Association between Combustible Cigarettes and Noncombustible Nicotine or Tobacco Products and Generalized Anxiety Disorder Based on Data from the 8th Korea National Health and Nutrition Examination Survey 2021.

Background: Despite the increasing prevalence of anxiety disorders in Korea, there have been no nationwide studies on the association between tobacco status and generalized anxiety disorder (GAD). Furthermore, despite the increasing number of people using noncombustible nicotine or tobacco products (NNTPs), the association between NNTP use and GAD remains unclear. Therefore, this study investigated the association between tobacco use and GAD. Methods: This nationwide study used data from the 8th Korea National Health and Nutrition Examination Survey (2021) and included 5,454 adults aged ≥19 years who self-reported on the tobacco use and mental health sections. Multivariable logistic regression analysis was performed to investigate the odds ratios (ORs) of GAD (Generalized Anxiety Disorder-7 score ≥10) according to tobacco status among Korean adults. The severity of anxiety was assessed using the Generalized Anxiety Disorder-7 scale. Results: Compared to never tobacco users, the ORs of GAD for combustible cigarette smokers and NNTP users were 2.74 (95% confidence interval [CI], 1.66-4.50) and 2.11 (95% CI, 1.16-3.83), respectively. The OR of GAD for former tobacco users was 1.63 (95% CI, 0.98-2.72). Conclusion: Tobacco use (combustible cigarettes and NNTP) was positively associated with GAD. However, in former tobacco users, there was no significant association with GAD when compared with never tobacco users. Given the OR of GAD among tobacco users, it is crucial to pay attention to screening for GAD and implement appropriate early interventions.

Efficient Somatic Embryogenesis, Regeneration and Acclimatization of Panax ginseng Meyer: True-to-Type Conformity of Plantlets as Confirmed by ISSR Analysis.

Panax ginseng Meyer grows in east Russia and Asia. There is a high demand for this crop due to its medicinal properties. However, its low reproductive efficiency has been a hindrance to the crop's widespread use. This study aims to establish an efficient regeneration and acclimatization system for the crop. The type of basal media and strength were evaluated for their effects on somatic embryogenesis, germination, and regeneration. The highest rate of somatic embryogenesis was achieved for the basal media MS, N6, and GD, with the optimal nitrogen content (≥35 mM) and NH4+/NO3- ratio (1:2 or 1:4). The full-strength MS medium was the best one for somatic embryo induction. However, the diluted MS medium had a more positive effect on embryo maturation. Additionally, the basal media affected shooting, rooting, and plantlet formation. The germination medium containing 1/2 MS facilitated good shoot development; however, the medium with 1/2 SH yielded outstanding root development. In vitro-grown roots were successfully transferred to soil, and they exhibited a high survival rate (86.3%). Finally, the ISSR marker analysis demonstrated that the regenerated plants were not different from the control. The obtained results provide valuable information for a more efficient micropropagation of various P. ginseng cultivars.

CRISPR/Cas9-Mediated Editing of AGAMOUS-like Genes Results in a Late-Bolting Phenotype in Chinese Cabbage (Brassica rapa ssp. pekinensis).

Due to the sudden change in temperature in spring, Chinese cabbage, a leafy vegetable cultivated for consumption, loses its commercial value due to the onset of bolting­the phenomenon of switching from vegetative to reproductive growth. In this study, we applied clustered regularly interspaced short palindromic repeats/(CRISPR)-associated system 9 (CRISPR/Cas9) technology to analyze AGAMOUS-like genes. We performed functional analysis of AGL19 and AGL24 genes related to bolting and flowering using CRISPR/Cas9-mediated Chinese cabbage transformation. Single-guide RNA (sgRNA) sequences were created with a low off-targeting probability to construct gene-editing vectors. Agrobacterium-mediated transformation was conducted, and tentative E0 AGL-edited lines were analyzed using molecular biotechnological methods. Two AGL19-edited lines with nucleotide sequence mutations in the target sequence of the AGL19 genes and four AGL24-edited lines with nucleotide sequence mutations in the target sequence of the AGL24 genes showed particularly late bolting compared to the inbred line 'CT001.' Generational progression using bud pollination obtained T-DNA-free E1 AGL-edited lines, which also showed late bolting. The loss of function of the AGL protein was caused by the occurrence of an indel mutation in the AGL19 and AGL24 genes, which results in an early stop codon. Furthermore, frameshift mutations led to structural changes and the introduction of an early stop codon in the AGL19 and AGL24 proteins. Our results indicate that CRISPR/Cas9-mediated editing of AGAMOUS-like genes results in a late-bolting phenotype and that CRISPR/Cas9 is a useful technology for analyzing gene function in Chinese cabbage (Brassica rapa ssp. pekinensis).

