J Mol Biol, 1995 Nov 10, 253(5), 726 - 38 A double mutation in subunit c of the Na(+)-specific F1F0-ATPase of Propionigenium modestum results in a switch from Na+ to H(+)-coupled ATP synthesis in the Escherichia coli host cells; Kaim G et al.; The in vivo synthesis of an F1F0-ATPase hybrid in Escherichia coli strain PEF42 which harbours the genes for the Propionigenium modestum subunits a, b, c, and delta, a gene for hybrid alpha subunit with the N-terminal portion (amino acids 1 to 173) of P . modestum and the C-terminal region (amino acids 176 to 513) from E . coli, and the genes for the E . coli subunits beta, gamma and epsilon, yielded a functional enzyme complex . This hybrid ATPase coupled ATP synthesis to Na+ transport and required Na+ for growth on succinate . After random mutagenesis of the P . modestum genes of strain PEF42, clones were selected that grew on succinate in the absence of Na+ . A double-mutation cPhe84Leu, cLeu87Val that was found in several of these clones, was introduced by site specific mutagenesis into the parent strain PEF42 . The resulting strain E . coli MPC8487 also exhibited Na(+)-independent growth on succinate, showing that the double mutation is the only reason for the new phenotype . The mutation causes a change of the coupling ions of the hybrid ATPase from Na+ in strain PEF42 to H+ in strain MPC8487 . This conclusion was supported by the biochemical properties of the ATPase from strain MPC8487 . Unlike the parent enzyme, the mutated ATPase was not activated by Na+, but retained activation by Li+ . The pH optimum of the mutated ATPase (in the absence of Na+ or Li+) was shifted from pH 6.5 to pH 7.5, and the specific ATPase activity of the cell membranes increased about fourfold over that found in membranes of the parent cells . The mutated ATPase pumped protons or Li+ after reconstitution into proteoliposomes, and the transport of both cations was not affected by Na+ . The double mutation in the c subunit thus results in the loss of Na+ binding, retention of Li+ binding and an improvement of H+ binding. J Med Chem, 1995 Nov 10, 38(23), 4637 - 47 A novel strategy for improving ligand selectivity in receptor-based drug design; Pastor M et al.; A major desirable characteristic of many drugs is their ability to interact specifically with only one variety of the target receptor among many others . It is remarkable that, even when accurate three dimensional structures for the target biomolecules are available, there is no well-established methodology to describe their differences and use them for the design of selectively-interacting compounds . This work presents a novel method that uses multivariate GRID descriptors and principal component analysis (PCA) with the aim of revealing the most relevant structural and physicochemical differences between biomacromolecules related to receptor selectivity . The methodology is described through an example involving the study of bacterial (Escherichia coli) and recombinant human varieties of the dihydrofolate reductase (EC 1.5.1.3, DHFR) enzyme . This analysis easily unveils the most important regions on these biomolecules which should be taken into consideration for the design of selectively interacting compounds. Nature, 1995 Nov 9, 378(6553), 212 - 6 A potential catalytic site revealed by the 1.7-A crystal structure of the amino-terminal signalling domain of Sonic hedgehog; Hall TM et al.; Within the past few years, members of the hedgehog (hh) family of secreted signalling proteins have emerged as the primary signals generated by certain embryonic patterning centres . In vertebrate embryos, for example, sonic hedgehog expression in the notochord appears to be responsible for the local and long-range induction of ventral cell types within the neural tube and somites (reviewed in refs 1, 2) . Protein products encoded by hh family members are synthesized as precursors that undergo autoprocessing to generate an amino-terminal domain that appears to be responsible for both local and long-range signalling activities, and a carboxy-terminal domain that contains the autoprocessing activity . As part of an effort to understand how hh family members participate in cell-to-cell signalling, we have determined and report here the crystal structure at 1.7 A of the amino-terminal domain of murine Sonic hedgehog (Shh-N) . The structure revealed a tetrahedrally coordinated zinc ion that appears to be structurally analogous to the zinc coordination sites of zinc hydrolases, such as thermolysin and carboxypeptidase A . This previously unsuspected catalytic site represents a distinct activity from the autoprocessing activity that resides in the carboxy-terminal domain. Biochemistry, 1995 Nov 7, 34(44), 14601 - 8 Specificity of minor-groove and major-groove interactions in a homeodomain-DNA complex; Ades SE et al.; To assess the importance of minor-groove and major-groove interactions in homeodomain-DNA recognition, the binding properties of variants of the altered-specificity engrailed homeodomain, containing Lys50, and its DNA site TAATCC were determined . This homeodomain contacts bases in the minor groove of the DNA using Arg3 and Arg5 from its N-terminal arm and contacts bases in the major groove of the DNA using Ile47, Lys50, and Asn51 from its third alpha-helix . Mutation of Arg3 or Ile47 to alanine reduces binding affinity 10-20-fold while mutation of Arg5, Asn51, or Lys50 to alanine reduces binding affinity > 100-fold, indicating that both minor-groove and major-groove interactions contribute to the overall binding energy . Binding site selections and affinity measurements show that the homeodomain can also discriminate among different base pairs in the minor groove and the major groove . However, the interactions between Lys50 of the recognition helix and the major-groove edges of base pairs 5 and 6 are more specific than interactions mediated by Arg3 and Arg5 in the N-terminal arm and the minor-groove edges of base pairs 1 and 2. Biochemistry, 1995 Nov 7, 34(44), 14581 - 7 Nature and consequences of GroEL-protein interactions; Itzhaki LS et al.; The importance of chaperonin-protein interactions has been investigated by analyzing the refolding of the barley chymotrypsin inhibitor 2 in the presence of GroEL . The chaperonin retards the rate of refolding of wild type and 32 representative point mutants . The retardation of the rate drops to a finite level at saturating concentrations of GroEL, being lowered by a factor of 3-100, depending on the mutation . It is seen qualitatively that truncation of large hydrophobic side chains to smaller side chains weakens binding . Analysis of the magnitude of the rates of retardation shows further that hydrophobic and positively charged side chains tend to interact favorably with GroEL whereas negatively charged side chains tend to repel . There is an inverse correlation between the strength of hydrophobic interactions and the rate constant for refolding of the GroEL-complexed protein: the better the binding, the slower the folding . This shows directly that hydrophobic (and other favorable) interactions between the chaperonin and substrate are weakened during the refolding process and implies that unfolding can be catalyzed by the gain of such interactions. Biochemistry, 1995 Nov 7, 34(44), 14573 - 80 Characterization of the slow folding reactions of trp aporepressor from Escherichia coli by mutational analysis of prolines and catalysis by a peptidyl-prolyl isomerase; Mann CJ et al.; Escherichia coli trp aporepressor (TR) is a highly helical, dimeric protein whose folding has been shown to involve three phases whose relaxation times range from 200 ms to 50 s at 25 degrees C and pH 7.6 {Gittelman, M . S., & Matthews, C . R . (1990) Biochemistry 29, 7011-7021} . All three phases are urea and protein concentration independent below 3 M urea, suggesting that cis/trans proline isomerization might limit the folding of TR under these conditions . This hypothesis was tested by measuring the sensitivity of the folding reaction to site-directed mutagenesis and to cyclophilin, a peptidyl-prolyl isomerase . Each of the four proline residues in TR was replaced singly as well as simultaneously, and the effects on the folding mechanism were assessed . All of these mutants, including the version lacking prolines (des-Pro TR), retain three slow, denaturant-independent folding phases similar to those observed for wild-type TR . However, the pattern of catalysis of the two slower folding phases in wild-type and mutant TRs by cyclophilin shows that cis/trans isomerization of the Thr44/Pro45 peptide bond can limit folding in proteins containing Pro45 . The observation of three urea-independent, slow folding phases in des-Pro TR demonstrates that proline isomerization is not solely responsible for this complex folding behavior . Other types of isomerization or conformational rearrangement reactions appear to limit the folding of this dimeric protein under strongly folding conditions. Biochemistry, 1995 Nov 7, 34(44), 14416 - 27 Solution structure of the CUUG hairpin loop: a novel RNA tetraloop motif; Jucker FM et al.; The solution structure of a uniformly 13C/15N-labeled CUUG RNA hairpin loop has been determined by multidimensional heteronuclear magnetic resonance spectroscopy in combination with distance geometry and restrained molecular dynamics calculations . The structure of this CUUG tetraloop represents a novel RNA loop motif where the first and last loop nucleotides form a standard Watson-Crick C-G base pair and the second loop nucleotide interacts directly with the closing base pair of the stem by folding into the minor groove . This structure helps explain why the closing base pair is phylogenetically conserved and indicates a six-nucleotide G(CUNG)C motif for the CUUG RNA tetraloop . Implications for the function of this CUUG tetraloop in ribosomal RNA and in RNA tertiary interactions are discussed. Biochemistry, 1995 Nov 7, 34(44), 14323 - 30 1.7 A structure of FR-1, a fibroblast growth factor-induced member of the aldo-keto reductase family, complexed with coenzyme and inhibitor; Wilson DK et al.; Murine FR-1 is a protein that is induced by fibroblast growth factor-1 and, therefore, may play a role in the regulation of the cell cycle . Sequence comparison indicates that it is a member of the NADPH-dependent aldo-keto reductase family . It bears 70% identity to human aldose reductase, an enzyme implicated in diabetic complications and a target for drug design . We have determined the 1.7 A resolution structure of the FR-1 in a ternary complex with NADPH and zopolrestat, a potent aldose reductase inhibitor . FR-1 folds into a (beta/alpha)8 barrel with an active site characterized by a preponderance of hydrophobic residues residing in a deep oblong cavity at the C-terminal end of the beta-barrel . The nicotinamide moiety of the coenzyme sits in the base of the cavity . Zopolrestat occupies the active site cavity and makes numerous contacts with several hydrophobic residues . The FR-1 ternary complex structure indicates that it uses the same general catalytic mechanism as aldose reductase and other members of the family whose structures have been determined . The protein exhibits reductase activity with DL-glyceraldehyde as a substrate and is strongly inhibited by zopolrestat . When compared with the structure of a similar ternary complex of aldose reductase, the binding site retains many of the interactions with the coenzyme and inhibitor from the conserved residues . Some differences in sequence, however, create a larger binding site that contains six more water molecules than in the aldose reductase ternary complex structure.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1995 Nov 7, 34(44), 14312 - 22 Site-directed mutagenesis as a tool for molecular modeling of cytochrome P450 2B1; Szklarz GD et al.; Prompted by our previous homology model of cytochrome P450 2B1 based on the 3-D structure of P450cam {Szklarz, G . D., Ornstein, R . L., & Halpert, J . R . (1994) J . Biomol . Struct . Dyn . 