Escherichia coli

FEBS Lett, 1997 Mar 10, 404(2-3), 179 - 84
Molecular cloning and expression of subunit 9 of the 26S proteasome; Hoffman L et al.; Seven peptides from subunit 9 (S9) of the human 26S proteasome were sequenced and this information was used to clone a HeLa cDNA that encodes the 46 kDa subunit . Rabbit polyclonal antisera were made against a ubiquitin fusion protein containing 12 amino acids from S9 and against a full-length S9 expressed in E . coli . Western blot analysis showed that the S9-specific antibodies bound the 26S proteasome and its regulatory complex separated on non-denaturing gels . In SDS-PAGE samples of the two complexes, the S9-specific antibodies bound a single 46 kDa subunit . Thus, a cDNA encoding a novel 26S protease subunit has been isolated, sequenced, and expressed.

FEBS Lett, 1997 Mar 10, 404(2-3), 140 - 2
COOH-terminal decamers in proteins are non-random; Berezovsky IN et al.; We have undertaken an exhaustive statistical analysis of the amino acid sequences at the carboxyl-terminal (C) ends of proteins . The composition of the C-terminal decapeptides differs from that expected for the given proteins from the overall amino acid composition . For E . coli, yeast, and H . sapiens it was shown that positively charged amino acid residues are over-represented while Gly residues are under-represented . The C-terminal bias, a novel feature of protein structure, should be taken into account when molecular evolution, spatial structure, translational termination and protein folding are concerned.

FEBS Lett, 1997 Mar 10, 404(2-3), 125 - 8
Amounts of proteins altered by mutations in the dnaA gene of Escherichia coli; Ohba A et al.; We identified proteins whose amounts were altered in a temperature-sensitive dnaA46 mutant of Escherichia coli . Proteins whose amounts were increased in the mutant were serine hydroxymethyltransferase, beta-ketoacyl {acyl carrier protein} synthase II, long-chain fatty acid transport protein, and UDP-glucose 4-epimerase, while the decreased ones were flagellin and D-ribose-binding protein . Transformation of the mutant with a plasmid containing the wild type dnaA gene complemented the phenotype . As pulse-labeling experiments revealed that the rates of synthesis of the proteins were altered in the mutant, DnaA protein may be involved in expression of these proteins.

J Immunol Methods, 1997 Mar 10, 202(1), 49 - 57
A murine model of pulmonary damage induced by lipopolysaccharide via intranasal instillation; Szarka RJ et al.; This study examines the intranasal instillation of lipopolysaccharide (LPS) into BALB/c mice causing acute pulmonary damage, due to neutrophil infiltration and sepsis . A dose response with LPS showed that an intranasal instillation of 167 microg/ml (10 microg/mouse) caused acute lung injury within 2-4 h and reached maximal damage at 24-48 h . We found the method of LPS administration for induction of acute pulmonary damage to be crucial . After 24 h post-LPS injection, a comparison showed a substantial increase in pulmonary damage with intranasal instillation of LPS . As for intravenous injection, it showed a baseline effect . This study indicates that LPS administered intranasally causes acute pulmonary damage, whereas with intravenous and intraperitoneal endotoxin administration a tissue-specific or similar degree of pulmonary injury may not develop.

J Cell Biol, 1997 Mar 10, 136(5), 1091 - 7
MAP kinase is required for the spindle assembly checkpoint but is dispensable for the normal M phase entry and exit in Xenopus egg cell cycle extracts; Takenaka K et al.; In Xenopus laevis egg cell cycle extracts that mimic early embryonic cell cycles, activation of MAP kinase and MAP kinase kinase occurs in M phase, slightly behind that of maturation promoting factor . To examine the possible role of MAP kinase in the in vitro cell cycle, we depleted the extracts of MAP kinase by using anti-Xenopus MAP kinase antibody . Like in the mock-treated extracts, the periodic activation and deactivation of MPF occurred normally in the MAP kinase-depleted extracts, suggesting that MAP kinase is dispensable for the normal M phase entry and exit in vitro . It has recently been reported that microtubule depolymerization by nocodazole treatment can block exit from mitosis in the extracts if enough sperm nuclei are present, and that the addition of MAP kinase-specific phosphatase MKP-1 overcomes this spindle assembly checkpoint, suggesting the involvement of MAP kinase in the checkpoint signal transduction . We show here that the spindle assembly checkpoint mechanism cannot operate in the MAP kinase-depleted extracts . But, adding recombinant Xenopus MAP kinase to the MAP kinase-depleted extracts restored the spindle assembly checkpoint . These results indicate unambiguously that classical MAP kinase is required for the spindle assembly checkpoint in the cell cycle extracts . In addition, we show that strong activation of MAP kinase by the addition of a constitutively active MAP kinase kinase kinase in the absence of sperm nuclei and nocodazole, induced mitotic arrest in the extracts . Therefore, activation of MAP kinase alone is sufficient for inducing the mitotic arrest in vitro.

J Cell Biol, 1997 Mar 10, 136(5), 1081 - 90
Mitochondrial association of a plus end-directed microtubule motor expressed during mitosis in Drosophila; Pereira AJ et al.; The kinesin superfamily is a large group of proteins (kinesin-like proteins {KLPs}) that share sequence similarity with the microtubule (MT) motor kinesin . Several members of this superfamily have been implicated in various stages of mitosis and meiosis . Here we report our studies on KLP67A of Drosophila . DNA sequence analysis of KLP67A predicts an MT motor protein with an amino-terminal motor domain . To prove this directly, KLP67A expressed in Escherichia coli was shown in an in vitro motility assay to move MTs in the plus direction . We also report expression analyses at both the mRNA and protein level, which implicate KLP67A in the localization of mitochondria in undifferentiated cell types . In situ hybridization studies of the KLP67A mRNA during embryogenesis and larval central nervous system development indicate a proliferation-specific expression pattern . Furthermore, when affinity-purified anti-KLP67A antisera are used to stain blastoderm embryos, mitochondria in the region of the spindle asters are labeled . These data suggest that KLP67A is a mitotic motor of Drosophila that may have the unique role of positioning mitochondria near the spindle.

J Mol Biol, 1997 Mar 7, 266(4), 733 - 44
Charged residues of the rotor protein FliG essential for torque generation in the flagellar motor of Escherichia coli; Lloyd SA et al.; The FliG protein of Escherichia coli is essential for assembly and function of the flagellar motor . Certain mutations in FliG give a non-motile, or Mot-, phenotype, in which flagella are assembled but do not rotate . Mutations with this property are clustered in a C-terminal segment of FliG that is stable when expressed alone, and thus probably constitutes an independently folded domain . Previously, we suggested that this domain forms the rotor portion of the active site for torque generation in the motor . In this work, we have used a mutational approach to identify the amino acid residues in the C-terminal domain of FliG that are most important for motor function . Site-directed mutagenesis was used to replace each of the conserved residues in this domain with alanine, and the effects on motor function were measured . Because charged residues have often been suggested to have important roles in torque generation, conserved charged residues were changed individually and in all pairwise combinations . The results show that three charged residues of FliG, Arg279, Asp286 and Asp287, are directly involved in torque generation . Mutations in these residues cause motility defects that suggest that they function jointly, in an active site whose most important property is a specific arrangement of charges . Two other charged residues, Lys262 and Arg295, may also be involved in torque generation, but are less critical than Arg279, Asp286 or Asp287 . Unchanged residues of the FliG motility domain do not appear to have direct roles in torque generation, although some are needed for the stability of the protein or for normal clockwise/ counter-clockwise switching . The Mot- mutations of fliG isolated previously by random mutagenesis do not alter the putative active-site residues, but render the proteins abnormally susceptible to proteolysis, suggesting significantly altered conformations or reduced stabilities.

J Mol Biol, 1997 Mar 7, 266(4), 703 - 10
Sequence-dependent modulation of nucleotide excision repair: the efficiency of the incision reaction is inversely correlated with the stability of the pre-incision UvrB-DNA complex; Delagoutte E et al.; The UvrABC excinuclease is involved in the nucleotide excision repair (NER) pathway . Sequence-dependent differences in repair efficiency have been reported for many different lesions, and it is often suggested that sites with poor repair contribute to the occurrence of mutation hot spots . However, guanine bases modified by N-2-acetylaminofluorence (AAF) within the NarI site (5'-G1G2CG3CC-3') are incised by the UvrABC excinuclease with different efficiencies in a pattern not correlated with the potency of mutation induction . To gain insight into the mechanism of sequence-dependent modulation of NER, we analyzed the formation, the structure and the stability of UvrB-DNA pre-incision complexes formed at all three positions of the AAF-modified NarI site . We show that the efficiency of release of UvrA2 from specific UvrA2B-DNA complexes is sequence-dependent and that the efficiency of incision is inversely related to the stability of the pre-incision complex . We propose that the pre-incision complex, {UvrB-DNA}, when formed upon dissociation of UvrA2, undergoes a conformational change (isomerization step) giving rise to an unstable but incision-competent complex that we call {UvrB-DNA}' . The {UvrB-DNA} complex is stable and unable to form an incision-competent complex with UvrC . As the release of UvrA2, this isomerization step is sequence-dependent . Both steps contribute to modulate NER efficiency.

J Mol Biol, 1997 Mar 7, 266(4), 677 - 87
Mechanistic insights into p53-promoted RNA-RNA annealing; Nedbal W et al.; The tumour suppressor protein p53 promotes the annealing of complementary nucleic acids in vitro . We observed an up to 1600-fold increase of RNA-RNA annealing by recombinant p53 protein which was shown to bind to RNA in sequence-independent way . Nuclease mapping experiments suggest that p53 binds to intramolecular duplex portions and only marginally changes the overall secondary structure of RNA at conditions of increased annealing . Thus, the mechanism of p53-promoted RNA-RNA annealing does not seem to be dependent on an activity that melts or changes RNA structure . The activation enthalpy of RNA-RNA annealing is decreased in the presence of p53, i.e . the p53 protein could stabilize the transition state whereas the activation entropy is unfavourable . A comparison with thermodynamic data measured for other facilitators strongly suggests that the mechanism of p53-promoted RNA-RNA annealing is distinct from the mechanism by which other facilitators work . The annealing activity of p53 is almost abolished in the presence of magnesium indicating that it can be sharply regulated in vitro and, in principle, could also be regulated in vivo.

J Theor Biol, 1997 Mar 7, 185(1), 97 - 118
A functional model for the ribosome; Wood PN; The "ice" structure for the E . coli ribosome clearly illustrates how the ribosome could work and it is used as the basis of a detailed account of ribosome function . There have been objections to this structure as it does not fit with current beliefs about the position of the T-site, for the arriving tRNA, or the Exit-site: these beliefs are wrong . The elongation factor EF-Tu is required at both the T-site and the E-site, and these two attachment areas were combined into the false site . The E-site has been placed at the L1-ridge because deacylated tRNAs attach to protein L1, but none of these experiments used a cognate codon to get a positive identification of this as the E-site . The reason L1 attaches to deacylated tRNAs is that it is at the E-site in polysomes, which contain closely stacked, or overlapping, ribosomes with the L1-ridge at the interface between ribosomes . The revised data allows the 1989 ribosome model to be reconciled with the most contradictory results and it is then used to develop a more detailed picture of the ribosome.

Biochim Biophys Acta, 1997 Mar 7, 1338(1), 77 - 92
Mobility of norbornane-type substrates and water accessibility in cytochrome P-450cam; Schulze H et al.; The behaviour of norbornane-type substrates bound to oxidised cytochrome P-450cam (CYP 101) in 60% (w/w) glycerol-containing phosphate buffer was investigated using electronic absorption spectroscopy . The high-pressure dependence study revealed that the value of the spin-state reaction-volume change decreased from -70 to -22.8 cm3/mol with decreasing high-spin state content from 99 to 63% . Simultaneously, the values for the enthalpy and entropy determined from the low-temperature dependence of the spin-state transition decreased from 73.7 to 24.3 kJ/mol and from 310.4 to 88.9 J/mol K, respectively . Under our experimental conditions the pH-value of the buffer remained at low temperatures and high pressures in the range of pH 7-8, in which no pH-value-induced spin-state conversion occurred . Therefore, the secondary effect of the temperature and pressure-induced pH change can be disregarded as being responsible for the observed spin-state transition effects . Substrate dissociation constants were determined . From the temperature-jump experiments (297 K to 180 K) we found a higher mobility in the active site for the substrates in the sequence (1R)-camphor, (1S)-camphor, camphane, (1R)- and (1S)-camphorquinone, norcamphor, and norbornane . Our findings can be explained by the incomplete fit of the methyl groups of the norbornane-type substrate to the protein, in particular to the I-helix, predominantly determining the substrate mobility and water accessibility to the protein.

J Biol Chem, 1997 Mar 7, 272(10), 6766 - 76
Cloning and functional expression of a mammalian gene for a peroxisomal sarcosine oxidase; Reuber BE et al.; Sarcosine oxidation in mammals occurs via a mitochondrial dehydrogenase closely linked to the electron transport chain . An additional H2O2-producing sarcosine oxidase has now been purified from rabbit kidney . A corresponding cDNA was cloned from rabbit liver and the gene designated sox . This rabbit sox gene encodes a protein of 390 amino acids and a molecular mass of 44 kDa identical to the molecular mass estimated for the purified enzyme . Sequence analysis revealed an N-terminal ADP-betaalphabeta-binding fold, a motif highly conserved in tightly bound flavoproteins, and a C-terminal peroxisomal targeting signal 1 . Sarcosine oxidase from rabbit liver exhibits high sequence homology (25-28% identity) to monomeric bacterial sarcosine oxidases . Both purified sarcosine oxidase and a recombinant fusion protein synthesized in Escherichia coli contain a covalently bound flavin, metabolize sarcosine, L-pipecolic acid, and L-proline, and cross-react with antibodies raised against L-pipecolic acid oxidase from monkey liver . Subcellular fractionation demonstrated that sarcosine oxidase is a peroxisomal enzyme in rabbit kidney . Transfection of human fibroblast cell lines and CV-1 cells (monkey kidney epithelial cells) with the sox cDNA resulted in a peroxisomal localization of sarcosine oxidase and revealed that the import into the peroxisomes is mediated by the peroxisomal targeting signal 1 pathway.

J Biol Chem, 1997 Mar 7, 272(10), 6733 - 40
Cloning and expression of the cDNA encoding the human homologue of the DNA repair enzyme, Escherichia coli endonuclease III; Hilbert TP et al.; We previously purified a bovine pyrimidine hydrate-thymine glycol DNA glycosylase/AP lyase . The amino acid sequence of tryptic bovine peptides was homologous to Escherichia coli endonuclease III, theoretical proteins of Saccharomyces cerevisiae and Caenorhabditis elegans, and the translated sequences of rat and human 3'-expressed sequence tags (3'-ESTs) (Hilbert, T . P., Boorstein, R . J., Kung, H . C., Bolton, P . H., Xing, D., Cunningham, R . P., Teebor, G . W . (1996) Biochemistry 35, 2505-2511) . Now the human 3'-EST was used to isolate the cDNA clone encoding the human enzyme, which, when expressed as a GST-fusion protein, demonstrated thymine glycol-DNA glycosylase activity and, after incubation with NaCNBH3, became irreversibly cross-linked to a thymine glycol-containing oligodeoxynucleotide, a reaction characteristic of DNA glycosylase/AP lyases . Amino acids within the active site, DNA binding domains, and {4Fe-4S} cluster of endonuclease III are conserved in the human enzyme . The gene for the human enzyme was localized to chromosome 16p13.2-.3 . Genomic sequences encoding putative endonuclease III homologues are present in bacteria, archeons, and eukaryotes . The ubiquitous distribution of endonuclease III-like proteins suggests that the 5,6-double bond of pyrimidines is subject to oxidation, reduction, and/or hydration in the DNA of organisms of all biologic domains and that the resulting modified pyrimidines are deleterious to the organism.

J Biol Chem, 1997 Mar 7, 272(10), 6671 - 6
The N-terminal moiety of CDC25(Mm), a GDP/GTP exchange factor of Ras proteins, controls the activity of the catalytic domain . Modulation by calmodulin and calpain; Baouz S et al.; This work describes the in vitro properties of full-length CDC25(Mm) (1262 amino acid residues), a GDP/GTP exchange factor (GEF) of H-ras p21 . CDC25(Mm), isolated as a recombinant protein in Escherichia coli and purified by various chromatographic methods, could stimulate the H-ras p21.GDP dissociation rate; however, its specific activity was 25 times lower than that of the isolated catalytic domain comprising the last C-terminal 285 residues (C-CDC25(Mm285)) and 5 times lower than the activity of the C-terminal half-molecule (631 residues) . This reveals a negative regulation of the catalytic domain by other domains of the molecule . Accordingly, the GEF activity of CDC25(Mm) was increased severalfold by the Ca2+-dependent protease calpain that cleaves around a PEST-like region (residues 798-853), producing C-terminal fragments of 43-56 kDa . In agreement with the presence of an IQ motif on CDC25(Mm) (residues 202-229), calmodulin interacted functionally with the exchange factor . Depending on the calmodulin concentration an inhibition up to 50% of the CDC25(Mm)-induced nucleotide exchange activity on H-ras p21 was observed, an effect requiring Ca2+ ions . Calmodulin also inhibited C-CDC25(Mm285) but with a approximately 100 times higher IC50 than in the case of CDC25(Mm) ( approximately 10 microM versus 0.1 microM, respectively) . Together, these results emphasize the role of the other domains of CDC25(Mm) in controlling the activity of the catalytic domain and support the involvement of calmodulin and calpain in the in vivo regulation of the CDC25(Mm) activity.

J Biol Chem, 1997 Mar 7, 272(10), 6539 - 47
Structure of recombinant human CPP32 in complex with the tetrapeptide acetyl-Asp-Val-Ala-Asp fluoromethyl ketone; Mittl PR et al.; The cysteine protease CPP32 has been expressed in a soluble form in Escherichia coli and purified to >95% purity . The three-dimensional structure of human CPP32 in complex with the irreversible tetrapeptide inhibitor acetyl-Asp-Val-Ala-Asp fluoromethyl ketone was determined by x-ray crystallography at a resolution of 2.3 A . The asymmetric unit contains a (p17/p12)2 tetramer, in agreement with the tetrameric structure of the protein in solution as determined by dynamic light scattering and size exclusion chromatography . The overall topology of CPP32 is very similar to that of interleukin-1beta-converting enzyme (ICE); however, differences exist at the N terminus of the p17 subunit, where the first helix found in ICE is missing in CPP32 . A deletion/insertion pattern is responsible for the striking differences observed in the loops around the active site . In addition, the P1 carbonyl of the ketone inhibitor is pointing into the oxyanion hole and forms a hydrogen bond with the peptidic nitrogen of Gly-122, resulting in a different state compared with the tetrahedral intermediate observed in the structure of ICE and CPP32 in complex with an aldehyde inhibitor . The topology of the interface formed by the two p17/p12 heterodimers of CPP32 is different from that of ICE . This results in different orientations of CPP32 heterodimers compared with ICE heterodimers, which could affect substrate recognition . This structural information will be invaluable for the design of small synthetic inhibitors of CPP32 as well as for the design of CPP32 mutants.

J Biol Chem, 1997 Mar 7, 272(10), 6406 - 15
The majority of stem cell factor exists as monomer under physiological conditions . Implications for dimerization mediating biological activity; Hsu YR et al.; Soluble Escherichia coli-derived recombinant human stem cell factor (rhSCF) forms a non-covalently associated dimer . We have determined a dimer association constant (Ka) of 2-4 x 10(8) M-1, using sedimentation equilibrium and size exclusion chromatography . SCF has been shown previously to be present at concentrations of approximately 3.3 ng/ml in human serum . Based on the dimerization Ka, greater than 90% of the circulating SCF would be in the monomeric form . When 125I-rhSCF was added to human serum and the serum analyzed by size exclusion chromatography, 72-49% of rhSCF was monomer when the total SCF concentration was in the range of 10-100 ng/ml, consistent with the Ka determination . Three SCF variants, SCF(F63C), SCF (V49L,F63L), and SCF(A165C), were recombinantly expressed in Escherichia coli, purified, and characterized . The dimer Ka values, biophysical properties, and biological activities of these variants were studied . Dimerization-defective variants SCF(F63C)S-CH2CONH2 and SCF(V49L,F63L) showed substantially reduced mitogenic activity, while the activity of the Cys165-Cys165 disulfide-linked SCF(A165C) dimer was 10-fold higher than that of wild type rhSCF . The results suggest a correlation between dimerization affinity and biological activity, consistent with a model in which SCF dimerization mediates dimerization of its receptor, Kit, and subsequent signal transduction.

J Biol Chem, 1997 Mar 7, 272(10), 6361 - 9
Assembly and full functionality of recombinantly expressed dihydrolipoyl acetyltransferase component of the human pyruvate dehydrogenase complex; Yang D et al.; The dihydrolipoyl acetyltransferase (E2) component of mammalian pyruvate dehydrogenase complex (PDC) consists of 60 COOH-terminal domains as an inner assemblage and sequentially via linker regions an exterior pyruvate dehydrogenase (E1) binding domain and two lipoyl domains . Mature human E2, expressed in a protease-deficient Escherichia coli strain at 27 degrees , was prepared in a highly purified form . Purified E2 had a high acetyltransferase activity, was well lipoylated based on its acetylation, and bound a large complement of bovine E1 . Electron micrographs demonstrated that the inner core was assembled in the expected pentagonal dodecahedron shape with E1 binding around the inner core periphery . With saturating E1 and excess dihydrolipoyl dehydrogenase (E3) but no E3-binding protein (E3BP), the recombinant E2 supported the overall PDC reaction at 4% of the rate of bovine E2.E3BP subcomplex . The lipoates of assembled human E2 or its free bilipoyl domain region were reduced by E3 at rates proportional to the lipoyl domain concentration, but those of the E2.E3BP were rapidly used in a concentration-independent manner consistent with bound E3 rapidly using a set of lipoyl domains localized nearby . Given this restriction and the need for E3BP for high PDC activity, directed channeling of reducing equivalents to bound E3 must be very efficient in the complex . The recombinant E2 oligomer increased E1 kinase activity by up to 4-fold and, in a Ca2+-dependent process, increased phospho-E1 phosphatase activity more than 15-fold . Thus the E2 assemblage fully provides the molecular intervention whereby a single E2-bound kinase or phosphatase molecule rapidly phosphorylate or dephosphorylate, respectively, many E2-bound E1 . Thus, we prepared properly assembled, fully functional human E2 that mediated enhanced regulatory enzyme activities but, lacking E3BP, supported low PDC activity.

J Biol Chem, 1997 Mar 7, 272(10), 6318 - 23
Excretion and uptake of putrescine by the PotE protein in Escherichia coli; Kashiwagi K et al.; The structure and function of the polyamine transport protein PotE was studied . Uptake of putrescine by PotE was dependent on the membrane potential . In contrast, the putrescine-ornithine antiporter activity of PotE studied with inside-out membrane vesicles was not dependent on the membrane potential (Kashiwagi, K., Miyamoto, S., Suzuki, F., Kobayashi, H., and Igarashi, K . (1992) Proc . Natl . Acad . Sci . U . S . A . 89, 4529-4533) . The Km values for putrescine uptake and for putrescine-ornithine antiporter activity were 1.8 and 73 microM, respectively . Uptake of putrescine was inhibited by high concentrations of ornithine . This effect of ornithine appears to be due to putrescine-ornithine antiporter activity because it occurs only after accumulation of putrescine within cells and because ornithine causes excretion of putrescine . Thus, PotE can function not only as a putrescine-ornithine antiporter to excrete putrescine but also as a putrescine uptake protein . Both the NH2 and COOH termini of PotE were located in the cytoplasm, as determined by the activation of alkaline phosphatase and beta-galactosidase by various PotE-fusion proteins . The activities of putrescine uptake and excretion were studied using mutated PotE proteins . It was found that glutamic acid 207 was essential for both the uptake and excretion of putrescine by the PotE protein and that glutamic acids 77 and 433 were also involved in both activities . These three glutamic acids are located on the cytoplasmic side of PotE, and the function of these three residues could not be replaced by other amino acids . Putrescine transport activities did not change significantly with mutations at the other 13 glutamic acid or aspartic acid residues in PotE.

J Biol Chem, 1997 Mar 7, 272(10), 6285 - 90
Active site topologies and cofactor-mediated conformational changes of nitric-oxide synthases; Gerber NC et al.; The active site topologies of neuronal (nNOS), endothelial (eNOS), and inducible (iNOS) nitric-oxide synthases heterologously expressed in Escherichia coli have been examined using three aryldiazene (Ar-N=NH) probes . The topological information derives from (a) the rate and extent of aryl-iron complex formation in the presence and absence of tetrahydrobiopterin (H4B), Ca2+-dependent calmodulin (CaM), and L-arginine, and (b) the N-phenylprotoporphyrin IX regioisomer ratios obtained upon migration of the phenyl of the phenyl-iron complex to the heme nitrogen atoms . The N-phenylprotoporphyrin ratios indicate that the three NOS isoforms have related active site topologies with unencumbered space above all four pyrrole rings but particularly above pyrrole ring D . H4B binds directly above the heme pyrrole ring D or causes a conformational change that constricts that region, because H4B markedly decreases phenyl migration to pyrrole ring D . Small CaM-dependent changes in the nNOS N-phenylporphyrin isomer pattern are consistent with a conformational link between the CaM and heme sites in this protein . The ceiling height directly above the heme iron atom differs among the isoforms and is lower than in the P450 enzymes because only nNOS and iNOS react with 2-naphthyldiazene, and none of the isoforms reacts with p-biphenyldiazene . L-Arg blocks access to the heme iron atom in all three NOS isoforms and nearly suppresses the phenyldiazene reaction . The data indicate that topological differences, including differences in the size of the active site, are superimposed on the structural similarities among the NOS active sites.

J Biol Chem, 1997 Mar 7, 272(10), 6220 - 5
Conformational and functional differences between recombinant human lens alphaA- and alphaB-crystallin; Sun TX et al.; Human and other mammalian lens proteins are composed of three major crystallins: alpha-, beta-, and gamma-crystallin . alpha-Crystallin plays a prominent role in the supramolecular assembly required to maintain lens transparency . With age, the crystallins, especially alpha-crystallin, undergo posttranslational modifications that may disrupt the supramolecular assembly, and the lens becomes susceptible to other stresses resulting in cataract formation . Because these modifications occur even at a relatively young age, it is difficult to obtain pure, unmodified crystallins for in vitro experiments . alpha-Crystallin is composed of two subunits, alphaA and alphaB . Before the application of recombinant DNA technology, these two alpha-crystallin subunits were separated from calf lens in the denatured state and reconstituted by the removal of the denaturant, but they were not refolded properly . In the present studies, we applied the recombinant DNA technology to prepare native, unmodified alphaA- and alphaB-crystallins for conformational and functional studies . The expressed proteins from Escherichia coli are in the native state and can be studied directly . First, alphaA and alphaB cDNAs were isolated from a human lens epithelial cell cDNA library . The cDNAs were cloned into a pAED4 expression vector and then expressed in E . coli strain BL21(DE3) . Pure recombinant alphaA- and alphaB-crystallins were obtained after purification by gel filtration and DEAE liquid chromatography . They were subjected to conformational studies involving various spectroscopic measurements and an assessment of chaperone-like activity . alphaA- and alphaB-crystallins have not only different secondary structure, but also tertiary structure . 1-Anilino-8-naphthalene sulfonate fluorescence indicates that alphaB-crystallin is more hydrophobic than alphaA-crystallin . The chaperone-like activity, as measured by the ability to protect insulin aggregation, is about 4 times greater for alphaB- than for alphaA-crystallin . The resulting data provide a base line for further studies of human lens alpha-crystallin.

Arch Microbiol, 1997 Mar 7, 167(2/3), 143 - 50
The kil gene of the ColE1 plasmid of Escherichia coli controlled by a growth-phase-dependent promoter mediates the secretion of a heterologous periplasmic protein during the stationary phase
Miksch G, Fiedler E, Dobrowolski P, Friehs K.
Heterologous gene products produced by Escherichia coli cells can be exported into the culture medium by the action of the kil gene of the ColE1 plasmid, which encodes a bacterial release protein . The kil gene was fused with the stationary-phase promoter of the fic gene of E . coli, and a secretion cassette (Kil-Km cassette) containing the regulated kil gene, the Km-resistance gene, and multiple cloning sites for the integration of target genes was constructed . Using the gene for beta-glucanase (bgl) as a target gene, it was shown that the protein produced was only secreted into the medium during the stationary phase . Quasi-lysis and lethality were not observed . The primary effect of the induction of the kil gene was the overproduction of beta-glucanase . The total amount produced per milliliter of bacterial culture was almost threefold higher than that of the corresponding Kil- control . The protein pattern of periplasm and culture medium was analyzed before and after induction of the kil gene expression, indicating that the release of periplasmic proteins is semiselective . This secretion system is the first to use a growth-phase-regulated promoter for the expression of the kil gene.

Arch Microbiol, 1997 Mar 7, 167(2/3), 126 - 36
Requirement of a large K+-uptake capacity and of extracytoplasmic protease activity for protamine resistance of Escherichia coli
Stumpe S, Bakker EP.
The effect of protamine on growing cells of Escherichia coli K-12 strains containing different K+-uptake systems was investigated . Immediately after the addition of the toxic peptide, growth ceased and all strains lost most of their K+ . In addition, these cells released a significant amount of their ATP into the medium, and the cytoplasmic volume of these cells decreased by 70% . Whereas cells without rapid K+-uptake systems did not recover, cells containing either the Trk systems or the overproduced Kup system slowly reversed the effects of protamine, and growth resumed after the cells had reached their original volume . Experiments with a set of strains carrying mutations in the K+-uptake gene trkA showed a reasonably satisfactory correlation between inhibition of net K+ uptake and the lag time for resumption of growth after addition of protamine . Cells carrying mutations in three extracytoplasmic proteases were hypersusceptible to protamine, suggesting that the toxic peptide is degraded by these proteases . Data on the effect of a second addition of protamine suggest that protamine degradation activity is inducible . These data are interpreted to mean that reaccumulation of K+ by protamine-treated cells triggers recovery of the cells, thereby allowing induction of extracytoplasmic proteases . These, in turn, degrade protamine, leading to complete recovery of the cells and resumption of growth . Cells that cannot take up K+ rapidly remain metabolically compromised to such an extent that extracytoplasmic protease activity is not induced, leading to a prolonged susceptibility of the cells to the toxic peptide.

Biochem Biophys Res Commun, 1997 Mar 6, 232(1), 231 - 5
Expression, purification, and partial characterization of HCV RNA polymerase; Yuan ZH et al.; The product of the NS5B gene of Hepatitis C Virus (HCV) has been expressed in Escherichia Coli both as a fusion protein with glutathione-S-transferase (GST) of molecular weight 91 KDa and at high level as a single protein of molecular weight 65 KDa . The protein was sequestered within inclusion bodies and a variety of procedures designed to minimize inclusion body formation proved unsuccessful . The method finally adopted involved the purification of inclusion bodies followed by the solubilization, purification, and refolding of the expressed protein . A good recovery and protein purity of the order of 80-90% were achieved . The purified protein was shown to possess RNA polymerase activity in an assay using polyA/oligoU as template . The enzymatic activity is rifampicin resistant, poly A dependent, and requires Mg++ . The availability of purified HCV RNA polymerase will allow the study of viral replication and constitute the basis for testing new anti-viral drugs.

Biochem Biophys Res Commun, 1997 Mar 6, 232(1), 198 - 203
Molecular cloning and expression of cDNAs encoding rat brain and liver cytosolic long-chain acyl-CoA hydrolases; Yamada J et al.; cDNAs encoding the long-chain acyl-CoA hydrolases (ACHs) from rat brain and liver, referred to as rBACH and rLACH1, respectively, were isolated and sequenced . The rBACH cDNA contained an open reading frame encoding a 338-amino acid polypeptide with a calculated molecular weight of 37,559, of which the deduced amino acid sequence matched partial amino acid sequences directly determined for peptides generated by tryptic digestion or CNBr cleavage of purified rBACH . The rLACH1 cDNA contained an open reading frame encoding a 343-amino acid polypeptide with a molecular weight of 38,240 . When expressed in Escherichia coli, these cDNAs produced palmitoyl-CoA hydrolase activity and 44-kDa proteins with molecular masses similar to those of purified rBACH and rLACH1 (43 kDa) . These expressed proteins and enzyme activity were immunoblotted and neutralized, respectively, by anti-rBACH or anti-rLACH1 antibodies . rLACH1 cDNA had 84 and 94% identity with rBACH cDNA at the nucleotide and amino acid levels, respectively . However, the 5'-end of the former cDNA which contained the N-terminal coding region of rLACH1 was entirely different from the corresponding region of rBACH cDNA, suggesting that these enzymes may be generated by alternative use of exons of the same gene . Northern blot analysis showed that ACH mRNA was expressed constitutively in the rat brain and testis, whereas its expression in the liver was inducible by treatment with the peroxisome proliferator . This study demonstrated the molecular diversity of ACH and suggested the presence of tissue-specific mechanisms to regulate the ACH gene expression.

Biochem Biophys Res Commun, 1997 Mar 6, 232(1), 130 - 5
Conformational transition of DnaA protein by ATP: structural analysis of DnaA protein, the initiator of Escherichia coli chromosome replication; Kubota T et al.; DnaA protein binds to the chromosomal origin (oriC) to initiate DNA replication . We developed an efficient system for purification of DnaA protein which will facilitate physicochemical analysis of the protein . The yield of DnaA protein was increased at least 6-fold compared to an available method being used, and over 22 mg of the protein were obtained from only 100 g of cells . DnaA protein purified by this procedure showed an indistinguishable affinity for ATP, and activity for in vitro replication of oriC plasmid . The process of denaturation of DnaA protein, which was blocked by ATP, was monitored by intrinsic fluorescence and circular dichroism . Analysis of circular dichroism revealed that DnaA protein is rich in alpha-helices, and that ATP-binding leads to a significant transition of protein conformation in that the content of alpha-helices is decreased . This is the first evidence indicating that ATP-binding profoundly affects conformation of DnaA protein.

Nature, 1997 Mar 6, 386(6620), 91 - 4
Visualization of a 4-helix bundle in the hepatitis B virus capsid by cryo-electron microscopy; Conway JF et al.; Despite the development of vaccines, the hepatitis B virus remains a major cause of human liver disease . The virion consists of a lipoprotein envelope surrounding an icosahedral capsid composed of dimers of a 183-residue protein, 'core antigen' (HBcAg) . Knowledge of its structure is important for the design of antiviral drugs, but it has yet to be determined . Residues 150-183 are known to form a protamine-like domain required for packaging RNA, and residues 1-149 form the 'assembly domain' that polymerizes into capsids and, unusually for a capsid protein, is highly alpha-helical . Density maps calculated from cryo-electron micrographs show that the assembly domain dimer is T-shaped: its stem constitutes the dimer interface and the tips of its arms make the polymerization contacts . By refining the procedures used to calculate the map, we have extended the resolution to 9 A, revealing major elements of secondary structure . In particular, the stem, which protrudes as a spike on the capsid's outer surface, is a 4-helix bundle, formed by the pairing of alpha-helical hairpins from both subunits.

Mutat Res, 1997 Mar 4, 374(1), 21 - 40
Statistical analysis of the lacI transgenic mouse mutagenicity assay; Fung KY et al.; The transgenic mouse assay is now widely used to test chemicals for genotoxic potential . In this article, we consider statistical tests for increasing trend in mutant frequency with increasing dose, along with statistical models that may be used to describe the observed dose-response relationships . The application of these methods is illustrated using data on 2-acetylaminofluorene, di(2-ethylhexyl)phthalate, heptachlor, and sodium phenobarbital . No strong evidence of extra-binomial variation was detected at the plate level, but greater evidence was noted when the data were aggregated to the package or animal level in liver, necessitating the use of statistical methods that allow for overdispersion relative to binomial variation . Clear increase on mutant frequency induced by 2-acetylaminofluorene was detected in both liver and bladder, but no apparent trends were noted with di(2-ethylhexyl)phthalate, heptachlor, and sodium phenobarbital . The exponential model provides a good fit to the observed dose-response relationship in liver, whereas a Weibull model provides a better fit for bladder.

Biochemistry, 1997 Mar 4, 36(9), 2622 - 36
Effects of buried charged groups on cysteine thiol ionization and reactivity in Escherichia coli thioredoxin: structural and functional characterization of mutants of Asp 26 and Lys 57; Dyson HJ et al.; To investigate the role of Asp 26 and Lys 57, two conserved, buried residues, in the redox mechanism of Escherichia coli thioredoxin (Trx), three mutant proteins, Asp 26 --> Ala (D26A), Lys 57 --> Met (K57M), and the double mutant D26A/K57M, were prepared, replacing the charged amino acids with hydrophobic residues with similar sizes . Both the oxidized (Trx-S2) and reduced {Trx-(SH)2} forms of the mutant thioredoxins are fully folded and similar in overall structure to the wild-type protein (wt) . The structure of the active site hydrophobic surface is unchanged by the mutation of Asp 26 and Lys 57, since DNA polymerase activity in the 1:1 complex of the T7 gene 5 protein and mutant Trx-(SH)2 shows similar Kd values (approximately 5 nM) for both mutants and wt . In contrast, redox reactions involving thioredoxin as a catalyst of the reduction of disulfides or oxidation of dithiols are strongly affected by the mutations . In the reaction of Trx-S2 with thioredoxin reductase at pH 8.0, the kcat/Km value for the D26A mutant is decreased by a factor of 10 from that of wt, while the value for the D26A/K57M mutant is reduced 40-fold . The activity of Trx-(SH)2 as a protein disulfide reductase was measured with insulin, using fluorescence to detect oxidation of thioredoxin . At 15 degrees C and pH 8.0, both the D26A and K57M mutants showed 5--10-fold decreases in rates of reaction compared to those of the wild type, and the pH-rate profiles for the mutants were shifted 1 (K57M) and 2 (D26A) units to higher pH compared with the wt curve . NMR measurements for the three mutant proteins indicate that the proteins have the same global fold as that of the wild type, although changes in the chemical shifts of a number of resonances indicate local structural changes in the active site region . The resonances of oxidized D26A and D26A/K57M are pH-independent between pH 6.0 and 10.0, confirming the identification of the active site group titrating with a pKa of 7.5 in wt Trx-S2 as Asp 26 . A profound change in the pKa of Asp 26, from 7.5 in the wild type to 9.4 in the mutant, is observed for K57M Trx-S2 . The pH-dependent behavior of the resonances is affected in all mutant Trx-(SH)2 proteins . A single pKa shifted to higher values is observed on both the Cys 32 and Cys 35 Cbeta resonances . Ultraviolet absorbance measurements (A240) as a function of pH for wt Trx-(SH)2 demonstrate that the cysteine thiols titrate with apparent pK(a)s of about 7.1 and 9.9 . The mutant proteins each show a single transition in the A240 measurements, with a midpoint at pH 7.8-8.0, consistent with the NMR results . The change in absorbance at 240 nm with increasing pH indicates that the number of thiols titrating in each mutant is greater than one but less than two . It is clear that both thiol pK(a)s have been significantly shifted by the mutations . The Cys 32 pKa is moved from 7.1 in wt to 7.8-8.0 in the mutants . The value of the Cys 35 pKa either is indistinguishable from that of Cys 32, thus accounting for more than one thiol titrating in the UV absorbance measurements or else is shifted to much higher pHs (> 10) where its transition is masked in both UV and NMR measurements by the effects of ionization of the tyrosine residues and unfolding of the protein . Our results strongly suggest that the buried Asp 26 carboxyl and Lys 57 epsilon-amino groups significantly affect the pK(a)s of the active site thiols, particularly that of the exposed low-pKa thiol Cys 32, thereby enhancing the rates of thiol-disulfide reactions at physiological pH.

Biochemistry, 1997 Mar 4, 36(9), 2577 - 85
Solution structure of an RNA.2'-O-methylated RNA hybrid duplex containing an RNA.DNA hybrid segment at the center; Nishizaki T et al.; The solution structure of an RNA.2'-O-methylated RNA hybrid duplex containing an RNA.DNA hybrid segment at its center, (ggagaugac).(GmUmCmATCTCmCm), where lowercase letters, capital letters, and capital letters with the subscript m are RNA, DNA, and 2'-O-methylated RNA residues, respectively, was determined by observing the NMR spectra and performing the full relaxation matrix refinement . The 2'-O-methylation gives several characteristic features to oligoribonucleotides . In addition, this hybrid duplex is cleaved at a specific position by Escherichia coli ribonuclease HI, and so the role of the tertiary structure during the substrate recognition by the enzyme is of interest . The NOE connectivities among the proton resonances revealed that the duplex was a right handed helix . The 2'-O-methylated RNA segments had a typical C3'-endo conformation, and the 2'-O-methyl groups were directed to the minor groove of this duplex, taking the torsion angles phi (C1'-C2'-02'-CH3) that were all gauche(+) . The DNA residues in the central RNA.DNA hybrid duplex formed the C3'-endo conformation, except for the middle thymine residue . No remarkable structural discontinuities were observed around the junction sites at either the 5'- or 3'-end of the DNA . The overall structure was close to the typical A-form duplex.

