Mechanism of saikogenin G against major depressive disorder determined by network pharmacology.

Many classic decoctions of Chinese medicine including Radix Bupleuri are used to treat major depressive disorder (MDD). Saikosaponin D is a representative bioactive ingredient discovered in Radix Bupleuri. The mechanism of saikogenin G (SGG) as a metabolite in MDD remains unclear to date. This study aims to elucidate the mechanism of SGG in treating MDD with network pharmacology. We evaluated the drug likeness of SGG with SwissADME web tool and predicted its targets using the SwissTargetPrediction and PharmMapper. MDD-related targets were identified from the following databases: DisGeNET, DrugBank, Online Mendelian Inheritance in Man, and GeneCards. The common targets of SGG and MDD were imported to the STRING11.0 database, and then a protein-protein interaction network was constructed. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment were analyzed with DAVID 6.8 database. The molecular weight of SGG was 472.7 g/mol, the topological polar surface area was 69.92 A2 <140 A2, the octanol/water partition coefficient (Consensus LogP0/W) was 4.80, the rotatable bond was 1, the hydrogen bond donors was 3, and the hydrogen bond acceptors was 4. A total of 322 targets of SGG were obtained and there were 1724 MDD-related targets. A total of 78 overlapping genes were selected as targets of MDD treatment including albumin, insulin-like growth factor I, mitogen-activated protein kinase 1, proto-oncogene tyrosine-protein kinase Src, and epidermal growth factor receptor. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis suggested that proteoglycans in cancer, pathways in cancer, prostate cancer, hypoxia-inducible factor-1, central carbon metabolism in cancer, estrogen, PI3K-Akt, ErbB, Rap1, and prolactin signaling pathways played an important role(P < .0001). This study showed that SGG exhibits good drug-like properties and elucidated the potential mechanisms of SGG in treating MDD with regulating inflammation, energy metabolism, monoamine neurotransmitters, neuroplasticity, phosphocreatine-creatine kinase circuits, and so on.

Nucleated red blood cells participate in myocardial regeneration in the toad Bufo Gargarizan Gargarizan.

Heart regeneration is negligible in humans and mammals but remarkable in some ectotherms. Humans and mammals lack nucleated red blood cells (NRBCs), while ectotherms have sufficient NRBCs. This study used Bufo gargarizan gargarizan, a Chinese toad subspecies, as a model animal to verify our hypothesis that NRBCs participate in myocardial regeneration. NRBC infiltration into myocardium was seen in the healthy toad hearts. Heart needle-injury was used as an enlarged model of physiological cardiomyocyte loss. It recovered quickly and scarlessly. NRBC infiltration increased during the recovery. Transwell assay was done to in vitro explore effects of myocardial injury on NRBCs. In the transwell system, NRBCs could infiltrate into cardiac pieces and could transdifferentiate toward cardiomyocytes. Heart apex cautery caused approximately 5% of the ventricle to be injured to varying degrees. In the mildly to moderately injured regions, NRBC infiltration increased and myocardial regeneration started soon after the inflammatory response; the severely damaged region underwent inflammation, scarring, and vascularity before NRBC infiltration and myocardial regeneration, and recovered scarlessly in four months. NRBCs were seen in the newly formed myocardium. Enzyme-linked immunosorbent assay and Western blotting showed that the levels of tumor necrosis factor-α, interleukin- 1ß, 6, and11, cardiotrophin-1, vascular endothelial growth factor, erythropoietin, matrix metalloproteinase- 2 and 9 in the serum and/or cardiac tissues fluctuated in different patterns during the cardiac injury-regeneration. Cardiotrophin-1 could induce toad NRBC transdifferentiation toward cardiomyocytes in vitro. Taken together, the results suggest that the NRBC is a cell source for cardiomyocyte renewal/regeneration in the toad; cardiomyocyte loss triggers a series of biological processes, facilitating NRBC infiltration and transition to cardiomyocytes. This finding may guide a new direction for improving human myocardial regeneration.

