J Biol Chem, 1998 Feb 6, 273(6), 3336 - 42 The transfer of retinol from serum retinol-binding protein to cellular retinol-binding protein is mediated by a membrane receptor; Sundaram M et al.; The hypothesis that the cellular uptake of retinol involves the specific interaction of a plasma membrane receptor with serum retinol-binding protein (RBP) at the extracellular surface followed by ligand transfer to cytoplasmic cellular retinol-binding protein (CRBP) has been investigated . The experimental system consisted of the {3H}retinol-RBP complex, Escherichia coli-expressed recombinant apo-CRBP containing the 10 amino acid long streptavidin-binding peptide sequence at its C terminus (designated as CRBP-Strep) and permeabilized human placental membranes . {3H}Retinol transfer from RBP to CRBP-Strep was monitored by measuring the radioactivity associated with CRBP-Strep retained by an immobilized streptavidin resin . Using this assay system, we have demonstrated that optimal retinol uptake is achieved with holo-RBP, the membrane receptor and apo-CRBP . The effects are specific: other binding proteins, including beta-lactoglobulin and serum albumin, despite their ability to bind retinol, failed to substitute for either RBP or apo-CRBP . The process is facilitated by membranes containing the native receptor suggesting that this protein is an important component in the transfer mechanism . Taken together, the data suggest that the RBP receptor, through specific interactions with the binding proteins, participates (either directly or via associated proteins) in the mechanism which mediates the transfer of retinol from extracellular RBP to intracellular CRBP. Biochem J, 1998 Feb 1, 329 ( Pt 3), 659 - 64 Cloning and expression of the gene for the active PPi-dependent phosphofructokinase of Entamoeba histolytica; Deng Z et al.; Pyrophosphate-dependent phosphofructokinase (PPi-PFK) from Entamoeba histolytica (HM-1) was purified from trophozoites . Oligonucleotide probes based on partial amino acid sequence were used to clone and sequence the gene and the cDNA of the enzyme . The molecular mass of the subunit was greater than, and the derived sequence significantly different from, that of the product of the PPi-PFK gene previously cloned from E . histolytica {Huang, Albach, Chang, Tripathi and Kemp (1995) Biochim . Biophys . Acta 1260, 215-217; Bruchhaus, Jacobs, Denart and Tannich (1996) Biochem . J . 316, 57-63} . The sequence identity between the two proteins was 17% . The sequence bore greater identity with the more phylogenetically advanced plant PPi-PFKs than with bacterial PPi-PFKs . The cloned cDNA was expressed and the protein purified . The kinetic properties were identical with those of the enzyme isolated from the organism . Furthermore, the specific activity was more than three orders of magnitude higher than that described for the product of the previously cloned E . histolytica PFK gene {Bruchhaus et al . (1996)} . The pH-dependence and apparent substrate affinities of the cloned enzyme were identical with those of the PPi-PFK in trophozoite extracts, indicating that the product of the cloned gene accounts for most if not all of the PFK activity in E . histolytica trophozoites. Biochem J, 1998 Feb 1, 329 ( Pt 3), 631 - 5 Human insulin production from a novel mini-proinsulin which has high receptor-binding activity; Chang SG et al.; To increase the folding efficiency of the insulin precursor and the production yield of insulin, we have designed a mini-proinsulin (M2PI) having the central C-peptide region replaced with a sequence forming a reverse turn . The mini-proinsulin was fused at the N-terminus to a 21-residue fusion partner containing a His10 tag for affinity purification . The gene for the fusion protein was inserted downstream of the T7 promoter of the expression plasmid pET-3a, and the fusion proteins were produced as inclusion bodies in the Escherichia coli cytoplasm at levels up to 25% of the total cell protein . The protein was sulphonated, cleaved by CNBr and the M2PI mini-proinsulin was purified using ion-exchange chromatography . The refolding yield of M2PI was 20-40% better than that of proinsulin studied at the same molar concentrations, indicating that the short turn-forming sequence is more effective in the refolding process than the much longer C-peptide . Native human insulin was successfully generated by subsequent enzymic conversion of mini-proinsulin . The mini-proinsulin exhibited high receptor-binding activity, about 50% as potent as insulin, suggesting that this single-chained mini-proinsulin may provide a foundation in understanding the receptor-bound structure of insulin as well as the role of C-peptide in the folding and activity of proinsulin. Biochem J, 1998 Feb 1, 329 ( Pt 3), 589 - 96 Selectivity of post-translational modification in biotinylated proteins: the carboxy carrier protein of the acetyl-CoA carboxylase of Escherichia coli; Reche P et al.; Biotin-dependent enzymes contain a biotinyl-lysine residue in a conserved sequence motif, MKM, located in a surface hairpin turn in one of the two beta-sheets that make up the domain . A sub-gene encoding the 82-residue C-terminal biotinyl domain from the biotin carboxy carrier protein of acetyl-CoA carboxylase from Escherichia coli as a fusion protein with glutathione S-transferase was created and over-expressed in E . coli . The biotinyl domain was readily released by cleavage with thrombin . Five mutant domains were created in which the conserved MKM motif was systematically replaced: by MAK and KAM, in which the target lysine is moved one place; by KKM and MKK, in which a second potential site for biotinylation is introduced; and by DKA, the motif found in the correspondingly conserved site of lipoylation in the structurally related lipoyl domains of 2-oxo acid dehydrogenase multienzyme complexes . No biotinylation of the MAK or KAM mutants was observed in vivo or by purified biotinyl protein ligase in vitro; in the KKM and MKK mutants, only one lysine residue, presumed to be that in its native position in the hairpin turn, was found to be biotinylated in vivo and in vitro . The DKA mutant was not biotinylated in vivo, but was partly lipoylated and octanoylated . It was also a poor substrate for lipoylation in vitro catalysed by the E . coli lipoyl protein ligase encoded by the lplA gene . The flanking sequence in the MKM motif is important, but not crucial, and appears to have been conserved in part to be compatible with the subsequent carboxylation reactions of biotin-dependent enzymes . The DKA motif, displayed in the hairpin loop, is sufficient to address lipoylation in E . coli but probably by a pathway different from that mediated by the lplA-dependent ligase . The recognition of the structurally homologous lipoyl and biotinyl domains by the appropriate ligase evidently has a major structural component to it, notably the positioning of the target lysine residue in the exposed hairpin loop, but there appear to be additional recognition sites elsewhere on the domains. Structure, 1997 Nov 15, 5(11), 1485 - 99 Structural foundation for the design of receptor antagonists targeting Escherichia coli heat-labile enterotoxin; Merritt EA et al.; BACKGROUND: Escherichia coli heat-labile enterotoxin (LT) is the causative agent of traveller's diarrhoea, and it is also responsible for the deaths of hundreds of thousands of children per year in developing countries . LT is highly homologous in sequence, structure and function to cholera toxin (CT) . Both toxins attack intestinal epithelial cells via specific binding to the branched pentasaccharide of ganglioside GM1 at the cell surface . A receptor-binding antagonist which blocked this interaction would potentially constitute a prophylactic drug conferring protection both against the severe effects of cholera itself and against the milder but more common disease caused by LT . RESULTS: Four derivatives of the simple sugar galactose, members of a larger series of receptor antagonists identified by computer modeling and competitive binding studies, have been co-crystallized with either the full LT AB5 holotoxin or the LT B pentamer . These crystal structures have provided detailed views of the toxin in complex with each of the four antagonists: melibionic acid at 2.8 A resolution, lactulose at 2.65 A resolution, metanitrophenylgalactoside (MNPG) at 2.2 A resolution and thiodigalactoside (TDG) at 1.7 A resolution . The binding mode of each galactose derivative was observed 5-15 times, depending on the number of crystallographically independent toxin B pentamers per asymmetric unit . There is a remarkable consistency, with one important exception, in the location and hydrogen-bonding involvement of well-ordered water molecules at the receptor-binding site . CONCLUSIONS: The bound conformations of these receptor antagonist compounds preserve the toxin-galactose interactions previously observed for toxin-sugar complexes, but gain additional favorable interactions . The highest affinity compound, MNPG, is notable in that it displaces a water molecule that is observed to be well-ordered in all other previous and current crystal structures of toxin-sugar complexes . This could be a favorable entropic factor contributing to the increased affinity . The highest affinity members of the present set of antagonists (MNPG and TDG) bury roughly half (400 A2) of the binding-site surface covered by the full receptor GM1 pentasaccharide, despite being considerably smaller . This provides an encouraging basis for the creation of subsequent generations of derived compounds that can compete effectively with the natural receptor. Infect Immun, 1998 Mar, 66(3), 980 - 6 A monoclonal antibody generated by antigen inoculation via tick bite is reactive to the Borrelia burgdorferi Rev protein, a member of the 2.9 gene family locus; Gilmore RD Jr et al.; Murine monoclonal antibodies directed against proteins of Borrelia burgdorferi B31 (low passage) were generated by the administration of antigen via the bite of borrelia-infected ticks . This strategy was employed as a mechanism to create antibodies against antigens presented by the natural route of tick transmission versus those presented by inoculation with cultured borreliae . One of the resultant antibodies reacted with a 17-kDa antigen from cultured B . burgdorferi, as seen by immunoblot analysis . This antibody was used to screen a B . burgdorferi genomic DNA lambda vector expression library, and an immunoreactive clone was isolated . DNA sequence analysis of this clone, containing a 2.7-kb insert, revealed several open reading frames . These open reading frames were found to be homologs of genes discovered as a multicopy gene family in the 297 strain of B . burgdorferi by Porcella et al . (S . F . Porcella, T . G . Popova, D . R . Akins, M . Li, J . D . Radolf, and M . V . Norgard, J . Bacteriol . 178:3293-3307, 1996) . By selectively subcloning genes found in this insert into an Escherichia coli plasmid expression vector, the observation was made that the rev gene product was the protein reactive with the 17-kDa-specific monoclonal antibody . The rev gene product was found to be expressed in low-passage, but not in high-passage, B . burgdorferi B31 . Correspondingly, the rev gene was not present in strain B31 genomic DNA from cultures that had been passaged >50 times . Serum samples from Lyme disease patients demonstrated an antibody response against the Rev protein . The generation of an anti-Rev response in Lyme disease patients, and in mice by tick bite inoculation, provides evidence that the Rev protein is expressed and immunogenic during the course of natural transmission and infection. Mol Cell Biol, 1998 Mar, 18(3), 1635 - 41 A naturally occurring hPMS2 mutation can confer a dominant negative mutator phenotype; Nicolaides NC et al.; Defects in mismatch repair (MMR) genes result in a mutator phenotype by inducing microsatellite instability (MI), a characteristic of hereditary nonpolyposis colorectal cancers (HNPCC) and a subset of sporadic colon tumors . Present models describing the mechanism by which germ line mutations in MMR genes predispose kindreds to HNPCC suggest a "two-hit" inactivation of both alleles of a particular MMR gene . Here we present experimental evidence that a nonsense mutation at codon 134 of the hPMS2 gene is sufficient to reduce MMR and induce MI in cells containing a wild-type hPMS2 allele . These results have significant implications for understanding the relationship between mutagenesis and carcinogenesis and the ability to generate mammalian cells with mutator phenotypes. Mol Cell Biol, 1998 Mar, 18(3), 1416 - 23 Physiological phosphorylation of protein kinase A at Thr-197 is by a protein kinase A kinase; Cauthron RD et al.; Phosphorylation of the catalytic subunit of cyclic AMP-dependent protein kinase, or protein kinase A, on Thr-197 is required for optimal enzyme activity, and enzyme isolated from either animal sources or bacterial expression strains is found phosphorylated at this site . Autophosphorylation of Thr-197 occurs in Escherichia coli and in vitro but is an inefficient intermolecular reaction catalyzed primarily by active, previously phosphorylated molecules . In contrast, the Thr-197 phosphorylation of newly synthesized protein kinase A in intact S49 mouse lymphoma cells is both efficient and insensitive to activators or inhibitors of intracellular protein kinase A . Using {35S}methionine-labeled, nonphosphorylated, recombinant catalytic subunit as the substrate in a gel mobility shift assay, we have identified an activity in extracts of protein kinase A-deficient S49 cells that phosphorylates catalytic subunit on Thr-197 . The protein kinase A kinase activity partially purified by anion-exchange and hydroxylapatite chromatography is an efficient catalyst of protein kinase A phosphorylation in terms of both a low Km for ATP and a rapid time course . Phosphorylation of wild-type catalytic subunit by the kinase kinase activates the subunit for binding to a pseudosubstrate peptide inhibitor of protein kinase A . By both the gel shift assay and a {gamma-32P}ATP incorporation assay, the enzyme is active on wild-type catalytic subunit and on an inactive