The recent development of a prosaposin -/- mouse model has allowed the investigation of the role of prosaposin in the development of the male reproductive organs. A morphometric analysis of the male reproductive system of 37 days old mice revealed that prosaposin ablation produced a 30 % reduction in size and weight of the testes, 37 % of the epididymis, 75 % of the seminal vesicles and 60 % of the prostate glands. Light microscopy (LM) showed that smaller testis size from homozygous mutant mice was associated with reduced spermiogenesis. Both, dorsal and ventral lobules of the prostate glands were underdeveloped in the homozygous mutant. LM analysis also showed that prostatic alveoli were considerably smaller and lined by shorter epithelial cells in the homozygous mutant. Smaller tubular diameter and shorter undifferentiated epithelial cells were also observed in seminal vesicles and epididymis. In the efferent ducts of the homozygous mutant mice, the epithelium was composed exclusively of ciliated cells in contrast to the heterozygotes, which showed the presence of nonciliated cells. Radioimmunoassays demonstrated that testosterone levels were normal or higher in mice with the inactivated prosaposin gene. Immunostaining of prostate sections with an anti-androgen receptor antibody showed that the epithelial cells lining the alveoli express androgen receptor in both the heterozygous and homozygous tissue. Similarly, sections immunostained with antibodies to the phosphorylated MAPKs and Akts strongly reacted with tall prostatic secretory cells in prostate from heterozygous mouse. On the other hand, the epithelial cells in the homozygous prostate remained unstained or weakly stained. These findings demonstrate that inactivation of the prosaposin gene affected the development of the prostate gland and some components of the MAPK pathway.
Animals ; Glycoproteins ; metabolism ; Humans ; MAP Kinase Signaling System ; physiology ; Male ; Prostate ; growth & development ; metabolism ; Protein-Serine-Threonine Kinases ; Proto-Oncogene Proteins ; metabolism ; Proto-Oncogene Proteins c-akt ; Saposins

BACKGROUNDTransient sublethal ischemia is known as ischemic preconditioning, which enables cells and tissues to survive subsequent prolonged lethal ischemic injury. Ischemic preconditioning exerts neuroprotection through phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Cbl-b belongs to the Casitas B-lineage lymphoma (Cbl) family, and it can regulate the cell signal transduction.The roles of ubiquitin ligase Cbl-b and PI3K/Akt pathway and the relationship between them in oxygen-glucose deprivation preconditioning (OGDPC) in PC12 cells were investigated in the present study.

METHODSOxygen and glucose deprivation (OGD) model in PC12 cells was used in the present study. The 3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, nuclear staining with Hoechst 33258, and Western blotting were applied to explore the roles of Cbl-b and PI3K/Akt pathway and the relationship between them in OGDPC in PC12 cells.

RESULTSCell viability was significantly changed by OGD and OGDPC. OGD significantly decreased cell viability compared with the control group (P < 0.05), and preconditioning could rescue this damage was demonstrated by the increase of cell viability (P < 0.05). The expression of Cbl-b was significantly increased after OGD treatment. However, the activation of Akt and GSK3β was greatly inhibited. Preconditioning could inhibit the increase of Cbl-b caused by OGD and increase the activation of Akt and GSK3β. LY294002, a specific inhibitor of PI3K, could effectively inhibit the increase of Akt and GSK3β after preconditioning treatment. It partly inhibited the decrease of Cbl-b expression after preconditioning treatment.

CONCLUSIONUbiquitin ligase Cbl-b and PI3K/Akt pathway are differently involved in OGDPC in PC12 cells.

Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Animals ; Cell Survival ; Glucose ; deficiency ; Ischemic Preconditioning ; Oxygen ; metabolism ; PC12 Cells ; Phosphatidylinositol 3-Kinase ; genetics ; metabolism ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; Proto-Oncogene Proteins c-cbl ; genetics ; metabolism ; Rats ; Signal Transduction ; physiology

Retinol-binding protein 4 (RBP4) is adipocyte-derived secreted adipokines and elevated RBP4 expression level was closely related to insulin resistance and type II diabetes mellitus. However, the exact mechanisms are unknown. To clarify the mechanism, RBP4 lentivirus particles were packaged to infect porcine preadipocytes. Then porcine preadipocytes were activated by insulin or induced model of insulin resistance. RBP4 interference efficiency and the gene expression of each treatment groups in PI3K/Akt pathways were examined by QRT-PCR and Western blotting. The result shows that RBP4 mRNA and protein expressions were suppressed more than 60% (P < 0.01). Furthermore, no matter under insulin stimulation or insulin resistance, RBP4 knockdown significantly increased the mRNA expressions of AKT2, PI3K, GLUT4 and IRS1 compared with the control. The protein phosphorylate levels of AKT2, PI3K, IRS1 arised, meanwhile enhanced the AKT2, PI3K, GLUT4 total protein expressions. Collectively, knockdown of RBP4 increased the insulin sensitivity through upregulated PI3K/Akt pathways related factors' expression and phosphorylation in porcine adipocytes. This research will provide a new idea to treat insulin resistance related diseases.
Adipocytes ; metabolism ; Animals ; Gene Knockdown Techniques ; Insulin Resistance ; physiology ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Retinol-Binding Proteins, Plasma ; genetics ; pharmacology ; Signal Transduction ; Swine

OBJECTIVETo study the ability of aqueous extract of Hericium erinaceus mushroom in the treatment of nerve injury following peroneal nerve crush in Sprague-Dawley rats.

METHODSAqueous extract of Hericium erinaceus was given by daily oral administration following peroneal nerve crush injury in Sprague-Dawley rats. The expression of protein kinase B (Akt) and mitogen-activated protein kinase (MAPK) signaling pathways; and c-Jun and c-Fos genes were studied in dorsal root ganglia (DRG) whereas the activity of protein synthesis was assessed in peroneal nerves by immunohistochemical method.

RESULTSPeripheral nerve injury leads to changes at the axonal site of injury and remotely located DRG containing cell bodies of sensory afferent neurons. Immunofluorescence studies showed that DRG neurons ipsilateral to the crush injury in rats of treated groups expressed higher immunoreactivities for Akt, MAPK, c-Jun and c-Fos as compared with negative control group (P <0.05). The intensity of nuclear ribonucleoprotein in the distal segments of crushed nerves of treated groups was significantly higher than in the negative control group (P <0.05).

CONCLUSIONH. erinaceus is capable of promoting peripheral nerve regeneration after injury. Potential signaling pathways include Akt, MAPK, c-Jun, and c-Fos, and protein synthesis have been shown to be involved in its action.

Agaricales ; chemistry ; Animals ; Axons ; pathology ; Female ; Ganglia, Spinal ; metabolism ; Glucans ; analysis ; MAP Kinase Signaling System ; Nerve Crush ; Nerve Regeneration ; physiology ; Peripheral Nerves ; enzymology ; physiology ; Peroneal Nerve ; physiology ; Protein Biosynthesis ; Proto-Oncogene Proteins c-akt ; metabolism ; Proto-Oncogene Proteins c-fos ; genetics ; metabolism ; Proto-Oncogene Proteins c-jun ; genetics ; metabolism ; Rats, Sprague-Dawley

OBJECTIVEFungal keratitis (FK) is a vision-threatening infection, whose treatment requires more effective and safer anti-fungal agent exploitation urgently. With this aim, we focused on the effect of an extracellular polysaccharide on fungal adhesion to human corneal epithelial cells.

METHODSWe performed the cytotoxicity assays of the extracellular polysaccharide EPS-II from an antarctic bacterium Pseudoaltermonas and evaluated its inhibitory effect on Candida albicans cells' adherence to human corneal epithelial cells (HCECs).

