Physicochemical properties and immune-enhancing activity of graded polysaccharides from the peels of stem lettuce (Lactuca sativa) by cascade membrane technology.

In this study, cascade membrane technology was applied to classify polysaccharides from the peels of stem lettuce (PPSLs), and three graded polysaccharides (PPSL100, PPSL10, and PPSL1) were obtained using ultrafiltration membranes of 100, 10, and 1 kDa in sequence. The physicochemical properties and immune-modulatory activity of three PPSLs fractions were analyzed and compared. Results showed that all three fractions have characteristic absorption peak of polysaccharides determined by FT-IR, and their monosaccharide composition only consisted of glucose determined by HPLC. PPSL10 had the highest contents of total sugar (88.09 ± 3.52%), uronic acid (2.55 ± 0.10%), and sulfate group (4.15 ± 0.20%). Besides, all three fractions exhibited immune-enhancing activities using RAW264.7 macrophages model, and PPSL10 was the best able to promote phagocytosis of neutral red and nitric oxide generation, which might relate to the high contents of above compositions and medium molecular weight (32 kDa). The findings indicated that PPSL10 could be developed as immune-modulator in the field of functional foods.

Overlapping Functions of the Paralogous Proteins AtPAP2 and AtPAP9 in Arabidopsis thaliana.

Arabidopsis thaliana purple acid phosphatase 2 (AtPAP2), which is anchored to the outer membranes of chloroplasts and mitochondria, affects carbon metabolism by modulating the import of some preproteins into chloroplasts and mitochondria. AtPAP9 bears a 72% amino acid sequence identity with AtPAP2, and both proteins carry a hydrophobic motif at their C-termini. Here, we show that AtPAP9 is a tail-anchored protein targeted to the outer membrane of chloroplasts. Yeast two-hybrid and bimolecular fluorescence complementation experiments demonstrated that both AtPAP9 and AtPAP2 bind to a small subunit of rubisco 1B (AtSSU1B) and a number of chloroplast proteins. Chloroplast import assays using [35S]-labeled AtSSU1B showed that like AtPAP2, AtPAP9 also plays a role in AtSSU1B import into chloroplasts. Based on these data, we propose that AtPAP9 and AtPAP2 perform overlapping roles in modulating the import of specific proteins into chloroplasts. Most plant genomes contain only one PAP-like sequence encoding a protein with a hydrophobic motif at the C-terminus. The presence of both AtPAP2 and AtPAP9 in the Arabidopsis genome may have arisen from genome duplication in Brassicaceae. Unlike AtPAP2 overexpression lines, the AtPAP9 overexpression lines did not exhibit early-bolting or high-seed-yield phenotypes. Their differential growth phenotypes could be due to the inability of AtPAP9 to be targeted to mitochondria, as the overexpression of AtPAP2 on mitochondria enhances the capacity of mitochondria to consume reducing equivalents.

Modulating the activities of chloroplasts and mitochondria promotes adenosine triphosphate production and plant growth.

Efficient photosynthesis requires a balance of ATP and NADPH production/consumption in chloroplasts, and the exportation of reducing equivalents from chloroplasts is important for balancing stromal ATP/NADPH ratio. Here, we showed that the overexpression of purple acid phosphatase 2 on the outer membranes of chloroplasts and mitochondria can streamline the production and consumption of reducing equivalents in these two organelles, respectively. A higher capacity of consumption of reducing equivalents in mitochondria can indirectly help chloroplasts to balance the ATP/NADPH ratio in stroma and recycle NADP+, the electron acceptors of the linear electron flow (LEF). A higher rate of ATP and NADPH production from the LEF, a higher capacity of carbon fixation by the Calvin-Benson-Bassham (CBB) cycle and a greater consumption of NADH in mitochondria enhance photosynthesis in the chloroplasts, ATP production in the mitochondria and sucrose synthesis in the cytosol and eventually boost plant growth and seed yields in the overexpression lines.

In planta study of photosynthesis and photorespiration using NADPH and NADH/NAD+ fluorescent protein sensors.

