Electrophoresis, 1996 May, 17(5), 932 - 7 Investigation on minor degraded derivatives of the recombinant hirudin variant HM2 from Hirudinaria manillensis isolated by isoelectric focusing in multicompartment electrolyzers; Bossi A et al.; On isoelectric focusing in immobilized pH gradients (IPG) a preparation of recombinant hirudin from Hirudinaria manillensis, purified to homogeneity, was found to still contain a total of 5% minor components: three with higher pI values (pIs 4.10, 4.25 and 4.31), one with a lower pI value (pI 3.98) as compared with the main form (pI 4.03) . Multicompartment electrolyzers with isoelectric membranes and micropreparative IPG gel slabs allowed the recovery of pure fractions of such minor components, which were further characterized by electrospray mass spectra, limited proteolysis, and sequence analysis . All four minor isoforms were found to be cleavage products of the parent, full-length hirudin molecule (molecular mass 6797 Da), as follows: the pI 4.31 (5032 Da) had lost sixteen amino acids from the N-terminus, the pI 4.25 (6212 Da) lacked five amino acids from the C-terminus, the pI 4.10 (2980 Da) was a cleavage product at residue Cys37, and the pI 3.98 (6610 Da) lacked the dipeptide Val-Ser at the N-terminus . Combining the extreme resolving power of IPGs with the high accuracy of mass spectra was found to be an attractive strategy in decoding post-synthetic modifications often encountered in r-DNA proteins. Environ Health Perspect, 1996 May, 104 Suppl 3, 683 - 6 Distinction of mutagenic carcinogens from a mutagenic noncarcinogen in the big blue transgenic mouse; Cunningham ML et al.; The aromatic amines 2,4-diaminotoluene (2,4-DAT) and 2,6-diaminotoluene (2,6-DAT) are structural isomers that have been extensively studied for their mutagenic and carcinogenic characteristics . Both compounds are rapidly absorbed after oral administration and are equally mutagenic in the Ames test; however, 2,4-DAT is a potent hepatocarcinogen, whereas 2,6-DAT does not produce an increased incidence of tumors in rats or mice at similar doses . The Big Blue transgenic B6C3F1 mouse carries multiple copies of the lacl mutational target gene . Our studies were designed to determine whether the Big Blue system could be used to detect differences in the vivo mutagenic activity between the carcinogen-noncarcinogen pair 2,4-DAT and 2,6-DAT and to determine whether the in vivo mutagenesis assay results correspond to the rodent carcinogen bioassay results . Male B6C3F1 transgenic mice were exposed to 2,4-DAT or 2,6-DAT at 0 or 1,000 ppm in the diet for 30 and 90 days or to dimethylnitrosamine as a positive control . Mutant frequencies were nearly identical for all three groups at 30 days, while at 90 days the mutant frequency for the hepatocarcinogen 2,4-DAT (12.1 +/- 1.4 x 10(-5)) was significantly higher (p < 0.01) as compared to both age-matched (spontaneous) controls (5.7 +/- 2.9 x 10(-5)) and the 2,6-DAT-exposed group (5.7 +/- 2.4 x 10(-5)) . Results from this study demonstrate that the Big Blue transgenic mutation assay can distinguish differences in vivo between the mutagenic responses of hepatic carcinogens ad a noncarcinogen; is sensitive to mutagens through subchronic dietary exposure; and yields a differential response depending upon the length of time mice are exposed to a mutagen. Environ Health Perspect, 1996 May, 104 Suppl 3, 557 - 62 Involvement of cytochrome P450, glutathione S-transferase, and epoxide hydrolase in the metabolism of aflatoxin B1 and relevance to risk of human liver cancer; Guengerich FP et al.; In recent years there has been considerable interest in the effect of variations in activities of xenobiotic-metabolizing enzymes on cancer incidence . This interest has accelerated with the development of methods for analyzing genetic polymorphisms . However, progress in epidemiology has been slow and the contributions of polymorphisms to risks from individual chemicals and mixtures are often controversial . A series of studies is presented to show the complexities encountered with a single chemical, aflatoxin B1 (AFB1) . AFB1 is oxidized by human cytochrome P450 enzymes to several products . Only one of these, the 8,9-exo-epoxide, appears to be mutagenic and the others are detoxication products . P450 3A4, which can both activate and detoxicate AFB1, is found in the liver and the small intestine . In the small intestine, the first contact after oral exposure, epoxidation would not lead to liver cancer . The (nonenzymatic) half-life of the epoxide has been determined to be approximately 1 sec at 23 degrees C and neutral pH . Although the half-life is short, AFB1-8,9-exo-epoxide does react with DNA and glutathione S-transferase . Levels of these conjugates have been measured and combined with the rate of hydrolysis in a kinetic model to predict constants for binding of the epoxide with DNA and glutathione S-transferase . A role for epoxide hydrolase in alteration of AFB1 hepatocarcinogenesis has been proposed, although experimental evidence is lacking . Some inhibition of microsome-generated genotoxicity was observed with rat epoxide hydrolase; further information on the extent of contribution of this enzyme to AFB1 metabolism is not yet available. J Neurochem, 1996 May, 66(5), 1819 - 25 A novel rat tyrosine hydroxylase mRNA species generated by alternative splicing; Laniece P et al.; Tyrosine hydroxylase (TH) catalyzes the first and rate-limiting step in the biosynthesis of catecholamines . Among the various mechanisms implicated in the regulation of TH activity, alternative splicing of TH primary transcript has been described as a characteristic of higher primates and Drosophila . We investigated whether there is such a regulatory mechanism in the rat . Reverse transcriptase-PCR experiments were performed with RNA from PC12 cells . A new TH mRNA species was evidenced, resulting from the use of an alternative donor site in exon 2 . RNase protection assays and in situ hybridization experiments detected this mRNA species in the adrenal medulla but not in the main catecholaminergic nuclei of the CNS . The corresponding putative protein lacks 33 amino acids in the N-terminal regulatory domain . A recombinant protein was produced in E . coli . Its in vitro specific activity was similar to that of the previously identified TH protein. Res Commun Mol Pathol Pharmacol, 1996 May, 92(2), 225 - 32 Copper replacement of cadmium-binding alpha-fragment of metallothionein expressed in Escherichia coli; Kurasaki M et al.; Titration studies of Cd-binding alpha-fragment, the carboxyl-terminal half of human metallothionein-2, which was independently expressed in Escherichia coli as a Cd-binding form, with Cu were performed . The Cu saturated alpha-fragment was not obtained when the alpha-fragment incubated with Cu (I), although completely saturated MT with Cu (I) was successfully obtained in the same conditions . The stoichiometry of the alpha-fragment replaced with Cu (I) was Cu 4 and Cd 1 . It is considered that the Cu-binding alpha-fragment required the presence of Cd (II) to form a stable structure . Our results could suggest that each fragment was independent in the metal binding processes, e.g . alpha-fragment for Cd-binding. J Hepatol, 1996 May, 24(5), 532 - 8 An ELISA for the detection of antibody to the core antigen of hepatitis C virus: use in diagnosis and analysis of indeterminate samples; Trowbridge R et al.; AIMS: In order to examine more carefully the natural history of hepatitis C virus infection and to determine a role for anti-core in the discrimination of indeterminate samples, a solid phase ELISA to detect antibody of the immunoglobulin G class to the hepatitis C virus core antigen was developed using purified protein expressed in Escherichia coli from a major portion of the core antigen coding region . METHODS/RESULTS: In a study which examined 651 samples submitted for routine testing by a commercial ELISA (Ortho), only 11 samples showed discrepant results; of these, 10 were Ortho ELISA positive, anti-core negative and one was Ortho ELISA negative anticore positive . Supplemental tests showed that 5/10 of these samples were anti-HCV negative by RIBA and the reciprocal 5 were negative for anti-C22 but positive for anti-C100 and anti-C33 . The Ortho ELISA negative, anticore positive sample was weakly positive for anti-C22 . The anti-core ELISA was then used to examine 67 indeterminate samples from the blood bank; 11/11 samples which were HCV-RNA positive were anti-core positive and 7/56 samples which were HCV-RNA negative were anti-core positive . The anti-core titre was then examined in two groups of indeterminate samples; group 1, polymerase chain reaction-positive, anti-core positive and group 2, polymerase chain reaction-negative, anti-core positive . The geometric mean anti-core titres in these groups were 1 x 10(-3.6) and 1 x 10(-2.3), respectively . Thus in this group of indeterminate samples, all samples (except one) with an anti-core titre > or = 1/200 were polymerase chain reaction-positive, confirming a close correlation between anti-core levels and hepatitis C viraemia . Anti-core was detected with equal efficiency in patients infected with genotypes which differed to that used to express the recombinant core antigen. Arch Pediatr, 1996 May, 3(5), 470 - 2 {Cystic adenomatoid malformation of the lung revealed in a newborn infant by an image of a lung abscess}; Kieffer F et al.; BACKGROUND: Cystic adenomatoid malformation, a rare pulmonary malformation, usually appears as a cystic mass, radiologically . It may be infected and confusion has also arisen in distinguishing it from pneumonia with pneumatoceles . CASE REPORTS: A full-term boy suffered from severe neonatal respiratory distress . Pregnancy had been uneventful despite the fact that his mother had insulin-dependent diabetes . Prenatal ultrasonographies did not reveal any abnormality . On day 2, X-rays showed a right pulmonary mass that appeared solid . The patient was treated for E Coli sepsis . Subsequently, the pulmonary mass became lacent, cystic, fluid-filled, resembling an abscess; the CT scan confirmed these features . As the lesion increased in volume, a limited resection was performed . Histologic examination showed adenomatoid proliferation of bronchiolar elements with formation of cysts and necrosis . CONCLUSION: Infection of cystic adenomatoid malformation may supervene the first days of life resulting in a lung abscess appearance. Plant Mol Biol, 1996 May, 31(2), 659 - 65 The cyanobacterial genome contains a single copy of the ffh gene encoding a homologue of the 54 kDa subunit of signal recognition particle; Packer JC et al.; Cyanobacteria possess thylakoid membranes that differ in their protein composition from the cytoplasmic membrane . To study possible pathways of protein targeting to these membranes, we have investigated whether or not cyanobacteria have a homologue or homologues of the signal recognition particlelike chaperone Ffh . We have amplified a fragment of ffh by polymerase chain reaction and established that ffh is present as a single copy in the genomes of three cyanobacterial species . We have cloned and sequenced ffh from Synechococcus sp . PCC7942 and predict that Ffh functions as a ribonucleoprotein in cyanobacteria and chloroplasts. Plant Mol Biol, 1996 May, 31(2), 405 - 12 Characterization of eight new members of the calmodulin-like domain protein kinase gene family from Arabidopsis thaliana; Hrabak EM et al.; A family of calcium-responsive protein kinases is abundant in plant cell extracts but has not been identified in animals and fungi . These enzymes have a unique structure consisting of a protein kinase catalytic domain fused to carboxy-terminal autoregulatory and calmodulin-like domains . In this report, we present the amino acid sequences for eight new Arabidopsis cDNA clones encoding isoforms of this enzyme . Three isoforms were expressed as fusion proteins in Escherichia coli and exhibited calcium-stimulated protein kinase activity . We propose CPK as the gene designation for this family of enzymes and describe a phylogenetic analysis for all known isoforms. Biokhimiia, 1996 May, 61(5), 786 - 99 {Cytochrome bd: structure and properties}; Borisov VB; Literary evidence concerning the arrangement and functioning of the cytochrome bd complex is reviewed with particular emphasis on ligand-binding properties of the enzyme . Some novel data on cytochrome bd interaction with carbon monoxide, cyanide and hydrogen peroxide are presented. Genome Res, 1996 May, 6(5), 414 - 30 A P1-based physical map of the Drosophila euchromatic genome; Kimmerly W et al.; A PCR-based sequence-tagged site (STS) content mapping strategy has been used to generate a physical map with 90% coverage of the 120-Mb euchromatic portion of the Drosophila genome . To facilitate map completion, the bulk of the STS markers was chosen in a nonrandom fashion . To ensure that all contigs were localized in relation to each other and the genome, these contig-building procedures were performed in conjunction with a large-scale in situ hybridization analysis of randomly selected clones from a Drosophila genomic library that had been generated in a P1 cloning vector . To date, the map consists of 649 contigs with an STS localized on average every 50 kb . This is the first whole genome that has been mapped based on a library constructed with large inserts in a vector that is maintained in Escherichia coli as a single-copy plasmid. J Cell Sci, 1996 May, 109 ( Pt 5), 1071 - 9 Activation of the Xenopus cyclin degradation machinery by full-length cyclin A; Jones C et al.; The entry into mitosis is dependent on the activation of mitotic forms of cdc2 kinase . In many cell types, cyclin A-associated kinase activity peaks just prior to that of cyclin B, although the precise role of cyclin A-associated kinase in the entry into mitosis is still unclear . Previous work has suggested that while cyclin B is capable of triggering cyclin destruction in Xenopus cell-free systems, cyclin A-associated kinase is not able to support this function . Here we have expressed a full-length human cyclin A in Escherichia coli and