Eur J Biochem, 1999 Jun, 262(2), 617 - 24 A single-chain antibody fragment is functionally expressed in the cytoplasm of both Escherichia coli and transgenic plants; Tavladoraki P et al.; Despite the well-known crucial role of intradomain disulfide bridges for immunoglobulin folding and stability, the single-chain variable fragment of the anti-viral antibody F8 is functionally expressed when targeted to the reducing environment of the plant cytoplasm . We show here that this antibody fragment is also functionally expressed in the cytoplasm of Escherichia coli . A gel shift assay revealed that the single-chain variable fragment (scFv) accumulating in the plant and bacterial cytoplasm bears free sulfhydryl groups . Guanidinium chloride denaturation/renaturation studies indicated that refolding occurs even in a reducing environment, producing a functional molecule with the same spectral properties of the native scFv(F8) . Taken together, these results suggest that folding and functionality of this antibody fragment are not prevented in a reducing environment . This antibody fragment could therefore represent a suitable framework for engineering recombinant antibodies to be targeted to the cytoplasm. Eur J Biochem, 1999 Jun, 262(2), 595 - 9 Changes in the proton-motive force in Escherichia coli in response to external oxidoreduction potential; Riondet C et al.; The pH homeostasis and proton-motive force (Deltap) of Escherichia coli are dependent on the surrounding oxidoreduction potential (ORP) . Only the internal pH value and, thus, the membrane pH gradient (DeltapH) component of the Deltap is modified, while the membrane potential (DeltaPsi) does not change in a significant way . Under reducing conditions (Eh < 50 mV at pH 7.0), E . coli decreases its Deltap especially in acidic media (21% decrease at pH 7.0 and 48% at pH 5.0 for a 850-mV ORP decrease) . Measurements of ATPase activity and membrane proton conductance (CH+m) depending on ORP and pH have shown that the internal pH decrease is due to an increase in membrane proton permeability without any modification of ATPase activity . We propose that low ORP values de-energize E . coli by modifying the thiol : disulfide balance of proteins, which leads to an increase in the membrane permeability to protons. Eur J Biochem, 1999 Jun, 262(2), 467 - 74 Characterization of MB-1 . A dimeric helical protein with a compact core; Hefford MA et al.; MB-1 is a de-novo protein designed to incorporate a large number of the nutritionally important amino acids methionine, lysine, leucine and threonine into a stable four-helix bundle protein . MB-1 has been expressed and purified from Escherichia coli, indicating it was resistant to intracellular proteases {Beauregard, M., Dupont, C., Teather, R.M . & Hefford, M.A . (1995) Bio/Technology 13, 974} . Here we report an analysis of the secondary, tertiary and quaternary structures in MB-1 using circular dichroism, fluorospectroscopy and size-exclusion chromatography . Our data indicate that the MB-1 structure is close to the target structure, an alpha-helical bundle, in many respects and is highly helical in solution . The single tyrosine incorporated into the designed protein as a spectrocopic probe of tertiary structure, is buried in a compact, folded core and becomes accessible on protein denaturation, as per design . Furthermore, MB-1 was found to be native-like in many respects: (a) protein denaturation induced by urea is cooperative and fully reversible; (b) its oligomeric state at moderate concentration is well defined; and (c) MB-1 has very low affinity for 8-anilino-1-naphthalenesulfonic acid (ANSA), leading to enhancement of ANSA fluorescence that resembles that of other native proteins . On the other hand, our analysis revealed two aspects that command further attention . The folding stability of MB-1 as assessed by urea and thermal denaturation is somewhat less than that found for natural globular proteins of similar size . Size-exclusion chromatography experiments and analysis of MB-1 denaturation indicate that MB-1 is dimeric, not monomeric as designed . In light of these results, the utility and the current limitations of our design approach are discussed. Eur J Biochem, 1999 Jun, 262(2), 417 - 25 Properties and molecular cloning of Ca2+/H+ antiporter in the vacuolar membrane of mung bean; Ueoka-Nakanishi H et al.; Kinetic and molecular properties of the Ca2+/H+ antiporter in the vacuolar membrane of mung bean hypocotyls were examined and compared with Ca2+-ATPase . Ca2+ transport activities of both transporters were assayed separately by the filtration method using vacuolar membrane vesicles and 45Ca2+ . Ca2+ uptake in the presence of ATP and bafilomycin A1, namely Ca2+-ATPase, showed a relatively low Vmax (6 nmol.min-1.mg-1 protein) and a low Km for Ca2+ . The Ca2+/H+ antiporter activity driven by H+-pyrophosphatase showed a high Vmax (25 nmol.min-1.mg-1) and a relatively high Km for Ca2+ . The cDNA for mung bean Ca2+/H+ antiporter (VCAX1) codes for a 444 amino-acid polypeptide . Two peptide-specific antibodies of the antiporter clearly reacted with a 42-kDa protein from vacuolar membranes and a cell lysate from a Escherichia coli transformant in which VCAX1 was expressed . These observations directly demonstrate that a low-affinity, high-capacity Ca2+/H+ antiporter and a high-affinity Ca2+-ATPase coexist in the vacuolar membrane . It is likely that the Ca2+/H+ antiporter removes excess Ca2+ in the cytosol to lower the Ca2+ concentration to micromolar levels after stimuli have increased the cytosolic Ca2+ level, the Ca2+-ATPase then acts to lower the cytosolic Ca2+ level further. Clin Exp Allergy, 1999 Jun, 29(6), 840 - 7 Molecular characterization of Dau c 1, the Bet v 1 homologous protein from carrot and its cross-reactivity with Bet v 1 and Api g 1; Hoffmann-Sommergruber K et al.; BACKGROUND: Up to 70% of patients with birch pollen allergy exhibit the so-called oral allergy syndrome, an IgE-mediated food allergy . The most frequent and therefore best characterized pollen-fruit syndrome is apple allergy in patients suffering from tree pollen-induced pollinosis . The occurrence of adverse reactions to proteins present in vegetables such as celery and carrots in patients suffering from pollen allergy has also been reported . cDNAs for Bet v 1 homologous proteins have been cloned from celery, apple and cherry . Objective The aim of the study was to identify Bet v 1 homologues from carrot (Daucus carota), to isolate the respective cDNA, to compare the IgE-binding capacity of the natural protein to the recombinant allergen and determine the cross-reactivity to Api g 1 and Bet v 1 . METHODS: Molecular characterization of the carrot allergen was performed using IgE-immunoblotting, cross-inhibition assays, N-terminal sequencing, PCR-based cDNA cloning and expression of the recombinant protein in Escherichia coli . RESULTS: A 16-kDa protein from carrot was identified as a major IgE-binding component and designated Dau c 1 . Sequencing corresponding cDNAs revealed three extremely similar sequences (Dau c 1.1, 1.2 and 1.3) with an open reading frame of 462 bp coding for 154 amino acid residues . CONCLUSIONS: Purified recombinant Dau c 1.2 was tested in immunoblots displaying IgE-binding capacity comparable to its natural counterpart . Cross-inhibition assays verified the existence of common B-cell epitopes present on Dau c 1, Api g 1 as well as on Bet v 1. J Biol Chem, 1999 May 28, 274(22), 15913 - 9 Recombinant SFD isoforms activate vacuolar proton pumps; Zhou Z et al.; The vacuolar proton pump of clathrin-coated vesicles is composed of two general sectors, a cytosolic, ATP hydrolytic domain (V1) and an intramembranous proton channel, V0 . V1 is comprised of 8-9 subunits including polypeptides of 50 and 57 kDa, termed SFD (Sub Fifty-eight-kDa Doublet) . Although SFD is essential to the activation of ATPase and proton pumping activities catalyzed by holoenzyme, its constituent polypeptides have not been separated to determine their respective roles in ATPase functions . Recent molecular characterization of these subunits revealed that they are isoforms that arise through an alternative splicing mechanism (Zhou, Z., Peng, S.-B., Crider, B.P., Slaughter, C., Xie, X.S., and Stone, D.K . (1998) J . Biol . Chem . 273, 5878-5884) . To determine the functional characteristics of the 57-kDa (SFDalpha)1 and 50-kDa (SFDbeta) isoforms, we expressed these proteins in Escherichia coli . We determined that purified recombinant proteins, rSFDalpha and rSFDbeta, when reassembled with SFD-depleted holoenzyme, are functionally interchangeable in restoration of ATPase and proton pumping activities . In addition, we determined that the V-pump of chromaffin granules has only the SFDalpha isoform in its native state and that rSFDalpha and rSFDbeta are equally effective in restoring ATPase and proton pumping activities to SFD-depleted enzyme . Finally, we found that SFDalpha and SFDbeta structurally interact not only with V1, but also withV0, indicating that these activator subunits may play both structural and functional roles in coupling ATP hydrolysis to proton flow. J Biol Chem, 1999 May 28, 274(22), 15615 - 21 A pathway for conformational diversity in proteins mediated by intramolecular chaperones; Shinde U et al.; Conformational diversity within unique amino acid sequences is observed in diseases like scrapie and Alzheimer's disease . The molecular basis of such diversity is unknown . Similar phenomena occur in subtilisin, a serine protease homologous with eukaryotic pro-hormone convertases . The subtilisin propeptide functions as an intramolecular chaperone (IMC) that imparts steric information during folding but is not required for enzymatic activity . Point mutations within IMCs alter folding, resulting in structural conformers that specifically interact with their cognate IMCs in a process termed "protein memory." Here, we show a mechanism that mediates conformational diversity in subtilisin . During maturation, while the IMC is autocleaved and subsequently degraded by the active site of subtilisin, enzymatic properties of this site differ significantly before and after cleavage . Although subtilisin folded by Ile-48 --> Thr IMC (IMCI-48T) acquires an "altered" enzymatically active conformation (SubI-48T) significantly different from wild-type subtilisin (SubWT), both precursors undergo autocleavage at similar rates . IMC cleavage initiates conformational changes during which the IMC continues its chaperoning function subsequent to its cleavage from subtilisin . Structural imprinting resulting in conformational diversity originates during this reorganization stage and is a late folding event catalyzed by autocleavage of the IMC. J Biol Chem, 1999 May 28, 274(22), 15598 - 604 Structure of the membrane domain of subunit b of the Escherichia coli F0F1 ATP synthase; Dmitriev O et al.; The structure of the N-terminal transmembrane domain (residues 1-34) of subunit b of the Escherichia coli F0F1-ATP synthase has been solved by two-dimensional 1H NMR in a membrane mimetic solvent mixture of chloroform/methanol/H2O (4:4:1) . Residues 4-22 form an alpha-helix, which is likely to span the hydrophobic domain of the lipid bilayer to anchor the largely hydrophilic subunit b in the membrane . The helical structure is interrupted by a rigid bend in the region of residues 23-26 with alpha-helical structure resuming at Pro-27 at an angle offset by 20 degrees from the transmembrane helix . In native subunit b, the hinge region and C-terminal alpha-helical segment would connect the transmembrane helix to the cytoplasmic domain . The transmembrane domains of the two subunit b in F0 were shown to be close to each other by cross-linking experiments in which single Cys were substituted for residues 2-21 of the native subunit and b-b dimer formation tested after oxidation with Cu(II)(phenanthroline)2 . Cys residues that formed disulfide cross-links were found with a periodicity indicative of one face of an alpha-helix, over the span of residues 2-18, where Cys at positions 2, 6, and 10 formed dimers in highest yield . A model for the dimer is presented based upon the NMR structure and distance constraints from the cross-linking data . The transmembrane alpha-helices are positioned at a 23 degrees angle to each other with the side chains of Thr-6, Gln-10, Phe-14, and Phe-17 at the interface between subunits . The change in direction of helical packing at the hinge region may be important in the functional interaction of the cytoplasmic domains. J Biol Chem, 1999 May 28, 274(22), 15367 - 74 ATP hydrolysis and DNA binding by the Escherichia coli RecF protein; Webb BL et al.; The Escherichia coli RecF protein possesses a weak ATP hydrolytic activity . ATP hydrolysis leads to RecF dissociation from double-stranded (ds)DNA . The RecF protein is subject to precipitation and an accompanying inactivation in vitro when not bound to DNA . A mutant RecF protein that can bind but cannot hydrolyze ATP (RecF K36R) does not readily dissociate from dsDNA in the presence of ATP . This is in contrast to the limited dsDNA binding observed for wild-type RecF protein in the presence of ATP but is similar to dsDNA binding by wild-type RecF binding in the presence of the nonhydrolyzable ATP analog, adenosine 5'-O-(3-thio)triphosphate (ATPgammaS) . In addition, wild-type RecF protein binds tightly to dsDNA in the presence of ATP at low pH where its ATPase activity is blocked . A transfer of RecF protein from labeled to unlabeled dsDNA is observed in the presence of ATP but not ATPgammaS . The transfer is slowed considerably when the RecR protein is also present . In competition experiments, RecF protein appears to bind at random locations on dsDNA and exhibits no special affinity for single strand/double strand junctions when bound to gapped DNA . Possible roles for the ATPase activity of RecF in the regulation of recombinational DNA repair are discussed. J Biol Chem, 1999 May 28, 274(22), 15329 - 35 A rat RuvB-like protein, TIP49a, is a germ cell-enriched novel DNA helicase; Makino Y et al.; We have isolated a novel nuclear protein with a molecular mass of 49 kDa (TIP49a) from