Escherichia coli

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 or a closely related peptide in cultured mouse cortical neurons.

J Bacteriol, 1995 Dec, 177(23), 6973 - 7
Strong function-related homology between the pore-forming colicins K and 5; Pilsl H et al.; Sequence determination of the Escherichia coli colicin K determinant revealed identity with the E . coli colicin 5 determinant in the immunity and lysis proteins, strong homologies in the pore-forming region (93.7%) and the Tsx receptor-binding region (77%) of the colicins, and low levels of homology (20.3%) in the N-terminal region of the colicins . This latter region is responsible for the Tol-dependent uptake of colicin K and the Ton-dependent uptake of colicin 5 in the respective colicins . During evolution, the DNA encoding colicin activity and binding to the Tsx receptor was apparently recombined with two different DNA fragments that determined different uptake routes, leading to the differences observed in colicin K and colicin 5 import.

J Bacteriol, 1995 Dec, 177(23), 6966 - 72
Evidence that the immunity protein inactivates colicin 5 immediately prior to the formation of the transmembrane channel; Pilsl H et al.; Determination and analysis of the nucleotide sequences of the activity, immunity, and lysis genes of colicin 5 assigned colicin 5 to the subclass of pore-forming colicins to which colicins 10, E1, Ia, Ib, and K belong . Mutational analysis of colicin 5 and exchange of DNA fragments between the most closely related colicins, colicins 5 and 10, and between their immunity proteins localized the regions that determine the reaction specificity between colicin 5 and its immunity protein to residues 405 to 424 of colicin 5, the region corresponding to the amphiphilic alpha-helix 6 of the similar colicins E1 and Ia . The specificity-conferring residues 55 to 58 and 68 to 75 of the immunity protein were localized in the cytoplasmic loop and the inner leaflet of the cytoplasmic membrane . The localization of the reactive regions of the immunity protein and the colicin close to the inner side of the cytoplasmic membrane suggests that the immunity protein inactivates colicin 5 shortly before the lethal colicin pores in the cytoplasmic membrane are opened.

J Bacteriol, 1995 Dec, 177(23), 6854 - 60
Principal sigma subunit of the Caulobacter crescentus RNA polymerase; Malakooti J et al.; We have identified the gene encoding the Caulobacter crescentus principal sigma subunit, RpoD . The rpoD gene codes for a polypeptide of 653 amino acids with a predicted molecular mass of 72,623 Da (sigma 73) . The C . crescentus sigma subunit has extensive amino acid sequence homology with the principal sigma factors of a number of divergent procaryotes . In particular, the segments designated region 2 that are involved in core polymerase binding and promoter recognition were identical among these bacteria despite the fact that the -10 region recognized by the C . crescentus sigma 73 differs significantly from that of the other bacteria . Thus, it appears that additional sigma factor regions must be involved in -10 region recognition . This conclusion was strengthened by a heterologous complementation assay in which C . crescentus sigma 73 was capable of complementing the Escherichia coli rpoD285 temperature-sensitive mutant . Furthermore, C . crescentus sigma 73 conferred new specificity on the E . coli RNA polymerase, allowing the expression of C . crescentus promoters in E . coli . Thus, the C . crescentus sigma 73 appears to have a broader specificity than does the sigma 70 of the enteric bacteria.

J Bacteriol, 1995 Dec, 177(23), 6832 - 5
Regulation of RNA polymerase sigma subunit synthesis in Escherichia coli: intracellular levels of sigma 70 and sigma 38; Jishage M et al.; The intracellular levels of two principal sigma subunits, sigma 70 (sigma D, the rpoD gene product) and sigma 38 (sigma s, the rpoS gene product), in Escherichia coli MC4100 were determined by a quantitative Western immunoblot analysis . Results indicate that the level of sigma 70 is maintained at 50 to 80 fmol per micrograms of total proteins throughout the transition from the exponential growth phase to the stationary phase, while the level of sigma 38 protein is below the detection level at the exponential growth phase but increases to 30% of the level of sigma 70 when cell growth stops to enter into the stationary phase . Beside the stationary phase, the increase in sigma 38 level was observed in two cases: exposure to heat shock at the exponential phase and osmotic shock at the stationary phase.

J Bacteriol, 1995 Dec, 177(23), 6782 - 90
Superoxide and the production of oxidative DNA damage; Keyer K et al.; The conventional model of oxidative DNA damage posits a role for superoxide (O2-) as a reductant for iron, which subsequently generates a hydroxyl radical by transferring the electron to H2O2 . The hydroxyl radical then attacks DNA . Indeed, mutants of Escherichia coli that lack superoxide dismutase (SOD) were 10-fold more vulnerable to DNA oxidation by H2O2 than were wild-type cells . Even the pace of DNA damage by endogenous oxidants was great enough that the SOD mutants could not tolerate air if enzymes that repair oxidative DNA lesions were inactive . However, DNA oxidation proceeds in SOD-proficient cells without the involvement of O2-, as evidenced by the failure of SOD overproduction or anaerobiosis to suppress damage by H2O2 . Furthermore, the mechanism by which excess O2- causes damage was called into question when the hypersensitivity of SOD mutants to DNA damage persisted for at least 20 min after O2- had been dispelled through the imposition of anaerobiosis . That behavior contradicted the standard model, which requires that O2- be present to rereduce cellular iron during the period of exposure to H2O2 . Evidently, DNA oxidation is driven by a reductant other than O2-, which leaves the mechanism of damage promotion by O2- unsettled . One possibility is that, through its well-established ability to leach iron from iron-sulfur clusters, O2- increases the amount of free iron that is available to catalyze hydroxyl radical production . Experiments with iron transport mutants confirmed that increases in free-iron concentration have the effect of accelerating DNA oxidation . Thus, O2- may be genotoxic only in doses that exceed those found in SOD-proficient cells, and in those limited circumstances it may promote DNA damage by increasing the amount of DNA-bound iron.

J Bacteriol, 1995 Dec, 177(23), 6766 - 72
Interactions of the origin of replication (oriV) and initiation proteins (TrfA) of plasmid RK2 with submembrane domains of Escherichia coli; Mei J et al.; It has been possible to locate a submembrane domain representing less than 10% of the total membrane that appears to be responsible for sequestering some essential components required for plasmid RK2 DNA replication . This subfraction, whose cellular location in the membrane prior to extraction is still unknown, is derived from the inner membrane fraction, since it possesses enzyme marker activity (NADH oxidase) exclusively associated with the inner membrane . The subfraction was detected by a modification of the methods of Ishidate et al . (K . Ishidate, E . S . Kreeger, J . Zrike, S . Deb, B . Glauner, T . MacAlister, and L . I . Rothfield, J . Biol . Chem . 261:428-443, 1986) in which low pressure in a French pressure cell and lysozyme were used to preserve the supercoil plasmid DNA template during cell disruption . This was followed by successive cycles of sucrose gradient sedimentation and flotation density gradient centrifugation to reveal a number of subfractions, including the one of interest . The characteristics of plasmid interaction with the subfraction include the presence of supercoil DNA after extraction, the binding of the origin of plasmid replication (oriV) in vitro, and the association of the two plasmid-encoded initiation (TrfA) proteins (encoded by overlapping genes) . However, another peak, the outer membrane fraction, also binds oriV in vitro, contains plasmid DNA in vivo, and associates with the TrfA initiation proteins . Nevertheless, it contains much less of the initiation proteins, and the specific activity of binding oriV is also much reduced compared with the other subfraction . There is a strong correlation between the association of the TrfA initiation proteins with a particular membrane fraction and the binding of oriV in vitro or plasmid DNA in vivo . Since the proteins are known to bind to repeated sequences in oriV (S . Perri, D . R . Helinski, and A . Toukdarian, J . Biol . Chem . 266:12536-1254, 1991; M . Pinkney, R . Diaz, E . Lanka, and C . M . Thomas, J . Mol . Biol . 203: 927-938, 1988), it appears that the initiation proteins themselves could be responsible, at least in part, for the association of plasmid DNA to the membrane.

J Bacteriol, 1995 Dec, 177(23), 6740 - 4
Mapping of the OxyR protein contact site in the C-terminal region of RNA polymerase alpha subunit; Tao K et al.; The Escherichia coli OxyR protein requires the C-terminal contact site I region of the RNA polymerase alpha subunit for cooperative interaction with and transcription activation at OxyR-dependent promoters, suggesting direct protein-protein contact between OxyR and the C-terminal region of the alpha subunit . To determine the precise location of the OxyR protein contact site(s) in this region, we carried out mutational analysis of the 3' half of E . coli rpoA, the gene encoding the alpha subunit of RNA polymerase . We isolated a number of rpoA mutants defective in oxyR-dependent transcription activation at the E . coli katG promoter . Nucleotide sequence analysis of the rpoA gene from these mutants revealed that the mutations showing clear phenotypes are all clustered at two narrow regions (amino acid residues 265 to 269 and 293 to 300) within the C terminus of the alpha subunit . Reconstituted RNA polymerases containing the mutant alpha subunits were unable to respond to transcription activation in vitro at the katG, ahpC, and oxyX promoters by OxyR . These results suggest that these two regions comprise the contact surfaces on the alpha subunit for OxyR.

J Bacteriol, 1995 Dec, 177(23), 6732 - 9
Altered (copy-up) forms of initiator protein pi suppress the point mutations inactivating the gamma origin of plasmid R6K; Urh M et al.; The R6K gamma origin core contains the P2 promoter, whose -10 and -35 hexamers overlap two of the seven binding sites for the R6K-encoded pi protein . Two mutations, P2-201 and P2-203, which lie within the -35 region of P2, are shown to confer a promoter-down phenotype . We demonstrate here that these mutations prevent replication of a gamma origin core plasmid . To determine whether or not the reduced promoter activity caused by these mutations is responsible for their effect on replication, we generated two new mutations (P2-245-6-7 and P2-246) in the -10 hexamer of the P2 promoter . Although these new mutations inhibit P2 activity as much as the P2-201 and P2-203 mutations, they do not prevent replication of the gamma origin core . Therefore, activity of the P2 promoter does not appear to be required for replication . We also show that the inability of the gamma origin to function in the presence of the P2-201 and P2-203 mutations is reversed by the hyperactive variants of pi protein called copy-up pi . This suppression occurs despite the fact that in vivo dimethyl sulfate methylation protection patterns of the gamma origin iterons are identical in cells producing wild-type pi and those producing copy-up pi variants . We discuss how the P2-201 and P2-203 mutations could inhibit replication of the gamma origin core and what mechanisms might allow the copy-up pi mutants to suppress this deficiency.

J Bacteriol, 1995 Dec, 177(23), 6704 - 10
Spacing requirements for transcription activation by Escherichia coli FNR protein; Wing HJ et al.; We cloned a consensus DNA site for the Escherichia coli FNR protein at different locations upstream of the E . coli melR promoter . FNR can activate transcription initiation at the melR promoter when the FNR binding site is centered around 41, 61, 71, 82, and 92 bp upstream from the transcription start . The SF73 positive control amino acid substitution in FNR interfered with transcription activation by FNR in each case . In contrast, the GA85 positive control substitution reduced activation only at the promoter, where the FNR binding site is 41 bp upstream of the transcript start . The SF73 substitution appears to identify an activating region of FNR that is important for transcription activation at promoters that differ in architecture . Experiments with oriented heterodimers showed that this activating region is functional in the upstream subunit of the FNR dimer at the promoter where FNR binds around 41 bp from the transcript start and in the downstream subunit at the promoters where FNR binds farther upstream.

J Bacteriol, 1995 Dec, 177(23), 6695 - 703
Growth of Escherichia coli K88 in piglet ileal mucus: protein expression as an indicator of type of metabolism; Blomberg L et al.; The physiological and molecular responses of enterotoxigenic Escherichia coli K88 strain Bd 1107/7508 during growth in piglet ileal mucus and lipids extracted from mucus were studied in terms of growth rate, protein expression, and rate of heat production . E . coli K88 multiplied at maximum speed in mucus and in lipids extracted from mucus . By two-dimensional gel electrophoresis of {35S}methionine-labelled cells, it was demonstrated that the synthesis of a subclass of 13 proteins was changed at least fourfold during exponential growth in mucus compared with growth in M9 minimal medium . Ten of these proteins were repressed, while three were induced, and one of the induced proteins was identified as heat shock protein GroEL . Furthermore, two-dimensional analysis of E . coli K88 cells grown on lipids extracted from mucus revealed a set of lipid utilization-associated proteins . None of these was induced fourfold during exponential growth in mucus . Microcalorimetric measurements (monitoring the rate of heat production) of E . coli K88 grown in mucus indicated metabolic shifts in the stationary phase, in which five of the lipid utilization-associated proteins were expressed at a higher level . An isogenic E . coli K88 fadAB mutant deficient in fatty acid degradation genes grew as well as the wild type on mucus and mucus lipids . The heat production rate curve of the mutant grown in mucus differed from that of the wild type only during the stationary phase . From these results it was concluded that protein expression is influenced when E . coli K88 is grown in piglet ileal mucus rather than in M9 minimal medium . Lipids extracted from ileal mucus can serve as a substrate for E . coli K88 but appear not to be utilized during exponential growth in mucus . Stationary-phase cells metabolize fatty acids; however, the functional purpose of this is unclear.

Infect Immun, 1995 Dec, 63(12), 4949 - 52
Monoclonal antibodies specific for the bundle-forming pilus of enteropathogenic Escherichia coli; Giron JA et al.; The bundle-forming pilus (BFP) produced by enteropathogenic Escherichia coli (EPEC) is associated with the presence of a large EPEC adherence factor plasmid and the formation of localized adherence clusters on tissue culture cells . Three mouse monoclonal antibodies (ICA2, ICA3, and ICA4) were produced against BFP purified from EPEC B171 (O111:NM) . These monoclonal antibodies reacted in immunoblots with different epitopes of the 19.5-kDa bundlin subunit of BFP of heterologous EPEC . These reagents could serve as diagnostic tools for the identification of EPEC as well as for studying the role of BfpA in the interaction of EPEC with eukaryotic cells.

Infect Immun, 1995 Dec, 63(12), 4946 - 8
Identification and functional characterization of thioredoxin of Mycobacterium tuberculosis; Wieles B et al.; We have previously described a Mycobacterium tuberculosis protein designated MPT46 that was present in culture filtrates . Here we report that the MPT46 protein is thioredoxin of M . tuberculosis . MPT46 is recognized by antibodies to thioredoxin (Trx) of Escherichia coli, and antibodies of MPT46 recognize Mycobacterium leprae Trx . Moreover, MPT46 was shown to have enzymatic activity identical to that of Trx of other species, such as its ability to reduce insulin . These findings identify MPT46 as a functionally active Trx.

Infect Immun, 1995 Dec, 63(12), 4917 - 20
Inhibition of S-fimbria-mediated adhesion to human ileostomy glycoproteins by a protein isolated from bovine colostrum; Ouwehand AC et al.; The aim of this study was to isolate and purify the component in bovine colostrum which is responsible for the inhibition of S-fimbria-mediated adhesion of Escherichia coli . Whey from defatted colostrum was fractionated by ultrafiltration, and the < 100K, < 30K, and < 10K fractions and the colostral whey were tested for inhibition of in vitro adhesion of radiolabelled S-fimbria-bearing E . coli to human ileostomy glycoproteins, which provide a model for human intestinal mucus . The inhibiting compound was purified from a dialyzed < 30K fraction with an anion exchange column which was eluted with a NaCl gradient (0 to 1.0 M) . The compound was found to be a heat-resistant but pepsin-sensitive protein with an Mr of approximately 18,000 and an isoelectric point of approximately 5.75 . The protein appears to block receptor sites for S-fimbriae on ileostomy glycoproteins, with steric hindrance being the most likely mechanism . Analysis of the amino acid sequence of the amino terminus of the 18K protein showed similarity with the sequence of beta-lactoglobulin.

Infect Immun, 1995 Dec, 63(12), 4849 - 56
Genes for CS2 pili of enterotoxigenic Escherichia coli and their interchangeability with those for CS1 pili; Froehlich BJ et al.; We have cloned and sequenced the DNA needed for production of CS2 pili in Escherichia coli K-12 . The four open reading frames, cotB, cotA, cotC, and cotD, show homology with the genes needed for production of CS1 and CFA/I pili, which are also found on enterotoxigenic E . coli associated with human diarrheal disease . We also report that CotA plus CotB interact with the CS1 gene products CooC and CooD to form pili that can be visualized by electron microscopy and, conversely, that the CS1 gene products CooA and CooB interact with CotC and CotD to form pili.

Infect Immun, 1995 Dec, 63(12), 4744 - 54
Characterization, genetic analysis, and expression of a protease antigen (PrpRI) of Porphyromonas gingivalis W50; Aduse-Opoku J et al.; Previous studies of the serum immunoglobulin G antibody response of periodontal patients have demonstrated significant reactivity to a cell surface or extracellular arginine-specific protease of Porphyromonas gingivalis which migrates as an approximately 50-kDa band on sodium dodecyl sulfate-polyacrylamide gels . In the present report, two forms of the enzyme (ArgI and ArgIA) with this electrophoretic behavior were isolated . ArgI is a heterodimer of alpha and beta subunits, and ArgIA is a monomer composed of the catalytically active alpha component alone . The gene encoding ArgI (prpR1 encoding protease polyprotein ArgI) was cloned from Sau3AI digests of P . gingivalis W50 DNA into pUC18 . Sequence analysis demonstrated that the alpha and beta components are contiguous on the initial translation product and are flanked by large N- and C-terminal extensions . prpR1 is 97.5% identical to the rgp-1 gene from P . gingivalis H66 . prpR1 expression in Escherichia coli demonstrated the presence of an internal transcription-translation initiation site which could permit independent expression of different regions of the polyprotein . Immunochemical analysis of P . gingivalis mid-logarithmic-phase cultures suggested that the processing of PrpRI may be closely coupled to its synthesis, with only the final stages taking place at the cell surface . Southern hybridization studies demonstrated that the prpR1 gene is widely distributed in other P . gingivalis strains and that a second homologous locus to the alpha component and at least two other homologous loci to the beta component are present on the P . gingivalis chromosome . These data indicate that the ArgI protease of P . gingivalis is a member of a family of sequence-related gene products which may share both functional and antigenic properties.

Infect Immun, 1995 Dec, 63(12), 4715 - 20
Contribution of individual disulfide bonds to biological action of Escherichia coli heat-stable enterotoxin B; Arriaga YL et al.; Heat-stable enterotoxins (STs) of Escherichia coli are peptides which alter normal gut physiology by stimulating the loss of water and electrolytes . The action of heat-stable toxin B (STb) is associated with an increase in levels of lumenal 5-hydroxytryptamine and prostaglandin E2, known mediators of intestinal secretion . In addition, the toxin is responsible for elevation of cytosolic calcium ion levels in cultured cells . STb is a 48-amino-acid basic peptide containing four cysteine residues and two disulfide bonds . Previous work indicates that disulfide bonds are required for intestinal secretory activity, and yet the relative contribution of the two bonds to toxin stability and action is presently unclear . Site-directed mutagenesis was used to alter the cysteine residues of STb to assess the role of the individual disulfide bonds in toxin activity . Our results indicate that loss of a single disulfide bond was sufficient to abolish the intestinal secretory and G protein-coupled calcium ion influx activities associated with STb toxicity . Loss of toxin action was not a function of increased sensitivity of STb mutants to proteolysis, since mutant toxins displayed proteolytic decay rates equivalent to that of wild-type STb . Circular dichroism spectroscopy of mutant STb toxins indicated that single-disulfide-bond elimination did not apparently affect the toxin secondary structure of one mutant, STbC33S,C71S . In contrast, the alpha-helical content of the other disulfide bond mutant, STbC44S,C59G, was significantly altered, as was that of reduced and alkylated authentic STb . Since both Cys-Cys mutant STbs were completely nontoxic, the absence of biological activity cannot be explained by dramatic secondary structural changes alone; keys to the conformational requirements for STb toxicity undoubtedly reside in the three-dimensional structure of this peptide.

Infect Immun, 1995 Dec, 63(12), 4613 - 8
Mapping of the delayed-type hypersensitivity-inducing epitope of secreted protein MPT64 from Mycobacterium tuberculosis; Oettinger T et al.; The gene encoding the immunogenic protein MPT64 found in culture filtrates of Mycobacterium tuberculosis H37Rv was expressed in Escherichia coli K-12 and purified as a recombinant protein . The purified recombinant MPT64 elicited delayed-type hypersensitivity (DTH) in outbred guinea pigs sensitized with Mycobacterium bovis BCG Tokyo . The skin reactions were comparable to those obtained with native MPT64 . No skin reactions were observed when either recombinant MPT64 or native MPT64 was used in guinea pigs sensitized with M . bovis BCG Danish 1331 . Amino- and carboxy-terminal deletion mutants of MPT64 were purified as fusion proteins for the mapping of DTH-inducing epitopes on recombinant MPT64 by use of the guinea pig skin test model . The part of the molecule responsible for the biological activity was located at the carboxy-terminal end . Further studies with overlapping synthetic peptides have pinpointed the biological activity at a single DTH-inducing epitope consisting of 15 residues between amino acids Gly-173 and Ala-187 . Screening by PCR of 56 clinical isolates of M . tuberculosis from Danish and Tanzanian patients demonstrated the presence of mpt64 in all of the strains . These results point to MPT64 as a possible candidate for a skin test reagent specific for diagnosis of human tuberculosis.

Cancer Res, 1995 Dec 1, 55(23), 5489 - 92
Mismatch repair, somatic mutations, and the origins of cancer; MacPhee DG; This paper outlines the basic properties of a newly recognized pathway that should enable somatic cells to generate double-stranded mutations in the complete absence of cell proliferation . Recognition of the existence of this pathway provides us with the basis for a better understanding of a number of important biological phenomena and, in particular, may help us to understand the origins of cancers in unselected human populations.

Am J Respir Cell Mol Biol, 1995 Dec, 13(6), 703 - 11
Accelerated binding of secretory leukoprotease inhibitor to human leukocyte elastase mediated by single-stranded sites in DNA from tracheobronchial mucus; Ying QL et al.; We have found that preparations of DNA isolated from purulent sputum possess a novel activity which accelerates and stabilizes the binding of human leukocyte elastase to secretory leukoprotease inhibitor, a major endogenous antielastase in the respiratory tract . DNA in sputum is derived from the nuclear debris of disintegrated inflammatory leukocytes, and can attain concentrations ranging from 10(2) to 10(4) micrograms/ml, depending on the severity of pulmonary infection and inflammation . In the presence of 23 micrograms/ml DNA, a concentration lower than those found in most purulent sputa, the rate constant for association of secretory leukoprotease inhibitor with elastase is increased to 1.1 x 10(8) M-1s-1, 44-fold greater than that in the absence of DNA . The equilibrium dissociation constant for the enzyme-inhibitor complex drops to 0.7 pM, two orders of magnitude lower than that in the absence of DNA . The accelerating effect of DNA is further increased by thermal denaturation or by modification with exonuclease III, while it is significantly reduced by digestion with S1 nuclease or by binding of Escherichia coli single-stranded DNA binding protein . The results from these experiments indicate that the structural elements in sputum DNA that are responsible for the accelerating effect have the characteristics of single-stranded sites . Similar kinetic effects on elastase inhibition were also observed with human placental DNA and genomic DNAs from a variety of other species . These findings suggest that DNA in pulmonary secretions may participate in antielastase defense by promoting the binding of secretory leukoprotease inhibitor to leukocyte elastase . The results may have important implications for use of nuclease preparations in mucolytic therapy for cystic fibrosis.

Arch Biochem Biophys, 1995 Dec 1, 324(1), 59 - 64
Purification of human matrilysin produced in Escherichia coli and characterization using a new optimized fluorogenic peptide substrate; Welch AR et al.; Human promatrilysin (matrix metalloproteinase-7) has been produced in Escherichia coli as an N-terminal fusion protein with ubiquitin . The insoluble product was solubilized, refolded, and activated with amino-phenylmercuric acetate . Activation of the fusion protein demonstrated kinetics and intermediates that were very similar to those observed during activation of promatrilysin produced in Chinese Hamster Ovary (CHO) cells . Following activation, matrilysin was purified to > 95% homogeneity using a Sepharose-Pro-Leu-Gly-NHOH affinity column . The matrilysin purified by this procedure is indistinguishable from the enzyme purified from CHO cells with respect to the kinetic parameters for hydrolysis of a peptide substrate and the ability to obtain diffraction quality crystals in the presence of an inhibitor of the enzyme . Additionally, to facilitate detailed kinetic analyses of matrilysin, a new fluorogenic peptide substrate with the optimized sequence Dnp-Arg-Pro-Leu-Ala-Leu-Trp-Arg-Ser (Dnp, dinitrophenyl) has been synthesized . This peptide is the best substrate developed for matrilysin thus far with Km and kcat values of 26 microM and 5.0 s-1, respectively.

Arch Biochem Biophys, 1995 Dec 1, 324(1), 153 - 8
Mutagenesis and reversion analysis of residue Met-209 of the beta-subunit of Escherichia coli ATP synthase; Wilke-Mounts S et al.; Residue beta-Met-209 is conserved in all known F1-ATPase sequences, and the mutation beta M209I in Escherichia coli causes profound inhibition of ATP synthesis and hydrolysis . Based on the properties of this mutant it had previously been proposed that residue beta-209 lies close to the site of catalysis . Two approaches were used to study this residue further . First, revertants were sought . Only wild-type and beta-Ser-209 were found; the Ser revertants involved a two-base change . Significantly, Ser is found at the equivalent position in the homologous vacuolar and archaebacterial ATPases . Second, all 20 natural amino acids were placed at position beta-209 by mutagenesis, and catalytic properties of the mutants were analyzed . The results showed that only a limited set of residues supported significant growth or ATPase activity, and that many of the mutations impacted severely on catalysis . X-ray structure analysis of the bovine enzyme has revealed that residue beta-Met-209 lies only 3.1 . A from residue beta-Glu-181, which has been proposed to act as catalytic base . The results reported here emphasize that, in this discrete region of the catalytic site, specific stereo-chemical constraints on structure are critical for catalysis.

Arch Biochem Biophys, 1995 Dec 1, 324(1), 143 - 52
Inactivation of human immunodeficiency virus type 1 reverse transcriptase by oltipraz: evidence for the formation of a stable adduct; Chavan SJ et al.; Oltipraz (5-pyrazinyl-4-methyl-1,2-dithiole-3-thione), which is undergoing clinical evaluation as an anticarcinogen, also inhibits HIV-1 replication (IC50 approximately equal to 10 microM) . The inactivation of RT appears to be a relevant antiviral mechanism since oltipraz blocks viral replication in acutely infected T-cell lines, but is ineffective in chronically infected ACH-2 cells (H . J . Prochaska, W . G . Bornmann, P . Baron, and B . Polsky (1995) Mol . Pharmacol . 48, 15-20) . Since a nucleophilic amino acid is a likely target for oltipraz, we assessed whether the conserved cysteine residues of HIV-1 RT (38Cys or 280Cys) were the target(s) for oltipraz, and we synthesized {Me 14C}oltipraz to determine if oltipraz forms a stable adduct with RT . Thus, HIV-2 RT as well as wild-type, 38Cys-->Ser, 280Cys-->Ser, and the Cys-->Ser double mutant of HIV-1 RT were purified from the lysates of transformed Escherichia coli strain DH5 alpha (A . Hizi, M . Shaharabany, R . Tal, and S . H . Hughes (1992) J . Biol . Chem . 267, 1293-1297) via a purification procedure that included (NH4)2SO4 fractionation followed by gel filtration, dye-ligand, and ion-exchange chromatographies . Procion yellow H4R was chosen as the dye-ligand chromatography since it was the most potent and selective inhibitor of RT among seventy reactive dyes that were screened . Mono Q anion-exchange chromatography with diethanolamine (pH 9) resulted in the generation of heterodimeric RT from a predominantly homodimeric enzyme preparation . Because the instability of dilute RT preparations at room temperature rendered the kinetic evaluation of inactivation difficult, we sought to identify conditions that prevent denaturation of these enzymes . High concentrations (25 mM) of MgCl2 had a stabilizing effect . Oltipraz behaved kinetically as an irreversible inhibitor of all RTs purified, and the kinetic constants for the inactivation of these enzymes were not significantly different from wild-type HIV-1 RT (Ki = 17.0 +/- 4.1 microM; k3 = 0.214 +/- 0.051 h-1) . In stark contrast, oltipraz neither inhibited nor inactivated the Klenow fragment of DNA polymerase I, whose subdomain structure is similar to the p66 subunit of RT . Wild-type RT was incubated with 60 microM {Me 14C}oltipraz for 4 h and was then subjected to gel filtration chromatography . The {14C} label comigrated with RT with a stoichiometry of binding of 0.88 +/- 0.05 oltipraz per inactivated RT subunit (N = 3 experiments), and the {14C} label remained bound after treatment with 4 M urea.(ABSTRACT TRUNCATED AT 400 WORDS)

J Urol, 1995 Dec, 154(6), 2179 - 84
Local expression of cytokine messenger RNA in rat model of Escherichia coli epididymitis; Tanaka K et al.; PURPOSE: In order to investigate whether cytokines play an important role in the regulation of host defense against bacterial epididymitis, we examined the local expression of cytokine mRNA in a rat model of Escherichia coli epididymitis . MATERIALS AND METHODS: Escherichia coli was inoculated into the spermatic cord of the rats and provoked epididymitis . At 6, 12, 24, 48, and 72 hours postinfection (p.i.) epididymides were harvested . We examined interleukin-6 (IL-6) and other inflammatory cytokine mRNAs in epididymides by Northern blotting and reverse transcriptase polymerase chain reaction (RT-PCR) . Histopathological findings and immunohistochemistry for IL-6 were also examined . RESULTS: Infected epididymides expressed IL-6 mRNA maximally at 6 hours p.i . and then the signal decreased by Northern blotting . mRNA for IL-1 beta and TNF-alpha also increased within 6 hours by RT-PCR . Histopathological examination showed that no inflammatory cells were observed at 6 hours p.i . and that inflammatory changes were most prominent at 72 hours p.i . Epididymal epithelium expressed IL-6 locally in response to local bacterial infection by immunohistochemistry . CONCLUSIONS: mRNA for IL-1 beta, IL-6, and TNF-alpha is expressed locally in response to bacterial infection before inflammatory cell infiltration . This rapid expression in the epithelial cells may be important in host defense to local genital infection.