mutant with Met substituted for Lys-72 but inactive on a mutant with Ala substituted for Thr-197 . Combined with the results from mutant subunits, phosphoamino acid analysis suggests that the enzyme is specific for phosphorylation of Thr-197. Infect Immun, 1998 Mar, 66(3), 1270 - 2 Expression of heat-stable enterotoxin STb by adherent Escherichia coli is not sufficient to cause severe diarrhea in neonatal pigs; Casey TA et al.; The role of Escherichia coli heat-stable enterotoxin B (STb) in neonatal porcine diarrhea caused by enterotoxigenic E . coli was examined by comparing adherent isogenic strains with or without STb . The cloned STb gene (in the plasmid pRAS1) was electroporated into a nonenterotoxigenic strain (226M) which expresses the F41 adhesin . Strain 226M pRAS1 adhered and expressed STb in vivo, causing fluid secretion in ligated ileal loops in neonatal pigs . Although strain 226M pRAS1 caused very mild diarrhea in some orally inoculated neonatal pigs, the weight loss in these pigs was similar to that caused by the parental strain without STb . We conclude that STb does not significantly contribute to diarrhea caused by enterotoxigenic E . coli in neonatal pigs. Infect Immun, 1998 Mar, 66(3), 1037 - 44 Antigenicity of recombinant proteins derived from rhoptry-associated protein 1 of Plasmodium falciparum; Fonjungo PN et al.; Rhoptry-associated protein 1 (RAP1) of Plasmodium falciparum is a potential component of a malaria vaccine . We have expressed in Escherichia coli eight recombinant RAP1 proteins representing almost the entire sequence of the mature protein and assessed the antigenicity of the proteins by immunization of mice . Antisera to six of the recombinant proteins reacted specifically with parasite-derived RAP1 (PfRAP1), as determined by indirect immunofluorescence and by immunoblotting . These proteins were then used in enzyme-linked immunosorbent assays to evaluate human antibody responses to RAP1 during naturally transmitted infections in The Gambia . Immunoglobulin G (IgG) antibodies specifically reactive with the recombinant RAP1 proteins are directed mostly towards fragments containing the N-terminal sequences of mature PfRAP1 . The most N-terminal segment (residues 23 to 175) contains only minor epitopes, while major epitopes are outside this region . Antibodies from malaria patients do not compete for a linear epitope recognized by an inhibitory anti-RAP1 monoclonal antibody . Analysis of IgG subclass distribution shows that human anti-RAP1 antibodies are predominantly IgG1. Infect Immun, 1998 Mar, 66(3), 944 - 9 Curli, fibrous surface proteins of Escherichia coli, interact with major histocompatibility complex class I molecules; Olsen A et al.; Curli are thin, coiled fibers expressed on the surface of Escherichia coli that bind several matrix and plasma proteins such as fibronectin, laminin, plasminogen, tissue plasminogen activator, and H-kininogen . In this work, we examined the interactions between curli-expressing E . coli and human major histocompatibility complex class I (MHC-I) and class II (MHC-II) molecules . Curliated E . coli was found to interact with an MHC-I-expressing lymphoma cell line as shown by scanning electron microscopy, whereas the binding to a mutant variant of this cell line expressing small amounts of MHC-I molecules was significantly lower . Moreover, curli-expressing E . coli bound purified radiolabeled MHC-I but not MHC-II molecules, whereas an isogenic curli-deficient mutant strain showed no affinity for either MHC-I or MHC-II . Purified insoluble curli could also bind 125I-labeled MHC-I molecules, and in Western blot experiments the 15-kDa curlin subunit protein bound intact MHC-I molecules as well as beta2-microglobulin, the light chain of MHC-I molecules . A direct interaction between monomeric MHC-I molecules and a bacterial surface protein has previously not been reported . The binding of curli to MHC-I molecules, which are present on virtually all cells in higher vertebrates, will provide curliated E . coli with ample opportunities to interact with a great variety of hosts and host cells . This should facilitate the adaptation of E . coli to different ecological niches, and in human infections the interaction between curli and MHC-I molecules could contribute to adherence and colonization. Infect Immun, 1998 Mar, 66(3), 907 - 11 D-lactate production and {14C}succinic acid uptake by adherent and nonadherent Escherichia coli; McCabe K et al.; Escherichia coli isolates of different adherence phenotypes produced different amounts of D-lactate . Alterations of culture conditions did not influence the amount of D-lactate produced . The observed pH decreases in tissue culture medium corresponded with increases in D-lactate concentration . Very little {14C}succinic acid was incorporated into cells during the in vitro incubation of adherent and nonadherent E . coli with HeLa cells, but the amounts of tracer removed from the culture medium by adherent and nonadherent strains differed . The results are further evidence of a difference in the metabolic behavior of adherent and nonadherent E . coli. Shock, 1998 Feb, 9(2), 95 - 100 Rapid and prolonged impairment of gut barrier function after thermal injury in mice; Eaves-Pyles T et al.; Loss of gut barrier function after burn injury can be important in the pathogenesis of systemic infections and organ dysfunction . The purpose of this study was to determine how rapidly impairment of gut barrier function occurs after burn injury and how long it persists . BALB/c mice were gavaged with 10(10) (111)In-oxine-labeled Escherichia coli 3 h before inflicting a 20% total body surface area burn . They were then killed at 5, 15, 30, 60, 120, or 240 min post-burn . Additional mice were given a 20% or 30% burn injury and were randomized into eight groups, which were killed at either 4 h or 1, 2, 4, 7, 14, or 21 days post-burn . Each mouse was gavaged with 10(10) (111)In-oxine-labeled E . coli 4 h before sacrifice to determine the magnitude of translocation . Gut barrier function was impaired as early as 5 min post-burn and was maximal by 4 h . Rapid improvement was observed by 24 h, followed by slow improvement, but with persistent abnormality through 21 days post-burn . Killing of translocated bacteria was impaired at 4 h and day 7 post-burn, according to the percentage of viable E . coli that remained alive in the tissues . The magnitude of gut dysfunction following burn injury is temporally related. Med Hypotheses, 1998 Jan, 50(1), 61 - 5 The etiology of benign prostatic hypertrophy; Roper WG; Benign prostatic hypertrophy (BPH) is a hyperplasia and not a hypertrophy . No cogent total explanation for the etiology of this ubiquitous disease has ever been given . There is such a vast cascade of incongruities in respect of hormonal influences, on the one hand, and the degree, variation, timing and rate of development of the condition, on the other hand, that a 'missing link' has constantly been envisaged in its etiology . In the search for this, two simple facts stand out: (a) BPH starts at some time after involution commences; (b) it is the transitional zone that is affected in the major extent . This hypothesis proposes that, owing to dynamic embryological differences between the transitional and the other zones, its secretions are differently affected by involution, resulting in stimulatory factors affecting known hormonal influences upon prostatic growth, further resulting in the development of BPH . These stimulatory factors are considered to be the 'missing link' in the etiology of BPH . It is further suggested that one of these stimulatory factors may be E . coli endotoxin, repeatedly released within a contained prostatic environment . This is considered secondary to intermittent colonization of the transitional zone acini and/or ducts by E . coli, followed by the destruction of these E . coli . This colonization is considered mandatory, if the known 95% occurrence of invasive infection of BPH is to be explained. Protein Eng, 1997 Oct, 10(10), 1205 - 11 Functional roles of the N-terminal amino acid residues in the Mn(II)-L-malate binding and subunit interactions of pigeon liver malic enzyme; Chou WY et al.; Pigeon liver malic enzyme has an N-terminal amino acid sequence of Met-Lys-Lys-Gly-Tyr-Glu- . In this work, various mutants of the enzyme with individual or combinational deletion (delta) or substitution at these amino acids were constructed and functionally expressed in Escherichia coli cells . A major protein band corresponding to an Mr of approximately 65000 was observed for all recombinant enzymes in sodium dodecyl sulfate polyacrylamide gel electrophoresis . However, when examining by polyacrylamide gel electrophoresis under native conditions, the recombinant enzymes were found to possess a tetrameric structure with Mr approximately 260000 or a mixture of tetramers and dimers with the exception of delta(K2K3G4) and delta(1-16) mutants, which existed exclusively as dimers at the protein concentration we employed . K3A and K3E also dissociated substantially . K(2,3)A was a tetramer but K(2,3)E essentially existed as dimers . All tetramers and dimers were enzymatically active in the gels . All mutants displayed a similar apparent Km value for NADP+ . The apparent Km for L-malate and Mn(II), on the other hand, was increased by 4-27-fold for the delta(K2/K3) and the delta(1-16) mutants . The small binding affinity of delta(K2/K3) with Mn(II)-L-malate was specific . With additional deletion at positions 3 and/or 4, the delta(K2K3), delta(K2G4/K3G4) or delta(K2K3G4) mutants exhibited similar kinetic properties for the wild type . The lysine residues at the positions 2 or 3 seem to be crucial for the correct active site conformation . The results indicate that the N-terminus of malic enzyme is located at the Mn(II)-L-malate binding domain of the active center and is also near the subunit's interface . These results were interpreted with our asymmetric double-dimer model for the enzyme in which the N-terminus was involved in the head-to-tail monomer-monomer interactions but not the dimer-dimer interactions. Life Sci, 1998, 62(3), 247 - 55 Glomerular endothelial injury associated with free radical production induced by a fungal cell wall component, (1-->3) beta-D glucan; Iwamoto N et al.; Clinical evidence suggests that microangiopathy may be induced by fungal infection . The present study evaluated the toxic effect of (1-->3) beta-D glucan, a major component of fungal cell wall, on cultured transformed glomerular endothelial cells (TF-GEN) . When TF-GEN were exposed to increasing concentrations of (1-->3) beta-D glucan (beta-DG; 115 to 430 pg/ml) for 1 to 3 hours, concentration- and time-dependent increases in hydroxyl radical production were demonstrated by electron paramagnetic resonance spectrometry using 5, 5-dimethyl-1-pyrrolyne-N-oxide as a spin trap agent . The amount of radicals induced by 230 or 430 pg/ml beta-DG was comparable to that induced by E . coli LPS (1 or 10 microg/ml) . The beta-DG-induced free radical production was associated with a subsequent increase in LDH release from TF-GEN . When TF-GEN pretreated with U78517F (0.1 or 1.0 microM), a lipophilic antioxidant, were stimulated with LPS (1 or 10 microg/ml) or beta-DG (230 pg/ml) for 3 hours, free radical production by TF-GEN was significantly reduced in cells pretreated with the higher concentration of U78517F . Thus, fungal (1-->3) beta-D glucan induces glomerular endothelial injury by stimulating cellular free radical production . Such a mechanism may underlie microangiopathy in systemic fungal infections. Photochem Photobiol, 1998 Feb, 67(2), 263 - 7 Fluorescence and photochemistry of recombinant phytochrome from the cyanobacterium Synechocystis; Sineshchekov V et al.; Fluorescence and photochemical properties of phytochrome from the cyanobacterium Synechocystis were investigated in the temperature interval from 293 to 85 K . The apoprotein was obtained by overexpression in Escherichia coli and assembled to a holophytochrome with phycocyanobilin (PCB) and phytochromobilin (P phi B), Syn(PCB)phy and Syn(P phi B)phy, respectively . Its red-absorbing form, Pr, is characterized at 85 K by the emission and excitation maxima at 682 and 666 nm in Syn(PCB)phy and at 690 and 674 nm in Syn(P phi B)phy . At room temperature, the spectra are blue shifted by 5-10 nm . The fluorescence intensity dropped down by approximately 15-20-fold upon warming from 85 to 293 K and activation energy of the fluorescence decay was estimated to be ca 5.4 and 4.9 kJ mol-1 in Syn(PCB)phy and Syn(P phi B)phy, respectively . Phototransformation of Pr upon red illumination was observed at temperatures above 160-170 K in Syn(PCB)phy and above 140-150 K in Syn(P phi B)phy with a 2-3 nm shift of the emission spectrum to the blue and increase of the intensity of its shorter wavelength part . This was interpreted as a possible formation of the photoproduct of the meta-Ra type of the plant phytochrome . At ambient temperatures, the extent of the Pr phototransformation to the far-red-absorbing form, Pfr, was ca 0.7-0.75 and 0.85-0.9 for Syn(PCB)phy and Syn(P phi B)phy, respectively . Fluorescence of Pfr and of the photoproduct similar to lumi-R was not observed . With respect to the photochemical parameters, Syn(PCB)phy and Syn(P phi B)phy are similar to each other and also to a small fraction of phyA (phyA") and to phyB . The latter were shown to have low photochemical activity at low temperatures in contrast to the major phyA pool (phyA'), which is distinguished by the high extent (ca 50%) of Pr phototransformation at 85 K . These photochemical features are interpreted in terms of different activation barriers for the photoreaction in the Pr excited state. Photochem Photobiol, 1998 Feb, 67(2), 184 - 91 Photophysical properties and photobiological activity of the furanochromones visnagin and khellin; Borges ML et al.; The larger photobiological activity of visnagin (VI) versus khellin (KH) toward several living organisms, including fungi, viruses, yeasts and bacteria, induced a detailed investigation of the photophysical properties of these naturally occurring furanochromones, using laser-flash-photolysis, photoacoustic calorimetry and fluorescence (steady-state and time-resolved) techniques in solvents with different polarity and content of water, including micelles and vesicles . The results have shown that the magnitude of all the three rate constants out of S1 (radiative, kf; internal conversion, kic and intersystem crossing, kisc) for VI and KH strongly depend on the solvent, namely on its hydrogen bonding ability and polarity . The changes of kf and kisc are due to the solvent-assisted mixing and/or inversion of the two first singlet excited states (1n, pi and 1 pi, pi), while kic increases with a decrease of the S0-S1 energy gap . As a consequence, the quantum yield of triplet formation (phi T) strongly decreases from values of approximately 0.8 in dioxane to < 0.05 in water for both compounds . The magnitude of solvent polarity/hydrogen bonding ability required, at which the state order is inverted and phi T starts to decrease, is greater for VI than for KH and consequently phi T (VI) >> phi T (KH) over a broad range of water content including that appropriate to the environment of the compounds in a living system . These facts account for the larger photobiological activity of VI with respect to KH, regarding both the fungus Fusarium culmorum L . and the wild strain of Escherichia coli, studied by us. Planta, 1998 Feb, 204(2), 242 - 51 Biochemical and molecular characterisation of xyloglucan endotransglycosylase from ripe kiwifruit; Schroder R et al.; Xyloglucan endotransglycosylase (XET) from the core tissue of ripe kiwifruit (Actinidia deliciosa {A . Chev.