rat liver . The rat TIP49a showed structural resemblance to several bacterial RuvBs and also displayed Walker A and B motifs . We overproduced the recombinant TIP49a in Escherichia coli and purified it to near homogeneity . Biochemical investigations demonstrated that TIP49a possessed ATPase activity that was stimulated by single-stranded DNA but neither by double-stranded DNA nor by any forms of RNA polymers tested . Moreover, a UV cross-linking assay indicated TIP49a specifically interacted with ATP . Interestingly, we found that DNA duplex was unwound by the recombinant TIP49a in the presence of ATP or dATP . Optimal concentrations of ATP and Mg2+ for the helicase activity were 1-2 mM and 0.25-1 mM, respectively . Displacement of the DNA strand occurred in the 3' to 5' direction with respect to the single-stranded DNA flanking the duplex . Western blot analysis revealed that TIP49a was abundantly expressed in testes and moderately in spleen, thymus, and lung . In mouse seminiferous tubules, the protein was restrictively observed in germ lineages from late pachytene spermatocytes to round spermatids . From these observations, we propose that TIP49a is a novel DNA helicase and may play a role in nuclear processes such as recombination and transcription. Anticancer Drug Des, 1998 Dec, 13(8), 995 - 1007 Synthesis of self-immolative glucuronide-based prodrugs of a phenol mustard; Lougerstay-Madec R et al.; The design and synthesis of the mustard pro-prodrugs which can be used in ADEPT is reported . Prodrugs 1 and 2 include a glucuronide group which is connected to the drug via an aromatic and/or aliphatic bis-carbamate spacer . The design of these new prodrugs takes advantage of a spontaneous 1,6-elimination and/or an intramolecular cyclization reaction after enzymatic cleavage . Thus, enzymatic-catalyzed hydrolysis of the glucuronyl moiety of 1 by Escherichia coli beta-glucuronidase results in the liberation of the parent mustard drug 20 with formation of CO2, 2-nitro-4-hydroxymethylphenol 19 and dimethylimidazolidinone 21 . Surprisingly, prodrug 2 was not cleaved under the same conditions . According to in vitro experiments, prodrugs 1 and 2 were approximately 50- and 80-fold less cytotoxic than the parent drug and, when treated with beta-glucuronidase, the level of cytotoxic activity of 1 became comparable to that of the drug . Stability of 1 in phosphate buffer was satisfactory . These results demonstrate that 1 is a prodrug that can be specifically activated to release the cytotoxic agent. Science, 1999 May 21, 284(5418), 1372 - 6 UDP-GlcNAc 2-epimerase: a regulator of cell surface sialylation; Keppler OT et al.; Modification of cell surface molecules with sialic acid is crucial for their function in many biological processes, including cell adhesion and signal transduction . Uridine diphosphate-N-acetylglucosamine 2-epimerase (UDP-GlcNAc 2-epimerase) is an enzyme that catalyzes an early, rate-limiting step in the sialic acid biosynthetic pathway . UDP-GlcNAc 2-epimerase was found to be a major determinant of cell surface sialylation in human hematopoietic cell lines and a critical regulator of the function of specific cell surface adhesion molecules. Biochem Biophys Res Commun, 1999 May 27, 259(1), 136 - 40 Role of tryptophanyl residues in tobacco acetolactate synthase; Chong CK et al.; Acetolactate synthase (ALS) catalyzes the first common step in the biosynthesis of valine, leucine, and isoleucine . ALS is the target of three classes of herbicides, the sulfonylureas, the imidazolinones, and the triazolopyrimidines . Five mutants (W266F, W439F, W490F, W503F, and W573F) of the ALS gene from Nicotiana tabacum were constructed and expressed in Escherichia coli, and the enzymes were purified . The W490F mutation abolished the binding affinity for cofactor FAD and inactivated the enzyme . The replacement of Trp573 by Phe yielded a mutant ALS resistant to the three classes of herbicides . The other three mutations, W266F, W439F, and W503F, did not significantly affect the enzymatic properties and the sensitivity to the herbicides . These results indicate that the Trp490 residue is essential for the binding of FAD and that Trp573 is located at the herbicide binding site . The data also suggest that the three classes of herbicides bind ALS competitively . Arch Biochem Biophys, 1999 Jun 1, 366(1), 116 - 24 Effects of human cytochrome b5 on CYP3A4 activity and stability in vivo; Voice MW et al.; Cytochrome P450s (P450) form a superfamily of membrane-bound proteins that play a key role in the primary metabolism of both xenobiotics and endogenous compounds such as drugs and hormones, respectively . To be enzymically active, they require the presence of a second membrane-bound protein, NADPH P450 reductase, which transfers electrons from NADPH to the P450 . Because of the diversity of P450 enzymes, much of the work on individual forms has been carried out on purified proteins, in vitro, which requires the use of complex reconstitution mixtures to allow the P450 to associate correctly with the NADPH P450 reductase . There is strong evidence from such reconstitution experiments that, when cytochrome b5 is included, the turnover of some substrates with certain P450s is increased . Here we demonstrate that allowing human P450 reductase, CYP3A4, and cytochrome b5 to associate in an in vivo-like system, by coexpressing all three proteins together in Escherichia coli for the first time, the turnover of both nifedipine and testosterone by CYP3A4 is increased in the presence of cytochrome b5 . The turnover of testosterone was increased by 166% in whole cells and by 167% in preparations of bacterial membranes . The coexpression of cytochrome b5 also resulted in the stabilization of the P450 during substrate turnover in whole E . coli, with 109% of spectrally active CYP3A4 remaining in cells after 30 min in the presence of cytochrome b5 compared with 43% of the original P450 remaining in cells in the absence of cytochrome b5 . Environ Mol Mutagen, 1999, 33(3), 249 - 56 Comparison of mutant frequencies at the transgenic lambda LacI and cII/cI loci in control and ENU-treated Big Blue mice; Zimmer DM et al.; We compared the lambda cII/cI transgenic mutation assay described by Jakubczak et al . {(1996): Proc Natl Acad Sci USA 93:9073-9078} to the previously established Big Blue assay . Genomic DNA isolated from liver, spleen, and lung tissue of control or ethylnitrosourea (ENU)-treated Big Blue mice (100 mg/kg i.p., single dose) was packaged into phage (five animals, two packagings per DNA sample) which were simultaneously plated for lacI and cII/cI mutant frequency (MF) and titer . Mean MF of control animals was higher for cII/cI than lacI for all three tissues examined (spontaneous cII/cI MF divided by spontaneous lacI MF = 2.9, 3.1, and 1.7 for liver, spleen, and lung, respectively) . The differences were statistically significant for liver and spleen, but not lung . The ENU-induced MF measured by subtracting control MFs from ENU-treated MFs was higher in the cII/cI assay than lacI (liver = 23.0 x 10(-5) for cII/cI vs . 15.1 x 10(-5) for lacI; spleen = 64.8 x 10(-5) for cII/cI vs . 36.1 x 10(-5) for lacI; lung = 17.1 x 10(-5) for cII/cI vs . 15.8 x 10(-5) for lacI) . Fold increase over control values measured by dividing MF of ENU-treated animals by appropriate control values was higher for lacI than cII/cI (liver = 4.4-fold for lacI vs . 2.7 for cII/cI; spleen = 13.1-fold for lacI vs . 8.4 for cII/cI; and lung = 5.6-fold for lacI vs . 4.0 for cII/cI) . Despite these differences, overall results were similar for the two mutational endpoints . These results suggest that the cII/cI assay may be an acceptable alternative to lacI where transgenic mutation studies are indicated. Genes Genet Syst, 1998 Dec, 73(6), 323 - 36 The euryarchaeotes, a subdomain of Archaea, survive on a single DNA polymerase: fact or farce? Ishino Y, Cann IK. Archaea is now recognized as the third domain of life . Since their discovery, much effort has been directed towards understanding the molecular biology and biochemistry of Archaea . The objective is to comprehend the complete structure and the depth of the phylogenetic tree of life . DNA replication is one of the most important events in living organisms and DNA polymerase is the key enzyme in the molecular machinery which drives the process . All archaeal DNA polymerases were thought to belong to family B . This was because all of the products of pol genes that had been cloned showed amino acid sequence similarities to those of this family, which includes three eukaryal DNA replicases and Escherichia coli DNA polymerase II . Recently, we found a new heterodimeric DNA polymerase from the hyperthermophilic archaeon, Pyrococcus furiosus . The genes coding for the subunits of this DNA polymerase are conserved in the euryarchaeotes whose genomes have been completely sequenced . The biochemical characteristics of the novel DNA polymerase family suggest that its members play an important role in DNA replication within euryarchaeal cells . We review here our current knowledge on DNA polymerases in Archaea with emphasis on the novel DNA polymerase discovered in Euryarchaeota. Nat Struct Biol, 1999 May, 6(5), 448 - 53 X-ray structure of the Escherichia coli periplasmic 5'-nucleotidase containing a dimetal catalytic site; Knofel T et al.; The crystal structure of 5'-nucleotidase (5'-NT) from E . coli, also known as UDP-sugar hydrolase, has been determined at 1.7 A resolution . Two zinc ions are present in the active site, which is located in a cleft between two domains . The dimetal center and a catalytic Asp-His dyad are the main players in the catalytic mechanism . Structure-based sequence comparisons show that the structure also provides a model for animal 5'-NTs, which together with other ectonucleotidases terminate the action of nucleotides as extracellular signaling substances in the nervous system. Mol Reprod Dev, 1999 Jun, 53(2), 149 - 58 Adenovirus-mediated introduction of DNA into pig sperm and offspring; Farre L et al.; The ability of adenoviral vectors to transfer DNA into boar spermatozoa and to offspring was tested . Exposure of spermatozoa to adenovirus bearing the E . coli lacZ gene resulted in the transfer of the gene to the head of the spermatozoa . Treatment did not affect either viability or acrosomal integrity of boar sperm . Of the 2-to 8-cell embryos obtained after in vitro fertilization with adenovirus-exposed sperm, 21.7% expressed the LacZ product . Four out of 56 piglets (about 7%) obtained after artificial insemination with adenovirus-exposed spermatozoa were positive in PCR analyses, even though none of the piglets showed the LacZ gene after southern blot analysis . RT-PCR analysis performed in tissues from two positive stillborn piglets showed the presence of the LacZ mRNA in all of the tissues tested . The offspring obtained after mating two positive animals did not show LacZ gene presence . Our results indicate that adenovirus could be a feasible mechanism for the delivery of DNA into spermatozoa, even though the transfer of the transgene may be limited to the first generation. Mol Reprod Dev, 1999 Jun, 53(2), 142 - 8 Computer assisted image analysis to assess colonization of recipient seminiferous tubules by spermatogonial stem cells from transgenic donor mice; Dobrinski I et al.; Transplantation of spermatogonial stem cells from fertile, transgenic donor mice to the testes of infertile recipients provides a unique system to study the biology of spermatogonial stem cells . To facilitate the investigation of treatment effects on colonization efficiency an analysis system was needed to quantify colonization of recipient mouse seminiferous tubules by donor stem cell-derived spermatogenesis . In this study, a computer-assisted morphometry system was developed and validated to analyze large numbers of samples . Donor spermatogenesis in recipient testes is identified by blue staining of donor-derived spermatogenic cells expressing the E . coli lacZ structural gene . Images of seminiferous tubules from recipient testes collected three months after spermatogonial transplantation are captured, and stained seminiferous tubules containing donor-derived spermatogenesis are selected for measurement based on their color by color thresholding . Colonization is measured as number, area, and length of stained tubules . Interactive, operator-controlled color selection and sample preparation accounted for less than 10% variability for all collected parameters . Using this system, the relationship between number of transplanted cells and colonization efficiency was investigated . Transplantation of 10(4) cells per testis only rarely resulted in colonization, whereas after transplantation of 10(5) and 10(6) cells per testis the extent of donor-derived spermatogenesis was directly related to the number of transplanted donor cells . It appears that about 10% of transplanted spermatogonial stem cells result in colony formation in the recipient testis . The present study establishes a rapid, repeatable, semi-interactive morphometry system to investigate treatment effects on colonization efficiency after spermatogonial transplantation in the mouse. Hypertension, 1999 May, 33(5), 1243 - 9 Role of increased production of superoxide anions by NAD(P)H oxidase and xanthine oxidase in prolonged endotoxemia; Brandes RP et al.; Superoxide anions (O2-) are supposedly involved in the pathogenesis of endothelial dysfunction . We investigated whether the enhanced formation of O2- is involved in the attenuation of endothelium-dependent relaxation induced by lipopolysaccharide (LPS) . Rats were injected with LPS (10 mg/kg IP), the aorta was removed after 12 or 30 hours, and generation of O2-, H2O2, and ONOO- was measured using chemiluminescence assays . Protein tyrosine nitration and expression of xanthine oxidase (XO), NAD(P)H oxidase, and manganese superoxide dismutase were determined by Western or Northern blotting, and endothelium-dependent relaxation in aortic rings was studied . LPS treatment increased vascular O2- (from 35+/-2 cpm/ring at baseline to 166+/-21 cpm/ring at 12 hours and 225+/-16 cpm/ring at 30 