RESULTSEPS-II, which displayed minor cytotoxicity but also promoted proliferation of HCECs, could inhibit the adherence of yeast cells to HCECs in a dose-dependent manner. EPS-II could also suppress the subsequent PI3K/AKT signaling pathway, and thereby decrease the expression of early inflammatory cytokines.

CONCLUSIONSExtracellular polysaccharide EPS-II was suggested as a new natural agent for attenuating FK.

Blotting, Western ; Candida ; drug effects ; physiology ; Cell Adhesion ; drug effects ; Cornea ; microbiology ; Humans ; Phosphorylation ; Polysaccharides, Bacterial ; pharmacology ; Proto-Oncogene Proteins c-akt ; metabolism ; Pseudoalteromonas ; metabolism

OBJECTIVETo study the changes of MAPK and Akt signaling pathways in hearts and placentas of aborted fetuses with congenital heart disease (CHD), and investigate their roles in the pathogenesis of CHD.

METHODSTen aborted fetuses with severe CHD (CHD group) and 7 gestational age-matched non-cardiac malformation aborted fetuses (control group) were enrolled. Western blot analysis was undertaken to assess the expression of p38, p38alpha, p-p38, MEF2, ERK, p-ERK, Akt, p-Akt(Ser473) and p-Akt(Thr308) in left ventricles and placentas of the fetuses, while semi-quantitative reverse transcription polymerase chain reaction analysis was used to detect the expression of p38alpha isoforms mRNA in hearts.

RESULTSCompared with the heart samples of the control group, the protein expression levels of p38 and its alpha isoform in 4 cases, p-p38 in 6 cases, MEF2 in 2 cases, p-ERK in 8 cases, Akt in 4 cases, p-Akt(Ser473) and p-Akt(Thr308) in 8 cases decreased, while the protein expression levels of p-p38 in 2 cases and p-Akt(Thr308) in 1 case increased. P-p38 protein level in 3 cases and p-ERK protein level in 2 cases decreased in placentas compared with the control group. The changes of protein expression of MAPK and Akt signaling pathway in hearts were not consistent with those in placentas in the CHD group. The expression of p38alpha isoform2 mRNA showed descent tendency in 4 heart samples with CHD, while the expression of other three p38alpha isoforms mRNA was reduced in only 1 sample compared with the control group.

CONCLUSIONSDysfunction of MAPK and Akt signaling pathways is tissue-specific in aborted fetuses with CHD. The perturbed two signaling pathways in hearts may contribute to the pathogenesis of human CHD.

Female ; Fetus ; metabolism ; Heart Defects, Congenital ; metabolism ; Humans ; MAP Kinase Signaling System ; physiology ; Myocardium ; metabolism ; Phosphatidylinositol 3-Kinases ; physiology ; Placenta ; metabolism ; Pregnancy ; Proto-Oncogene Proteins c-akt ; physiology ; Signal Transduction ; physiology ; p38 Mitogen-Activated Protein Kinases ; physiology