12, 61-78}, we constructed 11 new site-directed mutants at positions 100, 111, 205, 209, 291, 477, and 480 and expressed the enzymes in Escherichia coli . The mutations at positions 209, 477, and 480 affected androstenedione and progesterone hydroxylation as predicted by the model . For example, the Ile-477-->Ala and Ile-480-->Ala mutants retained < or = 5% activity with androstenedione and progesterone but were active with benzphetamine, whereas the Leu-209-->Ala mutant catalyzed 21-hydroxylation of progesterone . Mutations at the other positions, i.e., 100, 111, 205, and 291, did not change enzyme activity, contrary to predictions . Therefore, an improved molecular model of cytochrome P450 2B1 was constructed . An alignment of the P450 2B1 sequence with P450 BM-3, P450cam, and P450terp was optimized using data from site-directed mutagenesis at 27 positions in various cytochromes P450 2B and docking of androstenedione into the active site of the known crystal structures . Because all three structures were found to be suitable templates for P450 2B1, the new model was formulated on the basis of the crystallographic coordinates of the three proteins using a consensus strategy, a modeling method based on distance geometry calculations . The new model provides a means to explain alterations in regio- and stereospecificity of steroid hydroxylation upon residue substitution at key amino acid positions, including positions 114, 206, 209, 290, 302, 363, 367, 477, 478, and 480 in P450 2B1. Biochemistry, 1995 Nov 7, 34(44), 14284 - 7 Folding of an enzyme into an active conformation while bound as peptidyl-tRNA to the ribosome; Kudlicki W et al.; Rhodanese bound to bacterial ribosomes as peptidyl-tRNA can be folded into an enzymatically active conformation by generating C-terminal extensions of the wild-type enzyme . Rhodanese was synthesized by coupled transcription/translation in a cell-free Escherichia coli system from plasmids containing the coding sequences for the wild-type enzyme or its C-terminally extended mutants . Two proteins with extensions of 23 amino acids or longer were enzymatically active while bound to the ribosomes whereas wild-type protein and a 13-amino acid extension were not . All forms of the enzyme were active after termination and release of the full-length protein from the ribosomes . All five of the bacterial chaperones were required to substantially increase the specific enzymatic activity of the extended rhodanese while the nascent protein was bound to ribosomes . The results provide direct support for the hypothesis that proteins acquire tertiary structure as they are formed in ribosomes. Gene, 1995 Nov 7, 165(1), 77 - 80 Cloning, sequencing and expression in Escherichia coli of a Streptomyces aureofaciens gene encoding glyceraldehyde-3-phosphate dehydrogenase; Kormanec J et al.; The structural gene (gap) encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) from Streptomyces aureofaciens (Sa) has been cloned and sequenced . The predicted gap product consists of 332 amino acids (aa) (35,312 Da), and has considerable homology (up to 52% aa identity) with other bacterial and eukaryotic gap genes . Sequence analysis of the regions flanking gap revealed two incomplete open reading frames encoding proteins similar to the AraC family of bacterial transcriptional regulators and delta (5)-3-ketosteroid isomerase . The Sa gap gene was expressed at a high level in Escherichia coli (Ec) . Transformation of the Ec strain resulted in an up to eightfold increase in specific GAPDH activity. Gene, 1995 Nov 7, 165(1), 71 - 5 Cloning, sequence analysis, overproduction in Escherichia coli and enzymatic characterization of the RNase HI from Mycobacterium smegmatis; Dawes SS et al.; Activity gel analysis of cell extracts from slow- and fast-growing mycobacteria confirmed the presence of several RNase H activities in both classes of organism . The rnhA gene from Mycobacterium smegmatis (Ms) was subsequently cloned using an internal gene segment probe {Mizrahi et al., Gene 136 (1993) 287-290} . The gene encodes a polypeptide of 159 amino acids that shares 50% identity with the RNase HI from Escherichia coli (Ec) . However, unlike its counterparts from Gram- bacteria, Ms rnhA does not form an overlapping divergent transcriptional unit with dnaQ (encoding the epsilon (proofreading) subunit of DNA polymerase III) . Ms RNase HI was overproduced in Ec as an enzymatically active maltose-binding protein (MBP) fusion protein which cleaved the RNA strand of an RNA.DNA hybrid with a similar site selectivity to that of its Ec homologue. Gene, 1995 Nov 7, 165(1), 39 - 43 A growth-defective kirromycin-resistant EF-Tu Escherichia coli mutant and a spontaneously evolved suppression of the defect; Zeef LA et al.; This study has investigated the cause of a growth-defect phenotype of a mutation in the elongation factor EF-Tu from Escherichia coli . An M13-based genetic retrieval system reported by Zeef and Bosch {Mol . Gen . Genet . 238 (1993) 252-260} was used to segregate and identify an extremely growth-defective kirromycin-resistant (KrR) tufA mutation, encoding Gln124-->Lys (Q124K), from a KrR parent strain . This original strain also contained mutations, 124com1 and 124com2, that appear to have evolved to suppress the Q124K tufA mutation . In this communication we present these M13-based genetic experiments together with additional genetic and protein characterization experiments to clarify the basis of this complementation . The data indicate that the serious growth defect of Q124K originates from a defective GTP/GDP interaction . The GTP/GDP binding and GTP hydrolysis characteristics of ET-Tu Q124K were different from wild-type EF-Tu and especially of another KrR EF-Tu mutant A375T . In line with this, 124com1 specifically complemented EF-Tu Q124K, whereas the growth defects of strains containing EF-Tu mutated at aa 375 were aggravated . We also show that strains containing the segregated tufA Q124K mutation formed filaments. Gene, 1995 Nov 7, 165(1), 149 - 50 New multifunctional Escherichia coli-Streptomyces shuttle vectors allowing blue-white screening on XGal plates; Wehmeier UF; Four new shuttle vectors for Escherichia coli (Ec) and Streptomyces, pUWL218, pUWL219, pUWL-SK and pUWL-KS, which permit recognition of recombinant (re-) plasmids on XGal plates in Ec, were constructed . These vectors contain the replication functions of the Streptomyces wide-host-range multicopy plasmid pIJ101, the tsr gene conferring resistance to thiostrepton in Streptomyces, the ColEI origin of replication from the pUC plasmids for replication in Ec and the bla gene conferring resistance to ampicillin in Ec . They possess multiple cloning sites with a number of unique restriction sites and allow direct sequencing of re-derivatives using the pUC sequencing primers. Gene, 1995 Nov 7, 165(1), 145 - 6 A novel Escherichia coli lipoprotein expression vector; Jones TS et al.; A novel Escherichia coli (Ec) lipoprotein expression plasmid, pSJLP, was constructed . The plasmid contains a truncated alkaline phosphatase gene (phoA) located downstream from the Lac repressor gene lacIq and the IPTG inducible Ptac promoter . The phoA gene was truncated by deleting the native phoA signal sequence and fusing the truncated phoA gene to the lipoprotein signal sequence of the major Ec lipoprotein LPP . The recombinant LPP::PhoA fusion protein is produced and processed as a lipoprotein and can therefore be used as substrate for a novel signal peptidase II assay. Proc Natl Acad Sci U S A, 1995 Nov 7, 92(23), 10777 - 81 Cytoplasmic free-Ca2+ level rises with repellents and falls with attractants in Escherichia coli chemotaxis; Tisa LS et al.; Cytoplasmic free-Ca2+ levels in Escherichia coli were measured by use of the fluorescent Ca(2+)-indicator dye fura-2 . Chemotactically wild-type E . coli regulated cytoplasmic free Ca2+ at approximately 100 nM when no stimuli were encountered, but changes in bacterial behavior correlated with changes in cytoplasmic free-Ca2+ concentration . For chemotactically wild-type E . coli, addition of a repellent resulted in cells tumbling and a transient increase in cytoplasmic free-Ca2+ levels . Conversely, addition of an attractant to wild-type cells caused running and produced a transient decrease in cytoplasmic free-Ca2+ levels . Studies with mutant strains showed that the chemoreceptors were required for the observed changes in cytoplasmic free-Ca2+ levels in response to chemical stimuli. Proc Natl Acad Sci U S A, 1995 Nov 7, 92(23), 10639 - 43 Modification of the substrate specificity of an acyl-acyl carrier protein thioesterase by protein engineering; Yuan L et al.; The plant acyl-acyl carrier protein (ACP) thioesterases (TEs) are of biochemical interest because of their roles in fatty acid synthesis and their utilities in the bioengineering of plant seed oils . When the FatB1 cDNA encoding a 12:0-ACP TE (Uc FatB1) from California bay, Umbellularia californica (Uc) was expressed in Escherichia coli and in developing oilseeds of the plants Arabidopsis thaliana and Brassica napus, large amounts of laurate (12:0) and small amounts of myristate (14:0) were accumulated . We have isolated a TE cDNA from camphor (Cinnamomum camphorum) (Cc) seeds that shares 92% amino acid identity with Uc FatB1 . This TE, Cc FatB1, mainly hydrolyzes 14:0-ACP as shown by E . coli expression . We have investigated the roles of the N- and C-terminal regions in determining substrate specificity by constructing two chimeric enzymes, in which the N-terminal portion of one protein is fused to the C-terminal portion of the other . Our results show that the C-terminal two-thirds of the protein is critical for the specificity . By site-directed mutagenesis, we have replaced several amino acids in Uc FatB1 by using the Cc FatB1 sequence as a guide . A double mutant, which changes Met-197 to an Arg and Arg-199 to a His (M197R/R199H), turns Uc FatB1 into a 12:0/14:0 TE with equal preference for both substrates . Another mutation, T231K, by itself does not effect the specificity . However, when it is combined with the double mutant to generate a triple mutant (M197R/R199H/T231K), Uc FatB1 is converted to a 14:0-ACP TE . Expression of the double-mutant cDNA in E . coli K27, a strain deficient in fatty acid degradation, results in accumulation of similar amounts of 12:0 and 14:0 . Meanwhile the E . coli expressing the triple-mutant cDNA produces predominantly 14:0 with very small amounts of 12:0 . Kinetic studies indicate that both wild-type Uc FatB1 and the triple mutant have similar values of Km,app with respect to 14:0-ACP . Inhibitory studies also show that 12:0-ACP is a good competitive inhibitor with respect to 14:0-ACP in both the wild type and the triple mutant . These results imply that both 12:0- and 14:0-ACP can bind to the two proteins equally well, but in the case of the triple mutant, the hydrolysis of 12:0-ACP is severely impaired . The ability to modify TE specificity should allow the production of additional "designer oils" in genetically engineered plants. FEBS Lett, 1995 Nov 6, 374(3), 356 - 62 Expression of a chimeric, cGMP-sensitive regulatory subunit of the cAMP-dependent protein kinase type I alpha; Wild N et al.; To study the fluctuations of cGMP in living cells through changes of energy transfer of dissociable fluorescence labeled subunits, we constructed a cGMP-sensitive probe by combining the N-terminus of the type I regulatory subunit of cAMP-dependent protein kinase (PKA) with the cGMP binding sites of cGMP-dependent protein kinase I alpha (PKG) . This chimeric regulatory subunit retained PKA-like dimerization and PKG-compatible cGMP binding constants (Kd = 53 nM) for both binding sites . High affinity interaction with the PKA catalytic subunit was verified by Surface Plasmon Resonance (Kd = 3.15 nM) . Additionally, the chimera inhibits the formation of wild-type holoenzyme with an apparent Ki of 1.05 nM . Furthermore, cGMP dissociated the mutant holoenzyme with an apparent activation constant of 146 nM . Thus, our construct provides all the requirements needed to investigate changes in intracellular cGMP concentrations. FEBS Lett, 1995 Nov 6, 374(3), 345 - 50 Cinnamate 4-hydroxylase from Catharanthus roseus, and a strategy for the functional expression of plant cytochrome P450 proteins as translational fusions with P450 reductase in Escherichia coli; Hotze M et al.; A PCR-based approach was used to isolate cDNAs for cinnamate 4-hydroxylase (C4H) from Catharanthus roseus cell cultures . The protein shared 75.9% identity with C4H from other plants, and the transcription was induced under various stress conditions . The cloned protein was used to investigate the functional expression of plant P450/P450-reductase fusions in E . coli . Fusions containing a modified N-terminal membrane anchor were located in the membrane and possessed C4H activity without solubilization or addition of other factors . The results indicate that the fusion protein strategy provides a useful tool to analyze the activities encoded in the rapidly increasing number of plant P450 sequences of uncertain or unknown function . We also discuss critical elements of the strategy: the choice of the E . coli host strain, the N-terminal membrane anchor, and the conditions for protein expression. J Biol Chem, 1995 Nov 3, 270(44), 26538 - 42 Enoyl-acyl carrier protein reductase (fabI) plays a determinant role in completing cycles of fatty acid elongation in Escherichia coli; Heath RJ et al.; The role of enoyl-acyl carrier protein (ACP) reductase (E.C . 1.3.1.9), the product of the fabI gene, was investigated in the type II, dissociated, fatty acid synthase system of Escherichia coli . All of the proteins required to catalyze one cycle of fatty acid synthesis from acetyl-CoA plus malonyl-CoA to butyryl-ACP in vitro were purified . These proteins were malonyl-CoA:ACP transacylase (fabD), beta-ketoacyl-ACP synthase III (fabH), beta-ketoacyl-ACP reductase (fabG), beta-hydroxydecanoyl-ACP dehydrase (fabA), and enoyl-ACP reductase (fabI) . Unlike the other enzymes in the cycle, FabA did not efficiently convert its substrate beta-hydroxybutyryl-ACP to crotonyl-ACP, but rather the equilibrium favored formation of beta-hydroxybutyryl-ACP over crotonyl-ACP by a ratio of 9:1 . The amount of butyryl-ACP formed depended on the amount of FabI protein added to the assay . Extracts from fabI(Ts) mutants accumulated beta-hydroxybutyryl-ACP, and the addition of FabI protein to the fabI(Ts) extract restored both butyryl-ACP and long-chain acyl-ACP synthesis . FabI was verified to be the only enoyl-ACP reductase required for the synthesis of fatty acids by demonstrating that purified FabI was required for the elongation of both long-chain saturated and unsaturated fatty acids . These results were corroborated by analysis of the intracellular ACP pool composition in fabI(Ts) mutants that showed beta-hydroxybutyryl-ACP and crotonyl-ACP accumulated at the nonpermissive temperature in the same ratio found in the fabI(Ts) extracts and in the in vitro reconstruction experiments that lacked FabI . We conclude that FabI is the only enoyl-ACP reductase involved in fatty acid synthesis in E . coli and that the activity of this enzyme plays a determinant role in completing cycles of fatty acid biosynthesis. J Biol Chem, 1995 Nov 3, 270(44), 26391 - 8 Evidence for an RNA binding region in the Escherichia coli processing endoribonuclease RNase E; Taraseviciene L et al.; The processing endoribonuclease RNase E (Rne), which is encoded by the rne gene, is involved in the maturation process of messenger RNAs and a ribosomal RNA . A number of deletions were constructed in order to assess functional domains of the rne gene product . The expression of the deletion constructs using a T7 promoter/RNA polymerase overproduction system led to the synthesis of truncated Rne polypeptides . The smallest gene fragment in this collection that was able to complement a temperature sensitive rnets mutation and to restore the processing of 9 S RNA was a 2.3-kilobase pair fragment with a 1.9-kilobase pair N-terminal coding sequence that mediated synthesis of a 70.8-kDa polypeptide . Antibodies