Biochemistry, 1997 Mar 4, 36(9), 2544 - 9
Protein dissection of the antiparallel coiled coil from Escherichia coli seryl tRNA synthetase; Oakley MG et al.; The alpha-helices of coiled-coil proteins are predominantly parallel, in contrast to the general preference for an antiparallel orientation of interacting alpha-helices found in globular proteins . One intriguing exception is the antiparallel, two-stranded coiled coil comprising the long helical arm of the bacterial seryl tRNA synthetases (SRS) . A recombinant 82-residue peptide corresponding to the helical arm of Escherichia coli SRS folds into a stable, monomeric, helical structure in the absence of the rest of the protein, as shown by circular dichroism (CD) and equilibrium sedimentation centrifugation . However, peptides corresponding to the individual helices of SRS are unstructured at neutral pH and do not associate appreciably at total peptide concentrations up to 100 microM . Covalent attachment of the the two peptides through a nonnatural, disulfide-containing linker restores structure and allows study of variants in which the individual helices are constrained to interact in either an antiparallel or a parallel orientation . We find that the antiparallel species are substantially more helical and more stable to thermal denaturation than their parallel counterpart . Thus, the SRS helical arm is an autonomously folding unit, and, unlike most other coiled coils, has an intrinsic preference for an antiparallel orientation of its constituent helices.

Biochemistry, 1997 Mar 4, 36(9), 2517 - 30
Solution structure of the 30 kDa N-terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system by multidimensional NMR; Garrett DS et al.; The three-dimensional solution structure of the 259-residue 30 kDa N-terminal domain of enzyme I (EIN) of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli has been determined by multidimensional nuclear magnetic resonance spectroscopy . Enzyme I, which is autophosphorylated by phosphoenolpyruvate, reversibly phosphorylates the phosphocarrier protein HPr, which in turn phosphorylates a group of membrane-associated proteins, known as enzymes II . To facilitate and confirm NH, 15N, and 13C assignments, extensive use was made of perdeuterated 15N- and 15N/13C-labeled protein to narrow line widths . Ninety-eight percent of the 1H, 15N, and 13C assignments for the backbone and first side chain atoms of protonated EIN were obtained using a combination of double and triple resonance correlation experiments . The structure determination was based on a total of 4251 experimental NMR restraints, and the precision of the coordinates for the final 50 simulated annealing structures is 0.79 +/- 0.18 A for the backbone atoms and 1.06 +/- 0.15 A for all atoms . The structure is ellipsoidal in shape, approximately 78 A long and 32 A wide, and comprises two domains: an alpha/beta domain (residues 1-20 and 148-230) consisting of six strands and three helices and an alpha-domain (residues 33-143) consisting of four helices . The two domains are connected by two linkers (residues 21-32 and 144-147), and in addition, at the C-terminus there is another helix which serves as a linker between the N- and C-terminal domains of intact enzyme I . A comparison with the recently solved X-ray structure of EIN {Liao, D.-I., Silverton, E., Seok, Y.-J., Lee, B . R., Peterkofsky, A., & Davies, D . R . (1996) Structure 4, 861-872} indicates that there are no significant differences between the solution and crystal structures within the errors of the coordinates . The active site His189 is located in a cleft at the junction of the alpha and alpha/beta domains and has a pKa of approximately 6.3 . His189 has a trans conformation about chi1, a g+ conformation about chi2, and its Nepsilon2 atom accepts a hydrogen bond from the hydroxyl proton of Thr168 . Since His189 is thought to be phosphorylated at the N epsilon2 position, its side chain conformation would have to change upon phosphorylation.

Biochemistry, 1997 Mar 4, 36(9), 2450 - 8
De novo design of native proteins: characterization of proteins intended to fold into antiparallel, rop-like, four-helix bundles; Betz SF et al.; The de novo design and characterization of a series of 51-residue helix-turn-helix peptides intended to dimerize into antiparallel four-stranded coiled coils is described . The sequence is based on a coiled coil heptad repeat Ncap-(Aa Zb Zc Ld Ze Zf Zg)3-turn- (Xa Zb Zc Ld Ze Zf Zg)3-Ccap-CONH2, where X is either Val or Ala . The overall topology was intended to be similar to that found in the Escherichia coli protein ROP . The design strategy included consideration of geometric complementarity of the packing of side chains within the hydrophobic core as well as the use of specific interfacial interactions, both of which were intended to favor the desired ROP-like topology . Additionally, the sequence was designed to destabilize potential alternative structures that might compete with the desired topology . The peptides (RLP-1, RLP-2, and RLP-3) assemble into stable alpha-helical dimers and exhibit the hallmarks of a native protein as judged by its spectroscopic properties, and the lack of binding to hydrophobic dyes . Also, the enthalpy and heat capacity changes upon denaturation were determined by measuring the temperature dependence of the CD spectra and confirmed by differential scanning calorimetry (DSC) . The values determined by the two methods are in excellent agreement and are in the range of those of naturally occurring proteins of this size . These results suggest that it is now possible to design native-like helical proteins that should serve as templates for the further design of functional proteins.

Biochemistry, 1997 Mar 4, 36(9), 2425 - 38
Ribonuclease P catalysis requires Mg2+ coordinated to the pro-RP oxygen of the scissile bond; Chen Y et al.; Ribonuclease P (RNase P) is an essential enzyme whose action produces the mature 5' termini of all cellular and organellar transfer RNA molecules . In bacteria, the catalytic subunit of RNase P is an RNA molecule which by itself can bind substrate pre-tRNA, select and hydrolyze the correct phosphodiester bond, and release product tRNA . The simple requirements of the reaction-a monovalent cation such as K+ or NH4+ and the divalent cation Mg2+ (or Mn2+)-have prompted proposals that all aspects of phosphodiester bond hydrolysis might be accomplished by one or more divalent metal cations coordinated to the enzyme or substrate . To precisely localize the ligands of catalytically-involved Mg2+, we assayed cleavage by Escherichia coli RNase P RNA of pre-tRNA in which specific pro-Rp phosphate oxygens were replaced with sulfur . RNase P cleavage was targeted to that bond, at or nearest to the normal cleavage site, at which Mg2+ or Mn2+ could be coordinated . Single-turnover kinetics demonstrated that the apparent rate constant for the hydrolysis event was determined quantitatively by the affinity of the divalent cation (Mg2+ or Mn2+) for the atom (O or S) at the pro-Rp position of the scissile phosphodiester bond . We propose a model for pre-tRNA cleavage in which an essential Mg2+ ion is coordinated directly to the pro-Rp phosphate oxygen and indirectly to two other ligands near the scissile bond: the upstream ribose 2'-hydroxyl and the downstream purine N7 . This catalytic Mg2+ ion most likely positions and deprotonates a water molecule for in-line nucleophilic attack on the scissile bond phosphorus.

Proc Natl Acad Sci U S A, 1997 Mar 4, 94(5), 2085 - 90
The MUR1 gene of Arabidopsis thaliana encodes an isoform of GDP-D-mannose-4,6-dehydratase, catalyzing the first step in the de novo synthesis of GDP-L-fucose; Bonin CP et al.; GDP-L-fucose is the activated nucleotide sugar form of L-fucose, which is a constituent of many structural polysaccharides and glycoproteins in various organisms . The de novo synthesis of GDP-L-fucose from GDP-D-mannose encompasses three catalytic steps, a 4,6-dehydration, a 3,5-epimerization, and a 4-reduction . The mur1 mutant of Arabidopsis is deficient in L-fucose in the shoot and is rescued by growth in the presence of exogenously supplied L-fucose . Biochemical assays of the de novo pathway for the synthesis of GDP-L-fucose indicated that mur1 was blocked in the first nucleotide sugar interconversion step, a GDP-D-mannose-4,6-dehydratase . An expressed sequence tag was identified that showed significant sequence similarity to proposed bacterial GDP-D-mannose-4,6-dehydratases and was tightly linked to the mur1 locus . A full-length clone was isolated from a cDNA library, and its coding region was expressed in Escherichia coli . The recombinant protein exhibited GDP-D-mannose-4,6-dehydratase activity in vitro and was able to complement mur1 extracts in vitro to complete the pathway for the synthesis of GDP-L-fucose . All seven mur1 alleles investigated showed single point mutations in the coding region for the 4,6-dehydratase, confirming that it represents the MUR1 gene.

Proc Natl Acad Sci U S A, 1997 Mar 4, 94(5), 1761 - 6
Site-specific mutagenesis reveals differences in the structural bases for tight binding of RNase inhibitor to angiogenin and RNase A; Chen CZ et al.; RNase inhibitor (RI) binds with extraordinary affinity (Ki approximately 10(-13)-10(-16) M) to diverse proteins in the pancreatic RNase superfamily . In the present study, the structural basis for the recognition of two RI ligands, human angiogenin (Ang) and bovine RNase A, has been investigated by site-specific mutagenesis of human RI and Ang . The RI residues examined were those that appear to form strong contacts with RNase A in the crystal structure of the porcine RI x RNase A complex {Kobe, B . & Deisenhofer, J . (1995) Nature (London) 374, 183-186} that would not be replicated in the Ang complex . Ala substitutions of five of these residues (Glu-287, Lys-320, Glu-401, Cys-408, and Arg-457) were found to have little or no effect on binding of RNase A . In contrast, replacements of Tyr-434, Asp-435, and Tyr-437 and deletion of the C-terminal residue Ser-460 substantially weakened affinity for RNase A: the losses of binding energy associated with the mutations were 5.9, 3.6, 2.6, and 3.5 kcal/mol, respectively . Thus these four residues, which are neighbors in the tertiary structure, appear to constitute a "hot spot" for the RNase A interaction . However, only one of them, Asp-435, was equally important for binding of Ang; the Ki increases produced by mutations of the others were 20- to 93-fold smaller for Ang than for RNase A . Consequently, Tyr-434 plays a significant but lesser role in the Ang complex, whereas Tyr-437 and Ser-460 make only minor contributions . Ala mutations of four Ang residues (His-8, Gln-12, Asn-68, and Glu-108) that correspond to RI contacts on RNase A produced no major changes in affinity for RI . These findings indicate that RI uses largely different interactions to achieve its extremely tight binding of RNase A and Ang.

Proc Natl Acad Sci U S A, 1997 Mar 4, 94(5), 1755 - 60
Transcriptional arrest: Escherichia coli RNA polymerase translocates backward, leaving the 3' end of the RNA intact and extruded; Komissarova N et al.; RNA polymerase (RNAP) may become arrested during transcript elongation when ternary complexes remain intact but further RNA synthesis is blocked . Using a combination of DNA and RNA footprinting techniques, we demonstrate that the loss of catalytic activity upon arrest of Escherichia coli RNAP is accompanied by an isomerization of the ternary complex in which the enzyme disengages from the 3' end of the transcript and moves backward along the DNA with concomitant reverse threading of the intact RNA through the enzyme . The reversal of RNAP brings the active center to the internal RNA position and thereby it represents a step in factor-facilitated transcript cleavage . Secondary structure elements or the 5' end of the transcript can prevent the isomerization by blocking the RNA threading . The described novel property of RNAP has far-reaching implications for the understanding of the elongation mechanism and gene regulation.

Proc Natl Acad Sci U S A, 1997 Mar 4, 94(5), 1745 - 8
Cytohesin-1, a cytosolic guanine nucleotide-exchange protein for ADP-ribosylation factor; Meacci E et al.; Cytohesin-1, a protein abundant in cells of the immune system, has been proposed to be a human homolog of the Saccharomyces cerevisiae Sec7 gene product, which is crucial in protein transport . More recently, the same protein has been reported to be a regulatory factor for the alphaLbeta2 integrin in lymphocytes . Overexpression of human or yeast ADP-ribosylation factor (ARF) genes rescues yeast with Sec7 defects, restoring secretory pathway function . ARFs, 20-kDa guanine nucleotide-binding proteins initially identified by their ability to stimulate cholera toxin ADP-ribosyltransferase activity and now recognized as critical components in intracellular vesicular transport, exist in an inactive cytosolic form with GDP bound (ARF-GDP) . Interaction with a guanine nucleotide-exchange protein (GEP) accelerates exchange of GDP for GTP, producing the active ARF-GTP . Both soluble and particulate GEPs have been described . To define better the interaction between ARF and Sec7-related proteins, effects of cytohesin-1, synthesized in Escherichia coli, on ARF activity were evaluated . Cytohesin-1 enhanced binding of 35S-labeled guanosine 5'-{gamma-thio}triphosphate {35S}GTP{gammaS} or {3H}GDP to ARF purified from bovine brain (i.e., it appeared to function as an ARF-GEP) . Addition of cytohesin-1 to ARF3 with {35S}GTP{gammaS} bound, accelerated {35S}GTP{gammaS} release to a similar degree in the presence of unlabeled GDP or GTP{gammaS} and to a lesser degree with GDP{betaS}; release was negligible without added nucleotide . Cytohesin-1 also increased ARF1 binding to a Golgi fraction, but its effect was not inhibited by brefeldin A (BFA), a drug that reversibly inhibits Golgi function . In this regard, it differs from a recently reported BFA-sensitive ARF-GEP that contains a Sec7 domain.

Proc Natl Acad Sci U S A, 1997 Mar 4, 94(5), 1733 - 8
Cloning of murine RNA polymerase I-specific TAF factors: conserved interactions between the subunits of the species-specific transcription initiation factor TIF-IB/SL1; Heix J et al.; Promoter selectivity for all three classes of eukaryotic RNA polymerases is brought about by multimeric protein complexes containing TATA box binding protein (TBP) and specific TBP-associated factors (TAFs) . Unlike class II- and III-specific TBP-TAF complexes, the corresponding murine and human class I-specific transcription initiation factor TIF-IB/SL1 exhibits a pronounced selectivity for its homologous promoter . As a first step toward understanding the molecular basis of species-specific promoter recognition, we cloned the cDNAs encoding the three mouse pol I-specific TBP-associated factors (TAFIs) and compared the amino acid sequences of the murine TAFIs with their human counterparts . The four subunits from either species can form stable chimeric complexes that contain stoichiometric amounts of TBP and TAFIs, demonstrating that differences in the primary structure of human and mouse TAFIs do not dramatically alter the network of protein-protein contacts responsible for assembly of the multimeric complex . Thus, primate vs . rodent promoter selectivity mediated by the TBP-TAFI complex is likely to be the result of cumulative subtle differences between individual subunits that lead to species-specific properties of RNA polymerase I transcription.

Proc Natl Acad Sci U S A, 1997 Mar 4, 94(5), 1709 - 14
The two alpha subunits of Escherichia coli RNA polymerase are asymmetrically arranged and contact different halves of the DNA upstream element; Murakami K et al.; RNA polymerase core enzyme of Escherichia coli is composed of two alpha subunits and one each of the beta and beta' subunits . The C-terminal domain of the RNA polymerase alpha subunit plays a key role in molecular communications with class I transcription factors and upstream (UP) elements of promoter DNA, using the same protein surface . To identify possible differences in the functional roles of the two alpha subunits, we have developed a reconstitution method for hybrid RNA polymerases containing two distinct alpha subunit derivatives in a defined orientation ("oriented alpha-heterodimer") . The binding sites of two alpha C-terminal domains on the UP element DNA were determined by hydroxyl radical-based DNA cleavage mediated by (p-bromoacetamidobenzyl)-EDTA x Fe, which was bound at Cys-269 on the UP recognition surface of one or both alpha subunits . The results clearly indicated that the two alpha subunits bind in tandem to two helix turns of the rrnBP1 UP element, and that the beta'-associated alpha subunit is bound to the promoter-distal region.

Proc Natl Acad Sci U S A, 1997 Mar 4, 94(5), 1663 - 8
"Peptabody": a new type of high avidity binding protein; Terskikh AV et al.; A new type of high avidity binding molecule, termed "peptabody" was created by harnessing the effect of multivalent interaction . A short peptide ligand was fused via a semi-rigid hinge region with the coiled-coil assembly domain of the cartilage oligomeric matrix protein, resulting in a pentameric multivalent binding molecule . In the first peptabody (Pab-S) described here, a peptide (S) specific for the mouse B-cell lymphoma BCL1 surface Ig idiotype, was selected from a phage display library . A fusion gene was constructed encoding peptide S, followed by the 24 aa hinge region from camel IgG and a modified 55 aa cartilage oligomeric matrix protein pentamerization domain . The Pab-S fusion protein was expressed in Escherichia coli in a soluble form at high levels and purified in a single step by metal-affinity chromatography . Pab-S specifically bound the BCL1 surface idiotype with an avidity of about 1 nM, which corresponds to a 2 x 10(5)-fold increase compared with the affinity of the synthetic peptide S itself . Biochemical characterization showed that Pab-S is a stable homopentamer of about 85 kDa, with interchain disulfide bonds . Pab-S can be dissociated under denaturing and reducing conditions and reassociated as a pentamer with full-binding activity . This intrinsic feature provides an easy way to combine Pab molecules with two different peptide specificities, thus producing heteropentamers with bispecific and/or chelating properties.

Proc Natl Acad Sci U S A, 1997 Mar 4, 94(5), 1651 - 6
Gene transfer by guanidinium-cholesterol cationic lipids into airway epithelial cells in vitro and in vivo; Oudrhiri N et al.; Synthetic vectors represent an attractive alternative approach to viral vectors for gene transfer, in particular into airway epithelial cells for lung-directed gene therapy for cystic fibrosis . Having recently found that guanidinium-cholesterol cationic lipids are efficient reagents for gene transfer into mammalian cell lines in vitro, we have investigated their use for gene delivery into primary airway epithelial cells in vitro and in vivo . The results obtained indicate that the lipid bis(guanidinium)-tren-cholesterol (BGTC) can be used to transfer a reporter gene into primary human airway epithelial cells in culture . Furthermore, liposomes composed of BGTC and dioleoyl phosphatidylethanolamine (DOPE) are efficient for gene delivery to the mouse airway epithelium in vivo . Transfected cells were detected both in the surface epithelium and in submucosal glands . In addition, the transfection efficiency of BGTC/DOPE liposomes in vivo was quantitatively assessed by using the luciferase reporter gene system.

Virology, 1997 Mar 3, 229(1), 279 - 82
Demonstration of equine herpesvirus-1 neuronal latency in murine olfactory bulbs using a novel combined in situ PCR and protein synthesis method; Marshall KR et al.; Equine herpesvirus-1 (EHV-1) latency in murine olfactory bulbs was demonstrated by a novel combined in situ PCR and in vitro protein synthesis method (in situ PS-PCR) . The Escherichia coli lacZ gene replacing a deletion in EHV-1 gene 71 (EUS4) was thus amplified and transcribed/translated in situ followed by enzymatic detection using X-Gal (5-bromo-4-chloro-3-indoyl-beta-D-galactopyranoside) . beta-Galactosidase was found to be concentrated over mitral/tufted neurons indicating those to be the sites of latency . Our results suggest that, in common with other alpha-herpesviruses, EHV-1 can establish latency in central nervous system neurons and that the unique membrane glycoprotein encoded by EHV-1 gene 71 is nonessential for infection of neural tissues.

Virology, 1997 Mar 3, 229(1), 201 - 11
Characterization of the African swine fever virus structural protein p14.5: a DNA binding protein; Martinez-Pomares L et al.; The gene encoding the structural protein p14.5 of African swine fever virus (ASFV) has been mapped and sequenced . This gene, designated E120R, is located in the Sa/l H/EcoRl E restriction fragment of the ASFV genome and is predicted to encode a protein of 120 amino acids with a molecular weight of 13.4 kDa . Northern-blot analysis showed that E120R is transcribed at late times during the viral replication cycle . The E120R gene product has been expressed in Escherichia coli, purified, and used as an antigen for antibody production . The antiserum anti-pE120R recognized a protein in infected cell extracts with an apparent molecular mass of 14.5 kDa, named p14.5 . This antiserum also detected protein p14.5 in purified virus particles . Protein p14.5 is synthesized late in infection and is located in viral factories . Immunoprecipitation analysis and binding-assay experiments have shown that protein p14.5 interacts with a protein that could correspond to the major structural protein p72 . Purified protein p14.5 interacts with DNA in a sequence-independent manner . It binds to both single-stranded and double-stranded DNA . A possible role of protein p14.5 in the encapsidation of ASFV DNA is suggested.

EMBO J, 1997 Mar 3, 16(5), 989 - 97
1.9 A crystal structure of interleukin 6: implications for a novel mode of receptor dimerization and signaling; Somers W et al.; Interleukin 6 (IL-6) has many biological activities in vivo, and deregulation has been implicated in many disease processes . IL-6, a 185 amino acid polypeptide was refolded, purified and crystallized . The crystals diffracted to beyond 1.9 A and the structure was solved using single isomorphous replacement . The X-ray structure of IL-6 is composed of a four helix bundle linked by loops and an additional mini-helix . 157 out of 185 residues are well defined in the final structure, with 18 N-terminal and 8 A-B loop amino acids displaying no interpretable electron density . The three-dimensional structure has been used to construct a model of IL-6 interacting with the IL-6 receptor (alpha-chain) and gp130 (beta-chain) that gives new insight into the process of molecular recognition and signaling . Based on this model, we predict a fourth binding site on IL-6, a low affinity IL-6-IL-6 interaction, which may be necessary for the sequential assembly of a functional hexameric IL-6 receptor complex.

FEBS Lett, 1997 Mar 3, 404(1), 65 - 9
Rab proteins of Drosophila melanogaster: novel members of the Rab-protein family; Satoh AK et al.; From a Drosophila head cDNA library, we isolated 9 cDNA clones, each of which encodes a different member of Rab-protein family . Seven of them (DRabs) have more than 80% amino acid identity to the corresponding members of mammalian Rab proteins . The other two proteins (DRabRP3 and 4) show low sequence similarity to any of the known Rab proteins . However, both contain all amino acids conserved in known Rabs . In addition, DRabRP4 has strong GTP-binding activity, when synthesized in E . coli cells . These results indicate that DRabRPs are novel members of the Rab-protein family . Molecular phylogenetic analysis also supported this conclusion.

FEBS Lett, 1997 Mar 3, 404(1), 45 - 50
Secondary structure of the IIB domain of the Escherichia coli mannose transporter, a new fold in the class of alpha/beta twisted open-sheet structures; Gschwind RM et al.; The mannose transporter of the Escherichia coli bacterial phosphotransferase system consists of three subunits: IIAB, IIC and IID . IIABMan transfers phosphoryl groups to the transported substrate via phosphohistidine intermediates . Its IIB domain was overexpressed and isotopically labelled with 13C, 15N and 2H . Heteronuclear 3D triple-resonance NMR experiments combined with a semi-automatic assignment procedure yielded the sequential assignment of the 1H, 13C and 15N backbone resonances . Based on the evaluation of conformationally sensitive parameters, the secondary structure of the IIBMan domain has been determined as an alpha/beta twisted open-sheet structure consisting of a six-stranded parallel beta-sheet with the novel strand order 3-2-4-1-5-6, six helices and a short two-stranded antiparallel beta-sheet.

FEBS Lett, 1997 Mar 3, 404(1), 15 - 8
Nucleotide occupancy of F1-ATPase catalytic sites under crystallization conditions; Lobau S et al.; Using site-directed tryptophan fluorescence we studied nucleotide occupancy of the catalytic sites of Escherichia coli F1-ATPase, under conditions used previously for crystallization and X-ray structure analysis of the bovine mitochondrial enzyme {Abrahams et al . (1994) Nature 370, 621-628} . We found that only two of the three catalytic sites were filled in the E . coli enzyme under these conditions (250 microM MgAMPPNP plus 5 microM MgADP), consistent with what was reported in the bovine F1 X-ray structure . However, subsequent addition of a physiological concentration of MgATP readily filled the third catalytic site . Therefore the enzyme form seen in the X-ray structure results from the fact that it is obtained under sub-saturating nucleotide conditions . The data show that the X-ray structure is compatible with a catalytic mechanism in which all three F1-ATPase catalytic sites must fill with MgATP to initiate steady-state hydrolysis {e.g . Weber and Senior (1996) Biochim . Biophys . Acta 1275, 101-104} . The data further demonstrate that the site-directed tryptophan fluorescence technique can provide valuable support for F1 crystallography studies.

Cytokines Cell Mol Ther, 1997 Mar, 3(1), 27 - 32
Incidence of antibodies to interferon-beta in patients treated with recombinant human interferon-beta 1a from mammalian cells; Abdul-Ahad AK et al.; Patients receiving recombinant human interferon-beta 1a (IFN-beta 1a) produced in Chinese hamster ovary (CHO) cells were tested for the formation of neutralizing antibodies (NABs) to IFN-beta . Samples were tested in an enzyme-linked immunosorbent assay (ELISA), and if positive were then tested for neutralization of antiviral activity in an IFN-beta bioassay . A total of 793 patients with viral diseases, premalignant and malignant diseases, and multiple sclerosis received IFN-beta 1a in clinical studies . Long-term studies included 56 patients with cancer treated for 6 or 12 months and 334 patients with multiple sclerosis (MS) at the end of one year of treatment . All of the NAB-positive patients were found in the latter . Positivity in a single specimen was found in 14.4% of the MS patients . The incidence of sustained neutralizing antibody titres (i.e . positive in two tests at least 6 months apart) was 6.9% in this group . Comparison with results from other studies suggests that CHO-derived IFN-beta 1a induces less neutralizing antibody than IFN-beta 1b produced in E . coli.

Biochemistry (Mosc), 1997 Mar, 62(3), 337 - 41
Stability and stabilization of recombinant peroxidase in reversed micelles; Klyachko NL et al.; Stability of recombinant peroxidase lacking carbohydrate residues on the surface of the protein molecule has been characterized in reversed micelles of Aerosol OT in octane . The enzyme stability was found to depend on the surfactant hydration degree (w0 = {H2O}/{AOT}) . Residual activity after 1 h incubation dropped to zero at w0 = 7 but was 54% at w0 = 25 . However, the residual activity levels at all values of hydration degree were definitely low compared to that of glycosylated wild-type horseradish peroxidase . The stability of the enzyme apparently depends on the presence of carbohydrate residues . Stabilization of recombinant peroxidase in reversed micellar system involved sugar-containing co-surfactants such as Tweens and Spans is proposed . As an example, addition of 1 mM Span 80 (1% relative to AOT concentration) increased the recombinant peroxidase stability up to that of wild-type peroxidase.

Biochemistry (Mosc), 1997 Mar, 62(3), 233 - 6
A hybrid mutant form of Escherichia coli inorganic pyrophosphatase; Velichko IS et al.; The inorganic pyrophosphatase of Escherichia coli is a tightly hexamer of identical subunits . Upon interaction of its two mutant forms in which the trimer-trimer contacts were weakened because of E20D and H136Q substitutions, a hybrid hexameric E20D/H136Q-PPase is formed . The catalytic activity of its constituent H136Q trimer is same of its hexamer, whereas metal-binding affinity is significantly decreased . These results point to an interdependence of two trimers in catalysis by hexameric pyrophosphatase.

J Magn Reson, 1997 Mar, 125(1), 34 - 42
Protein heteronuclear NMR assignments using mean-field simulated annealing; Buchler NE et al.; A computational method for the assignment of the NMR spectra of larger (21 kDa) proteins using a set of six of the most sensitive heteronuclear multidimensional nuclear magnetic resonance experiments is described . Connectivity data obtained from HNC alpha, HN(CO)C alpha, HN(C alpha)H alpha, and H alpha (C alpha CO)NH and spin-system identification data obtained from CP-(H)CCH-TOCSY and CP-(H)C(C alpha CO)NH-TOCSY were used to perform sequence-specific assignments using a mean-field formalism and simulated annealing . This mean-field method reports the resonance assignments in a probabilistic fashion, displaying the certainty of assignments in an unambiguous and quantitative manner . This technique was applied to the NMR data of the 172-residue peptide-binding domain of the E . coli heat-shock protein, DnaK . The method is demonstrated to be robust to significant amounts of missing, spurious, noisy, extraneous, and erroneous data.

Mikrobiologiia, 1997 Mar-Apr, 66(2), 179 - 84
{Release of protein into the extracellular space as a nonspecific response to stress in Escherichia coli}; Roshchina EK et al.; This paper is concerned with the kinetics of excretion of fluorescent tryptophan-containing proteins from Escherichia coli cells kept in physiological saline at room temperature or incubated at 42, 48, and 55 degrees C . The kinetic curves of the extracellular concentration of protein can be described by parameters T and C, where C is the stationary extracellular concentration of the protein and T is the time in which the given concentration is reached . T was found to be a variable, and C was a constant independent of the type and strength of the stress . During the protein release, the viability of the cells was maintained at the initial level, but, after the concentration of the protein reached a stationary value, the culture cells died exponentially . All this allows the protein release into extracellular medium to be considered as a nonspecific response of E . coli to stress . The protein excretion was analyzed with reference to the data on the kinetics of release of other UV-absorbing compounds from the cells.

Int J Biochem Cell Biol, 1997 Mar, 29(3), 485 - 91
Recombinant rat liver S-adenosyl-L-methionine synthetase tetramers and dimers are in equilibrium; Mingorance J et al.; Rat liver S-adenosyl-L-methionine synthetase is present in two oligomeric forms, tetramers and dimers, with different substrate kinetics and regulation . In vivo the relative amounts of both forms may change in some instances . The basis of this regulatory mechanism is not known . When rat liver cDNA was used to express the protein in Escherichia coli the two oligomeric forms were found . Gel filtration chromatography of the purified recombinant enzyme suggested that these two isoforms might be in equilibrium . This was confirmed by kinetic experiments which showed that the specific activity of the enzyme was dependent on the protein concentration . From these experiments, apparent equilibrium constants of (5.6 +/- 0.4) x 10(5) M-1 and (3.5 +/- 0.9) x 10(5) M-1 were obtained at 2mM and 60 microM methionine concentrations, respectively . Using hydrophobic chromatography on phenyl-Sepharose to separate the tetrameric and dimeric forms, an equilibrium constant of (4.9 +/- 0.7) x 10(5) M-1 was calculated . A rate constant for the dissociation of the tetramer of k-1 = (8.1 +/- 0.4) x 10(-4) s-1 at 4 degrees C was also calculated using the same approach . In summary, we have shown that the rat liver S-adenosyl-L-methionine synthetase produced in bacterial cells is present in two oligomeric forms, tetramers and dimers, which are in equilibrium . This system might be useful for studying the dynamics and the regulation of the distribution of oligomeric forms in the mammalian liver.

Biol Trace Elem Res, 1997 Mar, 56(3), 295 - 309
Enhancement of adriamycin toxicity by iron chelates is not a free radical mechanism; Gelvan D; The possible involvement of metal ions and free radicals in the cytotoxic mechanism of Adriamycin (ADR) was investigated, using a model system of Escherichia coli cells . It is shown that E . coli mediated the production of free radicals under anaerobic (ADR-semiquinone) and aerobic (superoxide) conditions . ADR-induced loss of colony-forming ability was enhanced by the addition of iron (Fe) chelates . These observations suggested that a Fenton-type free radical mechanism was responsible for ADR toxicity . However, the mortality rate was essentially unchanged by the exclusion of oxygen . It was also unaffected by the addition of H2O2, catalase, or chelating agents . Cu(II), Zn(II) or Mg(II) had no effect on ADR toxicity . ADR and iron chelates did not induce measurable amounts of DNA strand-breaks . These observations suggest a mechanism of ADR-induced cell killing that is enhanced by Fe chelates, but does not directly involve oxygen-derived free radicals.

Bioorg Khim, 1997 Mar, 23(3), 200 - 4
{Regulation of translation of the distal lacZ gene in polycistronic mRNA by the ribosome stream from the proximal gene}; Nikolenko GN et al.; Four series of plasmids (pNSI, pNSII, pNLI, and pNLII) with artificial polycistrons containing the lacZ test gene were constructed . These plasmids coded for polycistronic mRNAs with two different types of cistron (orfZ and lacZ) coupling: in pNSI and pNLI, the orfZ termination codon and the lacZ initiation codon overlapped (type I); in pNSII and pNLII, the orfZ termination codon, was located upstream of the lacZ SD sequence . The length of the orfZ cistron was 60 bp in pNSI and pNSII or 300 bp in pNLI and pNLII . Plasmids with the same type of cistron coupling contained the same lacZ translation initiation region, whereas the structure of the orfZ translation initiation region varied, thereby providing varying efficiency of the orfZ gene translation . The effect of these variations on the efficiency of the lacZ gene translation was evaluated by direct measurement of the beta-galactosidase activity in Escherichia coli cells transformed with the corresponding plasmids . We found that the level of translation of the distal lacZ gene depended on the ribosome stream from the proximal gene and was maximal at the optimal ribosome stream level, which, in turn, depended on the type of cistron coupling.

Bioorg Khim, 1997 Mar, 23(3), 183 - 90
{Cloning and expression of cDNA for tobacco rab1, and structural-functional analysis of the protein}; Andreeva AV et al.; rab1 cDNA coding for a small GTP binding protein Rab1 was isolated from cDNA library of tobacco (Nicotiana tabacum) leaves . The primary structure of this protein was deduced from the rab1 structure . Tobacco rab1 cDNA was expressed in Escherichia coli, and the product was purified and shown to exhibit GTPase activity . A set of Rab1 mutants with altered GTP binding and/or GTPase activities was obtained . Polyclonal antipeptide antibodies were raised against a sequence in the C-terminal region of the tobacco Rab1 capable of recognizing this protein.

C R Acad Sci III, 1997 Mar, 320(3), 207 - 14
Human deoxycytidine kinase as a conditional mutator in Escherichia coli; Bouzon M et al.; The chemical diversification of DNA precursors was undertaken in Escherichia coli by expressing the human gene for deoxycytidine kinase, and supplying such recombinant strains with nucleoside analogues bearing an altered base or sugar . Arabinocytidine and dideoxycytidine thus became highly toxic to E . coli in the sub-millimolar range . Deoxynucleosides bearing isoadenine (2-aminopurine) and isoguanine (2-hydroxy-6-aminopurine) showed a high mutagenic potency towards the recombinant strains, to an extent comparable to that of the most efficient mutator alleles (dnaQ) . These findings open the way to the propagation of chemically remodelled nucleic acids and to the controlled hypermutagenesis of plasmids in vivo.

Biol Chem, 1997 Mar-Apr, 378(3-4), 321 - 6
Mode of action of cystathionine beta-lyase; Clausen T et al.; Cystathionine beta-lyase (CBL) is a member of the gamma-family of pyridoxal-5'-phosphate (PLP)-dependent enzymes (Alexander et al., 1994) that cleave C(beta,gamma)-S bonds of a broad variety of substrates . Recently, we reported the X-ray crystal structures of CBL and the CBL-trifluoroalanine inactivation complex at 1.83 A and 2.3 A resolution, respectively . The structures explicitly reveal the cofactor and substrate binding pockets . Spectral analysis of substrate turnover indicates a change of hydrophobicity in the microenvironment of the aldimine bond . In combination with further spectroscopic data, crystallographic evidence permits the formulation of a likely reaction mechanism.

Biol Chem, 1997 Mar-Apr, 378(3-4), 131 - 40
New insights into the mechanisms and importance of the proteasome in intracellular protein degradation; Goldberg AL et al.; Recent studies of the 20S proteasome from Thermoplasma acidophilum have uncovered some fundamental new properties of its catalytic mechanism . Unlike conventional proteases, 20S and 26S proteasomes degrade protein substrates in a highly processive fashion . They cleave a protein substrate to small peptides before attacking another substrate molecule . This processive behavior is an inherent feature of the 20S particle not requiring cofactors or ATP hydrolysis . Recently, we have described a proteasome-like particle, HslVU, in Escherichia coli . HslVU is a two-component ATP-dependent protease composed of the proteasome-related peptidase HslV (beta-subunit) and the ATPase HslU . In active HslVU complex, cleavage of small peptides and proteins requires the presence of ATP . EM analysis revealed that HslV and HslU are both ring-shaped particles and that the active HslVU complex is a cylindrical four-ring structure, composed of HslV, a two-ring dodecamer, sandwiched between HslU rings . Elucidation of its mode of action may help us understand the role of ATP in function of the 26S proteasome . Several proteasome-specific inhibitors have been recently identified which block the function of proteasome in vivo . These agents have proven very useful to clarify the intracellular function of the proteasome . In mammalian cells, both the rapid degradation of short-lived regulatory proteins and of abnormal polypeptides and the slower degradation of long-lived proteins are blocked by these agents . Thus, in mammalian cells, the proteasome is the site for the degradation of most cell proteins . In contrast, in budding yeast, proteasome inhibitors block the degradation of short-lived proteins but not the breakdown of long-lived proteins, which can be blocked by inhibitors of vacuolar proteases . The inhibition of proteasome function in yeast and mammalian cells, presumably by causing an accumulation of unfolded proteins, triggers the expression of heat shock proteins and concomitantly increases cell resistance to high temperature and various toxic insults.

J Radiat Res (Tokyo), 1997 Mar, 38(1), 37 - 43
Effects of 60Co gamma-rays, ultraviolet light, and mitomycin C on Halobacterium salinarium and Thiobacillus intermedius; Shahmohammadi HR et al.; Lethal effects of 60Co gamma-rays, UV light, and mitomycin C on two kinds of bacteria, Halobacterium salinarium which grows in highly concentrated salt media and Thiobacillus intermedius which requires reduced sulfur compounds, were studied and compared with those on Escherichia coli B/r . D37 values for H . salinarium, T . intermedius and E . coli B/r were 393, 150, and 92 Gy, respectively, by exposure to 60Co gamma-rays . They were 212, 38, and 10 J/m2, respectively, by exposure to UV light and 2.36, 0.25, and 0.53 microgram/ml/h, respectively, by exposure to mitomycin C . Against these agents, H . salinarium was much more resistant than T . intermedius and E . coli B/r.

J Radiat Res (Tokyo), 1997 Mar, 38(1), 27 - 36
Spectrum of spontaneous mutations in the cyclic AMP receptor protein gene on chromosomal DNA of Escherichia coli; Takimoto K et al.; We determined 46 spontaneous mutations occurring in the cyclic AMP receptor protein gene (crp) on the chromosomal DNA of Escherichia coli by the use of PCR cloning . Of 24 base substitutions, 17 were transversions and 7 transitions including all types of base substitutions . The frequency of the changes of A:T base pairs was similar to that of G:C pairs, suggesting that A:T pair is also a target for base substitution . Frameshifts including seven-1 and four +1 frameshifts occurred at the sites of a run of identical bases . Deletions extending 18 and 172 bases occurred at the sites where the deleted sequences were flanked by short repeated sequences at the junction . The insertions of IS2 element or its inverted sequence were detected in two and six mutations, respectively . The assay system of the mutation used here is available for the determination of the mutational spectrum of base substitutions.

Immunotechnology, 1997 Mar, 3(1), 45 - 59
Humanization of an antibody recognizing a breast cancer specific epitope by CDR-grafting; Fiorentini S et al.; BACKGROUND: Muc1-H23 is a cell surface mucin that is expressed on normal breast luminal epithelial cells and over-expressed in most breast tumors . In addition, Muc-1 expressed by malignant cells is glycosylated differently than Muc-1 expressed by normal cells . This difference in glycosylation exposes a peptide epitope on malignant cells which is not exposed on normal cells . Murine monoclonal antibody H23 recognizes this epitope and stains 91% of breast cancers, but only 1/56 non-malignant breast tissue samples . OBJECTIVE: To create a human antibody that was equivalent to H23 for potential uses in imaging and/or the therapy of breast cancer . STUDY DESIGN: We decided to humanize H23 by CDR-grafting using overlap PCR, and to this end, designed and constructed a bacterial expression vector that would allow V-regions, cloned via unique restriction sites, to be expressed as Fab fragments . In this way, we hoped to be able to rapidly evaluate Fab constructs for binding to Muc-1 and to cells and tissue sections that expressed the antigen . RESULTS: A fully humanized Fab fragment was able to bind Muc-1 peptide, as well as breast cancer cells known to express the epitope and tissue sections, generally showing the same reactivity as the native antibody . In addition, an analysis of sFab expressed with a {His}6 tag preceded by a factor Xa proteolytic cleavage site suggested that E . coli periplasmic signal peptidase was able to cleave the factor Xa site, thereby removing the {His}6 tag . CONCLUSION: We have generated a human antibody that is capable of recognizing a tumor specific epitope expressed by 91% of breast cancers.

Protein Eng, 1997 Mar, 10(3), 217 - 22
Prediction of the biologically active sites in eclosion hormone from the silkworm, Bombyx mori; Kikuchi T et al.; The structure-activity relationship of eclosion hormone from the silkworm, Bombyx mori, was analyzed . First, the probable active residues in silkworm eclosion hormone and also tobacco hornworm eclosion hormone were predicted by the average distance map method . To examine the contributions of those residues to the activity of silkworm eclosion hormone, Gly-substituted mutants for those predicted residues were produced by site-directed mutagenesis and their activities were evaluated by a bioassay . Finally, Glu12, Met24 and Phe25 were estimated to be the crucial residues for the eclosion hormone activity . The possibility of the development of a blocker of an eclosion hormone receptor on the basis of the present work is also discussed.

Protein Eng, 1997 Mar, 10(3), 285 - 90
Expression and epitope tagging of the membrane anchor subunit (DmsC) of Escherichia coli dimethyl sulfoxide reductase; Turner RJ et al.; Escherichia coli dimethyl sulfoxide reductase is a heterotrimer comprising a catalytic subunit (DmsA), an electron transfer subunit (DmsB) and an integral membrane anchor subunit (DmsC) . DmsC is not antigenic and the production of antibodies to this subunit has not been successful . We have tagged DmsC at the C-terminus with a dystrophin-specific amino acid sequence (dysp) to which antibodies are readily available . We were able to use this tagging technique to monitor expression and localization of DmsC in E . coli and non-muscle eukaryotic cells . Growth properties of wild-type E . coli, strain HB101, overexpressing DmsC:dysp suggest that the expression of DmsC is lethal to E . coli . The lethality could be overcome by utilizing an E . coli F0F1 ATPase mutant as the host . Growth conditions of culture density, duration of induction, temperature of incubation after induction and media conditions were investigated to optimize DmsC:dysp accumulation levels . In order to alleviate the problem arising from the toxicity of DmsC, expression in eukaryotic tissue culture was also explored . A plasmid expressing DmsC:dysp was transfected into COS-1 or McA-RH777 cells . The presence of expressed DmsC:dysp was confirmed using specific anti-dysp antibodies and immunofluorescence microscopy analysis revealed that the DmsC:dysp was localized to the endoplasmic reticulum . Expression of DmsC:dysp did not appear to be toxic to the eukaryotic cells . These data suggest methodologies to overcome lethality problems associated with the overexpression of integral membrane proteins like DmsC.

