Enhancing La(III) biosorption and biomineralization with Micromonospora saelicesensis: Involvement of phosphorus and formation of monazite nano-minerals.

The release of rare earth elements (REEs) from mining wastes and their applications has significant environmental implications, necessitating the development of effective prevention and reclamation strategies. The mobility of REEs in groundwater due to microorganisms has garnered considerable attention. In this study, a La(III) resistant actinobacterium, Micromonospora saelicesensis KLBMP 9669, was isolated from REE enrichment soil in GuiZhou, China, and evaluated for its ability to adsorb and biomineralize La(III). The findings demonstrated that M. saelicesensis KLBMP 9669 immobilized La(III) through the physical and chemical interactions, with immobilization being influenced by the initial La(III) concentration, biomass, and pH. The adsorption kinetics followed a pseudo-second-order rate model, and the adsorption isotherm conformed to the Langmuir model. La(III) adsorption capacity of this strain was 90 mg/g, and removal rate was 94 %. Scanning electron microscope (SEM) coupled with energy dispersive X-ray spectrometer (EDS) analysis revealed the coexistence of La(III) with C, N, O, and P. Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) investigations further indicated that carboxyl, amino, carbonyl, and phosphate groups on the mycelial surface may participate in lanthanum adsorption. Transmission electron microscopy (TEM) revealed that La(III) accumulation throughout the M. saelicesensis KLBMP 9669, with some granular deposits on the mycelial surface. Selected area electron diffraction (SAED) confirmed the presence of LaPO4 crystals on the M. saelicesensis KLBMP 9669 biomass after a prolonged period of La(III) accumulation. This post-sorption nano-crystallization on the M. saelicesensis KLBMP 9669 mycelial surface is expected to play a crucial role in limiting the bioimmobilization of REEs in geological repositories.

Anti-oxidant and Anti-inflammatory Effects of Ethanol Extract from Polygala sibirica L. var megalopha Fr. on Lipopolysaccharide-Stimulated RAW264.7 Cells.

OBJECTIVE: To investigate the anti-oxidant and anti-inflammatory effects of ethanol extract of Polygala sibirica L. var megalopha Fr. (EEP) on RAW264.7 mouse macrophages. METHODS: RAW264.7 cells were pretreated with 0-200 µg/mL EEP or vehicle for 2 h prior to exposure to 1 µg/mL lipopolysaccharide (LPS) for 24 h. Nitric oxide (NO) and prostaglandin (PGE2) production were determined by Griess reagent and enzyme-linked immunosorbent assay (ELISA), respectively. The mRNA levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor α (TNF-α), interleukin-1beta (IL-1ß), and IL-6 were determined using reverse transcription polymerase chain reaction (RT-PCR). Western blot assay was used to determine the protein expressions of iNOS, COX-2, phosphorylation of extracellular regulated protein kinases (ERK1/2), c-Jun N-terminal kinase (JNK), inhibitory subunit of nuclear factor Kappa B alpha (Iκ B-α) and p38. Immunofluorescence was used to observe the nuclear expression of nuclear factor-κ B p65 (NF-κ B p65). Additionally, the anti-oxidant potential of EEP was evaluated by reactive oxygen species (ROS) production and the activities of catalase (CAT) and superoxide dismutase (SOD). The 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl (OH), superoxide anion (O2-) radical and nitrite scavenging activity were also measured. RESULTS: The total polyphenol and flavonoid contents of EEP were 23.50±2.16 mg gallic acid equivalent/100 g and 43.78±3.81 mg rutin equivalent/100 g. With EEP treatment (100 and 150 µg/mL), there was a notable decrease in NO and PGE2 production induced by LPS in RAW264.7 cells by downregulation of iNOS and COX-2 mRNA and protein expressions (P<0.01 or P<0.05). Furthermore, with EEP treatment (150 µg/mL), there was a decrease in the mRNA expression levels of TNF-α, IL-1ß and IL-6, as well as in the phosphorylation of ERK, JNK and p38 mitogen-activated protein kinase (MAPK, P<0.01 or P<0.05), by blocking the nuclear translocation of NF-κ B p65 in LPS-stimulated cells. In addition, EEP (100 and 150 µg/mL) led to an increase in the anti-oxidant enzymes activity of SOD and CAT, with a concomitant decrease in ROS production (P<0.01 or P<0.05). EEP also indicated the DPPH, OH, O2- radical and nitrite scavenging activity. CONCLUSION: EEP inhibited inflammatory responses in activated macrophages through blocking MAPK/NF-κ B pathway and protected against oxidative stress.