J Leukoc Biol, 1995 Dec, 58(6), 675 - 82
An analogue of lipid A and LPS from Rhodobacter sphaeroides inhibits neutrophil responses to LPS by blocking receptor recognition of LPS and by depleting LPS-binding protein in plasma; Aida Y et al.; When incubated with lipopolysaccharide (LPS) in the presence of plasma, neutrophils become primed for enhanced release of superoxide in response to triggering by formyl-Met-Leu-Phe (fMLP) . The effect of LPS on phagocytes is inhibited by a synthetic lipid A precursor, LA-14-PP (lipid IVa) or by LPS from Rhodobacter sphaeroides (Rs) . We studied the mechanisms by which LA-14-PP or Rs-LPS inhibited LPS-induced responses . When neutrophils were exposed to LA-14-PP or Rs-LPS for 3 min and then to Escherichia coli-LPS, the antagonists inhibited priming for superoxide release, and also blocked up-regulation of CD11b and adherence . This inhibition was dependent on plasma, was not overcome by higher amounts of E . coli-LPS or plasma, and was not observed at 0 degrees C, suggesting that E . coli-LPS was not able to interact with its receptor or other cellular recognition molecule in neutrophils that had been exposed to the antagonists . The alternative possibility that LA-14-PP or Rs-LPS depleted a plasma cofactor, resulting in inhibition of priming, was investigated by using LPS from Porphyromonas gingivalis (Pg) and Bordetella pertussis (Bp) . These LPS primed neutrophils in a plasma-dependent and CD14-dependent manner, but were not blocked by LA-14-PP or Rs-LPS . When sub-optimal concentrations of plasma were exposed to LA-14-PP or Rs-LPS, and then mixed with Pg-LPS or Bp-LPS, followed by incubation with neutrophils, priming and up-regulation of CD11b were inhibited, and this inhibition was overcome by increasing the concentration of plasma . Binding of LPS-binding protein (LBP) in plasma to immobilized E . coli-LPS was inhibited by pre-incubation of plasma with LA-14-PP or Rs-LPS . Together with the result that treatment of plasma with anti-LBP antibody abolished the cofactor activity of plasma, these results indicated that LA-14-PP and Rs-LPS depleted LBP from plasma, resulting in inability of LPS to act on neutrophils . Thus LA-14-PP and Rs-LPS inhibited the action of LPS on neutrophils by at least two mechanisms, blocking of LPS receptor recognition and depletion of the cofactor LBP.

J Biol Chem, 1995 Dec 1, 270(48), 28989 - 94
Diversity of the pyruvate dehydrogenase kinase gene family in humans; Gudi R et al.; Recent evidence from this laboratory indicates that at least two isoenzymic forms of pyruvate dehydrogenase kinase (PDK1 and PDK2) may be involved in the regulation of enzymatic activity of mammalian pyruvate dehydrogenase complex by phosphorylation (Popov, K.M., Kedishvili, N.Y., Zhao, Y., Gudi, R., and Harris, R.A . (1994) J . Biol . Chem . 269, 29720-29724) . The present study was undertaken to further explore the diversity of the pyruvate dehydrogenase kinase gene family . Here we report the deduced amino acid sequences of three isoenzymic forms of PDK found in humans . In terms of their primary structures, two isoenzymes identified in humans correspond to rat PDK1 and PDK2, whereas a third gene (PDK3) encodes for a new isoenzyme that shares 68% and 67% of amino acid identities with PDK1 and PDK2, respectively . PDK3 cDNA expressed in Eschierichia coli directs the synthesis of a polypeptide with a molecular mass of approximately 45,000 Da that possesses catalytic activity toward kinase-depleted pyruvate dehydrogenase . PDK3 appears to have the highest specific activity among the three isoenzymes tested as recombinant proteins . Tissue distribution of all three isoenzymes of human PDK was characterized by Northern blot analysis . The highest amount of PDK2 mRNA was found in heart and skeletal muscle, the lowest amount in placenta and lung . Brain, kidney, pancreas, and liver expressed an intermediate amount of PDK2 (brain > kidney = pancreas > liver) . The tissue distribution of PDK1 mRNA differs markedly from PDK2 . The message for PDK1 was expressed predominantly in heart with only modest levels of expression in other tissues (skeletal muscle > liver > pancreas > brain > placenta = lung > kidney) . In contrast to PDk1 and PDK2, which are expressed in all tissues tested, the message for PDK3 was found almost exclusively in heart and skeletal muscle, indicating that PDK3 may serve specialized functions characteristic of muscle tissues . In all tissues tested thus far, the level of expression of PDK2 mRNA was essentially higher than that of PDK1 and PDK3, consistent with the idea that PDK2 is a major isoenzyme responsible for regulation of pyruvate dehydrogenase in human tissues.

J Biol Chem, 1995 Dec 1, 270(48), 28917 - 23
A novel cis-acting element in a liver cytochrome P450 3A gene confers synergistic induction by glucocorticoids plus antiglucocorticoids; Quattrochi LC et al.; The induction by dexamethasone of rat liver CYP3A1 differs from classical glucocorticoid gene regulation in part because both glucocorticoids and antiglucocorticoids such as pregnenolone 16 alpha-carbonitrile (PCN) induce CYP3A1 through transcriptional gene activation . In the present study, we transiently expressed in primary cultures of rat hepatocytes plasmids consisting of CYP3A1 5'-flanking sequences fused to a chloramphenicol acetyltransferase reporter plasmid . Deletional analysis identified a 78-base pair (bp) element located approximately 135 bp upstream of the transcriptional start site that was inducible by treatment of the cultures with dexamethasone or PCN and was induced synergistically by dexamethasone plus PCN . Nuclear extract from control rat liver protected two regions within the 78-bp sequence against digestion with DNase I . The same two regions were protected when nuclear extracts from dexamethasone-treated animals were used . Analysis of both of the "footprints" (FP1 and FP2) failed to reveal a classical sequence for the glucocorticoid-responsive element . A 33-bp element that includes FP1 sequences inserted into the chloramphenicol acetyltransferase reporter plasmid and transiently expressed in rat hepatocytes conferred a profile of dexamethasone and PCN induction similar to that of the 78-bp element . However, an Escherichia coli expressed glucocorticoid receptor protein failed to protect sequences within FP1 in DNase I footprinting experiments and failed to change its mobility in gel shift assays . Moreover, as judged by the gel shift assay, the specific protein binding to this fragment was the same whether nuclear extracts from the liver of untreated or dexamethasone-treated rats were used . We conclude that the activation of CYP3A1 gene transcription by glucocorticoids may involve proteins already bound to the controlling element in the CYP3A1 gene through a mechanism in which GR in the presence of hormone does not bind directly to CYP3A1 DNA.

J Biol Chem, 1995 Dec 1, 270(48), 28839 - 47
Cloning, expression, and characterization of the TATA-binding protein (TBP) promoter binding factor, a transcription activator of the Acanthamoeba TBP gene; Huang W et al.; TATA-binding protein (TBP) gene promoter binding factor (TPBF) is a transactivator which binds to the TBP promoter element (TPE) sequence of the Acanthamoeba TBP gene promoter and stimulates transcription in vitro . We have isolated a cDNA clone encoding TPBF . TPBF is a polypeptide of 327 amino acids with a calculated molecular mass of 37 kDa . The predicted amino acid sequence of TPBF shows no significant homology to other proteins . TPBF has two potential coiled-coil regions, a basic region, a proline-rich region, a histidine-rich N terminus, and a nuclear targeting sequence . The recombinant protein has an apparent molecular mass of 50 kDa, identical with that of TPBF purified from Acanthamoeba . Recombinant TPBF is able to bind DNA and activate transcription with the same specificity as natural Acanthamoeba TPBF, demonstrating the authenticity of the clone . Mobility shift assays of co-translated TPBF polypeptides and chemical cross-linking demonstrate that TPBF is tetrameric in solution and when bound to DNA . Analyses of TPBF mutants show that Coiled-coil II is essential for DNA binding, but Coiled-coil I and the basic region are also involved . TPBF is thus a novel DNA-binding protein with functional similarity to the tumor suppressor protein p53.

J Biol Chem, 1995 Dec 1, 270(48), 28635 - 41
Thioredoxin-linked "thiol peroxidase" from periplasmic space of Escherichia coli; Cha MK et al.; Three different molecular masses (24, 22, and 20 kDa) of antioxidant proteins were purified in Escherichia coli . These proteins exhibited the preventive effects against the inactivation of glutamine synthetase activity and the cleavage of DNA by a metal-catalyzed oxidation system capable of generating reactive oxygen species . Their antioxidant activities were supported by a thiol-reducing equivalent such as dithiothreitol . Analysis of the amino-terminal amino acid sequences and the immunoblots between 24- and 22-kDa proteins indicates that the 24-kDa protein is an intact form of the 22-kDa protein that was previously identified 22-kDa subunit (AhpC) of E . coli alkyl hydroperoxide reductase (AhpC/AhpF) . We isolated and sequenced an E . coli genomic DNA fragment that encodes 20-kDa protein . Comparison of the deduced amino acid sequence of the 20-kDa protein with that of AhpC revealed no sequence homology . A search of a data bank showed that the 20-kDa protein is a new type of antioxidant enzyme . The synthesis of this novel 20-kDa protein was increased in response to oxygen stress during growth . The 20-kDa protein resides mainly in the periplasmic space of E . coli, whereas the 24-kDa AhpC resides mainly in the matrix . The 20-kDa protein was functionally linked to the thioredoxin as an in vivo thiol-regenerating system and exerted a peroxidase activity . This 20-kDa protein is thus named "thiol peroxidase," which could act as an antioxidant enzyme removing peroxides or H2O2 within the catalase- and peroxidase-deficient periplasmic space of E . coli.

J Biol Chem, 1995 Dec 1, 270(48), 28629 - 34
Triabin, a highly potent exosite inhibitor of thrombin; Noeske-Jungblut C et al.; Triabin, a new thrombin inhibitor, has been purified from the saliva of Triatoma pallidipennis, a blood-sucking triatomine bug . It forms a noncovalent complex with thrombin at a molar ratio of 1:1, inhibits thrombin-induced platelet aggregation, and prolongs thrombin clotting time and activated partial thromboplastin time . However, it only minimally suppresses the amidolytic activity of thrombin, as measured by a chromogenic peptide substrate assay . It completely blocks trypsin-catalyzed cleavage of thrombin, probably via protection of the anion-binding exosite and inhibits the effect of thrombomodulin on thrombin in a dose-dependent fashion . These results indicate that the inhibitor is directed toward the anion-binding exosite of thrombin . The protein was partially sequenced and the information used to isolate cDNA clones from a T . pallidipennis salivary gland library . Four slightly polymorphic variants coding for mature proteins of 142 amino acids preceded by a putative leader sequence were obtained . The recombinant protein expressed in the periplasmic space of Escherichia coli has a biological activity similar to that of salivary triabin, as tested in a thrombin-induced platelet aggregation assay . In addition, recombinant triabin inhibits thrombin-catalyzed hydrolysis of fibrinogen with a Ki of about 3 pM.

J Biol Chem, 1995 Dec 1, 270(48), 28609 - 16
Interaction of deoxyinosine 3'-endonuclease from Escherichia coli with DNA containing deoxyinosine; Yao M et al.; By using a band mobility shift assay, deoxyinosine 3'-endonuclease, an Escherichia coli enzyme which recognizes deoxyinosine, AP site, urea residue, and base mismatches in DNA, was shown to bind tightly to deoxyinosine-containing oligonucleotide duplexes . Two distinct protein-DNA complexes were observed, the faster migrating complex (complex I, Kd = 4 x 10(-9) M) contained one molecule of deoxyinosine 3'-endonuclease, while the slower migrating complex (complex II, Kd = 4 x 10(-7) M) contained two molecules of the protein bound to every molecule of duplex DNA . The endonucleolytic activity of deoxyinosine 3'-endonuclease paralleled the formation of the complex I . Interestingly, deoxyinosine 3'-endonuclease exhibited similar affinities for both the substrate and the nicked duplex product and thus remained bound to the DNA after the cleavage reaction . The formation of a stable complex required the presence of a duplex structure 5' to the deoxyinosine residue . DNase I footprinting revealed that deoxyinosine 3'-endonuclease protected 4-5 nucleotides 5' to the deoxyinosine, and when complex II was formed, at least 13 nucleotides 3' to deoxyinosine were protected . Based on these results, a model is proposed for the interaction of deoxyinosine 3'-endonuclease with DNA containing deoxyinosine.

J Biol Chem, 1995 Dec 1, 270(48), 28586 - 94
Chemical modification of the N-10 ribityl side chain of flavins . Effects on properties of flavoprotein disulfide oxidoreductases; Murthy YV et al.; Three flavin derivatives modified at the 2'-position of the flavin N-10 ribityl side chain were synthesized: arabinoflavin, 2'-F-2'-deoxyarabinoflavin, and 2'-deoxyriboflavin . These were converted to the FAD level with FAD synthetase . Apoproteins of lipoamide dehydrogenase, glutathione reductase, and mercuric reductase, a family of flavoprotein oxidoreductases, were reconstituted with these flavins . Significant reduction of the catalytic activities was observed with the modified enzymes . During anaerobic reduction of the modified enzymes with substrate or dithiothreitol, decreased thermodynamic stability of the two-electron reduced enzyme forms (EH2) and the accumulation of the four-electron reduced forms (EH4) noted . This effect was more pronounced in case of arabino-FAD-reconstituted enzymes than with the other two . It was found that NAD+ binding influences the interaction between the flavin and the reduced disulfide in the 2'-F-arabino-FAD-lipoamide dehydrogenase, presumably by altering the relative oxidation-reduction potentials . 19F NMR data were obtained for different forms of the 2'-F-arabino-FAD-lipoamide dehydrogenase, which suggest marked conformational changes from one form to the other . The 19F NMR data for the oxidized forms of all three 2'-F-arabino-FAD proteins suggest that the fluorine experiences very similar chemical environments at the active sites.

J Biol Chem, 1995 Dec 1, 270(48), 28565 - 9
Cyanide-binding site of bd-type ubiquinol oxidase from Escherichia coli; Tsubaki M et al.; We extended our investigation on the structure of the redox centers of bd-type ubiquinol oxidase from Escherichia coli using cyanide as a monitoring probe . We found that addition of cyanide to the air-oxidized O2-bound enzyme caused appearance of an infrared C-N stretching band at 2161 cm-1 and concomitant disappearance of the 647 nm absorption band of the cytochrome d (Fe2+)-O2 species . Addition of cyanide to the air-oxidized CO-bound enzyme also resulted in disappearance of the 635 nm absorption band and the 1983.4 cm-1 C-O infrared band of the cytochrome d (Fe2+)-CO species . The resulting species had a derivative-shaped electron paramagnetic resonance signal at g = 3.15 . Upon partial reduction with sodium dithionite, this species was converted partly to a transient heme d (Fe3+)-C = N species having an electron paramagnetic resonance signal at gz = 2.96 and a C-N infrared band at 2138 cm-1 . These observations suggest that the active site of the enzyme has a heme-heme binuclear metal center distinct from that of the heme-copper terminal oxidase and that the treatment of the air-oxidized enzyme with cyanide resulted in a cyanide-bridging species with "heme d(Fe3+)-C = N-heme b595(Fe3+)" structure.

J Biol Chem, 1995 Dec 1, 270(48), 28515 - 8
Identification of the site of interaction of the 14-3-3 protein with phosphorylated tryptophan hydroxylase; Ichimura T et al.; The 14-3-3 protein family plays a role in a wide variety of cell signaling processes including monoamine synthesis, exocytosis, and cell cycle regulation, but the structural requirements for the activity of this protein family are not known . We have previously shown that the 14-3-3 protein binds with and activates phosphorylated tryptophan hydroxylase (TPH, the rate-limiting enzyme in the biosynthesis of neurotransmitter serotonin) and proposed that this activity might be mediated through the COOH-terminal acidic region of the 14-3-3 molecules . In this report we demonstrate, using a series of truncation mutants of the 14-3-3 eta isoform expressed in Escherichia coli, that the COOH-terminal region, especially restricted in amino acids 171-213, binds indeed with the phosphorylated TPH . This restricted region, which we termed 14-3-3 box I, is one of the structural regions whose sequence is highly conserved beyond species, allowing that the plant 14-3-3 isoform (GF14) could also activate rat brain TPH . The 14-3-3 box I is the first functional region whose activity has directly been defined in the 14-3-3 sequence and may represent a common structural element whereby 14-3-3 interacts with other target proteins such as Raf-1 kinase . The result is consistent with the recently published crystal structure of this protein family, which suggests the importance of the negatively charged groove-like structure in the ligand binding.

Hypertension, 1995 Dec, 26(6 Pt 2), 1046 - 50
Heterogeneity of adenovirus-mediated gene transfer in cultured thoracic aorta and renal artery of rats; Yao A et al.; Replication-deficient recombinant adenovirus vectors have been used to transfer foreign genes effectively to a wide variety of cell types in vivo and in vitro . We have now used adenovirus containing either the Escherichia coli beta-galactosidase (beta-gal) gene (AdHCMVsp1LacZ) or the firefly luciferase gene (Ad5-luc3) to test the hypothesis that efficiencies of adenovirus-mediated gene delivery into organ cultures of smooth muscle differ according to the anatomic origin of the muscle . Thoracic aorta and renal artery were isolated from 9-week-old male Sprague-Dawley rats and exposed to adenovirus after 16 hours of incubation with serum-free medium (Dulbecco's modified Eagle's medium) . With the use of histochemical methods, beta-gal staining was noted in both endothelial and adventitial cells but not in the muscular media of thoracic aorta and renal artery exposed to AdHCMVsp1LacZ . The efficiency of the transfection, assessed either by counting of beta-gal-stained cells in intact vessels or by measurement of beta-gal activity in tissue extracts, was higher in renal artery than thoracic aorta (P < .05) . Consistent with this result, luciferase activity in renal artery exposed to Ad5-luc3 (15.9 +/- 2.1 x 10(6) relative light units per milligram protein) was higher than that in thoracic aorta (8.3 +/- 2.0 x 10(6), P < .05) . To determine whether increased efficiency of adenovirus-mediated gene transfer into renal artery is a function of the replication status of vessels, we assessed {3H}thymidine incorporation . {3H}Thymidine uptake by thoracic aorta was only 63% of that in renal artery (P < .05), indicating that more proliferating cells are present in renal artery . We conclude that the efficiency of adenovirus-mediated gene transfer into cultured renal artery is enhanced compared with that into thoracic aorta and propose that the increase in efficiency is related to the higher proliferative activity of renal artery.

Genes Dev, 1995 Dec 1, 9(23), 2986 - 96
Genetic strategy for analyzing specificity of dimer formation: Escherichia coli cyclic AMP receptor protein mutant altered in its dimerization specificity; Joung JK et al.; Many transcriptional regulators function in homo- or heterodimeric combinations . The same protein can carry out distinct regulatory functions depending on the partner with which it associates . Here, we describe a mutant of the Escherichia coli cAMP receptor protein (CRP) that has an altered dimerization specificity; that is, mutant/mutant homodimers form preferentially over wild-type/mutant heterodimers . CRP dimerization involves the formation of a parallel coiled-coil structure, and our CRP mutant bears an amino acid substitution affecting the first "d" position residue within the alpha-helix that mediates CRP dimerization . The genetic strategy we used to isolate this CRP altered dimerization specificity (ADS) mutant is generalizable and could be utilized to isolate ADS mutants of other dimeric transcriptional regulators.

Exp Hematol, 1995 Dec, 23(13), 1341 - 6
Localization of an essential ligand binding determinant of the human erythropoietin receptor to a domain N-terminal to the WSXWS motif: implications for soluble receptor function; Schimmenti LA et al.; The interaction of erythropoietin (Epo) with the erythropoietin receptor (EpoR) supports erythropoiesis . The EpoR is a member of the well-recognized cytokine receptor superfamily characterized by four conserved cysteines and a WSXWS domain in the extracellular portion of the molecule . To localize ligand-binding determinants of the EpoR near the WSXWS domain, we tested the ligand-binding ability of the wild-type human EpoR extracellular domain (EREx), two truncated and three chimeric constructs with the interleukin-2 receptor beta subunit (IL2R beta) . Constructs were expressed in E . coli as GST fusion proteins linked to a solid-phase support and assayed for binding to 125I Epo . As previously shown, Epo bound specifically to the expressed extracellular domain, EREx . Epo did not bind to truncated receptors lacking either the entire fifth exon or the WSXWS domain . Epo also did not bind to chimeric receptors that had the amino acids encoded by the fifth exon replaced by IL2R beta or that had the amino acids subsequent to asparagine residue 209 replaced by IL2R beta . Specific binding was demonstrated for a construct in which the WSXWS was replaced by that of IL2R beta . We conclude that the amino acids encoded by this 5' portion of exon 5 of the EpoR are necessary for ligand binding and that the WSXWS domain is necessary for Epo binding but is not involved in ligand-binding specificity . We also speculate that if the putative soluble form of the EpoR is expressed (predicted to lack exon 5), it does not bind Epo and therefore may serve a physiologic purpose other than ligand binding.

Crit Care Med, 1995 Dec, 23(12), 2008 - 14
Ketamine attenuates endotoxin-induced leukocyte adherence in rat mesenteric venules; Schmidt H et al.; OBJECTIVES: To determine the influence of ketamine on endotoxin-induced leukocyte adherence and venular microhemodynamics . DESIGN: Randomized, controlled trial . SETTING: Experimental laboratory . SUBJECTS: Thirty male Wistar rats . INTERVENTIONS: The rats were pretreated with ketamine (10 mg/kg iv) or 0.9% saline, and both groups were given endotoxin (Escherichia coli lipopolysaccharide; 5 mg/kg iv) . The control group received two doses of 0.9% saline . MEASUREMENTS AND MAIN RESULTS: The rates of leukocyte adherence and changes in microhemodynamics were monitored in rat mesenteric venules, using in vivo video microscopy . The number of adherent leukocytes was determined on-line in 10-min intervals from 60 mins before until 2 hrs after endotoxin administration . Venular diameters, red blood cell velocity, volumetric blood flow, and the venular wall shear rate were monitored before and at 10, 30, and 60 mins after endotoxin exposure . A 6.3-fold increase in the number of adherent leukocytes was observed 10 mins after administration of endotoxin when compared with control animals (5.87 +/- 0.69 vs . 0.93 +/- 0.21 adherent cells/100 microns; p < .001) . This increase remained unchanged for 120 mins . In ketamine-pretreated rats, a 2.6-fold increase in leukocyte adherence occurred during the first 20 mins after endotoxin exposure (2.40 +/- 0.46 vs . 0.93 +/- 0.21 adherent cells/100 microns; p < .01) . However, no difference in the number of adherent leukocytes between ketamine-pretreated and control animals was found after this 20-min period . In animals of the control group, no increase in leukocyte adherence occurred during the entire observation time . Diameters of mesenteric venules did not change after endotoxin exposure in any of the groups . Red blood cell velocity and venular blood flow in the endotoxin-treated groups decreased 10 mins after the injection of endotoxin when compared with controls, but these values did not show any difference when they were compared between ketamine and saline-pretreated animals . Similarly, venular wall shear rate in the endotoxin-treated groups decreased 10 and 30 mins after injection of endotoxin . However, no significant difference occurred between ketamine and saline-pretreated animals . CONCLUSIONS: Pretreatment with ketamine attenuates endotoxin-induced leukocyte adherence by a shear rate-independent mechanism, suggesting reduced expression of adhesion molecules . These results indicate that ketamine exerts an anti-inflammatory effect, which might be beneficial in septic patients.

Radiat Res, 1995 Dec, 144(3), 301 - 9
Neutron and gamma-radiation sensitivity of plasmid DNA of varying superhelical density; Swenberg CE et al.; Several families of negatively supercoiled topoisomers of plasmid pIBI30 were prepared by a modification of the procedure of Singleton and Wells (Anal . Biochem . 122, 253-257, 1982) . The average superhelical density (sigma) was determined by two-dimensional agarose gel electrophoresis and varied from -0.010 to -0.067, corresponding to a change in the number of supercoils from 3 to 19 and an effective volume change from 1.6 x 10(8) to 4 x 10(8) A3 . Samples were exposed to either fission-neutron or 60Co gamma radiation and assayed for single-strand breaks by agarose gel electrophoresis . Form I DNA for all topoisomers decreased exponentially with increasing dose . The D37 values for both neutron and gamma radiation increased monotonically with increasing magnitude of sigma . Using a branched plectonemic (interwound) form for DNA over the range of sigma studied and standard (single-hit) target theory, a quantitative linear fit to (D37)-1 as a function of the effective DNA radius, S(A), was obtained . The model predicts that both the slope (a) and the intercept (b) of (D37)-1 as a function of S(A) are directly proportional to the length of DNA and the radiation fluence . Furthermore, the ratio b/a (= ro) at sigma = 0 depends only on the ionic strength of the medium and is independent of the radiation source parameters . Our results support the model and we calculate ro = 13.4 +/- 1.4 nm, a value consistent with other investigations . Our results are consistent with studies using 137Cs (Milligan et al., Radiat.(ABSTRACT TRUNCATED AT 250 WORDS)

Orig Life Evol Biosph, 1995 Dec, 25(6), 565 - 89
Two types of aminoacyl-tRNA synthetases could be originally encoded by complementary strands of the same nucleic acid; Rodin SN et al.; The lack of even a marginal similarity between the two aminoacyl-tRNA synthetase (aaRS) classes suggests their independent origins (Eriani et al., 1990; Nagel and Doolittle, 1991) . Yet, this independence is a puzzle inconsistent with the common origin of transfer RNAs, the coevolutionary theory of the genetic code (Wong, 1975, 1981) and other associated data and ideas . We present here the results of antiparallel 'class I versus class II' comparisons of aaRSs within their signature sequences . The two main HIGH- and KMSKS-containing motifs of class I appeared to be complementary to the class II motifs 2 and 1, respectively . The above sequence complementarity along with the mirror-image between crystal structures of complexes formed by the opposite aaRSs and their cognate tRNAs (Ruff et al., 1991), and the generally mirror ('head-to-tail') mapping of the basic functional sites in the sequences of aaRSs from the opposite two classes led us to conclude that these two synthetases emerged synchronously as complementary strands of the same primordial nucleic acid . This conclusion, combined with the hypothesis of tRNA concerted origin (Rodin et al., 1993a,b), may explain many intriguing features of aaRSs and favor the elucidation of the origin of the genetic code.

J Virol, 1995 Dec, 69(12), 8173 - 7
An aspartic acid at amino acid 108 is required to rescue infectious virus after transfection of a poliovirus cDNA containing a CGDD but not SGDD amino acid motif in 3Dpol; Walker DE et al.; The poliovirus RNA-dependent RNA polymerase (3Dpol) contains a region of homology centered around the amino acid motif YGDD (amino acids 326 to 329), which has been postulated to be involved in the catalytic activity of the enzyme . Previous studies from this laboratory have used oligonucleotide site-directed mutagenesis to substitute the tyrosine amino acid at this motif with other amino acids (S . A . Jablonski and C . D . Morrow, J . Virol . 67:373-381, 1993) . The viruses recovered with 3Dpol genes with a methionine mutation also contained a second mutation at amino acid 108 resulting in a glutamic acid-to-aspartic acid change (3D-E-108 to 3D-D-108) in the poliovirus RNA polymerase . On the basis of these results, we suggested that the amino acid at position 108 might interact with the YGDD region of the poliovirus polymerase . To further investigate this possibility, we have constructed a series of constructs in which the poliovirus RNA polymerases contained a mutation at amino acid 108 (3D-E-108 to 3D-D-108) as well as a mutation in which the tyrosine amino acid (3D-Y-326) was substituted with cysteine (3D-C-326) or serine (3D-S-326) . The mutant 3Dpol polymerases were expressed in Escherichia coli, and in vitro enzyme activity was analyzed . Enzymes containing the 3D-D-108 mutation with the wild-type amino acid (3D-Y-326) demonstrated in vitro enzyme activity similar to that of the wild-type enzyme containing 3D-E-108 . In contrast, enzymes with the 3D-C-326 or 3D-S-326 mutation had less in vitro activity than the wild type . The inclusion of the second mutation at amino acid 3D-D-108 did not significantly affect the in vitro activity of the polymerases containing 3D-C-326 or 3D-S-326 mutation . Transfections of poliovirus cDNAs containing the substitution at amino acid 326 with or without the second mutation at amino acid 108 were performed . Consistent with previous findings, we found that transfection of poliovirus cDNAs containing the 3D-C-326 or 3D-S-326 mutation in 3Dpol did not result in the production of virus . Surprisingly, transfection of the poliovirus cDNAs containing the 3D-D-108/C-326 double mutation, but not the 3D-D-108/S-326 mutation, resulted in the production of virus . The virus obtained from transfection of polio-virus cDNAs containing 3D-D-108/C-326 mutation replicated with kinetics similar to that of the wild-type virus . RNA sequence analysis of the region of the 3Dpol containing the 3D-C-326 mutation revealed that the codon for cysteine (UGC) reverted to the codon for tyrosine (UAC) . The results of these studies establish that under the appropriate conditions, poliovirus has the capacity to revert mutations within the YGDD amino acid motif of the poliovirus 3Dpol gene and further strengthen the idea that interaction between amino acid 108 and the YGDD region of 3Dpol is required for viral replication.

