J Virol, 1999 Jul, 73(7), 5411 - 21 Identification of the RNA-binding, dimerization, and eIF4GI-binding domains of rotavirus nonstructural protein NSP3; Piron M et al.; The rotavirus nonstructural protein NSP3 is a sequence-specific RNA binding protein that binds the nonpolyadenylated 3' end of the rotavirus mRNAs . NSP3 also interacts with the translation initiation factor eIF4GI and competes with the poly(A) binding protein . Deletion mutations and point mutations of NSP3 from group A rotavirus (NSP3A), expressed in Escherichia coli, indicate that the RNA binding domain lies between amino acids 4 and 149 . Similar results were obtained with NSP3 from group C rotaviruses . Data also indicate that a dimer of NSP3A binds one molecule of RNA and that dimerization is necessary for strong RNA binding . The dimerization domain of NSP3 was mapped between amino acids 150 and 206 by using the yeast two-hybrid system . The eukaryotic initiation factor 4 GI subunit (eIF-4GI) binding domain of NSP3A has been mapped in the last 107 amino acids of its C terminus by using a pulldown assay and the yeast two-hybrid system . NSP3 is composed of two functional domains separated by a dimerization domain. J Virol, 1999 Jul, 73(7), 5309 - 19 In vitro assembly of alphavirus cores by using nucleocapsid protein expressed in Escherichia coli; Tellinghuisen TL et al.; The production of the alphavirus virion is a multistep event requiring the assembly of the nucleocapsid core in the cytoplasm and the maturation of the glycoproteins in the endoplasmic reticulum and the Golgi apparatus . These components associate during the budding process to produce the mature virion . The nucleocapsid proteins of Sindbis virus and Ross River virus have been produced in a T7-based Escherichia coli expression system and purified . In the presence of single-stranded but not double-stranded nucleic acid, the proteins oligomerize in vitro into core-like particles which resemble the native viral nucleocapsid cores . Despite their similarities, Sindbis virus and Ross River virus capsid proteins do not form mixed core-like particles . Truncated forms of the Sindbis capsid protein were used to establish amino acid requirements for assembly . A capsid protein starting at residue 19 {CP(19-264)} was fully competent for in vitro assembly, whereas proteins with further N-terminal truncations could not support assembly . However, a capsid protein starting at residue 32 or 81 was able to incorporate into particles in the presence of CP(19-264) or could inhibit assembly if its molar ratio relative to CP(19-264) was greater than 1:1 . This system provides a basis for the molecular dissection of alphavirus core assembly. J Biol Chem, 1999 Jun 18, 274(25), 18040 - 8 Identification of a general transcription factor TFIIAalpha/beta homolog selectively expressed in testis; Upadhyaya AB et al.; In this paper we describe the isolation of a cDNA that encodes a human TFIIAalpha/beta-like factor (ALF) . The open reading frame of ALF predicts a protein of 478 amino acids that contains characteristic N- and C-terminal conserved domains separated by an internal nonconserved domain . In addition, a rare ALF-containing cDNA, which possesses an extended N terminus (Stoned B/TFIIAalpha/beta-like factor; SALF) has also been identified . The results of Northern and dot blot analyses show that ALF is expressed almost exclusively in testis; in contrast, TFIIAalpha/beta and TFIIAgamma are enriched in testis but are also widely expressed in other human tissues . Recombinant ALF (69 kDa) and TFIIAgamma (12 kDa) polypeptides produced in Escherichia coli form an ALF/gamma complex that can stabilize TBP-TATA interactions in an electrophoretic mobility shift assay . The ALF/gamma complex is also able to restore transcription from the adenovirus major late promoter using HeLa cell nuclear extracts that have been depleted of TFIIA . Overall, the data show that ALF is a functional homolog of human general transcription factor TFIIAalpha/beta that may be uniquely important to testis biology. J Biol Chem, 1999 Jun 18, 274(25), 17924 - 33 The plasmid ColIb-P9 antisense Inc RNA controls expression of the RepZ replication protein and its positive regulator repY with different mechanisms; Asano K et al.; The autonomous replication region of plasmid ColIb-P9 contains repZ encoding the RepZ replication protein, and inc and repY as the negative and positive regulators of repZ translation, respectively . inc encodes the antisense Inc RNA, and repY is a short open reading frame upstream of repZ . Translation of repY enables repZ translation by inducing formation of a pseudoknot containing stem-loop I, which base pairs with the sequence preceding the repZ start codon . Inc RNA inhibits both repY translation and formation of the pseudoknot by binding to the loop I . To investigate control of repY expression by Inc RNA, we isolated a number of mutations that express repY in the presence of Inc RNA . One class of mutations delete a part of another stem-loop (II), which derepresses repY expression by initiating translation at codon 10 (GUG), located within this structure . Point mutations in stem-loop II can also derepress repY translation, and the introduction of compensatory base-changes restores control of repY translation . These results not only indicate that suppressing a cryptic start codon by secondary structure is important for maintaining the translational control of repZ but also demonstrate that the position of start site for repY translation is critical for its control by Inc RNA . Thus, Inc RNA controls repY translation by binding in the vicinity of the start codon, in contrast to the control of repZ expression at the level of loop-loop interaction. J Biol Chem, 1999 Jun 18, 274(25), 17918 - 23 A critical DnaA box directs the cooperative binding of the Escherichia coli DnaA protein to the plasmid RK2 replication origin; Doran KS et al.; The requirement of DnaA protein binding for plasmid RK2 replication initiation the Escherichia coli was investigated by constructing mutations in the plasmid replication origin that scrambled or deleted each of the four upstream DnaA boxes . Altered origins were analyzed for replication activity in vivo and in vitro and for binding to the E . coli DnaA protein using a gel mobility shift assay and DNase I footprinting . Most strikingly, a mutation in one of the boxes, box 4, abolished replication activity and eliminated stable DnaA protein binding to all four boxes . Unlike DnaA binding to the E . coli origin, oriC, DnaA binding to two of the boxes (boxes 4 and 3) in the RK2 origin, oriV, is cooperative with box 4 acting as the "organizer" for the formation of the DnaA-oriV nucleoprotein complex . Interestingly, the inversion of box 4 also abolished replication activity, but did not result in a loss of binding to the other boxes . However, DnaA binding to this mutant origin was no longer cooperative . These results demonstrate that the sequence, position, and orientation of box 4 are crucial for cooperative DnaA binding and the formation of a nucleoprotein structure that is functional for the initiation of replication. J Biol Chem, 1999 Jun 18, 274(25), 17771 - 6 Structure and function of human prepro-orexin gene; Sakurai T et al.; Orexin-A and -B are recently identified potent orexigenic peptides that are derived from the same precursor peptide and are highly specifically localized in neurons located in the lateral hypothalamic area, a region classically implicated in feeding behavior . We cloned the whole length of the human prepro-orexin gene and corresponding cDNA . The human prepro-orexin mRNA was predicted to encode a 131-residue precursor peptide (prepro-orexin) . The human prepro-orexin gene consists of two exons and one intron distributed over 1432 base pairs . The 143-base pair first exon includes the 5'-untranslated region and a small part of the coding region that encodes the first seven residues of the secretory signal sequence . The second exon contains the remaining portion of the open reading frame and 3'-untranslated region . The 3.2 kilobase pairs of the 5'-upstream region from a cloned human prepro-orexin gene promoter is sufficient to direct the expression of the Escherichia coli beta-galactosidase (lacZ) gene in transgenic mice to neurons in the lateral hypothalamic area and adjacent regions . The lacZ-positive neurons were positively stained with anti-orexin antibody but not with anti-melanin-concentrating hormone antibody . These findings suggest that this genomic fragment contains all the necessary elements for appropriate expression of the gene and will be useful for the targeted expression of the exogenous gene in orexin-containing neurons . These mice might also be useful for examining the molecular mechanisms by which orexin gene expression is regulated. J Biol Chem, 1999 Jun 18, 274(25), 17696 - 704 The iron-oxygen reconstitution reaction in protein R2-Tyr-177 mutants of mouse ribonucleotide reductase . Epr and electron nuclear double resonance studies on a new transient tryptophan radical; Potsch S et al.; The ferrous iron/oxygen reconstitution reaction in protein R2 of mouse and Escherichia coli ribonucleotide reductase (RNR) leads to the formation of a stable protein-linked tyrosyl radical and a mu-oxo-bridged diferric iron center, both necessary for enzyme activity . We have studied the reconstitution reaction in three protein R2 mutants Y177W, Y177F, and Y177C of mouse RNR to investigate if other residues at the site of the radical forming Tyr-177 can harbor free radicals . In Y177W we observed for the first time the formation of a tryptophan radical in protein R2 of mouse RNR with a lifetime of several minutes at room temperature . We assign it to an oxidized neutral tryptophan radical on Trp-177, based on selective deuteration and EPR and electron nuclear double resonance spectroscopy in H2O and D2O solution . The reconstitution reaction at 22 degrees C in both Y177F and Y177C leads to the formation of a so-called intermediate X which has previously been assigned to an oxo (hydroxo)-bridged Fe(III)/Fe(IV) cluster . Surprisingly, in both mutants that do not have successor radicals as Trp . in Y177W, this cluster exists on a much longer time scale (several seconds) at room temperature than has been reported for X in E . coli Y122F or native mouse protein R2 . All three mouse R2 mutants were enzymatically inactive, indicating that only a tyrosyl radical at position 177 has the capability to take part in the reduction of substrates. J Biol Chem, 1999 Jun 18, 274(25), 17559 - 66 Characterization of mouse nNOS2, a natural variant of neuronal nitric-oxide synthase produced in the central nervous system by selective alternative splicing; Iwasaki T et al.; Mouse neuronal nitric-oxide synthase 2 (nNOS2) is a unique natural variant of constitutive neuronal nitric-oxide synthase (nNOS) specifically expressed in the central nervous system having a 105-amino acid deletion in the heme-binding domain as a result of in-frame mutation by specific alternative splicing . The mouse nNOS2 cDNA gene was heterologously expressed in Escherichia coli, and the resultant product was characterized spectroscopically in detail . Purified recombinant nNOS2 contained heme but showed no L-arginine- and NADPH-dependent citrulline-forming activity in the presence of Ca2+-promoted calmodulin, elicited a sharp electron paramagnetic resonance (EPR) signal at g = 6.0 indicating the presence of a high spin ferriheme as isolated and showed a peak at around 420 nm in the CO difference spectrum, instead of a 443-nm peak detected with the recombinant wild-type nNOS1 enzyme . Thus, although the heme domain of nNOS2 is capable of binding heme, the heme coordination geometry is highly abnormal in that it probably has a proximal non-cysteine thiolate ligand both in the ferric and ferrous states . Moreover, negligible spectral perturbation of the nNOS2 ferriheme was detected upon addition of either L-arginine or imidazole . These provide a possible rational explanation for the inability of nNOS2 to catalyze the cytochrome P450-type monooxygenase reaction. J Biol Chem, 1999 Jun 18, 274(25), 17505 - 10 Effectors of the stringent response target the active site of Escherichia coli adenylosuccinate synthetase; Hou Z et al.; Guanosine 5'-diphosphate 3'-diphosphate (ppGpp), a pleiotropic effector of the stringent response, potently inhibits adenylosuccinate synthetase from Escherichia coli as an allosteric effector and/or as a competitive inhibitor with respect to GTP . Crystals of the synthetase grown in the presence of IMP, hadacidin, NO3-, and Mg2+, then soaked with ppGpp, reveal electron density at the GTP pocket which is consistent with guanosine 5'-diphosphate 2':3'-cyclic monophosphate . Unlike ligand complexes of the synthetase involving IMP and GDP, the coordination of Mg2+ in this complex is octahedral with the side chain of Asp13 in the inner sphere of the cation . The cyclic phosphoryl group interacts directly with the side chain of Lys49 and indirectly through bridging water molecules with the side chains of Asn295 and Arg305 . The synthetase either directly facilitates the formation of the cyclic nucleotide or scavenges trace amounts of the cyclic nucleotide from solution . Regardless of its mode of generation, the cyclic nucleotide binds far more tightly to the active site than does ppGpp . Conceivably, synthetase activity in vivo during the stringent response may be sensitive to the relative concentrations of several effectors, which together exercise precise control over the de novo synthesis of AMP. J Biol Chem, 1999 Jun 18, 274(25), 17471 - 7 Identification of the folate binding sites on the Escherichia coli T-protein of the glycine cleavage system; Okamura-Ikeda K et al.; T-protein is a component of the glycine cleavage system and catalyzes the tetrahydrofolate-dependent reaction . To determine the folate-binding site on the enzyme, 14C-labeled methylenetetrahydropteroyltetraglutamate (5,10-CH2-H4PteGlu4) was enzymatically synthesized from methylenetetrahydrofolate (5, 10-CH2-H4folate) and {U-14C}glutamic acid and subjected to cross-linking with the recombinant Escherichia coli T-protein