs were purified from developing pisa seeds using DEAE-cellulose, Superose-6 FPLC and C4 reversed phase HPLC chromatographic methods . In contrast, only a single form of ACP was present in ground nut seeds which was purified by anion-exchange and activated thiol-Sepharose 4B affinity chromatography . The two isoforms of ACPs from pisa showed nearly the same specific activity of 6,706 and 7,175 pmol per min per mg protein while ground nut ACP showed a specific activity of 3,893 pmol per min per mg protein when assayed using E . coli acyl-ACP synthetase and {1-14C}palmitic acid . When compared with E . coli ACP, the purified ACPs from both the seeds showed considerable difference in their mobility in native PAGE, but showed similar mobility in SDS-PAGE under reducing conditions . In the absence of reducing agents formation of dimers was quite prominent . The ACPs from both the seed sources were acid- and heat-stable . The major isoform of pisa seed ACP and the ground nut ACP contain 91 amino acids with M(r) 11,616 and 1,228 respectively . However, there is significant variation in their amino acid composition . A comparison of the amino acid sequence in the N-terminal region of pisa and ground nut seed ACPs showed considerable homology between themselves and with other plant ACPs but not with E . coli ACP. Comput Appl Biosci, 1995 Jun, 11(3), 321 - 9 Co-inertia analysis of amino-acid physico-chemical properties and protein composition with the ADE package; Thioulouse J et al.; A multivariate analysis method called co-inertia analysis was used to determine the main relationships between two data tables having identical rows . This method is available in the ADE multivariate analysis package for Macintosh micro-computers . It was applied to two data sets, one containing the amino-acid composition of 999 E . coli proteins, and the other the values of 402 physico-chemical properties for the 20 natural amino-acids . There were strong relationships between amino-acid physico-chemical properties and the composition of proteins . The first common factor was hydrophobicity; it is linked to the biological environment of proteins, either in the cytoplasm (or outside the cell), or in the nonpolar environment of the phospholipid bilayer of biological membranes . The second factor linked the expressivity of protein genes and the propensity of amino-acids to form alpha helix/beta sheets . The third factor showed that heavy, aromatic amino-acids tend to be avoided, except when they are needed for structural or functional reasons . These results are discussed in terms of selective pressure acting on amino-acid composition of proteins. Br J Pharmacol, 1995 Jun, 115(3), 498 - 502 Attenuation by nitrosothiol NO donors of acute intestinal microvascular dysfunction in the rat; Laszlo F et al.; 1 . The effects of the nitric oxide (NO) donors, S-nitroso-glutathione (SNOG) and S-nitroso-N-acetyl-penicillamine (SNAP), on the acute intestinal microvascular dysfunction induced by NG-nitro-L-arginine methyl ester (L-NAME) in combination with low doses of endotoxin were investigated in the anaesthetized rat . 2 . Administration of L-NAME (5 mg kg-1, s.c.) concurrently with E . coli lipopolysaccharide (LPS, 3 mg kg-1, i.v.) provoked the leakage of radiolabelled albumin in the ileum and colon, as a measure of microvascular damage, determined 1 h after challenge . 3 . Intravenous infusion of SNOG or SNAP (1-10 micrograms kg-1 min-1) dose-dependently attenuated the microvascular leakage induced by L-NAME and LPS . 4 . Infusion of the lowest doses of SNOG or SNAP (1 microgram kg-1 min-1, i.v.) that significantly reduced the albumin leakage, did not affect the increase in blood pressure in response to L-NAME in LPS-treated rats . Higher doses of SNOG or SNAP (5-10 micrograms kg-1 min-1, i.v.) dose-dependently reduced this increase in blood pressure . 5 . In control studies, intravenous infusion of glutathione (10 micrograms kg-1 min-1) or N-acetyl-penicillamine (10 micrograms kg-1 min-1) had no effect on microvascular leakage in the ileum and colon induced by LPS and L-NAME . 6 . Pretreatment with rabbit anti-rat neutrophil serum (0.4 ml kg-1, i.p., 4 h before challenge), which reduced the neutrophil count in peripheral arterial blood, also inhibited the microvascular leakage in the ileum and colon.(ABSTRACT TRUNCATED AT 250 WORDS) FEMS Immunol Med Microbiol, 1995 Jun, 11(3), 197 - 205 A peptide, ALTTE, within the fimbrial subunit protein from Porphyromonas gingivalis, induces production of interleukin 6, gene expression and protein phosphorylation in human peripheral blood mononuclear cells; Ogawa T et al.; Porphyromonas gingivalis 381 fimbriae and a synthetic peptide composed of residues 69-73 (ALTTE) of the fimbrial subunit protein, FP381(69-73), function in the induction of interleukin 6 (IL-6) production, IL-6 mRNA expression, and tyrosine and serine/threonine phosphorylation of several proteins in human peripheral blood mononuclear cells (PBMC) . Herbimycin A and H-7, inhibitors of tyrosine kinases and protein kinase C (PKC), markedly inhibited IL-6 production, gene expression, and tyrosine and serine/threonine phosphorylation of proteins . An inactive analog of synthetic peptide replaced alanine to glycine at position 69 in FP381(69-73), GLTTE, exhibited an antagonistic effect on the IL-6 production induced by the fimbriae . These results suggest that the peptide ALTTE functions as an agent in inflammatory reactions and immune responses in the inflamed gingival and periodontal tissues, in which the participation of protein phosphorylation by tyrosine kinases and PKC in signal transduction may be considered. FEMS Immunol Med Microbiol, 1995 Jun, 11(3), 171 - 80 Prophylactic use of human endotoxin-core hyperimmune gammaglobulin to prevent endotoxaemia in colostrum-deprived, gnotobiotic lambs challenged orally with Escherichia coli; Hodgson JC et al.; The efficacy of human IgG polyclonal antibody to endotoxin-core in preventing endotoxaemia and subsequent disease was studied in colostrum-deprived gnotobiotic lambs challenged orally at about 5 h old with 10(9) cfu Escherichia coli . Human endotoxin-core hyperimmune gammaglobulin was given intravenously to 5 lambs at 1.9 g IgG/kg bodyweight prior to challenge . Human albumin was given intravenously to 3 control lambs . Bacteraemia was observed in all lambs, but the incidence was lower (P < 0.01) and the onset later (P < 0.05) in gammaglobulin pre-treated lambs . These lambs showed no signs of disease, whereas clinical endotoxaemia, manifesting as watery mouth disease, was diagnosed in 2 of the 3 control lambs which were killed between 18 and 22 h after challenge . Thus, prophylactic treatment of colostrum-deprived lambs with human IgG enriched in endotoxin-core antibodies was effective in reducing the degree of bacteraemia and preventing endotoxaemia, leukopenia and clinical disease following oral challenge with E . coli. FEMS Immunol Med Microbiol, 1995 Jun, 11(3), 163 - 9 Immunoaffinity chromatographic isolation of a high molecular weight seroreactive protein from Mycobacterium leprae cell sonicate; Deshpande RG et al.; The purpose of this study was to isolate Mycobacterium leprae antigen(s) by immunoaffinity chromatography using immunoglobulins from leprosy patients and from rabbit anti-M . leprae hyperimmune serum coupled to CNBr-Sepharose 4B . A high molecular weight (M(r)) M . leprae protein (MLP) with a subunit M(r) of 22,000 was isolated . MLP was recognized by monoclonal antibody MMPII1G4 which is known to react with MMPII, a 22 kDa protein of M . leprae . The N-terminal sequence of the 22 kDa subunit (Met-gln-gly-asp-pro-asp-val-leu-arg-leu-leu-asn-glu-gln-leu-thr) was identical to MMPII and to antigen D (bacterioferritin) of M . paratuberculosis . It showed 44% homology with N-terminal end of E . coli bacterioferritin . In ELISA, MLP showed 100% and 60% positivity with leprosy and TB sera respectively as compared to normal healthy sera . The role of bacterioferritin in M . leprae and the importance of MLP as an immunogen has been discussed. Int J Dev Biol, 1995 Jun, 39(3), 501 - 10 Analysis of polysulfate-binding domains in porcine proacrosin, a putative zona adhesion protein from mammalian spermatozoa; Jansen S et al.; Proacrosin is one of the major proteins found within the acrosomal vesicle of mammalian spermatozoa . Previous work has shown that it binds non-enzymatically and with high affinity to polysulfate groups on zona pellucida glycoproteins (ZPGPs) thereby leading to the hypothesis that at fertilization it functions as a secondary ligand molecule to retain acrosome-reacted spermatozoa on the surface of the egg . In the present work we have investigated the nature and extent of the polysulfate binding domain on boar sperm proacrosin using a combination of group-specific modifying reagents, fragmentation analysis, peptide synthesis and expression of deletion recombinants in E . coli bacteria . Taken overall, our results show that arginine, lysine and histidine residues located between Gly 93 and Ala 275, together with the participation of His 47 and Arg 50, are necessary for maximum polysulfate binding activity . The secondary and tertiary structure of this central peptide domain is also important to ensure correct alignment of basic residues with complementary sulfate groups on ZPGPs . Proacrosin, therefore, has many properties in common with other polysulfate binding proteins, such as antithrombin III and sea urchin sperm binding, in having a conformation-dependent domain containing basic amino acids that mediates specific protein-protein interactions . These observations strengthen the hypothesis that proacrosin is a multifunctional protein with a major role as a ligand molecule at fertilization. Int J Dev Biol, 1995 Jun, 39(3), 469 - 75 Desmin transgene expression in mouse somites requires the presence of the neural tube; Lee KK et al.; Transgenic mice were used to study the effect of the neural tube on somite myogenesis . These mice express a transgene in which the 1 kb DNA 5' regulatory sequence of the desmin gene is linked to a reporter gene which codes for E . coli beta-galactosidase . In order to determine whether the developmental fate of cells, specifically the prospective myogenic population, in newly developed somites was pre-determined, newly formed somites were isolated from the caudal region of day 9.5 transgenic embryos and transplanted into 8.5 day non-transgenic host embryos . Even though the implanted somites were not oriented in the host embryos, all the specimens examined developed