J Virol, 1995 Dec, 69(12), 7942 - 50
Neurons differentially control expression of a herpes simplex virus type 1 immediate-early promoter in transgenic mice; Mitchell WJ; The immediate-early proteins of herpes simplex virus control the cascade of viral gene expression during lytic infection . It is not known which viral or host proteins control the reactivation of the viral genome in latently infected neurons . To determine whether neuronal proteins can regulate a herpes simplex virus immediate-early promoter in vivo, transgenic mice containing the promoter regulatory region of the herpes simplex virus type 1 immediate-early gene (ICP4) fused to the bacterial beta-galactosidase gene were generated . Two lines of mice, in the absence of viral proteins, displayed ICP4 promoter activity in neurons in specific locations in the central nervous system . The anatomic locations of these neurons were the hippocampus, cerebellar cortex, superior colliculus, indusium griseum, mammillary nucleus, cerebral cortex, and the dorsal laminae of the dorsal horns of the spinal cord . Additional subsets of neurons expressed the ICP4 promoter at lower levels; these included trigeminal ganglia and retinas . In a third line of mice, lower levels of expression were present in many of the above-described neurons . Many types of neurons, nearly all nonneuronal cells in the central nervous system, and some non-nervous system tissues were negative . Viral proteins including VP16 are not necessary to induce transcription from the ICP4 promoter in many neurons and some other cell types but may be required in most cells in vivo . An approximately 100-fold-greater number of neurons in the trigeminal ganglia expressed ICP4 promoter activity in newborn mice compared with adults . These data provide direct evidence that host proteins are sufficient to activate a herpes simplex virus immediate-early promoter in neurons in vivo and that a differential expression pattern for this promoter exists within different neuronal phenotypes and between the same neurons in different ages of mice.

J Virol, 1995 Dec, 69(12), 7888 - 98
Retention of oncogenicity by a Marek's disease virus mutant lacking six unique short region genes; Parcells MS et al.; We previously reported the construction of Marek's disease virus (MDV) strains having mutations in various genes that map to the unique short (US) region of the viral genome (J.L . Cantello, A.S . Anderson, A . Francesconi, and R.W . Morgan, J . Virol . 65:1584-1588, 1991; M.S . Parcells, A.S . Anderson, and R.W . Morgan, Virus Genes 9:5-13, 1994; M.S . Parcells, A.S . Anderson, and R.W . Morgan, J . Virol . 68:8239-8253, 1994) . These strains were constructed by using a high-passage-level serotype 1 MDV strain which grew well in chicken embryo fibroblasts . Despite the growth of the parent and mutant viruses in cell culture, in vivo studies were limited by poor growth of these strains in chickens . One of the mutants studied lacked 4.5 kbp of US region DNA and contained the lacZ gene of Escherichia coli inserted at the site of the deletion . The deletion removed MDV homologs to the US1, US2, and US10 genes of herpes simplex virus type 1 as well as three MDV-specific open reading frames . We now report the construction of a mutant MDV containing a similar deletion in the US region of the highly oncogenic RB1B strain . This mutant, RB1B delta 4.5lac, had a growth impairment in established chicken embryo fibroblasts similar to that described previously for MDVs lacking a functional US1 gene . In chickens, RB1B delta 4.5lac showed decreased early cytolytic infection, mortality, tumor incidence, and horizontal transmission . Several lymphoblastoid cell lines were established from RB1B delta 4.5lac-induced tumors, and virus reactivated from these cell lines was LacZ+ . These results indicate that the deleted genes are nonessential for the transformation of chicken T cells or for the establishment and maintenance of latency . On the basis of the growth impairment observed for RB1B delta 4.5lac in cell culture and in vivo, we conclude that deletion of these genes affects the lytic replication of MDV . This is the first MDV mutant constructed in the RB1B oncogenic strain, and the methodology described herein provides for the direct examination of MDV-encoded determinants of oncogenicity.

J Virol, 1995 Dec, 69(12), 7807 - 13
In vivo and in vitro association of hsc70 with polyomavirus capsid proteins; Cripe TP et al.; Members of the 70-kDa family of cellular stress proteins assit in protein folding by preventing inappropriate intra- and intermolecular interactions during normal protein synthesis and transport and when cells are exposed to a variety of environmental stresses . During infection of A31 mouse fibroblasts with polyomavirus, the constitutive form of hsp70, hsc70, coimmunoprecipitated with all three viral capsid proteins (VP1, VP2, and VP3) . In addition, the subcellular location of hsc70 changed from cytoplasmic to nuclear late in polyomavirus infection, coincident with the nuclear localization of the viral capsid proteins . VP1 and VP2 expressed in Sf9 insect cells with recombinant baculovirus vectors also coimmunoprecipitated with an hsp70-like protein, and VP1 expressed in Escherichia coli coimmunoprecipitated with the hsp70 homolog DnaK . Capsid proteins expressed by in vitro translation coimmunoprecipitated with the hsc70 protein present in the reticulocyte translation extract . Therefore, the polyomavirus capsid proteins associate with hsc70 during virus infection as well as in recombinant protein expression systems . This association may play a role in preventing the premature assembly of capsids in the cytosol and/or in facilitating the nuclear transport of capsid protein complexes.

J Virol, 1995 Dec, 69(12), 7734 - 42
Expression of the polyomavirus minor capsid proteins VP2 and VP3 in Escherichia coli: in vitro interactions with recombinant VP1 capsomeres; Delos SE et al.; The polyomavirus VP2 and VP3 capsid proteins were expressed in Escherichia coli . The majority of the expressed proteins were in an insoluble fraction, and they were extracted and initially purified in 8 M urea before renaturation . Soluble VP2 and VP3 were mixed with purified recombinant VP1 capsomeres, and their interactions were assayed by immunoprecipitation and ion-exchange chromatography . Coimmunoprecipitation could be demonstrated with antibodies to either VP1 or VP2/VP3 . Mixing recombinant VP1 with VP2 and VP3 modified the recognition of VP1 by domain-specific antipeptide antibodies and altered the chromatographic behavior of the individual proteins . Similar results were observed when a truncated VP1 protein, delta NCOVP1, with 62 amino acids deleted from the carboxy terminus was mixed with VP2/VP3 . After the mixing, equilibrium dissociation constants for their binding to either VP1 or delta NCOVP1 were determined to be 0.37 +/- 0.23 microM for VP2 and 0.18 +/- 0.21 microM for VP3 . These studies demonstrate that the recombinant VP2 and VP3 proteins interact with VP1 to affect the biochemical properties of VP1 capsomeres and to change the epitope accessibility of VP1 pentamers . These changes may reflect conformational alterations in VP1 capsomeres which are necessary for viral genome encapsidation.

J Virol, 1995 Dec, 69(12), 7682 - 7
Replication efficiency of bovine papillomavirus type 1 DNA depends on cis-acting sequences distinct from the replication origin; Pierrefite V et al.; The viral elements required for the initiation of replication of bovine papillomavirus type 1 DNA include the origin region and two trans-acting factors, the E1 and E2 proteins . We now report that the replication efficiency of a DNA molecule which contains these three elements is modulated by other viral sequences . By measuring the extent of replication of deleted viral genomes in transfected mouse cells, we identified sequences required for maximal efficiency . Addition of these sequences to a construct carrying only the minimal origin region increased its replication . Among these cis-active elements, we identified a 69-bp fragment (nucleotides 4921 to 4990) which contains at least two binding sites for cellular proteins . One of them is the murine protein termed CDEBP, which recognizes the octameric motif ATCACGTG, identical to the yeast CDEI element . Either deletions affecting this CDEI box or a point mutation which impairs binding of CDEBP markedly decreased the extent of viral DNA replication . They had no detectable effect on viral transcription.

J Virol, 1995 Dec, 69(12), 7483 - 8
Assembly and catalytic properties of retrovirus integrase-DNA complexes capable of efficiently performing concerted integration; Vora AC et al.; The in vitro assembly process for forming nucleoprotein complexes containing linear retrovirus-like DNA and integrase (IN) was investigated . Solution conditions that allowed avian myeloblastosis virus IN to efficiently pair two separate linear DNA fragments (each 487 bp in length) containing 3' OH recessed long terminal repeat termini were established . Pairing of the viral termini by IN during preincubation on ice permitted these nucleoprotein complexes to catalyze the concerted insertion of the two termini into a circular DNA target (full-site reaction), mimicking the in vivo reaction . The three major solution determinants were high concentrations of NaCl (0.33 M), 1,4-dioxane, and polyethylene glycol . The aprotic solvent dioxane (15%) was significantly better (sixfold) than 15% dimethyl sulfoxide for forming complexes capable of full-site rather than half-site integration events . Half-site reactions by IN involved the insertion of a single donor terminus into circular pGEM . Although NaCl was essential for the efficient promotion of the concerted integration reaction, dioxane was necessary to prevent half-site reactions from occurring at high NaCl concentrations . Under optimal solution conditions, the concerted integration reaction was directly proportional to a sixfold range of IN . The complexes appeared not to turn over, and few half-site donor-donor molecules were produced . In the presence of 0.15 or 0.35 M NaCl, dioxane prevented efficient 3' OH trimming of a blunt-ended donor by IN, suggesting that the complexes formed by IN with blunt-ended donors were different from those formed with donors containing 3' OH recessed termini for strand transfer . The results suggest that IN alone was capable of protein-protein and protein-DNA interactions that efficiently promote the in vitro concerted integration reaction.

Cancer Res, 1995 Dec 1, 55(23 Suppl), 5983s - 5989s
Targeting c-erbB-2 expressing tumors using single-chain Fv monomers and dimers; Tai MS et al.; Single-chain Fv proteins containing a COOH-terminal cysteine (sFv') were constructed by using an antidigoxin 26.10 sFv and an anti-c-erbB-2 741F8 sFv . The fully active sFv' proteins were prepared by expression in Escherichia coli as insoluble inclusion bodies, followed by in vitro refolding using glutathione redox buffers and purification . The COOH-terminal cysteines of the refolded sFv' proteins were protected by a blocking group presumed to be the glutathionyl peptide, which was easily and selectively removed by gentle reduction . Air oxidation of the reduced sFv' monomers resulted in the efficient formation of disulfide-linked sFv' homodimers, designated (sFv')2, which were stable under oxidizing conditions and relatively slow to be disrupted under reducing conditions . The (26-10-1 sFv')-(741F8-1 sFv') heterodimer was prepared and possessed dual-antigen specificity; the active bispecific (sFv')2 dimerized under native conditions, apparently as a manifestation of self-association by the 741F8 sFv' subunit . Biodistribution and imaging studies that were performed on mice bearing human SK-OV-3 tumor xenografts that express the c-erbB-2 as a cell surface antigen were reviewed . Radioiodinated 741F8-2 (sFv')2 homodimer localized to the tumors with high specificity, as evidenced by excellent tumor:normal tissue ratios . Sagittal section autoradiography of whole animals 24 h after administration of antibody species revealed that 741F8 (sFv')2 produced a stronger tumor image than comparable doses of the 741F8 Fab, monomeric sFv', and the 26-10 (sFv')2 control without the high nonspecific background distribution of the 741F8 IgG.

Cancer Res, 1995 Dec 1, 55(23 Suppl), 5957s - 5967s
Biological properties of chimeric domain-deleted anticarcinoma immunoglobulins; Slavin-Chiorini DC et al.; CC49 is a second-generation monoclonal antibody (MAb) that has high affinity for the tumor-associated pancarcinoma antigen tumor-associated glycoprotein-72 . In clinical trials using gamma scanning, radiolabeled CC49 has facilitated the detection of more than 90% of carcinomas . We report here the development of a constant heavy-chain 2 (CH2) domain-deleted chimeric (c) CC49 MAb by transfecting an expression construct consisting of the CC49 murine variable region and a CH2 domain-deleted human IgG1 constant region into cCC49 kappa producing SP2/0 murine myeloma cells . As determined by SDS-PAGE, the intact cCC49 delta CH2 has a molecular weight of 153,000 and, under reducing conditions, molecular weights of 43,000 and 27,000 . The plasma clearance and tumor-targeting properties of cCC49 delta CH2 were evaluated and compared with those of mouse/human chimeric forms cCC49 delta CH1 and intact cCC49 . Previous studies have shown that the in vitro antigen-binding properties of cCC49 delta CH1 are similar to those of cCC49 . Biodistribution studies reported here, using 131I-labeled cCC49 delta CH1 and 125I-labeled cCC49 in athymic mice bearing human colon carcinoma xenografts, demonstrated that both cMAbs localized to the tumor and cleared from the normal tissues similarly . However, in comparison with 125I-labeled cCC49, 131I-labeled cCC49 delta CH2 localized to tumors earlier and had a significantly lower percentage of the injected dose of cMAb/g (%ID/g) in normal tissues than cCC49 . Immunoscintigraphy of 131I-labeled cCC49 delta CH2 and 125I-labeled cCC49 in athymic mice bearing human tumor xenografts demonstrated a clear image of the tumor by 24 h after i.v . administration of the delta CH2 cMAb versus the 72 h required for cCC49 . Biodistribution studies using 177Lu-conjugated cCC49 delta CH1 and cCC49 showed no significant difference between the radiolocalization indices (% ID/g in tumor divided by % ID/g in normal tissue) . 177Lu-conjugated cCC49 delta CH2, however, had lower % ID/g values in tumor xenografts and lower radiolocalization indices than either 177Lu-conjugated cCC49 delta CH1 or 177Lu-conjugated cCC49 . Pharmacokinetic studies in non-tumor-bearing athymic mice using cCC49 delta CH1 and cCC49 revealed no significant difference between these cMAbs . However, the plasma clearance of cCC49 delta CH2 in non-tumor-bearing mice was significantly faster than that of cCC49 . These results were similar when the cMAbs were labeled with either iodine or lutetium . In nonhuman primates, 131I-labeled cCC49 delta CH2 cleared significantly faster than 125I-labeled cCC49 . The similar plasma clearance and tumor localization of cCC49 and cCC49 delta CH1 suggest that these two cMAbs may be used in similar clinical settings . However, because of the unique pharmacokinetics and tumor targeting of cCC49 delta CH2 versus cCC49 or cCC49 delta CH1, this chimeric immunoglobulin form may be useful in clinical settings that require efficient tumor targeting and rapid serum and whole-body clearance.

Arch Surg, 1995 Dec, 130(12), 1266 - 72
Liposomes modulate Kupffer cell endotoxin response; Bankey P et al.; OBJECTIVES: To test the hypothesis that pretreatment with liposomes enriched with the omega 3 fatty acid docosahexaenoic acid (22:6 omega 3) will alter the Kupffer's cell and systemic cytokine (tumor necrosis factor and interleukin-6) response to endotoxin challenge, and to demonstrate alterations in Kupffer's cell phospholipid fatty acid composition after in vivo liposome treatment . DESIGN: Nonrandomized controlled laboratory investigation in Wistar rats . INTERVENTIONS: Animals were assigned to three pretreatment groups: no liposomes; liposomes, 100 mg/kg; or liposomes, 400 mg/kg given by bolus intravenous injection with the animals under inhalation anesthesia . Eighteen hours after liposome treatment, each group was challenged with Escherichia coli lipopolysaccharide (3 mg/kg intraperitoneally in 10 mL of lactated Ringer's solution) or lactated Ringer's solution only . In a separate set of experiments, Kupffer's cells were obtained from animals pretreated with liposome, 400 mg/kg, or controls and challenged with lipopolysaccharide (1, 100, or 10(4) ng/mL) in vitro . OUTCOME MEASURES: Serum and Kupffer's cell supernatant tumor necrosis factor and interleukin-6 bioactivity, Kupffer's cell phospholipid fatty acid composition, survival, and liver histologic findings . RESULTS: In vivo liposome pretreatment (400 mg/kg) resulted in significant increases in serum tumor necrosis factor and interleukin-6 levels 90 minutes after intraperitoneal lipopolysaccharide challenge (P < .05 vs no liposomes) . Kupffer's cells isolated from liposome-treated animals (400 mg/kg) compared with untreated controls release significantly more tumor necrosis factor and interleukin-6 after lipopolysaccharide stimulation in vitro in a dose-dependent response (P < .05) . Liposome treatment increased total polyunsaturated fatty acid, total omega 3, and docosahexaenoic acid 22:6 omega 3 content in Kupffer's cell phospholipids compared with untreated controls . Survival 24 hours after lipopolysaccharide challenge was reduced by liposome (400 mg/kg) pretreatment (P < .05 by chi 2 test) . Livers from each treatment group demonstrated focal areas of hepatocyte necrosis and inflammatory cells . CONCLUSION: Liposome pretreatment increases the circulating and Kupffer's cell cytokine response to endotoxemia, increases Kupffer's cell polyunsaturated fatty acid content, and is associated with reduced survival.

Science, 1995 Dec 1, 270(5241), 1495 - 7
Solution structure of the activator contact domain of the RNA polymerase alpha subunit; Jeon YH et al.; The structure of the carboxyl-terminal domain of the Escherichia coli RNA polymerase alpha subunit (alpha CTD), which is regarded as the contact site for transcription activator proteins and for the promoter UP element, was determined by nuclear magnetic resonance spectroscopy . Its compact structure of four helices and two long arms enclosing its hydrophobic core shows a folding topology distinct from those of other DNA-binding proteins . The UP element binding site was found on the surface comprising helix 1, the amino-terminal end of helix 4, and the preceding loop . Mutation experiments indicated that the contact sites for transcription activator proteins are also on the same surface.

J Mol Biol, 1995 Dec 1, 254(3), 431 - 46
Refined crystal structures of unligated adenylosuccinate synthetase from Escherichia coli; Silva MM et al.; Crystal structures of unligated adenylosuccinate synthetase from Escherichia coli in space groups P2(1) and P2(1)2(1)2(1) have been refined to R-factors of 0.199 and 0.206 against data to 2.0 and 2.5 A, respectively . Bond lengths and angles deviate from expected values by 0.011 A and 1.7 degrees for the P2(1) crystal form and by 0.015 A and 1.7 degrees for the P2(1)2(1)2(1) crystal form . The fold of the polypeptide chain is dominated by a central beta-sheet, which is composed of nine parallel strands and a tenth antiparallel strand . Extending off from this central beta-sheet are four subdomains . The four subdomains contribute loops of residues that are disordered or have high thermal parameters . At least three of these loops (residues 42 to 52, 120 to 131 and 298 to 304) contribute essential residues to the putative active site of the synthetase . In the absence of ligands, much of the active site of the synthetase exists in an ill-defined conformational state . Two, nearly independent regions contribute residues to the interface between polypeptide chains of the synthetase dimer . A pair of helices (H4 and H5) interact with their symmetry-equivalent mates by way of residues that are not conserved amongst the known sequences of the synthetase . The second interface region involves conserved residues belonging to structural elements that connect strands of the central beta-sheet . Residues putatively involved in the binding of IMP lie at or near the interface between polypeptide chains of the dimer . Of the four sequence elements putatively common to all GTP hydrolases, the synthetase has only the guanine recognition element and a glycine-rich loop (P-loop) . Although the base recognition element is essentially identical with those of the p21 ras and G alpha proteins, the P-loop of the synthetase is extended in size relative to the P-loops of other GTP hydrolases . The P-loop has two acid residues (Asp13 and Glu14), which are found in the P-loops of only the synthetase family . Glu14 may be involved in the stabilization of the enlarged P-loop of the synthetase, whereas Asp13 may play a role in catalysis and in the coordination of Mg2+ . The structural elements of the p21 ras and G alpha proteins responsible for binding Mg2+ are either absent from the synthetase or unavailable for the coordination of metal cations.

J Mol Biol, 1995 Dec 1, 254(3), 364 - 71
Electron microscopic visualization of RecT protein and its complexes with DNA; Thresher RJ et al.; Electron microscopy has been used to examine Escherichia coli RecT protein alone and in the complexes it forms with DNA substrates, with which it catalyzes strand exchange in vitro . Negative staining has revealed that the 33 kDa RecT protein monomers form open C-shaped and closed O-shaped particles . RecT protein monomers assemble into donut-shaped oligomers containing seven or eight protein monomers and rod-like structures . When bound to single-stranded DNA, RecT forms highly twisted nucleoprotein filaments that are 18 nm in diameter and have a helical pitch of 10 nm . When added to linear duplex DNA in the presence of active RecE protein (exonuclease VIII), filamentous nucleoprotein complexes are formed on the DNA ends and the DNA molecules are frequently cyclized through protein-protein interactions.

J Mol Biol, 1995 Dec 1, 254(3), 342 - 9
Functional map of the alpha subunit of Escherichia coli RNA polymerase: amino acid substitution within the amino-terminal assembly domain; Kimura M et al.; The alpha subunit of Escherichia coli RNA polymerase plays a key role in assembly of the core enzyme . In previous studies the amino-terminal domain consisting of 215 amino acid residues between positions 21 and 235 was identified to be involved in this assembly, and the sites for beta and beta' association were suggested to be located within or near the two conserved regions in this amino-terminal assembly domain of alpha . For detailed functional mapping, Ala was substituted for 26 highly conserved amino acids around residues 40, 80 and 170 to 210 . The alpha-point mutants were analyzed in vitro for their abilities to form dimers and to assemble beta beta' subunits . New types of assembly-deficient mutants were identified: alpha-R45A (having substituted Ala for Arg at residue 45) dimerized but did not assemble beta (and beta') subunits; and alpha-L48A showed a decreased level of alpha 2 beta subassembly formation, indicating that this region (residues 45 to 48) is responsible for beta-binding . Isolation of two mutants, alpha-K86A and alpha-V173A, both forming alpha 2 beta but not alpha 2 beta beta' complex, confirmed our previous conclusion that two separated regions participate in beta'-binding.

J Mol Biol, 1995 Dec 1, 254(3), 337 - 41
The gene for nucleoside diphosphate kinase functions as a mutator gene in Escherichia coli; Lu Q et al.; Nucleoside diphosphate (NDP) kinase is a key enzyme in the control of cellular concentrations of nucleoside triphosphates, and has been shown to play important roles in various cellular activities such as developmental control, signal transduction and metastasis in eukaryotic systems . In this study, the gene for NDP kinase of Escherichia coli (ndk) was disrupted and surprisingly found to be dispensable without any discernible effects on cell growth or morphology . However, a mutator phenotype was found in ndk-disruption strains; frequencies of spontaneous mutations to rifampicin resistance and nalidixic acid resistant significantly increased . A higher frequency in reversion mutations was observed with use of an amber mutation in the kanamycin-resistance gene in an ndk-disruption strain . Imbalance in dNTP pools, in particular a significant increase of the dCTP content was observed, which is likely to result in the higher spontaneous mutation rates . These results suggest that NDP kinase, although not essential, plays an important role in the appropriate balance of intracellular dNTP pools to maintain a high DNA replication fidelity . Strains with ndk- pykA- pykF- as well as ndk- scs- were constructed without any discernible effect on cell growth, indicating that there is yet another enzyme(s) catalyzing nucleoside triphosphate synthesis, in addition to NDP kinase, pyruvate kinases and succinyl CoA synthetase.

Hepatology, 1995 Dec, 22(6), 1648 - 55
Proteolytic activity of NS3 serine proteinase of hepatitis C virus efficiently expressed in Escherichia coli; Shoji I et al.; The serine proteinase of hepatitis C virus (HCV) non-structural protein NS3 was efficiently expressed in an active form as a fused protein with oligohistidine in Escherichia coli . The recombinant fusion protein was purified to near homogeneity by affinity chromatography on a metal chelation column . Trans-cleavage activity of this protein was investigated by using the substrate NS5 protein expressed in insect cells . The purified serine proteinase trans-cleaved the partially purified NS5 protein . In contrast, the NS3 proteins with mutations at the proposed catalytic site, Ser1165 or His1083, lost the trans-cleavage activity . Analysis of the authentic enzyme and variants with site-directed mutations provides a useful tool for understanding the structure-function relationship of the NS3 serine proteinase . We then developed an in vivo trans-cleavage assay system by coexpression of the NS3 proteinase and the NS5 substrate in E coli, and examined the effect of known inhibitors of serine proteinase . Inhibition of its proteolytic activity by N-p-tosyl-L-lysine chloromethyl ketone (TLCK) was observed, but only at high concentrations . The in vitro and in vivo trans-cleavage assays for NS3 serine proteinase will facilitate efficient testing for inhibitors of the replication of HCV and specific treatment for hepatitis C.

Arterioscler Thromb Vasc Biol, 1995 Dec, 15(12), 2246 - 53
Quantitative analysis of repeat adenovirus-mediated gene transfer into injured canine femoral arteries; Ueno H et al.; We quantitatively evaluated the effectiveness of a repeat administration of a recombinant adenoviral vector expressing bacterial Escherichia coli lacZ into the same arterial site of a relatively large animal, the dog . The replication-defective adenoviral vector was introduced percutaneously into balloon-injured femoral arteries through a double-balloon catheter . After a single dose of adenoviral vector, up to 90% of surface (73 +/- 16%, n = 7) and smooth muscle cells in multiple layers of the media showed transgene expression as evaluated by 5-bromo-4-chloro-3-indoyl beta-D-galactopyranoside histostaining without extralocal expression, as assessed by polymerase chain reaction . High-level expression (measured as beta-galactosidase activity) peaked 7 days after transfer and was transient, although it was retained for a month . Second does of the same adenovirus to the same arterial site were given 1, 2, 5, or 8 weeks after the first administration . At 1 week the second dose significantly enhanced lacZ expression . At 2, 5, or 8 weeks the second dose reinduced lacZ expression at 25% to 30% of the full expression . lacZ expression was also detected in preimmuned dogs, although the expression levels correlated inversely to the titer of neutralizing antibodies in their serum . These results demonstrate that arterial gene expression can be enhanced by a second administration of the same adenovirus after a short interval and that a repeat dose after a long interval partially but significantly reinduces gene expression despite the presence of an immune response . These data may provide an additional scientific foundation for the use of adenovirus-mediated arterial gene transfer in future clinical practice.

Radiology, 1995 Dec, 197(3), 665 - 9
Inflammation: imaging with methoxy poly(ethylene glycol)-poly-L-lysine-DTPA, a long-circulating graft copolymer; Gupta H et al.; PURPOSE: To test whether a nontargeted, long-circulating, synthetic polymer accumulates in areas of inflammation, with high capillary permeability and increased regional blood flow . MATERIALS AND METHODS: Methoxy poly(ethylene glycol)-poly-L-lysine (PL)-diethylenetriaminepentaacetic acid (MPEG-PL-DTPA) was labeled with technetium-99m for scintigraphy and with gadolinium for magnetic resonance (MR) imaging . Eleven Escherichia coli-infected rats were injected with 1.0 mCi (37 MBq) of Tc-99m-labeled MPEG-PL-DTPA for scintigraphy . Twelve rats underwent 1.5-T MR imaging after intravenous injection of gadolinium-labeled MPEG-PL-DTPA (35 mumol/kg) . RESULTS: Tc-99m-labeled MPEG-PL-DTPA demonstrated nearly eight-fold higher accumulation in infected muscle when compared with normal muscle . Scintigrams and MR images showed areas of inflammation with peak accumulation at 24 hours after injection of Tc-99m- or gadolinium-labeled MPEG-PL-DTPA . CONCLUSION: Nontargeted, long-circulating, copolymers can efficiently accumulate in sites of inflammation and thus represent an alternative to inflammation-specific agents.

Mol Cell Endocrinol, 1995 Nov 30, 115(1), 87 - 93
Expression of extracellular domain peptides of the FSH receptor and their effect on receptor-ligand interactions in vitro; Sharma SC et al.; Follicle-stimulating hormone receptor (FSH-R) displays considerable homology to luteinizing hormone receptor (LH-R) in structure and amino acid sequence . Comparison of the sequences of the extracellular domains (ECD) of the receptors reveals two regions (amino acids 4-56 and 265-319 in FSH-R) that share relatively little amino acid sequence similarity . This suggests that these variable regions may be important in providing specificity of ligand binding . We have expressed overlapping ECD peptides containing one or both of these regions (RFI, amino acids 5-125; RF2, amino acids 201-319; and RF3, amino acids 5-319) as fusion proteins in E . coli using pRSET vector . The presence of polyhistidine at the N-terminal end allowed substantial purification of the expressed proteins by a single step of affinity chromatography . The purified peptides were characterized for direct binding of hormone and their ability to block the binding of FSH and LH to the receptors . None of the peptides bound labelled hormone, while all peptides inhibited the binding of FSH to its receptor in a dose-dependent manner . However, only RF2 peptide inhibited ligand binding in a hormone-specific manner . These data suggest there is a site between amino acids 201-319 of the FSH-R ECD that is involved in FSH binding.

Carbohydr Res, 1995 Nov 30, 278(1), 155 - 65
NMR investigation of the 6-deoxy-L-talose-containing O45, O45-related (O45rel), and O66 polysaccharides of Escherichia coli; Jann B et al.; The structures of the 6-deoxytalose-containing O-specific polysaccharides from the O45 antigen, an O45-related antigen (O45rel), and the O66 antigen (lipopolysaccharides, LPSs) of Escherichia coli were elucidated by chemical characterization and by one- and two-dimensional 1H and 13C NMR spectroscopy . The O45 and O45-related polysaccharides have the following general structure: {formula: see text} For the O45 antigen, X is alpha-D-FucpNAc and for the O45-related antigen, X is beta-D-GlcpNAc . The structure of the O66 polysaccharide is {formula: see text}

Philos Trans R Soc Lond B Biol Sci, 1995 Nov 29, 350(1332), 189 - 202
The Leeuwenhoek Lecture, 1995 . Adaptation to life without oxygen; Guest JR; The Earth was populated by anaerobic organisms for at least a thousand million years before the atmosphere became oxygenated and aerobes could evolve . Many bacteria like Escherichia coli retain the ability to grow under both aerobic and anaerobic conditions . Recent studies have revealed some global regulatory mechanisms for activating or repressing the expression of relevant genes in response to oxygen availability . These mechanisms ensure that the appropriate metabolic mode is adopted when bacteria switch between aerobic and anaerobic environments.

