Lysine-specific demethylase 1 (LSD1) participate in porcine early embryonic development by regulating cell autophagy and apoptosis through the mTOR signaling pathway.

Lysine-specific demethylase 1 (LSD1) stands as the pioneering histone demethylase uncovered, proficient in demethylating H3K4me1/2 and H3K9me1/2, thereby governing transcription and participating in cell apoptosis, proliferation, or differentiation. Nevertheless, the complete understanding of LSD1 during porcine early embryonic development and the underlying molecular mechanism remains unclear. Thus, we investigated the mechanism by which LSD1 plays a regulatory role in porcine early embryos. This study revealed that LSD1 inhibition resulted in parthenogenetic activation (PA) and in vitro fertilization (IVF) embryo arrested the development, and decreased blastocyst quality. Meanwhile, H3K4me1/2 and H3K9me1/2 methylase activity was increased at the 4-cell embryo stage. RNA-seq results revealed that autophagy related biological processes were highly enriched through GO and KEGG pathway analyses when LSD1 inhibition. Further studies showed that LSD1 depletion in porcine early embryos resulted in low mTOR and p-mTOR levels and high autophagy and apoptosis levels. The LSD1 deletion-induced increases in autophagy and apoptosis could be reversed by addition of mTOR activators. We further demonstrated that LSD1 inhibition induced mitochondrial dysfunction and mitophagy. In summary, our research results indicate that LSD1 may regulate autophagy and apoptosis through the mTOR pathway and affect early embryonic development of pigs.

Preparation and Performance Study of High-Strength and Corrosion-Resistant Cement-Based Materials Applied in Coastal Acid Rain Areas.

Investigations regarding the preparation and durability of cement-based materials applied in specific coastal acid rain environments are scarce, particularly those involving the addition of four auxiliary cementitious materials (ACMs) to cement for modification. To improve the durability of concrete structures in coastal acid rain areas, a systematic study was conducted regarding the preparation of high-strength and corrosion-resistant cement-based materials using ACM systems composed of fly ash (FA), granulated blast furnace slag (GBFS), silica fume (SF), and desulfurization gypsum (DG) instead of partial cement. Through an orthogonal experimental design, the effect of the water-binder ratio, cementitious ratio, and replacement cement ratio on the compressive strength, corrosion resistance coefficient, and chloride ion permeability coefficient of the materials were analyzed and the mix proportions of the materials were evaluated and optimized using the comprehensive scoring method. The results show that implementing a FA:GBFS:SF:DG ratio of 2:6:1:1 to replace 60% of cement allows the consumption of calcium hydroxide crystals generated through cement hydration, promotes the formation of ettringite, optimizes the pore structures of cementitious materials, and improves the compressive strength, acid corrosion resistance, and chloride ion permeability of the materials. This study provides a reference for selecting concrete materials for buildings in coastal acid rain environments.

Exploring long-term global environmental impacts of chlorinated paraffins (CPs) in waste: Implications for the Stockholm and Basel Conventions and the global plastic treaty.

Chlorinated paraffins (CPs), mainly short-chain CPs (SCCPs) and medium-chain CPs (MCCPs), are currently the most produced and used industrial chemicals related to persistent organic pollutants (POPs) globally. These chemicals are widely detected in the environment and in the human body. As the release of SCCPs and MCCPs from products represents only a small fraction of their stock in products, the potential long-term release of CPs from a large variety of products at the waste stage has become an issue of great concern. The results of this study showed that, by 2050, SCCPs and MCCPs used between 2000 and 2021 will cumulatively generate 226.49 Mt of CP-containing wastes, comprising 8610.13 kt of SCCPs and MCCPs. Approximately 79.72 Mt of CP-containing wastes is predicted to be generated abroad through the international trade of products using SCCPs and MCCPs. The magnitude, distribution, and growth of CP-containing wastes subject to environmentally sound disposal will depend largely on the relevant provisions of the Stockholm and Basel Conventions and the forthcoming global plastic treaty. According to multiple scenarios synthesizing the provisions of the three conventions, 26.6-101.1 Mt of CP-containing wastes will be subject to environmentally sound disposal as POP wastes, which would pose a great challenge to the waste disposal capacity of China, as well as for countries importing CP-containing products. The additional 5-year exemption period for MCCPs is expected to see an additional 10 Mt of CP-containing wastes subject to environmentally sound disposal. Thus, there is an urgent need to strengthen the Stockholm and Basel Conventions and the global plastic treaty.

