Proteins, 1997 May, 28(1), 137 - 9 Crystallization and preliminary X-ray studies of I-CreI: a group I intron-encoded endonuclease from C . reinhardtii; Stephens KM et al.; Group I intron endonuclease I-CreI is encoded by an open reading frame contained within a self-splicing intron in the Chlamydomonas reinhardtii chloroplast 23S rRNA gene . I-CreI initiates the lateral transfer or homing of this intron by specifically recognizing and cleaving a pseudopalindromic 19-24 bp homing site in chloroplast 23S rRNA genes that lack the intron . The gene encoding this enzyme has been subcloned, and the protein product has been purified and crystallized . The crystals belong to space group P321, with unit cell dimensions a = b = 78.2 A, c = 67.4 A . The crystal unit cell is consistent with an asymmetric unit consisting of the enzyme monomer . The specific volume of this unit cell is 3.3 A3/Da . The crystals diffract to at least 3.0 A resolution after flash-cooling, when using a rotating anode x-ray source and an RAXIS image plate detector. Proteins, 1997 May, 28(1), 135 - 6 Crystallization and preliminary X-ray analysis of Escherichia coli peptidyl-tRNA hydrolase; Schmitt E et al.; Peptidyl-tRNA hydrolase from Escherichia coli, a monomer of 21 kDa, was overexpressed from its cloned gene pth and crystallized by using polyethylene glycol as precipitant . The crystals are orthorhombic and have unit cell parameters a = 47.24 A, b = 63.59 A, and c = 62.57 A . They belong to space group P2(1)2(1)2(1) and diffract to better than 1.2 A resolution . The structure is being solved by multiple isomorphous replacement. Protein Sci, 1997 May, 6(5), 1110 - 3 Identification and structural influence of a differentially modified N-terminal methionine in human S100b; Smith SP et al.; The calcium-binding protein S100b is a homodimer comprised of two identical 91-residue beta-subunits . Recombinant S100b is a heterogeneous protein, although the basis of this heterogeneity has not been established . We have used mass spectrometry and NMR spectroscopy to determine that heterogeneity in S100b arises from a mixture of formyl-S100b and desformyl-S100b when expressed in Escherichia coli . Reversed-phase HPLC purification of these two forms of S100b has allowed the differences in N-terminal composition to be used as a probe for tertiary contacts in the protein . The presence or absence of the N-terminal formyl group affected the chemical shifts of sequence neighboring residues and those in the linker of the protein (residues 40-43), indicating that these two regions are close in space. Protein Sci, 1997 May, 6(5), 1038 - 46 Design, synthesis, expression, and characterization of the genes for mouse Fc gamma RIIb1 and Fc gamma RIIb2 cytoplasmic regions; Chen L et al.; The cytoplasmic regions of the mouse low-affinity Fc gamma RII isoforms, mFc gamma RIIb1, and mFc gamma RIIb2, play a key role in signal transduction by mediating different cellular functions . mFc gamma RIIb1 has a 94-residue cytoplasmic region, whereas mFc gamma RIIb2 has a 47-residue cytoplasmic region . Genes encoding the cytoplasmic regions of mFc gamma RIIb1 (b1-94) and mFc gamma RIIb2 (b2-47) were designed, synthesized, and expressed as fusion proteins in Escherichia coli . A sequence-specific protease, thrombin, was used to release the b1-94 peptide, which was purified by using HPLC . The b2-47 peptide was synthesized chemically . CD spectropolarimetry was employed to examine the secondary structures of b1-94 and b2-47 . These studies were conducted in aqueous solution, in mixtures of water and trifluoroethanol or methanol, and as a function of temperature . The results indicate that the b1-94 and b2-47 structures are sensitive functions of the solvent environment, and that nonaqueous solvents induce significant alpha-helical structure. Protein Sci, 1997 May, 6(5), 1031 - 7 The role of divalent cations in structure and function of murine adenosine deaminase; Cooper BF et al.; For murine adenosine deaminase, we have determined that a single zinc or cobalt cofactor bound in a high affinity site is required for catalytic function while metal ions bound at an additional site(s) inhibit the enzyme . A catalytically inactive apoenzyme of murine adenosine deaminase was produced by dialysis in the presence of specific zinc chelators in an acidic buffer . This represents the first production of the apoenzyme and demonstrates a rigorous method for removing the occult cofactor . Restoration to the holoenzyme is achieved with stoichiometric amounts of either Zn2+ or Co2+ yielding at least 95% of initial activity . Far UV CD and fluorescence spectra are the same for both the apo- and holoenzyme, providing evidence that removal of the cofactor does not alter secondary or tertiary structure . The substrate binding site remains functional as determined by similar quenching measured by tryptophan fluorescence of apo- or holoenzyme upon mixing with the transition state analog, deoxycoformycin . Excess levels of adenosine or N6- methyladenosine incubated with the apoenzyme prior to the addition of metal prevent restoration, suggesting that the cofactor adds through the substrate binding cleft . The cations Ca2+, Cd2+, Cr2+, Cu+, Cu2+, Mn2+, Fe2+, Fe3+, Pb2+, or Mg2+ did not restore adenosine deaminase activity to the apoenzyme . Mn2+, Cu2+, and Zn2+ were found to be competitive inhibitors of the holoenzyme with respect to substrate and Cd2+ and Co2+ were noncompetitive inhibitors . Weak inhibition (Ki > or = 1000 microM) was noted for Ca2+, Fe2+, and Fe3+. Protein Sci, 1997 May, 6(5), 1016 - 23 Identification of a novel bond between a histidine and the essential tyrosine in catalase HPII of Escherichia coli; Bravo J et al.; A bond between the N delta of the imidazole ring of His 392 and the C beta of the essential Tyr 415 has been found in the refined crystal structure at 1.9 A resolution of catalase HPII of Escherichia coli . This novel type of covalent linkage is clearly defined in the electron density map of HPII and is confirmed by matrix-assisted laser desorption/ionization mass spectrometry analysis of tryptic digest mixtures . The geometry of the bond is compatible with both the sp3 hybridization of the C beta atom and the planarity of the imidazole ring . Two mutated variants of HPII active site residues, H128N and N201H, do not contain the His 392-Tyr 415 bond, and their crystal structures show that the imidazole ring of His 392 was rotated, in both cases, by 80 degrees relative to its position in HPII . These mutant forms of HPII are catalytically inactive and do not convert heme b to heme d, suggesting a relationship between the self-catalyzed heme conversion reaction and the formation of the His-Tyr linkage . A model coupling the two processes and involving the reaction of one molecule of H2O2 on the proximal side of the heme with compound 1 is proposed. Clin Diagn Lab Immunol, 1997 May, 4(3), 375 - 9 Standardization of measurement of immunoglobulin-secreting cells in human peripheral circulation; Baqar S et al.; A sensitive, and at times the most sensitive, measurement of human vaccine immunogenicity is enumeration of antibody-secreting cells (ASC) in peripheral blood . However, this assay, which is inherently capable of measurement of the absolute number of antigen-specific ASC, is not standardized . Thus, quantitative comparison of results between laboratories is not currently possible . To address this issue, isotype-specific ASC were enumerated from paired fresh and cryopreserved mononuclear cell (MNC) preparations from healthy adult volunteers resident in either the United States (US group) or Egypt (EG group) . Analysis of fresh cells from US volunteers revealed mean numbers of ASC per 10(6) MNC of 617, 7,738, and 868 for immunoglobulin M (IgM), IgG, and IgA, respectively, whereas EG volunteers had 2,086, 7,580, and 1,677 ASC/10(6) MNC for the respective isotypes . Cryopreservation resulted in a slight reduction in group mean IgM, IgG, and IgA ASC (maximum reduction in group mean, 14%), but in no instance were results obtained with cryopreserved cells significantly lower than those obtained with fresh cells . To determine if cryopreservation affected the number of bacterial antigen-specific ASC detected, cells from a group of US adult volunteers who received a single oral dose of a mutated Escherichia coli heat-labile enterotoxin (LT(R192G)) were tested . There was no significant difference (P > 0.05) in the number of antigen-specific IgA or IgG ASC detected between fresh and cryopreserved MNC . The results support the views that ASC assays can be standardized to yield quantitative results and that the methodology can be changed to make the test more practical. Hum Gene Ther, 1997 May 1, 8(7), 843 - 9 In vitro assembly of SV40 virions and pseudovirions: vector development for gene therapy; Sandalon Z et al.; SV40 is an attractive potential vector with high-efficiency gene transfer into a wide variety of human tissues, including the bone marrow, a critical target organ for the cure of many diseases . In the present study, the three SV40 capsid proteins, VP1, VP2, and VP3, were produced in Spodoptera frugiperda (Sf9) insect cells . Their co-production led to spontaneous assembly of SV40-like particles . Nuclear extracts containing the three proteins were allowed to interact with purified SV40 DNA, or with plasmid DNA produced and purified from Escherichia coli . The experiments demonstrated a physical association between the DNA and capsid proteins, protection from DNase I digestion, and the formation of infectious particles . The results indicate that intact, supercoiled DNA is being packaged and transmitted into the target cells . The transmitted DNA is biologically functional in gene expression and replication . The process, which utilizes naked DNA, is not dependent on the SV40 packaging signal ses . The procedure allows packaging of plasmids significantly larger than SV40 and permits the inclusion of potent regulatory signals, such as beta-globin locus control region (LCR) elements . These studies are the first step in the development of purified, in vitro-constructed pseudovirions for experimental and medical use. Arch Biochem Biophys, 1997 May 1, 341(1), 170 - 6 Lamprey fructose-1,6-bisphosphate aldolase: characterization of the muscle-type and non-muscle-type isozymes; Zhang R et al.; To study evolutionary aspects of fructose-1,6-bisphosphate (Fru-1,6-P2) aldolase during deuterostomian evolution, we have purified and characterized aldolases from the muscle and liver of lamprey (Entosphenus japonicus) . Aldolase from the skeletal muscle and liver was identified to be the muscle-type isozyme and the non-muscle-type isozyme that was encoded by cDNAs M8 and L3, respectively, as described previously (Zhang, R., Yatsuki, H., Kusakabe, T., Iwabe, Miyata, T., Imai, T., Yoshida, M., and Hori, K., J . Biochem . 117, 545-553, 1995) . The muscle-type isozyme has properties similar to vertebrate aldolase A, while the non-muscle-type isozyme shows a similarity to bacterial class I aldolase and vertebrate aldolase C but not to aldolase B, the liver-type aldolase, in terms of kinetic parameters: the Kcat values toward Fru-1,6-P2 and Fru-1-P, the Fru-1,6-P2/Fru-1-P activity ratio, and the Km values toward these substrates . The two enzymes have tetrameric forms with a molecular mass of approximately 160,000 and have similar pH optimum . The muscle-type and non-muscle-type isozymes from the tissues show different electrophoretic mobility; the muscle-type isozyme moves much faster than the non-muscle-type isozyme toward anodic side . The recombinant muscle-type and non-muscle-type aldolases gave similar characteristics as those from the tissues . The results presented in this paper, together with the data presented in the previous paper, strongly suggest that in lamprey it is possible to have two types of aldolase isozymes rather than one or three isozymes. Arch Biochem Biophys, 1997 May 1, 341(1), 98 - 103 The human glycinamide ribonucleotide transformylase domain: purification, characterization, and kinetic mechanism; Caperelli CA et al.; Glycinamide ribonucleotide transformylase catalyzes the third reaction of de novo purine biosynthesis, namely, the conversion of glycinamide ribonucleotide to N-formylglycinamide ribonucleotide, with concomitant conversion of 10-formyltetrahydrofolate to tetrahydrofolate . This activity has been shown to be a target for cancer chemotherapy, which has generated renewed interest in both the enzyme and the pathway . Moreover, in higher eukaryotes this activity constitutes the C-terminal domain of a monomeric protein which also catalyzes two additional reactions of de novo purine biosynthesis . In this study, the human glycinamide ribonucleotide transformylase domain has been expressed to high levels in Escherichia coli and purified to homogeneity . Our improved expression-purification system produces the desired activity exclusively in a soluble form and in higher abundance than previously achieved . The kinetic constants have been determined and the kinetic mechanism has been established as ordered-sequential, with the folate substrate binding first . The correspondence of these data to those obtained for the glycinamide ribonucleotide transformylase activity of the mammalian trifunctional enzyme indicates that the recombinant enzyme is fully functional. Arch Biochem Biophys, 1997 May 1, 341(1), 62 - 9 Potency comparison of peptidomimetic inhibitors against HIV-1 and HIV-2 proteinases: design of equipotent lead compounds; Weber J et al.; HIV-1 and HIV-2 proteinases (PR) are responsible for the processing of viral polyproteins, a step that is crucial for the formation of infectious virus particles . PR represents one of the most important targets for antiviral chemotherapy . Inhibitors of HIV-1 PR usually exhibit a 10- to 100-fold weaker affinity for HIV-2 PR . In order to design subnanomolar inhibitors for both HIV-1 and HIV-2 PRs, we prepared a series of compounds varying in the type of scissile bond replacement as well as in the P1, P1', and P2' side chains . While inhibitors containing reduced amide, hydroxyethylamine and statine isosteres had Ki values in the range of 10(-10)-10(-9) M against HIV-1 PR; their activities against HIV-2 PR were several orders of magnitude lower . Glutamic acid was identified to be the optimal P2' residue for both PRs . HIV-2 PR was shown to be more sensitive to P2' Glu-->Gln replacement . Using this data set we were able to design and prepare hydroxyethylene isostere containing inhibitors that were equipotent against both PRs. J Neuroimmunol, 1997 May, 75(1-2), 28 - 34 2',3'-cyclic nucleotide 3'-phosphodiesterase: a novel candidate autoantigen in demyelinating diseases; Rosener M et al.; Autoaggressive T-cells specific for myelin proteins like proteolipid protein (PLP) and myelin basic protein (MBP) are thought to play a major role in the pathogenesis of demyelinating diseases of the central nervous system (CNS) . 