Escherichia coli

Biochemistry, 1997 Sep 9, 36(36), 10867 - 71
Identification of essential amino acids in phenylalanine ammonia-lyase by site-directed mutagenesis; Langer B et al.; The postulated precursor of the prosthetic dehydroalanine of phenylalanine ammonia-lyase (PAL), serine 202, was changed to cysteine by site-directed mutagenesis . After cloning and heterologous expression in Escherichia coli, the gene product was assayed for PAL activity . Mutant S202C showed full catalytic activity, and its kinetic constants and the amount of thiol groups were identical to those of wild-type PAL . It must be concluded that in a posttranslational modification both water and hydrogen sulfide can be eliminated from the amino acid in position 202 to form dehydroalanine . In an attempt to identify further amino acids essential either for the posttranslational modification or for catalysis, arginine 174, glutamine 425, and lysine 499 were changed to isoleucine . Analysis of the heterologously expressed mutated gene products revealed that only the R174I mutant showed a significantly lower Vmax value (1/450) identifying this arginine as important . This finding was supported by treatment of wild-type PAL and mutant R174I with phenylglyoxal and 2,3-butandione . Both react specifically with the guanidino group of arginine . They irreversibly inhibited wild-type PAL but had no influence of the Vmax value of mutant R174I . Preincubation with l-phenylalanine protected wild-type PAL from inhibition by phenylglyoxal indicating that arginine 174 is close to the active site . Incubation with KCN irreversibly abolished the remaining activity of mutant R174I leading to the conclusion that arginine 174 is important in catalysis.

J Biol Chem, 1997 Sep 5, 272(36), 22924 - 8
Kringle 5 of plasminogen is a novel inhibitor of endothelial cell growth; Cao Y et al.; Angiostatin is a potent angiogenesis inhibitor which has been identified as an internal fragment of plasminogen that includes its first four kringle modules . We have recently demonstrated that the anti-endothelial cell proliferative activity of angiostatin is also displayed by the first three kringle structures of plasminogen and marginally so by kringle 4 (Cao, Y., Ji, R.-W., Davidson, D., Schaller, J., Marti, D., Sohndel, S., McCance, S . G., O'Reilly, M . S . , Llinas, M., and Folkman, J . (1996) J . Biol . Chem . 271, 29461-29467) . We now report that the kringle 5 fragment of human plasminogen is a specific inhibitor for endothelial cell proliferation . Kringle 5 obtained as a proteolytic fragment of human plasminogen displays potent inhibitory effect on bovine capillary endothelial cells with a half-maximal concentration (ED50) of approximately 50 nM . Thus, kringle 5 would appear to be more potent than angiostatin on inhibition of basic fibroblast growth factor-stimulated capillary endothelial cell proliferation . Appropriately folded recombinant mouse kringle 5 protein, expressed in Escherichia coli, exhibits a comparable inhibitory effect as the proteolytic kringle 5 fragment . Thus, kringle 5 domain of human plasminogen is a novel endothelial inhibitor that is sufficiently potent to block the growth factor-stimulated endothelial cell growth.

J Biol Chem, 1997 Sep 5, 272(36), 22891 - 7
Efficient assembly of functional cytochrome P450 2C2 requires a spacer sequence between the N-terminal signal anchor and catalytic domains; Chen CD et al.; Cytochromes P450 (P450) are anchored to the endoplasmic reticulum membrane by an N-terminal transmembrane sequence with the catalytic domain facing the cytoplasmic side . Within the peptide sequence linking these two domains in P450 2C2 is a glycine-rich region from residues 22 to 28 . To examine the role of this region, deletion and substitution mutations were constructed, and the activities and spectral properties were determined for the mutant proteins expressed in COS-1 cells, insect cells, and bacteria . Deletion of residues 22 to 28 or substitution of 7 valines for this region inactivated the proteins in COS-1 cells, and no P450 species was detected for these mutations in bacteria or insect cells . Substitution of the three glycine residues with alanine or proline or the entire sequence from 22 to 28 with 7 alanines did not reduce lauric acid hydroxylase activity of the proteins expressed in COS-1 cells . Reducing the number of alanines substituted to 4, 3, and 2 progressively decreased activity in COS-1 cells to undetectable levels when 2 alanines were substituted . The loss of activity in COS-1 cells correlated with decreased expression of hemoprotein with a reduced difference spectrum of 450 nm (P450 species) and a corresponding increase in the inactive P420 species in insect cells and bacteria . The activities expressed per nanomole of P450 in insect microsomes were similar for P450 2C2 and the alanine substitution mutants, including the mutant with 2 alanines which was inactive in COS-1 cells . The rates of conversion of P450 to P420 resulting from incubation at 48 degrees C in vitro were not changed sufficiently to explain the increase in expressed P420 observed for the mutants with 3 or 7 alanines substituted . These data are consistent with a role for the residue 22-28 region as a linker that facilitates the folding of P450; however, once the protein is properly folded into the functional P450 species, this region has little influence on the stability and activity of the enzyme.

J Biol Chem, 1997 Sep 5, 272(36), 22809 - 16
Human lysyl-tRNA synthetase accepts nucleotide 73 variants and rescues Escherichia coli double-defective mutant; Shiba K et al.; The nucleotide 73 (N73) "discriminator" base in the acceptor stem is a key element for efficient and specific aminoacylation of tRNAs and of microhelix substrates derived from tRNA acceptor stems . This nucleotide was possibly one of the first to be used for differentiating among groups of early RNA substrates by tRNA synthetases . In contrast to many other synthetases, we report here that the class II human lysyl-tRNA synthetase is relatively insensitive to the nature of N73 . We cloned, sequenced, and expressed the enzyme, which is a close homologue of the class II yeast aspartyl-tRNA synthetase whose co-crystal structure (with tRNAAsp) is known . The latter enzyme has a strong requirement for G73, which interacts with 4 of the 14 residues within the "motif 2" loop of the enzyme . Even though eukaryotic lysine tRNAs also encode G73, the motif 2 loop sequence of lysyl-tRNA synthetase differs at multiple positions from that of the aspartate enzyme . Indeed, the recombinant human lysine enzyme shows little preference for G, and even charges human tRNA transcripts encoding the A73 found in E . coli lysine tRNAs . Moreover, while the lysine enzyme is the only one in E . coli to be encoded by two separate genes, a double mutant that disables both genes is complemented by a cDNA expressing the human protein . Thus, the sequence of the loop of motif 2 of human lysyl-tRNA synthetase specifies a structural variation that accommodates nucleotide degeneracy at position 73 . This sequence might be used as a starting point for obtaining highly specific interactions with any given N73 by simple amino acid replacements.

J Biol Chem, 1997 Sep 5, 272(36), 22766 - 70
The UL8 subunit of the heterotrimeric herpes simplex virus type 1 helicase-primase is required for the unwinding of single strand DNA-binding protein (ICP8)-coated DNA substrates; Falkenberg M et al.; The Herpes simplex virus type 1 primosome consists of three subunits that are the products of the UL5, UL8, and UL52 genes . The heterotrimeric enzyme has DNA-dependent ATPase, helicase, and primase activities . Earlier studies show that a subassembly consisting of the UL5 and UL52 gene products was indistinguishable from the heterotrimeric enzyme in its helicase and primase activities . We demonstrate here that the UL8 protein is required for the helicase activity of the UL5/52 subassembly on long duplex DNA substrates (>30 nucleotides) with a single-stranded DNA loading site fully coated with the virus-encoded single strand DNA binding protein, ICP8 . The Escherichia coli single strand DNA binding protein cannot substitute for ICP8, suggesting a specific physical interaction between ICP8 and the UL8 protein . Surface plasmon resonance measurements demonstrated an interaction between ICP8 and the UL5/52/8 heterotrimer but not with the UL5/52 subassembly or the UL8 protein alone . At a subsaturating level of ICP8, the UL5/52 subassembly does show helicase activity, suggesting that the subassembly can bind to single-stranded DNA but not to ICP8-coated DNA.

J Biol Chem, 1997 Sep 5, 272(36), 22728 - 35
Expression and efficient export of enzymatically active Mycobacterium tuberculosis glutamine synthetase in Mycobacterium smegmatis and evidence that the information for export is contained within the protein; Harth G et al.; We have investigated the expression and extracellular release of active, recombinant Mycobacterium tuberculosis glutamine synthetase (EC 6.3.1.2), an enzyme that is a potentially important determinant of M . tuberculosis infection and whose extracellular release is correlated with pathogenicity . The M . tuberculosis glutamine synthetase gene encodes a polypeptide of 478 amino acids; 12 such subunits comprise the active enzyme . Northern blot, nuclease S1, and primer extension analyses revealed glutamine synthetase specific transcripts of approximately 1,550 and 1,650 nucleotides produced under low and high nitrogen conditions, respectively . Expression of recombinant M . tuberculosis glutamine synthetase in Escherichia coli YMC21E, a glutamine synthetase deletion mutant, led to transcomplementation of the mutant but not to release of active enzyme . Expression in Mycobacterium smegmatis 1-2c, from the gene's own promoter, resulted in the release of >95% of all recombinant enzyme . No hybrid molecules containing M . tuberculosis and M . smegmatis glutamine synthetase subunits were detected . Native and recombinant exported and intracellular glutamine synthetase molecules were indistinguishable from one another by mass, N-terminal amino acid sequence, antibody reactivity, and enzymatic activity . Since M . tuberculosis glutamine synthetase is similar to other, strictly intracellular, bacterial glutamine synthetases and the DNA sequence upstream of the structural gene does not encode a leader peptide, the information to target the protein for export must be contained in its amino acid sequence and/or conformation.

J Biol Chem, 1997 Sep 5, 272(36), 22714 - 20
Methyl-directed repair of mismatched small heterologous sequences in cell extracts from Escherichia coli; Fang W et al.; The methyl-directed DNA repair efficiency of a set of M13mp18 heteroduplexes containing 1-8 or 22 unpaired bases was determined by using an in vitro DNA mismatch repair assay . The unpaired bases of each heteroduplex residing at overlapping recognition sites of two restriction endonucleases allow independent assay of repair on either DNA strand . Our results showed that the repair of small nucleotide heterologies in Escherichia coli extracts was very similar to base-base mismatch repair, being strand-specific and highly biased to the unmethylated strand . The in vitro activity was also dependent on products of mutH, mutL, mutS, and uvrD loci and was equally efficient on nucleotide insertions and deletions . The repair levels of small heterologies were affected by base composition of the heterologies . However, the extent of repair of heteroduplexes containing small heterologous sequences was found to decrease with an increase in the number of unpaired bases . Heteroduplexes containing an extra nucleotide of 22 bases provoked very low level of methyl-directed repair.

J Biol Chem, 1997 Sep 5, 272(36), 22648 - 53
The X-ray structure of the PurR-guanine-purF operator complex reveals the contributions of complementary electrostatic surfaces and a water-mediated hydrogen bond to corepressor specificity and binding affinity; Schumacher MA et al.; The purine repressor, PurR, is the master regulatory protein of de novo purine nucleotide biosynthesis in Escherichia coli . This dimeric transcription factor is activated to bind to cognate DNA operator sites by initially binding either of its physiologically relevant, high affinity corepressors, hypoxanthine (Kd = 9.3 microM) or guanine (Kd = 1.5 microM) . Here, we report the 2.5-A crystal structure of the PurR-guanine-purF operator ternary complex and complete the atomic description of 6-oxopurine-induced repression by PurR . As anticipated, the structure of the PurR-guanine-purF operator complex is isomorphous to the PurR-hypoxanthine-purF operator complex, and their protein-DNA and protein-corepressor interactions are nearly identical . The former finding confirms the use of an identical allosteric DNA-binding mechanism whereby corepressor binding 40 A from the DNA-binding domain juxtaposes the hinge regions of each monomer, thus favoring the formation and insertion of the critical minor groove-binding hinge helices . Strikingly, the higher binding affinity of guanine for PurR and the ability of PurR to discriminate against 2-oxopurines do not result from direct protein-ligand interactions, but rather from a water-mediated contact with the exocyclic N-2 of guanine, which dictates the presence of a donor group on the corepressor, and the better electrostatic complementarity of the guanine base and the corepressor-binding pocket.

J Biol Chem, 1997 Sep 5, 272(36), 22576 - 82
Overexpression of nreB, a new GATA factor-encoding gene of Penicillium chrysogenum, leads to repression of the nitrate assimilatory gene cluster; Haas H et al.; To investigate the mechanism of nitrogen metabolite repression in the biotechnologically important fungus Penicillium chrysogenum a polymerase chain reaction approach was employed to identify transcription factors involved in this regulatory circuit, leading to the isolation of a new gene (nreB) encoding a 298 amino acid protein . Despite a low overall amino acid sequence identity of approximately 30%, it shares several features with Dal80p/Uga43p and Gzf3p/Nil2p, both repressors in nitrogen metabolism in Saccharomyces cerevisiae . All three proteins contain an N-terminal GATA-type zinc finger motif, displaying 86% amino acid sequence identity, and a putative leucine zipper motif in the C terminus . Northern blot analysis revealed the presence of two nreB transcripts, 1.8 and 1.5 kilobases in length, that differ in polyadenylation sites . The steady state level of both transcripts is subject to nitrogen metabolite repression . The putative DNA binding domain of NREB, expressed as a fusion protein in Escherichia coli, binds in vitro to GATA sites of its own 5'-upstream region as well as in the promoter of the nitrate assimilation gene cluster . Consistent with a role in the regulation of nitrogen metabolism, overexpression of nreB leads to repression of nitrate assimilatory genes . Hence, the simple view of nitrogen regulation by four GATA factors in yeast, but only one key regulator in filamentous ascomycetes seems no longer valid.

J Biol Chem, 1997 Sep 5, 272(36), 22509 - 13
Negatively charged anabaena flavodoxin residues (Asp144 and Glu145) are important for reconstitution of cytochrome P450 17alpha-hydroxylase activity; Jenkins CM et al.; Catalysis by microsomal cytochromes P450 requires the membrane-bound enzyme NADPH-cytochrome P450 reductase (P450 reductase), which transfers electrons to the P450 heme via a flavodoxin-like domain . Previously, we reported that Escherichia coli flavodoxin (Fld), a soluble electron transfer protein, directly interacts with bovine cytochrome P450 17alpha-hydroxylase/17,20-lyase (P450c17) and donates electrons to this enzyme when reconstituted with NADPH-ferredoxin (flavodoxin) reductase (FNR) (Jenkins, C . M., and Waterman, M . R . (1994) J . Biol . Chem . 269, 27401-27408) . To investigate whether flavodoxins can serve as useful models of the analogous domain in P450 reductase, we have examined the FNR-Fld system from the cyanobacterium Anabaena . Mutagenesis of two acidic Anabaena Fld residues (D144A and E145A) significantly decreased flavodoxin-supported P450c17 progesterone 17alpha-hydroxylase activity . Specifically, D144A exhibited only 15% of the activity of wild-type Fld, whereas the adjacent mutation, E145A, caused a 40% loss in activity . P450-dependent hydrogen peroxide/superoxide production by wild-type FNR-Fld was measurably higher than that generated by FNR-D144A or FNR-E145A, indicating that the mutations do not lead to P450 heme-mediated electron uncoupling . Interestingly, the D144A and E145A mutants bind with equal or even greater affinity to P450c17 than wild-type Fld . Furthermore, these mutations (D144A and E145A) actually increased cytochrome c reductase activity (35 and 100% higher than wild type) . Anabaena Fld residues Asp144 and Glu145 align closely with rat P450 reductase residue Asp208, which has been shown by mutagenesis to be important in electron transfer to P4502B1 but not to cytochrome c (Shen, A . L., and Kasper, C . B . (1995) J . Biol . Chem . 270, 27475-27480) . Thus, these residues in flavodoxins and P450 reductase appear to have similar functions in P450 recognition and/or electron transfer, supporting the hypothesis that flavodoxins represent valid models for the FMN-binding domain of P450 reductase.

J Biol Chem, 1997 Sep 5, 272(36), 22417 - 24
Cysteine sulfinate desulfinase, a NIFS-like protein of Escherichia coli with selenocysteine lyase and cysteine desulfurase activities . Gene cloning, purification, and characterization of a novel pyridoxal enzyme; Mihara H et al.; Selenocysteine lyase (EC 4.4.1.16) exclusively decomposes selenocysteine to alanine and elemental selenium, whereas cysteine desulfurase (NIFS protein) of Azotobacter vinelandii acts indiscriminately on both cysteine and selenocysteine to produce elemental sulfur and selenium respectively, and alanine . These proteins exhibit some sequence homology . The Escherichia coli genome contains three genes with sequence homology to nifS . We have cloned the gene mapped at 63.4 min in the chromosome and have expressed, purified to homogeneity, and characterized the gene product . The enzyme comprises two identical subunits with 401 amino acid residues (Mr 43,238) and contains pyridoxal 5'-phosphate as a coenzyme . The enzyme catalyzes the removal of elemental sulfur and selenium atoms from L-cysteine, L-cystine, L-selenocysteine, and L-selenocystine to produce L-alanine . Because L-cysteine sulfinic acid was desulfinated to form L-alanine as the preferred substrate, we have named this new enzyme cysteine sulfinate desulfinase . Mutant enzymes having alanine substituted for each of the four cysteinyl residues (Cys-100, Cys-176, Cys-323, and Cys-358) were all active . Cys-358 corresponds to Cys-325 of A . vinelandii NIFS, which is conserved among all NIFS-like proteins and catalytically essential (Zheng, L., White, R . H., Cash, V . L., and Dean, D . R . (1994) Biochemistry 33, 4714-4720), is not required for cysteine sulfinate desulfinase . Thus, the enzyme is distinct from A . vinelandii NIFS in this respect.

J Biol Chem, 1997 Sep 5, 272(36), 22409 - 16
The sequence of the alternatively spliced sixth exon of alpha-tropomyosin is critical for cooperative actin binding but not for interaction with troponin; Hammell RL et al.; Tropomyosins, a family of highly conserved coiled-coil actin binding proteins, can differ as a consequence of alternative expression of several exons (Lees-Miller, J., and Helfman, D . (1991) BioEssays 13, 429-437) . Exon 6, which encodes residues 189-213 in long, 284-residue tropomyosins, has two alternative forms, exon 6a or 6b, both highly conserved throughout evolution . In alpha-tropomyosin, exon 6a or 6b is not specific to any one of the nine isoforms . Exon 6b encodes part of a putative Ca2+-sensitive troponin binding site in striated muscle tropomyosins, suggesting that the exon 6-encoded region may be specialized for certain tropomyosin functions . A series of recombinant, unacetylated tropomyosin exon 6 deletion and substitution mutants and chimeras was expressed in Escherichia coli to determine the requirements of exon 6 for tropomyosin function . Functional properties of the tropomyosins were defined by actin affinity measured by cosedimentation, troponin T affinity using a newly developed biosensor assay, and regulation of the actomyosin MgATPase . The region of tropomyosin encoded by exon 6 affects actin affinity but not thin filament assembly, troponin T binding, or regulation with troponin . The tropomyosins with exon 6a or 6b function normally whether a striated muscle exon 9a or smooth/non-muscle exon 9d is present . However, the effect of deleting 21 amino acids encoded by exon 6 or replacing it with a GCN4 leucine zipper sequence depends on the COOH-terminal sequence.

Nature, 1997 Sep 4, 389(6646), 96 - 100
Structure of the inhibitory receptor for human natural killer cells resembles haematopoietic receptors; Fan QR et al.; Abnormal cells deficient in class I major histocompatibility complex (MHC) expression are lysed by a class of lymphocytes called natural killer (NK) cells . This lysis provides a defence against pathogens and tumour cells that downregulate MHC expression to avoid an MHC-restricted, T-cell immune response . Normal cells escape lysis because their MHC molecules are recognized by NK-cell inhibitory receptors, which inhibit lysis . Several such inhibitory receptor families have been described in humans and mice . In the human killer-cell inhibitory receptor family, individual p58 members are specific for a subset of class I human leukocyte antigen (HLA)-C molecules . The human p58 natural killer-cell inhibitory receptor clone 42 recognizes HLA-Cw4, -Cw2 and -Cw6, but not HLA-Cw3, -Cw2, -Cw7 or -Cw8, which are recognized by p58 killer-cell inhibitor receptor clone 43 . We have determined the X-ray structure of the p58 NK-cell inhibitory receptor clone 42 at 1.7-A resolution . The structure has tandem immunoglobulin-like domains positioned at an acute, 60-degree angle . Loops on the outside of the elbow between the domains form a binding site projected away from the NK-cell surface . The topology of the domains and their arrangement relative to each other reveal a relationship to the haematopoietic receptor family, with implications for the signalling mechanism in NK cells.

Nature, 1997 Sep 4, 389(6646), 93 - 6
Reversal in the direction of movement of a molecular motor; Henningsen U et al.; Kinesin and non-claret disjunctional (ncd) are molecular motors of the kinesin superfamily that move in opposite directions along microtubules . The molecular basis underlying the direction of movement is unclear, although it is thought to be an intrinsic property of the motor domain, a conserved region about 330 amino acids in length . The motor domain is found at the amino terminus in conventional kinesins, but at the carboxy terminus in ncd . Here we report on a chimaera composed of the motor domain of the minus-end-directed kinesin of Neurospora crassa . The bacterially expressed fusion protein was tested in motility assays using polarity-marked microtubules . Surprisingly, the chimaera moved towards the plus end, demonstrating that the polarity of force generation of the ncd motor domain has been reversed . This finding indicates that the domain organization, particularly the position of the motor domain, is of fundamental importance for the polarity of force production . It also demonstrates that the direction of microtubule movement is not controlled solely by the motor domain.

Proc Natl Acad Sci U S A, 1997 Sep 2, 94(18), 9637 - 42
The 2.0-A resolution crystal structure of a trimeric antibody fragment with noncognate VH-VL domain pairs shows a rearrangement of VH CDR3; Pei XY et al.; The 2.0-A resolution x-ray crystal structure of a novel trimeric antibody fragment, a "triabody," has been determined . The trimer is made up of polypeptides constructed in a manner identical to that previously described for some "diabodies": a VL domain directly fused to the C terminus of a VH domain-i.e., without any linker sequence . The trimer has three Fv heads with the polypeptides arranged in a cyclic, head-to-tail fashion . For the particular structure reported here, the polypeptide was constructed with a VH domain from one antibody fused to the VL domain from an unrelated antibody giving rise to "combinatorial" Fvs upon formation of the trimer . The structure shows that the exchange of the VL domain from antibody B1-8, a Vlambda domain, with the VL domain from antibody NQ11, a Vkappa domain, leads to a dramatic conformational change in the VH CDR3 loop of antibody B1-8 . The magnitude of this change is similar to the largest of the conformational changes observed in antibody fragments in response to antigen binding . Combinatorial pairing of VH and VL domains constitutes a major component of antibody diversity . Conformationally flexible antigen-binding sites capable of adapting to the specific CDR3 loop context created upon VH-VL pairing may be employed by the immune system to maximize the structural diversity of the immune response.

Proc Natl Acad Sci U S A, 1997 Sep 2, 94(18), 9602 - 7
Identification of an intramolecular interaction between small regions in type V adenylyl cyclase that influences stimulation of enzyme activity by Gsalpha; Scholich K et al.; Using the full-length and two engineered soluble forms (C1-C2 and Cla-C2) of type V adenylyl cyclase (ACV), we have investigated the role of an intramolecular interaction in ACV that modulates the ability of the alpha subunit of the stimulatory GTP-binding protein of AC (Gsalpha) to stimulate enzyme activity . Concentration-response curves with Gsalpha suggested the presence of high and low affinity sites on ACV, which interact with the G protein . Activation of enzyme by Gsalpha interaction at these two sites was most apparent in the C1a-C2 form of ACV, which lacks the C1b region (K572-F683) . Yeast two-hybrid data demonstrated that the C1b region interacted with the C2 region and its 64-aa subdomain, C2I . Using peptides corresponding to the C2I region of ACV, we investigated the role of the C1b/C2I interaction on Gsalpha-mediated stimulation of C1-C2 and full-length ACV . Our data demonstrate that a 10-aa peptide corresponding to L1042-T1051 alters the profile of the activation curves of full-length and C1-C2 forms of ACV by different Gsalpha concentrations to mimic the activation profile observed with C1a-C2 ACV . The various peptides used in our studies did not alter forskolin-mediated stimulation of full-length and C1-C2 forms of ACV . We conclude that the C1b region of ACV interacts with the 10-aa region (L1042-T1051) in the C2 domain of the enzyme to modulate Gsalpha-elicited stimulation of activity.

Proc Natl Acad Sci U S A, 1997 Sep 2, 94(18), 9585 - 9
The yeast peptide-methionine sulfoxide reductase functions as an antioxidant in vivo; Moskovitz J et al.; A gene homologous to methionine sulfoxide reductase (msrA) was identified as the predicted ORF (cosmid 9379) in chromosome V of Saccharomyces cerevisiae encoding a protein of 184 amino acids . The corresponding protein has been expressed in Escherichia coli and purified to homogeneity . The recombinant yeast MsrA possessed the same substrate specificity as the other known MsrA enzymes from mammalian and bacterial cells . Interruption of the yeast gene resulted in a null mutant, DeltamsrA::URA3 strain, which totally lost its cellular MsrA activity and was shown to be more sensitive to oxidative stress in comparison to its wild-type parent strain . Furthermore, high levels of free and protein-bound methionine sulfoxide were detected in extracts of msrA mutant cells relative to their wild-type parent cells, under various oxidative stresses . These findings show that MsrA is responsible for the reduction of methionine sulfoxide in vivo as well as in vitro in eukaryotic cells . Also, the results support the proposition that MsrA possess an antioxidant function . The ability of MsrA to repair oxidative damage in vivo may be of singular importance if methionine residues serve as antioxidants.

Proc Natl Acad Sci U S A, 1997 Sep 2, 94(18), 9556 - 61
Localization of the C terminus of the assembly domain of hepatitis B virus capsid protein: implications for morphogenesis and organization of encapsidated RNA; Zlotnick A et al.; The capsid protein of hepatitis B virus, consisting of an "assembly" domain (residues 1-149) and an RNA-binding "protamine" domain (residues 150-183), assembles from dimers into icosahedral capsids of two different sizes . The C terminus of the assembly domain (residues 140-149) functions as a morphogenetic switch, longer C termini favoring a higher proportion of the larger capsids, it also connects the protamine domain to the capsid shell . We now have defined the location of this peptide in capsids assembled in vitro by engineering a mutant assembly domain with a single cysteine at its C terminus (residue 150), labeling it with a gold cluster and visualizing the cluster by cryo-electron microscopy . The labeled protein is unimpaired in its ability to form capsids . Our density map reveals a single undecagold cluster under each fivefold and quasi-sixfold vertex, connected to sites at either end of the undersides of the dimers . Considering the geometry of the vertices, the C termini must be more crowded at the fivefolds . Thus, a bulky C terminus would be expected to favor formation of the larger (T = 4) capsids, which have a greater proportion of quasi-sixfolds . Capsids assembled by expressing the full-length protein in Escherichia coli package bacterial RNAs in amounts equivalent to the viral pregenome . Our density map of these capsids reveals a distinct inner shell of density-the RNA . The RNA is connected to the protein shell via the C-terminal linkers and also makes contact around the dimer axes.

Biochemistry, 1997 Sep 2, 36(35), 10793 - 800
Kinetic evidence for peptide-induced oligomerization of the molecular chaperone DnaK at heat shock temperatures; Farr CD et al.; The pre-steady-state kinetics of the binding of a fluorescent peptide (dansyl-KLIGVLSSLFRPK, fVSV13) to the Escherichia coli molecular chaperone DnaK were investigated over a range of temperatures (25-42 degrees C) . At 42 degrees C, over a wide range of peptide concentrations, the fVSV13 peptide bound to DnaK with biphasic kinetics: a rapid burst in the DnaK-fVSV13 signal in the first 5 s was followed by a gradual reduction in the signal over the next 100 s . The descending portion of each biphasic trace followed the equation F(t) = DeltaF exp(-kdt) + Finfinity, where DeltaF, kd, and Finfinity are the amplitude, the apparent first-order rate constant, and the fluorescence end point, respectively . Both DeltaF and kd increased with increasing concentrations of DnaK, which suggests that the loss of the DnaK-fVSV13 signal is caused by a bimolecular reaction . We propose that (i) the fVSV13 peptide binds to and induces a conformational change in the DnaK monomer {E + P right harpoon over left harpoon (EP)*}; and (ii) the conformational change promotes the formation of oligomeric DnaK-peptide complexes {En + (EP)* right harpoon over left harpoon En-EP} . The term (EP)* denotes a monomeric DnaK-peptide complex in which the bound peptide is fluorescent; En-EP denotes an oligomeric DnaK-peptide complex in which the fluorescence of the bound peptide is quenched . Numerical fitting of the stopped-flow data to reactions (i) and (ii) yielded values for the four rate constants . When the proposed kinetic model was tested by conducting experiments in the presence of excess peptide or excess ATP&sbd;conditions which inhibit oligomerization&sbd;DnaK-fVSV13 complex formation proceeded to stable asymptotes, with no reduction in the DnaK-fVSV13 signal at long times.

Biochemistry, 1997 Sep 2, 36(35), 10685 - 95
Molecular structures of the S124A, S124T, and S124V site-directed mutants of UDP-galactose 4-epimerase from Escherichia coli; Thoden JB et al.; UDP-galactose 4-epimerase plays a critical role in sugar metabolism by catalyzing the interconversion of UDP-galactose and UDP-glucose . Originally, it was assumed that the enzyme contained a "traditional" catalytic base that served to abstract a proton from the 4'-hydroxyl group of the UDP-glucose or UDP-galactose substrates during the course of the reaction . However, recent high-resolution X-ray crystallographic analyses of the protein from Escherichia coli have demonstrated the lack of an aspartate, a glutamate, or a histidine residue properly oriented within the active site cleft for serving such a functional role . Rather, the X-ray crystallographic investigation of the epimerase.NADH.UDP-glucose abortive complex from this laboratory has shown that both Ser 124 and Tyr 149 are located within hydrogen bonding distance to the 4'- and 3'-hydroxyl groups of the sugar, respectively . To test the structural role of Ser 124 in the reaction mechanism of epimerase, three site-directed mutant proteins, namely S124A, S124T, and S124V, were constructed and crystals of the S124A.NADH.UDP, S124A.NADH.UDP-glucose, S124T . NADH.UDP-glucose, and S124V.NADH.UDP-glucose complexes were grown . All of the crystals employed in this investigation belonged to the space group P3221 with the following unit cell dimensions: a = b = 83.8 A, c = 108.4 A, and one subunit per asymmetric unit . X-ray data sets were collected to at least 2.15 A resolution, and each protein model was subsequently refined to an R value of lower than 19.0% for all measured X-ray data . The investigations described here demonstrate that the decreases in enzymatic activities observed for these mutant proteins are due to the loss of a properly positioned hydroxyl group at position 124 and not to major tertiary and quaternary structural perturbations . In addition, these structures demonstrate the importance of a hydroxyl group at position 124 in stabilizing the anti conformation of the nicotinamide ring as observed in the previous structural analysis of the epimerase.NADH . UDP complex.

Biochemistry, 1997 Sep 2, 36(35), 10675 - 84
Mechanistic roles of tyrosine 149 and serine 124 in UDP-galactose 4-epimerase from Escherichia coli; Liu Y et al.; Synthesis and overexpression of a gene encoding Escherichia coli UDP-galactose 4-epimerase and engineered to facilitate cassette mutagenesis are described . General acid-base catalysis at the active site of this epimerase has been studied by kinetic and spectroscopic analysis of the wild-type enzyme and its specifically mutated forms Y149F, S124A, S124V, and S124T . The X-ray crystal structure of Y149F as its abortive complex with UDP-glucose is structurally similar to that of the corresponding wild-type complex, except for the absence of the phenolic oxygen of Tyr 149 . The major effects of mutations are expressed in the values of kcat and kcat/Km . The least active mutant is Y149F, for which the value of kcat is 0.010% of that of the wild-type epimerase . The activity of S124A is also very low, with a kcat value that is 0.035% of that of the native enzyme . The values of Km for Y149F and S124A are 12 and 21% of that of the wild-type enzyme, respectively . The value of kcat for S124T is about 30% of that of the wild-type enzyme, and the value of Km is similar to that of the native enzyme . The reactivities of the mutants in UMP-dependent reductive inactivation by glucose are similarly affected, with kobs being decreased by 6560-, 370-, and 3.4-fold for Y149F, S124A, and S124T, respectively . The second-order rate constants for reductive inactivation by NaBH3CN, which does not require general base catalysis, are similar to that for the native enzyme in the cases of S124A, S124T, and S124V . However, Y149F reacts with NaBH3CN 12-20-fold faster than the wild-type enzyme at pH 8.5 and 7.0, respectively . The increased rate for Y149F is attributed to the weakened charge-transfer interaction between Phe 149 and NAD+, which is present with Tyr 149 in the wild-type enzyme . The charge-transfer band is present in the serine mutants, and its intensity at 320 nm is pH-dependent . The pH dependencies of A320 showed that the pKa values for Tyr 149 are 6.08 for the wild-type epimerase, 6.71 for S124A, 6.86 for S124V, and 6.28 for S124T . The low pKa value for Tyr 149 is attributed mainly to the positive electrostatic field created by NAD+ and Lys 153 (4.5 kcal mol-1) and partly to hydrogen bonding with Ser 124 (1 kcal mol-1) . The pKa of Tyr 149 is the same as the kinetic pKa for the Bronsted base that facilitates hydride transfer to NAD+ . We concluded that Tyr 149 provides the driving force for general acid-base catalysis, with Ser 124 playing an important role in mediating proton transfer.

Biochemistry, 1997 Sep 2, 36(35), 10609 - 19
Characterization of the inducible nitric oxide synthase oxygenase domain identifies a 49 amino acid segment required for subunit dimerization and tetrahydrobiopterin interaction; Ghosh DK et al.; The oxygenase domain of inducible NO synthase (residues 1-498, iNOSox) is the enzyme's catalytic center . Its active form is a homodimer that contains heme and tetrahydrobiopterin (H4biopterin) and binds l-arginine {Ghosh, D . K., & Stuehr, D . J . (1995) Biochemistry 34, 801} . To help identify protein residues involved in prosthetic group and dimeric interaction, we expressed H4biopterin-free iNOSox in Escherichia coli . The iNOSox was 80% dimeric but contained a low-spin heme iron that bound DTT as a sixth ligand . The iNOSox bound H4biopterin or L-arginine with high affinity, which displaced DTT from the heme and caused spectral changes consistent with a closing up of the heme pocket . The H4biopterin-replete iNOSox could catalyze conversion of Nomega-hydroxyarginine to citrulline and NO in a H2O2-supported reaction . Limited trypsinolysis of the H4biopterin-free iNOSox dimer cut the protein at a single site in its N-terminal region (K117) . H4biopterin protected against the cleavage whereas l-arginine did not . The resulting 40 kDa protein contained thiol-ligated low-spin heme, was monomeric, catalytically inactive, showed no capacity to bind H4biopterin or l-arginine, and did not dimerize when provided with these molecules, indicating that residues 1-117 were important for iNOSox dimerization and H4biopterin/l-arginine interaction . A deletion mutant missing residues 1-114 was partially dimeric but otherwise identical to the 40 kDa protein regarding its spectral and catalytic properties and inability to respond to l-arginine and H4biopterin, whereas a deletion mutant missing residues 1-65 was equivalent to wild-type iNOSox, narrowing the region of importance to amino acids 66-114 . Mutation of a conserved cysteine in this region (C109A) decreased H4biopterin affinity without compromising iNOSox dimeric structure, L-arginine binding, or catalytic function . These results suggest that residues 66-114 of iNOSox are involved in productive H4biopterin interaction and subunit dimerization . H4biopterin binding appears to stabilize the protein structure in this region, and through doing so activates iNOS for NO synthesis.

Nat Genet, 1997 Sep, 17(1), 114 - 8
Female embryonic lethality in mice nullizygous for both Msh2 and p53; Cranston A et al.; The mutator hypothesis of tumorigenesis suggests that loss of chromosomal stability or maintenance functions results in elevated mutation rates, leading to the accumulation of the numerous mutations required for multistep carcinogenesis . The human DNA mismatch repair (MMR) genes are highly conserved homologues of the Escherichia coli MutHLS system, which contribute to genomic stability by surveillance and repair of replication misincorporation errors and exogenous DNA damage . Mutations in one of these MMR genes, hMSH2, account for about half of all cases of genetically linked hereditary non-polyposis colorectal cancer . Loss of function of p53 has also been proposed to increase cellular hypermutability, thereby accelerating carcinogenesis, although a clear role for p53 in genomic instability remains controversial . p53 is mutated frequently in a wide range of human cancers, including colonic tumours . Both Msh2- and p53-targeted knockout mice are viable and susceptible to cancer . Here we demonstrate that combined Msh2 and p53 ablation (Msh2-/-p53-/-) results in developmental arrest of all female embryos at 9.5 days . In contrast, male Msh2-/-p53-/- mice are viable, but succumb to tumours significantly earlier (t1-2 is 73 days) than either Msh2-/- or p53-/- littermates . Furthermore, the frequency of microsatellite instability (MSI) in tumours from Msh2-/-p53-/- mice is not significantly different than in Msh2-/- mice . Synergism in tumorigenesis and independent segregation of the MSI phenotype suggest that Msh2 and p53 are not genetically epistatic.

J Bacteriol, 1997 Sep, 179(17), 5648 - 53
Protein expression in response to folate stress in Escherichia coli; Huang EY et al.; Interruption of folate metabolism by trimethoprim results in the elevated expression of folate stress proteins in Escherichia coli . E . coli grown in culture medium supplemented with the folate-dependent metabolites glycine, methionine, and the purine nucleoside inosine shows reduced expression of folate stress proteins . The folate stress proteins include the universal stress protein, the ferric uptake regulatory repressor, and possibly, lipoamide dehydrogenase, the L protein component of the glycine cleavage enzyme complex.

J Bacteriol, 1997 Sep, 179(17), 5621 - 4
BglG, the response regulator of the Escherichia coli bgl operon, is phosphorylated on a histidine residue; Amster-Choder O et al.; We have shown previously that the activity of BglG, the response regulator of the bgl system, as a transcriptional antiterminator is modulated by the sensor BglF, which reversibly phosphorylates BglG . We show here that the phosphoryl group on BglG is present as a phosphoramidate, based on the sensitivity of phosphorylated BglG to heat, hydroxylamine, and acidic but not basic conditions . By analyzing the products of base-hydrolyzed phosphorylated BglG by thin-layer chromatography, we show that the phosphorylation occurs on a histidine residue . This result supports the notion that the bgl system is a member of a new family of bacterial sensory systems.

J Bacteriol, 1997 Sep, 179(17), 5582 - 4
Division pattern of a round mutant of Escherichia coli; Cooper S; A round mutant of Escherichia coli, when grown in Methocel medium, forms chains of cells and does not form tetrads . This implies that successive division planes of the round mutant are parallel rather than perpendicular . These results differ from a previous proposal that division planes in this round mutant are perpendicular to the prior division plane (W . D . Donachie, S . Addinall, and K . Begg, Bioessays 17:569-576, 1995).

J Bacteriol, 1997 Sep, 179(17), 5570 - 3
Lactose carrier mutants of Escherichia coli with changes in sugar recognition (lactose versus melibiose); Varela MF et al.; The purpose of this research was to identify amino acid residues that mediate substrate recognition in the lactose carrier of Escherichia coli . The lactose carrier transports the alpha-galactoside sugar melibiose as well as the beta-galactoside sugar lactose . Mutants from cells containing the lac genes on an F factor were selected by the ability to grow on succinate in the presence of the toxic galactoside beta-thio-o-nitrophenylgalactoside . Mutants that grew on melibiose minimal plates but failed to grow on lactose minimal plates were picked . In sugar transport assays, mutant cells showed the striking result of having low levels of lactose downhill transport but high levels of melibiose downhill transport . Accumulation (uphill) of melibiose was completely defective in all of the mutants . Kinetic analysis of melibiose transport in the mutants showed either no change or a greater than normal apparent affinity for melibiose . PCR was used to amplify the lacY DNA of each mutant, which was then sequenced by the Sanger method . The following six mutations were found in the lacY structural genes of individual mutants: Tyr-26-->Asp, Phe-27-->Tyr, Phe-29-->Leu, Asp-240-->Val, Leu-321-->Gln, and His-322-->Tyr . We conclude from these experiments that Tyr-26, Phe-27, Phe-29 (helix 1), Asp-240 (helix 7), Leu-321, and His-322 (helix 10) either directly or indirectly mediate sugar recognition in the lactose carrier of E . coli.

J Bacteriol, 1997 Sep, 179(17), 5551 - 9
Analysis of the interaction of FtsZ with itself, GTP, and FtsA; Wang X et al.; The interaction of FtsZ with itself, GTP, and FtsA was examined by analyzing the sensitivity of FtsZ to proteolysis and by using the yeast two-hybrid system . The N-terminal conserved domain consisting of 320 amino acids bound GTP, and a central region of FtsZ, encompassing slightly more than half of the protein, was cross-linked to GTP . Site-directed mutagenesis revealed that none of six highly conserved aspartic acid and asparagine residues were required for GTP binding . These results indicate that the specificity determinants for GTP binding are different than those for the GTPase superfamily . The N-terminal conserved domain of FtsZ contained a site for self-interaction that is conserved between FtsZ proteins from distantly related bacterial species . FtsZ320, which was truncated at the end of the conserved domain, was a potent inhibitor of division although it expressed normal GTPase activity and could polymerize . FtsZ was also found to interact directly with FtsA, and this interaction could also be observed between these proteins from distantly related bacterial species.

J Bacteriol, 1997 Sep, 179(17), 5543 - 50
A fragment liberated from the Escherichia coli CheA kinase that blocks stimulatory, but not inhibitory, chemoreceptor signaling; Morrison TB et al.; CheA, a cytoplasmic histidine autokinase, in conjunction with the CheW coupling protein, forms stable ternary complexes with the cytoplasmic signaling domains of transmembrane chemoreceptors . These signaling complexes induce chemotactic movements by stimulating or inhibiting CheA autophosphorylation activity in response to chemoeffector stimuli . To explore the mechanisms of CheA control by chemoreceptor signaling complexes, we examined the ability of various CheA fragments to interfere with receptor coupling control of CheA . CheA{250-654}, a fragment carrying the catalytic domain and an adjacent C-terminal segment previously implicated in stimulatory control of CheA activity, interfered with the production of clockwise flagellar rotation and with chemotactic ability in wild-type cells . Epistasis tests indicated that CheA{250-654} blocked clockwise rotation by disrupting stimulatory coupling of CheA to receptors . In vitro coupling assays confirmed that a stoichiometric excess of CheA{250-654} fragments could exclude CheA from stimulatory receptor complexes, most likely by competing for CheW binding . However, CheA{250-654} fragments, even in vast excess, did not block receptor-mediated inhibition of CheA, suggesting that CheA{250-654} lacks an inhibitory contact site present in native CheA . This inhibitory target is most likely in the N-terminal P1 domain, which contains His-48, the site of autophosphorylation . These findings suggest a simple allosteric model of CheA control by ternary signaling complexes in which the receptor signaling domain conformationally regulates the interaction between the substrate and catalytic domains of CheA.

J Bacteriol, 1997 Sep, 179(17), 5465 - 70
Outer membrane localization of murein hydrolases: MltA, a third lipoprotein lytic transglycosylase in Escherichia coli; Lommatzsch J et al.; Lytic transglycosylases are a unique lysozyme-like class of murein hydrolases believed to be important for growth of Escherichia coli . A membrane-bound lytic transglycosylase with an apparent molecular mass of 38 kDa, which was designated Mlt38, has previously been purified and characterized (A . Ursinus and J.-V . Holtje, J . Bacteriol . 176:338-343, 1994) . On the basis of four tryptic peptides, the gene mltA was mapped at 63 min on the chromosomal map of E . coli K-12 and cloned by reverse genetics . The open reading frame was found to contain a typical lipoprotein consensus sequence, and the lipoprotein nature of the gene product was demonstrated by {3H}palmitate labeling . On the basis of the distribution of MltA in membrane fractions obtained by sucrose gradient centrifugation, a localization in the outer membrane is indicated . Overexpression of MltA at 30 degrees C, the optimal temperature for enzyme activity, but not at 37 degrees C results in the formation of spheroplasts . Not only a deletion mutant in mltA, but also double mutants in mltA and one of the two other well-characterized lytic transglycosylases (either sltY or mltB), as well as a triple mutant in all three enzymes, showed no obvious phenotype . However, dramatic changes in the structure of the murein sacculus indicate that lytic transglycosylases are involved in maturation of the murein sacculus.

