Establishment of a Ciliogenesis-Associated Signaling Model for Polycystic Kidney Disease.

BACKGROUND: Polycystic kidney disease (PKD) represents the most prevalent inherited progressive kidney disorder in humans. Due to complexity of the genetic network behind the disease, the molecular mechanisms of PKD are still poorly understood yet. OBJECTIVES: This study aimed to develop a ciliogenesis-associated gene network for PKD patients and comprehensively understand the molecular mechanisms underlying the disease. METHOD: The potential hub genes were selected based on the differential expression analysis from the GEO database. Meanwhile, the primary hub genes were further elucidated by both in vivo and in vitro experiments. RESULTS: In this study, we established a comprehensive differentially expressed genes profile (including GNAS, PI4KB, UMOD, SLC7A13, and MIOX) for PKD patients compared with the control specimen. At the same time, enrichment analysis was utilized to demonstrate that the G-protein-related signaling and cilia assembling signaling pathways were closely associated with PKD development. The further investigations of the interaction between 2 genes (GNAS and PI4KB) with in vivo and in vitro analyses revealed that PI4KB functioned as a downstream factor for GNAS and spontaneously activated the phosphorylation of Akt into p-Akt for ciliogenesis in PKD formation. The PI4KB depletion mutant zebrafish model displayed a PKD phenotype as well as absence of primary cilia in the kidney. CONCLUSIONS: Collectively, our work discovered an innovative potential signaling pathway model for PKD formation, which provided a valuable insight for future study of the mechanism of this disease.

A narrow T cell receptor repertoire instructs thymic differentiation of MHC class Ib-restricted CD8+ regulatory T cells.

Although most CD8+ T cells are equipped to kill infected or transformed cells, a subset may regulate immune responses and preserve self-tolerance. Here, we describe a CD8 lineage that is instructed to differentiate into CD8 T regulatory cells (Tregs) by a surprisingly restricted set of T cell receptors (TCRs) that recognize MHC-E (mouse Qa-1) and several dominant self-peptides. Recognition and elimination of pathogenic target cells that express these Qa-1-self-peptide complexes selectively inhibits pathogenic antibody responses without generalized immune suppression. Immunization with synthetic agonist peptides that mobilize CD8 Tregs in vivo efficiently inhibit antigraft antibody responses and markedly prolong heart and kidney organ graft survival. Definition of TCR-dependent differentiation and target recognition by this lineage of CD8 Tregs may open the way to new therapeutic approaches to inhibit pathogenic antibody responses.

Tyroservatide therapy for tumor invasion and metastasis of human ovarian carcinoma and colon carcinoma.

Tyroservatide (YSV) is an active, low-molecular-weight peptide shown to have antimetastasic effects on experimental melanoma and lung carcinoma. This study was carried out to evaluate the therapeutic effects of YSV on tumor invasion and metastasis of ovarian carcinoma and colon carcinoma and explore its antitumor mechanism of action. In vivo, three metastasis models were established, and YSV inhibited abdominal cavity metastasis of human ovarian carcinoma, and liver metastasis after spleen implantation of human colon carcinoma, in mice. In vitro, YSV inhibited the proliferation, promoted the death of SKOV-3, HT-29, and SW620 cells, and inhibited the adhesion and invasion of these three types of cells. Through zymography, western blot, and reverse transcription-PCR methods, YSV was found to reduce the activity, expression, and mRNA level of matrix metalloproteinase (MMP)-2 and MMP-9. The results showed that YSV can inhibit tumor growth and metastasis of human ovarian carcinoma SKOV-3 and human colon carcinoma HT-29 and SW620. The mechanism of action of YSV may involve the inhibition of proliferation, promotion of death, inhibition of the adhesion and invasion of SKOV-3, HT-29, and SW620 cells by downregulating the expression and activity of MMP-2 and MMP-9.

Exploration of differentially expressed mRNAs and miRNAs for pediatric acute myeloid leukemia.