hours) and H2O2 formation, which was partially sensitive to the NAD(P)H oxidase inhibitor diphenylene iodonium at both time points studied and to the XO inhibitor oxypurinol only 30 hours after LPS treatment . Expression of XO and NAD(P)H oxidase (p22phox, p67phox, and gp91phox) were increased by LPS in a time-dependent manner, as were protein tyrosine nitration and ONOO- formation . LPS also induced expression of the oxidative stress-sensitive protein manganese superoxide dismutase . Endothelium-dependent relaxation was impaired after LPS treatment and could not be restored by inhibition of inducible NO synthase . Inhibition of O2- with superoxide dismutase, oxypurinol, tiron, or the superoxide dismutase mimetic Mn(III)tetrakis(4-benzoic acid)porphyrin chloride did not restore but further deteriorated the relaxation of LPS-treated rings . In summary, treatment of rats with LPS enhances vascular expression of XO and NAD(P)H oxidase and increases formation of O2- and ONOO- . Because removal of O2- compromised rather than restored endothelium-dependent relaxation, a direct role of O2- in the induction of endothelial dysfunction is unlikely . Other mechanisms, such as prolonged protein tyrosine nitration by peroxynitrite (which is formed from NO and O2-) or downregulation of the NO effector pathway, are more likely to be involved. J Clin Oncol, 1999 May, 17(5), 1568 - 73 Cerebrospinal fluid asparagine concentrations after Escherichia coli asparaginase in children with acute lymphoblastic leukemia; Woo MH et al.; PURPOSE: The CNS is an important sanctuary site in childhood acute lymphoblastic leukemia (ALL) . CSF asparagine concentration reflects asparaginase systemic pharmacodynamics . We evaluated the time course of CSF asparagine depletion in children with ALL during and after a course of Escherichia coli asparaginase . PATIENTS AND METHODS: Thirty-one children (24 newly diagnosed and seven at relapse) received E coli asparaginase 10,000 IU/m2 intramuscularly three times weekly for six and nine doses, respectively, as part of multiagent induction chemotherapy . CSF asparagine levels were measured before, during, and after asparaginase dosing . RESULTS: The percentage of patients with undetectable (< 0.04 micromol/L) CSF asparagine was 3.2% (one of 31 patients) at baseline, 73.9% (17 of 23) during asparaginase therapy, and 56.3% (nine of 16) 1 to 5 days, 43.8% (seven of 16) 6 to 10 days, 20.0% (two of 10) 11 to 30 days and 0% (zero of 21) more than 30 days after asparaginase therapy . The proportion of patients with depleted CSF asparagine was higher during asparaginase therapy than at baseline (P < .001), 11 to 30 days (P = .003), and more than 30 days after asparaginase therapy (P < .001) . Median CSF asparagine concentrations were 4.42 micromol/L before, less than 0.04 micromol/L during, and less than 0.04 micromol/L at 1 to 5 days, 1.63 micromol/L at 6 to 10 days, 1.70 micromol/L at 11 to 30 days, and 5.70 micromol/L at more than 30 days after asparaginase therapy, respectively . CSF depletion was more common in patients with low baseline CSF asparagine concentrations (P = .003) . CONCLUSION: CSF asparagine concentrations are depleted by conventional doses of E coli asparaginase in the majority of patients, but they rebound once asparaginase therapy is completed. J Interferon Cytokine Res, 1999 Apr, 19(4), 383 - 92 Recombinant chicken IFN-gamma expressed in Escherichia coli: analysis of C-terminal truncation and effect on biologic activity; Michalski WP et al.; Interferon-gamma (IFN-gamma) possesses potent immunostimulatory properties, and it has recently been shown to have potential therapeutic properties . Recombinant protein technology is frequently used for commercial production of therapeutics, such as IFN . Biologically active recombinant chicken IFN-gamma (rChIFN-gamma) constructs bearing an N-terminal poly-His tag were expressed in Escherichia coli . Preparations of rChIFN-gamma contained varying ratios of a full-length and a truncated protein species (18 and 16 kDa, respectively) . Amino acid sequence analysis of the full-length protein corroborated the sequence previously predicted from the cDNA sequence . Full-length rChIFN-gamma contains two cysteine residues at the C-terminus, and these were labeled by reduction and subsequent specific alkylation with fluorescent tag (5-I-AEDANS) to distinguish between full-length and C-terminally truncated forms of rChIFN-gamma . Comparative peptide mapping, amino acid sequencing, and mass spectrometry revealed that the 16 kDa protein was truncated at Lys133 . It was also observed that the 18 kDa rChIFN-gamma protein was infrequently contaminated with small quantities of protein truncated at Arg141 . A truncated recombinant construct (His1-Lys133) was also expressed in E . coli and had biologic activity comparable with that of the full-length construct . The 3-D structure of rChIFN-gamma was deduced by comparative modeling with bovine and human IFN-gamma crystallographic structures . Analysis of sequences and comparison of structures have revealed that the 3-D structure of rChIFN-gamma is similar to those of bovine and human molecules despite an overall amino acid identity of only 32%. RNA, 1999 May, 5(5), 670 - 7 Crystal structure of acceptor stem of tRNA(Ala) from Escherichia coli shows unique G.U wobble base pair at 1.16 A resolution; Mueller U et al.; The acceptor stem of Escherichia coli tRNA(Ala), rGGGGCUA.rUAGCUCC (ALAwt), contains the main identity element for the correct aminoacylation by the alanyl tRNA synthetase . The presence of a G3.U70 wobble base pair is essential for the specificity of this reaction, but there is a debate whether direct minor-groove contact with the 2-amino group of G3 or a distortion of the acceptor stem induced by the wobble pair is the critical feature recognized by the synthetase . We here report the structure analysis of ALAwt at near-atomic resolution using twinned crystals . The crystal lattice is stabilized by a novel strontium binding motif between two cis-diolic O3'-terminal riboses . The two independent molecules in the asymmetric unit of the crystal show overall A-RNA geometry . A comparison with the crystal structure of the G3-C70 mutant of the acceptor stem (ALA(C70)) determined at 1.4 A exhibits a modulation in ALAwt of helical twist and slide due to the wobble base pair, but no recognizable distortion of the helix fragment distant from the wobble base pair . We suggest that a highly conserved hydration pattern in both grooves around the G3.U70 wobble base pair may be functionally significant. Diabetes, 1999 Feb, 48(2), 371 - 6 Hyperglycemia-induced embryonic dysmorphogenesis correlates with genomic DNA mutation frequency in vitro and in vivo; Lee AT et al.; Congenital malformations affecting multiple organ systems are at least three times more common in infants of mothers with IDDM than in infants born to nondiabetic mothers . Numerous studies have confirmed the teratogenic effect of hyperglycemia on the developing embryo, although no direct mechanism has been determined . In this study, we aimed to correlate the frequency of lacI mutations with degree of hyperglycemic exposure and severity of malformations in mouse embryos from in vitro cultures . Day 8 transgenic mouse embryos cultured in 30 or 50 mmol/l glucose for 48 h exhibited a higher incidence of morphological abnormalities, as well as an increase in lacI mutation frequency, compared with embryos cultured in 10 mmol/l glucose with no abnormalities and a lower frequency of lacI mutations . We also used a transgenic lacI rat system to evaluate the relationship between abnormal embryonic development and DNA mutation frequency in day 11 embryos of severely diabetic rats (serum glucose >20 mmol/l) . Compared with control embryos, the embryos from diabetic rats displayed significantly more malformations, shorter crown-rump lengths, fewer somites, and more than six times greater genomic DNA mutation frequency . Genetic analysis of the mutated lacI gene from both in vitro cultured mouse embryos and in vivo developed rat embryos revealed that the majority of mutations were due to base substitutions (transitions and transversions), but that the rate of large DNA mutations tended to increase in embryos exposed to a diabetic environment . Our results support the interrelationship between increased rates of congenital malformations and DNA mutations in the offspring of diabetic pregnancy. Carcinogenesis, 1999 May, 20(5), 905 - 9 A study of endonuclease III-sensitive sites in irradiated DNA: detection of alpha-particle-induced oxidative damage; Prise KM et al.; An important difference between chemical agents that induce oxidative damage in DNA and ionizing radiation is that radiation-induced damage is clustered locally on the DNA . Both modelling and experimental studies have predicted the importance of clustering of lesions induced by ionizing radiation and its dependence on radiation quality . With increasing linear energy transfer, it is predicted that complex lesions will be formed within 1-20 bp regions of the DNA . As well as strand breaks, these sites may contain multiple damaged bases . We have compared the yields of single strand breaks (ssb) and double strand breaks (dsb) along with those produced by treatment of irradiated DNA with the enzyme endonuclease III, which recognizes a number of oxidized pyrimidines in DNA and converts them to strand breaks . Plasmid DNA was irradiated under two different scavenging conditions to test the involvement of OH* radicals with either 60Co gamma-rays or alpha-particles from a 238Pu source . Under low scavenging conditions (10 mM Tris) gamma-irradiation induced 7.1 x 10(-7) ssb Gy/bp, which increased 3.7-fold to 2.6 x 10(-6) ssb Gy/bp with endo III treatment . In contrast the yields of dsb increased by 4.2-fold from 1.5 x 10(-8) to 6.3 x 10(-8) dsb Gy/bp . This equates to an additional 2.5% of the endo III-sensitive sites being converted to dsb on enzyme treatment . For alpha-particles this increased to 9% . Given that endo III sensitive sites may only constitute approximately 40% of the base lesions induced in DNA, this suggests that up to 6% of the ssb measured in X- and 22% in alpha-particle-irradiated DNA could have damaged bases associated with them contributing to lesion complexity. Genes Genet Syst, 1998 Dec, 73(6), 407 - 13 The molecular basis of the instability of a crp- mutation in Escherichia coli; Umemoto A et al.; We have described a rapid spontaneous conversion in the stationary phase of Escherichia coli strain DOO (crp-) cells as a whole population to crp+ state (Sugino and Morita, 1994) . In this paper we have tried to elucidate the molecular basis of this unidirectional conversion by cloning and sequencing of the crp gene in their crp+ and crp- states . We have found that in the original crp- strain, an IS2 element has been inserted between its original promoter and the coding region of the crp gene in the so-called orientation II (Ahmed et al., 1981), accompanied by an 11 bp deletion . Unexpectedly, the crp+ "revertants" derived from the crp- mutant had no difference in sequence from the crp-, either in the coding or the regulatory region . This suggests that a change at another locus, such that this change somehow activates the expression of the crp gene to the level of a normal crp+, is responsible for the apparent reversion from crp- to crp+. Acta Microbiol Pol, 1998, 47(4), 409 - 13 Application of polymerase chain reaction for determination of toxins in Escherichia coli strains isolated from pigs with diarrhea; Osek J; The PCR method was used for the determination of LTI, STI, STII enterotoxins and Stx2e toxin genes in E . coli strains isolated from pigs with diarrhea . It was shown that most of the strains (77.3%) were enterotoxigenic, producing mainly LTI and STII (30.8%) or STI and STII (21.3%) toxins . It was found that 10.6% of isolates possessed the stx2e genes responsible for the elaboration of shiga toxin 2e. Acta Microbiol Pol, 1998, 47(4), 335 - 43 Characterisation of a new host-vector system for fast-growing mycobacteria; Golanska E et al.; In this paper we describe the development of a host-vector system for genetic studies of fast-growing mycobacteria able to biotransform sterols . A wild strain Mycobacterium smegmatis SN38 and a biotechnological mutant Mycobacterium vaccae B3805 were transformed by electroporation with the pSMT3 E . coli-Mycobacterium shuttle plasmid harbouring the hygromycin resistance gene . Both, the pSMT3 plasmid and its derivative pSMT3-ksdD carrying the 3-ketosteroid-delta 1-dehydrogenase gene (ksdD) from Arthrobacter simplex were stably maintained in M . vaccae B3805 . The presence of the pSMT3 vector did not affect biotransformation activities of the host strain . We consider the M . vaccae B3805 strain and the pSMT3 plasmid to be a good host-vector system for cloning in mycobacteria genes coding enzymes involved in steroid degradation pathway. Gene, 1999 May 17, 232(1), 59 - 68 Molecular characterization of a chemotaxis operon in the oral spirochete, Treponema denticola; Greene SR et al.; A chemotaxis gene cluster from Treponema denticola (Td), a pathogenic spirochete associated with human periodontal diseases, was cloned, sequenced, and analyzed . The gene cluster contained three chemotaxis (che) genes (cheA, cheW, and cheY) and an open reading frame (cheX) that is homologous with Treponema pallidum (Tp) and Borrelia burgdorferi (Bb) cheX . The Td che genes have the same transcriptional orientation with a sigma 70-like promoter located upstream of cheA and a stem-loop structure characteristic of a Rho-independent transcriptional terminator downstream of cheY . Primer extension analysis identified a transcriptional start point six nucleotides (nt) downstream of the -10 (TAAAAA) promoter sequence . Reverse-transcriptase-polymerase chain reaction (RT-PCR) data indicated that cheA through cheY are co-transcribed and suggested that transcription is terminated after cheY . The gene organization of the Td che operon is identical to that of the Tp che operon . Southern blot analysis indicated the presence of one copy of each che gene on the Td genome . The cheA, cheW, cheX, and cheY genes are 2403, 1332, 462, and 438nt long, respectively, and encode proteins with predicted molecular masses of 88.2, 49.7, 16.8, and 16 . 