Chemical Composition, Antibacterial, and Anti-Inflammatory Activities of Essential Oils from Flower, Leaf, and Stem of Rhynchanthus beesianus.

Rhynchanthus beesianus is a medicinal, ornamental, and edible plant, and its essential oil has been used as an aromatic stomachic in China. In this study, the chemical constituents, antibacterial, and anti-inflammatory properties of flower essential oil (F-EO), leaf essential oil (L-EO), and stem essential oil (S-EO) of R. beesianus were investigated for the first time. According to the GC-FID/MS assay, the F-EO was mainly composed of bornyl formate (21.7%), 1,8-cineole (21.6%), borneol (9.7%), methyleugenol (7.7%), ß-myrcene (5.4%), limonene (4.7%), camphene (4.5%), linalool (3.4%), and α-pinene (3.1%). The predominant components of L-EO were bornyl formate (33.9%), borneol (13.2%), 1,8-cineole (12.1%), methyleugenol (8.0%), camphene (7.8%), bornyl acetate (6.2%), and α-pinene (4.3%). The main components of S-EO were borneol (22.5%), 1,8-cineole (21.3%), methyleugenol (14.6%), bornyl formate (11.6%), and bornyl acetate (3.9%). For the bioactivities, the F-EO, L-EO, and S-EO exhibited significant antibacterial property against Bacillus subtilis, Enterococcus faecalis, Staphylococcus aureus, Proteus vulgaris, Pseudomonas aeruginosa, and Escherichia coli with the inhibition zones (7.28-9.69 mm), MIC (3.13-12.50 mg/mL), and MBC (6.25-12.50 mg/mL). Besides, the F-EO, L-EO, and S-EO significantly inhibited the production of proinflammatory mediator nitric oxide (NO) (93.15-94.72%) and cytokines interleukin-6 (IL-6) (23.99-77.81%) and tumor necrosis factor-α (TNF-α) (17.69-24.93%) in LPS-stimulated RAW264.7 cells at the dose of 128 µg/mL in the absence of cytotoxicity. Hence, the essential oils of R. beesianus flower, leaf, and stem could be used as natural antibacterial and anti-inflammatory agents with a high application potential in the pharmaceutical and cosmetic fields.

Knockdown of EMMPRIN (OX47) in MRMT-1 Carcinoma Cells Inhibits Tumor Growth and Decreases Cancer-Induced Bone Destruction and Pain.

PURPOSE: Bone destruction and pain caused by cancer is one of the most devastating complications of cancer patients with bone metastases, and it seriously affects the quality of patients' life. Extracellular matrix metalloproteinase inducer (EMMPRIN) is a cell adhesion molecule with increased expression in a variety of tumors. This study focused to clarify the specific function of EMMPRIN in bone metastasis of breast cancer. MATERIALS AND METHODS: Adenovirus with shRNA-EMMPRIN was transfected into MRMT-1 rat breast carcinoma cells, and the MRMT-1 cells with different expression levels of EMMPRIN were implanted into the bone marrow cavity of rat tibia. Next, the effect of down-regulation of EMMPRIN was evaluated as follows: bone damage was detected by X-ray radiological and tartrate-resistant acid phosphatase staining; the tumor burden was evaluated by hematoxylin and eosin staining; the test of pain-related behaviors was assessed used the bilateral paw withdrawal mechanical threshold; and the levels of secretory factors in tumor conditioned medium were determined by using enzyme-linked immunosorbent assay. RESULTS: We found that down-regulation of EMMPRIN in tumor cells can simultaneously reduce tumor burden, relieve cancer-induced bone destruction and pain. CONCLUSION: EMMPRIN is expected to be a therapeutic target for relieving bone metastasis of breast cancer and alleviating cancer-induced bone destruction and pain. The method of targeting EMMPRIN may be a promising strategy for the treatment of cancer in the future.

