Escherichia coli

Eur J Clin Microbiol Infect Dis, 1997 Jun, 16(6), 458 - 60
In vitro activity of trimethoprim against Borrelia burgdorferi; Reisinger EC et al.; A new culture medium has been developed to evaluate the activity of trimethoprim and sulfamethoxazole against Borrelia burgdorferi in vitro . In this specially modified Barbour-Stoenner-Kelly medium, in which antagonizing substances were reduced to a minimum, trimethoprim was more active against Borrelia burgdorferi than against a sensitive strain of Escherichia coli, but sulfamethoxazole was not active against Borrelia burgdorferi.

C R Acad Sci III, 1997 Jun, 320(6), 427 - 34
Human deoxycytidine kinase as a conditional mutator in Escherichia coli; Bouzon M et al.; The chemical diversification of DNA precursors was undertaken in Escherichia coil by expressing the human gene for deoxycytidine kinase, and supplying such recombinant strains with nucleoside analogues bearing an altered base or sugar . Arabinocytidine and dideoxycytidine thus became highly toxic to E . coli in the sub-millimolar range . Deoxynucleosides bearing isoadenine (2-aminopurine) and isoguanine (2-hydroxy-6-aminopurine) showed a high mutagenic potency towards the recombinant strains, to an extent comparable to that of the most efficient mutator alleles (dnaQ) . These findings open the way to the propagation of chemically remodelled nucleic acids and to the controlled hypermutagenesis of plasmids in vivo.

Z Ernahrungswiss, 1997 Jun, 36(2), 155 - 60
Is there any possibility of detecting the use of genetic engineering in processed foods?
Greiner R, Konietzny U, Jany KD.
To elucidate if there is any possibility to identify highly processed foods as produced through genetic engineering, beer, soya bean oil, processed tomato (ketch-up, paste, pizza tomatoes, peeled tomatoes, soup) and potato (french fries, crisps, mashed potatoes, flour, starch, fried potatoes) products as well as an enzyme preparation (Natuphos) were investigated by PCR . In pizza tomatoes, peeled tomatoes, french fries, fried potatoes, potato flour and potato crisps DNA suitable for PCR was found . Therefore, it is possible to identify these products as produced through genetic engineering . Such an identification is impossible in certain beers (pilsener, export, Nutfield lyte), soya bean oil, tomato soup, potato starch, mashed potatoes and Natuphos since PCR-analysis gave no indication of the presence of DNA in these products . As it was shown by adding Escherichia coli DNA the used method is, in principle, capable of detecting specifically small amounts of DNA in such products.

Int J Radiat Biol, 1997 Jun, 71(6), 667 - 74
What will molecular biology contribute to our understanding of radiation-induced cell killing and carcinogenesis?
Hall EJ.
The vast body of radiobiological data accumulated with mammalian systems in vitro and in vivo has had an enormous impact on radiotherapy . However, while quantitative, these data are essentially phenomenological, and it is only in the last decade or so that the techniques of molecular biology allow basic mechanisms to be understood . This will be illustrated by two examples, one involving cell killing and the other carcinogenesis . The identification and sequencing of repair and checkpoint control genes in the yeast S . pombe allow the mechanism of sensitivity/ resistance to radiation to be understood at the molecular level . The development of techniques to identify mutations in mismatch repair genes have made it possible to show that such mutations are associated with a wide range of human cancers and are a likely mechanism of radiation induced malignancies . Tikvah Alper would have been delighted to see the central role that micro-organisms have played in these recent developments.

Toxicon, 1997 Jun, 35(6), 879 - 88
Cloning and expression of a cysteine-rich venom protein from Trimeresurus mucrosquamatus (Taiwan habu); Chang TY et al.; A full-length cDNA for cysteine-rich venom protein (CRVP) was constructed by immunoscreening and 5'-rapid amplification of cDNA ends from a cDNA library of venom gland of Trimeresurus mucrosquamatus . The predicted CRVP consisted of 183 amino acid residues including a putative signal peptide of 21 residues . Northern blot hybridization suggested the tissue-specific expression in venom gland and its corresponding length of cDNA . The predicted amino acid sequence of CRVP was homologous to a rat epididymal metalloprotein and a lizard helothermine . Amino acid sequence analysis suggested that CRVP may be a venom metalloprotein targeted against ryanodine receptors and Ca2+ release . Moreover, CRVP expressed in Escherichia coli exhibited the same antigenicity as their native venom forms of T . mucrosquamatus . This is the first report in the cloning and expression of a CRVP from the venom gland of T . mucrosquamatus.

Mol Hum Reprod, 1997 Jun, 3(6), 459 - 66
Relaxin-like factor: a highly specific and constitutive new marker for Leydig cells in the human testis; Ivell R et al.; The complete protein-coding region of the human relaxin-like factor (RLF; formerly Ley-I-L) was cloned by reverse transcription-polymerase chain reaction from human testis and subcloned into a bacterial expression plasmid for the production of recombinant human RLF in Escherichia coli . Polyclonal antibodies were raised against the recombinant RLF, as well as against a peptide epitope from the B-domain of the RLF polypeptide . Antibodies were used for immunohistochemistry of Bouin-fixed, paraffin-embedded samples of human testis tissues . Specific immunoreactivity was located exclusively in the Leydig cells with a consistent high intensity of staining, showing similar spatial distribution to other Leydig cell markers, such as the luteinizing hormone (LH) receptor and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), and to the pattern of RLF mRNA shown by in-situ transcript hybridization . In biopsy samples from patients with severe disturbances of spermatogenesis, RLF staining intensity was consistently high in all cases, unlike staining for 3 beta-HSD which varied considerably between patients . Immunostaining for RLF would thus appear to offer an interesting new marker for Leydig cells in human testis samples.

Biochem Mol Biol Int, 1997 Jun, 42(2), 409 - 17
Cloning, expression and purification of the coat protein of encephalitis virus (DIEV) infecting Dicentrarchus labrax; Sideris DC; The coat protein gene from encephalitis virus infecting Dicentrarhus labrax (DIEV) has been cloned by gene amplification, sequenced and expressed in Escherichia coli . DNA sequencing has revealed an open reading frame of 1017 bases encoding a polypeptide of 338 amino acids . The sequence similarities between the DIEV coat protein gene and the same gene in five encephalitis viruses infected other fish species were over 71.5% at the nucleotide level and over 79.5% at the amino acid level . These results indicate that the nodaviruses that cause encephalopathy and retinopathy in fishes are very closed related . E . coli cells harbouring the plasmid containing the DIEV gene can produce the viral coat protein . An efficient purification scheme using a Sepharore-Ni+2 column is presented . This, gives approx . 10 mg of more than 95% pure protein per gr of E . coli culture.

Eur J Cardiothorac Surg, 1997 Jun, 11(6), 1023 - 8; discussion 1029
Adenovirus mediated gene transfer into rat lung grafts at the time of harvest; Schmid RA et al.; OBJECTIVE: New methods to introduce genetic material into cells in vivo may revolutionize current treatment modalities . Expression of functional genes in lung allografts could be used as a prophylactic strategy for reperfusion injury and rejection . We studied the feasibility of ex vivo adenovirus mediated transfection of rat lung allografts . METHODS: In group I (n = 3) donor rat lungs (Fisher) were flushed with Low Potassium Dextran Glucose (LPDG) solution (20 ml, 20 degrees C) . 4 x 10(11) viral particles of adenovirus 5 containing the E . coli lacZ reporter gene coding for beta-galactosidase (AdCMV-beta-Gal) were added to the last milliliter of the flush solution . Lung grafts were stored for 3.5 h at room temperature followed by syngenic orthotopic transplantation (Fisher to Fisher) using a microsurgical cuff technique . On postoperative day 5 the heart lung block was extracted and flushed with x-Gal (beta-Gal substrate) and kept in x-Gal for 3 h at 37 degrees C . Color development was observed macroscopically and plastic embedded sections were used for histologic examination . Group II grafts (n = 3) served as controls and were flushed without adenovirus . RESULTS: X-Gal stained the transfected lung grafts blue, indicating high reporter gene expression . Control lungs did not stain with x-Gal . In group I histological examination demonstrated transfection predominantly in type II pneumocytes . Surprisingly endothelial cells showed no beta-Gal activity . CONCLUSION: This study demonstrates that ex vivo transfection of lung grafts at the time of harvest is a feasible method of gene transfer and results in gene expression after transplantation.

Immunotechnology, 1997 Jun, 3(2), 137 - 44
Enzyme immunoassays using bispecific diabodies; Kontermann RE et al.; BACKGROUND: Bispecific antibodies with a first binding specificity to a target antigen and a second to an enzyme have great potential in enzyme immunoassays . As bispecific antibodies are difficult to make, the use of recombinant bispecific antibody fragments may provide a breakthrough . OBJECTIVES: To make bispecific antibody fragments directed against an enzyme and to demonstrate their application in enzyme immunoassays . STUDY DESIGN: Bispecific antibody fragments were assembled as diabodies (Holliger P., Prospero T., Winter G . Proc . Natl . Acad . Sci . USA 90, 1993, 6444-6448) directed to an enzyme, E . coli beta-galactosidase, and to each of three target antigens, hen-egg lysozyme (HEL), carcinoembryonic antigen (CEA), and HIV gpl20 (HIV) . The diabodies were then evaluated in immunoassays . RESULTS: The HEL diabody was shown to recruit beta-galactosidase in a microtiter plate immunoassay in which diabody and enzyme were co-incubated with antigen, washed and enzyme substrate added . The CEA diabody was shown to detect CEA by immunocytochemical staining of transfected, CEA-expressing HeLa cells and of adenocarcinoma colon tissue sections, and the HIV diabody to detect gpl20 in immunoblots of total cell extracts . CONCLUSION: The results illustrate the diagnostic potential of diabodies in enzyme immunoassays.

Immunotechnology, 1997 Jun, 3(2), 127 - 36
Cloning and cytotoxicity of a human pancreatic RNase immunofusion; Zewe M et al.; BACKGROUND: Immunotoxins based on plant and bacterial proteins are usually very immunogenic . Human ribonucleases could provide an alternative basis for the construction of less immunogenic reagents . Two members of the human RNase family, angiogenin and eosinophil-derived neurotoxin (EDN), have been fused to a single chain antibody against the transferrin receptor, which is known to be internalised by endocytosis . The fusion proteins proved to be very efficient inhibitors of protein synthesis using various cell lines . It is not yet known whether the side effects of angiogenin and EDN will compromise their potential use as immunotoxins . OBJECTIVES: The goal of this work was to construct a human immunotoxin with no harmful side effects . Bovine pancreatic ribonuclease has been shown to be as potent as ricin at abolishing protein synthesis on injection into oocytes . We therefore decided to clone its human analogue, which is fairly ubiquitous and per se non-toxic . An immunofusion of human pancreatic RNase with a single chain antibody against the transferrin receptor was tested for its ability to inhibit protein synthesis in three different human tumor cell lines . STUDY DESIGN: DNA coding for the human pancreatic RNase was cloned partially from a human fetal brain cDNA library and then completed by PCR using a human placental cDNA library as a template . The RNase gene was then fused with a DNA coding for an single chain antibody against the transferrin receptor (CD71) . After expressing the fusion protein in E . coli, the gene product was isolated from inclusion bodies and tested for cytotoxicity . RESULTS: This fusion protein inhibited the protein synthesis of three human tumor cell lines derived from a melanoma, a renal carcinoma and a breast carcinoma, with IC50s of 8, 5 and 10 nM, respectively . These values were comparable with those using a similar fusion protein constructed with eosinophil derived neurotoxin (EDN) as the toxic moiety (IC50s of 8, 1.2 and 3 nM, respectively) . The slightly lower activities of the human pancreatic RNase-scFv (pancRNase-scFv) with two of the cell lines suggests that fewer molecules are reaching the cytoplasmic compartment, since it was twice as active as EDN-scFv in inhibiting the protein synthesis of a rabbit reticulocyte lysate . CONCLUSION: These results demonstrate that the human pancreatic RNase, which is expected to have a very low immunogenic potential in humans with no inherent toxicity, may be a potent cytotoxin for tumor cells after antibody targeting.

Immunotechnology, 1997 Jun, 3(2), 83 - 105
New protein engineering approaches to multivalent and bispecific antibody fragments; Pluckthun A et al.; Multivalency is one of the hallmarks of antibodies, by which enormous gains in functional affinity, and thereby improved performance in vivo and in a variety of in vitro assays are achieved . Improved in vivo targeting and more selective localization are another consequence of multivalency . We summarize recent progress in engineering multivalency from recombinant antibody fragments by using miniantibodies (scFv fragments linked with hinges and oligomerization domains), spontaneous scFv dimers with short linkers (diabodies), or chemically crosslinked antibody fragments . Directly related to this are efforts of bringing different binding sites together to create bispecific antibodies . For this purpose, chemically linked fragments, diabodies, scFv-scFv tandems and bispecific miniantibodies have been investigated . Progress in E . coli expression technology makes the amounts necessary for clinical studies now available for suitably engineered fragments . We foresee therapeutic advances from a modular, systematic approach to optimizing pharmacokinetics, stability and functional affinity, which should prove possible with the new recombinant molecular designs.

Mol Gen Genet, 1997 Jun, 255(2), 180 - 6
Molecular cloning and characterization of the cDNA encoding the largest subunit of mouse RNA polymerase I; Seither P et al.; We describe the cloning and analysis of mRPA1, the cDNA encoding the largest subunit (RPA194) of murine RNA polymerase I . The coding region comprises an open reading frame of 5151 bp that encodes a polypeptide of 1717 amino acids with a calculated molecular mass of 194 kDa . Alignment of the deduced protein sequence reveals homology to the beta' subunit of Escherichia coli RNA polymerase in the conserved regions a-h present in all large subunits of RNA polymerases . However, the overall sequence homology among the conserved regions of RPA1 from different species is significantly lower than that observed in the corresponding beta'-like subunits of class II and III RNA polymerase . We have raised two types of antibodies which are directed against the conserved regions c and f of RPA194 . Both antibodies are monospecific for RPA194 and do not cross-react with subunits of RNA polymerase II or III . Moreover, these antibodies immunoprecipitate RNA polymerase I both from murine and human cell extracts and, therefore, represent an invaluable tool for the identification of RNA polymerase I-associated proteins.

Ocul Immunol Inflamm, 1997 Jun, 5(2), 117 - 28
Secretion of proinflammatory cytokines by human conjunctival epithelial cells; Gamache DA et al.; The production of cytokines by human conjunctival epithelial cells following stimulation was investigated . Primary cultures of human conjunctival epithelial cells were characterized by morphology and keratin expression . Cultured epithelial cells were treated with varying concentrations of lipopolysaccharide, interleukin (IL)-1 beta, calcium ionophore A23187, or phorbol myristate acetate, and cytokine secretion was determined over specified intervals . Culture supernatants and cell lysates were analyzed by ELISA for IL-1 beta, IL-3, IL-4, IL-5, IL-6, IL-8, IL-11, IL-1 receptor antagonist (IL-1ra), tumor necrosis factor-alpha (TNF-alpha), and granulocyte-macrophage colony stimulating factor (GM-CSF) . With the exception of IL-1ra, unstimulated conjunctival epithelial cells produced cytokines at relatively low or undetectable levels . IL-1ra was detected in both culture supernatants and cell lysates under basal conditions . In response to stimuli, conjunctival epithelial cells secreted the proinflammatory cytokines TNF-alpha, IL-6, IL-8, and GM-CSF in a dose- and time-dependent fashion . After stimulation, the intracellular levels of IL-1ra increased in these cells but the supernatant-associated levels remained unchanged . None of the other cytokines evaluated (IL-1 beta, IL-3, IL-4, IL-5, and IL-11) were detected in supernatants or lysates of resting or stimulated cells . These findings suggest that conjunctival epithelial cells may contribute to the pathogenesis of human ocular diseases by production of proinflammatory cytokines . Further evaluation of these cells as targets of therapy is warranted.

J Vet Med Sci, 1997 Jun, 59(6), 483 - 5
Evaluation of blood acid-base balance after experimental administration of endotoxin in adult cow; Ohtsuka H et al.; Esherichia coli endotoxin was administered intravenously to 7 Holstein adult cows, to evaluate the effect of endotoxin on acid-base balance . Endotoxin shock was observed immediately after the administration of endotoxin . A loss of appetite and depression of digestive tract motility continued for about 120 hr after the challenge . Metabolic alkalosis following hypochloremia and hypokalemia were particularly pronounced at 12 to 72 hr after the administration of endotoxin.

J Vet Med Sci, 1997 Jun, 59(6), 471 - 2
Metabolic alkalosis in coliform mastitis; Ohtsuka H et al.; Values of blood gas, serum chloride, and potassium were tabulated for 21 dairy cows with coliform mastitis . Severe cases showed marked clinical signs such as loss of appetite and depression of digestive tract motility, and metabolic alkalosis such as an increase in blood pH, hypochloremia and hypokalemia compared with normal and mild cases (p < 0.01) . The results showed that metabolic alkalosis can be detected more easily than acidosis in cases of severe coliform mastitis.

Mol Gen Genet, 1997 Jun, 255(1), 54 - 9
Lack of correlation between binding of EcoRII methyltransferase to DNA duplexes containing mismatches and the promotion of C to T mutations; Sheluho D et al.; The cytosine methyltransferases (MTases) M . HhaI and M . HpaII bind substrates in which the target cytosine is replaced by uracil or thymine, i.e . DNA containing a U:G or a T:G mismatch . We have extended this observation to the EcoRII MTase (M . EcoRII) and determined the apparent Kd for binding . Using a genetic assay we have also tested the possibility that MTase binding to U:G mismatches may interfere with repair of the mismatches and promote C:G to T:A mutations . We have compared two mutants of M . EcoRII that are defective for catalysis by the wild-type enzyme for their ability to bind DNA containing U:G or T:G mismatches and for their ability to promote C to T mutations . We find that although all three proteins are able to bind DNAs with mismatches, only the wild-type enzyme promotes C:G to T:A mutations in vivo . Therefore, the ability of M . EcoRII to bind U:G mismatched duplexes is not sufficient for their mutagenic action in cells.

Am J Physiol, 1997 Jun, 272(6 Pt 1), L1046 - 52
Adenovirus E1A upregulates interleukin-8 expression induced by endotoxin in pulmonary epithelial cells; Keicho N et al.; Adenovirus E1A DNA and proteins are frequently detected in lungs of patients with chronic obstructive pulmonary disease . Because adenovirus E1A can regulate host gene expression by interacting with cellular transcription factors, we postulate that E1A enhances synthesis of inflammatory mediators . To examine this possibility, we measured the expression of inflammatory cytokines in E1A-producing A549 human pulmonary epithelial cells and control cells . Interleukin (IL)-8 mRNA was markedly induced by lipopolysaccharide (LPS) in E1A-producing cells but not in controls . IL-8 protein levels were elevated in parallel . In both cell types, monocyte chemotactic and activating factor mRNA induced by LPS was low, and transforming growth factor-beta 1 mRNA was not affected . IL-1 beta, IL-6, granulocyte macrophage colony-stimulating factor, and granulocyte colony-stimulating factor mRNAs were too low to show any effect of E1A . We conclude that the LPS responsiveness of A549 pulmonary epithelial cells is altered by adenoviral E1A by upregulating IL-8 . We speculate that this mechanism may be important in the amplification of the inflammatory process of lungs to other irritants.

Am J Physiol, 1997 Jun, 272(6 Pt 1), G1581 - 6
Kupffer cells contain a glycine-gated chloride channel; Ikejima K et al.; Here the effect of glycine on intracellular Ca2+ concentration ({Ca2+}i) in cultured Kupffer cells stimulated with lipopolysaccharide (LPS) was investigated to assess the possibility that they contain a glycine-gated chloride channel . LPS (10 micrograms/ml) increased {Ca2+}i rapidly, with peak values reaching 307 +/- 29 nM . Glycine (1 mM) prevented this increase nearly completely . Low concentrations of strychnine (1 microM), a glycine receptor antagonist, reversed the inhibitory effect of glycine completely; however, high concentrations of strychnine (1 mM) mimicked glycine . The effects of glycine and high-dose strychnine were prevented when cells were incubated in chloride-free buffer . Furthermore, potassium (25 mM) and LPS depolarized the Kupffer cell plasma membrane, whereas glycine caused hyperpolarization and prevented depolarization due to potassium and LPS . Moreover, tumor necrosis factor-alpha (TNF-alpha) production in cultured Kupffer cells due to LPS was decreased significantly by glycine . Therefore, it is concluded that Kupffer cells contain a glycine-gated chloride channel similar to that described previously in the central nervous system . Prevention of increases in {Ca2+}i due to LPS by activation of chloride influx reduced synthesis and release of toxic mediators by Kupffer cells.

Am J Physiol, 1997 Jun, 272(6 Pt 1), C1995 - 2004
Differential processing of guanylyl cyclase C along villus-crypt axis of rat small intestine; Scheving LA et al.; Many strains of enterotoxigenic Escherichia coli produce a heat-stable peptide enterotoxin (STa) that binds to the intestinal receptor guanylyl cyclase C (GC-C) . STa receptors are structurally heterogeneous, but the molecular events causing this heterogeneity remain obscure . We examined the influence of cell position along the villus-crypt axis on STa receptor heterogeneity by fractionating EDTA-dissociated cells that detached in a villus-to-crypt direction . STa affinity labeling experiments revealed that the initially released villus "tip" fraction had four major STa binding proteins (STBPs), with relative molecular weight (M(r)) of 150,000, 135,000, 125,000, and 95,000, that did not react with a GC-C carboxy-terminal antibody . Yet succeeding villus cell fractions had major immunoreactive STBPs with M(r) of 275,000 and 250,000 . Limited proteolysis of these larger GC-C isoforms produced 1) smaller STBPs that had M(r) similar to those in the initial villus fraction, 2) a 65,000 M(r) protein GC-C isoform that did not bind STa, and 3) elevated basal and STa-induced cyclase activity . Our data show that STBP structural heterogeneity in the intact intestine arises largely from multisite proteolytic processing of GC-C.

J Pharm Biomed Anal, 1997 Jun, 15(9-10), 1417 - 26
Flow injection analysis for measurement of activity of matrix metalloproteinase-7 (MMP-7); Itoh M et al.; A simple and convenient method for measuring the activity of a recombinant human matrix metalloproteinase 7 (MMP-7, matrilysin) was developed by flow injection analysis (FIA) . For this method, purified recombinant MMP-7 zymogen expressed in E . coli and the substrate peptide (MOCAc-Pro-Leu-Gly-Leu-A2pr(DNP)-Ala-Arg-NH2) were used . Following the incubation of substrate peptide with activated r-proMMP-7, the resulting fluorescent product peptide (MOCAc-Pro-Leu-GLY) was monitored with a fluorescence detector (lambda ex 328 mm, lambda em 393 mm) without chromatographic separation . In this FIA system, the analysis time is 2 min and the standard curve is linear from 5 to 100 pmol of the product peptide injected . In order to use this FIA system as a method for screening inhibitors against MMP-7, the effects of CaCl2, EDTA and of the tissue inhibitor of metalloproteinase-1, and -2, were tested . A synthetic PRCGXPD-containing peptide (BS-10) was also observed to inhibit MMP-7 activity, with an IC50 value of 104 microM . Thus, it was concluded that the activity of r-MMP-7 can be reliably measured by the proposed system . Furthermore, to confirm the utility of this FIA system as a screening method, the inhibitory activity of the MMP-related substance in Joro spider (Nephilia clavata) venom was measured by this method . This inhibitory activity was observed in an extract of a venom diluted 1000-fold . Thus, the FIA method is not only simple and quick, but also sensitive enough to screen and analyze the inhibitory properties of a large number of test compounds.

Vet Q, 1997 Jun, 19(2), 41 - 7
The effect of discontinuation of postmilking teat disinfection in low somatic cell count herds . I . Incidence of clinical mastitis; Lam TJ et al.; Results are described of a split-udder trial on the effect of discontinuation of postmilking teat disinfection on the incidence of clinical mastitis in seven dairy herds with a low bulk milk somatic cell count and a high incidence of clinical mastitis . Overall incidence of clinical mastitis was non-significantly lower (18%), whereas the incidence of the most prevalent pathogen associated with clinical mastitis, Escherichia coli, was significantly lower in quarters for which postmilking teat disinfection was discontinued . We concluded that discontinuation of postmilking teat disinfection may decrease the incidence of clinical Escherichia coli mastitis in herds for which standard mastitis prevention measures are executed adequately, bulk milk somatic cell count is low, and incidence of clinical mastitis is high . However, because an increase in intramammary infections with contagious pathogens may occur, care is recommended when advising discontinuation of postmilking teat disinfection.

Biol Chem, 1997 Jun, 378(6), 545 - 51
Overproduction of Sac7d and Sac7e reveals only Sac7e to be a DNA-binding protein with ribonuclease activity from the extremophilic archaeon Sulfolobus acidocaldarius; Kulms D et al.; Genomic DNA from Sulfolobus acidocaldarius was screened using a degenerate oligodeoxyribonucleotide, derived from the sequence of 16 N-terminal amino acids from SaRD protein . SaRD protein was previously isolated in our laboratory and identified as a protein from S . acidocaldarius exhibiting ribonuclease activity as well as DNA-binding properties . On the basis of Southern hybridization analysis two genes from S . acidocaldarius have been cloned, sequenced and overproduced in Escherichia coli . The deduced amino acid sequences revealed that one gene encodes Sac7d and the other one Sac7e; two small, previously described basic proteins from S . acidocaldarius, and furthermore the N-termini of Sac7e and SaRD are identical . Northern blot analysis demonstrated that the genes are transcribed separately . After expression of sac7d and sac7e genes in E . coli it was shown that only recombinant Sac7e protein exhibits RNase activity and is catalytically indistinguishable from SaRD protein . Western blot analysis using a polyclonal antiserum raised against purified SaRD protein further confirmed that Sac7e and SaRD are identical proteins endowed with RNase activity and DNA-binding properties . A new RNA cleavage mechanism has to be postulated for Sac7e since, in contrast to common RNases (e.g . RNase A and T1), no histidines are present in the amino acid sequence . Differences between the very closely related 7 kDa proteins from two Sulfolobus strains converting DNA-binding proteins into RNases are pointed out and discussed, whereas substitutions of Glu by Gln (S . solfataricus) or by Lys (S . acidocaldarius) seem to be crucial.

Eur J Gastroenterol Hepatol, 1997 Jun, 9(6), 569 - 73
Santorini's duct--risk factor for acute pancreatitis or protective morphologic variant? Experiments in rabbits; Arendt T et al.; BACKGROUND AND AIMS: Gallstone pancreatitis is assumed to result from stone passage through the choledochoduodenal junction . Stone impactions may either result in the obstruction of the pancreatic duct or occur below the confluence of the biliary tract and the pancreatic duct and, thus, may favour bile reflux into the pancreatic duct . We studied effects of a patent Santorini's duct upon secretory flow and pancreas morphology under both conditions . METHODS: A catheter in the distal rabbit pancreatic duct created a second outlet for pancreatic juice and, thus, mimicked a patent Santorini's duct . A second catheter was introduced into the proximal pancreatic duct and into the common bile duct . This catheter mimicked a common channel behind a papillary obstruction . Clamping of this catheter mimicked a stone obstruction of the pancreatic duct . A catheter in the cystic duct allowed for the infection of bile with 10(7) Escherichia coli bacteria/ml . The flow direction of bile and pancreatic juice was directly observed . Pancreatic histology was analysed after 24 h . RESULTS: Pancreatic duct obstruction produced an oedema of the gland . Creation of a patent Santorini's duct prevented development of the histological changes caused by pancreatic duct obstruction . In rabbits in which a common channel obstruction was mimicked, Santorini's duct produced flow of bile along the pancreatic duct system . Flow of sterile bile along the duct did not cause pancreatic inflammatory lesions . Bile that was infected with E . coli bacteria produced an acute interstitial-oedematous pancreatitis . CONCLUSIONS: (1) A patient Santorini's duct protects the gland from the effects of main pancreatic duct obstruction; (2) Santorini's duct promotes biliary pancreatic reflux during obstruction of the common channel and subsequent development of pancreatitis caused by infected choledochal secretions; (3) Santorini's duct may thus be both a protective morphological variant and a risk factor for pancreatitis dependent upon the site of stone impaction within the choledochoduodenal junction.

Bioorg Med Chem, 1997 Jun, 5(6), 1037 - 42
Modulation of RNase H activity by modified DNA probes: major groove vs minor groove effects; Daniher AT et al.; We have previously prepared ribozyme mimics and chemical nucleases from modified DNA containing pendant bipyridine and terpyridine groups . The ability of these modified DNA probes to support RNase H cleavage of complementary RNA is described . DNA/RNA duplexes were formed using DNA probes designed to deliver metal complexes via either the major groove or the minor groove of the duplex . The duplexes were treated with Escherichia coli RNase H . Modifications in the major groove produced the same RNA cleavage pattern as unmodified DNA probes . However, minor groove substituents inhibited RNA cleavage over a four-base region . Comparison was made with a DNA probe containing a 2'-OMe modification . Our results support enzyme binding in the minor groove of a DNA/RNA duplex . We do not observe cleavage directly across from the modified nucleoside . The RNA cleavage efficiency effected by RNase H and a DNA probe decreases as follows: unmodified DNA > or = C-5 modified DNA >> c2'-modified DNA > C1'-modified DNA . Results with 28-mer RNA substrates roughly parallel those obtained with a 159-mer RNA target . The differences observed between low and high MW RNA substrates can be explained by a much higher enzyme-substrate binding constant for the high MW target.

Bioorg Med Chem, 1997 Jun, 5(6), 1021 - 35
Context dependent RNA-RNA recognition in a three-dimensional model of the 16S rRNA core; Masquida B et al.; A 3-D model of the core of the 16S rRNA of Escherichia coli containing 328 residues has been built in the protein map derived from neutron scattering data with the help of all the available phylogenetic, biochemical, and cross-linking data . The three pseudoknots of the 16S-core cluster, through the arrangement of complex three-, four- and five-way junctions, around the neck and at the subunit interface . The roles in assembly, initiation or elongation of the three pseudoknots in ribosomal dynamics are emphasized . The 530-loop, localized on the periphery of the 30S particle, could be built with and without a pseudoknot independently of the state of the particle . The pseudoknot of the central domain controls the dynamics of an helix connected to the subunit interface which could trigger some mechanism during translation . The process of the model construction is compatible with a folding scenario in which the 5'-terminal pseudoknot controls the assembly of the central junction and the subsequent folding of the 3'-major domain . The modelling, together with the phylogenetic analysis and the experimental data, point to several potential RNA-RNA contacts which depend on the structural and sequence context in which they occur.

Bioorg Med Chem, 1997 Jun, 5(6), 1011 - 9
A strategy of tRNA recognition that includes determinants of RNA structure; Hamann CS et al.; Recognition of tRNAs by aminoacyl tRNA synthetases establishes the connection between amino acids and anticodon triplets of the genetic code . Although anticodons and nucleotides adjacent to the amino acid attachment site are generally important, the tertiary structural framework of tRNAs has recently been implicated to have a role in tRNA recognition . A G15:G48 tertiary hydrogen base pair of E . coli tRNA(Cys) is important for recognition of the tRNA by cysteine tRNA synthetase . This base pair is proposed to consist of N2:N3, rather than N1:O6, hydrogen bonds . The reproduction of the hydrogen pairing scheme of tRNA(Gly) . This reproduction required an A13:A22 mismatch in the dihyrouridine stem . To determine if A13:A22 is a determinant of the structural features of G15:G48, we investigated the A15:U48 and A15:A48 variants of tRNA(Gly) which harbored specific substitutions of A13:A22 . We show here that introduction of A13:A22 to both tRNA frameworks confers structural features similar to those of G15:G48 in E . coli tRNA(Cys) . These structural features are accompanied by efficient recognition of both tRNAs by cysteine tRNA synthetase . Substitution of A13:A22 with U13:A22 alters the structural features at 15:48 and impairs tRNA recognition . The dependence on A13:22 for tRNA recognition has a distinct similarity to that of E . coli tRNA(Cys) and to that of the G15:G48 variant of tRNA(Gly) . The results have implications for the design and manipulation of RNA structural elements as the basis for tRNA recognition.

Cell Mol Biol (Noisy-le-grand), 1997 Jun, 43(4), 509 - 19
Immunochemical localization of the phylogenetically oldest receptor tyrosine kinase: existence in the marine sponge Geodia cydonium; Skorokhod A et al.; Until now molecular data, elucidating the basis of invertebrate immunity are lacking . Previously both the gene and different cDNAs, coding for the ancestor of metazoan receptor tyrosine kinases (RTK), have been isolated from the marine sponge Geodia cydonium . The sponge RTK shows high polymorphism in the coding as well as in the non-coding parts of the gene . To further elucidate if the sponge RTK might be a molecule involved in self/non-self recognition the intracellular portion of the sponge RTK was expressed in Escherichia coli . The 59 kDa recombinant protein was used to raise monoclonal antibodies (McAb) . The McAb recognized three polypeptides of sizes 135, 68 and 26 kDa by Western blotting . The McAb recognized only the plasma membranes of sponge cells as analyzed by immunohisto- and cytochemical studies . Northern blotting analysis revealed that the expression of the RTK gene depends on environmental conditions and on seasonal variations . Based on the high degree of polymorphism and on the immunochemical data we suggest that the RTK in G . cydonium might be involved in sponge immunity.

Mol Microbiol, 1997 Jun, 24(5), 1071 - 82
Relating primary structure to function in the Escherichia coli XerD site-specific recombinase; Spiers AJ et al.; XerC and XerD are related 298-amino-acid site-specific recombinases, each of which is responsible for the exchange of one pair of strands in Xer recombination . Both recombinases encode functions necessary for sequence-specific DNA-binding, co-operative XerC/D interactions, synapsis and catalysis . These functions were related to the primary amino acid sequence by constructing and analysing internal and C-terminal XerD deletions . An XerD derivative containing residues 1-233 was proficient in specific DNA binding, but did not interact co-operatively with XerC . Deletion of a further five C-terminal amino acids abolished binding to DNA . Proteins deleted for residues 32-88 and for residues 145-159 were deficient in DNA binding . Deletion of residues 244-281, a region containing amino acids necessary for catalysis, gave a protein that bound to DNA . An XerD derivative containing residues 1-268 retained co-operative interactions with XerC; nevertheless, it did not support XerC strand exchange and was defective in XerD catalysis . Residues 1-283 retain a functional catalytic active site, though a protein lacking the five C-terminal amino acids was still unable to mediate normal in vivo recombination, indicating that these residues are needed for a function that is not directly related to DNA binding or catalysis.

Mol Microbiol, 1997 Jun, 24(5), 1039 - 48
Involvement of the amino-terminal phosphorylation module of UhpA in activation of uhpT transcription in Escherichia coli; Webber CA et al.; The UhpA protein is required for expression of the sugar phosphate transporter UhpT in Escherichia coli and is regulated by phosphate transfer from the transmembrane UhpBC sensor kinase complex . UhpA action requires the sensor kinase complex and the site of phosphorylation, Asp-54, under normal conditions, but not when UhpA is overexpressed . Directed mutagenesis of the uhpA gene allowed examination of the role of several residues of UhpA in response to phosphorylation and in transcription activation . Residues Asp-9, Asp-54, and Lys-101 are highly conserved and required for function in other response regulators . Changes at any of these residues in UhpA resulted in complete loss of phosphorylation-dependent activity, but did not affect the high-level, constitutive, UhpBC-independent expression when the UhpA variants were overexpressed . Thus, these residues are important for the response to the phosphorylation pathway but not for transcription activation . Eight independent uhpA mutants selected for activity in the absence of UhpBC function carried the F17-->V or H170-->Y substitutions . Other substitutions for Phe-17 conferred various phenotypes, ranging from inducible to high-level constitutive behaviour . Residues in helix-1 flanking Phe-17 were converted to Ala or other residues . Alanine substitutions at Val-13, Arg-14, and Leu-20 resulted in complete loss of phosphorylation-dependent activation . Change of Gly-16 to Ala had no effect, but changes to other residues resulted in loss of function . Alanine substitutions at Phe-17 and at Gln-19 resulted in high-level constitutive expression, and changes at Ala-18 and Leu-21 had only modest effects . Most interesting was the L20-->A substitution, which conferred low uhpT expression when overexpressed and interfered with action of the wild-type chromosomal allele . The combination of the L20-->A change with changes at Phe-17, Asp-54 and His-170 indicated that the trans-dominant action of L20-->A occurred at several steps . The observations that UhpA can activate uhpT transcription in its unphosphorylated state are consistent with its occupancy of low-affinity binding sites necessary for promoter function . We propose that the effect of phosphorylation of UhpA is to enhance its oligomerization on the DNA surface to extend to the low-affinity sites, and that helix-1 participates in the process of oligomer formation.

Mol Microbiol, 1997 Jun, 24(5), 1001 - 12
Regulation of the Escherichia coli K5 capsule gene cluster by transcription antitermination; Stevens MP et al.; The expression of the Escherichia coli K5 (group II) capsular polysaccharide requires the rfaH gene . By reverse transcriptase-polymerase chain reaction (RT-PCR) it was possible to demonstrate that RfaH increases the transcription of region 2 genes by readthrough transcription from the region 3 promoter . A mutation in the rfaH gene reduced this readthrough transcription from the region 3 promoter by 10-fold as measured by quantitative RT-PCR . The region 3 promoter was mapped to 741 bp 5' of the initiation codon of the kpsM gene . Deletion and insertion mutagenesis of the JUMPstart sequence, which is 28 bp 5' of kpsM and is conserved upstream of RfaH-regulated operons and other polysaccharide biosynthesis genes, confirmed that this sequence was required for expression of the K5 antigen and for the antitermination activity of RfaH . The JUMPstart sequence could cause RfaH-dependent antitermination at other Rho-dependent terminators, suggesting that the JUMPstart sequence may function in a manner analogous to a lambda nut site . On the basis of these results we propose a model by which RfaH regulates expression of E . coli group II capsule gene clusters by allowing readthrough transcription to proceed from region 3 into region 2 and that sequences within the JUMPstart sequence are essential for this process.

Mol Microbiol, 1997 Jun, 24(5), 991 - 1000
DNA-binding activity of the A-factor receptor protein and its recognition DNA sequences; Onaka H et al.; The A-factor receptor protein (ArpA) containing an alpha-helix-turn-alpha-helix DNA-binding consensus sequence at its N-terminal portion plays a key role in the regulation of secondary metabolism and cell differentiation in Streptomyces griseus . A binding site forming a palindrome 24bp in length was initially recovered from a pool of random-sequence oligonucleotides by rounds of a binding/immunoprecipitation/amplification procedure with histidine-tagged ArpA and anti-ArpA antibody . By means of further binding/gel retardation/amplification experiments on the basis of the recovered sequence, a 22 bp palindromic binding site with the sequence 5'-GG(T/C)CGGT(A/T)(T/C)G(T/G)-3' as one half of the palindrome was deduced as a consensus sequence recognized and bound by ArpA . ArpA did not bind to the binding site in the presence of its ligand, A-factor . In addition, exogenous addition of A-factor to the ArpA-DNA complex induced immediate release of ArpA from the DNA . All of these data are consistent with the idea, obtained from previous genetic studies, that ArpA acts as a repressor-type regulator for secondary metabolism and cellular differentiation by preventing the expression of a certain key gene(s) during the early growth phase . A-factor, produced in a growth-dependent manner, releases ArpA from the DNA, thus switching on the expression of the key gene(s), leading to the onset of secondary metabolism and aerial mycelium formation.

Mol Microbiol, 1997 Jun, 24(5), 927 - 36
Contribution of individual promoters in the ddlB-ftsZ region to the transcription of the essential cell-division gene ftsZ in Escherichia coli; Flardh K et al.; The essential cell-division gene ftsZ is transcribed in Escherichia coli from at least six promoters found within the coding regions of the upstream ddlB, ftsQ, and ftsA genes . The contribution of each one to the final yield of ftsZ transcription has been estimated using transcriptional lacZ fusions . The most proximal promoter, ftsZ2p, contributes less than 5% of the total transcription from the region that reaches ftsZ . The ftsZ4p and ftsZ3p promoters, both located inside ftsA, produce almost 37% of the transcription . An ftsAp promoter within the ftsQ gene yields nearly 12% of total transcription from the region . A large proportion of transcription (approximately 46%) derives from promoters ftsQ2p and ftsQ1p, which are located inside the upstream ddlB gene . Thus, the ftsQAZ genes are to a large extent transcribed as a polycistronic mRNA . However, we find that the ftsZ proximal region is necessary for full expression, which is in agreement with a recent report that mRNA cleavage by RNase E at the end of the ftsA cistron has a significant role in the contol of ftsZ expression.

Hybridoma, 1997 Jun, 16(3), 227 - 33
Primary structure and functional scFv antibody expression of an antibody against the human protooncogen c-myc; Fuchs P et al.; The immunoglobulin heavy- and light-chain variable region (Vh and Vl) genes were isolated from Myc1-9E10 hybridoma cells, which secreted monoclonal antibody against human oncogen c-myc . The expression vector pOPE52-c-myc was constructed for the recombinant production in E . coli . A 30 kDa single chain fragment (scFv) expression product was found in the periplasmic space by SDS-PAGE and immunoblotting . A significant fraction was processed correctly as demonstrated with an antiserum recognizing the processed aminoterminus only . The specific binding of the scFv fragment to the peptide epitope of the maternal monoclonal antibody was demonstrated and the primary sequence of the variable regions was determined . Sequence comparison with previously published partial Vh and Vl sequences from this hybridoma cell line revealed a genetic heterogeneity for the light chain variable region . The potential use of this scFv as a new tool for detection and purification of tagged proteins, for adding costimulatory signals to the surface of cancer cells as well as for analyzing c-myc function in the living cell by cytoplasmic expression is discussed.

EMBO J, 1997 Jun, 16(12), 3731 - 43
Action of site-specific recombinases XerC and XerD on tethered Holliday junctions; Arciszewska LK et al.; In Xer site-specific recombination, two related recombinases, XerC and XerD, mediate the formation of recombinant products using Holliday junction-containing DNA molecules as reaction intermediates . Each recombinase catalyses the exchange of one pair of specific strands . By using synthetic Holliday junction-containing recombination substrates in which two of the four arms are tethered in an antiparallel configuration by a nine thymine oligonucleotide, we show that XerD catalyses efficient strand exchange only when its substrate strands are 'crossed' . XerC also catalyses very efficient strand exchange when its substrate strands are 'crossed', though it also appears to be able to mediate strand exchange when its substrate strands are 'continuous' . By using chemical probes of Holliday junction structure in the presence and absence of bound recombinases, we show that recombinase binding induces unstacking of the bases in the centre of the recombination site, indicating that the junction branch point is positioned there and is distorted as a consequence of recombinase binding.