Biochemistry, 1995 Nov 28, 34(47), 15592 - 8
Kinetics of inorganic phosphate release during the interaction of p21ras with the GTPase-activating proteins, p120-GAP and neurofibromin; Nixon AE et al.; The rate of GTP hydrolysis on p21ras is accelerated by approximately 10(5) times by the catalytic domains of GTPase-activating proteins (GAPs), p120-GAP (GAP-344) or neurofibromin (NF1-334) . The kinetic mechanism of this activation has been investigated by following the release of inorganic phosphate (Pi), using a fluorescent probe that is sensitive to Pi {Brune, M., Hunter, J., Corrie, J . E . T., & Webb, M . R . (1994) Biochemistry 33, 8262-8271} . Measurements were made in real time with a stopped-flow apparatus, in which the p21ras complex with the 2',3'-methanthraniloyl analogue of GTP (mantGTP) was mixed with the GAP in the presence of this Pi probe . The results show that Pi release is fast and that the overall hydrolysis is controlled by the cleavage itself or a conformational change preceding the cleavage . The time courses were single exponentials over a range of {GAP-344} and were modeled to show that a single step controlled Pi release . The maximum rate constant was 15 s-1 (all data at 30 degrees C, pH 7.6, low ionic strength) in experiments in which GAP-344 underwent a single turnover, compared with 5 s-1 for multiple-turnover experiments, and possible causes of this discrepancy were investigated and discussed . With NF1-334 the time courses were more complex, showing a lag prior to rapid release of Pi . The results were consistent with a Kd of 0.04 microM for NF1-344 affinity is some 3 orders of magnitude tighter than that of GAP-344.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1995 Nov 28, 34(47), 15574 - 82
Large activation energy barriers to chaperone-peptide complex formation and dissociation; Farr CD et al.; To probe the mechanism of chaperone substrate selection, we have investigated the kinetics of complex formation and dissociation between the molecular chaperone DnaK and a short peptide (Cro, representing amino acids 1-12 of the cro repressor protein) . The Cro protein was N-terminally labeled with the environmentally sensitive fluorophore dansyl chloride (Cro*), and steady-state and stopped-flow fluorescence spectroscopies and fluorescence-detected high-performance size exclusion chromatography (HPSEC) were used to monitor complex formation and dissociation over a range of temperatures in the absence of ATP . The results are summarized as follows: (i) Cro* binds to DnaK with a second-order rate constant, k(on), which varies from 8 to 200 M-1 s-1 between 15 and 37 degrees C . The slow on-rate is a consequence of a large activation energy barrier . The activation enthalpy (delta H*) and the prefactor {omega exp delta S*/R)} are 26 kcal mol-1 and 7 x 10(20) M-1 s-1, respectively . (ii) Once formed, DnaK-Cro* complexes are long-lived, especially at low temperatures (T < 15 degrees C) . The off-rate is unusually temperature-sensitive, for example, there is a 478-fold increase in k(off) from 2.3 x 10(6) to 1.1 x 10(-3) s-1 over a range of only 30 degrees C (5-35 degrees C) . The steep temperature-dependence of the off-rate is a consequence of a very large activation energy barrier to DnaK-Cro* complex dissociation {delta H* = 34.6 kcal mol-1 and omega exp (delta S*/R) = 2 x 10(21) s-1} . The relatively low affinity of the Cro* peptide for DnaK is due to a large kinetic barrier to binding . We discuss possible causes for these large kinetic barriers.

Biochemistry, 1995 Nov 28, 34(47), 15545 - 52
Effects of zinc finger mutations on the nucleic acid binding activities of Xenopus transcription factor IIIA; Zang WQ et al.; Transcription factor IIIA (TFIIIA) is required for the activation of 5S RNA gene transcription as well as the storage of 5s RNA as a 7S ribonucleoprotein particle . Interaction with both nucleic acids is mediated through nine C2H2 zinc fingers . In order to determine amino acid regions necessary for nucleic acid interaction, a series of substitution mutants Xenopus laevis TFIIIA have been constructed and expressed as recombinant proteins in Escherichia coli . The mutant proteins were purified to homogeneity and analyzed for 5S RNA gene and 5S RNA binding activities using a nitrocellulose filter binding assay . All of the mutant TFIIIA proteins retained full 5S RNA binding activity . Substitution of fingers 2, 3, and 4-6 of TFIIIA with zinc finger sequences from other proteins significantly reduced the interaction of the protein with the 5S RNA gene . In contrast, substitution of finger 1 or finger 7 had little effect on the interaction of TFIIIA with the 5S RNA gene . The results of scanning substitution mutagenesis within the first three zinc fingers of TFIIIA suggested that DNA contacts made by the alpha-helical regions of finger 2 and particularly of finger 3 provide the majority of the free energy of the TFIIIA-DNA interaction . Basic amino acids found at the same position within the alpha-helices of fingers 2 and 3 of TFIIIA are required for high-affinity DNA binding activity . The identification of amino acid residues critical for the formation of a TFIIIA-DNA complex contributes to our understanding of zinc finger protein-nucleic acid interactions.

Biochemistry, 1995 Nov 28, 34(47), 15539 - 44
Mechanism-based inactivation of tRNA-guanine transglycosylase from Escherichia coli by 2-amino-5-(fluoromethyl)pyrrolo{2,3-d}pyrimidin-4 (3H)-one; Hoops GC et al.; In Escherichia coli, tRNA-guanine transglycosylase (TGT) catalyzes the incorporation of the queuine precursor preQ1 {2-amino-5-(aminomethyl)pyrrolo{2,3-d}pyrimidin-4(3H)-one} into tRNA . This precursor is further elaborated to queuine by two subsequent enzymic reactions {Slany, R . K., & Kersten, H . (1994) Biochimie 76, 1178-1182} . Our previous studies {Hoops, G . C., Townsend, L . B., & Garcia, G . A., (1995) Biochemistry (in press)} on a series of synthetic 5- and 6-substituted 2-aminopyrrolo{2,3-d}pyrimidin-4(3H) -ones have revealed that the E . coli TGT tolerates a wide diversity of substituents (isosteric, or nearly so, to the aminomethyl group of preQ1) at the 5 position . We report here that 2-amino-5-(fluoromethyl)pyrrolo{2,3-d}pyrimidin-4 (3H)-one (FMPP) inactivates TGT in a time- and concentration-dependent manner with k(inact) = 0.074 min-1 and KI = 136 microM . A competitive inhibitor (7-methyl-preQ1), with respect to preQ1, of TGT {Hoops, G.C., Townsend, L.B., & Garcia, G.A . (1995) Biochemistry (in press)} protects the enzyme from inactivation by FMPP . FMPP also acts as a competitive inhibitor (KI = 114 microM) of TGT under initial velocity conditions . The rate of fluoride release from FMPP is slightly faster (0.064 min-1) than the k(inact) (0.053 min-1) at 300 microM FMPP, consistent with fluoride release preceding inactivation . FMPP appears to partition between "normal" turnover (kcat = 0.461 min-1 and Km = 152 microM), inactivation, and an alternative processing to an unidentified, fluoride-released product.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1995 Nov 28, 34(47), 15504 - 11
Reaction of cytochrome bo3 with oxygen: extra redox center(s) are present in the protein; Wang J et al.; The reaction of oxygen with cytochrome bo3, a quinol oxidase from Escherichia coli, has been studied by resonance Raman scattering after initiation of the reaction by CO photolysis in a continuous flow apparatus and by directly mixing the enzyme with oxygen . The high-frequency region of the spectrum was monitored to determine the time evolution of the spin, oxidation, and coordination states of heme O and the oxidation state of heme B by using newly established marker lines for each heme . Three phases of the reaction were detected . In phase I, complete in 75 microseconds, O2 reacted with heme O and formed a low-spin ferric or ferryl adduct without significant oxidation of heme B . In phase II, between 75 and 120 microseconds, a small fraction of heme B was oxidized . In phase III, at approximately 1 s, the majority of heme B was oxidized and heme O reverted to a high-spin ferric state . The high rate of oxygen reduction at heme O to the three- or four-electron reduced level, despite a very low rate of heme B oxidation, indicates that there are electron donors active in the enzyme other than the metal centers . Assays of our enzyme preparations rule out a quinol in the tight binding (QH) site as a possible donor but instead suggest electron donation from the protein matrix, such as from tryptophans or tyrosine.(ABSTRACT TRUNCATED AT 250 WORDS)

Cancer Lett, 1995 Nov 27, 98(1), 9 - 17
Adenoviral-mediated gene transfer into primary human and mouse mammary epithelial cells in vitro and in vivo; Yang J et al.; Primary mammary epithelial cells from both the human and mouse mammary glands can be genetically altered under a variety of situations using the replication-defective adenoviral vector containing a marker gene encoding the E . coli beta-galactosidase . Primary human and mouse mammary epithelial cells in monolayer culture and in three-dimensional collagen gel culture systems were transduced by adenovector at high efficiency . Successful gene transfer was also accomplished in situ and in vivo . In the mouse mammary gland, anatomically restricted gene transfer and expression was demonstrated by micro-injection of adenoviral vector directly into the main duct of the mammary gland . Injection of adenoviral vector directly into the human mammary tissues from reduction mammoplasty specimens, into the mouse mammary gland-free fat pad containing the previously transplanted dissociated human mammary epithelial cells, and intratumorally into the human breast cancer xenografts in nude mice, all resulted in successful gene transfer to human mammary epithelial cells . High efficiency introduction of genetic material into primary mammary epithelial cells is important in the study of mammary carcinogenesis and potentially for gene therapy of human breast cancer.

FEBS Lett, 1995 Nov 27, 376(1-2), 53 - 7
Identification of the Zn2+ binding region in calreticulin; Baksh S et al.; Calreticulin binds Zn2+ with the relatively high affinity/low capacity . To determine the location of the Zn2+ binding site in calreticulin different domains of the protein were expressed in E . coli, using the glutathione S-transferase fusion protein system, and their Zn(2+)-dependent interaction with Zn(2+)-IDA-agarose were determined . Three distinct domains were used in this study: the N + P-domain (the first 290 residues); the N-domain (residues 1-182) and the proline-rich P-domain (residues 180-273) . The N + P-domain bound to the Zn(2+)-IDA-agarose and were eluted with an increasing concentration of imidazole . The N-domain also bound 65Zn2+ as measured by the overlay method . The P-domain did not interact with the Zn(2+)-IDA-agarose and it did not bind any detectable amount of Zn2+ . Chemical modification of calreticulin with diethyl pyrocarbonate indicated that five out of seven histidines were protected in the presence of Zn2+ but they were modified by diethyl pyrocarbonate in the absence of Zn2+ suggesting that these residues may be involved in Zn2+ binding to calreticulin . We conclude that Zn2+ binding sites in calreticulin are localized to the N-domain of the protein, region that is not involved in Ca2+ binding to calreticulin.

FEBS Lett, 1995 Nov 27, 376(1-2), 19 - 23
Characterisation of the RANTES/MIP-1 alpha receptor (CC CKR-1) stably transfected in HEK 293 cells and the recombinant ligands; Proudfoot AE et al.; The CC chemokines RANTES and MIP-1 alpha are known to activate certain leucocytes and leucocytic cell lines . We have produced and fully characterised the recombinant proteins expressed in E . coli . They induce chemotaxis of the pro-monocytic cell line, THP-1 and T cells . THP-1 cells express three of the known CC chemokine receptors . In order to study the activation of a single receptor, we have expressed the shared receptor (CC CKR-1) for RANTES and MIP-1 alpha stably in the HEK 293 cell line . We have examined the effects of RANTES and MIP-1 alpha on the CC CKR-1 transfectants by equilibrium binding studies and in a chemotaxis assay . RANTES competes for {125I}RANTES with an IC50 of 0.6 +/- 0.23 nM, whereas MIP-1 alpha competes for its radiolabelled counterpart with an IC50 of 10 +/- 1.6 nM in the transfectants . These affinities are the same as those measured on the THP-1 cell line . The stably transfected HEK 293 cells respond to both these chemokines in the chemotaxis assay with the same EC50 values as those measured for THP-1 cells . This indicates that this cellular response can be mediated through the CC CKR-1 receptor.

FEBS Lett, 1995 Nov 27, 376(1-2), 103 - 7
Cre1, the carbon catabolite repressor protein from Trichoderma reesei; Strauss J et al.; In order to investigate the mechanism of carbon catabolite repression in the industrially important fungus Trichoderma reesei, degenerated PCR-primers were designed to amplify a 0.7-bp fragment of the cre1 gene, which was used to clone the entire gene . It encodes a 402-amino acid protein with a calculated M(r) of 43.6 kDa . Its aa-sequence shows 55.6% and 54.7% overall similarity to the corresponding genes of Aspergillus nidulans and A . niger, respectively . Similarity was restricted to the aa-region containing the C2H2 zinc finger and several aa-regions rich in proline and basic amino acids, which may be involved in the interaction with other proteins . Another aa-region rich in the SPXX-motif that has been considered analogous to a region of yeast RGR1p, was instead identified as a domain occurring in several eucaryotic transcription factors . The presence of the cre1 translation product was demonstrated with polyclonal antibodies against Cre1, which identified a protein of 43 (+/- 2) kDa in cell-free extracts from T . reesei . A Cre1 protein fragment from the two zinc fingers to the region similar to the aa-sequence of eucaryotic transcription factors, was expressed in Escherichia coli as a fusion protein with glutathione S-transferase . EMSA and in vitro footprinting revealed binding of the fusion protein to the sequence 5'-GCGGAG-3', which matches well with the A . nidulans consensus sequence for CreA binding (5'-SYGGRG-3') . Cell-free extracts of T . reesei formed different complexes with DNA-fragments carrying this binding sites, and the presence of Cre1 and additional proteins in these complexes was demonstrated . We conclude that T . reesei Cre1 is the functional homologue of Aspergillus CreA and that it binds to its target sequence probably as a protein complex.

Mol Gen Genet, 1995 Nov 27, 249(3), 328 - 35
Effects of different growth conditions on the in vivo activity of the tandem Escherichia coli ribosomal RNA promoters P1 and P2; Liebig B et al.; We have analyzed the relative activities of the Escherichia coli ribosomal RNA promoters P1 and P2 in vivo under different physiological conditions . Promoter efficiencies were determined by quantitative comparison of the transcript-specific primer extension products obtained from total RNA preparations . Cells were analyzed at different stages of the growth cycle, at different growth rates, and under conditions of stringent control . In addition, the rRNA gene dosage was altered by transformation with plasmids containing additional rrnD or rrnB transcription units, or rRNA operons in which one of the tandem promoters (P1) had been deleted . Under conditions of amino acid starvation (stringent control) we observed the expected strong reduction in P1-directed transcription . In contrast to the previous assumption that the P2 promoter is not regulated, we simultaneously noticed a smaller but significant repression of P2-directed transcription . In strains in which the rRNA gene dosage was increased by transformation with plasmids bearing rRNA transcription units, a similar degree of repression was observed . Repression of the P1 promoter activity was increased, however, when cells contained extra rRNA operons with P2 promoters only . As demonstrated under stringent control conditions, changes in the growth cycle also affected the activity of promoters P1 and P2 . A greater proportion of P2-derived transcripts was observed when cells changed from exponential to stationary growth or if cultures were grown in minimal medium . Under steady-state, slow growth conditions (minimal medium) we obtained evidence showing that the ratio of P1/P2 transcription products is much lower for cells with extra rrnB as compared to extra rrnD operons or cells lacking extra rRNA operons, implying an operon-specific regulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleic Acids Res, 1995 Nov 25, 23(22), 4690 - 7
Transcription termination at the Escherichia coli thra terminator by spinach chloroplast RNA polymerase in vitro is influenced by downstream DNA sequences; Chen LJ et al.; We have investigated the mechanism of transcription termination in vitro by spinach chloroplast RNA polymerase using templates encoding variants of the transcription-termination structure (attenuator) of the regulatory region of the threonine (thr) operon of Escherichia coli . Fourteen sequence variants located within its d(G+C) stem-loop and d(A+T)-rich regions were studied . We found that the helix integrity in the stem-loop structure is necessary for termination but that its stability is not directly correlated with termination efficiency . The sequence of the G+C stem-loop itself also influences termination . Moreover, the dA template stretch at the 3' end of the terminator plays a major role in termination efficiency, but base pairing between the A and U tract of the transcript does not . From the studies using deletion variants and a series of mutants that alter the sequences immediately downstream from the transcription termination site, we found that termination of transcription by spinach chloroplast RNA polymerase was also modulated by downstream DNA sequences in a sequence-specific manner . The second base immediately following the poly(T) tract is crucial for determining the termination efficiency by chloroplast RNA polymerase, but not of the T7 or E.coli enzymes.

Nucleic Acids Res, 1995 Nov 25, 23(22), 4683 - 9
Identification of a very early promoter of insect Hz-1 virus using a novel dual-expression shuttle vector; Lee ST et al.; Very early promoters of viruses control the proper cascade expression of viral genes and are essential for completion of virus life cycles . These promoters are usually rare and weak and do not encode structural proteins . As a result, they are difficult to identify . In order to identify and clone the very early promoters of a large eukaryotic DNA virus, the Hz-1 virus, a novel cloning strategy was applied . This strategy is based on a dual-expression shuttle vector containing a promoter-less lacZ gene . Insertion of eukaryotic promoters upstream permits the efficient expression of LacZ in bacteria cells . The function of the putative promoters was then confirmed by their proper expression in insect cells . The first two productive infection-specific promoters of Hz-1 virus, contained within the shuttle vectors pTSV-2-129 and pTSV-2-49, were cloned from the HindIII-K and HindIII-A fragments of the Hz-1 viral genome, respectively . By primer extension analysis, an immediate and constitutive expression of the promoter in clone pTSV-2-129 was detected after viral infection . Identification of the productive infection-specific promoters has laid down important groundwork for future studies on the molecular mechanism of the transcriptional switch between productive and persistent infections of Hz-1 virus.

Nucleic Acids Res, 1995 Nov 25, 23(22), 4635 - 41
The ribosomal neighbourhood of the central fold of tRNA: cross-links from position 47 of tRNA located at the A, P or E site; Osswald M et al.; The naturally occurring nucleotide 3-(3-amino-3-carboxy-propyl)uridine (acp3U) at position 47 of tRNA(Phe) from Escherichia coli was modified with a diazirine derivative and bound to ribosomes in the presence of suitable mRNA analogues under conditions specific for the ribosomal A, P or E sites . After photo-activation at 350 nm the cross-links to ribosomal proteins and RNA were identified by our standard procedures . In the 30S subunit protein S19 (and weakly S9 and S13) was the target of cross-linking from tRNA at the A site, S7, S9 and S13 from the P site and S7 from the E site . Similarly, in the 50S subunit L16 and L27 were cross-linked from the A site, L1, L5, L16, L27 and L33 from the P site and L1 and L33 from the E site . Corresponding cross-links to rRNA were localized by RNase H digestion to the following areas: in 16S rRNA between positions 687 and 727 from the P and E sites, positions 1318 and 1350 (P site) and 1350 and 1387 (E site); in the 23S rRNA between positions 865 and 910 from the A site, 1845 and 1892 (P site), 1892 and 1945 (A site), 2282 and 2358 (P site), 2242 and 2461 (P and E sites), 2461 and 2488 (A site), 2488 and 2539 (all three sites) and 2572 and 2603 (A and P sites) . In most (but not all) cases, more precise localizations of the cross-link sites could be made by primer extension analysis.

Nucleic Acids Res, 1995 Nov 25, 23(22), 4620 - 7
DNA strand transfer catalyzed by the 5'-3' exonuclease domain of Escherichia coli DNA polymerase I; Zhang W et al.; A protein which promotes DNA strand transfer between linear double-stranded M13mp19 DNA and single-stranded viral M13mp19 DNA has been isolated from recA- E.coli . The protein is DNA polymerase I . Strand transfer activity residues in the small fragment encoding the 5'-3' exonuclease and can be detected using a recombinant protein comprising the first 324 amino acids encoded by polA . Either the recombinant 5'-3' exonuclease or intact DNA polymerase I can catalyze joint molecule formation, in reactions requiring only Mg2+ and homologous DNA substrates . Both kinds of reactions are unaffected by added ATP . Electron microscopy shows that the joint molecules formed in these reactions bear displaced single strands and therefore this reaction is not simply promoted by annealing of exonuclease-gapped molecules . The pairing reaction is also polar and displaces the 5'-end of the non-complementary strand, extending the heteroduplex joint in a 5'-3' direction relative to the displaced strand . Thus strand transfer occurs with the same polarity as nick translation . These results show that E.coli, like many eukaryotes, possesses a protein which can promote ATP-independent strand-transfer reactions and raises questions concerning the possible biological role of this function.

Nucleic Acids Res, 1995 Nov 25, 23(22), 4608 - 15
Synthesis of cysteine-containing dipeptides by aminoacyl-tRNA synthetases; Jakubowski H; Arginyl-tRNA synthetase (ArgRS) catalyses AMP- and PPi-independent deacylation of Arg-tRNAArg in the presence of cysteine . A dipeptide, Arg-Cys, is a product of this deacylation reaction . Similar reaction with homocysteine yields Arg-Hcy . Arginine is a noncompetitive inhibitor of the cysteine-dependent deacylation which indicates that cysteine binds to the enzyme-Arg-tRNAArg complex at a site separate from the arginine binding site . In the presence of arginine, {14C}Arg-tRNAArg is deacylated at a rate similar to the rate of its spontaneous deacylation in solution and {14C}arginine is a product . Experiments with cysteine derivatives indicate that the -SH group is essential for the reaction whereas -NH2 and -COOH groups are not . Thioesters of arginine are formed with 3-mercaptopropionic acid, N-acetyl-L-cysteine and dithiothreitol . These data suggest that formation of the dipeptide Arg-Cys involves a thioester intermediate, S-(L-arginyl)-L-cysteine, which is not observed because of the rapid rearrangement to form a stable peptide bond . Facile intramolecular reaction results from the favorable geometric arrangement of the alpha-amino group of cysteine with respect to the thioester formed in the initial reaction . Similar reactions, yielding Ile-Cys and Val-Cys, are catalyzed by isoleucyl- and valyl-tRNA synthetases, respectively.

Nucleic Acids Res, 1995 Nov 25, 23(22), 4533 - 41
Structure of open promoter complexes with Escherichia coli RNA polymerase as revealed by the DNase I footprinting technique: compilation analysis; Ozoline ON et al.; Footprinting data for 33 open promoter complexes with Escherichia coli RNA polymerase, as well as 17 ternary complexes with different regulators, have been compiled using a computer program FUTPR . The typical and individual properties of their structural organization are analyzed . Promoters are subgrouped according to the extent of the polymerase contact area . A set of alternative sequence elements that could be responsible for RNA polymerase attachment in different promoter groups is suggested on the basis of their sequence homology near the hyperreactive sites . The model of alternative pathways used for promoter activation is discussed.

Mech Ageing Dev, 1995 Nov 24, 85(2-3), 65 - 72
Effect of different ambient temperature and lipopolysaccharide administration on the circadian rhythm of rectal temperature and hypothalamic PGE2 production in aged male rats; Shemi D et al.; Old and young male rats (22 and 7 months respectively) were exposed to ambient temperatures of 4, 22, 27 and 35 degrees C . The rats' rectal temperatures (RTs) were measured periodically, after exposure to the varying temperatures at different hours during the day . The mean circadian value of RTs in the aged rats was different from that of the young rats . Whereas exposure to low temperatures caused a decrease of 2.0 degrees C in the RTs of the old rats, exposure to heat (35 degrees C) caused an increase of 1 degree C in their RTs . An injection of 200 micrograms (intraperitoneally) of E . coli lipopolysaccharide caused them to experience a long period of hypothermia . Elevation in the RTs after the hypothermic period ended was significantly lower in the old rats . However no significant differences in hypothalamic PGE2 production were to be found between the old and young groups 24 h after pyrogen administration.

J Biol Chem, 1995 Nov 24, 270(47), 28381 - 6
Stepwise assembly of a relaxosome at the F plasmid origin of transfer; Howard MT et al.; A central step in the transfer of genetic information during bacterial conjugation of the Escherichia coli F plasmid involves the formation of a protein-DNA complex, called the relaxosome, at the origin of transfer . During conjugation, the relaxosome introduces a site- and strand-specific nick from which the physical transfer of a single strand of DNA is initiated . At least two F-encoded proteins, TraIp (traI gene product) and TraYp (traY gene product), and one host-encoded protein, integration host factor, are involved in this process . In this report, we use DNase I protection and electron microscopic techniques to investigate the mechanism of relaxosome formation . Our results show that TraYp and integration host factor form a protein-DNA complex that facilitates the binding of TraIp to assemble a relaxosome capable of introducing a site- and strand-specific nick at the origin of transfer . This nick is identical to that observed during conjugation.

J Biol Chem, 1995 Nov 24, 270(47), 28374 - 80
The traY gene product and integration host factor stimulate Escherichia coli DNA helicase I-catalyzed nicking at the F plasmid oriT; Nelson WC et al.; F plasmid conjugative transfer is initiated by the introduction of a site- and strand-specific nick within the plasmid origin of transfer (oriT) . Genetic studies have shown nick formation to be dependent on both the traI and traY genes . However, highly purified TraIp, the traI gene product, nicks oriT in a site- and strand-specific manner in the absence of the traY gene product (TraYp) in vitro (Matson, S.W., and Morton, B.S . (1991) J . Biol . Chem . 266, 16232-16237) . Analysis of the oriT region has revealed binding sites for TraYp and the host protein integration host factor (IHF) . To explore possible interactions occurring at oriT, highly purified TraIp, TraYp, and IHF were incubated with a supercoiled oriT-containing DNA substrate . A marked enhancement of the nicking reaction catalyzed by TraIp was observed in a reaction that required both TraYp and IHF . In addition, TraIp was able to nick a linear oriT-containing double-stranded DNA substrate when IHF and TraYp were present in the reaction; such a substrate is not nicked by TraIp alone . Individual protein concentration requirements for the supercoiled and linear nicking reactions were similar, and the reactions occurred at equal velocity, suggesting that they are biochemically identical . Concentrations of TraYp and IHF that yield half-maximal activity in the nicking assays compare well with the reported KD values for the IHF and TraYp binding sites in oriT . These data, coupled with data presented in the accompanying report, suggest that TraYp and IHF bind independent of one another, forming a nucleo-protein complex with oriT that can be recognized and nicked by TraIp.

J Biol Chem, 1995 Nov 24, 270(47), 28357 - 63
Cell cycle phase-specific phosphorylation of human topoisomerase II alpha . Evidence of a role for protein kinase C; Wells NJ et al.; Type II topoisomerases are essential for faithful cell division in all organisms . In human cells, the alpha isozyme of topoisomerase II has been implicated in catalyzing mitotic chromosome segregation via its action as a DNA unlinking enzyme . Here, we have shown that the enzymatic activity of topoisomerase II alpha protein purified from HeLa cell nuclei was strongly enhanced following phosphorylation by protein kinase C . We have investigated the possibility that this kinase is involved in cell cycle phase-specific phosphorylation of topoisomerase II alpha in HeLa cells . Two-dimensional tryptic phosphopeptide mapping revealed that topoisomerase II alpha protein immunoprecipitated from metabolically labeled HeLa cells was differentially phosphorylated during the G2/M phases of the cell cycle . To identify sites of phosphorylation, and the kinase(s) responsible for this modification, oligohistidine-tagged recombinant domains of topoisomerase II alpha protein were overexpressed in Escherichia coli and purified by affinity chromatography . Phosphorylation of a short fragment of the N-terminal ATPase domain of topoisomerase II alpha by protein kinase C in vitro generated two phosphopeptides that co-migrated with prominent G2/M phase-specific phosphopeptides from the HeLa cell-derived topoisomerase II alpha protein . Site-directed mutagenesis studies indicated that phosphorylation of serine 29 generated both of these phosphopeptides . Our results implicate protein kinase C in the cell cycle phase-dependent modulation of topoisomerase II alpha enzymatic activity in human cells.

J Biol Chem, 1995 Nov 24, 270(47), 28282 - 8
Membrane topology analysis of the sensor kinase KdpD of Escherichia coli; Zimmann P et al.; The expression of the kdpFABC operon, coding for the K(+)-translocating Kdp-ATPase, is under the control of the two regulatory proteins KdpD and KdpE, which belong to the group of sensor kinase/response regulator systems . The topology of the KdpD protein in the cytoplasmic membrane was investigated using LacZ and PhoA fusions at different sites within the polypeptide chain and by treating spheroplasts in the presence or absence of Triton X-100 with the protease kallikrein . The results revealed that KdpD has four membrane-spanning segments in the middle of the polypeptide chain, whereas N and C terminus are both cytoplasmic.

J Biol Chem, 1995 Nov 24, 270(47), 28199 - 203
Kinetics of folding and membrane insertion of a beta-barrel membrane protein; Surrey T et al.; We have studied the kinetics of folding and membrane insertion of the outer membrane protein OmpA of Escherichia coli . In the native structure, its membrane-inserted domain forms a beta-barrel . The protein was unfolded in solubilized form in water/urea, and refolding was induced by dilution of urea and simultaneous addition of lipid vesicles . Three transitions along the folding pathway could be distinguished . Their characteristic times lie below a second, in the range of minutes, and in the range of an hour . The fast process corresponds to the transition from the unfolded state in water/urea to a misfolded state in water, the moderately slow process to a transition from the misfolded state to a partially folded state in the membrane, and the slow process to the transition from the partially folded to the native state . The partially folded state in the membrane is interpreted as the analogue of the molten globule state of soluble proteins.

