Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 2. Treatment of some ROS- and Age-related diseases (heart arrhythmia, heart infarctions, kidney ischemia, and stroke)

Effects of 10-(6′-plastoquinonyl) decyltriphenylphosphonium (SkQ1) and 10-(6′-plastoquinonyl) decylrhod-amine 19 (SkQR1) on rat models of H₂O₂- and ischemia-induced heart arrhythmia, heart infarction, kidney ischemia, and stroke have been studied ex vivo and in vivo. In all the models listed, SkQ1 and/or SkQR1 showed pronounced protective effect. Supplementation of food with extremely low SkQ1 amount (down to 0.02 nmol SkQ1/kg per day for 3 weeks) was found to abolish the steady heart arrhythmia caused by perfusion of isolated rat heart with H₂O₂ or by ischemia/reperfusion. Higher SkQ1 (125–250 nmol/kg per day for 2–3 weeks) was found to decrease the heart infarction region induced by an in vivo ischemia/reperfusion and lowered the blood levels of lactate dehydrogenase and creatine kinase increasing as a result of ischemia/reperfusion. In single-kidney rats, ischemia/reperfusion of the kidney was shown to kill the majority of the animals in 2–4 days, whereas one injection of SkQ1 or SkQR1 (1 μmol/kg a day before ischemia) saved lives of almost all treated rats. Effect of SkQR1 was accompanied by decrease in ROS (reactive oxygen species) level in kidney cells as well as by partial or complete normalization of blood creatinine and of some other kidney-controlled parameters. On the other hand, this amount of SkQ1 (a SkQ derivative of lower membrane-penetrating ability than SkQR1) saved the life but failed to normalize ROS and creatinine levels. Such an effect indicates that death under conditions of partial kidney dysfunction is mediated by an organ of vital importance other than kidney, the organ in question being an SkQ1 target. In a model of compression brain ischemia/reperfusion, a single intraperitoneal injection of SkQR1 to a rat (1 μmol/kg a day before operation) effectively decreased the damaged brain area. SkQ1 was ineffective, most probably due to lower permeability of the blood-brain barrier to this compound. Electronic Supplementary Material  Supplementary material is available for this article at and is accessible for authorized users. Published in Russian in Biokhimiya, 2008, Vol. 73, No. 12, pp. 1607–1621.     

Mitochondria-targeted plastoquinone derivative SkQ1 increases early reproduction of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> at the cost of early survival

It was previously found that 20 pM SkQ1 (10-(6′-plastoquinonyl) decyltriphenylphosphonium) solution increased lifespan of virgin females and males of the fruit fly Drosophila melanogaster with maximal and highly reproducible effect on early survival of females. In this paper we demonstrate that SkQ1 solution of the same concentration does not increase lifespan of mated females and males, early effect on female survival being absent, whereas early fertility and the total number of progeny are elevated in treated flies. Increase in fertility observed in young mated females instead of increase of survival typical for young virgin females might illustrate the trade-off between the fly’s lifespan and reproduction.     

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 3. Inhibitory effect of SkQ1 on tumor development from p53-deficient cells