Ecological risk of phenol on typical biota of the northern Chinese river from an integrated probability perspective: the Hun River as an example.

Phenol, known for its bioaccumulative nature and severe toxicity to riverine organisms, poses complex challenges for ecological risk assessment. To tackle this issue, we developed a three-stage incremental assessment method, providing an integrated perspective on phenol toxicity risk for aquatic organisms. The findings indicated that phenol concentrations were generally higher in the aquatic environments of northern rivers, such as the Hun River, Taizi River, and Liao River, compared to those in southern China. The evaluation results at individual points showed that the ecological risk of phenol to aquatic organisms ranked from high to low during rainy, dry, and normal seasons, showing seasonal variation characteristics. Regarding spatial variation along the river, the ecological risk of phenol gradually increased from upper reaches, peaked in the middle reaches, and then decreased in the lower reaches. Considering the different species types, fish face a higher risk of toxic effects of phenol than invertebrates when exposed to phenol over a long period of time, probably due to the bioaccumulative nature of phenol. To address ecological risk control at the watershed scale, there is an urgent need to revise China's current river water quality standards. It is essential to increase the emphasis on ecological risk control for aquatic organisms. Developing more targeted and refined ecological risk control strategies for river phenols is crucial to maintain a healthier and more vibrant river ecosystem.

Dynamic Source Distribution and Emission Inventory of a Persistent, Mobile, and Toxic (PMT) Substance, Melamine, in China.

Persistent, mobile, and toxic (PMT) substances are affecting the safety of drinking water and are threatening the environment and human health. Many PMT substances are used in industrial processing or consumer products, but their sources and emissions mostly remain unclear. This study presents a long-term source distribution and emission estimation of melamine, a high-production-volume PMT substance of emerging global concern. The results indicate that in China, approximately 1858.7 kilotonnes (kt) of melamine were released into the water (∼58.9%), air (∼27.0%), and soil systems (∼14.1%) between 1995 and 2020, mainly from its production and use in the decorative panels, textiles, and paper industries. The textile and paper industries have the highest emission-to-consumption ratios, with more than 90% emissions per unit consumption. Sewage treatment plants are the largest source of melamine in the environment for the time being, but in-use products and their wastes will serve as significant melamine sources in the future. The study prompts priority action to control the risk of PMT substances internationally.

Ecological risk evaluation and sensitivity analysis of heavy metals on soil organisms under human activities in the Tibet Plateau, China.

The Tibetan Plateau (TP), once considered a pristine environment, is now facing increased heavy metal pollution due to human activities, causing unprecedented ecological risks to soil organisms. However, little is known about the sensitivity and tolerance of different soil organisms to heavy metal toxicity in the high-altitude areas of the TP under the background of human activity intensity and future risk control priorities. In this study, we conducted an ecological risk assessment and threshold calculation for 10 heavy metals in soil for typical soil organisms, including Cd, Co, Cr, Cu, Ni, Pb, Zn, Mn, Sb, and Sn, using the species sensitivity distribution (SSD) method in the zone between Ranwu town and Renlongba glacier on the TP. The results revealed that most sampling sites had average levels of Cd, Sb and Ni exceeding their regional soil environmental background values and were the major contaminants. Impressively, the hitherto undeveloped Renlungba Glacier showed relatively high contamination levels of Sb and Ni. From the view of sensitivity differences, the toxicity risks of Cd, Cr, Cu, Ni and Pb were higher for terrestrial plants than for soil invertebrates based on the fitted heavy metal SSD curve trends. In terms of the ecological risk level, the average potentially affected fraction values of Zn and Ni reached 18.5% and 17.0%, respectively, with significant ecological risk at a few sampling sites. In terms of ecological risk thresholds, the Cd concentration at the 5% hazard concentration (HC5) control level was 0.05 mg/kg, which was the heavy metal with the highest risk in this study. Comparing the HC5 values of each heavy metal with the limit values in the current Chinese soil environmental quality standards, the existing administrative policies as a whole lack a powerful prevention of the potential ecological risk posed by heavy metals to soil organisms.