EMBO J, 1997 Jun, 16(12), 3724 - 30
Negative control of DNA replication by hydrolysis of ATP bound to DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli; Mizushima T et al.; DnaA protein, the initiation factor for chromosomal DNA replication in Escherichia coli, is activated by ATP . ATP bound to DnaA protein is slowly hydrolyzed to ADP, but the physiological role of ATP hydrolysis is unclear . We constructed, by site-directed mutagenesis, mutated DnaA protein with lower ATPase activity, and we examined its function in vitro and in vivo . The ATPase activity of purified mutated DnaA protein (Glu204-->Gln) decreased to one-third that of the wild-type DnaA protein . The mutation did not significantly affect the affinity of DnaA protein for ATP or ADP . The mutant dnaA gene showed lethality in wild-type cells but not in cells growing independently of the function of oriC . Induction of the mutated DnaA protein in wild-type cells caused an overinitiation of DNA replication . Our results lead to the thesis that the intrinsic ATPase activity of DnaA protein negatively regulates chromosomal DNA replication in E . coli cells.

EMBO J, 1997 Jun, 16(12), 3666 - 74
Repressor induced site-specific binding of HU for transcriptional regulation; Aki T et al.; Transcription from two overlapping gal promoters is repressed by Gal repressor binding to bipartite gal operators, O(E) and O(I), which flank the promoters . Concurrent repression of the gal promoters also requires the bacterial histone-like protein HU which acts as a co-factor . Footprinting experiments using iron-EDTA-coupled HU show that HU binding to gal DNA is orientation specific and is specifically dependent upon binding of GalR to both O(E) and O(I) . We propose that HU, in concert with GalR, forms a specific nucleoprotein higher order complex containing a DNA loop . This way, HU deforms the promoter to make the latter inactive for transcription initiation while remaining sensitive to inducer . The example of gal repression provides a model for studying how a 'condensed' DNA becomes available for transcription.

EMBO J, 1997 Jun, 16(12), 3416 - 25
Crystal structure of UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase from Escherichia coli; Bertrand JA et al.; UDP-N-acetylmuramoyl-L-alanine:D-glutamate ligase (MurD) is a cytoplasmic enzyme involved in the biosynthesis of peptidoglycan which catalyzes the addition of D-glutamate to the nucleotide precursor UDP-N-acetylmuramoyl-L-alanine (UMA) . The crystal structure of MurD in the presence of its substrate UMA has been solved to 1.9 A resolution . Phase information was obtained from multiple anomalous dispersion using the K-shell edge of selenium in combination with multiple isomorphous replacement . The structure comprises three domains of topology each reminiscent of nucleotide-binding folds: the N- and C-terminal domains are consistent with the dinucleotide-binding fold called the Rossmann fold, and the central domain with the mononucleotide-binding fold also observed in the GTPase family . The structure reveals the binding site of the substrate UMA, and comparison with known NTP complexes allows the identification of residues interacting with ATP . The study describes the first structure of the UDP-N-acetylmuramoyl-peptide ligase family.

Mol Microbiol, 1997 Jun, 24(6), 1303 - 10
Proteolysis of the phage lambda CII regulatory protein by FtsH (HflB) of Escherichia coli; Shotland Y et al.; Rapid proteolysis plays an important role in regulation of gene expression . Proteolysis of the phage lambda CII transcriptional activator plays a key role in the lysis-lysogeny decision by phage lambda . Here we demonstrate that the E . coli ATP-dependent protease FtsH, the product of the host ftsH/hflB gene, is responsible for the rapid proteolysis of the CII protein . FtsH was found previously to degrade the heat-shock transcription factor sigma32 . Proteolysis of sigma32 requires, in vivo, the presence of the DnaK-DnaJ-GrpE chaperone machine . Neither DnaK-DnaJ-GrpE nor GroEL-GroES chaperone machines are required for proteolysis of CII in vivo . Purified FtsH carries out specific ATP-dependent proteolysis of CII in vitro . The degradation of CII is at least 10-fold faster than that of sigma32 . Electron microscopy revealed that purified FtsH forms ring-shaped structures with a diameter of 6-7 nm.

Mol Microbiol, 1997 Jun, 24(6), 1263 - 73
ftsW is an essential cell-division gene in Escherichia coli; Boyle DS et al.; In the absence of exogenous promoters, plasmid-mediated complementation of the temperature-sensitive ftsW201 allele requires the presence of the full coding sequence of ftsW plus upstream DNA encompassing the C-terminus of mraY and the full coding sequence of murD . We used molecular and genetic techniques to introduce an insertional inactivation into the chromosomal copy of ftsW, in the presence of the plasmid-borne wild-type ftsW gene under the control of P(BAD) . In the absence of arabinose, the ftsW-null strain is not viable, and a shift from arabinose- to glucose-containing liquid medium resulted in a block in division, followed by cell lysis . Immunofluorescence microscopy revealed that in ftsW-null filaments, the FtsZ ring is absent in 50-60% of filaments, whilst between one and three Z-rings per filament can be detected in the remainder of the population, with the majority of these containing only one Z-ring per filament . We also demonstrated that the expression of only ftsWS (the smaller of two ftsW open reading frames) from P(BAD) is sufficient for complementation of the ftsW-null allele . We conclude that FtsW is an essential cell-division protein in Escherichia coli, and that it plays a role in the stabilization of the FtsZ ring during cell division.

Mol Microbiol, 1997 Jun, 24(6), 1225 - 34
Isolation of a dnaE mutation which enhances RecA-independent homologous recombination in the Escherichia coli chromosome; Bierne H et al.; The mechanism of recombination of tandem repeats in the chromosome of Escherichia coli was investigated by genetic means . Tandem repeats 624 bp long were introduced into the lacZ gene of E . coli and the efficiency of deletion of one repeat was compared in different recombination mutants . No effects of the recA, recBC, recF, ruvA or ruvA recG mutations were detected . Hence, tandem repeat deletion appears to not proceed via the RecBCD or RecF homologous recombination pathways . A new mutant in which RecA-independent recombination is increased 15-fold was isolated . The mutation lies in the dnaE gene coding for the alpha subunit of polymerase III: it is a Gly to Asp change at codon 133 . Another dnaE mutation, dnaE486, was tested and also shown to stimulate RecA-independent recombination . It is proposed that tandem-repeat recombination occurs by a replication slippage mechanism . RecA-independent recombination is also enhanced in a rep mutant, in which chromosomal replication is slowed down by the absence of the Rep helicase, suggesting that replication pausing may facilitate slippage.

Mol Microbiol, 1997 Jun, 24(6), 1201 - 13
Cloning and analysis of a Borrelia burgdorferi membrane-interactive protein exhibiting haemolytic activity; Guina T et al.; We cloned the gene encoding a membrane-interactive protein of Borrelia burgdorferi by means of its haemolytic activity in Escherichia coli . The haemolytic activity was erythrocyte-species specific, with progressively decreasing activity for erythrocytes from horse, sheep, and rabbit, respectively . Genetic analysis of the haemolytic determinant revealed two borrelia haemolysin genes, blyA and blyB, that are part of a predicted four-gene operon which is present in multiple copies on the 30 kb circular plasmid(s) of B . burgdorferi B31 . blyA encodes a predicted alpha-helical 7.4 kDa protein with a hydrophobic central region and a positively charged C-terminus, which is structurally homologous to a large group of pore-forming toxins with cytolytic activity . blyB encodes a soluble protein which stabilized BlyA and enhanced haemolytic activity . While the majority of BlyA in E . coli was membrane-associated, only soluble protein was haemolytically active . The haemolytic activity was shown to be highly protease sensitive, heat labile, independent of divalent cations, and extremely dependent on protein concentration, consistent with a requirement for oligomerization as the mechanism of action . BlyA was highly purified from E . coli in a single step utilizing Triton X-114 phase partitioning . Genetic analysis of blyA and blyB mutants indicated that the stability, membrane association, and activity of BlyA was dependent on subtle changes in its sequence and on the BlyB protein . The bly genes were found to be expressed at a very low level in cultured B . burgdorferi.

Mol Microbiol, 1997 Jun, 24(6), 1157 - 68
Genes involved in conjugative DNA processing of plasmid R6K; Nunez B et al.; The conjugative transfer region of the IncX plasmid R6K (TRA(x)) was analysed by transposon mutagenesis and DNA sequencing . Tn5tac1 insertional mutations localized TRA(x) to a 14.8 kb segment containing the alpha origin of transfer (oriT alpha), genes involved in conjugative DNA-processing (Dtr(x)) and genes involved in pilus synthesis and assembly (Mpf(x)) . A second functional oriT, oriTbeta, was located at a distance of 5.3 kb from oriT alpha and was outside TRA(x) . Mpf(x) occupied a segment of 10kb, as judged by the location of insertions conferring resistance to infection by the X pilus-specific phage X-2 . At both sides of Mpf(x) there were insertions that were Tra but X-2 sensitive, suggesting that the mutations were in Dtr(x) . This region was sequenced and three genes were identified: taxA, taxB, and taxC . The overall organization was oriT alpha-taxA-taxC-Mpf(x)-taxB . taxC coded for a oriT-relaxase that belongs to the VirD2 family . taxA coded for a protein of 181 amino acids that showed similarity to TraY of F-like plasmids and to the Arc-repressor superfamily . TaxB showed similarity to TraG-like proteins, a protein superfamily probably involved in coupling the relaxosome to the DNA-transport apparatus . TaxA and TaxC are required for oriT nicking in vivo . The nicking reaction was mistakenly assumed by Flashner et al . (1996) to represent a feature of the vegetative replication origins . However, insertions or deletions disrupting taxA and taxC affected conjugation but not replication of R6K . Conversely, protein pi, which is absolutely required for replication of R6K, was not required for conjugative transfer . In addition, protein DDP3, which is also assumed to have a role in replication, was found to be a positive modulator of bacterial conjugation . Taken together, these results rule out a direct and essential involvement of conjugation proteins in R6K vegetative replication, and also rule out the requirement of replication protein pi for conjugation.

Mol Microbiol, 1997 Jun, 24(6), 1143 - 56
DNA binding of Escherichia coli arginine repressor mutants altered in oligomeric state; Chen SH et al.; The Escherichia coli arginine repressor (ArgR) controls expression of the arginine biosynthetic genes and acts as an accessory protein in Xer site-specific recombination at cer and related plasmid recombination sites . The hexameric wild-type protein shows L-arginine-dependent DNA binding . In this work, ArgR mutants that are defective in trimer-trimer interactions and bind DNA as trimers in an L-arginine-independent manner are isolated and characterized . Whereas the wild-type ArgR hexamer exhibits high-affinity binding to two repeated ARG boxes separated by 3 bp (each ARG box containing two identical dyad symmetrical 9 bp half-sites), the trimeric mutants bind to and footprint three adjacent half-sites of this 'idealized' substrate . Trimeric ArgR is impaired in its ability to repress the arginine biosynthetic genes and in Xer site-specific recombination . In the absence of L-arginine, residual wild-type ArgR-binding occurs as trimers . The binding of an N-terminal 77-amino-acid DNA-binding domain to idealized ARG boxes is also characterized.

Exp Neurol, 1997 Jun, 145(2 Pt 1), 546 - 54
Delivery of recombinant tetanus-superoxide dismutase proteins to central nervous system neurons by retrograde axonal transport; Figueiredo DM et al.; The nontoxic C fragment of tetanus toxin (TC) can transport other proteins from the circulation to central nervous system (CNS) motor neurons . Increased levels of CuZn superoxide dismutase (SOD) are protective in experimental models of stroke and Parkinson's disease, whereas mutations in SOD can cause motor neuron disease . We have linked TC to SOD and purified the active recombinant proteins in both the TC-SOD and SOD-TC orientations . Light microscopic immunohistochemistry and quantitative enzyme-linked immunosorbant assays (ELISA) of mouse brainstem, after intramuscular injection, demonstrate that the fusion proteins undergo retrograde axonal transport and transsynaptic transfer as efficiently as TC alone.

J Struct Biol, 1997 Jun, 119(1), 28 - 34
Crystallization and preliminary X-ray analysis of the single-headed and double-headed motor protein kinesin; Kozielski F et al.; Crystals of the single-headed and double-headed kinesin motor domains of Rattus norvegicus have been grown by vapor diffusion using ammonium sulfate as the precipitant . Both crystal systems belong to the orthorhombic space group P2(1)2(1)2(1) . Double-headed kinesin crystallized with unit cell constants of a = 72.2 A, b = 91.9 A, and c = 141.7 A, and so far the best crystals diffracted to a maximum resolution of 2.7 A . Using ammonium sulfate single-headed kinesin crystallized in two different crystal forms with cell constants of a = 73.1 A, b = 73.2 A, c = 84.0 A and a = 73.4 A, b = 74.1 A, c = 74.7 A, respectively . They were found to diffract to 2.1 A resolution . Crystals of monomeric kinesin were also obtained with lithium sulfate as precipitant . They have cell constants of a = 71.6 A, b = 73.7 A, and c = 74.1 A and diffract up to 1.7 A resolution.

Eur J Neurosci, 1997 Jun, 9(6), 1105 - 16
Cloning and characterization of a neural cell recognition molecule on axons of the retinotectal system and spinal cord; Brummendorf T et al.; Immunoglobulin superfamily molecules in the brain are involved in distinct aspects of nervous system histogenesis, for example neuronal migration and axonal growth . To identify novel members of this superfamily in the chick nervous system, we developed a polymerase chain reaction-based approach making use of sequence motifs of immunoglobulin-like domains . In the present study, we report the molecular cloning of three isoforms, the biochemical analysis and the immunohistochemical characterization of one of the proteins identified in this screen . This molecule has 91% sequence identity with the limbic system-associated membrane protein (LAMP) characterized in the rat and is therefore referred to as the chicken homologue of the latter (chLAMP) . The molecule is a glycosylphosphatidyl-inositol-anchored 60 kDa protein with three immunoglobulin-like domains and contains 40% N-linked carbohydrate . We identify three different mRNA forms of chLAMP and show that two forms with distinct 5'-termini are differentially transcribed in neural development . In addition, we demonstrate using a fusion protein expressed in eukaryotic cells that chLAMP has homophilic binding activity . The protein was found on a subset of axons in the central and peripheral nervous system and is likely to be involved in specific cell-cell interactions in neurohistogenesis.

J Lipid Res, 1997 Jun, 38(6), 1139 - 48
Cloning, expression, and chromosomal localization of mouse liver bile acid CoA:amino acid N-acyltransferase; Falany CN et al.; A mouse liver lambda Zap XR cDNA library was screened using the coding region of human bile acid CoA:amino acid N-acyltransferase (BAT) cDNA as a probe . Ten positive clones were isolated and purified, two of which apparently possessed complete open reading frames for BAT based on sequence analysis of the ends of the cDNAs . One clone (mBAT#9) was selected for sequence analysis and characterization . mBAT#9 is 1869 basepairs in length and the full-length cDNA possesses a 189 basepair 5'-nontranslated region, an open-reading frame of 1260 basepairs, and a 404 basepair 3'-nontranslated region followed by a poly(A) tail . The open-reading frame codes for a 420 amino acid protein with a calculated molecular mass of 46,525 daltons . The structural gene for mBAT was mapped to mouse Chromosome 4 . The amino acid sequence of mBAT is 69% identical and 84% similar to that of hBAT, and 86% identical and 95% similar to that of kan-1, a putative rat liver BAT . Enzymatically active mBAT was expressed in E . coli using the bacterial expression vector pKK233-2 . Immunoblot analysis of expressed mBAT with rabbit anti-human BAT polyclonal antibodies detected a single protein with a molecular mass of approximately 45,000 daltons . Cytosol from cells transformed with mBAT#9/pKK233-2 possessed significant amounts of BAT-catalyzed conjugating activity with taurine as substrate but the expressed enzyme did not use glycine or fluoro-beta-alanine as substrates . The K(m) value for taurine was 1.9 mM +/- 0.1 mM in reactions with cholyl CoA as a cosubstrate . The specificity of mBAT for taurine as a substrate was confirmed by the demonstration, using HPLC-electrospray ionization mass spectrometry, that mouse gallbladder bile contained only taurine conjugates of bile acids . The identification of the types of amino acid conjugates of bile acids present in mouse bile had not been previously reported . These results indicate that a taurine-specific form of BAT has been cloned and expressed from mouse liver.

J Allergy Clin Immunol, 1997 Jun, 99(6 Pt 1), 781 - 7
Mapping of IgE-binding epitopes on the recombinant major group I allergen of velvet grass pollen, rHol l 1; Schramm G et al.; BACKGROUND: New and more successful approaches to diagnosis and therapy of allergic diseases require a more subtle understanding of the structure and the epitopes on the allergen molecule . OBJECTIVE: This study was done to obtain more information on the structure and the IgE-binding epitopes of a major allergen of velvet grass pollen, Hol l 1 . METHODS: We cloned Hol l 1 from a complementary DNA library and performed B-cell epitope mapping with 21 recombinant fragments expressed as fusion proteins in Escherichia coli . The fragments were analyzed by Western blotting with sera from 50 different patients . RESULTS: The patients' sera individually recognized at least four different IgE-binding regions (amino acids 1 to 27, 61 to 76, 84 to 105, and 158 to 240) . According to their binding patterns with these epitopes, they were divided into five groups . Most sera (92%) bound to the C-terminal peptide (158 to 240), which consists of more than 80 amino acids, whereas there was virtually no binding to smaller fragments covering this region . In contrast to the C-terminal peptide, the IgE-binding peptides on the N terminus and on the middle region of the molecule were of a smaller size (15 to 30 amino acids) . CONCLUSIONS: The major group I allergen of velvet grass bears at least four different IgE-binding epitopes, which were individually recognized by sera from different patients . The C terminus represents the major IgE-binding region and contains at least one discontinuous IgE-binding epitope, whereas the N terminus and middle region of Hol l 1 seem to contain continuous IgE-binding epitopes.

Biosci Biotechnol Biochem, 1997 Jun, 61(6), 1041 - 3
Cloning, expression, and mutagenesis of trypsin inhibitor ETIb from Erythrina variegata seeds; Kouzuma Y et al.; Erythrina variegata trypsin inhibitors designated ETIa and ETIb belong to the Kunitz family trypsin inhibitor, but ETIa is unique in its ability to inhibit tissue-type plasminogen activator, while ETIb is not . The cDNA clone encoding ETIb was isolated from the seed cDNA library constructed in the lambda phage lambda gt11 . The ETIb cDNA insert consists of 765 bp, including an open reading frame of 606 pb from ATG to TGA codons . The deduced amino acid sequence consists of 202 amino acids, having the signal peptides of 22 amino acids in the N-terminus and 2 amino acids in C-terminus . The cDNA fragment encoding the mature form of ETIb was introduced into an expression vector, pET-22b, and expressed in Escherichia coli BL21 (ED3) in a functional form . Furthermore, the ETIb mutant bP61R/F62L, in which Pro61 and Phe62 in ETIb were changed to the corresponding amino acid residues Arg and Leu, respectively, as in ETIa, was constructed, and its inhibitory potency toward tPA was assayed . This mutant showed significant tPA inhibitory activity, albeit less than ETIa . The result demonstrates that the Arg61 and Leu62 residues in ETIa are important in inhibiting tPA, and also suggest that beside these two residues, the other amino acid(s) or other structural element may be involved in interaction of ETIa with tPA.

Virus Res, 1997 Jun, 49(2), 147 - 54
Analysis of the conformational change of recombinant human papilloma virus type 18 E7 protein induced by metal binding; Kang JH et al.; Human papillomavirus (HPV) type 18 E7 gene was isolated by polymerase chain reaction (PCR) amplification from tissues of Korean cervical cancer patients and cloned into a plasmid vector, pET-3a, for the expression of recombinant E7 protein (rE7) in Escherichia coli . The rE7 protein was purified to the homogeneity and its purity was confirmed by HPLC . The purified protein was analyzed for the metal-binding properties by UV spectroscopy and it was shown that two Cd2+ or Zn2+ ions bind to one E7 protein by the metal-sulfur ligand formation via two Cys-X-X-Cys motifs in E7 protein . When the change of intrinsic fluorescence of tryptophan residue was analyzed for rE7-Zn complex, the blue shift of emission wavelength and the decrease in maximum intensity of emission were observed compared with rE7 . These results suggest that Zn(2+)-bound rE7 has undergone conformational change, in which a tryptophan residue located in the second Cys-X-X-Cys motif was moved into solvent-inaccessible or hydrophobic environment . The rE7-Zn complex was found to be resistant to chymotrypic digestion by comparing the digestion patterns of rE7 . Therefore, we showed the folding status of HPV 18 E7 could be changed by metal binding resulting in a different conformation in which a tryptophan residue was driven into more hydrophobic environment and the resistancy to chymotryptic digestion was conferred.

Curr Genet, 1997 Jun, 31(6), 525 - 9
Disparate sequence characteristics of the Erysiphe graminis f.sp . hordei glyceraldehyde-3-phosphate dehydrogenase gene; Christiansen SK et al.; The Erysiphe graminis f.sp . hordei (Egh) glyceraldehyde-3-phosphate dehydrogenase (gpd) gene was isolated and characterized . It contains typical promoter elements and has three introns, one of which is positioned in the 5' untranslated region of the gene . The deduced amino-acid sequence has 87% similarity to gpd genes from other Ascomycete fungi . This is at the same level as previously estimated among these fungi . Comparison at the DNA level reveal similarities of only around 70%, which is 10% lower than previously reported . In an evolutionary tree based on the sequences from 18 fungal gpd genes, Egh falls into the group of Ascomycetes located at a basal position . The regulatory region of the Egh gpd gene has no homology to corresponding sequences in other filamentous Ascomycetes . Codon usage was determined for the four characterized Egh genes (tub2, Egh7, Egh16 and gpd) and found to be similar for all four genes . The results of the codon-usage analysis suggest that Egh is more flexible than other fungi in the choice of nucleotides at the wobble position . Codon-usage preferences in Egh and barley genes indicate a level of difference which may be exploited to discriminate between fungal and plant genes in sequence mixtures . The Egh gpd promoter appears to be superior to that of the Egh beta-tubulin gene (tub2) for driving the E . coli beta-glucuronidase (GUS) gene in transformation experiments.

Eur J Biochem, 1997 Jun 1, 246(2), 565 - 73
Structural studies of the O-antigenic polysaccharide from Escherichia coli O167; Linnerborg M et al.; The structure of the O-antigenic polysaccharide from Escherichia coli O167:H5 has been investigated . Sugar and methylation analyses, fast-atom-bombardment mass spectrometry and 1H- and 13C-NMR spectroscopy were the main methods used . The structure of the repeating unit of the polysaccharide was found to be: {formula in text} . Oligosaccharide derivatives of the polysaccharide were obtained by HF solvolysis and by a Smith degradation . Furthermore, base treatment of the polysaccharide led to a degraded polymeric material . For the methylated polysaccharide the amide linkage between alanine and the galacturonic acid residue was reductively cleaved with LiBD4 in ethanol, to give, among other things, a 3-O-methyl galactose derivative.

Eur J Biochem, 1997 Jun 1, 246(2), 548 - 56
Biochemical characterization of purified, human recombinant Lys304-->Glu medium-chain acyl-CoA dehydrogenase containing the common disease-causing mutation and comparison with the normal enzyme; Kieweg V et al.; Recombinant, normal human medium-chain acyl-CoA dehydrogenase (MCADH) and the common, human disease-causing K304E mutant ({Glu304}MCADH) protein were expressed in Escherichia coli using an optimized system, and the enzymes were purified to apparent homogeneity . The crucial factor leading to the production of active {Glu304}MCADH protein is the expression in E . coli cells at reduced temperature (28 degrees C) . Expression in the same system at 37 degrees C results in very low amounts of active mutant protein . Several catalytic and physicochemical parameters of these two proteins have been determined and were compared to those of purified pig kidney MCADH . Although {Glu304}MCADH has approximately the same rate of substrate reduction with dodecanoyl-CoA and the same V(max) as human MCADH with the best substrate for the latter, octanoyl-CoA, the K(m) in the mutant MCADH is fourfold higher, which generates a correspondingly lower catalytic efficiency . Importantly, V(max) obtained using the natural acceptor, electron transfer flavoprotein, is only a third that for human MCADH . The V(max)/K(m) versus chain-length profile of the mutant shows a maximum with dodecanoyl-CoA which differs markedly from that of human MCADH, which has maximal efficiency with octanoyl-CoA . The substrate specificity of the mutant is broader with a less pronounced activity peak resembling long-chain acyl-CoA dehydrogenase . The purified mutant enzyme exhibits a reduced thermal stability compared to human wild-type MCADH . The major difference between the two proteins expressed in E . coli is the more pronounced lability of the K304E mutant in crude extracts, which suggests a higher susceptibility to attack by endogenous proteases . Differences between tetrameric {Glu304}MCADH which survives the first step(s) of purification and corresponding MCADH are minor . The overall differences in properties of {Glu304}MCADH together with its impaired folding and tetramer assembly may contribute to the generation of the abnormalities observed in patients homozygous for the K304E mutation.

Eur J Biochem, 1997 Jun 1, 246(2), 452 - 60
Evolution of the enzymatic characteristics of C4 phosphoenolpyruvate carboxylase--a comparison of the orthologous PPCA phosphoenolpyruvate carboxylases of Flaveria trinervia (C4) and Flaveria pringlei (C3); Svensson P et al.; C4 phosphoenolpyruvate (P-pyruvate) carboxylases have evolved from ancestral C3 P-pyruvate carboxylases during the evolution of C4 photosynthesis (Lepiniec et al., 1994) . To meet the requirements of a new metabolic pathway, the C4 enzymes have gained distinct kinetic and regulatory properties compared to C3 enzymes . Our interest is to deduce the structure responsible for these C4-specific properties . As a model system, the orthologous ppcA P-pyruvate carboxylases of Flaveria trinervia (C4) and Flaveria pringlei (C3) were investigated by expressing them in Escherichia coli using their cDNAs . The K(m) (P-pyruvate) was about ten times higher for the C4 enzyme (650 microM) than for the C3 enzyme (60 microM) . The activation by glucose 6-phosphate, which was shown by a decrease in the K(m) (P-pyruvate), was about twice for the C4 enzyme and three times for the C3 enzyme . The C3 enzyme showed a very high sensitivity to L-malate with an I(0.5) (50% inhibition) value of 80 microM malate, whereas the C4 enzyme was much less sensitive with a I(0.5) value of 1.2 mM malate . To locate the structural positions responsible for these differences in kinetic and regulatory properties, chimeras of these 95% identical enzymes were made . In this study, the first 437 residues of the 966-amino-acid protein were interchanged . The results showed that the N-terminal part of the enzyme was responsible for a small but significant part of the kinetic difference observed between these two isoenzymes . Additionally, the results suggest that the N-terminus was the site for glucose 6-phosphate activation and was also responsible for the observed difference in activation by this sugar phosphate . The difference in inhibition by L-malate, however, is suggested to originate mainly from the C-terminal part of the enzyme.

Eur J Biochem, 1997 Jun 1, 246(2), 425 - 32
Cloning and characterization of a testis-specific, developmentally regulated A-kinase-anchoring protein (TAKAP-80) present on the fibrous sheath of rat sperm; Mei X et al.; cAMP is important for the initiation of mammalian sperm motility . Previously we established that a type II cAMP-dependent protein kinase is tightly associated with the fibrous sheath of rat sperm . This unique cytoskeletal structure surrounds the 9+2 axonemal network in the principal piece of the flagellum . Association of the kinase to the fibrous sheath is mediated via its regulatory subunit, RII . An RII-binding overlay procedure was used to document that RII could specifically associate with fibrous sheath polypeptides of 120 and 80 kDa . In this study, we report the cloning of a rat testis-specific, developmentally regulated, RII-binding protein (TAKAP-80) . A 1.2-kb cDNA clone, isolated by screening a rat testis expression library with 32P-labeled RII, hybridized to a 1.8-kb mRNA transcript present exclusively in testis . This transcript appeared at detectable levels at 30 days after birth . Over the next 10 days the mRNA levels increased greatly . This time interval corresponds to the initiation of spermiogenesis . The complete nucleotide sequence of TAKAP-80 cDNA was obtained by polymerase chain reaction and contained a continuous open reading frame of 502 amino acids . The deduced amino acid sequence showed a clear demarcation of charged and hydrophobic amino acid residues . Amino acids 1-147 of the protein contained 45% charged residues, with lysine and arginine predominating . Similarly, amino acids 268-502 also contained a high percentage of charged amino acids (35%) . In contrast, amino acids 148-267 were mostly hydrophobic and contained clusters of a repeating PXXP motif where X was predominantly valine and alanine or sometimes proline . The 1.2-kb cDNA clone was inserted into the pRSET vector and expressed as a His6 tag fusion protein in Escherichia coli . The recombinant protein was soluble and bound RIIalpha, RIIbeta and type IIalpha holoenzyme by the RII-binding overlay procedure . Deletion analysis revealed that the high-affinity interaction site for RII was contained within amino acids 258-378 of TAKAP-80 . Antibodies prepared against the fusion protein recognized an 80-kDa protein present in the urea-insoluble particulate fraction of rat testis and in purified fibrous sheath preparations isolated from rat epididymal sperm . Levels of the 80-kDa immunoreactive protein were significantly higher in mature (60 days old) compared with immature (30 days old) rat testis, correlating with the mRNA levels.

Eur J Biochem, 1997 Jun 1, 246(2), 350 - 9
Expression of properly folded human glutamate decarboxylase 65 as a fusion protein in Escherichia coli; Papouchado ML et al.; Autoantibodies to the islet-cell 65-kDa variant of glutamate decarboxylase (GAD65) are found in most insulin-dependent diabetes mellitus (IDDM) patients many years before the appearance of clinical symptoms of the disease . As IDDM-preventive therapies may be available in the future, an international effort is taking place to develop widely applicable anti-GAD immunochemical tests . These tests would help to detect individuals at risk before the full installation of the disease and to enroll them in prevention programs . Autoantibodies to GAD65 are mostly directed to conformational epitopes, and the enzyme is a complex molecule with a prosthetic group and 15 cysteine residues . Thus, the conformational integrity of GAD65 is essential for an appropriate anti-GAD assay . Isolation of large amounts of GAD65 from pancreas or other tissues is impractical, and no successful production of properly folded GAD65 has been reported in bacteria . Native recombinant GAD65 for immunochemical tests is usually obtained from eukaryotic expression systems . Since the large-scale production of a recombinant protein in an eukaryotic system is expensive and technically difficult, we investigated the expression of GAD65 in Escherichia coli as an alternative . A number of DNA constructs intended to export the enzyme to the periplasmic space or to improve its cytoplasmic solubility were designed and tested . Our results provide a solution to the two main problems associated with the expression of GAD65 in E . coli: misfolding, leading to the formation of inclusion bodies; and the presence of alternative initiation sites for translation that causes the preferential production of truncated variants of GAD65 . We describe here the production of properly folded, fully active, and immunochemically competent GAD65 as an N-terminal fusion protein with thioredoxin . An account of the reactivity of the produced protein with sera of six IDDM patients is also presented.

Eur J Biochem, 1997 Jun 1, 246(2), 301 - 10
1H, 15N and 13C NMR assignments, secondary structure and overall topology of the Escherichia coli GlgS protein; Beglova N et al.; GlgS is a 7892-Da protein which is involved in glycogen biosynthesis in bacteria . We report the 1H, 15N and 13C NMR assignments of the backbone and side-chain resonances at 25 degrees C and pH 6.7 from two-dimensional homonuclear and three-dimensional heteronuclear NMR experiments . The secondary structure of the protein was determined using sequential and medium-range NOE correlations, vicinal 3J(NH-H alpha) coupling values and amide proton exchange rates . The secondary structure obtained is consistent with the secondary chemical shifts of 1H alpha, 13C alpha and 13C = O . It was found that the secondary structure of GlgS comprises two amphipathic helices (Asn10-Met21 and Glu39-Arg60), one short highly hydrophobic helix (Ile30-Val33), a short extended beta-strand-like fragment (Arg26-Asp29) and two type I beta-turns (His22-Gly25 and Thr34-Met37) . An overall topology of GlgS is suggested based on long-range NOEs . The elements of secondary structure form a sandwich in which the beta-strand and the short hydrophobic helix are positioned between the two amphipathic helices.

Semin Oncol, 1997 Jun, 24(3 Suppl 9), S9 - 41-S9-51
Molecular and biologic characterization of recombinant interferon-alpha2b; Bordens R et al.; Recombinant IFN-alpha2b (Intron A, Schering-Plough Corp, Kenilworth, NJ), derived from Escherichia coli, is currently approved worldwide for the therapy of various cancers and chronic hepatitis B and C . We describe here the purity and identity tests for both physicochemical properties and bioactivity that have been developed for the characterization of highly purified IFN-alpha2b with a specific activity of 2.5 x 10(8) IU/mg protein . The data indicate that a product of high purity can be consistently produced without DNA contamination . The antiviral bioassay chosen to measure potency is based on the ability of IFN-alpha2b to protect human foreskin fibroblast FS-71 cells from the cytopathic effects of encephalomyocarditis virus . Various immunoassays are described that have been used to quantitate the presence of binding and neutralizing antibodies in patients treated with IFN-alpha2b . Overall, the available data indicate a very low incidence of neutralizing antibody formation . We also summarize the current state of knowledge with respect to IFN reference standards for the biologic assay as well as recommendations for the calculation of antibody neutralization titers.

Eur J Pediatr, 1997 Jun, 156(6), 493 - 8
Inhibition of enteropathogenic Escherichia coli adhesion to HEp-2 cells by colostrum and milk from mothers delivering low-birth-weight neonates; Delneri MT et al.; Breast milk samples from three groups of Brazilian women were evaluated for their inhibitory effect on enteropathogenic Escherichia coli (EPEC) adhesion to HEp-2 cells: G1, mothers delivering preterm babies of appropriate birth weight (n = 12); G2, mothers delivering term babies of low birth weight (n = 11); G3, the control group, mothers delivering term babies of appropriate birth weight (n = 39) . Colostrum samples were obtained at 48-72 h and milk samples on the 7th, 30th and 60th days after delivery . All samples showed strong inhibitory activity (66%-100%), without significant differences among the three groups and four periods . Total IgA and anti-EPEC IgA concentrations were significantly higher in colostrum than in milk samples in the three groups studied . The levels of colostral IgA and anti-EPEC IgA observed in G1 and G2 were significantly higher compared to the control group . Western blotting assays showed that individual samples as well as pools of colostrum or milk samples contain IgA antibodies to many EPEC outer membrane proteins . A 94 kDa band with molecular weight consistent with the EPEC adhesin named intimin; was recognized by all samples analysed . Bands of different molecular weight were also recognized by some samples of colostrum and milk, such as a band of approximately 18.4 kDa, with molecular weight equivalent to bundle-forming pilus subunits . CONCLUSION: Our results suggest that colostrum and milk from mothers of premature and small-for-date term neonates are as effective in protecting the newborn against EPEC infections as those from mothers of term babies of appropriate birth weight.

Br J Pharmacol, 1997 Jun, 121(4), 695 - 704
Effect of calpain inhibitor I, an inhibitor of the proteolysis of I kappa B, on the circulatory failure and multiple organ dysfunction caused by endotoxin in the rat; Ruetten H et al.; 1 . We compared the effects of calpain inhibitor I (inhibitor of the proteolysis of I kappa B and, hence, of the activation of nuclear factor kappa B (NF kappa B) and dexamethasone on (i) the circulatory failure, (ii) multiple organ dysfunction and (iii) induction of the inducible isoforms of nitric oxide (NO) synthase (iNOS) and cyclo-oxygenase (COX-2) in anaesthetized rats with endotoxic shock . 2 . Injection of lipopolysaccharide (LPS, E . coli, 10 mg kg-1, i.v.) resulted in hypotension and a reduction of the pressor responses elicited by noradrenaline . This circulatory dysfunction was attenuated by pretreatment of LPS-rats with calpain inhibitor I (10 mg kg-1, i.v., 2 h before LPS) or dexamethasone (1 mg kg-1, i.v.) . 3 . Endotoxaemia also caused rises in the serum levels of (i) urea and creatinine (renal dysfunction), (ii) alanine aminotransferase (ALT), aspartate aminotransferase (AST) (hepatocellular injury), bilirubin and gamma-glutamyl transferase (gamma GT) (liver dysfunction), (iii) lipase (pancreatic injury) and (iv) lactate . Calpain inhibitor I and dexamethasone attenuated the liver injury, the pancreatic injury, the lactic acidosis as well as the hypoglycaemia caused by LPS . Dexamethasone, but not calpain inhibitor I, reduced the renal dysfunction caused by LPS . 4 . Endotoxaemia for 6 h resulted in a substantial increase in iNOS and COX-2 protein and activity in lung and liver, which was attenuated in LPS-rats pretreated with calpain inhibitor I or dexamethasone . 5 . Thus, calpain inhibitor I and dexamethasone attenuate (i) the circulatory failure, (ii) the multiple organ dysfunction (liver and pancreatic dysfunction/injury, lactic acidosis, hypoglycaemia), as well as (iii) the induction of iNOS and COX-2 protein and activity in rats with endotoxic shock . We propose that prevention of the activation of NF-kappa B in vivo may be useful in the therapy of circulatory shock or of disorders associated with local or systemic inflammation.

Epidemiol Infect, 1997 Jun, 118(3), 199 - 205
Origin and characteristics of enteroinvasive strains of Escherichia coli (EIEC) isolated in Germany; Beutin L et al.; Thirty-five E . coli strains belonging to O-serogroups with enteroinvasive types of Escherichia coli (EIEC) isolated in Germany between 1989 and 1995 were investigated for invasivity-associated DNA sequences . Only 11 strains were positive for ipaH and thus confirmed as EIEC . All 11 EIEC isolates originated from human infections which were imported to Germany from Eastern Europe . EIEC O124 were most frequent and originated from asymptomatic Romanians arriving at Rostock, Germany in 1992 and 1993 . In January 1993, EIEC O124 were isolated from faeces of a laboratory technician with diarrhoea working at the enteric pathogen department of the Institute of Hygiene in Rostock . By comparing her E . coli O124 isolate with recently imported O124 strains for Xba I restriction fragment length polymorphisms (RFLP) the probable source of infection could be determined . Four major RFLP patterns were found in the group of O124 strains . O124 strains with identical RFLP patterns were found in the group of 0124 strains . 0124 strains with identical RFLP patterns were isolated from people who were in close contact to each other.

Curr Opin Struct Biol, 1997 Jun, 7(3), 343 - 7
The conformation of ribosomes and rRNA; Moore PB; During the past 18 months, electron microscopists have published two reconstructions of the Escherichia coli ribosome, independently derived from images of unstained particles . The resolutions of their images are 20-25 A-much higher than any previously available . During the same time, NMR spectroscopists have provided an atomic-resolution model for the A-site region of 16S rRNA complexed with paromomycin that explains much of what is known about the interaction of aminoglycoside antibiotics with ribosomes.

J Endocrinol, 1997 Jun, 153(3), 373 - 84
Rabbit sex hormone binding globulin: primary structure, tissue expression, and structure/function analyses by expression in Escherichia coli; Lee WM et al.; Sex hormone binding globulin (SHBG) is a homodimeric plasma protein found in mammals that binds sex steroids with high affinity and regulates their bioavailability . The protein is identical in structure and properties to the androgen binding protein (ABP) found in the male reproductive tract . We have isolated a 1245-base pair rabbit SHBG cDNA encoding a reading frame for a signal peptide followed by a protein of 367 amino acids, which shares 79.0, 68.1 and 63.2% amino acid identity with the corresponding human, rat and mouse proteins respectively . Northern blot and hot-nested PCR analyses indicated that rabbit SHBG is produced from a 1.6 kilobase mRNA in the liver of both sexes and in the testis . The rabbit SHBG cDNA was inserted into pGEX-1 lambda T for expression of a glutathione S-transferase/SHBG fusion protein in Escherichia coli . The bacterial product bound 5 alpha-dihydrotestosterone (DHT) in the same manner as the corresponding protein in serum . The dissociation constants (Kd) for rabbit and human SHBGs produced in E . coli were 11.1 +/- 1.1 nM and 2.1 +/- 0.6 nM respectively, and rabbit SHBG formed a less stable protein-steroid complex (t1/2 = 5 min) than human SHBG (t1/2 > 60 min) . Unlike human SHBG, rabbit SHBG does not bind estradiol with high affinity . To aid in the identification of differences in the sequences of rabbit and human SHBG, which determine species differences in steroid-binding affinity and specificity, chimeras containing the 5'-terminal half of SHBG from one species and 3'-terminal half of SHBG from the other species were constructed and expressed . It was found that the chimeric proteins assumed similar steroid-binding affinity and specificity as the wild-type proteins when the amino (N)-terminal half of SHBG was derived from the same species . Replacement of the carboxyl (C)-terminal half of rabbit SHBG by the corresponding region of the human molecule increased the integrity of its steroid-protein complex . This supports the concept that amino acids within the N-terminal half of SHBG constitute the steroid-binding domain while the C-terminal half of the molecule may provide structural stability to the protein and its steroid-binding site.

Lett Appl Microbiol, 1997 Jun, 24(6), 498 - 502
Efficiency of the polymerase chain reaction amplification of the uid gene for detection of Escherichia coli in contaminated water; Iqbal S et al.; Direct detection of Escherichia coli from polluted river water was achieved using polymerase chain reaction (PCR) amplification of the uid gene . Amplification using DNA from environmental samples resulted in non-specific DNA fragments . Specific amplification was achieved through use of the touch-down PCR procedure . Targeting the uidA structural region of the gene gave reproducibly better amplification than targeting the uidR regulatory region . The data demonstrate conditions for optimal specific detection.

