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PubMed2004

Molecular analysis of cytolysin A (ClyA) in pathogenic Escherichia coli strains.

Cytolysin A (ClyA) of Escherichia coli is a pore-forming hemolytic protein encoded by the clyA (hlyE, sheA) gene that was first identified in E. coli K-12. In this study we examined various clinical E. coli isolates with regard to the presence and integrity of clyA. PCR and DNA sequence analyses demonstrated that 19 of 23 tested Shiga toxin-producing E. coli (STEC) strains, all 7 tested enteroinvasive E. coli (EIEC) strains, 6 of 8 enteroaggregative E. coli (EAEC) strains, and 4 of 7 tested enterotoxigenic E. coli (ETEC) strains possess a complete clyA gene. The remaining STEC, EAEC, and ETEC strains and 9 of the 17 tested enteropathogenic E. coli (EPEC) strains were shown to harbor mutant clyA derivatives containing 1-bp frameshift mutations that cause premature termination of the coding sequence. The other eight EPEC strains and all tested uropathogenic and new-born meningitis-associated E. coli strains (n = 14 and 3, respectively) carried only nonfunctional clyA fragments due to the deletion of two sequences of 493 bp and 204 or 217 bp at the clyA locus. Expression of clyA from clinical E. coli isolates proved to be positively controlled by the transcriptional regulator SlyA. Several tested E. coli strains harboring a functional clyA gene produced basal amounts of ClyA when grown under standard laboratory conditions, but most of them showed a clyA-dependent hemolytic phenotype only when SlyA was overexpressed. The presented data indicate that cytolysin A can play a role only for some of the pathogenic E. coli strains.

Amino Acid Sequence↗
PubMed1995

Degradation of c-Fos by the 26S proteasome is accelerated by c-Jun and multiple protein kinases.

c-Fos is associated with c-Jun to increase the transcription of a number of target genes and is a nuclear proto-oncoprotein with a very short half-life. This instability of c-Fos may be important in regulation of the normal cell cycle. Here we report a mechanism for degradation of c-Fos. Coexpression of c-Fos and c-Jun in HeLa cells caused marked increase in the instability of c-Fos, whereas v-Fos, the retroviral counterpart of c-Fos, was stable irrespective of the coexpression of c-Jun. Interestingly, deletion of the C-terminal PEST region of c-Fos, which is altered in v-Fos by a frameshift mutation, greatly enhanced its stability, with loss of the effect of c-Jun on its stability. c-Fos synthesized in vitro was degraded by the 26S proteasome in a ubiquitin-dependent fashion. Simple association with c-Jun had no effect on the degradation of c-Fos, but the additions of three protein kinases, mitogen-activated protein kinase, casein kinase II, and CDC2 kinase, resulted in marked acceleration of its degradation by the proteasome-ubiquitin system, though only in the presence of c-Jun. In contrast, v-Fos and c-Fos with a truncated PEST motif were not degraded, suggesting that they escaped from down-regulation by breakdown. These findings indicate a new oncogenic pathway induced by acquisition of intracellular stability of a cell cycle modulatory factor.

Amino Acid Sequence↗
PubMed2004

Frequency of NOD2/CARD15 variants in both sporadic and familial cases of Crohn's disease across Italy. An Italian Group for Inflammatory Bowel Disease Study.

BACKGROUND: Three variants of the NOD2/CARD15 gene are strongly associated with susceptibility to Crohn's disease; however, striking racial and geographic differences of their frequency have been described. AIMS: We have compared the allele frequencies of familial cases of Crohn's disease recruited in a multicentre study across Italy, in order to disclose possible geographic heterogeneity. Moreover, we also compared the allele frequencies in sporadic cases of Crohn's disease and healthy controls from Southern Italy with those reported in other two populations from Central and Northern Italy. SUBJECTS AND METHODS: A total of 731 subjects were genotyped for the polymorphism of three main variants (R702W, G908R and 1007 fs): 152 patients were familial cases of Crohn's disease, 183 were healthy first-degree relatives, 180 were sporadic cases of Crohn's disease, and 216 were unrelated healthy subjects. RESULTS: The frequency of the frameshift mutation (1007 fs) was significantly higher in both familial and sporadic cases of Crohn's disease (P = 0.000001), and healthy first-degree relatives (P = 0.0001) compared to controls. At least one risk allele was found in 44% of familial Crohn's disease patients, compared to 7% of healthy controls (OR = 4; CI = 2-6.5). Two risk alleles were found in 14% of familial Crohn's disease, compared to less than 1% of controls (OR = 26: CI = 4-129). CONCLUSIONS: Our data confirm the strong correlation between the 1007 fs variant and Crohn's disease, in both familial and sporadic cases. Moreover, no significant difference of allele frequencies was detected in familial cases, sporadic cases and healthy controls among different geographic areas of Italy.