using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, a zero-length cross-linker between amino and carboxyl groups . The cross-linked product was digested with lysylendopeptidase, and the resulting peptides were separated by reversed-phase high performance liquid chromatography . Amino acid sequencing of the labeled peptides revealed that three lysine residues at positions 78, 81, and 352 were involved in the cross-linking with polyglutamate moiety of 5, 10-CH2-H4PteGlu4 . The comparable experiment with 5,10-CH2-H4folate revealed that Lys-81 and Lys-352 were also involved in cross-linking with the monoglutamate form . Mutants with single or multiple replacement(s) of these lysine residues to glutamic acid were constructed by site-directed mutagenesis and subjected to kinetic analysis . The single mutation of Lys-352 caused similar increase (2-fold) in Km values for both folate substrates, but that of Lys-81 affected greatly the Km value for 5,10-CH2-H4PteGlu4 rather than for 5,10-CH2-H4folate . It is postulated that Lys-352 may serve as the primary binding site to alpha-carboxyl group of the first glutamate residue nearest the p-aminobenzoic acid ring of 5,10-CH2-H4folate and 5,10-CH2-H4PteGlu4, whereas Lys-81 may play a key role to hold the second glutamate residue through binding to alpha-carboxyl group of the second glutamate residue. Arterioscler Thromb Vasc Biol, 1999 Jun, 19(6), 1447 - 55 A novel mutant, ApoA-I nichinan (Glu235-->0), is associated with low HDL cholesterol levels and decreased cholesterol efflux from cells; Han H et al.; A novel variant of apolipoprotein (apo) A-I associated with low high density lipoprotein (HDL) cholesterolemia has been identified in a Japanese family during screening for apoA-I variants by isoelectric focusing (IEF) gel analysis . ApoA-I (Glu235-->0) Nichinan was caused by a 3-bp deletion of nucleotides 1998 through 2000 in exon 4 of the apoA-I gene . Four subjects in the family were heterozygous carriers for this mutation; the mean plasma concentrations of apoA-I and HDL cholesterol of affected family members were 30% and 32% lower, respectively, than those of unaffected family members . There were no differences in the levels of very low density lipoprotein and low density lipoprotein cholesterol, triglycerides, and other apolipoproteins between the carriers and the noncarrier family members . In the proband, plasma lecithin:cholesterol acyltransferase activity was normal . Functional consequences of the mutation were examined by expressing the mutated and wild-type proapoA-I cDNAs in Escherichia coli . Cholesterol efflux to recombinant proapoA-I Nichinan from mouse peritoneal macrophages loaded with {3H}cholesterol-labeled acetylated low density lipoprotein was decreased by 54% when compared that of normal recombinant proapoA-I . In vivo turnover studies in normal rabbits demonstrated that the recombinant proapoA-I Nichinan was rapidly cleared (22% faster) compared with normal recombinant proapoA-I . We conclude that apoA-I (Glu235-->0) Nichinan induced a critical structural change in the carboxyl-terminal domain of apoA-I for cellular cholesterol efflux and increased the catabolism of apoA-I, resulting in low HDL cholesterol levels. Cancer Res, 1999 Jun 1, 59(11), 2570 - 6 Identification and characterization of human MT5-MMP, a new membrane-bound activator of progelatinase a overexpressed in brain tumors; Llano E et al.; A cDNA encoding a new member of the membrane-type (MT) matrix metalloproteinase (MMP) family has been identified and cloned from a human brain cDNA library . The isolated cDNA encodes a polypeptide of 645 amino acids that displays a similar domain organization as other MMPs, including a predomain with the activation locus, a zinc-binding site, and a hemopexin domain . The deduced amino acid sequence contains a COOH-terminal extension, rich in hydrophobic residues and similar in size to the equivalent domains identified in MT-MMPs . Immunofluorescence and Western blot analysis of COS-7 cells transfected with the isolated cDNA revealed that the encoded protein is localized in the plasma membrane . On the basis of these features, this novel human MMP has been called MT5-MMP because it represents the fifth member of the MT-MMP subfamily of MMPs . Fluorescent in situ hybridization experiments showed that the human MT5-MMP gene (MMP-24) maps to 20q11.2, a region frequently amplified in tumors from diverse sources . Northern blot analysis demonstrated that MT5-MMP is predominantly expressed in brain, kidney, pancreas, and lung . In addition, MT5-MMP transcripts were detected at high levels compared to normal brain tissue in a series of brain tumors, including astrocytomas and glioblastomas . The catalytic domain of MT5-MMP, produced in Escherichia coli as a fusion protein with glutathione S-transferase, exhibits a potent proteolytic activity against progelatinase A, leading to the generation of the Mr 62,000 active form of this enzyme . These data suggest that MT5-MMP may contribute to the activation of progelatinase A in tumor tissues, in which it is overexpressed, thereby facilitating tumor progression. Hum Immunol, 1999 Apr, 60(4), 282 - 90 The antigen binding domain of non-idiotypic human anti-F(ab')2 autoantibodies: study of their interaction with IgG hinge region epitopes; Welschof M et al.; In previous studies we described a natural human IgG-anti-F(ab')2 autoantibody family with immunoregulatory properties . Genes coding for the variable regions of the heavy and light chains of the Abs were isolated from a natural Ig gene library and scFv Abs were expressed in E . coli . The scFv Abs bound to F(ab')2 but not to Fab fragments . This points to an epitope located in the hinge region since Fab fragments are lacking most of the hinge . In order to verify our hypothesis, double chain peptides comprising the lower-, middle-, and part of the upper hinge subregion of IgG1-IgG4 were synthesized on cellulose membranes and tested for binding to the Abs . The results show binding of Abs to IgG1 and IgG4 hinge region peptides . In order to identify the key residues of the discontinuous epitopes we carried out complete substitutional analyses in which each amino acid of the wt peptides was substituted by all other amino acids except cysteine . The exchange of proline in the IgG1 or IgG4 middle hinge region abrogated the binding, revealing the importance of this subregion for epitope expression . No binding to the IgG2 or IgG3 hinge was detected . These results indicate that scFv anti-F(ab')2 Abs recognize the hinge region of IgG1 and IgG4 and that the expression of the epitope depends on an intact middle hinge subregion. Med Microbiol Immunol (Berl), 1999 May, 187(4), 213 - 9 Osp17, a novel immunodominant outer surface protein of Borrelia afzelii: recombinant expression in Escherichia coli and its use as a diagnostic antigen for serodiagnosis of Lyme borreliosis; Jauris-Heipke S et al.; Western blot analyses of the human humoral response of patients with Lyme borreliosis have shown that a 17-kDa protein is an immunodominant protein in late disease . Immune electron microscopy with a monoclonal antibody against this protein revealed that the 17-kDa protein is abundantly expressed on the surface of Borrelia afzelii strain PKo . Therefore, the protein has been renamed outer surface protein (Osp) 17 . Recombinant Osp 17 of strain PKo was expressed in Escherichia coli and purified by chromatography . Immunoblot analysis of human sera showed a comparable sensitivity with recombinant and natural proteins . The DNA sequences of the osp17 genes from different B . afzelii strains were determined . The DNA sequences of the different osp 17 homologues (six isolates from skin, three isolates from CSF and one isolate from synovial fluid) had high sequence identities of at least 94% . Using a polyclonal antibody against recombinant Osp 17, it was shown that Osp 17 expression varied considerably among the investigated B . afzelii strains . As previously also observed for OspA- and OspC-encoding genes, the osp 17 gene is present in strains not expressing the respective protein . It has been shown that OspA and OspC expression varies in different environments such as tick and vertebrate host . Studies are underway to examine whether this is also true for Osp 17 . For diagnostic purposes the use of recombinant Osp 17 has the advantage that the amount of Osp 17 antigen can be easily standardized for immunoblotting, and that this antigen can be used in a protein-specific enzyme-linked immunosorbent assay. Jpn J Cancer Res, 1999 Apr, 90(4), 460 - 8 Ex vivo delivery of suicide genes into melanoma cells using epidermal growth factor receptor-specific Fab immunogene; Ohtake Y et al.; The Fab fragment of monoclonal antibody B4G7 against human epidermal growth factor (EGF) receptor was conjugated with cationic poly-L-lysine and the resulting conjugate was further complexed with reporter genes or therapeutic genes . This Fab/DNA complex was designated as "Fab immunogene." The Fab immunogene transfer in vitro was mediated through the EGF receptors in two melanoma cell lines . The frequency of cells expressing beta-galactosidase (beta-Gal) reporter gene was approximately 1% . The induction of suicide effects after Fab immunogene transfer of herpes simplex virus thymidine kinase (TK) or Escherichia coli cytosine deaminase (CD) gene was quite remarkable, and the growth of melanoma cells was inhibited for over 7 days in the presence of ganciclovir (GCV) or 5-fluorocytosine (5-FC) . Similarly, when melanoma cells treated in vitro with the Fab immunogene carrying TK or CD were transplanted into the back of nude mouse, subsequent systemic administration of GCV or 5-FC effectively suppressed the growth of tumors, indicating the occurrence of in vivo suicide effects. Plant J, 1999 Apr, 18(2), 163 - 71 A minor form of starch branching enzyme in potato (Solanum tuberosum L.) tubers has a major effect on starch structure: cloning and characterisation of multiple forms of SBE A; Jobling SA et al.; Full length cDNAs encoding a second starch branching enzyme (SBE A) isoform have been isolated from potato tubers . The predicted protein has a molecular mass of 101 kDa including a transit peptide of 48 amino acids . Multiple forms of the SBE A gene exist which differ mainly in the length of a polyglutamic acid repeat at the C-terminus of the protein . Expression of the mature protein in Escherichia coli demonstrates that the gene encodes an active SBE . Northern analysis demonstrates that SBE A mRNA is expressed at very low levels in tubers but is the predominant isoform in leaves . This expression pattern was confirmed by Western analysis using isoform specific polyclonal antibodies raised against E . coli expressed SBE A . SBE A protein is found predominantly in the soluble phase of tuber extracts, indicating a stromal location within the plastid . Transgenic potato plants expressing an antisense SBE A RNA were generated in which almost complete reductions in SBE A were observed . SBE activity in the leaves of these plants was severely reduced, but tuber activity was largely unaffected . Even so, the composition and structure of tuber starch from these plants was greatly altered . The proportion of linear chains was not significantly increased but the average chain length of amylopectin was greater, resulting in an increase in apparent amylose content as judged by iodine binding . In addition, the starch had much higher levels of phosphorous. Anticancer Drug Des, 1999 Feb, 14(1), 61 - 70 Polybenzamide mustards: structure-activity relationships for DNA sequence-specific alkylation; Turner PR et al.; A series of cytotoxic polybenzamide mustards targeted to the minor groove of DNA were used to define structure-activity relationships for sequence-specific DNA alkylation . Compounds with an annular structure closely matched to the minor groove of DNA, and with concave-facing, potentially H-bonding NH groups, had a strong preference for alkylating adenines in sequences possessing four or more consecutive adenines . Two compounds whose annular structure matched that of the minor groove better when at least one carboxamide NH group faced outwards showed a high specificity for the consensus sequence (A/T)A(G/C) (A/T)N . Several compounds also alkylated specific guanines, presumably at the N3 position . Modelling studies suggest the most important contribution to sequence-specific alkylation is the H-bonds formed between these compounds and DNA, with factors such as the degree and positioning of cationic charge being less influential. Anticancer Drug Des, 1999 Feb, 14(1), 11 - 8 The role of base excision repair in the repair of DNA adducts formed by a series of nitrogen mustard-containing analogues of distamycin of increasing binding site size; Brooks N et al.; The role of base excision repair in the repair of alkylation damage produced by a series of sequence specific oligopyrrole-containing analogues of distamycin A that tether benzoic acid mustard (BAM) has been examined . Whereas BAM alkylates and cross-links in the major groove of DNA, attachment to pyrrole units produces monoalkylations in the minor groove of DNA at AT tracts . Both sequence specificity of alkylation and cytotoxicity increase from one to three attached pyrrole units (compounds 1-3), and with 3 alkylation is selective for purine-N3 in the sequence 5'-TTTTGPu (where Pu = guanine or adenine) . In a model bacterial (Escherichia coli) system repair of the sequence specific minor groove alkylations produced by 2 and 3 does not appear to involve BER, since neither a formamidopyrimidine-DNA glycosylase repair deficient E . coli mutant (BH 20, fpg- mutant) nor a 3-methyladenine-DNA glycosylase repair deficient mutant (GC 4803, tag-alkA- mutant) showed increased cytotoxicity to 2 or 3 compared with the wild type, AB 1157 . The monopyrrole compound 1 was, however, approximately 4-fold more cytotoxic to the GC 4803 mutant compared with wild type and BH 20, suggesting a role for the 3-methyladenine-DNA glycosylase in the recognition and excision of the adducts formed by 1 . In contrast, increased sensitivity (> 10-fold) was observed for the conventional nitrogen mustard BAM in the BH 20 strain, suggesting a role for the formamidopyrimidine-DNA glycosylase in the repair of the lesions produced by the agent . In a cell-free system the E . coli 3-methyladenine-DNA glycosylase (AlkA) was shown to remove alkylations at 5'-TTTTGPu sequences . However, the efficiency in removing the adducts formed by the oligopyrrole compounds decreased dramatically from compound 1 to compound 3 . Increasing the size of the DNA adduct formed in the minor groove therefore decreased the efficiency of recognition and removal of the adduct by the DNA glycosylase. Biochem Biophys Res Commun, 1999 Jun 7, 259(2), 483 - 8 Escherichia coli cafA gene encodes a novel RNase, designated as RNase G, involved in processing of the 5' end of 16S rRNA; Wachi M et al.; We found that the Escherichia coli cafA::cat mutant accumulated a precursor of 16S rRNA . This precursor migrated to the same position with 16.3S precursor found in the BUMMER strain that is known to be deficient in the 5' end processing of 16S rRNA . Accumulation of 16 . 