Biokhimiia, 1996 Mar, 61(3), 464 - 71 {Reactions of decarboxylated and side transamination during interaction of glutamate decarboxylase from Escherichia coli with substrate analogs, modified through C3 and C4 atoms}; Khristoforov RR et al.; The interaction of glutamate decarboxylase with the aspartate and glutamate analogues modified at C3 and C4 was studied . 3-Arsonoalanine, 3-phosphonoalanine, 2-amino-4-arsonobutyric acid, 2-amino-4-phosphonobutyric acid, a mixture of diastereoisomers of 4-(methylthio) glutamic acid and erythro-4-(methylthio) glutamic acid were shown to be poor substrates for the enzyme . Their decarboxylation was accompanied by transamination of the coenzyme (PLP) to pyridoxamine phosphate (PMP) which reversibly inactivated the enzyme . With arsonoalanine only part of PLP was converted into PMP and another part irreversibly formed a complex . 4-(Methylsulfonyl)-L-glutamic and 4-{(phenyl)(hydroxy)phosphoryl}-L-glutamic acids did not react with the glutamate decarboxylase. Biofizika, 1996 Mar-Apr, 41(2), 377 - 83 {Character of K+ absorption and its interaction with membrane protein pumps in Escherichia coli, grown under anaerobic conditions in the presence of nitrate}; Bagramian KA et al.; The character of H(+)-K(+)-exchange in E.coli, grown in anaerobic conditions in the presence of sodium nitrate and performed a nitrate respiration, has been studied . The K+ uptake has been shown to occur by one step, to be not inhibited by N,N'-dicyclohexylcarbodiimide, but to be stopped by arsenate and protonophore . It has K(m) of 4.5 mM, K+ accumulation in cell is more than 200 mM and K+ distribution between the cytoplasm and the medium is more than 10(3) (K+ equilibrium potential is achieved 190 mV) . To switch on a mechanism of K+ uptake is depended on osmotic shock and carried out upon positive as well as negative shocks in spheroplasts . A H+ efflux occurs with constant rate while glucose is in the medium and a stoichiometry for the initial H+ to K+ fluxes is variable upon a different experimental conditions . In spite of H(+)-K(+)-exchange in anaerobically grown E.coli, a production of H2 in bacteria is not observed, an ATPase activity of isolated membranes sensitive, to N,N'-dicyclohexylcarbodiimide is not stimulated by K+ and lost in E.coli mutant with an unc-deletion . It is concluded that H+ and K+ transfer through the membranes in E.coli performed a nitrate respiration, occur through different systems-a respiration chain and the TrkA system of K+ uptake . The latest operates itself, has no ATPase activity and interacts with membrane proton pumps indirectly using transmembrane proton gradient (delta mu H+) as a driving force as well as ATP as a regulator of activity . A sensitivity of this system to osmotic shock is lost under destruction of periplasmic space. Biofizika, 1996 Mar-Apr, 41(2), 369 - 76 {Role of components of formate-hydrogen-lyase in forming molecular hydrogen and their connection with proton-potassium exchange in anaerobically grown Escherichia coli}; Bagramian KA et al.; It is shown that -2H+/K(+)-exchange through the H(+)-K(+)-pump, formed by the F0F1-ATPase and the Trk H system, H(+)-K(+)-exchange via H(+)-K(+)-antiporter, formed by the F0 and the Trk G (core) system {1-2}, and production of H2 in anaerobically grown E.coli are changed in the mutants with defects in components of formate hydrogen lyase complex, oxidizing formate to CO2 and H2 . 2H+/K(+)-exchange and H2 production are destroyed, but H(+)-K(+)-exchange with a variable stoichiometry for N,N'-dicyclohexyl-carbodiimide-sensitive ion fluxes is displayed in the fdhF mutant E.coli FM911, where formate dehydrogenase(H) is absent . 2H+/K(+)-exchange does not occur, but H(+)-K(+)-exchange with variable stoichiometry for N,N'-dicyclohexylcarbodiimide-sensitive ion fluxes and H2 production are observed in the uncD mutant E.coli AN817 with defect in beta subunit of the F1 . Deletion of the hyc-operon in mutant E.coli HD700, led to absence of hydrogenase 3, destroys H(+)-K(+)-exchange and H2 production . H2 evaluation is shown in the E.coli K12(lambda) protoplasts, treated with toluene, by adding of NADH into the medium, containing ATP and K+ . It is inhibited by N,N'-dicyclohexylcarbodiimide . H2 production is increased by adding of dithiothreitol, when NADH is changed by formate . It is lost in the mutants with defects in the F0 (E.coli AN936) or in the Trk A protein (E.coli TK2242) . Dehydrogenase(H) and hydrogenase 3 are assumed to link mutually with a H(+)-K(+)-pump operation, reducing equivalents, necessary for a dithiol-disulfide interconversion within a mechanism of pump, are transferred from formate by means of dehydrogenase(H) to hydrogenase 3 through the F0F1 and the Trk H system to produce H2 . It is assumed that hydrogenase 3 can interact with a mechanism of H(+)-K(+)-antiporter, NADH could serve as a donor of reducing equivalents . A role of thiol-groups and dithiol-disulfide interconversion in a functions of both mechanism for H(+)-K(+)-exchange is confirmed. Biol Chem Hoppe Seyler, 1996 Mar, 377(3), 211 - 5 Heterologous expression and characterisation of mouse brain fatty acid binding protein; Schnutgen F et al.; A novel brain-type member of the fatty acid binding protein family (B-FABP) was heterologously expressed in Escherichia coli, either as inclusion bodies at 37 degrees C or in soluble form at 22 degrees C . Both B-FABP renatured from inclusion bodies and the solubly expressed protein could be purified to homogeneity by anion exchange chromatography and gel filtration in a functional conformation as they bound oleic acid with high affinity . None of the five cysteines of B-FABP was involved in disulphide bond formation . Isoelectric focusing revealed heterogeneity of the renatured protein but not of the solubly expressed protein . By Western blotting using affinity purified rabbit antibodies raised against the recombinant B-FABP it was demonstrated that in adult mice, B-FABP is predominantly expressed in the olfactory bulb. Biol Chem Hoppe Seyler, 1996 Mar, 377(3), 175 - 86 Structure of recombinant human parathyroid hormone in solution using multidimensional NMR spectroscopy; Gronwald W et al.; The solution structure of human parathyroid hormone, in the form of recombinant prolyl-hPTH(1-84), has been investigated by multidimensional NMR spectroscopy under conditions (aqueous trifluoroethanol) which favour the structured-state of the protein . Spin systems were identified from 3D 1H DQF (double-quantum filtered)-COSY and TOCSY spectra and sequence-specific assignments were from 2D 1H phase-sensitive NOESY spectra . Signal overlap was resolved in a 3D-NOESY-TOCSY spectrum and assignments were confirmed with 2D NOESY-15N-HMQC (heteronuclear multiple-quantum coherence) spectra taken of a sample universally labeled with 15N . A satisfactory set of final structures was calculated from the quantitative NOE data using restrained molecular dynamics and energy minimization calculations . The N-terminus is dominated by three, well defined helices between Ser-3 to Asn-10, Ser-17 to Lys-27 and Asp-30 to Leu-37, while the most significant structural features in the C-terminus are a short, less-well defined helix between Asn-57 to Ser-62 and a series of loose turns . These two terminal units are joined by an unstructured mid-region . The molecule shows a tendency towards tertiary structure, defined by a number of long-range NOEs . A detailed RMS deviation analysis allowed the final refined structures to be classified into a limited ensemble of stable conformations that reflect the inherent flexibility of the hormone in solution. Plant Cell, 1996 Mar, 8(3), 529 - 37 The SAL1 gene of Arabidopsis, encoding an enzyme with 3'(2'),5'-bisphosphate nucleotidase and inositol polyphosphate 1-phosphatase activities, increases salt tolerance in yeast; Quintero FJ et al.; A cDNA library in a yeast expression vector was prepared from roots of Arabidopsis exposed to salt and was used to select Li(+)-tolerant yeast transformants . The cDNA SAL1 isolated from one of these transformants encodes a polypeptide of 353 amino acid residues . This protein is homologous to the HAL2 and CysQ phosphatases of yeast and Escherichia coli, respectively . Partial cDNA sequences in the data bases indicate that rice produces a phosphatase highly homologous to SAL1 and that a second gene homologous to SAL1 exists in Arabidopsis . The SAL1 protein expressed in E . coli showed 3'(2'),5'-bisphosphate nucleotidase and inositol polyphosphate 1-phosphatase activities . In yeast, SAL1 restored the ability of a hal2/met22 mutant to grow on sulfate as a sole sulfur source, increased the intracellular Li+ tolerance, and modified Na+ and Li+ effluxes . We propose that the product of SAL1 participates in the sulfur assimilation pathway as well as in the phosphoinositide signaling pathway and that changes in the latter may affect Na+ and Li+ fluxes. Yakugaku Zasshi, 1996 Mar, 116(3), 209 - 16 {Properties of glycyrrhizin in Kampo extracts including licorice root and changes in the blood concentration of glycyrrhetic acid after oral administration of Kampo extracts}; Miyamura M et al.; We investigated in vitro the properties of glycyrrhizin (GL), such as dissolution, absorption and resolution, using a Sho-Seiryu-To extract, a Sho-Saiko-To extract, both including a licorice root, and licorice extract . The dissolution of GL differed with the pH of the solvent . The absorption (partition coefficient) of GL decreased with an increase in pH, and increased in the presence of other active constituents, such as baicalin, baicalein, and ephedrine . In the case of the Sho-Saiko-To extract, the conversion from GL to glycyrrhetic acid (GA) by beta-glucuronidase originated from E . coli occurred slowly . It was also suppressed by adding baicalin . We determined in vivo the pharmacokinetics of GA after oral administration of Kampo extracts in healthy volunteers . In each Kampo extract, the time of administration had no influence on the mean maximum blood concentration (Cmax) and the area under the blood concentration-time curve (AUC) . Tmax was delayed in the case of the administration after meal (p < 0.05). Yakugaku Zasshi, 1996 Mar, 116(3), 175 - 91 {Polyamine transport in Escherichia coli and eukaryotic cells}; Kashiwagi K; The polyamine content in cells is regulated by both polyamine biosynthesis and its transport . We recently obtained and characterized three clones of polyamine transport genes (pPT104, pPT79 and pPT71) in Escherichia coli . The system encoded by pPT104 was the spermidine-preferential uptake system and that encoded by pPT79 the putrescine-specific uptake system . Furthermore, these two systems were ABC (ATP binding cassette) transporters consisting of four kinds of proteins: pPT104 clone encoded PotA, -B, -C, and -D proteins and pPT79 clone encoded PotF, -G, -H, and I proteins . PotD and -F proteins were periplasmic substrate binding proteins and PotA and -G proteins membrane associated proteins having the nucleotide binding site . PotB and -C proteins, and PotH and -I proteins were transmembrane proteins probably forming channels for spermidine and putrescine, respectively . Their amino acid sequences in the corresponding proteins were similar to each other . The functions of PotA and -D proteins in the spermidine-preferential uptake system encoded by pPT104 clone were studied in detail through a combined biochemical and genetic approach . In contrast, the putrescine transport system encoded by pPT71 consisted of one membrane protein (PotE protein) having twelve transmembrane segments, and was active in both the uptake and excretion of putrescine . The uptake was dependent on the membrane potential, and the excretion was due to the exchange reaction between putrescine and ornithine . In mouse mammary carcinoma FM3A cells, it was shown that the antizyme, which negatively regulates the amount of ornithine decarboxylase, also negatively regulates the activity of polyamine transport. J Eukaryot Microbiol, 1996 Mar-Apr, 43(2), 150 - 8 Chemosensory responses of Acanthamoeba castellanii: visual analysis of random movement and responses to chemical signals; Schuster FL et al.; A visual assay slide chamber was used in conjunction with time-lapse videomicroscopy to analyze chemotactic behavior of axenically grown Acanthamoeba castellanii . Data were collected and analyzed as vector scatter diagrams and cell tracks . Amebas responded to a variety of bacterial products or potential bacterial products by moving actively toward the attractant . Responses to the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP), lipopolysaccharide, and lipid A were statistically significant (P < or = 0.03), as was the response to fMLP benzylamide (P < or = 0.05) . Significant responses to cyclic AMP, lipoteichoic acid, and N-acetyl glucosamine were also found . Chemotactic peptide antagonists, mannose, mannosylated bovine serum albumin, and N-acetyl muramic acid all yielded nonsignificant responses (P > 0.05) . There was no single optimal concentration for response to any of the attractants tested, and amebas responded equally over the range of concentrations tested . Pretreatment of amebas with chemotactic peptides, bacterial products, and bacteria reduced the directional response to attractants . Amebas that had been grown in the presence of bacteria appeared more responsive to chemotactic peptides . Treatment of amebas with trypsin reduced the response of cells to chemotactic peptides, though sensitivity was restored within a couple of hours . This suggests the ameba membrane may have receptors, sensitive to these bacterial substances, which are different from the mannose receptors involved in binding bacteria to the membrane during phagocytosis . The rate of movement was relatively constant (ca . 