Adult↗
PubMed1999

Ferric enterochelin transport in Yersinia enterocolitica: molecular and evolutionary aspects.

Yersinia enterocolitica is well equipped for siderophore piracy, encompassing the utilization of siderophores such as ferrioxamine, ferrichrome, and ferrienterochelin. In this study, we report on the molecular and functional characterization of the Yersinia fep-fes gene cluster orthologous to the Escherichia coli ferrienterochelin transport genes (fepA, fepDGC, and fepB) and the esterase gene fes. In vitro transcription-translation analysis identified polypeptides of 30 and 35 kDa encoded by fepC and fes, respectively. A frameshift mutation within the fepA gene led to expression of a truncated polypeptide of 40 kDa. The fepD, fepG, and fes genes of Y. enterocolitica were shown to complement corresponding E. coli mutants. Insertional mutagenesis of fepD or fes genes abrogates enterochelin-supported growth of Y. enterocolitica on iron-chelated media. In contrast to E. coli, the fep-fes gene cluster in Y. enterocolitica consists solely of genes required for uptake and utilization of enterochelin (fep) and not of enterochelin synthesis genes such as entF. By Southern hybridization, fepDGC and fes sequences could be detected in Y. enterocolitica biotypes IB, IA, and II but not in biotype IV strains, Yersinia pestis, and Yersinia pseudotuberculosis strains. According to sequence alignment data and the coherent structure of the Yersinia fep-fes gene cluster, we suggest early genetic divergence of ferrienterochelin uptake determinants among species of the family Enterobacteriaceae.

Base Sequence↗
PubMed1997

Rhizobium sp. strain NGR234 NodZ protein is a fucosyltransferase.

Rhizobium sp. strain NGR234 produces a large family of lipochitooligosaccharide Nod factors carrying specific substituents. Among them are 3-O- (or 4-O-) and 6-O-carbamoyl groups, an N-methyl group, and a 2-O-methylfucose residue which may bear either 3-O-sulfate or 4-O-acetyl substitutions. Investigations on the genetic control of host specificity revealed a number of loci which directly affect Nod factor structure. Here we show that insertion and frameshift mutations in the nodZ gene abolish fucosylation of Nod factors. In vitro assays using GDP-L-fucose as the fucose donor show that fucosyltransferase activity is associated with the nodZ gene product (NodZ). NodZ is located in the soluble protein fraction of NGR234 cells. Together with extra copies of the nodD1 gene, the nodZ gene and its associated nod box were introduced into ANU265, which is NGR234 cured of the symbiotic plasmid. Crude extracts of this transconjugant possess fucosyltransferase activity. Fusion of a His6 tag to the NodZ protein expressed in Escherichia coli yielded a protein able to fucosylate both nonfucosylated NodNGR factors and oligomers of chitin. NodZ is inactive on monomeric N-acetyl-D-glucosamine and on desulfated Rhizobium meliloti Nod factors. Kinetic analyses showed that the NodZ protein is more active on oligomers of chitin than on nonfucosylated NodNGR factors. Pentameric chitin is the preferred substrate. These data suggest that fucosylation occurs before acylation of the Nod factors.

Acetylglucosamine↗
PubMed1996

Duplication and divergence of the genes of the alpha-esterase cluster of Drosophila melanogaster.