Protein Eng, 1997 Mar, 10(3), 279 - 83
Effects of non-conservative changes to tyrosine 76, a key DNA binding residue of DNase I, on phosphodiester bond cleavage and DNA hydrolysis selectivity; Warren MA et al.; Non-conservative changes, consisting of Y76E, Y76L, Y76Q and Y76W, have been made to tyrosine 76, one of the key DNA binding residues in DNase I . Normally Y76 inserts into the minor groove of DNA and makes an unusual, hydrophobic, stacking interaction with one of the sugars . All four mutants bind to DNA more tightly than the wild type, but cut it more slowly as assessed by Kunitz assays . This gives a rather small decrease in the specificity constants (Vmax/K(m)) for the hydrolysis of DNA, which is roughly paralleled by the loss of activity towards the non-DNA small chromophoric substrate, thymidine-3',5'-di(p-nitrophenyl)phosphate . These non-conservative mutants, therefore, show different behaviour to Y76A and Y76G, studied previously {Doherty A.J., Worrall A.F . and Connolly B.A . (1995) J: Mol . Biol., 251, 366-377} . These two mutants both bind to and cut DNA poorly, resulting in large decreases in Vmax/K(m) values . However, they showed little reduction in rates with the chromophoric substrate . It is likely that the altered side chains in the non-conservative mutants are still able to interact productively with the DNA and contribute to the observed DNA distortion that is essential for efficient catalysis . However, these mutations disrupt the active site, most probably by interference with the hydrogen bonded Y76-E78-H134 triad . H134 is a critical hydrolytic residue of DNase I that is essential for catalysis . The DNA cleavage selectivity of the Y76E, Y76L, Y76Q and Y76W mutants were little altered as compared with the wild-type enzyme as measured using the cutting patterns of a 160 base-pair Escherichia coli Tyr T promoter DNA fragment . This confirms earlier observations, with Y76F, Y76A and Y76G, that showed that this tyrosine has little role in DNA cleavage specificity.

Protein Eng, 1997 Mar, 10(3), 263 - 72
Effects of insertions and deletions in a beta-bulge region of Escherichia coli dihydrofolate reductase; Dion-Schultz A et al.; The role of a beta-bulge in Escherichia coli dihydrofolate reductase (DHFR) has been explored by a series of insertion and deletion mutations . Insertion of a seven amino acid sequence from a structurally equivalent 'beta-blowout' sequence from human DHFR destabilizes E . coli DHFR by 3.6 kcal/mol and decreases catalytic efficiency (kcat/K(m)) 34-fold . Deletion of F137, delta 137, the looped out residue in the bulge, also destabilizes E . coli DHFR by 2.8 kcal/mol but only decreases catalytic efficiency threefold . Concurrent deletion of F137 and mutation of, V136 to proline to try and maintain the strand twist associated with the beta-bulge decreases protein stability by 3.4 kcal/mol and decreases catalytic efficiency 84-fold . These insertion/deletion mutations were also constructed in a D27S DHFR background . The D27S mutation has been described previously and proposed to remove the catalytic acid from the active site . The delta 137 mutation partially suppresses the effect of the D27S mutation as it decreases the K(m) for substrate, dihydrofolate, twofold . Non-additive effects are observed for the insertion/deletion mutations in wild-type versus D27S DHFR backgrounds, consistent with structural changes.

Electrophoresis, 1997 Mar-Apr, 18(3-4), 588 - 98
Two-dimensional electrophoretic analysis of human breast carcinoma proteins: mapping of proteins that bind to the SH3 domain of mixed lineage kinase MLK2; Rasmussen RK et al.; MLK2, a member of the mixed lineage kinase (MLK) family of protein kinases, first reported by Dorow et al . (Eur . J . Biochem . 1993, 213, 701-710), comprises several distinct structural domains including an src homology-3 (SH3) domain, a kinase catalytic domain, a unique domain containing two leucine zipper motifs, a polybasic sequence, and a cdc42/rac interactive binding motif . Each of these domains has been shown in other systems to be associated with a specific type of protein interaction in the regulation of cellular signal transduction . To study the role of MLK2 in recruiting specific substrates, we constructed a recombinant cDNA encoding the N-terminal 100 amino acids of MLK2 (MLK2N), including the SH3 domain (residues 23-77), fused to glutathione S-transferase . This fusion protein was expressed in Escherichia coli, purified using gluthathione-Sepharose affinity chromatography and employed in an affinity approach to isolate MLK2-SH3 domain binding proteins from lysates of 35S-labelled MDA-MB231 human breast tumour cells . Electrophoretic analysis of bound proteins revealed that two low-abundance proteins with a molecular weights (Mr) of approximately 31,500 and approximately 34,000, bound consistently to the MLK2N protein . To establish accurately the Mt / isoelectric point (pI) loci of these MLK2-SH3 domain binding proteins, a number of abundant proteins in a two-dimensional electrophoresis (2-DE) master gel were identified to serve as triangulation marker points . Proteins were identified by (i) direct Edman degradation following electroblotting onto polyvinylidene difluoride (PVDF) membranes, (ii) Edman degradation of peptides generated by in-gel proteolysis and fractionation by rapid (approximately 12 min) microbore column (2.1 mm ID) reversed-phase high performance liquid chromatography (HPLC), (iii) mass spectrometric methods including peptide-mass fingerprinting and electrospray (ESI)-mass spectrometry (MS)-MS utilizing capillary (0.2-0.3 mm ID) column chromatography, or (iv) immunoblot analysis . Using this information, a preliminary 2-DE protein database for the human breast carcinoma cell line MDA-MB231, comprising 21 identified proteins, has been constructed and can be accessed via the World Wide Web (URL address: l).

Electrophoresis, 1997 Mar-Apr, 18(3-4), 498 - 501
Large-scale protein modelling and integration with the SWISS-PROT and SWISS-2DPAGE databases: the example of Escherichia coli; Peitsch MC et al.; Knowledge-based molecular modelling of proteins has proven useful in many instances, including the rational design of mutagenesis experiments, but it has generally been limited by the availability of expensive computer hardware and software . To overcome these limitations, we developed the SWISS-MODEL server for automated knowledge-based protein modelling . The SWISS-MODEL server uses the Brookhaven Protein Data Bank as a source of structural information and automatically generates protein models for sequences which share significant similarities with at least one protein of known three-dimensional structure . We have now used the software framework of the server to generate large collections of protein models, and established the SWISS-MODEL Repository, a new database for automatically generated and theoretical protein models . This repository is directly integrated with the SWISS-PROT and SWISS-2DPAGE databases through the ExPASy World Wide Web server (URL is Here we present an illustration of this process by an application to the Escherichia coli sequences.

Vaccine, 1997 Mar, 15(4), 423 - 32
Subgroup specific protection of mice from respiratory syncytial virus infection with peptides encompassing the amino acid region 174-187 from the G glycoprotein: the role of cysteinyl residues in protection; Simard C et al.; We identified subgroup specific protective epitopes represented by the amino acid regions 174-187 and 171-187 of the G glycoproteins from respiratory syncytial virus (RSV), subgroups A and B . Mice immunized with coupled synthetic peptides corresponding to either the region 174-187 containing a Cys186-->Ser substitution or to the native region 171-187 were completely resistant to RSV infection but only to the respective virus . The protective activities of the peptides 174-187 were dependent on the Cys186-->Ser substitution . In addition, a recombinant protein representing the region 125-203 of the A subgroup G glycoprotein expressed in Escherichia coli was capable without further treatment to completely protect animals against RSV subgroup A infection . We show that the combinations of cysteinyl residues (positions 173, 176, 182, and 186) retained within either synthetic peptides or the recombinant protein G125-203 greatly influenced their protective activities . This indicates that the region 171-187 is essential for the protection conferred by the G125-203 protein . Furthermore, our results strongly suggest that the peptides' and recombinant protein's potencies are a function of a loop-like structure which is stabilized by intramolecular disulfide linkages between Cys176-Cys182 and Cys173-Cys186 . This is further supported by the observation that chemical blocking of the sulfidryl groups in synthetic peptides completely eliminated their protective activity.

Vaccine, 1997 Mar, 15(4), 370 - 6
Novel intranasal immunization techniques for antibody induction and protection of mice against gastric Helicobacter felis infection; Weltzin R et al.; Intranasal (i.n.) delivery of antigen can be highly effective for generating circulating and secretory antibody responses . Mice were immunized i.n . with two antigens, human IgA, and Helicobacter pylori urease in the presence or absence of mucosal adjuvant . To restrict antigen delivery to the upper airways, protein solutions were administered in a small volume without anesthesia . Repeated daily i.n . administration of antigen without adjuvant elicited high levels of specific IgG in serum and IgA in serum, saliva, and feces . Once weekly i.n . immunization with co-administration of cholera toxin or Escherichia coli heat-labile toxin as adjuvant elicited somewhat lower levels of antibody to urease . When challenged with Helicobacter felis, only mice immunized with urease in the presence of adjuvant were protected against gastric infection.

Jpn J Cancer Res, 1997 Mar, 88(3), 296 - 305
Efficient retrovirus-mediated cytokine-gene transduction of primary-cultured human glioma cells for tumor vaccination therapy; Wakimoto H et al.; In order to realize a novel vaccination treatment for malignant gliomas using tumor cells genetically modified to express certain cytokines, it is essential to achieve an efficient gene transduction into primary cultured cells . We investigated the feasibility of preparing a glioma vaccine through retrovirus-mediated gene transduction . Glioma cells were cultured primarily from surgically resected tumor tissues of six patients . We obtained more than 1000-fold proliferation of cultures within eight weeks in all six cases . In vitro infection with a recombinant retrovirus GKlacZ carrying an Escherichia coli beta-galactosidase marker gene resulted in over 65% gene transfer to the primary cultured glioma cells . Further enrichment (approximately 90%) of transduced cells was possible by employing repeated infections or using vectors with neo' marker gene . Two cytokine genes, granulocyte-macrophage colony-stimulating factor and interleukin-4, were introduced into glioma cells by sequential transduction with two single-expression GK vectors . The transduced glioma cells produced high levels of both cytokines . We also evaluated simultaneous introduction of two genes with double-expression GK vectors containing internal ribosomal entry site (IRES) or internal promoter (PGK) . Although the cells transduced with double-expression vectors secreted both cytokines, the level of the gene product following IRES or PGK was 10 times lower than that of the upstream gene product . The transduced cells retained cytokine secretion in vitro for 14 days after 100 Gy irradiation . Our data indicate the feasibility of retrovirus-mediated preparation of gene-modified tumor vaccines from clinical glioma materials, which could be useful for potentiating antitumor immunity in glioma patients.

J Med Virol, 1997 Mar, 51(3), 159 - 66
Mutated epitopes of hepatitis B surface antigen fused to the core antigen of the virus induce antibodies that react with the native surface antigen; Shiau AL et al.; Fusion of peptide epitopes to the core antigen (HBcAg) of hepatitis B virus (HBV) enhances their immunogenicity, both quantitatively and qualitatively . In a number of vaccine-induced mutants of HBV, glycine145 of the surface antigen S polypeptide (HBsAg) has been replaced by arginine, resulting in loss of cross-reactivity with antibodies to normal (wild-type) HBsAg . HBcAg fusion proteins carrying the immunodominant epitope of HBsAg, in which glycine145 was replaced by arginine, glutamic acid, or lysine, were produced in Escherichia coli and formed particles that displayed HBc antigenicity and immunogenicity similar to that of HBcAg itself . The fusion proteins also elicited T-cell proliferative responsiveness to HBcAg and HBsAg . Fusions carrying either wild-type or mutated epitopes of HBsAG showed HBs antigenicity in immunoblot analysis and antigen-capture immunoradiometric assay, but both mutant and wild-type derivatives induced antibodies that cross-reacted with wild-type HBsAG . The results emphasise the potential for HBcAg fusion proteins in vaccines by broadening the antibody response in a way that could confer protection against both wild-type and variant form of HBV.

Biophys J, 1997 Mar, 72(3), 1247 - 57
Evidence for phospholipid microdomain formation in liquid crystalline liposomes reconstituted with Escherichia coli lactose permease; Lehtonen JY et al.; The well-characterized integral membrane protein lactose (lac) permease from Escherichia coli was reconstituted together with trace amounts (molar fraction X = 0.005 of the total phospholipid) of different pyrene-labeled phospholipid analogs into 1-palmitoyl-2-oleoyl-sn-glycero-3-sn-glycero-3-phospho-rac'-glycerol (POPG) liposomes . Effects of lac permease on bilayer lipid dynamics were investigated by measuring the excimer-to-monomer fluorescence intensity ratio IE/IM . Compared to control vesicles, the presence of lac permease (at a protein:phospholipid stoichiometry P/L of 1:4.000) increased the rate of excimer formation by 1-palmitoyl-2{6-(pyren-1-yl)}decanoyl-sn-glycero-3-phosphocholine (PPDPC) by approximately fivefold . Decreasing P/L from approximately 1:4.000 to 1:7.600 decreased the IE/IM for PPDPC from 0.16 to 0.05, respectively . An increase in bilayer fluidity due to permease is unlikely, thus implying that the augmented IE/IM should arise from partial lateral segregation of PPDPC in the vesicles . This notion is supported by the further 38% increase in IE/IM observed for the pyrene-labeled Cys-148 lac permease reconstituted into POPG vesicles at P/L 1:4000 . The importance of the length of the lipid-protein boundary is implicated by the reduction in IE/IM resulting from the aggregation of the lac permease in vesicles by a monoclonal antibody . Interestingly, excimer formation by 1-palmitoyl-2{6-(pyren-1-yl)hexanoyl-sn-glycero-3-phosphocholine (PPHPC) was enhanced only fourfold in the presence of lac permease . Results obtained with the corresponding pyrenyl phosphatidylglycerols and -methanols were qualitatively similar to those above, thus indicating that lipid headgroup-protein interactions are not involved . Inclusion of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamino-N-(5-fluoresce inthio- carbamoyl) (DPPF, X = 0.005) into reconstituted lactose permease vesicles containing PPDPC caused a nearly 90% decrease in excimer fluorescence, whereas in control vesicles lacking the reconstituted protein only 40% quenching was evident . The addition of 1,2-dipalmitoyl-sn-glycero-3-phospho-rac'-glycerol (DPPG) decreased IE/IM for PPDPC, revealing the driving force for the lateral segregation of this probe to become attenuated . More specifically for protein-free bilayers at XDPPG = 0.10 the rate of lateral diffusion of PPDPC in POPG is diminished, as evidenced by the 24% decrement in IE/IM, under these conditions the increase in IE/IM due to lac permease was strongly reduced, by approximately 84% . The present data are interpreted in terms of the hydrophobic mismatch theory, which predicts that integral membrane proteins will draw lipids of similar hydrophobic thickness into their vicinity . In brief, the approximate lengths of most of the predicted 12 hydrophobic, membrane-spanning alpha-helical segments of lactose permease range between 28.5 and 37.5 A and thus exceed the hydrophobic thickness of POPG of approximately 25.8 A . Therefore, to reduce the free energy of the assembly, longer lipids such as PPDPC and DPPF are accumulated in the immediate vicinity of lactose permease in fluid, liquid crystalline POPG bilayers.

Biophys J, 1997 Mar, 72(3), 1109 - 26
A novel method for structure-based prediction of ion channel conductance properties; Smart OS et al.; A rapid and easy-to-use method of predicting the conductance of an ion channel from its three-dimensional structure is presented . The method combines the pore dimensions of the channel as measured in the HOLE program with an Ohmic model of conductance . An empirically based correction factor is then applied . The method yielded good results for six experimental channel structures (none of which were included in the training set) with predictions accurate to within an average factor of 1.62 to the true values . The predictive r2 was equal to 0.90, which is indicative of a good predictive ability . The procedure is used to validate model structures of alamethicin and phospholamban . Two genuine predictions for the conductance of channels with known structure but without reported conductances are given . A modification of the procedure that calculates the expected results for the effect of the addition of nonelectrolyte polymers on conductance is set out . Results for a cholera toxin B-subunit crystal structure agree well with the measured values . The difficulty in interpreting such studies is discussed, with the conclusion that measurements on channels of known structure are required.

Biophys J, 1997 Mar, 72(3), 1031 - 46
Molecular dynamics simulations of six different fully hydrated monomeric conformers of Escherichia coli re-lipopolysaccharide in the presence and absence of Ca2+; Obst S et al.; Six previously published conformational models of Escherichia coli Re lipopolysaccharide (ReLPS) were subjected to molecular dynamics simulations using the CHARMM force field . The monomers of ReLPS were completely immersed in a water box . The dynamic behavior of the solvated models in the presence and absence of calcium cations was compared . The structure of the solvent shell was analyzed in terms of radial distribution functions . Diffusion coefficients and mean residence times were analyzed to characterize the dynamic behavior of the solvent . Order parameters and number of gauche defects were used for the description of the dynamics of the acyl chains . The cations are preferentially located between the carboxylate and phosphate groups of the headgroup . Their presence leads to a rigidification of the headgroup structure and alters the conformation of the backbone, thus influencing the structure and flexibility of the hydrophobic region as well . The effect of calcium on the backbone flexibility was measured in terms of glycosidic torsion angles . The six fatty acid chains of each ReLPS monomer adopt a highly ordered micromembrane structure . The packing parameter indicates that aggregation of these ReLPS monomers will lead to lamellar structures . Evaluation of all data enables us to present one conformation, C, which is thought to best represent the average structure of the ReLPS conformers.

J Biochem (Tokyo), 1997 Mar, 121(3), 456 - 63
The tissue-type plasminogen activator inhibitor ETIa from Erythrina variegata: structural basis for the inhibitory activity by cloning, expression, and mutagenesis of the cDNA encoding ETIa; Kouzuma Y et al.; Erythrina variegata trypsin inhibitor ETIa belongs to the Kunitz inhibitor family, but is unique in its ability to bind and inhibit tissue-type plasminogen activator (tPA) . A cDNA clone encoding ETIa was isolated from the lambda gt11 cDNA library using specific antiserum as a probe and characterized by nucleotide sequencing . The cloned ETIa cDNA consists of 762 nucleotides and includes an open reading frame encoding a polypeptide of 198 amino acids . Comparison of the deduced protein sequence and the determined protein sequence indicated the presence of two signal peptides composed of 24 and 2 amino acids at the N- and C-termini, respectively . The cDNA encoding mature ETIa was amplified by polymerase chain reaction (PCR), ligated into the expression vector pET-22b, and expressed in Escherichia coli BL21(DE3) . The recombinant ETIa (rETIa) was expressed in E . coli as inclusion bodies; it was purified to homogeneity by gel filtration on Sephadex G-75 . The rETIa exhibited almost the same inhibitory activity toward trypsin and tPA as ETIa . Six mutants, in which the amino acids Arg61, Leu62, Arg63, and Ala65 were replaced by Pro, Phe, Leu/Asp, and Tyr, respectively, were constructed by site-specific mutagenesis and expressed in E . coli . The site-specific mutation of Arg63 to Leu (aR63L) or Asp (aR63D) in ETIa resulted in abolition of the inhibitory activities toward both trypsin and tPA . The mutants aR61P and aL62F showed significantly reduced tPA-inhibitory activity, and furthermore the double mutant aR61P/L62F lacked tPA-inhibitory activity, despite retaining the trypsin-inhibitory activity . In contrast, the mutant aA65Y exhibited tPA-inhibitory activity to the same extent as rETIa . This result suggests that Arg61 and Leu62 in ETIa, in addition to Arg63, may play an important role in the interaction with tPA.

Plant Mol Biol, 1997 Mar, 33(4), 753 - 6
Characterization of the plant homologue of prohibitin, a gene associated with antiproliferative activity in mammalian cells; Snedden WA et al.; This report describes the cloning and characterization of a plant cDNA coding for a protein which shows high amino acid sequence similarity with prohibitin, whose gene is associated with antiproliferative activity in mammalian cells . Arabidopsis thaliana and Nicotiana tabacum prohibitin complete cDNAs were isolated, and the expression pattern of prohibitin was examined using polyclonal antibodies raised against the Arabidopsis recombinant prohibitin expressed in Escherichia coli . A single immunoreactive protein was detected in various plant species and in all Arabidopsis organs examined . Subcellular fractionation using tobacco leaves revealed prohibitin in a mitochondrial-enriched fraction . Phylogenetic conservation of prohibitin's amino acid sequence and subcellular localization suggests a similar function in plants, yeast and mammals.

Plant Mol Biol, 1997 Mar, 33(4), 745 - 51
Cloning and characterisation of a gene encoding an antiviral protein from Clerodendrum aculeatum L; Kumar D et al.; The Clerodendrum aculeatum-systemic resistance inducing (CA-SRI) protein, a 34 kDa basic protein, plays a key role in inducing strong systemic resistance in susceptible plants against various plant viruses {22} . We have cloned the cDNA encoding the CA-SRI from C . aculeatum leaves using antibodies raised against the purified protein and degenerate oligonucleotide probes derived from microsequencing of the CA-SRI protein . The full-length cDNA consisted of 1218 nucleotides with an open reading frame of 906 bp . The deduced amino acid sequence of CA-SRI protein showed varying homology (ranging from 11 to 54%) to the ribosome inactivating proteins (RIPs) from other plant species . CA-SRI inhibited in vitro protein synthesis both in rabbit reticulocyte lysate and wheat germ lysate but not in Escherichia coli in vitro translation system . The CA-SRI open reading frame was expressed in an E . coli expression vector and the purified recombinant protein inhibited protein synthesis in rabbit reticulocyte lysate . Southern blot analysis indicated that the CA-SRI gene may be present in low copy number.

Plant Mol Biol, 1997 Mar, 33(4), 723 - 8
Expression of the Arabidopsis G-protein GP alpha1: purification and characterisation of the recombinant protein; Wise A et al.; The Arabidopsis G alpha subunit, GP alpha1, was expressed within Escherichia coli by co-transformation with the expression vector and the dnaY gene which encodes tRNA(Arg)(AGA/AGG) Isolation of the recombinant GP alpha1 in a highly pure form could be achieved by a combination of anion exchange and dye affinity chromatography or by a single step affinity procedure via chromatography on 4-amino-anilido-GTP agarose . The recombinant protein yielded by both procedures was highly active and bound GTPgammaS with an apparent Kd in the nM range . GTPgammaS binding was stimulated two-fold in the presence of Zn2+ compared with that in the presence of Mg2+, Mn2+ or Ca2+.

Plant Mol Biol, 1997 Mar, 33(4), 679 - 89
Characterization of two cDNAs encoding auxin-binding proteins in Nicotiana tabacum; Leblanc N et al.; The isolation and the characterization of two tobacco cDNAs, Nt-ERabp1 and Nt-ERabp2, homologous to Zm-ERabp1, encoding the major auxin-binding protein from maize coleoptiles, are described . Their predicted amino acid sequences correspond to proteins of ca . 21 kDa, in which the characteristic regions common to ABP1-related polypeptides are well-conserved . Southern analysis indicates that the genes corresponding to Nt-ERabp1 cDNA and Nt-ERabp2 cDNA derive respectively from Nicotiana tomentosiformis and Nicotiana sylvestris, the diploid progenitors of Nicotiana tabacum . Analysis of mRNA distribution in tobacco plants indicates that these two genes are preferentially expressed in flowers and growing seedlings . Whatever the tissue tested, Nt-ERabp1 mRNA is more abundant than Nt-ERabp2 mRNA . Furthermore, RT-PCR reveals developmental and organ-specific expression of these two genes in flower parts of tobacco plants . In particular, regulation of Nt-ERabp1 mRNA accumulation appears to be correlated with elongation growth of each floral organ . Recombinant Nt-ERabp1, produced in Escherichia coli, is recognized by antibodies raised against Zm-ERabp1.

Plant Mol Biol, 1997 Mar, 33(4), 625 - 33
Differential accumulation of two glycine-rich proteins during cold-acclimation alfalfa; Ferullo JM et al.; Two mRNAs, MsaCiA and MsaCiB, encoding for proteins harboring glycine-rich motifs, accumulate in alfalfa during cold acclimation . Fusion polypeptides containing the amino acid sequences deduced from these mRNAs were produced in Escherichia coli and used to raise antibodies . Each antibody cross-reacted specifically with soluble polypeptides, MSACIA-32 and MSACIB, respectively . These polypeptides were detectable only in crowns of cold-acclimated plants, even though MsaCiA mRNA accumulated in both crows and leaves during cold acclimation . The analysis of parietal proteins showed that several MSACIA-related proteins, with a molecular mass of 32, 41 and 68 kDa, did accumulate in leaf cell walls and one of 59 kDa crown cell walls . This diversity is most probably due to a tissue-specific maturation of MSACIA . A discrepancy was found between the time-course of accumulation of MSACIB and the one of the corresponding transcript . These results indicate that timing and localization of MSACIA and MSACIB expression are different, and suggest that this differential expression involves both transcriptional and post-transcriptional events . Comparisons made among six cultivars of contrasting freezing tolerance suggest that low tolerance could be explained by failure to accumulate proteins like MSACIA and MSACIB at a sufficient level.

Plant Mol Biol, 1997 Mar, 33(4), 583 - 91
Analysis of type 1 metallothionein cDNAs in Vicia faba; Foley RC et al.; In animals and fungi, small cysteine-rich proteins called metallothioneins (MTs) play a role in heavy metal tolerance . MT genes have been isolated in plants, but their function remains to be elucidated . We have isolated two distinct Vicia faba MT genes that belong to the type 1 group of plant MT genes in contrast to a MT gene we previously isolated that belongs to type 2 . We found similarities and differences between the V . faba MT genes . The RNA expression patterns differed and this was most pronounced in roots, which contained high MT1 but very low MT2 RNA levels . Like MT2, MT1 transcript levels were not significantly affected by treatment with Cd, Cu, Fe and Zn, at least under the experimental conditions . MT RNA levels varied in leaves and stem internodes of different developmental ages, with the highest expression in the older tissue . The levels of MT RNA correlated inversely with endogenous Cd, Cu and Fe levels within different internodes, but not with a number of other metals tested (including Zn) . The three bean MTs were expressed in Escherichia coli and found to bind Cd, Cu and Zn but not to Fe . The MTs were tested to determine if they differed in their ability to bind a specific metal but no significant differences in binding were observed.

Cell Mol Biol (Noisy-le-grand), 1997 Mar, 43(2), 211 - 25
Multiple regulatory regions control the expression of Ets-1 in the developing mouse: vascular expression conferred by intron I; Jorcyk CL et al.; Ets-1, a developmentally-regulated protooncogene, is expressed in multiple tissues during different stages of mouse development and cellular differentiation including high levels in lymphoid organs and endothelium . The putative roles of this DNA-binding protein in lymphoid development and maturation, as well as in angiogenesis and tumor vascularization, suggest that the regulation of Ets-1 may be critical to understanding these important developmental processes . We have cloned the mouse Ets-1 5' flanking region which shows significant homology to the human 5' flanking region, including potential transcription factor binding sites . Various amounts of mouse Ets-1 5' flanking, exon and intron sequences have been fused to the E . coli lacZ reporter gene and introduced into the mouse germline to identify genomic regions which regulate the developmental and tissue-specific expression of Ets-1 . The 2.4 kb 5' flanking region of Ets-1 directs lacZ expression to the folding neural tube of embryos at gestational day 8.5 which is identical to the endogenous expression pattern of Ets-1 . However, at later times in gestation, up to 5.3 kb of 5' flanking region results only in aberrant expression and is not able to confer lacZ expression in lymphoid or vascular tissues . When the first exon and 9 kb of the first intron are included with 5' flanking sequences, using an enhancer-trap-strategy, lacZ expression is observed in developing vessels, meninges and choroid plexus which correlates to endogenous Ets-1 expression . Further characterization of the vascular-specific element contained within intron I will provide important insights into the mechanisms controlling gene expression during angiogenesis.

Genes Dev, 1997 Mar 1, 11(5), 571 - 81
The recombination hot spot chi is a regulatory element that switches the polarity of DNA degradation by the RecBCD enzyme; Anderson DG et al.; Homologous recombination in Escherichia coli is stimulated at DNA sequences known as chi sites . Stimulation requires the multifunctional RecBCD enzyme, which is both a helicase and a 3' --> 5' exonuclease . Upon recognition of a properly oriented chi site, the 3' --> 5' exonuclease activity is attenuated . Here we show that in addition to attenuation of the 3' --> 5' exonuclease activity, recognition of chi by the RecBCD enzyme also up-regulates a nuclease activity of the opposite polarity, resulting in an enzyme that now preferentially degrades 5' --> 3' . These results demonstrate that chi is a unique regulatory element that converts the antirecombinogenic form of the RecBCD enzyme into a recombinogenic form by causing two distinct enzymatic changes: attenuation of the 3' --> 5' nuclease activity, and up-regulation of the 5' --> 3' nuclease activity . The consequence of chi recognition is the production of a recombination intermediate possessing a 3'-ssDNA overhang terminating at the chi sequence . This processing of a dsDNA end to a 3'-ssDNA overhang parallels that which occurs during the initation of homologous recombination in other pathways in E . coli, and in other organisms such as the yeast Saccharomyces cerevisiae.

Eur J Biochem, 1997 Mar 1, 244(2), 658 - 63
Effects of tyrosine ring fluorination on rates and equilibria of formation of intermediates in the reactions of carbon-carbon lyases; Phillips RS et al.; The interactions of ring fluorinated analogs of tyrosine with tyrosine phenol-lyase and tryptophan indole-lyase (tryptophanase) were studied by rapid-scanning stopped-flow spectrophotometry . The reaction of L-tyrosine with tyrosine phenol-lyase resulted in rapid formation of a small absorbance peak at 500 nm, attributed to a quinonoid intermediate . The reaction of 3-fluoro-L-tyrosine with tyrosine phenol-lyase resulted in a peak at 500 nm with much higher absorbance, as did the reaction of 3,5-difluoro-L-tyrosine, due to increased accumulation of quinonoid intermediates . In constrast, complexes with 2-fluoro-L-tyrosine, 2,3-difluoro-L-tyrosine, 2,5-difluoro-L-tyrosine, and 2,6-difluoro-L-tyrosine exhibited much lower absorbance intensity at 500 nm . The rate constant for quinonoid intermediate formation from 3-fluoro-L-tyrosine was comparable to that for L-tyrosine . However, 3,5-difluoro-L-tyrosine reacted to form a quinonoid intermediate at about half the rate of L-tyrosine, while 2,3-difluoro-L-tyrosine reacted at twice the rate of L-tyrosine . In addition, the 2-substituted difluorotyrosines exhibited an intermediate, which was formed rapidly, absorbing strongly at about 340 nm, which is likely due to a gem-diamine intermediate . Tyrosine is not a substrate for tryptophan indole-lyase; the reaction of tryptophan indole-lyase with L-tyrosine resulted in formation of external aldimine, which absorbed at 420 nm, and a very small absorbance peak at 500 nm . 3-Fluoro-L-tyrosine reacted with tryptophan indole-lyase to produce a prominent quinonoid absorbance peak at 500 nm, whereas L-tyrosine, 2-fluoro-L-tyrosine, and all difluoro-L-tyrosines, had a much reduced intensity for this peak . Thus, the presence of ring fluorine substituents in L-tyrosine that are remote from the site of the chemical transformation has significant effects on the rates and equilibria of intermediate formation in the reactions with both tyrosine phenol-lyase and tryptophan indole-lyase . Although it is commonly thought that fluorine substitution will not result in any significant steric effects, our results suggest that the effects of fluorine substitution in the reactions of fluorinated tyrosines with tyrosine phenol-lyase and tryptophan indole-lyase are due to a combination of steric and electronic effects.

Eur J Biochem, 1997 Mar 1, 244(2), 653 - 7
Membrane-type-2 matrix metalloproteinase can initiate the processing of progelatinase A and is regulated by the tissue inhibitors of metalloproteinases; Butler GS et al.; Membrane-type-1 matrix metalloproteinase has been identified as an activator of the matrix metalloproteinase progelatinase A at cell surfaces . We report here that a soluble active form of membrane-type-2 matrix metalloproteinase can also process progelatinase A in a comparable fashion to the type-1 at rates which are dependent on the concentration of the proenzyme . Activation is inhibited by tissue inhibitors of metalloproteinases TIMP-2 and TIMP-3, but only partially by TIMP-1 . These results suggest that cellular activation of progelatinase A may be initiated by different members of the membrane-type matrix metalloproteinase family depending on tissue distribution.

Eur J Biochem, 1997 Mar 1, 244(2), 627 - 34
Identification of amino acid residues at nucleotide-binding sites of chaperonin GroEL/GroES and cpn10 by photoaffinity labeling with 2-azido-adenosine 5'-triphosphate; Bramhall EA et al.; Although the chaperonin GroEL/GroES complex binds and hydrolyzes ATP, its structure is unlike other known ATPases . In order to better characterize its nucleotide binding sites, we have photolabeled the complex with the affinity analog 2-azido-ATP . Three residues of GroEL, Pro137, Cys138 and Thr468, are labeled by the probe . The location of these residues in the GroEL crystal structure {Braig, K., Otwinowski, Z., Hedge, R., Boisvert, D., Joachimiak, A., Horwich, A . & Sigler, P . (1994) Nature 371, 578-586: Boisvert, D . C., Wang, J., Otwinowski, Z., Horwich, A . L . & Sigler, P . B . (1996) Nat . Struct . Biol . 3, 170-177} suggests that 2-azido-ATP binds to an alternative conformer of GroEL in the presence of GroES . The labeled site appears to be located at the GroEL/GroEL subunit interface since modification of Pro137 and Cys138 is most readily explained by attack of a probe molecule bound to the adjacent GroEL subunit . Labeling of the co-chaperonin, GroES, is clearly demonstrated on gels and the covalent tethering of nucleotide allows detection of a GroES dimer in the presence of SDS . However, no stable peptide derivative of GroES could be purified for sequencing . In contrast, the GroES homolog, yeast cpn10, does give a stable derivative . The modified amino acid is identified as the conserved Pro13, which corresponds to Pro5 in Escherichia coli GroES.

Eur J Biochem, 1997 Mar 1, 244(2), 613 - 8
Mutations in the 1.1 subdomain of Escherichia coli sigma factor sigma70 and disruption of its overall structure; Gopal V et al.; Among various group I sigma factors, two amino acids, Val55 and Ala59 are the conserved amino acids in the 1.1 hydrophobic subdomain . These two sites have been mutated to generate variants designated as {Gly55}sigma70 and {Gly59}sigma70, where glycine replaces valine and alanine, respectively . The function of these sigma mutants is reported here . The molecular mass of these proteins determined on denaturing gels was 70 kDa, which is the expected calculated molecular mass; wild-type sigma70 has an apparent molecular mass of 87 kDa . However, {Gly434}sigma70, which contains a mutation at the DNA-binding rpoD box region, also migrates as a 70-kDa protein on SDS/PAGE . Circular dichroism spectral analysis indicated that both {Gly55}sigma70 and {Gly59}sigma70 have reduced helicity (20%) compared to wild-type sigma70 (50%) . Binding of sigma factors with the hydrophobic, surface active probe 1-anilinonapthalene-8-sulphonate, has shown that more hydrophobic surfaces are available/exposed in {Gly55}sigma70, {Gly59}sigma70 as well as in {Gly434}sigma70 in comparison to wild-type sigma70 . Time-resolved emission spectroscopic studies have suggested transient binding between these mutants and DNA . The different holoenzyme RNA polymerases generated upon reconstituting these mutants independently with core RNA polymerase (alpha2beta beta') have shown reduced transcriptional activity in comparison to the enzyme containing wild-type sigma factor . However, another mutation (Val-->Gly) in the hydrophobic subdomain 1.2 at position 83, which is designated as {Gly83}sigma70, has similar properties as the wild-type with respect to its mobility on denaturing gels, circular dichroism profile, and transcriptional activity when reconstituted with core RNA polymerase . It appears that the 1.1 subdomain in sigma70 may interact hydrophobically with the 2.3/2.4 DNA-binding region.

Eur J Biochem, 1997 Mar 1, 244(2), 604 - 12
A serine/threonine protein kinase from Mycobacterium tuberculosis; Peirs P et al.; Genomic DNA sequencing in the vicinity of the pstA-1 gene from Mycobacterium tuberculosis allowed us to clone, sequence and identify a gene encoding a 70-kDa protein . The size of the protein was confirmed by in vitro coupled transcription/translation . Its N-terminal domain shows extensive sequence similarity with the catalytic domain of eukaryotic serine/threonine protein kinases, and the protein was therefore called Mbk (mycobacterial protein kinase) . The deduced amino acid sequence contains two transmembrane segments, which flank a highly repetitive region, suggesting a receptor-like anchoring . The mbk gene was overexpressed in Escherichia coli and the gene product (Mbk) was purified as a fusion protein with gluthatione S-transferase . Recombinant Mbk was found to be autophosphorylated on threonine residues and capable of phosphorylating myelin basic proteins from bovine brain and histones from calf thymus on serine residues, both in a manganese-dependent manner . The phosphorylation of myelin basic proteins by Mbk was inhibited by calcium and by staurosporine, a widely used inhibitor of eukaryotic protein serine/threonine kinases . A similar gene was found in Mycobacterium bovis BCG DNA by Southern blot analysis . Its expression was detected in cultures of M . bovis BCG by reverse transcriptase/PCR . Although its biological role is unknown, it is the first serine/threonine protein kinase characterized in Mycobacteria.

Eur J Biochem, 1997 Mar 1, 244(2), 544 - 51
Dissection of the domain architecture of the alpha2macroglobulin-receptor-associated protein; Ellgaard L et al.; The alpha2macroglobulin-receptor-associated protein (RAP) binds to the alpha2macroglobulin receptor/low-density lipoprotein receptor-related protein (alpha2MR/LRP), a multi-functional cell surface receptor known to bind and internalize several macromolecular ligands . RAP has been shown to inhibit binding of all known alpha2MR/LRP ligands . Mutational studies have implicated distinct parts of RAP as specifically involved in inhibition of binding of a multitude of ligands . In the present paper we provide experimental evidence allowing assignment of elements of triplicate internal sequence similarity in RAP, noted previously {Warshawsky, I., Bu, G . & Schwartz, A . L . (1995) Sites within the 39-kDa protein important for regulating ligand binding to the low-density lipoprotein receptor-related protein, Biochemistry 34, 3404-3415}, to three structural domains, 1, 2 and 3, comprising residues 18-112, 113-218 and 219-323 of RAP, respectively . Structural analysis by 1H-NMR spectroscopy shows that domains 1 and 2 as separate domains have similar secondary structures, consisting almost exclusively of alpha-helices, whereas domain 3 as a separate domain appears only to be marginally stable . Ligand competition titration of recombinant RAP domains 1, 2 and 3 and double domains 1+2 and 2+3 against 125I-RAP and 125I-alpha2M* (methylamine-activated alpha2M) for binding to alpha2MR/LRP demonstrated (a) that functional integrity in single domains is largely preserved, and (b) that important determinants for the inhibition of test ligands reside in the C-terminal regions of domains 1 and 3.

Eur J Biochem, 1997 Mar 1, 244(2), 449 - 53
Structural analysis of the O-antigenic polysaccharide from the enteropathogenic Escherichia coli O142; Landersjo C et al.; The polysaccharide part of the lipopolysaccharide obtained from the enteropathogenic Escherichia coli O142 has been isolated, and its structure determined . Together with 1H-NMR and 13C-NMR spectroscopy, sugar and methylation analyses show that the polysaccharide is composed of repeating pentasaccharide units . Sequential information on the O-polysaccharide was obtained by two-dimensional NMR techniques, namely heteronuclear-multiple-bond-connectivity and NOESY experiments . The repeating unit of the O-polysaccharide of E . coli strain O142 has the following structure: {structure: see text}.

Eur J Biochem, 1997 Mar 1, 244(2), 414 - 25
A possible role for cathepsins D, E, and B in the processing of beta-amyloid precursor protein in Alzheimer's disease; Mackay EA et al.; Formation of the 4-kDa peptides, which are essential constituents of the extracellular plaques in Alzheimer's disease, involves the sequential cleavage of the amyloid precursor protein (APP) by beta- and gamma-secretases . The carboxy-terminal 99-amino-acid peptide which is liberated from APP by beta-secretase was used as a potential native substrate of the gamma-secretase(s) . With the addition of an initiator Met and a FLAG sequence at the C-terminus (betaAPP100-FLAG), it was expressed in Escherichia coli under the control of the T7 promotor . The preferred site(s) of cleavage in the N-terminal 40-amino-acid beta-amyloid peptide and betaAPP100-FLAG by potential gamma-secretase(s) were rapidly identified using matrix-assisted laser-desorption/ionization time-of-flight mass spectroscopy in addition to peptide mapping followed by protein sequence analysis . Since gamma-secretases seem to be active at acidic pH, three cathepsins (D, E and B) were selected for testing . Studies using different detergents indicated that the cleavage preference of cathepsin D for the betaAPP100-FLAG is highly dependent on the surfactant used to solubilize this substrate . All three cathepsins were found to be capable of catabolizing both beta-amyloid peptides and the betaAPP100-FLAG . As cathepsin D was found to cleave the betaAPP100-FLAG in the vicinity of the C-terminus of the beta-amyloid peptides and cathepsin B has a high carboxypeptidase activity at low pH, the possibility cannot be excluded that cathepsins D and B are involved in the amyloidogenic processing of APP.

