Active O-acetylserine-(thiol) lyase A and B confer improved selenium resistance and degrade l-Cys and l-SeCys in Arabidopsis.

The roles of cytosolic O-acetylserine-(thiol)-lyase A (OASTLA), chloroplastic OASTLB, and mitochondrial OASTLC in plant selenate resistance were studied in Arabidopsis. Impairment in OASTLA and OASTLB resulted in reduced biomass, chlorophyll and soluble protein content compared with selenate-treated OASTLC-impaired and wild-type plants. The generally lower total selenium (Se), protein-Se, organic-sulfur and protein-sulfur (S) content in oastlA and oastlB compared with wild-type and oastlC leaves indicated that Se accumulation was not the main cause for the stress symptoms in these mutants. Notably, the application of selenate positively induced S-starvation markers and the OASTLs, followed by increased sulfite reductase, sulfite oxidase activities, and increased sulfite and sulfide concentrations. Taken together, our results indicate a futile anabolic S-starvation response that resulted in lower glutathione and increased oxidative stress symptoms in oastlA and oastlB mutants. In-gel assays of l-cysteine and l-seleno-cysteine, desulfhydrase activities revealed that two of the three OASTL activity bands in each of the oastl single mutants were enhanced in response to selenate, whereas the impaired proteins exhibited a missing activity band. The absence of differently migrated activity bands in each of the three oastl mutants indicates that these OASTLs are major components of desulfhydrase activity, degrading l-cysteine and l-seleno-cysteine in Arabidopsis.

Cytokinin is involved in TPS22-mediated selenium tolerance in Arabidopsis thaliana.

Background and Aims: Excess selenium (Se) is toxic to plants, but relatively little is known about the regulatory mechanism of plant Se tolerance. This study explored the role of the TPS22 gene in Se tolerance in Arabidopsis thaliana. Methods: Arabidopsis wild type and XVE mutant seeds were grown on half-strength MS media containing Na2SeO3 for screening of the Se-tolerant mutant tps22. The XVE T-DNA-tagged genomic sequence in tps22 was identified by TAIL-PCR. The TPS22 gene was transformed into the mutant tps22 and wild type plants using the flower infiltration method. Wild type, tps22 mutant and transgenic seedlings were cultivated on vertical plates for phenotype analysis, physiological index measurement and gene expression analysis. Key Results: We identified an Arabidopsis Se-tolerant mutant tps22 from the XVE pool lines, and cloned the gene which encodes the terpenoid synthase (TPS22). TPS22 was downregulated by Se stress, and loss-of-function of TPS22 resulted in decreased Se accumulation and enhanced Se tolerance; by contrast, overexpression of TPS22 showed similar traits to the wild type under Se stress. Further analysis revealed that TPS22 mediated Se tolerance through reduction of Se uptake and activation of metabolism detoxification, which decreased transcription of high-affinity transporters PHT1;1, PHT1;8 and PHT1;9 and significantly increased transcription of selenocysteine methyltransferase (SMT), respectively. Moreover, loss-of-function of TPS22 resulted in reduced cytokinin level and repression of cytokinin signalling components AHK3 and AHK4, and upregulation of ARR3, ARR15 and ARR16. Exogenous cytokinin increased transcription of PHT1;1, PHT2;1 and SMT and decreased Se tolerance of the tps22 mutant. In addition, enhanced Se resistance of the tps22 mutant was associated with glutathione (GSH). Conclusions: Se stress downregulated TPS22, which reduced endogenous cytokinin level, and then affected the key factors of Se uptake and metabolism detoxification. This cascade of events resulted in reduced Se accumulation and enhanced Se tolerance.

Concurrent Overexpression of Arabidopsis thaliana Cystathionine γ-Synthase and Silencing of Endogenous Methionine γ-Lyase Enhance Tuber Methionine Content in Solanum tuberosum.