J Bacteriol, 1997 Sep, 179(17), 5458 - 64
Mutation of a single MalK subunit severely impairs maltose transport activity in Escherichia coli; Davidson AL et al.; The maltose transport system of Escherichia coli, a member of the ABC transport superfamily of proteins, consists of a periplasmic maltose binding protein and a membrane-associated translocation complex that contains two copies of the ATP-binding protein MalK . To examine the need for two nucleotide-binding domains in this transport complex, one of the two MalK subunits was inactivated by site-directed mutagenesis . Complexes with mutations in a single subunit were obtained by attaching a polyhistidine tag to the mutagenized version of MalK and by coexpressing both wild-type MalK and mutant (His)6MalK in the same cell . Hybrid complexes containing one mutant (His)6MalK subunit and one wild-type MalK subunit were separated from those containing two mutant (His)6MalK proteins based on differential affinities for a metal chelate column . Purified transport complexes were reconstituted into proteoliposome vesicles and assayed for maltose transport and ATPase activities . When a conserved lysine residue at position 42 that is involved in ATP binding was replaced with asparagine in both MalK subunits, maltose transport and ATPase activities were reduced to 1% of those of the wild type . When the mutation was present in only one of the two subunits, the complex had 6% of the wild-type activities . Replacement of a conserved histidine residue at position 192 in MalK with arginine generated similar results . It is clear from these results that two functional MalK proteins are required for transport activity and that the two nucleotide-binding domains do not function independently to catalyze transport.

J Bacteriol, 1997 Sep, 179(17), 5429 - 35
In vitro phosphorylation study of the arc two-component signal transduction system of Escherichia coli; Georgellis D et al.; The ArcB and ArcA proteins constitute a two-component signal transduction system that plays a broad role in transcriptional regulation . Under anoxic or environmentally reducing conditions, the sensor kinase (ArcB) is stimulated to autophosphorylate at the expense of ATP and subsequently transphosphorylates the response regulator (ArcA) . ArcB is a complex, membrane-bound protein comprising at least three cytoplasmic domains, an N-terminal transmitter domain with a conserved His292 residue (H1), a central receiver domain with a conserved Asp576 residue (D1), and a C-terminal alternative transmitter domain with a conserved His717 residue (H2) . To study the phosphoryl transfer pathways of the Arc system, we prepared the following His-tagged proteins: H1, D1, H2, H1-D1, D1-H2, H1-D1-H2, and ArcA . Incubations of various combinations of Arc proteins with {gamma-32P}ATP indicated that H1, but not D1 or H2, catalyzes autophosphorylation; that H1-P transfers the phosphoryl group to D1 much more rapidly than to ArcA; and that D1 accelerates the transphosphorylation of H2 . Finally, ArcA is phosphorylated much more rapidly by H2-P than by H1-P . Available data are consistent with a signal transduction model in which (i) reception of a membrane signal(s) triggers autophosphorylation of H1 at His292, (ii) the phosphoryl group can migrate to D1 at Asp576 and subsequently to H2 at His717, and (iii) ArcA receives the phosphoryl group from either His292 or His717, the relative contribution of which is regulated by cytosolic effectors.

J Bacteriol, 1997 Sep, 179(17), 5380 - 90
A new type of illegitimate recombination is dependent on restriction and homologous interaction; Kusano K et al.; Illegitimate (nonhomologous) recombination requires little or no sequence homology between recombining DNAs and has been regarded as being a process distinct from homologous recombination, which requires a long stretch of homology between recombining DNAs . Under special conditions in Escherichia coli, we have found a new type of illegitimate recombination that requires an interaction between homologous DNA sequences . It was detected when a plasmid that carried 2-kb-long inverted repeats was subjected to type II restriction in vitro and type I (EcoKI) restriction in vivo within a delta rac recBC recG ruvC strain . Removal of one of the repeats or its replacement with heterologous DNA resulted in a reduction in the level of recombination . The recombining sites themselves shared, at most, a few base pairs of homology . Many of the recombination events joined a site in one of the repeats with a site in another repeat . In two of the products, one of the recombining sites was at the end of one of the repeats . Removal of one of the EcoKI sites resulted in decreased recombination . We discuss the possibility that some structure made by homologous interaction between the long repeats is used by the EcoKI restriction enzyme to promote illegitimate recombination . The possible roles and consequences of this type of homologous interaction are discussed.

J Bacteriol, 1997 Sep, 179(17), 5333 - 9
Leaderless polypeptides efficiently extracted from whole cells by osmotic shock; Thorstenson YR et al.; Three molecular foldases, DsbA, DsbC, and rotamase (ppiA), exhibited the unusual property of accumulating in an osmotically sensitive cellular compartment of Escherichia coli when their signal sequences were precisely removed by mutation . A mammalian protein, interleukin-1 (IL-1) receptor antagonist, behaved in a similar fashion in E . coli when its native signal sequence was deleted . These leaderless mutants (but not two control proteins overexpressed in the same system) were quantitatively extractable from whole cells by a variety of methods generally employed in the recovery of periplasmic proteins . A series of biochemical and genetic experiments showed that (i) leaderless DsbA (but not the wild type) was retained in a nonperiplasmic location; (ii) beta-galactosidase fusions to leaderless DsbA (but not to the wild type) exhibited efficient alpha complementation; (iii) none of the leaderless mutant proteins were substantially associated with cell membranes, even when they were overexpressed in cells; and (iv) leaderless DsbA was not transported to an osmotically sensitive compartment via a secA- or ftsZ-dependent mechanism . The observation that these proteins transit to some privileged cellular location by a previously undescribed mechanism(s)--absent their normal mode of (signal sequence-dependent) translocation--was unexpected . DsbA, rotamase, and IL-1, whose tertiary structures are known, appear to be structurally unrelated proteins . Despite a lack of obvious homologies, these proteins apparently have a common mechanism for intracellular localization . As this (putative) bacterial mechanism efficiently recognizes proteins of mammalian origin, it must be well conserved across evolutionary boundaries.

Genetics, 1997 Sep, 147(1), 297 - 304
Activation of the lac repressor in the transgenic mouse; Scrable H et al.; We have introduced sequences encoding the lac repressor of Escherichia coli into the genome of the mouse . One sequence was derived from the bacterial lac operon and the other was created by re-encoding the amino acid sequence of lacI with mammalian codons . Both versions are driven by an identical promoter fragment derived from the human beta-actin locus and were microinjected into genetically identical pronuclear stage embryos . All transgenes utilizing the bacterial coding sequence were transcriptionally silent in all somatic tissues tested . The sequence re-encoded with mammalian codons was transcriptionally active at all transgene loci and expressed ubiquitously . Using methylation-sensitive enzymes, we have determined the methylation status of lac repressor transgenes encoded by either the bacterial or mammalian sequence . The highly divergent bacterial sequence was hypermethylated at all transgene loci, while the mammalian sequence was only hypermethylated at a high copy number locus . This may reflect a normal process that protects the genome from acquiring new material that has an abnormally divergent sequence or structure.

Genetics, 1997 Sep, 147(1), 7 - 17
Genetic evidence that recognition of cosQ, the signal for termination of phage lambda DNA packaging, depends on the extent of head filling; Cue D et al.; Packaging a phage lambda chromosome involves cutting the chromosome from a concatemer and translocating the DNA into a prohead . The cutting site, cos, consists of three subsites: cosN, the nicking site; cosB, a site required for packaging initiation; and cosQ a site required for termination of packaging . cosB contains three binding sites (R sequences) for gpNu1, the small subunit of terminase . Because cosQ has sequence identity to the R sequences, it has been proposed that cosQ is also recognized by gpNu1 . Suppressors of cosB mutations were unable to suppress a cosQ point mutation . Suppressors of a cosQ mutation (cosQ1) were isolated and found to be of three sorts, the first affecting a base pair in cosQ . The second type of cosQ suppression involved increasing the length of the phage chromosome to a length near to the maximum capacity of the head shell . A third class of suppressors were missense mutations in gene B, which encodes the portal protein of the virion . It is speculated that increasing DNA length and altering the portal protein may reduce the rate of translocation, thereby increasing the efficiency of recognition of the mutant cosQ . None of the cosQ suppressors was able to suppress cosB mutations . Because cosQ and cosB mutations are suppressed by very different types of suppressors, it is concluded that cosQ and the R sequences of cosB are recognized by different DNA-binding determinants.

Proc Assoc Am Physicians, 1997 Sep, 109(5), 462 - 9
Autoantibodies in pernicious anemia type I patients recognize sequence 251-256 in human intrinsic factor; Gueant JL et al.; Pernicious anemia is an organ-specific autoimmune disease characterized by cobalamin deficiency, megaloblastic anemia, neuropathy, and autoimmune gastritis with anti-intrinsic factor autoantibodies . Type 1 anti-intrinsic factor autoantibodies block the cobalamin binding site of the intrinsic factor, a gastric protein required for the assimilation of cobalamin . The aim of our study was to identify the epitope domain of type 1 antibodies . Different series of peptides derived from the intrinsic factor sequence were synthesized and tested for antibody binding in enzyme-linked immunosorbent assay, radioisotope assay, gel filtration, and SDS-PAGE autoradiography . One of these peptides, named IF-R7 (the intrinsic factor aminoacid sequence 251-265), showed a type 1 antibody binding activity and inhibited, in vitro, their blocking activity with Ki at 2.3 microM . The cross-linking of IF-R7 to beta-lactoglobulin produced type 1 anti-intrinsic factor antibodies in immunized sheep . In vivo Schilling tests performed on guinea pigs also revealed IF-R7 peptide inhibition of type 1 antibody blocking activity . 256Ser, 258Lys, 262Tyr and 265Val of the IF-R7 were essential for the epitope recognition . Reactivity with type 1 antibodies was found in IF-R7 homologous peptides from herpesvirus Saimiri and from pathogenic Escherichia coli . In conclusion, the epitope of type 1 anti-intrinsic factor autoantibodies is located in the 251-265 amino acid sequence of the protein . The identification of this epitope will enable the definition of an experimental animal model of anti-IF autoimmunity in order to study the pathogenesis of pernicious anemia.

Hum Mol Genet, 1997 Sep, 6(9), 1457 - 64
Expression and kinetic characterization of methylmalonyl-CoA mutase from patients with the mut- phenotype: evidence for naturally occurring interallelic complementation; Janata J et al.; L-Methylmalonyl-CoA mutase (MUT) is an adenosylcobalamin (AdoCbl)-requiring mitochondrial matrix enzyme that catalyzes the isomerization of L-methylmalonyl-CoA to succinyl-CoA . Inherited defects in the gene encoding this enzyme result in the mut forms of methylmalonic acidemia . Expression of mature human MUT cDNA in Escherichia coli at a post-induction cultivation temperature of 12 degrees C, rather than 37 degrees C, led to the folding of the majority of the synthesized protein to a soluble form, with an activity of 0.2-0.3 U/mg protein in the cell-free extract, 10-15 times higher than that in human liver homogenate . Six missense mutations, producing the amino acid changes G94V, Y231N, R369H, G623R, H678R and G717V, were detected in MUT cDNA of patients suffering from the mut- form of methylmalonic acidemia, resulting from defective AdoCbl binding . Two (G623R and G717V) had been reported in other patients . Three (G94V, Y231N and R369H) are the first changes in the NH2-terminal part of the enzyme reported to cause the mut- phenotype . Enzymes with the mutations were individually expressed, and their kinetic parameters were generally in accord with published biochemical data from extracts of fibroblasts from these patients . The mutations increased the K(m) for AdoCbl by 40- to 900-fold, while V(max) values varied from 0.2% to nearly 100% of that of wild-type protein . In one case of a doubly heterozygous cell line, however, neither of the constituent mutant enzymes had a K(m) corresponding to the lower of the two estimated from the extract data . This finding may reflect the natural occurrence of interallelic complementation in vivo in this cell line.

Curr Biol, 1997 Sep 1, 7(9), R576 - 9
DNA repair: caretakers of the genome?; Cunningham RP; Recent results show that the 8-oxoguanine DNA repair system is functionally conserved in bacteria and mammals . The bacterial system protects the genome from the mutagenic effects of oxidative stress; the role of the mammalian system is expected to be similar and defects in it may increase susceptibility to cancer.

Am J Vet Res, 1997 Sep, 58(9), 1010 - 3
Effects of acute endotoxemia on serum somatotropin and insulin-like growth factor I concentrations in prepubertal gilts; Hevener W et al.; OBJECTIVE: To evaluate effects of endotoxemia on serum somatotropin (ST) and insulin-like growth factor I (IGF-I) concentrations in finishing pigs . ANIMALS: Eight female pigs (98 +/- 2 kg) randomly assigned to IV administration (time 0) of saline solution (n = 4) or Escherichia coli lipopolysaccharide (LPS; 5 micrograms/kg of body weight; n = 4) . PROCEDURE: Serum ST concentration was determined in serum samples obtained at 20-minute intervals for 6 hours after treatment . Serum IGF-I concentration was determined in samples collected at 1-hour intervals for 6 hours and at 12, 15, 18, 24, 48, 72, and 96 hours after treatment . RESULTS: One distinct pulse of ST (peak 10.5 +/- 0.5 ng/ml) was observed at 40 minutes in each pig after administration of LPS . Control pigs had 2.25 +/- 0.48 ST pulses during the 6 hours of frequent sample collection; however, magnitude of the ST pulses was similar between gilts given LPS and control gilts . A temporal association between ST pulses and saline administration was not evident . Serum IGF-I concentration was similar between gilts of the LPS and control groups prior to treatment . The IGF-I concentration was similar between gilts of the LPS and control groups prior to treatment . The IGF-I concentration was lower (P < 0.01) in gilts of the LPS group (44 +/- 5 ng/ml) than in gilts of the control group (157 +/- 4 ng/ml) at 24 hours . The difference in IGF-I concentrations between groups was evident for 96 hours . CONCLUSIONS: Immediate release of ST was attributed to stress associated with acute endotoxemia and stimulation of the pituitary gland; immune stimulation by LPS may have contributed to the changes in IGF-I concentration . Because feed consumption was similar between the 2 groups of pigs, suppression of IGF-I concentration for 96 hours after administration of LPS was attributable to factors in addition to transient feed restriction . Thus, acute endotoxemia altered the positive association between ST and IGF-I, and provided evidence for a potential mechanism of impaired growth in endotoxemic animals.

Neuroscience, 1997 Sep, 80(2), 501 - 9
Identification and characterization of a novel gene (neurorep 1) expressed in nerve cells and up-regulated after axotomy; Uwabe K et al.; A novel gene, designated neurorep 1, was isolated by differential hybridization screening from a complementary DNA library constructed from the rat facial nucleus whose nerve had been transected seven days before sampling . In situ hybridization revealed that this gene was up-regulated in the repair stage after axotomy . The deduced protein, Neurorep 1, consists of 293 amino acid residues, and its molecular mass is approximately 34,000 . Protein sequence motif search indicates that this protein has an ecto-5'-nucleotidase consensus sequence at the carboxyl terminal region . In vitro studies showed that Neurorep 1 significantly increased the activity of ecto-5'-nucleotidase, which is considered to be involved in regeneration and repair of the central nervous system . Neurorep 1 might play a significant role in the repair process of nerve tissues by its regulation of ecto-5'-nucleotidase activity.

Biophys J, 1997 Sep, 73(3), 1579 - 92
Effects of fluorine substitution on the structure and dynamics of complexes of dihydrofolate reductase (Escherichia coli); Lau EY et al.; Fluorine NMR experiments with a protein containing fluorinated amino acid analogs can often be used to probe structure and dynamics of the protein as well as conformational changes produced by binding of small molecules . The relevance of NMR experiments with fluorine-containing materials to characteristics of the corresponding native (nonfluorinated) proteins depends upon the extent to which these characteristics are altered by the presence of fluorine . The present work uses molecular dynamics simulations to explore the effects of replacement of tryptophan by 6-fluorotryptophan in folate and methotrexate complexes of the enzyme dihydrofolate reductase (DHFR) (Escherichia coli) . Simulations of the folate-native enzyme complex produce local correlation times and order parameters that are generally in good agreement with experimental values . Simulations of the corresponding fluorotryptophan-containing system indicate that the structure and dynamics of this complex are scarcely changed by the presence of fluorinated amino acids . Calculations with the pharmacologically important methotrexate-enzyme complex predict dynamical behavior of the protein similar to that of the folate complex for both the fluorinated and native enzyme . It thus appears that, on the time scale sampled by these computer simulations, substitution of 6-fluorotryptophan for tryptophan has little effect on either the structures or dynamics of DHFR in these complexes.

Infect Immun, 1997 Sep, 65(9), 3961 - 5
A nonsubstituted primary hydroxyl group in position 6' of free lipid A is required for binding of lipid A monoclonal antibodies; Brade L et al.; Lipid A monoclonal antibodies, which require for binding the presence of the bisphosphorylated D-glucosamine disaccharide lipid A backbone, were tested against synthetic lipid A precursor Ia and compound B 1047 by enzyme immunoassay . The last-named compound is a precursor Ia analog with a methoxy instead of a hydroxy group at C6' and was chosen to determine why these antibodies failed to recognize the bound lipid A present in lipopolysaccharide (LPS) . Whereas all antibodies tested bound to precursor Ia, none of them bound to compound B 1047 or Escherichia coli Re LPS to a significant extent . Compared to the natural substituent at C6', i.e., 3-deoxy-D-manno-octulosonic acid (Kdo), the methoxy group is neither bulky nor charged . Thus, the data suggest that it is not hindrance by Kdo but rather the generation of a neoantigen that endows lipid A with immunoreactivity upon liberation from LPS by acid hydrolysis.

Infect Immun, 1997 Sep, 65(9), 3788 - 93
Peyer's patch adherence of enteropathogenic Escherichia coli strains in rabbits; Von Moll LK et al.; RDEC-1 (serotype O15) is an attaching and effacing strain of rabbit enteropathogenic Escherichia coli (REPEC) that causes diarrhea in postweanling rabbits . It expresses AF/R1 pili that mediate Peyer's patch M-cell adherence . We investigated Peyer's patch adherence, the presence of virulence genes, ileal brush border aggregation, and pilus expression in 9 strains representing several serotypes of REPEC as well as in two commensal strains . Postweanling rabbits were inoculated with 10(6) organisms and sacrificed at 24 h, and tissues were prepared for examination by light microscopy . Strains B10 and RDEC-1 were also studied at 12 and 72 h postinoculation . All REPEC strains were eaeA positive, expressed pili, and adhered to ileal brush borders . Both commensal strains expressed pili, and one strain adhered to brush borders . All REPEC strains demonstrated some degree of Peyer's patch lymphoid follicle adherence, ranging from diffuse coverage to small patches covering two to three dome epithelial cells . Strains C102 and C110 had genes homologous with the structural subunit gene of the AF/R1 pilus (afrA) of RDEC-1, which correlated with greater degrees of lymphoid follicle adherence and lesser degrees of ileal villus adherence . The observation that all REPEC strains adhere to Peyer's patch epithelium suggests the possibility that human strains of enteropathogenic E . coli (EPEC) might do likewise . EPEC strains might thus serve as mucosal vaccine vectors in humans . Better understanding of the molecular mechanism of REPEC adherence should provide a model for the targeting of the Peyer's patch in humans.

Infect Immun, 1997 Sep, 65(9), 3577 - 84
Serum factors, cell membrane CD14, and beta2 integrins are not required for activation of bovine macrophages by lipopolysaccharide; Jungi TW et al.; The role of serum factors such as lipopolysaccharide (LPS)-binding protein (LBP) and of macrophage-expressed CD14 and beta2 integrins in the activation of bovine macrophages by LPS was investigated . Macrophage activation was determined by measuring tumor necrosis factor production, NO generation, and upregulation of procoagulant activity by LPS (Escherichia coli O55:B5) at concentrations of 100 pg/ml to 100 ng/ml . The 50% effective dose for LPS was 1 order of magnitude higher than that for activating human macrophages . Macrophages were activated by LPS in the presence of serum or in the presence of albumin demonstrated to be free of LBP . The capacity to react to LPS in the absence of LBP was not due to the acquisition of LBP during a previous culture in serum . It was then established which CD14-specific antibodies block LPS binding to monocytes . Among the CD14-specific antibodies recognizing bovine mononuclear phagocytes (60bca, 3C10, My4, CAM36, VPM65, CMRF31, and TUK4), the first four blocked the binding of LPS-fluorescein isothiocyanate to bovine monocytes at low concentrations . Anti-CD14 antibodies did not block LPS-mediated activation of bovine bone marrow-derived macrophages, monocyte-derived macrophages, and alveolar macrophages . This was observed in experiments in which anti-CD14 concentrations exceeded the 50% inhibitory dose by >30-fold (3C10 and My4) or >300-fold (60bca), as defined in the binding assay described above . Monocyte-derived macrophages from an animal deficient in beta2 integrins and control macrophages were activated by similar concentrations of LPS, suggesting that beta2 integrins are not important bovine LPS receptors . Thus, in bovine macrophages, LPS recognition pathways which are independent of exogenous LBP, of membrane-expressed CD14, and of beta2 integrins may exist.

Infect Immun, 1997 Sep, 65(9), 3547 - 55
Characterization of two virulence proteins secreted by rabbit enteropathogenic Escherichia coli, EspA and EspB, whose maximal expression is sensitive to host body temperature; Abe A et al.; Enteropathogenic Escherichia coli (EPEC) and rabbit EPEC (RDEC-1) cause unique histopathological features on intestinal mucosa, including attaching/effacing (A/E) lesions . Due to the human specificity of EPEC, RDEC-1 has been used as an animal model to study EPEC pathogenesis . At least two of the previously identified EPEC-secreted proteins, EspA and EspB, are required for triggering host epithelial signal transduction pathways, intimate adherence, and A/E lesions . However, the functions of these secreted proteins and their roles in pathogenesis have not been characterized . To investigate the function of EspA and EspB in RDEC-1, the espA and espB genes were cloned and their sequences were compared to that of EPEC O127 . The EspA proteins showed high similarity (88.5% identity), while EspB was heterogeneous in internal regions (69.8% identity) . However, RDEC-1 EspB was identical to that of enterohemorrhagic E . coli serotype O26 . Mutations in RDEC-1 espA and espB revealed that the corresponding RDEC-1 gene products are essential for triggering of host signal transduction pathways and invasion into HeLa cells . Complementation with plasmids containing EPEC espA or/and espB genes into RDEC-1 mutant strains demonstrated that they were functionally interchangeable, although the EPEC proteins mediated higher levels of invasion . Furthermore, maximal expression of RDEC-1 and EPEC-secreted proteins occurred at their respective host body temperatures, which may contribute to the lack of EPEC infectivity in rabbits.

Biol Reprod, 1997 Sep, 57(3), 532 - 8
Sequence of complementary deoxyribonucleic acid encoding bonnet monkey (Macaca radiata) zona pellucida glycoprotein-ZP1 and its high-level expression in Escherichia coli; Gupta SK et al.; Zona pellucida (ZP) glycoproteins have been proposed as candidate antigens for immunocontraception . Studies on this potential use can be facilitated by the availability of recombinant proteins . A cDNA lambda gt11 library was constructed using poly(A)+ mRNA isolated from bonnet monkey (Macaca radiata) ovaries and was screened for bonnet monkey ZP1 using a 404-basepair (bp) human ZP1 fragment (nucleotides 818-1221) as probe . Bonnet monkey ZP1 cDNA comprises 1617 nucleotides and encodes a polypeptide of 539 amino acid residues that share 92.0% identity with human ZP1 . The major difference between bonnet monkey ZP1 and human ZP1 is the deletion of a 28-amino acid domain (amino acid residues 100-127 corresponding to human ZP1) . An internal fragment (1317 bp) of bonnet monkey ZP1, excluding the N-terminus signal sequence and the C-terminus transmembrane-like domain, was amplified by polymerase chain reaction . The amplified Sac I and Kpn I restricted fragment was cloned in a frame downstream of the T5 promoter under the lac operator control for expression in the pQE-30 vector . Recombinant ZP1 (r-ZP1) was expressed as a polyhistidine fusion protein in Escherichia coli strains SG13009{pREP4} and ompT and Ion protease-deficient BL21 (plysS) . SDS-PAGE analysis and immunoblotting with a murine monoclonal antibody, MA-410 (raised against porcine ZP3alpha--a homologue of bonnet monkey ZP1--and cross-reactive with bonnet monkey zona pellucida), revealed major bands of 51 and 40 kDa besides truncated fragments . Optimum expression of r-ZP1 was observed at 0.5 mM isopropyl beta-D-thiogalactopyranoside (IPTG) . Immunization of male rabbits with r-ZP1 purified on nickel-nitrilotriacetic acid (NTA) resin under denaturing conditions and of female rabbits with r-ZP1 conjugated with diphtheria toxoid-generated antibodies reactive with r-ZP1 in ELISA . Moreover, immune sera, when tested by indirect immunofluorescence on bonnet monkey ovarian sections, showed positive fluorescence with zona pellucida . The information on the sequence of bonnet monkey ZP1 and the availability of the recombinant protein will help toward better understanding and evaluation of the contraceptive potential of homologous immunization in a nonhuman primate model.

Virology, 1997 Sep 1, 235(2), 342 - 51
Membrane permeability changes induced in Escherichia coli by the SH protein of human respiratory syncytial virus; Perez M et al.; The small hydrophobic (SH) protein of human respiratory syncytial virus (HRSV) has been efficiently expressed in Escherichia coli . In analogy to small hydrophobic proteins encoded by other RNA viruses, membrane permeability changes to low-molecular-weight compounds were detected in bacteria expressing HRSV SH protein . These changes implied, at least, the entry of both the protein synthesis inhibitor hygromycin B and the beta-galactoside substrate o-nitrophenyl-beta-d-galactopyranoside and the exit of preloaded {3H}uridine from bacterial cells . Site-directed mutagenesis indicated that the C-terminal end of SH is needed for induction of membrane permeability changes . In addition, amino acid substitution at residue 32 (Ile to Lys) abolished that activity . This was correlated with a drastic increase in SH electrophoretic mobility and a decrease of the predicted values of alpha-helix for all residues of the SH transmembrane domain . Other sequence changes have either partial effect or no effect on the membrane permeability changes induced by the SH protein . However, none of the mutations abrogated the association of SH protein with bacterial membranes, indicating that incorporation of SH protein to membranes is not sufficient to induce the observed changes . Membrane permeability changes then might provide a useful test for the identification of key amino acid residues in this unique HRSV gene product.

Mol Biochem Parasitol, 1997 Sep, 88(1-2), 215 - 24
The antimalarial drug, chloroquine, interacts with lactate dehydrogenase from Plasmodium falciparum; Menting JG et al.; We have previously shown that a radioiodinated photoreactive analogue of chloroquine, {125I}N-(4-(4-diethylamino-1-methylbutylamino)quinolin-6-yl) -4-azido-2-hydroxybenzamide ({125I}ASA-Q), specifically labels two proteins in Plasmodium falciparum with apparent molecular weights (Mr) of 42 and 33 kDa (Foley M, Deady LW, Ng K, Cowman AF, Tilley L . J Biol Chem 1994:269:6955-6961) . We now report the identification of the 33 kDa protein . The 33 kDa protein was purified from Plasmodium falciparum using photoaffinity labeling with {125I}ASA-Q to monitor the enrichment process . N-terminal sequence analysis of the purified protein revealed exact identity of the first 35 amino acids with P . falciparum lactate dehydrogenase (PfLDH) . The plasmodial enzyme was cloned and expressed in E . coli and the recombinant protein used to produce a rabbit antiserum . Immunoprecipitation using affinity-purified anti-PfLDH antibodies confirmed the identity of the 33 kDa CQ-binding protein . The enzyme activity of purified PfLDH was not significantly affected by chloroquine indicating that PfLDH is not a direct target of CQ . PfLDH was, however, shown to be exquisitely sensitive to inhibition by free heme and chloroquine protected against this inhibitory effect.

Mol Biochem Parasitol, 1997 Sep, 88(1-2), 187 - 202
Localization and functional analysis of the cytosolic and extracellular CuZn superoxide dismutases in the human parasitic nematode Onchocerca volvulus; Henkle-Duhrsen K et al.; This study describes the histological localization of two CuZn superoxide dismutases (SOD1 and SOD2) in the parasitic nematode Onchocerca volvulus, and a functional characterization of the 'extracellular' form of this enzyme (SOD2) which provides evidence that it is involved in the defense against environmental superoxide anion radicals . These essential enzymes are detected in larval and adult stages of the parasite, determined at the mRNA and protein levels by in situ hybridization and immunolocalization studies . These proteins are distributed throughout the worm, at various concentrations with particularly high levels produced in the hypodermis . In vitro maintenance of parasites indicated that SOD2 was secreted outside the parasite into the medium . Baculovirus constructs designed to test the ability of the SOD2 hydrophobic N-terminal region to function in processing and secretion confirmed the ability of this polypeptide sequence to direct the secretion of a marker protein, as well as of the mature SOD2 enzyme . Analyses of the native, mature SOD2 enzyme molecular mass, and the primary and quaternary structure, indicate that unlike other extracellular SODs, the SOD2 is active as a non-glycosylated dimer, rather than as a tetrameric glycoprotein . The detection of SOD2 outside of the parasite maintained in vitro, and the confirmation that the SOD2 is a secreted enzyme, indicate that this enzyme plays a role in the interactive biology of parasitic nematodes with their hosts.

Mol Biochem Parasitol, 1997 Sep, 88(1-2), 137 - 49
Molecular cloning of the gene encoding the 83 kDa amastigote surface protein and its identification as a member of the Trypanosoma cruzi sialidase superfamily; Low HP et al.; Amastigote surface proteins of Trypanosoma cruzi are likely targets of both humoral and cell-mediated immune responses, however, few such molecules have been well studied . In this study, we have used modified RACE (rapid amplification of cDNA ends) and SOE (gene splicing by overlap extension) polymerase chain reaction strategies to clone the gene for the previously described 83 kDa amastigote surface protein of T . cruzi . Of the several clones obtained, only one clone, clone 4, was found to encode the 20 amino acid sequence originally reported by Pan and McMahon-Pratt (J Immunol 1989;143:1001-1008) . The identity of the cloned gene with the 83 kDa amastigote surface protein was further confirmed by the reactivity of polyclonal antisera against the purified 83 kDa protein with the gene product expressed in E . coli . Sequence analyses revealed that this amastigote surface protein (ASP-2) has two conserved aspartic acid box motifs and the highly conserved VTVxNVxLYNR motif characteristic of bacterial and viral sialidases and the type III module of fibronectin, respectively . ASP-2 thus joins ASP-1 as a member of the amastigote surface expressed family of sialidase-like molecules having strong homology with family 2 of the sialidase/trans-sialidase gene superfamily of T . cruzi.

Mol Biochem Parasitol, 1997 Sep, 88(1-2), 95 - 104
Molecular characterization and ultrastructural localization of Plasmodium falciparum Hsp 60; Das A et al.; Heat shock proteins (Hsp) are a group of highly conserved proteins which are widely represented phylogenetically . Genes for members of the Hsp 70, 90 and 60 families have been cloned from the human malaria parasite Plasmodium falciparum . In this study, we have cloned and expressed the P . falciparum Hsp 60 (PfHsp60) in E . coli . The sequence analysis identified a previously unknown intron of 257 bp beginning after the nucleotide 142 in the coding sequence . Antisera raised against the recombinant PfHsp60 was employed in immunoprecipitation studies with biosynthetically labeled parasite extracts to investigate regulation of expression of PfHsp60 at various temperatures . In contrast to the three to four fold accumulation of PfHsp60 transcripts in heat shocked parasites (37-40 degrees C), the expression of PfHsp60 was not induced in the blood stages of P . falciparum . On the other hand, the effect of heat induction on PfHsp70 was seen both at the level of specific mRNA and protein . In these studies we also observed co-immunoprecipitation of a number of other cellular proteins suggesting possible interaction with PfHsp60 . Immunofluorescence analysis indicated the presence of PfHsp60 in the cytoplasm of all the various stages of the parasite . In addition, immunoelectron microscopic analysis distinctly localized PfHsp60 in the mitochondrion of P . falciparum . This study suggests that different mechanisms are involved in the regulation of expression of various members of the heat shock proteins in the parasite.

Mol Biochem Parasitol, 1997 Sep, 88(1-2), 63 - 72
Molecular cloning and characterisation of a putative aspartate proteinase associated with a gut membrane protein complex from adult Haemonchus contortus; Longbottom D et al.; A cDNA was isolated from an adult Haemonchus contortus cDNA expression library the deduced amino acid sequence of which showed significant homology to mammalian pepsinogen sequences . The library was screened with antisera raised against Haemonchus galactose-containing glycoprotein complex, a gut membrane protein complex with aspartyl proteinase activity which has shown considerable potential as a protective antigen . The amino acid sequence obtained corresponded very closely in part to the N-terminal amino acid sequences of two polypetides within the complex . The enzyme was shown to be almost exclusively expressed by the blood-feeding parasite stages . The cDNA was expressed in E . coli, and antibody produced to the recombinant protein bound to the luminal surface of the gut in the adult parasite . The proteinase may play a central role in digesting the blood meal and is considered a potential sub-unit vaccine candidate.

Mol Biochem Parasitol, 1997 Sep, 88(1-2), 43 - 52
Cloning and characterization of actin depolymerizing factor from Toxoplasma gondii; Allen ML et al.; We determined the predicted amino acid sequence of actin depolymerizing factor (ADF) from Toxoplasma gondii by sequencing the full-length cDNA . T . gondii ADF consists of 118 amino acids (calculated molecular weight 13,400) and shares a high degree of sequence similarity to other low molecular weight actin monomer sequestering proteins, especially Acanthamoeba actophorin, plant ADFs and yeast and vertebrate cofilin . ADF from T . gondii is smaller and does not contain a nuclear localization sequence like the related vertebrate proteins . Southern blot analysis indicates that T . gondii ADF is a single-copy gene . Homogeneous recombinant T . gondii ADF purified from E . coli is active in binding actin monomers and depolymerizing F-actin . Localization of ADF by immunofluorescence and immunoelectron microscopy indicates ADF is scattered throughout the cytoplasm and prominently localized beneath the plasma membrane in T . gondii.

Mol Biochem Parasitol, 1997 Sep, 88(1-2), 1 - 11
Characterization of Trypanosoma brucei pyridoxal kinase: purification, gene isolation and expression in Escherichia coli; Scott TC et al.; Pyridoxal kinase catalyzes the ATP-dependent phosphorylation of vitamin B6, generating pyridoxal-5'-phosphate, an important cofactor for many enzymatic reactions . Pyridoxal kinase was purified 4300-fold to homogeneity from Trypanosoma brucei and peptides generated by proteolysis were subjected to amino acid sequence analysis . The peptide sequence information was used to generate a partial clone of T . brucei pyridoxal kinase by polymerase chain reaction (PCR), which in turn was used to screen a T . brucei genomic library for a full length clone . The 903-bp gene was sequenced and found to encode a 300-amino acid protein . The deduced amino acid sequence contains all of the peptide sequences obtained from the proteolytic cleavage of the native enzyme and shares 28% sequence identity with a putative Escherichia coli pyridoxal kinase, identified for its ability to compliment pyridoxal kinase deficient cells . The T . brucei pyridoxal kinase gene was expressed in E . coli and the purified enzyme was found to have pyridoxal kinase activity, confirming that this gene encodes the functional T . brucei enzyme . Native and recombinant pyridoxal kinase have a monomer molecular weight of 37 kDa by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and are dimers in solution . Native T . brucei pyridoxal kinase catalyzes the phosphorylation of pyridoxal with a specific activity of 990 nmol min(-1) per mg and apparent Km values for pyridoxal and ATP of 22 and 9 microM . respectively . Substrate inhibition is observed for pyridoxal . Similar results were obtained for the recombinant enzyme.

Mol Cell Biol, 1997 Sep, 17(9), 5437 - 52
Clusters of S1 nuclease-hypersensitive sites induced in vivo by DNA damage; Legault J et al.; DNA end-labeling procedures were used to analyze both the frequency and distribution of DNA strand breaks in mammalian cells exposed or not to different types of DNA-damaging agents . The 3' ends were labeled by T4 DNA polymerase-catalyzed nucleotide exchange carried out in the absence or presence of Escherichia coli endonuclease IV to cleave abasic sites and remove 3' blocking groups . Using this sensitive assay, we show that DNA isolated from human cells or mouse tissues contains variable basal levels of DNA strand interruptions which are associated with normal bioprocesses, including DNA replication and repair . On the other hand, distinct dose-dependent patterns of DNA damage were assessed quantitatively in cultured human cells exposed briefly to menadione, methylmethane sulfonate, topoisomerase II inhibitors, or gamma rays . In vivo induction of single-strand breaks and abasic sites by methylmethane sulfonate was also measured in several mouse tissues . The genomic distribution of these lesions was investigated by DNA cleavage with the single-strand-specific S1 nuclease . Strikingly similar cleavage patterns were obtained with all DNA-damaging agents tested, indicating that the majority of S1-hypersensitive sites detected were not randomly distributed over the genome but apparently were clustered in damage-sensitive regions . The parallel disappearance of 3' ends and loss of S1-hypersensitive sites during post-gamma-irradiation repair periods indicates that these sites were rapidly repaired single-strand breaks or gaps (2- to 3-min half-life) . Comparison of S1 cleavage patterns obtained with gamma-irradiated DNA and gamma-irradiated cells shows that chromatin structure was the primary determinant of the distribution of the DNA damage detected.

Mol Cell Biol, 1997 Sep, 17(9), 5369 - 76
Conformational changes induced in Hoxb-8/Pbx-1 heterodimers in solution and upon interaction with specific DNA; Sanchez M et al.; Two classes of homeodomain proteins, Hox and Pbx gene products, have the ability to bind cooperatively to DNA . In Hox proteins, the homeodomain and a highly conserved hexapeptide are required for cooperative DNA binding . In Pbx, the homeodomain and a region immediately C terminal of the homeodomain are essential for cooperativity . Using fluorescence and circular dichroism spectroscopy, we demonstrated that Hox and Pbx proteins interact in the absence of DNA . The interaction in solution is accompanied by conformational changes . Furthermore, upon interaction with specific DNA, additional conformational changes are induced in the Pbx-1/Hoxb-8 heterodimer . These data indicate that prior to DNA binding, Hox-Pbx interaction in solution is accompanied by structural alterations . We propose that these conformational changes modulate the DNA binding properties of these proteins, ultimately resulting in cooperative DNA binding.

Mol Cell Biol, 1997 Sep, 17(9), 5359 - 68
Characterization of strand exchange activity of yeast Rad51 protein; Namsaraev E et al.; The Saccharomyces cerevisiae RAD51 gene product takes part in genetic recombination and repair of DNA double strand breaks . Rad51, like Escherichia coli RecA, catalyzes strand exchange between homologous circular single-stranded DNA (ssDNA) and linear double-stranded DNA (dsDNA) in the presence of ATP and ssDNA-binding protein . The formation of joint molecules between circular ssDNA and linear dsDNA is initiated at either the 5' or the 3' overhanging end of the complementary strand; joint molecules are formed only if the length of the overhanging end is more than 1 nucleotide . Linear dsDNAs with recessed complementary or blunt ends are not utilized . The polarity of strand exchange depends upon which end is used to initiate the formation of joint molecules . Joint molecules formed via the 5' end are processed by branch migration in the 3'-to-5' direction with respect to ssDNA, and joint molecules formed with a 3' end are processed in the opposite direction.

Mol Cell Biol, 1997 Sep, 17(9), 5165 - 75
Characterization of the maize Mutator transposable element MURA transposase as a DNA-binding protein; Benito MI et al.; The autonomous MuDR element of the Mutator (Mu) transposable element family of maize encodes at least two proteins, MURA and MURB . Based on amino acid sequence similarity, previous studies have reported that MURA is likely to be a transposase . The functional characterization of MURA has been hindered by the instability of its cDNA, mudrA, in Escherichia coli . In this study, we report the first successful stabilization and expression of MURA in Saccharomyces cerevisiae . Gel mobility shift assays demonstrate that MURA is a DNA-binding protein that specifically binds to sequences within the highly conserved Mu element terminal inverted repeats (TIRs) . DNase I and 1,10-phenanthroline-copper footprinting of MURA-Mu1 TIR complexes indicate that MURA binds to a conserved approximately 32-bp region in the TIR of Mu1 . In addition, MURA can bind to the same region in the TIRs of all tested actively transposing Mu elements but binds poorly to the diverged Mu TIRs of inactive elements . Previous studies have reported a correlation between Mu transposon inactivation and methylation of the Mu element TIRs . Gel mobility shift assays demonstrate that MURA can interact differentially with unmethylated, hemimethylated, and homomethylated TIR substrates . The significance of MURA's interaction with the TIRs of Mu elements is discussed in the context of what is known about the regulation and mechanisms of Mutator activities in maize.

Exp Lung Res, 1997 Sep-Oct, 23(5), 393 - 404
Neutrophils pretreated with granulocyte colony-stimulating factor (G-CSF) are not related to the severity of endotoxin-induced lung injury; Koizumi T et al.; Neutrophils play an important role in mediating acute lung injury that is characteristic of adult respiratory distress syndrome . Granulocyte colony-stimulating factor (G-CSF) has been shown to increase neutrophil counts and to enhance their biological functions . This study investigated the effects of neutrophils pretreated with G-CSF on endotoxin-induced lung injury in conscious sheep . Nineteen sheep were chronically instrumented with a lung lymph fistula and vascular catheters for monitoring . Sheep were randomly allocated into three groups: group 1-sheep were infused only with endotoxin; group 2-G-CSF (250 micrograms/day) was administered intravenously for 5 days prior to endotoxin; and group 3-G-CSF (125 micrograms) was administered just before endotoxin . In each group, sheep received E . coli endotoxin (1 microgram/kg) for 30 min and observations were made for 5 h after endotoxin administration . Circulating leukocyte counts before endotoxin markedly increased in group 2 and significantly decreased in group 3, when compared with the level in group 1 (9700 +/- 900 (SEM) in group 1, 49,900 +/- 10,000 in group 2, and 3600 +/- 600/microL in group 3) . In each group, circulating leukocyte counts significantly decreased 1 h after endotoxin administration and then returned to baseline values . However, there were no significant differences in either pulmonary hemodynamic or lung lymph responses to endotoxin among the groups . The results indicate that G-CSF does not adversely affect physiologic responses of the lung to endotoxin in sheep.

J Virol, 1997 Sep, 71(9), 7114 - 8
Expression and characterization of recombinant murine cytomegalovirus protease; Sloan JH et al.; The protease domain of the murine cytomegalovirus (MCMV) M80 open reading frame was expressed in and purified from Escherichia coli . The recombinant enzyme was recovered as a mixture of active one- and two-chain forms . The two-chain enzyme was formed by internal cleavage of the one-chain enzyme at the I site . Activity measurements showed that MCMV protease cleaves R- and M-site peptide mimics with kinetics similar to those of recombinant human cytomegalovirus (HCMV) protease . Both the MCMV and HCMV proteases cleave I-site peptide substrates very poorly, but the crystal structure of the HCMV protease indicates that the cytomegalovirus I site likely resides on a solvent-exposed loop close to the active site.

J Virol, 1997 Sep, 71(9), 7030 - 8
The protease and the assembly protein of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8); Unal A et al.; A genomic clone encoding the protease (Pr) and the assembly protein (AP) of Kaposi's sarcoma-associated herpesvirus (KSHV) (also called human herpesvirus 8) has been isolated and sequenced . As with other herpesviruses, the Pr and AP coding regions are present within a single long open reading frame . The mature KSHV Pr and AP polypeptides are predicted to contain 230 and 283 residues, respectively . The amino acid sequence of KSHV Pr has 56% identity with that of herpesvirus salmiri, the most similar virus by phylogenetic comparison . Pr is expressed in infected human cells as a late viral gene product, as suggested by RNA analysis of KSHV-infected BCBL-1 cells . Expression of the Pr domain in Escherichia coli yields an enzymatically active species, as determined by cleavage of synthetic peptide substrates, while an active-site mutant of this same domain yields minimal proteolytic activity . Sequence comparisons with human cytomegalovirus (HCMV) Pr permitted the identification of the catalytic residues, Ser114, His46, and His134, based on the known structure of the HCMV enzyme . The amino acid sequences of the release site of KSHV Pr (Tyr-Leu-Lys-Ala*Ser-Leu-Ile-Pro) and the maturation site (Arg-Leu-Glu-Ala*Ser-Ser-Arg-Ser) show that the extended substrate binding pocket differs from that of other members of the family . The conservation of amino acids known to be involved in the dimer interface region of HCMV Pr suggests that KSHV Pr assembles in a similar fashion . These features of the viral protease provide opportunities to develop specific inhibitors of its enzymatic activity.

