Escherichia coli

J Biol Chem, 1995 Mar 17, 270(11), 6081 - 7
Immunological characterization and chloroplast localization of the tryptophan biosynthetic enzymes of the flowering plant Arabidopsis thaliana; Zhao J et al.; In order to study the tryptophan biosynthetic enzymes of the plant Arabidopsis thaliana, polyclonal antibodies were raised against five of the tryptophan biosynthetic pathway proteins: anthranilate synthase alpha subunit, phosphoribosylanthranilate transferase, phosphoribosylanthranilate isomerase, and the tryptophan synthase alpha and beta subunits . Immunoblot analysis of Arabidopsis leaf protein extracts revealed that the antibodies identify the corresponding proteins that are enriched in Arabidopsis chloroplast fractions . Precursors of phosphoribosylanthranilate isomerase and tryptophan synthase alpha subunit were synthesized by in vitro translation . The precursors were efficiently imported and processed by isolated spinach chloroplasts, and the cleavage sites within the precursors were determined . These results provide the first direct evidence that the tryptophan biosynthetic enzymes from Arabidopsis are synthesized as higher molecular weight precursors and then imported into chloroplasts and processed into their mature forms.

J Biol Chem, 1995 Mar 17, 270(11), 5963 - 78
Inhibition of HIV-1 replication and activation of RNase L by phosphorothioate/phosphodiester 2',5'-oligoadenylate derivatives; Sobol RW et al.; 2',5'-Oligoadenylate (2-5A) derivatives have been designed to act distal to the human immunodeficiency virus-1 (HIV-1)-induced blockade in the 2-5A synthetase/RNase L antiviral pathway . Stereochemical modification of individual internucleotide linkages of the 2-5A molecule was accomplished by phosphoramidite and phosphotriester chemical syntheses . Phosphorothioate/phosphodiester trimer and tetramer 2-5A derivatives revealed differences in the stereodynamics of activation of RNase L and inhibition of HIV-1 replication . The first and second internucleotide linkages are critical for activation of recombinant, human RNase L; A(Rp)ApA, A(Sp)ApA and ApA(Rp)A are agonists (IC50 = 2 x 10(-7), 2 x 10(-6) and 8 x 10(-6) M); ApA(Sp)A is an antagonist . The second and third internucleotide linkages are crucial for activation of murine RNase L; ApA(Rp)A, ApA(Rp)ApA, and ApApA(Rp)A are agonists (IC50 = 5 x 10(-7) M); ApA(Sp)A, ApA(Sp)ApA, and ApApA(Sp)A are antagonists . Inhibition of HIV-1-induced syncytia formation by the phosphorothioate/phosphodiester derivatives is specific for derivatives with substitution at the 2',3'-terminus . ApA(Rp)A, ApA(Sp)A, ApApA(Rp)A, and ApApA(Sp)A are potent inhibitors of HIV-1-induced syncytia formation (80-, 10-, 40-, and 15-fold more inhibitory, respectively, than solvent control) . HIV-1 infection results in enhanced uptake and accumulation of ApA(Rp)A and ApA(Sp)A (7- and 10-fold, respectively) . These stereochemically modified 2-5A derivatives are taken up preferentially by HIV-1-infected cells and show promise in anti-HIV-1 chemotherapy.

J Biol Chem, 1995 Mar 17, 270(11), 5818 - 22
Inorganic polyphosphate in mammalian cells and tissues; Kumble KD et al.; Inorganic polyphosphate (polyP), a linear polymer of hundreds of orthophosphate (Pi) residues linked by high-energy, phosphoanhydride bonds, has been identified and measured in a variety of mammalian cell lines and tissues by unambiguous enzyme methods . Subpicomole amounts of polyP (0.5 pmol/100 micrograms of protein) were determined by its conversion to ATP by Escherichia coli polyphosphate kinase and, alternatively, to Pi by Saccharomyces cerevisiae exopolyphosphatase . Levels of 25 to 120 microM (in terms of Pi residues), in chains 50 to 800 residues long, were found in rodent tissues (brain, heart, kidneys, liver, and lungs) and in subcellular fractions (nuclei, mitochondria, plasma membranes, and microsomes) . PolyP in brain was predominantly near 800 residues and found at similar levels pre- and postnatally . Conversion of Pi into polyP by cell lines of fibroblasts, T-cells, kidney, and adrenal cells attained levels in excess of 10 pmol per mg of cell protein per h . Synthesis of polyP from Pi in the medium bypasses intracellular Pi and ATP pools suggesting the direct involvement of membrane component(s) . In confluent PC12 (adrenal pheochromocytoma) cells, polyP turnover was virtually complete in an hour, whereas in fibroblasts there was little turnover in four hours . The ubiquity of polyP and variations in its size, location, and metabolism are indicative of a multiplicity of functions for this polymer in mammalian systems.

J Biol Chem, 1995 Mar 17, 270(11), 5805 - 11
Characterization of the Elk-1 ETS DNA-binding domain; Shore P et al.; The ETS domain family of transcription factors is comprised of several important proteins that are involved in controlling key cellular events such as proliferation, differentiation, and development . One such protein, Elk-1, regulates the activity of the c-fos promoter in response to extracellular stimuli . Elk-1 is representative of a subgroup of ETS domain proteins that utilize a bipartite recognition mechanism that is mediated by both protein-DNA and protein-protein interactions . In this study, we have overexpressed, purified, and characterized the ETS DNA-binding domain of Elk-1 (Elk-93) . Elk-93 was expressed in Escherichia coli as a fusion protein with glutathione S-transferase and purified to homogeneity from both the soluble and insoluble fractions using a two-column protocol . A combination of CD, NMR, and fluorescence spectroscopy demonstrates that Elk-93 represents an independently folded domain of mixed alpha/beta structure in which the three conserved tryptophans appear to contribute to the hydrophobic core of the protein . Moreover, DNA binding studies demonstrate that Elk-93 binds DNA with both high affinity (Kd approximately 0.85 x 10(-10)M) and specificity . Circular permutation analysis indicates that DNA binding by Elk-93 does not induce significant bending of the DNA . Our results are discussed with respect to predictive models for the structure of the ETS DNA-binding domain.

J Biol Chem, 1995 Mar 17, 270(11), 5764 - 71
Protein synthesis initiation factor eIF-1A is a moderately abundant RNA-binding protein; Wei CL et al.; Eukaryotic initiation factor (eIF) 1A (formerly called eIF-4C) is a small protein that promotes dissociation of 80 S ribosomes into subunits, stabilizes methionyl-tRNA binding to 40 S ribosomal subunits, and is required for the binding of mRNA to ribosomes . The sequence of eIF-1A derived from its cloned cDNA possesses a high frequency of basic residues and acidic residues at its N and C termini, respectively . Northwestern blotting with a fragment of mRNA indicates that eIF-1A binds RNA . Overexpression of the human eIF-1A cDNA in Escherichia coli and subsequent purification enabled us to prepare large quantities of active factor . The level of eIF-1A in HeLa cells determined by Western immunoblotting is 0.01% of total protein, which corresponds to 0.2 molecules of eIF-1A/ribosome . The moderate abundance means that eIF-1A is equal to or in excess of native 40 S subunits and suggests that the factor may not be limiting for protein synthesis, a conclusion reinforced by the failure of overproduced eIF-1A to stimulate translation rates in transiently transfected COS-1 cells . S1 nuclease protection and primer extension analyses show that eIF-1A mRNA possesses an unusually long 5'-untranslated leader that is very G/C-rich (72%) . Unexpectedly, the mRNA is efficiently translated in HeLa cells as judged by polysome profile analyses.

J Biol Chem, 1995 Mar 17, 270(11), 5748 - 55
The purification, cloning, and high level expression of a glutaredoxin-like protein from the hyperthermophilic archaeon Pyrococcus furiosus; Guagliardi A et al.; A protein has been purified to homogeneity from crude extracts of the hyperthermophilic archaeon Pyrococcus furiosus based on its ability to catalyze the reduction of insulin disulfides in the presence of dithiothreitol; the protein has a molecular mass of 24.8 kDa and a pI of 4.9, and it is highly heat-stable . The first 29 amino acid residues at the N terminus of the P . furiosus protein were determined by Edman degradation, and its gene was cloned in Escherichia coli . The amino acid sequence derived from the DNA sequence contains the CPYC sequence, which is typical of the active site of glutaredoxin (also called thioltransferase) . The C-terminal portion of the P . furiosus protein, containing the conserved sequence, shows sequence similarity with glutaredoxins from different sources . The P . furiosus protein can reduce disulfide bonds in L-cystine in the presence of GSH (the thioltransferase activity) with an optimum pH of 8.0 . The expression of the P . furiosus protein, with full activity, in E . coli at a very high level (21% of total soluble protein) is described; the recombinant protein was purified to homogeneity by merely two successive heat treatments and gel filtration chromatography . The features of the P . furiosus protein here described are discussed in light of the current knowledge about the ubiquitous family of protein disulfide oxidoreductases.

J Med Chem, 1995 Mar 17, 38(6), 967 - 72
Quantitative structure-activity relationships of the inhibition of Pneumocystis carinii dihydrofolate reductase by 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(X-phenyl)-s-triazines; Marlowe CK et al.; The inhibitory activities of 60 4,6-diamino-1,2-dihydro-2,2-dimethyl-1- (X-phenyl)-s-triazines versus purified, recombinant Pneumocystis carinii (Pc) dihydrofolate reductase (DHFR) have been determined at pH 7.0 . Utilization of these Kiapp values has led to the formulation of appropriate quantitative structure-activity relationships (QSAR's) for both meta- and parasubstituted derivatives . The QSAR's from Pc are compared with other triazine QSAR's derived versus chicken, murine tumor, Escherichia coli, and particularly human DHFR . Selectivity indices indicate that hydrophobic triazines are particularly effective versus Pc DHFR; they have lower Ki values for Pc DHFR than for human DHFR.

J Med Chem, 1995 Mar 17, 38(6), 869 - 74
1-Thiomethoxsalen and 1-thiopsoralen: synthesis, photobiological properties, and site specific reaction of thiopsoralens with DNA; Jakobs AE et al.; The sulfur analogues of psoralen and 8-methoxypsoralen (8-MOP) in the pyrone moiety were synthesized and compared to the parent compounds in terms of photoreactivity with viral M13mp19 RF DNA . The damaged viral DNA was transfected into Escherichia coli and scored for infectivity toward Ca-treated wild-type E . coli . This allowed a comparative study of the sulfur and oxygen analogues to be made in terms of photoreactivity . Furthermore, the DNA sequence specificity for the formation of monoadducts and cross-links of the four analogues was determined with 32P-labeled oligonucleotides containing thymidine in different sequences . The most site specific of the studied psoralens is 8-MOP, while 1-thiopsoralen is the most reactive analogue . This new thio analogue of psoralen leads to the efficient formation of monoadducts and cross-links in any pyrimidine-purine site.

Biochem Biophys Res Commun, 1995 Mar 17, 208(2), 714 - 20
Cloning, expression and purification of full length Rep78 of adeno-associated virus as a fusion protein with maltose binding protein in Escherichia coli; Batchu RB et al.; The adeno-associated virus (AAV) Rep78 protein is required for many aspects of AAV's life cycle including its DNA replication and the regulation of its gene expression . Because of increasing utilization of AAV as a gene therapy vector and its possible use as an anti-cancer/anti-viral agent, the complete characterization of its Rep78 regulatory protein is important . In order to study various functional aspects of Rep78, we have cloned and expressed the Rep78 gene in Escherichia coli using an inducible expression plasmid . The entire Rep78 open reading frame (nt 321 to 2185) was cloned into the LacZ inducible expression vector pMALc2 . Upon induction of the Ptac promoter with isopropyl thio-beta-D-galactopyranoside (IPTG), Rep78 is produced as a fusion protein with maltose binding protein (MBP) . This recombinant MBP-Rep78 protein displayed all the biochemical activities which are described for the wild type protein including binding to the AAV terminal repeats (TR), endonuclease activity, and helicase activity . Furthermore, for the first time, ATP binding by Rep78 is demonstrated.

J Biol Chem, 1995 Mar 17, 270(11), 6298 - 307
Self-coded 3'-extension of run-off transcripts produces aberrant products during in vitro transcription with T7 RNA polymerase; Triana-Alonso FJ et al.; More than 70% of the RNA synthesized by T7 RNA polymerase during run-off transcription in vitro can be incorrect products, up to twice as long as the expected transcripts . Transcriptions with model templates indicate that false transcription is mainly observed when the correct product cannot form stable secondary structures at the 3'-end . Therefore, the following hypothesis is tested: after leaving the DNA template, the polymerase can bind a transcript to the template site and the 3'-end of the transcript to the product site and extend it, if the 3'-end is not part of a stable secondary structure . Indeed, incubation of purified transcripts with the polymerase in transcription conditions triggers a 3'-end prolongation of the RNA . When two RNAs of different lengths are added to the transcription mix, both generate distinct and specific patterns of prolonged RNA products without any interference, demonstrating the self-coding nature of the prolongation process . Furthermore, sequencing of the high molecular weight transcripts demonstrates that their 5'-ends are precisely defined in sequence, whereas the 3'-ends contain size-variable extensions which show complementarity to the correct transcript . Surprisingly, a reduction of the UTP concentration to 0.2-1.0 mM in the presence of 3.5-4.0 mM of the other NTPs leads to faithful transcription and good yields, irrespective of the nucleotide composition of the template.

Eur J Pharmacol, 1995 Mar 16, 292(3-4), 257 - 63
Mechanisms of interleukin-2-induced depression of hepatic cytochrome P-450 in mice; Cantoni L et al.; Interleukin-2 (15 micrograms/mouse, i.p . twice daily for 4 days and once on the 5th day) significantly lowered cytochrome P-450 and heme content and increased heme oxygenase mRNA accumulation; the activities of 7-ethoxycoumarin O-deethylase, ethoxy- and pentoxyphenoxazone O-dealkylases were decreased . The activity of the type O form of hepatic xanthine oxidase increased, but there was no increase in lipid peroxide, expressed in terms of microsomal malondialdehyde . In vivo inactivation of xanthine oxidase activity by feeding mice with tungstate did not substantially change the degree of interleukin-2-induced cytochrome P-450 depression, suggesting that the two processes are not causally linked . Induction of tolerance to endotoxin by a 4-day pretreatment with lipopolysaccharide resulted in 50% protection against this depression despite inhibition of the interleukin-2 induced formation of tumor necrosis factor . This suggests that the release of tumor necrosis factor per se does not fully account for the depression of cytochrome P-450 . Dexamethasone, already used in patients to reduce the toxicity of interleukin-2 therapy, provided full protection against the cytochrome P-450 depression.

Nature, 1995 Mar 16, 374(6519), 287 - 90
Site-specific RNase E cleavage of oligonucleotides and inhibition by stem-loops; McDowall KJ et al.; The enzyme RNase E (ref . 1) cuts RNA at specific sites within single-stranded segments . The role of adjacent regions of secondary structure in such cleavages is controversial . Here we report that 10-13-nucleotide oligomers lacking any stem-loop but containing the RNase E-cleaved sequence of RNA I, the antisense repressor of replication of ColE1-type plasmids, are cut at the same phosphodiester bond as, and 20 times more efficiently than, RNA I . These findings indicate that, contrary to previous proposals, stem-loops do not serve as entry sites for RNase E, but instead limit cleavage at potentially susceptible sites . Cleavage was reduced further by mutations in a non-adjacent stem-loop, suggesting that distant conformational changes can also affect enzyme access . Modulation of RNase E cleavages by stem-loop regions and to a lesser extent by higher-order structure may explain why this enzyme, which does not have stringent sequence specificity, cleaves complex RNAs at a limited number of sites.

Structure, 1995 Mar 15, 3(3), 239 - 43
Thioredoxin structure and mechanism: conformational changes on oxidation of the active-site sulfhydryls to a disulfide; Holmgren A; The recent high-resolution solution structures of human and Escherichia coli thioredoxin in their oxidized and reduced states support a catalytic model of protein disulfide reduction involving binding of a target protein and nucleophilic attack by the active-site Cys32 thiolate to form a transition state mixed disulfide.

FEMS Microbiol Lett, 1995 Mar 15, 127(1-2), 35 - 9
Use of conjugative and thermosensitive cloning vectors for transposon delivery to Mycobacterium smegmatis; Gavigan JA et al.; Conjugative, thermosensitive shuttle plasmids capable of transfer from Escherichia coli to Mycobacterium smegmatis were constructed . They contain both an E . coli replicon and a thermosensitive derivative of the pAL5000 mycobacterial replicon . Using a temperature shift protocol, the conjugative plasmid, pJAZ11 was used to deliver the transposon Tn611 from E . coli into the chromosome of M . smegmatis . Analysis of transconjugants revealed the random insertion of the transposon.

FEMS Microbiol Lett, 1995 Mar 15, 127(1-2), 133 - 8
Characterization of eukaryotic-like kinase activity in Escherichia coli using the gene-protein database; Sweeney ST et al.; The gene-protein database was used to obtain the two-dimensional polyacrylamide gel coordinates of proteins phosphorylated in extracts of Escherichia coli including those phosphorylated by eukaryotic-like kinase activities . These suggest that the phosphoproteins correspond to, or co-migrate with, the product of an open reading frame at 1.3 min (Orf80), Enzyme 1 of the phosphoenolpyruvate-dependent phosphotransferase system (PtsI), the tRNA synthetase for histidine (HisS), and proteins involved in the response to carbon starvation and quinone treatment.

Eur J Biochem, 1995 Mar 15, 228(3), 863 - 9
Expression and characterization of Geotrichum candidum lipase I gene . Comparison of specificity profile with lipase II; Bertolini MC et al.; Despite tremendous progress in the elucidation of three-dimensional structures of lipases, the molecular basis for their observed substrate preference is not well understood . In an effort to correlate the lipase structure with its substrate preference and to clarify the contradicting reports in the literature, we have compared the enzymic characteristics of two closely related recombinant lipases from the fungus Geotrichum candidum . These enzymes were expressed in the yeast Saccharomyces cerevisiae as fusions with an N-terminal poly(His) tag and were purified in a single step by metal-affinity chromatography . Their specific activities against a series of triacylglycerol substrates were compared using a titrimetric assay . The substrates varied in fatty acyl chain length, number of double bonds and their position along the chain . G . candidum lipases I and II (GCL I and GLC II) are markedly different with respect to their substrate preferences . For unsaturated substrates having long fatty acyl chains (C18:2 cis-9, cis-12 and C18:3 cis-9, cis-12, cis-15), GCL I showed higher specific activity than GCL II, whereas GCL II showed higher specific activity against saturated substrates having short fatty acid chains (C8, C10, C12 and C14) . We have constructed a hybrid molecule containing the N-terminal portion of GCL I (including the flap covering the active site) linked to the C-terminal portion of GCL II . The hybrid molecule showed a substrate preference pattern identical to that of GCL II . These results indicate that sequence variation within the N-terminal 194 amino acids of G . candidum lipases do not contribute to the observed variation in efficiency by which the lipases hydrolyze their substrates . Moreover, it also shows that the flap region in GCL is not directly involved in substrate differentiation, even though this region is thought to be involved in recognition of the interface and in the activation of the enzyme.

Eur J Biochem, 1995 Mar 15, 228(3), 745 - 52
Site-directed mutagenesis of the redox-active cysteines of Trypanosoma cruzi trypanothione reductase; Borges A et al.; The gene for trypanothione reductase from the Silvio strain of Trypanosoma cruzi has been cloned, sequenced and overexpressed in Escherichia coli using the constitutive lpp promoter on the expression plasmid pBSTNAV . Up to 13% of the total soluble protein is enzymically active trypanothione reductase with kinetic properties similar to the enzyme purified from T . cruzi . In order to assess the catalytic role of the putative active-site cysteine residues (C53 and C58), three mutant proteins have been constructed by site-directed mutagenesis substituting alanine or serine residues for cysteine; {C53A}trypanothione reductase, {C53S}trypanothione reductase and {C58S}trypanothione reductase . Although the purified, recombinant mutant proteins were catalytically inactive with NADPH and trypanothione disulphide as substrates, all showed comparable levels of transhydrogenase activity between NADPH and thio-NADP+, suggesting that the mutant proteins had correctly folded in vivo . All three mutants showed substantially different catalytic parameters for thio-NADP+ than the wild-type enzyme, presumably as a consequence of modifying the environment of the enzyme-bound flavin, thereby altering its chemical reactivity . The purified {C58S}trypanothione reductase showed spectral properties similar to the oxidised wild-type enzyme but, unlike the wild-type enzyme, did not acquire the characteristic charge-transfer complex of the EH2 form on addition of NADPH . In contrast, in the absence of NADPH both {C53A}trypanothione reductase and {C53S}trypanothione reductase showed spectral properties similar to the EH2 form of the wild-type enzyme . These data indicate that both C53 and C58 are essential for overall catalysis, with the thiolate anion of C58 interacting with the enzyme-bound FAD and C53 interacting with the disulphide substrate . These mutants should be useful in crystallographic studies of reaction intermediates which cannot be obtained with the catalytically active native enzyme.

Eur J Biochem, 1995 Mar 15, 228(3), 739 - 44
The gag homologue of retrotransposon Ty1 assembles into spherical particles in Escherichia coli; Luschnig C et al.; Expression of TyA (reading frame A) of the yeast retrotransposon Ty1 in Escherichia coli is possible by using efficient transcriptional and translational initiation signals . When expressed in E . coli, the gag homologue of Ty1 assembles into spherical particles similar, but not identical to virus-like particles in the natural host of Ty1, Saccharomyces cerevisiae . Deletion analysis reveals a domain in the C-terminus of TyA that is essential for the assembly process . These findings indicate that an early step of the retroelement life cycle, assembly of the gag homologue into spherical particles, does not depend on specific host factors . The experiments also demonstrate that Ty1 Gag fusion proteins, potential tools for immunization, can be produced in E . coli, an organism that lacks endogenous retrotransposons.

Eur J Biochem, 1995 Mar 15, 228(3), 531 - 50
Signal recognition particle (SRP), a ubiquitous initiator of protein translocation; Lutcke H; In higher eukaryotes, most secretory and membrane proteins are synthesised by ribosomes which are attached to the membrane of the rough endoplasmic reticulum (RER) . This allows the proteins to be translocated across that membrane already during their synthesis . The ribosomes are directed to the RER membrane by a cytoplasmic ribonucleoprotein particle, the signal recognition particle (SRP) . SRP fulfills its task by virtue of three distinguishable activities: the binding of a signal sequence which, being part of the nascent polypeptide to be translocated, is exposed on the surface of a translating ribosome; the retardation of any further elongation; and the SRP-receptor-mediated binding of the complex of ribosome, nascent polypeptide and SRP to the RER membrane which results in the detachment of SRP from the signal sequence and the ribosome and the insertion of the nascent polypeptide into the membrane . Evidence is accumulating that SRP is not restricted to eukaryotes: SRP-related particles and SRP-receptor-related molecules are found ubiquitously and may function in protein translocation in every living organism . This review focuses on the mammalian SRP . A brief discussion of its overall structure is followed by a detailed description of the structures of its RNA and protein constituents and the requirements for their assembly into the particle . Homologues of SRP components from organisms other than mammals are mentioned to emphasize the components' conserved or less conserved features . Subsequently, the functions of each of the SRP constituents are discussed . This sets the stage for a presentation of a model for the mechanism by which SRP cyclically assembles and disassembles with translating ribosomes and the RER membrane . It may be expected that similar mechanisms are used by SRP homologues in organisms other than mammals . However, the mammalian SRP-mediated translocation mechanism may not be conserved in its entirety in organisms like Escherichia coli whose SRP lack components required for the function of the mammalian SRP . Possible translocation pathways involving the rudimentary SRP are discussed in view of the existence of alternative, chaperone-mediated translocation pathways with which they may intersect . The concluding two sections deal with open questions in two areas of SRP research . One formulates basic questions regarding the little-investigated biogenesis of SRP . The other gives an outlook over the insights into the mechanisms of each of the known activities of the SRP that are to be expected in the short and medium-term future.

Biochem J, 1995 Mar 15, 306 ( Pt 3), 865 - 9
Free calcium transients in chemotactic and non-chemotactic strains of Escherichia coli determined by using recombinant aequorin; Watkins NJ et al.; Intracellular Ca2+ has been previously implicated in the chemotactic response of Escherichia coli . However, no correlative measurements of intracellular free Ca2+ have been made during bacterial chemotaxis, essential if this is to be established . In order to monitor internal free Ca2+ in E . coli during challenge with chemotactic agents, the Ca(2+)-activated photoprotein aequorin was expressed in a chemotactic strain (AB1157) and a non-chemotactic strain {BL21(DE3)} of E . coli . Repellents were found to cause an increase (50-150 nM) in intracellular free Ca2+, whereas attractants caused a small but consistent decrease in intracellular free Ca2+ . These data are in agreement with the proposed model that an increase in intracellular free Ca2+ causes tumbling . The effect of increasing external Ca2+ on the regulation of intracellular free Ca2+ in both strains was monitored by using aequorin . The resting level of free Ca2+ in E . coli (AB1157) was found to be 100 nM, which agrees with previous data {Gangola and Rosen (1987) J . Biol . Chem . 262, 12570-12574} . As these results also show differences in the regulation of intracellular free Ca2+ between the two strains in the presence of high external Ca2+ concentrations, this may have implications for the effect of high-Ca2+ environments on E . coli.

Biochem J, 1995 Mar 15, 306 ( Pt 3), 627 - 30
Formation in vitro of the 3,4,6-trihydroxyphenylalanine quinone cofactor; Hanlon SP et al.; An Escherichia coli K-12 2-phenylethylamine oxidase gene with a mutated leader sequence region produced a largely inactive form of the enzyme in the cytoplasm . This form of the enzyme was activated 30-50-fold on incubation at 30 degrees C in the absence of any added cofactors . After activation the enzyme contained a quinone which was not detected in the non-activated form . This is the first report of the formation in vitro of any quinoenzyme cofactor.

Biochim Biophys Acta, 1995 Mar 15, 1247(2), 284 - 92
NMR studies of binding of 5-FdUDP and dCDP to ribonucleoside-diphosphate reductase from Escherichia coli; Roy B et al.; 5-Fluoro-2'-deoxyuridine-5'-diphosphate (5-FdUDP) has been synthesised using an original route, previously applied to the synthesis of natural nucleoside diphosphates . The interaction between 5-FdUDP and the enzyme ribonucleoside-diphosphate reductase (EC 1.17.4.1) has been studied with 19F-NMR . The product analogue is shown to be in fast exchange with substrate binding sites on protein subunit 1 (R1) of ribonucleoside-diphosphate (NDP) reductase . The number of binding sites is reduced to half when the complete holoenzyme R1R2 is formed . The temperature dependence of the line broadening of 5-FdUDP was studied using 19F-NMR, and of dCDP and dUDP using 1H-NMR . The temperature dependences are complex and a molecular model in which R1 is in a temperature dependent equilibrium between at least two conformations is suggested in order to explain the observed behaviour . Binding of a ligand to the substrate binding sites affects the conformational equilibrium in a ligand specific way . Formation of the holoenzyme R1R2 also affects the equilibrium.

Biochim Biophys Acta, 1995 Mar 15, 1247(2), 265 - 71
Purification and characterization of the pyridoxol-5'-phosphate:oxygen oxidoreductase (deaminating) from Escherichia coli; Notheis C et al.; The E . coli gene pdxH encoding pyridoxol-5'-phosphate:oxygen oxidoreductase (deaminating) (EC 1.4.3.5, PdxH) was cloned, located to phage 20B5 of the library of Kohara et al . (Kohara, Y, Akiyama, K . and Isono K . (1987) Cell 50, 495-508) and assigned to a stretch between 36.0 and 36.1 min of the E . coli chromosome . The gene was overexpressed as a MBP/PdxH fusion protein . The fusion protein was purified by affinity chromatography on an amylose resin and hydrolyzed in the presence of protease 'factor Xa' resulting in homogeneous PdxH protein after another column chromatography . Both the MBP/PdxH fusion protein and the PdxH protein were characterized . Both enzymes are FMN-dependent enzymes which oxidize pyridoxol phosphate and pyridoxamine phosphate in the presence of oxygen to pyridoxal phosphate . Km values of both proteins were similar ranging from 350 to 400 microM for the two substrates . The enzymes did not accept non-phosphorylated substrates . Kinetic data indicate that the enzyme (MBP/PdxH) is product inhibited (Ki 8 microM) by pyridoxal phosphate as a mixed type inhibitor . As revealed by gel exclusion chromatography a minor fraction of the fusion protein formed a dimer, whereas the bulk amount of protein was a monomer . No indication was found that the PdxH protein forms a dimer . The monomer was shown to be catalytically active.

Biochim Biophys Acta, 1995 Mar 15, 1247(2), 253 - 9
Avian cytosolic 3-hydroxy-3-methylglutaryl-CoA synthase: evaluation of the role of cysteines in reaction chemistry; Misra I et al.; The pH dependence of avian cytosolic HMG-CoA synthase activity is fit by a titration curve with a pK = 8.6 . The observation of optimal activity at alkaline pH and the insensitivity of pK to divalent cation concentration suggest that the pK reflects ionization of an amino-acid side chain (e.g., cysteinyl sulfhydryl) rather than substrate enolization . Upon reaction of 3-chloropropionyl-CoA with HMG-CoA synthase C129S, an enzyme variant lacking the sulfhydryl group normally targeted by this mechanism-based inhibitor, stoichiometric modification occurs . Amino-acid analysis indicates that cysteine is the principal target in C129S enzyme, demonstrating the presence of a second reactive cysteine within this enzyme . To test whether another cysteine functions in reaction chemistry, conserved cysteines were identified by sequence homology analysis . Five cysteine residues (C59, C69, C224, C232, C268), invariant in the nine sequences available for various eukaryotic HMG-CoA synthase isozymes, were individually replaced by alanine in a series of mutant enzymes . Kinetic analyses of the isolated mutant HMG-CoA synthases indicate that none of these is crucial to the chemistry that results in production of HMG-CoA . These results further distinguish the HMG-CoA synthase reaction from the related condensation of acyl-CoA substrates catalyzed by beta-ketothiolase.

Biochim Biophys Acta, 1995 Mar 15, 1247(2), 179 - 84
Refolding of alpha 1-antitrypsin expressed as inclusion bodies in Escherichia coli: characterization of aggregation; Kwon KS et al.; Recombinant alpha 1-antitrypsin (alpha 1AT) produced as inclusion bodies in Escherichia coli was purified via several steps including solubilization of the inclusion bodies in 8 M urea and refolding by direct dilution of denaturant, followed by ion-exchange chromatography . The purified recombinant alpha 1AT has an activity comparable to human plasma alpha 1AT . During refolding, prolonged incubation of the alpha 1AT polypeptides at intermediate urea concentration favored production of inactive but soluble aggregates, which could regain activity after denaturation and renaturation . Nondenaturing polyacrylamide gel electrophoresis of the aggregates revealed the existence of dimers and higher oligomers . Immunological approach to characterize conformation showed that the oligomers were distinct from the native, the cleaved, or the denatured form, but was similar to the polymers induced from the native structure in mild denaturing condition . These results suggest that the oligomers are formed through specific interactions between aggregation-competent species which are stabilized at intermediate denaturant concentration.

Biochim Biophys Acta, 1995 Mar 14, 1261(1), 35 - 43
Expression and mutagenesis of recombinant human and murine erythropoietins in Escherichia coli; Bill RM et al.; Expression of the polypeptide hormone erythropoietin (EPO) in Escherichia coli by four bacterial expression vectors was examined . Complementary DNAs encoding human and murine EPO were amplified by polymerase chain reaction (PCR) and cloned into the glutathione-S-transferase (GST) fusion vector, pGEX-2T . Human EPO DNA was also cloned into the vectors, pET14b, pIN III-Omp A2 and pT7/7 . Expression of human and murine EPO was obtained using constructs based on pGEX-2T . For constructs based on the other vectors, expression of EPO was absent or occurred at low levels, despite attempts to optimise conditions . Human and murine EPO, expressed as fusion proteins with GST, were partially soluble and displayed EPO bioactivity . Soluble GST-EPO fusion proteins were affinity purified on immobilised glutathione . Insoluble protein could also be purified by elution from gel slices following SDS-PAGE to yield either fusion protein or, after treatment with thrombin, unmodified EPO which was both soluble and bioactive . The pGEX expression system was evaluated as a means of analysing the structure-function relationships of EPO by in vitro mutagenesis . Three human and three murine EPO mutants were constructed and expressed as GST fusion proteins . Following purification, biological activity was evaluated using assays for bioactivity, immunoactivity and GST activity . The pGEX expression system complements eukaryotic systems described previously for expression of EPO and should provide much useful information about the structure-function relationships of the hormone.

Proc Natl Acad Sci U S A, 1995 Mar 14, 92(6), 2333 - 7
HypB protein of Bradyrhizobium japonicum is a metal-binding GTPase capable of binding 18 divalent nickel ions per dimer; Fu C et al.; Bradyrhizobium japonicum hypB encodes a protein containing an extremely histidine-rich region (24 histidine residues within a 39-amino-acid stretch) and guanine nucleotide-binding domains . The product of the hypB gene was overexpressed in Escherichia coli and purified by Ni(2+)-charged metal chelate affinity chromatography (MCAC) in a single step . In SDS/PAGE, HypB migrated at 38 kDa--slightly larger than the calculated molecular mass (32.8 kDa) . Purified HypB has GTPase activity with a kcat of 0.18 min-1 and a Km for GTP of 7 microM, and it has dGTPase activity as well . HypB exists as a dimer of molecular mass 78 kDa in native solution as determined by fast protein liquid chromatography on Superose 12 . It binds 9.0 +/- 0.14 divalent nickel ions per monomer (18 Ni2+ per dimer) with a Kd of 2.3 microM; it also binds Zn2+, Cu2+, Co2+, Cd2+, and Mn2+ . In-frame deletion of the histidine-rich region (deletion of 38 amino acids including 23 histidine residues) resulted in a truncated HypB that did not bind to the MCAC column, whereas in-frame deletion of 14 amino acids including 8 histidine residues within HypB resulted in a truncated HypB that still bound to the column . The results indicate that the histidine residues within the histidine-rich region of HypB are involved in metal binding.

Proc Natl Acad Sci U S A, 1995 Mar 14, 92(6), 2328 - 32
Cloning, expression, and characterization of cDNAs encoding Arabidopsis thaliana squalene synthase; Nakashima T et al.; We have isolated and characterized two overlapping cDNA clones for Arabidopsis thaliana squalene synthase . Their nucleotide sequences contained an open reading frame for a 410-amino acid polypeptide (calculated molecular mass, 47 kDa) . The deduced amino acid sequence of the Arabidopsis polypeptide was significantly homologous (42-44% identical) to the sequences of known squalene synthases of several species, from yeast to man, but it was much less homologous to that of tomato phytoene synthase . To express the Arabidopsis enzyme in Escherichia coli, the entire coding region was subcloned into an expression vector . A cell-free extract of E . coli transformed with the recombinant plasmid, in the presence of NADPH and Mg2+, efficiently converted {14C}farnesyl diphosphate into squalene . On the other hand, in the absence of NADPH and the presence of Mn2+, the cell-free extract formed dehydrosqualene as a secondary product . Another E . coli extract expressing mouse squalene synthase showed the same activity as the Arabidopsis enzyme . Therefore, both the structure and reaction mechanism of squalene synthases are markedly conserved in taxonomically remote eukaryotes.

Proc Natl Acad Sci U S A, 1995 Mar 14, 92(6), 2298 - 302
Nuclear foci of mammalian Rad51 recombination protein in somatic cells after DNA damage and its localization in synaptonemal complexes; Haaf T et al.; Rad51 protein of Saccharomyces cerevisiae is a structural homolog of the Escherichia coli recombination enzyme RecA . In yeast, the Rad51 protein is required for mitotic and meiotic recombination and for repair of double-strand breaks in DNA . We have used antibodies raised against the homologous human protein, HsRad51, expressed in E . coli, to visualize the spatial distribution of the protein in mammalian somatic and meiotic cells . In cultured human cells, the HsRad51 protein is concentrated in multiple discrete foci in the nucleoplasm; it is largely absent from cytoplasm and nucleoli . After treatment of cells with methyl methanesulfonate, ultraviolet irradiation, or 137Cs irradiation, the percentage of cells with HsRad51 protein immunofluorescence increases; the same cells show unscheduled DNA synthesis . Induction of Rad51 foci is blocked by inhibitors of transcription . In mouse pachytene spermatocytes, the mouse homolog of Rad51 protein is highly enriched in synaptonemal complexes that are formed between the paired homologous chromosomes during meiotic prophase . We conclude that the mammalian proteins homologous to yeast Rad51 are involved in repair of DNA damage and recombinational repair during meiosis.

Proc Natl Acad Sci U S A, 1995 Mar 14, 92(6), 2229 - 33
A region in the cytosolic domain of the epidermal growth factor receptor antithetically regulates the stimulatory and inhibitory guanine nucleotide-binding regulatory proteins of adenylyl cyclase; Sun H et al.; Epidermal growth factor (EGF) stimulates adenylyl cyclase in the heart via activation of the stimulatory GTP-binding protein Gs . Therefore, employing peptides corresponding to regions in the cytosolic domain of the EGF receptor, we have investigated the ability of sequences within the EGF receptor to activate Gs . A 13-aa peptide (EGFR-13) corresponding to the juxtamembrane region in the cytosolic domain of the EGF receptor stimulated GTP binding and GTPase activity of Gs . This peptide did not stimulate GTP binding to Gi but increased the GTPase activity of this protein . Additionally, phosphorylation of the protein kinase C site (threonine residue) within EGFR-13 decreased the ability of the peptide to stimulate Gs and increase GTPase activity of Gi . Further, in functional assays of Gs employing S49 cyc- cell membranes, EGFR-13 increased the ability of Gs to stimulate adenylyl cyclase; phospho-EGFR-13 and a 14-aa peptide corresponding to a sequence in the cytosolic domain of the EGF receptor did not alter the functional activity of Gs . Hence, the juxtamembrane region of the EGF receptor can activate Gs and, by stimulating GTPase activity of Gi, inactivates this latter G protein . Phosphorylation of the threonine residue within this region attenuates the activity of the peptide as a modulator of G-protein function.

Proc Natl Acad Sci U S A, 1995 Mar 14, 92(6), 2204 - 8
Site-specific rates of excision repair of benzo{a}pyrene diol epoxide adducts in the hypoxanthine phosphoribosyltransferase gene of human fibroblasts: correlation with mutation spectra; Wei D et al.; When populations of repair-proficient diploid human fibroblasts were treated with (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo{a}pyrene (BPDE) during early S phase, just as the hypoxanthine phosphoribosyltransferase gene (HPRT) was being replicated, 5% of the induced base substitutions were found at nt 212, and 5% of the substitutions were found at nt 229 in exon 3 . However, when the population was treated in early G1 phase to allow at least 12 hr for repair before the onset of S phase, 21% of the substitutions were found at nt 212, and 10% were found at nt 229 . No such cell-cycle-dependent difference in distribution of base substitutions occurred in excision-repair-deficient cells . To test whether the increase in the relative frequency of mutations resulted from inefficient repair at these sites, we adapted ligation-mediated PCR to measure the rates of removal of BPDE adducts from individual sites in exon 3 of the HPRT gene . Cells were treated with 0.5 microM BPDE in early G1 phase and harvested immediately or after 10, 20, and 30 hr for repair . the nontranscribed strand of exon 3 was analyzed for the original distribution of adducts and those remaining after repair, using Escherichia coli UvrABC excinuclease to excise the adducts and annealing a 5' biotinylated gene-specific primer to the DNA and extending it with Sequenase 2.0 to generate a blunt end at the site of each cut . A linker was ligated to the blunt end, and the desired fragments were isolated from the rest of the genomic DNA by using magnetic beads, amplified by PCR, and analyzed on a sequencing gel . The distribution of fragments of particular lengths indicated the relative number of BPDE adducts initially formed or remaining at specific sites . The rates of repair at individual sites varied widely along exon 3 of the HPRT gene and were very slow at nt 212 and 229, strongly supporting the hypothesis that inefficient DNA repair plays an important role in the formation of mutation hotspots.

Proc Natl Acad Sci U S A, 1995 Mar 14, 92(6), 2136 - 40
Partial rescue of human carbonic anhydrase II frameshift mutation by ribosomal frameshift; Hu PY et al.; A single-base-pair deletion in exon 7 of the human carbonic anhydrase II gene was found to be the molecular defect in a group of independently ascertained, clinically heterogeneous, Hispanic carbonic anhydrase II-deficient patients, all of whom had ancestors from the Caribbean islands . This mutation predicts a +1 frameshift at codon 227 and incorporation of 12 missense amino acids before an early stop codon at position 239 produces a 27-kDa truncated carbonic anhydrase II . Expression of the Hispanic mutant cDNA in bacteria produced predominantly the 27-kDa protein, which was inactive . However, a minor 29-kDa polypeptide species was also produced that had 10% the specific activity of the wild-type enzyme after affinity purification . Amino acid sequencing showed that the 29-kDa mutant protein was produced by two frameshift events: a +1 frameshift at codon 227 due to the single-base deletion and a -1 ribosomal frameshift at codon 237 that restored the original reading frame after 11 missense amino acids were incorporated . Antibody against the 11-amino acid frameshift peptide detected the 29-kDa mutant protein in lysates of transfected COS cells . These results indicate that ribosomal frameshift can partially rescue the human carbonic anhydrase II frameshift mutation and suggest a mechanism whereby a compensatory ribosomal frameshift can ameliorate the consequences of certain frameshift mutations . Whether individual differences in efficiency of ribosomal frameshift contribute to clinical heterogeneity in patients with such mutations deserves further study.