Potatoes (Solanum tuberosum) are deficient in methionine, an essential amino acid in human and animal diets. Higher methionine levels increase the nutritional quality and promote the typically pleasant aroma associated with baked and fried potatoes. Several attempts have been made to elevate tuber methionine levels by genetic engineering of methionine biosynthesis and catabolism. Overexpressing Arabidopsis thaliana cystathionine γ-synthase (AtCGS) in S. tuberosum up-regulates a rate-limiting step of methionine biosynthesis and increases tuber methionine levels. Alternatively, silencing S. tuberosum methionine γ-lyase (StMGL), which causes decreased degradation of methionine into 2-ketobutyrate, also increases methionine levels. Concurrently enhancing biosynthesis and reducing degradation were predicted to provide further increases in tuber methionine content. Here we report that S. tuberosum cv. Désirée plants with AtCGS overexpression and StMGL silenced by RNA interference are morphologically normal and accumulate higher free methionine levels than either single-transgenic line.

Kinetic characterization of the human O-phosphoethanolamine phospho-lyase reveals unconventional features of this specialized pyridoxal phosphate-dependent lyase.

Human O-phosphoethanolamine (PEA) phospho-lyase is a pyridoxal 5'-phosphate (PLP) dependent enzyme that catalyzes the degradation of PEA to acetaldehyde, phosphate and ammonia. Physiologically, the enzyme is involved in phospholipid metabolism and is expressed mainly in the brain, where its expression becomes dysregulated in the course of neuropsychiatric diseases. Mechanistically, PEA phospho-lyase shows a remarkable substrate selectivity, strongly discriminating against other amino compounds structurally similar to PEA. Herein, we studied the enzyme under steady-state and pre-steady-state conditions, analyzing its kinetic features and getting insights into the factors that contribute to its specificity. The pH dependence of the catalytic parameters and the pattern of inhibition by the product phosphate and by other anionic compounds suggest that the active site of PEA phospho-lyase is optimized to bind dianionic groups and that this is a prime determinant of the enzyme specificity towards PEA. Single- and multiple-wavelength stopped-flow studies show that upon reaction with PEA the main absorption band of PLP (λmax  = 412 nm) rapidly blue-shifts to ~ 400 nm. Further experiments suggest that the newly formed and rather stable 400-nm species most probably represents a Michaelis (noncovalent) complex of PEA with the enzyme. Accumulation of such an early intermediate during turnover is unusual for PLP-dependent enzymes and appears counterproductive for absolute catalytic performance, but it can contribute to optimize substrate specificity. PEA phospho-lyase may hence represent a case of selectivity-efficiency tradeoff. In turn, the strict specificity of the enzyme seems important to prevent inactivation by other amines, structurally resembling PEA, that occur in the brain.

Successful fertilization requires the presence of at least one major O-acetylserine(thiol)lyase for cysteine synthesis in pollen of Arabidopsis.

The synthesis of cysteine (Cys) is a master control switch of plant primary metabolism that coordinates the flux of sulfur with carbon and nitrogen metabolism. In Arabidopsis (Arabidopsis thaliana), nine genes encode for O-acetylserine(thiol)lyase (OAS-TL)-like proteins, of which the major isoforms, OAS-TL A, OAS-TL B, and OAS-TL C, catalyze the formation of Cys by combining O-acetylserine and sulfide in the cytosol, the plastids, and the mitochondria, respectively. So far, the significance of individual OAS-TL-like enzymes is unresolved. Generation of all major OAS-TL double loss-of-function mutants in combination with radiolabeled tracer studies revealed that subcellular localization of OAS-TL proteins is more important for efficient Cys synthesis than total cellular OAS-TL activity in leaves. The absence of oastl triple embryos after targeted crosses indicated the exclusiveness of Cys synthesis by the three major OAS-TLs and ruled out alternative sulfur fixation by other OAS-TL-like proteins. Analyses of oastlABC pollen demonstrated that the presence of at least one functional OAS-TL isoform is essential for the proper function of the male gametophyte, although the synthesis of histidine, lysine, and tryptophan is dispensable in pollen. Comparisons of oastlABC pollen derived from genetically different parent plant combinations allowed us to separate distinct functions of Cys and glutathione in pollen and revealed an additional role of glutathione for pollen germination. In contrast, female gametogenesis was not affected by the absence of major OAS-TLs, indicating significant transport of Cys into the developing ovule from the mother plant.