Nocardioides potassii sp. nov., isolated from weathered potash tailings soil.

A Gram-positive, aerobic actinomycete, designated strain KLBMP 9356T, was isolated from weathered potash tailings soil sampled in Xuzhou, Jiangsu Province, PR China. The colonies were cream-coloured, convex and rounded. The optimal growth conditions of strain KLBMP 9356T were 1 % (w/v) NaCl, 28 °C and pH 7. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain KLBMP 9356T showed the highest similarity to Nocardioides zhouii CGMCC 1.11084T (98.9 %) and Nocardioides glacieisoli CGMCC 1.11097T (98.7 %). Results from two tree-making algorithms supported the position that strain KLBMP 9356T forms a stable clade with N. zhouii CGMCC 1.11084T and N. glacieisoli CGMCC 1.11097T. Strain KLBMP 9356T exhibited low digital DNA-DNA hybridization values with N. zhouii CGMCC 1.11084T (27.6 %) and N. glacieisoli CGMCC 1.11097T (31.4 %). The average nucleotide identity values between strain KLBMP 9356T and N. zhouii CGMCC 1.11084T and N. glacieisoli CGMCC 1.11097T were 83.8% and 85.9%, respectively. The peptidoglycan in the cell wall of the novel strain was ll-2,6-diaminopimelic acid and the predominant menaquinone was MK-8(H4). The major fatty acids (>10 %) were C17:1ω8c and C18:1ω9c. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, lyso-phospatidylglycerol and phosphatidylinositol. The genomic DNA G+C content was 71.6 mol%. Based on its morphological, chemotaxonomic and phylogenetic characteristics, strain KLBMP 9356T represents a novel species of the genus Nocardioides, for which the name Nocardioides potassii sp. nov. is proposed. The type strain is KLBMP 9356T (=CGMCC 4.7738T=NBRC 115493T).

Antribacter soli sp. nov., a novel actinobacterium isolated from soils of weathering dolomite.

Strain KLBMP 9083T, a novel actinobacterium, was isolated from weathered soils collected from a karst area in Anshun, Guizhou Province, PR China. The taxonomic position of strain KLBMP 9083T was studied using the polyphasic approach. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain KLBMP 9083T formed a stabilized monophyletic clade with its closest relative strain Antribacter gilvus CGMCC 1.13856T (98.4 % 16S rRNA gene sequence similarity). The peptidoglycan hydrolysates contained alanine, glutamic acid, threonine and lysine. The polar lipids were composed of diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside, an unidentified phosphoglycolipid, an unidentified phospholipid and an unidentified glycolipid. The predominant menaquinones were MK-9(H8) (87.1 %), MK-9(H6) (7.3 %) and MK-9(H4) (5.6 %). The major fatty acids (>10 %) were anteiso-C15 : 0 and iso-C15 : 0. The genomic DNA G+C content was 72.3 mol%. The digital DNA-DNA hybridization and average nucleotide identity values between strain KLBMP 9083T and A. gilvus CGMCC 1.13856T were 23.4 and 79.9 %, respectively. On the basis of morphological, chemotaxonomic and phylogenetic characteristics, strain KLBMP 9083T represents a novel species of the genus Antribacter, for which the name Antribacter soli sp. nov. is proposed. The type strain is KLBMP 9083T (=CGMCC 4.7737T=NBRC 115577T).

Yinghuangia soli sp. nov., isolated from soil of weathering dolomite in a karst area.