0.40 microns/s), indicating that the locomotor response to these signals is a taxis, or possibly a klinokinesis, but not an orthokinesis . Studies of the population diffusion rate in the absence of signals indicate that the basic population motility follows the pattern of a Levy walk, rather than the more familiar Gaussian diffusion . This suggests that the usual mathematical models of ameboid dispersion may need to be modified. Plant Mol Biol, 1996 Mar, 30(6), 1169 - 79 Expression of the type 2 metallothionein-like gene MT2 from Arabidopsis thaliana in Zn(2+)-metallothionein-deficient Synechococcus PCC 7942: putative role for MT2 in Zn2+ metabolism; Robinson NJ et al.; Zn2+ proteins pervade metabolism and are essential for gene expression . However, no proteins have been ascribed the central roles of Zn2+ donation to, or removal from, metalloproteins, or Zn2+ storage in vegetative plant tissue . In animals, such functions have been proposed for metallothioneins . Plants contain multiple metallothionein-like genes but their predicted products, which differ significantly from animal metallothioneins, remain to be isolated from vegetative tissue and their roles are uncertain . The type 2 metallothionein-like gene from Arabidopsis, MT2, was expressed under the control of Zn2+-responsive elements derived from the cyanobacterial metallothionein divergon, smt . Zn2+-dependent expression of MT2 transcripts in Synechococcus PCC 7942 was confirmed by northern analysis . The Arabidopsis MT2 gene partly complemented Zn2+ hypersensitivity in mutants of Synechococcus PCC 7942 which are functionally deficient in an endogenous Zn2+-metallothionein gene, smtA . MT2 was also expressed as a recombinant fusion protein in Escherichia coli, purified and shown to bind Zn2+ in vitro . The mean pH of half displacement of Zn2+ from MT2 was estimated to be 5.05 . This suggests that MT2 has a greater affinity for Zn2+ than phytochelatins . The results presented here reveal that MT2 is capable of binding Zn2+ in vitro, conferring tolerance to elevated {Zn2+} in vivo within cyanobacteria and is likely to compete with other polypeptides for cellular Zn2+ in planta. Plant Mol Biol, 1996 Mar, 30(6), 1139 - 51 Molecular cloning, in vitro expression and characterization of a plant squalene synthetase cDNA; Hanley KM et al.; Squalene synthetase (farnesyl-diphosphate:farnesyl-diphosphate farnesyltransferase, EC 2.5.1.21) catalyzes the first committed step for sterol biosynthesis and is thought to play an important role in the regulation of isoprenoid biosynthesis in eukaryotes . Using degenerate oligonucleotides based on a conserved region found in yeast and human squalene synthetase genes, a cDNA was cloned from the plant Nicotiana benthamiana . The cloned cDNA contained an open reading frame of 1234 bp encoding a polypeptide of 411 amino acids (M(r) 47002) . Northern blot analysis of poly(A)+ mRNA from N . benthamiana and N . tabacum cv . MD609 revealed a single band of ca . 1.6 kb in both Nicotiana species . The identity and functionality of the cloned plant squalene synthetase cDNA was further confirmed by expression of the cDNA in Escherichia coli and in a squalene synthetase-deficient erg9 mutant of Saccharomyces cerevisiae . Antibodies raised against a truncated form of the protein recognized an endogenous plant protein of appropriate size as well as the full-length bacterially expressed protein as detected by western analysis . Comparison of the deduced primary amino acid sequences of plant, yeast, rat and human squalene synthetase revealed regions of conservation that may indicate similar functions within each polypeptide. J Membr Biol, 1996 Mar, 150(1), 27 - 35 Characterization of a large conductance, cation-selective channel from sea urchin eggs that is sensitive to sulfhydryl reducing agents; Lii T et al.; Vesicles containing large conductance cation selective channels were isolated from sea urchin (Strongylocentrotus purpuratus) eggs . Addition of the vesicles to one side of lipid bilayer led to the rapid appearance of 200 or more identical channels . These channels would then inactivate within 2 to 10 min . The inactivation could be prevented by the addition of sulfhydryl reducing agents (e.g., dithiothreitol or glutathione) to the cis side of the membrane . Only one channel type is present . The channel is cation selective, with a conductance of 572 ps in symmetrical 0.5 M KCl . The relative cation selectivity is K (1.0) > Cs (0.53) approximately < Na (0.52) > Li (0.2) . The permeability ratio (Px/Pk) is 1.37 (Li) > 1.27 (Na) > 0.57 (Cs) . Most organic cations (choline, tetraethylamine, tetrabutylamine, gallamine, lysine, histidine, arginine, etc.) and multivalent cations (La+3, alkali earth family, Zn+2, Eu+3, etc.) produced a significant channel block . The highest observed affinity was for La+3 which produced a 50% decrease in conductance in 500 mM KCl at a concentration of 8 microM . The biophysical properties of this channel are similar to those of a non-selective channel found in ascidian egg plasma membrane (Dale & DeFelice, 1984) . A soluble extract of the egg supernatant can also prevent the inactivation of the channels . Using deactivated channels reconstituted into a planar lipid bilayer as an assay, this factor was partially purified . It is heat and acetone stable with a molecular weight of between 10 and 20 K . One of the major bands remaining in the purest fraction cross reacted with antibodies raised against E . coli thioredoxin. J Radiat Res (Tokyo), 1996 Mar, 37(1), 29 - 37 Effect of NaN3 on oxygen-dependent lethality of UV-A in Escherichia coli mutants lacking active oxygen-defence and DNA-repair systems; Yamada K et al.; Escherichia coli mutants which lack defence systems against such active oxygen forms as OxyR (delta oxyR), superoxide dismutase (SOD) (sodA and sodB) and catalase (katE and katG) are sensitive to UV-A lethality under aerobic conditions, whereas OxyR- and SOD-mutants have resistance under anaerobic conditions and in the presence of sodium azide (NaN3) during irradiation . UV-A induces lipid peroxidation in the delta oxyR mutant, which is suppressed by NaN3 . These results suggest that UV-A generates 1O2 or the hydroxyl radical to produce lipid peroxides intracellularly in the delta oxyR mutant and that O2- stress may be generated in the sodAB mutant after 8hr of exposure to UV-A . The sensitivities of such DNA repair-deficient mutants as recA(ind-) and uvrA to UV-A also were examined and compared . These mutants are sensitive to UV-A lethality under aerobic conditions but show only slight resistance under anaerobic conditions or in the presence of NaN3 during irradiation . We conclude that NaN3 protects these mutant cells from oxygen-dependent UV-A lethality. East Afr Med J, 1996 Mar, 73(3), 176 - 8 Ten years experience with chronic prostatitis in Africans; Magoha GA; This is a prospective study of seventy three patients with chronic prostatitis over a ten year period (1984-1994 . The study was carried out at various hospitals in Lagos Nigeria and Nairobi Kenya . The mean age was 39.3 years . Chronic bacterial prostatitis was diagnosed in 15 patients (20.5%) of which 11 patients (73.3%) had Escherichia coli as the causative pathogen . Four of these patients (36.4%) were symptom and culture free after 12 weeks therapy with trimethoprimsulfamethoxazole . Four of the other seven patients not responding to trimethoprim (57.1%) became symptom and culture free after four weeks therapy with ciproflaxacin . Non bacterial prostatitis including prostatodynia was diagnosed in 58 patients (79.5%) . Only 15 of these patients (25.8%) reported some subjective relief of symptoms on emperic therapy with doxycycline with complete relapse on discontinuation of therapy . Further therapy with non steroidal anti-inflammatory ibuprofen and anticholinergic oxybutinin chloride proved effective in alleviating symptoms in 40 patients (68.96%), but all relapsed on discontinuation of therapy emphasizing the ineffective and unsatisfactory nature of the present emperic treatment regimens as the cause of non bacterial prostatitis remains unknown. Shock, 1996 Mar, 5(3), 217 - 22 Endotoxin-induced oxidative stress in the rat small intestine: role of nitric oxide; Chamulitrat W et al.; Reactive oxygen species have been implicated in the gastrointestinal pathogenesis of septic and endotoxic shock . The objective of this study was to investigate the role of inducible nitric oxide synthase during endotoxin-induced formation of oxidants by cells of the small intestine . After intravenous Escherichia coli lipopolysaccharide (LPS) (1 mg/kg) injection, nitric oxide production was measured as nitrosyl complex formation in the ileum using electron paramagnetic resonance spectroscopy . Oxidative stress biomarkers were determined as duodenal mucosal-reduced thiols, the ileal lipid peroxidation and luminal free radical production using spin trapping methodology . Demonstration of nitrosyl complex formation commenced at 3 h and diminished 24 h post-LPS . Mucosal thiol levels were decreased at 3, 6, 12, and 18 h post-LPS treatment . At these time point, the ileal lipid peroxidation also increased as did luminal formation of hydroxyl radical adduct . Nitric oxide synthase inhibitors reversed the elevation of hydroxyl radical formation and reversed the decrease in mucosal-reduced thiol levels in the LPS-treated rats . Our data indicate that nitric oxide or its oxidant product(s), such as peroxynitrite, contribute to oxidative injury in the small intestine of rats treated with endotoxin. Shock, 1996 Mar, 5(3), 208 - 12 Release site of TNF alpha after intravenous and intraperitoneal injection of LPS from Escherichia coli in rats; Asari Y et al.; Lipopolysaccharide (LPS) concentrations in the portal vein after intraperitoneal (i.p.) injection were slightly higher than those in the arteries . The tumor necrosis factor (TNF alpha) levels in arterial serum were higher after i.p . injection than after i.v . injection and rose to a peak at 90 min after some delay . Infusion of LPS into the portal vein increased the TNF alpha levels in the arterial serum . Pretreatment with indomethacin further increased the arterial levels of TNF alpha after portal infusion, but did not after them after i.p . injection, because of the reduction by indomethacin of LPS absorption after i.p . Injection of LPS . TNF alpha was also generated in the peritoneal cavity after i.p . injection of LPS . The TNF alpha concentrations in the arterial serum and in the peritoneal cavity were accelerated by mast cell degradation . In conclusion, TNF alpha was generated mainly in the liver, but also in the peritoneal cavity, after i.p . injection of LPS, and was negatively regulated by prostaglandins. Exp Eye Res, 1996 Mar, 62(3), 221 - 9 Purification and characterization of a new enzyme dipeptidase from human lens; Sulochana KN et al.; A new enzyme dipeptidase has been purified to homogeneity from human lens tissue adopting isoelectric focusing, preparative electrophoresis and gel filtration HPLC . The purified enzyme hydrolyses a wide variety of dipeptides containing aliphatic as well as aromatic amino acids but does not act on tripeptides and proteins . The identity of this enzyme as a dipeptidase has been confirmed by the use of dipeptides with modified amino or carboxyl groups . The optimum temperature and pH for this enzyme are 25 +/- 2 degrees C and 5.5 respectively and pI is 6.5 . The Km for different dipeptides varied from 0.04 mM to 4.2 mM . The molecular weight of the native enzyme as determined by gel permeation HPLC is 52 kDa . Preparative electrophoresis, followed by HPLC gave two active proteins, with molecular weights of 52 kDa and 13 kDa . That with the molecular weight of 52 kDa was found to be the tetramer of the other by SDS-PAGE, and peptide mapping of tryptic digests . Properties of this enzyme have been compared with those reported for other proteinases and peptidases of the lens and dipeptidases of Escherichia coli and mouse tumour cells and they render additional support to the finding that this is a new enzyme . The physiological function of this enzyme is also discussed. Bioorg Khim, 1996 Mar, 22(3), 163 - 7 {An effective method of purifying recombinant human tumor necrosis factor alpha}; Tikhonov RV et al.; An efficient and productive isolation method for human recombinant tumor necrosis factor alpha from Escherichia coli cells was developed . The method includes a membrane filtration step, two steps of ion-exchange chromatography, and gel filtration on a Sephadex G-25 column . The target product was obtained with approximately 50% total yield and greater than 95% purity according to PAGE and HPLC. Anticancer Res, 1996 Mar-Apr, 16(2), 693 - 708 Xeroderma pigmentosum and molecular cloning of DNA repair genes; Boulikas T; Human cells from patients suffering with xeroderma pigmentosum (XP) characterized by extreme sensitivity to UV light and a high incidence of skin tumors fall into seven complementation groups, XPA to XPG, and are lacking a functional helicase, endonuclease, or lesion-recognizing protein involved in the initial steps during nucleotide excision repair (NER); a number of proteins involved in DNA repair are termed XPA to XPG depending on which one is defective in a particular complementation group of XP and include: (i) proteins involved in the recognition of (6-4) photoproducts (XPE) and of a broad range of lesions such as pyrimidine dimers (XPA); (ii) proteins that are DNA helicases and integral parts of the general transcription factor TFIIH functioning in both transcription and repair (XPB, XPD); (iii) endonucleases that perform the two incisions, the XPG incising six nucleotides (nt) to the 3' side from a photodimer and the ERCC1-XPF protein complex incising 22 nt to the 5' side of the lesion; and (iv) single-strand DNA-binding proteins (XPC) . The ERCC6 helicase is largely responsible for coupling transcription to repair whereas XPC seems to be responsible for the repair of the inactive parts of the genome as well as for the repair of the nontranscribed strand in active genes . p53 recognizes insertion/deletion mismatches as well as free ends of DNA produced by ionizing radiation to arrest the cell cycle . Most of the human DNA repair proteins have their counterparts in both budding and fission yeasts and some of them also in E . coli evoking an evolutionary conservation of DNA repair pathways . Accumulation of mutations within repair genes in single cells followed by their escape from the immune surveillance and in clonal expansion may greatly contribute to the appearance and development of human cancers. Anticancer Res, 1996 Mar-Apr, 16(2), 651 - 60 Selective induction of mucosal immune responses to 2-acetylaminofluorene; Silbart LK et al.; Mucosal vaccination with chemical carcinogens coupled to enterotoxins such as cholera toxin (CT) can elicit carcinogen-specific immunoglobulin secretion into the intestinal lumen . The present study examines the ability of several related bacterial enterotoxins and their subunits to act as adjuvants or carrier proteins in stimulating an intestinal secretory IgA (S-IgA) response to 2-acetylaminofluorene (AAF) . Using Thiry-Vella loops in rabbits, CT, cholera toxin B subunit (CTB) and the recombinant B subunit of the heat labile enterotoxin from E . coli (rLTB) were all found to be effective carrier proteins and adjuvants for eliciting S-IgA anti-AAF . However, marked differences in the ratio of mucosal S-IgA to serum IgG production were observed . CT elicited the highest luminal S-IgA anti-AAF titers as well as the highest ratio of intestinal S-IgA/serum IgG when used as an adjuvant . Conversely, rLTB elicited a high serum IgG anti-AAF titer but only a modest intestinal S-IgA response . Dialysis studies using monoclonal IgA versus IgG anti-AAF on opposing sides of a semipermeable membrane demonstrated the potential importance of the intestinal S-IgA/serum IgG ratio . A high "intestinal" IgA/"serum" IgG ratio abolished carcinogen transfer to the "serum" side of the membrane, while a low ratio enhanced transfer . Thus, to generate an active mucosal immune response capable of blocking carcinogen absorption, the carrier protein or adjuvant should be selected to optimize the intestinal S-IgA/serum IgG ratio. Res Vet Sci, 1996 Mar, 60(2), 190 - 2 Induction of L-arginine-dependent production of nitric oxide in bovine monocytes by interferon gamma and lipopolysaccharide; Zhao BY et al.; Bovine monocytes freshly isolated from peripheral blood were induced to produce nitric oxide by exposing them to recombinant bovine interferon gamma (rbIFN-y) and Escherichia coli lipopolysaccharide (LPS) in vitro . Moderate amounts of nitric oxide were induced by rbIFN-gamma alone, but larger amounts were induced by rbIFN-gamma and LPS together, the amount being dependent on the quantity of rbIFN-gamma added . Reactive nitrogen intermediates (RNI) were produced within six hours, their concentration peaked at four days and they were detectable for at least eight days after the cells had been stimulated with rbIFN-gamma and LPS . The production of RNI was diminished by the addition of NG-monomethyl-L-arginine . The data suggest that bovine monocytes can produce RNI via a pathway involving an inducible nitric oxide synthase. J Am Anim Hosp Assoc, 1996 Mar-Apr, 32(2), 139 - 45 Bronchopulmonary and disseminated granulomatous disease associated with Aspergillus fumigatus and Candida species infection in a golden retriever; Clercx C et al.; A seven-year-old, female golden retriever was referred for a paroxysmal, chronic cough and dyspnea, dysphagia, facial pruritus, anterior uveitis, and deteriorating general condition . A severe, mixed interstitial and alveolar pattern, with poorly defined amorphous lesions, was seen on thoracic radiographs . Multiple, whitish nodules disseminated on the hyperemic respiratory mucosa were noted on bronchoscopy . Escherichia coli and Aspergillus fumigatus were cultured from the bronchoalveolar lavage . Granulomatous lesions in numerous organs were identified during necropsy, and Aspergillus fumigatus and Candida spp . were cultured from lung and kidney tissues . Microscopic granulomatous lesions were compatible with mycotic infection; however fungal organisms were not observed. Biotechniques, 1996 Mar, 20(3), 492 - 7 Preparation of pure plasmid or cosmid DNA using single-strand affinity matrix and gel-filtration spin columns; Pham TT et al.; A rapid method has been developed for ultrapure plasmid or cosmid DNA isolation from ten-mL to several hundred-mL cultures of Escherichia coli (midi to maxi prep) . A cleared lysate is prepared by alkaline lysis, followed by a quick alcohol precipitation step . Denatured bacterial DNA and RNA having at least 20 nucleotides of single-stranded regions are removed from the supercoiled plasmid by binding strongly to the single-strand affinity matrix (SSAMTM) . Plasmid DNA is then effectively purified on a gel-filtration spin column to remove SSAM, proteins, small RNA and salts . This method produces consistent yields of high-quality plasmids that are suitable for use in many molecular biology applications . In addition, recombinant cosmids of approximately 46 kb can be purified intact, free of chromosomal DNA. Biotechniques, 1996 Mar, 20(3), 460 - 9 Generation of high-titer defective HSV-1 vectors using an IE 2 deletion mutant and quantitative study of expression in cultured cortical cells; Lim F et al.; Vectors based on herpes simplex virus type 1 (HSV-1) show promise for gene transfer into mammalian cells because of their wide host range, efficient infection and ability to deliver genes to nondividing cells . Defective HSV-1 vectors, or amplicons, are plasmid vectors which are unable to propagate on their own but contain specific HSV-1 sequences that, in the presence of helper virus, support DNA replication and subsequent packaging into virus particles . We compared three replication-incompetent HSV-1 mutants (KOS strain 5dl1.2, strain 17 D30EBA, KOS strain d120) as the helper virus for packaging the prototype defective HSV-1 vector, pHSVlac, which uses the HSV-1 immediate-early (1E) 4/5 promoter to regulate expression of the Escherichia coli lacZ gene . Use of 5dl1.2, which contains a deletion in the IE 2 gene, consistently produced virus stocks that contained a high level of vector, undetectable levels of wild-type HSV-1 and a ratio of vector to helper greater than 1 . Virus stocks prepared using 5dl1.2 were superior to those prepared using helper viruses that harbor a deletion in the IE 3 gene, either D30EBA or dl20, and supported more efficient gene transfer than possible with previously published procedures . Lactate dehydrogenase efflux assays in rat cortical cultures showed that 5dl1.2 was no more cytotoxic than either D30EBA or dl20, despite the expression of more viral genes . Rat cortical cultures infected with pHSVlac packaged with either 5dl1.2 or D30EBA were used to quantify the stability of vector expression . Our results show a decrease in the number of cells with detectable levels of beta-galactosidase to 30% of peak levels after one week, irrespective of the helper virus used . However, simultaneous superinfection with 5dl1.2, but not with either D30EBA or dl20, produced a transient increase in the number of cells expressing beta-galactosidase . Superinfection with 5dl1.2 at 9 days after gene transfer increased the number of cells expressing detectable beta-galactosidase back to peak levels, most probably because of reactivation of the IE 4/5 promoter in pHSVlac . These results thus provide the first quantitative demonstration of long-term persistence of defective HSV-1 vectors in neurons. Biotechniques, 1996 Mar, 20(3), 446 - 8, 450-1 Consecutive cycles of precise, unidirectional 14-bp deletions using a BseRI/BsgI trimming plasmid; Ariazi EA et al.; A straightforward method for generating precise, consecutive, unidirectional 14-bp deletions into cloned DNA, adopted from the trimming principle developed by Szybalski and his colleagues, is presented . The method utilizes pTRIM14, a plasmid constructed with the class-IIS restriction enzyme recognition sites for BseRI and BsgI arranged in the form of a cassette, just upstream from the cloned DNA . Class-IIS restriction enzymes cleave DNA downstream of their recognition sites . pTRIM14, containing the cloned DNA, is processed through a trimming cycle that involves sequential restriction digestions with BsgI and then BseRI, followed by treatment with Mung Bean nuclease and then with ligase . One trimming cycle results in a net 14-bp deletion . We demonstrate precise, consecutive deletions at very high efficiency. Biotechniques, 1996 Mar, 20(3), 433 - 8 Simple procedure for creation of in-frame deletion mutations throughout an open reading frame; Ludes-Meyers JH et al.; A general method is presented for randomly mutagenizing open reading frames (ORF) to generate in-frame deletions and insertions . The protocol requires expression of the ORF of interest as a hybrid ORF-beta-galactosidase fusion protein . This allows colorimetric screening for beta-galactosidase activity during subsequent mutagenesis steps . Consequently, proteins with no suitable phenotypic selection or screening properties can be readily screened for mutations that disrupt and subsequently restore the reading frame of the hybrid protein . In addition, this system provides gene expression for subsequent biochemical analysis of the mutant proteins . The bovine papillomavirus type 1 (BPV-1) EI ORF has been mutagenized using this method as an example. Neuroendocrinology, 1996 Mar, 63(3), 219 - 26 Involvement of central histamine in the early phase of ACTH and corticosterone responses to endotoxin in rats; Givalois L et al.; The involvement of histaminergic transmission in the rapid and sustained plasma ACTH and corticosterone (CORT) responses induced in conscious rats by intra-arterial infusions of 25 micrograms.kg-1 Escherichia coli lipopolysaccharide (LPS) was investigated . LPS challenge produced a rapid and transient increase (+ 62%) in the amount of histamine (HA) in the median eminence 15 min after LPS administration, which contrasted with constant concentrations of plasma HA throughout the entire study (up to 480 min) . Blockade of histaminergic receptors by intra-arterial pretreatment with H1 or H2 antagonists (mepyramine, 1 mg/rat, and cimetidine, 2 mg/rat), administered separately, did not affect either ACTH or CORT responses to LPS . Pretreatment with the same doses of the two antagonists in combination very significantly but transiently impaired the earliest phase (30 min) of the ACTH and CORT responses, without any apparent effect on the late phase of these responses . Pretreatment of the animals with an H3-receptor agonist (R alpha-methylhistamine dihydrochloride, 1 mg/rat) similarly blunted the early corticotropic responses to LPS, and also slightly depressed the long-lasting CORT response . These findings support the view that activated central HA transmission may be a key intermediate mechanism triggering the CRH41-ACTH-CORT responses to LPS, in addition to the previously demonstrated activating role of catecholaminergic afferences to the CRH41 neurons during this early complex phase of corticotropic response to LPS. Mol Immunol, 1996 Mar-Apr, 33(4-5), 417 - 26 Common IgE-epitopes of recombinant Phl p I, the major timothy grass pollen allergen and natural group I grass pollen isoallergens; Laffer S et al.; Grass pollen allergens are potent elicitors of Type I allergy . More than 95% of grass pollen allergic patients display IgE-cross-reactivity to group I grass pollen allergens of different grass species . A cDNA coding for the major timothy grass pollen allergen, Phl p I, was isolated previously . To investigate the presence of common IgE-epitopes among naturally occurring group I grass pollen isoallergens, Phl p I was expressed in Escherichia coli and used for IgE-absorption experiments . Recombinant Phl p I was able to inhibit IgE-binding to most of group I isoallergens from seven grass species as identified by two dimensional electrophoresis . When tested in competitive ELISA experiments, recombinant Phl p I bound a high percentage of grass pollen specific IgE . The results indicate that recombinant Phl p I shares many of the IgE-epitopes with natural group I grass pollen allergens and hence may represent a useful tool for specific diagnosis and therapy of grass pollen allergy. Cell Biol Int, 1996 Mar, 20(3), 193 - 203 Distribution of annexin I during non-pathogen or pathogen phagocytosis by confocal imaging and immunogold electron microscopy; Harricane MC et al.; Annexin I is an abundant protein in U937 cells differentiated towards a macrophagic phenotype . These cells become able to kill Escherichia coli, however, the intracellular pathogen Brucella suis, known to interfere with phagosome maturation, multiply in these differentiated cells . We have analysed by confocal and electron microscopy the cellular localization of annexin I during phagocytosis of yeast, non-pathogenic E . coli and the intracellular pathogen B . suis . Using immunocytochemical detections annexin I was found mainly as patches in the cytoplasm of uninfected cells . Upon phagocytosis of yeast or E . coli organisms, annexin I rapidly translocated and concentrated around phagosomes . On the other hand, annexin I was never detected around live B . suis-containing phagosomes . However, when dead brucellae were used, annexin I did translocate to the periphagosomal region . Our results suggest that annexin I could play a role in the molecular mechanism of phagosome maturation, which is impaired by some intracellular pathogens. Biochem J, 1996 Mar 1, 314 ( Pt 2), 695 - 700 The delta- and epsilon-subunits of bovine F1-ATPase interact to form a heterodimeric subcomplex; Orriss GL et al.; The delta-subunit of bovine F1-ATPase was expressed from a bacterial vector at fairly high level in Escherichia coli, but the yield of bovine epsilon-subunit was rather low under similar conditions . However, co-expression of the proteins from a dicistronic operon delta-epsilon in the same expression vector, produced both of them in good yield in a soluble form in the bacterial cytoplasm, and by chromatography it was found that the delta- and epsilon-subunits were associated in a stable complex . The amino groups in the complex were labelled exhaustively by chemical reaction