purified the protein to homogeneity by virtue of an N-terminal histidine tag . We have found that when added to Xenopus cell-free extracts free of cyclin B and incapable of protein synthesis, the temporal pattern of cyclin A-associated cdc2 kinase activity showed distinct differences that were dependent on the concentration of cyclin A added . When cyclin A was added to a concentration that generated levels of cdc2 kinase activity capable of inducing nuclear envelope breakdown, the histone H1 kinase activity profile was bi-phasic, consisting of an activation phase followed by an inactivation phase . Inactivation was found to be due to cyclin destruction, which was prevented by mos protein . Cyclin destruction was followed by nuclear reassembly and an additional round of DNA replication, indicating that there is no protein synthesis requirement for DNA replication in this embryonic system . It has been suggested that the evolutionary recruitment of cyclin A into an S phase function may have necessitated the loss of an original mitotic ability to activate the cyclin destruction pathway . The results presented here indicate that cyclin A has not lost the ability to activate its own destruction and that cyclin A-mediated activation of the cyclin destruction pathway permitted destruction of cyclin B1 as well as cyclin A, indicating that there are not distinct cyclin A and cyclin B destruction pathways . Thus the ordered progression of the cell cycle requires the careful titration of cyclin . A concentration in order to avoid activation of the cyclin destruction pathway before sufficient active cyclin B/cdc2 kinase has accumulated. Eur J Cell Biol, 1996 May, 70(1), 42 - 53 Immunodetection of alpha 1-3 fucosyltransferase (FucT-V); Borsig L et al.; The fucosyltransferases constitute a family of glycosyltransferases incorporating fucose residues into glycoprotein or glycolipid glycans . They afford one of the possible termination steps of glycoconjugate biosynthesis creating the sialyl Lewisx or sialyl Lewisa determinant, which play an important role in cell-cell interaction . While cDNA, chromosomal localization and kinetic properties of a number of fucosyltransferases are known, immunocytochemical localization and trafficking studies have been delayed because of the lack of specific antibodies due to the pronounced homology of alpha 1, 3 fucolsyltransferases III, V and VI . Here we report development and characterization of monospecific polyclonal antibodies to alpha 1-3 fucosyltransferase V (FucT-V) and their application for immunodetection in transfected cells . Antisera against FucT-V were raised in two different ways: first by producing a fusion protein beta-galactosidase-FucT-V in Escherichia coli, and by synthesizing a peptide stretch specific for FucT-V . Polyclonal antisera were raised against each of both antigens and characterized by enzyme-linked immunosorbent assay, neutralization of activity, immunoblotting, immunofluorescence and immunoprecipitation of metabolically labeled COS cells, transiently transfected with cDNA encoding FucT-V . Both antibodies recognized only FucT-V . No cross-reactivity to FucT-III or FucT-VI was observed . FucT-V was localized mainly to the Golgi apparatus by colocalization with beta 1, 4-galactosyltransferase, and to the cell surface of COS, CHO and HeLa cells . Expression of FucT-V in COS cells revealed three enzyme forms of 58, 53 and 50 kDa, respectively . These size differences arose by post-translational modifications, as shown by pulse-chase experiments . Our results indicate that alpha 1-3 fucosyltransferase is a Golgi-associated enzyme and suggest its possible occurrence on the cell surface. Inflamm Res, 1996 May, 45(5), 254 - 8 Nitric oxide enhances cyclooxygenase activity in articular cartilage; Manfield L et al.; Nitric oxide (NO) is a small messenger molecule synthesized by a family of enzymes, the nitric oxide synthases . Cyclooxygenases are a group of proinflammatory enzymes that release prostaglandins including prostaglandin E2 (PGE2) . Both nitric oxide synthase and cyclooxygenase are involved in the inflammatory cascade of arthritis . However, the relationship between these two enzymes and their products has not been explored in articular cartilage . Here we show that in cultured bovine chondrocytes and explants of human osteoarthritic cartilage both nitric oxide synthase and cyclooxygenase activities were induced by the inflammatory mediators, lipopolysaccharide, and interleukin-1 beta or tumor necrosis factor-alpha . When nitric oxide synthase activity was inhibited, PGE2, synthesis was inhibited . NO donors also induced PGE2 synthesis and NO scavengers inhibited cyclooxygenase activity . Taken together, these results support the concept that PGE2 synthesis is directly related to NO formation and that NO may modulate cyclooxygenase activity in articular cartilage. Inflamm Res, 1996 May, 45(5), 246 - 53 Human whole blood assays for inhibition of prostaglandin G/H synthases-1 and -2 using A23187 and lipopolysaccharide stimulation of thromboxane B2 production; Young JM et al.; When freshly drawn, heparinized human whole blood is incubated with 50 microM calcium ionophore A23187, platelets are stimulated to produce thromboxane B2 (TxB2) by activation of prostaglandin G/H synthase-l (PGHS-1) . TxB2 concentration, as measured by immunoassay, is maximal at 20-30 min and declines thereafter . Addition of acetylsalicylic acid (IC50 = 2.8 microM) or other nonsteroidal antiinflammatory drugs (NSAIDs) 15 min or 4.5h prior to 30 min stimulation with ionophore results in concentration dependent inhibition of TxB2 production . When blood is incubated with 0.01-10 micrograms/ml E . colilipopolysaccharide (LPS), PGHS-2 is induced and TxB2 levels become detectable at 3h and continue to increase through 24h . Using a 5h incubation with 10 micrograms/ml LPS, aspirin (10 microM added at 0 h), which is rapidly metabolized to salicylic acid, had no effect on 10 micrograms/ml LPS-induced TxB2, but inhibited TxB2 production by ionophore A23187 added at 4.5h through acetylation of pre-existing PGHS-1 . In a 5h assay, NSAIDs added at 0 h were compared for inhibition of TxB2 production stimulated by addition of ionophore A23187 at 4.5h (PGHS-1), or by addition of LPS at 0 h (PGHS-2) . Most NSAIDs were more potent against PGHS-1 than PGHS-2 . Diclofenac, naproxen and flufenamic acid were equipotent or slightly selective for PGHS-2 . Diflunisal and nimesulide were > 4-fold selective for PGHS-2, and NS-398 was > 30-fold selective for PGHS-2. Mol Microbiol, 1996 May, 20(3), 645 - 55 Identification and characterization of FrzZ, a novel response regulator necessary for swarming and fruiting-body formation in Myxococcus xanthus; Trudeau KG et al.; The frz genes of Myxococcus xanthus constitute a signal-transduction pathway that processes chemotactic information in a manner analogous to that found in enteric bacteria . Ultimately, these genes regulate the frequency of individual cell reversal . We report here the identification of a novel component of this signal-transduction pathway, designated frzZ, which was discovered as an open reading frame located 5' to the frz operon but transcribed in the opposite orientation . The translational start site of frzZ is 170 base pairs from that of frzA.frzZ utilizes a promoter similar to the sigma 70 promoters of Escherichia coli, and encodes a 290-amino-acid soluble protein, FrzZ (M(r) 30,500) . FrzZ contains two domains, both of which show strong homology to CheY and other members of the response-regulator family . Linking these domains is a 39-amino-acid region that is very rich in alanine and proline (38% Ala and 33% Pro) . A frzZ null mutant showed abnormally low reversal rates when compared to the wild-type control and was unable to form fruiting bodies on starvation medium, but it did form 'frizzy' aggregates . In addition, the frzZ mutant was defective in swarming, particularly on soft agar (0.3% w/v) . However, unlike most frz mutants, the frzZ mutant was able to respond to attractants and repellents in the spatial chemotaxis assay . The discovery of FrzZ demonstrates that the M . xanthus frz signal-transduction pathway utilizes multiple response-regulator (CheY-like) proteins. Mol Microbiol, 1996 May, 20(3), 621 - 32 Analysis of nitrate regulatory protein NarL-binding sites in the fdnG and narG operon control regions of Escherichia coli K-12; Darwin AJ et al.; During anaerobic growth, expression of the fdnGHI and narGHJI operons of Escherichia coli is induced by the NarL protein in response to nitrate . The fdnG operon control region contains four NarL-binding sites (termed NarL heptamers) between positions -70 and -130 . The two central NarL heptamers of fdnG are arranged as an inverted repeat and are essential for regulation by NarL . We used mutational analysis of these central heptamers to investigate the precise sequence requirements for NarL-dependent induction . Mutations were examined for their effects on NarL-dependent expression in vivo . Substitutions at position 1 of either heptamer had the strongest effect whereas substitutions at position 7 had the weakest effect . For some positions, alterations in both heptamers had a stronger effect than either of the single changes . The 2 bp spacing between these NarL heptamers was also important for normal nitrate induction . The narG operon control region has at least eight NarL heptamers arranged in two groups . Previous work has shown that nucleotide substitutions in two of these heptamers, centred at positions -195 and -89, severely reduce nitrate induction of narG operon expression in vivo and significantly interfere with NarL-DNA interactions in vitro . Substitutions in heptamers -185 and -101 affected narG operon induction only when the concentration of phospho-NarL was low (during growth in the presence of nitrite) . Changes in each of the other four NarL heptamers studied had little or no effect on nitrate or nitrite induction of narG operon expression or on NarL-DNA interactions in vitro. Mol Microbiol, 1996 May, 20(3), 613 - 20 Accessory proteins impose site selectivity during ColE1 dimer resolution; Guhathakurta A et al.; The cer-Xer dimer resolution system of plasmid ColE1 is highly selective, acting only at sites on the same molecule and in direct repeat . Recombination requires the XerCD recombinase and accessory proteins ArgR and PepA . The Escherichia coli chromosome dimer resolution site dif and the type II hybrid site use the same recombinase but are independent of ArgR and PepA and show no site selectivity . This has led to the proposal that ArgR and PepA are responsible for the imposition of constraint . We describe here the characterization of a novel class of "conditionally constrained' multimer resolution sites whose properties support this hypothesis . In the presence of ArgR and PepA, plasmids containing conditionally constrained sites are monomeric, but in their absence, extensive multimerisation is seen . A mutant ArgR derivative (ArgR110), which is defective in cer-mediated dimer resolution, remains able to prevent plasmid multimerisation by a conditionally constrained site . This implies that the accessory factors block recombination in trans rather than ensuring rapid multimer resolution . When the distance between the ArgR and XerCD binding sites in a conditionally constrained site was altered by a non-integral number of helical turns, the site became unconstrained . Constraint was restored by the insertion of a full helical turn. Mol Microbiol, 1996 May, 20(3), 549 - 58 Regulation of the expression of the traM gene of the F sex factor of Escherichia coli; Penfold SS et al.; Conjugative F-plasmid transfer is mediated by the transfer (tra) region which encodes nearly 40 genes, 25 of which are essential for this process in Escherichia coli . TraM is required for conjugation and is encoded on a separate operon between the origin of transfer and the traJ gene . The traJ gene product is the positive regulator of transcription of the 30 kb tra operon, the first gene of which is traY . Using primer-extension assays and immunoblots on the F plasmid itself and its derivatives, we demonstrate that F TraM regulates its own expression from two promoters and that it requires TraY as well as expression of the tra operon for maximal traM transcription . traY is the first gene in the tra operon under the control of the TraJ regulator, which is in turn negatively regulated by the antisense RNA, FinP, and the FinO protein . Thus, a control circuit has been established whereby traM is negatively regulated by the FinOP fertility inhibition system through its repression of TraJ expression, which adversely affects transcription of the traY gene. Mol Microbiol, 1996 May, 20(3), 489 - 96 Phosphorylation-dependent binding of BvgA to the upstream region of the cyaA gene of Bordetella pertussis; Karimova G et al.; In Bordetella pertussis, transcription of virulence-associated genes is regulated by the BvgS and BvgA proteins, members of the bacterial two-component signal-transduction family . BvgS is the transmembrane sensor and BvgA, in its phosphorylated form, is believed to be the key transcriptional activator in B . pertussis . However, the BvgA recognition sites in most virulence promoters have not yet been identified . To investigate the interaction of BvgA with the upstream region of cyaA, the gene encoding adenylate cyclase haemolysin, we have produced large amounts of BvgA in Escherichia coli . The protein was purified from inclusion bodies and then phosphorylated by acetyl phosphate . Using electrophoretic mobility-shift and footprinting assays, we provide evidence that BvgA cannot bind to the cyaA promoter unless it is phosphorylated . The phosphorylated form of BvgA (BvgA-P) is able to bind specifically to the upstream region of cyaA . Analysis of this region revealed that an unexpectedly large sequence, from -137 to -51, appears to be the target for BvgA-P binding, and probably contains multiple binding sites. Mol Microbiol, 1996 May, 20(3), 467 - 73 Very short patch repair: reducing the cost of cytosine methylation; Lieb M et al.; In Escherichia coli and related bacteria, the product of gene dcm methylates the second cytosine of 5'-CCWGG sequences (where W is A or T) . Deamination of 5-methylcytosine (5meC) results in C to T mutations . The mutagenic potential of 5meC is reduced by a system called very short patch (VSP) repair, which replaces T with C . T:G and U:G mispairs in the methylatable sequence and in related sequences are recognized by the product of vsr, a gene adjacent to dcm . Vsr creates a nick just 5' of the mispaired pyrimidine to initiate the repair . Additional products known to be required for VSP repair are DNA polymerase I and DNA ligase . MutS and MutL have a stimulatory role but are not required . The ability of Vsr to recognize T:G mispairs in sequences related to CCWGG is probably responsible for over- and under-representation of certain tetranucleotides in the E . coli genome . Although VSP repair reduces spontaneous mutations at 5meCs in replicating bacteria, mutation hot-spots persist at these sites . Under conditions that more accurately mimic the natural environment of E . coli, VSP repair appears to be effective in preventing mutation at 5meC. Br J Pharmacol, 1996 May, 118(1), 198 - 204 Effects of the endothelin receptor antagonist, SB 209670, on circulatory failure and organ injury in endotoxic shock in the anaesthetized rat; Ruetten H et al.; 1 . This study investigates the effects of the non-selective ETA/ETB receptor antagonist, SB 209670, on systemic haemodynamics, renal function, liver function, acid-base balance and survival in a rat model of endotoxic shock . 