Disruption of a mitochondrial MutS DNA repair enzyme homologue confers drug resistance in the parasite Toxoplasma gondii

MutS homologues (MSHs) are critical components of the eukaryotic mismatch repair machinery. In addition to repairing mismatched DNA, mismatch repair enzymes are known in higher eukaryotes to directly signal cell cycle arrest and apoptosis in response to DNA-damaging agents. Accordingly, mammalian cells lacking certain MSHs are resistant to chemotherapeutic drugs. Interestingly, we have discovered that the disruption of TgMSH-1 , an MSH in the pathogenic parasite, Toxoplasma gondii , confers drug resistance. Through a genetic selection for T. gondii mutants resistant to the antiparasitic drug monensin, we have isolated a strain that is resistant not only to monensin but also to salinomycin and the alkylating agent, methylnitrosourea. We have shown that this phenotype is due to the disruption of TgMSH-1 as the multidrug-resistance phenotype is complemented by a wild-type copy of TgMSH-1 and is recapitulated by a directed disruption of this gene in a wild-type strain. We have also shown that, unlike previously described MSHs involved in signalling, TgMSH-1 localizes to the parasite mitochondrion. These results provide the first example of a mitochondrial MSH that is involved in drug sensitivity and implicate the induction of mitochondrial stress as a mode of action of the widely used drug, monensin.     

Targeted nucleotide repair of cyc1 mutations in Saccharomyces cerevisiae directed by modified single-stranded DNA oligonucleotides

Modified single-stranded DNA oligonucleotides have been used to direct base changes in the CYC1 gene of Saccharomyces cerevisiae. In this process, the oligonucleotide is believed to hybridize to the target site through the action of a DNA recombinase and, once bound, DNA repair enzymes act to excise the nucleotide, replace it, and revert the gene to wild-type status. Nucleotide exchange exhibits a strand bias as, in most cases, a higher level of base reversal appears in cells in which the oligonucleotide is designed to hybridize to the nontemplate strand. But, in one case, a higher level was observed when an oligonucleotide complementary to the transcribed strand was used. Mutant haploid and diploid strains are reverted to wild type at this locus with approximately the same frequency and all strains take up the oligonucleotide with approximately equal efficiency. Some repair preference for certain base mismatches was observed; for example, T/T and C/C mispairs exhibited the highest degree of reactivity. Finally, we demonstrate that proteins involved in DNA pairing can enhance the repair activity up to 22-fold, while others affect the reaction minimally. Taken together, these results confirm the importance and versatility of yeast as a model system to elucidate the factors regulating the frequency of nucleotide exchange directed by oligonucleotides.     

Investigation of the stability of yeast rad52 mutant proteins uncovers post-translational and transcriptional regulation of Rad52p

We investigated the stability of the Saccharomyces cerevisiae Rad52 protein to learn how a cell controls its quantity and longevity. We measured the cellular levels of wild-type and mutant forms of Rad52p when expressed from the RAD52 promoter and the half-lives of the various forms of Rad52p when expressed from the GAL1 promoter. The wild-type protein has a half-life of 15 min. rad52 mutations variably affect the cellular levels of the protein products, and these levels correlate with the measured half-lives. While missense mutations in the N terminus of the protein drastically reduce the cellular levels of the mutant proteins, two mutations--one a deletion of amino acids 210-327 and the other a missense mutation of residue 235--increase the cellular level and half-life more than twofold. These results suggest that Rad52p is subject to post-translational regulation. Proteasomal mutations have no effect on Rad52p half-life but increase the amount of RAD52 message. In contrast to Rad52p, the half-life of Rad51p is >2 hr, and RAD51 expression is unaffected by proteasomal mutations. These differences between Rad52p and Rad51p suggest differential regulation of two proteins that interact in recombinational repair.     