under denaturing conditions with ethyl-{1-14C}acetimidate . The alpha-amino groups of the proteins were unmodified, but complete reaction of all epsilon-amino groups in both proteins was demonstrated by determination of the molecular masses of the modified proteins by electrospray MS . The modified subunits were separated by denaturing gel electrophoresis, and from measurements of the ratio of incorporated radioactivities and the lysine contents of the proteins, it was calculated that the subcomplex contains equimolar amounts of the two proteins . As the apparent molecular mass of the complex determined by gel filtration was 29 kDa, it appears that the complex contains one copy of each protein . It is likely that the delta- and epsilon subunits are associated in a similar manner in the bovine F1-ATPase complex, and that, like a bacterial homologue of the delta-subunit, they interact with the gamma- and beta-subunits. Biochem J, 1996 Mar 1, 314 ( Pt 2), 463 - 7 Optimized heterologous expression of the human zinc enzyme glyoxalase I; Ridderstrom M et al.; DNA coding for human glyoxalase I was isolated from a HeLa cell cDNA library by means of PCR . The deduced amino acid sequence differs form previously isolated sequences in that a glutamic acid replaces an alanine in position 111 . This variant cDNA may represent the more acidic isoform of glyoxalase I originally identified at the protein level . An expression clone was constructed for high-level production of glyoxalase I in Escherichia coli . For optimal yield of the recombinant protein, silent random mutations were introduced in the cDNA coding region . Antisera against human glyoxalase I were used to select a high-level expression clone . This clone afforded 60 mg of purified enzyme per litre of culture medium . Addition of a zinc salt to the culture medium was essential to obtain an active enzyme and a stoicheiometric metal content . The functional characterization of the recombinant enzyme included determination of kinetic constants for methylglyoxal, phenylglyoxal and p-phenylphenylglyoxal, as well as inhibition studies . The kinetic properties of recombinant glyoxalase I were indistinguishable from those of the enzyme purified from human tissues. Biochem J, 1996 Mar 1, 314 ( Pt 2), 391 - 5 Protein biotinylation in higher plants: characterization of biotin holocarboxylase synthetase activity from pea (Pisum sativum) leaves; Tissot G et al.; Biotin holocarboxylase synthetase was partially purified from pea leaves by a sequence of ammonium sulphate fractionation and DEAE 52-cellulose chromatography . Enzyme activity was assayed using apo-(biotin carboxyl carrier protein) from an Escherichia coli bir A mutant affected in biotin holocarboxylase synthetase activity . Conditions for optimal catalytic activity and biochemical parameters of the plant enzyme were determined . This is the first direct evidence of the existence of biotin holocarboxylase synthetase activity in plants. J Membr Biol, 1996 Mar, 150(2), 127 - 32 Ion channels formed by NB, an influenza B virus protein; Sunstrom NA et al.; The influenza B virus protein, NB, was expressed in Escherichia coli, either with a C-terminal polyhistidine tag or with NB fused to the C-terminus of glutathione S-transferase (GST), and purified by affinity chromatography . NB produced ion channel activity when added to artificial lipid bilayers separating NaCl solutions with unequal concentrations (150-500 mM cis, 50 mM trans) . An antibody to a peptide mimicking the 25 residues at the C-terminal end of NB, and amantadine at high concentration (2-3 mM), both depressed ion channel activity . Ion channels had a variable conductance, the lowest conductance observed being approximately 10 picosiemens . At a pH of 5.5 to 6.5, currents reversed at positive potentials indicating that the channel was more permeable to sodium than to chloride ions (PNa/PCl approximately 9) . In asymmetrical NaCl solutions at a pH of 2.5, currents reversed closer to the chloride than to the sodium equilibrium potential indicating that the channel had become more permeable to chloride than to sodium ions (PCl/PNa approximately 4) . It was concluded that, at normal pHs, NB forms cation-selective channels. J Membr Biol, 1996 Mar, 150(2), 143 - 52 Effect of mutation of potassium-efflux system, KefA, on mechanosensitive channels in the cytoplasmic membrane of Escherichia coli; Cui C et al.; The effect of a kefA mutation on the mechanosensitive channels in the cytoplasmic membrane of Escherichia coli was established by introducing a mutation of the kefA gene into wild-type E . coli by P1 transduction . The mutation of the kefA gene not only made the cells sensitive to K+ in the medium but also changed the mechanosensitive channel activity . The kefA mutation did not change the conductances of the two mechanosensitive channels in the cytoplasmic membrane of E . coli, but it prolonged the channel open time . Also, the kefA mutation made the cells more sensitive to pressure in comparison to wild-type cells . The high sensitivity to pressure of the kefA mutant was not modulated by betaine or by the potassium gradient across the membrane . The effect of the kefA mutation on mechanosensitive channels was not due to a membrane fluidity change . KefA might be a regulator for mechanosensitive channels. J Surg Res, 1996 Mar, 61(2), 496 - 502 Impairment of the brain beta-adrenergic system during experimental endotoxemia; Kadoi Y et al.; Brain dysfunction is observed clinically in patients suffering from prolonged endotoxic shock . However, the etiology of brain dysfunction during sepsis is not clear . Certain researchers have reported that the decrease in brain catecholamines concentration during septic shock might be etiologically important in brain dysfunction . Therefore, we hypothesized that the beta-adrenergic receptor system undergoes a change during septic shock, and plays a role in the pathogenesis of septic encephalopathy . In this study, we examined two models of septic shock in rats, each of which has a different time course for the shock state . Male Wistar rats were divided into four groups: (1) Control--0.9% saline vehicle, (2) Lipopolysaccharide (LPS) i.v.-- Escherichia coli endotoxin 1.0 mg/ml i.v . bolus, (3) Sham-operated, and (4) Cecal ligation and puncture (CLP) model . The rats were killed by decapitation at 3, 12, or 24 hr after the treatments, and the brains were removed and subdivided into three areas: the forebrain, cerebellum, and brain stem . In the LPS i.v . group, the brain tissue norepinephrine (NE) concentration had decreased in the forebrain and brain stem and the tissue epinephrine (E) concentration had decreased in the brain stem by 3 hr after treatment . In the CLP group, the brain tissue NE concentration had decreased in the forebrain, cerebellum, and brain stem (P < 0.05), and the tissue E concentration had decreased in the forebrain and brain stem by 24 hr after treatment (P < 0.05) . An alteration in beta-adrenergic receptor density in the forebrain was observed at 24 hr in the CLP group (control, 237.0 +/- 14.0 fmole/mg protein; LPS i.v., 233.2 +/- 3.0 fmole/mg protein; sham-operated, 236.0 +/- 3.0 fmole/mg protein; CLP, 177.0 +/- 4.2 fmole/mg protein) . These alterations in transmitter concentrations and beta-adrenergic density in the forebrain may be an important factor in septic encephalopathy. J Surg Res, 1996 Mar, 61(2), 311 - 6 A new approach to the treatment of experimental septic shock; Hardaway RM et al.; Previous work has shown that disseminated intravascular coagulation (DIC) may produce multiple organ failure, including adult respiratory distress syndrome, by obstruction of visceral micro circulation by microclots DIC can be produced by sepsis . This study tests the ability of a plasminogen activator to prevent death from an intravenous injection of killed Escherichia coli by causing lysis of the microclots . Subjects were two groups of 8 pigs each with body weight of 60-70 lbs . Killed Escherichia coli were injected IV in 16 pigs . Invasive monitoring was used to record physiologic data during the 5.0-hr experimental period . Urokinase injected 20 min after the injection of Escherichia coli organisms significantly prevented mortality, acidosis, and development of blood incoagulability . We conclude that plasminogen activator can significantly prevent fatal Escherichia coli (septic) shock without causing bleeding. J Ind Microbiol, 1996 Mar, 16(3), 197 - 203 Purification and characterization of recombinant Streptomyces clavuligerus isopenicillin N synthase produced in Escherichia coli; Durairaj M et al.; Recombinant isopenicillin N synthase from Streptomyces clavuligerus was produced in the form of inactive inclusion bodies in Escherichia coli . These inclusion bodies were solubilized by treatment with 5 M urea under reducing conditions . Optimization of refolding conditions to recover active isopenicillin N synthase indicated that a dialysis procedure carried out at a protein concentration of about 1.0 mg ml(-1) gave maximal recovery of active isopenicillin N synthase . Solubilized isopenicillin N synthase of more than 95% purity was obtained by passing this material through a DEAE-Trisacryl ion exchange column . Expression studies conducted at different temperatures indicated that isopenicillin N synthase was produced predominantly in a soluble, active form when expression was conducted at 20 degrees C, and accounted for about 20% of the total soluble protein . This high-level production facilitated the purification of soluble isopenicillin N synthase to near homogeneity in four steps . Characterization of the purified soluble and solubilized isopenicillin N synthase revealed that they are very similar. FASEB J, 1996 Mar, 10(4), 461 - 70 The Leloir pathway: a mechanistic imperative for three enzymes to change the stereochemical configuration of a single carbon in galactose; Frey PA; The biological interconversion of galactose and glucose takes place only by way of the Leloir pathway and requires the three enzymes galactokinase, galactose-1-P uridylyltransferase, and UDP-galactose 4-epimerase . The only biological importance of these enzymes appears to be to provide for the interconversion of galactosyl and glucosyl groups . Galactose mutarotase also participates by producing the galactokinase substrate alpha-D-galactose from its beta-anomer . The galacto/gluco configurational change takes place at the level of the nucleotide sugar by an oxidation/reduction mechanism in the active site of the epimerase NAD+ complex . The nucleotide portion of UDP-galactose and UDP-glucose participates in the epimerization process in two ways: 1) by serving as a binding anchor that allows epimerization to take place at glycosyl-C-4 through weak binding of the sugar, and 2) by inducing a conformational change in the epimerase that destabilizes NAD+ and increases its reactivity toward substrates . Reversible hydride transfer is thereby facilitated between NAD+ and carbon-4 of the weakly bound sugars . The structure of the enzyme reveals many details of the binding of NAD+ and inhibitors at the active site . The essential roles of the kinase and transferase are to attach the UDP group to galactose, allowing for its participation in catalysis by the epimerase . The transferase is a Zn/Fe metalloprotein, in which the metal ions stabilize the structure rather than participating in catalysis . The structure is interesting in that it consists of single beta-sheet with 13 antiparallel strands and 1 parallel strand connected by 6 helices . The mechanism of UMP attachment at the active site of the transferase is a double displacement, with the participation of a covalent UMP-His 166-enzyme intermediate in the Escherichia coli enzyme . The evolution of this mechanism appears to have been guided by the principle of economy in the evolution of binding sites. Leukemia, 1996 Mar, 10(3), 439 - 46 Mutations in the gene for human dihydrofolate reductase: an unlikely cause of clinical relapse in pediatric leukemia after therapy with methotrexate; Spencer HT et al.; Resistance to methotrexate (MTX) in some sublines of mammalian cells is reported to be due to one of the following amino acid substitutions in dihydrofolate reductase (DHFR) that lower inhibition by MTX: Gly15 to Trp, Leu22 to Arg or Phe or Phe31 to Trp or Ser . We have produced variants of human DHFR (hDHFR) with these substitutions by directed mutagenesis . Recombinant hDHFR variants expressed in Escherichia coli have greatly decreased inhibition by MTX, but decreased catalytic efficiency, and in one case decreased stability . When a retroviral vector encoding wild-type (wt) hDHFR or one of these variants was introduced into murine fibroblasts or bone marrow progenitors, modest protection from MTX was conferred, even by wt . Relapsed pediatric patients with acute lymphoblastic leukemia who have received multiple courses of high-dose MTX seem most likely to develop such MTX resistance . cDNA was reverse transcribed from blast mRNA from 17 of these patients . However, upon amplification and sequencing of DHFR cDNA, no resistance mutation was found . The explanation for this probably lies in the need for considerable gene amplification to offset lowered catalytic efficiency, and the need for two-base changes for most substitutions, both of which are probably infrequent events. J Exp Med, 1996 Mar 1, 183(3), 1037 - 44 Role of the ceramide-signaling pathway in cytokine responses to P-fimbriated Escherichia coli; Hedlund M et al.; Escherichia coli express fimbriae-associated adhesins through which they attach to mucosal cells and activate a cytokine response . The receptors for E . coli P fimbriae are the globoseries of glycosphingolipids; Gal alpha 1-->4Gal beta-containing oligosaccharides bound to ceramide in the outer leaflet of the lipid bilayer . The receptors for type 1 fimbriae are mannosylated glycoproteins rather than glycolipids . This study tested the hypothesis that P-fimbriated E . coli elicit a cytokine response through the release of ceramide in the receptor-bearing cell . We