raised against a truncated 110-kDa polypeptide cross-reacted with the intact rne gene product and with all of the shorter C-terminal truncated polypeptides, indicating that the N-terminal part of the molecule contained strong antigenic determinants . Furthermore, by analyzing the Rne protein and the truncated polypeptides for their ability to bind substrate RNAs, we were able to demonstrate that the central part of the Rne molecule encodes an RNA binding region . Binding to substrate RNAs correlated with the endonucleolytic activity . RNAs that are not substrates for RNase E did not bind to the protein . The two mutated Rne polypeptides expressed from the cloned gene containing either the rne-3071 or ams1 mutation also had the ability to bind 9 S RNA, while their enzymatic function was completely abolished . The data presented here suggest that the endonucleolytic activity is encoded by the N-terminal part of the Rne protein molecule and that the central part of it possesses RNA binding activity. J Biol Chem, 1995 Nov 3, 270(44), 26382 - 90 Biochemical, structural, and transglutaminase substrate properties of human loricrin, the major epidermal cornified cell envelope protein; Candi E et al.; Loricrin is the major protein of the cornified cell envelope of terminally differentiated epidermal keratinocytes which functions as a physical barrier . In order to understand its properties and role in cornified cell envelope, we have expressed human loricrin from a full-length cDNA clone in bacteria and purified it to homogeneity . We have also isolated loricrin from newborn mouse epidermis . By circular dichroism and fluorescence spectroscopy, the in vivo mouse and bacterially expressed human loricrins possess no alpha or beta structure but have some organized structure in solution associated with their multiple tyrosines and can be reversibly denatured by either guanidine hydrochloride or temperature . The transglutaminase (TGase) 1, 2, and 3 enzymes expressed during epidermal differentiation utilized loricrin in vitro as a complete substrate, but the types of cross-linking were different . The TGase 3 reaction favored certain lysines and glutamines by forming mostly intrachain cross-links, whereas TGase 1 formed mostly large oligomeric complexes by interchain cross-links involving different lysines and glutamines . Together, the glutamines and lysines used in vitro are almost identical to those seen in vivo . The data support a hypothesis for the essential and complementary roles of both TGase 1 and TGase 3 in cross-linking of loricrin in vivo . Failure to cross-link loricrin by TGase 1 may explain the phenotype of lamellar ichthyosis, a disease caused by mutations in the TGase 1 gene. J Biol Chem, 1995 Nov 3, 270(44), 26377 - 81 A new factor from Escherichia coli affects translocation of mRNA; Ganoza MC et al.; Reconstitution of protein synthesis from purified translation factors on ribosomes from Escherichia coli has revealed the requirement for a protein, W, that affects chain elongation and is essential to reconstitute the process (Ganoza, M . C., Cunningham, C., and Green, R . M . (1985) Proc . Natl . Acad . Sci . U.S.A . 82, 1648-1652) . We report that W has no effect on initiation complex formation by 30 or 70 S ribosomes or on the association of ribosomal subunits, peptide bond synthesis, or binding Ala-tRNA, which is the second amino acid of the coat protein of the MS2 RNA virion . W has a pronounced effect on tripeptide synthesis, and is obligatory for the synthesis of the coat protein or of the hexapeptide encoded by f2am3 RNA . Extracts from a temperature-sensitive mutant of the translocase, EF-G, were purified free of the W protein and were used to score for translocation defects . W is required for binding Ser-tRNA, the third N-terminal amino acid of the MS2 or f2 RNA coat protein to ribosomes bearing fMet-Ala-tRNA, as well as for the ejection of deacyl-tRNA from ribosomes, which occurred concomitant with the binding of the Ser-tRNA . We propose that W functions by ejecting tRNAs from ribosomes in a step that precedes the movement of mRNA during translocation. J Biol Chem, 1995 Nov 3, 270(44), 26352 - 7 Characterization of the iron-binding site in mammalian ferrochelatase by kinetic and Mössbauer methods; Franco R et al.; All organisms utilize ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) to catalyze the terminal step of the heme biosynthetic pathway, which involves the insertion of ferrous ion into protoporphyrin IX . Kinetic methods and Mossbauer spectroscopy have been used in an effort to characterize the ferrous ion-binding active site of recombinant murine ferrochelatase . The kinetic studies indicate that dithiothreitol, a reducing agent commonly used in ferrochelatase activity assays, interferes with the enzymatic production of heme . Ferrochelatase specific activity values determined under strictly anaerobic conditions are much greater than those obtained for the same enzyme under aerobic conditions and in the presence of dithiothreitol . Mossbauer spectroscopy conclusively demonstrates that, under the commonly used assay conditions, dithiothreitol chelates ferrous ion and hence competes with the enzyme for binding the ferrous substrate . Mossbauer spectroscopy of ferrous ion incubated with ferrochelatase in the absence of dithiothreitol shows a somewhat broad quadrupole doublet . Spectral analysis indicates that when 0.1 mM Fe(II) is added to 1.75 mM ferrochelatase, the overwhelming majority of the added ferrous ion is bound to the protein . The spectroscopic parameters for this bound species are delta = 1.36 +/- 0.03 mm/s and delta EQ = 3.04 +/- 0.06 mm/s, distinct from the larger delta EQ of a control sample of Fe(II) in buffer only . The parameters for the bound species are consistent with an active site composed of nitrogenous/oxygenous ligands and inconsistent with the presence of sulfur ligands . This finding is in accord with the absence of conserved cysteines among the known ferrochelatase sequences . The implications these results have with regard to the mechanism of ferrochelatase activity are discussed. J Biol Chem, 1995 Nov 3, 270(44), 26326 - 31 Structural and mechanistic studies of galactoside acetyltransferase, the Escherichia coli LacA gene product; Lewendon A et al.; Escherichia coli galactoside acetyltransferase (GAT) is a member of a large family of acetyltransferases that O-acetylate dissimilar substrates but share limited sequence homology . Steady-state kinetic analysis of over-expressed GAT demonstrated that it accepted a range of substrates, including glucosides and lactosides which were acetylated at rates comparable to galactosides . GAT was shown to be a trimeric acetyltransferase by cross-linking with dimethyl suberimidate . Fluorometric analysis of coenzyme A binding showed that there is a fluorescence quench associated with acetyl-CoA binding whereas CoA has no effect . This difference was exploited to measure dissociation rates for both CoA and acetyl-CoA by stopped-flow fluorometry . The rate of dissociation of CoA (2500 s-1) is at least 170-fold faster than kcat for any substrate tested . The fluorescence response to acetyl-CoA binding is entirely due to Trp-139 since replacement by phenylalanine completely abolished the fluorescence quench . Treatment of GAT by {14C}iodoacetamide resulted in complete inactivation of the enzyme and the incorporation of label into histidyl and cysteinyl residues to approximately equal extents . Following replacement of His-115 by alanine, label was incorporated solely into cysteinyl residues . Furthermore, the substitution results in an 1800-fold decrease in kcat suggesting that His-115 has an important catalytic role in GAT. J Biol Chem, 1995 Nov 3, 270(44), 26282 - 5 GrpE alters the affinity of DnaK for ATP and Mg2+ . Implications for the mechanism of nucleotide exchange; Skowyra D et al.; DnaK, DnaJ, and GrpE heat shock proteins of Escherichia coli activate site-specific DNA binding by the RepA replication initiator protein of plasmid P1 in a reaction dependent on ATP and Mg2+ . We previously showed that GrpE is essential for in vitro RepA activation specifically at about 1 microM free Mg2+ . In this paper, we demonstrate that GrpE lowers the requirement of DnaK ATPase for Mg2+, resulting in a large stimulation of ATP hydrolysis at about 1 microM Mg2+ with and without DnaJ and RepA . In contrast to its effect on the Mg2+ requirement, GrpE increases the ATP requirement for DnaK ATPase and dramatically lowers the affinity of DnaK for ATP in the absence of Mg2+ . We propose that GrpE not only lowers the affinity of DnaK for nucleotide but, by increasing affinity of DnaK for Mg2+, also weakens the interactions of Mg2+ with nucleotide prior to its release. J Biol Chem, 1995 Nov 3, 270(44), 26257 - 64 Tetrameric hemoglobin expressed in Escherichia coli . Evidence of heterogeneous subunit assembly; Hernan RA et al.; Recombinant alpha 2 beta 2 tetrameric Hb expressed and assembled in Escherichia coli has been characterized extensively . Electrospray mass spectrometry and optical and electron paramagnetic resonance spectroscopy suggest that the overexpressed protein is identical to native human Hb . Although the functional properties of this recombinant Hb are nearly identical to native Hb, crucial differences exist between the two molecules . The recombinant Hb expressed in E . coli has a lower Hill coefficient even though oxygen equilibrium binding studies indicate cooperative binding . The most significant difference observed between the recombinant and native Hb is the loss of oxygen affinity regulation by 2,3-diphosphoglyerate and protons . CO binding to the deoxy tetramer was found to be biphasic with both phases sensitive to the presence of allosteric effectors . The recombinant chains were isolated, and the ligand binding properties demonstrated that the recombinant chains behave in a similar fashion to native alpha-sh and beta-sh . To investigate whether the chains were capable of forming a well behaved tetramer, the isolated chains were reassembled into a tetramer and purified to homogeneity . Oxygen binding properties of the reassembled recombinant Hb now show an increased Hill coefficient of 2.5, close to, but still slightly lower than, that observed for native Hb . Additionally, reassembly of recombinant Hb produces a protein that is subject to regulation by allosteric effectors . Furthermore, CO binding to the reassembled recombinant deoxy tetramer was found to be monophasic under all conditions. J Biol Chem, 1995 Nov 3, 270(44), 26159 - 67 Mycobacterium tuberculosis chaperonin 10 forms stable tetrameric and heptameric structures . Implications for its diverse biological activities; Fossati G et al.; The chaperonin activity of sequence-related chaperonin 10 proteins requires their aggregation into heptameric structures . We describe size-exclusion chromatography and ultracentrifugation studies that reveal that while Escherichia coli chaperonin 10 exists as a heptamer, the Mycobacterium tuberculosis chaperonin 10 is tetrameric in dilute solutions and in whole M . tuberculosis lysate . At high protein concentration and in the presence of saturating amounts of divalent ions, the protein is heptameric . Human chaperonin 10 is predominantly heptameric, although smaller oligomers were detected . These differences in structural assembly between species may explain differences in biological activity such as antigenicity . Using C-terminal and N-terminal fragments, sequence 1-25 was identified as indispensable for aggregation . CD spectroscopy studies revealed that (i) a minimum at 202-204 nm correlates with aggregation and characterizes not only the spectrum of the mycobacterial protein, but also those of E . coli and human chaperonin 10 proteins; (ii) the interactions between subunits are of the hydrophobic type; and (iii) the anti-parallel beta-pleated sheet is the main secondary structure element of subunits in both tetrameric and heptameric proteins. J Mol Biol, 1995 Nov 3, 253(4), 604 - 17 Mutations at positions 153 and 328 in Escherichia coli alkaline phosphatase provide insight towards the structure and function of mammalian and yeast alkaline phosphatases; Murphy JE et al.; In order to understand some of the differences between human placental, human, Saccharomyces cerevisiae and Escherichia coli alkaline phosphatases in specific activity, activation by magnesium, and pH versus activity profiles, the X-ray crystal structures of three mutant E . coli alkaline phosphatases have been determined . The aligned sequences of alkaline phosphatases from mammalian, yeast and E . coli show that 25 to 30% of the amino acids are absolutely conserved and the active site residues are completely conserved with the exception of residues 153, 328 and 155 . The bacterial enzyme has a salt-bridge, Asp153/Lys328, near the third metal binding site which, based on sequence homology, is apparently absent in the yeast and mammalian enzymes . The human enzymes have histidine at positions 153 and 328, and the yeast enzyme has histidine at position 328 . In the E . coli enzyme, Asp153 was replaced by histidine (D153H), Lys328 was replaced by histidine (K328H), and a double mutant (DM) was constructed containing both mutations . The structure of the K328H enzyme was refined using cross-validation to a resolution of 2.3 A with a working R-factor of 0.181 and a free R-factor of 0.249 . The DM structure was determined to a resolution of 2.5 A with a working R-factor of 0.166 and a free R-factor of 0.233 . The structure of the D135H enzyme, which has been reported to a resolution of 2.4 A, has been re-refined using cross-validation to a working R-factor of 0.179 and a free R-factor of 0.239 for controlled comparisons with the two new structures . In all three structures the most significant changes are related to the bound phosphate inhibitor and the identity of the metal ion in the third binding site . The changes in the position of the phosphate group and the alterations at the third metal binding site indicate the structural basis for the variations in the