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Background: Osteoporosis (OP) is a chronic skeletal disorder characterized by low bone mass and microarchitectural deterioration of bone tissue, resulting in increased bone fragility and a higher risk of fractures. It is a significant public health concern, particularly among postmenopausal women and older adults. The imbalance between bone formation and resorption is the fundamental cause of OP. Current clinical drugs for OP have limited efficacy and can cause side effects. Therefore, there is a need to explore alternative treatments and investigate their mechanisms to improve OP management. The Xianling Gubao capsule, a traditional Chinese medicine, is commonly used to treat OP by tonifying the kidney. However, the specific mechanism of action of the Xianling Gubao capsule in improving OP remains unclear, necessitating further research in this area. Methods: The N6-methyladenosine (m6A) content was evaluated by dot blot and m6A ribonucleic acid (RNA) methylation assay kit. The contents of methyltransferase-like 3 (METTL3), runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), and bone gamma-carboxyglutamate protein (BGLAP) were appraised by quantitative Reverse Transcription polymerase chain reaction (qRT-PCR) and western blot. The bilateral ovariectomy (OVX) method was used to establish an animal model of OP. OP bone marrow mesenchymal stem cells (OP-BMSCs) were extracted from mice in the OVX group by the whole bone marrow method. METTL3 overexpression and control vectors were transfected to OP-BMSCs using X-tremeGENE HP DNA Transfection Reagent. The ALP activity in OP-BMSCs was assessed by ALP staining. The calcium nodules in OP-BMSCs were detected by Alizarin Red S (ARS) assay. The Xianling Gubao capsule solution was employed to gavage mice, and the drug-containing serum was used to treat OP-BMSCs. Dot blot allows for the assessment of relative levels of m6A modification. The m6A RNA methylation assay kit is a specialized kit designed to quantitatively measure m6A levels in RNA samples. qRT-PCR allows for the measurement of mRNA levels of target genes. Western blot is used to detect and quantify specific proteins in a sample, and provides information about protein expression levels. OVX mimics the hormonal changes occurring in postmenopausal women and leads to bone loss and osteoporotic conditions in animals. This model allows for the investigation of the effects of the Xianling Gubao capsule on OP in a controlled experimental setting. Results: The m6A modification and METTL3, RUNX2, ALP, and BGLAP levels were reduced in bone samples of patients with OP and OVX mice compared with the corresponding control groups. Upregulated METTL3 enhanced the osteogenic ability of OP-BMSCs. METTL3 overexpression obviously increased m6A modification and METTL3, RUNX2, ALP, and BGLAP levels in OP-BMSCs. Xianling Gubao capsule treatment could weaken the impact of OP in mice by regulating the m6A modification and METTL3, RUNX2, ALP, and BGLAP levels. Serum containing Xianling Gubao capsule could enhance the osteogenic capability of OP-BMSCs and boost METTL3, RUNX2, ALP, and BGLAP levels. Treatment with the Xianling Gubao capsule shows promising effects in attenuating the impact of OP. The capsule is found to regulate m6A modification and increase the levels of METTL3, RUNX2, ALP, and BGLAP in OP-BMSCs. This indicates that the Xianling Gubao capsule may rescue the diminished osteogenic capability of OP-BMSCs by modulating METTL3. These findings suggest that the Xianling Gubao capsule has the potential to be an effective drug for the treatment of OP. Conclusion: Taken together, the m6A modification and contents of osteogenic-related factors were reduced in OP. Upregulated METTL3 improved the osteogenic ability, m6A modification, and osteogenic-related factor abundances in OP-BMSCs. Xianling Gubao capsule rescued the diminished osteogenic capability of OP-BMSCs by modulating METTL3 and might serve as an effective drug for OP. The Xianling Gubao capsule, as a traditional Chinese medicine, could potentially complement existing therapeutic approaches for OP. By targeting the m6A modification pathway and promoting osteogenic differentiation, the capsule may help to expedite bone formation and repair, which are critical for managing OP and reducing the risk of fractures.