Inhibition of Arabidopsis O-acetylserine(thiol)lyase A1 by tyrosine nitration.

The last step of sulfur assimilation is catalyzed by O-acetylserine(thiol)lyase (OASTL) enzymes. OASTLs are encoded by a multigene family in the model plant Arabidopsis thaliana. Cytosolic OASA1 enzyme is the main source of OASTL activity and thus crucial for cysteine homeostasis. We found that nitrating conditions after exposure to peroxynitrite strongly inhibited OASTL activity. Among OASTLs, OASA1 was markedly sensitive to nitration as demonstrated by the comparative analysis of OASTL activity in nitrated crude protein extracts from wild type and different oastl mutants. Furthermore, nitration assays on purified recombinant OASA1 protein led to 90% reduction of the activity due to inhibition of the enzyme, as no degradation of the protein occurred under these conditions. The reduced activity was due to nitration of the protein because selective scavenging of peroxynitrite with epicatechin impaired OASA1 nitration and the concomitant inhibition of OASTL activity. Inhibition of OASA1 activity upon nitration correlated with the identification of a modified OASA1 protein containing 3-nitroTyr(302) residue. The essential role of the Tyr(302) residue for the catalytic activity was further demonstrated by the loss of OASTL activity of a Y302A-mutated version of OASA1. Inhibition caused by Tyr(302) nitration on OASA1 activity seems to be due to a drastically reduced O-acetylserine substrate binding to the nitrated protein, and also to reduced stabilization of the pyridoxal-5'-phosphate cofactor through hydrogen bonds. This is the first report identifying a Tyr nitration site of a plant protein with functional effect and the first post-translational modification identified in OASA1 enzyme.

The effects of enhanced methionine synthesis on amino acid and anthocyanin content of potato tubers.

BACKGROUND: Potato is a staple food in the diet of the world's population and also being used as animal feed. Compared to other crops, however, potato tubers are relatively poor in the essential amino acid, methionine. Our aim was to increase the methionine content of tubers by co-expressing a gene involved in methionine synthesis with a gene encoding a methionine-rich storage protein in potato plants. RESULTS: In higher plants, cystathionine gamma-synthase (CgS) is the first enzyme specific to methionine biosynthesis. We attempted to increase the methionine content of tubers by expressing the deleted form of the Arabidopsis CgS (CgSDelta90), which is not regulated by methionine, in potato plants. To increase the incorporation of free methionine into a storage protein the CgSDelta90 was co-transformed with the methionine-rich 15-kD beta-zein. Results demonstrated a 2- to 6-fold increase in the free methionine content and in the methionine content of the zein-containing protein fraction of the transgenic tubers. In addition, in line with higher methionine content, the amounts of soluble isoleucine and serine were also increased. However, all of the lines with high level of CgSDelta90 expression were phenotypically abnormal showing severe growth retardation, changes in leaf architecture and 40- to 60% reduction in tuber yield. Furthermore, the colour of the transgenic tubers was altered due to the reduced amounts of anthocyanin pigments. The mRNA levels of phenylalanine ammonia-lyase (PAL), the enzyme catalysing the first step of anthocyanin synthesis, were decreased. CONCLUSION: Ectopic expression of CgSDelta90 increases the methionine content of tubers, however, results in phenotypic aberrations in potato. Co-expression of the 15-kD beta-zein with CgSDelta90 results in elevation of protein-bound methionine content of tubers, but can not overcome the phenotypical changes caused by CgSDelta90 and can not significantly improve the nutritional value of tubers. The level of PAL mRNA and consequently the amount of anthocyanin pigments are reduced in the CgSDelta90 transgenic tubers suggesting that methionine synthesis and production of anthocyanins is linked.