2 . Injection of E . coli lipopolysaccharide (LPS, 10 mg kg-1, i.v.) resulted in increases in the serum levels of tumour necrosis factor-alpha (TNF-alpha, maximum 60 min after LPS), endothelin-1, (ET-1; maximum 120 min after LPS), and interferon-gamma (IFN-gamma, maximum 180 min after LPS) . 3 . Injection of LPS also resulted in a fall in blood pressure from 113 +/- 3 mmHg (time = 0) to 84 +/- 4 mmHg at 360 min (n = 15) as well as a hyporeactivity to the vasoconstrictor responses elicited by noradrenaline (NA, 1 microgram kg-1, i.v.) . Pretreatment of rats with a continuous infusion of SB 209670 (3 mg kg-1, i.v . bolus + 100 micrograms kg-1, i.v . infusion commencing 15 min prior to LPS) significantly augmented the hypotension as well as the vascular hyporeactivity to NA caused by endotoxaemia . 4 . Pretreatment of LPS-rats with SB 209670 (3 mg kg-1, i.v . bolus given 15 min prior to LPS) or infusion of SB 209670 (bolus dose and infusion as above) resulted in a reduction in 6 h-survival from 71% (control) to 30% and 13%, respectively . 5 . Endotoxaemia for 4 h resulted in rises in the serum levels of urea and creatinine (indicators of renal failure), but not in the serum levels of bilirubin, GPT and GOT (indicators of liver dysfunction and/or hepatocellular injury) . Pretreatment of LPS-rats with SB 209670 (3 mg kg-1, i.v . bolus 15 min prior to LPS) significantly augmented the serum levels of creatinine, bilirubin, GPT and GOT caused by endotoxin . In addition, endotoxaemia caused, within 15 min, an acute metabolic acidosis (falls in pH, HCO3- and base excess) which was compensated by hyperventilation (fall in PaCO2) . Pretreatment of LPS-rats with SB 209670 (3 mg kg-1, i.v . bolus) significantly augmented the metabolic acidosis caused by LPS . 6 . Thus, the non-selective ETA/ETB receptor antagonist, SB 209670, augments the degree of (i) hypotension, (ii) vascular hyporeactivity to noradrenaline, (iii) renal dysfunction and (iv) metabolic acidosis caused by endotoxin in the anaesthetized rat . In contrast to rats treated with LPS alone, LPS-rats treated with SB 209670 exhibited liver dysfunction and hepatocellular injury . We propose that the release of endogenous ET-1 serves to maintain blood pressure and subsequently organ perfusion in septic shock. Br J Pharmacol, 1996 May, 118(1), 179 - 85 Nitric oxide synthase-cyclo-oxygenase pathways in organum vasculosum laminae terminalis: possible role in pyrogenic fever in rabbits; Lin JH et al.; 1 . Fever was induced in rabbits by administration of Escherichia coli endotoxin (lipopolysaccharide; LPS; 0.001-10 micrograms) into the organum vasculosum laminae terminalis (OVLT) . Deep body temperature was evaluated over a period of 7 h . 2 . The LPS-induced febrile response was mimicked by intra-OVLT injection of the nitric oxide (NO) donors, S-nitroso-acetylpenicillamine (SNAP, 1-10 micrograms), sodium nitroprusside (SNP, 50 micrograms), or hydroxylamine (10 micrograms), the cyclic GMP analogue 8-bromo-cyclic GMP (8-Br-cyclic GMP, 10-100 micrograms), or prostaglandin E2 (PGE2, 0.2 micrograms) . 3 . Dexamethasone (Dex, a potent inhibitor of the transcription of inducible NO synthase, iNOS, 10 micrograms), anisomycin (a protein synthesis inhibitor, 100 micrograms), L-N5-(1-iminoethyl)ornithine (L-NIO; an irreversible NOS inhibitor, 10-200 micrograms), aminoguanidine (a specific iNOS inhibitor, 1000 micrograms), or NG-methyl-L-arginine acetate (L-NMMA, a NOS inhibitor, 100 micrograms) inhibited fever induced by LPS when injected into the OVLT 1 h before LPS injection . An intra-OVLT dose of 1000 micrograms of NG-nitro-L-arginine methyl ester (L-NAME, a potent inhibitor of constitutive NOS) did not exhibit antipyretic effects . 4 . Methylene blue (an inhibitor of NOS and soluble guanylate cyclase, 1-10 micrograms), 6-(phenylamino)-5,8-quinolinedione (LY-83583; an inhibitor of soluble guanylate cyclase and NO release, 20 micrograms), or indomethacin (an inhibitor of cyclo-oxygenase, COX, 400 micrograms) inhibited fever induced by LPS when injected into the OVLT 1 h before LPS injection . Pretreatment with methylene blue or haemoglobin (a NO scavenger, 100 micrograms) attenuated the fever induced by intra-OVLT injection of SNAP . 5 . The PGE2-induced fever was potentiated, rather then attenuated, by pretreatment with an intra-OVLT dose of animoguanidine (1000 micrograms), L-NMMA (100 micrograms) or L-NIO (200 micrograms) . 6 . These results suggest that iNOS-COX pathways in the OVLT represent an important mechanism for modulation of pyrogenic fever in rabbits. Protein Sci, 1996 May, 5(5), 914 - 22 A novel family of phospholipase D homologues that includes phospholipid synthases and putative endonucleases: identification of duplicated repeats and potential active site residues; Ponting CP et al.; Phosphatidylcholine-specific phospholipase D (PLD) enzymes catalyze hydrolysis of phospholipid phosphodiester bonds, and also transphosphatidylation of phospholipids to acceptor alcohols . Bacterial and plant PLD enzymes have not been shown previously to be homologues or to be homologous to any other protein . Here we show, using sequence analysis methods, that bacterial and plant PLDs show significant sequence similarities both to each other, and to two other classes of phospholipid-specific enzymes, bacterial cardiolipin synthases, and eukaryotic and bacterial phosphatidylserine synthases, indicating that these enzymes form an homologous family . This family is suggested also to include two Poxviridae proteins of unknown function (p37K and protein K4), a bacterial endonuclease (nuc), an Escherichia coli putative protein (o338) containing an N-terminal domain showing similarities with helicase motifs V and VI, and a Synechocystis sp . putative protein with a C-terminal domain likely to possess a DNA-binding function . Surprisingly, four regions of sequence similarity that occur once in nuc and o338, appear twice in all other homologues, indicating that the latter molecules are bi-lobed, having evolved from an ancestor or ancestors that underwent a gene duplication and fusion event . It is suggested that, for each of these enzymes, conserved histidine, lysine, aspartic acid, and/or asparagine residues may be involved in a two-step ping pong mechanism involving an enzyme-substrate intermediate. Protein Sci, 1996 May, 5(5), 904 - 13 Enzymatic and fluorescence studies of four single-tryptophan mutants of rat testis fructose 6-phosphate,2-kinase:fructose 2,6-bisphosphatase; Watanabe F et al.; In order to determine environments around four tryptophan residues, located in the N-terminus, in the kinase and in the phosphatase domains of rat testis Fru 6-P,2-kinase:Fru 2,6-bisphosphatase, mutant enzymes containing a single tryptophan were constructed by site-directed mutagenesis . The kinetic constants of these mutant enzymes were similar to those of the wild-type enzyme . The sum of the fluorescence intensities of the enzymes was 1.5 x that of the wild-type enzyme, and Trp 299, Trp 64, Trp 15, and Trp 320 contributed 38%, 28%, 17%, and 17%, respectively . The fluorescence polarization of the wild-type enzyme was significantly lower than any of the mutant enzymes, suggesting proximity of two tryptophan residues in the wild-type enzyme . The polarization in the presence of Fru 6-P affected only Trp 15, which suggested that it is located near the Fru 6-P binding site, but Trp 64 is not . Inactivation of both enzyme activities and unfolding of these enzymes in guanidine were monitored by activity assays and fluorescence intensities and maxima . Both Fru 6-P,2-kinase and Fru 2,6-bisphosphatase activities of all these enzymes were inactivated between 0.7 and 1 M guanidine . Enzymes containing Trp 64 or Trp 15 showed biphasic fractional unfolding curves, but those of Trp 299 or Trp 320 showed gradual steady changes . Fluorescence quenching by iodide indicated that Trp 64 was not accessible and that other Trp residues were only slightly accessible to solvent . These results suggest that all the Trp residues are in heterogeneous environments and that none are exposed on the protein surface. Protein Sci, 1996 May, 5(5), 814 - 24 Sequence replacements in the central beta-turn of plastocyanin; Ybe JA et al.; The role of beta-turns in dictating the structure of a beta-barrel protein is assessed by probing the tolerance of the central beta-turn of poplar plastocyanin to substitution by arbitrary sequences . Native plastocyanin binds copper and is colored bright blue . However, when the wild-type Pro47-Ser48-Gly49-Val50 turn sequence is replaced by arbitrary tetrapeptides, the vast majority (92/98 = 94%) of mutant proteins cannot fold into the native blue structure . Characterization of the colorless mutant proteins demonstrates that the majority of substitutions in this type II beta-turn disrupt the native structure severely . Gross structural changes are indicated by major differences in the CD spectra of the mutants relative to the wild-type protein, and by the much larger apparent size of mutant proteins in gel filtration experiments . These mutant proteins do not bind copper . Furthermore, Cys84 forms a disulfide bond readily in the colorless mutant proteins, indicating that it has moved away from the buried position it occupies in the native copper binding site and has become exposed . These results indicate that the central beta-turn in plastocyanin is not merely a default structure arising in response to the surrounding context; rather, sequence information in this turn plays an active role in dictating the location of a chain reversal in the beta-barrel structure . These findings are discussed in terms of their implications for the folding of natural proteins, as well as the design of de novo proteins. J Inorg Biochem, 1996 May 1, 62(2), 89 - 102 Reactivity of the N-terminal cysteine residues in active and inactive forms of FNR, and O2-responsive, Fe containing transcriptional regulator of Escherichia coli; Six S et al.; FNR, the O2-responsive gene regulator of anaerobic respiratory genes in Escherichia coli, contains an N-terminal cluster of four cysteine residues (Cys16-X3-Cys20-X2-Cys23-X5-Cys29), three of which are thought to be involved in the binding of an iron cofactor . The accessibility of the cysteine residues for iodoacetate is known to increase upon switch from the active (anaerobic) to the inactive (aerobic or metal depleted) state . It was analyzed which residues become accessible under either condition . Up to four modified forms, FNR-I, FNR-II, FNR-III, and FNR-IV, containing approximately 1, 2, 3.5, and 5 carboxymethyl groups, were obtained either by reaction in vivo and in vitro under conditions of aerobiosis, anaerobiosis, or iron limitation . By N-terminal sequencing, the carboxymethylated cysteine residues were identified . The amount of label in each of the four cysteine residues increased rather uniformly and gradually from FNR-I to FNR-IV irrespective of the condition of labeling; only Cys16 was preferentially labeled to some extent . It is concluded that the four essential cysteine residues change their accessibility in a similar way in the switch from active to inactive (aerobic or metal depleted) FNR, without specific differences in their reaction or function . Potential modes of Fe-binding by the cysteine residues are discussed . In addition, a different type of interaction of Fe(II) with FNR is described . The interaction occurred also in FNR carboxymethylated at approximately three cysteine residues. Proteins, 1996 May, 25(1), 139 - 41 Crystallization and preliminary X-ray analysis of Escherichia coli methionyl-tRNA(fMet) formyltransferase; Schmitt E et al.; Methionyl-tRNA(fMet) formyltransferase from Escherichia coli, a monomer of 34kDa, was overexpressed from its cloned gene fmt (Guillon, J.M., Mechulam, Y., Schmitter, J.M., Blanquet, S., and Fayat, G., J . Bacteriol . 