Population genetics of the Mediterranean corn borer (Sesamia nonagrioides) differs between wild and cultivated plants.

The population genetic structure of crop pest populations gives information about their spatial ecology, which helps in designing management strategies. In this paper, we investigated the genetic structure of the Mediterranean Corn Borer (MCB), Sesamia nonagrioides Lefèbvre (Lepidoptera: Noctuidae), one of the most important maize pests in the Mediterranean countries, using microsatellite markers for the first time in this species. Insects were collected in twenty-five locations in southwest and southeast France from cultivated and wild host plants (Zea mays, Sorghum halepense and Typha domingensis). Contrary to what has been reported so far in France, we found that MCB populations could be locally abundant on wild poales plants. Analysis was carried out at 11 polymorphic microsatellite markers. Molecular variance was significantly determined by geography, then by host plant, with 17% and 4%, respectively, when considered as a major effect, and with 14% and 1%, respectively, when considered as a marginal effect in permutational analysis. Multidimensional scaling (MDS) and GENELAND Bayesian clustering suggested that populations infecting wild plants (T. domingensis and S. halepense) were more structured locally than those affecting cultivated maize. In S. halepense, significant Isolation By Distance (IBD) indicated that this factor could explain genetic differentiation of the moth populations. In T. domingensis, local population differentiation was strong but did not depend on distance. The implication of this absence of population structure in maize and the heterogeneity of population genetics patterns in wild plants are discussed in the context of the population dynamics hypothesis and population management strategies.

Role of egg-laying behavior, virulence and local adaptation in a parasitoid's chances of reproducing in a new host.

Understanding the ability of parasitoid insects to succeed in new host populations is a relevant question for biological control and adaptive mechanisms. Cotesia typhae is an African parasitoid specialized on the moth Sesamiae nonagrioides, also called the Mediterranean corn borer. Two Kenyan strains of C. typhae differ in their virulence against a new host population from France. We explored behavioral and physiological hypotheses about this differentiation. Cotesia genus belongs to a group of Hymenoptera in which females inject a domesticated virus in their host to overcome its resistance. Since viral particles are injected along with eggs and since the strain with the higher virulence injects more eggs, we hypothesized that virulence could be explained by the quantity of virus injected. To test this assumption, we measured the injected quantities of eggs and viral particles (estimated by viral DNA segments) of each parasitoid strain along several ovipositions, to vary these quantities. Unexpectedly, results showed that virulence against the French host was not correlated to the injected quantities of eggs or viral segments, indicating that virulence differentiation is explained by other causes. The virulence against the respective natural hosts of the two C. typhae strains was also measured, and results suggest that local adaptation to a more resistant natural host may explain the pre-adaptation of one strain to the new host population. We also identified a differentiation of oviposition strategy and subsequent offspring number between the parasitoid strains, which is important in a biocontrol perspective.

Relationship between oviposition, virulence gene expression and parasitism success in Cotesia typhae nov. sp. parasitoid strains.

Studying mechanisms that drive host adaptation in parasitoids is crucial for the efficient use of parasitoids in biocontrol programs. Cotesia typhae nov. sp. (Fernández-Triana) (Hymenoptera: Braconidae) is a newly described parasitoid of the Mediterranean corn borer Sesamia nonagrioides (Lefebvre) (Lepidoptera: Noctuidae). Braconidae are known for their domesticated bracovirus, which is injected with eggs in the host larva to overcome its resistance. In this context, we compared reproductive success traits of four Kenyan strains of C. typhae on a French and a Kenyan populations of its host. Differences were found between the four strains and the two most contrasted ones were studied more thoroughly on the French host population. Parasitoid offspring size was correlated with parasitism success and the expression of bracovirus virulence genes (CrV1 and Cystatin) in the host larva after parasitism. Hybrids between these two parasitoid strains showed phenotype and gene expression profiles similar to the most successful parental strain, suggesting the involvement of dominant alleles in the reproductive traits. Ovary dissections revealed that the most successful strain injected more eggs in a single host larva than the less successful one, despite an equal initial ovocyte number in ovaries. It can be expected that the amount of viral particles increase with the number of eggs injected. The ability to bypass the resistance of the allopatric host may in consequence be related to the oviposition behaviour (eggs allocation). The influence of the number of injected eggs on parasitism success and on virulence gene expression was evaluated by oviposition interruption experiments.