The alpha-esterase cluster of D. melanogaster contains 11 esterase genes dispersed over 60 kb. Embedded in the cluster are two unrelated open reading frames that have sequence similarity with genes encoding ubiquitin-conjugating enzyme and tropomyosin. The esterase amino acid sequences show 37-66% identity with one another and all but one have all the motifs characteristic of functional members of the carboxyl/cholinesterase multigene family. The exception has several frameshift mutations and appears to be a pseudogene. Patterns of amino acid differences among cluster members in relation to generic models of carboxyl/cholinesterase protein structure are broadly similar to those among other carboxyl/cholinesterases sequenced to date. However the alpha-esterases differ from most other members of the family in: their lack of a signal peptide; the lack of conservation in cysteines involved in disulfide bridges; and in four indels, two of which occur in or adjacent to regions that align with proposed substrate-binding sites of other carboxyl/cholinesterases. Phylogenetic analyses clearly identify three simple gene duplication events within the cluster. The most recent event involved the pseudogene which is located in an intron of another esterase gene. However, relative rate tests suggest that the pseudogene remained functional after the duplication event and has become inactive relatively recently. The distribution of indels also suggests a deeper node in the gene phylogeny that separates six genes at the two ends of the cluster from a block of five in the middle.

Amino Acid Sequence↗
PubMed1997

Studies on the role of plasminogen activator in systemic infection by virulent Yersinia pestis strain C092.

Plasminogen activator is an outer membrane protease of Yersinia pestis encoded by the pla gene on plasmid pPst. Pla of the KIM-10 strain of Y. pestis appears to be required for the virulence from a subcutaneous (sc) but not an intraperitoneal (ip) or intravenous (iv) route of infection in mice. However, other strains of Y. pestis are highly virulent by the sc route yet lack pPst and pla. In this study, the pPst- Pestoides F strain was lethal to mice inoculated sc, with an LD50 (3 cfu), equal to that of C092, a virulent pPst+ strain. To analyse further the role of Pla in invasive infection, isogenic derivatives of C092, including one harboring pla with a frameshift mutation and another cured of pPst, were made. Although the ip LD50 of pPst- C092 and of the pla mutant were nearly identical to that of the wild type, the subcutaneous LD50 of the cured and mutant strains were 4 to 6 logs greater than that of wild type. Thus, pPst appears to be required for development of a lethal infection by some strains after sc inoculation but not after direct ip inoculation. Pla-associated virulence did not appear to be mediated by interference with the phagocyte chemoattractant C5a, as shown by the lack of correlation of C5a production with susceptibility to Y. pestis in C5a+ and C5a- congenic mice. In a footpad model of the early host response to subcutaneous infection, pPst- C092 proliferated at the subcutaneous injection site to a similar extent as did the wild type parent strain, and elicited a similarly large, local inflammatory response. However, the wild type was present at higher concentrations at more distant sites such as the popliteal lymph node and spleen.

Animals↗
PubMed1999

Multiple heterologies increase mitotic double-strand break-induced allelic gene conversion tract lengths in yeast.

Spontaneous and double-strand break (DSB)-induced allelic recombination in yeast was investigated in crosses between ura3 heteroalleles inactivated by an HO site and a +1 frameshift mutation, with flanking markers defining a 3.4-kbp interval. In some crosses, nine additional phenotypically silent RFLP mutations were present at approximately 100-bp intervals. Increasing heterology from 0.2 to 1% in this interval reduced spontaneous, but not DSB-induced, recombination. For DSB-induced events, 75% were continuous tract gene conversions without a crossover in this interval; discontinuous tracts and conversions associated with a crossover each comprised approximately 7% of events, and 10% also converted markers in unbroken alleles. Loss of heterozygosity was seen for all markers centromere distal to the HO site in 50% of products; such loss could reflect gene conversion, break-induced replication, chromosome loss, or G2 crossovers. Using telomere-marked strains we determined that nearly all allelic DSB repair occurs by gene conversion. We further show that most allelic conversion results from mismatch repair of heteroduplex DNA. Interestingly, markers shared between the sparsely and densely marked interval converted at higher rates in the densely marked interval. Thus, the extra markers increased gene conversion tract lengths, which may reflect mismatch repair-induced recombination, or a shift from restoration- to conversion-type repair.

Alleles↗
PubMed1995

Replication slippage between distant short repeats in Saccharomyces cerevisiae depends on the direction of replication and the RAD50 and RAD52 genes.