A Gram-stain-positive, aerobic actinomycete strain, designated KLBMP 8922T, was isolated from a soil sample collected from weathering dolomite crust in Guizhou Province, PR China. KLBMP 8922T showed the 16S rRNA gene similarities to Yinghuangia seranimata CCTCC AA 206006T (98.7 %), Yinghuangia catbensis VN07A0015T (98.3 %) and Yinghuangia aomiensis M24DS4T (98.2 %). The taxonomic status of this strain was investigated by using a polyphasic approach. The aerial mycelia of KLBMP 8922T formed spore chains, and spores were cylindrical with smooth surfaces. The whole-cell sugars were ribose, mannose and galactose with traces of glucose and xylose. The diagnostic amino acids of the cell wall were ll-diaminopimelic acid, alanine and glutamic acid. The predominant menaquinones were MK-9(H6) and MK-9(H8). The diagnostic phospholipids were diphosphatidylglycerol, phosphatidylinositol, phosphatidylinositolmannoside, phosphatidylethanolamine, an unidentified phospholipid and an unidentified lipid. The major cellular fatty acids (>10 %) were iso-C15 : 0, iso-C16 : 0 and iso-C16 : 1H. The genomic DNA G+C content was 72.0 mol%. The digital DNA-DNA hybridization (dDDH) value between KLBMP 8922T and Y. seranimata CCTCC AA 206006T was 24.1 %, and the average nucleotide identity (ANI) value was 81.0 %. On the basis of a combination of morphological, chemotaxonomic and phylogenetic characteristics, strain KLBMP 8922T represents a novel species of the genus Yinghuangia for which the name Yinghuangia soli sp. nov. is proposed. The type strain was KLBMP 8922T (= CGMCC 1.19360T = NBRC 115572T).

Anthocyanin Extract from Purple Sweet Potato Exacerbate Mitophagy to Ameliorate Pyroptosis in Klebsiella pneumoniae Infection.

Given the rise of morbidity and mortality caused by Klebsiella pneumoniae (KP), the increasing number of strains resistant to antibiotics, and the emergence of hypervirulent Klebsiella pneumonia, treatment of KP infection becomes difficult; thus, novel drugs are necessary for treatment. Anthocyanins, or natural flavonoids, have an extensive effect against bacterial infection. However, few studies on anti-KP are identified. Here, we evaluated the therapeutic effect of purple sweet potato anthocyanins (PSPAs) on KP, containing 98.7% delphinidin 3-sambubioside. Results showed that KP-infected mice after PSPAs treatment manifested decreased mortality, weakened lung injury, dampened inflammatory responses, and reduced bacterial systemic dissemination in vivo. In Vitro, PSPAs significantly suppressed pyroptosis and restricted NLRP3 inflammasome activation in alveolar macrophages infected with KP. As for the mechanism, PSPAs promote mitophagy by recruiting Parkin to the mitochondria. PSPAs-conferred mitophagy increased mitochondrial membrane potential and decreased mitochondrial reactive oxygen species and mitochondrial DNA, resulting in impaired NLRP3 inflammasome activation. In addition, the promotion of mitophagy by PSPAs required the Nrf2 signaling pathway. Collectively, these findings suggest that PSPAs are a potential option for the treatment of KP infection.

Cochlioquinone derivative CoB1 induces cytostatic autophagy in lung cancer through miRNA-125b and Foxp3.