The challenge of monitoring in planta dynamic changes of NADP(H) and NAD(H) redox states at the subcellular level is considered a major obstacle in plant bioenergetics studies. Here, we introduced two circularly permuted yellow fluorescent protein sensors, iNAP and SoNar, into Arabidopsis thaliana to monitor the dynamic changes in NADPH and the NADH/NAD+ ratio. In the light, photosynthesis and photorespiration are linked to the redox states of NAD(P)H and NAD(P) pools in several subcellular compartments connected by the malate-OAA shuttles. We show that the photosynthetic increases in stromal NADPH and NADH/NAD+ ratio, but not ATP, disappear when glycine decarboxylation is inhibited. These observations highlight the complex interplay between chloroplasts and mitochondria during photosynthesis and support the suggestions that, under normal conditions, photorespiration supplies a large amount of NADH to mitochondria, exceeding its NADH-dissipating capacity, and the surplus NADH is exported from the mitochondria to the cytosol through the malate-OAA shuttle.

ATP compartmentation in plastids and cytosol of Arabidopsis thaliana revealed by fluorescent protein sensing.

Matching ATP:NADPH provision and consumption in the chloroplast is a prerequisite for efficient photosynthesis. In terms of ATP:NADPH ratio, the amount of ATP generated from the linear electron flow does not meet the demand of the Calvin-Benson-Bassham (CBB) cycle. Several different mechanisms to increase ATP availability have evolved, including cyclic electron flow in higher plants and the direct import of mitochondrial-derived ATP in diatoms. By imaging a fluorescent ATP sensor protein expressed in living Arabidopsis thaliana seedlings, we found that MgATP2- concentrations were lower in the stroma of mature chloroplasts than in the cytosol, and exogenous ATP was able to enter chloroplasts isolated from 4- and 5-day-old seedlings, but not chloroplasts isolated from 10- or 20-day-old photosynthetic tissues. This observation is in line with the previous finding that the expression of chloroplast nucleotide transporters (NTTs) in Arabidopsis mesophyll is limited to very young seedlings. Employing a combination of photosynthetic and respiratory inhibitors with compartment-specific imaging of ATP, we corroborate the dependency of stromal ATP production on mitochondrial dissipation of photosynthetic reductant. Our data suggest that, during illumination, the provision and consumption of ATP:NADPH in chloroplasts can be balanced by exporting excess reductants rather than importing ATP from the cytosol.

FdC1 and Leaf-Type Ferredoxins Channel Electrons From Photosystem I to Different Downstream Electron Acceptors.

Plant-type ferredoxins in Arabidopsis transfer electrons from the photosystem I to multiple redox-driven enzymes involved in the assimilation of carbon, nitrogen, and sulfur. Leaf-type ferredoxins also modulate the switch between the linear and cyclic electron routes of the photosystems. Recently, two novel ferredoxin homologs with extra C-termini were identified in the Arabidopsis genome (AtFdC1, AT4G14890; AtFdC2, AT1G32550). FdC1 was considered as an alternative electron acceptor of PSI under extreme ferredoxin-deficient conditions. Here, we showed that FdC1 could interact with some, but not all, electron acceptors of leaf-type Fds, including the ferredoxin-thioredoxin reductase (FTR), sulfite reductase (SiR), and nitrite reductase (NiR). Photoreduction assay on cytochrome c and enzyme assays confirmed its capability to receive electrons from PSI and donate electrons to the Fd-dependent SiR and NiR but not to the ferredoxin-NADP+ oxidoreductase (FNR). Hence, FdC1 and leaf-type Fds may play differential roles by channeling electrons from photosystem I to different downstream electron acceptors in photosynthetic tissues. In addition, the median redox potential of FdC1 may allow it to receive electrons from FNR in non-photosynthetic plastids.

Stomatin-like protein 2 is overexpressed in cervical cancer and involved in tumor cell apoptosis.