Background: To establish a comprehensive differential gene profile for pediatric acute myeloid leukemia patients (pAML) based on two independent databases and verify the differentially expressed genes using in vitro and in vivo analyses. Methods: The mRNA and miRNA sequencing information of GSE2191 and GSE35320, clinically recruited pAML individuals, and human AML cell line (NB4 cells) were utilized in the study. Results: Compared with the control sample, pAML patients demonstrated a total of 778 differentially expressed genes, including 565 upregulated genes and 213 downregulated genes. The genes including ZC3H15, BCLAF1, PPIG, DNTTIP2, SRSF11, KTN1, UBE3A, PRPF40A, TMED5, and GNL2 were the top 10 potential hub genes. At the same time, 12 miRNAs demonstrated remarkable differential expressions in pAML individuals compared with control individuals, as five upregulated and seven downregulated miRNAs. The hsa-miR-133, hsa-miR-181, and hsa-miR-195 were significantly downregulated. Building a miRNA-mRNA regulatory network, hsa-miR-133 regulated ZC3H15, BCLAF1, SRSF11, KTN1, PRPF40A, and GNL2. Using the NB4 cell model, hsa-miR-133 treatment inhibited cell proliferation capacity, which could be attenuated by a single mRNA transfection or a combination of ZC3H15 and BCLAF1. At the same time, hsa-miR-133 mimic treatment could significantly accelerate cell apoptosis in NB4 cells, which was also ZC3H15- and BCLAF1-dependent. The concentrations of ZC3H15 and BCLAF1 were investigated in peripheral blood using the ELISA method for the clinical control and pAML samples. In pAML samples, the expression levels of ZC3H15 and BCLAF1 were significantly enhanced (p < 0.01), regardless of the classification. Conclusion: Collectively, this study hypothesized several promising candidates for pAML formation.

A Pathogenic Role for FcγRI in the Immune Response against Chlamydial Respiratory Infection.

FcγRI is an important cell surface receptor reported to be involved in multiple immune responses, although it has not yet been extensively studied in intracellular bacterial infections. Here, using a mouse model of C. muridarum respiratory infection, we were able to determine how FcγRI regulates the host resistance against chlamydial invasion. According to our findings, the chlamydial loads and pulmonary pathology were both reduced in FcγRI deficient (Fcgr1-/-) animals. Being infected, monocytes, macrophages, neutrophils, DCs, CD4+/CD8+ T cells, and effector Th1 subsets displayed increased FcγRI expression patterns. Altered infiltration of these cells in the lungs of Fcgr1-/- mice further demonstrated the regulation of FcγRI in the immune system and identified Th1 cells and macrophages as its target cell populations. As expected, we observed that the Th1 response was augmented in Fcgr1-/- mice, while the pro-inflammatory M1 macrophage polarization was constrained. These findings might indicate FcγRI as a potential regulator for host immunity and inflammatory response during chlamydial infection.

Expression, purification, and activity assay of peptide deformylase from Escherichia coli and Staphylococcus aureus.

Peptide deformylase (PDF) is considered an attractive target for screening novel antibiotics. The PDF from Escherichia coli and Staphylococcus aureus are representative of the gram-negative species type of PDF (type I PDF) and the gram-positive species type of PDF (type II PDF), respectively. They could be used for screening broad-spectrum antibiotics. Herein, we cloned the def gene by PCR, inserted it into plasmid pET-22b-def, and transformed the plasmid into E. coli BL21 (DE3) cells, then the cells were induced by IPTG to express PDF. E. coli Ni(2+)-PDF was extracted and purified by ion-exchange chromatography and gel filtration chromatography. S. aureus PDFs were extracted and purified using the MagExtractor kit. The nickel form of S. aureus PDF was obtained by adding NiCl(2) to all reagents used for purification. Iron-enriched S. aureus PDF was obtained by adding FeCl(3) to the growth medium for E. coli BL21 (DE3) cells and adding FeCl(3) and catalase to all reagents used for purification. The activities of PDFs were analyzed, compared, and grouped according to the experimental conditions that produced optimal activity, and we used actinonin as an inhibitor of PDF and calculated the IC(50) value. We obtained high expression of E. coli and S. aureus PDF with high activity and stability. The function of PDFs was inhibited by actinonin in a dose-dependent manner. Results may be helpful for future mechanistic investigations of PDF as well as high-throughput screening for other PDF inhibitors.

Therapeutic effects of tyroserleutide on lung metastasis of human hepatocellular carcinoma SK-HEP-1 and its mechanism affecting ICAM-1 and MMP-2 and -9.