BACKGROUND: Lung cancer is the leading cause of cancer death worldwide, yet no effective medication for this disease is available. Cochlioquinone B derivative (CoB1), purified from Salvia miltiorrhiza endophytic Bipolaris sorokiniana, affects the defense against pulmonary pathogens by regulating inflammatory responses. However, the effect of CoB1 on lung cancer and the underlying molecular mechanisms remain unknown. In the present study, we investigate the protective effects of CoB1 on lung cancer and explore its underlying mechanism. METHOD: We examined the inhibitory effect of CoB1 on lung cancer cells (A549 cells) by MTT and colony formation assay. The effect of CoB1 on cytostatic autophagy in lung cancer cells was verified by Western blot, transmission electron microscopy, and confocal microscopy. The differentially expressed miRNAs were identified using quantitative RT-PCR. Luciferase assay and Northern blot were performed to verify the correlation between miRNA-125b and Foxp3. Protein expression in autophagy-related pathways was detected by Western blot. Xenograft tumor models were constructed to explore the inhibitory effect of CoB1 and the role of miRNA-125b as a suppressor in lung cancer in vivo. RESULT: CoB1 inhibited lung cancer cell proliferation by inducing cytostatic autophagy both in vitro and in vivo. CoB1-induced autophagy was related to blocking of the PI3K/Akt1/mTOR signaling pathway. In addition, CoB1 induced miR-125b expression via activating the TAK1/MKK4/JNK/Smad axis, thereby reducing Foxp3 expression and further inducing autophagy. CONCLUSION: This study is the first to report the specific inhibitory function of CoB1 purified from Salvia miltiorrhiza endophytic Bipolaris sorokiniana in lung cancer, which may be due to the induction of autophagy. This study provides evidence and novel insights into the anticancer efficacy of CoB1.

Contribution of traditional Chinese medicine combined with conventional western medicine treatment for the novel coronavirus disease (COVID-19), current evidence with systematic review and meta-analysis.

This study provides current evidence for efficacy and safety of treating COVID-19 with combined traditional Chinese medicine (TCM) and conventional western medicine (CWM). Six databases were searched from January 1 to December 31, 2020. Randomized controlled trials (RCTs), case-control studies (CCTs), and cohort studies on TCM or TCM combined with CWM treatment for COVID-19 were included. The quality of included RCTs was assessed by Cochrane risk of bias tool, and the Newcastle-Ottawa Scale (NOS) was used to assess the quality of cohort studies and CCTs. Review Manager 5.4 software was used to perform meta-analysis. The quality of evidence was assessed using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach. A total of 35 studies (3,808 patients) composing 19 RCTs and 16 observational studies were included. The results of meta-analysis revealed that comparing with CWM alone, integrated TCM and CWM had significant improvement in total effective rate, improvement rate of chest CT, the rate of disease progression, as well as improvement of fever, fatigue and cough. The overall quality of evidence was very low to moderate. In conclusion, TCM combined with CWM was a potential treatment option for increasing clinical effective rate, improving the clinical symptoms, and preventing disease progression in COVID-19 patients. High-quality clinical trials are required in the further.

Effect of Heat Treatment on the Anticancer Activity of Houttuynia cordata Thunb Aerial Stem Extract in Human Gastric Cancer SGC-7901 Cells.

Gastric cancer is one of the most common malignant tumors in the world, and prevention through diet is one of the ways to control. Houttuynia cordata thunb.(HCT) is a plant having medicine and food function, has many biological properties. However, the effect of food style on the anticancer activity of HCT is not clear. So, we investigate the effect of heat treatment on anticancer activity of HCT. HCT extracts (heated aerial stem, heated subterraneous stem, heated leaves defined as HAS, HSS, HL, respectively, and not heated defined as NAS, NSS, NL, respectively) were obtained, and their inhibited activity were detected by alamar blue assay. The cell apoptosis was detected by DAPI staining and flow cytometry analysis. Western blot was performed to test the expression of apoptotic related protein. HCT showed the anticancer activity in four human tumor cell lines. Interestingly, heat treatment could increase the anticancer activity. In SCG-7901 cells, heat treatment increased anticancer activity of AS by 2-14 folds and induced apoptosis through regulating the intrinsic signaling pathways. Intriguingly, the caspase nine specific inhibitor blocked AS-reduced cell viability. Heat treatment increased the anticancer activity of HCT, and can be used as a dietary style for prevention of gastric cancer.

A Novel Cochlioquinone Derivative, CoB1, Regulates Autophagy in Pseudomonas aeruginosa Infection through the PAK1/Akt1/mTOR Signaling Pathway.

