Unveiling Cryptic Species Diversity and Genetic Variation of Lasiodiplodia (Botryosphaeriaceae, Botryosphaeriales) Infecting Fruit Crops in Taiwan.

The genus Lasiodiplodia, a member of the family Botryosphaeriaceae, is an important fungal disease genus in agriculture. However, the Lasiodiplodia species survey and genetic diversity in Taiwan remain unclear. This study aimed to investigate the Lasiodiplodia species associated with various fruit species to explore the cryptic Lasiodiplodia species diversity, validate species delimitation, and unveil cryptic genetic diversity. Overall, six Lasiodiplodia species were identified, with several new records of infection identified. Additionally, phylogenetic analyses indicated that the relations of all isolates of L. theobromae might be paraphyletic. They were grouped with L. brasiliense based on Automatic Barcode Gap Discovery (ABGD), Automatic Partitioning (ASAP) and structure-based clustering analyses. These analyses did not provide conclusive evidence for L. brasiliensis as a stable species. It may be necessary to gather more information to clarify the species delineation. The multiple new records of Lasiodiplodia species with high genetic diversity and differentiation revealed that the diversity of Lasiodiplodia in Taiwan was underestimated in the past. We found that L. theobromae has the highest number of haplotypes but the lowest number of haplotype and nucleotide diversities, indicating a recent population expansion. This was supported by the significant negative Tajima's D and Fu and Li's D* tests. The high genetic diversity, low gene flow, and host-associated differentiation of Lasiodiplodia species indicate that they might harbour powerful evolutionary potential in Taiwan. This study provided critical insights into genetic variation, host-associated differentiation, and demography of Lasiodiplodia species, which would be helpful for disease management of related pathogens.

Structured Populations of Critically Endangered Yellow Water Lily (Nuphar shimadai Hayata, Nymphaeaceae).

Yellow water lily (Nuphar shimadai Hayata) is a critically endangered species in Taiwan. Here, we examined genetic structures of four extant populations, WP, GPa, GPb and GPn, using 39 simple sequence repeat (SSR) markers. Positive genetic correlation was observed within 50 m, beyond which no correlation was detected due to isolation by distance according to Mantel correlogram. This suggests a significant genetic structuring of the species. Besides, multilocus genotype (MLG) analysis revealed that GPa was a panmictic population and the species' putative center of origin. Genetic exchange was observed between GPa and GPb populations, which likely resulted from their geographic proximity. Nevertheless, there was a strong asymmetric migration detected from GPa to WP, but a recent genetic barrier prevented dispersal further northward (WP). Geneland estimated the best number of clusters as K = 2, where WP distinctly separated from the rest of the populations. In STRUCTURE output of K = 3, a third cluster was abundant only in WP. We suggest to consider GPn and WP as separate conservation units, being far from GPa. There is indeed a need to investigate these populations; as predicted, Ne = 1.6 to 3.0 is considered low and that may put the species at risk of extinction.

The genetic relationships of Indian jujube (Ziziphus mauritiana Lam.) cultivars using SSR markers.

The genetic relationships among 24 Indian jujube cultivars (Ziziphus mauritiana Lam.) were evaluated by genotyping the microsatellite loci using simple sequence repeat (SSR) markers. The SSR loci were scored by fluorescent labelling and automated detection systems for the high-throughput capillary electrophoresis and high-resolution gel electrophoresis. Out of the 29 newly characterized SSR loci, 26 were considered as polymorphic with a total of 181 alleles obtained. The number of alleles ranged from 2-12, while the polymorphism information content ranged from 0.08-0.83, and the expected and observed heterozygosity were 0.04-0.83 and 0.04-0.82, respectively. The allele pattern of Indian jujube for all SSR loci confirmed its karyotype as tetraploid. Similarity coefficients and UPGMA dendrogram revealed that the Taiwanese cultivars consisted of a large 'A' clade, which is further divided into 'A1' and 'A2' groups, and the 'B' clade where both are rooted by the wild accession, 'Chad native'. These four genetic clusters were supported by the results of PCoA and the assignment test. The excess of heterozygotes based on F-statistics was attributed to its mating system as outcrossing and self-incompatible, and the introgression of the presumed mutation-derived cultivars with genetic admixture. Based on this study, SSR markers offer valuable information on the genetic relationship of this tropical fruit tree which is basically in agreement with the genealogy of its breeding history.