} C.F . Liang et A.R . Ferguson var . deliciosa cv . Hayward) was purified 3000-fold to homogeneity . The enzyme has a molecular weight of 34 kDa, is N-glycosylated, and is active between pH 5.0 and 8.0, with an optimum between 5.5 and 5.8 . The Km was 0.6 mg.mL-1 for kiwifruit xyloglucan and 100 microM for {3H}XXXG-ol, a reduced heptasaccharide derived from kiwifruit xyloglucan . Kiwifruit core XET was capable of depolymerising xyloglucan in the absence of {3H}XXXG-ol by hydrolysis, and in the presence of {3H}XXXG-ol by hydrolysis and endotransglycosylation . Six cDNA clones (AdXET1-6) with homology to other reported XETs were isolated from ripe kiwifruit mRNA . The six cDNA clones share 93-99% nucleotide identity and appear to belong to a family of closely related genes . Peptide sequencing indicated that ripe kiwifruit XET was encoded by AdXET6 . Northern analysis indicated that expression of the AdXET1-6 gene family was induced in ripening kiwifruit when endogenous ethylene production could first be detected, and peaked in climacteric samples when fruit were soft . A full-length cDNA clone (AdXET5) was overexpressed in E . coli to produce a recombinant protein that showed endotransglycosylase activity when refolded. Appl Microbiol Biotechnol, 1998 Jan, 49(1), 31 - 8 Purification and characterization of recombinant spider silk expressed in Escherichia coli; Arcidiacono S et al.; A partial cDNA clone, from the 3' end of the dragline silk gene was isolated from Nephila clavipes major ampullate glands . This clone contains a 1.7-kb insert, consisting of a repetitive coding region of 1.4-kb and a 0.3-kb nonrepetitive coding region; 1.5-kb of the 1.7-kb fragment was cloned into Escherichia coli and a 43-kDa recombinant silk protein was expressed . Characterization of the purified protein by Western blot, amino acid composition analysis, and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry confirms it to be spider dragline silk. Pharm Res, 1998 Jan, 15(1), 116 - 21 Terplex DNA delivery system as a gene carrier; Kim JS et al.; PURPOSE: To characterize the physical and biochemical properties of the DNA terplex delivery system, which has previously been shown to deliver and express pSV-beta-gal plasmid efficiently in cultured smooth muscle cells (SMC) (1) . METHODS: Atomic force microscopy (AFM), zeta-potential measurement (ZP), gel electrophoresis (GE), circular dichroism (CD), fluorescence quenching and 1H-NMR spectrometry were used . RESULTS: AFM showed that the plasmid DNA of about 600 nm long in its extended state was condensed to the size of about 100 nm by terplex formation . The DNA condensing effect of the terplex system was as good as unmodified PLL, as shown by an ethidium bromide displacement assay . Zeta-potential measurement showed that the terplex system exerts a slightly positive surface charge (+2 mV) at a 1:1:1 weight ratio of DNA:LDL:stearyl-PLL, which showed the best transfection efficiency on SMC . GE indicated that electrophoretic mobility of the terplex system decreased with increasing amounts of stearyl-PLL, indicating that the surface charge of the terplex system became more positive as more stearyl-PLL was added . Results from CD showed that there was no significant changes in tertiary structure of plasmid DNA from the terplex formation . Presence of strong hydrophobic interaction between stearyl-PLL and LDL was confirmed by 1H-NMR, where about a 30% decrease in epsilon-methylene peak of PLL backbone was observed when stearyl-PLL was mixed with LDL, but this phenomenon was not observed when unmodified PLL was used . CONCLUSIONS: Our results indicate that the plasmid DNA, when formulated with the stearyl-PLL and LDL, forms a stable and hydrophobicity/charge balanced terplex system of optimal size for efficient cellular uptake and the DNA is still intact after the terplex formation . This information is expected to be utilized for the development of much improved transfection vectors for in vivo gene therapy. East Afr Med J, 1997 Aug, 74(8), 499 - 502 Individual and combined genotoxic response of boric acid and aflatoxin B1 in Escherichia coli PQ37; Odunola OA; The genotoxic potentials of boric acid in Escherichia coli PQ37 was assessed along in the presence of aflatoxin B1 using SOS chromotest . Boric acid induced beta-galactosidase synthesis on the tester bacteria both on the presence and absence of S9 activation mixture . Therefore, the inorganic acid may not require metabolic activation to be genotoxic for this bacteria . When present together with aflatoxin B1 in the assay medium, boric acid increased the degree of beta-galactosidase synthesis induced by the maximal inducing concentration of aflatoxin B1 before the toxin's activation . However, the degree of enzyme synthesis induced was significantly decreased when boric acid was present after aflatoxin activation . This suggests that boric acid may not interfere with nor block the expoxidation of aflatoxin B1 . It may however interact with the expoxide thereby inhibiting its activity . Boric acid may be a genotoxin and could possibly act as a syngenotoxic and/or a cogenotoxic agent. Tumori, 1997 Jul-Aug, 83(4 Suppl 2), S3 - 15 {Erythropoietin: biochemical characteristics, biologic effects, indications and results of use in hematology}; Marmont AM; This review has two objects: a brief recapitulation of the biological background of erythropoietin (EPO), and a review of its clinical utilization in hematology . EPO, both in its naturally occurring and recombinant form (rH-EPO), is a single chain glycoprotein with an approximate molecular weight of 30.000 to 34.000 kD . Its heavy glycosilation is essential for its activity in vivo, since asialoEPO is readily cleared by the heptic asialoglycoprotein receptor . This impedes the recombinant molecule's synthesis in biologic cultures other than mammalian cells (Chinese hamster's ovary cells), and inevitably increases costs . If in vitro glycosilation of E . coli-derived rH-EPO could be achieved, the clinical utilization of the product would be considerably enhanced, most especially when very high doses are necessary, as discussed later . There is no antigenic diversity between natural and recombinant EPO, so that out of the enormous clinical experience only one single case of immunization has been recorded . Almost paradoxically there are however three published cases of pure red cell aplasia (PRCA) caused by immunization against autologous EPO . It is now established that in adults EPO is synthetized in renal peritubular interstitial cells, although some residual activity remains in the liver . Hypoxia results in a rapid induction of EPO expression, although the role of the oxygen sensor system is still debated . Cellular targets are notoriously erythroid progenitors and precursors (BFU-E, CFU-E, early and intermediate erythroblasts) . The global erythropoietic activity resulted in various effects (proliferation, differentiation, survival), but most probably each single effect is integrated with and complementary of the others . The utilization of rH-EPO in hematologic diseases came much later than its dramatic success in renal anemia . A variety of tools useful for assessing the possible beneficial effects of rH-EPO in clinical hematology has been proposed, among which a low level of endogenous EPO is a good predictor for therapeutic success . 'Hemopathic' anemia can be subdivided into three categories: patients with normal erythropoiesis due to inadequate EPO production (anemia of prematurity), patients with depressed but nonclonal erythropoiesis (chemotherapy, lymphoid malignancies such as multiple myeloma-MM and chronic lymphatic leukemia-CCL) and patients with at least partially clonal anemia, such as paroxysmal nocturnal hemoglobinuria (PNH), hemoglobinopaties, myelodysplastic syndromes (MDS) and others . Results in the first category of patients are, as expected, prompt and satisfactory with physiologic doses . Although therapeutic strategy for MM is moving fast to curative intents, the utilization of rH-EPO is indicated for the control of anemia in conservatively-treated patients . In the third category the most important and controversial area is MDS . Significant erythropoietic results are generally obtained in about 20% of patients; however, the association with G-CSF has considerably enhanced the response rate . In the field of bone marrow transplantation there is an inadequate production of endogenous EPO in the allogeneic setting, and randomized studies have shown the benefits of rH-EPO in this situation . However, the most important results have been obtained in post-major-ABO incompatible PRCA, when the removal of the recipient's isohemagglutinins does not resolve the anemia . High and very high doses of rH-EPO (even over 500 UI/kg/day for 2-4 weeks) may resolve this occasionally quite refractory condition . Although extremely expensive, this treatment may be life-saving when an otherwise successful allogeneic transplant is at the risk of failure because of this relatively uncommon but severe immunohematologic complication. Nature, 1998 Feb 19, 391(6669), 811 - 4 Role of the histone deacetylase complex in acute promyelocytic leukaemia; Lin RJ et al.; Non-liganded retinoic acid receptors (RARs) repress transcription of target genes by recruiting the histone deacetylase complex through a class of silencing mediators termed SMRT or N-CoR . Mutant forms of RARalpha, created by chromosomal translocations with either the PML (for promyelocytic leukaemia) or the PLZF (for promyelocytic leukaemia zinc finger) locus, are oncogenic and result in human acute promyelocytic leukaemia (APL) . PML-RARalpha APL patients achieve complete remission following treatments with pharmacological doses of retinoic acids (RA); in contrast, PLZF-RARalpha patients respond very poorly, if at all . Here we report that the association of these two chimaeric receptors with the histone deacetylase (HDAC) complex helps to determine both the development of APL and the ability of patients to respond to retinoids . Consistent with these observations, inhibitors of histone deacetylase dramatically potentiate retinoid-induced differentiation of RA-sensitive, and restore retinoid responses of RA-resistant, APL cell lines . Our findings suggest that oncogenic RARs mediate leukaemogenesis through aberrant chromatin acetylation, and that pharmacological manipulation of nuclear receptor co-factors may be a useful approach in the treatment of human disease. Nature, 1998 Feb 19, 391(6669), 795 - 9 HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C; Braud VM et al.; The protein HLA-E is a non-classical major histocompatibility complex (MHC) molecule of limited sequence variability . Its expression on the cell surface is regulated by the binding of peptides derived from the signal sequence of some other MHC class I molecules . Here we report the identification of ligands for HLA-E . We constructed tetramers in which recombinant HLA-E and beta2-microglobulin were refolded with an MHC leader-sequence peptide, biotinylated, and conjugated to phycoerythrin-labelled Extravidin . This HLA-E tetramer bound to natural killer (NK) cells and a small subset of T cells from peripheral blood . On transfectants, the tetramer bound to the CD94/NKG2A, CD94/NKGK2B and CD94/NKG2C NK cell receptors, but did not bind to the immunoglobulin family of NK cell receptors (KIR) . Surface expression of HLA-E was enough to protect target cells from lysis by CD94/NKG2A+ NK-cell clones . A subset of HLA class I alleles has been shown to inhibit killing by CD94/NKG2A+ NK-cell clones . Only the HLA alleles that possess a leader peptide capable of upregulating HLA-E surface expression confer resistance to NK-cell-mediated lysis, implying that their action is mediated by HLA-E, the predominant ligand for the NK cell inhibitory receptor CD94/NKG2A. Am J Physiol, 1998 Feb, 274(2 Pt 2), H580 - 90 Endotoxemia-induced myocardial dysfunction is not associated with changes in myofilament Ca2+ responsiveness; Rigby SL et al.; Myocardial contractile function is depressed after onset of endotoxemia and is intrinsic to the ventricular myocyte . We tested the hypothesis that decreased Ca2+ responsiveness of the contractile myofilaments underlies this inotropic depression . Specifically, we evaluated the relationship between Ca2+ and unloaded cell shortening and isometric tension development of skinned guinea pig ventricular myocytes . Myocytes were isolated 4 h after intraperitoneal injection of 4 mg/kg Escherichia coli lipopolysaccharide (LPS) or saline (control; Ctl) . Myofilament Ca2+ responsiveness assessed by image analysis of shortening of skinned