The Cotesia sesamiae story: insight into host-range evolution in a Hymenoptera parasitoid and implication for its use in biological control programs.

This review covers nearly 20 years of studies on the ecology, physiology and genetics of the Hymenoptera Cotesia sesamiae, an African parasitoid of Lepidoptera that reduces populations of common maize borers in East and South Africa. The first part of the review presents studies based on sampling of C. sesamiae from maize crops in Kenya. From this agrosystem including one host plant and three main host borer species, studies revealed two genetically differentiated populations of C. sesamiae species adapted to their local host community, and showed that their differentiation involved the joint evolution of virulence genes and sensory mechanisms of host acceptance, reinforced by reproductive incompatibility due to Wolbachia infection status and natural inbreeding. In the second part, we consider the larger ecosystem of wild Poales plant species hosting many Lepidoptera stem borer species that are potential hosts for C. sesamiae. The hypothesis of other host-adapted C. sesamiae populations was investigated based on a large sampling of stem borer larvae on various Poales across sub-Saharan Africa. The sampling provided information on the respective contribution of local hosts, biogeography and Wolbachia in the genetic structure of C. sesamiae populations. Molecular evolution analyses highlighted that several bracovirus genes were under positive selection, some of them being under different selection pressure in C. sesamiae populations adapted to different hosts. This suggests that C. sesamiae host races result from co-evolution acting at the local scale on different bracovirus genes. The third part considers the mechanisms driving specialization. C. sesamiae host races are more or less host-specialized. This character is crucial for efficient and environmentally-safe use of natural enemies for biological control of pests. One method to get an insight in the evolutionary stability of host-parasite associations is to characterize the phylogenetic relationships between the so-called host-races. Based on the construction of a phylogeny of C. sesamiae samples from various host- and plant species, we revealed three main lineages. Mechanisms of differentiation are discussed with regard to the geography and ecology of the samples. One of the lineage presented all the hallmarks of a distinct species, which has been morphologically described and is now studied in the perspective of being used as biological control agent against Sesamia nonagrioides Lefèbvre (Lepidoptera: Noctuidae), a major maize pest in West Africa and Mediterranean countries (see Benoist et al. 2017). The fourth part reviews past and present use of C. sesamiae in biological control, and points out the interest of such molecular ecology studies to reconcile biodiversity and food security stakes in future biological control.

ß-catenin downregulates Dicer to promote ovarian cancer metastasis.

Ovarian cancer is a nearly uniform lethal disease and its highly aggressive metastatic phenotype portends a poor prognosis. Lack of a well-controlled, relevant experimental model has been a major obstacle to identifying key molecules causing metastasis. Here we describe the creation of a new isogenic model of spontaneous human ovarian cancer metastasis exhibiting opposite phenotypes-highly metastatic (HM) and non-metastatic (NM)-both in vitro and in vivo. HM was unique in its ability to metastasize consistently to the peritoneum, mimicking the major dissemination route of human ovarian cancer. In contrast, NM failed to form detectable metastases, although it was equally tumorigenic. Using comparative label-free quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS), we identified ß-catenin, which we demonstrated for the first time as having a direct role in the pathogenesis of ovarian cancer metastasis. Our studies also revealed a previously unrecognized role of ß-catenin in the downregulation of multiple microRNAs (miRNAs) through attenuating miRNA biogenesis by targeting Dicer, a key component of the miRNA-processing machinery. One such downregulated miRNAs was miR-29s involved in epithelial-to-mesenchymal transition and subsequent stem cell traits. Silencing ß-catenin or overexpressing Dicer or miR-29 mimics in HM significantly reduced the ability of these cells to migrate. ß-catenin-knockdown cells also failed to metastasize in an orthotopic model of ovarian cancer. Meta-analysis revealed an increase in CTNNB1 and a decrease in DICER1 expression levels in the high-risk group. These results uncover ß-catenin as a critical factor in promoting ovarian cancer aggressiveness and a new mechanism linking between ß-catenin and miRNA downregulation underlying this process.