BACKGROUND: Tyroserleutide (YSL) inhibits the growth and metastasis of human hepatocellular carcinoma (HCC). This paper studied the effect of YSL on metastasis of human HCC and investigated its mechanisms. METHODS: In vivo, experimental lung metastasis models of human HCC SK-HEP-1 cells in nude mice were established, and In vitro, the proliferation, adhesion and invasion of SK-HEP-1 cells were detected. RESULTS: In vivo, YSL significantly inhibited the metastasis of human HCC. In vitro, YSL significantly inhibited the proliferation, adhesion and invasion of SK-HEP-1 cells. Through analyses with reverse transcription PCR (RT-PCR) and Western blot, we observed that YSL significantly inhibited the expressions of ICAM-1 in SK-HEP-1 cells. Through RT-PCR, Western blot and zymography methods, YSL was discovered to decrease the mRNA level, protein expression and activity of MMP-2 and -9 in SK-HEP-1 cells. CONCLUSION: We concluded that YSL could inhibit tumor growth and metastasis of human HCC SK-HEP-1 cells.

[Tyroservatide inhibits the growth of human hepatocarcinoma in nude mice].

OBJECTIVE: To investigate the inhibitory effects of tyroservatide and its amino acid mixture on growth of hepatocarcinoma. METHODS: Hepatocarcinoma in nude mice was induced by implantation of cells of human hepatocarcinoma cell line BEL-7402. The inhibition of hepatocarcinoma growth was determined by calculating the tumor volume and measuring the tumor weight. The effects of tyroservatide on tumor cells in nude mice were assessed by immunohistochemical staining of proliferating cell nuclear antigen (PCNA), electron microscopic observation of ultrastructure, and apoptosis of tumor cells using terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL). RESULTS: Tyroservatide significantly inhibited the growth of human hepatocarcinoma in nude mice, with an inhibiting rate more than 60%. But the mixture of amino acid did not show a significant inhibitory effect on the tumor growth. Tyroservatide also induced apoptosis of tumor cells and decreased the expression of PCNA in tumor cells. CONCLUSION: Tyroservatide may significantly inhibit the growth of human hepatocarcinoma in nude mice by inducing apoptosis and inhibiting proliferation of tumor cells.

Preliminary investigation of the inhibitory effects of the tyroservaltide (YSV) tripeptide on human hepatocarcinoma BEL-7402.

This study aimed to investigate the inhibitory effect of tyroservaltide (YSV) on the human hepatocarcinoma BEL-7402 transplanted into nude mice and to explore its possible anti-tumor mechanism. Nude mice bearing xenografts of the human BEL-7402 hepatoma were given daily i.p. injections of YSV or saline (as a control) after the tumor were transplanted. Calculating tumor volume and measuring tumor weight determined the extent of inhibition of xenografts. The ultrastructure of tumor cells was observed by electron microscopy. Proliferating cell nuclear antigen (PCNA) expression in tissues of the YSV-treated group was observed by immunohistochemistry. Apoptosis of tumor tissue cells was assayed by the terminal transferase uridyl nick end labeling (TUNEL) method. At doses of 80 microg/kg/d and 160 microg/kg/d, YSV could significantly inhibit growth of tumors transplanted into nude mice, with inhibition rates of 60% and 64%, respectively, compared with that of the controls (P < 0.05). Moreover, YSV changed the ultrastructure of tumor cells, resulting in necrosis and apoptosis of the tumor cells. Compared with the saline group, the expression of PCNA in tumor tissue decreased and the count of apoptotic cell increased. Therefore, YSV can significantly inhibit the growth of human hepatocarcinoma BEL-7402 in nude mice, decrease the expression of PCNA in tumor tissue, and induce tumor cell apoptosis.

Tyroserleutide tripeptide affects calcium homeostasis of human hepatocarcinoma BEL-7402 cells.

This study aimed to observe the effects of tyroserleutide (tyrosyl-seryl-leucine, YSL) on the growth of human hepatocarcinoma BEL-7402 that was transplanted into nude mice, and explore its anti-tumor mechanism preliminarily. YSL, at doses of 80 microg x kg(-1) x d(-1), 160 microg x kg(-1) x d(-1) and 320 microg x kg(-1) x d(-1) significantly inhibited the growth of the human hepatocarcinoma BEL-7402 tumor in nude mice, producing inhibition of 21.66%, 41.34%, and 34.78%, respectively. Ultra structure of BEL-7402 tumor in nude mice showed that YSL could induce tumor cells apoptosis and necrosis, cell organelle mitochondria and endoplasmic reticulum damage, and calcium overload. By confocal laser scanning microscopy and flow cytometry, we found that 10 microg/mL YSL rapidly induced an increase of the concentration of cytoplasmic free calcium in BEL-7402 cells in vitro, and maintained high concentrations of cytoplasmic free calcium for 1 h. Then the calcium concentration began to decrease after 2 h, and was lower than that of the control group at 4 h and 24 h (p < 0.05). YSL also decreased the mitochondrial transmembrane potential of BEL-7402 cells in vitro, but had no effect on the calcium homeostasis or mitochondrial transmembrane potential of Chang liver hepatocytes. So affecting calcium homeostasis, then inducing apoptosis and necrosis may be a mechanism by which YSL inhibits the tumor growth in animal model.