used the A498 human kidney cell line, which expressed functional receptors for P and type 1 fimbriae and secreted higher levels of interleukin (IL)-6 when exposed to the fimbriated strains than to isogenic nonfimbriated controls . P-fimbriated E . coli caused the release of ceramide and increased the phosphorylation of ceramide to ceramide 1-phosphate . The IL-6 response to P-fimbriated E . coli was reduced by inhibitors of serine/threonine kinases but not by other protein kinase inhibitors . In contrast, ceramide levels were not influenced by type 1-fimbriated E . coli, and the IL-6 response was insensitive to the serine/threonine kinase inhibitors . These results demonstrate that the ceramide-signaling pathway is activated by P-fimbriated E . coli, and that the receptor specificity of the P fimbriae influences this process . We propose that this activation pathway contributes to the cytokine induction by P-fimbriated E . coli in epithelial cells. Infect Immun, 1996 Mar, 64(3), 974 - 9 Induction of antigen-specific antibodies in vaginal secretions by using a nontoxic mutant of heat-labile enterotoxin as a mucosal adjuvant; Di Tommaso A et al.; Immunization of the female reproductive tract is important for protection against sexually transmitted diseases and other pathogens of the reproductive tract . However, intravaginal immunization with soluble antigens generally does not induce high levels of secretory immunoglobulin A (IgA) . We recently developed safe mucosal adjuvants by genetically detoxifying Escherichia coli heat-labile enterotoxin, a molecule with a strong mucosal adjuvant activity, and here we describe the use of the nontoxic mutant LTK63 to induce a response in the mouse vagina against ovalbumin (Ova) . We compared intravaginal and intranasal routes of immunization for induction of systemic and vaginal responses against LTK63 and Ova . We found that LTK63 is a potent mucosal immunogen when given by either the intravaginal or intranasal route . It induces a strong systemic antibody response and IgG and long-lasting IgA in the vagina . The appearance of vaginal IgA is delayed in the intranasally immunized mice, but the levels of vaginal anti-LTK63 IgA after repeated immunizations are higher in the intranasally immunized mice than in the intravaginally immunized mice . LTK63 also acts as a mucosal adjuvant, inducing a serum response against Ova, when given by both the intravaginal and intranasal routes . However, vaginal IgA against Ova is stimulated more efficiently when LTK63 and antigen are given intranasally . In conclusion, our results demonstrate that LTK63 can be used as a mucosal adjuvant to induce antigen-specific antibodies in vaginal secretions and show that the intranasal route of immunization is the most effective for this purpose. Infect Immun, 1996 Mar, 64(3), 842 - 8 A Legionella pneumophila gene that promotes hemin binding; O'Connell WA et al.; The ability to bind and utilize hemin is a trait common to many human pathogens . Nevertheless, the relationship between Legionella pneumophila, the agent of Legionnaires' disease, and hemin has received little attention . Thus, we explored the capacity of a virulent, serogroup 1 strain of L . pneumophila to bind hemin and use it as an iron source . Hemin, but not protoporphyrin IX, restored bacterial growth in iron-limiting media, indicating that it can serve as an iron source for L . pneumophila . In support of this idea, we observed that wildtype legionellae were able to bind 50 to 60% of added hemin, a binding capacity that was comparable to those of other pathogens . To begin to identify proteins involved in hemin acquisition, we identified a Legionella locus that conferred hemin binding upon Escherichia coli . Subcloning and nucleotide sequence analysis determined that a single open reading frame, which was designated hbp for hemin-binding promotion, was responsible for this binding activity . The hbp gene was predicted to encode a secreted, 15.5-kDa protein . To ascertain the importance of this gene in L . pneumophila biology, we used allelic exchange to construct an hbp mutant . Importantly, the mutant displayed a 42% reduction in hemin binding, confirming that hbp potentiates hemin acquisition by L . pneumophila . However, the strain was unaltered in its ability to grow within macrophage-like cells and freshwater amoebae, indicating that hbp is not required for intracellular infection . Despite this, Southern hybridization analysis and database searches demonstrated that hbp is nearly exclusive to the L . pneumophila species. Infect Immun, 1996 Mar, 64(3), 810 - 7 Characterization of two conformational epitopes of the Chlamydia trachomatis serovar L2 DnaK immunogen; Birkelund S et al.; Chlamydia trachomatis DnaK is an important immunogen in chlamydial infections . DnaK is composed of a conserved N-terminal ATP-binding domain and a variable C-terminal peptide-binding domain . To locate the immunogenic part of C . trachomatis Dnak, we generated monoclonal antibodies (MAbs) against this protein . By use of recombinant DNA techniques, we located the epitopes for two MAbs in the C-terminal variable part . Although the antibodies reacted in an immunoblot assay, it was not possible to map the epitopes completely by use of 16-mer synthetic peptides displaced by one amino acid corresponding to the C-terminal part of C . trachomatis DnaK . To determine the limits of the epitopes, C . trachomatis DnaK and glutatione S-transferase fusion proteins were constructed and affinity purified . The purified DnaK fusion proteins were used for a fluid-phase inhibition enzyme-linked immunosorbent assay with the two antibodies . The epitopes were found not to overlap . To obtain DnaK fragments recognized by the antibodies with the same affinity as native C . trachomatis DnaK, it was necessary to express, respectively, regions of 127 and 77 amino acids . The MAbs described in this study thus recognized conformational epitopes of C . trachomatis DnaK. Infect Immun, 1996 Mar, 64(3), 775 - 81 Enhanced protection by use of a combination of anticapsule and antilipopolysaccharide monoclonal antibodies against lethal Escherichia coli O18K5 infection of mice; Frasa H et al.; To study antibody-mediated protection against Escherichia coli peritonitis in BALB/c mice, monoclonal antibodies (MAbs) were generated against the capsule (K5) and the lipopolysaccharide (O18) of E . coli . Flow cytometric analysis with two selected immunoglobulin M MAbs revealed that bacteria were antigenically heterogeneous . Arbitrarily, three subpopulations in E . coli O18K5 cultures could be distinguished by double immunofluorescence . A subpopulation bound only the anti-K5 MAb, and another subpopulation bound only the anti-O18 MAb . An intermediate subpopulation, however, bound both MAbs . In agreement with this result, combinations of both MAbs enhanced phagocytosis of fluorescein isothiocyanate-labeled bacteria by human polymorphonuclear leukocytes and mouse macrophage J774 cells as well . In protection experiments, combinations of both MAbs, preincubated with 3 50% lethal doses of E . coli O18K5, protected all mice upon intraperitoneal challenge . Relatively high doses of either MAb alone proved to be not fully protective in this infection model . Protection of mice by the combination of MAbs was associated with significantly lower (P < 0.02) tumor necrosis factor levels in serum 90 min after challenge compared with any other treatment group . Similarly, prophylactic administration of MAbs yielded significantly lower (P < 0.01) tumor necrosis factor levels in mice that received the combination of MAbs than in any other treatment group. Infect Immun, 1996 Mar, 64(3), 1075 - 7 Glycans of bovine lactoferrin function as receptors for the type 1 fimbrial lectin of Escherichia coli; Teraguchi S et al.; Bovine lactoferrin strongly inhibited the hemagglutination activity of type 1 fimbriated Escherichia coli . In addition, it agglutinated these bacteria . The agglutination reaction was specifically inhibited by glycopeptides derived from bovine lactoferrin or alpha-methyl-D-mannoside . These observations indicate that the glycans of bovine lactoferrin can serve as receptors for type 1 fimbrial lectin. Haematologica, 1996 Mar-Apr, 81(2), 127 - 31 Influence of two different Escherichia coli asparaginase preparations on fibrinolytic proteins in childhood ALL; Nowak-Gottl U et al.; BACKGROUND . Alterations in hemostasis have frequently been observed in patients with leukemia, and thrombotic events are well documented in patients receiving L-asparaginase (ASP) as a single agent or in combination with vincristine, prednisone (sometimes complemented by an anthracycline) . The present study was designed to evaluate prospectively fibrinolytic parameters in leukemic children receiving different E . coli ASP preparations (Kyowa ASP, n = 20; Bayer ASP, n = 20), and to relate changes in the fibrinolytic system to serum ASP activity . MATERIALS AND METHODS . Blood samples for coagulation studies were obtained together with serum samples for pharmacokinetic monitoring in the same venipuncture (before the first and 6th-7th doses of ASP) . RESULTS . Patients receiving Kyowa ASP showed significantly (0.0001) enhanced ASP-activity compared to children treated with the Bayer preparation . Significantly decreased values of fibrinogen (p < 0.001), plasminogen (p < 0.0002) and alpha 2-antiplasmin (p < 0.0003) were found in the Kyowa group, along with significantly enhanced thrombin generation (F1 + 2; p < 0.001), t-P (p < 0.01) and D-dimer levels (p < 0.05) . In contrast, PAI 1 activity demonstrated no significant difference in the two E . coli ASP administered . CONCLUSIONS . Changes in fibrinogen, plasminogen, alpha 2-antiplasmin and D-dimer are clearly associated with ASP activity during the course of ASP administration in children with ALL. Cancer Res, 1996 Mar 1, 56(5), 1050 - 5 High-efficiency in vivo gene transfer using intraarterial plasmid DNA injection following in vivo electroporation; Nishi T et al.; A novel method for high-efficiency and region- controlled in vivo gene transfer was developed by combining in vivo electroporation and intraarterial plasmid DNA injection . A mammalian expression plasmid for the Escherichia coli lacZ gene (driven with a SV40 early promoter) was injected into the internal carotid artery of rats whose brain tumors (from prior inoculation) had been electroporated between two electrodes . The lacZ gene was efficiently transferred and expressed in the tumor cell 3 days after plasmid injection . However, neither any gene transfers nor any elevated lacZ activity was detected in tissues outside the electrodes . The plasmid was not transferred without electroporation . Human monocyte chemoattractant protein-1 cDNA was also transferred by this method, and its long-lasting (3 weeks) expression was confirmed by using the Epstein-Barr virus episomal replicon system . The expressed monocyte chemoattractant protein-1 protein was functional, as evident by the presence of a large number of monocytes in tumor tissue . This method, inverted question markelectrogene therapy, inverted question mark which does not require viral genes or particles, allows genes to be transferred and expressed in desired organs or tissues, and it may lead to the development of a new type of highly effective gene therapy. Plant Mol Biol, 1996 Mar, 30(5), 983 - 94 Molecular analysis of two functional homologues of the S3 allele of the Papaver rhoeas self-incompatibility gene isolated from different populations; Walker EA et al.; The S3 allele of the S gene has been cloned from Papaver rhoeas cv . Shirley . The sequence predicts a hydrophilic protein of 14.0 kDa, showing 55.8% identity with the previously cloned S1 allele, preceded by an 18 amino acid signal sequence . Expression of the S3 coding region in Escherichia coli produced a form of the protein, denoted S3e, which specifically inhibited S3 pollen in an in vitro bioassay . The recombinant protein was ca . 