Eur J Biochem, 1997 Mar 1, 244(2), 407 - 13
Reversible folding of UDP-galactose 4-epimerase from Escherichia coli; Dutta S et al.; UDP-galactose 4-epimerase from Escherichia coli is a homodimer of 39-kDa subunits having 1 or 2 molecules of NAD bound non-covalently/dimer . The enzyme can be dissociated and denatured by 8 M urea at pH 7.0 to a state having only 15% of residual secondary structure . Dilution of the denaturant by 20 mM potassium phosphate, pH 8.5, leads to functional reconstitution of the enzyme . No addition of extraneous NAD is required for reactivation, indicating a strong affinity of the cofactor for refolded molecule . The reactivation follows a second-order kinetics (k = 1.2 +/- 0.07 X 10(3) M(-1) s(-1) at 25 degrees C) with an energy of activation of 23.79 +/- 0.33 kJ/mol . The native, denatured and renatured states of the enzyme were characterized by far-ultraviolet CD spectra for secondary structure: protein fluorescence, interaction with extrinsic fluorescence probe ANS (1-anilino 8-naphthalene sulfonic acid) and ultraviolet absorption spectra for tertiary structure and size-exclusion HPLC, gel-filtration chromatography and light-scattering for quaternary structure . The folding process could be broadly divided into two distinct steps: (a) regain of secondary structure and dimerization were fast and were complete within 2 min and 9 min, respectively, and (b) regain of catalytic activity was slow and was complete by 45 min . No active holoenzyme could be identified . It appears that generation of the NAD-binding site and subsequent assembly of NAD is the rate-limiting step expressing catalytic activity.

Eur J Biochem, 1997 Mar 1, 244(2), 384 - 99
NMR assignments, secondary structure and hydration of oxidized Escherichia coli flavodoxin; Ponstingl H et al.; Recombinant flavodoxin from Escherichia coli was uniformly enriched with 15N and 13C isotopes and its oxidized form in aqueous solution investigated by three-dimensional NMR spectroscopy . Nearly complete 1H, 15N and 13C resonance assignments were obtained . The secondary structure was determined from chemical shift, NOE and 3J(HNH alpha) coupling constant data . Like homologous long-chain flavodoxins, E . coli flavodoxin contains a five-stranded parallel beta-sheet and five helices . The beta-strands were found to comprise the residues 3-8, 29-34, 48-56, 80-89, 114-116 and 141-145 . The helices comprise residues 12-25, 40-45, 62-73, 98-108 and 152-166 . The FMN-binding site was determined by intermolecular NOEs and low-field shifted amide proton resonances induced by the phosphoester group of the cofactor . The data are in good agreement with a previously predicted model of E . coli flavodoxin {Havel, T . F . (1993) Mol . Sim . 10, 175-210} . The analysis of of water-flavodoxin NOEs revealed the presence of two, possibly three, buried hydration water molecules which are located at sites, where homologous flavodoxins from Anacystis nidulans and Anabena 7120 contain conserved hydration water molecules . One of these water molecules mediates hydrogen bonds between the protein backbone and the ribityl chain of the FMN cofactor.

Eur J Biochem, 1997 Mar 1, 244(2), 301 - 9
Some DNA targets of the yeast CYP1 transcriptional activator are functionally asymmetric--evidence of two half-sites with different affinities; Nait-Kaoudjt R et al.; CYP1 protein is a yeast transcriptional regulator which contains a zinc cluster in its DNA-binding domain . It was recently shown by selecting random CYP1 binding sites that CYP1 protein recognizes with a higher affinity targets containing the CGGNNNTANCGG consensus sequence . Notably, this ideal sequence is however not found in wild-type CYP1 target sites . In order to investigate how CYP1 protein actually binds to its targets, mutations were introduced in three of them (UAS1-A/CYC1, UAS1-A/CYB2, UAS/CYC7) and the consequences towards the binding of purified CYP1-(1-200)-peptide were analyzed . Our data support the following conclusions: (a) When the sequence element contains two CGGs and no TA, both CGGs are essential for binding . (b) If the sequence element contains only the right CGG and the TA, both are sufficient but indispensable for the binding of CYP1 . (c) When two CGCs and the TA are present, the right CGC, and not the left one, is essential for the binding of CYP1 . (d) CYP1-(1-200)-peptide is usually a monomer in solution but binds DNA as a dimer . Finally, we found evidence for the presence of two half-sites with different measured affinities in the asymmetric sequences of some CYP1 targets.

Eur J Biochem, 1997 Mar 1, 244(2), 286 - 93
Role of ribonucleotide reductase and deoxynucleotide pools in the oxygen-dependent control of DNA replication in Ehrlich ascites cells; Brischwein K et al.; Cultured Ehrlich ascites cells were exposed to different oxygen tensions (ranging from nearly complete anoxia to 95% O2 at 10(5) Pa) and to transient (5-10 h) hypoxia (0.02% O2 at 10(5) Pa) . Treated cells were examined with respect to the intracellular concentration of the M2-specific tyrosyl free radical of ribonucleotide reductase by EPR spectroscopy, and with respect to the pool sizes of all four deoxynucleoside triphosphates by an enzymatic assay employing DNA polymerase I of Escherichia coli . From 2% to 0.02% O2, the free radical level decreased continually from a normal value to just above detectability by the EPR measurement employed, and quickly recovered when hypoxic cells were resupplied with atmospheric O2 . Concurrently, analogous changes of the size of the dCTP pool occurred, whereas the pool sizes dATP and dGTP underwent no changes, and the size of the dTTP pool only moderate changes . The changes of the free radical concentration and of the dCTP pool correlated well with the suppression or reactivation of DNA replication under the respective O2 conditions . The results consistently support the hypothesis of a fast-acting regulatory pathway that controls the rate of DNA replication in proliferating cells according to sufficient availability of O2 . Therefore, ribonucleotide reductase may serve, in addition to providing DNA building blocks, as a pO2 sensor, which transmits the signal in the form of an altered intracellular dCTP concentration, directly or indirectly, to the nuclear-replication machinery.

Crit Care Med, 1997 Mar, 25(3), 504 - 11
Myocardial and vascular adrenergic alterations in a rat model of endotoxin shock: reversal by an anti-tumor necrosis factor-alpha monoclonal antibody; Boillot A et al.; OBJECTIVES: a) To investigate responsiveness to exogenous catecholamines in rat endotoxin shock by studying both myocardial and vascular functional parameters, and to determine the relationship of these parameters with other relevant biological parameters of the adrenergic pathway, such as myocardial beta-adrenergic receptors and cyclic adenosine monophosphate (cAMP); b) to investigate the role of tumor necrosis factor (TNF)-alpha via prophylactic anti-TNF-alpha monoclonal antibody administration . DESIGN: Experimental, comparative hospital . SETTING: Laboratory in a university hospital . SUBJECTS: Male Sprague-Dawley rats, weighing 280 to 340 g . INTERVENTIONS: Intravenous injection of Escherichia coli endotoxin (5 mg/100 g) in the first group; injection of the same dose of endotoxin preceded by 2 mg/100 g of anti-TNF-alpha monoclonal antibody in the second group; injection of saline in the third (control) group . MEASUREMENTS AND MAIN RESULTS: TNF-alpha concentration was measured before and during the first 3 hrs in all three groups . Myocardial and vascular functional parameters were obtained, respectively, from Langendorff perfused hearts and isolated aortic rings . Adrenergic biochemical parameters (catecholamines, density and affinity of beta-receptors, and isoproterenol-stimulated myocardial cAMP) were determined 3 hrs after injections in the three groups . After endotoxin injection, serum TNF-alpha concentrations peaked at 60 mins (2496 +/- 412 pg/mL) and returned slowly to control values at 3 hrs; serum TNF-alpha concentrations remained under the limit of detection in the other two groups . When compared with the control group, plasma concentrations of epinephrine and norepinephrine were significantly (p < .05) increased . Baseline values for differential left ventricular pressure and coronary flow were significantly (p < .001, p < .01, respectively) reduced in the endotoxin group; heart rate remained unchanged . In the endotoxin and control groups, isoproterenol induced a similar increase in differential left ventricular pressure and in heart rate . Anti-TNF-alpha antibody increased cardiac response by partially preventing the decrease by endotoxin in differential left intraventricular pressure . Maximal specific binding of 125iodocyanopindolol and myocardial cAMP accumulation were significantly (p < .01) reduced in the endotoxin group in comparison with the control group . Anti-TNF-alpha antibody prevented the endotoxin-induced decrease in cAMP synthesis (p < .05) but did not modify the density of receptors . Affinity of receptors was similar in the three groups . In aortic rings, endotoxin administration significantly (p < .01) shifted the dose-response curve to norepinephrine to the right, both in the presence and absence of endothelium . NG-monomethyl-L-arginine significantly increased the contractions to attain the control level: p < .001 in the presence of endothelium; p < .05 in the absence of endothelium . Anti-TNF-alpha antibody did not prevent endotoxin-induced vascular hyporeactivity to norepinephrine in either endothelium-intact or -denuded rings, but partially attenuated the decrease in maximal response . CONCLUSIONS: In ex vivo experiments, 3 hrs after endotoxin injection, vascular responsiveness was sharply decreased . This impaired response was improved in vitro by the inhibition of nitric oxide . The heart response to isoproterenol, nevertheless, was maintained, even though there was an obvious decrease in receptor density and an impaired myocardial accumulation of cAMP . Anti-TNF-alpha antibody partially prevented the alteration of both myocardial pressure response to isoproterenol and biochemical parameters, and was not efficacious in preventing vascular hyporeactivity to vasoconstrictor agents.

Crit Care Med, 1997 Mar, 25(3), 452 - 9
Effects of N omega-nitro-L-arginine methyl ester on the endotoxin-induced disseminated intravascular coagulation in porcine septic shock; Jourdain M et al.; OBJECTIVES: Nitric oxide is known to prevent platelet aggregation and clot formation . Inhibitors of nitric oxide synthase might promote or enhance endotoxin disseminated intravascular coagulation . The present study was designed to evaluate the effects of the arginine analog, N omega-nitro-L-arginine methyl ester (L-NAME), on the endotoxin-induced disseminated intravascular coagulation in a porcine model of septic shock . DESIGN: Prospective, comparative, experimental study . SETTING: Laboratory at a large university hospital . SUBJECTS: Sixteen female piglets, weighing 20 to 28 kg . INTERVENTIONS: Three groups of animals were studied: a control group (n = 6); a lipopolysaccharide (LPS)-treated group (n = 5) receiving Escherichia coli endotoxin (5 micrograms/kg/min over 30 mins); and an LPS + L-NAME group (n = 5) receiving endotoxin and, 1 hr after, a bolus of L-NAME (25 mg/kg) . MEASUREMENTS AND MAIN RESULTS: Hemodynamic changes, usual coagulation parameters, and plasma concentrations of thrombin-antithrombin complexes, antithrombin III activity (At III), tissue plasminogen activator, plasminogen activator inhibitor type 1, and von Willebrand factor were measured at baseline, and at 30, 60, 90, 120, 180, 240, and 300 mins . After euthanasia or death, lungs and kidneys were withdrawn for histologic study . The extent of microvascular thrombosis was assessed by a semiquantitative disseminated intravascular coagulation score . In both septic endotoxin group, administration of LPS resulted in hemodynamic changes typical of severe septic shock, with disseminated intravascular coagulation and histologic changes characterized by adult respiratory distress syndrome and kidney microthrombosis . L-NAME administration normalized mean arterial pressure with a dramatic increase in systemic vascular resistances and a marked decrease in cardiac index . The changes in usual coagulation parameters, AT III, tissue plasminogen activator, and plasminogen-activator inhibitor type 1 concentrations were not different between both septic groups . However, in the LPS + L-NAME group, thrombin-antithrombin complexes and von Willebrand factor were higher and associated with a higher histologic disseminated intravascular coagulation score . CONCLUSION: In this model of endotoxin septic shock, L-NAME administration resulted in histologic and coagulation changes consistent with an increased activation of intravascular coagulation.

DNA Cell Biol, 1997 Mar, 16(3), 281 - 9
Isolation and expression of an isoform of human CDP-diacylglycerol synthase cDNA; Weeks R et al.; Phosphatidic acid (PA) is a phospholipid involved in signal transduction and in glycerolipid biosynthesis . CDP-diacylglycerol synthase (CDS) or CTP:phosphatidate cytidylyltransferase (EC 2.7.7.41) catalyzes the conversion of PA to CDP-diacylglycerol (CDP-DAG), an important precursor for the synthesis of phosphatidylinositol, phosphatidylglycerol, and cardiolipin . We describe in this study the isolation and characterization of a human cDNA clone that encodes amino acid sequences homologous to Escherichia coli, yeast, and Drosophila CDS sequences . Expression of this human cDNA under the control of a GAL1 promoter in a null cds1 mutant yeast strain complements its growth defect and produces CDS activity when induced with galactose . Transfection of this cDNA into mammalian cells leads to increased CDS activity in cell-free extracts using an in vitro assay that measures the conversion of {alpha-32P}CTP to {32P}CDP-DAG . This increase in CDS activity also leads to increased secretion of tumor necrosis factor-alpha and interleukin-6 from endothelial ECV304 cells upon stimulation with interleukin-1beta, suggesting that CDS overexpression may amplify cellular signaling responses from cytokines.

Chem Biol, 1997 Mar, 4(3), 203 - 7
Expression of a functional non-ribosomal peptide synthetase module in Escherichia coli by coexpression with a phosphopantetheinyl transferase; Ku J et al.; BACKGROUND: Non-ribosomal peptide synthetases (NRPSs) found in bacteria and fungi are multifunctional enzymes that catalyze the synthesis of a variety of biologically important peptides . These enzymes are composed of modular units, each responsible for the activation of an amino acid to an aminoacyl adenylate and for the subsequent formation of an aminoacyl thioester with the sulfhydryl group of a 4'-phosphopantetheine moiety . Attempts to express these modules in Escherichia coli have resulted in recombinant proteins deficient in 4'-phosphopantetheine . The recent identification of a family of phosphopantetheinyl transferases (P-pant transferases) associated with NRPS have led us to investigate whether coexpression of NRPS modules with P-pant transferases in E . coli would lead to the incorporation of 4'-phosphopantetheine . RESULTS: A truncated module of gramicidin S synthetase, PheAT(His6), was expressed as a His6 fusion protein in E . coli with and without Gsp, the P-pant transferase associated with gramicidin S synthetase . Although PheAT(His6) expressed alone in E . coli catalyzed Phe-AMP formation from Phe and ATP, <1% was converted to the Phe thioester . In contrast, >80% of the PheAT(His6) that was coexpressed with Gsp could form the Phe thioester in the presence of Phe and ATP . CONCLUSIONS: Our finding indicates the presence of an almost equimolar amount of 4'-phosphopantetheine covalently bound to the NRPS module PheAT(His6), and that the functional expression of NRPS modules in E . coli is possible, provided that they are coexpressed with an appropriate P-pant transferase.

Appl Microbiol Biotechnol, 1997 Mar, 47(3), 241 - 5
Development of HIV-1 protease expression methods using the T7 phage promoter system; Komai T et al.; New and simple human immunodeficiency virus type 1 (HIV-1) protease expression methods in Escherichia coli were developed using the T7 phage promoter system . In order to suppress leaky HIV-1 protease expression under the control of the T7 polymerase, two new methods were tested . One involved the introduction of supplementary T7 promoter regions into host cells {E . coli BL-21 (DE3)} containing the HIV-1 protease gene under the control of the T7 promoter . It was expected that the supplementary T7 promoter regions would compete with the HIV-1 protease expression vector for the T7 polymerase binding . The other involved the infection of late-log-phase cultures of E . coli JM109 harboring the same HIV-1 protease expression vector with the M13 phage expressing T7 polymerase . Both methods were effective, and transformants with the mature HIV-1 protease expression vector showed ten times higher HIV-1 protease activity than activities obtained with the autoprocessing vector . The expression systems described here are convenient and are also easily applicable for the expression of other proteins toxic for E . coli.

Comp Biochem Physiol B Biochem Mol Biol, 1997 Mar, 116(3), 353 - 8
Evaluation of a heterologous metallothionein gene promoter in transfected mosquito cells; Wu CC et al.; Mosquito cells from the C7-10 Aedes albopictus line were transfected with a recombinant plasmid containing the Escherichia coli galactokinase gene under control of the promoter from the Drosophila melanogaster metallothionein gene, Mtn . Consistent with what has been observed with heterologous metallothionein promoters in several vertebrate systems, treatment of transiently transfected mosquito cells with CuSO4 or CdCl2 induced a 2- to 5-fold increase in galactokinase gene expression . Levels of enzyme activity were not increased in tests using stably transformed lines despite wide ranges in the number of transfected gene copies detected in Southern blots . The importance of comparative studies with gene constructs that may eventually be used to produce genetically modified mosquitoes is underscored by the apparent variability in activity of heterologous promoters from D . melanogaster in different mosquito cell lines.

Acta Anaesthesiol Scand, 1997 Mar, 41(3), 399 - 407
Inhaled nitric oxide does not prevent endotoxin-induced lung injury in rabbits; Nishina K et al.; BACKGROUND: Neutrophils, platelets, and cytokines are thought to play a pivotal role in the pathogenesis of endotoxin-induced lung injury which resembles features of acute respiratory distress syndrome (ARDS) . For initiation of this pathological process, neutrophils and platelets are activated and adhere to pulmonary endothelium . Nitric oxide (NO) inhibits adhesion and activation of these cells and decreases the cytokine level in bronchoalveolar lavage (BAL) fluid obtained from patients with ARDS . Limited data are available on the effect of NO treatment before and after endotoxin on the development and advance of ARDS . The aim of the current study was to determine whether NO inhalation prevents acute lung injury . METHODS: Thirty-two male anaesthetized rabbits were randomly assigned to receive one of four treatments (n = 8 each); Group S-N received saline with nitrogen (N2), Group S-NO received saline infusion with NO (20 p.p.m.) inhalation, Group E-N received an infusion of Escherichia coli endotoxin 100 micrograms/ kg over 60 min with inhalation of N2, and Group E-NO received endotoxin with NO (20 p.p.m.) inhalation . The lungs of the rabbits were ventilated with 40% oxygen until 6 h after the start of endotoxin or saline administration . Haemodynamics and PaO2 were recorded during the ventilation period . After observation, the lung wet-to dry-(W/D) weight ratio, lung mechanics, and cell fraction, activated complements, cytokines, arachidonic acid metabolites, and albumin concentrations in the BAL fluid were measured and analysed . Light microscopic findings were compared among the four groups . RESULTS: Pulmonary hypertension and deterioration of oxygenation by endotoxin were less pronounced in rabbits receiving NO . The lung compliance after endotoxin was similar in Groups E-NO and E-N . The W/D weight ratio and neutrophils and albumin concentrations in the BAL fluid increased in Groups E-NO and E-N . The BAL fluid concentrations of interleukin-8, thromboxane A2, and prostacyclin were similar in the two endotoxin-treated groups . Endotoxin caused extensive morphologic lung damage regardless of NO inhalation . CONCLUSIONS: The increase in pulmonary arterial pressure and deterioration of oxygenation were less in endotoxin-exposed rabbits receiving NO inhalation compared with those receiving N2 . Accumulation of neutrophils and platelets in the lung, morphological lung damage, and the release of cytokines and prostanoids were observed in the E-NO group . However, we are unable to extrapolate these results directly to the human clinical setting because of the short observation period, the use of only one dose of NO, and the species difference.

Antonie Van Leeuwenhoek, 1997 Mar, 71(3), 249 - 55
The base analog 6-N-hydroxylaminopurine (HAP) mutagenesis is dependent on the integrity of the uvsE, uvsF and uvsB genes in Aspergillus nidulans; Babudri N et al.; Most of the available data in lower eukaryotes are consistent with the idea that base analogs-induced mutagenesis is due to the mis-pairing properties of these compounds, which, in turn, is due to a shift in the tautomeric equilibrium of the molecule . A tautomeric shift may in fact lead to mismatches which, at least in Escherichia coli, can be repaired by genes involved in the post-replicative mismatch repair whose activity is necessary to control spontaneous mutagenesis . In filamentous fungi, such as Aspergillus nidulans, nothing is known about the repair of base pairing mistakes after base analogs treatment . For this reason, we have decided to screen UV-sensitive Aspergillus nidulans mutants for their mutagenic response to 6-N-hydroxylaminopurine (HAP) . We have shown that three mutations (uvsB, uvsC and uvsE), which enhance the UV-sensitivity of germinating conidia, cause a lower mutagenic response to HAP . On the other hand, the uvsH mutation, has no effect on HAP-induced mutagenesis.

Antonie Van Leeuwenhoek, 1997 Mar, 71(3), 231 - 7
Differences in isoenzyme patterns of axenically and monoxenically grown Acanthamoeba and Hartmannella; Weekers PH et al.; Axenically and monoxenically grown Acanthamoeba castellanii, Acanthamoeba polyphaga and different isolates of Hartmannella vermiformis strains were examined by polyacrylamide isoelectric focusing in the pH range 3-10 . Isoenzyme patterns of acid phosphatase (AP), propionyl esterase (PE), malate dehydrogenase (MDH), alcohol dehydrogenase (ADH), glucose phosphate isomerase (GPI) and phosphoglucomutase (PGM) were compared . Zymograms were used to reveal differences in typical isoenzyme patterns between axenically and monoxenically grown amoebae and to compare axenically grown A . castellanii, A . polyphaga and H . vermiformis . Comparison of zymograms for AP, PE and MDH between axenically grown Acanthamoeba and Hartmannella strains revealed different isoenzyme patterns . Acanthamoeba showed strong bands for ADH and extremely weak bands for GPI and PGM, while Hartmannella lacked ADH but possessed bands for GPI and PGM . Comparison of zymograms from axenically and monoxenically grown amoebae revealed a lower intensity and even lack of typical isoenzyme bands in lysates from monoxenic cultures . The observed changes in typical isoenzyme patterns induced by the bacterial substrate can influence the correct isoenzymatic typing of different strains in clinical and phylogenetic studies.

Antonie Van Leeuwenhoek, 1997 Mar, 71(3), 217 - 30
The growth rate control in Escherichia coli at near to maximum growth rates: the A-stat approach; Paalme T et al.; The growth characteristics of Escherichia coli K-12 in the continuous culture with a smooth increase in the dilution rate (A-stat) of various carbon sources (glucose, acetate, succinate, glycerol, lactate, acetate + succinate, casamino, acids + glucose) were studied . For all substrates studied the maximum value of specific respiration rate, QO2, remained between 14-18 mmol O2 h-1 g dwt-1 and the maximum growth rate varied from 0.22 h-1 on acetate to 0.77 h-1 on glucose + casamino acids . After the respiratory capacity of the cells was exhausted at growth rates mu > mu crit, the growth yield YXO2, increased slightly when the dilution rate increased . The maximum growth rate of Escherichia coli K12 was dependent on growth yield, respiratory capacity and glycolytic capacity of the strain . Analysis of the cultivation data using a stoichiometric flux model indicated that ATP synthesis in E . coli exceeds by two-fold that (theoretically) required to build up biomass . The experimental value of mATP < 4 mmol ATP h-1 g dwt-1 determined from A-stat cultivation data was low compared with the calculated 'unproductive hydrolysis' of ATP (64-103 mmole ATP g dwt-1).

Res Virol, 1997 Mar-Apr, 148(2), 161 - 4
An improved phage display vector for antibody repertoire cloning by construction of combinatorial libraries; Burioni R et al.; Phagemid pComb3 is a widely used vector for molecular cloning of the antibody repertoire and for production of phage display libraries . However, in practical use, the utilization of this vector has some drawbacks . In this work we describe the construction of pComb3/TIG, an improved, easily manipulated vector for the cloning and display of antibody fragment libraries on the surface of filamentous phage . The two small "stuffer" fragments at the cloning sites were replaced with long DNA fragments, for easier differentiation of the correctly cut forms of the vector . Moreover, in pComb3/TIG the fragment at the heavy-chain-fragment cloning site contains an acid phosphatase-encoding gene . This feature allows the easy distinction of the Escherichia coli cells containing the unmodified form of the phagemid instead of the heavy-chain fragment coding cDNA in a simple plate histochemical assay.

Mol Biochem Parasitol, 1997 Mar, 85(1), 41 - 51
A putative Plasmodium falciparum exported serine/threonine protein kinase; Kun JF et al.; An 8kb gene coding for a putative serine/threonine protein kinase from Plasmodium falciparum has been cloned and sequenced . It is arranged in two exons: exon I is 2 kb and exon II is 5.6 kb . The gene codes for a large protein of 2510 amino acids . Antibodies raised against a fusion protein were used to localize the putative kinase . By immunofluorescence microscopy, it was found in the cytoplasm of infected red cells . By immunoelectron microscopy it was associated with membranous structures in the red cell and with the red cell membrane, particularly at parasite-induced knobs . This is the first putative protein kinase of P . falciparum to be exported from the parasite into its host cell.

Plant J, 1997 Mar, 11(3), 429 - 41
Cinnamoyl CoA reductase, the first committed enzyme of the lignin branch biosynthetic pathway: cloning, expression and phylogenetic relationships; Lacombe E et al.; Cinnamoyl CoA:NADP oxidoreductase (CCR, EC 1.2.1.44) catalyzes the conversion of cinnamoyl CoA esters to their corresponding cinnamaldehydes, i.e . the first specific step in the synthesis of the lignin monomers . The cloning of a cDNA encoding CCR in Eucalyptus gunnii (EUCCR) is reported here . The identity of the EUCCR cDNA was demonstrated by comparison with peptide sequence data from purified CCR and functional expression of the recombinant enzyme in Escherichia coli . Sequence analysis revealed remarkable homologies with dihydroflavonol-4-reductase (DFR), the first enzyme of the anthocyanin biosynthetic pathway . Moreover, significant similarities were found with mammalian 3 beta-hydroxysteroid dehydrogenase and bacterial UDP-galactose-4-epimerase, suggesting that CCR shared a common ancestor with these enzymes and can therefore be considered as a new member of the mammalian 3 beta-hydroxysteroid dehydrogenase/ plant dihydroflavonol reductase superfamily . In Eucalyptus gunnii, CCR is encoded by one gene containing four introns whose positions are similar to those of introns I, II, III and V in DFR genes from dicots . In agreement with the involvement of CCR in lignification, the CCR transcript was shown to be expressed in lignified organs, i.e . root and stem tissues, and was localized mainly in young differentiating xylem . On the other hand, its abundance in Eucalyptus leaves suggests that monolignols may be precursors of end products other than lignins . This first characterization of a gene corresponding to CCR opens new possibilities to genetically engineer plants with lower lignin content . This is particularly important for woody plants such as Eucalyptus which are used for pulp making.

Plant Mol Biol, 1997 Mar, 33(5), 931 - 3
Soybean DapA mutations encoding lysine-insensitive dihydrodipicolinate synthase; Silk GW et al.; In plants, the rate-limiting step in the pathway for lysine synthesis is catalyzed by the enzyme dihydrodipicolinate synthase (DS), which is encoded by the DapA gene . We previously cloned the soybean (Glycine max cv . Century) DapA gene in Escherichia coli to express functional soybean DS protein . Like the wild-type soybean DS enzyme, the DS activity encoded by the cloned gene was extremely sensitive to feedback inhibition by micromolar concentrations of lysine . Three mutants of the soybean DapA gene were constructed using PCR: one with a single amino acid substitution at codon 104, another with a single amino acid substitution at codon 112, and a mutant containing both modifications . When expressed in E . coli, the mutant DS activities were insensitive to lysine at concentrations up to 1 mM.

Mol Psychiatry, 1997 Mar, 2(2), 122 - 4
Regulation of ICE activity and ICE isoforms by LPS; Tingsborg S et al.; Interleukin 1 beta (IL-1 beta) is a highly inducible proinflammatory cytokine . It is processed to its mature, secreted 17-kDa form by a cysteine endoprotease; the interleukin 1 beta converting enzyme (ICE) . Regulation of IL-1 beta levels can be achieved both at transcriptional and translational level and in particular at the posttranslational, ICE catalysed, level . Thus, we examined ICE activity in rats under conditions of systemic stimulation by intraperitoneal (i.p.) injections of lipopolysaccharide (LPS) from E . coli, which are known to dramatically alter IL-1 beta mRNA and protein levels . ICE mRNA levels and endoprotease activity have also been found to be differentially regulated in the rat adrenal gland and rat brain after i.p . injections of LPS . An induction in ICE mRNA levels could be seen in the adrenal gland, the pituitary and in the hypothalamus after LPS treatment as measured by reverse transcription-polymerase chain reaction (RT-PCR), whereas the ICE endoprotease activity was increased in the pituitary and decreased in the hippocampus and in the adrenal gland . The discrepancy between increased mRNA level for ICE and decreased enzyme activity in the adrenals might be explained by the induction of ICE isoforms, some of which might be inhibitory for the enzyme activity and induced by LPS, yielding as a net effect a suppression of ICE activity.

Mol Microbiol, 1997 Mar, 23(6), 1303 - 15
Autoregulation of the Escherichia coli replication initiator protein, DnaA, is indirect; Smith RW et al.; The expression of dnaA is autoregulated, in that transcription of the gene increases when DnaA is inactivated (and initiation of replication prevented) and decreases when DnaA is supplied in excess . However, the inactivation of DnaA does not necessarily lead to increased DnaA production, as dnaA(Ts; temperature sensitive) strains which are integratively suppressed by derivatives of the plasmid R1 do not show temperature-induced derepression . Several possible explanations for this unanticipated behaviour were considered and ruled out . We suggest here that the completion of a critical step in initiation may prevent dnaA derepression: although DnaA would be required to complete this step at oriC, DnaA(Ts) would be sufficient at the R1 origin . Autoregulation of dnaA has been attributed to the binding of DnaA at a consensus binding site in the dnaA promoter region . We show here, using reporter systems, that this DnaA-binding site is not required for the autoregulatory response . We find, further, that replacement of the chromosomal dnaA gene with one containing a mutated binding site causes no demonstrable phenotypic change: cells with the mutant gene show no disadvantage in competition with dnaA+ cells.

Mol Microbiol, 1997 Mar, 23(6), 1215 - 20
Replication of minichromosomes in a host in which chromosome replication is random; Eliasson A et al.; Minichromosomes are plasmids with the origin of chromosome replication, oriC, as their only origin of replication . In Escherichia coli, minichromosomes are compatible with the chromosome and replicate in a cell-cycle-specific manner at the same time as oriC located on the chromosome initiates replication . In int strains, oriC has been inactivated and replaced by a plasmid origin . Because plasmids control their own replication, chromosome replication is uncoupled from the normal cell-cycle control and is random with respect to the cell cycle in the int strains . We have used an intP1 strain to address the question of whether minichromosome replication is coupled to the replication of the chromosome or is governed by cell-cycle-specific signals . Minichromosome replication was analysed by density-shift experiments and found not to be random in the randomly replicating intP1 host . This suggests that the cell-cycle-specific control functions of oriC replication are operating also in the intP1 strain.

Mol Microbiol, 1997 Mar, 23(6), 1193 - 202
DnaA protein stimulates polA gene expression in Escherichia coli; Quinones A et al.; The polA gene of Escherichia coli encodes DNA polymerase I that is involved in DNA replication and repair . Despite the wide knowledge about structure and function of DNA polymerase I, there is little insight into the regulatory mechanisms involved in polA expression . DnaA is the initiator protein for DNA replication in E . coli . There are two putative DnaA-binding sites within the extended promoter region of polA . In this work we studied the influence of altered levels of DnaA protein on polA expression . We found that DnaA overproduction increases polA expression in stationary-phase cultures . The stimulation effect was independent of rpoS, which encodes the sigma factor for stationary-phase-inducible genes . However, it was modulated by ppGpp . Comparative S1 analyses revealed that the induction was based on transcriptional stimulation . Footprinting experiments demonstrated that DnaA binds only to the proximal DnaA box near the polA promoter . These results suggest an additional role for DnaA as transcriptional activator of polA at least under certain physiological conditions.

Mol Microbiol, 1997 Mar, 23(6), 1181 - 91
Maltose-binding protein interacts simultaneously and asymmetrically with both subunits of the Tar chemoreceptor; Gardina PJ et al.; The Tar chemotactic signal transducer of Escherichia coli mediates attractant responses to L-aspartate and to maltose . Aspartate binds across the subunit interface of the periplasmic receptor domain of a Tar homodimer . Maltose, in contrast, first binds to the periplasmic maltose-binding protein (MBP), which in its ligand-stabilized closed form then interacts with Tar . Intragenic complementation was used to determine the MBP-binding site on the Tar dimer . Mutations causing certain substitutions at residues Tyr-143, Asn-145, Gly-147, Tyr-149, and Phe-150 of Tar lead to severe defects in maltose chemotaxis, as do certain mutations affecting residues Arg-73, Met-76, Asp-77, and Ser-83 . These two sets of mutations defined two complementation groups when the defective proteins were co-expressed at equal levels from compatible plasmids . We conclude that MBP contacts both subunits of the Tar dimer simultaneously and asymmetrically . Mutations affecting Met-75 could not be complemented, suggesting that this residue is important for association of MBP with each subunit of the Tar dimer . When the residues involved in interaction with MBP were mapped onto the crystal structure of the Tar periplasmic domain, they localized to a groove at the membrane-distal apex of the domain and also extended onto one shoulder of the apical region.

Mol Microbiol, 1997 Mar, 23(6), 1133 - 45
Different structures of selected and unselected araB-lacZ fusions; Maenhaut-Michel G et al.; Formation of araB-lacZ coding-sequence fusions is a key adaptive mutation system . Eighty-four independent araB-lacZ fusions were sequenced . All fusions carried rearranged MuR linker sequences between the araB and lacZ domains indicating that they arose from the standard intermediate of the well-characterized Mu DNA rearrangement process, the strand transfer complex (STC) . Five non-standard araB-lacZ fusions isolated after indirect sib selection had novel structures containing back-to-back inverted MuR linkers . The observation that different isolation procedures gave rise to standard and non-standard fusions indicates that cellular physiology can influence late steps in the multi-step biochemical sequence leading to araB-lacZ fusions . Each araB-lacZ fusion contained two novel of DNA junctions . The MuR-lacZ junctions showed 'hot-spotting' according to established rules for Mu target selection . The araB-MuR and MuR-MuR junctions all involved exchanges at regions of short sequence homology . More extensive homology between MuR and araB sequences indicates potential STC isomerization a resolvable four-way structure analogous to a Holliday junction . These results highlight the molecular complexity of araB-lacZ fusion formation, which may be thought of as a multi-step cell biology process rather than a unitary biochemical reaction.

Arzneimittelforschung, 1997 Mar, 47(3), 329 - 34
Absorption, kinetics, antibody-bound and free serum determination of a 14C-labeled Escherichia coli extract after single oral administration in rats; van Dijk A et al.; The bioavailability of the major fraction of a 14C-labeled Escherichia coli extract (OM-89) of high molecular mass > 30 kD (HEC) was investigated in rats after a single oral administration of a dose of 100 mg/kg b.w . The determination of radioactivity in blood and plasma showed that HEC was absorbed by the digestive tract, either in its original form or partly degraded . In plasma, the radioactivity reached a Cmax value after 4 h, equivalent to an extract concentration of 31.7 micrograms/g of plasma with an elimination half live of 33 h . High molecular weight molecules of HEC (> 30 kD) were found by radiochromatography in rat serum 4 h after administration . Immunoprecipitation in the serum showed that HEC retains intact determinants throughout the digestive and absorptive processes . It is therefore absorbed either in its original form or partly degraded . A high incorporation of radiolabel was found in liver, secretory glands (adrenals, thyroid gland, pancreas), and in lymphoid organs (spleen, mesenteric lymph nodes).

Arzneimittelforschung, 1997 Mar, 47(3), 325 - 8
Absorption kinetics of a 14C-labelled Escherichia coli extract after oral administration in mice; Burckhart MF et al.; The fate of orally administered total 14C-labelled Escherichia coli extract (TEC, OM-89) was compared to that of its major 14C-labeled high molecular weight fractions (HEC, > 30 kD) in mice . High molecular weight substances (> 30 kD) were observed in blood 1 h after oral administration of the products, TEC and HEC . This suggests absorption from the digestive tract of the total E . coli extract . Blood clearance after 24 h indicates that the product was rapidly eliminated or taken up by the tissues . Its highest organ distribution among the measured organs was found in the liver, followed by the spleen and the Peyer's patches . Low molecular weight fractions (LEC, < 30 kD) were also used as control . This pilot study provides a rationale for in vivo pharmacological investigations with oral administration of the E . coli immunomodulating extract OM-89.

Arzneimittelforschung, 1997 Mar, 47(3), 316 - 9
Genotoxicity testing of an aqueous mistletoe extract in vitro; Mengs U et al.; An aqueous mistletoe extract (AME, the active principle of Lektinol) was investigated for its genotoxic potential in a wide range of in vitro studies, according to the actual international standard guidelines . Gene mutation tests on bacteria and mammalian cells in vitro all gave negative results . From the findings of cytogenetic studies, there was no indication of a clastogenic activity in human lymphocytes . A cell transformation assay revealed no malignant changes of Syrian hamster embryo cell cultures . In summary, the results suggest that AME has no genotoxic potential in vitro.

Biotechnol Prog, 1997 Mar-Apr, 13(2), 144 - 50
Effect of inclusion body contaminants on the oxidative renaturation of hen egg white lysozyme; Maachupalli-Reddy J et al.; The effect of typical contaminants in inclusion body preparations such as DNA, ribosomal RNA, phospholipids, lipopolysaccharides, and other proteins on renaturation rate and yield of hen egg white lysozyme was investigated . Separate experiments were conducted in which known amounts of individual contaminants were added to test their effect on renaturation kinetics . On the basis of a simplified model for the kinetic competition between folding and aggregation, it was found that none of the above contaminants had an effect on the rate of the folding reaction, but some of them significantly affected the rate of the aggregation reaction and, thus, the overall renaturation yield . While ribosomal RNA did not seem to affect the aggregation reaction, plasmid DNA and lipopolysaccharides increased the aggregation rate, resulting in a decrease of about 10% in the overall renaturation yield . Phospholipids were found to improve refolding yields by about 15% by decreasing the overall rate of the aggregation reaction without affecting the rate of the folding reaction . Proteinaceous contaminants which aggregate upon folding, such as beta-galactosidase and bovine serum albumin, were found to significantly decrease renaturation yields by promoting aggregation . This effect was strongly dependent on the concentration of the proteinaceous impurity . On the other hand, the presence of refolding ribonuclease A, which does not significantly aggregate upon folding under the conditions tested in this work, did not affect the renaturation kinetics of lysozyme, even at concentrations as high as 0.7 mg/mL.

Biotechnol Prog, 1997 Mar-Apr, 13(2), 132 - 43
Mathematical model of the lac operon: inducer exclusion, catabolite repression, and diauxic growth on glucose and lactose; Wong P et al.; A mathematical model of the lactose (lac) operon was developed to study diauxic growth on glucose and lactose . The model includes catabolite repression, inducer exclusion, lactose hydrolysis to glucose and galactose, and synthesis and degradation of allolactose . Two models for catabolite repression were tested: (i) cyclic AMP (cAMP) synthesis inversely correlated with the external glucose concentration and (ii) synthesis inversely correlated with the glucose transport rate . No significant differences in the two models were observed . In addition to synthesis, degradation and secretion of cAMP were also included in the model . Two models for the phosphorylation of the glucose produced from lactose hydrolysis were also tested: (i) phosphorylation by intracellular hexokinase and (ii) secretion of glucose and subsequent phosphorylation upon transport back into the cell . The latter model resulted in weak catabolite repression when the glucose produced from lactose was transported out of the cell, whereas the former model showed no catabolite repression during growth on lactose . Parameter sensitivity analysis indicates the importance of key parameters to lac operon expression and cell growth: the lactose and allolactose transformation rates by beta-galactosidase and the glucose concentrations that affect catabolite repression and inducer exclusion . Large values of the allolactose hydrolysis rate resulted in low concentrations of allolactose, low-level expression of the lac operon, and slow growth due to limited import and metabolism of lactose; small values resulted in a high concentration of allolactose, high-level expression of the lac operon, and slow growth due to a limiting concentration of glucose 6-phosphate formed from allolactose . Changes in the rates of all beta-galactosidase-catalyzed reactions showed similar behavior, but had more drastic effects on the growth rate . Changes in the glucose concentration that inhibited lactose transport could extend or contract the diauxic growth period during growth in the presence of glucose and lactose . Moreover, changes in the glucose concentration that affected catabolite repression affected the cAMP levels and lac operon expression, but had a lesser effect on the growth rate.