Effects of Tyroserleutide on phosphatidylinositol 3'-kinase/AKT pathway in human hepatocellular carcinoma cell.

Tyroserleutide (YSL) is an active, low-molecular-weight polypeptide with in vitro and in vivo anticancer effects on human hepatocellular carcinoma BEL-7402 cells. In this study, we studied the effects of YSL on PI3K/AKT in the BEL-7402 cells to explore its anti-tumor mechanism. Results showed that YSL could up-regulate the mRNA and protein expression of tumor suppressor PTEN and increase their activities, meanwhile inhibited the mRNA and protein expression of oncogene AKT and decreased the kinase activities of AKT and PDK1. The resuming balance effect of YSL between PTEN and AKT could prevent the transmission of tumor cell proliferation signals in the PI3K/AKT pathway. Inhibition of AKT would change the status of downstream effectors in the PI3K/AKT pathway: (1) inhibition of AKT up-regulated expression of cell cycle regulatory factors of downstream - P21 and P27 which repressed cell cycle and inhibited proliferation of tumor cells. (2) Inhibition of AKT decreased the phosphorylation level of MDM2, and then increased the protein level of P53 which would accelerate death proceeding of tumor cells. (3) Inactivation of AKT removed its inhibition effect on phosphorylation of Bad, which might decrease protein level of apoptosis inhibitor Bcl-2 and Bcl-XL, damaging mitochondria of tumor cells and inducing apoptosis.

Subcellular location of antitumor tripeptide-tyroserleutide in human hepatocellular carcinoma cells.

Tyroserleutide (YSL) is a tripeptide compound that exhibits potent antitumor activity in human tumor xenografts and tumor cell lines. However, the target of YSL on which it exerts its antitumor activity has yet to be identified. Therefore, our study aimed to investigate the subcellular location of YSL in BEL-7402 human hepatocellular carcinoma cells. Using methods of fluorescent tracing and confocal colocalization, we provide evidence that when BEL-7402 cells are treated with YSL, YSL is distributed in the cytoplasm and colocalized with the mitochondria of cancer cells. Furthermore, we observed the effect of YSL on the isolated mitochondria. Using fluorescence spectrophotometry to monitor the Δψ collapse of mitochondria isolated from BEL-7402 cells by reversion of the quenching of tetramethylrhodamine methyl ester (TMRM), we found that the isolated mitochondria reversed the quenching of the fluorescence in the solution containing TMRM and YSL. This indicates that YSL decreases the Δψ of the isolated mitochondria. Another photometry method was used to observe the effect on mitochondrial swelling when YSL acted directly on the isolated mitochondria. We reveal that YSL directly causes mitochondrial swelling in 60 min. In conclusion, this study encloses a preliminary facet of the pharmacological target of YSL, and we speculate that YSL may act directly on the mitochondria to exert its antitumor activity.

The new immunosuppressant PLNPK prolongs allograft survival in mice.

The pentapeptide PLNPK (Pro-Leu-Asn-Pro-Lys) is extracted from the spleen. Preliminary studies have shown that PLNPK could inhibit T lymphocyte transformation and antibody production. In the present study, we detected the inhibitory effect of PLNPK on one-way mixed leukocyte reaction (MLR) in vitro and observed the effect of PLNPK on the duration of allograft survival in mouse models of skin or cardiac transplantation. Pathological damage and T cell infiltration of the grafts were also detected. Results showed that PLNPK could significantly inhibit T lymphocyte proliferation, with an optimized inhibition of 40%. Also PLNPK could significantly prolong the mean survival time of skin allograft and cardiac allograft, producing survival rates of 42% and 38.7%, respectively. PLNPK at a dose of 200 µg/kg/d or 100 µg/kg/d could significantly suppress ConA-induced T cell proliferation and T cell IL-2 secretion in transplant recipient mice, compared to the saline group (P<0.05). This information suggests that PLNPK can effectively antagonize transplant rejection, possibly by reducing IL-2 secretion by T cells and inhibiting T cell proliferation and activation.