Phytochemical Analysis, Antioxidant, Antibacterial, Cytotoxic, and Enzyme Inhibitory Activities of Hedychium flavum Rhizome.

Hedychium flavum Roxb., a medicinal, edible, and ornamental plant, is widely cultivated throughout China, India, and Southeast Asia. The rhizome from this plant has been used for food flavoring and in traditional Chinese medicine to treat diverse diseases, but the detailed constituents and bioactivities are still limited known. Therefore, phytochemical analysis by GC-MS and UHPLC-Q-Orbitrap-MS, and antioxidant, antibacterial, cytotoxic, and enzyme inhibitory activities tests have been conducted in the current study. Based on the GC-MS results, the essential oil (EO) of rhizome was mainly composed of coronarin E (20.3%), ß-pinene (16.8%), E-nerolidol (11.8%), and linalool (8.5%). Among them, coronarin E was reported in H. flavum EO firstly. Furthermore, the spectrophotometric indicated rhizome had high total phenolic content (TPC, 50.08-57.42 mg GAEs/g extract) and total flavonoid content (TFC, 12.45-21.83 mg REs/g extract), no matter in water extract (WE) or in 70% ethanol extract (EE). UHPLC-Q-Orbitrap-MS was applied to further characterize composition, and 86 compounds were putatively identified from WE and EE, including 13 phenolic components. For the bioactivities, both WE and EE showed remarkable antioxidant activity by DPPH and ABTS tests, being superior to the positive control (butylated hydroxytoluene, BTH). EO revealed significant antibacterial activity against Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, and Proteus vulgaris with DIZ (10.34-24.43 mm), MIC (78.13-312.50 µg/mL), and MBC (156.25-625.00 µg/mL). Moreover, EO exhibited a considerable selectivity to human tumor cell K562 (IC50 = 27.16 µg/mL), and its toxicity was more than 3.5-fold different from that of non-cancerous MRC-5 cell (IC50 = 95.96 µg/mL) and L929 cell (IC50 = 129.91 µg/mL). A series of apoptosis analysis demonstrated that EO induced apoptosis against K562 cells in a dose-dependent manner. In enzyme inhibitory effect assays, WE and EE showed strong α-glucosidase inhibition activity, being superior to the positive control (acarbose). Besides, the EO, WE, and EE didn't show a promising inhibition on tyrosinase (19.30-32.51 mg KAEs/g sample) and exhibited a weak inhibitory effect on cholinesterase. Based on the current results, H. flavum could be considered as a source of bioactive compounds and has high exploitation potential in the cosmetics, food, and pharmaceutical industries.

HOXD3 was negatively regulated by YY1 recruiting HDAC1 to suppress progression of hepatocellular carcinoma cells via ITGA2 pathway.

OBJECTIVES: HOXD3 is associated with progression of multiple types of cancer. This study aimed to identify the association of YY1 with HOXD3-ITGA2 axis in the progression of hepatocellular carcinoma. MATERIALS AND METHODS: Bioinformatics assay was used to identify the effect of YY1, HOXD3 and ITGA2 expression in HCC tissues. The function of YY1 and HOXD3 in HCCs was determined by qRT-PCR, MTT, apoptosis, Western blotting, colony formation, immunohistochemistry, and wound-healing and transwell invasion assays. The relationship between YY1 and HOXD3 or HOXD3 and ITGA2 was explored by RNA-Seq, ChIP-PCR, dual luciferase reports and Pearson's assays. The interactions between YY1 and HDAC1 were determined by immunofluorescence microscopy and Co-IP. RESULTS: Herein, we showed that the expression of YY1, HOXD3 and ITGA2 associated with the histologic and pathologic stages of HCC. Moreover, YY1, recruiting HDAC1, can directly target HOXD3 to regulate progression of HCCs. The relationship between YY1 and HOXD3 was unknown until uncovered by our present investigation. Furthermore, HOXD3 bound to promoter region of ITGA2 and up-regulated the expression, thus activating the ERK1/2 signalling and inducing HCCs proliferation, metastasis and migration in the vitro and vivo. CONCLUSIONS: Therefore, HOXD3, a target of YY1, facilitates HCC progression via activation of the ERK1/2 signalling by promoting ITGA2. This finding provides a new whole way to HCC therapy by serving YY1-HOXD3-ITGA2 regulatory axis as a potential therapeutic target for HCC therapy.