Small direct repeats, which are frequent in all genomes, are a potential source of genome instability. To study the occurrence and genetic control of repeat-associated deletions, we developed a system in the yeast Saccharomyces cerevisiae that was based on small direct repeats separated by either random sequences or inverted repeats. Deletions were examined in the LYS2 gene, using a set of 31- to 156-bp inserts that included inserts with no apparent potential for secondary structure as well as two quasipalindromes. All inserts were flanked by 6- to 9-bp direct repeats of LYS2 sequence, providing an opportunity for Lys+ reversion via precise excision. Reversions could arise by extended deletions involving either direct repeats or random sequences and by -1-or +2-bp frameshift mutations. The deletion breakpoints were always associated with short (3- to 9-bp) perfect or imperfect direct repeats. Compared with the POL+ strain, deletions between small direct repeats were increased as much as 100-fold, and the spectrum was changed in a temperature-sensitive DNA polymerase delta pol3-t mutant, suggesting a role for replication. The type of deletion depended on orientation relative to the origin of replication. On the basis of these results, we propose (i) that extended deletions between small repeats arise by replication slippage and (ii) that the deletions occur primarily in either the leading or lagging strand. The RAD50 and RAD52 genes, which are required for the recombinational repair of many kinds of DNA double-strand breaks, appeared to be required also for the production of up to 90% of the deletions arising between separated repeats in the pol3-t mutant, suggesting a newly identified role for these genes in genome stability and possibly replication.

Base Sequence↗
PubMed1991

An in vitro approach to identifying specificity determinants of mutagenesis mediated by DNA misalignments.

In vitro, misalignments of the newly synthesized (primer) strand during DNA polymerization lead to deletion and/or complex frameshift mutations. In vivo, similar misalignments of repeated and quasipalindromic DNA sequences are predicted to be intermediates of mutagenesis. The mutagenic misalignments are mediated by complementary pairing between the sequence at the 3'-OH end of the newly synthesized DNA strand and sequences in the template or in the newly synthesized DNA. Mutant sequences are produced when the misaligned primers act as substrates for DNA polymerization. The misalignments responsible for detected mutant sequences were compared to similar misalignments that were not implicated in mutagenesis, and all misalignment possibilities were compared to the position of pausing during polymerization by Escherichia coli polymerase I or its Klenow fragment. These comparisons revealed three characteristics of in vitro misalignment specificity. First, the termini produced by pausing are likely to be precursors to mutagenic misalignments. Second, the absence of some potential misalignments from the detected spectrum is explained well by the predicted undetectability of the mutant sequences they produce. Third, factors distinct from pausing and mutant detectability are responsible for differences in the specificity of misalignment mutagenesis mediated by E. coli DNA polymerase I and Klenow polymerase during in vitro synthesis.

Base Sequence↗
PubMed2002

A novel association of the SMN protein with two major non-ribosomal nucleolar proteins and its implication in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is caused by the loss of functional survival motor neuron 1 (SMN1) protein. This ubiquitously expressed protein is a component of a novel complex immunodetected in both the cytoplasm and the nucleus, which is associated with complexes involved in mRNA splicing, ribosome biogenesis and transcription. Here, we study a mutant protein corresponding to the N-terminal half of the protein that is encoded by the SMA frameshift mutation SMN 472del5. We show by confocal microscopy that the resulting mutant protein exhibits various distribution patterns in different transiently transfected COS cells. The mutant distributes into the nucleoplasm and/or the nucleolus, whereas the normal SMN protein accumulates at discrete nucleocytoplasmic dot-like structures previously named gems/Cajal bodies. The cell population with the nucleolar distribution is enriched upon treatment with mimosine, a synchronizing drug in late G(1) phase. Co-immunoprecipitation studies carried out on nuclear extracts reveal that both the endogenous SMN and mutant proteins are associated with complexes containing two major non-ribosomal nucleolar proteins, namely nucleolin and protein B23, and that the association is mediated, by among other things, RNA moieties. Both the association of the SMN protein with nucleolin-containing complexes and the nucleolin/B23 complex are disrupted in fibroblasts derived from a type I SMA patient harboring a homozygous SMN1 gene deletion. These findings suggest that altered assembly and/or stability of ribonucleoprotein complexes may contribute to the pathophysiological processes in SMA.