Quantitative detection of hepadnavirus-infected lymphoid cells by in situ PCR combined with flow cytometry: implications for the study of occult virus persistence

The detection of small amounts of viral pathogens in infected cells by classical PCR is hampered by a partial loss of virus nucleic acid due to extraction and by difficulties in discrimination between truly intracellular virus genome material and that possibly adhered to the cell surface. These impediments limit reliable identification of virus traces within infected cells, which are typically encountered in latent and persistent occult infections. In this study, hepadnavirus-specific in situ PCR combined with the enzymatic elimination of extracellular virus and flow cytometry permitted detection of viral genomes in lymphoid cells without nucleic acid isolation and allowed quantification of infected cells during the course of persistent infection with woodchuck hepatitis virus (WHV). The validity of the procedure was confirmed by hybridization analysis of the in situ-amplified viral sequences. The results showed that hepadnavirus can be directly detected within lymphoid cells not only in serologically accountable infection, but also years after recovery from viral hepatitis and in the course of primary occult virus carriage. Percentages of infected peripheral lymphoid cells in symptomatic WHV hepatitis fluctuate between 3.4 and 20.4% (mean +/- standard error of the mean, 9.6% +/- 1.7%), whereas those in persistent, serologically mute WHV infection range from 1.1 to 14.6% (mean +/- standard error of the mean, 4.8% +/- 0.8%) (P = 0.005). The data obtained provide further evidence that WHV infection continues indefinitely in the lymphatic system independently of whether it is symptomatic or concealed. They document that hepadnavirus can be detected in a significant proportion of circulating lymphoid cells in both immunovirologically apparent as well as occult persistent infection.     

Pregnancy zone protein is a carrier and modulator of placental protein-14 in T-cell growth and cytokine production

A successful pregnancy can only occur when the maternal immune system fails to attack the allogeneic fetus. Two plasma proteins with described immunoregulatory activities, pregnancy zone protein (PZP) and placental protein-14 (PP14; also known as glycodelin-A), increase dramatically during pregnancy, prompting us to examine their potential role in mediating fetal protection. First, we demonstrated that both native PZP and its receptor-recognized monoamine-activated form (MA-PZP) bound non-covalently and specifically to PP14, exhibiting K(d) values greater than 3 microM, as determined by surface plasmon resonance. Our evidence further suggests that PZP is potentially a more effective carrier of PP14 than its relative alpha2-macroglobulin. Second, we found that T-cell activation, as measured by increased proliferation and IL-2 production, was inhibited by either PZP or PP14 in a dose-dependent manner. However, when PZP and PP14 were combined, they acted synergistically to inhibit T cell proliferation and IL-2 production. Interestingly, the combination of PZP and PP14 had little effect on the production of T(H)2 cytokine, IL-4. Based upon these findings, we hypothesize that PZP and PP14 form a stable complex in the plasma of pregnant women and together act synergistically to selectively modulate T-cell activation. Mechanistically, this activity appears to be independent of the PZP receptor (CD91) or PZP's anti-proteinase activity.     

The Arabidopsis phenylalanine insensitive growth mutant exhibits a deregulated amino acid metabolism

Amino acids and amino acid analogs have been used in numerous genetic screens to isolate mutants deficient in amino acid biosynthetic pathways or in the regulation of amino acid metabolism. Several of these mutants exhibit relaxed feedback control of branched amino acid biosynthetic pathways and are thus resistant to accumulation of pathway end products. For example, feedback-regulated enzymes of the shikimate pathway are anthranilate synthase on the branch leading to Trp and chorismate mutase on the branch leading to Phe and Tyr. A feedback-insensitive mutant of anthranilate synthase alpha, trp5-1, is resistant to toxic Trp analogs. Mutants resistant to Phe have not previously been reported, and this article describes the isolation of the recessive Arabidopsis Phe insensitive growth mutant pig1-1 by a forward genetic screen. pig1-1 was not only tolerant to Phe, Tyr, and Trp, but also to other, not biosynthetically related amino acids. Amino acid contents in pig1-1 were significantly elevated with respect to wild-type controls but, in contrast to the wild type, dramatically decreased when plants were supplemented with 2 mm Phe. Protein contents were similar in the mutant and the wild type at all tested conditions. Phe catabolism was similar to the wild type in pig1-1 roots but was significantly increased in pig1-1 shoots. Phenylalanine uptake into the root, its root-to-shoot translocation, and Phe and phenylpropanoid contents were unaltered in pig1-1, indicating that pig1-1 is not affected in amino acid translocation or the shikimate pathway. Instead, the response of pig1-1 toward amino acid feeding indicates that amino acid metabolism is generally deregulated in pig1-1.     