Proc Natl Acad Sci U S A, 1995 Mar 14, 92(6), 2111 - 5
A method for probing the topography and interactions of proteins: footprinting of myoglobin; Zhong M et al.; We describe a procedure for mapping residues on the surface of a protein molecule to its sequence, using a scheme that is analogous to nucleic acid footprinting . The protein is end labeled radioactively and subjected to limited proteolysis, and the products are analyzed by denaturing polyacrylamide gel electrophoresis and autoradiography . The method is tested with the heme protein myoglobin and applied to mapping the (unknown) surface of the molecule lacking the heme group: apomyoglobin . Sites of protein-protein interaction can be identified, as illustrated by footprinting the association between myoglobin and an anti-myoglobin monoclonal antibody.

Proc Natl Acad Sci U S A, 1995 Mar 14, 92(6), 1950 - 4
Restoration of mismatch repair to nuclear extracts of H6 colorectal tumor cells by a heterodimer of human MutL homologs; Li GM et al.; Hypermutable H6 colorectal tumor cells are defective in strand-specific mismatch repair and bear defects in both alleles of the hMLH1 gene . We have purified to near homogeneity an activity from HeLa cells that complements H6 nuclear extracts to restore repair proficiency on a set of heteroduplex DNAs representing the eight base-base mismatches as well as a number of slipped-strand, insertion/deletion mispairs . This activity behaves as a single species during fractionation and copurifies with proteins of 85 and 110 kDa . Microsequence analysis demonstrated both of these proteins to be homologs of bacterial MutL, with the former corresponding to the hMLH1 product and the latter to the product of hPMS2 or a closely related gene . The 1:1 molar stoichiometry of the two polypeptides and their hydrodynamic behavior indicate formation of a heterodimer, which we have designated hMutL alpha . These observations indicate that interactions between members of the family of human MutL homologs may be restricted.

Proc Natl Acad Sci U S A, 1995 Mar 14, 92(6), 1945 - 9
GTP consumption of elongation factor Tu during translation of heteropolymeric mRNAs; Rodnina MV et al.; The stoichiometry of elongation factor Tu (EF-Tu) and GTP in the complex with aminoacyl-tRNA and the consumption of GTP during peptide bond formation on the ribosome were studied in the Escherichia coli system . The ribosomes were programmed either with two different heteropolymeric mRNAs coding for Met-Phe-Thr-Ile .. . (mMFTI) or Met-Phe-Phe-Gly .. . (mMFFG) or with poly(U) . The composition of the complex of EF-Tu, GTP, and Phe-tRNA(Phe) was studied by gel chromatography . With equimolar amounts of factor and Phe-tRNA(Phe), a pentameric complex, (EF-Tu.GTP)2.Phe-tRNA(Phe), was observed, whereas the classical ternary complex, EF-Tu.GTP.Phe-tRNA(Phe), was found only when Phe-tRNA(Phe) was in excess . Upon binding of the purified pentameric complex to ribosomes carrying fMet-tRNA(fMet) in the peptidyl site and exposing a Phe codon in the aminoacyl site, only one out of two GTPs of the pentameric complex was hydrolyzed per Phe-tRNA bound and peptide bond formed, regardless of the mRNA used . In the presence of EF-G, the stoichiometry of one GTP hydrolyzed per peptide bond formed was found on mMFTI when one or two elongation cycles were completed . In contrast, on mMFFG, which contains two contiguous Phe codons, UUU-UUC, two GTP molecules of the pentameric complex were hydrolyzed per Phe incorporated into dipeptide, whereas the incorporation of the second Phe to form tripeptide consumed only one GTP . Thus, generally one GTP is hydrolyzed by EF-Tu per aminoacyl-tRNA bound and peptide bond formed, and more than one GTP is hydrolyzed only when a particular mRNA sequence, such as a homopolymeric stretch, is translated . The role of the additional GTP hydrolysis is not known; it may be related to frameshifting of peptidyl-tRNA during translocation.

Proc Natl Acad Sci U S A, 1995 Mar 14, 92(6), 1812 - 6
The N-terminal portion of domain E of retinoic acid receptors alpha and beta is essential for the recognition of retinoic acid and various analogs; Ostrowski J et al.; Utilizing a strategy involving domain exchange between retinoic acid receptors alpha and beta (RAR alpha and RAR beta) and monitoring the transcriptional activity of the resulting chimeric receptors with receptor-selective retinoids, we identified a 70-aa region within the N-terminal portion of the RAR alpha and -beta domain E which is important for an RAR alpha- or RAR beta-specific response . Two amino acid residues within this region, serine-232 (S232) and threonine-239 (T239) in RAR alpha and the corresponding alanine-225 (A225) and isoleucine-232 (I232) in RAR beta, were found to be essential for this effect . In addition, binding studies using the chimeric receptors expressed in Escherichia coli showed that the N-terminal portion of domain E was also important for the characteristic binding profile of t-RA and various retinoids with RAR alpha or RAR beta . Structural predictions of the primary amino acid sequence in this region indicate the presence of an amphipathic helix-turn-helix structure with five hydrophobic amino acids that resemble a leucine zipper motif . The amino acid residues identified by domain swapping, S232 and T239 in RAR alpha and A225 and I232 in RAR beta, were found within the hydrophobic face of an alpha-helix in close proximity to this zipper motif, suggesting that the ligand may interact with the receptor in the region adjacent to a surface involved in protein-protein interactions . This finding may link ligand binding to other processes important for transcriptional activation.

Biochemistry, 1995 Mar 14, 34(10), 3430 - 7
Insertion of the polytopic membrane protein lactose permease occurs by multiple mechanisms; Zen KH et al.; The lactose permease of Escherichia coli has 12 transmembrane hydrophobic domains in probable alpha-helical conformation connected by hydrophilic loops . Previous studies {Consler, T . G., Persson, B., et al . (1993) Proc . Natl . Acad . Sci . U.S.A . 90, 6934-6938} demonstrate that a peptide fragment (the XB domain) containing a factor Xa protease site immediately upstream of a biotin acceptor domain can be engineered into the permease, thereby allowing rapid purification to a high state of purity . Here we describe the use of the XB domain to probe topology and insertion . Cells expressing permease with the XB domain at the N terminus, at the C terminus, or in loop 6 or 10 on the cytoplasmic face of the membrane catalyze active transport, although only the chimeras with the XB domain at the C terminus or in loop 6 are biotinylated . In contrast, chimeras with the XB domain in periplasmic loop 3 or 7 are inactive, but strikingly, both constructs are biotinylated . Furthermore, the XB domain in all the constructs, particularly in the loop 3 and loop 7 chimeras, is accessible from the cytoplasmic face of the membrane, as evidenced by factor Xa proteolysis or avidin binding studies with spheroplasts and disrupted membrane preparations . Finally, alkaline phosphatase fusions one loop downstream from each periplasmic XB domain exhibit high phosphatase activity . Thus, the presence of the XB domain in a periplasmic loop apparently blocks translocation of a discrete segment of the permease consisting of the loop and the two adjoining helices without altering insertion of the remainder of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1995 Mar 14, 34(10), 3386 - 95
Characterization of a Thermomonospora fusca exocellulase; Zhang S et al.; The exocellulase E3 gene was cloned on a 7.1 kb NotI fragment from Thermomonospora fusca genomic DNA into Escherichia coli and expressed in Streptomyces lividans . The E3 gene was sequenced and encoded a 596 residue peptide . The molecular masses of the native and cloned E3s were determined by mass spectrometry, and the value for E . coli E3, 59,797 Da, agreed well with that predicted from the DNA sequence, 59,646 Da . The value of 61,200 Da for T . fusca E3 is consistent with E3 being a glycoprotein . E3 is thermostable, retaining full activity after 16 h at 55 degrees C . It also has a broad pH optimum around 7-8, retaining 90% of its maximal activity between pH 6 and 10 . The cloned E3s were identical to the native enzyme in their activity, cellulose binding, and thermostability . Papain digestion produced a 45.7 kDa catalytic domain with 77% of the native activity on amorphous cellulose and 33% on crystalline cellulose . E3 belongs to cellulase family B and retains the residues that have been identified to be crucial for catalytic activity in Trichoderma reesei cellobiohydrolase II and T . fusca E2 . The E3 gene contains a 14 bp inverted repeat regulatory sequence 212 bp before the translational start codon instead of the 30-70 bp found for the other T . fusca cellulase genes . An additional copy of this sequence with one base changed is 314 bp before the translational start codon . The transcriptional start site of the E3 gene was shown to be between these two inverted repeats.

Biochemistry, 1995 Mar 14, 34(10), 3344 - 51
Critical side-chain interactions at a subunit interface in the Arc repressor dimer; Milla ME et al.; In the Arc repressor dimer, the side chains of Ile37 and Val41 in alpha-helix B pack against each other and against the symmetry-related side chains of Ile37' and Val41' in alpha-helix B' to form part of the hydrophobic core and the dimer interface . Following combinatorial mutagenesis of these positions, only the wild-type combination of hydrophobic residues was recovered as a fully active protein, and only a few conservative replacements were recovered as stably folded or partially active proteins . Equilibrium and kinetic studies of the folding of purified mutants show that the delta-CH3 groups of Ile37 and Ile37' contribute approximately 2 kcal/mol of dimer to protein stability and are involved in interactions that are only partially formed in the transition state for protein folding . Alanine substitution at either position 37 or 41 results in proteins which differ from wild type in being monomeric at a concentration of 10 microM, having reduced secondary structure, having solvent-exposed tryptophans, and showing non-cooperative thermal and urea denaturation transitions . These mutants appear to exist in a physiologically denatured state that is similar in many ways to the molten globule state.

Biochemistry, 1995 Mar 14, 34(10), 3319 - 24
Site-directed mutagenesis of Lys36 in human thioredoxin: the highly conserved residue affects reduction rates and growth stimulation but is not essential for the redox protein's biochemical or biological properties; Oblong JE et al.; Previous studies have demonstrated that a recombinant form of the human redox protein thioredoxin can stimulate the growth rate of Swiss 3T3 murine fibroblasts and that this ability to promote cellular proliferation was dependent upon a redox-active form . A site-directed mutagenesis study of the highly conserved Lys36 adjacent to the two active site cysteines of thioredoxin was performed to determine whether the basic residue was essential for the biochemical and mitogenic properties of human thioredoxin . Two mutants were generated in which the lysine residue was replaced with either glutamic acid (K36E) or leucine (K36L) . While K36E and K36L were both redox-active in a thioredoxin-specific assay, the mutants exhibited decreased affinities for thioredoxin reductase relative to wild-type thioredoxin since their respective KM values increased by a factor of 5 and 7 . Examination of the secondary structure of the variants by circular dichroism spectroscopy revealed that both mutants had minor variations in the overall structural content when compared to thioredoxin, with K36L being most similar to the wild-type protein . Thermal equilibrium denaturation studies of the variants showed that K36E had a TM of 69.5 degrees C . A TM value for thioredoxin and K36L could not be established because the absence of a plateau above 83 degrees C rendered it difficult to establish an upper base line and, hence, the TM . The two mutants were able to stimulate cellular proliferation, albeit with reduced efficiency when compared with wild-type thioredoxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1995 Mar 14, 34(10), 3310 - 8
Recombinant Desulfovibrio vulgaris rubrerythrin . Isolation and characterization of the diiron domain; Gupta N et al.; The gene encoding Desulfovibrio (D.) vulgaris rubrerythrin (Prickril, B . C., Kurtz, D . M., Jr., LeGall, J., & Voordouw, G . (1991) Biochemistry 30, 1118), a protein of unknown function containing both FeS4 and (mu-oxo)diiron sites, was cloned and overexpressed in Escherichia coli . Upon cell lysis, the overexpressed protein was found in an insoluble form deficient in iron . Iron was incorporated in vitro by dissolving the protein in 3 M guanidinium chloride, adding Fe(II) anaerobically and diluting the denaturant . This recombinant rubrerythrin was found to have properties very similar to those of rubrerythrin isolated from D . vulgaris, except that the recombinant rubrerythrin contained six rather than four (or five) iron atoms per 44 kDa homodimer . Analyses of UV-vis, Mossbauer, and EPR spectra showed that the six iron atoms in recombinant rubrerythrin are organized as two FeS4 and two (mu-oxo/hydroxo)diiron sites . In order to allow examination of the diiron sites in the absence of the FeS4 sites, a truncated gene encoding the N-terminal 152 residues of D . vulgaris rubrerythrin was also cloned and overexpressed as an insoluble protein in E . coli, and iron was incorporated by a procedure analogous to that for recombinant rubrerythrin . This so-called "chopped" rubrerythrin (CRr) was found to consist of an approximately 35 kDa homodimer containing four iron atoms . Spectroscopic characterization indicated that the four iron atoms in CRr are organized as two diiron sites, the majority of which closely resemble the (mu-oxo)diiron(III) sites in E . coli ribonucleotide reductase R2 protein, and a minor fraction of which resemble the mixed-valent diiron(II,III) site in methane monooxygenase hydroxylase.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1995 Mar 14, 34(10), 3286 - 99
Thermodynamics of denaturation of barstar: evidence for cold denaturation and evaluation of the interaction with guanidine hydrochloride; Agashe VR et al.; Isothermal guanidine hydrochloride (GdnHCl)-induced denaturation curves obtained at 14 different temperatures in the range 273-323 K have been used in conjunction with thermally-induced denaturation curves obtained in the presence of 15 different concentrations of GdnHCl to characterize the thermodynamics of cold and heat denaturation of barstar . The linear free energy model has been used to determine the excess changes in free energy, enthalpy, entropy, and heat capacity that occur on denaturation . The stability of barstar in water decreases as the temperature is decreased from 300 to 273 K . This decrease in stability is not accompanied by a change in structure as monitored by measurement of the mean residue ellipticities at both 222 and 275 nm . When GdnHCl is present at concentrations between 1.2 and 2.0 M, the decrease in stability with decrease in temperature is however so large that the protein undergoes cold denaturation . The structural transition accompanying the cold denaturation process has been monitored by measuring the mean residue ellipticity at 222 nm . The temperature dependence of the change in free energy, obtained in the presence of 10 different concentrations of GdnHCl in the range 0.2-2.0 M, shows a decrease in stability with a decrease as well as an increase in temperature from 300 K . Values of the thermodynamic parameters governing the cold and the heart denaturation of barstar have been obtained with high precision by analysis of these bell-shaped stability curves . The change in heat capacity accompanying the unfolding reaction, delta Cp, has a value of 1460 +/- 70 cal mol-1 K-1 in water . The dependencies of the changes in enthalpy, entropy, free energy, and heat capacity on GdnHCl concentration have been analyzed on the basis of the linear free energy model . The changes in enthalpy (delta Hi) and entropy (delta Si), which occur on preferential binding of GdnHCl to the unfolded state, vis-a-vis the folded state, both have a negative value at low temperatures . With an increase in temperature delta Hi makes a less favorable contribution, while delta Si makes a more favorable contribution to the change in free energy (delta Gi) due to this interaction . The change in heat capacity (delta CPi) that occurs on preferential interaction of GdnHCl with the unfolded form has a value of only 53 +/- 36 cal mol-1 K-1 M-1 . The data validate the linear free energy model that is commonly used to analyze protein stability.

Biochemistry, 1995 Mar 14, 34(10), 3248 - 52
Local structural preferences in the alpha-lactalbumin molten globule; Peng ZY et al.; Molten globules have been proposed to be general intermediates in protein folding . Despite numerous studies, a detailed description of the structure of a molten globule remains elusive . Recently, we showed that the molten globule formed by the helical domain of alpha-lactalbumin (alpha-LA) has a native-like backbone topology . Here we probe local structural preferences in the helical domain of the alpha-LA molten globule by analyzing a set of native and nonnative single disulfide bond variants using a combination of circular dichroism spectroscopy and determination of the equilibrium constant for disulfide bond formation . We find that the region surrounding the 28-111 disulfide bond has a high preference to adopt a native-like structure . Formation of other native or nonnative disulfide bonds is significantly less favorable . Our results suggest that molten globules contain regions with varying degrees of specificity for native-like structure and that the core region surrounding the 28-111 disulfide bond plays an important role in alpha-LA folding by stabilizing the molten globule intermediate.

Biochemistry, 1995 Mar 14, 34(10), 3239 - 47
Phosphorylation site mutants of the mannitol transport protein enzyme IImtl of Escherichia coli: studies on the interaction between the mannitol translocating C-domain and the phosphorylation site on the energy-coupling B-domain; Boer H et al.; Mannitol binding and translocation catalyzed by the C domain of the Escherichia coli mannitol transport protein enzyme IImtl is influenced by domain B . This interaction was studied by monitoring the effects of mutating the B domain phosphorylation site, C384, on the kinetics of mannitol binding to the C domain . The dissociation constants for mannitol to the C384 mutants in inside-out membrane vesicles varied from 45 nM for the wild-type enzyme to 306 nM for the mutants . The rate constants pertinent to the binding equilibrium were also altered by the mutations . The association rate of mannitol to the cytoplasmic binding site in the mutants was accelerated for all mutants . The exchange rate of bound mannitol on the wild-type enzyme was shown to be pH dependent with a pKa of approximately 8 and increasing rates at higher pH . This rate was increased for all the mutants, but the pKas differed for the various mutants . The exchange rate for binding to the isolated IICmtl, however, was not pH dependent and exhibited a low rate . Exchange measured at 4 degrees C showed that, of the two steps, binding and occlusion, involved in binding to wild-type EIImtl in inside-out vesicles, only one could be detected for the C384E and C384L mutants . This suggests that the mutations increased the rate of the occlusion step so that it was no longer separable from the initial binding step or that the mutations eliminated the occlusion step altogether . The change in the mannitol binding kinetics of the C domain indicates that the B and C domains of EIImtl influence each other's conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1995 Mar 14, 34(10), 3231 - 8
Resolution of the steroid-binding and dimerization domains of human sex hormone-binding globulin by expression in Escherichia coli; Hildebrand C et al.; To determine the minimal sequence requirements for steroid binding and dimerization of human sex hormone-binding globulin (SHBG), the SHBG polypeptide and various SHBG deletion mutants were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli . Fusion proteins containing the complete SHBG sequence, or the first 177 N-terminal residues of SHBG, bound steroids with high affinity and specificity . Further deletions from the C-terminus severely compromised steroid-binding activity, as did N-terminal deletions beyond residue 18 in the SHBG sequence . Thus, residues 18-177 in SHBG encompass a region required for its steroid-binding activity, and a disulfide bridge normally present between Cys-164 and Cys-188 in SHBG is not obviously essential for steroid binding . Most of the GST/SHBG fusion proteins undergo cleavage at 4 degrees C, releasing immunoreactive polypeptides that correspond approximately in size to their respective SHBG sequences . The 23-kDa immunoreactive cleavage product released from the fusion protein containing residues 1-205 in the SHBG sequence (SHBG 1-205) has a 50-fold greater steroid-binding capacity but a 7.5-fold lower affinity than its parent fusion protein . In addition, the 22-kDa immunoreactive polypeptide released from SHBG(1-194) binds steroid, and its dimerization is promoted by steroid ligands that bind SHBG with high affinity . These data suggest that the N-terminal region of SHBG dimerizes readily in the absence of GST and in doing so acquires steroid-binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1995 Mar 14, 34(10), 3212 - 21
Thermodynamic evaluation of binding interactions in the methionine repressor system of Escherichia coli using isothermal titration calorimetry; Hyre DE et al.; The binding interactions of the methionine repressor protein, MetJ, from Escherichia coli with its cognate, metbox DNA sequence and corepressor S-adenosylmethionine were examined using calorimetric methods . A detailed thermodynamic characterization of this system which exhibits the recently reported (beta alpha alpha)2 binding motif provides values for delta G, delta H, and delta S for each step in the repressor binding cycle . These studies show that, in the presence of corepressor, MetJ binds to a single metbox operator site with delta G = -7.7 kcal.mol-1, whereas in the absence of corepressor, the free energy of interaction with a single site is -5.8 kcal.mol-1 . Cooperative interactions between two repressor molecules bound to two adjacent sites contribute an additional free energy of -1.3 kcal.mol-1 to binding at the second site . Binding is enthalpically unfavorable in the absence of the corepressor with delta H = +2.6 kcal.mol-1 but becomes exothermic with delta H = -4.6 kcal.mol-1 when corepressor is present . The heat capacity for the system decreases significantly by delta Cp = -290 cal.mol-1.K-1 on a per site basis when the protein binds to DNA, and interactions between repressor molecules bound to adjacent sites contribute a delta Cp = -800 cal.mol-1.K-1, indicating that solvent exclusion plays a significant role in binding in this system . The corepressor binds to the unbound repressor protein with a free energy of delta G = -6.0 kcal.mol-1 and to the MetJ-operator complex with delta G = -6.95 kcal.mol-1 . Repressor binding to random-sequence DNA was estimated to occur with a free energy of -5.7 kcal.mol-1 in the presence of corepressor . These data clearly indicate that MetJ repressor dimer binds specifically to the central region of its 8 bp cognate metbox operator but recognizes partial operator sequences as short as 6 bp . Cooperativity in binding of adjacent MetJ dimers to a double metbox sequence is demonstrated to be important in determining the energetics of the interaction . Finally, the corepressor S-adenosylmethionine enhances the affinity of MetJ for its recognition site DNA by a factor of 25 and contributes significantly to the net exothermicity of repressor binding.

Biochemistry, 1995 Mar 14, 34(10), 3183 - 92
Electrostatic effects of surface acidic amino acid residues on the oxidation-reduction potentials of the flavodoxin from Desulfovibrio vulgaris (Hildenborough); Zhou Z et al.; The flavodoxin from Desulfovibrio vulgaris (Hildenborough) is a member of a family of small, acidic proteins that contain a single noncovalently bound flavin mononucleotide (FMN) cofactor . These proteins function as low-potential one-electron transferases in bacteria . A distinguishing feature of these flavoproteins is the dramatic decrease in the midpoint potential of the semiquinone/hydroquinone couple of the FMN upon binding to the apoprotein (-172 mV for FMN free in solution versus -443 mV when bound), a perturbation thought to be essential for physiological function . The structural basis of this phenomenon is not yet thoroughly understood . In this study, the contribution of six acidic residues (Asp62, Asp63, Glu66, Asp95, Glu99, and Asp106) to the perturbation of the redox properties of the cofactor has been investigated . These residues are clustered about the FMN binding site within 13 A of the N(1) atom of the cofactor . Using oligonucleotide-directed mutagenesis, these residues were neutralized in various combinations through the substitution of asparagine for aspartate and glutamine for glutamate . Seventeen mutant flavodoxins were generated in which one to all six acidic residues were systematically neutralized, often in various spatial configurations . There was no obvious correlation between the midpoint potentials for the oxidized/semiquinone couple and general electrostatic environment, although some differences were noted . However, the midpoint potential for the semiquinone/hydroquinone couple for each of the mutants was less negative than that of the wild type . These increases are strongly correlated with the number of acid to amide substitutions, with an average contribution of about 15 mV per substitution . Collectively, the unfavorable electrostatic environment provided by these acidic residues accounts for approximately one-third of the large midpoint potential shift for the semiquinone/hydroquinone couple that typifies the flavodoxin family, apparently through the destabilization of the flavin hydroquinone anion.

Biochemistry, 1995 Mar 14, 34(10), 3172 - 82
Mechanism of adenylate kinase . The "essential lysine" helps to orient the phosphates and the active site residues to proper conformations; Byeon L et al.; Although how Lys21 interacts with the substrate MgATP of muscle adenylate kinase (AK) can now be deduced from the crystal structure of Escherichia coli AK.MgAP5A {P1,P5-bis(5'-adenosyl) pentaphosphate} {Muller, C . W., & Schulz, G . E . (1992) J . Mol . Biol . 224, 159-177}, its contribution to catalysis has not yet been demonstrated by functional studies since the proton NMR of the K21M mutant was shown to be perturbed significantly {Tian, G., Yan., H., Jiang, R.-T., Kishi, F., Nakazawa, A., & Tsai, M.-D . (1990) Biochemistry 29, 4296-4304} . We therefore undertook further structural and functional analyses of a conservative mutant K21R and a nonconservative mutant K21A . In addition to kinetic analyses, the structures of the mutants were analyzed by one- and two-dimensional proton NMR spectroscopy and (1H, 15N) heteronuclear multiple-quantum coherence (HMQC) experiments . Detailed assignments were performed in reference to the total backbone assignments of the WT AK.MgAP5A complex {Byeon, I.-J . L., Yan, H., Edison, A . S., Mooberry, E . S., Abildgaard, F., Markley, J . L., & Tsai, M.-D . (1993) Biochemistry 32, 12508-12521} . The analysis showed that the residues located near the active site (Gly15, Thr23, Arg97, Gln101, Arg128, Arg132, Asp140, Asp141, and Tyr153) exhibit greater changes in 1H-15N chemical shifts . Finally, two-dimensional 31P-31P COSY experiments were used to examine the effects of the lysine side chain on the phosphate groups in the bound AP5A . Our data have led to the following conclusions independent of the crystal structure: (i) Because the perturbations in the conformation of the mutants are not global and are mainly localized at active site residues and Tyr153, the side chain of Lys21 can be concluded to stabilize the transition state in the catalysis of AK by up to 7 kcal/mol on the basis of the 10(5)-fold decreases in the kcat/Km of mutants . (ii) The results of 31P NMR analyses suggest that Lys21 functions by orienting the triphosphate chain of MgATP to a proper conformation required for catalysis . (iii) The interaction between Lys21 and the phosphate chain in turn dictates the interactions between the substrates and the active site residues . In the K21R.MgATP complex, the NH chemical shifts of many of the active site residues are perturbed . (iv) The catalytic functions of Lys21 cannot be replaced by a conservative residue arginine . In addition, since K21A and K21R behave similarly, the catalytic function of Lys21 should not be merely a charge effect.

Biochemistry, 1995 Mar 14, 34(10), 3140 - 3
A native tertiary interaction stabilizes the A state of cytochrome c; Marmorino JL et al.; Certain kinetic intermediates in protein folding are similar to the molten globule, or A state, an equilibrium state of many proteins that is populated under high salt and low pH conditions . Many A states are nearly as compact as native proteins and have native-like secondary structure, but the extent to which nonlocal interactions stabilize the A state is unclear . In this study, thermal denaturation, monitored by circular dichroism, was used to determine the free energy of denaturation of the A state (delta GA<-->D) for Saccharomyces cerevisiae iso-1-ferricytochrome c . Specifically, we examined the wild-type protein, seven variants with amino acid substitutions at the interface between the N- and C-terminal helices, and two variants with mutations at a position close to, but not involved in, the interface . A plot of delta GA<-->D versus delta GN<-->D (the free energy of denaturation of the native state) has a slope near unity, showing that the evolutionarily conserved helix-helix interaction stabilizes the A state to the same degree that it stabilizes the native state.

Proc Natl Acad Sci U S A, 1995 Mar 14, 92(6), 2086 - 90
The N terminus of phosducin is involved in binding of beta gamma subunits of G protein; Xu J et al.; Phosducin is a soluble phosphoprotein found in retinal photoreceptor cells and in the pineal gland . It binds to the beta gamma subunits of guanine nucleotide-binding proteins (G proteins) (G beta gamma) and may regulate G-protein function . In this study, the ability of specific regions of phosducin to bind G beta gamma was characterized . A series of deletion mutants were made in bovine phosducin . They were tested in cotransfection assays for their ability to inhibit G beta gamma-mediated phospholipase C beta 2 isoform activation . Overexpression of the N-terminal half of phosducin showed inhibition, whereas overexpression of the C-terminal half did not . The first 63 amino acid residues were required for inhibition . A tryptophan-to-valine substitution at residue 29, which is part of a well conserved 11-amino acid sequence, severely impaired phosducin inhibitory function . Glutathione S-transferase-phosducin fusion proteins were expressed in Escherichia coli to study phosducin-G beta gamma interaction in vitro . The N-terminal 63-amino acid fragment was able to bind to G beta gamma . In contrast, the C-terminal half failed to bind to G beta gamma . The substitution mutants showed little or no binding . Furthermore, direct measurements of interaction between G beta gamma and fragments of phosducin, using surface plasmon resonance technology, confirmed the assignment of binding activity to the 63-amino acid fragment and the importance of the tryptophan residue.

Proc Natl Acad Sci U S A, 1995 Mar 14, 92(6), 2003 - 7
A small RNA acts as an antisilencer of the H-NS-silenced rcsA gene of Escherichia coli; Sledjeski D et al.; The regulation of capsular polysaccharide synthesis in Escherichia coli K-12 depends on the level of an unstable positive regulator, RcsA . The amount of RcsA protein is limited both by its rapid degradation by Lon, an ATP-dependent protease, and by its low level of synthesis . We have found that the low level of expression from the rcsA promoter is due to transcriptional silencing by the histone-like protein H-NS; this silencing is sensitive to both sequence and context in a region upstream of the -35 region of the promoter . A small (85-nt) RNA, DsrA, when overproduced, activates transcription of rcsA::lacZ fusions by counteracting H-NS silencing . DsrA RNA does not show any extended homology with the rcsA promoter or other sequenced regions of E . coli . Since the stimulation of rcsA transcription by this small RNA does not depend on any sequences from within the rcsA transcript, DsrA acts, either directly or indirectly, on rcsA transcription initiation.

Proc Natl Acad Sci U S A, 1995 Mar 14, 92(6), 1896 - 900
SopB protein-mediated silencing of genes linked to the sopC locus of Escherichia coli F plasmid; Lynch AS et al.; Expression of a high level of F-plasmid-encoded SopB protein in Escherichia coli is found to repress genes linked to sopC, a sequence element of F consisting of 12 tandemly joined imperfect repeats of a 43-bp motif . Repression of a gene can occur over a distance of at least 10 kb from the sopC element and is not affected by the relative orientation of sopC . In the repressed state, accessibility of intracellular DNA to cellular proteins is greatly reduced in the region containing sopC, as monitored by the trapping of the covalent intermediate between DNA and DNA gyrase and by Dam methylase-catalyzed DNA methylation . These results signify the formation of a nucleoprotein structure emanating from sopC and are discussed in terms of position-dependent silencing of genes in general and the IncG type of plasmid incompatibility in particular.

Proc Natl Acad Sci U S A, 1995 Mar 14, 92(6), 1807 - 11
Polyadenylylation helps regulate mRNA decay in Escherichia coli; O'Hara EB et al.; As part of our genetic analysis of mRNA decay in Escherichia coli K-12, we examined the effect of the pcnB gene {encoding poly(A) polymerase I} on message stability . Eliminating poly(A) polymerase I (delta pcnB) dramatically stabilized the lpp, ompA, and trxA transcripts . The half-lives of individual mRNAs were increased in both a delta pcnB single mutant and a delta pcnB pnp-7 rnb-500 rne-1 multiple mutant . We also found mRNA decay intermediates in delta pcnB mutants that were not detected in control strains . By end-labeling total E . coli RNA with {32P}pCp and T4 RNA ligase and then digesting the RNA with RNase A and T1, we showed that many RNAs in a wild-type strain contained poly(A) tails ranging from 10 nt to > 50 nt long . When polynucleotide phosphorylase, RNase II, and RNase E were absent, the length (> 100 nt) and number (10- to 20-fold) of the poly(A) tails increased . After transcription initiation was stopped with rifampicin, polyadenylylation apparently continued . Deleting the structural gene for poly(A) polymerase I (pcnB) reduced the amount of 3'-terminal poly(A) sequences by > 90% . We propose a model for the role of polyadenylylation in mRNA decay.

Biochemistry, 1995 Mar 14, 34(10), 3404 - 15
Sites within the 39-kDa protein important for regulating ligand binding to the low-density lipoprotein receptor-related protein; Warshawsky I et al.; A 39-kDa protein copurifies with the low-density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP) and inhibits the binding and/or cellular uptake of ligands by this receptor . We recently utilized glutathione S-transferase (GST)-39-kDa fusion protein constructs to demonstrate that constructs encoding amino-terminal residues 1-114 and carboxy-terminal residues 115-319 of the 39-kDa protein independently bind to purified LRP and to LRP on hepatoma cells with similar affinities as the full-length GST-39-kDa protein (Kd approximately 8-10 nM) . These regions, however, inhibit ligand binding to LRP differently: GST/1-114 inhibits both tissue-type plasminogen activator (t-PA) and alpha 2-macroglobulin-methylamine (alpha 2M*) binding whereas GST/115-319 only potently inhibits t-PA binding . Four domains, containing residues 18-24 and 100-107 within amino-terminal constructs and residues 200-225 and 311-319 within carboxy-terminal constructs, are required for inhibition of ligand binding . In the present study, we generated additional 39-kDa protein constructs to precisely define residues within each domain required for inhibition of t-PA and alpha 2M* binding to LRP . The potential importance of these residues in mediating direct binding both to purified LRP and to LRP on hepatoma cells was examined . Within amino-terminal residues 1-114, alanine 103 and leucine 104 are required for inhibition of t-PA and alpha 2M* binding . These residues, however, are not required for binding either to purified LRP or to LRP on hepatoma cells . Within domain 18-24, arginine 21 is required for inhibition of t-PA and alpha 2M* binding as well as for the direct binding of amino-terminal constructs to LRP . Within carboxy-terminal domains 200-225 and 311-319, leucine 222 and leucine 319 are both required for inhibition of t-PA binding . Deletion of leucine 319 changes the ligand specificity from inhibition of t-PA binding to inhibition of alpha 2M* binding . Thus, leucine 319 is not required for direct binding to LRP whereas leucine 222 is required for high-affinity binding to LRP.

FEBS Lett, 1995 Mar 13, 361(1), 55 - 60
In vitro dissociation of self-assembly of three chaperonin 60s: the role of ATP; Lissin NM; A comparative study has investigated the in vitro dissociation and self-assembly of chaperonin 60 14-mers isolated from E . coli (GroEL), yeast mitochondria and pea chloroplasts . In all cases Mg2+ inhibits, and low temperature stimulates, the urea-induced dissociation . ATP or ADP in the presence of Mg2+ enhance the dissociation of the chaperonins . Re-assembly of the 14-mers from their monomers shows different efficiencies between the three proteins . In all cases, however, self-assembly is stimulated by Mg-adenine nucleotides . Surprisingly, effective self-assembly of GroEL is promoted by 20% glycerol in the absence of ATP . The role of Mg-adenine nucleotides in the dissociation and assembly of the chaperonins is discussed.

FEBS Lett, 1995 Mar 13, 361(1), 25 - 8
Effects of mutations at position 36 of tRNA(Glu) on missense and nonsense suppression in Escherichia coli; Gregory ST et al.; Mutations in the anticodon of tRNA(Glu) (UUC) were isolated or constructed and characterized for their ability to suppress cognate nonsense or missense mutations in vivo . The C36-to-A36 transversion mutation was isolated as an ochre and an amber suppressor, while the G36 transversion was selected as a CAG missense suppressor . tRNA(Glu) suppressors of an AAG missense mutation could not be isolated, and a U36 transition mutation introduced into tRNA(Glu) in vitro conferred no suppressor phenotype . Over-expression of glutamyl-tRNA synthetase did not increase the activity of the U36 mutant tRNA(Glu), suggesting a defect at the level of translation rather than at the level of synthetase recognition.

FEBS Lett, 1995 Mar 13, 361(1), 111 - 4
Site-directed mutagenesis of histidine residues in the delta 12 acyl-lipid desaturase of Synechocystis; Avelange-Macherel MH et al.; In the cyanobacterium Synechocystis sp . PCC 6803, there are four acyl-lipid desaturases that are, respectively, specific to the delta 6, delta 9, delta 12 and omega 3 positions of fatty acids . The desA gene for the delta 12 acyl-lipid desaturase was modified by site-directed mutagenesis, such that four of the histidine residues that are conserved in the four desaturases and one histidine residue that is not conserved were replaced by arginine, and the mutated desA genes were overexpressed in Escherichia coli . All of these mutations eliminated the delta 12 desaturase activity . These results demonstrate that the five histidine residues are essential for the activity of the delta 12 desaturase, perhaps by providing the ligands for the catalytic Fe center.

J Immunol Methods, 1995 Mar 13, 180(1), 69 - 79
Immunochemical analyses of human plasma fibronectin-cytosolic transglutaminase interactions; Achyuthan KE et al.; Fibronectin is a glycoprotein involved in cell adhesion, tissue organization and wound healing . Transglutaminase binding and covalent cross-linking of fibronectin are physiologically important reactions . We describe microtiter plate-based immunochemical methods to analyze cytosolic transglutaminase-human plasma fibronectin interactions . The method was sensitive, specific, species-independent and capable of simultaneously analyzing 96 samples for binding . Binding was time-, temperature- and concentration-dependent and demonstrable with either protein immobilized to the plastic . The assay detected 1-5 ng transglutaminase or 50 pg fibronectin and was comparable in sensitivity to enzyme-linked immunosorbent assays . CaCl2 (8 mM) enhanced transglutaminase binding by two-fold . Molar concentrations of NaCl or millimolar concentrations of chloride salts of barium, copper or zinc inhibited binding by 50-60% . The binding was also competitively blocked by soluble fibronectin (IC50 = 2.3 nM) or by anti-fibronectin IgG (IC50 = 0.5 microM) . Inclusion of dithiothreitol or 2-mercaptoethanol during binding resulted in a concentration-dependent inhibition of transglutaminase-fibronectin interactions (IC50 = 1.5 mM and 20 mM, respectively) . A complex of {anti-transglutaminase IgG-transglutaminase-fibronectin-anti- fibronectin IgG} suggested that the binding sites and antibody epitopes could overlap, but are distinct and surface-exposed in the two proteins . Liver transglutaminase bound fibronectin 30-50% less compared to erythrocyte transglutaminase . Fibronectin-transglutaminase affinity was adequate for quantitating either antigen in lysates of lung fibroblasts, breast carcinomas or Escherichia coli . These immunochemical analyses will be useful for determining the affinity and mapping the domains involved in antibody recognition or protein-protein interactions using recombinant molecules of transglutaminase and fibronectin.

Nucleic Acids Res, 1995 Mar 11, 23(5), 869 - 75
Characterization of a specific interaction between Escherichia coli thymidylate synthase and Escherichia coli thymidylate synthase mRNA; Voeller DM et al.; Previous studies have shown that human TS mRNA translation is controlled by a negative autoregulatory mechanism . In this study, an RNA electrophoretic gel mobility shift assay confirmed a direct interaction between Escherichia coli (E.coli) TS protein and its own E.coli TS mRNA . Two cis-acting sequences in the E.coli TS mRNA protein-coding region were identified, with one site corresponding to nucleotides 207-460 and the second site corresponding to nucleotides 461-807 . Each of these mRNA sequences bind TS with a relative affinity similar to that of the full-length E.coli TS mRNA sequence (IC50 = 1 nM) . A third binding site was identified, corresponding to nucleotides 808-1015, although its relative affinity for TS (IC50 = 5.1 nM) was lower than that of the other two cis-acting elements . E.coli TS proteins with mutations in amino acids located within the nucleotide-binding region retained the ability to bind RNA while proteins with mutations at either the nucleotide active site cysteine (C146S) or at amino acids located within the folate-binding region were unable to bind TS mRNA . These studies suggest that the regions on E.coli TS defined by the folate-binding site and/or critical cysteine sulfhydryl groups may represent important RNA binding domains . Further evidence is presented which demonstrates that the direct interaction with TS results in in vitro repression of E.coli TS mRNA translation.

Nucleic Acids Res, 1995 Mar 11, 23(5), 827 - 34
Promoter determinants for Escherichia coli RNA polymerase holoenzyme containing sigma 38 (the rpoS gene product); Tanaka K et al.; Sequence determinants responsible for promoter recognition by RNA polymerase holoenzyme containing sigma 38, the rpoS gene product, were analyzed . In a previous study {Tanaka et al . (1993) Proc . Natl . Acad . Sci . USA, 90, 3511-3515}, Escherichia coli promoters were classified into three groups: promoters recognized only by RNA polymerase holoenzyme containing sigma 70 (E sigma 70); promoters recognized preferentially by that containing sigma 38 (E sigma 38); promoters recognized by both E sigma 70 and E sigma 38 . As representatives of each group of promoter, we chose the alaS, fic and lacUV5 promoters . Making use of a restriction enzyme site inserted between the -10 and -35 hexamer sequences, promoters were divided into the upstream (UE) and downstream (DE) elements . These UEs and DEs were combined in all possible combinations and used for in vitro transcription reactions . Promoters containing DE from the fic or lacUV5 promoter were found to be recognized by E sigma 38, while those containing DE from the alaS promoter were not . Moreover, fic DE alone functioned as an efficient promoter for E sigma 38 . Thus we conclude that the discrimination signal resides within the DE sequence . To test the activator response of E sigma 38, in vitro transcription reactions were also performed with the gal and lac promoters . For both CRP-responsive P1 promoters, E sigma 38 was found to be activated by the CRP-cAMP complex.