3S rRNA in the BUMMER mutant was complemented by introduction of a plasmid carrying the cafA gene . The mutant type cafA gene cloned from the BUMMER strain had a 11-bp deletion in its coding region . A small amount of the mature 16S rRNA was still formed in the cafA::cat mutant . This residual activity was found to be due to RNase E encoded by the rne/ams gene by rifampicin-chase experiments of the cafA::cat ams1 double mutant . These results indicated that the cafA gene encodes a novel RNase responsible for processing of the 5' end of 16S rRNA . Biochem Biophys Res Commun, 1999 Jun 7, 259(2), 262 - 70 Characterization of a 3'-5' exonuclease associated with VDJP; Zhu L et al.; VDJP (V(D)J RSS Dependent DNA Joining Protein) was cloned based on binding to the nonamer portion of the V(D)J recombinational signal sequence (RSS), and genetic analysis revealed that VDJP is encoded by the same gene as the large subunit of Replication Factor C (RF-C) . Recombinant VDJP has a site directed DNA joining activity and is capable of forming a covalent bond between DNA fragments containing an RSS element near their ends and exhibits 3' to 5' exonuclease activity . In this report, we examine the biochemical properties of the VDJP exonuclease activity such as directionality of nuclease action (3' to 5' or 5' to 3'), single-strand substrate preference, cleavage products, dependence on cofactors and metal cations, and optimal reaction conditions . From this analysis, we conclude that VDJP has an intrinsic 3'-5' exonuclease activity that produces mononucleotide products . Biochem Biophys Res Commun, 1999 Jun 7, 259(2), 244 - 9 Selenocysteine-containing thioredoxin reductase in C . elegans; Gladyshev VN et al.; Mammalian thioredoxin reductases contain a TGA-encoded C-terminal penultimate selenocysteine (Sec) residue, and show little homology to bacterial, yeast, and plant thioredoxin reductases . Here we show that the nematode, Caenorhabditis elegans, contains two homologs related to the mammalian thioredoxin reductase family . The gene for one of these homologs contains a cysteine codon in place of TGA, and its product, designated TR-S, was previously suggested to function as thioredoxin reductase . The other gene contains TGA and its product is designated TR-Se . This Sec-containing thioredoxin reductase lacks a canonical Sec insertion sequence element in the 3'-untranslated area of the gene . TR-Se shows greater sequence similarity to mammalian thioredoxin reductase isozymes TR1 and TR2, whereas TR-S is more similar to TR3 . TR-Se was identified as a thioredoxin reductase selenoprotein by labeling C . elegans with 75Se and characterizing the resulting 75Se-labeled protein by affinity and other column chromatography and gel-electrophoresis . TR-Se was expressed in Escherichia coli as a selenoprotein when a bacterial SECIS element was introduced downstream of the Sec TGA codon . The data show that TR-Se is the major naturally occurring selenoprotein in C . elegans, and suggest an important role for selenium and the thioredoxin system in this organism . Crit Care Med, 1999 May, 27(5), 880 - 6 Increase in endotoxin-induced mucosal permeability is related to increased nitric oxide synthase activity using the Ussing chamber; Mishima S et al.; OBJECTIVE: To determine if nitric oxide production is associated with increased intestinal permeability after endotoxin challenge using the ex vivo Ussing chamber . SUBJECTS: Ileal mucosal membranes harvested from normal rats weighing 300 to 420 g . INTERVENTIONS: Endotoxin (lipopolysaccharide), 1, 10, 100 microg/ mL, or saline was placed on the serosal side of ileal mucosal membranes mounted in Ussing chambers after 10(9) Escherichia coli C-25 had been placed on the mucosal side of the ileal membranes (n = 6-7/group) . In a second set of experiments, ileal membranes were exposed to 100 microg/mL lipopolysaccharide with or without the addition of the nitric oxide synthase inhibitor, N(G)-monomethyl-L-arginine at a concentration of 10 mM (n = 7-8/group) . MAIN OUTCOME MEASURE: Bacterial translocation of E . coli C-25 from the mucosal to the serosal side of the ileal membrane was measured every hour during the 3-hr experimental period, as were serial measurements of the potential difference and resistance values of the ileal membranes . At the conclusion of the 3-hr period, the ileal membranes were harvested and levels of inducible nitric oxide synthase and constitutive nitric oxide synthase activity were measured . RESULTS: The incidence of E . coli C-25 passage across the ileal membranes mounted in the Ussing chambers was significantly increased in the ileal membranes exposed to 10 or 100 microg/mL of lipopolysaccharide (71% and 86%, respectively) vs . the control membranes (0%) or the membranes exposed to 1 microg/mL of lipopolysaccharide (0%) (p < .05) . This increase in E . coli C-25 passage in the ileal membranes exposed to 10 or 100 microg/mL of lipopolysaccharide was associated with a decrease in ileal membrane resistance and an increase in inducible nitric oxide synthase activity (p < .05) . The addition of N(G)-monomethyl-L-arginine protected against lipopolysaccharide-induced bacterial translocation and prevented the lipopolysaccharide-induced increase in ileal membrane inducible nitric oxide synthase activity . CONCLUSION: These results indicate that lipopolysaccharide induction of increased ileal inducible nitric oxide synthase activity is necessary for lipopolysaccharide-induced E . coli C-25 translocation to occur in normal ileal mucosal membranes tested in the Ussing chamber system. FEMS Microbiol Lett, 1999 Jun 1, 175(1), 119 - 25 Construction and immunologic evaluation of a Porphyromonas gingivalis subsequence peptide fused to hepatitis B virus core antigen; Dawson JA et al.; Several proteins of Porphyromonas gingivalis contain multiple copies of a 47 amino acid conserved repeated sequence . A fusion protein was constructed in which the P . gingivalis peptide was fused to the carboxy terminus of the hepatitis B core protein . This fusion protein was expressed in Escherichia coli, purified, and used to vaccinate mice that were later challenged with P . gingivalis W83 using the mouse abscess model . Although the mice were not protected against bacterial challenge, Western blot analysis showed that sera from the mice and from rabbits immunized with the fusion protein reacted with a number of vesicle proteins from P . gingivalis W83 . These data suggested that this peptide is recognized by the host's immune system but that the antibodies are not protective. Biosci Biotechnol Biochem, 1999 Apr, 63(4), 779 - 83 Expression of a functional single-chain antibody against GA24/19 in transgenic tobacco; Shimada N et al.; An anti-gibberellin A24/19 single-chain Fv gene was constructed from gamma and kappa genes cloned from a hybridoma cell line producing monoclonal antibody against gibberellin A24/19, biosynthetic precursors of gibberellin A4/1 which are biologically active per se . The single-chain Fv gene was introduced into tobacco plants after the binding activity of the single-chain Fv expressed in Escherichia coli was confirmed . When the single-chain Fv expression is targeted to endoplasmic reticulum, the plants could accumulate the single-chain Fv protein with the antigen binding activity up to 3.6% of the total soluble protein . On the other hand, when the expression is targeted to cytosol, accumulation of the single-chain Fv protein was not detected at all . The dwarf phenotype of the transgenic plants expressing the single-chain Fv protein, together with the preliminary analytical data indicating a decreased level of gibberellin A1 in the dwarf transgenics, suggested that the single-chain Fv decreased the concentration of bioactive gibberellins by trapping and inhibiting the metabolism of gibberellin A24 and/or A19 to gibberellin A4 and/or A1. Biosci Biotechnol Biochem, 1999 Apr, 63(4), 776 - 8 Construction and characterization of Escherichia coli disruptants defective in the yaeM gene; Kuzuyama T et al.; Escherichia coli disruptants defective in the yaeM gene, which is located at 4.2 min on the chromosome map, were constructed and characterized . The disruptants showed auxotrophy for 2-C-methylerythritol, a free alcohol of 2-C-methyl-D-erythritol 4-phosphate that is a biosynthetic precursor in the nonmevalonate pathway . This result clearly shows that the yaeM gene is indeed involved in this pathway in E . coli. Biosci Biotechnol Biochem, 1999 Apr, 63(4), 710 - 8 High-multiplicity of chitinase genes in Streptomyces coelicolor A3(2); Saito A et al.; Six different genes for chitinase from ordered cosmids of the chromosome of Streptomyces coelicolor A3(2) were identified by hybridization, using the chitinase genes from other Streptomyces spp . as probes, and cloned . The genes were sequenced and analyzed . The genes, together with an additional chitinase gene obtained from the data bank, can be classified into either family 18 or family 19 of the glycosyl hydrolase classification . The five chitinases that fall into family 18 show diversity in their multiple domain structures as well as in the amino acid sequences of their catalytic domains . The remaining two chitinases are members of family 19 chitinases, since their C-terminus shares more than 70% identity with the catalytic domain of ChiC of Streptomyces griseus, the sole gene for family 19 chitinase so far found in an organism other than higher plants. Biosci Biotechnol Biochem, 1999 Apr, 63(4), 703 - 9 Production of recombinant Der fI (a major mite allergen) by Aspergillus oryzae; Shoji H et al.; Der fI is a major mite allergen . To produce Der fI by Aspergillus oryzae, we placed a DNA fragment encoding precursor-type recombinant Der fI E(-1)K (reDer fI E(-1) K), which had the C-terminal amino acid of the pro-sequence (Glu) changed to Lys, downstream of the glaA gene promoter and introduced it into Aspergillus oryzae . In liquid culture, most of the reDer fI E(-1)K produced by the transformants was degraded when culture was shaken vigorously . However, the degradation of reDer fI E(-1)K was suppressed when it was shaken gently . The processed reDer fI E(-1)K could be obtained after lysylendopeptidase and endoglycosidase Hf (Endo Hf) treatment . The yield of processed reDer fI E(-1)K was 8 mg/l . When the transformant was grown on a wheat bran culture, the yield of processed reDer fI E(-1)K reached 48 mg/kg . Because processed reDer fI E(-1)Ks obtained from both cultures had almost the same IgE-binding activity and elicited the same skin reaction as native Der fI, they could be very useful for diagnostic purposes or immunotherapy. Biosci Biotechnol Biochem, 1999 Apr, 63(4), 666 - 71 Expression of elongation factor 1 beta' in Escherichia coli and its interaction with elongation factor 1 alpha from silk gland; Kamiie K et al.; Silk gland elongation factor 1 (EF-1) consists of four subunits: alpha, beta, beta', and gamma . EF-1 beta beta' gamma catalyzes the exchange of GDP for GTP on EF-1 alpha and stimulates the binding of EF-1 alpha-dependent aminoacyl-tRNA to ribosomes . The carboxy-terminal regions of the EF-1 beta subunits from various species are highly conserved . We examined the region of EF-1 beta' that binds to EF-1 alpha by in vitro binding assays, and examined the GDP/GTP exchange activity using deletion mutants of a GST-EF1 beta' fusion protein . We thereby suggested a pivotal amino acid region, residues 189-222, of EF-1 beta' for binding to EF-1 alpha. Biosci Biotechnol Biochem, 1999 Apr, 63(4), 648 - 54 Preparation and application of anti-idiotypic antibody against anti-gibberellin A4 antibody; Suzuki Y et al.; A monoclonal anti-idiotypic antibody was raised against anti-gibberellin A4 (GA4) antibody, which recognizes biologically active gibberellins such as GA1 and GA4 specifically . Amino acid sequences of variable regions of both anti-GA4 and anti-idiotypic antibodies were analyzed . By using the property of the anti-idiotypic antibody to compete with GA1/4 in binding to the anti-GA4 antibody, we successfully applied the anti-idiotypic antibody to ELISA as a tracer for measuring GA1/4 . The single-chain Fv (scFv) gene of the anti-idiotypic antibody was constructed, and scFv expressed in E . coli showed binding activity to anti-GA4 antibody . These results suggest the possible application of anti-idiotypic antibody as a handy and stable source of an enzymatic tracer for ELISA by production of fusion protein of the scFv and an appropriate enzyme. Mol Microbiol, 1999 Jun, 32(5), 1090 - 102 Multiple hok genes on the chromosome of Escherichia coli; Pedersen K et al.; The hok/sok locus of plasmid R1 mediates plasmid stabilization by the killing of plasmid-free cells . Many bacterial plasmids carry similar loci . For example, the F plasmid carries two hok homologues, flm and srnB, that mediate plasmid stabilization by this specialized type of programmed cell death . Here, we show that the chromosome of E . coli K-12 codes for five hok homologous loci, all of which specify Hok-like toxins . Three of the loci appear to be inactivated by the insertion elements IS150 or IS186 located close to but not in the toxin-encoding reading frames (i.e . hokA, hokC