0.8 kDa larger than the native stigmatic form, indicating post-translational modifications in planta, as was previously suggested for the S1 protein . In contrast to other S proteins identified to date, S3 protein does not appear to be glycosylated . Of particular significance is the finding that despite exhibiting a high degree of sequence polymorphism, secondary structure predictions indicate that the S1 and S3 proteins may adopt a virtually identical conformation . Sequence analysis also indicates that the S1 and S3 proteins may adopt a virtually identical conformation . Sequence analysis also indicates that the P . rhoeas S alleles share some limited homology with the SLG and SRK genes from Brassica oleracea . Previously, cross-classification of different populations of P . rhoeas had revealed a number of functionally identical alleles . Probing of Western blots of stigma proteins from plants derived from a wild Spanish population which contained an allele functionally identical to the Shirley S3 allele with antiserum raised to S3e, revealed a protein (S3s) which was indistinguishable in pI and Mr from that in the Shirley population . A cDNA encoding S3s was isolated, nucleotide sequencing revealing a coding region with 99.4% homology with the Shirley-derived clone at the DNA level, and 100% homology at the amino acid level. Plant Mol Biol, 1996 Mar, 30(5), 863 - 72 Expression analysis of a sucrose synthase gene from sugar beet (Beta vulgaris L.); Hesse H et al.; To investigate the expression pattern of sucrose synthase, a cDNA from tap roots of sugar beet (Beta vulgaris L.) was isolated using a heterologous sucrose synthase cDNA from potato . The 2762 bp long cDNA clone designated SBSS 1 encodes for a 822 amino acid polypeptide of a predicted molecular mass of 93.7 kDa . The deduced amino acid sequence of sugar beet sucrose synthase has homologies of 65-70% when compared to predicted amino acid sequences of sucrose synthases from other species . RNA blot analysis shows that SBSS1 is expressed most predominant in tap root under normal growth conditions . Cold treatment and anaerobiosis lead to an increase in the steady-state levels of SBSS 1 mRNA in leaf and root tissue . In tap root slices, sugars in various concentrations had no influence on the SBSS 1 transcript level . On the other hand, wounding resulted in a decreased transcript level. Plant Mol Biol, 1996 Mar, 30(5), 1041 - 9 Cloning and characterization of an Arabidopsis thaliana cDNA clone encoding an organellar isoform of serine acetyltransferase; Roberts MA et al.; We have cloned an Arabidopsis thaliana cDNA encoding serine acetyltransferase (EC 2.3.1.30) by functional complementation of the Escherichia coli cysE mutant JM15 . The cDNA clone Sat-1 conferred serine acetyltransferase activity (with apparent Km for the two substrates acetyl CoA and L-serine of 0.043 and 3.47 mmol/dm3 respectively) on the cysE mutant . The 1515 bp full-length cDNA encodes a deduced protein of 391 amino acids which includes a putative chloroplastic targeting presequence . Northern analysis revealed a single message of 1.5 kb, while Southern hybridisation suggests a small multigene family of related sequences. Z Naturforsch {C}, 1996 Mar-Apr, 51(3-4), 233 - 42 A cytotoxic principle of Tamarindus indica, di-n-butyl malate and the structure-activity relationship of its analogues; Kobayashi A et al.; Bioassay-guided fractionation of the methanolic extract of Tamarindus indica fruits led to the isolation of L-(-)-di-n-butyl malate which exhibited a pronounced cytotoxic activity against sea urchin embryo cells . In order to study structure-activity relationships, close-structure relatives of di-n-butyl malate were synthesized using D-(+)- and L-(-)-malic acid as starting materials, and their cytotoxic activities were examined for the sea urchin embryo assay . L-(-)-Di-n-pentyl malate was the most effective inhibitor to the development of the fertilized eggs . Significant inhibitory activity was not seen in the esters of D-(-)-isomer. Am J Physiol, 1996 Mar, 270(3 Pt 1), C778 - 85 Role of nitric oxide and phosphodiesterase isoenzyme II for reduction of endothelial hyperpermeability; Suttorp N et al.; Regulation of endothelial permeability is poorly understood . Previous studies have shown that endothelial cells contain phosphodiesterase (PDE) isoenzymes II-IV and that simultaneous adenylate cyclase activation and/or PDE inhibition blocked endothelial hyperpermeability (J.Clin.Invest . 91: 1421-1428, 1993) . We now focused on a possible role for guanosine 3',5'-cyclic monophosphate (cGMP)-dependent mechanisms and studied H2O2-exposed porcine pulmonary artery endothelial cell monolayers . Pretreatment of cells with different nitric oxide (NO) donors or atrial natriuretic peptide (ANP) increased endothelial cGMP-content severalfold and blocked H2O2-related effects on permeability; opposite results were obtained with a NO synthase inhibitor . Determination of cGMP degradation in nitroprusside-exposed endothelial cells identified PDE II as the major cGMP metabolizing pathway, whereas PDE III and IV contributed little or nothing . Inhibition of PDE II reduced H2O2-related endothelial hyperpermeability, an effect that could be enhanced synergistically by simultaneous guanylate cyclase activation . In summary, these studies indicate that cGMP-dependent mechanisms (NO donors, ANP, and dibutyryl-cGMP) blocked H2O2-related increases in endothelial permeability . The major cGMP degrading pathway in endothelial cells was PDE II, thereby substituting the missing PDE V in these cells . Simultaneous guanylate cyclase activation and/or PDE II inhibition may be a valuable approach to treat endothelial hyperpermeability. Science, 1996 Mar 1, 271(5253), 1247 - 54 Crystal structure of the lactose operon repressor and its complexes with DNA and inducer; Lewis M et al.; The lac operon of Escherichia coli is the paradigm for gene regulation . Its key component is the lac repressor, a product of the lacI gene . The three-dimensional structures of the intact lac repressor, the lac repressor bound to the gratuitous inducer isopropyl-beta-D-1-thiogalactoside (IPTG) and the lac repressor complexed with a 21-base pair symmetric operator DNA have been determined . These three structures show the conformation of the molecule in both the induced and repressed states and provide a framework for understanding a wealth of biochemical and genetic information . The DNA sequence of the lac operon has three lac repressor recognition sites in a stretch of 500 base pairs . The crystallographic structure of the complex with DNA suggests that the tetrameric repressor functions synergistically with catabolite gene activator protein (CAP) and participates in the quaternary formation of repression loops in which one tetrameric repressor interacts simultaneously with two sites on the genomic DNA. DNA Cell Biol, 1996 Mar, 15(3), 255 - 62 Quantification of 3'OH DNA breaks by random oligonucleotide-primed synthesis (ROPS) assay; Basnakian AG et al.; A simple and precise assay is presented for quantification of the relative number of 3'OH ends (breaks) present in DNA molecules . The assay is based on the ability of the Klenow fragment polymerase to initiate random oligonucleotide-primed synthesis from the reannealed 3'OH ends of single-stranded (ss) DNA . After a denaturation-reassociation step, the ssDNA serves as its own primer by randomly reassociating itself or to other ssDNA molecules . Under strictly defined reaction conditions (time, temperature, concentration of precursors) the incorporation of {32P}dNTP into newly synthesized DNA will be proportional to the initial number of 3'OH ends (breaks) . The assay is specific for the detection of 3'OH ends and requires only 0.25 micrograms of DNA for analysis . It has application for the detection of the relative number of breaks per DNA molecule generated in vitro by endonucleases or in vivo during normal processes of DNA repair and also for the detection of DNA strand breaks from genotoxic DNA damaging agents . Although specific for 3'OH DNA ends, the assay can be adapted to measure 3'P (5'OH) DNA ends or breaks induced by oxidative DNA damaging agents by pretreatment of the DNA with alkaline phosphatase or Escherichia coli exonuclease III . The assay is capable of quantifying first several breaks per 10(5) bp. Am J Obstet Gynecol, 1996 Mar, 174(3), 983 - 9 Gestational pyelonephritis--associated Escherichia coli isolates represent a nonrandom, closely related population; Hart A et al.; OBJECTIVE: A select group of Escherichia coli strains known as uropathogenic cause pyelonephritis in nonpregnant individuals . We investigated whether Escherichia coli from gestational pyelonephritis represent a random population or possess common uropathogenic characteristics . STUDY DESIGN: Repetitive element sequence-based polymerase chain reaction, plasmid profiles, hemolysin, and O serotypes were assayed from Escherichia coli isolates of 57 pregnant patients with acute pyelonephritis at different gestational ages . RESULTS: The majority of the first trimester isolates fell primarily into repetitive element sequence-based patterns 1 and 3 and O6, O15, and O75 serotypes . Second-trimester isolates had multiple patterns with high-frequency repetitive element sequence-based polymerase chain reaction 1 and 5 and an unknown (OX) serotype . Pattern 3, predominantly O75 serotype, was found primarily among third-trimester isolates . CONCLUSION: It is likely that Escherichia coli associated with acute pyelonephritis during different trimesters of pregnancy represents nonrandom closely related isolates, and some of these strains may be characteristic in pregnant patients only. J Bacteriol, 1996 Mar, 178(5), 1465 - 8 Induction of RpoS-dependent functions in glucose-limited continuous culture: what level of nutrient limitation induces the stationary phase of Escherichia coli? Notley L, Ferenci T. treA and osmY expression and RpoS protein levels were investigated in glucose-limited continuous culture . The level of induction of these stationary-phase markers became as high during growth at a D of 0.1 to 0.2 h(-1) as in carbon-starved batch cultures but only in rpoS+ bacteria . The stress protectant trehalose was actually produced at higher levels at low growth rates than in stationary-phase cultures . The pattern of induction of RpoS-dependent activities could be separated from those regulated by cyclic AMP (cAMP) or endoinduction, and the induction occurred at extreme glucose limitation . Escherichia coli turns to a protective stationary-phase response when nutrient levels fall below approximately 10(-7) M glucose, which is insufficient to saturate scavenger transporters regulated by cAMP plus endoinducers, and this response is optimally expressed at 10(-6) M glucose . The high-level induction of protective functions also explains the maintenance energy requirement of bacterial growth at low dilution rates. J Bacteriol, 1996 Mar, 178(5), 1420 - 9 Plasmid RK2 toxin protein ParE: purification and interaction with the ParD antitoxin protein; Johnson EP et al.; The parDE operon, located within the 3.2-kb stabilization region of plasmid RK2, encodes antitoxin (ParD) and toxin (ParE) proteins that stabilize the maintenance of this broad-host-range plasmid via a postsegregational killing mechanism . A ParE protein derivative, designated ParE', was purified by construction of a fusion protein, GST-ParE, followed by glutathione-agarose binding and cleavage of the fusion protein . ParE' has three additional amino acids on the N terminus and a methionine residue in place of the native leucine residue . The results of glutathione-agarose affinity binding and glutaraldehyde cross-linking indicate that ParE' exists as a dimer in solution and that it binds to the dimeric form of ParD to form a tetrameric complex . The formation of this complex is presumably responsible for the ability of ParD to neutralize ParE toxin activity . Previous studies demonstrated that the parDE operon is autoregulated as a result of the binding of the ParD protein to the parDE promoter . ParE' also binds to the parDE promoter but only in the presence of the autoregulatory ParD protein . ParE', in the presence or absence of the ParD protein, does not bind to any other part of the 3.2-kb stabilization region . The binding of the ParE' protein to ParD did not alter the DNase I footprint pattern obtained as a result of ParD binding to the parDE promoter . The role of ParE in binding along with ParD to the promoter, if any, remains unclear. J Bacteriol, 1996 Mar, 178(5), 1394 - 400 Inorganic polyphosphate supports resistance and survival of stationary-phase Escherichia coli; Rao NN et al.; The Escherichia coli mutant (ppk) lacking the enzyme polyphosphate kinase, which makes long chains of inorganic polyphosphate (poly P), is deficient in functions expressed in the stationary phase of growth . After 2 days of growth in a medium limited in carbon sources, only 7% of the mutants survived compared with nearly 100% of the wild type; the loss in viability of the mutant was even more pronounced in a rich medium . The mutant showed a greater sensitivity to heat, to an oxidant (H2O2), to a redox-cycling agent (menadione), and to an osmotic challenge with 2.5 M NaCl . After a week or so in the stationary phase, mutant survivors were far fewer in number and were replaced by an outgrowth of a small-colony-size variant with a stable genotype and with improved viability and resistance to heat and H2O2; neither polyphosphate kinase nor long-chain poly P was restored . Suppression of the ppk feature of heat sensitivity by extra copies of rpoS, the gene encoding the RNA polymerase sigma factor that regulates some 50 stationary-phase genes, further implicates poly P in promoting survival in the stationary phase. J Bacteriol, 1996 Mar, 178(5), 1347 - 50 Kinetics of pyrimidine(6-4)pyrimidone photoproduct repair in Escherichia coli; Koehler DR et al.; We compared the removal of pyrimidine(6-4)pyrimidone photoproducts {(6-4) photoproducts} and cyclobutane pyrimidine dimers (CPDs) from the genome of repair-proficient Escherichia coli, using monoclonal antibodies specific for each type of lesion . We found that (6-4) photoproducts were removed at a higher rate than CPDs in the first 30 min following a moderate UV dose (40 J/m2) . The difference in rates was less than that typically reported for cultured mammalian cells, in which the removal of (6-4) photoproducts is far more rapid than that of CPDs. J Bacteriol, 1996 Mar, 178(5), 1328 - 34 Topological characterization of the essential Escherichia coli cell division protein FtsN; Dai K et al.; Genetic and biochemical approaches were used to analyze a topological model for FtsN, a 36-kDa protein with a putative transmembrane segment near the N terminus, and to ascertain the requirements of the putative cytoplasmic and membrane-spanning domains for the function of this protein . Analysis of FtsN-PhoA fusions revealed that the putative transmembrane segment of FtsN could act as a translocation signal . Protease accessibility studies of FtsN in spheroblasts and inverted membrane vesicles confirmed that FtsN had a simple bitopic topology with a short cytoplasmic amino terminus, a single membrane-spanning domain, and a large periplasmic carboxy terminus . To ascertain the functional requirements of the N-terminal segments of FtsN, various constructs were made . Deletion of the N-terminal cytoplasmic and membrane-spanning domains led to intracellular localization of the carboxy domain, instability,and loss of function . Replacement of the N-terminal cytoplasmic and membrane-spanning domains with a membrane-spanning domain from MalG restored subcellular localization and function . These N-terminal domains of FtsN could also be replaced by the cleavable MalE signal sequence with restoration of subcellular localization and function . It is concluded that the N-terminal, cytoplasmic, and transmembrane domains of FtsN are not required for function of the carboxy domain other than to transport it to the periplasm . FtsQ and FtsI were also analyzed. J Bacteriol, 1996 Mar, 178(5), 1248 - 57 