Evaluation of the metal phytoextraction potential of crop legumes. Regulation of the expression of O-acetylserine (thiol)lyase under metal stress.

The metal phytoextraction potential of three legumes belonging to different genera has been studied under greenhouse conditions. Legumes accumulate As and metals mainly in roots, although translocation to shoot is observed. Alfalfa did accumulate the highest concentrations of As and metals in shoots and aerial biomass was less affected by the toxic elements, indicating its good behaviour in phytoextraction. Clover accumulated less metal, but showed larger biomass. EDTA addition enhanced Pb phytoextraction up to levels similar to those described for plants proposed in phytoremediation. The regulation of O-acetylserine (thiol)lyase from legumes under metal stress has been analysed to test the possibility of establishing a possible correlation between the expression of OASTL in the presence of the metals and the metal accumulation in legume plant tissues. Cd and Pb(EDTA) produce the strongest increases of OASTL activity, with the higher enhancement seen in roots, in parallel with the higher metal accumulation. Arsenic produced an increase of root enzyme activity, whereas Cu produced a decrease, mainly in shoots. Western blots using antibodies against an A. THALIANA cytosolic OAS-TL recognised up to five protein bands in crude extracts from LOTUS and clover. A low molecular weight isoform of 32 kDa was induced in the presence of Cd and Pb. A partial RT-PCR sequence from clover has been obtained, showing 86 - 97 % identity with other described OASTLs. The PCR fragment has been used to analyse OASTL mRNA levels of legumes under metal stress. OASTL transcripts were increased by As, Cd, and Pb, especially in roots, where metal accumulation was maximal, while Cu produced a decrease in the transcript levels.

Impact of reduced O-acetylserine(thiol)lyase isoform contents on potato plant metabolism.

Plant cysteine (Cys) synthesis can occur in three cellular compartments: the chloroplast, cytoplasm, and mitochondrion. Cys formation is catalyzed by the enzyme O-acetylserine(thiol)lyase (OASTL) using O-acetylserine (OAS) and sulfide as substrates. To unravel the function of different isoforms of OASTL in cellular metabolism, a transgenic approach was used to down-regulate specifically the plastidial and cytosolic isoforms in potato (Solanum tuberosum). This approach resulted in decreased RNA, protein, and enzymatic activity levels. Intriguingly, H(2)S-releasing capacity was also reduced in these lines. Unexpectedly, the thiol levels in the transgenic lines were, regardless of the selected OASTL isoform, significantly elevated. Furthermore, levels of metabolites such as serine, OAS, methionine, threonine, isoleucine, and lysine also increased in the investigated transgenic lines. This indicates that higher Cys levels might influence methionine synthesis and subsequently pathway-related amino acids. The increase of serine and OAS points to suboptimal Cys synthesis in transgenic plants. Taking these findings together, it can be assumed that excess OASTL activity regulates not only Cys de novo synthesis but also its homeostasis. A model for the regulation of Cys levels in plants is proposed.

Exploring the selenium phytoremediation potential of transgenic Indian mustard overexpressing ATP sulfurylase or cystathionine-gamma-synthase.

Indian mustard (Brassica juncea) plants overexpressing ATP sulfurylase (APS transgenics) were previously shown to have higher shoot selenium (Se) levels and enhanced Se tolerance compared to wild type when supplied with selenate in a hydroponic system. Other transgenic Indian mustard overexpressing cystathionine-gamma-synthase (CGS) showed a higher Se volatilization rate, lower shoot Se levels, and higher Se tolerance than wild type, also in hydroponic studies. In the present study, these APS and CGS transgenics were evaluated for their capacity to accumulate Se from soil that is naturally rich in Se. Wild-type Indian mustard and the Se hyperaccumulator Stanleya pinnata were included for comparison. After 10 weeks on Se soil, the APS transgenics contained 2.5-fold higher shoot Se levels than wild type Indian mustard, similar to those of S. pinnata. The CGS transgenics contained 40% lower shoot Se levels than wild type. Shoot biomass was comparable for all Indian mustard types and higher than that of S. pinnata. These results obtained with these transgenics on soil are in agreement with those obtained earlier using hydroponics. The significance of these findings is that they are the first report on the performance of transgenic plants on Se in soil and show the potential of genetic engineering for phytoremediation.

