Br J Pharmacol, 1995 Dec, 116(7), 2845 - 51 The multiple organ dysfunction syndrome caused by endotoxin in the rat: attenuation of liver dysfunction by inhibitors of nitric oxide synthase; Thiemermann C et al.; 1 . We have investigated whether (i) endotoxaemia caused by E . coli lipopolysaccharide in the anaesthetized rat causes a multiple organ dysfunction syndrome (MODS; e.g . circulatory failure, renal failure, liver failure), and (ii) an enhanced formation of nitric oxide (NO) due to induction of inducible NO synthase (iNOS) contributes to the MODS . In addition, this study elucidates the beneficial and adverse effects of aminoethyl-isothiourea (AE-ITU), a relatively selective inhibitor of iNOS activity, and NG-methyl-L-arginine (L-NMMA), a non-selective inhibitor of NOS activity on the MODS caused by endotoxaemia . 2 . In the anaesthetized rat, LPS caused a fall in mean arterial blood pressure (MAP) from 117 +/- 3 mmHg (time 0) to 97 +/- 4 mmHg at 2 h (P < 0.05, n = 15) and 84 +/- 4 mmHg at 6 h (P < 0.05, n = 15) . The pressor effect of noradrenaline (NA, 1 micrograms kg-1, i.v.) was also significantly reduced at 1 to 6 h after LPS (vascular hyporeactivity) . Treatment of LPS-rats with AE-ITU (1 mg kg-1, i.v . plus 1 mg kg-1 h-1 starting at 2 h after LPS) caused only a transient rise in MAP, but significantly attenuated the delayed vascular hyporeactivity seen in LPS-rats . Infusion of L-NMMA (3 mg kg-1, i.v . plus 3 mg kg-1 h-1) caused a rapid and sustained rise in MAP and attenuated the delayed vascular hyporeactivity to NA . Neither AE-ITU nor L-NMMA had any effect on either MAP or the pressor effect elicited by NA in rats infused with saline rather than LPS . 3 . Endotoxaemia for 6 h was associated with a significant rise in the serum levels of aspartate or alanine aminotransferase (i.e . GOT or GPT), gamma-glutamyl-transferase (gamma GT), and bilirubin, and hence, liver dysfunction . Treatment of LPS-rats with AE-ITU significantly attenuated this liver dysfunction (rise in GOT, GPT, gamma GT and bilirubin) (P < 0.05, n = 10) . In contrast, L-NMMA reduced the increase in the serum levels of gamma GT and bilirubin, but not in GOT and GPT (n = 5) . Injection of LPS also caused a time-dependent, but rapid (almost maximal at 2 h), increase in the serum levels of urea and creatinine, and hence, renal dysfunction . This renal dysfunction was not affected by either AE-ITU (n = 10) or L-NMMA (n = 5) . In rats infused with saline rather than LPS, neither AE-ITU (n = 4) nor L-NMMA (n = 4) had any significant effect on the serum levels of GOT, GPT, gamma GT, bilirubin, creatinine or urea . 4 . Endotoxaemia for 6 h resulted in a 4.5 fold rise in the serum levels of nitrite (9.13 +/- 0.77 microM, P < 0.01, n = 15), which was significantly reduced by treatment with AE-ITU (6.32 +/- 0.48 microM, P < 0.05, n = 10) or L-NMMA (5.10 +/- 0.40 microM, P < 0.05, n = 5) . In addition, endotoxaemia for 6 h was also associated with a significant increase in iNOS activity in lung and liver homogenates, which was significantly reduced in lung or liver homogenates obtained from LPS-rats treated with either AE-ITU or L-NMMA . 5 . Thus, AE-ITU or L-NMMA (i) inhibits iNOS activity in LPS-rats without causing a significant increase in MAP in rats infused with saline and, hence inhibition of endothelial NOS activity, and (ii) attenuates the delayed circulatory failure as well as the liver dysfunction caused by endotoxaemia in the rat . Thus, an enhanced formation of NO may contribute to the development of liver failure in endotoxic shock. AIDS Res Hum Retroviruses, 1995 Dec, 11(12), 1459 - 65 Genetically engineered HIV type 1 peptides as tools for the determination of anti-V3 loop antibody titers in sera from HIV type 1-infected patients; Von Brunn A et al.; Recombinant peptides from Escherichia coli encoding the principal neutralizing domain (PND) and surrounding sequences of gp120 of human immunodeficiency virus type 1 (HIV-1) with a C-terminal polyhistidine tag were expressed and purified on Ni(2+)-nitrilotriacetate agarose . High yields of more than 99% pure protein were obtained . Their serological reactivity with anti-HIV-positive and -negative human sera was compared to chemically synthesized V3 loop peptides . Overall the genetic PND peptides of the HIV-1MN isolate showed higher and broader reactivity patterns (84%) with HIV-positive sera from German patients than the chemically synthesized peptides (74%) . By their higher reactivity and easy way of production and purification, recombinant peptides seem to be highly preferable for the determination of antibody titers to the PND of HIV-infected patients. Vet Med (Praha), 1995 Dec, 40(12), 365 - 70 {Combined parenteral and oral immunization against enterotoxigenic Escherichia coli diarrhea in weaned piglets}; Alexa P et al.; Experiments were focused on diarrhea prevention in weaned piglets caused by enterotoxigenic strains of Escherichia coli (ETEC) with colonizing factor 8813 . An immunization procedure consisted of intramuscular application of ETEC strain bacterin a day before weaning and a peroral administration of a live culture of nontoxic E . coli strain with the same colonizing factor on the day of weaning . In an experiment on the litter of 10 piglets (six were immunized, four were controls), their intestines were colonized by the nontoxic E . coli strain for 4-7 days (Fig . 1) . The challenge peroral infection by virulent ETEC strain demonstrated the protection of immunized piglets from the disease as well as from intestinal colonization by the administered ETEC strain . The same immunization procedure was tested on three pig farms with enzootic occurrence of diarrheas in weaned piglets . On these farms, besides ETEC strain with colonizing factor 8813 (F18) ETEC strains with other colonizing factors (K88, F not specified) were found out in the weanlings - Tab . I . Immunization effect was evaluated according to the rate of mortality of immunized and nonimmunized piglets within a fortnight after weaning . Out of 222 immunized piglets on S farm (Tab . II), 25 piglets died (11.3%), out of 232 nonimmunized animals it was 39 that died (16.8%) . As for T farm (Tab . III), 22 piglets (8.6%) died out of 255 immunized animals while 71 out of control 274 piglets died (25.7%) . A total of 3,692 were immunized on V farm (Tab . IV) . Ninety-four animals died among them (2.5%) . Mortality rate in the control group of 6,301 animals was 523 piglets (8.3%). Mol Immunol, 1995 Dec, 32(17-18), 1405 - 12 Production and characterization of bispecific single-chain antibody fragments; De Jonge J et al.; We report the construction, expression and purification of a bispecific single-chain Fv antibody fragment produced in Escherichia coli . The protein possesses a dual specificity: the single-chain FvB1 portion is directed to the Idiotype of BCL1 lymphoma cells, the single-chain Fv2C11 moiety binds to the CD3 marker on T cells . The two domains are joined by a flexible peptide linker . Using Immobilized Metal Affinity Chromatography, the recombinant protein was purified from bacterial insoluble membrane fractions . After refolding of the bispecific protein, it was affinity-purified . As demonstrated by flow cytometry, both binding sites are retained in the refolded protein . Retargeted cytotoxicity and T cell proliferation assays further prove the biological activity and specificity of the bispecific single-chain Fv . Thus, these bispecific molecules show a potential anti-tumor activity. Plant Mol Biol, 1995 Dec, 29(6), 1157 - 65 A cDNA clone encoding an IgE-binding protein from Brassica anther has significant sequence similarity to Ca(2+)-binding proteins; Toriyama K et al.; Thirteen cDNA clones encoding IgE-binding proteins were isolated from expression libraries of anthers of Brassica rapa L . and B . napus L . using serum IgE from a patient who was specifically allergic to Brassica pollen . These clones were divided into two groups, I and II, based on the sequence similarity . All the group I cDNAs predicted the same protein of 79 amino acids, while the group II predicted a protein of 83 amino acids with microheterogeneity . Both of the deduced amino acid sequences contained two regions with sequence similarity to Ca(2+)-binding sites of Ca(2+)-binding proteins such as calmodulin . However flanking sequences were distinct from that of calmodulin or other Ca(2+)-binding proteins . RNA-gel blot analysis showed the genes of group I and II were preferentially expressed in anthers at the later developmental stage and in mature pollen . The recombinant proteins produced in Escherichia coli was recognized in immunoblot analysis by the IgE of a Brassica pollen allergic patient, but not by the Ige of a non-allergic patient . The cDNA clones reported here, therefore, represent pollen allergens of Brassica species. Biosci Biotechnol Biochem, 1995 Dec, 59(12), 2351 - 4 Formation of 1:1 complex of the cytokine receptor homologous region of granulocyte colony-stimulating factor receptor with ligand; Hiraoka O et al.; The cytokine receptor homologous (CRH) region of the murine granulocyte colony-stimulating factor (G-CSF) receptor was secreted using a Escherichia coli maltose binding protein (MBP) fusion system . The CRH region was prepared from the periplasmic fraction by G-CSF affinity column chromatography and restriction protease factor Xa digestion, and was purified to homogeneity . The purified CRH region specifically bound G-CSF, with an apparent dissociation constant (kd) of about 1.5 x 10(-9) M . A 1:1 CRH.G-CSF complex was established by gel-filtration high pressure liquid chromatography (HPLC) . However, a 2:1 stoichiometric complex was not established, as in the case of the growth hormone (GH) receptor {Recent Prog . Hormone Res., 48, 233-275 (1993)}. Shock, 1995 Dec, 4(6), 455 - 60 Arteriolar reactivity of endotoxin-tolerant rats after hemorrhage and reinfusion; Baker CH et al.; Adrenergic and endothelium-dependent arteriolar reactivity are greatly reduced in hemorrhagic shock . However, development of tolerance to endotoxin may prevent the decrease . The reactivity of cremaster muscle arterioles was tested in pentobarbital-anesthetized endotoxin-tolerant (ENDT-T) and nontolerant control rats . Tolerance was developed by sublethal intraperitoneal injections of Escherichia coli endotoxin for 4 days (n = 9) . Controls received saline (n = 9) . Mean arterial pressure (MAP), arteriolar diameter-response curves to topical norepinephrine (NE) (10-9M to 10-3M) and responses to 10-3M acetylcholine (ACh) were obtained as follows 1) at control, 2) following hemorrhage to 40 mmHg . 3) after uptake of 25% of bled volume with the remainder infused, and 4) at 240 min post-hemorrhage . The A1, A2, and A3 arterioles were constricted following hemorrhage in the ENDT-T group and in the saline group . After reinfusion and in late shock, vessel diameters remained constricted . MAP increased to control levels (106 +/- 5 and 101 +/- 4 mmHg, respectively) following re-infusion in both groups but in late shock it decreased until death in the nontolerant group and decreased only minimally (96 +/- 4 mmHg) in the ENDT-T group . The nontolerant group NE ED50 increased from pre-hemorrhage to late shock (p < .05) . The ENDT-T group ED50 was unchanged . The bleeding volumes of the two groups were not different . The survival time of the nontolerant group was 234 +/- 36 min, whereas the ENDT-T group all survived and were sacrificed at 427 +/- 30 min . The response to endothelium-dependent ACH vasodilation in late shock was significantly reduced in the saline group but was unchanged in the ENDT-T group . Alpha 1 receptor activity was maintained in both groups . Alpha 2 receptor activity was attenuated pre-hemorrhage and at 240 min post-hemorrhage in ENDT-T rats . In late shock, alpha 2 receptor activity was attenuated in nontolerant rats . The development of endotoxin tolerance prevents the loss of arteriolar responsiveness to NE and ACh . ENDT-T rats have attenuated alpha 2 receptor activity but not alpha 1 receptor activity. J Dent Res, 1995 Dec, 74(12), 1837 - 44 Functional comparison of native and recombinant human salivary histatin 1; Driscoll J et al.; Histatin 1 is a histidine-rich phosphoprotein present in human parotid saliva that possesses candidacidal activity and functions in mineralization by adsorbing to hydroxyapatite . The objective of the present study was to develop a system for recombinant production of histatin 1 and to examine the role of phosphorylation in the functional activities of this molecule . Native histatin 1 (containing a phosphoserine at residue 2) was purified from parotid saliva, whereas a bacterial expression system was used to produce a recombinant form of histatin 1 (re-Hst1) that lacked phosphorylated serine . Histatin 1 cDNA was inserted into the vector pGEX-3X, which expresses foreign genes as soluble fusion proteins attached to the carboxyl-terminus of glutathione S-transferase (GST) . The GST/re-Hst1 fusion protein was isolated from cell lysates by affinity chromatography on glutathione (GSH)-Sepharose and digested with cyanogen bromide to separate re-Hst1 from the GST fusion partner . The digest was subjected to reversed-phase high-performance liquid chromatography on a C18 column, and re-Hst1 was eluted as a well-defined peak . The yield of re-Hst1 was 4 mg/L of bacterial culture . Amino-terminal sequencing and amino acid analysis confirmed the final product as re-Hst1 . Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that native histatin 1 and re-Hst1 had the same apparent molecular weights, while cationic PAGE showed that re-Hst1 was more basic . Phosphate analysis indicated 1 mol phosphate/mol of native histatin 1, while re-Hst1 lacked any detectable phosphate . Re-Hst1 demonstrated candidacidal activity comparable to that of native histatin 1, but displayed substantially lower binding to hydroxyapatite . These results show that phosphorylation of histatin 1 at residue 2 contributes significantly to its ability to bind to hydroxyapatite. Biophys J, 1995 Dec, 69(6), 2703 - 9 Structures of revertant signal sequences of Escherichia coli ribose binding protein; Chi SW et al.; Recently we reported (Yi et al., 1994) that the alpha-helical content of the signal peptide of Escherichia coli ribose binding protein, when determined by circular dichroism (CD) and two-dimensional NMR in trifluoroethanol/water solvent, is higher than that of its nonfunctional mutant signal peptide . In the present investigation, the structures of the signal peptides of two revertant ribose binding proteins in the same solvent were also determined with CD and two-dimensional 1H NMR spectroscopy . According to the CD results, both of these revertant signal peptides showed an intermediate helicity between those of wild-type and mutant signal peptides, the helical content of the revertant peptide with higher recovery of the translocation capability being higher . On the other hand, the alpha-helix regions of the wild-type and the revertant peptides as determined by NMR were shown to be the same . This discrepancy may be due to the difference in stability between identical alpha-helical stretches in wild-type and revertant peptides . A good correlation was observed between the helical content of these four ribose binding protein signal peptides in TFE/water as studied by CD and their in vivo translocation activities . It appears, therefore, that both the proper length of the helix and the stability are of functional significance. Appl Microbiol Biotechnol, 1995 Dec, 44(3-4), 484 - 8 Protein substrates and heat shock reduce the DNA-binding ability of Escherichia coli Lon protease; Sonezaki S et al.; Interaction between the fusion protein MBP-Lon, formed by maltose-binding protein and Lon protease, and the plasmid pBR322 was studied to clarify the DNA-binding behavior of the Lon protease . Since the MBP-Lon fusion protein that was bound to the plasmid was strongly adsorbed by amylose resin, complex formation and dissociation were determined by quantifying the unadsorbed plasmid using agarose gel electrophoresis . The autolysis of MBP-Lon fusion protein was suppressed when the protein was bound to the plasmid . The plasmid was completely dissociated from MBP-Lon fusion protein by the addition of the protein substrates of Lon protease (i.e . alpha-casein and denatured bovine serum albumin) . In addition, at high temperatures, MBP-Lon fusion protein lost its plasmid-binding ability, although it fully retained ATP-dependent protease activity . These results suggest that Lon protease loses DNA-binding ability when cells are exposed to abnormal conditions and the amount of damaged proteins increases . On the other hand, DNA probably plays an important role in controlling the Lon protease activity in cells under normal conditions by entrapping the enzyme. Appl Microbiol Biotechnol, 1995 Dec, 44(3-4), 425 - 31 Cloning, characterization and overproduction of nuclease S1 gene (nucS) from Aspergillus oryzae; Lee BR et al.; The nuclease S1 gene (nucS) from Aspergillus oryzae was isolated using a polymerase-chain-reaction-amplified DNA fragment as a probe, and a 2.6-kb SalI-EcoRI fragment containing the nucS gene was sequenced . It was deduced that the nucS gene had two short introns, 49 and 50 nucleotides in length . The nucS gene had an open-reading frame of 963 base pairs and coded for a protein of 287 amino acid residues, comprising the signal peptide of 20 amino acids and a mature protein of 267 amino acids . The deduced amino acid sequence agreed well with the published amino acid sequence except for one substitution . Southern hybridization analysis showed that the nucS gene existed as a single copy in the A . oryzae chromosome . When the structural gene of nucS was fused with the promoter of the glaA gene and introduced into A . oryzae, the yield of secreted nuclease S1 increased about 100-fold compared with the recipient strain. Anat Rec, 1995 Dec, 243(4), 466 - 78 Surface coat of sheep pulmonary intravascular macrophages: reconstitution, and implication of a glycosyl-phosphatidylinositol anchor; Singh B et al.; BACKGROUND: Pulmonary intravascular macrophages (PIMs) of sheep have a globular surface coat that facilitates endocytosis of tracer particles and Escherichia coli lipopolysaccharide, and is disrupted by the heparin and Brefeldin A treatments . The present study investigated the in vivo dynamics of the coat globules following heparin-mediated removal, and the mechanism of globule organization on the plasma membrane of PIMs in vitro . METHODS: Sheep were administered heparin at a dose of 50 IU/kg body weight IV, and euthanised at 30 min, 3, 6, 12, 48, and 120 hr (n = 2 for each treatment) after the treatment . Control sheep (n = 2) were injected with normal saline solution . The tissues were processed for an ultrastructural examination and acid phosphatase (ACPase) cytochemistry . Heparin-treated lungs were subjected to morphometric analysis of the coat globules . Lung tissues from normal sheep (n = 2) were incubated with phosphatidylinositol-specific-phospholipase C (PIPLC; 2 IU/ml PBS) in vitro for 30 and 75 min . RESULTS: Heparin study: The ultrastructural and morphometric data showed that the coat globules were removed at 30 min and reconstituted within 48 hr of the treatment . The PIMs showed prominent Golgi complexes associated with secretory vesicles, microtubules, and centriole between 3-12 hr of heparin treatment . Acid phosphatase cytochemistry also demonstrated secretory activity in the Golgi complexes of PIMs during the coat reconstitution . PIPLC study: The coat globules of PIMs were removed in a time-dependent mode by the PIPLC treatment without damage to other cell organelles . CONCLUSIONS: This study demonstrates a time-dependent reconstitution of the coat of PIMs in conjunction with secretory activity following heparin-mediated removal, probably through sequestration of the globules from blood . This ability is of functional significance as the coat mediates particle endocytosis by the PIMs . The results also suggest the presence of a glycosyl-phosphatidylinositol (GPI) anchor in tethering of globules on the plasma membrane of PIMs to offer a structural basis for their integrity in pulmonary vascular flow. Toxicol Lett, 1995 Dec, 82-83, 577 - 89 Multidimensional NMR spectroscopy of DNA-binding proteins: structure and function of a transcription factor; Hsu VL et al.; The solution structure of a type II DNA-binding protein (DBPII), transcription factor 1 (TF1), has been determined using NMR spectroscopy . A multidimensional, heteronuclear strategy was employed to overcome assignment ambiguities due to resonance overlap and broadened crosspeaks . This approach involved the use of selectively deuteriated, 13C- and 15N-labeled samples and 'isotopic heterodimers' to distinguish between intra- and intermonomeric NOEs . A comparison with the crystal structure and NMR analysis of the E . coli HU protein suggests that other homologous proteins in this family will possess similar tertiary structures . This NMR strategy is applicable to the study of other proteins and their biomolecular complexes. Curr Genet, 1995 Dec, 29(1), 88 - 95 Transformation of Sordaria macrospora to hygromycin B resistance: characterization of transformants by electrophoretic karyotyping and tetrad analysis; Walz M et al.; The ascomycete Sordaria macrospora was transformed using different plasmid molecules containing the bacterial hygromycin B resistance gene (hph) under the control of different expression signals . The highest transformation frequency was obtained with vector pMW1 . On this plasmid molecule, expression of the hph gene is directed by the upstream region of the isopenicillin N synthetase gene (pcbC) from the deuteromycete Acremonium chrysogenum . Southern analysis suggests that the vector copies are integrated as tandem repeats into the S . macrospora chromosomes and that duplicated sequences are most probably not inactivated by methylation during meiosis . Furthermore, the hygromycin B resistance (hygR) is not correlated with the number of integrated vector molecules . Electrophoretic karyotyping was used to further characterize S . macrospora transformants . Five chromosomal bands were separated by pulsed-field gel electrophoresis (PFGE) representing seven chromosomes with a total genome size of 39.5Mb . Hybridization analysis revealed ectopic integration of vector DNA into different chromosomes . In a few transformants, major rearrangements were detected . Transformants were sexually propagated to analyze the fate of the heterologous vector DNA . Although the hygR phenotype is stably maintained during mitosis, about a third of all lines tested showed loss of the resistance marker gene after meiosis . However, as was concluded from electrophoretic karyotyping, the resistant spores showed a Mendelian segregation of the integrated vector molecules in at least three consecutive generations . Our data indicate that heterologous marker genes can be used for transformation tagging, or the molecular mapping of chromosomal loci in S . macrospora. Curr Genet, 1995 Dec, 29(1), 66 - 72 Integrative and replicative genetic transformation of Aureobasidium pullulans; Thornewell SJ et al.; A hybrid selectable marker for transformation was constructed by placing the promoter (TEF1p) from the gene encoding the Aureobasidium pullulans translation elongation factor 1-alpha (TEF1) adjacent to the 5' end of the Escherichia coli hygromycin B phosphotransferase gene (HPT) . Plasmids containing this hybrid gene (TEF1p/HPT) transformed A . pullulans strain R106 to a hygromycin B-resistant (HmBR) phenotype . A PCR-generated DNA fragment consisting of the TEF1p/HPT resistance marker flanked by 41bp of homologous DNA has also been shown to transform A . pullulans to HmBR . Linearized plasmid DNA consistently produced more transformants than circular plasmid DNA . Analyses of 23 HmBR transformants revealed integration of the plasmid in only eight of these transformants . In two transformants, integration into the largest chromosome (VIII) resulted in an alteration of the molecular karyotype . In four other transformants, integration occurred in chromosome VI (the chromosome containing TEF1) but only one was the result of homologous recombination with the genomic copy of the TEF1 promoter . The remainder of the transformants contained replicative plasmids that could be visualized on an agarose gel by ethidium bromide staining . These plasmids were generally 7-8kb in size . One transformant appeared to contain four plasmids ranging in size from 4 to 8kb, suggesting rearrangement of the transforming DNA . One plasmid obtained from a HmBR A . pullulans transformant was able to transform E . coli to ampicillin resistance . However, after recovery from E . coli, this plasmid (approximately 4kb) was unable to transform A . pullulans to HmBR. RNA, 1995 Dec, 1(10), 1018 - 28 The ribosomal environment of tRNA: crosslinks to rRNA from positions 8 and 20:1 in the central fold of tRNA located at the A, P, or E site; Rinke-Appel J et al.; The naturally occurring nucleotide 3-(3-amino-3-carboxy-propyl) uridine ("acp3U") at position 20:1 of lupin tRNAMet was coupled to a photoreactive diazirine derivative . Similarly, the 4-thiouridine at position 8 of Escherichia coli tRNAPhe was modified with an aromatic azide . Each of the derivatized tRNAs was bound to E . coli ribosomes in the presence of suitable mRNA analogues, under conditions specific for the A, P, or E sites . After photoactivation of the diazirine or azide groups, the sites of crosslinking from the tRNAs to 16S or 23S rRNA were analyzed by our standard procedures, involving a combination of ribonuclease H digestion and primer extension analysis . The crosslinked ribosomal proteins were also identified . The results for the rRNA showed a well-defined series of crosslinks to both the 16S and 23S molecules, the most pronounced being (1) an entirely A-site-specific crosslink from tRNA position 20:1 to the loop-end region (nt 877-913) of helix 38 of the 23S RNA (a region that has not so far been associated at all with tRNA binding), and (2) a largely P-site-specific crosslink from tRNA position 8 to nt 2111-2112 of the 23S RNA (nt 2112 being a position that has previously been identified in footprinting studies as belonging to the ribosomal E site) . The data are compared with results from a parallel study of crosslinks from position 47 (also in the central fold of the tRNA), as well as with previously published crosslinks from the anticodon loop (positions 32, 34, and 37) and the CCA-end region (position 76, and the aminoacyl residue). Am J Physiol, 1995 Dec, 269(6 Pt 2), H2090 - 9 Select dietary fatty acids attenuate cardiopulmonary dysfunction during acute lung injury in pigs; Murray MJ et al.; We examined the effect of substituting linoleic acid (LA) with eicosapentaenoic acid (EPA) and gamma-linolenic acid (gamma-LA), precursors of trienoic and monoenoic eicosanoids, respectively, on acute lung injury (ALI) . Three groups (n = 8/group) of pigs were fed enteral diets containing LA (diet A), EPA (diet B), or EPA+gamma-LA (diet C) for 8 days . ALI was then induced with a 0.1 mg/kg bolus of Escherichia coli endotoxin followed by a continuous infusion for 4 h (0.075 mg.kg-1.h-1) . Pulmonary arterial and capillary wedge pressures, cardiac index (CI), arterial blood gases, arterial O2 content, and plasma thromboxane B2 (TxB2) were measured . Arterial PO2 decreased at 20 min in animals fed diet A . This change was attenuated with diets B and C . The EPA- and EPA + gamma-LA-enriched diets attenuated the fall in O2 delivery at 20 min, an improvement that was sustained throughout the 4-h study period with the EPA+gamma-LA-enriched diet only . This improvement in O2 delivery was due not only to the improved arterial PO2, but also to the maintenance of CI at 20 min in animals fed diets B and C and throughout the 4-h study period in animals fed diet C . At 4 h, TxB2 increased 10-fold over baseline in animals fed diet A, whereas in animals fed diets B and C the increase was only 3-fold . These decreased TxB2 levels in