Identification and Characterization of PTE-2, a Stowaway-like MITE Activated in Transgenic Chinese Cabbage Lines.

Transposable elements (TEs) are DNA fragments that can be replicated or transposed within a genome. TEs make up a high proportion of the plant genome and contribute to genetic diversity and evolution, affecting genome structure or gene activity. Miniature inverted-repeat transposable elements (MITEs) are short, non-autonomous class II DNA transposable elements. MITEs have specific sequences, target site duplications (TSDs), and terminal inverted repeats(TIRs), which are characteristics of the classification of MITE families. In this study, a Stowaway-like MITE, PTE-2, was activated in transgenic Chinese cabbage lines. PTE-2 was revealed by in silico analysis as the putative activated element in transgenic Chinese cabbage lines. To verify the in silico analysis data, MITE insertion polymorphism (MIP) PCR was conducted and PTE-2 was confirmed to be activated in transgenic Chinese cabbage lines. The activation tendency of the copy elements of PTE-2 at different loci was also analyzed and only one more element was activated in the transgenic Chinese cabbage lines. Analyzing the sequence of MIP PCR products, the TSD sequence and TIR motif of PTE-2 were identified and matched to the characteristics of the Stowaway-like MITE family. In addition, the flanking region of PTE-2 was modified when PTE-2 was activated.

Function Analysis of the PR55/B Gene Related to Self-Incompatibility in Chinese Cabbage Using CRISPR/Cas9.

Chinese cabbage, a major crop in Korea, shows self-incompatibility (SI). SI is controlled by the type 2A serine/threonine protein phosphatases (PP2As). The PP2A gene is controlled by regulatory subunits that comprise a 36 kDa catalyst C subunit, a 65 kDa regulatory A subunit, and a variety of regulatory B subunits (50-70 kDa). Among them, the PP2A 55 kDa B regulatory subunit (PR55/B) gene located in the A05 chromosome has 13 exons spanning 2.9 kb, and two homologous genes, Bra018924 and Bra014296, were found to be present on the A06 and A08 chromosome, respectively. In this study, we performed a functional analysis of the PR55/B gene using clustered regularly interspaced short palindromic repeats/CRISPR-associated system 9 (CRISPR/Cas9)-mediated gene mutagenesis. CRISPR/Cas9 technology can be used to easily introduce mutations in the target gene. Tentative gene-edited lines were generated by the Agrobacterium-mediated transfer and were selected by PCR and Southern hybridization analysis. Furthermore, pods were confirmed to be formed in flower pollination (FP) as well as bud pollination (BP) in some gene-edited lines. Seed fertility of gene-edited lines indicated that the PR55/B gene plays a key role in SI. Finally, self-compatible T-DNA-free T2 gene-edited plants and edited sequences of target genes were secured. The self-compatible Chinese cabbage developed in this study is expected to contribute to Chinese cabbage breeding.

Using Transcriptome Analysis to Explore Gray Mold Resistance-Related Genes in Onion (Alliumcepa L.).