174:4294-4301, 1992) and crystallized using ammonium sulphate as precipitant . The crystals are trigonal and have unit cell parameters a = b = 151.0 A, c = 81.8 A . They belong to space group P3(2)21 and diffract to 2.0 A resolution . The structure is being solved by multiple isomorphous replacement. Proteins, 1996 May, 25(1), 137 - 8 Crystallization and preliminary crystallographic analysis of RepA1, a replication control protein of the RepFIC replicon of enterotoxin plasmid EntP307; Song H et al.; RepA1 protein is essential for replication of the RepFIC replicon of enterotoxin plasmid EntP307 and is thought to interact directly with the origin of replication . We have purified RepA1 from an over-producing expression system and have prepared single crystals using a macroseeding technique . The crystals belong to space group P2(1)2(1)2(1) or P2(1)2(1)2, with cell dimensions a = 61 A, b = 67 A, and c = 243 A . They diffract X-rays to 3.3 A resolution and probably contain two 40,000 molecular weight RepA1 molecules per asymmetric unit. Proteins, 1996 May, 25(1), 112 - 9 Purification, stabilization, and crystallization of a modular protein: Grb2; Guilloteau JP et al.; We report here the purification and the crystallization of the modular protein Grb2 . The protein was expressed as a fusion with glutathione-S-transferase and purified by affinity chromatography on glutathione agarose . It was apparent from reverse phase chromatography that the purified protein was conformationally unstable . Instability was overcome by the addition of 100 mM arginine to the buffers . Because Grb2 appeared to be extremely sensitive to oxidation, crystallization experiments were performed with a dialysis button technique involving daily addition of fresh DTT to the reservoirs . The presence of 8 to 14% glycerol was necessary to obtain monocrystals . These results are discussed in relation with the modular nature of Grb2. Proteins, 1996 May, 25(1), 79 - 88 Study of global motions in proteins by weighted masses molecular dynamics: adenylate kinase as a test case; Elamrani S et al.; The weighted masses molecular dynamics (WMMD) technique is applied to the protein adenylate kinase . A novel set of restraints has been developed to allow the use of this technique with proteins . The WMMD simulation is successful in predicting the flexibility of the two mobile domains of the protein . The end product of the simulation is similar to the known open and AMP bound forms of the enzyme . The biological relevance of the restraints used and potential methods of improving the technique are discussed. Proteins, 1996 May, 25(1), 48 - 78 The three-dimensional structure of Escherichia coli porphobilinogen deaminase at 1.76-A resolution; Louie GV et al.; Porphobilinogen deaminase (PBGD) catalyses the polymerization of four molecules of porphobilinogen to form the 1-hydroxymethylbilane, preuroporphyrinogen, a key intermediate in the biosynthesis of tetrapyrroles . The three-dimensional structure of wild-type PBGD from Escherichia coli has been determined by multiple isomorphous replacement and refined to a crystallographic R-factor of 0.188 at 1.76 A resolution . the polypeptide chain of PBGD is folded into three alpha/beta domains . Domains 1 and 2 have a similar overall topology, based on a five-stranded, mixed beta-sheet . These two domains, which are linked by two hinge segments but otherwise make few direct interactions, form an extensive active site cleft at their interface . Domain 3, an open-faced, anti-parallel sheet of three strands, interacts approximately equally with the other two domains . The dipyrromethane cofactor is covalently attached to a cysteine side-chain borne on a flexible loop of domain 3 . The cofactor serves as a primer for the assembly of the tetrapyrrole product and is held within the active site cleft by hydrogen-bonds and salt-bridges that are formed between its acetate and propionate side-groups and the polypeptide chain . The structure of a variant of PBGD, in which the methionines have been replaced with selenomethionines, has also been determined . The cofactor, in the native and functional form of the enzyme, adopts a conformation in which the second pyrrole ring (C2) occupies an internal position in the active site cleft . On oxidation, however, this C2 ring of the cofactor adopts a more external position that may correspond approximately to the site of substrate binding and polypyrrole chain elongation . The side-chain of Asp84 hydrogen-bonds the hydrogen atoms of both cofactor pyrrole nitrogens and also potentially the hydrogen atom of the pyrrole nitrogen of the porphobilinogen molecule bound to the proposed substrate binding site . This group has a key catalytic role, possibly in stabilizing the positive charges that develop on the pyrrole nitrogens during the ring-coupling reactions . Possible mechanisms for the processive elongation of the polypyrrole chain involve: accommodation of the elongating chain within the active site cleft, coupled with shifts in the relative positions of domains 1 and 2 to carry the terminal ring into the appropriate position at the catalytic site; or sequential translocation of the elongating polypyrrole chain, attached to the cofactor on domain 3, through the active site cleft by the progressive movement of domain 3 with respect to domains 1 and 2 . Other mechanisms are considered although the amino acid sequence comparisons between PBGDs from all species suggest they share the same three-dimensional structure and mechanism of activity. Cancer Gene Ther, 1996 May-Jun, 3(3), 163 - 7 Recombinant vaccinia expressing interleukin-2 for cancer gene therapy; Qin H et al.; Use of a recombinant vaccinia virus expressing human interleukin-2 (IL-2) was evaluated for preparation of tumor vaccines . A/J mice were immunized against neuroblastoma (C1300) cells using a preparation of C1300 cells infected/transfected with the recombinant virus, vCF13, expressing IL-2 . A second recombinant vaccinia, vSC8, expressing Escherichia coli beta-galactosidase, was used as a control . After three weekly immunizations with virus-transfected cells, the mice were challenged with 1 x 10(6) unmodified C1300 cells and tumor development was monitored . Tumor development in the mice was inhibited by immunization with vCF13-transfected cells, compared to those vaccinated with vSC8-transfected cells (P < .008) . A group of mice (7/15) immunized with vCF13-transfected cells followed by tumor challenge survived more than 60 days, at which time all mice immunized with the control vaccine were dead (p < .006) . Five of the mice treated with the vCF13 vaccine were alive for more than 75 days (P < .05), after which they were rechallenged with another dose of 1 x 10(6) unmodified tumor cells . Tumor development was not apparent in these mice for more than 45 days following the second challenge, suggesting that these mice were completely protected by this immunization . These results demonstrate that recombinant vaccinia virus expressing IL-2 may be useful for cancer gene therapy. Cancer Gene Ther, 1996 May-Jun, 3(3), 155 - 62 Escherichia coli gpt gene sensitizes rat glioma cells to killing by 6-thioxanthine or 6-thioguanine; Tamiya T et al.; Genes that encode enzymes that convert inactive "prodrugs" into anticancer metabolites may be therapeutically useful against brain tumors . Unlike other genes tested to date in brain tumor models, the Escherichia coli gpt gene is unique in that it not only sensitizes cells to the prodrug 6-thioxanthine (6TX) but also encodes resistance to a different regimen (mycophenolic acid, xanthine, and hypoxanthine), thus providing a means to select for gpt-positive cells . In the present study, rat C6 glioma cells were infected with a retrovirus vector that transduces this gene . A clonal line (C6GPT-7) was derived that exhibited significant 6TX susceptibility in vitro with an ID50 of 2.5 mumol/L, whereas 50% growth inhibition of parental C6 cells was not achieved at concentrations tested (up to 50 mumol/L) . This line also exhibited significant sensitivity to 6-thioguanine (6TG), with an ID50 of 0.05 mumol/L, whereas 50% growth inhibition of parental C6 cells was achieved at 0.5 mumol/L . In a "bystander" assay, C6GPT-7 tumor cells efficiently transferred 6TX sensitivity to C6 cells at ratios as low as 1:9 (C6GPT-7:C6) . This in vitro bystander effect was abrogated when C6GPT-7 and C6 cells were separated by a microporous membrane, suggesting that it was not mediated by highly diffusible metabolites . In vivo both 6TX and 6TG significantly inhibited the growth of subcutaneously transplanted C6GPT-7 cells but not that of C6 cells in athymic mice . In an intracerebral model, both 6TX and 6TG exhibited significant antiproliferative effects against tumors formed by C6GPT-7 cells . These findings provide a basis for exploring further gene therapy strategies based on in vivo transfer of the E coli gpt gene to provide chemosensitivity against 6TX and 6TG. Biotechniques, 1996 May, 20(5), 862 - 4, 866-8 Sequencing homopolymer tracts and repetitive elements; Robbins CM et al.; We investigated the use of Taq dye primer and Taq terminator sequencing chemistry to optimize the quality of sequence data obtained from templates containing homopolymer tracts and repetitive elements . In direct side-by-side comparisons using the Applied Biosystems Model 373A Fluorescent Sequencer, the Taq terminator sequencing chemistry gave much cleaner and more consistent results on long homopolymer tracts and dinucleotide repeats . We also investigated various thermal cycling conditions and determined that higher annealing temperatures and longer denaturation times improved the ability to sequence through these problem templates. Am J Vet Res, 1996 May, 57(5), 659 - 63 Attempt to pharmacologically modulate procoagulant activity of lipopolysaccharide-stimulated adherent bovine alveolar macrophages; Olchowy TW et al.; OBJECTIVE--To investigate the effects of anti-inflammatory drugs on lipopolysaccharide-induced procoagulant activity of bovine alveolar macrophages . DESIGN--Procoagulant activity was induced in bovine alveolar macrophages from 4 healthy Holstein calves aged 6 to 16 weeks by incubation with lipopolysaccharide . 3 anti-inflammatory drugs were used at 4 concentrations and 3 times to pretreat the alveolar macrophages . Results were analyzed to determine whether drug, concentration, or exposure period had a significant (P > 0.05) effect . PROCEDURE--Bovine alveolar macrophages, harvested by volume-controlled bronchoalveolar lavage, were pretreated for 30, 60, or 120 minutes with an anti-inflammatory compound (dexamethasone, flunixin meglumine, or phenylbutazone) at several concentrations ( 0, 1, 10, and 100 microM) . Bovine alveolar macrophages were exposed to lipopolysaccharide (Escherichia coli O55:B5) in the presence and absence of fetal bovine serum for 4 hours . Procoagulant activity was measured, using a chromogenic assay . RESULTS--None of the drugs was associated with a modification of procoagulant activity expression . CONCLUSION--Use of these 3 anti-inflammatory drugs is unlikely to modify the extent of the fibrinous reaction commonly observed in cases of acute bovine respiratory tract disease complex . CLINICAL RELEVANCE--The alveolar macrophage has a key role in fibrin production . Assuming in vivo events mimic the in vitro model, is appears unlikely that administration of anti-inflammatory drugs will reduce the procoagulant activity of the bovine alveolar macrophages and the directly associated pulmonary fibrosis. J Neurosci Res, 1996 May 1, 44(3), 235 - 42 Lipopolysaccharide stimulates differential expression of myristoylated protein kinase C substrates in murine microglia; Rose SD et al.; Microglia rapidly respond to lipoplysaccharide (LPS) by transformation from resting to active states and secretion of several neuro- and immuno-regulators including tumour necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta), and interleukin 6 (IL-6) . With longer LPS treatment, microglia are converted to reactive or phagocytic states with characteristics similar to macrophages in inflammation and injury processes . We have investigated LPS-mediated changes in two myristoylated substrates of protein kinase C (PKC): MARCKS (myristoylated alaninerich C kinase substrate) and MRP (MARCKS-related protein) . Within 6 hours of addition, LPS induced a twofold increase in {3H}myristoylated and immunoreactive MARCKS protein and a sevenfold increase in MRP . The differential effect of LPS on expression of MRP vs . MARCKS was even more dramatic at the level of transcription: S1 nuclease protection assays revealed a 40-fold increase in MRP mRNA levels (maximum at 4-6 hours), whereas a threefold increase was observed for MARCKS . TNF alpha and colony-stimulating factor 1 (CSF-1), two cytokines which are induced by LPS, did not reproduce the observed effect of LPS on MARCKS and MRP gene transcription . CSF-1 also induced differential transcription of MRP, but of lower magnitude (threefold) and more sustained than by LPS . Accordingly, these two substrates for PKC are differentially up-regulated by LPS, apparently independent of TNF alpha or CSF-1. Genetics, 1996 May, 143(1), 27 - 36 Orientation dependence in homologous recombination; Yamamoto K et al.; Homologous recombination was investigated in Escherichia coli with two plasmids, each carrying the homologous region (two defective neo genes, one with an amino-end deletion and the other with a carboxyl-end deletion) in either direct or inverted orientation . Recombination efficiency was measured in recBC sbcBC and recBC sbcA strains in three ways . First, we measured the frequency of cells carrying neo+ recombinant plasmids in stationary phase . Recombination between direct repeats was much more frequent than between inverted repeats in the recBC sbcBC strain but was equally frequent in the two substrates in the recBC sbcA strain . Second, the fluctuation test was used to exclude bias by a rate difference between the recombinant and parental plasmids and led to the same conclusion . Third, direct selection for recombinants just after transformation with or without substrate double-strand breaks yielded essentially the same results . Double-strand breaks elevated recombination in both the strains and in both substrates . These results are consistent with our previous findings that the major route of recombination in recBC sbcBC strains generates only one recombinant DNA from two DNAs and in recBC sbcA strains generates two recombinant DNAs from two DNAs. Genetics, 1996 May, 143(1), 15 - 26 Long-term experimental evolution in Escherichia coli . IV . Targets of selection and the specificity of adaptation; Travisano M et al.; This study investigates the physiological manifestation of adaptive evolutionary change in 12 replicate populations of Escherichia coli that were propagated for 2000 generations in a glucose-limited