It was proposed that increased level of mitochondrial reactive oxygen species (ROS), mediating execution of the aging program of an organism, could also be critical for neoplastic transformation and tumorigenesis. This proposal was addressed using new mitochondria-targeted antioxidant SkQ1 (10-(6′-plastoquinonyl) decyltriphenylphosphonium) that scavenges ROS in mitochondria at nanomolar concentrations. We found that diet supplementation with SkQ1 (5 nmol/kg per day) suppressed spontaneous development of tumors (predominantly lymphomas) in p53^-/- mice. The same dose of SkQ1 inhibited the growth of human colon carcinoma HCT116/p53^-/- xenografts in athymic mice. Growth of tumor xenografts of human HPV-16-associated cervical carcinoma SiHa was affected by SkQ1 only slightly, but survival of tumor-bearing animals was increased. It was also shown that SkQ1 inhibited the tumor cell proliferation, which was demonstrated for HCT116 p53^-/- and SiHa cells in culture. Moreover, SkQ1 induced differentiation of various tumor cells in vitro. Coordinated SkQ1-initiated changes in cell shape, cytoskeleton organization, and E-cadherin-positive intercellular contacts were observed in epithelial tumor cells. In Ras- and SV40-transformed fibroblasts, SkQ1 was found to initiate reversal of morphological transformation of a malignant type, restoring actin stress fibers and focal adhesion contacts. SkQ1 suppressed angiogenesis in Matrigel implants, indicating that mitochondrial ROS could be important for tumor angiogenesis. This effect, however, was less pronounced in HCT116/p53^-/- tumor xenografts. We have also shown that SkQ1 and related positively charged antioxidants are substrates of the P-glycoprotein multidrug resistance pump. The lower anti-tumor effect and decreased intracellular accumulation of SkQ1, found in the case of HCT116 xenografts bearing mutant forms of p53, could be related to a higher level of P-glycoprotein. The effects of traditional antioxidant N-acetyl-L-cysteine (NAC) on tumor growth and tumor cell phenotype were similar to the effects of SkQ1 but more than 1,000,000 times higher doses of NAC than those of SkQ1 were required. Extremely high efficiency of SkQ1, related to its accumulation in the mitochondrial membrane, indicates that mitochondrial ROS production is critical for tumorigenesis at least in some animal models. Electronic Supplementary Material  Supplementary material is available for this article at and is accessible for authorized users. Published in Russian in Biokhimiya, 2008, Vol. 73, No. 12, pp. 1622–1640.     

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 4. Age-related eye disease. SkQ1 returns vision to blind animals

Mitochondria-targeted cationic plastoquinone derivative SkQ1 (10-(6′-plastoquinonyl) decyltriphenylphosphonium) has been investigated as a potential tool for treating a number of ROS-related ocular diseases. In OXYS rats suffering from a ROS-induced progeria, very small amounts of SkQ1 (50 nmol/kg per day) added to food were found to prevent development of age_induced cataract and retinopathies of the eye, lipid peroxidation and protein carbonylation in skeletal muscles, as well as a decrease in bone mineralization. Instillation of drops of 250 nM SkQ1 reversed cataract and retinopathies in 3-12-month-old (but not in 24-month-old) OXYS rats. In rabbits, experimental uveitis and glaucoma were induced by immunization with arrestin and injections of hydroxypropyl methyl cellulose to the eye anterior sector, respectively. Uveitis was found to be prevented or reversed by instillation of 250 nM SkQ1 drops (four drops per day). Development of glaucoma was retarded by drops of 5 μM SkQ1 (one drop daily). SkQ1 was tested in veterinarian practice. A totally of 271 animals (dogs, cats, and horses) suffering from retinopathies, uveitis, conjunctivitis, and cornea diseases were treated with drops of 250 nM SkQ1. In 242 cases, positive therapeutic effect was obvious. Among animals suffering from retinopathies, 89 were blind. In 67 cases, vision returned after SkQ1 treatment. In ex vivo studies of cultivated posterior retina sector, it was found that 20 nM SkQ1 strongly decreased macrophagal transformation of the retinal pigmented epithelial cells, an effect which might explain some of the above SkQ1 activities. It is concluded that low concentrations of SkQ1 are promising in treating retinopathies, cataract, uveitis, glaucoma, and some other ocular diseases. Electronic Supplementary Material  Supplementary material is available for this article at and is accessible for authorized users. Published in Russian in Biokhimiya, 2008, Vol. 73, No. 12, pp. 1641–1654.     