J Biol Chem, 1995 Nov 24, 270(47), 28188 - 92
Granulocyte-colony stimulating factor and lipopolysaccharide regulate the expression of interleukin 8 receptors on polymorphonuclear leukocytes; Lloyd AR et al.; Interleukin 8 (IL-8) is a potent chemoattractant and activating factor for human polymorphonuclear leukocytes (PMN) and hence plays a critical role in the pathogenesis of acute inflammation . Two unique but homologous receptors for IL-8 have been cloned (IL-8RA and -B), each of which binds the IL-8 ligand with high affinity . PMN stimulated by cytokines or lipopolysaccharide (LPS) exhibit changes in IL-8R mRNA and 125I-IL-8 binding . Granulocyte-colony stimulating factor (G-CSF) treatment of PMN enhances, and LPS inhibits, IL-8R mRNA expression . Similarly, 125I-IL-8 ligand binding to PMN is increased by G-CSF and decreased by LPS treatment . The stimulatory effect of G-CSF on IL-8R expression is transcriptional as it is inhibited by actinomycin D and is evident in nuclear run-on analyses . In contrast, LPS down-regulates IL-8R by both transcriptional and post-transcriptional mechanisms . The alterations in IL-8R expression are associated with similar changes in the IL-8-induced chemotactic responses of PMN . In conclusion, the two types of IL-8 receptor differ in their cellular distribution and are regulated in response to cytokines and LPS . Regulation of IL-8R expression by endogenous and exogenous immunomodulators may be important in the in vivo control of PMN effector functions in inflammation.

J Biol Chem, 1995 Nov 24, 270(47), 28177 - 82
Unfolding properties of tryptophan-containing alpha-subunits of the Escherichia coli tryptophan synthase; Choi SG et al.; The urea-induced unfolding of the Escherichia coli tryptophan synthase alpha-subunit is examined via fluorescence measurements with tryptophan-containing alpha-subunit mutants, constructed by in vitro mutagenesis . Early unfolding studies with urea and guanidine suggested that the wild type protein unfolded in a two-step process with a stable intermediate composed of a native alpha-1 folding unit (residues 1-188) and a completely unfolded alpha-2 folding unit (residues 189-268) . Recently, more detailed spectroscopic and calorimetric data from the Matthews and Yutani groups indicate that such a structure for the intermediates seems unlikely . Previously, we described the introduction of Trp residues as unfolding reporter groups separately into each of the folding domains and showed that these proteins are wild type enzymatically and in their stability to urea . The unfolding behavior of these alpha-subunits, monitored by fluorescence intensity changes at the discrete emission lambda max for each, in both equilibrium and kinetic experiments, suggest that: (a) both folding units commence unfolding simultaneously (near 2 M urea); (b) the larger alpha-1 unit unfolds in a multistep process, initially yielding a partially unfolded intermediate form which subsequently appears to unfold progressively to completion; and (c) the smaller alpha-2 unit unfolds in a single step event . These results are also clearly incompatible with the early proposals on the structure of the intermediate . It is suggested here that the intermediate is heterogeneous, consisting of a stable, partially unfolded form of alpha-1 attached to either a completely folded or completely unfolded form of alpha-2 . These results are consistent with and provide an added dimension to the recent description of the proposed structure of the intermediate.

J Biol Chem, 1995 Nov 24, 270(47), 28165 - 8
Proton-translocating nicotinamide nucleotide transhydrogenase . Reconstitution of the extramembranous nucleotide-binding domains; Yamaguchi M et al.; The nicotinamide nucleotide transhydrogenase of bovine mitochondria is a homodimer of monomer M(r) = 109,065 . The monomer is composed of three domains, an NH2-terminal 430-residue-long hydrophilic domain I that binds NAD(H), a central 400-residue-long hydrophobic domain II that is largely membrane intercalated and carries the enzyme's proton channel, and a COOH-terminal 200-residue-long hydrophilic domain III that binds NADP(H) . Domains I and III protrude into the mitochondrial matrix, where they presumably come together to form the enzyme's catalytic site . The two-subunit transhydrogenase of Escherichia coli and the three-subunit transhydrogenase of Rhodospirillum rubrum have each the same overall tridomain hydropathy profile as the bovine enzyme . Domain I of the R . rubrum enzyme (the alpha 1 subunit) is water soluble and easily removed from the chromatophore membranes . We have isolated domain I of the bovine transhydrogenase after controlled trypsinolysis of the purified enzyme and have expressed in E . coli and purified therefrom domain III of this enzyme . This paper shows that an active bidomain transhydrogenase lacking domain II can be reconstituted by the combination of purified bovine domains I plus III or R . rubrum domain I plus bovine domain III.

J Biol Chem, 1995 Nov 24, 270(47), 28118 - 25
Modified ligands to FA and FB in photosystem I . II . Characterization of a mixed ligand {4Fe-4S} cluster in the C51D mutant of PsaC upon rebinding to P700-Fx cores; Yu L et al.; A Photosystem I (PS I) complex reconstituted with PsaC-C51D (aspartate in lieu of cysteine in position 51) shows light-induced EPR signals with g values, line widths, and photoreduction behavior characteristic of FB . Contrary to an earlier report, a {3Fe-4S} cluster was not located in the reconstituted PS I complex . Instead, a second set of resonances with g values of 2.044, 1.942, and 1.853 becomes EPR-visible when the C51D-PS I complex is measured at 4.2 K . This fast relaxing center, termed FA' is likely to represent a {4Fe-4S} cluster in the mixed ligand (3Cys.1Asp) site . Redox studies show that the Em of FA' and FB are -630 mV and -575 mV, respectively . Room temperature optical studies support the presence of two functioning electron acceptors subsequent to Fx, and NADP+ photoreduction rates mediated by ferredoxin and flavodoxin are nearly equivalent to the wild type . In addition to {3Fe-4S} clusters and S = 1/2 ground state {4Fe-4S} clusters, the free PsaC-C51D protein shows resonances near g = 5.5, which may represent a population of high spin (S = 3/2) {4Fe-4S} clusters in the mixed ligand FA' site . Similar to the C14D-PS I mutant complex, it is proposed that the P700-Fx core selectively rebinds those free PsaC-C51D proteins that contain two {4Fe-4S} clusters . These studies show that primary photochemistry and electron transfer rates in PS I are relatively unaffected by the presence of a highly reducing, mixed ligand cluster in the FA' site.

J Biol Chem, 1995 Nov 24, 270(47), 28049 - 54
Using modified nucleotides to map the DNA determinants of the Tus-TerB complex, the protein-DNA interaction associated with termination of replication in Escherichia coli; Duggan LJ et al.; A series of modified nucleotides was used to map the hydrogen-bonding and hydrophobic sites of the TerB DNA required for Tus interaction . Each of four consensus guanine residues in the TerB-binding site was replaced by 7-deazaguanine, 2-aminopurine, or inosine nucleobase analogues, and each thymine by a uracil analogue . The observable equilibrium dissociation constant for the Tus protein-TerB DNA complex was measured at pH 7.5, 25 degrees C, and 150 mM potassium glutamate using a competition binding method . Substitutions made at position 10 with a 7-deazaguanine, 2-aminopurine, or inosine analogue had a large effect on the stability of the complex, approximately +3 kcal/mol in each case . Substitutions made at positions 13 and 17 had a varied response . For uracil substitutions, potential hydrophobic sites were identified at six positions in the TerB DNA . The energetic penalty for the removal of a single methyl group ranged between +1 and +2 kcal/mol . Rate dissociation measurements agree with these results . Overall, major and minor groove determinants are required for binding . An unusual result was that the conserved nucleotide at position 6 did not significantly affect in vitro binding of the complex.

J Biol Chem, 1995 Nov 24, 270(47), 28006 - 9
The essential function of protein-disulfide isomerase is to unscramble non-native disulfide bonds; Laboissiere MC et al.; Protein-disulfide isomerase (PDI) is an abundant protein of the endoplasmic reticulum that catalyzes dithiol oxidation and disulfide bond reduction and isomerization using the active site CGHC . Haploid pdi1 delta Saccharomyces cerevisiae are inviable, but can be complemented with either a wild-type rat PDI gene or a mutant gene coding for CGHS PDI (shufflease) . In contrast, pdi1 delta yeast cannot be complemented with a gene coding for SGHC PDI . In vitro, shufflease is an efficient catalyst for the isomerization of existing disulfide bonds but not for dithiol oxidation or disulfide bond reduction . SGHC PDI catalyzes none of these processes . These results indicate that in vivo protein folding pathways contain intermediates with non-native disulfide bonds, and that the essential role of PDI is to unscramble these intermediates.

J Mol Biol, 1995 Nov 24, 254(2), 175 - 83
Mapping of the active site zinc ligands of peptide deformylase; Meinnel T et al.; A set of 50 site-directed mutants of the Escherichia coli fms gene was constructed to delineate the residues of the active site of peptide deformylase, including the ligands of the zinc ion . In particular, because zinc is usually coordinate by Asp, Cys, Glu or His residues, all the corresponding codons were individually changed . The functional consequence of the substitutions was assessed by complementation of a fms-null strain with the help of vectors expressing the mutate genes . In addition to the mutations of the Cys90 codon, only those of the three conserved residues of the 132HEXXH136 motif of peptide deformylase prevented the indicator strain growing . Most enzyme variants were purified to homogeneity in a second step . Their characterization in vitro showed that the defects in complementation as observed in vivo corresponded to huge decreases of deformylation efficiency . The change of Glu88 also led to a significant decrease in catalytic rate . Unexpectedly, upon substitutions of Glu79 or of Glu83, the enzymes exhibited a strongly increased catalytic efficiency . The measurement of the content of zinc in each purified variant indicated that Cys90, His132 and His136 bound the metal ion . Zinc-free variants mutated at these positions were obtained and shown to display an increased sensitivity to proteolytic attack . Altogether, the data showed that both the presence of zinc and the conserved residues of the HEXXH motif were crucial for the activity of deformylase . This behaviour identified the enzyme as a member of the zinc metalloproteases superfamily . However, the unexpected participation in the binding of the zinc atom of Cys90, upstream from the HEXXH motif, suggested that peptide deformylase could be representative of a new sub-family, distinct from those of thermolysin and astacin.

J Mol Biol, 1995 Nov 24, 254(2), 163 - 74
An interactive framework for RNA secondary structure prediction with a dynamical treatment of constraints; Gaspin C et al.; A novel approach aiding in the prediction of RNA secondary structures is presented . Although phylogenetic methods are the most successful at deriving RNA secondary structures, the are not applicable when the number of sequences or the sequence variability is too low . Methods based on energy minimization are therefore of great interest . However, some of the suboptimal RNA secondary structures computed with classic methods are unsaturated structures, i.e . some structures are included into others . Thus, the incorporation of constraints during the process of folding is not possible, while the incorporation of constraints before the process of folding often introduces a bias into the energy function . This paper describes a new procedure which allows for the incorporation of constraints before and during the process of RNA folding . SAPSSARN is an interactive program which offers a framework, both to specify a secondary structure through a set of folding constraints and to compute all the supoptimal saturated RNA secondary structures which satisfy all the folding constraints . At the start, it relies on the computation of the probabilities of pairing of each base with all others according to McCaskill's algorithm . The constraint satisfaction formulation of the problem deals dynamically with a chosen set of folding constraints and, finally, a search algorithm computes all the suboptimal saturated secondary structures which satisfy those folding constraints . Within such a framework, it is possible to test new ideas about RNA folding and secondary structures, including pseudoknots, can be computed . The program is illustrated with RNA sequences on which we obtained results in agreement with known structures by using a protocol which mimics the hierarchical folding of RNA molecules.

J Mol Biol, 1995 Nov 24, 254(2), 150 - 62
The DNA-binding domain of the hexameric arginine repressor; Grandori R et al.; The arginine repressor of Escherichia coli is a classical feedback regulator, signalling the availability of L-arginine inside the cell . It differs from most other bacterial repressors in functioning as a hexamer, but structural details have been lacking and its shares no clear sequence homologies with other transcriptional regulators . Analysis of the amino acid residue sequence and proteolytic cleavage pattern of the repressor was used to identify a region predicted to house the DNA-binding function . When this protein fragment is overexpressed from a clone of the corresponding gene fragment, it represses ornithine transcarbamylase levels in vivo, and binds to the operator DNA in vitro, both in an arginine-independent manner . Sedimentation equilibrium and gel filtration indicate that the purified protein fragment is a monomer in solution . The results thus define the domain organization of the repressor at low resolution, suggesting that the N and C-terminal portions of the polypeptide chain are separated by a structural and functional border that decouples hexamerization and arginine binding from DNA binding.

Carbohydr Res, 1995 Nov 22, 277(2), 283 - 90
Structure of the O-specific side chain of the Escherichia coli O128 lipopolysaccharide; Sengupta P et al.; The O-specific polysaccharide isolated from Escherichia coli O128 lipopolysaccharide contains D-galactose, L-fucose, and 2-acetamido-2-deoxy-D-galactose in the molar ratios 2:1:2 . The primary structure of the O-specific polysaccharide from E . coli was established by compositional analysis, methylation analysis, together with 1H and 13C NMR spectroscopy including two-dimensional shift-correlated and one-dimensional NOE spectroscopy . The polysaccharide moiety was found to consist of a tetrasaccharide backbone containing D-galactose and 2-acetamido-2-deoxy-D-galactose, with L-fucose as a side chain in a branched pentasaccharide repeating unit having the following structure: {formula: see text}

Biochem Biophys Res Commun, 1995 Nov 22, 216(3), 881 - 91
Sequence analysis of frog alpha B-crystallin cDNA: sequence homology and evolutionary comparison of alpha A, alpha B and heat shock proteins; Lu SF et al.; alpha-Crystallin is a major lens protein present in the lenses of all vertebrate species . Recent studies have revealed that bovine alpha-crystallins possess genuine chaperone activity similar to small heat-shock proteins . In order to facilitate the determination of the primary sequence of amphibian alpha B-crystallin, cDNA encoding alpha B subunit chain was amplified using a new "Rapid Amplification of cDNA Ends" (RACE) protocol of Polymerase Chain Reaction (PCR) . PCR-amplified product corresponding to alpha B subunit was then subcloned into pUC18 vector and transformed into E . coli strain JM109 . Plasmids purified from the positive clones were prepared for nucleotide sequencing by the automatic fluorescence-based dideoxynucleotide chain-termination method . Sequencing more than five clones containing DNA inserts coding for alpha B-crystallin subunit constructed only one complete full-length reading frame of 522 base pairs similar to that of alpha A subunit, covering a deduced protein sequence of 173 amino acids including the universal translation-initiating methionine . The frog alpha B crystallin shows 69, 66 and 56% whereas alpha A crystallin shows 83, 81 and 69% sequence similarity to the homologous chains of bovine, chicken and dogfish, respectively, revealing a more divergent structural relationship among these alpha B subunits as compared to alpha A subunits . Structural analysis and comparison of alpha A- and alpha B-crystallin subunits from eye lenses of different classes of vertebrates also shed some light on the evolutionary relatedness between alpha B/alpha A crystallins and the small heat-shock proteins.

Biochem Biophys Res Commun, 1995 Nov 22, 216(3), 814 - 20
Affinity of Bandeiraea (Griffonia) simplicifolia lectin-I, isolectin B4 for Gal alpha 1-->4 Gal ligand; Wu AM et al.; The affinity of Bandeiraea (Griffonia) simplicifolia lectin-I isolectin B4 (BSI-B4) for the isomer of human blood group B active disaccharide (B, Gal alpha 1-->3Gal), the Gal alpha 1-->4Gal galabiose ligand, was studied by quantitative precipitin (QPA) and precipitin-inhibition assays . When human blood group B, P1 and H active glycoproteins were tested by OPA . BSI-B4 reacted strongly with both the B active glycoprotein purified from human ovarian cyst fluid and a P1 active glycoprotein isolated from sheep hydatid fluid and precipitated over 86% of the lectin nitrogen added . The P1 active glycoprotein-BSI-B4 interaction was inhibited by both Gal alpha 1-->3Gal alpha 1-->methyl and Gal alpha 1-->4Gal disaccharide indicating that BSI-B4 is not only reacting with Gal alpha 1-->3Gal disaccharide, but also recognizing Gal alpha 1-->4Gal . The galabiose sequence is frequently found in the carbohydrate chains of many glycosphingolipids located at the mammalian cell membranes such as intestinal and red blood cell membranes, for E . coli ligand binding and toxin attachment.

Biochem Biophys Res Commun, 1995 Nov 22, 216(3), 1101 - 9
Mouse rhodanese gene (Tst): cDNA cloning, sequencing, and recombinant protein expression; Dooley TP et al.; Rhodanese (thiosulfate sulfurtransferase) is expressed at high levels in liver and is involved in the detoxification of cyanide . The full-length cDNA corresponding to the mouse rhodanese gene (Tst), which is located on chromosome 15, was cloned by PCR amplification of a liver cDNA library and subjected to DNA sequencing . Alignment of the rhodanese cDNA sequences from mouse and rat, which we previously cloned (Biochem . J . 275:227-231), revealed 97.3 percent identity at the protein level and 94.6 percent identity at the DNA level . When the mouse and rat cDNAs were expressed under the control of IPTG-inducible promoters in E . coli, the cell extracts exhibited cyanide-metabolizing activity, indicating that both genes encode functional rhodanese molecules.

Biochem Biophys Res Commun, 1995 Nov 22, 216(3), 1088 - 94
cDNA sequence analysis and expression of alpha-bungarotoxin from Taiwan banded krait (Bungarus multicinctus); Kuo KW et al.; The cDNA encoding alpha-bungarotoxin (alpha-BuTx) was constructed from the cellular RNA isolated from the venom glands of Bungarus multicintus by RT-PCR . The primers deduced from the N-, C-terminal amino acid residues and the conserved middle segment of the sequence, i.e., R25KMWC29 in alpha-BuTx could specifically amplify cDNAs encoding amino acid residues from 1 to 29 and 25 to 74 of alpha-BuTx . The complete cDNA was obtained by linking sufficient amounts of the two cDNA fragments by PCR . The amino acid sequence translated from the alpha-BuTx cDNA sequence was identical to that of native toxin . The PCR product was then cloned into pGEX vector and transformed in DH5 E . coli strain . The fused protein expressed in host strain exhibited the same antigenicity as native alpha-BuTx . This communication is the first report for the cloning and expression of a long alpha-neurotoxin from the venom glands.

J Biotechnol, 1995 Nov 21, 43(1), 63 - 70
Bioluminescent detection of RNA with sequence-specificity using RNA binding protein-luciferase fusion protein; Kajita Y et al.; A novel bifunctional reagent has been synthesized for RNA detection by genetic fusion of a sequence-specific RNA binding protein and firefly luciferase . The RNA binding protein used in this study recognizes the oligoribonucleotide rH4 which contains four (UUAGGG)-repeat sequence, while luciferase works as a bioluminescent marker . The constructed fusion protein exhibited both sequence-specific RNA binding and bioluminescent activities . The rH4 and rECGF, which has an unrelated sequence having the same length as rH4, were immobilized on a nylon membrane . The membrane was pre-treated by 5% bovine serum albumin and soaked into a fusion protein solution . Bioluminescent detection has successfully been performed at more than 50 pmol of rH4, so the detection limit using this protein was 50 pmol . However, no appreciable bioluminescence was induced by rECGF.

J Biotechnol, 1995 Nov 21, 43(1), 41 - 4
Effect of PQQ glucose dehydrogenase overexpression in Escherichia coli on sugar-dependent respiration; Sode K et al.; Pyrroloquinoline quinone glucose dehydrogenase (PQQGDH) was overexpressed in Escherichia coli, and its impact on sugar-dependent respiration was investigated . Sugar-dependent respiration patterns under PQQGDH overexpression can be devided into two types . The first type involves D-glucose and D-mannose, which are utilized by the phosphotransferase system (PTS) and are also the substrates of PQQGDH . As a result of PQQGDH overexpression, the apparent Km value of sugar-dependent respiration shifted to higher concentration compared with E . coli parental cells . The second type included D-xylose and D-galactose, which are the substrates of PQQGDH, but not the PTS sugars . PQQGDH overexpressing cells showed much higher respiration than parental cells . These results suggested that PQQGDH overexpression may alter sugar utilization preferences in E . coli, suggesting further possible applications in metabolic engineering for carbon source utilization.

Biochemistry, 1995 Nov 21, 34(46), 15381 - 7
tRNA-guanine transglycosylase from Escherichia coli: structure-activity studies investigating the role of the aminomethyl substituent of the heterocyclic substrate PreQ1; Hoops GC et al.; A series of 5-substituted 2-aminopyrrolo{2,3-d}pyrimidin-4(3H)-ones have been synthesized in order to study the substrate specificity of the tRNA-guanine transglycosylase (TGT) from Escherichia coli . A number of these compounds were initially examined as inhibitors of radiolabeled guanine incorporation into tRNA catalyzed by TGT {Hoops, G . C., Garcia, G . A., & Townsend, L . B . (1992) 204th National Meeting of the American Chemical Society, Washington, DC, August 23-28, 1992, Division of Medicinal Chemistry, Abstract 113} . The kinetic parameters of these analogues as substrates in the TGT reaction have been determined by monitoring the loss of radiolabeled guanine from 8-{14C}G34-tRNA . This study reveals that the tRNA-guanine transglycosylase from E . coli will tolerate a wide variety of substituents at the 5-position . The role of the 5-substituent appears to be entirely in binding/recognition with no apparent effects upon catalysis . A correlation between N7 pKa and Vmax suggests the deprotonation of N7 during the reaction, which must occur prior to subsequent glycosidic bond formation, appears to be partially rate-determining for the natural substrate . Comparison of the Kis of 7-methyl-substituted competitive inhibitors to the Kms of their corresponding substrates suggests that some substrates (including preQ1) are kinetically "sticky" (i.e., Km is equivalent to Kd) and other substrates have Kms that reflect catalytic rates as well as binding.

Biochemistry, 1995 Nov 21, 34(46), 15375 - 80
Alanine-scanning mutagenesis of human transcript elongation factor TFIIS; Cipres-Palacin G et al.; TFIIS is a transcription elongation factor that binds to RNA polymerase II and allows it to transcribe through a variety of transcriptional blockages by inducing cleavage near the 3' end of the nascent transcript . Although this cleavage reaction plays a key role in the process of reactivation of transcription by TFIIS, the exact mechanism by which TFIIS promotes readthrough by RNA polymerase II is not completely understood . We therefore undertook a systematic mutagenesis of the C-terminal half of TFIIS (delta TFIIS) to evaluate the contribution of charged residues in this region to induce transcript cleavage and promote readthrough in vitro . Twenty-two delta TFIIS alanine-scanning mutants were constructed by substitution of alanine for each amino acid in clusters of charged residues in the C-terminal half of HeLa TFIIS . The ability to induce transcript cleavage and readthrough of these mutants was tested in vitro using RNA polymerase II ternary elongation complexes arrested at a block to elongation . This alanine-scanning mutagenesis analysis allowed the identification of regions or residues important for the activity of TFIIS . Many of the mutants were reduced alike in both cleavage and readthrough activities . However, in several cases there was no simple correlation between these activity reductions.

Biochemistry, 1995 Nov 21, 34(46), 15328 - 33
A peptide derived from a tissue factor loop region functions as a tissue factor--factor VIIa antagonist; Paborsky LR et al.; Tissue factor (TF) is a transmembrane protein that functions in the initiation of blood coagulation in vivo . At sites of vascular injury, TF serves as a cell-surface receptor for the serine protease factor VIIa (FVIIa), forming an enzyme--cofactor complex and enhancing the catalytic activity of FVIIa . Tissue factor, along with the receptors for alpha- and gamma-interferons, is a member of the class 2 cytokine receptor superfamily . Crystallographic analysis demonstrated that the extracellular domain of TF consists of two immunoglobulin-like domains joined by a linker region . Each domain is comprised of two antiparallel beta-sheets containing seven conserved beta-strands separated by more variable loop regions . Extensive mutagenesis has been performed in order to map the FVIIa binding site on TF . Results indicated that the discontinuous binding site for FVIIa lies at the domain--domain interface and includes residues from extended loops and beta-strands within both the N- and C-terminal domains . Our previous study provided evidence that three consecutive residues (D44, W45, K46) within the TF loop region between beta-strands C and C' of the N-terminal domain were important for interactions with FVIIa . We have presently extended our alanine-scanning mutagenesis to include the residues within the flanking beta-strands . Thirteen sTF mutants were screened for their ability to enhance FVIIa activity . Three residues within strand C (Y34, Q37, I38) and two residues within C' (K48, Y51) were shown to be important for TF cofactor function.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1995 Nov 21, 34(46), 15307 - 14
Phospholipase A2 engineering . The roles of disulfide bonds in structure, conformational stability, and catalytic function; Zhu H et al.; Site-directed mutagenesis was used to probe the contribution of each of the seven disulfide bonds of bovine pancreatic phospholipase A2 (PLA2, overexpressed in Escherichia coli) to the structure, conformational stability, and catalytic function of the enzyme . Each of the seven disulfide bonds, C11-C77, C27-C123, C29-C45, C44-C105, C51-C98, C61-C91, and C84-C96, was deleted separately by changing both cysteine (C) residues to alanine (A) . The structural properties of the mutants were analyzed by 1D and 2D proton NMR, the conformational stability by guanidine hydrochloride-induced denaturation, and the catalytic property by measuring kinetic parameters toward DC8PC (1,2-dioctanoyl-sn-glycero-3-phosphocholine) micelles . The results led to the following significant findings: (i) All but one (C84A-C96A) mutants have been refolded and purified by use of the same procedure for wild-type PLA2 . Thus, the disulfide bonds are generally not important to the folding pathway of PLA2 . (ii) The disulfide bond C11-C77 is most important to the conformation and conformational stability of the enzyme since deletion of this disulfide bond resulted in greatly perturbed NMR properties and in a decrease of 6.2 kcal/mol in conformational stability . However, the C11A-C77A mutant displayed little change in catalytic function . (iii) The effects of deleting disulfide bonds on the catalytic function of PLA2 are small, except the disulfide bond C29-C45 which connects the calcium binding loop with the helix C . However, the conformation and conformational stability of the C29A-C45A mutant was found to decrease by a factor of 10 or greater . (ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1995 Nov 21, 34(46), 15191 - 203
Conversion of cytochrome b562 to c-type cytochromes; Barker PD et al.; Cytochrome b562 from the periplasm of Escherichia coli is the only member of a family of cytochromes sharing the 4-alpha-helical bundle structural motif that does not have a covalently bound heme . We have introduced cysteine residues into the amino acid sequence of cytochrome b562 in positions homologous to those found in the other members of the family, generating the ubiquitous heme-binding peptide (-C-X-Y-C-H-) found in virtually all c-type cytochromes . The resulting single-cysteine-containing mutants, R98C and Y101C, together with the double mutant combining both of these mutations have been expressed into the periplasm of E . coli . The apo- and holoprotein products of each mutation have been isolated, and all the mutants produce multiple species with covalently attached heme . Results from ion exchange chromatograph, optical spectroscopy, SDS gel electrophoresis, and electrospray mass spectrometry identified those species that appear to be cytochrome b562 holoprotein with thioether covalent linkages to the heme as the only difference in chemical composition between them and the wild-type protein . Results from 1H-NMR experiments prove the existence of the expected c-type covalent bonds in each of these proteins and show that the structure of the heme pocket is not significantly perturbed by the covalent modification(s) . These proteins all have perturbed optical spectra, compared with those of the wild-type protein, that are consistent with the modifications but are still characteristic of six-coordinate, low-spin cytochromes with Met-His ligation to the heme iron in both oxidation states.

Biochemistry, 1995 Nov 21, 34(46), 15182 - 90
The actin-binding protein hisactophilin binds in vitro to partially charged membranes and mediates actin coupling to membranes; Behrisch A et al.; The interaction of the actin-binding protein hisactophilin from Dictyostelium discoideum amoebae to partially charged lipid membranes composed of mixtures of L-alpha-dimyristoylphosphatidylcholine (DMPC) with L-alpha-dimyristoylphosphatidylglycerol (DMPG) and L-alpha-phosphatidylinositol 4,5-bisphosphate (PIP2) is studied by film balance experiments, microfluorescence, and lateral diffusion measurements at low ionic strengths (approximately 20 mM) . Excess surface concentrations and adhesion energies of the protein are evaluated by the application of Gibbs law of surface excess as a function of charged lipid content . Protein expressed in E . coli lacking a myristic acid chain (EC-HIS) and natural protein with a fatty acid (DIC-HIS) isolated from Dictyostelium cells are compared . For mixtures of DMPG and DMPC, protein binding leads to an increase in lateral pressure of the monolayer (at constant area) and causes strong lipid immobilization pointing to partial penetration of the protein into the lipid layer . The natural protein causes a much stronger immobilization than does EC-HIS . For a given bulk concentration, the adsorbed protein/lipid molar ratio increases with the molar fraction chi PG of charged lipid but saturates at about 50 mol% of DMPG . Natural hisactophilin (DIC-HIS) binding to PIP2-containing monolayers is purely electrostatic at low bulk concentration cb, and protein penetration dominates only at cb > 68 nM . Fluorescence experiments demonstrate that the natural protein (DIC-HIS) can mediate the binding of monomeric actin or very small oligomers to membranes, showing that the adsorbed protein remains functional . In contrast, the recombinant hisactophilin (EC-HIS) can mediate only the membrane coupling of larger actin structures.