Gray mold disease caused by Botrytis in onions (Allium cepa L.) during growth and storage negatively affects their yield and quality. Exploring the genes related to gray mold resistance in onion and their application to the breeding of resistant onion lines will support effective and ecological control methods of the disease. Here, the genetic relationship of 54 onion lines based on random amplified polymorphic DNA (RAPD) and in vitro-cultured onion lines infected with gray mold were used for screening resistance and susceptibility traits. Two genetically related onion lines were selected, one with a resistant and one with a susceptible phenotype. In vitro gray mold infection was repeated with these two lines, and leaf samples were collected for gene expression studies in time series. Transcript sequences obtained by RNA sequencing were subjected to DEG analysis, variant analysis, and KEGG mapping. Among the KEGG pathways, 'α-linoleic acid metabolism' was selected because the comparison of the time series expression pattern of Jasmonate resistant 1 (JAR1), Coronatine-insensitive protein 1 (COI 1), and transcription factor MYC2 (MYC2) genes between the resistant and susceptible lines revealed its significant relationship with gray-mold-resistant phenotypes. Expression pattern and SNP of the selected genes were verified by quantitative real-time PCR and high-resolution melting (HRM) analysis, respectively. The results of this study will be useful for the development of molecular marker and finally breeding of gray-mold-resistant onions.

CRISPR/Cas9-Mediated Mutagenesis of BrLEAFY Delays the Bolting Time in Chinese Cabbage (Brassica rapa L. ssp. pekinensis).

Chinese cabbage has unintended bolting in early spring due to sudden climate change. In this study, late-bolting Chinese cabbage lines were developed via mutagenesis of the BrLEAFY (BrLFY) gene, a transcription factor that determines floral identity, using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system. Double-strand break of the target region via gene editing based on nonhomologous end joining (NHEJ) was applied to acquire useful traits in plants. Based on the 'CT001' pseudomolecule, a single guide RNA (sgRNA) was designed and the gene-editing vector was constructed. Agrobacterium-mediated transformation was used to generate a Chinese cabbage line in which the sequence of the BrLFY paralogs was edited. In particular, single base inserted mutations occurred in the BrLFY paralogs of the LFY-7 and LFY-13 lines, and one copy of T-DNA was inserted into the intergenic region. The selected LFY-edited lines displayed continuous vegetative growth and late bolting compared to the control inbred line, 'CT001'. Further, some LFY-edited lines showing late bolting were advanced to the next generation. The T-DNA-free E1LFY-edited lines bolted later than the inbred line, 'CT001'. Overall, CRISPR/Cas9-mediated mutagenesis of the BrLFY gene was found to delay the bolting time. Accordingly, CRISPR/Cas9 is considered an available method for the molecular breeding of crops.

DNA Methylation Level Changes in Transgenic Chinese Cabbage (Brassica rapa ssp. pekinensis) Plants and Their Effects on Corresponding Gene Expression Patterns.

Plant tissue culture is an in vitro technique used to manipulate cells, tissues, or organs, and plays an important role in genetic transformation. However, plants cultured in vitro often exhibit unintended genetic and epigenetic variations. Since it is important to secure the stability of endogenous and exogenous gene expressions in transgenic plants, it is preferable to avoid the occurrence of such variations. In this study, we focused on epigenetic variations, exclusively on methylation level changes of DNA, in transgenic Chinese cabbage (Brassica rapa ssp. pekinensis) plants. To detect these methylation level changes of DNA, bisulfite sequencing was performed and the obtained sequences were compared with the 'CT001' reference genome. Differentially methylated regions (DMRs) of DNA between the non-transgenic and transgenic lines were detected by bisulfite sequencing, and ten DMRs located in exonic regions were identified. The regions with methylation variations that were inherited and consistently maintained in the next generation lines were selected and validated. We also analyzed the relationship between methylation status and expression levels of transformant-conserved DMR (TCD) genes by quantitative reverse transcription-PCR. These results suggested that the changes in methylation levels of these DMRs might have been related to the plant transformation process, affecting subsequent gene expression. Our findings can be used in fundamental research on methylation variations in transgenic plants and suggest that these variations affect the expression of the associated genes.

Identification and Validation of Genetic Variations in Transgenic Chinese Cabbage Plants (Brassica rapa ssp. pekinensis) by Next-Generation Sequencing.