Characterization of 39 microsatellite markers from Nuphar shimadai (Nymphaeaceae) and cross-amplification in two related taxa.

PREMISE OF THE STUDY: Microsatellite loci were developed for Nuphar shimadai (Nymphaeaceae) to evaluate the population genetic dynamics for conservation purposes. The species is an endemic aquatic species in Taiwan that is endangered by anthropogenic activities. METHODS AND RESULTS: A magnetic bead enrichment protocol was used to identify 72 potential microsatellite loci and develop 39 microsatellite markers from N. shimadai. The number of alleles per locus ranged from one to 10 per locus, with levels of observed heterozygosity ranging from 0 to 1.0 within populations. As a result of inbreeding within isolated populations, 65% of loci significantly deviated from Hardy-Weinberg equilibrium within populations. CONCLUSIONS: These novel markers should be valuable tools to evaluate the genetic diversity within the endangered aquatic taxon N. shimadai for conservation and reintroduction purposes in Taiwan.

Screening transferable microsatellite markers across genus Phalaenopsis (Orchidaceae).

BACKGROUND: Molecular identification based on microsatellite loci is an important technology to improve the commercial breeding of the moth orchid. There are more than 30,000 cultivars have been enrolled at the Royal Horticultural Society (RHS). In this study, genomic microsatellite primer sets were developed from Phalaenopsis aphrodite subsp. formosana to further examine the transferability of across 21 Phalaenopsis species. METHODS AND RESULTS: Twenty-eight polymorphic microsatellite markers were obtained using the magnetic bead enrichment method, with high transferability of the 21 species of the genus Phalaenopsis, especially in the subgenus Phalaenopsis. The 28 newly developed polymorphic microsatellite markers with high polymorphism information content values. The best and second fit grouping (K) are inferred as two and four by the ΔK evaluation in the assignment test. This result indicates that these microsatellite markers are discernible to subgenus Phalaenopsis. CONCLUSIONS: Our results indicate that these new microsatellite markers are useful for delimiting species within genus Phalaenopsis. As expected, the genetic relationships between species of subgenus Phalaenopsis can be well distinguished based on the assignment test. These molecular markers could apply to assess the paternity of Phalaenopsis as well as investigating hybridization among species of genus Phalaenopsis.

Direct LAMP Assay without Prior DNA Purification for Sex Determination of Papaya.

Papaya (Carica papaya L.) is an economically important tropical fruit tree with hermaphrodite, male and female sex types. Hermaphroditic plants are the major type used for papaya production because their fruits have more commercial advantages than those of female plants. Sex determination of the seedlings, or during the early growth stages, is very important for the papaya seedling industry. Thus far, the only method for determining the sex type of a papaya at the seedling stage has been DNA analysis. In this study, a molecular technique-based on DNA analysis-was developed for detecting male-hermaphrodite-specific markers to examine the papaya's sex type. This method is based on the loop-mediated isothermal amplification (LAMP) and does not require prior DNA purification. The results show that the method is an easy, efficient, and inexpensive way to determine a papaya's sex. This is the first report on the LAMP assay, using intact plant materials-without DNA purification-as samples for the analysis of sex determination of papaya. We found that using high-efficiency DNA polymerase was essential for successful DNA amplification, using trace intact plant material as a template DNA source.

Characterization of Genomic Inheritance of Intergeneric Hybrids between Ascocenda and Phalaenopsis Cultivars by GISH, PCR-RFLP and RFLP.