Vet Microbiol, 1997 Mar, 54(3-4), 321 - 8
Colonization antigens of enterotoxigenic Escherichia coli strains isolated from piglets in Spain; Garabal JI et al.; Eighty-eight enterotoxigenic E.coli strains isolated from 69 pigs with enteric infections (diarrhoea or oedema disease) were investigated for the presence of F4 (K88), F5 (K99), F6 (987P) and F41 colonization antigens . The commonest colonization antigen was F6 (987P), which was detected in ETEC strains from 31.9% pigs, followed by F5 (K99) 11.6%, F4 (K88) 10.1% and F41 8.7% . Presence of F6 (987P) and F5 (K99) fimbriae was statistically associated (0.025 > p < 0.005) with diarrhoeic piglets younger than 15 days . F4 (K88) colonization antigen was only expressed by ETEC isolated from piglets older than 15 days . 90.5% of ETEC isolated from 90.0% of piglets younger than 15 days expressed F5 (K99), F6 (987P) or F41 antigens, whereas only 31.3% ETEC isolated from piglets older than 15 days were positive for F4 (K88), F5 (K99), F6 (987P) or F41 antigens (p < 0.001) . None of the ETEC pigs with oedema disease produced any of the four colonization antigens . ETEC bearing colonization antigens were associated with particular serogroups and toxic phenotypes, whereas 4P- ETEC strains showed diverse phenotypic characteristics.

Biosci Biotechnol Biochem, 1997 Mar, 61(3), 545 - 6
High-level expression of maize C4-type phosphoenolpyruvate carboxylase in Escherichia coli and its rapid purification; Dong LY et al.; Maize C4-type phosphoenolpyruvate carboxylase (PEPC) was expressed in E . coli with the pET32 system . The expressed fusion PEPC was active and its amount comprised more than 10% of total soluble protein . The specific activity increased by about 45-fold, compared with our previous system {S . Yanagisawa and K . Izui, Agric . Biol . Chem., 54, 241-243 (1990)} . The fusion PEPC was rapidly purified with His bind metal chelation resin, showing a single band on SDS-PAGE . Moreover, the tag domain fused at the N-terminus did not have any effect on catalytic and regulatory properties of PEPC.

Biosci Biotechnol Biochem, 1997 Mar, 61(3), 506 - 9
Substitutions of alanine for cysteine at a reactive thiol site and for lysine at a pyridoxal phosphate binding site of 1-aminocyclopropane-1-carboxylate deaminase; Murakami T et al.; 1-Aminocyclopropane-1-carboxylate (ACC) deaminase catalyzes the cyclopropane ring fragmentation and deamination of ACC . Replacement of cysteine with alanine at a reactive thiol site, Cys-162, of ACC deaminase did not affect the enzyme activity, in spite of the previous result that modification of Cys-162 caused complete loss of the enzyme activity . Substitution of glycine or valine for the cysteine residue gave a higher Km for ACC without a significant change of the K0, indicating that changes of the amino acid side chain had structural effects on substrate binding . Replacement of lysine with alanine at the pyridoxal phosphate (PLP) binding site of the ACC deaminase caused a lower content of PLP and loss of detectable activity of ACC deamination . This mutant enzyme, K51A, showed absorption peaks at 330 nm and 405 nm . The peak at 405 nm was shifted to about 425 nm by the addition of ACC, D-, L-alanine, and D-, L-serine . The formation of aldimine complexes indicated by the spectral shift was reversible . It is suggested that lysine 51 affects the formation of holoenzyme and is important in catalysis.

Biochem Mol Biol Int, 1997 Mar, 41(3), 547 - 54
Behavior of uridine phosphorylase from Escherichia coli K-12 in hydrated reversed micelles of surfactant in organic solvent; Kurganov BI et al.; The catalytic activity of uridine phosphorylase from Escherichia coli K-12 entrapped in hydrated reversed micelles of aerosol OT (AOT) in octane has been studied as a function of the degree of hydration of micelles . It was shown that the catalytic activity reaches maximum values at ratios {H2O}/{AOT} equal to 8.4, 12.8, 16.1, and 18.6 . On the basis of sedimentation data the conclusion has been made that the maximums of the catalytic activity of uridine phosphorylase correspond to monomeric, dimeric, trimeric, and tetrameric forms of the enzyme.

Int Immunol, 1997 Mar, 9(3), 451 - 9
Stimulation of human cytotoxic T cells with HIV-1-derived peptides presented by recombinant HLA-A2 peptide complexes; Walter JB et al.; HLA-A2 heavy chain and beta 2-microglobulin were expressed in Escherichia coli, and refolded in the presence of peptides derived from HIV-1 RT and gag proteins . When recombinant HLA-A2 molecules were attached to cells lacking HLA-A2, the cells became susceptible to lysis by HLA-A2-restricted cytotoxic T lymphocyte (CTL) clones specific for peptides derived from RT and gag proteins . Limiting dilution analyses of peripheral blood mononuclear cells from HIV-1-infected individuals showed that the recombinant HLA-A2 peptide complexes covalently immobilized on microspheres stimulated the development of HLA-A2 peptide-specific CTL . Preformed HLA-peptide complexes may provide an alternative to immunization procedures that depend upon intracellular processing of antigen to elicit T cell responses.

Cell Biol Toxicol, 1997 Mar, 13(3), 141 - 53
Synthetic hispidin, a PKC inhibitor, is more cytotoxic toward cancer cells than normal cells in vitro; Gonindard C et al.; The trypanocidal activity of naturally occurring 6-(3,4-dihydroxystyryl)-4-hydroxy-2-pyrone (hispidin) prompted us to examine its cytotoxic activity toward normal and cancerous cells in culture . Hispidin synthesized in our laboratory to a high degree of purity (checked by 1H and 13C NMR spectroscopy) was shown to be cytotoxic (between 10(-3) mol/L and 10(-7) mol/L) toward normal human MRC-5 fibroblasts, human cancerous keratinocytes (SCL-1 cell line), and human cancerous pancreatic duct cells (Capan-1 cell line) . Interestingly, addition of hispidin in three successive doses (between 10(-5) mol/L and 10(-7) mol/L) led to a 100-fold increase in activity with an enhanced activity on cancer cells compared to normal cells (50%) . Synthetic hispidin was found to inhibit isoform beta of protein kinase C (IC50 of 2 x 10(-6) mol/L), but not E . coli and placental type XV alkaline phosphatases . The enhanced activity of hispidin toward the cancerous cell lines is discussed.

Am J Physiol, 1997 Mar, 272(3 Pt 2), R857 - 61
Local cytokine induction by LPS in the rat air pouch and its relationship to the febrile response; Miller AJ et al.; Peripheral induction of cytokines is a critical event in the induction of febrile responses . The sequence of induction and site of action of these cytokines, however, remain unclear . The objective of the present study was to investigate the kinetics of local and systemic production of interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF), with the aim of identifying the relationship between these cytokines and the febrile response induced by injection of lipopolysaccharide (LPS) into a subcutaneous air pouch in the rat . Intrapouch injection of LPS induced dose-dependent fevers and increases in the concentration of bioactive IL-6 in the plasma . Further studies using 100 microg/kg LPS demonstrated significant increases in local (air pouch) concentrations of bioactive IL-1, TNF and IL-6, and circulating IL-6 . No significant increases in TNF or IL-1 were detected in the plasma of the same animals . Local TNF was induced rapidly and peaked 1 h after LPS injection . The kinetics of local IL-1 and IL-6 induction were similar and both peaked after 3 h . The rise in local IL-6 preceded that of plasma IL-6 and reached a peak concentration that was 25-fold higher than that observed in the plasma . The data indicate that IL-1 and TNF act locally at the site of inflammation and that locally induced IL-6 is the important systemic mediator of the response.

Rinsho Byori, 1997 Mar, 45(3), 224 - 8
{Gene diagnosis with an affinity sensor, BIACORE--principle and applications}; Gotoh M et al.; We are developing new techniques for detecting point mutations by DNA-DNA hybridization and DNA-protein interaction analysis with an affinity sensor, BIACORE . To detect point mutations by the hybridization method using synthetic oligonucleotides, we already found that the length of the probe and the location of mismatches were important . The PCR products of the N-ras gene derived from Hep G2 cells, which have heterozygous point mutations at codon 61 in the gene, were analyzed and the point mutations were detected with 13-mer probes . We suggest that the detection method using DNA-DNA hybridization is useful for detecting known point mutations . However, detect unknown mutations, E . coli mismatch recognition protein, MutS, was employed . All mismatches in immobilized 20 base pairs of double-strand DNA could be detected by MutS binding . We have started to apply of the MutS to the detection of point mutations in PCR products.

Nippon Rinsho, 1997 Mar, 55(3), 747 - 50
{A novel marker of development of HUS associated with enterohemorrhagic Escherichia coli infection--thrombomodulin levels in the blood}; Nagayama K; Vero toxins-mediated vascular endothelial cell damage is thought to play a central and important role in the pathogenesis of HUS associated with EHEC infection . Thrombomodulin (TM) is an endothelial membrane protein inhibiting procoagulant activities of thrombin . Elevation of soluble TM in the blood is regarded a possible marker for endothelial cell damage . To test whether serum TM acts as an indicator for HUS, we evaluated serum TM in 21 children with HUS, 31 children with hemorrhagic colitis(HC), and 25 healthy age-matched controls . TM levels in acute HUS patients (6.89 +/- 1.91 ng/ml) were significantly higher when compared with in acute HC patients(3.15 +/- 0.37 ng/ml) and in the controls (2.77 +/- 0.42 ng/ml) (p < 0.001, respectively) . This result indicated that serum TM may be an useful marker for the development of HUS.

Nippon Rinsho, 1997 Mar, 55(3), 741 - 6
{Rapid detection of the Escherichia cori verotoxin gene using fluorescence polarization}; Tsuruoka M et al.; The effects of NaCl concentration, temperature and base-pair mismatches on the hybridization of two complementary single-stranded DNA 24-mers were investigated using a fluorescence polarization method . Over a temperature range of 46 degrees C to 56 degrees C in 0.8 M NaCl it was found that hybridization was essentially complete in under 10 minutes and that rapid in vitro determination of the target DNA was possible when the sample DNA had three or less base-pair mismatches in the 24-mer sequence . Polarization measurements on positive and negative samples showed excellent agreement with results obtained from electrophoresis . Under our optimized conditions, a 23 base-pair single-stranded DNA sequence of the Verotoxin gene (VT2) of Escherichia coli, previously multiplied using PCR (40 cycles), could be detected within 10 minutes.

Nippon Rinsho, 1997 Mar, 55(3), 736 - 40
{Brain lesions in rabbits given an intravenous injection of verotoxin 2 and protection by anti-VT2 antibody}; Fujii J et al.; Magnetic resonance imaging was obtained to determine the effects of VT2 toxemia on the rabbit's CNS . The first lesion was noted at 24 h in the hypothalamic area of all experimental animals . The rabbits accompanied with the brain stem lesion were dead within 6 days . We examined the integrity of cerebrospinal fluid-brain barrier (CBB) using a tracer . The tracer was detected throughout the cytoplasm of the ependymal cell layer covering the third ventricle after intrathecal injection of the tracer, which means a deterioration of CBB . Furthermore, we examined whether anti-VT2 antibody injected intrathecally protects rabbits from brain damage . All the rabbits survived when they were given an intrathecal injection of rabbit anti-VT2 antibody 2h before the intravenous injection of VT2.

Nippon Rinsho, 1997 Mar, 55(3), 731 - 5
{Verotoxin induced hemolytic uremic syndrome: pathophysiology of neurological involvement}; Takagi C et al.; Hemolytic uremic syndrome (HUS) is caused by endothelial cell damages . Ninety percent of children with HUS have verotoxin-producing E.coli infection . Verotoxin binds to glycolipid receptors globotriaosyl ceramide (Gb3), and the difference of Gb3 expression level in each organ would lead to specific organ involvement . The receptors are expressed in human renal cortex and medulla . The expression level of Gb3 in normal human brain has not been characterized completely . However involvement of central nervous system is a severe complication of HUS . Spreading of microvascular thrombosis caused by combined effects of lipopolysaccharide, cytokine, enhanced shear stress, and verotoxin would play a major role in the development of central nervous dysfunction.

Nippon Rinsho, 1997 Mar, 55(3), 721 - 5
{Renal pathology of VTEC related HUS}; Shigematsu H; Renal histopathology of verotoxin producing escherichia coli(VTEC) related hemolytic uremic syndrome (HUS) was described . Endothelial damage is expressed in renal small arteries and glomeruli as luminal narrowing due to endothelial swelling and subendothelial edema . Extracellular matrix shows reticularization and/or mesangiolysis in glomeruli . Although HUS and thrombotic thromocytopenic purpura (TTP) is recently considered as a single entity, the tissue expression is different . In the latter endothelial proliferation is profound resulting in arteriolar deteriolation such as glomeruloid structure.

Nippon Rinsho, 1997 Mar, 55(3), 715 - 20
{Advances in the treatment of hemolytic uremic syndrome (HUS)}; Ito K et al.; The hemolytic uremic syndrome (HUS) is the end result of a variety of etiologic agents that can induce endothelial cell injury and thrombotic microangiopathy (TMA) mostly within the kidney . The typical, post-diarrheal verocytotoxin associated HUS (D + HUS) is the major cause of acute renal failure in children worldwide . In the course of HUS treatment, fluid overload is usually the result of overhydration in the context of oliguria or anuria which cause edema, hypertension, worsening of neurologic signs and cardiac failure . Appropriate and timely use of dialysis has dramatically reduced complications of renal failure and extra-renal complications are now the main causes of mortality and morbidity in D + HUS . The reasons for treatment by infusion of fresh frozen plasma and/or plasmapheresis for D + HUS are theoretical and their therapeutic effects are inconclusive . We believe that plasma administration for regular D + HUS has no value and is potentially harmful . Until new strategies become available in clinical practice, the general consensus for the moment is that careful supportive management with patience is still the most appropriate form of D + HUS therapy.

Nippon Rinsho, 1997 Mar, 55(3), 641 - 5
{Genes involved in the virulence of enterohemorrhagic Escherichia coli}; Izumiya H et al.; Some of the virulent genes of enterohemorrhagic E . coli(EHEC) are described . stx, bfpA, eaeA and ehxA encode an enterocytotoxin homologous to Shiga toxin, bundle-forming pilus, intimin and an RTX toxin, respectively . In pathogenesis, although bfp and eae cause diarrhea, and ehx and stx seem to cause hemorrhagic colitis and hemolytic uremic syndrome, many problems remain for prevention and treatment of these symptoms . In evolution, it is intriguining that the virulent genes of EHEC might be mediated by phages, plasmids and so on, because stx is derived from a bacterio-phage, bfpA is located in a plasmid in enteropathogenic E . coli(EPEC) though it has not yet been cloned in EHEC, eaeA is one of the genes in LEE(locus of enterocyte effacement), a putative genetic cassette ubiquitous for EPEC and EHEC, and ehxA is in a plasmid.

Pediatr Pathol Lab Med, 1997 Mar-Apr, 17(2), 267 - 74
Markers of virulence among prospectively acquired putative enteropathogenic Escherichia coli serogroups; Cimolai N et al.; We assessed the frequency of proposed enteropathogenic virulence factor genes (eaeA and eaf) by genetic amplification for a series of prospectively collected putative enteropathogenic Escherichia coli serogroup isolates that were acquired from the stool specimens of children . Among 102 isolates, eaeA and eaf markers were determined among 27.5% and 4.9%, respectively . Eaf positivity was found to be coexisting in only a minority of eaeA+ E . coli; the eaeA+/eaf- genotype was most common among strains that had evidence of at least one virulence marker . When clinical variables were compared for two groups of patients whose strains did or did not possess eaeA, the eaeA+ group was more likely to have had an acute diarrheal illness (P = .05) and less likely to have had an underlying chronic illness (P = .03) . Localized adherence in vitro was easily recognized for eaeA+/eaf+ E . coli but eaeA+/eaf- isolates were less consistent in manifesting this phenotype . The availability of genetic amplification technologies has the potential to rekindle diagnostic interests in this area, although a rational approach has yet to be defined.

Exp Parasitol, 1997 Mar, 85(3), 249 - 63
Leishmania major: molecular cloning, sequencing, and expression of the heat shock protein 60 gene reveals unique carboxy terminal peptide sequences; Rey-Ladino JA et al.; Heat shock proteins (HSP) in the size range of M(r) 60,000 are major targets of the immune response in vivo . The leishmania heat-inducible proteins of M(r) 65-67,000 are expressed at relatively high levels in infected macrophages (Infection and Immunity 1993, 61, 3265-3272) and may be important targets of the host response . To facilitate further studies concerned with these proteins, the HSP60 gene of Leishmania major was cloned, sequenced, and expressed . A lambdaEMBL-3 L . major genomic library was screened with a PCR-generated DNA probe derived from a highly conserved region of the leishmania HSP60 gene . A single clone that hybridized strongly was characterized . Sequence analysis revealed an open reading frame of 1770 bp encoding a putative polypeptide of 589 amino acids with a predicted size of M(r) 64,790 and with the highest degree of amino acid sequence similarity (56%) to HSP60 from Trypanosoma cruzi . Less extensive amino acid sequence similarity (48%) was observed between that leishmania HSP60 and the corresponding human protein . Notably, significant regions of sequence dissimilarity between the leishmania and human proteins were identified principally within the carboxy-terminal regions of the proteins . The entire coding region of the leishmania HSP60 gene was subcloned into the pET-3a vector and expressed in Escherichia coli . Purified recombinant protein was used to examine sera from patients with tegumentary leishmaniasis from Colombia for the presence of antibodies to HSP60 . Unlike sera from healthy, uninfected controls, sera from patients reacted strongly with recombinant leishmania HSP60 . This recognition had specificity in that these same sera showed little or no reactivity with either recombinant mycobacterial HSP65 or recombinant human HSP60 . These findings indicate that patients with tegumentary forms of leishmaniasis have humoral responses to leishmania HSP60 . Further studies of this protein will clarify its importance as a target of the immune response and as a potential antigen for serodiagnosis.

Exp Parasitol, 1997 Mar, 85(3), 241 - 8
Expression of the microfilarial sheath protein 2 (shp2) of the filarial parasites Litomosoides sigmodontis and Brugia malayi; Conraths FJ et al.; The microfilarial sheaths of the filarial parasites Brugia malayi, Brugia pahangi, and Litomosoides sigmodontis consist of several parasite proteins, probably ranging between 7 and 10 . The gene encoding sheath protein 2 (shp2), which is the object of this study, is transcribed in embryos and in the uterine epithelium; at least in B . malayi, it is translated in both tissues . Apparently, shp2 is synthesized as a monomer, exported by the respective cells, and integrated into the microfilarial sheath . In the sheath, it exists as a highly polymerized molecule cross-linked by cysteine formation and other covalent bonds, presumably epsilon-(gamma-glutamyl)-lysine links.

Plant Physiol, 1997 Mar, 113(3), 997 - 1002
Complementary DNAs encoding eukaryotic-type cytidine-5'-diphosphate-diacylglycerol synthases of two plant species; Kopka J et al.; Cytidine diphosphate (CDP)-diacylglycerol synthase (cytidine triphosphate:phosphatidate cytihyltransferase, EC 2.7.7.41) catalyzes the formation of CDP-diacylglycerol, which is the precursor of phosphatidylinositol, phosphatidylglycerol, and cardiolipin . We report the first cloning, to our knowledge, of two plant cDNAs, StCDS1 and AtCDS1, coding for CDP-diacylglycerol synthase from potato (Solanum tuberosum) and Arabidopsis thaliana, respectively . The two proteins belong to the eukaryotic type of CDP-diacylglycerol synthase and contain eight predicted transmembrane-spanning domains . We analyzed gene expression in shoot and root tissues of potato plants and demonstrated enzyme activity by expression of N-terminally truncated, recombinant StCDS1 in Escherichia coli.

Biol Pharm Bull, 1997 Mar, 20(3), 224 - 9
Characterization of human p33/41 (annexin IV), a Ca2+ dependent carbohydrate-binding protein with monoclonal anti-annexin IV antibodies, AS11 and AS17; Satoh A et al.; p33/41 (annexin IV) is a member of the family of Ca(2+)-dependent phospholipid binding proteins known as annexins . We previously described that bovine kidney p33/41 (annexin IV) has Ca(2+)-dependent carbohydrate binding activity . In this study, we purified human p33/41 (annexin IV) from the HT29, human colon adenocarcinoma cell line, as well as the bovine kidney annexin by affinity chromatography . Then, we prepared recombinant human p33/41 (annexin IV) expressed in Escherichia coli . The apparent size and the Ca(2+)-dependent carbohydrate binding properties of purified recombinant p33/41 (annexin IV) were indistinguishable from those of the bovine kidney protein . We also performed inhibition assays of carbohydrate binding and of phosphatidylserine/phosphatidylcholine liposome binding of recombinant p33/41 (annexin IV) with anti-p33/41 monoclonal antibodies (AS11 and AS17) . We determined the epitopes recognized by the monoclonal antibodies by Western blot analysis using deleted-recombinant p33/41 (annexin IV) . The monoclonal antibodies recognized domain 1 and/or 2 of p33/41 (annexin IV) . The results of the inhibition assays and the determination of the epitope showed that a carbohydrate binding site is located at domains 3 and 4 of p33/41 (annexin IV) and on the cell surface.

Microbiology, 1997 Mar, 143 ( Pt 3), 937 - 45
Determination of the pathway for rhamnose biosynthesis in mycobacteria: cloning, sequencing and expression of the Mycobacterium tuberculosis gene encoding alpha-D-glucose-1-phosphate thymidylyltransferase; Ma Y et al.; The mycobacterial cell wall core consists of an outer lipid layer of mycolic acids connected, via arabinogalactan polysaccharide, to an inner peptidoglycan layer . An alpha-L-rhamnopyranosyl residue has been shown to be a key component linking the mycolated arabinogalactan to the peptidoglycan and, therefore, the biosynthesis of L-rhamnose (Rha) in mycobacteria was investigated as the first step of developing inhibitors of its biosynthesis . Biochemical assays were used to show that dTDP-Rha was synthesized in Mycobacterium smegmatis from alpha-D-glucose 1-phosphate (alpha-D-Glc-1-P) and dTTP by the same four enzymic steps used by Escherichia coli and other bacteria . PCR primers based on consensus regions of known sequences of the first enzyme in this series, alpha-D-Glc-1-P thymidylytransferase (RfbA) were used to amplify rfbA DNA from M . tuberculosis . The entire rfbA gene was then cloned and sequenced . The deduced amino acid sequence revealed a 31362 Da putative protein product which showed similarity to RfbA proteins of other bacteria (59% identity to that found in E . coli) . Sequencing of DNA flanking the rfbA gene did not reveal any of the other rfb genes required for dTDP-Rha biosynthesis . Therefore, the four Rha biosynthetic genes are not clustered in M . tuberculosis . The enzymic activity of the sequenced gene product was confirmed by transformation of E . coli with pBluescript KS(-) containing the rfbA gene from M . tuberculosis . Analysis of enzyme extracts prepared from this transformant revealed an 11-fold increase in alpha-D-Glc-1-P thymidylyltransferase activity.

Microbiology, 1997 Mar, 143 ( Pt 3), 921 - 8
Identification of Mycobacterium paratuberculosis gene expression signals; Bannantine JP et al.; Mycobacterium paratuberculosis promoter-containing clones were isolated from a genomic DNA library constructed in the transcriptional-translational fusion vector pYUB76 . The promoter-containing DNA fragments were identified in the surrogate host Mycobacterium smegmatis by expression of the promoterless lacZ reporter gene of pYUB76 . The expression signals exhibited a wide range of strengths, as indicated by their corresponding beta-galactosidase activities . Eight clones were sequenced and characterized further . Predicted open reading frames and codon usage were identified by computer analysis . Database searching for related sequences using the BLAST method revealed no homologies . Transcriptional activity was measured by slot-blot hybridization with steady-state RNA isolated from lacZ+ M . smegmatis clones . Primer extension analysis identified the transcription start sites within the cloned fragments . The promoter regions characterized in this study were used to establish a consensus promoter sequence for M . paratuberculosis . M . paratuberculosis consensus hexanucleotide sequences of TGMCGT and CGGCCS centred approximately 35 and 10 bp upstream from the transcription startpoints do not correspond to the consensus hexanucleotides of Escherichia coli promoters.

Microbiology, 1997 Mar, 143 ( Pt 3), 847 - 54
Reversion rates in a leuB auxotroph of Escherichia coli K-12 correlate with ppGpp levels during exponential growth; Wright BE et al.; Two isogenic strains of Escherichia coli K-12 differing only in relA, as well as two spoT transductants of the relA- strain, were examined with respect to ppGpp levels and reversion rates of a leuB- allele under nine different conditions . A positive correlation was established between reversion rates and the steady-state concentration of ppGpp during exponential growth . The leuB genes from two leuB- strains (isogenic except for relA) were cloned and sequenced and found to contain a single mutation, namely, a C-to-T transition at nucleotide 857 . This mutation resulted in a serine-to-leucine substitution at amino acid residue 286 of the LeuB protein . PCR products that encompassed the leuB lesion were generated from 53 revertants and then sequenced . Of these revertants, 36 were found to contain nucleotide substitutions that would result in a serine (wild type), valine or methionine at amino acid residue 286 of LeuB, and nearly all of them exhibited generation times similar to wild type . Seventeen of the analysed revertants were found to be suppressors that retained the encoded leucine at residue 286 . The majority of the suppressor mutants exhibited generation times that were significantly longer than wild type.

Microbiology, 1997 Mar, 143 ( Pt 3), 785 - 92
Cra-mediated regulation of Escherichia coli adenylate cyclase; Crasnier-Mednansky M et al.; In Escherichia coli, expression of certain genes and operons, including the fructose operon, is controlled by Cra, the pleiotropic catabolite repressor/activator protein formerly known as FruR . In this study we have demonstrated that cra mutant strains synthesize 10-fold less cAMP than isogenic wild-type strains, specifically when grown in fructose-containing minimal media . The glucose-specific IIA protein (IIAglc) of the phosphotransferase system, which activates adenylate cyclase when phosphorylated, is largely dephosphorylated in cra but not wild-type strains growing under these conditions . Dephosphorylation of IIAglc in cra strains apparently results from enhanced fructose operon transcription and fructose uptake . These conclusions were supported by showing that fructose-grown cra strains possess 2.5-fold higher fructose-1-phosphate kinase activity than fructose-grown wild-type strains . Moreover, artificially increasing fructose operon expression in cells transporting fructose dramatically decreased the activity of adenylate cyclase . The results establish that Cra indirectly regulates the activity of adenylate cyclase by controlling the expression of the fructose operon in cells growing with fructose as the sole carbon source.

Microbiology, 1997 Mar, 143 ( Pt 3), 775 - 83
Overlapping promoters modulate Fnr- and ArcA-dependent anaerobic transcriptional activation of the focApfl operon in Escherichia coli; Kaiser M et al.; The recently identified P6A promoter of the anaerobically inducible focApfl operon of Escherichia coll overlaps the Fnr (fumarate-nitrate reduction regulator)-dependent P6 promoter . The Fnr-binding site of P6 and the -35 hexamer sequence of P6A are shared between the promoters . Inactivation of P6A, through introduction of a -10 hexamer mutation, resulted in enhanced anaerobic induction of operon expression . The dependence on the ArcA (aerobic respiration control regulator) and Fnr transcription factors for anaerobic induction was tested for several focA-lacZ and pfl-lacZ gene fusions . Anaerobic induction became more dependent on Fnr in derivatives lacking a functional P6A promoter compared with wild-type constructs . Moreover, aerobic expression of the focA gene was reduced by the p6A mutation, as was the dependence on ArcA for anaerobic induction . Inactivation of P6 severely reduced Fnr-dependent anaerobic induction, in accord with previous findings . Transcription analyses demonstrated that a mutation in the -10 hexamer sequence of either P6A or P6 did not adversely affect transcription from the remaining promoter . Taken together, these results indicate that the P6A promoter moderates the Fnr-dependent activation of P6 through competition for RNA polymerase binding.

Eur J Pediatr, 1997 Mar, 156(3), 204 - 6
Disseminated Mycobacterium avium infection in a child with decreased tumour necrosis factor production; Tuerlinckx D et al.; Disseminated atypical mycobacterium infection is essentially reported in cellular immunodeficient children . Cell-mediated immunity including cytokines like tumour necrosis factor alpha (TNF) and gamma interferon (IFN) is the most important factor allowing control of the dissemination of Mycobacterium . We report a child with disseminated Mycobacterium avium infection without classical immunodeficiency or HIV infection . Immunological studies revealed a defect of TNF production when the monocytes of the patient were primed with endotoxin (Escherichia coli) . CONCLUSION: This patient represents a further case of possible macrophage defect explaining the susceptibility to intracellular pathogens.

Cancer Gene Ther, 1997 Mar-Apr, 4(2), 113 - 7
Enzyme/prodrug gene therapy approach for breast cancer using a recombinant adenovirus expressing Escherichia coli cytosine deaminase; Li Z et al.; A recombinant adenovirus expressing Escherichia coli cytosine deaminase (AdCD) was constructed with the purpose of exploring its utility for the treatment of breast cancer . Infection of the human breast cancer cell line, MDA-MB-231, with AdCD resulted in high levels of cytosine deaminase enzyme activity . MDA-MB-231 cells infected with AdCD were 1000-fold more sensitive to 5-fluorocytosine (5-FC) than cells infected with a control adenovirus . Cell mixing experiments indicated that only 10% of AdCD-infected cells in a population were needed to induce complete cytotoxicity of noninfectious cells exposed to 5-FC . This suggests that bystander effects play an important role in AdCD-mediated cytotoxicities . Direct injection of AdCD into human breast MDA-MB-231-derived tumors grown as xenografts in nude mice, followed by daily intraperitoneal injection 5-FC was sufficient to inhibit tumor growth . These results suggest that in vivo gene therapy for breast cancer using AdCD is feasible.

J Anim Sci, 1997 Mar, 75(3), 720 - 6
Effect of dietary energy source and immunological challenge on growth performance and immunological variables in growing pigs; Spurlock ME et al.; Forty-eight growing pigs (23 kg BW) were assigned to four treatments (n = 12) arranged as a 2 x 2 factorial . Dietary energy source (conventional {CON} vs high-oil corn {HOC}), with or without an immunological challenge (IC) regimen constituted main effects . The IC regimen consisted of injection of endotoxin (E . coli lipopolysaccharide {LPS}) and vaccination for porcine respiratory and reproductive syndrome (PRRS) . Growth performance data were collected over a 5-wk period and are presented as prechallenge (d 1 to 14; d 1 was the 1st d of the study), challenge (d 15 to 21), and postchallenge (d 22 to 36) periods, and overall . Overall, the pigs fed HOC consumed less feed (P < .11) and gained more efficiently (P < .03) . During the immunological challenge period, ADG was depressed 21% and feed intake 15% (P < .01) . The IC resulted in lower (P < .01) serum alpha-1-acid glycoprotein (AGP) concentrations on d 22, and the magnitude of the reduction was greater in the pigs fed the CON diet (energy source x immune challenge, P < .10) . Serum AGP concentrations remained lower (P < .08) in challenged pigs on d 36 . Immunoreactive prostaglandin concentrations were higher (55%, P < .08) in the pigs fed HOC immediately following the IC period (d 22) . The data reported herein indicate that the performance of pigs fed HOC is satisfactory, and that feeding HOC does not compromise growth performance during or after an immunological challenge.

Photochem Photobiol, 1997 Mar, 65(3), 543 - 9
Near ultraviolet radiation (UVA and UVB) causes a formamidopyrimidine glycosylase-dependent increase in G to T transversions; Palmer CM et al.; In contrast to far-UV (< 290 nm) DNA damage, a large fraction of the DNA damage caused by near-UV is oxygen-dependent, suggesting the involvement of reactive oxygen species (ROS) . The oxidized base 8-oxo-7,8-dihydroguanine (GO) is characteristic of ROS-induced DNA damage and is removed by Fapy (formamidopyrimidine) glycosylase . We have recently shown that Escherichia coli strains deficient in Fapy glycosylase (fpg) are hypersensitive to the lethal effects of UVA but not far-UV (UVC), suggesting lesions recognized by this enzyme may be important premutagenic or lethal lesions generated by near-UV radiation . In this study, we have found that while the far-UV-induced mutation rates of Fapy-deficient and wild-type strains were similar, near-UV (UVA and UVB) was hypermutagenic to a Fapy-deficient strain, causing a dose-dependent increase in induced mutation relative to wild type (up to five-fold at 200 kJ/m2) . Using a plasmid back mutation assay, the predominant near-UV-induced mutations in both wild-type and Fapy-deficient strains were found to be C-->T transitions and G -->T transversions . The former is probably due to replicative bypass of pyrimidine dimers or (6-4) photoproducts that are known to be generated by near-UV, whereas the latter may be due to mispairing of GO lesions with adenine during replication . Consistent with this, the frequency of near-UV-induced G-->T transversions was 16-fold higher in a Fapy-deficient strain than a wild-type strain.

Mol Microbiol, 1997 Mar, 23(5), 987 - 95
Isolation and properties of enhancer-bypass mutants of sigma 54; Syed A et al.; The N-terminal activation domain of Escherichia coli sigma 54 was randomly mutated to provide a library of changes that might allow the required enhancer function to be bypassed . Five clones harbouring mutant sigma factors were obtained that exhibited this property in that they enhanced growth under nitrogen-limiting conditions in cells lacking NtrC . DNA sequence analysis located all mutations to four leucines in a small region between amino acids 25 and 31 . No mutant sigma factors retained the hydrophobic character of the leucine residues . Mutant sigma factors were shown to transcribe in vitro without the need for enhancer binding activator or ATP hydrolysis, confirming the in vivo phenotype . These and other data suggest that a very small set of leucines is critical for keeping polymerase function in check, allowing high responsiveness to physiological induction via enhancer proteins such as NtrC.

Mol Microbiol, 1997 Mar, 23(5), 967 - 77
HoxA is a transcriptional regulator for expression of the hup structural genes in free-living Bradyrhizobium japonicum; Van Soom C et al.; A chromosomally integrated Bradyrhizobium japonicum hoxA mutant is unable to oxidize hydrogen in free-living conditions . Derepressing conditions that induce hydrogenase activity in free-living, wild-type B . japonicum cells cannot induce expression of the hydrogenase structural genes in the hoxA mutant . The DNA-binding capacity of HoxA at the hup promoter region was studied by means of gel retardation . Both heterotrophically growing cells and cells induced to express hydrogenase activity contain a protein that specifically binds to the hup promoter region . Crude protein extracts isolated from a B . japonicum hoxA mutant do not contain this binding compound . The HoxA protein was overexpressed in E . coli and isolated in the form of a maltose-binding protein (MBP)-HoxA fusion . The MBP-HoxA hybrid protein specifically bound to a 50 bp region of the hupSL promoter known to be important for regulation of hupSL expression.

Mol Microbiol, 1997 Mar, 23(5), 909 - 20
The N-terminal domain of colicin E3 interacts with TolB which is involved in the colicin translocation step; Bouveret E et al.; Colicins use two envelope multiprotein systems to reach their cellular target in susceptible cells of Escherichia coli: the Tol system for group A colicins and the TonB system for group B colicins . The N-terminal domain of colicins is involved in the translocation step . To determine whether it interacts in vivo with proteins of the translocation system, constructs were designed to produce and export to the cell periplasm the N-terminal domains of colicin E3 (group A) and colicin B (group B) . Producing cells became specifically tolerant to entire extracellular colicins of the same group . The periplasmic N-terminal domains therefore compete with entire colicins for proteins of the translocation system and thus interact in situ with these proteins on the inner side of the outer membrane . In vivo cross-linking and co-immunoprecipitation experiments in cells producing the colicin E3 N-terminal domain demonstrated the existence of a 120 kDa complex containing the colicin domain and TolB . After in vitro cross-linking experiments with these two purified proteins, a 120 kDa complex was also obtained . This suggests that the complex obtained in vivo contains exclusively TolB and the colicin E3 domain . The N-terminal domain of a translocation-defective colicin E3 mutant was found to no longer interact with TolB . Hence, this interaction must play an important role in colicin E3 translocation.

J Hepatol, 1997 Mar, 26(3), 567 - 73
Decreased endotoxin-binding capacity of whole blood in patients with alcoholic liver disease; Schafer C et al.; BACKGROUND/AIMS: The proinflammatory effects of endotoxemia, which is often observed in alcohol-abusing patients with various degrees of liver disease, may be modulated by changes in the concentration of endotoxin binding factors . Therefore, the plasma endotoxin concentration and the overall endotoxin binding capacity of whole blood were measured in these patients . METHODS: Patients with minor (A1; n=27), more pronounced (A2; n=13), cirrhotic alcoholic liver disease (A3; n=18), and non-alcoholic cirrhosis (NC; n=6), and 15 healthy control persons (HC) were included in the study . Endotoxin plasma levels were determined using a standardized limulus assay . A modified assay was applied to additionally detect tightly bound endotoxin . To measure the endotoxin-binding capacity, aliquots of whole blood were incubated with serial dilutions of endotoxin, supernatants were obtained, and endotoxin retrieval was estimated by addition of limulus lysate, followed by photometric measurement of the maximal reaction velocity (dODmax) . Endotoxin binding capacity equals the endotoxin concentration at which dODmax reaches a predefined threshold . RESULTS: All groups of alcohol abusers had significantly elevated endotoxin plasma levels with a considerable portion of 'bound' endotoxin . Conversely, the endotoxin binding capacity was markedly diminished, mainly in patients with more advanced liver disease (A1: 85.8% of the control value {non-significant vs . controls}; A2: 25.4% {p<0.05}; A3: 43.6% {p<0.02}, NC: 43.2%) . CONCLUSIONS: The endotoxin-binding capacity is diminished in patients with alcoholic and non-alcoholic cirrhosis, as well as in less advanced alcoholic liver disease . Reduced endotoxin binding may contribute to the adverse effects of endotoxemia.

Nephrol Dial Transplant, 1997 Mar, 12(3), 543 - 9
In vitro biocompatibility evaluation of a novel bicarbonate-buffered amino-acid solution for peritoneal dialysis; Jorres A et al.; BACKGROUND: Conventional lactate-buffered peritoneal dialysis fluids containing glucose as the osmotic agent have been shown to compromise important peritoneal host defence functions . The current study employed an in vitro model using activated peripheral blood mononuclear leukocytes (PBMC) for the preclinical biocompatibility assessment of a novel bicarbonate-buffered peritoneal dialysis fluid containing 1.0% amino acids as the osmotic agent . METHODS: PBMC (5 x 10(6)/ml) were pre-exposed (10-30 mm, 37 degrees C) to bicarbonate-buffered 1% amino-acid solution, bicarbonate- or lactate-buffered 1.5% glucose fluid, or control medium (RPMI) . The cells were then washed and stimulated for 2 h at 37 degrees C in RPMI containing 100 ng/ml E.coli endotoxin from strain O55:B5 . The cytokines IL-6 and TNF alpha in cell supernatants were assessed using specific enzyme immunoassays, cytokine mRNA expression by reverse transcription polymerase chain reaction . RESULTS: Short, i.e . 10 min, exposure to conventional, lactate-buffered glucose fluid resulted in a significant and time-dependent inhibition of cytokine release and mRNA expression by activated PBMC, whereas the cytokine response was improved even following prolonged (up to 2 h) exposure to bicarbonate-buffered 1% amino-acid solution or bicarbonate-buffered 1.5% glucose fluid . CONCLUSIONS: Our results suggest that very short, i.e . potentially clinically relevant, exposure to conventional dialysis fluid impairs the cytokine response by activated leukocytes . In this respect, the use of bicarbonate-buffered solutions containing 1.0% amino acids or 1.5% glucose may result in improved biocompatibility properties.

J Crit Care, 1997 Mar, 12(1), 7 - 12
Splanchnic buffering of metabolic acid during early endotoxemia; Kellum JA et al.; PURPOSE: We sought to determine the sites of metabolic acid production and clearance during acute endotoxemia . MATERIALS AND METHODS: In 10 pentobarbital-anesthetized dogs, flow was measured (ultrasonic probes) for the protal vein, hepatic artery, and renal artery . Catheters were inserted into the hepatic vein, pulmonary artery, renal vein and portal vein . Measurements of blood gases and strong ions were obtained from each site during control conditions and after 30 minutes of intravenous infusion of 1 mg/kg of Escherichia coli endotoxin . The total metabolic acid flux across each organ was calculated using the standard base excess formula and the effective strong ion difference method . PaCO2 was maintained by controlled ventilation . RESULTS: Mean arterial pH decreased from 7.34 to 7.22 with acute endotoxemia . Although transvisceral pH gradients revealed net acid release, the source of this was purely respiratory (carbon dioxide) . During early endotoxemia, the gut significantly increased metabolic acid uptake (36.60 +/- 6.60 mmol/h, P < .05) . CONCLUSIONS: We conclude that during early endotoxemia in the dog, the gut is a major site of metabolic acid removal.

Br J Haematol, 1997 Mar, 96(4), 675 - 81
Dose reduction of asparaginase under pharmacokinetic and pharmacodynamic control during induction therapy in children with acute lymphoblastic leukaemia; Ahlke E et al.; The enzyme asparaginase is an important element in the therapy of acute lymphoblastic leukaemia (ALL) . The usual asparaginase dose as prescribed in the ALL-BFM-86/90 treatment protocol for the therapy of ALL is 10,000 IU/m2 at 3 d intervals and had been developed on the basis of the E . coli asparaginase preparation Crasnitin from the Bayer company . Using the described schedule the E . coli asparaginase preparation from the Medac company shows significantly higher biological activity than the Bayer preparation . These findings prompted an attempt to reduce the dose of the Asparaginase medac under careful pharmacokinetic and pharmacodynamic monitoring . At the first step of dose reduction in ALL treatment protocol I, 11 children received 5000 IU/m2 of Asparaginase medac . Another 15 children were given 2500 IU/m2 of the enzyme at the second step of dose reduction . Prior to each asparaginase dose, blood samples were taken to determine amino acids and trough enzyme activity . Concurrent with the asparaginase monitoring, the coagulation parameters were measured . 96% of samples from the first step of dose reduction (5000 IU/m2 every third day) showed complete L-asparagine depletion (< 0.1 microM), the median trough enzyme activity was 265 IU/l . At the second step of dose reduction (2500 IU/m2) complete L-asparagine depletion was seen in 97% of samples, and the median trough enzyme activity was 102 IU/l . Cerebrospinal fluid (CSF) depletion was complete in all samples tested (11/11) . We concluded that an Asparaginase medac dose reduced from the usual 10000 IU/m2 down to 5000 IU/ m2 or 2500 IU/m2, applied at 3 d intervals, was sufficient to achieve complete L-asparagine depletion in serum . Changes of the fibrinogen levels was significantly less pronounced in the group on 2500 IU.