Transgenic plants are usually produced through tissue culture, which is an essential step in Agrobacterium-mediated plant transformation. However, genomic variations, termed somaclonal variations, have been detected in transgenic plants cultured in vitro. The occurrence of these variations should be as low as possible to secure the stability of transgenic crops. Determining the cause and mechanism of somaclonal variations in tissue culture-derived plants will help reduce the rate of variation and promote the stable expression of genes in transgenic plants. In order to determine the genetic variability in transgenic Chinese cabbage plants, we performed whole-genome resequencing and compared the sequencing data with the 'CT001' reference genome. The variation candidates that were expected to consistently occur in the transgenic lines were selected and validated. The single nucleotide polymorphism (SNP) and insertion and deletion (InDel) candidates were identified using the resequencing data and validated by reverse transcription (RT)-PCR analysis. The deduced amino acid sequences were used to determine whether the variations caused changes in the resulting polypeptide, and the annotations of the mutated genes were analyzed to predict the possible effects of the SNPs on gene function. In conclusion, we selected and validated the genetic variations identified in transgenic Chinese cabbage plants. Their genomes were expected to be affected by the process of Agrobacterium-mediated transformation. The findings of our study will provide a genetic basis for transgenic plant research.

Construction of pseudomolecule sequences of Brassica rapa ssp. pekinensis inbred line CT001 and analysis of spontaneous mutations derived via sexual propagation.

Chinese cabbage (Brassica rapa ssp. pekinensis) is a major crop that is widely cultivated, especially in Korea, Japan, and China. With the advent of next generation sequencing technology, the cost and time required for sequencing have decreased and the development of genome research accelerated. Genome sequencing of Chinese cabbage was completed in 2011 using the variety Chiifu-401-42, and since then the genome has been continuously updated. In the present study, we conducted whole-genome sequencing of Chinese cabbage inbred line CT001, a line widely used in traditional or molecular breeding, to improve the accuracy of genetic polymorphism analysis. The constructed CT001 pseudomolecule represented 85.4% (219.8 Mb) of the Chiifu reference genome, and a total of 38,567 gene models were annotated using RNA-Seq analysis. In addition, the spontaneous mutation rate of CT001 was estimated by resequencing DNA obtained from individual plants after sexual propagation for six generations to estimate the naturally occurring variations. The CT001 pseudomolecule constructed in this study will provide valuable resources for genomic studies on Chinese cabbage.

Aberrant promoter methylation occurred from multicopy transgene and SU(VAR)3-9 homolog 9 (SUVH9) gene in transgenic Nicotiana benthamiana.

The chemical modification of DNA by methylation is a heritable trait and can be subsequently reversed without altering the original DNA sequence. Methylation can reduce or silence gene expression and is a component of a host's defence response to foreign nucleic acids. In our study, we employed a plant transformation strategy using Nicotiana benthamiana Domin to study the heritable stability of the introduced transgenes. Through the introduction of the cauliflower mosaic virus (CaMV) 35S promoter and the green fluorescent protein (GFP) reporter gene, we demonstrated that this introduced promoter often triggers a homology-dependent gene-silencing (HDGS) response. These spontaneous transgene-silencing phenomena are due to methylation of the CaMV 35S promoter CAAT box during transgenic plant growth. This process is catalysed by SU(VAR)3-9 homologue 9 (SUVH9), histone deacetylase 1 (HDA1) and domains rearranged methylase 2 (DRM2). In particular, we showed from our data that SUVH9 is the key regulator of methylation activity in epigenetically silenced GFP transgenic lines; therefore, our findings demonstrate that an introduced viral promoter and transgene can be subject to a homology-dependent gene-silencing mechanism that can downregulate its expression and negatively influence the heritable stability of the transgene.

Knock-out mutations of Arabidopsis SmD3-b induce pleotropic phenotypes through altered transcript splicing.