Stomatin-like protein 2 (SLP-2) is overexpressed in numerous types of human cancer and previous studies revealed that SLP-2 may function in mitochondria. The purpose of the present study was to evaluate the expression of SLP-2 in cervical cancer and the association between SLP-2 expression and clinical features, in addition to investigating the role of SLP-2 in the apoptosis of cervical cancer cells. The expression profile of SLP-2 was determined by quantitative polymerase chain reaction, western blotting and immunohistochemical staining. The effect of SLP-2 on cell apoptosis induced by chemotherapeutics in cervical cancer cells was evaluated using Annexin V staining and terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL) assays. The results indicated that SLP-2 expression in cervical cancer was significantly upregulated at the mRNA and protein levels, compared with that in normal cervical tissues. Immunohistochemical analysis revealed significant correlation between SLP-2 protein expression and clinical characteristics, including the squamous cell carcinoma antigen (P=0.003), deep stromal invasion (P=0.021), lymphovascular space involvement (P=0.044) and pelvic lymph node metastasis (P<0.001), which served as independent prognostic factors for predicting the shortening of overall survival time in patients with early-stage cervical cancer. In addition, TUNEL and Annexin V binding assays revealed that silencing SLP-2 expression significantly enhanced the sensitivity of cervical cancer cells to apoptosis induced by chemotherapeutics. Taken together, the results of the present study suggest that SLP-2 may be a progressive gene in the development of cervical cancer. Overexpression of SLP-2 serves an important role in the apoptosis of human cervical cancer cells.

Dihydromyricetin Reduces TGF-ß Via P53 Activation-dependent Mechanism in Hepatocellular Carcinoma HepG2 Cells.

Natural antineoplastic drug development is crucial to treatment of clinical oncology. Dihydromyricetin, a bioactive flavonoid compound was extracted from the stems and leaves of Ampelopsis grossedentata. It exhibited anticancer activity and induced apoptosis in human hepatocellular carcinoma cells according to our previous studies. In this study, we demonstrated that DHM could significantly inhibit proliferation and induce apoptosis in HepG2 cells with MTT and Flow Cytometry methods. It is very interesting that we found DHM could regulate TGF-ß signal pathway and which has a crosstalk with P53, Smad3 and P-Smad2/3 proteins. Meanwhile, we confirmed that DHM showed antitumor activity by regulating the activation of the p53-dependent pathways (MDM2, P-MDM2, BAX and Bcl-2). These findings defined and supported a novel mechanism that DHM could induce cell apoptosis by reducing TGF-ß via p53 signal pathway in HepG2 cells.

Treatment of medullary thyroid carcinoma with apatinib: A case report and literature review.

RATIONALE: Medullary thyroid carcinoma (MTC) is a rare type thyroid carcinoma originating from the thyroid parafollicular cells (C cells). Chemotherapy has a limited efficacy for treating persistent or recurrent MTC. PATIENT CONCERNS: A 46-year-old woman who underwent thyroidectomy for MTC in December 2007. She began experience recurring diarrhea in January 2015 and started to cough and feel shortness of breath in March 2016. DIAGNOSES: A chestcomputed tomography (CT) scan showed metastases in the bilateral lungs, pulmonary hilum, and mediastinal lymph nodes. Percutaneous biopsy of the pulmonary occupying lesions performed on March 21, 2016 indicated medullary carcinoma metastases at the right pulmonary hilum. INTERVENTIONS: This patient was treated with oral apatinib (500 mg daily). OUTCOMES: The patient's symptoms of diarrhea, coughing, and shortness of breath disappeared. CT reexaminations for efficacy assessment at 1, 2, and 3 months after the treatment indicated partial remission. Systemic migrating bone and joint pains occurred during the treatment, which were considered to be adverse events of apatinib. LESSONS: Treatment of MTC with apatinib has been shown to be effective in our case. Tyrosine kinase inhibitors (TKIs) that suppress rearranged during transfection (RET) and vascular endothelial growth factor receptor (VEGFR) should be considered as a effective therapeutic approaches.

AtPAP2 modulates the import of the small subunit of Rubisco into chloroplasts.