steady-state kinetic parameters previously reported for these enzymes. J Mol Biol, 1995 Nov 3, 253(4), 530 - 46 A model for transmembrane signalling by the aspartate receptor based on random-cassette mutagenesis and site-directed disulfide cross-linking; Maruyama IN et al.; Extracellular information is transduced by transmembrane receptors into the inside of the cell across a membrane barrier . To understand the molecular basis of transmembrane signalling, we replaced the transmembrane segment 2 (TM2) of the Escherichia coli aspartate receptor, Tar, with random sequences that are 21 amino acid residues in length and consist of Arg, Gly, Ser, Cys, Val, Leu, Ile and Phe at each position . From this ensemble for recombinant molecules, functional receptors were recovered as clones that could bind aspartate and transmit a signal to the intracellular domain . Restricted average hydrophobicity values were observed for functional transmembrane domains, and support the observation that transmembrane segments typically have hydrophobicity values greater than 1.6 . However, non-functional transmembrane domains with greater hydrophobicity than 1.6 indicate that hydrophobicity is not a sole determinant for its function . Fourier transform analysis of the functional TM2 sequences suggests that the transmembrane segment has an alpha-helical structure with three distinct faces . Cross-linking of the faces to transmembrane segment 1 (TM1) mimics the "locked" signalling phenotypes of the wild-type receptor . The results are consistent with a model in which TM2 rotates in the plane of the lipid bilayer, and the rotation becomes locked at one face of the alpha-helix in the presence of attractant and at another face in the presence of repellent . This dynamic movement of the transmembrane domain may be a common signalling mechanism of homologous membrane receptor molecules such as the insulin receptor . Random-cassette mutagenesis and disulfide cross-linking provide powerful strategies for examining the structure and function of transmembrane segments. Biochem Biophys Res Commun, 1995 Nov 2, 216(1), 390 - 8 Cloning of the cDNA for a brain glycine-, glutamate- and thienylcyclohexylpiperidine-binding protein; Kumar KN et al.; Polyclonal antibodies (Ab's) were raised against a 43-kDa component of a protein complex that has ligand recognition sites similar to those of brain N-methyl-D-aspartate (NMDA) receptors . The Ab's were used to immunopurify from brain synaptic membranes a 60-kDa glycine (Gly), glutamate (Glu) and thienylcyclohexylpiperidine (TCP)-binding protein and to screen a rat hippocampal cDNA expression library . A 1.85-kb clone, pGlyBP, coding for a protein of 470 amino acids (52.7 kDa) was identified . Northern blot analyses performed on poly(A+) RNA from brain revealed hybridization of the labeled cDNA probes to transcripts of 1.9 kb . E . coli transformed with the pGyBP expressed a protein that was recognized by the anti-43 kDa Ab's and had recognition sites for Gly, Glu and TCP . The cloned protein has 2 glycosylation sites, 3 hydrophobic domains, 4 cysteine-rich motifs (C-X2-C-X16-20-C-X5-11), and 2 regions homologous to the NR1 receptor protein. Biochem Biophys Res Commun, 1995 Nov 2, 216(1), 367 - 74 Endotoxin antagonism by a synthetic lipid A analogue, DT-5461, with low endotoxicity in human peripheral blood monocytes; Sato K et al.; We examined the molecular mechanism of DT-5461-induced LPS antagonism in human peripheral blood monocytes . Dose-response studies revealed that LPS-induced IL-1 and TNF-alpha production was apparently totally suppressed in a competitive manner by a 10-fold excess of DT-5461 . A 10-fold excess of DT-5461 significantly blocked the binding of FITC-LPS to the monocytes . DT-5461 suppressed IL-1 and TNF-alpha mRNA expression in LPS-activated monocytes . Western blots showed that DT-5461 suppressed the LPS-induced tyrosine phosphorylation of p42mapk/ERK2 . These results suggested that the competitive binding inhibition and repression of early intracellular signaling involved in DT-5461-mediated LPS antagonism. Biochem Biophys Res Commun, 1995 Nov 2, 216(1), 329 - 37 Stabilisation of purified human collagenase by site-directed mutagenesis; O'Hare MC et al.; During purification, human fibroblast collagenase breaks down into two major forms, an N-terminal 22000/25000-Mr fragment and a C-terminal 27000-Mr fragment; the most likely mechanism being autolysis . The cleavage site has been identified (Pro269- Ile270) and in an attempt to obtain full-length human collagenase (i.e., Mr 42570), this cleavage site and another potential cleavage site (Ala258- Ile259) have been mutated by PCR- directed mutagenesis: Ile270Ser and Ile259Leu . The mutated cDNA was then cloned into the expression vector, pGEX2T, and expressed in Escherichia coli as a fusion protein with glutathione-S-transferase (GST) . After cleavage with factor Xa, the mutated collagenase was purified on a peptide hydroxamic acid affinity column . The mutated recombinant collagenase is stable, remains full length and retains the ability to cleave collagen. Nature, 1995 Nov 2, 378(6552), 92 - 6 A eukaryotic transcriptional repressor with carboxypeptidase activity; He GP et al.; Adipocyte differentiation involves the transcriptional activation of several genes in triglyceride metabolism, including the adipose P2 (aP2 or 422) gene that encodes the adipocyte lipid-binding protein ALBP . Within the mouse aP2 promoter region, the AE-1 sequence functions as either a positive or a negative element in the regulation of aP2 gene expression . The AE-1 sequence is the binding site for the positive murine (3T3) adipocyte factor C/EBP-alpha, several human preadipocyte factors, and a 3T3 preadipocyte factor(s) that has been implicated as a repressor of aP2 gene expression . Here we report the cloning of new complementary DNAs that encode the 3T3 preadipocyte factor (termed AEBP1) and demonstrate that AEBP1 expression is abolished during adipocyte differentiation . Furthermore, we show that an activity of a carboxypeptidase associated with AEBP1 is important in the transcriptional repression function of AEBP1 . Thus AEBP1 might represent a new type of transcription factor that regulates transcription by cleavage of factors involved in transcription. J Mol Recognit, 1995 Nov-Dec, 8(6), 327 - 33 Metabolic engineering of a non-allosteric citrate synthase in an Escherichia coli citrate synthase mutant; Evans CT; This study examined the organization of the Krebs tricarboxylic acid (TCA) cycle by metabolic engineering and high-resolution 13C NMR . The oxidation of {1,2,3-13C}propionate to glutamate via the TCA cycle was measured in wild-type (WT) and a citrate synthase mutant (CS-) strain of Escherichia coli transformed with allosteric E . coli citrate synthase (ECCS) or non-allosteric pig citrate synthase (PCS) . The 13C fractional enrichment in glutamate C-2, C-3, and C-4 in ECCS and PCS were similar; although quantitative differences in total citrate synthase activity and total C-4 labeling of glutamate were observed in ECCS and PCS . Allosteric ECCS cells contained 10-fold less total enzyme activity than PCS but only 50% less total labeling in glutamate C-4 and equivalent doubling times . The observed spectra were mathematically fitted using an iterative procedure (TCACALC) and yielded an acetate/succinyl-CoA flux ratio of 10 for both ECCS and PCS, a result that is in agreement with the isotopomer analyses of the 13C spectra of cells presented with {3-13C}propionate or {2-13C}propionate . The results are consistent with the presence of an allosteric citrate synthase in ECCS and a non-allosteric citrate synthase in PCS . The former maintains TCA cycle flux via alternative propionate pathways activated by positive allosteric mechanisms and the latter via elevated enzyme levels. Biol Chem Hoppe Seyler, 1995 Nov, 376(11), 681 - 4 Production of active recombinant human chymase from a construct containing the enterokinase cleavage site of trypsinogen in place of the native propeptide sequence; Wang ZM et al.; Human chymase, a chymotrypsin-like proteinase found in mast cells, was produced in an enzymatically active recombinant form . The protein was expressed in Escherichia coli as part of an insoluble fusion protein which was solubilized and renatured . The structure of the fusion protein was NH2-ubiquitin-enterokinase cleavage site-chymase-COOH . The enterokinase cleavage site of trypsinogen replaced the native propeptide sequence of chymase, allowing for activation by a readily available proteinase (enterokinase) of known specificity . Characterization of refolded-activated recombinant chymase with substrates and inhibitors demonstrated properties identical to that of the native proteinase isolated from skin. Biol Chem Hoppe Seyler, 1995 Nov, 376(11), 651 - 60 Probing the presumed catalytic triad of selenium-containing peroxidases by mutational analysis of phospholipid hydroperoxide glutathione peroxidase (PHGPx); Maiorino M et al.; Single and double site mutants affecting the presumed catalytic centre of the selenoenzyme PHGPx were subjected to functional analysis . The rate constants k+1 and k'+2, for the oxidation and the regeneration of the ground state enzyme were estimated, respectively . Moreover, the alkylation rate of the reactive centre by iodoacetate (kinact.) was also analysed . The substitution of the catalytically competent selenocysteine 46 by cysteine (PHGPxcys46) decreased k+1 and k'+2 by about three orders of magnitude, although leaving unaffected kinact. . Furthermore, mutations of PHGPxcys46 involving the other residues of the triad decreased both kinact . and k+1, thus highlighting the involvement of Gln 81 and Trp 136 in the dissociation/activation of the nucleophilic cysteine thiol . In general, substitutions of Gln 81 or Trp 136 by acidic residues in PHGPxcys46 most dramatically depressed the k+1 values, because they practically prevented the dissociation of the thiol group, while neutral or positively charged residues in these positions allowed an intermediate dissociation and induced a corresponding reactivity of the thiol . Our data, for the first time, reveal that the presumed triad of selenocysteine, glutamine and tryptophan residues represents a novel type of catalytic centre, whose integrity is essential for the full catalytic function of glutathione peroxidases. Biol Chem Hoppe Seyler, 1995 Nov, 376(11), 637 - 41 Topological requirements for recognition and cleavage of DNA by ribosome-inactivating proteins; Ling J et al.; Ribosome-inactivating proteins (RIPs) were demonstrated to exhibit a unique enzymatic activity on cleaving supercoiled double-stranded DNA into the nicked or linear form . Although there is an interaction between supercoiled DNA and RIP, no sequence-specific recognition was involved . Instead, RIPs recognize supercoiled DNA by conformational specificity . Negatively supercoiled DNA is the preferential conformation in the action of RIPs . When double-stranded DNA occurs in the supercoiled form, even if with lower linking number, RIPs can still convert it into nicked or linear form . Terminal-labelling experiments indicated that radioactivity was incorporated into putative 5'-ends of nicked or linear DNA generated by RIPs . We conclude that RIPs act as a novel supercoil-dependent endonuclease when they cleavage supercoiled DNA . The impossibility that contaminating enzymes in the RIP preparations cleaved the supercoiled DNA is briefly discussed. Acta Pol Pharm, 1995 Nov-Dec, 52(6), 471 - 6 Esters of cephalosporins . Part III . Separation and properties of the R and S isomers of the 1-acetoxyethyl ester of cefuroxime; Oszczapowicz I et al.; Two separation methods for the R and S isomers of the 1-acetoxyethyl ester of cefuroxime {I} were evaluated . Physico-chemical and biological properties of these isomers were studied . It was found that the R isomer after oral administration to rats is absorbed better and faster than the isomer S. East Afr Med J, 1995 Nov, 72(11), 699 - 702 Effect of human immunodeficiency virus on local immunity in children with diarrhoea; Kakai R et al.; The purpose of this study was to determine the relationship between intestinal mucosal immunity and diarrhoea . Stools were tested for total IgA by radial immunodiffusion, cultured for bacteria and examined for ova/cysts by microscopy . Peripheral blood was screened for HIV-1 antibody by ELISA, CD4 and CD8 enumerated by flow cytometry and phagocytic activity by C . albicans engulfment . A total of 271 children were enrolled with a mean age of 20.3 m (range 0.3-60.0 m) . HIV exposed (born to HIV seropositive mothers) had more episodes of diarrhoea than HIV unexposed (born to HIV seronegative mothers) children in the first six months of life (26.0% versus 5.5%, p = 0.002) . Exposed children had severe (16/44 versus 6/29, p = 0.02) and prolonged diarrhoea lasting more than nine days (11.0% versus 1.4%, p = 0.03) than unexposed . CD8 counts were significantly higher in exposed than unexposed children (1837.0 versus 1373.0 cells/mm3, p = p.01) . Among children aged 15 months and over, HIV seropositive children had severe diarrhoea (4/6 versus 11/32, p<0.01), reduced phagocytic activity (phagocytic index 15.4 versus 28.9, p<0.01), total intestinal IgA (0.2 versus 0.7 mg/ml, p = 0.04) and CD4 counts (624.2 versus 1345.1 cells/mm3, p = 0.01) than seronegative . Reduction of CD4 was more significant in HIV seropositive children with severe diarrhoea (298.7 versus 1318.5 cells/mm3, p = 0.01) . Isolation of enteric pathogens was independent of either maternal or child's HIV serostatus although E . coli was more frequent in children with low CD4 counts . These results highlight the importance of mucosal immunity in the intestinal infections . Exposure to HIV, reduced CD4 counts and IgA were associated with diarrhoea probably due to impaired intestinal mucosal immunity. Plasmid, 1995 Nov, 34(3), 236 - 9 Site-specific mutations in the traI relaxase and upstream region of plasmid RP4; Cole SP et al.; The relaxase of RP4 nicks the double-stranded plasmid at the oriT site and binds