Nucleic Acids Res, 1995 Mar 11, 23(5), 819 - 26
Selectivity of the Escherichia coli RNA polymerase E sigma 38 for overlapping promoters and ability to support CRP activation; Kolb A et al.; A series of gal promoter mutants has been used to compare the in vitro selectivities of the two forms of Escherichia coli RNA polymerase, E sigma 38 and E sigma 70 . In the absence of the CRP-cAMP complex, E sigma 38 shows a strong preference for the ga/P1 promoter, whereas E sigma 70 preferentially initiates transcription from the ga/P2 promoter . E sigma 38 selectivity is not affected by the nature and position of the upstream sequences or by the phasing between synthetic upstream curved sequences and the -10 regions . In fact, all effects of mutations in the extended -10 region can be accounted for without evoking strong new sequence preferences for E sigma 38 . Finally, both E sigma 38 and E sigma 70 initiate transcription from the ga/P1 promoter in the presence of CRP-cAMP complex and support direct cAMP-CRP activation at several CRP-dependent promoters.

Nucleic Acids Res, 1995 Mar 11, 23(5), 785 - 7
A motif conserved among the type I restriction-modification enzymes and antirestriction proteins: a possible basis for mechanism of action of plasmid-encoded antirestriction functions; Belogurov AA et al.; Antirestriction proteins Ard encoded by some self-transmissible plasmids specifically inhibit restriction by members of all three families of type I restriction-modification (R-M) systems in E.coli . Recently, we have identified the amino acid region, 'antirestriction' domain, that is conserved within different plasmid and phage T7-encoded antirestriction proteins and may be involved in interaction with the type I R-M systems . In this paper we demonstrate that this amino acid sequence shares considerable similarity with a well-known conserved sequence (the Argos repeat) found in the DNA sequence specificity (S) polypeptides of type I systems . We suggest that the presence of these similar motifs in restriction and antirestriction proteins may give a structural basis for their interaction and that the antirestriction action of Ard proteins may be a result of the competition between the 'antirestriction' domains of Ard proteins and the similar conserved domains of the S subunits that are believed to play a role in the subunit assembly of type I R-M systems.

Nucleic Acids Res, 1995 Mar 11, 23(5), 761 - 6
Misincorporation of dAMP opposite 2-hydroxyadenine, an oxidative form of adenine; Kamiya H et al.; Nucleotide incorporation opposite an oxidative form of adenine, 2-hydroxyadenine (2-OH-Ade) was investigated . When a primed template with 2-OH-Ade was treated with an exonuclease-deficient Klenow fragment of Escherichia coli DNA polymerase I (KFexo-), recombinant rat DNA polymerase beta (pol beta) or calf thymus DNA polymerase alpha (pol alpha), incorporation of dTMP and dAMP was observed . In addition, KFexo- inserted dGMP as well . A steady-state kinetic study indicated that the insertion of dAMP and dTMP opposite the DNA lesion occurred with similar frequency with KFexo- and pol beta . Insertion of dTMP opposite 2-OH-Ade was favored to that of dAMP by pol alpha . Chain extension from the A.2-OH-Ade pair is less favored than that from the T.2-OH-Ade pair by all three DNA polymerase . Analysis of full-length products of in vitro DNA synthesis showed that dTMP and dAMP were incorporated by DNA polymerases and that exonuclease-proficient and -deficient Klenow fragments also inserted dGMP opposite 2-OH-Ade . These results suggest that formation of 2-OH-Ade from A in DNA will induce A-->T and A-->C transversions in cells.

Nucleic Acids Res, 1995 Mar 11, 23(5), 803 - 10
Mutational sensitivity patterns define critical residues in the palm subdomain of the reverse transcriptase of human immunodeficiency virus type 1; Chao SF et al.; We have analyzed 154 single amino acid replacement mutants within a 40 amino acid region (residues 164-203) of the reverse transcriptase (RT) from human immunodeficiency virus type 1 (HIV-1) . This region consists of two antiparallel beta-strands (strands 9 and 10) flanked by two alpha helices (E and F) . The structure of this region of the 'palm' subdomain is conserved in a variety of DNA and RNA polymerases, indicating a critical role in enzyme structure and function . Functional assays were performed by screening RT activity of mutants expressed in E . coli . A functionally important region corresponding closely to beta-strands 9 and 10 and the loop joining them was revealed by its mutational sensitivity . Structural analysis of mutants was performed by using Western blots to assay correct folding, which is required for processing to produce the mature p66 and p51 RT species . This analysis indicates that beta-strand 10 is a structurally important region . Combined analysis of these two assays revealed diagnostic patterns of mutational sensitivity which identify key positions in the RT sequence at which a specific amino acid side chain is critical, either for structure or function, as well as residues which are external to the RT structure . This work illustrates the utility of large-scale mutagenesis in relating primary sequence to significant features of protein structure and function.

Life Sci, 1995 Mar 10, 56(16), 1343 - 6
Is hypothalamic prostaglandin E2 involved in avian fever?
Fraifeld V, Blaicher-Kulick R, Degen AA, Kaplanski J.
In chickens, the effect of Escherichia coli lipopolysaccharide (LPS) on body temperature and ex vivo hypothalamic prostaglandin E2 (PGE2) production was examined to test the possible involvement of PGE2 in mechanisms of avian fever . PGE2 is reported to be the major central mediator of fever in mammals; it has not been examined in birds . An intraperitoneal injection of LPS caused an elevation of body temperature but not an elevation of hypothalamic PGE2 production . It seems that: (a) hypothalamic PGE2 is not involved in the development of the febrile response in birds; (b) central mechanisms of avian fever differ from those in mammals.

Arch Biochem Biophys, 1995 Mar 10, 317(2), 348 - 56
Enhancement of Escherichia coli H(+)-ATPase caused by binding of monoclonal antibodies is attributed to structural changes of Leu-456 and Ser-440 in the alpha subunit; Kanazawa H et al.; Five monoclonal antibodies against the alpha subunit of F1-ATPase from Escherichia coli alpha 104, alpha 105, alpha 107, alpha 109, and alpha 110 were prepared . The monoclonal antibodies alpha 104 and alpha 110 enhanced the F1-ATPase activity maximally to 1.6- and 1.7-fold that of the wild-type, respectively, while alpha 105 did not . Both antibodies bound to a peptide corresponding to the region between residues 354 and 513 . Mutations in this region which caused reduced binding of the alpha subunit to the antibodies were identified at residues Ser-440, Leu-456, Leu-471, Leu-482, Met-483, and Ser-506 for alpha 104 and residues Ser-440, Leu-456, Leu-471, Asp-476, Leu-482, Met-483, and Ser-506 for alpha 110 . These residues are possibly involved in the epitopes for the antibodies and are located close together on the surface of the alpha subunit . Among the mutations, Leu-456 to Pro and Ser-440 to Pro mutations caused increase of the F1-ATPase activity up to 1.9 and 1.2 times that of the wild-type, respectively, while Leu-471 to Pro mutation caused a defect in assembly of the F1-ATPase on the membrane . The other mutations caused no significant change in ATPase activity . These results suggested that Ser-440 and Leu-456 have an important role in regulating catalysis by the F1-ATPase, but that the neighboring residue Leu-471 has an important role in assembly of the F1-ATPase complex . It was also suggested that binding of the monoclonal antibodies alpha 104 and alpha 110 to residues Ser-440 and Leu-456 caused local conformational changes, leading to enhancing effects on F1-ATPase activity similar to the Ser-440 to Pro and Leu-456 to Pro mutations.

Arch Biochem Biophys, 1995 Mar 10, 317(2), 343 - 7
The role of cytochrome b5 in the biosynthesis of androgens by human P450c17; Katagiri M et al.; Human cytochrome b5 has a profound effect on the 17,20-lyase activities catalyzed by purified, human cytochrome P450c17 . It enhances the conversion of 17 alpha-hydroxypregnenolone to dehydroepiandrosterone by 13-fold and the conversion of 17 alpha-hydroxyprogesterone to androstenedione by at least 10-fold . This latter activity is virtually undetectable in the absence of cytochrome b5 . Other activities catalyzed by P450c17 include 17 alpha-hydroxylation of progesterone and pregnenolone and are much less influenced by cytochrome b5 . The conversion of pregnenolone to 17 alpha-hydroxypregnenolone is increased by 2-fold, while that of progesterone to 17 alpha-hydroxyprogesterone is unchanged . These studies using purified systems suggest that cytochrome b5 plays a role in regulating the activities of P450c17 to optimize the balance between sex hormone synthesis and glucocorticoid synthesis . In particular, they indicate that in human testes which contains a high b5/P450 ratio, 17 alpha-hydroxyprogesterone can serve as an intermediate in testosterone production, rather than being a dead-end product, or stated another way, because of the relatively high concentration of cytochrome b5 in the human testis, both the delta 4 and the delta 5 steroidogenic pathways can lead to testosterone production.

J Biol Chem, 1995 Mar 10, 270(10), 5642 - 8
Cloning and sequencing of an intronless mouse S-adenosylmethionine decarboxylase gene coding for a functional enzyme strongly expressed in the liver; Persson K et al.; A genomic clone for a mouse S-adenosylmethionine decarboxylase (AdoMetDC) gene was isolated from a cosmid library . Surprisingly, the gene proved to be intronless . With the exception of three base substitutions (changing 2 amino acids in the deduced protein), the 1002-nucleotide sequence of the open reading frame was identical to that of mouse AdoMetDC cDNA . Moreover, the gene contained a poly(dA) tract at the 3' end and was flanked by 13-base pair direct repeats . Our findings suggest that this gene has arisen by retroposition, in which a fully processed AdoMetDC mRNA has been reverse transcribed into a DNA copy and inserted into the genome . By polymerase chain reaction, we positively identified the intronless gene in the mouse genome, and, by primer extension analysis, we proved the gene to be functional . Thus, its transcripts were found in many cell lines and tissues of the mouse and were particularly abundant in the liver . When the open reading frame of the intronless gene was expressed in Escherichia coli HT551, a strain with no AdoMetDC activity, it was found to encode a 38-kDa protein, corresponding to AdoMetDC proenzyme . Although the change of methionine 70 to isoleucine was close to the cleavage site at serine 68, this protein underwent proenzyme processing, generating a 31-kDa alpha subunit and an 8-kDa beta subunit . Importantly, the protein encoded by the intronless gene was functional, i.e . it catalyzed the decarboxylation of S-adenosylmethionine, and its specific activity was comparable with that of recombinant human AdoMetDC purified according to the same procedure.

J Biol Chem, 1995 Mar 10, 270(10), 5606 - 13
Escherichia coli DNA polymerase III holoenzyme subunits alpha, beta, and gamma directly contact the primer-template; Reems JA et al.; Escherichia coli DNA polymerase III holoenzyme forms a stable initiation complex with RNA-primed template in the presence of ATP . To determine the linear arrangement of the holoenzyme subunits along the primer-template duplex region, we cross-linked holoenzyme to a series of photo-reactive primers . Site-specific photo-cross-linking revealed that the alpha, beta, and gamma subunits formed ATP-dependent contacts with the primer-template . The alpha-polymerase catalytic subunit covalently attached to nucleotide positions -3, -9, and -13 upstream of the primer terminus, with the most efficient adduct formation occurring at position -9 . The gamma subunit contacted the primer at positions -13, -18, and -22, with the strongest gamma-primer interactions occurring at position -18 . The beta subunit predominated in cross-linking at position -22 . Thus, within the initiation complex, alpha contacts roughly the first 13 nucleotides upstream of the 3'-primer terminus followed by gamma at -18 and beta at -22, and the gamma subunit remains a part of the initiation complex, bridging the alpha and beta subunits . Analyses of the interaction of photo-activatible primer-templates with the preinitiation complex proteins (gamma-complex (gamma-delta-delta'-chi-psi) and beta subunit) revealed the gamma subunit within the preinitiation complex covalently attached to primer at position -3 . However, addition of core DNA polymerase III to preinitiation complex, fully reconstituting holoenzyme resulted in replacement of gamma by alpha at the primer terminus . These data indicate that assembly of holoenzyme onto a primer-template can occur in distinct stages and results in a structural rearrangement during initiation complex formation.

J Biol Chem, 1995 Mar 10, 270(10), 5578 - 86
Alternate binding of actin and calmodulin to multiple sites on dystrophin; Jarrett HW et al.; Mouse dystrophin protein sequence 1-385 and various deletion mutants were expressed in Escherichia coli as fusion proteins, and the binding of actin, calmodulin, and troponin C were characterized . The fusion protein-containing sequence 1-385 bound actin with an apparent dissociation constant of 129 +/- 65 nM as measured using a solid-phase immunoassay . High affinity was also observed with ultracentrifuge cosedimentation assays and biotinylated-actin binding assays . Results with deletion mutants and analysis based upon sequence homology were consistent with two or three high affinity F-actin-binding sequences within this region of dystrophin at sequence positions 18-37 (ABS 1), 128-149 (ABS 2), and potentially at a new region called ABS 3 (86-120) . A fusion protein lacking these sequences but containing dystrophin triple-helix sequences also bound actin but with reduced affinity . Calmodulin binds to dystrophin sequence 1-385 in a Ca(2+)-dependent manner and competitively inhibits F-actin binding . Results were consistent with two Ca(2+)-calmodulin-binding sites in this region of dystrophin at approximate sequence positions 18-42 (CBS 1) and 104-125 (CBS 2) with calmodulin affinities of 2.1 +/- 1 and 1.6 +/- 1.2 microM, respectively . Troponin C can substitute for calmodulin, although it binds with about 2-fold lower affinity . These results suggest that calmodulin (or troponin C) binding alternates with and may regulate F-actin binding by dystrophin much as has been postulated for other cytoskeletal proteins which are homologous to dystrophin.

J Biol Chem, 1995 Mar 10, 270(10), 5519 - 26
Product of a new gene, syd, functionally interacts with SecY when overproduced in Escherichia coli; Shimoike T et al.; A mutant form of SecY, SecY-d1, was previously suggested to sequester a component(s) of the protein translocator complex . Its synthesis from a plasmid leads to interference with protein export in Escherichia coli . SecE is a target of this sequestration, and its overproduction cancels the export interference . We now report that overexpression of another gene, termed syd, also suppresses secY-d1 . The nucleotide sequence of syd predicted that it encodes a protein of 181 amino acid residues, which has been identified by overproduction, purification, and determination of the amino-terminal sequence . Cell fractionation experiments suggested that Syd is loosely associated with the cytoplasmic surface of the cytoplasmic membrane . SecY may be involved in the membrane association of Syd since the association is saturable, the extent of which depends on the overproduction of SecY . SecY is rapidly degraded in vivo unless its primary partner, SecE, is sufficiently available . Overproduction of Syd was found to stabilize oversynthesized SecY . However, Syd cannot stabilize the SecY-d1 form of SecY . Thus, in the presence of both secY+ and secY-d1, Syd increases the effective SecY+/SecY-d1 ratio in the cell and cancels the dominant interference by the latter . We also found that overproduction of Syd dramatically inhibits protein export in the secY24 mutant cell in which SecY-SecE interaction has been weakened . These results indicate that Syd, especially when it is overproduced, has abilities to interact with SecY . Possible significance of such interactions is discussed in conjunction with the apparent lack of phenotypic consequences of genetic disruption of syd.

J Biol Chem, 1995 Mar 10, 270(10), 5495 - 505
Cloning and identification of amino acid residues of human phospholipase C delta 1 essential for catalysis; Cheng HF et al.; In vitro single point mutagenesis, inositol phospholipid hydrolysis, and substrate protection experiments were used to identify catalytic residues of human phosphatidylinositide-specific phospholipase C delta 1 (PLC delta 1) isolated from a human aorta cDNA library . Invariant amino acid residues containing a functional side chain in the highly conserved X region were changed by in vitro mutagenesis . Most of the mutant enzymes were still able to hydrolyze inositol phospholipid with activity ranging from 10 to 100% of levels in the wild type enzyme . Exceptions were mutants with the conversion of Arg338 to Leu (R338L), Glu341 to Gly (E341G), or His356 to Leu (H356L), which made the enzyme severely defective in hydrolyzing inositol phospholipid . Phospholipid vesicle binding experiments showed that these three cleavage-defective mutant forms of PLC delta 1 could specifically bind to phosphatidylinositol 4,5-bisphosphate (PIP2) with an affinity similar to that of wild type enzyme . Western blotting analysis of trypsin-treated enzyme-PIP2 complexes revealed that a 67-kDa major protein fragment survived trypsin digestion if the wild type enzyme, E341G, or H356L mutant PLC delta 1 was preincubated with 7.5 microM PIP2, whereas if it was preincubated with 80 microM PIP2, the size of major protein surviving was comparable to that of intact enzyme . However, mutant enzyme R338L was not protected from trypsin degradation by PIP2 binding . These observations suggest that PLC delta 1 can recognize PIP2 through a high affinity and a low affinity binding site and that residues Glu341 and His356 are not involved in either high affinity or low affinity PIP2 binding but rather are essential for the Ca(2+)-dependent cleavage activity of PLC.

J Biol Chem, 1995 Mar 10, 270(10), 5469 - 75
Carboxyl-terminal domain truncation alters apolipoprotein A-I in vivo catabolism; Schmidt HH et al.; Apolipoprotein A-I (apoA-I), the major protein of high density lipoproteins, facilitates reverse cholesterol transport from peripheral tissue to liver . To determine the structural motifs important for modulating the in vivo catabolism of human apoA-I (h-apoA-I), we generated carboxyl-terminal truncation mutants at residues 201 (apoA-I201), 217 (apoA-I217), and 226 (apoA-I226) by site-directed mutagenesis . ApoA-I was expressed in Escherichia coli as a fusion protein with the maltose binding protein, which was removed by factor Xa cleavage . The in vivo kinetic analysis of the radioiodinated apoA-I in normolipemic rabbits revealed a markedly increased rate of catabolism for the truncated forms of apoA-I . The fractional catabolic rates (FCR) of 9.10 +/- 1.28/day (+/- S.D.) for apoA-I201, 6.34 +/- 0.81/day for apoA-I217, and 4.42 +/- 0.51/day for apoA-I226 were much faster than the FCR of recombinant intact apoA-I (r-apoA-I, 0.93 +/- 0.07/day) and h-apoA-I (0.91 +/- 0.34/day) . All the truncated forms of apoA-I were associated with very high density lipoproteins, whereas the intact recombinant apoA-I (r-apoA-I) and h-apoA-I associated with HDL2 and HDL3 . Gel filtration chromatography revealed that in contrast to r-apoA-I, the mutant apoA-I201 associated with a phospholipid-rich rabbit apoA-I containing particle . Analysis by agarose gel electrophoresis demonstrated that the same mutant migrated in the pre-beta position, but not within the alpha position as did r-apoA-I . These results indicate that the carboxyl-terminal region (residue 227-243) of apoA-I is critical in modulating the association of apoA-I with lipoproteins and in vivo metabolism of apoA-I.

J Biol Chem, 1995 Mar 10, 270(10), 5388 - 94
Stability of the asymmetric Escherichia coli chaperonin complex . Guanidine chloride causes rapid dissociation; Todd MJ et al.; The chaperonin proteins, GroEL14 and GroES7, inhibit protein aggregation and assist in protein folding in a potassium/ATP-dependent manner . In vitro, assays for chaperonin activity typically involve adding a denatured substrate protein to the chaperonins and measuring the appearance of correctly folded substrate protein . The influence of denaturant is generally ignored . Low concentrations of guanidinium chloride (< 100 mM) had a profound effect on the activity/structure of the chaperonins . Guanidinium decreased the ATPase activity of GroEL and attenuated the inhibition of GroEL ATP hydrolysis by GroES . The stable, asymmetric chaperonin complex which forms in the presence of GroES and ADP (GroES7.ADP7.GroEL7-GroEL7) rapidly dissociated upon addition of 80 mM guandinium chloride . Dissociation was enhanced at high ionic strength, but rapid dissociation was guanidinium-specific . Accelerated release of the GroES from the complex was also demonstrated . Unfolded proteins alone had no effect on complex stability . Residual guanidinium depressed the rate of Rhodospirillum rubrum ribulose-1,5-bisphosphate carboxylase (Rubisco) folding; an increased aggregation rate also decreased the yield of folded Rubisco . Chaperonin-assisted folding is therefore best studied using proteins denatured by means other than guanidinium chloride.

J Biol Chem, 1995 Mar 10, 270(10), 5367 - 74
DsbA-mediated disulfide bond formation and catalyzed prolyl isomerization in oxidative protein folding; Frech C et al.; The interrelationship between the acquisition of ordered structure, prolyl isomerization, and the formation of the disulfide bonds in assisted protein folding was investigated by using a variant of ribonuclease T1 (C2S/C10N-RNase T1) with a single disulfide bond and two cis-prolyl bonds as a model protein . The thiol-disulfide oxidoreductase DsbA served as the oxidant for forming the disulfide bond and prolyl isomerase A as the catalyst of prolyl isomerization . Both enzymes are from the periplasm of Escherichia coli . Reduced C2S/C10N-RNase T1 is unfolded in 0 M NaCl, but native-like folded in > or = 2 M NaCl . Oxidation of 5 microM C2S/C10N-RNase T1 by 8 microM DsbA (at pH 7.0, 25 degrees C) is very rapid with a t1/2 of about 10 s (the second-order rate constant is 7 x 10(3) s-1 M-1), irrespective of whether the reduced molecules are unfolded or folded . When they are folded, the product of oxidation is the native protein . When they are denatured, first the disulfide bond is formed in the unfolded protein chains and then the native structure is acquired . This slow reaction is limited in rate by prolyl isomerization and catalyzed by prolyl isomerase . The efficiency of this catalysis is strongly decreased by the presence of the disulfide bond . Apparently, the rank order of chain folding, prolyl isomerization, and disulfide bond formation can vary in the oxidative folding of ribonuclease T1 . Such a degeneracy could generally be an advantage for protein folding both in vitro and in vivo.

J Biol Chem, 1995 Mar 10, 270(10), 5326 - 30
Stereoselective hydroxylation of norcamphor by cytochrome P450cam . Experimental verification of molecular dynamics simulations; Loida PJ et al.; The stereoselectivity of cytochrome P450cam hydroxylation has been investigated with the enantiomerically pure substrate analog norcamphor . (1R)- and (1S)-norcamphor (> 92 enantiomeric excess) were characterized in the hydroxylation reaction with cytochrome P450cam with respect to the product profile, steady state kinetics, coupling efficiency, and free energy of substrate dissociation . The experimental results demonstrate regiospecificity that is enantiomer-specific and confirm our previously reported prediction that (1R)-norcamphor is hydroxylated preferentially at the 5-carbon and (1S)-norcamphor at the 6-carbon (Bass, M . B., and Ornstein, R . L . (1993) J . Comput . Chem . 14, 541-548); these simulation results are now compared with simulations involving a ferryl oxygen intermediate . Hydroxylation of (1R)-norcamphor was found at the 5-, 6-, and 3-carbons in a ratio of 65:30:5 (respectively), whereas (1S)-norcamphor was oxidized to produce a 28:62:10 ratio of the same products . With the exception of the regiospecificity, all of the reaction and physical parameters are similar for each enantiomer of norcamphor . These results show that the position of the carbonyl group on the hydrocarbon skeleton of norcamphor plays a role in determining the average orientation of this substrate in the active site and suggests that hydrogen bonding interactions can aid in directing the regiospecificity and stereospecificity of the hydroxylation reaction catalyzed by cytochrome P450cam.

J Biol Chem, 1995 Mar 10, 270(10), 5258 - 65
Functional reconstitution of the purified mannose phosphotransferase system of Escherichia coli into phospholipid vesicles; Mao Q et al.; The mannose transporter complex acts by a mechanism which couples translocation with phosphorylation of the substrate . It consists of a hydrophilic subunit (IIABMan) and two transmembrane subunits (IICMan, IIDMan) . The purified complex was reconstituted into phospholipid vesicles by octyl glucoside dilution . Glucose export was measured with proteoliposomes which were loaded with radiolabeled glucose and to which purified IIABMan, cytoplasmic phosphorylcarrier proteins, and P-enolpyruvate were added from the outside . Vectorial transport was accompanied by stoichiometric phosphorylation of the transported sugar . Glucose added to the outside of the proteoliposomes was also phosphorylated rapidly but did not compete with vectorial export and phosphorylation of internal glucose . Glucose uptake was measured with proteoliposomes which were loaded with the cytoplasmic phosphoryl carrier proteins and P-enolpyruvate and to which glucose was added from the outside . Vectorial import and phosphorylation occurred with a higher specificity (Km 30 +/- 6 microM, kcat 401 +/- 32 pmol of Glc/micrograms of IICDMan/min) than nonvectorial phosphorylation (Km 201 +/- 43 microM, kcat 975 +/- 88 pmol of Glc/micrograms of IICDMan/min) . A new plasmid pTSHIC9 for the controlled overexpression of the cytoplasmic phosphoryl carrier proteins, enzyme I, HPr, and IIAGlc, and a simplified procedure for the purification of these proteins are also described.

J Biol Chem, 1995 Mar 10, 270(10), 5198 - 206
Galectin-1, a beta-galactoside-binding lectin in Chinese hamster ovary cells . I . Physical and chemical characterization; Cho M et al.; We report our studies on the characterization of an approximately 14-kDa lectin, termed galectin-1 that we have found to be expressed by Chinese hamster ovary (CHO) cells . cDNA for galectin-1 from CHO cells was prepared and sequenced, and a recombinant form (rGal-1) was expressed in Escherichia coli . A mutated form of the protein that fully retained activity was also constructed (termed C2SrGal-1) in which Cys-2 was changed to Ser-2 . rGal-1 was stable in the presence of reducing agent, but it quickly lost all activity in the absence of reducing agent . In contrast, glycoprotein ligands, such as basement membrane laminin, stabilized the activity of rGal-1 in the absence of reducing agent (t1/2 = 2 weeks) . C2SrGal-1 was stable in the presence or absence of either ligand or reducing agent . Unexpectedly, galectin-1 was found to exist in a reversible and active monomer-dimer equilibrium with a Kd approximately 7 microM and an equilibration time of t1/2 approximately 10 h . Addition of haptenic sugars did not affect this equilibrium . Galectin-1 isolated from the cytosol of CHO cells was found to exist as monomers and dimers . These studies demonstrate that galectin-1 binding to a biological ligand stabilizes its activity and that the monomer/dimer state of the protein is regulated by lectin concentration.

J Biol Chem, 1995 Mar 10, 270(10), 5115 - 21
Structural basis for the biological specificity of cystatin C . Identification of leucine 9 in the N-terminal binding region as a selectivity-conferring residue in the inhibition of mammalian cysteine peptidases; Hall A et al.; The structural basis for the biological specificity of human cystatin C has been investigated . Cystatin C and other inhibitors belonging to family 2 of the cystatin superfamily interact reversibly with target peptidases, seemingly by independent affinity contributions from a wedge-shaped binding region built from two loop-forming inhibitor segments and a binding region corresponding to the N-terminal segment of the inhibitor . Human cystatin C variants with Gly substitutions for residues Arg-8, Leu-9, and/or Val-10 of the N-terminal binding region, and/or the evolutionarily conserved Trp-106 in the wedge-shaped binding region, were produced by site-directed mutagenesis and Escherichia coli expression . A total of 10 variants were isolated, structurally verified, and compared to wild-type cystatin C with respect to inhibition of the mammalian cysteine peptidases, cathepsins B, H, L, and S . Varying contributions from the N-terminal binding region and the wedge-shaped binding region to cystatin C affinity for the four target peptidases were observed . Interactions from the side chains of residues in the N-terminal binding region and Trp-106 are jointly responsible for the major part of cystatin C affinity for cathepsin L and are also of considerable importance for cathepsin B and H affinity . In contrast, for cathepsin S inhibition these interactions are of lesser significance, as reflected by a Ki value of 10(-8) M for the cystatin C variant devoid of Arg-8, Leu-9, Val-10, and Trp-106 side chains . The side chain of Val-10 is responsible for most of the affinity contribution from the N-terminal binding region, for all four enzymes . The contribution of the Arg-8 side chain is minor, but significant for cystatin C interaction with cathepsin B . The Leu-9 side chain confers selectivity to the inhibition of the target peptidases; it contributes to cathepsin B and L affinity by factors of 200 and 50, respectively, to cathepsin S binding by a factor of 5 only, and results in a 10-fold decreased affinity between cystatin C and cathepsin H.

J Biol Chem, 1995 Mar 10, 270(10), 5048 - 56
Biochemical and functional characterization of a recombinant GTPase, Rab5, and two of its mutants; Hoffenberg S et al.; Biochemical, structural, and functional properties of Rab5 wild-type (WT) protein were compared with those of Q79L and N133I mutants . The detergent 3-{(3-cholamidopropyl)dimethylammonio}-1-propanesulfonate increased guanine nucleotide binding to Rab5 WT approximately 10-fold . The single-step catalytic rate of Rab5 WT exceeded that of Q79L 12.2-fold, but the steady-state GTPase rate was only 2.8-fold greater because GDP dissociation was rate-limiting and GDP dissociation was 3.6-fold slower than for Q79L . In contrast, dissociation rates of GTP were indistinguishable . Binding to Rab5 N133I was not detectable . GTP protected Rab5 WT and Q79L from any apparent proteolysis by trypsin . A 20-kDa fragment was the major product of digestion in the presence of GDP, and 12- and 8-kDa fragments were the major products in the absence of added guanine nucleotides . Rab5 N133I underwent no apparent proteolysis with 10 mM GTP or GDP, suggesting a "triphosphate" conformation may be induced in Rab5 N133I by either GTP or GDP . Partially geranylgeranylated Rab5 WT stimulated endosome fusion in vitro, whereas unmodified Rab5 WT did not . Processed Rab5 Q79L failed to inhibit endosome fusion, and Rab5 N133I could not be geranylgeranylated . These findings identify biochemical and structural features of Rab5 proteins, providing data for the interpretation of functional assays.

J Biol Chem, 1995 Mar 10, 270(10), 4990 - 5000
In vivo and in vitro replication consequences of stereoisomeric benzo{a}pyrene-7,8-dihydrodiol 9,10-epoxide adducts on adenine N6 at the second position of N-ras codon 61; Chary P et al.; Benzo{a}pyrene-7,8-dihydrodiol 9,10-epoxide (BPDE), a metabolite of the widespread environmental pollutant benzo{a}pyrene, is a mutagenic in both bacterial and mammalian systems . Toward understanding the mutagenic effects of different stereoisomers of BPDE at specific sites in DNA, six stereochemically defined BPDE adducts were constructed on adenine N6 at position 2 of the human N-ras 61 codon within an 11-base oligonucleotide fragment . Both the nonadducted and BPDE-adducted N-ras 61 11-mers were inserted into a unique EcoRI site in single-stranded M13mp7L2 DNA and utilized for in vivo studies . The ligation efficiencies of BPDE-adducted 11-mers into the single-stranded vector were determined by Southern hybridization and confirmed by electron microscopy . Repair-deficient AB2480 E . coli cells were transformed with adducted and non-adducted DNA samples . The resultant plaque-forming abilities were used to evaluate the replication competence of the various BPDE adducts with respect to the nonadducted 11-mer . Point mutations due to aberrant replication at the adducted site were identified by the technique of differential DNA hybridization . All of the six BPDE adducts examined were mutagenic in vivo, generating exclusively A-->G mutations at frequencies ranging from 0.26 to 1.20% . In vitro replication studies using these BPDE-adducted 11-mers involved primer extension assays with Klenow fragment . All of the BPDE-modified templates demonstrated distinct blockage at the adducted site and/or 1 base 3' to the adducted site, allowing essentially no translesion synthesis to form fully extended polymerization products in vitro.

J Biol Chem, 1995 Mar 10, 270(10), 4975 - 8
Identification of alpha-syntrophin binding to syntrophin triplet, dystrophin, and utrophin; Yang B et al.; Syntrophin represents three cytoplasmic components of the dystrophin-glycoprotein complex that links the cytoskeleton to the extracellular matrix in skeletal muscle . alpha-Syntrophin has now been translated in vitro and shown to associate directly with all three components of the syntrophin triplet and with dystrophin . The in vitro translated 71-kDa non-muscle dystrophin isoform, containing the cystein-rich/C-terminal domain, can also interact with the syntrophin triplet . The syntrophin binding motif in dystrophin was localized to exons 73 and 74 including amino acids 3447-3481 by comparing the interactions of alpha-syntrophin and seven overlapping human dystrophin fusion proteins . More than one syntrophin interaction site in this binding motif was suggested . alpha-Syntrophin also interacts directly with a C-terminal utrophin fusion protein . alpha-Syntrophin is localized to the muscle sarcolemma as well as to the neuromuscular junction in control mouse muscle . However, similar to utrophin, alpha-syntrophin is only present at the neuromuscular junction in mdx mouse muscle in which dystrophin is absent . Our data suggest that alpha-syntrophin binds all syntrophin isoforms, and syntrophin directly interacts with dystrophin through more than one binding site in dystrophin exons 73 and 74 including amino acids 3447-3481.

Gene, 1995 Mar 10, 154(2), 159 - 66
Total synthesis and expression in Escherichia coli of a gene encoding human tropoelastin; Martin SL et al.; To elucidate the structural features and interactions of tropoelastin (TEL) molecules which assist in giving the elastic fibre its physical properties, a 2210-bp synthetic human TEL-encoding gene (SHEL) was constructed for expression in Escherichia coli . To this end, a model of codon adjustment was tested which better suits the polypeptide biosynthetic needs of E . coli than the human sequence, where over one-third of this natural sequence contains expression-limiting rare codons and 4 amino acids alone account for 75% of the resulting polypeptide . This large synthetic TEL gene was expressed at a high level as the recombinant counterpart of human TEL and as a C-terminal fusion with glutathione S-transferase . This demonstrates that a synthetic approach based upon matching codon usage to that of the host organism can support significant expression of recombinant sequences . The synthetic gene incorporates the facility for simple cassette replacement in future insertion, deletion and mutagenesis experiments, including the introduction and removal of exon homologues . The resulting soluble polypeptide is easily purified and displays properties expected for this protein.

Cell, 1995 Mar 10, 80(5), 787 - 93
Branch migration during homologous recombination: assembly of a RuvAB-Holliday junction complex in vitro; Hiom K et al.; The RuvA and RuvB proteins of E . coli promote the branch migration or movement of Holliday junctions during genetic recombination and DNA repair . Using small synthetic Holliday junctions in which the crossover point is confined near one end of the DNA molecule, we show that RuvAB-mediated branch migration occurs with a defined polarity . The assembly of RuvA and RuvB on the Holliday junction has been investigated by sedimentation analysis and by DNase I footprinting . We find that RuvA protein binds and protects all four strands of DNA at the crossover point, whereas RuvB protein binds the DNA asymmetrically . The polarity of branch migration is defined by the asymmetric assembly of the RuvAB branch migration complex relative to the junction and is consistent with a model in which RuvAB drives branch migration by passing the DNA through the hexameric rings of RuvB.

Virology, 1995 Mar 10, 207(2), 495 - 502
Chimeric macaque/human Fab molecules neutralize simian immunodeficiency virus; Samuelsson A et al.; A collection of simian immunodeficiency virus (SIV) neutralizing recombinant Fab fragments was generated using the combinatorial antibody library approach . Functional antibody fragments efficiently expressed in Escherichia coli were identified only in the form of chimeric macaque heavy chain gamma 1 and human light chain kappa . The gamma 1 and kappa chains were derived from a clinically healthy long-term surviving SIVsm-infected cynomolgus macaque and from an asymptomatic HIV-2 seropositive individual, respectively . The combinatorial library was constructed on the surface of filamentous phage using the pComb3 phagemid vector and screened against purified SIVsm surface glycoprotein (gp148) . Twelve chimeric clones reacting with the antigen were isolated . Six of these clones showed a pronounced neutralizing activity against SIVsm with effects at concentrations of 0.01-0.1 micrograms/ml . All neutralizing Fab fragments were clonally unrelated as demonstrated by nucleic acid sequencing . These potent neutralizing reagents will be used for prophylactic and therapeutic immune intervention of lentivirus infection in macaques and to map neutralizing determinants of SIV.

Virology, 1995 Mar 10, 207(2), 486 - 94
In vitro assembly of cowpea chlorotic mottle virus from coat protein expressed in Escherichia coli and in vitro-transcribed viral cDNA; Zhao X et al.; The small spherical plant virus, cowpea chlorotic mottle virus (CCMV), provides an ideal system to examine spherical virus assembly . We have modified the CCMV in vitro assembly system to produce virions from coat protein expressed in Escherichia coli and viral RNA transcribed in vitro from full-length cDNAs . Examination of the in vitro-assembled particles with cryoelectron microscopy and image reconstruction techniques demonstrates that the particles are indistinguishable from plant purified particles at 2.5 nm resolution . Mutational analysis of the coat protein N- and C-terminal extensions demonstrate their respective roles in virus assembly . The N-terminus is required for assembly of RNA containing particles but not for the assembly of empty virions . The C-terminus is essential for coat protein dimer formation and particle assembly.

Virology, 1995 Mar 10, 207(2), 475 - 85
A side chain at position 48 of the human immunodeficiency virus type-1 protease flap provides an additional specificity determinant; Moody MD et al.; Substitution of glycine with glutamic acid at position 48 of the human immunodeficiency virus protease resulted in an enzyme with reduced activity on one of the protease processing sites in the viral Pol polyprotein precursor . Cleavage at this site was restored by a second-site substitution in the substrate replacing an aspartic acid with either glycine or asparagine . These results suggest that the glutamic acid side chain in the mutant protease has an unfavorable charge-charge interaction with this position in the substrate . Cleavage of a processing site in the viral Gag polyprotein precursor with the mutant enzyme was enhanced, and this enhancement was dependent on the presence of an arginine residue in the substrate, again suggesting a charge-charge interaction . The potential for such interactions was confirmed using molecular modeling . The effect of the position 48 substitution was attributed to a 10-fold increase in Km for the processing site in Pol . These results indicate that the addition of a side chain at position 48 can alter the specificity of the HIV-1 protease to substrate in a sequence specific manner and that compensatory changes can be made in the substrate.

Virology, 1995 Mar 10, 207(2), 345 - 53
Cucumber mosaic virus 3a protein potentiates cell-to-cell trafficking of CMV RNA in tobacco plants; Ding B et al.; Contrary to a previous report, electron microscopic studies on the Fny strain of cucumber mosaic virus (CMV)-infected tobacco tissues revealed that plasmodesmata were not structurally modified during CMV infection, nor were virions ever observed in plasmodesmata connecting infected cells . To further explore the basis of CMV infection, experiments were performed on the CMV 3a ORF . The 3a protein of CMV was expressed in and purified from Escherichia coli . The purified protein was labeled with fluorescein isothiocyanate (FITC) and subsequently microinjected into mesophyll cells of mature leaves of Nicotiana tabacum cv . Turkish Samsun NN . Within a brief period (as little as 1 sec), the microinjected FITC-labeled CMV 3a protein moved into neighboring cells . Co-injection of unlabeled CMV 3a protein with 9.4-kDa fluorescein-conjugated dextran (F-dextran) resulted in extensive cell-to-cell movement (diffusion) of the F-dextran, indicating that the 3a protein can interact with and dilate plasmodesmata . Furthermore, co-injection of unlabeled 3a protein with fluorescently labeled infectious CMV RNA molecules resulted in rapid and extensive cell-to-cell transport . In contrast, a mutant form of the 3a protein was unable to traffic from cell to cell, to increase the size exclusion limit of plasmodesmata, or to potentiate cell-to-cell trafficking of CMV RNA molecules . Microinjection studies performed on transgenic tobacco plants expressing the CMV 3a protein indicated that fluorescently labeled CMV RNA moved out of the target cell into the surrounding mesophyll tissue . In addition, expression of the CMV 3a protein also potentiated the cell-to-cell movement of 9.4-kDa F-dextran . Collectively, these results provide direct experimental evidence that the CMV 3a protein functions as the movement protein of CMV . These findings are consistent with the hypothesis that CMV moves from cell-to-cell in the form of a ribonucleoprotein complex.

J Mol Biol, 1995 Mar 10, 246(5), 618 - 27
Solution dynamics of the trp repressor: a study of amide proton exchange by T1 relaxation; Gryk MR et al.; The amide proton exchange rates of Escherichia coli trp repressor have been measured through their effects on the longitudinal relaxation rates of the amide protons . Three types of exchange regimes have been observed: (1) slow exchange (on a minute/hour time-scale), measurable by isotope exchange, but not by relaxation techniques in the core of the molecule; (2) relatively rapid exchange, with the rates on a T1 relaxation time-scale (seconds) in the DNA-binding region and (3) very fast exchange at the N and C termini . The results have been analyzed in terms of the two-site exchange model originally proposed by Linderstrom-Lang, and of a three-site extension of the model . The values of the intrinsic exchange rates calculated using the two-state model agree with the values expected from the studies of Englander and co-workers for the very fast case of the chain terminals, but disagree with the literature values by two orders of magnitude in the intermediate case found in the DNA-binding region . The implication of these findings is that the "open" state of the two-state model in the DNA-binding region is not completely open and has an intrinsic exchange rate different from that of a random coil peptide . Alternatively, if the literature values of the intrinsic exchange rates are assumed to apply to the open states in all parts of the repressor molecule, two "closed" helical states have to be postulated, in slow exchange with each other, with only one of them in rapid exchange with the open state and hence with the solvent . Kinetically, the two models are indistinguishable.

