Organic solvents-free and ambient-pressure drying melamine formaldehyde resin aerogels with homogeneous structures, outstanding mechanical strength and flame retardancy.

Atmospheric drying method for fabricating aerogels is considered the most promising way for casting aerogels on a large scale. However, the organic solvent exchange, remaining environmental pollution risk, is a crucial step in mitigating the impact of surface tension during the atmospheric drying process, especially for wet gel formed through the alkoxy-derived sol-gel process, such as melamine-formaldehyde resin (MF) aerogel. Herein, a tough polymer-assisted in situ polymerization was proposed to fabricate MF resin aerogel with a combination of mechanical toughness and strength, enabling it to withstand the capillary force during water evaporation. The monolithic MF resin aerogel through the sol-gel method can be directly prepared without additional network strengthening or organic solvent exchange. The resulting MF resin aerogel exhibits a homogeneous as well as hierarchical structure with macropores and mesopores (~6 µm and ~5 nm), high compressive modulus of 31.8 MPa, self-extinguishing property, and high-temperature thermal insulation with 97 % heat decrease for butane flame combustion. This work presents a straightforward and environmentally friendly method for fabricating MF resin aerogels with nanostructures and excellent performance in open conditions, exhibiting various applications.

Catalytic polymer self-cleavage for CO2 generation before combustion empowers materials with fire safety.

Polymeric materials, rich in carbon, hydrogen, and oxygen elements, present substantial fire hazards to both human life and property due to their intrinsic flammability. Overcoming this challenge in the absence of any flame-retardant elements is a daunting task. Herein, we introduce an innovative strategy employing catalytic polymer auto-pyrolysis before combustion to proactively release CO2, akin to possessing responsive CO2 fire extinguishing mechanisms. We demonstrate that potassium salts with strong nucleophilicity (such as potassium formate/malate) can transform conventional polyurethane foam into materials with fire safety through rearrangement. This transformation results in the rapid generation of a substantial volume of CO2, occurring before the onset of intense decomposition, effectively extinguishing fires. The inclusion of just 1.05 wt% potassium formate can significantly raise the limiting oxygen index of polyurethane foam to 26.5%, increase the time to ignition by 927%, and tremendously reduce smoke toxicity by 95%. The successful application of various potassium salts, combined with a comprehensive examination of the underlying mechanisms, underscores the viability of this strategy. This pioneering catalytic approach paves the way for the efficient and eco-friendly development of polymeric materials with fire safety.

Recombinant Human HAPLN1 Mitigates Pulmonary Emphysema by Increasing TGF-ß Receptor I and Sirtuins Levels in Human Alveolar Epithelial Cells.

Chronic obstructive pulmonary disease (COPD) will be the third leading cause of death worldwide by 2030. One of its components, emphysema, has been defined as a lung disease that irreversibly damages the lungs' alveoli. Treatment is currently unavailable for emphysema symptoms and complete cure of the disease. Hyaluronan (HA) and proteoglycan link protein 1 (HAPLN1), an HA-binding protein linking HA in the extracellular matrix to stabilize the proteoglycan structure, forms a bulky hydrogel-like aggregate. Studies on the biological role of the full-length HAPLN1, a simple structure-stabilizing protein, are limited. Here, we demonstrated for the first time that treating human alveolar epithelial type 2 cells with recombinant human HAPLN1 (rhHAPLN1) increased TGF-ß receptor 1 (TGF-ß RI) protein levels, but not TGF-ß RII, in a CD44-dependent manner with concurrent enhancement of the phosphorylated Smad3 (p-Smad3), but not p-Smad2, upon TGF-ß1 stimulation. Furthermore, rhHAPLN1 significantly increased sirtuins levels (i.e., SIRT1/2/6) without TGF-ß1 and inhibited acetylated p300 levels that were increased by TGF-ß1. rhHAPLN1 is crucial in regulating cellular senescence, including p53, p21, and p16, and inflammation markers such as p-NF-κB and Nrf2. Both senile emphysema mouse model induced via intraperitoneal rhHAPLN1 injections and porcine pancreatic elastase (PPE)-induced COPD mouse model generated via rhHAPLN1-containing aerosols inhalations showed a significantly potent efficacy in reducing alveolar spaces enlargement. Preclinical trials are underway to investigate the effects of inhaled rhHAPLN1-containing aerosols on several COPD animal models.