Owing to multiple antibiotic resistance, Pseudomonas aeruginosa causes the most intractable infections to human beings worldwide, thus exploring novel drugs to defend against this bacterium remains of great importance. In this study, we purified a novel cochlioquinone B derivative (CoB1) from Salvia miltiorrhiza endophytic Bipolaris sorokiniana and reveal its role in host defense against P. aeruginosa infection by activating cytoprotective autophagy in alveolar macrophages (AMs) both in vivo and in vitro. Using a P. aeruginosa infection model, we observed that CoB1-treated mice manifest weakened lung injury, reduced bacterial systemic dissemination, decreased mortality, and dampened inflammatory responses, compared with the wild type littermates. We demonstrate that CoB1-induced autophagy in mouse AMs is associated with decreased PAK1 expression via the ubiquitination-mediated degradation pathway. The inhibition of PAK1 decreases the phosphorylation level of Akt, blocks the Akt/mTOR signaling pathway, and promotes the release of ULK1/2-Atg13-FIP200 complex from mTOR to initiate autophagosome formation, resulting in increased bacterial clearance capacity. Together, our results provide a molecular basis for the use of CoB1 to regulate host immune responses against P. aeruginosa infection and indicate that CoB1 is a potential option for the treatment of infection diseases.

Saccharothrix deserti sp. nov., an actinomycete isolated from desert soil.

A Gram-stain-positive, aerobic actinomycete, designated strain BMP B8144T, was isolated from desert soil, in Xinjiang province, northwest China. The isolate produced scanty aerial mycelium and fragmented substrate mycelium on most tested media. Cell-wall hydrolysates contained meso-diaminopimelic acid, galactose and mannose. The diagnostic phospholipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylhydroxylethanolamine, phosphatidylinositol, and phosphatidylinositol mannosides. The major fatty acids included iso-C16 : 0, C17 : 1 ω8c and iso-C15 : 0. The predominant menaquinones were MK-9(H4) and MK-10(H4). The DNA G+C content was 70.4 mol% (genome). Based on the 16S rRNA gene sequence analysis on EzBioCloud server, strain BMP B8144T showed the closest similarities to Saccharothrix lopnurensis YIM LPA2hT (98.9 %) and 'Saccharothrix yanglingensis' Hhs.015 (98.6 %). However, it can be distinguished from the closest strains based on the low levels of DNA-DNA relatedness (59.3±1.8 and 47.9±2.3 %, respectively). A combination of morphological, chemotaxonomic and phylogenetic characteristics, strain BMP B8144T represents a novel species of the genus Saccharothrix, for which the name Saccharothrix deserti sp. nov. is proposed. The type strain is BMP B8144T (=CGMCC 4.7490T=KCTC 49001T).

[Expression of ß-catenin in Skin Lesions of Patients with Scleroderma and Its Effect on Epithelial-Mesenchymal Transition of Human Epidermal Keratinocytes].

OBJECTIVE: To investigate the expression of ß-catenin in the skin lesions of patients with systemic scleroderma (SSc) and its effect on epithelial-mesenchymal transition (EMT) of human epidermal keratinocytes. METHODS: The expression of ß-catenin, Snail1 and E-cadherin in the skin lesions sample of 45 SSc patients and normal skin sample from 20 healthy adults was detected with SP immunohistochemistry. HaCaT, the human epidermal keratinocytes, were treated with different concentrations of Wnt10b (0 ng/mL (control), 2 ng/mL and 4 ng/mL) for 48 h. then detected the localization of ß-catenin in HaCaT cells by immunofluorescence assay, determined the mRNA levels of Snail1 and Snail2 in HaCaT cells by real-time fluorescent quantitative PCR, detected the proteins expression of ß-catenin, Vimentin, N-cadherin and E-cadherin in HaCaT cells by Western blot. RESULTS: The positive rates of ß-catenin, Snail1 and E-cadherin in skin lesions of SSc patients were 100%, 88.89% and 2.22% respectively, while in healthy adult skin, the corresponding positive rates were 0%, 10.00%, and 95.00%. The difference between the two groups was significant. Compared with control group, treatment with different concentrations of Wnt10b (2 ng/mL and 4 ng/mL) induced up-regulation of ß-catenin expression and promoted translocation of ß-catenin from cytoplasm to nucleus, increased the mRNA levels of Snail1 and Snail2 (P < 0.05), and up-regulated the proteins expression of Vimentin, N-cadherin, down-regulated the E-cadherin protein expression in HaCaT cells (P < 0.05). CONCLUSIONS: Abnormally activated Wnt/ß-catenin signaling pathway and abnormally expressed EMT-related proteins are observed in SSc lesions. Activation of Wnt/ß-catenin signaling pathway may promote EMT in HaCaT cells.