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 1. Cationic plastoquinone derivatives: Synthesis and in vitro studies

Synthesis of cationic plastoquinone derivatives (SkQs) containing positively charged phosphonium or rhodamine moieties connected to plastoquinone by decane or pentane linkers is described. It is shown that SkQs (i) easily penetrate through planar, mitochondrial, and outer cell membranes, (ii) at low (nanomolar) concentrations, posses strong antioxidant activity in aqueous solution, BLM, lipid micelles, liposomes, isolated mitochondria, and cells, (iii) at higher (micromolar) concentrations, show pronounced prooxidant activity, the “window” between anti- and prooxidant concentrations being very much larger than for MitoQ, a cationic ubiquinone derivative showing very much lower antioxidant activity and higher prooxidant activity, (iv) are reduced by the respiratory chain to SkQH₂, the rate of oxidation of SkQH₂ being lower than the rate of SkQ reduction, and (v) prevent oxidation of mitochondrial cardiolipin by OH^·. In HeLa cells and human fibroblasts, SkQs operate as powerful inhibitors of the ROS-induced apoptosis and necrosis. For the two most active SkQs, namely SkQ1 and SkQR1, C _1/2 values for inhibition of the H₂O₂-induced apoptosis in fibroblasts appear to be as low as 1·10⁻¹¹ and 8·10⁻¹³ M, respectively. SkQR1, a fluorescent representative of the SkQ family, specifically stains a single type of organelles in the living cell, i.e. energized mitochondria. Such specificity is explained by the fact that it is the mitochondrial matrix that is the only negatively-charged compartment inside the cell. Assuming that the Δψ values on the outer cell and inner mitochondrial membranes are about 60 and 180 mV, respectively, and taking into account distribution coefficient of SkQ1 between lipid and water (about 13,000: 1), the SkQ1 concentration in the inner leaflet of the inner mitochondrial membrane should be 1.3·10⁸ times higher than in the extracellular space. This explains the very high efficiency of such compounds in experiments on cell cultures. It is concluded that SkQs are rechargeable, mitochondria-targeted antioxidants of very high efficiency and specificity. Therefore, they might be used to effectively prevent ROS-induced oxidation of lipids and proteins in the inner mitochondrial membrane in vivo. Electronic Supplementary Material  Supplementary material is available for this article at and is accessible for authorized users. Published in Russian in Biokhimiya, 2008, Vol. 73, No. 12, pp. 1589–1606. This and the following four articles were written by the request of the Editorial Board of Biochemistry (Moscow).     

An attempt to prevent senescence: A mitochondrial approach

Antioxidants specifically addressed to mitochondria have been studied to determine if they can decelerate senescence of organisms. For this purpose, a project has been established with participation of several research groups from Russia and some other countries. This paper summarizes the first results of the project. A new type of compounds (SkQs) comprising plastoquinone (an antioxidant moiety), a penetrating cation, and a decane or pentane linker has been synthesized. Using planar bilayer phospholipid membrane (BLM), we selected SkQ derivatives with the highest permeability, namely plastoquinonyl-decyl-triphenylphosphonium (SkQ1), plastoquinonyl-decyl-rhodamine 19 (SkQR1), and methylplastoquinonyldecyltriphenylphosphonium (SkQ3). Anti- and prooxidant properties of these substances and also of ubiquinonyl-decyl-triphenylphosphonium (MitoQ) were tested in aqueous solution, detergent micelles, liposomes, BLM, isolated mitochondria, and cell cultures. In mitochondria, micromolar cationic quinone derivatives were found to be prooxidants, but at lower (sub-micromolar) concentrations they displayed antioxidant activity that decreases in the series SkQ1 = SkQR1 > SkQ3 > MitoQ. SkQ1 was reduced by mitochondrial respiratory chain, i.e. it is a rechargeable antioxidant. Nanomolar SkQ1 specifically prevented oxidation of mitochondrial cardiolipin. In cell cultures, SkQR1, a fluorescent SkQ derivative, stained only one type of organelles, namely mitochondria. Extremely low concentrations of SkQ1 or SkQR1 arrested H₂O₂-induced apoptosis in human fibroblasts and HeLa cells. Higher concentrations of SkQ are required to block necrosis initiated by reactive oxygen species (ROS). In the fungus Podospora anserina, the crustacean Ceriodaphnia affinis, Drosophila, and mice, SkQ1 prolonged lifespan, being especially effective at early and middle stages of aging. In mammals, the effect of SkQs on aging was accompanied by inhibition of development of such age-related diseases and traits as cataract, retinopathy, glaucoma, balding, canities, osteoporosis, involution of the thymus, hypothermia, torpor, peroxidation of lipids and proteins, etc. SkQ1 manifested a strong therapeutic action on some already pronounced retinopathies, in particular, congenital retinal dysplasia. With drops containing 250 nM SkQ1, vision was restored to 67 of 89 animals (dogs, cats, and horses) that became blind because of a retinopathy. Instillation of SkQ1-containing drops prevented the loss of sight in rabbits with experimental uveitis and restored vision to animals that had already become blind. A favorable effect of the same drops was also achieved in experimental glaucoma in rabbits. Moreover, the SkQ1 pretreatment of rats significantly decreased the H₂O₂ or ischemia-induced arrhythmia of the isolated heart. SkQs strongly reduced the damaged area in myocardial infarction or stroke and prevented the death of animals from kidney ischemia. In p53^−/− mice, 5 nmol/kg × day SkQ1 decreased the ROS level in the spleen and inhibited appearance of lymphomas to the same degree as million-fold higher concentration of conventional antioxidant NAC. Thus, SkQs look promising as potential tools for treatment of senescence and age-related diseases.     