Novel Effect of Hyaluronan and Proteoglycan Link Protein 1 (HAPLN1) on Hair Follicle Cells Proliferation and Hair Growth.

Hair loss is a common condition that can have a negative impact on an individual's quality of life. The severe side effects and the low efficacy of current hair loss medications create unmet needs in the field of hair loss treatment. Hyaluronan and Proteoglycan Link Protein 1 (HAPLN1), one of the components of the extracellular matrix, has been shown to play a role in maintaining its integrity. HAPLN1 was examined for its ability to impact hair growth with less side effects than existing hair loss treatments. HAPLN1 was predominantly expressed in the anagen phase in three stages of the hair growth cycle in mice and promotes the proliferation of human hair matrix cells. Also, recombinant human HAPLN1 (rhHAPLN1) was shown to selectively increase the levels of transforming growth factor-ß receptor II in human hair matrix cells. Furthermore, we observed concomitant activation of the ERK1/2 signaling pathway following treatment with rhHAPLN1. Our results indicate that rhHAPLN1 elicits its cell proliferation effect via the TGF-ß2-induced ERK1/2 pathway. The prompt entering of the hair follicles into the anagen phase was observed in the rhHAPLN1-treated group, compared to the vehicle-treated group. Insights into the mechanism underlying such hair growth effects of HAPLN1 will provide a novel potential strategy for treating hair loss with much lower side effects than the current treatments.

A Novel Role of Hyaluronic Acid and Proteoglycan Link Protein 1 (HAPLN1) in Delaying Vascular Endothelial Cell Senescence.

Cardiovascular diseases (CVDs) are the most common cardiovascular system disorders. Cellular senescence is a key mechanism associated with dysfunction of aged vascular endothelium. Hyaluronic acid and proteoglycan link protein 1 (HAPLN1) has been known to non-covalently link hyaluronic acid (HA) and proteoglycans (PGs), and forms and stabilizes HAPLN1-containing aggregates as a major component of extracellular matrix. Our previous study showed that serum levels of HAPLN1 decrease with aging. Here, we found that the HAPLN1 gene expression was reduced in senescent human umbilical vein endothelial cells (HUVECs). Moreover, a recombinant human HAPLN1 (rhHAPLN1) decreased the activity of senescence-associated ß-gal and inhibited the production of senescence-associated secretory phenotypes, including IL-1ß, CCL2, and IL-6. rhHAPLN1 also down-regulated IL-17A levels, which is known to play a key role in vascular endothelial senescence. In addition, rhHAPLN1 protected senescent HUVECs from oxidative stress by reducing cellular reactive oxygen species levels, thus promoting the function and survival of HUVECs and leading to cellular proliferation, migration, and angiogenesis. We also found that rhHAPLN1 not only increases the sirtuin 1 (SIRT1) levels, but also reduces the cellular senescence markers levels, such as p53, p21, and p16. Taken together, our data indicate that rhHAPLN1 delays or inhibits the endothelial senescence induced by various aging factors, such as replicative, IL-17A, and oxidative stress-induced senescence, thus suggesting that rhHAPLN1 may be a promising therapeutic for CVD and atherosclerosis.

Hyaluronic acid and proteoglycan link protein 1 suppresses platelet­derived growth factor-BB-induced proliferation, migration, and phenotypic switching of vascular smooth muscle cells.