Actinomadura deserti sp. nov., isolated from desert soil.

A Gram-positive, strictly aerobic actinobacterium, designated BMP B8004T, was isolated from desert soil collected in Xinjiang Province, Northwest China. It produced an extensively branched non-fragmenting substrate mycelium, and very scanty aerial mycelium that formed a short hooked chain of arthrospores with a smooth surface. Optimum growth occurred at 28 °C, pH 7.0 and 0 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain BMP B8004T formed a distinct phyletic lineage within the genus Actinomadura. It shared the highest 16S rRNA gene sequence similarity to Actinomadura apis IM17-1T (99.2 %) and Actinomadura rifamycini NBRC 14183T (98.6 %). However, it could be distinguished from the two closest strains based on the low levels of DNA-DNA relatedness (52.7±0.7 and 45±1.8 %, respectively). Chemotaxonomic characteristics, including the main phospholipids, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannosides, the major menaquinones MK-9(H6) and MK-9(H8), the predominant fatty acids iso-C16 : 0, C16 : 0, C18 : 0 10-methyl and C18 : 1ω9c, were also consistent with the properties of the genus Actinomadura. The DNA G+C content of strain BMP B8004T was 71.9 mol%. Based on phenotypic and genotypic features, strain BMP B8004T is considered to represent a novel species of the genus Actinomadura, for which the name Actinomadura deserti sp. nov. is proposed. The type strain is BMP B8004T (=CGMCC 4.7432T=KCTC 39998T).

Effect of Hsp27 on early embryonic development in the mouse.

Previous studies by this study group have showed that heat shock protein 27 (Hsp27), expressed in the oocyte of growing follicles, is down-regulated in polycystic ovary syndrome ovaries and that down-regulation of Hsp27 improves the maturation of mouse oocytes and increases early apoptosis of oocytes. In this study, the effect of Hsp27 on early embryo development in the mouse was observed. Following microinjection of AdCMV-Hsp27 or AdsiRNA-Hsp27 into the cytoplasm of mouse zygotes, blastocyst morphology was observed and cell apoptosis of blastocysts was detected by TUNEL. After culture in vitro for 96h, blastocysts were analysed for Hsp27 expression by real-time PCR and immunofluorescence. The blastocyst formation rate and embryo quality were evaluated. The expression of Hsp27 was significantly increased in embryos with Hsp27 overexpression (AdCMV-Hsp27), while it was significantly suppressed by 75% in embryos with the gene silenced (AdsiRNA-Hsp27; both P<0.05). Cell apoptosis in blastocysts, blastocyst formation rate and embryo quality were unaffected by Hsp27 overexpression or gene silencing. In conclusion, overexpression or down-regulation of Hsp27 in zygotes, as a single factor, does not significantly affect the subsequent embryonic development.

Effects of upregulation of Hsp27 expression on oocyte development and maturation derived from polycystic ovary syndrome.

Heat shock protein 27 (Hsp27) is a heat shock protein family member which can inhibit apoptosis. Our previous studies reported down-regulated Hsp27 in ovarian tissue derived from women with polycystic ovary syndrome (PCOS) however, the exact effect of Hsp27 on oocyte maturation and developmental competence in PCOS is unclear. The effect of Hsp27 over-expression was studied in vitro using oocytes derived from PCOS patients. An artificial GFP-plasmid was injected into human oocyte to increase Hsp27 protein level. Oocyte maturation was evaluated by morphological observation. Mature oocytes were fertilized by intracytoplasmic sperm injection (ICSI) and embryonic developmental competence was evaluated. Critical apoptotic factors and cytokines were measured at both the mRNA and protein level. Our results revealed that Overexpression of HSP27 lowered the maturation rate of oocytes derived from PCOS patients. Meanwhile, fertilization rate and high quality embryo rate were similar between the Hsp27 overexpressing group and controls; however, the blastocyst formation rate in this group was significantly higher than control. Expression analysis revealed that the oocyte-secreted factors, BMP15 and GDF9, and the apoptotic-related regulators, Caspase 3, 8 and 9, were all significantly decreased in Hsp27 overexpressing oocytes. In conclusion, upregulation of Hsp27 inhibits oocyte maturation from PCOS patients, but improves embryonic developmental potential.