myocytes at pH 7.0 was not different between Ctl{pCa value that resulted in half-maximal shortening (pCa50): 5.78 +/- 0.04} and LPS (pCa50: 5.72 +/- 0.02) . Similarly, myofilament Ca2+ responsiveness measured by isometric tension of skinned myocytes was not different between Ctl (pCa50: 5.73 +/- 0.02) and LPS (pCa50: 5.76 +/- 0.02) . Maximal tension generated by LPS myocytes (2.89 +/- 0.23 g/mm2) was significantly less (P < 0.05) than Ctl (3.75 +/- 0.34 g/mm2) . However, when myocytes were isolated and skinned in the presence of protease inhibitors, maximal tension generated by LPS myocytes (3.53 +/- 0.98 g/mm2) was similar to Ctl (3.01 +/- 0.80 g/mm2) . We conclude that in vivo administration of LPS resulting in endotoxemia without shock does not alter myofilament Ca2+ responsiveness of ventricular myocytes . Rather, reduced contractility is more likely a result of decreased Ca2+ availability because systolic Ca2+ transients of fura 2-loaded LPS myocytes were significantly decreased (P < 0.05) compared with Ctl myocytes. Am J Physiol, 1998 Feb, 274(2 Pt 1), C319 - 32 pH-independent retrograde targeting of glycolipids to the Golgi complex; Schapiro FB et al.; A small fraction of the molecules internalized by endocytosis reaches the Golgi complex through a retrograde pathway that is poorly understood . In the present work, we used bacterial toxins to study the retrograde pathway in Vero cells . The recombinant B subunit of verotoxin 1B (VT1B) was labeled with fluorescein to monitor its progress within the cell by confocal microscopy . This toxin, which binds specifically to the glycolipid globotriaosyl ceramide, entered endosomes by both clathrin-dependent and -independent pathways, reaching the Golgi complex . Once internalized, the toxin-receptor complex did not recycle back to the plasma membrane . The kinetics of internalization and the subcellular distribution of VT1B were virtually identical to those of another glycolipid-binding toxin, the B subunit of cholera toxin (CTB) . Retrograde transport of VT1B and CTB was unaffected by addition of weak bases in combination with concanamycin, a vacuolar-type ATPase inhibitor . Ratio imaging confirmed that these agents neutralized the luminal pH of the compartments where the toxin was located . Therefore, the retrograde transport of glycolipids differs from that of proteins like furin and TGN38, which require an acidic luminal pH . Additional experiments indicated that the glycolipid receptors of VT1B and CTB are internalized independently and not as part of lipid "rafts" and that internalization is cytochalasin insensitive . We conclude that glycolipids utilize a unique, pH-independent retrograde pathway to reach compartments of the secretory system and that assembly of F-actin is not required for this process. FEMS Microbiol Lett, 1998 Feb 1, 159(1), 93 - 7 Effect of environmental factors on the dominant lethality caused by expression of a mutated DnaA protein with decreased intrinsic ATPase activity; Sakamoto K et al.; Induction of a mutant DnaA protein (DnaA E204Q) with decreased intrinsic ATPase activity in cells was previously shown to cause overinitiation of chromosomal DNA replication and a dominant lethal phenotype . Here it is shown that the dominant lethality required incubation at high temperatures; cells harboring the expression plasmid of DnaA E204Q showed very weak colony formation ability (less than 1/10(5) that of the wild-type DnaA) at 42 degrees C, whereas they showed a normal colony formation ability at 28 degrees C on LB agar plates . Overinitiation of chromosomal DNA replication caused by expression of DnaA E204Q also required incubation at high temperatures in LB medium . When the incubation was performed in synthetic (Tanaka) medium at 42 degrees C, neither the dominant lethality nor overinitiation caused by expressing DnaA E204Q was observed . These results suggest that the dominant lethality and the overinitiation caused by expressing DnaA E204Q require culture conditions that provide a high potential for cell growth. Proteins, 1997, Suppl 1, 38 - 42 Protein modeling by multiple sequence threading and distance geometry; Aszodi A et al.; The application of homology modeling is often limited by the lack of known structures with sufficiently high sequence similarity to the target protein . The recent development of threading methods now enable the identification of likely folding patterns in a number of cases where the structural relatedness between target and template(s) is not detectable at the sequence level . We devised a hybrid method in which fold recognition was performed using the Multiple Sequence Threading (MST) method . The structural equivalences deduced from the threading output were used to guide the distance geometry program DRAGON in the construction of low-resolution C alpha/C beta models . The initial structures were converted to full-atom representation and refined using the general-purpose molecular modeling package QUANTA . The performance of the approach is illustrated on the CASP2 target T0004 (polyribonucleotide nucleotidyl-transferase S1 motif (PNS1) from Escherichia coli, PDB code: 1SRO) for which no obvious homologues with known structure were available . The correct fold of PNS1 was successfully identified, and the model was found to be more similar to the experimental PNS1 structure than the scaffold (C alpha RMSD of 6.2 A compared with 6.4 A) . Our results indicate that a sensitive fold recognition algorithm coupled with a distance geometry program capable of rapidly generating initial structures can successfully complement high-resolution homology modeling methods in cases where sequential similarity is low. Biochemistry, 1998 Feb 17, 37(7), 2004 - 16 Conformational states in denaturants of cytochrome c and horseradish peroxidases examined by fluorescence and circular dichroism; Tsaprailis G et al.; Steady-state fluorescence and circular dichroism (CD) were used to examine the unfolding in denaturants of recombinant cytochrome c peroxidase {CCP(MI)} and horseradish peroxidase (HRP) in their ferric forms . CCP(MI) unfolds in urea and in guanidine hydrochloride (GdHCl) at pH 7.0, while HRP loses its secondary structure only in the presence of GdHCl . CCP(MI) unfolds in urea by two distinct steps as monitored by fluorescence, but the loss of its secondary structure as monitored by UV/CD occurs in a single step between 3.4 and 5 M urea and 1.5 and 2.5 M GdHCl . The localized changes detected by fluorescence involve the CCP(MI) heme cavity since the Soret maximum red-shifts from 408 to 416 nm, and the heme CD changes examined in urea are biphasic . The polypeptide of HRP also loses secondary structure in a single step between 1.2 and 2.7 M GdHCl as monitored by UV/CD, and a fluorescence-monitored transition involving conformational change in the Trp117-containing loop occurs above 4 M GdHCl . Free energies of denaturation extrapolated to 0 M denaturant (delta Gd,aq) of approximately 6 and approximately 4 kcal/mol were calculated for CCP(MI) and HRP, respectively, from the UV/CD data . The refolding mechanisms of the two peroxidases differ since heme capture in CCP(MI) is synchronous with refolding while apoHRP captures heme after refolding . Thus, the denatured form of apoHRP does not recognize heme and has to correctly refold prior to heme capture . The half-life for unfolding of native HRP in 6 M GdHCl is slow (519 s) compared to that for CCP(MI) (14.3 s), indicating that HRP is kinetically much more stable than CCP(MI) . Treatment with EDTA and DTT greatly destabilizes HRP, and unfolding in 4 M GdHCl occurs with t1/2 = 0.42 s. Biochemistry, 1998 Feb 17, 37(7), 1979 - 88 Protein anatomy: C-tail region of human tau protein as a crucial structural element in Alzheimer's paired helical filament formation in vitro; Yanagawa H et al.; Tau is a microtubule-associated protein in mammalian brain . In Alzheimer's disease, this protein is present in the somatodendritic compartment of certain nerve cells, where it forms a portion of paired helical filament, the major constituent of the neurofibrillary tangle . For clarification of the mechanism of this formation, recombinant human tau and its fragments (N-terminal half, C-terminal half, and 4-repeats) expressed in Escherichia coli were prepared, eight peptide fragments (C-tails 1-8) of the C-tail region were synthesized, and the conformation and capacity for aggregation essential for filamentous structure formation in vitro were examined . Recombinant full-length tau, the N-terminal half, 4-repeats, and the C-terminal half did not form filamentous structures in aqueous solution after standing at 20 degrees C . Peptides corresponding to the C-tail region of tau, C-tail 5, C-tail 7, and C-tail 8, produced the paired filament or single straight filament in acidic solution . The rate of filament formation by each peptide was followed by circular dichroism, which showed the C-tails to have predominantly random coil structures immediately following dissolution in aqueous solution and be gradually converted to the beta-sheet structure . The kinetics of aggregation were characterized by a delay period during which the solution remained clear, followed by a nucleation event which led to a growth phase, whose negative peak intensity at 218 nm in circular dichroism increased due to filamentous structure formation . This delay was eliminated by seeding supersaturated solution of preformed filaments . C-tails interacted with recombinant full-length tau to form definite single straight filament . The C-tail region of tau is thus shown indispensable to the formation of paired helical filament and nucleation to reduce the rate of paired helical filament formation in amyloidogenesis in vitro . These findings may provide some clarification of the pathogenesis of Alzheimer's disease. Biochemistry, 1998 Feb 17, 37(7), 1917 - 25 Polyamide nucleic acid-DNA chimera lacking the phosphate backbone are novel primers for polymerase reaction catalyzed by DNA polymerases; Misra HS et al.; A peptide nucleic acid (PNA) oligomer, an analogue of DNA, was examined for its ability to function as a primer or a template to support DNA synthesis catalyzed by DNA polymerases . The analogue, (PNA)19-TpG-OH, comprised of 19 bases in the form of PNA followed by a dinucleotide (TpG-OH) with a single phosphate and a free 3'OH terminus, was recognized as a bona fide primer by 2 reverse transcriptases and also by the Klenow fragment of E . coli DNA polymerase I . The 21-mer PNA chimera is extended on both RNA and DNA templates by all three polymerases . The specificity of binding of the PNA chimeric primer/DNA template at the template-primer binding site of the enzyme was shown by its photo-cross-linking ability to the enzyme which could be effectively competed out by another TP but not by template or primer alone . Furthermore, the chimeric TP-enzyme covalent complex was found to be catalytically active as judged by its ability to incorporate one nucleotide onto the 3'OH terminus of the immobilized primer . PNA sequences were also recognized as template when annealed with a DNA primer . These observations are in variance with the conventional suggestion that the phosphate backbone in the duplex region is essential for recognition and binding by DNA polymerases . The efficient extension of (PNA)19-TpG-OH suggests that the diameter of the duplex region of template primer rather than the phosphate backbone may be sufficient for recognition by DNA polymerases. Biochemistry, 1998 Feb 17, 37(7), 1905 - 9 The human LON protease binds to mitochondrial promoters in a single-stranded, site-specific, strand-specific manner; Fu GK et al.; LON proteases, which are ATP-dependent and exhibit ATPase activity, are found in bacteria, yeast, and humans . In Escherichia coli, LON is known to regulate gene expression by targeting specific regulatory proteins for degradation . The yeast and human LON proteins are encoded in the nucleus but localize to the mitochondrial matrix . In yeast, LON has been shown to be essential for the maintenance of the integrity of the mitochondrial genome . E . coli Lon has long been known to bind DNA, but we have only recently demonstrated that it binds preferentially to a specific TG-rich double-stranded sequence . We now show that human LON recognizes a very similar site in both the light and heavy chain promoters of the mitochondrial genome, in a region which is involved in regulating both DNA replication and transcription . Unlike E . coli Lon, however, human LON specifically binds to the TG-rich element only when it is presented in the context of a single DNA strand . These findings suggest that the human LON protease might regulate mitochondrial DNA replication and/or gene expression using site-specific, single-stranded DNA binding to target the degradation of regulatory proteins binding to adjacent sites in mitochondrial promoters. Biochemistry, 1998 Feb 17, 37(7), 1861 - 7 Characterization of the overproduced NADH dehydrogenase fragment of the NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli; Braun M et al.; The proton-pumping NADH:ubiquinone oxidoreductase of Escherichia coli is composed of 14 different subunits and contains one FMN and up to nine iron-sulfur clusters as prosthetic groups . By use of salt treatment, the complex can be split into an NADH dehydrogenase fragment, a connecting fragment and a membrane fragment . The water-soluble NADH dehydrogenase fragment has a molecular mass of approximately 170,000 Da and consists of the subunits NuoE, F, and G . The fragment harbors the FMN and probably six iron-sulfur clusters, four of them being observable by EPR spectroscopy . Here, we report that the fully assembled fragment can be overproduced in E . coli when the genes nuoE, F, and G were simultaneously overexpressed with the genes nuoB, C, and D . Furthermore, riboflavin, sodium sulfide, and ferric ammonium citrate have to be added to the culture medium . The fragment was purified from the cytoplasm by means of ammonium sulfate fractionation and chromatographic steps . The preparation contains one noncovalently bound FMN per molecule . Two binuclear (N1b and N1c) and two tetranuclear (N3 and N4) iron-sulfur clusters were