animals fed diets B and C correlate with an attenuation in the increase in pulmonary vascular resistance that was observed at 20 min after endotoxin infusion in animals fed diet A . These data suggest that specialized enteral diets enriched in EPA+gamma-LA improve gas exchange and O2 delivery, presumably in part through a modification of TxB2 production with a decrease in pulmonary vascular resistance and an increase in CI, during ALI. FEMS Microbiol Lett, 1995 Dec 1, 134(1), 39 - 44 Role of rpoS in the regulation of glutathione oxidoreductase (gor) in Escherichia coli; Becker-Hapak M et al.; RpoS (sigma-38) is the major regulator of genes for survival of Escherichia coli in the stationary phase . OxyR is a transcriptional regulator that responds to H2O2 induced stress in exponential phase . Once considered to act independently of each other, they are now known to be integrally involved in the expression of several oxidative stress genes . While it is known that in the exponential phase, OxyR is the transcriptional regulator of gor, this study has shown that RpoS regulates gor in the stationary phase . beta-Galactosidase activity of a gor::lacZ promoter fusion showed no induction in a oxyR rpoS double mutant . Challenge of a gor mutant to several oxidants showed that the gene product was not functioning as a classic antioxidant. Mol Biol Cell, 1995 Dec, 6(12), 1875 - 85 Nuclear and nucleolar targeting of human ribosomal protein S6; Schmidt C et al.; Chimeric proteins were constructed to define the nuclear localization signals (NLSs) of human ribosomal protein S6 . The complete cDNA sequence, different cDNA fragments and oligonucleotides of the human ribosomal proteins S6, respectively, were joined to the 5' end of the entire LacZ gene of Escherichia coli by using recombinant techniques . The hybrid genes were transfected into L cells, transiently expressed, and the intracellular location of the fusion proteins was determined by their beta-galactosidase activity . Three NLSs were identified in the C-terminal half of the S6 protein . Deletion mutagenesis demonstrated that a single NLS is sufficient for targeting the corresponding S6-beta-galactosidase chimera into the nucleus . Removal of all three putative NLSs completely blocked the nuclear import of the resulting S6-beta-galactosidase fusion protein, which instead became evenly distributed in the cytoplasm . Chimeras containing deletion mutants of S6 with at least one single NLS or unmodified S6 accumulated in the nucleolus . Analysis of several constructs reveals the existence of a specific domain that is essential but not sufficient for nucleolar accumulation of S6. Melanoma Res, 1995 Dec, 5(6), 403 - 11 Generation and selection of monoclonal antibodies, single-chain Fv and antibody fusion phage specific for human melanoma-associated antigens; Kupsch JM et al.; A panel of 13 murine monoclonal antibodies (mAbs) recognizing antigens on human melanoma cells but not on melanocytes was generated . Two mAbs (LHM3 and LHM5) stained sections of melanoma but not normal tissues . mAbs LHM2 and LHM8 stained only a minority of normal tissues . The mAbs differed further in their staining patterns on melanoma cell lines HMB2, DX3 and SK23 in FACS . The mAbs recognize antigens of 34, 38, 57, 94, 190-200 and > 200 kD . One mAb each bound to each of the antigens HLA DR (LHM4) and high molecular weight proteoglycan (LHM2) . The high molecular weight proteoglycan-specific mAb was used to construct a single-chain Fv (scFv) antibody fragment and an antibody fusion phage in Escherichia coli . Both the scFv and the fusion phage were shown to bind specifically to melanoma cells . A method for the selection of melanoma cell-binding phages from phage libraries is described. Arch Microbiol, 1995 Dec, 164(6), 396 - 405 Expression of the cbbLcbbS and cbbM genes and distinct organization of the cbb Calvin cycle structural genes of Rhodobacter capsulatus; Paoli GC et al.; Rhodobacter capsulatus fixes CO2 via the Calvin reductive pentose phosphate pathway and, like some other nonsulfur purple bacteria, is known to synthesize two distinct structural forms of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) . Cosmid clones that hybridized to form I (cbbLcbbS) and form II (cbbM) RubisCO gene probes were isolated from a genomic library of R . capsulatus strain SB1003 . Southern blotting and hybridization analysis with gene-specific probes derived from Rhodobacter sphaeroides revealed that R . capsulatus cbbM is clustered with genes encoding other enzymes of the Calvin cycle, including fructose 1,6/sedoheptulose 1,7-bisphosphatase (cbbF), phosphoribulokinase (cbbP), transketolase (cbbT), glyceraldehyde-3-phosphate dehydrogenase (cbbG), and fructose 1,6-bisphosphate aldolase (cbbA), as well as a gene (cbbR) encoding a divergently transcribed LysR-type regulatory protein . Surprisingly, a cosmid clone containing the R . capsulatus form I RubisCO genes (cbbL and cbbS) failed to hybridize to the other cbb structural gene probes, unlike the situation with the closely related organism R . sphaeroides . The form I and form II RubisCO genes were cloned into pUC-derived vectors and were expressed in Escherichia coli to yield active recombinant enzyme in each case . Complementation of a RubisCO-deletion strain of R . sphaeroides to photosynthetic growth by R . capsulatus cbbLcbbS or cbbM was achieved using the broad host-range vector, pRK415, and R . sphaeroides expression vector pRPS-1. J Mol Evol, 1995 Dec, 41(6), 703 - 11 Partition of aminoacyl-tRNA synthetases in two different structural classes dating back to early metabolism: implications for the origin of the genetic code and the nature of protein sequences; Delarue M; We describe, on the molecular level, a possible fuzzy and primordial translation apparatus capable of synthesizing polypeptides from nucleic acids in a world containing a mixture of coevolving molecules of RNA and proteins already arranged in metabolic cycles (including cofactors) . Close attention is paid to template-free systems because they are believed to be the immediate ancestors of this primordial translation apparatus . The two classes of aminoacyl-tRNA synthetases (aaRSs), as seen today, are considered as the remnants of such a simple imprecise translation apparatus and are used as guidelines for the construction of the model . Earlier theoretical work by Bedian on a related system is invoked to show how specificity and stability could have been achieved automatically and rather quickly, starting from such an imprecise system, i.e., how the encoded synthesis of proteins could have appeared . Because of the binary nature of the underlying proto-code, the first genetically encoded proteins would then have been alternating copolymers with a high degree of degeneracy, but not random . Indeed, a clear signal for alternating hydrophobic and hydrophilic residues in present-day protein sequences can be detected . Later evolution of the genetic code would have proceeded along lines already discussed by Crick . However, in the initial stages, the translation apparatus proposed here is in fact very similar to the one postulated by Woese, only here it is given a molecular framework . This hypothesis departs from the paradigm of the RNA world in that it supposes that the origin of the genetic code occurred after the apparition of some functional (statistical) proteins first . Implications for protein design are also discussed. J Mol Evol, 1995 Dec, 41(6), 1048 - 56 Glutathione S-transferase and S-crystallins of cephalopods: evolution from active enzyme to lens-refractive proteins; Tomarev SI et al.; Our previous studies have shown that the S-crystallins of cephalopod (Ommastrephes sloani pacificus) eye lenses comprise a family of at least ten members which are evolutionarily related to glutathione S-transferase (GST, EC 2.5.1.18) . Here we show by cDNA cloning that there are at least 24 different S-crystallins that are 46-99% identical to each other by amino acid sequence in the squid Loligo opalescens . In each species, all but one S-crystallin (SL11 in O . pacificus and Lops4 in L . opalescens) examined has an inserted central peptide of variable length and sequence . cDNA expression studies conducted in Escherichia coli showed that squid GST (which is expressed little in the lens) has very high enzymatic activity using 1-chloro-2, 4-dinitrobenzene (CDNB) as a substrate; by contrast, SL20-1 of O . pacificus and Lops12 of L . opalescens (which are encoded by abundant lens mRNAs) have no GST activity . Interestingly, SL11 and Lops4 have some enzymatic activity with the CDNB substrate . Site-specific mutations at Y7 or W38, both residues essential for activity of vertebrate GSTs, or insertion of the central peptide present in the inactive SL20-1, reduced the specific activity of squid GST by 30- to 100-fold . These data indicate that the S-crystallins consist of a family of enzymatically inactive proteins (when using CDNB as a substrate) which is considerably larger than previously believed and that GST activity was lost by gradual drift in sequence as well as by insertion of an extra peptide by exon shuffling . The results are also consistent with the idea that SL11 and Lops4 are orthologous crystallins representing the first descendants of the ancestral GST gene in the pathway which gave rise to the extensive S-crystallin family of lens proteins. J Clin Microbiol, 1995 Dec, 33(12), 3174 - 8 Clonal relatedness of Shiga-like toxin-producing Escherichia coli O101 strains of human and porcine origin; Franke S et al.; Shiga-like toxin (SLT)-producing Escherichia coli (SLTEC) O101 has recently been associated with hemorrhagic colitis and hemolytic-uremic syndrome in humans . In this study, SLTEC O101 strains from humans and pigs were characterized for clonal relatedness by nucleotide sequence analysis of their slt genes, DNA finger-printing of genomic DNA, and determination of virulence factors . The slt genes of five E . coli O101 strains were cloned and sequenced . For all strains, the deduced amino acid sequences of the B subunits were identical to those of the SLT-IIe present in the classical SLTEC O139 strains that cause edema disease in pigs . The A subunit revealed more than 99% homology to that of SLT-IIe . DNA fingerprinting revealed a high degree of genetic relatedness between the human and porcine O101 isolates . None of the O101 strains investigated had virulence factors frequently found in porcine (F107 fimbriae or heat-stable or heat-labile enterotoxins) or human SLTEC strains (eaeA or enterohemorrhagic E . coli hemolysin) . The absence of virulence factors typical of SLT-I- and SLT-II-producing E . Coli together with the presence of SLT-IIe, a toxin previously seen only in porcine E . coli, suggests a new pathogenic mechanism for E . coli O101 infection of humans . For diagnostic purposes, we recommend the use of PCR primers and DNA probes complementary to slt-IIe to correctly identify such strains and to further evaluate their role in human diseases. Plant J, 1995 Dec, 8(6), 963 - 72 Isolation of two novel myb-like genes from Arabidopsis and studies on the DNA-binding properties of their products; Li SF et al.; Two novel myb-like genes (atmyb6 and atmyb7) were isolated from an Arabidopsis thaliana cDNA library . The entire proteins or the Myb domains encoded by the genes were expressed as fusion proteins in Escherichia coli . The DNA-binding domain of the murine c-Myb was also expressed in the same way for use in comparative studies . The fusion proteins were examined for their DNA-binding activity using the animal c-Myb DNA-binding site (MBS) and the binding site of the maize P gene product (PBS) . The Myb domain of Atmyb6 bound to PBS more efficiently than to MBS . Complete Atmyb6 and Atmyb7 proteins preferentially bound to PBS but not MBS . This suggests that the in vitro binding consensus sequences for both Atmyb6 and Atmyb7 are similar to PBS . The binding of the Myb domain of Atmyb6 to both PBS and MBS raises the possibility that the protein recognizes multiple sequences in vivo . The third alpha-helix and three adjacent amino acids in the third repeat (R3) of c-Myb was replaced with the analogous sequence of Atmyb6 to create a chimeric Myb protein . This chimeric protein bound to PBS with a low affinity but failed to bind to MBS . Thus the binding pattern of the chimeric Myb protein is similar to that of the Atmyb6 . This result suggests that the last 20 amino acids in the R3 repeat of Atmyb6 play a major role in DNA-binding. Insect Biochem Mol Biol, 1995 Dec, 25(10), 1093 - 100 Expression and characterization of recombinant Manduca sexta serpin-1B and site-directed mutants that change its inhibitory selectivity; Jiang H et al.; Hemolymph of Manduca sexta contains a number of serine proteinase inhibitors from the serpin superfamily . During formation of a stable complex between a serpin and a serine proteinase, the enzyme cleaves a specific peptide bond in an exposed loop (the reactive-site region) at the surface of the serpin . The amino acid residue on the amino-terminal side of this scissile bond, the P1 residue, is important in defining the selectivity of a serpin for inhibiting different types of serine proteinases . M . sexta serpin-1B, with alanine at the position predicted from sequence alignments to be the P1 residue, was previously named alaserpin . This alanyl residue was changed by site-directed mutagenesis to lysine (A343K) and phenylalanine (A343F) . The serpin-1B cDNA and its mutants were inserted into an expression vector, H6pQE-60, and the serpin proteins were expressed in Escherichia coli . Affinity-purified recombinant serpins selectively inhibited mammalian serine proteinases: serpin-1B inhibited elastase; serpin-1B(A343K) inhibited trypsin, plasmin, and thrombin; serpin-1B(A343F) inhibited chymotrypsin as well as trypsin . All three serpins inhibited human cathepsin G . This insect serpin and its site-directed mutants associated with mammalian serine proteinases at rates similar to those reported for mammalian serpins . Serpin-1B and its mutants