normally at the graft site forming a dermatome, myotome and sclerotome in the correct anatomical positions . The myotome even expressed the desmin transgene . In addition, we isolated the 3 most caudal somites, that is, the most recently developed somites, from day 9.5 transgenic embryos and maintained them on gelatin-coated coverslips in culture for up to 4 days . While these somite explants did not develop myoblasts, it was possible to induce myogenesis by introducing pieces of neural tube into the explant cultures . These results suggest that the developmental fate of cells within the newly formed somite is not predetermined, but is dependent on the microenvironment surrounding the developing somite. Biol Chem Hoppe Seyler, 1995 Jun, 376(6), 385 - 8 Expression of full-length human procathepsin L cDNA in Escherichia coli and refolding of the expression product; Dolinar M et al.; From human embrional lung fibroblasts mRNA was obtained and converted to cDNA . The procathepsin L coding region was amplified by PCR, inserted into pALTER and, after checking the nucleotide sequence, transferred into pET81F1+ . Procathepsin L was expressed by induction of recombinant E . coli strain BL21{DE3}(pLysS) with IPTG and was found to be deposited into inclusion bodies . These were isolated and solubilized in guanidinium hydrochloride . The soluble proteins were sulphonated and procathepsin L was obtained after gel filtration . Purified proenzyme was refolded by dialysis and autoactivated into a form of the expected size and enzymatic activity against a fluorogenic substrate. Bioessays, 1995 Jun, 17(6), 527 - 36 Overview of controls in the Escherichia coli cell cycle; Vinella D et al.; The harmonious growth and cell-to-cell uniformity of steady-state bacterial populations indicate the existence of a well-regulated cell cycle, responding to a set of internal signals . In Escherichia coli, the key events of this cycle are the initiation of DNA replication, nucleoid segregation and the initiation of cell division . The replication initiator is the DnaA protein . In nucleoid segregation, the MukB protein, required for proper partitioning, may be a member of the myosin-kinesin superfamily of mechanoenzymes . In cell division, the FtsZ protein has a tubulin motif, is a GTPase and polymerizes in a ring around midcell during septation; the FtsA protein has an actin-like structure . The nature of the internal signals triggering these events is not known but candidates include cell mass, the superhelical density of the chromosome and the concentration of two regulatory nucleotides, cyclic AMP and ppGpp . The involvement of cytoskeletal-like proteins in key cycle events encourages the notion of a fundamental biological unity in cell cycle regulation in all organisms. Int J Dev Neurosci, 1995 Jun-Jul, 13(3-4), 167 - 78 Modulation of interleukin-1 receptors in the neuro-endocrine-immune axis; Takao T et al.; Interleukin-1 (IL-1) receptors with kinetics, pharmacological and biochemical characteristics of type I IL-1 receptors have been identified in the mouse neuro-endocrine-immune axis . In the present study, we examined the in-vitro and in-vivo modulation of IL-1 receptors by stress and endotoxin treatment . The treatment of AtT-20 mouse pituitary adenoma cells for 24 hr with neuro-endocrine mediators of stress such as corticotropin releasing factor (CRF) and catecholamine (beta 2 adrenergic) receptor agonists produced a dose-dependent increase in cAMP and {125I}IL-1 alpha binding . In contrast, somatostatin and dexamethasone significantly inhibited CRF-stimulated cAMP production and decreased both basal and CRF-mediated increase of {125I}IL-1 alpha binding . Furthermore, in keeping with the effects of stress mediators to upregulate IL-1 receptors in AtT-20 cells, ether-laparotomy stress in mice resulted in a significant increase in {125I}IL-1 alpha binding in the pituitary with no significant alterations observed in the brain; in contrast, {125I}oCRF binding in the pituitary was significantly decreased after the ether-laparotomy stress . Next, we investigated the modulation of IL-1 beta levels and {125I}IL-1 alpha binding following endotoxin lipopolysaccharide (LPS) treatment . IL-1 beta levels were dramatically increased in the peripheral tissues (pituitary, testis and spleen) at 2-6 hr after a single LPS injection (30 micrograms LPS/mouse) . However, no significant changes were observed in brain (hippocampus and hypothalamus) . {125I}IL-1 alpha binding in the pituitary gland, liver, spleen and testis was significantly decreased at 2 hr following a single administration of both low (30 micrograms LPS/mouse) and high (300 micrograms LPS/mouse) doses of endotoxin . {125I}IL-1 alpha binding in the hippocampus was not significantly altered at 2 hr by a low dose of LPS and was significantly decreased by high dose administration of LPS (300 micrograms/mouse) . Following two LPS injections (at 0 and 12 hr), dramatic increases in IL-1 beta concentrations in the hypothalamus, hippocampus, spleen and testis were observed at 2 hr after the second LPS injection; a small but statistically nonsignificant change was evident in the pituitary . Moreover, dramatic decreases in {125I}IL-1 alpha binding were seen after two injections of 30 micrograms LPS/mouse in both central and peripheral tissues . These data provide further support for a role for IL-1 in co-ordinating neuro-endocrine-immune responses to stress and infection. Ultramicroscopy, 1995 Jun, 58(3-4), 381 - 91 Automatic particle picking from electron micrographs; Lata KR et al.; A computer program for automatic particle picking based on textural methods is proposed . The technique relies on the evaluation of certain textural parameters for data windows containing single particles, and those containing undesirable material . These parameters are manipulated by a discriminant analysis routine for determining the rules of classification between the different categories . The effectiveness of the method was demonstrated by application to electron micrographs of 70S Escherichia coli ribosomes. Proteins, 1995 Jun, 22(2), 81 - 99 LINUS: a hierarchic procedure to predict the fold of a protein; Srinivasan R et al.; We describe LINUS, a hierarchic procedure to predict the fold of a protein from its amino acid sequence alone . The algorithm, which has been implemented in a computer program, was applied to large, overlapping fragments from a diverse test set of 7 X-ray-elucidated proteins, with encouraging results . For all proteins but one, the overall fragment topology is well predicted, including both secondary and supersecondary structure . The algorithm was also applied to a molecule of unknown conformation, groES, in which X-ray structure determination is presently ongoing . LINUS is an acronym for Local Independently Nucleated Units of Structure . The procedure ascends the folding hierarchy in discrete stages, with concomitant accretion of structure at each step . The chain is represented by simplified geometry and folds under the influence of a primitive energy function . The only accurately described energetic quantity in this work is hard sphere repulsion--the principal force involved in organizing protein conformation {Richards, F . M . Ann . Rev . Biophys . Bioeng . 6:151-176, 1977} . Among other applications, the method is a natural tool for use in the human genome initiative. Proteins, 1995 Jun, 22(2), 191 - 2 Crystallization of UDP-N-acetylglucosamine O-acyltransferase from Escherichia coli; Pfitzner U et al.; Crystals of UDP-N-acetylglucosamine O-acyltransferase (lpxA) from Escherichia coli have been obtained from solutions of sodium/potassium phosphate and dimethylsulfoxide . These crystals belong to the cubic space group P2(1)3 (a = 99.0 A), diffract X-rays to approximately 2.5 A resolution and contain one subunit of the enzyme in the asymmetric unit. Proteins, 1995 Jun, 22(2), 187 - 90 Crystallization and preliminary X-ray investigation of recombinant human interleukin 10; Cook WJ et al.; Crystals of recombinant human interleukin 10 have been grown from solutions of ammonium sulfate . The crystals are tetragonal, space group P4(1)2(1)2 or P4(3)2(1)2; the unit cell axes are a = 36.5 A and c = 221.9 A . There is the equivalent of one polypeptide chain in the asymmetric unit . The crystals are stable to X-rays and diffract to at least 2.5 A resolution. Comp Immunol Microbiol Infect Dis, 1995 Jun, 18(3), 209 - 14 Influence of the temperature upon the proliferative response of lymphocytes of tench (Tinca tinca) during winter and summer; Collazos ME et al.; The influence of incubation temperature upon proliferation of Tinca tinca lymphocytes induced by phytohemagglutinin (PHA) and E . coli lipopolisaccharide (LPS) mitogens was studied during the summer and the winter . The cultures were performed in vitro at 22 degrees C in both summer and winter, and at 12 and 30 degrees C in winter and summer respectively . The proliferative response at 22 degrees C was higher than that at 12 degrees C during the winter, and a small increase was observed at 30 degrees C respective to 22 degrees C in summer . These results indicate that in vitro lymphocyte proliferation requires temperatures higher than those in the fish environment. Comp Immunol Microbiol Infect Dis, 1995 Jun, 18(3), 179 - 88 Virulence factors associated with strains of Escherichia coli from cases of sudden infant death syndrome (SIDS); Bettelheim KA et al.; Strains of Escherichia coli isolated from cases of Sudden Infant Death Syndrome, healthy infants and infants that died of other causes were subjected to a series of tests with particular reference to serotyping, toxigenicity and adherence factors . E . coli from SIDS infants tended to have a low hydrophobicity and high toxigenicity, compared to those from healthy infants, while no notable differences in haemagglutination patterns were observed between these two groups of strains. J Interferon Cytokine Res, 1995 Jun, 15(6), 547 - 55 Amino acid sequence analysis, gene construction, cloning, and expression of gelonin, a toxin derived from Gelonium multiflorum; Rosenblum MG et al.; The plant toxin gelonin is an extremely potent inhibitor of protein synthesis, similar in action to ricin . The mature protein primary sequence was obtained using conventional sequencing techniques . Gelonin was found to be composed of 258 amino acids and contains 21 lysine residues . This toxin shares approximately 33% sequence homology with trichosanthin and ricin A chain . A 774 bp synthetic gene encoding gelonin was synthesized and expressed