Mol Gen Genet, 1995 Mar 10, 246(5), 648 - 56
A new phenotype for sbcB mutations in Escherichia coli: RecA-dependent increase in plasmid-borne gene expression; Jayashree P et al.; A new chromosomal mutation (cpeA), that causes increased expression of plasmid-borne genes in Escherichia coli, was identified and mapped to the sbcB locus . The effect of the mutation on plasmid transcription was non-specific with respect to the various promoters that were studied, but was more pronounced for an IncW low-copy-number plasmid than for ColE1- or p15A-based, high-copy-number plasmids . The mutant phenotype was observed even in recB+C+D+ strains, but not in recA mutants . The increased-expression phenotype was also observed in sbcB15 but not in xonA1 (another sbcB allele) mutants, suggesting that the expression of this phenotype is mediated by genes of the so-called RecF pathway family . Consistent with this interpretation was the observation that the cpeA mutant phenotype was less pronounced in recF, recJ and recO mutants . The increased-expression phenotype was also correlated with increased recovery of plasmid DNA from the cpeA/sbcB mutant strains, but there was no evidence for the occurrence of linear plasmid multimers in these strains.

Mol Gen Genet, 1995 Mar 10, 246(5), 605 - 9
A cluster of cell division genes maps to the terC region of the chromosome of Escherichia coli K-12; Ben-Neria T et al.; Thirty-nine cell division mutants were isolated in Escherichia coli K-12 and were mapped in the terminus region of the chromosome, between 33.5 and 36 min . They were obtained by two different approaches involving specific mutagenesis of the terC region . The mutants could be divided into eight classes (I to VIII) based on their map position and phenotype at the restrictive temperature, and constitute a new cell division gene cluster.

J Biol Chem, 1995 Mar 10, 270(10), 5320 - 5
Lipopolysaccharide (LPS) signal transduction and clearance . Dual roles for LPS binding protein and membrane CD14; Gegner JA et al.; Under physiological conditions, lipopolysaccharide (LPS) activation of cells involves the LPS binding protein (LBP) and either membrane or soluble CD14 . We find LPS forms a ternary complex with LBP and membrane CD14 (mCD14) . Subsequent to complex formation and distinct from signal transduction, LBP and LPS internalize . Internalization can be separated from signal transduction with the anti-LBP antibody 18G4 and the anti-CD14 antibody 18E12 . 18G4 inhibits LBP binding to mCD14 without blocking signal transduction or LPS transfer to soluble CD14; 18E12 inhibits signal transduction without affecting LPS binding and uptake . These data show that while LPS signal transduction and LPS clearance utilize both LBP and mCD14, the pathways bifurcate after LPS binding to mCD14.

Nature, 1995 Mar 9, 374(6518), 180 - 3
RNA degradation in Escherichia coli regulated by 3' adenylation and 5' phosphorylation; Xu F et al.; Although polyadenylation has commonly been regarded as a special feature of eukaryotic messenger RNA, there are many reports of polyA tails on bacterial RNA (for example, refs 3-8) . In Escherichia coli, adenylation mediated by the pcnB gene greatly accelerates decay of RNA I, an antisense repressor of replication of ColE1 type plasmids that resembles highly structured transfer RNA but shows the rapid turnover characteristic of mRNA . Here we report that both 3' adenylation and 5' phosphorylation affect the rate of digestion of RNA I by the 3' exonuclease, polynucleotide phosphorylase; conversely, mutation of the polynucleotide phosphorylase-encoding pnp gene affects ribonuclease acting at the 5' end . Together these findings indicate that enzymes attacking RNA I at its separate termini can interact functionally . Additionally, our discovery that adenylation-mediated degradation by polynucleotide phosphorylase imparts an mRNA-like half-like to RNA I suggests a possible mechanism to account for the rapid decay of mRNA in E . coli.

Biochem Biophys Res Commun, 1995 Mar 8, 208(1), 68 - 74
CD 69 antigen of human lymphocytes is a calcium-dependent carbohydrate-binding protein; Bezouska K et al.; CD69 is a signal transducing molecule of hematopoietic cells . Previous molecular cloning of CD69 has revealed a type II transmembrane orientation and the presence of an extracellular domain related to the Ca(2+)-dependent (C-type) animal lectins . As the predicted amino acid sequence for the lectin-like domain is highly divergent from those of other C-type lectin-like proteins - a feature shared with NKR-P1 of natural killer cells - CD69 and NKR-P1 are among proteins assigned to a separate group, group V . To initiate ligand identification studies, we have prepared soluble forms of CD69 protein by bacterial expression of its extracellular portion . We show that cysteine 68 located in the short membrane-proximal neck region of CD69 which adjoins the C-terminal lectin-like domain is a critical element for dimerization . We have evidence that the soluble dimeric CD69 has a tight association with calcium, a feature shared with NKR-P1, and that it is a carbohydrate-binding protein with N-acetyl-D-glucosamine and N-acetyl-D-galactosamine as the best inhibitors: 4-8 x 10(-5) M giving 50% inhibition of binding to N-acetyl-D-glucosamine neoglycoprotein . Thus, the tight association with calcium and high affinities for carbohydrate binding appear to be features of at least two members of the C-type lectin group V.

Biochem Biophys Res Commun, 1995 Mar 8, 208(1), 260 - 6
Functional activity of a biotinylated human neurokinin 1 receptor fusion expressed in the Semliki Forest virus system; Lundstrom K et al.; The 1.3 S biotinylatable subunit of Proprionibacterium shermanii transcarboxylase complex was fused to the C-terminus of the human neurokinin 1 receptor gene and introduced into the Semliki Forest virus expression vector pSFV1 . RNA transcribed from pSFV1-NK1-biot and pSFV-Helper2 was coelectroporated into BHK cells permitting in vivo packaging of recombinant virus . Infection of BHK and CHO cells with SFV-NK1-biot virus yielded high level of the fusion receptor as detected by metabolic labeling, immunoblotting with streptavidin alkaline phosphatase and binding to substance P . Like native receptor, the biotinylated receptor fusion was able to stimulate Ca2+ mobilization in infected CHO cells, indicating functional coupling to guanine-nucleotide-binding proteins.

Biochem Biophys Res Commun, 1995 Mar 8, 208(1), 216 - 22
Effect of dexamethasone on alpha 1-proteinase inhibitor synthesis in human cells of monocytic origin; Cichy J et al.; In this study we provide evidence for downregulation of alpha 1-Proteinase Inhibitor (alpha 1-PI) synthesis in cells of monocytic origin by the glucocorticoid analog, dexamethasone . This factor significantly reduced the basal level of alpha 1-PI expression as well as antagonized the effect of lipopolysaccharide and interleukin-6, stimulators of alpha 1-PI synthesis in cells of monocyte/macrophage lineage . Since increased levels of all of these mediators are observed in both acute infections and inflammatory reactions, their combination may affect the contribution of monocytes to the synthesis of alpha 1-PI.

Biochemistry, 1995 Mar 7, 34(9), 3121 - 32
Combinatorial mutagenesis of the four domains of annexin IV: effects on chromaffin granule binding and aggregating activities; Nelson MR et al.; This study addresses the roles of individual annexin IV domains in calcium-dependent membrane binding and aggregation through an analysis of the activities of mutant annexin IV proteins in which critical residues in one or more domains have been altered . The consensus sequence for high-affinity Ca(2+)-binding pockets obtained from the annexin V crystal structure is GXGT-38 residues-D/E {Huber, R., et al . (1992) J . Mol . Biol . 223, 683-704} . Site-directed mutagenesis was used to change the conserved acidic residues (D/E) in these sequences to alanine residues in each of the four domains of bovine annexin IV, singly or in combinations . Fourteen mutants with one, two, three, or four mutated domains were constructed and expressed in Escherichia coli . Purified recombinant product was evaluated for Ca(2+)-dependent binding to and aggregation of bovine chromaffin granules . Increases in the number of mutated domains resulted in increased Ca2+ requirements for both granule binding and aggregation . Further analysis revealed that mutations in individual domains had preferential effects on the binding or aggregating activities of the protein . For example, mutation of the first or fourth domains had a greater effect on membrane binding than aggregation, while conversely, mutation of the second domain had a more dramatic effect on membrane aggregation . Mutation of the third domain was largely silent in these assays . An additional mutation was made in the fourth domain to substitute a serine for a highly conserved arginine residue (Arg274) present at the C-terminus of the fourth endonexin fold . This mutation increased the calcium requirement for membrane binding 2-fold and for membrane aggregation 3-fold . This mutant protein was found to be an effective inhibitor of membrane aggregation by native annexin IV at intermediate levels of calcium.

Biochemistry, 1995 Mar 7, 34(9), 3056 - 65
Reversible dissociation and unfolding of the Escherichia coli aspartate receptor cytoplasmic fragment; Wu J et al.; The thermal denaturation of a 31-kDa soluble fragment derived from the Escherichia coli aspartate receptor cytoplasmic region (c-fragment) was found to be reversible . Denaturation monitored by differential scanning calorimetry (DSC) and circular dichroism (CD) was typically over 90% reversible in pH 7.0 buffer . The wild-type c-fragment exhibited one transition (Tm = 51 degrees C), which was taken as the main denaturation transition . c-Fragments derived from signaling mutants, shown to form oligomers by gel filtration chromatography (GFC), displayed a second low-temperature transition that correlated with the disappearance of the oligomeric form in the GFC traces over the same temperature range . The CD and DSC experiments also indicated that oligomers were more folded than monomers, observations that may provide an explanation for the structural basis of the smooth-swimming signaling state of the receptor . Octyl glucoside (OG), phospholipid (PL), and glycerol were added to characterize factors that contribute to c-fragment stability . At 10 mg/mL OG, the van't Hoff enthalpy of unfolding was reduced ca . 10-fold, although at room temperature the CD spectrum indicated little change in the secondary structure . The van't Hoff enthalpy was not affected by 35% (w/v) glycerol, but the Tm increased by ca . 18 degrees C . Cooperative transitions were detected in buffer containing OG, PL, and glycerol (10 mg/mL, 2 mg/mL, 35%, respectively) . The correlation between conditions where cooperative transitions are observed, and where aspartate-modulated receptor signaling has been previously observed, provides an explanation for the inhibition of signaling in OG-containing buffers without glycerol and PL.

Biochemistry, 1995 Mar 7, 34(9), 3048 - 55
Large amplitude twisting motions of an interdomain hinge: a disulfide trapping study of the galactose-glucose binding protein; Careaga CL et al.; The galactose--glucose binding protein possesses two structural domains bordering a ligand binding cleft, with three polypeptide strands serving as a flexible hinge connecting the two domains . The hinge is known to bend, enabling the cleft to open by an angle of at least 18 degrees . Here the twisting motions of the hinge were examined by placing pairs of engineered cysteines on the perimeter of the cleft to generate six stable di-cysteine proteins . Each cysteine pair introduced reactive sulfhydryls into both rims of the cleft, one in the N-terminal domain and the other in the C-terminal domain . Collisions between sulfhydryls in different domains were trapped by disulfide formation, yielding sensitive detection of large amplitude domain rotations . When the cleft was occupied by the ligand D-glucose, counterclockwise hinge twist rotations were detected with amplitudes up to 36 degrees, and frequencies ranging from 10(1) to 10(3) collisions s-1 . Removal of ligand from the cleft increased the range of twist angles 3-fold and the frequency of motions up to 10(2)-fold . Thus, in this representative hinged cleft protein, large amplitude hinge twist motions occur on biologically relevant timescales . The functional implications of such motions are discussed.

Biochemistry, 1995 Mar 7, 34(9), 2998 - 3008
Protein folding intermediates with rapidly exchangeable amide protons contain authentic hydrogen-bonded secondary structures; Guijarro JI et al.; Recent studies on protein folding intermediates by pulsed amide proton exchange and by far-ultraviolet circular dichroism have shown important discrepancies between the secondary structure contents estimated by these two methods at early folding stages . To solve these apparent discrepancies, structural studies have been performed on the isolated, 101 residue long, C-terminal proteolytic domain (F2) of the Escherichia coli tryptophan synthase beta chain, which had previously been reported to behave as an early folding intermediate {Chaffotte, A . F., Cadieux, C., Guillou, Y., & Goldberg, M . E . (1992) Biochemistry 31, 4303-4308} . The secondary structure of F2 has been investigated by far-UV circular dichroism (CD), Fourier transform infrared (FTIR) spectroscopy, and NMR . The CD and FTIR spectra clearly indicate that isolated F2 has about 30-45% of its residues involved in secondary structures stabilized by conventional hydrogen bonds . The characteristics of the NMR spectrum (line broadening, absence of structure-induced chemical shifts, absence of nuclear Overhauser effects in the amide region, few dipolar interactions between the side-chain protons) suggest that isolated F2 is oscillating between several conformations in rapid equilibrium . The rate of amide proton exchange has been studied by one-dimensional NMR, which indicates a significant extent of proton protection, with, however, protection factors that can be estimated to be at most 60 and more probably closer to 10 . Thus, F2 appears to exist as a molten globule that exhibits very low amide proton protection and yet contains a large fraction of its residues involved in authentic secondary structures stabilized by hydrogen bonds . Such a state is likely to correspond to the earliest structured folding intermediates thus far characterized.

Biochemistry, 1995 Mar 7, 34(9), 2956 - 64
A single HMG domain in high-mobility group 1 protein binds to DNAs as small as 20 base pairs containing the major cisplatin adduct; Chow CS et al.; Proteins containing a relatively new DNA-binding motif known as the high-mobility group (HMG) domain bind specifically to DNA modified by the anticancer drug cisplatin, but not to unmodified DNA (McA'Nulty & Lippard, 1995) . Southwestern-blot analyses of the binding of proteolytic fragments of HMG1 to a 123-bp globally platinated DNA demonstrate that the HMG domains A and B of HMG1 are responsible for its specific interactions with cisplatin-modified DNA . An 81 amino acid recombinant protein representing a single HMG motif, HMG1 domain B, binds with an affinity (Kd = 10(-7) M) equal to that of HMG1 itself to 92- and 100-bp DNAs containing the major adduct of cisplatin, a cis-{Pt(NH3)2-{d(GpG)-N7(1), -N7(2)}} intrastrand cross-link, at a specific site . The isolated HMG domain B binds with comparable affinity to cisplatin-modified DNAs having as few as 20 bp . The related human mitochondrial HMG domain protein mtTFA also recognizes the 123-bp globally platinated DNA, providing further evidence that HMG domains are responsible for modulating binding of this class of proteins to cisplatin-modified DNA . This work provides direct biochemical evidence in support of conclusions drawn previously from analyses of sequence conservation (Bruhn et al., 1992) that HMG domains are the key elements in protein binding to cisplatin-modified DNA.

Biochemistry, 1995 Mar 7, 34(9), 2946 - 55
DNA binding specificity of the Mu Ner protein; Strzelecka TE et al.; Binding of purified phage Mu Ner protein to a series of DNA fragments was investigated in order to determine the length requirements for tight specific binding . Gel retardation experiments with wild-type 307 base pair (bp) Mu DNA and shorter, synthetic oligonucleotides were performed, and apparent dissociation constants (KappD) were determined from the half-saturation point . While Ner formed four complexes with the 307 bp DNA fragment, only one complex was observed with the shorter DNAs . The 50 and 30 bp fragments had KappD values of 5 and 20 nM, respectively . Ner binding was progressively weaker with decreasing size of the DNA fragments, with no binding observed for 12mers . The shortest DNA fragments which bound well were two 18 bp fragments for which KappD values were in the range of 50-100 nM . The stoichiometry of Ner complexes with the 30 and 18 bp fragments was determined using a modified Ferguson method . Ner was found to form a tetramer on the 30 bp DNA and a dimer on the 18 bp DNA, which makes the latter a good candidate for the study of a Ner-DNA complex by NMR . In order to clarify which DNA regions were important for Ner-DNA binding, hydroxyl radical footprinting was performed for a range of Ner concentrations from 30 to 500 nM . The footprint revealed that Ner contacts the DNA backbone every 12-13 bp, on both strands of the DNA . The order in which protected regions appeared with increasing protein concentration indicated that two Ner monomers bound to DNA simultaneously . A model of Ner binding to DNA is proposed on the basis of these results.

Biochemistry, 1995 Mar 7, 34(9), 2901 - 7
Use of a biosensor with surface plasmon resonance detection for the determination of binding constants: measurement of interleukin-6 binding to the soluble interleukin-6 receptor; Ward LD et al.; The interaction of recombinant human interleukin-6 (IL-6) with the soluble extracellular form of its receptor (sIL-6R) has been characterized by the application of expressions developed for quantitative affinity chromatography to results obtained with a biosensor based on surface plasmon resonance detection . First, the interaction of sIL-6R with IL-6 covalently attached to the biosensor-chip was characterized from the dependence of the surface plasmon resonance response upon the concentration of receptor injected into the biosensor . A binding constant for the interaction between sIL-6R and IL-6 was then determined from the biosensor response observed for mixtures of IL-6 and receptor--a procedure that is shown to provide unequivocal characterization of the competing reaction, irrespective of the model used to describe the biphasic interaction between partitioning receptor and immobilized IL-6 . A binding constant of 5 x 10(7) M-1 has been obtained for the interaction of sIL-6R with two equivalent and independent sites on an essentially dimeric IL-6 preparation produced using the pUC vector system, and also for the interaction of sIL-6R with a monomeric IL-6 preparation that was univalent in its interaction with receptor.

Biochemistry, 1995 Mar 7, 34(9), 2768 - 76
Active site mapping of Escherichia coli D-Ala-D-Ala ligase by structure-based mutagenesis; Shi Y et al.; Eleven Escherichia coli D-Ala-D-Ala ligase (DdlB) mutants, at K144, K215, and E270 in the ATP binding site, at E15, S150, H63, and R255 in the first D-Ala subsite, and at Y216, S281, L282, and D257 in the second D-Ala subsite, were constructed, purified, and examined for steady-state kinetic parameters, kcat and Kms for ATP, and both first (D-Ala1), and second (D-Ala2) D-alanines . Of these, E270Q, K215A, R255A, and D257N retained very low or no detectable activity consistent with X-ray structure based predictions for roles in Mg2+ coordination to beta, gamma-P of ATP (E270), coordination to transferring gamma-PO3 of ATP (K215), and coordination/orientation of nucleophilic COO- of D-Ala1 that attacks gamma-PO3 of ATP (R255, D257) and the side chain of R255, respectively . The substantial retention of activity in the Y216F mutant argues against the possibility that Y216 may be a catalytic base that deprotonates the alpha-NH3+ of D-Ala2 to attack the acyl phosphate form of D-Ala1 . While all seven mutants that retain activities have a 300-2000 fold elevation in Km for D-Ala1 (1-2 microM in wild type), the S281A mutant has a 500-fold elevation in Km for D-Ala2, consistent with a proposed interaction with the COO- of D-Ala2 . Similarly, the kinetics of inhibition by a slow-binding phosphinate inhibitor in the presence of ATP are most altered in the S281A mutant.

Biochemistry, 1995 Mar 7, 34(9), 2731 - 8
The energetics and dynamics of molecular recognition by calmodulin; Ehrhardt MR et al.; Amide hydrogen exchange has been used to examine the structural dynamics and energetics of the interaction of a peptide corresponding to the calmodulin binding domain of smooth muscle myosin light chain kinase with calcium-saturated calmodulin . Heteronuclear NMR 15N-1H correlation techniques were used to quantitate amide proton exchange rates of both 15N-labeled and unlabeled amide protons of the smMLCK peptide complexed to calmodulin . Hydrogen exchange slowing factors were determined for 18 of the 19 amide hydrogens and found to span 6 orders of magnitude . The first six residues of the bound peptide were found to have slowing factors near 1 and are considered not to be hydrogen bonded, consistent with the previously reported model for the structure of the peptide . The pattern of hydrogen exchange of hydrogen-bonded amide hydrogens is indicative of end-fraying behavior characteristic of helix-coil transitions . The effective statistical mechanical parameters revealed by the end fraying are consistent with exchange from a highly solvated state . However, the slowing factors of the first hydrogen-bonded amide hydrogens are large, indicating the requirement for a reorganization of the calmodulin-peptide complex before the helix-coil transitions leading to exchange can occur . Taken together, these observations suggest that the collapsed complex reorganizes with an associated free energy change of 5.5 kcal/mol to a more open state where the helical peptide is highly solvated and undergoes helix-coil transitions leading to exchange . The free energy difference between the most and least stable intrahelical amide hydrogen bonds of the bound peptide is estimated to be approximately 2.5 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)

Regul Pept, 1995 Mar 7, 56(1), 71 - 88
Recombinant human chromogranin A: expression, purification and characterization of the N-terminal derived peptides; Taupenot L et al.; Chromogranin A (CGA) is an ubiquitous 48 kDa secretory protein stored and released from most endocrine cells and is present in nanomolar concentration in the human vascular system . Recent data suggest that CGA may be the precursor of several peptides with a defined biological activity . The present report describes the expression of human CGA in Escherichia coli using the pET3a vector system, the purification and characterization of the recombinant protein and the production of antibody against the expressed protein . The expressed CGA was purified by a multi-step protocol including heat treatment, gel filtration and high performance-anion exchange chromatography and two-dimensional gel electrophoresis . Two major forms of recombinant human CGA (rhCGA) were purified from the bacterial cytosol: a 70 kDa form which corresponded to the native full-length CGA and a major proteolytic 63 kDa product recognized by antibodies raised against the 70 kDa rhCGA or to synthetic peptides localized in the N-terminal part of the bovine CGA sequence . This E . coli expression system provides a method for producing a suitable protein which will permit the identification of CGA-derived peptides with defined biological function in human . Fragments containing the N-terminal domain were generated by acidic cleavage of the two forms of rhCGA . A two-step purification using high-performance reverse-phase chromatography yielded 6 peptide bands ranging in apparent molecular mass from 7 to 18 kDa . Four components (molecular mass range 12-18 kDa) were immunostained with antibodies directed against synthetic sequences of bovine vasostatin II (bCGA1-113) while the two others (molecular mass range 7-8 kDa) were immunostained only with antibodies directed against vasostatin I (bCGA1-76) . From protein staining the ratio vasostatins II/I was 10:1 . The vasoinhibitory activity of this preparation was examined on isolated human saphenous vein segments . An inhibitory effect was obtained in paired vessel segments from 7 patients undergoing surgery for coronary artery bypass, however with low potency for supression of the endothelin-1 evoked sustained tension in these vessels.

Biochemistry, 1995 Mar 7, 34(9), 2978 - 84
Permutation of a pair of tertiary nucleotides in a transfer RNA; Hou YM et al.; The tertiary nucleotides at positions 15 and 48 in a tRNA establish non-Watson-Crick hydrogen interactions that connect the dihydrouridine (D) loop with the variable loop and stabilize the "L"-shaped tRNA structure . Although the majority of tRNAs have G15.C48 or A15.U48, all of the 16 possible nucleotide pairs at positions 15 and 48 can be found in the existing cytoplasmic and mitochondrial tRNA sequences . Because tRNAs contain a variety of slightly different sets of tertiary nucleotides, this complexity raises the question of whether a given tRNA sequence framework can accommodate all of the 16 compositions at positions 15 and 48 . In this work, G15 and C48 in an Escherichia coli alanine amber suppressor tRNA were permuted, and variants were tested for biological activity in vivo . All but an A15.A48 variant were functional, indicating substantial flexibility at positions 15 and 48 to accommodate nucleotide variations . Analysis of the A15.A48 variant with chemical probes showed that this mutant harbors a defect that specifically changes the conformation of the anticodon sequence . Interestingly, human tRNA(Ala) has A15.A48 . Additional nucleotide substitutions in E . coli A15.A48 tRNA(Ala) that recreate the D loop sequence of human tRNA(Ala) restored the biological activity to this tRNA by reestablishing the wild-type conformation of the anticodon sequence . The results suggest a distal relationship between the D and the anticodon loops in a tRNA and delineate covariation of specific nucleotides in the evolution of tRNA(Ala) from E . coli to human.

FEBS Lett, 1995 Mar 6, 360(3), 255 - 60
Lipid specificity for membrane mediated partial unfolding of cytochrome c; de Jongh HH et al.; In this study we investigated the lipid specificity for destabilization of the native structure of horse heart cytochrome c by model membranes . From (i) the enhanced release of deuterium from deuterium-labelled cytochrome c and (ii) the increased proteolytic digestion of the protein in the presence of anionic lipids, it was concluded that these lipids are able to destabilize the native structure of cytochrome c . Changes in the absorbance at 695 nm indicated that the destabilization was accompanied by a diminished ligation of Met-80 to the heme . Beef heart cardiolipin was found to be more effective than DOPS, DOPG or DOPA, while no protein destabilization was observed in the presence of the zwitterionic lipid DOPC or, surprisingly, in the presence of E . coli cardiolipin . Experiments with mitoplasts showed that the protein can also be destabilized in its native structure by a biological membrane.

Life Sci, 1995 Mar 3, 56(15), 1243 - 9
Short-term exposure to lipopolysaccharide is associated with microvascular contractile dysfunction in vivo; Gao XP et al.; The purpose of this study was to determine whether short-term exposure of resistance arterioles to lipopolysaccharide in situ is associated with changes in vasomotor tone . Using intravital microscopy, we found that suffusion of Escherichia coli lipopolysaccharide (3 micrograms/ml) over hamster cheek pouch arterioles for 1 h was associated with a significant immediate biphasic response: vasoconstriction followed by vasodilation (p < 0.05) . The former was attenuated by indomethacin, and the latter by SK&F 108566, a selective, non-peptide angiotension II receptor antagonist (p < 0.05) . The nitric oxide synthase inhibitor, NG-L-nitro arginine, had no significant effects on lipopolysaccharide-induced responses . Allopurinol, a scavenger of reactive oxygen species, significantly attenuated lipopolysaccharide-induced vasodilation . Acetylcholine- and nitroglycerin-induced vasodilation were significantly potentiated after lipopolysaccharide . These responses were recorded in the absence of any significant changes in systemic arterial blood pressure . Collectively, these data suggest that short-term exposure of the peripheral microcirculation to lipopolysaccharide in situ is associated with an ischemia-reperfusion-like injury . These changes may contribute to end organ failure observed several hours after exposure to lipopolysaccharide.

J Biol Chem, 1995 Mar 3, 270(9), 4890 - 5
Structure and function of transcription-repair coupling factor . II . Catalytic properties; Selby CP et al.; The transcription repair coupling factor (TRCF) of Escherichia coli has the so-called helicase motifs, is a DNA-, RNA Pol-, and UvrA-binding protein, and is required for the coupling of repair to transcription . We investigated the potential helicase, transcription termination, and transcription-repair coupling activities of TRCF on various substrates . We found that TRCF does not have a helicase activity on any of the substrates tested . However, the TRCF releases both RNA Pol and the truncated transcript from a transcriptional road block caused by a lesion, a "missing base," or a DNA-bound protein . It does not have any effect on rho-dependent or rho-independent transcriptional termination . However, some premature terminations were induced by TRCF at other sites . The coupling of transcription to repair occurs with supercoiled and relaxed circular DNA and with linear DNA . However, the coupling with linear DNA is strongly affected by the length of the DNA and does not occur with fragments in which the lesion is closer than 90 nucleotides to the 5' terminus of the template strand . Under transcription conditions the repair of lesions in the promoter region and up to the eleventh transcribed base is inhibited even in the presence of TRCF . Stimulation of repair in the transcribed strand starts at lesions at +15 nucleotides . Stimulation of repair occurs via facilitating the delivery of the A2B1 complex to the lesion site by the TCRF and can be inhibited by excess UvrA which binds to the TRCF off DNA . In vitro, strand-specific repair is not dependent on the MutL and MutS proteins which have recently been implicated in preferential repair in vivo.

J Biol Chem, 1995 Mar 3, 270(9), 4882 - 9
Structure and function of transcription-repair coupling factor . I . Structural domains and binding properties; Selby CP et al.; The 130-kDa mfd gene product is required for coupling transcription to repair in Escherichia coli . Mfd displaces E . coli RNA polymerase (Pol) stalled at a lesion, binds to the damage recognition protein UvrA, and increases the template strand repair rate during transcription . Here, the interactions of Mfd (transcription-repair coupling factor, TRCF) with DNA, RNA Pol, and UvrA were investigated . TRCF bound nonspecifically to double stranded DNA; binding to DNA produced alternating DNase I-protected and -hypersensitive regions, suggesting possible wrapping of the DNA around the enzyme . Weaker binding to single stranded DNA and no binding to single stranded RNA were observed . DNA binding required ATP, and hydrolysis of ATP promoted dissociation . Removal of a stalled RNA Pol also requires ATP hydrolysis . Apparently, TRCF recognizes a stalled elongation complex by directly interacting with RNA Pol, since binding to a synthetic transcription bubble was no stronger than binding to double stranded DNA, and binding to free RNA Pol holoenzyme and to initiation and elongation complexes in the absence of adenosine 5'-O-(thiotriphosphate) were observed . Structure-function analysis showed that residues 379-571 are involved in binding to a stalled RNAP . The helicase motifs region, residues 571-931, binds to ATP and duplex polynucleotide (DNA:DNA or DNA:RNA) . Dissociation of the ternary complex upon hydrolysis of ATP also requires the carboxyl terminus of TRCF . Finally, residues 1-378 bind to UvrA and deliver the damage recognition component of the excision nuclease to the lesion.

J Biol Chem, 1995 Mar 3, 270(9), 4870 - 4
Recombinant human insulin receptor substrate-1 protein . Tyrosine phosphorylation and in vitro binding of insulin receptor kinase; Siemeister G et al.; Insulin receptor substrate-1 (IRS-1) is a major endogenous substrate of the insulin receptor . To study the interaction of the insulin receptor with IRS-1 in vitro, we expressed in Escherichia coli the amino acids 516-777 of human IRS-1 (hIRS-p30) covering five potential tyrosine phosphorylation sites within YXXM motifs . Kinetic data for tyrosine phosphorylation of hIRS-p30 by partially purified insulin receptor and insulin-like growth factor I receptor and by baculovirus-expressed insulin receptor kinase domain were determined . Native insulin receptor demonstrated the highest affinity to hIRS-p30 (Km = 6.8 +/- 0.6 microM), followed by the insulin-like growth factor I receptor (Km = 9.9 +/- 1.0 microM) . We used the soluble recombinant insulin receptor kinase domain, which phosphorylated hIRS-p30 with high affinity (Km = 11.9 +/- 0.8 microM), and affinity columns prepared by coupling hIRS-p30 to NHS-activated Sepharose for binding assays . The insulin receptor kinase domain phosphorylated the hIRS-p30 on the column, was bound by the immobilized hIRS-p30, and was eluted with high salt buffer . Autophosphorylated and EDTA-inactivated insulin receptor kinase domain was bound only by immobilized hIRS-p30 protein that has been prephosphorylated . Our results indicate that the recombinant hIRS-p30 protein is a high affinity substrate for insulin receptor and insulin-like growth factor I receptor in vitro . Moreover, we show that only tyrosine-phosphorylated hIRS-p30 is able to bind to the insulin receptor.

J Biol Chem, 1995 Mar 3, 270(9), 4822 - 39
Novel proteins of the phosphotransferase system encoded within the rpoN operon of Escherichia coli . Enzyme IIANtr affects growth on organic nitrogen and the conditional lethality of an erats mutant; Powell BS et al.; Two rpoN-linked delta Tn10-kan insertions suppress the conditionally lethal erats allele . One truncates rpoN while the second disrupts another gene (ptsN) in the rpoN operon and does not affect classical nitrogen regulation . Neither alter expression of era indicating that suppression is post-translational . Plasmid clones of ptsN prevent suppression by either disruption mutation indicating that this gene is important for lethality caused by erats . rpoN and six neighboring genes were sequenced and compared with sequences in the database . Two of these genes encode proteins homologous to Enzyme IIAFru and HPr of the phosphoenolpyruvate:sugar phosphotransferase system . We designate these proteins IIANtr (ptsN) and NPr (npr) . Purified IIANtr and NPr exchange phosphate appropriately with Enzyme I, HPr, and Enzyme IIA proteins of the phosphoenolpyruvate: sugar phosphotransferase system . Several sugars and tricarboxylic acid cycle intermediates inhibited growth of the ptsN disruption mutant on medium containing an amino acid or nucleoside base as a combined source of nitrogen, carbon, and energy . This growth inhibition was relieved by supplying the ptsN gene or ammonium salts but was not aleviated by altering levels of exogenously supplied cAMP . These results support our previous proposal of a novel mechanism linking carbon and nitrogen assimilation and relates IIANtr to the unknown process regulated by the essential GTPase Era.

J Biol Chem, 1995 Mar 3, 270(9), 4784 - 91
Studies on the structure and function of glycosylated and nonglycosylated neu differentiation factors . Similarities and differences of the alpha and beta isoforms; Lu HS et al.; Comparative analyses of both glycosylated and nonglycosylated neu differentiation factor (NDF) isoforms revealed significant similarities and differences of their overall structures and functions . Biophysical analyses confirmed that all NDF isoforms are monomeric, but have an extended ellipsoidal shape in solution . All full-length NDFs are similar in secondary and tertiary structures and they contain no alpha-helix but are abundant in beta-strand structures . A small NDF fragment containing only the epidermal growth factor domain is also rich in beta-strand structures, but exhibits tertiary structure different from the long NDF forms . Monoclonal antibodies that selectively recognize epidermal growth factor domains of human NDF-alpha and -beta can specifically bind the respective NDF-alpha and -beta isoforms independent of NDF origins . Western blot analysis and quantitative binding assays further identify that an NDF preparation produced naturally from Rat1-EJ cells contains both alpha and beta isoforms in a 3 to 2 ratio . In receptor-binding competition experiments, human and rat NDF-beta isoforms have higher affinity than NDF-alpha isoforms . NDF-beta isoforms can dramatically enhance the stimulation of DNA synthesis for transfected NIH3T3 cells that overexpress HER-3 and HER-4 receptors, while NDF-alpha isoforms can only stimulate proliferation of HER-4-transfected cells with lower activity . Taken together, NDF-alpha and -beta isoforms share similar gross protein conformations but are biologically distinct.

J Biol Chem, 1995 Mar 3, 270(9), 4753 - 8
The effect of internal autocleavage on kinetic properties of the human cytomegalovirus protease catalytic domain; O'Boyle DR 2nd et al.; The 28-kilodalton (kDa) catalytic domain of the human cytomegalovirus (HCMV) protease undergoes autoproteolytic cleavage at an internal site (I site), yielding amino-terminal 15-kDa (N15) and carboxyl-terminal 13-kDa (C13) fragments . I site autocleavage has been postulated to inactivate the protease and provide a mechanism for the negative regulation of enzyme activity during viral infection . We purified recombinant enzymes to demonstrate I site autocleavage in vitro and used site-directed mutagenesis of the I site to stabilize the protease . No difference in the kinetic properties of wild type and stabilized mutant proteases were observed in an in vitro peptide cleavage assay . The consequences of I site cleavage on enzyme activity were investigated two ways . First, autodigestion of the wild type enzyme converted the intact protease to N15 and C13 autocleavage products without a corresponding loss in enzyme activity . Second, genetic constructs encoding the N15 and C13 autocleavage products were generated and expressed separately in Escherichia coli, and each fragment was purified . An active enzyme was reconstituted by refolding a mixture of the purified fragments in vitro to form a noncovalent complex . The kinetic properties of this complex were very similar to the wild type and stabilized enzymes under optimal reaction conditions . We concluded from these studies that I site cleavage does not inactivate the HCMV protease, in the absence of other virally induced factors, and that limited potential exists for the regulation of catalytic activity by I site cleavage.

J Biol Chem, 1995 Mar 3, 270(9), 4312 - 7
Cloning and expression of four novel isoforms of human interleukin-1 beta converting enzyme with different apoptotic activities; Alnemri ES et al.; To understand the mechanism of interleukin-1 beta converting enzyme (ICE) activation in apoptosis, we analyzed the expression of ICE mRNA in two human cell lines by reverse transcription-polymerase chain reaction technique . This resulted in the identification and cloning of four alternatively spliced ICE mRNA isoforms . Although all the alternative splicing events were within the coding sequence of ICE, the four ICE isoforms maintained open reading frames and were designated as ICE beta, gamma, delta, and epsilon . In ICE gamma, most of the propeptide (amino acids 20-112) is deleted, which suggests that it may function as a catalyst for ICE autoprocessing in vivo . In ICE delta, amino acids 288-335, which contain the cleavage sites between the p20 and p10 subunits of ICE, are deleted thus resulting in its inactivation . Intriguingly, in ICE epsilon amino acids 20-335, which encompass most of the propeptide and the p20 subunit, are deleted resulting in the formation of a molecule that is homologous to the p10 subunit . Examination of the ability of these four ICE isoforms to cause apoptosis revealed that only the parental ICE alpha and isoforms beta and gamma, but not isoforms delta and epsilon, can induce apoptosis when overexpressed in Sf9 insect cells . In addition, coexpression of the p20 and p10 but not the p20 and ICE epsilon in Sf9 cells results in apoptosis . Interestingly, expression of ICE epsilon and to a lesser degree ICE delta resulted in extension of the survival of baculovirus-infected cells in a manner similar to expression of BCL2 . The ability of ICE epsilon to extend the survival of Sf9 cells suggests that baculovirus-induced apoptosis in these cells is mediated by an ICE-like protease . We show that ICE epsilon can bind to the p20 subunit of ICE and potentially may compete with the p10 subunit to form an inactive ICE complex . Therefore, by acting as a dominant inhibitor of ICE activity, ICE epsilon may regulate ICE activation in vivo.

J Biol Chem, 1995 Mar 3, 270(9), 4216 - 9
Defective export of a periplasmic enzyme disrupts regulation of fatty acid synthesis; Cho H et al.; Escherichia coli thioesterase I (TesA) encoded by the tesA gene is located in the cellular periplasm . The tesA gene was modified by deletion of the leader sequence such that the mature enzyme was instead localized to the cellular cytosol . Production of thioesterase I in the cytosol results in striking changes in the pattern of E . coli lipid synthesis . In contrast to normal E . coli cells, cells producing cytosolic TesA synthesize large amounts of free fatty acid at all stages of growth . Moreover, cultures of the cytosolic TesA-producing strain continue lipid synthesis (as free fatty acid) in stationary phase whereas lipid synthesis is normally strongly inhibited in such cultures . Surprisingly, production of cytosolic thioesterase I gave only modest inhibition of membrane phospholipid synthesis . These results demonstrate that internalization of a normally secreted enzyme can disrupt normal cellular regulatory mechanisms.

Masui, 1995 Mar 3, 44(3), 342 - 8
{The function of red blood cells during experimental hemorrhagic and endotoxic shock}; Matsumoto Y; The changes of P50, 2, 3-DPG, intracellular ATP, and erythrocyte deformability as function of red blood cells during hemorrhagic and endotoxic shock were studied in 12 anesthetized mongrel dogs . Hypotension due to hemorrhagic shock was induced by removing 40 ml.kg-1 of blood in 30 minutes . The function of red blood cells remained normal during hemorrhagic shock, because P50 was unchanged and 2, 3-DPG decreased slightly, whereas intracellular ATP increased . Endotoxin shock was induced using a 30-minute continuous infusion of Escherichia coli 055 endotoxin 5 mg.kg-1 . Levels of 2, 3-DPG, and intracellular ATP decreased significantly, and P50 tended to decrease . These data suggest that the disturbance in the function of red blood cells may be causing dysfunction of oxygen metabolism in tissues in these dogs.

J Biol Chem, 1995 Mar 3, 270(9), 4255 - 61
Importance of residues 2-9 in the immunoreactivity, subunit interactions, and activity of the beta 2 subunit of Escherichia coli tryptophan synthase; Navon A et al.; The epitope recognized by a monoclonal antibody (mAb19) directed against the beta 2 subunit of Escherichia coli tryptophan synthase was found to be carried by residues 2-9 of the beta chain . The affinities of mAb19 for peptides of different lengths containing the 2-9 sequence were close to 0.6 x 10(9) M-1, the affinity of mAb19 for native beta 2 . In view of these results, a model is proposed to account for the kinetics of appearance of the epitope during in vitro renaturation of beta 2 (Murry-Brelier, A., and Goldberg, M.E . (1988) Biochemistry 27, 7633-7640) . A mutant producing beta chains lacking residues 1-9 (beta delta 1-9) was prepared . The beta delta 1-9 protein was able to fold into a heat stable homodimer resembling wild type beta 2 . Isolated beta delta 1-9 had no detectable enzymatic activity . It could bind alpha chains extremely weakly and be slightly activated . In the presence of the 1-9 peptide, the beta delta 1-9 protein could bind alpha chains much more strongly and generate a 50% active enzyme . Thus, although having little role in the overall folding and stability of the protein, the 1-9 sequence of the beta chain appears strongly involved in the alpha-beta interactions and in the enzymatic activity.