Identification and characterization of the caiF gene encoding a potential transcriptional activator of carnitine metabolism in Escherichia coli; Eichler K et al.; Expression of the Escherichia coli caiTABCDE and fixABCX operons involved in carnitine metabolism is induced by both carnitine and anaerobiosis . When cloned into a multicopy plasmid, the 3' region adjacent to the caiTABCDE operon was found to increase levels of carnitine dehydratase activity synthesized from the chromosomal caiB gene . The nucleotide sequence was determined, and it was shown to contain an open reading frame of 393 bp named caiF which is transcribed in the direction opposite that of the cai operon . This open reading frame encodes a protein of 131 amino acids with a predicted molecular mass of 15,438 Da which does not have any significant homology with proteins available in data libraries . In vivo overexpression consistently led to the synthesis of a 16-kDa protein . The caiF gene was transcribed as a monocistronic mRNA under anaerobiosis independently of the presence of carnitine . Primer extension analysis located the start site of transcription to position 82 upstream of the caiF initiation codon . It was preceded by a cyclic AMP receptor protein motif centered at position -41.5 . Overproduction of CaiF resulted in the stimulation of transcription of the divergent cai and fix operons in the presence of carnitine . This suggested that CaiF by interacting with carnitine plays the role of an activator, thereby mediating induction of carnitine metabolism . Moreover, CaiF could complement in trans the regulatory defect of laboratory strain MC4100 impaired in the carnitine pathway . Expression of a caiF-lacZ operon fusion was subject to FNR regulator-mediated anaerobic induction and cyclic AMP receptor protein activation . The histone-like protein H-NS and the NarL (plus nitrate) regulator acted as repressors . Because of the multiple controls to which the caiF gene is subjected, it appears to be a key element in the regulation of carnitine metabolism. J Bacteriol, 1996 Mar, 178(5), 1242 - 7 Rifampin-induced initiation of chromosome replication in dnaR-deficient Escherichia coli cells; Sakakibara Y; The dnaR130 mutant of Escherichia coli, which was thermosensitive in initiation of chromosome replication, was capable of thermoresistant DNA synthesis in the presence of rifampin at a low concentration that allowed almost normal RNA synthesis . The DNA synthesis in the presence of the drug depended on protein synthesis at the high temperature . The protein synthesis in the dnaR-deficient cells provided a potential for thermoresistant DNA synthesis to be induced at a high dose of the drug that almost completely prevented RNA synthesis . The induced synthesis was synchronously initiated from oriC and proceeded semiconservatively toward terC . The replication depended on the dnaA function, which was essential for normal initiation of replication from oriC . The capability for drug-induced replication was abolished by certain rifampin resistance mutations in the beta subunit of RNA polymerase . Thus, the drug can induce the dnaA-dependent initiation of replication in the dnaR-deficient cells through its effect on RNA polymerase . This result implies that the dnaR product is involved in the transcription obligatory for the initiation of replication of the bacterial chromosome. Development, 1996 Mar, 122(3), 795 - 804 Polyembryonic development: insect pattern formation in a cellularized environment; Grbic M et al.; THe polyembryonic wasp Copidosoma floridanum produces up to 2000 individuals from a single egg . During the production of individual embryos the original anteroposterior axis of the egg is lost and axial patterning must subsequently be reestablished within each embryo . The mechanism by which this occurs is unknown . In most insects, egg polarity is established during oogenesis and early development takes place in a syncytium . In Drosophila melanogaster, the syncytium is considered essential for establishing the morphogenetic gradients that initiate segmental patterning . However, we found that development of C . floridanum occurs almost exclusively in a cellularized environment . To determine whether the D . melanogaster patterning cascade is conserved in the absence of a syncytium, we analyzed the expression of Even-skipped, Engrailed and Ultrabithorax/Abdominal-A during polyembryonic development . Here we show that in spite of the absence of a syncytium, the elements of the D . melanogaster segmentation hierarchy are conserved . The segment-polarity gene Engrailed and the homeotic genes Ultrabithorax/Abdominal-A are expressed in a conserved pattern relative to D . melanogaster . However, we detect an alteration in the expression of the Even-skipped antigen . Even-skipped is initially expressed in segmentally reiterated stripes and not in the pair-rule pattern as it is in D . melanogaster . We also observe that the expression of these regulatory proteins does not occur during the early proliferative phases of polyembryony . Our results indicate that a syncytium is not required for segmental patterning in this insect. J Virol, 1996 Mar, 70(3), 2008 - 13 Purification and characterization of herpes simplex virus type 1 alkaline exonuclease expressed in Escherichia coli; Bronstein JC et al.; The alkaline exonuclease (AE) encoded by the herpes simplex virus type 1 (HSV-1) UL12 open reading frame was inducibly expressed in Escherichia coli and purified without the use of chromatographic separation . This recombinant AE was found to exhibit the same biochemical properties as the virus-encoded protein and was used to confirm the existence of a weak endonucleolytic activity in the enzyme . Antisera raised against the recombinant protein recognized several forms of the AE in HSV-1-infected cells . This expression and purification strategy will provide an economical and easily accessible alternative source of HSV-1 AE for future in vitro studies. J Virol, 1996 Mar, 70(3), 1493 - 504 Comparing transcriptional activation and autostimulation by ZEBRA and ZEBRA/c-Fos chimeras; Kolman JL et al.; The lytic cycle of Epstein-Barr virus (EBV) can be activated by transfection of the gene for ZEBRA, a viral basic-zipper (bZip) transcriptional activator . ZEBRA and cellular AP-1 bZip activators, such as c-Fos, have homologous DNA-binding domains, and their DNA-binding specificities overlap . Moreover, EBV latency can also be disrupted by phorbol esters, which act, in part, through AP-1 activators . It is not known whether ZEBRA and AP-1 factors play equivalent roles in the initial stages of reactivation . Here the contribution of ZEBRA's basic DNA recognition domain to disruption of latency was analyzed by comparing ZEBRA with chimeric mutants in which the DNA recognition domain of ZEBRA was replaced with the analogous domain of c-Fos . Chimeric ZEBRA/c-Fos proteins overexpressed in Escherichia coli bound DNA with the specificity of c-Fos; they bound a heptamer AP-1 site and an octamer TPA response element (TRE) . ZEBRA bound the AP-1 site and an array of ZEBRA response elements (ZREs) . In assays with reporter genes, both ZEBRA and ZEBRA/c-Fos chimeric mutants activated transcription from Zp, a promoter of the ZEBRA gene (BZLF1) that contains the TRE and multiple ZREs . However, despite their capacity to activate reporters bearing Zp, neither ZEBRA nor the c-Fos chimeras activated transcription from Zp in the context of the intact latent viral genome . In contrast, ZEBRA but not ZEBRA/c-Fos chimeras activated Rp, a second viral promoter that controls ZEBRA expression . Hence, transcriptional autostimulation by transfected ZEBRA occurred preferentially at Rp . Both ZEBRA and the ZEBRA/c-Fos chimeras activated transcription from reporters with multimerized AP-1 sites . However, in the context of the virus, only ZEBRA activated the promoters of two early lytic cycle genes, BMRF1 and BMLF1, that contain an AP-1 site . Thus, overexpression of an activator that recognized AP-1 and TRE sites was not sufficient to activate EBV early lytic cycle genes. J Infect Dis, 1996 Mar, 173(3), 627 - 35 Mechanisms for mucosal immunogenicity and adjuvancy of Escherichia coli labile enterotoxin; Takahashi I et al.; Escherichia coli labile toxin (LT) was assessed as mucosal immunogen and as adjuvant for tetanus toxoid (TT) in mice . After oral administration of LT, C57BL/6 (H-2b) and BALB/c(H-2d) mice were high mucosal and serum antibody responders, while C3H/HeN (H-2k) mice were low responders . High responders exhibited mainly serum IgG (including IgG1, IgG2a, and IgG2b), as well as IgM and IgA, while mucosal responses were IgA . Analysis of LT-B-specific CD4+ T helper (Th) cells from Peyer's patches (PP) or from spleen revealed a mixed Th1 (interferon-gamma) and Th2 (interleukin-4 and -5) cell pattern . Oral LT given with TT induced TT-specific response patterns identical to LT-B . Analysis of mRNA from TT-specific PP CD4+ Th cells also revealed a mixed Th1- and Th2- type response . Thus, antibody response profiles induced by LT are regulated by both CD4+ Th1 and Th2 cell types. J Bacteriol, 1996 Mar, 178(6), 1770 - 3 SurA assists the folding of Escherichia coli outer membrane proteins; Lazar SW et al.; Many proteins require enzymatic assistance in order to achieve a functional conformation . One rate-limiting step in protein folding is the cis-trans isomerization of prolyl residues, a reaction catalyzed by prolyl isomerases . SurA, a periplasmic protein of Escherichia coli, has sequence similarity with the prolyl isomerase parvulin . We tested whether SurA was involved in folding periplasmic and outer membrane proteins by using trypsin sensitivity as an assay for protein conformation . We determined that the efficient folding of three outer membrane proteins (OmpA, OmpF, and LamB) requires SurA in vivo, while the folding of four periplasmic proteins was independent of SurA . We conclude that SurA assists in the folding of certain secreted proteins. J Bacteriol, 1996 Mar, 178(6), 1671 - 9 The organization of the outside end of transposon Tn5; Jilk RA et al.; The end sequences of the IS50 insertion sequence are known as the outside end (OE) and inside end . These complex ends are related but nonidentical 19-bp sequences that serve as substrates for the activity of the Tn5 transposase . Besides providing the binding site of the transposase, the end sequences of a transposon contain additional types of information necessary for transposition . These additional properties include but are not limited to host protein interaction sites and sites that program synapsis and cleavage events . In order to delineate the properties of the IS50 ends,the base pairs involved in the transposase binding site have been defined . This has been approached through performing a variety of in vitro analyses: a ++hydroxyl radical missing-nucleoside interference experiment, a dimethyl sulfate interference experiment, and an examination of the relative binding affinities of single-site end substitutions . These approaches have led to the conclusion that the transposase binds to two nonsymmetrical regions of the OE, including positions 6 to 9 and 13 to 19 . Proper binding occurs along one face of the helix, over two major and minor grooves, and appears to result in a significant bending of the DNA centered approximately 3 bp from the donor DNA-OE junction. J Bacteriol, 1996 Mar, 178(6), 1663 - 70 DNA-binding properties of the BetI repressor protein of Escherichia coli: the inducer choline stimulates BetI-DNA complex formation; Rkenes TP et al.; The betT and betIBA genes govern glycine betaine synthesis from choline in Escherichia coli . In an accompanying paper we report that the betT and betI promoters are divergently organized and partially overlapping and that both are negatively regulated by BetI in response to choline . (T . Lamark, T.P . Rokenes, J . McDougall, and A.R . Strom, J . Bacteriol . 178:1655-1662, 1996) . In this paper, we report that the in vivo synthesis rate of the BetI protein constituted only 10% of that of BetA and BetB dehydrogenase proteins, indicating the existence of a posttranscriptional control of the betIBA operon . A genetically modified BetI protein called BetI*, which carries 7 extra N-terminal amino acids, was purified as a glutathione S-transferase fusion protein . Gel mobility shift assays showed that BetI* formed a complex with a 41-bp DNA fragment containing the -10 and -35 regions of both promoters . Only one stable complex was detected with the 41-bp fragment and all larger promoter-containing fragments tested . In DNase I footprinting, BetI* protected a region of 21 nucleotides covering both the -35 boxes . Choline stimulated complex formation but did not change the binding site of BetI* . We conclude that in vivo BetI is bound to its operator in both repressed and induced cells and that BetI represents a new type of repressor. J Bacteriol, 1996 Mar, 178(6), 1655 - 62 The complex bet promoters of Escherichia coli: regulation by oxygen (ArcA), choline (BetI), and osmotic stress; Lamark T et al.; The bet regulon allows Escherichia coli to synthesize the osmoprotectant glycine betaine from choline . It comprises a regulatory gene, betI, and three structural genes: betT (choline porter), betA (choline dehydrogenase), and betB (betaine aldehyde dehydrogenase) . The bet genes are regulated by oxygen, choline, and osmotic stress . Primer extension analysis identified two partially overlapping promoters which were responsible for the divergent expression of the betT and betIBA transcripts . The transcripts were initiated 61 bp apart . Regulation of the promoters was investigated by using cat (chloramphenicol acetyltransferase) and lacZ (beta-galactosidase) operon fusions . Mutation of betI on plasmid F'2 revealed that BetI is a repressor which regulates both promoters simultaneously in response to the inducer choline . Both promoters remained inducible by osmotic stress in a betI mutant background . On the basis of experiments with hns and hns rpoS mutants, we conclude