detected by EPR in the NADH reduced preparation with spectral characteristics identical with those of the corresponding clusters in complex I . The preparation fulfills all prerequisites for crystallization of the fragment. Biochemistry, 1998 Feb 17, 37(7), 1828 - 38 The recombinant alpha subunit of glutamate synthase: spectroscopic and catalytic properties; Vanoni MA et al.; As part of our studies of Azospirillum brasilense glutamate synthase, a complex iron-sulfur flavoprotein, we have overproduced the two enzyme subunits separately in Escherichia coli . The beta subunit (53.2 kDa) was demonstrated to contain the site of NADPH oxidation of glutamate synthase and the FAD cofactor, which was identified as Flavin 1 of glutamate synthase, the flavin located at the site of NADPH oxidation . We now report the overproduction of the glutamate synthase alpha subunit (162 kDa), which is purified to homogeneity in a stable form . This subunit contains FMN as the flavin cofactor which exhibits the properties of Flavin 2 of glutamate synthase: reactivity with sulfite to yield a flavin-N(5)-sulfite addition product (Kd = 2.6 +/- 0.22 mM), lack of reactivity with NADPH, reduction by L-glutamate, and reoxidation by 2-oxoglutarate and glutamine . Thus, FMN is the flavin located at the site of reduction of the iminoglutarate formed on the addition of glutamine amide group to the C(2) carbon of 2-oxoglutarate . The glutamate synthase alpha subunit contains the {3Fe-4S} cluster of glutamate synthase, as shown by low-temperature EPR spectroscopy experiments . The glutamate synthase alpha subunit catalyzes the synthesis of glutamate from L-glutamine and 2-oxoglutarate, provided that a reducing system (dithionite and methyl viologen) is present . The FMN moiety but not the {3Fe-4S} cluster of the subunit appears to participate in this reaction . Furthermore, the isolated alpha subunit of glutamate synthase exhibits a glutaminase activity, which is absent in the glutamate synthase holoenzyme . These findings support a model for glutamate synthase according to which the enzymes prepared from various sources share a common glutamate synthase function (the alpha subunit of the bacterial enzyme, or its homologous polypeptide forming the ferredoxin-dependent plant enzyme) but differ for the chosen electron donor . The pyridine nucleotide-dependent forms of the enzyme have recruited a FAD-dependent oxidoreductase (the bacterial beta subunit) to mediate electron transfer from the NAD(P)H substrate to the glutamate synthase polypeptide . However, it appears that the presence of the enzyme beta subunit and/or of the additional iron-sulfur clusters (Centers II and III) of the bacterial glutamate synthase is required for communication between Center I (the {3Fe-4S} center) and the FMN moiety within the alpha subunit, and for ensuring coupling of glutamine hydrolysis to the transfer of the released ammonia molecule to 2-oxoglutarate in the holoenzyme. Biochemistry, 1998 Feb 17, 37(7), 1789 - 99 Modeling the carbohydrate-binding specificity of pig edema toxin; Cummings MD et al.; The wild-type binding pentamer of Shiga-like toxin IIe (SLT-IIe) binds both the globotriaosylceramide (Gb3) and globotetraosylceramide (Gb4) cell surface glycolipids, whereas the double mutant GT3 (Q65E/K67Q) exhibits a marked preference for Gb3 {Tyrrell, G . J., et al . (1992) Proc . Natl . Acad . Sci . U.S.A . 89, 524-528} . We modeled three unique sites (sites 1-3) for binding of the carbohydrate moiety of Gb3 to GT3 and SLT-IIe, on the basis of the three sites observed for the SLT-I pentamer {Ling, H., et al . (1998) Biochemistry 37, 1777-1788} . Examination of the three sites in light of various mutation and binding data strongly suggested that one of the binding sites plays a role in the change of specificity observed for the GT3 mutant . We applied several modeling techniques, and developed a model for binding of the carbohydrate moiety of Gb4 to this site of the SLT-IIe binding pentamer . This model is consistent with a wide variety of mutation and binding data and clearly shows the importance of the terminal GalNAc residue of Gb4, as well as that of the two mutated residues of GT3, to the intermolecular interaction. Biochemistry, 1998 Feb 17, 37(7), 1777 - 88 Structure of the shiga-like toxin I B-pentamer complexed with an analogue of its receptor Gb3; Ling H et al.; Shiga-like toxin I (SLT-I) is a virulence factor of Escherichia coli strains that cause disease in humans . Like other members of the Shiga toxin family, it consists of an enzymatic (A) subunit and five copies of a binding subunit (the B-pentamer) . The B-pentamer binds to a specific glycolipid, globotriaosylceramide (Gb3), on the surface of target cells and thereby plays a crucial role in the entry of the toxin . Here we present the crystal structure at 2.8 A resolution of the SLT-I B-pentamer complexed with an analogue of the Gb3 trisaccharide . The structure reveals a surprising density of binding sites, with three trisaccharide molecules bound to each B-subunit monomer of 69 residues . All 15 trisaccharides bind to one side of the B-pentamer, providing further evidence that this side faces the cell membrane . The structural model is consistent with data from site-directed mutagenesis and binding of carbohydrate analogues, and allows the rational design of therapeutic Gb3 analogues that block the attachment of toxin to cells. Mol Microbiol, 1998 Jan, 27(2), 493 - 505 Regulation of transcription initiation at the Escherichia coli nir operon promoter: a new mechanism to account for co-dependence on two transcription factors; Wu H et al.; Expression from the Escherichia coli nir promoter is co-dependent on Fnr (a transcription factor triggered by oxygen starvation) and on NarL or NarP (transcription factors triggered by nitrite and nitrate ions) . Fnr binds to a single DNA site centred between basepairs 41 and 42 upstream from the nir transcript start, whereas NarL and NarP bind to a site upstream, centred between basepairs 69 and 70 . A novel mechanism to account for co-dependence on Fnr and NarL/NarP is suggested from experiments in which the spacing between the DNA sites for Fnr and NarL/NarP was altered . DNA sequence elements located upstream of the NarL/NarP-binding site are the targets for two or more proteins that act to repress Fnr-dependent activation of the nir promoter . This inhibition is counteracted by NarL or NarP . The model has been corroborated by the effects of several deletions and single base substitutions in the nir promoter upstream sequences: these deletions and substitutions prevent the binding of the repressor proteins . One of these repressors has been identified as the Fis protein, that binds to a site located 135-149bp upstream of the nir transcript start: the binding of Fis is suppressed by a single base substitution at position -146 . The other repressor protein(s) have yet to be identified, but appear to bind downstream of the DNA site for Fis: binding is suppressed by a single base substitution at position -99. Mol Microbiol, 1998 Jan, 27(2), 477 - 92 Novel keto acid formate-lyase and propionate kinase enzymes are components of an anaerobic pathway in Escherichia coli that degrades L-threonine to propionate; Hesslinger C et al.; An immunological analysis of an Escherichia coli strain unable to synthesize the main pyruvate formate-lyase enzyme Pfl revealed the existence of a weak, cross-reacting 85 kDa polypeptide that exhibited the characteristic oxygen-dependent fragmentation typical of a glycyl radical enzyme . Polypeptide fragmentation of this cross-reacting species was shown to be dependent on Pfl activase . Cloning and sequence analysis of the gene encoding this protein revealed that it coded for a new enzyme, termed TdcE, which has 82% identity with Pfl . On the basis of RNA analyses, the tdcE gene was shown to be part of a large operon that included the tdcABC genes, encoding an anaerobic threonine dehydratase, tdcD, coding for a propionate kinase, tdcF, the function of which is unknown, and the tdcG gene, which encodes a L-serine dehydratase . Expression of the tdcABCDEFG operon was strongly catabolite repressed . Enzyme studies showed that TdcE has both pyruvate formate-lyase and 2-ketobutyrate formate-lyase activity, whereas the TdcD protein is a new propionate/acetate kinase . By monitoring culture supernatants from various mutants using 1H nuclear magnetic resonance (NMR), we followed the anaerobic conversion of L-threonine to propionate . These studies confirmed that 2-ketobutyrate, the product of threonine deamination, is converted in vivo by TdcE to propionyl-CoA . These studies also revealed that Pfl and an as yet unidentified thiamine pyrophosphate-dependent enzyme(s) can perform this reaction . Double null mutants deficient in phosphotransacetylase (Pta) and acetate kinase (AckA) or AckA and TdcD were unable to metabolize threonine to propionate, indicating that propionyl-CoA and propionyl-phosphate are intermediates in the pathway and that ATP is generated during the conversion of propionyl-P to propionate by AckA or TdcD. Mol Microbiol, 1998 Jan, 27(2), 469 - 76 Correlation between requirement for SecA during export and folding properties of precursor polypeptides; de Cock H et al.; The structural complexity of a ligand in association with the molecular chaperones SecB and SecA was investigated using three species of precursor maltose-binding protein, which differ in their stability as a result of an amino acid substitution in each that affects the rate of folding of the polypeptide . In the presence of high concentrations of both SecB and SecA, the precursors were translocated in vitro with indistinguishable kinetics . However, when SecA was limiting, the translocation was more rapid for precursor species, which had lower stability in the native state relative to the stability of the wild-type precursor . We propose that, when in complex with SecB, precursors can form an element of tertiary structure and that these tertiary contacts are blocked when SecA is bound. Mol Microbiol, 1998 Jan, 27(2), 455 - 68 MvaL1 autoregulates the synthesis of the three ribosomal proteins encoded on the MvaL1 operon of the archaeon Methanococcus vannielii by inhibiting its own translation before or at the formation of the first peptide bond; Mayer C et al.; The control of ribosomal protein synthesis has been investigated extensively in Eukarya and Bacteria . In Archaea, only the regulation of the MvaL1 operon (encoding ribosomal proteins MvaL1, MvaL10 and MvaL12) of Methanococcus vannielii has been studied in some detail . As in Escherichia coil, regulation takes place at the level of translation . MvaL1, the homologue of the regulatory protein L1 encoded by the L11 operon of E . coli, was shown to be an autoregulator of the MvaL1 operon . The regulatory MvaL1 binding site on the mRNA is located about 30 nucleotides downstream of the ATG start codon, a sequence that is not in direct contact with the initiating ribosome . Here, we demonstrate that autoregulation of MvaL1 occurs at or before the formation of the first peptide bond of MvaL1 . Specific interaction of purified MvaL1 with both 23S RNA and its own mRNA is confirmed by filter binding studies . In vivo expression experiments reveal that translation of the distal MvaL10 and MvaL12 cistrons is coupled to that of the MvaL1 cistron . A mRNA secondary structure resembling a canonical L10 binding site and preliminary in vitro regulation experiments had suggested a co-regulatory function of MvaL10, the homologue of the regulatory protein L10 of the beta-operon of E . coil . However, we show that MvaL10 does not have a regulatory function. Mol Microbiol, 1998 Jan, 27(2), 415 - 24 Genetic analysis of the stationary phase-induced mcb operon promoter in Escherichia coli; Mao W et al.; A combination of deletion analysis and random mutagenesis was used to identify regulatory elements in Pmcb, the stationary phase-induced promoter of the mcb operon . Our results indicate that Pmcb is controlled by at least three different factors, two previously identified and at least one unknown factor, which act at four different sites in the promoter . Sequences between -344 and -164 upstream of the transcriptional start site were required for wild-type levels of mcb transcription in stationary phase . More dramatic reductions in both exponential and stationary phase expression were observed when sequences from -164 to -54 were deleted . Point mutations located between -105 and -138 decreased both exponential and stationary phase expression . All but one of these mutations decreased OmpR-dependent activation of Pmcb transcription . EmrR, also known as MprA, acts directly or indirectly at sequences downstream of -54 to repress Pmcb . A minimal promoter containing sequences from -34 to +79 was still induced > or = 10-fold in stationary phase . Point mutations within this region identified sequences at -8, -11, -30, -31 and -32 as important for Pmcb activity . These bases are in the gearbox sequence, present in Pmcb and several other stationary phase-induced Escherichia coli promoters. Mol Microbiol, 1998 Jan, 27(2), 381 - 90 Negative transcriptional regulation of a positive regulator: the expression of malT, encoding the transcriptional activator of the maltose regulon of Escherichia coli, is negatively controlled by Mlc; Decker K et al.; The maltose regulon consists of 10 genes encoding a multicomponent and binding protein-dependent ABC transporter for maltose and maltodextrins as well as enzymes necessary for the degradation of these sugars . MalT, the transcriptional activator of the system, is necessary for the transcription of all mal genes . MalK, the energy-transducing subunit of the transport system, acts phenotypically as repressor, particularly when overproduced . We isolated an insertion mutation that strongly reduced the repressing effect of overproduced MalK . The affected gene was sequenced and identified as mlc, a known gene encoding a protein of unknown function with homology to the Escherichia coli NagC protein . The loss of Mlc function led to a threefold increase in malT expression, and the presence of mlc on a multicopy plasmid reduced malT expression . By