Influence of plant silicon in Busseola fusca (Lepidoptera: Noctuidae) larvae - Poaceae interactions.

The noctuid stem borer Busseola fusca is an important pest of maize and sorghum in Sub-Saharan Africa. The presence of this species occurred mostly on cultivated than on wild habitats. Busseola fusca is oligophagous having a narrow range of a wild grass species. This might be due, in part, to differences in silicon (Si) content in plant tissues between cultivated and wild grasses. In the present study, we have tested this hypothesis by studying the survival and the relative growth rate (RGR) expressed as daily weight gains of B. fusca larvae on maize and six wild host plants, mostly present in the natural habitat where B. fusca occurred, and correlated with their Si contents. Survival and RGR of B. fusca larvae were considerably higher on maize and wild sorghum than on the other grass species, and they were negatively related to plant Si content. This was corroborated with results on RGR from artificial diets amended with increasing levels of Si. In addition, if Si was added to maize growing substrate B. fusca larval growth was significantly reduced confirming the involvement of Si in B. fusca larvae - Poaceae interactions. The results provide insight into the possible mechanisms of oligophagy of B. fusca and provide a correlative support for a physical role of plant endogenous Si in impeding feeding of B. fusca larvae.

Effect of temperature on the phenology of Chilo partellus (Swinhoe) (Lepidoptera, Crambidae); simulation and visualization of the potential future distribution of C. partellus in Africa under warmer temperatures through the development of life-table parameters.

Maize (Zea mays) is a major staple food in Africa. However, maize production is severely reduced by damage caused by feeding lepidopteran pests. In East and Southern Africa, Chilo partellus is one of the most damaging cereal stem borers mainly found in the warmer lowland areas. In this study, it was hypothesized that the future distribution and abundance of C. partellus may be affected greatly by the current global warming. The temperature-dependent population growth potential of C. partellus was studied on artificial diet under laboratory conditions at six constant temperatures (15, 18, 20, 25, 28, 30, 32 and 35 °C), relative humidity of 75±5% and a photoperiod of L12:L12 h. Several non-linear models were fitted to the data to model development time, mortality and reproduction of the insect species. Cohort updating algorithm and rate summation approach were stochastically used for simulating age and stage structure populations and generate life-table parameters. For spatial analysis of the pest risk, three generic risk indices (index of establishment, generation number and activity index) were visualized in the geographical information system component of the advanced Insect Life Cycle modeling (ILCYM) software. To predict the future distribution of C. partellus we used the climate change scenario A1B obtained from WorldClim and CCAFS databases. The maps were compared with available data on the current distribution of C. partellus in Kenya. The results show that the development times of the different stages decreased with increasing temperatures ranging from 18 to 35 °C; at the extreme temperatures, 15 and 38 °C, no egg could hatch and no larvae completed development. The study concludes that C. partellus may potentially expands its range into higher altitude areas, highland tropics and moist transitional regions, with the highest maize potential where the species has not been recorded yet. This has serious implication in terms of food security since these areas produce approximately 80% of the total maize in East Africa.

Phylogeography in continuous space: coupling species distribution models and circuit theory to assess the effect of contiguous migration at different climatic periods on genetic differentiation in Busseola fusca (Lepidoptera: Noctuidae).

Current population genetic models fail to cope with genetic differentiation for species with large, contiguous and heterogeneous distribution. We show that in such a case, genetic differentiation can be predicted at equilibrium by circuit theory, where conductance corresponds to abundance in species distribution models (SDMs). Circuit-SDM approach was used for the phylogeographic study of the lepidopteran cereal stemborer Busseola fuscaFüller (Noctuidae) across sub-Saharan Africa. Species abundance was surveyed across its distribution range. SDMs were optimized and selected by cross-validation. Relationship between observed matrices of genetic differentiation between individuals, and between matrices of resistance distance was assessed through Mantel tests and redundancy discriminant analyses (RDAs). A total of 628 individuals from 130 localities in 17 countries were genotyped at seven microsatellite loci. Six population clusters were found based on a Bayesian analysis. The eastern margin of Dahomey gap between East and West Africa was the main factor of genetic differentiation. The SDM projections at present, last interglacial and last glacial maximum periods were used for the estimation of circuit resistance between locations of genotyped individuals. For all periods of time, when using either all individuals or only East African individuals, partial Mantel r and RDA conditioning on geographic distance were found significant. Under future projections (year 2080), partial r and RDA significance were different. From this study, it is concluded that analytical solutions provided by circuit theory are useful for the evolutionary management of populations and for phylogeographic analysis when coalescence times are not accessible by approximate Bayesian simulations.