environment . Representative genotypes from each population were assayed for fitness relative to their common ancestor in the experimental glucose environment and in 11 novel single-nutrient environments . After 2000 generations, the 12 derived genotypes had diverged into at least six distinct phenotypic classes . The nutrients were classified into four groups based upon their uptake physiology . All 12 derived genotypes improved in fitness by similar amounts in the glucose environment, and this pattern of parallel fitness gains was also seen in those novel environments where the limiting nutrient shared uptake mechanisms with glucose . Fitness showed little or no consistent improvement, but much greater genetic variation, in novel environments where the limiting nutrient differed from glucose in its uptake mechanisms . This pattern of fitness variation in the novel nutrient environments suggests that the independently derived genotypes adapted to the glucose environment by similar, but not identical, changes in the physiological mechanisms for moving glucose across both the inner and outer membranes. Genetics, 1996 May, 143(1), 5 - 13 Differential suppression of priA2::kan phenotypes in Escherichia coli K-12 by mutations in priA, lexA, and dnaC; Sandler SJ et al.; First identified as an essential component of the phi X174 in vitro DNA replication system, PriA has ATPase, helicase, translocase, and primosome-assembly activities . priA1::kan strains of Escherichia coli are sensitive to UV irradiation, deficient in homologous recombination following transduction, and filamentous . priA2::kan strains have eightfold higher levels of uninduced SOS expression than wild type . We show that (1) priA1::kan strains have eightfold higher levels of uninduced SOS expression, (2) priA2::kan strains are UVS and Rec-, (3) lexA3 suppresses the high basal levels of SOS expression of a priA2::kan strain, and (4) plasmid-encoded priA300 (K230R), a mutant allele retaining only the primosome-assembly activity of priA+, restores both UVR and Rec+ phenotypes to a priA2::kan strain . Finally, we have isolated 17 independent UVR Rec+ revertants of priA2::kan strains that carry extragenic suppressors . All 17 map in the C-terminal half of the dnaC gene . DnaC loads the DnaB helicase onto DNA as a prelude for primosome assembly and DNA replication . We conclude that priA's primosome-assembly activity is essential for DNA repair and recombination and that the dnaC suppressor mutations allow these processes to occur in the absence of priA. Gut, 1996 May, 38(5), 747 - 52 Lipopolysaccharide induced apoptosis of rat pancreatic acinar cells; Laine VJ et al.; BACKGROUND--Bacterial lipopolysaccharide (LPS) has been proposed to participate in the pathogenesis of pancreatic inflammatory disease . AIMS--This study investigated the role of endotoxaemia in the pathogenesis of pancreatic acinar cell injury . METHODS--Sixty eight male Spraque-Dawley rats were used in the study . Escherichia coli LPS (5 mg/kg) was injected into the peritoneal cavity of the rats . The concentration of pancreatic phospholipase A2 (PLA2) in plasma was measured and pancreatic tissue examined by histology, in situ detection of free DNA 3'-ends, and electrophoretic DNA analysis . RESULTS--The concentration of pancreatic PLA2 increased in plasma and the catalytic activity of PLA2 increased in pancreatic tissue after an LPS injection . Apoptosis in pancreatic acinar cells and fragmentation of DNA typical of apoptosis in pancreatic tissue was seen 24 hours after an LPS injection . Pancreatic acinar atrophy was seen 72 hours after the LPS injection . CONCLUSIONS--These data show that LPS causes release of pancreatic PLA2 into blood plasma, activation of PLA2 in pancreatic tissue, and apoptosis of acinar cells. Crit Care Med, 1996 May, 24(5), 807 - 14 Effect of human hemoglobin on systemic and regional hemodynamics in a porcine model of endotoxemic shock; Aranow JS et al.; OBJECTIVE: Excessive release of nitric oxide has been implicated as being an important factor contributing to systemic arterial hypotension in septic shock . Hemoglobin is an effective nitric oxide scavenger . The purpose of this study was to test the hypothesis that treatment with cross-linked human hemoglobin can ameliorate systemic arterial hypotension and improve organ perfusion in a porcine model of normodynamic endotoxemic shock . DESIGN: Prospective, randomized, controlled trial . SETTING: Laboratory at a university medical center . SUBJECTS: Fourteen, male, random-bred swine . INTERVENTIONS: All animals were challenged with Escherichia coli lipopolysaccharide (400 microg/kg) infused from t = 0 to 90 mins . Pigs in group 1 (n = 7) were infused with cross-linked human hemoglobin (150 mg/kg) at t = 30 mins . Pigs in group 2 (n = 7) were infused at t = 30 mins with 150 mg/kg of dextran (average molecular weight 70,000 daltons) as a 5% (weight per volume) solution . MEASUREMENTS AND MAIN RESULTS: After infusion of endotoxin, mean arterial pressure decreased significantly (p < .05) but baseline cardiac index was maintained in both groups . In hemoglobin-treated pigs (group 1), mean arterial pressure was higher than in controls (group 2) from t = 60 to 120 mins (p < .05) . There were no significant differences between the two groups in systemic vascular resistance index, renal blood flow, mesenteric blood flow, systemic oxygen delivery, or systemic oxygen extraction . Ileal mucosal blood flow was lower (p < .07) in group 1 than in group 2 . Mean pulmonary arterial pressure increased relative to baseline in both groups, but was significantly greater in group 1 as compared with group 2 . Compared with controls, infusion of hemoglobin significantly exacerbated endotoxin-induced arterial hypoxemia (p < .05) . CONCLUSIONS: Treatment with hemoglobin improved mean arterial pressure in endotoxemic swine without significantly impairing blood flow to the renal or mesenteric vascular beds . Infusion of hemoglobin, however, significantly exacerbated endotoxin-induced pulmonary hypertension and arterial hypoxemia . Additional pharmacologic strategies may be necessary to ameliorate the potential adverse pulmonary effects of administering hemoglobin solutions to patients with sepsis. Clin Diagn Lab Immunol, 1996 May, 3(3), 355 - 7 Recombinant Toxoplasma gondii surface antigen 1 (P30) expressed in Escherichia coli is recognized by human Toxoplasma-specific immunoglobulin M (IgM) and IgG antibodies; Harning D et al.; The immunodominant surface antigen of Toxoplasma gondii, surface antigen 1 (SAG1), was expressed in Escherichia coli as a fusion protein containing a majority of the SAG1 protein supplied with six histidyl residues in the N-terminal end . The recombinant protein was purified on a Ni-chelate column and then on a fast-performance liquid chromatography column and was in a nonreduced condition . It was recognized by T . gondii-specific human immunoglobulin G (IgG) and IgM antibodies as well as by a mouse monoclonal antibody (S13) recognizing only nonreduced native SAG1 . Antibodies induced in mice by the recombinant SAG1 recognized native SAG1 from the T . gondii RH isolate in culture . Recombinant SAG1 is suitable for use in diagnostic systems for detecting anti-SAG1-specific IgG and IgM antibodies. Microbiology, 1996 May, 142 ( Pt 5), 1141 - 8 Reactions of the Escherichia coli flavohaemoglobin (Hmp) with NADH and near-micromolar oxygen: oxygen affinity of NADH oxidase activity; Poole RK et al.; The soluble flavohaemoglobin (Hmp) of Escherichia coli, product of the hmp gene, contains haem B and FAD in a single polypeptide of molecular mass 44 kDa . The function of this protein (and of the similar proteins identified in several bacteria and yeast) is unknown, but the observation that the binding of oxygen to haem modulates the reduction level of FAD has suggested that Hmp could act as an oxygen sensor . Here, stopped-flow, rapid-scan spectroscopy has shown that the oxidized protein reacts rapidly with NADH to form an oxygenated species, even when efforts are made to reduce oxygen concentrations to sub-micromolar levels, suggesting a high affinity for this ligand . As is the case at high oxygen concentrations (130 microM), oxygenated species formation was kinetically and spectrally heterogeneous . Between 12 ms and 1 s after mixing, following transient formation of the deoxy form and its reaction with dioxygen, a steady-state level of the oxygenated species was attained . During the oxygenated steady state, the flavin remained largely oxidized, as observed previously at 130 microM oxygen . Hmp is an NADH oxidase; on exhaustion of oxygen by reduction (in < 10 s under these conditions), the oxygenated species disappeared to generate the deoxy Fe(II) haem, whereupon the flavin was reduced . The affinity for oxygen during NADH oxidation was measured by continuous dual-wavelength monitoring of the deoxygenation of oxymyoglobin . The Km for oxygen was 2.6 microM, much higher than the Km values determined, using the same method, for the membrane-bound terminal oxidases cytochromes bo' and bd . These results show that the oxidase activity of Hmp, but not necessarily oxygen binding, would be minimal at oxygen concentrations that limit terminal oxidase function. Acta Crystallogr A, 1996 May 1, 52 ( Pt 3), 480 - 9 Direct phasing in protein electron crystallography--phase extension and the prospects for ab initio determinations; Dorset DL; Zonal diffraction amplitudes and crystallographic phases, derived from an averaged electron micrograph of two-dimensionally crystalline E . coli Omp F outer membrane porin (plane group p31m, a = 72 A), embedded in glucose, were used as a model data set to test the feasibility of direct phase extension and ab initio direct phase determination . If 17 phase terms derived from e.g . a 10 A (diffraction) resolution image are expanded to 6 A by the Sayre-Hughes equation, the unknown phases are found with reasonable accuracy (mean error 43 degrees for 25 reflections) . This, however, is not the most optimal starting point . As a function of initial image resolution, the accuracy of the phase extension to 6 A is approximately a parabolic function . That is, an optimal basis resolution, found at 11 A (i.e . 14 defined reflections), produces a least mean error of 18 degrees for 28 new reflections . In addition, ab initio phase determination is possible via a multisolution technique, using a test for density flatness as a figure of merit . The success of the determination, again is sensitive to the size of the starting basis set generated from the permuted unknown reflections . If an annealing step is used to improve the basis set, the test for flatness will identify which reflections should be changed in phase . However, this figure of merit is not absolutely reliable for finding the exact value of the unknown phases. Phytochemistry, 1996 May, 42(2), 321 - 4 Effect of cucurbitacins on mRNA coding for laccase in Botrytis cinerea; Gonen L et al.; The effect of cucurbitacin and of Ecballium extract on the formation of mRNA coding for laccase was examined in cultures of Botrytis cinerea grown with inducers of laccase formation, in the presence or absence of the inhibitory compounds . RNA was isolated from the cultures and probed with specific DNA probes for laccase . As an internal control, the RNA was probed for Botrytis beta-tubulin mRNA . From an analysis of the results it is clear that cucurbitacin I and Ecballium extract specifically repress the amount of mRNA coding for laccase . This could account for the previously observed repression of laccase formation by cucurbitacins. Plant Physiol, 1996 May, 111(1), 61 - 71 A putative Mg chelatase subunit from Arabidopsis thaliana cv C24 . Sequence and transcript analysis of the gene, import of the protein into chloroplasts, and in situ localization of the transcript and protein; Gibson LC et al.; We have isolated and sequenced a cDNA from Arabidopsis thaliana cv C24 that encodes a putative Mg chelatase subunit . The deduced amino acid sequence shows a very high level of identity to a gene previously characterized from Antirrhinum majus (olive and also high similarity to bchH, a bacterial gene involved in the Mg chelatase reaction of bacteriochlorophyll biosynthesis . We suggest that this gene be called CHL H . Northern blot analyses were used to investigate the expression of CHL H, another putative Mg chelatase gene, ch-42, and ferrochelatase . The CHL H transcript was observed to undergo a dramatic diurnal variation, rising almost to its maximum level by the end of the dark period, then increasing slightly at the onset of the light and declining steadily to a minimum by the end of the light period; in contrast, transcripts for ch-42 and ferrochelatase remained constant . A model is proposed in which the CHL H protein plays a role in regulating the levels of chlorophyll during this cycle . In situ hybridization revealed that the transcripts are located over the surface of the chloroplasts, a feature in common with transcripts for the ch-42 gene . The CHL H protein was imported into the stromal compartment of the chloroplast and processed in an in vitro assay . Immunoblotting showed that the distribution of CHL H protein between the stroma and chloroplast membranes varies depending on the concentration of Mg+ . In situ immunofluorescence was used to establish that the CHL H and CH-42 proteins are localized within the chloroplast in vivo. Leuk Res, 1996 May, 20(5), 369 - 74 Trisomy 12 is a rare cytogenetic finding in typical chronic lymphocytic leukemia; Woessner S et al.; We have studied 61 cases of B-chronic lymphocytic leukemia (CLL), combining cytological features, conventional cytogenetics and in situ hybridization (ISH) . The comparison of these results constitutes the main subject of this study . The patients were cytologically classified according to the FAB criteria as: chronic lymphocytic leukemia (CLL) typical type (48 cases) and CLL atypical types (13 cases) . Chromosome analysis was carried out on lymphoid cells from peripheral blood . The following mitogens were used: phytohemagglutinin (PHA) 5%, pokeweed (PWM) and lipopolysaccharide