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The occurrence of cyanobacterial blooms in summer are frequently accompanied by the succession of phytoplankton communities in freshwater. However, little is known regarding the roles of viruses in the succession, such as in huge reservoirs. Here, we investigated the viral infection characteristics of phytoplankton and bacterioplankton during the summer bloom succession in Xiangxi Bay of Three Gorges Reservoir, China. The results indicated that three distinct bloom stages and two successions were observed. From cyanobacteria and diatom codominance to cyanobacteria dominance, the first succession involved different phyla and led to a Microcystis bloom. From Microcystis dominance to Microcystis and Anabaena codominance, the second succession was different Cyanophyta genera and resulted in the persistence of cyanobacterial bloom. The structural equation model (SEM) showed that the virus had positive influence on the phytoplankton community. Through the Spearman's correlation and redundancy analysis (RDA), we speculated that both the increase of viral lysis in the eukaryotic community and the increase of lysogeny in cyanobacteria may contributed to the first succession and Microcystis blooms. In addition, the nutrients supplied by the lysis of bacterioplankton might benefit the second succession of different cyanobacterial genera and sustain the dominance of cyanobacteria. Based on hierarchical partitioning method, the viral variables still have a marked effect on the dynamics of phytoplankton community, although the environmental attributes were the major factors. Our findings suggested that viruses played multiple potential roles in summer bloom succession and may help the blooms success of cyanobacteria in Xiangxi Bay. Under the background of increasingly serious cyanobacterial blooms worldwide, our study may have great ecological and environmental significance for understanding the population succession in phytoplankton and controlling the cyanobacterial blooms.
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Microplastics (MPs) are ubiquitous in freshwater ecosystems, but knowledge of their effects on extracellular polymeric substance (EPS) produced by algae is poorly understood. The components in specific EPS fractions of Microcystis respond when exposed to MPs is also still unclear. In this study, the responses of Microcystis aeruginosa under polystyrene (PS) microplastic exposure were studied over 17 days of cultivation, using 0.1 µm and 1.0 µm sized PS at three concentration gradients (1, 10 and 100 mg/L). Results indicate that algal growth significantly increased using the 0.1 and 1.0 µm PS at a high concentration (100 mg/L) on day 17, with growth rates of 74.71% ± 0.94% and 35.87% ± 1.23%, respectively. All tested PS had a maximum inhibitory effect on the photosynthesis on day 5, but the inhibition of photosynthetic activity by 0.1 µm PS alleviated after 13 days of exposure, indicating recovery of microalgae from the toxic environment. The two PS sizes at 100 mg/L concentration triggered EPS release in the latter stage of the experiment; meanwhile, fluorescence EEM analysis showed that smaller-sized PS (0.1 µm) at various doses noticeably increased humic acid-like substances in tightly bound EPS (TB-EPS) fractions on day 17. Our findings showed that EPS release and humic acid-like substances secretion of Microcystis likely can resist MPs exposure. The results provide new insights into the toxicity mechanism of MPs on freshwater microalgae, as well as understanding the ecological risks of microplastics.
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Microalgas , Microcystis , Ecosistema , Matriz Extracelular de Sustancias Poliméricas/química , Sustancias Húmicas/análisis , Microalgas/metabolismo , Microcystis/metabolismo , Microplásticos/toxicidad , Plásticos/metabolismo , Poliestirenos/análisisRESUMEN
When changing surface wettability and nanostructure size, condensation behavior displays distinct features. In this work, we investigated evaporation on a flat hydrophilic surface and condensation on both hydrophilic and hydrophobic nanostructured surfaces at the nanoscale using molecular dynamics simulations. The simulation results on hydrophilic surfaces indicated that larger groove widths and heights produced more liquid argon atoms, a quicker temperature response, and slower potential energy decline. These three characteristics closely relate to condensation areas or rates, which are determined by groove width and height. For condensation heat transfer, when the groove width was small, the change of groove height had little effect, while change of groove height caused a significant variation in the heat flux with a large groove width. When the cold wall was hydrophobic, the groove height became a significant impact factor, which caused no vapor atoms to condense in the groove with a larger height. The potential energy decreased with the increase of the groove height, which demonstrates a completely opposing trend when compared with hydrophilic surfaces.
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BACKGROUND: Magnaporthe grisea (Hebert) ME Barr infection is one of the most serious diseases for cultivated rice in the world. Sterol 14alpha-demethylase (CYP51) is an important drug target for microbial pathogenic infections. To exploit specific and effective fungicides for M. grisea better, the authors have analysed the characteristics of interaction between sterol 14alpha-demethylase from M. grisea (MGCYP51) and azoles. MGCYP51 with truncation of N-terminal residues was cloned and expressed in E. coli, difference binding spectra of MGCYP51 induced by addition of four commercial azoles were determined and molecular modelling of MGCYP51 based on the crystal structure of Mycobacterium tuberculosis Lehmann & Newman and docking with the azoles were performed. RESULTS: The affinity of the azoles for MGCYP51 was positively correlated with their hydrophobicity. Amino acid residues Tyr112, Phe120, Phe220, His308 and Phe497 of MGCYP51, forming a large hydrophobic cavity, are the key residues interacting with azole fungicides. Furthermore, Phe220 and Phe497 are fungus and species specific respectively. CONCLUSION: The results suggest that the more potent azole fungicides for MGCYP51 should possess more hydrophobic groups interacting with residues Phe220 and Phe497.