Effect of a mitochondria-targeted antioxidant on ageing of <Emphasis Type="Italic">Podospora anserina</Emphasis>: Ultrastructural study

A beneficial effect of mitochondria targeted antioxidant (MTA) SkQ1 added to the culture medium on life span of Podospora anserina was revealed. As was shown earlier, optimal concentration was 400 nM. SkQ1 was shown to increase P. anserina life span 2.4 times, at the same time maintaining native cell ultrastructure: degradative alterations were not revealed. Significant reorganization of P. anserina cell ultrastructure was detected. These changes were not described earlier. The greatest structural changes proceeded in mitochondria. Specific character of these alterations suggests an essential role of oxidative stress in senescence of P. anserina.     

Acarbose, 17‐α‐estradiol,and nordihydroguaiaretic acid extend mouse lifespan preferentially in males

Four agents — acarbose (ACA), 17‐α‐estradiol (EST), nordihydroguaiaretic acid (NDGA), and methylene blue (MB) — were evaluated for lifespan effects in genetically heterogeneous mice tested at three sites. Acarbose increased male median lifespan by 22% (P < 0.0001), but increased female median lifespan by only 5% (P = 0.01). This sexual dimorphism in ACA lifespan effect could not be explained by differences in effects on weight. Maximum lifespan (90th percentile) increased 11% (P < 0.001) in males and 9% (P = 0.001) in females. EST increased male median lifespan by 12% (P = 0.002), but did not lead to a significant effect on maximum lifespan. The benefits of EST were much stronger at one test site than at the other two and were not explained by effects on body weight. EST did not alter female lifespan. NDGA increased male median lifespan by 8–10% at three different doses, with P‐values ranging from 0.04 to 0.005. Females did not show a lifespan benefit from NDGA, even at a dose that produced blood levels similar to those in males, which did show a strong lifespan benefit. MB did not alter median lifespan of males or females, but did produce a small, statistically significant (6%, P = 0.004) increase in female maximum lifespan. These results provide new pharmacological models for exploring processes that regulate the timing of aging and late‐life diseases, and in particular for testing hypotheses about sexual dimorphism in aging and health.     

Effect of the mitochondria-targeted antioxidant SkQ1 on development of spontaneous tumors in BALB/c mice

The mitochondria-targeted antioxidant SkQ1 (10-(6′-plastoquinonyldecyl)triphenylphosphonium) is a new pharmaceutical substance with a wide spectrum of effects including increase in lifespan of laboratory animals (for example, of BALB/c mice males) and inhibition of development of some experimental tumors and also of tumor cell growth. In this work, the effects of SkQ1 on development of spontaneous tumors in female and male BALB/c mice housed in an SPF-class vivarium were studied. We found that the addition of SkQ1 to drinking water at the dose of 1 and 30 nmol/kg body weight per day throughout the lifespan modified the spectrum of spontaneous tumors in the female mice, decreasing the incidence of follicular lymphomas. SkQ1 at the dose of 1 nmol/kg per day also suppressed the dissemination of these neoplasms, but it did not significantly influence the overall incidence of benign and malignant tumors (including primary multiple tumors) or the lifespan of the tumor-bearing mice (both males and females). Hence, the previously described ability of SkQ1 to increase the lifespan of laboratory BALB/c mice is not related to its anticarcinogenic activity.     