Arabidopsis thaliana purple acid phosphatase 2 (AtPAP2) is the only phosphatase that is dual-targeted to both chloroplasts and mitochondria. Like Toc33/34 of the TOC and Tom 20 of the TOM, AtPAP2 is anchored to the outer membranes of chloroplasts and mitochondria via a hydrophobic C-terminal motif. AtPAP2 on the mitochondria was previously shown to recognize the presequences of several nuclear-encoded mitochondrial proteins and modulate the import of pMORF3 into the mitochondria. Here we show that AtPAP2 binds to the small subunit of Rubisco (pSSU) and that chloroplast import experiments demonstrated that pSSU was imported less efficiently into pap2 chloroplasts than into wild-type chloroplasts. We propose that AtPAP2 is an outer membrane-bound phosphatase receptor that facilitates the import of selected proteins into chloroplasts.

Phosphorylation and Dephosphorylation of the Presequence of Precursor MULTIPLE ORGANELLAR RNA EDITING FACTOR3 during Import into Mitochondria from Arabidopsis.

The nucleus-encoded mitochondria-targeted proteins, multiple organellar RNA editing factors (MORF3, MORF5, and MORF6), interact with Arabidopsis (Arabidopsis thaliana) PURPLE ACID PHOSPHATASE2 (AtPAP2) located on the chloroplast and mitochondrial outer membranes in a presequence-dependent manner. Phosphorylation of the presequence of the precursor MORF3 (pMORF3) by endogenous kinases in wheat germ translation lysate, leaf extracts, or STY kinases, but not in rabbit reticulocyte translation lysate, resulted in the inhibition of protein import into mitochondria. This inhibition of import could be overcome by altering threonine/serine residues to alanine on the presequence, thus preventing phosphorylation. Phosphorylated pMORF3, but not the phosphorylation-deficient pMORF3, can form a complex with 14-3-3 proteins and HEAT SHOCK PROTEIN70. The phosphorylation-deficient mutant of pMORF3 also displayed faster rates of import when translated in wheat germ lysates. Mitochondria isolated from plants with altered amounts of AtPAP2 displayed altered protein import kinetics. The import rate of pMORF3 synthesized in wheat germ translation lysate into pap2 mitochondria was slower than that into wild-type mitochondria, and this rate disparity was not seen for pMORF3 synthesized in rabbit reticulocyte translation lysate, the latter translation lysate largely deficient in kinase activity. Taken together, these results support a role for the phosphorylation and dephosphorylation of pMORF3 during the import into plant mitochondria. These results suggest that kinases, possibly STY kinases, and AtPAP2 are involved in the import of protein into both mitochondria and chloroplasts and provide a mechanism by which the import of proteins into both organelles may be coordinated.

[A randomized phase II trial of docetaxel and doxorubicin in treatment for patients with non-small-cell lung cancer who have failed previous platinum-based chemotherapy].

OBJECTIVE: The aim of this study was to evaluate the efficacy, toxicity and safety of doxorubicin combined with domestically produced docetaxel versus with taxotere, and to investigate whether these two regimens result in similar outcomes in the treatment for non-small-cell lung cancer (NSCLC) patients who failed previous platinum-based chemotherapy. METHODS: Eighty-eight NSCLC patients were enrolled into this clinical phase II trial. The patients randomly received either domestic docetaxel (study arm) or taxotere (control arm) at a dose of 70 mg/m2 on D2, while doxorubicin at a dose of 40 mg/m2 on D1 was administered in both groups. It was repeated every 3 weeks, totally for three cycles. No granulocyte colony-stimulating factor was used to prevent granulocytopenia. The response rate and toxicity were evaluated using World Health Organization toxicity scale and Karnofsky performance status scale. RESULTS: Of the 88 patients, 81 were evaluable in terms of efficacy. There was no complete responder in this series. The response rate (RR) was 17.1% in the study arm versus 7.5% in the control arm, and the clinical benefit rate (CBR) was 80.5% in the study group versus 72.5% in the control group. The most frequent grade 3 or 4 toxicities were neutropenia, leucopenia and gastrointestinal symptoms. Other toxicities such as alopecia and vomiting were mild and generally well tolerated. No fluid retention was noticed. CONCLUSION: The administration of doxorubicin 40 mg/m2 on D1 combined with domestic docetaxel 70 mg/m2 on D2 is proved to be as effective and tolerable as with taxotere. The domestic drug docetaxel may be considered as an alternative for patients with non-small-cell lung cancer who failed previous platinum-based chemotherapy.