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Azoles/metabolismo , Sistema Enzimático del Citocromo P-450/química , Proteínas Fúngicas/química , Fungicidas Industriales/metabolismo , Expresión Génica , Magnaporthe/enzimología , Aminoácidos , Azoles/química , Azoles/farmacología , Sitios de Unión , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fungicidas Industriales/química , Fungicidas Industriales/farmacología , Interacciones Hidrofóbicas e Hidrofílicas , Magnaporthe/química , Magnaporthe/efectos de los fármacos , Magnaporthe/genética , Modelos Moleculares , Datos de Secuencia Molecular , Oryza/microbiología , Enfermedades de las Plantas/microbiología , Unión Proteica , Alineación de Secuencia , Esterol 14-DesmetilasaRESUMEN
The experimental control of gene expression in specific tissues or cells at defined time points is a useful tool for the analysis of gene function. GAL4/VP16-UAS enhancer trap lines can be used to selectively express genes in specific tissues or cells, and an ethanol-inducible system can help to control the time of expression. In this study, the combination of the two methods allowed the successful regulation of gene expression in both time and space. For this purpose, a binary vector, 962-UAS::GUS, was constructed in which the ALCR activator and beta-glucuronidase (GUS) reporter gene were placed under the control of upstream activator sequence (UAS) elements and the alcA response element, respectively. Three different GAL4/VP16-UAS enhancer trap lines of Arabidopsis were transformed, resulting in transgenic plants in which GUS activity was detected only on ethanol induction and exclusively in the predicted tissues of the enhancer trap lines. As a library of different enhancer trap lines with distinct green fluorescent protein (GFP) patterns exist, transformation with a similar vector, in which GUS is replaced by another gene, would enable the control of the time and place of transgene expression. We have constructed two vectors for easy cloning of the gene of interest, one with a polylinker site and one that is compatible with the GATEWAY vector conversion system. The method can be extended to other species when enhancer trap lines become available.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Elementos de Facilitación Genéticos , Etanol/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , TransgenesRESUMEN
Double-stranded RNA (dsRNA)-dependent protein kinase (PKR) is an interferon-induced protein kinase that plays a central role in the anti-viral process. Due to its pro-apoptotic and anti-proliferative action, there is an increased interest in PKR modulation as an anti-tumor strategy. PKR is overexpressed in breast cancer cells; however, the role of PKR in breast cancer cells is unclear. The expression/activity of PKR appears inversely related to the aggressiveness of breast cancer cells. The current study investigated the role of PKR in the motility/migration of breast cancer cells. The activation of PKR by a synthesized dsRNA (PIC) significantly decreased the motility of several breast cancer cell lines (BT474, MDA-MB231 and SKBR3). PIC inhibited cell migration and blocked cell membrane ruffling without affecting cell viability. PIC also induced the reorganization of the actin cytoskeleton and impaired the formation of lamellipodia. These effects of PIC were reversed by the pretreatment of a selective PKR inhibitor. PIC also activated p38 mitogen-activated protein kinase (MAPK) and its downstream MAPK-activated protein kinase 2 (MK2). PIC-induced activation of p38 MAPK and MK2 was attenuated by the PKR inhibitor and the PKR siRNA, but a selective p38 MAPK inhibitor (SB203580) or other MAPK inhibitors did not affect PKR activity, indicating that PKR is upstream of p38 MAPK/MK2. Cofilin is an actin severing protein and regulates membrane ruffling, lamellipodia formation and cell migration. PIC inhibited cofilin activity by enhancing its phosphorylation at Ser3. PIC activated LIM kinase 1 (LIMK1), an upstream kinase of cofilin in a p38 MAPK-dependent manner. We concluded that the activation of PKR suppressed cell motility by regulating the p38 MAPK/MK2/LIMK/cofilin pathway.