J Periodontol, 1997 Jun, 68(6), 531 - 5
Reactive change in proliferative activity of the junctional epithelium after topical application of lipopolysaccharide; Takata T et al.; IT IS WELL ESTABLISHED THAT apical migration of junctional epithelium (JE) along a root surface is an important factor in periodontal pocket formation and deepening . However, the exact mechanism and, more specifically, the role of inflammatory products in influencing the activity of cells within the JE is not known . To address this issue lipopolysaccharide (LPS) was applied topically into rat molar gingival sulcus and then tissues evaluated immunohistochemically for expression of proliferating cell nuclear antigen (PCNA) . Tissues were prepared for histological analysis at designated times . Histologically, infiltration of neutrophils with associated edema was noted in JE and gingival connective tissues 6 hours after LPS application and was prominent at 12 hours . These inflammatory changes persisted in the 2- and 3-day specimens, and disappeared at 5 days . In normal gingiva, before the LPS application, the JE showed few PCNA positive cells, while almost all cells in the basal and suprabasal cell layers of the oral gingival epithelium and the oral sulcular epithelium were PCNA positive . No increase in the number of PCNA positive cells in the JE beyond zero time was observed at 6 and 12 hours after LPS application . One day after LPS application, PCNA positive cells appeared in the basal cell layer of the JE, with a continued increase number of PCNA positive cells in the JE continued at 2 and 3 days . By day 5 the number of PCNA positive cells were decreasing with return to a normal range by 7 days . These results showed that 1) under normal physiological conditions, cells within the JE have minimal mitotic activity and 2) the JE cells can enter the proliferating cell cycle when exposed to LPS, and suggest that the enhanced proliferating activity in the JE is an important factor for the deepening of the periodontal pocket, if the connective tissue attachment is broken down.

Microbiology, 1997 Jun, 143 ( Pt 6), 2085 - 95
The aldA gene of Escherichia coli is under the control of at least three transcriptional regulators; Limon A et al.; Expression studies on the aldA gene encoding aldehyde dehydrogenase in Escherichia coli showed induction by two types of molecule (hydroxyaldehydes and 2-oxoglutarate), carbon catabolite repression and respiration dependence . Promoter deletion analysis showed that the proximal operator, which includes inducer-regulator complex and catabolite repression protein (Crp) recognition sites, was necessary for induction by either type of inducer, and that full induction by aldehydes required the cooperation of distal operator sequences beyond position -119 . Interactions of the regulator protein with the -59 to -6 fragment were shown by DNA mobility shift assays . Fusions of different deletions of the aldA promoter to lacZ indicated that a Crp site proximal to the transcriptional start point (tsp) was functional in the cAMP-dependent catabolite repression of this system, whereas a distal control site was likely to operate in a cAMP-independent catabolite repression . DNA mobility shift and footprint analyses showed that only the tsp proximal site was bound by pure Crp with a Kd of 5.4 x 10(-7) M . As shown by an Arc-defective strain, the aldA gene seems to be repressed by the Arc system under anaerobiosis, displaying its physiological full induction and activity in the presence of oxygen.

Microbiology, 1997 Jun, 143 ( Pt 6), 2079 - 84
Metabolic regulation of lrp gene expression in Escherichia coli K-12; Chen CF et al.; Expression of the lrp gene is regulated in part by the nutrients available to the cell, and is decreased in rich medium, in glucose minimal media enriched with amino acids, and in minimal medium with alternative carbon sources, such as acetate and succinate . When Lrp production is increased in a given medium, expression of its target genes is also increased . However, when the medium is changed from glucose to acetate, the response of the target genes is governed by many factors.

Microbiology, 1997 Jun, 143 ( Pt 6), 2039 - 46
The genetic structure of Escherichia coli populations in feral house mice; Gordon DM; Escherichia coli was isolated from feral house mice (Mus domesticus) during the course of a mouse plague in the state of Victoria, Australia . Two farms were sampled over a period of 7 months and a total of 447 isolates were collected . The isolates were characterized using the techniques of randomly amplified polymorphic DNA and multi-locus enzyme electrophoresis . The mean genetic diversity of this E . coli population (H = 0.24) was found to be substantially lower than the diversity of an E . col population reported elsewhere for a single human host . Analysis of the allozyme data revealed that there were significant differences in the relative abundance of genotypes between the two localities sampled and among sample dates . Overall, however, spatial and temporal effects accounted for less than 5% of the genotypic diversity . Allele frequencies and the relative abundance of the more common genotypes did not differ between male and female hosts . The number of genotypes and genotype diversity increased as the age of the host increased, suggesting that the mice are continuing to acquire new E . coli clones throughout their life . The frequency of some alleles changed with respect to host age, which indicates that clone acquisition may not be a random process . It is argued that the low level of genetic diversity observed in this population of E . coli reflects the boom and bust nature of mouse population density in this region of Australia.

Microbiology, 1997 Jun, 143 ( Pt 6), 1909 - 18
The relationship between external glucose concentration and cAMP levels inside Escherichia coli: implications for models of phosphotransferase-mediated regulation of adenylate cyclase; Notley-McRobb L et al.; The concentration of glucose in the medium influences the regulation of cAMP levels in Escherichia coli . Growth in minimal medium with micromolar glucose results in 8- to 10-fold higher intracellular cAMP concentrations than observed during growth with excess glucose . Current models would suggest that the difference in cAMP levels between glucose-rich and glucose-limited states is due to altered transport flux through the phosphoenolpyruvate: glucose phosphotransferase system (PTS), which in turn controls adenylate cyclase . A consequence of this model is that cAMP levels should be inversely related to the saturation of the PTS transporter . To test this hypothesis, the relationship between external glucose concentration and cAMP levels inside E . coli were investigated in detail, both through direct cAMP assay and indirectly through measurement of expression of cAMP-regulated genes . Responses were followed in batch, dialysis and glucose-limited continuous culture . A sharp rise in intracellular cAMP occurred when the nutrient concentration in minimal medium dropped to approximately 0.3 mM glucose . Likewise, addition of > 0.3 mM glucose, but not < 0.3 mM glucose, sharply reduced the intracellular cAMP level of starving bacteria . There was no striking shift in growth rate or {14C} glucose assimilation in bacteria passing through the 0.5 to 0.3 mM concentration threshold influencing cAMP levels, suggesting that neither metabolic flux nor transporter saturation influenced the sensing of nutrient levels . The (IIA/IIBC)Glc PTS is 96-97% saturated at 0.3 mM glucose so these results are not easily reconcilable with current models of cAMP regulation . Aside from the transition in cAMP levels initiated above 0.3 mM, a second shift occurred below 1 muM glucose . Approaching starvation, well below saturation of the PTS, cAMP levels either increased or decreased depending on unknown factors that differ between common E . coli K-12 strains.

Microbiology, 1997 Jun, 143 ( Pt 6), 1837 - 46
Construction and properties of aconitase mutants of Escherichia coli; Gruer MJ et al.; Escherichia coli contains two genes (acnA and acnB) encoding aconitase activities . An acnB mutant was engineered by replacing the chromosomal acnB gene by an internally deleted derivative containing a tetR cassette . An acnB double mutant was then made by transducing a previously constructed acnA::kanR mutation into the acnB::tetR strain . Western blotting confirmed that the AcnA and AcnB proteins were no longer produced by the corresponding mutants and PCR analysis showed that the chromosomal acnB gene had been replaced by the disrupted gene . Aerobic and anaerobic growth in glucose minimal medium were impaired but not abolished by the acnB mutation, indicating that the lesion is partially complemented by the acnA+ gene, and growth was enhanced by glutamate . The acnAB double mutant would not grow on unsupplemented glucose minimal medium and although it responded to glutamate like a typical auxotroph under anaerobic conditions, under aerobic conditions no response to glutamate was observed before it was over-grown by 'revertants' lacking citrate synthase (acnAB gltA) . The acnAB double mutant retained a low but significant aconitase activity (< or = 5% of wild-type), designated AcnC . Enzymological and regulatory studies with acn-lacZ fusions indicated that AcnB is the major aconitase, which is synthesized earlier in the growth cycle than AcnA, and subject to catabolite and anaerobic repression.

Microbiology, 1997 Jun, 143 ( Pt 6), 1797 - 804
Escherichia coli LT enterotoxin subunit A demonstrates partial toxicity independent of the nicking around Arg192; Tsuji T et al.; A study was conducted into whether or not nicking of the A subunit of Escherichia coli LT enterotoxin at position Arg192 or its neighbouring amino acids Arg192 to The195 is required for its toxicity . The toxic activity of mutants created by substitution or deletion at this position, which lacked ADP-ribosyltransferase activity in vitro, was not completely obliterated and cyclic AMP was partially induced in the target cells, showing that they still displayed enzymic activity in vivo . Moreover, although the A subunit possesses three potential sites for cleavage by furin, furin was not involved in the partial toxicity and cyclic AMP induction observed . These data suggest that target cells have a nick mechanism that operates at sites other than those around Arg192 or those recognized by furin, which generates an active fragment by processing the A subunit after toxin binding to the cell membrane.

Crit Care Med, 1997 Jun, 25(6), 1051 - 8
Effect of NG-nitro-L-arginine-methyl-ester on cardiopulmonary function and biosynthesis of cyclooxygenase products during porcine endotoxemia; Hellyer PW et al.; OBJECTIVE: To determine if inhibition of nitric oxide synthase with NG-nitro-L-arginine-methyl-ester (L-NAME) potentiates endotoxin-induced cardiopulmonary dysfunction and release of cyclooxygenase products in a porcine model of endotoxemia . DESIGN: Prospective, multiple group, controlled experimental study . SETTING: Physiologic research laboratory at a veterinary medicine college . SUBJECTS: Fifty-seven domestic pigs (mean 28.7 +/- 0.8 {SEM} kg) . INTERVENTIONS: Pentobarbital-anesthetized pigs were intubated and mechanically ventilated to normocapnia with room air . A ther-modilution cardiac output catheter was advanced into the pulmonary artery . Additional catheters were inserted into the jugular and femoral veins and femoral artery . The pigs received the following infusions: saline (control, n = 5); L-NAME (0.1, 0.5, 2.2, or 5.5 mg/ kg/hr, from -0.5 to 2 hrs, n = 16); Escherichia coli endotoxin (5 micrograms/ kg from 0 to 1 hr followed by 2 micrograms/kg from 1 to 2 hrs, i.v., n = 14); L-NAME plus endotoxin (n = 9); indomethacin plus endotoxin (n = 6); or L-NAME indomethacin plus endotoxin (n = 7) . MEASUREMENTS AND MAIN RESULTS: L-NAME significantly (p < .05) worsened endotoxin-induced hypoxemia and enhanced the increases in pulmonary vascular resistance index and systemic vascular resistance index at 30 to 60 mins . Endotoxin increased (p < .05) plasma concentrations of thromboxane B2 by seven- to eight-fold at 30 to 120 mins and 6-keto-prostaglandin F1 alpha by 16- to 24-fold at 60 to 120 mins . L-NAME enhanced (additive effect) endotoxin-induced increases in plasma concentrations of thromboxane B2 (60 mins) and significantly (p < .05) potentiated the increases in 6-keto-prostaglandin F1 alpha (120 mins) . At 120 mins of endotoxemia, indomethacin (cyclooxygenase inhibitor) plus L-NAME markedly increased (p < .05, synergistic effect) systemic vascular resistance index compared with endotoxemic pigs pretreated with either L-NAME or indomethacin . CONCLUSIONS: During endotoxemia, inhibition of nitric oxide synthase with L-NAME may be deleterious to cardiopulmonary function, as evidence by potentiation of endotoxin-induced systemic and pulmonary vasoconstriction, impairment of gas exchange, and enhanced biosynthesis of cyclooxygenase products . Moreover, during endotoxemia, the concomitant inhibition of two important vasodilators (i.e., nitric oxide and prostacyclin) is associated with a potentiated (p < .05) increase in systemic vascular resistance index.

Mol Biochem Parasitol, 1997 Jun, 86(2), 187 - 97
Molecular cloning and characterization of two iron superoxide dismutase cDNAs from Trypanosoma cruzi; Ismail SO et al.; Two cDNAs (FeSODA and FeSODB cDNAs) corresponding to superoxide dismutase (1.15.1.1., SOD) were isolated from a Trypanosoma cruzi cDNA library . Comparison of the deduced amino acid sequences with previously reported SOD protein sequences revealed that the T . cruzi open reading frames had considerable homology with FeSODs . The coding region of the T . cruzi FeSODB cDNA has been expressed in fusion with glutathione-S-transferase using an Escherichia coli mutant QC779, lacking both MnSOD and FeSOD genes (sodA sodB) . Staining of native polyacrylamide gels for SOD activity of T cruzi crude lysate and the recombinant SOD suggests that this protein is an FeSOD . The recombinant enzyme also protected the E . coli mutant QC779 from paraquat toxicity . Northern blot analysis showed that FeSODB is differentially expressed, showing a higher level at the epimastigote stage of T . cruzi development; whereas, FeSODA is constitutively expressed at a lower level in all developmental stages . Furthermore, Southern hybridization shows that both FeSODA and FeSODB genes appear to be present in the T . cruzi genome as multiple repeating units (multi-copy gene family).

Cytokine, 1997 Jun, 9(6), 412 - 5
Induction of gelatinase B and MCP-2 in baboons during sublethal and lethal bacteraemia; Paemen L et al.; Intravenous injection of sublethal or lethal doses of Escherichia coli in baboons resulted in increased serum levels of the matrix metalloprotease gelatinase B and the chemokine monocyte chemotactic protein 2 (MCP-2) . In both animal models, gelatinase B appeared faster than MCP-2 . After sublethal challenge, serum levels of gelatinase B and MCP-2 were found to be correlated, reaching peak levels between 2 and 4 h after bacterial challenge . After lethal challenge, however, MCP-2 tended to increase until 10 h . The kinetics of appearance suggest induction of release of gelatinase B and de novo synthesis and secretion of MCP-2, both by endotoxin.

Biotechnol Appl Biochem, 1997 Jun, 25 ( Pt 3), 223 - 33
Parameters influencing the expression of mature glial-cell-line-derived neurotrophic factor in Escherichia coli; Liew OW et al.; Factors governing expression in Escherichia coli, namely promoters, fusion partners, targeting signals, host strains, growth temperature of cultures and inducer concentrations, were investigated to elucidate their influence on the accumulation of mature glial-cell line-derived neurotrophic factor (GDNF) . The present study provided evidence indicating that translational and/or post-translational events were more important in determining overall accumulation of the target protein than was transcription . Under the control of the strong inducible tac or T7 promoter, no direct correlation between transcript abundance and final yield of recombinant protein was observed . GDNF was also recalcitrant to being produced in a soluble form in E . coli . Direct expression resulted in the exclusive localization of GDNF in inclusion bodies, regardless of whether the protein was produced in the cytoplasm or targeted to the periplasm . The fusion approach was found to be the most efficient method, as it resulted not only in the highest level of GDNF produced, albeit primarily in inclusion bodies, but also in the accumulation of small amounts of soluble proteins . Using different host strains, low inducer concentration or sub-optimal growth temperature did not result in any detectable shift towards solubility . Persistent localization in inclusion bodies, low levels of expression as a native protein and in vivo proteolysis of soluble fusion forms appeared to be influenced by structural features located at the N-terminus of GDNF . Deletion of this region was found to result in substantial alleviation of these problems.

J Clin Oncol, 1997 Jun, 15(6), 2222 - 30
Augmented Berlin-Frankfurt-Munster therapy abrogates the adverse prognostic significance of slow early response to induction chemotherapy for children and adolescents with acute lymphoblastic leukemia and unfavorable presenting features: a report from the Children's Cancer Group; Nachman J et al.; PURPOSE: Compared with previous Children's Cancer Group (CCG) acute lymphoblastic leukemia (ALL) trials, therapy based on the Berlin-Frankfurt-Munster (BFM) 76 trial has effected an improvement in event-free survival (EFS) . In an attempt to improve EFS further, CCG investigators formulated an augmented BFM (A-BFM) regimen that provides prolonged, intensified postinduction chemotherapy relative to the CCG-modified BFM regimen . PATIENTS AND METHODS: We tested A-BFM in 101 patients with ALL and unfavorable presenting features that showed slow early response (SER) to induction therapy who attained remission on day 28 . Their outcome was compared with that of 251 concurrent patients with unfavorable presenting features, a rapid early response to therapy (RER), and remission by day 28, treated with CCG-BFM with or without cranial radiation (CRT) . RESULTS: The 4-year EFS rate from the end of induction for SER patients treated with A-BFM was 70.8% +/- 4.6% . Seventeen patients remain in continuous remission beyond 5 years . Vincristine (VCR) neurotoxicity developed in 50% of patients, but was rarely debilitating . Allergies to Escherichia coli L-asparaginase (L-ASP) occurred in 35% of patients . Avascular necrosis of bone (AVN) developed in 9% of patients . In comparison, a concurrent RER group treated with standard BFM +/- CRT had a 4-year EFS rate of 73.1% +/- 4.6% . CONCLUSION: The toxicity of A-BFM is significant, but acceptable . Compared with historical control SER patients treated with CCG-modified BFM, A-BFM therapy appears to produce a significant improvement in EFS . This is the first study to show that intensive chemotherapy, as given in the A-BFM regimen, can abrogate the adverse prognostic significance of SER.

Am J Respir Crit Care Med, 1997 Jun, 155(6), 1965 - 71
Effects of norepinephrine on regional blood flow and oxygen extraction capabilities during endotoxic shock; Zhang H et al.; We explored the effects of norepinephrine on blood flow distribution and oxygen extraction capabilities during hyperdynamic endotoxic shock . Twelve anesthetized and mechanically ventilated dogs received 2 mg/kg of Escherichia coli endotoxin followed by a general saline infusion and were then randomly divided into two groups: six received norepinephrine (1 microg/kg/min), and six served as control subjects . The norepinephrine group maintained higher mean arterial pressure, cardiac index, left ventricular stroke work index, and hepatic arterial blood flow without altering blood flow to portal, mesenteric, and renal beds . When cardiac tamponade was induced to study tissue oxygen extraction capabilities, the norepinephrine group had a lower critical oxygen delivery in whole body (11.5 +/- 5.2 versus 14.3 +/- 1.4 ml/kg/min, p < 0.05) and in liver (25.0 +/- 11.3 versus 38.0 +/- 9.0 ml/min, p = NS) and a higher critical oxygen extraction ratio in whole body (53.8 +/- 17.7 versus 32.0 +/- 6.1%, p < 0.05), and in liver (57.0 +/- 11.9 versus 35.2 +/- 4.3%, p < 0.05) . We conclude that during endotoxic shock in dogs, norepinephrine hardly influences blood flow distribution and could even increase hepatic artery blood flow, and it can also improve whole body and liver oxygen extraction capabilities.

Alcohol Clin Exp Res, 1997 Jun, 21(4), 576 - 80
A transketolase assembly defect in a Wernicke-Korsakoff syndrome patient; Wang JJ et al.; Thiamine deficiency, a frequent complication of alcoholism, contributes significantly to the development of damage in various organ systems, including the brain . The molecular mechanisms that underlie the differential vulnerabilities to thiamine deficiency of tissue and cell types and among individuals are not understood . Investigations into these mechanisms have examined potential variations in thiamine utilizing enzymes . Transketolase is a homodimeric enzyme containing two molecules of noncovalently bound thiamine pyrophosphate . In the present study, we examined a his-tagged human transketolase that was produced in and purified from Escherichia coli cells . Previous findings demonstrated that purified his-transketolase had a Km app for cofactor and a thiamine pyrophosphate-dependent lag period for attaining steady-state kinetics that was similar to transketolase purified from human tissues . Interestingly, the time of the lag period, which is normally independent of enzyme concentration, was found herein to be dependent on the concentration of the recombinant protein . This atypical behavior was due to production in E . coli . Generation of the normal, enzyme concentration-independent state required a cytosolic factor(s) derived from human cells . Importantly, the required factor(s) was found to be defective in a Wernicke-Korsakoff patient whose cells in culture show an enhanced sensitivity to thiamine deficiency.

Surg Laparosc Endosc, 1997 Jun, 7(3), 228 - 31
Laparoscopy and septic dissemination caused by perioperative perforation of the occluded small bowel: an experimental study; Bustos B et al.; Based on the observation of septic shock and severe respiratory impairment in two patients subjected to laparoscopic surgery for small bowel occlusion, an experimental study was carried out in rabbits to evaluate the effect of intra-abdominal CO2 hyperpressure on massive bacterial spread . Increased bacterial access to the blood was observed as a result of the mechanical effect of the hyperpressure associated with the highly septic contents of the occluded bowel . The important risk of bacterial dissemination following accidental peroperative perforation requires extreme caution in the laparoscopic management of late occlusions of the small intestine.

Protein Sci, 1997 Jun, 6(6), 1343 - 6
Characterization and crystallization of human uroporphyrinogen decarboxylase; Phillips JD et al.; The cytosolic enzyme uroporphyrinogen decarboxylase (URO-D) catalyzes the fifth step in the heme biosynthetic pathway, converting uroporphyrinogen to coproporphyrinogen by decarboxylating the four acetate side chains of the substrate . Recombinant human URO-D has been expressed in Escherichia coli with a histidine tag and has been purified to homogeneity . Purified protein was determined to be a monodisperse dimer by dynamic light scattering . Equilibrium sedimentation analysis confirmed that the protein is dimeric, with a dissociation constant of 0.1 microM . URO-D containing an amino-terminal histidine tag was crystallized in space group P3(1)21 or its enantiomer P3(2)21 with unit cell dimensions a = b = 103.6 A, c = 75.2 A . There is one molecule in the asymmetric unit, consistent with generation of the dimer by the twofold axis of this crystallographic operator . Native data have been collected to 3.0 a resolution.

Protein Sci, 1997 Jun, 6(6), 1325 - 32
The adaptability of Escherichia coli thioredoxin to non-conservative amino acid substitutions; O'Brien R et al.; The adaptability of Escherichia coli thioredoxin to the substitution of a series of non-natural amino acids has been investigated . Different thiosulfonated alkyl groups were inserted into the hydrophobic core of the protein in position 78 via disulfide bonding with a buried cysteine residue as previously described (Wynn R, Richards FM . 1993 . Unnatural amino acid packing mutants of Escherichia coli thioredoxin produced by combined mutagenesis/chemical modification techniques . Protein Sci 2:395-403) . The side chains added to the cysteine included methyl, ethyl, n-propyl, n-butyl, n-pentyl, and cyclo-pentyl derivatives . The side chains appear to exploit the presence of the large cavities to incorporate these variant forms, enabling the protein to fold and have some activity . Solution structural and kinetic data suggested that these substitutions had little effect on the overall fold of the protein . Thermodynamic data revealed that the entropic effect of restricting the side chains in the folded protein has an effect on the stability . The variant forms of thioredoxin have different propensities to form dimers despite the limited structural perturbations . Molecular modeling studies allow the conformation of the side chains to be assessed.

Protein Sci, 1997 Jun, 6(6), 1237 - 47
Secondary and tertiary structural changes in gamma delta resolvase: comparison of the wild-type enzyme, the I110R mutant, and the C-terminal DNA binding domain in solution; Pan B et al.; gamma delta Resolvase is a site-specific DNA recombinase (M(r) 20.5 kDa) in Escherichia coli that shares homology with a family of bacterial resolvases and invertases . We have characterized the secondary and tertiary structural behavior of the cloned DNA binding domain (DBD) and a dimerization defective mutant in solution . Low-salt conditions were found to destabilize the tertiary structure of the DBD dramatically, with concomitant changes in the secondary structure that were localized near the hinge regions between the helices . The molten tertiary fold appears to contribute significantly to productive DNA interactions and supports a mechanism of DNA-induced folding of the tertiary structure, a process that enables the DBD to adapt in conformation for each of the three imperfect palindromic sites . At high salt concentrations, the monomeric I110R resolvase shows a minimal perturbation to the three helices of the DBD structure and changes in the linker segment in comparison to the cloned DBD containing the linker . Comparative analysis of the NMR spectra suggest that the I110R mutant contains a folded catalytic core of approximately 60 residues and that the segment from residues 100 to 149 are devoid of regular structure in the I110R resolvase . No increase in the helicity of the linker region of I110R resolvase occurs on binding DNA . These results support a subunit rotation model of strand exchange that involves the partial unfolding of the catalytic domains.

Protein Sci, 1997 Jun, 6(6), 1228 - 36
Characterization of the receptor binding determinants of granulocyte colony stimulating factor; Young DC et al.; We performed a series of experiments using alanine-scanning mutagenesis to locate side chains within human granulocyte colony-stimulating factor (G-CSF) that are involved in human G-CSF receptor binding . We constructed a panel of 28 alanine mutants that examined all surface exposed residues on helices A and D, as well as all charged residues on the surface of G-CSF . The G-CSF mutants were expressed in a transiently transfected mammalian cell line and quantitated by a sensitive biosensor method . We measured the activity of mutant proteins using an in vitro proliferation assay and an ELISA binding competition assay . These studies show that there is a region of five charged residues on helices A and C employed by G-CSF in binding its receptor, with the most important residue in this binding patch being Glu 19 . Both wild-type G-CSF and the E19A mutant were expressed in E . coli . The re-folded proteins were found to have proliferative activities similar to the analogous proteins from mammalian cells: furthermore, biophysical analysis indicated that the E19A mutation does not cause gross structural perturbations in G-CSF . Although G-CSF is likely to signal through receptor homo-dimerization, we found no compelling evidence for a second receptor binding region . We also found no evidence of self-antagonism at high G-CSF concentrations, suggesting that, in contrast to human growth hormone (hGH) and erythropoietin (EPO), G-CSF probably does not signal via a pure 2:1 receptor ligand complex . Thus, G-CSF, while having a similar tertiary structure to hGH and EPO, uses different areas of the four helix bundle for high-affinity interaction with its receptor.

Protein Sci, 1997 Jun, 6(6), 1148 - 56
The uncharged surface features surrounding the active site of Escherichia coli DsbA are conserved and are implicated in peptide binding; Guddat LW et al.; DsbA is a protein-folding catalyst from the periplasm of Escherichia coli that interacts with newly translocated polypeptide substrate and catalyzes the formation of disulfide bonds in these secreted proteins . The precise nature of the interaction between DsbA and unfolded substrate is not known . Here, we give a detailed analysis of the DsbA crystal structure, now refined to 1.7 A, and present a proposal for its interaction with peptide . The crystal structure of DsbA implies flexibility between the thioredoxin and helical domains that may be an important feature for the disulfide transfer reaction . A hinge point for domain motion is identified-the type IV beta-turn Phe 63-Met 64-Gly 65-Gly 66, which connects the two domains . Three unique features on the active site surface of the DsbA molecule-a groove, hydrophobic pocket, and hydrophobic patch-form an extensive uncharged surface surrounding the active-site disulfide . Residues that contribute to these surface features are shown to be generally conserved in eight DsbA homologues . Furthermore, the residues immediately surrounding the active-site disulfide are uncharged in all nine DsbA proteins . A model for DsbA-peptide interaction has been derived from the structure of a human thioredoxin:peptide complex . This shows that peptide could interact with DsbA in a manner similar to that with thioredoxin . The active-site disulfide and all three surrounding uncharged surface features of DsbA could, in principle, participate in the binding or stabilization of peptide.

Plant Physiol, 1997 Jun, 114(2), 669 - 77
Broad-range and binary-range acyl-acyl-carrier protein thioesterases suggest an alternative mechanism for medium-chain production in seeds; Voelker TA et al.; In the current model of medium-chain (C8-14) fatty acid biosynthesis in seeds, specialized FatB acyl-acyl-carrier-protein (ACP) thioesterases are responsible for the production of medium chains . We have isolated and characterized FatB cDNAs from the maturing seeds of elm (Ulmus americana) and nutmeg (Myristica fragrans), which accumulate predominantly caprate (10:0)- and myristate (14:0)-containing oils, respectively . In neither species were we able to find cDNAs encoding enzymes specialized for these chain lengths . Nutmeg FatB hydrolyses C14-18 substrates in vitro and expression in Brassica napus seeds leads to an oil enriched in C14-18 saturates . Elm FatB1 displays a binary specificity: one activity is centered on 10:0-ACP, and a second is centered on palmitate (16:0)-ACP . After expression in B . napus seeds the oil is enriched in C10-18 saturates, predominantly 16:0, 14:0, and 10:0 . The composition of free fatty acids produced by elm FatB1 in Escherichia coli shifts from C14-16 to mostly C8-10 by increasing the rate of chain termination by this enzyme . These results suggest the existence of an alternative mechanism used in the evolution of medium-chain production, a model of which is presented.

Plant Physiol, 1997 Jun, 114(2), 643 - 52
Light regulation of the abundance of mRNA encoding a nucleolin-like protein localized in the nucleoli of pea nuclei; Tong CG et al.; A cDNA encoding a nucleolar protein was selected from a pea (Pisum sativum) plumule library, cloned, and sequenced . The translated sequence of the cDNA has significant percent identity to Xenopus laevis nucleolin (31%), the alfalfa (Medicago sativa) nucleolin homolog (66%), and the yeast (Saccharomyces cerevisiae) nucleolin homolog (NSR1) (28%) . It also has sequence patterns in its primary structure that are characteristic of all nucleolins, including an N-terminal acidic motif, RNA recognition motifs, and a C-terminal Gly- and Arg-rich domain . By immunoblot analysis, the polyclonal antibodies used to select the cDNA bind selectively to a 90-kD protein in purified pea nuclei and nucleoli and to an 88-kD protein in extracts of Escherichia coli expressing the cDNA . In immunolocalization assays of pea plumule cells, the antibodies stained primarily a region surrounding the fibrillar center of nucleoli, where animal nucleolins are typically found . Southern analysis indicated that the pea nucleolin-like protein is encoded by a single gene, and northern analysis showed that the labeled cDNA binds to a single band of RNA, approximately the same size and the cDNA . After irradiation of etiolated pea seedlings by red light, the mRNA level in plumules decreased during the 1st hour and then increased to a peak of six times the 0-h level at 12 h . Far-red light reversed this effect of red light, and the mRNA accumulation from red/far-red light irradiation was equal to that found in the dark control . This indicates that phytochrome may regulate the expression of this gene.

Plant Physiol, 1997 Jun, 114(2), 623 - 30
Evidence for transcriptional regulation of plastid photosynthesis genes in Arabidopsis thaliana roots; Isono K et al.; Mechanisms underlying suppressed levels of transcripts for plastid photosynthesis genes in nongreen tissues such as roots and calli were analyzed in Arabidopsis thaliana, a plant suitable for further genetic dissection . A region encoding promoters of rbcL, the gene encoding the large subunit of ribulose-1,5-biphosphate carboxylase/oxygenase, and the atpB/E operon for the beta and epsilon subunits of coupling factor one were cloned and sequenced . Transcripts for rbcL, atpB/E, and psbA, the gene for the D1 protein in the photosystem II reaction center, were barely detectable in roots of A . thaliana, whereas 16S rRNA was detected at a low level . The run-on transcription experiment revealed that expression of rbcL, atpB/E, and psbA was regulated at transcription . The copy number of plastid DNA in roots was one-fifth that in green leaves on the basis of total cellular DNA, suggesting that in the latter the DNA copy-number regulation also exists in plastid gene expression . Digestion of DNA with methyl-sensitive and -insensitive isoschizomeric endonucleases and subsequent polymerase chain reaction, as well as in vitro transcription of plastid DNAs with Escherichia coli RNA polymerase, resulted in no evidence of regulation by DNA modification . In spite of predominant suppression of expression of rbcL, atpB/E, and psbA at transcription in roots and calli, 16S rRNA levels were decreased because of low RNA stability.

Plant Physiol, 1997 Jun, 114(2), 493 - 500
Molecular cloning of the cowpea leghemoglobin II gene and expression of its cDNA in Escherichia coli . Purification and characterization of the recombinant protein; Arredondo-Peter R et al.; Cowpea (Vigna unguiculata) nodules contain three leghemoglobins (LbI, LbII, and LbIII) that are encoded by at least two genes . We have cloned and sequenced the gene that encodes for LbII (lbII), the most abundant Lb in cowpea nodules, using total DNA as the template for PCR . Primers were designed using the sequence of the soybean lbc gene . The lbII gene is 679 bp in length and codes for a predicted protein of 145 amino acids . Using sequences of the cowpea lbII gene for the synthesis of primers and total nodule RNA as the template, we cloned a cDNA for LbII into a constitutive expression vector (pEMBL19+) and then expressed it in Escherichia coli . Recombinant LbII (rLbII) and native LbII (nLbII) from cowpea nodules were purified to homogeneity using standard techniques . Properties of rLbII were compared with nLbII by partially sequencing the proteins and by sodium dodecyl sulfate- and isoelectric focusing polyacrylamide gel electrophoresis, western-blot analysis using anti-soybean Lba antibodies, tryptic and chymotryptic mapping, and spectrophotometric techniques . The data showed that the structural and spectral characteristics of rLbII and nLbII were similar . The rLbII was reversibly oxygenated/deoxygenated, showing that it is a functional hemoglobin.

Genes Dev, 1997 Jun 1, 11(11), 1422 - 34
The an11 locus controlling flower pigmentation in petunia encodes a novel WD-repeat protein conserved in yeast, plants, and animals; de Vetten N et al.; In petunia flowers, the loci an1, an2, and an11 control the pigmentation of the flower by stimulating the transcription of anthocyanin biosynthetic genes . The an1 and an2 locus were recently cloned and encode a basic helix-loop-helix (bHLH) and MYB-domain transcriptional activator, respectively . Here, we report the isolation of the an11 locus by transposon tagging . RNA gel blot experiments show that an11 is expressed independently from an1 and an2 throughout plant development, as well as in tissues that do not express the anthocyanin pathway . It encodes a novel WD-repeat protein that is highly conserved even in species that do not produce anthocyanins such as yeast, nematodes, and mammals . The observation that the human an11 homolog partially complements the an11 petunia mutant in transient assays shows that sequence similarity reflects functional conservation . Overexpression of an2 in an11- petals restored the activity of a structural anthocyanin gene in transient assays, indicating that AN11 acts upstream of AN2 . Cell fractionation experiments show that the bulk of the AN11 protein is localized in the cytoplasm . Taken together, this indicates that AN11 is a cytoplasmic component of a conserved signal transduction cascade that modulates AN2 function in petunia, thereby linking cellular signals with transcriptional activation.

Biochem Mol Biol Int, 1997 Jun, 42(1), 211 - 5
Half-life of Escherichia coli polyadenylated lipoprotein mRNA; Taljanidisz J et al.; In the light of recent evidence that the half-life of bacterial mRNA may be modulated by polyadenylation at the 3' end, we determined the half-life of polyadenylated lpp mRNA, which is an abundant and comparatively stable message encoding a major lipoprotein of the outer membrane . Messenger RNAs were pulse-labeled with {3H} adenosine and poly(A) RNA was isolated at various times after pulse-labeling by affinity chromatography on oligo(dT) cellulose . The amount of lpp mRNA remaining was quantitated by hybridization with the antisense strand of the lpp gene cloned in M13mp18 . The observed half-life for polyadenylated lpp mRNA was 12 min . This represents the first half-life measurement for a specific polyadenylated mRNA in E . coli, and is slightly longer than the half-life of total lpp RNA reported earlier . It coincides with the functional half-life for lpp mRNA determined by Inouye and coworkers by measuring the rate of lipoprotein synthesis at various times after rifampicin addition . This suggest that polyadenylated lpp RNA is the predominant and translationally active form of lpp mRNA within the cell.

Biochem Mol Biol Int, 1997 Jun, 42(1), 173 - 81
Selective excitation of tryptophans in OmpF: a fluorescence emission study; Pattnaik BR et al.; The fluorescence studies of E . coli porin OmpF at pH 7.5 were carried out using excitation at 280 and 305 nm . Similar studies were performed in presence of a denaturant (urea) and a quencher (KI) . Results show that both the tryptophans present in OmpF (residue numbers 61 and 214) contribute to fluorescence at 280 nm excitation, whereas only one residue shows fluorescence emission when excited at 305 nm . Based on these findings and the available crystal structure, it is speculated that tryptophan 61 of OmpF is selectively excited at 305 nm . The present studies point out some interesting features of the tryptophan microenvironments in OmpF.

J Gen Virol, 1997 Jun, 78 ( Pt 6), 1265 - 70
Maize streak virus coat protein binds single- and double-stranded DNA in vitro; Liu H et al.; Maize streak virus (MSV) coat protein (CP) is required for virus movement within the plant . Deletion or mutation of MSV CP does not prevent virus replication in single cells or protoplasts but leads to a loss of infectivity in the inoculated plant . The mechanism by which MSV CP mediates the transfer of MSV DNA from cell to cell and through the vascular bundle is still unknown . Towards understanding the role of MSV CP in virus movement, the interaction of the CP with viral DNA was investigated using the 'south-western' assay . Wild-type and truncated MSV CPs were expressed in E . coli and the expressed CPs were used to investigate interactions with single-stranded (ss) and double-stranded (ds) DNA . The results showed that MSV CP bound ss and ds viral and plasmid DNA in a sequence non-specific manner . The binding domain was mapped to within the 104 N-terminal amino acids of the MSV CP . We propose that the binding of CP to MSV DNA is involved in viral DNA nuclear transport as well as encapsidation and thus may have a role in intra- and inter-cellular movement as well as systemic infection.

Brain Res Mol Brain Res, 1997 Jun, 46(1-2), 243 - 55
The melanin-concentrating hormone gene in human: flanking region analysis, fine chromosome mapping, and tissue-specific expression; Viale A et al.; Genomic sequences encoding the human melanin-concentrating hormone (MCH) were isolated from a YAC library and subcloned in pUC vector using a novel E . coli transformation method . A 4.1-kb fragment encompassing approximately 1.0 kb of the 5'-end-flanking region, the three exons-two introns of the coding region and approximately 1.7 kb of the 3'-end-flanking region, was sequenced . Comparison with the rat MCH gene indicated strong conservation in the 5'-flanking region, in particular over the putative TATA box, CAAT box, GRE and AP-1 elements that could potentially regulate MCH gene expression . FISH with a fluorescent MCH genomic probe on human chromosomes and PCR analysis of a YAC panel mapped MCH to chromosome 12q23.1 in a region flanked by D12S1074 and D12S1030 markers . Expression of the MCH RNA species and pro-MCH-derived peptides (MCH and NEI) was investigated in human tissues by combining Northern blotting, RT-PCR, in situ hybridization, immunohistochemistry and RIA . In the human brain, MCH mRNA and MCH/NEI peptides were predominantely expressed in the lateral hypothalamus in agreement with the known distribution of MCH expression in rat . In addition, MCH gene products were detected in extra-hypothalamic sites, such as the pallidum, neocortex and cerebellum . In peripheral tissues, MCH mRNA was identified in several organs, including the thymus, brown adipose tissue, duodenum and testis . An additional shorter MCH gene transcript, likely the result of alternate splicing, was revealed in several brain areas and peripheral tissues . While only fully processed MCH and NEI were found in hypothalamus, a different peptide form, bearing MCH and NEI epitopes, was detected in peripheral organs . This represents the first evidence for differential processing of pro-MCH in mammals.

J Bacteriol, 1997 Jun, 179(12), 4075 - 9
A signal transducer for aerotaxis in Escherichia coli; Bibikov SI et al.; The newly discovered aer locus of Escherichia coli encodes a 506-residue protein with an N terminus that resembles the NifL aerosensor and a C terminus that resembles the flagellar signaling domain of methyl-accepting chemoreceptors . Deletion mutants lacking a functional Aer protein failed to congregate around air bubbles or follow oxygen gradients in soft agar plates . Membranes with overexpressed Aer protein also contained high levels of noncovalently associated flavin adenine dinucleotide (FAD) . We propose that Aer is a flavoprotein that mediates positive aerotactic responses in E . coli . Aer may use its FAD prosthetic group as a cellular redox sensor to monitor environmental oxygen levels.

J Bacteriol, 1997 Jun, 179(12), 4039 - 42
Multiple transcribed elements control expression of the Escherichia coli btuB gene; Franklund CV et al.; Repression by vitamin B12 of the cobalamin transport protein BtuB in the outer membrane of Escherichia coli operates at both the transcriptional and translational levels and is controlled by transcribed sequences within the leader and proximal portion of the btuB coding sequence . The effects of deletions from either end of this region on repression and expression were determined with lac fusions . An element at the 5' end of the transcript and the putative attenuator within the coding sequence were required for transcriptional repression . The presence of either element caused a marked reduction in btuB-lacZ expression which was reversed by the presence of a conserved sequence element in the leader, suggesting the importance of long-range interactions in the btuB leader for expression and regulation.

J Bacteriol, 1997 Jun, 179(12), 3981 - 8
Translation of the leaderless Caulobacter dnaX mRNA; Winzeler E et al.; The expression of the Caulobacter crescentus homolog of dnaX, which in Escherichia coli encodes both the gamma and tau subunits of the DNA polymerase III holoenzyme, is subject to cell cycle control . We present evidence that the first amino acid in the predicted DnaX protein corresponds to the first codon in the mRNA transcribed from the dnaX promoter; thus, the ribosome must recognize the mRNA at a site downstream of the start codon in an unusual but not unprecedented fashion . Inserting four bases in front of the AUG at the 5' end of dnaX mRNA abolishes translation in the correct frame . The sequence upstream of the translational start site shows little homology to the canonical Shine-Dalgarno ribosome recognition sequence, but the region downstream of the start codon is complementary to a region of 16S rRNA implicated in downstream box recognition . The region downstream of the dnaX AUG, which is important for efficient translation, exhibits homology with the corresponding region from the Caulobacter hemE gene adjacent to the replication origin . The hemE gene also appears to be translated from a leaderless mRNA . Additionally, as was found for hemE, an upstream untranslated mRNA also extends into the dnaX coding sequence . We propose that translation of leaderless mRNAs may provide a mechanism by which the ribosome can distinguish between productive and nonproductive templates.