J Virol, 1997 Sep, 71(9), 6996 - 7004
Mutational analysis of the adeno-associated virus type 2 Rep68 protein helicase motifs; Walker SL et al.; The adeno-associated virus type 2 (AAV) Rep78 and Rep68 proteins are required for viral replication . These proteins are encoded by unspliced and spliced transcripts, respectively, from the p5 promoter of AAV and therefore have overlapping amino acid sequences . The Rep78 and Rep68 proteins share a variety of activities including endonuclease, helicase, and ATPase activities and the ability to bind AAV hairpin DNA . The part of the amino acid sequence which is identical in Rep78 and Rep68 contains consensus helicase motifs that are conserved among the parvovirus replication proteins . In the present study, we mutated highly conserved amino acids within these helicase motifs . The mutant proteins were synthesized as maltose binding protein-Rep68 fusions in Escherichia coli cells and affinity purified on amylose resin . The fusion proteins were assayed in vitro, and their activities were directly compared to those of the fusion protein MBP-Rep68 delta, which contains most of the amino acid sequences common to Rep78 and Rep68 and was demonstrated previously to have all of the in vitro activities of wild-type Rep78 and Rep68 . Our analysis showed that almost all mutations in the putative helicase motifs severely reduced or abolished helicase activity in vitro . Most mutants also had ATPase activity less than one-eighth of the wild-type levels and lacked endonuclease activity.

J Virol, 1997 Sep, 71(9), 6576 - 81
Molecular characterization of the type-specific gamma-determinant located on the adenovirus fiber; Eiz B et al.; The fiber knob carries the type-specific gamma-antigen which can be demonstrated in hemagglutination inhibition tests . In order to characterize the gamma-determinant we selected subgenus DI adenovirus serotypes 9 and 19 (Ad9 and Ad19) which exhibited 29 amino acid exchanges in the knob domain . Like all subgenus DI adenoviruses they showed a complete hemagglutination pattern with rat and human erythrocytes . We constructed a total of 14 chimeric Ad9/Ad19 and Ad19/Ad9 fiber proteins, which possessed fiber knobs with progressively exchanged Ad9 and Ad19 amino acids . Furthermore, we created 39 fiber proteins with distinct amino acid exchanges in the knob regions by primer-directed mutagenesis . The proteins were expressed in Escherichia coli and tested in hemagglutination and hemagglutination inhibition tests . From our results we can conclude that the type-specific gamma-determinant is not restricted to a distinct region on the adenovirus fiber knob but is composed of at least 17 amino acids . Most of the amino acids contributing to the Ad9 and Ad19 gamma-determinants are located on the fiber knob loops.

Nucleic Acids Res, 1997 Sep 1, 25(17), 3503 - 7
Expression of bovine mitochondrial tRNASer GCU derivatives in Escherichia coli; Hayashi I et al.; By replacing a stretch of five A-U base pairs in the acceptor stem with G-C pairs, mitochondrial tRNA-SerGCU lacking a D arm could be expressed in Escherichia coli cells in considerable amounts . The expressed tRNA with no modified nucleoside was serylated in vitro with the mitochondrial enzyme . The tRNASerGCU derivatives carrying identity elements for alanine tRNA and the related anticodons were expressed . However, this expression event did not affect cell growth, probably because the expression started from the late log phase, which suggests that these mitochondrial tRNA derivatives are not involved in E.coli gene expression systems . Although there are some restrictions in the secondary structure of tRNAs that can be expressed by this method, it could prove useful for preparing large amounts of heterologous tRNAs in vivo.

Cell Tissue Res, 1997 Sep, 289(3), 537 - 45
The separation and characterisation of haemocytes from the mussel Mytilus edulis; Pipe RK et al.; The separation of haemocytes from the mussel Mytilus edulis was carried out on continuous Percoll gradients . The haemocytes separated into three distinct layers, the first comprised 97% basophilic cells, the third comprised 84% eosinophilic cells and the middle layer was a mixture of eosinophilic and basophilic cells . Enzyme cytochemistry demonstrated arylsulphatase, phenol oxidase and peroxidase associated with the haemocytes from the third layer . Lectin-binding studies showed differential binding of lectins to the separated cells . The ultrastructural morphology demonstrated that the first layer of cells was composed predominantly of small agranular cells with a high nucleus to cytoplasm ratio . The second layer comprised a mixture of cells with the majority being granular cells with small granules . The third layer was almost exclusively composed of granular cells with small and large granules . Assays to assess the function of the different cells demonstrated that respiratory burst activity, measured as the reduction of cytochrome-c, was carried out almost entirely by the eosinophilic haemocytes . Similarly, levels of phagocytosis, measured as uptake of Escherichia coli, were much higher in the eosinophilic haemocytes . Of the potential mitogenic factors investigated, concanavalin A and pokeweed mitogen showed some evidence of inducing haemocyte proliferation.

J Mol Biol, 1997 Aug 29, 271(4), 656 - 68
Consideration of the pH-dependent inhibition of dihydrofolate reductase by methotrexate; Cannon WR et al.; Poisson-Boltzmann calculations were used to determine the pKa of protein functional groups in the unliganded dihydrofolate reductase enzyme, and the pKa of protein and ligand groups in methotrexate-enzyme complexes . The results reported here are in conflict with two fundamental tenets of dihydrofolate reductase inhibition by methotrexate: (1) Asp27 is not expected to be protonated near pH 6.5 in the apoenzyme as previously proposed based on fitting of empirical equations to binding data, and (2) the calculated pKa for the pteridine N1 of the inhibitor while bound to the protein is significantly lower than that estimated for this group from interpretation of NMR data (>10) . In fact, the electrostatic calculations and complementary quantum chemical calculations indicate that Asp27 is likely protonated when methotrexate is bound, resulting in a neutral dipole-dipole interaction rather than a salt-bridge between the enzyme and the inhibitor . Reasons for this discrepancy with the experimental data are discussed . Furthermore, His45 and Glu17 in the Escherichia coli enzyme are proposed to be in part responsible for the pH dependence of the conformational degeneracy in the inhibitor-enzyme complex.

J Mol Biol, 1997 Aug 29, 271(4), 645 - 55
Ca2+-loaded spherulin 3a from Physarum polycephalum adopts the prototype gamma-crystallin fold in aqueous solution; Rosinke B et al.; Spherulin 3a is the most abundantly expressed cytosolic protein in spherulating plasmodia of the slime mold Physarum polycephalum . High yields of unlabeled, uniformly 15N and uniformly 13C/15N-labeled recombinant spherulin 3a from Escherichia coli could be produced by a simple protocol described here . The three-dimensional solution structure of Ca2+-loaded spherulin 3a was determined by homo- and heteronuclear NMR spectroscopy . The structure of monomeric spherulin 3a consists of two pleated beta-sheets plus a short alpha-helix arranged into the gamma-crystallin fold . The beta-sheets comprise two intertwined Greek-key motifs . An additional N-terminal beta-strand is unique to spherulin 3a . Complexation of calcium ions greatly enhances overall conformational stability of the protein . The average atomic root-mean-square deviations (r.m.s.d.) for heavy atoms in beta-strands were 0.34(+/-0.16) A for the backbone atoms and 0.73(+/-0.40) A for all atoms . The corresponding r.m.s.d . values for heavy atoms in the whole protein were 0.62(+/-0.42) A for the backbone atoms and 0.99(+/-0.65) A for all atoms . We show the structural relationship between spherulin 3a, a myxomycete dormancy protein, and crystallins from the vertebrate eye lens . Since spherulin 3a has a structure corresponding to one domain of bovine gammaB(II)-crystallin, it represents a hypothetical ancestral gamma-crystallin precursor structure.

J Mol Biol, 1997 Aug 29, 271(4), 602 - 18
Solution scattering structural analysis of the 70 S Escherichia coli ribosome by contrast variation . II . A model of the ribosome and its RNA at 3.5 nm resolution; Svergun DI et al.; Selectively deuterated 70 S E . coli ribosomes and isolated 30 S and 50 S subunits were analyzed by X-ray and neutron solution scattering . The resulting contrast variation data set (42 curves in total) was proven to be consistent in describing the ribosome as a four-phase system composed of the protein and rRNA moieties of both subunits . This data set thus provides ten times more information than a single scattering curve . A solid body four-phase model of the 70 S ribosome at low resolution was built from the envelope functions of the 30 S and 50 S subunits and of those of the corresponding RNA moieties . The four envelopes were parameterized at a resolution of 3.5 nm using spherical harmonics and taking into account interface layers between the phases . The initial approximation for the envelopes of the subunits was taken from electron microscopic data presented recently by J . Frank and co-workers (Albany); the rRNA envelopes were initially approximated by spheres . The optimization and the refinement of the model proceeded by non-linear least squares minimization fitting the available experimental data . The refined envelopes of the subunits differ by about 10% from the starting approximation and the shape of the final 70 S model lies between the outer envelopes of the models by Frank and by M . von Heel & R . Brimacombe (Berlin) . The rRNA moiety in the 30 S subunit is more anisometric than the subunit itself, whereas the rRNA of the 50 S subunit forms a compact core . The rRNAs protrude to the surfaces of the subunits and occupy approximately 30 to 40% of the corresponding surface areas . X-ray scattering curves of the two main functional elongation 70 S complexes (pre- and post-translocational) differ only marginally from those of the non-programmed ribosomes, suggesting that the low resolution four-phase model is also valid for the elongating 70 S ribosome.

J Mol Biol, 1997 Aug 29, 271(4), 588 - 601
Solution scattering structural analysis of the 70 S Escherichia coli ribosome by contrast variation . I . Invariants and validation of electron microscopy models; Svergun DI et al.; Solutions of selectively deuterated 70 S Escherichia coli ribosomes and of free 30 S and 50 S subunits were studied by neutron scattering using contrast variation . The integrity of the partially deuterated particles was controlled by parallel X-ray measurements . Integral parameters of the entire ribosome, of its subunits and of the protein and rRNA moieties were evaluated . The data allow an experimental validation of the two most recent electron microscopy reconstructions of the 70 S ribosome presented by the groups of J . Frank (Albany) and of M . van Heel & R . Brimacombe (Berlin) . For each reconstruction, integral parameters and theoretical scattering curves from the 70 S and its subunits were calculated and compared with the experimental data . Although neither of the two models yields a comprehensive agreement with the experimental data, Frank's model provides a better fit . For the 50 S subunit of van Heel & Brimacombe's model the fit with the experimental data improves significantly when the internal channels and tunnels are filled up . The poorer fit of the latter model is thus caused by its "sponge"-like structure which may partly be due to an enhancement of high frequency contributions in some of the steps of the three-dimensional image reconstruction . It seems therefore unlikely that the ribosome has a "sponge"-like structure with a pronounced network of channels.

J Mol Biol, 1997 Aug 29, 271(4), 545 - 65
A new model for the three-dimensional folding of Escherichia coli 16 S ribosomal RNA . II . The RNA-protein interaction data; Mueller F et al.; The map of the mass centres of the 21 proteins from the Escherichia coli 30 S ribosomal subunit, as determined by neutron scattering, was fitted to a cryoelectron microscopic (cryo-EM) model at a resolution of 20 A of 70 S ribosomes in the pre-translocational state, carrying tRNA molecules at the A and P sites . The fit to the 30 S moiety of the 70 S particles was accomplished with the help of the well-known distribution of the ribosomal proteins in the head, body and side lobe regions of the 30 S subunit, as determined by immuno electron microscopy (IEM) . Most of the protein mass centres were found to lie close to the surface (or even outside) of the cryo-EM contour of the 30 S subunit, supporting the idea that the ribosomal proteins are arranged peripherally around the rRNA . The ribosomal protein distribution was then compared with the corresponding model for the 16 S rRNA, fitted to the same EM contour (described in an accompanying paper), in order to analyse the mutual compatibility of the arrangement of proteins and rRNA in terms of the available RNA-protein interaction data . The information taken into account included the hydroxyl radical and base foot-printing data from Noller's laboratory, and our own in situ cross-linking results . Proteins S1 and S14 were not considered, due to the lack of RNA-protein data . Among the 19 proteins analysed, 12 (namely S2, S4, S5, S7, S8, S9, S10, S11, S12, S15, S17 and S21) showed a fit to the rRNA model that varied from being excellent to at least acceptable . Of the remaining 7, S3 and S13 showed a rather poor fit, as did S18 (which is considered in combination with S6 in the foot-printing experiments) . S16 was difficult to evaluate, as the foot-print data for this protein cover a large area of the rRNA . S19 and S20 showed a bad fit in terms of the neutron map, but their foot-print and cross-link sites were clustered into compact groups in the rRNA model in those regions of the 30 S subunit where these proteins have respectively been located by IEM studies.

J Mol Biol, 1997 Aug 29, 271(4), 524 - 44
A new model for the three-dimensional folding of Escherichia coli 16 S ribosomal RNA . I . Fitting the RNA to a 3D electron microscopic map at 20 A; Mueller F et al.; Recently published models of the Escherichia coli 70 S ribosome at 20 A resolution, obtained by cryo-electron microscopy (cryo-EM) combined with computerized image processing techniques, exhibit two features that are directly relevant to the in situ three-dimensional folding of the rRNA molecules . First, at this level of resolution many fine structural details are visible, a number of them having dimensions comparable to those of nucleic acid helices . Second, in reconstructions of ribosomes in the pre- and post-translocational states, density can be seen that corresponds directly to the A and P site tRNAs, and to the P and E site tRNAs, respectively, thus enabling the decoding region on the 30 S subunit to be located rather precisely . Accordingly, we have refined our previous model for the 16 S rRNA, based on biochemical evidence, by fitting it to the cryo-EM contour of ribosomes carrying A and P site tRNAs . For this purpose, the most immediately relevant evidence consists of new site-directed cross-linking data in the decoding region, which define sets of contacts between the 16 S rRNA and mRNA, or between 16 S rRNA and tRNA at the A, P and E sites; these contact sites can be correlated directly with the tRNA positions in the EM structure . The model is extended to other parts of the 16 S molecule by fitting individual elements of the well-established secondary structure of the 16 S rRNA into the appropriate fine structural elements of the EM contour, at the same time taking into account other data used in the previous model, such as intra-RNA cross-links within the 16 S rRNA itself . The large body of available RNA-protein cross-linking and foot-printing data is also considered in the model, in order to correlate the rRNA folding with the known distribution of the 30 S ribosomal proteins as determined by neutron scattering and immuno-electron microscopy . The great majority of the biochemical data points involve single-stranded regions of the rRNA, and therefore, in contrast to most previous models, the single-stranded regions are included in our structure, with the help of a specially developed modelling programme, ERNA-3D . This allows the various biochemical data sets to be displayed directly, in this and in the accompanying papers, on diagrams of appropriate parts of the rRNA structure within the cryo-EM contour.

J Mol Biol, 1997 Aug 29, 271(4), 499 - 510
Long-range effects in a supercoiled DNA domain generated by transcription in vitro; Wang Z et al.; The translocation of a transcription complex can transiently introduce positive and negative superhelical windings into the template DNA . To gain further insight into this dynamic DNA supercoiling mechanism and its possible involvement in biological processes, we employed an in vitro system in which site-specific recombination by gammadelta resolvase is topologically coupled to transcription-induced negative supercoiling . Our kinetic experiments suggest that recombination is closely linked to the process of supercoiling by transcription . We utilized the known high speed at which two resolvase-bound recombination sites can pair to form a synaptic complex in kinetic experiments with DNA substrates containing three recombination sites . Our data provide evidence for the existence of a transient gradient of negative supercoiling . Such a gradient seems to be predominantly a consequence of DNA double helix rotation behind a translocating RNA polymerase and originates within a broad region up to two kilobase-pairs upstream of the transcriptional start site . We further demonstrate that the topological coupling between transcription and recombination is not affected when the DNA-bending protein integration host factor from E . coli is bound to multiple sites within the phage lambda attachment region . We discuss implications of our in vitro findings with respect to possible in vivo functions of the dynamic nature of transcription-induced supercoiling.

J Mol Biol, 1997 Aug 29, 271(4), 491 - 8
Reported translational bypass in a trpR'-lacZ' fusion is accounted for by unusual initiation and +1 frameshifting; Wills NM et al.; I . Benhar and H . Engelberg-Kulka reported that a 55 nucleotide translational bypass occurs in decoding a fusion of the Escherichia coli tryptophan repressor, trpR, and lacZ genes . The start of the bypass occurred in the trpR gene and coding resumed in the lacZ gene . It was considered that bypassing likely occurred in expression of trpR itself to produce an additional 10 kDa product which may be biologically important . We report here that bypass is undetectable in the same and related trpR'-lacZ' fusions . The beta-galactosidase activity derived from the fusions is accounted for by unusual internal initiation and +1 frameshifting, both of which occur in the lacZ part of the fusion . The 10 kDa product reportedly encoded by the trpR gene was not detectable to a level of 1% of the full-length 12 kDa tryptophan repressor product, at least when expressed from a T7 promoter.

Science, 1997 Aug 29, 277(5330), 1262 - 7
A conformational switch in Escherichia coli 16S ribosomal RNA during decoding of messenger RNA; Lodmell JS et al.; Direct evidence is presented for a conformational switch in 16S ribosomal RNA (rRNA) that affects tRNA binding to the ribosome and decoding of messenger RNA (mRNA) . These data support the hypothesis that dynamic changes in rRNA structure occur during translation . The switch involves two alternating base-paired arrangements apparently facilitated by ribosomal proteins S5 and S12, and produces significant changes in the rRNA structure . Chemical probing shows reciprocal enhancements or protections at sites in 16S rRNA that are at or very near sites that were previously crosslinked to mRNA . These data indicate that the switch affects codon-anticodon arrangement and proper selection of tRNA at the ribosomal A site, and that the switch is a fundamental mechanism in all ribosomes.

J Biol Chem, 1997 Aug 29, 272(35), 22272 - 7
Interactions of forskolin and ATP with the cytosolic domains of mammalian adenylyl cyclase; Dessauer CW et al.; Fragments of the two cytoplasmic domains of mammalian adenylyl cyclases can be synthesized independently (and abundantly) as soluble proteins; Gsalpha- and forskolin-stimulated enzymatic activity is restored upon their mixture . We have utilized this system to characterize the interactions of adenylyl cyclase with forskolin and its substrate, ATP . In the presence of Gsalpha, adenylyl cyclase is activated in response to occupation of only one forskolin-binding site . A single binding site for forskolin was identified by equilibrium dialysis; its Kd (0.1 microM) corresponds to the EC50 for enzyme activation . The affinity of forskolin for adenylyl cyclase is greatly reduced in the absence of Gsalpha ( approximately 40 microM) . Binding of forskolin to the individual cytoplasmic domains of the enzyme was not detected . A single binding site for the ATP analog, alpha,beta-methylene ATP (Ap(CH2)pp), was also detected by equilibrium dialysis . Such binding was not observed with the individual domains . Binding of Ap(CH2)pp was unaffected by P-site inhibitors of adenylyl cyclase . A modified P-loop sequence located near the carboxyl terminus of adenylyl cyclase has been implicated in ATP binding . Mutation of the conserved, non-glycine residues within this region caused no significant changes in the Km for ATP or the Ki for Ap(CH2)pp . It thus seems unlikely that this region is part of the active site . However, a mutation in the C1 domain (E518A) causes a 10-fold decrease in the binding affinity for Ap(CH2)pp . This residue and the active site of the enzyme may lie at the interface between the two cytosolic domains.

J Biol Chem, 1997 Aug 29, 272(35), 22253 - 8
Protein-protein interaction specificity of Im9 for the endonuclease toxin colicin E9 defined by homologue-scanning mutagenesis; Li W et al.; The colicin DNase-specific immunity proteins interact with the endonuclease domain of the bacterial toxin colicin E9 with dissociation constants that span the millimolar to femtomolar affinity range . Among the non-cognate interactions Im2 shows the strongest binding toward the E9 DNase domain with a Kd of 10(-8) M, 6 orders of magnitude weaker than that of the cognate immunity protein Im9 . Based on a NMR structure of Im9 that shows it to be a 4-helix protein, we have conducted a mutagenic scan in which elements of Im9 secondary structure were substituted into Im2 to precisely delineate regions that define specificity . Eleven chimeras were constructed, and their biological cross-reactivity toward colicins E2 and E9 was evaluated . From this set of mutants seven proteins were purified, and the Kd for their interaction with the E9 DNase domain was measured by a combination of stopped-flow fluorescence and subunit exchange kinetics . Our results show that immunity specificity is dominated by residues on helix II, accounting for 5 orders of magnitude binding specificity relative to Im2, and that packing interactions of helix II with its neighbor helix I and the loop connecting helix III with helix IV play minor roles . The conformational stability of these chimeric proteins was also determined . Proteins displaying an Im9 phenotype were all more stable than the parent Im2 protein, and surprisingly some chimeras were significantly more stable than either Im2 or Im9.

J Biol Chem, 1997 Aug 29, 272(35), 22125 - 33
Biochemical characterization and mass spectrometric disulfide bond mapping of periplasmic alpha-amylase MalS of Escherichia coli; Spiess C et al.; Periplasmic alpha-amylase of Escherichia coli, the malS gene product, hydrolyzes linear maltodextrins . The purified enzyme exhibited a Km of 49 microM and a Vmax of 0.36 micromol of p-nitrophenylhexaoside hydrolyzed per min per mg of protein . Amylase activity was optimal at pH 8 and was dependent on divalent cations such as Ca2+ . MalS exhibited altered migration on SDS-polyacrylamide gel electrophoresis under nonreducing conditions . Analytical ultracentrifugation and electrospray mass spectrometry indicated that MalS is monomeric . The four cysteine residues are involved in intramolecular disulfide bonds . To map disulfide bonds, MalS was proteolytically digested . The resulting peptides were separated by reverse phase-high performance liquid chromatography, and matrix-assisted laser desorption/ionization mass spectrometry analysis indicated the presence of two disulfide bonds, i.e . Cys40-58 and Cys104-520 . The disulfide bond at Cys40-58 is located in an N-terminal extension of about 160 amino acids which has no homology to other amylases but to the proposed peptide binding domain of GroEL, the Hsp60 of E . coli . The N-terminal extension is linked to the C-terminal amylase domain via disulfide bond Cys104-520 . Reduction of disulfide bonds by dithiothreitol treatment led to aggregation suggesting that the N terminus of MalS may represent an internal chaperone domain.

J Biol Chem, 1997 Aug 29, 272(35), 22097 - 102
Ricin A chain can transport unfolded dihydrofolate reductase into the cytosol; Beaumelle B et al.; Ricin is a heterodimeric protein toxin . The ricin A chain is able to cross the membrane of intracellular compartments to reach the cytosol where it catalytically inactivates protein synthesis . It is linked via a disulfide bond to the B chain, a galactose-specific lectin, which allows ricin binding at the cell surface and endocytosis . To examine the potential of ricin A to carry proteins into the cytosol and the requirement for unfolding of the passenger protein, we connected mouse dihydrofolate reductase (DHFR) to ricin A by gene fusion via a spacer peptide . DHFR-ricin A expressed in Escherichia coli displayed the biological activities of the parent proteins and associated quantitatively with ricin B to form DHFR-ricin . The resulting toxin was highly cytotoxic to cells (4-8-fold less than recombinant ricin) . DHFR-ricin cytotoxicity was inhibited by methotrexate, a DHFR inhibitor stabilizing DHFR-ricin A in a folded conformation . The DHFR moiety of DHFR ricin bound to the plasma membrane . Although methotrexate prevented this binding, it did not significantly affect DHFR-ricin endocytosis, which proceeded via ricin B chain . Intoxication kinetics data and a cell-free translocation assay demonstrated that protection of cells from DHFR-ricin cytotoxicity resulted from a selective inhibition by methotrexate of DHFR-ricin A translocation . We conclude that ricin A is a potential carrier of proteins to the cytosol, provided that the passenger protein is able to unfold for transmembrane transport.

J Biol Chem, 1997 Aug 29, 272(35), 22092 - 6
Comparison of the DNA association kinetics of the Lac repressor tetramer, its dimeric mutant LacIadi, and the native dimeric Gal repressor; Hsieh M et al.; The rates of association of the tetrameric Lac repressor (LacI), dimeric LacIadi (a deletion mutant of LacI), and the native dimeric Gal repressor (GalR) to DNA restriction fragments containing a single specific site were investigated using a quench-flow DNase I "footprinting" technique . The dimeric proteins, LacIadi and GalR, and tetrameric LacI possess one and two DNA binding sites, respectively . The nanomolar protein concentrations used in these studies ensured that the state of oligomerization of each protein was predominantly either dimeric or tetrameric, respectively . The bimolecular association rate constants (ka) determined for the LacI tetramer exceed those of the dimeric proteins . The values of ka obtained for LacI, LacIadi, and GalR display different dependences on {KCl} . For LacIadi and GalR, they diminish as {KCl} increases from 25 mM to 200 mM, approaching rates predicted for three-dimensional diffusion . In contrast, the ka values determined for the tetrameric LacI remain constant up to 300 mM {KCl}, the highest salt concentration that could be investigated by quench-flow footprinting . The enhanced rate of association of the tetramer relative to the dimeric proteins can be modeled by enhanced "sliding" (Berg, O . G., Winter, R . B., and von Hippel, P . H . (1981) Biochemistry 20, 6929-6948) of the LacI tetramer relative to the LacIadi dimer or a combination of enhanced sliding and the superimposition of "direct transfer" mediated by the bidentate DNA interactions of the tetramer.

J Biol Chem, 1997 Aug 29, 272(35), 22023 - 9
The isolated RNase H domain of murine leukemia virus reverse transcriptase . Retention of activity with concomitant loss of specificity; Zhan X et al.; Retroviral RNases H are similar in sequence and structure to Escherichia coli RNase HI and yet have differences in substrate specificities, metal ion requirements, and specific activities . Separation of reverse transcriptase (RT) into polymerase and RNase H domains yields an active RNase H from murine leukemia virus (MuLV) but an inactive human immunodeficiency virus (HIV) RNase H . The "handle region" present in E . coli RNase HI but absent in HIV RNase H contributes to the binding to its substrate and when inserted into HIV RNase H results in an active enzyme retaining some degree of specificity . Here, we show MuLV protein containing the C-terminal 175 amino acids with its own handle region or that of E . coli RNase HI has the same specific activity as the RNase H of RT, retains a preference for Mn2+ as the cation required for activity, and has association rate (KA) 10% that of E . coli RNase HI . However, with model substrates, specificities for removal of the tRNAPro primer and polypurine tract stability are lost, indicating specificity of RNase H of MuLV requires the remainder of the RT . Differences in KA, while significant, appear insufficient to account for the differences in specific activities of the bacterial and viral RNases H.

J Biol Chem, 1997 Aug 29, 272(35), 22015 - 22
Kinetic and stoichiometric analysis for the binding of Escherichia coli ribonuclease HI to RNA-DNA hybrids using surface plasmon resonance; Haruki M et al.; To understand how ribonucleases H recognize RNA-DNA hybrid substrates, we analyzed kinetic parameters of binding of Escherichia coli RNase HI to RNA-DNA hybrids ranging in length from 18 to 36 base pairs (bp) using surface plasmon resonance (BIAcoreTM) . The kon and koff values for the binding of the enzyme to the 36-bp substrate were 1.5 x 10(6) M-1 s-1 and 3.2 x 10(-2) s-1, respectively . Similar values were obtained with the shorter substrates . Using uncleavable 2'-O-methylated RNA-DNA substrates, values for kon and koff were 2.1 x 10(5) M-1 s-1 and 1.3 x 10(-1) s-1 in the absence of Mg2+ that were further reduced in the presence of Mg2+ to 7.4 x 10(3) M-1 s-1 and 2.6 x 10(-2) s-1 . Kinetic parameters similar to the wild-type enzyme were obtained using an active-site mutant enzyme, Asp134 replaced by Ala, whereas a greatly reduced on-rate was observed for another inactive mutant enzyme, in which the basic protrusion is eliminated, thereby distinguishing between poor catalysis and inability to bind to the substrate . Stoichiometric analyses of RNase HI binding to substrates of 18, 24, 30, and 36 bp are consistent with previous reports suggesting that RNase HI binds to 9-10 bp of RNA-DNA hybrid.

J Biol Chem, 1997 Aug 29, 272(35), 21977 - 81
Expression, purification, and characterization of inosine 5'-monophosphate dehydrogenase from Borrelia burgdorferi; Zhou X et al.; Inosine 5'-monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in de novo guanine nucleotide biosynthesis . IMPDH converts IMP to xanthosine 5'-monophosphate with concomitant conversion of NAD+ to NADH . All IMPDHs characterized to date contain a 130-residue "subdomain" that extends from an N-terminal loop of the alpha/beta barrel domain . The role of this subdomain is unknown . An IMPDH homolog has been cloned from Borrelia burgdorferi, the causative agent of Lyme disease (Margolis, N., Hogan, D., Tilly, K., and Rosa, P . A . (1994) J . Bacteriol . 176, 6427-6432) . This homolog has replaced the subdomain with a 50-residue segment of unrelated sequence . We have expressed and characterized the B . burgdorferi IMPDH homolog . This protein has IMPDH activity, which unequivocally demonstrates that the subdomain is not required for catalytic activity . The monovalent cation and dinucleotide binding sites of B . burgdorferi IMPDH are significantly different from those of human IMPDH . Therefore, these sites are targets for the design of specific inhibitors for B . burgdorferi IMPDH . Such inhibitors might be new treatments for Lyme disease.

J Biol Chem, 1997 Aug 29, 272(35), 21956 - 63
Role of domains in Escherichia coli and mammalian mitochondrial elongation factor Ts in the interaction with elongation factor Tu; Zhang Y et al.; Bovine mitochondrial elongation factor Ts (EF-Tsmt) stimulates the activity of Escherichia coli elongation factor Tu (EF-Tu) . In contrast, E . coli EF-Ts is unable to stimulate mitochondrial EF-Tu . EF-Tsmt forms a tight complex with E . coli EF-Tu governed by an association constant of 8.6 x 10(10) . This value is 100-fold stronger than the binding constant for the formation of the E . coli EF-Tu.Ts complex . To test which domain of EF-Tsmt is important for its strong binding with EF-Tu, chimeras were made between E . coli EF-Ts and EF-Tsmt . Replacing the N-terminal domain of E . coli EF-Ts with that of EF-Tsmt increases its binding to E . coli EF-Tu 2-3-fold . Replacing the N-terminal domain of EF-Tsmt with the corresponding region of E . coli EF-Ts decreases its binding to E . coli EF-Tu approximately 4-5-fold . A chimera consisting of the C-terminal half of E . coli EF-Ts and the N-terminal half of EF-Tsmt binds to E . coli EF-Tu as strongly as EF-Tsmt . A chimera in which Subdomain N of the core of EF-Ts is replaced by the corresponding region of EF-Tsmt binds E . coli EF-Tu approximately 25-fold more tightly than E . coli EF-Ts . Thus, the higher strength of the interaction between EF-Tsmt and EF-Tu can be localized primarily to a single subdomain.

J Biol Chem, 1997 Aug 29, 272(35), 21950 - 5
Biphasic binding kinetics between FepA and its ligands; Payne MA et al.; The Escherichia coli FepA protein is an energy- and TonB-dependent, ligand-binding porin that functions as a receptor for the siderophore ferric enterobactin and colicins B and D . We characterized the kinetic and thermodynamic parameters associated with the initial, energy-independent steps in ligand binding to FepA . In vivo experiments produced Kd values of 24, 185, and 560 nM for ferric enterobactin, colicin B, and colicin D, respectively . The siderophore and colicin B bound to FepA with a 1:1 stoichiometry, but colicin D bound to a maximum level that was 3-fold lower . Preincubation with ferric enterobactin prevented colicin B binding, and preincubation with colicin B prevented ferric enterobactin binding . Colicin B release from FepA was unexpectedly slow in vivo, about 10-fold slower than ferric enterobactin release . This slow dissociation of the colicin B.FepA complex facilitated the affinity purification of FepA and FepA mutants with colicin B-Sepharose . Analysis of a fluorescent FepA derivative showed that ferric enterobactin and colicin B adsorbed with biphasic kinetics, suggesting that both ligands bind in at least two distinct steps, an initial rapid stage and a subsequent slower step, that presumably establishes a transport-competent complex.

J Biol Chem, 1997 Aug 29, 272(35), 21932 - 7
Active-site Arg --> Lys substitutions alter reaction and substrate specificity of aspartate aminotransferase; Vacca RA et al.; Arg386 and Arg292 of aspartate aminotransferase bind the alpha and the distal carboxylate group, respectively, of dicarboxylic substrates . Their substitution with lysine residues markedly decreased aminotransferase activity . The kcat values with L-aspartate and 2-oxoglutarate as substrates under steady-state conditions at 25 degrees C were 0.5, 2.0, and 0.03 s-1 for the R292K, R386K, and R292K/R386K mutations, respectively, kcat of the wild-type enzyme being 220 s-1 . Longer dicarboxylic substrates did not compensate for the shorter side chain of the lysine residues . Consistent with the different roles of Arg292 and Arg386 in substrate binding, the effects of their substitution on the activity toward long chain monocarboxylic (norleucine/2-oxocaproic acid) and aromatic substrates diverged . Whereas the R292K mutation did not impair the aminotransferase activity toward these substrates, the effect of the R386K substitution was similar to that on the activity toward dicarboxylic substrates . All three mutant enzymes catalyzed as side reactions the beta-decarboxylation of L-aspartate and the racemization of amino acids at faster rates than the wild-type enzyme . The changes in reaction specificity were most pronounced in aspartate aminotransferase R292K, which decarboxylated L-aspartate to L-alanine 15 times faster (kcat = 0.002 s-1) than the wild-type enzyme . The rates of racemization of L-aspartate, L-glutamate, and L-alanine were 3, 5, and 2 times, respectively, faster than with the wild-type enzyme . Thus, Arg --> Lys substitutions in the active site of aspartate aminotransferase decrease aminotransferase activity but increase other pyridoxal 5'-phosphate-dependent catalytic activities . Apparently, the reaction specificity of pyridoxal 5'-phosphate-dependent enzymes is not only achieved by accelerating the specific reaction but also by preventing potential side reactions of the coenzyme substrate adduct.

J Biol Chem, 1997 Aug 29, 272(35), 21865 - 71
The amino-terminal 118 amino acids of Escherichia coli trigger factor constitute a domain that is necessary and sufficient for binding to ribosomes; Hesterkamp T et al.; Escherichia coli trigger factor has prolyl-isomerase and chaperone activities and associates with nascent polypeptide chains . Trigger factor has a binding site on ribosomes, which is a prerequisite for its efficient association with nascent chains and its proposed function as a cotranslational folding catalyst . We set out to identify the domain of trigger factor that mediates ribosome binding . Of a series of recombinant fragments, the amino-terminal fragments, TF (1-144) and TF (1-247), cofractionated with ribosomes from cell extracts and rebound to isolated ribosomes in vitro . They competed efficiently with full-length trigger factor for stoichiometric binding to a single site on the large ribosomal subunit . However, TF (1-144) and TF (1-247) differed from full-length trigger factor in that their association with ribosomes was not strengthened by the presence of nascent chains, indicating a role for carboxyl-terminal trigger factor segment in sensing the translational status . The domain responsible for ribosome binding was further investigated by limited proteolysis of recombinant fragments . A stable domain comprising the amino-terminal 118 residues was identified that was still capable of ribosome binding and thus represents a novel structural and functional element of trigger factor.

J Biol Chem, 1997 Aug 29, 272(35), 21855 - 64
Identification of the gene encoding the Escherichia coli lipid A 4'-kinase . Facile phosphorylation of endotoxin analogs with recombinant LpxK; Garrett TA et al.; The genes for seven of nine enzymes needed for the biosynthesis of Kdo2-lipid A (Re endotoxin) in Escherichia coli have been reported . We have now identified a novel gene encoding the lipid A 4'-kinase (the sixth step of the pathway) . The 4'-kinase transfers the gamma-phosphate of ATP to the 4'-position of a tetraacyldisaccharide 1-phosphate intermediate (termed DS-1-P) to form tetraacyldisaccharide 1,4'-bis-phosphate (lipid IVA) . The 4'-phosphate is required for the action of distal enzymes, such as Kdo transferase and also renders lipid A substructures active as endotoxin antagonists or mimetics . Lysates of E . coli generated using individual lambda clones from the ordered Kohara library were assayed for overproduction of 4'-kinase . Only one clone, {218}E1D1, which directed 2-2.5-fold overproduction, was identified . This construct contains 20 kilobase pairs of E . coli DNA from the vicinity of minute 21 . Two genes related to the lipid A system map in this region: msbA, encoding a putative translocator, and kdsB, the structural gene for CMP-Kdo synthase . msbA forms an operon with a downstream, essential open reading frame of unknown function, designated orfE . orfE was cloned into a T7 expression system . Washed membranes from cells overexpressing orfE display approximately 2000-fold higher specific activity of 4'-kinase than membranes from cells with vector alone . Membranes containing recombinant, overexpressed 4'-kinase (but not membranes with wild-type kinase levels) efficiently phosphorylate three DS-1-P analogs: 3-aza-DS-1-P, base-treated DS-1-P, and base-treated 3-aza-DS-1-P . A synthetic hexaacylated DS-1-P analog, compound 505, can also be phosphorylated by membranes from the overproducer, yielding {4'-32P} lipid A (endotoxin) . The overexpressed lipid A 4'-kinase is very useful for making new 4'-phosphorylated lipid A analogs with potential utility as endotoxin mimetics or antagonists . We suggest that orfE is the structural gene for the 4'-kinase and that it be redesignated lpxK.

J Biol Chem, 1997 Aug 29, 272(35), 21818 - 23
Borna disease virus P-protein is phosphorylated by protein kinase Cepsilon and casein kinase II; Schwemmle M et al.; Borna disease virus (BDV) is a newly classified nonsegmented negative-strand RNA virus (order of Mononegavirales) that persistently infects specific brain regions and circuits of warm-blooded animals to cause behavioral disturbances . Viruses within the order of Mononegavirales have phosphoproteins that typically serve as transcription factors and are modulated in functional activity through phosphorylation . To identify the kinases involved in BDV phosphoprotein (BDV-P) phosphorylation, in vitro phosphorylation assays were performed using recombinant phosphoprotein produced in Escherichia coli as substrate and cytoplasmic extracts from a rat glioma cell line (C6) or rat brain extracts as sources of kinase activity . These experiments revealed that BDV-P was phosphorylated predominantly by protein kinase C (PKC) and to a lesser extent by casein kinase II . Partial purification of the PKC from rat brain extract suggested that the BDV-P phosphorylating kinase is PKCepsilon . A role for PKC phosphorylation in vivo was confirmed by using the PKC-specific inhibitor GF109203X . Furthermore, peptide mapping studies indicated that BDV-P is phosphorylated at the same sites in vitro as it is in vivo . Mutational analysis identified Ser26 and Ser28 as sites for PKC phosphorylation and Ser70 and Ser86 as sites for casein kinase II phosphorylation . The anatomic distribution of PKCepsilon in the central nervous system may have implications for BDV neurotropism and pathogenesis.

J Biol Chem, 1997 Aug 29, 272(35), 21784 - 92
Monoterpene synthases from grand fir (Abies grandis) . cDNA isolation, characterization, and functional expression of myrcene synthase, (-)-(4S)-limonene synthase, and (-)-(1S,5S)-pinene synthase; Bohlmann J et al.; Grand fir (Abies grandis) has been developed as a model system for studying defensive oleoresin formation in conifers in response to insect attack or other injury . The turpentine fraction of the oleoresin is a complex mixture of monoterpene (C10) olefins in which (-)-limonene and (-)-alpha- and (-)-beta-pinene are prominent components; (-)-limonene and (-)-pinene synthase activities are also induced upon stem wounding . A similarity based cloning strategy yielded three new cDNA species from a wounded stem cDNA library that appeared to encode three distinct monoterpene synthases . After expression in Escherichia coli and enzyme assay with geranyl diphosphate as substrate, subsequent analysis of the terpene products by chiral phase gas chromatography and mass spectrometry showed that these sequences encoded a (-)-limonene synthase, a myrcene synthase, and a (-)-pinene synthase that produces both alpha-pinene and beta-pinene . In properties and reaction stereochemistry, the recombinant enzymes resemble the corresponding native monoterpene synthases of wound-induced grand fir stem . The deduced amino acid sequences indicated the limonene synthase to be 637 residues in length (73.5 kDa), the myrcene synthase to be 627 residues in length (72.5 kDa), and the pinene synthase to be 628 residues in length (71.5 kDa); all of these monoterpene synthases appear to be translated as preproteins bearing an amino-terminal plastid targeting sequence . Sequence comparison revealed that these monoterpene synthases from grand fir resemble sesquiterpene (C15) synthases and diterpene (C20) synthases from conifers more closely than other monoterpene synthases from angiosperm species . This similarity between extant monoterpene, sesquiterpene, and diterpene synthases of gymnosperms is surprising since functional diversification of this enzyme class is assumed to have occurred over 300 million years ago . Wound-induced accumulation of transcripts for monoterpene synthases was demonstrated by RNA blot hybridization using probes derived from the three monoterpene synthase cDNAs . The availability of cDNA species encoding these monoterpene synthases will allow an understanding of the regulation of oleoresin formation in conifers and will ultimately permit the transgenic manipulation of this defensive secretion to enhance resistance to insects . These cDNAs also furnish tools for defining structure-function relationships in this group of catalysts that generate acyclic, monocyclic, and bicyclic olefin products.

J Biol Chem, 1997 Aug 29, 272(35), 21726 - 34
Dissection of pathways implicated in integrin-mediated actin cytoskeleton assembly . Involvement of protein kinase C, Rho GTPase, and tyrosine phosphorylation; Defilippi P et al.; A panel of antibodies to the alphaIIbbeta3 integrin was used to promote adhesion of Chinese hamster ovary cells transfected with the alphaIIbbeta3 fibrinogen receptor . While some alphaIIbbeta3 antibodies were not able to induce p125 focal adhesion kinase (p125FAK) tyrosine phosphorylation, all the antibodies equally support cell adhesion but not spreading and assembly of actin stress fibers . Absence of stress fibers was also obtained by plating on antibodies directed to the hamster beta1 integrin . In contrast, cells plated on matrix proteins spread organizing actin stress fibers . Treatment with phorbol esters phorbol 12-myristate 13-acetate (PMA) induced cells to spread on antibodies-coated dishes but not to organize actin in stress fibers . The combination of PMA and cytotoxic necrotizing factor 1 (CNF1), a specific Rho activator, induced cell spreading and organization of stress fibers . PMA or the combination of PMA and CNF1 also increases tyrosine phosphorylation of p125FAK in response to antibodies that were otherwise unable to trigger this response . These data show that: 1) matrix proteins and antibodies differ in their ability to induce integrin-dependent actin cytoskeleton organization (while matrix induced stress fibers formation, antibodies did not); 2) p125FAK tyrosine phosphorylation is insufficient per se to trigger actin stress fibers formation since antibodies that activate p125FAK tyrosine phosphorylation did not lead to actin stress fibers assembly; and 3) the inability of anti-integrin antibodies to trigger stress fibers organization is overcome by concomitant activation of the protein kinase C (PKC) and Rho pathways; PKC activation leads to cell spreading and Rho activation is required to organize actin stress fibers.

J Biol Chem, 1997 Aug 29, 272(35), 21720 - 5
Probing the interactions of putidaredoxin with redox partners in camphor P450 5-monooxygenase by mutagenesis of surface residues; Holden M et al.; The role of surface amino acid residues in the interaction of putidaredoxin (Pdx) with its redox partners in the cytochrome P450cam (CYP101) system was investigated by site-directed mutagenesis . The mutated Pdx genes were expressed in Escherichia coli, and the proteins were purified and studied in vitro . Activity of the complete reconstituted P450cam system was measured, and kinetic parameters were determined . Partial assays were also conducted to determine the effect of the mutations on interactions with each redox partner . Some mutations altered interactions of Pdx with one redox partner but not the other . Other mutations affected interactions with both redox partners, suggesting some overlap in the binding sites on Pdx for putidaredoxin reductase and CYP101 . Cysteine 73 of Pdx was identified as important in the interaction of Pdx with putidaredoxin reductase, whereas aspartate 38 serves a critical role in the subunit binding and electron transfer to CYP101.

J Biol Chem, 1997 Aug 29, 272(35), 21692 - 9
Elimination of all charged residues in the vicinity of the active-site helix of the disulfide oxidoreductase DsbA . Influence of electrostatic interactions on stability and redox properties; Jacobi A et al.; Disulfide oxidoreductases are structurally related proteins that share the thioredoxin fold and a catalytic disulfide bond that is located at the N terminus of an alpha-helix . The different redox potentials of these enzymes varying from -270 mV for thioredoxin to -125 mV for DsbA have been attributed to the lowered pKa values of their nucleophilic, active-site cysteines and the difference in thermodynamic stability between their oxidized and reduced forms (DeltaDeltaGox/red) . The lowered pKa of the nucleophilic cysteine thiols was proposed to result from favorable interactions with the helix dipole and charged residues in their vicinity . In this study, we have eliminated all charged residues in the neighborhood of the active-site disulfide of DsbA from Escherichia coli to analyze their contribution to the physicochemical properties of the protein . We show that the conserved charge network among residues Glu24, Glu37, and Lys58 stabilizes the oxidized form of DsbA and thus does not cause the high redox potential of the enzyme . The pKa values of the nucleophilic cysteine (Cys30) and the redox potentials of the DsbA variants E24Q, E37Q, K58M, E24Q/K58M, E37Q/K58M, E24Q/E37Q, E24Q/E37Q/K58M, and E24Q/E37Q/E38Q/K58M are similar to those of DsbA wild type . The redox potentials of the variants neither correlate with the Cys30 pKa values nor with the DeltaDeltaGox/red values, demonstrating that the relationship between these parameters is far more complex than previously thought.