Enhancement of the primary flavor compound methional in potato by increasing the level of soluble methionine.

The primary flavor compound in potato, methional, is synthesized from methionine by the Strecker degradation reaction. A major problem associated with potato processing is the loss of methional. Methional or its precursor, methionine, is not added back during potato processing due to high costs of production. A novel approach to enhance the methional level in processed potato would be to increase the production of its precursor, soluble methionine (Met). Cystathionine gamma-synthase (CGS) is a key enzyme regulating methionine biosynthesis in plants. To increase the level of soluble methionine in potato, Arabidopsis thaliana CGS cDNA was introduced under transcriptional control of the cauliflower mosaic virus 35S promoter into Russet Burbank potato by Agrobacterium-mediated transformation. Ten different transgenic potato lines (CGS1-10) were analyzed. Immunoblot analysis demonstrated that Arabidopsis CGS is expressed in the leaves, tubers, and roots of transgenic potato plants. CGS enzymatic activity was higher in the leaves and roots of the transgenic potato lines compared to the wild-type potato. Methionine levels in the leaves, roots and tubers of transgenic potato lines were enhanced as high as 6-fold compared to those in wild type potato plants. The methional level in baked tubers of field-grown transgenic potato lines was increased between 2.4- and 4.4-fold in lines CGS1, CGS2, and CGS4. The increase observed in methional levels correlated with the soluble methionine level in the tubers from the same lines measured before processing. These results provide the first evidence that the methional level can be enhanced in processed potatoes by increasing the production of its precursor, methionine.

Functional analysis of cystathionine gamma-synthase in genetically engineered potato plants.

In plants, metabolic pathways leading to methionine (Met) and threonine diverge at the level of their common substrate, O-phosphohomoserine (OPHS). To investigate the regulation of this branch point, we engineered transgenic potato (Solanum tuberosum) plants affected in cystathionine gamma-synthase (CgS), the enzyme utilizing OPHS for the Met pathway. Plants overexpressing potato CgS exhibited either: (a) high transgene RNA levels and 2.7-fold elevated CgS activities but unchanged soluble Met levels, or (b) decreased transcript amounts and enzyme activities (down to 7% of wild-type levels). In leaf tissues, these cosuppression lines revealed a significant reduction of soluble Met and an accumulation of OPHS. Plants expressing CgS antisense constructs exhibited reductions in enzyme activity to as low as 19% of wild type. The metabolite contents of these lines were similar to those of the CgS cosuppression lines. Surprisingly, neither increased nor decreased CgS activity led to visible phenotypic alterations or significant changes in protein-bound Met levels in transgenic potato plants, indicating that metabolic flux to Met synthesis was not greatly affected. Furthermore, in vitro feeding experiments revealed that potato CgS is not subject to feedback regulation by Met, as reported for Arabidopsis. In conclusion, our results demonstrate that potato CgS catalyzes a near-equilibrium reaction and, more importantly, does not display features of a pathway-regulating enzyme. These results are inconsistent with the current hypothesis that CgS exerts major Met metabolic flux control in higher plants.

Engineering of cysteine and methionine biosynthesis in potato.