Methylated actinomycin D, a novel actinomycin D analog induces apoptosis in HepG2 cells through Fas- and mitochondria-mediated pathways.

Actinomycin D (Act D), a well known of clinical antitumor drug, has been used for the treatment of some highly malignant tumors, however, the clinical application was limited by its extreme cytotoxicity. In the present study, we reported that methylated actinomycin D (mAct D), a novel actinomycin D analog isolated from Streptomyces sp. KLBMP 2541 in our previous study, could not only exert stronger inhibitory effects on several human cancer cells than Act D in dose- and time-dependent manner at ng concentrations, especially on HepG2 cells, but also lower cytotoxicity in normal cells (HL-7702). Base on these results, HepG2 cells were treated for further study to illustrate the potential mechanism of mAct D. The results of nuclei morphology examination, DNA fragmentation detection, sub-G1 analysis, annexin V-FITC/PI staining and activation of caspase-3 indicated mAct D significantly induced HepG2 cells apoptosis. Semiquantitative RT-PCR and Western blot analysis revealed that mAct D induced apoptosis in HepG2 cells through mitochondria-dependent pathway by increasing levels of caspase-9, Bax, Bak while decreasing levels of Bcl-2, Bid, and Fas-dependent pathway by increasing levels of Fas, FasL, FADD, and caspase-8. Subsequently, pretreatment with specific inhibitor of caspase-8 Z-LEHD-FMK and caspase-9 Z-LEHD-FMK significantly attenuated caspase-3 activity, the cleavage of caspase-3 and PARP, meanwhile increased the cell viability. In addition, p53 and mitochondrial transcription factor A (mtTFA) were also upregulated. Taken together, ng concentrations mAct D induces the apoptosis of HepG2 through Fas- and mitochondria-mediated pathway and presents a potential novel alternative agent for the treatment of human hepatic carcinoma.

[Role of Wnt 2, Wnt 3a and ß-catenin in skin lesions of patients with scleroderma].

OBJECTIVE: To study the role of abnormally activated Wnt/ß-catenin signal pathway in the pathogenesis of scleroderma (SD) and its association with the clinical classification of SD. METHODS: The expression and distribution of Wnt 2, Wnt 3a, and ß-catenin in the skin lesions of 45 SD patients, including 25 with systemic sclerosis (SSc) and 20 with localized scleroderma (LSc), were detected with SP immunohistochemistry, using 20 samples from healthy skin tissues as normal control. RESULTS: In the dermis and epidermis of the SD skin lesions, Wnt 2 and Wnt 3a were located in the cytoplasm and cell nuclei, respectively; ß-catenin was distributed in the nuclei of dermal fibroblast-like cells, glandular epithelium cells and infiltrating lymphocytes, and on the cell membrane in normal and a part of the affected epidermis. The skin lesions of SD patients showed obviously increased staining intensity of cytoplasmic Wnt 2, nuclear Wnt 3a and ß-catenin, but markedly lowered cell membrane staining of ß-catenin than normal skins (P<0.01). Both Wnt 2 and Wnt 3a were positively correlated with nuclear ß-catenin deposition (r=0.663 and 0.654, P<0.01) and negatively with cell membrane ß-catenin staining (r=-0.532 and -0.529, P<0.01). No significant difference was found in the staining intensities of the 3 proteins between SSc and LSc (P>0.05). CONCLUSION: Abnormal activation of Wnt/ß-catenin pathway occurs in the skin lesions of SD patients, which may play an important role in the pathogenesis of SD. SSc and LSc represent the two opposite ends of the SD spectrum rather than two separate diseases.