formed SDS-stable complexes with the enzymes they inhibited . The scissile bond was determined to be between residues 343 and 344 in wild-type serpin-1B and in serpin-1B with mutations at residue 343 . These results demonstrate that the P1 alanine residue defines the primary selectivity of serpin-1B for elastase-like enzymes, and that this selectivity can be altered by mutations at this position. Protein Sci, 1995 Dec, 4(12), 2616 - 8 Recombinant protein sequences can trigger methylation of N-terminal amino acids in Escherichia coli; Apostol I et al.; Recombinant human hemoglobin rHb1.1 has been genetically engineered with the replacement of the wild-type valine residues at all N-termini with methionine, an Asn 108 Lys substitution on the beta globins, and a fusion of the two alpha globins with a glycine linker . When rHb1.1 was expressed in Escherichia coli, methylation of the N-terminal methionine of the alpha globin was discovered . Another mutant has been engineered with the alpha globin gene coding for N-terminal methionine followed by an insertion of alanine . Characterization of expressed hemoglobin from this variant revealed a methylated N-terminal alanine that occurred through two posttranslational events: initial excision of the N-terminal methionine, followed by methylation of alanine as the newly generated N-terminus . No methylation was observed for variants expressed with wild-type valine at the N-terminus of the alpha globin . The methylation of N-terminal amino acids was attributed to a specific protein sequence that can trigger methylation of proteins expressed in E . coli . Here we demonstrate that proline at position 4 in the protein sequence of alpha globin seems an essential part of that signaling . Although N-terminal methylation has been observed previously for native E . coli proteins with similar N-terminal sequences, methylation of the recombinant globins has allowed further delineation of the recognition sequence, and indicates that methylation of heterologous proteins can occur in E . coli. Protein Sci, 1995 Dec, 4(12), 2587 - 93 Nuclear magnetic resonance characterization of the N-terminal thioredoxin-like domain of protein disulfide isomerase; Kemmink J et al.; A genetically engineered protein consisting of the 120 residues at the N-terminus of human protein disulfide isomerase (PDI) has been characterized by 1H, 13C, and 15N NMR methods . The sequence of this protein is 35% identical to Escherichia coli thioredoxin, and it has been found also to have similar patterns of secondary structure and beta-sheet topology . The results confirm that PDI is a modular, multidomain protein . The last 20 residues of the N-terminal domain of PDI are some of those that are similar to part of the estrogen receptor, yet they appear to be an intrinsic part of the thioredoxin fold . This observation makes it unlikely that any of the segments of PDI with similarities to the estrogen receptor comprise individual domains. Protein Sci, 1995 Dec, 4(12), 2510 - 6 Conservative substitutions in the hydrophobic core of Rhodobacter sphaeroides thioredoxin produce distinct functional effects; Assemat K et al.; The internal residue Phe 25 in Rhodobacter sphaeroides thioredoxin was changed to five amino acids (Ala, Val, Leu, Ile, Tyr) by site-directed mutagenesis, and the mutant proteins were characterized in vitro and in vivo using the mutant trxA genes in an Escherichia coli TrxA- background . The substitution F25A severely impaired the functional properties of the enzyme . Strains expressing all other mutations can grow on methionine sulfoxide with growth efficiencies of 45-60% that of the wild type at 37 degrees, and essentially identical at 42 degrees . At both temperatures, however, strains harboring the substitutions F25V and F25Y had lower growth rates and formed smaller colonies . In another in vivo assay, only the wild type and the F25I substitution allowed growth of phage T3/7 at 37 degrees, demonstrating that subtle modifications of the protein interior at position 25 Ile/Leu or Phe/Tyr) can produce significant biological effects . All F25 mutants were good substrates for E . coli thioredoxin reductase . Although turnover rates and apparent Km values were significantly lower for all mutants compared to the wild type, catalytic efficiency of thioredoxin reductase was similar for all substrates . Determination of the free energy of unfolding showed that the aliphatic substitutions (Val, Leu, Ile) significantly destabilized the protein, whereas the F25Y substitution did not affect protein stability . Thus, thermodynamic stability of R . sphaeroides thioredoxin variants is not correlated with the distinct functional effects observed both in vivo and in vitro. Appl Biochem Biotechnol, 1995 Dec, 55(3), 167 - 74 Preparation of Tyr-C-peptide from genetically altered human insulin precursor; Sun H et al.; C-peptide radioimmunoassay (C-peptide RIA) is widely used in determination of pancreatic B-cell secretion activity . 125I labeled Tyr-C-peptide is indispensable in C-peptide RIA kit . Herein we discuss a way of obtaining recombinant Tyr-C-peptide . Arg32Tyr human pro-insulin mutant (R32Y-proinsulin) gene was constructed by site-directed mutagenesis and overexpressed in Escherichia coli . Purified R32Y-proinsulin was converted to insulin and Tyr-C-peptide by trypsin and carboxypeptidase B codigestion . Tyr-C-peptide was isolated through reverse-phase HPLC (RP-HPLC) and identified by C-peptide RIA and amino acid analysis. Development, 1995 Dec, 121(12), 4371 - 82 Early even-skipped stripes act as morphogenetic gradients at the single cell level to establish engrailed expression; Fujioka M et al.; even-skipped (eve) has been proposed to set up parasegment borders at the anterior edge of each of its seven stripes by providing a sharp expression boundary, where engrailed is activated on one side and wingless on the other . By expressing bell-shaped early eve stripes without the sharp boundary provided by narrow, late stripes, we find that the early gradient is sufficient for generating stable parasegment borders . Based on several lines of evidence, we propose that the anterior portion of each early stripe has morphogenic activity, repressing different target genes at different concentrations . These distinct repression thresholds serve to both limit and subdivide a narrow zone of paired expression . Within this zone, single cell rows express either engrailed, where runt and sloppy-paired are repressed, or wingless, where they are not . While the early eve gradient is sufficient to establish parasegmental borders without refined, late expression, late eve expression has a role in augmenting this boundary to provide for strong, continuous stripes or engrailed expression . In addition, we show that the early eve gradient is sufficient, at its posterior edge, for subdividing the ftz domain into engrailed expressing and non-expressing cells. Development, 1995 Dec, 121(12), 4303 - 8 Segmental patterning of heart precursors in Drosophila; Lawrence PA et al.; The mesoderm of Drosophila embryos is segmented; for instance there are segmentally arranged clusters of cells (some of which are heart precursors) that express even-skipped . Expression of even-skipped depends on Wingless, a secreted molecule . In principle, Wingless could act directly in the mesoderm or it could induce the pattern after crossing from ectoderm to mesoderm . Using mosaic embryos, we show that Wingless produced in the mesoderm is sufficient for even-skipped expression . This proves that induction is not essential . However, induction can occur: when patches of wingless mutant mesoderm are overlaid by wild-type ectoderm, they do express even-skipped . We therefore believe that Wingless from both the ectoderm and mesoderm may contribute to patterning the mesoderm . Using the UAS/Gal4 system, we made embryos in which the Wingless protein is uniformly expressed . This is sufficient to rescue the repeated clusters of even-skipped expressing cells, although they are enlarged . We conclude that the mesoderm is segmented in some way not dependent on the distribution of Wingless, suggesting a more permissive and less instructive role for the protein in this instance. Microbiology, 1995 Dec, 141 ( Pt 12), 3119 - 26 Starvation yields a drastic decrease in outer-membrane permeability to a periplasmic foreign protein in Myxococcus xanthus; Laval-Favre K et al.; A recombinant Myxococcus xanthus strain was constructed that constitutively produces two proteins from Escherichia coli, the cytoplasmic beta-galactosidase and the periplasmic pH 2.5 acid phosphatase (AppA protein) . We have previously shown that during vegetative growth, AppA protein is partly accumulated in the periplasm of M . xanthus and partly released into the medium . We demonstrate here that during starvation-induced development, release of periplasmic AppA protein to the medium did not occur over a period of 20 h . This was coincident with, but not caused by, the arrest of the synthesis of the foreign proteins . We have shown that this lack of secretion could be attributed to starvation per se and did not depend on the ability of the cells to undergo development . Our findings suggest that protein secretion which occurs during the first hours of starvation-induced development might therefore take place via a different route from that which occurs in vegetative cells. Microbiology, 1995 Dec, 141 ( Pt 12), 3029 - 37 Mycobacterium smegmatis DNA gyrase: cloning and overexpression in Escherichia coli; Madhusudan K et al.; The cloning and characterization of DNA gyrase genes from Mycobacterium smegmatis is described . The DNA sequence of 5119 bp encoding both gyrB and gyrA genes was determined . The gene gyrB precedes gyrA with a short intergenic region of 29 nucleotides . The proteins encoded, GyrB and GyrA, exhibit 45-80% identity to gyrase polypeptides from other bacteria . The genes were further engineered for overexpression in Escherichia coli . Both genes were individually cloned into a phage T7 expression system and overexpressed . The expressed GyrB and GyrA proteins had molecular masses of 75 and 95 kDa, respectively, in agreement with that calculated from the ORFs . The extracts from the overexpressing clones were fractionated to enrich the subunits and assayed for enzyme activity . While the individual extracts showed no detectable activity, the combined extract exhibited a strong DNA supercoiling activity . This activity was ATP-dependent and novobiocin-sensitive . The identity of the genes was also confirmed by complementation analysis. Biotechnol Appl Biochem, 1995 Dec, 22 ( Pt 3), 269 - 80 Production of bovine-pancreatic-trypsin-inhibitor homologues in Escherichia coli and their characterization; Chesshyre JA et al.; Biologically active bovine pancreatic trypsin inhibitor (BPTI) was produced in Escherichia coli using an OmpA leader-peptide fusion-protein system, and BPTI homologues were generated by cassette mutagenesis . Amino acids in the reactive loop of alpha 1-proteinase inhibitor (alpha 1-PI) were incorporated into the reactive loop of BPTI in a stepwise approach such that the contribution of individual amino acids could be assessed . The introduction of mutations into BPTI diminished the yield of heterologous protein relative to wild-type BPTI . However, for three BPTI homologues sufficient material was isolated to allow characterization of the proteins by electrospray MS and N-terminal peptide sequencing. Am J Physiol, 1995 Dec, 269(6 Pt 1), G874 - 82 Genistein and tyrphostin 47 stimulate CFTR-mediated Cl- secretion in T84 cell monolayers; Sears CL et al.; The involvement of tyrosine phosphorylation in the regulation of epithelial cell Cl- secretion is unknown . Therefore, the purpose of these studies was to determine if tyrosine kinase activation was involved in the regulation of Cl- secretion, using the tyrosine kinase inhibitors, genistein and tyrphostin 47, and human intestinal epithelial cells (T84 cells) as an intestinal Cl- secretory model . Genistein rapidly but reversibly stimulated sustained apical Cl- secretion in monolayers of T84 cells without increasing intracellular cyclic nucleotides or Ca2+ levels . Tyrphostin 47 also stimulated Cl- secretion in T84 monolayers, although it was short-lived . Transfection experiments in 3T3 fibroblasts and IEC-6 intestinal cells utilizing wild-type cystic fibrosis transmembrane conductance regulator (CFTR) showed that genistein and tyrphostin 47 stimulated 125I efflux only in CFTR-transfected cells and not in CFTR-negative cells . Thus genistein- and tyrphostin 47-stimulated Cl- secretion involved CFTR . Genistein also acted synergistically with the Ca(2+)- and protein kinase C-dependent acetylcholine analogue, carbachol, to stimulate Cl- secretion in T84 monolayers . However, the Cl- secretory response to saturating concentrations of the adenosine 3',5'-cyclic monophosphate (cAMP) agonist, forskolin, or the guanosine 3',5'-cyclic monophosphate (cGMP) agonist, Escherichia coli heat-stable enterotoxin, was not further enhanced by genistein . Although the mechanism of activation of Cl- secretion is unclear, these data suggest that tyrosine kinase activity limits basal Cl- secretion in T84 cells and that inhibition of T84 cell tyrosine kinase(s) stimulates apical membrane Cl- secretion, most likely through activation of the CFTR-Cl- channel . Moreover, genistein does not itself act through cAMP or cGMP elevation but appears to share a common Cl- secretory pathway with cyclic nucleotide-dependent agonists, whereas it augments the secretory responses to a Ca(2+)- and protein kinase C-dependent agonist. Cell Biochem Funct, 1995 Dec, 13(4), 251 - 7 Glucocorticoid-induced changes in liver: inhibition of nuclear Ca2+, Mg(2+)-dependent endonuclease activity in response to dexamethasone administration; Goodlad GA et al.; The endogenous Ca2+, Mg(2+)-dependent endonuclease activity in nuclei from livers of rats receiving daily injections of the synthetic glucocorticoid dexamethasone was examined with respect to the production of both single and