[Docetaxel in the treatment of advanced breast cancer ].

OBJECTIVE: To evaluate the efficacy, toxicity and safety of an new domestic docetaxel in the treatment of pretreated advanced breast cancer. METHODS: Fourty-four breast cancer patients who had failed in first-line chemotherapy were included in this trial. They received docetaxel as the second-line chemotherapy. Docetaxel was administered alone at a dose of 70 mg/m2 every 3 weeks. The use of granulocyte colony-stimulating factor to prevent granulocytopenia was not permitted. The response rate and toxicity were evaluated by World Health Organization toxicity scale and performance status by Karnofsky scale. RESULTS: Of the 41 evaluable patients, 4 achieved complete response and 14 partial remission, with a response rate and clinical benefit rate of 43.9% and 85.4%, respectively. Grade 3 or grade 4 neutropenia developed in 42.9%, alopecia in 7.1% and vomiting in 4.8% of these patients. Fluid retention was not observed in this series. CONCLUSION: Three-week administration of docetaxel alone at a dose of 70 mg/m2 is effective and tolerable. It provides an alternative for the pretreated advanced breast cancer patients.

[Phase II multicenter clinical trial of nedaplatin in the treatment of malignant tumors].

OBJECTIVE: To evaluate and compare the efficacy and safety of Nedaplatin (NDP)-based regimen and cisplatin (DDP)-based regimen for head and neck squamous cell carcinoma (HNSCC), non-small cell lung cancer (NSCLC), esophageal cancer and ovary epithelial cell carcinoma. METHODS: Single agent group: NDP was administered at a dose of 100 mg/m(2) on D1, every 3 weeks for at least 2 cycles. Combination chemotherapy group: combined with 5-Fu, NVB, VDS + 5-Fu, PTX or CTX respectively, NDP 80 mg/m(2) on D1 or DDP 30 mg/m(2) on D1-3, every 3 weeks for at least 2 cycles was given. RESULTS: Of 237 patients in this trial, 37 were treated by single Nedaplatin, 139 by NDP-based regimen, 61 by DDP-based regimen in the control group. The response rate of single Nedaplatin chemotherapy for advanced NSCLC was 10.5% (2/19), for ovary carcinoma (1/3) and HNSCC (1/1). For NSCLC and ovary carcinoma patients who had failed in the previous DDP-based chemotherapy, the response rates by single NDP chemotherapy were still 9.1% and 33.3%. The response rate of NDP-based combination regimen for NSCLC, ovary carcinoma, HNSCC and esophageal cancer was 33.9% (21/62), 44.8% (13/29), 20.0% (3/15) and 18.2% (4/22), respectively, which was not statistically different from the rate of controlled group treated by DDP-based regimen. For chemonaive NSCLC, the effect of NDP-based combination regimen (35.7%) was significantly superior to the effect of DDP-based regimen (17.1%) (P = 0.045). The most common adverse events of nedaplatin were myelosuppression (leukopenia, thrombocytopenia, anemia), nausea and vomiting. The myelosuppression and renal toxicity of NDP-based regimen were similar to that of DDP-based regimen, but vomiting was milder than that of DDP-based regimen (54% vs. 75.4%), and grade I/II liver toxicity was more common in the NDP-based regimen than in DDP-based regimen (10.8% vs. 0). CONCLUSION: Nedaplatin is effective in the treatment for HNSCC, NSCLC and ovary carcinoma. Compared with the control group treated by DDP-based regimen, nedaplatin-based combination chemotherapy has similar effect on HNSCC, NSCLC, ovary carcinoma and esophageal cancer. Gastrointestinal reaction of nedaplatin is milder than that of cisplatin but the liver function during chemotherapy must be monitored closely.