from E . coli . The ISH was performed with a biotin-labeled, chromosome 12-specific alpha satellite DNA probe, pSP12-1 . Trisomy 12 was not found in any of the 48 patients with the typical type of CLL and in contradistinction it was present in some patients with atypical types . This study emphasizes the great importance of a closer link between hematological morphology and the cytogenetic approach. Transgenic Res, 1996 May, 5(3), 171 - 7 A model for the mechanism of precise integration of a microinjected transgene; McFarlane M et al.; A unique transgenic mouse line has undergone transgene integration in a very precise fashion . The phenotype displayed by mice of the line followed the predicted inheritance patterns for X-linked transgene insertion which has been confirmed . In order to investigate the mechanism of integration the DNA sequence of the transgene and cellular junctions have been determined . A comparison between wild type and transgenic mutant sequences at the site of insertion revealed that there was no loss or rearrangement of cellular DNA upon integration of the transgene . The cellular sequences at the transgene 5' and 3' joins are contiguous in the wild type . The integrant exists as a head to tail tandem dimer with minimal loss of sequence compared with the injected monomer . Analysis of the site of insertion has revealed a 5 bp homology between the 5' end of the transgene and the cellular sequences . In addition, adjacent to the site of insertion within the cellular sequences, there are several sequence motifs implicated in recombination events including a clustering of strong consensus sites of DNA topoisomerase type I and a region of homology to the human minisatellite consensus core sequence, the Escherichia coli Chi site and the meiotic recombination hotspot within the E beta gene of the murine major histocompatibility complex . This clustering of features is likely to have been factorial in the integrity of the insertion event . A model depicting the mechanism of this precise integration is proposed. Int Immunol, 1996 May, 8(5), 731 - 6 Cross-linking of cell surface ganglioside GM1 induces the selective apoptosis of mature CD8+ T lymphocytes; Nashar TO et al.; Gangliosides are glycosphingolipids found ubiquitously on the surface of mammalian cells . They contain a ceramide tail that is inserted into the membrane and exposed carbohydrate and sialic acid moieties . The non-toxic B subunit oligomer (EtxB) of Escherichia coli heat-labile enterotoxin (Etx) is a potent immunogen in vivo and has profound modulatory effects on EtxB-primed lymphocytes in vitro, properties which are dependent on its ability to bind to GM1 ganglioside receptors . Here, it is shown that cross-linking GM1 by EtxB causes a differential effect on mature CD4(+) and CD8(+) T cells from lymph node cultures proliferating in response to an unrelated antigen, ovalbumin . Addition of EtxB to such cultures led to the complete depletion of CD8(+) T cells compared with enhanced activation of CD4(+) cells {as measured by expression of CD25 (IL-2Ralpha)} . By contrast, addition of a mutant EtxB, EtxB(G33D), which does not bind to GM1, failed to trigger CD8(+) T cell depletion . When EtxB was added to isolated non-immune CD8(+) lymphocytes rapid (12-18 h) alterations in nuclear morphology and the appearance of sub-G0/G1 levels of DNA were induced; properties which are characteristic of cells undergoing apoptosis . EtxB(G33D) failed to trigger apoptosis, indicating that the induction of the apoptotic signal was dependent on the binding of GM1 . These findings provide an insight into the potent immunogenicity and immunomodulatory properties of E . coli enterotoxins as well as heralding a novel method for the selective induction of apoptosis in mature CD8(+) T lymphocytes. J Natl Med Assoc, 1996 May, 88(5), 306 - 9 Deceptive prothrombin and activated partial thromboplastin times in alcoholic cirrhosis; Sirikonda PR et al.; It is believed that perioperative hemorrhage, in the hepatoportal area, results from a coagulopathy . This study determined if this could be quantitated by a modified recalcification time (MRT) test developed in our laboratory . Unlike prothrombin (PT) and activated partial thromboplastin times (APTT), the MRT is performed with whole blood to ensure the role of blood cells and chemicals (particularly tissue factor, a potent procoagulant) in the coagulation process . Candidates for liver transplantation (n = 11) were studied . Samples (5 mL) of citrated venous blood were obtained from the patients . Aliquots (1 mL) from these samples were divided into groups of vials labeled C, S, and E . Groups C and S received 20 microL saline and group E, 20 microL of saline containing 10 micrograms of Escherichia coli endotoxin (055: B5W) . Vial C was incubated for 10 minutes and vials S and E for 120 minutes, all at 37 degrees C . Then, the MRT was determined on 300 microL of blood from each vial after adding 40 microL of 0.1M calcium chloride . Mean MRT values (minutes +/- standard deviation) for C (MRTC), for S (MRTS), and for E (MRTE) were compared with like values from healthy controls (n = 29) . Despite prolonged PT and APTT values, MRT values were shortened in patients with cirrhosis . This hypercoagulability detected by the MRT exonerates a hemorrhagic coagulopathy and possibly implicates widened and thinned gaps in the walls of the portal venous tributaries as the cause of perioperative hemorrhage. RNA, 1996 May, 2(5), 463 - 72 Phylogenetic comparative mutational analysis of the base-pairing between RNase P RNA and its substrate; Svard SG et al.; We have studied the base-pairing between the 3'-terminal CCA motif of a tRNA precursor and RNase P RNA by a phylogenetic mutational comparative approach . Thus, various derivatives of the Escherichia coli tRNA(Ser)Su1 precursor harboring all possible substitutions at either the first or the second C of the 3'-terminal CCA motif were generated . Cleavage site selection on these precursors was studied using mutant variants of M1 RNA, the catalytic subunit of E . coli RNase P, carrying changes at positions 292 or 293, which are involved in the interaction with the 3'-terminal CCA motif . From our data we conclude that these two C's in the substrate interact with the well-conserved G292 and G293 through canonical Watson-Crick base-pairing . Cleavage performed using reconstituted holoenzyme complexes suggests that this interaction also occurs in the presence of the C5 protein . Furthermore, we studied the interaction using various derivatives of RNase P RNAs from Mycoplasma hyopneumoniae and Mycobacterium tuberculosis . Our results suggest that the base-pairing between the 3'-terminal CCA motif and RNase P is present also in other bacterial RNase P-substrate complexes and is not limited to a particular bacterial species. Curr Genet, 1996 May, 29(6), 582 - 6 A small insertion in the SSU rDNA of the lichen fungus Arthonia lapidicola is a degenerate group-I intron; Grube M et al.; Insertions of less than 100 nt occurring in highly conserved regions of the small subunit ribosomal DNA (SSU rDNA) may represent degenerate forms of the group-I introns observed at the same positions in other organisms . A 63-nt insertion at SSU rDNA position 1512 (relative to the Escherichia coli SSU rDNA) of the lichen-forming fungus Arthonia lapidicola can be folded into a secondary structure with two stem loops and a pairing of the insertion and flanking sequences . The two stem loops may correspond to the P1 and P2, and the insertion-flanking pairing to the P10, of a group-I intron . Considering these small insertions as degenerate introns provides important clues to the evolution and catalytic function of group-I introns.Keywords Ribosomal DNA middle dot Small subunit middle dot 18s middle dot Degenerate introns middle dot Ascomycetes J Mol Evol, 1996 May, 42(5), 525 - 36 Codon usage and base composition in Rickettsia prowazekii; Andersson SG et al.; Codon usage and base composition in sequences from the A + T-rich genome of Rickettsia prowazekii, a member of the alpha Proteobacteria, have been investigated . Synonymous codon usage patterns are roughly similar among genes, even though the data set includes genes expected to be expressed at very different levels, indicating that translational selection has been ineffective in this species . However, multivariate statistical analysis differentiates genes according to their G + C contents at the first two codon positions . To study this variation, we have compared the amino acid composition patterns of 21 R . prowazekii proteins with that of a homologous set of proteins from Escherichia coli . The analysis shows that individual genes have been affected by biased mutation rates to very different extents: genes encoding proteins highly conserved among other species being the least affected . Overall, protein coding and intergenic spacer regions have G + C content values of 32.5% and 21.4%, respectively . Extrapolation from these values suggests that R . prowazekii has around 800 genes and that 60-70% of the genome may be coding. Arch Microbiol, 1996 May, 165(5), 333 - 41 Analysis of the hydA locus of Escherichia coli: two genes (hydN and hypF) involved in formate and hydrogen metabolism; Maier T et al.; The hydA locus of Escherichia coli is known to encode some function necessary for formation of hydrogenase activity . The locus contains two open reading frames, hydN and hypF . In this communication, an analysis of the regulation of these two genes and of the phenotype of respective mutants is presented, Both genes were expressed in a T7 promoter/polymerase system, yielding a 19-kDa (HydN) and a 81-kDa (HypF) protein . In-frame deletions were constructed for each gene and transferred to the chromosome by homologous recombination . The mutation in hydN led to a decrease of the activity of formate dehydrogenase H (FDH-H) in crude extracts, but the activity and maturation of hydrogenases were nearly unaffected . In contrast, a deletion in hypF resulted in the loss of hydrogenase activity and in the synthesis of the large subunits of the hydrogenase isoenzymes 1, 2 and 3 in the inactive precursor form . For hydrogenase 3, it was shown that this is due to a lack of incorporation of nickel into the large subunit . hydN and hypF are organised in an operon that is a member of the formate regulon . Transcription was shown to be dependent on sigma 54 and Fh1A, and an Fh1A-binding site upstream of hydN was identified . The sigma 54-dependent promoter shows a rare deviation from the consensus at positions -24/-12, namely GG/GA instead of GG/GC . In conclusion, the product of hydN appears to have some role in electron flow from or to FDH-H, and the product of hypF is connected with maturation of all three hydrogenases of E . coli. Mamm Genome, 1996 May, 7(5), 349 - 52 Mouse uroporphyrinogen decarboxylase: cDNA cloning, expression, and mapping; Wu C et al.; Uroporphyrinogen decarboxylase (URO-decarboxylase; EC 4.1.1.37), the heme biosynthetic enzyme responsible for the conversion of uroporphyrinogen III to coproporphyrinogen III, is the enzymatic defect in porphyria cutanea tarda, the most common porphyria . The mouse URO-decarboxylase cDNA was isolated from a mouse adult liver cDNA library . The longest clone of 1.5 kb, designated pmUROD-1, had 5' and 3' untranslated sequences of 281 and 97 bp, respectively, and an open reading frame of 1104 bp encoding a 367-amino acid polypeptide with a predicted molecular mass of 40,595 Da . The mouse and human coding sequences had 87.8% and 90.0% nucleotide and amino acid identity, respectively . The authenticity of the mouse cDNA was established by expression of the active enzyme in Escherichia coli . In addition, the analysis of two sets of multilocus genetic crosses localized the mouse gene, Urod, on Chromosome (Chr) 4, consistent with the map location of the human gene to a position of conserved synteny on Chr 1 . The availability of the mouse URO-decarboxylase should facilitate studies of the structure and organization of the mouse genomic sequence and the development of a mouse model of this inherited porphyria. J Membr Biol, 1996 May, 151(2), 175 - 87 Multiple mechanosensitive ion channels from Escherichia coli, activated at different thresholds of applied pressure; Berrier C et al.; Mechanosensitive ion channels from Escherichia coli were studied in giant proteoliposomes reconstituted from an inner membrane fraction, or in giant round cells in which the outer membrane and the cell wall had been disrupted by a lysozyme-EDTA treatment and a mild osmotic shock . Patch-clamp experiments revealed the presence in these two preparations of an array of different conductances (100 to 2,300 pS in 0.1 M KCl) activated by stretch . The electrical activity induced by stretch in the native membrane was complex, due to the activation of several different conductances . In contrast, patches of proteoliposomes generally contained clusters of identical conductances, which differed from patch to patch . These experiments are consistent with the notion that these different conductances correspond to different proteins in the plasma membrane of E . coli, which segregate into clusters of identical channels on dilution involved in reconstitution in proteoliposomes . These conductances could be grouped into three subfamilies of poorly selective channels . In both preparations, the higher the conductance, the higher was the negative pressure needed for activation . We discuss the putative role of these channels as parts of a multicomponent osmoregulatory system. Hum Genet, 1996 May, 97(5), 557 - 60 A novel point mutation in congenital erythropoietic porphyria in two members of Japanese family; Tanigawa K et al.; The molecular basis of the uroporphyrinogen III synthase (UROIIIS) deficiency was investigated in two members of a Japanese family . This defect in heme biosynthesis is responsible for a rare autosomal recessive disease: congenital erythropoietic porphyria (CEP) or Gnther's disease . The first patient was homoallelic for a novel missense mutation: a T to C transition of nucleotide 634 that predicted a serine to proline substitution