Glycine supplementation extends lifespan of male and female mice

Diets low in methionine extend lifespan of rodents, though through unknown mechanisms. Glycine can mitigate methionine toxicity, and a small prior study has suggested that supplemental glycine could extend lifespan of Fischer 344 rats. We therefore evaluated the effects of an 8% glycine diet on lifespan and pathology of genetically heterogeneous mice in the context of the Interventions Testing Program. Elevated glycine led to a small (4%–6%) but statistically significant lifespan increase, as well as an increase in maximum lifespan, in both males (p = 0.002) and females (p < 0.001). Pooling across sex, glycine increased lifespan at each of the three independent sites, with significance at p = 0.01, 0.053, and 0.03, respectively. Glycine‐supplemented females were lighter than controls, but there was no effect on weight in males. End‐of‐life necropsies suggested that glycine‐treated mice were less likely than controls to die of pulmonary adenocarcinoma (p = 0.03). Of the 40 varieties of incidental pathology evaluated in these mice, none were increased to a significant degree by the glycine‐supplemented diet. In parallel analyses of the same cohort, we found no benefits from TM5441 (an inhibitor of PAI‐1, the primary inhibitor of tissue and urokinase plasminogen activators), inulin (a source of soluble fiber), or aspirin at either of two doses. Our glycine results strengthen the idea that modulation of dietary amino acid levels can increase healthy lifespan in mice, and provide a foundation for further investigation of dietary effects on aging and late‐life diseases.     

Effects of flask sealing and growth regulators on in vitro propagation of neem (Azadirachta indica A. Juss.)

In vitro propagation of neem (Azadirachta indica A. Juss.) may offer an efficient alternative to seed propagation of this species. For optimization of in vitro propagation, different basal salt formulations, growth regulators, and culture container sealants (polytetrafluoroethylene hydrophobic membranes [PTFE]) were evaluated. Nodal segments cultured on Murashige and Skoog (MS) medium showed the highest shoot formation per explant (1.67). Explants cultured in flasks containing MS medium with 0.5 mg L⁻¹ benzyladenine, 0.5 mg L⁻¹ kinetin, and 0.05 mg L⁻¹ naphthaleneacetic acid, and sealed with two PTFE membranes, produced the highest number of shoots (4.04). In contrast, explants cultured in flasks without membranes showed leaf chlorosis and senescence. For plant recovery, regenerants were acclimatized in a substrate of coconut fiber and eucalyptus bark (1:1) and showed 80% survival. Our results indicated that the use of flasks with vents was beneficial for in vitro propagation of this important plant.     

Predicted highly expressed genes in Nocardia farcinica and the implication for its primary metabolism and nocardial virulence

Nocardia farcinica is a Gram positive, filamentous bacterium, and is considered an opportunistic pathogen. In this study, the highly expressed genes in N. farcinica were predicted using the codon adaptation index (CAI) as a numerical estimator of gene expressivity. Using ribosomal protein (RP) genes as references, the top ∼ ∼10% of the genes were predicted to be the predicted highly expressed (PHX) genes in N. farcinica using a CAI cutoff of greater than 0.73. Consistent with earlier analysis of Streptomyces genomes, most of the PHX genes in N. farcinica were involved in various ‘house-keeping’ functions important for cell growth. However, 15 genes putatively involved in nocardial virulence were predicted as PHX genes in N. farcinica, which included genes encoding four Mce proteins, cyclopropane fatty acid synthase which is involved in the modification of cell wall which may be important for nocardia virulence, polyketide synthase PKS13 for mycolic acid synthesis and a non-ribosomal peptide synthetase involved in biosynthesis of a mycobactin-related siderophore. In addition, multiple genes involved in defense against reactive oxygen species (ROS) produced by the phagocyte were predicted with high expressivity, which included alkylhydroperoxide reductase (ahpC), catalase (katG), superoxide dismutase (sodF), thioredoxin, thioredoxin reductase, glutathione peroxidase, and peptide methionine sulfoxide reductase, suggesting that combating against ROS is essential for survival of N. farcinica in host cells. The study also showed that the distribution of PHX genes in the N. farcinica circular chromosome was uneven, with more PHX genes located in the regions close to replication initiation site. The results provided the first estimates of global gene expression patterns in N.␣farcinica, which will be useful in guiding experimental design for further investigations. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Superoxide scavenging activity of plastoquinone derivative 10-(6′-plastoquinonyl)decyltriphenylphosphonium (SkQ1)