double strand breaks in chromatin DNA . The ability to form single strand breaks was measured by means of a nick translation assay and double strand breaks by following the appearance of nucleosomal ladders . A fall in the activity causing double strand breaks to approximately 50 per cent of the control value was apparent at 12 h after the first injection of the steroid . A fall of 25-30 per cent was also observed in the nicking activity but this was not apparent until 24 h after the first steroid injection . Both endonuclease activities remained at these lower levels for the remainder of the period of treatment . Nuclear extracts from dexamethasone-treated rats also showed a reduced ability to produce nucleosomal ladders when incubated with rat muscle nuclei, indicating that the inhibition observed in intact nuclei from treated animals was independent of any changes in chromatin structure . On the other hand the nick translation activity of the two extracts was the same when calf thymus DNA was used as the substrate suggesting that steroid-induced alterations in chromatin structure may be a critical factor in the reduced level of this activity observed in intact nuclei. APMIS, 1995 Dec, 103(12), 869 - 77 The Legionella micdadei flagellin: expression in Escherichia coli K 12 and DNA sequence of the gene; Bangsborg JM et al.; To study the structure and function of the Legionella flagellum, we screened a genomic L . micdadei library in Escherichia coli for expression of the flagellin (Fla) subunit . One recombinant clone, JM105 (pHI5588), producing a truncated Fla protein of 40.5 kDa was identified . The plasmid pHI5588 carried a L . micdadei DNA insert of 5 kb, containing ca 95% of the fla gene . The complete DNA sequence of the L . micdadei fla gene was obtained by combining sequence data from pHI5588 with results using a polymerase chain reaction-based system for genome walking (vectorette PCR) . The L . micdadei fla gene shared a high degree of homology with other flagellin genes in the amino- and carboxy termini, whereas the central region was found to be nonconserved . The fla sequence will facilitate the cloning of Fla proteins from other Legionella species and the study of flagella in the pathogenesis of Legionnaires' disease. J Biotechnol, 1995 Dec 1, 43(2), 139 - 43 Amplification of lambda plasmids in Escherichia coli relA mutants; Wegrzyn G; It was previously demonstrated that, contrary to wild-type stringent (rel+) strains of Escherichia coli, in amino acid-starved relaxed (relA) mutants the replication of lambda plasmid proceeds for several hours . The replication leads to amplification of lambda plasmid DNA . Here, the conditions for this amplification have been optimized . The amplification efficiency depends on the temperature as well as on the nature of amino acid starvation, but it is only little or totally not dependent on the pH value of the medium in a range from 6.0 to 8.0 . It seems that the most efficient amplification can be achieved by overnight cultivation of E . coli relA arg strain harbouring lambda plasmid at 36-39 degrees C in minimal medium containing Casamino acids . Under these conditions, the copy number of lambda plasmid increases from about 40 to about 300 per cell giving greater than 7-fold amplification. J Biotechnol, 1995 Dec 1, 43(2), 133 - 8 Use of the Escherichia coli chromosomal DHFR gene as selection marker in mammalian cells; Asselbergs FA et al.; The folA gene, the chromosomal dhfr gene of Escherichia coli, was engineered for expression in mammalian cells . In contrast to plasmid-derived bacterial dhfr genes previously used as selection markers in mammalian cells, the folA gene product is inhibitable by methotrexate (MTX) and trimethoprim (TMP) . Therefore, this dhfr may present an alternative to mammalian dhfr species currently used as amplifiable selection markers . Transfected E . coli folA dhfr could complement the lack of endogenous DHFR in Chinese hamster ovary (CHO) cells lacking a functional dhfr gene . Both MTX and TMP inhibited growth of E . coli folA dhfr-transfected CHO cells . Expression of E . coli folA DHFR could be visualized by incubating the transfected cells with a fluorescent methotrexate derivative (F-MTX) . Binding of F-MTX to E . coli folA DHFR was inhibitable as by both MTX and TMP, whereas MTX but not TMP blocked binding of F-MTX to recombinant mouse DHFR. Epidemiol Infect, 1995 Dec, 115(3), 455 - 63 Isolation of enterotoxigenic Escherichia coli from British troops in Saudi Arabia; Willshaw GA et al.; Specimens from 181 patients with diarrhoea were examined by a Military General Hospital in a 3-month period during deployment of troops to Saudi Arabia in 1990/1 . DNA probes for heat labile (LT) and heat stable (ST) enterotoxin genes identified enterotoxigenic Escherichia coli (ETEC) in 47 of the specimens (26%) and 49 ETEC strains were isolated . The majority (55%) belonged to a novel ETEC serotype having the O-antigen 159 and a flagellar antigen designated as a provisional new type . They produced ST and the coli surface associated antigen (CS)6 . Strains of serotype O6:H16 represented 22% of the ETEC examined . They produced ST, LT and CS3 together with either CS1 or CS2 . The remaining ETEC belonged to seven O:H serotypes . Overall, ST was the only enterotoxin gene identified in 73% of the ETEC and 67% of the strains expressed CS6 in the absence of other colonization antigens . Resistance to three or more antibiotics was observed in 53% of the ETEC, including most of the O159 strains. Br J Biomed Sci, 1995 Dec, 52(4), 317 - 20 Detection of the heat-stable toxin coding gene (ST-gene) in enterotoxigenic Escherichia coli: development of a colour amplified PCR detection system; Fanning S et al.; Screening biological samples using the polymerase chain reaction (PCR) has obvious advantages compared with current molecular analytical methods based on gel electrophoresis and/or hybridisation, both of which are expensive and time-consuming, therefore the development of a PCR assay format that is applicable to large sample numbers and that can readily use equipment commonly found in diagnostic laboratories would be advantageous . This report describes the development of a colour amplified PCR detection system which is simple in design and could be universally applied to the detection of any DNA template . As an example, the system has been applied in the detection of the heat-stable toxin coding gene (ST-gene) from enterotoxigenic Escherichia coli (ETEC) . The assay is sensitive, detecting 10 fg of a purified DNA template and 270 cfu of an ST-gene-positive ETEC strain. Plant Mol Biol, 1995 Dec, 29(5), 1039 - 55 Functional analysis of isolated cpn10 domains and conserved amino acid residues in spinach chloroplast co-chaperonin by site-directed mutagenesis; Bertsch U et al.; The possibilities of independent function of the two chaperonin 10 (cpn10) domains of the cpn10 homologue from spinach chloroplasts and the role of five conserved amino acid residues in the N-terminal cpn10 unit were investigated . Recombinant single domain proteins and complete chloroplast cpn10 proteins carrying amino acid exchanges of conserved residues in their N-terminal cpn10 domain were expressed in Escherichia coli and partially purified . The function of the recombinant proteins was tested using GroEL as chaperonin 60 (cpn60) partner for in vitro refolding of denatured ribulose-1,5-bisphosphate carboxylase (Rubisco) . Interaction with cpn60 was also monitored by the ability to inhibit GroEL ATPase activity . In vitro both isolated cpn10 domains were found to be incapable of co-chaperonin function . All mutants were also severely impaired in cpn10 function . The results are interpreted in terms of an essential role of the exchanged amino acid residues for the interaction between co-chaperonin and cpn60 partner and in terms of a functional coupling of both cpn10 domains . To test the function of mutant chloroplast cpn10 proteins in vivo the cpn10 deficiency of E . coli strain CG712 resulting in an inability to assemble lambda-phage was exploited in a complementation assay . Transformation with plasmids directing the expression of mutant chloroplas cpn10 proteins in two cases restored lambda-phage assembly in this bacterial strain to the same extent as did transformation with a plasmid encoding wild-type cpn10 protein . In contrast a plasmid encoded third mutant and truncated forms of chloroplast cpn10 showed significantly reduced complementation efficiencies. Plant Mol Biol, 1995 Dec, 29(5), 1027 - 38 A simplified procedure for the subtractive cDNA cloning of photoassimilate-responding genes: isolation of cDNAs encoding a new class of pathogenesis-related proteins; Herbers K et al.; Transgenic tobacco plants (ppa-1) constitutively expressing Escherichia coli pyrophosphatase behind the 35S CaMV promoter accumulate high levels of soluble sugars in their leaves {27} . These plants were considered a tool to study adaptation of leaves to photoassimilate accumulation at the molecular level . By differential hybridization of a subtractive library enriched for transcripts present in the transgenic plants 12 different cDNAs were isolated . By sequence analysis four cDNAs could be identified as 1-aminocyclopropane-1-carboxylate-oxidase and as three different pathogenesis-related proteins (PR-1b, PR-Q and SAR 8.2) . Two cDNAs were homologous to a calmodulin-like protein from Arabidopsis and a human ribosomal protein L19 while six cDNA clones remained unknown . One of these clones (termed PAR-1 for photoassimilate-responsive) displayed features similar to pathogenesis-related proteins: Hybridizing transcripts, 1.2 and 1.0 kb in length, were strongly inducible by salicylate and accumulated in tobacco plants after infection with potato virus Y (PVY) both in infected and uninfected systemic leaves . PAR-1 transcripts also accumulated in wildtype leaves upon floating on glucose and sucrose whereas sorbitol and polyethylene glycol had no effect . Rescreening of the ppa-1 cDNA library with the PAR-1 cDNA as probe resulted in 25 hybridizing cDNAs which by homology were found to fall into three classes (PAR-1a, b, c) . The cDNAs coding for PAR-1a and b were 90.6% homologous on the DNA level while both were less related to the PAR-1c cDNA (70.5% and 75.2% homologous, respectively) . One open reading frame was identified in all three PAR-1 cDNA classes . Translation would result in proteins with a theoretical molecular mass of about 20 kDa . The N-terminal amino acid sequences resemble a signal peptide which would direct the proteins to the secretory pathway . Using selective 3' hybridization probes of the three PAR-1 cDNAs it was possible to discriminate the different transcripts . Both PAR-1a and PAR-1c mRNAs are induced in plants treated with PVY. Cancer Gene Ther, 1995 Dec, 2(4), 263 - 71 Effectiveness of three ribozymes for cleavage of an RNA transcript from human papillomavirus type 18; Chen Z et al.; We tested three hammerhead ribozymes for their ability to bind and cleave RNA transcripts derived from the E6 and E7 genes of human papillomavirus (HPV) type-18 . Targets were located at nucleotides (nt) 123, 309, and 671 of the viral transcript . In vitro each ribozyme hybridized to its target site when the ribozyme:target ratio was 20:1 or greater and achieved maximal hybridization within 1 hour . HPV RNA from the HeLa cervical cancer cell line was cleaved effectively by each ribozyme . When HPV RNA and a ribozyme were expressed simultaneously in Escherichia coli, each ribozyme produced a significant reduction in the intracellular concentration of HPV RNA . In each assay the ribozyme directed to nt 309 was the most effective . A noncatalytic antisense molecule was used as a control and did not digest HPV RNA or reduce its concentration . The data imply that three different ribozymes each have potential for use in gene therapy of human tumors that express HPV-18 but that the ribozyme targeted to nt 309 is likely to be most effective. Mutat Res, 1995 Dec, 348(4), 183 - 6 PM2 DNA damage induced by 3-chloro-4-(dichloromethyl)-5-hydroxy-2 (5H)-furanone (MX); Hyttinen JM et al.; 3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) is a potent direct-acting mutagen found in chlorinated drinking water . In the present study, the induction of DNA strand breaks and apurinic/apyrimidinic (AP) sites by MX in supercoiled PM2 DNA was examined using exonuclease III, which specifically cleaves the DNA at AP sites . The results showed that MX induced AP sites in great excess of direct strand breaks . In view of the known mutagenicity of AP sites, these results provide insight into the mechanism of MX-induced mutagenesis. Mutat Res, 1995 Dec, 348(4), 147 - 52 Genotoxicity assay of chloral hydrate and chloropicrine; Giller S et al.; The chlorination by-products chloral hydrate and chloropicrine were assayed for genotoxicity in three short-term tests . Chloropicrine was 100-fold more potent than chloral in inducing mutations in strain TA100 of S . typhimurium (fluctuation test) and, at variance with chloral, was positive in the SOS chromotest using strain PQ37 of E . coli . On the other hand, only chloral caused a significant increase in the frequency of micronucleated erythrocytes following in vivo exposure of the amphibian Pleurodeles waltl newt larvae. J Allergy Clin Immunol, 1995 Dec, 96(6 Pt 1), 951 - 9 IgE cross-reactivities against albumins in patients allergic to animals; Spitzauer S et al.; BACKGROUND: Type I allergic symptoms and severe asthma in particular are frequently caused by animal hair/dander proteins, among which albumins are possible cross-sensitizing allergenic components . METHODS: The significance and degree of IgE-cross-reactivities against various albumins were studied in a representative number (n = 200) of patients allergic to animals with hair/dander extracts, purified albumins from different animals, and a recombinant dog albumin fragment expressed in lysogenic Escherichia coli Y1089 and purified as a beta-galactosidase fusion protein . RESULTS: Despite a high degree of sequence