Ecological risk characteristics of sediment-bound heavy metals in large shallow lakes for aquatic organisms: The case of Taihu Lake, China.

Heavy metal contamination in the surface sediments of large shallow lakes in China is becoming increasingly serious. However, more attention has been paid to the human health risk of heavy metals in the past, while little consideration has been given to aquatic organisms. Taking Taihu Lake as an example, we explored the spatial and temporal heterogeneity of the potential ecological risks of seven heavy metals (Cd, As, Cu, Pb, Cr, Ni, and Zn) to species at different taxonomic scales using an improved species sensitivity distribution (SSD) method. The results showed that all six heavy metals, except Cr, were exceeded to some extent compared to background levels, with Cd being the most severe exceedance. Based on the hazardous concentration for 5% of the species (HC5), Cd had the lowest HC5 value, implying the highest ecological risk of toxicity. Ni and Pb had the highest HC5 values and the lowest risk. Cu, Cr, As and Zn were at a relatively moderate levels. For the different groups of aquatic organisms, the ecological risk of most heavy metals was generally lower for vertebrates than for the whole species. The risk for invertebrates and algae was higher than that for all species. Zn and Cu had the highest potentially affected fractions (PAFs) for all classification cases, with mean PAFs of 30.25% and 47.2%, respectively. Spatially, the high ecological risk of sediment heavy metals was significantly related to the spatial characteristics of the type and intensity of human activities in the catchment. Administratively, the environmental quality standards for freshwater sediments proposed by America and Canada are insufficient to protected against the ecological risks of heavy metals in Taihu Lake. In the absence of such standards, China urgently needs to establish an approptiate system of environmental quality standards for heavy metals in lake sediments.

[Research Status and Trend Analysis of Environmental and Health Risk and Control of Persistent, Mobile, and Toxic Chemicals].

Persistent, mobile, and toxic or very persistent and very mobile (PMT/vPvM) chemicals have been widely detected in surface water, groundwater, and drinking water around the world and are important emerging contaminants that may significantly affect human health and the environment in the future. According to the identification criteria proposed by the European Union, there are thousands of PMT/vPvM substances in existing chemicals, covering a wide range of applications, including dozens of high-yield industrial chemicals such as melamine. PMT/vPvM chemicals can be discharged into the environment through farmland runoff, industrial wastewater, and domestic sewage, and sewage treatment plants are currently considered to be their main discharge route. It is difficult to effectively remove PMT/vPvM chemicals through the current conventional water treatment technology; they can exist in the water circulation system of the urban human settlement environment for a long time, endangering the safety of drinking water and the ecosystem. The European Union has taken the lead in introducing PMT/vPvM chemicals specifically into the priority areas of the current chemical risk management system. At present, there are still many potential PMT/vPvM chemicals in the environment, and their monitoring methods need to be further improved. It will take time for the identification of substances, the scope of categories, and the establishment of lists. Studies on the environmental fate and exposure of PMT/vPvM in various regions of the world are still very limited, and research on the potential, long-term ecotoxicity, and human health hazard effects remains scarce. At the same time, the research and development of substitute or alternative technologies, as well as environmental engineering treatment technologies such as sewage treatment and contaminated site remediation, will become an urgent need for future PMT/vPvM risk scientific research and management decisions.

Emission and environmental distribution of decabromodiphenyl ethane (DBDPE) in China from 2006 to 2026: Retrospection, forecasting, and implications for assessment and management.