Biochemistry, 1997 Aug 26, 36(34), 10558 - 65
Altering the binuclear manganese cluster of arginase diminishes thermostability and catalytic function; Scolnick LR et al.; Arginase is a thermostable (Tm = 75 degrees C) binuclear manganese metalloenzyme which hydrolyzes l-arginine to form l-ornithine and urea . The three-dimensional structures of native metal-depleted arginase, metal-loaded H101N arginase, and metal-depleted H101N arginase have been determined by X-ray crystallographic methods to probe the roles of the manganese ion in site A (Mn2+A) and its ligand H101 in catalysis and thermostability . We correlate these structures with thermal stability and catalytic activity measurements reported here and elsewhere {Cavalli, R . C., Burke, C . J., Kawamoto, S., Soprano, D . R., and Ash, D . E . (1994) Biochemistry 33, 10652-10657} . We conclude that the substitution of a wild-type histidine ligand to Mn2+A compromises metal binding, which in turn compromises protein thermostability and catalytic function . Therefore, a fully occupied binuclear manganese metal cluster is required for optimal catalysis and thermostability.

Biochemistry, 1997 Aug 26, 36(34), 10526 - 37
Structure and function of ubiquitin conjugating enzyme E2-25K: the tail is a core-dependent activity element; Haldeman MT et al.; Individual members of the conserved family of ubiquitin conjugating enzymes (E2s) mediate the ubiquitination and turnover of specific substrates of the ubiquitin-dependent degradation pathway . E2 proteins have a highly conserved core domain of approximately 150 amino acids which contains the active-site Cys . Certain E2s have unique terminal extensions, which are thought to contribute to selective E2 function by interacting either with substrates or with trans-acting factors such as ubiquitin-protein ligases (E3s) . We used the mammalian ubiquitin conjugating enzyme E2-25K in a biochemical test of this hypothesis . The properties of two truncated derivatives show that the 47-residue tail of E2-25K is necessary for three of the enzyme's characteristic properties: high activity in the synthesis of unanchored K48-linked polyubiquitin chains; resistance of the active-site Cys residue to alkylation; and an unusual discrimination against noncognate (nonmammalian) ubiquitin activating (E1) enzymes . However, the tail is not sufficient to generate these properties, as shown by the characteristics of a chimeric enzyme in which the tail of E2-25K was fused to the core domain of yeast UBC4 . These and other results indicate that the specific biochemical function of the tail is strongly dependent upon unique features of the E2-25K core domain . Thus, divergent regions within the conserved core domains of E2 proteins may be highly significant for function . Expression of truncated E2-25K as a glutathione S-transferase (GST) fusion protein resulted in the apparent recovery of E2-25K-specific properties, including activity in chain synthesis . However, the catalytic mechanism utilized by the truncated fusion protein proved to be distinct from the mechanism utilized by the wild-type enzyme . The unexpected properties of the fusion protein were due to GST-induced dimerization . These results indicate the potential for self-association to modulate the polyubiquitin chain synthesis activities of E2 proteins, and indicate that caution should be applied in interpreting the activities of GST fusion proteins.

Biochemistry, 1997 Aug 26, 36(34), 10517 - 25
Noncoded amino acid replacement probes of the aspartate aminotransferase mechanism; Park Y et al.; The primary role of Tyr225 in the aspartate aminotransferase mechanism is to provide a hydrogen bond to stabilize the 3'O- functionality of bound pyridoxal phosphate . The strength of this hydrogen bond is perturbed by replacement of Tyr225 with 3-fluoro-L-tyrosine (FlTyr) by in vitro transcription/translation . This mutant enzyme exhibits kcat/values that are near to those of wild type enzyme; however, the kcat/vs pH profile is much sharper with similar pKas of approximately 7.5 for both the ascending and descending limbs . The pKas are assigned to the endocyclic proton of the internal aldimine and to the bridging hydrogen bond, respectively . The pKas in the kcat vs pH profile of 7.2 and 8.7 are assigned to the epsilon-NH3+ of lysine 258 and to the endocyclic protons of the ketimine complex, respectively . Arginine 292 forms a salt bridge with the beta-COOH of the substrate, aspartate . An improvement on the earlier attempt to invert the substrate charge specificity via R292D mutation-induced arginine transaminase activity {Cronin, C . N., & Kirsch, J . F . (1988) Biochemistry 27, 4572-4579} is described . Here Arg292 is replaced with homoglutamate (R292hoGlu) . This construct exhibits 6.8 x 10(4)-fold greater activity for the cationic substrate D,L-{Calpha-3H}-alpha-amino-beta-guanidinopropionic acid (D,L-{Calpha-3H}AGPA) than does wild type enzyme . The gain in selectivity for this substrate is at least 4500-fold greater than that achieved in the 1988 experiment, i.e., {(kcat/KM)R292hoGlu/(kcat/KM)WT (D,L-{Calpha-3H}AGPA)} >/= 4500 x {(kcat/KM)R292D/(kcat/KM)WT (L-arginine)} . The value of (kcat/KM)R292D is 0.43 M-1 s-1 with L-Arg while (kcat/KM)R292hoGlu is 29 M-1 s-1 with D,L-{Calpha-3H}AGPA (it is assumed that the D-enantiomer is unreactive) . The latter value is the lower limit because of the uncertain value of 3H kinetic isotope effect.

Biochemistry, 1997 Aug 26, 36(34), 10414 - 21
Engineering the independent folding of the subtilisin BPN' prodomain: analysis of two-state folding versus protein stability; Ruvinov S et al.; In complex with subtilisin BPN', the 77 amino acid prodomain folds into a stable compact structure comprising a four-stranded antiparallel beta-sheet and two three-turn alpha-helices . When isolated from subtilisin, the prodomain is 97% unfolded even under optimal folding conditions . Traditionally, to study stable proteins, denaturing cosolvents or temperatures are used to shift the equilibrium from folded to unfolded . Here we manipulate the folding equilibrium of the unstable prodomain by introducing stabilizing mutations generated by design . By sequentially introducing three stabilizing mutations into the prodomain we are able to shift the equilibrium for independent folding from 97% unfolded to 65% folded . Spectroscopic and thermodynamic analysis of the folding reaction was carried out to assess the effect of stability on two-state behavior and the denatured state . The denatured states of single and combination mutants are not discernably different in spite of a range of DeltaGunfolding from -2.1 to 0.4 kcal/mol . Conclusions about the nature of the denatured state of the prodomain are based on CD spectral data and calorimetric data . Two state folding is observed for a combination mutant of marginal stability (DeltaG = 0) . Evidence for its two-state folding is based on the observed additivity of individual mutations to the overall DeltaGunfolding and the conformity of DeltaGunfolding vs T to two-state assumptions as embodied in the Gibbs-Helmholz equation . We believe our success in stabilizing the two-state folding reaction of the prodomain originates from the selection of mutations with improved ability to fold subtilisin rather than selection for increase in secondary structure content . The fact that a small number of mutations can stabilize the independent folding of the prodomain implies that most of the folding information already exists in the wild-type amino acid sequence in spite of the fact that the unfolded state predominates.

Biochemistry, 1997 Aug 26, 36(34), 10393 - 405
Tertiary structure of RBD2 and backbone dynamics of RBD1 and RBD2 of the human U1A protein determined by NMR spectroscopy; Lu J et al.; The human U1A protein has two putative RNA binding domains, one at the N-terminal region of the protein (RBD1) and the other at the C-terminal end (RBD2) . RBD1 binds tightly and specifically to one of the stem loops of the U1 snRNA, as well as to its own 3'-UTR . In contrast, RBD2 does not appear to associate with any RNA . The two domains share 25% amino acid identity, and both have the same betaalphabeta-betaalphabeta secondary structure fold . In this work, 13C/15N/1H multidimensional NMR methods were used to obtain side-chain assignments for RBD2, and then the tertiary structure was calculated using a distance geometry/simulated annealing algorithm that employs pairwise Gaussian metrization . RBD2 is shown to fold into an alpha/beta sandwich with a four-stranded antiparallel beta-sheet, which is the typical global topology of these domains . Specific structural features of RBD2 include a beta-bulge in beta2, N-capping boxes for both alpha-helices, and an extremely shallow twist of its beta-sheet . The 15N backbone dynamics of these two structurally homologous RBDs are significantly different, compared using order parameters and T2 exchange terms in the Lipari and Szabo model-free formalism . Conformational exchange observed in RBD1, which is absent in RBD2, may correlate to the mechanism of RNA binding.

Exp Cell Res, 1997 Aug 25, 235(1), 145 - 54
N-Myristoyltransferase overexpression in human colorectal adenocarcinomas; Raju RV et al.; Modification of proteins by myristoylation has been proposed as a chemotherapeutic target against colon cancer because it is important in the function of various signal transduction proteins . Recently we reported that the enzyme that catalyzes this modification, N-myristoyltransferase (NMT), is elevated in colorectal adenocarcinomas {Magnuson, B . A., Raju, R . V . S., Moyana, T . N., and Sharma, R . K . (1995) J . Natl . Cancer . Inst . 87, 1630-1635} . The purpose of the present study was to investigate whether the elevated activity of NMT in colorectal adenocarcinomas is due to an increase in the production of NMT or a change in the structure of the preexisting enzyme . The expression of NMT in normal colonic mucosa and adenocarcinomas from human colorectal surgical specimens was studied by immunoblotting, and its localization was confirmed by immunohistochemistry . The molecular weight of NMT was determined by fast protein liquid chromatography . In both normal mucosa and colorectal adenocarcinomas, NMT with a molecular mass of 48.5 kDa was identified with anti-human NMT and anti-peptide antibody . However, the expression of NMT was found to be higher in the colorectal tumors . This finding was further confirmed by immunohistochemical studies which showed stronger cytoplasmic staining in the tumors . These findings represent the first description of NMT overexpression in colorectal adenocarcinomas . This has implications with regard to (i) the design of chemotherapeutic drugs and (ii) prognosis, for instance, in monitoring colorectal cancer recurrence or metastases.

J Neurosci Methods, 1997 Aug 22, 75(2), 137 - 45
Semithin cryosections as a tool to perform high resolution immunofluorescence and in situ hybridization analysis of the nervous tissue: a study in the supraoptic nucleus; Lafarga M et al.; Immunofluorescence and fluorescence in in situ hybridization represent powerful approaches to correlate biochemical and molecular data with the structural organization of cells and tissues . However, the analysis of tissues by fluorescence microscopy is limited by the fact that most methods currently used to preserve the morphological integrity of sectioned samples at high resolution do not allow access of the labeled probes to the target molecules . Here we have made use of semithin cryosections obtained from rat supraoptic nucleus to perform immunofluorescence with antibodies directed against cytoplasmic and nuclear antigens, as well as fluorescence in situ hybridization with antisense oligonucleotide probes complementary to the poly(A) tail of mRNA and to specific mRNAs . In addition, DNA was visualized by incubation of sections with digoxigenin-labeled nucleotides in the presence of Escherichia coli DNA polymerase I . The high resolution of this DNA staining in combination with immunolabeling for nuclear antigens provides a powerful tool to analyze the structural and functional compartmentalization of neuronal cell nuclei . The major conclusion from this study is that performing fluorescence microscopy on 1 micron-thick cryosections provides an important tool to accurately localize proteins, DNA and RNA within nervous tissue in general and particularly in the model of supraoptic nucleus . Moreover, the cryosectioning technique appears particularly suited to the study of the localization of specific mRNA species in the neuronal cytoplasm and represents a useful approach to addressing the functional significance of mRNA localization in protein targeting.

J Mol Biol, 1997 Aug 22, 271(3), 472 - 87
Amide hydrogen exchange and internal dynamics in the chemotactic protein CheY from Escherichia coli; Lacroix E et al.; The backbone internal dynamics of the wild-type 129 amino acid alpha/beta parallel protein CheY and its double mutant F14N/P110G are analysed here by the hydrogen-exchange method . The F14N mutation is known to stabilise the protein and to accelerate refolding while P110G is destabilising and accelerates unfolding . We first assigned and characterised the double mutant by nuclear magnetic resonance (NMR), to try and discover any possible conformational change induced by the two mutations . The main difference between the two proteins is a favourable N-capping interaction of the newly introduced Asn14 side-chain at the beginning of the first alpha-helix (alpha-helix A) . Second, we have measured the exchange rates in the wild-type and mutant CheY . In the first case the observed protection factors are slightly dispersed around an average value . According to their distribution in the structure, protein stability is highest on one face of the central beta-sheet, in the surroundings of the main hydrophobic core formed by side-chains of residues in beta-strands I, II and III and helices A and E . The mutations in the double mutant protein affect two distinct subdomains differently (from beta-strand I to III and from alpha-helix C to the end) . In the second subdomain the number of protected protons is reduced with respect to those in the wild-type . This differential behaviour can be explained by a selective decrease in stability of the second folding subdomain produced by the P110G mutation and the opposite effect in the first subdomain, produced by the F14N mutation . alpha-Helix A, which is involved together with beta-strands I and III in the folding nucleus of CheY, shows the largest protection factors in both proteins.

J Mol Biol, 1997 Aug 22, 271(3), 362 - 73
Tn5 transposase mutants that alter DNA binding specificity; Zhou M et al.; Tn5 transposase (Tnp) binds to Tn5 and IS50 end inverted repeats, the outside end (OE) and the inside end (IE), to initiate transposition . We report the isolation of four Tnp mutants (YH41, TP47, EK54 and EV54) that increase the OE-mediated transposition frequency and enhance the binding affinity of Tnp for OE DNA . In addition, two of the Tnp mutants (TP47 and EK54) appear to be change-of-specificity mutants, since they alter the recognition of OE versus IE relative to the wild-type Tnp . EK54 enhances OE recognition but decreases IE recognition . TP47 enhances both OE and IE recognition but with a much greater enhancement for IE than for OE . This change-of-specificity effect of TP47 is observed only when TP47 Tnp is synthesized in cis to the DNA that contains the ends . We propose that Lys54 makes a favorable interaction with an OE-specific nucleotide pair(s), while Pro47 may cause a more favorable interaction with an IE-specific nucleotide pair(s) than it does with the corresponding OE-specific nucleotide pair(s) . A model to explain the preference of TP47 Tnp for the IE in cis but not in trans is proposed.

J Biol Chem, 1997 Aug 22, 272(34), 21582 - 8
Characterization of the precursor of prostate-specific antigen . Activation by trypsin and by human glandular kallikrein; Takayama TK et al.; The precursor or zymogen form of prostate-specific antigen (pro-PSA) is composed of 244 amino acid residues including an amino-terminal propiece of 7 amino acids . Recombinant pro-PSA was expressed in Escherichia coli, isolated from inclusion bodies, refolded, and purified . The zymogen was readily activated by trypsin at a weight ratio of 50:1 to generate PSA, a serine protease that cleaves the chromogenic chymotrypsin substrate 3-carbomethoxypropionyl-L-arginyl-L-prolyl-L-tyrosine-p-nitroanili ne- HCl (S-2586) . In this activation, the amino-terminal propiece Ala-Pro-Leu-Ile-Leu-Ser-Arg was released by cleavage at the Arg-Ile peptide bond . The recombinant pro-PSA was also activated by recombinant human glandular kallikrein, another prostate-specific serine protease, as well as by a partially purified protease(s) from seminal plasma . The recombinant PSA was inhibited by alpha1-antichymotrypsin, forming an equimolar complex with a molecular mass of approximately 100 kDa . The recombinant PSA failed to activate single chain urokinase-type plasminogen activator, in contrast to the recombinant hK2, which readily activated single chain urokinase-type plasminogen activator . These results indicate that pro-PSA is converted to an active serine protease by minor proteolysis analogous to the activation of many of the proteases present in blood, pancreas, and other tissues . Furthermore, PSA is probably generated by a cascade system involving a series of precursor proteins . These proteins may interact in a stepwise manner similar to the generation of plasmin during fibrinolysis or thrombin during blood coagulation.

J Biol Chem, 1997 Aug 22, 272(34), 21364 - 72
Proteolytic activity of the ATP-dependent protease HslVU can be uncoupled from ATP hydrolysis; Huang H et al.; HslVU is a new Escherichia coli ATP-dependent protease composed of two multimeric complexes: the HslU ATPase and the HslV peptidase . Prior studies indicated that HslVU requires ATP hydrolysis for the cleavage of peptides and proteins . We show here that ATP concentrations that activate hydrolysis of benzyloxycarbonyl-Gly-Gly-Leu-7-amido-4-methylcoumarin are 50-100 fold lower than those necessary for degradation of proteins (e.g . casein) . Also, the nonhydrolyzable analogs of ATP, 5'-adenylyl beta, gamma-imidodiphosphate (AMP-PNP) and adenosine 5'-(alpha, beta-methylene)triphosphate, can support peptide hydrolysis, but only after an initial time lag not seen with ATP . This delay decreased at higher temperatures and with higher HslU or HslV concentrations and was eliminated by preincubation of HslU and HslV together . Thus, ATP hydrolysis accelerates the association of HslU and HslV, which occurs slowly with the nonhydrolyzable analog . The addition of KCl stimulated 4-6-fold the peptidase activity with AMP-PNP present and eliminated the time lag, but KCl had no stimulatory effect with ATP . NH4+ and Cs+ had similar effects as K+, but Na+ and Li+ were ineffective . AMP-PNP by itself supported hydrolysis of casein and other polypeptides only 20% as well as ATP, but in the presence of K+, Cs+, or NH4+, AMP-PNP activated casein degradation even better than ATP, although it was not hydrolyzed . In addition, MgCl2, MnCl2, and CaCl2 allowed some peptidase and caseinase activity in the absence of any nucleotide . However, Mn2+ and Ca2+, unlike Mg2+, abolished ATP hydrolysis and prevented further activation by ATP or AMP-PNP . These findings indicate that ATP binding to a high affinity site triggers the formation of an active state capable of peptide cleavage, although ATP hydrolysis facilitates this process . Rapid degradation of proteins requires a distinct state of the enzyme, which is normally reached through ATP hydrolysis at low affinity sites . However, AMP-PNP binding together with K+ can induce a form of HslVU that degrades proteins without energy consumption.

J Biol Chem, 1997 Aug 22, 272(34), 21240 - 3
Guanosine tetra- and pentaphosphate promote accumulation of inorganic polyphosphate in Escherichia coli; Kuroda A et al.; High levels of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), generated in response to amino acid starvation in Escherichia coli, lead to massive accumulations of inorganic polyphosphate (polyP) . Inasmuch as the activities of the principal enzymes that synthesize and degrade polyP fluctuate only slightly, the polyP accumulation can be attributed to a singular and profound inhibition by pppGpp and/or ppGpp of the hydrolytic breakdown of polyP by exopolyphosphatase, thereby blocking the dynamic turnover of polyP . The Ki values of 10 microM for pppGpp and 200 microM for ppGpp are far below the concentrations of these nucleotides in nutritionally stressed cells . In the complex metabolic network of pppGpp and ppGpp, the greater inhibitory effect of pppGpp (compared with ppGpp) leading to the accumulation of polyP, may have some significance in the relative roles played by these regulatory compounds.

J Biol Chem, 1997 Aug 22, 272(34), 21233 - 9
Dimerization interactions of the b subunit of the Escherichia coli F1F0-ATPase; McLachlin DT et al.; Site-directed mutagenesis and N-terminal truncations were used to examine dimerization interactions in the b subunit of Escherichia coli F1F0-ATPase . Individual cysteine residues were incorporated into bsyn, a soluble form of the protein lacking the membrane-spanning N-terminal domain, in two main areas: the heptad repeat region and the hydrophobic region which begins at residue Val-124 . The tendencies of these cysteine residues to form disulfide bonds with the corresponding cysteine in the bsyn dimer were tested using disulfide exchange by glutathione and air oxidation catalyzed by Cu2+ . Within the heptad repeat region, only cysteines at residues 59 and 60, which occupy the b and c positions of the heptad repeat, showed significant tendencies to form disulfides, a result inconsistent with a coiled-coil model for bsyn . Mixed disulfide formation most readily occurred with the S60C + L65C and A61C + L65C pairs . Cysteines at positions 124, 128, 132, and 139 showed strong tendencies to form disulfides with their mates in the dimer, suggesting a parallel alpha-helical interaction between the subunits in this region . Deletion of residues N-terminal to either Glu-34 or Asp-53 had no apparent effect on dimerization as determined by sedimentation equilibrium, while deletion of all residues N-terminal to Lys-67 produced a monomeric form . These results imply that residues 53-66 but not 24-52 are essential for bsyn dimerization . Taken together the results are consistent with a model in which the two b subunits interact in more than one region, including a parallel alignment of helices containing residues 124-139.

J Biol Chem, 1997 Aug 22, 272(34), 21195 - 200
Inhibition of thymidine transport in dnaA mutants of Escherichia coli; Mizushima T et al.; DnaA protein is the initiator of chromosomal DNA replication in Escherichia coli . We report here our evidence that thymidine transport across cytoplasmic membranes in temperature-sensitive dnaA mutants is greatly decreased at a permissive temperature for growth of the mutants . Complementation analysis with a plasmid containing the wild type dnaA gene and P1 phage-mediated transduction confirmed that mutations in the dnaA gene were responsible for the phenotype . A low level of nucleoside transport in the dnaA mutant was specific for thymidine; transport activities for other nucleosides were much the same as those in wild type cells . Membrane vesicles prepared from the dnaA mutant showed much the same activity of thymidine transport as did those from the wild type cells . No significant difference in the activity of thymidine kinase, which is known to facilitate thymidine transport, was seen between the mutant and the wild type cells . An increase in the pool of dTTP, a negative regulator for thymidine kinase, was observed in the dnaA mutant . Based on these observations, we suggest that inhibition of thymidine transport in dnaA mutants is caused by increases in the dTTP pool.

J Biol Chem, 1997 Aug 22, 272(34), 21084 - 9
Ligand interactions of the ArsC arsenate reductase; Liu J et al.; ArsC encoded by Escherichia coli plasmid R773 catalyzes the reduction of arsenate to arsenite . The enzymatic reaction requires reduced glutathione and glutaredoxin . In this study a direct association between ArsC and glutaredoxin was demonstrated . An arsC gene with six histidine codons added to the 5' end of the gene was constructed, and the resulting ArsC enyzme was shown to be functional . Interaction of the histidine-tagged ArsC and glutaredoxin was examined by Ni2+ affinity chromatography . The association required the presence of reduced glutathione and either the substrate arsenate or a competitive inhibitor, phosphate or sulfate . A free thiolate on glutathione was not required . A tryptophan residue was introduced into ArsC at the 11th position, immediately adjacent to the active site Cys-12 . Trp-11 fluorescence was quenched upon addition of arsenate . Addition of reduced glutathione after arsenate resulted in a rapid increase in fluorescence followed by a slower decay of the signal . These spectroscopic signals were specific for arsenate and reduced glutathione; neither competitive inhibitors nor non-thiol glutathione analogs produced this effect . Cys-12 thiolate was also required . Thus the intrinsic fluorescence of Trp-11 provides a useful probe to investigate the mechanism of this novel reductase.

Nature, 1997 Aug 21, 388(6644), 792 - 8
Distinct actions of cis and trans ATP within the double ring of the chaperonin GroEL; Rye HS et al.; The chaperonin GroEL is a double-ring structure with a central cavity in each ring that provides an environment for the efficient folding of proteins when capped by the co-chaperone GroES in the presence of adenine nucleotides . Productive folding of the substrate rhodanese has been observed in cis ternary complexes, where GroES and polypeptide are bound to the same ring, formed with either ATP, ADP or non-hydrolysable ATP analogues, suggesting that the specific requirement for ATP is confined to an action in the trans ring that evicts GroES and polypeptide from the cis side . We show here, however, that for the folding of malate dehydrogenase and Rubisco there is also an absolute requirement for ATP in the cis ring, as ADP and AMP-PNP are unable to promote folding . We investigated the specific roles of binding and hydrolysis of ATP in the cis and trans rings using mutant forms of GroEL that bind ATP but are defective in its hydrolysis . Binding of ATP and GroES in cis initiated productive folding inside a highly stable GroEL-ATP-GroES complex . To discharge GroES and polypeptide, ATP hydrolysis in the cis ring was required to form a GroEL-ADP-GroES complex with decreased stability, priming the cis complex for release by ATP binding (without hydrolysis) in the trans ring . These observations offer an explanation of why GroEL functions as a double-ring complex.

Nature, 1997 Aug 21, 388(6644), 741 - 50
The crystal structure of the asymmetric GroEL-GroES-(ADP)7 chaperonin complex; Xu Z et al.; Chaperonins assist protein folding with the consumption of ATP . They exist as multi-subunit protein assemblies comprising rings of subunits stacked back to back . In Escherichia coli, asymmetric intermediates of GroEL are formed with the co-chaperonin GroES and nucleotides bound only to one of the seven-subunit rings (the cis ring) and not to the opposing ring (the trans ring) . The structure of the GroEL-GroES-(ADP)7 complex reveals how large en bloc movements of the cis ring's intermediate and apical domains enable bound GroES to stabilize a folding chamber with ADP confined to the cis ring . Elevation and twist of the apical domains double the volume of the central cavity and bury hydrophobic peptide-binding residues in the interface with GroES, as well as between GroEL subunits, leaving a hydrophilic cavity lining that is conducive to protein folding . An inward tilt of the cis equatorial domain causes an outward tilt in the trans ring that opposes the binding of a second GroES . When combined with new functional results, this negative allosteric mechanism suggests a model for an ATP-driven folding cycle that requires a double toroid.

Proc Natl Acad Sci U S A, 1997 Aug 19, 94(17), 9208 - 13
Translational coupling by modulation of feedback repression in the IF3 operon of Escherichia coli; Chiaruttini C et al.; A pseudoknot formed by a long-range interaction in the mRNA of the initiation factor 3 (IF3) operon is involved in the translational repression of the gene encoding ribosomal protein L35 by another ribosomal protein, L20 . The nucleotides forming the 5' strand of the key stem of the pseudoknot are located within the gene for IF3, whereas those forming the 3' strand are located 280 nt downstream, immediately upstream of the Shine-Dalgarno sequence of the gene for L35 . Here we show that premature termination of IF3 translation at a nonsense codon introduced upstream of the pseudoknot results in a substantial enhancement of L20-mediated repression of L35 expression . Conversely, an increase of IF3 translation decreases repression . These results, in addition to an analysis of the effect of mutations in sequences forming the pseudoknot, indicate that IF3 translation decreases L20-mediated repression of L35 expression . We propose that ribosomes translating IF3 disrupt the pseudoknot and thereby attenuate repression . The result is a novel type of translational coupling, where unfolding of the pseudoknot by ribosomes translating IF3 does not increase expression of L35 directly, but alleviates its repression by L20.

Proc Natl Acad Sci U S A, 1997 Aug 19, 94(17), 9075 - 9
Proof for a nonproteinaceous calcium-selective channel in Escherichia coli by total synthesis from (R)-3-hydroxybutanoic acid and inorganic polyphosphate; Das S et al.; Traditionally, the structure and properties of natural products have been determined by total synthesis and comparison with authentic samples . We have now applied this procedure to the first nonproteinaceous ion channel, isolated from bacterial plasma membranes, and consisting of a complex of poly(3-hydroxybutyrate) and calcium polyphosphate . To this end, we have now synthesized the 128-mer of hydroxybutanoic acid and prepared a complex with inorganic calcium polyphosphate (average 65-mer), which was incorporated into a planar lipid bilayer of synthetic phospholipids . We herewith present data that demonstrate unambiguously that the completely synthetic complex forms channels that are indistinguishable in their voltage-dependent conductance, in their selectivity for divalent cations, and in their blocking behavior (by La3+) from channels isolated from Escherichia coli . The implications of our finding for prebiotic chemistry, biochemistry, and biology are discussed.

Proc Natl Acad Sci U S A, 1997 Aug 19, 94(17), 9022 - 7
Structural kinetics of transcription activation at the malT promoter of Escherichia coli by UV laser footprinting; Eichenberger P et al.; We have studied the kinetics of transcriptional initiation and activation at the malT and malTp1 promoters of Escherichia coli using UV laser footprinting . Contrary to previous studies and because of the very rapid signal acquisition by this technique, we can obtain structural information about true reaction intermediates of transcription initiation . The consequences of adding a transcriptional activator, the cAMP receptor protein/cAMP complex (CRP), are monitored in real time, permitting us to assign specific interactions to the activation of discrete steps in transcription initiation . Direct protein-protein contacts between CRP and the RNA polymerase appeared very rapidly, followed by DNA melting around the -10 hexamer . CRP slightly increased the rate of this isomerization reaction but, more importantly, favored the establishment of additional contacts between the DNA upstream of the CRP binding site and RNA polymerase subsequent to open complex formation . These contacts make a major contribution to transcriptional activation by stabilizing open forms of the promoter complex, thereby indirectly accelerating promoter escape . The ensemble of the kinetic, structural signals demonstrated directly that CRP exerts most of its activating effects on the late stages of transcriptional initiation at the malT promoter.

Proc Natl Acad Sci U S A, 1997 Aug 19, 94(17), 9011 - 6
Significance of chaperonin 10-mediated inhibition of ATP hydrolysis by chaperonin 60; Dubaquie Y et al.; Chaperonins are essential for the folding of proteins in bacteria, mitochondria, and chloroplasts . We have functionally characterized the yeast mitochondrial chaperonins hsp60 and hsp10 . In the presence of ADP, one molecule of hsp10 binds to hsp60 with an apparent Kd of 0.9 nM and a second molecule of hsp10 binds with a Kd of 24 nM . In the presence of ATP, the purified yeast chaperonins mediate the refolding of mitochondrial malate dehydrogenase . Hsp10 inhibits the ATPase activity of hsp60 by about 40% . Hsp10(P36H) is a point mutant of hsp10 that confers temperature-sensitive growth to yeast . Consistent with the in vivo phenotype, refolding of mitochondrial malate dehydrogenase in the presence of purified hsp10(P36H) and hsp60 is reduced at 25 degrees C and abolished at 30 degrees C . The affinity of hsp10(P36H) to hsp60 as well as to Escherichia coli GroEL is reduced . However, this decrease in affinity does not correlate with the functional defect, because hsp10(P36H) fully assists the GroEL-mediated refolding of malate dehydrogenase at 30 degrees C . Refolding activity, rather, correlates with the ability of hsp10(P36H) to inhibit the ATPase of GroEL but not that of hsp60 . Based on our findings, we propose that the inhibition of ATP hydrolysis is mechanistically coupled to chaperonin-mediated protein folding.

Proc Natl Acad Sci U S A, 1997 Aug 19, 94(17), 9006 - 10
A role for TFIIH in controlling the activity of early RNA polymerase II elongation complexes; Dvir A et al.; TFIIH is a multifunctional RNA polymerase II transcription factor that possesses DNA-dependent ATPase, DNA helicase, and protein kinase activities . Previous studies have established that TFIIH enters the preinitiation complex and fulfills a critical role in initiation by catalyzing ATP-dependent formation of the open complex prior to synthesis of the first phosphodiester bond of nascent transcripts . In this report, we present direct evidence that TFIIH also controls RNA polymerase II activity at a postinitiation stage of transcription, by preventing premature arrest by very early elongation complexes just prior to their transition to stably elongating complexes . Unexpectedly, we observe that TFIIH is capable of entering the transcription cycle not only during assembly of the preinitiation complex but also after initiation and synthesis of as many as four to six phosphodiester bonds . These findings shed new light on the role of TFIIH in initiation and promoter escape and reveal an unanticipated flexibility in the ability of TFIIH to interact with RNA polymerase II transcription intermediates prior to, during, and immediately after initiation.

Proc Natl Acad Sci U S A, 1997 Aug 19, 94(17), 8954 - 8
Three functional luciferase domains in a single polypeptide chain; Li L et al.; We report a unique case of a gene containing three homologous and contiguous repeat sequences, each of which, after excision, cloning, and expression in Escherichia coli, is shown to code for a peptide catalyzing the same reaction as the native protein, Gonyaulax polyedra luciferase (Mr = 137) . This enzyme, which catalyzes the light-emitting oxidation of a linear tetrapyrrole (dinoflagellate luciferin), exhibits no sequence similarities to other luciferases in databases . Sequence analysis also reveals an unusual evolutionary feature of this gene: synonymous substitutions are strongly constrained in the central regions of each of the repeated coding sequences.

Biochemistry, 1997 Aug 19, 36(33), 10320 - 6
Strand specificity in the interactions of Escherichia coli primary replicative helicase DnaB protein with a replication fork; Jezewska MJ et al.; The interactions of the Escherichia coli primary replicative helicase DnaB protein, with synthetic DNA replication fork substrates, having either a single arm or both arms, have been studied using the thermodynamically rigorous fluorescence titration techniques . This approach allows us to obtain absolute stoichiometries of the formed complexes and interaction parameters without any assumptions about the relationship between the observed signal (fluorescence) and the degree of binding . Subsequently, the formation of the complexes, with different replication fork substrates, has also been characterized using the sedimentation velocity technique . To our knowledge, this is the first quantitative characterization of interactions of a hexameric helicase with replication fork substrates . In the presence of the ATP nonhydrolyzable analog, AMP-PNP, the E . coli DnaB helicase preferentially binds to the 5' arm of the single-arm fork substrate with an intrinsic affinity 6-fold higher than its affinity for the 3' arm . ATP hydrolysis is not necessary for formation of the helicase-fork complex . The asymmetric interactions are consistent with the 5' --> 3' directionality of the helicase activity of the DnaB protein and most probably reflects a preferential 5' --> 3' polarity in the helicase binding to ssDNA, with respect to the ssDNA backbone . The double-stranded part of the fork contributes little to the free energy of binding . The data indicate a rather passive role of the duplex part of the fork in the binding of the helicase . This role seems to be limited to impose steric hindrance in the formation of nonproductive complexes of the enzyme with the fork . Quantitative analysis of binding of the helicase to the two-arm fork substrate shows that two DnaB hexamers can bind to the fork, with each single hexamer associated with a single arm of the fork . In this complex, the intrinsic affinity of the DnaB hexamer for the 5' arm in a two-arm fork is not affected by the presence of the 3' arm . Moreover, the results show that the 3' arm is in a conformation which makes it easily available for the binding of the next DnaB hexamer . Because of the large size of the DnaB hexamer, the data indicate that the 3' arm is separated from the 5' arm . The separation of both arms must be to such an extent that the 3' arm can bind an additional large DnaB hexamer . These results reveal that the 3' arm is not engaged in thermodynamically stable interactions with the helicase hexamer, when it is bound in its stationary complex to the 5' arm of the fork . The significance of the these results for a mechanistic model of the hexameric DnaB helicase action is discussed.

Biochemistry, 1997 Aug 19, 36(33), 10292 - 300
Single-step kinetics of HIV-1 reverse transcriptase mutants responsible for virus resistance to nucleoside inhibitors zidovudine and 3-TC; Krebs R et al.; Two mutants of HIV-1 reverse transcriptase (RT) associated with high-level resistance of the virus to AZT (RT-AZT: D67N, K70R, T215Y, K219Q, and M41L) or 3-TC (RT-3TC: M184V) were expressed in Escherichia coli and purified . None of these mutants showed significant changes in the affinity and kinetics of binding to a DNA/DNA primer/template . RT-AZT was investigated in detail with respect to its kinetics of incorporation of nucleotides . No change in the relative rates of TMP and AZTMP incorporation could be detected for RT-AZT with respect to wild type RT . These results imply that there is no increased discrimination against AZTTP in the mutant . This was found for DNA/DNA and DNA/RNA primer/template . Additionally, rapid kinetics of incorporation of 3'-amino-3'-deoxythymidine 5'-monophosphate (a possible metabolite of AZT) were investigated and compared with TMP incorporation, but no difference in its relative rates of incorporation between wild type RT and RT-AZT was detected . In contrast, the already very slow rate of incorporation of 3-TCMP seen with wild type enzyme was drastically reduced (by a factor of 23 and 36 with DNA/DNA primer/template and DNA/RNA primer/template, respectively) for RT-3TC, showing a clear correlation between in vitro and in vivo effects . The affinity of 3-TCTP to the RT-3TC-primer/template complex was not affected by the mutation M184V . A 1.6-fold cross-resistance to ddATP, the converted form of the prodrug ddI, could also be shown for RT-3TC, but no cross-resistance to ddCTP was detected . Additionally, rapid kinetics of AZTMP incorporation by RT-3TC were investigated . There was an indication of a slightly higher rate of incorporation of AZTMP by RT-3TC than wild type RT.

Biochemistry, 1997 Aug 19, 36(33), 10256 - 61
The major, N2-dG adduct of (+)-anti-B{a}PDE shows a dramatically different mutagenic specificity (predominantly, G --> A) in a 5'-CGT-3' sequence context; Shukla R et al.; Mutations induced by the (+)-anti diol epoxide of benzo{a}pyrene {(+)-anti-B{a}PDE} were described previously in the supF gene of the Escherichia coli plasmid pUB3 {Rodriguez et al.(1993) Biochemistry, 32, 1759} . (+)-anti-B{a}PDE induced a complex pattern of mutations, including insertions, deletions, frameshifts, as well as base substitution mutations, which for G:C base pairs alone included a significant fraction of G:C --> T:A, A:T and C:G mutations . A variety of results suggest that most of these mutations arise from the major adduct ({+ta}-B{a}P-N2-dG), raising the question how can a single adduct induce different kinds of mutations? Our working hypothesis in this regard is that (1) an adduct can adopt multiple conformations; (2) different conformations cause different mutations; and (3) adduct conformation is controlled by various factors, such as DNA sequence context . To investigate what conformation is associated with what mutation, it is essential to find examples where {+ta}-B{a}P-N2-dG induces principally one kind of mutation as a prelude to the study in that same context of the conformation(s) potentially relevant to mutagenesis . Earlier work indicated that (+)-anti-B{a}PDE gave a preponderance of G --> A mutations in a 5'-CGT-3 sequence context, and herein it is shown that these mutations are likely to be attributable to the major adduct, since in this same sequence context {+ta}-B{a}P-N2-dG studied site specifically also induces principally G --> A mutations ( approximately 82%) . Previously, {+ta}-B{a}P-N2-dG was shown to induce principally G --> T mutations (approximately 97%) in a 5'-TGC-3' sequence context . Thus, by simply altering its surrounding sequence context this adduct can give a preponderance of either G --> A or G --> T mutations . This is the most dramatic change in base substitution mutagenic specificity for an adduct described to date and illustrates that the qualitative pattern of mutagenesis by a bulky adduct can be remarkably diverse.

Biochemistry, 1997 Aug 19, 36(33), 10192 - 9
Raman study of the polarizing forces promoting catalysis in 4-chlorobenzoate-CoA dehalogenase; Clarkson J et al.; The enzyme 4-chlorobenzoate-CoA dehalogenase catalyzes the hydrolysis of 4-chlorobenzoate-CoA (4-CBA-CoA) to 4-hydroxybenzoyl-CoA (4-HBA-CoA) . In order to facilitate electrophilic catalysis, the dehalogenase utilizes a strong polarizing interaction between the active site residues and the benzoyl portion of the substrate {Taylor, K . L., et al . (1995) Biochemistry 34, 13881} . As a result of this interaction, the normal modes of the benzoyl moiety of the bound 4-HBA-CoA undergo a drastic rearrangement as shown by Raman spectroscopy . Here, we present Raman difference spectroscopic data on the product-enzyme complex where the product's benzoyl carbonyl is labeled with 18O (C=18O) or 13C (13C=O) or where the 4-OH group is labeled with 18O . The data demonstrate that the carbonyl group participates in the most intense normal modes occurring in the Raman spectrum in the 1520-1560 cm-1 region . The substrate analog 4-methylbenzoate-CoA (4-MeBA-CoA) has also been characterized by Raman difference spectroscopy in its free form and bound to the dehalogenase . Upon binding, the 4-MeBA-CoA shows evidence of polarization within the delocalized pi-electrons, but to a lesser extent compared to that seen for the product . The use of 4-MeBA-CoA labeled with 18O at the carbonyl enables us to estimate the degree of electron polarization within the C=O group of the bound 4-MeBA-CoA . The C=O stretching frequency occurs near 1663 cm-1 in non-hydrogen bonding solvents such as CCl4, near 1650 cm-1 in aqueous solution, and near 1610 cm-1 in the active site of dehalogenase . From model studies, we can estimate that in the active site the carbonyl group behaves as though it is being polarized by hydrogen bonds approximately 57 kJ mol-1 in strength . Major contributions to this polarization come from hydrogen bonds from the peptide NHs of Gly114 and Phe64 . However, an additional contribution, which may account for up to half of the observed shift in nuC=O, originates in the electrostatic field due to the alpha-helix dipole from residues 121-114 . The helix which terminates at Gly114, near the C=O group of the bound benzoyl, provides a dipolar electrostatic component which contributes to the polarization of the C=O bond and to the polarization of the entire benzoyl moiety . The effect of both the helix dipole and the hydrogen bonds on the C=O is a "pull" of electrons onto the carbonyl oxygen, which, in turn, polarizes the electron distribution within the benzoyl pi-electron system . The ability of these two factors to polarize the electrons within the benzoyl moiety is increased by the environment about the benzoyl ring; it is surrounded by hydrophobic residues which provide a low-dielectric constant microenvironment . Electron polarization promotes catalysis by reducing electron density at the C4 position of the benzoyl ring, thereby assisting attack by the side chain of Asp145 . An FTIR study on the model compound 4-methylbenzoyl S-ethyl thioester, binding to a number of hydrogen bonding donors in CCl4, is described and is used to relate the observed shift of the C=O stretching mode of 4-MeBA-CoA in the active site to the hydrogen bonding strength value . Since the shift of the C=O frequency upon binding is due to hydrogen bonding and helix dipole effects, we refer to this bonding strength as the effective hydrogen bonding strength.

Biochemistry, 1997 Aug 19, 36(33), 10168 - 77
Identification of cysteine-523 in the aspartate binding site of Escherichia coli asparagine synthetase B; Boehlein SK et al.; The site-directed chemical modifier {p-(fluorosulfonyl)benzoyl}adenosine (5'-FSBA) inactivates Escherichia coli asparagine synthetase B activity following pseudo-first-order kinetics, with ATP providing specific protection, with a Kd of 12 microM . The 5'-FSBA modification appears to be covalent, even though a nonstoichiometric amount (less than 10%) of radiolabeled 5'-FSBA was associated with a totally inactivated enzyme . However, the inactivation by 5'-FSBA could be reversed upon the addition of dithiothreitol . These results are indicative of 5'-FSBA-induced disulfide bond formation, which requires the presence of at least two cysteine residues in the proximity of the ATP binding site . Identification of the critical cysteine residue was accomplished by sequential replacement of each cysteine in the protein by site-directed mutagenesis . Cys 523 was identified as the key residue involved in the formation of the 5'-FSBA-induced disulfide bond . Detailed kinetic analyses and comparison with similar enzymes, suggest that this cysteine residue, while in close proximity to the ATP binding site, is actually involved in aspartate binding in asparagine synthetase B.

Biochemistry, 1997 Aug 19, 36(33), 10161 - 7
Functionally linked hydration changes in Escherichia coli aspartate transcarbamylase and its catalytic subunit; LiCata VJ et al.; Aspartate transcarbamylase (ATCase) is a highly regulated, dodecameric enzyme that catalyzes the first committed step in pyrimidine biosynthesis . Upon ligation, ATCase undergoes a conformational transition from a low-activity T-state to a high-activity R-state . This transition involves major changes in the molecular architecture, including structural rearrangements of several intersubunit interfaces and a 12 A expansion of the molecule along its 3-fold axis . Solute-induced osmotic stress experiments report that approximately 208 solvent waters are taken up by ATCase as it binds substrate . Solvent-accessible surface area calculations conducted on the T and R conformers of ATCase agree very well with this result, predicting that approximately 189 waters are taken up during this conformational change . Both osmotic stress measurements and surface area calculations on the catalytic trimer of ATCase predict water release upon ligation of the trimer . Specific aspects of the application of osmotic stress to ATCase are also discussed, including solute size effects, and an assessment of potential alternative explanations for these results.