SmD3 is a core protein of small nuclear ribonucleoprotein (snRNP) essential for splicing of primary transcripts. To elucidate function of SmD3 protein in plants, phenotypes and gene expression of SmD3 knock-out and overexpressing mutants in Arabidopsis have been analyzed. smd3-a knock-out mutant or SmD3-a and SmD3-b overexpressors did not show phenotypic alteration. Knock-out of SmD3-b resulted in the pleotropic phenotypes of delayed flowering time and completion of life cycle, reduced root growth, partially defective leaf venation, abnormal numbers of trichome branches, and changed numbers of floral organs. Microarray data revealed that the smd3-b mutant had altered expression of genes related to the above phenotypes, indirectly suggesting that changed splicing of these genes may cause the observed phenotypes. Splicing of selected genes was either totally blocked or reduced in the smd3-b mutant, indicating the important role of SmD3-b in the process. A double knock-out mutant of smd3-a and smd3-b could not be generated, indicating possible redundant function of these two genes. All data indicate that SmD3-b may be major component of the spliceosomal snRNP in Arabidopsis, but the function of SmD3-a may be redundant.

PCR-linked reverse DNA hybridization using oligonucleotide-specific probes of rpoB for identification of Mycobacterium avium and Mycobacterium intracellulare.

A PCR-linked reverse DNA hybridization method using two different specific rpoB DNA probes (Avp and Intp) of Mycobacterium avium and Mycobacterium intracellulare, respectively, were evaluated for the differentiation and identification of M. avium and M. intracellulare culture isolates. Among the 504 culture isolates tested by this method, 48 strains showed positive results for M. avium and 60 strains showed positive results for M. intracellulare. The other 396 culture isolates showed negative results for both M. avium and M. intracellulare. These results were consistent with those obtained from partial rpoB (306 bp) sequence analysis and biochemical tests. The negative strains obtained by this DNA hybridization method were identified as M. tuberculosis (366 strains), M. peregrinum (11 strains), M. abscessus (9 strains), M. fortuitum (8 strains), and M. flavescens (2 strains) by rpoB DNA sequence analysis. Due to the high sensitive and specific result obtained by this assay, we suggest that this PCR-linked reverse DNA hybridization method using two different specific rpoB DNA probes of M. avium and M. intracellulare would be used for the rapid and precise method for differentiation and identification of M. avium and M. intracellulare.

An insertional mutagenesis system for analyzing the Chinese cabbage genome using Agrobacterium T-DNA.

In this study, we applied insertional mutagenesis using Agrobacterium transfer DNA to functionally characterize the gene of Brassica rapa L. ssp. pekinensis. The specific objectives were to: (i) develop and apply a gene tagging system using plasmid rescue and inverse PCR, (ii) select and analyze mutant lines, and (iii) analyze the phenotypic characteristics of mutants. A total of 3,400 insertional mutant lines were obtained from the Chinese cabbage cultivar, 'seoul', using optimized condition. Plasmid rescue was performed successfully for transgenic plants with multiple T-DNA insertions, and inverse PCR was performed for plants with a single copy. The isolated flanking DNA sequences were blasted against the NCBI database and mapped to a linkage map. We determined the genetic loci in B. rapa with two methods: RFLP using the rescue clones themselves and sequence homology analysis to the B. rapa sequence database by queries of rescued clones sequences. Compared to wild type, the T(1) progenies of mutant lines showed variable phenotypes, including hairless and wrinkled leaves, rosette-type leaves, and chlorosis symptoms. T-DNA inserted mutant lines were the first population that we developed and will be very useful for functional genomics studies of Chinese cabbage.

Metabolic engineering of aliphatic glucosinolates in Chinese cabbage plants expressing Arabidopsis MAM1, CYP79F1, and CYP83A1.