in E . coli . Recombinant gelonin (approximately 28 kD) expression was monitored and demonstrated by western analysis . Purification and functional activity studies demonstrated that this protein behaves identically to that of the natural product . Recombinant gelonin (RG) thus joins a growing list of recombinant toxins currently available for use in the construction of recombinant immunotoxins composed of gelonin fused to binding domains of antibodies, growth factors, or other cytokines. Biol Pharm Bull, 1995 Jun, 18(6), 900 - 2 Enzyme immunoassay of elcatonin in human plasma; Takeyama M et al.; A sensitive and specific double-antibody enzyme immunoassay (EIA) for detecting an elcatonin-like immunoreactive substance (ECT-IS) in human plasma has been developed . In competitive reactions, the ECT antibody was incubated with a plasma sample (or ECT standard) and beta-D-galactosidase-linked synthetic ECT . Free and antibody-bound enzymes were separated using an anti-rabbit IgG-coated immunoplate . Enzyme activity on the plate was determined by fluorescence analysis . This immunoassay allows the detection of 20 to 300 fmol/ml (67 to 1000 pg/ml) of ECT . The EIA was applied to determine the pharmacokinetic behavior of ECT after a single intramuscular administration (20 IU) . The maximum level was achieved 30 min after administration, at approximately 30 pg ECT/ml of plasma. Biol Pharm Bull, 1995 Jun, 18(6), 854 - 8 Oral tolerance to ovalbumin in mice as a model for detecting modulators of the immunologic tolerance to a specific antigen; Kim JH et al.; Oral tolerance is thought to have a role in preventing allergic responses and immune-mediated diseases . Modulation of this tolerance by drugs and chemicals can cause or suppress them . An improved model of oral tolerance to ovalbumin (OVA) in mice was developed to detect modulators of the tolerance and to apply it to selected immunomodulating substances, cyclophosphamide (CP), Escherichia coli lipopolysaccharide (LPS) and cadmium chloride (Cd) . Male C3H/HeN mice given an oral administration of 20 mg OVA were immunized 7 d later with an i.p . injection of 0.1 mg OVA in complete Freund's adjuvant . Effects of oral OVA and agents on systemic immunity were assessed by enzyme-linked immunosorbent assay (ELISA) of immunoglobulin (Ig) levels in serum collected 7 or 14 d after immunization . Oral tolerance was adequately induced on day 7 after immunization and was more effective in C3H/HeN mice than in BALB/c mice . It was primarily associated with the decreased serum levels of anti-OVA IgG (including both IgG1 and IgG2a subclasses regulated differently by T-helper subpopulations, Th2 and Th1 cells, respectively) . The C3H model of oral tolerance was further examined to detect modulators of the tolerance . An i.p . injection of CP prior to oral OVA, or 5 consecutive daily oral administrations of LPS after oral OVA elevated or reduced serum levels of anti-OVA IgG in C3H mice hyposensitized by the oral OVA, respectively . Concerning IgG subclasses, CP restored anti-OVA IgG2a but not IgG1 levels, while LPS caused greater suppression of both anti-OVA IgG1 and IgG2a levels . Oral administrations of Cd for 5 d after oral OVA also suppressed anti-OVA IgG1 levels further.(ABSTRACT TRUNCATED AT 250 WORDS) Biol Pharm Bull, 1995 Jun, 18(6), 797 - 801 Activity of artificial mutant variants of human growth hormone changes in charged residues around 62-67; Uchida E et al.; Our previous work has shown that the amino acid residues around 62-67 located in the connecting loop between helix I and II of human growth hormone (hGH) are important in eliciting the differentiation of preadipose 3T3-F442A cells to adipocytes . In this study, we evaluated the role of the charged residues around 62-67 in receptor binding and biological activity . Eight artificial mutant variants of hGH were prepared in Escherichia coli by site-directed mutagenesis . Replacement of Arg64 with Tyr (R64Y variant) resulted in a significant loss of binding to the somatogenic receptors on 3T3-F442A cells, but retained full adipose conversion activity on these cells . Replacement of Arg64 with Glu (R64E) produced a considerable loss in receptor binding and a significant loss in biological activity . hGH variants in which either Glu65 or Glu66 was replaced with Asp (E65D and E66D) and with Gln (E65Q and E66Q) showed a slight loss in binding activity and retained almost a full adipogenic activity . An E65P variant (replacement of Glu65 with Pro) possessed the same binding activity as hGH, although it failed to induce full biological activity . The insertion of Ala between Asn63 and Arg64 (63NAR) caused a marked loss in both activities . These results indicate that the positively charged Arg64 is important for receptor binding and thereby in eliciting the biological activity of hGH, while negatively charged Glu65 and Glu66 are less important . In addition, our findings confirm that the conformation and size of the loop region around Arg64 is important for the adipose conversion activity of hGH. Protein Sci, 1995 Jun, 4(6), 1118 - 23 Interaction of SecB with intermediates along the folding pathway of maltose-binding protein; Diamond DL et al.; SecB, a molecular chaperone involved in protein export in Escherichia coli, displays the remarkable ability to selectively bind many different polypeptide ligands whose only common feature is that of being nonnative . The selectivity is explained in part by a kinetic partitioning between the folding of a polypeptide and its association with SecB . SecB has no affinity for native, stably folded polypeptides but interacts tightly with polypeptides that are nonnative . In order to better understand the nature of the binding, we have examined the interaction of SecB with intermediates along the folding pathway of maltose-binding protein . Taking advantage of forms of maltose-binding protein that are altered in their folding properties, we show that the first intermediate in folding, represented by the collapsed state, binds to SecB, and that the polypeptide remains active as a ligand until it crosses the final energy barrier to attain the native state. Protein Sci, 1995 Jun, 4(6), 1100 - 7 Quantitative approaches to utilizing mutational analysis and disulfide crosslinking for modeling a transmembrane domain; Lee GF et al.; The transmembrane domain of chemoreceptor Trg from Escherichia coli contains four transmembrane segments in its native homodimer, two from each subunit . We had previously used mutational analysis and sulfhydryl cross-linking between introduced cysteines to obtain data relevant to the three-dimensional organization of this domain . In the current study we used Fourier analysis to assess these data quantitatively for periodicity along the sequences of the segments . The analyses provided a strong indication of alpha-helical periodicity in the first transmembrane segment and a substantial indication of that periodicity for the second segment . On this basis, we considered both segments as idealized alpha-helices and proceeded to model the transmembrane domain as a unit of four helices . For this modeling, we calculated helical crosslinking moments, parameters analogous to helical hydrophobic moments, as a quantitative way of condensing and utilizing a large body of crosslinking data . Crosslinking moments were used to define the relative separation and orientation of helical pairs, thus creating a quantitatively derived model for the transmembrane domain of Trg . Utilization of Fourier transforms to provide a quantitative indication of periodicity in data from analyses of transmembrane segments, in combination with helical crosslinking moments to position helical pairs should be useful in modeling other transmembrane domains. Curr Opin Genet Dev, 1995 Jun, 5(3), 382 - 95 Identification of mismatch repair genes and their role in the development of cancer; Fishel R et al.; Mismatched base pairs are generated by damage to DNA, by damage to nucleotide precursors, by errors that occur during DNA replication, and during the formation of intermediates in genetic recombination . Enzyme systems that faithfully repair these DNA aberrations have been identified in a wide variety of organisms . At lease some of the components of these repair systems have been conserved, both structurally and functionally, throughout evolutionary time . In humans, defective mismatch repair genes have been linked to hereditary nonpolyposis colon cancer as well as to sporadic cancers that exhibit length polmorphisms in simple repeat (microsatellite) DNA sequences . The involvement of mismatch repair defects in microsatellite instability and tumorigenesis suggests that a generalized mutator phenotype is responsible for the large number of genetic alterations observed in tumors. Tuber Lung Dis, 1995 Jun, 76(3), 240 - 7 Comparison of the ability of Mycobacterium avium, M . smegmatis and M . tuberculosis to invade and replicate within HEp-2 epithelial cells; Bermudez LE et al.; OBJECTIVE: Previous studies have demonstrated that mycobacteria can interact with epithelial cells, a property which can be important for establishing infection . In this study we investigated comparatively the ability of Mycobacterium avium, M . tuberculosis and M . smegmatis to invade and multiply within HEp-2 epithelial cells . DESIGN: The ability to invade and to multiply intracellularly in HEp-2 cells was examined using a virulent strain of M . avium, a virulent (H37Rv) and an attenuated (H37Ra) strain of M . tuberculosis and a strain of M . smegmatis . The locus responsible for M . avium invasion was also cloned in Escherichia coli and M . smegmatis . RESULTS: It was observed that M . avium invaded HEp-2 cells with greater efficiency than M . tuberculosis and M . smegmatis, while the H37Rv strain of M . tuberculosis was more efficient in invading HEp-2 than H37Ra and M . smegmatis . Both M . avium and M . tuberculosis were capable of multiplying within HEp-2 cells, while M . smegmatis was not . E . coli K12 and M . smegmatis were transformed with M . avium DNA . The invasive locus of M . avium provided E . coli K12 and M . smegmatis strains S5M101-1 and S5M101-2 with the ability to invade HEp-2 epithelial cells . Transformed M . smegmatis strains were able to grow intracellularly . CONCLUSION: 'Virulent' strains of M . avium and M . tuberculosis were shown to invade and to multiply within HEp-2 epithelial cells . This property was transferred to E . coli K12 and M . smegmatis by transformation with the invasive locus of M . avium . The ability of certain