FEBS Lett, 1997 Aug 18, 413(2), 282 - 8
NMR characterization of the full-length recombinant murine prion protein, mPrP(23-231); Riek R et al.; The recombinant murine prion protein, mPrP(23-231), was expressed in E . coli with uniform 15N-labeling . NMR experiments showed that the previously determined globular three-dimensional structure of the C-terminal domain mPrP(121-231) is preserved in the intact protein, and that the N-terminal polypeptide segment 23-120 is flexibly disordered . This structural information is based on nearly complete sequence-specific assignments for the backbone amide nitrogens, amide protons and alpha-protols of the polypeptide segment of residues 121-231 in mPrP(23-231) . Coincidence of corresponding sequential and medium-range nuclear Overhauser effects (NOE) showed that the helical secondary structures previously identified in mPrP(121-231) are also present in mPrP(23-231), and near-identity of corresponding amide nitrogen and amide proton chemical shifts indicates that the three-dimensional fold of mPrP(121-231) is also preserved in the intact protein . The linewidths in heteronuclear 1H-15N correlation spectra and 15N{1H}-NOEs showed that the well structured residues 126-230 have correlation times of several nanoseconds, as is typical for small globular proteins, whereas correlation times shorter than 1 nanosecond were observed for all residues of mPrP(23-231) outside of this domain.

FEBS Lett, 1997 Aug 18, 413(2), 277 - 81
Recombinant full-length murine prion protein, mPrP(23-231): purification and spectroscopic characterization; Hornemann S et al.; The cellular prion protein of the mouse, mPrP(C), consists of 208 amino acids (residues 23-231) . It contains a carboxy-terminal domain, mPrP(121-231), which represents an autonomous folding unit with three alpha-helices and a two-stranded antiparallel beta-sheet . We expressed the complete amino acid sequence of the prion protein, mPrP(23-231), in the cytoplasm of Escherichia coli . mPrP(23-231) was solubilized from inclusion bodies by 8 M urea, oxidatively refolded and purified to homogeneity by conventional chromatographic techniques . Comparison of near-UV circular dichroism, fluorescence and one-dimensional 1H-NMR spectra of mPrP(23-231) and mPrP(121-231) shows that the amino-terminal segment 23-120, which includes the five characteristic octapeptide repeats, does not contribute measurably to the manifestation of three-dimensional structure as detected by these techniques, indicating that the residues 121-231 might be the only polypeptide segment of PrP(C) with a defined three-dimensional structure.

FEBS Lett, 1997 Aug 18, 413(2), 191 - 3
Drosophila alcohol dehydrogenase: evaluation of Ser139 site-directed mutants; Cols N et al.; Drosophila alcohol dehydrogenase (DADH) belongs to the large and highly heterogeneous (15-30% residue identity) short-chain dehydrogenase/reductase family (SDR) . It is the only reported member that oxidizes mainly ethanol and 2-propanol among other alcohols . To confirm the role of Ser139 we constructed two site-directed mutants, Ser139Ala and Ser139Cys, which show no enzymatic activity . Molecular replacement and data from crystallographically refined 3D structures confirm the position of Ser139, whose hydroxyl group faces the cleft of the presumed catalytic pocket, very close to Tyr152 and Lys156 . Thus, consistent with the constitution of the catalytic triad of other SDR, our results suggest that Ser139 of DADH is directly involved in the catalytic reaction.

Biochem Biophys Res Commun, 1997 Aug 18, 237(2), 325 - 30
Limited in vivo proteolysis of aggregated proteins; Corchero JL et al.; Degradation pathways of insoluble proteins have been analyzed in Escherichia coli by using a N-terminal beta-galactosidase fusion protein (VP1LAC) that aggregates immediately after its synthesis . In recombinant E . coli cells, lower molecular mass products, antigenically related to the entire fusion, accumulate together with the entire fusion . In absence of protein synthesis, the insoluble intact protein declines, suggesting that degradation of the recombinant protein also affects aggregated protein . Time course analysis of both soluble and insoluble cell fractions has revealed a limited proteolysis of the insoluble protein that removes the heterologous domain and permits the resulting beta-galactosidase fragments to refold and solubilize . Further extensive degradation occurs exclusively on soluble protein . The restricted proteolysis of misfolded, insoluble protein is the initiating event of a subsequent degradative pathway in which rate-limiting steps permit the accumulation of stable degradative intermediates.

Biochem Biophys Res Commun, 1997 Aug 18, 237(2), 205 - 10
Direct demonstration of the bifunctional property of Tetrahymena 14-nm filament protein/citrate synthase following expression of the gene in Escherichia coli; Takeda T et al.; Tetrahymena 14-nm filament protein/citrate synthase (49K protein) is a bifunctional protein with roles in the cytoskeleton and as a citrate synthase . Though previous studies have shown that the 49K protein is derived from a single transcript of a single gene, direct demonstration of the 49K protein's bifunctional property remained to be elucidated . In this study, a recombinant 49K protein was expressed in Escherichia coli, purified and characterized . The citrate synthase activity of the recombinant 49K protein was comparable to that of the 49K protein purified from Tetrahymena . The recombinant 49K protein formed 14-nm filaments, but only of short length . The filaments were elongated in the presence of a soluble fraction of Tetrahymena . These results suggest that the 49K protein itself is bifunctional, but some co-factor(s) is necessary for elongation of filaments.

FEMS Microbiol Lett, 1997 Aug 15, 153(2), 465 - 73
Colorimetric protein phosphatase inhibition assay of laboratory strains and natural blooms of cyanobacteria: comparisons with high-performance liquid chromatographic analysis for microcystins; Ward CJ et al.; Microcystins are cyclic heptapeptide hepatotoxins commonly produced by bloom-forming genera of cyanobacteria . These toxins are potent and specific inhibitors of protein phosphatases 1 and 2A . We have optimised a rapid, simple and sensitive colorimetric protein phosphatase 1 inhibition assay, utilising the activity of protein phosphatase 1 as expressed in a recombinant strain of Escherichia coli, towards the chromogenic substrate, p-nitrophenyl phosphate . A standard curve for the inhibition of protein phosphatase 1 by microcystin-LR was constructed with an IC50 of about 38 ng ml-1 and a limit of detection of 10-20 ng ml-1 . Twenty-three laboratory-grown strains and 25 natural bloom samples of cyanobacteria were analysed by high-performance liquid chromatography for microcystins and by the protein phosphatase 1 inhibition assay . Agreement for the microcystin contents of the samples detected by high-performance liquid chromatography and the protein phosphatase 1 inhibition assay showed good correlation (R2 > 0.93, P < 0.0001) . The suitability of the colorimetric protein phosphatase 1 inhibition assay as a screen for cyanobacterial microcystins is discussed.

FEMS Microbiol Lett, 1997 Aug 15, 153(2), 311 - 9
Specific in vivo thiol-labeling of the FhuA outer membrane ferrichrome transport protein of Escherichia coli K-12: evidence for a disulfide bridge in the predicted gating loop; Bos C et al.; The multifunctional FhuA protein of Escherichia coli K-12 forms a channel that is closed by a loop, tentatively designated the 'gating loop', which is also the principal binding site for all FhuA ligands . In this report, it is shown by in vivo labeling that the two cysteines in the gating loop form a disulfide bridge, and they react weakly after reduction with biotin-maleimide, as determined by streptavidin-beta-galactosidase bound to biotin . The two cysteines close to the C-terminus of FhuA also form a disulfide bridge and react with the thiol reagents only after heat denaturation of FhuA in SDS . Replacement of the existing cysteines by serine did not alter the sensitivity of cells to the FhuA ligands tested (T5, phi 80, T1, colicin M, and albomycin) and supported growth on ferrichrome as sole iron source . The cysteines in the gating loop play no specific functional role; they are largely buried in the interior of the loop, and the disulfide bridges are not essential for maintaining the conformation of FhuA . The C-terminal cysteines are in the interior of FhuA and are also not important for the structure of FhuA . The method used allows the identification of free cysteines and disulfides in surface exposed protein regions.

Cancer Res, 1997 Aug 15, 57(16), 3402 - 6
CYP2D6 catalyzes tamoxifen 4-hydroxylation in human liver; Dehal SS et al.; The major metabolites of tamoxifen (tam) formed by animal and human liver microsomes are mono-N-demethylated tam, 4-hydroxy-tam (4-OH-tam), and tam-N-oxide . The N-desmethylated-tam and 4-OH-tam are formed by P450s, whereas the N-oxide is primarily formed by flavin-containing monooxygenase . Because 4-OH-tam is a highly potent antiestrogen (and possibly is the active anticancer tam metabolite) and is on the path of formation of the reactive intermediate that binds covalently to proteins and DNA, it was of importance to identify the P450(s) catalyzing its formation . In the current study, three different preparations of expressed human P450s in Escherichia coli, lymphoblastoma cells, and insect cell line and livers from several human donors were used to identify the P450 isoform catalyzing the 4-hydroxylation (preliminary results were reported by Dehal et al., Eleventh International Symposium on Microsomes and Drug Oxidations, p . 71 . Los Angeles, 1996) . Tam metabolism was examined with human CYP2C8, 2C9, 2C18, 2C19, and 2D6 expressed in E . coli . These P450s were reconstituted with P450 reductase and lipid and were incubated with 50 microM {3H}tam and NADPH at 37 degrees C for 60 min . Essentially all of the recombinant P450s catalyzed the N-demethylation to various degrees; however, only 2D6 yielded detectable levels of 4-OH-tam . The inclusion of cytochrome b5 in the reconstituted system of 2D6 and 2C9 did not significantly affect the rate of 4-hydroxylation, indicating that b5 is not essential for this activity . Tam metabolism by CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2D6, 2E1, and 3A4, expressed in lymphoblastoma cells, revealed that only 2D6 significantly catalyzed the 4-hydroxylation . Tam metabolism by CYP2D6 coexpressed with P450 reductase in a baculovirus infected insect cell line ("supersomes") exhibited marked tam 4-hydroxylation . In an experiment with human liver microsomes, the inclusion of quinidine, a specific 2D6 inhibitor, resulted in approximately 50% inhibition of tam 4-hydroxylation without affecting N-demethylation . Polyclonal antibodies raised against 2D6 moderately inhibited (approximately 30%) the 4-hydroxylation in human liver microsomes . These results demonstrate a significant contribution by CYP2D6 to the catalysis of tam-4-hydroxylation by human liver.

J Mol Biol, 1997 Aug 15, 271(2), 258 - 65
Crystal structure of cytoplasmic Escherichia coli peptidyl-prolyl isomerase: evidence for decreased mobility of loops upon complexation; Edwards KJ et al.; The structure of the unliganded form of the Escherichia coli cytoplasmic peptidyl-prolyl isomerase (ppiB gene product) in a new crystal form was determined by the molecular replacement method and refined to an R-factor of 16.1% at 2.1 A resolution . The enzyme crystallized in the orthorhombic C2221 space group with unit cell dimensions of a=44.7 A, b=68.2 A and c=102.0 A . Comparison with the reported structure of the enzyme complexed with the tripeptide substrate succinyl-Ala-Pro-Ala-p-nitroanilide revealed subtle changes that occur upon complex formation . There is evidence to suggest that two surface loops have significantly reduced mobility in the complexed structure.

J Mol Biol, 1997 Aug 15, 271(2), 195 - 208
A Xenopus zinc finger protein that specifically binds dsRNA and RNA-DNA hybrids; Finerty PJ Jr et al.; Proteins containing C2H2 type zinc finger motifs represent one of the largest classes of nucleic acid-binding proteins found in nature . We describe a novel zinc finger protein, dsRBP-ZFa, isolated by screening an expression library with dsRNA . The dsRBP-ZFa cDNA encodes a protein containing seven zinc finger motifs and an acidic C-terminal domain . Mobility shift experiments demonstrate that dsRBP-ZFa binds dsRNA and RNA-DNA hybrids with nanomolar dissociation constants and in a sequence independent manner . We also show that DNA and single stranded RNA fail to compete with dsRNA for binding suggesting dsRBP-ZFa prefers to bind an A-form helix . Using western analyses we have localized dsRBP-ZFa primarily to the nucleus of Xenopus laevis oocytes . The identification of dsRBP-ZFa provides the first example of a zinc finger protein that is specific for dsRNA . In addition, dsRBP-ZFa does not contain the previously described dsRNA binding motif, suggesting certain zinc fingers may provide an alternative way to recognize the A-form helix.

J Mol Biol, 1997 Aug 15, 271(2), 168 - 77
RecA protein assisted selection reveals a low fidelity of recognition of homology in a duplex DNA by an oligonucleotide; Malkov VA et al.; We have developed an in vitro selection procedure to elucidate the specificity of RecA assisted oligonucleotide recognition of double stranded DNA . The procedure was based on formation of a synaptic complex between an oligonucleotide-RecA filament and a supercoiled plasmid bearing a homologous partially degenerate region . The specificity of the selection depended on the reaction conditions: starting with a population that had, on average, 2.8 randomly distributed mismatches out of 27 bp, a population selected in the presence of 100 mM KCl had on average 1.0 mismatches, while a population selected at low ionic strength was less specific and had, on average, 2.0 mismatches . From the distributions of mismatches observed we calculated that the average destabilization free energy for one mismatch is 1.7(+/-0.5) kcal/mol . This is substantially less than the free energy for the incorporation of one mismatch in naked DNA duplex or a Py-Pu-Py triplex . Thus, RecA has an ability to decrease the fidelity of the homologous pairing reaction and minimize the cost of pairing between similar but not identical sequences . This "antiproofreading" activity of RecA protein does not require ATP hydrolysis.

J Biol Chem, 1997 Aug 15, 272(33), 20936 - 44
The p38/reactivating kinase mitogen-activated protein kinase cascade mediates the activation of the transcription factor insulin upstream factor 1 and insulin gene transcription by high glucose in pancreatic beta-cells; Macfarlane WM et al.; Insulin upstream factor 1 (IUF1), a transcription factor present in pancreatic beta-cells, binds to the sequence C(C/T)TAATG present at several sites within the human insulin promoter . Here we isolated and sequenced cDNA encoding human IUF1 and exploited it to identify the signal transduction pathway by which glucose triggers its activation . In human islets, or in the mouse beta-cell line MIN6, high glucose induced the binding of IUF1 to DNA, an effect mimicked by serine/threonine phosphatase inhibitors, indicating that DNA binding was induced by a phosphorylation mechanism . The glucose-stimulated binding of IUF1 to DNA and IUF1-dependent gene transcription were both prevented by SB 203580, a specific inhibitor of stress-activated protein kinase 2 (SAPK2, also termed p38 mitogen-activated protein kinase, reactivating kinase, CSBP, and Mxi2) but not by several other protein kinase inhibitors . Consistent with this finding, high glucose activated mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP kinase-2) (a downstream target of SAPK2) in MIN6 cells, an effect that was also blocked by SB 203580 . Cellular stresses that trigger the activation of SAPK2 and MAPKAP kinase-2 (arsenite, heat shock) also stimulated IUF1 binding to DNA and IUF1-dependent gene transcription, and these effects were also prevented by SB 203580 . IUF1 expressed in Escherichia coli was unable to bind to DNA, but binding was induced by incubation with MgATP, SAPK2, and a MIN6 cell extract, which resulted in the conversion of IUF1 to a slower migrating form . SAPK2 could not be replaced by p42 MAP kinase, MAPKAP kinase-2, or MAPKAP kinase-3 . The glucose-stimulated activation of IUF1 DNA binding and MAPKAP kinase-2 (but not the arsenite-induced activation of these proteins) was prevented by wortmannin and LY 294002 at concentrations similar to those that inhibit phosphatidylinositide 3-kinase . Our results indicate that high glucose (a cellular stress) activates SAPK2 by a novel mechanism in which a wortmannin/LY 294002-sensitive component plays an essential role . SAPK2 then activates IUF1 indirectly by activating a novel IUF1-activating enzyme.

J Biol Chem, 1997 Aug 15, 272(33), 20907 - 12
Induction of IgE antibody responses by glutathione S-transferase from the German cockroach (Blattella germanica); Arruda LK et al.; We report that a major 23-kDa allergen from German cockroach (Blattella germanica) is a glutathione S-transferase (EC 2.5.1.18; GST) . Natural B . germanica GST, purified from cockroach body extracts by glutathione affinity chromatography, and recombinant protein expressed in Escherichia coli using the pET21a vector, showed excellent IgE antibody binding activity . B . germanica GST caused positive immediate skin tests in cockroach-allergic patients using as little as 3 pg of recombinant protein . The NH2-terminal sequence of the natural protein and the deduced amino acid sequence from cDNA were identical except for one substitution (Phe9 --> Cys) . Assignment of this protein to the GST superfamily was based on binding to glutathione and sequence identity (42-51%) to the GST-2 subfamily from insects, including Anopheles gambiae and Drosophila melanogaster . B . germanica GST contained 18 of the 26 invariable residues identified in mammalian GST by x-ray crystallography and exhibited enzymic activity against a GST substrate . Our results show that cockroach GST causes IgE antibody responses and is associated with asthma . The data strongly support the view that the immune response to GST plays an important role in allergic diseases.

J Biol Chem, 1997 Aug 15, 272(33), 20531 - 7
Trimeresurus stejnegeri snake venom plasminogen activator . Site-directed mutagenesis and molecular modeling; Zhang Y et al.; The specific plasminogen activator from Trimeresurus stejnegeri venom (TSV-PA) is a serine proteinase presenting 23% sequence identity with the proteinase domain of tissue type plasminogen activator, and 63% with batroxobin, a fibrinogen clotting enzyme from Bothrops atrox venom that does not activate plasminogen . TSV-PA contains six disulfide bonds and has been successfully overexpressed in Escherichia coli (Zhang, Y., Wisner, A., Xiong, Y . L., and Bon, C . (1995) J . Biol . Chem . 270, 10246-10255) . To identify the functional domains of TSV-PA, we focused on three short peptide fragments of TSV-PA showing important sequence differences with batroxobin and other venom serine proteinases . Molecular modeling shows that these sequences are located in surface loop regions, one of which is next to the catalytic site . When these sequences were replaced in TSV-PA by the equivalent batroxobin residues none generated either fibrinogen-clotting or direct fibrinogenolytic activity . Two of the replacements had little effect in general and are not critical to the specificity of TSV-PA for plasminogen . Nevertheless, the third replacement, produced by the conversion of the sequence DDE 96a-98 to NVI, significantly increased the Km for some tripeptide chromogenic substrates and resulted in undetectable plasminogen activation, indicating the key role that the sequence plays in substrate recognition by the enzyme.

J Biol Chem, 1997 Aug 15, 272(33), 20414 - 9
Localization of a binding site for the proteoglycan decorin on collagen XIV (undulin); Ehnis T et al.; Through its ability to bind extracellular matrix constituents and growth factors the small leucine-rich chondroitin/dermatan sulfate proteoglycan decorin which is present in many types of connective tissues may play an important biological role in remodeling and maintenance of extracellular matrices during inflammation, fibrosis, and cancer growth . In this study we investigated the known binding of decorin to human collagen XIV . This binding was unaffected when the small collagenous moiety of collagen XIV was removed with collagenase . Therefore, fragments covering the large noncollagenous domain NC3 of collagen XIV were expressed in Escherichia coli, each fused to a 26-kDa fragment of glutathione S-transferase . Using radioiodinated decorin as ligand for the immobilized fusion proteins, a binding site that interacted with the decorin core protein could be assigned to the NH2-terminal fibronectin type III repeat of collagen XIV . In addition, an auxiliary binding site located COOH-terminal to this fibronectin type III repeat interacted with the glycosaminoglycan component of decorin.

Nucleic Acids Res, 1997 Aug 15, 25(16), 3310 - 7
2',5'-linked oligo-3'-deoxyribonucleoside phosphorothioate chimeras: thermal stability and antisense inhibition of gene expression; Bhan P et al.; 2',5'-Linked oligo-3'-deoxyribonucleotides bind selectively to complementary RNA but not to DNA . These oligonucleotides (ODNs) do not recognize double-stranded DNA by Hoogsteen triplex formation and the complexes formed by these ODNs with RNA are not substrates for Escherichia coli RNase H . Substitution of the 2',5'-phosphodiester backbone by phosphorothioate linkages gives 2',5'-linked oligo-3'-deoxynucleoside phosphorothioate ODNs that exhibit significantly less non-specific binding to cellular proteins or thrombin . Incorporation of a stretch of seven contiguous 3',5'-linked oligo-2'-deoxynucleoside phosphorothioate linkages in the center of 2',5'-linked ODNs (as a putative RNase H recognition site) afford chimeric antisense ODNs that retain the ability to inhibit steroid 5alpha-reductase (5alphaR) expression in cell culture.

Nucleic Acids Res, 1997 Aug 15, 25(16), 3218 - 27
A two unit antisense RNA cassette test system for silencing of target genes; Engdahl HM et al.; This communication describes a two unit antisense RNA cassette system for use in gene silencing . Cassettes consist of a recognition unit and an inhibitory unit which are transcribed into a single RNA that carries sequences of non-contiguous complementarity to the chosen target RNA . The recognition unit is designed as a stem-loop for rapid formation of long- lived binding intermediates with target sequences and resembles the major stem-loop of a naturally occurring antisense RNA, CopA . The inhibitory unit consists of either a sequence complementary to a ribosome binding site or of a hairpin ribozyme targeted at a site within the chosen mRNA . The contributions of the individual units to inhibition was assessed using the lacI gene as a target . All possible combinations of recognition and inhibitory units were tested in either orientation . In general, inhibition of lacI expression was relatively low . Fifty per cent inhibition was obtained with the most effective of the constructs, carrying the recognition stem-loop in the antisense orientation and the inhibitory unit with an anti-RBS sequence . Several experiments were performed to assess activities of the RNAs in vitro and in vivo : antisense RNA binding assays, cleavage assays, secondary structure analysis as well as Northern blotting and primer extension analysis of antisense and target RNAs . The problems associated with this antisense RNA approach as well as its potential are discussed with respect to possible optimization strategies.

Nucleic Acids Res, 1997 Aug 15, 25(16), 3212 - 7
Increased polymerase fidelity of the 3TC-resistant variants of HIV-1 reverse transcriptase; Oude Essink BB et al.; Human immunodeficiency virus type 1 (HIV-1) variants with resistance mutations in the reverse transcriptase (RT) gene appear during drug therapy with the nucleoside analogue 2',3'-dideoxy-3'-thiacytidine (3TC) . These 3TC-resistant RT variants have a single point mutation that changes the 184Met residue into either Val or Ile . Both codon 184 variants are frequently observed in 3TC-treated patients and can also be selected in cell culture infections . We demonstrated previously that the 184Ile and 184Val RT enzymes exhibit a processivity defect in in vitro assays, with 184Ile being the least processive enzyme {Met(wt) >Val >Ile} . In this study, we measured the polymerase fidelity of the wild-type (184Met) and 3TC-resistant RT enzymes (184Ile and 184Val) on DNA and RNA templates . Both virion- extracted and Escherichia coli -expressed recombinant RT enzymes were used to measure the nucleotide misinsertion and mispair extension efficiencies . The 3TC-resistant enzymes were more accurate than the wild-type RT protein in both type of assays . The order of accuracy observed for the codon 184 variants {Ile >Val >Met(wt)} may suggest an inverse correlation between the fidelity and processivity properties of these enzymes.

Nucleic Acids Res, 1997 Aug 15, 25(16), 3187 - 95
Comparative sequence analysis of ribonucleases HII, III, II PH and D; Mian IS; Escherichia coli ribonucleases (RNases) HII, III, II, PH and D have been used to characterise new and known viral, bacterial, archaeal and eucaryotic sequences similar to these endo- (HII and III) and exoribonucleases (II, PH and D) . Statistical models, hidden Markov models (HMMs), were created for the RNase HII, III, II and PH and D families as well as a double-stranded RNA binding domain present in RNase III . Results suggest that the RNase D family, which includes Werner syndrome protein and the 100 kDa antigenic component of the human polymyositis scleroderma (PMSCL) autoantigen, is a 3'-->5' exoribonuclease structurally and functionally related to the 3'-->5' exodeoxyribonuclease domain of DNA polymerases . Polynucleotide phosphorylases and the RNase PH family, which includes the 75 kDa PMSCL autoantigen, possess a common domain suggesting similar structures and mechanisms of action for these 3'-->5' phosphorolytic enzymes . Examination of HMM-generated multiple sequences alignments for each family suggest amino acids that may be important for their structure, substrate binding and/or catalysis.

Oncogene, 1997 Aug 14, 15(7), 845 - 50
Biochemical characterization of C3G: an exchange factor that discriminates between Rap1 and Rap2 and is not inhibited by Rap1A(S17N); van den Berghe N et al.; A catalytically active fragment of the Rap-specific guanine-nucleotide exchange factor C3G was expressed in E coli . It was purified and its interaction with GTP-binding proteins was investigated using fluorescence spectroscopy . C3G stimulates GDP dissociation from Rap1, but not from Rap2, neither from Bud1, which is believed to be the yeast homologue of Rap1 nor from all other proteins of the human Ras-subfamily . Like the corresponding fragment from CDC25Mm, the increase in the GDP dissociation rate is linear with increasing concentration of Rap1A x GDP up to 100 microM, indicating an apparent K(M) higher than 100 microM . Unlike the Ras-CDC25Mm system, the Rap1A(S17N) mutant does not inhibit the C3G-activated guanine nucleotide dissociation from wild-type Rap1A in vitro . These data suggest that Rap1A(S17N) is unlikely to titrate away C3G in vivo, the proposed mechanism by which S17N-mutants exert their dominant negative effects.

Biochemistry, 1997 Aug 12, 36(32), 9941 - 9
Construction and characterization of monomeric tryptophan repressor: a model for an early intermediate in the folding of a dimeric protein; Shao X et al.; Tryptophan repressor (TR) from Escherichia coli is a homodimer whose highly helical subunits intertwine in a complex fashion . A monomeric version of Trp repressor has been constructed by introducing a pair of polar amino acids at the hydrophobic dimer interface . Analytical ultracentrifugation was used to show that the replacement of leucine at position 39 with glutamic acid results in a monomer/dimer equilibrium whose dissociation constant is 1.11 x 10(-)4 M at 25 degrees C and pH 7.6 . Tryptophan fluorescence, both near- and far-UV circular dichroism, and NMR spectroscopies demonstrated that, at the micromolar concentrations where the monomer predominates, secondary and tertiary structure are present . Hydrophobic dye-binding experiments showed that nonpolar surface is accessible in the monomeric form . The urea-induced equilibrium unfolding of monomeric L39E TR was monitored by circular dichroism, fluorescence, and absorbance spectroscopies . Coincident transitions show that the urea denaturation process follows a simple two-state model involving monomeric native and unfolded forms . The free energy at standard state in the absence of denaturant was estimated to be 2.37 +/- 0.15 kcal mol-1, and the sensitivity of the unfolding transition to denaturant, the m value, was 0.86 +/- 0.04 kcal mol-1 M(urea)-1 at pH 7.6 and 25 degrees C . The thermal denaturation transition occurred over a broad temperature range, suggesting either that the enthalpy change is small or that intermediates may exist . Kinetic studies showed that both the refolding and unfolding of the monomer were complete in the mixing dead time of stopped-flow CD and fluorescence spectroscopy, 5 ms . These structural, thermodynamic, and kinetic results are very similar to those previously reported for an early, monomeric intermediate in the folding of the wild-type TR dimer {Mann, C . J., & Matthews, C . R . (1993) Biochemistry 32, 5282-5290} . The construction of a stable, monomeric form of TR that strongly resembles a transient folding intermediate should provide useful insights into the nature of the early events in the folding of TR.

Biochemistry, 1997 Aug 12, 36(32), 9917 - 26
Optimizing the metal binding parameters of an EF-hand-like calcium chelation loop: coordinating side chains play a more important tuning role than chelation loop flexibility; Drake SK et al.; In calcium signaling pathways regulated by the EF-hand Ca2+ binding motif, proper regulation requires that the equilibrium and kinetics of Ca2+ binding to the EF-hand chelation loop be precisely optimized for each physiological application . Studies of small-molecule organic chelators have shown that metal binding parameters can be tuned both by the nature of the coordinating ligands and by the structural framework to which these ligands are attached . By analogy, the present study tests the relative importance of (i) coordinating side chains and (ii) backbone torsion angle constraints to the tuning of an EF-hand-like Ca2+ chelation loop . A series of engineered chelation loops are generated by modifying Ca2+ binding site of the Escherichia coli galactose binding protein . The resulting loops, each containing an altered coordinating side chain or a Gly substitution, are compared with respect to their metal binding affinities, specificities, and dissociation kinetics . The Gly variants examined include substitutions which eliminate or introduce a Gly at each of the nine chelation loop positions . The results reveal that Gly is not tolerated at loop positions 1, 3, 5, or 8 or at the external coordinating position, where the removal of a key coordinating or hydrophobic side chain destabilizes the protein . In contrast, Gly residues at loop positions 2, 4, 6, and 7, none of which is required for side chain coordination, have little effect on Ca2+ affinity and the ability to discriminate between cations of different size and charge . Kinetic measurements show that some of these Gly residues measurably alter the rates of metal ion association and dissociation, but in each case the two rates are changed by approximately the same factor so that the effects on equilibrium are minor . Overall, Gly residues yield surprisingly small effects at loop positions 2, 4, 6, and 7, especially when compared to the larger equilibrium and kinetic effects observed for coordinating side chain substitutions . It follows that the conserved Gly at position 6 is not required for Ca2+ binding and that constraints on the backbone torsion angles at the non-coordinating side chain positions 2, 4, 6, and 7 play a relatively minor role in tuning metal binding parameters . Instead, specific coordinating side chains optimize the metal binding parameters of the GBP chelation loop for its protein context and biological application.

Biochemistry, 1997 Aug 12, 36(32), 9766 - 73
Mutagenesis of the Mn2+-binding site of manganese peroxidase affects oxidation of Mn2+ by both compound I and compound II; Whitwam RE et al.; The present study investigates whether compound I and compound II of manganese peroxidase from the white-rot fungus Phanerochaete chrysosporium utilize the same Mn-binding site for catalysis . Manganese peroxidase was expressed from its cDNA in Escherichia coli and refolded from inclusion bodies to yield fully active enzyme . Three mutants of the enzyme were generated by site-directed mutagenesis . Each of the three amino acid residues proposed to be involved in Mn2+ binding, E35, D179, and E39, was mutated . The acidic side chains of E35 and E39 were shortened by one carbon to the acidic group D, and the acidic side chain of D179 was shortened by one carbon to the alkyl group A . These mutants, E35D, D179A, and E39D, were used to determine whether Mn2+ reacts at the same site with both compound I and compound II of manganese peroxidase and to determine whether phenolic substrates for the enzyme react at this site . Our results conclusively demonstrate that E35 and D179 residues are involved not only in Mn2+ binding but also in electron transfer from Mn2+ to the enzyme for both compound I and compound II . In contrast, E39 is not critically important to either process . None of the three residues is involved in reactions with phenolic substrates or with H2O2.

Biochemistry, 1997 Aug 12, 36(32), 9655 - 62
A double mutation at the tip of the dimer interface loop of triosephosphate isomerase generates active monomers with reduced stability; Schliebs W et al.; Triosephosphate isomerase (TIM) is a very stable dimer . In order to understand better the importance of dimerization for stability and catalytic activity, we have constructed a monomeric double-mutation variant . The dimer interface residues Thr75 and Gly76, which are at the tip of loop 3, have been substituted by an arginine and a glutamate, respectively . In wild type TIM, these two residues are at a distance of 27 A from the active site (as measured within the same subunit) . This new variant, RE-TIM, was expressed in Escherichia coli, purified to homogeneity, and biochemically characterized . Sedimentation equilibrium ultracentrifugation runs showed that RE-TIM is a monomer in solution . Far-UV CD spectra indicate that this new variant is folded properly and that the secondary structure contents of RE-TIM are similar to those of wild type TIM . The monomeric RE-TIM has residual TIM activity . The thermal stability of RE-TIM is lower than that for wild type TIM . CD melting curves for RE-TIM and wild type TIM show Tm values of 52 and 57 degrees C, respectively, in the presence of the active site ligand 2-phosphoglycolate at 1 mM . Previously, we have characterized two other monomeric forms of TIM: monoTIM and H47N-TIM . The properties of RE-TIM, H47N-TIM, and monoTIM are compared, and it is argued that the properties of RE-TIM will be very similar to those of wild type monomeric subunits . This implies that wild type monomeric subunits have some stability and are catalytically active . It is also inferred that these monomeric subunits have flexible loops which rigidify at the dimer interface on dimerization, causing a 1000-fold increase of kcat and a 10-fold decrease of Km.

FEBS Lett, 1997 Aug 11, 413(1), 109 - 14
In vitro membrane integration of leader peptidase depends on the Sec machinery and anionic phospholipids and can occur post-translationally; van Klompenburg W et al.; A cell-free system based on a lysate and membrane vesicles from Escherichia coli is used to study characteristics of the membrane integration reaction of the polytopic membrane protein leader peptidase (Lep) . Integration into inverted inner membrane vesicles was detected by partial protection against externally added protease . Integration is most efficient when coupled to translation but can also occur post-translationally and depends on the action of the proteinaceous Sec machinery and availability of anionic phospholipids . Lep is the first example of a membrane protein without cleavable signal sequence which requires anionic lipids for integration in vitro.

Biochem Biophys Res Commun, 1997 Aug 8, 237(1), 192 - 201
Isolation and analysis of mutated histidyl-tRNA synthetases from Escherichia coli; Ruhlmann A et al.; Amino terminally deleted and point-mutated histidyl-tRNA synthetases were purified from E . coli via betaGal fusion proteins . A hinge region proximal and distal to the factor Xa cleavage region was necessary to cut the betaGal-fusion proteins efficiently under very mild nondenaturing conditions . N-terminal addition of either methionine or valine to this enzyme (its starting N-formyl-methionine is in vivo post-translationally removed) or the deletion of 6 amino terminal amino acids decreased the specific aminoacylation activity 2- to 7-fold . Further N-terminal deletions of 10 or 17 amino acids caused significantly reduced aminoacylation (100-fold) and ATP/PPi exchange (10-fold) activities, and a reduced binding affinity for histidine . Removal of 18 or more amino acids from the N-terminus thereby removing residues from MOTIF 1 resulted in inactive histidyl-tRNA synthetase mutants . Two point mutations within the histidyl-adenylate binding pocket, R259Q and R259K, also blocked histidyl-tRNA synthetase activity without affecting histidine or ATP binding . The experiments shown identify a highly conserved N-terminal R/KG-patch in front of MOTIF 1 as well as R259 as vital for full enzymatic activity.

Biochem Biophys Res Commun, 1997 Aug 8, 237(1), 84 - 9
A novel zebrafish gene expressed specifically in the photoreceptor cells of the retina; Chang H et al.; We have identified and characterized a novel protein from adult zebrafish retina, which we named ES1 . Database search revealed that the ES1 gene has significant similarity to two genes with unknown functions: the Escherichia coli sigma cross-reacting protein 27a (scrp27a) and the human KNP-I/GT335 . In situ hybridization and immunohistochemistry experiments showed that both ES1 mRNA and protein are expressed specifically in adult photoreceptor cells . ES1 seems to be a cytoplasmic protein . An ES1-like antigen was also detected in photoreceptor cells of goldfish with anti-ES1 antibodies . The retina specific expression and the evolutionary conservation suggest that ES1 protein may be important for maintaining normal retina structure and function.

J Biol Chem, 1997 Aug 8, 272(32), 20259 - 62
A specific role for the phosphorylation of mammalian acidic ribosomal protein P2; Vard C et al.; The acidic ribosomal proteins P1-P2 from rat liver were overproduced for the first time by expression of their cDNA in Escherichia coli . They were tested for their ability to reactivate inactive P1-P2-deficient core particles derived from 60 S ribosomal subunits treated with dimethylmaleic anhydride, in poly(U)-directed poly(Phe) synthesis . The recombinant P1-P2 were unable to reactivate these core particles although they could bind to them . When recombinant P1-P2 had been phosphorylated first with casein kinase II, they were as efficient in the reactivation process as P1-P2 extracted with ethanol/KCl from the 60 S subunits . Reconstitution experiments were carried out using all possible combinations of the two recombinant proteins phosphorylated or not . Reactivation of the core particles required the presence of both P1 and P2 with the latter in its phosphorylated form . These experiments reveal a distinct role for P1 and P2 in protein synthesis . Phosphorylated P2 produced a partial quenching of the intrinsic fluorescence of eukaryotic elongation factor 2, which was not observed with the unphosphorylated protein . This result demonstrates the existence of an interaction between phosphorylated P2 and eukaryotic elongation factor 2 . P2 also quenched part of the intrinsic fluorescence of P1, due to the interaction between the two proteins.

J Biol Chem, 1997 Aug 8, 272(32), 20173 - 8
Role of TrfA and DnaA proteins in origin opening during initiation of DNA replication of the broad host range plasmid RK2; Konieczny I et al.; The Escherichia coli protein DnaA and the plasmid RK2-encoded TrfA protein are required for initiation of replication of the broad host range plasmid RK2 . The TrfA protein has been shown to bind to five 17-base pair repeat sequences, referred to as iterons, at the minimal replication origin (oriV) . Using DNase I footprinting and a gel mobility shift assay, purified DnaA protein was found to bind to four DnaA consensus binding sequences immediately upstream of the five iterons at the RK2 origin of replication . Binding of the TrfA protein to the iterons results in localized strand opening within the A+T-rich region of the replication origin as determined by reactivity of the top and bottom strands to potassium permanganate (KMnO4) . The presence of either the E . coli DnaA or HU protein is required for the TrfA-mediated strand opening . Although the DnaA protein itself did not produce an RK2 open complex, it did enhance and/or stabilize the TrfA-induced strand opening.

J Biol Chem, 1997 Aug 8, 272(32), 20088 - 95
A dual involvement of the amino-terminal domain of ezrin in F- and G-actin binding; Roy C et al.; Human recombinant ezrin, or truncated forms, were coated in microtiter plate and their capacity to bind actin determined . F-actin bound ezrin with a Kd of 504 +/- 230 nM and a molecular stoichiometry of 10.6 actin per ezrin . Ezrin bound both alpha- and beta/gamma-actin essentially as F-form . F-actin binding was totally prevented or drastically reduced when residues 534-586 or 13-30 were deleted, respectively . An actin binding activity was detected in amino-terminal constructs (ezrin 1-310 and 1-333) provided the glutathione S-transferase moiety of the fusion protein was removed . Series of carboxyl-terminal truncations confirmed the presence of this actin-binding site which bound both F- and G-actin . The F- and G-actin-binding sites were differently sensitive to various chemical effectors and distinct specific ezrin antibodies . The internal actin-binding site was mapped between residues 281 and 333 . The association of ezrin amino-terminal fragment to full-length ezrin blocked F-actin binding to ezrin . It is proposed that, in full-length ezrin, the F-actin-binding site required the juxtaposition of the distal-most amino- and carboxyl-terminal residues of the ezrin molecule.

J Biol Chem, 1997 Aug 8, 272(32), 20082 - 7
In vitro analysis of the stop-transfer process during translocation across the cytoplasmic membrane of Escherichia coli; Sato K et al.; In this study, using a derivative of proOmpA containing an artificial stop-transfer sequence (proOmpA2xH1), we analyzed the process of stop-transfer during translocation across the cytoplasmic membrane of Escherichia coli . ProOmpA2xH1 did not interfere with the transit of wild-type proOmpA . When proOmpA2xH1 was anchored in the membrane, membrane-inserted SecA was deinserted with the reversion of the inverted topology of SecG . Cross-linking experiments revealed that the anchored proOmpA2xH1 that does not interact with either SecY or SecA . These results, taken together, suggest that proOmpA2xH1 leaves the translocation pathway by means of a specific interaction between the stop-transfer sequence and the translocational channel.

J Biol Chem, 1997 Aug 8, 272(32), 20044 - 8
Dimerization of the N-terminal amphipathic alpha-helix domain of the fungal immunomodulatory protein from Ganoderma tsugae (Fip-gts) defined by a yeast two-hybrid system and site-directed mutagenesis; Lin WH et al.; A fungal immunomodulatory protein (Fip-gts) was purified from Ganoderma tsugae . The DNA encoding Fip-gts was isolated from a cDNA library of G . tsugae by reverse transcriptase-polymerase chain reaction . The complete amino acid sequence of Fip-gts, deduced from the nucleotide sequence of the cDNA, was the same as LZ-8 isolated from Ganodermn lucidum . Recombinant Fip-gts was expressed as a glutathione S-transferase fusion protein in Escherichia coli with a yield of 20 mg/liter of culture . Recombinant Fip-gts, purified to homogeneity, had the same blast formation stimulatory activity to human peripheral blood lymphocytes as native Fip-gts . The yeast two-hybrid system and site-directed mutagenesis were used to determine whether dimerization of Fip-gts occurred . Deletion analysis of the N-terminal amphipathic alpha-helix domain of Fip-gts identified a sequence of about 10 amino acids responsible for inducing immunomodulatory activity . Non-functional Fip-gts deletion mutants did not form dimers, whereas wild type Fip-gts did as determined by gel filtration . A mutant with deletions at Leu-5, Phe-7, and Leu-9 lost the amphipathic characteristics of the N-terminal domain and the ability to form dimers as well as its immunomodulatory activity . Fusion of Fip-gts with the DNA binding and the transactivation domains of GAL4 resulted in the activation of the lacZ activator gene, indicating the interaction of Fip-gts with it itself . The dimerization domain was further defined by analyzing the ability of the N-terminal 13 amino acids or Leu-5, Phe-7, and Leu-9 deletion mutants of Fip-gts to interact with the wild type Fip-gts . These experiments confirmed the N-terminal amphipathic alpha-helix as the dimerization domain and suggest that the dimerization of Fip-gts may play an important role in Fip-gts immunomodulatory activity.

J Biol Chem, 1997 Aug 8, 272(32), 19913 - 8
Activation by fusion of the glutaminase and synthetase subunits of Escherichia coli carbamyl-phosphate synthetase; Guy HI et al.; Escherichia coli carbamyl-phosphate synthetase consists of two subunits that act in concert to synthesize carbamyl phosphate . The 40-kDa subunit is an amidotransferase (GLN subunit) that hydrolyzes glutamine and transfers ammonia to the 120-kDa synthetase subunit (CPS subunit) . The enzyme can also catalyze ammonia-dependent carbamyl phosphate synthesis if provided with exogenous ammonia . In mammalian cells, homologous amidotransferase and synthetase domains are carried on a single polypeptide chain called CAD . Deletion of the 29-residue linker that bridges the GLN and CPS domains of CAD stimulates glutamine-dependent carbamyl phosphate synthesis and abolishes the ammonia-dependent reaction (Guy, H . I., and Evans, D . R . (1997) J . Biol . Chem . 272, 19906-19912), suggesting that the deletion mutant is trapped in a closed high activity conformation . Since the catalytic mechanisms of the mammalian and bacterial proteins are the same, we anticipated that similar changes in the function of the E . coli protein could be produced by direct fusion of the GLN and CPS subunits . A construct was made in which the intergenic region between the contiguous carA and carB genes was deleted and the sequences encoding the carbamyl-phosphate synthetase subunits were fused in frame . The resulting fusion protein was activated 10-fold relative to the native protein, was unresponsive to the allosteric activator ornithine, and could no longer use ammonia as a nitrogen donor . Moreover, the functional linkage that coordinates the rate of glutamine hydrolysis with the activation of bicarbonate was abolished, suggesting that the protein was locked in an activated conformation similar to that induced by the simultaneous binding of all substrates.

J Biol Chem, 1997 Aug 8, 272(32), 19906 - 12
Trapping an activated conformation of mammalian carbamyl-phosphate synthetase; Guy HI et al.; The amidotransferase or glutaminase domain (GLN domain) of mammalian carbamyl-phosphate synthetase II (CPSase II) catalyzes glutamine hydrolysis and transfers ammonia to the synthetase domain (CPS domain), where carbamyl phosphate formation is catalyzed in three consecutive reactions . The GLN and CPS domains are part of a single polypeptide and are connected via a 29-amino acid chain segment (GC linker) . In contrast, the two comparable domains of Escherichia coli CPSase are not fused, but are separate, noncovalently associated subunits . To establish the function of the GC linker in mammalian CPSase, it was deleted, and the two domains were directly fused . The deletion mutant not only catalyzed glutamine-dependent carbamyl phosphate synthesis, but was activated 10-fold relative to its wild-type counterpart . However, ammonia-dependent synthesis of carbamyl phosphate was abolished, indicating that ammonia no longer had access to the active site on the CPS domain . The mutant was still sensitive to inhibition by the allosteric effector UTP, but was no longer activated by the allosteric effector phosphoribosyl pyrophosphate, although evidence indicated that the latter could bind to the enzyme . The linker appears to serve as a spacer that allows the complex to cycle between two conformations, an open low activity form in which the ammonia site on the CPS domain is accessible and an activated conformation in which the ammonia generated in situ from glutamine is directly channeled to the CPS active site and access to exogenous ammonia is blocked.