Decabromodiphenyl ethane (DBDPE) is the main alternative to decabromodiphenyl ether (deca-BDE) in commercial use. However, there is increasing evidence show that DBDPE is a potential persistent organic pollutant, and it has been found ubiquitously in environmental media across China in recent years. Monitoring studies have not been able to determine the overall levels and temporal trends of DBDPE contamination in China, and have been unable to explain how emission patterns can affect their environmental distribution. Therefore, this study estimated the temporal variance of DBDPE emissions and environmental concentrations in five regions of China from 2006 to 2026 using the PROduction-To-EXposure (PROTEX) mass balance model. The results showed that Guangdong Province was the greatest DBDPE pollution hotspot in China due to emissions from plastics manufacturing and e-waste disposal; there was also severe pollution in Shandong Province, where almost all the DBDPE in China is produced. The DBDPE concentrations in indoor and outdoor environments increased substantially in all regions during 2006-2021. Furthermore, in Guangdong Province and Shandong Province, the ratio of indoor/outdoor air concentrations was greater than or close to 1, indicative of significant outdoor emission sources of DBDPE. In contrast, the ratios for the Beijing-Tianjin-Hebei region, East China, and Southwest China were below 1 due to the indoor use of electronic equipment containing DBDPE. The temporal trends of these ratios indicated that DBDPE contamination has gradually spread from high-concentration environments with strong emission sources to low-concentration environments. The outcomes of this study have important implications for the risk assessment of DBDPE use in China and can be used to establish contamination-mitigation actions.

Global Historical Production, Use, In-Use Stocks, and Emissions of Short-, Medium-, and Long-Chain Chlorinated Paraffins.

The last few decades have seen ubiquitous and increasing contamination of chlorinated paraffins (CPs) worldwide. Here, we develop the first global inventories of production, use, in-use stocks, and emissions of total CPs, including the short-, medium- and long-chain components (SCCPs, MCCPs, and LCCPs) during 1930-2020 using a dynamic substance flow analysis model named Chemical in Products Comprehensive Anthroposhpheric Fate Estimation. The model estimates that a total of ∼33 million metric tons of CPs have been produced and used globally, ∼40% of which still resided in in-use products by 2020 and is available for long-term emissions in the next decades. Global cumulative emissions of CPs have increased to ∼5.2 million metric tons by 2020, with SCCPs, MCCPs, and LCCPs accounting for ∼30, 40, and 30%, respectively. While the production, use, and emissions of CPs started declining in regions such as Western Europe, they remain high in China. The model also suggests that homologues with 10, 14, and 22-23 carbons were predominant in the cumulatively produced and emitted SCCPs, MCCPs, and LCCPs, respectively. The emission estimates were evaluated by generating environmental concentrations that are comparable to literature-reported environmental monitoring data. Our estimates provide opportunities to link the environmental fate and occurrence of CPs to emission sources and lay the basis for future risk-reduction strategies of CPs around the world.

Chlorogenic Acid Ameliorates Damage Induced by Fluorene-9-Bisphenol in Porcine Sertoli Cells.

4,4'-(9-Fluorenylidene) diphenol (BPFL, also known as BHPF and fluorene-9-bisphenol) is a novel bisphenol A substitute that is used in the plastics industry as an organic synthesis intermediate and is a potential endocrine disruptor. However, the deleterious effects of BPFL on porcine Sertoli cells (SCs) and the possible underlying mechanisms are still unclear. Chlorogenic acid (CA) is a free radical scavenger in the cellular antioxidant system that prevents oxidative damage and apoptosis. In the present research, we found that BPFL induced impairments in porcine SCs in a dose-dependent manner and that CA protected porcine SCs against BPFL exposure-induced impairments. Cell viability, proliferation and apoptosis assay results revealed that BPFL exposure could inhibit porcine SC proliferation and induce apoptosis, while CA supplementation ameliorated the effects of BPFL. Further analysis revealed that BPFL exposure induced oxidative stress, mitochondrial membrane potential dysfunction and DNA damage accumulation. Transcriptome analysis and further real-time quantitative PCR and Western blot results showed that BPFL exposure induced endoplasmic reticulum stress and apoptosis. Supplementation with CA dramatically ameliorated these phenotypes in BPFL-exposed porcine SCs. Overall, the present research reveals the possible underlying mechanisms by which BPFL exposure induced impairments and CA supplementation protected against these impairments in porcine SCs.

Glycine alleviates fluoride-induced oxidative stress, apoptosis and senescence in a porcine testicular Sertoli cell line.