Purification and characterization of a fibrinolytic enzyme from Streptomyces sp. XZNUM 00004.

A fibrinolytic enzyme (SFE1) from Streptomyces sp. XZNUM 00004 was purified to electrophoretic homogeneity with the methods including ammonium sulfate precipitation, polyacrylamide gel, DEAE-Sepharose Fast Flow anion exchange and gel-filtration chromatography. The molecular weight of SFE1 was estimated to be 20 kDa by SDS-PAGE, fibrin zymography, and gel filtration chromatography. The isoelectric point was 4.9. K (m) and V (max) values were 0.96 mg/ml and 181.8 unit/ml, respectively. It was very stable at pH 5.0-8.0 and below 65 °C. The optimum pH for enzyme activity was 7.8. The optimum temperature was 35 °C. The fibrinolytic activity of SFE1 was enhanced by Na(+), K(+), Mn(2+), Mg(2+), Zn(2+) and Co(2+). Conversely, Cu(2+) showed strong inhibition. Furthermore, the fibrinolytic activity was strongly inhibited by PMSF, and partly inhibited by EDTA and EGTA. SFE1 rapidly hydrolyzed the Aα-chain of fibrinogen, followed by the Bß-chain and finally the γ-chain. The first 15 amino acids of the N-terminal sequence were APITLSQGHVDVVDI. Additionally, SFE1 directly digested fibrin and not by plasminogen activators in vitro. SFE1 can be further developed as a potential candidate for thrombolytic therapy.

Reversal effect of Tween-20 on multidrug resistance in tumor cells in vitro.

Multidrug resistance (MDR) is a major barrier for chemotherapy of many cancers. Non-ionic surfactants have great potential to reverse the MDR by preventing onset or delay progression of the carcinogenic process. However, the role of Tween-20 in the development of MDR remains unknown. The aim of this study was to explore the reversal effect and potential mechanism of Tween-20 on tumor cells in vitro. Alamar Blue assay was used to examine the reversal index of Tween-20 to vincristine (VCR), doxorubicin (DOX) and 5-fluorouracil (5-FU) in KBv200, HepG2/R and Bel-7402/5-FU, respectively. Morphological change was determined by Gimsa and Hoechst 33258 staining. The acumulation of DOX was confirmed by spectrofluorimetric assay. Cell cycle analysis was performed using flow cytometry. The mRNA and protein expression levels of MDR were assessed by semiquantitative RT-PCR and dot blot, respectively. The results showed that Tween-20 at concentrations of 0.0025%, 0.005%, 0.01% had little cytotoxicity. When combined with the cancer drugs, it significantly promoted the sensitivity of MDR cells. Fluorescence staining confirmed that the percentage of apoptotic cell increased when combined with Tween-20. This notion was further supported by the observation that Tween-20 treatment potentiated VIN-induced G2/M arrest of the cell cycle. Furthermore, Tween-20 treatment increased significantly intracellular accumulation of DOX. RT-PCR and dot blot revealed that Tween-20 could downregulate the expression of MDR and P-glycoprotein. Low concentrations of Tween-20 can efficiently reverse the multidrug resistance phenotype by enhancing accumulation of the anticancer drugs. The potential mechanism may be via inhibiting the multidrug-resistant gene expression.

Synthesis and antitumor activity of conjugates of 5-Fluorouracil and emodin.

A series of conjugates of 5-Fluorouracil (5-FU) and emodin were synthesized by coupling trimethyl emodin with N(1), N(3) dialkylated 5-FU. The 5-FU moiety contained various substituents at the N(3)-position were linked to the 2-position of trimethyl emodin via a methylene linkage. Their cytotoxicity against three cancer cell lines and one noncancerous cell were studied. The results revealed that some of conjugates exhibited better or comparable in vitro antitumor activity to 5-FU and emodin and low toxicity in the normal cell. The structure-activity relationship study showed N(3)-aromatic substituent was important for their cytotoxic activity.