A distinct class of plant and animal viral proteins that disrupt mitosis by directly interrupting the mitotic entry switch Wee1-Cdc25-Cdk1.

Many animal viral proteins, e.g., Vpr of HIV-1, disrupt host mitosis by directly interrupting the mitotic entry switch Wee1-Cdc25-Cdk1. However, it is unknown whether plant viruses may use this mechanism in their pathogenesis. Here, we report that the 17K protein, encoded by barley yellow dwarf viruses and related poleroviruses, delays G2/M transition and disrupts mitosis in both host (barley) and nonhost (fission yeast, Arabidopsis thaliana, and tobacco) cells through interrupting the function of Wee1-Cdc25-CDKA/Cdc2 via direct protein-protein interactions and alteration of CDKA/Cdc2 phosphorylation. When ectopically expressed, 17K disrupts the mitosis of cultured human cells, and HIV-1 Vpr inhibits plant cell growth. Furthermore, 17K and Vpr share similar secondary structural feature and common amino acid residues required for interacting with plant CDKA. Thus, our work reveals a distinct class of mitosis regulators that are conserved between plant and animal viruses and play active roles in viral pathogenesis.

Norepinephrine transporter promotes the invasion of human colon cancer cells.

Epidemiological studies suggested the use of antidepressants to be associated with decreased risk of colorectal cancer (CRC). However, the underlying mechanism through which this decreased risk occurs remains elusive. The norepinephrine transporter (NET) is a target of antidepressants that maintains noradrenergic transmission homeostasis; however, little is known about its function in human CRC cells. The present study, using public datasets and immunohistochemistry approaches, revealed that NET was highly expressed in human CRC tissues with metastasis and in human colon cancer cells. Furthermore, knockdown of NET inhibited the invasive capability of human colon cancer cells. Additionally, epithelial (E)-cadherin expression was increased and Notch1 signaling was inhibited in NET-depleted colon cancer cells. These findings suggest that NET is highly expressed in human colon cancer, which is associated with the invasion of human colon cancer cells by influencing cell-cell adhesion through the Notch1-E-cadherin pathway. Thus, the present study revealed a novel function for NET and its downstream effectors in colon cancer cells, which will be valuable for future studies in a clinical setting.

Chemical Composition, Antibacterial, Anti-Inflammatory, and Enzyme Inhibitory Activities of Essential Oil from Rhynchanthus beesianus Rhizome.

Rhynchanthus beesianus W. W. Smith, an edible, medicinal, and ornamental plant, is mainly cultivated in China and Myanmar. The essential oil (EO) from R. beesianus rhizome has been used as an aromatic stomachic in China. The chemical composition and biological activities of EO from R. beesianus rhizome were reported for the first time. Based on gas chromatography with flame ionization or mass selective detection (GC-FID/MS) results, the major constituents of EO were 1,8-cineole (47.6%), borneol (15.0%), methyleugenol (11.2%), and bornyl formate (7.6%). For bioactivities, EO showed a significant antibacterial activity against Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, and Proteus vulgaris with the diameter of the inhibition zone (DIZ) (8.66-10.56 mm), minimal inhibitory concentration (MIC) (3.13-6.25 mg/mL), and minimal bactericidal concentration (MBC) (6.25-12.5 mg/mL). Moreover, EO (128 µg/mL) significantly inhibited the production of proinflammatory mediators nitric oxide (NO) (92.73 ± 1.50%) and cytokines tumor necrosis factor-α (TNF-α) (20.29 ± 0.17%) and interleukin-6 (IL-6) (61.08 ± 0.13%) in lipopolysaccharide (LPS)-induced RAW264.7 macrophages without any cytotoxic effect. Moreover, EO exhibited significant acetylcholinesterase (AChE) inhibitory activity (the concentration of the sample that affords a 50% inhibition in the assay (IC50) = 1.03 ± 0.18 mg/mL) and moderate α-glucosidase inhibition effect (IC50 = 11.60 ± 0.25 mg/mL). Thus, the EO could be regarded as a bioactive natural product and has a high exploitation potential in the cosmetics and pharmaceutical industries.