Glycine is a well-known free radical scavenger in the cellular antioxidant system that prevents oxidative damage and apoptosis. Excessive fluoride exposure is associated with multiple types of cellular damage in humans and animals. The objective of the present study was to investigate the protective effects of glycine on sodium fluoride (NaF) exposure and the possible underlying mechanisms in a porcine testicular Sertoli cell line model. Cellular viability and proliferation were examined following NaF exposure and glycine supplementation, and glycine dramatically ameliorated the decreases in NaF-induced porcine testicular Sertoli cell viability and proliferation. Further investigations revealed that glycine decreased NaF-induced intracellular reactive oxygen species production, DNA fragment accumulation and the apoptosis incidence in the porcine testicular Sertoli cell line; in addition, glycine improved mitochondrial function and ATP production. Notably, results of the SPiDER-ß-Gal analysis suggested that glycine alleviated NaF-induced cellular senescence and downregulated P53, P21, HMGA2 and P16INK4a gene expression in the porcine testicular Sertoli cell line. Collectively, the beneficial effects of glycine alleviate NaF-induced oxidative stress, apoptosis and senescence, and together with our previous findings, support the hypothesis that glycine plays an important role in protecting against NaF exposure-induced impairments in the porcine testicular Sertoli cell line.

Emission and fate of antibiotics in the Dongjiang River Basin, China: Implication for antibiotic resistance risk.

Antibiotics used for human and veterinary purposes are released into the environment, resulting in potential adverse effects, including the development and spread of antibiotic resistant bacteria. Here we investigated the dynamic fate of 36 antibiotics in a large river basin Dongjiang in South China, and discussed their potential antibiotic resistance selection risk. Based on the usage, excretion rate, wastewater treatment rate, human population and animal numbers the emissions of 36 frequently detected antibiotics were estimated for the Dongjiang River Basin. The total usage of the 36 antibiotics in the basin was 623.4 tons, which included 37% for human use and the rest for veterinary purposes. After being metabolized and partially treated, the amount of antibiotics excreted and released into the environment decreased to 267.6 tons. By allocating the high-precision antibiotic discharge inventory to 42 sewage plants and 17 livestock farms, an improved GREAT-ER (Geography referenced Regional Exposure Assessment Tool for European Rivers) model for the Dongjiang River Basin, with a well calibration river flow network based on the SWAT (Soil and Water Assessment Tool), was established to simulate the dynamic fate of 36 antibiotics. The simulation results showed that antibiotics contaminated >50% of the river sections. The modelled concentrations in water were almost within an order of magnitude of the measured concentrations. Antibiotic contamination in the dry season was obviously higher than that in the wet season. The concentrations of the antibiotics were always higher at the discharge zones and lower reaches of the river basin than the other reaches. The antibiotic resistance risk assessment showed that 23 out of the 36 antibiotics (nearly 65%) could pose high risks in the river basin. For those river reaches with high risks, the risk levels could mostly be reduced to low risk levels with a certain distance (15 km) from the pollution source. Therefore, more attention should be paid to those impact zones in term of antibiotic resistance.

Emission estimation and fate modelling of three typical pesticides in Dongjiang River basin, China.

Pesticides are widely and intensively used in the world for crops protection. High pesticide loadings can potentially pollute the water resource. However, little is known about the usage, environmental emission and fate of pesticides in river basins. Here, we firstly established a pesticide emission estimation method, and investigated the environmental fate of three commonly used pesticides (chlorpyrifos, triazophos, and isoprothiolane) in Dongjiang River basin, southern China using mathematical modelling approach in combination with field monitoring. The distributed hydrological model SWAT (Soil and Water Assessment Tool) was applied to model the emission of the target pesticides from farmland to stream water, and their fate in the basin. A satisfactory model calibration for flow and suspended sediment was obtained based on eight-year observation data of four hydrological monitoring stations in Dongjiang River basin. The differences between the simulation and observation of pesticides were almost within an order of magnitude, including more than 53% differences within 0.5 order of magnitude. In the river basin, 78860 kg of chlorpyrifos, 54990 kg of triazophos and 35320 kg of isoprothiolane were sprayed onto the crops, the estimated annual emissions of the basin come up to 1801 kg, 3779 kg, and 2330 kg under the conditions of rainfall, surface runoff and percolation. After a series of environmental processes including settlement and degradation within the channels, the predicted export masses for chlorpyrifos, triazophos and isoprothiolane were reduced to 266 kg, 1858 kg, 1350 kg, respectively. Successful prediction suggests that the reliable estimation method combing the SWAT modelling can help us understand the source, concentration levels and fate of pesticides in river basin in different scales. Combing the method of emission and fate modelling method we proposed, countries and regions lacking pesticide-application database can facilitate better management of pesticides.

miR-196a Promotes Proliferation and Inhibits Apoptosis of Immature Porcine Sertoli Cells.