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) is the third most abundant myelin protein in the CNS . Due to lack of supply with enough CNPase of sufficient purity its immunologic properties have not been studied yet . We subcloned a human CNPase cDNA and expressed human recombinant CNPase (rh-CNPase) in E . coli . Purification of the protein was achieved by Ni(2+)-chelating chromatography . Furthermore we describe for the first time several rh-CNPase specific T-cell lines from a multiple sclerosis patient and a healthy control. Appl Environ Microbiol, 1997 May, 63(5), 1689 - 95 Manipulation of independent synthesis and degradation of polyphosphate in Escherichia coli for investigation of phosphate secretion from the cell; Van Dien SJ et al.; The genes involved in polyphosphate metabolism in Escherichia coli were cloned behind different inducible promoters on separate plasmids . The gene coding for polyphosphate kinase (PPK), the enzyme responsible for polyphosphate synthesis, was placed behind the Ptac promoter . Polyphosphatase, a polyphosphate depolymerase, was similarly expressed by using the arabinose-inducible PBAD promoter . The ability of cells containing these constructs to produce active enzymes only when induced was confirmed by polyphosphate extraction, enzyme assays, and RNA analysis . The inducer concentrations giving optimal expression of each enzyme were determined . Experiments were performed in which ppk was induced early in growth, overproducing PPK and allowing large amounts of polyphosphate to accumulate (80 mumol in phosphate monomer units per g of dry cell weight) . The ppx gene was subsequently induced, and polyphosphate was degraded to inorganic phosphate . Approximately half of this polyphosphate was depleted in 210 min . The phosphate released from polyphosphate allowed the growth of phosphate-starved cells and was secreted into the medium, leading to a down-regulation of the phosphate-starvation response . In addition, the steady-state polyphosphate level was precisely controlled by manipulating the degree of ppx induction . The polyphosphate content varied from 98 to 12 mumol in phosphate monomer units per g of dry cell weight as the arabinose concentration was increased from 0 to 0.02% by weight. Anesth Analg, 1997 May, 84(5), 1097 - 103 ONO-5046, an elastase inhibitor, attenuates endotoxin-induced acute lung injury in rabbits; Nishina K et al.; Endotoxin causes acute lung injury resembling acute respiratory distress syndrome . Elastase, as well as reactive oxygen species released from activated neutrophils, are thought to play pivotal roles in the pathogenesis of this lung injury . This study investigated whether ONO-5046, a specific elastase inhibitor, can attenuate acute lung injury induced by endotoxin in rabbits . Thirty-two male anesthetized rabbits were randomly assigned to receive one of four treatments (n = 8 for each group): infusion of saline without ONO-5046 treatment (Group S-S), infusion of saline with ONO-5046 (Group S-O), infusion of Escherichia coli endotoxin (100 micrograms/kg over 60 min) without ONO-5046 (Group E-S), and infusion of endotoxin with ONO-5046 (Group E-O) . Fifteen minutes before the infusion of endotoxin (Groups E-O and E-S) or saline (Groups S-S and S-O), the animals received a bolus injection of ONO-5046 (10 mg/kg) followed by continuous infusion (10 mg.kg-1.h-1: Groups S-O and E-O) or saline (Groups S-S and E-S) . The lungs of the rabbits were ventilated with 40% oxygen . Hemodynamics, peripheral leukocyte and platelet counts, and PaO2 were recorded during the ventilation period (6 h) . Lung mechanics, cell fraction of the bronchoalveolar lavage fluid (BALF), activated complement, cytokines, and arachidonic acid metabolite concentrations in the BALF were measured at 6 h . The wet- to dry (W/D)-weight ratio of the lung and albumin concentrations in BALF were analyzed as indices of pulmonary edema . Endotoxin decreased lung compliance and PaO2, and increased the W/D weight ratio, neutrophil counts, and albumin concentration in the BALF . Concentrations of activated complement C5a, interleukin-6, interleukin-8, and thromboxane B2 in the BALF were increased by the infusion of endotoxin . ONO-5046 treatment attenuated these changes . Endotoxin caused extensive morphologic lung damage, which was lessened by ONO-5046 . In conclusion, intravenous ONO-5046 pretreatment attenuated endotoxin-induced lung injury in rabbits . This beneficial effect of ONO-5046 may be due, in part, to a reduction in the levels of mediators that activate neutrophils, in addition to the direct inhibitory effect on elastase. Phytochemistry, 1997 May, 45(2), 313 - 20 Dihydroisocoumarins from fungi: isolation, structure elucidation, circular dichroism and biological activity; Krohn K et al.; Five known and three new dihydroisocoumarins were isolated from different fungi . The new isocoumarins are 5-chloro-6-hydroxymellein, 5-chloro-4,6-dihydroxymellein and 5,6-dihydroxymellein . The absolute configuration of these secondary metabolites was confirmed by CD measurements and in two cases by X-ray structure analysis. J Bacteriol, 1997 May, 179(9), 3053 - 7 Nucleotide sequence of the Mycobacterium leprae katG region; Nakata N et al.; Synthetic oligonucleotide primers based on the DNA sequence data of the Escherichia coli, Mycobacterium tuberculosis, and Mycobacterium intracellulare katG genes encoding the heme-containing enzyme catalase-peroxidase were used to amplify and analyze the Mycobacterium leprae katG region by PCR . A 1.6-kb DNA fragment, which hybridized to an M . tuberculosis katG probe, was obtained from an M . leprae DNA template . Southern hybridization analysis with a probe derived from the PCR-amplified fragment showed that the M . leprae chromosome contains only one copy of the putative katG sequence in a 3.4-kb EcoRI-BamHI DNA segment . Although the nucleotide sequence of the katG region of M . leprae was approximately 70% identical to that of the M . tuberculosis katG gene, no open reading frame encoding a catalase-peroxidase was detectable in the whole sequence . Moreover, two DNA deletions of approximately 100 and 110 bp were found in the M . leprae katG region, and they seemed to be present in all seven M . leprae isolates tested . These results strongly suggest that M . leprae lacks a functional katG gene and catalase-peroxidase activity. J Bacteriol, 1997 May, 179(9), 2976 - 86 Characterization of pURB500 from the archaeon Methanococcus maripaludis and construction of a shuttle vector; Tumbula DL et al.; The complete sequence of the 8,285-bp plasmid pURB500 from Methanococcus maripaludis C5 was determined . Sequence analysis identified 18 open reading frames as well as two regions of potential iterons and complex secondary structures . The shuttle vector, pDLT44, for M . maripaludis JJ was constructed from the entire pURB500 plasmid and pMEB.2, an Escherichia coli vector containing a methanococcal puromycin-resistance marker (P . Gernhardt, O . Possot, M . Foglino, L . Sibold, and A . Klein, Mol . Gen . Genet . 221:273-279, 1990) . By using polyethylene glycol transformation, M . maripaludis JJ was transformed at a frequency of 3.3 x 10(7) transformants per microg of pDLT44 . The shuttle vector was stable in E . coli under ampicillin selection but was maintained at a lower copy number than pMEB.2 . Based on the inability of various restriction fragments of pURB500 to support maintenance in M . maripaludis JJ, multiple regions of pURB500 were required . pDLT44 did not replicate in Methanococcus voltae. J Bacteriol, 1997 May, 179(9), 2944 - 8 Roles of histidine-103 and tyrosine-235 in the function of the prolipoprotein diacylglyceryl transferase of Escherichia coli; Sankaran K et al.; Phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt) is the first enzyme in the posttranslational sequence of reactions resulting in the lipid modification of lipoproteins in bacteria . A previous comparison of the primary sequences of the Lgt enzymes from phylogenetically distant bacterial species revealed several highly conserved amino acid sequences throughout the molecule; the most extensive of these was the region 103HGGLIG108 in the Escherichia coli Lgt (H.-Y . Qi, K . Sankaran, K . Gan, and H . C . Wu, J . Bacteriol . 177:6820-6824, 1995) . These studies also revealed that the kinetics of inactivation of E . coli Lgt with diethylpyrocarbonate were consistent with the modification of a single essential histidine or tyrosine residue . The current study was conducted in an attempt to identify this essential amino acid residue in order to further define structure-function relationships in Lgt . Accordingly, all of the histidine residues and seven of the tyrosine residues of E . coli Lgt were altered by site-directed mutagenesis, and the in vitro activities of the altered enzymes, as well the abilities of the respective mutant lgt alleles to complement the temperature-sensitive phenotype of E . coli SK634 defective in Lgt activity, were determined . The data obtained from these studies, in conjunction with additional chemical inactivation studies, support the conclusion that His-103 is essential for Lgt activity . These studies also indicated that Tyr-235 plays an important role in the function of this enzyme . Although other histidine and tyrosine residues were not found to be essential for Lgt activity, alterations of His-196 resulted in a significant reduction of in vitro activity. J Bacteriol, 1997 May, 179(9), 2930 - 7 Identification and characterization of the nifV-nifZ-nifT gene region from the filamentous cyanobacterium Anabaena sp . strain PCC 7120; Stricker O et al.; The nifV and leuA genes, which encode homocitrate synthase and alpha-isopropylmalate synthase, respectively, were cloned from the filamentous cyanobacterium Anabaena sp . strain PCC 7120 by a PCR-based strategy . Since the N-terminal parts of NifV and LeuA from other bacteria are highly similar to each other, a single pair of PCR primers was used to amplify internal fragments of both Anabaena strain 7120 genes . Sequence analysis of cloned PCR products confirmed the presence of two different nifV-like DNA fragments, which were subsequently used as nifV- and leuA-specific probes, respectively, to clone XbaI fragments of 2.1 kbp (pOST4) and 2.6 kbp (pOST2) . Plasmid pOST4 carried the Anabaena strain 7120 nifV-nifZ-nifT genes, whereas pOST2 contained the leuA and dapF genes . The nifVZT genes were not located in close proximity to the main nif gene cluster in Anabaena strain 7120, and therefore nifVZT forms a second nif gene cluster in this strain . Overlaps between the nifV and nifZ genes and between the nifZ and nifT genes and the presence of a 1.8-kb transcript indicated that nifVZT might form one transcriptional unit . Transcripts of nifV were induced not only in a nitrogen-depleted culture but also by iron depletion irrespective of the nitrogen status . The nifV gene in Anabaena strain 7120 was interrupted by an interposon insertion (mutant strain BMB105) and by a plasmid integration via a single crossover with a nifV internal fragment as a site for recombination (mutant strain BMB106) . Both mutant strains were capable of diazotrophic growth, and their growth rates were only slightly impaired compared to that of the wild type . Heterologous complementation of the Rhodobacter capsulatus nifV mutant R229I by the Anabaena strain 7120 nifV gene corroborated the assumption that Anabaena strain 7120 nifV also encodes a homocitrate synthase . In contrast, the Anabaena strain 7120 leuA gene did not complement the nifV mutation of R229I efficiently. J Bacteriol, 1997 May, 179(9), 2922 - 9 A mycobacterial extracytoplasmic function sigma factor involved in survival following stress; Wu QL et al.; The extracytoplasmic function (ECF) sigma factors constitute a diverse group of alternative sigma factors that have been demonstrated to regulate gene expression in response to environmental conditions in several bacterial species . Genes encoding an ECF sigma factor of Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium smegmatis, designated sigE, were cloned and analyzed . Southern blot analysis demonstrated the presence of a single copy of this gene in these species and in Mycobacterium bovis BCG, Mycobacterium leprae, and Mycobacterium fortuitum . Sequence analysis showed the sigE gene to be highly conserved among M . tuberculosis, M . avium, M . smegmatis, and M . leprae . Recombinant M . tuberculosis SigE, when combined with core RNA polymerase from M . smegmatis, reconstituted specific RNA polymerase activity on sigE in vitro, demonstrating that this gene encodes a functional sigma factor . Two in vivo transcription start sites for sigE were also identified in M . smegmatis and M . bovis BCG . Comparison of wild-type M . smegmatis with a sigE mutant strain demonstrated decreased survival of the mutant under conditions of high-temperature heat shock, acidic pH, exposure to detergent, and oxidative stress . An inducible protective response to oxidative stress present in the wild type was absent in the mutant . The mycobacterial SigE protein, although nonessential for viability in vitro, appears to play a role in the ability of these organisms to withstand a variety of stresses. J Bacteriol, 1997 May, 179(9), 2900 - 6 Three different putative phosphate transport