Biochemistry, 1995 Nov 21, 34(46), 15150 - 6
Solution conformation of the extracellular domain of the human tumor necrosis factor receptor probed by Raman and UV-resonance Raman spectroscopy: structural effects of an engineered PEG linker; Tuma R et al.; The solution structure of the Escherichia coli-expressed extracellular domain, residues 12-172, of the human 55 kDa type I tumor necrosis factor receptor (TNFR) has been probed by Raman (514.5 nm) and ultraviolet-resonance Raman (244 nm) excitations . The Raman spectra have been collected from both the free TNFR domain and an engineered "dumbbell-like" derivative, consisting of two mutant receptor moieties linked by a 20 kDa polyethylene glycol (PEG) tether . The results demonstrate a TNFR secondary structure which is rich in beta-sheet and deficient in alpha-helix, consistent with the reported X-ray crystal structure of baculovirus expressed receptor complexed with factor beta {Banner, D . W., D'Arcy, A., Janes, W., Gentz, R., Schoenfeld, H.-J., Broger, C., Loetscher, H., & Lesslauer, W . (1993) Cell 73, 431-445} . Conversely, the solution structure of TNFR differs from the crystal structure in its distribution of disulfide rotamers and in the orientation of its unique indole side chain (tryptophan-107) . These differences are attributed, respectively, to N-terminal truncation and factor binding in the TNFR crystal structure . The tryptophan configuration, which is easily monitored in both Raman and UVRR spectra, is proposed as a potential signal of receptor/factor recognition and binding . Application of the Raman probes to the engineered TNFR dumbbell, which is of interest as a potential therapeutic, shows that TNFR moieties of the dumbbell exhibit secondary structures and side chain environments which are indistinguishable from those of the native, wild-type moiety . The results suggest that the PEGylated dumbbell may function as an effective TNFR drug delivery system without the consequence of a deleterious antigenic response.

Biochemistry, 1995 Nov 21, 34(46), 14997 - 5005
Solution structure of the MutT enzyme, a nucleoside triphosphate pyrophosphohydrolase; Abeygunawardana C et al.; The MutT enzyme (129 residues) catalyzes the hydrolysis of normal and mutagenic nucleoside triphosphates, such as 8-oxo-dGTP, by substitution at the rarely attacked beta-P, to yield NMP and pyrophosphate . Previous heteronuclear NMR studies of MutT have shown the secondary structure to consist of a five-stranded mixed beta-sheet connected by the loop I-alpha-helix I--loop II motif, by two tight turns, and by loop III, and terminated by loop IV--alpha-helix II {Abeygunawardana et al . (1993) Biochemistry 32, 13071-13080; Weber et al . (1993) Biochemistry 32, 13081-13087) . Complete side-chain assignments of 1H and 13C resonances have now been made by 3D C(CO)NH and HCCH-TOCSY experiments . A total of 1461 interproton proximities (11 per residue), obtained by 3D 15N-resolved NOESY-HSQC and 3D 13C-resolved NOESY-HSQC spectra, including 372 long-range NOEs, as well as 65 dihedral angle (phi) restraints and 34 backbone hydrogen bond restraints were used to determine the tertiary structure of MutT by distance geometry, simulated annealing, and energy minimization with the program X-PLOR . The structure is globular and compact with the parallel portion of the beta-sheet sandwiched between the two alpha-helices, forming an alpha+beta fold . The essential divalent cation has previously been shown to bind near residues Gly-37, Gly-38, Lys-39, and Glu-57, and nucleotides have been shown to bind near residues Leu-54 and Val-58 by NMR relaxation methods {Frick et al . (1995) Biochemistry 34, 5577-5586}.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1995 Nov 21, 34(46), 14955 - 62
A trimeric subdomain of the simian immunodeficiency virus envelope glycoprotein; Blacklow SC et al.; Previous attempts to define the oligomeric state of the HIV and SIV envelope glycoproteins have yielded conflicting results . We have produced in Escherichia coli a recombinant model for the ectodomain of the SIV envelope protein gp41 and have identified a small, trimeric subdomain by proteolytic digestion of this gp41 fragment . The subdomain assembles from two peptide fragments, spanning residues 28-80 (N28-80) and residues 107-149 (C107-149) of SIV gp41 . Each of these peptides contains a 4,3-hydrophobic repeat, the hallmark of coiled-coil sequences . Upon mixing, the peptides form a highly helical, trimeric complex {3(N+C)} that resists proteolysis and has a melting temperature (Tm) above 90 degrees C in physiological buffer . The N- and C-terminal fragments are antiparallel to each other in the complex, as judged by the observation that digestion of a variant recombinant protein truncated at the amino terminus yields a C-terminal fragment shortened at its carboxy terminus . The N28-80 peptide contains more positions within the heptad repeat than C107-149 that are predominantly hydrophobic, suggesting that N28-80 is buried in the interior of the complex . We propose that the complex consists of a parallel, trimeric coiled-coil of the N-terminal peptide, encircled by three C-terminal peptide helices arranged in an antiparallel fashion, and that this complex forms a core within the gp41 extracellular domain.

Proc Natl Acad Sci U S A, 1995 Nov 21, 92(24), 11289 - 93
Invariant chain made in Escherichia coli has an exposed N-terminal segment that blocks antigen binding to HLA-DR1 and a trimeric C-terminal segment that binds empty HLA-DR1; Park SJ et al.; Invariant chain (Ii), a membrane glycoprotein, binds class II major histocompatibility complex (MHC) glycoproteins, probably via its class II-associated Ii peptide (CLIP) segment, and escorts them toward antigen-containing endosomal compartments . We find that a soluble, trimeric ectodomain of Ii expressed and purified from Escherichia coli blocks peptide binding to soluble HLA-DR1 . Proteolysis indicates that Ii contains two structural domains . The C-terminal two-thirds forms an alpha-helical domain that trimerizes and interacts with empty HLA-DR1 molecules, augmenting rather than blocking peptide binding . The N-terminal one-third, which inhibits peptide binding, is proteolytically susceptible over its entire length . In the trimer, the N-terminal domains act independently with each CLIP segment exposed and free to bind an MHC class II molecule, while the C-terminal domains act as a trimeric unit.

Proc Natl Acad Sci U S A, 1995 Nov 21, 92(24), 11115 - 9
Artificial regulation of gene expression in Escherichia coli by RNase P; Guerrier-Takada C et al.; Plasmids encoding various external guide sequences (EGSs) were constructed and inserted into Escherichia coli . In strains harboring the appropriate plasmids, the expression of fully induced beta-galactosidase and alkaline phosphatase activity was reduced by more than 50%, while no reduction in such activity was observed in strains with non-specific EGSs . The inhibition of gene expression was virtually abolished at restrictive temperatures in strains that were temperature-sensitive for RNase P (EC 3.1.26.5) . Northern blot analysis showed that the steady-state copy number of EGS RNAs was several hundred per cell in vivo . A plasmid that contained a gene for M1 RNA covalently linked to a specific EGS reduced the level of expression of a suppressor tRNA that was encoded by a separate plasmid . Similar methods can be used to regulate gene expression in E . coli and to mimic the properties of cold-sensitive mutants.

Proc Natl Acad Sci U S A, 1995 Nov 21, 92(24), 11095 - 9
Restriction-modification systems as genomic parasites in competition for specific sequences; Kusano K et al.; Restriction-modification (RM) systems are believed to have evolved to protect cells from foreign DNA . However, this hypothesis may not be sufficient to explain the diversity and specificity in sequence recognition, as well as other properties, of these systems . We report that the EcoRI restriction endonuclease-modification methylase (rm) gene pair stabilizes plasmids that carry it and that this stabilization is blocked by an RM of the same sequence specificity (EcoRI or its isoschizomer, Rsr I) but not by an RM of a different specificity (PaeR7I) on another plasmid . The PaeR7I rm likewise stabilizes plasmids, unless an rm gene pair with identical sequence specificity is present . Our analysis supports the following model for stabilization and incompatibility: the descendants of cells that have lost an rm gene pair expose the recognition sites in their chromosomes to lethal attack by any remaining restriction enzymes unless modification by another RM system of the same specificity protects these sites . Competition for specific sequences among these selfish genes may have generated the great diversity and specificity in sequence recognition among RM systems . Such altruistic suicide strategies, similar to those found in virus-infected cells, may have allowed selfish RM systems to spread by effectively competing with other selfish genes.

Proc Natl Acad Sci U S A, 1995 Nov 21, 92(24), 10964 - 8
Rotation of subunits during catalysis by Escherichia coli F1-ATPase; Duncan TM et al.; During oxidative and photo-phosphorylation, F0F1-ATP synthases couple the movement of protons down an electrochemical gradient to the synthesis of ATP . One proposed mechanistic feature that has remained speculative is that this coupling process requires the rotation of subunits within F0F1 . Guided by a recent, high-resolution structure for bovine F1 {Abrahams, J . P., Leslie, A . G., Lutter, R . & Walker, J . E . (1994) Nature (London) 370, 621-628}, we have developed a critical test for rotation of the central gamma subunit relative to the three catalytic beta subunits in soluble F1 from Escherichia coli . In the bovine F1 structure, a specific point of contact between the gamma subunit and one of the three catalytic beta subunits includes positioning of the homolog of E . coli gamma-subunit C87 (gamma C87) close to the beta-subunit 380DELSEED386 sequence . A beta D380C mutation allowed us to induce formation of a specific disulfide bond between beta and gamma C87 in soluble E . coli F1 . Formation of the crosslink inactivated beta D380C-F1, and reduction restored full activity . Using a dissociation/reassembly approach with crosslinked beta D380C-F1, we incorporated radiolabeled beta subunits into the two noncrosslinked beta-subunit positions of F1 . After reduction of the initial nonradioactive beta-gamma crosslink, only exposure to conditions for catalytic turnover results in similar reactivities of unlabeled and radiolabeled beta subunits with gamma C87 upon reoxidation . The results demonstrate that gamma subunit rotates relative to the beta subunits during catalysis.

Anal Biochem, 1995 Nov 20, 232(1), 98 - 106
Single-run separation and detection of multiple metabolic intermediates by anion-exchange high-performance liquid chromatography and application to cell pool extracts prepared from Escherichia coli; Bhattacharya M et al.; A method is described for analysis of the intracellular concentrations of metabolic intermediates of the Embden-Meyerhof-Parnas pathway, the Entner-Doudoroff pathway, the pentose phosphate pathway, and the tricarboxylic acid cycle in cell pool extracts of Escherichia coli . A single anion-exchange HPLC run of 40 min allowed resolution of 27 anionic metabolite standards . Detection limits of 0.1 nmol per injection were achieved by use of a conductivity detector equipped with an anion self-regenerating suppressor and a uv detector . A boiling water extraction procedure was used to prepare cell pool extracts . Cochromatography of cell pool extracts and metabolite standards was used to confirm the identities of metabolites in the cell pool . As many as 16 metabolites could be detected and quantified in the cell pool extracts by using the described HPLC method . An analysis of metabolite concentrations in E . coli showed the dynamics of glucose metabolism during a 2-min transition from starvation to steady-state metabolism following addition of glucose . The ease and power of this method suggests general utility for in vivo metabolite analysis in a variety of experimental systems.

Gene, 1995 Nov 20, 165(2), 307 - 11
Cloning and characterization of the human 5,10-methenyltetrahydrofolate synthetase-encoding cDNA; Dayan A et al.; Methenyltetrahydrofolate synthetase (MTHFS) catalyses the obligatory initial metabolic step in the intracellular conversion of 5-formyltetrahydrofolate to other reduced folates . We have isolated and sequenced a human MTHFS cDNA which is 872-bp long and codes for a 203-amino-acid protein of 23,229 Da . Escherichia coli BL21(DE3), transfected with pET11c plasmids containing an open reading frame encoding MTHFS, showed a 100-fold increase in MTHFS activity in bacterial extracts after IPTG induction . Northern blot studies of human tissues determined that the MTHFS mRNA was expressed preferentially in the liver and Southern blot analysis of human genomic DNA suggested the presence of a single-copy gene.

Gene, 1995 Nov 20, 165(2), 303 - 6
High-level production and one-step purification of biologically active human growth hormone in Escherichia coli; Mukhija R et al.; A plasmid has been constructed to direct the synthesis of recombinant human growth hormone (re-hGH) in Escherichia coli as a fusion protein containing a His6 tag at the N-terminus under the control of the T5 promoter . The re-hGH was synthesized in large amounts and accumulated in the form of inclusion bodies upon induction with IPTG . Inclusion bodies were solubilized in 6 M guanidine.HCl and the re-hGH was purified by single-step affinity chromatography on Ni(2+)-nitrilotriacetic acid (NTA) agarose . At the shake flask level, the purified re-hGH was obtained with a yield of 30 mg/l of culture . The re-hGH was biologically active in a node rat lymphoma (Nb2) cell bioassay.

Gene, 1995 Nov 20, 165(2), 223 - 7
Cloning and characterization of a cDNA encoding an 18.0-kDa class-I low-molecular-weight heat-shock protein from rice; Lee YL et al.; A novel cDNA clone, Oshp18.0 cDNA, encoding a rice (Oryza sativa L . cv . Tainong 67) 18.0-kDa heat-shock protein (HSP), was isolated from a cDNA library of heat-shocked rice seedlings by use of the rice HSP cDNA, Oshsp17.3 cDNA, as a probe . The sequence showed that Oshsp18.0 cDNA contains a 749-bp insert encoding an ORF of 160 amino acids, with a predicted molecular mass of 18.0 kDa and a pI of 7.3 . Sequence comparison reveals that Oshsp18.0 cDNA is highly homologous to other low-molecular-weight (LMW) HSP cDNAs . Also, the results of hybrid-selected in vitro translation clearly establish that Oshsp18.0 cDNA is the rice 18.0-kDa LMW HSP-encoding cDNA clone . The recombinant Oshsp18.0 fusion protein produced in Escherichia coli was of the size predicted, and was recognized by the class-I rice 16.9-kDa HSP antiserum . The results suggest that Oshsp18.0 cDNA is an 18.0-kDa class-I LMW HSP- encoding cDNA clone from rice.

FEBS Lett, 1995 Nov 20, 375(3), 294 - 8
Amino acid sequence and expression of the hepatic glycogen-binding (GL)-subunit of protein phosphatase-1; Doherty MJ et al.; A full-length cDNA encoding the putative hepatic glycogen-binding (GL) subunit of protein phosphatase-1 (PP1) was isolated from a rat liver library . The deduced amino acid sequence (284 residues, 32.6 kDa) was 23% identical (39% similar) to the N-terminal region of the glycogen-binding (GM) subunit of PP1 from striated muscle . The similarities between GM and GL were most striking between residues 63-86, 144-166 and 186-227 of human GM (approximately 40% identity), nearly all the identities with the putative yeast homologue GAC1 being located between 144-166 and 186-227 . The cDNA was expressed in E . coli, and the expressed protein transformed the properties of PP1 to those characteristic of the hepatic glycogen-associated enzyme . These experiments establish that the cloned protein is GL.

FEBS Lett, 1995 Nov 20, 375(3), 273 - 6
Thermostable peroxidase activity with a recombinant antibody L chain-porphyrin Fe(III) complex; Takagi M et al.; In order to engineer a new type of catalytic antibody, we attempt to use a monoclonal antibody L chain as a host protein for a porphyrin . TCPP (meso-tetrakis(4-carboxyphenyl)porphyine) was chemically synthesized and Balb/c mice were immunized using TCPP as a hapten . Two hybridoma cells (03-1, 13-1), that produce monoclonal antibody against TCPP, were obtained . Genes for both H and L chains of monoclonal antibodies were cloned, sequenced and overexpressed using E . coli as a host . ELISA and fluorescence quenching method show that the independent antibody L chains from both Mab03-1 and Mab13-1 have specific interaction with TCPP . Furthermore, the recombinant antibody L chain from Mab13-1 exhibits much higher peroxidase activity than TCPP Fe(III) alone . The enzyme activity was detectable with pyrogallol and ABTS (2,2-azinobis-3-ethylbenzthiazolin-6-sulfonic acid) but not with catechol . This new catalytic antibody was extremely thermostable . Optimum temperature of the peroxidase reaction by the complex of 13-1L chain and TCPP Fe(III) was 90 degrees C, while that the TCPP Fe(III) alone was 60 degrees C.

FEBS Lett, 1995 Nov 20, 375(3), 235 - 8
Expression, purification and kinetic behaviour of fission yeast low M(r) protein-tyrosine phosphatase; Modesti A et al.; A gene named stp1+, coding for a 17.5-kDa protein, that rescues cdc25-22 when overexpressed, has been previously isolated from fission yeast . Here we describe the expression and purification of Stp1 protein as a fusion with the glutathione S-transferase in E . coli and its kinetic characterisation . Stp1 deduced protein sequence shows an high homology to members of a class of cytosolic low M(r) protein phosphatase previously known to exist only in mammalian species . Stp1 has a kinetic behaviour that appears to be intermediate with respect to the two isoenzymatic forms of low M(r) protein tyrosine phosphatases present in mammalian tissues . These differing kinetic characteristics are mainly due to the sequence 45-56 that is spatially close to the active site pocket.

FEBS Lett, 1995 Nov 20, 375(3), 211 - 4
Synthesis of chloramphenicol acetyltransferase in a coupled transcription-translation in vitro system lacking the chaperones DnaK and DnaJ; Vysokanov AV; A trimeric enzyme chloramphenicol acetyltransferase (CATI) has been synthesized in the Zubay system genetically depleted from DnaK and DnaJ . Most of CAT formed in the system fail to assemble into an active trimer . Instead CAT is accumulated in either a GroEL-bound complex or as an inactive monomer . Addition of purified DnaK and DnaJ to the system prior to the start of protein synthesis leads to the increase of the specific activity of formed CAT . A portion of exogenous DnaK and DnaJ added to the system associate with nascent polypeptide chains in the ribosomes . DnaK also comigrates with 50S-ribosomal subunits.

Tidsskr Nor Laegeforen, 1995 Nov 20, 115(28), 3493 - 5
{Nitrogen oxide in the treatment of oxygenation failure in neonates}; Hansen TW; Oxygenation problems are common in very sick infants, and involve a high risk of mortality or residual morbidity . We describe two infants with severe oxygenation difficulties . In one baby these were caused by meconium aspiration, while in the second, premature (1,500 g birth weight) infant Escherichia coli septicemia had precipitated persistent pulmonary hypertension . Inhaled nitric oxide led to immediate improvement in oxygenation, and the one infant was weaned off mechanical ventilatory support after four days, the other after ten days . There were no discernible side effects in either baby . Nitric oxide treatment should be considered for infants with life-threatening oxygenation problems who do not respond adequately to conventional management.

Neurosci Lett, 1995 Nov 17, 200(2), 144 - 6
Human ramified microglial cells produce nitric oxide upon Escherichia coli lipopolysaccharide and tumor necrosis factor alpha stimulation; Colasanti M et al.; This study shows that human ramified microglial cells derived from fetal brain primary cultures, are able to produce nitric oxide (NO) . In fact, stimulation with Escherichia coli lipopolysaccharide (LPS) (1 microgram ml-1) or tumor necrosis factor alpha (TNF alpha) (500 U ml-1) enhances nitrite release in cell supernatants, as determined by the Griess reaction . A synergistic effect is achieved following treatment with LPS plus TNF alpha, this effect being inhibited by pretreating cells with NOS inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) . Using reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot analysis, we also found that LPS/TNF alpha produce an increase of inducible NO synthase (iNOS) mRNA expression.

J Biol Chem, 1995 Nov 17, 270(46), 27937 - 41
Transient accumulation of heme O (cytochrome o) in the cytoplasmic membrane of semi-anaerobic Anacystis nidulans . Evidence for oxygenase-catalyzed heme O/A transformation; Peschek GA et al.; Incubation of obligately photoautotrophic and aerobic cyanobacterium Anacystis nidulans (Synechococcus sp . PCC 6301) in the light in the presence of the photo-system II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea and equilibrated with approximately 1% (v/v) O2 in N2 (10 microM O2 in solution) led to a decrease of the heme A content of isolated cytoplasmic membranes and to the appearance of heme O . The latter was not seen in membranes from fully aerated cells (> 210 microM dissolved O2) . Non-covalently bound hemes extracted from the membranes were identified by reversed phase high performance liquid chromatography . Heme A and O contents of the membranes changed in a reversible fashion solely depending on the ambient oxygen regime . Both hemes A and O combine with the same apoprotein as suggested by immunoblotting . CO/reduced-minus-reduced optical difference spectra, photoaction spectra of CO-inhibited O2 uptake by the membranes, and pyridine hemochrome spectra pointed to either heme belonging to a functional form of the terminal oxidase . The NADH:O2 oxidoreductase reaction catalyzed by membranes from both high O2 and low O2 cells was strictly dependent on the addition of catalytic amounts of cytochrome c, fully inhibited by 1.2 microM KCN, and insensitive to 5 microM 2-n-heptyl-4-hydroxyquinoline-N-oxide . O2 uptake by the membranes was effectively catalyzed by N,N,N',N'-tetramethyl-p-phenylenediamine but not 2-methylnaphthoquinol or plastoquinol-1 as artificial substrates . Therefore we conclude that the cyanobacterial respiratory oxidase, irrespective of the type of heme in its O2-reducing center, is a cytochrome c rather than a quinol oxidase.

J Biol Chem, 1995 Nov 17, 270(46), 27920 - 31
Triggering of the human interleukin-6 gene by interferon-gamma and tumor necrosis factor-alpha in monocytic cells involves cooperation between interferon regulatory factor-1, NF kappa B, and Sp1 transcription factors; Sanceau J et al.; We investigated the molecular basis of the synergistic induction by interferon-gamma (IFN-gamma)/tumor necrosis factor-alpha (TNF-alpha) of human interleukin-6 (IL-6) gene in THP-1 monocytic cells, and compared it with the basis of this induction by lipopolysaccharide (LPS) . Functional studies with IL-6 promoter demonstrated that three regions are the targets of the IFN-gamma and/or TNF-alpha action, whereas only one of these regions seemed to be implicated in LPS activation . The three regions concerned are: 1) a region between -73 and -36, which is the minimal element inducible by LPS or TNF-alpha; 2) an element located between -181 and -73, which appeared to regulate the response to IFN-gamma and TNF-alpha negatively; and 3) a distal element upstream of -224, which was inducible by IFN-gamma alone . LPS signaling was found to involve NF kappa B activation by the p50/p65 heterodimers . Synergistic induction of the IL-6 gene by IFN-gamma and TNF-alpha, in monocytic cells, involved cooperation between the IRF-1 and NF kappa B p65 homodimers with concomitant removal of the negative effect of the retinoblastoma control element present in the IL-6 promoter . This removal occurred by activation of the constitutive Sp1 factor, whose increased binding activity and phosphorylation were mediated by IFN-gamma.

J Biol Chem, 1995 Nov 17, 270(46), 27845 - 51
Ligand binding characteristics of the carboxyl-terminal domain of the cytokine receptor homologous region of the granulocyte colony-stimulating factor receptor; Anaguchi H et al.; The carboxyl-terminal domain (BC domain, roughly 100 amino acid residues) of the cytokine receptor homologous region in the receptor for murine granulocyte colony-stimulating factor was secreted as a maltose binding protein fusion into the Escherichia coli periplasm . The murine BC domain was prepared from the fusion protein by restriction protease factor Xa digestion and was purified to homogeneity . The purified BC domain specifically and stoichiometrically bound granulocyte colony-stimulating factor . This result indicates that the BC domain is also critical for ligand binding, as shown for the amino-terminal domain of the cytokine receptor homologous region (Hiraoka, O., Anaguchi, H., Yamasaki, K., Fukunaga, R., Nagata, S., and Ota, Y . (1994) J . Biol . Chem . 269, 22412-22419) . The tertiary folding and the beta-sheet structure of the BC domain were confirmed by NMR spectroscopy . The disulfide bond pattern suggested from peptide mapping was Cys224-Cys271 and Cys242-Cys285 . Disruption of the disulfide bonds suggested that both bonds are critical for maintaining the folding of the BC domain, although a BC domain lacking the second bond still retained ligand binding activity . Mutational analysis of the WSXWS sequence conserved in the cytokine receptor family suggested that this motif is critical for protein folding rather than for ligand binding.

J Biol Chem, 1995 Nov 17, 270(46), 27646 - 52
Inhibition of lipopolysaccharide biosynthesis and cell growth following inactivation of the kdtA gene in Escherichia coli; Belunis CJ et al.; The enzyme 3-deoxy-D-manno-octulosonic acid (Kdo) transferase is encoded by the kdtA gene in Escherichia coli . The enzyme is a single polypeptide that catalyzes the transfer of two Kdo residues to a tetraacyldisaccharide-1,4'-bisphosphate precursor of lipid A, designated lipid IVA (Belunis, C.J., and Raetz, C.R.H . (1992) J . Biol . Chem . 267, 9988-9997) . To determine if Kdo transfer to lipid IVA is required for growth, we constructed a strain of E . coli with a chromosomal kdtA::kan insertion mutation . In mutants carrying the kdtA::kan allele on the chromosome, cell growth and Kdo transferase activity were dependent upon a copy of the intact kdtA gene on a plasmid . When the kdtA-bearing plasmid was itself temperature sensitive for replication, the growth of these strains was inhibited after several hours at 44 degrees C, and Kdo transferase activity in extracts became undetectable . Concomitantly, the cells accumulated massive amounts of lipid IVA, the precursor of (Kdo)2-lipid IVA . The kdtA::kan mutation could also be complemented by hybrid plasmids bearing the gseA gene of Chlamydia trachomatis . gseA specifies a distinct Kdo transferase that adds three Kdo moieties to lipid IVA . Lipopolysaccharide from E . coli kdtA::kan constructs complemented by gseA reacts strongly with antibodies directed against the genus-specific epitope of Chlamydia, whereas lipopolysaccharide from parental E . coli K-12 does not . Our studies prove that Kdo attachment during lipid A biosynthesis is essential for cell growth and accounts for the conditional lethality associated with mutations in Kdo biosynthesis.

J Biol Chem, 1995 Nov 17, 270(46), 27615 - 21
A conserved region of c-Ha-Ras is required for efficient GTPase stimulation by GTPase activating protein but not neurofibromin; Yoder-Hill J et al.; The effector binding domain and the switch II region of c-Ha-Ras are necessary for p120GAP-stimulated GTP hydrolysis . We report a third region of c-Ha-Ras located within the alpha 3 helix (amino acids 101-103) which is also required for efficient p120GAP, but not neurofibromin-mediated hydrolysis . This highly conserved region of the Ras protein was investigated using an insertion-deletion mutant (Ras-100LIR104) originally characterized by Willumsen et al . (Willumsen, B . M., Adari, H., Zhang, K., Papageorge, A . G., Stone, J . C., McCormick, F., and Lowy, D . R (1989) in The Guanine Nucleotide Binding Proteins; Common Structural and Functional Properties (Bosch, L., Kraal, B., and Parmeggiani, A., eds) pp . 165-178, Plenum Press, New York) . The 100LIR104 substitution did not alter the intrinsic hydrolytic rate of the protein . The p120GAP-stimulated hydrolysis of Ras-100LIR104, however, was decreased by 2-3-fold compared to wild type Ras . This decrease in p120GAP-stimulated hydrolysis was not due to its inability to physically associate with Ras-100LIR104 . GTP (as determined by competitive binding assays) . Surprisingly, neurofibromin-stimulated GTP hydrolysis was unaltered by the mutation . Finally, no differences were observed in the ability of either the p120GAP catalytic domain or the neurofibromin GRD to accelerate Ras-100LIR104 GTPase activity, indicating that the amino-terminal noncatalytic GAP region is critical for p120GAP-stimulated GTP hydrolysis . This is the first report of a Ras mutation which differentiates between p120GAP and neurofibromin activity.

J Biol Chem, 1995 Nov 17, 270(46), 27481 - 8
Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) functions as an IGF-reversible inhibitor of IGFBP-4 proteolysis; Fowlkes JL et al.; Previous studies have shown that insulin-like growth factor (IGF)-binding protein-4 (IGFBP-4) is degraded only in the presence of exogenous IGFs; however, we found that cation-dependent proteinase activity present in conditioned medium of MC3T3-E1 osteoblasts degrades 125I-recombinant human (rh)IGFBP-4 in the absence of IGFs . Addition of IGF-I, IGF-II, or insulin to conditioned medium had little affect on 125I-rhIGFBP-4 proteolysis, while extraction of IGFs resulted in only a approximately 10% reduction in proteinase activity . Since factors other than IGFs appeared to be involved in regulating IGFBP-4 proteolysis, we hypothesized that IGFBP-3, an IGFBP produced by many cell lines, but not MC3T3-E1 cells, might function as an inhibitor of IGFBP-4 proteolysis . Addition of rhIGFBP-3 to conditioned media inhibited 125I-rhIGFBP-4 proteolysis by 90%, while IGF-I and IGF-II reversed the inhibitory effects of rhIGFBP-3 in a dose-dependent manner . 125I-rhIGFBP-4 proteolysis was not inhibited by N-terminal rhIGFBP-3 fragments that bind IGFs, but was inhibited by two synthetic peptides corresponding to sequences contained in the mid-region or C-terminal region of IGFBP-3 . Both inhibitory peptides contain highly basic, putative heparin-binding domains and heparin partially reversed the inhibitory effects of rhIGFBP-3 on 125I-rhIGFBP-4 proteolysis . These data demonstrate that rhIGFBP-3 inhibits IGFBP-4-degrading proteinase activity and binding of IGFs or glycosaminoglycans to IGFBP-3 may induce conformational changes in the binding protein, causing disinhibition of the proteinase.