DNasel protection assay, we found that Mlc protected a DNA region comprising positions +1 to +23 of the malT transcriptional start point . Using a mlc-lacZ fusion in a mlc and mlc+ background, we found that Mlc represses its own expression . As Mlc also regulates another operon (manXYZ, see pages 369-379 of this issue), it may very well constitute a new global regulator of carbohydrate utilization. Mol Microbiol, 1998 Jan, 27(2), 369 - 80 Control of the expression of the manXYZ operon in Escherichia coli: Mlc is a negative regulator of the mannose PTS; Plumbridge J; The manXYZ operon of Escherichia coli encodes a sugar transporter of the phosphoenol pyruvate (PEP)-dependent phosphotransferase system, which is capable of transporting many sugars, including glucose, mannose and the aminosugars, glucosamine and N-acetylglucosamine . Transcription of manX is strongly dependent on cyclic AMP (cAMP)/cAMP receptor protein (CAP) . A cAMP/CAP binding site is located at -40.5, and activation by cAMP/CAP is shown to be typical of a class II promoter . The 5' end of a transcript, potentially encoding two proteins, is expressed divergently from the manXYZ operon . Previously, two binding sites for the NagC repressor were detected upstream of manX, but a mutation in nagC has very little effect on manX expression . However, a mutation in the mlc gene, encoding a homologue of nagC, results in a threefold derepression of manX expression, suggesting that this protein is a more important regulator of manX expression than NagC . The Mlc protein binds to the NagC operators, binding preferentially to the promoter-proximal operator . Plasmids overproducing either the NagC protein or the Mlc protein repress the expression of manX, but the effect of the Mlc protein is stronger . The mlc gene is shown to be allelic with the previously characterized dgsA mutation affecting the mannose phosphoenolpyruvate-dependent phosphotransferase system (PTS). Mol Microbiol, 1998 Jan, 27(2), 257 - 68 Cell division is required for resolution of dimer chromosomes at the dif locus of Escherichia coli; Steiner WW et al.; The dif locus is a RecA-independent resolvase site in the terminus region of the chromosome of Escherichia coli . The locus reduces dimer chromosomes, which result from sister chromatid exchange, to monomers . A density label assay demonstrates that recombination occurs at dif, and that it requires XerC and XerD . The frequency of this recombination is approximately 14% per site per generation, which is doubled in polA12 mutants . We have determined that recombination occurs late in the cell cycle, and that resolution is blocked if cell division is inhibited with cephalexin or by a ftsZts mutation . Fluorescence microscopy has demonstrated that abnormal nucleoids are present in cells incubated in cephalexin, and this is increased in polA12 mutants. Br J Pharmacol, 1998 Jan, 123(1), 5 - 12 Internally applied endotoxin and the activation of BK channels in cerebral artery smooth muscle via a nitric oxide-like pathway; Hoang LM et al.; 1 . In this study the role of nitric oxide synthase (NOS) in the acute activation of large conductance, Ca2+-activated K+ channels (BK channels) by internally applied E . coli lipopolysaccharide (LPS, endotoxin) was examined in vascular smooth muscle cells . 2 . Cerebrovascular smooth muscle cells (CVSMCs) were enzymatically dispersed from the middle, posterior communicating and posterior cerebral arteries of adult Wistar rats and maintained at 4 degrees C for 2-4 days before recording with standard patch-clamp techniques . 3 . Acute application of LPS (100 microg ml(-1)) to inside-out patches of CVSMC membrane isolated in a cell-free environment rapidly and reversibly increased the open probability, Po of BK channels in these patches by 3.3+/-0.30 fold . 4 . Acute application of the nitric oxide (NO) donor sodium nitroprusside (SNP, 100 microM) to inside-out patches of CVSMC membrane, studied in the presence of intact cells, also reversibly increased Po, by some 1.8+/-0.2 fold over control . 5 . Kinetic analysis showed that both LPS and SNP increased Po by accelerating the rate of BK channel reopening, rather than by retarding the closure of open channels . 6 . Neither LPS nor SNP altered the reversal potential or conductance of BK channels . 7 . The NOS substrate L-arginine (1 microM) potentiated the acute activation of BK channels by LPS, while the synthetic enantiomer D-arginine (1 microM) inhibited the action of LPS on BK channels . 8 . The acute activation of BK channels by LPS was suppressed by pre-incubation of cells with N(omega)-nitro-L-arginine (50 microM) or N(omega)-nitro-L-arginine methyl ester (1 mM), two competitive antagonists of nitric oxide synthases . N(omega)-nitro-D-arginine (50 microM), a poor inhibitor of NOS in in vitro assays, had no effect on BK channel activation by LPS . 9 . These results indicate that excised, inside-out patches of CVSMC membrane exhibit a NOS-like activity which is acutely activated when LPS is present at the cytoplasmic membrane surface . Possible relationships between this novel mechanism and the properties of known isoforms of nitric oxide synthase are discussed. Br J Cancer, 1998 Feb, 77(4), 537 - 46 Design, characterization and anti-tumour cytotoxicity of a panel of recombinant, mammalian ribonuclease-based immunotoxins; Deonarain MP et al.; Bovine seminal ribonuclease (BSRNase) is an unusual member of the ribonuclease superfamily, because of its remarkable anti-tumour and immunosuppressive properties . We describe here the construction, expression, purification and characterization of a panel of six immunotoxins based upon this enzyme and show that we can increase its anti-tumour activity by over 2 x 10(4)-fold . This is achieved by improving tumour cell targeting using a single-chain Fv (scFv) directed against the oncofetal antigen placental alkaline phosphatase . As well as the simple scFv-BSRNase fusion protein, we have constructed five other derivatives with additional peptides designed to improve folding and intracellular trafficking and delivery . We find that the molecule most cytotoxic to antigen (PLAP)-positive cells in vitro is one that contains a C-terminal 'KDEL' endoplasmic reticulum retention signal and a peptide sequence derived from diphtheria toxin . All these molecules are produced in Escherichia coli (E . coli) as insoluble inclusion bodies and require extensive in vitro processing to recover antigen binding and ribonuclease activity . Despite incomplete ribonuclease activity and quaternary assembly, these molecules are promising reagents for specific chemotherapy of cancer and are potentially less harmful and immunogenic than current immunotoxins. Biochem Pharmacol, 1998 Feb 1, 55(3), 313 - 7 Mouse steroid sulfotransferases: substrate specificity and preliminary X-ray crystallographic analysis; Kakuta Y et al.; Three mouse cytosolic sulfotransferases were expressed in Escherichia coli cells in order to study their substrate specificities toward natural as well as synthetic steroid hormones . The Km and Vmax values confirmed the high substrate specificity of estrogen and hydroxysteroid sulfotransferases toward estradiol and dehydroepiandrosterone, respectively . In sharp contrast, the synthetic estrogen diethylstilbestrol was metabolized efficiently by both enzymes to its disulfate ester . These sulfotransferases display highly stereospecific sulfotransferase activity for sulfating only the trans-isomer of diethylstilbestrol . Crystals suitable for high-resolution structure determination of estrogen sulfotransferase were grown with polyethylene glycol . The crystals belong to the orthorhombic space group P2(1)2(1)2, and diffracted to 2.5 A. Oncogene, 1998 Feb 19, 16(7), 883 - 90 Regulation of the specific DNA binding activity of Xenopus laevis p53: evidence for conserved regulation through the carboxy-terminus of the protein; Bessard AC et al.; Recombinant human p53 isolated either from E . coli or from insect cells is poorly active for binding to DNA but it can be dramatically stimulated by phosphorylation, antibody binding to the carboxy-terminal negative regulatory domain, short peptides derived from this negative regulatory domain or short single strands of DNA . We report here that Xenopus p53 has a very similar behavior . Using a new set of monoclonal antibodies directed either to the amino- or the carboxy-terminus of Xenopus p53, we demonstrate that the frog protein can be activated by specific carboxy-terminus monoclonal antibodies in order to bind to human p53 DNA response element . In addition, we report that such activation of both humans and frogs protein can also be achieved by small peptides derived from the carboxy-terminus of both p53 . Although, the sequence of this region is not conserved in the various p53 species, the presence of conserved basic residues indicates that such activation is charge-dependent . This is confirmed by the finding that small poly-lysine peptides can activate both human and Xenopus p53 . In vivo expression of Xenopus p53 indicates that this protein is able to transactivate a wide variety of human p53 response elements as long as the experiments are performed at 32 degrees C since activity at 37 degrees C, a temperature well above the natural temperature of Xenopus, is lost . Finally, we demonstrate that human mdm2 is able to down regulate the transcriptional activity of Xenopus p53. Plant Mol Biol, 1998 Feb, 36(3), 393 - 405 Cloning of three A-type cytochromes P450, CYP71E1, CYP98, and CYP99 from Sorghum bicolor (L.) Moench by a PCR approach and identification by expression in Escherichia coli of CYP71E1 as a multifunctional cytochrome P450 in the biosynthesis of the cyanogenic glucoside dhurrin; Bak S et al.; A cDNA encoding the multifunctional cytochrome P450, CYP71E1, involved in the biosynthesis of the cyanogenic glucoside dhurrin from Sorghum bicolor (L.) Moench was isolated . A PCR approach based on three consensus sequences of A-type cytochromes P450- (V/I)KEX(L/F)R, FXPERF, and PFGXGRRXCXG-was applied . Three novel cytochromes P450 (CYP71E1, CYP98, and CYP99) in addition to a PCR fragment encoding sorghum cinnamic acid 4-hydroxylase were obtained . Reconstitution experiments with recombinant CYP71E1 heterologously expressed in Escherichia coli and sorghum NADPH-cytochrome P450-reductase in L-alpha-dilaurylphosphatidyl choline micelles identified CYP71E1 as the cytochrome P450 that catalyses the conversion of p-hydroxyphenylacetaldoxime to p-hydroxymandelonitrile in dhurrin biosynthesis . In accordance to the proposed pathway for dhurrin biosynthesis CYP71E1 catalyses the dehydration of the oxime to the corresponding nitrile, followed by a C-hydroxylation of the nitrile to produce p-hydroxymandelonitrile . In vivo administration of oxime to E . coli cells results in the accumulation of the nitrile, which indicates that the flavodoxin/flavodoxin reductase system in E . coli is only able to support CYP71E1 in the dehydration reaction, and not in the subsequent C-hydroxylation reaction . CYP79 catalyses the conversion of tyrosine to p-hydroxyphenylacetaldoxime, the first committed step in the biosynthesis of the cyanogenic glucoside dhurrin . Reconstitution of both CYP79 and CYP71E1 in combination with sorghum NADPH-cytochrome P450-reductase resulted in the conversion of tyrosine to p-hydroxymandelonitrile, i.e . the membranous part of the biosynthetic pathway of the cyanogenic glucoside dhurrin . Isolation of the cDNA for CYP71E1 together with the previously isolated cDNA for CYP79 provide important tools necessary for tissue-specific regulation of cyanogenic glucoside levels in plants to optimize food safety and pest resistance. Plant Mol Biol, 1998 Jan, 36(1), 43 - 54 Stress responses in alfalfa (Medicago sativa L) . XXII . cDNA cloning and characterization of an elicitor-inducible isoflavone 7-O-methyltransferase; He XZ et al.; Medicarpin, the major phytoalexin in alfalfa, is synthesized via the isoflavonoid branch of phenylpropanoid metabolism . The methyl group at the 9 position of medicarpin is generally accepted to arise via the methylation of the 4' position (B-ring) of daidzein . Surprisingly, the isoflavone-O-methyltransferase (IOMT), which is induced along with other enzymes involved in medicarpin biosynthesis, methylates the A-ring 7-hydroxyl group of daidzein in vitro, a reaction that probably does not occur in vivo . Utilizing internal amino acid sequence information from purified alfalfa IOMT, we have isolated three full-length IOMT cDNA clones . A search of the protein databases revealed sequence similarities to O-methyltransferases from various sources . The highest match (50.5% identity) was found between IOMT8 and 6a-hydroxymaackiain 3-O-methyltransferase from Pisum sativum . The molecular weight of alfalfa IOMT expressed in Escherichia coli was similar to that of purified IOMT from alfalfa cell cultures (41 kDa by SDS-PAGE) . The recombinant enzyme catalyzed the O-methylation of A-ring hydroxyl group(s) of isoflavones, and could also methylate the pterocarpan (+) 6a-hydroxymaackiain . Alfalfa contains multiple IOMT genes, and closely related sequences are present in the genomes of chickpea and cowpea, species that also produce B-ring methylated isoflavonoids in vivo . Northern blot analysis indicated that IOMT transcripts are rapidly induced following elicitation, prior to the increase in IOMT activity and medicarpin accumulation . The possible role of the isoflavone 7-OMT in the synthesis of formononetin in vivo is discussed. Plant Mol Biol, 1998 Mar, 36(4), 553 - 63 RNaseI from Escherichia coli cannot substitute for S-RNase in rejection of Nicotiana plumbaginifolia pollen; Beecher B et al.; Unilateral incompatibility often occurs between self-incompatible (SI) species and their self-compatible (SC) relatives . For example, SI Nicotiana alata rejects pollen from SC N . plumbaginifolia, but the reciprocal pollination is compatible . This interspecific pollen rejection system closely resembles intraspecific S-allele-specific pollen rejection . However, the two systems differ in degree of specificity . In SI, rejection is S-allele-specific, meaning that only a single S-RNase causes rejection of pollen with a