and hokE), one system is probably inactivated by point mutation (hokB), whereas the fifth system is inactivated by a major genetic rearrangement (hokD) . In the ECOR collection of wild-type E . coli strains, we identified hokA and hokC loci without IS elements . A molecular and a genetic analysis show that the hokA and hokC loci specify unstable antisense RNAs and stable toxin-encoding mRNAs that are processed at their 3' ends . An alignment of the mRNA sequences reveals all the regulatory elements known to be required for correct folding and refolding of the plasmid-encoded mRNAs . The conserved elements include fbi that ensure a long-range interaction in the full-length mRNAs, and tac and antisense RNA target stem-loops that are required for translation and rapid antisense RNA binding of the processed mRNAs . Consistently, we find that the chromosome-encoded mRNAs are processed at their 3' ends, resulting in the presumed translationally active mRNAs . Despite the presence of all of the regulatory elements, the chromosome-encoded loci do not mediate plasmid stabilization by killing of plasmid-free cells . The chromosome-encoded mRNAs are poorly translated in vitro, thus yielding an explanation for the lacking phenotype . These observations suggest that the chromosomal hok-like genes may be induced by an as yet unknown signal. Mol Microbiol, 1999 Jun, 32(5), 1031 - 42 C-terminal interactions between the XerC and XerD site-specific recombinases; Spiers AJ et al.; Studies of the site-specific recombinase Cre suggest a key role for interactions between the C-terminus of the protein and a region located about 30 residues from the C-terminus in linking in a cyclical manner the four recombinase monomers present in a recombination complex, and in controlling the catalytic activity of each monomer . By extrapolating the Cre DNA recombinase structure to the related site-specific recombinases XerC and XerD, it is predicted that the extreme C-termini of XerC and XerD interact with alpha-helix M in XerD and the equivalent region of XerC respectively . Consequently, XerC and XerD recombinases deleted for C-terminal residues, and mutated XerD proteins containing single amino acid substitutions in alphaM or in the C-terminal residues were analysed . Deletion of C-terminal residues of XerD has no measurable effect on co-operative interactions with XerC in DNA-binding assays to the recombination site dif, whereas deletion of 5 or 10 residues of XerC reduces co-operativity with XerD some 20-fold . Co-operative interactions between pairs of truncated proteins during dif DNA binding are reduced 20- to 30-fold . All of the XerD mutants, except one, were catalytically proficient in vitro; nevertheless, many failed to mediate a recombination reaction on supercoiled plasmid in vivo or in vitro, implying that the ability to form a productive recombination complex and/or mediate a controlled recombination reaction is impaired. Mol Microbiol, 1999 Jun, 32(5), 953 - 60 The identification of three biologically relevant globotriaosyl ceramide receptor binding sites on the Verotoxin 1 B subunit; Bast DJ et al.; The Verotoxin 1 (VT1) B subunit binds to the glycosphingolipid receptor globotriaosylceramide (Gb3) . Receptor-binding specificity is associated with the terminally linked Galalpha(1-4) Galbeta disaccharide sequence of the receptor . Recently, three globotriose (Galalpha{1-4} Galbeta {1-4} Glcbeta) binding sites per B-subunit monomer were identified by crystallography . Two of these sites (sites I and II) are located adjacent to phenylalanine-30 . Site I was originally predicted as a potential Gb3 binding site on the basis of sequence conservation, and site II was additionally predicted based on computer modelling and receptor docking . The third (site III) was also identified by crystallography and is located at the N-terminal end of the alpha-helix . To determine the biological significance of sites II and III, and to support our previous findings of the significance of site I, we examined the binding properties and cytotoxicity of VT1 mutants designed to block Gb3 binding at each site selectively . The Scatchard analysis of saturation-binding data for each mutant revealed that only the amino acid substitutions predicted to affect site I (D-17E) or site II (G-62T) caused reductions in the binding affinity and capacity of VT1 for Gb3 . Similarly, those mutations at sites I and II also caused significant reductions in both Vero and MRC-5 cell cytotoxicity (by seven and five logs, respectively, for G-62T and by four and two logs, respectively, for D-17E) . In contrast, the substitution of alanine for W-34 at site III did not reduce the high-affinity binding of the B subunit, despite causing a fourfold reduction in the receptor-binding capacity . The corresponding mutant W-34A holotoxin had a two-log reduction in cytotoxicity on Vero cells and no statistically significant reduction on MRC-5 cells . We conclude that the high-affinity receptor binding most relevant for cell cytotoxicity occurs at sites I and II . In contrast, site III appears to mediate the recognition of additional Gb3 receptor epitopes but with lower affinity . Our results support the significance of the indole ring of W-34 for binding at this site. Mol Microbiol, 1999 May, 32(4), 851 - 67 In vivo and in vitro studies of major surface loop deletion mutants of the Escherichia coli K-12 maltoporin: contribution to maltose and maltooligosaccharide transport and binding; Andersen C et al.; The trimeric protein LamB of Escherichia coli K-12 (maltoporin) specifically facilitates the diffusion of maltose and maltooligosaccharides through the outer membrane . Each monomer consists of an 18-stranded antiparallel beta-barrel with nine surface loops (L1 to L9) . The effects on transport and binding of the deletion of some of the surface loops or of combinations of several of them were studied in vivo and in vitro . In vivo, single-, DeltaL4, DeltaL5, DeltaL6, and double-loop deletions, DeltaL4 + DeltaL5 and DeltaL5 + DeltaL6, abolished maltoporin functions, but not the double deletion DeltaL4 + DeltaL6 and the triple deletion DeltaL4 + DeltaL5 + DeltaL6 . While deletion of the central variable portion of loop L9 (DeltaL9v) affected maltoporin function only moderately, the combination of DeltaL9v with the double deletion of loops L4 and L6 (triple deletion DeltaL4 + DeltaL6 + DeltaL9v) strongly impaired maltoporin function and resulted in sensitivity to large hydrophilic antibiotics without change in channel size as measured in vitro . In vitro, the carbohydrate-binding properties of the different loop mutants were studied in titration experiments using the asymmetric and symmetric addition of the mutant porins and of the carbohydrates to one or both sides of the lipid bilayer membranes . The deletion of loop L9v alone (LamBDeltaL9v), of two loops L4 and L6 (LamBDeltaL4 + DeltaL6), of three loops L4, L5 and L6 (LamBDeltaL4 + DeltaL5 + DeltaL6) or of L4, L6 and L9v (LamBDeltaL4 + DeltaL6 + DeltaL9v) had relatively little influence on the carbohydrate-binding properties of the mutant channels, and they had approximately similar binding properties for carbohydrate addition to both sides compared with only one side . The deletion of one of the loops L4 (LamBDeltaL4) or L6 (LamBDeltaL6) resulted in an asymmetric carbohydrate binding . The in vivo and in vitro results, together with those of the purification across the starch column, suggest that maltooligosaccharides enter the LamB channel from the cell surface side with the non-reducing end in advance . The absence of some of the loops leads to obstruction of the channel from the outside, which results in a considerable difference in the on-rate of carbohydrate binding from the extracellular side compared with that from the periplasmic side. Mol Microbiol, 1999 May, 32(4), 789 - 98 The stationary-phase morphogene bolA from Escherichia coli is induced by stress during early stages of growth; Santos JM et al.; The Escherichia coli morphogene bolA causes round morphology when overexpressed . The expression of bolA is mainly regulated by a sigmas-dependent gearbox promoter bolA1p . Such regulation results in increased relative levels of expression at slow growth rates, as seen with those attained at the onset of stationary phase . We demonstrate that bolA1p is also induced during early logarithmic growth in response to several forms of stress, and that this induction can be partially sigmas independent . Sudden carbon starvation results in a 17-fold increase in mRNA levels derived from bolA1p 1 h after stress imposition . Increased osmolarity results in a more than 20-fold increase after the same period . Considerable increases in bolA1p mRNA levels were also detected as a result of heat shock, acidic stress and oxidative stress, which has been shown to inhibit sigmas translation . The orders of magnitude of bolA1p induction in log phase due to sudden starvation, osmotic shock and oxidative stress surpass the levels reached in stationary phase . Under sudden carbon starvation and osmotic shock, the cells changed their morphology, resembling those cells in which bolA is overexpressed in stationary phase . Increased expression and morphological changes due to sudden carbon starvation and osmotic shock still occur when sigmaS is not present in a rpoS- background . The results show that expression of bolA is not confined to stationary phase, but it can also play an important role in general stress response . We propose that bolA1p stress induction overrides the normal regulation imposed by growth rate, which is strictly the result of sigmaS-directed transcription. Mol Microbiol, 1999 May, 32(4), 777 - 88 The role of the trehalose system in regulating the maltose regulon of Escherichia coli; Decker K et al.; The maltose regulon consists of 10 genes encoding an ABC transporter for maltose and maltodextrins as well as enzymes necessary for their degradation . MalK, the energy-transducing subunit of the transport system, acts phenotypically as a repressor of MalT, the transcriptional activator of the mal genes . Using MacConkey maltose indicator plates we isolated an insertion mutation that strongly reduced the repressing effect of overproduced MalK . The insertion had occurred in treR encoding the repressor of the trehalose system . The loss of TreR function led to derepression of treB encoding an enzymeIITre of the PTS for trehalose and of treC encoding TreC, the cytoplasmic trehalose-6-phosphate hydrolase . Further analysis revealed that maltose can enter the cell by facilitated diffusion through enzymeIITre, thus causing induction of the maltose system . In addition, derepression of TreC by itself caused induction of the maltose system, and a mutant lacking TreC was reduced in the uninduced level of mal gene expression indicating synthesis of endogenous inducer by TreC . Extracts containing TreC transformed {14C}-maltose into another 14C-labelled compound (preliminarily identified as maltose 1-phosphate) that is likely to be an alternative inducer of the maltose system. Clin Exp Immunol, 1999 Jun, 116(3), 462 - 7 Depletion of liver and splenic macrophages reduces the lethality of Shiga toxin-2 in a mouse model; Palermo MS et al.; The haemolytic uraemic syndrome (HUS) is a clinical syndrome consisting of haemolytic anaemia, thrombocytopenia, and acute renal insufficiency . HUS is the most frequent cause of acute renal failure in childhood . It has been previously suggested that the presence of Shiga toxin (Stx) is necessary but not sufficient for HUS development, and cytokines such as tumour necrosis factor-alpha (TNF-alpha) and IL-1beta appear to be necessary to develop the syndrome . Since the mononuclear phagocytic system (MPS) is the major source of these cytokines, macrophages might be one of the relevant targets for Stx action in the pathophysiology of HUS . In this study our objective was to examine the role of the hepatic and splenic macrophages in a mouse model of HUS induced by injection of Shiga toxin type-2 (Stx2) or Stx2 plus lipopolysaccharide (LPS) . For this purpose, depletion of mice macrophages by liposome-encapsulated clodronate (lip-clod), followed by injection of STx2 or Stx2 plus LPS, was assayed . In this study we show that depletion of hepatic and splenic macrophages by clodronate treatment induces a survival of 50% in animals treated with Stx2 alone or in presence of LPS . This maximal effect was observed when lip-clod was injected 48-72 h before Stx2 injection . Biochemical and histological parameters show characteristics of the lesion produced by Stx2, discarding non-specific damage due to LPS or lip-clod . In addition, we determined that the toxic action of Stx2 is similar in BALB/c and N:NIH nude mice, indicating the T cell compartment is not involved in the Stx2 toxicity . Briefly, we demonstrate that macrophages play a central role in the pathophysiology of HUS, and that the systemic production of cytokines by liver and/or spleen is for Stx2 to manifest its full cytotoxic effect . In addition, the toxicity of Stx2 alone, or in presence of LPS, is independent of the T cell compartment. Fungal Genet Biol, 1999 Apr, 26(3), 253 - 69 Carbon regulation of ribosomal genes in Neurospora crassa occurs by a mechanism which does not require Cre-1, the homologue of the Aspergillus carbon catabolite repressor, CreA; de la Serna I et al.; Transcription of the ribosomal protein and 40S rRNA genes is coordinately regulated during steady state growth and carbon shifts in Neurospora crassa . Recognition sequences for the Aspergillus nidulans carbon catabolite repressor, CreA, overlap transcriptional elements of a 40S rRNA gene and the crp-2 ribosomal protein gene . They also occur in similar locations in the promoters of several other ribosomal protein genes . Substitutions encompassing the -74 and -167 CreA consensus sequences in the crp-2 promoter result in a decrease in transcription . A cDNA encoding the N . crassa homologue of CreA was cloned and designated Cre-1 . The Cre-1 protein is 45% identical to CreA from A . nidulans . Cre-1 protein produced in Escherichia coli binds to the CreA sites in the promoters of the 40S rRNA and crp-2 genes . An amino