Methionine and cysteine, two amino acids containing reduced sulfur, are not only an important substrate of protein biosynthesis but are also precursors of various other metabolites such as glutathione, phytochelatines, S-adenosylmethionine, ethylene, polyamines, biotin, and are involved as methyl group donor in numerous cellular processes. While methionine is an essential amino acid due to an inability of monogastric animals and human beings to synthesise this metabolite, animals are still able to convert methionine consumed with their diet into cysteine. Thus, a balanced diet containing both amino acids is necessary to provide a nutritionally favourable food or feed source. Because the concentrations of methionine and cysteine are often low in edible plant sources, e.g. potato, considerable efforts in plant breeding and research have been and are still performed to understand the physiological, biochemical, and molecular mechanisms that contribute to their synthesis, transport, and accumulation in plants. During the last decade molecular tools have enabled the isolation of most of the genes involved in cysteine and methionine biosynthesis, and the efficient plant transformation technology has allowed the creation of transgenic plants that are altered in the activity of individual genes. The physiological analysis of these transgenic plants has contributed considerably to our current understanding of how amino acids are synthesised. We focused our analysis on potato (Solanum tuberosum cv. Désirée) as this plant provides a clear separation of source and sink tissues and, for applied purposes, already constitutes a crop plant. From the data presented here and in previous work we conclude that threonine synthase and not cystathionine gamma-synthase as expected from studies of Arabidopsis constitutes the main regulatory control point of methionine synthesis in potato. This article aims to cover the current knowledge in the area of molecular genetics of sulfur-containing amino acid biosynthesis and will provide new data for methionine biosynthesis in solanaceous plants such as potato.

Antisense inhibition of threonine synthase leads to high methionine content in transgenic potato plants.

Methionine (Met) and threonine (Thr) are members of the aspartate family of amino acids. In plants, their biosynthetic pathways diverge at the level of O-phosphohomo-serine (Ser). The enzymes cystathionine gamma-synthase and Thr synthase (TS) compete for the common substrate O-phosphohomo-Ser with the notable feature that plant TS is activated through S-adenosyl-Met, a metabolite derived from Met. To investigate the regulation of this branch point, we engineered TS antisense potato (Solanum tuberosum cv Désirée) plants using the constitutive cauliflower mosaic virus 35S promoter. In leaf tissues, these transgenics exhibit a reduction of TS activity down to 6% of wild-type levels. Thr levels are reduced to 45% wild-type controls, whereas Met levels increase up to 239-fold depending on the transgenic line and environmental conditions. Increased levels of homo-Ser and homo-cysteine indicate increased carbon allocation into the aspartate pathway. In contrast to findings in Arabidopsis, increased Met content has no detectable effect on mRNA or protein levels or on the enzymatic activity of cystathionine gamma-synthase in potato. Tubers of TS antisense potato plants contain a Met level increased by a factor of 30 and no reduction in Thr. These plants offer a major biotechnological advance toward the development of crop plants with improved nutritional quality.

The cytosolic O-acetylserine(thiol)lyase gene is regulated by heavy metals and can function in cadmium tolerance.

Regulation of the expression of the cytosolic O-acetylserine(thiol)lyase gene (Atcys-3A) from Arabidopsis thaliana under heavy metal stress conditions has been investigated. Northern blot analysis of Atcys-3A expression shows a 7-fold induction after 18 h of cadmium treatment. Addition of 50 microm CdCl(2) to the irrigation medium of mature Arabidopsis plants induces a rapid accumulation of the mRNA throughout the leaf lamina, the root and stem cortex, and stem vascular tissues when compared with untreated plants, as observed by in situ hybridization. High pressure liquid chromatography analysis of GSH content shows a transient increase after 18 h of metal treatment. Our results are compatible with a high cysteine biosynthesis rate under heavy metal stress required for the synthesis of GSH and phytochelatins, which are involved in the plant detoxification mechanism. Arabidopsis-transformed plants overexpressing the Atcys-3A gene by up to 9-fold show increased tolerance to cadmium when grown in medium containing 250 microm CdCl(2), suggesting that increased cysteine availability is responsible for cadmium tolerance. In agreement with these results, exogenous addition of cystine can, to some extent, also favor the growth of wild-type plants in cadmium-containing medium. Cadmium accumulates to higher levels in leaves of tolerant transformed lines than in wild-type plants.

cDNA cloning, expression and functional characterization of an Arabidopsis thaliana homologue of the Escherichia coli DNA repair enzyme endonuclease III.