J Biol Chem, 1997 Aug 8, 272(32), 19880 - 3
Synthesis and characterization of selenolipoylated H-protein of the glycine cleavage system; Fujiwara K et al.; H-protein of the glycine cleavage system has a lipoic acid prosthetic group . Selenolipoic acid is a lipoic acid analog in which both sulfur atoms are replaced by selenium atoms . Two isoforms of bovine lipoyltransferase that are responsible for the attachment of lipoic acid to H-protein had an affinity for selenolipoyl-AMP and transferred the selenolipoyl moiety to bovine apoH-protein comparable to lipoyl-AMP . Selenolipoylated H-protein was overexpressed in Escherichia coli and purified . Selenolipoylated H-protein was 26% as effective as lipoylated H-protein in the glycine decarboxylation reaction, in which reduction of the diselenide bond of selenolipoylated H-protein is catalyzed by P-protein . The diselenide form of selenolipoylated H-protein was a poor substrate for L-protein, and the rate of reduction was 0.5% of that of lipoylated H-protein . The rate of the overall glycine cleavage reaction with selenolipoylated H-protein was <1% of that with lipoylated H-protein . These results are consistent with the difference in the redox potential between the diselenide and disulfide bonds . In contrast, selenolipoylated H-protein showed three times as high glycine-14CO2 exchange activity as lipoylated H-protein, presumably because the rate of reoxidation of reduced selenolipoylated H-protein is much higher than that of lipoylated H-protein.

J Biol Chem, 1997 Aug 8, 272(32), 19851 - 7
An internal cysteine is involved in the thioredoxin-dependent activation of sorghum leaf NADP-malate dehydrogenase; Ruelland E et al.; The chloroplastic NADP-malate dehydrogenase is activated by thiol/disulfide interchange with reduced thioredoxins . Previous experiments showed that four cysteines located in specific N- and carboxyl-terminal extensions were implicated in this process, leading to a model where no internal cysteine was involved in activation . In the present study, the role of the conserved four internal cysteines was investigated . Surprisingly, the mutation of cysteine 207 into alanine yielded a protein with accelerated activation time course, whereas the mutations of the three other internal cysteines into alanines yielded proteins with unchanged activation kinetics . These results suggested that cysteine 207 might be linked in a disulfide bridge with one of the four external cysteines, most probably with one of the two amino-terminal cysteines whose mutation similarly accelerates the activation rate . To investigate this possibility, mutant malate dehydrogenases (MDHs) where a single amino-terminal cysteine was mutated in combination with the mutation of both carboxyl-terminal cysteines were produced and purified . The C29S/C365A/C377A mutant MDH still needed activation by reduced thioredoxin, while the C24S/C365A/C377A mutant MDH exhibited a thioredoxin-insensitive spontaneous activity, leading to the hypothesis that a Cys24-Cys207 disulfide bridge might be formed during the activation process . Indeed, an NADP-MDH where the cysteines 29, 207, 365, and 377 are mutated yielded a permanently active enzyme very similar to the previously created permanently active C24S/C29S/C365A/C377A mutant . A two-step activation model involving a thioredoxin-mediated disulfide isomerization at the amino terminus is proposed.

J Biol Chem, 1997 Aug 8, 272(32), 19819 - 26
Overexpression, purification, and characterization of the SbcCD protein from Escherichia coli; Connelly JC et al.; The sbcC and sbcD genes mediate palindrome inviability in Escherichia coli . The sbcCD operon has been cloned into the plasmid pTrc99A under the control of the strong trc promoter and introduced into a strain carrying a chromosomal deletion of sbcCD . The SbcC and SbcD polypeptides were overexpressed to 6% of total cell protein, and both polypeptides copurified in a four-step purification procedure . Purified SbcCD is a processive double-strand exonuclease that has an absolute requirement for Mn2+ and uses ATP as a preferred energy source . Gel filtration chromatography and sedimentation equilibrium analyses were used to show that the SbcC and SbcD polypeptides dissociate at some stage after purification and that this dissociation is reversed by the addition of Mn2+ . We demonstrate that SbcD has the potential to form a secondary structural motif found in a number of protein phosphatases and suggest that it is a metalloprotein that contains the catalytic center of the SbcCD exonuclease.

J Biol Chem, 1997 Aug 8, 272(32), 19746 - 51
Dihydrolipoamide dehydrogenase-binding protein of the human pyruvate dehydrogenase complex . DNA-derived amino acid sequence, expression, and reconstitution of the pyruvate dehydrogenase complex; Harris RA et al.; Protein X, recently renamed dihydrolipoamide dehydrogenase-binding protein (E3BP), is required for anchoring dihydrolipoamide dehydrogenase (E3) to the dihydrolipoamide transacetylase (E2) core of the pyruvate dehydrogenase complexes of eukaryotes . DNA and deduced protein sequences for E3BP of the human pyruvate dehydrogenase complex are reported here . With the exception of only a single lipoyl domain, the protein has a segmented multi-domain structure analogous to that of the E2 component of the complex . The protein has 46% amino acid sequence identity in its amino-terminal region with the second lipoyl domain of E2, 38% identity in its central region with the putative peripheral subunit-binding domain of E2, and 50% identity in its carboxyl-terminal region with the catalytic inner core domain of E2 . The similarity in the latter domain stands in contrast to E3BP of Saccharomyces cerevisiae, which is quite different from its homologous transacetylase in this region . The putative catalytic site histidine residue present in the inner core domains of all dihydrolipoamide acyltransferases is replaced by a serine residue in human E3BP; thus, catalysis of coenzyme A acetylation by this protein is unlikely . Coexpression of cDNAs for E3BP and E2 resulted in the formation of an E2.E3BP subcomplex that spontaneously reconstituted the pyruvate dehydrogenase complex in the presence of native E3 and recombinant pyruvate decarboxylase (E1).

J Biol Chem, 1997 Aug 8, 272(32), 19645 - 8
Conformational changes at the nucleotide binding of GroEL induced by binding of protein substrates . Luminescence studies; Churchich JE; 2'-Deoxy-3'-anthraniloyl adenosine-5-triphosphate (ANT-dATP) coordinated to Tb3+ was used as an environmentally sensitive probe of the nucleotide-binding site of GroEL . Tb3+.ANT-dATP recognizes the nucleotide-binding site of GroEL and inhibits ATPase activity . Sensitized luminescence, arising from resonance energy transfer from the anthraniloyl moiety to Tb3+, is substantially enhanced in the presence of GroEL . Binding of denatured mitochondrial malate dehydrogenase to the apical domain of GroEL causes a red shift in the fluorescence emitted by anthraniloyl and further enhancement in the phosphorescence emitted by Tb3+ upon excitation at 320 nm . It is suggested that binding of the protein substrate initiates domain movement, which is extended to the nucleotide-binding site . The luminescence results are discussed in reference to the structure of GroEL derived from x-ray crystallographic studies.

Proc Natl Acad Sci U S A, 1997 Aug 5, 94(16), 8907 - 11
Mendel's dwarfing gene: cDNAs from the Le alleles and function of the expressed proteins; Martin DN et al.; The major gibberellin (GA) controlling stem elongation in pea (Pisum sativum L.) is GA1, which is formed from GA20 by 3beta-hydroxylation . This step, which limits GA1 biosynthesis in pea, is controlled by the Le locus, one of the original Mendelian loci . Mutations in this locus result in dwarfism . We have isolated cDNAs encoding a GA 3beta-hydroxylase from lines of pea carrying the Le, le, le-3, and led alleles . The cDNA sequences from le and le-3 each contain a base substitution resulting in single amino acid changes relative to the sequence from Le . The cDNA sequence from led, a mutant derived from an le line, contains both the le "mutation" and a single-base deletion, which causes a shift in reading frame and presumably a null mutation . cDNAs from each line were expressed in Escherichia coli . The expression product for the clone from Le converted GA9 to GA4, and GA20 to GA1, with Km values of 1.5 microM and 13 microM, respectively . The amino acid substitution in the clone from le increased Km for GA9 100-fold and reduced conversion of GA20 to almost nil . Expression products from le and le-3 possessed similar levels of 3beta-hydroxylase activity, and the expression product from led was inactive . Our results suggest that the 3beta-hydroxylase cDNA is encoded by Le . Le transcript is expressed in roots, shoots, and cotyledons of germinating pea seedlings, in internodes and leaves of established seedlings, and in developing seeds.

Proc Natl Acad Sci U S A, 1997 Aug 5, 94(16), 8652 - 7
Mutagenicity in Escherichia coli of the major DNA adduct derived from the endogenous mutagen malondialdehyde; Fink SP et al.; The spectrum of mutations induced by the naturally occurring DNA adduct pyrimido{1,2-alpha}purin-10(3H)-one (M1G) was determined by site-specific approaches using M13 vectors replicated in Escherichia coli . M1G was placed at position 6256 in the (-)-strand of M13MB102 by ligating the oligodeoxynucleotide 5'-GGT(M1G)TCCG-3' into a gapped-duplex derivative of the vector . Unmodified and M1G-modified genomes containing either a cytosine or thymine at position 6256 of the (+)-strand were transformed into repair-proficient and repair-deficient E . coli strains, and base pair substitutions were quantitated by hybridization analysis . Modified genomes containing a cytosine opposite M1G resulted in roughly equal numbers of M1G-->A and M1G-->T mutations with few M1G-->C mutations . The total mutation frequency was approximately 1%, which represents a 500-fold increase in mutations compared with unmodified M13MB102 . Transformation of modified genomes containing a thymine opposite M1G allowed an estimate to be made of the ability of M1G to block replication . The (-)-strand was replicated >80% of the time in the unadducted genome but only 20% of the time when M1G was present . Correction of the mutation frequency for the strand bias of replication indicated that the actual frequency of mutations induced by M1G was 18% . Experiments using E . coli with different genetic backgrounds indicated that the SOS response enhances the mutagenicity of M1G and that M1G is a substrate for repair by the nucleotide excision repair complex . These studies indicate that M1G, which is present endogenously in DNA of healthy human beings, is a strong block to replication and an efficient premutagenic lesion.

Proc Natl Acad Sci U S A, 1997 Aug 5, 94(16), 8551 - 6
Complete resolution of the solid-state NMR spectrum of a uniformly 15N-labeled membrane protein in phospholipid bilayers; Marassi FM et al.; Complete resolution of the amide resonances in a three-dimensional solid-state NMR correlation spectrum of a uniformly 15N-labeled membrane protein in oriented phospholipid bilayers is demonstrated . The three orientationally dependent frequencies, 1H chemical shift, 1H-15N dipolar coupling, and 15N chemical shift, associated with each amide resonance are responsible for resolution among resonances and provide sufficient angular restrictions for protein structure determination . Because the protein is completely immobilized by the phospholipids on the relevant NMR time scales (10 kHz), the linewidths will not degrade in the spectra of larger proteins . Therefore, these results demonstrate that solid-state NMR experiments can overcome the correlation time problem and extend the range of proteins that can have their structures determined by NMR spectroscopy to include uniformly 15N-labeled membrane proteins in phospholipid bilayers.

Proc Natl Acad Sci U S A, 1997 Aug 5, 94(16), 8509 - 14
Molecular cloning of a peroxisomal Ca2+-dependent member of the mitochondrial carrier superfamily; Weber FE et al.; A cDNA from a novel Ca2+-dependent member of the mitochondrial solute carrier superfamily was isolated from a rabbit small intestinal cDNA library . The full-length cDNA clone was 3,298 nt long and coded for a protein of 475 amino acids, with four elongation factor-hand motifs located in the N-terminal half of the molecule . The 25-kDa N-terminal polypeptide was expressed in Escherichia coli, and it was demonstrated that it bound Ca2+, undergoing a reversible and specific conformational change as a result . The conformation of the polypeptide was sensitive to Ca2+ which was bound with high affinity (Kd approximately 0.37 microM), the apparent Hill coefficient for Ca2+-induced changes being about 2.0 . The deduced amino acid sequence of the C-terminal half of the molecule revealed 78% homology to Grave disease carrier protein and 67% homology to human ADP/ATP translocase; this sequence homology identified the protein as a new member of the mitochondrial transporter superfamily . Northern blot analysis revealed the presence of a single transcript of about 3,500 bases, and low expression of the transporter could be detected in the kidney but none in the liver . The main site of expression was the colon with smaller amounts found in the small intestine proximal to the ileum . Immunoelectron microscopy localized the transporter in the peroxisome, although a minor fraction was found in the mitochondria . The Ca2+ binding N-terminal half of the transporter faces the cytosol.

Proc Natl Acad Sci U S A, 1997 Aug 5, 94(16), 8491 - 6
A glutamate residue in the catalytic center of the yeast chorismate mutase restricts enzyme activity to acidic conditions; Schnappauf G et al.; Chorismate mutase acts at the first branchpoint of aromatic amino acid biosynthesis and catalyzes the conversion of chorismate to prephenate . Comparison of the x-ray structures of allosteric chorismate mutase from the yeast Saccharomyces cerevisiae with Escherichia coli chorismate mutase/prephenate dehydratase suggested conserved active sites between both enzymes . We have replaced all critical amino acid residues, Arg-16, Arg-157, Lys-168, Glu-198, Thr-242, and Glu-246, of yeast chorismate mutase by aliphatic amino acid residues . The resulting enzymes exhibit the necessity of these residues for catalytic function and provide evidence of their localization at the active site . Unlike some bacterial enzymes, yeast chorismate mutase has highest activity at acidic pH values . Replacement of Glu-246 in the yeast chorismate mutase by glutamine changes the pH optimum for activity of the enzyme from a narrow to a broad pH range . These data suggest that Glu-246 in the catalytic center must be protonated for maximum catalysis and restricts optimal activity of the enzyme to low pH.

Proc Natl Acad Sci U S A, 1997 Aug 5, 94(16), 8485 - 90
Interaction of the human androgen receptor transactivation function with the general transcription factor TFIIF; McEwan IJ et al.; The human androgen receptor (AR) is a ligand-activated transcription factor that regulates genes important for male sexual differentiation and development . To better understand the role of the receptor as a transcription factor we have studied the mechanism of action of the N-terminal transactivation function . In a protein-protein interaction assay the AR N terminus (amino acids 142-485) selectively bound to the basal transcription factors TFIIF and the TATA-box-binding protein (TBP) . Reconstitution of the transactivation activity in vitro revealed that AR142-485 fused to the LexA protein DNA-binding domain was competent to activate a reporter gene in the presence of a competing DNA template lacking LexA binding sites . Furthermore, consistent with direct interaction with basal transcription factors, addition of recombinant TFIIF relieved squelching of basal transcription by AR142-485 . Taken together these results suggest that one mechanism of transcriptional activation by the AR involves binding to TFIIF and recruitment of the transcriptional machinery.

Proc Natl Acad Sci U S A, 1997 Aug 5, 94(16), 8450 - 5
Structural flexibility in transcription complex formation revealed by protein-DNA photocrosslinking; Cleary MA et al.; The Oct-1 POU domain binds diverse DNA-sequence elements and forms a higher-order regulatory complex with the herpes simplex virus coregulator VP16 . The POU domain contains two separate DNA-binding domains joined by a flexible linker . By protein-DNA photocrosslinking we show that the relative positioning of the two POU DNA-binding domains on DNA varies depending on the nature of the DNA target . On a single VP16-responsive element, the POU domain adopts multiple conformations . To determine the structure of the Oct-1 POU domain in a multiprotein complex with VP16, we allowed VP16 to interact with previously crosslinked POU-domain-DNA complexes and found that VP16 can associate with multiple POU-domain conformations . These results reveal the dynamic potential of a DNA-binding domain in directing transcriptional regulatory complex formation.

Proc Natl Acad Sci U S A, 1997 Aug 5, 94(16), 8445 - 9
In vivo kinetics of a redox-regulated transcriptional switch; Ding H et al.; SoxR is a transcription activator governing a cellular response to superoxide and nitric oxide in Escherichia coli . SoxR protein is a homodimer, and each monomer has a redox-active {2Fe-2S} cluster . Oxidation and reduction of the {2Fe-2S} clusters can reversibly activate and inactivate SoxR transcriptional activity . Here, we use electron paramagnetic resonance spectroscopy to follow the redox-switching process of SoxR protein in vivo . SoxR {2Fe-2S} clusters were in the fully reduced state during normal aerobic growth, but were completely oxidized after only 2-min aerobic exposure of the cells to superoxide-generating agents such as paraquat . The oxidized SoxR {2Fe-2S} clusters were rapidly re-reduced in vivo once the oxidative stress was removed . The in vivo kinetics of SoxR {2Fe-2S} cluster oxidation and reduction exactly paralleled the increase and decrease of transcription of soxS, the target gene for SoxR . The kinetic analysis also revealed that an oxidative stress-linked decrease in soxS mRNA stability contributes to the rapid attainment of a new steady state after SoxR activation . Such a redox stress-related change in soxS mRNA stability may represent a new level of biological control.

Proc Natl Acad Sci U S A, 1997 Aug 5, 94(16), 8411 - 6
Three-dimensional structure of NADPH-cytochrome P450 reductase: prototype for FMN- and FAD-containing enzymes; Wang M et al.; Microsomal NADPH-cytochrome P450 reductase (CPR) is one of only two mammalian enzymes known to contain both FAD and FMN, the other being nitric-oxide synthase . CPR is a membrane-bound protein and catalyzes electron transfer from NADPH to all known microsomal cytochromes P450 . The structure of rat liver CPR, expressed in Escherichia coli and solubilized by limited trypsinolysis, has been determined by x-ray crystallography at 2.6 A resolution . The molecule is composed of four structural domains: (from the N- to C- termini) the FMN-binding domain, the connecting domain, and the FAD- and NADPH-binding domains . The FMN-binding domain is similar to the structure of flavodoxin, whereas the two C-terminal dinucleotide-binding domains are similar to those of ferredoxin-NADP+ reductase (FNR) . The connecting domain, situated between the FMN-binding and FNR-like domains, is responsible for the relative orientation of the other domains, ensuring the proper alignment of the two flavins necessary for efficient electron transfer . The two flavin isoalloxazine rings are juxtaposed, with the closest distance between them being about 4 A . The bowl-shaped surface near the FMN-binding site is likely the docking site of cytochrome c and the physiological redox partners, including cytochromes P450 and b5 and heme oxygenase.

Biochemistry, 1997 Aug 5, 36(31), 9571 - 80
Repositioning the catalytic triad aspartic acid of haloalkane dehalogenase: effects on stability, kinetics, and structure; Krooshof GH et al.; Haloalkane dehalogenase (DhlA) catalyzes the hydrolysis of haloalkanes via an alkyl-enzyme intermediate . The covalent intermediate, which is formed by nucleophilic substitution with Asp124, is hydrolyzed by a water molecule that is activated by His289 . The role of Asp260, which is the third member of the catalytic triad, was studied by site-directed mutagenesis . Mutation of Asp260 to asparagine resulted in a catalytically inactive D260N mutant, which demonstrates that the triad acid Asp260 is essential for dehalogenase activity . Furthermore, Asp260 has an important structural role, since the D260N enzyme accumulated mainly in inclusion bodies during expression, and neither substrate nor product could bind in the active-site cavity . Activity for brominated substrates was restored to D260N by replacing Asn148 with an aspartic or glutamic acid . Both double mutants D260N+N148D and D260N+N148E had a 10-fold reduced kcat and 40-fold higher Km values for 1,2-dibromoethane compared to the wild-type enzyme . Pre-steady-state kinetic analysis of the D260N+N148E double mutant showed that the decrease in kcat was mainly caused by a 220-fold reduction of the rate of carbon-bromine bond cleavage and a 10-fold decrease in the rate of hydrolysis of the alkyl-enzyme intermediate . On the other hand, bromide was released 12-fold faster and via a different pathway than in the wild-type enzyme . Molecular modeling of the mutant showed that Glu148 indeed could take over the interaction with His289 and that there was a change in charge distribution in the tunnel region that connects the active site with the solvent . On the basis of primary structure similarity between DhlA and other alpha/beta-hydrolase fold dehalogenases, we propose that a conserved acidic residue at the equivalent position of Asn148 in DhlA is the third catalytic triad residue in the latter enzymes.

Biochemistry, 1997 Aug 5, 36(31), 9486 - 92
Replication of M13 single-stranded viral DNA bearing single site-specific adducts by escherichia coli cell extracts: differential efficiency of translesion DNA synthesis for SOS-dependent and SOS-independent lesions; Wang G et al.; In order to characterize mutagenic translesion DNA synthesis in UVM-induced Escherichia coli, we have developed a high-resolution DNA replication system based on E . coli cell extracts and M13 genomic DNA templates bearing mutagenic lesions . The assay is based on the conversion of M13 viral single-stranded DNA (ssDNA) bearing a single site-specific DNA lesion to the double-stranded replicative form (RF) DNA, and permits one to quantitatively measure the efficiency of translesion synthesis . Our data indicate that DNA replication is most strongly inhibited by an abasic site, a classic SOS-dependent noninstructive lesion . In contrast, the efficiency of translesion synthesis across SOS-independent lesions such as O6-methylguanine and DNA uracil is around 90%, very close to the values obtained for control DNA templates . The efficiency of translesion synthesis across 3,N4-ethenocytosine and 1, N6-ethenoadenine is around 20%, a value that is similar to the in vivo efficiency deduced from the effect of the lesions on the survival of transfected M13 ssDNA . Neither DNA polymerase I nor polymerase II appears to be required for the observed translesion DNA synthesis because essentially similar results are obtained with extracts from polA- or polB-defective cells . The close parallels in the efficiency of translesion DNA synthesis in vitro and in vivo for the five site-specific lesions included in this study suggest that the assay may be suitable for modeling mutagenesis in an accessible in vitro environment.

Biochemistry, 1997 Aug 5, 36(31), 9464 - 77
Reductive half-reaction of thioredoxin reductase from Escherichia coli; Lennon BW et al.; Thioredoxin reductase is a homodimeric flavoenzyme containing a flavin adenine dinucleotide (FAD) and a redox-active disulfide in each subunit . Structural work on the enzyme from Escherichia coli suggests that thioredoxin reductase exists in two conformations, both of which are necessary for catalysis {Waksman, G., Krishna, T . S . R., Williams, C . H., Jr., & Kuriyan, J . (1994) J . Mol . Biol . 236, 800-816} . These factors make it likely that the mechanism of this enzyme is complex . The rapid reaction of enzyme with nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) (the reductive half-reaction), proceeds in three phases . The first phase represents the formation of an NADPH-FAD charge transfer complex . The second phase involves FAD reduction, with loss of the NADPH-FAD charge transfer band . The third phase shows a slower decrease in absorbance at 456 nm and the formation of a reduced flavin-NADP+ charge transfer band . These and other results indicate that NADP+ and NADPH compete for the single binding site on oxidized and fully reduced enzyme and that NADP+ release does not limit the third phase of reduction . Experiments that include examination of the reductive half-reactions of active-site mutants, having the active-site disulfide removed by mutating one or both of the active-site cysteines, indicate that the third phase does not represent reduction by a second equivalent of NADPH . Comparison of the rate constants and temperature dependence of the reductive half-reaction with those of turnover show that the reductive half-reaction is not solely rate-limiting in catalysis . The results suggest that wild type and each altered enzyme exists in a unique equilibrium of conformers . It is proposed that the third phase of the reductive half-reaction represents a flavin reduction event largely limited by the conformational change proposed in the structural work.

Biochemistry, 1997 Aug 5, 36(31), 9323 - 31
Effects of decyl-aurachin D and reversed electron transfer in cytochrome bd; Junemann S et al.; Decyl-aurachin D is a near-stoichiometric inhibitor of cytochrome bd from Azotobacter vinelandii . Interaction of decyl-aurachin D with the oxidase induces a redshift of the alpha-band and Soret band of a b-type cytochrome, probably b-558, suggesting close proximity of the inhibitor binding site to this haem and hence to the proposed quinol binding domain . The compound does not affect the oxygen binding site directly as judged from unchanged CO recombination kinetics to haem d in dithionite-reduced enzyme . Although in the presence of ubiquinol-1 a decyl-aurachin D containing sample generates levels of haem reduction and catalytic intermediates similar to the control, the approach to this steady state is severely inhibited . In addition to the spectral effect on b-558, decyl-aurachin D raises the midpoint potential of haem b-558, but also lowers that of haem b-595 . Consistent with the shift in midpoint potentials, electron backflow from haem d to the b-type haems can be observed in decyl-aurachin D inhibited samples following photolysis of the mixed-valence CO-ligated form of the enzyme . The data show that decyl-aurachin D acts on the donor side of haem b-558 without substantially affecting internal electron transfer rates or the oxygen reduction site.

FEBS Lett, 1997 Aug 4, 412(3), 592 - 6
Novel components and enzymatic activities of the human erythrocyte plasma membrane calcium pump; Reusch RN et al.; The plasma membrane Ca2+ pump is essential for the maintenance of cystolic calcium ion concentration levels in eukaryotes . Here we show that the Ca2+-ATPase, purified from human erythrocytes, contains two homopolymers, poly(3-hydroxybutyrate) (PHB) and inorganic polyphosphate (polyP), which form voltage-activated calcium channels in the plasma membranes of Escherichia coli and other bacteria . Furthermore, we demonstrate that the plasma membrane Ca2+-ATPase may function as a polyphosphate kinase, i.e . it exhibits ATP-polyphosphate transferase and polyphosphate-ADP transferase activities . These findings suggest a novel supramolecular structure for the functional Ca2+-ATPase, and a new mechanism of uphill Ca2+ extrusion coupled to ATP hydrolysis.

Eur J Biochem, 1997 Aug 1, 247(3), 1143 - 50
The heat-shock protein HslVU from Escherichia coli is a protein-activated ATPase as well as an ATP-dependent proteinase; Seol JH et al.; HslVU in Escherichia coli a new two-component ATP-dependent protease composed of two heat-shock proteins, the HslU ATPase and the HslV peptidase which is related to proteasome beta-type subunits . Here we show that the reconstituted HslVU enzyme degrades not only certain hydrophobic peptides but also various polypeptides, including insulin B-chain, casein, and carboxymethylated lactalbumin . Maximal proteolytic activity was obtained with a 1:2 molar ratio of HslV (a 250-kDa complex) to HslU (a 450-kDa complex) . By itself, HslV could slowly hydrolyze these polypeptides, but its activity was stimulated 20-fold by HslU in the presence of ATP . The ATPase activity of HslU was stimulated up to 50% by the protein substrates, but not by nonhydrolyzed proteins, and this stimulation further increased 2-3-fold in the presence of HslV . Concentrations of insulin B-chain that maximally stimulated the ATPase allowed maximal rates of the B-chain hydrolysis . Furthermore, addition of increasing amounts of ADP or N-ethylmaleimide reduced ATP and protein or peptide hydrolysis in parallel . Thus, HslVU is a protein-activated ATPase as well as an ATP-dependent proteinase, and these processes appear linked . Surprisingly, the protein and peptide substrates do not compete with each other for hydrolysis . Lactacystin strongly inhibits protein degradation, but has little effect on peptide hydrolysis, while the peptide aldehydes are potent inhibitors of hydrolysis of small peptides, but have little effect on proteins . Thus, the functional requirements for ATP-dependent hydrolysis of peptides and proteins appear different.

Eur J Biochem, 1997 Aug 1, 247(3), 1136 - 42
Characterization of two F-actin-binding and oligomerization sites in the cell-contact protein vinculin; Huttelmaier S et al.; Vinculin, a structural protein of animal cells, is critically involved in the assembly of microfilament/plasma membrane junctions at cell contacts . To understand its role in organizing the distal portions of microfilaments into specific, morphologically distinct structures at these sites in more detail, we characterized its interaction with filamentous actin and with itself by means of in vitro assays . Using recombinant proteins comprising different parts of the vinculin tail fused to the maltose-binding protein of Escherichia coli, we show in sedimentation assays that this part of vinculin harbors two discrete sites that can bind to actin independently . They reside within amino acid residues 893-985 and 1016-1066 of the 1066-residue polypeptide chain . However, both sites are necessary to cross-link or bundle actin filaments, as demonstrated by low shear viscometry . Crosslinking and bundling are alternatives determined by the molar ratio of fusion protein to F-actin . Both actin-binding sequences are capable of oligomer formation, as shown in chemical-cross-linking and dot-overlay assays . These data allow us to propose a possible role for vinculin in organizing the distal ends of microfilaments at the plasma membrane into the point-like structure characteristic for cell-matrix contacts.

Eur J Biochem, 1997 Aug 1, 247(3), 1111 - 7
The subunit f of mitochondrial yeast ATP synthase--characterization of the protein and disruption of the structural gene ATP17; Spannagel C et al.; The subunit f of the yeast F1F0ATP synthase has been isolated from the purified enzyme . Amino acid composition, protein and peptide sequencing were performed . The data are in agreement with the sequence of the predicted product of the gene D9481.21 identified on the Saccharomyces cerevisiae chromosome IV . A 303-bp open reading frame encoding a 101-amino acid polypeptide is described . The deduced amino acid sequence from the ATP17 gene is 6 amino acids longer than the mature protein, which displays a molecular mass of 10567 Da . The protein is basic with a short hydrophobic segment located in the C-terminal part of the subunit . Subunit f remained associated with other F0 subunits upon sodium bromide treatment of the whole enzyme . A null mutant was constructed . The disrupted strain was unable to grow on glycerol medium and the mutation was recessive; rho- cells arose spontaneously . The null mutant mitochondria were devoid of oligomycin-sensitive ATPase, but still contained an active F1, while the subunits f, 6 and 8 were absent.

Eur J Biochem, 1997 Aug 1, 247(3), 1083 - 90
All-transglycolytic synthesis and characterization of sialyl(alpha2-3)galactosyl(beta1-4)xylosyl-p-nitrophenyl(beta1-), an oligosaccharide derivative related to glycosaminoglycan biosynthesis; Vetere A et al.; Beta-D-Xylopyranosides, such as p-nitrophenyl-beta-D-xylopyranoside (Xyl-Np) or 4-methylumbelliferyl-beta-D-xylopyranoside (Xyl-MeUmb), when added to the culture medium of human skin fibroblasts have previously been shown to produce some Np- or MeUmb-oligosaccharides related to the regulation of glycosaminoglycan biosynthesis . Among these oligosaccharide derivatives, we synthesized the trisaccharide derivative NeuAc(alpha2-3)Gal(beta1-4)Xyl-Np(beta1- as a potential inhibitor of human skin fibroblast glycosaminoglycan biosynthesis . This synthesis was achieved by sequential use of transglycosylating activities of Escherichia coli beta-galactosidase and Trypanosoma cruzi trans-sialidase . The structure of the oligosaccharide obtained was determined by HPLC, ion-spray mass spectrometry, and NMR.

Eur J Biochem, 1997 Aug 1, 247(3), 1038 - 45
Isolation of the gene encoding Pyrococcus furiosus ornithine carbamoyltransferase and study of its expression profile in vivo and in vitro; Roovers M et al.; The gene coding for ornithine carbamoyltransferase (OTCase, argF) in the hyperthermophilic archaea Pyrococcus furiosus was cloned by complementation of an OTCase mutant of Escherichia coli . The cloned P . furiosus argF gene also complemented a similar mutant of Saccharomyces cerevisiae . Sequencing revealed an open reading frame of 314 amino acids homologous to known OTCases and preceded by a TATA box showing only limited similarity with the Euryarchaeota consensus sequence . This is in accordance with the comparatively low in vitro promoter activity observed in a cell-free purified transcription system . Transcription initiates in vivo as well as in vitro at a guanine, 22 nucleotides downstream of the TATA box . Upstream from argF is a putative gene for diphthine synthetase, a eukaryotic enzyme assumed to occur also in archaea but not in bacteria.

Eur J Biochem, 1997 Aug 1, 247(3), 1029 - 37
cDNA cloning, recombinant expression, and site-directed mutagenesis of bovine liver carnitine octanoyltransferase--Arg505 binds the carboxylate group of carnitine; Cronin CN; The cDNA for bovine liver carnitine octanoyltransferase (COT) has been cloned by a combination of lambda gt11 library screening and 3' rapid amplification of cDNA ends (3'-RACE) . The cDNA comprises 338 bases of 5' non-coding sequence, a reading frame of 1839 bases including the stop codon, and 820 bases of 3' non-coding DNA . The deduced amino acid sequence of 612 residues predicts a protein with a calculated mass of 70263 Da and pI 6.28 . The enzyme was expressed in recombinant soluble form in Escherichia coli and was purified by a two-step procedure to near-homogeneity with a yield of purified protein of 2-3 mg/l culture . Recombinant COT had similar kinetic properties to those of the enzyme isolated directly from beef liver . Arg505 in COT, conserved in all reported carnitine acyltransferase sequences but replaced by asparagine or isoleucine in the choline acetyltransferases, was converted to asparagine by site-directed mutagenesis . This single mutation resulted in a greater than 1650-fold increase in the Km value for COT towards carnitine, but had little effect on the value of k(cat) or the Km value for the acyl-CoA substrate . In addition, although choline was an extremely poor substrate for COT, the k(cat)/Km ratio towards this substrate was increased fourfold as a result of the mutation . These data support the notion that Arg505 in COT, and other carnitine acyltransferases, contributes to substrate binding by forming a salt bridge with the carboxylate moiety of carnitine.

Eur J Biochem, 1997 Aug 1, 247(3), 1009 - 18
Expression, purification, mass spectrometry, crystallization and multiwavelength anomalous diffraction of selenomethionyl PvuII DNA methyltransferase (cytosine-N4-specific); O'Gara M et al.; The type II DNA-methyltransferase (cytosine N4-specific) M.PvuII was overexpressed in Escherichia coli, starting from the internal translation initiator at Met14 . Selenomethionine was efficiently incorporated into this short form of M.PvuII by a strain prototrophic for methionine . Both native and selenomethionyl M.PvuII were purified to apparent homogeneity by a two-column chromatography procedure . The yield of purified protein was approximately 1.8 mg/g bacterial paste . Mass spectrometry analysis of selenomethionyl M.PvuII revealed three major forms that probably differ in the degree of selenomethionine incorporation and the extent of selenomethionine oxidation . Amino acid sequencing and mass spectrometry analysis of selenomethionine-containing peptides suggests that Met30, Met51, and Met261 were only partially replaced by selenomethionine . Furthermore, amino acid 261 may be preferentially oxidized in both native and selenomethionyl form . Selenomethionyl and native M.PvuII were crystallized separately as binary complexes of the methyl donor S-adenosyl-L-methionine in the monoclinic space group P2(1) . Two complexes were present per asymmetric unit . Six out of nine selenium positions (per molecule), including the three that were found to be partially substituted, were identified crystallographically.

Eur J Biochem, 1997 Aug 1, 247(3), 990 - 9
Guanosine 3',5'-bis(diphosphate) (ppGpp)-dependent inhibition of transcription from stringently controlled Escherichia coli promoters can be explained by an altered initiation pathway that traps RNA polymerase; Heinemann M et al.; An in vitro analysis was performed to investigate the inhibitory mechanism of the global regulatory substances guanosine 3',5'-bis(diphosphate) (ppGpp) and guanosine 3'-diphosphate 5'-triphosphate (pppGpp) during initiation of transcription . Three promoters with well known differential ppGpp sensitivities in vivo were studied: the Escherichia coli rrnB P2 promoter that is only weakly ppGpp dependent; a P2 base change variant (P2F) that confers both stringent and growth rate regulation; and the completely unregulated PtacI promoter . The in vivo ppGpp dependency for all three promoters was verified in vitro in multiple round transcription reactions, reflecting a combination of the effects at initiation, promoter clearance, and elongation . In the main part of our study, we concentrated on the contribution of initiation complex formation to the overall inhibition of transcription . Kinetic measurements of complex association and dissociation revealed that at sensitive promoters (p)ppGpp triggered an alternative initiation pathway by RNA polymerase . This involved the stabilization of the initial closed complexes, and impeded open complex formation . Subsequently formed ternary complexes were structurally altered . Based on the above findings, we propose a model which suggests that ppGpp-altered RNA polymerases are preferentially bound and enter the alternative pathway . Thus, discrimination is obtained at early steps of initiation, which causes efficient inhibition at later steps of the transcription cycle probably involving promoter clearance and elongation.

Eur J Biochem, 1997 Aug 1, 247(3), 890 - 5
Expression of natural and synthetic genes encoding herpes simplex virus 1 protease in Escherichia coli and purification of the protein; Apeler H et al.; An attractive target for anti-herpes chemotherapy is the herpes simplex virus 1 (HSV-1) protease encoded by the UL26 gene . Studies with HSV-1 strains that harbour mutations in the protease gene have demonstrated that the protease is essential for DNA packaging and virus maturation . The UL26 translation product is 635 amino acids long and undergoes autoproteolytic processing between residues Ala247/Ser248 and Ala610/Ser611 . The N-terminal processing product (amino acids 1-247) contains the protease domain . To perform crystallization studies and high throughput screening for potent inhibitors, large amounts of the HSV-1 protease are required . However, expression of the natural HSV-1 protease gene in Escherichia coli using a T7-promoter-regulated system is low and does not allow for the efficient production of larger amounts of highly purified enzyme . In this report, we describe the use of a synthetic protease gene with optimized E . coli codon usage . The level of protease expression was at least 20 times higher with the synthetic gene as compared to the natural UL26 gene . The HSV-1 protease was purified to homogeneity in three steps using mixed-bed ion-exchange chromatography, affinity chromatography, and hydroxyapatite chromatography.

Eur J Biochem, 1997 Aug 1, 247(3), 884 - 9
Studies on the omega subunit of Escherichia coli RNA polymerase--its role in the recovery of denatured enzyme activity; Mukherjee K et al.; Highly purified Escherichia coli RNA polymerase contains a small subunit termed omega that has a molecular mass of 10,105 Da and is comprised of 91 amino acids . To elucidate the function of omega, whose role is as yet undefined, the subunit was purified to over 95% purity from an overproducing strain {BL21 (pGP1-2, pE3C-2)} . Purified omega was then reconstituted with RNA polymerase isolated from an omega-less mutant . Externally added omega inhibited promoter-specific transcriptional activity at all promoters tested . Renaturation of fully denatured omega-less RNA polymerase in the presence of excess omega yielded maximum recovery of activity suggesting a structural rather than functional role for omega.

Eur J Biochem, 1997 Aug 1, 247(3), 826 - 32
Cloning and biochemical characterization of an anionic peroxidase from Zea mays; Teichmann T et al.; We have isolated, cloned and characterized a cDNA from Zea mays L., denoted ZmAP1, coding for an anionic peroxidase . The open reading frame of ZmAP1 starting 72 residues from the 5' end of the cDNA predicts a 37,778 dalton protein of 356 amino acid residues . The protein has high similarity to other peroxidases and contains two peroxidase motifs that carry two highly conserved histidines in the active center . We expressed recombinant ZmAP1 protein in E . coli as a fusion with maltose-binding protein . The fusion protein was biochemically active after addition of hemin to the apoprotein . The maize peroxidase ZmAP1 has a pH optimum at pH 4.0 and a Km of 0.2 mM for the substrate 2,2'-azino-bis-(3-ethyl-benzothiazolin-6-sulfonic acid) at this pH . In maize seedlings the ZmAP1 gene is expressed predominantly in roots, the mesocotyl, the coleoptile and to a lower extent in the node, whereas no expression in the primary leaf was found . In situ hybridization shows that the expression of ZmAP1 in the young maize root is confined to the epidermis, hypodermis and the pericycle.

Eur J Biochem, 1997 Aug 1, 247(3), 820 - 5
Subunit c from the sodium-ion-translocating F1F0-ATPase of Propionigenium modestum--production, purification and properties of the protein in dodecylsulfate solution; Matthey U et al.; Escherichia coli strain PEF42 produces a sodium-ion-dependent hybrid F1F0-ATPase consisting of the Propionigenium modestum subunits a, b, c and delta, of a hybrid alpha subunit and of the E . coli subunits beta, gamma and epsilon . The gene encoding subunit c of the P . modestum F1F0-ATPase was cloned into the pT7-7 expression vector to yield plasmid pT7c . E . coli PEF42 was transformed with plasmid pT7c together with plasmid pGP1-2, which harbours the gene for the T7 RNA polymerase . The production of the P . modestum subunit c was induced by a temperature shift from 30 degrees C to 42 degrees C for 30 min and led to an increased concentration of this protein in the membrane of the host strain . The c subunit produced in E . coli moved as a monomer in dodecylsulfate electrophoresis . The protein was extracted from the cells with chloroform/methanol, purified and incorporated into sodium dodecylsulfate micelles . Circular dichroism of subunit c in sodium dodecylsulfate showed a temperature-stable spectrum (between 20-60 degrees C) with a high proportion of alpah-helical structure . Upon incubation of subunit c with {14C}dicyclohexylcarbodiimide the protein became labelled in a sodium-ion-dependent manner, similar to the labelling observed if the purified F1F0-ATPase of P . modestum, was treated with the radioactive carbodiimide . The Na+-specific site was therefore retained in the isolated c subunit dissolved in dodecylsulfate.

Eur J Biochem, 1997 Aug 1, 247(3), 770 - 5
Oligomeric states and siderophore binding of the ligand-gated FhuA protein that forms channels across Escherichia coli outer membranes; Locher KP et al.; The channel-forming FhuA protein, which translocates ferrichrome across Escherichia coli outer membranes, binds 1 mol ligand/mol monomer in detergent solution . The protein is homogenous and migrates as a single band with a mobility corresponding to 77 kDa in SDS/PAGE electrophoresis . Analytical ultracentrifugation revealed a monodisperse species (s(20,w) = 3.8 S) with a mass of 77,800 +/- 3200 Da . The properties of ligand binding, determined by two independent methods, revealed one binding site/monomer, but are complicated by a pronounced convexity of the Scatchard plot and a Hill coefficient calculated to be 2.5 . This strongly suggests that oligomeric species are present . Cross-linking agents revealed the existence of possibly transient, mostly dimeric and trimeric species . The difference between the FhuA protein in detergent solution and in its native membrane environment may be related to the removal of lateral pressure that exists in situ.

Mutat Res, 1997 Aug 1, 378(1-2), 127 - 37
Use of a postlabelling assay to examine the removal of radiation-induced DNA lesions by purified enzymes and human cell extracts; Weinfeld M et al.; We have used a 32P-postlabelling assay to examine the activity of purified Esherichia coli endonuclease IV, human apurinic/apyrimidinic endonuclease I and human cell-free extracts towards irradiated DNA . The assay can detect thymine glycols, 3'-phosphoglycolate groups and at least one other major lesion that has yet to be fully characterized . It was observed that endonuclease IV removed the phosphoglycolates and the uncharacterized lesion(s) suggesting that the latter are abasic sites with modified deoxyribose residues . The purified human enzyme acted only on the phosphoglycolate residues . Cell-free extract, prepared from A549 lung carcinoma cells by sonication or treatment with toluene, efficiently removed the phosphoglycolate and unknown lesions, but was less reactive towards thymine glycols . The extract was completely inactivated by heating at 60 degrees C for 10 min . Removal of the unknown product and phosphoglycolate did not require magnesium, but 1 mM EDTA did inhibit release of the latter . The cell-free extract exhibited substantially more activity towards native than heat-denatured DNA . A comparison of extracts prepared from 4 cell lines displaying a range of radiosensitivities, including an ataxia telangiectasia cell line, showed that all contained similar levels of repair activity towards the detectable lesions.

Surgery, 1997 Aug, 122(2), 394 - 402; discussion 402-3
Endotoxin-mediated synthesis of nitric oxide is dependent on Gq protein signal transduction; Kuo PC et al.; BACKGROUND: Nitric oxide (NO) is a ubiquitous multifunctional free radical produced during sepsis, shock, reperfusion injury, and allograft rejection . Many studies are presently evaluating the functional roles of NO production in these settings . However, the signal transduction mechanisms underlying initiation of NO production are largely unknown . This study defines the cell surface receptor proteins that mediate endotoxin-induced NO synthesis in ANA-1 murine macrophages . METHODS: Endotoxin (LPS, 10 micrograms/ml) was added to ANA-1 macrophages to induce NO synthesis . In selected instances guanosine 5'-O-(2-thiodiphosphate)-trilithium salt (GOTP), pertussis toxin, cholera toxin, or suramin were added as inhibitors of specific subclasses of heterotrimeric G proteins . Calphostin was added as a protein kinase C inhibitor, and ET-OCH3 was added as a phospholipase C-beta inhibitor . NO release was quantified by measurement of the NO metabolite, nitrite . Membrane guanosine triphosphatase (GTPase) activity was also analyzed . Steady-state levels of inducible nitric oxide synthase (iNOS) mRNA were determined by using reverse transcription-polymerase chain reaction analysis . RESULTS: Inhibition of G protein function by suramin or GOTP significantly decreased synthesis of NO and expression of iNOS mRNA . Pertussis and cholera toxin did not alter NO synthesis, suggesting that the Gi and Gs classes are not involved . Inhibition of protein kinase C or upstream phospholipase C-beta activity decreased NO synthesis, implicating the Gq class of heterotrimeric G proteins . CONCLUSIONS: In ANA-1 macrophages, endotoxin-mediated NO synthesis is dependent on heterotrimeric Gq protein-phospholipase C-beta-protein kinase C signal transduction.