The development of atherosclerotic cardiovascular disease is associated with the phenotypic switching of vascular smooth muscle cells (SMCs) from a contractile to a synthetic state, leading to cell migration and proliferation. Platelet­derived growth factor­BB (PDGF­BB) modulates this de-differentiation by initiating a number of biological processes. In this study, we show that gene expression of hyaluronic acid (HA) and proteoglycan link protein 1 (HAPLN1) was upregulated during differentiation of human aortic SMCs (HASMCs) into a contractile state, but downregulated upon during PDGF-BB-induced dedifferentiation. This is the first study showing that the treatment of HASMCs with full-length recombinant human HAPLN1 (rhHAPLN1) significantly reversed PDGF-BB-induced decrease in the protein levels of contractile markers (SM22α, α-SMA, calponin, and SM-MHC), and inhibited the proliferation and migration of HASMCs induced by PDGF-BB. Furthermore, our results show that rhHAPLN1 significantly inhibited the phosphorylation of FAK, AKT, STAT3, p38 MAPK and Raf mediated by the binding of PDGF-BB to PDGFRß. Together, these results indicated that rhHAPLN1 can suppress the PDGF-BB-stimulated phenotypic switching and subsequent de-differentiation of HASMCs, highlighting its potential as a novel therapeutic target for atherosclerosis and other vascular diseases. [BMB Reports 2023; 56(8): 445-450].

Purification and Characterization of Mitochondrial Mg2+-Independent Sphingomyelinase from Rat Brain.

Sphingomyelinase (SMase) catalyzes ceramide production from sphingomyelin. Ceramides are critical in cellular responses such as apoptosis. They enhance mitochondrial outer membrane permeabilization (MOMP) through self-assembly in the mitochondrial outer membrane to form channels that release cytochrome c from intermembrane space (IMS) into the cytosol, triggering caspase-9 activation. However, the SMase involved in MOMP is yet to be identified. Here, we identified a mitochondrial Mg2+-independent SMase (mt-iSMase) from rat brain, which was purified 6,130-fold using a Percoll gradient, pulled down with biotinylated sphingomyelin, and subjected to Mono Q anion exchange. A single peak of mt-iSMase activity was eluted at a molecular mass of approximately 65 kDa using Superose 6 gel filtration. The purified enzyme showed optimal activity at pH of 6.5 and was inhibited by dithiothreitol and Mg2+, Mn2+, N2+, Cu2+, Zn2+, Fe2+, and Fe3+ ions. It was also inhibited by GW4869, which is a non-competitive inhibitor of Mg2+-dependent neutral SMase 2 (encoded by SMPD3), that protects against cytochrome c release-mediated cell death. Subfractionation experiments showed that mt-iSMase localizes in the IMS of the mitochondria, implying that mt-iSMase may play a critical role in generating ceramides for MOMP, cytochrome c release, and apoptosis. These data suggest that the purified enzyme in this study is a novel SMase.

Polyelectrolyte Complex with Controllable Viscosity by Doping Cu2+ Protects Nylon-Cotton Fabric against Fire.

Nylon-cotton (NC) blend fabrics are widely used in military and industrial applications, but their high flammability still remains a serious problem. In an effort to effectively and quickly impart flame retardancy to the NC fabric, it was treated by simply blade coating with a Cu2+-doped polyelectrolyte complex (CPEC) that consists of ammonium polyphosphate (APP), polyethylenimine (PEI), and copper sulfate. The viscosity of the CPEC can be adjusted by altering the content of CuSO4, which controls the amount of extrinsic and intrinsic ion pairs. By adjusting the proportion and content of PEI, APP, and CuSO4, CPEC suitable for treating the NC fabric was obtained. Only 0.067 wt % Cu2+ was needed to adjust the viscosity and impart self-extinguishing behavior in a vertical burning test. This simple two-step treatment provides a promising technology to protect flammable polymeric substrates with ultralow metal-doped polyelectrolyte complexes.

A Novel Therapeutic Effect of a New Variant of CTLA4-Ig with Four Antennas That Are Terminally Capped with Sialic Acid in the CTLA4 Region.