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Neoplasias de la Mama/enzimología , eIF-2 Quinasa/metabolismo , Factores Despolimerizantes de la Actina/metabolismo , Mama/citología , Mama/enzimología , Mama/patología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Membrana Celular/enzimología , Membrana Celular/patología , Movimiento Celular , Activación Enzimática , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Quinasas Lim/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Both epidemiological and experimental studies indicate that ethanol exposure enhances tumor progression. Ethanol exposure promotes cancer cell invasion and is implicated in tumor metastasis. Metastasis consists of multiple processes involving intravasation and extravasation of cancer cells across the blood vessel walls. The integrity of the vascular endothelial barrier that lines the inner surface of blood vessels plays a critical role in cancer cell intravasation/extravasation. We examined the effects of ethanol on the endothelial integrity in vitro. Ethanol at physiologically relevant concentrations did not alter cell viability but disrupted the endothelial monolayer integrity, which was evident by a decrease in the electric resistance and the appearance of intercellular gaps in the endothelial monolayer. The effect of ethanol was reversible once ethanol was removed. The disruption of the endothelial monolayer integrity was associated with an increased invasion of cancer cells through the endothelial monolayer. Ethanol induced the formation of stress fibers; stabilization of actin filaments by jasplakinolide prevented ethanol-induced disruption of endothelial integrity and cancer cell invasion. VE-cadherin is a critical component of the adherens junctions, which regulates vascular endothelial integrity. Ethanol induced the endocytosis of VE-cadherin and the effect was blocked by jasplakinolide. Our results indicate that ethanol may facilitate cancer metastasis by disrupting the vascular endothelial barrier.
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Depresores del Sistema Nervioso Central/toxicidad , Etanol/toxicidad , Uniones Comunicantes/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Actinas/metabolismo , Antineoplásicos/farmacología , Cadherinas/metabolismo , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Depsipéptidos/farmacología , Progresión de la Enfermedad , Impedancia Eléctrica , Endocitosis/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Invasividad Neoplásica , Metástasis de la Neoplasia/tratamiento farmacológico , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Fibras de Estrés/efectos de los fármacosRESUMEN
Subchronic exposure to arsenic increases the incidence of human cancers such as skin, lung, colon, and rectal cancer. The mechanism for arsenic-induced tumorigenesis is still not clear. It is generally believed that DNA damage and genomic instability, generated by arsenic-promoted oxidative stress, account largely for this process. The major sources of reactive oxygen species (ROS) are arsenic-damaged mitochondria. Autophagy is a catabolic process functioning in turnover of long-lived proteins and dysfunctional organelles such as mitochondria. Defects of autophagy under stress conditions promote genomic instability and increase the risk of tumorigenesis. In the present study using a human bronchial epithelial cell line, BEAS-2B cells, we investigated the role of autophagy in arsenic-induced cell transformation, an important step in arsenic tumorigenesis. Our results show that subchronic arsenic exposure induces BEAS-2B cell transformation accompanied with increased ROS generation and autophagy activation. However, the patterns for ROS and autophagy alteration are different. Arsenic exposure generated a prolonged and steady increase of ROS levels, whereas the activation of autophagy, after an initial boost by arsenic administration, decreases in response to subchronic arsenic exposure, although the activity is still higher than a nontreated control. Further stimulation of autophagy increases mitochondria turnover and decreases ROS generation and arsenic-induced cell transformation. Contrarily, inhibition of autophagy activity decreases mitochondria turnover and enhances arsenic-induced ROS generation and cell transformation. In addition, the mammalian target of rapamycin signaling pathway is involved in arsenic-mediated autophagy activation. Our results suggest that autophagy is a cell self-protective mechanism against arsenic-induced cell transformation.
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Arsenitos/toxicidad , Autofagia/efectos de los fármacos , Transformación Celular Neoplásica/inducido químicamente , Células Epiteliales/efectos de los fármacos , Neoplasias Pulmonares/inducido químicamente , Mucosa Respiratoria/efectos de los fármacos , Compuestos de Sodio/toxicidad , Animales , Línea Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Desnudos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/patología , Estrés Oxidativo/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/metabolismo , Factores de Tiempo , Transfección , Carga TumoralRESUMEN
Ethanol is a neuroteratogen and neurodegeneration is the most devastating consequence of developmental exposure to ethanol. The mechanisms underlying ethanol-induced neurodegeneration are complex. Ethanol exposure produces reactive oxygen species (ROS) which cause oxidative stress in the brain. We hypothesized that ethanol would activate autophagy to alleviate oxidative stress and neurotoxicity. Our results indicated that ethanol increased the level of the autophagic marker Map1lc3-II (LC3-II) and upregulated LC3 puncta in SH-SY5Y neuroblastoma cells. It also enhanced the levels of LC3-II and BECN1 in the developing brain; meanwhile, ethanol reduced SQSTM1 (p62) levels. Bafilomycin A(1), an inhibitor of autophagosome and lysosome fusion, increased p62 levels in the presence of ethanol. Bafilomycin A(1) and rapamycin potentiated ethanol-increased LC3 lipidation, whereas wortmannin and a BECN1-specific shRNA inhibited ethanol-promoted LC3 lipidation. Ethanol increased mitophagy, which was also modulated by BECN1 shRNA and rapamycin. The evidence suggested that ethanol promoted autophagic flux. Activation of autophagy by rapamycin reduced ethanol-induced ROS generation and ameliorated ethanol-induced neuronal death in vitro and in the developing brain, whereas inhibition of autophagy by wortmannin and BECN1-specific shRNA potentiated ethanol-induced ROS production and exacerbated ethanol neurotoxicity. Furthermore, ethanol inhibited the MTOR pathway and downregulation of MTOR offered neuroprotection. Taken together, the results suggest that autophagy activation is a neuroprotective response to alleviate ethanol toxicity. Ethanol modulation of autophagic activity may be mediated by the MTOR pathway.