Surgery, 1997 Aug, 122(2), 303 - 12
The novel chemokine mob-1: involvement in adult respiratory distress syndrome; Abdullah F et al.; BACKGROUND: Using differential display reverse transcriptase-polymerase chain reaction we have recently identified mob-1, the novel rat homologue of the human alpha-chemokine IP-10, as a highly inducible gene in adult respiratory distress syndrome (ARDS) lungs . The present study aimed to further implicate mob-1 in the pathogenesis of ARDS . METHODS: Pulmonary mob-1 mRNA up-regulation was confirmed by Northern blot analysis in three different rat models of ARDS-like lung injury and localized to pulmonary macrophages by using in situ hybridization . Also, Escherichia coli-derived recombinant mob-1 (rmob-1) was tested for its properties in relationship to lung injury . RESULTS: In vivo, intratracheal injection of rmob-1 (50 micrograms/rat) induced pulmonary leukosequestration (myeloperoxidase +93% +/- 8% versus control, p < 0.05) with preferential accumulation of neutrophils in bronchoalveolar lavage fluid (36.0% +/- 1.0% versus 0.1% +/- 0.1% in controls, p < 0.01) . In vitro, transwell migration studies demonstrated chemotactic activity of rmob-1 (50 to 100 ng/ml) toward human monocytes (+151% +/- 34% versus rmob-1 vehicle, p < 0.01) and only weak chemotaxis for human neutrophils (+15% +/- 0% versus rmob-1 vehicle, p < 0.01) . Utilizing a rat aortic ring model ex vivo, rmob-1 at 100 ng/ml exerted a very potent inhibitory effect on angiogenesis (-78.7% +/- 6.3% versus rmob-1 vehicle, p < 0.01), a major component of the resolution phase of ARDS . CONCLUSIONS: Taken together, these data support the involvement of mob-1 in the pathogenic mechanisms of ARDS possibly through chemotaclic actions on inflammatory cells and modulation of angiogenesis in the recovery phase of the disease.

Surgery, 1997 Aug, 122(2), 204 - 11; discussion 211-2
Protein kinase C regulates macrophage tumor necrosis factor secretion: direct protein kinase C activation restores tumor necrosis factor production in endotoxin tolerance; West MA et al.; BACKGROUND: Macrophages pretreated in vitro with endotoxin (LPSp) secrete less tumor necrosis factor (TNF) in response to a second LPS activating (LPSa) stimulus . Protein kinase C (PKC) is required for TNF secretion in a macrophage stimulated with LPSa . In these experiments we examined the role of PKC in TNF signal transduction in naive and tolerant macrophages . METHODS: Murine macrophages were cultured +/- LPSp for 24 hours . Cultures were washed and treated for 1 hour with PKC inhibitors or phorbol myristate acetate (PMA), a direct PKC activator . Cells were then stimulated with a range of LPSa for 6 hours, and TNF was determined by bioassay . RESULTS: LPSa-stimulated TNF secretion by nontolerant macrophages was inhibited by LPSp in the absence of PMA . PKC inhibitors decreased TNF by naive macrophages and exaggerated inhibition in tolerant cells . Depletion of PKC by 24 hours of PMA decreased TNF production by both naive and tolerant macrophages . PKC activation with PMA 1 hour before LPSa augmented TNF secretion in naive cells and reversed TNF inhibition of tolerant cells . CONCLUSIONS: Direct PKC activation with PMA restored TNF secretion in LPS-tolerant macrophages . Endotoxin tolerance may alter the LPSa signal transduction pathway between the LPS receptor and PKC activation.

Surgery, 1997 Aug, 122(2), 163 - 71; discussion 171-2
Impaired apoptotic death signaling in inflammatory lung neutrophils is associated with decreased expression of interleukin-1 beta converting enzyme family proteases (caspases); Watson RW et al.; BACKGROUND: Fas and tumor necrosis factor receptor 1 (TNFR1) are membrane proteins that signal for apoptotic cell death by downstream activation of proteins of the interleukin-1 beta converting enzyme (ICE) family . Spontaneous apoptosis is delayed in neutrophils activated by transmigration into an inflammatory focus . In this study we evaluated the effects of transmigration on Fas and TNFR1-induced apoptosis and apoptotic gene expression . METHODS: Sprague-Dawley rats were killed 4 hours after intratracheal challenge with 500 micrograms lipopolysaccharide (LPS) . Neutrophils isolated from the systemic circulation (circulation) or bronchoalveolar lavage fluid (lung) were incubated with or without an agonistic antibody to Fas (clone CH-11, 100 ng/ml) or TNF (10 ng/ml) for 24 hours . Apoptosis and Fas expression were assessed by flow cytometry . Expression of the antiapoptotic protein Bcl-2 and proapoptotic proteins ICE and CPP32 were measured by Western blots . RESULTS: Neutrophils transmigrating into the lung in response to LPS showed delayed apoptosis compared with circulating neutrophils and failed to undergo apoptosis in response to anti-Fas antibody or TNF-alpha . Fas expression was unaltered; however, TNFR1 expression was reduced . Bcl-2 was not detected in either group; both the pro- and active forms of ICE and active CPP32 were significantly decreased in lung neutrophils . The specific ICE inhibitor, YVAD-CMK, partially blocked the increased rates of apoptosis resulting from engagement of Fas or TNFR1 . CONCLUSIONS: Neutrophil transmigration retards apoptosis through engagement of the death receptors Fas and TNFR1 . This refractory state is associated with reduced levels of proapoptotic proteins . Blunted responsiveness to physiologic apoptotic stimuli prolongs neutrophil functional survival during acute inflammation and may contribute to the tissue injury associated with acute respiratory distress syndrome.

Surgery, 1997 Aug, 122(2), 153 - 62
Integrin regulation of polymorphonuclear leukocyte apoxis during hypoxia is primarily dependent on very late activation antigens 3 and 5; Leuenroth S et al.; BACKGROUND: Apoptosis is thought to be a central mechanism that leads to resolution of the inflammatory response . The regulation of polymorphonuclear leukocyte (PMN) apoptosis during hypoxia has not been previously characterized, and we hypothesized that integrin signaling by matrix proteins (laminin) would regulate PMN apoptosis . METHODS: PMNs at 1 x 10(5)/ml were adhered on plastic or laminin for 12 hours during normoxia or hypoxia . Apoptosis was determined both by cellular histologic evaluation and the TUNEL assays (Tdt) . Phagocytosis in apoptotic PMNs was determined with two-color flow cytometric analyses with rhodamine-labeled heat-killed Escherichia coli (511 nm) and the Tdt reagent (563 nm) . Western blot analyses were performed on nine apoptotic regulatory proteins with monoclonal antibodies directed against each protein, and tyrosine phosphorylation was assessed after integrin receptor cross-linkage . RESULTS: Adherence of PMNs to laminin reduced apoptosis by cellular histologic evaluation and the Tdt method (%apoptosis = 19 +/- 1.0 versus 63 +/- 4.2 by histologic evaluation, 38 +/- 3.8 versus 60 +/- 10.5 by flow cytometry +/- adherence to laminin) . Apoptosis-positive PMNs exhibited significantly greater phagocytosis than apoptosis-negative PMNs +/- laminin . Western blot analyses demonstrated increased p53 expression after 2 and 4 hours of hypoxia . Cross-linkage of very late activation antigen-3 (alpha 3/beta 1) resulted in the phosphorylation of 53 kd, 44 kd, and 39 kd proteins at 30 seconds . CONCLUSIONS: (1) Chemotaxis of PMNs into the interstitium during hypoxia not only provides a means of ensuring PMN-pathogen contact but also provides a mechanism for improved survival by reducing apoptosis . (2) The reduction of apoptosis is mediated primarily by very late activation antigen-3, which leads to a subsequent increase in the intracellular expression of p53 and increased bacterial phagocytosis.

Plant Cell, 1997 Aug, 9(8), 1435 - 43
Mendel's stem length gene (Le) encodes a gibberellin 3 beta-hydroxylase; Lester DR et al.; We describe the isolation of the Le gene of pea, which controls internode elongation and originally was described by Mendel . Heterologous screening of a pea cDNA library yielded a partial clone that was 61% identical to coding regions of the putative Arabidopsis gibberellin 3 beta-hydroxylase gene, GA4 . DNA gel blot analysis with this cDNA revealed a HindIII restriction fragment length polymorphism between pea isolines differing at Mendel's Le locus . Genomic clones of the GA4-related gene were isolated from the Le and le isolines . Polymerase chain reaction combined with restriction fragment length polymorphism analysis were used to show that the gene mapped to the Le locus . A cDNA containing a complete open reading frame of the pea GA4-related gene was amplified by polymerase chain reaction from each isoline . Recombinant expression in Escherichia coli demonstrated that the product of the Le cDNA was a gibberellin 3 beta-hydroxylase that is able to convert GA20 to the bioactive GA1 . Substantially reduced levels of gibberellin 3 beta-hydroxylase activity were measured, after expression of the le cDNA, by using identical methods . This reduced activity was associated with an alanine-to-threonine substitution in the predicted amino acid sequence of the enzyme near its proposed active site.

Vaccine, 1997 Aug, 15(11), 1200 - 8
Broadly reactive antibodies against a gp120 V3 loop multi-epitope polypeptide neutralize different isolates of human immunodeficiency virus type 1 (HIV-1); Montero M et al.; A gene encoding for a novel multi-epitope polypeptide (TAB4) was synthesized and expressed in Escherichia coli . The protein was composed of 15 amino acid fragments derived from the V3 loop of HIV-1 isolates MN, IIIB, RF, JY1, BRVA and LR150, joined by five-amino-acid linkers . Immunogenicity of TAB4 in rabbits was studied, and the antibody response against individual peptides investigated . TAB4 was shown to be immunogenic in Complete Freund's Adjuvant in a dose-dependent manner, and was able to elicit a humoral response against all V3 epitopes included on the protein . Sera from some of the animals were able to neutralize the replication of viral strain MN, and in one case IIIB, with moderate titers . Some sera also neutralized several Cuban clinical strains, isolated in peripheral blood mononuclear cells, after one round of amplification in MT4 cells.

Graefes Arch Clin Exp Ophthalmol, 1997 Aug, 235(8), 530 - 4
The in vitro effect of platelet-derived growth factor isoforms on the proliferation of bovine corneal stromal fibroblasts depends on cell density; Denk PO et al.; PURPOSE: Recently, it has been shown that corneal stromal fibroblasts express the mRNA for PDGF-beta-type receptors, while corneal epithelial cells express the mRNA for the PDGF B-chain, suggesting a role of PDGF isoforms in the regulation of corneal homeostasis and wound healing via an unidirectional epithelial to stromal paracrine interaction . The purpose of this study was to characterize the proliferative response of cultured bovine corneal stromal fibroblasts to PDGF isoforms . METHODS: Bovine corneal stromal fibroblasts were seeded at a cell density of 60 cells/mm2 (low density) and 120 cells/mm2 (high density) and were cultured under serum-free conditions . Except for corresponding controls, PDGF AA, BB and AB (obtained by separate expression of cloned genes in E . coli) were added in concentrations ranging from 3.125 to 100 ng/ml . Cell numbers were determined after an incubation period of 6 days using a cell counter . RESULTS: Stromal fibroblasts, when cultured at a high density, revealed constant cell numbers during the whole incubation period . Under these culture conditions, stimulation with PDGF AA, BB and AB led to a significant dose-dependent increase in cell proliferation . When cultured at a low cell density, stromal fibroblasts revealed a significant reduction of cell numbers after 6 days of incubation . This reduction was prevented by PDGF AA and AB isoforms in a dose-dependent manner . In contrast, PDGF BB was not effective . CONCLUSION: The results of the "high-density" assays suggest that PDGF isoforms act as mitogens for stromal fibroblasts during wound healing, when density of fibroblasts is high . The results of the "low-density" assays support the idea that PDGF AA and AB can prevent cell loss during corneal homeostasis when density of keratocytes is low.

Biochem Mol Biol Int, 1997 Aug, 42(5), 1045 - 50
Residues K128-Q175 of human interleukin-6 are essential for its biological activity; Liu H et al.; Internal deletion K128-Q175 of the human interleukin-6 (hIL-6) has been generated at the cDNA level . With pBV220 as expressing vector, the recombinant pBV*-DIL-6 encoding the deletion mutant (12 kD) of hIL-6 has been constructed . The resulted recombinant plasmids were then used to transform E . coli strain HB101, and the expression in the PLPR promoter system, which is temperature-regulatable, was achieved . After purification and renaturation, the biological activity of the expressed product, designated as DM120, was measured by MTT method in an IL-6-dependent cell line 7TD1 . The results show that the amino acid residues of IL-6 128 to 175 are crucial for IL-6 activity . Receptor binding assay in vitro indicates that the entire region is not involved in forming the receptor binding surface.

Biochem Mol Biol Int, 1997 Aug, 42(5), 891 - 9
NMR characterization of physicochemical properties of rat D-dopachrome tautomerase; Yoshida H et al.; D-dopachrome tautomerase (DOPD) catalyzes the conversion of D-dopachrome to 5,6-dihydroxyindole . DOPD was found to have amino acid sequence homology with macrophage migration inhibitor factor (MIF), suggesting that DOPD functions as a proinflammatory cytokine . We here demonstrate the physicochemical properties of DOPD by nuclear magnetic resonance (NMR) . The native molecular weight of rat DOPD was about 37 kDa as calculated from Sephadex G-100 column chromatography . Since the deduced molecular weight of its cDNA (117 amino acid residues) is 12.5 kDa, it is assumed that DOPD exists as a homotrimer in native form . Since several methyl proton resonances were observed in the high magnetic field region of the one-dimensional 1H-NMR spectrum, DOPD appeared to have a hydrophobic core in which methyl groups and aromatic groups are located close to each other . From a simple integration of one-dimensional 1H-NMR spectra, the contents of the alpha-helix, beta-strand, and random coil were calculated to be 16%, 68%, and 16%, respectively . Since the denaturation temperature of DOPD is exceedingly high at the range between 90-100 degrees C, it is considered that the high beta-strand content may contribute to its heat stability.

Biochem Mol Biol Int, 1997 Aug, 42(5), 881 - 9
Isoform-specific monoclonal antibodies against HSP90; Nemoto T et al.; The purpose of this study was to develop monoclonal antibodies (mAbs) that distinguish between the two isoforms of human 90-kDa heat shock protein (HSP90), i.e., HSP90 alpha and beta . Human HSP90 alpha and beta isoforms expressed in Escherichia coli were separately used as antigens for developing the mAbs . Twenty-three and ten mAbs were obtained by immunization of mice with HSP90 alpha and beta, respectively . Among them, ten and three mAbs specifically recognized HSP90 alpha and beta isoforms, respectively, on the criteria of both enzyme-linked immunosorbent and immunoblotting analyses . Immunochemical analysis by use of the mAbs revealed that both of the HSP90 isoforms were present in human cells even under unstressed conditions and that the expression of HSP90 alpha was more strongly induced when the cells were exposed to arsenate . This is the first report of the development of the mAbs discriminating between the two isoforms of HSP90 . The mAbs specific for HSP90 isoforms should be useful for the regulational and functional analyses of HSP90 isoforms.

Acta Med Okayama, 1997 Aug, 51(4), 195 - 206
cDNA cloning, sequence analysis and expression of a mouse 44-kDa nuclear protein copurified with DNA repair factors for acid-depurinated DNA; Nakagawa Y et al.; We purified a 44-kDa nuclear protein from salt-extract of permeable mouse ascites sarcoma cells in an effort to isolate factors involved in the repair of acid-depurinated DNA . It was copurified with a major AP endonuclease (APEX nuclease) by sequential column chromatography then further purified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis as a possible DNA repair support factor . Its partial amino acid sequences were determined, and a cDNA clone for the protein was isolated from a mouse T-cell cDNA library using long degenerate oligonucleotide probes deduced from the amino acid sequence . The complete nucleotide sequence of the cDNA (1.7 kilobases) was determined . Northern hybridization using this cDNA detected two transcripts: 1.8 kb being the major one and 2.6 kb being the minor one . The complete amino acid sequence for the protein predicted from the nucleotide sequence of the cDNA indicates that the 44-kDa protein consists of 394 amino acids with a calculated molecular weight of 43,698 . In tests performed thus far, the recombinant 44-kDa protein expressed in Escherichia coli has not expressed any repair-support activity . It remains to be analyzed whether the protein attains this activity after appropriate posttranslational modifications . Most parts of the 44-kDa protein cDNA and the deduced amino acid sequence were found to be identical to those of the protein p38-2G4, recently reported as a cell cycle-specifically modulated nuclear protein of 38kDa . The p38-2G4 may be a truncated form of the present 44-kDa protein.

Clin Pharmacol Ther, 1997 Aug, 62(2), 171 - 80
Pharmacodynamics of subcutaneous recombinant human interleukin-10 in healthy volunteers; Huhn RD et al.; Interleukin-10 inhibits T-lymphocyte activation and proliferation and lipopolysaccharide-induced monocyte production of proinflammatory cytokines . Fifty-four healthy volunteers received single doses of recombinant human interleukin-10 (1.0, 2.5, 5.0, 10, 25, or 50 micrograms/kg) or placebo by subcutaneous injection (randomized double-blind assignment) . Clinical adverse events were infrequent at doses below 50 micrograms/kg (five of six subjects had mild flu-like syndrome) . Mean serum interleukin-10 concentrations were dose related . The mean terminal-phase half-life ranged from 2.7 to 4.5 hours, and the apparent volume of distribution ranged from 0.70 to 1.35 L/kg . Hematologic changes included transient mild to moderate increases of neutrophil counts, decreases of lymphocyte counts, and a delayed decrease of platelet counts . Recombinant human interleukin-10 significantly suppressed production of the proinflammatory cytokines interleukin-1 beta and tumor necrosis factor-alpha by whole blood stimulated ex vivo with Escherichia coli lipopolysaccharide.

Comput Appl Biosci, 1997 Aug, 13(4), 439 - 44
A computer program for the analysis of protein complex formation; Lay S et al.; MOTIVATION: We needed an efficient way to explore the binding reactions leading to protein complexes of known composition and structure . RESULTS: A new program is described that allows the user to define a set of protein elements and to link these elements into an oligomeric 'ball-and-stick' assembly in a graphical interface . Once the structure of the oligomer has been defined, the program then employs a novel algorithm to deduce the binding reactions and intermediate complexes needed to make the oligomer from its starting protein components . The program also finds the equilibrium state of the system, using either default starting concentrations and Kd values or data supplied by the user.

Hear Res, 1997 Aug, 110(1-2), 95 - 106
Transcripts encoding three types of guanylyl-cyclase-coupled trans-membrane receptors in inner ear tissues of guinea pigs; Krause G et al.; The distribution of membrane-bound guanylyl cyclase (GC) transcription in inner ear tissues of the guinea pig was addressed by a reverse transcription-PCR approach using consensus primers flanking a region of about 630 bp in the intracellular domains in the target sequences . Restriction mapping of such amplificates obtained from cochlear and vestibular specimens permitted us to demonstrate GC-A, GC-B, and GC-C expression by differentiating overall PCR signals . This assay indicated that GC-A was expressed in the cochlea and vestibular organ . PCR products resulting from transcripts of the GC-B gene were obtained at considerably lower abundance than amplificates typical of the GC-A gene . The consensus primer approach with subsequent restriction mapping provided the opportunity to examine at the same time expression of GC-C in the inner ear and revealed the occurrence of GC-C transcripts in both inner ear compartments under investigation . The distribution pattern found by analysing the intracellular domains of membrane-bound guanylyl cyclases was confirmed by demonstrating transcription of the corresponding extracellular receptor domains . In addition, single-strand conformation polymorphism analysis of cDNA amplificates comprising the catalytic domain of guanylyl cyclases also indicated the presence of GC-C expression in the inner ear tissues examined . The GC-C transcripts detected in inner ear tissues appeared to correlate with functional receptor expression, since the production of cyclic GMP catalyzed by cochlear and vestibular specimens was stimulated by 1 microM of heat-stable enterotoxin to 18 and 80% above basal levels, respectively . Thus, GC-C may be involved in the fluid regulation by typical ligands (e.g., the peptide hormone guanylin or the toxins causing travellers' diarrhea), not only in the intestine but also in the organs responsible for hearing and gravitational orientation.

Virus Res, 1997 Aug, 50(2), 159 - 73
High level expression of equine herpesvirus 1 glycoproteins D and H and their role in protection against virus challenge in the C3H (H-2Kk) murine model; Stokes A et al.; N and C-terminal truncated forms of equine herpesvirus 1 (EHV 1) glycoproteins gD and gH were expressed in baculovirus resulting in the production of secreted recombinant proteins . A carboxy-terminal histidine tag was included on each of the genes for protein isolation by nickel affinity chromatography . Recombinant gD was recognized by three gD specific monoclonal antibodies, 20C4, 5H6 and F3132 . F3132 is a conformationally dependent monoclonal antibody with virus neutralizing activity . Expression of gH was confirmed by reacting the protein with the gH peptide specific antiserum R319 . The truncated gD gene was also expressed as a beta-galactosidase fusion protein which was purified from E . coli by nickel affinity chromatography . C3H mice were inoculated with purified recombinant gD or gH or insect cells which had been infected with recombinant baculoviruses . Mice were subsequently challenged with EHV 1 . Purified recombinant baculovirus gD provided the most protection and produced high levels of virus neutralizing antibodies . The gD fusion protein was less effective at protecting mice and insect cells infected with either of the recombinant baculoviruses or purified recombinant gH were poor at conferring protection . The results emphasize the importance of using purified proteins in vaccine formulations and of including EHV 1 gD as a component of a subunit vaccine.

J Appl Microbiol, 1997 Aug, 83(2), 208 - 18
Rapid quantitative analysis of binary mixtures of Escherichia coli strains using pyrolysis mass spectrometry with multivariate calibration and artificial neural networks; Timmins EM et al.; Pyrolysis mass spectrometry (PyMS) and multivariate calibration were used to show the high degree of relatedness between Escherichia coli HB101 and E . coli UB5201 . Next, binary mixtures of these two phenotypically closely related E . coli strains were prepared and subjected to PyMS . Fully interconnected feedforward artificial neural networks (ANNs) were used to analyse the pyrolysis mass spectra to obtain quantitative information representative of level of E . coli UB5201 in E . coli HB101 . The ANNs exploited were trained using the standard back propagation algorithm, and the nodes used sigmoidal squashing functions . Accurate quantitative information was obtained for mixtures with > 3% E . coli UB5201 in E . coli HB101 . To remove noise from the pyrolysis mass spectra and so lower the limit of detection, the spectra were reduced using principal components analysis (PCA) and the first 13 principal components used to train ANNs . These PCA-ANNs allowed accurate estimates at levels as low as 1% E . coli UB5201 in E . coli HB101 to be predicted . In terms of bacterial numbers, it was shown that the limit of detection of PyMS in conjunction with ANNs was 3 x 10(4) E . coli UB5201 cells in 1.6 x 10(7) E . coli HB101 cells . It may be concluded that PyMS with ANNs provides a powerful and rapid method for the quantification of mixtures of closely related bacterial strains.

Mol Cell Probes, 1997 Aug, 11(4), 293 - 6
Acute intermittent porphyria: the in vitro expression of mutant hydroxymethylbilane synthase; Ong PM et al.; Acute intermittent porphyria (AIP) is an inborn error of haem biosynthesis caused by a variety of mutations in the gene coding for hydroxymethylbilane synthase (HMB-S) . The entire coding sequence of this gene, from each of three South African AIP patients, was therefore screened for mutations using chemical cleavage mismatch (CCM) analysis and any changes detected characterized by DNA sequencing . Three single base changes were identified; a G77 to A in exon 3, a C346 to T in exon 8 and a G518 to A in exon 10 . These missense mutations, previously reported to be present in other populations, are known to be responsible for the structurally deleterious amino acid replacements R26H, R116W and R173Q, respectively . The in vitro expression of the enzymes containing these mutations and the subsequent measurement of their specific activities revealed a reduction to approximately 4% of normal activity.

Drug Metab Dispos, 1997 Aug, 25(8), 1001 - 7
Characterization of two human flavin-containing monooxygenase (form 3) enzymes expressed in Escherichia coli as maltose binding protein fusions; Brunelle A et al.; To examine the possibility for drug metabolism polymorphism, adult human flavin-containing monooxygenases (form 3) (EC 1.14.13.8) that differ at one amino acid were expressed in Escherichia coli as maltose binding protein fusions . The cDNA that was first reported during the cloning of adult human flavin-containing monooxygenase was designated the wild type lys158 enzyme . A second cDNA has been identified as a common polymorphism in some human populations and was designated the glu158 enzyme . The cDNA that encodes both enzymes was subcloned into a high yield protein fusion expression system, expressed, and the protein was partially purified by affinity chromatography and characterized for enzyme activity with selective functional substrate probes . N- and S-oxygenation activity of both enzymes was determined with 10-(N,N-dimethylaminopentyl)-2-(trifluoromethyl)phenothiazine and methyl p-tolyl sulfide, respectively . It was found that expression of both lys158 and glu158 enzymes of the human flavin-containing monooxygenase form 3 as fusions with the maltose binding protein resulted in an enzyme that was soluble and greatly stabilized and had a reduced requirement for detergent during enzyme purification and during the assay for activity . Expression of the fusion proteins has allowed the preparation of stable and highly active enzyme at greater purity than was readily possible in the past . With the exception of the stability and solubility characteristics, the physical and chemical properties of lys158 and glu158 maltose binding fusion proteins of human flavin-containing monooxygenase form 3 variants resembled that of flavin-containing monooxygenase enzyme activity associated with human liver microsomes and enzyme isolated from a previous Escherichia coli expression system that lacked the protein fusion . Comparison of the catalytic activity of the two fusion proteins showed that while both forms were active, there were differences in their substrate specificities . Expression of the adult human flavin-containing monooxygenase form 3 as a maltose binding protein has allowed considerable advances over the previously reported cDNA-expressed enzyme systems and may provide the basis for examining the role of the flavin-containing monooxygenase in human xenobiotic or drug metabolism.

J Vet Pharmacol Ther, 1997 Aug, 20(4), 267 - 75
Pharmacokinetics of oxolinic acid in sea-bass, Dicentrarchus labrax (L., 1758), after a single rapid intravascular injection; Poher I et al.; The pharmacokinetics of oxolinic acid was studied in sea-bass (Dicentrarchus labrax) . The fish were kept in seawater at 15.2 degrees C with a 12 h/12 h photoperiod . Oxolinic acid was injected in the caudal vein of anaesthetized sea-bass in a single rapid intravascular administration at a dose of 10 mg/kg of body weight . Plasma concentrations of oxolinic acid were determined using two analytical methods, a classic plate diffusion bioassay using Escherichia coli and a high performance liquid chromatography (HPLC) using solid phase extraction with an internal standard and a U.V . detection . The mean recoveries were 99.6% and 110.8% and determination limits were 0.04 microg/mL and 0.02 microg/mL, for the bioassay and the HPLC respectively . Compared to other fish species, the oxolinic acid was rapidly (absorption half life, t(a1/2) = 0.69 h) distributed to body tissues outside the blood volume (volume of central compartment, Vc = 0.4 L/kg) and presented a large volume of distribution (Vdss = 2.55 L/kg) . Considering its disappearance from the central compartment (rate constant: central-eliminated, k10 = 0.16 h{-1}) and its total body clearance (Cl{t} = 0.066 L/kg x h), the elimination phase of the oxolinic acid in sea-bass was shorter than in trout kept in freshwater, and longer than in salmon in seawater . Consequently, the area under the concentration-time curve (AUC = 157 microg x h/mL) and the mean residence time (MRT = 42 h) were relatively low and short, respectively.

Neurosci Lett, 1997 Aug 1, 231(1), 25 - 8
Lipopolysaccharide rapidly activates K+ channels at the intracellular membrane face of rat cerebral artery smooth muscle cells; Hoang LM et al.; Lipopolysaccharide (LPS) may accumulate inside mammalian cells through endocytotic uptake or during the replication in invasive bacterial strains . However, the effects of intracellular LPS on cell function remain unknown . This study therefore examined the action of intracellularly applied E . coli LPS on large-conductance, Ca2+-dependent K+ channels (BK channels) in the membrane of rat cerebrovascular smooth muscle cells (CVSMCs) . LPS (10-100 microg/ml) rapidly increased the open probability of BK channels when applied to the cytoplasmic face of CVSMC membrane patches . This response was reversible, dose-dependent and reflected an enhanced rate of channel opening in the presence of LPS . These results show for the first time that LPS can alter the gating behavior of ionic channels when applied to the cytoplasmic face of a eukaryotic cell membrane.

J Vasc Surg, 1997 Aug, 26(2), 313 - 8
Molecular cloning of the complementary DNA for an additional member of the family of aortic aneurysm antigenic proteins; Hirose H et al.; PURPOSE: We have purified and partially sequenced a protein from the adventitia of the human aorta (aortic aneurysm antigenic protein 40 kDa; AAAP-40) that has homologies to bovine aortic microfibril-associated glycoprotein (MAGP-36) . It is immunoreactive with immunoglobulin G (IgGs) purified from the serum and aortic wall of patients with abdominal aortic aneurysms . AAAP-40 and MAGP-36 have fibrinogen-like and vitronectin-like motifs . Screening an expression library constructed from human aortic adventitial messenger RNA has resulted in the cloning of three complementary DNAs whose gene products are immunoreactive with immunoglobulin G from patients with abdominal aortic aneurysms . Two strongly resemble each other and have been described separately . The purpose of this article is to report the third clone . METHODS: Messenger RNA from a specimen of human aortic aneurysmal adventitia was reverse-transcribed for insertion into the phagemid Uni Zap XR (Stratagene) . A strain of Escherichia coli, engineered for expression (XL 1-Blue MFR', Stratagene), was transfected, and rabbit antihuman vitronectin antibody as used to identify positive clones . Sequencing of the positive clones was performed by the Core Laboratories at Columbia University . RESULTS: The hypothetical protein of rAAAP-CL4 (clone 4) shares sequence motifs with known microfibril-associated glycoproteins (MAGPs) . The recombinant protein (rAAAP-CL4) is immunoreactive with serum from patients (three of four abdominal aortic aneurysm sera) . In addition, similarities have been detected with immunoglobulins of the kappa family and with a protein from cytomegalovirus that is a potential molecular mimic . CONCLUSIONS: There may several members of a novel family of human aortic autoantigenic proteins implicated in abdominal aortic aneurysm disease.

Biochem Pharmacol, 1997 Aug 1, 54(3), 419 - 24
Mutagenic consequences of the incorporation of 6-thioguanine into DNA; Uribe-Luna S et al.; 6-Thioguanine (S6G) has been used in the treatment of acute leukemias because of its cytotoxic effect on proliferating leukemic cells . The cytotoxicity of S6G is thought to derive from its incorporation into DNA in place of guanine . The deoxyribonucleoside triphosphate of S6G, SdGTP, is a good substrate for bacterial and human DNA polymerases (Ling et al., Mol Pharmacol 40: 508-514, 1991) . Since SdGTP was observed to misincorporate in place of adenine at a greater frequency than did dGTP, it appeared plausible that this analog could produce more subtle effects (mutations) due to mispairing with thymine . To assess whether such mutations occur, SdGTP was incorporated into the lacI gene of phage M13lacISaXb in reactions that omitted dGTP (-G) or dATP (-A) . LacI mutation frequency was determined by beta-galactosidase colorimetric staining (inactivation of the lac repressor results in blue plaques in the absence of inducer) . When a high concentration of SdGTP (24 microM) was used in the DNA polymerase reaction, phage infectivity was inhibited . When a relatively low concentration (2.4 nM) was added to the -G and -A reactions, mutagenic effects were observed . DNA sequencing of mutant progeny arising from the -G + S6G reaction revealed C-to-T base transitions and some C-to-A transversions . Similarly, the presence of SdGTP in the -A reactions led to mutants with T-to-C transitions . No insertions or deletions were observed . These data indicate that mispairing of S6G with thymine leads to mutagenic effects in this assay.

Biochem Pharmacol, 1997 Aug 1, 54(3), 381 - 90
Expression and purification of recombinant human N-formyl-L-leucyl-L-phenylalanine (FMLP) receptor: generation of polyclonal antibody against FMLP receptor; Lala A et al.; The recombinant formyl peptide receptor has been successfully expressed and purified, utilizing an Escherichia coli expression system . Purification of formyl peptide receptor was performed using gel filtration chromatography and affinity chromatography, and the purified protein migrated at an apparent molecular mass of 36,000 Da . The purified recombinant receptor retained functional activity as determined by a ligand binding assay . The yield of the recombinant purified receptor was approximately 1 mg/2 L of culture, and the binding activity was determined to be approximately 8 nM, which suggests the conclusion that glycosylation does not affect significantly ligand binding of the N-formyl-L-leucyl-L-phenylalanine (FMLP) receptor molecule . The recombinant receptor protein yield was found to be significantly higher than that obtained from neutrophils . The purified recombinant receptor was then utilized to generate antibody against the same . The reaction of the antibody against recombinant formylpeptide receptor and against native formylpeptide receptor on neutrophils was confirmed by western blot analysis and flow cytometric analysis, respectively . The antibody was also used successfully to detect recombinant formylpeptide receptor expression on transfected 293 cells . These results describe for the first time the expression, purification, and characterization of recombinant FMLP receptor with ligand binding activity and the generation of polyclonal antibody against the same . This work also provides a foundation for future biophysical studies of the FMLP receptor molecule, which have not been possible until now.

Am J Physiol, 1997 Aug, 273(2 Pt 2), R777 - 83
Febrile responsiveness of vagotomized rats is suppressed even in the absence of malnutrition; Romanovsky AA et al.; The repeatedly observed attenuation of fever in vagotomized rats has been accepted as evidence of an essential role of vagal afferents in the transduction of pyrogenic signals from the periphery to the brain . If, however, the general condition of a vagotomized animal is poor (the usual case) and accompanied by malnutrition and body mass loss (common complications of vagotomy), the febrile responsiveness can be suppressed not because of the lack of vagal afferentation, but rather secondarily to a malnutrition-associated thermogenic incompetence . In the present study, we addressed this dilemma . Male Wistar rats were subjected to subdiaphragmatic vagotomy (or sham surgery) and, 24 days later, catheterized in the jugular vein . Postsurgically, the rats were closely watched and fed highly palatable food . Their febrile responsiveness {colonic (Tc) and tail skin (Tsk) temperature responses} to Escherichia coli lipopolysaccharide (LPS: 1 microgram/kg i.v.) was tested on day 27 postvagotomy . To verify the completeness of vagotomy, each rat was food deprived for 24 h and then euthanized; its stomach's evacuatory function was assessed by weighing the organ . One month postsurgery, both food consumption and body mass of the vagotomized rats (33 +/- 2 g/day and 313 +/- 4 g, respectively) were similar to the control values (30 +/- 1 g/day and 315 +/- 8 g) . In the sham rats, LPS induced a monophasic Tc rise of 0.5 +/- 0.3 degree C at 70 min postinjection (peak), preceded by a fall in Tsk . Neither this Tsk fall (tail skin vasoconstriction) nor the resultant fever occurred in the vagotomized rats; at 70 min, Tc change was -0.1 +/-0.1 degree C . The gastric mass (4.1 +/- 0.5 g in the vagotomized vs . 1.8 +/- 0.1 g in sham rats) indicated the effectiveness of vagotomy . In sum, although the vagotomy-associated malnutrition was successfully prevented with special perioperative care, the vagotomized animals still did not respond to LPS with fever . Malnutrition is, therefore, unlikely to constitute the main reason of the febrile irresponsiveness of vagotomized rats.

Am J Physiol, 1997 Aug, 273(2 Pt 1), E433 - 7
Tumor necrosis factor activity of pancreatic islets; Leeper-Woodford SK et al.; Tumor necrosis factor (TNF) is involved in the pathogenesis of acute sepsis-induced organ injury and has been implicated as a mediator of metabolic alterations observed during sepsis . Pancreatic islet cell function may be significantly compromised during sepsis or endotoxemia, and sepsis also increases plasma levels of epinephrine, a modifier of islet insulin secretion . We proposed that islets exposed to bacterial lipopolysaccharide (LPS) produce TNF and that epinephrine attenuates islet secretory activity . We monitored the effects of LPS and epinephrine on TNF and insulin activity of isolated Wistar-Furth rat islets (pancreas digested with collagenase, islets isolated using Ficoll gradients; n = 4 islet populations, each with 632 +/- 11 islets/2.5 ml culture medium) . Islets were incubated (37 degrees C, 5% CO2) 3 days . LPS (Escherichia coli, 1 microgram/ml) and epinephrine (14 micrograms/ml) were added to the islets, and incubations were continued for 1-4 h . Glucose (Beckman Glucose Analyzer), insulin (radioimmunoassay), and TNF (L929 cytotoxicity assay) were measured in the islet medium samples at 1- to 4-h time points . In the conditioned medium, glucose decreased (P < 0.05), insulin increased (P < 0.05), and exposure to LPS did not alter these levels {P = not significant (NS)} but did increase TNF activity by 400% (P < 0.05) . Epinephrine reduced insulin by 38-43% (P < 0.05) and TNF by 20-25% (P < 0.05) but had no effect on glucose levels (P = NS) . We conclude that insulin is secreted from isolated islets and that exposure to LPS acutely increases islet-derived TNF activity, whereas epinephrine modifies TNF and insulin secretion of rat pancreatic islets.

Am J Physiol, 1997 Aug, 273(2 Pt 1), E328 - 35
Involvement of capsaicin-sensitive nerves in regulating the hormone and glucose metabolic response to endotoxin; Morgan AE et al.; This study investigated the role that sensory nerves play in mediating the hormone and glucose metabolic response to endotoxin {lipopolysaccharide (LPS)} . Adult rats were pretreated subcutaneously with capsaicin to selectively destroy primary sensory afferent nerve fibers . Ten days later, {3-3H}glucose was infused intravenously to assess whole body glucose flux before and after the intravenous injection of Escherichia coli LPS (100 micrograms/100 g body wt) . Control animals responded to LPS with characteristic increases in the plasma concentration of glucose (91%) and lactate (threefold) and elevations in the rates of glucose appearance and disappearance (77%) . In capsaicin-treated rats, the maximal LPS-induced increase in these parameters was attenuated by 50-60% . In addition, these animals were hypoglycemic at the conclusion of the experiment . Control animals demonstrated early and sustained elevations in circulating levels of corticosterone, glucagon, and catecholamines . In contrast, the early LPS-induced elevation in epinephrine and norepinephrine, and to a lesser extent glucagon, was completely absent or greatly impaired by capsaicin pretreatment . In a separate study, the epinephrine-induced increase in glucose flux was blunted by 75% in capsaicin-treated rats . These data indicate that sensory afferent neurons play a critical role in the early secretory response of glucagon and catecholamines, the maintenance of tissue catecholamine responsiveness, and the stimulation of glucose production after LPS.

Photochem Photobiol, 1997 Aug, 66(2), 214 - 23
Photoreaction of new psoralen analogs with DNA: sequence and mutation specificity in the Escherichia coli lacZ gene; Collet M et al.; New thio- and seleno-analogs of psoralen were synthesized and analyzed for their photoreactivity toward DNA . Using oligonucleotides of defined sequence, we first showed that these derivatives predominantly generated interstrand crosslinks at 5'-TpA sites . We also observed a surprisingly high reactivity of 7H-thiopyrano{3,2-f}{1}benzofuran-7-one (PSO{O-S}) with the BamHI and PstI oligomers, giving rise to the formation of crosslinks at 5'-ApT sites and of the thymidine-psoralen-cytosine type . Next, the sequence specificity in the photochemical binding of all the compounds was investigated in two DNA fragments encompassing the lacZ gene of Escherichia coli, using the T4 DNA polymerase sequencing methodology . Resulting maps demonstrated that thio- and seleno-analogs of psoralen preferentially photoreacted with thymine and cytosine residues . The AT-rich sequences proved to be particularly reactive sites as did adjacent thymines, especially at C-surrounding residues . Likewise, photoaddition at cytosines in CA/AC context was observed . It was highly significant that all of the derivatives exhibited similar sequence specificities with only minor differences . However, PSO(O-S) differed from the other heteropsoralens . Photoadducts occurred with a higher frequency at AC and CA dinucleotides, and new sites were detected . A comparison with 8-methoxypsoralen photobinding is also reported . Finally, the mutagenic consequences of photoadducts induced in M13mp19 DNA by PSO(O-S) were determined in a forward system that detects all classes of mutagenic events . The high phototoxicity exhibited by PSO(O-S) could be attributed to crosslinks, and the comparison of the observed mutational specificity with the photoadduct distribution within the same gene showed that mutations were targeted at potential monoadduct sites where photolesions were detected in our footprinting experiments.

Plant Physiol, 1997 Aug, 114(4), 1169 - 75
High-yield expression of pea thioredoxin m and assessment of its efficiency in chloroplast fructose-1,6-bisphosphatase activation; Lopez Jaramillo J et al.; A cDNA clone encoding pea (Pisum sativum L.) chloroplast thioredoxin (Trx) m and its transit peptide were isolated from a pea cDNA library . Its deduced amino acid sequence showed 70% homology with spinach (Spinacia oleracea L.) Trx m and 25% homology with Trx f from pea and spinach . After subcloning in the Ndel-BamHI sites of pET-12a, the recombinant supplied 20 mg Trx m/L . Escherichia coli culture . This protein had 108 amino acids and was 12,000 D, which is identical to the pea leaf native protein . Unlike pea Trx f, pea Trx m showed a hyperbolic saturation of pea chloroplast fructose-1,6-bisphosphatase (FBPase), with a Trx m/ FBPase molar saturation ratio of about 60, compared with 4 for the Trx f/FBPase quotient . Cross-experiments have shown the ability of pea Trx m to activate the spinach chloroplast FBPase, results that are in contrast with those in spinach found by P . Schurmann, K . Maeda, and A . Tsugita ({1981} Eur J Biochem 116: 37-45), who did not find Trx m efficiency in FBPase activation . This higher efficiency of pea Trx m could be related to the presence of four basic residues (arginine-37, lysine-70, arginine-74, and lysine-97) flanking the regulatory cluster; spinach Trx m lacks the positive charge corresponding to lysine-70 of pea Trx m . This has been confirmed by K70E mutagenesis of pea Trx m, which leads to a 50% decrease in FBPase activation.

Carcinogenesis, 1997 Aug, 18(8), 1609 - 15
Sequence context is an important determinant in the mutagenic potential of 1,N6-ethenodeoxyadenosine (epsilonA): formation of epsilonA basepairs and elongation in defined templates; Litinski V et al.; Many laboratories have obtained data on mutagenicity of modified bases in naturally occurring DNA sequences . It has often been noted that mutation is favored in certain sequence contexts, sometimes termed 'hot spots' . This approach to the contribution of neighboring sequences does not permit a systematic study of both the qualitative and quantitative mutational frequencies . In the present experiments we have chosen to use the exocyclic adduct, 1,N6-etheno A (epsilonA), site-specifically placed in a defined 25-mer oligonucleotides in which epsilonA is flanked by differing 5' and 3' tandem bases . Mutation was assessed using an in vitro replication assay and five polymerases of varying fidelity . The relevant central sequences were 3' --> 5' -CC-epsilonA-CC-, -GG-epsilonA-GG-, -TT-epsilonA-TT-, -AA-epsilonA-AA-, -GG-epsilonA-TT-, -TT-epsilonA-AA-, -AT-epsilonA-TT- and -TA-epsilonA-TA- . Using the Klenow fragment (Kf) (exo+ or exo-) of E . coli Pol I, it was found the epsilonA is an ambiguous base and, with varying efficiencies, all four dNTPs could be inserted opposite epsilonA in all sequences . However, only 3' --> 5' -TT-epsilonA-TT-, -GG-epsilonA-TT- and -AT-epsilonA-TT- were fully extended to a significant extent . The only sequences essentially blocked at the position of epsilonA were -AA-epsilonA-AA- and -TT-epsilonA-AA- . The others were intermediate . When replication was performed with Sequenase, MMLV RT or HIV RT, different patterns were observed, in which replication terminated one base prior to epsilonA, at epsilonA, or one base after epsilonA without further extension . In favored sequences, using the Klenow fragment, an epsilonA x N pair could be extended to form normal basepairs . No extension could be demonstrated in sequences in which tandem adenines were 5' to epsilonA . Kinetic data showed that two of the epsilonA x N pairs, epsilonA x A and epsilonA x C, could form at 10 microM or less dNTP . Which bases were preferentially inserted opposite epsilonA was a function of the flanking bases . Under the kinetic conditions used, epsilonA x T did not form even at 1 mM dTTP . These results indicate that the chemical structure of an adduct is not the only determinant of mutagenic efficiency . It is likely that the effect of the adduct on replication is due to the changes in the structural environment conferred by the flanking bases.