covalently to DNA at the 5' end of the nick . The 80-kDa relaxase (TraI) is encoded on an operon with several overlapping open reading frames (ORFs) . The importance in conjugation of a short ORF (traX) with a start site overlapping the 5' terminus of traI was investigated, as well as the effects of specific mutations in the relaxase . Elimination of TraX reduced the transfer efficiency by approximately 50% in several intergeneric matings, especially when Escherichia coli was the donor . While TraI was essential for transfer to occur, deletion of the C-terminus of TraI decreased, but did not eliminate plasmid transfer . Mutation of the active site tyrosine resulted in residual transfer associated with amino acid misincorporation. Plasmid, 1995 Nov, 34(3), 175 - 83 Trapping developmental promoters in Dictyostelium; Chang WT et al.; Recently an insertional mutagenesis procedure has been developed to permit cloning of genes affected in developmental mutants of Dictyostelium discoideum (Kuspa and Loomis, Proc . Natl . Acad . Sci . USA 89, 8803-8807, 1992) . In this procedure a plasmid bearing the URA (pyr5-6) gene is linearized with a restriction enzyme and electroporated into URA- amoebae (auxotrophic for uracil) together with the corresponding restriction enzyme . Transformants that can grow without uracil are screened for developmental defects resulting from insertion of the plasmid into a gene of developmental importance . We have modified this procedure to permit characterization of the promoters and structural sequences of genes that would be missed by the standard procedure because their disruption produces no obvious phenotype . Constructs carrying a promoter-less Escherichia coli lacZ gene were designed so that expression of lacZ requires insertion into an active host transcription unit . By screening restriction enzyme-generated transformants we have isolated several strains in which lacZ is under the control of a developmentally activated promoter and have cloned the 5' flanking DNA adjacent to the insertion site . Sequencing the junction between plasmid and host genome has confirmed in-frame fusion with the lacZ gene, and reintroduction of the cloned plasmids into parental cells has shown that the cloned sequences do actually contain the relevant promoters . This procedure should give ready access to a wide range of developmental promoters without the need for prior identification of the developmental genes involved. Protein Eng, 1995 Nov, 8(11), 1163 - 9 Generation of a Ni(II) binding site by introduction of a histidine cluster in the structure of human glutathione transferase A1-1; Yilmaz S et al.; Two mutant forms of human glutathione transferase (GST) A1-1 with affinity for metal ions were constructed by introduction of His residues by site-directed mutagenesis . A mutant, 2-His, contained the mutations Lys84Gln, Asp85His and Glu88His, and another, 5-His, contained the mutations Tyr79His, Asn80His, Lys84His, Asp85His and Glu88His . The mutant proteins were obtained in good yields (40-150 mg per 3 l culture) by heterologous expression in Escherichia coli . The mutant enzymes possessed novel binding affinities for Ni(II) and Zn(II) ions, as demonstrated by immobilized metal ion affinity chromatography . The mutant with two novel His residues (2-His mutant) did not bind as tightly to immobilized Ni(II) as did the mutant with five novel His residues (5-His mutant) . When tested for affinity to immobilized Zn(II), only the 5-His mutant remained bound to the column . The affinity of the 5-His mutant for Ni(II) ions in solution was determined by binding experiments in an aqueous polymeric two-phase system . Analysis of the binding curve showed two binding sites per enzyme subunit and a dissociation constant of 6.7 +/- 1.6 mu M . The kinetic constants kcat, Km and kcat/Km for the reaction with glutathione and 1-chloro-2,4-dinitrobenzene were determined by steady-state kinetic analysis and the parameter values for the mutant forms were found to be indistinguishable from those obtained for the wild-type GST A1-1 . The differences in surface charge in the mutant proteins as compared with the wild-type enzyme did not alter the pH dependence of kcat . The results provide an alternative method for purification of fully active recombinant GST A1-1 by the introduction of novel metal binding sites . The data also showed that two His residues are sufficient for Ni(II) binding. Protein Eng, 1995 Nov, 8(11), 1153 - 61 Effects of amino acid substitutions in the hydrophobic core of alpha-lactalbumin on the stability of the molten globule state; Uchiyama H et al.; Five mutant alpha-lactalbumins, with one or two amino acid substitution(s) in the B helix, were engineered to examine the relation between the stability of the molten globule state and the hydrophobicity of these amino acids . The mutation sites (Thr29, Ala30 and Thr33) have been chosen on the basis of comparison of the amino acid sequences of goat, bovine and gunea pig alpha-lactalbumin, in which the guinea pig protein shows a remarkably more stable molten globule than the other proteins . The recombinant proteins were expressed Escherichia coli and then purified and refolded efficiently to produce the active proteins . The stability of the molten globule state of these engineered proteins has been investigated by urea-induced unfolding transition under an acidic condition (pH 2.0), where the molten globule state is stable in the absence of urea . The results show that the molten globule state is stabilized by the amino acid substitutions which raise the hydrophobicity of the residues, suggesting that the hydrophobic core in a globular protein plays an important role in the stability of the molten globule state . The change in stabilization free energy of the molten globule state caused by each amino acid substitution has been evaluated, and molecular mechanisms of stabilization of the molten globule state are discussed. Mol Microbiol, 1995 Nov, 18(4), 661 - 70 Expression of two csg operons is required for production of fibronectin- and congo red-binding curli polymers in Escherichia coli K-12; Hammar M et al.; Two divergently transcribed operons in Escherichia coli required for the expression of fibronectin- and Congo red-binding curli polymers were identified and characterized by transposon mutagenesis, sequencing and transcriptional analyses, as well as for their ability to produce the curli subunit protein . The csgBA operon encodes CsgA, the major subunit protein of the fibre, and CsgB, a protein with sequence homology to CsgA . A non-polar csgB mutant is unaffected in its production of CsgA, but the subunit protein is not assembled into insoluble fibre polymers . A third open reading frame, orfC, positioned downstream of csgA may affect some functional property of curli since an insertion in this putative gene abolishes the autoagglutinating ability typical of curliated cells without affecting the production of the fibre . The promoter for the oppositely transcribed csgDEFG operon was identified by primer extension and shown, like the csgBA promoter, to be dependent upon the alternate stationary phase-specific sigma factor sigma s in wild-type cells, but not in mutants lacking the nucleoid associated protein H-NS . Insertions in csgD abolish completely trancription from the csgBA promoter . Therefore, any regulatory effect on the csgBA promoter might be secondary to events controlling the csgDEFG promoter and/or activation of CsgD . Insertions in csgE, csgF and csgG abolish curli formation but allow CsgA expression suggesting that one or more of these gene products are involved in secretion/assembly of the CsgA subunit protein . No amino acid sequence homologies were found between the CsgE, CsgF and CsgG proteins and secretion/assembly proteins for other known bacterial fibres, suggesting that the formation of curli follows a novel pathway. Mol Microbiol, 1995 Nov, 18(4), 641 - 7 Expression of the gltP gene of Escherichia coli in a glutamate transport-deficient mutant of Rhodobacter sphaeroides restores chemotaxis to glutamate; Jacobs MH et al.; Rhodobacter sphaeroides is chemotactic to glutamate and most other amino acids . In Escherichia coli, chemotaxis involves a membrane-bound sensor that either binds the amino acid directly or interacts with the binding protein loaded with the amino acid . In R . sphaeroides, chemotaxis is thought to require both the uptake and the metabolism of the amino acid . Glutamate is accumulated by the cells via a binding protein-dependent system . To determine the role of the binding protein and transport in glutamate taxis, mutants were created by Tn5 insertion mutagenesis and selected for growth in the presence of the toxic glutamine analogue gamma-glutamyl-hydrazide . One of the mutants, R . sphaeroides MJ7, was defective in glutamate uptake but showed wild-type levels of binding protein . The mutant showed no chemotactic response to glutamate . Both glutamate uptake and chemotaxis were recovered when the gltP gene, coding for the H(+)-linked glutamate carrier of E . coli, was expressed in R . sphaeroides MJ7 . It is concluded that the chemotactic response to glutamate strictly requires uptake of glutamate, supporting the view that intracellular metabolism is needed for chemotaxis in R . sphaeroides. Mol Microbiol, 1995 Nov, 18(4), 615 - 22 Benefit of transcription-coupled nucleotide excision repair for gene expression in u.v.-damaged Escherichia coli; Li BH et al.; Expression of the lactose operon upon induction by IPTG was studied with Escherichia coli B/r and K-12 strains as a function of exposure to ultraviolet light . Patterns of expression inactivation were compared in cells with wild-type UvrABC nucleotide excision repair, with transcription-coupled excision repair (TCR) specifically defective because of a defect at mfd, or with excision repair (ER) and TCR eliminated by defects at uvrA or uvrC . Sets of inactivation patterns were also determined for cells expressing the lactose operon via the "UV5' promoter, an alternative to the wild-type promoter that eliminates dependence of expression on negative DNA supercoiling . The results demonstrated a major contribution by TCR to successful gene expression . Gene expression was more sensitive to u.v . inactivation when TCR was defective and similarly more sensitive when both ER and TCR were defective . Thus, TCR may be the only means of repairing transcription-blocking damage at active genes . Contrasting results with wild-type and UV5 promoters suggested that relaxed supercoiling might accompany repair and reduce expression even though a template lesion is removed . A test of mismatch repair defects on ultraviolet inactivation of gene expression found only limited interference with TCR as it benefits gene expression. Methods Find Exp Clin Pharmacol, 1995 Nov, 17 Suppl C, 21 - 4 Role of histamine produced by macrophages in mouse bone marrow; Takamatsu S et al.; Promyelocytic HL-60 cells differentiated into mature cells when they were cultured in the presence of dimaprit (10(-4) M), a histamine H2 agonist . An injection of Escherichia coli lipopolysaccharide increased the activity of histidine decarboxylase in bone marrow cells in C3H/HeN mice to a much greater extent than in C3H/HeJ mice, which are resistant to various effects of lipopolysaccharide . Histamine production increased concomitantly . In WBB6/F1 (W/W(v)) mice, which are genetically deficient in mast cells, histidine decarboxylase activity increased more than in C3H/HeN mice . Pure (>99% nonspecific esterase, CD14 and Mac-1 positive) macrophage populations were obtained from long-term culture of the bone marrow cells (bone marrow-derived macrophages, BMDM) . Culture of the cells in the presence of lipopolysaccharide caused a slight, but dose-dependent increase in histidine decarboxylase-associated histamine synthesis . Granulocyte/macrophage colony-stimulating factor (rmGM-CSF) or interleukin 3 (rmIL-3) potently increased lipopolysaccharide-induced histamine formation. Genome Res, 1995 Nov, 5(4), 404 - 7 A highly efficient procedure for site-specific mutagenesis of full-length plasmids using Vent DNA polymerase; Byrappa S et al.; Careful titration of Vent polymerase activity allows efficient amplification of full-length plasmids (12 kb) . The high processivity and fidelity of this enzyme made oligonucleotide-directed site-specific mutagenesis of plasmids a straight-forward process . Using only two primers, a mutagenic and a complementary, single-base mutants of recombinant plasmids were obtained consistently with > 90% efficiency from a single round of PCR . This procedure also made site-specific deletion, insertion, and several bases mutagenesis facile and efficient. J Neurosci Methods, 1995 Nov, 62(1-2), 213 - 9 Generating a phage display antibody library against an identified neuron; Merz DC et al.; The generation of monoclonal antibodies by conventional hybridoma technology is limited by the diversity of the clones and the difficulties of screening the antibodies and of their large-scale production from isolated clones . As an alternative approach, we have investigated the suitability of phage display libraries for the production of recombinant antibodies against an identified neuron . Mice were immunized with isolated leech Retzius (R) neurons . The spleen poly A+ RNA was isolated and first-strand cDNA was prepared . The variable regions of light- and heavy-chain IgG molecules were amplified by the polymerase chain reaction (PCR) and separate libraries of each were constructed and combined in the pComb8 vector to yield a combinatorial library of approximately 10(7) transformants . Single R neurons that were plated in culture bound approximately 100 phages on a first screen and several thousand when the first batch was re-screened . Of these, 96 individual phage colonies were isolated and used for immunocytochemistry with leech CNS ganglia: 41 exhibited general staining, 20 showed no detectable staining and 30 stained selective subsets of neurons including (but not specific for) the R neuron . The phage display library approach