J Bacteriol, 1997 Jun, 179(12), 3845 - 50
Size of cotA and identification of the gene product in Synechocystis sp . strain PCC6803; Sonoda M et al.; cotA of Synechocystis sp . strain PCC6803 is a gene involved in light-induced proton extrusion (A . Katoh, M . Sonoda, H . Katoh, and T . Ogawa, J . Bacteriol . 178:5452-5455, 1996) . There are two possible initiation codons in cotA, and either long (L-) or short (S-) cotA encoding a protein of 440 or 247 amino acids could be postulated . To determine the gene size, we inserted L-cotA and S-cotA into the genome of a cotA-less mutant (M29) to construct M29(L-cotA) and M29(S-cotA), respectively . M29(L-cotA) showed essentially the same net proton movement profile as the wild type, whereas no light-induced proton extrusion was observed with M29(S-cotA) . Two kinds of antibodies were raised against partial gene products of the N- and C-terminal regions of L-cotA, respectively, fused to glutathione S-transferase expressed in Escherichia coli . Both antibodies cross-reacted with a band at 52 kDa in both cytoplasmic and thylakoid membrane fractions of the wild-type cells . The same cross-reacting band was present in the membranes of M29(L-cotA) but not in M29 or M29(S-cotA) . These antibodies cross-reacted more strongly with the cytoplasmic membrane fraction than with the thylakoid membrane fraction . The antibody against NrtA, a nitrate transporter protein present only in the cytoplasmic membrane, also cross-reacted with the thylakoid membrane fraction strongly . Based on these results we concluded that CotA of 440 amino acids (51 kDa) is located in the cytoplasmic membrane . Whether CotA is absent in the thylakoid membrane remains to be solved.

Mol Biol Evol, 1997 Jun, 14(6), 666 - 73
Nucleotide polymorphism in colicin E2 gene clusters: evidence for nonneutral evolution; Tan Y et al.; To explore the molecular mechanisms behind the diversification of colicin gene clusters, we examined DNA sequence polymorphism for the colicin gene clusters of 14 colicin E2 (ColE2) plasmids obtained from natural isolates of Escherichia coli . Two types of ColE2 plasmids are revealed, with type II gene clusters generated by recombination between type I ColE2 and ColE7 gene clusters . The levels and patterns of DNA polymorphism are different between the two types . Type I polymorphism is distributed evenly along the gene cluster, while type II accumulates polymorphism at an elevated rate in the 5' end of the colicin gene . These differences may be explained by recombinational origins of type II gene clusters . The pattern of divergence between the ColE2 gene cluster and its close relative ColE9 is not correlated with the pattern of polymorphism within ColE2, suggesting that this gene cluster is not evolving in a neutral fashion . A statistical test confirms significant departures from the predictions of neutrality . These data lend further support to the hypothesis that colicin gene clusters may evolve under the influence of nonneutral forces.

Photochem Photobiol, 1997 Jun, 65(6), 964 - 8
Photoreactivating enzyme for (6-4) photoproducts in cultured goldfish cells; Uchida N et al.; We previously reported that when cultured goldfish cells are illuminated with fluorescent light, photorepair ability for both cyclobutane pyrimidine dimers and (6-4) photoproducts increased . In the present study, it was found that the duration of the induced photorepair ability for cyclobutane pyrimidine dimers was longer than that for (6-4) photoproducts, suggesting the presence of different photolyases for repair of these two major forms of DNA damage . A gel shift assay was then performed to show the presence of protein(s) binding to (6-4) photoproducts and its dissociation from (6-4) photoproducts under fluorescent light illumination . In addition, at 8 h after fluorescent light illumination of the cell, the binding of protein(s) to (6-4) photoproducts increased . The restriction enzymes that have recognition sites containing TT or TC sequences failed to digest the UV-irradiated DNA photoreactivated by using Escherichia coli photolyase for cyclobutane pyrimidine dimers, indicating that restriction enzymes could not function because (6-4) photoproducts remained in recognition sites . But, when UV-irradiated DNA depleted of cyclobutane pyrimidine dimers was incubated with extract of cultured goldfish cells under fluorescent light illumination, it was digested with those restriction enzymes . These results suggested the presence of (6-4) photolyase in cultured goldfish cells as in Drosophila, Xenopus and Crotalus.

Microb Pathog, 1997 Jun, 22(6), 331 - 41
Localization of the in vivo expression of P and F1 fimbriae in chickens experimentally inoculated with pathogenic Escherichia coli; Pourbakhsh SA et al.; Escherichia coli causing septicemia in poultry often possess F1 (type 1) and/or P fimbriae which may be involved in bacterial colonization and infection . To investigate the expression of these fimbriae in vivo, two pathogenic E . coli strains with different fimbrial profiles, TK3 (fim+/pap+) and MT78 (fim+/pap-), were administered to 2-week-old chickens by either the intratracheal or caudal thoracic air sac inoculation route . Antibodies specific for native F1 fimbriae were detected by ELISA and immunodot in the serum of chickens inoculated with either strain MT78 or strain TK3, irrespective of the route of inoculation . Antibodies specific for P fimbriae of serotype F11 were detected by ELISA and immunoblotting in the serum of chickens inoculated by either route with strain TK3 . F1, but not P fimbriae, were expressed by bacteria colonizing the trachea of chickens inoculated by the air sac route with strain MT78 or TK3, as demonstrated by examination of frozen tissue sections using immunofluorescence . F1 fimbriae were also expressed by bacteria colonizing the air sacs and lungs, but not by bacteria in the blood or other internal organs, of chickens inoculated with either strain . P fimbriae were expressed by bacteria colonizing the air sacs, lungs, kidney, blood, and pericardial fluid, but not by bacteria colonizing the trachea, of chickens inoculated with strain TK3 . Fimbriae-like structures were observed by electron microscopy on bacteria adhering to the epithelial cells of the air sacs of chickens inoculated with strain TK3 . These results demonstrate that both strains MT78 and TK3 undergo in vivo phase variation with respect to their fimbrial profiles and site of bacterial colonization in different organs of infected chickens and suggest that F1 fimbriae are important for initial bacterial colonization of the upper respiratory tract whereas P fimbriae are important for later stages of the infection.

Cancer Res, 1997 Jun 1, 57(11), 2151 - 6
Cloning and characterization of mammalian 8-hydroxyguanine-specific DNA glycosylase/apurinic, apyrimidinic lyase, a functional mutM homologue; Aburatani H et al.; 8-Hydroxyguanine (8-OH-G) is one of the major DNA oxidation products implicated in mutagenesis induced by oxygen radical-forming agents, including ionizing radiation . It is also believed to be involved in spontaneous mutation induced by metabolically produced oxygen radicals . A mammalian homologue of 8-OH-G glycosylase/apurinic, apyrimidinic lyase (mutM homologue, MMH) has been identified in the EST database (for expressed sequence tags) through a homology search with yeast OGG1 protein . The human MMH protein (hMMH), 34% identical to the yeast OGG1 protein, is a member of the DNA repair protein superfamily . The hMMH gene was composed of seven exons, with the alternate last exon, exon 8, producing three major alternative splicing isoforms, because splicing of the sixth intron was optional . The hMMH protein expressed in Escherichia coli revealed the glycosylase activity and apurinic, apyrimidinic lyase activity on duplex DNA containing 8-OH-G . The hMMH protein can rescue a spontaneous mutator strain of E . coli lacking mutM and mutY . By the introduction of recombinant hMMH, the rate of mutation, the formation of rifampicin-resistant revertants, was reduced by 4-7 fold . Genomic structure analysis showed that 3' exons of the hMMH gene are transcribed on the antisense strand of the calcium-dependent calmodulin kinase 1 gene.

Am J Vet Res, 1997 Jun, 58(6), 601 - 7
Resazurin reduction as a function of respiratory burst in bovine neutrophils; Fang W et al.; OBJECTIVE: To determine whether the respiratory burst of neutrophils from bovine blood and milk can be analyzed by use of a fluorometric resazurin reduction assay . SAMPLE POPULATION: Neutrophils were obtained from EDTA-anticoagulated blood of 7 dairy cows . Neutrophils also were isolated from milk samples of a cow intramammarily challenge exposed with Escherichia coli lipopolysaccharide . PROCEDURE: The respiratory burst of neutrophils was analyzed in parallel, using the conventional luminol-enhanced luminometric procedure and a novel fluorometric procedure with resazurin as the fluorogenic substrate . Opsonized zymosan and phorbol myristate acetate were used as stimulants . The mechanism of the fluorescent response was analyzed, using metabolic inhibitors to various cell functions . Luminometry and fluorometry were carried out in parallel, using microtitration tray-reading instruments . RESULTS: Stimulation of neutrophils induced resazurin reduction to resorufin and a fluorescent response . The luminescent response was transient, but the fluorescent response (build-up of fluorescent resorufin) was cumulative . Therefore, a single end-point measurement can be used for the fluorometric assay . CONCLUSIONS: The proposed fluorometric microtitration tray technology is simple and has a high throughput capacity . The fluorometric and luminometric assays seem to have similar potential in the analysis of phagocyte functions.

Am J Vet Res, 1997 Jun, 58(6), 594 - 600
Effect of growth hormone or chromium picolinate on swine metabolism and inflammatory cytokine production after endotoxin challenge exposure; Myers MJ et al.; OBJECTIVE: To determine whether recombinant porcine somatotropin (PST) or chromium picolinate (CrP) affected cytokine production and metabolism in swine after endotoxin challenge exposure . ANIMALS: 20 Poland China X Landrace pigs, 5/group . PROCEDURE: Pigs were given CrP-supplemented feed at body weight of 20 kg; PST treatment began at 60 kg, and both treatments continued through body weight of 90 kg . At 90 kg, pigs were challenge exposed with 20 micrograms of lipopolysaccharide (LPS)/kg of body weight . Blood samples were obtained at various times through 24 hours after LPS challenge exposure . RESULTS: In all pigs not given PST, glucose concentration decreased 2 to 4 hours after LPS . In PST-treated pigs, blood glucose concentration was decreased at 6 to 8 hours after LPS . Plasma insulin concentration paralleled changes in glucose concentration . Nonesterified fatty acid concentration was high 2 to 24 hours after LPS in pigs not given PST and at 6 to 24 h in PST-treated pigs . Plasma urea nitrogen concentration was high at 6 to 24 hours after LPS in pigs not given PST . The urea nitrogen values in PST-treated pigs were lower at all times . Serum aspartate transaminase activity was high 6 to 24 hours after LPS in pigs not given PST, whereas PST treatment prevented the increase in this enzyme activity . In untreated (PST) pigs, plasma bilirubin (total and direct) concentrations were high 4 to 8 hours after LPS and returned to normal at 24 hours . The PST- and CrP-treated pigs maintained normal plasma bilirubin concentrations . Interleukin 6 activity was unaffected by CrP and PST treatments . Treatment with CrP and PST decreased the tumor necrosis factor alpha response to LPS, compared with that in control pigs . CONCLUSIONS: PST, and to a lesser extent CrP, provide protection against the adverse metabolic effects of LPS-induced septic shock.

Arch Biochem Biophys, 1997 Jun 1, 342(1), 187 - 9
Assignment of the beta-subunit of wheat eIF2 by protein and DNA sequence analysis and immunoanalysis; Metz AM et al.; Wheat germ initiation factor 2 (eIF2), like mammalian and yeast eIF2, contains three nonidentical subunits . The estimated molecular weights for the wheat subunits are 38,000 (p38), 42,000 (p42), and 50,000 (p50) . Peptide sequence was obtained for the p38 subunit of wheat eIF2 and the resulting amino acid sequence suggested that it was actually the equivalent of the mammalian beta-subunit . A wheat sprout cDNA expression library was screened with antibody affinity purified to the p38 subunit . The DNA sequence of the clones obtained also indicated that the p38 subunit was the equivalent to the mammalian beta-subunit . The wheat p38 subunit was then expressed in Escherichia coli and antibodies raised to the purified recombinant protein . Only the p38 subunit of purified wheat germ eIF2 reacted with the antisera . The p38 subunit of wheat eIF2 is therefore the equivalent of mammalian eIF2beta.

Arch Biochem Biophys, 1997 Jun 1, 342(1), 92 - 8
Comparing the properties of Escherichia coli branching enzyme and maize branching enzyme; Guan H et al.; Escherichia coli glycogen branching enzyme (GBE) and maize starch branching enzymes I (SBEI) and II (SBEII) were expressed in E . coli and purified . E . coli GBE branched amylose at a higher rate than did SBEII, but branched amylose at a lower rate than did SBEI . Similar to SBEI, GBE branched amylopectin at a lower rate than did SBEII . High-performance anion-exchange chromatography analysis of the branched products produced by BE revealed the minimum chain length (cl) required for branching . While GBE and SBEII showed the same minimum cl {degree of polymerization (dp) 12} required for branching, SBEI had a slightly higher minimum cl (dp 16) requirement for branching . The major differences between GBE and SBE are their specificities in terms of the size of chains transferred . In comparison with SBE, GBE had a much narrower size range of chains transferred and transferred mainly shorter chains . While SBEI and SBEII produced a large number of chains ranging from dp 6 to over dp 30, GBE predominantly transferred chains ranging from dp 5 to 16 and produced only a very small number of long chains with dp greater than 20 . Although it has been reported that SBEI and SBEII preferentially transfer longer and shorter chains, respectively (1), this study further defines the differences between SBEI and SBEII in the size of chains transferred . SBEI predominantly transfers longer chains with dp greater than 10, while producing few shorter chains with dp 3 to 5 . In contrast, SBEII preferentially transfers smaller chains with dp 3 to 9, with the most abundant chains being dp 6 and 7 . The significance of minimum chain-length requirement by SBE is discussed in setting the invariant size of amylopectin cluster size (9 nm).

Arch Biochem Biophys, 1997 Jun 1, 342(1), 58 - 67
Activity, peroxide compound formation, and heme d synthesis in Escherichia coli HPII catalase; Obinger C et al.; Wild-type Escherichia coli HPII catalase (heme d containing) has 15% the activity of beef liver enzyme per heme . The rate constant for compound I formation with H2O2 is 1.3 x 10(6) M(-1) s(-1) . HPII compound I reacts with H2O2 to form O2 with a rate constant of 1.8 x 10(6) M(-1) s(-1) . Forty percent of HPII hemes are in the compound I state during turnover . Compound I is reduced by ethanol and formate at rates of 5 and 13 M(-1) s(-1) (pH 7.0), respectively . Incubation of HPII compound I with ferrocyanide and ascorbate does not form a compound II species . Mutation of His128 to alanine or asparagine gives inactive protoheme proteins . Mutation of Asn201 gives partially active heme d forms . Asn201Ala has 24%, Asn201Asp 10%, and Asn201Gln 0.4% of wild-type activity . Asn201His contains protoheme when isolated and converts this via protoheme compound I to a heme d species . Both distal heme cavity residues His128 and Asn201 are implicated in catalytic activity, compound I formation, and in situ heme d biosynthesis . HPII Asn201, like the corresponding residue in protoheme catalases, may promote H+ transfer to His128 imidazole, facilitating (i) peroxide anion binding to heme and (ii) stabilization of a transition state for heterolytic cleavage of the O-O bond.

Microbiol Mol Biol Rev, 1997 Jun, 61(2), 212 - 38
Stable DNA replication: interplay between DNA replication, homologous recombination, and transcription; Kogoma T; Chromosome replication in Escherichia coli is normally initiated at oriC, the origin of chromosome replication . E . coli cells possess at least three additional initiation systems for chromosome replication that are normally repressed but can be activated under certain specific conditions . These are termed the stable DNA replication systems . Inducible stable DNA replication (iSDR), which is activated by SOS induction, is proposed to be initiated from a D-loop, an early intermediate in homologous recombination . Thus, iSDR is a form of recombination-dependent DNA replication (RDR) . Analysis of iSDR and RDR has led to the proposal that homologous recombination and double-strand break repair involve extensive semiconservative DNA replication . RDR is proposed to play crucial roles in homologous recombination, double-strand break repair, restoration of collapsed replication forks, and adaptive mutation . Constitutive stable DNA replication (cSDR) is activated in mhA mutants deficient in RNase HI or in recG mutants deficient in RecG helicase . cSDR is proposed to be initiated from an R-loop that can be formed by the invasion of duplex DNA by an RNA transcript, which most probably is catalyzed by RecA protein . The third form of SDR is nSDR, which can be transiently activated in wild-type cells when rapidly growing cells enter the stationary phase . This article describes the characteristics of these alternative DNA replication forms and reviews evidence that has led to the formulation of the proposed models for SDR initiation mechanisms . The possible interplay between DNA replication, homologous recombination, DNA repair, and transcription is explored.

Arthritis Rheum, 1997 Jun, 40(6), 1012 - 9
In vivo suppression of early experimental osteoarthritis by interleukin-1 receptor antagonist using gene therapy; Pelletier JP et al.; OBJECTIVE: This study explored the therapeutic effect of interleukin-1 receptor antagonist (IL-1Ra), administered by gene transfer, on the progression of osteoarthritic (OA) lesions in an experimental dog model . METHODS: Seventeen mature mongrel dogs were divided into 3 groups . Group 1 (n = 7) had an anterior cruciate ligament (ACL) section of the right knee through a stab wound incision . Groups 2 and 3 (n = 5 per group), had an ACL section of the right knee and partial synovectomy of the left knee . Each dog's synovium was subjected to enzymatic digestion, and the synovial fibroblasts were propagated in monolayer culture . Synovial cells from each dog were transduced in vitro using the retrovirus MFG with either the Escherichia coli beta-galactosidase (lac Z) gene (group 2) or the human IL-1Ra gene (group 3) . Two days after surgery, the dogs received intraarticular injections as follows: group 1 phosphate buffered saline (PBS) (2 ml); group 2 autologous cells (60 x 10(6) cells/2 ml of PBS) transduced with the lac Z gene; group 3 autologous cells transduced with the IL-1Ra gene . Synovial fluid was aspirated at 2 weeks and 4 weeks . All dogs were euthanized at 4 weeks postsurgery . The right knees were dissected, and lesions were scored for macroscopic and microscopic changes . Synovial explants were dissected and representative specimens were used for histology or were cultured for 48 hours . The levels of IL-1Ra in synovial fluid and synovial explant conditioned medium were measured by specific enzyme-linked immunosorbent assay . RESULTS: The level of IL-1Ra in synovial fluid of group 3 was 202.8 +/- 131.5 ng/ml (mean +/- SEM) at 2 weeks and 2.8 +/- 2.2 ng/ml at 4 weeks after surgery . Membrane explants isolated from dogs that received synovial cells transduced with the IL-1Ra gene (group 3) actively produced IL-1Ra (4.0 +/- 2.0 ng/gm of tissue wet weight) . The severity of OA cartilage lesions was similar in groups 1 and 2 . In contrast, group 3 dogs had a marked reduction in macroscopic lesion severity on the tibial plateaus (P < 0.01 for grade; P < 0.04 for size) and femoral condyles . Moreover, the histologic lesion severity was decreased on both plateaus (P < 0.06) and condyles . CONCLUSION: This study showed that a local increase in IL-1Ra production in OA knee joints by intraarticular injection of transduced synovial cells can reduce the progression of experimentally induced lesions.

Biochem J, 1997 Jun 1, 324 ( Pt 2), 681 - 7
Properties of a cysteine-free proton-pumping nicotinamide nucleotide transhydrogenase; Meuller J et al.; Nicotinamide nucleotide transhydrogenase from Escherichia coli was investigated with respect to the roles of its cysteine residues . This enzyme contains seven cysteines, of which five are located in the alpha subunit and two are in the beta subunit . All cysteines were replaced by site-directed mutagenesis . The final construct (alphaC292T, alphaC339T, alphaC395S, alphaC397T, alphaC435S, betaC147S, betaC260S) was inserted normally in the membrane and underwent the normal NADPH-dependent conformational change of the beta subunit to a trypsin-sensitive state . Reduction of NADP+ by NADH driven by ATP hydrolysis or respiration was between 32% and 65% of the corresponding wild-type activities . Likewise, the catalytic and proton pumping activities of the purified cysteine-free enzyme were at least 30% of the purified wild-type enzyme activities . The H+/H- ratio for both enzymes was 0.5, although the cysteine-free enzyme appeared to be more stable than the wild-type enzyme in proteoliposomes . No bound NADP(H) was detected in the enzymes . Modification of transhydrogenase by diethyl pyrocarbonate and the subsequent inhibition of the enzyme were unaffected by removal of the cysteines, indicating a lack of involvement of cysteines in this process . Replacement of cysteine residues in the alpha subunit resulted in no or little change in activity, suggesting that the basis for the decreased activity was probably the modification of the conserved beta-subunit residue Cys-260 or (less likely) the non-conserved beta-subunit residue Cys-147 . It is concluded that the cysteine-free transhydrogenase is structurally and mechanistically very similar to the wild-type enzyme, with minor modifications of the properties of the NADP(H) site, possibly mediated by the betaC260S mutation . The cysteine-free construct will be a valuable tool for studying structure-function relationships of transhydrogenases.

Biochem J, 1997 Jun 1, 324 ( Pt 2), 619 - 26
A soluble 3-hydroxy-3-methylglutaryl-CoA reductase in the protozoan Trypanosoma cruzi; Pena-Diaz J et al.; We report the isolation and characterization of a genomic clone containing the open reading frame sequence for 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase from Trypanosoma cruzi, the causative agent of Chagas' disease . The protozoan gene encoded for a smaller polypeptide than the rest of the genes described from eukaryotic organisms and the deduced amino acid sequence could be aligned with the C-terminal half of animal and plant reductases exhibiting pronounced similarity to other eukaryotic counterparts . Further examination of the 5' flanking region by cDNA analysis and establishment of the splice acceptor sites clearly indicated that the corresponding mRNA apparently lacks sequences encoding a membrane N-terminal domain . The reductase gene is a single copy and is located on a chromosome of 1.36 Mb as determined by contour-clamped homogeneous electric field electrophoresis . The overall cellular distribution of enzymic activity was investigated after differential centrifugation of Trypanosoma cell extracts . Reductase activity was primarily associated with the cellular soluble fraction because 95% of the total cellular activity was recovered in the supernatant and was particularly sensitive to proteolytic inactivation . Furthermore the enzyme can be efficiently overexpressed in a highly active form by using the expression vector pET-11c . Thus Trypanosoma cruzi HMG-CoA reductase is unique in the sense that it totally lacks the membrane-spanning sequences present in all eukaryotic HMG-CoA reductases so far characterized.

Biochem J, 1997 Jun 1, 324 ( Pt 2), 591 - 5
Interaction of truncated human interferon gamma variants with the interferon gamma receptor: crucial importance of Arg-129; Haelewyn J et al.; Recombinant human interferon gamma (IFN-gamma), produced in Escherichia coli, was selectively truncated at its C-terminus with chymotrypsin, clostripain or plasmin . The C-terminal amino acid residues of the three truncated IFN-gamma variants were identified as Phe136, Arg129 and Lys128, indicating the removal of 7, 14 and 15 amino acid residues from the full-length molecule . The absence of seven C-terminal residues did not influence the binding of IFN-gamma to its receptor . In contrast, the truncation of 14 residues resulted in a decrease in the Ka value to 1/24, as determined by surface plasmon resonance analysis . The removal of one additional amino acid residue from the C-terminal region of IFN-gamma led to a marked loss of receptor-binding capacity and biological activity . These observations demonstrate that Arg129 is an essential part of a functionally important C-terminal IFN-gamma sequence that is involved in receptor interaction.

Nat Biotechnol, 1997 Jun, 15(6), 581 - 5
Manipulating the aggregation and oxidation of human SPARC in the cytoplasm of Escherichia coli; Schneider EL et al.; Human SPARC (secreted protein acidic and rich in cysteine), an extracellular matrix protein containing 14 cysteine residues, was found to partition equally between soluble and insoluble cellular fractions when overexpressed in the Escherichia coli cytoplasm . While the growth temperature and medium pH had little effect on inclusion body formation, co-overproduction of the dnaKJ operon, but not of the groE operon, suppressed aggregation at the expense of intracellular accumulation . Although both forms of the protein were fully reduced in wild-type cells, 70% to 85% of soluble and insoluble SPARC could be converted into oxidized species in a thioredoxin reductase (trxB) null mutant following incubation on ice . Approximately 15% to 20% of SPARC exhibited the electrophoretic mobility of the biologically active protein . Overproduction of the dnaKJ operon in trxB cells decreased the formation of disulfide-bonded SPARC multimers in the aggregated material but not in its soluble counterpart . Our results suggest that the activity responsible for disulfide bond formation in trxB mutants acts at the post-translational level and is able to freely diffuse within inclusion bodies.

Nat Biotechnol, 1997 Jun, 15(6), 553 - 7
Yeast surface display for screening combinatorial polypeptide libraries; Boder ET et al.; Display on the yeast cell wall is well suited for engineering mammalian cell-surface and secreted proteins (e.g., antibodies, receptors, cytokines) that require endoplasmic reticulum-specific post-translational processing for efficient folding and activity . C-terminal fusion to the Aga2p mating adhesion receptor of Saccharomyces cerevisiae has been used for the selection of scFv antibody fragments with threefold decreased antigen dissociation rate from a randomly mutated library . A eukaryotic host should alleviate expression biases present in bacterially propagated combinatorial libraries . Quantitative flow cytometric analysis enables fine discrimination of kinetic parameters for protein binding to soluble ligands.

Protein Expr Purif, 1997 Jun, 10(1), 141 - 53
Overexpression and large-scale purification of recombinant hamster polymorphic arylamine N-acetyltransferase as a dihydrofolate reductase fusion protein; Sticha KR et al.; N-Acetyltransferases (NATs) are enzymes that catalyze the detoxification and/or bioactivation of a variety of xenobiotics . Rapid kinetic, biophysical, structural, and bioactivation studies on NATs require quantities of purified enzyme capable of being obtained only through recombinant DNA technology . This laboratory has previously developed a protein expression and purification system in which NATs are expressed as proteins fused to a FLAG octapeptide followed by a thrombin-cleavage site to allow liberation of the rNAT . Typically, however, only 0.5-1.5 mg of the recombinant NAT's could be readily purified in a single isolation sequence by immunoaffinity chromatography . Therefore, the expression system was modified by inserting the L54F dihydrofolate reductase (DHFR) mutant gene sequence between the FLAG octapeptide and the thrombin-cleavage site . Expression was carried out with TOPP3 Escherichia coli cells . The new purification methodology utilizes the unique pH dependence of binding to a methotrexate (MTX)-affinity column by the L54F DHFR mutant . Unfortunately, the affinity chromatography strategy did not work satisfactorily . Although the specific activity of the purified rNAT2 was comparable to that of NAT2 obtained from hamster tissue, only 3% of the activity was recovered . The apparent cause of the low recovery is the unanticipated irreversible binding of rNAT2 to MTX . Ion-exchange chromatography was investigated as an alternative purification method . An initial DEAE anion-exchange column resulted in partial purification of the fusion protein . The fusion protein was cleaved with thrombin and reapplied to a DEAE anion-exchange column . The second DEAE column resulted in not only the separation of rNAT2-70D from FLAG-L54F DHFR, but also the purification of rNAT2-70D to near homogeneity . Application of the nearly homogeneous rNAT2-70D to a gel-filtration column resulted in recovery of homogeneous protein . The ion-exchange method of purifying rNAT2-70D is inexpensive and simple and yields more than 8 mg of pure enzyme from 1 liter of cell culture.

Protein Expr Purif, 1997 Jun, 10(1), 123 - 31
In vitro folding and functional analysis of an anti-insect selective scorpion depressant neurotoxin produced in Escherichia coli; Turkov M et al.; The selective toxicity of depressant scorpion neurotoxins to insects is useful in studying insect sodium channel gating and has an applied potential . In order to establish a genetic system enabling a structure-activity approach, the functional expression of such polypeptides is required . By engineering the cDNA encoding the depressant scorpion neurotoxin, LahIT2, behind the T7 promoter, large amounts of recombinant insoluble and nonactive toxin were obtained in Escherichia coli . Following denaturation and reduction, the recombinant protein, constructed with an additional N-terminal methionine residue, was subjected to renaturation . Optimal conditions for reconstitution of a functional toxin, having a dominant fold over many other possible isoforms, were established . The recombinant active toxin was purified by RP-HPLC and characterized . Toxicity (ED50) to insects, binding affinity (IC50) to an insect receptor site, and electrophysiological effect on an insect axonal preparation were found to be similar to those of the native toxin . Substitution of the C-terminal glycine by a Gly-Lys-Lys triplet did not abolish folding but affected toxicity (3.5-fold decrease) of LqhIT2 . Apparently, this efficient bacterial expression system (500 micrograms HPLC-purified toxin/1 liter E . coli culture) provides the means for studying structure/ activity relationship and the molecular basis for the phylogenetic selectivity of scorpion depressant neurotoxins.

Protein Expr Purif, 1997 Jun, 10(1), 100 - 6
cDNA cloning, expression, and rapid purification of a Kunitz-type winged bean chymotrypsin inhibitor; Ghosh S et al.; A 183-residue Kunitz-type winged bean chymotrypsin inhibitor (WbCI), inhibits its cognate protease at a molar ratio of 1:2, instead of the usual ratio of 1:1 common to other members of the family . From the cDNA pool obtained by reverse transcription of the poly(A)+ RNA of the developing winged bean seeds, the structural gene of WbCI has been amplified by PCR using primers designed to delete the 24-residue signal peptide and introduce EcoRI and SalI sites at the ends of the amplified DNA . The latter is cloned in pBluescript and the insert has been sequenced to confirm its authenticity . Subcloning it in pTrc99A, a high-expression vector for Escherichia coli has generated a chimeric plasmid, pTrc-WbCI, which has a reading frame for a recombinant protein (rWbCI), having an additional tripeptide (M-E-F) fused to the N-terminus of WbCI . The expression of rWbCI has been ascertained by immunoblot analysis using rabbit anti-WbCI immune sera and quantitated by ELISA . The optimal conditions for the induction of the protein by IPTG, avoiding complications of protein-body formation, have also been standardized . rWbCI has been purified by a simple and rapid procedure of immunoaffinity chromatography, with an overall yield of 1.3 mg/g wet cell . SDS-PAGE analysis shows the presence of a single protein band, attesting to the homogeneity of the preparation; functionally, it is indistinguishable from WbCI since they inhibit alpha-chymotrypsin in an identical manner.

Protein Expr Purif, 1997 Jun, 10(1), 80 - 8
High-level expression of the prohormones proenkephalin, pro-neuropeptide Y, proopiomelanocortin, and beta-protachykinin for in vitro prohormone processing; Hook VY et al.; Prohormone substrates are required for investigation of the proteolytic processing of prohormones and proproteins into active peptide hormones and neurotransmitters . However, the lack of prohormone proteins has been a limiting factor in elucidating proteolytic mechanisms for conversion of prohormones into active peptides . Therefore, in this study, cloned cDNAs encoding the prohormones proenkephalin (PE), pro-neuropeptide Y (pro-NPY), pro-opiomelanocortin (POMC), and beta-protachykinin (beta-PT) were utilized to express recombinant prohormones in Escherichia coli . High-level expression of milligrams of prohormones was achieved with the pET3c expression vector utilizing the T7 promoter for production of PE, pro-NPY, and POMC, as demonstrated by SDS-PAGE gel electrophoresis, Western blots, and 35S-methionine labeling . In addition, beta-PT was expressed at high levels as fusion proteins with the maltose-binding protein and glutathione S-transferase by the pMAL-c and pGEX-2T expression vectors, respectively . Relative rates of processing by the established processing proteases "prohormone thiol protease" (PTP), 70-kDa aspartyl protease, and PC1/ 3 and PC2 (PC, prohormone convertase) were examined with purified PE, pro-NPY, and POMC . Distinct preferences of processing enzymes for different prohormones was demonstrated . PTP preferred PE and pro-NPY substrates, whereas little processing of POMC was detected . In contrast, the 70-kDa aspartyl protease cleaved POMC more readily than pro-NPY or PE . However, PC1/3 and PC2 prefer POMC as substrate . Demonstration of selectivity of processing enzymes for prohormone substrates illustrates the importance of expressing recombinant prohormones for in vitro processing studies.

Protein Expr Purif, 1997 Jun, 10(1), 51 - 4
Expression, purification, and characterization of the recombinant NAD-malic enzyme from Ascaris suum; Chooback L et al.; The cDNA encoding the 65-kDa subunit of malic enzyme from Ascaris suum was cloned into the bacterial expression vector pKK223-3 and overproduced in Escherichia coli . A protein with a subunit molecular mass of 65,000 was expressed at a level of up to 3% of the total soluble protein in JM109, as judged by SDS-PAGE . The enzyme was purified using column chromatography on phenyl-Sepharose followed by orange-A agarose . The purification procedure resulted in a 32-fold purification with an overall yield of 51% . The bacterially expressed enzyme exhibits kinetic constants identical to those measured for native A . suum NAD-malic enzyme.

Protein Expr Purif, 1997 Jun, 10(1), 42 - 50
Large-scale isolation of proteins of the large subunit from Escherichia coli ribosomes; Diedrich G et al.; A strategy has been developed and optimized that allows the isolation of proteins of the large subunit from Escherichia coli ribosomes and combines the following advantages: speed, applicability for the isolation of milligram amounts of a single protein, and preservation of the biological activity of the proteins . The method consists of the following steps: ion-exchange chromatography on MonoS and MonoQ, gel filtration on Sephadex 75, and salt washes . Eleven proteins can be purified by a single chromatographic step, and a combination of two steps enables the isolation of the other proteins.

Protein Expr Purif, 1997 Jun, 10(1), 10 - 26
Purification and characterization of human and mouse recombinant alpha-fetoproteins expressed in Escherichia coli; Boismenu R et al.; Alpha-fetoprotein (AFP) is a tumor-associated embryonic molecule whose precise biological function(s) remains unclear . A more complete analysis of the physiological activities of this oncofetal protein has, until now, been severely limited by the lack of an appropriate source from which to obtain pure AFP in any sizeable quantity . In the present investigation, we obviate this problem by cloning and efficiently overexpressing mature mouse and human AFP cDNA's in Escherichia coli . For recombinant mouse AFP (rMoAFP), large segments of the coding region were excised from the preexisting plasmids pAFP1 and pAFP2, which together encompass 90% of the AFP sequence . The mouse cDNA was made complete by the addition of N- and C-terminal encoding oligonucleotides . Mouse AFP cDNA was expressed directly as a full-length molecule in vector pTrp4 or as fusion proteins in plasmids pMALc and pRX1 under the transcriptional control of trp or tac promoters . Accumulation of rMoAFP was significantly increased in protease-deficient E . coli strains over nonprotease-deficient strains, > or = 10% of total cell protein . Of the gene fusion proteins examined, none offered significant advantage over the direct expression product in terms of recombinant protein stability, overall levels of synthesis, or facilitated purification . Recombinant AFP polypeptides expressed by pTrp4 were as expected, deposited in bacterial inclusion bodies . Subsequent to resolubilization/refolding, rMoAFP was first enriched by passage over Q-Sepharose resin followed by final purification using immobilized copper-chelate affinity chromatography . Protein sequencing of the N-terminus revealed that purified rMoAFP had a deletion of the first nine amino acids coded for by the full-length mouse AFP cDNA . Similar N-terminal deletions are observed with AFP isolates originating from natural sources . A complete human AFP cDNA was generated from a fetal liver cDNA library and was cloned into vector pTrp4 . Recombinant human AFP (rHuAFP) was expressed under the identical conditions employed for rMoAFP but purification had to be modified to include preparative Mono Q anion exchange chromatography . N-terminal sequencing, amino acid compositional analysis, and electrospray mass spectrometry revealed that purified rHuAFP was intact and unaltered and that the initiator methionine was completely removed . The biological activity of recombinant AFP, as judged by its inhibitory effects on in vitro lymphocyte proliferation, was equivalent to that of the native protein . The availability of large quantities of mouse and human recombinant AFP molecules should now permit detailed structure-function analyses of this important oncofetal protein to proceed in a manner unimpeded by previous limitations in both quantity and quality of the native proteins.

J Lab Clin Med, 1997 Jun, 129(6), 634 - 7
Phagocytosis and burst activity of granulocytes and monocytes after stem cell transplantation; Miyagawa B et al.; In addition to a low number of leukocytes, an impairment of leukocyte function can also contribute to the increased susceptibility to bacterial and fungal infection in marrow transplant recipients . Phagocytosis and oxidative burst activity were measured in patients at various stages after transplant by using assay systems that are based on the quantification of the immunofluorescence of ingested bacteria . Although phagocytosis was normal in most transplant recipents, the oxidative burst of granulocytes was significantly impaired in recipients early after autologous and allogeneic transplants . Patients who underwent tests in later stages after transplant had normal leukocyte function test results . These observations are compatible with the notion that recipients early after bone marrow transplant may be able to phagocytize bacteria readily but that their ability to inactivate them via the oxidative burst might be compromised.

Genetics, 1997 Jun, 146(2), 471 - 9
Long-term experimental evolution in Escherichia coli . VI . Environmental constraints on adaptation and divergence; Travisano M; The effect of environment on adaptation and divergence was examined in two sets of populations of Escherichia coli selected for 1000 generations in either maltose-or glucose-limited media . Twelve replicate populations selected in maltose-limited medium improved in fitness in the selected environment, by an average of 22.5% . Statistically significant among-population genetic variation for fitness was observed during the course of the propagation, but this variation was small relative to the fitness improvement . Mean fitness in a novel nutrient environment, glucose-limited medium, improved to the same extent as in the selected environment, with no statistically significant among-population genetic variation . In contrast, 12 replicate populations previously selected for 1000 generations in glucose-limited medium showed no improvement, as a group, in fitness in maltose-limited medium and substantial genetic variation . This asymmetric pattern of correlated responses suggests that small changes in the environment can have profound effects on adaptation and divergence.

Genetics, 1997 Jun, 146(2), 457 - 70
Enhanced deletion formation by aberrant DNA replication in Escherichia coli; Saveson CJ et al.; Repeated genes and sequences are prone to genetic rearrangements including deletions . We have investigated deletion formation in Escherichia coli strains mutant for various replication functions . Deletion was selected between 787 base pair tandem repeats carried either on a ColE1-derived plasmid or on the E . coli chromosome . Only mutations in functions associated with DNA Polymerase III elevated deletion rates in our assays . Especially large increases were observed in strains mutant in dnaQ the epsilon editing subunit of Pol III, and dnaB, the replication fork helicase . Mutations in several other functions also altered deletion formation: the alpha polymerase (dnal;), the gamma clamp loader complex (holC, dnaX), and the beta clamp (dnaN) subunits of Pol III and the primosomal proteins, dnaC and priA . Aberrant replication stimulated deletions through several pathways . Whereas the elevation in dnaB strains was mostly recA- and lexA-dependent, that in dnaQ strains was mostly recA- and lexA-independent . Deletion product analysis suggested that slipped mispairing, producing monomeric replicon products, may be preferentially increased in a dnaQ mutant and sister-strand exchange, producing dimeric replicon products, may be elevated in dnaE mutants . We conclude that aberrant Polymerase III replication can stimulate deletion events through several mechanisms of deletion and via both recA-dependent and independent pathways.

Clin Immunol Immunopathol, 1997 Jun, 83(3), 310 - 7
Screening of SLE sera using purified recombinant Sm-D1 protein from a baculovirus expression system; Ou Y et al.; The Sm-D1 polypeptide is a major target of autoantibodies diagnostic for systemic lupus erythematosus (SLE) . The cDNA encoding the human antigen was expressed as a full-length, nonfusion protein using a eukaryotic baculovirus expression system . This recombinant version of Sm-D1 (rSm-D1) was purified to apparent homogeneity by a combination of differential extraction steps and FPLC chromatography . A direct antibody-binding ELISA was developed using the purified antigen . There was 96% correlation between the rSm-D1 and bona fide Sm-D1 from either HeLa cells or rabbit thymus when tested against Sm-positive patient sera by ELISA . The baculovirus-expressed Sm-D1 is reactive not only with patient anti-Sm sera, but also with anti-Sm monoclonal antibodies . Our results suggest that this rSm-D1 mimics the bona fide sources, providing a valuable addition to the roster of antigens available for SLE screening, epitope mapping and overall structure study.

Hum Mol Genet, 1997 Jun, 6(6), 963 - 8
'Late onset' ornithine transcarbamylase deficiency: function of three purified recombinant mutant enzymes; Morizono H et al.; Although many mutations in the ornithine transcarbamylase gene have been correlated with 'late onset' of hyperammonemia in patients, the effects of these mutations on enzyme function are largely unknown . Three recurrent mutations (R40H, R277W and R277Q) found in patients with 'late onset' disease were incorporated into 'mature' human ornithine transcarbamylase cDNA and overexpressed in Escherichia coli . The three recombinant mutant enzymes were purified to homogeneity on an affinity column and their biochemical characteristics were compared to the wild type enzyme . The R277W and R277Q mutants display markedly reduced affinity for L-ornithine, loss of substrate inhibition, alkaline shift of pH optimum, and reduced thermal stability compared to the wild type enzyme . These differences, particularly the reduced affinity for L-ornithine, are sufficient to account for their biochemical effects . In contrast, the 'mature' R40H mutant was biochemically indistinguishable from the wild type enzyme in vitro.

RNA, 1997 Jun, 3(6), 613 - 23
The protein cofactor allows the sequence of an RNase P ribozyme to diversify by maintaining the catalytically active structure of the enzyme; Kim JJ et al.; To study the effect proteins have on the catalysis and evolution of RNA enzymes, we simulated evolution of RNase P catalytic M1 RNA in vitro, in the presence and absence of its C5 protein cofactor . In the presence of C5, functional M1 sequence variants (not catalytically active in the absence of C5) were selected in addition to those identical to M1 . C5 maintains the catalytically active structure of the variants and allows for an enhanced spectrum of M1 molecules to function in the context of a ribonucleoprotein (RNP) complex . The generation of an RNP enzyme, requiring both RNA and protein components, from a catalytically active RNA molecule has implications for how modern RNP complexes evolved from ancestral RNAs.

RNA, 1997 Jun, 3(6), 602 - 12
Exact determination of UV-induced crosslinks in 16S ribosomal RNA in 30S ribosomal subunits; Wilms C et al.; Escherichia coli 30S ribosomal subunits were UV-irradiated to induce intramolecular crosslinks in the 16S rRNA . Intact 16S rRNA was purified and subjected to gel electrophoresis, under denaturing conditions, to separate molecules on the basis of the crosslinked loop size . Molecules separated this way were enriched for specific crosslinks and could be analyzed by the reverse transcription arrest assay to determine exact crosslinking sites . Thirteen crosslinking sites have been identified at single nucleotide resolution . Of these, eight are within or adjacent to secondary structure elements: one of these (C582 x G760) involves an interaction between nucleotides within an interior loop, one (C1402 x X1501) involves an interaction between nucleosides in adjacent base pairs, and the others involve interactions between nucleotides that are within junction regions (A441 x G494, U562 x U884, C934 x U1345, and U991 x U1212) or are interactions between nucleotides (C54 x A353 and U1052 x C1200) that somehow cross known base pairs . Five other crosslinks connect sites distant in the secondary structure and provide global constraints for the arrangement of RNA regions within RNA domains I and II (U244 x G894, G894 x A1468, C967 x C1400) and within domain III (U1126 x C1281 and A1093 x G1182) . These crosslinks, known at single-nucleotide resolution, are useful in the prediction of local RNA regions, as well as the global structure.