receptors are encoded by the Mycobacterium tuberculosis genome and are present at the surface of Mycobacterium bovis BCG; Lefevre P et al.; A gene encoding a protein homologous to the periplasmic ABC phosphate binding receptor PstS from Escherichia coli was cloned and sequenced from a lambda gt11 library of Mycobacterium tuberculosis by screening with monoclonal antibody 2A1-2 . Its degree of similarity to the E . coli PstS is comparable to those of the previously described M . tuberculosis phosphate binding protein pab (Ag78, Ag5, or 38-kDa protein) and another M . tuberculosis protein which we identified recently . We suggest that the three M . tuberculosis proteins share a similar function and could be named PstS-1, PstS-2, and PstS-3, respectively . Molecular modeling of their three-dimensional structures using the structure of the E . coli PstS as a template and their inducibility by phosphate starvation support this view . Recombinant PstS-2 and PstS-3 were produced and purified by affinity chromatography . With PstS-1, these proteins were used to demonstrate the specificity of three groups of monoclonal antibodies . Using these antibodies in flow cytometry and immunoblotting analyses, we demonstrate that the three genes are expressed and their protein products are present and accessible at the mycobacterial surface as well as in its culture filtrate . Together with the M . tuberculosis genes encoding homologs of the PstA, PstB, and PstC components we cloned before, the present data suggest that at least one, and possibly several, related and functional ABC phosphate transporters exist in mycobacteria . It is hypothesized that the mycobacterial gene duplications presented here may be a subtle adaptation of intracellular pathogens to phosphate starvation in their alternating growth environments. J Bacteriol, 1997 May, 179(9), 2872 - 8 Unbalanced membrane phospholipid compositions affect transcriptional expression of certain regulatory genes in Escherichia coli; Inoue K et al.; The amount of porin protein OmpF in the outer membrane of Escherichia coli was reduced to one-third by the pgsA3 mutation that diminishes the amount of phosphatidylglycerol and cardiolipin in the membrane, whereas a cls (cardiolipin synthase) null mutation had no effect . Osmoregulation of OmpF was functional in the pgsA3 mutant . As assessed by the beta-galactosidase activities of lacZ fusions, the ompF expression was not reduced at the transcriptional level but was reduced about threefold at the posttranscriptional level by pgsA3 . This reduction was mostly restored by a micF null mutation, and the micF RNA that inhibits the ompF mRNA translation was present 1.3 to 1.4 times more in the pgsA3 mutant, as assayed by RNase protection and Northern blot analyses . Elevation of the level of micF RNA was not restricted to acidic-phospholipid deficiency: OmpF was hardly detected and micF RNA was present 2.7 to 2.8 times more in a pssA null mutant that lacked phosphatidylethanolamine . Other common phenotypes of pgsA3 and pssA null mutants, reduced rates of cell growth and phospholipid synthesis, were not the cause of micF activation . Salicylate, which activates micF expression and inhibits cell motility, did not repress the flagellar master operon . These results imply that an unbalanced phospholipid composition, rather than a decrease or increase in the amount of specific phospholipid species, induces a phospholipid-specific stress signal to which certain regulatory genes respond positively or negatively according to their intrinsic mechanisms. J Bacteriol, 1997 May, 179(9), 2857 - 62 Lethality of the covalent linkage between mislocalized major outer membrane lipoprotein and the peptidoglycan of Escherichia coli; Yakushi T et al.; The major outer membrane lipoprotein (Lpp) of Escherichia coli possesses serine at position 2, which is thought to function as the outer membrane sorting signal, and lysine at the C terminus, through which Lpp covalently associates with peptidoglycan . Arginine (R) is present before the C-terminal lysine in the wild-type Lpp (LppSK) . By replacing serine (S) at position 2 with aspartate (D), the putative inner membrane sorting signal, and by deleting lysine (K) at the C terminus, Lpp mutants with a different residue at either position 2 (LppDK) or the C terminus (LppSR) or both (LppDR) were constructed . Expression of LppSR and LppDR little affected the growth of E . coli . In contrast, the number of viable cells immediately decreased when LppDK was expressed . Prolonged expression of LppDK inhibited separation of the inner and outer membranes by sucrose density gradient centrifugation, whereas short-term expression did not . Pulse-labeled LppDK and LppDR were localized in the inner membrane, indicating that the amino acid residue at position 2 functions as a sorting signal for the membrane localization of Lpp . LppDK accumulated in the inner membrane covalently associated with the peptidoglycan and thus prevented the separation of the two membranes . Globomycin, an inhibitor of lipoprotein-specific signal peptidase II, was lethal for E . coli only when Lpp possessed the C-terminal lysine . Taken together, these results indicate that the inner membrane accumulation of Lpp per se is not lethal for E . coli . Instead, a covalent linkage between the inner membrane Lpp having the C-terminal lysine and the peptidoglycan is lethal for E . coli, presumably due to the disruption of the cell surface integrity. J Bacteriol, 1997 May, 179(9), 2835 - 9 Mutagenic properties of the T-C cyclobutane dimer; Horsfall MJ et al.; G x C-->A x T transitions within T-C or C-C bipyrimidine sequences are by far the most frequent class of mutation induced by 254-nm UV irradiation in most genes and species investigated, but the reason for the high degree of mutability and specificity at these sites is uncertain . Some data implicate the deamination of cytosine to uracil as a possible cause, but other results appear to indicate that the rate of deamination is too low for this to be significant in Escherichia coli . If deamination is not the cause, the high degree of mutability must presumably reflect the inherent properties of T-C and C-C dimers . We investigated this question by transfecting excision-deficient and excision-proficient strains of E . coli with single-stranded vectors that carried a site-specific cis-syn T-C cyclobutane dimer and by analyzing the nucleotide sequences of replicated vector products . We found that replication past the T-C dimer, like replication past its T-T and U-U counterparts, is in fact >95% accurate and that the frequencies of bypass are also very similar for these photoproducts . Since the T-C dimer appears to be only weakly mutagenic, the high frequency of UV-induced mutations at T-C sites presumably depends on some other process, such as deamination, although the mechanism remains to be established. Nature, 1997 May 1, 387(6628), 101 - 5 Essential role for diacylglycerol in protein transport from the yeast Golgi complex; Kearns BG et al.; Yeast phosphatidylinositol transfer protein (Sec14p) is required for the production of secretory vesicles from the Golgi . This requirement can be relieved by inactivation of the cytosine 5'-diphosphate (CDP)-choline pathway for phosphatidylcholine biosynthesis, indicating that Sec14p is an essential component of a regulatory pathway linking phospholipid metabolism with vesicle trafficking (the Sec14p pathway) . Sac1p (refs 7 and 8) is an integral membrane protein related to inositol-5-phosphatases such as synaptojanin, a protein found in rat brain . Here we show that defects in Sac1p also relieve the requirement for Sec14p by altering phospholipid metabolism so as to expand the pool of diacylglycerol (DAG) in the Golgi . Moreover, although short-chain DAG improves secretory function in strains with a temperature-sensitive Sec14p, expression of diacylglycerol kinase from Escherichia coli further impairs it . The essential function of Sec14p may therefore be to maintain a sufficient pool of DAG in the Golgi to support the production of secretory vesicles. Cytometry, 1997 May 1, 28(1), 36 - 41 Verapamil inhibition of enzymatic product efflux leads to improved detection of beta-galactosidase activity in lacZ-transfected cells; Poot M et al.; The beta-galactosidase activity encoded by the lacZ gene of Escherichia coli is widely used to monitor successful expression of transfected genes . Fluorogenic substrates allow detection of enzyme activity in viable cells, which, subsequently, can be selected for further study on the basis of fluorescence emission . We analyzed three fluorogenic substrates (FDG, C12FDG, and CMFDG), all of which incorporate fluorescein as their fluorophore, regarding intensity of fluorescent signal and selectivity toward the transfected beta-galactosidase activity vs . lysosomal enzyme activity . Among these substrates, 5-chloromethylfluoresecein di-beta-D-galactopyranoside (CMFDG) showed the strongest selectivity toward the lacZ-encoded enzyme activity . An attempt to improve this selectivity by alkalinization of the lysosomal pH with chloroquine, such that the endogenous enzyme would be exposed to a suboptimal pH, led to significant cell death . In contrast, inhibition of dye efflux with verapamil enhanced the selectivity of CMFDG toward the lacZ-encoded enzyme activity by approximately threefold . Incubation with probenecid, on the other hand, showed little effect. J Cell Biochem, 1997 May, 65(2), 186 - 97 Analysis of epitope structure of PSP94 (prostate secretory protein of 94 amino acids): (II) . Epitope mapping by monoclonal antibodies; Xuan JW et al.; PSP94 has shown potential to be a serum biomarker for evaluating prostate cancer . Studies of the epitope structure is crucial for this endeavour . In this article, we have used 15 different monoclonal antibodies (MAb) to analyse the epitope structure of PSP94 and to compare with the results obtained from our previous work using polyclonal antibody and recombinant PSP94 . Firstly, we determined the relative activities of the 15 MAb population by direct and competitive ELISA . The two predominant MAbs (MAb PSP-6 and -19) in 15 MAbs were selected for further studies of the epitope structure . By comparing the binding activities of recombinant GST-PSP94 and natural PSP94 with MAbs, and by comparing their affinity with MAbs in an in vitro denaturing experiment, PSP94 was shown to have a similar, prevalently linear epitope structure as we demonstrated by polyclonal antibody . Using recombinant GST fusion protein with PSP94 and with each half of the N- and C-terminal 47 amino acids (GST-PSP-N47/C47) in E . coli cells, the different epitopes recognized by 15 monoclonal antibodies were delineated and the polar distribution of the epitope structure of PSP94 was characterized . Results of direct ELISA of recombinant N47 and C47 and their competitive binding against natural PSP94 (competitive ELISA) showed that the N- and C-termini represent the immuno-dominant and immuno-recessive area separately . A majority of the monoclonal antibodies (12/15) showed preferential binding of the N-terminal sequence of the PSP94 protein . Using GST-PSP-N47 as a standard protein, an epitope map of the 15 monoclonal antibodies was obtained . The results of this study will help to define the clinical utility of PSP94. J Cell Biochem, 1997 May, 65(2), 172 - 85 Analysis of epitope structure of PSP94 (prostate secretory protein of 94 amino acids): (I) . Immuno-dominant and immuno-recessive area; Xuan JW et al.; PSP94 is a potential biomarker for evaluating patients with prostate carcinoma . We have systematically studied the epitope structure of PSP94 by using a polyclonal antibody against human PSP94 . Results of peptide mapping and ELISA tests of dose response to rabbit antiserum against human PSP94 protein showed that only the N-terminal peptides (N30 and M23) are immunoreactive while all the synthetic peptides (C28, C10) located closer to the C-terminus are completely devoid of antigenic activity with the polyclonal antibody . These results were confirmed by analysis of reciprocal competitive binding of PSP94 polyclonal antibody by the N-terminal peptides (N30 and M23) v . either recombinant GST-PSP94 fusion protein, purified recombinant PSP94, or natural PSP94 protein . To further delineate the antigenic activity of the N- and C-termini, we have also expressed N- and C-terminal half of the whole PSP94 (each 47 peptides) using the E . coli GST expression system . The recombinant N47/C47 peptides were released by thrombin cleavage from the GST fusion protein and characterized by Western blotting experiments . Dose response of the recombinant GST-PSP-N47 and -C47 peptides to PSP94 polyclonal antibody showed differential binding activities . Competitive binding of these recombinant N47/C47 proteins against the GST-PSP94 protein demonstrates that the polyclonal antibody has a higher affinity for the N47 peptide than the C47 peptide . Based on the immunological studies of both synthetic peptides and recombinant PSP94- N/C terminal proteins, we propose an epitope structure of human PSP94 with an immuno-dominant N-terminus and an immuno-recessive C-terminus. Genetics, 1997 May, 146(1), 295 - 307 Natural selection and the frequency distributions of "silent" DNA polymorphism in Drosophila; Akashi H et al.; In Escherichia coli, Saccharomyces cerevisiae, and Drosophila melanogaster, codon bias may be maintained by a balance among mutation pressure, genetic drift, and natural selection favoring translationally superior codons . Under such an evolutionary model, silent mutations fall into two fitness categories: preferred mutations that increase codon bias and unpreferred changes in the opposite direction . This prediction can be tested by comparing the frequency spectra of synonymous changes segregating within populations; natural selection will elevate the frequencies of advantageous mutations relative to that of deleterious changes . The frequency distributions of preferred and unpreferred mutations differ in the predicted direction among 99 alleles of two D . pseudoobscura genes and five alleles of eight