MeCP2 Promotes Gastric Cancer Progression Through Regulating FOXF1/Wnt5a/ß-Catenin and MYOD1/Caspase-3 Signaling Pathways.

Methyl-CpG binding protein 2 (MeCP2) has recently been characterized as an oncogene frequently amplified in several types of cancer. However, its precise role in gastric cancer (GC) and the molecular mechanism of MeCP2 regulation are still largely unknown. Here we report that MeCP2 is highly expressed in primary GC tissues and the expression level is correlated with the clinicopathologic features of GC. In our experiments, knockdown of MeCP2 inhibited tumor growth. Molecular mechanism of MeCP2 regulation was investigated using an integrated approach with combination of microarray analysis and chromatin immunoprecipitation sequencing (ChIP-Seq). The results suggest that MeCP2 binds to the methylated CpG islands of FOXF1 and MYOD1 promoters and inhibits their expression at the transcription level. Furthermore, we show that MeCP2 promotes GC cell proliferation via FOXF1-mediated Wnt5a/ß-Catenin signaling pathway and suppresses apoptosis through MYOD1-mediated Caspase-3 signaling pathway. Due to its high expression level in GC and its critical function in driving GC progression, MeCP2 represents a promising therapeutic target for GC treatment.

Identification of differentially expressed genes in salivary adenoid cystic carcinoma cells associated with metastasis.

INTRODUCTION: Salivary adenoid cystic carcinoma (SACC) is a frequent type of salivary gland cancer which is characterized by slow growth but high incidence of distant metastasis. We aimed to identify therapeutic targets which are associated with metastasis of SACC. MATERIAL AND METHODS: Total RNA was isolated from a low metastatic SACC cell line (ACC-2) and a highly metastatic SACC cell line (ACC-M), which was screened from ACC-2 by combination of in vivo selection and cloning in vitro. Then the total RNA was subjected to microarray analysis. Differentially expressed genes (DEGs) were screened from ACC-M compared with ACC-2, followed by Gene Ontology function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Function annotation for DEGs also was performed. A protein-protein interaction network (PPI) was constructed for DEGs. RESULTS: A total of 1128 DEGs were identified from ACC-M cells compared with ACC-2 cells. Both up- and down-regulated DEGs were enriched in different functions in biological process (BP), cellular component (CC) and molecular function (MF). Additionally, down-regulated DEGs were mainly enriched in "Apoptosis" and "Cytokine-cytokine receptor interaction" pathways which involved IFN-α1, NTRK1 and TGF-ß1. In the PPI network, PIK3CA, PTPN11 and PIK3R1 had a number of nodes greater than 10. CONCLUSIONS: Transforming growth factor ß1 might play a pivotal role during lung metastasis of SACC and be selected as a candidate target for treatment of metastatic SACC. IFNA1, NTRK1 and PIK3CA were also associated with tumor metastasis.

Distinct Regulatory Changes Underlying Differential Expression of TEOSINTE BRANCHED1-CYCLOIDEA-PROLIFERATING CELL FACTOR Genes Associated with Petal Variations in Zygomorphic Flowers of Petrocosmea spp. of the Family Gesneriaceae.