homology among different albumins, a remarkable variability of IgE cross-reactivities was observed, indicating that some patients were sensitized preferentially against certain albumins . Most of the patients allergic to albumins, however, reacted to dog, cat, and horse albumin, which also bound a high percentage of albumin-specific IgE . CONCLUSION: The purified recombinant dog albumin fragment, representing 265 amino acids of the mature protein, bound IgE from all 15 patients allergic to albumin tested suggesting its potential usefulness for diagnosis and perhaps therapy. Exp Parasitol, 1995 Dec, 81(4), 592 - 9 Molecular cloning and characterization of the filarial LIM domain proteins AvL3-1 and OvL3-1; Oberlander U et al.; A full-length cDNA of the filarial nematode Acanthocheilonema viteae was isolated from a cDNA library of female worms, using a partial cDNA of the OvL3-1 gene of Onchocerca volvulus as a probe . The AvL3-1 cDNA contained an open reading frame which encoded for a protein with a theoretical molecular weight of 64 kDa . The deduced protein contained a predicted signal sequence, a short repetitive motive of unknown function, and three LIM domains . The structure of the LIM domains was identical to those of zyxin, a cytoskeleton-associated protein of chicken fibroblasts, suggesting that AvL3-1 has a similar role in filarial nematodes . The sequence information was used to isolate the homologous cDNA of O . volvulus by PCR from a cDNA library of female O . volvulus, which showed an overall identity of 76.9% to AvL3-1 on the protein level . AvL3-1 was expressed in Escherichia coli and the affinity-purified fusion free protein was used to immunized jirds (Meriones unguiculatus) . Immunization together with the adjuvant STP or with Freund's adjuvant induced IgG and IgM antibody responses, but no significant protection against a challenge infection with L3 of A . viteae, compared to appropriate control groups. Exp Parasitol, 1995 Dec, 81(4), 536 - 45 Immunological characterization and expression in Escherichia coli and baculovirus systems of a Trypanosoma vivax antigen detected in the blood of infected animals; Masake RA et al.; A monoclonal antibody (MAb)Tv27 employed in an antigen-detection enzyme immunosorbent assay (Ag-ELISA) for diagnosis of Trypanosoma vivax infection was shown to react with a T . vivax-specific protein of an approximate molecular weight of 10 kDa . This protein is diffusely distributed throughout the cytosol and nucleus of metacyclic forms, bloodstream forms, and procyclic-like elongated trypomastigotes, but is not detectable in epimastigotes of T . vivax . The T . vivax-specific antigen prepared from parasite lysates appeared to be of lower molecular mass than the form expressed in either Escherichia coli or in baculovirus-infected silkworm insect cells . In the recombinant baculovirus-infected cells, the protein was expressed mostly as an 18-kDa peptide with less abundant forms of 13 and 12 kDa, while the protein expressed in E . coli was approximately 14 kDa . Both the low- and higher-molecular-weight proteins are recognized by the MAb Tv27 in Western blots and in Ag-ELISA . Although the crude preparations of the protein produced by the insect cells are labile when kept for more than 2 hr at 24 degrees C, they retained reactivity at temperatures below 4 degrees C for several weeks . The proteins expressed in both the insect cells and E . coli captured anti-T . vivax antibodies in sera prepared from trypanosome-infected animals . Since the recombinant protein expressed in the baculovirus-infected cells is available in large homogeneous quantities, it would serve as a positive control in Ag-ELISA and is also usable for antibody detection assays. Can J Microbiol, 1995 Dec, 41(12), 1057 - 62 Comparative study of two trehalase activities from Fusarium oxysporum var . lini; Amaral FC et al.; Acid and neutral trehalase activities (optimum pH of 4.6 and 6.8, respectively) from Fusarium oxysporum var . lini were studied separately through partial isolation by ammonium sulfate precipitation followed by ion-exchange chromatography on DEAE-Sephacel for neutral enzyme, or using some of their differential properties . Acid activity was unaffected by 1 mM of Ca2+, Mg2+, Mn2+, Ba2+, or EDTA . Contrarily, the neutral enzyme was activated by Ca2+ with an apparent Ka of 0.15 mM; was inhibited by EDTA, Zn2+, Hg2+, or Mg(2+)-ATP; and showed an increase in activity by the raise of buffer ionic strength or by the addition of 100 mM KCl . Acid and neutral enzymes have, respectively, an apparent optimum temperature of 45 and 30 degrees C, an apparent Km for trehalose of 0.43 and 8.45 mM, and an apparent M(r) of 160,000 and 100,000 (by glycerol gradient ultracentrifugation) . Acid trehalase was specifically inhibited by acetate buffer and more stable at 50 degrees C than the neutral enzyme . Neutral enzyme exhibited a pI of 6.2 by isoelectric focusing . Contrary to neutral trehalases from other fungi, the enzyme from Fusarium oxysporum var . lini was not activated in crude extract by treatment with Mg(2+)-ATP in the presence of cAMP and not inactivated by alkaline phosphatase from Escherichia coli. Plant Physiol, 1995 Dec, 109(4), 1379 - 88 Molecular dissection of the epsilon subunit of the chloroplast ATP synthase of spinach; Cruz JA et al.; The gene encoding the epsilon subunit (atpE) of the chloroplast ATP synthase of Spinacia oleracea has been overexpressed in Escherichia coli . The recombinant protein can be solubilized in 8 M urea and directly diluted into buffer containing ethanol and glycerol to obtain epsilon that is as biologically active as epsilon purified from chloroplast-coupling factor 1 (CF1) . Recombinant epsilon folded in this manner inhibits the ATPase activity of soluble and membrane-bound CF1 deficient in epsilon and restores proton impermeability to thylakoid membranes reconstituted with CF1 deficient in epsilon . Site-directed mutagenesis was used to generate truncations and single amino acid substitutions in the primary structure of epsilon . In the five mutants tested, alterations that weaken ATPase inhibition by recombinant epsilon affect its ability to restore proton impermeability to a similar extent, with one exception . Substitution of histidine-37 with arginine appears to uncouple ATPase inhibition and the restoration of proton impermeability . As in the case of E . coli, it appears that N-terminal truncations of the epsilon subunit have more profound effects than C-terminal deletions on the function of epsilon . Recombinant epsilon with six amino acids deleted from the C terminus, which is the only region of significant mismatch between the epsilon of spinach and the epsilon of Pisum sativum, inhibits ATPase activity with a reduced potency similar to that of purified pea epsilon . Four of the six amino acids are serine or threonine . These hydroxylated amino acids may be important in epsilon-CF1 interactions. Eur J Biochem, 1995 Dec 1, 234(2), 598 - 602 Production of recombinant human brain type I inositol-1,4,5-trisphosphate 5-phosphatase in Escherichia coli . Lack of phosphorylation by protein kinase C; Erneux C et al.; The dephosphorylation of inositol 1,4,5-trisphosphate (InsP3) to inositol 1,4-bisphosphate is catalyzed by InsP3 5-phosphatase . The coding region of human brain type I InsP3 5-phosphatase was expressed as a fusion protein with the maltose-binding protein (MBP) in Escherichia coli, using the pMAL-cR1 vector . The relative molecular mass of the purified fusion protein (MBP-InsP3-5-phosphatase) was approximately M(r) 85,000 as analysed by SDS/PAGE . The yield was about 10 mg fusion protein/l lysate . After cleavage from MBP with factor Xa, the specific activity of recombinant 5-phosphatase was 120-250 mumol.mg-1.min-1 . The molecular mass of purified protein by SDS/PAGE was M(r) 43,000 . The activity was inactivated by p-hydroxymercuribenzoate . The possibility that protein kinase C might phosphorylate InsP3 5-phosphatase was tested on the purified 43,000 M(r) protein . In this study, we show that recombinant 5-phosphatase is not a substrate of protein kinase C. Eur J Biochem, 1995 Dec 1, 234(2), 397 - 405 Human monoclonal antibodies to cytomegalovirus . Characterization and recombinant expression of a glycoprotein-B-specific antibody; Boldicke T et al.; Human monoclonal antibodies (mAb) to human cytomegalovirus (HCMV) were established from spleen cells of a HCMV-positive donor . The antibodies (gamma 3, lambda) secreted from a stable heterohybridoma cell line were further characterized by immunoprecipitation and immune-fluorescence microscopy using HCMV infected cells and recombinant cell lines expressing HCMV glycoprotein B . The antibody reacted with the entire glycoprotein B or the extracellular domain expressed as glycoprotein-B--beta-galactosidase fusion protein in the native state, but the antibody was not neutralizing HCMV . Denatured and reduced forms of glycoprotein B were not recognized by this antibody, however, native glycoprotein B on the surface of infected cells was detected efficiently . The genes encoding the Fab part of the antibody were cloned and expressed in Escherichia coli . Recombinant Fab fragments specifically binding the extracellular domain of glycoprotein B could easily be isolated from the periplasmic space . Recombinant antibodies provide the opportunity to modify effector functions and to add tags to diagnostic antibodies for more efficient detection of CMV-infected cells. Appl Environ Microbiol, 1995 Dec, 61(12), 4147 - 51 Entry of Escherichia coli into stationary phase is indicated by endogenous and exogenous accumulation of nucleobases; Rinas U et al.; Endogenous and exogenous accumulation of nucleobases was observed when Escherichia coli entered the stationary phase . The onset of the stationary phase was accompanied by excretion of uracil and xanthine . Except for uracil and xanthine, other nucleobases (except for minor amounts of hypoxanthine), nucleosides, and nucleotides (except for cyclic AMP) were not detected in significant amounts in the culture medium . In addition to exogenous accumulation of nucleobases, stationary-phase cells increased the endogenous concentrations of free nucleobases . In contrast to extracellular nucleobases, hypoxanthine was the dominating intracellular nucleobase and xanthine was present only in minor concentrations inside the cells . Excretion of nucleobases was always connected to declining growth rates . It was observed in response to entry into the stationary phase independent of the initial cause of the cessation of cell growth (e.g., starvation for essential nutrients) . In addition, transient accumulation of exogenous nucleobases was observed during perturbations of balanced growth conditions such as energy source downshifts . The nucleobases uracil and xanthine are the final breakdown products of pyrimidine (uracil and cytosine) and purine (adenine and guanine) bases, respectively . Hypoxanthine is the primary degradation product of adenine, which is further oxidized to xanthine . The endogenous and exogenous accumulation of these nucleobases in response to entry into the stationary phase is attributed to degradation of rRNA. Gene, 1995 Dec 1, 166(1), 73 - 6 Sequence of the Escherichia coli C homoprotocatechuic acid degradative operon completed with that of the 2,4-dihydroxyhept-2-ene-1,7-dioic acid aldolase-encoding gene (hpcH); Stringfellow JM et al.; The homoprotocatechuic acid (HPC) pathway is a typical catabolic sequence for converting peripheral metabolites into intermediates of central metabolism . How the pathway enzymes that catalyse such natural sequences have arisen is as yet uncertain, but the explanation is likely to be of interest in devising pathways to catabolise the man-made chemicals that are increasingly found in the environment . The nucleotide (nt) sequence of the Escherichia coli C 2,4-dihydroxyhept-2-ene-1,7-dioic acid (HHED) aldolase-encoding gene (hpcH) reported here completes the sequencing of the HPC pathway genes, and so makes it possible to assess the relatedness of all the pathway enzymes . There were no striking amino acid (aa) sequence identities between any of the pathway enzymes, suggesting that they had not arisen by duplication of an ancestral gene, with subsequent divergence . The HHED aldolase showed no striking identity (16-22%) with the aldolases from five other bacteria catalysing the analogous reaction in the catechol meta-fission pathway . However, there was significant aa identity (47.8%) with an E . coli K-12 open reading frame (ORF) of as yet unknown function, suggesting that this ORF may encode an aldolase of some kind. Gene, 1995 Dec 1, 166(1), 181 - 2 Color selection with a hygromycin-resistance-based Escherichia coli-mycobacterial shuttle vector; Howard NS et al.; Hygromycin-resistance (HyR)-based Escherichia coli-mycobacterial shuttle plasmids have high efficiencies of transformation and a broad mycobacterial host range . We have introduced a lacZ alpha (encoding the alpha-polypeptide fragment of beta-galactosidase (beta Gal))-multiple cloning site cassette into a HyR-based shuttle vector to generate a plasmid with nine unique cloning sites and the added feature of beta Gal color selection in appropriate E . coli host strains. Gene, 1995 Dec 1, 166(1), 177 - 8 Escherichia coli expression vectors containing a protein kinase recognition motif, His6-tag and hemagglutinin epitope; Kelman Z et al.; Escherichia coli expression vectors, based on the pET system, were constructed to allow fusion of a protein kinase (PK) recognition motif, a hemagglutinin (HG) epitope-tag and a His6-tag at the N-terminal portion of a protein of interest . The fusion proteins, that result from expression using these vectors, can be phosphorylated in vitro using cAMP-dependent PK, immunoprecipitated using monoclonal antibody against the HG-epitope, and can be rapidly purified using a Ni2+ column. Gene, 1995 Dec 1, 166(1), 173 - 4 Multiple cloning sites carrying loxP and FRT recognition sites for the Cre and Flp site-specific recombinases; Snaith MR et