D . simulans genes . This result confirms the existence of fitness classes of silent mutations . Maximum likelihood estimates suggest that selection intensity at silent sites is, on average, very weak in both D . pseudoobscura and D . simulans (magnitude of NS approximately 1) . Inference of evolutionary processes from within-species sequence variation is often hindered by the assumption of a stationary frequency distribution . This assumption can be avoided when identifying the action of selection and tested when estimating selection intensity. Genetics, 1997 May, 146(1), 9 - 26 Genetic recombination through double-strand break repair: shift from two-progeny mode to one-progeny mode by heterologous inserts; Takahashi NK et al.; Double-strand break repair models of genetic recombination propose that a double-strand break is introduced into an otherwise intact DNA and that the break is then repaired by copying a homologous DNA segment . Evidence for these models has been found among lambdoid phages and during yeast meiosis . In an earlier report, we demonstrated such repair of a preformed double-strand break by the Escherichia coli RecE pathway . Here, our experiments with plasmids demonstrate that such reciprocal or conservative recombination (two parental DNAs resulting in two progeny DNAs) is frequent at a double-strand break even when there exists the alternative route of nonreciprocal or nonconservative recombination (two parental DNAs resulting in only one progeny DNA) . The presence of a long heterologous DNA at the double-strand break, however, resulted in a shift from the conservative (two-progeny) mode to the nonconservative (one-progeny) mode . The product is a DNA free from the heterologous insert containing recombinant flanking sequences . The potential ability of the homology-dependent double-strand break repair reaction to detect and eliminate heterologous inserts may have contributed to the evolution of homologous recombination, meiosis and sexual reproduction. Nat Biotechnol, 1997 May, 15(5), 436 - 8 Molecular evolution of an arsenate detoxification pathway by DNA shuffling; Crameri A et al.; Functional evolution of an arsenic resistance operon has been accomplished by DNA shuffling, involving multiple rounds of in vitro recombination and mutation of a pool of related sequences, followed by selection for increased resistance in vivo . Homologous recombination is achieved by random fragmentation of the PCR templates and reassembly by primerless PCR . Plasmid-determined arsenate resistance from plasmid pl258 encoded by genes arsR, arsB, and arsC was evolved in Escherichia coli . Three rounds of shuffling and selection resulted in cells that grew in up to 0.5 M arsenate, a 40-fold increase in resistance . Whereas the native plasmid remained episomal, the evolved operon reproducibly integrated into the bacterial chromosome . In the absence of shuffling, no increase in resistance was observed after four selection cycles, and the control plasmid remained episomal . The integrated ars operon had 13 mutations . Ten mutations were located in arsB, encoding the arsenite membrane pump, resulting in a fourfold to sixfold increase in arsenite resistance . While arsC, the arsenate reductase gene, contained no mutations, its expression level was increased, and the rate of arsenate reduction was increased 12-fold . These results show that DNA shuffling can improve the function of pathways by complex and unexpected mutational mechanisms that may be activated by point mutation . These mechanisms may be difficult to explain and are likely to be overlooked by rational design. Int Arch Allergy Immunol, 1997 May-Jul, 113(1-3), 246 - 8 Division of the major birch pollen allergen, Bet v 1, into two non-anaphylactic fragments; Vrtala S et al.; We have expressed in Escherichia coli two halves of the major birch pollen allergen, Bet v 1 . Both fragments representing the complete 17-kD allergen were purified to homogeneity . In contrast to the complete recombinant, Bet v 1, the fragments had almost completely lost their IgE-binding capacity and exhibited a random coil structure as analyzed by circular dichroism . The ability of the recombinant fragments to trigger histamine release from allergic patients' basophils as well as their capacity to elicit skin reactions were also largely abolished . Both non-anaphylactic Bet v 1 fragments carried the majority of T cell epitopes and may therefore be considered as safe tools for immunotherapy of tree pollen and associated food allergy. Int Arch Allergy Immunol, 1997 May-Jul, 113(1-3), 240 - 2 Characterization of recombinant shrimp allergen Pen a 1 (tropomyosin); Reese G et al.; Tropomyosin (Pen a 1) from brown shrimp, Penaeus aztecus, has been identified as the only major shrimp allergen . Since beef, pork and chicken are other tropomyosin-containing foods that are not very allergenic, tropomyosins can serve to investigate the contribution of the structural properties of a protein to its allergenicity . The aim of this study was to determine the primary structure of Pen a 1 and to identify IgE-binding epitopes . The screening of a unidirectional expression cDNA library from shrimp tail muscle with the Pen-a-1-specific monoclonal antibody 4.9.5 resulted in 4 positive Escherichia coli clones . Immunoblot analysis with human sera from shrimp-allergic subjects demonstrated IgE binding of all 4 recombinant shrimp proteins . Three of 4 expressed recombinant proteins have a molecular weight of approximately 36 kD, consistent with the molecular weight of natural Pen a 1 . The DNA sequence analysis identified these recombinant shrimp proteins as tropomyosin and could be aligned with the sequence of greasyback shrimp (Metapenaeus ensis) tropomyosin (Met e 1) . In order to characterize contiguous IgE-binding epitopes of Pen a 1, a peptide library (Novagen epitope mapping system) expressing 10-30 amino-acid-residue-long recombinant Pen a 1 peptides was constructed and screened with human IgE . Four recombinant, IgE-reactive Pen a 1 peptides were selected and sequenced . They show various degrees of sequence identity with tropomyosins of other arthropods, such as fruitfly and house dust mite, helminths and vertebrates. Int Arch Allergy Immunol, 1997 May-Jul, 113(1-3), 227 - 30 Inhibition of IgE antibody formation by plasmid DNA immunization is mediated by both CD4+ and CD8+ T cells; Lee DJ et al.; BACKGROUND: We previously showed that immunization of mice with plasmid DNA (pDNA) encoding the Escherichia coli beta-galactosidase gene (pCMV-LacZ) induces a Th1 response, whereas beta-galactosidase (beta-gal) in saline or alum induces a Th2 response . Furthermore, the Th1 response dominates over the Th2 response and downregulates preexisiting IgE antibody formation . Here, we determined by passive transfer of CD4+ or CD8+ lymphocytes and by immunizing beta2-microglobulin knockout (beta2-M KO) mice whether CD4+ and/or CD8+ cells from pDNA-immunized mice suppress IgE antibody production . METHODS: BALB/c mice were injected with either CD4+ or CD8+ lymphocytes from naive beta-gal-in-alum or pCMV-LacZ-immunized mice, then immunized with beta-gal in alum, and the IgE antibody formation was determined . Second, C57BL/6 wild-type (WT) or beta2-M KO mice were immunized with beta-gal orpCMV-LacZ, and the IgE antibody production was assessed . RESULTS: Passive transfer of both CD4+ and CD8+ lymphocytes from pDNA-immunized mice suppressed the IgE antibody response by 90% compared to transfer of CD4+ T cells from naive or beta-galin-alum immunized mice . beta2-M KO mice produced 3 times more IgE than the WT control mice both in the primary and secondary response . CONCLUSION: Both CD4+ and CD8+ subsets of T cells from pDNA-immunized mice can suppress IgE antibody production by affecting the primary response and/or by propagating the Th1 memory response in a passive cell transfer system . Immunization with pDNA-encoding allergens may be an effective new form of immunotherapy for atopic diseases. Int Arch Allergy Immunol, 1997 May-Jul, 113(1-3), 213 - 5 Cloning, production, characterization and IgE cross-reactivity of different manganese superoxide dismutases in individuals sensitized to Aspergillus fumigatus; Mayer C et al.; BACKGROUND: Manganese superoxide dismutase (MnSOD) from Aspergillus fumigatus has been demonstrated to be an allergen showing a high degree of homology with phylogenetically distant MnSODs . We describe cloning, production and characterization of MnSODs from different species . METHODS: MnSODs were cloned by PCR, expressed as inclusion body protein in Escherichia coli, purified by Ni2+-chelate affinity chromatography and characterized . RESULTS: The MnSODs from A . fumigatus, man, yeast, Drosophila melanogaster and E . coli show about 50% identity and 70% similarity on the primary structure . All proteins were produced at a high level in E . coli and refolded to achieve enzymatic activity . The proteins were able to bind IgE from sera of individuals sensitized to the A . fumigatus MnSOD . CONCLUSIONS: MnSOD represents a novel pan-allergen restricted to individuals sensitized to A . fumigatus. Int Arch Allergy Immunol, 1997 May-Jul, 113(1-3), 170 - 2 T cell receptor CDR3 sequences and recombinant T cell receptors . Prerequisites for developing ligands to interfere with T cell receptor binding to the MHC/peptide complex; Sowka S et al.; BACKGROUND: The interaction of T cell receptors (TCRs) with peptide fragments bound to major histocompatibility complex (MHC) molecules is central to the initiation and propagation of most immune responses . In order to understand and control the molecular interactions underlying T cell recognition of MHC/peptide complexes, recent efforts have focused on the production of recombinant soluble forms of the TCR heterodimer . METHODS: TCRA variable (TCRAV) and TCRBV sequences used by human T cell clones were amplified by PCR, cloned and sequenced . The deduced amino acid sequences of the complementarity determining region (CDR) 3 loops of TCRs specific for the same allergenic epitope were compared . V region genes of a selected TCR were expressed as a single-chain (sc) molecule in the periplasm of Escherichia coli . RESULTS: Conserved amino acid motifs specific for allergenic peptides of Bet v 1 and Phl p 1 were identified in CDR3 sequences of TCRs . A recombinant scTCR was produced . The ratio of insoluble to soluble material was 1:1 . The recombinant protein was of the correct size and showed no signs of degradation . CONCLUSIONS: Conservation of amino acid motifs in CDR3 loops of TCRs specific for the same allergen fragments indicated that the three-dimensional structure of the CDR3 was determined by the presented peptide . The recombinant scTCR will be used to identify ligands for the CDR3s from random peptide libraries to interfere with TCR binding to the MHC/peptide complex. Immunopharmacol Immunotoxicol, 1997 May, 19(2), 165 - 74 The prostaglandin analogs, misoprostol and SC-46275, potently inhibit cytokine release from activated human monocytes; Widomski D et al.; Inflammatory mediator release is one of the body's responses to tissue injury and inflammation . These mediators, such as interleukin-1 beta (I1-1 beta), tumor necrosis factor (TNF-alpha), and products of arachidonic acid metabolism, are themselves proinflammatory . Purified human monocytes stimulated in vitro with E . coli-derived lipopolysaccharide (LPS) will release these key cytokines along with various other eicosanoid mediators . Monocytes incubated with LPS and the prostaglandin E-1 analog, misoprostol, released significantly lower levels of cytokines compared to monocytes incubated with LPS alone . Eicosanoid release was also affected by misoprostol . SC-46275, a more potent mucosal protective PGE1 analog, also altered the release of cytokines and eicosanoids from human monocytes . However SC-46275 inhibited I1-1 beta release with an IC50 value of 9 microM compared to 75 microM for misoprostol . SC-46275 and misoprostol both inhibited TNF-alpha release . These data suggest there is a potential immunomodulatory role for prostaglandin analogs in the therapeutic treatment of inflammatory diseases such as ulcerative colitis, Crohn's disease, and autoimmune inflammatory diseases of the central nervous system. Biophys J, 1997 May, 72(5), 2217 - 25 Influence of the intrinsic membrane protein bacteriorhodopsin on gel-phase domain topology in two-component phase-separated bilayers; Schram V et al.; We have investigated the effect of the intrinsic membrane protein bacteriorhodopsin of Halobacterium halobium on the lateral organization of the lipid phase structure in the coexistence region of an equimolar mixture of dimyristoylphos-phatidylcholine and distearoylphosphatidylcholine . The fluorescence recovery after photobleaching (FRAP) technique was used to monitor the diffusion of both a lipid analog (N-(7-nitrobenzoxa-2,3-diazol-4-yl)-dimyristoylphosphatidyle thanolamine, NBD-DMPE) and fluorescein-labeled bacteriorhodopsin (Fl-BR) . In the presence of bacteriorhodopsin, the mobile fractions of the two fluorescent probes display a shift of the percolation threshold toward lower temperatures (larger gel-phase fractions), independent of the protein concentration, from 43 degrees C (without bacteriorhodopsin) to 39 degrees C and 41 degrees C for NBD-DMPE and Fl-BR, respectively . Moreover, in the presence of bacteriorhodopsin, the gel-phase domains are much less efficient in restricting the diffusion of both probes than they are in the absence of the protein in the two-phase coexistence region . Bacteriorhodopsin itself, however, obstructs diffusion of NBD-DMPE and Fl-BR to about the same extent in the fluid phase of the two-phase region as it does in the homogeneous fluid phase . These observations suggest that 1) the protein induces the formation of much larger and/or more centrosymmetrical gel-phase domains than those formed in its absence, and 2) bacteriorhodopsin partitions almost equally between the coexisting fluid and gel phases . Although the molecular mechanisms involved are not clear, this phenomenon is fully consistent with the effect of the transmembrane peptide