BACKGROUND: The intergeneric hybrids between Ascocenda John De Biase 'Blue' and Phalaenopsis Chih Shang's Stripes have been generated to introduce the blue color into the Phalaenopsis germplasm in prior study. In order to confirm the inheritance in hybrid progenies, genomic in situ hybridization (GISH) and restriction fragment length polymorphism (RFLP) analysis were conducted to confirm the intergeneric hybridization status. METHODS/RESULTS: GISH analysis showed the presence of both maternal and paternal chromosomes in the cells of the putative hybrids indicating that the putative hybrid seedlings were intergeneric hybrids of the two parents. Furthermore, twenty-seven putative hybrids were randomly selected for DNA analysis, and the external transcribed spacer (ETS) regions of nrDNA were analyzed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and RFLP analyses to identify the putative hybrids. RFLP analysis showed that the examined seedlings were intergeneric hybrids of the two parents. However, PCR-RFLP analysis showed bias to maternal genotype. CONCLUSIONS: Both GISH and RFLP analyses are effective detection technology to identify the intergeneric hybridization status of putative hybrids. Furthermore, the use of PCR-RFLP analysis to identify the inheritance of putative hybrids should be carefully evaluated.

RNA-Seq SSRs of Moth Orchid and Screening for Molecular Markers across Genus Phalaenopsis (Orchidaceae).

BACKGROUND: The moth orchid (Phalaenopsis species) is an ornamental crop that is highly commercialized worldwide. Over 30,000 cultivars of moth orchids have been registered at the Royal Horticultural Society (RHS). These cultivars were obtained by artificial pollination of interspecific hybridization. Therefore, the identification of different cultivars is highly important in the worldwide market. METHODS/RESULTS: We used Illumina sequencing technology to analyze an important species for breeding, Phalaenopsis aphrodite subsp. formosana and develop the expressed sequence tag (EST)-simple sequence repeat (SSR) markers. After de novo assembly, the obtained sequence covered 29.1 Mb, approximately 2.2% of the P. aphrodite subsp. formosana genome (1,300 Mb), and a total of 1,439 EST-SSR loci were detected. SSR occurs in the exon region, including the 5' untranslated region (UTR), coding region (CDS), and 3'UTR, on average every 20.22 kb. The di- and tri-nucleotide motifs (51.49% and 35.23%, respectively) were the two most frequent motifs in the P. aphrodite subsp. formosana. To validate the developed EST-SSR loci and to evaluate the transferability to the genus Phalaenopsis, thirty tri-nucleotide motifs of the EST-SSR loci were randomly selected to design EST-SSR primers and to evaluate the polymorphism and transferability across 22 native Phalaenopsis species that are usually used as parents for moth orchid breeding. Of the 30 EST-SSR loci, ten polymorphic and transferable SSR loci across the 22 native taxa can be obtained. The validated EST-SSR markers were further proven to discriminate 12 closely related Phalaenopsis cultivars. The results show that it is not difficult to obtain universal SSR markers by transcriptome deep sequencing in Phalaenopsis species. CONCLUSIONS: This study supported that transcriptome analysis based on deep sequencing is a powerful tool to develop SSR loci in non-model species. A large number of EST-SSR loci can be isolated, and about 33.33% EST-SSR loci are universal markers across the Phalaenopsis breeding germplasm after preliminary validation. The potential universal EST-SSR markers are highly valuable for identifying all of Phalaenopsis cultivars.

Structure elucidation and cytotoxic evaluation of new polyacetylenes from a marine sponge Petrosia sp.

The sponge Petrosia sp. yielded five polyacetylenic compounds (1-5), including two new polyacetylenes, petrosianynes A (1) and B (2). The structures of these compounds were elucidated by detailed spectroscopic analysis and by comparison with the physical and spectral data of related known analogues. Compounds 1-5 exhibited significant cytotoxic activity against a limited panel of cancer cell lines.

Cracking the cytotoxicity code: apoptotic induction of 10-acetylirciformonin B is mediated through ROS generation and mitochondrial dysfunction.