J Biol Chem, 1995 Nov 17, 270(46), 27407 - 10
PTB domains of IRS-1 and Shc have distinct but overlapping binding specificities; Wolf G et al.; PTB domains are non-Src homology 2 (SH2) phosphotyrosine binding domains originally described in the receptor tyrosine kinase substrate, Shc . By serial truncation, we show that a 174-residue region of Shc p52 (33-206) has full PTB activity . We also show that a 173-residue region of insulin receptor substrate-1 (IRS-1; residues 144-316) has related PTB activity . In vitro both domains bind directly to activated insulin receptors . Binding is abrogated by substitution of Tyr-960 and selectively inhibited by phosphopeptides containing NPXY sequences . Phosphopeptide assays developed to compare PTB domain specificities show that the Shc PTB domain binds with highest affinity to psi XN beta 1 beta 2 pY motifs derived from middle T (mT), TrkA, ErbB4, or epidermal growth factor receptors (psi = hydrophobic, beta = beta-turn forming); the IRS-1 PTB domain does not bind with this motif . In contrast, both the Shc and IRS-1 PTB domains bind psi psi psi XXN beta 1 beta 2pY sequences derived from insulin and interleukin 4 receptors, although specificities vary in detail . Shc and IRS-1 are phosphorylated by distinct but overlapping sets of receptor-linked tyrosine kinases . These differences may be accounted for by the inherent specificities of their respective PTB domains.

Oncogene, 1995 Nov 16, 11(10), 2113 - 20
Regulation of c-Myb through protein phosphorylation and leucine zipper interactions; Ramsay RG et al.; c-Myb function is modulated in part by a negative regulation domain which encompasses a leucine zipper (LZ) . When E . coli-expressed c-Myb with wild type or mutated LZ proteins are assessed for DNA binding activity, the mutant form is substantially better at DNA binding than the wild type (WT) form . In contrast, the DNA binding activity of the WT protein is increased to an equivalent level of activity of the LZ-mutant when both are expressed in rabbit reticulocyte lysates (RRL) or insect cells . The possibility that phosphorylation overcomes the negative influence of the LZ was investigated . E . coli-expressed mutant, but not wild type c-Myb proteins, were shown to be substrates for Casein Kinase II (CKII) and cAMP-dependent Protein Kinase (PKA) . The phosphorylation sites for CKII and PKA were serines 11 and 12, and 8 and 116, respectively . Serines 11 and 12 were found to be phosphorylated in recombinant wild type and mutant c-Myb expressed in insect cells and DNA binding was markedly reduced following phosphatase treatment . Substitution of serines 11 and 12 with glutamic acid and alanine in E . coli-expressed Myb demonstrated that these amino terminal residues influence the negative effect on DNA binding exerted by the LZ . Collectively, these observations support the notion that phosphorylation of serines 11 and 12 positively modulate DNA binding.

Structure, 1995 Nov 15, 3(11), 1217 - 24
Structural comparison of the free and DNA-bound forms of the purine repressor DNA-binding domain; Nagadoi A et al.; BACKGROUND: The purine repressor (PurR) regulates genes that encode enzymes for purine biosynthesis . PurR has a two domain structure with an N-terminal DNA-binding domain (DBD) and a C-terminal corepressor-binding domain (CBD) . The three dimensional structure of a ternary complex of PurR bound to both corepressor and a specific DNA sequence has recently been determined by X-ray crystallography . RESULTS: We have determined the solution structure of the PurR DBD by NMR . It contains three helices, with the first and second helices forming a helix-turn-helix motif . The tertiary structure of the three helices is very similar to that of the corresponding region in the ternary complex . The structure of the hinge helical region, however, which makes specific base contacts in the minor groove of DNA, is disordered in the DNA-free form . CONCLUSION: The stable formation of PurR hinge helices requires PurR dimerization, which brings the hinge regions proximal to each other . The dimerization of the hinge helices is likely to be controlled by the CBD dimerization interface, but is induced by specific-DNA binding.

Structure, 1995 Nov 15, 3(11), 1171 - 84
Crystal structure of a quinoenzyme: copper amine oxidase of Escherichia coli at 2 A resolution; Parsons MR et al.; BACKGROUND: Copper amine oxidases are a ubiquitous and novel group of quinoenzymes that catalyze the oxidative deamination of primary amines to the corresponding aldehydes, with concomitant reduction of molecular oxygen to hydrogen peroxide . The enzymes are dimers of identical 70-90 kDa subunits, each of which contains a single copper ion and a covalently bound cofactor formed by the post-translational modification of a tyrosine side chain to 2,4,5-trihydroxyphenylalanine quinone (TPQ) . RESULTS: The crystal structure of amine oxidase from Escherichia coli has been determined in both an active and an inactive form . The only structural differences are in the active site, where differences in copper coordination geometry and in the position and interactions of the redox cofactor, TPQ, are observed . Each subunit of the mushroom-shaped dimer comprises four domains: a 440 amino acid C-terminal beta sandwich domain, which contains the active site and provides the dimer interface, and three smaller peripheral alpha/beta domains (D1-D3), each of about 100 amino acids . D2 and D3 show remarkable structural and sequence similarity to each other and are conserved throughout the quinoenzyme family . In contrast, D1 is absent from some amine oxidases . The active sites are well buried from solvent and lie some 35 A apart, connected by a pair of beta hairpin arms . CONCLUSIONS: The crystal structure of E . coli copper amine oxidase reveals a number of unexpected features and provides a basis for investigating the intriguing similarities and differences in catalytic mechanism of members of this enzyme family . In addition to the three conserved histidines that bind the copper, our studies identify a number of other conserved residues close to the active site, including a candidate for the catalytic base and a fourth conserved histidine which is involved in an interesting intersubunit interaction.

Structure, 1995 Nov 15, 3(11), 1127 - 9
Copper amine oxidase: a novel use for a tyrosine; Fontecave M et al.; The first three-dimensional structure of copper amine oxidase demonstrates that one tyrosine residue is converted into 2,4,5-trihydroxyphenylalanine quinone (TPQ) . TPQ binds to copper in the inactive form of the enzyme but not in the active form.

Eur J Biochem, 1995 Nov 15, 234(1), 358 - 62
Identification of Trp300 as an important residue for Escherichia coli leader peptidase activity; Kim YT et al.; We previously reported that leader peptidase from Escherichia coli was extensively inactivated by reaction with N-bromosuccinimide with concomitant and selective modification of the Trp300 and Trp310 residues {Kim, Y.-T., Muramatsu, T . & Takahashi, K . (1995) J . Biochem . (Tokyo) 117, 535-544} . This indicated that one or both of these tryptophan residues are important for the activity of the enzyme . In order to define further the role of individual tryptophan residues in the activity of leader peptidase, site-directed mutagenesis studies were performed to replace each tryptophan residue with phenylalanine and/or alanine . The replacements of Trp20, Trp59, Trp261, Trp284, and Trp310 with phenylalanine hardly affected the enzyme activity toward a synthetic peptide substrate and the ability to complement the temperature sensitivity of the mutant leader peptidase in E . coli IT41 . In contrast, the activity toward the synthetic substrate was significantly decreased by replacement of Trp300 with phenylalanine or alanine . The kcat values of the W300F and W300A mutant enzymes were reduced to 42% and 22%, respectively, of that of the wild-type enzyme, whereas the Km values of these mutant enzymes were almost identical with that of the wild-type enzyme . Moreover, the complementing ability in E . coli IT41 was lost (almost) completely when Trp300 was replaced with phenylalanine or alanine . These results strongly indicate that Trp300 in leader peptidase is important for the catalytic mechanism and/or the construction of the active site structure of the enzyme.

Eur J Biochem, 1995 Nov 15, 234(1), 350 - 7
Functional properties of a recombinant chimeric protein with combined thrombin inhibitory and plasminogen-activating potential; Lijnen HR et al.; A chimeric protein (rscu-PA-40-kDa/Hir), consisting of the C-terminal amino acids 53-65 of hirudin (Hir), fused via a 14-amino-acid linker sequence to the C-terminal of a 40-kDa fragment (Ser47-Leu411) of recombinant (r) single-chain (sc) urokinase-type plasminogen activator (rscu-PA), was produced by expression of the corresponding chimeric cDNA in Escherichia coli cells . The thrombin inhibitory potential of purified rscu-PA-40-kDa/Hir was confirmed by complete inhibition of the coagulant activity of thrombin at 20-30-fold molar excess of the chimera, and by the resistance of rscu-PA-40-kDa/Hir to proteolytic cleavage by thrombin, rscu-PA-40-kDa/Hir prolonged the thrombin time of normal human plasma in a dose-dependent way (reduction of the apparent thrombin concentration to 50% with 95 nM chimeric protein as compared to 4.7 nM hirudin), and inhibited thrombin-mediated platelet aggregation (reduction of the apparent thrombin concentration to 50% with 40 nM chimeric protein) . The chimera had a specific activity on fibrin films of 57,000 IU/mg as compared to 95,000 IU/mg for rscu-PA . The urokinase-like amidolytic activity of the single-chain protein was only 220 IU/mg but increased to 169,000 IU/mg after treatment with plasmin, which resulted in quantitative conversion to a two-chain (tc) derivative (rtcu-PA-40-kDa/Hir) . Corresponding values for rscu-PA were 270 and 226,000 IU/mg . The catalytic efficiencies for plasmin-mediated conversion to two-chain molecules were comparable for rscu-PA-40-kDa/Hir and rscu-PA (0.63 and 0.65 microM-1.s-1, respectively) . The plasminogen-activating potential of the single-chain chimera was comparable to that of rscu-PA; the catalytic efficiencies for plasminogen activation by their two-chain counterparts were also similar (0.55 and 0.73 microM-1.s-1, respectively) . In 2 h, 50% lysis of 125I-fibrin-labeled clots prepared from platelet-poor human plasma and immersed in normal plasma was obtained with 1.3 micrograms/ml rscu-PA-40-kDa/Hir and with 0.67 micrograms/ml rscu-PA, with corresponding residual fibrinogen levels of 74% and 87%, respectively . In the absence of fibrin, 50% fibrinogenolysis in 2 h in normal human plasma required 2.1 micrograms/ml rscu-PA, but 7.9 micrograms/ml rscu-PA-40-kDa/Hir . Thus, the chimera rscu-PA-40-kDa/Hir has maintained the specific fibrinolytic and plasminogen activating activity of rscu-PA as well as its fibrinolytic potency in plasma, whereas it displayed a similar or somewhat better fibrin specificity.(ABSTRACT TRUNCATED AT 250 WORDS)

Eur J Biochem, 1995 Nov 15, 234(1), 336 - 42
Molecular cloning of the human glucose-regulated protein ERp57/GRP58, a thiol-dependent reductase . Identification of its secretory form and inducible expression by the oncogenic transformation; Hirano N et al.; Recently it was shown that putative phospholipase C-alpha cDNA does not code for an isotype of the phospholipase C superfamily but for one of the glucose-regulated proteins (GRPs), ERp57/GRP58 . We have isolated human ERp57/GRP58 cDNA from human placenta . Sequence analysis showed that ERp57/GRP58 has two Trp-Cys-Gly-His-Cys-Lys motifs completely conserved among the mammals . Bacterially expressed recombinant ERp57/GRP58 protein contained a thiol-dependent reductase activity which was completely abolished when Ser residues were substituted for Cys residues in both of the two motifs . Furthermore, we have identified a soluble form of ERp57/GRP58 by Western blotting and biosynthetic labeling . In v-onc transformants of normal rat kidney cells, the expression level of ERp57/GRP58 was elevated at the protein level . In NIH3T3 cells transformed with v-src, activated c-src (Y527F) or c-src, the expression level of ERp57/GRP58 was upregulated in proportion to their transforming abilities . These results indicate that a soluble form of ERp57/GRP58 exists and that this protein may control both extracellular and intracellular redox activities through its thiol-dependent reductase activity . Moreover, it is likely that ERp57/GRP58 is involved in the oncogenic transformation.

Eur J Biochem, 1995 Nov 15, 234(1), 323 - 8
Expression of a recombinant human glycosyltransferase from a synthetic gene and its utilization for synthesis of the human blood group B trisaccharide; Seto NO et al.; A 1034-bp synthetic gene encoding the human blood group B glycosyltransferase, which catalyzes the transfer of galactose from UDP-Gal to Fuc alpha(1-2)Gal beta-OR to give the blood group B determinant Gal alpha(1-3){Fuc alpha(1-2)}Gal beta-OR (where R is a glycoprotein or glycolipid), has been expressed in Escherichia coli by replacing its membrane-anchoring domain with an ompA bacterial secretory signal . The active enzyme was purified from the periplasm using UDP-hexanolamine affinity chromatography and used in the synthesis of preparative amounts of the human blood group B trisaccharide antigen . The substrate specificity and kinetics of the recombinant enzyme were comparable to the enzyme from human sera . Thus we have achieved the construction of a completely synthetic glycosyltransferase gene and its successful expression.

Eur J Biochem, 1995 Nov 15, 234(1), 148 - 59
Sequential 1H and 15N nuclear magnetic resonance assignments and secondary structure of the lipoyl domain of the 2-oxoglutarate dehydrogenase complex from Azotobacter vinelandii . Evidence for high structural similarity with the lipoyl domain of the pyruvate dehydrogenase complex; Berg A et al.; A 79-amino-acid polypeptide, corresponding to the lipoyl domain of the succinyltransferase component of the 2-oxoglutarate dehydrogenase multienzyme complex from Azotobacter vinelandii, has been sub-cloned and produced in Escherichia coli . Complete sequential 1H and 15N resonance assignments for the lipoyl domain have been obtained by using homo- and hetero-nuclear NMR spectroscopy . Two antiparallel beta-sheets of four strands each were identified from characteristic NOE connectivities and 3JHN alpha values . The lipoyl-lysine residue is found in a type-I turn connecting two beta-strands . The secondary structure of the lipoyl domain very much resembles the secondary solution structure of the N-terminal lipoyl domain of the A . vinelandii pyruvate dehydrogenase complex, despite the sequence identity of 25% . A detailed comparison of the NMR-derived parameters of both lipoyl domains, i.e . chemical shifts, NH-exchange rates, NOEs, and 3JHN alpha values suggests a high structural similarity in solution between the two lipoyl domains . Preliminary tertiary-structure calculations confirm that these lipoyl domains have very similar overall folds . The observed specificity of the 2-oxo acid dehydrogenase components of both complexes for these lipoyl domains is discussed in this respect.

FEMS Microbiol Lett, 1995 Nov 15, 133(3), 225 - 31
Identification of the genes in multicopy plasmids affecting ompC and ompF expression in Escherichia coli; Jin T et al.; Osmoregulation of the porin genes, ompF and ompC of Escherichia coli, occurs at the level of transcription through the action of EnvZ and OmpR proteins as well as at the level of translation through micF antisense RNA . In this study, we used a genetic screening approach to identify new genes which interfere with the expression of ompC or ompF . Using an E . coli genomic library in pUC19, we identified three clones whose products altered expression of ompC and ompF in response to medium osmolarity . One clone carrying the secB gene was found to block ompC and inhibit ompF expression . One clone carrying gcvA, a transcriptional regulator for the gvcA operon, was found to block ompF expression at high osmolarity and elevate ompC expression at low osmolarity . One clone carrying rbsR, a repressor for the rbs operon, was found to block ompF expression at both low and high osmolarities and elevate ompC expression at low osmolarity . These results suggest that ompF and ompC expression is associated with other physiological regulating systems in addition to osmoregulation.

FEMS Microbiol Lett, 1995 Nov 15, 133(3), 209 - 13
micF RNA is a substrate for RNase E; Schmidt M et al.; Ribonuclease E (RNase E) is known to play an important role in mRNA decay and RNA processing in Escherichia coli . While several substrates for RNase E have been identified, the specificity for the recognition and cleavage sites has not been completely determined . In this study, micF RNA, an antisense RNA found in E . coli and related bacteria, was found to be a substrate for RNase E in vitro . Two cleavage sites were mapped, and both are found in the sequence context UA/UUU and are located within 10 nucleotides upstream of stem-loop structures . These results help define a generalized RNase E cleavage/recognition pattern.

EMBO J, 1995 Nov 15, 14(22), 5506 - 13
Non-bilayer lipids are required for efficient protein transport across the plasma membrane of Escherichia coli; Rietveld AG et al.; The construction of a mutant Escherichia coli strain which cannot synthesize phosphatidylethanolamine provides a tool to study the involvement of non-bilayer lipids in membrane function . This strain produces phosphatidylglycerol and cardiolipin (CL) as major membrane constituents and requires millimolar concentrations of divalent cations for growth . In this strain, the lipid phase behaviour is tightly regulated by adjustment of the level of CL which favours a nonbilayer organization in the presence of specific divalent cations . We have used an in vitro system of inverted membrane vesicles to study the involvement of non-bilayer lipids in protein translocation in the secretion pathway . In this system, protein translocation is very low in the absence of divalent cations but can be enhanced by inclusion of Mg2+, Ca2+ or Sr2+ but not by Ba2+ which is unable to sustain growth of the mutant strain and cannot induce a non-bilayer phase in E . coli CL dispersions . Alternatively, translocation in cation depleted vesicles could be increased by incorporation of the non-bilayer lipid DOPE (18:1) but not by DMPE (14:0) or DOPC (18:1), both of which are bilayer lipids under physiological conditions . We conclude that non-bilayer lipids are essential for efficient protein transport across the plasma membrane of E . coli.

EMBO J, 1995 Nov 15, 14(22), 5485 - 93
A complex of the signal sequence binding protein and the SRP RNA promotes translocation of nascent proteins; Hauser S et al.; Translocation of proteins across the endoplasmic reticulum membrane is initiated by the signal recognition particle (SRP), a cytoplasmic ribonucleoprotein complex consisting of a 7S RNA and six polypeptides . To investigate the functions of the SRP components, we have tested the activities of several SRP subparticles . We show that the SRP GTPase (SRP54) alone binds a signal sequence and discriminates it from a non-signal sequence . Although SRP54 alone is unable to promote translocation, SRP54 in a complex with SRP RNA is both necessary and sufficient to promote translocation of an elongation-arrested nascent protein in a GTP-regulated manner . For co-translational translocation, additional SRP components are required . We discuss the implications of our results for the function of the Escherichia coli SRP which is homologous to the SRP54/SRP-RNA complex.

J Immunol, 1995 Nov 15, 155(10), 4899 - 908
Lipopolysaccharide-induced change of phosphorylation of two cytosolic proteins in human monocytes is prevented by inhibitors of ADP-ribosylation; Heine H et al.; Interaction of LPS with human monocytes causes altered phosphate labeling of cytosolic proteins of 36 kDa and 38 kDa (p36/38) . This property, determined by in vitro studies, is shared by other monocyte activators . Phosphorylated p36/38 are distinct from p38, 42-kDa, and 44-kDa isoforms of mitogen-activated protein kinases expressed in monocytes . Occupation of LPS binding sites by a LPS antagonist, the synthetic tetraacylated bisphosphate precursor of Escherichia coli lipid A (also known as compound 406, lipid IVa, or precursor Ia), prevents LPS-induced changes in the phosphate labeling of the two proteins . Abs against CD14 inhibit protein phosphorylation induced by low concentrations of LPS (10 ng/ml), whereas at high concentrations (1 microgram/ml), the Abs fail to prevent phosphorylation . In addition to phosphorylation, ADP-ribosylation of proteins has been implicated in a number of biologic processes . Here we show that inhibitors of ADP-ribosylation, namely meta-iodobenzylguanidine and nicotinamide, inhibit LPS-initiated altered phosphorylation of p36/38 . This loss of phosphate labeling of p36/38 is accompanied by an inhibition of TNF-alpha and Il-6 mRNA and protein production . The synthesis of IL-1 is not affected . This suggests that the inhibitors interfere with specific steps in IL-6 and TNF-alpha production, which are not required for IL-1 synthesis . Taken together, the data indicate that ADP-ribosylation may be involved in LPS-induced alteration of the phosphorylation state of two cytosolic proteins (p36/38) and that these proteins modulate cellular processes leading to TNF-alpha and IL-6 release.

Genes Dev, 1995 Nov 15, 9(22), 2859 - 69
Roles of topoisomerase IV and DNA gyrase in DNA unlinking during replication in Escherichia coli; Zechiedrich EL et al.; For a cell to complete DNA replication, every link between the Watson-Crick strands must be removed by topoisomerases . Previously, we reported that the inhibition of topoisomerase IV (topo IV) leads to the accumulation of catenated plasmid replicons to a steady-state level of approximately 10% . Using pulse labeling with {3H}thymidine in Escherichia coli, we have found that in the absence of topo IV activity, nearly all newly synthesized plasmid DNA is catenated . Pulse-chase protocols revealed that catenanes are metabolized even in the absence of topo IV and that the residual turnover is carried out by DNA gyrase at a rate of approximately 0.01/sec . Using extremely short pulse-labeling times, we identified significant amounts of replication catenanes in wild-type cells . The rate of catenane unlinking in wild-type cells by the combined activities of topo IV and DNA gyrase was approximately 1/sec . Therefore, gyrase is 100-fold less efficient than topo IV in plasmid replicon decatenation in vivo . This may explain why a fully functional gyrase cannot prevent the catenation of newly synthesized plasmid DNA and the partition phenotype of topo IV mutants . We conclude that catenanes are kinetic intermediates in DNA replication and that the essential role of topo IV is to unlink daughter replicons.

Blood, 1995 Nov 15, 86(10), 3789 - 96
Extracellular epitopes of platelet glycoprotein Ib alpha reactive with serum antibodies from patients with chronic idiopathic thrombocytopenic purpura; He R et al.; Glycoproteins (GPs) IIb/IIIa and Ib/IX are principal targets of autoantibodies (autoAbs) in idiopathic thrombocytopenic purpura (ITP) . Platelet-associated Abs against GPIIb/IIIa primarily recognize discontinuous or nonlinear epitopes (Fujisawa et al, Blood 81:1284, 1993) . This study focused on whether Abs against the extracellular domain of GPIb/IX might react with short linear amino acid (aa) sequences of GPIb alpha . Complementary DNAs (cDNAs) coding for two overlapping fragments of GPIb alpha were amplified, cloned into pFLAG.2 plasmids, and expressed in Escherichia coli DH5 alpha competent cells as FLAG fusion proteins, which were purified by anti-FLAG immunoaffinity chromatography . Of 16 selected ITP sera containing anti-GPIb/IX, 6 reacted in microtiter radioimmunoassays (RIAs) with recombinant protein fragment 2 (aas 240 to 485); 1 also with fragment 1 (aas 1 to 247) . When synthetic peptides corresponding to 4 segments of fragment 2 with high antigenic indices (P1 to P4) were used as targets in RIAs, all 6 sera reacted with P2 (aas 326 to 346); 1 also reacted with P4 (aas 389 to 412) . P2 was shown to be present on the surface of intact platelets by adsorption studies, and anti-P2 was detected in direct eluates of platelets from ITP patients . Glycocalicin in solution effectively competed with immobilized P2 for anti-P2; P2 in solution was a less effective competitor . Epitope scanning with a panel of synthetic 15-mer peptides localized the P2 epitope to the sequence, TKEQTTFPP . Epitope definition may offer insight into the pathophysiology of and more specific treatments for ITP.

Mol Gen Genet, 1995 Nov 15, 249(2), 202 - 8
A new Escherichia coli gene, fdrA, identified by suppression analysis of dominant negative FtsH mutations; Akiyama Y et al.; An Escherichia coli membrane protein, FtsH, has been implicated in several cellular processes, including integration of membrane proteins, translocation of secreted proteins, and degradation of some unstable proteins . However, how it takes part in such diverse cellular events is largely unknown . We previously isolated dominant negative ftsH mutations and proposed that FtsH functions in association with some other cellular factor(s) . To test this proposal we isolated multicopy suppressors of dominant negative ftsH mutations . One of the multicopy suppressor clones contained an N-terminally truncated version of a new gene that was designated fdrA . The FdrA fragment suppressed both of the phenotypes--increased abnormal translocation of a normally cytoplasmic domain of a model membrane protein and retardation of protein export--caused by dominant negative FtsH proteins . The intact fdrA gene (11.9 min on the chromosome) directed the synthesis of a 60 kDa protein in vitro.

Mol Gen Genet, 1995 Nov 15, 249(2), 139 - 46
Partial inhibition of protein synthesis accelerates the synthesis of porphyrin in heme-deficient mutants of Escherichia coli; Nakayashiki T et al.; Mutants of Escherichia coli defective in the HemA protein grow extremely poorly as the result of heme deficiency . A novel hemA mutant was identified whose rate of growth was dramatically enhanced by addition to the medium of low concentrations of translational inhibitors, such as chloramphenicol and tetracycline . This mutant (H110) carries mutation at position 314 in the hemA gene, which resulted in diminished activity of the encoded protein . Restoration of growth of H110 upon addition of the drugs mentioned above was due to activation of the synthesis of porphyrin . However, this activation was not characteristic exclusively of cells with this mutant hemA gene since it was also observed in a heme-deficient strain bearing the wild-type hemA gene . The activation did not depend on the promoter activity of the hemA gene, as indicated by studies with fusion genes . It appears that partial inhibition of protein synthesis via inhibition of peptidyltransferase can promote the synthesis of porphyrin by providing an increased supply of glutamyl-tRNA for porphyrin synthesis . Glutamyl-tRNA is the common substrate for peptidyltransferase and HemA.

Biochemistry, 1995 Nov 14, 34(45), 14942 - 53
Recombinant human liver medium-chain acyl-CoA dehydrogenase: purification, characterization, and the mechanism of interactions with functionally diverse C8-CoA molecules; Peterson KL et al.; We offer a large scale purification procedure for the recombinant human liver medium-chain acyl-CoA dehydrogenase (HMCAD) . This procedure routinely yield 100-150 mg of homogeneous preparation of the enzyme from 80 L of the Escherichia coli host cells . A comparative investigation of kinetic properties of the human liver and pig kidney enzymes revealed that, except for a few minor differences, both of these enzymes are nearly identical . We undertook detailed kinetic and thermodynamic investigations for the interaction of HMCAD-FAD with three C8-CoA molecules (viz., octanoyl-CoA, 2-octenoyl-CoA, and 2-octynoyl-CoA), which differ with respect to the extent of unsaturation of the alpha-beta carbon center; octanoyl-CoA and 2-octenoyl-CoA serve as the substrate and product of the enzyme, respectively, whereas 2-octynoyl-CoA is known to inactivate the enzyme . Our experimental results demonstrate that all three C8-CoA molecules first interact with HMCAD-FAD to form corresponding Michaelis complexes, followed by two subsequent isomerization reactions . The latter accompany either subtle changes in the electronic structures of the individual components (in case of 2-octenoyl-CoA and 2-octynoyl-CoA ligands), or a near-complete reduction of the enzyme-bound flavin (in case of octanoyl-CoA) . The rate and equilibrium constants intrinsic to the above microscopic steps exhibit marked similarity with different C8-CoA molecules . However, the electronic structural changes accompanying the 2-octynoyl-CoA-dependent inactivation of enzyme is 3-4 orders of magnitude slower than the above isomerization reactions . Hence, the octanoyl-CoA-dependent reductive half-reaction and the 2-octynoyl-CoA-dependent covalent modification of the enzyme occur during entirely different microscopic steps . Arguments are presented that the origin of the above difference lies in the protein conformation-dependent orientation of Glu-376 in the vicinity of the C8-CoA binding site.

Biochemistry, 1995 Nov 14, 34(45), 14909 - 17
Membrane topology of helices VII and XI in the lactose permease of Escherichia coli studied by lacY-phoA fusion analysis and site-directed spectroscopy; Ujwal ML et al.; The use of lactose permease-alkaline phosphatase fusions (lacY-phoA) demonstrates that the lactose permease of Escherichia coli contains 12 transmembrane domains and that approximately half of a transmembrane domain is required to translocate alkaline phosphatase to the periplasmic surface of the membrane {Calamia, J., & Manoil, C . (1990) Proc . Natl . Acad . Sci . U.S.A . 87, 4937-4941} . We have now used fusion analysis in combination with site-directed spectroscopy to examine more precisely the topology of putative helices VII and XI which contain the interacting residues Asp237 and Lys358, respectively . For this purpose, alkaline phosphatase was fused to alternate amino acid residues in transmembrane domains VII and XI . A sharp increase in alkaline phosphatase activity is observed as the fusion junction proceeds from Try228 to Ile230 in helix VII and from Phe354 to Phe356 in helix XI, suggesting that these residues approximate the middle of the corresponding transmembrane helices . Analysis of fluorescence quenching of the pyrene-labeled single-Cys mutants Asp237 --> Cys or Lys358 --> Cys, as well as measurement of collision frequencies between freely diffusing paramagnetic probes and a nitroxide spin-label at these sites, also indicates that Asp237 and also Asp240, which interacts with Lys319 (helix X), are located in transmembrane domains . However, Asp237 and Asp240 are accessible both from the aqueous phase and from within the membrane . The results provide more direct evidence that the three residues are located within transmembrane helices and suggest that Asp237 and Asp240 are either located near the periplasmic surface of the membrane or exposed within a solvent-filled cleft in the permease.

Biochemistry, 1995 Nov 14, 34(45), 14861 - 7
A suggested mechanism for the catalytic cycle of cytochrome bd terminal oxidase based on kinetic analysis; Junemann S et al.; The apparent oxygen affinity of cytochrome bd from Escherichia coli and Azotobacter vinelandii has been measured using oxymyoglobin as a sensitive monitor of oxygen concentration . In membrane preparations, the Km(O2) and respiratory rate varied with the nature of the primary substrate used (malate, lactate, reduced nicotinamide adenine dinucleotide (NADH), or ubiquinol-1) . At maximum respiratory rates, the Km(O2) for cytochrome bd from A . vinelandii was 4.1 microM, approximately 2 times higher than the corresponding value for the E . coli enzyme . There were no significant differences between the Km(O2) values for membrane-bound and purified cytochrome bd from A . vinelandii when ubiquinol-1 was used as primary substrate . The kinetic parameters Km(O2) and Vmax provide a value of 2.8 x 10(8) M-1 s-1 for the bimolecular rate constant for oxygen reaction with the enzyme, suggesting that this reaction is diffusion-controlled . Kinetic analysis indicates a mechanism involving a ternary complex . A scheme for the reaction mechanism of cytochrome bd is proposed.