Two sugar isomers influence host plant acceptance by a cereal caterpillar pest.

Plant sugars are often considered as primary feeding stimuli, conditioning host plant acceptance by herbivorous insects. Of the nine sugars identified from methanolic extracts of seven grass species, only turanose, a sucrose isomer, was negatively correlated with the survival and growth of the noctuid larva of cereal stemborer, Busseola fusca. Sucrose was the most abundant sugar, although it did not vary significantly in concentration among the plant species studied. Using Styrofoam™ cylinders impregnated with increasing concentrations of turanose or sucrose, the two sugars had opposing effects: turanose appeared phagodeterrent while sucrose was phagostimulatory. Electrophysiological studies indicated that B. fusca larvae were able to detect both sugars via their styloconic sensilla located on the mouthparts. The findings indicate that, whereas sucrose is a feeding stimulant and positively influences food choice by B. fusca larvae, turanose negatively contributes to larval food choice. The balance in concentrations of both sugars, however, somehow influences the overall host plant choice made by the larvae. This can partly explain host plant suitability and choice by this caterpillar pest in the field.

Systematics and molecular phylogeny of two African stem borer genera, Sciomesa Tams & Bowden and Carelis Bowden (Lepidoptera: Noctuidae).

Currently, the systematics of the African noctuid stem borers of the subtribe Sesamiina, which include major pests of cereals, is confused. In addition, their ecology is poorly known, as are the factors influencing their evolution. In this paper, we address these shortcomings for two genera of the Sesamiina, Sciomesa Tams & Bowden and Carelis Bowden. Mixed Bayesian phylogenetic analysis, which included their host plants and two mitochondrial genes, showed the genus Sciomesa to be polyphyletic. Two new genera were created, Pirateolea and Feraxinia. The genus Carelis proved to be paraphyletic and was subdivided into two sub-genera. The genera Sciomesa, Carelis and Pirateolea (named the 'Sciomesa genus group') share morphological traits, and the phylogenetic analysis showed that they had a common ancestor living on Cyperaceae and that they were distant from the genus Feraxinia belonging to another clade which had an ancestor living on Poaceae. Seven new species were described: Sciomesa gnosia sp. n., Sciomesia bua sp. n., Pirateolea nola gen. n, sp. n., Feraxinia serena gen. n., sp. n., Carelis australis sp. n., Carelis transversa sp. n. and Carelis agnae sp. n. Ten species were sunk as synonyms: Sciomesa mesoscia (Hampson) syn. n., Sciomesa mirifica Laporte syn. n., Sciomesa constantini Laporte syn. n. and Sciomesa etchecopari Laporte syn. n. are synonyms of Sciomesa mesophaea (Aurivillius); Acrapex sparsipucta Laporte syn. n. is a synonym of Sciomesa excelsa (Laporte) comb. n.; Acrapex congitae Laporte syn. n., Sesamia minuta Laporte syn. n. and Sesamia minuscula Laporte syn. n. are synonyms of Sciomesa boulardi (Laporte) comb. n.; Acrapex bryae Laporte syn. n. and Acrapex fayei Laporte syn. n. are synonyms of Feraxinia jemjemensis (Laporte) comb. n. Eleven new combinations were created: Sciomesa excelsa (Laporte) comb. n., Sciomesa boulardi (Laporte) comb. n., Sciomesa punctipennis (Krüger) comb. n., Pirateolea piscator (Fletcher) comb. n., Pirateolea argocyma (Fletcher) comb. n., Pirateolea cyclophora (Fletcher) comb. n., Pirateolea ochroneura (Fletcher) comb. n., Pirateolea funebris (Krüger) comb. n., Feraxinia nyei (Fletcher) comb. n., Feraxinia jemjemensis (Laporte) comb. n. and Carelis biluma (Nye) comb. n.

