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 d