Nature, 1995 Mar 2, 374(6517), 88 - 91
A highly conserved ATPase protein as a mediator between acidic activation domains and the TATA-binding protein; Swaffield JC et al.; Biochemical and genetic studies suggest the existence of mediators that work between the activation domains (ADs) of regulatory proteins and the basic transcriptional machinery . We have previously shown genetically that Sug1 interacts with the AD of the yeast activator Ga14 . Here we provide evidence that the Sug1 protein of yeast binds directly to the ADs of Ga14 and the viral activator, VP16 . Sug1 protein is associated with the TATA-binding protein in vivo and binds to it in vitro, consistent with a mediator function . We also demonstrate that Sug1 is not a component of the 26S proteasome, contrary to previous reports . Sug1 is a member of a large, highly conserved family of ATPases, implying a role for ATP hydrolysis in the activation of transcription.

Zentralbl Veterinarmed A, 1995 Mar, 42(1), 1 - 8
Endotoxin stimulates secretion of cortisol, but not ACTH in heifers pretreated with suppressive doses of a glucocorticoid; Bosu WT et al.; The ability of triamcinolone acetonide (TA) to suppress actions of lipopolysaccharide (LPS) in the pituitary-adrenal axis was examined in Holstein heifers . The study was carried out using repeated measure/split plot factorial design involving four heifers which were repeatedly used in four experimental groups thus yielding eight experimental units (EU) . Each EU received two treatments, the first at zero hour and the second at 28 h . Heifers in Groups I (n = 2) and II (n = 2) were given sterile saline as treatment (TRT) 1 . For TRT 2, group I was given sterile saline, and group II received Escherichia coli lipopolysaccharide (LPS) . In group III (n = 2) and group IV (n = 2) triamcinolone acetonide was given as TRT 1 . Sterile saline (SAL) was TRT 2 for GP III, and group IV heifers received LPS . Administration of LPS elicited increases in concentrations of the stress hormones ACTH (P < 0.05) and cortisol (P < 0.001) in SAL-pretreated heifers . However, in TA-pretreated animals, the endotoxin could only cause increase (P < 0.001) in concentrations of cortisol, but not ACTH . Therefore, the significant response of cortisol to LPS stimulation suggest that, at doses used in this study, triamcinolone acetonide did not suppress LPS-triggered cortisol secretion from adrenal zonae fasciculata and reticularis cells . The LPS-induced ACTH response appears to have been blunted by prior administration of triamcinolone acetonide.

Mol Med, 1995 Mar, 1(3), 325 - 32
Immunogenicity and in vivo efficacy of recombinant Plasmodium falciparum merozoite surface protein-1 in Aotus monkeys; Kumar S et al.; BACKGROUND: The carboxy-terminus of the merozoite surface protein-1 (MSP1) of Plasmodium falciparum has been implicated as a target of protective immunity . MATERIALS AND METHODS: Two recombinant proteins from the carboxy-terminus of MSP1, the 42 kD fused to GST (bMSP1(42)) and the 19 kD (yMSP1(19)), were expressed in Escherichia coli and secreted from Saccharomyces cerevisiae, respectively . To determine if vaccination with these recombinant proteins induces protective immunity, we conducted a randomized, blinded vaccine trial in two species of Aotus monkeys, A . nancymai and A . vociferans . After three injections using Freund's adjuvant, the monkeys were challenged with the virulent Vietnam Oak Knoll (FVO) strain of P . falciparum . RESULTS: All three control monkeys required treatment by Day 19 . Two of three monkeys vaccinated with bMSP1(42) required treatment by Day 17, whereas the third monkey controlled parasitemia for 28 days before requiring treatment . In contrast, both of the A . nancymai vaccinated with yMSP1(19) self-resolved an otherwise lethal infection . One of the two yMSP1(19)-vaccinated A . vociferans had a prolonged prepatent period of > 28 days before requiring treatment . No evidence of mutations were evident in the parasites recovered after the prolonged prepatent period . Sera from the two A . nancymai that self-cured had no detectable effect on in vitro invasion . CONCLUSIONS: Vaccination of A . nancymai with yMSP1(19) induced protective immune responses . The course of recrudescing parasitemias in protected monkeys suggested that immunity is not mediated by antibodies that block invasion . Our data indicate that vaccine trials with the highly adapted FVO strain of P . falciparum can be tested in A . nancymai and that MSP1(19) is a promising anti-blood-stage vaccine for human trials.

J Gen Virol, 1995 Mar, 76 ( Pt 3), 723 - 6
Repair of phage lambda DNA damaged by near ultraviolet light plus 8-methoxypsoralen; Dye K et al.; Treatment of phage lambda with 8-methoxypsoralen plus near ultraviolet light (PUVA) and its subsequent infection and growth on various mutant and non-mutant hosts were investigated . A number of Escherichia coli DNA repair-deficient mutants, particularly those deficient in genes producing proteins known to participate in interstrand crosslink repair, were used as hosts to assess the roles of these gene products in the activation of phage affected by PUVA . Results show that puvA, uvrA, uvrD, recA, recO, sulA and recN of E . coli are involved in the repair process . Based on the data presented it is proposed that phage lambda DNA is repaired, following PUVA damage, using the recombinational repair process . This may be in agreement with the recombinational model of the repair of E . coli DNA.

J Gen Virol, 1995 Mar, 76 ( Pt 3), 541 - 50
Identification and characterization of the protein product of gene 67 in equine herpesvirus type 1 strain Ab4; Sun Y et al.; Equine herpesvirus type 1 (EHV-1) strain Ab4 gene 67 has no counterpart in any herpesvirus sequenced to date . To identify and characterize the product of EHV-1 gene 67, we have expressed the putative amino acids 11 to 260 encoded by gene 67 as a beta-galactosidase fusion protein in Escherichia coli . The expressed fusion protein has been used to generate an antiserum raised against the gene 67 product . Immunoblotting and immunoprecipitation experiments have revealed that the anti-67 serum specifically recognizes a polypeptide with an M(r) of 36,000 (the 36K polypeptide) in infected cell extracts . The gene 67 protein is regulated as an early polypeptide in EHV-1 strain Ab4 infected cells and post-translational modification experiments have revealed that the protein is phosphorylated, but not glycosylated . The gene 67 protein has been transiently expressed in BHK-21/C13 cells using plasmid pCMV67, which contains the putative gene 67 ORF under the control of the cytomegalovirus immediate early promoter . Immunoblotting experiments with anti-67 have shown that the 36K protein is expressed at high levels in transfected cells . From both immunofluorescence and cellular fractionation experiments it is concluded that the gene 67 protein is associated with intracellular membranes and produces novel ribbon or filament-like structures within the cytoplasm of infected cells . We have demonstrated that the gene 67 product is a component of the virion nucleocapsid/tegument.

EMBO J, 1995 Mar 1, 14(5), 995 - 1003
Mitotic destruction of the cell cycle regulated NIMA protein kinase of Aspergillus nidulans is required for mitotic exit; Pu RT et al.; NIMA is a cell cycle regulated protein kinase required, in addition to p34cdc2/cyclin B, for initiation of mitosis in Aspergillus nidulans . Like cyclin B, NIMA accumulates when cells are arrested in G2 and is degraded as cells traverse mitosis . However, it is stable in cells arrested in mitosis . NIMA, and related kinases, have an N-terminal kinase domain and a C-terminal extension . Deletion of the C-terminus does not completely inactivate NIMA kinase activity but does prevent functional complementation of a temperature sensitive mutation of nimA, showing it to be essential for function . Partial C-terminal deletion of NIMA generates a highly toxic kinase although the kinase domain alone is not toxic . Transient induction experiments demonstrate that the partially truncated NIMA is far more stable than the full length NIMA protein which likely accounts for its toxicity . Unlike full length NIMA, the truncated NIMA is not degraded during mitosis and this affects normal mitotic progression . Cells arrested in mitosis with non-degradable NIMA are able to destroy cyclin B, demonstrating that the arrest is not due to stabilization of p34cdc2/cyclin B activity . The data establish that NIMA degradation during mitosis is required for correct mitotic progression in A . nidulans.

EMBO J, 1995 Mar 1, 14(5), 884 - 93
The allele-specific synthetic lethality of prlA-prlG double mutants predicts interactive domains of SecY and SecE; Flower AM et al.; The secretion of proteins from the cytoplasm of Escherichia coli requires the interaction of two integral inner membrane components, SecY and SecE . We have devised a genetic approach to probe the molecular nature of the SecY-SecE interaction . Suppressor alleles of secY and secE, termed prlA and prlG, respectively, were analyzed in pair-wise combinations for synthetic phenotypes . From a total of 115 combinations, we found only seven pairs of alleles that exhibit a synthetic defect when present in combination with one another . The phenotypes observed are not the result of additive defects caused by the prl alleles, nor are they the consequence of multiple suppressors functioning within the same strain . In all cases, the synthetic defect is recessive to wild-type secY or secE provided in trans . The recessive nature argues for a defective interaction between the Prl suppressors . The extreme allele specificity and topological coincidence of the mutations represented by these seven pairs of alleles identify domains of interaction between SecY/PrlA and SecE/PrlG.

EMBO J, 1995 Mar 1, 14(5), 866 - 75
The translocation of negatively charged residues across the membrane is driven by the electrochemical potential: evidence for an electrophoresis-like membrane transfer mechanism; Cao G et al.; The role of the membrane electrochemical potential in the translocation of acidic and basic residues across the membrane was investigated with the M13 procoat protein, which has a short periplasmic loop, and leader peptidase, which has an extended periplasmically located N-terminal tail . For both proteins we find that the membrane potential promotes membrane transfer only when negatively charged residues are present within the translocated domain . When these residues are substituted by uncharged amino acids, the proteins insert into the membrane independently of the potential . In contrast, when a positively charged residue is present within the N-terminal tail of leader peptidase, the potential impedes translocation of the tail domain . However, an impediment was not observed in the case of the procoat protein, where positively charged residues in the central loop are translocated even in the presence of the membrane potential . Intriguingly, several of the negatively charged procoat proteins required the SecA and SecY proteins for optimal translocation . The studies reported here provide insights into the role of the potential in membrane protein assembly and suggest that electrophoresis can play an important role in controlling membrane topology.

EMBO J, 1995 Mar 1, 14(5), 1032 - 42
rpoE, the gene encoding the second heat-shock sigma factor, sigma E, in Escherichia coli; Rouviere PE et al.; In Escherichia coli, the heat shock response is under the control of two alternative sigma factors: sigma 32 and sigma E . The sigma 32-regulated response is well understood, whereas little is known about that of sigma E, except that it responds to extracytoplasmic immature outer membrane proteins . To further understand this response, we located the rpoE gene at 55.5' and analyzed the role of sigma E . sigma E is required at high temperature, and controls the transcription of at least 10 genes . Some of these might contribute to the integrity of the cell since delta rpoE cells are more sensitive to SDS plus EDTA and crystal violet . sigma E controls its own transcription from a sigma E-dependent promoter, indicating that rpoE transcription plays a role in the regulation of E sigma E activity . Indeed, under steady-state conditions, the transcription from this promoter mirrors the levels of E sigma E activity in the cell . However, it is unlikely that the rapid increase in E sigma E activity following induction can be accounted for solely by increased transcription of rpoE . Based upon homology arguments, we suggest that a gene encoding a negative regulator of sigma E activity is located immediately downstream of rpoE and may function as the target of the E sigma E inducing signal.

Biochem J, 1995 Mar 1, 306 ( Pt 2), 589 - 97
Expression of recombinant human phenylalanine hydroxylase as fusion protein in Escherichia coli circumvents proteolytic degradation by host cell proteases . Isolation and characterization of the wild-type enzyme; Martinez A et al.; Recombinant human phenylalanine hydroxylase (hPAH) was produced in high yields in Escherichia coli using the pET and pMAL expression vectors . In the pMAL system, hPAH was fused through the target sequences of the restriction protease factor Xa (IEGR) or enterokinase (D4K) to the C-terminal end of the highly expressed E . coli maltose-binding protein (MBP) . The recombinant hPAH, recovered in soluble forms, revealed a high specific activity even in crude extracts and was detected as a homogeneous band by Western-blot analysis using affinity-purified polyclonal rabbit anti-(rat PAH) antibodies . The enzyme expressed in the pET system was subject to limited proteolysis by host cell proteases and was difficult to purify with a satisfactory yield . By contrast, when expressed as a fusion protein in the pMAL system, hPAH was resistant to cleavage by host cell proteases and was conveniently purified by affinity chromatography on an amylose resin . Catalytically active tetramer-dimer (in equilibrium) forms of the fusion protein were separated from inactive, aggregated forms by size-exclusion h.p.l.c . After cleavage by restriction protease, factor Xa or enterokinase, hPAH was separated from uncleaved fusion protein, MBP and restriction proteases by hydroxylapatite or ion-exchange (DEAE) chromatography . The yield of highly purified hPAH was approx . 10 mg/l of culture . The specific activity of the isolated recombinant enzyme was high (i.e . 1440 nmol of tyrosine.min-1.mg-1 with tetrahydrobiopterin as the cofactor) and its catalytic and physicochemical properties are essentially the same as those reported for the enzyme isolated from human liver . The recombinant enzyme, both as a fusion protein and as purified full-length hPAH, was phosphorylated in vitro by the catalytic subunit of cyclic AMP-dependent protein kinase . The phosphorylated from of hPAH electrophoretically displayed an apparently higher molecular mass (approximately 51 kDa) than the non-phosphorylated (approximately 50 kDa) form.

Biochem J, 1995 Mar 1, 306 ( Pt 2), 437 - 43
Human alpha-tocopherol transfer protein: cDNA cloning, expression and chromosomal localization; Arita M et al.; alpha-Tocopherol transfer protein (alpha TTP), which specifically binds this vitamin and enhances its transfer between separate membranes, was previously isolated from rat liver cytosol . In the current study we demonstrated the presence of alpha TTP in human liver by isolating its cDNA from a human liver cDNA library . The cDNA for human alpha TTP predicts 278 amino acids with a calculated molecular mass of 31,749, and the sequence exhibits 94% similarity with rat alpha TTP at the amino acid level . The recombinant human alpha TTP expressed in Escherichia coli exhibits both alpha-tocopherol transfer activity in an in vitro assay and cross-reactivity to the anti-(rat alpha TTP) monoclonal antibody . Northern blot analysis revealed that human alpha TTP is expressed in the liver like rat alpha TTP . The human and rat alpha TTPs show structural similarity with other apparently unrelated lipid-binding/transfer proteins, i.e . retinaldehyde-binding protein present in retina, and yeast SEC14 protein, which possesses phosphatidylinositol/phosphatidylcholine transfer activity . Both Southern-blot hybridization of human-hamster somatic cell hybrid lines and fluorescence in situ hybridization revealed a single alpha TTP gene corresponding to the 8q13.1-13.3 region of chromosome 8, which is identical to the locus of a recently described clinical disorder, ataxia with selective vitamin E deficiency (AVED) . The relationship between alpha TTP and AVED will be discussed.

Mutat Res, 1995 Mar, 336(2), 153 - 9
Mutations of a shuttle vector plasmid, pZ189, in Escherichia coli induced by boron neutron captured beam (BNCB) containing alpha-particles; Nakano T et al.; A shuttle vector, pZ189, carrying a bacterial suppressor tRNA marker gene (supF) was dissolved in Tris-EDTA buffer containing 0.3 M 10B-enriched boric acid and then irradiated with boron neutron captured beam (BNCB) produced by the nuclear reaction 10B (n,alpha) 7Li with thermal neutrons . A DNA repair-deficient mutant, KS46 (uvrA-), of Escherichia coli was transformed with the plasmid DNA, and the transformants carrying mutations on the supF gene were selected as nalidixic acid-resistant colonies . The mutation frequency (2.4 x 10(-4)) of pZ189 at the D10 dose was about 70 times greater than the spontaneous rate (3.5 x 10(-6)) . The plasmid mutations were analyzed using DNA sequencers; 88% of them were base substitutions . A few minus-one frameshifts (7%) and deletions (5%) were detected . Among these base substitutions, transversions of G:C to T:A (42%) and G:C to C:G (29%) predominated . Twenty-seven percent of the base substitutions were G:C to A:T transitions; no A:T to G:C transitions were detected.

J Med Microbiol, 1995 Mar, 42(3), 196 - 9
The chemotactic response of blood neutrophils and monocytes to strains of Escherichia coli with different virulence characteristics; Beeken W et al.; Chemotactic responses of blood neutrophils and monocytes to media conditioned by eight strains of Escherichia coli with different virulence characteristics were measured in modified Boyden assay chambers to determine if these characteristics were associated with differences in leucocyte mobility . Responding neutrophils and monocytes were prepared on conventional density gradients, and in three instances, the chemotaxis of eosinophils isolated on metrizamide gradients was also studied . Media conditioned by enteroinvasive and nonenteroinvasive E . coli strains were tested as chemo-attractants and compared to the formylated peptide standard attractant . Chemotactic activity of neutrophils was greater than that of monocytes and eosinophils, and migration by all populations was significantly greater to conditioned media than to the control medium . Chemotactic responses to media conditioned by non-enteroinvasive E . coli and strains lacking virulence factors was greater than to media conditioned by plasmid- and Sereny-positive enteroinvasive organisms . The results suggest that virulence factors of E . coli that determine invasiveness did not augment the chemotactic responses of the leucocyte populations tested in vitro, and give no support to the hypothesis that they induce mucosal inflammation by directly increasing chemotaxis in vivo.

J Clin Invest, 1995 Mar, 95(3), 1009 - 17
Modulation of neutrophil influx in glomerulonephritis in the rat with anti-macrophage inflammatory protein-2 (MIP-2) antibody; Feng L et al.; The role of the chemokine, macrophage inflammatory protein-2 (MIP-2), during anti-glomerular basement membrane (GBM) antibody (Ab) glomerulonephritis (GN) was studied . Rat MIP-2 cDNA had been cloned previously . Recombinant rat MIP-2 (rMIP-2) from Escherichia coli exhibited neutrophil chemotactic activity and produced neutrophil influx when injected into the rat bladder wall . By using a riboprobe derived from the cDNA and an anti-rMIP-2 polyclonal Ab, MIP-2 was found to be induced in glomeruli with anti-GBM Ab GN as mRNA by 30 min and protein by 4 h, with both disappearing by 24 h . The expression of MIP-2 correlated with glomerular neutrophil influx . A single dose of the anti-MIP-2 Ab 30 min before anti-GBM Ab was effective in reducing neutrophil influx (40% at 4 h, P < 0.01) and periodic acid-Schiff deposits containing fibrin (54% at 24 h, P < 0.01) . The anti-rMIP-2 Ab had no effect on anti-GBM Ab binding (paired-label isotope study) . Functional improvement in the glomerular damage was evidenced by a reduction of abnormal proteinuria (P < 0.05) . These results suggest that MIP-2 is a major neutrophil chemoattractant contributing to influx of neutrophils in Ab-induced glomerular inflammation in the rat.

J Bacteriol, 1995 Mar, 177(6), 1627 - 9
A reassessment of the relationship between aroK- and aroL-encoded shikimate kinase enzymes of Escherichia coli; Whipp MJ et al.; In the course of sequencing the aroK gene, a number of errors were found in the published sequence . The corrected sequence alters the length of the aroK coding region such that the AroK and AroL proteins are now of comparable length and the homology between them extends the entire length of the two enzymes.

J Bacteriol, 1995 Mar, 177(6), 1620 - 3
A mutant phosphoenolpyruvate carboxykinase in Escherichia coli conferring oxaloacetate decarboxylase activity; Hou SY et al.; The phosphoenolpyruvate carboxykinase in Escherichia coli (encoded by pck) catalyzes the conversion from oxaloacetate (OAA) to phosphoenolpyruvate under gluconeogenic conditions . We report here the characterization of two mutant alleles, pck-51 and pck-53, both of which are point mutations leading to single amino acid changes (D to N at position 268 and G to S at position 284, respectively) . Pck51 is an altered-activity mutant that catalyzes the conversion from OAA to pyruvate (OAA decarboxylase activity) . This new activity was not detected from the wild-type Pck, and it complements the pck null mutation only in a pps+ background . Pck53 is a reduced-activity mutant that complements the pck null mutation in a strain-dependent fashion.

J Bacteriol, 1995 Mar, 177(6), 1585 - 8
Widespread protein sequence similarities: origins of Escherichia coli genes; Labedan B et al.; To learn more about the evolutionary origins of Escherichia coli genes, we surveyed systematically for extended sequence similarities among the 1,264 amino acid sequences encoded by chromosomal genes of E . coli K-12 in SwissProt release 26 by using the FASTA program and imposing the following criteria: (i) alignment of segments at least 100 amino acids long and (ii) at least 20% amino acid identity . Altogether, 624 extended alignments meeting the two criteria were identified, corresponding to 577 protein sequences (45.6% of the 1,264 E . coli protein sequences) that had an extended alignment with at least one other E . coli protein sequence . To exclude alignments of questionable biological significance, we imposed a high threshold on the number of gaps allowed in each of the 624 extended alignments, giving us a subset of 464 proteins . The population of 464 alignments has the following characteristics expressed as median values of the group: 254 amino acids in the alignment, representing 86% of the length of the protein, 33% of the amino acids in the alignment being identical, and 1.1 gaps introduced per 100 amino acids of alignment . Where functions are known, nearly all pairs consist of functionally related proteins . This implies that the sequence similarity we detected has biological meaning and did not arise by chance . That a major fraction of E . coli proteins form extended alignments strongly suggests the predominance of duplication and divergence of ancestral genes in the evolution of E . coli genes . The range of degrees of similarity shows that some genes originated more recently than others . There is no evidence of genome doubling in the past, since map distances between genes of sequence-related proteins show no coherent pattern of favored separations.

J Bacteriol, 1995 Mar, 177(6), 1497 - 504
Gratuitous overexpression of genes in Escherichia coli leads to growth inhibition and ribosome destruction; Dong H et al.; We attempted to test the idea that the relative abundance of each individual tRNA isoacceptor in Escherichia coli can be altered by varying its cognate codon concentration . In order to change the overall codon composition of the messenger pool, we have expressed in E . coli lacZ with the aid of T7 RNA polymerase so that their respective gene products individually accounted for 30% of the total bacterial protein . Unexpectedly, the maximum expression of either test gene has no specific effect on the relative rates of synthesis of the tRNA species that we studied . Instead, we find that there is a cumulative breakdown of rRNAs, which results in a loss of ribosomes and protein synthetic capacity . After either of the test genes is maximally induced, there is a growing fraction of protein synthesis invested in beta-galactosidase or delta tufB that is matched by a comparable decrease of the fraction of normal protein synthesis . We have also observed enhanced accumulation of two heat shock proteins during overexpression . Finally, after several hours of overexpression of either test protein, the bacteria are no longer viable . These results are relevant to the practical problems of obtaining high expression levels for cloned proteins.

J Bacteriol, 1995 Mar, 177(6), 1477 - 84
The Escherichia coli G-fimbrial lectin protein participates both in fimbrial biogenesis and in recognition of the receptor N-acetyl-D-glucosamine; Saarela S et al.; The gafD gene encoding the N-acetyl-D-glucosamine-specific fimbrial lectin (adhesin) protein GafD of uropathogenic Escherichia coli was cloned and subjected to genetic analysis . The corresponding gene product was isolated as a MalE fusion protein . The lectin gene was identified with the aid of deletion mutagenesis; mutations in gafD impaired either receptor binding or both receptor binding and fimbria production, depending on the mutation created . All mutants converted to wild-type expressors when complemented in trans with the cloned intact gafD gene . The predicted 354-amino-acid sequence of GafD, deduced from the nucleotide sequence, is closely related to those of the fimbria-associated F17-G and F17b-G proteins coded for by enterotoxigenic and invasive E . coli strains . Isolated GafD was shown to recognize N-acetyl-D-glucosamine by virtue of specific binding to an immobilized receptor, thus proving directly that GafD is a sugar-binding protein . Our results indicate that GafD as such is sufficient for receptor recognition and that the protein also participates in fimbrial biogenesis.

Clin Exp Immunol, 1995 Mar, 99(3), 376 - 83
Tumour cell binding by a human monoclonal IgM antibody from the spleen of a non-tumour-associated patient is due to somatic mutations in the VH gene; Bohn J et al.; Recently we described the occurrence of B cells producing polyspecific natural IgM with anti-tumour specificity in the spleen of non-tumour-bearing individuals as well as in fetal organisms . Immunoprecipitation and 2-D electrophoresis showed the binding of such antibodies to a 55-kD (pI 6.0) membrane surface glycoprotein . In vitro cultivation of human cancer cell lines in the presence of the purified IgM antibodies resulted in growth inhibition and complement-mediated cell lysis . Furthermore, the antibodies were shown to be able to induce MHC class I molecule expression on tumour cells . Because of this, a role for naturally occurring antibodies with anti-tumour specificity in preventing neoplasias had been suggested . We have constructed and expressed in Escherichia coli single-chain fragments (scFv: VH-linker-VL) derived from a polyspecific human monoclonal IgM autoantibody produced by a human x mouse heterohybridoma which was obtained from the spleen of an autoimmune patient . The mutated complementarity determining region (CDR) gene segments were replaced by the equivalent germ-line sequences and the CDR3 region was swapped for that from another polyspecific human natural antibody with no binding to tumours . Using these four scFv constructs for binding analyses and in vitro cultivation experiments we found: (i) scFv containing the mutated VH region of the original antibody were able to bind to tumour cells, to induce MHC class I molecule expression, and to inhibit tumour growth in a way similar to what had been described for the complete antibody; (ii) replacement of the mutated by the germ-line VH gene independently of the CDR3 to which it had been recombined, resulted in failure to bind to tumour cells . Nevertheless, other antigens (ssDNA, tetanus toxin) were still recognized, although with lower affinity . We discuss the significance of the replacement mutations in the VH gene CDRs, selected probably by B cell contact to an (auto)antigen, for generating a tumour binding capacity, not encoded by the germ-line gene.

Am J Respir Crit Care Med, 1995 Mar, 151(3 Pt 1), 854 - 66
SP-A deficiency in primate model of bronchopulmonary dysplasia with infection . In situ mRNA and immunostains; Coalson JJ et al.; The surfactant protein secretory cells in airway and alveolar epithelium were studied in premature baboons with bronchopulmonary dysplasia and superimposed infection . PRN animals were delivered by hysterotomy at 140 d gestational age and ventilated on clinically appropriate oxygen for a 16-d experimental period . To assess 0 time and sacrifice time gestational parameters, 140 and 156 d were studied . BPD animals were delivered at 140 d and ventilated with positive-pressure ventilation and an FIO2 of 1.0 for 11 d followed by 5 d of oxygen sufficient to maintain PAO2 at 40 to 50 mm Hg . BPD-infected animals were comparably ventilated and treated like the BPD group except that 10(8) E . coli organisms were endotracheally instilled on Day 11 . In situ hybridization studies for mRNA expression of SP-A, SP-B, and SP-C revealed that an SP-A mRNA deficiency, present at 140 d, persisted in the BPD and BPD-infected groups, whereas SP-A mRNA was abundant in PRN and 156 d gestation control groups . SP-B and SP-C mRNA expression in the two hyperoxically injured groups was particularly extensive in cells around peribronchiolar and perivasicular sites . Immunostaining with SP-A, SP-B, and SP-C antibodies showed variable staining patterns . The study clearly demonstrates that a deficiency of SP-A mRNA expression persists in chronic lung injury and that variable protein staining patterns are manifested depending upon the underlying pathology.

Am J Respir Crit Care Med, 1995 Mar, 151(3 Pt 1), 780 - 4
The early pattern of conjugated dienes in liver and lung after endotoxin exposure; Clements NC Jr et al.; If lipid peroxidation (LP) contributes to organ dysfunction in sepsis rather than simply reflecting established injury, it should occur soon after the onset of the septic insult, and it may not progress uniformly in all organs . We assessed whether LP occurs within 90 min after onset of continuous intravenous endotoxin (E . coli 055:B5) infusion in rats, using second-derivative spectroscopy to semiquantitatively assess conjugated dienes (CD) in lung, liver, and plasma phospholipids . Measurements were also made after 90-min infusions with saline or 1 mM H2O2 . Both the quantity and spectrophotometric patterns of CD differed between the three groups . Compared with saline controls, lung lipid CD increased after both H2O2 and endotoxin . Venous plasma CD were elevated only after H2O2, while arterial plasma and liver lipid CD were not different between the three groups . Exhaled ethane (an indicator of peroxidation of omega-3 fatty acids) did not differ between groups . Wet-to-dry lung weights were significantly increased after endotoxin compared with that after saline controls . Our results indicate that tissue-specific LP occurs within 90 min of endotoxin or H2O2 intravenous infusion.

J Nutr, 1995 Mar, 125(3), 520 - 30
Tissue protein turnover is altered during catch-up growth following Escherichia coli infection in weanling rats; Samuels SE et al.; Infection in young growing animals is manifested by poor tissue protein accretion; during subsequent catch-up growth this is reversed . To account for these changes, protein synthesis and degradation were measured in vivo in skeletal muscle, skin, liver and small intestine in weanling rats during catch-up growth after Escherichia coli infection . Observations were made at d 4, 6, 8, 11 and 14, when infected rats had elevated nitrogen balance . Liver protein mass and turnover were not affected by treatment . Although protein mass of small intestine fell during infection, catch-up was achieved before d 4, suggesting a high priority for protein repletion in this tissue . On d 4, protein mass was lower (P < 0.05) in muscle (-19%) and skin (-23%) in infected vs . control rats . Thereafter growth rates of skeletal muscle and skin were higher (P < 0.001) in infected rats compared with controls . Catch-up growth was most pronounced early, but continued throughout the study . During catch-up growth, protein synthesis (mg/d) in muscle and skin was not different between control and infected animals . Protein synthesis was maintained in muscle because RNA mass was maintained . During catch-up growth in muscle and skin of infected rats, there was lower protein degradation (mg/d) than in controls (P < 0.05) . We conclude that alterations in protein turnover during catch-up growth are tissue and time dependent and are different from those described in other hyperanabolic states.

J Cell Biol, 1995 Mar, 128(5), 761 - 8
Chromokinesin: a DNA-binding, kinesin-like nuclear protein; Wang SZ et al.; Microtubule-associated mechanoenzymes have been proposed to play a fundamental role in chromosome movement . We have cloned and characterized the cDNA for a novel protein, named Chromokinesin, that fulfills several of the criteria expected of a mitotic motor . Chromokinesin contains both a kinesin motor-like domain and an unusual basic-leucine zipper DNA-binding domain . Its mRNA is readily detectable in proliferating cells, but not in postmitotic cells . Immunocytochemical analysis with antibodies directed against the nonconserved COOH-terminal region of Chromokinesin indicates that the protein is localized in the nucleus, and primarily associated with chromosome arms in mitotic cells . These data suggest that Chromokinesin is likely to function as a microtubule-based mitotic motor with DNA as its cargo.

Hepatology, 1995 Mar, 21(3), 855 - 62
Acute endotoxin tolerance downregulates superoxide anion release by the perfused liver and isolated hepatic nonparenchymal cells; Bautista AP et al.; This work is based on the hypothesis that low-dose lipopolysaccharide (LPS) suppresses the stimulatory and priming effects of a subsequent high-dose endotoxin on the formation of toxic oxygen-derived radicals by the perfused liver and isolated hepatic nonparenchymal cells . Such effects may in turn contribute to hyposensitivity to the lethal effect of large doses of endotoxin . Male Sprague-Dawley rats received a nonlethal ("low-dose") intravenous injection of Escherichia coli LPS (0.5 mg/kg body weight) 12 to 120 hours before they were challenged by a "large dose" of endotoxin (10 mg/kg) . Three hours after LPS challenge, the livers were perfused, and superoxide release was determined . Nonparenchymal cells were also isolated for the determination of superoxide anion formation in vitro . There was a low rate (0.14 +/- 0.1 nmol/min/g liver weight) of superoxide generated by the perfused livers from rats that received the low-dose LPS 1 to 5 days previously . Control livers generated less than 0.08 nmol superoxide . A high rate (1.3 +/- 0.1 nmol/min/g) of superoxide release was measured in the perfused liver 4 hours after treatment of previously untreated control rats with large-dose LPS . This was attenuated to 0.7 +/- 0.04 nmol/min/g by an injection of low-dose LPS before challenge . This attenuation was time dependent; it failed to manifest at 12, 24, or 120 hours after low-dose LPS . Isolated endothelial cells, Kupffer cells, and sequestered hepatic neutrophils from rats given a high-dose LPS also generated significant amounts of superoxide both in the presence or absence of agonists, i.e., phorbol myristate acetate or opsonized zymosan.(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatology, 1995 Mar, 21(3), 752 - 9
Ex vivo hepatic gene transfer in mouse using a defective herpes simplex virus-1 vector; Lu B et al.; A defective amplicon herpes simplex virus-1 (HSV-1) vector, HSVlac, was used to transfer an E . coli lacZ reporter gene into primary hepatocytes . The lacZ gene was driven by the HSV immediate early (IE) 4/5 promoter . Use of the HSVlac vector resulted in highly efficient gene transfer . Because difficulties in culturing primary hepatocytes impose limitations in ex vivo gene therapy, we sought to determine whether use of the HSVlac vector could simplify gene transfer . Therefore, we incubated HSVlac with primary hepatocytes in suspension and found that the lacZ gene was still transferred with great rapidity and efficiency . To examine lacZ expression in transduced hepatocytes in vivo, we used a mouse hepatocyte transplantation system . In congeneic recipients of primary hepatocytes transduced with HSVlac in suspension, the lacZ gene was expressed in liver and spleen up to 2 weeks . However, survival of transplanted hepatocytes, as well as persistence of HSVlac genome in recipient organs, was demonstrated for up to an 11-week duration of the experiment . These findings suggest that in vivo regulation of the HSV IE4/5 promoter was responsible for the short-term expression of lacZ, which should be overcome by the use of liver-specific promoters . Therefore, our results indicate the feasibility of hepatic gene transfer with a defective HSV-1 vector.

Radiat Res, 1995 Mar, 141(3), 244 - 51
Ultraviolet (193, 216 and 254 nm) photoinactivation of Escherichia coli strains with different repair deficiencies; Gurzadyan GG et al.; Escherichia coli K12 bacteria strains AB1157 (repair-deficient wild-type, uvrA+ recA+), AB1886 (urvA-), AB2463 (recA-) and AB2480 (urvA- recA-) were exposed to 254 nm germicidal UV and 216 or 193 nm laser radiation . The mean lethal incident dose (D37) for AB1157 does not change significantly with wavelength, whereas it increases for the other strains on going from lambda irr = 254 to 193 nm, e.g . eightfold for AB2480 . Quantum yields for inactivation, due to light absorbed by the chromosomal DNA, have been estimated from the D37 values . The large differences in D37 between uvrA+ and uvrA- strains indicate a significant contribution of pyrimidine dimers and (6-4) photoproducts to photodamage of DNA in the cells . The formation of dimers even with lambda irr = 193 nm is supported by the result that the photoreactivable sector is larger than 63% . Inactivation of E . coli upon irradiation at 193 and 216 nm is therefore due to damage to intracellular DNA rather than to membrane or protein damage as is the case for mammalian cells . The ratio of the lethal doses for AB1157 vs AB2480 is approximately 900 with lambda irr = 254 nm, but only 160 with lambda irr = 193 nm . We propose that this is due to the better repair of photodimers and (6-4) photoproducts than of damage induced by photoionization via upper excited states . Irradiation at 193 nm inactivates AB1157 mainly by damage due to photoionization and AB2480 by damage due to photodimers in the chromosomal DNA.

Mutat Res, 1995 Mar, 327(1-2), 67 - 73
Interlaboratory comparison: liver spontaneous mutant frequency from lambda/lacI transgenic mice (Big Blue) (II); Young RR et al.; Spontaneous mutant frequency in livers of two transgenic mouse strains, each carrying identical lambda shuttle vectors with a lacI target gene, was evaluated by two laboratories . These studies investigated variability in spontaneous mutant frequency between animals and as a function of the number of phage screened . Liver DNA was independently isolated from 7-11 week old C57BL/6 and B6C3F1 Big Blue transgenic mice . At least 500,000 phage were screened for mutation at lacI for each animal using standardized assay procedures . In the two labs, the C57BL/6 liver spontaneous mutant frequency was 45 +/- 9 x 10(-6) and 41 +/- 7 x 10(-6) . The B6C3F1 liver spontaneous mutant frequency was 42 +/- 10 x 10(-6) at one lab and 43 +/- 12 x 10(-6) and 41 +/- 8 x 10(-6) in two trials at the second lab . Mean mutant frequency data from both labs, calculated in increments of 100,000 plaque forming units (pfu) scored for each mouse strain, show stabilized mean mutant frequency and standard deviation after approximately 200,000-300,000 pfu screened . The frequency of spontaneous lacI mutants was reproducible both within and between labs and was comparable between the two transgenic mouse strains.

Mutat Res, 1995 Mar, 327(1-2), 57 - 66
Intralaboratory optimization and standardization of mutant screening conditions used for a lambda/lacI transgenic mouse mutagenesis assay (I); Rogers BJ et al.; A lambda/lacI shuttle vector transgenic mouse mutagenesis assay has been optimized and standardized for reproducible mutant detection . The mutagenic endpoints are blue lacI- phage plaques on a bacterial lawn resulting from the de-repression of beta-galactosidase activity acting on the chromogenic substrate X-gal . Non-mutant lacI phage plaques remain colorless . Factors demonstrated to affect mutant detection include X-gal concentration per assay tray, plaque density per assay tray, pH of plating agar, incubation time at 37 degrees C and the use of a red translucent screening filter over a light source to enhance mutant plaque visibility . In vivo mutant frequencies for liver in untreated animals using standard protocols and internal controls were repeatable in separate experiments using lambda/lacI B6C3F1 mice (4.3 +/- 1.2 x 10(-5) and 4.1 +/- 0.8 x 10(-5)) . These studies analyze the use of internal controls to monitor the level of mutant phage plaque detection in a given experiment and evaluate the repeatability of observed mutant frequencies obtained when using standardized procedures.

Mutat Res, 1995 Mar, 327(1-2), 201 - 8
Transgenic lambda/lacI mutagenicity assay: statistical determination of sample size; Callahan JD et al.; Statistical analysis of the lambda/lacI transgenic mutagenicity assay was used to determine optimal sample size and resource allocation in terms of number of animals and number of recovered target genes (recovered phage) required to demonstrate a statistically significant induction in mutant frequency . Statistical assumptions as applied to mutagenicity data are discussed for a number of frequently used statistical analyses . Log transformations are suggested as a means of meeting statistical assumptions and examples are given on interpreting results of analyses of log transformed data . The data analyzed in this study indicate that 300,000 lambda plaques from each of five animals should be analyzed per treatment group in order to detect a doubling of mutant frequencies . Additional sensitivity is gained primarily through increase of animal number and not the number of phage rescued, due to inherent animal-to-animal variability.

J Immunol, 1995 Mar 1, 154(5), 2198 - 208
Domain interactions and antigen binding of recombinant anti-Z-DNA antibody variable domains . The role of heavy and light chains measured by surface plasmon resonance; Polymenis M et al.; The heavy (H) and light (L) chain V domains of anti-Z-DNA mouse mAb Z22 were expressed separately in bacteria . When mixed in vitro, the V domains associated stoichiometrically to reconstitute the Ag binding site of Z22, as judged by specific reactivity with Z-DNA and anti-Z22 ld Abs . The apparent Kd of the Z22 VH-VL association was 5.47 x 10(-8) M, measured by surface plasmon resonance . A replacement at VL position 96, which reduced Ag binding affinity of a single chain Fv (clone LZ1-2) by two orders of magnitude, did not reduce the affinity of interaction between the VH and VL domains (apparent Kd = 1.93 x 10(-8) M for VH association with LZ1-2) . Fab prepared from native Z22 bound specifically to a 30-bp Z-DNA oligonucleotide with an apparent Kd = 1.56 x 10(-8) M . The VH domain alone bound Z-DNA specifically with an affinity similar to that of the Fab or Fv's of Z22 (Kd = 1.68 x 10(-8) M), whereas Z22 VL domain alone did not interact with nucleic acids . Z22 VH binding to Ag was inhibited by association with the mutant LZ1-2 VL . These results indicate that the Z22 H chain makes important contributions to specific binding of Z-DNA . Although the L chain does not add greatly to the binding energy, an appropriate L chain is required to permit Ag binding in the Fv domain . These in vitro results resemble the in vivo modulation of H chain autoreactivity that occurs with L chain substitution in receptor editing.

J Bacteriol, 1995 Mar, 177(5), 1393 - 8
Reshuffling of Rhs components to create a new element; Zhao S et al.; RhsF has been identified as the fourth member of the RhsABCF subfamily of genetic elements . This new element is found in Escherichia coli ECOR-50 and several other strains but not in strain K-12 . A novel feature of RhsF is that it represents a new arrangement of components previously uniquely associated with RhsA and RhsC of strain K-12.

J Bacteriol, 1995 Mar, 177(5), 1388 - 92
Escherichia coli NusG protein stimulates transcription elongation rates in vivo and in vitro; Burova E et al.; The rate of transcription elongation in Escherichia coli was reduced when cells were depleted of NusG . In a purified in vitro system, NusG accelerated the transcription elongation rate . The stimulation of the rate of transcription elongation by NusG appears to result from the suppression of specific transcription pause sites.