CYCLOIDEA (CYC)-like genes, belonging to the plant-specific TCP transcription factor family that is named after TEOSINTE BRANCHED1 (TB1) from maize (Zea mays), CYC from Antirrhinum majus, and the PROLIFERATING CELL FACTORS (PCF) from rice (Oryza sativa), have conserved dorsal identity function in patterning floral zygomorphy mainly through specific expression in dorsal petals of a flower. Their expression changes are usually related to morphological diversity of zygomorphic flowers. However, it is still a challenge to elucidate the molecular mechanism underlying their expression differentiation. It is also unknown whether CINCINNATA (CIN)-like TCP genes, locally controlling cell growth and proliferation, are involved in the evolution of floral zygomorphy. To address these questions, we selected two closely related species, i.e. Petrocosmea glabristoma and Petrocosmea sinensis, with distinct petal morphology to conduct expression, hybridization, mutant, and allele-specific expression analyses. The results show that the size change of the dorsal petals between the two species is mainly mediated by the expression differentiation of CYC1C and CYC1D, while the shape variation of all petals is related to the expression change of CIN1. In reciprocal F1 hybrids, the expression of CYC1C, CYC1D, and CIN1 conforms to an additive inheritance mode, consistent with the petal phenotypes of hybrids. Through allele-specific expression analyses, we find that the expression differentiation of these TCP genes is underlain by distinctly different types of regulatory changes. We suggest that highly redundant paralogs with identical expression patterns and interspecific expression differentiation may be controlled by remarkably different regulatory pathways because natural selection may favor different regulatory modifications rather than coding sequence changes of key developmental genes in generating morphological diversity.

miR-15b-5p induces endoplasmic reticulum stress and apoptosis in human hepatocellular carcinoma, both in vitro and in vivo, by suppressing Rab1A.

In human hepatocellular carcinoma (HCC), aberrant expression of miRNAs correlates with tumor cell proliferation, apoptosis, invasion, and migration by targeting downstream proteins. miR-15b levels are reported increased in HCC tissues; however, they negatively correlate to HCC recurrence. Our aim was to understand the reason for this phenomenon. We used the reverse transcription-polymerase chain reaction (RT-PCR) to measure miR-15b-5p expression in both HCC tissues and HCC cell lines. Our results were consistent with the report. Using bioinformatics and luciferase reporter assays, we identified Rab1A as a novel and direct target of miR-15b-5p. Inhibiting the function of Rab1A with shRab1A also inhibited the growth of HCC cells and induced endoplasmic reticulum stress (ERS) and apoptosis. Similarly, suppressing Rab1A by overexpression of miR-15b-5p also inhibited cell growth and induced ERS and apoptosis. Moreover, re-expression of Rab1A rescued the miR-15b-5p-induced ERS, apoptosis, and growth inhibition in HCC cells. In vivo assays were further performed to testify them. Taken together, our data suggest that miR-15b-5p induces ERS, apoptosis, and growth inhibition by targeting and suppressing Rab1A, acting as a tumor suppressor gene in HCC. This finding suggests a novel relation among Rabs, miRNAs, and apoptosis.

Identification of microRNA profiles in salivary adenoid cystic carcinoma cells during metastatic progression.