Reactive oxygen species (ROS) are ubiquitous DNA-damaging agents, and the repair of oxidative DNA lesions is essential to prevent mutations and cell death. Escherichia coli endonuclease III is the prototype repair enzyme for removal of oxidized pyrimidines from DNA. A database homology search identified a genomic sequence in Arabidopsis thaliana encoding a predicted protein with sequence similarity to E. coli endonuclease III. We cloned, sequenced and expressed the corresponding cDNA, which encodes a 39.1 kDa protein containing several sequence motifs conserved in endonuclease III homologues, including an iron-sulfur cluster domain and critical residues at the active site. The protein, designated AtNTH1, was over-expressed in E. coli and purified to apparent homogeneity. AtNTH1 exhibits DNA-glycosylase activity on different types of DNA substrates with pyrimidine damage, being able to release both urea and thymine glycol from double-stranded polydeoxyribonucleotides. The enzyme also possesses an apurinic/apyrimidinic lyase activity on UV- and gamma-irradiated DNA substrates. The AtNTH1 gene contains 10 introns and 11 exons and is widely expressed in different plant tissues. Our results suggest that AtNTH1 is a structural and functional homologue of endonuclease III and probably plays a major role in plant defence against oxidative DNA damage.

Three to four-year-old nonpassaged EGF-responsive neural progenitor cells: proliferation, apoptosis, and DNA repair.

Epidermal growth factor responsive (EGFr) neural progenitor (NP) cells have been shown to be a potential alternative tissue source for neural transplantation and for developmental study. We have shown that nonpassaged EGFr NP cells can self-renew for 2 years in neurospheres and can robustly differentiate into glia and a number of neuronal cell types. We are now attempting to investigate if the EGFr NP cells will die or continue to live beyond the life span of the donor. In addition, we and other investigators have also found that EGFr NP cells, after transplant, retain only a small number of cells in the transplant site. In this study, we investigate the plasticity and fate of the EGFr NP cells. Using the nonpassaged method, we found EGFr NP cells live in the EGF supplement medium for over 4 years-the longest-lived EGFr NP cells ever reported. The 4-year-old striatal or cortical EGFr neurospheres, when subplated with substrate coating, migrate out of neurospheres and have robust growth with many processes. Furthermore, when nucleotide marker bromodeoxyuridine (BrdU) was added 3 days prior to the subplating, the EGFr NP cells were labeled positively with BrdU in the nucleus, indicating active proliferation activity. Meanwhile two other events were also found in the long-term EGFr NP cells. In the midst of the proliferation, apoptosis occurred. A subpopulation of EGFr NP cells are undergoing programmed cell death as indicated by the cell morphology and the TUNEL staining for DNA strand breaks. The TUNEL fluorescein-staining indicates that over 50% of EGFr NP cells are positive in the nuclei. On the other hand, we have also found that the major base excision repair enzyme, APE/ref-1, which is responsible for recognizing and repairing baseless sites in DNA, was present in the progenitor cells. However, in those cells undergoing apoptosis, APE/ref-1 levels were dramatically reduced or missing, and only a small percentage of cells were TUNEL and APE/ref-1 positive. These observations indicate that EGFr neural progenitor cells can live beyond the life span of the donor animal. The longevity of these cells in culture may be enhanced due to decreased apoptosis and the retention of normal DNA repair capacity.

The food of sweet and bitter fancy.

The MAP30 ribosomal inactivating protein structure has been determined by NMR spectroscopy. This anti-HIV and anti-cancer protein is an RNA and DNA glycosylase as well as a DNA apurinic/apyrimidinic (AP) lyase.