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Autofagia/efectos de los fármacos , Citoprotección/efectos de los fármacos , Etanol/toxicidad , Neuronas/efectos de los fármacos , Neuronas/patología , Androstadienos/farmacología , Animales , Antioxidantes/farmacología , Proteínas Reguladoras de la Apoptosis/metabolismo , Beclina-1 , Biomarcadores/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/enzimología , Encéfalo/crecimiento & desarrollo , Caspasa 3/metabolismo , Línea Celular Tumoral , Activación Enzimática/efectos de los fármacos , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/metabolismo , Estrés Oxidativo/efectos de los fármacos , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/metabolismo , WortmaninaRESUMEN
Ultraviolet B (UVB) exposure causes damage to skin and represents the primary etiological agent for skin cancer formation. UVB induces DNA damage and apoptosis in epidermal cells. In this study, we demonstrated that UVB activated autophagy in JB6 epidermal cells, which was evident by the formation of LC3 puncta, the induction of LC3 lipidation, the increase in beclin 1 expression, and the decrease in the levels of p62. Autophagy appeared to be a protective response to UVB-induced damage because inhibition of autophagy exacerbated UVB-induced cell death, and stimulation of autophagy offered protection. Furthermore, we demonstrated that glycogen synthase kinase 3ß (GSK3ß) was involved in UVB-induced autophagy. UVB inhibited GSK3ß activation by simultaneously enhancing phosphorylation at Ser9 and suppressing Tyr216 phosphorylation. GSK3ß negatively regulated autophagy; overexpression of wildtype or S9A (constitutive-active) GSK3ß mutant inhibited UVB-mediated autophagy, while overexpression of a dominant-negative K85R mutant enhanced UVB-mediated autophagy. Inhibition of GSK3ß also offered protection against UVB-mediated damage. UVB activated AMP-activated protein kinase (AMPK), an important regulator of autophagy through the inhibition of GSK3ß. Taken together, our results suggest that UVB-stimulated autophagy is a protective response for epidermal cells and is mediated by the GSK3ß/AMPK pathway.
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Autofagia/efectos de la radiación , Epidermis/metabolismo , Células Epiteliales/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Transducción de Señal/efectos de la radiación , Rayos Ultravioleta , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Línea Celular , Epidermis/efectos de la radiación , Células Epiteliales/efectos de la radiación , Glucógeno Sintasa Quinasa 3 beta , Ratones , Fosforilación/efectos de la radiaciónRESUMEN
Sterol 14alpha-demethylase (CYP51), the most widely distributed member of the P450 superfamily, is the key enzyme in sterol biosynthesis pathway. CYP51 is not only an important model for fundamental P450 structure/function studies, but also an important target protein of cholesterol-lowering agents, antifungal drugs and herbicides. This article reviewed the research advances in CYP51 at various aspects, including sequence characteristics, physiological roles, catalytic properties in vitro, protein structure, structure-function relationships and inhibition of CYP51. The problems remained in current research and designations of CYP51 inhibitors are also discussed.
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Sistema Enzimático del Citocromo P-450 , Inhibidores Enzimáticos del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/fisiología , Mutagénesis Sitio-Dirigida , Análisis de Secuencia de Proteína , Esterol 14-DesmetilasaRESUMEN
Agroinfiltration is employed as a fast way to directly create marker-free transgenic tobacco plants. As an example for the efficiency of the method, Agrobacterium cells harboring a marker-free vector coding for beta-glucuronidase (GUS) were infiltrated into the leaf discs of Nicotiana tabacum, which were then used as explants for marker-free plant regeneration by tissue culture. Through GUS staining, a large number of small calli were shown to be stably transformed on the treated leaf discs at 17 days after agroinfiltration. Most importantly, after continuous culture of the leaf discs until shoot regeneration, about 15% of the regenerants were proven to be transformants by polymerase chain reaction (PCR) analysis.