Three Arabidopsis cDNAs, MAM1, CYP79F1, and CYP83A1, required for aliphatic glucosinolate biosynthesis were introduced into Chinese cabbage by Agrobacterium tumefaciens-mediated transformation. The transgenic lines overexpressing MAM1 or CYP83A1 showed wild-type phenotypes. However, all the lines overexpressing CYP79F1 displayed phenotypes different from wild type with respect to the stem thickness as well as leaf width and shape. Glucosinolate contents of the transgenic plants were compared with those of wild type. In the MAM1 line M1-1, accumulation of aliphatic glucosinolates gluconapin and glucobrassicanapin significantly increased. In the CYP83A1 line A1-1, all the aliphatic glucosinolate levels were increased, and the levels of gluconapin and glucobrassicanapin were elevated by 4.5 and 2 fold, respectively. The three CYP79F1 transgenic lines exhibited dissimilar glucosinolate profiles. The F1-1 line accumulated higher levels of gluconapoleiferin, glucobrassicin, and 4-methoxy glucobrassicin. However, F1-2 and F1-3 lines demonstrated a decrease in the levels of gluconapin and glucobrassicanapin and an increased level of 4-hydroxy glucobrassicin.

Delayed flowering time in Arabidopsis and Brassica rapa by the overexpression of FLOWERING LOCUS C (FLC) homologs isolated from Chinese cabbage (Brassica rapa L.: ssp. pekinensis).

Chinese cabbage plants remain in the vegetative growth phase until they have experienced prolonged exposure to cold temperature, known as vernalization. This inhibition of flowering is caused by the high levels of FLOWERING LOCUS C (FLC) expression. To increase the product value of Chinese cabbage by inhibiting the floral transition, three genes (BrFLC1, BrFLC2, and BrFLC3) homologous to the AtFLC gene, which encodes a floral repressor, were isolated from the Chinese cabbage 'Chiifu'. These genes showed high similarity to AtFLC, although the putative BrFLC1 protein contained ten more residues than AtFLC. The BrFLC genes were expressed ubiquitously, except that BrFLC3 was not expressed in roots. BrFLC1 and BrFLC2 showed stronger expression than BrFLC3 in unvernalized and vernalized Chinese cabbage. The expression levels of the three BrFLC genes were lower in an early-flowering Chinese cabbage, suggesting that the BrFLC transcript level was associated with flowering time. Constitutive expression of the BrFLC genes in Arabidopsis significantly delayed flowering, which was also observed in transgenic Chinese cabbage overexpressing BrFLC3. These results suggest that the BrFLC genes act similarly to AtFLC. Our results provide a technique for controlling flowering time in Chinese cabbage and other crops to produce high yields of vegetative tissues.

Methylene chloride fraction of Scutellaria barbata induces apoptosis in human U937 leukemia cells via the mitochondrial signaling pathway.

BACKGROUND: Scutellaria barbata D.Don has been applied to treat cancers, inflammation and urinary disease. However, its antitumor mechanism still remains unclear. METHODS: With methylene chloride fraction of Herba Scutellariae barbatae (MCSB), apoptosis-related experiments were carried out on human U937 leukemia cells by (a) 2,3-bis[2-4-nitro-5-sulphophenyl]2H-tetrazolium-5-carboxanilide (XTT) assay for cytotoxicity; (b) terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL) assay for morphological changes; (c) cell cycle analysis; (d) Western blot analysis of poly(ADP-ribose) polymerase (PARP), caspase-8, caspase-9, caspase-3 and Bax, Bcl-2 and cytochrome c expressions for apoptosis signaling pathway. RESULTS: MCSB inhibited the proliferation of human U937 leukemia cells in a dose-dependent manner (IC50 = approximately 10 microg/ml). MCSB dose-dependently increased the sub-G1 DNA contents by cell cycle analysis. DNA fragments indicating induction of apoptosis were observed in MCSB-treated U937 cells by TUNEL assay. Caspase-9 and caspase-3 were activated while caspase-8 was intact by MCSB. Similarly, MCSB effectively cleaved PARP, increased the ratio of Bax/Bcl-2 and released the cytochrome c from mitochondria during apoptosis in U937 cells. CONCLUSIONS: Our results suggest that MCSB can induce apoptosis via the mitochondria-mediated signaling pathway.

Paeonol inhibits anaphylactic reaction by regulating histamine and TNF-alpha.