Rheumatoid arthritis (RA) is a multifactorial immune-mediated disease, the pathogenesis of which involves different cell types. T-cell activation plays an important role in RA. Therefore, inhibiting T-cell activation is one of the current therapeutic strategies. Cytotoxic T-lymphocyte antigen 4-immunoglobulin (CTLA4-Ig), also known as abatacept, reduces cytokine secretion by inhibiting T-cell activation. To achieve a homeostatic therapeutic effect, CTLA4-Ig has to be administered repeatedly over several weeks, which limits its applicability in RA treatment. To overcome this limitation, we increased the number of sialic acid-capped antennas by genetically engineering the CTLA4 region to increase the therapeutic effect of CTLA4-Ig. N-acetylglucosaminyltransferase (GnT) and α2,6-sialyltransferase (α2,6-ST) were co-overexpressed in Chinese hamster ovary (CHO) cells to generate a highly sialylated CTLA4-Ig fusion protein, named ST6. The therapeutic and immunogenic effects of ST6 and CTLA4-Ig were compared. ST6 dose-dependently decreased paw edema in a mouse model of collagen-induced arthritis and reduced cytokine levels in a co-culture cell assay in a similar manner to CTLA4-Ig. ST6- and CTLA4-Ig-induced T cell-derived cytokines were examined in CD4 T cells isolated from peripheral blood mononuclear cells after cell killing through irradiation followed by flow- and magnetic-bead-assisted separation. Interestingly, compared to CTLA4-Ig, ST6 was substantially less immunogenic and more stable and durable. Our data suggest that ST6 can serve as a novel, less immunogenic therapeutic strategy for patients with RA.

Computer vision applied to dual-energy computed tomography images for precise calcinosis cutis quantification in patients with systemic sclerosis.

BACKGROUND: Although treatments have been proposed for calcinosis cutis (CC) in patients with systemic sclerosis (SSc), a standardized and validated method for CC burden quantification is necessary to enable valid clinical trials. We tested the hypothesis that computer vision applied to dual-energy computed tomography (DECT) finger images is a useful approach for precise and accurate CC quantification in SSc patients. METHODS: De-identified 2-dimensional (2D) DECT images from SSc patients with clinically evident lesser finger CC lesions were obtained. An expert musculoskeletal radiologist confirmed accurate manual segmentation (subtraction) of the phalanges for each image as a gold standard, and a U-Net Convolutional Neural Network (CNN) computer vision model for segmentation of healthy phalanges was developed and tested. A validation study was performed in an independent dataset whereby two independent radiologists manually measured the longest length and perpendicular short axis of each lesion and then calculated an estimated area by assuming the lesion was elliptical using the formula long axis/2 × short axis/2 × π, and a computer scientist used a region growing technique to calculate the area of CC lesions. Spearman's correlation coefficient, Lin's concordance correlation coefficient with 95% confidence intervals (CI), and a Bland-Altman plot (Stata V 15.1, College Station, TX) were used to test for equivalence between the radiologists' and the CNN algorithm-generated area estimates. RESULTS: Forty de-identified 2D DECT images from SSc patients with clinically evident finger CC lesions were obtained and divided into training (N = 30 with image rotation × 3 to expand the set to N = 120) and test sets (N = 10). In the training set, five hundred epochs (iterations) were required to train the CNN algorithm to segment phalanges from adjacent CC, and accurate segmentation was evaluated using the ten held-out images. To test model performance, CC lesional area estimates calculated by two independent radiologists and a computer scientist were compared (radiologist 1 vs. radiologist 2 and radiologist 1 vs. computer vision approach) using an independent test dataset comprised of 31 images (8 index finger and 23 other fingers). For the two radiologists', and the radiologist vs. computer vision measurements, Spearman's rho was 0.91 and 0.94, respectively, both p < 0.0001; Lin's concordance correlation coefficient was 0.91 (95% CI 0.85-0.98, p < 0.001) and 0.95 (95% CI 0.91-0.99, p < 0.001); and Bland-Altman plots demonstrated a mean difference between radiologist vs. radiologist, and radiologist vs. computer vision area estimates of - 0.5 mm2 (95% limits of agreement - 10.0-9.0 mm2) and 1.7 mm2 (95% limits of agreement - 6.0-9.5 mm2, respectively. CONCLUSIONS: We demonstrate that CNN quantification has a high degree of correlation with expert radiologist measurement of finger CC area measurements. Future work will include segmentation of 3-dimensional (3D) images for volumetric and density quantification, as well as validation in larger, independent cohorts.

Microwave-assisted periselective annulation of triarylphosphenes with aldehydes and ketones.