Our previous study showed that the expression of miR-196a was significantly higher in immature porcine testes than in mature porcine testes. However, the role of miR-196a in immature Sertoli cells remains unclear. In this study, miR-196a mimics, miR-196a inhibitor, and microRNA-small hairpin negative control (miRNA-ShNC) were transfected into immature Sertoli cells, respectively. Subsequently, the cell cycle and apoptosis rate of the immature Sertoli cells were measured by flow cytometry, and the viability of the Sertoli cells was measured by the MTS assay. Furthermore, the candidate target genes of miR-196a were analyzed by bioinformatics, and the target genes were validated by dual luciferase reporter assays, then the expression of target genes was detected by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot assays. The results showed that miR-196a promotes the proliferation and inhibits the apoptosis of immature Sertoli cells. miR-196a directly binds the 3' untranslated region (3' UTR) of RCC2 and ABCB9. The expression of miR-196a was shown to be negatively correlated with the messenger RNA and protein levels of the RCC2 and ABCB9 genes. The study demonstrates that miR-196a regulates immature Sertoli cell proliferation and apoptosis and inhibits the expression of RCC2 and ABCB9.

MiR-375 induces ROS and apoptosis in ST cells by targeting the HIGD1A gene.

HIGD1A can reduce ROS and apoptosis in cells under low-glucose or hypoxic conditions, and HIGD1A is one of the target genes for miR-375, according to our previous studies. However, it is not known whether miR-375 can indirectly regulate ROS and apoptosis in porcine Sertoli cells. To answer this question, HIGD1A and miR-375 were overexpressed in porcine Sertoli cells, and ROS and apoptosis were assayed. The results showed that ROS levels and expression levels of CASPASE3 in HIGD1A-overexpressing cells were significantly lower than those in the control cells. However, ROS levels and CASPASE3 expression in miR-375-overexpressing cells were significantly higher than those in the control cells. The rate of apoptosis in HIGD1A-overexpressing cells was significantly lower than that in miR-375-overexpressing cells. Considering that the HIGD1A gene is a target of miR-375, these findings suggest that miR-375 can induce an increase in ROS levels and apoptosis by inhibiting HIGD1A in porcine ST cells.

INDETERMINATE SPIKELET1 Recruits Histone Deacetylase and a Transcriptional Repression Complex to Regulate Rice Salt Tolerance.

Perception and transduction of salt stress signals are critical for plant survival, growth, and propagation. Thus, identification of components of the salt stress-signaling pathway is important for rice (Oryza sativa) molecular breeding of salt stress resistance. Here, we report the identification of an apetala2/ethylene response factor transcription factor INDETERMINATE SPIKELET1 (IDS1) and its roles in the regulation of rice salt tolerance. By genetic screening and phenotype analysis, we demonstrated that IDS1 conferred transcriptional repression activity and acted as a negative regulator of salt tolerance in rice. To identify potential downstream target genes regulated by IDS1, we conducted chromatin immunoprecipitation (ChIP) sequencing and ChIP-quantitative PCR assays and found that IDS1 may directly associate with the GCC-box-containing motifs in the promoter regions of abiotic stress-responsive genes, including LEA1 (LATE EMBRYOGENESIS ABUNDANT PROTEIN1) and SOS1 (SALT OVERLY SENSITIVE1), which are key genes regulating rice salt tolerance. IDS1 physically interacted with the transcriptional corepressor topless-related 1 and the histone deacetylase HDA1, contributing to the repression of LEA1 and SOS1 expression. Analyses of histone H3 acetylation status and RNA polymerase II occupation on the promoters of LEA1 and SOS1 further defined the molecular foundation of the transcriptional repression activity of IDS1. Our findings illustrate an epigenetic mechanism by which IDS1 modulates salt stress signaling as well as salt tolerance in rice.