Animals↗
PubMed1994

Deletion and duplication sequences induced in CHO cells by teniposide (VM-26), a topoisomerase II targeting drug, can be explained by the processing of DNA nicks produced by the drug-topoisomerase interaction.

Frameshift mutations induced by acridines in bacteriophage T4 have been shown to be due to the ability of these mutagens to cause DNA cleavage by the type II topoisomerase of T4 and the subsequent processing of the 3' ends at DNA nicks by DNA polymerase or its associated 3' exonuclease followed by ligation of the processed end to the original 5' end. An analysis of the ability of nick-processing models is presented here to test the ability of nick processing to account for the DNA sequences of duplications and deletions induced in the aprt gene of CHO cells by teniposide (VM-26) [Han et al. (1993) J. Mol. Biol., 229, 52]. Although teniposide is not an acridine, it induces topoisomerase II-mediated DNA cutting in aprt sequences in vitro and mutagenesis in vivo. Although the previous study noted a correlation between mutation sites and nearby DNA discontinuities induced by the enzyme in vitro, neither the nick-processing model responsible for T4 mutations, nor double-strand break models alone were able to account for most of the mutant sequences. Thus, no single model explained the correlation between teniposide-induced DNA cleavage and mutagenic specificity. This report describes an expanded analysis of the ways that nick-processing models might be related to mutagenesis and demonstrates that a modified nick-processing model provides a biochemical rationale for the mutant specificities. The successful nick-processing model proposes that either 3' ends at nicks are elongated by DNA polymerase and/or that 5' ends of nicks are subject to nuclease activity; 3'-nuclease activity is not implicated. The mutagenesis model for nick-processing of teniposide-induced nicks in CHO cells when compared to the mechanism of nick-processing in bacteriophage T4 at acridine-induced nicks provides a framework for considering whether the differences may be due to cell-specific modes of DNA processing and/or due to the precise characteristics of topoisomerase-DNA intermediates created by teniposide or acridine that lead to mutagenesis.

Acridines↗
PubMed2000

Uterine dysfunction and genetic modifiers in centromere protein B-deficient mice.

Centromere protein B (CENP-B) binds constitutively to mammalian centromere repeat DNA and is highly conserved between humans and mouse. Cenpb null mice appear normal but have lower body and testis weights. We demonstrate here that testis-weight reduction is seen in male null mice generated on three different genetic backgrounds (denoted R1, W9.5, and C57), whereas body-weight reduction is dependent on the genetic background as well as the gender of the animals. In addition, Cenpb null females show 31%, 33%, and 44% reduced uterine weights on the R1, W9.5, and C57 backgrounds, respectively. Production of "revertant" mice lacking the targeted frameshift mutation but not the other components of the targeting construct corrected these differences, indicating that the observed phenotype is attributable to Cenpb gene disruption rather than a neighbouring gene effect induced by the targeting construct. The R1 and W9.5 Cenpb null females are reproductively competent but show age-dependent reproductive deterioration leading to a complete breakdown at or before 9 months of age. Reproductive dysfunction is much more severe in the C57 background as Cenpb null females are totally incompetent or are capable of producing no more than one litter. These results implicate a further genetic modifier effect on female reproductive performance. Histology of the uterus reveals normal myometrium and endometrium but grossly disrupted luminal and glandular epithelium. Tissue in situ hybridization demonstrates high Cenpb expression in the uterine epithelium of wild-type animals. This study details the first significant phenotype of Cenpb gene disruption and suggests an important role of Cenpb in uterine morphogenesis and function that may have direct implications for human reproductive pathology.

Animals↗
PubMed1998

Transgenic plants expressing potato virus X ORF2 protein (p24) are resistant to tobacco mosaic virus and Ob tobamoviruses.