A marine furanoterpenoid derivative, 10-acetylirciformonin B (10AB), was found to inhibit the proliferation of leukemia, hepatoma, and colon cancer cell lines, with selective and significant potency against leukemia cells. It induced DNA damage and apoptosis in leukemia HL 60 cells. To fully understand the mechanism behind the 10AB apoptotic induction against HL 60 cells, we extended our previous findings and further explored the precise molecular targets of 10AB. We found that the use of 10AB increased apoptosis by 8.9%-87.6% and caused disruption of mitochondrial membrane potential (MMP) by 15.2%-95.2% in a dose-dependent manner, as demonstrated by annexin-V/PI and JC-1 staining assays, respectively. Moreover, our findings indicated that the pretreatment of HL 60 cells with N-acetyl-l-cysteine (NAC), a reactive oxygen species (ROS) scavenger, diminished MMP disruption and apoptosis induced by 10AB, suggesting that ROS overproduction plays a crucial rule in the cytotoxic activity of 10AB. The results of a cell-free system assay indicated that 10AB could act as a topoisomerase catalytic inhibitor through the inhibition of topoisomerase IIα. On the protein level, the expression of the anti-apoptotic proteins Bcl-xL and Bcl-2, caspase inhibitors XIAP and survivin, as well as hexokinase II were inhibited by the use of 10AB. On the other hand, the expression of the pro-apoptotic protein Bax was increased after 10AB treatment. Taken together, our results suggest that 10AB-induced apoptosis is mediated through the overproduction of ROS and the disruption of mitochondrial metabolism.

Development and characterization of 16 polymorphic microsatellite markers from Taiwan cow-tail fir, Keteleeria davidiana var. formosana (Pinaceae) and cross-species amplification in other Keteleeria taxa.

BACKGROUND: Keteleeria davidiana var. formosana (Pinaceae), Taiwan cow-tail fir, is an endangered species listed on the IUCN Red List of Threatened Species and only two populations remain, both on the Taiwan Island. Sixteen polymorphic microsatellite loci were developed in an endangered and endemic gymnosperm species, Keteleeria davidiana var. formosana, and were tested in an additional 6 taxa, K. davidiana var. calcarea, K. davidiana var. chienpeii, K. evelyniana, K. fortunei, K. fortunei var. cyclolepis, and K. pubescens, to evaluate the genetic variation available for conservation management and to reconstruct the phylogeographic patterns of this ancient lineage. FINDINGS: Polymorphic primer sets were developed from K. davidiana var. formosana using the modified AFLP and magnetic bead enrichment method. The number of alleles ranged from 3 to 16, with the observed heterozygosity ranging from 0.28 to 1.00. All of the loci were found to be interspecifically amplifiable. CONCLUSIONS: These polymorphic and transferable loci will be potentially useful for future studies that will focus on identifying distinct evolutionary units within species and establishing the phylogeographic patterns and the process of speciation among closely related species.

Characterization of 42 microsatellite markers from poison ivy, Toxicodendron radicans (Anacardiaceae).

Poison ivy, Toxicodendron radicans, and poison oaks, T. diversilobum and T. pubescens, are perennial woody species of the Anacardiaceae and are poisonous, containing strong allergens named urushiols that cause allergic contact dermatitis. Poison ivy is a species distributed from North America to East Asia, while T. diversilobum and T. pubescens are distributed in western and eastern North America, respectively. Phylogreography and population structure of these species remain unclear. Here, we developed microsatellite markers, via constructing a magnetic enriched microsatellite library, from poison ivy. We designed 51 primer pairs, 42 of which successfully yielded products that were subsequently tested for polymorphism in poison oak, and three subspecies of poison ivy. Among the 42 loci, 38 are polymorphic, while 4 are monomorphic. The number of alleles and the expected heterozygosity ranged from 1 to 12 and from 0.10 to 0.87, respectively, in poison ivy, while varied from 2 to 8 and, from 0.26 to 0.83, respectively in poison oak. Genetic analysis revealed distinct differentiation between poison ivy and poison oak, whereas slight genetic differentiation was detected among three subspecies of poison ivy. These highly polymorphic microsatellite fingerprints enable biologists to explore the population genetics, phylogeography, and speciation in Toxicodendron.