al.; Plasmids were constructed carrying the loxP and FRT recognition sites for the Cre and Flp site-specific recombinases, respectively, within multiple cloning sites . Vectors carrying single and tandemly repeated targets are available with various flanking restriction enzyme sites . In addition, a series of plasmids carrying both loxP and FRT sites is available . These vectors facilitate construction of target molecules for these site-specific recombinases which are becoming increasingly important tools for the in vivo manipulation of DNA. Gene, 1995 Dec 1, 166(1), 139 - 43 An abnormally acidic TATA-binding protein from a hyperthermophilic archaeon; Rashid N et al.; The gene encoding the TATA-binding protein (PkTBP) from a hyperthermophilic archaeon, Pyrococcus sp . KOD1 (Pk), was cloned and sequenced . An open reading frame with homology to the conserved C-terminal core region of eukaryotic TBP was expressed in Escherichia coli . Specific DNA-binding activity of the recombinant PkTBP (190 amino acids, 21.36 kDa) was also demonstrated . Although it was composed of a structurally direct repeat sequence which is similar to eukaryotic TBP, the total net charge of archaeal TBP was amazingly negative (calculated isoelectric point (pI) was 4.66 and experimentally estimated pI was 4.8) . A series of five Glu residues was found at the C terminus of archaeal TBP . These data strongly suggest that a positively charged protein is also involved in the transcription initiation event which might stabilize the structure of the genomic DNA under high-growth-temperature conditions. Gene, 1995 Dec 1, 166(1), 127 - 32 Cloning, sequencing and expression of the ilvBNC gene cluster from Streptomyces avermitilis; De Rossi E et al.; The metabolism of the branched-chain amino acids (BCAA) isoleucine, leucine and valine is correlated to the production of polyketide antibiotics in many streptomycetes . Despite its significance, this biosynthetic pathway is poorly understood in Streptomyces . In order to develop a better understanding of Streptomyces BCCA biosynthesis, two genes, ilvBN and ilvC, encoding acetohydroxy acid synthase (AHS) and acetohydroxy acid isomeroreductase (IR), respectively, were cloned from Streptomyces avermitilis, a strain producing avermectins, potent antiparasitic compounds . The genes were isolated by applying a combination of PCR and genomic library screening . The deduced amino-acid sequences revealed significant homology to the AHS and IR proteins from other bacterial species . The ilvBN gene, expressed in Escherichia coli (Ec) by using the expression vector pGEX-4T-1, complemented the ilv- mutation of Ec PS1283 . Ec transformants produced high levels of AHS, whose activity was feedback inhibited by valine. Gene, 1995 Dec 1, 166(1), 111 - 6 Repeated sequences isolated from Bordetella pertussis induce DNA rearrangements and deletions at high frequency; Kirillov MYu et al.; Two repeated sequences (RS) from Bordetella pertussis were cloned in Escherichia coli and sequenced . The RS, called RSBP1 and RSBP3, are highly homologous to other B . pertussis RS . The recombinant plasmids containing RSBP1 and RSBP3 or transposon-like structures of these elements were not stable but segregated plasmids with deletions or rearranged DNA . RS of B . pertussis seem to be able to stimulate both intra- and inter-genomic RecA-independent recombination events . In at least one case, the observed deletion had occurred precisely between the RS terminus and a site with sequence homology to the terminus . The high frequency rearrangements associated with the RS imply that the RS are transposable elements. Gene, 1995 Dec 1, 166(1), 1 - 9 New tools for integrated genetic and physical analyses of the Escherichia coli chromosome; Rode CK et al.; Genetic and biophysical techniques have traditionally been applied to genome mapping independently of one another . We present a series of Escherichia coli mini-Tn10 insertions that contain the rare-cutting polylinker 1 (RCP1) of rare restriction sites {including BlnI/AvrII, SpeI, NheI, XbaI, NotI, PacI and SfiI; Mahillon and Kleckner, Gene 116 (1992) 69-74} which allows them to be used not just for genetic mapping, but also for rapid physical mapping and integrated physical and genetic mapping of the E . coli chromosome . Their isolation and their physical and genetic coordinates in K-12 strain MG1655 are presented . Also, their use in purifying insertion-delimited DNAs from E . coli K-12 and in macrorestriction mapping of a pathogenic strain's chromosome is demonstrated . These insertions allow integration of (i) different macrorestriction patterns of a single strain's chromosome, (ii) the physical map of a single strain's chromosome with the genetic map of the species, and (iii) the physical maps of different strains' chromosomes. Biochem J, 1995 Dec 1, 312 ( Pt 2), 599 - 608 Kinetic characteristics of Escherichia coli RNase H1: cleavage of various antisense oligonucleotide-RNA duplexes; Crooke ST et al.; 1 . The effects of variations in substrates on the kinetic properties of Escherichia coli RNase H were studied using antisense oligonucleotides of various types hybridized to complementary oligoribonucleotides . The enzyme displayed minimal sequence preference, initiated cleavage through an endonucleolytic mechanism near the 3' terminus of the RNA in a DNA-RNA chimera and then was processively exonucleolytic . Phosphorothioate oligodeoxynucleotides hybridized to RNA supported cleavage more effectively than phosphodiester oligodeoxynucleotides . Oligonucleotides comprised of 2'-methoxy-, 2'-fluoro- or 2'-propoxy-nucleosides did not support RNase H1 activity . 2 . The Km and Vmax . of cleavage of RNA duplexes with full phosphorothioate oligodeoxynucleotides were compared with methoxy-deoxy 'gapmers', i.e.; oligonucleotides with 2'-methoxy wings surrounding a deoxynucleotide centre . Such structural modifications resulted in substantial increases in affinity, but significant reductions in cleavage efficiency . The initial rates of cleavage increased as the deoxynucleotide gap size was increased . Multiple deoxynucleotide gaps increased the Vmax . but had little effect on Km . 3 . The effects of several base modifications on the site of initial cleavage, processivity and initial rate of cleavage were also studied. Biochem J, 1995 Dec 1, 312 ( Pt 2), 527 - 33 Altering kinetic mechanism and enzyme stability by mutagenesis of the dimer interface of glutathione reductase; Bashir A et al.; In wild-type glutathione reductase from Escherichia coli residues Val421 and Ala422 are located in an alpha-helix in a densely packed and hydrophobic region of the dimer interface, with their side chains packed against those of residues Ala422' and Val421' in the second subunit . A series of mutant glutathione reductases was constructed in which the identities of the residues at positions 421 and 422 were changed . Mutations were designed so as to present like charges (mutants Val421-->Glu:Ala422-->Glu and Val421-->Lys:Ala422-->Lys) or opposite charges (mutant Val421-->Lys:Ala422-->Glu) across the dimer interface to assess the role of electrostatic interactions in dimer stability . A fourth mutant (Val421-->His:Ala422-->His) was also constructed to investigate the effects of introducing a potentially protonatable bulky side chain into a crowded region of the dimer interface . In all cases, an active dimeric enzyme was found to be assembled but each mutant protein was thermally destabilized . A detailed steady-state kinetic analysis indicated that each mutant enzyme no longer displayed the Ping Pong kinetic behaviour associated with the wild-type enzyme but exhibited what was best described as a random bireactant ternary complex mechanism . This leads, depending on the chosen substrate concentration, to apparent sigmoidal, hyperbolic or complex kinetic behaviour . These experiments, together with others reported previously, indicate that simple mutagenic changes in regions distant from the active site can lead to dramatic switches in steady-state kinetic mechanism. Biochem J, 1995 Dec 1, 312 ( Pt 2), 505 - 10 Two different isoschizomers of the type-II restriction endonuclease Taq I (T/CGA) within the same Thermus isolate: Tsp32 I, an enzyme with similar heat stability properties to the prototype enzyme Taq I, and Tsp32 II, a hyperthermostable isoschizomer of Taq I; Welch SG et al.; We have recently screened 112 separate isolates of the genus Thermus, collected from neutral and alkaline hot water springs on four continents, for the presence of the Type-II restriction endonuclease Taq I (T/CGA) . One particular isolate from the Azores (strain 32) was found to contain high levels of a restriction endonuclease with the same recognition and cleavage site as Taq I . Initial studies revealed that the partially purified enzyme from strain 32 was considerably more resistant to heat inactivation than the prototype enzyme Taq I, being able to withstand temperatures at least 10 degrees C higher than Taq I, before showing evidence of heat inactivation . Subsequently it became clear that the partially purified extract from strain 32 contains two separate enzymes, both of which are isoschizomers of Taq I . One of the enzymes, Tsp32 I, has similar thermal stability characteristics to Taq I, whereas the second Taq I isoschizomer, Tsp32 II, found in the same Thermus isolate as Tsp32 I, is considerably more thermostable than Taq I, retaining full enzyme activity up to a temperature of 85 degrees C . Tsp32 I and Tsp32 II were further distinguished by virtue of their different requirements for magnesium ions. Biochem J, 1995 Dec 1, 312 ( Pt 2), 465 - 9 Glutathionylspermidine metabolism in Escherichia coli; Smith K et al.; Intracellular levels of glutathione and glutathionylspermidine conjugates have been measured throughout the growth phases of Escherichia coli . Glutathionylspermidine was present in mid-log-phase cells, and under stationary and anaerobic growth conditions accounted for 80% of the total glutathione content . N1,N8-bis(glutathionyl)spermidine (trypanothione) was undetectable under all growth conditions . The catalytic constant kcat/Km of recombinant E . coli glutathione reductase for glutathionylspermidine disulphide was approx . 11,000-fold lower than that for glutathione disulphide . The much higher catalytic constant for the mixed disulphide of glutathione and glutathionylspermidine (11% that of GSSG), suggests a possible explanation for the low turnover of trypanothione disulphide by E . coli glutathione reductase, given the apparent lack of a specific glutathionylspermidine disulphide reductase in E . coli. Biochem J, 1995 Dec 1, 312 ( Pt 2), 385 - 92 Identification of the ferroxidase centre of Escherichia coli bacterioferritin; Le Brun NE et al.; The bacterioferritin (BFR) of Escherichia coli takes up iron in the ferrous form and stores it within its central cavity as a hydrated ferric oxide mineral . The mechanism by which oxidation of iron (II) occurs in BFR is largely unknown, but previous studies indicated that there is ferroxidase activity associated with a site capable of forming a dinuclear-iron centre within each subunit {Le Brun, Wilson, Andrews, Harrison, Guest, Thomson and Moore (1993) FEBS Lett . 333, 197-202} . We now report site-directed mutagenesis experiments based on a putative dinuclear-metal-ion-binding site located within the BFR subunit . The data reveal that this dinuclear-iron centre is located at a site within the four-alpha-helical bundle of each subunit of BFR, thus identified as the ferroxidase centre of BFR . The metal-bound form of the centre bears a remarkable similarity to the dinuclear-iron sites of the hydroxylase subunit of methane mono-oxygenase and the R2 subunit of ribonucleotide reductase . Details of how the dinuclear centre of BFR is involved in the oxidation mechanism were investigated by studying the inhibition of iron (II) oxidation by zinc (II) ions . Data indicate that zinc (II) ions bind at the ferroxidase centre of apo-BFR in preference to iron (II), resulting in a dramatic reduction in the rate of oxidation . The mechanism of iron (II) oxidation is discussed in the light of this and previous work. Virology, 1995 Dec 1, 214(1), 40 - 9 Strain variability and localization of important epitopes on the major structural protein (VP2) of infectious pancreatic necrosis virus; Heppell J et al.; Infectious pancreatic necrosis virus (IPNV), a birnavirus, is an important pathogen in fish farms . Analyses of viral proteins showed that VP2 is the major structural and immunogenic polypeptide of the virus . All neutralizing monoclonal antibodies (mAbs) against IPNV are specific to VP2 and bind to continuous or discontinuous epitopes . In order to determine which parts of the protein are involved in antigenic variations, five IPNV strains were sequenced over the VP2 coding region . Comparison of the sequences obtained with three previously published strains revealed a central variable domain (positions 183 to 335) which encompasses two hydrophilic hypervariable segments . Viral mutants which escaped neutralization were then selected with anti-VP2 mAbs directed against discontinuous epitopes . Sequencing of three mutants revealed a single amino acid mismatch in each of them . All of these substitutions occurred in the hypervariable segments, suggesting that these regions are involved in the formation of a discontinuous epitope . Finally, expression of different truncated VP2s in Escherichia coli allowed localization of the binding site for neutralizing mAbs which recognize continuous epitopes . One of these mAbs bound to the region adjacent to the C-terminus of the variable domain of VP2, while two others reacted with the central and C-terminal parts of the variable domain . No antibody reacted with the N-terminus of VP2 . These results suggest that the variable domain of VP2 and the 20 adjacent amino acids of the conserved C-terminal part are the most important in inducing an immune response for the protection of animals. Virology, 1995 Dec 1, 214(1), 110 - 7 Antigenic sites on the receptor-binding