The p24 protein, one of the three proteins implicated in local movement of potato virus X (PVX), was expressed in transgenic tobacco plants (Nicotiana tabacum Xanthi D8 NN). Plants with the highest level of p24 accumulation exhibited a stunted and slightly chlorotic phenotype. These transgenic plants facilitate the cell-to-cell movement of a mutant of PVX that contained a frameshift mutation in p24. Upon inoculation with tobacco mosaic virus (TMV), the size of necrotic local lesions was significantly smaller in p24+ plants than in nontransgenic, control plants. Systemic resistance to tobamoviruses was also evidenced after inoculation of p24+ plants with Ob, a virus that evades the hypersensitive response provided by the N gene. In the latter case, no systemic symptoms were observed, and virus accumulation remained low or undetectable by Western immunoblot analysis and back-inoculation assays. In contrast, no differences were observed in virus accumulation after inoculation with PVX, although more severe symptoms were evident on p24-expressing plants than on control plants. Similarly, infection assays conducted with potato virus Y showed no differences between control and transgenic plants. On the other hand, a considerable delay in virus accumulation and symptom development was observed when transgenic tobacco plants containing the movement protein (MP) of TMV were inoculated with PVX. Finally, a movement defective mutant of TMV was inoculated on p24+ plants or in mixed infections with PVX on nontransgenic plants. Both types of assays failed to produce TMV infections, implying that TMV MP is not interchangeable with the PVX MPs.

Animals↗
PubMed1995

Structure of the carboxy-terminal region of thyroid hormone nuclear receptors and its possible role in hormone-dependent intermolecular interactions.

The thyroid hormone nuclear receptors (TRs) are ligand-dependent transcription factors. To understand the molecular basis of ligand-dependent transactivation, we studied the structure of their carboxy-terminal activation domain. We analyzed the structures of the peptides derived from the C-terminal sequences of human TR subtypes beta 1 (h-TR beta 1) and alpha 1 (h-TR alpha 1) and a human TR mutant, PV, by circular dichroism (CD). Mutant PV has a C-terminal frameshift mutation and does not bind to the thyroid hormone, 3,3',5-triiodo-L-thyronine (T3). Analyses of the secondary structures of the peptides by CD indicate that five amino acids, EVFED, are part of an amphipathic alpha-helix which is required to maintain the structural integrity of the hormone binding domain. A monoclonal antibody, C4 (mAb C4), which recognizes both h-TR beta 1 and h-TR alpha 1 was developed. Using a series of truncated mutants and synthetic peptides, we mapped the epitope of mAb C4 to the conserved C-terminal amino acids, EVFED. Analysis of the binding data indicates that binding of T3 to either h-TR beta 1 or h-TR alpha 1 was competitively inhibited by mAb C4. Deletion of C-terminal amino acids including EVFED led to a total loss of T3 binding activity. Thus, part of the T3 binding site is located in this five amino acid segment. T3 may transduce its hormonal signal to the transcriptional machinery via interaction with EVFED at the C-terminus of TRs.

Amino Acid Sequence↗
PubMed2001

Expression of hMLH1 is inactivated in the gastric adenomas with enhanced microsatellite instability.

Microsatellite instability (MSI) and frameshift mutations in the genes containing coding nucleotide repeats have been reported in a subset of gastric adenomas, however the inactivation profiles of DNA mismatch repair genes in MSI-positive gastric adenomas have not been characterized. To address the origin of MSI in gastric adenomas, expressions of hMLH1 and hMSH2 were explored in 86 gastric adenomas. Gastric carcinomas, of which 16 were MSI-positive and 22 MSI-negative, were used as controls. MSI was found in 15 (17%) of gastric adenomas. Absent or decreased hMLH1 expression by immunohistochemistry was noted in most of the MSI-positive adenomas (13/15, 87%) and carcinomas (14/16, 88%), and all of these tumours showed methylation of the hMLH1 gene promoter. In contrast, rare inactivation of hMLH1 expression was found in MSI-negative adenomas (3/71, 4%) and carcinomas (2/22, 9%). Intense expression of hMSH2 gene product was observed in most of the gastric adenomas and carcinomas regardless of MSI status. These findings indicate that the inactivation of hMLH1 gene expression by promoter methylation is an early event and might be the origin of MSI-positive gastric adenomas.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Differential mutagenic, antimutagenic and cytotoxic responses induced by apomorphine and its oxidation product, 8-oxo-apomorphine-semiquinone, in bacteria and yeast.