at residue 212 (S212P) . The second patient appeared heteroallelic, carrying the same missense mutation and a nonsense mutation: a C to T change at nucleotide 745, resulting in a premature stop at codon 249, instead of a glutamine (Q249X) . The corresponding mutated proteins were expressed in Escherichia coli and no residual activity was observed . A family study was also performed to determine the carrier status. Genes Dev, 1996 May 1, 10(9), 1162 - 71 Use of a transposon (Tndif) to obtain suppressing and nonsuppressing insertions of the dif resolvase site of Escherichia coli; Kuempel P et al.; The dif locus is a RecA-independent recombination site, located in the terminus region of the chromosome of Escherichia coli . This site functions to reduce circular dimer chromosomes to monomers before cell division . Strains lacking this site exhibit the Dif phenotype, in which a fraction of the cells form extended filaments with abnormal nucleoids, and the SOS system is induced . We have used a transposon (Tndif), as well as linear transformation, to position dif in 19 locations around the chromosome . All of the suppressing insertions that we obtained were within 10 kb of the normal site, even in strains in which the normal symmetry, between the origin of replication and dif had been altered by 200 kb . We also observed that the nonsuppressing insertions in the terminus region became suppressing if a deletion occurred that extended from the ectopic site up to or past the normal location of dif . We propose that dif is normally located at the center of converging polarities in the terminus region and that deletions that restore suppression do so by placing ectopic sites once again at the center of this polarity . Similar results and conclusions are described in this issue. Genes Dev, 1996 May 1, 10(9), 1152 - 61 Restriction of the activity of the recombination site dif to a small zone of the Escherichia coli chromosome; Cornet F et al.; The recombination site dif is the target on the Escherichia coli chromosome of the site-specific recombinases XerC and XerD . The dif/XerC-D system plays a role during the cell cycle, probably by favoring sister chromosome monomerization or separation . A phenomenon of regional control over dif activity, also analyzed in this issue, is demonstrated here by translocation of dif to a series of loci close to the normal locus . We found that the site is physiologically active only within a narrow zone around its natural position . Competence for dif activity does not depend on the sequence of the normal dif activity zone (DAZ), because delta(dif) deletions larger than the DAZ result in Dif+ bacteria when dif is reinserted at the junction point . Although dif maps where replication normally terminates, termination of replication is not the elicitor . A strain with a large inversion that places dif and its surrounding region close to oriC remains Dif+, even when a Tus- mutation allows replication to terminate far away from it . Preliminary data suggest the possibility that specialized sequences separate the competent zone from the rest of the chromosome . We suspect that these sequences are members of a set of sequences involved in a polarized process of postreplicative reconstruction of the nucleoid structure . We propose that this reconstruction forces catenation links between sister chromosomes to accumulate within the DAZ, where they eventually favor recombination at dif. Genes Dev, 1996 May 1, 10(9), 1143 - 51 The RNA-binding protein HF-I, known as a host factor for phage Qbeta RNA replication, is essential for rpoS translation in Escherichia coli; Muffler A et al.; The rpoS-encoded sigma(S) subunit of RNA polymerase in Escherichia coli is a global regulatory factor involved in several stress responses . Mainly because of increased rpoS translation and stabilization of sigma(S), which in nonstressed cells is a highly unstable protein, the cellular sigma(S) content increases during entry into stationary phase and in response to hyperosmolarity . Here, we identify the hfq-encoded RNA-binding protein HF-I, which has been known previously only as a host factor for the replication of phage Qbeta RNA, as an essential factor for rpoS translation . An hfq null mutant exhibits strongly reduced sigma(S) levels under all conditions tested and is deficient for growth phase-related and osmotic induction of sigma(S) . Using a combination of gene fusion analysis and pulse-chase experiments, we demonstrate that the hfq mutant is specifically impaired in rpoS translation . We also present evidence that the H-NS protein, which has been shown to affect rpoS translation, acts in the same regulatory pathway as HF-I at a position upstream of HF-I or in conjunction with HF-I . In addition, we show that expression and heat induction of the heat shock sigma factor sigma(32) (encoded by rpoH) is not dependent on HF-I, although rpoH and rpoS are both subject to translational regulation probably mediated by changes in mRNA secondary structure . HF-I is the first factor known to be specifically involved in rpoS translation, and this role is the first cellular function to be identified for this abundant ribosome-associated RNA-binding protein in E . coli. Genes Dev, 1996 May 1, 10(9), 1120 - 30 Posterior patterning by the Caenorhabditis elegans even-skipped homolog vab-7; Ahringer J; Patterning of the posterior end in animals is not well understood . Homologs of Drosophila even-skipped (eve) have a similar posterior expression pattern in many animals, and in vertebrates they are linked physically to the "posterior" ends of homeotic clusters (HOM-C), suggesting a conserved role in posterior development . However, the function of this posterior expression is not known . Here I show that the Caenorhabditis elegans gene vab-7 encodes an eve homolog that is required for posterior development and expressed in a pattern strikingly similar to that of vertebrate eve genes . Using a four-dimensional recording system, I found that posterior body muscles and the posterior epidermis are patterned abnormally in vab-7 mutants, but commitment to muscle and epidermal fates is normal . Furthermore, vab-7 activity is required for the complete expression of the most posterior HOM-C gene egl-5 in muscle cells, supporting the idea that eve homologs may act with the HOM-C to determine posterior cell fates . The conservation of sequence and expression pattern between vab-7 and eve homologs in other animals argues that most eve genes have posterior mesodermal and ectodermal patterning functions. Genes Dev, 1996 May 1, 10(9), 1073 - 83 The Caenorhabditis elegans cell-death protein CED-3 is a cysteine protease with substrate specificities similar to those of the human CPP32 protease; Xue D et al.; The Caenorhabditis elegans cell-death gene ced-3 encodes a protein similar to mammalian interleukin-1beta-converting enzyme (ICE), a cysteine protease implicated in mammalian apoptosis . We show that the full-length CED-3 protein undergoes proteolytic activation to generate a CED-3 cysteine protease and that CED-3 protease activity is required for killing cells by programmed cell death in C . elegans . We developed an easy and general method for the purification of CED-3/ICE-like proteases and used this method to facilitate a comparison of the substrate specificities of four different purified cysteine proteases . We found that in its substrate preferences CED-3 was more similar to the mammalian CPP32 protease than to mammalian ICE or NEDD2/ICH-1 protease . Our results suggest that different mammalian CED-3/ICE-like proteases may have distinct roles in mammalian apoptosis and that CPP32 is a candidate for being a mammalian functional equivalent of CED-3. Plant J, 1996 May, 9(5), 671 - 81 Reduction of the cytosolic fructose-1,6-bisphosphatase in transgenic potato plants limits photosynthetic sucrose biosynthesis with no impact on plant growth and tuber yield; Zrenner R et al.; Sucrose produced in source leaves is the predominant carbon source for developing sink tissues in most higher plants . Consequently the rate of sucrose synthesis is likely to be important for sink development and final crop yield . Two sucrose biosynthetic enzymes are believed to possess regulatory properties with respect to the rate of sucrose synthesis: (i) cytosolic FBPase and (ii) sucrose phosphate synthase . To study the impact of reduced photosynthetic sucrose biosynthesis on plant growth and crop yield a cDNA clone encoding cytosolic FBPase was isolated from a potato leaf cDNA library and used for antisense experiments in transgenic potato plants . The cDNA clone cy-F1, containing an open reading frame of 1020 bp highly homologous (85%) to other known sequences of plant cytosolic FBPases, was cloned in reversed orientation between the 35S CaMV promoter and the octopine synthase polyadenylation signal . Out of 75 independent transformants five transgenic lines having 9 to 55% of the wild-type FBPase activity were chosen for further analysis . A 45% reduction of the cytosolic FBPase activity did not cause any measurable change in metabolite concentrations, growth behaviour or photosynthetic parameters of the transgenic plants . Inhibition of cytosolic FBPase activity below 20% of the wild-type activity led to an accumulation of 3-PGA, triose-phosphates and fructose-1,6-biphosphate in source leaves . This resulted in a reduced light-saturated rate of assimilation measured via gas exchange and a decreased photosynthetic rate under conditions of the leaf disc electrode with saturating light and CO2 . Measuring photosynthetic carbon fluxes by labelling leaf discs with 14CO2 revealed a 53-65% reduction of sucrose synthesis whereas starch synthesis decreased only by 18-24% . The flux into the anionic and cationic fraction was not altered . Despite these changes steady-state sucrose concentrations were not effected in source leaves from transgenic plants . Starch accumulated by more than a factor of 3 compared with wild-type leaves and was degraded during the night . This provides strong evidence for the hypothesis that hexoses and/or hexosephosphates are exported out of the chloroplasts, thereby circumventing the limitation of sucrose biosynthesis caused by the inhibition of cytosolic FBPase in the dark . Accordingly, plant growth and potato tuber yield remained unaltered . From these data it can be concluded that a reduced photosynthetic sucrose biosynthetic capacity can be efficiently compensated without any reduction in crop yield under greenhouse or growth chamber conditions by changing carbon export strategy . Whether the same holds true for field conditions remains to be elucidated. Nucleic Acids Res, 1996 May 1, 24(9), 1753 - 7 Accumulation of a mRNA decay intermediate by ribosomal pausing at a stop codon; Bjornsson A et al.; A RNA fragment which is protected from degradation by ribosome pausing at a stop codon has been identified in growing Escherichia coli . The fragment is 261 nt long and corresponds to the 3'-end of the mRNA expressed from a semi-synthetic model gene . The 5'-end of the RNA fragment, denoted rpRNA (ribosomal pause RNA), is located 13 bases upstream of the stop codon . In vivo decay of the complete mRNA and accumulation of rpRNA are dependent on the nature of the stop codon and its codon context . The data indicate that the rpRNA fragment arises from interrupted decay of the S3A'mRNA in the 5'-->m3'direction, in connection with a ribosomal pause at the stop codon . RF-2 decoding of UGA is less efficient than RF-1 decoding of UAG in identical codon contexts, as judged from rpRNA steady-state levels . The half-life of UGA-containing rpRNAs is at least 5 min, indicating that ribosomal pausing can be a major factor in stabilising downstream regions of messenger RNAs. Nucleic Acids Res, 1996 May 1, 24(9), 1747 - 52 Identification of promoter and stringent regulation of transcription of the fabH, fabD and fabG genes encoding fatty acid biosynthetic enzymes of Escherichia coli; Podkovyrov SM et al.; In Escherichia coli, amino acid starvation results in the coordinate inhibition of a variety of metabolic activities, including fatty acid and phospholipid biosynthesis . By using primer extension analysis we identified the fabH promoter responsible for transcription of the fabH, fabD and fabG genes encoding fatty acid biosynthetic enzymes . The response of the fabH promoter to amino acid starvation was determined in vivo . Transcripts originating from the fabH promoter were quantified by employing a ribonuclease protection assay . The fabH promoter was subject to relA-dependent stringent control and was repressed approximately 4-fold upon amino acid starvation . The results suggest that inhibition of transcription initiation of lipid biosynthetic genes in starved cells contributes to the stringent control of lipid biosynthesis. Nucleic Acids Res, 1996 May 1, 24(9), 1742 - 6 Reduction of the toxicity and mutagenicity of aziridine in mammalian cells harboring the Escherichia coli fpg gene; Cussac C et al.; Aziridine (ethyleneimine) reacts with DNA in vitro, mainly at the N7 position of guanine and N3 of adenine, then imidazole ring opening of the modified guanine results in formation of formamidopyrimidine (FaPy) residues . The Escherichia coli fpg gene encodes a DNA glycosylase that removes FaPy residues from DNA . To determine whether aziridine produces FaPy lesions in mammalian cells we have expressed the E.coli fpg gene in CHO cells . The transfected cells, expressing high levels of the bacterial protein, are more resistant to the toxic and mutagenic effects of aziridine than the control population . Less DNA damage was measured by quantitative PCR analysis in transfected than in control cells treated with equimolar concentrations of aziridine . The results suggest that aziridine produces in vivo FaPy residues that could account for the deleterious effects of this compound. Nucleic Acids Res, 1996 May 1, 24(9), 1710 - 8 Differences in mutagenesis during minus strand, plus strand and strand transfer (recombination) synthesis of the HIV-1 gene in vitro; Wu W et al.; We have developed an HIV nef-Escherichia coli lacZ fusion system in vitro that