specific S genotype . Rejection of N . plumbaginifolia pollen is less specific, occurring in response to almost any S-RNase . Here, we have tested whether a non-S-RNase can cause rejection of N . plumbaginifolia pollen . The Escherichia coli rna gene encoding RNAseI was engineered for expression in transgenic (N . plumbaginifolia x SC N . alata) hybrids . Expression levels and pollination behavior of hybrids expressing E . coli RNaseI were compared to controls expressing SA2-RNase from N . alata . Immunoblot analysis and RNase activity assays showed that RNaseI and SA2-RNase were expressed at comparable levels . However, expression of SA2-RNase caused rejection of N . plumbaginifolia pollen, whereas expression of RNaseI did not . Thus, in this system, RNase activity alone is not sufficient for rejection of N . plumbaginifolia pollen . The results suggest that S-RNases may be specially adapted to function in pollen rejection. Plant Mol Biol, 1998 Mar, 36(4), 521 - 8 Soluble, highly fluorescent variants of green fluorescent protein (GFP) for use in higher plants; Davis SJ et al.; Green fluorescent protein (GFP) from Aequorea victoria has rapidly become a standard reporter in many biological systems . However, the use of GFP in higher plants has been limited by aberrant splicing of the corresponding mRNA and by protein insolubility . It has been shown that GFP can be expressed in Arabidopsis thaliana after altering the codon usage in the region that is incorrectly spliced, but the fluorescence signal is weak, possibly due to aggregation of the encoded protein . Through site-directed mutagenesis, we have generated a more soluble version of the codon-modified GFP called soluble-modified GFP (smGFP) . The excitation and emission spectra for this protein are nearly identical to wild-type GFP . When introduced into A . thaliana, greater fluorescence was observed compared to the codon-modified GFP, implying that smGFP is 'brighter' because more of it is present in a soluble and functional form . Using the smGFP template, two spectral variants were created, a soluble-modified red-shifted GFP (smRS-GFP) and a soluble-modified blue-fluorescent protein (smBFP) . The increased fluorescence output of smGFP will further the use of this reporter in higher plants . In addition, the distinct spectral characters of smRS-GFP and smBFP should allow for dual monitoring of gene expression, protein localization, and detection of in vivo protein-protein interactions. Plant Mol Biol, 1998 Mar, 36(4), 509 - 20 Expression of the tobacco anionic peroxidase gene is tissue-specific and developmentally regulated; Klotz KL et al.; Transcriptionally regulated expression of tobacco anionic peroxidase was investigated with regard to tissue specificity and developmental regulation . Two tobacco species, Nicotiana sylvestris and Nicotiana tabacum cv . Xanthi, were stably transformed with a gene chimera composed of 3 kb of the tobacco anionic peroxidase promoter, the Escherichia coli beta-glucuronidase (GUS) coding region and the nopaline synthase terminator . Gene expression was regulated spatially and developmentally in all organs, and generally increased with age and maturity of the plant, tissue or organ . In the aerial portions of the plant, GUS activity was strongly expressed in trichomes and epidermis at nearly all developmental stages . In later stages of development, activity was also detected in ground tissue and parenchyma cells associated with vascular tissues . Activity in roots was limited to cortical cells and vascular-associated parenchyma cells . In reproductive tissue, expression was observed in sepals and petals before anthesis, and in all floral organs after anthesis . Expression was never detected in vascular tissue and was poorly correlated with lignification except in the cells surrounding primary xylem and pericyclic fibers in N . sylvestris . These studies suggest that this peroxidase isoenzyme is only limitedly involved in lignification but may be important in plant defense, growth and development. Plant Mol Biol, 1998 Jan, 36(2), 307 - 14 The sequence and structure of the 3'-untranslated regions of chloroplast transcripts are important determinants of mRNA accumulation and stability; Rott R et al.; A general characteristic of the 3'-untranslated regions (3' UTRs) of plastid mRNAs is an inverted repeat (IR) sequence that can fold into a stem-loop structure . These stem-loops are RNA 3'-end processing signals and determinants of mRNA stability, not transcription terminators . Incubation of synthetic RNAs corresponding to the 3' UTRs of Chlamydomonas chloroplast genes atpB and petD with a chloroplast protein extract resulted in the accumulation of stable processing products . Synthetic RNAs of the petA 3' UTR and the antisense strand of atpB 3' UTR were degraded in the extract . To examine 3' UTR function in vivo, the atpB 3' UTR was replaced with the 3' UTR sequences of the Chlamydomonas chloroplast genes petD, petD plus trnR plus trnR, rbcL, petA and E . coli thrA by biolistic transformation of Chlamydomonas chloroplasts . Each 3' UTR was inserted in both the sense and antisense orientations . The accumulation of both total atpB mRNA and ATPase beta-subunit protein in all transformants was increased compared to a strain in which the atpB 3' UTR had been deleted . However, the level of discrete atpB transcripts in transformants containing the antisense 3' UTR sequences was reduced to approximately one-half that of transformants containing the 3' UTRs in the sense orientation . These results imply that both the nucleotide sequences and the stem-loop structures of the 3' UTRs are important for transcript 3'-end processing, and for accumulation of the mature mRNAs. Plant Mol Biol, 1998 Jan, 36(2), 219 - 27 A flavonoid 7-O-methyltransferase is expressed in barley leaves in response to pathogen attack; Christensen AB et al.; We have shown previously that transcripts corresponding to the cDNA clone pBH72-F1, with similarities to O-methyltransferases (OMT), accumulated in barley leaves in response to attack by the pathogenic fungus Blumeria graminis (Plant Mol Biol 26 (1994) 1797) . To investigate the accumulation pattern in the defence response and the organ localization of the pBH72-F1-encoded polypeptide (F1-OMT), an antiserum was raised against Escherichia coli expressed F1-OMT . The 43 kDa protein was absent in normal leaves but accumulated strongly in response to pathogen attack . The F1-OMT protein accumulated faster in barley lines inoculated with an avirulent B . graminis isolates compared to a virulent isolate . Additionally, F1-OMT related proteins were detected in developing kernels . F1-OMT was expressed as a functional enzyme in E . coli and the substrate specificity was investigated . The enzyme exhibited OMT activity towards flavonoid aglycones with the highest activity against apigenin (4',5,7-trihydroxyflavone) . In contrast, caffeic acid did not serve as substrate for F1-OMT . The product of F1-OMT was analyzed by HPLC and GC-MS and found to be genkwanin (4',5-dihydroxy-7-methoxyflavone) . Initial velocity data were best represented by a sequential bi-bi mechanism, and kinetic parameters of KSAM = 10.9 microM, Kapigenin = 4.6 microM and a specific activity of 0.45 mukat/g were obtained . Barley F1-OMT, apigenin 7-O-methyltransferase, is suggested to be involved in the production of a methylated flavonoid phytoalexin. Plant Mol Biol, 1998 Jan, 36(2), 195 - 204 The activities of acidic and glutamine-rich transcriptional activation domains in plant cells: design of modular transcription factors for high-level expression; Schwechheimer C et al.; The aim of this work was to design strong transcriptional activators that can be used to regulate plant gene expression . The contribution of different components in a transcription factor and target gene system was assayed by measuring transcriptional activation . Each component was optimised to achieve maximal reporter gene expression in transient protoplast transformation assays . The DNA-binding domain of the yeast transcriptional activator GAL4 was studied in the context of fusion proteins with activation domains of the herpes simplex virus protein VP16 or the tomato Myb-like activator THM18 . Multimerisation of the activation domain and insertion of a homopolymeric glutamine stretch was used to increase transcription factor potency . Evidence is presented that these modifications can result in even more active transcription factors when they are combined . Finally, it was demonstrated using competition experiments that transcription factors with acidic activation domains can mutually suppress their activation potentials when expressed at high levels. Biochemistry, 1998 Feb 10, 37(6), 1722 - 30 Reversible folding of Ada protein (O6-methylguanine-DNA methyltransferase) of Escherichia coli; Bhattacharyya D et al.; The multifunctional 39 kDa Escherichia coli Ada protein (O6-methylguanine-DNA methyltransferase) (EC 2.1.1.63), product of the ada gene, is a monomeric globular polypeptide with two distinct alkylacceptor activities located in two domains . The two domains are of nearly equal size and are connected by a hinge region . The Ada protein accepts stoichiometrically the alkyl group from O6-alkylguanine in DNA at the Cys-321 residue and from alkyl phosphotriester at the Cys-69 residue . This protein functions in DNA repair by direct dealkylation of mutagenic O6-alkylguanine . The protein methylated at Cys-69 becomes a transcriptional activator of the genes in the ada regulon, including its own . Each of the two domains functions independently as an alkyl acceptor . The purified homogeneous protein is unstable at 37 degrees C and spontaneously loses about 30% of its secondary structure in less than 30 min concomitant with a complete loss of activity . However, sedimentation equilibrium studies indicated that the inactive protein remains in the monomeric form without aggregation . Furthermore, electrospray mass spectroscopic analysis indicated the absence of oxidation of the inactive protein . This temperature-dependent inactivation of the Ada protein is inhibited by DNA . In the presence of increasing concentrations of urea or guanidine, the protein gradually loses more than 80% of its structure . The two alkyl acceptor activities appear to be differentially sensitive to unfolding and the phosphotriester methyltransferase activity is resistant to 7 M urea . The partial or complete unfolding induced by urea or guanidine is completely reversed within seconds by removal of the denaturant . The heat-coagulated protein can also be restored to full activity by cycling it through treatment with 8 M urea or 6 M guanidine . These results suggest that the nascent or unfolded Ada polypeptide folds to a metastable form which is active and that the thermodynamically stable structure is partially unfolded and inactive. Biochemistry, 1998 Feb 10, 37(6), 1697 - 705 Influence of excision of a methylene group from Glu-376 (Glu376-->Asp mutation) in the medium chain acyl-CoA dehydrogenase-catalyzed reaction; Peterson KL et al.; The human liver medium chain acyl-CoA dehydrogenase (MCAD)-catalyzed reaction proceeds via abstraction of an alpha-proton from the acyl-CoA substrates by the carboxyl group of Glu-376 . By using the methods of site-directed mutagenesis, we replaced Glu-376 by Asp (E376D mutation), expressed the wild-type and mutant enzymes in Escherichia coli, purified them to homogeneity, and compared their kinetic properties . The steady-state kinetic data revealed that the E376D mutation impaired (by about 15-20-fold) the turnover rate of the enzyme as well as its inactivation by 2-octynoyl-CoA . There was no selective solvent deuterium isotope effect on enzyme catalysis . These results lead to the suggestion that the carboxyl group of Asp-376 does not serve as efficient catalytic base as the carboxyl group of Glu-376 . The E376D mutation impaired the octanoyl-CoA-dependent reductive half-reaction such that the rate-limiting step of enzyme catalysis shifted from the product dissociation step (in the case of the wild-type enzyme) to the flavin reduction step, and abolished the previously noted kinetic and thermodynamic correspondences between the octanoyl-CoA-dependent reductive half-reaction and the enzyme-octenoyl-CoA interaction {Kumar, N . R., and Srivastava, D . K . (1994) Biochemistry 33, 8833-8841} . Arguments are presented that the Glu-376-->Asp mutation results in uncoupling between the proton transfer and protein conformational change steps during enzyme catalysis. Biochemistry, 1998 Feb 10, 37(6), 1632 - 9 Substitutions of conserved aromatic amino acid residues in subunit I perturb the metal centers of the Escherichia coli bo-type ubiquinol oxidase; Mogi T et al.; Cytochrome bo is a four-subunit quinol oxidase in the aerobic respiratory chain of Escherichia coli and functions as a redox-coupled proton pump . Subunit I binds all the redox metal centers, low-spin heme b, high-spin heme o, and CuB, whose axial ligands have been identified to be six invariant histidines . This work explored the possible roles of the aromatic amino acid residues conserved in the putative transmembrane helices (or at the boundary of the membrane) of subunit I . Sixteen aromatic amino acid residues were individually substituted by Leu, except for Tyr61 and Trp282 by Phe and Phe415 by Trp . Leu substitutions of Trp280 and Tyr288 in helix VI, Trp331 in loop VII-VIII, and Phe348 in helix VIII reduced the catalytic activity, whereas all other mutations did not affect the in vivo activity . Spectroscopic analyses of the purified mutant enzymes revealed that the defects were attributable to perturbations of the binuclear center . On the basis of these findings and recent crystallographic studies on cytochrome c oxidases, we discuss the possible roles of the conserved aromatic amino acid residues in subunit I of the heme-copper terminal oxidases. Neuroscience, 1998 Apr, 83(3), 691 - 700 Transgenic mice with cerebral expression of human immunodeficiency virus type-1 coat protein gp120 show divergent changes in short- and long-term potentiation in CA1 hippocampus; Krucker T et al.; The human immunodeficiency virus type-1 envelope glycoprotein