strains of mycobacteria to invade epithelial cells (bronchial, alveolar, intestinal) may represent an important phenotypic characteristic and could be directly related to pathogenicity. Chem Res Toxicol, 1995 Jun, 8(4), 580 - 5 Stereoselective catalysis of a retro-Michael reaction by class mu glutathione transferases . Consequences for the internal distribution of products in the active site; Chen J et al.; The reaction of glutathione (GSH) with trans-4-phenyl-3-buten-2-one (PBO) is readily reversible in aqueous solution with an apparent (pH-dependent) equilibrium constant at pH 8 of 6.4 x 10(2) M-1 . Two class mu isoenzymes of GSH transferase from rat (M1-1 and M2-2) and two site specific mutants (M1-1/V9I and M2-2/I9V) catalyze the addition of GSH to PBO and the elimination of GSH from the two diastereomeric products (isomers A and B) of 4-(S-glutathionyl)-4-phenyl-2-butanone with varying degrees of efficiency and stereoselectivity, with the major kinetic product in the addition reaction (isomer A) being the preferred substrate for the elimination reaction . The kinetic stereoselectivity of the addition reaction and the steady-state kinetics of the elimination reactions with product isomers A and B are used to estimate internal stereochemical equilibrium constants in which product isomer B is predominant . This result is consistent with the internal equilibrium constants measured under conditions of enzyme in excess . The results can be used to construct reaction coordinate diagrams for the interconversion of central complexes in the enzyme-catalyzed reactions . The possible metabolic consequences of the reversibility of additions of GSH to alpha, beta-unsaturated carbonyl compounds are discussed. Chem Res Toxicol, 1995 Jun, 8(4), 574 - 9 Escherichia coli expression of site-directed mutants of cytochrome P450 2B1 from six substrate recognition sites: substrate specificity and inhibitor selectivity studies; He YQ et al.; Cytochrome P450 2B1 wild-type and eight site-directed mutations at positions 114, 206, 236, 302, 363, 367, and 478 have been expressed in an Escherichia coli system . Solubilized membrane preparations yielded 100-180 nmol of P450/L of culture . The metabolism of a number of substrates including androstenedione, progesterone, (benzyloxy)resorufin, pentoxyresorufin, and benzphetamine was analyzed . The E . coli-expressed enzymes displayed the same androstenedione metabolite profiles previously observed with a COS cell expression system . Several of the mutants exhibited an increased rate of progesterone hydroxylation, possibly as the result of an enlarged substrate binding pocket and increased D-ring alpha-face binding . (Benzyloxy)resorufin and pentoxyresorufin O-dealkylation by the P450 2B1 mutants exhibited activities ranging from 10% to 99% and 3% to 71% of wild-type, respectively . Interestingly, the Val-363-->Leu mutant showed markedly suppressed pentoxyresorufin but unaltered (benzyloxy)resorufin dealkylase activity . Benzphetamine N-demethylase activities ranged from 28% to 110% of wild-type . Mechanism-based inactivation of the P450 2B1 mutants showed that susceptibility to inactivation by chloramphenicol and D-erythro- and L-threo-chloramphenicol was abolished in the Val-367-->Ala mutant . The Val-363-->Leu mutant was refractory to L-threo-chloramphenicol . Studies of chloramphenicol covalent binding and metabolism by the Val-367-->Ala mutant showed that its resistance to inactivation is largely attributable to an inability to bioactivate the inhibitor . The expression of P450 2B1 wild-type and mutants in E . coli provides an excellent opportunity to study structure/function relationships by site-directed mutagenesis. Mech Dev, 1995 Jun, 51(2-3), 199 - 215 Patterns of conservation and divergence at the even-skipped locus of Drosophila; Sackerson C; The even-skipped (eve) gene of Drosophila melanogaster has been intensively studied as a model for spatial and temporal control of gene expression, using in vitro and transgenic techniques . Here, the study of eve is extended, using evolutionary conservation of DNA sequences . Conservation of much of the protein, and of known regulatory elements, supports models for eve function and regulation that have previously been advanced, and extensive conservation found in noncoding sequences predicts that functional elements exist that have yet to be defined . In contrast, a part of the protein implicated in transcriptional repression has diverged extensively while preserving overall amino acid composition, highlighting potentially essential features of this domain . Also, the basal promoter has diverged extensively, indicating evolutionary flexibility of promoter function. J Virol Methods, 1995 Jun, 53(2-3), 235 - 44 Comparable sensitivities for detection of HIV-1 reverse transcriptase (RT) and other polymerases by RT assays requiring no radioisotopic materials; Sano K et al.; An improved non-radioisotopic (Non-RI) reverse transcriptase (RT) assay with a template-primer-immobilized microtiter plate is described, which has greater sensitivity than the former Non-RI RT assay previously described . Non-RI and commercially available non-radioactive (Non-RA) RT assays were compared for their ability to detect various polymerases . Two RTs from Rous-associated virus 2 (RAV-2) and avian myeloblastosis virus (AMV), one polymerase from Escherichia coli (Pol-I) and one recombinant RT of human immunodeficiency virus type 1 (HIV-1) were assessed . Two HIV-1 samples in a culture supernatant and pelleted virion suspended in Triton X-100 solution were measured . The Non-RI RT assay was one hundred times more sensitive by RAV-2 and Pol-I polymerases, and one thousand times more sensitive by the Non-RA assay than by the AMV RT . The Non-RI RT assay was 10, 16 and 64 times more sensitive than the Non-RA assay for measuring recombinant HIV-1 RT, pelleted virus and virus suspended in culture medium, respectively . To explain the discrepancy, it is shown that free biotin, such as in culture medium, disturbs the assay system of the Non-RA RT assay, but not the Non-RI assay . The present assay can be used to clarify the inhibitory mechanism of an anti-HIV-1 substance. Microbiology, 1995 Jun, 141 ( Pt 6), 1321 - 9 Transcriptional and translational regulation of the expression of the major outer surface proteins in Lyme disease Borrelia strains; Jonsson M et al.; The major outer surface proteins of Lyme disease spirochaetes are differentially expressed in different isolates . Borrelia afzelii strain F1 expresses none, or very low amounts, of the OspA and OspB proteins . To elucidate the mechanisms that control the expression of these abundant surface proteins the ospAB operon of B . afzelii F1 was cloned, sequenced and compared to the previously sequenced ospAB operon of B . afzelii ACAI and Borrelia burgdorferi B31 . The two B . afzelii strains showed almost 100% identity at the DNA level, although Coomassie-stained gels and Western blot analyses showed significant variation in the Osp protein content . Transcriptional analysis revealed that the amount of ospAB mRNA produced in B . afzelii F1 varies more than the amount of protein, suggesting that the expression of OspA and OspB proteins is regulated at both the transcriptional and the translational level . Furthermore, the inverse relationship between the transcription of ospC and the ospAB operon could indicate coregulation of these separately encoded operons. J Clin Pathol, 1995 Jun, 48(6), 525 - 30 Activated phenotype in neutrophils and monocytes from patients with primary proliferative polycythaemia; Westwood NB et al.; AIM--To investigate whether monocytes and neutrophils from patients with primary proliferative polycythaemia (PPP) exhibit increased expression of markers of cell activation and, if so, whether they are associated with the phagocytic activity of these cells and concentrations of circulating cytokines . METHODS--Expression of CD11b, CD14, CD18, and CD64 on monocytes and neutrophils was assessed by flow cytometry . Phagocytosis was analysed using immunoglobulin opsonised Escherichia coli . Serum concentrations of granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF) and macrophage CSF (M-CSF) were determined by bioassays, and interferon-gamma (IFN-gamma) by enzyme linked immunosorbent assay (ELISA) . RESULTS--Patients with PPP (n = 18), when compared with normal subjects (n = 10), had increased percentages of CD64+ monocytes (52% v 36%) and neutrophils (42% v 11%) and of CD14+ neutrophils (36% v 18%) . Monocytes from patients with PPP exhibited increased expression of CD64 (47 v 26) and of CD11b (65 v 36) . These abnormalities were not found in patients with secondary (n = 8) or apparent (n = 13) polycythaemia . The percentage of neutrophils undergoing phagocytosis was higher in patients with PPP (mean 64%; n = 6) than in normal subjects (mean 42%; n = 5) . G-CSF, GM-CSF and IFN-gamma concentrations in patients' serum samples were comparable with normal; M-CSF was not detected in any of the samples . There was no correlation between cytokine concentrations and the expression of CD11b, CD14, CD18, and CD64 on patients' phagocytes . CONCLUSIONS--Increased expression of CD11b and CD64 by monocytes, increased percentages of CD14+ and CD64+ neutrophils and the high phagocytic activity of neutrophils suggests that these cells are activated in vivo in patients with PPP . The phenotypic changes of PPP phagocytes were not associated with increased concentrations of circulating cytokines and probably reflect intrinsic abnormalities within the neoplastic PPP clone. Biokhimiia, 1995 Jun, 60(6), 874 - 82 {Recombinant reverse transcriptase from Rous sarcoma virus . Kinetics and inhibition of DNA polymerase activity}; Chernov AP et al.; Preparations of Rous sarcoma virus reverse transcriptase isolated from a culture of E . coli HB101 (pMF14) and purified to homogeneity were used to study the steady state kinetics of DNA polymerization and inhibition of DNA-polymerase activity . DNA synthesis was examined using a system of poly(rA) as template, oligo(dT) as primer and dTTP as nucleotide substrate . Kinetic constants for steady state conditions were determined . The substrate initial velocity patterns point to an ordered mechanism which results in the formation of a ternary complex, in which the template-primer is the first to bind to the enzyme . Inhibition of the DNA-polymerase activity of the enzyme by various inhibitors was studied . Analysis of final products of the DNA-polymerase reaction revealed the presence of distribution syntheses