Noncompetitive antagonist binding sites in the torpedo nicotinic acetylcholine receptor ion channel. Structure-activity relationship studies using adamantane derivatives

We used a series of adamantane derivatives to probe the structure of the phencyclidine locus in either the resting or desensitized state of the nicotinic acetylcholine receptor (AChR). Competitive radioligand binding and photolabeling experiments using well-characterized noncompetitive antagonists such as the phencyclidine analogue [piperidyl-3,4-(3)H(N)]-N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine ([(3)H]TCP), [(3)H]ethidium, [(3)H]tetracaine, [(14)C]amobarbital, and 3-(trifluoromethyl)-3-(m-[(125)I]iodophenyl)diazirine ([(125)I]TID) were performed. Thermodynamic and structure-function relationship analyses yielded the following results. (1) There is a good structure-function relationship for adamantane amino derivatives inhibiting [(3)H]TCP or [(3)H]tetracaine binding to the resting AChR. (2) Since the same derivatives inhibit neither [(14)C]amobarbital binding nor [(125)I]TID photoincorporation, we conclude that these positively charged molecules preferably bind to the TCP locus, perhaps interacting with alphaGlu(262) residues at position M2-20. (3) The opposite is true for the neutral molecule adamantane, which prefers the TID (or barbiturate) locus instead of the TCP site. (4) The TID site is smaller and more hydrophobic (it accommodates neutral molecules with a maximal volume of 333 +/- 45 A(3)) than the TCP locus, which has room for positively charged molecules with volumes as large as 461 A(3) (e.g., crystal violet). This supports the concept that the resting ion channel is tapering from the extracellular mouth to the middle portion. (5) Finally, although both the hydrophobic environment and the size of the TCP site are practically the same in both states, there is a more obvious cutoff in the desensitized state than in the resting state, suggesting that the desensitization process constrains the TCP locus. A plausible location of neutral and charged adamantane derivatives is shown in a model of the resting ion channel.     

Techniques for monitoring cranberry tipworm (Diptera: Cecidomyiidae) in rabbiteye and southern highbush blueberries

Several monitoring techniques were evaluated for their effectiveness, based on the highest mean captures of cranberry tipworm, Dasineura oxycoccana (Johnson), in detecting D. oxycoccana in rabbiteye, Vaccinium ashei Reade, and southern highbush, V. corymbosum L. x V. darrowi Camp, blueberry plantings. There were no significant differences in captures of D. oxycoccana adults on unbaited sticky board traps, regardless of color (yellow, white, green, or blue). In a separate experiment, three monitoring techniques, yellow unbaited sticky boards, larval/adult emergence from infested buds, and bud dissection, were evaluated for detecting D. oxycoccana, eggs, larvae, and adults. In total, four bud types were examined, including rabbiteye floral, rabbiteye leaf, southern highbush floral, and southern highbush leaf. The emergence monitoring technique detected significantly more D. oxycoccana adults than the other techniques evaluated. Emergence and dissection techniques performed equally well for detecting D. oxycoccana larvae. Dissection was the only technique capable of detecting D. oxycoccana eggs. Overall, the highest numbers of D. oxycoccana eggs were detected in southern highbush leaf buds. However, larval infestation was lower for southern highbush leaf buds compared with other bud types sampled. Hypotheses to explain this phenomenon are discussed. The fewest number of eggs was recorded for southern highbush flower buds, potentially because these buds develop before peak emergence of D. oxycoccana. Managing D. oxycoccana in infested plantings can be improved by incorporating monitoring techniques, specifically bud dissection to search for eggs, that will aid growers in making timely insecticide applications.