J Bacteriol, 1995 Mar, 177(5), 1374 - 9
Heat shock-dependent transcriptional activation of the metA gene of Escherichia coli; Biran D et al.; In Escherichia coli, the growth rate at elevated temperatures is controlled by the availability of endogenous methionine, which is limited because of the temperature sensitivity of the metA gene product, homoserine transsuccinylase (HTS) . In order to determine the relationship between this control mechanism and the heat shock response, we estimated the cellular levels of HTS during heat shock by Western (immunoblot) analysis and found an increase following induction by temperature shift and by addition of ethanol or cadmium ions . The elevated level of HTS was a result of transcriptional activation of the metA gene . This activation was heat shock dependent, as it did not take place in rpoH mutants, and probably specific to the metA gene, as another gene of the methionine regulon (metE) was not activated . These results suggest a metabolic link between the two systems that control the response of E . coli to elevated temperatures: the metA gene, which codes for the enzyme responsible for regulating cell growth as a function of temperature elevation (HTS), is transcriptionally activated by the heat shock response.

J Bacteriol, 1995 Mar, 177(5), 1326 - 35
DNA helicases in recombination and repair: construction of a delta uvrD delta helD delta recQ mutant deficient in recombination and repair; Mendonca VM et al.; DNA helicases play pivotal roles in homologous recombination and recombinational DNA repair . They are involved in both the generation of recombinogenic single-stranded DNA ends and branch migration of synapsed Holliday junctions . Escherichia coli helicases II (uvrD), IV (helD), and RecQ (recQ) have all been implicated in the presynaptic stage of recombination in the RecF pathway . To probe for functional redundancy among these helicases, mutant strains containing single, double, and triple deletions in the helD, uvrD, and recQ genes were constructed and examined for conjugational recombination efficiency and DNA repair proficiency . We were unable to construct a strain harboring a delta recQ delta uvrD double deletion in a recBC sbcB(C) background (RecF pathway), suggesting that a delta recQ deletion mutation was lethal to the cell in a recBC sbcB(C) delta D background . However, we were able to construct a triple delta recQ delta uvrD Delta helD mutant in the recBC sbcB(C) background . This may be due to the increased mutator frequency in delta uvrD mutants which may have resulted in the fortuitous accumulation of a suppressor mutation(s) . The triple helicase mutant recBC sbcB(C) delta uvrD delta recQ delta helD severely deficient in Hfr-mediated conjugational recombination and in the repair of methylmethane sulfonate-induced DNA damage . This suggests that the presence of at least one helicase--helicase II, RecQ helicase, or helicase IV--is essential for homologous recombination and recombinational DNA repair in a recBC sbcB(C) background . The triple helicase mutant was recombination and repair proficient in a rec+ background . Genetic analysis of the various double mutants unmasked additional functional redundancies with regard to conjugational recombination and DNA repair, suggesting that mechanisms of recombination depend both on the DNA substrates and on the genotype of the cell.

J Bacteriol, 1995 Mar, 177(5), 1292 - 8
Formyltetrahydrofolate hydrolase, a regulatory enzyme that functions to balance pools of tetrahydrofolate and one-carbon tetrahydrofolate adducts in Escherichia coli; Nagy PL et al.; The enzyme encoded by Escherichia coli purU has been overproduced, purified, and characterized . The enzyme catalyzes the hydrolysis of 10-formyltetrahydrofolate (formyl-FH4) to FH4 and formate . Formyl-FH4 hydrolase thus generates the formate that is used by purT-encoded 5'-phosphoribosylglycinamide transformylase for step three of de novo purine nucleotide synthesis . Formyl-FH4 hydrolase, a hexamer with 32-kDa subunits, is activated by methionine and inhibited by glycine . Heterotropic cooperativity is observed for activation by methionine in the presence of glycine and for inhibition by glycine in the presence of methionine . These results, along with previous mutant analyses, lead to the conclusion formyl-FH4 hydrolase is a regulatory enzyme whose main function is to balance the pools of FH4 and C1-FH4 in response to changing growth conditions . The enzyme uses methionine and glycine to sense the pools of C1-FH4 and FH4, respectively.

J Bacteriol, 1995 Mar, 177(5), 1275 - 84
Mutational analysis of the redox-sensitive transcriptional regulator OxyR: regions important for oxidation and transcriptional activation; Kullik I et al.; OxyR is a redox-sensitive transcriptional regulator of the LysR family which activates the expression of genes important for the defense against hydrogen peroxide in Escherichia coli and Samonella typhimurium . OxyR is sensitive to oxidation and reduction, and only oxidized OxyR is able to activate transcription of its target genes . Using site-directed mutagenesis, we found that one cysteine residue (C-199) is critical for the redox sensitivity of OxyR, and a C-199-->S mutation appears to lock the OxyR protein in the reduced form . We also used a random mutagenesis approach to isolate eight constitutively active mutants . All of the mutations are located in the C-terminal half of the protein, and four of the mutations map near the critical C-199 residue . In vivo as well as in vitro transcription experiments showed that the constitutive mutant proteins were able to activate transcription under both oxidizing and reducing conditions, and DNase I footprints showed that this activation is due to the ability of the mutant proteins to induce cooperative binding of RNA polymerase . Unexpectedly, RNA polymerase was also found to reciprocally affect OxyR binding.

J Bacteriol, 1995 Mar, 177(5), 1268 - 74
Alteration of lysine 178 in the hinge region of the Escherichia coli ada protein interferes with activation of ada, but not alkA, transcription; Saget BM et al.; The ada gene of Escherichia coli K-12 encodes the 39-kDa Ada protein, which consists of two domains joined by a hinge region that is sensitive to proteolytic cleavage in vitro . The amino-terminal domain has a DNA methyltransferase activity that repairs the S-diastereoisomer of methylphosphotriesters while the carboxyl-terminal domain has a DNA methyltransferase activity that repairs O6-methylguanine and O4-methylthymine lesions . Transfer of a methyl group to Cys-69 by repair of a methylphosphotriester lesion converts Ada into a transcriptional activator of the ada and alkA genes . Activation of ada, but not alkA, requires elements contained within the carboxyl-terminal domain of Ada . In addition, physiologically relevant concentrations of the unmethylated form of Ada specifically inhibit methylated Ada-promoted ada transcription both in vitro and in vivo and it has been suggested that this phenomenon plays a pivotal role in the down-regulation of the adaptive response . A set of site-directed mutations were generated within the hinge region, changing the lysine residue at position 178 to leucine, valine, glycine, tyrosine, arginine, cysteine, proline, and serine . All eight mutant proteins have deficiencies in their ability to activate ada transcription in the presence or absence of a methylating agent but are proficient in alkA activation . AdaK178P (lysine 178 changed to proline) is completely defective for the transcriptional activation function of ada while it is completely proficient for transcriptional activation of alkA . In addition, AdaK178P possesses both classes of DNA repair activities both in vitro and in vivo . Transcriptional activation of ada does not occur if both the amino- and carboxyl-terminal domains are produced separately within the same cell . The mutation at position 178 might interfere with activation of ada transcription by changing a critical contact with RNA polymerase, by causing a conformational change of Ada, or by interfering with the communication of conformational information between the amino- and the carboxyl-terminal domains . These results indicate that the hinge region of Ada is important for ada but not alkA transcription and further support the notion that the mechanism(s) by which Ada activates ada transcription differs from that by which it activates transcription at alkA.

J Bacteriol, 1995 Mar, 177(5), 1247 - 53
bor gene of phage lambda, involved in serum resistance, encodes a widely conserved outer membrane lipoprotein; Barondess JJ et al.; bor is one of two recently identified genes of phage lambda which are expressed during lysogeny and whose products display homology to bacterial virulence proteins . bor is closely related to the iss locus of plasmid CoIV,I-K94, which promotes bacterial resistance to serum complement killing in vitro and virulence in animals . bor has a similar in vitro effect . We show here that the bor gene product is a lipoprotein located in the Escherichia coli outer membrane . We also find that antigenically related proteins are expressed by lysogens of a number of other lambdoid coliphage, in cells carrying the cloned iss gene, and in several clinical isolates of E . coli . These results demonstrate that bor sequences are widespread and present a starting point for mechanistic analysis of bor-mediated serum resistance.

Infect Immun, 1995 Mar, 63(3), 954 - 60
Molecular and immunological characterization of the highly conserved antigen 84 from Mycobacterium tuberculosis and Mycobacterium leprae; Hermans PW et al.; Crossed immunoelectrophoresis (CIE) has been used to develop a reference system for classifying mycobacterial antigens . The subsequent use of specific antibodies allowed further determination of antigens by molecular weight . The monoclonal antibody F126-2, originally raised against a 34-kDa antigen of Mycobacterium kansasii, reacted with antigen 84 (Ag84) in the CIE reference system for Mycobacterium bovis BCG and Mycobacterium tuberculosis . To characterize Ag84, we screened a lambda gt11 gene library from M . tuberculosis with antibody F126-2 and identified the encoding gene . The corresponding Mycobacterium leprae Ag84 gene was subsequently selected from a cosmid library, using the M . tuberculosis gene as a probe . Both genes were expressed as 34-kDa proteins in Escherichia coli, and the recombinant proteins indeed corresponded to Ag84 in the CIE reference system . The derived amino acid sequences of the M . tuberculosis and M . leprae proteins showed 85% identity, which indicates that Ag84 constitutes a group of highly conserved mycobacterial antigens . Antibodies of almost 60% of lepromatous leprosy patients responded to Ag84, indicating that the protein is highly immunogenic following infection in multibacillary leprosy.

Infect Immun, 1995 Mar, 63(3), 745 - 50
Involvement of 5-hydroxytryptamine and prostaglandin E2 in the intestinal secretory action of Escherichia coli heat-stable enterotoxin B; Harville BA et al.; The intestinal secretory action of Escherichia coli heat-stable enterotoxin B (STb) is poorly defined . Previous work indicates that STb causes loss of intestinal fluid and electrolytes by a mechanism independent of elevated levels of cyclic nucleotides, the hallmark of other E . coli cytotonic enterotoxins . In the work described in this report, we observed that treatment of ligated rat intestinal loops with purified STb of E . coli resulted in a dose-dependent rise in intestinal secretion concomitant with dose-related increases in levels of serotonin (5-hydroxytryptamine {5-HT}) and prostaglandin E2 (PGE2) . Treatment of rats with the 5-HT2 receptor antagonist ketanserin prior to STb challenge resulted in significant (P < 0.05) reduction in intestinal secretion . Blockage of 5-HT2 receptors with ketanserin also reduced (P < 0.05) the level of PGE2 observed following STb treatment, indicating that at least a portion of the PGE2 was formed in response to 5-HT2 receptor stimulation . In a similar fashion, indomethacin, an inhibitor of cyclooxygenase activity, significantly reduced the level of secretion (P < 0.05) observed following STb treatment yet had no effect on 5-HT levels . Treatment of rats with both ketanserin and indomethacin further reduced STb-mediated secretion to a level not attained by either drug alone . Taken together, our data suggest that secretion due to STb involves both 5-HT and PGE2 as intestinal secretagogues . Furthermore, PGE2 formation appears to arise through both 5-HT-dependent and 5-HT-independent pathways.

Infect Immun, 1995 Mar, 63(3), 1122 - 6
Lipopolysaccharide-induced apoptosis in swine lymphocytes in vivo; Norimatsu M et al.; The in vivo effects of bacterial lipopolysaccharide (LPS) on the immune systems of piglets were investigated . Intravenous injection of 0.5 mg of LPS per kilogram of body weight induced apoptosis, which was characterized by nuclear chromatin condensation and fragmentation and a ladder formation of nucleosomal DNA in lymphocytes both in the cortex of the thymus and in the germinal centers and paracortical areas of mesenteric lymph nodes at 24 h postinjection . The levels of endotoxin, tumor necrosis factor alpha, and cortisol in serum increased, generally according to the dose of LPS . These findings suggest that LPS can induce in vivo apoptosis of lymphocytes in piglets and support the notion that cytokine and endocrine responses may play an important role in LPS-induced apoptosis in the immune system.

Infect Immun, 1995 Mar, 63(3), 1004 - 12
In vivo growth characteristics of leucine and methionine auxotrophic mutants of Mycobacterium bovis BCG generated by transposon mutagenesis; McAdam RA et al.; Insertional mutagenesis in Mycobacterium bovis BCG, a member of the slow-growing M . tuberculosis complex, was accomplished with transposons engineered from the Mycobacterium smegmatis insertion element IS1096 . Transposons were created by placing a kanamycin resistance gene in several different positions in IS1096, and the resulting transposons were electroporated into BCG on nonreplicating plasmids . These analyses demonstrated that only one of the two open reading frames was necessary for transposition . A library of insertions was generated . Southern analysis of 23 kanamycin-resistant clones revealed that the transposons had inserted directly, with no evidence of cointegrate formation, into different restriction fragments in each clone . Sequence analysis of nine of the clones revealed junctional direct 8-bp repeats with only a slight similarity in target sites . These results suggest that IS1096-derived transposons transposed into the BCG genome in a relatively random fashion . Three auxotrophs, two for leucine and one for methionine, were isolated from the library of transposon insertions in BCG . They were characterized by sequencing and found to be homologous to the leuD gene of Escherichia coli and a sulfate-binding protein of cyanobacteria, respectively . When inoculated intravenously into C57BL/6 mice, the leucine auxotrophs, in contrast to the parent BCG strain or the methionine auxotroph, showed an inability to grow in vivo and were cleared within 7 weeks from the lungs and spleen.

Mol Cell Biol, 1995 Mar, 15(3), 1642 - 50
BC1 RNA: transcriptional analysis of a neural cell-specific RNA polymerase III transcript; Martignetti JA et al.; Rodent BC1 RNA represents the first example of a neural cell-specific RNA polymerase III (Pol III) transcription product . By developing a rat brain in vitro system capable of supporting Pol III-directed transcription, we showed that the rat BC1 RNA intragenic promoter elements, comprising an A box element and a variant B box element, as well as its upstream region, containing octamer-binding consensus sequences and functional TATA and proximal sequence element sites, are necessary for transcription . The BC1 B box, lacking the invariant A residue found in the consensus B boxes of tRNAs, represents a functionally related and possibly distinct promoter element . The transcriptional activity of the BC1 B box element is greatly increased, in both a BC1 RNA and a chimeric tRNA(Leu) gene construct, when the BC1 5' flanking region is present and is appropriately spaced . Moreover, a tRNA consensus B-box sequence can efficiently replace the BC1 B box only if the BC1 upstream region is removed . These interactions, identified only in a homologous in vitro system, between upstream Pol II and intragenic Pol III promoters suggest a mechanism by which the tissue-specific BC1 RNA gene and possibly other Pol III-transcribed genes can be regulated.

Mol Cell Biol, 1995 Mar, 15(3), 1602 - 12
Analysis of protein-DNA and protein-protein interactions of centromere protein B (CENP-B) and properties of the DNA-CENP-B complex in the cell cycle; Kitagawa K et al.; We previously reported that centromere protein B (CENP-B) forms a stable complex (designated complex A) containing two alphoid DNAs in vitro . Domains in the CENP-B polypeptide involved in the formation of complex A were determined in the present study with truncated derivatives expressed in Escherichia coli and in rabbit reticulocyte lysates . It was revealed by gel mobility shift analyses that polypeptides containing the NH2-terminal DNA-binding domain bind a DNA molecule as a monomer, while dimerizing at a novel hydrophobic domain in the COOH-terminal region of 59 amino acid residues . This polypeptide dimerization activity at the COOH-terminal region was also confirmed with the two-hybrid system in Saccharomyces cerevisiae cells . The results thus proved that CENP-B polypeptides form a homodimer at the COOH-terminal hydrophobic domain, each binding a DNA strand at their NH2-terminal domains . The dimerization and DNA-binding domains fall into two of the three completely conserved sequences found in human and mouse CENP-B, and complex A-forming activity was also detected in nuclear extracts of mouse cells . Metaphase-specific phosphorylation of CENP-B was also detected, but this had no effect on its complex A-forming activity . On the basis of the present results, we propose that CENP-B plays an important role in the assembly of specific centromere structures by forming unique DNA-protein complexes at the sites of CENP-B boxes on the centromeric repetitive DNA both in interphase nuclei and on mitotic chromosomes.

Mol Cell Biol, 1995 Mar, 15(3), 1422 - 30
Cloning and characterization of a Xenopus poly(A) polymerase; Gebauer F et al.; During oocyte maturation and early embryogenesis in Xenopus laevis, the translation of several mRNAs is regulated by cytoplasmic poly(A) elongation, a reaction catalyzed by poly(A) polymerase (PAP) . We have cloned, sequenced, and examined several biochemical properties of a Xenopus PAP . This protein is 87% identical to the amino-terminal portion of bovine PAP, which catalyzes the nuclear polyadenylation reaction, but lacks a large region of the corresponding carboxy terminus, which contains the nuclear localization signal . When injected into oocytes, the Xenopus PAP remains concentrated in the cytoplasm, suggesting that it is a specifically cytoplasmic enzyme . Oocytes contain several PAP mRNA-related transcripts, and the levels of at least the one encoding the putative cytoplasmic enzyme are relatively constant in oocytes and early embryos but decline after blastulation . When expressed in bacteria and purified by affinity and MonoQ-Sepharose chromatography, the protein has enzymatic activity and adds poly(A) to a model substrate . Importantly, affinity-purified antibodies directed against Xenopus PAP inhibit cytoplasmic polyadenylation in egg extracts . These data suggest that the PAP described here could participate in cytoplasmic polyadenylation during Xenopus oocyte maturation.

Mol Cell Biol, 1995 Mar, 15(3), 1405 - 21
Binding of disparate transcriptional activators to nucleosomal DNA is inherently cooperative; Adams CC et al.; To investigate mechanisms by which multiple transcription factors access complex promoters and enhancers within cellular chromatin, we have analyzed the binding of disparate factors to nucleosome cores . We used a purified in vitro system to analyze binding of four activator proteins, two GAL4 derivatives, USF, and NF-kappa B (KBF1), to reconstituted nucleosome cores containing different combinations of binding sites . Here we show that binding of any two or all three of these factors to nucleosomal DNA is inherently cooperative . Thus, the binuclear Zn clusters of GAL4, the helix-loop-helix/basic domains of USF, and the rel domain of NF-kappa B all participated in cooperative nucleosome binding, illustrating that this effect is not restricted to a particular DNA-binding domain . Simultaneous binding by two factors increased the affinity of individual factors for nucleosomal DNA by up to 2 orders of magnitude . Importantly, cooperative binding resulted in efficient nucleosome binding by factors (USF and NF-kappa B) which independently possess little nucleosome-binding ability . The participation of GAL4 derivatives in cooperative nucleosome binding required only DNA-binding and dimerization domains, indicating that disruption of histone-DNA contacts by factor binding was responsible for the increased affinity of additional factors . Cooperative nucleosome binding required sequence-specific binding of all transcription factors, appeared to have spatial constraints, and was independent of the orientation of the binding sites on the nucleosome . These results indicate that cooperative nucleosome binding is a general mechanism that may play a significant role in loading complex enhancer and promoter elements with multiple diverse factors in chromatin and contribute to the generation of threshold responses and transcriptional synergy by multiple activator sites in vivo.

Mol Cell Biol, 1995 Mar, 15(3), 1210 - 9
Novel CDC34 (UBC3) ubiquitin-conjugating enzyme mutants obtained by charge-to-alanine scanning mutagenesis; Pitluk ZW et al.; CDC34 (UBC3) encodes a ubiquitin-conjugating (E2) enzyme required for transition from the G1 phase to the S phase of the budding yeast cell cycle . CDC34 consists of a 170-residue catalytic N-terminal domain onto which is appended an acidic C-terminal domain . A portable determinant of cell cycle function resides in the C-terminal domain, but determinants for specific function must reside in the N-terminal domain as well . We have explored the utility of "charge-to-alanine" scanning mutagenesis to identify novel N-terminal domain mutants of CDC34 that are enzymatically competent with respect to unfacilitated (E3-independent) ubiquitination but that nevertheless are defective with respect to its cell cycle function . Such mutants may reveal determinants of specific in vivo function, such as those required for interaction with substrates or trans-acting regulators of activity and substrate selectivity . Three of 18 "single-scan" mutants (in which small clusters of charged residues were mutated to alanine) were compromised with respect to in vivo function . One mutant (cdc34-109, 111, 113A) targeted a 12-residue segment of the Cdc34 protein not found in most other E2s and was unable to complement a cdc34 null mutant at low copy numbers but could complement a null mutant when overexpressed from an induced GAL1 promoter . Combining adjacent pairs of single-scan mutants to produce "double-scan" mutants yielded four additional mutants, two of which showed heat and cold sensitivity conditional defects . Most of the mutant proteins expressed in Escheria coli displayed unfacilitated (E3-independent) ubiquitin-conjugating activity, but two mutants differed from wild-type and other mutant Cdc34 proteins in the extent of multiubiquitination they catalyzed during an autoubiquitination reation-conjugating enzyme function and have identified additional mutant alleles of CDC34 that will be valuable in further genetic and biochemical studies of Cdc34-dependent ubiquitination.

J Virol, 1995 Mar, 69(3), 1842 - 50
Human immunodeficiency virus type 1 Nef protein inhibits activation pathways in peripheral blood mononuclear cells and T-cell lines; Greenway A et al.; Human immunodeficiency virus type 1 (HIV-1) Nef protein causes the loss of cell surface CD4 and interleukin-2 (IL-2) receptor (Tac) from peripheral blood mononuclear cells (PBMC) and CD4+ T-cell lines . As both CD4 and the IL-2 receptor play crucial roles in antigen-driven helper T-cell signalling and T-cell proliferation, respectively, the role of Nef in the viral life cycle may be to perturb signalling pathways emanating from these receptors . However, the intracellular targets for Nef that result in receptor down-regulation are unknown . Using a recombinant glutathione S-transferase-full-length 27 kDa Nef (Nef27) fusion protein, produced in Escherichia coli by translation from the first start codon of HIV-1 nef clone pNL4-3, as an affinity reagent to probe cytoplasmic extracts of MT-2 cells and PBMC, we have shown interaction with at least seven host cell protein species ranging from 24 to 75 kDa . Immunoblotting identified four of these proteins as p56lck, CD4, p53, and p44mapk/erk1, all of which are intimately involved in intracellular signalling . To assess the relevance of these interactions and further define the biochemical activity of Nef in signal transduction pathways, highly purified Nef27 protein was introduced directly into PBMC by electroporation . Nef27-treated PBMC showed reduced proliferative responsiveness to exogenous recombinant IL-2 . Normally, stimulation of T-cells by IL-2 or phorbol 12-myristate 13-acetate provokes both augmentation of p56lck activity and corresponding posttranslational modification of p56lck . These changes were also inhibited by treatment of PBMC with Nef, suggesting that Nef interferes with activation of p56lck and as a consequence of signalling via the IL-2 receptor . Further evidence for Nef interfering with cell proliferation was the decreased production of the proto-oncogene c-myb, which is required for cell cycle progression, in Nef-treated MT-2 cells . In contrast to the binding characteristics and biological effects of Nef27, the alternate 25-kDa isoform of Nef (Nef25) produced by translation from the second start codon of HIV nef pNL4-3 (57 nucleotide residues downstream) was shown to interact with only three cellular proteins of approximately 26, 28, and 56 kDa from PBMC and MT-2 cells, one of which was identified as p56lck . Also, proliferation and posttranslational modification of p56lck in response to IL-2 stimulation were not profoundly affected by treatment of PBMC with Nef25 compared with Nef27.(ABSTRACT TRUNCATED AT 400 WORDS)

J Virol, 1995 Mar, 69(3), 1819 - 26
Vaccinia virus gene H5R encodes a protein that is phosphorylated by the multisubstrate vaccinia virus B1R protein kinase; Beaud G et al.; Vaccinia virus gene B1R encodes a protein kinase, the previously identified substrates of which include the proteins S2 and Sa of 40S ribosomal subunits . This work characterizes another substrate of the B1R kinase: a 36-kDa protein induced at the early stage of infection . Partially purified 36-kDa protein, eluted from a single-stranded DNA-cellulose column by 0.5 M NaCl, was separated by two-dimensional gel electrophoresis . Phosphorylation in vitro yielded multiple forms of the 36-kDa protein with approximate isoelectric points (pI) of 5.5, 5.7, 5.9, and 6.3, in addition to the apparently unphosphorylated form with a pI of approximately 6.8 . The tryptic peptides derived from 36-kDa proteins with pI values of 5.7, 5.9, and 6.3 yielded almost identical high-pressure liquid chromatography profiles, strongly suggesting that the 36-kDa protein was modified by the phosphorylation of at least four sites, which were characterized as threonine residues . The amino acid sequence of several tryptic peptides derived from the 36-kDa protein showed that the 36-kDa protein was encoded by gene H5R of vaccinia virus . Consistent with this, the B1R kinase--either expressed in Escherichia coli or highly purified from HeLa cells--phosphorylated a recombinant trpE-H5R fusion protein in vitro . Fingerprints of the trpE-H5R and 36-kDa proteins phosphorylated by recombinant B1R kinase revealed common sites of phosphorylation, although some tryptic peptides were specific to either protein . Comparison was made of fingerprints of tryptic phosphopeptides derived from 36-kDa single-stranded DNA-binding protein labelled in vivo or in vitro . A common subset of peptides was observed, suggesting that some sites on H5R protein are phosphorylated by the B1R kinase in infected cells . These results suggest that some of the multiple threonine sites in the H5R protein are phosphorylated in vivo by the B1R protein kinase.

J Virol, 1995 Mar, 69(3), 1785 - 93
Characterization of a ubiquitinated protein which is externally located in African swine fever virions; Hingamp PM et al.; An antiserum was raised against the African swine fever virus (ASFV)-encoded ubiquitin-conjugating enzyme (UBCv1) and used to demonstrate by Western blotting (immunoblotting) and immunofluorescence that the enzyme is present in purified extracellular virions, is expressed both early and late after infection of cells with ASFV, and is cytoplasmically located . Antiubiquitin serum was used to identify novel ubiquitin conjugates present during ASFV infections . This antiserum stained virus factories late after infection, suggesting that virion proteins may be ubiquitinated . This possibility was confirmed by Western blotting, which identified three major antiubiquitin-immunoreactive proteins with molecular masses of 5, 18, and 58 kDa in purified extracellular virions . The 18-kDa protein was solubilized from virions at relatively low concentrations of the detergent n-octyl-beta-D-glucopyranoside, indicating that it is externally located and is possibly in the virus capsid . The 18-kDa protein was purified, and N-terminal amino acid sequencing confirmed that the protein was ubiquitinated and was ASFV encoded . The ASFV gene encoding this protein (PIG1) was sequenced, and the encoded protein expressed in an Escherichia coli expression vector . Recombinant PIG1 was ubiquitinated in the presence of E . coli expressed UBCv1 in vitro . These results suggest that PIG1 may be a substrate for UBCv1 . The predicted molecular masses of the PIG1 protein and recombinant ubiquitinated protein were larger than the 18-kDa molecular mass of the ubiquitinated protein present in virions . Therefore, during viral replication, a precursor protein may undergo limited proteolysis to generate the ubiquitinated 18-kDa protein.

J Virol, 1995 Mar, 69(3), 1727 - 33
Cleavage specificity of purified recombinant hepatitis A virus 3C proteinase on natural substrates; Schultheiss T et al.; Hepatitis A virus (HAV) 3C proteinase expressed in Escherichia coli was purified to homogeneity, and its cleavage specificity towards various parts of the viral polyprotein was analyzed . Intermolecular cleavage of the P2-P3 domain of the HAV polyprotein gave rise to proteins 2A, 2B, 2C, 3ABC, and 3D, suggesting that in addition to the primary cleavage site, all secondary sites within P2 as well as the 3C/3D junction are cleaved by 3C . 3C-mediated processing of the P1-P2 precursor liberated 2A and 2BC, in addition to the structural proteins VP0, VP3, and VP1-2A and the respective intermediate products . A clear dependence on proteinase concentration was found for most cleavage sites, possibly reflecting the cleavage site preference of 3C . The most efficient cleavage occurred at the 2A/2B and 2C/3A junctions . The electrophoretic mobility of processing product 2B, as well as cleavage of the synthetic peptide KGLFSQ*AKISLFYT, suggests that the 2A/2B junction is located at amino acid position 836/837 of the HAV polyprotein . Furthermore, using suitable substrates we obtained evidence that sites VP3/VP1 and VP1/2A are alternatively processed by 3C, leading to either VP1-2A or to P1 and 2A . The results with regard to intermolecular cleavage by purified 3C were confirmed by the product pattern derived from cell-free expression and intramolecular processing of the entire polyprotein . We therefore propose that polyprotein processing of HAV relies on 3C as the single proteinase, possibly assisted by as-yet-undetermined viral or host cell factors and presumably controlled in a concentration-dependent fashion.

J Virol, 1995 Mar, 69(3), 1532 - 9
Mutation of the aspartic acid residues of the GDD sequence motif of poliovirus RNA-dependent RNA polymerase results in enzymes with altered metal ion requirements for activity; Jablonski SA et al.; The poliovirus RNA-dependent RNA polymerase, 3Dpol, is known to share a region of sequence homology with all RNA polymerases centered at the GDD amino acid motif . The two aspartic acids have been postulated to be involved in the catalytic activity and metal ion coordination of the enzyme . To test this hypothesis, we have utilized oligonucleotide site-directed mutagenesis to generate defined mutations in the aspartic acids of the GDD motif of the 3Dpol gene . The codon for the first aspartate (3D-D-328 {D refers to the single amino acid change, and the number refers to its position in the polymerase}) was changed to that for glutamic acid, histidine, asparagine, or glutamine; the codons for both aspartic acids were simultaneously changed to those for glutamic acids; and the codon for the second aspartic acid (3D-D-329) was changed to that for glutamic acid or asparagine . The mutant enzymes were expressed in Escherichia coli, and the in vitro poly(U) polymerase activity was characterized . All of the mutant 3Dpol enzymes were enzymatically inactive in vitro when tested over a range of Mg2+ concentrations . However, when Mn2+ was substituted for Mg2+ in the in vitro assays, the mutant that substituted the second aspartic acid for asparagine (3D-N-329) was active . To further substantiate this finding, a series of different transition metal ions were substituted for Mg2+ in the poly(U) polymerase assay . The wild-type enzyme was active with all metals except Ca2+, while the 3D-N-329 mutant was active only when FeC6H7O5 was used in the reaction . To determine the effects of the mutations on poliovirus replication, the mutant 3Dpol genes were subcloned into an infectious cDNA of poliovirus . The cDNAs containing the mutant 3Dpol genes did not produce infectious virus when transfected into tissue culture cells under standard conditions . Because of the activity of the 3D-N-329 mutant in the presence of Fe2+ and Mn2+, transfections were also performed in the presence of the different metal ions . Surprisingly, the transfection of the cDNA containing the 3D-N-329 mutation resulted in the production of virus at a low frequency in the presence of FeSO4 or CoCl2 . The virus derived from transfection in the presence of FeSO4 grew slowly, while the viruses recovered from transfection in CoCl2 grew at a rate which was similar to that of the wild-type poliovirus.(ABSTRACT TRUNCATED AT 400 WORDS)

J Virol, 1995 Mar, 69(3), 1377 - 88
Cloning and characterization of herpes simplex virus type 1 oriL: comparison of replication and protein-DNA complex formation by oriL and oriS; Hardwicke MA et al.; The herpes simplex virus type 1 genome contains three origins of DNA replication: two copies of oriS and one copy of oriL . Although oriS has been characterized extensively, characterization of oriL has been severely limited by the inability to amplify oriL sequences in an undeleted form in Escherichia coli . We report the successful cloning of intact oriL sequences in an E . coli strain, SURE, which contains mutations in a series of genes involved in independent DNA repair pathways shown to be important in the rearrangement and deletion of DNA containing irregular structures such as palindromes . The oriL-containing clones propagated in SURE cells contained no deletions, as determined by Southern blot hybridization and DNA sequence analysis, and were replication competent in transient DNA replication assays . Deletion of 400 bp of flanking sequences decreased the replication efficiency of oriL twofold in transient assays, demonstrating a role for flanking sequences in enhancing replication efficiency . Comparison of the replication efficiencies of an 822-bp oriS-containing plasmid and an 833-bp oriL-containing plasmid demonstrated that the kinetics of replication of the two plasmids were similar but that the oriL-containing plasmid replicated 60 to 70% as efficiently as the oriS-containing plasmid at both early and late times after infection with herpes simplex virus type 1 . The virus-specified origin-binding protein (OBP) and a cellular factor(s) (OF-1) have been shown in gel mobility shift experiments to bind specific sequences in oriS (C.E . Dabrowski, P . Carmillo, and P.A . Schaffer, Mol . Cell . Biol . 14:2545-2555, 1994; C.E . Dabrowski and P.A . Schaffer, J . Virol . 65:3140-3150, 1991) . Although the nucleotides required for the binding of OBP to OBP binding site I in oriL and oriS are the same, a single nucleotide difference distinguishes OBP binding site III in the two origins . The nucleotides adjacent to oriS sites I and III have been shown to be important for the binding of OF-1 to oriS site I . Several nucleotide differences exist in these sequences in oriL and oriS . Despite these minor nucleotide differences, the protein-DNA complexes that formed with oriL and oriS sites I and III were indistinguishable when extracts of infected and uninfected cells were used as the source of protein . Furthermore, the results of competition analysis suggest that the proteins involved in protein-DNA complex formation with sites I and III of the two origins are likely the same.

East Afr Med J, 1995 Mar, 72(3), 150 - 4
Breastfeeding and immunity to intestinal infections; Kakai R et al.; The purpose of this study was to compare immune response in breast and non breastfed children presenting with diarrhoea at Paediatric Observation Ward, Kenyatta National Hospital (KNH-POW) and Maternal and Child Health Clinic, Pumwani Maternity Hospital (PMH-MCH) . Blood and stool samples were collected from the first four consecutive children aged 5 years and below per day, presenting with or without diarrhoea from January to December, 1992 . The stools were tested for total IgA by single radial immunodiffusion (SRID) and specific IgA by enzyme linked immunosorbent assay (ELISA) . Peripheral blood CD4 and CD8 enumeration was done by flow cytometry . Stools were cultured for bacteria on selective media while ova and cysts of parasites were identified by wet preparation microscopy . A total of 457 children were enrolled into the study, 69.6% of whom presented with diarrhoea . Breastfed children tended to have a shorter duration of diarrhoea than either mixed fed or bottle fed (8.3 vs 9.8 vs 11.2 days, p = 0.2) . In general, E . coli were more commonly isolated from breastfed than mixed fed or bottle fed (56.7% vs 43.9% vs 28.9%, p = 0.004) while intestinal parasites were mostly in bottle fed than mixed or breastfed children (28.8% vs 8.2 vs 0.8, p < 0.004) . However, when children with diarrhoea were considered, E . coli was more frequently isolated from bottle fed children who presented with diarrhoea than without (26.7% vs 7.7%, p = 0.03).(ABSTRACT TRUNCATED AT 250 WORDS)

Hum Mol Genet, 1995 Mar, 4(3), 359 - 66
Tissue-specific expression of a FMR1/beta-galactosidase fusion gene in transgenic mice; Hergersberg M et al.; Fragile X syndrome is one of the most common genetic causes of mental retardation, yet the mechanisms controlling expression of the fragile X mental retardation gene FMR1 are poorly understood . To identify sequences regulating FMR1 transcription, transgenic mouse lines were established using a fusion gene consisting of an E.coli beta-galactosidase reporter gene (lacZ) linked to a 2.8 kb fragment spanning the 5'-region of FMR1 . Five transgenic mouse lines showed lacZ expression in brain, in particular in neurons of the hippocampus and the granular layer of the cerebellum . Expression of the reporter gene was also detected in Leydig cells and spermatogonia in the testis, in many epithelia of adult mice, and in the two other steroidogenic cell types, adrenal cortex cells and ovarian follicle cells . Embryonic tissues which showed strong activity of the reporter gene included the telencephalon, the genital ridge, and the notochord . This expression pattern closely resembles the endogenous one, indicating that the 5' FMR1 gene promoter region used in this study contains most cis-acting elements regulating FMR1 transcription.

Protein Sci, 1995 Mar, 4(3), 538 - 41
Deletion mutants of tyrosine hydroxylase identify a region critical for heparin binding; Daubner SC et al.; Phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase constitute a family of tetrahydropterin-dependent aromatic amino acid hydroxylases . It has been proposed that each hydroxylase is composed of a conserved C-terminal catalytic domain and an unrelated N-terminal regulatory domain . Of the three, only tyrosine hydroxylase is activated by heparin and binds to heparin-Sepharose . A series of N-terminal deletion mutants of tyrosine hydroxylase has been expressed in Escherichia coli to identify the heparin-binding site . The mutants lacking the first 32 or 68 amino acids bind to heparin-Sepharose . The mutant lacking 76 amino acids binds somewhat to heparin-Sepharose and the proteins lacking 88 or 128 do not bind at all . Therefore, an important segment of the heparin-binding site must be composed of the region from residues 76 to 90 . All of the deletion mutants are active, and the Michaelis constants for pterins and tyrosine are similar among all the mutant and wild-type enzymes.

Protein Sci, 1995 Mar, 4(3), 534 - 7
Structural features of the uniporter/symporter/antiporter superfamily; Goswitz VC et al.; The uniporter/symporter/antiporter superfamily is an evolutionarily related group of solute transporters . For the entire superfamily, we have used a new predictive program to identify the transmembrane domains . These transmembrane domains were then analyzed with regard to their overall hydrophobicity and amphipathicity . In addition, the lengths of the hydrophilic loops connecting the transmembrane domains were calculated . These data, together with structural information in the literature, were collectively used to produce a general model for the three-dimensional arrangement of the transmembrane domains.

Protein Sci, 1995 Mar, 4(3), 421 - 32
Recombinant immunoglobulin variable domains generated from synthetic genes provide a system for in vitro characterization of light-chain amyloid proteins; Stevens PW et al.; The primary structural features that render human monoclonal light chains amyloidogenic are presently unknown . To gain further insight into the physical and biochemical factors that result in the pathologic deposition of these proteins as amyloid fibrils, we have selected for detailed study three closely homologous protein products of the light-chain variable-region single-gene family VkIV . Two of these proteins, REC and SMA, formed amyloid fibrils in vivo . The third protein, LEN, was excreted by the patient at levels of 50 g/day with no indication of amyloid deposits . Sequences of amyloidogenic proteins REC and SMA differed from the sequence of the nonpathogenic protein LEN at 14 and 8 amino acid positions, respectively, and these amino acid differences have been analyzed in terms of the three-dimensional structure of the LEN dimer . To provide a replenishable source of these human proteins, we constructed synthetic genes coding for the REC, SMA, and LEN variable domains and expressed these genes in Escherichia coli . Immunochemical and biophysical comparisons demonstrated that the recombinant VkIV products have tertiary structural features comparable to those of the patient-derived proteins . This well-defined set of three clinically characterized human kIV light chains, together with the capability to produce these kIV proteins recombinantly, provide a system for biophysical and structural comparisons of two different amyloidogenic light-chain proteins and a nonamyloidogenic protein of the same subgroup . This work lays the foundation for future investigations of the structural basis of light-chain amyloidogenicity.

Reprod Toxicol, 1995 Mar-Apr, 9(2), 169 - 74
Opioid peptides involvement in endotoxin-induced suppression of LH secretion in ovariectomized Holstein heifers; Kujjo LL et al.; Three groups of ovariectomized Holstein heifers were used in this study . Group I heifers (n = 6) were pretreated with saline (SAL), followed 3 h later by three injections of naloxone hydrochloride (NLX) given 1 h apart . In the same sequence, group II heifers (n = 3) received lipopolysaccharide (LPS), followed by the opioid antagonist NLX . Group III heifers (n = 3) received LPS followed by SAL . Concentrations of cortisol and progesterone increased (P < 0.05) following LPS injections in both groups II and III, whereas luteinizing hormone (LH) concentrations were suppressed (P < 0.05) . Administration of NLX to heifers pretreated with LPS elicited significant increases (P < 0.05) in LH concentrations, whereas SAL infusion had no effect . These results indicate that the inhibitory actions of opiate mu-delta receptors, and possibly other POMC gene products, were at least partially involved in LPS-induced suppression of the gonadotropic hormone . These results are discussed, including the possibility that feedback suppression by LPS-triggered adrenal gland steroids may have interfered with complete restoration of LH secretion by the opioid antagonist.