Population genetics of Eldana saccharina Walker (Lepidoptera: Pyralidae) and the implications for management using biocontrol.

The African sugarcane stalk borer, Eldona saccharina Walker (Lepidoptera: Pyralidae), is widely distributed throughout sub-Saharan Africa and is an important insect pest of maize and sugarcane. The insect shows significant variation in behaviour, host plant and natural enemy guild in different regions. Several attempts to redistribute the natural enemies of E. saccharina from West Africa to South Africa were unsuccessful. The significant behavioural, host plant and natural enemy variations as well as failures of biocontrol attempts evoked a hypothesis of genetic diversification. To evaluate this hypothesis a molecular analysis was conducted on geographically isolated populations of E. saccharina from East, North, South and West Africa, using the cytochrome c oxidase subunit I (COI) region of the mitochondrial genome. The results revealed that E. saccharina populations are separated into four major units corresponding to the West Africa, Rift Valley, South/East Africa and southern African populations. Mitochondrial DNA divergence among the four populations ranged from 1% to 4.98%. To examine the impact of the observed genetic variation on the fertility of inter-population crosses, a mating experiment was conducted between the Rift valley and South African population to produce an F1 generation, and these were backcrossed with the South African parent population. Fertility of eggs produced by the F1/parent population cross was significantly reduced when compared to fertility of the "true" South African line, and the F1/F1 cross. The contributions of the observed genetic differences and inter population incompatibility for the failure of previous biocontrol attempts are discussed and recommendations on future biocontrol practices are given.

Variability in pheromone communication among different haplotype populations of Busseola fusca.

The relationship between pheromone composition and mitochondrial haplotype clades was investigated by coupling DNA analyses with pheromone identification and male mate searching behavior among different geographic populations of Busseola fusca. The within-population variations in pheromone blend were as great as those observed between geographic populations, suggesting that the female sex pheromone blend was not the basis of reproductive isolation between the geographic clades. Furthermore, while data from wind tunnel experiments demonstrated that most of the tested males were sensitive to small variations in pheromone mixture, there was considerable within-population variability in the observed response. The study identified a new pheromone component, (Z)-11-hexadecen-1-yl acetate, which when added to the currently used three-component synthetic blend resulted in significantly higher traps catches. The new recommended blend for monitoring flight phenology and for timing control measures for optimal efficacy of B. fusca is (Z)-11-tetradecen-1-yl acetate (62%), (E)-11-tetradecen-1-yl acetate (15%), (Z)-9-tetradecen-1-yl acetate (13%), and (Z)-11-hexadecen-1-yl acetate (10%).

Phylogeography and population genetics of the maize stalk borer Busseola fusca (Lepidoptera, Noctuidae) in sub-Saharan Africa.

The population genetics and phylogeography of African phytophagous insects have received little attention. Some, such as the maize stalk borer Busseola fusca, display significant geographic differences in ecological preferences that may be congruent with patterns of molecular variation. To test this, we collected 307 individuals of this species from maize and cultivated sorghum at 52 localities in West, Central and East Africa during the growing season. For all collected individuals, we sequenced a fragment of the mitochondrial cytochrome b. We tested hypotheses concerning the history and demographic structure of this species. Phylogenetic analyses and nested clade phylogeographic analyses (NCPA) separated the populations into three mitochondrial clades, one from West Africa, and two--Kenya I and Kenya II--from East and Central Africa. The similar nucleotide divergence between clades and nucleotide diversity within clades suggest that they became isolated at about the same time in three different refuges in sub-Saharan Africa and have similar demographic histories. The results of mismatch distribution analyses were consistent with the demographic expansion of these clades. Analysis of molecular variance (amova) indicated a high level of geographic differentiation at different hierarchical levels. NCPA suggested that the observed distribution of haplotypes at several hierarchical levels within the three major clades is best accounted for by restricted gene flow with isolation by distance. The domestication of sorghum and the introduction of maize in Africa had no visible effect on the geographic patterns observed in the B. fusca mitochondrial genome.