gp120 is shed from the virus and from infected cells and thus can diffuse and interact with a variety of central nervous system cells . Transgenic mice constitutively expressing glial fibrillary acidic protein-driven gp120 from brain astrocytes display neuronal and glial changes resembling abnormalities in human immunodeficiency virus type-1-infected human brains . To assess the neurophysiology of these transgenic mice and determine whether gp120 expression impairs synaptic plasticity, we examined CA1 population excitatory postsynaptic potentials in hippocampal slices from transgenic mice and from non-transgenic controls, using a double-blind protocol . Compared with slices from non-transgenic littermate controls, slices from gp120 transgenic mice showed four significant alterations: (i) increased mean slopes of normalized population excitatory postsynaptic potentials; (ii) larger paired-pulse facilitation after induction of long-term potentiation at 50 ms interpulse intervals; (iii) markedly elevated short-term potentiation after 10 and 20 shocks at 100 Hz; and (iv) a significant reduction in the magnitude of CA1 long-term potentiation . In slices from transgenic mice expressing Escherichia coli beta-galactosidase from the same promoter, paired-pulse facilitation and long-term potentiation were normal . These results indicate that brain slice preparations from gp120 transgenic mice can be used to assess pathophysiological effects of gp120 on neuronal networks . Because short-term potentiation involves presynaptic mechanisms, our results suggest that gp120 expression in these mice enhances either presynaptic glutamate release or postsynaptic glutamate receptor function, or both . These changes could lead to increased Ca2+ influx, thereby contributing to neuronal dysfunction and injury . As long-term potentiation is a cellular model of learning and memory, our results may be relevant to memory (cognitive) impairments seen in patients with AIDS. Mech Ageing Dev, 1997 Dec 30, 99(3), 257 - 71 Transgenic mouse models for studying mutations in vivo: applications in aging research; Vijg J et al.; To study mutation accumulation in the DNA of somatic cells and tissues during aging in vivo, a transgenic mouse model has been constructed . The model harbors plasmid vectors, containing the lacZ reporter gene, integrated head to tail at various chromosomal locations . Procedures have been worked out to efficiently recovery the plasmids into E . coli host cells . A positive selection system, permitting only E . coli cells with a lacZ mutated plasmid to grow, allows for the accurate determination of mutation frequencies as the ratio of mutant colonies versus the total number of transformants, i.e., the total number of plasmid copies recovered . Results obtained from a life span study of plasmid mice with vector clusters on chromosome 3 and 4 indicated age-related mutation accumulation in the liver, but not in the brain . Comparison of the mutational spectra revealed a significantly larger proportion of large size-change mutations in liver than in brain. Int Arch Allergy Immunol, 1998 Feb, 115(2), 150 - 6 Involvement of the N-terminus of Der p 2 in IgE and monoclonal antibody binding; Hakkaart GA et al.; The major house dust mite allergen Der p 2 was expressed as a recombinant fusion protein in Escherichia coli either with glutathione-S-transferase as fusion partner or with a poly-histidine tag . Both recombinant fusion proteins failed to react with 3/14 Der p 2-specific monoclonal antibodies (mAbs) . When Der p 2 was expressed in yeast with one alanine linked N-terminally to the allergen, no reactivity was observed . When expressed without any fusion partner, all 14 mAbs showed reactivity . The addition of a single N-terminal alanine also disrupted an important epitope for IgE . In RAST inhibitions, an average decrease in inhibitory potency of 72+/-32% was observed (n = 16) with a maximum decrease of 91% . These observations suggest that the N-terminus of Der p 2 is involved in an important epitope for IgE that is disrupted by the addition of one single amino-acid . Recombinant Der p 2 molecules should therefore preferably lack any fusion peptide. Nucl Med Commun, 1997 Dec, 18(12), 1189 - 93 The value of 67Ga-citrate scanning in psoas abscess; Chiu NT et al.; Psoas abscess is a serious health problem which presents with non-specific symptoms and signs . To reduce morbidity and mortality, it is important to diagnose the presence and extent of a psoas abscess accurately using imaging studies . Because the 67Ga scan may facilitate the early diagnosis of insidious infection and assist CT-guided percutaneous drainage of the abscess, we examined the value of 67Ga scans in 18 patients with psoas abscess . The imaging results of 67Ga scans (18 patients), computed tomography (CT) (16 patients) and bone scans (13 patients) were analyzed . In this series, concomitant infections were very common (94%) in patients with psoas abscess . For detecting psoas abscess, the sensitivity of 67Ga scanning (92%) and CT (91%) was similar . However, 67Ga scanning is superior to CT in demonstrating concomitant infectious foci at other sites . Bone scanning is a sensitive tool for depicting osteomyelitis, which was common in this series of patients . We also found that increased vascularity in the psoas area was demonstrated by three-phase bone scanning in 60% of patients. Comp Immunol Microbiol Infect Dis, 1997 Sep, 20(4), 299 - 307 Role of iron and iron chelation therapy in oxygen free radical mediated tissue injury in an ascending mouse model of chronic pyelonephritis; Gupta R et al.; The contribution of iron towards the free radical generation leading to renal tissue damage was assessed using a non-obstructive ascending mouse model for chronic pyelonephritis . The parameters studied include luminol dependent chemiluminescence (LDCL), histopathology and some biochemical investigations . We found that iron enhanced the renal tissue damage and led to renal scarring, and end point in chronic renal inflammation, irrespective of the bacterial strain studied . In addition a role of iron chelation therapy as a treatment for chronic renal inflammation is also suggested. Biochem Biophys Res Commun, 1998 Feb 13, 243(2), 538 - 44 Molecular cloning of human D-dopachrome tautomerase cDNA: N-terminal proline is essential for enzyme activation; Nishihira J et al.; D-Dopachrome tautomerase converts 2-carboxy-2,3-dihydroindole-5,6-quinone (D-dopachrome) into 5,6-dihydroxyindole . This protein has an amino acid sequence that is highly homologous with that of macrophage migration inhibitory factor (MIF), which has the potential to catalyze D-dopachrome to 5,6-dihydroxyindole-2-carboxylic acid and is an important cytokine for T-lymphocyte activation . We isolated and sequenced a 566 bp-long cDNA encoding human D-dopachrome tautomerase . The cDNA contains an open reading frame encoding 118 amino acids, including the initiator methionine . The amino acid sequence of the protein shares 80% homology with that of the rat enzyme . Northern blot analysis demonstrated that mRNA of D-dopachrome tautomerase is expressed in a large amount in the liver, and to lesser extent in other organs, including the heart, lung and pancreas . After purification of D-dopachrome tautomerase expressed in E . coli, we confirmed that the recombinant protein catalyzes the conversion of D-dopachrome to 5,6-dihydroxyindole . Its catalytic mechanism is not well understood . We found that the protein completely lost the enzyme activity when the N-terminal proline residue was replaced with alanine by site-directed mutagenesis . This fact suggests that the N-terminal proline is essential for the catalytic mechanism . Although the precise pathophysiological function of D-dopachrome tautomerase remains to be elucidated, the present results could contribute to further understanding of isomerase activity in relation to the immune response. Biochem Biophys Res Commun, 1998 Feb 13, 243(2), 438 - 43 Expression, purification, and characterization of a recombinant 5-lipoxygenase from potato tuber; Chen X et al.; We have isolated a full length 5-LOX cDNA clone from potato cDNA library using degenerate primers designed from conserved sequences of LOXs . Sequence analysis and comparison of the deduced amino acid sequence revealed high homology to other plant LOXs . We have expressed the cDNA in Escherichia coli and purified the recombinant protein to electrophoretic homogeneity by anion exchange liquid chromatography followed by HPLC on a Mono-Q column . Substrate specificity of the purified recombinant protein revealed LOX activity towards linoleic, linolenic acid, arachidonic acids as substrates with linoleic acid being the best substrate . The relative LOX activity as well as the product profiles for the recombinant L1 5-LOX are comparable to values determined for the purified potato tuber 5-LOX . When the recombinant L1 5-LOX and the native peak-2 5-LOX (the most abundant isozyme) were compared on SDS-PAGE, single bands of apparently identical mass 97,000 Da, was observed, which agrees well with the L1 molecular mass calculated from amino acid sequences. Biochem Biophys Res Commun, 1998 Feb 13, 243(2), 372 - 6 Role of glycosylation in the lipid-binding activity of the exchangeable apolipoprotein, apolipophorin-III; Soulages JL et al.; Non-glycosylated recombinant Locusta migratoria apolipophorin-III, apoLp-III, was expressed in E . coli and its physical-chemical properties were compared to those of the glycosylated native apoLp-III . Fluorescence quantum yield and acrylamide quenching studies indicated a slightly higher accessibility of the Trp residues in the recombinant apoLp-III . Far-UV CD spectroscopy indicated that the recombinant apoLp-III has a lower alpha-helical content than the glycosylated apoLp-III . Both proteins spontaneously formed discoidal recombinant lipoprotein particles when incubated with dimyristoylphosphatidylcholine (DMPC) . Interaction with lipid promotes an increase in alpha-helical content . CD and fluorescence studies indicate that both proteins adopt the same conformation in the lipid-bound state . However, the kinetics of association of the recombinant protein with DMPC is 5-fold faster than that of the native protein . The results suggest that glycosylation inhibits the lipid binding activity by preventing the exposure of hydrophobic domains and/or decreasing the conformational flexibility of the protein. Schweiz Arch Tierheilkd, 1997, 139(11), 495 - 9 {Current resistance status of Escherichia coli strains from bovine mastitis milk samples}; Stephan R et al.; Between December 1996 and March 1997, 95 E.coli strains were isolated from mastitis milk samples from 95 different animals . In 29.5% resistance could be observed against one or several of the examined antibiotics . However, Cefoperazone, Polymyxin B, Colistin and Gentamycin proved effective against the majority of these strains . Between 0 and 23% of E.coli were resistant against the single tested antibiotics . As opposed to earlier investigations a smaller number of Chloramphenicol-resistant strains could be found . One strain with multiple resistance was also resistant against Gentamycin . However, in the treatment of mastitis the resistance of a particular agent is only one among several contributing factors. Genetika, 1997 Nov, 33(11), 1487 - 93 {Genetic study of the mechanism of tandem duplication formation in conjugational crosses in Escherichia coli K-12}; Sukhodolets VV; The formation of heterozygous tandem duplications of the deo operon was studied in conjugational matings HfrH deoA deoB::Tn5 thyA x HfrH deoC deoD thyA . When the HfrH deoC deoD thr::Tn9 car::Tn10 thyA donor strain was used, the thr::Tn9 and car::Tn10 transposon insertions linked to the deo operon were integrated into some duplications . Duplications carrying the recipient thr+ and car+ alleles in the duplicated state were not found among duplications that did not include the thr::Tn9 and car::Tn10 donor markers . These data indicate that heterozygous deo operon duplications are primarily formed directly during the recombinational interaction between chromosomes in the merozygote, but not on the basis of preexisting sister duplications in the recipient strain . Thus, genetic recombination occurring during the process of conjugation in E . coli induces both symmetrical and unequal crossing over . Nevertheless, when the HfrH deoA deoB::Tn5 thr::Tn9 car::Tn10 thyA strain was used as a recipient, a deo operon duplication containing thr::Tn9 and car::Tn10 markers was found in the homozygous state . Consequently, some heterozygous duplications can also be formed on the basis of preexisting sister duplications. Science, 1998 Feb 6, 279(5352), 873 - 6 Conjugative transfer by the virulence system of Legionella pneumophila; Vogel JP et al.; Legionella pneumophila, the causative agent of Legionnaires' pneumonia, replicates within alveolar macrophages by preventing phagosome-lysosome fusion . Here, a large number of mutants called dot (defective for organelle trafficking) that were unable to replicate intracellularly because of an inability of the bacteria to alter the endocytic pathway of macrophages were isolated . The dot virulence genes encoded a large putative membrane complex that functioned as a secretion system that was able to transfer plasmid DNA from one cell to another. J Biol Chem, 1998 Jan 30, 273(5), 3076 - 81 Human small intestinal maltase-glucoamylase cDNA cloning . Homology to sucrase-isomaltase; Nichols BL et al.; It has been hypothesized that human mucosal glucoamylase (EC 3.2.1 . 20 and 3.2.1.3) activity serves as an alternate pathway for starch digestion when luminal alpha-amylase activity is reduced because of immaturity or malnutrition and that maltase-glucoamylase plays a unique role in the digestion of malted dietary oligosaccharides used in food manufacturing . As a first step toward the testing of this hypothesis, we have cloned human small intestinal maltase-glucoamylase cDNA to permit study of the individual catalytic and binding sites for maltose and starch enzyme hydrolase activities in subsequent expression experiments . Human maltase-glucoamylase was purified by immuno