of the DNA chain by the alpha alpha-subunit form of the enzyme. J Allergy Clin Immunol, 1995 Jun, 95(6), 1229 - 35 Natural and recombinant enzymatically active or inactive bee venom phospholipase A2 has the same potency to release histamine from basophils in patients with Hymenoptera allergy; Forster E et al.; BACKGROUND: A complementary DNA encoding the major bee venom allergen phospholipase A2 (PLA) has been characterized recently . Recombinant PLA was produced in Escherichia coli and purified to apparent homogeneity . Natural PLA was compared with recombinant PLA in its ability to release histamine from blood basophils . METHODS: A synthetic gene encoding the mature form of PLA was expressed in E . coli, and the polypeptide was purified to homogeneity by affinity chromatography and refolded, yielding fully enzymatically active PLA . In addition, we have produced a genetically engineered enzymatically inactive variant by substitution of a single amino acid residue in the catalytic center . A standard histamine release assay was used to compare the potency of natural PLA with correctly folded enzymatically active and inactive recombinant PLA to release histamine from blood basophils of nine patients with bee venom allergy . RESULTS: Recombinant enzymatically active PLA and purified natural protein were equally effective in releasing histamine from sensitized basophils . By comparing the histamine-releasing capacity of enzymatically active and inactive recombinant allergen, we further demonstrate that catalytic activity is not a requirement for allergenicity in the effector phase . Denaturation of natural PLA or incorrect folding of recombinant protein resulted in a total loss of allergenic potency . CONCLUSION: We demonstrate the feasibility of producing native-like recombinant allergens with or without enzymatic activity . We also provide evidence for the requirement of correct three-dimensional structure of PLA to induce histamine release from basophils and thus evidence for its recognition by IgE. EMBO J, 1995 Jun 1, 14(11), 2483 - 90 Secretion by Trypanosoma cruzi of a peptidyl-prolyl cis-trans isomerase involved in cell infection; Moro A et al.; Macrophage infectivity potentiators are membrane proteins described as virulence factors in bacterial intracellular parasites, such as Legionella and Chlamydia . These factors share amino acid homology to eukaryotic peptidyl-prolyl cis-trans isomerases that are inhibited by FK506, an inhibitor of signal transduction in mammalian cells with potent immunosuppressor activity . We report here the characterization of a protein released into the culture medium by the infective stage of the protozoan intracellular parasite Trypanosoma cruzi . The protein possesses a peptidyl-prolyl cis-trans isomerase activity that is inhibited by FK506 and its non-immunosuppressing derivative L-685,818 . The corresponding gene presents sequence homology with bacterial macrophage infectivity potentiators . The addition of the protein, produced heterologously in Escherichia coli, to cultures of trypomastigotes and simian epithelial or HeLa cells enhances invasion of the mammalian cells by the parasites . Antibodies raised in mice against the T.cruzi isomerase greatly reduce infectivity . A similar reduction of infectivity is obtained by addition to the cultures of FK506 and L-685,818 . We concluded that the T.cruzi isomerase is involved in cell invasion. Virology, 1995 Jun 1, 209(2), 684 - 7 Mapping of a serotype specific epitope of the major capsid protein VP2 of infectious pancreatic necrosis virus; Liao L et al.; A serotype-specific, conformational epitope of VP2 of infectious pancreatic necrosis virus (IPNV) (Jasper), has been mapped by restriction enzyme site-specific deletions of cloned viral cDNA . Subclones encoding fragments of VP2 were expressed in Escherichia coli followed by polyacrylamide gel electrophoresis and Western blot analysis . The epitope (between amino acid residues 242 and 325) reacted with monoclonal anti-VP2 antibodies that neutralized the homologous (Jasper), but not the heterologous (Sp) serotype of IPNV. Blood, 1995 Jun 1, 85(11), 3066 - 76 A phase I trial of recombinant human interleukin-6 in patients with myelodysplastic syndromes and thrombocytopenia; Gordon MS et al.; To evaluate the hematologic effects of recombinant human interleukin-6 (rhIL-6, Escherichia coli, SDZ ILS 969, IL-6), and determine its toxicity profile, we performed a phase I trial of IL-6 in 22 patients with various myelodysplastic syndromes (MDS), platelet counts < 100,000/microL, and < 5% bone marrow (BM) blasts . Patients received one of four doses of IL-6 (1.0, 2.5, 3.75, and 5.0 micrograms/kg/d) as a subcutaneous injection on day 1, followed by a 7-day wash-out period, and then 28 days of IL-6 therapy . Dose-limiting toxicities of fatigue, fever, and elevated alkaline phosphatase were seen at 5.0 micrograms/kg/d; the maximum tolerated dose was 3.75 micrograms/kg/d . All patients experienced at least grade II fever and all had an increase in acute phase proteins . Eight patients (36%) experienced at least a transient improvement in platelet counts; three fulfilled the criteria for response, whereas five others had clinically significant increases that failed to meet response criteria . Various IL-6-related toxicities prevented more than three patients from receiving maintenance therapy . Two of the three patients who received maintenance IL-6 therapy had a persistent increase in platelet counts, during 3 and 12 months of IL-6 therapy, respectively . Laboratory studies indicated that IL-6 increased the frequency of higher ploidy megakaryocytes but did not significantly increase the number of assayable megakaryocytic progenitor cells, suggesting that IL-6 acts as a maturational agent rather than a megakaryocyte colony-stimulating factor . Although IL-6 therapy can promote thrombopoiesis in some MDS patients, its limited activity and significant therapy-related toxicity preclude its use as a single agent in this patient population . Further studies, combining low doses of IL-6 with other hematopoietic growth factors, are underway. J Virol, 1995 Jun, 69(6), 3542 - 8 A maltose-binding protein/adeno-associated virus Rep68 fusion protein has DNA-RNA helicase and ATPase activities; Wonderling RS et al.; The adeno-associated virus type 2 (AAV) Rep68 protein produced in Escherichia coli as a fusion protein with maltose-binding protein (MBP-Rep68 delta) has previously been shown to possess DNA-DNA helicase activity, as does the purified wild-type Rep68 . In the present study, we demonstrate that MBP-Rep68 delta also catalyzes the unwinding of a DNA-RNA hybrid . MBP-Rep68 delta-mediated DNA-RNA helicase activity required ATP hydrolysis and the presence of Mg2+ ions and was inhibited by high ionic strength . The efficiency of the DNA-RNA helicase activity of MBP-Rep68 delta was comparable to its DNA-DNA helicase activity . However, MBP-Rep68 delta lacked the ability to unwind a blunt-ended DNA-RNA substrate and RNA-RNA duplexes . We have also demonstrated that MBP-Rep68 delta has ATPase activity which is enhanced by the presence of single-stranded DNA but not by RNA . The MBP-Rep68 delta NTP mutant protein, which has a lysine-to-histidine substitution at amino acid 340 in the putative nucleoside triphosphate-binding site of Rep68, not only lacks DNA-RNA helicase and ATPase activities but also inhibits the helicase activity of MBP-Rep68 delta . DNA-RNA helicase activity of Rep proteins might play a pivotal role in the regulation of AAV gene expression by AAV Rep proteins. Genetics, 1995 Jun, 140(2), 435 - 42 Mutations in the mitochondrial ATP synthase gamma subunit suppress a slow-growth phenotype of yme1 yeast lacking mitochondrial DNA; Weber ER et al.; In Saccharomyces cerevisiae, inactivation of the nuclear gene YME1 causes several phenotypes associated with impairment of mitochondrial function . In addition to deficiencies in mitochondrial compartment integrity and respiratory growth, yme1 mutants grow extremely slowly in the absence of mitochondrial DNA . We have identified two genetic loci that, when mutated, act as dominant suppressors of the slow-growth phenotype of yme1 strains lacking mitochondrial DNA . These mutations only suppressed the slow-growth phenotype of yme1 strains lacking mitochondrial DNA and had no effect on other phenotypes associated with yme1 mutations . One allele of one linkage group had a collateral respiratory deficient phenotype that allowed the isolation of the wild-type gene . This suppressing mutation was in ATP3, a gene that encodes the gamma subunit of the mitochondrial ATP synthase . Recovery of two of the suppressing ATP3 alleles and subsequent sequence analysis placed the suppressing mutations at strictly conserved residues near the C terminus of Atp3p . Deletion of the ATP3 genomic locus resulted in an inability to utilize nonfermentable carbon sources . atp3 deletion strains lacking mitochondrial DNA grew slowly on glucose media but were not as compromised for growth as yme1 yeast lacking mitochondrial DNA. RNA, 1995 Jun, 1(4), 437 - 48 A dual-specificity pseudouridine synthase: an Escherichia coli synthase purified and cloned on the basis of its specificity for psi 746 in 23S RNA is also specific for psi 32 in tRNA(phe); Wrzesinski J et al.; An Escherichia coli pseudouridine (psi) synthase, which forms both psi 746 in E . coli 23S ribosomal RNA and psi 32 in tRNA(Phe), has been isolated and cloned . The enzyme contains 219 amino acids and has a calculated MW of 24,432 Da . Amino acid sequence comparison with the three other psi synthases that have been cloned to date, two for tRNA and one for 16S RNA, did not reveal any common sequence motifs, despite the catalysis of a common reaction . The gene was cloned behind a (His)6 leader for affinity purification . Upon overexpression, most of the enzyme remained soluble in the cell cytoplasm and could be purified to homogeneity on a Ni(2+)-containing resin . The enzyme reacted with both full-length 23S RNA or a fragment from residues 1-847, forming 1 mol psi/mol RNA at position 746, a normal site for psi . The enzyme has no dependence on Mg2+ . The same yield was obtained in 1 mM EDTA as in 10 mM Mg2+, and the rate was faster in EDTA than in Mg2+ . Full-length 16S RNA or fragments 1-526 or 1-678, as well as tRNA(Val) transcripts, were not modified in either EDTA or Mg2+ . tRNA(Phe) transcripts, however, were modified with a yield of 1 mol psi/mol transcript at a rate in EDTA like that of 23S RNA . Sequencing showed all of the psi to be at