thus simplifies the screening of large libraries with small numbers of (and even single) cells . However, the combinatorial antibodies with binding activity must still be tested individually by immunocytochemistry, which is further limited by their apparently low affinity. J Biochem (Tokyo), 1995 Nov, 118(5), 1083 - 9 Significance of Thr182 in the nucleotide-exchange and GTP-hydrolysis reactions of the alpha subunit of GTP-binding protein Gi2; Nishina H et al.; The crystal structures of the GTP- and GDP-bound alpha subunits of heterotrimeric GTP-binding proteins were recently determined, and a conserved Thr residue in the G2 (linker 2) region of the alpha subunits, which corresponds to Thr182 in Gi2 alpha, was deduced to interact with the gamma-phosphate of GTP and Mg2+ . To investigate biochemically the significance of the Thr residue, we produced a mutant Gi2 alpha, in which Thr182 was substituted for Ala (T182A), in Escherichia coli . The rate of guanosine 5'-(gamma-thio)tri-phosphate (GTP gamma S) binding to T182A was higher than that to the wild-type Gi2 alpha, especially with a high concentration (10 mM) of Mg2+ . The rate of dissociation of bound GDP from T182A was also much faster than that from the wild-type with the high Mg2+ concentration . Moreover, T182A had much lower GTPase activity than the wild-type, like the gip mutant (R179C) of Gi2 alpha found in human endocrine tumors . The ability of T182A to interact with beta gamma subunits and membrane-bound receptors was the same as that of the wild-type alpha subunit . T182A could take on a GTP-bound active conformation, as judged from its sensitivity to tryptic digestion . These results indicated that Thr182 plays an important role not only in the Mg(2+)-sensitive GDP-GTP exchange reaction but also in the GTPase activity of Gi2 alpha . The T182A mutant of Gi2 alpha, characterized by the faster GDP release and the slower GTP hydrolysis, would be a novel mutant that retains the ability to interact with receptors and beta gamma subunits. JPEN J Parenter Enteral Nutr, 1995 Nov-Dec, 19(6), 477 - 81 Inhibition of pulmonary microvascular endothelial glutamine transport by glucocorticoids and endotoxin; Pan M et al.; BACKGROUND: During septic states, the lungs produce increased amounts of glutamine, an event that is mediated by both endotoxin and glucocorticoid hormones and is presumed to be due to accelerated intracellular glutamine biosynthesis . Because enhanced net glutamine release in vivo could also be due to a decrease in cellular uptake, we assayed glutamine transport in cultured rat microvascular pulmonary endothelial cells . METHODS: The effect of Escherichia coli endotoxin (LPS, 1 microgram/mL), various cytokines, and dexamethasone (DEX, 0.1 mumol/L) on glutamine transport activity was studied in rat lung microvascular endothelial cells grown in varying glutamine concentrations (0, 0.1, 0.5, and 2 mmol/L) . Experiments were also performed in cells treated with cycloheximide, actinomycin D, or chelerythrine chloride . RESULTS: More than 90% of glutamine transport was mediated by the Na+ -dependent transport system ASC . DEX and LPS inhibited endothelial glutamine uptake in a time- and dose-dependent manner, a response that was only observed with incubation medium contained the lower concentrations of glutamine . Neither DEX nor LPS altered transport activity in cells cultured in medium containing 2 mmol glutamine/L . There was no synergistic or additive effect when both compounds were added together . The cytokines tumor necrosis factor alpha, interleukin (IL) 1, IL-2, and IL-6 did not alter glutamine transport . both DEX and LPS inhibited glutamine transport by decreasing transporter maximal transport velocity (Vmax) without affecting transporter affinity (Km) . Cycloheximide and actinomycin D abrogated the inhibition of transport activity that was observed in DEX- or LPS-treated cells, whereas the protein kinase C inhibitor chelerythrine chloride had no effect on either control or stimulated glutamine transport . CONCLUSIONS: These data suggest that DEX and LPS "down-regulate" glutamine uptake by lung microvascular endothelial cells by inducing the synthesis of an inhibitory protein that modulates the activity of the system ASC protein . This response in vitro appears to be influenced by the extracellular glutamine concentration . This decrease in microvascular endothelial glutamine transport may be one mechanism by which net lung glutamine release is enhanced during critical illness. Mol Microbiol, 1995 Nov, 18(3), 507 - 20 Evidence for in vivo but not in vitro expression of a Borrelia burgdorferi outer surface protein F (OspF) homologue; Akins DR et al.; Protein export signals from the low-passage 297 strain of Borrelia burgdorferi were cloned as fusions with an Escherichia coli alkaline phosphatase (PhoA) reporter lacking a signal sequence . One PhoA+ clone (BbK2.10-PhoA) was derived from a borrelial lipoprotein . Although the polypeptide encoded by the full-length bbk2.10 gene had 76% similarity and 56% identity to outer surface protein F (OspF) from B . burgdorferi strain N40, antibodies directed against recombinant forms of the two proteins revealed that they were not cross-reactive . The nucleotide sequences of bbk2.10 and ospF from the N40 and 297 strains, respectively, were determined to confirm that the N40 and 297 strains each contained both genes . Southern blot analysis revealed that bbk2.10 is a single-copy gene and that the B . burgdorferi strain 297 and N40 genomes appeared to contain one other gene more closely related to ospF than bbk2.10 . It was particularly noteworthy that ospF, but not bbk2.10, was expressed in vitro while B . burgdorferi-infected mice generated antibodies reactive with both lipoproteins . To help confirm that the BbK2.10-reactive antibodies produced by the B . burgdorferi-infected mice were specific for that protein, a second gene, bbk2.11, which hybridized with the ospF probe was cloned; the corresponding polypeptide reacted strongly with OspF antisera but failed to react with BbK2.10-specific antisera . Taken together, these data demonstrate that BbK2.10, BbK2.11, and OspF comprise a B . burgdorferi lipoprotein family and that at least one member (BbK2.10) appears to be expressed only during infection. Mol Microbiol, 1995 Nov, 18(3), 491 - 505 Mutational activation of the Cpx signal transduction pathway of Escherichia coli suppresses the toxicity conferred by certain envelope-associated stresses; Cosma CL et al.; The processing-defective outer membrane porin protein LamBA23D (Carlson and Silhavy, 1993) and a tripartite fusion protein, LamB-LacZ-PhoA (Snyder and Silhavy, 1995), are both secreted across the cytoplasmic membrane of Escherichia coli, where they exert an extracytoplasmic toxicity . Suppressors of these toxicities map to a previously characterized gene, cpxA, that encodes the sensor kinase protein of a two-component regulatory system . These activated cpxA alleles, designated as cpxA*, stimulate transcription of the periplasmic protease DegP (Danese et al., 1995), which in turn catalyses degradation of the tripartite fusion protein . In contrast, degradation of precursor LamBA23D is not significantly stimulated in a cpxA* suppressor background . In fact, increased levels of DegP in a wild-type background stabilized this protein . While a functional degP gene is required for full cpxA*-mediated suppression of both toxic envelope proteins, residual suppression is seen in cpxA* degP::Tn10 double mutants . Furthermore, cpxA* mutations suppress the toxicity conferred by the LamB-LacZ hybrid protein, which exerts its effects in the cytoplasm, sequestered from DegP . Together, these observations suggest that the activated Cpx pathway regulates additional downstream targets that contribute to suppression . A subset of these targets may constitute a regulon involved in relieving extracytoplasmic and/or secretion-related stress. Mol Microbiol, 1995 Nov, 18(3), 437 - 48 Deletion mutagenesis of Tn 10 Tet repressor--localization of regions important for dimerization and inducibility in vivo; Berens C et al.; The gene for the Tn 10 Tet repressor (TetR) was subjected to deletion mutagenesis . Screening for a transdominant operator-binding negative phenotype yielded 10 mutants with internal deletions . Three deletions extend from residue D5 to residues L41, W75, or Q76, respectively, and two contain deletions of the alpha-helix-turn-alpha-helix DNA-binding motif . Five deletions range from residue K84 to residues between R87 and K98 . Since residues from the N-terminus up to position 98 are not necessary for dimerization, this must take place in the C-terminal half of the protein . Ability to dimerize was probed by introducing ochre nonsense codons (oc) at residues G138, H151, E159, I174, or K202 . Koc202 shows wild-type in vivo operator-binding and inducibility by tetracycline indicating that the six C-terminal residues of TetR are not important for activity . Mutants with longer C-terminal truncations are inactive and not transdominant . They show reduced steady-state protein levels and are probably impaired in folding and degraded in vivo . Two mutants (delta151-166, delta164-166) with deletions in a region variable in primary structure and length among Tet repressors from different resistance determinants bind tet operator efficiently, but are not inducible by tetracycline . This result indicates that these residues are not important for dimer formation in the operator-binding form. Res Commun Mol Pathol Pharmacol, 1995 Nov, 90(2), 289 - 300 Effect of pentobarbital anesthesia on the pressor response to agonists in vivo in normal and endotoxemic rats; Hauser GJ et al.; Studies of cardiovascular physiology are frequently performed under barbiturate anesthesia even though the effect of barbiturates on the pressor response to catecholamines is controversial, and their effect on the response to other agonists is unknown . The effect of pentobarbital (PB) anesthesia on the pressor and heart rate (HR) dose responses to norepinephrine (NE), angiotensin II (AII), vasopressin (VP) and neuropeptide Y (NPY) was studied in vivo in normal and endotoxemic rats . Four groups of rats (5-6 rats/group) were studied for each agonist: 1) anesthetized/endotoxemic, 2) anesthetized/control, 3) conscious/endotoxemic, and 4) conscious/control . Anesthesia was maintained with 10 mg/kg of PB i.v . q 45 minutes . Endotoxemia was established by infusion of a non-hypotensive dose of E . coli lipopolysaccharide 0127:B8, (LPS, 10 micrograms/10 microliters/min) throughout the experiment . One hour after the LPS (or saline control) infusion was started, dose response curves of the pressor and HR responses to agonists were established . LPS infusion resulted in marked suppression of the pressor response to NE, AII, and VP in both conscious and anesthetized rats . LPS infusion suppressed the response to NPY in conscious, but not in anesthetized rats . LPS did not affect the baroreceptor reflex . In both normal and endotoxemic rats, PB anesthesia suppressed the pressor response and attenuated the baroreceptor reflex to AII and NPY, enhanced the pressor response without affecting the heart rate response to NE, and attenuated the baroreceptor reflex to VP . The pressor response to VP was suppressed by anesthesia in normal, but not in endotoxemic rats . PB anesthesia interferes with the cardiovascular effects of different agonists in a variable manner, depending on the agonist tested and the presence or absence of endotoxemia, indicating their different modes of action . These effects should be considered when planning in vivo experiments with these and other agonists. J Protein Chem, 1995 Nov, 14(8), 665 - 78 Extensive modifications for methionine enhancement in the beta-barrels do not alter the structural stability of the bean seed storage protein phaseolin; Dyer JM et al.; Common beans are widely utilized as a food source, yet are low in the essential amino acid methionine . As an initial step to overcome this defect the methionine content of the primary bean seed storage protein phaseolin was increased by replacing 20 evolutionarily variant hydrophobic residues with methionine and inserting short, methionine-rich sequences into turn and loop regions of the protein structure . Methionine enhancement ranged from 5 to 30 residues . An Escherichia coli expression system was developed to characterize the structural stability of the mutant proteins . Proteins of expected sizes were obtained for all constructs except for negative controls, which were rapidly degraded in E . coli . Thermal denaturation of the purified proteins demonstrated that both wild-type and mutant phaseolin proteins denatured reversibly at approximately 61 degrees C . In addition, urea denaturation experiments of the wild-type and a mutant protein (with 30 additional methionines) confirmed that the structural stability of the proteins was very similar . Remarkably, these results indicate that the phaseolin protein tolerates extensive modifications, including 20 substitutions and two loop inserts for methionine enhancement in the beta-barrel and loop structures, with extremely small effects on protein stability. Biochem Cell Biol, 1995 Nov-Dec, 73(11-12), 1199 - 207 Cotranslational folding of nascent proteins on Escherichia coli ribosomes; Hardesty B et al.; Evidence is presented for cotranslational folding of rhodanese or ricin during its synthesis on Escherichia coli ribosomes . During transcription-translation, full-length but enzymatically inactive polypeptides accumulated as peptidyl-tRNA on the ribosomes . These polypeptides were activated and released by subsequent incubation with the bacterial chaperones and with release factor (RF-2) . Coumarin was incorporated cotranslationally at the N-terminus of the nascent protein from fluorophore-S-Ac-Met-tRNAf . Changes in fluorescence indicated that DnaJ bound to the nascent proteins and to a fluorescently labeled synthetic peptide corresponding to the N-terminal 17 amino acids of bovine rhodanese . This peptide also bound to 70S ribosomes or 