Inhibition by tyroserleutide (YSL) on the invasion and adhesion of the mouse melanoma cell.

Tyroserleutide (YSL) is an active, low-molecular-weight polypeptide, comprised of three amino acids, that has shown antitumor effects on human hepatocarcinoma BEL-7402 in vitro and in vivo. In this study, we evaluated the inhibition of YSL on invasion and adhesion of the mouse B16-F10 melanoma cell line by injecting B16-F10 cells into the tail veins of C57BL/6 mice to establish an experimental lung metastasis model. YSL inhibited B16-F10 cell metastasis to lung, reducing the number and area of metastasis lesions. When we treated B16-F10 cells with YSL (0.01, 0.1, 1, 10, or 100 microg/mL) in vitro, we found that YSL inhibited the proliferation of B16-F10 cells with a 28.11% rate of inhibition. YSL significantly decreased the adhesiveness of B16-F10 cells to Matrigel with a 29.15% inhibition rate; YSL also significantly inhibited the invasion of B16-F10 cells, producing an inhibition of 35.31%. By analyses with Western blot and real-time RT-PCR, we found that YSL markedly inhibited the expression of ICAM-1 in B16-F10 cells. These data suggest that YSL inhibits the growth, invasion, and adhesion of B16-F10 cells.

Tyroservatide therapy for tumor growth, invasion and metastasis of Lewis lung carcinoma and human lung carcinoma A549.

OBJECTIVES: The tripeptide tyroservatide (tyrosyl-seryl-valine, p-Tyr-Ser-Val-NH(2), YSV) has been shown to have anti-tumor effects on experimental hepatocarcinoma. The study was conducted to evaluate the therapeutic effects of YSV on tumor growth, invasion and metastasis of lung cancers. METHODS: Anti-tumor and anti-metastatic effects of YSV were evaluated in three experimental systems. In C57BL/6 mice, a spontaneous metastasis model of Lewis lung cancer was used to study the anti-tumor and anti-metastasis effects of YSV. A549 human lung carcinoma was used to create an orthotopic model in nude mice to investigate the anti-metastasis effect of YSV. Finally, an in vitro model using the B16F10 melanoma cell line was selected to observe the effect of YSV on adhesion and invasion, and to use immunocytochemistry to assay the expression of ICAM-1. RESULTS: YSV inhibited subcutaneous tumor growth of Lewis lung carcinoma (p < 0.05) and markedly decreased lung metastases in the spontaneous metastasis model of Lewis lung cancer and the orthotopic model of A549 human lung carcinoma. In vitro, YSV reduced adhesion and invasion as well as the expression of ICAM-1 in tumor cells. CONCLUSION: YSV was able to inhibit tumor growth and metastasis in lung cancer.

[Inhibitory effect of tripeptide compound tyroservaltide on invasion and metastasis of mouse melanoma cell line B16-F10].

BACKGROUND & OBJECTIVE: Tripeptide compound tyroservaltide (YSV) has obvious inhibitory effect on experimental liver cancer. This study was to evaluate the inhibitory effect of YSV on the invasion and metastasis of mouse melanoma cell line B16-F10. METHODS: The cytotoxic effect of YSV on B16-F10 cells was assessed by MTT assay, and the effects of YSV on the adhesiveness and invasiveness of B16-F10 cells were determined using Matrigel and a transwell system. B16-F10 cells were injected into the tail veins of C57BL/6 mice to establish an experimental lung metastasis model, and the effect of YSV on lung metastasis was observed. The effect of YSV on the expression of intercellular adhesion molecule-1 (ICAM-1) in lung tissue was detected by immunohistochemistry. RESULTS: YSV (100 microg/ml, 48 h) inhibited the proliferation of B16-F10 cells, with an inhibitory rate of 24.36%; YSV (100 microg/ml, 24 h) inhibited the adhesiveness of B16-F10 cells to Matrigel, with an inhibitory rate of 36.51%; YSV (10 microg/ml, 48 h) inhibited the invasiveness of B16-F10 cells, with an inhibitory rate of 36.53%; YSV [640 microg.(kg.d)-1] inhibited lung metastasis by B16-F10 cells, with an inhibitory rate of 62.21%. The expression of ICAM-1 in lung tissue was lower in YSV group than in normal saline group. CONCLUSION: YSV can inhibit the growth, invasion, and metastasis of B16-F10 cells.