RNA, 1997 Jun, 3(6), 561 - 76
Analysis of the tertiary structure of the ribonuclease P ribozyme-substrate complex by site-specific photoaffinity crosslinking; Harris ME et al.; Bacterial ribonuclease P (RNase P), an endonuclease involved in tRNA maturation, is a ribonucleoprotein containing a catalytic RNA . The secondary structure of this ribozyme is well-established, and a low-resolution model of the three-dimensional structure of the ribozyme-substrate complex has been proposed based on site-specific crosslinking and phylogenetic comparative data {Harris ME et al., 1994 EMBO J 13:3953-3963} . However, several substructures of that model were poorly constrained by the available data . In the present analysis, additional constraints between elements within the Escherichia coli RNase P RNA-pre-tRNA complex were determined by intra- and intermolecular crosslinking experiments . Circularly permuted RNase P RNAs were used to position an azidophenacyl photoactive crosslinking agent specifically at strategic sites within the ribozyme-substrate complex . Crosslink sites were mapped by primer extension and confirmed by analysis of the mobility of the crosslinked RNA lariats on denaturing acrylamide gels relative to circular and linear RNA standards . Crosslinked species generally retained significant catalytic activity, indicating that the results reflect the native ribozyme structure . The crosslinking results support the general configuration of the structure model and predicate new positions and orientations for helices that were previously poorly constrained by the data set . The expanded library of crosslinking constraints was used, together with secondary and tertiary structure identified by phylogenetic sequence comparisons, to refine significantly the model of RNase P RNA with bound substrate pre-tRNA . The crosslinking results and data from chemical-modification and mutational studies are discussed in the context of the current structural perspective on this ribozyme.

Parasitology, 1997 Jun, 114 ( Pt 6), 605 - 13
Characterization of two cDNAs encoding cysteine proteinases from the soybean cyst nematode Heterodera glycines; Urwin PE et al.; Two cDNAs encoding cysteine proteinases were isolated from a cDNA library constructed from feeding females of Heterodera glycines . The library was screened with a cysteine proteinase gene fragment originally amplified from cDNA of H . glycines . Database searches predict that 1 cDNA (hgcp-I) encodes a cathepsin L-like proteinase, while the second (hgcp-II) encodes a cathepsin S-like enzyme . Both predicted proteins contain a short secretion signal sequence, a long propeptide and a mature protein of 219 amino acids . Southern blot analysis suggests that the cathepsin S-like enzyme, HGCP-II, is encoded by a single-copy gene in contrast to the cathepsin L-like proteinase, HGCP-I which may have 2 homologues . The regions encoding the mature proteinases were cloned into an expression vector and recombinant protein produced in E . coli . HGCP-I was shown, after refolding, to cleave the synthetic peptide Z-Phe-Arg-AMC, and this activity could be inhibited by the engineered rice cystatin Oc-I delta D86 . HGCP-II showed no activity against the synthetic substrates tested . The knowledge gained from these studies will improve our understanding of plant nematode proteinases and aid the development of a rational proteinase inhibitor-based approach to plant nematode resistance.

Parasitology, 1997 Jun, 114 ( Pt 6), 507 - 16
Mapping of the antigenic determinants of the Leishmania infantum gp63 protein recognized by antibodies elicited during canine visceral leishmaniasis; Morales G et al.; The gp63 gene encoding the major surface antigen of Leishmania infantum has been cloned and sequenced . In spite of the overall sequence homology with the gp63 genes from other Leishmania species, particularly with the constitutively expressed Leishmania chagasi Gp63 gene, the carboxy-terminal ends of these genes are clearly divergent (62% homology) . To study the prevalence of anti-gp63 antibodies in the sera from dogs with visceral leishmaniasis, a recombinant L . infantum gp63 protein was expressed in Escherichia coli . It was found that 100% of the sera from these dogs recognized the recombinant gp63 protein, suggesting that it must function as a potent B cell immunogen during natural canine visceral leishmaniasis . However, heterogeneity in the level of response was observed . Fine mapping of the antigenic determinants was performed by means of 6 overlapping subfragments of the gp63 protein and by the use of a library of synthetic peptides . The data showed that there is some degree of immunological restriction in the recognition of the protein since reactivity was observed preferentially against the most divergent region . The epitope mapping of this region showed 2 immunodominant peptides the response to which seems to be preferentially of the IgG2 type.

Appl Environ Microbiol, 1997 Jun, 63(6), 2472 - 6
Characterization of recombinant glutamine synthetase from the hyperthermophilic archaeon Pyrococcus sp . strain KOD1; Adul Rahman RN et al.; The glnA gene encoding glutamine synthetase was cloned from the hyperthermophilic archaeon Pyrococcus sp . strain KOD1, and its nucleotide sequence was determined . The glnA gene was expressed in Escherichia coli ME8459 (glnA mutant strain), and the protein was purified to homogeneity and shown to be functional in a dodecameric from (637,000 Da), exhibiting both transferase and synthetase activities . However, kinetic studies indicated that the enzyme possessed low biosynthetic activity, suggesting that the reaction was biased towards glutamate production . The optimum temperature for both activities was 60 degrees C, which was lower than the optimal growth temperature of KOD1 . Recombinant KOD1 GlnA exhibited different optimum pHs depending on the reaction employed (pH 7.8 for the synthetase reaction and pH 7.2 for the transferase reaction) . Of the various nucleoside triphosphates tested, GTP as well as ATP was involved in the synthetase reaction.

Appl Environ Microbiol, 1997 Jun, 63(6), 2403 - 10
Conjugative plasmids and the degradation of arylsulfonates in Comamonas testosteroni; Junker F et al.; Comamonas testosteroni T-2 degrades p-toluenesulfonate (TSA) via p-sulfobenzoate (PSB) and protocatechuate and degrades toluenecarboxylate via terephthalate (TER) and protocatechuate . The appropriate genes are expressed in at least five regulatory units, some of which are also found in C . testosteroni PSB-4 (F . Junker, R . Kiewitz, and A . M . Cook, J . Bacteriol . 179:919-927, 1997) . C . testosteroni T-2 was found to contain two plasmids, pTSA (85 kbp) and pT2T (50 kbp); a TSA- mutant (strain TER-1) contained only plasmid pT2T . C . testosteroni PSB-4, which does not degrade TSA, contained one plasmid, pPSB (85 kbp) . The type strain contained no plasmids . Conjugation experiments showed that plasmid pTSA (possibly in conjunction with pT2T) was conjugative, and the single copy of the TSA operon (tsaMBCD) with its putative regulator gene (tsaR) in strain T-2 was found on plasmid pTSA, which also carried the PSB genes (psbAC) and presumably transport for both substrates . Plasmid pTSA was assigned to the IncP1 beta group and was found to carry two copies of insertion element IS1071 . Plasmid pPSB (of strain PSB-4), which could be maintained in strains with plasmid pTSA or pT2T, was also conjugative and was found to carry the PSB genes as well as to contain two copies of IS1071 . In attempted conjugations with the type strain, no plasmid was recovered, but the PSB+ transconjugant carried two copies of IS1071 in the chromosome . We presume the PSB genes to be located in a composite transposon . The genes encoding the putative TER operon and degradation of protocatechuate, with the meta cleavage pathway, were attributed a chromosomal location in strains T-2 and PSB-4.

Appl Environ Microbiol, 1997 Jun, 63(6), 2355 - 60
Modelling the growth rate of Escherichia coli as a function of pH and lactic acid concentration; Presser KA et al.; The growth rate responses of Escherichia coli M23 (a nonpathogenic strain) to suboptimal pH and lactic acid concentration were determined . Growth rates were measured turbidimetrically at 20 degrees C in the range of pH 2.71 to 8.45 . The total concentration of lactic acid was fixed at specific values, and the pH was varied by the addition of a strong acid (hydrochloric) or base (sodium hydroxide) to enable the determination of undissociated and dissociated lactic acid concentrations under each condition . In the absence of lactic acid, E . coli grew at pH 4.0 but not at pH 3.7 and was unable to grow in the presence of > or = 8.32 mM undissociated lactic acid . Growth rate was linearly related to hydrogen ion concentration in the absence of lactic acid . In the range 0 to 100 mM lactic acid, growth rate was also linearly related to undissociated lactic acid concentration . A mathematical model to describe these observations was developed based on a Belehradek-like model for the effects of water activity and temperature . This model was expanded to describe the effects of pH and lactic acid by the inclusion of novel terms for the inhibition due to the presence of hydrogen ions, undissociated lactic acid, and dissociated lactic acid species . Preliminary data obtained for 200 and 500 mM total lactic acid concentrations show that the response to very high lactic acid concentrations was less well described by the model . However, for 0 to 100 mM lactic acid, the model described well the qualitative and quantitative features of the response.

J Bacteriol, 1997 Jun, 179(11), 3818 - 21
The Escherichia coli flagellar transcriptional activator flhD regulates cell division through induction of the acid response gene cadA; Pruss BM et al.; FlhD is a positive regulator of cadA . A mutant with a transposon-mediated lacZ fusion to cadA exhibited a cell division phenotype similar to that of the flhD mutant and had FlhD-dependent beta-galactosidase activity . Under different growth conditions, the cell division rate correlated with the level of expression of cadA.

J Bacteriol, 1997 Jun, 179(11), 3813 - 7
Formation of pH and potential gradients by the reconstituted Azotobacter vinelandii cytochrome bd respiratory protection oxidase; Kolonay JF Jr et al.; To directly characterize the bioenergetic properties of the cytochrome bd terminating branch of the Azotobacter vinelandii electron transport chain, the purified cytochrome bd oxidase was reconstituted into a phospholipid environment consisting of phosphatidylethanolamine and phosphatidylglycerol (3:1) . The average diameter of the proteoliposomes after extrusion through a polycarbonate membrane was 94 +/- 4 nm . Initiation of respiration upon the addition of 20 microM ubiquinone-1 to proteoliposomes loaded with the pH-sensitive dye pyranine resulted in an immediate alkalization of the vesicle lumen by an average pH change of 0.11 unit . This pH gradient was readily collapsed upon the addition of nigericin, carbonyl cyanide p-(tri-fluoromethoxy) phenyl-hydrazone, gramicidin, Triton X-100, or 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) . Proteoliposomal respiration initiated in the presence of the potentiometric membrane dye rhodamine 123 caused the generation of a transmembrane potential; the potential was collapsed upon the addition of either valinomycin or HQNO . The formation of both pH and potential gradients during turnover demonstrates that the A . vinelandii cytochrome bd oxidase is coupled to energy conservation in vivo.

J Bacteriol, 1997 Jun, 179(11), 3793 - 6
Maximum activity of recombinant ribulose 1,5-bisphosphate carboxylase/oxygenase of Anabaena sp . strain CA requires the product of the rbcX gene; Li LA et al.; Filamentous cyanobacteria of the genus Anabaena contain a unique open reading frame, rbcX, which is juxtaposed and cotranscribed with the genes (rbcL and rbcS) encoding form I ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) . Plasmid constructions containing the genes from Anabaena sp . strain CA were prepared, and expression studies in Escherichia coli indicated that the product of the rbcX gene mimicked the ability of chaperonin proteins to facilitate the proper folding of recombinant RubisCO proteins . The purified recombinant Anabaena sp . strain CA RubisCO, much like the RubisCO enzymes from other cyanobacteria, was shown not to undergo inhibition of activity during a time course experiment, and the properties of this chaperoned recombinant protein appear to be consistent with those of the enzyme isolated from the native organism.

J Bacteriol, 1997 Jun, 179(11), 3783 - 5
Characterization of endonuclease III (nth) and endonuclease VIII (nei) mutants of Escherichia coli K-12; Saito Y et al.; The nth and nei genes of Escherichia coli affect the production of endonuclease III and endonuclease VIII, respectively, glycosylases/apurinic lyases that attack DNA damaged by oxidizing agents . Here, we provide evidence that oxidative lethal lesions are repaired by both endonuclease III and endonuclease VIII and that spontaneous mutagenic lesions are repaired mainly by endonuclease III.

J Bacteriol, 1997 Jun, 179(11), 3773 - 82
Escherichia coli endonuclease VIII: cloning, sequencing, and overexpression of the nei structural gene and characterization of nei and nei nth mutants; Jiang D et al.; Escherichia coli possesses two DNA glycosylase/apurinic lyase activities with overlapping substrate specificities, endonuclease III and endonuclease VIII, that recognize and remove oxidized pyrimidines from DNA . Endonuclease III is encoded by the nth gene . Endonuclease VIII has now been purified to apparent homogeneity, and the gene, nei, has been cloned by using reverse genetics . The gene nei is located at 16 min on the E . coli chromosome and encodes a 263-amino-acid protein which shows significant homology in the N-terminal and C-terminal regions to five bacterial Fpg proteins . A nei partial deletion replacement mutant was constructed, and deletion of nei was confirmed by genomic PCR, activity analysis, and Western blot analysis . nth nei double mutants were hypersensitive to ionizing radiation and hydrogen peroxide but not as sensitive as mutants devoid of base excision repair (xth nfo) . Single nth mutants exhibited wild-type sensitivity to X rays, while nei mutants were consistently slightly more sensitive than the wild type . Double mutants lacking both endonucleases III and VIII exhibited a strong spontaneous mutator phenotype (about 20-fold) as determined by a rifampin forward mutation assay . In contrast to nth mutants, which showed a weak mutator phenotype, nei single mutants behaved as the wild type.

J Bacteriol, 1997 Jun, 179(11), 3729 - 35
Function of conserved histidine-243 in phosphatase activity of EnvZ, the sensor for porin osmoregulation in Escherichia coli; Hsing W et al.; EnvZ and OmpR are the sensor and response regulator proteins of a two-component system that controls the porin regulon of Escherichia coli in response to osmolarity . Three enzymatic activities are associated with EnvZ: autokinase, OmpR kinase, and OmpR-phosphate (OmpR-P) phosphatase . Conserved histidine-243 is critical for both autokinase and OmpR kinase activities . To investigate its involvement in OmpR-P phosphatase activity, histidine-243 was mutated to several other amino acids and the phosphatase activity of mutated EnvZ was measured both in vivo and in vitro . In agreement with previous reports, we found that certain substitutions abolished the phosphatase activity of EnvZ . However, a significant level of phosphatase activity remained when histidine-243 was replaced with certain amino acids, such as tyrosine . In addition, the phosphatase activity of a previously identified kinase- phosphatase+ mutant was not abolished by the replacement of histidine-243 with asparagine . These data indicated that although conserved histidine-243 is important for the phosphatase activity, a histidine-243-P intermediate is not required . Our data are consistent with a previous model that proposes a common transition state with histidine-243 (EnvZ) in close contact with aspartate-55 (OmpR) for both OmpR phosphorylation and dephosphorylation . Phosphotransfer occurs from histidine-243-P to aspartate-55 during phosphorylation, but water replaces the phosphorylated histidine side chain leading to hydrolysis during dephosphorylation.

J Bacteriol, 1997 Jun, 179(11), 3721 - 8
Insertion mutagenesis of the lac repressor and its implications for structure-function analysis; Nelson BD et al.; We recently developed a simple technique for the generation of relatively large (31-codon) insertion mutations in cloned genes . To test whether the analysis of such mutations could provide insight into structure-function relationships in proteins, we examined a set of insertion mutants of the Escherichia coli lac repressor (LacI) . Representatives of several LacI mutant classes were recovered, including mutants which exhibit fully active, inducer-insensitive, or weak dominant-negative phenotypes . The various properties of the recovered mutants agree with previous biophysical, biochemical, and genetic data for the protein . In particular, the results support the prior designation of mutationally tolerant spacer regions of LacI as well as proposed differences in dimerization interactions among regions of the protein core domain . These findings suggest that the analysis of 31-codon insertion mutations may provide a simple approach for characterizing structure-function relationships in proteins for which high-resolution structures are not available.

J Bacteriol, 1997 Jun, 179(11), 3697 - 705
Domains of Escherichia coli acyl carrier protein important for membrane-derived-oligosaccharide biosynthesis; Tang L et al.; Acyl carrier protein participates in a number of biosynthetic pathways in Escherichia coli: fatty acid biosynthesis, phospholipid biosynthesis, lipopolysaccharide biosynthesis, activation of prohemolysin, and membrane-derived oligosaccharide biosynthesis . The first four pathways require the protein's prosthetic group, phosphopantetheine, to assemble an acyl chain or to transfer an acyl group from the thioester linkage to a specific substrate . By contrast, the phosphopantetheine prosthetic group is not required for membrane-derived oligosaccharide biosynthesis, and the function of acyl carrier protein in this biosynthetic scheme is currently unknown . We have combined biochemical and molecular biological approaches to investigate domains of acyl carrier protein that are important for membrane-derived oligosaccharide biosynthesis . Proteolytic removal of the first 6 amino acids from acyl carrier protein or chemical synthesis of a partial peptide encompassing residues 26 to 50 resulted in losses of secondary and tertiary structure and consequent loss of activity in the membrane glucosyltransferase reaction of membrane-derived oligosaccharide biosynthesis . These peptide fragments, however, inhibited the action of intact acyl carrier protein in the enzymatic reaction . This suggests a role for the loop regions of the E . coli acyl carrier protein and the need for at least two regions of the protein for participation in the glucosyltransferase reaction . We have purified acyl carrier protein from eight species of Proteobacteria (including representatives from all four subgroups) and characterized the proteins as active or inhibitory in the membrane glucosyltransferase reaction . The complete or partial amino acid sequences of these acyl carrier proteins were determined . The results of site-directed mutagenesis to change amino acids conserved in active, and altered in inactive, acyl carrier proteins suggest the importance of residues Glu-4, Gln-14, Glu-21, and Asp-51 . The first 3 of these residues define a face of acyl carrier protein that includes the beginning of the loop region, residues 16 to 36 . Additionally, screening for membrane glucosyltransferase activity in membranes from bacterial species that had acyl carrier proteins that were active with E . coli membranes revealed the presence of glucosyltransferase activity only in the species most closely related to E . coli . Thus, it seems likely that only bacteria from the Proteobacteria subgroup gamma-3 have periplasmic glucans synthesized by the mechanism found in E . coli.

J Bacteriol, 1997 Jun, 179(11), 3691 - 6
Characterization of a thermosensitive Escherichia coli aspartyl-tRNA synthetase mutant; Martin F et al.; The Escherichia coli tls-1 strain carrying a mutated aspS gene (coding for aspartyl-tRNA synthetase), which causes a temperature-sensitive growth phenotype, was cloned by PCR, sequenced, and shown to contain a single mutation resulting in substitution by serine of the highly conserved proline 555, which is located in motif 3 . When an aspS fragment spanning the codon for proline 555 was transformed into the tls-1 strain, it was shown to restore the wild-type phenotype via homologous recombination with the chromosomal tls-1 allele . The mutated AspRS purified from an overproducing strain displayed marked temperature sensitivity, with half-life values of 22 and 68 min (at 42 degrees C), respectively, for tRNA aminoacylation and ATP/PPi exchange activities . Km values for aspartic acid, ATP, and tRNA(Asp) did not significantly differ from those of the native enzyme; thus, mutation Pro555Ser lowers the stability of the functional configuration of both the acylation and the amino acid activation sites but has no significant effect on substrate binding . This decrease in stability appears to be related to a conformational change, as shown by gel filtration analysis . Structural data strongly suggest that the Pro555Ser mutation lowers the stability of the Lys556 and Thr557 positions, since these two residues, as shown by the crystallographic structure of the enzyme, are involved in the active site and in contacts with the tRNA acceptor arm, respectively.

J Bacteriol, 1997 Jun, 179(11), 3683 - 90
The TolA protein interacts with colicin E1 differently than with other group A colicins; Schendel SL et al.; The 421-residue protein TolA is required for the translocation of group A colicins (colicins E1, E2, E3, A, K, and N) across the cell envelope of Escherichia coli . Mutations in TolA can render cells tolerant to these colicins and cause hypersensitivity to detergents and certain antibiotics, as well as a tendency to leak periplasmic proteins . TolA contains a long alpha-helical domain which connects a membrane anchor to the C-terminal domain, which is required for colicin sensitivity . The functional role of the alpha-helical domain was tested by deletion of residues 56 to 169 (TolA delta1), 166 to 287 (TolA delta2), or 54 to 287 (TolA delta3) of the alpha-helical domain of TolA, which removed the N-terminal half, the C-terminal half, or nearly the entire alpha-helical domain of TolA, respectively . TolA and TolA deletion mutants were expressed from a plasmid in an E . coli strain producing no chromosomally encoded TolA . Cellular sensitivity to the detergent deoxycholate was increased for each deletion mutant, implying that more than half of the TolA alpha-helical domain is necessary for cell envelope stability . Removal of either the N- or C-terminal half of the alpha-helical domain resulted in a slight (ca . 5-fold) decrease in cytotoxicity of the TolA-dependent colicins A, E1, E3, and N compared to cells producing wild-type TolA when these mutants were expressed alone or with TolQ, -R, and -B . In cells containing TolA delta3, the cytotoxicity of colicins A and E3 was decreased by a factor of >3,000, and K+ efflux induced by colicins A and N was not detectable . In contrast, for colicin E1 action on TolA delta3 cells, there was little decrease in the cytotoxic activity (<5-fold) or the rate of K+ efflux, which was similar to that from wild-type cells . It was concluded that the mechanism(s) by which cellular uptake of colicin E1 is mediated by the TolA protein differs from that for colicins A, E3, and N . Possible explanations for the distinct interaction and unique translocation mechanism of colicin E1 are discussed.

J Bacteriol, 1997 Jun, 179(11), 3649 - 54
Stimulatory effect of trehalose on formation and activity of Escherichia coli RNA polymerase E sigma38 holoenzyme; Kusano S et al.; The intracellular concentration of trehalose increases in the stationary-phase cells of Escherichia coli . The effects of trehalose on transcription in vitro by E . coli RNA polymerase were compared for two holoenzymes, E sigma70 and E sigma38, which were reconstituted from purified core enzyme and either sigma70 (the major sigma at the exponential growth phase) or sigma38 (the essential sigma at the stationary growth phase), respectively . The optimum trehalose concentration giving maximum transcription by E sigma38 was higher than that by E sigma70 . Transcription activation by trehalose was attributed to both increased formation of E sigma38 holoenzyme and increased transcription initiation by E sigma38 from sigma38-dependent promoters . The activation of E sigma38 by trehalose was additive with the transcription enhancement by decreased superhelicity of template DNA prepared from stationary-phase cells . We thus propose that the selective activation of transcription by E sigma38 holoenzyme takes place in the presence of specific conditions and factors present under stress conditions.

J Bacteriol, 1997 Jun, 179(11), 3613 - 8
Overexpression and characterization of a prolyl endopeptidase from the hyperthermophilic archaeon Pyrococcus furiosus; Harwood VJ et al.; The maltose-regulated mlr-2 gene from the hyperthermophilic archaeon Pyrococcus furiosus having homology to bacterial and eukaryal prolyl endopeptidase (PEPase) was cloned and overexpressed in Escherichia coli . Extracts from recombinant cells were capable of hydrolyzing the PEPase substrate benzyloxycarbonyl-Gly-Pro-p-nitroanilide (ZGPpNA) with a temperature optimum between 85 and 90 degrees C . Denaturing gel electrophoresis of purified PEPase showed that enzyme activity was associated with a 70-kDa protein, which is consistent with that predicted from the mlr-2 sequence . However, an apparent molecular mass of 59 kDa was obtained from gel permeation studies . In addition to ZGPpNA (K(Mapp) of 53 microM), PEPase was capable of hydrolyzing azocasein, although at a low rate . No activity was detected when ZGPpNA was replaced by substrates for carboxypeptidase A and B, chymotrypsin, subtilisin, and neutral endopeptidase . N-{N-(L-3-trans-Carboxirane-2-carbonyl)-L-Leu}-agmatine (E-64) and tosyl-L-Lys chloromethyl ketone did not inhibit PEPase activity . Both phenylmethylsulfonyl fluoride and diprotin A inhibited ZGPpNA cleavage, the latter doing so competitively (K(lapp) of 343 microM) . At 100 degrees C, the enzyme displayed some tolerance to sodium dodecyl sulfate treatment . Stability of PEPase over time was dependent on protein concentration; at temperatures above 65 degrees C, dilute samples retained most of their activity after 24 h while the activity of concentrated preparations diminished significantly . This decrease was found to be due, in part, to autoproteolysis . Partially purified PEPase from P . furiosus exhibited the same temperature optimum, molecular weight, and kinetic characteristics as the enzyme overexpressed in E . coli . Extracts from P . furiosus cultures grown in the presence of maltose were approximately sevenfold greater in PEPase activity than those grown without maltose . Activity could not be detected in clarified medium obtained from maltose-grown cultures . We conclude that mlr-2, now called prpA, encodes PEPase; the physiological role of this protease is presently unknown.

J Bacteriol, 1997 Jun, 179(11), 3588 - 93
Isolation and characterization of multiple adenylate cyclase genes from the cyanobacterium Anabaena sp . strain PCC 7120; Katayama M et al.; Adenylate cyclase genes, designated cyaA, cyaB1, cyaB2, cyaC, and cyaD, were isolated from the filamentous cyanobacterium Anabaena sp . strain PCC 7120 by complementation of a strain of Escherichia coli defective for the presence of cya . These genes encoded polypeptides consisting of 735, 859, 860, 1,155, and 546 amino acid residues, respectively . Deduced amino acid sequences of the regions near the C-terminal ends of these cya genes were similar to those of catalytic domains of eukaryotic adenylate cyclases . The remaining part of each cya gene towards its N-terminal end showed a characteristic structure . CyaA had two putative membrane-spanning regions . Both CyaB1 and CyaB2 had regions that were very similar to the cyclic GMP (cGMP)-binding domain of cGMP-stimulated cGMP phosphodiesterase . CyaC consisted of four distinct domains forming sequentially from the N terminus: a response regulator-like domain, a histidine kinase-like domain, a response regulator-like domain, and the catalytic domain of adenylate cyclase . CyaD contained the forkhead-associated domain in its N-terminal region . Expression of these genes was examined by reverse transcription-PCR . The transcript of cyaC was shown to be predominant in this cyanobacterium . The cellular cyclic AMP level in the disruptant of the cyaC mutant was much lower than that in the wild type.

J Bacteriol, 1997 Jun, 179(11), 3528 - 33
In vivo supercoiling of plasmid and chromosomal DNA in an Escherichia coli hns mutant; Mojica FJ et al.; We have used trimethylpsoralen to measure localized levels of unconstrained DNA supercoiling in vivo . The data provide direct evidence that plasmid and chromosomal DNA supercoiling is altered in vivo in an hns mutant . This increase in supercoiling is independent of transcription or changes in the activity of topoisomerase I . These data have implications for the mechanisms by which the chromatin-associated protein H-NS may influence chromosome organization and gene expression.

J Bacteriol, 1997 Jun, 179(11), 3519 - 27
Enhancing transcription through the Escherichia coli hemolysin operon, hlyCABD: RfaH and upstream JUMPStart DNA sequences function together via a postinitiation mechanism; Leeds JA et al.; Escherichia coli hlyCABD operons encode the polypeptide component (HlyA) of an extracellular cytolytic toxin as well as proteins required for its acylation (HlyC) and sec-independent secretion (HlyBD) . The E . coli protein RfaH is required for wild-type hemolysin expression at the level of hlyCABD transcript elongation (J . A . Leeds and R . A . Welch, J . Bacteriol . 178:1850-1857, 1996) . RfaH is also required for the transcription of wild-type levels of mRNA from promoter-distal genes in the rfaQ-K, traY-Z, and rplK-rpoC gene clusters, supporting the role for RfaH in transcriptional elongation . All or portions of a common 39-bp sequence termed JUMPStart are present in the untranslated regions of RfaH-enhanced operons . In this study, we tested the model that the JUMPStart sequence and RfaH are part of the same functional pathway . We examined the effect of JUMPStart deletion mutations within the untranslated leader of a chromosomally derived hlyCABD operon on hly RNA and HlyA protein levels in either wild-type or rfaH null mutant E . coli . We also provide in vivo physical evidence that is consistent with RNA polymerase pausing at the wild-type JUMPStart sequences.

J Bacteriol, 1997 Jun, 179(11), 3494 - 9
The Escherichia coli histone-like protein HU affects DNA initiation, chromosome partitioning via MukB, and cell division via MinCDE; Jaffe A et al.; Escherichia coli hupA hupB double mutants, lacking both subunits (HU1 and HU2) of the histone-like protein HU, accumulate secondary mutations . In some genetic backgrounds, these include mutations in the minCDE operon, inactivating this system of septation control and resulting in the formation of minicells . In the course of the characterization of hupA hupB mutants, we observed that the simultaneous absence of the HU2 subunit and the MukB protein, implicated in chromosome partitioning, is lethal for the bacteria; the integrity of either HU or MukB thus seems to be essential for bacterial growth . The HU protein has been shown to be involved in DNA replication in vitro; we show here that its inactivation in the hupA hupB double mutant disturbs the synchrony of replication initiation in vivo, as evaluated by flow cytometry . Our results suggest that global nucleoid structure, determined in part by the histone-like protein HU, plays a role in DNA replication initiation, in proper chromosome partitioning directed by the MukFEB proteins, and in correct septum placement directed by the MinCDE proteins.

J Bacteriol, 1997 Jun, 179(11), 3458 - 69
Maximization of transcription of the serC (pdxF)-aroA multifunctional operon by antagonistic effects of the cyclic AMP (cAMP) receptor protein-cAMP complex and Lrp global regulators of Escherichia coli K-12; Man TK et al.; The arrangement of the Escherichia coli serC (pdxF) and aroA genes into a cotranscribed multifunctional operon allows coregulation of two enzymes required for the biosynthesis of L-serine, pyridoxal 5'-phosphate, chorismate, and the aromatic amino acids and vitamins . RNase T2 protection assays revealed two major transcripts that were initiated from a promoter upstream from serC (pdxF) . Between 80 to 90% of serC (pdxF) transcripts were present in single-gene mRNA molecules that likely arose by Rho-independent termination between serC (pdxF) and aroA . serC (pdxF)-aroA cotranscripts terminated at another Rho-independent terminator near the end of aroA . We studied operon regulation by determining differential rates of beta-galactosidase synthesis in a merodiploid strain carrying a single-copy lambda{phi(serC {pdxF}'-lacZYA)} operon fusion . serC (pdxF) transcription was greatest in bacteria growing in minimal salts-glucose medium (MMGlu) and was reduced in minimal salts-glycerol medium, enriched MMGlu, and LB medium . serC (pdxF) transcription was increased in cya or crp mutants compared to their cya+ crp+ parent in MMGlu or LB medium . In contrast, serC (pdxF) transcription decreased in an lrp mutant compared to its lrp+ parent in MMGlu . Conclusions obtained by using the operon fusion were corroborated by quantitative Western immunoblotting of SerC (PdxF), which was present at around 1,800 dimers per cell in bacteria growing in MMGlu . RNase T2 protection assays of serC (pdxF)-terminated and serC (pdxF)-aroA cotranscript amounts supported the conclusion that the operon was regulated at the transcription level under the conditions tested . Results with a series of deletions upstream of the P(serC (pdxF)) promoter revealed that activation by Lrp was likely direct, whereas repression by the cyclic AMP (cAMP) receptor protein-cAMP complex (CRP-cAMP) was likely indirect, possibly via a repressor whose amount or activity was stimulated by CRP-cAMP.

J Bacteriol, 1997 Jun, 179(11), 3383 - 90
Extracellular secretion of Escherichia coli heat-stable enterotoxin I across the outer membrane; Yamanaka H et al.; Escherichia coli heat-stable enterotoxin Ip (STIp) is an extracellular toxin consisting of 18 amino acid residues that is synthesized as a precursor of pre (amino acid residues 1 to 19), pro (amino acid residues 20 to 54), and mature (amino acid residues 55 to 72) regions . The precursor synthesized in the cytoplasm is translocated across the inner membrane by the general export pathway consisting of Sec proteins . The pre region functions as a leader peptide and is cleaved during translocation . However, it remains unknown how the resulting peptide (pro-mature peptide) translocates across the outer membrane . In this study, we investigated the structure of the STIp that passes through the outer membrane to determine how it translocates through the outer membrane . The results showed that the pro region is cleaved in the periplasmic space . The generated peptide becomes the mature form of STIp, which happens to have disulfide bonds, which then passes through the outer membrane . We also showed that STIp with a carboxy-terminal peptide consisting of 3 amino acid residues passes through the outer membrane, whereas STIp with a peptide composed of 37 residues does not . Amino acid analysis of mutant STIp purified from culture supernatant revealed that the peptide composed of 37 amino acid residues was cleaved into fragments of 5 amino acid residues . In addition, analyses of STIps with a mutation at the cysteine residue and the dsbA mutant strain revealed that the formation of an intramolecular disulfide bond within STIp is not absolutely required for the mature region of STIp to pass through the outer membrane.

J Neurosurg, 1997 Jun, 86(6), 998 - 1006
Chymopapain-induced reduction of proinflammatory phospholipase A2 activity and amelioration of neuropathic behavioral changes in an in vivo model of acute sciatica; Sawin PD et al.; The mechanism of action underlying chymopapain (Chymodiactin) chemonucleolysis remains obscure . Radiographic studies suggest that chymopapain does not alter disc fragment size acutely; nonetheless, patients often report symptom resolution within a few days, even hours, of treatment . The authors postulate that, in addition to its chemonucleolytic action, chymopapain may possess antiinflammatory properties . To test this hypothesis, the authors assessed the ability of chymopapain to modulate the activity of the proinflammatory enzyme phospholipase A2 (PLA2) and to ameliorate behavioral changes associated with inflammatory neuropathy in an in vivo model of sciatica . Thirty-nine male Fischer rats were randomly assigned to one of three treatment groups: 1) saline, 2) betamethasone, or 3) chymopapain . All of the rats underwent unilateral sciatic nerve ligation with loose chromic gut suture to induce inflammatory mononeuropathy . The animals were tested for thermal and mechanical hyperalgesia on Days 0 (preoperation), 7 (pretreatment), and 14 (prior to death) . Three animals were killed on Day 0 to determine the baseline PLA2 activity within unmanipulated rat sciatic nerves . On Day 7, three animals from each group were killed to assess PLA2 activity prior to treatment . The remainder were given a single infusion of saline, betamethasone (0.3 mg/kg), or chymopapain (100 pKat U) around the inflamed nerve . On Day 14, the remaining animals were killed and their sciatic nerves were removed . The tissue was homogenized and the PLA2 activity was determined using {14C}arachidonate-labeled Escherichia coli phospholipid membrane as a substrate . Lipids were extracted and separated by thin-layer chromatography . All animals developed behavioral changes consistent with inflammatory mononeuropathy 24 to 72 hours postoperatively; these included gait disturbance, flexion deformity, and hyperalgesia of the involved hindlimb . The degree of mechanical and thermal hyperalgesia was comparable between groups at Day 7 . By Day 14, the thermal hyperalgesia had resolved; the mechanical hyperalgesia was less evident in the betamethasone- and chymopapain-treated groups than in the saline-treated controls (p = 0.003; saline- vs . chymopapain-treated groups p = 0.004; saline- vs . betamethasone-treated groups p = 0.008) . The mean PLA2 activity at baseline (Day 0) was 11.6 +/- 4.9 nmol phospholipid hydrolyzed per minute per milligram of protein . The PLA2 activity at Day 7 was 74.4 +/- 18.2 (ligated side) and 21.2 +/- 11.7 (nonligated side) . At Day 14, PLA2 activity was reduced in the chymopapain- (47.8 +/- 12.3) and betamethasone- (39.7 +/- 9.5) treated groups compared with the saline control group (62.3 +/- 11.2), (saline- vs . chymopapain-treated groups p < 0.05; saline- vs . betamethasone-treated groups p < 0.01) . The PLA2 activity in nonligated specimens was 18.6 +/- 10.1 . These data indicate that chymopapain exhibits antiinflammatory properties in vivo, reducing PLA2 activity and ameliorating mechanical hyperalgesia in this model of inflammatory sciatic neuropathy.

Infect Immun, 1997 Jun, 65(6), 2233 - 9
Prelytic and lytic conformations of erythrocyte-associated Escherichia coli hemolysin; Moayeri M et al.; Flow cytometry was developed as a method to assess the conformation of erythrocyte-bound Escherichia coli hemolysin polypeptide (HlyA) . Topology of membrane-associated hemolysin (HlyA(E)) was investigated by testing surface accessibility of HlyA regions in lytic and nonlytic bound states, using a panel of 12 anti-HlyA monoclonal antibodies (MAbs) . Hemolysin associates nonlytically with erythrocytes at 0 to 2 degrees C . To test the hypothesis that the nonlytic HlyA(E) conformation at 0 to 2 degrees C differs from the lytic conformation at 23 degrees C, MAb epitope reactivity profiles at the two temperatures were compared by flow cytometry . Four MAbs have distinctly increased reactivity at 0 to 2 degrees C compared to 23 degrees C . HlyA requires HlyC-dependent acylation at lysine residues 563 and 689 for lytic function . Toxin with cysteine substitution mutations at each lysine (HlyA(K563C) and HlyA(K689C)) as well as the nonacylated form of hemolysin made in a HlyC-deficient strain were examined by flow cytometry at 0 to 2 and 23 degrees C . The three mutants bind erythrocytes at wild-type toxin levels, but there are conformational changes reflected by altered MAb epitope accessibility for six of the MAbs . To test further the surface accessibility of regions in the vicinity of MAb-reactive epitopes, HlyA(E) was proteolytically treated prior to testing for MAb reactivity . Differences in protease susceptibility at 0 to 2 degrees and 23 degrees C for the reactivities of three of the MAbs further support the model of two distinct conformations of cell-associated toxin.

Infect Immun, 1997 Jun, 65(6), 2218 - 24
Pleiotropic effects of a mutation in rfaC on Escherichia coli hemolysin; Bauer ME et al.; Several genes involved in the lipopolysaccharide (LPS) biosynthetic pathway have been shown to affect the expression or activity of Escherichia coli hemolysin (Hly), a secreted cytotoxin that is the prototype of the RTX family of toxins . To further study this relationship, E . coli K-12 strains harboring mutations in the LPS biosynthetic genes rfaS, rfaQ, rfaJ, rfaP, and rfaC were transformed with a recombinant plasmid harboring the hlyCABD operon and examined for their effects on extracellular expression and hemolytic activity . A mutation in rfaC that affected both extracellular expression and activity of Hly was studied in greater detail . This mutation led to a growth-phase-dependent decrease up to 16-fold in the steady-state level of extracellular HlyA, although transcription and secretion of HlyA were decreased no more than 2-fold . Specific hemolytic activity in toxin produced from the rfaC mutant strain was significantly reduced, in a growth-phase-dependent manner . With the rfaC gene supplied in trans, both the decreased expression and activity of Hly were restored to wild-type levels . Hly from the rfaC mutant strain exhibited much slower kinetics of hemolysis, a more rapid rate of decay of activity, and greater formation of apparently inactive HlyA-containing aggregates in culture supernatants than was exhibited in the wild-type strain . A model is proposed for a physical interaction between LPS and Hly in which LPS with intact inner core participates in forming or maintaining an active conformation of Hly and helps to protect it from aggregation or degradation.

Infect Immun, 1997 Jun, 65(6), 2211 - 7
A third secreted protein that is encoded by the enteropathogenic Escherichia coli pathogenicity island is required for transduction of signals and for attaching and effacing activities in host cells; Lai LC et al.; Enteropathogenic Escherichia coli strains are able to signal host cells, cause dramatic cytoskeletal rearrangements, and adhere intimately to the cell surface in a process known as the attaching and effacing effect . A pathogenicity island of 35 kb known as the locus of enterocyte effacement (LEE) is necessary and sufficient for this effect . The LEE encodes an outer membrane adhesin called intimin, a type III secretion apparatus, and the EspA and EspB secreted proteins . The DNA sequence of the region between espA and espB revealed a new gene, espD . The product of espD was demonstrated by using a T7 expression system . We constructed a nonpolar mutation in espD and found that the mutant is incapable of the signal transduction events that lead to activation of the putative intimin receptor in host cells and that the mutant fails to induce the attaching and effacing effect . These phenotypes were restored to the mutant by complementation with a plasmid containing the cloned espD locus . We demonstrated by immunoblotting and microsequencing that the EspD protein is secreted via the type III apparatus . Thus, we describe a novel locus encoding a secreted protein that is required for attaching and effacing activity.

Infect Immun, 1997 Jun, 65(6), 2183 - 9
Identification of proteins of Francisella tularensis induced during growth in macrophages and cloning of the gene encoding a prominently induced 23-kilodalton protein; Golovliov I et al.; The adaptation of facultative intracellular bacteria to host macrophages involves regulation of the synthesis of bacterial proteins . We analyzed the protein synthesis of Francisella tularensis LVS growing intracellularly in the macrophage-like murine cell line J774 and extracellularly in culture medium . After pulse-labeling with {35S} methionine and separation by one- and two-dimensional polyacrylamide gel electrophoresis, induction of a few proteins during intracellular growth was demonstrated . One of them, a 23-kDa protein, was prominently induced in the macrophages and also when extracellularly growing F . tularensis was exposed to hydrogen peroxide . After isolation of the 23-kDa protein from a preparative two-dimensional gel, a 22-amino-acid N-terminal peptide and two peptides obtained by trypsin digestion were sequenced . Based on the sequences, degenerate oligonucleotides were constructed for use as primers in a PCR . Hybridization of amplified DNA to XbaI-digested LVS DNA identified the gene of the 23-kDa protein in a 1.3-kb DNA fragment . Nucleotide sequence analysis revealed an open reading frame encoding a putative protein of a calculated molecular mass of 22.2 kDa . The open reading frame was preceded by a sequence typical of ribosome-binding sites in Escherichia coli . The amplified gene was successfully expressed by the pTrc99A vector in E . coli under control of the trc promoter . The gene product showed the same mobility and immunoreactivity as the 23-kDa protein of F . tularensis . The deduced amino acid sequence showed no significant homology with protein sequences in current data banks . Thus, intracellular growth of F . tularensis in macrophages was associated with prominent upregulation of a novel 23-kDa protein.