acid change from histidine (92) to threonine changed the Cre-1 binding specificity from (5'G/CC/TGGG/AG3') to (5'G/CC/TGGCG3') . Base substitutions in the Cre-1 binding sites of the crp-2 promoter disrupted binding of wildtype Cre-1 in vitro but had no effect on transcription during steady state growth or carbon shifts, indicating that regulation of ribosomal genes by carbon source is not mediated by Cre-1, but via different proteins binding the Cre-1 sites and the Dde boxes . Anal Biochem, 1999 Jun 15, 271(1), 81 - 5 Real-time detection and quantification of adenosine triphosphate sulfurylase activity by a bioluminometric approach; Karamohamed S et al.; A real-time, sensitive, and simple assay for detection and quantification of adenosine triphosphate sulfurylase (ATP:sulfate adenylytransferase, EC 2.7.7.4) activity has been developed . The method is based on detection of ATP generated in the ATP sulfurylase reaction between APS and PPi by the firefly luciferase system . For the Saccharomyces cerevisiae ATP sulfurylase, the concentrations of APS and PPi at the half-maximal rate were found to be about 0.5 and 7 microM, respectively . The assay is sensitive and yields linear response between 0.1 microU and 50 mU . The method can be used for monitoring and quantification of recombinant ATP sulfurylase activity in Escherichia coli lysate, as well as for detection of the activity during different purification procedures . Nature, 1999 May 27, 399(6734), 384 - 8 Structure of the small G protein Cdc42 bound to the GTPase-binding domain of ACK; Mott HR et al.; The proteins Cdc42 and Rac are members of the Rho family of small GTPases (G proteins), which control signal-transduction pathways that lead to rearrangements of the cell cytoskeleton, cell differentiation and cell proliferation . They do so by binding to downstream effector proteins . Some of these, known as CRIB (for Cdc42/Rac interactive-binding) proteins, bind to both Cdc42 and Rac, such as the PAK1-3 serine/threonine kinases, whereas others are specific for Cdc42, such as the ACK tyrosine kinases and the Wiscott-Aldrich-syndrome proteins (WASPs) . The effector loop of Cdc42 and Rac (comprising residues 30-40, also called switch I), is one of two regions which change conformation on exchange of GDP for GTP . This region is almost identical in Cdc42 and Racs, indicating that it does not determine the specificity of these G proteins . Here we report the solution structure of the complex of Cdc42 with the GTPase-binding domain ofACK . Both proteins undergo significant conformational changes on binding, to form a new type of G-protein/effector complex . The interaction extends the beta-sheet in Cdc42 by binding an extended strand from ACK, as seen in Ras/effector interactions, but it also involves other regions of the G protein that are important for determining the specificity of effector binding. Nature, 1999 May 27, 399(6734), 379 - 83 Structure of Cdc42 in complex with the GTPase-binding domain of the 'Wiskott-Aldrich syndrome' protein; Abdul-Manan N et al.; The Rho-family GTP-hydrolysing proteins (GTPases), Cdc42, Rac and Rho, act as molecular switches in signalling pathways that regulate cytoskeletal architecture, gene expression and progression of the cell cycle . Cdc42 and Rac transmit many signals through GTP-dependent binding to effector proteins containing a Cdc42/Rac-interactive-binding (CRIB) motif . One such effector, the Wiskott-Aldrich syndrome protein (WASP), is postulated to link activation of Cdc42 directly to the rearrangement of actin . Human mutations in WASP cause severe defects in haematopoletic cell function, leading to clinical symptoms of thrombocytopenia, immunodeficiency and eczema . Here we report the solution structure of a complex between activated Cdc42 and a minimal GTPase-binding domain (GBD) from WASP . An extended amino-terminal GBD peptide that includes the CRIB motif contacts the switch I, beta2 and alpha5 regions of Cdc42 . A carboxy-terminal beta-hairpin and alpha-helix pack against switch II . The Phe-X-His-X2-His portion of the CRIB motif and the alpha-helix appear to mediate sensitivity to the nucleotide switch through contacts to residues 36-40 of Cdc42 . Discrimination between the Rho-family members is likely to be governed by GBD contacts to the switch I and alpha5 regions of the GTPases . Structural and biochemical data suggest that GBD-sequence divergence outside the CRIB motif may reflect additional regulatory interactions with functional domains that are specific to individual effectors. Protein Eng, 1999 May, 12(5), 429 - 35 Expression of a synthetic gene encoding canine milk lysozyme in Escherichia coli and characterization of the expressed protein; Koshiba T et al.; A high-expression plasmid of the canine milk lysozyme, which belongs to the family of calcium-binding lysozymes, was constructed in order to study its physico-chemical properties . Because the cDNA sequence of the protein has not yet been determined, a 400 base-pair gene encoding canine milk lysozyme was first designed on the basis of the known amino acid sequence . The gene was constructed by an enzymatic assembly of 21 chemically synthesized oligonucleotides and inserted into an Escherichia coli expression vector by stepwise ligation . The expression plasmid thus constructed was transformed into BL21(DE3)/pLysS cells . The gene product accumulated as inclusion bodies in an insoluble fraction . Recombinant canine milk lysozyme was obtained by purification and refolding of the product and showed the same characteristics in terms of bacteriolytic activity and far- and near-UV circular dichroism spectra as the authentic protein . The NMR spectra of refolded lysozyme were also characteristic of a native globular protein . It was concluded that recombinant canine milk lysozyme was folded into the correct native structure . Moreover, the thermal unfolding profiles of the refolded recombinant lysozyme showed a stable equilibrium intermediate, indicating that the molten globule state of this protein was extraordinarily stable . This expression system of canine milk lysozyme will enable biophysical and structural studies of this protein to be extended. Protein Eng, 1999 May, 12(5), 423 - 8 Construction, expression, purification and functional analysis of recombinant NFkappaB p50/p65 heterodimer; Chen FE et al.; NFkappaB plays an important role in mediating the gene expression of numerous cellular processes such as growth, development, the inflammatory response and virus proliferation . The p50/p65 heterodimer is the most abundant form of the NFkappaB dimers and plays a more elaborate role in gene regulation . Biochemical research on p50/p65 NFkappaB has not benefited however from the availability of easily purified recombinant protein . We report two methods for the large scale expression and purification of recombinant NFkappaB p50/p65 heterodimer . The first utilizes a bacterial double expression vector which contains two ribosomal binding sites to facilitate the coexpression of the polypeptides in the p50/p65 NFkappaB heterodimer . The second method uses a mixed protein refolding strategy . Both methods yield crystallizable protein . Electrophoretic mobility shift assays confirm that the DNA binding affinity is independent of the method used to purify the protein . These methods will facilitate the numerous studies on various NFkappaB/Rel family members. Biochemistry, 1999 Jun 8, 38(23), 7509 - 16 Orientation of LamB signal peptides in bilayers: influence of lipid probes on peptide binding and interpretation of fluorescence quenching data; Voglino L et al.; The orientation in lipid bilayers of the signal sequence of the bacterial protein LamB was studied using binding, circular dichroism, and fluorescence quenching experiments . Measurements were made of binding modifications caused by the incorporation of lipid probes (brominated or nitroxide-labeled phospholipids) used in the parallax fluorescence quenching method of determining peptide penetration depth {Abrams, F . S., and London, E . (1992) Biochemistry 31, 5312-5322} . The signal peptide bound to a similar extent to neutral bilayers composed of either egg phosphatidylcholine (EPC) or phosphatidylcholines brominated at various positions on their acyl chains . The fluorescence of a tryptophan in either the 18 or 24 position of the peptide was quenched more by bromines in the 6 and 7 than in the 9 and 10 positions on the lipid hydrocarbon chain . Parallax calculations showed that tryptophan-18 was located only 4 A from the hydrocarbon-water interface, consistent with the peptide adopting a "hammock" configuration in the bilayer, with both termini exposed to the aqueous phase and the central alpha-helix located near the hydrocarbon-water interface . In contrast, the incorporation of 10% nitroxide-labeled lipids into EPC bilayers modified peptide binding in a manner dependent on the position of the nitroxide on the hydrocarbon chain; 7-Doxyl PC reduced the percent peptide bound by about one-half, whereas 12-Doxyl PC had little effect on binding . These binding differences modified tryptophan quenching by these probes, making parallax analysis problematical . In the presence of the positively charged LamB peptide, the incorporation of negatively charged phospholipids into EPC bilayers increased the level of peptide binding and modified tryptophan quenching by nitroxide probes . These results suggest that the nitroxide probe could be partially excluded from negatively charged lipid domains where the peptide preferentially bound . Quite different binding and quenching results were obtained with a negatively charged peptide analogue, showing that the charge on both the peptide and bilayer affects peptide-nitroxide probe interactions. Biochemistry, 1999 Jun 8, 38(23), 7498 - 508 Apoptosis-linked gene product ALG-2 is a new member of the calpain small subunit subfamily of Ca2+-binding proteins; Lo KW et al.; ALG-2 is a newly discovered Ca2+-binding protein which has been demonstrated to be directly linked to apoptosis . Structurally, ALG-2 is expressed as a single polypeptide chain corresponding to a 22 kDa protein containing five putative EF-hand Ca2+-binding sites . In this work, we have developed an efficient expression and purification scheme for recombinant ALG-2 . Utilizing this protocol, we can routinely obtain purified recombinant protein with a yield of approximately 100 mg per liter of bacterial cell cultures . Gel filtration and chemical cross-linking experiments have shown that Ca2+-free ALG-2 forms a weak homodimer in solution . Biochemical and spectroscopic studies of truncated and point mutants of ALG-2 demonstrated that the fifth EF-hand Ca2+-binding motif is likely to participate in the formation of the dimer complex . Experimentally, both the amino- and carboxyl-terminal truncated mutants of ALG-2 have shown their ability to retain the structural, as well as, Ca2+-binding integrity when individually expressed in bacteria . In this respect, the N-terminal domain encompasses the first two EF-hands, and the C-terminal domain contains the remaining three EF-hands . Combining mutagenesis and spectroscopic studies, we showed that ALG-2 possesses two strong Ca2+-binding sites . Employing fluorescence spectroscopy and circular dichroism, we showed that the binding of Ca2+ to ALG-2 induced significant conformational changes in both the N-terminal and C-terminal domains of the protein . Furthermore, our studies demonstrated that Ca2+ binding to both strong Ca2+-binding sites of ALG-2 is required for ion-induced aggregation of the protein . We also report here the expression, purification, and partial characterization of a Ca2+-binding-deficient ALG-2 mutant (Glu47Ala/Glu114Ala) . In light of its much decreased affinity for Ca2+, this mutant could prove to be instrumental in elucidating the Ca2+-mediated function of ALG-2 within the context of its cellular environment. Biochemistry, 1999 Jun 8, 38(23), 7431 - 43 Design and synthesis of a globin fold; Isogai Y et al.; We propose a simple method to find an amino acid sequence that is foldable into a globular protein with a desired structure based on a knowledge-based 3D-1D compatibility function . An asymmetric alpha-helical single-domain structure of sperm whale myoglobin consisting of 153 amino acid residues was chosen for the design target . The optimal sequence to fit the main-chain framework has been searched by recursive generation of the protein 3D profile . The heme-binding site was designed by fixing His64 and His93 at the distal and proximal positions, respectively, and by penalizing residues that protrude into the space with a repulsive function . The apparent bumps among side chains in the computer model of the converged, self-consistent sequence were removed by replacing some of the bumping residues with smaller ones according to the final 3D profile . The finally obtained sequence shares 26% of sequence with the natural myoglobin . The designed globin-1 (DG1) with the artificial sequence was obtained by expression of the synthetic gene in Escherichia coli . Analyses using size-exclusion chromatography, circular dichroism spectroscopy, and solution X-ray scattering showed that DG1 folds into a monomeric, compact, highly helical, and globular form with an overall molecular shape similar to the target structure in an aqueous solution . Furthermore, it binds a single heme per protein molecule, which exhibited well-defined spectroscopic properties . The radius of gyration of DG1 was determined to be 20.6 A, slightly larger than that of natural apoMb, and decreased to 19.5 A upon heme binding based on X-ray scattering analysis . However, the heme-bound DG1 did not stably bind molecular oxygen as natural globins do, possibly due to high conformational diversity of side-chain structures observed in the NMR and denaturation experiments . These results give insight into the relationship between the sequence selection and the structural uniqueness of natural proteins to achieve biological functions. Biochemistry, 1999 Jun 8, 38(23), 7413 - 20 Escherichia coli primase zinc is sensitive to substrate and cofactor binding; Powers L et al.; The ligation state of the single zinc