Antimicrob Agents Chemother, 1995 Mar, 39(3), 672 - 6
Effect of cefodizime and ceftriaxone on phagocytic function in patients with severe infections; Wenisch C et al.; Thirty patients with severe bacterial infections were treated with 50 mg of cefodizime per kg of body weight once daily or 50 mg of ceftriaxone per kg once daily for 10 +/- 3 days . The effect of cefodizime and ceftriaxone on the phagocytic capacity and generation of reactive oxygen intermediates after phagocytosis by granulocytes was assessed prior to, during, and after therapy . Flow cytometry was used to study phagocytic capacity by measuring the uptake of fluorescein-labeled bacteria . The generation of reactive oxygen intermediates after phagocytosis was estimated by the quantification of the intracellular conversion of dihydrorhodamine 123 to rhodamine 123 . Prior to therapy, patients in both groups exhibited a decreased capacity to phagocytize Escherichia coli and subsequently to generate reactive oxygen intermediates . Granulocyte function increased after the initiation of therapy and normalized within 7 days for the ceftriaxone-treated patients and within 3 days for the cefodizime group (P < 0.05) . In the cefodizime group, an enhancement of phagocytic capacity was observed 14 days after the initiation of therapy (P < 0.05) . Prior to therapy, phagocytic capacity was significantly correlated with the generation of reactive oxygen products (r = 0.674 and P < 0.005).

Psychosom Med, 1995 Mar-Apr, 57(2), 97 - 104
Effects of sleep on the production of cytokines in humans; Uthgenannt D et al.; The restorative functions of sleep may affect immunologic functioning . The present study examined the effects of sleep on stimulated cytokine release in 13 healthy men . The subjects spent 2 experimental nights in the sleep laboratory . In one condition, lights were turned off at 11:00 PM to enable sleep for 3.5 hours . Thereafter, they stayed awake till 7:00 AM . In the other condition, conversely, subjects stayed awake between 11:00 PM and 3:00 AM . Then, lights were turned off for a 3.5-hour phase of sleep . Blood was sampled every 30 minutes between 11:00 PM and 7:00 AM . Sleep was monitored by polysomnographic recordings . Release of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) was determined after stimulation of mononuclear cells with lipopolysaccharide from Escherichia coli . The release of IL-2 was stimulated with phytohemagglutinin . Compared with wakefulness, after 3 hours of sleep, production of TNF-alpha and IL-1 beta was substantially diminished (p < .01) . Production of IL-2 was enhanced during sleep (p < .05), with this effect being limited to the second nocturnal sleep phase after 3:00 AM . Sleep-dependent changes in stimulated cytokine release were independent of changes in plasma cortisol concentrations . These results indicate a specific reducing effect of sleep (vs . wakefulness) on cytokine production by monocytes (TNF-alpha and IL-1 beta) . The rather slow development of the effects calls for further studies to establish the exact time course of the influence of sleep on cytokine production.

Abdom Imaging, 1995 Mar-Apr, 20(2), 169 - 72
Emphysematous pyelonephritis with resultant emphysematous cholecystitis secondary to hematogenous dissemination; Lee HM et al.; Both emphysematous pyelonephritis and emphysematous cholecystitis are uncommon, but potentially fatal, clinical entities . The simultaneous diagnosis of these two entities in the same patient has not previously been reported . In this paper, we describe a 68-year-old diabetic male who presented acutely with emphysematous pyelonephritis and emphysematous cholecystitis . This case demonstrates several important diagnostic and treatment considerations . Additionally, the unique circumstances of this case offer support for the proposal that emphysematous cholecystitis may often be secondary to hematogenous seeding/embolic phenomena rather than obstruction of the cystic duct . Prompt diagnosis is essential, as prompt intervention can minimize mortality and morbidity.

Virus Res, 1995 Mar, 35(3), 291 - 305
Characterization of a protein kinase gene from two Chlorella viruses; Que Q et al.; An open reading frame (ORF) with strong homology to eukaryotic serine/threonine protein kinases was found in the two Chlorella viruses SC-1A and PBCV-1 . The deduced molecular weights of each putative protein kinase were 35 kDa and the predicted amino acid sequences of the two proteins were 95% identical . The ORF encoding the SC-1A protein kinase was over-expressed as a fusion protein in Escherichia coli . The recombinant fusion protein had autophosphorylation activity and could phosphorylate certain exogenous proteins . Antiserum against the recombinant fusion protein reacted with a 35 kDa protein plus three larger proteins from virus infected cells . The 35 kDa protein was a late protein; however, the 35 kDa protein was not packaged in the virion, even though virions contain protein kinase activity.

Virus Res, 1995 Mar, 35(3), 263 - 75
Expression in Escherichia coli and purification of biologically active L proteinase of foot-and-mouth disease virus; Piccone ME et al.; The foot-and-mouth disease virus (FMDV) Lb gene was cloned into bacterial expression vectors under the control of a T7 RNA polymerase promoter . The Lb protein was expressed in both an in vitro transcription-translation system and in Escherichia coli . In vitro expression of a construct containing the Lb gene fused to a portion of the VP4 and 3D genes demonstrated cis cleavage activity that could be blocked by the thiol protease inhibitor E-64 . Lb expressed in E . coli was purified from the soluble fraction by metal chelation chromatography . Purified Lb had trans cleavage activity at the L/P1 junction and cleaved the p220 component of the cap-binding protein complex.

Mol Biol (Mosk), 1995 Mar-Apr, 29(2), 398 - 406
{Production of biologically active recombinant ricin B-chain}; Tonevitskii AG et al.; Escherichia coli cells transformed with plasmids containing ricin B-chain coding sequences are shown to express this heterologous protein in inclusion bodies . After denaturation and renaturation of the product in the presence of glutathione and lactose, the recombinant ricin B-chain is soluble, biologically active and stable . Cytotoxicity of heterodimer containing this protein and ricin A-chain is found to be only ten times lower, than that of native ricin . Recombinant B-chain alone was nontoxic to cells (ID50 > 10(-6) M) . Our data suggest that ricin B-chain oligosaccharides are essential for stability preserving protein from proteolytic degradation in cells.

Mol Biol (Mosk), 1995 Mar-Apr, 29(2), 308 - 16
{Cloning and analysis of the nucleotide sequence of the segment in the Mycoplasma gallisepticum genome containing the gene for the ATP-binding subunit of DNA topoisomerase type II (topIIB)}; Skamrov AV et al.; M . gallisepticum genome fragment carrying complete coding sequence for ATP-binding subunit of topoisomerase II (topIIB), partial coding sequence for N-terminal part of A-subunit of topoisomerase II and region upstream of topIIB gene (open reading frame encoding 99 amino acids) was sequenced . The nucleotide sequence of topIIB has significant homology with previously reported gyrase genes and parE gene of E . coli . No protein sequence significantly similar to the open reading frame upstream from the gene topIIB was found in GeneBank data.

Mol Biol (Mosk), 1995 Mar-Apr, 29(2), 301 - 7
{The mutagenic activity of N4-(4-aminobutoxy)-2'-deoxycytidine-5'-triphosphate}; Tat'kov SI et al.; We have shown that deoxycytidine-5'-triphosphate modified by O-(4-aminobutyl)hydroxylamine in the pyrimidine ring, is effectively incorporated into DNA synthesizing in vitro, replacing deoxythymidine-5'-triphosphate or deoxycytidine-5'-triphosphate and inducing A-->G and G-->A transitions, respectively . UV spectroscopy and NMR spectroscopy have shown that the modified cytidine-5'-triphosphate is identical to N4-(4-aminobutoxy)-2'-deoxycytidine-5'-triphosphate . When the modified deoxycytidine-5'-triphosphate was inserted into DNA in vitro by DNA polymerase I of E . coli Klenow fragment, retardation sites correlating with poly-A sites (when the modified triphosphate replaced deoxythymidine-5'-triphosphate) or with poly-G sites (when it replaced deoxycytidine-5'-triphosphate) were revealed . Our data show high mutagenic effect of the modified deoxycytidine-5'-triphosphate inserted into DNA, allowing us to recommend this compound for localized static mutagenesis.

Mol Biol (Mosk), 1995 Mar-Apr, 29(2), 287 - 93
{Determination and analysis of the primary structure of a genomic sequence adjacent to the 3'-end of the human tissue plasminogen activator gene}; Sarafanov AG et al.; Primary structure was determined for the recently cloned f1/BglII-fragment {19} containing 2102 b.p . of the human tissue plasminogen activator (tPA) gene 3' end and adjacent DNA region . Computer analysis has revealed an Alu-repeat 820 b.p . downstream the tPA gene; the sequence proved to have a considerable homology (86-88%) with the Alus from the 3'-untranslated regions (3'UTRs) of cytochrome P-450, lysozyme and p53 protein human mRNAs . The same homology was estimated for this Alu in reversed orientation and Alus from the 3'UTRs of some other human mRNAs . In contrast, the homology between this 3' end tPA gene flanking Alu-repeat and other Alus dispersed throughout the gene introns either direct or reversed, was less than 70% . The polyadenylation signal AATAAA downstream the Alu and two nearby signals CACAG and GTGTT resembling consensus sequences CACAG and YGTGTTYY, respectively, were also detected . The two latter motifs located close to the 3' ends in most mammalian genes are likely to regulate mature mRNA formation . The comparison of the sequenced spaser flank adjacent to the tPA gene with short homologous sequence from the same genomic region primary structure reported previously has revealed discrepancies (substitutions, deletions or insertions) in 21 nucleotide positions . The nucleotide sequence of E . coli uvrB gene fragment (980 b.p.) is also reported . This E . coli gene fragment was cloned accidentally within the f1/BglII-fragment being an artifact of the host-vector system used.

Glia, 1995 Mar, 13(3), 174 - 84
Indicator expression directed by regulatory sequences of the glial fibrillary acidic protein (GFAP) gene: in vivo comparison of distinct GFAP-lacZ transgenes; Johnson WB et al.; An increase in the expression of the glial fibrillary acidic protein (GFAP) gene by astrocytes appears to constitute a crucial component of the brain's response to injury because it is seen in many different species and features prominently in diverse neurological diseases . Previously, we have used a modified GFAP gene (C-339) to target the expression of beta-galactosidase (beta-gal) to astrocytes in transgenic mice (Mucke et al.; New Biol 3:465-474 1991) . To determine to what extent the in vivo expression of GFAP-driven fusion genes is influenced by intragenic GFAP sequences, the E . coli lacZ reporter gene was either placed downstream of approximately 2 kb of murine GFAP 5' flanking region (C-259) or ligated into exon 1 of the entire murine GFAP gene (C-445) . Transgenic mice expressing C-259 versus C-445 showed similar levels and distributions of beta-gal activity in their brains . Exclusion of intragenic GFAP sequences from the GFAP-lacZ fusion gene did not diminish injury-induced upmodulation of astroglial beta-gal expression or increase beta-gal expression in non-astrocytic brain cells . These results demonstrate that 2 kb of murine GFAP 5' flanking region is sufficient to restrict transgene expression primarily to astrocytes and to mediate injury-responsiveness in vivo . This sequence therefore constitutes a critical target for mediators of reactive astrocytosis . While acute penetrating brain injuries induced focal increases in beta-gal expression around the lesion sites in C-259, C-445, and C-339 transgenic mice, infection of C-339 transgenic mice with scrapie led to a widespread upmodulation of astroglial beta-gal expression . Hence, GFAP-lacZ transgenic mice can be used to monitor differential patterns of astroglial activation in vivo . These and related models should facilitate the assessment of strategies aimed at the in vivo manipulation of GFAP expression and astroglial activation.

Curr Biol, 1995 Mar 1, 5(3), 275 - 82
A small peptide inhibitor of DNA replication defines the site of interaction between the cyclin-dependent kinase inhibitor p21WAF1 and proliferating cell nuclear antigen; Warbrick E et al.; BACKGROUND: p21WAF1 is a potent inhibitor of the cell-cycle regulatory cyclin-dependent kinases (Cdks) . It acts on Cdks in the G1 and S phases of the cell cycle, and also binds to proliferating cell nuclear antigen (PCNA), blocking DNA replication in vitro . Transcription of p21WAF1 can be induced by the human tumour suppressor protein p53, suggesting that the action of p21WAF1 may be important in cancer prevention . We have investigated the interaction between p21WAF1 and PCNA using a genetic two-hybrid screen and with arrays of synthetic peptides derived from the p21WAF1 protein sequence . RESULTS: We have established that the carboxy-terminal region of p21WAF1 interacts with PCNA in a yeast two-hybrid screen . Interaction with p21WAF1 involves the central loop of PCNA, which connects the two domains of the PCNA monomer . The interaction was finely mapped using peptides derived from the entire sequence of the p21WAF1 protein, and the critical residues were found to be QTSMTDFY (amino acids 144-151 of p21WAF1) . Remarkably, a 20-residue peptide containing this sequence inhibited replication of simian virus 40 (SV40) DNA in vitro and could capture PCNA from whole cell extracts, demonstrating that small molecules can retain the biological activity characteristic of the whole protein . Sequential alanine-scan mutations of the peptide demonstrated that its ability to block replication correlates with its affinity for binding PCNA . CONCLUSIONS: We have shown that PCNA and the cell-cycle regulator p21WAF1 interact in vivo, and that this interaction requires the central loop of PCNA and an eight amino-acid motif from the carboxyl terminus of p21WAF1.(ABSTRACT TRUNCATED AT 250 WORDS)

Chin Med Sci J, 1995 Mar, 10(1), 20 - 4
The use of a shuttle plasmid to study nontargeted mutagenesis and its sequence specificity; Zhang X et al.; Intact pZ189 DNA was replicated in monkey kidney vero cells which had been pretreated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) . The mutants were selected in E . coli MBM7070 and the mutation frequencies involving mutants with unchanged electrophoretic mobility of their plasmid DNA were scored . When compared to the spontaneous mutation frequency, the mutation frequencies were increased by 5.8 and 2.9-fold in cells pretreated with 0.2 and 2 mumol/L MNNG, respectively . The supF genes of these mutants were sequenced, and it was found that the types of base substitution and the sites of frameshifts differed from findings in studies of spontaneous and targeted mutagenesis . The results suggest that nontargeted mutagenesis occurs in mammalian cells and may have a sequence specificity.

Biotechniques, 1995 Mar, 18(3), 458 - 64
Multisite oligonucleotide-mediated mutagenesis: application to the conversion of a mitochondrial gene to universal genetic code; Sutherland L et al.; Using multisite oligonucleotide-mediated mutagenesis in conjunction with a mutagenesis selection procedure and rapid screening by allele-specific oligonucleotide hybridization has allowed us to develop a reliable protocol that enables a large number of base changes to be introduced rapidly into a piece of DNA, with the minimum number of manipulations . We have applied this protocol to generate synthetic versions of four mouse mitochondrial genes capable of being expressed in the nucleus/cytosol.

Vet Surg, 1995 Mar-Apr, 24(2), 87 - 96
Alterations of endothelium-dependent digital vascular responses in horses given low-dose endotoxin; Baxter GM; Low doses of endotoxin cause vasoconstriction and hypoperfusion of the digit, small intestine, and cecum in horses . To determine the potential cause of these vascular alterations, in vitro vascular responses of palmar digital arteries and veins were determined in 8 horses after intravenous (IV) infusion of 1 L 0.9% NaCl (control) and 0.1 microgram/kg Escherichia coli 055:B5 endotoxin in 1 L of 0.9% NaCl (endotoxin-treated) . Vessels were surgically removed under general anesthesia, cut into 4-mm vascular rings, suspended in tissue baths, and attached to force displacement transducers for measurement of vascular tension . Cumulative concentration response curves to acetylcholine, bradykinin, nitroprusside, norepinephrine, 5-hydroxytryptamine (serotonin), and endothelin were determined . Maximal relaxation or contraction and the concentrations needed to produce 50% maximal relaxation or contraction were determined . Palmar digital arteries from endotoxin-treated horses relaxed significantly less in response to acetylcholine and bradykinin (endothelium-dependent), but not to nitroprusside (endothelium-independent) when compared with arteries from control horses . Digital arteries from endotoxin-treated horses also contracted significantly more with norepinephrine but less with serotonin . Digital veins responded less than digital arteries . In another study, vascular reactivity experiments documented that acetylcholine and bradykinin were endothelium-dependent vasodilators (endothelium-denuded vessels relaxed less than control vessels) in palmar digital vessels . Additionally, maximal relaxations for both vasodilators were significantly inhibited by N-nitro-L-arginine methyl ester (L-NAME), a nitric oxide antagonist, suggesting that acetylcholine and bradykinin cause relaxation through the nitric oxide pathway . The data from these studies indicate that low dose endotoxin impairs endothelium-dependent relaxation and augments adrenergic contraction of palmar digital arteries in horses.

J Appl Physiol, 1995 Mar, 78(3), 784 - 92
Effects of endotoxin on isolated porcine liver: pressure-flow analysis; Brienza N et al.; The peripheral vascular response to sepsis is characterized by a vasodilatation of the systemic arterial vessels . Pulmonary hypertension with an increase in resistance and back pressure to flow defined by pressure-flow (P-Q) relationships has been reported in experimental sepsis . We hypothesized that endotoxin can induce differential alterations in resistance and back pressure to flow in the liver venous and arterial beds . Ninety minutes after endotoxin administration in intact anesthetized pigs (n = 8), the liver was vascularly isolated and perfused . Steady-state P-Q relationships in both the portal vein (PV) and hepatic artery (HA) were generated at multiple outflow pressures (Pout; 0, 5, 10, and 15 mmHg) and compared with those obtained in control livers (n = 6) . Extrapolated zero-flow pressure intercepts (Pback) and slopes of the P-Q relationships were obtained by least squares linear regression analysis . Endotoxemia increased PV Pback (P < 0.05), and Pback always exceeded Pout (P < 0.05) when the latter was raised . In contrast, in controls, no difference was observed between Pback and Pout when the latter was raised . Endotoxemia also increased the PV slope compared with control . Raising Pout from 0 to 15 mmHg decreased PV slope in the endotoxin group to a greater degree than in controls (P < 0.05) . In the HA, endotoxin caused a decrease in slope but did not alter Pback . The simultaneous increase in the PV Pback and slope that occurs with endotoxemia decreases splanchnic venous return, pooling blood in the splanchnic compartment for a given total blood volume.(ABSTRACT TRUNCATED AT 250 WORDS)

Shock, 1995 Mar, 3(3), 224 - 34
Amrinone combined with dobutamine improves hemodynamics and oxygen delivery without down-regulation of cardiac beta-adrenergic receptor density in porcine endotoxemia; Jones JL et al.; Effects of amrinone (AMR), a phosphodiesterase inhibitor, alone and in combination with dobutamine (DOB), on hemodynamics and O2 delivery were studied during porcine endotoxemia . Pentobarbital-anesthetized pigs were randomly administered either Escherichia coli lipopolysaccharide (endotoxin) or equivolumetric .9% NaCl (control) as a continuous infusion for 4 h . From 2 to 4 h (T = 120-240 min) of endotoxin infusion, pigs were randomly administered one of the following treatments; AMR infusion (40 micrograms/kg/min) (AMRlow); DOB (10 micrograms/kg/min) (DOB); AMR infusion (40 micrograms/kg/min) + DOB (AMRlow+DOB); AMR bolus (.75 mg/kg) followed by AMR infusion (40 micrograms/kg/min) (AMRhigh); or AMR bolus (.75 mg/kg) followed by infusion (40 micrograms/kg/min) + DOB (AMRhigh+DOB) . Myocardial samples were obtained at the end of the experiment and flash-frozen for beta-adrenergic receptor analysis . Endotoxin significantly (p < .05) decreased cardiac index, right ventricular ejection fraction, stroke volume index, maximum rate of rise of left ventricular pressure (dP/dtmax), mean arterial pressure, and O2 delivery, and increased pulmonary vascular resistance and mean pulmonary arterial pressure (p < .05) . AMRlow+DOB significantly (p < .05) increased cardiac index, dP/dtmax, right ventricular ejection fraction, stroke volume index, O2 delivery and consumption, and decreased mean pulmonary arterial pressure, pulmonary vascular resistance, mean arterial pressure, and systemic vascular resistance . beta-Adrenergic receptor density (Bmax) and binding equilibrium dissociation constant (KD) for {3H}dihydroalprenolol were not affected by endotoxin or any treatment (p < .05) . Endotoxin-induced hemodynamic deterioration and decreased O2 delivery was attenuated by AMRlow+DOB . Potential applications of this combination may exist in treatment of septic patients with inadequate myocardial performance and reduction in O2 delivery complicated by pulmonary hypertension.

Shock, 1995 Mar, 3(3), 173 - 8
Tumor necrosis factor antibody treatment of septic baboons reduces the production of sustained T-cell suppressive factors; Junger WG et al.; Post-traumatic septic complications result from impaired cell-mediated immune function, which is caused in part by circulating T-cell suppressive factors (TSFs) . We examined whether tumor necrosis factor alpha (TNF-alpha) antibody treatment in a baboon sepsis model influences the production of TSFs, including interleukin-10 (IL-10) and transforming growth factor-beta (TGF-beta) . Sepsis was induced in anesthetized baboons by Escherichia coli infusion, and caused an increase in plasma levels of TNF, TSF activity, IL-10, and active TGF-beta, as well as a decrease in latent TGF-beta . TNF antibody pretreatment reduced TNF levels by 98% . Transient TSF activity (0-4 h) was only marginally influenced, while sustained TSF activity (8-24 h) was markedly reduced . TSF activity at 24 h correlated with peak TNF levels . IL-10 levels, coinciding with early TSF activity, remained unchanged by anti-TNF treatment . Levels of active TGF-beta and the drop in latent TGF-beta were decreased . We conclude that anti-TNF treatment reduces sustained TSF activity and may partially restore impaired cell-mediated immune function.

Nat Struct Biol, 1995 Mar, 2(3), 199 - 204
Conformational constraints in protein degradation by the 20S proteasome; Wenzel T et al.; Conformationally stabilized peptides and unfolding intermediates of bovine alpha-lactalbumin have been used to define the degree of unfolding required for degradation by 20S proteasomes . It appears that complete unfolding and the absence of disulphide bonds are prerequisites for degradation, suggesting that a relatively narrow opening controls access to the inner proteolytic compartment of the barrel-shaped proteasome . This is corroborated by electron microscopy studies showing that the insulin B-chain, which is otherwise easily degraded, cannot pass the orifice of this putative peptide channel when a Nanogold particle with a diameter of approximately 2 nm is covalently attached to it.

Biochem Mol Biol Int, 1995 Mar, 35(3), 567 - 74
Alteration of the microenvironment in plasma membranes of rat enterocytes after Escherichia coli heat stable enterotoxin treatment: effect on protein kinase C activity; Chaudhuri AG et al.; Plasma membranes isolated from Escherichia coli heat stable enterotoxin (STa) treated rat enterocytes were studied in respect to protein kinase C activity and fluidity change . Pretreatment of enterocytes with STa increased the membrane bound protein kinase C activity about 5 fold as compared to control . STa treatment made the membrane more fluid as evident from a higher phospholipid/cholesterol ratio and greater unsaturated fatty acid levels . Moreover, the phase transition temperature of the STa treated membrane appeared to be significantly lower than that of the corresponding control membrane, thereby further indicating a rise in fluidity of the membrane in the former case . Our results, therefore, suggested that following STa enterotoxin treatment an appropriate fluid environment in the rat intestinal cell membrane was essential for the activation of protein kinase C.

J Virol Methods, 1995 Mar, 52(1-2), 111 - 9
Highly specific confirmatory western blot test for African swine fever virus antibody detection using the recombinant virus protein p54; Alcaraz C et al.; A Western blot technique using a recombinant protein has been developed to confirm positive results obtained in African swine fever (ASF)-specific antibody detection by ELISA . The new confirmatory Western blot is based on the use of protein p54, one of the most antigenic ASF virus structural proteins, expressed in Escherichia coli fused to the N-terminus of MS2 polymerase . The recombinant Western blot assay was highly specific and equally sensitive for ASF virus-infected pigs detection as the conventional Western blot, which uses virus-induced proteins ranging in molecular weight between 23 and 35 kDa . The novel Western blot assay provides a simpler interpretation of the test, eliminates the possibility of false-positive reactions produced by cellular compounds that contaminate the antigen employed in the conventional technique, and avoids the use of live virus in antigen production.

Genetics, 1995 Mar, 139(3), 1123 - 48
Conjugational recombination in Escherichia coli: genetic analysis of recombinant formation in Hfr x F- crosses; Lloyd RG et al.; The formation of recombinants during conjugation between Hfr and F- strains of Escherichia coli was investigated using unselected markers to monitor integration of Hfr DNA into the circular recipient chromosome . In crosses selecting a marker located approximately 500 kb from the Hfr origin, 60-70% of the recombinants appeared to inherit the Hfr DNA in a single segment, with the proximal exchange located > 300 kb from the selected marker . The proportion of recombinants showing multiple exchanges increased in matings selecting more distal markers located 700-2200 kb from the origin, but they were always in the minority . This effect was associated with decreased linkage of unselected proximal markers . Mutation of recB, or recD plus recJ, in the recipient reduced the efficiency of recombination and shifted the location of the proximal exchange(s) closer to the selected marker . Mutation of recF, recO or recQ produced recombinants in which this exchange tended to be closer to the origin, though the effect observed was rather small . Up to 25% of recombinant colonies in rec+ crosses showed segregation of both donor and recipient alleles at a proximal unselected locus . Their frequency varied with the distance between the selected and unselected markers and was also related directly to the efficiency of recombination . Mutation of recD increased their number by twofold in certain crosses to a value of 19%, a feature associated with an increase in the survival of linear DNA in the absence of RecBCD exonuclease . Mutation of recN reduced sectored recombinants in these crosses to approximately 1% in all the strains examined, including recD . A model for conjugational recombination is proposed in which recombinant chromosomes are formed initially by two exchanges that integrate a single piece of duplex Hfr DNA into the recipient chromosome . Additional pairs of exchanges involving the excised recipient DNA, RecBCD enzyme and RecN protein, can subsequently modify the initial product to generate the spectrum of recombinants normally observed.

Genetics, 1995 Mar, 139(3), 1107 - 21
On the clustered exchanges of the RecBCD pathway operating on phage lambda; Stahl FW et al.; Lytic cycle crosses of Red- Gam- phage lambda were conducted in rec+ Escherichia coli carrying one or another plasmid with homology to lambda . Lambda x lambda recombinants and lambda x plasmid recombinants were formed by RecBCD-mediated recombination . We showed previously that the act of recombining with a plasmid alters the disposition of selected lambda x lambda exchanges . This work reports that the relationships between the lambda x plasmid and the lambda x lambda exchanges is unaltered by the removal from one lambda parent of the homology shared with the plasmid . This result supports our view that a reciprocal exchange, allowing for cointegrate formation, is associated with but mechanistically separable from a (presumably) nonreciprocal lambda x lambda exchange . The nature of this relationship is independent of lambda's Rap function, which is shown to alter the ratio of cointegrate formation (splices) to marker pick-up (patches) in lambda x plasmid recombination mediated by the RecBCD pathway.

Clin Chest Med, 1995 Mar, 16(1), 13 - 28
The pathology of nosocomial pneumonia; Coalson JJ; Of patients who die in the intensive care unit (ICU), the mortality rate is significantly higher in those who develop nosocomial bacterial pneumonias . Pneumonia is difficult to diagnose clinically in patients with acute lung injury, so the "gold standard" for a pneumonia diagnosis has been its histopathologic presence in autopsy lung specimens . The potential pitfalls in the proper sampling of the lung at autopsy and how the histopathological appearance may be altered in a diffusely injured lung are discussed . Pathologists are encouraged to carefully analyze and evaluate bronchoalveolar lavage (BAL) and protected specimen brush (PSB) specimens obtained from patients before death to help determine if pneumonia is present.

Plant Cell Physiol, 1995 Mar, 36(2), 273 - 9
Differential regulation of the expression in transgenic tobacco of the gene for beta-glucuronidase under the control of the 5'-upstream regions of two catalase genes from castor bean; Suzuki M et al.; The regulatory functions of the 5'-flanking regions of two genes for catalase (cat1 and cat2) from castor bean were analyzed in transgenic tobacco plants that carried fusion constructs that included the gene for beta-glucuronidase (GUS) for Escherichia coli . Dry mature seeds from transgenic plants carrying the CAT1-GUS or CAT2-GUS constructs, in which the GUS gene was fused to the 5'-flanking region of cat1 or cat2, respectively, contained significant GUS activity, indicating that the promoters of cat1 and cat2 were active during seed development . GUS activity increased in response to germination in the seeds of transgenic tobacco that carried CAT1-GUS, as well as in those that carried CAT2-GUS . During the post-germinative stage the GUS activity directed by CAT2-GUS increased still further, whereas that directed by CAT1-GUS decreased . The changes in GUS activity in the transgenic tobacco plants that carried CAT1-GUS and CAT2-GUS were similar to the changes in the levels of transcripts of cat1 and cat2, respectively, in castor bean . The results suggest that the expression of cat1 and cat2 in the germinating seeds and post-germinative seedlings is regulated mainly at the level of transcription . However, the distribution of GUS activity among the organs of the transgenic tobacco seedlings and plantlets, which was examined by histochemical staining and by enzymatic assays of tissue extracts, was not identical to that of transcripts of cat1 and cat2 in castor bean . Histochemical analysis also revealed the interesting spatial regulation of the expression of the promoter of cat2 in the transgenic tobacco seedlings.

Plant Mol Biol, 1995 Mar, 27(6), 1227 - 33
Isolation, characterisation and expression of a cDNA clone encoding plastid aspartate aminotransferase from Arabidopsis thaliana; Wilkie SE et al.; A clone encoding aspartate aminotransferase (AAT, EC 2.6.1.1) was isolated from an Arabidopsis thaliana leaf cDNA library . This clone contains a 1365 bp open reading frame encoding a polypeptide of 49.8 kDa, designated Ataat1 . The clone was shown to contain a chloroplastic isoenzyme as an in organellar protein import assay demonstrated that a radiolabelled transcription/translation product of 49.8 kDa was imported into viable pea chloroplasts and was subsequently processed to yield a mature protein of 45 kDa . The open reading frame corresponding to the predicted mature AAT was manipulated into an expression construct (pEC14) . Transformed Escherichia coli cells containing pEC14 expressed up to 16 times more AAT activity than vector only controls, thus demonstrating conclusively that the clone encoded AAT.

Plant Mol Biol, 1995 Mar, 27(6), 1215 - 20
T-protein of the glycine decarboxylase multienzyme complex: evidence for partial similarity to formyltetrahydrofolate synthetase; Kopriva S et al.; We have isolated and sequenced cDNA clones encoding T-protein of the glycine decarboxylase complex from three plant species, Flaveria pringlei, Solanum tuberosum and Pisum sativum . The predicted amino acid sequences of these clones are at least 87% identical and all are similar to the predicted sequences of the bovine, human, chicken and Escherichia coli T-proteins . Alignment of all these sequences revealed conserved domains, one of which showed a significant similarity to a part of the formyltetrahydrofolate synthetases from procaryotes and eucaryotes . This suggests that the T-protein sequence is not as unique as previously thought.

Plant Mol Biol, 1995 Mar, 27(6), 1183 - 8
Structure of a maize tryptophan synthase alpha subunit gene with pith enhanced expression; Kramer VC et al.; A cDNA derived from an abundant maize pith mRNA transcript and its corresponding genomic equivalent have been isolated and characterized . High transcript levels are seen in the pith and young leaves of maize plants, while no transcript is detected in seed tissue of any age . The protein encoded by the isolated gene has considerable homology with tryptophan synthase alpha subunit (trpA) from other organisms and the cDNA clone can complement an E . coli trpA mutant . These data support the conclusion that this cDNA and the corresponding genomic clone encode a maize trpA protein.

Plant Mol Biol, 1995 Mar, 27(6), 1173 - 81
Import of a new chloroplast inner envelope protein is greatly stimulated by potassium phosphate; Hirsch S et al.; A cDNA clone encoding a major chloroplast inner envelope membrane protein of 96 kDa (IEP96) was isolated and characterized . The protein is synthesized as a larger-molecular-weight precursor (pIEP96) which contains a cleavable N-terminal transit sequence of 50 amino acids . The transit peptide exhibits typical stromal targeting information . It is cleaved in vitro by the stromal processing peptidase, though the mature protein is clearly localized in the inner envelope membrane . Translocation of pIEP96 into chloroplasts is greatly stimulated in the presence of 80 mM potassium phosphate which results in an import efficiency of about 90% . This effect is specific for potassium and phosphate, but cannot be ascribed to a membrane potential across the inner envelope membrane . Protein sequence analysis reveals five stretches of repeats of 26 amino acids in length . The N-terminal 300 amino acids are 45% identical (76% similarity) to the 35 kDa alpha-subunit of acetyl-CoA carboxyl-transferase from Escherichia coli . The C-terminal 500 amino acids share significant similarity (69%) with USOI, a component of the cytoskeleton in yeast.

Plant Mol Biol, 1995 Mar, 27(6), 1119 - 31
Molecular cloning and characterization of RGA1 encoding a G protein alpha subunit from rice (Oryza sativa L . IR-36); Seo HS et al.; A cDNA clone, RGA1, was isolated by using a GPA1 cDNA clone of Arabidopsis thaliana G protein alpha subunit as a probe from a rice (Oryza sativa L . IR-36) seedling cDNA library from roots and leaves . Sequence analysis of genomic clone reveals that the RGA1 gene has 14 exons and 13 introns, and encodes a polypeptide of 380 amino acid residues with a calculated molecular weight of 44.5 kDa . The encoded protein exhibits a considerable degree of amino acid sequence similarity to all the other known G protein alpha subunits . A putative TATA sequence (ATATGA), a potential CAAT box sequence (AGCAATAC), and a cis-acting element, CCACGTGG (ABRE), known to be involved in ABA induction are found in the promoter region . The RGA1 protein contains all the consensus regions of G protein alpha subunits except the cysteine residue near the C-terminus for ADP-ribosylation by pertussis toxin . The RGA1 polypeptide expressed in Escherichia coli was, however, ADP-ribosylated by 10 microM {adenylate-32P} NAD and activated cholera toxin . Southern analysis indicates that there are no other genes similar to the RGA1 gene in the rice genome . Northern analysis reveals that the RGA1 mRNA is 1.85 kb long and expressed in vegetative tissues, including leaves and roots, and that its expression is regulated by light.

Plant Mol Biol, 1995 Mar, 27(6), 1085 - 95
Characterization of a soybean cDNA clone encoding the mitochondrial isozyme of aspartate aminotransferase, AAT4; Wadsworth GJ et al.; A soybean leaf cDNA clone, pSAT2, was isolated by hybridization to a carrot aspartate aminotransferase (EC 2.6.1.1.; AAT) cDNA clone at low stringency . pSAT2 contained an open reading frame encoding a 47640 Da protein . The protein encoded by pSAT2 showed significant sequence similarity to AAT proteins from both plants and animals . It was most similar to two Panicum mitochondrial AATs, 81.5% and 82.0% identity . Alignment of the pSAT2-encoded protein with other mature AAT enzymes revealed a 25 amino acid N-terminal extension with characteristics of a mitochondrial transit peptide . A plasmid, pEXAT2, was constructed to encode the mature pSAT2 protein lacking the putative mitochondrial transit peptide . Escherichia coli containing the plasmid expressed a functional AAT isozyme which comigrated with the soybean AAT4 isozyme during agarose gel electrophoresis . Equilibrium sucrose gradient sedimentation of soybean extracts demonstrated that AAT4 specifically cofractionated with mitochondria . Antibodies raised against the pEXAT2-encoded AAT protein reacted with AAT4 of soybean and not with other AAT isozymes detected in soybean tissues, providing further evidence that clone pSAT2 encodes the soybean mitochondrial isozyme AAT4.

Plant Mol Biol, 1995 Mar, 27(5), 943 - 51
Biochemical properties of rice adenylate kinase and subcellular location in plant cells; Kawai M et al.; Previously, we characterized nucleotide sequences of two cDNAs encoding adenylate kinase from rice plants (Oryza sativa L.) . Each cDNA (Adk-a or Adk-b) was cloned into the expression vector pET 11d-GST to produce GST-AK fusion proteins in Escherichia coli . Recombinant proteins were cleaved by thrombin, and GST-free adenylate kinase proteins were obtained . Enzyme activity profiles of different pH and inhibition effects to the enzyme by Ap5A (adenosine-5'-pentaphospho-5'-adenosine) indicates that both adenylate kinase proteins have similar biochemical characteristics . Among the nucleoside monophosphates (AMP, CMP, GMP and UMP) investigated, only AMP reacted with ATP . Furthermore, using the antiserum against the rice adenylate kinase proteins, the cellular location of adenylate kinase proteins was examined by immunomicroscopic analysis in combination with a subcellular fractionation method . The results indicated that adenylate kinase proteins were distributed largely in cytosol of rice cells.

Plant Mol Biol, 1995 Mar, 27(5), 875 - 86
Modification of Brassica napus seed oil by expression of the Escherichia coli fabH gene, encoding 3-ketoacyl-acyl carrier protein synthase III; Verwoert II et al.; The Escherichia coli fabH gene encoding 3-ketoacyl-acyl carrier protein synthase III (KAS III) was isolated and the effect of overproduction of bacterial KAS III was compared in both E . coli and Brassica napus . The change in fatty acid profile of E . coli was essentially the same as that reported by Tsay et al . (J Biol Chem 267 (1992) 6807-6814), namely higher C14:0 and lower C18:1 levels . In our study, however, an arrest of cell growth was also observed . This and other evidence suggests that in E . coli the accumulation of C14:0 may not be a direct effect of the KAS III overexpression, but a general metabolic consequence of the arrest of cell division . Bacterial KAS III was expressed in a seed- and developmentally specific manner in B . napus in either cytoplasm or plastid . Significant increases in KAS III activities were observed in both these transformation groups, up to 3.7 times the endogenous KAS III activity in mature seeds . Only the expression of the plastid-targeted KAS III gene, however, affected the fatty acid profile of the storage lipids, such that decreased amounts of C18:1 and increased amounts of C18:2 and C18:3 were observed as compared to control plants . Such changes in fatty acid composition reflect changes in the regulation and control of fatty acid biosynthesis . We propose that fatty acid biosynthesis is not controlled by one rate-limiting enzyme, such as acetyl-CoA carboxylase, but rather is shared by a number of component enzymes of the fatty acid biosynthetic machinery.

Plant Mol Biol, 1995 Mar, 27(5), 1053 - 8
Expression of functional proteins of cDNA encoding rice nucleoside diphosphate kinase (NDK) in Escherichia coli and organ-related alteration of NDK activities during rice seed germination (Oryza sativa L.); Yano A et al.; The GST (glutathione S-transferase)-NDK (nucleoside diphosphate kinase) fusion protein was expressed in Escherichia coli . The GST-NDK protein was capable of transferring gamma-phosphate from ATP to nucleoside diphosphates such as GDP, CDP, TDP and UDP . Western blot analysis using anti-NDK antibody indicated that NDK in endosperm gradually decreased during 36 h of imbibition . On the contrary, NDK in embryo increased during the same period . NDK activities in both tissues were in accord with these observations . Whereas the NDK protein in roots of rice seedlings during 7 days of imbibition remained constant, in shoots it declined after 5 days of imbibition . Thus, NDK may play a significant role in the cellular event modulated by adenylate energy charge level.

Phytochemistry, 1995 Mar, 38(5), 1119 - 26
Expression in Escherichia coli and partial characterization of two tyrosine/dopa decarboxylases from opium poppy; Facchini PJ et al.; Two tyrosine/dopa decarboxylases (TYDC1 and TYDC2) from opium poppy (Papaver somniferum) were heterologously expressed in Escherichia coli and partially characterized . TYDC1 and TYDC2 are representative members of the two major isoform sub-classes of genes found in opium poppy which share less than 75% amino acid identity . Although both enzymes exhibit a marginal preference in vitro for L-dopa over L-tyrosine, the apparent Kms of both TYDC1 and TYDC2 in total protein extracts for either substrate were equal (Kms = 1 mM) at pH 7.2 . Both TYDC1 and TYDC2 exhibited a similar broad pH optimum in the range 7.5-8.5, and their activity was enhanced in the presence of pyridoxal phosphate co-factor . The Vmax values for TYDC1 with either tyrosine or dopa as substrate were virtually identical (Vmax = 0.59 fkat mg-1 protein), whereas, the Vmax for TYDC2 was two-fold greater with dopa (Vmax = 0.21 fkat mg-1 protein) than with tyrosine (Vmax = 0.12 fkat mg-1 protein) as substrate . Bacterial cell cultures expressing the TYDC1 polypeptide accumulated up to 350 micrograms ml-1 tyramine and 360 micrograms ml-1 dopamine in the medium within 8 hr after the addition of exogenous tyrosine or dopa, respectively . In contrast, cultures expressing the TYDC2 polypeptide accumulated 160 micrograms ml-1 tyramine and 110 micrograms ml-1 dopamine 8 hr after adding tyrosine or dopa, respectively . The higher in vivo conversion rates by bacterial cultures expressing TYDC1 relative to bacteria expressing TYDC2 is consistent with the higher specific activity of TYDC1 measured in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Sci China B, 1995 Mar, 38(3), 313 - 9
Nucleotide sequence of maize dwarf mosaic virus capsid protein gene and its expression in Escherichia coli; Sai J et al.; The 3'-terminal 1,279 nucleotide sequence of maize dwarf mosaic virus (MDMV) genome has been determined . This sequence contains an open reading frame of 1,023 nucleotides and a 3'-non-coding region of 256 nucleotides . The open reading frame includes all of the coding regions for the viral capsid protein (CP) and part of the viral nuclear inclusion protein (NIb) . The predicted viral CP consists of 313 amino acid residues with a calculated molecular weight of 35,400 . The amino acid sequence of the viral CP derived from MDMV cDNA shows about 47%-54% homology to that of 4 other potyviruses . The viral CP gene was constructed in frame with the lacZ gene in pUC19 plasmid and expressed in E . coli cells . The fusion polypeptide positively reacted in Western blot with an antiserum prepared against the native viral CP.