Infect Immun, 1997 Jun, 65(6), 2034 - 40
Virulence properties and clonal structures of strains of Escherichia coli O119 serotypes; Goncalves AG et al.; A total of 110 Escherichia coli strains of serogroup O119 were examined for the presence of virulence properties characteristic of enteropathogenic E . coli (EPEC) . Three virulence patterns were distinguished based on the detection of a chromosomal gene mediating intimate attachment (eaeA) and plasmid DNA involved in localized adherence (EAF and bfpA) . The first pattern, represented by strains which hybridized with three gene probes, was the most common (68%) and, with a single exception, included only O119:H6 strains . Of these strains, 90% showed a typical localized adherence (LA) pattern in HEp-2 cells and 96% were positive for intimate attachment in a fluorescent-actin staining test with a 3-h incubation period . The second pattern was represented by strains which hybridized with the eaeA gene only . Most (89.5%) of these strains showed the LA phenotype but only after 6 h of incubation (LA-like phenotype) . The third pattern consisted of strains which were positive for eaeA and bfpA but did not hybridize with the EAF probe . Most (80%) of these strains exhibited the LA-like phenotype . Analysis of several eaeA+ bfpA+ strains for the expression of the pilin subunit (BfpA) of the bundle-forming pili demonstrated that all LA strains expressed BfpA whereas the LA-like strains did not . The study of the clonal relationships, carried out by multilocus enzyme electrophoresis in 79 representative strains, defined 11 distinct electrophoretic types (ETs) . ET1 included 66% of the strains, most of which displayed the eaeA+ bfpA+ EAF+ pattern and were serotyped as O119:H6 or O119:H- . The remaining 10 ETs were each represented by no more than five strains and, with the exception of ET8, included strains of a single serotype . The genetic relatedness of the ETs revealed two main clusters, with most strains in cluster A having the eaeA+ bfpA+ EAF+ combination and a O119:H6 serotype . Cluster B was represented by atypical EPEC strains with only the eaeA+ and the eaeA+ bfpA+ virulence pattern.

Infect Immun, 1997 Jun, 65(6), 2019 - 28
Toxicity and immunogenicity of a verotoxin 1 mutant with reduced globotriaosylceramide receptor binding in rabbits; Bast DJ et al.; The verotoxins (VT1 and VT2), produced by strains of enterohemorrhagic Escherichia coli, have been implicated in the pathogenesis of hemorrhagic colitis and the hemolytic uremic syndrome . To better understand the role of globotriaosylceramide (Gb3) receptor binding by the verotoxins in disease production, we examined the clinicopathologic effects of an intravenously (i.v.) administered verotoxin 1 mutant holotoxin (Phe30Ala) in rabbits . The substitution of alanine for phenylalanine 30 in the VT1 B subunit has been shown previously to reduce both Gb3 binding affinity and capacity in vitro . This reduction in receptor binding corresponded to a 10(5)-fold reduction in the toxic activity of VT1 on a Vero cell monolayer . In this study, purified 125I-labeled Phe30Ala was administered i.v . to rabbits to determine its specific distribution in rabbit tissues . In contrast to the rapid elimination of i.v . administered 125I-VT1 from the bloodstream, 125I-Phe30Ala had a 52-fold-longer half-life in serum and failed to localize preferentially in the gastrointestinal tract and central nervous system (CNS) . Rabbits challenged with Phe30Ala at a dose equivalent to 10 times the 50% lethal dose (LD50) of VT1 showed no visible clinical symptoms typical of VT effect after 7 days . Administration of Phe30Ala at a dose equivalent to 100 times the LD50 of VT1, however, caused both clinical and histopathologic features indistinguishable from VT1 toxemia in rabbits, although the onset of symptoms was delayed . Rabbits were immunized with Phe30Ala and challenged i.v . with either 125I-VT1 or 125I-VT2 . The specific uptake of 125I-VT1 in the gastrointestinal tract and CNS was totally inhibited in Phe30Ala immune rabbits . Only a partial decrease in target organ uptake was observed in Phe30Ala immune rabbits challenged with 125I-VT2 . From this study, we conclude that Gb3 binding is responsible for target organ localization of VT1 and disease production in the rabbit . The ability of Phe30Ala to induce both strong antibody and protective responses against VT1 suggests that VT mutants with reduced receptor binding properties may be useful in vaccine strategies . A further reduction in the toxicity of Phe30Ala would be required for its use as a natural toxoid to protect against human verotoxigenic E . coli infections.

Biophys J, 1997 Jun, 72(6), 2710 - 8
Dichroic ratios in polarized Fourier transform infrared for nonaxial symmetry of beta-sheet structures; Marsh D; The transition moments for the amide bands from beta-sheet peptide structures generally do not exhibit axial symmetry about the director in linearly polarized Fourier transform infrared (FTIR) measurements on oriented systems . The angular dependences of the dichroic ratios of the amide bands are derived for beta-sheet structures in attenuated total reflection (ATR) and polarized transmission experiments on samples that are oriented with respect to the normal to the substrate and are randomly distributed with respect to the azimuthal angle in the plane of the orienting substrate . The orientational distributions of both the beta-strands and the beta-sheets are considered, and explicit expressions are given for the dichroic ratios of the amide I and amide II bands . The dichroic ratio of the amide II band, which is parallel polarized, can yield the orientation of the beta-strands directly, but to specify the orientations of the beta-sheets completely requires measurement of the dichroic ratios of both the amide I and amide II bands, or generally two bands with parallel and perpendicular polarizations . A random distribution in tilt of the planes of the beta-sheets does not give rise to equal dichroic ratios for bands with perpendicular and parallel polarizations, such as the amide I and amide II bands . The results are applied to previous ATR and polarized transmission FTIR measurements on a potassium channel-associated peptide, the Escherichia coli outer membrane protein OmpA, and the E . coli OmpF porin protein in oriented membranes.

Biophys J, 1997 Jun, 72(6), 2688 - 701
Escherichia coli diacylglycerol kinase: a case study in the application of solution NMR methods to an integral membrane protein; Vinogradova O et al.; Diacylglycerol kinase (DAGK) is a 13-kDa integral membrane protein that spans the lipid bilayer three times and which is active in some micellar systems . In this work DAGK was purified using metal ion chelate chromatography, and its structural properties in micelles and organic solvent mixtures studies were examined, primarily to address the question of whether the structure of DAGK can be determined using solution NMR methods . Cross-linking studies established that DAGK is homotrimeric in decyl maltoside (DM) micelles and mixed micelles . The aggregate detergent-protein molecular mass of DAGK in both octyl glucoside and DM micelles was determined to be in the range of 100-110 kDa-much larger than the sum of the molecular weights of the DAGK trimers and the protein-free micelles . In acidic organic solvent mixtures, DAGK-DM complexes were highly soluble and yielded relatively well-resolved NMR spectra . NMR and circular dichroism studies indicated that in these mixtures the enzyme adopts a kinetically trapped monomeric structure in which it irreversibly binds several detergent molecules and is primarily alpha-helical, but in which its tertiary structure is largely disordered . Although these results provide new information regarding the native oligomeric state of DAGK and the structural properties of complex membrane proteins in micelles and organic solvent mixtures, the results discourage the notion that the structure of DAGK can be readily determined at high resolution with solution NMR methods.

Biophys J, 1997 Jun, 72(6), 2660 - 8
Fluorescence quenching and electron spin resonance study of percolation in a two-phase lipid bilayer containing bacteriorhodopsin; Piknova B et al.; The effect of bacteriorhodopsin (BR) on the percolation properties of dimyristoylphosphatidylcholine/distearoylphosphatidylcholine bilayers was examined by studying the quenching of a lipid-bound fluorophore by a lipid-bound quencher, and by spin-spin interactions of a nitroxide-labeled lipid using electron spin resonance (ESR) . At the low concentrations of BR used, differential scanning calorimetry showed that although the transition enthalpy was reduced in a concentration-dependent manner by incorporation of BR, the solidus and fluidus phase boundaries and overall shape of the heat capacity profiles were essentially unchanged . However, fluorescence quenching and spin-label ESR data showed that the domain topology, as reflected in the percolation properties, is strongly affected by the protein . In contrast to our previous fluorescence data for the pure lipid mixtures, quenching in the coexistence region is independent of the fluid phase fraction when BR is present . In addition, the percolation threshold estimated by spin-label ESR is shifted in the presence of BR to a higher gel phase fraction at a given lipid composition . Both the fluorescence quenching and spin-label ESR data, together with the results of earlier simulations, strongly suggest that the fluid phase domains are substantially larger and/or less ramified in the presence of BR than in its absence . We have previously reported a similar effect of a transmembrane peptide, pOmpA (Escherichia coli outer membrane protein A signal peptide), on fluid domain connectivity in binary phosphatidylcholine mixtures.

Blood, 1997 Jun 1, 89(11), 4078 - 84
7E3 F(ab')2, a monoclonal antibody to the platelet GPIIb/IIIa receptor, protects against microangiopathic hemolytic anemia and microvascular thrombotic renal failure in baboons treated with C4b binding protein and a sublethal infusion of Escherichia coli; Taylor FB et al.; We have used our previously described baboon model of infusion of both a sublethal dose of Escherichia coli and C4b binding protein to assess the impact of inhibiting platelet function with the F(ab')2 fragment of the monoclonal antibody 7E3, directed against the platelet glycoprotein (GP)IIb/IIIa receptor, on the characteristic microvascular changes . At a dose of 0.25 to 0.35 mg/kg bolus plus an infusion of 0.25 to 0.35 mg/kg over 6 hours, c7E3 F(ab')2 had only a minimal impact on fibrinogen consumption and delayed but did not prevent, the development of thrombocytopenia . Treatment with 7E3 F(ab')2, however, produced significant protection from the development of microangiopathic hemolysis and renal insufficiency . Histologic examination supported these observations, with treated animals having fewer schistocytes on blood smear and less evidence of ischemic renal changes . Treated animals also had more rapid recovery of peripheral white blood counts, suggesting a possible protective effect of treatment on ischemic damage to the bone marrow . These data indicate that potent inhibition of platelet function via GPIIb/IIIa receptor blockade can decrease ischemic organ damage in this animal model that has features similar to those found in diffuse intravascular coagulation, hemolytic uremic syndrome, and thrombotic thrombocytopenic purpura.

Diabetes, 1997 Jun, 46(6), 937 - 40
Treatment with neutralizing antibodies specific for IL-1beta prevents cyclophosphamide-induced diabetes in nonobese diabetic mice; Cailleau C et al.; Interleukin-1 (IL-1) has been shown to be involved in the pathogenesis of IDDM, but it is not clear which form, IL-1alpha or IL-1beta, is predominantly implicated . In this study, we have evaluated the contribution of IL-1beta by treating diabetes-prone nonobese diabetic (NOD) mice with specific neutralizing antibodies . First, we assessed the neutralizing potential of these antibodies in C57BL/6 mice under acute septic shock by measuring IL-1beta in sera 4 h after lipopolysaccharide injection . One milligram and 0.1 mg of anti-IL-1beta antibodies (Abs) were capable of neutralizing the IL-1beta produced, and the effect persisted for at least 5 days . Second, we evaluated the role of IL-1beta in the cyclophosphamide (CY)-accelerated model of diabetes . Nondiabetic male NOD mice were injected with 200 mg/kg CY and treated twice weekly with anti-IL-1beta Ab . The incidence of diabetes reached 76 and 100% in the control groups treated with 0.25 and 0.1 mg rabbit IgG, respectively . In contrast, only 34% of mice treated with 0.25 mg of anti-IL-1beta Ab became diabetic . In the group treated with 0.1 mg of anti-IL-1beta Ab, 89% of the mice became diabetic in the same period of time, demonstrating that the protective effect was dose dependent . Our results show that IL-1beta is a critical effector molecule in this model of IDDM and that its specific inhibition could be an attractive target for therapeutic intervention.

J Clin Microbiol, 1997 Jun, 35(6), 1639 - 41
Coli surface antigens associated with enterotoxigenic Escherichia coli strains isolated from persons with traveler's diarrhea in Asia; Serichantalergs O et al.; Enterotoxigenic Escherichia coli (ETEC) strains were isolated from travelers or military personnel who developed diarrhea after visiting Nepal or who were deployed to Thailand, Indonesia, or the Philippines . ETEC isolates were examined for colonization factor antigen (CFA) . CFAs were identified on 59% (40 of 68) of the isolates examined . The lack of a detectable CFA on 41% (28 of 68) of the isolates is of concern for the development of an effective ETEC vaccine.

J Clin Microbiol, 1997 Jun, 35(6), 1521 - 5
Random amplification of polymorphic DNA reveals serotype-specific clonal clusters among enterotoxigenic Escherichia coli strains isolated from humans; Pacheco AB et al.; The genetic diversity of 47 enterotoxigenic Escherichia coli (ETEC) strains of serotypes O6:H16, O27:H7, O29:H21, O128ac:H12, and O153:H45, previously isolated from diarrheic patients in Brazil over a period of 15 years, was investigated by random amplification of polymorphic DNA (RAPD) . Informative band arrays were obtained with three 10-mer primers with G+C contents of 50, 60, and 70% . Based on the combination of the band profiles generated by the three primers 22 RAPD types were detected, and 5 major clonal clusters, each one with at least 80% identical bands, were established . The clonal clusters corresponded to strains having the same serotype which, in most cases, also had the same virulence factors (colonization factors and toxin types) and outer membrane protein and lipopolysaccharide sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles . The results suggested a correlation between phenotypic properties and genetic relatedness of ETEC isolates of human origin and indicated that a reduced number of clonally related strains are found in areas of ETEC endemicity in Brazil . Moreover, the RAPD technique revealed intraserotype-specific variations, undetectable by the combination of several phenotypic typing methods, among the ETEC strains analyzed . These results show that RAPD typing represents a useful tool for population genetics as well as for epidemiological studies of ETEC.

J Clin Microbiol, 1997 Jun, 35(6), 1404 - 10
Analysis of incidence of infection with enterotoxigenic Escherichia coli in a prospective cohort study of infant diarrhea in Nicaragua; Paniagua M et al.; Diarrheal episodes with enterotoxigenic Escherichia coli (ETEC) were prospectively monitored during the first 2 years of life in a cohort of 235 infants from Leon, Nicaragua . ETEC was an etiological finding in 38% (310 of 808) of diarrheal episodes and in 19% (277 of 1,472) of samples taken as asymptomatic controls at defined age intervals (P = <0.0001) . The majority of diarrheal episodes (80%) occurred before 12 months of age . The major ETEC type was characterized by colonization factor CFA I and elaboration of both heat-labile enterotoxin and heat-stable enterotoxin (ST) . The proportion of E . coli strains with CFA I was significantly higher in cases with diarrhea (P = 0.002) . The second most prevalent type showed putative colonization factor PCFO166 and production of ST . The prevalence of PCFO166 was approximately 20%, higher than reported before . Children with a first CFA I episode contracted a second ETEC CFA I infection 24% of the time, compared with 46% for ETEC strains of any subtype . Most of the ETEC episodes were of moderate severity, and only 5% (15 of 310) were characterized as severe . In conclusion, our results give valuable information for the planning of intervention studies using ETEC vaccines.

Mol Cell Biol, 1997 Jun, 17(6), 3164 - 72
Overexpression of human release factor 1 alone has an antisuppressor effect in human cells; Le Goff X et al.; Two eukaryotic proteins involved in translation termination have recently been characterized in in vitro experiments . Eukaryotic release factor 1 (eRF1) catalyzes the release of the polypeptide chain without any stop codon specificity . The GTP-binding protein eRF3 confers GTP dependence to the termination process and stimulates eRF1 activity . We used tRNA-mediated nonsense suppression at different stop codons in a cat reporter gene to analyze the polypeptide chain release factor activities of the human eRF1 and eRF3 proteins overexpressed in human cells . In a chloramphenicol acetyltransferase assay, we measured the competition between the suppressor tRNA and the human release factors when a stop codon was present in the ribosomal A site . Whatever the stop codon (UAA, UAG, or UGA) present in the cat open reading frame, the overexpression of human eRF1 alone markedly decreased translational readthrough by suppressor tRNA . Thus, like the procaryotic release factors RF1 and RF2 in Escherichia coli, eRF1 seems to have an intrinsic antisuppressor activity in human cells . Levels of antisuppression of overexpression of both eRF3 and eRF1 were almost the same as those of overexpression of eRF1 alone, suggesting that eRF1-eRF3 complex-mediated termination may be controlled by the expression level of eRF1 . Surprisingly, when overexpressed alone, eRF3 had an inhibitory effect on cat gene expression . The results of cat mRNA stability studies suggest that eRF3 inhibits gene expression at the transcriptional level . This indicates that in vivo, eRF3 may perform other functions, including the stimulation of eRF1 activity.

Neuroscience, 1997 Jun, 78(3), 703 - 13
Intrastriatal grafts of embryonic mesencephalic rat neurons genetically modified using an adenovirus encoding human Cu/Zn superoxide dismutase; Barkats M et al.; Intrastriatal grafting of embryonic dopamine-containing neurons is a promising approach for treating clinical and experimental Parkinson's disease . However, neuropathological analyses of grafted patients and transplanted rats have demonstrated that the survival of grafted dopamine neurons is relatively poor . In the present study, we pursued a strategy of transferring a potentially neuroprotective gene into rat embryonic mesencephalic rat cells in vitro, before grafting them into the denervated striatum of 6-hydroxydopamine-lesioned rats . We performed intrastriatal grafts of embryonic day 14 mesencephalic cells infected with replication-defective adenoviruses bearing either the human copper-zinc superoxide dismutase gene or, as a control, the E . coli lac Z marker gene . The transgenes were expressed in the grafts four days after transplantation and the expression persisted for at least five weeks thereafter . After five weeks postgrafting, there was more extensive functional recovery in the superoxide dismutase group as compared to the control (uninfected cells) and beta-galactosidase groups . The functional recovery was significantly correlated with the number of tyrosine hydroxylase-positive cells in the grafts, although the clear trend to increased survival of the dopamine neurons in the superoxide dismutase grafts did not reach statistical significance . Only a moderate inflammatory reaction was revealed by OX-42 immunostaining in all groups, suggesting that ex vivo gene transfer using adenoviral vectors is a promising method for delivering functional proteins into brain grafts.

Nucleic Acids Res, 1997 Jun 1, 25(11), 2174 - 81
CCAAT binding NF-Y-TBP interactions: NF-YB and NF-YC require short domains adjacent to their histone fold motifs for association with TBP basic residues; Bellorini M et al.; Both the TATA and CCAAT boxes are widespread promoter elements and their binding proteins, TBP and NF-Y, are extremely conserved in evolution . NF-Y is composed of three subunits, NF-YA, NF-YB and NF-YC, all necessary for DNA binding . NF-YB and NF-YC contain a putative histone-like motif, a domain also present in TBP-associated factors (TAFIIs) and in the subunits of the transcriptional repressor NC2 . Immunopurification of holo-TFIID with anti-TBP and anti-TAFII100 antibodies indicates that a fraction of NF-YB associates with TFIID in the absence of NF-YA . Sedimentation velocity centrifugation experiments confirm that two pools of NF-YB, and most likely NF-YC, exist: one associated with NF-YA and binding to the CCAAT box; another involved in high molecular weight complexes . We started to dissect NF-Y-TFIID interactions by showing that: (i) NF-YB and NF-YC interact with TBP in solution, both separately and once bound to each other; (ii) short stretches of both NF-YB and NF-YC located within the evolutionary conserved domains, adjacent to the putative histone fold motifs, are necessary for TBP binding; (iii) TBP single amino acid mutants in the HS2 helix, previously shown to be defective in NC2 binding, are also unable to bind NF-YB and NF-YC.

Nucleic Acids Res, 1997 Jun 1, 25(11), 2153 - 60
DNA binding and phasing analyses of Tn5 transposase and a monomeric variant; York D et al.; Both full-length Tn 5 transposase and a COOH-terminal truncated monomeric form of the protein,n369, have been shown to specifically bind end sequences at comparable affinities . In addition, both proteins distort the target sequence in a similar manner, as determined by a circular permutation assay . In this study,nEK54, a derivative ofn369 with a single amino acid substitution that significantly enhances binding activity, is used in further binding and bending studies along with full-length transposase . Phasing analysis has shown that distortion of the end sequences upon binding of full-length transposase and nEK54 protein is due in part to a protein-induced bend oriented towards the major groove . Because the center of transposase-induced bending maps to the extreme leftward end of the 19 bp consensus sequence, we examined the possibility that optimal protein binding requires additional upstream nucleotide contacts . Experiments presented here show that 9-10 nucleotides are needed upstream of +1 of the 19 bp sequence for efficient binding and this requirement can be met by either single-stranded or double-stranded DNA.

Nucleic Acids Res, 1997 Jun 1, 25(11), 2106 - 13
Functional characterization of the T4 DNA ligase: a new insight into the mechanism of action; Rossi R et al.; ATP-dependent DNA ligases are essential enzymes in both DNA replication and DNA repair processes . Here we report a functional characterization of the T4 DNA ligase . One N-terminal and two C-terminal deletion mutants were expressed in Escherichia coli as histidine- tagged proteins . An additional mutant bore a substitution of Lys159 in the active site that abolished ATP binding . All the proteins were tested in biochemical assays for ATP-dependent self-adenylation, DNA binding, nick joining, blunt-end ligation and AMP- dependent DNA relaxation . From this analysis we conclude that binding to DNA is mediated by sequences at both protein ends and plays a key role in the reaction . The enzyme establishes two different complexes with DNA: (i) a transient complex (T.complex) involving the adenylated enzyme; (ii) a stable complex (S.complex) requiring the deadenylated T4 DNA ligase . The formation of an S . complex seems to be relevant during both blunt-end ligation and DNA relaxation . Moreover the inactive His-K159L substitution mutant, although unable to self-adenylate, still possesses AMP-dependent DNA nicking activity.

Nucleic Acids Res, 1997 Jun 1, 25(11), 2083 - 90
Structure of the acceptor stem of Escherichia coli tRNA Ala: role of the G3.U70 base pair in synthetase recognition; Ramos A et al.; The fidelity of translation of the genetic code depends on accurate tRNA aminoacylation by cognate aminoacyl-tRNA synthetases . Thus, each tRNA has specificity not only for codon recognition, but also for amino acid identity; this aminoacylation specificity is referred to as tRNA identity . The primary determinant of the acceptor identity of Escherichia coli tRNAAlais a wobble G3.U70 pair within the acceptor stem . Despite extensive biochemical and genetic data, the mechanism by which the G3.U70 pair marks the acceptor end of tRNAAla for aminoacylation with alanine has not been clarified at the molecular level . The solution structure of a microhelix derived from the tRNAAla acceptor end has been determined at high precision using a very extensive set of experimental constraints (approximately 32 per nt) obtained by heteronuclear multidimensional NMR methods . The tRNAAla acceptor end is overall similar to A-form RNA, but important differences are observed . The G3.U70 wobble pair distorts the conformation of the phosphodiester backbone and presents the functional groups of U70 in an unusual spatial location . The discriminator base A73 has extensive stacking overlap with G1 within the G1.C72 base pair at the end of the double helical stem and the -CCA end is significantly less ordered than the rest of the molecule.

J Virol, 1997 Jun, 71(6), 4626 - 37
Long-term gene delivery into the livers of immunocompetent mice with E1/E4-defective adenoviruses; Dedieu JF et al.; We have compared the in vitro and in vivo behaviors of a set of isogenic E1- and E1/E4-defective adenoviruses expressing the lacZ gene of Escherichia coli from the Rous sarcoma virus long terminal repeat . Infection of tumor-derived established cell lines of human origin with the doubly defective adenoviruses resulted in (i) a lower replication of the viral backbone that correlated with reduced levels of E2A-specific RNA and protein, (ii) a significant shutoff of late gene and protein expression, and (iii) no apparent virus-induced cytotoxicity . Independently of the extent of the deletion, the additional inactivation of E4 from the viral backbone therefore drastically disabled the virus in vitro, with no apparent effect on transgene expression . A lacZ-transgenic model was used to compare the different recombinant adenoviruses in the livers of C57BL/6 mice . The immune response to the virally encoded beta-galactosidase was minimal in this model, as infusion of the E1-defective adenovirus resulted in a time course of transgene expression that mimicked that in immunodeficient (nu/nu) mice, with very little inflammation and necrosis in the liver . Administration of a doubly defective adenovirus to the transgenic animals led to long-term extrachromosomal persistence of viral DNA in the liver, with no detectable methylation of CpG dinucleotides . However, transient transgene expression was observed independently of the extent of the E4 deletion, suggesting that the choice of the promoter may be critical to maintain transgene expression from these attenuated adenovirus vectors.

J Virol, 1997 Jun, 71(6), 4502 - 8
Derepression of prophage P2 by satellite phage P4: cloning of the P4 epsilon gene and identification of its product; Liu T et al.; Escherichia coli phage P4 lacks all of the genetic information necessary for capsid, tail, and lysis functions . P4 is therefore dependent on a helper phage, such as P2, for lytic propagation . During P4 superinfection of a P2 lysogen, the P2 prophage is derepressed by the action of the P4-encoded epsilon gene . We have cloned the epsilon gene and identified the 10-kDa E protein . The epsilon gene product is the only P4 protein required to derepress prophage P2, which leads to in situ P2 DNA replication . A two-plasmid derepression assay system has been developed to examine the derepression activity of E . The reporter plasmid contains the two face-to-face promoters, Pe and Pc, involved in the lysis-lysogeny transcriptional switch of phage P2 and the immunity repressor C . The Pe promoter is coupled to a cat reporter gene . In the construct, the C repressor is transcribed from the Pc promoter and represses the Pe promoter, which mimics the in situ-repressed P2 prophage . The E protein is supplied in trans from a compatible plasmid in which the epsilon gene is under the control of the T7 promoter . We show here that in the two-plasmid assay system, induction of the E protein derepresses the Pe promoter . The ash9 mutation, which is located upstream of the epsilon gene, enhances the E-mediated derepression of the Pe promoter . The purified E protein shows no specific DNA binding activity, and the implications of this are discussed.

J Virol, 1997 Jun, 71(6), 4425 - 35
In vitro assembly of virus-like particles with Rous sarcoma virus Gag deletion mutants: identification of the p10 domain as a morphological determinant in the formation of spherical particles; Campbell S et al.; Retroviruses are unusual in that expression of a single protein, Gag, leads to budding of virus-like particles into the extracellular space . We have developed conditions under which virus-like particles are formed spontaneously in vitro from fragments of Rous sarcoma virus (RSV) Gag protein purified after expression in Escherichia coli . The CA-NC fragment of Gag was shown previously to assemble into hollow cylinders (S . Campbell and V . M . Vogt, J . Virol . 69:6487-6497, 1995) . We have now extended these studies to larger Gag proteins . In every case examined, assembly into regular structures required RNA . A nearly full-length Gag missing only the C-terminal PR domain, as well as similar proteins missing in addition the N-terminal half of MA, the C-terminal half of MA, the entire MA sequence, or the entire p2 sequence, all assembled into spherical particles resembling RSV in size . By contrast, proteins missing p10 assembled into cylindrical particles like those formed by CA-NC alone . Thin section electron microscopy showed that each of these Gag proteins formed in the expressing E . coli cells particles similar in shape to those seen in vitro . We conclude from these results that neither the sequences required for membrane binding in vivo, near the N terminus of Gag, nor the sequences required for a late step in budding, in the p2 portion of Gag, are essential for formation of virus-like particles in this system . Furthermore, we postulate the existence of a shape-determining sequence in p10, which provides or facilitates interactions required for the growing particle to be constrained to a spherical shape.

J Virol, 1997 Jun, 71(6), 4264 - 71
Expression and purification of vesicular stomatitis virus N-P complex from Escherichia coli: role in genome RNA transcription and replication in vitro; Gupta AK et al.; The nucleocapsid protein (N) and phosphoprotein (P) genes of vesicular stomatitis virus (VSV), Indiana serotype, were coexpressed in Escherichia coli BL21(DE3) by using the expression vector pET-3a . The coexpression resulted in the formation of N-P complex . The purified N-P complex was found to inhibit transcription in vitro mediated by viral ribonucleoprotein (RNP) complex in a dose-dependent manner . However, addition of uninfected mammalian cell extracts together with the N-P complex to the transcribing RNP resulted in the synthesis of full-length negative-strand genome RNA . These results indicate that the N-P complex regulated transcription and a cellular factor(s) in combination with the N-P complex may switch the RNA polymerase from transcription to replication mode.

J Virol, 1997 Jun, 71(6), 4254 - 63
The antiretrovirus drug 3'-azido-3'-deoxythymidine increases the retrovirus mutation rate; Julias JG et al.; It was previously observed that the nucleoside analog 5-azacytidine increased the spleen necrosis virus (SNV) mutation rate 13-fold in one cycle of retrovirus replication (V . K . Pathak and H . M . Temin, J . Virol . 66:3093-3100, 1992) . Based on this observation, we hypothesized that nucleoside analogs used as antiviral drugs may also increase retrovirus mutation rates . We sought to determine if 3'-azido-3'-deoxythymidine (AZT), the primary treatment for human immunodeficiency virus type 1 (HIV-1) infection, increases the retrovirus mutation rate . Two assays were used to determine the effects of AZT on retrovirus mutation rates . The strategy of the first assay involved measuring the in vivo rate of inactivation of the lacZ gene in one replication cycle of SNV- and murine leukemia virus-based retroviral vectors . We observed 7- and 10-fold increases in the SNV mutant frequency following treatment of target cells with 0.1 and 0.5 microM AZT, respectively . The murine leukemia virus mutant frequency increased two- and threefold following treatment of target cells with 0.5 and 1.0 microM AZT, respectively . The second assay used an SNV-based shuttle vector containing the lacZ alpha gene . Proviruses were recovered as plasmids in Escherichia coli, and the rate of inactivation of lacZ alpha was measured . The results indicated that treatment of target cells increased the overall mutation rate two- to threefold . DNA sequence analysis of mutant proviruses indicated that AZT increased both the deletion and substitution rates . These results suggest that AZT treatment of HIV-1 infection may increase the degree of viral variation and alter virus evolution or pathogenesis.

Biochim Biophys Acta, 1997 May 30, 1352(2), 203 - 12
A novel type of dimerization motif, related to leucine zippers, is present in plant homeodomain proteins; Palena CM et al.; Sunflower HAHR1 is a homeodomain protein presumably involved in some aspects of root development . In the present work, we have studied the oligomerization properties of HAHR1 . A protein containing the entire homeodomain plus adjacent C-terminal sequences (amino acids 86-325) behaves as a dimer in gel filtration experiments . When a fragment C-terminal to the homeodomain (amino acids 151-263) is fused to the N-terminal domain of the lambda phage repressor, it is able to confer binding efficiency to this domain, as judged by protection from lambda superinfection and repression of beta-galactosidase expression under the control of the P(R) promoter . A smaller fragment (amino acids 151-184) confers only conditional repression . GSH transferase fusion proteins containing the entire homeodomain of HAHR1 plus the above-mentioned adjacent sequences bind with similar efficiency a mixture of oligonucleotides selected from a random population . The smaller protein, however, loses its binding capacity when separated from the GSH transferase moiety . Retention of a labelled HAHR1 protein synthesized in vitro by GSH transferase fusions containing different protein fragments adjacent to the homeodomain and bound to GSH agarose suggests that a portion from amino acids 151-263 is required for efficient interaction . The results obtained indicate that HAHR1 interacts with DNA as a dimer and that its dimerization domain is located immediately C-terminal to the homeodomain . We define two regions, the first of which confers non-efficient dimerization; this region would be stabilized by the presence of the second one through putative mutual interactions . A similar motif is present in other related plant homeodomain proteins.

Biochim Biophys Acta, 1997 May 30, 1352(2), 193 - 202
Green fluorescent protein as a reporter of gene expression in transgenic mice; Chiocchetti A et al.; We used the green fluorescent protein (GFP) from the jellyfish Aequorea victoria as a reporter of gene expression in transgenic mice . The GFP coding sequence was placed under the control of the human hemopexin and the mouse beta1 integrin promoter that were previously studied in transgenic mice using the lacZ reporter gene . We showed that GFP has a higher degree of sensitivity compared to the lacZ reporter gene allowing to identify cells with low and otherwise undetectable beta-galactosidase activity . Thus we showed the potentiality of GFP in replacing lacZ as a reporter gene to investigate promoter mapping and gene regulation in transgenic mice.

Biochim Biophys Acta, 1997 May 30, 1352(2), 161 - 6
Identification and analysis of the rpoS-dependent promoter of katE, encoding catalase HPII in Escherichia coli; Tanaka K et al.; The rpoS gene of Escherichia coli encodes an alternative sigma factor of RNA polymerase sigma38 (or sigma(s)) that is required for transcription of katE encoding catalase HPII . The transcription start site of the single katE transcript identified by ribonuclease protection has been determined by primer extension analysis to be either 53 or 54 bp (depending on the strain used) upstream of the open reading frame . A series of promoter fragments were constructed and fused to lacZ to confirm the start site location . A - 10 sequence similar to that found in other sigma70- and sigma38-dependent E . coli promoters was identified 8 or 7 bp upstream of the start site but a sigma70-dependent -35 sequence was not evident.

Stat Med, 1997 May 30, 16(10), 1151 - 66
Comparison of non parallel immunoassay curves resulting from mixtures of competing antigens; Kaiser MS et al.; Relative potency is a measure that has been used for many years to summarize the comparison of dose-response curves in parallel line bioassays . When response curves for two preparations are not parallel the traditional definition of relative potency no longer applies . We review the concept of relative potency and show that, in some situations, it can be given meaning for non-parallel curves as the ratio of biological activity in full strength assay preparations . Under an assumption that non-parallel curves result from the competition of mixtures of antigens for receptor binding sites, estimation of relative potency for non-parallel curves can be accomplished . We show that estimation of models for both parallel curve and response attenuation situations may be accomplished within the framework of generalized linear models . This estimation depends on the ability to deal with non-linear parameters appearing in the link function, and an iterative algorithm depending on direct parameter updates is outlined . The topics discussed are illustrated with the analysis of data from two immunoassays conducted with veterinary vaccines . The models developed here depend in an essential way on the assumption of response attenuation by competing antigens . Our methods may not be appropriate for non-parallel curves caused by other phenomena.

J Biol Chem, 1997 May 30, 272(22), 14465 - 9
The role of alpha- and epsilon-amino groups in the glycation-mediated cross-linking of gammaB-crystallin . Study of three site-directed mutants; Zhao HR et al.; In the previous report we demonstrated that gammaB-crystallin is glycated predominantly at the N-terminal alpha-amino group (Casey, E . B., Zhao, H . R., and Abraham, E . C . (1995) J . Biol . Chem . 270, 20781-20786) . To investigate the possible role of alpha- and epsilon-amino groups of gammaB-crystallin in glycation-mediated cross-linking, Lys-2 or Lys-163, or both, were mutated to threonine by site-directed mutagenesis in bovine gammaB-crystallin cDNA . Wild type and mutant gammaB-crystallins were expressed in Escherichia coli cells . Cross-linking studies were performed by incubating wild type and mutant gammaB-crystallins with glyceraldehyde, ribose, and galactose followed by SDS-polyacrylamide gel electrophoresis under reducing conditions . When both of the lysines of gammaB-crystallin were mutated to threonines (gammaB-K2T/K163T), the quantity of cross-linked products was greatly reduced, indicating that, despite the fact that the alpha-amino group is a major glycated site, epsilon-amino groups play a predominant role in cross-linking . Therefore, cross-linking ability depends not only upon the level of glycation but also upon which amino group is glycated . Steric hindrance may decrease the cross-linking ability of the alpha-amino group . Our results also show that Lys-2 and Lys-163 play almost equal roles in cross-linking of gammaB-crystallin . By incubating carbonic anhydrase, a protein with a blocked N terminus, and our novel "no lysine" gammaB (gammaB-K2T/K163T) with sugar, we were able to show for the first time that significant cross-linking occurs between lysines and non-lysine sites . The fact that pentosidine and imidazolysine, formed from ribose and methylglyoxal, respectively, were present in the cross-linked gammaB-crystallins revealed the existence of Lys-Arg and Lys-Lys cross-linking.

J Biol Chem, 1997 May 30, 272(22), 14454 - 8
Identification of a palindromic sequence that is responsible for the up-regulation of NAPDH-ferredoxin reductase in a ferredoxin I deletion strain of Azotobacter vinelandii; Yannone SM et al.; Azotobacter vinelandii ferredoxin I (AvFdI) is one member of a class of 7Fe ferredoxins found in a variety of organisms that are all capable of aerobic growth . Disruption of the fdxA gene, which encodes AvFdI, leads to overexpression of its redox partner, NADPH-ferredoxin reductase (FPR) . In this study the mechanism of FdI-mediated regulation of FPR was investigated . Northern analysis has shown that regulation is at the level of fpr transcription, the start site for transcription has been identified, and it is preceded by a canonical sigma 70-type bacterial promoter . Gel mobility shift assays show that there is a putative regulatory protein in A . vinelandii that binds specifically upstream of the -35 region . That protein is not AvFdI . A palindromic sequence was identified as a putative binding site, and randomization of that sequence completely eliminates binding of the putative regulatory protein . A luciferase reporter gene was placed under control of the A . vinelandii fpr promoter and introduced into wild type and FdI- strains of A . vinelandii . Luciferase activity was enhanced 7-fold in the FdI- mutant relative to the wild type . Alteration of the palindromic sequence reduced the luciferase levels in the FdI- strain to those of the wild type, demonstrating that FdI regulates FPR through the palindrome and that the reaction is an activation rather than a repression . The identified palindrome is approximately 50% identical to the SoxS binding site upstream of Escherichia coli fpr, suggesting that A . vinelandii may have a SoxS-like regulatory system and that the function of FdI might be to specifically inactivate that system.

J Biol Chem, 1997 May 30, 272(22), 14327 - 33
Calcium binding, but not a calcium-myristoyl switch, controls the ability of guanylyl cyclase-activating protein GCAP-2 to regulate photoreceptor guanylyl cyclase; Olshevskaya EV et al.; Guanylyl cyclase-activating protein 2 (GCAP-2) is a recoverin-like calcium-binding protein that regulates photoreceptor guanylyl cyclase (RetGC) (Dizhoor, A . M., and Hurley, J . B . (1996) J . Biol . Chem . 271, 19346-19350) . It was reported that myristoylation of a related protein, GCAP-1, was critical for its affinity for RetGC (Frins, S., Bonigk, W., Muller, F., Kellner, R., and Koch, K.-W . (1996) J . Biol . Chem . 271, 8022-8027) . We demonstrate that the N terminus of GCAP-2, like those of other members of the recoverin family of Ca2+-binding proteins, is fatty acylated . However, unlike other proteins of this family, more GCAP-2 is present in the membrane fraction at low Ca2+ than at high Ca2+ concentrations . We investigated the role of the N-terminal fatty acyl residue in the ability of GCAP-2 to regulate RetGCs . Myristoylated or nonacylated GCAP-2 forms were expressed in Escherichia coli . Wild-type GCAP-2 and the Gly2 --> Ala2 GCAP-2 mutant, which is unable to undergo N-terminal myristoylation, were also expressed in mammalian HEK293 cells . We found that compartmentalization of GCAP-2 in photoreceptor outer segment membranes is Ca2+- and ionic strength-sensitive, but it does not require the presence of the fatty acyl group and does not necessarily directly reflect GCAP-2 interaction with RetGC . The lack of myristoylation does not significantly affect the ability of GCAP-2 to stimulate RetGC . Nor does it affect the ability of the Ca2+-loaded form of GCAP-2 to compete with the GCAP-2 mutant that constitutively activates RetGC . We conclude that while Ca2+ binding plays a major regulatory role in GCAP-2 function, it does not operate through a calcium-myristoyl switch similar to the one found in recoverin.

J Biol Chem, 1997 May 30, 272(22), 14257 - 62
Metalloregulatory properties of the ArsD repressor; Chen Y et al.; The plasmid-encoded arsenical resistance (ars) operon of plasmid R773 produces resistance to trivalent and pentavalent salts of the metalloids arsenic and antimony in cells of Escherichia coli . The first two genes in the operon, arsR and arsD, were previously shown to encode trans-acting repressor proteins . ArsR controls the basal level of expression of the operon, while ArsD controls maximal expression . Thus, action of the two repressors form a homeostatic regulatory circuit that maintains the level of ars expression within a narrow range . In this study, we demonstrate that ArsD binds to the same site on the ars promoter element as ArsR but with 2 orders of magnitude lower affinity . The results of gel shift assays demonstrate that ArsD is released from the ars DNA promoter by phenylarsine oxide, sodium arsenite, and potassium antimonyl tartrate (in order of effectiveness), the same inducers to which ArsR responds . Using the quenching of intrinsic tryptophan fluorescence to measure the affinity of the repressor for inducers, apparent Kd values for Sb(III) and As(III) of 2 and 60 microM, respectively, were obtained . These results demonstrate that the arsR-arsD pair provide a sensitive mechanism for sensing a wide range of environmental heavy metals.

J Biol Chem, 1997 May 30, 272(22), 14220 - 6
Two oligomeric forms of plasma ficolin have differential lectin activity; Ohashi T et al.; Ficolins are plasma proteins with binding activity for carbohydrates, elastin, and corticosteroids . The ficolin polypeptide has a collagen-like domain that presumably brings three subunits together in a triple helical rod, a C-terminal fibrinogen-like domain (fbg) similar to that of tenascin, which presumably has the binding activities, and a small N-terminal domain that we find to be the primary site for forming the ficolin oligomer . By sedimentation equilibrium we determined that the main plasma form, which we call big ficolin, had mass of 827,000 Da, consistent with 24 subunits . Little ficolin, about half this size, was obtained after binding to a GlcNAc affinity column . Electron microscopy of little ficolin showed a parachute-like structure, with a small globe at one end, corresponding to the 12 N-terminal domains, and the fbg domains clustered together at the ends of the collagen rods . Big ficolin was formed by the face to face fusion of the fbg domains of two little ficolins, leaving the rods and N-terminal domains projecting at opposite ends . Little ficolin maintained a high affinity for the GlcNAc column, and big ficolin had a low affinity or none . The binding sites for ligands may be obscured in this big ficolin oligomer, providing a regulation of their activity.