Apomorphine (APO) is considered to be a classical mixed type dopamine D(1) and D(2) receptor agonist. It has been used in the therapy of Parkinson's disease and, more recently, for the treatment of erectile dysfunction. Like other catechols (e.g. dopamine), APO easily autoxidizes, producing quinone and semiquinone derivatives that may lead to the formation of reactive oxygen species and induce neurotoxicity. We assayed mutagenicity, antimutagenicity, and cytotoxicity of these compounds by means of the Salmonella/microsome assay, WP2 Mutoxitest and sensitivity assay in Saccharomyces cerevisiae yeast strains lacking antioxidant defenses. In the absence of S9 mix both compounds Apomorphine and its oxidation derivative, 8-oxo-apomorphine-semiquinone (8-OASQ), both at doses ranging from 20 to 80 microg per plate, induced frameshift mutations in TA98 and TA97 S. typhimurium strains, with 8-OASQ being up to two times more mutagenic. However, for strains which detect oxidative mutagens, 8-OASQ acted as a mutagen while APO was an antimutagen, inhibiting H(2)O(2) and t-BOOH-induced mutagenicity in TA102 S. typhimurium and WP2-derived E. coli strains. The S9 mix inhibited all mutagenic effects, probably either by conjugation of APO and 8-OASQ to proteins or by quenching reactive oxygen species. In sensitivity assays with S. cerevisiae, APO was only clearly cytotoxic to some strains at higher doses (200 and 400 microg/ml), whereas 8-OASQ dose-dependently sensitized all the strains, mainly the mutants lacking catalase (deltactt1), superoxide dismutase (deltasod1) and Yap1 transcription factor (deltayap1), suggesting that 8-OASQ cytotoxicity towards S. cerevisiae results from its pro-oxidant properties. APO also tended to protect S. cerevisiae strains against oxidative damage induced by high concentrations of H(2)O(2) and t-BOOH, while 8-OASQ enhanced pro-oxidant effects and induced adaptation responses to these agents. These results suggest that the 8-OASQ oxidation product of APO might induce cytotoxic and genotoxic effects.

Antimutagenic Agents↗
PubMed1996

Regulation of colony-stimulating factor 1 receptor signaling by the SH2 domain-containing tyrosine phosphatase SHPTP1.

SHPTP1 (PTP1C, HCP, SHP) is an SH2 domain-containing tyrosine phosphatase expressed predominantly in hematopoietic cells. A frameshift mutation in the SHPTP1 gene causes the motheaten (me/me) mouse. These mice are essentially SHPTP1 null and display multiple hematopoietic abnormalities, most prominently hyperproliferation and inappropriate activation of granulocytes and macrophages. The me/me phenotype suggests that SHPTP1 negatively regulates macrophage proliferative pathways. Using primary bone marrow-derived macrophages from me/me mice and normal littermates, we examined the role of SHPTP1 in regulating signaling by the major macrophage mitogen colony-stimulating factor 1 (CSF-1) (also known as macrophage colony-stimulating factor). Macrophages from me/me mice hyperproliferate in response to CSF-1. In the absence of SHPTP1, the CSF-1 receptor (CSF-1R) is hyperphosphorylated upon CSF-1 stimulation, suggesting that SHPTP1 dephosphorylates the CSF-1R. At least some CSF-1R-associated proteins also are hyperactivated. SHPTP1 is associated constitutively, via its SH2 domains, with an unidentified 130-kDa phosphotyrosyl protein (P130). P130 and SHPTP1 are further tyrosyl phosphorylated upon CSF-1 stimulation. Tyrosyl-phosphorylated SHPTP1 binds to Grb2 via the Grb2 SH2 domain. Moreover, in me/me macrophages, Grb2 is associated, via its SH3 domains, with several tyrosyl phosphoproteins. These proteins are hyperphosphorylated on tyrosyl residues in me/me macrophages, suggesting that Grb2 may recruit substrates for SHPTP1. Our results indicate that SHPTP1 is a critical negative regulator of CSF-1 signaling in vivo and suggest a potential new function for Grb2.

Animals↗