Microsatellite primers for the endangered beech tree, Fagus hayatae (Fagaceae).

PREMISE OF THE STUDY: Microsatellite primers were developed for the endangered species Fagus hayatae (Fagaceae) to investigate the genetic diversity of the population and to investigate species delimitation within Fagus. METHODS AND RESULTS: Ten polymorphic simple sequence repeat markers were developed for F. hayatae using a magnetic bead enrichment method. The primers amplified trinucleotides, hexanucleotides, and complex repeats with six to 16 alleles per locus. The observed and expected heterozygosities across loci varied, with a range of 0.05-0.71 and 0.63-0.91, respectively. Most of the primers also amplified DNA from F. crenata, F. grandifolia, F. japonica, F. longipetiolata, F. lucida, F. orientalis, and F. sylvatica. CONCLUSIONS: These results indicate that these polymorphic loci of F. hayatae will be potentially useful for future studies of the population genetic diversity within the species. In addition, the interspecific amplification indicates that these transferable microsatellite markers will also be useful for future phylogeographic and speciation studies among close Fagus species.

Characterization of microsatellite loci from Litsea hypophaea (Lauraceae), a tree endemic to Taiwan.

PREMISE OF THE STUDY: Microsatellite primers were developed for the endemic tree Litsea hypophaea (Lauraceae) in Taiwan to investigate its genetic diversity and population genetic structure and to investigate species delimitation within Litsea. METHODS AND RESULTS: Fifteen new simple sequence repeat markers were developed from L. hypophaea with a magnetic bead enrichment method. Most loci were also amplified from three closely related species, L. coreana, L. lii, and L. acutivena. The number of alleles and observed and expected heterozygosities across loci varied with a range of 1-25, 0.000-1.000, and 0.000-0.956, respectively. CONCLUSIONS: The application of these microsatellite markers of L. hypophaea provides a tool for understanding genetic diversity and population differentiation. In addition, interspecific amplification suggests that these markers will also be useful for species identification of related taxa within Litsea in Taiwan.

Characterization of 24 transferable microsatellite loci in four skullcaps (Scutellaria, Labiatae).

PREMISE OF THE STUDY: Transferable polymorphic microsatellite loci for four skullcaps, Scutellaria indica, S. taiwanensis, S. austrotaiwanensis, and S. playfairii, were developed for future studies of the mating system and population structure of these species. Interspecific amplification was also tested in various Scutellaria species. METHODS AND RESULTS: Twelve novel polymorphic microsatellite loci were isolated from four S. taiwanensis specimens, and seven are interspecifically transferable. Microsatellite loci developed from S. austrotaiwanensis in a previous study were also analyzed in the other three species, and 12 loci were found to be transferable. Allele numbers of the total 24 loci for S. indica, S. taiwanensis, S. playfairii, and S. austrotaiwanensis are two to four, two, two to five, and two to three, respectively, with an expected heterozygosity ranging from 0.114-0.661, 0.062-0.499, 0.280-0.730, and 0.268-0.662, respectively. The interspecies transferability of these 24 loci was further tested in another 10 Scutellaria species, including three species native to Taiwan. Seventeen loci were found to be interspecifically amplifiable, especially among the Taiwan native species. CONCLUSIONS: These highly polymorphic and transferable loci will be useful for future studies of the mating system of closely related Scutellaria species.

Isolation of 16 polymorphic microsatellite markers from an endangered and endemic species, Podocarpus nakaii (Podocarpaceae).

PREMISE OF THE STUDY: Sixteen polymorphic microsatellite markers were isolated and characterized from the endangered evergreen tree Podocarpus nakaii to evaluate the population structure for conservation efforts. METHODS AND RESULTS: Based on a modified amplified fragment length polymorphism and magnetic bead enrichment method, 16 polymorphic primer sets were developed for this endangered insular species. Allele numbers ranged from five to seven, with an observed heterozygosity ranging from 0.29 to 0.88. Most primers were able to amplify DNA from the endemic P. fasciculus and the widely distributed P. macrophyllus var. macrophyllus, P. macrophyllus var. maki, and P. costalis. CONCLUSIONS: The results reported here indicate the usefulness of codominant markers for future studies of the population genetics of P. nakaii. In addition, the markers are useful for further phytogeographic and speciation studies in P. fasciculus, P. macrophyllus var. macrophyllus, and P. macrophyllus var. maki, which are closely related species.