The reaction of diazo(aryl)methyl(diaryl)phosphine oxides with aldehydes and ketones generates benzo-δ-phosphinolactones in low to good yields with 1,1-diarylalk-1-enes as byproducts under microwave irradiation. Diazo(aryl)methyl(diaryl)phosphine oxides first undergo a Wolff rearrangement to form diaryl(aryl)phosphenes, which further react with aldehydes and ketones to afford benzo-δ-phosphinolactones and ß-phosphinolactones. The latter are unstable under heating and fragment into the corresponding 1,1-diarylalk-1-enes and arylphosphine dioxides under reaction conditions. The arylphosphine dioxides become arylphosphonic acids during workup. The periselectivity in the annulation shows that the reaction of diaryl(aryl)phosphenes with most aldehydes and ketones favors phosphene phenyl participation in (4 + 2) annulation over (2 + 2) annulation.

[Consistency evaluation of human serum 25-hydroxyvitamin D using liquid chromatography-tandem mass spectrometry and enzyme-linked immunosorbent assay].

OBJECTIVE: Comparison of liquid chromatography-tandem mass spectrometry(LC-MS/MS) and enzyme-linked immunosorbent assay(ELISA) for the detection of 25(OH)D concentration in human serum and the diagnosis of vitamin D deficiency. METHODS: In the serum pool of "National Institute for Nutrition and Health, Chinese Center for Disease Control and Prevention", 500 serum samples of women aged 18 to 45 years old were randomly selected, and 25(OH)D levels were measured by ELISA and LC-MS/MS for the same serum sample, respectively. The LC-MS/MS column was Waters XBridge BEH C_(18)(2. 1 mm×50 mm, 2. 5 µm). The correlation between the two method was tested by correlation analysis, regression analysis and consistency test. The Endocrine Society and Institute of Medicine recommendations were used to determine the deficiency of vitamin D, and the McNemar test, Kappa coefficient and diagnostic test were used to diagnose the consistency of vitamin D deficiency. RESULTS: The regression equation for the 25(OH)D concentration measured by the two method was y_(LC-MS/MS)=-0. 035+1. 007×x_(ELISA)(r=0. 877), and the average deviation between the two was 4. 48% and the intraclass correlation coefficient was 0. 87. The 25(OH)D concentration was less than 12 and 20 ng/mL as a criterion for vitamin D deficiency, and the Kappa coefficients were greater than 0. 60(0. 64 and 0. 74). CONCLUSION: When serum 25(OH)D level was detected by LC-MS/MS and ELISA, the correspondence of the two method was fine. Taking the "gold standard" LC-MS/MS method as a reference, the ELISA method was used to determine human vitamin D deficiency with good sensitivity and specificity, which can be used for the accurate and rapid detection of large-scale population samples.

Transition metal-free access to 3,4-dihydro-1,2-oxaphosphinine-2-oxides from phosphonochloridates and chalcones through tandem Michael addition and nucleophilic substitution.

A novel and transition metal-free synthesis of 3,4-dihydro-1,2-oxaphosphinine 2-oxides was developed. LiHMDS-mediated tandem Michael addition and nucleophilic substitution of readily available phosphonochloridates and chalcones afforded a variety of valuable 3,4-dihydro-1,2-oxaphosphinine 2-oxides bearing diverse functionalities in excellent yields and satisfactory to good diastereoselectivity (up to 99% yield and up to 99 : 1 dr).

Chronic Food Antigen-specific IgG-mediated Hypersensitivity Reaction as A Risk Factor for Adolescent Depressive Disorder.

Major depressive disorder (MDD) is the most common nonfatal disease burden worldwide. Systemic chronic low-grade inflammation has been reported to be associated with MDD progression by affecting monoaminergic and glutamatergic neurotransmission. However, whether various proinflammatory cytokines are abnormally elevated before the first episode of depression is still largely unclear. Here, we evaluated 184 adolescent patients who were experiencing their first episode of depressive disorder, and the same number of healthy individuals was included as controls. We tested the serum levels of high-sensitivity C-reactive protein (hs-CRP), tumor necrosis factor-α (TNF-α), IgE, 14 different types of food antigen-specific IgG, histamine, homocysteine, S100 calcium-binding protein B, and diamine oxidase. We were not able to find any significant differences in the serum levels of hs-CRP or TNF-α between the two groups. However, the histamine level of the patients (12.35 µM) was significantly higher than that of the controls (9.73 µM, P < 0.001, Mann-Whitney U test). Moreover, significantly higher serum food antigen-specific IgG positive rates were also found in the patient group. Furthermore, over 80% of patients exhibited prolonged food intolerance with elevated levels of serum histamine, leading to hyperpermeability of the blood-brain barrier, which has previously been implicated in the pathogenesis of MDD. Hence, prolonged high levels of serum histamine could be a risk factor for depressive disorders, and antihistamine release might represent a novel therapeutic strategy for depression treatment.