Am J Respir Cell Mol Biol, 1997 Mar, 16(3), 317 - 24
Synthesis of 4- and 5-series leukotrienes in the lung microvasculature challenged with Escherichia coli hemolysin: critical dependence on exogenous free fatty acid supply; Grimminger F et al.; Escherichia coli hemolysin (HlyA) has been identified as a potent inductor of phosphoinositide hydrolysis and related metabolic responses in neutrophils (Grimminger and colleagues, 1991, J . Clin . Invest . 88:1531-1539) . In isolated perfused rabbit lungs, which harbor a large number of entrapped microvascular leukocytes, we investigated the effect of a low dose of HlyA on lipoxygenase product formation in the presence of exogenous free arachidonic acid (AA), eicosapentaenoic acid (EPA), or both precursor fatty acids . Leukotrienes (LT) and hydroxyeicosatetra(penta)enoic acids (HET{P}E) in the recirculating perfusate were quantified using high-performance liquid chromatography techniques . In the absence of exogenous precursor fatty acid supply, 0.02 hemolytic units/ml HlyA elicited only minor amounts of LTs and 5-HETE . AA, 10 microM, provoked the generation of limited quantities of LTB4, LTE4, and 5-HETE . Combined application of HlyA and AA caused a manifold amplification of 4-series LT and 5-HETE generation, with predominance of cysteinyl-LTs . EPA, 10 microM, elicited the synthesis of 5-series LTs accompanied by marked quantities of 5-HEPE . Dual stimulation with HlyA and EPA provoked exclusive generation of excessive quantities of all 5-series 5-lipoxygenase products . When HlyA was administered in the presence of both AA (10 microM) and EPA (10 microM), the n-3 fatty acid clearly turned out to be the preferred substrate, with ratios of the various 5-series to 4-series products ranging between 1.8 and 14.5 . Moreover, the absolute quantities of AA-derived metabolites and the total sum of all 5-lipoxygenase products was markedly reduced under these conditions . We conclude that the HlyA-evoked 5-lipoxygenase product formation in the pulmonary vasculature of the rabbit is critically dependent on the presence of free precursor fatty acids . The profile of LTs suggests neutrophil (PMN)-related transcellular eicosanoid synthesis as a major underlying metabolic pathway . EPA represents the preferred substrate as compared with AA, resulting in a marked suppression of AA metabolite formation . Therapeutic attempts to provide n-3 fatty acids via the intravenous route may have a major impact on lipid mediator profiles in PMN-related inflammatory events.

Protein Sci, 1997 Mar, 6(3), 722 - 4
Crystallization and preliminary X-ray crystallographic analysis of squalene-hopene cyclase from Alicyclobacillus acidocaldarius; Wendt KU et al.; The membrane-associated protein squalene-hopene cyclase from Alicyclobacillus acidocaldarius was overexposed in Escherichia coli and purified by ion exchange and gel permeation chromatography . Crystals of three interrelated forms were grown by vapor diffusion under identical conditions . The crystals diffract to about 2.3 A resolution, but they are unstable in the X-ray beam . An interpretable heavy-atom derivative was obtained.

Protein Sci, 1997 Mar, 6(3), 569 - 79
Importance of the A-helix of the catalytic subunit of cAMP-dependent protein kinase for stability and for orienting subdomains at the cleft interface; Herberg FW et al.; All eukaryotic protein kinases share a conserved catalytic core . In the catalytic (C) subunit of cAMP-dependent protein kinase (cAPK) this core is preceded by a myristylation motif followed by a long helix with Trp 30 at the end of this A-helix filling a hydrophobic cavity between the two lobes of the core . To understand the importance of the A-helix, the myristylation motif (delta 1-14) as well as the entire N-terminal segment (delta 1 -39) were deleted . In addition, Trp 30 was replaced with both Tyr and Ala . All proteins were overexpressed in E . coli and purified to homogeneity . rC(delta 1-14), rC(W30Y), and rC(W30A) all had reduced thermostability, but were catalytically indistinguishable from wild-type C . Based on Surface Plasmon Resonance, all three also formed stable holoenzyme complexes with the RI-subunit, although the appKds were reduced by more than 10-fold due to decrease in the association rate . Surprisingly, however, the holoenzymes were even more thermostable than wild-type holoenzyme . To obtain active enzyme, it was necessary to purify rC(delta 1-39) as a fusion protein with glutathione-S-transferase (GST-rC(delta 1-39), although its thermostability (Tm) was decreased by 12.5 degrees C, was catalytically similar to wild-type C and was inhibited by both the type I and II R-subunits and the heat-stable protein kinase inhibitor (PKI) . The Tm for holoenzyme II formed with GST-rC(delta 1-39) was 16.5 degrees C greater than the Tm for free GST-rC(delta 1-39), and the Ka(cAMP) was increased nearly 10-fold . These mutants point out striking and unanticipated differences in how the RI and RII subunits associate with the C-subunit to form a stable holoenzyme and indicate, furthermore, that this N-terminal segment, far from the active site cleft, influences those interactions . The importance of the A-helix and Trp 30 for stability correlates with its location at the cleft interface where it orients the C-helix in the small lobe and the activation loop in the large so that these subdomains are aligned in a way that allows for correct configuration of residues at the active site . This extensive network of contacts that links the A-helix directly to the active site in cAPK is compared to other kinases whose crystal structures have been solved.

J Bacteriol, 1997 Mar, 179(6), 2089 - 91
A suppressor of mutations in the region adjacent to iterons of pSC101 ori; Ohkubo S et al.; Some single-base changes in a 14-bp region (the downstream region) adjacent to three repeated sequences (iterons) in pSC101 ori are very deleterious for replication . We isolated a host suppressor mutation for one of these mutations and found that the suppressor suppressed all the mutations tested in the downstream region . The nucleotide sequence of the suppressor revealed that the suppressor gene was identical to dksA, which encodes a multicopy suppressor of the heat shock gene dnaK.

J Bacteriol, 1997 Mar, 179(6), 2086 - 8
Growth rate-related regulation of the ilvGMEDA operon of Escherichia coli K-12 is a consequence of the polar frameshift mutation in the ilvG gene of this strain; Parekh BS et al.; In Escherichia coli K-12 the intracellular levels of threonine deaminase and transaminase B, products of ilvA and ilvE, respectively, in the ilvGMEDA operon, increase with increasing growth rates (S . Pedersen, P . L . Bloch, S . Reeh, and F . C . Neidhardt, Cell 14:179-190, 1978) . However, the transcriptional activities of the upstream ilvpG and the internal ilvpE promoters do not increase . Therefore, the growth rate-related expression of this operon is not regulated at the level of transcription initiation . Unlike other wild-type E . coli strains, E . coli K-12 contains a polar frameshift mutation in the ilvG gene (R . P . Lawther, D . H . Calhoun, C . W . Adams, C . A . Hauser, J . Gray, and G . W . Hatfield, Proc . Natl . Acad . Sci . USA 78:922-925, 1981) . In an E . coli K-12 (IlvG+) derivative strain, where the reading frame of the ilvG gene is restored, no growth rate-related expression of the ilvGMEDA operon is observed . Thus, the growth rate-related expression of the ilvGMEDA operon in E . coli K-12 is the fortuitous consequence of the polar frameshift mutation in the ilvG gene of this strain.

J Bacteriol, 1997 Mar, 179(6), 2077 - 80
Characterization of the tol-pal and cyd region of Escherichia coli K-12: transcript analysis and identification of two new proteins encoded by the cyd operon; Muller MM et al.; Sequence analysis showed that the cyd operon is immediately upstream of the tol-pal region . Northern (RNA) blot analysis demonstrated that the transcript for the cyd operon terminates just before the promoter for transcription of the tol genes . The cyd transcript contains cydA cydB followed by two open reading frames: orfC, encoding a 37-residue peptide, and orfD, encoding a 97-residue peptide . Both OrfC and OrfD are synthesized in minicells.

J Bacteriol, 1997 Mar, 179(6), 2073 - 6
A new periplasmic protein of Escherichia coli which is synthesized in spheroplasts but not in intact cells; Hagenmaier S et al.; The gene spy from Escherichia coli has been cloned and sequenced . It encodes a precursor of a so far unknown 139-residue, rather basic periplasmic protein . It was not detectable immunologically in intact cells but was produced abundantly in spheroplasts . It could be a stress protein specific for spheroplasting.

J Bacteriol, 1997 Mar, 179(6), 1998 - 2005
Reduction of conjugal transfer efficiency by three restriction activities of Anabaena sp . strain PCC 7120; Elhai J et al.; The efficiency of conjugal transfer of plasmids from Escherichia coli to the cyanobacterium Anabaena sp . strain PCC 7120 was quantitated as a function of the number of restriction sites for the restriction enzymes carried by the recipient . In addition to the previously recognized isoschizomers of AvaI and AvaII, PCC 7120 was found to possess an isoschizomer of AvaIII . Plasmids modified in E . coli with methylases that protect in vitro against restriction by the three enzymes were transferred with high efficiency, nearly independent of the number of restriction sites on the plasmid . Plasmids left unprotected against one of the three restriction enzymes were transferred with lower efficiencies . For low numbers of sites, the efficiency of conjugal transfer decreased as an exponential function of the number of unprotected sites . The methods presented may be used to increase the efficiency of conjugal transfer into restriction-competent bacteria.

J Bacteriol, 1997 Mar, 179(6), 1931 - 9
sfi-independent filamentation in Escherichia coli Is lexA dependent and requires DNA damage for induction; Hill TM et al.; In Escherichia coli, damage to DNA induces the expression of a set of genes known collectively as the SOS response . Part of the SOS response includes genes that repair DNA damage, but another part of the response coordinates DNA replication and septation to prevent untimely cell division . The classic SOS gene product that inhibits cell division is SfiA (or SulA), which binds to FtsZ and prevents septum formation until the DNA damage has been repaired . However, another pathway acts to coordinate DNA replication and cell division when sfiA, or the sfi-dependent pathway, is inoperative . Until recently, little was known of this alternative pathway, which is called the sfi-independent pathway . We report here that sfi-independent filamentation is suppressed by lexA(Ind-) mutations, suggesting that derepression of the LexA regulon is necessary for sfi-independent induction . However, expression of LexA-controlled genes is not sufficient; DNA damage is also required to induce this secondary pathway of cell division inhibition . Furthermore, we postulate that loss of the common regulatory circuitry of the sfi-dependent and sfi-independent pathways by recA or lexA mutants uncouples cell division and DNA replication.

J Bacteriol, 1997 Mar, 179(6), 1898 - 908
Isolation and characterization of a hemin-regulated gene, hemR, from Porphyromonas gingivalis; Karunakaran T et al.; An hemR (hemin-regulated) gene from Porphyromonas gingivalis ATCC 53977 has been isolated and characterized . This gene is present downstream from the prtT gene, previously cloned in this laboratory . In addition, another putative gene, ORF1, was identified between hemR and prtT . The complete nucleotide sequences of ORF1 and hemR were determined, and the deduced amino acid sequence of ORF1 and HemR proteins corresponded to 16- and 48-kDa proteins, respectively . The amino termini of the HemR protein exhibited significant homology with iron-regulated, TonB-dependent outer membrane receptor proteins from various bacteria, while the carboxyl terminus of the HemR protein displayed almost complete identity with a P . gingivalis PrtT protease domain . PCR analyses confirmed the existence of such extensive homology between the carboxyl termini of both the prtT and hemR genes on the P . gingivalis chromosome . Northern blots indicated that ORF1 was part of a 1.0-kb mRNA and was positively regulated by hemin levels . On the other hand, the hemR gene was apparently a part of a 3.0-kb polycistronic message and was negatively regulated at the transcriptional level by hemin . Primer extension analysis of the hemR gene revealed that the transcription start site was at a C residue located within ORF1 . An examination of HemR::lacZ constructs in both Escherichia coli and P . gingivalis confirmed hemin repression of hemR expression in both organisms . Moreover, the HemR protein expressed in E . coli was detected by an antiserum from a periodontitis patient heavily colonized with P . gingivalis but not by serum from a periodontally healthy patient or by antisera against hemin-grown P . gingivalis cells . Therefore, it is likely that the 48-kDa HemR protein can be expressed only under hemin-restricted conditions . These results suggest that we have isolated a hemin-regulated gene, hemR, which encodes a 48-kDa protein that may be a TonB-dependent outer membrane protein.

J Bacteriol, 1997 Mar, 179(6), 1867 - 71
Analysis of DNA inversions in the shufflon of plasmid R64; Gyohda A et al.; The shufflon, a multiple DNA inversion system in the plasmid R64, consists of four DNA segments flanked and separated by seven 19-bp repeat sequences . Site-specific recombinations mediated by the rci product occur between each inverted repeat sequence, resulting in inversions of the four segments independently or in groups . The seven 19-bp repeat sequences are classified into four types (repeat-a, -b, -c, and -d), according to their 3-bp variable sequences . We individually cloned A, B, and C segments of the R64 shufflon and determined the in vivo inversion frequency of each segment . The inversion frequencies of three segments differed greatly . The inversion frequency declined in the following order: segments A, B, and C . Synthetic 19-mer oligonucleotides corresponding to both strands of repeat-a, -b, -c, and -d sequences were inserted into appropriate sites of pBR322 . The rci-mediated DNA inversion occurred between two synthetic inverted repeats, indicating that the 19-bp inverted repeat sequences are the sole elements required in cis for the shufflon system . The inversion frequencies of DNA segments flanked by various sequences indicate that the four types of repeat sequences determine the inversion frequency of the four DNA segments of the R64 shufflon . Deletion of a DNA segment flanked by direct repeat sequences could not be detected.

Trends Biochem Sci, 1997 Mar, 22(3), 81 - 5
The evolution of ribonucleotide reduction; Reichard P; Ribonucleotide reduction was essential for the transition from RNA to DNA by supplying deoxyribonucleotide precursors . The reaction requires free radical chemistry . Three quite different classes of ribonucleotide reductases are known today . All three are proteins containing a stable free radical amino acid, but each uses a different mechanism for its generation . Did they evolve from a common ancestor, with the arrival of atmospheric oxygen providing the driving force for their divergence, or was each a separate evolutionary invention?

FEMS Microbiol Lett, 1997 Mar 1, 148(1), 35 - 42
The CS6 colonization factor of human enterotoxigenic Escherichia coli contains two heterologous major subunits; Wolf MK et al.; The genes encoding the CS6 colonization factor were cloned from two human enterotoxigenic Escherichia coli strains of different serotypes . The DNA sequences from both clones were nearly identical and contained four open reading frames . Two of them have homology to genes encoding molecular chaperones and ushers found in many other operons encoding colonization factors . The two remaining open reading frames encode two heterologous major subunit proteins which makes CS6 unique because other colonization factors have only one major subunit . Upstream and downstream of the CS6 operon the DNA sequences of the clones diverged abruptly.

Thromb Haemost, 1997 Mar, 77(3), 585 - 90
Haemostatic properties of human pulmonary and cerebral microvascular endothelial cells; Grau GE et al.; Little is known on the haemostatic profiles of human microvascular endothelial cells (MVEC) from different tissues . In addition it is not known whether MVEC from patients display the same haemostatic pattern as MVEC coming from healthy controls . To address these questions MVEC from human lung and brain were isolated and stimulated with tumour necrosis factor alpha (TNF) and E . coli lipopolysaccharide (LPS) for 24 h . The level and the kinetics of procoagulant activity (PCA) and thrombomodulin (TM) expression were found to be different depending on the tissue of origin and on the agonist used . In particular, the inducible PCA was higher in lung than in brain MVEC, an observation that may be related to the frequency of lung involvement in septic shock . Differences were also observed for tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) with MVEC supernatants or cell lysates . These variables were then measured in lung MVEC purified from patients with acute respiratory distress syndrome (ARDS) and compared to controls . Cells from ARDS patients constitutively expressed more PCA and PAI-1 than controls . The fibrinolytic potential, expressed as t-PA/PAI-1 ratio, was lower in ARDS than in lung MVEC . It is concluded that MVEC display different haemostatic features depending on the tissue they come from and that lung MVEC from ARDS patients present a procoagulant profile when compared with those from controls.

Biochem J, 1997 Mar 1, 322 ( Pt 2), 663 - 9
Differential plasma clearance of murine acute-phase serum amyloid A proteins SAA1 and SAA2; Kluve-Beckerman B et al.; Serum amyloid A (SAA) proteins SAA1 and SAA2 are prominent acute-phase reactants which circulate in association with the high-density-lipoprotein (HDL) fraction of plasma . Plasma levels of SAA1 and SAA2 increase dramatically, by as much as 1000-fold, within 24 h of tissue injury and then rapidly decrease with cessation of the inflammatory stimulus, suggesting that SAA clearance and/or catabolism is important to the re-establishment of homoeostasis . In this context, aberrant SAA catabolism has long been considered a potential factor in the pathogenesis of reactive amyloidosis . To initiate studies aimed at understanding the differential regulation of SAA metabolism, we have produced 35S-labelled murine SAA1 and SAA2 in Escherichia coli, bound them individually to HDL, and then compared the plasma-clearance characteristics of SAA1 and SAA2 under normal and acute-phase conditions . When bound to normal HDL, SAA2 {half-life (t1/2) = 30 min} was cleared significantly faster than SAA1 (t1/2 = 75 min) . Clearance of SAA1 and SAA2 was significantly slower when each was bound to acute-phase HDL as opposed to normal HDL, when clearance rates were determined in acute-phase mice versus normal mice, and when normal HDL was remodelled to contain both recombinant isotypes rather than just one of the isotypes . Thus it appears that an increased amount of SAA on HDL, or possibly the combined presence of both isotypes on HDL, is associated with a prolongation in the plasma half-life of SAA.

Biochem J, 1997 Mar 1, 322 ( Pt 2), 543 - 50
Release of calreticulin from neutrophils may alter C1q-mediated immune functions; Kishore U et al.; Calreticulin is an abundant intracellular protein which is involved in a number of cellular functions . During cytomegalovirus infection, as well as inflammatory episodes in autoimmune disease, calreticulin can be released from cells and detected in the circulation, where it may act as an immunodominant autoantigen in diseases such as systemic lupus erythematosus . Calreticulin is known to bind to the molecules of innate immunity, such as C1q, the first subcomponent of complement . However, the functional implications of C1q-calreticulin interactions are unknown . In the present study we sought to investigate, in greater detail, the interaction between these two proteins following the release of calreticulin from neutrophils upon stimulation . In order to pinpoint the regions of interaction, recombinant calreticulin and its discrete domains (N-, P- and C-domains) were produced in Escherichia coli . Both the N- and P-domains of calreticulin were shown to bind to the globular head regions of C1q . Calreticulin also appeared to alter C1q-mediated immune functions . Binding of calreticulin to C1q inhibited haemolysis of IgM-sensitized erythrocytes . Both the N- and P-domains of calreticulin were found to contain sites involved in the inhibition of C1q-induced haemolysis . Full-length calreticulin, and its N- and P-domains, were also able to reduce the C1q-dependent binding of immune complexes to neutrophils . We conclude that calreticulin, once released from neutrophils during inflammation, may not only induce an antigenic reaction, but, under defined conditions, may also interfere with C1q-mediated inflammatory processes.

Biochem J, 1997 Mar 1, 322 ( Pt 2), 511 - 7
Cloning and characterization of a cDNA encoding a maize seedling phytase; Maugenest S et al.; During germination, maize seedlings express a phytase able to hydrolyse the large amount of phytin stored in the dry seed . Previous studies allowed purification and characterization of this enzyme as a homodimer of 38 kDa subunits {Laboure, Gagnon and Lescure, Biochem . J . (1993) 295, 413-419} . In the present work, an antibody against the purified maize phytase has been used to screen a maize seedling cDNA expression library . Several positive clones containing an insert of about 1400 bp were isolated . The nucleotide sequence of the insert of one of these clones has been established . This cDNA, called phy S11, was 1335 bp long and contained an open reading frame of 387 amino acids . The sequence of N-terminal residues (23 amino acids) of the purified phytase has been established . These residues are found at positions 19-41 of the amino acid sequence encoded by phy S11 . This confirms that this cDNA codes for the maize phytase . The deduced amino acid sequence appears to be very different from those of published Aspergillus niger phytases; however, an homologous region of 33 amino acids was detected . This region of the fungal sequence contains the RHGxRxP consensus motif found in various high molecular mass acid phosphatases and believed to be the acceptor site for phosphate . Expression of the phy S11 cDNA in Escherichia coli allowed the production of the phytase subunit and its assembly to give a protein of the same size as the native phytase . The time course of phy S11 mRNA accumulation during germination showed that no transcript was present in dry seeds . The mRNA accumulated during the first day of germination, to reach a maximum after 2 days (radicle protrusion), and then decreased in young seedlings . Genomic Southern blot analyses suggest the existence of at least two genes and genetic mapping reveals two loci separated by 1 cM on chromosome 3 of maize . The cloning of this first cDNA coding for a plant phytase, will allow the isolation of the corresponding genes and the study of their regulation during germination.

Biochem J, 1997 Mar 1, 322 ( Pt 2), 449 - 54
Molecular cloning and characterization of the thiolesterase glyoxalase II from Arabidopsis thaliana; Ridderstrom M et al.; cDNA encoding glyoxalase II from Arabidopsis thaliana has been cloned and sequenced . The isolated 894 bp segment included a sequence of 774 bp encoding a protein with a calculated molecular mass of 28,791 Da . The amino acid sequence deduced from the A . thaliana cDNA showed 54% identity with that of the human enzyme . Searches in databanks identified seven additional DNA sequences from different species with high similarity to glyoxalase II . Certain limited regions, one rich in histidine residues, shared 100% identity . A 29 kDa protein with an isoelectric point of 6.2 was obtained by heterologous expression of the A . thaliana cDNA in Escherichia coli . Homogeneous enzyme was obtained by affinity purification and its catalytic parameters with thiolesters of glutathione were similar to those for human glyoxalase II . The structural and functional similarities between glyoxalase II from A . thaliana and from human tissues suggest a common evolutionary origin.

Nat Biotechnol, 1997 Mar, 15(3), 273 - 7
Generation of catalytic RNAs by rolling transcription of synthetic DNA nanocircles; Daubendiek SL et al.; Small catalytic RNAs are commonly produced either by transcription of promoter-driven linear DNA templates or by stepwise chemical synthesis on solid supports . We describe a different approach, in which very small chemically synthesized circular DNAs serve as efficient templates for generation of catalytic RNAs in vitro . The circles are 83 nucleotides in size, are single stranded, and contain no canonical RNA polymerase promoters . Despite this, T7 and Escherichia coli RNA polymerases transcribe the circles by a rolling mechanism, producing long concatemeric RNAs (approximately 7,500 nt) . During the transcription reaction, the repeating RNAs self-cleave, ultimately reaching monomer length . Despite having self-complementary sequences at their substrate-binding domains, these monomeric 83-nt RNAs are shown to be catalytically active ribozymes that sequence-specifically cleave RNA targets in trans . In addition, a circular vector encoding a repeating (non-self-processing) ribozyme is described; the resulting multimeric ribozyme, targeted to a sequence in the HIV-1 genome, is also catalytically active in trans . This novel approach to the synthesis of catalytic RNAs offers a number of differences and potential advantages over current approaches to RNA synthesis.

Biochim Biophys Acta, 1997 Mar 1, 1355(3), 303 - 14
Characterization of a cartilage-derived 66-kDa protein (RGD-CAP/beta ig-h3) that binds to collagen; Hashimoto K et al.; A 66-kDa collagen fiber-associated protein (RGD-CAP) was isolated from a fiber-rich fraction of pig cartilage by ultrafiltration and collagen-affinity chromatography . Amino acid sequencing and cDNA cloning indicated that the RGD-CAP is identical or closely related to beta ig-h3 protein which is induced in human adenocarcinoma cells by transforming growth factor-beta (TGF-beta) (Skonier, J., Neubauer, M., Madisen, L., Bennett, K., Plowman, G.D., and Purchio, A.F . (1992) DNA Cell . Biol . 11, 511-522) . The RGD-CAP, as well as beta ig-h3, has the RGD sequence in the C-terminal region . The native RGD-CAP bound to type I, II, and IV collagens even in the presence of 1 M NaCl . A recombinant preparation of RGD-CAP expressed in Escherichia coli cells also bound to collagen but not to gelatin . The RGD-CAP mRNA was expressed in chondrocytes throughout all stages, although the expression level was highest during the prehypertrophic stage . In addition, TGF-beta increased the RGD-CAP mRNA level in chondrocyte cultures . Since RGD-CAP transcripts were found in most tissues, this novel collagen-binding protein may play an important role in cell-collagen interactions in various tissues including developing cartilage.

J Rheumatol, 1997 Mar, 24(3), 576 - 8
Antinuclear antibody positivity in pediatric patients with autoimmune thyroid disease; Inamo Y et al.; OBJECTIVE: The incidence of antinuclear antibody (ANA) positivity was investigated in pediatric patients with autoimmune thyroid diseases . METHODS: Subjects were 21 untreated patients with Graves' disease, 12 untreated patients with Hashimoto's disease, and 16 untreated patients with non-autoimmune thyroid disease, including one patient with Plummer's disease, 11 patients with simple goiter (probable), and 4 patients with cretinism . Patients with Graves' disease were treated with either propylthiouracil or methimazole . ANA was measured using HEp-2 cells . Anti-dsDNA antibody was measured using double stranded DNA derived from an Escherichia coli plasmid . RESULTS: The incidence of ANA positivity was significantly higher in patients with untreated Graves' disease (n = 21; age range 9-15 yrs) than in Hashimoto's thyroiditis (n = 12; age range 7-15 yrs) (p < 0.03, Fisher's exact probability test) . However, the 2 diseases were not significantly different with respect to anti-dsDNA antibody positivity . Most of the ANA positive patients with Graves' disease required treatment for more than 2 years, unlike the 6 ANA negative patients (p < 0.002, Fisher's exact probability test) . CONCLUSION: We conclude that ANA positivity may predict a poor response to antithyroid drugs in Graves' disease.

RNA, 1997 Mar, 3(3), 255 - 68
The RNA binding site of S8 ribosomal protein of Escherichia coli: Selex and hydroxyl radical probing studies; Moine H et al.; The RNA binding site of ribosomal protein S8 of Escherichia coli is confined to a small region within the stem of a hairpin in 16S rRNA (nt 588-605/633-651), and thus represents a model system for understanding RNA/protein interaction rules . The S8 binding site on 16S rRNA was suspected to contain noncanonical features difficult to prove with classical genetical or biochemical means . We performed in vitro iterative selection of RNA aptamers that bind S8 . For the different aptamers, the interactions with the protein were probed with hydroxyl radicals . Aptamers that were recognized according to the same structural rules as wild-type RNA, but with variations not found in nature, were identified . These aptamers revealed features in the S8 binding site that had been concealed during previous characterizations by the high base conservation throughout evolution . Our data demonstrate that the core structure of the S8 binding site is composed of three interdependent bases (nt 597/641/643), with an essential intervening adenine nucleotide (position 642) . The other elements important for the binding site are a base pair (598/640) above the three interdependent bases and a bulged base at position 595, the identity of which is not important . Possible implications on the geometry of the S8 binding site are discussed with the help of a three-dimensional model.

Protein Expr Purif, 1997 Mar, 9(2), 295 - 300
Expression, purification, and characterization of a highly soluble N-terminal-truncated form of the neuron-specific membrane-associated phosphoprotein SCG10; Antonsson B et al.; SCG10 is a neuron-specific growth-associated protein with high sequence homology to the ubiquitous phosphoprotein stathmin/Op18 . The main structural difference between the two proteins is the 34-amino-acid N-terminal extension of SCG10, which is responsible for the membrane attachment . Full length SCG10 has been purified and shows limited solubility, in contrast to stathmin, which is a highly soluble protein . In order to obtain a more soluble form of SCG10 which would be better suited for biochemical and structural studies, we deleted the N-terminal extension and expressed the C-terminal portion of the protein . Two forms of N-terminal-truncated SCG10 (delta SCG10 and delta SCG10r) were purified to homogeneity in a four-step purification procedure . delta SCG10 starts at amino acid 35 and delta SCG10r at amino acid 48 in the SCG10 sequence, giving proteins of 16,899 and 15,189 kDa, respectively . The truncated SCG10 was highly soluble up to concentrations of 20 mg/ml . The proteins were like the full length SCG10 substrate for serine/threonine protein kinases, including MAP kinase, PKA, and p34cdc2 kinase . With these highly soluble forms of SCG10 biochemical and structural studies of this multiphosphoprotein become feasible.

Protein Expr Purif, 1997 Mar, 9(2), 288 - 94
Expression in Escherichia coli and purification of soluble forms of the F protein of bovine respiratory syncytial virus; Naval J et al.; Six fragments of the F gene from bovine respiratory syncytial virus (BRSV) were engineered into the pMAL-c2 Escherichia coli expression vector and expressed as C-terminal maltose-binding protein (MBP) fusion products . The resulting polypeptides were partially soluble and single-step purified by affinity chromatography . These fusion proteins were recognized in Western blots by several MAbs directed against human respiratory syncytial virus F protein . In addition, rabbit polyclonal antisera raised against two purified MBP-derived proteins reacted with the BRSV-F protein.

Protein Expr Purif, 1997 Mar, 9(2), 279 - 87
Human carbonic anhydrase IV: in vitro activation and purification of disulfide-bonded enzyme following expression in Escherichia coli; Waheed A et al.; Human carbonic anhydrase IV (CA IV) expressed in Escherichia coli was refolded and activated in cell extracts with the help of endogenous periplasmic protein disulfide isomerase, DsbA, in the presence of oxidized glutathione . The refolding and activation were inhibited by bacitracin but not affected by known cofactors or activators of other chaperones . Although the yield of the purified CA IV recovered from cell extracts was maximal when activated at 4 degrees C in the presence of 2 mM oxidized glutathione, the rate of refolding and activation was much more rapid at 25 and 37 degrees C . The enzyme purified from the E . coli cell extracts following activation in vitro showed similar structural stability and functional properties as CA IV purified from secretion medium from a stably transfected CHO cell line . These studies suggest that the soluble truncated form of human CA IV expressed in E . coli, which is disulfide-bonded zinc metalloenzyme, can provide a useful model enzyme for studies of protein folding and enzyme activation in vitro . Furthermore, the procedure described for recovery of CA IV following expression in E . coli may be useful for in vitro activation and subsequent purification of other disulfide-containing proteins.

Protein Expr Purif, 1997 Mar, 9(2), 262 - 78
Phospholipase C isoforms delta 1 and delta 3 from human fibroblasts . High-yield expression in Escherichia coli, simple purification, and properties; Ghosh S et al.; Phospholipase C isoforms delta 1 and delta 3 (PLC delta 1 and delta 3) were expressed in Escherichia coli using the cDNA sequences from human fibroblasts . The enzymes were also expressed with the sequence Met-Gly-His6-Ser-Gly-Leu-Phe-Lys-Arg, a hexahistidine sequence followed by a Kex2 protease cleavage site, denoted as "-H6K2," attached to their amino termini . PLC delta 1, PLC delta 1-H6K2, PLC delta 3, PLC delta 3-H6K2 all expressed in highly active form . The H6K2-bearing isoforms were each purified to homogeneity in a single step, with yields of 90-100%, using agarose-iminodiacetic acid-Ni columns and imidazole buffer as eluting agent . Yields in terms of activity increased as the temperature of expression was decreased . Expression at 16 degrees C for 72 h yielded 33 mg of pure PLC delta 1-H6K2 and 13 mg of pure PLC delta 3-H6K2 per liter of culture . Removal of H6K2 from both isoforms with Kex2 protease resulted in little or no loss of activity . Expression of PLC isoforms bearing -H6K2 at the amino terminus resulted in about 12 times more activity than expression of the isoforms lacking -H6K2 . PLC delta 3 is much less stable than PLC delta1 . Successful purification and storage of PLC delta 3 depends on a suitable stabilizing medium . Both isoforms require 0.3 microM calcium ion for half-maximum activity . The specific activities of the isoforms expressed with and without -H6K2 are the same, as are their calcium saturation curves.

Protein Expr Purif, 1997 Mar, 9(2), 253 - 61
Overexpression, purification, and characterization of tyrosine-sensitive 3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase from Escherichia coli; Ramilo CA et al.; An overexpression system (pCR105) for DAHP synthase (Tyr) was constructed by cloning the aroF gene at the NdeI site of the pET-22b(+) translation vector, a plasmid expression vector that contains the T7 lac promoter . The enzyme was overexpressed, purified to > 90% purity (by SDS-polyacrylamide gel electrophoresis), and characterized . The protein was overexpressed at a level of 58% the total soluble cell protein (based on enzymatic activities) . About 244 mg of pure enzyme was obtained from a 2-liter cell culture . So far, this is the highest yield reported for the isozyme DAHP synthase (Tyr) . The enzyme showed a bell-shaped pH-activity profile, with a pH optimum at pH 7.0-7.5 and pK values of 6.10 and 8.92 . Inhibition of the enzyme by tyrosine was specific with 50% inhibition observed at 9 microM tyrosine, pH 7.0 . The specific activity of the enzyme increased with added metal and metal sensitivity increased with purity of the enzyme . Only substoichiometric amounts of Cu, Fe, and Zn were found in the pure enzyme and this result is consistent with sensitivity of the enzyme to added metal . Although treatment with EDTA inactivated the enzyme almost completely, the activity of the apoenzyme was restored to differing extents by a variety of metals including Mn2+, Cd2+, Co2+, Fe2+, Cu2+, Mg2+, and Zn2+ . Both Fe2+ and Cu2+ only partially reactivated EDTA-treated enzyme . Reconstitution of EDTA-treated enzyme with either Cd2+ or Mn2+ gave 1 mol of metal per mole of enzyme monomer . KCN inactivated the enzyme to only 80% and added metals reactivated the CN-treated enzyme only to a small extent . These results confirm the importance of the metal in the enzymatic reaction.

Protein Expr Purif, 1997 Mar, 9(2), 246 - 52
Soluble expression in Escherichia coli, one-step purification, and characterization of Chinese hamster dihydrofolate reductase; Fan YX et al.; Chinese hamster dihydrofolate reductase (ch-DHFR) was overexpressed in Escherichia coli DH5 alpha under the transcriptional control of PRPL promoters regulated by temperature-sensitive repressors . The desired recombinant product is soluble and constitutes about 30% of the total soluble proteins of the bacterial cell . With repeated cycles of freezing and thawing as a first step, the purification of the recombinant ch-DHFR to homogeneity requires only one further step, gel filtration on a Sephadex G-75 column with 85-90% enzyme recovery, two to three times higher than that obtained with the commonly used affinity chromatography on a methotrexate-Sepharose column . The purified enzyme migrates as a single protein band on SDS-polyacrylamide gel electrophoresis with approximate mass of 23 kDa, in accord with that calculated from the DNA sequence . The initiation methionine residue at the N-terminus of the enzyme is completely removed by E . coli methionine aminopeptidase as judged by amino-terminal analysis . The steady-state kinetic parameters, dissociation constants for binary complexes of dihydrofolate, NADPH, and methotrexate with ch-DHFR, and the inhibitor constant of methotrexate have also been determined . The enzyme is activated about 4-fold in 3 M urea and about 2.5-fold in 0.5 M guanidine hydrochloride.

Protein Expr Purif, 1997 Mar, 9(2), 211 - 8
Expression of aggregation-prone recombinant proteins at low temperatures: a comparative study of the Escherichia coli cspA and tac promoter systems; Vasina JA et al.; The aggregation-prone fusion protein preS2-S'-beta-galactosidase was used as a model system to compare the efficiencies of the IPTG-inducible tac promoter and the low-temperature-inducible cspA promoter in directing the expression of soluble recombinant polypeptides at reduced growth temperatures in Escherichia coli . At 37 degrees C, the fusion protein was produced at high levels from the tac promoter, but aggregated quantitatively in a biologically inactive form . In contrast, little preS2-S'-beta-galactosidase was synthesized from the cspA promoter at this temperature, presumably due to transcript instability . The highest yields of active enzyme were obtained following temperature downshift from 37 to 30 degrees C for the tac promoter and 25 degrees C for the cspA promoter . At 25 degrees C, the kinetics of accumulation of beta-galactosidase activity, ratios of soluble to insoluble fusion protein, and synthesis rates of preS2-S'-beta-galactosidase were virtually identical for both promoters for a period of 2 h postinduction . Thereafter, the cspA promoter became repressed, whereas synthesis of the fusion protein continued with the tac system . Following transfer to 10 degrees C, the tac promoter was almost completely inhibited while the cspA promoter was able to direct the synthesis of soluble preS2-S'-beta-galactosidase for up to 2 h . However, the levels of active enzyme produced were approximately threefold lower than those measured at 25 degrees C . Overexpression of native CspA had no effect on the accumulation of active preS2-S'-beta-galactosidase from the cspA promoter . It is therefore unlikely that CspA acts as it own positive inducer . Our results indicate that the cspA promoter can efficiently substitute for the tac system at 25 degrees C and may be particularly valuable for the expression of highly aggregation-prone or unstable gene products at 10 degrees C.

Protein Expr Purif, 1997 Mar, 9(2), 182 - 90
Overexpression, purification, and characterization of recombinant Dictyostelium discoideum calcium-regulated 34,000-dalton F-actin bundling protein from Escherichia coli; Lim RW et al.; The Dictyostelium discoideum 34-kDa protein is an F-actin bundling protein that demonstrates diverse distributions in the cell during cell shape changes and cell movement . The protein is expressed at a very low level in the amoeba, just 0.4% of the total cell protein . This presents a challenging problem when purifying sufficient protein for structural and biochemical studies . The purification procedure is lengthy and yields only a few milligrams of protein . An alternative protein expression system, that of the bacterial T7 expression system, was used to produce large quantities of recombinant 34-kDa protein (r34-kDa) . The soluble r34-kDa protein constitutes up to a quarter of the total bacterial protein, and was purified to homogeneity by a modification of the purification procedure for the native D . discoideum 34-kDa protein (N34-kDa) . The r34-kDa possesses all the same functional characteristics as the N34-kDa protein with respect to its interactions with F-actin in vitro: it bound to and cross-linked F-actin, mediated F-actin bundle formation, directly bound calcium, and demonstrated calcium-sensitive F-actin binding activities.

Protein Expr Purif, 1997 Mar, 9(2), 171 - 81
Chick calretinin: purification, composition, and metal binding activity of native and recombinant forms; Stevens J et al.; Chick calretinin has been previously expressed in Escherichia coli and purified to homogeneity {Cheung, W-T., Richards, D.E., and Rogers, J.H . (1993) Eur . J . Biochem . 215, 401-410} . In the present study we have developed an improved purification procedure, involving a heat precipitation step followed by DEAE-cellulose chromatography with calcium-dependent elution . Native calretinin was purified from chick brainstem using the same method as for the recombinant protein but with an added affinity chromatography step . Typically 30 g of brainstem yielded 350 micrograms of protein . Several differences between the two forms imply that the native protein is acetylated at the N-terminus but otherwise unmodified . The calcium binding activities of both forms of calretinin were measured by equilibrium dialysis with 45Ca in Ca2+/EGTA buffers . The recombinant form bound 4.9 +/- 0.12 calcium ions with Kd = 0.38 +/- 0.02 microM and the native form was not significantly different . Recombinant calretinin was used to study its interaction with other cations present in cells and it was found that calcium binding was affected by Mg2+ . Calretinin appears to bind 4.69 +/- 0.13 magnesium ions with Kd = 4.5 mM . Mg2+ increased the apparent dissociation constant for Ca2+ . The shift is consistent with competitive binding of Ca2+ and Mg2+ to the same five sites, but Mg2+ binding is too weak to interfere significantly with Ca2+ binding under physiological conditions.

Protein Expr Purif, 1997 Mar, 9(2), 153 - 8
Expression in Escherichia coli: purification and characterization of cyclin H, a subunit of the human general transcription/DNA repair factor TFIIH; Poterszman A et al.; The human cyclin H, a protein normally associated with the cyclin-dependent kinase cdk7, was overexpressed in Escherichia coli using a T7 RNA polymerase expression system and further purified to apparent homogeneity . The purified recombinant cyclin H is similar to the endogenous one according to the following criteria: molecular weight, microsequencing and mass spectra studies, ability to interact with cdk7, and regulatory kinase activity . The scale-up of cyclin H purification is described.