0kDa, respectively . Functional domains of the T . denticola CheA and CheY proteins are highly conserved with those of the Escherichia coli (Ec) CheA and CheY proteins . Phylogenetic analysis of Td CheY indicated that it is closely related to Tp CheY and Bb CheY3. J Protein Chem, 1999 Feb, 18(2), 187 - 92 Biosynthesis and characterization of 4-fluorotryptophan-labeled Escherichia coli arginyl-tRNA synthetase; Zhang QS et al.; Escherichia coli 4-fluorotryptophan-substituted arginyl-tRNA synthetase was biosynthetically prepared and purified from a tryptophan auxotroph which could overproduce this enzyme . A method was developed to separate 4-fluorotryptophan from tryptophan and to determine accurately their contents in the 4-fluorotryptophan-containing proteins . It was confirmed that more than 95% of the tryptophan residues in the purified 4-fluorotryptophan-substituted arginyl-tRNA synthetase were replaced by 4-fluorotryptophan . Studies on the effect of the 4-fluorotryptophan replacement on properties of the enzyme showed that, when compared with the native enzyme, both the specific activity and the first-order rate constant of the fluorinated enzyme decreased by approximately 20% with just slightly higher Km values . CD studies, however, did not reveal any difference between the secondary structure of the native and fluorinated enzymes . In addition, thermal unfolding studies showed that the 4-fluorotryptophan replacement did not significantly affect the thermal stability of the enzyme . We may conclude that the substitution of 4-fluorotryptophan in arginyl-tRNA synthetase had no substantial effect on the structure and function of the enzyme . Finally, a preliminary study of 19F nuclear magnetic resonance spectroscopy of the fluorinated enzyme has shown promising prospect for further investigation of its structure and function with NMR. Eur J Gastroenterol Hepatol, 1999 Mar, 11(3), 329 - 35 Biliary pancreatic reflux-induced acute pancreatitis--myth or possibility? Arendt T, Nizze H, Monig H, Kloehn S, Stuber E, Folsch UR. OBJECTIVE: The mechanism whereby gallstone passage through the choledochoduodenal junction initiates acute pancreatitis is not known . We mimicked different patterns of stone impaction at the choledochoduodenal junction in a rabbit model and studied whether these result in biliary pancreatic reflux and the initiation of pancreatic inflammation . METHODS: In rabbits, catheters were introduced into the common bile duct (CBD) and the pancreatic duct . In five experiments, obstruction of these catheters at various time intervals mimicked different patterns of stone obstruction of both ducts prior to a stone impaction at the papilla of Vater: experiment I--no obstruction of the pancreatic duct and the CBD; experiment II--separate obstruction of the CBD and the pancreatic duct; experiment III--selective obstruction of the CBD; experiment IV--separate obstruction of the CBD and the pancreatic duct and subsequent decompression of the pancreatic duct; experiment V--obstruction pattern as in experiment IV associated with a bacterial infection of bile (10(8) E . coli/ml) . Ductal pressures were recorded for 24 h . In order to study the effects of a subsequent impaction of the stone at the papilla of Vater, the catheters in the CBD and in the pancreatic duct were connected and mimicked a common channel behind a papillary stone . The flow direction of bile and pancreatic juice was directly observed . Pancreatic histology was analysed 24 h later . RESULTS: In experiments I-III, neither biliary pancreatic reflux nor acute pancreatitis was observed . In experiments IV and V, obstruction of the CBD caused an increase in the biliary pressure to 17 +/- 3 cm H2O, whereas the pancreatic duct pressure dropped to subnormal levels following obstruction and selective decompression (2 +/- 0.5 cm H2O) . After the creation of a 'common channel', biliary pancreatic reflux was observed for 118 +/- 21 min . Flow of sterile bile into the pancreas was not harmful to the gland . Infected biliary pancreatic reflux initiated acute pancreatitis . CONCLUSIONS: 1 . Bile flow into the pancreas may occur . 2 . Biliary pancreatic reflux may initiate acute pancreatitis . 3 . Bile reflux-induced acute pancreatitis requires previous biliary hypertension, temporary pancreatic duct obstruction, and the bacterial infection of choledochal secretions. J Biomol Struct Dyn, 1999 Apr, 16(5), 989 - 1002 Intermolecular contacts between the lambda-Cro repressor and the operator DNA characterized by nuclear magnetic resonance spectroscopy; Tochio H et al.; The specific interaction between lambda phage Cro repressor and the DNA fragment bearing the consensus sequence of operators has been studied using nuclear magnetic resonance (NMR) . Using both 15N- and 13C/15N- labeled lambda-Cro in complex with unlabeled DNA, chemical shift assignments of the lambda-Cro-DNA complex were obtained using heteronuclear NMR experiments . Inter-molecular contacts between the protein and DNA were identified using heteronuclear filtered NOESY experiments . The inter-molecular contacts were supplemented with intra-protein and intra-DNA NOE constraints to dock lambda-Cro to the bent B-form DNA using restrained molecular dynamics . The structure of one of the subunits of lambda-Cro in the complex is essentially the same as that of the unbound form . In the complex, inter-molecular NOEs were observed between the "helix-turn-helix" region comprising the alpha2 and alpha3 helices of the lambda-Cro protein and the major groove of the DNA . The methyl group of Thr17 forms a hydrophobic contact with the methyl group of the thymine at base pair 1 in the DNA, and Val25 and Ala29 make hydrophobic contacts with the methyl group of the thymine at base pair 5 . The presence and the absence of these contacts can explain the difference in the affinity of lambda-Cro to several variants of the operator sequence. Antiviral Res, 1999 May, 42(1), 35 - 46 Guanosine nucleotide analogs as inhibitors of alphavirus mRNA capping enzyme; Lampio A et al.; The two virus-specific reactions in the capping of alphavirus RNAs, catalyzed by the replicase protein nsP1, are promising targets for developing virus-specific inhibitors . In this report, we have studied the effect of over 50 cap analogs on the guanine-7-methyltransferase and guanylyltransferase activities of Semliki Forest virus nsP1 . Recombinant nsP1 was expressed in Escherichia coli and partially purified by flotation in a discontinuous sucrose gradient . The methyltransferase activity had a pH optimum between pH 6.5 and 7.1, and the apparent Km values were 1.9 mM for GTP, 6.0 microM for S-adenosyl-L-methionine and 170 microM for Mg2+ . NsP1 methyltransferase was able to methylate efficiently GTP (relative activity 100%), GDP (16%), GpppG (35%), GppppG (50%) and less efficiently GpppA (12%), m2GTP (9%), and m2,2GTP (25%), but not m7GppG . The most potent inhibitors for nsP1 methyltransferase were et2m7GMP (Ki value 42 microM), m2,7GMP, (64 microM), m2,7GpppG (82 microM), m2et7GMP (105 microM), m2(2-phet)7GMP (194 microM) and m2GMP (386 microM) . Of these compounds, m2GMP, m2et7GMP and m2(2-phet)7GMP showed competitive inhibition, whereas the others showed mixed type inhibition . All compounds that inhibited the methyltransferase activity inhibited also the guanylyltransferase activity of nsP1. J Mol Biol, 1999 May 21, 288(5), 989 - 98 Crystal structure of the hydrogenase maturating endopeptidase HYBD from Escherichia coli; Fritsche E et al.; The maturation of {NiFe} hydrogenases includes formation of the nickel metallocenter, proteolytic processing of the metal center carrying large subunit, and its assembling with other hydrogenase subunits . The hydrogenase maturating enzyme HYBD from Escherichia coli, a protease of molecular mass 17.5 kDa, specifically cleaves off a 15 amino acid peptide from the C terminus of the precursor of the large subunit of hydrogenase 2 in a nickel-dependent manner . Here we report the crystal structure of HYBD at 2.2 A resolution . It consists of a twisted five-stranded beta-sheet surrounded by four and three helices, respectively, on each side . A cadmium ion from the crystallization buffer binds to the proposed nickel-binding site and is penta-coordinated by Glu16, Asp62, His93, and a water molecule in a pseudo-tetragonal arrangement . HYBD is topologically related to members of the metzincins superfamily of zinc endoproteinases, sharing the central beta-sheet and three helices . In contrast to the metzincins, the metal-binding site of HYBD is localized at the C-terminal end of the beta-sheet . Three helical insertions unique to HYBD pack against one side of the sheet, build up the active site cleft, and provide His93 as ligand to the metal . From this structure, we derive molecular clues into how the protease HYBD is involved in the hydrogenase maturation process . Mol Cell Biochem, 1999 Feb, 192(1-2), 95 - 103 Role of acylCoA binding protein in acylCoA transport, metabolism and cell signaling; Knudsen J et al.; Long chain acylCoA esters (LCAs) act both as substrates and intermediates in intermediary metabolism and as regulators in various intracellular functions . AcylCoA binding protein (ACBP) binds LCAs with high affinity and is believed to play an important role in intracellular acylCoA transport and pool formation and therefore also for the function of LCAs as metabolites and regulators of cellular functions {1} . The major factors controlling the free concentration of cytosol long chain acylCoA ester (LCA) include ACBP {2}, sterol carrier protein 2 (SCP2) {3} and fatty acid binding protein (FABP) {4} . Additional factors affecting the concentration of free LCA include feed back inhibition of the acylCoA synthetase {5}, binding to acylCoA receptors (LCA-regulated molecules and enzymes), binding to membranes and the activity of acylCoA hydrolases {6}. Mol Cell Biochem, 1999 Mar, 193(1-2), 153 - 7 Activation of toxin ADP-ribosyltransferases by eukaryotic ADP-ribosylation factors; Moss J et al.; ADP-ribosylation factors (ARFs) are members of a multigene family of 20-kDa guanine nucleotide-binding proteins that are regulatory components in several pathways of intracellular vesicular trafficking . The relatively small (approximately 180-amino acids) ARF proteins interact with a variety of molecules (in addition to GTP/GDP, of course) . Cholera toxin was the first to be recognized, hence the name . Later it was shown that ARF also activates phospholipase D . Different parts of the molecule are responsible for activation of the two enzymes . In vesicular trafficking, ARF must interact with coatomer to recruit it to a membrane and thereby initiate vesicle budding . ARF function requires that it alternate between GTP- and GDP-bound forms, which involves interaction with regulatory proteins . Inactivation of ARF-GTP depends on a GTPase-activating protein or GAP . A guanine nucleotide-exchange protein or GEP accelerates release of bound GDP from inactive ARF-GDP to permit GTP binding . Inhibition of GEP by brefeldin A (BFA) blocks ARF activation and thereby vesicular transport . In cells, it causes apparent disintegration of Golgi structure . Both BFA-sensitive and insensitive GEPs are known . Sequences of peptides from a BFA-sensitive GEP purified in our laboratory revealed the presence of a Sec7 domain, a sequence of approximately 200 amino acids that resembles a region in the yeast Sec7 gene product, which is involved in Golgi vesicular transport . Other proteins of unknown function also contain Sec7 domains, among them a lymphocyte protein called cytohesin-1 . To determine whether it had GEP activity, recombinant cytohesin-1 was synthesized in E . coli . It preferentially activated class I ARFs 1 and 3 and was not inhibited by BFA but failed to activate ARF5 (class II) . There are now five Sec7 domain proteins known to have GEP activity toward class I ARFs . It remains to be determined whether there are other Sec7 domain proteins that are GEPs for ARFs 4, 5, or 6. Mol Cell Biochem, 1999 Mar, 193(1-2), 149 - 52 Function of poly(ADP-ribose) polymerase in response to DNA damage: gene-disruption study in mice; Masutani M et al.; To elucidate the biological functions of poly(ADP-ribose) polymerase (PARP, {EC 2.4.2.30}) in DNA damage responses, genetic and biochemical approaches were undertaken . By disrupting exon 1 of the mouse PARP gene by a homologous recombination, PARP-deficient mouse embryonic stem (ES) cell lines and mice could be produced without demonstrating lethality . PARP-/- ES cells showed complete loss of PARP activity and increased sensitivity to gamma-irradiation and an alkylating agents, indicating a physiological role for PARP in the response to DNA damage . p53, a key molecule in cellular DNA damage response, was found to stimulate PARP activity and became poly(ADP-ribosyl)ated in the presence of damaged DNA . However, PARP-/- ES cells showed p21 and Mdm-2 mRNA induction following gamma-irradiation, indicating that PARP activity is not indispensable for p21 and Mdm-2 mRNA induction in the established p53-cascade . On the other hand, in a reconstituted reaction system, purified PARP from human placenta suppressed the pRB-phosphorylation activity in the presence of NAD and damaged DNA . Human PARP expressed in E . coli showed a similar effect on pRB-phosphorylation activity of cdk2 . These findings suggest a direct involvement of PARP in the regulation of cdk activity for cell-cycle arrest. Mol Cell Biochem, 1999 Mar, 193(1-2), 109 - 13 Characterization of NAD:arginine ADP-ribosyltransferases; Moss J et al.; NAD:arginine mono-ADP-ribosyltransferases catalyze the transfer of ADP-ribose from NAD to the guanidino group of arginine on a target protein . Deduced amino acid sequences of one family (ART1) of mammalian ADP-ribosyltransferases, cloned from muscle and lymphocytes, show hydrophobic amino and carboxyl termini consistent with glycosylphosphatidylinositol (GPI)-anchored proteins . The proteins, overexpressed in mammalian cells transfected