J Biol Chem, 1997 May 30, 272(22), 14133 - 8
Sequential interchange of four amino acids from blood group B to blood group A glycosyltransferase boosts catalytic activity and progressively modifies substrate recognition in human recombinant enzymes; Seto NO et al.; The human blood group A and B glycosyltransferase enzymes are highly homologous and the alteration of four critical amino acid residues (Arg-176 --> Gly, Gly-235 --> Ser, Leu-266 --> Met, and Gly-268 --> Ala) is sufficient to change the enzyme specificity from a blood group A to a blood group B glycosyltransferase . To carry out a systematic study, a synthetic gene strategy was employed to obtain their genes and to allow facile mutagenesis . Soluble forms of a recombinant glycosyltransferase A and a set of hybrid glycosyltransferase A and B mutants were expressed in Escherichia coli in high yields, which allowed them to be kinetically characterized extensively for the first time . A functional hybrid A/B mutant enzyme was able to catalyze both A and B reactions, with the kcat being 5-fold higher for the A donor . Surprisingly, even a single amino acid replacement in glycosyltransferase A with the corresponding residue from glycosyltransferase B (Arg-176 --> Gly) produced enzymes with glycosyltransferase A activity only, but with very large (11-fold) increases in the kcat and increased specificity . The increases observed in kcat are among the largest obtained for a single amino acid change and are advantageous for the preparative scale synthesis of blood group antigens.

J Biol Chem, 1997 May 30, 272(22), 14080 - 6
How GroES regulates binding of nonnative protein to GroEL; Sparrer H et al.; At present, it is still enigmatic how the reaction cycle by which the Escherichia coli GroE chaperones mediate protein folding in the cell is coordinated with respect to the sequential order of binding and release of GroES, nucleotide, and nonnative protein . It is generally assumed that the asymmetric GroEL.GroES complex is the acceptor state for substrate protein . Nevertheless, this species is poorly understood in its binding characteristics for nucleotide and nonnative protein . We show here that this species has a high affinity binding site for nonnative protein . In addition to this, binding of nucleotide to one GroEL ring is strongly favored by GroES binding to the other ring . However, the slow rate of release of substrate protein from the unproductive trans-position kinetically favors the binding of a second GroES, thereby forming a symmetric GroEL14.(GroES7)2 complex and simultaneously ensuring that substrate protein is sequestered in a position underneath GroES . Our results demonstrate that the intrinsic binding characteristics of the trans-bullet complex determine the sequence of events during the reaction cycle.

J Biol Chem, 1997 May 30, 272(22), 14045 - 50
Critical role of human bisphosphoglycerate mutase Cys22 in the phosphatase activator-binding site; Ravel P et al.; The enzymatic activities catalyzed by bisphosphoglycerate mutase (BPGM, EC 5.4.2.4) have been shown to occur at a unique active site, with distinct binding sites for diphosphoglycerates and monophosphoglycerates . The physiological phosphatase activator (2-phosphoglycolate) binds to BPGM at an undetermined site . BPGM variants were constructed by site-directed mutagenesis of three amino acid residues in the active site to identify residues specifically involved in the binding of the monophosphoglycerates and 2-phosphoglycolate . Substitution of Cys22 by functionally conservative residues, Thr or Ser, caused a great decrease in 2-phosphoglycolate-stimulated phosphatase activity and in the Ka value of the activator, whereas it caused no change in other catalytic activities or in the Km values of 2,3-diphosphoglycerate (2,3-DPG) and glycerate 3-phosphate (3-PG, EC 1.1.1.12), indicating that Cys22 is specifically involved either directly or indirectly in 2-phosphoglycolate binding . Kinetic experiments showed that the Ka of the cofactor and the Km of 3-PG were affected by the substitution of Ser23 indicating that this residue is necessary for the fixation of both 3-PG and 2-phosphoglycolate . The R89K variant has previously been shown to have a modified Km value for monophosphoglycerates, however, its affinity for 2-phosphoglycolate is unaltered, suggesting that Arg89 is specifically involved in monophosphoglycerates binding . CD spectroscopic studies of substrates and cofactor binding showed that 2,3-DPG induced structural modifications of normal and mutated enzymes which could be due to protein phosphorylation . Addition of 2-phosphoglycolate to phosphorylated proteins with normal affinity for the cofactor produced spectra with the same characteristics as unphosphorylated species . In summary, monophosphoglycerates and 2-phosphoglycolate have partially distinct binding sites in human BPGM . The specific implication of the Cys22 residue in 2-phosphoglycolate binding is of great significance in the design of analogs of therapeutic benefit.

Science, 1997 May 30, 276(5317), 1420 - 2
Role of the major antigen of Mycobacterium tuberculosis in cell wall biogenesis; Belisle JT et al.; The dominant exported proteins and protective antigens of Mycobacterium tuberculosis are a triad of related gene products called the antigen 85 (Ag85) complex . Each has also been implicated in disease pathogenesis through its fibronectin-binding capacities . A carboxylesterase domain was found within the amino acid sequences of Ag85A, B, and C, and each protein acted as a mycolyltransferase involved in the final stages of mycobacterial cell wall assembly, as shown by direct enzyme assay and site-directed mutagenesis . Furthermore, the use of an antagonist (6-azido-6-deoxy-alpha, alpha'-trehalose) of this activity demonstrates that these proteins are essential and potential targets for new antimycobacterial drugs.

Int J Cancer, 1997 May 29, 71(5), 874 - 80
Release of replication-deficient retroviruses from a packaging cell line: interaction with glioma tumor spheroids in vitro; Thorsen F et al.; The present study describes how various growth conditions affect gene expression and virus production from a retroviral packaging cell line (Liz 9), grown as monolayers and as multicellular spheroids . In addition, to study the direct interaction between packaging cells and tumor tissue of glioma origin, Liz 9 spheroids were confronted with tumor spheroids derived from a human glioma cell line, GaMg . The results show a progressive gene transfer into the tumor tissue, with 9% transfection efficacy after 5 days of co-culture . In comparison, no gene transfer was observed when the Liz 9 spheroids were confronted with normal brain-cell aggregates . The Liz 9 spheroids established from early-passage cultures (passages 7-14) showed limited growth during 28 days, whereas those initiated from late-passage monolayer cultures (passages 39-49) showed extensive growth . Flow-cytometric DNA profiles of monolayers and of spheroids indicated no difference in cell-cycle distribution or ploidy between early and late passages . A cell-viability assay using scanning confocal microscopy revealed mostly viable cells in the Liz 9 spheroids, with only a few dead cells scattered within the structures . The lacZ-gene expression was maintained in early- and in late-passage cultures . In comparison, in Liz 9 early-passage monolayers, the virus titer was 3.1 x 10(4) +/- 0.4 x 10(4) CFU/ml, whereas no virus titer was found in late-passage cultures . The virus titer from the Liz 9 spheroids was found to be between 10(3) and 10(4) CFU/ml . It is concluded that the virus production from packaging cells may vary, depending on passage number and tissue-culture conditions . In the present study, this is demonstrated by a complete loss in virus titer during prolonged culture of packaging cells . In addition, the 3-dimensional confrontation system described allows direct visualization of how packaging cells interact with tumor tissue . Thus, the co-culture system represents a model for studying the efficiency of packaging cells in transfecting heterogeneous tumor tissue in vitro.

Biochem Biophys Res Commun, 1997 May 29, 234(3), 564 - 7
Expression of the Escherichia coli thioredoxin gene is negatively regulated by cyclic AMP; Sa JH et al.; Regulation of the Escherichia coli thioredoxin gene (trxA) was studied using trxA-lac translational fusion constructed in the vector pMC1403 . Synthesis of beta-galactosidase from the trxA-lac fusion was found to be repressed in the presence of lactose . A switch of carbon source from glucose to lactose and an addition of cyclic AMP (cAMP) caused a decrease in beta-galactosidase synthesis from the trxA-lac fusion . The repression effect of exogenous cAMP was not observed in the crp mutant strain . The beta-galactosidase synthesis from the trxA-lac fusion lacking a plausible cAMP-CRP binding site was not lowered in the presence of lactose or in the addition of cAMP . Expression of the chromosomal trxA gene was reduced by exogenous cAMP . These findings indicate that the expression of the trxA gene is controlled by cAMP in a negative manner.

Biochem Biophys Res Commun, 1997 May 29, 234(3), 549 - 53
Soluble overexpression in Escherichia coli, and purification and characterization of wild-type recombinant tobacco acetolactate synthase; Chang SI et al.; Acetolactate synthase (ALS) is the first common enzyme in the biosynthesis of L-leucine, L-isoleucine, and L-valine . The wild-type ALS gene from Nicotiana tabacum was cloned into the bacterial expression vector pGEX-2T . The resulting recombinant plasmid pGEX-ALS2 was used to transform Escherichia coli strain XL1-Blue, and the wild-type tobacco ALS (wALS) was expressed in the bacteria as a protein fused with glutathione S-transferase (GST) . The fusion product GST-wALS was purified in a single step on a glutathione-Sepharose column . The purified GST-wALS was sensitive to a sulfonylurea herbicide, and was lost its sensitivity to end products, L-valine, L-leucine and L-isoleucine . These results suggest that the purified recombinant tobacco ALS was functionally active, and that the sulfonylureas may not bind to the feedback regulatory site on the plant ALS.

Biochemistry, 1997 May 27, 36(21), 6437 - 47
Glutathione reductase turned into trypanothione reductase: structural analysis of an engineered change in substrate specificity; Stoll VS et al.; Trypanosoma and Leishmania, pathogens responsible for diseases such as African sleeping sickness, Chagas' heart disease, or Oriental sore, are two of the very few genera that do not use the ubiquitous glutathione/glutathione reductase system to keep a stable cellular redox balance . Instead, they rely on trypanothione and trypanothione reductase to protect them from oxidative stress . Trypanothione reductase (TR) and the corresponding host enzyme, human red blood cell glutathione reductase (GR), belong to the same flavoprotein family . Despite their closely related three-dimensional structures and although their natural substrates share the common structural glutathione core, the two enzymes are mutually exclusive with respect to their disulfide substrates . This makes the parasite enzyme a potential target for antitrypanosomal drug design . While a large body of structural data on GR complexes is available, information on TR-ligand interactions is very limited . When the two amino acid changes Ala34Glu and Arg37Trp are introduced into human GR, the resulting mutant enzyme (GRTR) prefers trypanothione 700-fold over its original substrate, effectively converting a GR into a TR {Bradley, M., Bucheler, U . S., & Walsh, C . T . (1991) Biochemistry 30, 6124-6127} . The crystal structure of GRTR has been determined at 2.3 A resolution and refined to a crystallographic R factor of 20.9% . We have taken advantage of the ease with which ligand complexes can be produced in GR crystals, a property that extends to the isomorphous GRTR crystals, and have produced and analyzed crystals of GRTR complexes with glutathione, trypanothione, glutathionylspermidine and of a true catalytic intermediate, the mixed disulfide between trypanothione and the enzyme . The corresponding molecular structures have been characterized at resolutions between 2.3 and 2.8 A with R factors ranging from 17.1 to 19.7% . The results indicate that the Ala34Glu mutation causes steric hindrance leading to a large displacement of the side chain of Arg347 . This movement combined with the change in charge introduced by the mutations modifies the binding cavity, forcing glutathione to adopt a nonproductive binding mode and permitting trypanothione and to a certain degree also the weak substrate glutathionylspermidine to assume a productive mode.

Biochemistry, 1997 May 27, 36(21), 6408 - 14
Binding of ligand or monoclonal antibody 4B1 induces discrete structural changes in the lactose permease of Escherichia coli; Frillingos S et al.; By using Cys-scanning mutagenesis with site-directed sulfhydryl modification in situ {Frillingos, S., & Kaback, H . R . (1996) Biochemistry 35, 3950-3956}, conformational changes induced by binding of ligand or monoclonal antibody (mAb) 4B1 in the lactose permease of Escherichia coli were studied . Out of 31 single-Cys replacement mutants in helices I, V, VII, VIII, X, or XI, 4B1 binding alters the reactivity of Val238-->Cys (helix VII), Val331-->Cys (helix X), or single-Cys355 (helix XI) permease with N-ethylmaleimide (NEM) in right-side-out membrane vesicles . In addition, site-directed fluorescence spectroscopy shows that mAb 4B1 binding causes position 331 (helix X) in the permease to experience a more hydrophobic environment . In contrast, ligand binding elicits more widespread changes, as evidenced by enhancement of the NEM reactivity of Ala244-->Cys, Thr248-->Cys (helix VII), Thr265-->Cys (helix VIII), Val315-->Cys (helix X), Gln359-->Cys, or Met362-->Cys (helix XI) permease, none of which are altered by 4B1 binding . Furthermore, no effect of 4B1 is observed on the reactivity of Cys148 (helix V), Val264-->Cys, Gly268-->Cys, or Asn272-->Cys (helix VIII), positions which probably make direct contact with substrate . With respect to the N-terminal half of the permease, 4B1 binding causes a small increase in the reactivity of mutants Pro28-->Cys or Pro31-->Cys (helix I), while ligand binding causes much greater increases in reactivity . The findings indicate that 4B1 binding induces a structural change in the permease that is much less widespread than that induced by ligand binding.

Biochemistry, 1997 May 27, 36(21), 6391 - 400
Quenching of tryptophan fluorescence by the active-site disulfide bridge in the DsbA protein from Escherichia coli; Hennecke J et al.; The disulfide oxidoreductase DsbA is a strong oxidant of protein thiols and required for efficient disulfide bond formation in the bacterial periplasm . The enzyme consists of a thioredoxin-like domain and a second, alpha-helical domain which is inserted into the thioredoxin motif . Reduction of the active-site disulfide in the thioredoxin domain causes a more than 3-fold increase in tryptophan fluorescence . However, both tryptophan residues of the protein, W76 and W126, are not in contact with the disulfide and located in the alpha-helical domain . Analysis of the variants W76F and W126F revealed that the fluorescence of W126 is fully quenched in every redox state of DsbA . W126 is also a sink for nonradiative energy transfer from W76 . In oxidized DsbA, W76 is quenched by an intramolecular, dynamic quenching process which involves energy transfer from W76 via F26 to the disulfide . The contributions of the disulfide bridge and the tryptophan residues to the near-UV CD spectra were also quantified . Analysis of the thermodynamic stabilities of the variants W76F and F26L revealed that the interdomain contact between W76 and F26 strongly contributes to the overall stability of DsbA, and selectively stabilizes its oxidized form . The DsbA variant F26L is the most oxidizing disulfide oxidoreductase known so far.

Biochemistry, 1997 May 27, 36(21), 6305 - 16
Structure of carbamoyl phosphate synthetase: a journey of 96 A from substrate to product; Thoden JB et al.; Carbamoyl phosphate synthetase catalyzes the production of carbamoyl phosphate from bicarbonate, glutamine, and two molecules of MgATP . As isolated from Escherichia coli, the enzyme has a total molecular weight of approximately 160K and consists of two polypeptide chains referred to as the large and small subunits . Here we describe the X-ray crystal structure of this enzyme determined to 2.8 A resolution in the presence of ADP, Mn2+, phosphate, and ornithine . The small subunit is distinctly bilobal with the active site residues located in the interface formed by the NH2- and COOH-terminal domains . Interestingly, the structure of the COOH-terminal half is similar to that observed in the trpG-type amidotransferase family . The large subunit can be envisioned as two halves referred to as the carboxyphosphate and carbamoyl phosphate synthetic components . Each component contains four distinct domains . Strikingly, the two halves of the large subunit are related by a nearly exact 2-fold rotational axis, thus suggesting that this polypeptide chain evolved from a homodimeric precursor . The molecular motifs of the first three domains observed in each synthetic component are similar to those observed in biotin carboxylase . A linear distance of approximately 80 A separates the binding sites for the hydrolysis of glutamine in the small subunit and the ATP-dependent phosphorylations of bicarbonate and carbamate in the large subunit . The reactive and unstable enzyme intermediates must therefore be sequentially channeled from one active site to the next through the interior of the protein.

Biochemistry, 1997 May 27, 36(21), 6294 - 304
Structural analysis of UDP-sugar binding to UDP-galactose 4-epimerase from Escherichia coli; Thoden JB et al.; UDP-galactose 4-epimerase from Escherichia coli catalyzes the interconversion of UDP-galactose and UDP-glucose through the transient reduction of the tightly bound cofactor NAD+ . The enzyme is unique among the NAD+-dependent enzymes in that it promotes stereospecific reduction of the cofactor but nonstereospecific hydride return during normal catalysis . In addition to hydride transfer, the reaction mechanism of epimerase involves two key features: the abstraction of a proton from the 4'-hydroxyl group of glucose or galactose by an active site base and the rotation of a 4-ketopyranose intermediate in the active site pocket . To address the second issue of movement within the active site, the X-ray structures of reduced epimerase complexed with UDP-mannose, UDP-4-deoxy-4-fluoro-alpha-D-galactose, or UDP-4-deoxy-4-fluoro-alpha-D-glucose have been determined and refined to 1.65, 1.8, and 1.65 A resolution, respectively . A comparison of these models to that of the previously determined epimerase/NADH/UDP-glucose abortive complex reveals that the active site accommodates the various sugars by simple rearrangements of water molecules rather than by large changes in side chain conformations . In fact, the polypeptide chains for all of the epimerase/NADH/UDP-sugar complexes studied thus far are remarkably similar and can be superimposed with root-mean-square deviations of not greater than 0.24 A . The only significant differences between the various enzyme/UDP-sugar models occur in two of the dihedral angles defining the conformation of the UDP-sugar ligands.

Proc Natl Acad Sci U S A, 1997 May 27, 94(11), 5967 - 72
Complete nucleotide sequence of the chloroplast genome from the green alga Chlorella vulgaris: the existence of genes possibly involved in chloroplast division; Wakasugi T et al.; The complete nucleotide sequence of the chloroplast genome (150,613 bp) from the unicellular green alga Chlorella vulgaris C-27 has been determined . The genome contains no large inverted repeat and has one copy of rRNA gene cluster consisting of 16S, 23S, and 5S rRNA genes . It contains 31 tRNA genes, of which the tRNALeu(GAG) gene has not been found in land plant chloroplast DNAs analyzed so far . Sixty-nine protein genes and eight ORFs conserved with those found in land plant chloroplasts have also been found . The most striking is the existence of two adjacent genes homologous to bacterial genes involved in cell division, minD and minE, which are arranged in the same order in Escherichia coli . This finding suggests that the mechanism of chloroplast division is similar to bacterial division . Other than minD and minE homologues, genes encoding ribosomal proteins L5, L12, L19, and S9 (rpl5, rpl12, rpl19, and rps9); a chlorophyll biosynthesis Mg chelating subunit (chlI); and elongation factor EF-Tu (tufA), which have not been reported from land plant chloroplast DNAs, are present in this genome . However, many of the new chloroplast genes recently found in red and brown algae have not been found in C . vulgaris . Furthermore, this algal species possesses two long ORFs related to ycf1 and ycf2 that are exclusively found in land plants . These observations suggest that C . vulgaris is closer to land plants than to red and brown algae.

Proc Natl Acad Sci U S A, 1997 May 27, 94(11), 5784 - 8
Antigens produced in plants by infection with chimeric plant viruses immunize against rabies virus and HIV-1; Yusibov V et al.; The coat protein (CP) of alfalfa mosaic virus was used as a carrier molecule to express antigenic peptides from rabies virus and HIV . The antigens were separately cloned into the reading frame of alfalfa mosaic virus CP and placed under the control of the subgenomic promoter of tobacco mosaic virus CP in the 30BRz vector . The in vitro transcripts of recombinant virus with sequences encoding the antigenic peptides were synthesized from DNA constructs and used to inoculate tobacco plants . The plant-produced protein (virus particles) was purified and used for immunization of mice . Both antigens elicited specific virus-neutralizing antibodies in immunized mice.

Proc Natl Acad Sci U S A, 1997 May 27, 94(11), 5733 - 8
SOS factors involved in translesion synthesis; Napolitano RL et al.; Mutations are permanent DNA sequence changes that can be induced when replication occurs on a damaged DNA template . In Escherichia coli, the process of translesion synthesis past a lesion that hinders replication requires the induction of SOS-controlled gene products, among which are those of the umuDC operon . To study translesion synthesis in vivo, we have constructed single-stranded vectors containing single 2-acetylaminofluorene adducts located within -1 and -2 frameshift mutation hot spots formed by short repetitive sequences . These adducts strongly hinder DNA replication as only 2-5% of the molecules give rise to progeny under non-SOS-induced conditions . Induction of the SOS response lead to a 10-fold increase in survival . Adducts present within repetitive sequences trigger the formation of misaligned primer/template replication intermediates which, upon elongation, will result in the fixation of frameshift errors (mutagenic translesion synthesis) . Surprisingly we find that elongation from the nonslipped intermediate depends upon functional umuDC+ gene products, whereas elongation from the slipped intermediate is umuDC+ independent but requires another, as yet biochemically uncharacterized, SOS function . These data are discussed in terms of the different steps involved during translesion synthesis through a replication-blocking lesion.

Proc Natl Acad Sci U S A, 1997 May 27, 94(11), 5617 - 21
Synergy between adjacent zinc fingers in sequence-specific DNA recognition; Isalan M et al.; Zif268-like zinc fingers are generally regarded as independent DNA-binding modules that each specify three base pairs in adjacent, but discrete, subsites . However, crystallographic evidence suggests that a contact also can occur from the second helical position of one finger to the subsite of the preceding finger . Here we show for the three-finger DNA-binding domain of the protein Zif268, and a panel of variants, that deleting the putative contact from finger 3 can affect the binding specificity for the 5' base in the adjoining triplet, which forms part of the binding site of finger 2 . This finding demonstrates that Zif268-like zinc fingers can specify overlapping 4-bp subsites, and that sequence specificity at the boundary between subsites arises from synergy between adjacent fingers . This has important implications for the design and selection of zinc fingers with novel DNA binding specificities.

Proc Natl Acad Sci U S A, 1997 May 27, 94(11), 5588 - 93
Animal fatty acid synthase: functional mapping and cloning and expression of the domain I constituent activities; Chirala SS et al.; Animal fatty acid synthase (FAS; EC 2.3.1.85) is a homodimer of a multifunctional subunit protein and catalyzes the synthesis of palmitate from acetyl-CoA, malonyl-CoA, and NADPH . The subunit (Mr approximately 270,000) carries seven distinct component activities and a site for the prosthetic group 4'-phosphopantetheine (acyl carrier protein) . Based on proteolytic mapping, the organization of the activity domains along the subunit polypeptide from the N terminus is as follows: beta-ketoacyl synthase, acetyl and malonyl transacylases, beta-hydroxyacyl dehydratase, enoyl reductase, beta-ketoacyl reductase, acyl carrier protein, and thioesterase . By comparing the amino acid sequences of the chicken, rat, and human synthases, we found that kallikrein cleavage sites occur in the least conserved regions of the FAS polypeptide subunit . Determining the amino acid sequences of the N-terminal end of the major kallikrein cleavage peptides helped delineate the most likely boundaries of the component activities in the cDNA-derived amino acid sequence . To confirm this organization, we cloned the chicken FAS cDNA coding for domain I and expressed it in Escherichia coli as a maltose-binding fusion protein . The isolated recombinant protein contained the activities of the acetyl and malonyl transacylases and the beta-hydroxyacyl dehydratase . Based on the boundaries of the acetyl and malonyl transacylases and the beta-hydroxyacyl dehydratase, we also cloned the appropriate cDNA fragments encoding the domains that contain the transacylases and the dehydratase in pET vectors and expressed them in E . coli as thioredoxin-6xHis fusion proteins . The purified recombinant proteins contained, respectively, the activities of the acetyl and malonyl transacylases and the dehydratase . These results not only confirmed the order of the component activities in domain I, but also paved the way for successful expression and characterization of the remaining activities.

Proc Natl Acad Sci U S A, 1997 May 27, 94(11), 5582 - 7
I2B is a small cytosolic protein that participates in vacuole fusion; Slusarewicz P et al.; Saccharomyces cerevisiae vacuole inheritance requires two low molecular weight activities, LMA1 and LMA2 . LMA1 is a heterodimer of thioredoxin and protease B inhibitor 2 (I2B) . Here we show that the second low molecular weight activity (LMA2) is monomeric I2B . Though LMA2/I2B was initially identified as a protease B inhibitor, this protease inhibitor activity is not related to its ability to promote vacuole fusion: (i) Low Mr protease B inhibitors cannot substitute for LMA1 or LMA 2, (ii) LMA1 and LMA2 promote the fusion of vacuoles from a strain that has no protease B, (iii) low concentrations of LMA2 that fully inhibit protease B do not promote vacuole fusion, and (iv) LMA1, in which I2B is complexed with thioredoxin, is far more active than LMA2/I2B in promoting vacuole fusion and far less active in inhibiting protease B . These studies establish a new function for I2B.

Proc Natl Acad Sci U S A, 1997 May 27, 94(11), 5574 - 81
Both an N-terminal 65-kDa domain and a C-terminal 30-kDa domain of SecA cycle into the membrane at SecYEG during translocation; Eichler J et al.; SecA, a 102-kDa hydrophilic protein, couples the energy of ATP binding to the translocation of preprotein across the bacterial inner membrane . SecA function and topology were studied with metabolically labeled {35S}SecA and with inner membrane vesicles from cells that overexpressed SecYEGDFyajC, the integral domain of preprotein translocase . During translocation in the presence of ATP and preprotein, a 65-kDa N-terminal domain of SecA is protected from proteolytic digestion through insertion into the membrane, as previously reported for a 30-kDa C-terminal domain {Economou, A . & Wickner, W . (1994) Cell 78, 835-843} . Insertion of both domains occurs at saturable SecYEGDFyajC sites and is rapidly followed by deinsertion . SecA also associates nonsaturably and unproductively with lipid . In the presence of ATP, yet without involvement of preprotein or SecYEG, lipid-bound SecA forms domains that are protease-resistant and that remain so even upon subsequent membrane disruption . Unlike the {35S}SecA that inserts into the membrane at SecYEGDFyajC as it promotes preprotein translocation, lipid-associated {35S}SecA does not chase from its protease-resistant state upon the addition of excess SecA . The finding that two domains of SecA (which together represent most regions of the polypeptide chain) cycle into the membrane during preprotein translocation, as well as the distinction between the membrane association of SecA at translocation sites of SecYEGDFyajC and at nonproductive lipid sites, are fundamental to the study of the role of SecA in preprotein movement.

Proc Natl Acad Sci U S A, 1997 May 27, 94(11), 5568 - 73
Characterization of the thyroid Na+/I- symporter with an anti-COOH terminus antibody; Levy O et al.; The Na+/I- symporter (NIS) is the plasma membrane protein that catalyzes active I- transport in the thyroid, the first step in thyroid hormone biogenesis . The cDNA encoding NIS was recently cloned in our laboratory and a secondary structure model proposed, suggesting that NIS is an intrinsic membrane protein (618 amino acids; approximately 65.2 kDa predicted molecular mass) with 12 putative transmembrane domains . Here we report the generation of a site-directed polyclonal anti-COOH terminus NIS antibody (Ab) that immunoreacts with a approximately 87 kDa-polypeptide present in membrane fractions from a rat thyroid cell line (FRTL-5) . The model-predicted cytosolic-side location of the COOH terminus was confirmed by indirect immunofluorescence experiments using anti-COOH terminus NIS Ab in permeabilized FRTL-5 cells . Immunoreactivity was competitively blocked by the presence of excess synthetic peptide . Treatment of membrane fractions from FRTL-5 cells, Xenopus laevis oocytes, and COS cells expressing NIS with peptidyl N-glycanase F converted the approximately 87 kDa-polypeptide into a approximately 50 kDa-species, the same relative molecular weight exhibited by NIS expressed in E . coli . Anti-NIS Ab immunoprecipitated both the NIS precursor molecule (approximately 56 kDa) and the mature approximately 87 kDa form . Furthermore, a direct correlation between circulating levels of thyroid-stimulating hormone and NIS expression in vivo was demonstrated.

Proc Natl Acad Sci U S A, 1997 May 27, 94(11), 5562 - 7
Construction of a catalytically active iron superoxide dismutase by rational protein design; Pinto AL et al.; The rational protein design algorithm DEZYMER was used to introduce the active site of nonheme iron superoxide dismutase (SOD) into the hydrophobic interior of the host protein, Escherichia coli thioredoxin (Trx), a protein that does not naturally contain a transition metal-binding site . Reconstitution of the designed protein, Trx-SOD, showed the incorporation of one high-affinity metal-binding site . The electronic spectra of the holoprotein and its N3- and F- adducts are analogous to those previously reported for native inverted question markFe3+ inverted question markSOD . Activity assays showed that inverted question markFe3+ inverted question markTrx-SOD is capable of catalyzing the dismutation of the superoxide anion; comparative studies with the unrelated wild-type E . coli iron SOD indicated that inverted question markFe3+ inverted question markTrx-SOD catalyzes the dismutation reaction at a rate on the order of 10(5) M-1s -1 . The ability to design catalytically competent metalloenzymes allows for the systematic investigation of fundamental mechanistic questions concerning catalysis at transition metal centers.

Proc Natl Acad Sci U S A, 1997 May 27, 94(11), 5556 - 61
Skn-1: evidence for a bipartite recognition helix in DNA binding; Pal S et al.; Skn-1 is a maternally expressed transcription factor that specifies the fate of certain blastomeres early in the development of Caenorhabditis elegans . This transcription factor contains a basic region, but it binds to DNA as a monomer . Because other transcription factors containing basic regions bind as dimers, this finding implied that Skn represents a new DNA recognition motif . It has been proposed that the basic region helix of Skn is stabilized for binding by tertiary contacts to other parts of the protein . We have tested this proposal by carrying out circular dichroism (CD) and NMR experiments on the Skn domain and five truncated proteins . Our results have shown that the basic region of Skn is unstructured in solution and does not contact other parts of the protein; like other basic region peptides, it folds into a helix only upon binding specifically to DNA . However, there is a stably folded helical module in the Skn domain, and one of the helices in this module terminates immediately before the start of the basic region . This pre-organized helix contains a surface rich in basic amino acids, and we propose that this helix contacts the DNA distal to the basic region proper, providing an extra long helical recognition surface which helps to stabilize monomeric binding . Homology between the Skn domain and several basic-region leucine zipper (bZIP) domains raises the possibility that the affinity and perhaps the specificity of DNA binding by bZIP proteins can be modulated by incorporating a stably folded helical segment that contacts the DNA just below the basic region proper.

Proc Natl Acad Sci U S A, 1997 May 27, 94(11), 5539 - 43
A molecular mechanism for energy coupling in a membrane transport protein, the lactose permease of Escherichia coli; Kaback HR; A mechanism for the coupled translocation of substrate and H+ by the lactose permease of Escherichia coli is proposed, based on a variety of experimental observations . The permease is composed of 12 alpha-helical rods that traverse the membrane with the N and C termini on the cytoplasmic face . Four residues are irreplaceable with respect to coupling, and the residues are paired-Arg-302 (helix IX) with Glu-325 (helix X) and His-322 (helix X) with Glu-269 (helix VIII) . In an adjacent region of the molecule at the interface between helices VIII and V is the substrate translocation pathway . Because of this arrangement, interfacial changes between helices VIII and V are transmitted to the interface between helices IX and X and vice versa . Upon ligand binding, a structural change at the interface between helices V and VIII disrupts the interaction between Glu-269 and His-322, Glu-269 displaces Glu-325 from Arg-302, and Glu-325 is protonated . Simultaneously, protonated Glu-325 becomes inaccessible to water, which drastically increases its pKa . In this configuration, the permease undergoes a freely reversible conformational change that corresponds to translocation of the ternary complex . To return to ground state after release of substrate, the Arg-302-Glu-325 interaction must be reestablished, which necessitates loss of H+ from Glu-325 . The H+ is released into a water-filled crevice between helices IX and X which becomes transiently accessible to both sides of the membrane due to a change in helix tilt, where it is acted upon equally by either the membrane potential or the pH gradient across the membrane.

FEBS Lett, 1997 May 26, 408(3), 337 - 40
Interaction between the CheY response regulator and the histidine-containing phosphotransfer (HPt) domain of the ArcB sensory kinase in Escherichia coli; Yaku H et al.; Bacteria have devised sophisticated His-Asp phosphorelay signaling systems for eliciting a variety of adaptive responses to their environment . The histidine-containing phosphotransfer (HPt) domain, found in many signal transduction protein, functions as a mediator of the His-Asp phosphorelay . The ArcB anaerobic sensor of E . coli contains such a HPt domain, although its function is not fully understood . In this study, we provide in vivo and in vitro evidence that the HPt domain is capable of interacting with the CheY receiver, which contains a phospho-accepting aspartate residue.

FEBS Lett, 1997 May 26, 408(3), 315 - 8
Aluminum fluoride associates with the small guanine nucleotide binding proteins; Ahmadian MR et al.; AlF4- has long been known to associate with and activate the GDP-bound alpha subunits of heterotrimeric G-proteins . Recently the small guanine nucleotide binding protein Ras has also been shown to associate with AlF4- in the presence of stoichiometric amounts of its GTPase activating protein (GAP) . Here we present the isolation of a stable Ras x GDP- x AlF4- x GAP ternary complex by gel filtration . In addition, we generalise the association of AlF4- with the small GTP-binding proteins by demonstrating ternary complex formation for the Cdc42, Rap and Ran proteins in the presence of their respective GAP proteins.

FEBS Lett, 1997 May 26, 408(3), 311 - 4
Identification and characterization of a cyanobacterial DnaX intein; Liu XQ et al.; A new intein is identified and characterized in the DnaX protein of Synechocystis sp . PCC6803 . This cyanobacterial DnaX protein is a homologue of the intein-less 71-kDa tau-subunit of Escherichia coli DNA polymerase III and is related to eukaryotic DNA replication factor C (RFC) . The 430-residue DnaX intein contains several putative intein sequence motifs and undergoes protein splicing when produced in E . coli cells . Its position in the DnaX protein is close to, but different from, positions of three inteins present in a DnaX-related RFC protein of Methanococcus jannaschii.

FEBS Lett, 1997 May 26, 408(3), 281 - 4
Human L-3-phosphoserine phosphatase: sequence, expression and evidence for a phosphoenzyme intermediate; Collet JF et al.; We report the sequence of the cDNA encoding human L-3-phosphoserine phosphatase . The encoded polypeptide contains 225 residues and shows 30% sequence identity with the Escherichia coli enzyme . The human protein was expressed in a bacterial expression system and purified . Similar to known L-3-phosphoserine phosphatases, it catalyzed the Mg2(+)-dependent hydrolysis of L-phosphoserine and an exchange reaction between L-serine and L-phosphoserine . In addition we found that the enzyme was phosphorylated upon incubation with L-{32P}phosphoserine, which indicates that the reaction mechanism proceeds via the formation of a phosphoryl-enzyme intermediate . The sensitivity of the phosphoryl-enzyme to alkali and to hydroxylamine suggests that an aspartyl- or a glutamyl-phosphate was formed . The nucleotide sequence of the cDNA described in this article has been deposited in the EMBL data base under accession number Y10275.

Virology, 1997 May 26, 232(1), 198 - 206
Phosphorylation of canine distemper virus P protein by protein kinase C-zeta and casein kinase II; Liu Z et al.; Transcription by nonsegmented negative-strand RNA viruses is mediated by the viral RNA-dependent RNA polymerase and transcriptional cofactor P . The P protein is activated by phosphorylation, an event initiated by cellular kinases . The kinase used differs among this group of RNA viruses; vesicular stomatitis virus and respiratory syncytial virus utilize casein kinase II (CKII), whereas human parainfluenza virus type 3 utilizes PKC isoform zeta (PKC-zeta) for activation of its P protein . To identify the cellular kinase(s) involved in the phosphorylation of the canine distemper virus (CDV) P protein, we used recombinant CDV P in phosphorylation assays with native kinase activities present in CV1 cell extracts or purified CKII and PKC isoforms . Here, we demonstrate that the CDV P protein is phosphorylated by two cellular kinases, where PKC-zeta has the major and CKII the minor activities . In contrast, the P protein of another member of the morbillivirus genus, measles virus, is phosphorylated predominantly by CKII, whereas PKC-zeta has only minor activity . Selective inhibition of PKC-zeta activity within CV1 cells eliminated permissiveness to CDV replication, indicating an in vivo role for PKC-zeta in the virus replication cycle . The broad tissue expression of PKC-zeta parallels the pantropic nature of CDV infections, suggesting that PKC-zeta activity is a determinant of cellular permissiveness to CDV replication.

Virology, 1997 May 26, 232(1), 32 - 43
A tobamovirus genome that contains an internal ribosome entry site functional in vitro; Ivanov PA et al.; Most eukaryotic mRNAs are translated by a "scanning ribosome" mechanism . We have found that unlike the type member of the genus Tobamovirus, translation of the 3'-proximal coat protein (CP) gene of a crucifer infecting tobamovirus (crTMV) (Dorokhov et al., 1993; 1994) occurred in vitro by an internal ribosome entry mechanism . Three types of synthetic dicistronic RNA transcripts were constructed and translated in vitro: (i) "MP-CP-3'NTR" transcripts contained movement protein (MP) gene, CP gene and the 3'-nontranslated region of crTMV RNA . These constructs were structurally equivalent to dicistronic subgenomic RNAs produced by tobamoviruses in vivo . (ii) "deltaNPT-CP" transcripts contained partially truncated neomycin phosphotransferase I gene and CP gene . (iii) "CP-GUS" transcripts contained the first CP gene and the gene of Escherichia coli beta-glucuronidase (GUS) at the 3'-proximal position . The results indicated that the 148-nt region upstream of the CP gene of crTMV RNA contained an internal ribosome entry site (IRES(CP)) promoting internal initiation of translation in vitro . Dicistronic IRES(CP), containing chimeric mRNAs with the 5'-terminal stem-loop structure preventing translation of the first gene (MP, deltaNPT, or CP), expressed the CP or GUS genes despite their 3'-proximal localization . The capacity of crTMV IRES(CP) for mediating internal translation distinguishes this CP tobamovirus from the well-known-type member of the genus, TMV UI . The equivalent 148-nt sequence from TMV RNA was incapable of mediating internal translation . Two mutants were used to study structural elements of IRES(CP) . It was concluded that integrity of IRES(CP) was essential for internal initiation . The crTMV provides a new example of internal initiation of translation, which is markedly distinct from IRESs shown for picornaviruses and other viral and eukaryotic mRNAs.

Biochim Biophys Acta, 1997 May 24, 1360(3), 211 - 21
Molecular bases of hexokinase deficiency; Bianchi M et al.; Hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1; HK) deficiency is a rare disease where the predominant clinical effect is nonspherocytic hemolytic anemia . We have previously shown that the only patient for which hexokinase deficiency has been so far investigated at molecular level is a double heterozygote carrying a T1667 --> C substitution on one HK type I allele and a 96 bp deletion (concerning nucleotides 577 to 672 in the HK cDNA sequence) in the other allele . To investigate whether these mutations found in the patient with the hexokinase variant referred to as 'HK-Melzo' could be associated with hexokinase deficiency, we have expressed in E . coli the wild-type human hexokinase type I and two different mutants carrying the T --> C nucleotide substitution at position 1667 and the nt 577-672 deletion, respectively . Wild-type human recombinant hexokinase is expressed in bacterial cells as a soluble catalytically active enzyme that, upon purification to homogeneity, exhibited the same kinetic properties of human placenta hexokinase type I . Both mutant hexokinases were also expressed as soluble recombinant proteins under the same conditions, but they showed an impaired catalytic activity with respect to the wild-type enzyme . In particular, the T1667 --> C substitution, causing the amino acid change from Leu529 to Ser, is responsible for the complete loss of the hexokinase catalytic activity, while the 96 bp deletion causes a drastic reduction of the hexokinase activity . Taken together, both mutations explain the hexokinase deficiency found in the patient with the 'HK-Melzo' variant.

Biochim Biophys Acta, 1997 May 23, 1339(2), 331 - 40
Effects of profilin-annexin I association on some properties of both profilin and annexin I: modification of the inhibitory activity of profilin on actin polymerization and inhibition of the self-association of annexin I and its interactions with liposomes; Alvarez-Martinez MT et al.; We have previously shown that annexin I, a member of a family of calcium-dependent phospholipid and membrane binding proteins, interacts with profilin with high specificity and affinity . This finding further suggests that annexin I is involved through profilin in the regulation of membrane-cytoskeleton organization . We have investigated the consequences of a complex formed by these two proteins on the functions of both profilin and annexin I . Annexin I is able to modify the inhibitory effect of profilin on actin polymerization . This action is partial and the mechanism involved appears to be complex . On the other hand, the association between annexin I and profilin is sufficiently strong to inhibit the self-association of annexin I . The binding capacity of annexin I to liposomes containing phosphatidylserine, which mimics annexin I binding to membranes, is also decreased by profilin . This binding is nevertheless restored when phosphatidylinositol 4,5-biphosphate (PtdInsP2) is included in the liposomes . Finally, the capacity of annexin I to aggregate liposomes is also modified . It is worthwhile mentioning that the liposomes-binding and liposomes-aggregating activities of annexin I are independently regulated . The cell localization and functions of annexin I and profilin suggest that interaction between these two proteins may be directly implicated in the regulation of membrane-cytoskeleton . The phospholipid composition of membranes may be one of the modulating factors.

Biochim Biophys Acta, 1997 May 23, 1339(2), 192 - 202
Characterization of the affinity of the G(M2) activator protein for glycolipids by a fluorescence dequenching assay; Smiljanic-Georgijev N et al.; The G(M2) activator protein is a substrate specific cofactor for degradation of G(M2) ganglioside by lysosomal beta-hexosaminidase A . Mutations in the gene encoding the activator result in the AB-variant form of G(M2) gangliosidosis . The activator protein contains at least three functional elements; a hydrophobic binding pocket, an oligosaccharide binding site(s), and an area that interacts with hexosaminidase A . In this report a fluorescence dequenching assay specific for only the hydrophobic binding pocket is evaluated and optimized . It is shown that various glycolipids inhibit the transport between liposomes of a self-quenching fluorescent lipid probe, octadecylrhodamine, by the activator protein . The level of inhibition produced by each glycolipid is then used to characterize the oligosaccharide-binding specificity of the activator . The fluorescence dequenching assay is also used to evaluate the functionality of a truncated form of the activator protein . Our results indicate that this simple assay can be used to determine structure-function relationships within the normal or mutant forms of the activator . The data suggest that the C-terminus of the activator is required to produce a functional hydrophobic binding pocket.