site in primase from Escherichia coli changes when various substrates and cofactors are added alone or in combination as determined by X-ray absorption spectroscopy . X-ray absorption spectroscopy (XAS) provides information about the local structure (approximately 5 A) of atoms surrounding the metal and has been widely used to characterize metalloproteins . The zinc site in native primase and in primase bound to low (30 mM) magnesium acetate was found to be tetrahedrally ligated by three sulfurs at an average distance of 2.36 +/- 0.02 A and one histidine nitrogen located at a distance of 2.15 +/- 0.03 A . When ATP, ATP and (dT)17, or ATP, low magnesium acetate and (dT)17 was added to primase, one (or two) additional nitrogen/oxygen ligands were coordinated to the zinc together with the histidine nitrogen at an average distance of 2.15 +/- 0.03 A . These additional ligands are likely from adjacent phosphates from ATP . Another structure was observed for the primase-(dT)17 complex in which an additional nitrogen/oxygen ligand likely from the phosphate backbone together with the histidine nitrogen was located at a significantly shorter average distance of 2.05 +/- 0.03 A . High magnesium acetate (300 mM) completely inactivates primase in a reversible manner such that the region near the zinc ligands becomes accessible to proteolytic digestion {Urlacher, T . M., and Griep, M . A . (1995) Biochemistry 34, 16708-16714} . In this inactive complex, additional oxygen/nitrogen ligands from acetate as well as the histidine nitrogen are located at a distance of 2.20 +/- 0.03 A from the zinc site . To test whether the catalytic magnesium was binding within approximately 5 A of the zinc, we incubated primase with high (300 mM) manganese acetate . The functional properties of magnesium and manganese are similar, but the larger atomic number of manganese enhances the X-ray backscattering, making it possible to identify . Since no significant difference was observed from the manganese-incubated sample, the catalytic metal-binding site is likely located >5 A from the zinc . These studies clearly show that primase zinc ligation changes upon binding substrates. Biochemistry, 1999 Jun 8, 38(23), 7407 - 12 Proximity between Glu126 and Arg144 in the lactose permease of Escherichia coli; Zhao M et al.; Evidence has been presented {Venkatesan, P., and Kaback, H . R . (1998) Proc . Natl . Acad . Sci . U.S.A . 95, 9802-9807} that Glu126 (helix IV) and Arg144 (helix V) which are critical for substrate binding in the lactose permease of Escherichia coli are charge paired and therefore in close proximity . To test this conclusion more directly, three different site-directed spectroscopic techniques were applied to permease mutants in which Glu126 and/or Arg144 were replaced with either His or Cys residues . (1) Glu126-->His/Arg144-->His permease containing a biotin acceptor domain was purified by monomeric avidin affinity chromatography, and Mn(II) binding was assessed by electron paramagnetic resonance spectroscopy . The mutant protein binds Mn(II) with a KD of about 40 microM at pH 7.5, while no binding is observed at pH 5.5 . In addition, no binding is detected with Glu126-->His or Arg144-->His permease . (2) Permease with Glu126-->Cys/Arg144-->Cys and a biotin acceptor domain was purified, labeled with a thiol-specific nitroxide spin-label, and shown to exhibit spin-spin interactions in the frozen state after reconstitution into proteoliposomes . (3) Glu126-->Cys/Arg144-->Cys permease with a biotin acceptor domain was purified and labeled with a thiol-specific pyrene derivative, and fluorescence spectra were obtained after reconstitution into lipid bilayers . An excimer band is observed with the reconstituted E126C/R144C mutant, but not with either single-Cys mutant or when the single-Cys mutants are mixed prior to reconstitution . The results provide strong support for the conclusion that Glu126 (helix IV) and Arg144 (helix V) are in close physical proximity. Ann Neurol, 1999 Jun, 45(6), 716 - 23 Brainstem mechanisms of autonomic dysfunction in encephalopathy-associated Shiga toxin 2 intoxication; Yamada Y et al.; Acute encephalopathy is the major determinant of death in an early stage of Shiga toxin (Stx)-producing Escherichia coli infection . Rapid progress toward refractory hypotension and dysfunction of breathing implies autonomic center dysfunction of patients . To clarify whether autonomic dysfunction becomes an ultimate cause of death in Shiga toxemia, we injected purified Stx2 (20 microg/kg) intravenously into rabbits, and monitored changes in cardiovascular and respiratory function together with renal sympathetic nerve activity (RSNA) in the conscious state . After an approximately 24-hour silent (lag) period, all rabbits given Stx2 developed hemorrhagic diarrhea (25.7 +/- 1.1 hours) and limb paralysis (31.2 +/- 1.3 hours) . This limb paralysis was observed initially in the hind legs, and then it gradually extended to the forelegs . After 23.2 +/- 2.3 hours, RSNA increased gradually, and arterial blood pressure was maintained within normal limits together with an increase in the maximum gain of baroreflex response . Severe hypotension developed within 34.8 +/- 2.2 hours, without any increase in heart rate; RSNA significantly increased by 39.5 +/- 0.9 hours . In the final stage, RSNA decreased concurrently with decreases in arterial blood pressure, heart rate, and baroreflex response, suggesting dysfunction of the baroreflex control system . Thereafter, all rabbits died within 47.8 +/- 1.2 hours after the intravenous Stx2 injection . Magnetic resonance imagings of the central nervous system (T2-weighted images) showed high-intensity areas in the dorsal two-thirds of the cervical spinal cord and brainstem 48 hours after Stx2 administration . These results show that the cause of death is circulatory failure caused by impairment of the cardiovascular center in the medulla . We believe that this animal model helps to clarify the mechanism of rapid progress to death of patients with Shiga toxin-producing E . coli infection. Mech Ageing Dev, 1999 Mar 15, 107(3), 323 - 32 Methionine residues may protect proteins from critical oxidative damage; Levine RL et al.; Cysteine and methionine are the two sulfur-containing residues normally found in proteins . Cysteine residues function in the catalytic cycle of many enzymes, and they form disulfide bonds which contribute to protein structure . In contrast, the key functions of methionine residues are not known . We propose that methionine residues constitute an important antioxidant defense mechanism . A variety of oxidants react readily with methionine to form methionine sulfoxide, and surface exposed methionine residues create an extremely high concentration of reactant, providing for efficient scavenging of oxidants . The effect of hydrogen peroxide exposure upon glutamine synthetase from Escherichia coli was studied as an in vitro model system . Eight of the sixteen methionine residues could be oxidized with little effect on activity . The oxidizable methionine residues were found to be relatively surface exposed while the intact residues were generally buried within the core of the protein . Further, the susceptible residues were physically arranged in an array which guarded the entrance to the active site . Methionine sulfoxide can be reduced back to methionine by the enzyme methionine sulfoxide reductase, providing a catalytic amplification of the antioxidant potential of each methionine residue . Given the importance of oxidative stress during aging, the potential function of methionine residues as antioxidants during aging should be investigated experimentally. J Lab Clin Med, 1999 Jun, 133(6), 541 - 50 Up-regulation of endothelial cell binding proteins/receptors for complement component C1q by inflammatory cytokines; Guo WX et al.; Endothelial cells express a variety of receptor systems involved in humoral defense, including receptors for the collagen-like and globular domains of the complement component C1q, designated cC1qR and gC1qR, respectively . In the present study a microvascular endothelial cell line was used to test the hypothesis that expression of these C1q-binding proteins may be affected by vascular inflammatory reactions . The results demonstrate that the expression of both cC1qR and gC1qR by bone marrow vascular endothelial cells is up-regulated by inflammatory mediators, interferon-gamma, tumor necrosis factor-alpha, and lipopolysaccharide (Escherichia coli, 055:B5) in a dose- and time-dependent manner, as detected by enzyme-linked immunosorbent assay . cC1qR and gC1qR expression increased significantly (P < .05) within 4 to 7 hours and doubled after 22 hours of stimulation . 3H-thymidine incorporation studies and direct cell counts confirmed that increased C1qR expression was not due to increased cell proliferation . Northern blot analysis revealed that the up-regulation of cC1qR and gC1qR protein expression was preceded by increases in corresponding mRNA levels, suggesting increased gene transcription . Indeed C1qR mRNA up-regulation was prevented by actinomycin D, and C1qR protein synthesis was inhibited by cycloheximide . Bone marrow vascular endothelial cell exposure to C1q, however, did not alter cC1qR or gC1qR expression, but up-regulation of the leukocyte adhesion molecule ICAM-1 was noted in the presence of aggregated C1q . The up-regulation of C1qR by inflammatory mediators and the ability of C1q itself to increase ICAM-1 expression suggest a potential role for these binding sites in vascular inflammation and immune injury. Mol Cell, 1999 May, 3(5), 601 - 9 Novel roles for classical factors at the interface between translation termination and initiation; Karimi R et al.; The pathway of bacterial ribosome recycling following translation termination has remained obscure . Here, we elucidate two essential steps and describe the roles played by the three translation factors EF-G, RRF, and IF3 . Release factor RF3 is known to catalyze the dissociation of RF1 or RF2 from ribosomes after polypeptide release . We show that the next step is dissociation of 50S subunits from the 70S posttermination complex and that it is catalyzed by RRF and EF-G and requires GTP hydrolysis . Removal of deacylated tRNA from the resulting 30S:mRNA:tRNA posttermination complex is then necessary to permit rapid 30S subunit recycling . We show that this step requires initiation factor IF3, whose role was previously thought to be restricted to promoting specific 30S initiation complex formation from free 30S subunits. Proc Natl Acad Sci U S A, 1999 Jun 8, 96(12), 7065 - 70 Guanosine triphosphatase stimulation of oncogenic Ras mutants; Ahmadian MR et al.; Interest in the guanosine triphosphatase (GTPase) reaction of Ras as a molecular drug target stems from the observation that, in a large number of human tumors, Ras is characteristically mutated at codons 12 or 61, more rarely 13 . Impaired GTPase activity, even in the presence of GTPase activating proteins, has been found to be the biochemical reason behind the oncogenicity of most Gly12/Gln61 mutations, thus preventing Ras from being switched off . Therefore, these oncogenic Ras mutants remain constitutively activated and contribute to the neoplastic phenotype of tumor cells . Here, we show that the guanosine 5'-triphosphate (GTP) analogue diaminobenzophenone-phosphoroamidate-GTP (DABP-GTP) is hydrolyzed by wild-type Ras but more efficiently by frequently occurring oncogenic Ras mutants, to yield guanosine 5'-diphosphate-bound inactive Ras and DABP-Pi . The reaction is independent of the presence of Gln61 and is most dramatically enhanced with Gly12 mutants . Thus, the defective GTPase reaction of the oncogenic Ras mutants can be rescued by using DABP-GTP instead of GTP, arguing that the GTPase switch of Ras is not irreversibly damaged . An exocyclic aromatic amino group of DABP-GTP is critical for the reaction and bypasses the putative rate-limiting step of the intrinsic Ras GTPase reaction . The crystal structures of Ras-bound DABP-beta,gamma-imido-GTP show a disordered switch I and identify the Gly12/Gly13 region as the hydrophobic patch to accommodate the DABP-moiety . The biochemical and structural studies help to define the requirements for the design of anti-Ras drugs aimed at the blocked GTPase reaction. Proc Natl Acad Sci U S A, 1999 Jun 8, 96(12), 6678 - 82 Lon and Clp family proteases and chaperones share homologous substrate-recognition domains; Smith CK et al.; Lon protease and members of the Clp family of molecular chaperones and protease regulatory subunits contain homologous regions with properties expected for substrate-binding domains . Fragments corresponding to these sequences are stably and independently folded for Lon, ClpA, and ClpY . The corresponding regions from ClpB and ClpX are unstable . All five fragments exhibit distinct patterns of binding to three proteins that are protease substrates in vivo: the heat shock transcription factor sigma32, the SOS mutagenesis protein UmuD, and Arc repressor bearing the SsrA degradation tag . Recognition of UmuD is mediated through peptide sequences within a 24-residue N-terminal region whereas recognition of both sigma32 and SsrA-tagged Arc requires sequences at the C terminus . These results indicate that the Lon and Clp proteases use the same mechanism of substrate discrimination and suggest that these related ATP-dependent bacterial proteases scrutinize accessible or disordered regions of potential substrates for the presence of specific targeting sequences. Proc Natl Acad Sci U S A, 1999 Jun 8, 96(12), 6666 - 71 Myosin light-chain kinase of smooth muscle stimulates myosin ATPase activity without phosphorylating myosin light chain; Ye LH et al.; Myosin light-chain kinase (MLCK) of smooth muscle is multifunctional, being composed of N-terminal actin-binding domain, central kinase domain, and C-terminal myosin-binding domain . The kinase domain is the best characterized; this domain activates the interaction of smooth-muscle myosin with actin by phosphorylating the myosin light chain . We have recently shown that the Met-1-Pro-41 sequence of MLCK binds to actin to inhibit this interaction . However, it is not known whether the myosin-binding domain modifies the actin-myosin interaction . We designed MLCK.cDNA to overexpress the Asp-777-Glu-972 sequence in Escherichia coli . The purified Asp-777-Glu-972 fragment, although devoid of the kinase activity, exerted a stimulatory effect on the ATPase activity of dephosphorylated myosin (Vmax = 7.36 +/- 0.44-fold, Km = 1.06 +/- 0 . 