Development of a Label-Free LC-MS/MS-Based Glucosylceramide Synthase Assay and Its Application to Inhibitors Screening for Ceramide-Related Diseases.

Ceramide metabolism is known to be an essential etiology for various diseases, such as atopic dermatitis and Gaucher disease. Glucosylceramide synthase (GCS) is a key enzyme for the synthesis of glucosylceramide (GlcCer), which is a main ceramide metabolism pathway in mammalian cells. In this article, we developed a liquid chromatography-tandem mass spectrometry (LCMS/MS) method to determine GCS activity using synthetic non-natural sphingolipid C8-ceramide as a substrate. The reaction products, C8-GlcCer for GCS, could be separated on a C18 column by reverse-phase high-performance liquid chromatography (HPLC). Quantification was conducted using the multiple reaction monitoring (MRM) mode to monitor the precursor-to-product ion transitions of m/z 588.6 → 264.4 for C8-GlcCer at positive ionization mode. The calibration curve was established over the range of 0.625-160 ng/mL, and the correlation coefficient was larger than 0.999. This method was successfully applied to detect GCS in the human hepatocellular carcinoma cell line (HepG2 cells) and mouse peripheral blood mononuclear cells. We also evaluated the inhibition degree of a known GCS inhibitor 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) on GCS enzymatic activity and proved that this method could be successfully applied to GCS inhibitor screening of preventive and therapeutic drugs for ceramide metabolism diseases, such as atopic dermatitis and Gaucher disease.

Activation of neutral sphingomyelinase 2 by starvation induces cell-protective autophagy via an increase in Golgi-localized ceramide.

Autophagy is essential for optimal cell function and survival, and the entire process accompanies membrane dynamics. Ceramides are produced by different enzymes at different cellular membrane sites and mediate differential signaling. However, it remains unclear which ceramide-producing pathways/enzymes participate in autophagy regulation under physiological conditions such as nutrient starvation, and what the underlying mechanisms are. In this study, we demonstrate that among ceramide-producing enzymes, neutral sphingomyelinase 2 (nSMase2) plays a key role in autophagy during nutrient starvation. nSMase2 was rapidly and stably activated upon starvation, and the enzymatic reaction in the Golgi apparatus facilitated autophagy through the activation of p38 MAPK and inhibition of mTOR. Moreover, nSMase2 played a protective role against cellular damage depending on autophagy. These findings suggest that nSMase2 is a novel regulator of autophagy and provide evidence that Golgi-localized ceramides participate in cytoprotective autophagy against starvation.

Protective effect of sesquiterpene lactone parthenolide on LPS-induced acute lung injury.

Acute lung injury (ALI) is a respiratory failure disease and the major source of mortality in the critically ill patients. The main pathological changes involved in ALI include the excessive recruitment and activation of neutrophils by increased pro-inflammatory mediators. However, any specific therapy for ALI has not been developed. The objective of this study was to investigate protective effects of parthenolide, a sesquiterpene lactone produced in feverfew, on LPS-induced lung injury. In the present study, parthenolide treatment reduced infiltration of inflammatory cells, airway permeability and production of pro-inflammatory cytokines in LPS-induced ALI mouse model. Further, LPS-stimulated phosphorylation of NF-κB, the key regulatory transcription factor in ALI, was inhibited by parthenolide treatment in lung epithelial BEAS-2B cells and alveolar macrophage MH-S cells. These results suggest that parthenolide may provide a beneficial therapeutic strategy for ALI.

P2X1 Receptor-Mediated Ca2+ Influx Triggered by DA-9801 Potentiates Nerve Growth Factor-Induced Neurite Outgrowth.