Arch Biochem Biophys, 1997 Mar 1, 339(1), 218 - 25
The function of recombinant cytochrome P450s in intact Escherichia coli cells: the 17 alpha-hydroxylation of progesterone and pregnenolone by P450c17; Shet MS et al.; Studies are reported showing that recombinant P450c17, coexpressed with rat NADPH-P450 reductase or expressed as a fusion protein containing the domain of the P450 linked to the domain of NADPH-P450 reductase, function effectively in intact Escherichia coli cells . Progesterone is rapidly hydroxylated by transformed E . coli cells at rates as rapid as 50 nmol of steroid hydroxylated/min/nmol of P450 at 37 degrees C . This rate measured in vivo equals or exceeds the best rates we have measured when reconstituting progesterone hydroxylase activity in vitro using purified recombinant bovine P450c17 and purified recombinant rat NADPH-P450 reductase . The limits imposed in vivo by the availability of reducing equivalents (NADPH) and molecular oxygen are identified by showing the nearly fivefold increase in hydroxylation activity when glucose is present and the tendency for the constitutive respiratory activity of E . coli to limit the availability of oxygen required for the P450-catalyzed reaction . The rate of progesterone metabolism is about 200 times faster by P450c17 coexpressed with NADPH-P450 reductase than when P450c17 functions with the constitutive electron transfer system of E . coli (flavodoxin and flavodoxin reductase) . Expression of the fusion protein, termed rF450{mBov17A/mRatOR}L1, results in a rate of progesterone metabolism in vivo at 37 degrees C of about 15 nmol of steroid hydroxylated/min/nmol of P450 . Pregnenolone is actively metabolized to dehydroepiandrosterone at rates similar to those seen when the P450 activity is reconstituted in vitro with cytochrome b5 . Experiments are described showing that the limited solubility of progesterone in water imposes a limit on the extent of steroid hydroxylated . The practicality of this type of P450-containing system for the bioconversion of large amounts of a chemical for the manufacture of speciality chemicals is discussed.

Arch Biochem Biophys, 1997 Mar 1, 339(1), 151 - 6
Nicked multifunctional loop of glutathione synthetase still protects the catalytic intermediate; Tanaka T et al.; A derivative of glutathione synthetase (GSHase) with the multifunctional loop cleaved (nicked GSHase) was compared to both a deletion mutant of the loop (loopless GSHase) and wild-type with the intact loop (wild-type GSHase) . The loop had been shown to be in a closed state in order to protect a catalytic intermediate and accelerate the reaction . Data indicated that cleavage of the loop resulted in a drastic decrease in glutathione synthetic activity which was similar to the results for the loop deletion . Kinetic analyses indicated that the manipulations of the loop impaired the substrate affinity, especially for glycine, and also catalytic efficiency . The nicked loop did not accelerate the reaction as fast as the intact loop; however, the catalytic intermediate was protected from hydrolysis by the cleaved loop as effectively as by the intact loop . These results suggest that the fragmental loop assumed the closed state . High concentrations of ATP showed some inhibitory effects on wild-type GSHase, while both nicked and loopless GSHase were not inhibited, indicating that the fragments of the nicked loop functioned independently . In conclusion, it is postulated that the two fragments of the nicked loop independently assumed the closed state to protect the catalytic intermediate and have lost the ability to accelerate glutathione synthesis.

Arch Biochem Biophys, 1997 Mar 1, 339(1), 107 - 14
Microsomal P450 2C3 is expressed as a soluble dimer in Escherichia coli following modification of its N-terminus; von Wachenfeldt C et al.; A hydrophobic segment present in the N-terminus of microsomal P450s is thought to serve as a membrane anchor . A variant of P450 2C3 was constructed, P450 2C3d, that lacked the putative membrane-spanning segment of the N-terminus, residues 3-20 . This construct also incorporated substitutions of an alanine for 2Asp to facilitate expression in Escherichia coli and of serines for 24His and 25Gly to introduce a restriction site . P450 2C3d is expressed at relatively high levels in E . coli, 800-1200 nmol/liter of culture medium . In contrast to P450 2C3mod, which retains a membrane-spanning N-terminal sequence modified for expression in E . coli, the subcellular distribution of P450 2C3d in E . coli is dependent on the ionic strength of the buffer used for cell disruption . In low ionic strength buffers, 2C3d was mainly localized in the membrane fraction, whereas in buffers containing 1 M NaCl or 0.5 M KPi, P450 2C3d was predominantly found in the soluble fraction, indicating that deletion of the hydrophobic segment converted the intrinsic membrane protein to an extrinsic one . P450 2C3d was further modified by the incorporation of four histidine residues at the C-terminus (P450 2C3dH), and this enzyme could be purified in the absence of detergent using immobilized metal affinity chromatography following extraction from isolated membranes in high salt buffers . The catalytic properties of the purified, modified enzymes are similar to those of the native enzyme . Size-exclusion chromatography indicated that 2C3dH and 2C3d are predominantly dimers, whereas 2C3 is a larger oligomer (> 8-mer) . Moreover, the detergents sodium cholate and Chaps each dissociate the dimers of 2C3dH to monomers at concentrations that do not alter the aggregation state of 2C3 . These modifications are likely to facilitate attempts to crystallize the catalytic domains of microsomal P450s.

Anal Biochem, 1997 Mar 1, 246(1), 52 - 61
Analysis of the lipidated recombinant outer surface protein A from Borrelia burgdorferi by mass spectrometry; Bouchon B et al.; The outer surface protein A, OspA, from the spirochete Borrelia burgdorferi is a lipoprotein of 25 kDa . The recombinant OspA (rOspA) expressed in Escherichia coli has been purified and analyzed by electrospray mass spectrometry (ESMS) . A heterogenous spectrum gave a measured mass of 28,462 +/- 9 Da for the major component compared to an expected mass of 28,445 Da (Deltam = +17 Da), and a measured mass of 28,228 +/- 7 Da for a minor component . The theoretical mass is based on the N-terminal being an S-{2,3-bis(palmitoyloxy)-(2R, S-{2,3-bis(palmitoyloxy)-(2R,S)-propyl}-N-palmitoylcysteine modification according to the model established by Hantke and Braun (Eur . J . Biochem . 34, 284-296, 1973) for bacterial lipoproteins . To determine whether rOspA conforms to this model, a complementary detailed analysis of this lipidation was necessary . The fatty acid content of the complete protein as analyzed by gas chromatography-mass spectrometry revealed saturated fatty acids ranging from C14 to C18 as well as C16 and C18 unsaturated fatty acids, with palmitate (C16:0) being the major component . Focusing on the lipid moieties, the N-terminal tryptic peptide was purified by normal phase HPLC using a silica column and a gradient of hexane in isopropanol . Analysis of the N-terminal peptide by ESMS and fast atom bombardment-mass spectrometry revealed a minor fraction of rOspA molecules which contained only two C16 residues and that in addition to partial oxidation, the major N-terminal peptide had a mass difference of -2 Da compared to a theoretical structure with three palmitate residues, indicating that one of the three fatty acid residues was unsaturated . Minor forms with mass differences of 28 Da were also observed, indicating that one of the three acyl residues was C14 in one case and C18 in the other, instead of C16 in the major form . Analysis of the rOspA peptide backbone revealed that the sole methionine at position 22 was partially oxidized to a methionine sulfoxide . Thus the mass analysis of the major mass is consistent with a mixed population of lipoprotein molecules containing variations not only in the lipid moiety contributing to an elevation in the mass of Deltam = 7 Da compared to the theoretical structure proposed, but also in the peptide chain . Partial oxidations at two points in the protein backbone (<30% of the population in each case) contribute to an additional augmentation in mass and thus can account for the remaining mass difference in the measured mass.

Can Vet J, 1997 Mar, 38(3), 159 - 62
Isolation of Escherichia coli from cellulitis and other lesions of the same bird in broilers at slaughter; Gomis SM et al.; Cellulitis results in substantial losses to the broiler industry due to condemnations at slaughter . This study was conducted to clarify the association between Escherichia coli isolated from cellulitis and other lesions caused by E . coli in individual birds . Fourteen flocks were sampled and 118 birds with cellulitis were examined . Escherichia coli was isolated from all but 2 of the cellulitis lesions, and serogroups O78, O1, and O2 predominated . Thirty-six birds had at least 1 other lesion in addition to the cellulitis lesion . Isolation of E . coli from cellulitis and other lesions occurred in 7 of the 14 flocks . Escherichia coli of the same serogroup were isolated from cellulitis and other lesions in some birds, suggesting that a single E . coli may sometimes be responsible for both types of lesions.

Am J Vet Res, 1997 Mar, 58(3), 260 - 4
Prevalence of intestinal chlamydial infection in pigs in the midwest, as determined by immunoperoxidase staining; Nietfeld JC et al.; OBJECTIVE: To determine prevalence of intestinal chlamydial infection in pigs and to compare prevalence of diarrhea in infected pigs with that in noninfected pigs to evaluate the importance of Chlamydia sp as causes of diarrhea in pigs . ANIMALS AND PROCEDURES: Intestines from 351 sick pigs submitted to 2 veterinary diagnostic laboratories and from 96 healthy pigs that were part of an Escherichia coli susceptibility study were examined by immunoperoxidase staining for chlamydial antigen . The proportion of Chlamydia-infected pigs in each group was calculated and compared . The proportion of Chlamydia-infected pigs with diarrhea was compared with the proportion of noninfected pigs with diarrhea . RESULTS: 15% of the sick and healthy pigs were infected with Chlamydia sp . Prevalence of diarrhea was equal between infected and noninfected pigs . Chlamydia sp were the third most common pathogens identified, and prevalence of chlamydial infection increased after 3 weeks of age . CONCLUSIONS AND CLINICAL RELEVANCE: Intestinal chlamydiosis is common in commercial pigs, but most, if not all, infections are subclinical Without collaborative evidence, simply identifying Chlamydia sp in feces or the intestinal tract of pigs with enteritis or diseases of other organ systems should not be considered proof that the organism caused the clinical signs of disease.

Nat Med, 1997 Mar, 3(3), 306 - 12
Recombinant adeno-associated virus for muscle directed gene therapy; Fisher KJ et al.; Although gene transfer with adeno-associated virus (AAV) vectors has typically been low, transduction can be enhanced in the presence of adenovirus gene products through the formation of double stranded, non-integrated AAV genomes . We describe the unexpected finding of high level and stable transgene expression in mice following intramuscular injection of purified recombinant AAV (rAAV) . The rAAV genome is efficiently incorporated into nuclei of differentiated muscle fibers where it persists as head-to-tail concatamers . Fluorescent in situ hybridization of muscle tissue suggests single integration sites . Neutralizing antibody against AAV capsid proteins does not prevent readministration of vector . Remarkably, no humoral or cellular immune responses are elicited to the neoantigenic transgene product E . coli beta-galactosidase . The favorable biology of rAAV in muscle-directed gene therapy described in this study expands the potential of this vector for the treatment of inherited and acquired diseases.

Appl Environ Microbiol, 1997 Mar, 63(3), 1058 - 65
Analysis of mechanisms regulating expression of the ver-1 gene, involved in aflatoxin biosynthesis; Liang SH et al.; Previous studies have shown that ver-1A encodes an enzyme which is directly involved in the conversion of versicolorin A to demethylsterigmatocystin during aflatoxin B1 (AFB1) biosynthesis in the filamentous fungus Aspergillus parasiticus . In this study, two different tools were utilized to study the regulation of ver-1A expression at the level of transcription and protein accumulation . First, a ver-1A cDNA was expressed in Escherichia coli with the vector pMAL-c2 . The resulting maltose-binding protein-Ver-1A fusion protein was purified and used to generate polyclonal antibodies . Western blot analyses showed that these antibodies specifically recognized the Ver-1 protein (approximately 28 kDa) in cell extracts of Aspergillus parasiticus SU1 . Second, a GUS (uidA; encodes beta-glucuronidase) reporter system was developed by fusing the ver-1A promoter and transcription terminator to the GUS gene . Reporter constructs were transformed into A . parasiticus, resulting in a single copy of the ver-1A-GUS reporter integrated adjacent to the wild-type ver-1A gene (3' end) in the chromosome . Western blot analysis, Northern hybridization analysis, and a GUS activity assay were used to analyze transformants . The timing of appearance and pattern of accumulation of GUS transcript and GUS protein in transformants were consistent with the timing of appearance and pattern of accumulation of ver-1 transcript and Ver-1 protein . These data suggested that the GUS gene was under the same regulatory control as the wild-type ver-1 gene and confirmed that transcriptional regulation plays an important role in ver-1A expression . Integration of the ver-1A-GUS reporter construct at the niaD locus resulted in 500-fold-lower GUS activity, but the temporal pattern of accumulation of GUS activity was not affected . Therefore, chromosomal location can play a role in determining the level of gene expression in A . parasiticus and should be an important consideration when analyzing promoter function in this organism.

Appl Environ Microbiol, 1997 Mar, 63(3), 990 - 5
Development of polyclonal antibodies for detection of aflatoxigenic molds involving culture filtrate and chimeric proteins expressed in Escherichia coli; Shapira R et al.; Polyclonal antibodies (PAb) were raised against an aflatoxigenic strain of Aspergillus parasiticus by using two different sources for antibody elicitation: (i) filtrate of a culture on which the fungus had been grown (ii) and two chimeric proteins, expressed in Escherichia coli as separate products, of the genes ver-1 and apa-2, which are involved in aflatoxin biosynthesis . The gene products were amplified by PCR, and each was cloned into the E . coli expression vector pGEX2T . Upon induction, the bacteria overexpressed 38- and 33-kDa chimeric proteins corresponding to the N-terminal domains of the genes ver-1 and apa-2, respectively . The chimeric proteins were isolated and affinity purified for use as antigens . The specificity of the raised antibodies was examined by enzyme-linked immunosorbent assay (ELISA) . The PAbs raised against the culture filtrate reacted with all the species of Aspergillus and Penicillium tested but not with Fusarium species or corn gain . However, the PAbs elicited against the chimeric proteins were highly specific, showing significantly higher ELISA absorbance values (A405) against A . parasiticus and A . flavus than against the other fungi tested and the corn grain . The approach of utilizing gene products associated with aflatoxin biosynthesis for antibody production therefore appears to be feasible . Such a multiantibody system combined with the PCR technique, could provide a useful tool for the rapid, sensitive, and accurate detection of aflatoxin producers present in grains and foods.

Appl Environ Microbiol, 1997 Mar, 63(3), 945 - 50
Escherichia coli mutants resistant to inactivation by high hydrostatic pressure; Hauben KJ et al.; Alternating cycles of exposure to high pressure and outgrowth of surviving populations were used to select for highly pressure-resistant mutants of Escherichia coli MG1655 . Three barotolerant mutants (LMM1010, LMM1020, and LMM1030) were isolated independently by using outgrowth temperatures of 30, 37, and 42 degrees C, respectively . Survival of these mutants after pressure treatment for 15 min at ambient temperature was 40 to 85% at 220 MPa and 0.5 to 1.5% at 800 MPa, while survival of the parent strain, MG1655, decreased from 15% at 220 MPa to 2 x 10(-8)% at 700 MPa . Heat resistance of mutants LMM1020 and LMM1030 was also altered, as evident by higher D values at 58 and 60 degrees C and reduced z values compared to those for the parent strain . D and z values for mutant LMM1010 were not significantly different from those for the parent strain . Pressure sensitivity of the mutants increased from 10 to 50 degrees C, as opposed to the parent strain, which showed a minimum around 40 degrees C . The ability of the mutants to grow at moderately elevated pressure (50 MPa) was reduced at temperatures above 37 degrees C, indicating that resistance to pressure inactivation is unrelated to barotolerant growth . The development of high levels of barotolerance as demonstrated in this work should cause concern about the safety of high-pressure food processing.

Genetics, 1997 Mar, 145(3), 697 - 705
Cytosolic ribosomal mutations that abolish accumulation of circular intron in the mitochondria without preventing senescence of Podospora anserina; Silar P et al.; The filamentous fungus Podospora anserina presents a degeneration syndrome called Senescence associated with mitochondrial DNA modifications . We show that mutations affecting the two different and interacting cytosolic ribosomal proteins (S7 and S19) systematically and specifically prevent the accumulation of senDNA alpha (a circular double-stranded DNA plasmid derived from the first intron of the mitochondrial cox1 gene or intron alpha) without abolishing Senescence nor affecting the accumulation of other usually observed mitochondrial DNA rearrangements . One of the mutant proteins is homologous to the Escherichia coli S4 and Saccharomyces cerevisiae S13 ribosomal proteins, known to be involved in accuracy control of cytosolic translation . The lack of accumulation of senDNA alpha seems to result from a nontrivial ribosomal alteration unrelated to accuracy control, indicating that S7 and S19 proteins have an additional function . The results strongly suggest that modified expression of nucleus-encoded proteins contributes to Senescence in P . anserina . These data do not fit well with some current models, which propose that intron alpha plays the role of the cytoplasmic and infectious Determinant of Senescence that was defined in early studies.

Hum Gene Ther, 1997 Mar 1, 8(4), 439 - 52
LacZ gene transfer to skeletal muscle using a replication-defective herpes simplex virus type 1 mutant vector; Huard J et al.; Herpes simplex virus type 1 (HSV-1) represents a promising new viral vector capable of efficient transduction of myofibers in vivo . Here we report on the use of a replication-defective HSV-1 mutant vector (DZ) deleted for the essential immediate early (IE) gene ICP4 for studies of reporter gene transfer and expression following direct inoculation of mouse skeletal muscle . The recombinant vector was engineered to contain the Escherichia coli lacZ gene under transcriptional control of the strong human cytomegalovirus (HCMV) IE promoter . The effect of vector cytotoxicity on the durability of transgene expression following infection of muscle cells in culture and myofibers in vivo revealed that this first-generation HSV vector was cytopathic, limiting the persistence of vector-transduced cells . UV irradiation of vector preparations reduced viral cytotoxicity for myoblasts in culture without reducing significantly beta-galactosidase production . Moreover, muscle cell viability and the durability of transgene expression was enhanced by several days following UV inactivated-vector infection in vivo . Nevertheless, the viral DNA was subsequently lost from vector-inoculated muscle tissue within 2 weeks . This observation indicated that vector toxicity alone did not account for the lack of persistent transgene expression . Longer-term vector transduction and transgene expression was observed, however, following inoculation of immunodeficient SCID mice, indicating that host immunocompetence played an important role in determining the duration of transgene expression in animals . To support this hypothesis, cells expressing CD4 and CD8 antigens have been found in the HSV-1 injected muscle of immunocompetent mice . These data demonstrated that both vector toxicity and vector-induced immunity are significant obstacles to the use of HSV-1 vectors for muscle gene transfer . These impediments must be overcome to further develop HSV vectors for muscle gene therapy applications.

Hum Genet, 1997 Mar, 99(3), 399 - 406
Coding versus intron variability: extremely polymorphic HLA-DRB1 exons are flanked by specific composite microsatellites, even in distant populations; Epplen C et al.; Although microsatellite typing is the dominant method in genome research and indirect gene diagnosis, precise relationships of exonic and adjacent simple repeat polymorphisms are not known . We investigated exon 2 sequences of HLA-DRB1 genes and their neighbouring (GT)n(GA)m repeats including the intervening single copy spacer . DRB1 is the most polymorphic protein-coding locus in man and all vertebrates investigated . The entire DRB1 variability exists in exon 2 . DRB1 genes in different haplotype groups (DR1, DR51, DR52, DR8 and DR53) are accompanied by characteristic modifications of the (GT)n(GA)m block (3' to group-specific single copy spacers) . Among more than 520 alleles analysed, > 100 different types of microsatellites were observed . The perfect (GT)n and (GA)m blocks vary in length and may be partly 'degenerated', mostly in a subgroup-specific manner . Interestingly, the extent of microsatellite diversity varies in given DRB1 alleles . While the microsatellites of the DR7, DR9 alleles and in the DR1 group are virtually invariant, in DR4 and DR13, in particular, simple repeats appear hypervariable with at least 15 or 17 different length alleles, respectively . Comparing Caucasians, Bushmen and South American Indians, the microsatellite variation in identical DRB1 alleles (e.g . DRB1*0102, 03011, 1302) is smaller than within any of the DR groups in Caucasians . Taken together, extremely polymorphic DRB1 exons evolve in concert with certain variants of an exceptionally well-preserved microsatellite.

Hum Genet, 1997 Mar, 99(3), 329 - 33
Isolation of a cDNA encoding the human homolog of COX17, a yeast gene essential for mitochondrial copper recruitment; Amaravadi R et al.; The COX17 gene of Saccharomyces cerevisiae codes for a cytoplasmic protein essential for the expression of functional cytochrome oxidase . This protein has been implicated in targeting copper to mitochondria . To determine if Cox17p is present in mammalian cells, a yeast strain carrying a null mutation in COX17 was transformed with a human cDNA expression library . All the respiratory competent clones obtained from the transformations carried a common cDNA sequence with a reading frame predicting a product homologous to yeast Cox17p . The cloning of a mammalian COX17 homolog suggests that the encoded product is likely to function in copper recruitment in eucaryotic cells in general . Its presence in humans provides a possible target for genetically inherited deficiencies in cytochrome oxidase.

J Neurochem, 1997 Mar, 68(3), 961 - 9
Defective herpes simplex virus vectors expressing the rat brain stress-inducible heat shock protein 72 protect cultured neurons from severe heat shock; Fink SL et al.; Recently, preinduction of the heat shock response has been shown to protect CNS neurons undergoing various stressful insults, e.g., heat, ischemia, or exposure to excitotoxins . However, it is not known which of the proteins induced by the heat shock response mediate the protective effects . Previous correlative evidence points to a role for the highly stress-induced 72-kDa heat shock protein (hsp72) . However, it is not known whether hsp72 expression alone can protect against a range of acute neuronal insults . We constructed a herpes simplex virus-1 vector carrying the rat brain stress-inducible hsp72 gene and the Escherichia coli lacZ (marker) gene . Infection with the vector caused hippocampal neurons to coexpress hsp72 and beta-galactosidase . Infection with a control vector led to marker gene expression only . Overexpression of hsp72 protected cultured hippocampal neurons against a heat shock but not against the metabolic toxin 3-nitropropionic acid or the excitotoxin glutamate . This is the first published report of protection following heat shock protein transfection in CNS neurons.

Circ Res, 1997 Mar, 80(3), 327 - 35
Expression and function of recombinant endothelial nitric oxide synthase gene in canine basilar artery; Chen AF et al.; Endothelial NO synthase (eNOS) is an enzyme responsible for the production of a potent vasodilator and a key regulator of vascular tone, NO . In peripheral arteries, expression of a recombinant eNOS gene increases production of NO in the blood vessel wall . This approach appears to be a promising strategy for gene therapy of cerebrovascular disease . The major objective of the present study was to determine whether a recombinant eNOS gene (AdCMVNOS) can be functionally expressed in cerebral arteries . Replication-defective recombinant adenovirus vectors encoding bovine eNOS and Escherichia coli beta-galactosidase (AdCMVLacZ) genes, driven by the cytomegalovirus promoter, were used for ex vivo gene transfer . Rings of canine basilar artery were incubated with increasing titers of the vectors in MEM . Twenty-four or forty-eight hours after gene transfer, expression and function of AdCMVNOS were evaluated by (1) immunohistochemical staining, (2) isometric tension recording, and (3) cGMP radioimmunoassay . Transfection with AdCMVNOS resulted in the expression of recombinant eNOS protein in the vascular adventitia and endothelium, associated with significantly reduced contractile responses to UTP and enhanced endothelium-dependent relaxation to calcium ionophore A23187 . Basal production of cGMP was significantly increased in the transfected vessels . The reduced contractions to UTP with increased cGMP production were reversed by a NOS inhibitor, N(G)-monomethyl-L-arginine . Contractions to UTP or production of cGMP were not affected in arteries transfected with AdCMVLacZ reporter gene . The results of the present study represent the first successful transfer and functional expression of recombinant eNOS gene in cerebral arteries . Our findings suggest that cerebral arterial tone can be modulated by recombinant eNOS expression in the vessel wall.

J Bacteriol, 1997 Mar, 179(5), 1828 - 31
Identification of the lrp gene in Bradyrhizobium japonicum and its role in regulation of delta-aminolevulinic acid uptake; King ND et al.; The heme precursor delta-aminolevulinic acid (ALA) is taken up by the dipeptide permease (Dpp) system in Escherichia coli . In this study, we identified a Bradyrhizobium japonicum genomic library clone that complemented both ALA and dipeptide uptake activities in E . coli dpp mutants . The complementing B . japonicum DNA encoded a product with 58% identity to the E . coli global transcriptional regulator Lrp (leucine-responsive regulatory protein), implying the presence of Dpp-independent ALA uptake activity in those cells . Data support the conclusion that the Lrp homolog induced the oligopeptide permease system in the complemented cells by interfering with the repressor activity of the endogenous Lrp, thus conferring oligopeptide and ALA uptake activities . ALA uptake by B . japonicum was effectively inhibited by a tripeptide and, to a lesser extent, by a dipeptide, and a mutant strain that expressed the lrp homolog from a constitutive promoter was deficient in ALA uptake activity . The data show that Lrp negatively affects ALA uptake in E . coli and B . japonicum . Furthermore, the product of the isolated B . japonicum gene is both a functional and structural homolog of E . coli Lrp, and thus the regulator is not restricted to enteric bacteria.

J Bacteriol, 1997 Mar, 179(5), 1819 - 23
In vivo evidence that acyl coenzyme A regulates DNA binding by the Escherichia coli FadR global transcription factor; Cronan JE Jr; In vitro experiments point to fatty acyl coenzymes A (acyl-CoAs) rather than unesterified fatty acids as the small-molecule ligands regulating DNA binding by the FadR protein of Escherichia coli . To provide an in vivo test of this specificity, unesterified fatty acids were generated within the cellular cytosol . These fatty acids were found to be efficient modulators of FadR action only when the acids could be converted to acyl-CoAs.

J Bacteriol, 1997 Mar, 179(5), 1787 - 95
Mutations in the alpha and sigma-70 subunits of RNA polymerase affect expression of the mer operon; Caslake LF et al.; The mercury resistance (mer) operon is transcribed from overlapping, divergent promoters: PR for the regulatory gene merR and P(TPCAD) for the structural genes merTPCAD . The dyadic binding site for MerR lies within the 19-bp spacer of the sigma70-dependent P(TPCAD) . Unlike typical repressors, MerR does not exclude RNA polymerase from P(TPCAD) but rather forms an inactive complex with RNA polymerase at P(TPCAD) prior to addition of the inducer, the mercuric ion Hg(II) . In this "active repression" complex, MerR prevents transcriptional initiation at merTPCAD until Hg(II) is added . When Hg(II) is added, MerR remains bound to the same position and activates transcription of merTPCAD by distorting the DNA of the spacer region . MerR also represses its own transcription from PR regardless of the presence or absence of Hg(II) . To explore the role of MerR-RNA polymerase in these processes, we examined mutations in the sigma70 and alpha subunits of RNA polymerase, mutations known to influence other activators but not to impair transcription generally . We assessed the effects of these sigma70 and alpha mutants on unregulated P(TPCAD) and PR transcription (i.e., MerR-independent transcription) and on the two MerR-dependent processes: repression of P(TPCAD) and of PR and Hg(ll)-induced activation of P(TPCAD) . Among the MerR-independent effects, we found that mutations in regions 2.1 and 4.2 of rpoD suppress the deleterious effects of nonoptimal promoter spacing . Some C-terminal rpoA mutants also have this property to a considerably lesser degree . Certain "spacer suppressor" variants of rpoA and of rpoD also interfere with the MerR-dependent repression of P(TPCAD) and PR . MerR-Hg(II)-mediated transcriptional activation of P(TPCAD) was also affected in an allele-specific manner by substitutions at position 596 of sigma70 and at positions 311 and 323 of alpha . Thus, certain changes in sigma70 or alpha render them either more or less effective in participating in the topologically novel transcriptional control effected by MerR at the divergent mer operons.

J Bacteriol, 1997 Mar, 179(5), 1774 - 9
Regulation of the Escherichia coli tna operon: nascent leader peptide control at the tnaC stop codon; Konan KV et al.; Expression of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and by tryptophan-induced transcription antitermination at Rho-dependent termination sites in the leader region of the operon . Tryptophan induction is dependent on translation of a short leader peptide coding region, tnaC, that contains a single, crucial tryptophan codon . Recent studies suggest that during induction, the TnaC leader peptide acts in cis on the translating ribosome to inhibit its release at the tnaC stop codon . In the present study we use a tnaC-UGA-'lacZ construct lacking the tnaC-tnaA spacer region to analyze the effect of TnaC synthesis on the behavior of the ribosome that translates tnaC . The tnaC-UGA-'lacZ construct is not expressed significantly in the presence or absence of inducer . However, it is expressed in the presence of UGA suppressors, or when the structural gene for polypeptide release factor 3 is disrupted, or when wild-type tRNATrP is overproduced . In each situation, tnaC-UGA-'lacZ expression is reduced appreciably by the presence of inducing levels of tryptophan . Replacing the tnaC UGA stop codon with a sense codon allows considerable expression, which is also reduced, although to a lesser extent, by the addition of tryptophan . Inhibition by tryptophan is not observed when Trp codon 12 of tnaC is changed to a Leu codon . Overexpression of tnaC in trans from a multicopy plasmid prevents inhibition of expression by tryptophan . These results support the hypothesis that the TnaC leader peptide acts in cis to alter the behavior of the translating ribosome.

J Bacteriol, 1997 Mar, 179(5), 1755 - 63
Nature of DNA binding and RNA polymerase interaction of the Bordetella pertussis BvgA transcriptional activator at the fha promoter; Boucher PE et al.; The expression of virulence factor genes in Bordetella pertussis is mediated by the BvgA-BvgS two-component signal transduction system . The response regulator, BvgA, acts directly as a transcriptional activator at the loci encoding pertussis toxin (ptx) and filamentous hemagglutinin (fha) . Previous studies have demonstrated that these two loci are differentially regulated by BvgA . As an initial step in gaining insight into the mechanism underlying this differential regulation, we initiated DNA binding and in vitro transcription analyses to examine the activities of BvgA and RNA polymerase (RNAP) purified from both B . pertussis and Escherichia coli at the fha promoter . We discovered that unphosphorylated BvgA binds to a single region (-100 to -70, relative to the start of transcription), whereas phosphorylated BvgA binds both this region and another, farther downstream, that extends to the -35 nucleotide . In the absence of BvgA, RNAP binds a region farther upstream than expected (-104 to -35) . However, occupation of both sites by BvgA phosphate repositions RNAP to the site used in vivo . The binding of BvgA phosphate to the downstream site correlates with in vitro transcriptional activity at the fha promoter . As the DNA binding and transcription activities of the E . coli-derived RNAP are similar to those observed for the B . pertussis enzyme, we employed several mutant E . coli proteins in in vitro transcription analyses . We observed that polymerases carrying either a deletion of the C-terminal domain of the alpha subunit or substitution of alanine at either of two critical residues within this domain were severely impaired in the ability to mediate BvgA-activated transcription at fha.

J Bacteriol, 1997 Mar, 179(5), 1727 - 33
The stpA gene form synechocystis sp . strain PCC 6803 encodes the glucosylglycerol-phosphate phosphatase involved in cyanobacterial osmotic response to salt shock; Hagemann M et al.; Mutations in a gene, stpA, had been correlated with the loss of tolerance to high NaCl concentrations in the cyanobacterium Synechocystis sp . strain PCC 6803 . Genetic, biochemical, and physiological evidence shows that stpA encodes glucosylglycerol-phosphate phosphatase . stpA mutants are salt sensitive and accumulate glucosylglycerol-phosphate, the precursor of the osmoprotectant glucosylglycerol necessary for salt adaptation of Synechocystis . The consensus motif present in acid phosphatases was found in StpA; however, the homology with other sugar phosphatases is very poor . The amount of stpA mRNA was increased by growth of the cells in the presence of NaCl concentrations above 170 mM . Expression of stpA in Escherichia coli allowed the production of a 46-kDa protein which exhibited glucosylglycerol-phosphate phosphatase activity . The StpA-specific antibody revealed a protein of similar size in extracts of Synechiocystis, and the amount of this protein was increased in salt-adapted cells . The protein produced in E . coli had lost the requirement for activation by NaCl that was observed for the genuine cyanobacterial enzyme.

J Bacteriol, 1997 Mar, 179(5), 1704 - 13
Examination of the Tn5 transposase overproduction phenotype in Escherichia coli and localization of a suppressor of transposase overproduction killing that is an allele of rpoH; Yigit H et al.; Tn5 transposase (Tnp) overproduction is lethal to Escherichia coli . Tnp overproduction causes cell filamentation, abnormal chromosome segregation, and an increase in anucleated cell formation . There are two simple explanations for the observed phenotype: induction of the SOS response or of the heat shock response . The data presented here show that overproduction of Tnp neither induces an SOS response nor a strong heat shock response . However, our experiments do indicate that induction of some sigma32-programmed function(s) (either due to an rpoH mutation, a deletion of dnaK, or overproduction of sigma32) suppresses Tnp overproduction killing . This effect is not due to overproduction of DnaK, DnaJ, or GroELS . In addition, Tnp but not deltall Tnp (whose overproduction does not kill the host cells) associates with the inner cell membrane, suggesting a possible correlation between cell killing and Tnp membrane association . These observations will be discussed in the context of a model proposing that Tnp overproduction titrates an essential host factor(s) involved in an early cell division step and/or chromosome segregation.

J Bacteriol, 1997 Mar, 179(5), 1636 - 45
Phospho-beta-glucosidase from Fusobacterium mortiferum: purification, cloning, and inactivation by 6-phosphoglucono-delta-lactone; Thompson J et al.; 6-Phosphoryl-beta-D-glucopyranosyl:6-phosphoglucohydrolase (P-beta-glucosidase, EC 3.2.1.86) has been purified from Fusobacterium mortiferum . Assays for enzyme activity and results from Western immunoblots showed that P-beta-glucosidase (Mr, 53,000; pI, 4.5) was induced by growth of F . mortiferum on beta-glucosides . The novel chromogenic and fluorogenic substrates, p-nitrophenyl-beta-D-glucopyranoside-6-phosphate (pNPbetaGlc6P) and 4-methylumbelliferyl-beta-D-glucopyranoside-6-phosphate (4MUbetaGlc6P), respectively, were used for the assay of P-beta-glucosidase activity . The enzyme hydrolyzed several P-beta-glucosides, including the isomeric disaccharide phosphates cellobiose-6-phosphate, gentiobiose-6-phosphate, sophorose-6-phosphate, and laminaribiose-6-phosphate, to yield glucose-6-phosphate and appropriate aglycons . The kinetic parameters for each substrate are reported . P-beta-glucosidase from F . mortiferum was inactivated by 6-phosphoglucono-delta-lactone (P-glucono-delta-lactone) derived via oxidation of glucose 6-phosphate . The pbgA gene that encodes P-beta-glucosidase from F . mortiferum has been cloned and sequenced . The first 42 residues deduced from the nucleotide sequence matched those determined for the N terminus by automated Edman degradation of the purified enzyme . From the predicted sequence of 466 amino acids, two catalytically important glutamyl residues have been identified . Comparative alignment of the amino acid sequences of P-beta-glucosidase from Escherichia coli and F . mortiferum indicates potential binding sites for the inhibitory P-glucono-delta-lactone to the enzyme from F . mortiferum.

J Bacteriol, 1997 Mar, 179(5), 1584 - 90
Molecular genetic characterization of the Escherichia coli gntT gene of GntI, the main system for gluconate metabolism; Porco A et al.; The Escherichia coli gntT gene was subcloned from the Kohara library, and its expression was characterized . The cloned gntT gene genetically complemented mutant E . coli strains with defects in gluconate transport and directed the formation of a high-affinity gluconate transporter with a measured apparent Km of 6 microM for gluconate . Primer extension analysis indicated two transcriptional start sites for gntT, which are separated by 66 bp and which give rise to what appears on a Northern blot to be a single, gluconate-inducible, 1.42-kb gntT transcript . Thus, it was concluded that gntT is monocistronic and is regulated by two promoters . Both of the promoters have - 10 and -35 sequence elements typical of sigma70 promoters and catabolite gene activator protein binding sites in appropriate locations to exert glucose catabolite repression . In addition, two putative gnt operator sites were identified in the gntT regulatory region . A search revealed the presence of nearly identical palindromic sequences in the regulatory regions of all known gluconate-inducible genes, and these seven putative gnt operators were used to derive a consensus gnt operator sequence . A gntT::lacZ operon fusion was constructed and used to examine gntT expression . The results indicated that gntT is maximally induced by 500 microM gluconate, modestly induced by very low levels of gluconate (4 microM), and partially catabolite repressed by glucose . The results also showed a pronounced peak of gntT expression very early in the logarithmic phase, a pattern of expression similar to that of the Fis protein . Thus, it is concluded that GntT is important for growth on low concentrations of gluconate, for entry into the logarithmic phase, and for cometabolism of gluconate and glucose.

J Bacteriol, 1997 Mar, 179(5), 1550 - 4
Nonadaptive mutations occur on the F' episome during adaptive mutation conditions in Escherichia coli; Foster PL; One of the most studied examples of adaptive mutation is a strain of Escherichia coli, FC40, that cannot utilize lactose (Lac-) but that readily reverts to lactose utilization (Lac+) when lactose is its sole carbon source . Adaptive reversion to Lac+ occurs at a high rate when the Lac- allele is on an F' episome and conjugal functions are expressed . It was previously shown that nonselected mutations on the chromosome did not appear in the Lac- population while episomal Lac+ mutations accumulated, but it remained possible that nonselected mutations might occur on the episome . To investigate this possibility, a second mutational target was created on the Lac- episome by mutation of a Tn1O element, which encodes tetracycline resistance (Tetr), to tetracycline sensitivity (Tets) . Reversion rates to Tetr during normal growth and during lactose selection were measured . The results show that nonselected Tetr mutations do accumulate in Lac- cells when those cells are under selection to become Lac+ . Thus, reversion to Lac+ in FC40 does not appear to be adaptive in the narrow sense of the word . In addition, the results suggest that during lactose selection, both Lac+ and Tetr mutations are created or preserved by the same recombination-dependent mechanism.

J Clin Microbiol, 1997 Mar, 35(3), 609 - 13
Comparison of the western blot assay with the neutralizing-antibody and enzyme-linked immunosorbent assays for measuring antibody to verocytotoxin 1; Reymond D et al.; A Western blot (immunoblot) assay (WBA) was developed to detect immunoglobulin G (IgG) antibodies against Escherichia coli Verocytotoxin 1 (VT1) by using a chemiluminescence detection system . The assay was compared with a VT1-neutralizing-antibody (VT1-NAb) assay and an anti-VT1 IgG enzyme-linked immunosorbent assay (ELISA) . When four human serum samples that were known to be positive by VT1-NAb assay and ELISA were titrated to the endpoint by the three assays, the WBA gave endpoint titers that were up to 8-fold higher than those by ELISA and up to 256-fold higher than those by the VT1-NAb assay . Of 32 serum samples that were known to be positive by VT1-NAb assay and ELISA, 31 (97%) were positive by WBA; the one sample with a discrepant result gave borderline results by the VT1-NAb assay and ELISA . Of 52 serum samples that were known to be negative by the VT1-NAb assay and ELISA, 50 (96%) were negative and 2 (4%) were positive by WBA . Of 44 serum samples that gave discrepant results by the VT1-NAb assay and ELISA, neither of the latter correlated with the results of WBA . In an investigation of 19 pairs of acute- and convalescent-phase serum samples from patients with hemolytic-uremic syndrome, 10 pairs that were positive by the VT1-NAb assay were also WBA positive, while 9 pairs that were NAb negative were also WBA negative . The WBA is inherently more specific and sensitive than either the NAb assay or the ELISA and may be used as a "gold standard" to detect IgG antibodies to VT1 . Like the NAb assay and the ELISA for detecting antibodies to VT1, the WBA has little to offer in the diagnostic setting but is expected to play an important role in seroepidemiological studies.

J Clin Microbiol, 1997 Mar, 35(3), 553 - 7
Evaluation of the recombinant 38-kilodalton antigen of Mycobacterium tuberculosis as a potential immunodiagnostic reagent; Wilkinson RJ et al.; The diagnosis of infection caused by Mycobacterium tuberculosis is of increased public health concern following increases in the number of cases in developed countries and major increases in developing countries associated with the spread of human immunodeficiency virus (HIV) infection . The specificity of purified protein derivative skin testing for the detection of infection is compromised by exposure to environmental mycobacteria . Examination of sputum detects the most infectious patients, but not those with extrapulmonary disease . The 38-kDa antigen of M . tuberculosis contains two M . tuberculosis-specific B-cell epitopes . We overexpressed the gene for this antigen in Escherichia coli and evaluated the recombinant product in in vitro assays of T-cell function and as a target for the antibody response in humans . The sensitivity and specificity of the antigen as a skin test reagent were also assessed in outbred guinea pigs . We found that 69% of healthy sensitized humans recognize the antigen in vitro, as manifested by both cell proliferation and the production of gamma interferon . Untreated patients initially have a lower frequency of response (38%); this recovers to 72% during therapy . A total of 292 patients (20 with HIV coinfection) and 58 controls were examined for production of antibody to the 38-kDa antigen by using a commercially available kit . The sensitivity of the test in comparison with that of culture was 72.6%, and the specificity was 94.9% . The antigen was also tested for its ability to induce skin reactions in outbred guinea pigs sensitized by various mycobacterial species . The antigen provoked significant skin reactions in M . tuberculosis-, M . bovis BCG-, and M . intracellulare-sensitized animals . The significance of these findings and the usefulness of this antigen in immunodiagnosis are discussed.