domain of human adenovirus type 2 fiber; Fender P et al.; The trimeric fiber of adenovirus type 2 (Ad2) mediates the first stage of virus-cell attachment, and the distal head region of the fiber has been implicated as the receptor-binding domain . To locate regions on the primary polypeptide sequence of the fiber which may be involved in virus-cell interaction, peptide-based epitope mapping was performed using (1) polyclonal antibodies prepared against both native Ad2 fiber and Ad2 head protein expressed in Escherichia coli and (2) 18 monoclonal antibodies prepared against trimeric Ad2 head protein expressed in baculovirus . The approach using polyclonal antibodies revealed eight domains on the primary sequence of the head which contain one or more continuous epitopes . At least two of these regions were also recognized by monoclonal antibodies reacting against both monomeric and trimeric fiber head protein . The majority of monoclonal antibodies which did not recognize Ad2 head-specific peptides in ELISA were also nonreactive against the monomeric form of protein in Western blot, suggesting that their recognition of trimer is due to the existence of as yet undefined discontinuous epitopes or to alterations in monomer configuration . Our results correspond well with the recently published X-ray crystallographic model of Ad5 fiber head (D . Xia, L.J . Henry, R.D . Gerard, and J . Deisenhofer, Structure 2, 1259-1270, 1994), since most antigenic determinants containing linear epitopes mapped to the outer loops or uppermost beta-sheets in this structure . Four of five neutralizing monoclonal antibodies recognized trimer only and none recognized linear peptides . This might suggest that the trimeric form of fiber is necessary for making contact with the receptor(s) and that discontinuous epitopes on the head domain may be involved in fiber-cell interaction. Mol Cell Biol, 1995 Dec, 15(12), 6729 - 35 RNA polymerase bypass at sites of dihydrouracil: implications for transcriptional mutagenesis; Liu J et al.; Dihydrouracil (DHU) is a major base damage product formed from cytosine following exposure of DNA to ionizing radiation under anoxic conditions . To gain insight into the DNA lesion structural requirements for RNA polymerase arrest or bypass at various DNA damages located on the transcribed strand during elongation, DHU was placed onto promoter-containing DNA templates 20 nucleotides downstream from the transcription start site . In vitro, single-round transcription experiments carried out with SP6 and T7 RNA polymerases revealed that following a brief pause at the DHU site, both enzymes efficiently bypass this lesion with subsequent rapid generation of full-length runoff transcripts . Direct sequence analysis of these transcripts indicated that both RNA polymerases insert primarily adenine opposite to the DHU site, resulting in a G-to-A transition mutation in the lesion bypass product . Such bypass and insertion events at DHU sites (or other types of DNA damages), if they occur in vivo, have a number of important implications for both the repair of such lesions and the DNA damage-induced production of mutant proteins at the level of transcription (transcriptional mutagenesis). Mol Cell Biol, 1995 Dec, 15(12), 6593 - 600 Context effects on misreading and suppression at UAG codons in human cells; Phillips-Jones MK et al.; The effect of the 3' codon context on the efficiency of nonsense suppression in mammalian tissue culture cells has been tested . Measurements were made following the transfection of cells with a pRSVgal reporter vector that contained the classical Escherichia coli lacZ UAG allele YA559 . The position of this mutation was mapped by virtue of its fortuitous creation of a CTAG MaeI restriction enzyme site . Determination of the local DNA sequence revealed a C-->T mutation at codon 600 of the lacZ gene: CAG-->TAG . Site-directed mutagenesis was used to create a series of vectors in which the base 3' to the nonsense codon was either A, C, G, or U . Suppression of the amber-containing reporter was achieved by cotransfection with genes for human tRNA(Ser) or tRNA(Gln) UAG nonsense suppressors and by growth in the translational error-promoting aminoglycoside drug G418 . Nonsense suppression was studied in the human cell lines 293 and MRC5V1 and the simian line COS-7 . Overall, the rank order for the effect of changes to the base 3' to UAG was C < G = U < A . This study confirms and extends earlier findings that in mammalian cells 3' C supports efficient nonsense suppression while 3' A is unsympathetic for read-through at nonsense codons . The rules for the mammalian codon context effect on nonsense suppression are therefore demonstrably different from those in E . coli. J Bacteriol, 1995 Dec, 177(24), 7261 - 4 Corrected gene assignments of Escherichia coli pro- mutations; Serebrijski I et al.; We have reevaluated the gene assignments of the proline mutant alleles of some known Pro- Escherichia coli strains . Of nine proline auxotrophs included in the study, five presented phenotypes inconsistent with their previously assigned genotypes . We discuss the possible sources and the consequences of these assignment errors. J Bacteriol, 1995 Dec, 177(24), 7141 - 9 Expression and characterization of the Escherichia coli fdo locus and a possible physiological role for aerobic formate dehydrogenase; Abaibou H et al.; In the presence of nitrate, the major anaerobic respiratory pathway includes formate dehydrogenase (FDH-N) and nitrate reductase (NAR-A), which catalyze formate oxidation coupled to nitrate reduction . Two aerobically expressed isoenzymes, FDH-Z and NAR-Z, have been recently characterized . Enzymatic analysis of plasmid subclones carrying min 88 of the Escherichia coli chromosome was consistent with the location of the fdo locus encoding FDH-Z between the fdhD and fdhE genes which are necessary for the formation of both formate dehydrogenases . The fdo locus produced three proteins (107, 34, and 22 kDa) with sizes similar to those of the subunits of the purified FDH-N . In support to their structural role, these polypeptides were recognized by antibodies specific to FDH-N . Expression of a chromosomal fdo-uidA operon fusion was induced threefold by aerobic growth and about twofold by anaerobic growth in the presence of nitrate . However, it was independent of the two global regulatory proteins FNR and ArcA, which control genes for anaerobic and aerobic functions, respectively, and of the nitrate response regulator protein NARL . In contrast, a mutation affecting either the nucleoid-associated H-NS protein or the CRP protein abolished the aerobic expression . A possible role for FDH-Z during the transition from aerobic to anaerobic conditions was examined . Synthesis of FDH-Z was maximal at the end of the aerobic growth and remained stable after a shift to anaerobiosis, whereas FDH-N production developed only under anaerobiosis . Furthermore, in an fnr strain deprived of both FDH-N and NAR-A activities, aerobically expressed FDH-Z and NAR-Z enzymes were shown to reduce nitrate at the expense of formate under anaerobic conditions, suggesting that this pathway would allow the cell to respond quickly to anaerobiosis. J Bacteriol, 1995 Dec, 177(24), 7112 - 8 Chemotactic properties of Escherichia coli mutants having abnormal Ca2+ content; Tisa LS et al.; The calA, calC, and calD mutants of Escherichia coli are known to be sensitive to Ca2+ (R . N . Brey and B . P . Rosen, J . Bacteriol . 139:824-834, 1979) . In the absence of any added stimuli for chemotaxis, both the calC and the calD mutants swam with a tumbly bias . Both the calC and the calD mutants were defective in chemotaxis as measured by computer analysis, use of swarm plates, and capillary assays . The calA mutant was only slightly defective in motility and only slightly impaired in chemotaxis . Chemotactically wild-type cells had an intra-cellular free-Ca2+ level of about 105 nM . The intracellular free-Ca2+ levels of the mutants, as determined by use of the fluorescent Ca2+ indicator dye fura-2 or fluo-3, were about 90, about 1,130, and about 410 nM for calA, calC, and calD, respectively . Lowering the intracellular free-Ca2+ levels in wild-type cells and in the tumbly cal mutants by use of Ca2+ chelators promoted running (smooth swimming) . Overexpression of CheZ (which causes dephosphorylation of CheY-phosphate) in the wild type and in the tumbly cal mutants decreased the level of tumbliness (which is caused by CheY-phosphate) . The calA mutant was 4- to 10-fold more resistant than the wild type to the inhibitory effect of omega-conotoxin on chemotaxis . omega-Conotoxin had no effect on Ca2+ extrusion by wild-type E . coli; that result suggests that omega-conotoxin affects Ca2+ transport at the point of entry instead of exit. J Bacteriol, 1995 Dec, 177(24), 7105 - 11 Characterization of the celB gene coding for beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus and its expression and site-directed mutation in Escherichia coli; Voorhorst WG et al.; The celB gene encoding the cellobiose-hydrolyzing enzyme beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus has been identified, cloned, and sequenced . The transcription and translation gene was overexpressed in Escherichia coli, resulting in high-level (up to 20% of total protein) production of beta-glucosidase that could be purified by a two-step purification procedure . The beta-glucosidase produced by E . coli had kinetic and stability properties similar to those of the beta-glucosidase purified from P . furiosus . The deduced amino acid sequence of CelB showed high similarity with those of beta-glycosidases that belong to glycosyl hydrolase family 1, implicating a conserved structure . Replacement of the conserved glutamate 372 in the P . furiosus beta-glucosidase by an aspartate or a glutamine led to a high reduction in specific activity (200- or 1,000-fold, respectively), indicating that this residue is the active site nucleophile involved in catalysis above 100 degrees C. Cell, 1995 Dec 1, 83(5), 783 - 91 Identification of double Holliday junctions as intermediates in meiotic recombination; Schwacha A et al.; During meiosis, branched DNA molecules containing information from both parental chromosomes occur in vivo at loci where meiosis-specific double-stranded breaks occur . We demonstrate here that these joint molecules are recombination intermediates: they contain single strands that have undergone exchange of information . Moreover, these joint molecules are resolved into both parental and recombinant duplexes when treated in vitro with Holliday junction-resolving endonucleases RuvC or T4 endo VII . Taken together with previous observations, these results strongly suggest that joint molecules are double Holliday junctions. Mol Carcinog, 1995 Dec, 14(4), 233 - 9 Mutational specificity of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea in the Escherichia coli lacl gene of O6-alkylguanine-DNA alkyltransferase-proficient and -deficient strains; Jurado J et al.; Forward mutations induced by 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) in the lacl gene of Escherichia coli were recovered from bacteria proficient (Ogt+ Ada+) and deficient (Ogt- Ada-) in O6-alkylguanine-DNA alkyltransferase activity . A CCNU dose of 1 mM was selected for DNA sequence analysis . A total of 245 induced mutations were characterized . The mutations were almost exclusively (95%) GC-->AT transitions, indicating that CCNU-induced mutations arose in bacteria primarily from misreplication of O6-chloroethylguanine, in total agreement with results obtained for monofunctional alkylating agents . The distribution of CCNU-induced GC-->AT mutations was significantly altered by the presence of DNA alkyltransferase activity (P = 0.01) . In the Ogt+ Ada+ mutational spectrum, guanines flanked on both sides by A:T base-pairs were on average 2.8 times more likely to mutate than those flanked by G:C base-pairs on at least one side . This bias disappeared in the Ogt- Ada- genetic background, thereby providing evidence that O6-chloroethylated guanines adjacent to G:C base-pairs are better targets for bacterial alkyltransferase than those not adjacent to G:C base-pairs . We recently reported a similar bias for ethyl methanesulfonate, strengthening the idea that CCNU is acting as a simple ethylating compound . In summary, this paper presents for the first time evidence that DNA repair by O6-alkylguanine-DNA alkyltransferases plays a major role in removing lesions responsible for GC-->AT transitions induced by CCNU, influencing their ultimate distribution with respect to sequence context. Acta Virol, 1994 Dec, 38(6), 311 - 5 Recombination between genetically modified and unmodified Autographa californica nuclear polyhedrosis virus in Trichoplusia ni larvae; Merryweather-Clarke AT et al.; Trichoplusia ni larvae have been injected with a mixture of wild-type Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) and a mutant derivative, AcRP8.UW1.lacZ, which lacks the polyhedrin gene, and has the p10 gene replaced by the Escherichia coli beta-galactosidase gene . Following plaque assay of the haemolymph and subsequent staining for beta-galactosidase activity and scoring for polyhedra, recombinant plaques were identified and the recombination frequency estimated as 6.6%. J Neurochem, 1995 Dec, 65(6), 2812 - 5 Phosphorylation of F1F0 ATPase delta-subunit is regulated by platelet-derived growth factor in mouse cortical neurons in vitro; Zhang FX et al.; The delta subunit of F1F0 ATPase (ATP synthase complex) is part of the stalk connecting the F1 and F0 moieties . Studies in Escherichia coli suggest that the analogous bacterial subunit, called epsilon, is essential for the ATPase assembly energy coupling . Platelet-derived growth factor (PDGF) is an important growth factor for various cell types, including neurons of the CNS . Using two-dimensional gel electrophoresis, microsequencing, western blot analysis, and immunoprecipitation techniques, we have found that PDGF induces phosphorylation of the delta subunit