Biochim Biophys Acta, 1997 May 23, 1339(2), 181 - 91
Characterization and mutational studies of equine infectious anemia virus dUTPase; Shao H et al.; The macrophage tropic lentivirus, equine infectious anemia virus (EIAV), encodes a dUTPase in the pol gene that is required for efficient replication in macrophages . Two naturally occurring variants of the enzyme were expressed as recombinant proteins in Escherichia coli; metal chelate affinity chromatography was used to purify histidine-tagged recombinant enzymes to greater than 80% homogeneity in a single chromatographic step . Biochemical and enzymatic analyses of these preparations suggest that this method yields dUTPase that is suitable for detailed mutational analysis . Specific activities of preparations ranged from 4 x 10(3) to 5 x 10(4) units/mg . Recombinant EIAV dUTPase was highly specific for dUTP with a Km in the range of 3 to 8 microM . The enzyme was sensitive to inhibition by dUDP with little inhibition by other nucleotides or the reaction products, dUMP and PPi . The subunit organization of recombinant EIAV dUTPase was probed by gel filtration, glycerol gradient centrifugation, and chemical cross-linking, and is a trimer . We have begun mutational analyses by targeting a conserved domain present at the carboxyl terminus of all dUTPases that shares high homology to the phosphate binding loops (P-loops) of a number of ATP- and GTP-binding phosphatases . The P-loop-like motif of dUTPases is glycine rich but lacks the invariant lysine found in authentic P-loops . Deletion of this motif leads to loss of dUTPase activity; a series of point mutations that have been shown to inactivate authentic P-loops also abolish EIAV dUTPase activity.

J Mol Biol, 1997 May 23, 268(5), 934 - 51
The rational construction of an antibody against cystatin: lessons from the crystal structure of an artificial Fab fragment; Schiweck W et al.; In a protein design study the artificial antibody M41 was modelled with its binding site complementary to the protease inhibitor cystatin, which was chosen as a structurally well-characterized "antigen" . The modelling of M41 took advantage of the crystal structure of the anti-lysozyme antibody HyHEL-10 as a structural template . Its combining site was reshaped by replacing 19 amino acid side-chains in the hypervariable loops . In addition, ten amino acid residues were substituted in the framework regions . The crystal structure of the corresponding antibody model M41, which was produced as an F(ab) fragment in Escherichia coli, was determined at a resolution of 1.95 A . The crystals exhibited symmetry of the space group P2(1)2(1)2(1) (a = 96.5 A; b = 103.5 A; c = 113.6 A) with two F(ab) fragments in the asymmetric unit, which were independently refined (final R-factor 21.7%) . The resulting coordinates were used for a detailed comparison with the modelled protein structure . It was found that the mutual arrangement of the six complementarity-determining regions as well as most of their backbone conformation had been correctly predicted . One major difference that was detected for the conformation of a five residue insertion in complementarity-determining region L1 could be explained by an erroneously defined segment in the structure of the antibody 4-4-20, which had been used as a template for this loop . In the light of more recent crystallographic data it appears that this segment adopts a new canonical structure . Apart from this region, most of the side-chains in the antigen-binding site had been properly placed in the M41 model . There was however one important exception concerning Trp H98, whose side-chain conformation had been kept as it appeared in HyHEL-10 . The differing orientation of this residue in the model compared with the crystal structure of the artificial F(ab) fragment M41 explains why an antigen affinity could not be detected so far . The detailed analysis of this and other, more subtle deviations suggests how to make this F(ab) fragment function by introducing a few additional amino acid changes into M41.

J Mol Biol, 1997 May 23, 268(5), 886 - 902
Structure of neurotoxin B-IV from the marine worm Cerebratulus lacteus: a helical hairpin cross-linked by disulphide bonding; Barnham KJ et al.; B-IV is a 55-residue, crustacean-selective, neurotoxin secreted by Cerebratulus lacteus, a large marine worm found along the northeastern coast of North America . The 3D structure of this molecule in aqueous solution has been determined by 1H NMR spectroscopy at 600 MHz . The molecule has a well-defined helical hairpin structure, with the branches of the hairpin linked by four disulphide bonds . The disulphide connectivities were established from the NMR data to be 1-8/2-7/3-6/4-5, which differed from those determined previously by chemical means, where 1-7 and 2-8 connectivities were found . Each branch of the hairpin is largely alpha-helical, with the helices in the N and C-terminal branches encompassing residues 11 to 23 and 34 to 49, respectively . The loop connecting the branches of the hairpin contains two inverse gamma-turns centred on residues 24 and 25, a type I beta-turn at residues 28 to 31 and a type II beta-turn at residues 30 to 33 . Arg17, -25 and -34, which are important for activity, are all on the same face of the molecule, while Trp30, which is also important for activity, is on the opposite face . Structure comparisons show that the B-IV structure is quite similar to those of Rop (ColE1 repressor of primer) and the heat-stable enterotoxin B from Escherichia coli . These structural similarities are discussed in relation to possible mechanisms of action of B-IV.

J Mol Biol, 1997 May 23, 268(5), 857 - 68
Protein evolution viewed through Escherichia coli protein sequences: introducing the notion of a structural segment of homology, the module; Riley M et al.; Paralogous genes are genes which descend from a progenitor gene which has duplicated as an ancestral gene, each copy having diverged prior to speciation . With comprehensive information available on functions of Escherichia coli proteins, analysis of sequence-related E . coli paralogous proteins can give information on the early ancestors of families of proteins now residing in many contemporary organisms, such as the enzymes of metabolism, some kinds of transport mechanisms and some kinds of regulatory mechanisms . In the first step, we have confirmed that E . coli contains a very high proportion of paralogous proteins . Next, we have defined two main classes of paralogous proteins . One class is formed of proteins which contain a unique structural segment homologous to a single set of related proteins . The other class corresponds to proteins which contain more than one structural segment of homology, each segment homologous to unrelated sets of proteins . We define such an independent structural segment of homology as a module . This modular structure (mean length equivalent to 209 amino acids) corresponds often to entire proteins, but there are also proteins that appear to be assembled from two or three independent modules having independent origins . Most multimodular proteins appear to have been formed early in their history, a minority appear to be relatively recent fusions of independent modules . Examining 1404 independent structural segments of homology, composed of both modules and entire proteins, we found that the segments of homology fell into 352 sequence-related groups or families . The majority of these families (ranging from 2 to 62 members) are functionally homogeneous . This strongly suggests that the 1404 present-day modules and proteins derive from a minimal set of 352 ancestral modules, each one being already of the same size and having a function similar to all members of its progeny.

J Mol Biol, 1997 May 23, 268(5), 803 - 8
In vitro trans translation mediated by alanine-charged 10Sa RNA; Himeno H et al.; 10Sa RNA is a bacterial small stable RNA, in which the 5' and 3'-terminal sequences can be folded into a tRNA-like secondary structure which can be aminoacylated with alanine . It was found that Escherichia coli 10Sa RNA facilitated the incorporation of alanine, tyrosine, aspartic acid and glutamic acid, but not valine, isoleucine, serine or arginine, into the growing polypeptide in vitro, depending on poly (U)-directed poly-phenylalanine synthesis . This result indicates that 10Sa RNA functions as an mRNA for the tag-peptide which has been found to be attached to the C termini of truncated polypeptides synthesized in vivo . Aminoacylation with alanine was required for tag-specific amino acid incorporation and for efficient association of 10Sa RNA with the ribosome, indicating that 10Sa RNA also functions as an alanine tRNA in the tag-peptide synthesis . The dual function of 10Sa RNA both as an mRNA and as a tRNA in vitro strongly supports the trans translation hypothesis.

Science, 1997 May 23, 276(5316), 1258 - 60
Promoter recognition as measured by binding of polymerase to nontemplate strand oligonucleotide; Marr MT et al.; In transcription initiation, the DNA strands must be separated to expose the template to RNA polymerase . As the closed initiation complex is converted to an open one, specific protein-DNA interactions involving bases of the nontemplate strand form and stabilize the promoter complex in the region of unwinding . Specific interaction between RNA polymerase and the promoter in Escherichia coli was detected and quantified as the binding affinity of nontemplate oligonucleotide sequences . The RNA polymerase subunit sigma factor 70 contacted the bases of the nontemplate DNA strand through its conserved region 2; a mutation that affected promoter function altered the binding affinity of the oligonucleotide to the enzyme.

Science, 1997 May 23, 276(5316), 1250 - 2
Discrete determinants in transfer RNA for editing and aminoacylation; Hale SP et al.; During translation errors of aminoacylation are corrected in editing reactions which ensure that an amino acid is stably attached to its corresponding transfer RNA (tRNA) . Previous studies have not shown whether the tRNA nucleotides needed for effecting translational editing are the same as or distinct from those required for aminoacylation, but several considerations have suggested that they are the same . Here, designed tRNAs that are highly active for aminoacylation but are not active in translational editing are presented . The editing reaction can be controlled by manipulation of nucleotides at the corner of the L-shaped tRNA . In contrast, these manipulations do not affect aminoacylation . These results demonstrate the segregation of nucleotide determinants for the editing and aminoacylation functions of tRNA.

J Biol Chem, 1997 May 23, 272(21), 13916 - 22
Translesional synthesis on DNA templates containing a single abasic site . A mechanistic study of the "A rule"; Shibutani S et al.; Site-specifically modified oligodeoxynucleotides containing a single natural abasic site or a chemically synthesized (tetrahydrofuran or deoxyribitol) model abasic site were used as templates for primer extension reactions catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I or by calf thymus DNA polymerase alpha . Analysis of the fully extended products of these reactions indicated that both polymerases preferentially incorporate dAMP opposite the natural abasic site and tetrahydrofuran, while DNA templates containing the ring-opened deoxyribitol moiety block translesional synthesis, promoting sequence context-dependent deletions . The frequency of nucleotide insertion opposite the three types of abasic sites follows the order dAMP > dGMP > dCMP > dTMP . The frequency of chain extension was highest when dAMP was positioned opposite a natural abasic site . The frequency of translesional synthesis past abasic sites follows the order tetrahydrofuran > deoxyribose > deoxyribitol . The Klenow fragment promotes blunt end addition of dAMP; this reaction was much less efficient than insertion of dAMP opposite an abasic site . We conclude that the miscoding potential of a natural abasic site in vitro closely resembles that of its tetrahydrofuran analog . Ring-opened abasic sites favor deletions . Studies with polymerase alpha in vitro predict preferential incorporation of dAMP at abasic sites in mammalian cells.

J Biol Chem, 1997 May 23, 272(21), 13911 - 5
Down-regulation of the filamentous actin cross-linking activity of cortactin by Src-mediated tyrosine phosphorylation; Huang C et al.; Cortactin, a prominent substrate for pp60(c-src), is a filamentous actin (F-actin) binding protein . We show here that cortactin can promote sedimentation of F-actin at centrifugation forces under which F-actin is otherwise not able to be precipitated . Electron microscopic analysis after negative staining further revealed that actin filaments in the presence of cortactin are cross-linked into bundles of various degrees of thickness . Hence, cortactin is also an F-actin cross-linking protein . We also demonstrate that the optimal F-actin cross-linking activity of cortactin requires a physiological pH in a range of 7.3-7.5 . Furthermore, pp60(c-src) phosphorylates cortactin in vitro, resulting in a dramatic reduction of its F-actin cross-linking activity in a manner depending on levels of tyrosine phosphorylation . In addition, pp60(c-src) moderately inhibits the F-actin binding activity of cortactin . This study presents the first evidence that pp60(c-src) can directly regulate the activity of its substrate toward the cytoskeleton and implies a role of cortactin as an F-actin modulator in tyrosine kinase-regulated cytoskeleton reorganization.

J Biol Chem, 1997 May 23, 272(21), 13823 - 8
ARD-1 cDNA from human cells encodes a site-specific single-strand endoribonuclease that functionally resembles Escherichia coli RNase E; Claverie-Martin F et al.; The human ARD-1 (activator of RNA decay) cDNA sequence can rescue mutations in the Escherichia coli rne gene, which specifies the essential endoribonuclease RNase E, resulting in RNase E-like cleavages in vivo in rne-defective bacteria and in vitro in extracts isolated from these cells (Wang, M., and Cohen, S . N . (1994) Proc . Natl . Acad . Sci . U . S . A . 91, 10591-10595) . Recent studies indicate that the 13.3-kDa protein encoded by ARD-1 cDNA is almost identical to the carboxyl-terminal end of the bovine protein NIPP-1, a nuclear inhibitor of protein phosphatase 1; separate transcripts formed by alternative splicing are proposed to encode the discrete ARD-1 and combined ARD-1/NIPP-1 products (Van Eynde, A., Wera, S., Beullens, M . , Torrekens, S., Van Leuven, F., Stalmans, W., and Bollens, M . (1995) J . Biol . Chem . 270, 28068-28074) . Here we show that affinity column-purified protein encoded by human ARD-1 cDNA in E . coli is a site-specific Mg2+-dependent endoribonuclease that binds in vitro to RNase E substrates, cleaves RNA at the same sites as RNase E, and, like RNase E, generates 5' phosphate termini at sites of cleavage . Our results indicate that the ARD-1 peptide can function as a ribonucleolytic analog of E . coli RNase E as well as a domain of the protein phosphatase inhibitor, NIPP-1.

J Biol Chem, 1997 May 23, 272(21), 13779 - 85
Binding of HIV-1 Nef to a novel thioesterase enzyme correlates with Nef-mediated CD4 down-regulation; Liu LX et al.; Nef is a 27-kDa myristoylated protein conserved in primate lentiviruses . In vivo, simian immunodeficiency virus Nef is required in macaques to produce a high viral load and full pathological effects . Nef has at least three major effects in vitro, induction of CD4 down-regulation, alteration of T cell activation pathways, and enhancement of viral infectivity . We have used the yeast two-hybrid system to identify cellular proteins that interact with HIV-1Lai Nef and could mediate Nef function . A human cDNA was isolated that encodes a new type of thioesterase, an enzyme that cleaves thioester bonds . This novel thioesterase is unlike the animal types I and II thioesterases previously cloned but is homologous to the Escherichia coli thioesterase II . Nef and this thioesterase interact in vitro and are co-immunoprecipitated by anti-Nef antibodies in CEM cells expressing Nef . Nef alleles from human immunodeficiency virus-1 (HIV-1) isolates unable to down-regulate CD4 do not react or react poorly with thioesterase . An HIV-1 NefLai mutant selected for its lack of interaction with thioesterase was also unable to down-regulate CD4 cell-surface expression . These observations suggest that this human thioesterase is a cellular mediator of Nef-induced CD4 down-regulation.

J Biol Chem, 1997 May 23, 272(21), 13676 - 82
Organ-specific transcription of the rrn operon in spinach plastids; Iratni R et al.; The spinach rrn operon is used as a model system to study transcriptional regulation in higher plant photosynthetic and non-photosynthetic plastids . We performed capping experiments to determine whether P1, PC, or P2 promoters are employed for rrn transcription start sites in cotyledon and root tissues . By using a new method of analysis of capped RNA we demonstrate for the first time that 1) in both organs the rrn operon is expressed in a constitutive manner by cotranscription with the preceding tRNA(GAC)Val gene, and 2) the PC transcription start site is used only in cotyledons and leaves, i.e . we demonstrate the organ-specific usage of a plastid promoter . Both start sites, PC and that of the tRNA(GAC)Val cotranscript, lack Escherichia coli-like consensus sequences . The cotranscript is initiated 457 base pairs upstream of the tRNA(GAC)Val gene . The PC-specific DNA-binding factor, CDF2, is not detectable in root tissues confirming its regulatory role in PC-initiated rrn expression and the organ specificity of PC expression . Furthermore, our results show that rrn operon expression patterns differ in spinach and tobacco indicating species-specific transcriptional regulation of plant plastid gene expression.

J Biol Chem, 1997 May 23, 272(21), 13660 - 5
SecE-depleted membranes of Escherichia coli are active . SecE is not obligatorily required for the in vitro translocation of certain protein precursors; Yang YB et al.; Membrane vesicles were prepared from Escherichia coli cells in which SecE was depleted to 2% of wild-type membranes . SecE depletion had pleiotropic effects; SecD, SecF, SecG, and SecY were decreased 4-6-fold, whereas SecA was increased about 16-fold over that of wild-type membranes . These membranes were substantially active in the in vitro translocation of proOmpA, which was mediated by the SecA pathway since it was inhibited by azide . Similar substantial translocation activities were observed for proLamB and proLpp in the SecE-depleted membranes . However, the translocation of proPhoA was more severely impaired . These data indicate that SecE may enhance but is not obligatorily required for the translocation of at least certain precursors, and suggest that the effects of the SecE depletion on protein translocation may be precursor-dependent.

J Biol Chem, 1997 May 23, 272(21), 13640 - 6
Accessory subunit of mitochondrial DNA polymerase from Drosophila embryos . Cloning, molecular analysis, and association in the native enzyme; Wang Y et al.; A full-length cDNA of the accessory (beta) subunit of mitochondrial DNA polymerase from Drosophila embryos has been obtained, and its nucleotide sequence was determined . The cDNA clone encodes a polypeptide with a deduced amino acid sequence of 361 residues and a predicted molecular mass of 41 kDa . The gene encoding the beta subunit lies within 4 kilobase pairs of that for the catalytic subunit in the Drosophila genome, on the left arm of chromosome 2 . The two genes have similar structural features and share several common DNA sequence elements in their upstream regions, suggesting the possibility of coordinate regulation . A human cDNA homolog of the accessory subunit was identified, and its nucleotide sequence was determined . The human sequence encodes a polypeptide with a predicted molecular mass of 43 kDa that shows a high degree of amino acid sequence similarity to the Drosophila beta subunit . Subunit-specific rabbit antisera, directed against the recombinant catalytic and accessory subunit polypeptides overexpressed and purified from Escherichia coli, recognize specifically and immunoprecipitate the native enzyme from Drosophila embryos . Demonstration of the physical association of the two subunits in the Drosophila enzyme and identification of a human accessory subunit homolog provide evidence for a common heterodimeric structure for animal mitochondrial DNA polymerases.

J Biol Chem, 1997 May 23, 272(21), 13562 - 9
Characterization of cyanobacterial biliverdin reductase . Conversion of biliverdin to bilirubin is important for normal phycobiliprotein biosynthesis; Schluchter WM et al.; The Synechocystis sp . PCC 6803 gene (bvdR) encoding biliverdin reductase was amplified by the polymerase chain reaction, cloned, and overexpressed in Escherichia coli as the native form and as a 6-histidine-tagged amino-terminal fusion . The latter form of the enzyme was purified by affinity chromatography and shown to have the appropriate molecular weight by electrospray mass spectrometry . Both forms of the enzyme reduced biliverdin IXalpha using NADPH or NADH, with NADPH as the preferred reductant . The His-tagged enzyme has a Km for biliverdin of 1.3 microM . The pH optimum for the NADPH-dependent activity is 5.8, whereas that for rat biliverdin reductase is at pH 8.7 . Absorbance spectra and high performance liquid chromatography retention times of the reaction product reaction match those of authentic bilirubin, the product of the reduction of biliverdin by the mammalian enzymes . These results provide the first evidence for the formation of bilirubin in bacteria . Fully segregated Synechocystis sp . PCC 6803 bvdR interposon mutants produce approximately 85% of the normal amount of phycobilisome cores containing allophycocyanin and other phycocyanobilin-bearing core polypeptides, but no detectable phycocyanin . Thus, surprisingly, the blockage of the conversion of biliverdin to bilirubin interferes with normal phycobiliprotein biosynthesis in cyanobacteria . Possible interpretations of this finding are presented.

J Biol Chem, 1997 May 23, 272(21), 13512 - 8
Catalytic properties of murine carbonic anhydrase IV; Hurt JD et al.; A cDNA encoding the murine carbonic anhydrase IV (mCA IV) gene, modified to resemble a form of mature human carbonic anhydrase IV (Okuyama, T., Waheed, A., Kusumoto, W., Zhu, X . L., and Sly, W . S . (1995) Arch . Biochem . Biophys . 320, 315-322), was expressed in Escherichia coli . Inactive inclusion bodies were collected and refolded, and active enzyme was purified; the resulting mCA IV was used to characterize the catalysis of CO2 hydration using stopped flow spectrophotometry and 18O exchange between CO2 and water . Unlike previously studied isozymes in this class of carbonic anhydrase, the pH profile for kcat for hydration of CO2 catalyzed by mCA IV could not be described by a single ionization, suggesting multiple proton transfer pathways between the zinc-bound water molecule and solution . A role for His64 in transferring protons between the zinc-bound water and solution was confirmed by the 100-fold lower activity of the mutant of mCA IV containing the replacement His64 --> Ala . The remaining activity in this mutant at pH levels near 9 suggested a second proton shuttle mechanism . The maximal turnover number kcat for hydration of CO2 catalyzed by mCA IV was 1.1 x 10(6) s-1 at pH > 9 . A pKa of 6.6 was estimated for the zinc-bound water molecule in mCA IV.

Biochim Biophys Acta, 1997 May 22, 1326(1), 157 - 65
Site-directed mutagenesis of hepatitis A virus protein 3A: effects on membrane interaction; Beneduce F et al.; Due to a stretch of hydrophobic amino acids, protein 3A of hepatitis A virus (HAV) has been suggested to act as a membrane anchor or a carrier of the genome-linked protein 3B (VPg) during viral RNA synthesis . Mutagenesis analysis was performed in order to elucidate the role of the N- and C-terminal tracts of protein 3A in cell membrane interaction . Expression of the mutated proteins in E . coli cells demonstrated that the presence of positively charged residues at the C-terminus is not required for membrane anchoring . Changes in the primary sequence involving charged amino acids at the N- and C-termini critically influenced the ability of the protein 3A of a cytopathic strain of HAV to change bacterial membrane permeability . This result demonstrates the strict correlation between the structure and pore-forming potential of HAV protein 3A.

Biochim Biophys Acta, 1997 May 22, 1326(1), 23 - 36
The hydrophobic region of signal peptides is involved in the interaction with membrane-bound SecA; Mori H et al.; The positive charges of signal peptides are important for the interaction with SecA, a translocation ATPase . To examine whether or not the hydrophobic region of signal peptides also interacts with SecA, we constructed model preproteins, proOmpF-Lpps, possessing no positively charged amino acid residues at the amino-terminus and different numbers of alanine/leucine residues in the hydrophobic region of signal peptides . When the hydrophobic stretch was sufficiently long, amino-terminal positively charged residues were not required for the translocation of preproteins across the cytoplasmic membrane of Escherichia coli both in vitro and in vivo . Chemical cross-linking between SecA and preproteins possessing no positively charged residues at the amino-terminus was observed only in the presence of liposomes containing acidic phospholipids . The degree of cross-linking increased as the length of the hydrophobic stretch increased irrespective of whether positively charged residues were present or not . A preprotein possessing no positively charged residues at the amino-terminus, which is competent in the presence of liposomes, competitively inhibited the cross-linking of wild-type proOmpF-Lpp with SecA under the same conditions . It is concluded that both the amino-terminal positive charges and central hydrophobic domains are involved in the interaction with SecA in the initial stage of translocation in addition to their possible roles in transmembrane movement of preproteins.

J Theor Biol, 1997 May 21, 186(2), 251 - 60
Mathematical model of the SOS response regulation of an excision repair deficient mutant of Escherichia coli after ultraviolet light irradiation; Aksenov SV et al.; A mathematical model for the development of the SOS signal in nucleotide-excision repair deficient Escherichia coli cells subjected to ultraviolet light irradiation is proposed, in which regions of single-stranded DNA (gaps) are created during replication of a damaged chromosome when the strand elongation stops at pyrimidine dimers . The concentration of single-stranded DNA of gaps as a function of time is obtained . The model for the interaction of the LexA and RecA proteins, a well-established key event in SOS regulation, is presented, resulting in a system of differential equations for the concentrations of LexA, RecA and activated RecA proteins . The simulated LexA protein kinetic curves agree with the experimental data for two excision repair deficient mutants: uvrA6 and dnaC28 uvrB(del), which is also a temperature-sensitive DNA replication initiation mutant . It is shown that the model can be used to quantitatively describe the kinetics of SOS response through the amount of the SOS signal (concentration of single-stranded DNA) in a cell as a function of time.

Int J Food Microbiol, 1997 May 20, 36(2-3), 171 - 8
Assessment of the hygienic performances of hamburger patty production processes; Gill CO et al.; The hygienic conditions of the hamburger patties collected from three patty manufacturing plants and six retail outlets were examined . At each manufacturing plant a sample from newly formed, chilled patties and one from frozen patties were collected from each of 25 batches of patties selected at random . At three, two or one retail outlet, respectively, 25 samples from frozen, chilled or both frozen and chilled patties were collected at random . Each sample consisted of 30 g of meat obtained from five or six patties . Total aerobic, coliform and Escherichia coli counts per gram were enumerated for each sample . The mean log (x) and standard deviation (s) were calculated for the log10 values for each set of 25 counts, on the assumption that the distribution of counts approximated the log normal . A value for the log10 of the arithmetic mean (log A) was calculated for each set from the values of x and s . A chi2 statistic was calculated for each set as a test of the assumption of the log normal distribution . The chi2 statistic was calculable for 32 of the 39 sets . Four of the sets gave chi2 values indicative of gross deviation from log normality . On inspection of those sets, distributions obviously differing from the log normal were apparent in two . Log A values for total, coliform and E . coli counts for chilled patties from manufacturing plants ranged from 4.4 to 5.1, 1.7 to 2.3 and 0.9 to 1.5, respectively . Log A values for frozen patties from manufacturing plants were between < 0.1 and 0.5 log10 units less than the equivalent values for chilled patties . Log A values for total, coliform and E . coli counts for frozen patties on retail sale ranged from 3.8 to 8.5, < 0.5 to 3.6 and < 0 to 1.9, respectively . The equivalent ranges for chilled patties on retail sale were 4.8 to 8.5, 1.8 to 3.7 and 1.4 to 2.7, respectively . The findings indicate that the general hygienic condition of hamburgers patties could be improved by their being manufactured from only manufacturing beef of superior hygienic quality, and by the better management of chilled patties at retail outlets.

Gene, 1997 May 20, 191(1), 115 - 21
A site-specific DNA endonuclease specified by one of two ORFs encoded by a group I intron in Dictyostelium discoideum mitochondrial DNA; Ogawa S et al.; The second intron (DdOX1/2.2) of Dictyostelium discoideum cytochrome oxidase subunit 1/2 fused gene has two free-standing ORF genes (Dd ai2a and Dd ai2b) in a loop, which have similar amino acid sequences and are homologous to aI4 DNA endonuclease (I-SceII) of Saccharomyces cerevisiae . To elucidate the functions of these ORFs, we cloned the ORFs into an expression vector and introduced the composite vectors into E . coli . The expression of Dd ai2a in E . coli caused growth inhibition and degradation of the E . coli genomic DNA . To determine whether Dd ai2a protein is a homing type DNA endonuclease, the ability to cleave the homing site of its intron in vivo was examined . Dd ai2a cleaved only one strand of intronless DNA sequence at the site which coincides with the I-SceII cleavage recognition site . We suppose that Dd ai2a functions actually as a homing type DNA endonuclease in D . discoideum mitochondria by virtue of other factors . To obtain further information about the relationship between the existence of introns and the mating system, we carried out in vitro self-splicing assay and polymerase chain reaction analysis using 13 strains of the cellular slime mold.

Gene, 1997 May 20, 191(1), 97 - 102
Molecular cloning of cDNA for lysenin, a novel protein in the earthworm Eisenia foetida that causes contraction of rat vascular smooth muscle; Sekizawa Y et al.; Lysenin, which causes contraction of rat vascular smooth muscle, is a protein that was isolated from the earthworm Eisenia foetida . A cDNA encoding lysenin was isolated by use of a partial cDNA probe that had been generated by the PCR with a primer designed by reference to an internal peptide sequence of lysenin . This clone had an ORF encoding 297 amino acid residues . The amino acid sequence deduced from the cDNA revealed the absence of any significant homology to those of previously characterized vasoactive substances . The recombinant lysenin was produced in Escherichia coli . This protein and native lysenin isolated from the earthworm had similar contractive activities when tested on rat aorta . Northern blot analysis of the RNA from various tissues of the earthworm indicated that lysenin is produced by the coelomocytes.

Mol Gen Genet, 1997 May 20, 254(5), 599 - 603
Roles of RpoS (sigmaS), IHF and ppGpp in the expression of the hmp gene encoding the flavohemoglobin (Hmp) of Escherichia coli K-12; Membrillo-Hernandez J et al.; The Escherichia coli K-12 gene hmp encodes the flavohemoglobin Hmp . A hmp promoter phi(hmp-lacZ)-operon fusion was constructed in the chromosome and its activity measured during the growth cycle . Logarithmically growing cultures had low levels of phi(hmp-lacZ) expression, which increased two-fold at the onset of stationary phase in rich medium . The effect was abolished in a strain carrying a null allele of the gene rpoS encoding the stationary phase-specific sigma subunit of RNA polymerase sigmaS . A himA mutation resulted in a 1.5-fold increase in expression of phi(hmp-lacZ) but did not affect growth phase-dependent regulation . A single transcriptionial start site was found for hmp, located 38 bp upstream of the initiation codon . Putative Fnr boxes at positions -2 to +11 occur in the hmp promoter region.

Mol Gen Genet, 1997 May 20, 254(5), 578 - 83
A silent mutation in the ftsH gene of Escherichia coli that affects FtsH protein production and colicin tolerance; Makino S et al.; Among Escherichia coli tolZ mutants tolerant to colicins E2, E3, and D, one, KHI10, had a high frequency of lambda lysogenization, like the tolZ21 mutant, but unlike tolZ21 KHI10 grew on nonfermentable carbon sources . The tolZ10 gene of KHI10 was cloned and sequenced, and found to have a silent mutation in the ftsH gene, causing an alteration of a minor codon, CUA, for Leu-5 to a suboptimal codon, CUC . In spite of the change in a minor codon, the amount of the FtsH protein present in mutant cells was much less than that in the parental strain . In vivo transcription of the tolZ10 gene was not decreased relative to the wild-type ftsH gene . Analysis of other silent mutations altering the Leu-5 codon (CUA) to CUG or CUU (optimal and suboptimal codons, respectively) revealed that the decrease in concentration of FtsH was seen only with the CUC (tolZ10) codon . Prediction of the mRNA secondary structure suggested that the change from A to C extends the base pairing region longer by one base pair at the root of the stem structure, thus sequestering the Shine-Dalgarno sequence and decreasing the rate of the translational initiation of FtsH.

Mol Gen Genet, 1997 May 20, 254(5), 548 - 54
HU protein binding to the replication origin of the rolling-circle plasmid pKYM enhances DNA replication; Yasukawa H et al.; The RepK protein, which is encoded by the rolling-circle plasmid pKYM, binds to the PR I site in the pKYM DNA replication origin . We have identified HU as a protein that binds to the PR II and PR III sites in the replication-enhancing region which is downstream of PR I . DNA footprinting assays show that HU binds to these two sites only when RepK is bound to PR I, and that HU also enhances the binding of RepK to PR I . In vivo, pKYM was unable to transform an HU null strain . Two mutant RepK proteins, RepKW179Y, which contains a Trp-to-Tyr exchange at position 179, and RepKD277L, which contains an Asp-to-Leu mutation at residue 277, initiate DNA replication in vivo in the absence of HU . In vitro, these mutant RepK proteins form more stable complexes with the pKYM origin region than does the wild-type RepK protein . These results indicate that HU plays a role in the formation of a stable RepK-origin complex, which is required for the initiation of pKYM DNA replication.

Eur J Pharmacol, 1997 May 20, 326(2-3), 183 - 90
L-canavanine and dexamethasone attenuate endotoxin-induced suppression of ischaemia-reperfusion arrhythmias; Iskit AB et al.; The role of inducible nitric oxide synthase in the antiarrhythmic effects of Escherichia coli endotoxin was examined in an anaesthetised rat model of myocardial ischaemia (7 min occlusion) and reperfusion (7 min) arrhythmias by using its specific blocker L-canavanine (100 mg/kg) and dexamethasone (5 mg/kg), which inhibits its expression . Endotoxin (1 mg/kg) or its solvent saline was administered intraperitoneally 4 h before the occlusion of the left coronary artery and L-canavanine or dexamethasone was administered 1 h before endotoxin or saline injection . The mean arterial blood pressure of rats receiving endotoxin was significantly lower than that of saline-treated controls, and neither L-canavanine nor dexamethasone prevented the hypotension exerted by endotoxin . However, during both the occlusion and reperfusion periods, endotoxin significantly reduced the total number of ectopic beats (e.g., during reperfusion, saline: 1177 +/- 183, n = 11; endotoxin: 248 +/- 91, n = 9; P < 0.005) and the duration of ventricular tachycardia (e.g., during occlusion, saline: 30.9 +/- 5.7 s; endotoxin: 1.8 +/- 0.9 s; P < 0.0001) while L-canavanine or dexamethasone treatment abolished the reduction exerted by endotoxin . Therefore we conclude that endotoxin possesses significant antiarrhythmic (protectant) effects in this rat model of ischaemia-reperfusion arrhythmias, and that its mechanism appears to involve the inducible nitric oxide synthase since both L-canavanine and dexamethasone inhibited this phenomenon.

Biochemistry, 1997 May 20, 36(20), 6230 - 42
Two populations of the estrogen receptor separated and characterized using aqueous two-phase partitioning; Fritsch M et al.; Two populations of the rat uterine estrogen receptor (ER) were separated and characterized using aqueous two-phase partitioning . Countercurrent distribution of rat uterine cytosolic ER allowed rapid and efficient separation of two populations, one population partitioned preferentially into the top phase (T, K(obs) = 3-6) and the other into the bottom phase (B, K(obs) = 0.01-0.03) . The majority of unoccupied cytosolic ER is in the T population . Upon estrogen binding and/or heating to 30 degrees C in vitro the T population is converted to the B population . The transition from T to B does not exclusively involve loss of heat shock protein 90 and does not alter the ligand binding ability of the steroid binding domain . Using the human ER steroid binding domain overproduced in Escherichia coli and the steroid binding domain generated by partial trypsinization of the rat uterine ER, we demonstrate that the characteristic distinguishing T and B populations is not localized to this domain alone but may be associated with the amino terminal half of the ER (the A/B and DNA binding domains) . The T to B transition of the ER also occurs in human MCF-7 breast cancer cells upon treatment with estrogen at 37 degrees C.

Biochemistry, 1997 May 20, 36(20), 6223 - 9
Conditionally lethal Escherichia coli murein mutants contain point defects that map to regions conserved among murein and folyl poly-gamma-glutamate ligases: identification of a ligase superfamily; Eveland SS et al.; Bacterial peptidoglycan biosynthesis includes four enzymatic reactions in which successive amino acid residues are ligated to uridine diphospho-N-acetylmuramic acid (UDP-MurNAc) . By comparing the amino acid sequences of MurC, -D, -E, and -F proteins from various bacterial genera, four regions of homology were identified . A profile search of Swissprot for related sequences revealed that these regional similarities were present in the folyl-gamma-polyglutamate ligases . These sequence homologies appear to track with catalytic function: both enzyme families proceed through an ordered kinetic mechanism and form product via an acyl phosphate intermediate . Two highly conserved residues in region II were examined through site-directed mutagenesis of the murein D-alanyl-D-alanine-adding enzyme from Escherichia coli (murF; E158 and H188) . All mutations were highly detrimental to activity with enzyme specific activity reductions of 200-4500-fold, validating the critical nature of these residues . DNA sequence analysis from three E . coli mutants harboring the murC3 (G344D), murE1 (G344K, A495S), and murF2 (A288T) mutations revealed the presence of point mutation(s) closely associated with the fourth of these aligned regions . The murF2 allele, expressed and purified as a glutathione S-transferase::MurF2 fusion, was 181-fold less catalytically active at 30 degrees C and was further reduced at the nonpermissive temperature (42 degrees C) . Thus the murF2 temperature-sensitive phenotype arises from a point mutation within a highly conserved region within this protein family . These data argue that these proteins comprise a superfamily of three substrate amide ligases that share significant structural and catalytic homologies.

Biochemistry, 1997 May 20, 36(20), 6069 - 79
Misincorporation of dNTPs opposite 1,N2-ethenoguanine and 5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazo{1,2-a}purine in oligonucleotides by Escherichia coli polymerases I exo- and II exo-, T7 polymerase exo-, human immunodeficiency virus-1 reverse transcriptase, and rat polymerase beta; Langouet S et al.; 1,N2-Ethenoguanine (1,N2-epsilon-Gua) and 5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazo{1,2-a}purine (HO-ethanoGua) are two modified bases formed in the reaction of DNA with 2-chlorooxirane, the epoxide derivative of vinyl chloride . The oligonucleotides (19-mers), 5'-CAGTGGGTG*TCCGAATTGA-3', were prepared, with each of these modified bases substituted for G at G* . HO-ethanodeoxyguanosine exists predominantly as a mixture of diastereomers of the closed cyclic hemiaminal form, 5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazo{1,2-a}purine, shown by H2(18)O experiments to be in equilibrium with the open form, N2-(2-oxoethyl)Gua . Both adducts retarded the 3'-extension of a complementary 10-mer primer by all of the polymerases examined, but in every case, some full-length product was obtained . Nucleotide sequence analysis indicated misincorporation of dGTP and dATP across from both 1,N2-epsilon-Gua and HO-ethanoGua, with the extent varying considerably among the polymerases . Similar results were obtained when the abilities of the polymerases to incorporate a single dNTP were evaluated . In addition, -1 and -2 base frame shifts were detected with both 1,N2-epsilon-Gua and HO-ethanoGua with some of the polymerases . Steady-state kinetic experiments with Escherichia coli polymerase I exo- and T7 polymerase exo-/thioredoxin showed large decreases in k(cat) for all dNTP incorporations compared to the normal G x dCTP pair and high misincorporation frequencies for dATP and dGTP with both adducts (compared to dCTP) . Collectively, the results indicate that both of these adducts have considerable miscoding potential with some of these polymerases, that there are a number of differences between the 1,N2-epsilon-Gua and HO-ethanoGua adducts (which formally differ only in the presence of the elements of water), and that misincorporation of dNTPs at a single modified base can vary considerably among different polymerases even in the absence of exonuclease activity.

Biochemistry, 1997 May 20, 36(20), 6059 - 68
Exploiting nucleotide thiophosphates to probe mechanistic aspects of Escherichia coli DNA gyrase; Cullis PM et al.; The interaction of DNA gyrase with ATP has been probed using a range of thiophosphate ATP analogs . ATP gammaS is not detectably hydrolyzed by gyrase but can support limited, probably catalytic, DNA supercoiling . ATP gammaS is a good inhibitor of both ATP hydrolysis and ATP-supported supercoiling . In contrast, both ATP alphaS(Rp) and ATP betaS(Rp) have been shown to be good substrates for the ATPase reaction of gyrase and to support catalytic DNA supercoiling . The corresponding Sp diastereoisomers do not support significant levels of supercoiling and are not readily hydrolyzed, but are shown to be reasonable inhibitors of gyrase . For ATP alphaS(Rp), the supercoiling and ATPase activities appear to be tightly coupled with the thionucleotide being apparently a better substrate than ATP in terms of both DNA supercoiling and nucleotide hydrolysis . In the case of ATP betaS(Rp), DNA supercoiling and nucleotide hydrolysis appear to be uncoupled in that ATP betaS(Rp) is almost as good a substrate as ATP for the ATPase reaction of both intact gyrase and the 43 kDa GyrB fragment, whereas it only supports slow DNA supercoiling; the mechanistic consequences of these observations are discussed in terms of a new model for energy coupling in gyrase . DNA gyrase has been shown to be capable of catalyzing DNA supercoiling in the presence of Mg2+, Ca2+, and Mn2+ but not Zn2+, Co2+, Ni2+, or Cd2+ . The pronounced diastereoselectivity seen in both the DNA supercoiling and ATPase activity with ATP alphaS and ATP betaS together with evidence from the X-ray structure of the 43 kDa GyrB-ADPNP-Mg complex is consistent with metal ion coordination at both of these sites, and probably to the gamma-phosphoryl center during turnover . Thus, the absolute configuration of the catalytically active Mg2+-ATP complex is likely to involve coordination to the pro-S oxygens at both P alpha and P beta, leading to the alpha,beta,gamma-tridentate Mg-ATP complex with the lambda-exo configuration.

Biochemistry, 1997 May 20, 36(20), 6009 - 16
Crystal structure at 2.0 A resolution of phosphoribosyl anthranilate isomerase from the hyperthermophile Thermotoga maritima: possible determinants of protein stability; Hennig M et al.; The structural basis of thermostability of proteins is of great scientific and biotechnological interest . Differences in the X-ray structues of orthologous proteins from hyperthermophilic and mesophilic organisms can indicate crucial stabilizing interactions . To this end the crystal structure of dimeric phosphoribosyl anthranilate isomerase from the hyperthermophile Thermotoga maritima (tPRAI) was determined using phases derived from the isomorphous replacement method and was refined at 2.0 A resolution . The comparison to the known 2.0 A structure of PRAI from Escherichia coli (ePRAI) shows that tPRAI has the complete TIM- or (beta alpha)8-barrel fold, whereas helix alpha5 in ePRAI is replaced by a loop . The subunits of tPRAI associate via the N-terminal faces of their central beta-barrels . Two long, symmetry-related loops that protrude reciprocally into cavities of the other subunit provide for multiple hydrophobic interactions . Moreover, the side chains of the N-terminal methionines and the C-terminal leucines of both subunits are immobilized in a hydrophobic cluster, and the number of salt bridges is increased in tPRAI . These features appear to be mainly responsible for the high thermostability of tPRAI . In contrast to other hyperthermostable enzymes, tPRAI at 25 degrees C is catalytically more efficient than ePRAI, mainly due to its small K(M) value for the substrate {Sterner, R., Kleemann, G . R., Szadkowski, H., Lustig, A., Hennig, M., & Kirschner, K . (1996) Protein Sci . 5, 2000-2008} . The increased number of hydrogen bonds between the phosphate ion and tPRAI compared to ePRAI could be responsible for this effect.

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