pOmpA of Escherichia coli investigated by electron spin resonance in the same lipid system. Biophys J, 1997 May, 72(5), 2094 - 102 Computer simulations of the OmpF porin from the outer membrane of Escherichia coli; Watanabe M et al.; Molecular dynamics simulations were used to study the structure and dynamics of the Escherichia coli OmpF porin, which is composed of three identical 16-stranded beta-barrels . Simulations of the full trimer in the absence of water and the membrane led to significant contraction of the channel in the interior of each beta-barrel . With very weak harmonic constraints (0.005 kcal/mol A2/atom) applied to the main-chain C alpha atoms of the beta-barrel, the structure was stabilized without alteration of the average fluctuations . The resulting distribution of the fluctuations (small for beta-strands, large for loops and turns) is in good agreement with the x-ray B factors . Dynamic cross-correlation functions showed the importance of coupling between the loop motions and barrel flexibility . This was confirmed by the application of constraints corresponding to the observed temperature factors to the barrel C alpha atoms . With these constraints, the beta-barrel fluctuations were much smaller than the experimental values because of the intrinsic restrictions on the atomic motions, and the loop motions were reduced significantly . This result indicates that considerable care is required in introducing constraints to keep proteins close to the experimental structure during simulations, as has been done in several recent studies . Loop 3, which is thought to be important in gating the pore, undergoes a displacement that shifts it away from the x-ray structure . Analysis shows that this arises from the breakdown of a hydrogen bond network, which appears to result more from the absence of solvent that from the use of standard ionization states for the side chains of certain beta-barrel residues. Blood, 1997 May 1, 89(9), 3219 - 27 A soluble tissue factor mutant is a selective anticoagulant and antithrombotic agent; Kelley RF et al.; One approach to developing safer and more efficacious agents for the treatment of thrombotic disease involves the design and testing of inhibitors that block specific steps in the coagulation cascade . We describe here the development of a mutant of human tissue factor (TF) as a specific antagonist of the extrinsic pathway of blood coagulation and the testing of this mutant in a rabbit model of arterial thrombosis . Alanine substitutions of Lys residues 165 and 166 in human TF have been shown previously to diminish the cofactor function of TF in support of factor X (FX) activation catalyzed by factor VIIa (FVIIa) . The K165A:K166A mutations have been incorporated into soluble TF (sTF; residues 1-219) to generate the molecule "hTFAA." hTFAA binds FVIIa with kinetics and affinity equivalent to wild-type sTF, but the hTFAA x FVIIa complex shows a 34-fold reduction in catalytic efficiency for FX activation relative to the activity measured for sTF x FVIIa . hTFAA inhibits the activation of FX catalyzed by the complex formed between FVIIa and relipidated TF(1-243) . hTFAA prolongs prothrombin time (PT) determined with human plasma and relipidated TF(1-243) or membrane bound TF, and has no effect on activated partial thromboplastin time, but is 70-fold less potent as an inhibitor of PT with rabbit plasma . The rabbit homologue of this mutant ("rTFAA") was produced and shown to have greater potency with rabbit plasma . Both hTFAA and rTFAA display an antithrombotic effect in a rabbit model of arterial thrombosis with rTFAA giving full efficacy at a lower dose than hTFAA . Compared to heparin doses of equal antithrombotic potential, hTFAA and rTFAA cause less bleeding as judged by measurements of the cuticle bleeding time . These results indicate that TF x FVIIa is a good target for the development of new anticoagulant drugs for the treatment of thrombotic disease. Pediatr Res, 1997 May, 41(5), 635 - 40 Ontogeny of nitric oxide synthase activity and endotoxin-mediated damage in the neonatal rat colon; Brown JF et al.; Nitric oxide (NO) is synthesized by most regions of the gastrointestinal tract and is an important regulator of mucosal function and integrity . In this study we examined the ontogenic appearance of constitutively expressed Ca2+-dependent NO synthase (cNOS) and inducible Ca2+-independent NO synthase (iNOS) activity, in the colon of neonatal rats . Furthermore, the susceptibility of the colon to damage after induction of iNOS activity following bacterial endotoxin treatment was also examined . Segments of distal colon were removed from either control rat pups (aged between 10 and 25 d) or from animals pretreated with the following agents: 1) Escherichia coli lipopolysaccharide {LPS; 3 mg/kg, intraperitoneally (i.p.), 4 h before sacrifice}, 2) dexamethasone (2 mg/kg, i.p., 1 h before administration of LPS) or aminoguanidine (25 mg/kg, i.p., at the same time as LPS) . NOS activity was measured via the conversion of L-{14C}arginine to L-{14C}citrulline . Samples of colon were assessed for damage by either light microscopy or by measurement of the malondialdehyde content to estimate lipid peroxidation . In untreated animals cNOS activity increased during the first 20 postnatal days and fell postweaning at 25 d . LPS treatment resulted in a significant increase in iNOS activity in all age groups examined, with maximal activity occurring between 10 and 15 d of age . This coincided with the greatest histologic damage score and lipid peroxidation . Dexamethasone or aminoguanidine attenuated the effects of LPS suggesting the involvement of iNOS in these responses . These data suggest that colonic cNOS activity in the neonatal rat may be important during development and maturation of that tissue . Furthermore, the colon of the preweaned rat is more susceptible to the detrimental effects of LPS-induced NO production than the colon of postweaned animals. Infect Immun, 1997 May, 65(5), 1640 - 3 Inhibition of lipopolysaccharide-induced transcription factor Sp1 binding by spectrally pure diphosphoryl lipid A from Rhodobacter sphaeroides, protein kinase inhibitor H-8, and dexamethasone; Jarvis BW et al.; The transcription factor Sp1 plays a crucial role in the monocyte-specific expression of CD14, a binding site (or putative receptor) for lipopolysaccharide (LPS) complexes with LPS-binding protein (LBP) . By using RAW 264.7 macrophages treated with spectrally pure deep-rough-chemotype hexa-acyl LPS from Escherichia coli D31m4, three inhibitors were found to block the binding activity of transcription factor Sp1, as measured by electrophoretic mobility shift assays . These inhibitors were diphosphoryl lipid A from Rhodobacter sphaeroides (10 microg/ml); the isoquinoline-sulfonamide H-8 (10 and 100 microM), which is thought to be a cGMP-dependent protein kinase inhibitor; and the anti-inflammatory agent dexamethasone (10 microM). Infect Immun, 1997 May, 65(5), 1606 - 14 Acquired immune responses to the N- and C-terminal regions of Plasmodium vivax merozoite surface protein 1 in individuals exposed to malaria; Soares IS et al.; In this study, we evaluated the naturally acquired immune response to Plasmodium vivax merozoite surface protein 1 (PvMSP1) in individuals with recent clinical episodes of malaria from the state of Para, Brazil . Ten recombinant proteins representing the first 682 amino acids (aa) of the N-terminal region and one representing the final 111 aa of the C-terminal region were expressed in Escherichia coli as glutathione S-transferase fusion proteins . Both of these regions have been suggested as candidates for development of a vaccine against Plasmodium sp . The total frequencies of individuals with antibodies and cellular immune responses to PvMSP1 were high (83.8 and 75%, respectively) . The recombinant proteins representing the N- and C-terminal regions were recognized by 51.4 and 64.1% of sera, respectively . The frequency of responders to the C-terminal region increased according to the number of previous malaria episodes, reaching 83.3% after four episodes . Cellular immune response was measured by in vitro proliferation and gamma interferon production . Peripheral blood mononuclear cells of 75 and 47.2% of individuals proliferated in response to stimulation by the N- and C-terminal regions, respectively . Also, we found that one protein representing the N terminus and a second representing the C terminus of PvMSP1 stimulated 54.5% of individuals to secrete gamma interferon . We concluded that PvMSP1 is immunogenic to a large proportion of individuals exposed to malaria . Our results also suggested that the C-terminal region of PvMSP1 containing the two epidermal growth factor-like domains is particularly immunogenic to antibodies and T cells during natural infection in humans. J Clin Microbiol, 1997 May, 35(5), 1209 - 15 Use of monoclonal antibodies to facilitate identification, cloning, and purification of Chlamydia trachomatis hsp10; LaVerda D et al.; As a requisite for a physiological and immunological investigation, reagents were developed that facilitated the identification and purification of Chlamydia trachomatis hsp10 (chsp10) . Monoclonal antibodies that specifically recognize chsp10 were generated with multiple-antigen peptides (MAPs) to promote recognition of Chlamydia-specific epitopes . MAP2, containing amino acids 54 to 69 of the hsp10 sequence, elicited strong antibody responses after immunization of BALB/c mice . Monoclonal antibodies from several cloned hybridomas reacted on immunoblots with an approximately 15-kDa chlamydial protein and recombinant chsp10 . Because of its strict specificity for chsp10, monoclonal antibody M1.2 was selected for routine use . M1.2 reacted by immunoblot with the hsp10s of several C . trachomatis strains but not with Chlamydia psittaci hsp10 or Escherichia coli homolog GroES, suggesting that M1.2 recognizes a species-specific epitope . Recombinant chsp10 was purified by immunoaffinity chromatography with M1.2 . For large-scale purification, chsp10 was appended with a C-terminal six-histidine tag for purification by nickel chelate affinity chromatography . The hypA gene encoding the chsp10 of C . trachomatis serovar E/Bour was cloned into the pQE-60 vector (QIAGEN, Inc.) following PCR amplification from genomic DNA . E . coli DH5 transformants were screened for chsp10 expression by colony immunoblotting with M1.2, were tested for nickel matrix binding, and were sequenced . The sequence of serovar E/Bour chsp10 was found to be closely homologous to those of hsp10s of other chlamydiae . Purified chsp10 and specific anti-chsp10 monoclonal antibodies will be useful for investigating the biological and immunological roles of hsp10 in chlamydial infections. J Clin Microbiol, 1997 May, 35(5), 1122 - 30 Serological diagnosis of hantavirus infections by an enzyme-linked immunosorbent assay based on detection of immunoglobulin G and M responses to recombinant nucleocapsid proteins of five viral serotypes; Elgh F et al.; Worldwide, hantaviruses cause more than 100,000 human infections annually . Rapid and accurate methods are important both in monitoring acute infections and for epidemiological studies . We and others have shown that the amino termini of hantavirus nucleocapsid proteins (Ns) are sensitive tools for the detection of specific antibodies in hantavirus disease . Accordingly, we expressed truncated Ns (amino acids 1 to 117) in Escherichia coli from the five hantaviruses known to be pathogenic to man; Hantaan (HTN), Seoul (SEO), Dobrava (DOB), Sin Nombre (SN), and Puumala (PUU) viruses . In order to obtain pure antigens for use in an enzyme-linked immunosorbent assay (ELISA), the recombinant proteins were purified by polyhistidine-metal chelate affinity chromatography . Polyclonal animal antisera and a panel of serum specimens from hantavirus-infected individuals from Scandinavia, Slovenia, Russia, Korea, China, and the United States were used to evaluate the usefulness of the method . With both human and animal sera, it was possible to designate the antibody response into two groups: those with HTN, SEO, and DOB virus reactivity on the one hand and those with SN and PUU virus reactivity on the other . In sera from Scandinavia, European Russia, and the United States, the antibody response was directed mainly to the PUU and SN virus group . The sera from Asia reacted almost exclusively with the HTN, SEO, and DOB types of viruses . This was true for both the immunoglobulin M (IgM) and IgG antibody responses, indicating that this type of discrimination can be done during the acute phase of hantavirus infections . Both the HTN, SEO, and DOB virus and the PUU and SN virus types of antibody response patterns were found in patients from the Balkan region (Solvenia). J Clin Microbiol, 1997 May, 35(5), 1103 - 7 Age-specific prevalence of Escherichia coli with localized and aggregative adherence in Venezuelan infants with acute diarrhea; Gonzalez R et al.; To evaluate the epidemiological significance of HEp-2 cell-adherent Escherichia coli isolates in diarrheal disease, we performed a study with 513 Venezuelan infants with diarrhea and 241 age-matched controls to determine the prevalence of enteropathogenic E . coli (enteroadherent E . coli, enterotoxigenic E . coli, enteroinvasive E . coli, and enterohemorrhagic E . coli) and their correlation with O:H serotypes . E . coli isolates exhibiting localized and aggregative adherence in the HEp-2 cell assay were significantly more frequently isolated from the patients (8.5 and 26.9%, respectively) than from the controls (1.7 and 15%, respectively) . This difference was significant for the group 0 to 2 months of age but for older infants . Regardless of age, E . coli isolates with diffuse adherence were found at similar frequencies in both the patients and the controls . A striking correlation between classic O serogroups and localized adherence was also observed . These findings confirm the pathogenic role of E . coli with localized and aggregative adherence in diarrheal disease, as well as the epidemiological importance of O:H serotyping