Salivary adenoid cystic carcinoma (SACC) is a common type of salivary gland cancer. The poor long-term prognosis of patients with SACC is primarily due to local recurrence, distant metastasis and perineural invasion. MicroRNAs (miRNAs) have been identified as important post-transcriptional regulators, which are involved in various biological processes. The aim of the present study was to identify the miRNA expression profiles that are involved in the metastatic progression of SACC. Therefore, microarray technology was employed to identify miRNA expression profiles in an SACC cell line, ACC-2, and a highly metastatic SACC cell line, ACC-M, which was screened from ACC-2 by a combination of in vivo selection and cloning in vitro. Differences in miRNA expression were assessed by quantitative polymerase chain reaction (qPCR) assay. In addition, the potential target genes that are regulated by selected miRNAs were analyzed by various target prediction tools. The microarray data revealed that the levels of 38 miRNAs significantly differed between the ACC-M cells and the control ACC-2 cells. Six miRNAs (miR-4487, -4430, -486-3p, -5191, -3131 and -211-3p) were selected to validate the microarray data via qPCR. The expression of two miRNAs (miR-4487 and -4430) was significantly upregulated in the ACC-M cells, while the expression of two other miRNAs (miR-5191 and -3131) was significantly downregulated in the ACC-M cells. The potential target genes that were identified to be controlled by the six selected miRNAs were divided into four groups according to function, as follows: Apoptosis and proliferation (46 genes), cell cycle (30 genes), DNA damage and repair (24 genes) and signaling pathway (30 genes). The identification of microRNA expression profiles in highly metastatic SACC cells may provide an improved understanding of the mechanisms involved in metastatic progression, which would aid in the development of novel strategies for the treatment of SACC.

A novel chronic stress-induced shift in the Th1 to Th2 response promotes colon cancer growth.

Epidemiological data have shown that stress and other psychological factors might influence cancer onset and progression. However, to date, the mechanisms are not well understood. In the present study, we used chronic exposure to a scream as a novel form of sound stress to explore the influence of the chronic stress burden on colon cancer progression, and changes in the immune system were observed. Chronic exposure to scream sound stress induced freezing behavior in the mice and decreased the bodyweight gain. It also caused changes in the adrenal gland and increased serum corticosterone and norepinephrine levels. Cytokine microarray analysis showed changes in the levels of Th1 and Th2 cytokines. The chronic scream sound stress caused a shift from the Th1 to the Th2 response both in the circulation and in tumor-infiltrated lymphocytes, and it promoted colon cancer progression significantly. Taken together, chronic scream sound stress can be conveniently used as a novel chronic stress model. Chronic stress contributes to colon cancer progression and induces a Th1/Th2 imbalance in the mouse immune system, which is considered critical during cancer progression.

Morphologic study of the drainage pathway using a tracer after a bypass filtering procedure in rabbit eyes.

BACKGROUND AND OBJECTIVE: To study the drainage pathway using a tracer after a modified filtering surgery in rabbit eyes. MATERIALS AND METHODS: The authors performed trabeculectomy combined with deep sclerectomy on one eye (TS group) and trabeculectomy on the other eye (T group) of 20 rabbits. Cationic ferritin was injected intracamerally just before killing the rabbits at postsurgical week 2, 4, 8, 12, or 24. The opposite side of the surgical site was used as the control. Histologic study was conducted under light microscopy. RESULTS: In both groups, significantly more intrascleral and episcleral drainage vessels were observed than in the paired controls (P < .001). Intrascleral drainage canals were more often manifested in the TS group than in the T group (P < .05), especially after week 12. CONCLUSION: Intrascleral and episcleral outflow were the main drainage routes after filtering surgeries in this model. Compared with trabeculectomy, the modified surgery was superior for forming an intrascleral "bleb" and seemed to be better maintained at a later stage.

Exogenous phosphatidylethanolamine induces apoptosis of human hepatoma HepG2 cells via the bcl-2/Bax pathway.