position 32, a normal site for psi in this tRNA . Both 23S rRNA psi 746 and tRNA psi 32 occur in single-stranded segments of the same sequence, psi UGAAAA, closed by a stem . Therefore, this synthase may require for recognition only a short stretch of primary sequence 3' to the site of pseudouridylation . This is the first example of a dual-specificity modifying enzyme for RNA, that is, one which is specific for a single site in one RNA, and equally site-specific in a second class of RNA . The essentiality of these psi residues can now be assessed by disruption of the synthase gene. RNA, 1995 Jun, 1(4), 418 - 24 Transfer RNA aminoacylation: identification of a critical ribose 2'-hydroxyl-base interaction; Yap LP et al.; To understand the relationship between tRNA architecture and specific aminoacylation by aminoacyl-tRNA synthetases, we performed kinetic assays of Escherichia coli tRNA(Pro) molecules containing single deoxynucleotide substitutions . We identified an important 2'-hydroxyl group at position U8 (of 22 positions probed) . Chemical modification studies showed that this 2'-hydroxyl interacts with either the N1 or the exocyclic amine of G46 in a hydrogen bonding interaction that contributes 1.8 kcal/mol to the free energy of activation for aminoacylation . Molecular modeling of tRNA(Pro) supports the existence of this interaction . This is the first study to identify a specific ribose 2'-hydroxyl-base interaction in the core region of a tRNA molecule that makes a thermodynamically significant contribution to aminoacylation. J Biochem (Tokyo), 1995 Jun, 117(6), 1209 - 17 Site-directed mutagenetic study on the role of negative patches on silene plastocyanin in the interactions with cytochrome f and photosystem I; Lee BH et al.; To investigate the role of two highly conserved negative patches, residues #42-45 and #59-61, on the surface of plant plastocyanin, six mutants were constructed by site-directed mutagenesis of the intermediate precursor gene from Silene pratensis . The mutants were designed systematically to incorporate positive charges into the negative patches, and the net charge on negative patches was modified from -4 to +1 . Upon expression in Escherichia coli, the mutant proteins were correctly processed to the mature size and accumulated as holo-proteins . Absorption spectra, EPR, and redox potentials of the purified mutant proteins were almost indistinguishable from those of the wild-type . It was found that the electron transfer rate from cytochrome f to plastocyanin decreased exponentially as the net charge on the negative patch (#42-45) was increased, whereas the modification of the other negative patch (#59-61) had no effect . Ionic strength dependence studies indicated that the rate constants at infinite ionic strength did not change significantly among the wild-type and the six mutants, and the electrostatic attraction energies between plastocyanin and cytochrome f decreased when residues #42-45 were modified, whereas the modification of residues #59-61 had no effect . These results clearly indicated that only one (#42-45) of the two negative patches is involved in the transient complex formation with cytochrome f . Essentially similar results were observed for the electron transfer from plastocyanin to the photosystem I reaction center (P700), although in this case, slight participation of the negative patch (#59-61) is suggested.(ABSTRACT TRUNCATED AT 250 WORDS) J Biochem (Tokyo), 1995 Jun, 117(6), 1196 - 200 Catalytic role of an arginine residue in the highly conserved and unique sequence of phosphoenolpyruvate carboxylase; Yano M et al.; Phosphoenolpyruvate carboxylase (PEPC) {EC 4.1.1.31} has a highly conserved and unique sequence, 578-FHGRGGSIGRGGAP-591 (on Escherichia coli, PEPC), in which a GRGG motif is repeated twice with two intervening residues . Since previous chemical modification studies suggested the functional importance of arginine residues, the invariant Arg587 in this region was replaced with Ser, and the enzymatic properties of the resulting mutant enzyme (R587S) were investigated . Replacement led to virtual loss of the catalytic activity to form oxaloacetate . The specific activity was 37 nmol.min-1.mg-1, which corresponds to 2 x 10(-4)-fold the activity of the wild-type enzyme . However, the activity of bicarbonate- and Mg(2+)-dependent hydrolysis of phosphoenolpyruvate (PEP) to pyruvate appeared for the mutant enzyme with a specific activity of 2.1 mumol.min-1.mg-1 . In view of the stepwise reaction mechanism proposed for PEPC, this activity can be attributed to impairment of the subsequent partial reaction(s) following the formation of the intermediate carboxyphosphate . The half-saturation concentration (S0.5) of HCO3- in R587S was about 100-fold that in the wild-type enzyme, whereas the respective values for PEP and Mg2+ were 20- and 15-fold, indicative of this residue participating in the binding of HCO3-. J Biochem (Tokyo), 1995 Jun, 117(6), 1148 - 50 A putative metal-binding site in the beta subunit of rat mitochondrial processing peptidase is essential for its catalytic activity; Kitada S et al.; Mitochondrial processing peptidase (MPP) consists of alpha- and beta-subunits (alpha-MPP and beta-MPP) . beta-MPP has a putative metal-binding sequence (HXXEH) . To determine whether the sequence of beta-MPP is essential for the enzymatic activity, we individually mutated the histidines and glutamic acid to arginines and glutamine, respectively . The wild-type and mutated beta-MPPs were co-expressed with alpha-MPP in Escherichia coli . All three mutants had completely lost the activity, whereas the lost activity was recovered on the addition of wild-type beta-MPP . The activity of the wild-type enzyme was reduced by the mutant beta-MPPs . We conclude from these observations that the HXXEH region is involved in the formation of the active site and that beta-MPP is the catalytic subunit of MPP. Prostaglandins, 1995 Jun, 49(6), 371 - 82 Regulation of two isozymes of prostaglandin endoperoxide synthase and thromboxane synthase in human monoblastoid cell line U937; Nanayama T et al.; The mechanism responsible for the rapid increase of thromboxane A2 synthesis by cells of the human monoblastoid cell line U937, which were differentiated with 12-O-tetradecanoyl-phorbol-13-acetate, induced by lipopolysaccharide (LPS) was studied . Both RNA blot and immunoblot analyses showed that LPS increased the levels of prostaglandin endoperoxide synthase-1 (PES-1) and -2 (PES-2) in a time-dependent manner, and the modes of induction of the two isozymes differed . The maximum PES-1 mRNA level was 1.6 times higher 36 h after than before stimulation by LPS, and that of PES-2 mRNA was elevated about 20-fold at its peak at 12 h after stimulation . Consequently, the immunoreactive PES-1 and PES-2 protein levels also increased time-dependently after LPS stimulation . However, the effects of LPS on the thromboxane synthase mRNA and protein levels were much less marked . These results indicate that LPS-induced thromboxane synthesis by the differentiated cells was regulated at the levels of the two PES isozymes, predominantly at the PES-2 level. Mol Microbiol, 1995 Jun, 16(5), 943 - 53 Negative control of fae (K88) expression by the 'global' regulator Lrp is modulated by the 'local' regulator FaeA and affected by DNA methylation; Huisman TT et al.; Expression of the K88 (fae) operon is negatively controlled by the co-operative binding of Lrp and FaeA to the fae regulatory region and is dependent on the methylation status of three GATC sites present in this region . In this paper, we describe the binding of Lrp to a T-rich DNA helix between GATC site I and site II . FaeA stabilized and modified the Lrp binding, thereby extending the Lrp footprint over GATC site I and site III . Methylation of GATC site I prevented the binding of Lrp/FaeA at this site and appeared to be essential for the cells, since mutation of this site into GTTC resulted in a lethal overproduction of K88 fimbriae . Methylation of GATC site II and site III reduced the stability of Lrp/FaeA binding . Moreover, methylation of GATC site III stimulated faeB promoter activity . The plasmid population in cells harbouring multiple copies of a K88 plasmid consisted of two differentially methylated forms . Form A plasmids with a methylated GATC site I and site III and a nonmethylated site II (+,-,+) represented 20% of the population and were responsible for high-level expression . Form B plasmids with a methylated GATC site I and a non-methylated site II and site III (+,-,-) represented 80% of the population and were responsible for low-level expression . Apparently, K88 fimbriae expression in vivo is balanced at its maximal possible level by modulation of the methylation status of GATC site III . The ratio (1:4) between these populations is stabilized by a constitutive synthesis of FaeA resulting from the presence of an IS1 insertion upstream of faeA . This IS1 insertion separates the faeA promoter from the FaeB-binding sites, thereby neutralizing the control by FaeB activity on expression of FaeA . Instead, faeA transcription is stimulated by binding of FaeA to the faeA promoter region. Mol Microbiol, 1995 Jun, 16(5), 817 - 24 Open complex formation by Escherichia coli RNA polymerase: the mechanism of polymerase-induced strand separation of double helical DNA; deHaseth PL et al.; Escherichia coli RNA polymerase is able to site-specifically melt 12 bp of promoter DNA at temperatures far below those normally associated with DNA melting . Here we consider several models to explain how RNA polymerase destabilizes duplex DNA . One popular model proposes that upon binding to the promoter, RNA polymerase untwists the spacer DNA between the -10 and -35 regions, which results in a destabilization of the -10 region at a TA base step where melting initiates . Promoter untwisting may result, in part, from extensive wrapping of the DNA around RNA polymerase . Formation of the strand-separated open complex appears to be facilitated by specific protein-DNA interactions which occur predominantly on the non-template strand . Recent evidence suggests that these include important contacts with sigma factor region 2.3, which we propose binds the displaced single strand of DNA. Mol Microbiol, 1995 Jun, 16(5), 1021 - 9 Structural requirements for the glycolipid receptor of human uropathogenic Escherichia coli; Striker R et al.; The binding of uropathogenic Escherichia coli to the globo series of glycolipids via P pili is a critical step in the infectious process that is mediated