allows the detection of low frequency mutations, including frameshifts, deletions and insertions . A portion of the nef gene that encompasses a hypervariable region was fused in-frame with a downstream lacZalpha peptide coding region . The resulting lacZalpha peptide fusion protein remained functional . Any frameshift mutations in the nef insert would put the downstream lacZ alpha peptide gene out of frame, eliminating alpha complementation . With this system we compared the error rates of frameshift mutations that arise during DNA-directed and RNA-directed DNA synthesis . Results showed that DNA-directed and RNA-directed DNA synthesis did not contribute equally to the generation of mutations . DNA-directed DNA synthesis generated frameshift mutations at a frequency approximately 10-fold higher than those arising from RNA-directed DNA synthesis . RNA-directed DNA synthesis in the presence of acceptor templates showed an increase in mutation rate and differences in the mutation spectrum . The enhancement of mutation rate was caused by the appearance of mutations at three new locations that correlated with likely recombination sites . Results indicate that recombination is another source of mutations during viral replication. Nucleic Acids Res, 1996 May 1, 24(9), 1608 - 15 The polypyrimidine tract binding (PTB) protein interacts with single-stranded DNA in a sequence-specific manner; Brunel F et al.; Polypyrimidine tract binding (PTB) protein is a cellular factor whose function is unknown . Various RNA or single-stranded DNA sequences have been shown to interact with PTB . In this paper, using laser UV crosslinking and electrophoretic mobility shift assays to probe DNA-protein interactions, we demonstrate that PTB binding at a single-stranded DNA target is highly sequence-specific . We provide data showing that PTB interacts with the top strand of the adenovirus major late promoter transcriptional initiator, a sequence rich in pyrimidine residues . We also demonstrate that PTB is organised into at least two different binding domains. Eur J Biochem, 1996 May 1, 237(3), 870 - 5 Cys5 and Cys214 of NAD(P)H:flavin oxidoreductase from Escherichia coli are located in the active site; Fieschi F et al.; The NAD(P)H:flavin oxidoreductase (NADPH:riboflavin oxidoreductase) from Escherichia coli, Fre, is a monomer of 26.1 kDa, which catalyzes the reduction of free flavins by NADPH or NADH . A sequential ordered mechanism, with NADPH binding first, operates . Fre is the prototype of a class of flavin reductases able to transfer electrons with no prosthetic group . It has been previously reported that several members of this family, including Fre, were inactivated by thiol reagents such as N-ethylmaleimide (MalNEt) . Amino acid sequence similarities among these enzymes reveal that one of the three cysteines residues of Fre is highly conserved . Altogether this suggested a crucial role of cysteine residues for catalysis . The three cysteine residues were mutated to serine residues . Single-mutant and double-mutant enzymes were as active as the wild-type and Km values for both substrates remained the same . Cysteine residues are thus not important for activity . Nevertheless, we showed that cysteines 5 and 214, but not cysteine 149, were responsible for MalNEt inactivation . In addition, it has been found that riboflavin, but not NADPH, can protect Fre from MalNEt inactivation . This strongly suggested that Cys5 and Cys214 are located at the flavin-binding site of Fre and that flavin can bind to the enzyme in the absence of NADPH. Eur J Biochem, 1996 May 1, 237(3), 827 - 32 Expression in Escherichia coli of Sin a 1, the major allergen from mustard; Gonzalez De La Pena MA et al.; Sin a 1, the major yellow mustard allergen, is a seed storage protein that belongs to the 2S albumin family . It is composed of two disulfide-bonded polypeptide chains . The cloning of this allergen has been carried out by means of the polymerase chain reaction using non-degenerate oligonucleotides encoding the N-terminal and C-terminal regions of the mature protein as primers . Five genomic nucleotide sequences have been analyzed, encoding both mature polypeptide chains linked by the internal processed fragment . The sequence data show the existence of microheterogeneities at ten positions, demonstrating the polymorphism exhibited by the natural protein . One of the genomic clones was expressed in Escherichia coli by fusion to glutathione S-transferase from Schistosoma japonicum . The resulting chimeric protein was purified by affinity chromatography on a glutathione-Sepharose 4B matrix, and digested with thrombin to release the recombinant allergen . The recombinant Sin a 1 is recognized by rabbit polyclonal and mouse monoclonal antisera raised against natural Sin a 1, as well as by the IgE of mustard-sensitive human sera . In addition, recombinant Sin a 1 possesses a high resistance to trypsin digestion, like the native mustard allergen. Eur J Biochem, 1996 May 1, 237(3), 800 - 8 Analysis of the specificity of the AMP-activated protein kinase by site-directed mutagenesis of bacterially expressed 3-hydroxy 3-methylglutaryl-CoA reductase, using a single primer variant of the unique-site-elimination method; Ching YP et al.; The specificity of protein kinases is usually examined using synthetic peptide substrates, either designed variants, or, more recently random peptide libraries . However not all protein kinases utilize synthetic peptides efficiently as substrates . Even among those that do, these approaches neglect effects caused by three-dimensional protein conformation, or the existence of determinants remote from the phosphorylation site . To follow up our previous peptide studies on the specificity of the AMP-activated protein kinase (AMPK) {Dale, S., Wilson, W . A., Edelman, A.M., & Hardie, D . G . (1995) FEBS Lett . 361, 191-195}, we have expressed the C-terminal, catalytic domain of Chinese hamster hydroxymethylglutaryl-CoA reductase in Escherichia coli . The domain was expressed with an N-terminal His6 tag which allowed rapid purification on Nj(2+)-agarose . The purified protein retained full enzymic activity, and as with the native enzyme, was totally inactivated by phosphorylation by AMPK at a single site corresponding to Ser871 . Using a novel modification of the unique-site elimination method (which allowed direct mutagenesis of the double-stranded expression vector using a single oligonucleotide primer) we expressed 18 mutations involving residues around Ser871 . The results broadly confirmed the recognition motif previously proposed on the basis of peptide studies . Three of the mutants were better substrates for AMPK than the wild type, and one of these (K872A) had hydroxymethylglutaryl-CoA reductase kinetic parameters virtually indistinguishable from the wild type . This suggests that hydroxymethylglutaryl-CoA reductase may have been selected to be a sub-optimal substrate for AMPK. Eur J Biochem, 1996 May 1, 237(3), 743 - 51 Systematic mutational analysis of the receptor-binding region of the human urokinase-type plasminogen activator; Magdolen V et al.; The amino-terminal fragment of human uPA (ATF; amino acids 1-135), which contains the binding site for the uPA receptor (uPAR, CD87) was expressed in the yeast Saccharomyces cerevisiae . Recombinant yeast ATF, modified and extended by an amino-terminal in-frame insertion of a His6 tract, was purified from total protein extracts by nickel chelate affinity chromatography and shown to be functionally active since it efficiently competes with uPA for binding to cell-surface-associated uPAR . The ATF expression plasmid served as a template for the construction of a series of site-directed mutants in order to define those amino acids that are important for binding to uPAR . All mutant ATF proteins but one (deletion of Ser26) were expressed in a stable form (about 20-30 ng/mg total protein) and the binding capacity of each mutant was tested by a uPA-ligand binding assay employing recombinant uPAR immobilized to a microtiter plate . Each of the 11 amino acids of loop B of the binding region of uPA (amino acids 20-30) were individually substituted with alanine . Lys23, Tyr24, Phe25, IIe28, and Trp30 were important determinants for uPAR binding . A systematic alanine scan was also performed with chemically synthesized linear peptides spanning amino acids 14-32 of ATF . Comparable results to those with the yeast ATF mutants were obtained . In a different set of experiments, those amino acids of the uPAR-binding region of uPA that are only conserved between man and baboon but not in other species were altered: whereas substitution of Thr18 by alanine or Asn32 by serine had hardly any effect, replacement of Asn22 by tyrosine and Trp30 by arginine (both positions are strictly conserved in other mammals) led to ATF variants incapable of interacting with human uPAR . Deletion of either Val20, Ser21, Lys23, His29 or Val20 plus Ser21, respectively, also generated non-reactive ATF mutants . Finally, Lys23 in ATF was substituted with certain amino acids: whereas the replacement of Lys23 by alanine, histidine or glutamine generated ATF variants with moderate uPAR-binding activity, the introduction of a negatively charged amino acid (exchange of Lys23 by glutamic acid) completely abolished uPAR-binding activity . The results presented for the ATF mutants and uPA-derived peptides may provide clues necessary to establish the nature of the physical interaction of uPA with its receptor and may help to develop uPA-derived peptide analogues as potential therapeutic agents to block tumor cell-associated uPA/uPAR interaction. Eur J Biochem, 1996 May 1, 237(3), 592 - 600 Flavin motion in p-hydroxybenzoate hydroxylase . Substrate and effector specificity of the Tyr22-->Ala mutant; van der Bolt FJ et al.; The side chain of Tyr222 in p-hydroxybenzoate hydroxylase interacts with the carboxy moiety of the substrate . Studies on the Tyr222-->Phe mutant, {F222}p-hydroxybenzoate hydroxylase, have shown that disruption of this interaction hampers the hydroxylation of 4-hydroxybenzoate . Tyr222 is possibly involved in flavin motion, which may facilitate the exchange of substrate and product during catalysis . To elucidate the function of Tyr222 in more detail, in the present study the substrate and effector specificity of the Tyr222-->Ala mutant, {A222}p-hydroxybenzoate hydroxylase, was investigated . Replacement of Tyr222 by Ala impairs the binding of the physiological substrate 4-hydroxybenzoate and the substrate analog 4-aminobenzoate . With these compounds, {A222}p-hydroxybenzoate hydroxylase mainly acts as a NADPH oxidase . {A222}p-hydroxybenzoate hydroxylase tightly interacts with 2,4-dihydroxybenzoate and 2-hydroxy-4-aminobenzoate . Crystallographic data {Schreuder, H.A., Mattevi, A., Oblomova, G., Kalk, K.H., Hol, W.G.J., van der Bolt, F.J.T . & van Berkel, W.J.H . (1994) Biochemistry 33, 10161-10170} suggest that this is due to motion of the flavin ring out of the active site, allowing hydrogen-bond interaction between the 2-hydroxy group of the substrate analogs and N3 of the flavin . {A222}p-Hydroxybenzoate hydroxylase produces about 0.6 mol 2,3,4-trihydroxybenzoate from 2,4-dihydroxybenzoate/mol NADPH oxidized . This indicates that reduction of the Tyr222-->Ala mutant shifts the equilibrium of flavin conformers towards the productive "in' position . {A222}p-Hydroxybenzoate hydroxylase converts 2-fluoro-4-hydroxybenzoate to 2-fluoro-3,4-dihydroxybenzoate . The regioselectivity of hydroxylation suggests that {A222}p-hydroxybenzoate hydroxylase binds the fluorinated substrate in the same orientation as wild-type . Spectral studies suggest that wild-type and {A222}p-hydroxybenzoate hydroxylase bind 2-fluoro-4-hydroxybenzoate in the phenolate form with the flavin ring preferring the "out' conformation . Despite activation of the fluorinated substrate and in contrast to the wild-type enzyme, {A222}p-hydroxybenzoate hydroxylase largely produces hydrogen peroxide . The effector specificity of p-hydroxybenzoate hydroxylase is not changed by the Tyr222-->Ala replacement . This supports the idea that the effector specificity is mainly dictated by the protein-substrate interactions at the re-side of the flavin ring. Int Arch Allergy Immunol, 1996 May, 110(1), 41 - 5 Cloning Aspergillus fumigatus allergens by the pJuFo filamentous phage display system; Crameri R et al.; A cDNA library from Aspergillus fumigatus has been displayed on the surface of filamentous phage M13 and screened for gene products binding to human serum IgE . The physical linkage of cDNA gene products to the genetic information required for their production, achieved by exploiting the high-affinity interaction of the Jun and Fos leucine zippers, allows rapid and easier screening of large libraries in semifluid systems . The pJuFo cloning vector is designed to display proteins on the surface of phage and allows selective isolation of genes by gene product-ligand interaction . Thus the system is applicable to clone cDNA that encodes proteins for which a ligand is available . Herein we show that phage expressing IgE binding proteins from A . fumigatus can be enriched and separated from nonspecific phage by using serum IgE from A . fumigatus-allergic individuals coated to plastic dishes . Subsequently, the proteins can be produced in high amounts in Escherichia coli and purified for usage in allergy testing. Biochem J, 1996 May 1, 315 ( Pt 3), 989 - 94 Bovine inositol monophosphatase: enzyme-metal-ion interactions studied by pre-equilibrium fluorescence spectroscopy; Thorne MR et al.; Stopped-flow fluorescence spectroscopy has been used to determine the on-rate (kass) and the off-rate (kdiss) for the equilibrium between inositol monophosphatase and Mg2+ ions . The dissociation constant (Kd) for the equilibrium calculated from these constants suggests that the ions interact at site 1 on the enzyme with a Kd typically around 450 microM, close to values determined by equilibrium studies (270-300 microM) . The affinity of this site on the wild-type enzyme for Mg2+ ions increases as the pH is increased . This is mediated almost entirely by change in the