Diversity of lepidopteran stem borers on monocotyledonous plants in eastern Africa and the islands of Madagascar and Zanzibar revisited.

Surveys were completed in Eritrea, Ethiopia, Kenya, Madagascar, Mozambique, Tanzania, Uganda and Zanzibar to assess the lepidopteran stem borer species diversity on wild host plants. A total of 24,674 larvae belonging to 135 species were collected from 75 species of wild host plants belonging to the Poaceae, Cyperaceae and Typhaceae. Amongst them were 44 noctuid species belonging to at least nine genera, 33 crambids, 15 pyralids, 16 Pyraloidea species not yet identified, 25 tortricids and three cossids. The noctuid larvae represented 73.6% of the total number of larvae collected, with 66.3, 3.5 and 3.8% found on Poaceae, Cyperaceae and Typhaceae, respectively. The Crambidae, Pyralidae, Tortricidae and Cossidae represented 19.8, 1.9, 2.5 and 0.1% of the total larvae collected, respectively, with 90.4% of the Crambidae and Pyralidae collected from Poaceae, and 99.7% of the Tortricidae collected from Cyperaceae. The lepidopteran stem borer species diversity in the wild host plants was far more diverse than previously reported.

Considerable haplotype diversity within the 23kb encompassing the ADH7 gene.

BACKGROUND: Of the seven known human alcohol dehydrogenase (ADH) genes, the non-liver expressed ADH7 gene codes for the enzyme with the highest maximal activity for ethanol. Previous study from our laboratory has suggested that ADH7 has an epistatic role for protection against alcoholism based on a single ADH7 SNP. METHODS: We have now studied seven SNPs, additional populations for the SNP previously examined, and six more new SNPs, across 23 kb of ADH7 in 38 population samples originating from different geographical regions of the world. RESULTS: The overall linkage disequilibrium is moderate to strong across this region even though considerable 7-SNP haplotype diversity is observed. This uncommonly high haplotype diversity is explained by high LD within each "half," the three upstream SNPs and the four downstream SNPs, but near randomization between the "halves." This division significantly simplified the haplotype pattern: only four major haplotypes account for almost all chromosomes in all populations in each "half." CONCLUSIONS: The low linkage disequilibrium between these two "halves" suggests multiple recombination(s) have occurred in this region, specifically, within intron 7. The absence of strong LD between the functional variation in ADH1B that is strongly associated with alcoholism and any of the variation in ADH7 supports the genetic independence of ADH7 in association studies. Thus, the previously observed epistatic effect of ADH7 cannot be explained by its linkage disequilibrium with a causative factor in ADH1B.

Different protective roles in vitro of alpha- and beta-domains of growth inhibitory factor (GIF) on neuron injuries caused by oxygen free radicals.

It was well known that beta-amyloid (Abeta) and tau protein play an important role in pathological procedure of Alzheimer's disease (AD), a senile dementia. The growth inhibitory factor (GIF, also named metallothionein-3, MT-3) had been demonstrated to inhibit the outgrowth of cortex neurons in the medium with extract of the AD patient brain. In our experiments, it was found that the neurons of cortex and the PC12 (pheochromocytoma) cells could be protected from the cytotoxicity of beta-amyloid 25-35 in presence of GIF and its domains. Additionally, GIF can scavenge the hydroxyl radical efficiently in CytC-VitC radical producing system and its alpha-domain shown more effective potentials than its beta-domain. The electron paramagnetic resonance spectra also show that the alpha-domain has more potential ability for eliminating reactive oxygen free radicals than its beta-domain. The results suggest that GIF could act as an efficient scavenger against free radicals in vitro and the alpha-domain in GIF molecule shows more potential in protecting against reactive oxygen species injury than the beta-domain.

[Genetic analysis of T1 progeny of transgenic tobacco with metallothionein gene and metallothionein domain mutant alpha alpha gene].