Ribonucleotide reductase is an effective target to overcome gemcitabine resistance in gemcitabine-resistant pancreatic cancer cells with dual resistant factors.

Gemcitabine is widely used for pancreatic, lung, and bladder cancer. However, drug resistance against gemcitabine is a large obstacle to effective chemotherapy. Nucleoside transporters, nucleoside and nucleotide metabolic enzymes, and efflux transporters have been reported to be involved in gemcitabine resistance. Although most of the resistant factors are supposed to be related to each other, it is unclear how one factor can affect the other one. In this study, we established gemcitabine-resistant pancreatic cancer cell lines. Gemcitabine resistance in these cells is caused by two major processes: a decrease in gemcitabine uptake and overexpression of ribonucleotide reductase large subunit (RRM1). Knockdown of RRM1, but not the overexpression of concentrative nucleoside transporter 1 (CNT1), could completely overcome the gemcitabine resistance. RRM1 knockdown in gemcitabine-resistant cells could increase the intracellular accumulation of gemcitabine by increasing the nucleoside transporter expression. Furthermore, a synergistic effect was observed between hydroxyurea, a ribonucleotide reductase (RR) inhibitor, and gemcitabine on the gemcitabine-resistant cells. Here we indicate that RR is one of the most promising targets to overcome gemcitabine resistance in gemcitabine-resistant cells with dual resistant factors.

Molecular basis for the induction of an angiogenesis inhibitor, thrombospondin-1, by 5-fluorouracil.

5-Fluorouracil (5-FU) is one of the most commonly used anticancer drugs in chemotherapy against various solid tumors. 5-FU dose-dependently increased the expression levels of intrinsic antiangiogenic factor thrombospondin-1 (TSP-1) in human colon carcinoma KM12C cells and human breast cancer MCF7 cells. We investigated the molecular basis for the induction of TSP-1 by 5-FU in KM12C cells. Promoter assays showed that the region with the Egr-1 binding site is critical for the induction of TSP-1 promoter activity by 5-FU. The binding of Egr-1 to the TSP-1 promoter was increased in KM12C cells treated with 5-FU. Immunofluorescence staining revealed that 5-FU significantly increased the level of Egr-1 in the nuclei of KM12C cells. The suppression of Egr-1 expression by small interfering RNA decreased the expression level of TSP-1. Furthermore, 5-FU induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and heat shock protein 27 (HSP27). Blockade of the p38 MAPK pathway by SB203580 remarkably inhibited the phosphorylation of HSP27 induced by 5-FU and decreased the induction of Egr-1 and TSP-1 by 5-FU in KM12C cells. These findings suggest that the p38 MAPK pathway plays a crucial role in the induction of Egr-1 by 5-FU and that induced Egr-1 augments TSP-1 promoter activity, with the subsequent production of TSP-1 mRNA and protein.

Molecular cloning and sequence analysis of the porcine precursor of endothelin-2.

The amino acid sequences of two of the three endothelin (ET) family peptides, ET-1 and ET-3, are identical among mammals, whereas for the other family member, ET-2 or vasoactive intestinal contractor (VIC), the mouse and rat sequences differ from the human counterpart ET-2 by one amino acid residue. To examine more deeply the structural diversity among ET-2/VIC orthologs (EDN2), we screened porcine ET-2/VIC-like cDNAs using the 5' rapid amplification of cDNA ends (RACE) method with degenerate primers based on ET-2/VIC mature peptides. Sequence analysis of the cDNAs showed that ET-2 is present in pig. The full-length cDNA sequence, produced by combining 5' RACE and 3' RACE products, revealed the porcine precursor protein of ET-2 (PPET-2). Porcine PPET-2, made up of 214 amino acids, includes a 26-residue putative signal sequence, big ET-2, mature ET-2, and ET-2-like peptide. The percent sequence identity of porcine PPET-2 with human PPET-2, and rat or mouse precursor protein of VIC runs between approximately 70% and 74%. ET-2, although expressed in intestine, has no anti-microbial activity.