The plastoquinone derivative 10-(6′-plastoquinonyl)decyltriphenylphosphonium (SkQ1) has the ability to scavenge superoxide anion radical. This ability is manifested both in vitro and in vivo in experiments with the bacterium Escherichia coli. The protective effect of SkQ1 in vivo significantly exceeds that of ascorbic acid.     

Controlling the oxidoreduction potential of the culture of <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> leads to an earlier initiation of solventogenesis,thus increasing solvent productivity

Fermentative production of solvents (acetone, butanol, and ethanol) by Clostridium acetobutylicum is generally a biphasic process consisting of acidogenesis and solventogenesis. We report that the biphasic metabolism of C. acetobutylicum could be changed by oxidoreduction potential (ORP) regulation. When using air to control the ORP of the fermentation broth at −290 mV, an earlier initiation of solventogenesis was achieved. Solvent production reached 25.6 g l⁻¹ (2.8 g acetone l⁻¹, 16.8 g butanol l⁻¹, 6.0 g ethanol l⁻¹), a 35% increase compared with the ORP uncontrolled process. Metabolic flux analysis revealed that there was a general increase of the central carbon flux in the first 24 h of fermentation when ORP was controlled at −290 mV, compared with the control. Specifically, the solvent ratio (acetone:butanol:ethanol) was changed from 25:64:11 to 11:66:23 at ORP level of −290 mV, which might have resulted from the rigidity at acetyl-CoA node and the flexibility at acetoacetyl-CoA and butyryl-CoA nodes in response to ORP regulation.     

Effect of relative humidity on population growth of <Emphasis Type="Italic">Apolygus lucorum</Emphasis> (Heteroptera: Miridae)

Apolygus lucorum (Meyer-Dür) (Heteroptera: Miridae) is a significant insect pest of cotton, Chinese dates, grapes and many other crops in China, and its populations typically increase after heavy rains. However, the intrinsic mechanism of the rainfall-dependent outbreak is not yet fully understood. In our study, the effect of different relative humidity (RH), 40, 50, 60, 70, and 80% RH, on population growth of A. lucorum was evaluated in the laboratory. High humidity (e.g. 70 and 80% RH) was observed to significantly increase egg and nymph survival, prolong adult life longevity, and improve female fecundity. However, low humidity (e.g. 40 and 50% RH) led to unfavorable effects on survival and fecundity. As a result, the intrinsic capacity for increase (r _m), net production (R _o), and the finite rate of increase (λ) of A. lucorum population greatly increased with increasing relative humidity. Additionally, the relationships between r _m and R _o and relative humidity were good fits to the logistic models y = 36.82/(1 + Exp(10.76 − 0.19x)) (p < 0.001) and y = 0.10/(1 + Exp(9.26 − 0.19x)) (p = 0.003), respectively. This study provides insight into the phenology of A. lucorum, and may contribute to modeling of its population dynamics.     