by a human-specific PapG adhesin . Three classes of PapG adhesins exist with different binding specificities to Gal alpha 4Gal-containing glycolipids . The structural basis for PapG recognition of the human glycolipid receptor globoside was investigated by using soluble saccharide analogues as inhibitors of bacterial haemagglutination . The minimum binding epitope was confirmed as the Gal alpha 4Gal moiety, but parts of the GalNAc beta and glucose residues, which flank the Gal alpha 4Gal in globoside (GbO4), were also shown to be important for strong binding . Furthermore, the same five hydroxyl groups of Gal alpha 4Gal in globotriasyl ceramide that were recognized by a previously characterized PapG variant were also recognized by the human-specific PapG in binding the GbO4 that dominates in the human kidney . Saccharide analogues that blocked haemagglutination also blocked the adherence of human uropathogenic E . coli to human kidney sections . Knowledge of the molecular details of the PapG-GbO4 interaction will make it possible to design antiadherence therapeutics. Mol Microbiol, 1995 Jun, 16(5), 1011 - 20 Molecular dissection of PapD interaction with PapG reveals two chaperone-binding sites; Xu Z et al.; P pili are composite adhesive fibres that allow uropathogenic Escherichia coli to gain a foothold in the host by binding to receptors present on the uroepithelium via the adhesin PapG . The assembly of P pili requires a periplasmic chaperone, PapD, that has an immunoglobulin-like three-dimensional structure . PapD-subunit complex formation involves a conserved anchoring mechanism in the chaperone cleft and a 'molecular zippering' to the extreme C-terminus of pilus subunits . A chaperone-binding assay was developed using fusions of the C-terminus of PapG to maltose-binding protein (MBP/G fusions) to investigate whether chaperone-subunit complex formation requires additional interactions . PapD bound strongly to an MBP/G fusion containing the C-terminal 140 amino acids of PapG (MBP/G175-314) but only weakly to the MBP/G234-314 fusion containing 81 C-terminal residues, arguing that the region between residues 175-234 contains additional information that is required for strong PapD-PapG interactions . PapD was shown to interact with a PapG C-terminal truncate containing residues 1-198 but not a truncate containing residues 1-145, suggesting the presence of a second, independent PapD interactive site . Four peptides overlapping the second site region were tested for binding to PapD in vitro to further delineate this motif . Only one of the peptides synthesized was recognized by PapD . The MBP/G fusion containing both binding sites formed a tight complex with PapD in vivo and inhibited pilus assembly by preventing chaperone-subunit complex formation. Biochemistry, 1995 May 30, 34(21), 7038 - 46 Function of conserved histidine residues in mammalian dihydroorotase; Zimmermann BH et al.; Dihydroorotase (DHOase, EC 3.5.2.3) catalyzes the reversible cyclization of carbamyl aspartate to form dihydroorotate, the third step in de novo pyrimidine biosynthesis . In mammals this activity is carried by the zinc-containing domain of the 243 kDa multifunctional protein CAD . We have replaced conserved residues in the cloned 46 kDa DHOase domain by site-directed mutagenesis . Mutants His1471Ala and His1473Ala lacked catalytic activity, judging by their failure to complement a DHOase-deficient Escherichia coli strain, and were unable to coordinate the active site zinc ion in zinc blotting experiments . This result confirmed earlier predictions . A mutant protein in which the third suspected zinc ligand was changed, Glu1512Asn, had a kcat similar to that of the intact CAD molecule and a Km similar to that of the wild-type recombinant DHOase, observations that argue against a role for glutamate 1512 in catalysis . Mutant His1590Asn had no measurable catalytic activity . This histidine residue was tentatively identified as the third zinc ligand by the failure of the mutant to bind the full complement of zinc in atomic absorption measurements . Mutant His1690Asn had a kcat 34-fold lower and a Km 9-fold higher than those of wild-type recombinant . The kinetic parameters of the mutant His1642Asn were also altered, but to a lesser extent . Diethyl pyrocarbonate (DEPC) was shown previously to inactivate mammalian DHOase . Spectroscopic studies and {14C}DEPC incorporation demonstrated that the loss of activity is associated with the modification of approximately two histidine residues located at or near the active site.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1995 May 30, 34(21), 7020 - 6 Characterization of the topa quinone cofactor in amine oxidase from Escherichia coli by resonance Raman spectroscopy; Moenne-Loccoz P et al.; The aromatic amine oxidase from Escherichia coli (ECAO) utilizes Cu(II) and 2,4,5-trihydroxyphenylalanine quinone (TPQ) as cofactors in enzymatic catalysis . The TPQ cofactor is clearly identified by a set of characteristic vibrational modes between 1200 and 1700 cm-1 in the resonance Raman (RR) spectrum of the native enzyme . This is the first report of a RR spectrum for an underivatized TPQ cofactor in an enzyme, showing that it is possible to study changes in the cofactor during the natural reaction cycle . The RR spectrum of ECAO closely matches that of a 2-hydroxy-1,4-benzoquinone model compound, particularly in the deprotonated state in aqueous solution . The principal in-phase C = O symmetric stretching mode of the quinone occurs at 1681 cm-1 in ECAO and at 1666 cm-1 in the model compound and, in both cases, undergoes a downshift of approximately 25 cm-1 upon substitution of one of the carbonyl oxygens with 18O . The overall similarity of the 18O and D shifts in their RR spectra shows that the TPQ cofactor and model compound have the same structure and reactivity, with oxygen exchange occurring at the carbonyl adjacent to the hydroxyl group . Substrate reduction of ECAO under anaerobic conditions leads to a stable semiquinone (lambda max at 442 and 468 nm) with a RR spectrum characteristic of an amine-substituted semiquinone.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1995 May 30, 34(21), 6947 - 55 Influence of alpha-deoxyadenosine on the stability and structure of DNA . Thermodynamic and molecular mechanics studies; Ide H et al.; The alpha anomer of deoxyadenosine (alpha) and an abasic site (tetrahydrofuran, F), which are DNA lesions produced by free radicals, were site-specifically incorporated in 9-mer duplexes d(TGAGXGTAC).d-(GTACNCTCA), where X = alpha or F and N = A, G, C, or T . Their influence on thermodynamic stability and structure of DNA was assessed by UV-melting measurements and molecular mechanics calculations . UV-melting studies revealed that a duplex containing an alpha T pair was as stable as the parental duplex containing an AT pair at the same site . Furthermore, the stability of duplexes containing alpha varied depending on the base opposite this lesion, with the Tm decreasing in the following order: alpha T > alpha C approximately alpha A > alpha G . On the contrary, an abasic site introduced in the same site showed a significantly greater destabilizing effect than alpha, but variation of Tm with the bases opposite F was less evident . To delineate the molecular mechanism of thermodynamic effects of an alpha lesion, molecular mechanics calculations were performed for the same duplexes as used for UV-melting measurements . The results suggest that the structural perturbation introduced into DNA by an alpha N pair is alpha G > alpha A > alpha C > alpha T, showing a parallel correlation with the destabilizing effects of alpha N pairs . On the basis of these results, it is discussed how the perturbations introduced by these DNA lesions may influence the selection of nucleotides opposite the lesions by DNA polymerases and the interaction with DNA repair enzymes such as Escherichia coli endonuclease IV and exonuclease III. FEBS Lett, 1995 May 29, 365(2-3), 214 - 8 Relevance of histidine-84 in the elongation factor Tu GTPase activity and in poly(Phe) synthesis: its substitution by glutamine and alanine; Scarano G et al.; Substitution of His-84 (-->Gln and -->Ala), a residue of the switch II region of E . coli elongation factor (EF) Tu, hardly affected the binding of GTP or GDP . The activity in poly(Phe) synthesis and GTP hydrolysis of EF-Tu H84Q were both reduced to about 35%, as compared to EF-Tu wt, whereas EF-Tu H84A was inactive in poly(Phe) synthesis but still showed a 10% residual GTPase activity . Phe-tRNAPhe exerted a similar inhibitory effect on the GTPase activity of EF-Tu wt and EF-Tu H84Q while abolishing that of EF-Tu H84A . Ribosomes enhanced the GTPase activity of EF-Tu H84Q, but not that of EF-Tu H84A, on which they even seemed to exert an inhibitory effect . The one-round GTP hydrolysis associated with the EF-TuH84Q-dependent binding of Phe-tRNAPhe to poly(U)-programmed ribosomes was less efficient than with EF-Tu wt . Kirromycin stimulated the GTPase activities of both mutants less than EF-Tu wt . The results of this work do not support a catalytic role of His-84 in the intrinsic GTPase of EF-Tu, but they emphasize the importance of its side-chain for polypeptide synthesis and GTP hydrolysis. FEBS Lett, 1995 May 29, 365(2-3), 159 - 63 Ecotin is a potent inhibitor of the contact system proteases factor XIIa and plasma kallikrein; Ulmer JS et al.; Ecotin, a serine protease inhibitor found in the periplasm of Escherichia coli, has been characterized as a potent reversible tight-binding inhibitor of the human contact activation proteases factor XIIa (FXIIa) and plasma kallikrein, having Ki values of 89 pM and 163 pM, respectively . Ecotin also inhibited human leukocyte elastase (HLE) with high affinity (Ki = 55 pM) . The association rate constants kon for FXIIa and kallikrein were 5.3 x 10(5) M-1.s-1 and 2.9 x 10(5) M-1.s-1, respectively . The dissociation rate constant koff for kallikrein, measured in the presence of HLE to prevent reassociation, was 6.3 x 10(-5) s-1; the koff for ecotin with FXIIa was 4.7 x 10(-5) s-1 . Both FXIIa and kallikrein cleaved ecotin slowly at pH 5.0, identifying Met-84 as the P1 residue . The potent anticoagulant effect by ecotin is explained by the coincident inhibition of FXIIa, kallikrein, and FXa and suggests that it may be useful in the study of inflammatory or thrombotic disorders such as sepsis or cardiopulmonary bypass. FEBS Lett, 1995 May 29, 365(2-3), 155 - 8 Mutational analysis of a putative polyphosphoinositide binding site in phospholipase