20 microM, n = 4) . When the N-terminal 39 residues of the fragment were deleted from the fragment, the resultant fragment, Met-816-Glu-972, lost the stimulatory activity . We synthesized the Ala-777-Ser-815 peptide that was deleted from the fragment and confirmed its stimulatory effect of the peptide (Vmax = 3.03 +/- 0 . 22-fold, Km = 6.93 +/- 1.61 microM, n = 3) . When this peptide was further divided into Asp-777-Met-795 and Ala-796-Ser-815 peptides, the stimulatory activity was found in the latter . We confirmed that the myosin phosphorylation did not occur during the experiments with the above fragments and peptides . Therefore, we suggest that phosphorylation is not obligatory for smooth-muscle myosin not to be active. Proc Natl Acad Sci U S A, 1999 Jun 8, 96(12), 6637 - 42 Interfacial membrane docking of cytosolic phospholipase A2 C2 domain using electrostatic potential-modulated spin relaxation magnetic resonance; Ball A et al.; The C2 domain of cytosolic phospholipase A2 (C2cPLA2) plays an important role in calcium-dependent transfer of the protein from the cytosol to internal cellular membranes as a prelude for arachidonate release from membrane phospholipids . By using a recently developed electron paramagnetic resonance approach together with 13 site-specifically nitroxide spin labeled C2cPLA2s and membrane-permeant and -impermeant spin relaxants, we have determined the orientation of C2cPLA2 with respect to the surface of vesicles of the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphomethanol . The structure reveals that the two calcium-binding regions on C2cPLA2 that display hydrophobic residues, CBR1 and CBR3, are partially inserted into the core of the membrane . CBR2 that contains predominantly hydrophilic residues is close to the membrane but not inserted . The long axis of the cylindrical C2cPLA2 molecule is tilted with respect to the bilayer normal, which brings a cluster of basic protein residues close to the phospholipid headgroups . Such an orientation places the two bound calcium ions close to the membrane surface . All together, the results provide structural support for previous proposals that binding of C2cPLA2 to the membrane interface is driven in part by insertion of hydrophobic surface loops into the membrane core . The results are contrasted with previous studies of the interfacial binding of the first C2 domain of synaptotagmin I, which has shorter surface loops that display basic residues for electrostatic interaction with the bilayer surface. Proc Natl Acad Sci U S A, 1999 Jun 8, 96(12), 6587 - 90 Cytochrome b562 folding triggered by electron transfer: approaching the speed limit for formation of a four-helix-bundle protein; Wittung-Stafshede P et al.; Ferrocytochrome b562 {Fe(II)cyt b562} folding can be triggered by photoinduced electron transfer to unfolded Fe(III)cyt b562 in 2-3 M guanidine hydrochloride solutions . The folding rates increase with decreasing guanidine hydrochloride; the extrapolated time constant for this folding process in the absence of denaturant (5 micros) is near the predicted value for intrachain diffusion . The relatively smooth energy landscape indicated for Fe(II)cyt b562 folding accords with the helical, highly symmetrical structure of the protein. Biochem J, 1999 Jun 15, 340 ( Pt 3), 639 - 47 Characterization of the membrane quinoprotein glucose dehydrogenase from Escherichia coli and characterization of a site-directed mutant in which histidine-262 has been changed to tyrosine; Cozier GE et al.; The requirements for substrate binding in the quinoprotein glucose dehydrogenase (GDH) in the membranes of Escherichia coli are described, together with the changes in activity in a site-directed mutant in which His262 has been altered to a tyrosine residue (H262Y-GDH) . The differences in catalytic efficiency between substrates are mainly related to differences in their affinity for the enzyme . Remarkably, it appears that, if a hexose is able to bind in the active site, then it is also oxidized, whereas some pentoses are able to bind (and act as competitive inhibitors), but are not substrates . The activation energies for the oxidation of hexoses and pentoses are almost identical . In a previously published model of the enzyme, His262 is at the entrance to the active site and appears to be important in holding the prosthetic group pyrroloquinoline quinone (PQQ) in place, and it has been suggested that it might play a role in electron transfer from the reduced PQQ to the ubiquinone in the membrane . The H262Y-GDH has a greatly diminished catalytic efficiency for all substrates, which is mainly due to a marked decrease in their affinities for the enzyme, but the rate of electron transfer to oxygen is unaffected . During the processing of the PQQ into the apoenzyme to give active enzyme, its affinity is markedly dependent on the pH, four groups with pK values between pH7 and pH8 being involved . Identical results were obtained with H262Y-GDH, showing that His262 it is not directly involved in this process. Biochem J, 1999 Jun 15, 340 ( Pt 3), 631 - 8 Colicin E1 forms a dimer after urea-induced unfolding; Steer BA et al.; Unfolding of the soluble colicin E1 channel peptide was examined with the use of urea as a denaturant; it was shown that it unfolds to an intermediate state in 8.5 M urea, equivalent to a dimeric species previously observed in 4 M guanidinium chloride . Single tryptophan residues, substituted into the peptide at various positions by site-directed mutagenesis, were employed as fluorescent probes of local unfolding . Unfolding profiles for specific sites within the peptide were obtained by quantifying the shifts in the fluorescence emission maxima of single tryptophan residues on unfolding and plotting them against urea concentration . Unfolding reported by tryptophan residues in the C-terminal region was not characteristic of complete peptide denaturation, as evidenced by the relatively blue-shifted values of the fluorescence emission maxima . Unfolding was also monitored by using CD spectroscopy and the fluorescent probe 2-(p-toluidinyl)-naphthalene 6-sulphonic acid; the results indicated that unfolding of helices is concomitant with the exposure of protein non-polar surface . Unfolding profiles were evaluated by non-linear least-squares curve fitting and calculation of the unfolding transition midpoint . The unfolding profiles of residues located in the N-terminal region of the peptide had lower transition midpoints than residues in the C-terminal portion . The results of unfolding analysis demonstrated that urea unfolds the peptide only partly to an intermediate state, because the C-terminal portion of the channel peptide retained significant structure in 8.5 M urea . Characterization of the peptide's global unfolding by size-exclusion HPLC revealed that the partly denatured structure that persists in 8.5 M urea is a dimer of two channel peptides, tightly associated by hydrophobic interactions . The presence of the dimerized species was confirmed by SDS/PAGE and intermolecular fluorescence resonance energy transfer. Med Pediatr Oncol, 1999 Jun, 32(6), 416 - 20 Serum antibody levels and avidities to Escherichia coli O antigens and poliovirus type 1 antigen are increased in children treated for malignant disease; Abrahamsson J et al.; BACKGROUND: Treatment of malignant disease in children is often associated with low serum immunoglobulin and reduced specific antibody levels . The aim of this study was to investigate if the functional affinity of specific antibodies in serum and saliva is reduced as well and to evaluate if antigenic exposure or treatment duration affects this antibody avidity . PROCEDURE: Serum samples were obtained from 45 children and salivary specimens from 30 children with malignant disease . The children were tested either prior to, during, or after chemotherapy . Levels of antibody to E . coli O and to poliovirus type 1 antigens were determined using an ELISA and isotype-specific relative antibody avidity was measured using thiocyanate to elute antibodies from solid-phase immobilized antigens . RESULTS: Children with malignant disease had higher levels and relative avidity indexes of serum antibodies to both antigens as compared to controls . The duration of treatment and type of malignant disease were unrelated to these parameters . In saliva, the level of antibodies to E . coli O antigens, but not to poliovirus type 1 antigen, increased during treatment . CONCLUSIONS: Both the amount and avidity of serum antibodies to these antigens are increased in children with malignant disease . This may be due to a dysregulation of the immune system caused by the malignancy and seems not to be dependent on exposure . In contrast, the avidity and levels of these antibodies in saliva seem to correlate with the presence of antigenic exposure. FEBS Lett, 1999 May 7, 450(3), 306 - 11 Effects of frontotemporal dementia FTDP-17 mutations on heparin-induced assembly of tau filaments; Goedert M et al.; Missense mutations and intronic mutations in the gene for microtubule-associated protein tau cause frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17) . Most missense mutations have as likely primary effect a reduced ability of tau to interact with microtubules . We report here an additional effect of several missense mutations, namely the stimulation of heparin-induced filament assembly of recombinant tau, despite the absence of any change in structure indicated by circular dichroism . These findings indicate that missense mutations in tau lead to frontotemporal dementia through potentially multiple mechanisms. Eur J Haematol, 1999 May, 62(5), 317 - 26 The antiplatelet activity of Escherichia coli lipopolysaccharide is mediated through a nitric oxide/cyclic GMP pathway; Sheu JR et al.; In this study, Escherichia coli LPS dose-dependently (100-500 microg/ml) and time-dependently (10-60 min) inhibited platelet aggregation in human and rabbit platelets stimulated by agonists . LPS also dose-dependently inhibited the intracellular Ca2+ mobilization in human platelets stimulated by collagen . In addition, LPS (200 and 500 microg/ml) significantly increased the formation of cyclic GMP but not cyclic AMP in platelets . LPS (200 microg/ml) significantly increased the production of nitrate within a 10-min incubation period . Furthermore, LPS also dose-dependently inhibited platelet aggregation induced by PDBu (30 nmol/l), a protein kinase C activator . These results indicate that the antiplatelet activity of E . coli LPS may be involved in the activation of a nitric oxide/cyclic GMP pathway in platelets, resulting in inhibition of platelet aggregation . Therefore, LPS-mediated alteration of platelet function may contribute to bleeding diathesis in septicemic and endotoxemic patients. Jpn J Cancer Res, 1999 Mar, 90(3), 349 - 54 Circumvention of 5-fluorouracil resistance in human stomach cancer cells by uracil phosphoribosyltransferase gene transduction; Inaba M et al.; A human stomach cancer cell line with acquired resistance to 5-fluorouracil (5-FU), NUGC-3/5FU/ L, has been found to possess reduced ability to convert 5-FU into active metabolites . We attempted in vitro gene therapy for this 5-FU-resistant cell line . NUGC-3 and NUGC-3/5FU/L cells were infected with recombinant adenovirus (Ad) containing Escherichia coli uracil phosphoribosyltransferase (UPRT) gene driven by CAG promoter (CA), AdCA-UPRT, and changes in their 5-FU metabolism and sensitivity were investigated . Activities of orotate phosphoribosyltransferase increased from 10.2 and 1.56 (nmol/mg protein/30 min) in the uninfected cells of NUGC-3 and NUGC-3/5FU/L to 216 and 237, respectively, after the transfection of UPRT gene . The 5-FU nucleotide level in the acid-insoluble fraction increased from 7.32 to 15.9 (pmol/mg protein) in NUGC-3 cells on infection with AdCA-UPRT, and in NUGC-3/5FU/L cells it increased from 1.91 to 21.4 . The 50% growth-inhibition concentration (IC50) was 12.7 micromol/liter for NUGC-3 and much higher than 100 micromol/liter for NUGC-3/5FU/L, indicating over 8-fold resistance . NUGC-3/ SFU/L transfected with the UPRT gene showed very high sensitivity to 5-FU with an IC50 of 3.2 micromol/liter . The high resistance in this metabolic activation-deficient cell line was thus completely reversed by transduction of an exogenous gene coding for a 5-FU-anabolizing enzyme. Vopr Virusol, 1999 Mar-Apr, 44(2), 89 - 92 {Diagnosis of the active form of cytomegalovirus infection based on determination of IgG antibodies to the immediate-early cytomegalovirus protein by lanthanide immunofluorescence analysis}; Pomelova VG et al.; A system for time-resolved fluoroimmunoassay (TR-FIA) is developed for detecting early IgG during the active stage of cytomegalovirus infection on the basis of CMV-control enzyme immunoassay system . The antigen is cloned and expressed in E . coli recombinant main immediate early protein of human CMV . The active form of CMV infection was detected additionally in 5 out of 25 women who gave birth to sick babies by means of the new test system in complex with other laboratory diagnostic methods . The test is recommended for prenatal diagnosis of active CMV infection in pregnant women and for predicting the risk of neonatal disease. Vopr Virusol, 1999 Mar-Apr, 44(2), 60 - 4 {Antigenic specificity of recombinant polypeptides, containing fragments of hepatitis E virus proteins}; Alatortseva GI et al.; Antigenic specificity of recombinant polypeptides HE40 and HE60 containing fragments of gene ORF2 and ORF3 protein products of hepatitis E, strain Burma, produced in E . coli cells, is analyzed . Blood sera from patients with acute hepatitis from an endemic region in Uzbekistan were tested for IgG to recombinant antigens by solid-phase enzyme immunoassay with a protein fragment coded by PRF3 gene, a synthetic peptide previously characterized in a commercial test system, as the positive control . 