Biochemistry, 1995 Nov 14, 34(45), 14752 - 7
A cytosine methyltransferase converts 5-methylcytosine in DNA to thymine; Yebra MJ et al.; Sites of cytosine methylation are known to be hot spots for C.G to T.A mutations in a number of systems, including human cells . Traditionally, spontaneous hydrolytic deamination of 5-methylcytosine to thymine has been invoked as the cause of this phenomenon . We show here that a bacterial cytosine methyltransferase can convert 5-methylcytosine in DNA to thymine and that this reaction creates a mutational hot spot at a site of DNA methylation . The reaction is fairly insensitive to the methyl donor in the reaction, S-adenosylmethionine . In many cancers, the most frequent class of mutations is C to T changes within CG dinucleotides of the tumor suppressor gene p53 . Because of the similarities of the reaction mechanisms of mammalian and bacterial enzymes and the physiology of the cancer cells, this reaction is expected to contribute to mutations at CG dinucleotides in precancerous cells.

Biochemistry, 1995 Nov 14, 34(45), 14703 - 11
The polypeptide chain of eukaryotic initiation factor 5A occurs in two distinct conformations in the absence of the hypusine modification; Joao HC et al.; Eukaryotic initiation factor 5A (eIF-5A) requires posttranslational modification of lysine at position 50 to hypusine for its biological activity . We have expressed an unmodified variant of eIF-5A in Escherichia coli and show that it has structural properties different from those of the native protein in terms of its near- and far-UV circular dichroism spectra and its equilibrium unfolding transition with guanidinium chloride . In contrast to the hypusine-modified protein, which unfolds in a two-state process, the complex unfolding transition of unmodified eIF-5A suggests that this variant occurs in two differently folded conformations, F1 and F2 . Both conformations are populated under near-physiological conditions at a ration of 60 to 40, respectively . Equilibrium unfolding consists of parallel events: unfolding of F1 to one or several intermediate states (I), and unfolding of F2 to the unfolded state (U) . Although the establishment of each of these individual equilibria is fast, the interconversion is slow at guanidinium chloride concentrations between 0 M and 3 M . Kinetic analysis reveals activation energies of 24.3 kcal mol-1 for the reaction of F1 and F2 and 24.1 kcal mol-1 for the reaction of F2 to F1 . Both F1 and F2 possess well-defined secondary and tertiary structure . However, the tertiary structures of the two conformations differ as indicated by their distinct near-UV circular dichroism spectra . These differences may be restricted to the C-terminal part of the protein as 2-dimensional 1H-NMR spectra of unmodified eIF-5A reveal no doubled set of proton resonances for aromatic amino acid and histidine residues, of which almost all are located in the N-terminal region.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1995 Nov 14, 34(45), 14687 - 92
Laser photolysis behavior of ferrous horseradish peroxidase with carbon monoxide and cyanide: effects of mutations in the distal heme pocket; Meunier B et al.; Native horseradish peroxidase and several forms with mutations in the distal heme pocket (His42Leu, His42Arg, and Arg38Leu) have been expressed in Escherichia coli . These enzymes have been purified and analyzed in terms of the room temperature recombination rate of carbon monoxide and cyanide after photolysis of the reduced forms . The recombinant wild-type ferrous form exhibited monophasic recombination of carbon monoxide with an observed bimolecular rate constant at pH 8.5 of 4.4 x 10(3) M-1 s-1 which is essentially the same as the natural glycosylated form . This recombination rate constant increases in the mutants in the order WT < H42R < H42L << R38L . The value for R38L (5 x 10(6) M-1 s-1) is increased by 3 orders of magnitude relative to the wild-type and is similar to that for human hemoglobin {Mims et al . (1983) J . Biol . Chem . 258, 14219-14232} . Cyanide recombination with the wild-type ferrous form at room temperature is biphasic at pH 6.5 but becomes more monophasic at pH 8.5, again similar to the behavior of the natural glycosylated form, although the Fe(2+)-cyano form of the recombinant enzyme appears to be more unstable at high pH . None of the mutant forms were able to bind cyanide in the ferrous state to any significant extent (Kdiss > 250 mM) when cyanide was added at a concentration (10-20 mM) sufficient to almost saturate the wild-type form (Kdiss approximately equal to 1 mM at pH 7) . This behavior contrasts with that of the oxidized forms of the mutants where increases in cyanide dissociation constants are smaller ( < 25 times).(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1995 Nov 14, 34(45), 14626 - 36
The response regulators CheB and CheY exhibit competitive binding to the kinase CheA; Li J et al.; The autophosphorylating kinase CheA of the bacterial chemosensory signaling pathway donates a phosphoryl group to either of two regulator proteins, CheY or the receptor methylesterase (CheB) . With isothermal titration calorimetry, it was demonstrated that CheA and CheA fragment composed of amino acid residues 1-233 (CheA1-233) bound to CheY with similar dissociation constants of 2.0 and 1.2 microM at 298 K, respectively, indicating that the CheY binding site is wholly within the 1-233 amino acid locus . CheB bound to CheA1-233 with a KD of 3.2 microM, and also bound to intact CheA with the same affinity . CheY was found to complete with CheB for binding to CheA1-233, in spite of the low level of sequence identity between CheY and the regulatory domain of CheB . The competitive nature of CheY and CheB binding was determined in two independent sets of experiments: titration experiments in which either a CheB-CheA1-233 complex was titrated with CheY or CheB was titrated with a CheY-CheA1-233 complex, and competitive affinity chromatography experiments that used a Ni-NTA-chelating resin as an affinity matrix for complexes of the histidine-tagged CheA1-233 fragment and CheY or CheB . The effects of phosphorylation, binding-site mutations, and active-site mutations were also studied to probe the influence of conformational changes in CheY as a regulatory mechanism of CheY-CheA Interactions . Phosphorylated CheY, in the presence of excess EDTA, was found to have a 2-fold lower affinity for CheA1-233, and 6 mM Mg2+ further reduced the affinity of phosphorylated CheY for CheA1-233 (ca . 3-fold), although Mg2+ on its own had no effect on the interactions of either CheB or CheY with CheA1-233 . The data thus indicate that phosphorylated CheY has a significantly lower affinity for CheA under physiological conditions . The idea that phosphorylation may induce a significant conformational change, reducing the strength of the CheY-CheA interaction, is supported by the relative values of the association constants measured for CheY active-site and binding-site mutants . A binding-site mutation (A103V) in CheY, which is remote from the site of phosphorylation produced a 10-fold reduction in Ka, whereas active-site mutations produced a modest (2-fold) reduction.

FEBS Lett, 1995 Nov 13, 375(1-2), 37 - 40
rap1 p21 regulates the interaction of ras p21 with RGL, a new effector protein of ras p21; Ikeda M et al.; We have recently found that ralGDS family members (RGL and ralGDS) are putative effector proteins of ras p21 . rap1 p21 is a small GTP-binding protein which has the same amino acid sequence as the effector loop of ras p21 . We examined the effect of rap1 p21 on the interaction of ras p21 with RGL . The GTP-bound form of rap1 p21 interacted with RGL as well as did ras p21 . rap1 p21 inhibited the interaction of ras p21 with RGL . RGL was phosphorylated by cyclic AMP-dependent protein kinase (protein kinase A) . Phosphorylation of RGL did not affect its binding to ras p21 and rap1 p21 under the conditions that phosphorylation of Raf-1 reduced its affinity for ras p21 . These results demonstrate that rap1 p21 but not protein kinase A regulates the interaction of ras p21 with RGL and suggest that rap1 p21 and protein kinase A may cooperate to distinguish the signal or ras p21 to RGL from that to Raf-1.

FEBS Lett, 1995 Nov 13, 375(1-2), 27 - 30
Mechanism of Lac repressor switch-off: orientation of the Lac repressor DNA-binding domain is reversed upon inducer binding; Kamashev DE et al.; Lac repressor's DNA-binding domains contain helix-turn-helix motif which, though similar to those of phage lambda Cro protein, are oriented differently with respect to DNA: in the specific complexes with Lac operator, N termini of the repressor's subunits are facing inwards . We demonstrate that, in the presence of an inducer, the repressor's N termini cross-link to the operator's outermost nucleotides . We suggest that the inducer fixes the repressor's DNA-binding domains in the Cro-type configuration and thus garbles its recognition surface . Since the Cro-type configuration is perfectly suitable for binding the DNA, this also explains how the switched-off repressor retains its non-specific DNA-binding.

Nucleic Acids Res, 1995 Nov 11, 23(21), 4275 - 82
A mutant HpaII methyltransferase functions as a mutator enzyme; Shen JC et al.; DNA (cytosine-5)-methyltransferases can cause deamination of cytosine when the cofactor S-adenosylmethionine (AdoMet) is limiting and thus function as sequence-specific C-->U mutator enzymes . Here we explored whether mutations causing inactivation of the cofactor binding activity of the HpaII methyltransferase, thus mimicking conditions of limiting AdoMet concentration, could convert a DNA methyltransferase to a C-->U mutator enzyme . We created two mutator enzymes from the HpaII methyltransferase (F38S and G40D) which both showed enhanced cytosine deamination activities in vitro and in vivo . Interestingly, the G:U mispairs generated by these enzymes were not repaired completely in bacteria equipped with uracil-DNA glycosylase-initiated repair machinery, giving rise to a potent mutator phenotype . This is the first report showing the creation of mutator enzymes from a DNA methyltransferase and the demonstration of their mutagenicity in living cells.

J Biol Chem, 1995 Nov 10, 270(45), 27358 - 65
Analysis of the binding of Xenopus ribosomal protein L5 to oocyte 5 S rRNA . The major determinants of recognition are located in helix III-loop C; Scripture JB et al.; Xenopus ribosomal protein L5 was expressed in Escherichia coli and exhibits high affinity (Kd = 2 nM) and specificity for oocyte 5 S rRNA . The pH dependence of the association constant for the complex reveals an ionization with a pK alpha value of 10.1, indicating that tyrosine and/or lysine residues are important for specific binding of L5 to the RNA . Formation of the L5.5 S rRNA complex is remarkably insensitive to ionic strength, providing evidence that nonelectrostatic interactions make significant contributions to binding . Together, these results suggest that one or more tyrosine residues may form critical contacts through stacking interactions with bases in the RNA . In order to locate recognition elements within 5 S rRNA, we measured binding of L5 to a collection of site-specific mutants . Mutations in the RNA that affected the interaction are confined to the hairpin structure comprised of helix III and loop C . Earlier experiments with a rhodium structural probe had shown that the two-nucleotide bulge in helix III and the intrinsic structure of loop C create sites in the major groove that are opened and accessible to stacking interactions with the metal complex . In the present studies, we detect a correlation between the intercalative binding of the rhodium complex to mutants in the hairpin and binding of L5, supporting the proposal that binding of the protein is mediated, in some part, by stacking interactions . Furthermore, the results from mutagenesis establish that, despite overlapping binding sites on 5 S rRNA, L5 and transcription factor IIIA utilize distinct structural elements for recognition.

J Biol Chem, 1995 Nov 10, 270(45), 27051 - 7
Phosphorylation of nodulin 26 on serine 262 affects its voltage-sensitive channel activity in planar lipid bilayers; Lee JW et al.; Nodulin 26 is an symbiosome membrane protein of soybean nodules that shows ion channel activity in planar lipid bilayers . Serine 262 of nodulin 26 is phosphorylated by calmodulin-like domain protein kinase . To study the effects of phosphorylation, nodulin 26 with Ser, Ala, or Asp at position 262 were expressed in Escherichia coli . The expressed protein possessed a histidine-rich leader sequence for purification by Ni2+ chelate fast protein liquid chromatography . Upon reconstitution into planar lipid bilayers, the recombinant proteins showed a large single channel conductance (3.1 nanosiemens (nS) in cis0.2M/trans1.0 M KCl and 1.6 nS in cis 0.2M/trans0.2 M KCl) and weak anion selectivity, similar to native soybean nodulin 26 . Nodulin 26 with Ser- or Ala-262 occupied the maximal open conductance state greater than 97% of the time (3.1 nS in cis0.2M/trans1.0 M KCl) regardless of applied voltage . However, nodulin 26 with Asp-262 showed increased gating and preferential occupancy of lower subconductance states (1.8 and 0.6 nS in cis0.2M/trans1.0 M KCl) at high applied voltages (e.g . 70 mV) . In situ phosphorylation of Ser-262 of nodulin 26 by calmodulin-like domain protein kinase also resulted in increased voltage-dependent gating and preferential occupancy of lower subconductance states . These results suggest that phosphorylation of serine 262 of nodulin 26 modulates channel activity by conferring voltage sensitivity.

J Biol Chem, 1995 Nov 10, 270(45), 26897 - 903
An isoform of the neuronal cyclin-dependent kinase 5 (Cdk5) activator; Tang D et al.; Neuronal Cdc2-like kinase is a heterodimer of Cdk5 and a 25-kDa subunit that is derived from a 35-kDa brain- and neuron-specific protein called the neuronal Cdk5 activator (p35/p25nck5a) (Lew, J., Huang, Q.-Q., Qi, Z., Winkfein, R . J., Aebersold, R., Hunt, T., and Wang, J . H . (1994) Nature 371, 423-426; Tsai, L . H., Delalle, I., Caviness, V . S., Jr., Chae, T., and Harlow, E . (1994) Nature 371, 419-423) . Upon screening of a human hippocampus library with a bovien Nck5a cDNA, we uncovered a distinct clone encoding a 39-kDa isoform of Nck5a . The isoform, designated the neuronal Cdk5 activator isoform (p39nck5ai), showed a high degree of sequence similarity to p35nck5a with 57% amino acid identity . Northern blot analysis detected its mRNA transcript in bovine and rat cerebrum and cerebellum, but not in any other rat tissues examined . In situ hybridization showed that Nck5ai was enriched in CA1 to CA3 of the hippocampus, but absent in the fimbria of hippocampal formation . Among seven cell lines in proliferating cultures, only PC12 and N2A, two cell lines capable of differentiating into neuron-like cells, were found to contain Nck5ai mRNA . A 30-kDa truncated form of Nck5ai expressed as a glutathione S-transferase fusion protein in Escherichia coli} was found to associate with Cdk5 to form an active Cdk5 kinase . Thus, the isoform shares many common characteristics with p35nck5a, including Ckd5 activating activity and brain- and neuron-specific expression . Both proteins show limited sequence homology to cyclins, suggesting that they define a new family of cyclin-dependent kinase-activating proteins.

J Biol Chem, 1995 Nov 10, 270(45), 26849 - 56
Tandem binding of six OmpR proteins to the ompF upstream regulatory sequence of Escherichia coli; Harlocker SL et al.; OmpR is a transcription factor in Escherichia coli whose function is modulated by phosphorylation in the presence of phosphorylated EnvZ, a transmembrane protein histidine kinase involved in osmosensing . Using a protein S-OmpR hybrid protein, we demonstrated that six OmpR molecules bind tandemly to the -100 to -39 sequence of ompF . This sequence consists of three 20-base pair units: F1, F2, and F3, each of which is bound by two OmpR proteins . Polymerase chain reaction selection of nine randomized base pairs within the F1 sequence revealed highly conserved C residues spaced 10 base pairs apart . Further mutational analysis of conserved bases indicated that two OmpR molecules bind tandemly to two direct repeats . Mobility shift assays showed that cooperative interactions play a role in enhancing binding of OmpR to lower affinity F2 and F3 sites . Activation and repression of ompF expression are thus regulated by a total of eight OmpR molecules, including two molecules that bind to a distal site (-380 to -361).

J Biol Chem, 1995 Nov 10, 270(45), 26833 - 9
Mechanism of regulation in yeast glycogen phosphorylase; Lin K et al.; The mechanism of yeast glycogen phosphorylase activation by covalent phosphorylation involves structural elements distinct from the mammalian homologs . To understand the role of the amino-terminal 39-residue extension in the phosphorylation control mechanism, mutants with 22 and 42 amino-terminal residues removed were expressed in Escherichia coli, and their properties were compared with the wild-type (WT) enzyme . The unphosphorylated WT enzyme had a specific activity of 0.1 unit/mg and was not activated significantly by the substrate, glucose 1-phosphate . Phosphorylation by protein kinase resulted in a 1300-fold activation . Glucose 6-phosphate inhibited the unphosphorylated enzyme more effectively than the phosphorylated form, and inhibition of the latter was cooperative . Glucose was a poor inhibitor for both the unphosphorylated and phosphorylated WT enzyme with Ki > 300 mM . The rate of phosphorylation by protein kinase depended on substrates and interactions of the amino terminus . Maltoheptaose increased the rate of phosphorylation of the WT enzyme by yeast phosphorylase kinase 5-fold . The 22-residue deletion mutant (Nd22) had overall kinetic properties similar to the WT enzyme, except that Nd22 was a better substrate for the protein kinase and the rate of phosphorylation was unaffected by maltoheptaose . The 42-residue deletion mutant (Nd42), which lacks the phosphorylation site, was measurably active, although much less active than phosphorylated WT . Sedimentation equilibrium analysis indicated that the WT, Nd22, and Nd42 exist as tetramer, partially dissociated tetramer, and dimer, respectively . Phosphorylation of the WT and Nd22 converted both to dimer . The results indicated that the amino terminus affects quaternary structure and mediates activity regulation through conformational transition.

J Biol Chem, 1995 Nov 10, 270(45), 26813 - 7
Replacements of histidine 226 of NhaA-Na+/H+ antiporter of Escherichia coli . Cysteine (H226C) or serine (H226S) retain both normal activity and pH sensitivity, aspartate (H226D) shifts the pH profile toward basic pH, and alanine (H226A) inactivates the carrier at all pH values; Rimon A et al.; We have previously shown that replacement of His-226 in the NhaA Na+/H+ antiporter of Escherichia coli to Arg (H226R) shifts the pH profile of the antiporter toward acidic pH and as a result of delta nhaA delta nhaB strain bearing this mutation is Na+ sensitive at alkaline pH (Gerchman, Y., Olami, Y., Rimon, A., Taglicht, D., Schuldiner, S . and Padan, E . (1993) Proc . Natl . Acad . Sci . U.S.A . 90, 1212-1216) . In the present work the role of His-226 in the response of NhaA to pH has been studied in detail . The Na+ sensitivity of the delta nhaA delta nhaB mutant bearing the H226R-NhaA plasmid at alkaline pH provided a very powerful tool to isolate revertants and suppressants of H226R growing on high Na+ at alkaline pH . With this approach cysteine (H226C) and serine (H226S) replacements were found to efficiently replace His-226 and yield an antiporter, which like the wild-type protein, is activated by pH between pH 7 and 8 . These results imply that polarity and/or hydrogen bonding, the common properties shared by these amino acid residues, are essential at position 226 for pH regulation of NhaA . This suggestion was substantiated by site-directed mutagenesis of His-226 either to alanine (H226A) or aspartate (H226D) . Whereas H226A-NhaA shows very low activity which is not activated by pH, H226D-NhaA is active and regulated by pH . The pH profile of H226D is shifted by half a pH unit toward alkaline pH, as opposed to the previously isolated mutant H226R which has a pH profile shift, to the same extent, but toward acidic pH . It is suggested that charge modifies the pH profile but is not essential for the pH regulation of NhaA.

Virology, 1995 Nov 10, 213(2), 581 - 9
Identification of the active-site residues of the 3C proteinase of foot-and-mouth disease virus; Grubman MJ et al.; To identify the active-site residues of the 3C proteinase of foot-and-mouth disease virus (FMDV), we introduced mutations into the 3C coding region and examined the activity of mutant enzymes on various substrates . Based on alignment of FMDV 3C with other picornavirus 3C proteinases and with the trypsin family of serine proteinases, mutations were introduced at residues presumed to be part of the catalytic triad, involved in substrate binding, or present in nonconserved regions . Wild-type and mutant 3C proteins were expressed in Escherichia coli and tested for their ability to cleave synthetic substrates corresponding to different portions of the viral genome . Substitutions at His-46 (catalytic triad), Asp-84 (catalytic triad), or His-181 (substrate binding) produced enzymes unable to process P1, P2, or P3 substrates in trans, whereas a change in the conserved Asp-98 had no effect on enzyme activity . Substitution of Ser for Cys-163 (catalytic triad) yielded an enzyme that retained activity on some substrates, while a substitution of Gly at this position resulted in a completely inactive enzyme . The kinetics of trans processing of translation products from a transcript encoding the P1 and P2 coding regions and the 2C/3A cleavage site with wild-type 3C or a transcript encoding P1 with 3C mutants revealed that the order of cleavage was VP3-VP1, VP0-VP3, VP1-2A, 2C-3A, and 2B-2C . Mutations in 3C that resulted in a partially active enzyme were individually introduced into full-length FMDV cDNA and RNA transcripts were translated in a cell-free system and used to transfect cells . In all cases the virus that was rescued had reverted to the wild-type 3C codon.

Virology, 1995 Nov 10, 213(2), 558 - 68
Molecular basis of antigenic variation between the glycoproteins C of respiratory bovine herpesvirus 1 (BHV-1) and neurovirulent BHV-5; Chowdhury SI; Herpesvirus glycoprotein C (gC) functions as a major virus attachment protein . The gC sequence of the neurovirulent bovine herpesvirus type 5 (BHV-5) virus was determined and compared with the gC sequence of the nonneurovirulent BHV-1 . Alignment of the predicted amino acid sequences of BHV-1 and BHV-5 gC ORFs showed that the amino-terminal third of the protein differed between the two viruses . Whole or subgenomic fragments of gC coding regions from both viruses were expressed as trpE-gC fusion proteins in Escherichia coli to map linear epitopes defined by type-specific murine monoclonal antibodies (MAbs) . Based on the reactivity of BHV-1-specific MAbs with the recombinant proteins, two epitopes were mapped between BHV-1 gC residues 22 and 172 . Undirectional deletion of these residues at the carboxy end mapped one within residues 22-69 and the other within residues 103-122 . Two BHV-5-specific MAbs identified an epitope coding region within BHV-5 gC residues 31-78 . Bovine antisera against BHV-1 and BHV-5 showed specificity to BHV-1 gC residues 22-69 and to BHV-5 gC residues 31-78, respectively, in a type-specific manner.

Virology, 1995 Nov 10, 213(2), 517 - 25
Expression, purification, and identification of a novel self-cleavage site of the Nla C-terminal 27-kDa protease of turnip mosaic potyvirus C5; Kim DH et al.; The gene encoding the C-terminal protease domain (27 kDa) of the nuclear inclusion protein a of turnip mosaic potyvirus C5 was cloned and expressed as a fusion protein with glutathione S-transferase in Escherichia coli XL1-blue . Two forms of the protease (27 and 25 kDa) were purified from the fusion protein by glutathione affinity chromatography and Mono S chromatography and exhibited the specific proteolytic activity when a synthetic undecapeptide, Glu-Pro-Thr-Val-Tyr-His-Gln-Thr-Leu-Asn-Glu, or an in vitro translation product of the polyprotein containing the cleavage site between the nuclear inclusion protein b and the capsid protein, was used as a substrate . The purified proteases showed a Km of 1.15 +/- 0.16 mM and a Vmax of 0.74 +/- 0.091 mumol/mg/min with the synthetic peptide substrate . The 25-kDa protein was found to be generated by the cleavage between Ser223 and Gly224 near the C-terminus of the 27-kDa protease and to retain the specific proteolytic activity . The point mutation of Asp81 or Cys151, two putative active site residues in the 27-kDa protease, to Asn or Ser, respectively, prevented the generation of the 25-kDa protein and diminished the proteolytic activity of the protease drastically, suggesting that the 27-kDa protease cleaves itself between Ser223 and Gly224 to generate the 25-kDa protein.

Virology, 1995 Nov 10, 213(2), 455 - 61
Differential subcellular localization of hepatitis C virus core gene products; Lo SY et al.; The expression of the core gene of two different hepatitis C virus (HCV) isolates was analyzed . In the presence of its downstream E1 envelope protein sequence, two major core protein products with molecular masses of 21 kDa (P21) and 19 kDa (P19) and a minor protein product with molecular mass of 16 kDa (P16) were detected . In the absence of its downstream E1 envelope protein sequence, P21 and P19 remained the major protein products expressed from the core gene of the HCV-RH isolate, whereas P16 became the major protein product of the core gene of the HCV-1 isolate . Analysis of the amino-terminal sequences of P21 and P16 expressed in Escherichia coli revealed that P21 and P16 were co-amino terminal . Deletion-mapping analysis indicated that P16 lacked the carboxy-terminal sequence of P21 . Immunofluorescence analysis of the subcellular localization of different HCV core proteins indicated that P21 and P19 displayed a reticular and punctate staining pattern typical of endoplasmic reticulum-associated proteins, while P16 was localized to the nucleus . The distinct subcellular localization of P16 raises the possibility that P16 may have a biological function very different from those of P21 and P19.

Arch Biochem Biophys, 1995 Nov 10, 323(2), 423 - 8
The effects of an atpE ribosome-binding site mutation on the stoichiometry of the c subunit in the F1F0 ATPase of Escherichia coli; Schemidt RA et al.; We tested the hypothesis that the stoichiometry of the c subunit in the F0 sector of the Escherichia coli F1F0 ATPase is dependent upon the level of atpE gene expression . F0 was purified from cells carrying plasmids encoding the F0 subunits with and without a ribosome-binding site mutation preceding atpE, the gene which codes for the c subunit . Subunit-specific antibodies were used to quantitate the relative amounts of the b and c subunits . The decreased expression of atpE resulted in a significantly decreased amount of the c subunit in the purified F0 . Immunoblot quantitation of the amounts of b and c subunits in F1F0 precipitated by anti-F1 antiserum also showed that the mutation produced significant differences in the stoichiometry of subunit c . The amount of c subunit assembled into the F1F0 synthesized from a plasmid carrying the atpE ribosome binding site mutation was 2-5 times less than the amount found in the F1F0 synthesized from a wild-type plasmid . Therefore, the stoichiometry of the c subunit assembled into the F1F0 complex appears to be variable, depending on the expression of atpE.

Arch Biochem Biophys, 1995 Nov 10, 323(2), 361 - 6
Analysis of the in vitro translation product of a novel-type Drosophila melanogaster aldolase mRNA in which two carboxyl-terminal exons remain unspliced; Sugimoto Y et al.; Drosophila melanogaster generates three different types of aldolase mRNAs from a single gene by selective usage of the triplicate exons 4 (4 alpha, 4 beta, and 4 gamma), which encode three different isozymes having respective carboxyl termini . We have found the presence of a novel-type mRNA (named alpha beta) in which two final exons, 4 alpha and 4 beta, were retained unspliced . Herein, a cDNA clone containing the alpha beta sequence was inserted into pINIII and expressed in an Escherichia coli system . The product, which exhibited aldolase activity, was found to be isozyme alpha from the primary structure and the enzymological properties, with the 4 alpha sequence alone being present as the carboxyl terminus . In tissues of D . melanogaster, the production of mRNA encoding exon 4 alpha is known to be restrained to a low level . This may be understood by the fact that the aldolase gene of this species does not have a typical poly(A) signal at the 3' end in exon 4 alpha . Instead, the transcript-encoding exons, 4 alpha and 4 beta, might be produced when AATATA, which resides downstream of the coding frame in exon 4 beta, is recognized as a poly(A) signal during RNA processing.

Arch Biochem Biophys, 1995 Nov 10, 323(2), 313 - 22
Early increase in choline kinase activity upon induction of the H-ras oncogene in mouse fibroblast cell lines; Ratnam S et al.; The effects of expression of the H-ras oncogene on phosphatidylcholine metabolism were examined in C3H10T1/2 and NIH3T3 cells expressing ras constitutively or under the control of inducible promoters . Cell lines expressing ras under the control of the mouse metallothionein promoter and the Escherichia coli lac operator/repressor system and an NIH3T3 cell line stably transfected with the ras oncogene were studied . Phosphocholine levels were elevated in the cells constitutively expressing ras and were increased 4-6 h upon induction in the inducible cell lines . Glycerophosphocholine, which is elevated five- to sixfold in constitutively transfected ras cells, did not increase at early times of induction, suggesting the absence of increased phosphatidylcholine degradation via a phospholipase A . Choline kinase activity increased within 4-6 h upon induction and correlated well with the increase in phosphocholine levels . This increase in phosphocholine levels could be prevented by the addition of hemicholinium-3, a competitive inhibitor of choline kinase . Expression of activated c-raf or v-raf also increased choline kinase activity, suggesting that the induction of choline kinase by ras is downstream of the ras/raf interaction . Long-term and short-term labeling experiments failed to detect evidence for increased phospholipase C activity . These results suggest that the increase in choline kinase activity observed in cells expressing ras is an early, integral part of ras transformation and is the main contributor to increased phosphocholine levels accompanying morphological changes.

Science, 1995 Nov 10, 270(5238), 992 - 4
Converting Escherichia coli RNA polymerase into an enhancer-responsive enzyme: role of an NH2-terminal leucine patch in sigma 54; Wang JT et al.; The protein sigma 54 associates with Escherichia coli core RNA polymerase to form a holoenzyme that binds promoters but is inactive in the absence of enhancer activation . Here, mutants of sigma 54 enabled polymerases to transcribe without enhancer protein and adenosine triphosphate . The mutations are in leucines within the NH2-terminal glutamine-rich domain of sigma 54 . Multiple leucine substitutions mimicked the effect of enhancer protein, which suggests that the enhancer protein functions to disrupt a leucine patch . The results indicate that sigma 54 acts both as an inhibitor of polymerase activity and as a receptor that interacts with enhancer protein to overcome this inhibition, and that these two activities jointly confer enhancer responsiveness.

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