with the transferase cDNAs, are released from the cell surface with phosphatidylinositol-specific phospholipase C (PI-PLC), and display immunological and biochemical characteristics consistent with a cell surface, GPI-anchored protein . In contrast, the deduced amino acid sequence of a second family (ART5) of transferases, cloned from murine lymphoma cells and expressed in high abundance in testis, displays a hydrophobic amino terminus, consistent with a signal sequence, but lacks a hydrophobic signal sequence at its carboxyl terminus, suggesting that the protein is destined for export . Consistent with the surface localization of the GPI-linked transferases, multiple surface substrates have been identified in myotubes and activated lymphocytes, and, notably, include integrin alpha subunits . Similar to the bacterial toxin ADP-ribosyltransferases, the mammalian transferases contain the characteristic domains involved in NAD binding and ADP-ribose transfer, including a highly acidic region near the carboxy terminus, which, when disrupted by in vitro mutagenesis, results in a loss of enzymatic activity . The carboxyl half of the protein, synthesized as a fusion protein in E . coli, possessed NADase, but not ADP-ribosyltransferase activity . These findings are consistent with the existence at the carboxyl terminus of ART1 of a catalytically active domain, capable of hydrolyzing NAD, but not of transferring ADP-ribose to a guanidino acceptor. Mol Cell Biochem, 1999 Mar, 193(1-2), 103 - 7 Functional analysis of poly(ADP-ribose) polymerase in Drosophila melanogaster; Miwa M et al.; Poly(ADP-ribose) polymerase (PARP) is conserved in eukaryotes . To analyze the function of PARP, we isolated and characterized the gene for PARP in Drosophila melanogaster . The PARP gene consisted of six translatable exons and spanned more than 50 kb . The DNA binding domain is encoded by exons 1-4 . Although the consensus cleavage site of CED-3 like protease during apoptosis is conserved from human to Xenopus laevis PARPs, it is neither conserved in the corresponding region of Drosophila nor Sarcophaga peregrina . There are two cDNAs species in Drosophila . One cDNA could encode the full length PARP protein (PARP I), while the other is a truncated cDNA which could encode a partial-length PARP protein (PARP II), which lacks the automodification domain and is possibly produced by alternative splicing . The expression of these two forms of PARP in E . coli demonstrated that while PARP II has the catalytic NAD-binding domain and DNA-binding domain it is enzymatically inactive . On the other hand PARP I is active . A deletion mutant of PARP gene could grow to the end of embryogenesis but did not grow to the adult fly . These results suggest that the PARP gene plays an important function during the development of Drosophila. Mol Cell Biochem, 1999 Mar, 193(1-2), 53 - 60 A dual approach in the study of poly (ADP-ribose) polymerase: in vitro random mutagenesis and generation of deficient mice; Trucco C et al.; A dual approach to the study of poly (ADP-ribose)polymerase (PARP) in terms of its structure and function has been developed in our laboratory . Random mutagenesis of the DNA binding domain and catalytic domain of the human PARP, has allowed us to identify residues that are crucial for its enzymatic activity . In parallel PARP knock-out mice were generated by inactivation of both alleles by gene targeting . We showed that: (i) they are exquisitely sensitive to gamma-irradiation, (ii) they died rapidly from acute radiation toxicity to the small intestine, (iii) they displayed a high genomic instability to gamma-irradiation and MNU injection and, (iv) bone marrow cells rapidly underwent apoptosis following MNU treatment, demonstrating that PARP is a survival factor playing an essential and positive role during DNA damage recovery and survival. J Korean Med Sci, 1999 Apr, 14(2), 187 - 92 DNA-mediated immunization of mice with plasmid encoding HBs antigen; Yoon SJ et al.; In order to develop an experimental DNA vaccine for the prevention and treatment of hepatitis B virus infection, hepatitis B virus surface antigen (HBsAg) DNA was subcloned into an E . coli-eukaryotic cell shuttle vector and was expressed in the Baculovirus expression system . Intramuscular, intradermal, and intraperitoneal injections of 30 microg of the plasmid DNA expressing HBsAg induced humoral and cellular immune responses in ICR mice . The first IgG antibodies were detected after ten days and specific IgG antibody titers peaked after two months of a single intramuscular DNA injection . Anti-HBs antibody titers gradually increased and peaked at four months following intradermal DNA injection, and in case of intraperitoneal injection they peaked at seven months . Generation of HBs-specific helper T lymphocytes was also investigated through the production of interleukin-2 by T helper cells . Boosting effects of HBs DNA were investigated without much results . In general, DNA-mediated HBs immunization induced humoral and cellular immune responses in mice that appears to simulate immune responses in human during the course of HBV vaccination. Mol Biol Evol, 1999 Jan, 16(1), 83 - 97 Evidence for genetic drift in endosymbionts (Buchnera): analyses of protein-coding genes; Wernegreen JJ et al.; Buchnera, the bacterial endosymbionts of aphids, undergo severe population bottlenecks during maternal transmission through their hosts . Previous studies suggest an increased effect of drift within these strictly asexual, small populations, resulting in an increased fixation of slightly deleterious mutations . This study further explores sequence evolution in Buchnera using three approaches . First, patterns of codon usage were compared across several homologous Escherichia coli and Buchnera loci, in order to test the prediction that selection for the use of optimal codons is less effective in small populations . A chi 2-based measure of codon bias was developed to adjust for the overall A + T richness of silent positions in the endosymbionts . In contrast to E . coli homologues, adaptive codon bias across Buchnera loci is markedly low, and patterns of codon usage lack a strong relationship with gene expression level . These data suggest that codon usage in Buchnera has been shaped largely by mutational pressure and drift rather than by selection for translational efficiency . One exception to the overall lack of bias is groEL, which is known to be constitutively overexpressed in Buchnera and other endosymbionts . Second, relative-rate tests show elevated rates of sequence evolution of numerous protein-coding loci across Buchnera, compared to E . coli . Finally, consistently higher ratios of nonsynonymous to synonymous substitutions in Buchnera loci relative to the enteric bacteria strongly suggest the accumulation of nonsynonymous substitutions in endosymbiont lineages . Combined, these results suggest a decreased effectiveness of purifying selection in purging endosymbiont populations of slightly deleterious mutations, particularly those affecting codon usage and amino acid identity. J Vet Med Sci, 1999 Mar, 61(3), 251 - 4 Expression of Recombinant bovine L-selectin in Escherichia coli and insect cells; Kashima T et al.; Bovine L-selectin was expressed in bacteria using pGEX vector and in insect cells infected with recombinant baculovirus in order to obtain recombinant protein for preparation of specific antiserum and its functional studies . In bacterial expression, L-selectin fusion protein with glutathione S-transferase was detected in the insoluble fraction with the expected molecular weight of 60 kDa by SDS-PAGE and reacted with anti-bovine CD62L monoclonal antibody in immunoblot analysis . In insect cells infected with recombinant baculovirus, a band corresponding to L-selectin was not observed in SDS-PAGE with protein staining, but they apparently reacted with anti-bovine CD62L monoclonal antibody in immunoblot analysis . Furthermore, the indirect immunofluorescence test revealed that bovine L-selectin was efficiently expressed on the surface of Sf9 cells infected with recombinant baculovirus, and flow cytometric analysis showed that the percentage of CD62L positive cells in bovine PBMC was about 66% and that most Sf9 cells infected with recombinant baculovirus had specific immunofluorescence. Hum Antibodies, 1999, 9(1), 23 - 36 Targeting cytokines to tumors to induce active antitumor immune responses by recombinant fusion proteins; Xiang J; Cytokines such as interleukin-2(IL-2), gamma interferon (IFN-gamma) and alpha tumor necrosis factor (TNF-alpha) are important mediators in immune responses against tumors . However, their therapeutic efficacy and clinical utilities in treatment of human malignancies are in large part limited due to the low concentrations of cytokine in tumors and the severe toxic side-effects derived from high-dose administration of cytokines . One critical issue to improve therapeutic efficacy is how to increase the local concentration of cytokine in tumors without causing severe side-effects . A series of recent reports demonstrated that the introduction of cytokine genes into tumor cells and subsequent local secretion can circumvent the limitations associated with the systemic cytokine administration . An alternative means of cytokine delivery is to target cytokines to tumor cells with tumor specific antibodies . Thereby, effective local cytokine concentrations can be achieved at the tumor sites without resorting to patient-specific therapy . With the advance in biotechnology, two structurally disparate domains of immunoglobulin and cytokine can be brought together into one fusion protein molecule by protein engineering These engineered antibody-cytokine fusion proteins combine the unique targeting ability of tumor-specific antibodies with the multifunctional activity of cytokines . In general, there are two commonly engineered fusion proteins, the F(ab')2/cytokine expressed in mammalian cells and the single-chain FV/cytokine expressed in Escherichia coli . Both the tumor-binding reactivity and the functional cytokine activity are maintained in most of fusion proteins . Therefore, these fusion proteins may be useful in targeting cytokine to tumors to stimulate immune destruction of tumors, while limiting severe toxic side-effects by the high dose of cytokine administration . Recent preclinical studies have shown that these fusion proteins are able to target cytokines to tumors expressing the tumor-associated antigen in vivo, and to inhibit both the primary and metastatic tumors in an immune competent animal model . Therefore, these recombinant fusion proteins may represent a new generation of novel immunotherapeutic reagents for the treatment of human malignant diseases. Anal Chem, 1999 May 1, 71(9), 1744 - 52 Use of evolutionary factor analysis in the spectroelectrochemistry of Escherichia coli sulfite reductase hemoprotein and a Mo/Fe/S cluster; Keesey RL et al.; The deconvolution of spectroelectrochemical data is often quite difficult if the spectra of intermediates are not known . Factor analysis, however, has been shown to be a powerful technique which can make it possible to deconvolute overlapping spectra . In this work, evolving factor analysis will be used to determine the number of intermediates and the spectra of those species for two typical spectroelectrochemical experiments: linear scan voltammetry and chronoabsorptometry in a thin-layer cell . The first system was the reduction of E . coli sulfite reductase hemoprotein (SiR-HP) . Principal factor analysis indicated that three species were present . By using evolving factor analysis, the potential regions where each of the species were present were identified, and their concentrations and spectra were determined by the use of the mass balance equation . The spectra of the one-electron (SiR-HP1-) and two-electron (SiR-HP2-) reduced product were compared with previous work . The second experiment was the chronoabsorptometry of Cl2FeS2MoS2FeCl2(2-) in methylene chloride . This experiment indicated that five species were present during the experiment . The entire set of 61 spectra were fit by assuming that there were 4 species present during the electrolysis . The rate constant for the appearance of subsequent species fit quite well with the rate constant for the disappearance of previous species . The spectra of the intermediates and final product were obtained using evolving factor analysis and a mass balance equation . Identification of the fifth species, which was probably the initial reduction product, Cl2FeS2MoS2FeCl2(3-), was difficult due to its low concentration and the fact that it was present in the same time region as the starting material. Biofizika, 1999 Jan-Feb, 44(1), 59 - 65 {Sulfur ligands within the cluster formations of copper at its strong'binding sites on the cytoplasmic membrane of Escherichia coli}; Volodina LA et al.; The analysis of the saturation curves of ESR signals revealed a decrease in the relaxation rate of fast-relaxation Cu(I) complexes on the cytoplasmic membrane of E . coli after the interaction of these bacteria with low concentrations of SH-reagents . It was concluded that the observed changes are associated with the reorganization of Cu clusters due to the binding of SH groups incorporated into the clusters N-ethylmaleimide or Ag(I). Acta Crystallogr D Biol Crystallogr, 1999 Jun, 55 ( Pt 6), 1226 - 8 Cubic crystals of phosphopantetheine adenylyltransferase from Escherichia coli; Izard T et al.; Phosphopantetheine adenylyltransferase (PPAT, E.C . 