Carcinogenesis, 1997 Aug, 18(8), 1561 - 7
Nitrosated peptides and polyamines as endogenous mutagens in O6-alkylguanine-DNA alkyltransferase deficient cells; Sedgwick B; Mutants of Escherichia coli and Saccharomyces cerevisiae that lack O6-alkylguanine-DNA alkyltransferase activities have increased spontaneous mutation rates, indicating the presence of a cellular metabolite that can alkylate DNA . Bacterially catalysed nitrosation has been implicated previously in producing the endogenous alkylating agent(s) . Here, nitrosated polyamines and azaserine, a model compound for nitrosated peptides, are shown to be mutagenic to E . coli ada ogt mutants deficient in O6-alkylguanine-DNA alkyltransferase activity . The mutagenicity of azaserine may be explained by its ability to methylate DNA, whereas nitrosated spermidine causes DNA damage that is susceptible to both nucleotide excision repair and O6-alkylguanine-DNA alkyltransferase activity, which indicates the generation of more bulky DNA adducts . Nitrosated peptides and polyamines are therefore potential endogenous mutagens that are harmful particularly in O6-alkylguanine-DNA alkyltransferase deficient cells.

J Virol Methods, 1997 Aug, 67(1), 93 - 101
Characterization of the double-stranded RNA genome segment S3 of avian reovirus; Yin HS et al.; The double-stranded RNA genome segment S3 of avian reovirus (ARV) S1133 was cloned following polyadenylation of both strands and cDNA synthesis of S3 RNA . The complete segment S3 nucleotide sequence was determined . S3 is 1196 base pairs long with one long open reading frame (ORF) . The ORF possesses the AUG initiation codon in an optimum context for translation and starts at the first initiation codon (residue 24) and extends for 367 codons, sufficient to encode a protein of the same size as the known S3 gene product, protein sigmaB, one of the major outer capsid proteins of avian reovirus (Mr 41471) . Protein sigmaB was subsequently expressed in Escherichia coli . The expressed protein sigmaB was indistinguishable from virion protein sigmaB as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot assay, and N-terminal amino acid sequencing of several peptides generated by Staphyloccus aureus V8 protease digestion . ARV S3 genome segment possesses a pentanucleotide UCAUC at the 3'-terminus of its plus strand . The pentanucleotide sequence is common to the other genome segment S1 of ARV and to ten genome segments of mammalian reovirus at the 3'-terminus of their plus strands . Amino acid sequence analysis revealed that ARV sigmaB does not contain a repeated basic amino acid motif as do the three serotypes of mammalian reovirus . The results of amino acid sequencing suggest that the most susceptible cleavage sites of sigmaB to V8 protease are located in a hydrophilic area between amino acids 95 and 140.

Development, 1997 Aug, 124(16), 3099 - 109
The role of the msh homeobox gene during Drosophila neurogenesis: implication for the dorsoventral specification of the neuroectoderm; Isshiki T et al.; Development of the Drosophila central nervous system begins with the delamination of neural and glial precursors, called neuroblasts, from the neuroectoderm . An early and important step in the generation of neural diversity is the specification of individual neuroblasts according to their position . In this study, we describe the genetic analysis of the msh gene which is likely to play a role in this process . The msh/Msx genes are one of the most highly conserved families of homeobox genes . During vertebrate spinal cord development, Msx genes (Msx1-3) are regionally expressed in the dorsal portion of the developing neuroectoderm . Similarly in Drosophila, msh is expressed in two longitudinal bands that correspond to the dorsal half of the neuroectoderm, and subsequently in many dorsal neuroblasts and their progeny . We showed that Drosophila msh loss-of-function mutations led to cell fate alterations of neuroblasts formed in the dorsal aspect of the neuroectoderm, including a possible dorsal-to-ventral fate switch . Conversely, ectopic expression of msh in the entire neuroectoderm severely disrupted the proper development of the midline and ventral neuroblasts . The results provide the first in vivo evidence for the role of the msh/Msx genes in neural development, and support the notion that they may perform phylogenetically conserved functions in the dorsoventral patterning of the neuroectoderm.

Am J Respir Cell Mol Biol, 1997 Aug, 17(2), 173 - 80
Expression of nitric oxide synthases and GTP cyclohydrolase I in the ventilatory and limb muscles during endotoxemia; Hussain SN et al.; Nitric oxide synthases (NOS) convert L-arginine to nitric oxide in the presence of tetrahydrobiopterin (BH4) . Two constitutive isoforms of NOS exist in normal skeletal muscle fibers, however, the existence of a third, the inducible isoform (iNOS), has never been detected in these fibers in vivo . Therefore, we assessed the influence of in vivo endotoxemia on skeletal muscle expression of constitutive and inducible NOS isoforms and GTP cyclohydrolase I, the rate limiting enzyme of BH4 synthesis . Two groups of rats were infused i.p . either with E . coli endotoxin (20 mg/kg, LPS group) or saline (saline group) . Animals were killed 6 h later and the ventilatory and limb muscles were quickly frozen . Endotoxin infusion elicited a significant rise in NOS activity of the diaphragm, intercostal, soleus and gastrocnemius muscles . Reverse transcription-polymerase chain reaction (RT-PCR) on muscle total RNA detected very low expression of iNOS and GTP cyclohydrolase I mRNA in the saline group, but significant upregulation of both enzymes was found in the ventilatory and limb muscles of the LPS group . Immunoblotting detected no iNOS protein in the saline group but a significant iNOS protein expression was found in the diaphragm, intercostal and soleus muscles and to a lesser extent, in the gastrocnemius of the LPS group . Endotoxemia was also associated with increased protein expression of constitutive NOS isoforms mainly in the diaphragm and to lesser extent in the intercostal, gastrocnemius and soleus muscles . We conclude that in vivo exposure to endotoxin leads to differential induction of both iNOS and GTP cyclohydrolase I in the ventilatory and limb muscles.

Genes Dev, 1997 Aug 1, 11(15), 2012 - 21
The response to extracytoplasmic stress in Escherichia coli is controlled by partially overlapping pathways; Connolly L et al.; The activity of the alternate sigma-factor sigmaE of Escherichia coli is induced by several stressors that lead to the extracytoplasmic accumulation of misfolded or unfolded protein . The sigmaE regulon contains several genes, including that encoding the periplasmic protease DegP, whose products are thought to be required for maintaining the integrity of the cell envelope because cells lacking sigmaE are sensitive to elevated temperature and hydrophobic agents . Selection of multicopy suppressors of the temperature-sensitive phenotype of cells lacking sigmaE revealed that overexpression of the lipoprotein NlpE restored high temperature growth to these cells . Overexpression of NlpE has been shown previously to induce DegP synthesis by activating the Cpx two-component signal transduction pathway, and suppression of the temperature-sensitive phenotype by NlpE was found to be dependent on the Cpx proteins . In addition, a constitutively active form of the CpxA sensor/kinase also fully suppressed the temperature-sensitive defect of cells lacking sigmaE . DegP was found to be necessary, but not sufficient, for suppression . Activation of the Cpx pathway has also been shown to alleviate the toxicity of several LamB mutant proteins . Together, these results reveal the existence of two partially overlapping regulatory systems involved in the response to extracytoplasmic stress in E . coli.

Biochem J, 1997 Aug 1, 325 ( Pt 3), 645 - 51
Molecular characterization and expression of Onchocerca volvulus glutathione reductase; Muller S et al.; Glutathione metabolism represents a potential target for anti-parasite drug design . The central role of glutathione reductase (GR) in maintenance of the thiol redox state and in anti-oxidative defence has to be evaluated in more detail in order to establish the essential function of this enzyme for the survival of the filarial parasite Onchocerca volvulus . The O . volvulus GR (OvGR) gene was cloned and sequenced . The gene is composed of 13 exons and 12 introns and spans 4065 bp . The first intron is located within the 5'-untranslated region of the gene, 16 nucleotides upstream of the translation initiation codon . Southern-blot analysis and structural characterization of the genomic sequence indicate that OvGR is encoded by a single-copy gene . Isolation of various cDNA clones revealed a polymorphism of polyadenylation initiation with no consensus polyadenylation sites in any of the cDNAs analysed . The entire cDNA is 1977 bp long and carries the nematode-specific spliced leader sequence SL1 at its 5' end, 236 nucleotides upstream of the first in-frame methionine . The cDNA codes for a polypeptide of 462 amino acids with 53.5% sequence identity with human GR (HsGR) . A total of 18 out of 19 residues contributing to glutathione binding are identical in OvGR and HsGR . However, one of the arginine residues (Arg-224 in HsGR) involved in discrimination between NADPH and NADH in all known GRs is substituted by tryptophan (Trp-207 in OvGR) . The coding region of OvGR was expressed in Escherichia coli as a histidine-fusion protein, and it was established that the parasite protein still favours the binding of NADPH (Km 10.9 microM) over NADH (Km 108 microM) . The histidine-fusion protein has a subunit size of 54 kDa and is active as a homodimer of 110 kDa.

Lipids, 1997 Aug, 32(8), 805 - 9
Recombinant antibody fragments that detect enoyl acyl carrier protein reductase in Brassica napus; Ziegler A et al.; Purified Brassica napus enoyl acyl carrier protein reductase (ENR) was used to select specific antibodies from a library of antibody fragments, single-chain Fv (scFv), displayed on filamentous phage . Analysis of the selected clones by BstNI fingerprinting and nucleotide sequencing showed that the scFv were derived from three different human VH germline genes . The binding specificities were confirmed by Western blots and ELISA . The scFv preparations reacted with B . napus ENR, but not with beta-keto reductase, nor enoyl reductase from Escherichia coli . Analysis of fragments generated by CNBr treatment indicates that the scFv 3.13 recognized an epitope located within the N-terminal 80 amino acids of the enzyme molecule . The scFv were used to detect ENR directly in extracts of B . napus seeds.

Trends Biochem Sci, 1997 Aug, 22(8), 307 - 12
Distinct and specific functions of cGMP-dependent protein kinases; Lohmann SM et al.; cGMP-dependent protein kinases I and II conduct signals from widespread signaling systems . Whereas the type I kinase mediates numerous effects of natriuretic peptides and nitric oxide in cardiovascular cells, the type II kinase transduces signals from the Escherichia coli heat-stable enterotoxin, STa, and from the endogenous intestinal peptide, guanylin, stimulating Cl- conductance of the cystic fibrosis transmembrane conductance regulator (CFTR) . Although the two kinases may be interchangeable for several functions, CFTR regulation specifically requires the type II kinase.

Hum Pathol, 1997 Aug, 28(8), 929 - 37
Newly marketed tissue markers for malignant mesothelioma: immunoreactivity of rabbit AMAD-2 antiserum compared with monoclonal antibody HBME-1 and a review of the literature on so-called antimesothelioma antibodies; Donna A et al.; A complementary DNA (cDNA) library was constructed from a human malignant mesothelioma (MM) cell line and a cDNA fragment encoding for a cytoplasmic mesothelial protein recognized by the polyclonal antibody AMAD-1 was then cloned and expressed in Escherichia coli . The purified recombinant protein was used to raise a novel antibody, named AMAD-2, in rabbits . This antibody reacted with normal mesothelium and most MM (15 of 17) on paraffin sections and featured a cytoplasmic labeling . Conversely, AMAD-2 immunostaining of normal and tumor tissues from body sites other than serosal membranes was limited with respect to the proportion of positive specimens and usually less conspicuous than in MM . AMAD-2 immunoreactivity was subsequently compared with staining for HBME-1, another newly marketed antimesothelial monoclonal antibody, concerning the ability to distinguish pleural MM from metastatic pleural tumors of epithelial type . A granular cytoplasmic immunoreactivity for AMAD-2 was present in 50% or more of tumor cells in all 84 MM, regardless of histological type, but also in 3 (7%) of 42 pleural metastases, albeit only focally . HBME-1 was shown in 63 of 66 epithelial MM and in the epithelial component of all 8 mixed MM, with a prevailingly membranous pattern, usually homogeneous and strong, whereas none of the 10 sarcomatous MM was positive . HBME-1 was also expressed in 6 (14%) of 42 pleural metastases in a cytoplasmic or membranous pattern . Compared with HBME-1, AMAD-2 showed a higher degree of specificity and sensitivity for MM . AMAD-2 still proved to be superior to HBME-1, also when sarcomatoid MM were excluded from the assessment . This finding supports the view that AMAD-2 is an antibody highly, although not entirely, specific for the mesothelial lineage, whereas HBME-1 is probably a cell marker more closely related to the epithelial differentiation of MM . Therefore, AMAD-2 is preferable as a positive tissue marker to be incorporated in the optimal immunohistochemical panel for the diagnosis of MM.

Protein Expr Purif, 1997 Aug, 10(3), 331 - 9
Amino-terminal charge affects the periplasmic accumulation of recombinant heregulin/EGF hybrids exported using the Escherichia coli alkaline phosphatase signal sequence; Campion SR et al.; An Escherichia coli expression system that exploits the bacterial alkaline phosphatase (PhoA) signal sequence to translocate recombinant human epidermal growth factor (hEGF) to the periplasm was used to evaluate how changes in the composition and sequence of amino acids near the PhoA-hEGF junction influence the periplasmic accumulation of recombinant protein . A series of chimeric structural genes was generated by in vitro replacement of hEGF sequence with analogous segments from the EGF-like domain of human heregulin (HRG), significantly altering the electrostatic character of the amino-terminal region of the mature protein . Quantitation of HRG/EGF protein in E . coli periplasmic extracts, by RP-HPLC, showed a fourfold decrease after one of two acidic residues located in the amino-terminal region of the mature hEGF, near the PhoA junction, was replaced . An additional threefold decrease was observed when the second acidic residue was replaced with a positively charged lysine . Further extension of the amino-terminal HRG sequence, beyond the first six residues, resulted in net neutralization of a more distant EGF acidic residue with no additional effect on protein yield . The importance of having a negatively charged group in the amino-terminal region of the mature protein was confirmed when insertion of an aspartic acid near the amino-terminus of two poorly expressed hybrid protein sequences resulted in a five- to eightfold increase in their recovery from the periplasm . This study demonstrates the importance of having negatively charged residues near the fusion junction of recombinant proteins expressed in E . coli using the PhoA signal sequence for protein export.

Protein Expr Purif, 1997 Aug, 10(3), 293 - 300
High-level production and isotope labeling of snake neurotoxins, disulfide-rich proteins; Drevet P et al.; The aim of this work was to produce and to label snake neurotoxins, disulfide-rich proteins . A mutant of a snake toxin, erabutoxin a, was used as a model . Its N-terminal part was fused to ZZ, a synthetic IgG-binding domain of protein A (B . Nilsson et al., 1987, Protein Eng . 1, 107-113), thus preventing degradation in the bacterial cytoplasm and providing a simple affinity-purification method on IgG Sepharose . A soluble fusion protein was obtained with a yield of 60 mg/L, corresponding to 20 mg/L toxin . The toxin moiety was folded on the column while the hybrid was still bound . The oxidoreducing conditions for the refolding were optimized and were found to be oxidative but with a need for reducing molecules . The concentration of the hybrid bound to the column could be increased up to 3.3 mg/ml without significantly altering the folding process . CNBr cleavage of the fusion protein followed by a purification step yielded about 2 mg of biologically active toxin mutant per gram of dry cell weight . This procedure was applied to produce 55 mg of a toxin uniformly labeled with 15N.

Crit Care Med, 1997 Aug, 25(8), 1371 - 7
Dobutamine improves gastrointestinal mucosal blood flow in a porcine model of endotoxic shock; Neviere R et al.; OBJECTIVE: To test the hypothesis that saline solution plus dobutamine increases gastrointestinal mucosal perfusion better than saline solution alone in a model of endotoxic shock . DESIGN: Prospective, randomized, unblinded study . SETTING: Animal research laboratory affiliated with a university teaching hospital . SUBJECTS: Twelve female pigs, weighing 30 to 32 kg . INTERVENTIONS: Animals were anesthetized, and their lungs were mechanically ventilated . Catheters were inserted into the right atrium, pulmonary artery, and carotid artery for blood sampling and blood pressure and cardiac output measurements . A tonometer and a laser Doppler probe were placed in the lumen of the stomach and the ileum for determination of mucosal acid-base status and measurement of mucosal blood flow . Group 1 animals (n = 6) received an infusion (T = 0 min) of 150 mcirog/kg Escherichia coli endotoxin and normal saline solution (0.3 mL/kg/min) . Group 2 animals (n = 6) received an infusion of endotoxin and were resuscitated with the same method as used in group 1, but an infusion of dobutamine (5 microg/kg/min) was begun at T = 60 mins, and continued for the duration of the experiment . MEASUREMENTS AND MAIN RESULTS: Both experimental regimens produced shock, with decreased mean arterial pressure and systemic vascular resistance, without change in cardiac output and oxygen delivery . Endotoxin plus saline infusion decreased gastrointestinal mucosal blood flow to <60% of baseline and decreased gastrointestinal pH . In contrast, gastrointestinal mucosal blood flow returned to baseline values, and intramucosal pH tended to normalize by the end of the saline solution plus dobutamine resuscitative protocol . CONCLUSION: Compared with saline solution alone, saline solution plus dobutamine increased blood flow to the gastrointestinal mucosa, and may have partially improved oxygenation.

Alcohol Clin Exp Res, 1997 Aug, 21(5), 779 - 83
Acute ethanol administration modulates leukocyte actin polymerization in endotoxic rats; Zhang P et al.; The effects of acute ethanol intoxication with or without endotoxemia were studied on chemoattractant-initiated actin polymerization in circulating and liver-recruited neutrophils {polymorphonuclear leukocytes (PMNs)} and Kupffer cells of age-matched male and female rats . In female rats, F-actin content in response to f-Met-Leu-Phe in circulating PMNs was significantly upregulated by ethanol+endotoxin (ET) treatment relative to saline+ET-treated animals . Male rats did not show this upregulated response . In liver-recruited PMNs, the F-actin response did not change significantly due to ethanol + ET treatment in cells of either gender . The actin polymerization response in Kupffer cells was smaller than in liver sequestered PMNs and also exhibited a gender difference . In Kupffer cells of female rats, only ethanol+ET treatment elicited an F-actin response to f-Met-Leu-Phe, whereas in cells of male rats similar increases in F-actin content were observed in both saline+ET and ethanol+ET groups . We conclude that ethanol-enhanced actin polymerization in rats is associated with gender differences . This may be one of the underlying mechanisms resulting in upregulated leukocyte activation in acutely ethanol-intoxicated endotoxic animals.

Alcohol Clin Exp Res, 1997 Aug, 21(5), 773 - 8
Acute ethanol intoxication suppresses the pulmonary inflammatory response in rats challenged with intrapulmonary endotoxin; Zhang P et al.; The effects of acute ethanol intoxication on the functional activities of circulating and lung-recruited polymorphonuclear leukocytes (PMNs) and alveolar macrophages (AMs) were determined in rats challenged with intratracheal endotoxin to elucidate the mechanisms underlying the defects of pulmonary host defenses caused by acute ethanol intoxication . Acute ethanol Intoxication was induced by an intraperitoneal injection of 20% ethanol at a dose of 5.5 g of ethanol/kg . The control animals were injected with an equal amount of saline . Thirty min after intraperitoneal injection, rats were challenged with intratracheal endotoxin (300 micrograms/kg in 0.5 ml of saline) or saline . The rats were killed 3 h after intratracheal injection . CD11b/c expression on PMNs and phagocytosis and hydrogen peroxide generation of PMNs and AMs were determined by flow cytometry . Cytokine-Induced neutrophil chemoattractant (CINC) level in bronchoalveolar lavage fluid was measured with a specific ELISA . Intratracheal endotoxin caused a significant PMN recruitment into the lung in control animals . Acute ethanol intoxication completely suppressed the endotoxin-induced pulmonary recruitment of PMNs . Pulmonary-recruited PMNs exhibited a significant upregulation (8-fold) of CD11b/c expression when compared with circulating PMNs . This upregulation of CD11b/c expression was abolished by ethanol intoxication . Ethanol intoxication suppressed hydrogen peroxide generation by AMs and lung-recruited PMNs, and the phagocytosis of circulating PMNs . In contrast, acute ethanol intoxication did not affect pulmonary CINC production . These data indicate that the antiinflammatory effects of alcohol seem to be primarily based on the effects of ethanol on the PMNs themselves and not on the generation of certain chemotactic stimuli . In addition to the impairment of PMN recruitment, the suppression of AM and PMN activities also contributes to the mechanisms underlying ethanol-induced defects of pulmonary host defenses.

J Gen Virol, 1997 Aug, 78 ( Pt 8), 2095 - 9
Mapping of the RNA-binding domain of the cucumber mosaic virus movement protein; Vaquero C et al.; A series of in-frame deletion mutants was used to identify a domain within the 3a protein of cucumber mosaic virus (CMV) that is required for RNA-binding activity . Deletions in the 3a gene were generated by PCR and restriction digestion, and the resulting mutated 3a sequences were cloned either in pT7-7 or in pGEX-5X3 expression vectors . The mutated 3a proteins or fusions with glutathione S-transferase (GST) were expressed in E . coli, purified, and their nucleic acid-binding activities analysed by photochemical UV cross-linking assays using digoxigenin-UTP-labelled RNA probes . Comparative analyses of seven mutated 3a proteins obtained from inclusion bodies and eight GST fusion proteins revealed that there is an RNA-binding domain located between amino acids 174 and 233 . This RNA-binding domain is able to bind single-stranded RNA out of the context of the complete 3a movement protein and is highly conserved within both subgroups of CMV.

J Gen Virol, 1997 Aug, 78 ( Pt 8), 2049 - 53
Mosaic hepatitis B virus core particles allow insertion of extended foreign protein segments; Koletzki D et al.; Because of its particular immunological properties, the core protein of hepatitis B virus (HBcAg) has become one of the favoured 'virus-like particles' for use as a carrier of foreign epitopes . A new strategy to construct core particles presenting extended foreign protein segments was established based on the introduction of a linker containing a translational stop codon between sequences encoding a C-terminally truncated HBcAg (HBcAg delta) and a foreign protein sequence . Expression in an Escherichia coli suppressor strain allowed the simultaneous synthesis of both HBcAg delta and a read-through fusion protein containing a part of the hantavirus nucleocapsid protein . After purification, the presence of core-like mosaic particles with HBc and hantavirus antigenicity was demonstrated by electron microscopy and immunological tests . This strategy of partial stop codon suppression should improve the use of HBcAg as a carrier of foreign epitopes by allowing insertion of long foreign sequences into particle-forming proteins . The resulting mosaic particles should be of general interest for further vaccine developments.

Curr Opin Struct Biol, 1997 Aug, 7(4), 537 - 42
Helix packing in polytopic membrane proteins: the lactose permease of Escherichia coli; Kaback HR et al.; Recent advances in protein engineering have facilitated the development of alternative approaches to determine helix packing in polytopic membrane proteins . Using the lac permease as a paradigm, several site-directed biophysical and biochemical techniques are described which should be generally applicable.

Biotechniques, 1997 Aug, 23(2), 296 - 9
Identification of endonuclease activity in HIV-1 gp120 preparations produced using baculovirus expression systems; Davidoff AN et al.; Experiments undertaken with commercially available recombinantly produced human immunodeficiency virus Type 1 (HIV-1) gp120 demonstrated that the resuspended lyophilized protein, a product of the baculovirus expression system, had intrinsic nuclease activity . This nuclease activity was distinguishable from the molecular-grade bovine serum albumin that it was constituted in . The activity was thermolabile in that if the preparation was heated to 100 degrees C for 10 min, the activity was abolished, although this did not happen when it was stored at -20 degrees C . The nuclease activity was also Ca+2- and Mg+2-dependent, and had endonuclease as opposed to exonuclease activity . Zn+2 ions were found to inhibit the enzyme . The intensity of nuclease activity varied from batch to batch . A lyophilized homogenate of Sf9 insect cells expressing the Rho baculovirus-derived red blood cell protein 4.2 (Pallidin) was also found to have nuclease activity on reconstitution . In contrast, most, though not all E . coli-produced recombinant proteins were found to be free of nuclease activity . The use of activity gels to identify the size of the nuclease contained in the gp120 preparation was limited, because despite the use of many renaturation methods, the enzyme in the gp120 preparation could not be functionally resuscitated following sodium dodecyl sulfate polyacrylamide gel electrophoresis . Immunoprecipitation studies were useful to demonstrate that nuclease activity in the gp120 preparation was functionally distinguishable from the gp120 itself . When mononuclear cells transformed with anti-CD3 were concurrently incubated with gp120 (5-40 micrograms/mL), internucleosomal DNA fragments characteristic of apoptosis were demonstrated in the supernatant by DNA gel electrophoresis . In the context of HIV-1 and AIDS, where the depletion of CD4+ T-cells has been found to be associated with apoptosis, nuclease activity intrinsic to the gp120 preparation used in experimentation may potentially alter experimental results.

Acta Anaesthesiol Scand, 1997 Aug, 41(7), 824 - 9
Vitamin A exerts potential therapeutic effects in the endotoxaemic pig; Eriksson M et al.; BACKGROUND: Pretreatment with an injection of vitamin A has beneficial effects on cardiac and pulmonary functions in normally bred endotoxaemic pigs . The present study was performed in order to elucidate whether the response of an ongoing infusion of E . coli can be modulated by a single injection of vitamin A . METHODS: Sixteen healthy (not vitamin A-depleted) pigs were anaesthetized, monitored, mechanically ventilated and subjected to an infusion of E . coli endotoxin (10 micrograms.kg-1.h-1) . This infusion resulted within 30 min in a progressive haemodynamic derangement . When the mean pressure in the pulmonary artery was twice the baseline value, vitamin A (460 IU.kg-1) or a corresponding volume of vehicle was injected intravenously . After sacrificing the animals, the right lung was excised and weighed, and biopsy specimens were taken from the left lung for microscopical examination . RESULTS: Mean arterial blood pressure was significantly less affected (P < 0.01) between the 1st and 6th hour in endotoxaemic pigs treated with vitamin A than in those given vehicle . The mean lung weight in the vitamin A-treated pigs was significantly lower than that in the vehicle group (164 +/- 5.3 vs 199 +/- 19.8 g; P < 0.01) . Microscopical examination showed significantly less oedema (0.93 +/- 0.17 vs 2.00 +/- 0.26; P < 0.01) and microatelectasis (0.56 +/- 0.17 vs 1.75 +/- 0.31; P < 0.01) in the vitamin A group . Endotoxaemia was also accompanied by an initial, steep decline in neutrophil counts in all animals; this decrease was significantly less pronounced (P < 0.05) in vitamin A-injected pigs than in the vehicle-injected group . The major difference was a more rapid restitution in the vitamin A group . CONCLUSION: Several manifestations of endotoxaemia were expressed less in the vitamin A group . Thus, vitamin A may turn out to be a tool in the management of endotoxaemia.

J Protein Chem, 1997 Aug, 16(6), 637 - 50
Expression, purification, characterization, and X-ray analysis of selenomethionine 215 variant of leukocyte collagenase; Pieper M et al.; Matrix metalloproteinases belong to the superfamily of metzincins containing, besides a similar topology and a strictly conserved zinc environment, a 1,4-tight turn with a strictly conserved methionine residue at position three (the so called Met-turn {Bode et . al . (1993) FEBS 331, 134-140; Stocker et al . (1995) Protein Sci . 4, 823-840} . The distal S-CH3 moiety of this methionine residue forms the hydrophobic basement of the three His residues liganding the catalytic zinc ion . To assess the importance of this methionine, we have expressed the catalytic domain of neutrophil collagenase (rHNC, residues Met80-Gly242) in the methionine auxotrophic Escherichia coli strain B834{DE3}(hsd metB), with the two methionine residues replaced by selenomethionine . Complete replacement was confirmed by amino acid analysis and electrospray mass spectrometry . The folded and purified enzyme retained its catalytic activity, but showed modifications which are reflected in change kinetic parameters . The Met215SeMet substitution caused a decrease in conformational stability upon area denaturation . The X-ray crystal structure of this selenomethionine rHNC was virtually identical to that of the wild-type catalytic domain except for a very faint local disturbance around the sulfur-seleno substitution site.

J Pharmacol Exp Ther, 1997 Aug, 282(2), 1044 - 54
Interaction of ethanol with inducible nitric oxide synthase messenger RNA and protein: direct effects on autacoids and endotoxin in vivo; Greenberg SS et al.; Inducible nitric oxide synthase (iNOS) mRNA is up-regulated in vivo by dibutyryl-cAMP (db-cAMP), the purine-2y receptor agonist 2-methylthio-ATP and Escherichia coli endotoxin lipopolysaccharide (LPS) . Ethanol and diethyldithiocarbamate inhibit LPS-stimulated iNOS mRNA . Their effects on db-cAMP- and 2-methylthio-ATP-stimulated iNOS mRNA remain undefined . We examined the effect of ethanol (4.5 g/kg intraperitoneal) and intratracheal diethyldithiocarbamate (5 mg/kg) on intratracheal LPS (0.6 mg/kg), db-cAMP (0.1 and 1 mg/kg) or 2-methylthio-ATP (5 mg/kg)-stimulated rat alveolar macrophage (AM) iNOS mRNA and protein, reactive nitrogen intermediates nitrite and nitrate anion (RNI) and nuclear transcription factor-kappaB (NF-kappaB) in vivo . LPS and the autacoids increased iNOS mRNA and protein in rat AM and RNI in bronchoalveolar lavage fluid and in ex vivo incubates of AM compared with these parameters in control rats (n = 6-21/group) . Only LPS up-regulated TNF-alpha mRNA and release of TNF-alpha in bronchoalveolar lavage fluid and AM . Ethanol inhibited LPS stimulation of the iNOS cascade at the level of transcription but inhibited only autacoid-stimulated iNOS protein and RNI . Diethyldithiocarbamate selectively inhibited the LPS-stimulated iNOS cascade at the level of transcription . Coadministration of ethanol and diethyldithiocarbamate inhibited LPS-stimulated iNOS mRNA, protein and RNI more than either inhibitor alone but did not differ from ethanol alone on autacoid-stimulated iNOS protein or RNI . LPS increased and db-cAMP did not affect NF-kappaB in AM . Ethanol inhibited LPS-stimulated NF-kappaB . Thus, two distinct pathways exist for induction of iNOS mRNA in rat AM in vivo: an NF-kappaB pathway for LPS and cytokines inhibitable by ethanol and diethyldithiocarbamate and an NF-kappaB-independent pathway, refractory to inhibition by ethanol and diethyldithiocarbamate for db-cAMP and 2-mes-ATP . Finally, ethanol inhibits iNOS at the level of transcription and at the level of the enzyme.

Glia, 1997 Aug, 20(4), 365 - 72
Localization of endothelial-monocyte-activating polypeptide II (EMAP II), a novel proinflammatory cytokine, to lesions of experimental autoimmune encephalomyelitis, neuritis and uveitis: expression by monocytes and activated microglial cells; Schluesener HJ et al.; Endothelial-Monocyte-Activating Polypeptide II (EMAP II) is a proinflammatory cytokine and chemoattractant of macrophages . In order to investigate the role of EMAP II in autoimmune lesions of the rat nervous system, we have used a synthetic gene to express EMAP II in E . coli and have produced monoclonal antibodies against EMAP II . Monoclonal antibodies are suited to demonstrate EMAP II in ELISAs, Western blots, and paraffin-embedded tissue sections . EMAP II was localized to monocytes/macrophages with rather selective staining of a minor rat monocyte subpopulation of lymphoid tissues such as spleen, lymph nodes or follicles of the gut . In the normal brain, cells of the perivascular but not parenchymal microglia were stained . We then investigated expression of EMAP II during experimental autoimmune encephalomyelitis (EAE), neuritis (EAN), and uveitis (EAU) . Within the local inflammatory lesions infiltrating macrophages are prominently stained . In the diseased brain, EMAP II-positive microglial cells are not only found in the direct vicinity of the inflammatory infiltrate, but widespread activation is seen in the parenchyma . This is the first demonstration that EMAP II is present in autoimmune lesions . Immunostaining of microglial cells is noteworthy, as these cells are strategically placed regulatory elements of CNS immunosurveillance . EMAP II might be a factor regulating monocyte chemoattraction, endothelial cell activation and a regulator of microglial cell reactivity in autoimmune inflammation of the central nervous system.

Biotechnol Appl Biochem, 1997 Aug, 26 ( Pt 1), 39 - 49
Use of the HIV-1 protease for excision of growth-hormone-releasing factor from synthetic and recombinant peptide precursors; Tomasselli AG et al.; An autolysis-resistant mutant of the HIV-I protease was employed for removal of metabolically stabilized and highly bioactive analogues of bovine growth-hormone-releasing factor (bGRF) from their larger either synthetic or recombinant precursors . The N-terminal four amino acids in two selected model GRF analogues, Y1IDAIFTSSYRKVLAQLSARKLLQDILSRQVF32-OH (I; GRF32) and Y1IDAIFTSSYRKVLAQLSARKLLQDILSRQ30-OH (IA; GRF30), conform well to the specificity of the HIV-I protease for residues in the P1' to P4' positions of its peptide substrates . A variety of amino acids were tried in the N-terminal extension (positions P4-P1) to fit the protease substrate specificity for the 8 amino acids in positions P4-P4' . A synthetic precursor of I, extended N-terminally with RQVF-, a sequence representing the four C-terminal residues in I, was effectively cleaved by the protease at the Phe-1-Tyr1 bond (.. . RQVF-decreases-YIDA ...) to release GRF32 . However, when several soluble fusion proteins linked to GRF32 by the RQVF sequence were expressed in Escherichia coli, attempts to cleave out the core GRF32 met with variable, and only limited, success . By random mutagenesis in a propeptide segment, {MGQSVAQVF}-decreases-GRF30, (II) was identified as a construct that showed reasonably high-level expression in E . coli and was effectively processed by the HIV-I protease . A yield of 5 mg of pure GRF30 was obtained/litre of culture medium after a single HPLC purification step.

Proteins, 1997 Aug, 28(4), 568 - 79
Flexible glycine rich motif of Escherichia coli deoxyuridine triphosphate nucleotidohydrolase is important for functional but not for structural integrity of the enzyme; Vertessy BG; Deoxyuridine triphosphate nucleotidohydrolase (dUTPase), a ubiquitous enzyme of DNA metabolism, has been implicated as a novel target of anticancer and antiviral drug design . This task is most efficiently accomplished by X-ray crystallography of the relevant protein-inhibitor complexes . However, the topic of the present investigation, a glycine-rich strictly conserved structural motif of dUTPases, could not be located in the crystal structure of the Escherichia coli enzyme, probably due to its increased flexibility . The present work shows that removal of a C-terminal 11-residue fragment, including this motif, by limited trypsinolysis strongly impairs catalytic activity . Kinetic analysis of the intact and digested variants showed that kcat decreases 40-fold, while KM increases less than twofold upon digestion . The tryptic site was identified by mass spectrometry, amino acid analysis and N-terminal sequencing . The shortened enzyme variant retains the secondary, tertiary, and quaternary (trimeric) structure of the intact species as suggested by UV absorption, fluorescence and circular dichroism spectroscopy, and analytical gel filtration . Moreover, binding affinity of the shortened variant toward the substrate analogue MgdUDP is identical to the one displayed by the intact enzyme . I conclude that the glycine-rich motif is functionally relevant for E . coli dUTPase . It may play a role in enzymatic catalysis by contributing to the formation of the catalytically potent enzyme-substrate complex.

Proteins, 1997 Aug, 28(4), 481 - 93
Accuracy and precision of NMR relaxation experiments and MD simulations for characterizing protein dynamics; Philippopoulos M et al.; Model-free parameters obtained from nuclear magnetic resonance (NMR) relaxation experiments and molecular dynamics (MD) simulations commonly are used to describe the intramolecular dynamical properties of proteins . To assess the relative accuracy and precision of experimental and simulated model-free parameters, three independent data sets derived from backbone 15N NMR relaxation experiments and two independent data sets derived from MD simulations of Escherichia-coli ribonuclease HI are compared . The widths of the distributions of the differences between the order parameters for pairs of NMR data sets are congruent with the uncertainties derived from statistical analyses of individual data sets; thus, current protocols for analyzing NMR data encapsulate random uncertainties appropriately . Large differences in order parameters for certain residues are attributed to systematic differences between samples for intralaboratory comparisons and unknown, possibly magnetic field-dependent, experimental effects for interlaboratory comparisons . The widths of distributions of the differences between the order parameters for two NMR sets are similar to widths of distributions for an NMR and an MD set or for two MD sets . The linear correlations between the order parameters for an MD set and an NMR set are within the range of correlations observed between pairs of NMR sets . These comparisons suggest that the NMR and MD generalized order parameters for the backbone amide N-H bond vectors are of comparable accuracy for residues exhibiting motions on a fast time scale (< 100 ps) . Large discrepancies between NMR and MD order parameters for certain residues are attributed to the occurrence of "rare" motional events over the simulation trajectories, the disruption of an element of secondary structure in one of the simulations, and lack of consensus among the experimental data sets . Consequently, (easily detectable) severe distortions of local protein structure and infrequent motional events in MD simulations appear to be the most serious artifacts affecting the accuracy and precision, respectively, of MD order parameters relative to NMR values . In addition, MD order parameters for motions on a fast (< 100 ps) timescale are more precisely determined than their NMR counterparts, thereby permitting more detailed dynamic characterization of biologically important residues by MD simulation than is sometimes possible by experimental methods . Proteins 28:481-493, 1997.

J Bacteriol, 1997 Aug, 179(16), 5232 - 7
Autogenous control of PspF, a constitutively active enhancer-binding protein of Escherichia coli; Jovanovic G et al.; Escherichia coli sigma54-dependent phage shock protein operon (pspA to -E) transcription is under the control of PspF, a constitutively active activator . Sigma70-dependent transcription of pspF is under autogenous control by wild-type PspF but not by a DNA-binding mutant, PspF deltaHTH . Negative autoregulation of PspF is continual and not affected by stimuli, like f1 pIV, that induce the pspA to -E operon . PspF production is independent of PspA (the negative regulator of the pspA to -E operon) and of PspB and -C (positive regulators).

J Bacteriol, 1997 Aug, 179(16), 5218 - 21
Characterization of Escherichia coli strains with gapA and gapB genes deleted; Seta FD et al.; We obtained a series of Escherichia coli strains in which gapA, gapB, or both had been deleted . Delta gapA strains do not revert on glucose, while delta gapB strains grow on glycerol or glucose . We showed that gapB-encoded protein is expressed but at a very low level . Together, these results confirm the essential role for gapA in glycolysis and show that gapB is dispensable for both glycolysis and the pyridoxal biosynthesis pathway.

J Bacteriol, 1997 Aug, 179(16), 5195 - 202
The IncP plasmid-encoded cell envelope-associated DNA transfer complex increases cell permeability; Daugelavicius R et al.; IncP-type plasmids are broad-host-range conjugative plasmids . DNA translocation requires DNA transfer-replication functions and additional factors required for mating pair formation (Mpf) . The Mpf system is located in the cell membranes and is responsible for DNA transport from the donor to the recipient . The Mpf complex acts as a receptor for IncP-specific phages such as PRD1 . In this investigation, we quantify the Mpf complexes on the cell surface by a phage receptor saturation technique . Electrochemical measurements are used to show that the Mpf complex increases cell envelope permeability to lipophilic compounds and ATP . In addition it reduces the ability of the cells to accumulate K+ . However, the Mpf complex does not dissipate the membrane voltage . The Mpf complex is rapidly disassembled when intracellular ATP concentration is decreased, as measured by a PRD1 adsorption assay.

J Bacteriol, 1997 Aug, 179(16), 5171 - 7
Subunit and amino acid interactions in the Escherichia coli mannitol permease: a functional complementation study of coexpressed mutant permease proteins; Saraceni-Richards CA et al.; Mannitol-specific enzyme II, or mannitol permease, of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system of Escherichia coli carries out the transport and phosphorylation of D-mannitol and is most active as a dimer in the membrane . We recently reported the importance of a glutamate residue at position 257 in the binding and transport of mannitol by this protein (C . Saraceni-Richards and G . R . Jacobson, J . Bacteriol . 179:1135-1142, 1997) . Replacing Glu-257 with alanine (E257A) or glutamine (E257Q) eliminated detectable mannitol binding and transport by the permease . In contrast, an E257D mutant protein was able to bind and phosphorylate mannitol in a manner similar to that of the wild-type protein but was severely defective in mannitol uptake . In this study, we have coexpressed proteins containing mutations at position 257 with other inactive permeases containing mutations in each of the three domains of this protein . Activities of any active heterodimers resulting from this coexpression were measured . The results show that various inactive mutant permease proteins can complement proteins containing mutations at position 257 . In addition, we show that both Glu at position 257 and His at position 195, both of which are in the membrane-bound C domain of the protein, must be on the same subunit of a permease dimer in order for efficient mannitol phosphorylation and uptake to occur . The results also suggest that mannitol bound to the opposite subunit within a permease heterodimer can be phosphorylated by the subunit containing the E257A mutation (which cannot bind mannitol) and support a model in which there are separate binding sites on each subunit within a permease dimer . Finally, we provide evidence from these studies that high-affinity mannitol binding is necessary for efficient transport by mannitol permease.

J Bacteriol, 1997 Aug, 179(16), 5131 - 7
Cloning and characterization of the gene (farA) encoding the receptor for an extracellular regulatory factor (IM-2) from Streptomyces sp . strain FRI-5; Waki M et al.; IM-2 is a butyrolactone autoregulator that controls production of blue pigment and nucleoside antibiotics in Streptomyces sp . strain FRI-5 . An IM-2-specific receptor gene, farA, was cloned from strain FRI-5, and nucleotide sequencing revealed that the farA gene consists of 666 bp encoding a 221-amino-acid protein of 24.3 kDa with an NH2-terminal amino acid sequence identical to that of purified native receptor . Another gene, farX, encoding a homolog of AfsA of Streptomyces griseus, was present upstream of farA . The monocistronic nature of the farA transcript was shown by Northern blot hybridization, and the transcript level increased upon addition of IM-2 . Recombinant FarA expressed in and purified from E . coli showed clear ligand specificity toward IM-2, with a dissociation constant (Kd) for {3H}IM-2-C5 of 18.2 nM . FarA showed high overall homology to BarA (virginiae butanolide receptor from S . virginiae) and ArpA (A-factor receptor from S . griseus) . Sequence alignment of the three receptor proteins revealed that the NH2-terminal region containing a helix-turn-helix DNA binding motif was highly conserved . The DNA binding motif is common in procaryotic repressors of the TetR family, suggesting that all the Streptomyces autoregulator receptors may act as transcriptional repressors.

J Bacteriol, 1997 Aug, 179(16), 5126 - 30
Growth-phase-dependent expression of cspD, encoding a member of the CspA family in Escherichia coli; Yamanaka K et al.; The cspD gene of Escherichia coli encodes a protein of high sequence similarity with the cold shock protein CspA, but cspD expression is not induced by cold shock . In this study, we analyzed the regulation of cspD gene expression . By using a cspD-lacZ fusion and primer extension analysis, the expression of cspD was found to be dramatically induced by stationary-phase growth . However, this induction does not depend on the stationary-phase sigma factor sigmaS . Moreover, the expression of cspD is inversely dependent on growth rates and induced upon glucose starvation . Using a (p)ppGpp-depleted strain, we found that (p)ppGpp is one of the positive factors for the regulation of cspD expression.

J Bacteriol, 1997 Aug, 179(16), 5037 - 45
Characterization by electron paramagnetic resonance of the role of the Escherichia coli nitrate reductase (NarGHI) iron-sulfur clusters in electron transfer to nitrate and identification of a semiquinone radical intermediate; Magalon A et al.; We have used Escherichia coli cytoplasmic membrane preparations enriched in wild-type and mutant (NarH-C16A and NarH-C263A) nitrate reductase (NarGHI) to study the role of the {Fe-S} clusters of this enzyme in electron transfer from quinol to nitrate . The spectrum of dithionite-reduced membrane bound NarGHI has major features comprising peaks at g = 2.04 and g = 1.98, a peak-trough at g = 1.95, and a trough at g = 1.87 . The oxidized spectrum of NarGHI in membranes comprises an axial {3Fe-4S} cluster spectrum with a peak at g = 2.02 (g(z)) and a peak-trough at g = 1.99 (g(xy)) . We have shown that in two site-directed mutants of NarGHI which lack the highest potential {4Fe-4S} cluster (B . Guigliarelli, A . Magalon, P . Asso, P . Bertrand, C . Frixon, G . Giordano, and F . Blasco, Biochemistry 35:4828-4836, 1996), NarH-C16A and NarH-C263A, oxidation of the NarH {Fe-S} clusters is inhibited compared to the wild type . During enzyme turnover in the mutant enzymes, a distinct 2-n-heptyl-4-hydroxyquinoline-N-oxide-sensitive semiquinone radical species which may be located between the hemes of NarI and the {Fe-S} clusters of NarH is observed . Overall, these studies indicate (i) the importance of the highest-potential {4Fe-4S} cluster in electron transfer from NarH to the molybdenum cofactor of NarG and (ii) that a semiquinone radical species is an important intermediate in electron transfer from quinol to nitrate.

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