Paeonol, a major phenolic component of Moutan Cortex, was known to have antiaggregatory, antioxidant and antiinflammatory activities. In the present study, we tried to elucidate the effects of paeonol on anaphylactic reaction and its mode of action. Paeonol significantly inhibited histamine release from the rat peritoneal mast cells (RPMCs) treated with compound 48/80, a mast cell degranulator. The release of tumor necrosis factor (TNF)-alpha mast cell activating cytokine was significantly suppressed in RBL-2H3 mast cells pretreated with anti-dinitrophenyl (DNP) immunoglobulin E (IgE) in a dose-dependent manner. Paeonol significantly inhibited IgE production in B cells activated by anti-CD40 mAb, recombinant interleukin-4 (rIL-4) and recombinant histamine releasing factor (rHRF). Paeonol effectively downregulated the expression of IL-4 in the activated B cells by reverse transcription-polymerase chain reaction (RT-PCR). We also confirmed that paeonol effectively inhibited anaphylactic shock in mice by 90% at a dose of 0.5 mg/mouse versus PBS treated control 2 h after the i.p. injection of compound 48/80. These results suggest that paeonol has antianaphylatic activity by regulating histamine and TNF-alpha.

Studies on the effects of a flanking repetitive sequence on the expression of single-copy transgenes in Nicotiana sylvestris and in N. sylvestris-N. tomentosiformis hybrids.

To test the influence of a Nicotiana tomentosiformis repetitive sequence (R8.3) on transgene expression in N. sylvestris and in N. sylvestris-N. tomentosiformis hybrids, the R8.3 sequence was placed upstream of a nopaline synthase promoter (NOSpro)-NPTII reporter gene in a T-DNA construct. A number of transgenic N. sylvestris lines were produced and in most, the NPTII gene was expressed. In one line, however, the NPTII gene became silenced and methylated in the NOSpro region. The silenced locus was able to trans-inactivate and induce methylation of two stably expressed transgene loci comprising a similar construct. Nucleotide sequence analyses of the three transgene loci revealed that they each contained a single incomplete copy of the T-DNA, which had sustained deletions of varying sizes in the R8.3 region. Paradoxically, the R8.3 DNA upstream of the two active, unmethylated NOSpro-NPTII genes was highly methylated, whereas the R8.3 DNA upstream of the silenced, methylated NOSpro-NPTII gene was less methylated. The methylated portions of the R8.3 sequence corresponded to retroelement remnants. An active NOSpro-NPTII gene downstream of a nearly intact R8.3 sequence did not become methylated in N. sylvestris-N. tomentosiformis hybrids. Thus, methylation in the R8.3 sequence did not spread into adjoining transgene promoters and the effect of the R8.3 dispersed repeat family on transgene expression was negligible. The silencing phenomena observed with the three single-copy transgene loci are discussed in the context of other possible triggers of silencing.

Effects of the ethyl acetate fraction of Spatholobi caulis on tumour cell aggregation and migration.

The ethyl acetate (EA) fraction obtained from a methanol extract of Spatholobi caulis (Leguminosae) has been investigated for anti-metastatic activities in vitro. The EA fraction of Spatholobi caulis inhibited platelet aggregation induced by B16BL6 melanoma cells with an IC(50) of 50 microgram/mL. The EA fraction significantly inhibited HT1080 cancer cell invasion through a matrigel-coated filter with an IC(50) of 25 microgram/mL. Messenger RNA expression of uPA was effectively decreased in HT1080 cells by the EA fraction of Spatholobi caulis with an IC(50) of 30 microgram/mL, but the expressions of MMP-2 (matrix metalloproteinase) and TIMPs (tissue inhibitors of metalloproteinases) were not changed. These findings indicated that the EA fraction suppressed tumour cell invasion by downregulation of uPA (urokinase-type plasminogen activator). Taken together, these results suggest that the EA fraction of Spatholobi caulis may have anti-metastatic activities by blocking tumour cell-induced platelet aggregation (TCIPA) and tumour cell invasion.