J Infect Dis, 1997 Mar, 175(3), 606 - 10
Vaginal colonization by Escherichia coli as a risk factor for very low birth weight delivery and other perinatal complications; Krohn MA et al.; This study evaluated the relationship of vaginal Escherichia coli colonization to birth weight <1500 g and other perinatal complications in a cross-sectional study of 2646 women at the University of Washington Medical Center, Seattle, between October 1992 and January 1995 . Vaginal E . coli colonization was more strongly associated with delivery at <34 weeks (relative risk {RR}, 1.7; 95% confidence interval {CI}, 1.3-2.3) and very low birth weight (RR, 1.9; 95% CI, 1.3-2.7) than with prematurity between 34 and 36 weeks or low birth weight . Heavy growth of E . coli had a higher risk of very low birth weight than light growth (RR, 2.4; 95% CI, 1.0-6.2) . It may be important to screen and treat pregnant women for genital tract colonization with E . coli during prenatal care.

Cancer Res, 1997 Mar 1, 57(5), 913 - 20
Characterization of a second human cyclin A that is highly expressed in testis and in several leukemic cell lines; Yang R et al.; In this study, we isolated and characterized a human cyclin A-like gene that we named cyclin A1 . Cyclin A1 has 48% identity with human cyclin A and is more related to the recently cloned murine cyclin A1 (84% identity) . The human cyclin A1 is specifically expressed in testis and brain among all of the normal tissues that we studied by Northern blot analysis; in addition, it is expressed in several myeloid leukemia cell lines, including ML-1, U937, NB4, KG-1, and THP1 . A sensitive reverse transcription-PCR-Southern blot method also detected low-level expression of this gene in many other hematopoietic and nonhematopoietic cell lines . The expression of cyclin A1 mRNA is differentiation- and cell cycle-regulated in the ML-1 cells . We raised polyclonal antibodies against a glutathione S-transferase-cyclin A1 fusion protein produced in Escherichia coli . In immunoblot analyses, the antibodies recognized the Mr 65,000 cyclin A1 protein in ML-1 cells . The anti-cyclin A1 also immunoprecipitated the Mr 65,000 cyclin A1, along with the Mr 33,000 cyclin-dependent kinase (CDK) 2 and other proteins at Mr 39,000, 42,000, 45,000, 95,000, and 110,000 . In an in vitro kinase assay, the CDK2-cyclin A1 complex precipitated by anti-cyclin A1 showed kinase activities against histone H1 . In a yeast two-hybrid assay, cyclin A1 can bind to CDK2 but not to CDC2, CDK4, and CDK5 . We mapped the human cyclin A1 gene to chromosome 13q12.3-q13, approximately 1000 kb from the sequence-tagged site marker WI-3374.

J Virol, 1997 Mar, 71(3), 1931 - 7
Characterization of an ATP-dependent DNA ligase encoded by Chlorella virus PBCV-1; Ho CK et al.; We report that Chlorella virus PBCV-1 encodes a 298-amino-acid ATP-dependent DNA ligase . The PBCV-1 enzyme is the smallest member of the covalent nucleotidyl transferase superfamily, which includes the ATP-dependent polynucleotide ligases and the GTP-dependent RNA capping enzymes . The specificity of PBCV-1 DNA ligase was investigated by using purified recombinant protein . The enzyme catalyzed efficient strand joining on a singly nicked DNA in the presence of magnesium and ATP (Km, 75 microM) . Other nucleoside triphosphates or deoxynucleoside triphosphates could not substitute for ATP . PBCV-1 ligase was unable to ligate across a 2-nucleotide gap and ligated poorly across a 1-nucleotide gap . A native gel mobility shift assay showed that PBCV-1 DNA ligase discriminated between nicked and gapped DNAs at the substrate-binding step . These findings underscore the importance of a properly positioned 3' OH acceptor terminus in substrate recognition and reaction chemistry.

Nucleic Acids Res, 1997 Mar 1, 25(5), 987 - 91
DNA binding and subunit interactions in the type I methyltransferase M.EcoR124I; Mernagh DR et al.; The type I DNA methyltransferase M.EcoR124I consists of two methylation subunits (HsdM) and one DNA recognition subunit (HsdS) . When expressed independently, HsdS is insoluble, but this subunit can be obtained in soluble form as a GST fusion protein . We show that the HsdS subunit, even as a fusion protein, is unable to form a discrete complex with its DNA recognition sequence . When HsdM is added to the HsdS fusion protein, discrete complexes are formed but these are unable to methylate DNA . The two complexes formed correspond to species with one or two copies of the HsdM subunit, indicating that blocking the N-terminus of HsdS affects one of the HsdM binding sites . However, removal of the GST moiety from such complexes results in tight and specific DNA binding and restores full methylation activity . The results clearly demonstrate the importance of the HsdM subunit for DNA binding, in addition to its catalytic role in the methyltransferase reaction.

Brain Res, 1997 Feb 28, 749(2), 344 - 6
In the mouse, the corticoid stress response depends on lateralization; Neveu PJ et al.; The influence of brain/behavioral lateralization on the neuroendocrine stress response was studied in the mouse . Using a paw preference test in a food reaching task, mice were classified as left-pawed, ambidextrous or right-pawed . Plasma levels of corticosterone (CS) were measured in basal conditions, 4 h after an intraperitoneal injection of lipopolysaccharide (LPS) or after a short period of restraint . In unstressed control mice, plasma levels of corticosterone were higher in left-pawed animals as compared to ambidextrous . LPS increased plasma levels of CS to similarly high levels, around 600 ng/ml, in the three experimental groups . By contrast after 1 h of restraint, the increased CS levels, lower to those observed after LPS injection, were higher in left-pawed mice as compared to right-pawed animals . These results are the first demonstration that activation of the hypothalamic-pituitary-adrenal axis observed during the stress response to a physical stimulus may be related to lateralization.

Biophys Chem, 1997 Feb 28, 64(1-3), 253 - 69
Quantitative analysis of ligand-macromolecule interactions using differential dynamic quenching of the ligand fluorescence to monitor the binding; Jezewska MJ et al.; Quantitative analyses of the thermodynamics and kinetics of ligand-macromolecule interactions in biological systems rely predominately on monitoring changes in the spectroscopic properties of the ligand or macromolecule, particularly fluorescence changes, which accompany the formation of the studied complexes . However, in many instances the interactions do not affect the fluorescence properties of interacting species and do not provide a resolution high enough to perform quantitative and rigorous measurements of the thermodynamic and/or kinetic parameters . In this communication, we describe the theoretical and experimental aspects of a method of studying complex, multiple ligand-macromolecule interactions by the fluorescence titration technique, when the intrinsic fluorescence changes accompanying binding do not provide a resolution necessary to perform quantitative analyses . The method is based on the fact that a fluorescent ligand, or binding sites of the macromolecule, can have different accessibility to the collisional (dynamic) quencher, when involved in the complex, rather than in the free, unbound state . The presence of an external dynamic quencher in solution, i.e., the presence of an extra collisional quenching process, transforms the fluorescence of the ligand or macromolecule, intrinsically independent of the complex formation, into a property which is dramatically different in the free state than in the bound state of the fluorophore . The approach is applicable to any model of noncooperative or cooperative ligand binding to a macromolecule and allows for the optimization of the resolution of the binding or kinetic studies for a given ligand-macromolecule system . The application of the method is illustrated by applying it to the study of the binding of the fluorescent derivative of a nucleotide cofactor, epsilon ADP, to the six interacting sites of the E . coli primary replicative helicase DnaB protein hexamer.

Mol Cells, 1997 Feb 28, 7(1), 110 - 4
Deletion analysis of the Escherichia coli rnpB terminators; Kim S et al.; The region for intrinsic transcription termination of the Escherichia coli rnpB gene coding for M1 RNA is repeated three times . Each region encodes an RNA-terminator hairpin and U-rich 3' tail . Most transcription is terminated at the first terminator (T1), but a complete termination requires the second terminator (T2) and the third (T3) . A deletion experiment, where deletions were extended from T3 into T1, was carried out to determine parameters affecting in vivo transcription termination at T1 . The deletion of T3, T2 or the downstream sequences up to near the termination site of T1 showed little or no significant effect on transcription termination at T1 . When the base corresponding to the acute termination position of T1 was included in the deletion, termination efficiency slightly decreased . For T1 bearing the deletion of the region encoding the RNA-terminator hairpin and U-rich 3' tail, termination was completely abolished . When T1 contained only the region encoding the RNA-termination hairpin without the U-rich 3' tail, termination still occurred even though the efficiency was low . Characteristics of rnpB termination were discussed with these results.

Mol Cells, 1997 Feb 28, 7(1), 72 - 7
Cross-competition for TATA-binding protein between TATA boxes of the selenocysteine tRNA{Ser}Sec promoter and RNA polymerase II promoters; Park JM et al.; In this study we show that the TATA-binding protein (TBP) interacts with the selenocysteine tRNA{Ser}Sec TATA element in a fashion analogous to the TBP-TATA interaction in RNA polymerase (Pol) II-transcribed genes even though the gene is transcribed by Pol III . Recombinant TBPs expressed in Escherichia coli bound to the tRNA{Ser}Sec TATA element . A factor was detected in Xenopus oocyte extracts which contain TBP and bind to the TATA boxes of the tRNA{Ser}Sec gene and various class II genes . Transcription of the microinjected tRNA{Ser}Sec gene was inhibited in Xenopus oocytes by coinjection with the TATA box of the adenovirus major late promoter (AdMLP) . Transcription of a 5S gene was not affected under these conditions . These results suggest that the tRNA{Ser}Sec gene recruits TBP in a manner similar to that of TATA-dependent Pol II-transcribed genes and differently from that of Pol III-transcribed genes lacking a TATA box.

Gene, 1997 Feb 28, 186(2), 227 - 35
Molecular cloning and characterization of Borrelia burgdorferi rpoB; Alekshun M et al.; Borrelia burgdorferi rpoB, the gene encoding the beta-subunit of RNA polymerase, has been cloned and sequenced . The full-length gene encodes a protein of 1154 amino acids with a calculated molecular mass of 129.8 kDa . The amino-acid sequence is 49% identical to the corresponding protein from Escherichia coli . B . burgdorferi rpoB is a component of a gene cluster, which includes rplJ, rplL and rpoC . A temperature-sensitive E . coli rpoB mutant could be complemented by introduction of the B . burgdorferi gene, indicating that the B . burgdorferi rpoB is expressed in E . coli and the beta-subunit can be assembled into functional holoenzyme . The wild-type amino-acid sequence of the B . burgdorferi beta-subunit is consistent with those of spontaneously arising rifampicin-resistant mutants of E . coli and Mycobacterium tuberculosis at certain critical residues . This suggests that the natural resistance of B . burgdorferi to rifampicin may be due to the primary amino-acid sequence of its beta-subunit.

Gene, 1997 Feb 28, 186(2), 219 - 26
Transforming DNA integrates at multiple sites in the dimorphic fungal pathogen Blastomyces dermatitidis; Hogan LH et al.; Blastomyces dermatitidis is a primary fungal pathogen of man and other mammals, but like many other human fungal pathogens, relatively little is known about the factors that account for its virulence and pathogenicity . We developed a transformation system to facilitate molecular genetic studies of putative virulence factors from B . dermatitidis . Transformation of the multinucleate yeasts was achieved by electroporation of DNAs containing a dominant selectable marker, hygromycin B (HygB) resistance . Southern analysis showed that transforming DNA invariably integrated ectopically into the chromosome . No evidence was found for extrachromosomal DNA . The HygB resistance could be expressed by either a 375-bp promoter fragment of the B . dermatitidis WI-1 gene encoding adhesin or an Aspergillus gpdA promoter placed 5' of the E . coli hph gene . Primer extension analysis showed that for plasmids containing the WI-1 promoter, transcription of the hph gene initiated within the 375-bp WI-1 promoter fragment . The combination of gene transfer and two promoters capable of independent transcription will allow us to restore or augment gene expression in appropriate strains and test an influence on virulence . Molecular genetic manipulation of B . dermatitidis represents a major advance in our ability to investigate the pathogenesis of blastomycosis and other similar fungal diseases.

J Mol Biol, 1997 Feb 28, 266(3), 621 - 32
Alkaline phosphatase-Strep tag fusion protein binding to streptavidin: resonant mirror studies; Hengsakul M et al.; The properties of a fusion protein comprising a streptavidin recognition sequence (Strep tag) fused to the C terminus of Escherichia coli alkaline phosphatase are described . The catalytic properties were determined with p-nitrophenyl phosphate and compared to those of the native E . coli alkaline phosphatase . It was found that the Km values were similar in both cases (8 microM for transferase and 2 microM for hydrolase activities) whilst the Vmax values were lower for the fusion protein, possibly due to the presence of misfolded forms . An optical biosensor based on the resonant mirror was used to determine the binding kinetics between the fusion protein and the immobilised streptavidin . The association and dissociation rate constants were determined to be 2.1(+/-0.3) x 10(-2) microM(-1) s(-1) and 11(+/-0.2) x 10(-3) s(-1), respectively, which results in an equilibrium dissociation constant of 0.5 microM . This is larger than previously reported affinities based on titration calorimetry and may be a consequence of the presence of two streptavidin binding sequences on the dimeric alkaline phosphatase simultaneously binding to two subunits of streptavidin.

J Mol Biol, 1997 Feb 28, 266(3), 610 - 20
Folding kinetics of Che Y mutants with enhanced native alpha-helix propensities; Lopez-Hernandez E et al.; In this work we study the folding kinetics of Che Y mutants in which the helical propensity of each of its five alpha-helices has been greatly enhanced by local interactions (between residues close in sequence) . This constitutes an experimental test on the role of local interactions in protein folding, as well as providing new information on the details of the folding pathway of the protein Che Y . With respect to the first issue, our results show that the enhancement of helical propensities by native-like local interactions in Che Y has the following general effects: (1) the energetics of the whole Che Y folding energy landscape (folded state, intermediate, denatured state and main transition state) are affected by the enhancement of helical propensities, thus, native-like local interactions appear to have a low specificity for the native conformation; (2) our results support the idea, proposed from thermodynamic analysis of the mutants, that the denatured state under native conditions becomes more compact upon enhancement of helical propensities; (3) the rate of folding in aqueous solution decreases in all the mutants, suggesting that the optimization of the folding rate in this protein requires low secondary structure propensities . Regarding the description of the folding pathway of Che Y, we find evidence that the folding transition state of Che Y is constituted by two sub-domains with different degree of helical structure . The first includes helices 1 and 2 which are rather structured, while the second encompasses the last three helices, which are very unstructured . On the other hand, the same analysis for the folding intermediate indicates that all the five alpha-helices are, on average, rather structured . Thus, suggesting that a large structural reorganization of the last three alpha-helices must take place before folding can be completed . This conclusion indicates that the folding intermediate of Che Y is a misfolded species.

J Mol Biol, 1997 Feb 28, 266(3), 525 - 37
Recombinase binding specificity at the chromosome dimer resolution site dif of Escherichia coli; Hayes F et al.; Xer site-specific recombination functions in Escherichia coli chromosome segregation and cell division apparently by resolving chromosome dimers, which arise through homologous recombination, to monomers . Xer recombination requires two closely related site-specific recombinases, XerC and XerD, which bind cooperatively to the recombination site dif and catalyse separate pairs of strand exchanges . The dif site is an imperfect palindrome whose left and right halves are bound by XerC and XerD, respectively . By using variant dif sites in which the symmetry between the XerC and XerD binding sites was increased incrementally, the determinants in the dif site that specifically direct binding of XerC and XerD to their cognate sites were elucidated . The primary specificity nucleotides in the XerC and XerD binding sites were identified and their relative contributions to specificity assessed . The biological affects of these mutations on site-specific recombination, chromosome segregation and cell division were examined . The specificity determinants are confined to the non-palindromic outer ends of the binding sites . Replacement of the wild-type dif site with mutated dif sites at the normal location in the replication terminus region of the chromosome revealed that the sequence of the dif site can be altered substantially while retaining apparently normal chromosome segregation activity.

Biochim Biophys Acta, 1997 Feb 28, 1350(3), 317 - 24
Cloning and expression of cDNA for a newly identified isozyme of bovine liver 3-hydroxyacyl-CoA dehydrogenase and its import into mitochondria; Furuta S et al.; cDNA for a heretofore undescribed mitochondrial 3-hydroxyacyl-CoA dehydrogenase, designated as the type II enzyme with different molecular and catalytic properties, compared to those of the classical mitochondrial beta-oxidation enzyme (type I enzyme), was cloned from a bovine liver cDNA library . Nucleotide sequence of the cDNA encoded 261 amino acids with a subunit molecular weight of 27,140 . The deduced primary structure of the type II enzyme showed no significant homology to the reported amino acid sequence of the classical 3-hydroxyacyl-CoA dehydrogenases . On SDS-PAGE, no differences in subunit molecular weights were observed among the in vitro translation products, the recombinant type II enzyme produced in Escherichia coli and the purified enzyme . NH2-terminal and COOH-terminal amino acid sequence analysis of the purified type II enzyme revealed that the mature enzyme had not been proteolytically processed . The in vitro translation products of the type II enzyme were efficiently incorporated into isolated rat liver mitochondria, without changes in size, thereby suggesting that unlike other mitochondrial enzymes of fatty acid beta-oxidation, the type II enzyme had no cleavable signal peptide upon import into mitochondria.

Biochim Biophys Acta, 1997 Feb 28, 1350(3), 293 - 305
Purification and characterization of full-length mammalian poly(A) polymerase; Wittmann T et al.; Bovine poly(A) polymerase was purified from overexpressing strains of Escherichia coli and from Spodoptera frugiperda Sf21 cells infected with a recombinant baculovirus . The E . coli-expressed enzyme had an apparent molecular mass of 85 kDa in SDS gels, as anticipated from the cDNA sequence . Poly(A) polymerase from insect cells consisted of several species with higher apparent molecular weights due to phosphorylation . The two preparations showed minor differences in their catalytic properties . The insect cell-expressed enzyme had a 5-fold higher Km for the primer in a nonspecific Mn(2+)-dependent polyadenylation reaction and a lower activity in specific AAUAAA-dependent polyadenylation and generated shorter poly(A) tails during the processive phase of polyadenylation . Both recombinant poly(A) polymerases stimulated 3'-cleavage of the SV40 late mRNA precursor . Neither preparation contained ATPase or poly(A) degrading activity . The enzyme polymerized adenosine 5'-O-(1-thiotriphosphate), SP-diastereomer, with inversion of configuration . Thus, poly(A) synthesis proceeds via an SN2-in-line mechanism without covalent intermediate.

Biochim Biophys Acta, 1997 Feb 28, 1350(3), 277 - 81
The sequence of a symbiotically essential Bradyrhizobium japonicum operon consisting of trpD, trpC and a moaC-like gene; Kuykendall LD et al.; The 2767 bp BamHI-HindIII fragment specifying the trpDC genes of B . japonicum I-110 was sequenced . The trpD and trpC genes each have three highly conserved 'Crawford' consensus sequences and are part of an operon with three open reading frames (ORFs) . The third ORF has a predicted product with 58% amino-acid sequence identity with the gene product of E . coli moaC, a gene encoding an enzyme involved in biosynthesis of the molybdenum cofactor required for the activity of nitrate reductase and other Mo cofactor-requiring enzymes.

J Med Chem, 1997 Feb 28, 40(5), 677 - 83
Structure-based design of substituted diphenyl sulfones and sulfoxides as lipophilic inhibitors of thymidylate synthase; Jones TR et al.; Six new diphenyl sulfoxide and five new diphenyl sulfones were designed, synthesized, and tested for their inhibition of human and Escherichia coli thymidylate synthase (TS) and of the growth of cells in tissue culture . The best sulfoxide inhibitor of human TS was 3-chloro-N-((3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl)-4- (phenylsulfinyl)-N-(prop-2-ynyl)-aniline (7c) that had a Ki of 27 nM . No sulfone improved on TS inhibition by the previously reported 4-(N-((3,4-dihydro-2-methyl-6-quinazolinyl)methyl)-N-prop-2- ynylamino)phenyl phenyl sulfone (Ki = 12 nM) . Nevertheless, one sulfone, 4-((2-chlorophenyl)sulfonyl)-N-((3,4-dihydro-2-methyl-4-oxo-6- quinazolinyl)methyl)-N-(prop-2-ynyl)aniline, was selected, on the basis of its inhibition of both TS and cell growth, for antitumor testing; it gave a 61% increase in life span to mice bearing the thymidino kinase-deficient L5178Y (TK-) lymphoma . A crystal structure of N-((3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)methyl)-4-((2- methylphenyl)sulfinyl)-N-(prop-2-ynyl)aniline complexed with E . coli TS was solved and revealed selective binding of one sulfoxide enantiomer . AMBER calculations showed that the enantioselection was due to asymmetric electrostatic effects at the mouth of the active site . In contrast, a similar crystal structure of the sulfoxide 7c, along with AMBER calculations, indicated that both enantiomers bound, but with different affinities . The side chain of Phe176 shifted in order to structurally accommodate the chlorine of the more weakly bound enantiomer.

J Biol Chem, 1997 Feb 28, 272(9), 5880 - 6
Short hydrophobic segments in the mature domain of ProOmpA determine its stepwise movement during translocation across the cytoplasmic membrane of Escherichia coli; Sato K et al.; Based on the finding that a series of engineered proOmpAs containing disulfide-bridged loops of different sizes at different positions exhibits a discontinuous mode of polypeptide transit across the cytoplasmic membrane of Escherichia coli, we suggested previously that the translocation of preproteins takes place at every 30 amino acid residues (Uchida, K., Mori, H., and Mizushima, S . (1995) J . Biol . Chem . 270, 30862-30868) . In the present study, we investigated the molecular mechanism underlying this stepwise translocation . Deletion or relocation of hydrophobic segments of the mature domain of proOmpA (H1, residues 233-237; H2, residues 261-265) significantly altered the pattern of the stepwise translocation . The stepwise mode of polypeptide insertion was also observed with reconstituted proteoliposomes comprising purified SecA, SecY, and SecE . Cross-linking experiments involving a photoactivable cross-linker revealed that SecY and SecA are the components which interact with the hydrophobic segment of proOmpA . The present results indicate that the hydrophobic segments of the mature domains of preproteins interact with membrane embedded translocase during polypeptide transit across the membrane, which causes a discontinuous mode of polypeptide movement.

J Biol Chem, 1997 Feb 28, 272(9), 5821 - 7
Segments in the C-terminal folding domain of lipoprotein lipase important for binding to the low density lipoprotein receptor-related protein and to heparan sulfate proteoglycans; Nielsen MS et al.; Lipoprotein lipase (LpL) can mediate cellular uptake of chylomicron and VLDL remnants via binding to heparan sulfate proteoglycans (HSPG) and the endocytic alpha2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha2MR/LRP) . Whereas it is established that the C-terminal folding domain binds to alpha2MR/LRP, it remains uncertain whether it binds to heparin and to HSPG . To identify segments important for binding to alpha2MR/LRP and to clarify possible binding to heparin, we produced constructs of the human C-terminal folding domain, LpL-(313-448), and of the fragment LpL-(347-448) in Escherichia coli . In addition to binding to alpha2MR/LRP, LpL-(313-448) displayed binding to heparin with an affinity similar to that of the LpL monomer, whereas it bound poorly to lipoprotein particles . Moreover, LpL-(313-448) displayed heparin sensitive binding to normal, but not to HSPG deficient Chinese hamster ovary cells . LpL-(313-448) and LpL-(347-448) showed similar affinities for binding to both purified alpha2MR/LRP and to heparin . Deletion of LpL residues 380-384 abolished the binding to LRP, whereas binding to heparin was unperturbed . The binding to both heparin and alpha2MR/LRP was essentially abolished following deletion of residues 404-430, and pretreatment of CHO cells with the peptide comprising aa 402-423 inhibited the binding of LpL-(313-448) . We conclude that the C-terminal folding domain of human LpL has a site for binding to heparin and to HSPG, presumably involving amino acids within residues 404-430 . Two segments of the domain are necessary for efficient binding to alpha2MR/LRP, one comprising residues 380-384 and another overlapping the segment important for binding to heparin.

J Biol Chem, 1997 Feb 28, 272(9), 5741 - 6
Identification and functional characterization of an active-site lysine in mevalonate kinase; Potter D et al.; We report the construction of an expression plasmid for rat mevalonate kinase and the overexpression of recombinant enzyme in Escherichia coli . The homogeneous enzyme had a specific activity of 30 units/mg and an observed subunit molecular mass of 42 kDa . The Michaelis constants (Km) for DL-potassium mevalonate (288 microM) and for ATP (1.24 mM) were in agreement with values reported for enzymes isolated from rat liver (Tanaka, R . D., Schafer, B . L., Lee, L . Y., Freudenberger, J . S., and Mosley, S . T . (1990) J . Biol . Chem . 265, 2391-2398) . Recombinant rat mevalonate kinase was inactivated by the lysine-specific reagent, pyridoxal phosphate (PLP) . ATP (5 mM) afforded protection against inactivation, suggesting reaction of PLP with an active-site lysine . Mapping, isolation, and Edman degradation of the ATP-protectable peptide from {3H}PLP-inactivated borohydride-reduced mevalonate kinase allow assignment of lysine 13, a residue invariant in known mevalonate kinase sequences, as the modification site . These results represent the first identification of an active-site residue in mevalonate kinase . The function of lysine 13 was evaluated by replacing this residue with methionine . Vm of the mutant protein is diminished by 56-fold, suggesting that lysine 13 facilitates catalysis . Kd values of wild-type and mutant proteins for ATP were determined in electron spin resonance competition experiments . The observed 56-fold diminution in affinity for the mutant enzyme supports an additional role for lysine 13 in stabilization of ATP binding.

J Biol Chem, 1997 Feb 28, 272(9), 5445 - 51
Side reactions catalyzed by ribulose-bisphosphate carboxylase in the presence and absence of small subunits; Morell MK et al.; The large subunit core of ribulose-bisphosphate carboxylase from Synechococcus PCC 6301 expressed in Escherichia coli in the absence of its small subunits retains a trace of carboxylase activity (about 1% of the kcat of the holoenzyme) (Andrews, T . J (1988) J . Biol . Chem . 263, 12213-12219) . During steady-state catalysis at substrate saturation, this residual activity diverted approximately 10% of the reaction flux to 1-deoxy-D-glycero-2,3-pentodiulose-5-phosphate as a result of beta elimination of inorganic phosphate from the first reaction intermediate, the 2,3-enediol form of ribulose bisphosphate . This indicates that the active site's ability to stabilize and/or retain this intermediate is compromised by the absence of small subunits . Epimerization and isomerization of the substrate resulting from misprotonation of the enediol intermediate were not significantly exacerbated by lack of small subunits . The residual carboxylating activity partitioned product between pyruvate and 3-phosphoglycerate in a ratio similar to that of the holoenzyme, indicating that stablization of the penultimate three-carbon aci-acid intermediate is not perturbed by lack of small subunits . The underlying instability of the five-carbon enediol intermediate was revealed, even with the holoenzyme, under conditions designed to lead to exhaustion of substrate CO2 (and O2) . When carboxylation (and oxygenation) stalled upon exhaustion of gaseous substrate, both spinach and Synechococcus holoenzymes continued slowly to beta eliminate inorganic phosphate from and to misprotonate the enediol intermediate . With carboxylation and oxygenation blocked, the products of these side reactions of the enediol intermediate accumulated to readily detectable levels, illustrating the difficulties attendant upon ribulose-P2 carboxylase's use of this reactive species as a catalytic intermediate.

Science, 1997 Feb 28, 275(5304), 1305 - 8
Crystal structure of formate dehydrogenase H: catalysis involving Mo, molybdopterin, selenocysteine, and an Fe4S4 cluster; Boyington JC et al.; Formate dehydrogenase H from Escherichia coli contains selenocysteine (SeCys), molybdenum, two molybdopterin guanine dinucleotide (MGD) cofactors, and an Fe4S4 cluster at the active site and catalyzes the two-electron oxidation of formate to carbon dioxide . The crystal structures of the oxidized {Mo(VI), Fe4S4(ox)} form of formate dehydrogenase H (with and without bound inhibitor) and the reduced {Mo(IV), Fe4S4(red)} form have been determined, revealing a four-domain alphabeta structure with the molybdenum directly coordinated to selenium and both MGD cofactors . These structures suggest a reaction mechanism that directly involves SeCys140 and His141 in proton abstraction and the molybdenum, molybdopterin, Lys44, and the Fe4S4 cluster in electron transfer.

Mol Gen Genet, 1997 Feb 27, 253(6), 734 - 44
The phytoene dehydrogenase gene of Phycomyces: regulation of its expression by blue light and vitamin A; Ruiz-Hidalgo MJ et al.; By using a polymerase chain reaction based cloning strategy we isolated the gene (carB) encoding the enzyme phytoene dehydrogenase from Phycomyces blakesleeanus . The deduced protein, a 583 residue polypeptide, showed great similarity to carotenoid dehydrogenases from other fungi and bacteria, especially in the amino-terminal region . The main conserved regions found in other phytoene dehydrogenases, which are thought to be essential for the enzymatic activity, are present in the sequence from Phycomyces . Heterologous expression of the Phycomyces gene in Escherichia coli showed that, as in other fungi and bacteria, a single polypeptide catalyzes the four dehydrogenations that convert phytoene to lycopene . RNA measurements indicated that the level of expression of the phytoene dehydrogenase gene in wild-type mycelia increased in response to blue light . The kinetics of this increase in transcription of the gene after blue light induction (0.1 and 0.4 W/m2) exhibit a two-step (biphasic) dependence on fluence rate, suggesting that there could be two separate components involved in the reception of the low and high blue light signal . The presence of vitamin A in the medium stimulated transcript accumulation in the wild type and in some carotenogenic mutant strains . Diphenylamine, a phytoene dehydrogenase inhibitor, did not affect the level of transcription of this gene.

Mol Gen Genet, 1997 Feb 27, 253(6), 695 - 702
Targeting of a functional Escherichia coli RecA protein to the nucleus of plant cells; Reiss B et al.; We have characterised a RecA protein fused to the simian virus 40 large T nuclear-localisation signal . The fusion protein was targeted to the nucleus in transgenic tobacco plants with high efficiency . By contrast, authentic RecA was not enriched in the nuclei of plant cells expressing comparable amounts of protein . For detailed characterisation of the strand-exchange activity of the nuclear-targeted RecA protein, a nearly identical protein was expressed in Escherichia coli and purified to homogeneity . This protein was found to bind to single-stranded DNA with the same stoichiometry and to promote the exchange of homologous DNA strands with the same kinetics as authentic RecA . It was concluded that the amino-terminal modification did not alter any of the essential properties of RecA and that the fusion protein is a fully functional strand-exchange protein . However, the ATPase activity of this protein was 20 times greater than that of RecA in the absence of single-stranded DNA . As with RecA, this activity was further stimulated by the addition of single-stranded DNA . Since ATPase activity is correlated with the ability of RecA to assume its high affinity state for DNA, the nuclear-targeted RecA protein might be regarded as a constitutively stimulated RecA variant, fully functional in promoting homologous recombination.

Biochemistry, 1997 Feb 25, 36(8), 2332 - 7
DNA synthesis arrest at C4'-modified deoxyribose residues; Hess MT et al.; Many genotoxic agents form base lesions that inhibit DNA polymerases . To study the mechanism underlying termination of DNA synthesis on defective templates, we tested the capacity of a model enzyme (Klenow fragment of Escherichia coli DNA polymerase I) to catalyze primer elongation across a series of C4' deoxyribose derivatives . A site with inverted C4' configuration or two different C4' deoxyribose adducts were introduced into the backbone of synthetic templates without modifying the chemistry of the corresponding bases . Inverted deoxyribose moieties may arise in cellular DNA as a product of C4' radical attack . We found that DNA synthesis by the Klenow polymerase was arrested transiently at the C4' inversion and was essentially blocked at C4' deoxyribose adducts . Major termination sites were located one position downstream of a C4' selenophenyl adduct and immediately 3' to or opposite a C4' pivaloyl adduct . Primer extension studies in the presence of single deoxyribonucleotides showed intact base pairing fidelity opposite all three C4' variants regardless of whether the Klenow fragment or its proofreading-deficient mutant was tested . These results imply that the coding ability of template bases is maintained at altered C4' deoxyribose moieties . However, their capacity to impede DNA polymerase progression indicates that backbone distortion and steric hindrance are important determinants of DNA synthesis arrest on damaged templates . The strong inhibition by C4' adducts suggests a potential target for new chemotherapeutic strategies.

Biochemistry, 1997 Feb 25, 36(8), 2323 - 31
Thermodynamics of unfolding for Kazal-type serine protease inhibitors: entropic stabilization of ovomucoid first domain by glycosylation; DeKoster GT et al.; A synthetic gene for chicken ovomucoid first domain (OMCHI1) has been overexpressed in Escherichia coli . The resulting recombinant protein, rOMCHI1, is expressed and correctly folded without the use of fusion proteins or export secretion signal peptides incorporated into the gene . The thermostability of rOMCHI1 has been compared to that of the naturally occurring glycosylated OMCHI1 (gOMCHI1) . The results of differential scanning calorimetry (DSC) studies show that the heat capacity change for unfolding, deltaCp, for both rOMCHI1 and gOMCHI1 is approximately 600 cal/(mol x K) . At any given pH, however, the presence of N-linked carbohydrate increases the Tm for thermal unfolding of gOMCHI1 over rOMCHI1 by 2-4 degrees C, without changing the enthalpy of unfolding, delta H(degree)m . This suggests that the increased thermal stability of gOMCHI1 is entropic . Comparison of the unfolding thermodynamics of rOMCHI1 with those of turkey ovomucoid third domain (OMTKY3), which is 36% identical to rOMCHI1, reveals similar deltaCp values for both proteins, about 600 cal/(mol x K), but a reduction in delta H(degree)m of about 5 kcal/mol for rOMCHI1 at all temperatures . Decreases in delta H(degree)m for rOMCHI1 versus OMTKY3 may be explained by an overall less ordered native state in rOMCHI1 . In the absence of a native structure for OMCHI1, the change in accessible surface area upon unfolding, deltaASA, was calculated using unfolding parameters and structural energetic relationships {Murphy & Freire (1992) Adv . Protein Chem . 43, 313-361; Murphy et al . (1993), Proteins: Struct., Funct., Genet . 15, 113-120} . These calculations suggest that the larger protein rOMCHI1 (Mr 7500) exposes less surface area than OMTKY3 (Mr 6100) upon thermal denaturation . Overall, structural energetic relationships may provide a useful framework for interpretation and comparison of thermodynamic data for structurally homologous proteins.

Biochemistry, 1997 Feb 25, 36(8), 2278 - 90
Ligand binding alters the backbone mobility of intestinal fatty acid-binding protein as monitored by 15N NMR relaxation and 1H exchange; Hodsdon ME et al.; The backbone dynamics of the liganded (holo) and unliganded (apo) forms of Escherichia coli-derived rat intestinal fatty acid-binding protein (I-FABP) have been characterized and compared using amide 15N relaxation and 1H exchange NMR measurements . The amide 1H/15N resonances for apo and holo I-FABP were assigned at 25 degrees C, and gradient- and sensitivity-enhanced 2D experiments were employed to measure l5N T1, T2, and {1H}15N NOE values and relative 1H saturation transfer rates . The 15N relaxation parameters were analyzed using five different representations of the spectral density function based on the Lipari and Szabo formalism . A majority of the residues in both apo and holo I-FABP were characterized by relatively slow hydrogen exchange rates, high generalized order parameters, and no conformational exchange terms . However, residues V26-N35, S53-R56, and A73-T76 of apo I-FABP were characterized by rapid hydrogen exchange, low order parameters, and significant conformational exchange . These residues are clustered in a single region of the protein where variability and apparent disorder were previously observed in the chemical shift analyses and in the NOE-derived NMR structures of apo I-FABP . The increased mobility and discrete disorder in the backbone of the apo protein may permit the entry of ligand into the binding cavity . We postulate that the bound fatty acid participates in a series of long-range cooperative interactions that cap and stabilize the C-terminal half of helix II and lead to an ordering of the portal region . This ligand-modulated order-disorder transition has implications for the role of I-FABP in cellular fatty acid transport and targeting.

Biochemistry, 1997 Feb 25, 36(8), 2139 - 46
Requirement for an additional divalent metal cation to activate protein tyrosine kinases; Sun G et al.; In addition to the magnesium ion needed to form the true phosphate-donating substrate (ATP-Mg complex), we have determined that at least one additional Mg2+ ion is essential for the activation of protein tyrosine kinases . This activation was investigated in detail using purified Csk, Src, and the fibroblast growth factor receptor kinase, which led to the following conclusions . (1) The catalytic activity of these kinases is dependent on the Mg2+ concentration present in the assay, approaching saturation at 5-8 mM MgCl2, while ATP was saturated at approximately 1 mM MgCl2 . (2) Extrapolation to zero free Mg2+ at a constant ATP-Mg concentration predicts zero activity, suggesting that free magnesium ion in excess of that needed to bind to ATP is essential for the activation of these enzymes . (3) The free magnesium ion activates Csk and Src kinase activity by increasing the Vmax but does not change their apparent Km(ATP-Mg) . In contrast, the free magnesium ion activates the fibroblast growth factor receptor kinase activity by increasing its Vmax and decreasing its apparent Km(ATP-Mg) . These and previous studies with the insulin receptor tyrosine kinase suggest that receptor-type protein tyrosine kinases respond to the concentration of free Mg2+ differently than soluble protein tyrosine kinases . (4) With the phosphate-accepting substrate as the variable ligand, increases in the concentration of free Mg2+ resulted in increases in the apparent Vmax for all tyrosine kinases examined, but the apparent Km response is dependent on the enzyme and the substrate used . While these studies do not pinpoint a single kinetic mechanism, they do suggest that additional magnesium ion(s) is(are) an essential activator for protein tyrosine kinases in addition to being a part of the ATP-Mg complex . The difference among protein tyrosine kinases in their kinetic response to the additional divalent metal cation and the potential biological significance of such are discussed.

FEBS Lett, 1997 Feb 24, 403(3), 294 - 8
Studies by site-directed mutagenesis of the carbohydrate-binding properties of a bark lectin from Robinia pseudoacacia; Nishiguchi M et al.; A bark lectin, RBL, from Robinia pseudoacacia (black locust), binds galactose-related sugars specifically . Recombinant RBL (rRBL) with a histidine tag was expressed in Escherichia coli, purified and characterized . rRBL agglutinated rabbit erythrocytes and the hemagglutination was inhibited by galactose and related sugars . To elucidate the mechanism of the binding of carbohydrate by RBL, 16 mutant rRBLs were produced by site-directed mutagenesis . The analysis of the mutants indicated that residues Phe130 and Asp87 play key roles in the binding of carbohydrate by RBL . When Thu215, Leu217 and Ser218 in the carboxy-terminal region were replaced by alanine, the respective replacements decreased the hemagglutinating activity . However, replacement by alanine of Glu219 did not decrease this activity . Three mutant rRBLs were generated by reference to the primary sequences of the proposed carbohydrate- and metal-binding regions of mannose-specific lectins . Although these rRBLs agglutinated rabbit erythrocytes, the hemagglutination was not inhibited by mannose . Substitution or insertion that yielded a partial sequence similar to those of L-fucose-specific lectins and hemagglutinin from Maackia amurensis resulted in a complete loss of the hemagglutinating activity of rRBL.

FEBS Lett, 1997 Feb 24, 403(3), 268 - 72
Deletion mutagenesis as a test of evolutionary relatedness of indoleglycerol phosphate synthase with other TIM barrel enzymes; Stehlin C et al.; The role of the extra helix alpha zero in the N-terminal extension of the eight-fold beta alpha barrel of indoleglycerol phosphate synthase was probed by point mutation and truncation . Replacing invariant leucine 5 by valine of the enzyme from Escherichia coli affected neither kcat nor Km, but deletion of 8 N-terminal residues decreased solubility strongly . The similarly truncated variant from the hyperthermophile Sulfolobus solfataricus was soluble, and had the same kcat value as the wild-type protein but a 220-fold greater Km value . These results suggest that the N-terminal portion of helix alpha zero provides for strong binding of the substrate, but is not essential for stabilizing the bound transition state . Thus, three enzymes of tryptophan biosynthesis operate essentially as canonical eight-fold beta alpha barrels, as required for their divergent evolution.

FEBS Lett, 1997 Feb 24, 403(3), 254 - 8
Identification of Raf-1 Ser621 kinase activity from NIH 3T3 cells as AMP-activated protein kinase; Sprenkle AB et al.; Raf-1 is extensively phosphorylated on Ser621 in both quiescent and mitogen-stimulated cells . To identify the responsible kinase(s), cytosolic fractions of NIH 3T3 cells were analyzed for Ser621 peptide kinase activity . One major peak of activity was detected and identified as AMP-activated protein kinase (AMPK) by immunodepletion experiments . AMPK phosphorylated the catalytic domain of Raf-1, expressed in Escherichia coli as a soluble GST fusion protein, to generate a single tryptic {32P}phosphopeptide containing exclusively phospho-Ser621 . AMPK also phosphorylated full-length, kinase-defective Raf-1 (K375M) to generate two {32P}phosphopeptides, one co-migrating with synthetic tryptic peptide containing phospho-Ser621 and the other with phospho-Ser259.

FEBS Lett, 1997 Feb 24, 403(3), 245 - 8
Ribonuclease T1 has different dimensions in the thermally and chemically denatured states: a dynamic light scattering study; Gast K et al.; Ribonuclease T1 can be unfolded and refolded without forming noticeable amounts of aggregates allowing to characterise the dimensions of a protein in different denatured states in terms of the Stokes radius RS . Upon thermal unfolding RS increases from 1.74 nm at 20 degrees C to 2.14 nm at 60 degrees C . By contrast, RS = 2.40 nm was obtained at 5.3 M guanidinium chloride (GuHCl) and 20 degrees C . Heating from 20 degrees C to 70 degrees C in the presence of 5.3 M GuHCl led to a 5% decrease in RS.

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