Biosci Biotechnol Biochem, 1995 Mar, 59(3), 501 - 6
Syntheses of 1-O-{5-(carboxy)pentanoyl}-2-deoxy-2-(2,2- difluorotetradecanamido)-3-O-{(R)-3-(tetradecanoyloxy)tetradecanoyl}-4- O- phosphono-alpha-D-glucopyranose and its analogues; Shiozaki M et al.; 1-O-{5-(Carboxy)pentanoyl}-2-deoxy-2-(2,2-difluorotetradecanamido) -3-O- {(R)-3-(tetradecanoyl-oxy)tetradecanoyl}-4-O-phosphono-alpha-D-glucop yranos e (13) and its analogues (16 and 19) were synthesized . Compound 13 showed strong LPS-agonistic activity.

Biosci Biotechnol Biochem, 1995 Mar, 59(3), 401 - 7
Synthesis of 2-(2-hydroxyalkylidene)cyclopentanones and their bio-antimutagenic activity; Iriye R et al.; 2-(2-Hydroxyalkylidene)cyclopentanones (1) and 5-(2-hydroxyoctylidene)-2-cyclopentenone (2) were synthesized by the aldol reaction of cyclopentanone-enolate (3)/cyclopentenone-enolate (19) with 2-bromoalkanals (4)/2-bromooctanal (4c) and subsequent treatment of the products with sodium acetate . The stereochemistry of the condensation, substitution and elimination was elucidated . The bio-antimutagenic activity of 1 increased as the 2-hydroxyalkylidene group increased in length up to = CHCH(OH)C8H17.

Biotechnol Prog, 1995 Mar-Apr, 11(2), 202 - 7
Extractive cultivation of Escherichia coli using poly(ethylene glycol)/phosphate aqueous two-phase systems to produce intracellular beta-galactosidase; Kuboi R et al.; Escherichia coli cells were found to grow in poly(ethylene glycol) (PEG)/phosphate aqueous two-phase systems by selecting the phase-forming components and their concentrations, the tie-line length, and the phase volume ratio properly . The cells cultivated up to an optical density at 660 nm of 1.0 were disrupted by ultrasonic irradiation to release and recover overproduced beta-galactosidase . The surviving cells were found to grow immediately after ultrasonic irradiation . The PEG/phosphate (KH2-PO4-K2HPO4) system with added Na2SO4 was the one optimized for extractive cultivation of E . coli cells, where beta-galactosidase was selectively partitioned to the top phase while total soluble proteins and cells partitioned to the bottom phase . This integrated process was extended to a semicontinuous operating mode, where the top phase containing beta-galactosidase was removed following intermittent ultrasonic irradiation and the bottom phase containing cells was recycled together with the new top phase solution to repeat production and recovery of beta-galactosidase.

Appl Microbiol Biotechnol, 1995 Mar, 42(6), 890 - 4
Mitomycin C stimulates thermally induced recombinant gene expression in Escherichia coli MC strains; Corchero JL et al.; The effects of mitomycin C on C1857-controlled recombinant gene expression have been explored in E . coli cultures when the drug was added simultaneously to the thermal induction . A significantly improved yield of homologous, heterologous and chimeric fusion proteins was observed in E . coli MC1061 and GE864 (a MC4100 derivative) thermoinduced cells . This feature was not detected in other E . coli strains and does not involve a gene dosage mechanism but a strain-dependent stimulation of gene expression unrelated to the RecA protease activity.

Appl Microbiol Biotechnol, 1995 Mar, 42(6), 884 - 9
Cloning, DNA sequencing and heterologous expression of the gene for thermostable N-acylamino acid racemase from Amycolatopsis sp . TS-1-60 in Escherichia coli; Tokuyama S et al.; The gene encoding the novel enzyme N-acylamino acid racemase (AAR) was cloned in recombinant phage lambda-4 from the DNA library of Amycolatopsis sp . TS-1-60, a rare actinomycete, using antiserum against the enzyme . The cloned gene was subcloned and transformed in Escherichia coli JM105 using pUC118 as a vector . The AAR gene consists of an open-reading frame of 1104 nucleotides, which specifies a 368-amino-acid protein with a molecular mass of 39411Da . The molecular mass deduced from the AAR gene is in good agreement with the subunit molecular mass (40kDa) of AAR from Amycolatopsis sp . TS-1-60 . The guanosine plus cytosine content of the AAR gene was about 70% . Although the AAR gene uses the unusual initiation codon GTG, the gene was expressed in Escherichia coli using the lac promoter of pUC118 . The amount of the enzyme produced by the transformant was 16 times that produced by Amycolatopsis sp . TS-1-60 . When the unusual initiation codon GTG was changed to ATG, the enzyme productivity of the transformant increased to more than 37 times that of Amycolatopsis sp . TS-1-60 . In the comparison of the DNA sequence and the deduced amino acid sequence of AAR with those of known racemases and epimerases in data bases, no significant sequence homology was found . However, AAR resembles mandelate racemase in that requires metal ions for enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Free Radic Res, 1995 Mar, 22(3), 275 - 83
Action of cystine in the cytotoxic response of Escherichia coli cells exposed to hydrogen peroxide; Cantoni O et al.; Cystine markedly enhanced the cytotoxic response of Escherichia coli cells to concentrations of hydrogen peroxide resulting in mode one killing, but displayed little effect in mode two killed cells . The effect of cystine was concentration-dependent over a range of 5-50 microM and did not further increase at higher levels . Cystine had similar effects in other bacterial systems . In order to sensitize the cells to the oxidative injury, the amino acid must be present during exposure to the oxidant since no enhancement of the cytotoxic response can be observed in cystine pre-loaded cells . In addition, no further enhancement of cytotoxicity could be detected when cystine was added before and left during challenge with the oxidant . The enhancing effect of cystine on oxidative injury of E . coli cells appears to be directly mediated by the amino acid and in fact cysteic acid, the most likely oxidation product, had no effect on the killing of bacterial cells elicited by hydrogen peroxide . Other disulfide compounds such as oxidized glutathione, cystamine and dithionitrobenzoic acid only slightly increased the susceptibility of bacteria to the oxidant . The effect of the disulfides was not concentration-dependent over a range of 200-800 microM and was statistically significant only for cystamine . Taken together, these results indicate that cystine markedly increases the cytotoxic response of bacteria to hydrogen peroxide and suggest that the amino acid might impair the cellular defence machinery against hydrogen peroxide . This effect may involve a thiol-disulfide exchange reaction at the cell membrane level.

J Crit Care, 1995 Mar, 10(1), 7 - 14
The role of platelet-activating factor in lipopolysaccharide-induced myocardial depression in guinea pigs; Heard SO et al.; PURPOSE: To determine if platelet-activating factor (PAF) is a key mediator of lipopolysaccharide (LPS)-induced myocardial depression in guinea pigs . METHODS: Hartley guinea pigs of either sex received intraperitoneal (IP) injections of either vehicle (n = 45) or one of three chemically dissimilar PAF receptor antagonists (n = 38) followed 30 to 60 minutes later by IP injections of either saline (0.8 mL, n = 33) or LPS (2 to 4 mg/kg, n = 50) . Left atria (LA) were harvested 16 hours later, suspended in Krebs-Henseleit buffer and attached to force-displacement transducers . Starling and force-frequency curves, contractile function in the potentiated and resting states, and inotropic response to either isoproterenol or phenylephrine were measured . RESULTS: LPS caused a significant reduction in LA contractile function . Two of the three PAF receptor antagonists failed to ameliorate LPS-induced alterations in cardiac function . The third antagonist, SR27417, was approximately 50% effective in preventing LA contractile dysfunction . However, this beneficial response appeared to be caused by a primary inotropic effect of SR27417 because LA from animals treated with SR27417 and saline showed significantly higher contractile function compared with LA from animals treated with vehicle and saline . In vitro tests confirmed this . Some LA from LPS-treated animals exhibited reduced contractile responses when in the potentiated state, a sign of impaired calcium release from the sarcoplasmic reticulum (SR) . The response of LA from endotoxic animals to isoproterenol was unchanged compared with controls whereas it was markedly impaired to phenylephrine . Use of SR27417 failed to improve this abnormal response . CONCLUSIONS: PAF does not appear to be a primary mediator of LPS-induced myocardial depression in guinea pigs . LPS may impair SR calcium release thereby causing cardiac dysfunction.

Plant J, 1995 Mar, 7(3), 491 - 501
Purification and cDNA cloning of anthranilate synthase from Ruta graveolens: modes of expression and properties of native and recombinant enzymes; Bohlmann J et al.; Ruta graveolens utilizes anthranilate synthase (AS) for the synthesis both of tryptophan in primary metabolism and acridone alkaloids in secondary metabolism . AS has been purified from plants and cell cultures of R . graveolens 670- and 1700-fold, respectively . Glutamine- and ammonia-dependent AS activities were strictly co-purified in all steps . Through cDNA cloning and complementation of Escherichia coli deletion mutants defective for AS, it is shown that young Ruta plants express two genes for functional AS alpha subunits, AS alpha 1 and AS alpha 2 . The data indicate that AS alpha from Ruta requires an AS beta subunit with a native molecular weight of 60-65 kDa for the glutamine-dependent reaction . Protein synthesized in vitro from cloned cDNA is processed upon import into isolated chloroplasts, indicating that mature AS alpha subunits are active in plastids in vivo . AS alpha 1 and AS alpha 2 are constitutively expressed in Ruta cell cultures, but AS alpha 1 steady-state mRNA levels are increased 100-fold 6 h subsequent to elicitation whereas AS alpha 2 expression remains constitutive . Increased AS alpha 1 transcription corresponds to elicitor-induced alkaloid accumulation . The data indicate that Ruta regulates anthranilate flux into primary and secondary metabolism through differential regulation of AS genes specific to these pathways.

Aktuelle Radiol, 1995 Mar, 5(2), 112 - 4
{Spondylogenic psoas abscess: long-term follow-up after percutaneous drainage}; Rieker O et al.; Iliopsoas abscesses originating from bacterial lumbar spondylodiscitis were successfully treated by CT-guided percutaneous abscess drainage, antibiotics, and immobilization in two patients . Vertebral fusion of the affected segments was observed in both patients in the follow-up period (1 and 6 years, respectively) . Percutaneous abscess drainage can replace surgery for iliopsoas abscesses following vertebral osteomyelitis.

J Clin Microbiol, 1995 Mar, 33(3), 701 - 5
Development of PCR for screening of enteroaggregative Escherichia coli; Schmidt H et al.; In this study, we determined the sequence of the EcoRI-PstI fragment of the plasmid pCVD432, also termed the enteroaggregative Escherichia coli (EAggEC) probe . A primer pair complementary to this probe was designed for PCR amplification of a 630-bp region . Comparison of the analysis of the EAggEC probe sequence with those in database libraries revealed no significant similarity to any known bacterial gene . Pure cultures of E . coli cells, as well as mixed cultures from stool specimens, were investigated with the PCR assay, the EAggEC probe test, and the adherence test . Of 50 E . coli strains which demonstrated aggregative adherence to HEp-2 cells, 43 (86%) were positive with the EAggEC PCR . All 43 of these strains reacted with the EAggEC probe . Six EAggEC strains gave negative results by both molecular techniques . In contrast, only 4 of 418 (0.96%) strains representing other categories of diarrheagenic E . coli demonstrated a positive PCR result . The PCR was also successful in screening for the presence of EAggEC in enriched cultures grown from stool specimens . Compared with cell culture assays and colony hybridization, our findings revealed that the PCR assay was more rapid, simple, and highly sensitive and can therefore be recommended as a screening method for EAggEC in the clinical laboratory.

J Clin Microbiol, 1995 Mar, 33(3), 680 - 3
Recombinant 46-kilodalton surface antigen (P46) of Mycoplasma hyopneumoniae expressed in Escherichia coli can be used for early specific diagnosis of mycoplasmal pneumonia of swine by enzyme-linked immunosorbent assay; Futo S et al.; The 46-kDa surface antigen (P46) is the early and species-specific immunogenic protein of Mycoplasma hyopneumoniae . Three TGA codons encoding tryptophan in the P46 gene were replaced with TGG by an in vitro mutagenesis technique . The mutated P46 gene was expressed in Escherichia coli by using the chelating peptide tag system . The purified recombinant P46 was successfully used in an enzyme-linked immunosorbent assay for detection of antibodies against M . hyopneumoniae in swine serum . It did not cross-react with sera from swine infected with Mycoplasma flocculate, Mycoplasma hyorhinis, or Mycoplasma hyosynoviae . With this method, mycoplasmal pneumonia of swine was detectable within 2 weeks after infection.

J Clin Microbiol, 1995 Mar, 33(3), 620 - 4
Study on reliability of commercially available hepatitis C virus antibody tests; Feucht HH et al.; The serodiagnosis of hepatitis C virus (HCV) infection was analyzed by a recombinant immunoblot assay (RIBA) with recombinant proteins encoded by the viral RNA isolated from our patients in Hamburg, Germany . The HCV RNA was amplified by PCR, and proteins encoded by the viral core and the NS3, NS4, and NS5 regions were expressed subsequently in Escherichia coli . The results obtained with our UKE RIBA were compared with the results of the Abbott HCV second-generation enzyme immunoassay (EIA) . Serum samples from 270 patients, which were sent to us on the suspicion of HCV hepatitis and which were negative for hepatitis A virus and hepatitis B virus antibodies, were examined . In 227 cases (84.1%), there were identical positive (204 cases, 75.6%) or negative (23 cases, 8.5%) results in both tests . In 32 cases (11.9%), the reactive Abbott second-generation HCV EIA results could not be confirmed by the UKE RIBA and the HCV PCR . In follow-up studies conducted over 1 year, these results did not change . In three cases (1.1%), the UKE RIBA presented a positive result while the Abbott second-generation HCV EIA was negative . All three cases were positive in the HCV PCR and showed seroconversion in an HCV EIA 4 to 6 weeks later . In addition, 33 patient serum samples were examined by UKE RIBA in parallel with the Ortho RIBA 2.0 . In three cases (9.1%), a positive Ortho RIBA 2.0 result could not be confirmed by the UKE RIBA and the HCV PCR . All three patients were free of complaints . The UKE RIBA showed also a smaller number of indeterminate results (3.0%) than the Ortho RIBA 2.0 (24.2%) . This comparison study demonstrates that the commercially available HCV antibody tests should be further improved.

Growth Regul, 1995 Mar, 5(1), 60 - 5
Somatogenic and lactogenic activity of the recombinant 22 kDa isoform of human placental growth hormone; Igout A et al.; During pregnancy, the human placenta secretes a variant of pituitary growth hormone (hGH-V) differing in sequence from the pituitary growth hormone (hGH-N) by 13 amino acids substitutions . HGH-V replaces hGH-N in the maternal bloodstream during the second half of pregnancy . HGH-V is produced in two, possibly three, isoforms: a 22 kDa form, a glycosylated 25 kDa form, and a probable 26 kDa form resulting from alternative splicing of the mRNA . Here we have studied the somatogenic and lactogenic activity of recombinant 22 kDa hGH-V isoform produced in Escherichia coli and compared it with the 22 kDa form of hGH-N . The two variants exert a similar somatogenic effect on rabbit and rat cells, but differ as regards their lactogenic activity in the rat.

Jpn J Cancer Res, 1995 Mar, 86(3), 270 - 6
Comparison of incorporation and extension of nucleotides in vitro opposite 8-hydroxyguanine (7,8-dihydro-8-oxoguanine) in hot spots of the c-Ha-ras gene; Kamiya H et al.; DNA templates with 8-hydroxyguanine (7,8-dihydro-8-oxoguanine, oh8Gua) at a site corresponding to the first or second position of codon 12 of the c-Ha-ras gene were prepared, and the nucleotides inserted opposite the modified base were compared . The Klenow fragment (KF) of Escherichia coli DNA polymerase I inserted C opposite oh8Gua at both positions . Taq DNA polymerase incorporated C and A opposite oh8Gua, and the ratio of C to A was higher at the first position than at the second position . DNA polymerase alpha (pol alpha) inserted A and C at the first position, and A at the second position of codon 12, indicating that the ratio of C to A was higher at the first position . Moreover, we studied the extensions of bases paired with oh8Gua by DNA polymerases with or without 3'-5' exonuclease activity . G and T opposite oh8Gua were removed, and subsequently C was inserted by KF . We found that an oh8Gua:A pair was recognized by the exonuclease activity of the enzyme and that A was partially substituted by C . On the other hand, pol alpha extended only C and A opposite oh8Gua . No difference was observed with oh8Gua at the two positions . These results indicate that the ratio of nucleotides incorporated opposite oh8Gua depends on the sequence context, while there is no particular difference in the extension of base pairs involving oh8Gua by DNA polymerases.

J Endocrinol, 1995 Mar, 144(3), 393 - 403
Extracellular domain of prolactin receptor from bovine mammary gland: expression in Escherichia coli, purification and characterization of its interaction with lactogenic hormones; Tchelet A et al.; The cDNA of the extracellular domain of bovine prolactin receptor (bPRLR-ECD) was cloned and expressed at high yield as an insoluble protein in Escherichia coli . This protein was solubilized, refolded and purified to > 98% homogeneity yielding 80 mg of monomeric fraction per 2 litres of induced culture . Its molecular mass was 25.7 kDa, as determined by SDS-PAGE in the absence of reducing agent and 24 kDa by gel filtration on a Superdex column . Binding experiments revealed that bPRLR-ECD binds to human (h) GH (hGH) with high affinity, whereas its affinity for ovine (o) or bovine (b) prolactins (PRLs) was lower and for bovine placental lactogen (bPL) very low . The affinity of bPRLR-ECD for the latter three hormones was, however, much higher than that of membrane-embedded or solubilized bPRLR . The stoichiometries of interaction of bPRLR-ECD with hGH, oPRL, bPRL and bPL were determined by gel-filtration chromatography . Even at a 3:1 ECD excess, only 1:1 complexes were detected at microM concentrations of ECD and ligand . At an up to 32-fold dilution, the complexes with oPRL, bPRL, and particularly bPL, underwent progressive dissociation, whereas the complex with hGH remained stable . Although all four hormones exhibited nearly identical activity in the Nb2 lymphoma cell bioassay, the ability of bPRLR-ECD to inhibit hormonal mitogenic activities differed, generally reflecting its affinity for the respective hormones . In view of these and previous results, we suggest that, unlike in the GH:GHR-ECD interaction, neither the stoichiometry of interaction of bovine or other PRLR-ECDs nor the affinity constants can predict the biological potency of the different lactogenic hormones.

Cytometry, 1995 Mar 1, 19(3), 209 - 16
High-speed photodamage cell selection using a frequency-doubled argon ion laser; Keij JF et al.; A flow cytometer was developed for the high-speed "sorting" of desired cells by selectively irradiating (zapping) the undesired cells from a population . After previous efforts to photoinactivate cells with photosensitizers had failed, it was decided to exploit the photosensitivity of the cell's DNA at 257 nm . It was shown that a 257 nm laser output power of 20-100 mW was sufficient to induce a 4.5 log cell kill after the cells were processed through a focused 257 nm laser beam . Experiments proved that the photodamage flow cytometer (ZAPPER) could selectively photoinactivate cells at rates over 22,000 events/s, and selection purities ranged from 81% to 100% . The yields of the desired cells depended on the selection mode . In the Enrichment mode, the zap laser was not aimed at the jet, and only undesired cells were exposed to a brief ultraviolet (UV) pulse after modulation of the UV laser beam . The yields of desired cells ranged from 95% to 105% . In the Purge mode, the zap laser beam was aimed onto the jet, and only desired cells were allowed to pass after deflection of the UV laser beam; the yields of desired cells ranged from 12% to 52% . The cause of the reduced yields in the PURGE mode was traced to the fact that the Electro-Optic Modulator was used to modulate the zap laser proved too slow for the intended application . The lifetime of the frequency-doubling crystal used for the generation of the 257 nm beam was found to be limited to several days.(ABSTRACT TRUNCATED AT 250 WORDS)

Microbiol Res, 1995 Mar, 150(1), 7 - 61
ECDC--a totally integrated and interactively usable genetic map of Escherichia coli K12; Wahl R et al.; A printed version of the interactively usable genetic map of Escherichia coli K12 is provided together with some statistical information about the actual status of the respective genome sequencing project . A total of 3,179,967 bp corresponding to 68.38% of the genome is available through the ECDC database . Contigs as well as individual DNA sequences for each gene or open reading frame are provided . Access to a number of other databases is possible using World Wide Web or local programs.

Biokhimiia, 1995 Mar, 60(3), 387 - 94
{Interaction of Escherichia coli RNA polymerase with promoters . The need for a classified approach in studying the code of promoter-polymerase recognition}; Kamzolova SG; The data on E . coli RNA polymerase (E sigma 70) interaction with its numerous promoters are reviewed . Two different approaches to the problem of promoter-polymerase recognition code are analyzed . One approach is consistent with the idea of a universal code on the basis of a consensus promoter . According to this approach, the unique active center in the enzyme is involved in interaction with all promoters . The second approach is based on the original concept suggesting the existence of several active centers in RNA polymerase, each of them interacting with its own group of promoters . This approach is based on the idea that promoter-polymerase recognition is coded by different programs for different groups of promoters; therefore, the problem should be contemplated from the standpoint of a classification analysis of promoters . Supporting evidence in favour of this concept is presented.

J Clin Pathol, 1995 Mar, 48(3), 260 - 6
Immunological factors and risk of infection in plateau phase myeloma; Hargreaves RM et al.; AIMS--A series of patients with myeloma were investigated to assess whether immunological risk factors predisposing to serious infection could be identified . METHODS--Patients (n = 102) with predominantly plateau phase myeloma were monitored prospectively for infections . Immunological parameters including total non-paraprotein immunoglobulins and specific antibody titres were measured in all patients and compared with a control population of healthy individuals of a similar age; response to immunisation with Pneumovax II, tetanus and diphtheria toxoids and IgG subclasses were measured in a subgroup of 41 patients . Other characteristics investigated for any association with infection included age, sex, paraprotein type, disease stage, and chemotherapy . RESULTS--Specific antibody titres to pneumococcal capsular polysaccharides and tetanus and diphtheria toxoids were significantly reduced compared with the control population . Low antipneumococcal and anti Escherichia coli titres correlated with risk of serious infection and low anti-pneumococcal titres with severity of non-paraprotein immunosuppression . In 41 immunised patients responses to Pneumovax II, tetanus and diphtheria toxoids were poor; IgG subclass levels were significantly reduced and a poor IgG response to Pneumovax II immunisation was associated with an increased risk of septicaemia and low IgG2 levels . The overall serious infection rate was 0.92 infections per patient year and was four times higher during periods of active disease (1.90) compared with plateau phase myeloma (0.49) . The predominant site of infection was the respiratory tract . Clinical and laboratory parameters showed only male sex and reduced non-paraprotein IgG and IgA levels to be significantly associated with at least one serious infection . CONCLUSIONS--A subgroup of patients with myeloma with poor IgG responses to exogenous antigens, who are at increased risk of serious infection, can be identified and may benefit from replacement immunoglobulin therapy to reduce the risk of infection.

Ther Umsch, 1995 Mar, 52(3), 179 - 82
{Diarrhea, coli infection, septic encephalopathy: escalation of a seemingly banal symptom}; Bodmann KF et al.; Septic encephalopathy is an early manifestation of sepsis . Changes in consciousness, focal or generalized seizures, multifocal myoclonus and/or varying hemiparesis are common clinical findings . All of these symptoms are reversible when sepsis has been successfully treated . Because there are no generally accepted criteria for the diagnosis of septic encephalopathy, it is a diagnosis of exclusion . We report the case of a 68-year-old patient who developed septic encephalopathy secondary to diarrhea and E . coli sepsis . In this case, symptoms of septic encephalopathy were fully reversed after the patient's E . coli sepsis had been adequately treated.

Mol Immunol, 1995 Mar, 32(4), 295 - 302
Mapping of the antigenic and allergenic epitopes of Lol p VB using gene fragmentation; Ong EK et al.; The recombinant proteins of Lol p VA and Lol p VB expressed in E . coli reacted with IgE antibodies from sera of allergic patients and mAbs FMC A7 and PpV1 . Cross-absorption analyses using these recombinant proteins showed that Lol p VA and Lol p VB possess both similar and unique IgE binding determinants . Gene fragmentation was utilized to localize the antigenic and allergenic determinants of Lol p VB . When full-length cDNA of Lol p VB was digested into three fragments and expressed as the fusions from the glutathione transferase of pGEX vectors, fragments Met1-Val196 and Asp197-Val339 bound IgE while fragment Met1-Pro96 did not . The data suggest that there are at least two IgE binding determinants in Lol p VB . In addition, only fragment Met1-Val196 reacted with mAb PpV1 . The localization of these determinants was further resolved using random fragment expression libraries . The mAb PpV1 determinant was near the N-terminal region of Lol p VB molecule . The IgE binding determinants were distributed in the central region: region I (amino acids 111-195) and II (199-254) . These IgE binding determinants are conserved in Lol p VA.

Mol Immunol, 1995 Mar, 32(4), 249 - 58
Preparation and characterization of a disulfide-stabilized Fv fragment of the anti-Tac antibody: comparison with its single-chain analog; Webber KO et al.; Recombinant DNA techniques now allow the production of "mini-antibodies" called Fv fragments . These have been produced either as the independent variable domains of the heavy and light chains non-covalently associated in one-to-one stoichiometry or as single-chain gene products with the two domains linked by an intervening peptide sequence . Although Fv fragments can have excellent binding properties, they are often difficult to produce in good yield and lack the characteristic stability of whole antibodies . To improve the stability of the Fv molecule, we have introduced a cysteine residue into conserved framework regions of both the heavy and light variable domains from the anti-Tac antibody at positions compatible with the formation of an interdomain disulfide linkage (i.e . VH-44 and VL-99) . The mutant subunits form a disulfide-bonded Fv molecule, which binds to the alpha-subunit of the IL2 receptor (IL2R alpha) with an affinity identical to that of humanized anti-Tac IgG . This disulfide-stabilized Fv (dsFv) proved to be substantially more resistant to denaturation by heat or urea treatment than the single-chain Fv (scFv) . Furthermore, the yield of dsFv is -four-fold higher than that of the single-chain analog.

Gene Ther, 1995 Mar, 2(2), 138 - 42
Efficient in vivo transduction of the neonatal mouse liver with pseudotyped retroviral vectors; Miyanohara A et al.; Ideal methods for human gene therapy will eventually include direct gene transfer to defective tissues in a patient in vivo . Toward that goal, we have used high titer, pseudotyped retroviral vectors expressing genes for the Escherichia coli beta-galactosidase (lacZ) or hepatitis B virus surface antigen (HBsAg) to infect mouse liver by in vivo direct injection into the liver parenchyma . We have found that a single percutaneous injection of small volumes of vectors into the newborn mouse liver leads to transduction of at least 25-30% of the hepatocytes throughout the liver, as judged by in situ staining of liver sections for beta-gal activity at 4 weeks after injection . We have demonstrated that stable levels of HBsAg were also detected in the circulation of injected mice up to 4 months after HBsAg-vector injection . We suggest that the high efficiency of in vivo transduction in the neonatal liver and subsequent stable transgene expression by high-titer pseudotyped retroviral vectors in the absence of an invasive partial hepatectomy may effectively be applied to gene therapy studies in a number of human liver disease {corrected}.

Photochem Photobiol, 1995 Mar, 61(3), 281 - 4
Unique DNA repair property of an ultraviolet-sensitive (radC) mutant of Dictyostelium discoideum; Okaichi K et al.; Dictyostelium discoideum is an organism that shows higher UV resistance than other organisms, such as Escherichia coli and human cultured cells . We examined the removal of cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts from DNA in the radC mutant and the wild-type strain using an enzyme-linked immunosorbent assay with monoclonal antibodies . Wild-type cells excised more than 90% of both CPD and 6-4 photoproducts within 4 h . Dictyostelium discoideum appeared to have a special repair system, because 6-4 photoproducts were repaired faster than CPD in E . coli and human cultured cells . In radC mutant cells, although only 50% of CPD were excised from DNA within 8 h, effective removal of 6-4 photoproducts (80% in 8 h) was observed . Excision repair-deficient mutants generally cannot remove both CPD and 6-4 photoproducts . Though the radC mutant shows deficient excision repair, it can remove 6-4 photoproducts to a moderate degree . These results suggest that D . discoideum has two kinds of repair systems, one mainly for CPD and the other for 6-4 photoproducts, and that the radC mutant has a defect mainly in the repair enzyme for CPD.

Trends Biochem Sci, 1995 Mar, 20(3), 109 - 13
How specific is the first recognition step of homologous recombination?
Rao BJ, Chiu SK, Bazemore LR, Reddy G, Radding CM.
The Escherichia coli RecA protein promotes homologous recognition in base triplets via non-Watson-Crick bonds that differ from those formed nonenzymically from DNA consisting of runs of purines or pyrimidines . Base substitutions reveal recognition to be permissive, consistent with a search for homology that achieves speed at the cost of precision.

Eur J Biochem, 1995 Mar 1, 228(2), 515 - 23
Molecular cloning of a cDNA that encodes the precursor to several exogastrula-inducing peptides, epidermal-growth-factor-related polypeptides of the sea urchin Anthocidaris crassispina; Yamasu K et al.; Complementary DNA clones for exogastrula-inducing peptides (EGIPs) of the sea urchin Anthocidaris crassispina, which are related to epidermal growth factor (EGF), were obtained from a cDNA library of late gastrula embryos using, as probe, the partial cDNA for one of the EGIP (EGIP-D) obtained by the reverse-transcription PCR method . The longest cDNA was composed of 1662 bp, and encoded a protein of approximately 36 kDa with a region that resembled a signal sequence . The deduced protein contains the sequences of EGIP-C, EGIP-D, and EGIP-A in that order, followed by the sequence for an unidentified EGIP-like polypeptide . When expressed in Escherichia coli as a fusion protein with beta-galactosidase, the product for the cDNA was specifically recognized by a rabbit antibody raised against EGIP-D that had been purified from embryos . Characteristic amino acid residues were found around the N-terminus and the C-terminus of each EGIP sequence, suggesting a specific processing mechanism for the generation of the individual EGIPs from the precursor . RNA-blot analysis revealed the presence of EGIP mRNA in unfertilized eggs . The level of this mRNA decreased gradually after fertilization, began to increase dramatically after the onset of gastrulation, and continued to increase through the pluteus stage . Genomic Southern-blot analysis suggested that this gene is present as a single copy . A homology search showed that the EGIP cDNA has a similarity to the cDNA for SpEGF2 which was cloned as a gastrula-specific gene in another sea urchin, Strongylocentrotus purpuratus.

Eur J Biochem, 1995 Mar 1, 228(2), 473 - 9
Cloning and expression of carbonyl reductase from rat testis; Wermuth B et al.; Carbonyl reductase is a cytosolic, monomeric, NADPH-dependent oxidoreductase with broad specificity for carbonyl compounds and a general distribution in human and many animal tissues . A carbonyl-reductase-like enzyme is exclusively expressed in rat reproductive tissues and adrenal glands . To understand the structural and functional relationships between the two enzymes, we cloned and sequenced the cDNA for the enzyme from rat testis and overexpressed the encoded protein in Escherichia coli . Three types of cDNA coding for the same protein but differing in their 5'-untranslated regions were isolated . The encoded protein had the same length as, and showed 86% positional identity with, carbonyl reductase from human placenta and liver . The recombinant enzyme exhibited the same substrate specificity and susceptibility to inhibitors as the enzyme isolated from rat testis, and also closely resembled human carbonyl reductase . Similar to the human enzyme, the recombinant rat enzyme catalyzed its own modification by pyruvate and 2-oxoglutarate, respectively . We conclude that carbonyl reductase from rat testis and human tissues represent species-specific forms of the same enzyme.

Eur J Biochem, 1995 Mar 1, 228(2), 395 - 402
1H and 15N resonance assignments and structure of the N-terminal domain of Escherichia coli initiation factor 3; Garcia C et al.; Initiation factor IF3 from Escherichia coli is composed of two domains connected by a hydrophilic peptide . In this study, the N-terminal domain (residues 7-83) has been overexpressed, 15N labelled and purified . NMR assignments for this domain have been obtained by two-dimensional and three-dimensional heteronuclear and homonuclear spectroscopy . Using distance geometry and simulated annealing, a three-dimensional solution structure was calculated using 506 NOE and 56 dihedral angle restraints . The resulting structure is composed of a five-stranded antiparallel beta sheet surrounded by two alpha helices . Since the heteronuclear 1H-15N correlation spectrum of the N-terminal domain of IF3 is an almost exact subset of that of the native protein, the assignments obtained and the structure calculated should be directly transposable to the full-length protein.

Eur J Biochem, 1995 Mar 1, 228(2), 381 - 7
Cloning, expression and tissue distribution of mouse tetrameric carbonyl reductase . Identity with an adipocyte 27-kDa protein; Nakanishi M et al.; We previously cloned a cDNA for pig lung tetrameric carbonyl reductase which shows significant similarity to a putative 27-kDa protein predicted from the cDNA for murine adipocyte RNA which had been increased in its differentiation {Nakanishi, M., Deyashiki, Y., Nakayama, T., Sato, K . & Hara, A . (1993) Biochem . Biophys . Res . Commun . 194, 1311-1316} . In this investigation, we isolated and sequenced a full-length cDNA for the tetrameric enzyme from a mouse lung cDNA library . It consisted of 984 bp and coded for a protein of 244 amino acid residues, of which 202 residues were identified by protein sequencing . The expression of the cDNA in Escherichia coli resulted in synthesis of a protein structurally and functionally similar to the enzyme purified from mouse lung . The nucleotide sequence of the cDNA was virtually identical to that of the cDNA for the adipocyte 27-kDa protein . Although Northern-blot analysis of mouse tissues showed that enzyme mRNA to be 1.1 kb only in lung, low expression of the mRNA in all the extrapulmonary tissues, including adipose tissue, was demonstrated by a reverse-transcription PCR method . Western-blot analysis also indicated the presence of the enzyme in the adipose tissue . This is the first report on an identification of the putative gene product of adipocytes as tetrameric carbonyl reductase, the expression of which is tissue-specifically regulated.

Bull Math Biol, 1995 Mar, 57(2), 247 - 76
Dynamical behaviour of biological regulatory networks--I . Biological role of feedback loops and practical use of the concept of the loop-characteristic state; Thomas R et al.; In the field of biological regulation, models dictated by experimental work are usually complex networks comprising intertwined feedback loops . In this paper the biological roles of individual positive loops (multistationarity, differentiation) and negative loops (homeostasis, with or without oscillations, buffering of gene dosage effect) are discussed . The relationship between feedback loops and steady states is then clarified, and the problem: "How can one conveniently disentangle complex networks?" is then considered . Initiated long ago, logical descriptions have been generalized from various viewpoints; these developments are briefly discussed . The recent concept of the loop-characteristic state, defined as the logical state located at the level of the thresholds involved in the loop, together with its application, are then presented . Biological applications are also discussed.

Mol Biol Evol, 1995 Mar, 12(2), 198 - 207
Dynamics of IS-related genetic rearrangements in resting Escherichia coli K-12; Naas T et al.; An analysis of restriction fragment length polymorphism (RFLP) using eight residential insertion sequence (IS) elements as hybridization probes reveals that the genome of resting bacteria is more dynamic than it was long believed . Escherichia coli strains stored in agar stabs for up to 30 yr accumulate a genetic variation which is correlated to time of storage . This spontaneous mutagenesis is often IS-specific, with particularly high activity for IS5, and thus suggests that transpositional DNA rearrangements are a major cause for the observed genetic polymorphism . The RFLP patterns indicate a burst of IS30 transposition to occur occasionally . Mutation rate is estimated by two different methods to roughly 10(-5) IS-related DNA rearrangements per bacterial chromosome per hour of storage for the eight IS elements studied . A pedigree derived from the RFLP data reveals that populations had evolved independently in each stab and showed no signs of convergence . Relics of an assumed ancestral population were still present in the stab cultures, but the elder stabs provided mostly mutants . These results indicate that cells placed under nutritional deprivation might have a highly plastic genome and suggest that such plasticity might play an adaptive role.

Cell Signal, 1995 Mar, 7(3), 225 - 33
Effect of the diglyceride lipase inhibitor, RG80267, on epithelial chloride secretion induced by various agents; Barrett KE; We previously reported that a diglyceride lipase inhibitor, RG80267, inhibits chloride secretion stimulated by adenosine agonists, stimuli whose effects appear unrelated to cAMP, cGMP or cytosolic calcium . Here, the effect of RG80267 on Cl- secretory responses to agents which do utilize these messengers was examined . RG80267 inhibited responses to vasoactive intestinal polypeptide, forskolin (cAMP-dependent) and E . coli heat stable enterotoxin (cGMP-dependent), but not to prostaglandin E1 or cholera toxin (cAMP-dependent) . RG80267 enhanced responses to histamine (calcium-dependent) . The inhibitory effect of RG80267 was not due to inhibition of cAMP accumulation . Arachidonic acid release may participate in chloride secretion . Vasoactive intestinal polypeptide, but not prostaglandin E1, released radiolabel from cells preloaded with {3H}arachidonic acid . There may thus be differences between mechanisms of various cyclic nucleotide-dependent chloride secretory responses . Arachidonic acid release may modulate the extent of secretion elicited by some secretagogues.

Hua Xi Yi Ke Da Xue Xue Bao, 1995 Mar, 26(1), 45 - 9
{Cloning and sequencing of CTP synthetase cDNA from Chinese hamster cells}; Zhai C et al.; The CTP synthetase cDNA was cloned from Chinese hamster lung cells V79 . By comparison of the amino acid sequence of CTP synthetase with the counterpart of other species published, it was show that the CTP synthetase was a highly conserved and stable gene among the mammalian cells, but more differences of the CTP synthetase sequence could be seen between procaryotic and eukaryotic cells . This phenomenon suggested that the catalysing model of the enzyme might have some small differences in the organisms.

Hua Xi Yi Ke Da Xue Xue Bao, 1995 Mar, 26(1), 1 - 5
{Identification of pathogenic and nonpathogenic leptospires by recombinant DNA probe}; Jiang N et al.; A gene bank of L . interrogans serovar lai strain 017 was constructed with plasmid vector pUC 18 . Recombinant plasmids designated pDJ 6 and pDJ 8 were screened from the gene bank . Inserted fragments of them are 1.9kb and 2.2kb respectively . Diglabelled 1.9kb inserted fragment of pDJ 6 . Results showed that the probe had hybridization with pathogenic leptospires, but it did not have hybridization with nonpathogenic leptospires such as L . biflexa strain Patoc I and Leptonema illini . The probe did not have hybridization with nonhomologous DNA (e.g . human leucocyte and E . coli JM 103), either . So, the recombinant probe is a good tool for distinguishing and identifying genus, species, pathogenic leptospires and nonpathogenic leptospires.

Mol Biochem Parasitol, 1995 Mar, 70(1-2), 19 - 31
Characterization of genes encoding members of the nuclear hormone receptor superfamily from Onchocerca volvulus; Yates RA et al.; Several lines of evidence suggest that molting in parasitic nematodes is controlled through the action of steroid molting hormones, or ecdysones . In other organisms, the central mediator of steroid hormone action is the hormone receptor . These receptor molecules are members of a superfamily of proteins called the nuclear hormone receptor family . Using an oligonucleotide derived from the amino-acid sequence of the Drosophila melanogaster ecdysone receptor, genes encoding homologues of the nuclear hormone receptor family were identified in the genome of the human filarial parasite Onchocerca volvulus . The O . volvulus genome contains at least three genes that encode putative members of the nuclear hormone receptor superfamily . A complete cDNA for one of these genes, designated OvNHR-1, has been isolated and characterized . The OvNHR-1 cDNA was 2378 bp in length, and contained a single open reading frame of 1104 bp . The open reading frame encoded a peptide with all of the features characteristic of a member of the nuclear hormone receptor superfamily of proteins . OVNHR-1 appeared to be encoded by a single-copy gene . Expression of the mRNA corresponding to OvNHR-1 was developmentally regulated, with maximal expression occurring during early embryogenesis . The polypeptide encoded by the OvNHR-1 open reading frame is antigenic in a minority of individuals exposed to O . volvulus.

Mol Biochem Parasitol, 1995 Mar, 70(1-2), 107 - 18
A member of the ClpB family of stress proteins is expressed during heat shock in Leishmania spp; Hubel A et al.; We have identified and isolated the Leishmania major homologue to the bacterial ClpB gene and to the yeast Hsp104 gene . ClpB in Leishmania major is a single-copy gene and encodes a low-abundance mRNA which is induced several-fold during a heat stress . We raised antibodies against the product of the recombinant gene and show that the leishmanial ClpB encodes a predominantly cytoplasmic protein of approx . 100 kDa which is detectable in Leishmania promastigotes of various species after exposure to elevated temperatures . We, therefore, term this protein Hsp100.

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