50S subunits but not to 30S subunits . It inhibited activation and RF-2-dependent release of the full-length ribosome-bound rhodanese . A deletion mutant of rhodanese lacking the N-terminal 23 amino acids was not accumulated on the ribosome but was synthesized very efficiently . However, the protein that was formed was enzymatically inactive . DnaJ did not bind to this deletion mutant on ribosomes . We conclude that the chaperone-mediated reactions facilitate binding of the N-terminal sequence of nascent proteins to a specific site on 50S ribosomal subunits where it blocks release . The ribosome-bound protein undergoes chaperone-mediated reactions that are required for folding into an enzymatically active conformation. Biochem Cell Biol, 1995 Nov-Dec, 73(11-12), 1141 - 9 The accuracy center of a eukaryotic ribosome; Liebman SW et al.; Mutations in yeast ribosomal proteins and ribosomal RNAs have been shown to affect translational fidelity . These mutations include: proteins homologous to Escherichia coli's S4, S5, and S12; a eukaryote specific ribosomal protein; yeast ribosomal rRNA alterations at positions corresponding to 517, 912, and 1054 in 16S E . coli rRNA and to 2658 in the sarcin-ricin domain of 23S E . coli rRNA . Overall there appears to be a remarkable conservation of the accuracy center throughout evolution. Biochem Cell Biol, 1995 Nov-Dec, 73(11-12), 1131 - 40 A pseudoknot is required for efficient translational initiation and regulation of the Escherichia coli rpsO gene coding for ribosomal protein S15; Ehresmann C et al.; Escherichia coli ribosomal protein S15 down regulates its own synthesis by binding to its mRNA in a region overlapping the ribosome binding site, called the translational operator . This binding stabilizes a pseudoknot structure that exists in equilibrium with two stem-loop structures . When synthesized in excess over 16S rRNA, S15 binds to its translational operator and traps the ribosome on its loading site in a transient state, preventing the formation of the active ternary (30S-mRNA-rRNA(f)Met) complex . This inhibition can be suppressed by 16S rRNA, which displaces S15 from the mRNA . An extensive mutational analysis showed that the pseudoknot is the structural element required for S15 recognition and in vivo translational control . Specific sequence determinants are located in limited regions of the structure formed by the pseudoknot . An unexpected result is that the pseudoknot can exist in a variety of topologically equivalent structures recognizable and shapable by S15 . Based on footprinting experiments and computer graphic modelling, S15 shields the two stems of the pseudoknot, sitting in the major groove of the coaxial stack. Biochem Cell Biol, 1995 Nov-Dec, 73(11-12), 1055 - 9 Translational bypassing: a new reading alternative of the genetic code; Groisman I et al.; The translation of the genetic code, once thought to be rigid, has been found to be quite flexible, and several alternatives in its reading have been described . An unusual alternative is translational bypassing, a frameshift event where the transition from frame 0 to another frame occurs by translational bypassing of an extended region of the mRNA sequence rather than by slippage past a single nucleotide, as has been described for most examples of frameshifting . Translational bypassing has been characterized in two cases, T4 gene 60 coding for a topoisomerase subunit and in a trpR-lac'Z fusion . The latter was discovered in our laboratory, and the unique bypass mechanism is investigated further in this study . Using a trpR+1-lac'Z fusion system, we show that the Gln codon at the beginning of lacZ end at the 3' side of the gap is required for bypassing to occur . The Gln codon is part of an mRNA segment that can (potentially) base pair with a segment at the 5' and of Escherichia coli 16S rRNA . A model of trpR+1-lac'Z bypassing is suggested in which the untranslated region of the mRNA is looped out through base pairing between a segment in the 5' end of the 16S rRNA and two sites in the mRNA . Translational bypassing is a newly discovered mechanism of gene expression, and trpR is the first cellular gene identified in which such a mechanism could operate . The understanding of this mechanism and its associated signals may be considered a paradigm for the expression of other genes by this alternative reading of the genetic code. Biochem Cell Biol, 1995 Nov-Dec, 73(11-12), 1041 - 7 Peptidyl transferase and beyond; Wower J et al.; The peptidyl transferase center of the Escherichia coli ribosome encompasses a number of 50S-subunit proteins as well as several specific segments of the 23S rRNA . Although our knowledge of the role that both ribosomal proteins and 23S rRNA play in peptide bond formation has steadily increased, the location, organization, and molecular structure of the peptidyl transferase center remain poorly defined . Over the past 10 years, we have developed a variety of photoaffinity reagents and strategies for investigating the topography of tRNA binding sites on the ribosome . In particular, we have used the photoreactive tRNA probes to delineate ribosomal components in proximity to the 3' end of tRNA at the A, P, and E sites . In this article, we describe recent experiments from our laboratory which focus on the identification of segments of the 23S rRNA at or near the peptidyl transferase center and on the functional role of L27, the 50S-subunit protein most frequently labeled from the acceptor end of A- and P-site tRNAs . In addition, we discuss how these results contribute to a better understanding of the structure, organization, and function of the peptidyl transferase center. Biochem Cell Biol, 1995 Nov-Dec, 73(11-12), 1023 - 31 Escherichia coli initiator tRNA: structure-function relationships and interactions with the translational machinery; Mangroo D et al.; We showed previously that the sequence and (or) structural elements important for specifying the many distinctive properties of Escherichia coli initiator tRNA are clustered in the acceptor stem and in the anticodon stem and loop . This paper briefly describes this and reviews the results of some recently published studies on the mutant initiator tRNAs generated during this work . First, we have studied the effect of overproduction of methionyl-tRNA transformylase (MTF) and initiation factors IF2 and IF3 on activity of mutant initiator tRNAs that are defective at specific steps in the initiation pathway . Overproduction of MTF rescued specifically the activity of mutant tRNAs defective in formylation but not mutants defective in binding to the P site . Overproduction of IF2 increased the activity of all mutant tRNAs having the CUA anticodon but not of mutant tRNA having the GAC anticodon . Overproduction of IF3 had no effect on the activity of any of the mutant tRNAs tested . Second, for functional studies of mutant initiator tRNA in vivo, we used a CAU --> CUA anticodon sequence mutant that can initiate protein synthesis from UAG instead of AUG . In contrast with the wild-type initiator tRNA, the mutant initiator tRNA has a 2-methylthio-N6-isopentenyl adenosine (ms2i6A) base modification next to the anticodon . Interestingly, this base modification is now important for activity of the mutant tRNA in initiation . In a miaA strain of E . coli deficient in biosynthesis of ms2i6A, the mutant initiator tRNA is much less active in initiation . The defect is specifically in binding to the ribosomal P site. Biochem Cell Biol, 1995 Nov-Dec, 73(11-12), 949 - 58 Location and domain structure of Escherichia coli ribosomal protein L7/L12: site specific cysteine crosslinking and attachment of fluorescent probes; Traut RR et al.; Five different variants of L7/L12 containing single cysteine substitutions, two in the N-terminal (NTD) and three in the C-terminal domain (CTD), were produced, modified with {125I}N-{4-(p-azidosalicylamido)butyl}-3-(2'-pyridyldithio) propionamide ({125I}APDP), a sulfhydryl-specific, heterobifunctional, cleavable photo-cross-linking reagent, and reconstituted into ribosomes . These were irradiated, the total proteins were extracted and reductively cleaved, and the cross-linked proteins were identified . The effect of zero-length disulfide cross-linking on binding and activity was also determined . The same sites in L7/L12 were used to attach a rhodamine dye . The formation of ground-state rhodamine dimers caused the appearance of a new absorption band at 518 nm that was used to estimate the extent of interaction of the probes in the free protein and in complexes with L10 . The three sites in the CTD, but not the N-terminal sites, cross-linked to L2 and L5 and to 30S proteins S2, S3, S7, S14, and S18 in a manner influenced by elongation factors . Binding to the ribosome and, therefore, function were blocked by zero-length cross-linking within the NTD, but not the CTD . Binding also disrupted rhodamine dimers in the NTD . No rhodamine dimers formed in the CTD. Biochem Cell Biol, 1995 Nov-Dec, 73(11-12), 933 - 47 Structure and evolution of mammalian ribosomal proteins; Wool IG et al.; Mammalian (rat) ribosomes have 80 proteins; the sequence of amino acids in 75 have been determined . What has been learned of the structure of the rat ribosomal proteins is reviewed with particular attention to their evolution and to amino acid sequence motifs . The latter include: clusters of basic or acidic residues; sequence repeats or shared sequences; zinc finger domains; bZIP elements; and nuclear localization signals . The occurrence and the possible significance of phosphorylated residues and of ubiquitin extensions is noted . The characteristics of the mRNAs that encode the proteins are summarized . The relationship of the rat ribosomal proteins to the proteins in ribosomes from humans, yeast, archaebacteria, and Escherichia coli is collated. Biochem Cell Biol, 1995 Nov-Dec, 73(11-12), 767 - 73 Getting closer to an understanding of the three-dimensional structure of ribosomal RNA; Mueller F et al.; Two experimentally unrelated approaches are converging to give a first low-resolution solution to the question of the three-dimensional organization of the ribosomal RNA from Escherichia coli . The first of these is the continued use of biochemical techniques, such as cross-linking, that provide information on the relative locations of different regions of the RNA . In particular, recent data identifying RNA regions that are juxtaposed to functional ligands such as mRNA or tRNA have been used to construct improved topographical models for the 16S and 23S RNA . The second approach is the application of high-resolution reconstruction techniques from electron micrographs of ribosomes in vitreous ice . These methods have reached a level of resolution at which individual helical elements of the ribosomal RNA begin to be discernible . The electron microscopic data are currently being used in our laboratory to refine the biochemically derived topographical RNA models. Biochem Cell Biol, 1995 Nov-Dec, 73(11-12), 757 - 65 A model of the translational apparatus based on a three-dimensional reconstruction of the Escherichia coli ribosome; Frank J et al.; The morphology of the Escherichia coli ribosome, i.e., its shape at moderate to low (20-40 A (1 A = 0.1 nm)) resolution, provides important constraints in modeling both the folding of ribosomal RNA and the translational process . A new reconstruction, obtained by low-dose cryoelectron microscopy and image processing of single ribosomes, contains clues to the way in which the ribosome interacts with the key functional ligands: the mRNA and the A- and P-site tRNAs . It also suggests possible pathways of the nascent polypeptide chain . From an interpretation of these clues in the light of existing knowledge, a plausible model for the locations and interactions of key components of protein synthesis is suggested. Biochem Cell Biol, 1995 Nov-Dec, 73(11-12), 751 - 6 Modeling the structure of the ribosome; Easterwood TR et al.; Considering the size and complexity of the ribosome and the growing body of data from a wide range of experiments on ribosomal structure, it is becoming increasingly important to develop tools that facilitate the development of reliable models for the ribosome . We use a combination of manual and computer-based approaches for building and refining models of the ribosome and other RNA-protein complexes . Our methods are aimed at determining the range of models compatible with the data, making quantitative statements about the positional uncertainties (resolution) of different regions, identifying conflicts in the data, establishing which regions of the ribosome need further experimental exploration, and, where possible, predicting the outcome of future experiments . Our previous low-resolution model for the small subunit of the Escherichia coli ribosome is briefly reviewed, along with progress on atomic resolution modeling of the mRNA-tRNA complex and its interaction with the decoding site of the 16S RNA. Mol Biochem Parasitol, 1995 Nov, 74(2), 189 - 200 The Leishmania promastigote surface antigen 2 complex is differentially expressed during the parasite life cycle; Handman E et al.; The promastigote surface antigen 2 (PSA-2) complex comprises a family of antigenically similar polypeptides of M(r) 96,000, 80,000 and 50,000, anchored to the membrane with glycosylphosphatidylinositol . Although PSA-2 was initially detected only in promastigotes, Northern blot analysis indicated that mRNA transcripts are also present in amastigotes . Unlike the situation in promastigotes, where at least four major transcripts (2.6-5.3 kb) were detected, only one major (2.6 kb) and two minor transcripts were present in amastigotes . A cDNA clone encoding a member of the PSA-2 family expressed in amastigotes was isolated using DNA probes . The predicted protein sequence of M(r) 40,000 is distinct from promastigote sequences, but shows significant similarity to previously described members of the family from L major and L amazonensis . Antibodies to the carboxyl terminal sequence conserved in all L major PSA-2 studied to