for characterizing localized-adhering E . coli. J Inorg Biochem, 1997 May 1, 66(2), 99 - 102 Inhibition of the GDP/GTP exchange reaction of ras p21 by aluminum ion; Landino LM et al.; In an effort to understand the biochemical mechanisms of aluminum-induced neurotoxicity, we investigated the effects of aluminum ion, Al3+, on the Mg(2+)- and nucleotide-dependent protein, ras p21 . Picomolar Al3+ concentrations inhibited the GTPase activity of ras p21 in an Mg(2+)-dependent manner, consistent with an Al3+/Mg2+ competition mechanism . GTPase activity was inhibited by 60% in the presence of 100 microM Mg2+ and 2.9 x 10(-10) M Al3+ . Kinetic studies demonstrated that the mode of Al(3+)-induced inhibition of ras p21 GTPase activity changed from competitive to mixed non-competitive as the number of ras p21 turnovers increased . Further dissection of the ras p21 cycle revealed that Mg(2+)-dependent GDP/GTP exchange was the Al(3+)-sensitive step. Mol Cell Biol, 1997 May, 17(5), 2844 - 50 Frameshift intermediates in homopolymer runs are removed efficiently by yeast mismatch repair proteins; Greene CN et al.; A change in the number of base pairs within a coding sequence can result in a frameshift mutation, which almost invariably eliminates the function of the encoded protein . A frameshift reversion assay with Saccharomyces cerevisiae that can be used to examine the types of insertions and deletions that are generated during DNA replication, as well as the editing functions that remove such replication errors, has been developed . Reversion spectra have been obtained in a wild-type strain and in strains defective for defined components of the postreplicative mismatch repair system (msh2, msh3, msh6, msh3 msh6, pms1, and mih1 mutants) . Comparison of the spectra reveals that yeast mismatch repair proteins preferentially remove frameshift intermediates that arise in homopolymer tracts and indicates that some of the proteins have distinct substrate or context specificities. Biopolymers, 1997 May, 41(6), 635 - 45 Solution conformations of peptides representing the sequence of the toxin pardaxin and analogues in trifluoroethanol-water mixtures: analysis of CD spectra; Thennarasu S et al.; The cytolytic activities and conformational properties of pardaxin (GFFALIPKIISSPLFKTLLSAVGSALSSSGEQE), a 33-residue linear peptide that exhibits unusual shark repellent and cytolytic activities, and its analogues have been examined in aqueous environment and trifluoroethanol (TFE) using CD spectroscopy . A peptide corresponding to the 1-26 segment and an analogue where P7 has been changed to A show greater hemolytic activity than pardaxin . While the peptide corresponding to the N-terminal 18-residue segment does not exhibit hemolytic activity, its analogue where P7 is replaced by A is hemolytic . The secondary structural propensities of the peptides were inferred by deconvolution of the experimental spectra into pure components . Pardaxin, its variant where proline at position 7 was replaced by alanine, and shorter peptides corresponding to N-terminal segments exist in multiple conformations in aqueous medium that are comprised of beta-turn, beta-sheet, and distorted helical structures . With increasing proportions of TFE, while helical conformation predominates in all the peptides, both distorted and the regular alpha-helices appear to be populated . Analysis of CD spectra by deconvolution methods appears to be a powerful tool for delineating multiple conformations in peptides, especially membrane-active peptides that encounter media of different polarity ranging from aqueous environment to one of low dielectric constant in the hydrophobic interior of membranes . Our study provides further insights into the structural requirements for the biological activity of pardaxin and related peptides. Nucleic Acids Res, 1997 May 1, 25(9), 1868 - 9 In situ activity gel for DNA repair 3'-phosphodiesterase; Sander M; An enzyme that plays an important role in the repair of oxidative DNA damage is the 3'-phosphodiesterase . This activity, which repairs damaged DNA 3'-termini,can be detected using several available biochemical assays . We present a method to detect 3'-phosphodiesterase activity of renatured proteins immobilized in polyacrylamide gels . The model substrate, labeled with {alpha-32P}dCTP, contains 3'-phosphoglycolate termini produced by bleomycin-catalyzed cleavage of the self-complementary alternating copolymer poly(dGdC) . The DNA substrate is incorporated into the gel matrix during standard SDS-PAGE . Active 3'-phosphodiesterase enzymes are detected visibly by the loss of radioactivity at a position corresponding to the mobility of the enzyme during SDS-PAGE . Using this procedure, two Escherichia coli 3'-phosphodiesterases, exonuclease III and endonuclease IV, are readily detected in crude cell extracts or as homogeneous purified proteins . Extracts of mutant cells lack activity at the positions of exonuclease III and endonuclease IV but retain activity in the position of a much larger protein (Mr approximately 100 kDa) . The identification of this novel 100 kDa E.coli 3'-phosphodiesterase demonstrates the potential value of the activity gel method described here. Nucleic Acids Res, 1997 May 1, 25(9), 1753 - 60 Herpes simplex virus: selection of origins of DNA replication; Hammarsten O et al.; A selection procedure was devised to study the role of cis -acting sequences at origins of DNA replication . Two regions in Herpes simplex virus oriS were examined: an AT-rich spacer sequence and a putative binding site, box III, for the origin binding protein . Plasmid libraries were generated using oligonucleotides with locally random sequences . The library, amplified in Escherichia coli , was used to transfect BHK cells followed by superinfection with HSV-1 . Replicated plasmids resistant to Dpn I cleavage were amplified in E . coli . The selection scheme was repeated . Plasmids were isolated at different stages of the procedure and their replication efficiency was determined . Efficiently replicating plasmids had a high AT content in the spacer sequence as well as a low helical stability of this region . In contrast, this was not seen using the box III library . We also noted that the wild type sequence invariably dominated the library after five rounds of selection . These plasmids arose from recombination between plasmids and viral DNA . Our results imply that a large group of sequences can mechanistically serve as origins of DNA replication . In a viral system, however, where the initiation process might be rate-limiting, this potentially large group of sequences would always converge towards the most efficient replicator. Nucleic Acids Res, 1997 May 1, 25(9), 1709 - 14 HIV-1 protease inhibits its homologous reverse transcriptase by protein-protein interaction; Bottcher M et al.; The reading frame of the HIV-1 pol gene, encoding protease (PR) and reverse transcriptase (RT), including RNase H as well as integrase, was fused to the bacterialbeta-galactosidase gene and overexpressed in Escherichia coli cells . The resulting fusion protein was cleaved autocatalytically leading to PR, RT and integrase . Immunoprecipitations of bacterial crude extracts with anti-RT antibodies precipitated both RT and PR . Co-precipitation of PR and RT was also observed with anti-PR antibodies, strongly suggesting a physical interaction between fully processed RT and PR within the bacterial cell . Physical interactions were confirmed with purified components by means of an ELISA assay . Furthermore, purified PR inhibited the DNA synthesis activity of purified RT, while its RNase H activity remained unaffected . The type of inhibition was uncompetitive with respect to poly(rA).oligo(dT); the inhibition constant was 50-100 nM . A possible physiological significance of this type of interaction is discussed. Nucleic Acids Res, 1997 May 1, 25(9), 1685 - 93 DNA binding and DNA bending by the MelR transcription activator protein from Escherichia coli; Bourgerie SJ et al.; The Escherichia coli melR gene encodes MelR protein which is a member of the AraC/XylS family of bacterial transcription activators . The function of MelR was investigated by making a targeted deletion in the melR gene of the Escherichia coli chromosome . MelR is a transcription activator essential for melibiose- dependent expression of the melAB operon which is needed for bacterial growth with melibiose as a carbon source . To investigate the interactions of MelR at the melAB promoter, both full length MelR and a shortened derivative, MelR173, containing the C-terminal DNA-binding domain, were purified as fusions to glutathione- S -transferase . Circular permutation studies show that both full-length MelR and MelR173 induce an apparent bend upon binding to target sites at the melAB promoter . Bound full-length MelR, but not MelR173, can oligomerise to form larger complexes that are likely to be involved in transcription activation. J Neurosci, 1997 May 1, 17(9), 3343 - 51 Antipyretic role of endogenous melanocortins mediated by central melanocortin receptors during endotoxin-induced fever; Huang QH et al.; Bacterial infection causes fever, an adaptive but potentially self-destructive response, in the host . Also activated are counterregulatory systems such as the pituitary-adrenal axis . Antipyretic roles have also been postulated for certain endogenous central neuropeptides, including the melanocortins (alpha-MSH-related peptides) . To test the hypothesis that endogenous central melanocortins have antipyretic effects mediated by central melanocortin receptors (MCRs), we determined the effect of intracerebroventricular injection of a synthetic MCR antagonist, Ac-Nle4,c-{Asp5,DNal(2')7,Lys10}alpha-MSH(4-10)-NH2 (SHU-9119) in endotoxin-challenged rats . The efficacy and specificity of SHU-9119 as an MCR antagonist in the rat was first validated in vitro and in vivo . In vitro, in heterologous cells expressing either rat MC3-R or MC4-R, the major MCR subtypes expressed in brain, SHU-9119 showed no intrinsic agonism, but it inhibited alpha-MSH-induced cAMP accumulation (IC50 = 0.48 +/- 0.19 and 0.41 +/- 0.28 nM, respectively) and {125I}-{Nle4,DPhe7}-alpha-MSH binding (IC50 = 1.0 +/- 0.1 and 0.9 +/- 0.3 nM, respectively) . In vivo, exogenous alpha-MSH (180 pmol) inhibited fever in rats when administered intracerebroventricularly 30 min after Escherichia coli lipopolysaccharide (LPS) (25 microg/kg, i.p.) . When co-injected with alpha-MSH, SHU-9119 (168 pmol, i.c.v.) prevented the antipyretic action of exogenous alpha-MSH . In contrast, neither alpha-MSH nor SHU-9119, alone or in combination, affected body temperatures in afebrile rats . In LPS-treated rats, intracerebroventricular injection of SHU-9119 significantly increased fever, whereas intravenous injection of the same dose of SHU-9119 had no effect . Neither intracerebroventricular nor intravenous SHU-9119 significantly affected LPS-stimulated plasma ACTH or corticosterone levels . The results indicate that endogenous central melanocortins exert an antipyretic influence during fever by acting on MCRs located within the brain, independent of any modulation of the activity of the pituitary-adrenal axis. J Virol, 1997 May, 71(5), 3992 - 7 Biosynthesis, purification, and characterization of the human coronavirus 229E 3C-like proteinase; Ziebuhr J et al.; Coronavirus gene expression involves proteolytic processing of the gene 1-encoded polyprotein(s), and a key enzyme in this process is the viral 3C-like proteinase . In this report, we describe the biosynthesis of the human coronavirus 229E 3C-like proteinase in Escherichia coli and the enzymatic properties, inhibitor profile, and substrate specificity of the purified protein . Furthermore, we have introduced single amino acid substitutions and carboxyl-terminal deletions into the recombinant protein and determined the ability of these mutant 3C-like proteinases to catalyze the cleavage of a peptide substrate . Using this approach, we have identified the residues Cys-3109 and His-3006 as being indispensable for catalytic activity . Our results also support the involvement of His-3127 in substrate recognition, and they confirm the requirement of the carboxyl-terminal extension found in coronavirus 3C-like proteinases for enzymatic activity . These data provide experimental evidence for the relationship of coronavirus 3C-like proteinases to other viral chymotrypsin-like enzymes, but they also show that the coronavirus proteinase has additional, unique properties. J Virol, 1997 May, 71(5), 3693 - 701 A model system for human cytomegalovirus-mediated modulation of human immunodeficiency virus type 1 long terminal repeat activity in brain cells; Moreno TN et al.; Previously, our laboratory showed that human cytomegalovirus (HCMV) activates human immunodeficiency virus type 1 (HIV-1) in brain-derived cells with limited HIV-1 gene expression but inhibits HIV-1 in cells fully permissive for replication of both viruses (F . M . Jault, S . A . Spector, and D . H . Spector, J . Virol . 