93% sera reacting with recombinant polypeptide HE60 and 32% reacting with HE40 reacted with the synthetic peptide . No antibodies to the studied polypeptides were detected in the sera of Moscow patients with hepatitis A, B, or C confirmed by laboratory findings . Antigenic specificity of recombinant polypeptide HE60 was confirmed by competitive enzyme immunoassay with the same peptide as the competitive antigen . Test system based on recombinant polypeptides HE40 and HE60 was used for deciphering the etiological structure of acute viral hepatitis which occurred in a hepatitis E endemic region of Uzbekistan in 1993. Acta Virol, 1998 Dec, 42(6), 369 - 74 Sequence and characterisation of the Z gene encoding ring finger protein of the lymphocytic choriomeningitis virus MX strain; Gibadulinova A et al.; We have cloned and characterised a cDNA encoding Z protein of recently identified MX strain of lymphocytic choriomeningitis virus (LCMV) persistently infecting human MaTu cells . Deduced amino acid sequence of LCMV MX Z protein showed 88.9% identity with that of the LCMV Armstrong (ARM) strain and 80.9% identity with that of the LCMV Traub (TRA) strain . It contained conserved zinc-binding RING finger domain and C-terminal proline-rich region . Northern blot analysis of total RNA from MaTu cells revealed presence of abundant truncated forms of L RNA . Z protein-specific rabbit antibodies were produced to glutathione S-transferase (GST)-Z fusion protein expressed in E . coli and used for the detection of Z protein in MaTu cells . Western blot and immunofluorescence analyses detected relatively high levels of Z protein indicating its role in maintenance of persistent LCMV. J Biol Chem, 1999 Jun 11, 274(24), 17290 - 6 Topological analysis of the membrane-localized redox-responsive sensor kinase PrrB from Rhodobacter sphaeroides 2.4.1; Ouchane S et al.; Photosynthesis gene expression in Rhodobacter sphaeroides is controlled in part by the two-component (Prr) regulatory system composed of a membrane-bound sensor kinase (PrrB) and a response regulator (PrrA) . Hydropathy profile-based computer analysis predicted that the PrrB polypeptide could contain six membrane-spanning domains at its amino terminus and a hydrophilic, cytoplasmic carboxyl terminus . Both the localization and the topology of the PrrB sensor kinase have been studied by generating a series of gene fusions with the Escherichia coli periplasmically localized alkaline phosphatase and the cytoplasmic beta-galactosidase . Eighteen prrB-phoA and five prrB-lacZ fusions were constructed and expressed in both E . coli and R . sphaeroides . Enzymatic activity assays and immunoblot analyses were performed to identify and to localize the different segments of PrrB in the membrane . The data obtained in E . coli generally correlated with the data obtained in R . sphaeroides and support the computer predictions . On the basis of the theoretical model and the results provided by these studies, a topological model for the membrane localization of the PrrB polypeptide is proposed. J Biol Chem, 1999 Jun 11, 274(24), 17267 - 74 Characterization of a novel giant scaffolding protein, CG-NAP, that anchors multiple signaling enzymes to centrosome and the golgi apparatus; Takahashi M et al.; A novel 450-kDa coiled-coil protein, CG-NAP (centrosome and Golgi localized PKN-associated protein), was identified as a protein that interacted with the regulatory region of the protein kinase PKN, having a catalytic domain homologous to that of protein kinase C . CG-NAP contains two sets of putative RII (regulatory subunit of protein kinase A)-binding motif . Indeed, CG-NAP tightly bound to RIIalpha in HeLa cells . Furthermore, CG-NAP was coimmunoprecipitated with the catalytic subunit of protein phosphatase 2A (PP2A), when one of the B subunit of PP2A (PR130) was exogenously expressed in COS7 cells . CG-NAP also interacted with the catalytic subunit of protein phosphatase 1 in HeLa cells . Immunofluorescence analysis of HeLa cells revealed that CG-NAP was localized to centrosome throughout the cell cycle, the midbody at telophase, and the Golgi apparatus at interphase, where a certain population of PKN and RIIalpha were found to be accumulated . These data indicate that CG-NAP serves as a novel scaffolding protein that assembles several protein kinases and phosphatases on centrosome and the Golgi apparatus, where physiological events, such as cell cycle progression and intracellular membrane traffic, may be regulated by phosphorylation state of specific protein substrates. J Biol Chem, 1999 Jun 11, 274(24), 17171 - 8 Oligomerization state influences the degradation rate of 3-hydroxy-3-methylglutaryl-CoA reductase; Cheng HH et al.; The steady-state level of the resident endoplasmic reticulum protein, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), is regulated, in part, by accelerated degradation in response to excess sterols or mevalonate . Previous studies of a chimeric protein (HM-Gal) composed of the membrane domain of HMGR fused to Escherichia coli beta-galactosidase, as a replacement of the normal HMGR cytosolic domain, have shown that the regulated degradation of this chimeric protein, HM-Gal, is identical to that of HMGR (Chun, K . T., Bar-Nun, S., and Simoni, R . D . (1990) J . Biol . Chem . 265, 22004-22010; Skalnik, D . G., Narita, H., Kent, C., and Simoni, R . D . (1988) J . Biol . Chem . 263, 6836-6841) . Since the cytosolic domain can be replaced with beta-galactosidase without effect on regulated degradation, it has been assumed that the cytosolic domain was not important to this process and also that the membrane domain of HMGR was both necessary and sufficient for regulated degradation . In contrast to our previous results with HM-Gal, we observed in this study that replacement of the cytosolic domain of HMGR with various heterologous proteins can have an effect on the regulated degradation, and the effect correlates with the oligomeric state of the replacement cytosolic protein . Chimeric proteins that are oligomeric in structure are relatively stable, and those that are monomeric are unstable . To test the hypothesis that the oligomeric state of the cytosolic domain of HMGR influences degradation, we use an "inducible" system for altering the oligomeric state of a protein in vivo . Using a chimeric protein that contains the membrane domain of HMGR fused to three copies of FK506-binding protein 12, we were able to induce oligomerization by addition of a "double-headed" FK506-like "dimerizer" drug (AP1510) and to monitor the degradation rate of both the monomeric form and the drug-induced oligomeric form of the protein . We show that this chimeric protein, HM-3FKBP, is unstable in the monomeric state and is stabilized by AP1510-induced oligomerization . We also examined the degradation rate of HMGR as a function of concentrations within the cell . HMGR is a functional dimer; therefore, its oligomeric state and, we predict, its degradation rate should be concentration-dependent . We observed that it is degraded more rapidly at lower concentrations. J Biol Chem, 1999 Jun 11, 274(24), 17080 - 7 A two-hybrid dual bait system to discriminate specificity of protein interactions; Serebriiskii I et al.; Biological regulatory systems require the specific organization of proteins into multicomponent complexes . Two hybrid systems have been used to identify novel components of signaling networks based on interactions with defined partner proteins . An important issue in the use of two-hybrid systems has been the degree to which interacting proteins distinguish their biological partner from evolutionarily conserved related proteins and the degree to which observed interactions are specific . We adapted the basic two-hybrid strategy to create a novel dual bait system designed to allow single-step screening of libraries for proteins that interact with protein 1 of interest, fused to DNA binding domain A (LexA), but do not interact with protein 2, fused to DNA binding domain B (lambda cI) . Using the selective interactions of Ras and Krev-1(Rap1A) with Raf, RalGDS, and Krit1 as a model, we systematically compared LexA- and cI-fused baits and reporters . The LexA and cI baitr reporter systems are well matched for level of bait expression and sensitivity range for interaction detection and allow effective isolation of specifically interacting protein pairs against a nonspecific background . These reagents should prove useful to refine the selectivity of library screens, to reduce the isolation of false positives in such screens, and to perform directed analyses of sequence elements governing the interaction of a single protein with multiple partners. J Biol Chem, 1999 Jun 11, 274(24), 17011 - 6 Defining the domain of binding of F1 subunit epsilon with the polar loop of F0 subunit c in the Escherichia coli ATP synthase; Hermolin J et al.; We have previously shown that the E31C-substituted epsilon subunit of F1 can be cross-linked by disulfide bond formation to the Q42C-substituted c subunit of F0 in the Escherichia coli F1F0-ATP synthase complex (Zhang, Y., and Fillingame, R . H . (1995) J . Biol . Chem . 270, 24609-24614) . The interactions of subunits epsilon and c are thought to be central to the coupling of H+ transport through F0 to ATP synthesis in F1 . To further define the domains of interaction, we have introduced additional Cys into subunit epsilon and subunit c and tested for cross-link formation following sulfhydryl oxidation . The results show that Cys, in a continuous stretch of residues 26-33 in subunit epsilon, can be cross-linked to Cys at positions 40, 42, and 44 in the polar loop region of subunit c . The results are interpreted, and the subunit interaction is modeled using the NMR and x-ray diffraction structures of the monomeric subunits together with information on the packing arrangement of subunit c in a ring of 12 subunits . In the model, residues 26-33 form a turn of antiparallel beta-sheet which packs between the polar loop regions of adjacent subunit c at the cytoplasmic surface of the c12 oligomer. J Biol Chem, 1999 Jun 11, 274(24), 16788 - 95 Differential regulation of Drosophila tyrosine hydroxylase isoforms by dopamine binding and cAMP-dependent phosphorylation; Vie A et al.; Tyrosine hydroxylase (TH) catalyzes the first step in dopamine biosynthesis in Drosophila as in vertebrates . We have previously reported that tissue-specific alternative splicing of the TH primary transcript generates two distinct TH isoforms in Drosophila, DTH I and DTH II (Birman, S., Morgan, B., Anzivino, M., and Hirsh, J . (1994) J . Biol . Chem . 269, 26559-26567) . Expression of DTH I is restricted to the central nervous system, whereas DTH II is expressed in non-nervous tissues like the epidermis . The two enzymes present a single structural difference; DTH II specifically contains a very acidic segment of 71 amino acids inserted in the regulatory domain . We show here that the enzymatic and regulatory properties of vertebrate TH are generally conserved in insect TH and that the isoform DTH II presents unique characteristics . The two DTH isoforms were expressed as apoenzymes in Escherichia coli and purified by fast protein liquid chromatography . The recombinant DTH isoforms are enzymatically active in the presence of ferrous iron and a tetrahydropteridine co-substrate . However, the two enzymes differ in many of their properties . DTH II has a lower Km value for the co-substrate (6R)-tetrahydrobiopterin and requires a lower level of ferrous ion than DTH I to be activated . The two isoforms also have a different pH profile . As for mammalian TH, enzymatic activity of the Drosophila enzymes is decreased by dopamine binding, and this effect is dependent on ferrous iron levels . However, DTH II appears comparatively less sensitive than DTH I to dopamine inhibition . The central nervous system isoform DTH I is activated through phosphorylation by cAMP-dependent protein kinase (PKA) in the absence of dopamine . In contrast, activation of DTH II by PKA is only manifest in the presence of dopamine . Site-directed mutagenesis of Ser32, a serine residue occurring in a PKA site conserved in all known TH proteins, abolishes phosphorylation of both isoforms and activation by PKA . We propose that tissue-specific alternative splicing of TH has a functional role for differential regulation of dopamine biosynthesis in the nervous and non-nervous tissues of insects. J Biol Chem, 1999 Jun 11, 274(24), 16727 - 35 Histidine 179 mutants of GTP cyclohydrolase I catalyze the formation of 2-amino-5-formylamino-6-ribofuranosylamino-4(3H)-pyrimidinone triphosphate; Bracher A et al.; GTP cyclohydrolase I catalyzes the conversion of GTP to dihydroneopterin triphosphate . The replacement of histidine 179 by other amino acids affords mutant enzymes that do not catalyze the formation of dihydroneopterin triphosphate . However, some of these mutant proteins catalyze the conversion of GTP to 2-amino-5-formylamino-6-ribofuranosylamino-4(3H)-pyrimidinone 5'-triphosphate as shown by multinuclear NMR analysis . The equilibrium constant for the reversible conversion of GTP to the ring-opened derivative is approximately 0.1 . The wild-type enzyme converts the formylamino pyrimidine derivative to dihydroneopterin triphosphate; the rate is similar to that observed with GTP as substrate . The data support the conclusion that the formylamino pyrimidine derivative is an intermediate in the overall reaction catalyzed by GTP cyclohydrolase I. Development, 1999 Jul, 126(13), 2841 - 53 The N-terminal domain of Sxl protein disrupts Sxl autoregulation in females and promotes female-specific splicing of tra in males; Deshpande G et al.; Sex determination in Drosophila depends upon the post-transcriptional regulatory activities of the Sex-lethal (Sxl) gene . Sxl maintains the female determined state and activates female differentiation pathways by directing the female-specific splicing of Sxl and tra pre-mRNAs . While there is compelling evidence that Sxl proteins regulate splicing by directly binding to target RNAs, previous studies indicate that the