2.7.7.3) catalyzes the penultimate step in coenzyme A (CoA) biosynthesis, transferring an adenylyl group from ATP to 4'-phosphopantetheine, and forming dephospho-CoA . Cubic crystals of native PPAT from Escherichia coli as well as PPAT in complex with its substrates were obtained . The crystals belong to space group I23 or I213 with unit-cell dimension a = 135.5 A . The crystals diffract to better than 1.8 A resolution on a Cu Kalpha rotating-anode generator . The asymmetric unit is likely to contain two molecules, corresponding to a packing density of 2.9 A3 Da-1. Acta Crystallogr D Biol Crystallogr, 1999 Jun, 55 ( Pt 6), 1222 - 5 Crystallization and preliminary X-ray crystallographic analysis of a periplasmic tetrahaem flavocytochrome c3 from Shewanella frigidimarina NCIMB400 which has fumarate reductase activity; Bamford V et al.; The fumarate reductase of Escherichia coli and other bacteria is a membrane-bound enzyme consisting of four subunits . A soluble periplasmic 64 kDa tetrahaem flavocytochrome c3 from Shewanella frigidimarina NCIMB400 which possesses a catalytic fumarate reductase activity has been crystallized . The crystals belong to space group P212121 with unit-cell parameters a = 72.4, b = 110.1, c = 230.2 A . Assuming a molecular dimer in the asymmetric unit, the crystals contain 65% solvent and, when cryocooled to 100 K, the crystals diffract to at least 3.0 A resolution . The crystals, however, display an inherent lack of isomorphism and the plausibility of a MAD phasing experiment has therefore been investigated by measuring the iron K absorption edge from a single crystal. Acta Crystallogr D Biol Crystallogr, 1999 Jun, 55 ( Pt 6), 1201 - 3 Purification, crystallization and preliminary X-ray analysis of Drosophila melanogaster ferrochelatase; Wang KF et al.; Ferrochelatase (protoheme ferrolyase, E.C . 4.99.1.1), the terminal enzyme in the heme biosynthetic pathway, catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme . In eukaryotes, the protein is associated with the inner surface of the inner mitochondrial membrane, and in higher animals the enzyme contains a {2Fe-2S} cluster . This cluster is highly sensitive to NO and is coordinated by four Cys residues whose spacing in the primary sequence is unique . Ferrochelatase from Drosophila melanogaster has been expressed in Escherichia coli with an amino-terminal six-histidine tag and purified to homogeneity . The protein has been crystallized with the {2Fe-2S} cluster intact . The crystals belong to space group I422, with unit-cell dimensions a = b = 158.1, c = 171.2 A and two molecules in the asymmetric unit, and diffract to 3 . 0 A resolution. Mol Cell Biol, 1999 Jun, 19(6), 4414 - 22 DIX domains of Dvl and axin are necessary for protein interactions and their ability to regulate beta-catenin stability; Kishida S et al.; The N-terminal region of Dvl-1 (a mammalian Dishevelled homolog) shares 37% identity with the C-terminal region of Axin, and this related region is named the DIX domain . The functions of the DIX domains of Dvl-1 and Axin were investigated . By yeast two-hybrid screening, the DIX domain of Dvl-1 was found to interact with Dvl-3, a second mammalian Dishevelled relative . The DIX domains of Dvl-1 and Dvl-3 directly bound one another . Furthermore, Dvl-1 formed a homo-oligomer . Axin also formed a homo-oligomer, and its DIX domain was necessary . The N-terminal region of Dvl-1, including its DIX domain, bound to Axin directly . Dvl-1 inhibited Axin-promoted glycogen synthase kinase 3beta-dependent phosphorylation of beta-catenin, and the DIX domain of Dvl-1 was required for this inhibitory activity . Expression of Dvl-1 in L cells induced the nuclear accumulation of beta-catenin, and deletion of the DIX domain abolished this activity . Although expression of Axin in SW480 cells caused the degradation of beta-catenin and reduced the cell growth rate, expression of an Axin mutant that lacks the DIX domain did not affect the level of beta-catenin or the growth rate . These results indicate that the DIX domains of Dvl-1 and Axin are important for protein-protein interactions and that they are necessary for the ability of Dvl-1 and Axin to regulate the stability of beta-catenin. Mol Cell Biol, 1999 Jun, 19(6), 4279 - 88 Role of phosphoinositide 3-kinase in activation of ras and mitogen-activated protein kinase by epidermal growth factor; Wennstrom S et al.; The paradigm for activation of Ras and extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinase by extracellular stimuli via tyrosine kinases, Shc, Grb2, and Sos does not encompass an obvious role for phosphoinositide (PI) 3-kinase, and yet inhibitors of this lipid kinase family have been shown to block the ERK/MAP kinase signalling pathway under certain circumstances . Here we show that in COS cells activation of both endogenous ERK2 and Ras by low, but not high, concentrations of epidermal growth factor (EGF) is suppressed by PI 3-kinase inhibitors; since Ras activation is less susceptible than ERK2 activation, PI 3-kinase-sensitive events may occur both upstream of Ras and between Ras and ERK2 . However, strong elevation of PI 3-kinase lipid product levels by expression of membrane-targeted p110alpha is by itself never sufficient to activate Ras or ERK2 . PI 3-kinase inhibition does not affect EGF-induced receptor autophosphorylation or adapter protein phosphorylation or complex formation . The concentrations of EGF for which PI 3-kinase inhibitors block Ras activation induce formation of Shc-Grb2 complexes but not detectable EGF receptor phosphorylation and do not activate PI 3-kinase . The activation of Ras by low, but mitogenic, concentrations of EGF is therefore dependent on basal, rather than stimulated, PI 3-kinase activity; the inhibitory effects of LY294002 and wortmannin are due to their ability to reduce the activity of PI 3-kinase to below the level in a quiescent cell and reflect a permissive rather than an upstream regulatory role for PI 3-kinase in Ras activation in this system. Mol Cell Biol, 1999 Jun, 19(6), 4191 - 9 A two-hit mechanism for vitamin D3-mediated transcriptional repression of the granulocyte-macrophage colony-stimulating factor gene: vitamin D receptor competes for DNA binding with NFAT1 and stabilizes c-Jun; Towers TL et al.; We previously described a control element in the granulocyte-macrophage colony-stimulating factor (GM-CSF) enhancer that is necessary and sufficient to mediate both transcriptional activation in response to T-cell stimuli and transcriptional repression by 1,25-dihydroxyvitamin D3 {1,25(OH)2D3} through the vitamin D3 receptor (VDR) . This DNA element is a composite site that is recognized by both Fos-Jun and NFAT1; it is directly bound by VDR in the absence of a retinoid X receptor as an apparent monomer, and it is bound in a unique tertiary conformation . We describe here the mechanism by which VDR elicits its transcriptional inhibitory effect . Firstly, VDR outcompetes NFAT1 for binding to the composite site . Overexpression of NFAT1 in vivo by transient transfection is able to relieve the 1,25(OH)2D3-dependent repression . Secondly, VDR stabilizes the binding of a Jun-Fos heterodimer to the adjacent AP-1 portion of the element . This appears to occur through a direct interaction between VDR and c-Jun, as demonstrated in vitro by direct glutathione S-transferase coprecipitation assays . In vivo, overexpression of c-Jun, but not c-Fos, leads to a rescue of the 1, 25(OH)2D3-mediated repression . Transfected FLAG-VDR bound to the NFAT1-AP-1 DNA binding element can be selectively precipitated from nuclear extracts that are made from cells treated with activating agents in the presence of 1,25(OH)2D3 . VDR is not detected in the complex in the absence of the ligand . Thus, VDR acts selectively on the two components required for activation of this promoter/enhancer: it competes with NFAT1 for binding to the composite site, positioning itself adjacent to Jun-Fos on the DNA . Co-occupancy apparently leads to an inhibitory effect on c-Jun's transactivation function . These two events mediated by VDR effectively block the NFAT1-AP-1 activation complex, resulting in an attenuation of activated GM-CSF transcription. Mol Cell Biol, 1999 Jun, 19(6), 4028 - 38 Targeting of p38 mitogen-activated protein kinases to MEF2 transcription factors; Yang SH et al.; Mitogen-activated protein (MAP) kinase-mediated signalling to the nucleus is an important event in the conversion of extracellular signals into a cellular response . However, the existence of multiple MAP kinases which phosphorylate similar phosphoacceptor motifs poses a problem in maintaining substrate specificity and hence the correct biological response . Both the extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) subfamilies of MAP kinases use a second specificity determinant and require docking to their transcription factor substrates to achieve maximal substrate activation . In this study, we demonstrate that among the different MAP kinases, the MADS-box transcription factors MEF2A and MEF2C are preferentially phosphorylated and activated by the p38 subfamily members p38alpha and p38beta2 . The efficiency of phosphorylation in vitro and transcriptional activation in vivo of MEF2A and MEF2C by these p38 subtypes requires the presence of a kinase docking domain (D-domain) . Furthermore, the D-domain from MEF2A is sufficient to confer p38 responsiveness on different transcription factors, and reciprocal effects are observed upon the introduction of alternative D-domains into MEF2A . These results therefore contribute to our understanding of signalling to MEF2 transcription factors and demonstrate that the requirement for substrate binding by MAP kinases is an important facet of three different subclasses of MAP kinases (ERK, JNK, and p38). Genome Res, 1999 May, 9(5), 463 - 70 Automated filtration-based high-throughput plasmid preparation system; Itoh M et al.; Current methods of plasmid preparation do not allow for large capacity automated processing . We have developed an automated high-throughput system that prepares plasmid DNA for large-scale sequencing . This system is based on our previously reported filtration method . In this method, cell harvesting, alkaline lysis, and plasmid purification occur in a single 96-well microtiter plate from which sequence-ready DNA samples are collected . The plates are designed to allow all reagents to be injected from above the wells and the spent reagents to be aspirated from below . This design has enabled us to build a linear process plasmid preparation system consisting of an automated filter plate stacker and a 21-stage automated plasmid preparator . The 96-well plates used are outfitted with glass-filters that trap Escherichia coli before the plates are stacked in the automated stacker . The plates move from the stacker to each of the 21 stages of the preparator . At specific stages, various reagents or chemicals are injected into the wells from above . Finally, the plates are collected in the second stacker . The optimal throughput of the preparator is 40,000 samples in 17.5 hr . Here, we describe a pilot experiment preparing 15,360 templates in 160 specially designed 96-well glass-filter plates . The prepared plasmids were subjected to restriction digestion, DNA sequencing, and transcriptional sequencing. Am J Physiol, 1999 May, 276(5 Pt 1), L736 - 43 Cytokine gene expression after inhalation of corn dust; Wohlford-Lenane CL et al.; To characterize the time course and localize the production of proinflammatory cytokines after inhalation of corn dust, we exposed mice (C3H/HeBFeJ) by inhalation challenge to sterile corn dust extract (CDE) and contrasted this response to inhalation of Escherichia coli 0111:B4 lipopolysaccharide (LPS) or pyrogen-free saline . After both CDE and LPS exposure, an increase in the concentration of bronchoalveolar lavage neutrophils was detected 1 h postinhalation and persisted for 48 h . Significant increases in the bronchoalveolar lavage concentration of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1alpha, and macrophage inflammatory protein (MIP)-2 resulted after inhalation of either CDE or LPS . Although the time courses of these cytokines were distinct, a similar pattern of release was observed after both CDE and LPS exposure . A single inhalation exposure of either CDE or LPS resulted in enhanced expression of mRNA for TNF-alpha, IL-1alpha, and MIP-2 that was evident and most pronounced within 1 h of the inhalation challenge . Although enhanced expression of mRNA for TNF-alpha was detectable 12 h after completion of the inhalation challenge, IL-1alpha and MIP-2 mRNA expression remained elevated through the 24-h time point . TNF-alpha, IL-1alpha, and MIP-2 expression was localized by in situ hybridization to inflammatory cells in the airways and alveoli from 1 to 24 h in both CDE- and LPS-exposed lungs . Interestingly, there was no convincing evidence that MIP-2 was substantially produced by airway epithelial cells . The pattern, timing, and location of expression of TNF-alpha, IL-1alpha, and MIP-2 mRNA after a single inhalation exposure of CDE in comparison with LPS is similar, supporting a common etiology and mechanism of inflammation in the lower respiratory tract . Moreover, our findings indicate that inhalation of corn dust or LPS results in an acute inflammatory process that is primarily mediated by inflammatory cells and appears to be self-limited. J Biol Chem, 1999 May 21, 274(21), 15167 - 72 Activation of a cyanobacterial adenylate cyclase, CyaC, by autophosphorylation and a subsequent phosphotransfer reaction; Kasahara M et al.; The CyaC protein, a cyanobacterial adenylate cyclase, has a unique primary structure composed of the catalytic domain of adenylate cyclase and the conserved domains of bacterial two-component regulatory systems, one transmitter domain and two receiver domains . In the present work, CyaC was produced in Escherichia coli as a histidine-tagged recombinant protein and purified to homogeneity . CyaC showed ability to autophosphorylate in vitro with the gamma-phosphate of {gamma-32P}ATP . CyaC derivatives were constructed by site-directed mutagenesis in which the highly conserved phosphorylation sites in the transmitter domain (His572) and receiver domains (Asp60 or Asp895) were replaced by glutamine and alanine residues, respectively . After autophosphorylation of the CyaC derivatives, the chemical stabilities of the phosphoryl groups bound to the derivatives were determined . It was found that His572 is the initial phosphorylation site and that the phosphoryl group once bound to His572 is transferred to Asp895 . The enzyme activities of the CyaC derivatives defective in His572 or Asp895 were considerably reduced . Asp895 is phosphorylated by acetyl {32P}phosphate, a small phosphoryl molecule, but Asp60 is not . Acetyl phosphate stimulates adenylate cyclase activity only when Asp895 is intact . These results suggest that the phosphorylation of Asp895 is essential for the activation of adenylate cyclase and that Asp60 functions differently from Asp895 in regulating the enzyme activity. J Biol Chem, 1999 May 21, 274(21), 14979 - 87 Mapping the phospholipid-binding surface and translocation determinants of the C2 domain from cytosolic phospholipase A2; Perisic O et al.; Cytosolic phospholipase A2 (cPLA2) plays a key role in the generation of arachidonic acid, a precursor of potent inflammatory mediators . Intact cPLA2 is known to translocate in a calcium-dependent manner from the cytosol to the nuclear envelope and endoplasmic reticulum . We show here that the C2 domain of cPLA2 alone is sufficient for this calcium-dependent translocation in living cells . We have identified sets of exposed hydrophobic residues in loops known as calcium-binding region (CBR) 1 and CBR3, which surround the C2 domain calcium-binding sites, whose mutation dramatically decreased phospholipid binding in vitro without significantly affecting calcium binding . Mutation of a residue that binds calcium ions (D43N) also eliminated phospholipid binding . The same mutations that prevent phospholipid binding of the isolated C2 domain in vitro abolished the calcium-dependent translocation of cPLA2 to internal membranes in vivo, suggesting that the membrane targeting is driven largely by direct interactions with the phospholipid bilayer . Using fluorescence quenching by spin-labeled phospholipids for a series of mutants containing a single tryptophan residue at various positions in the cPLA2 C2 domain, we show that two of the calcium-binding loops, CBR1 and CBR3, penetrate in a calcium-dependent manner into the hydrophobic core of the phospholipid bilayer, establishing an anchor for docking the domain onto the membrane. J Biol Chem, 1999 May 21, 274(21), 14918 - 25 A human RNA viral cysteine proteinase that depends upon a unique Zn2+-binding finger connecting the two domains of a papain-like fold ; Herold J et al.; A cysteine proteinase, papain-like proteinase (PL1pro), of the human coronavirus 229E (HCoV) regulates the expression of the replicase polyproteins, pp1a and ppa1ab, by cleavage between Gly111 and Asn112, far upstream of its own catalytic residue Cys1054 . In this report, using bioinformatics tools, we predict that, unlike its distant cellular homologues, HCoV PL1pro and its coronaviral relatives have a poorly conserved Zn2+ finger connecting the left and right hand domains of a papain-like fold . Optical emission spectrometry has been used to confirm the presence of Zn2+ in a purified and proteolytically active form of the HCoV PL1pro fused with the Escherichia coli maltose-binding protein . In denaturation/renaturation experiments using the recombinant protein, its activity was shown to be strongly dependent upon Zn2+, which could be partly substituted by Co2+ during renaturation . The reconstituted, Zn2+-containing PL1pro was not sensitive to 1,10-phenanthroline, and the Zn2+-depleted protein was not reactivated by adding Zn2+ after renaturation . Consistent with the proposed essential structural role of Zn2+, PL1pro was selectively inactivated by mutations in the Zn2+ finger, including replacements of any of four conserved Cys residues predicted to co-ordinate Zn2+ . The unique domain organization of HCoV PL1pro provides a potential framework for regulatory processes and may be indicative of a nonproteolytic activity of this enzyme. J Biol Chem, 1999 May 21, 274(21), 14909 - 17 The RACK1 signaling scaffold protein selectively interacts with the cAMP-specific phosphodiesterase PDE4D5 isoform; Yarwood SJ et al.; The WD-repeat protein receptor for activated C-kinase (RACK1) was identified by its interaction with the cyclic AMP-specific phosphodiesterase (PDE4) isoform PDE4D5 in a yeast two-hybrid screen . The interaction was confirmed by co-immunoprecipitation of native RACK1 and PDE4D5 from COS7, HEK293, 3T3-F442A, and SK-N-SH cell lines . The interaction was unaffected by stimulation of the cells with the phorbol ester phorbol 2-myristate 3-acetate . PDE4D5 did not interact with two other WD-repeat proteins, beta'-coatomer protein and Gsbeta, in two-hybrid tests . RACK1 did not interact with other PDE4D isoforms or with known PDE4A, PDE4B, and PDE4C isoforms . PDE4D5 and RACK1 interacted with high affinity (Ka approximately 7 nM) {corrected} when they were expressed and purified from Escherichia coli, demonstrating that the interaction does not require intermediate proteins . The binding of the E . coli-expressed proteins did not alter the kinetics of cAMP hydrolysis by PDE4D5 but caused a 3-4-fold change in its sensitivity to inhibition by the PDE4 selective inhibitor rolipram . The subcellular distributions of RACK1 and PDE4D5 were extremely similar, with the major amount of both proteins (70%) in the high speed supernatant (S2) fraction . Analysis of constructs with specific deletions or single amino acid mutations in PDE4D5 demonstrated that a small cluster of amino acids in the unique amino-terminal region of PDE4D5 was necessary for its interaction with RACK1 . We suggest that RACK1 may act as a scaffold protein to recruit PDE4D5 and other proteins into a signaling complex. J Biol Chem, 1999 May 21, 274(21), 14779 - 85 Functional interactions of mitochondrial DNA polymerase and single-stranded DNA-binding protein . Template-primer DNA binding and initiation and elongation of DNA strand synthesis; Farr CL et al.; Functional interactions between mitochondrial DNA polymerase (pol gamma) and mitochondrial single-stranded DNA-binding protein (mtSSB) from Drosophila embryos have been evaluated with regard to the overall activity of pol gamma and in partial reactions involving template-primer binding and initiation and idling in DNA strand synthesis . Both the 5' --> 3' DNA polymerase and 3' --> 5' exonuclease in pol gamma are stimulated 15-20-fold on oligonucleotide-primed single-stranded DNA by native and recombinant forms of mtSSB . That the extent of stimulation is similar for both enzyme activities over a broad range of KCl concentrations suggests their functional coordination and a similar mechanism of stimulation by mtSSB . At the same time, the high mispair specificity of pol gamma in exonucleolytic hydrolysis is maintained, indicating that enhancement of pol gamma catalytic efficiency is likely not accompanied by increased nucleotide turnover . DNase I footprinting of pol gamma.DNA complexes and initial rate measurements show that mtSSB enhances primer recognition and binding and stimulates 30-fold the rate of initiation of DNA strands . Dissociation studies show that productive complexes of the native pol gamma heterodimer with template-primer DNA are formed and remain stable in the absence of replication accessory proteins. J Biol Chem, 1999 May 21, 274(21), 14768 - 72 A nifS-like gene, csdB, encodes an Escherichia coli counterpart of mammalian selenocysteine lyase . Gene cloning, purification, characterization and preliminary x-ray crystallographic studies; Mihara H et al.; Selenocysteine lyase is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the exclusive decomposition of L-selenocysteine to L-alanine and elemental selenium . An open reading frame, named csdB, from Escherichia coli encodes a putative protein that is similar to selenocysteine lyase of pig liver and cysteine desulfurase (NifS) of Azotobacter vinelandii . In this study, the csdB gene was cloned and expressed in E . coli cells . The gene product was a homodimer with the subunit Mr of 44,439, contained 1 mol of PLP as a cofactor per mol of subunit, and catalyzed the release of Se, SO2, and S from L-selenocysteine, L-cysteine sulfinic acid, and L-cysteine, respectively, to yield L-alanine; the reactivity of the substrates decreased in this order . Although the enzyme was not specific for L-selenocysteine, the high specific activity for L-selenocysteine (5.5 units/mg compared with 0.019 units/mg for L-cysteine) supports the view that the enzyme can be regarded as an E . coli counterpart of mammalian selenocysteine lyase . We crystallized CsdB, the csdB gene product, by the hanging drop vapor diffusion method . The crystals were of suitable quality for x-ray crystallography and belonged to the tetragonal space group P43212 with unit cell dimensions of a = b = 128.1 A and c = 137.0 A . Consideration of the Matthews parameter Vm (3.19 A3/Da) accounts for the presence of a single dimer in the crystallographic asymmetric unit . A native diffraction dataset up to 2.8 A resolution was collected . This is the first crystallographic analysis of a protein of NifS/selenocysteine lyase family. J Biol Chem, 1999 May 21, 274(21), 14573 - 8 An arginine residue is essential for stretching and binding of the substrate on UDP-glucose-4-epimerase from Escherichia coli . Use of a stacked and quenched uridine nucleotide fluorophore as probe; Bhattacharyya U et al.; In the previous paper we demonstrated that uridine-5'-beta-1-(5-sulfonic acid) naphthylamidate (UDPAmNS) is a stacked and quenched fluorophore that shows severalfold enhancement of fluorescence in a stretched conformation . UDPAmNS was found to be a powerful competitive inhibitor (Ki = 0.2 mM) for UDP-glucose-4-epimerase from Escherichia coli . This active site-directed fluorophore assumed a stretched conformation on the enzyme surface, as was evidenced by full enhancement of fluorescence in saturating enzyme concentration . Complete displacement of the fluorophore by UDP suggested it to bind to the substrate binding site of the active site . Analysis of inactivation kinetics in presence of alpha,beta-diones such as phenylglyoxal, cyclohaxanedione, and 2,3-butadione suggested involvement of the essential arginine residue in the overall catalytic process . From spectral analysis, loss of activity could also be directly correlated with modification of only one arginine residue . Protection experiments with UDP showed the arginine residue to be located in the uridyl phosphate binding subsite . Unlike the native enzyme, the modified enzyme failed to show any enhancement of fluorescence with UDPAmNS clearly demonstrating the role of the essential arginine residue in stretching and binding of the substrate . The potential usefulness of such stacked and quenched nucleotide fluorophores has been discussed. J Biol Chem, 1999 May 21, 274(21), 14537 - 40 Zinc content of Escherichia coli-expressed constitutive isoforms of nitric-oxide synthase . Enzymatic activity and effect of pterin; Miller RT et al.; Recently, we obtained x-ray crystallographic data showing the presence of a ZnS4 center in the structure of Escherichia coli-expressed bovine endothelial nitric-oxide synthase (eNOS) and rat neuronal nitric-oxide synthase (nNOS) . The zinc atom is coordinated by two CXXXXC motifs, one motif being contributed by each NOS monomer (cysteine 326 through cysteine 331 in rat nNOS) . Mutation of the nNOS cysteine 331 to alanine (C331A) results in the loss of NO . synthetic activity and also results in an inability to bind zinc efficiently . Although prolonged incubation of the C331A mutant of nNOS with high concentrations of L-arginine results in a catalytically active enzyme, zinc binding is not restored . In this study, we investigate the zinc stoichiometry in wild-type nNOS and eNOS, as well as in the C331A-mutated nNOS, using a chelation assay and electrothermal vaporization-inductively coupled plasma-mass spectrometry . The data reveal an approximate 2:1 stoichiometry of heme to zinc in (6R)-5,6,7,8-tetrahydro-L-biopterin-replete, wild-type nNOS and eNOS and show that the reactivated C331A mutant of nNOS has a limited ability to bind zinc . The present study substantiates that the zinc in NOS is structural rather than catalytic and is important for maintaining optimally functional, enzymatically active, constitutive NOSs. EMBO J, 1999 May 17, 18(10), 2878 - 85 RNase G (CafA protein) and RNase E are both required for the 5' maturation of 16S ribosomal RNA; Li Z et al.; In Escherichia coli, rRNA operons are transcribed as 30S precursor molecules that must be extensively processed to generate mature 16S, 23S and 5S rRNA . While it is known that RNase III cleaves the primary transcript to separate the individual rRNAs, there is little information about the secondary processing reactions needed to form their mature 3' and 5' termini . We have now found that inactivation of the endoribonuclease RNase E slows down in vivo maturation of 16S RNA from the 17S RNase III cleavage product . Moreover, in the absence of CafA protein, a homolog of RNase E, formation of 16S RNA also slows down, but in this case a 16.3S intermediate accumulates . When both RNase E and CafA are inactivated, 5' maturation of 16S rRNA is completely blocked . In contrast, 3' maturation is essentially unaffected . The 5' unprocessed precursor that accumulates in the double mutant can be assembled into 30S and 70S ribosomes . Precursors also can be processed in vitro by RNase E and CafA . These data indicate that both RNase E and CafA protein are required for a two step, sequential maturation of the 5' end of 16S rRNA, and that CafA protein is a new ribonuclease . We propose that it be renamed RNase G. EMBO J, 1999 May 17, 18(10), 2673 - 82 Structure and selectivity in post-translational modification: attaching the biotinyl-lysine and lipoyl-lysine swinging arms in multifunctional enzymes; Reche P et al.; The post-translational attachment of biotin and lipoic acid to specific lysine residues displayed in protruding beta-turns in homologous biotinyl and lipoyl domains of their parent enzymes is catalysed by two different ligases . We have expressed in Escherichia coli a sub-gene encoding the biotinyl domain of E.coli acetyl-CoA carboxylase, and by a series of mutations converted the protein from the target for biotinylation to one for lipoylation, in vivo and in vitro . The biotinylating enzyme, biotinyl protein ligase (BPL), and the lipoylating enzyme, LplA, exhibited major differences in the recognition