68:959-973, 1994) . To investigate these effects further, we developed a model system that uncouples HIV-1 gene expression from long terminal repeat (LTR) activity . Two monoclonal U373-MG astrocytoma/glioblastoma cell lines (LTRIG and LIGHIVDC) were generated, each containing an integrated copy of an LTR-chloramphenicol acetyltransferase (CAT) construct and the Escherichia coli lacI gene . LIGHIVDC also has an inducible HIV-1 genome controlled by a Rous sarcoma virus promoter with lac operator sequences . Basal LTR-mediated CAT activity is 65-fold higher in LIGHIVDC than in LTRIG, and this activity is further increased (20-fold) following incubation of LIGHIVDC with isopropyl-beta-D-thiogalactopyranoside (IPTG) . Tat protein can be detected by immunostaining in LIGHIVDC . However, Rev-mediated transport and subsequent translation of the singly spliced and unspliced HIV-1 mRNAs is inefficient . In the absence of Tat, HCMV stimulated CAT activity approximately 20-fold, and this activation required HCMV gene expression but not viral DNA replication . LTR-directed transcription was unaffected by HCMV infection in LIGHIVDC but was inhibited in these cells when they contained increased Tat levels following IPTG induction . These results support the hypothesis that HCMV can induce the HIV-1 LTR when HIV-1 gene expression is minimal and that a threshold level of HIV-1 gene products is necessary for HCMV to inhibit this promoter. Arch Microbiol, 1997 May, 167(5), 280 - 3 Efficient production of heat-labile enterotoxin mutant proteins by overexpression of dsbA in a degP-deficient Escherichia coli strain; Wulfing C et al.; Escherichia coli heat-labile enterotoxin (LT) mutants containing Val60-->Gly or Ser114-->Lys substitutions in the A subunit do not produce the A subunit efficiently in E . coli . These mutants accumulate mostly the B pentamer devoid of the A subunit in the periplasmic space . Here we show that overproduction of the periplasmic chaperone DsbA, which is involved in disulfide bond formation, in a strain deficient in the periplasmic protease DegP allows efficient production of the mutant LT molecules . Our results suggest that the formation of the oligomeric toxin is influenced by DsbA, which helps protein folding, and by DegP, which removes the folded intermediates that can be untoxic for the cell. Mol Cells, 1997 Apr 30, 7(2), 237 - 43 Reassembly and reconstitution of separate alpha and beta chains of human leukocyte antigen DR4 molecule isolated from Escherichia coli; Kang JH et al.; The class II major histocompatibility complex molecules play a major role in presentation of exogenous antigenic peptides to the CD4 positive helper T cell . These are heterodimeric cell surface glycoproteins consisting of alpha- and beta-chains . In the present study, we cloned and expressed the alpha- and beta-chain of HLA-DR4 lacking the transmembrane and cytoplasmic domain separately in Escherichia coli using the pET-5a expression vector system . The expressed alpha- and beta-chains were purified in a denaturing condition by an ion exchange chromatography on Q-Sepharose and a gel filtration chromatography on Sephacryl S-200, respectively . The recombinant proteins were refolded and reassembled by removing the denaturing agent and concomitant reoxidation of the disulfide bond . The refolded heterodimeric rDR4 molecule was resolved by 12.5% SDS-PAGE in a nonreducing condition and confirmed by Western blot using polyclonal antibody against DR-alpha and the monoclonal antibody (L243) for the conformationally correct DR molecule . The rDR4 molecules were reconstituted with a 50-fold molar excess biot-HA (307-319), and the bound peptides to the heterodimer complex were determined by a microplate assay coated with L243 antibody using Extravidin-HRP conjugate. Mol Cells, 1997 Apr 30, 7(2), 226 - 30 Molecular characterization of a rab-related small GTP binding protein cDNA from rice (Oryza sativa L . IR-36); Kim HY et al.; To study physiological roles of plant small GTP binding proteins, we isolated a cDNA clone (ORrab2) encoding the rab-related small GTP binding protein from rice (Oryza sativa L . IR-36) by using human cDNA rab2 as a probe . The deduced amino acid sequence of the ORrab2 gene shared all the conserved regions, important for GTPase/GTP binding activities, with those of other small GTP binding proteins . ORrab2 is a 1028 bp long cDNA, encodes a 23.2 kDa protein which shows 85.2% similarity on the amino acid sequence level to the Hrab2 protein, and was used as a probe . Through Southern and Northern blot analyses, we found that ORrab2 is a single copy gene and actively expressed at the stages of cell division and elongation . We investigated GTP binding abilities by a filter assay procedure . Deletion of a binding motif, GDTGVGKS, within an ORrab2 protein showed a significant decrease of GTP binding affinity, suggesting its important role in nucleotide binding. Gene, 1997 Apr 29, 190(1), 145 - 50 Expression of lacZ reporter gene under the control of the polyhedrin promoter of Spodoptera litura nuclear polyhedrosis virus; Behera AK et al.; Promoter function of the putative polyhedrin-encoding gene (polh) of Spodoptera litura nuclear polyhedrosis virus (S1MNPV) was determined by transferring it to the Autographa californica nuclear polyhedrosis virus (AcMNPV) through the AcNPV polh based vector, pVL1393 . Three transfer vectors pCBT2, pCBT3 and pCBT4 were constructed by substituting the promoter and the neighbouring sequences of AcNPV in pVL1393 by that of S1NPV . The Escherichia coli lacZ gene was placed downstream from the S1NPV polh promoter in the hybrid transfer vector (pCBT) constructs . Co-transfection of Spodoptera frugiperda cells (Sf9) with each of the pCBTlacZ vector and wild-type AcNPV DNAs led to synthesis of beta-galactosidase (beta Gal) . The plaque-purified recombinant viruses (S1AcNPV.lacZ) expressing lacZ under the polh promoter of S1NPV are stable . The highest beta Gal activity was obtained with S1AcNPV4.lacZ . Production of beta Gal with recombinant virus, S1AcNPV3.lacZ in which S1NPV polh promoter is in the reverse orientation in the AcNPV genome, is 83% of that produced by S1AcNPV4.lacZ . These results indicate that the S1NPV polh promoter is active in the genetic environment of AcNPV; the polh of S1NPV is phylogenetically related to AcNPV like other baculoviruses. Gene, 1997 Apr 29, 190(1), 131 - 7 Purification and characterization of secreted human leptin produced in baculovirus-infected insect cells; Churgay LM et al.; The product of the human ob (obesity) gene, leptin, appears to function in the maintenance of body weight in vivo . When injected into mice, this hormone reduces food consumption and causes weight loss . This work has been done with recombinant leptin (re-leptin) purified and renatured from inclusion bodies in Escherichia coli . We have expressed the human obesity gene encoding the predicted full-length leptin in Spodoptera frugiperda (Sf-9) cells by infection with the recombinant baculovirus system . Protein corresponding to re-leptin was secreted into the culture medium and purified in sufficient quantity for testing biological activity . The secreted re-protein was characterized and found to be unmodified except for correct cleavage of the signal peptide during export from the cells . The resulting molecule is expected to be properly folded and has been purified to a high level of homogeneity . The re-leptin secreted from Sf-9 cells should be an appropriate source of protein for study of the native structure. Gene, 1997 Apr 29, 190(1), 37 - 44 Construction of shuttle vectors for genetic manipulation and molecular analysis of mycobacteria; Jain S et al.; Two novel shuttle vectors for mycobacteria are described which have been derived from the expression system pSD5 developed in our laboratory . Plasmid pSD5B is a promoter-selection vector containing a promoterless lacZ gene and allows the identification of mycobacterial promoters by the blue colour of the colonies on solid media containing XGal . Moreover, the chronological order of appearance of blue colonies and intensity of colour provide a qualitative index of transcriptional strengths of the cloned promoters . Plasmid pSD5C has been designed to construct mycobacterial genomic libraries and express the cloned DNA inserts as fusion proteins with maltose binding protein in mycobacteria . Libraries in pSD5C provide feasibility for their screening with either DNA probes or specific antisera for identifying the genes of interest and for isolation of specific genetic loci by complementation of Escherichia coli and mycobacterial mutants . These vectors combine the ease of working in E . coli with the advantage of directly propagating them in mycobacteria without further manipulations . Finally, we demonstrate that these vectors function efficiently both in fast growing Mycobacterium smegmatis and slow growing mycobacteria including Mycobacterium tuberculosis and Mycobacterium bovis BCG. Gene, 1997 Apr 29, 190(1), 31 - 5 Overproduction of fungal ribotoxin alpha-sarcin in Escherichia coli: generation of an active immunotoxin; Rathore D et al.; alpha-Sarcin is a ribonucleolytic protein secreted by the mold Aspergillus giganteus . DNA encoding alpha-sarcin was isolated from the host and cloned into T7 promoter based E . coli expression vectors . Using bacterial outer membrane protein A (OmpA) signal sequence, properly processed recombinant (re-) protein was secreted into the culture medium while in the absence of a signal sequence protein remained insoluble in the bacterial inclusion bodies . The re-alpha-sarcin was purified to homogeneity by simple chromatographic techniques both from the insoluble and soluble sources with respective yields of 40-50 microg/ml and 2-3 microg/ml . The re-ribotoxin was functionally as active as the native toxin and preserved its specificity . The re-alpha-sarcin was used in the construction of an active immunotoxin targeted at the human cancer cells overexpressing transferrin receptor (TFR). Gene, 1997 Apr 29, 190(1), 27 - 30 Human immunodeficiency virus type-1 p24 sequence from an Indian strain: expression in Escherichia coli and implications in diagnostics; Gupta SK et al.; A 637-bp fragment, corresponding to the p24 human immunodeficiency virus (HIV) core protein from the gag ORF, was PCR amplified from DNA isolated from peripheral blood mononuclear lymphocytes (PBML) of an asymptomatic HIV-1 seropositive human subject from Bombay and cloned into PCRScript SK(+) . The nucleotide sequence revealed highest homology (98.6%) with the consensus sequence of the HIV-1 B subtype . The 637-bp KpnI-HindIII fragment was cloned downstream from a His6 tag in the pQE30 vector under the control of phage T5 promoter leading to production of a 6XHis-p24 fusion protein in Escherichia coli . It showed an approx . 24-kDa band by SDS-PAGE . The recombinant p24 reacted with serum samples from HIV-infected subjects when tested by Western blot and ELISA. Biochemistry, 1997 Apr 29, 36(17), 5149 - 56 Nucleotide-dependent complex formation between the Escherichia coli chaperonins GroEL and GroES studied under equilibrium conditions; Behlke J et al.; Binding of heptameric GroES to the tetradecameric chaperonin GroEL in the absence or presence of nucleotides was investigated by analytical ultracentrifugation . In the absence of nucleotides, the association constant for the binding of GroES to GroEL, K1, was found to be approximately equal to 3 x 10(5) M(-1) . The binding of a second GroES heptamer with only one-fourth the affinity of the first one can be neglected at subequimolecular concentrations relative to GroEL . Under these conditions, mainly an asymmetric "bullet"-shaped complex is formed {see also Schmidt et al . (1994) Science 265, 656-659} . In the presence of ADP or ATP analogues such as ATP-gamma-S or AMP-PNP, the affinity to bind GroES increases by at least 2 orders of magnitude depending on the nucleotide concentration . With increasing GroES:GroEL ratios in the presence of 1 mM ATP analogue, up to two GroES oligomers were bound to one GroEL oligomer, forming the symmetrical "American football"-shaped complex with apparently high affinity for the first GroES ring and considerably lower for the second one . These are the first results that provide an accurate and quantitative description of the equilibrium between asymmetrical and symmetrical complexes at relatively high concentrations of GroEL and GroES that are proposed to exist in vivo . We suggest that the increased affinity of GroEL for GroES plays a role in releasing substrate proteins from the central cavity of GroEL after folding under "non-permissive" conditions. Biochemistry, 1997 Apr 29, 36(17), 5097 - 103 Mutagenesis of acidic residues in the oxygenase domain of inducible nitric-oxide synthase identifies a glutamate involved in arginine binding; Gachhui R et al.; The oxygenase domain of the mouse cytokine-inducible nitric-oxide synthase (iNOSox, amino acids 1-498) binds heme, tetrahydrobiopterin, and the substrate Arg and is the domain responsible for catalyzing nitric oxide synthesis and maintaining the enzyme's active dimeric structure . To further understand iNOSox structure-function, we carried out alanine point mutagenesis on 15 conserved acidic residues located within a region of iNOSox (amino acids 352-473) that shares sequence homology with the pterin-binding module in dihydrofolate reductases and may be important for iNOSox subunit dimerization and/or Arg binding . Five point mutants were identical or nearly identical to wild-type, while 10 exhibited a range of defects that included low heme content (2), heme ligand instability (2), defective dimerization (2), and poor Arg and/or tetrahydrobiopterin binding (4) . Mutations that caused defective tetrahydrobiopterin binding were also associated with other defects . In contrast, two mutants (E371A and D376A) exhibited an exclusive defect in Arg binding . These mutants were dimeric, indicating that dimerization of iNOSox in Escherichia coli does not require Arg . In one case (E371A), the defect in Arg binding was absolute, as assessed by spectral perturbation, radioligand binding, and catalytic studies . We conclude that mutagenesis of conserved acidic residues within this region of iNOSox can lead to exclusive defects in dimerization and in Arg binding . Modeling considerations predict that the E371 carboxylate may participate in Arg binding by interacting with its guanidine moiety. Proc Natl Acad Sci U S A, 1997 Apr 29, 94(9), 4681 - 5 Mutagenicity of 5-aza-2'-deoxycytidine is mediated by the mammalian DNA methyltransferase; Jackson-Grusby L et al.; The cytosine analog 5-aza-2'-deoxycytidine has been used clinically to reactivate genes silenced by DNA methylation . In particular, patients with beta-thalassemia show fetal globin expression after administration of this hypomethylating drug . In addition, silencing of tumor suppressor gene expression by aberrant DNA methylation in tumor cells may potentially be reversed by a similar regimen . Consistent with its function in maintaining tumor suppressor gene expression, 5-aza-2'-deoxycytidine significantly reduces intestinal tumor multiplicity in the predisposed Min mouse strain . Despite its utility as an anti-cancer agent, the