that osmoregulation of the bet promoters was hns independent . The bet promoters were repressed by ArcA under anaerobic growth conditions . An 89-bp promoter fragment, as well as all larger fragments tested, which included both transcriptional start points, displayed osmotic induction and BetI-dependent choline regulation when linked with a cat reporter gene on plasmid pKK232-8 . Flanking DNA, presumably on the betT side of the promoter region, appeared to be needed for ArcA-dependent regulation of both promoters. J Bacteriol, 1996 Mar, 178(6), 1640 - 5 RNases involved in ribozyme degradation in Escherichia coli; Wang JY et al.; Hammerhead ribozymes are small catalytic RNA molecules that can be designed to specifically cleave other RNAs . These ribozymes have exhibited low efficiency when examined inside cells, perhaps in part because of their sensitivity to intracellular RNases . In an effort to better understand intracellular degradation of small, foreign RNAs and to develop more stable ribozymes, the ability of Escherichia coli RNase mutants to digest ribozymes was examined . In soluble extracts, most (80 to 90%) of the endonucleolytic activity was due to RNases I and I*, since degradative activity was inhibited by Mg2+ and by the rna-2 mutation . Degradation by exonucleolytic activities was temperature sensitive in extracts from an rna pnp rnb(Ts) triple mutant but not in extracts from an rna rnb(Ts) double mutant . Thus, the products of rnb and pnp, RNase II and polynucleotide phosphorylase, respectively, appear to be the major exonucleases that degrade hammerhead ribozymes . Examination of intracellular degradation revealed that RNases I and I* contributed to about half of the degradative activity as judged by comparison of the rate of ribozyme decay in wild-type and rna-2 mutant cells . Little additional effect was observed in rne(RNase E) and rnc (RNaseIII) mutants . Taken together, these data indicate that hammerhead ribozymes are digested largely by the degradative class of RNase (RNases I, I* and II and polynucleotide phosphorylase). J Bacteriol, 1996 Mar, 178(6), 1614 - 22 Analysis of CRP-CytR interactions at the Escherichia coli udp promoter; Brikun I et al.; Multiprotein complexes regulate the transcription of certain bacterial genes in a sensitive, physiologically responsive manner . In particular, the transcription of genes needed for utilization of nucleosides in Escherichia coli is regulated by a repressor protein, CytR, in concert with the cyclic AMP (cAMP) activated form of cAMP receptor protein (CRP) . We studied this regulation by selecting and characterizing spontaneous constitutive mutations in the promoter of the udp (uridine phosphorylase) gene, one of the genes most strongly regulated by CytR . We found deletions, duplications, and point mutations that affect key regulatory sites in the udp promoter, insertion sequence element insertions that activated cryptic internal promoters or provided new promoters, and large duplications that may have increased expression by udp gene amplification . Unusual duplications and deletions that resulted in constitutive udp expression that depended on the presence of CytR were also found . Our results support the model in which repression normally involves the binding of CytR to cAMP-CRP to form a complex which binds to specific sites in the udp promoter, without direct interaction between CytR protein and a specific operator DNA sequence, and in which induction by specific inducer cytidine involves dissociation of CytR from cAMP-CRP and the RNA polymerase interaction with cAMP-CRP bound to a site upstream of then transcription start point . The stimulation of udp expression by CytR in certain mutants may reflect its stabilization of cAMP-CRP binding to target DNA and illustrates that only modest evolutionary changes could allow particular multiprotein complexes to serve as either repressors or transcriptional activators. J Bacteriol, 1996 Mar, 178(6), 1607 - 13 Posttranscriptional osmotic regulation of the sigma(s) subunit of RNA polymerase in Escherichia coli; Muffler A et al.; The sigma(s) subunit of RNA polymerase (encoded by the rpoS gene) is a master regulator in a complex regulatory network that governs the expression of many stationary-phase-induced and osmotically regulated genes in Escherichia coli . rpoS expression is itself osmotically regulated by a mechanism that operates at the posttranscriptional level . Cells growing at high osmolarity already exhibit increased levels of sigma(s) during the exponential phase of growth . Osmotic induction of rpoS can be triggered by addition of NaCl or sucrose and is alleviated by glycine betaine . Stimulation of rpoS translation and a change in the half-life of sigma(s) from 3 to 50 min both contribute to osmotic induction . Experiments with lacZ fusions inserted at different positions within the rpoS gene indicate that an element required for sigma(s) degradation is encoded between nucleotides 379 and 742 of the rpoS coding sequence. J Bacteriol, 1996 Mar, 178(6), 1491 - 7 Nucleotide sequence and characterization of the trbABC region of the IncI1 Plasmid R64: existence of the pnd gene for plasmid maintenance within the transfer region; Furuya N et al.; A 6.72-kb DNA sequence between the exc gene and the oriT operon within the transfer region of IncI1 plasmid R64 was sequenced and characterized . Three novel transfer genes, trbA, trbB, and trbC, were found in this region, along with the pnd gene responsible for plasmid maintenance . The trbABC genes appear to be organized into an operon located adjacent to the oriT operon in the opposite orientation . The trbA and trbC genes were shown to be indispensable for R64 plasmid transfer, while residual transfer activity was detected in the case of R64 derivatives carrying the trbB++ deletion mutation . The T7 RNA polymerase-promoter system revealed that the trbB gene produced a 43-kDa protein and the trbC gene produced an 85-kDa protein . The nucleotide sequence of the pnd gene is nearly identical to that of plasmid R483, indicating a function in plasmid maintenance . The plasmid stability test indicated that the mini-R64 derivatives with the pnd gene are more stably maintained in Escherichia coli cells under nonselective conditions than the mini-R64 derivatives without the pnd gene . It was also shown that the R64 transfer system itself is involved in plasmid stability to a certain degree . Deletion of the pnd gene from the tra+ mini-R64 derivative did not affect transfer frequency . DNA segments between the exc and trbA genes for IncI1 plasmids R64, Colb-P9, and R144 were compared in terms of their physical and genetic organization. Crit Care Med, 1996 Mar, 24(3), 488 - 94 Aerosolized and instilled surfactant therapies for acute lung injury caused by intratracheal endotoxin in rats; Tashiro K et al.; OBJECTIVE: To compare the effects of surfactant replacement by aerosol inhalation and bolus instillation on acute lung injury caused by the intratracheal injection of endotoxin in rats . DESIGN: Prospective, randomized study . SETTING: University Laboratory . SUBJECTS: Male Wistar rats weighing 368 +/- 31 (SD) g . Interventions: Escherichia coli endotoxin (57 +/- 20 mg/kg) was injected into the tracheas of 36 anesthetized and mechanically ventilated rats (FIO of 1.0) . When the PaO2 had decreased to <200 torr (<26.7 kPa), the rats were randomly assigned to one of three groups: a control group (n=12)given no material; a bolus group (n=12) given a modified natural surfactant suspension (100 mg/kg in 2.0 mL/kg saline) by bolus instillation into the trachea; and an aerosol group (n=12) given surfactant aerosolized with an ultra-sonic nebulizer for 60 mins . MEASUREMENTS AND MAIN RESULTS: Bolus instillation transiently decreased the mean blood pressure by approximately 30% . However, aerosol inhalation did not . The PaO2 values of the control group remained <90 torr (<12.0 kPa) until the end of the experiment (180 mins) . In contrast, the PaO2 of the bolus group increased to 387 +/- 134 torr (51.6 +/- 17.9 kPa; p<.05 vs . other groups) 15 mins after surfactant replacement, and remained at approximately 400 torr (approximately 53.3 kPa) throughout the experiment . The PaO2 values of the aerosol group increased slowly, peaked at 240 +/- 109 torr (32.0 +/- 14.5 kPa; p<.05 vs . the control group) 60 mins after the start of surfactant replacement, and remained at approximately 200 torr (approximately 26.7 kPa) . CONCLUSIONS: Bolus instillation was superior to aerosol inhalation concerning maximum efficacy, the rapid onset of therapeutic effects, and the necessary dose of surfactant . However, aerosol that does not cause hypotension may be of use in the treatment of adult respiratory distress syndrome in patients with circulatory instability. Mol Cell Biol, 1996 Mar, 16(3), 907 - 13 Amber suppression in mammalian cells dependent upon expression of an Escherichia coli aminoacyl-tRNA synthetase gene; Drabkin HJ et al.; As an approach to inducible suppression of nonsense mutations in mammalian and in higher eukaryotic cells, we have analyzed the expression of an Escherichia coli glutamine-inserting amber suppressor tRNA gene in COS-1 and CV-1 monkey kidney cells . The tRNA gene used has the suppressor tRNA coding sequence flanked by sequences derived from a human initiator methionine tRNA gene and has two changes in the coding sequence . This tRNA gene is transcribed, and the transcript is processed to yield the mature tRNA in COS-1 and CV-1 cells . We show that the tRNA is not aminoacylated in COS-1 cells by any of the endogenous aminoacyl-tRNA synthetases and is therefore not functional as a suppressor . Concomitant expression of the E . coli glutaminyl-tRNA synthetase gene results in aminoacylation of the suppressor tRNA and its functioning as a suppressor . These results open up the possibility of attempts at regulated suppression of nonsense codons in mammalian cells by regulating expression of the E . coli glutaminyl-tRNA synthetase gene in an inducible, cell-type specific, or developmentally regulated manner. J Biol Chem, 1996 Mar 1, 271(9), 5171 - 6 Structure of aquaporin-2 vasopressin water channel; Bai L et al.; Aquaporin-2 (AQP-2) is a vasopressin-regulated water channel in the kidney collecting duct . AQP-2 is selectively permeable to water molecule and is translocated between the apical membrane and subapical endosomes in response to vasopressin . To investigate the localization and structure of the aqueous pathway of the AQP-2 water channel, a series of site-directed mutants was constructed and functionally analyzed . Insertion of N-glycosylation reporter sequence into each hydrophilic loop (HL) indicated that AQP-2 has a six-membrane spanning topology and that insertional mutations in HL-2 or HL-5 do not alter water channel function . Mercury-sensitive site of AQP-2 is located near the second asparagine-proline-alanine (NPA) domain at cysteine 181, but not near the first NPA domain . Replacement of HL-3 or HL-4 with the corresponding part of Escherichia coli glycerol facilitator abolished water channel function without changing plasma membrane expression of the channel protein . Introduction of cysteine residues in His-122, Asn-123, Gly-154, Asp-155, or Asn-156 induced partial mercury sensitivity, and point mutations in asparagine 123 significantly altered water permeability . Our results implicate that the structure of AQP-2 is different from models previously proposed for AQP-1 and that HL-3 and HL-4 are closely located to the aqueous pathway. J Biol Chem, 1996 Mar 1, 271(9), 5125 - 30 In vivo and in vitro iron-replaced zinc finger generates free radicals and causes DNA damage; Conte D et al.; The estrogen receptor (ER) is a ligand-activated transcription factor whose DNA-binding domain (ERDBD) has eight cysteines, which coordinate two zinc atoms, forming two zinc finger-like structures . We demonstrate the capability of iron to replace zinc in zinc finger (hereby referred to as iron finger) both in vivo (using Escherichia coli BL21 (DE3)) and in vitro . Iron has the ability to substitute for zinc in the ERDBD as demonstrated by mobility shift and methylation interference assays of iron finger, which show specific recognition of the estrogen response element . The DNA binding constants for both in vivo and in vitro iron-replaced zinc fingers were similar to that of the native finger . Atomic absorption analysis revealed a ratio of 2:1 iron atoms/mol of ERDBD protein, as found for zinc in the crystal structure of native ERDBD . More importantly, we demonstrate that iron finger in the presence of H2O2 and ascorbate generates highly reactive free radicals, causing a reproducible cleavage pattern to the proximate DNA, the estrogen response element . The deoxyribose method, used to detect free radical species generated, and the resultant cleaved DNA ends, caused by iron finger, suggest that the free radicals generated are hydroxyl radicals . Due to the close proximity of the zinc finger to DNA, we postulate that iron-substituted zinc finger may generate free radicals while bound to genetic regulatory response elements, leading to adverse consequences such as iron-induced toxicity and/or carcinogenesis. J Biol Chem, 1996 Mar 1, 271(9), 5059 - 65 Histidine patch thioredoxins . Mutant forms of thioredoxin with metal chelating affinity that provide for convenient purifications of thioredoxin fusion proteins; Lu Z et al.; A cluster of surface amino acid residues on Escherichia coli thioredoxin were systematically mutated in order to provide the molecule with an ability to chelate metal ions . The combined effect of two histidine mutants, E30H and Q62H, gave thioredoxin the capacity to bind to nickel ions immobilized on iminodiacetic acid- and nitrilotriacetic acid-Sepharose resins . Even though these two histidines were more than 30 residues apart in thioredoxin's primary sequence, they were found to satisfy the geometric constraints for metal ion coordination as a result of the thioredoxin tertiary fold . A third histidine mutation, S1H, provided additional metal ion che