Upregulation of estrogen receptor alpha and mediation of 17beta-estradiol vasoprotective effects via estrogen receptor alpha in basilar arteries in rats after experimental subarachnoid hemorrhage.

OBJECT: The authors previously demonstrated that 17beta-estradiol benzoate (E2) treatment prevents subarachnoid hemorrhage (SAH)-induced cerebral vasospasm and preserves endothelial nitric oxide synthase (eNOS) in male rats. Changes in the expression of estrogen receptor (ER) subtypes ERalpha and -beta and their roles in the E2-mediated preservation of eNOS in SAH remain unknown. In the present study the effects of SAH on the expression of ERalpha and -beta in the cerebral arteries were clarified, and the receptor roles in the E2-mediated preservation of eNOS expression in SAH were differentiated. METHODS: A 2-hemorrhage SAH model was induced by 2 autologous blood injections into the cisterna magna of adult male rats. The effect of SAH on ERalpha and -beta expression was evaluated. Other rats subcutaneously received implanted Silastic tubes containing corn oil with E2 and daily injections of various doses of an ERalpha- (methyl-piperidinopyrazole [MPP]) or ERbeta-selective antagonist (R,R-tetrahydrochrysene) after the first hemorrhage. The protein levels of ERalpha, ERbeta, eNOS, and inducible nitric oxide synthase (iNOS) from basilar arteries were examined using Western blot analysis, and their mRNAs were evaluated by reverse transcription-polymerase chain reaction. RESULTS: The ERalpha but not the ERbeta was upregulated in the basilar artery after SAH. Treatment with MPP eliminated E2-mediated effects in SAH, relieved cerebral vasospasm, preserved eNOS expression, and suppressed iNOS expression. CONCLUSIONS: Estrogen receptor alpha is upregulated in the basilar artery after SAH. Note that E2 exerts its protective effects through ERalpha-dependent pathways to relieve cerebral vasospasm and preserve eNOS expression. A selective ERalpha agonist may be the drug of choice for the treatment of patients with SAH.

Attenuation of experimental subarachnoid hemorrhage--induced cerebral vasospasm by the adenosine A2A receptor agonist CGS 21680.

OBJECT: Impaired endothelium-dependent relaxation is present in vasospastic cerebral vessels after subarachnoid hemorrhage (SAH) and may result from deficient production of endothelial nitric oxide synthase (eNOS) or increased production and/or activity of inducible NOS (iNOS). Accumulating evidence demonstrates that adenosine A2A receptors increase the production of NO by human and porcine arterial endothelial cells, which in turn leads to vasodilation. This study was designed to examine the effects of an adenosine A2A receptor agonist, (2(4-[2-carboxyethyl]phenyl)ethylamino)-5'-N-ethylcarboxamidoadenosine (CGS 21680), in the prevention of SAH-induced vasospasm. METHODS: . Experimental SAH was induced in Sprague-Dawley rats by injecting 0.3 ml of autologous blood into the cisterna magna of each animal. Intraperitoneal injections of CGS 21680 or vehicle were administered 5 minutes and 24 hours after induction of SAH. The degree of vasospasm was determined by averaging measurements of cross-sectional areas of the basilar artery (BA) 48 hours after SAH. Expression of eNOS and iNOS in the BA was also evaluated. Prior to perfusion-fixation, there were no significant differences among animals in the control and treated groups in any physiological parameter that was recorded. The CGS 21680 treatment significantly attenuated SAH-induced vasospasm. Induction of iNOS mRNA and protein in the BA by the SAH was significantly diminished by administration of CGS 21680. The SAH-induced suppression of eNOS mRNA and protein was also relieved by the CGS 21680 treatment. CONCLUSIONS: This is the first evidence that adenosine A2A receptor agonism is effective in preventing SAH-induced vasospasm without significant complications. The beneficial effect of adenosine A2A receptor agonists may be, at least in part, related to the prevention of augmented expression of iNOS and the preservation of normal eNOS expression following SAH. Adenosine A2A receptor agonism holds promise in the treatment of cerebral vasospasm following SAH and merits further investigation.