drug is highly mutagenic by an unknown mechanism . To gain insight into how 5-aza-2'-deoxycytidine induces mutations in vivo, we examined the mutational spectrum in an Escherichia coli lac I transgene in colonic DNA from 5-aza-2'-deoxycytidine-treated mice . Mutations induced by 5-aza-2'-deoxycytidine were predominantly at CpG dinucleotides, which implicates DNA methyltransferase in the mutagenic mechanism . C:G-->G:C transversions were the predominant class of mutations observed . We suggest a model for how the mammalian DNA methyltransferase may be involved in facilitating these mutations . The observation that 5-aza-2'-deoxycytidine-induced mutations are mediated by the enzyme suggests that novel inhibitors of DNA methyltransferase, which can inactivate the enzyme before its interaction with DNA, are needed for chemoprevention or long term therapy. Proc Natl Acad Sci U S A, 1997 Apr 29, 94(9), 4560 - 5 Double mutagenesis of a positive charge cluster in the ligand-binding site of the ferric enterobactin receptor, FepA; Newton SM et al.; Siderophores and colicins enter bacterial cells through TonB-dependent outer membrane proteins . Using site-directed substitution mutagenesis, we studied ligand recognition by a prototypic Escherichia coli siderophore receptor, FepA, that binds the iron chelate ferric enterobactin and colicins B and D . These genetic experiments identified a common binding site for two of the three ligands, containing multiple positive charges, within cell surface residues of FepA . Elimination of single residues in this region did not impair the adsorption or transport of ferric enterobactin, but double mutagenesis in the charge cluster identified amino acids (Arg-286 and Arg-316) that participate in siderophore binding and function in FepA-mediated killing by colicins B and D . Ferric enterobactin binding, furthermore, prevented covalent modification of FepA within this domain by either a fluorescent probe or an arginine-specific reagent, corroborating the involvement of this site in ligand recognition . These results identify, for the first time, residues in a TonB-dependent outer membrane protein that participate in ligand binding . They also explain the competition between ferric enterobactin and the colicins on the bacterial cell surface: all three ligands interact with the same arginine residues within FepA during their penetration through the outer membrane. Proc Natl Acad Sci U S A, 1997 Apr 29, 94(9), 4504 - 9 Directed evolution of a fucosidase from a galactosidase by DNA shuffling and screening; Zhang JH et al.; An efficient beta-fucosidase was evolved by DNA shuffling from the Escherichia coli lacZ beta-galactosidase . Seven rounds of DNA shuffling and colony screening on chromogenic fucose substrates were performed, using 10,000 colonies per round . Compared with native beta-galactosidase, the evolved enzyme purified from cells from the final round showed a 1,000-fold increased substrate specificity for o-nitrophenyl fucopyranoside versus o-nitrophenyl galactopyranoside and a 300-fold increased substrate specificity for p-nitrophenyl fucopyranoside versus p-nitrophenyl galactopyranoside . The evolved cell line showed a 66-fold increase in p-nitrophenyl fucosidase specific activity . The evolved fucosidase has a 10- to 20-fold increased kcat/Km for the fucose substrates compared with the native enzyme . The DNA sequence of the evolved fucosidase gene showed 13 base changes, resulting in six amino acid changes from the native enzyme . This effort shows that the library size that is required to obtain significant enhancements in specificity and activity by reiterative DNA shuffling and screening, even for an enzyme of 109 kDa, is within range of existing high-throughput technology . Reiterative generation of libraries and stepwise accumulation of improvements based on addition of beneficial mutations appears to be a promising alternative to rational design. Proc Natl Acad Sci U S A, 1997 Apr 29, 94(9), 4451 - 6 Cloning and characterization of human karyopherin beta3; Yaseen NR et al.; Nuclear import of classical nuclear localization sequence-bearing proteins is mediated by karyopherin alpha/beta1 heterodimers . A second nuclear import pathway, mediated by karyopherin beta2 (transportin), recently was described for mRNA-binding proteins . Here we report the cloning and characterization of human karyopherin beta3, which may be involved in a third pathway for nuclear import . Karyopherin beta3 was localized mainly to the cytosol and the nucleus, particularly the nuclear rim . It bound to several of the repeat-containing nucleoporins (Nup358, Nup214, Nup153, Nup98, and p62) in overlay and solution-binding assays and was competed away by karyopherin beta1 . For Nup98, we localized this binding to the peptide repeat-containing region . Karyopherin beta3 contains two putative Ran-binding homology regions and bound to Ran-GTP in a solution-binding assay with much higher affinity than to Ran-GDP . Furthermore, it interacted with two ribosomal proteins in an overlay assay . We suggest that karyopherin beta3 is a nuclear transport factor that may mediate the import of some ribosomal proteins into the nucleus. Proc Natl Acad Sci U S A, 1997 Apr 29, 94(9), 4366 - 71 The rational design of allosteric interactions in a monomeric protein and its applications to the construction of biosensors; Marvin JS et al.; Rational protein design is an emerging approach for testing general theories of structure and function . The ability to manipulate function rationally also offers the possibility of creating new proteins of biotechnological value . Here we use the design approach to test the current understanding of the structural principles of allosteric interactions in proteins and demonstrate how a simple allosteric system can form the basis for the construction of a generic biosensor molecular engineering system . We have identified regions in Escherichia coli maltose-binding protein that are predicted to be allosterically linked to its maltose-binding site . Environmentally sensitive fluorophores were covalently attached to unique thiols introduced by cysteine mutations at specific sites within these regions . The fluorescence of such conjugates changes cooperatively with respect to maltose binding, as predicted . Spatial separation of the binding site and reporter groups allows the intrinsic properties of each to be manipulated independently . Provided allosteric linkage is maintained, ligand binding can therefore be altered without affecting transduction of the binding event by fluorescence . To demonstrate applicability to biosensor technology, we have introduced a series of point mutations in the maltose-binding site that lower the affinity of the protein for its ligand . These mutant proteins have been combined in a composite biosensor capable of measuring substrate concentration within 5% accuracy over a concentration range spanning five orders of magnitude. Mol Gen Genet, 1997 Apr 28, 254(4), 433 - 9 Indolepyruvate ferredoxin oxidoreductase from Pyrococcus sp . KOD1 possesses a mosaic structure showing features of various oxidoreductases; Siddiqui MA et al.; Indolepyruvate ferredoxin oxidoreductase (IOR) catalyzes the oxidative decarboxylation of arylpyruvates . Gene cloning and sequencing analysis of the IOR gene from the hyperthermophilic archaeon Pyrococcus sp . KOD1 was performed . Two genes, iorA and iorB . encoding alpha and beta subunits of IOR were found to be tandemly arranged, which suggests that gene expression is translationally coupled . Sequence analysis showed the C-terminal region of the alpha subunit to have a typical ferredoxin-type {4Fe-4S} cluster motif (CXXCXXCXXCXXXCP), which is similar to that present in the delta subunits of other oxidoreductases such as pyruvate ferredoxin oxidoreductase (POR) and 2-ketoisovalerate ferredoxin oxidoreductase (VOR) . We suggest that the alpha subunit of KOD1-IOR has a mosaic structure composed of features characteristic of the alpha, beta and delta subunits from POR and VOR . KOD1-IOR was overproduced in anaerobically incubated Escherichia coli cells and the crude enzyme was extracted under anaerobic conditions . The optimal temperature for activity of recombinant IOR was 70 degrees C and the half-life of this enzyme in the presence of air was 15 min at 25 degrees C. Mol Gen Genet, 1997 Apr 28, 254(4), 351 - 7 Functional dissection of a cell-division inhibitor, SulA, of Escherichia coli and its negative regulation by Lon; Higashitani A et al.; SulA is induced in Escherichia coli by the SOS response and inhibits cell division through interaction with FtsZ . To determine which region of SulA is essential for the inhibition of cell division, we constructed a series of N-terminal and C-terminal deletions of SulA and a series of alanine substitution mutants . Arginine at position 62, leucine at 67, tryptophan at 77 and lysine at 87, in the central region of SulA, were all essential for the inhibitory activity . Residues 3-27 and the C-terminal 21 residues were dispensable for the activity . The mutant protein lacking N-terminal residues 3-47 was inactive, as was that lacking the C-terminal 34 residues . C-terminal deletions of 8 and 21 residues increased the growth-inhibiting activity in lon+ cells, but not in lon- cells . The wild-type and mutant SulA proteins were isolated in a form fused to E . coli maltose-binding protein, and tested in vitro for sensitivity to Lon protease . Lon degraded wild-type SulA and a deletion mutant lacking the N-terminal 93 amino acids, but did not degrade the derivative lacking 21 residues at the C-terminus . Furthermore, the wild-type SulA and the N-terminal deletion mutant formed a stable complex with Lon, while the C-terminal deletion did not . MBP fused to the C-terminal 20 residues of SulA formed a stable complex with, but was not degraded by Lon . When LacZ protein was fused at its C-terminus to 8 or 20 amin acid residues from the C-terminal region of SulA the protein was stable in lon+ cells . These results indicate that the C-terminal 20 residues of SulA permit recognition by, and complex formation with, Lon, and are necessary, but not sufficient, for degradation by Lon. Toxicol Lett, 1997 Apr 28, 91(2), 121 - 7 Comparison of ribosome-inactivating proteins in the induction of apoptosis; Williams JM et al.; The aim of this study was to evaluate the ability of verocytotoxin-1 (VT1), VT1 B chain alone, ricin and a hybrid toxin (RASTA2) consisting of ricin A chain linked to VT1 B chain to inhibit protein synthesis and to induce apoptosis . The lethal effects of the toxins were compared using vero cells (originating from green African monkey kidney tissue) . As previously described cell death occurred through apoptosis which was quantified using the diphenylamine assay . DNA fragmentation was seen with VT1 at 10 pg/ml but there was no effect with B chain alone . Fragmentation with ricin was seen at 10 ng/ml and with RASTA2 at 1 ng/ml . Protein synthesis inhibition was measured by {(35)S}methionine incorporation . VT1 had an IC50 of 0.0024 ng/ml, B chain alone was ineffective at inhibiting protein synthesis . Ricin had an IC50 of 0.39 ng/ml and RASTA2 of 1.7 ng/ml . In vero cells the B chain of these toxins does not participate in cell killing. Biochem Biophys Res Commun, 1997 Apr 28, 233(3), 627 - 30 DnaJ potentiates the interaction between DnaK and alpha-helical peptides; de Crouy-Chanel A et al.; Molecular chaperones bind selectively to nascent, unfolded, misfolded, or aggregated polypeptides, and are involved in protein folding, protein targeting to membranes, and protein renaturation after stress . The DnaK chaperone of Escherichia coli is known to interact preferentially with positively charged hydrophobic peptides in an extended conformation . Accordingly, we show in the present study that DnaK has a low affinity for alpha-helical peptides . In the presence of its co-chaperone DnaJ and ATP, however, DnaK interacts more efficiently with alpha-helical peptides . This suggests that DnaJ triggers a conformational change in DnaK which improves its interaction with these peptides . The ability of the DnaK/DnaJ/GrpE chaperone machine to interact with alpha-helical peptides (which represent the most frequent secondary structure in proteins) should be an important part of its role in protein folding and renaturation. FEBS Lett, 1997 Apr 28, 407(2), 184 - 90 Comparative mutational analysis of peptidyl prolyl cis/trans isomerases: active sites of Escherichia coli trigger factor and human FKBP12; Tradler T et al.; A low degree of amino acid sequence similarity to FK506-binding proteins (FKBPs) has been obtained for the peptidyl prolyl cis/trans isomerase (PPIase) domain of E . coli trigger factor (TF) that was thought to be significant with regard to the enzymatic properties of the bacterial enzyme . We examined whether the alteration of a negatively charged side-chain at position 37 (FKBP numbering) and a phenylalanine at position 99, both highly conserved through both types of enzymes, leads to parallel effects on the catalytic activity of both FKBP12 and TF-PPIase domain in a series of tetrapeptide substrates with different P1 subsites . For the latter enzyme, substitution of Glu178 by Val or Lys, which aligns to Asp37 in human FKBP12, enhanced the PPIase activity, whereas a strongly decreased enzymatic activity was determined for the Asp37Leu and Asp37Val variants of FKBP12 . Regardless of the P1 subsite of the substrate used for the assay, mutation of Phe233Tyr generated a protein variant of the TF-PPIase domain with about 1% of the wild type PPIase activity . Dependent on the substrate nature, a moderate decrease as well as a 4.8-fold increase in k(cat)/K(M) could be determined for the corresponding Phe99Tyr FKBP12 variant . Neither of the mutations of the TF-PPIase domain was able to implant FK506 inhibition found as a major characteristic of the FKBP family of PPIases. Virology, 1997 Apr 28, 231(1), 20 - 7 An equine herpesvirus-1 gene 71 deletant is attenuated and elicits a protective immune response in mice; Marshall KR et al.; The pathogenesis of pulmonary infection and the imm