C-beta 2; Simoes AP et al.; The phosphatidylinositol 4,5-bisphosphate (PtdIns-P2)-regulated actin-binding protein gelsolin and most phosphoinositide-specific phospholipases C (PLCs) comprise a basic amino acid motif ((K/R)xxxKxK(K/R); x denotes any amino acid) which was previously suggested to represent a PtdInsP2-binding site commonly present in these proteins . We have challenged this hypothesis for PLC beta 2 by replacing one or several residues of this motif (KILIKNKK; residues 457-464) and examining the functional consequences of these alterations . The results show that the integrity of the basic motif is important for PtdInsP2 hydrolysis by PLC beta 2 . Replacement of lysines 463 or 461 by arginine led to reduction or complete loss, respectively, of enzyme activity . The results provide further support to the concept that the function of the basic motif within the various PLCs is to bind the enzyme substrate PtdInsP2. Gene, 1995 May 26, 158(1), 51 - 4 The pTugA and pTugAS vectors for high-level expression of cloned genes in Escherichia coli; Graham RW et al.; Plasmids pTugA and pTugAS, designed for expression of cloned genes in Escherichia coli, possess the features of high-level inducible transcription, enhanced RNA translation, portability, high copy number, stability and versatility . In addition, pTugAS can be used to produce fusion proteins comprising a target protein and a cellulose-binding domain . Such fusion proteins can be purified in a single step by affinity chromatography on cellulose . Expression of two model gene fusions using the pTug plasmids resulted in yields of 500 mg of intracellular and 250 mg of extracellular recombinant protein per liter. Gene, 1995 May 26, 158(1), 41 - 50 Analysis of a family of ypt genes and their products from Chlamydomonas reinhardtii; Dietmaier W et al.; Small G-proteins encoded by the ras-like ypt genes are ubiquitous in eukaryotic cells . They have been shown to play an essential role in membrane vesicle transport . We have isolated four ypt genes, yptC1, yptC4, yptC5 and yptC6, from Chlamydomonas reinhardtii (Cr) genomic and cDNA libraries . Three of them, yptC1, yptC4 and yptC5, are close homologues of ypt genes previously found in the multicellular alga Volvox carteri (Vc), the fourth, yptC6, is new . Each yptC gene is present as a single copy in the genome . Comparisons of genomic and cDNA sequences revealed that the coding regions are interrupted by five (yptC5), six (yptC6), seven (yptC4) and eight (yptC1) introns, respectively . Cr ypt genes and the closely related Vc ypt genes have identical exon-intron structures, but the corresponding intron sequences are completely different . Polyadenylation is signalled by UAUAA, UGUAG and UGUAA . The deduced amino acid (aa) sequence of YptC6 exhibited 79% identity with HRab2; YptC1, YptC4 and YptC5 exhibited over 90% identity with their Vc homologues . Primary structures of the 9-aa 'effector domain' and the contiguous 'helix3-loop7' motif (approx . 30 aa) are 'diagnostic' features for functional assignment . Recombinant YptC proteins, overproduced in Escherichia coli and purified to near homogeneity, displayed strong and specific binding of GTP, but not of GMP or ATP . The four Cr Ypt proteins showed immunochemical cross reactions to their Vc counterparts . Moreover, Western blots demonstrated at least six types of Ypt in both Cr and Vc, suggesting that these Ypt are used for household functions responsible for vesicle transport rather than for cellular differentiation. Gene, 1995 May 26, 158(1), 125 - 8 Cloning, characterization and functional expression of an endoglucanase-encoding gene from the phytopathogenic fungus Macrophomina phaseolina; Wang H et al.; An endoglucanase-encoding clone (egl2) was isolated from the phytopathogenic soilborne deuteromycete fungus Macrophomina phaseolina (Mp) . Clones were obtained from a cDNA library by functional expression in Escherichia coli . The egl2 clone hybridized to a 1.3-kb mRNA . Expression is induced by carboxymethylcellulose (CMC) and repressed by glucose . The deduced amino acid (aa) sequence revealed strong similarity to the egl3 from Trichoderma reesei (Tr) (72% for identical residues and 81% with conservative substitution over a span of 324 aa) . The Mp egl2 lacks the cellulose-binding domain and linker region found in the Tr egl3 . Different codon usage between the two fungi resulted in a much shorter span of nucleotide homology . The Egl2 protein cleaves cellodextrins with continguous beta, 1-4 linkages of four and larger, and shows activity against CMC and birchwood xylan. Biochem Pharmacol, 1995 May 26, 49(11), 1641 - 7 Virtual cofactors for an Escherichia coli nitroreductase enzyme: relevance to reductively activated prodrugs in antibody directed enzyme prodrug therapy (ADEPT); Knox RJ et al.; A nitroreductase enzyme has been isolated from Escherichia coli that has the unusual property of being equally capable of using either NADH or NADPH as a cofactor for the reduction of its substrates which include menadione as well as 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) . This property is shared with the mammalian enzyme, DT diaphorase . The nitroreductase can, like DT diaphorase, also use simple reduced pyridinium compounds as virtual cofactors . The intact NAD(P)H molecule is not required and the simplest quaternary (and therefore reducible) derivative of nicotinamide, 1-methylnicotinamide (reduced), is as effective as NAD(P)H in its ability to act as an electron donor for the nitroreductase . The structure-activity relationship is not identical to that of DT diaphorase and nicotinic acid riboside (reduced) is selective, being active only for the nitroreductase . Irrespective of the virtual cofactor used, the nitroreductase formed the same reduction products of CB 1954 (the 2- and 4-hydroxylamino derivatives in equal proportions) . Nicotinic acid riboside (reduced), unlike NADH, was stable to metabolism by serum enzymes and had a plasma half-life of seven minutes in the mouse after an i.v . bolus administration . NADH had an unmeasurably short half-life . Nicotinic acid riboside (reduced) could also be produced in vivo by administration of nicotinic acid 5'-O-benzoyl riboside (reduced) . These results demonstrate that the requirement for a cofactor need not be a limitation in the use of reductive enzymes in antibody directed enzyme prodrug therapy (ADEPT) . It is proposed that the E . coli nitroreductase would be a suitable enzyme for ADEPT in combination with CB 1954 and a synthetic, enzyme-selective, virtual cofactor such as nicotinic acid riboside (reduced). J Med Chem, 1995 May 26, 38(11), 1892 - 903 Synthesis and biological evaluation of novel 2,6-diaminobenz{cd}indole inhibitors of thymidylate synthase using the protein structure as a guide; Varney MD et al.; The design, synthesis, and biochemical and biological evaluations of a novel series of 2,6-diaminobenz{cd}indole-containing inhibitors of human thymidylate synthase (TS) are described . The compounds are characterized by having either a pyridine or pyridazine ring in place of the (phenylsulfonyl)morpholinyl group of the known inhibitor N6-{4-(morpholinosulfonyl)benzyl}-N6-methyl-2,6-diaminobenz{ cd}indole glucuronate (i) . Active compounds from this series showed human TS inhibition constants below the 10 nM level and were potent, selective submicromolar antitumor agents in cell culture . The compounds were synthesized by reductive alkylation of a substituted 6-aminobenz{cd}indole or reductive cyclization of a substituted 1-cyano-8-nitronaphthalene. J Mol Biol, 1995 May 26, 249(1), 29 - 44 Functional characterization of the RNA-binding domain and motif of the double-stranded RNA-dependent protein kinase DAI (PKR); Schmedt C et al.; The double-stranded (ds) RNA-activated protein kinase, DAI (also known as PKR), contains an RNA-binding domain comprising two tandem repeats of a motif, the dsRBM, which is shared with a number of other proteins that interact with structured RNAs . We have expressed the entire domain and the first copy of the motif in Escherichia coli and purified the two proteins, p20 and p10, to apparent homogeneity in order to study their interactions with RNA and with the intact kinase enzyme . Both p20 and p10 bound preferentially to structured RNA molecules . Competition assays showed that in both cases the order of affinity is dsRNA > VA RNA > tRNA, but the isolated motif bound much less tightly than the entire domain . Measurement of the dissociation constants for dsRNA by quantitative gel mobility shift analysis gave apparent Kd values of 4 x 10(-9) M and 3.8 x 10(-7) M for p20 and p10, respectively . The binding of p20 molecules to dsRNA appeared to be cooperative . Multiple complexes were formed between the intact domain and dsRNA, saturating at a density of about one p20 molecule/11.25 base-pairs (or one turn) of duplex, whereas p10 achieved only about half of this packing density . The apparent Kd for the p20-VA RNA interaction was estimated as 3.5 x 10(-7) M and at least three complexes were detected, but no distinct complexes were visualized for the interaction between p10 and VA RNA . Both p20 and p10 inhibited autophosphorylation of intact DAI, probably by binding the dsRNA activator . Once activated, DAI could phosphorylate both p10 and p20, suggesting that intermolecular phosphorylation can occur. J Mol Biol, 1995 May 26, 249(1), 153 - 75 Towards structure-based drug design: crystal structure of a multisubstrate adduct complex of glycinamide ribonucleotide transformylase at 1.96 A resolution; Klein C et al.; An inhibitor complex structure of glycinamide ribonucleotide transformylase (GAR-Tfase; EC 2.1.2.2) from Escherichia coli has been determined with a multisubstrate adduct BW1476U89 to an R-value of 19.1% at 1.96 A resolution . The structure was determined by a combination of molecular and single isomorphous replacement using data from two different monoclinic crystal lattices and collecting data from crystals soaked in 20% (w/v) methyl-pentanediol as cryoprotectant for shock-freezing at -150 degrees C . The multisubstrate adduct is bound in an extended crevice at the interface between the two functional domains of the enzyme . This inhibitor is positioned in the binding site by three sets of tight interactions with its phosphate, glutamate and pyrimidone ring moieties, while its interventing linker atoms are more flexible and adopt two distinct sets of conformations . The highly conserved Arg103, His108 and Gln170 residues that are key in ligand binding and catalysis (His108), h