17beta-Estradiol inhibits subarachnoid hemorrhage-induced inducible nitric oxide synthase gene expression by interfering with the nuclear factor kappa B transactivation.

BACKGROUND AND PURPOSE: Previously, we showed that 17beta-estradiol (E(2)) treatment prevented the subarachnoid hemorrhage (SAH)-induced cerebral vasospasm in male rats. The aim of this study was designed to further delineate the molecular mechanisms underlying E(2)-induced inhibition of inducible nitric oxide synthase (iNOS) upregulation and relief of vasospasm caused by SAH. METHODS: The 2-hemorrhage SAH model was induced by 2 autologous injections of blood into the cisterna magna of adult male rats. The rats were then subcutaneously implanted of a Silastic tube containing corn oil with or without 17beta-estradiol benzoate and received daily intraperitoneal injections of various doses of ICI 182,780, a nonselective estrogen receptor (ER) antagonist, for 7 days after the first hemorrhage. Basilar arteries were then removed for protein extraction, RNA isolation, and gel mobility assay. The protein levels of iNOS, p65, and ER were examined by Western blot analysis, and that iNOS mRNA expression was evaluated by reverse-transcription polymerase chain reaction. RESULTS: E(2) prevented the SAH-induced vasospasm and increases of the levels of iNOS protein and mRNA in basilar artery through an ER-dependent mechanism. Treatment of the SAH rat with E(2) did not affect the nuclear translocation of p65 subunit of nuclear factor kappaB, but caused an increase of the association of p65/ER, and reversed the SAH-induced increase of the p65 binding on iNOS promoter. CONCLUSIONS: E(2) inhibits the SAH-induced increase of iNOS by increasing the association of p65/ER, which in turn inhibits the binding of p65 to iNOS DNA. Our data suggest the potential applications of E(2) in the treatment of SAH patient.

The effect of 17beta-estradiol in attenuating experimental subarachnoid hemorrhage-induced cerebral vasospasm.

OBJECT: Sex differences in the outcome of aneurysmal subarachnoid hemorrhage (SAH) are controversial, and the potential influence of estradiol on vasodilation is unclear. In the present study the authors evaluate the effect and possible mechanism of 17beta-estradiol (E2) on SAH-induced vasospasm in a two-hemorrhage rodent model of SAH. METHODS: A 30-mm Silastic tube filled with E2 in corn oil (0.3 mg/ml) was subcutaneously implanted in male rats. Serum levels of E2 were measured on Days 0, 1, 2, 3, 4, and 7 postimplantation. The degree of vasospasm was determined by averaging the cross-sectional areas of the basilar artery (BA) 7 days after the first SAH. Expressions of endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS) in the BA were also evaluated. Serum levels of E2 in the E2-treated rats were at physiological levels (56-92 pg/ml) and were significantly higher than those in the control and vehicle-treated groups. Treatment with E2 significantly (p < 0.01) attenuated SAH-induced vasospasm. Induction of iNOS messenger (m)RNA and protein in the BA by SAH was significantly diminished by the E2 treatment but not by vehicle treatment. The SAH-induced suppression of eNOS mRNA and protein was relieved by E2 treatment. CONCLUSIONS: These results suggest that continuous treatment with E2 at physiological levels prevents cerebral vasospasm following SAH. The beneficial effect of E2 may be in part related to the prevention of augmentation of iNOS expression and the preservation of normal eNOS expression after SAH. Treatment with E2 holds therapeutic promise in the treatment of cerebral vasospasm following SAH and merits further investigation.