The chimeric gene containing a cloned mouse metallothionein processed gene or a cloned mouse metallothionein domain mutant alpha alpha gene was respectively introduced into tobacco (Nicotiana tobacum L. cv. NC89) on a disarmed Ti-plasmid of Agrobacterium tumefaciens. T1 seeds from self-fertilized transgenic tobacco were germinated on media containing cadmium or herbicide PPT. The PPT tolerance trait followed Mendelian inheritance and co-segregated with heavy metal tolerance. Meanwhile Southern blot and Western blot verified the existence of the MT gene and alpha alpha mutant gene in the T1 generation plants which keep tolerance to heavy metal. All the results demonstrated the stable integration and inheritance of exotic genes. In addition, assay of the root length and fresh weight of T1 seedlings indicate that transgenic tobacco plants with alpha alpha mutant gene still have a little higher tolerance than that with matural MT gene.

Phosphorylation of tau by glycogen synthase kinase 3beta in intact mammalian cells influences the stability of microtubules.

Tau is a neuronal microtubule-associated protein found predominantly in axons. Hyperphosphorylation of tau reduces the stability of microtubules, which may be a pathogenic mechanism in Alzheimer's disease. To understand the different effects between tau and glycogen synthase kinase 3beta (GSK-3beta) phosphorylated tau on the organization and stability of microtubules, we performed transfection studies on 3T3 cells using EGFP-tau (Enhanced Green Fluorescence Protein-tau) and GSK-3beta to quantify the stability of microtubules. Laser confocal microscope observation revealed that thick and thin microtubule bundles could be induced by tau and GSK-3beta phosphorylated tau. The bundles appeared either to be relatively straight or to form a ring around the circumference of the cell. Both the thick and thin microtubule bundles were resistant to colchicine-induced dissociation, with thick bundles more resistant than thin bundles. The bundles induced by GSK-3beta phosphorylated tau were sensitive to colchicine, and could be reversed by the addition of LiCl, an inhibitor of GSK-3beta.

[Application of a gel chromatography renaturing way on low molecular single-chain urokinase mutant(DscuPA-32K)].

A recombinant mutant gene with thrombolytic and antithrombolytic bifunction was expressed in E. coli. Owing to two reasons of high molecular weight and over expression, dscuPA existed in inclusion body form. The protein of inclusion body was inactive protein. In order to obtain active protein, inclusion bodies should be denatured and then renatured. We performed a novel way named gel-chromatography column renaturation way. Compare with traditional renaturation way, this refolding approach had some obvious advantages, such as low cost and high recovery, and accomplished the preliminary purification step of desired protein(DscuPA-32K). Especially to proteins that easily became inactive and degradation, this approach might have good prospect.

Characterization of a recombinant chimeric plasminogen activator with enhanced fibrin binding.

A recombinant chimeric plasminogen activator (GHRP-scu-PA-32K), consisting of the tetrapeptide Gly-His-Arg-Pro fused to the N-terminus of the low-molecular single-chain urokinase-type plasminogen activator (Leu144-Leu411), was produced by expression in CHO cells. The stable expression cell line was selected for large-scale expression. The product was purified by antibody-Sepharose affinity chromatography with a recovery of 67%. The apparent molecular weight of purified GHRP-scu-PA-32K was 33 kDa according to SDS-PAGE. Its specific activity was 150000 IU/mg protein according to fibrin plate determination. The conversion of single-chain to two-chain molecules mediated by plasmin was comparable for GHRP-scu-PA-32K (K(m)=4.9 microM, k(2)=0.35 s(-1)) and scu-PA-32K. The activation of plasminogen by GHRP-scu-PA-32K (K(m)=1.02 microM, k(2)=0.0028 s(-1)) was also similar to that of scu-PA-32K. The fibrin binding of GHRP-scu-PA-32K was 2.5 times higher than that of scu-PA-32K at a fibrin concentration of 3.2 mg/ml. In contrast to scu-PA-32K in vitro 125I-fibrin-labeled plasma clot lysis, GHRP-scu-PA had a higher thrombolytic potency, whereas it depleted less fibrinogen in plasma. These results show that GHRP-scu-PA-32K as expected is a potential thrombolytic agent.