Nerve growth factor (NGF)-induced neuronal regeneration has emerged as a strategy to treat neuronal degeneration-associated disorders. However, direct NGF administration is limited by the occurrence of adverse effects at high doses of NGF. Therefore, development of a therapeutic strategy to promote the NGF trophic effect is required. In view of the lack of understanding of the mechanism for potentiating the NGF effect, this study investigated molecular targets of DA-9801, a well-standardized Dioscorea rhizome extract, which has a promoting effect on NGF. An increase in intracellular calcium ion level was induced by DA-9801, and chelation of extracellular calcium ions with ethylene-bis(oxyethylenenitrilo)tetraacetic acid (EGTA) suppressed the potentiating effect of DA-9801 on NGF-induced neurite outgrowth. In addition, EGTA treatment reduced the DA-9801-induced phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), the major mediators of neurite outgrowth. To find which calcium ion-permeable channel contributes to the calcium ion influx induced by DA-9801, we treated PC12 cells with various inhibitors of calcium ion-permeable channels. NF449, a P2X1 receptor selective antagonist, significantly abolished the potentiating effect of DA-9801 on NGF-induced neurite outgrowth and abrogated the DA-9801-induced ERK1/2 phosphorylation. In addition, transfection with siRNA of P2X1 receptor significantly reduced the DA-9801-enhanced neurite outgrowth. In conclusion, calcium ion influx through P2X1 receptor mediated the promoting effect of DA-9801 on NGF-induced neurite outgrowth via ERK1/2 phosphorylation.

Paraquat Induces Apoptosis through a Mitochondria-Dependent Pathway in RAW264.7 Cells.

Paraquat dichloride (N,N-dimethyl-4-4'-bipiridinium, PQ) is an extremely toxic chemical that is widely used in herbicides. PQ generates reactive oxygen species (ROS) and causes multiple organ failure. In particular, PQ has been reported to be an immunotoxic agrochemical compound. PQ was shown to decrease the number of macrophages in rats and suppress monocyte phagocytic activity in mice. However, the effect of PQ on macrophage cell viability remains unclear. In this study, we evaluated the cytotoxic effect of PQ on the mouse macrophage cell line, RAW264.7 and its possible mechanism of action. RAW264.7 cells were treated with PQ (0, 75, and 150 µM), and cellular apoptosis, mitochondrial membrane potential (MMP), and intracellular ROS levels were determined. Morphological changes to the cell nucleus and cellular apoptosis were also evaluated by DAPI and Annexin V staining, respectively. In this study, PQ induced apoptotic cell death by dose-dependently decreasing MMP. Additionally, PQ increased the cleaved form of caspase-3, an apoptotic marker. In conclusion, PQ induces apoptosis in RAW264.7 cells through a ROS-mediated mitochondrial pathway. Thus, our study improves our knowledge of PQ-induced toxicity, and may give us a greater understanding of how PQ affects the immune system.

Paraquat reduces natural killer cell activity via metallothionein induction.

Paraquat (PQ), one of the most widely used herbicides, has been used for several decades in agriculture. Some studies suggest that PQ has effects on the immune system. Moreover, previous studies have shown that PQ imparted some immunosuppressive effects. In the present study, cytotoxicity assays using splenic NK cells from mice treated for 28 days with PQ (at 0.2, 1, and 5 mg/kg) were performed to determine whether PQ altered the function of NK cells. Given that PQ was expected to induce an immunosuppressive effect, it was hypothesized that a gene involved in cellular metal ion homeostasis, metallothionein-1 (MT-1), could play an important role in this outcome. This belief was based on the fact that MT1 encodes a protein responsible for zinc ion homeostasis, and that a reduction in free zinc ion levels impairs NK cell function. The results showed that PQ treatments led to increased MT expression in several organs (liver, kidneys, testes) and in splenocytes, caused a reduction of both free zinc ions in sera and in free intracellular zinc, and reduced the expression of GATA-3, a zinc-finger transcription factor important for maturation and activity of T-cells and NK cells. These results provide a basis for a new molecular mechanism to describe potential immunosuppressive effects of PQ in vivo.