process . LplA accepted the highly conserved MKM motif that houses the target lysine residue in the biotinyl domain beta-turn, but was responsive to structural cues in the flanking beta-strands . BPL was much less sensitive to changes in these beta-strands, but could not biotinylate a lysine residue placed in the DKA motif characteristic of the lipoyl domain beta-turn . The presence of a further protruding thumb between the beta2 and beta3 strands in the wild-type biotinyl domain, which has no counterpart in the lipoyl domain, is sufficient to prevent aberrant lipoylation in E.coli . The structural basis of this discrimination contrasts with other forms of post-translational modification, where the sequence motif surrounding the target residue can be the principal determinant. Am J Pathol, 1999 May, 154(5), 1489 - 501 Cysteine-rich domain of human ADAM 12 (meltrin alpha) supports tumor cell adhesion; Iba K et al.; The ADAMs (A disintegrin and metalloprotease) comprise a family of membrane-anchored cell surface proteins with a putative role in cell-cell and/or cell-matrix interactions . By immunostaining, ADAM 12 (meltrin alpha) was up-regulated in several human carcinomas and could be detected along the tumor cell membranes . Because of this intriguing staining pattern, we investigated whether human ADAM 12 supports tumor cell adhesion . Using an in vitro assay using recombinant polypeptides expressed in Escherichia coli, we examined the ability of individual domains of human ADAM 12 and ADAM 15 to support tumor cell adhesion . We found that the disintegrin-like domain of human ADAM 15 supported adhesion of alphavbeta3-expressing A375 melanoma cells . In the case of human ADAM 12, however, recombinant polypeptides of the cysteine-rich domain but not the disintegrin-like domain supported cell adhesion of a panel of carcinoma cell lines . On attachment to recombinant polypeptides from the cysteine-rich domain of human ADAM 12, most tumor cell lines, such as MDA-MB-231 breast carcinoma cells, were rounded and associated with numerous actin-containing filopodia and used a cell surface heparan sulfate proteoglycan to attach . Finally, we demonstrated that authentic full-length human ADAM 12 could bind to heparin Sepharose . Together these results suggest a novel role of the cysteine-rich domain of ADAM 12 -- that of supporting tumor cell adhesion. Virology, 1999 May 10, 257(2), 511 - 23 Vaccinia virus DNA polymerase promotes DNA pairing and strand-transfer reactions; Willer DO et al.; Vaccinia virus infection results in the synthesis of a protein that promotes joint molecule formation and strand-transfer reactions in vitro . We show here that this activity is also expressed by vaccinia DNA polymerase (gpE9L) . Recombinant vaccinia polymerase was produced using a hybrid vaccinia/T7 expression system and purified to homogeneity . This protein catalyzed joint molecule formation and strand transfer in vitro in reactions containing single-stranded circular and linear duplex DNAs . The reaction required homologous substrates and magnesium ions and was stimulated by DNA aggregating agents such as spermidine HCl and Escherichia coli single-strand DNA binding protein . There was no requirement for a nucleoside triphosphate cofactor . The reaction ceased when approximately 20% of the double-stranded substrate had been incorporated into joint molecules and required stoichiometric quantities of DNA polymerase (0.5-1 molecules of polymerase per double-stranded DNA end) . Electron microscopy showed that the joint molecules formed during these reactions contained displaced strands and thus represented the products of a strand-exchange reaction . We also reexamined the link between replication and recombination using a luciferase-based transfection assay and cells infected with DNA polymerase Cts42 mutant viruses . These data substantiate the claim that there exists an inextricable link between replication and recombination in poxvirus-infected cells . Together, these biochemical and genetic data suggest a way of linking poxviral DNA replication with genetic recombination . Virology, 1999 May 10, 257(2), 449 - 59 Antigenic profile of African horse sickness virus serotype 4 VP5 and identification of a neutralizing epitope shared with bluetongue virus and epizootic hemorrhagic disease virus; Martinez-Torrecuadrada JL et al.; African horse sickness virus (AHSV) causes a fatal disease in horses . The virus capsid is composed of a double protein layer, the outermost of which is formed by two proteins: VP2 and VP5 . VP2 is known to determine the serotype of the virus and to contain the neutralizing epitopes . The biological function of VP5, the other component of the capsid, is unknown . In this report, AHSV VP5, expressed in insect cells alone or together with VP2, was able to induce AHSV-specific neutralizing antibodies . Moreover, two VP5-specific monoclonal antibodies (MAbs) that were able to neutralize the virus in a plaque reduction assay were generated . To dissect the antigenic structure of AHSV VP5, the protein was cloned in Escherichia coli using the pET3 system . The immunoreactivity of both MAbs, and horse and rabbit polyclonal antisera, with 17 overlapping fragments from VP5 was analyzed . The most immunodominant region was found in the N-terminal 330 residues of VP5, defining two antigenic regions, I (residues 151-200) and II (residues 83-120) . The epitopes were further defined by PEPSCAN analysis with 12mer peptides, which determined eight antigenic sites in the N-terminal half of the molecule . Neutralizing epitopes were defined at positions 85-92 (PDPLSPGE) for MAb 10AE12 and at 179-185 (EEDLRTR) for MAb 10AC6 . Epitope 10AE12 is highly conserved between the different orbiviruses . MAb 10AE12 was able to recognize bluetongue virus VP5 and epizootic hemorrhagic disease virus VP5 by several techniques . These data will be especially useful for vaccine development and diagnostic purposes . J Struct Biol, 1999 Jun 1, 126(1), 80 - 3 Crystallographic characterization of a stress-induced multifunctional protein, rat HBP-23; Hirotsu S et al.; HBP-23 is a stress-induced multifunctional rat protein that belongs to a novel family of antioxidant proteins, referred to as peroxiredoxins, and exhibits heme-binding and inhibition of c-Abl protein tyrosine kinase . Recombinant HBP-23 was crystallized by a hanging-drop vapor-diffusion method . The crystals belong to space group P41212 or P43212 with unit-cell dimensions of a = b = 73.47 A, c = 210.37 A and contain two protein molecules in the asymmetric unit . A data set at 2.7-A resolution was collected with a cryo-crystallographic technique . Crystals of selenomethionyl HBP-23 were also obtained under the same conditions . J Struct Biol, 1999 Jun 1, 126(1), 76 - 9 Crystallization of recombinant Crithidia fasciculata tryparedoxin; Alphey MS et al.; Recombinant tryparedoxin, a thioredoxin homologue from Crithidia fasciculata, has been purified from an Escherichia coli expression system and used in crystallization trials . Orthorhombic needles in space group P212121, with unit cell dimensions of a = 38.63, b = 51 . 47, and c = 73.41 A, have been obtained . The crystals present a monomer of approximate molecular mass 16 kDa in the asymmetric unit and diffract to 1.8-A resolution using synchrotron radiation . Structure determination will be carried out to further the understanding of the role tryparedoxin plays in regulating oxidative stress in parasitic trypanosomatids . Biochem Biophys Res Commun, 1999 May 19, 258(3), 808 - 11 Manganese is essential for catalytic activity of Escherichia coli agmatinase; Carvajal N et al.; Purified Escherichia coli agmatinase (EC 3.5.3.11) expressed the same activity in the absence or presence of added Mn2+ (0-5mM) . However, it was strongly inhibited by Co2+, Ni2+, and Zn2+ and almost half inactivated by EDTA . Partial inactivation by EDTA yielded enzyme species containing 0.85 +/- 0.1 Mn2+/subunit, and it was accompanied by a decrease in intensity of fluorescence emission and a red shift from the emission maximum of 340 nm to 346 nm, indicating the movement of tryptophane residues to a more polar environment . The activity and fluorescence properties of fully activated agmatinase were restored by incubation of dialysed species with Mn2+ . Manganese-free species, obtained by treatment with EDTA and guanidinium chloride (3 M), were active only in the presence of added Mn2+ . Results obtained, which represent the first demonstration of the essentiality of Mn2+ for agmatinase activity, are discussed in connection with a possible binuclear metal center in the enzyme . Biochem Biophys Res Commun, 1999 May 19, 258(3), 768 - 71 Cloning and expression of a new isotype of the peroxiredoxin gene of Chinese cabbage and its comparison to 2Cys-peroxiredoxin isolated from the same plant; Choi YO et al.; A cDNA encoding a newly identified isotype of peroxiredoxin (Prx) was isolated from a Chinese cabbage flower bud cDNA library and designated CPrxII . Database searches using the predicted CPrxII amino acid sequence revealed no substantial homology to other proteins with the exception of the yeast type II Prx with which CPrxII shares 27.8% sequence identity . Recombinant CPrxII expressed in Escherichia coli was able to protect glutamine synthetase from inactivation in a metal-catalyzed oxidation system and to reduce H2O2 with electrons provided by thioredoxin . This specific antioxidant activity of CPrxII was about 6-fold higher than that of 2Cys-Prx of the same plant . In contrast to 2Cys-Prx, which is predominantly expressed in leaf tissue of cabbage seedlings, CPrxII is highly expressed in root tissue as revealed by Northern and Western blot analyses . The CPrxII gene exists as a small multigene family in the cabbage genome . Biochem Biophys Res Commun, 1999 May 19, 258(3), 605 - 10 Human MMH (OGG1) type 1a protein is a major enzyme for repair of 8-hydroxyguanine lesions in human cells; Monden Y et al.; 8-Hydroxyguanine (8-OH-G) is the site of a frequent mutagenic lesion of DNA, produced by oxidative damage . MutM of E . coli and OGG1 of Saccharomyces cervisiae are known to possess 8-OH-G glycosylase activity and apurinic (AP) site lyase activity to repair 8-OH-G lesions . Recently, cDNA clones of human OGG1 homologues (hMMH) of four isoforms (type 1a, type 1b, type 1c, and type 2) were isolated . However, it is unknown whether expression of endogenous hMMH proteins actually occurs in mammalian cells . Here using hMMH type 1a-specific antibody and cells overexpressing tag-fused hMMH type 1a, we show the expression of hMMH type 1a protein in many types of human cells and show that endogenous hMMH type 1a protein has 8-OH-G glycosylase/AP lyase activity . Furthermore, we show that upon depletion of hMMH type 1a protein in a whole cell extract by its antibody, most of the AP lyase activity is lost, indicating that hMMH type 1a protein is a major enzyme for repair of 8-OH-G lesions in human cells . Biochem Biophys Res Commun, 1999 May 10, 258(2), 416 - 24 Analysis of DNA compaction profile and intracellular contents of archaeal histones from Pyrococcus kodakaraensis KOD1; Higashibata H et al.; Two histone genes, hpkA and hpkB, from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 strain were cloned, sequenced, and expressed in Escherichia coli cells . Both hpkA and hpkB genes encoded a protein of 67 amino acids, however they possessed the different molecular weight (HpkA, 7,378:HpkB, 7,167) . Deduced amino acid sequences of HpkA and HpkB were homologous to other archaeal histones and eucaryal core histones (H2A, H4) . Gel mobility shift assays by purified proteins demonstrated that HpkB possessed higher affinity to DNA and more extensive ability to compact DNA than HpkA . HpkB prevented double stranded DNA from thermal denaturation in less amount than HpkA in vitro . In order to investigate intracellular contents of HpkA and HpkB in KOD1 cells, immunoblot analysis was performed by using anti-HpkA antisera obtained from immunized BALB/c mice, showing that HpkA was less abundantly expressed than HpkB in KOD1 cells . These results suggest that HpkB plays a major role to protect double stranded DNA from thermal denaturation in vivo . Biochem Biophys Res Commun, 1999 May 10, 258(2), 341 - 4 Heat shock protein stimulates in vitro transcription of nitrogen regulator gene glnL of Escherichia coli; Takahashi T; The in vitro transcription of glnL, initiated at the gene's specific promoter, was stimulated by adding a proteinaceous fraction purified from a culture at 42 degrees C, while the corresponding fraction from a culture at 28 degrees C was not stimulating . The stimulating fraction was not effective on in vitro transcription of some other genes . These stimulating and nonstimulating fractions were further purified and comparatively analyzed in sodium dodecyl sulfate polyacrylamide gel electrophoresis, which revealed that the amounts of 84 kD protein had predominantly increased in the stimulating fraction, but not in the nonstimulating fraction . Exp Parasitol, 1999 May, 92(1), 12 - 8 Plasmodium falciparum: variations in the C-terminal cysteine-rich region of the merozoite surface protein-1 in field samples among Indian isolates; Lalitha PV et al.; The cysteine-rich C-terminal region of the merozoite surface protein-1, MSP-119, of Plasmodium falciparum has been the most promising vaccine target antigen to date, based on protective immunization studies with recombinant proteins in mice and monkey models . To be further developed as a vaccine candidate, it is essential to study its sequence heterogeneity in field isolates from diverse geographical areas . We have analyzed the DNA sequences encoding the C-terminal region of P . falciparum MSP-1 (1526-1744 aa, corresponding to part of the 16th and all of the 17th blocks) of 16 isolates from different regions in India . The PNG-MAD20 type of MSP-1 sequence predominated in this subcontinent . The MSP-119 region as usual was found to be highly conserved, with amino acid variations at four positions . Based on these variations, only three MSP-119 forms (Q-KNG, E-KNG, and E-TSG, a novel variant) were detected among these isolates