date, as well as antibodies affinity purified on the amastigote cDNA-derived polypeptide recognized a major M(r) 50,000 amastigote polypeptide . Immuno-electron microscopy localized both promastigote and amastigote PSA-2 to the cell surface . The expression of PSA-2 polypeptides during the transformation of amastigotes into promastigotes was ordered in a time-dependent manner, with the promastigote M(r) 80000 polypeptide appearing first, followed by the M(r) 96000 polypeptide . In contrast to the glycosylphosphatidylinositol anchor of promastigote PSA-2, which could be hydrolysed by phosphatidylinositol-specific phospholipase C, the amastigote form was resistant to this enzyme. Eur J Obstet Gynecol Reprod Biol, 1995 Nov, 63(1), 91 - 4 Delayed interval delivery after intrauterine infection and immature birth of twin 1--a case report and literature review; de Jong MW et al.; We report a case of delayed interval delivery in a twin pregnancy complicated by rupture of membranes, intrauterine infection and birth of one twin at 21 weeks gestation . Tocolysis combined with antibiotics and corticosteroids successfully prolonged pregnancy for 73 days, allowing the second twin to mature and reach viability . At 31.5 weeks gestation, a 1890 g healthy male neonate was born with good Apgar scores . His postnatal course was uneventful . A literature review of several other cases of delayed interval delivery is presented . When multifetal pregnancies are complicated by immature birth of one fetus, delayed interval delivery may offer survival chances and favourable outcome for the remaining fetus(es). Eur J Obstet Gynecol Reprod Biol, 1995 Nov, 63(1), 61 - 3 Treatment of Bartholin's cyst and abscess: excision versus silver nitrate insertion; Mungan T et al.; In a prospective randomized study, intracavitary silver nitrate (AgNO3) stick insertion (n = 25) was compared to the conventional excision technique (n = 25) for Bartholin's cyst or abscess . Two groups were similar with regard to age, previous Bartholin's cyst/abscess and size of the cyst . Operation and healing time was significantly shorter in the AgNO3 group (P < 0.001, P < 0.001, respectively) . Chemical burning in the vulva was observed in one patient in the AgNO3 group, whereas hematoma on the operation site occurred in two patients in the excision group . Scar formation was found in two patients in whom excision was performed . Patients were followed for a period of 2 years and recurrence was not found in any of the cases in both groups . We conclude that, AgNO3 insertion treatment for Bartholin's cyst and abscess is as effective as excision and is associated with fewer complications . Because it is simple and inexpensive, it is an attractive alternative treatment modality for this common gynecological disease. Bioorg Khim, 1995 Nov, 21(11), 834 - 7 {Study of the role of histidine residues in the function of uridine phosphorylase from Escherichia coli K-12 by protein engineering}; Veiko VP et al.; Using site-directed mutagenesis, mutant genes of the E.coli UDPase that coded proteins with point substitutions of histidine residues (i.e., H8N, H47N, H101N, H122N, H152N, H179N, and H240N) were constructed . Study of the enzymatic activity of mutant UDPases showed that histidine-asparagine substitutions at the positions 47, 101, 152, 179, and 240 do not affect protein functioning . Whereas H122N and H8N substitutions inhibit the activity of UDPase by 60 and 100%, respectively . This evidences the important functional role of the His122 and His8 residues for the formation of the active site fo the enzyme. Biochem Soc Trans, 1995 Nov, 23(4), 767 - 70 Conformational changes in the gamma and epsilon subunits are integral to the functioning of the Escherichia coli H(+)-pumping ATPase (ECF1F0); Capaldi RA et al.; ATP synthesis and ATP hydrolysis by F1F0-type ATPases involve conformational changes transmitted from the catalytic site regions to the proton channel, a distance of more than 100 A . Our studies focus attention on the gamma and epsilon subunits that provide a part of the stalk region in the energy-coupling process within the complex . There are conformational changes in the gamma subunit, and translocations of the epsilon unit, linked to nucleotide-binding changes in catalytic sites, which might be expected to alter the interaction of this subunits with c subunits and, hence, be linked to proton translocation. Eur J Clin Microbiol Infect Dis, 1995 Nov, 14(11), 1004 - 8 Two cases of infected atherosclerotic aneurysms and a comparison with infective endocarditis; Peacock SJ et al.; Two cases of infected atherosclerotic aneurysms thought to have arisen following haematogenous seeding of atheromatous lesions are described . Although infective endocarditis also arises by the haematogenous route, there are striking contrasts in both the range and therefore likely source of organisms, together with a perceived difference in the rate of blood-culture positivity . This case report provides a focus for discussion of these observations. Biochem Mol Biol Int, 1995 Nov, 37(5), 975 - 82 Peroxide complex of cytochrome bd: kinetics of generation and stability; Borisov V et al.; Hydrogen peroxide reacts with the isolated fully oxidized cytochrome bd from Escherichia coli bringing about spectral changes characterized by increased absorption at 680 nm, disappearance of a charge transfer band at 740 nm and a red shift in the Soret band . Only one type of spectral changes is observed throughout the entire range of H2O2 concentration studied, 5 - 5000 microM . The absorption changes are consistent with peroxide binding to heme d and do not show any evidence for reaction with heme b-595 . The spectral response saturates at increased H2O2 concentration with apparent Kd of 30 microM and is reversed by catalase . Stopped-flow measurements show the reaction to be first order with respect to H2O2 with a second order rate constant Kon = 600M-1s-1 . Decay of the H2O2-induced spectral changes upon addition of catalase (k approximately 0.001 s-1) is about 20-fold slower than expected for dissociation of peroxide from the complex with heme d assuming a simple reversible binding of H2O2 with Kd and Kon values give above (Koff = Kon) . We suggest that the reaction of H2O2 with cytochrome bd may be in fact irreversible, the initial binding followed by a cleavage of the O-O bond and formation of the oxoferryl complex of heme d . Upon removal of excess peroxide, the oxoferryl compound could decay being reduced to the ferric state by endogenous reductants. Ann Oncol, 1995 Nov, 6(9), 871 - 81 Folate-based thymidylate synthase inhibitors as anticancer drugs; Jackman AL et al.; The enzyme, thymidylate synthase (TS) is considered an important target for the development of new anticancer agents . Moreover, the folate-binding site in TS is believed to offer better opportunities for the design of highly specific inhibitors than the pyrimidine (dUMP) binding site . This belief led to the design of N10-propargyl-5,8-dideazafolic acid (CB3717), a quinazoline-based drug which had antitumour activity in clinical studies . Occasional, but serious nephrotoxicity led to the withdrawal of CB3717 from further clinical study . More water-soluble and non-nephrotoxic analogues were developed with an interesting diversity in biochemical profile, particularly with respect to interactions with the reduced-folate cell membrane carrier (RFC) and folylpolyglutamate synthetase (FPGS) . An example of a compound that uses both of these processes well is the quinazoline, ZD1694 (Tomudex), a drug which is about to complete phase III evaluation for colorectal cancer . High chain length polyglutamates are formed that are up to 70-fold more potent TS inhibitors than the parent drug (Ki tetraglutamate = 1 nM) . Furthermore they are retained in cells/tissues for a prolonged period . A number of other novel folate-based TS inhibitors are currently in pre-clinical or clinical study . For example, LY231514 is a pyrrolopyrimidine analogue in phase I study and, although less potent as a TS inhibitor, has biochemical properties similar to ZD1694 . Another compound in phase I study is the benzoquinazoline, BW1843U89 which has somewhat different properties . It is a very potent TS inhibitor (Ki = 0.09 nM) and an excellent substrate for the RFC (human) and FPGS, but polyglutamation proceeds to diglutamate only and is not accompanied by increased TS inhibition . Another highly water-soluble compound in pre-clinical development is ZD9331 which was specifically designed to use the RFC but not be a substrate for FPGS . Potent TS inhibition (Ki = 0.4 nM) was achieved through a rational programme of computerised molecular modelling of the active site of TS and a large database of structure-activity relationships . Two lipophilic compounds were designed to be devoid of interactions with either the RFC or FPGS . High resolutions crystal complexes of E . coli TS were central to obtaining potent TS inhibitors and both AG337 (Ki human recombinant TS = 16 nM) and AG331 (Ki = 12 nM) are in clinical studies . This portfolio of novel compounds therefore comprehensively addresses the potential of TS as a target for cancer chemotherapy. Bioconjug Chem, 1995 Nov-Dec, 6(6), 673 - 82 Fluorescent labeling of cysteine 39 on Escherichia coli primase places the dye near an active site; Griep MA et al.; Cysteine 39 of Escherichia coli primase is the most chemically reactive cysteine . Its high chemical reactivity is likely due to its proximity to primase's zinc, which is probably ligated to the adjacent residues 40-62 . The zinc may stabilize the deprotonated form of cysteine 39 to make it chemically reactive . Primase is rapidly, site-specifically modified by fluorescein maleimide (FM) at this cysteine . Modification with FM at this residue does not lead to any activity loss in a coupled RNA/DNA synthesis assay, indicating that it is not a catalytically essential residue . In contrast, iodoacetamidefluorescein (IAF) modifies cysteine 39 more slowly and stoichiometrically inhibits activity . It was not clear why these two similar fluorescent dyes should have such different inhibitory effects when attached to the same cysteine . The IAF inhibition must be due to some property of the link between the fluorescein and the cysteine because that is how it differs from FM . The pKa's of the fluoresceins from both FM- and IAF-modified primase are strongly shifted to lower values (approximately 5.4) compared to free fluorescein . These results strongly suggest that the deprotonated form of the fluoresceins are stabilized on primase by a strong interaction with the adjacent zinc in the zinc finger motif . The ability to place a noninhibitory FM at this site will be of great assistance in fluorescence energy transfer studies since the distances established to cysteine 39 will reflect the distance to the essential zinc finger motif. Vet Microbiol, 1995 Nov, 47(1-2), 1 - 7 Isolation and identification of fimbriae and toxin production by Escherichia coli strains from cows with clinical mastitis; Lipman LJ et al.; A total of 20 Escherichia coli strains isolated from cases of bovine mastitis were examined for fimbriae production, for the presence of genes coding for enterotoxins (LT and ST1), verotoxins (VT), and for the production of cytotoxic necrotizing factors (CNF1 and CNF2) . Fimbriae could be isolated from four strains . The N-terminal amino acid sequence of the fimbriae from two strains, was determined . The two sequences were almost identical and homologous to that of the major subunit of E . coli F17 fimbriae . A DNA probe was derived from this N-terminal sequence and used as probe in hybridization experiments with chromosomal DNA of the 20 strains . To test if the strains contained genes that code for the F17 adhesin and the F17 major subunit, a Polymerase Chain Reaction (PCR) assay was performed with primers based on the nucleotide sequence of the genes . Eleven of 20 strains contained sequences that were homologous with sequences for the F17 fimbrial subunit and the F17 adhesin . Strains were tested directly for toxin production on Hela cells and by PCR for the presence of toxin genes . One of the twenty strains, produced a CNF toxin . No strains reacted positive in the PCR for LT, ST1 and verotoxin genes. Neurosci Res, 1995 Nov, 23(4), 327 - 33 Preparation and a structure-function analysis of human ciliary neurotrophic factor; He C et al.; Ciliary neurotrophic factor (CNTF) is a trophic protein that promotes survival and/or differentiation of a variety of neuronal cell types including sensory, sympathetic, and motor neurons . CNTF, leukemia inhibitory factor (LIF), interleukin-6 (IL-6) and oncostatin M (OSM) share a predicted common helical framework and partially identical receptor components . In this study, we present the preparation and structure--functional analysis of recombinant human CNTF . The human CNTF gene was expressed under the control of the PL promoter in Escherichia coli, and the mutants were constructed by insertion, deletion and site-directed mutagenesis . The recombinant proteins were purified from bacteria via DEAE A-50 and Sephacryl S-200 chromatography, and their survival promoting activities were determined using cultures of embryonic chick dorsal root ganglion (DRG) neurons . Insertion at position 23 with APGL, or at position 79 with PRGA, or substitution of 162L163Q for PIDG resulted in proteins with no neurotrophic activity . However, insertion at position 186 with PRGI did not alter human CNTF activity . Deletion of the carboxy-terminal amino acid 186-200 did not reduce the biological activity, but elimination of the amino acid 162-186 abolished the activity . The mutant substituting of 17 Cys for Ser was found to display a biological activity equivalent to that of the wild type . Our data provided experimental confirmation for the structural prediction of CNTF. J Rheumatol, 1995 Nov, 22(11), 2114 - 9 Induction of release of secretory nonpancreatic phospholipase A2 from human articular chondrocytes; Pruzanski W et al.; OBJECTIVE: Secretory nonpancreatic phospholipase A2