Micro-scale chlorophyll analysis and developmental expression of a cytokinin oxidase/dehydrogenase gene during leaf development and senescence

Chlorophyll content is a baseline measurement in many plant studies. With the miniaturization of many molecular and biochemical assays the sample size needed for these assays has been greatly reduced. Chlorophyll is commonly determined by spectrophotometry. Traditionally this analysis has been carried out in 10 mm cuvettes with volumes of 0.2–2.0 ml. The NanoDrop™ spectrophotometer requires a sample volume of 1–2 μl which provides the opportunity to analyze far smaller samples. Chlorophyll analyses are a critical part of any study of senescence and the cytokinins are considered to play a key role in regulating senescence. The tissue specific levels of cytokinins are thought to be regulated, at least in part, by expression of the cytokinin degradation enzyme cytokinin oxidase/dehydrogenase (CKX). Here an assay is described for the spectral determination of chlorophyll using the NanoDrop™ spectrophotometer that allows the determination of chlorophyll from part samples of clover leaves while leaving sufficient tissue for quantitative real-time PCR study of CKX expression. The expression of TrCKX2 increased during leaf development along the stolon of Trifolium repens but was stable during the onset and progression of senescence suggesting that TrCKX2 was not an initiator of leaf senescence but may have facilitated the progression.     

Genetic analysis of extended lifespan in Drosophila melanogaster III. On the relationship between artificially selected and wild stocks

Adult lifespans, age‐specific survival, age‐specific mortality, survival times on paraquat, and survival times on DDT were assayed in seven lines of Drosophila melanogaster, including two genetically heterogeneous wild lines recently collected from nature, and three inbred and recombinant inbred lines derived from an artificial selection experiment for increased lifespan. Survival on paraquat is positively correlated with adult lifespan. DDT resistance is uncorrelated with either paraquat resistance or lifespan. The wild lines are unexceptional with respect to average lifespan, paraquat resistance, age‐specific survivorship, and leveling off of mortality rates at advanced ages, but have high levels of DDT resistance. Cluster analysis groups the wild lines with three unselected laboratory stocks in one cluster, while two long‐lived elite recombinant inbred lines form a second cluster. Long‐lived laboratory‐adapted lines are quantitatively differentiated from the wild stocks, both with respect to average adult lifespans and resistance to an oxidizing agent. We reject the ‘recovery’ hypothesis, which proposes that Drosophila artificially selected for long life have phenotypes that merely recover the wild state. This revised version was published online in July 2006 with corrections to the Cover Date.     

Transformation of carotenoid biosynthetic genes using a micro-cross section method in kiwifruit (Actinidia deliciosa cv. Hayward)

Genetic transformation using a micro-cross section (MCS) technique was conducted to improve the carotenoid content in kiwifruit (Actinidia deliciosa cv. Hayward). The introduced carotenoid biosynthetic genes include geranylgeranyl diphosphate synthase (GGPS), phytoene desaturase (PDS), ζ-carotene desaturase (ZDS), β-carotene hydroxylase (CHX), and phytoene synthase (PSY). The transformed explants were selected on half-strength MS medium containing 0.001 mg l⁻¹ of 2,4-D and 0.1 mg l⁻¹ of zeatin, either 5 mg l⁻¹ hygromycin or 25 mg l⁻¹ kanamycin, and 500 mg l⁻¹ cefotaxime. The genomic PCR, genomic Southern blot analysis, and RT-PCR were performed to confirm the integration and expression of the transgenes. The transformation efficiencies of either kanamycin- or hygromycin-resistant shoots ranged from 2.9 to 22.1% depending on the target genes, and from 2.9 to 24.2% depending on the reporter genes. The selection efficiencies ranged from 66.7 to 100% for the target genes and from 95.8 to 100% for the reporter genes. Changes of carotenoid content in the several PCR-positive plants were determined by UPLC analysis. As a result, transgenic plants expressing either GGPS or PSY increased about 1.2- to 1.3-fold in lutein or β-carotene content compared to non-transgenic plants. Our results suggest that the Agrobacterium-mediated transformation efficiency of kiwifruit can be greatly increased by this MCS method and that the carotenoid biosynthetic pathway can be modified in kiwifruit by genetic transformation. Our results further suggest that GGPS and PSY genes could be major target genes to increase carotenoid contents in kiwifruit.