AIM: To investigate the signaling pathways implicated in phosphatidylethanolamine (PE)-induced apoptosis of human hepatoma HepG2 cells. METHODS: Inhibitory effects of PE on human hepatoma HepG2 cells were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell cycle, apoptosis and mitochondrial transmembrane potential (DeltaPsi m) were analyzed by flow cytometry. Immunocytochemical assay and Western blotting were used to examine Bcl-2, Bax and caspase-3 protein levels in HepG2 cells treated with PE. RESULTS: PE inhibited the growth of HepG2 cells in a dose- and time- dependent manner. It did not affect the cell cycle, but induced apoptosis. PE significantly decreased DeltaPsi m at 0.25, 0.5 and 1 mmol/L, respectively, suggesting that PE induces cell apoptosis by decreasing the mitochondrial transmembrane potential. The Bcl-2 expression level induced by different concentrations of PE was lower than that in control groups. However, the Bax expression level induced by PE was higher than that in the control group. Meanwhile, PE increased the caspase-3 expression in a dose- and time-dependent manner. CONCLUSION: Exogenous PE induces apoptosis of human hepatoma HepG2 cells via the bcl-2/bax pathway.

[Effects of progesterone on intracellular free Ca2+ concentration in the spermatozoa of fertile men and patients with unexplained infertility].

OBJECTIVE: To investigate the difference in the responsiveness of intracellular free Ca2+ concentration ([Ca2+]i) to progesterone in the spermatozoa of normal fertile men and patients with unexplained infertility. METHODS: Nine normal fertile men and 10 patients with unexplained infertility were selected in this study. After swim-up separation of the motile fraction and 2-hour in vitro capacitation, the spermatozoa were loaded with the fluorescent calcium indicator Fluo-3/AM (8.85 micromol/L) for 40 minutes away from the light, and then the sperm suspension was mixed with equal amount of 20% gelatin to immobilize the spermatozoa. The basal intracellular free [Ca2+]i and that induced by 10 micromol/L progesterone in the individual sperm were assessed by laser scanning confocal microscopy. RESULTS: The infertile patients had a significantly lower basal level of [Ca2+]i in the capacitated sperm than the fertile men (P < 0.01). The sperm from the normal controls responded to progesterone by exhibiting a rapid but transient rise in [Ca2+]i, with the peak level significantly higher than the basal level (P < 0.05), while those from the infertile patients by showing a slight increase, with no significant difference between the peak and basal levels (P > 0.05). Both the peak of the progesterone-induced [Ca2+]i and its increase amplitude expressed as the difference between the peak and basal levels were significantly higher in the normal fertile group than in the infertile patients (P < 0.01). CONCLUSION: The responsiveness of [Ca2+]i to progesterone is reduced in the spermatozoa of patients with unexplained infertility, which suggests a functional defect in the non-genomic sperm membrane progesterone receptor responsible for calcium influx.

Gene expression profile of salivary adenoid cystic carcinoma associated with perineural invasion.

Adenoid cystic carcinoma (ACC) is a common salivary gland malignancy characterized by slow but progressive clinical course, proclivity for hematogenous spread and perineural invasion (PNI) that exhibits inherent resistance to complete surgical resection, systemic chemotherapy and conventional radiotherapy. The molecular alterations that underlie its PNI are poorly characterized. We report the combined use of laser capture microdissection (LCM) and high-throughput cDNA microarray to monitor in vivo gene expression profile of salivary ACC and to correlate the profile with PNI. Consecutive section staining with hematoxylin & eosin was applied to 15 cancerous tissues, among which 6 were judged as PNI. Pure cancer cells adjacent to the nerve tracts from 6 cancerous tissues judged as PNI were laser captured, and pure cancer cells from the same 6 tumors distant from the nerve tracts were also procured. Total RNA was extracted, amplified and subjected to cDNA microarray-based expression analysis. The patterns of gene expression were verified by quantitative real-time PCR and immunohistochemistry. As to the result of 6 arrays, a total of 53 genes were identified as being 2-fold or more differentially expressed in PNI cancer cell group as compared to non-PNI cancer cell control. Out of the 53 genes found consistently differentially expressed, 38 were up-regulated and 15 down-regulated. The combined use of LCM and cDNA microarray analysis provides a powerful new approach to monitor the in vivo molecular events of PNI in salivary ACC. These identified novel genes deserve further investigations to elucidate their clinicopathological significance.