OBJECTIVES: Liver cancer is the sixth most common malignant tumor in the world. Hepatocellular carcinoma (HCC) accounts for 85%-90% of all patients with liver cancer. It possesses the characteristics of insidious onset, rapid progression, early recurrence, easy drug resistance, and poor prognosis. NIMA related kinase 2 (NEK2) is a cell cycle regulating kinases, which regulates cell cycle in mitosis. Cellular senescence is a complex heterogeneous process, and is a stable form of cell cycle arrest that limits the proliferative potential of cells. This study aims to investigate the relationship between the expression level of NEK2 and the senescence in hepatoma cells, and to explore the effect of NEK2 expression on hepatoma cell senescence and the underlying molecular mechanism. METHODS: A total of 581 senescence-relevant genes were obtained from the GenAge website. The gene expression data of tumor tissues of 370 HCC patients were downloaded from the Cancer Genome Atlas database. The co-expression of NEK2 and aging-related genes was analyzed by R-package. KEGG was used to analyze the significant gene enrichment pathway of differentially expressed genes in NEK2 overexpression HEK293. The stable transfected cell lines with overexpression and knockdown of NEK2 were constructed in hepatoma cell line SMMC-7721 and HepG2, and senescence-associated β-galactosidase (SA-β-gal) staining was used to detect senescence, the cell proliferation was detected by CCK-8 method and clone formation experiment, the cell cycle was analyzed by flow cytometry, and the expression of proteins related to p53/p21, p16/Rb, and phosphatase and tensin homolog deleted on chromosome ten (PTEN)/Akt signal transduction pathway was detected by Western blotting. RESULTS: There were 320 senescence related genes co-expressed with NEK2. KEGG analysis showed that the senescence signaling pathway was significantly enriched in HEK293 cells with overexpression of NEK2.Compared with SMMC-7721 or HepG2 without knockdown of NEK2, the senescent cells of SMMC-7721 and HepG2 with knockdown of NEK2 were increased, cell proliferation and clone formation were decreased significantly, the percentage of cells in G₀/G₁ phase was increased, the expression levels of phospho-Akt (p-Akt) and phospho-Rb (p-Rb) protein were decreased significantly, and the expression level of p16 protein was increased significantly (all P<0.05). Compared with SMMC-7721 or HepG2 transfected with blank plasmid, the senescent cells of SMMC-7721 and HepG2 overexpressing NEK2 were decreased, the cell proliferation and clone formation were increased significantly, the percentage of cells in G₀/G₁ phase were decreased, the expression levels of p-Akt and p-Rb protein were increased significantly, and the expression level of p16 protein was decreased significantly (all P<0.05). CONCLUSIONS NEK2 may mediate the anti-aging effect of hepatoma cells through p16/Rb and PTEN/Akt signal transduction pathways, which provides a new theoretical basis for NEK2 to promote the progress of liver cancer and a new idea for the targeting treatment for liver cancer.
Carcinoma, Hepatocellular/pathology* ; Cell Line, Tumor ; Cell Proliferation/physiology* ; Cellular Senescence/genetics* ; HEK293 Cells ; Humans ; Liver Neoplasms/pathology* ; NIMA-Related Kinases/genetics* ; Proto-Oncogene Proteins c-akt/metabolism*

AIMTo investigate the chronic effect of palmitic acid (PA) on apoptosis of pancreatic islet beta-cells and the possible mechanism.

METHODSInsulinoma cell line (MIN6 cells) were used in this study. After being incubated in PA (0.1 - 1.6 mml/L) for 24 and 48 hours, MTT method was used to evaluate the livability. After being incubated for 48 h, Hoechst-PI and Annexin-V-FTTC/PI FACS were used to estimate the apoptosis in each group, Western-blotting assay was used to estimate the protein level of p-Akt, Akt, Bax and Bcl-2.

RESULTSChronic PA dose-dependently (1) decreased the availability and increased the apoptosis of MIN6 cells; (2) decreased the phosphorylation of Akt and Bcl-2, but had no significant effects on Akt and Bax.

CONCLUSIONChronic PA dose-dependently induced apoptosis of MIN6 cells, and this effect was possibly regulated by Akt/Bcl-2.

Animals ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cells, Cultured ; Insulinoma ; pathology ; Islets of Langerhans ; pathology ; Mice ; Oxidative Stress ; physiology ; Palmitic Acid ; pharmacology ; Proto-Oncogene Proteins ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; Signal Transduction ; drug effects ; physiology ; bcl-2-Associated X Protein ; metabolism

BACKGROUNDIncreased reactive oxygen species (ROS) formation, which in turn promotes cardiomyocytes apoptosis, is associated with the pathogenesis and progression of various cardiac diseases such as ischemia and heart failure. Recent studies have shown that over expression of heat shock protein 27 (Hsp27) confers resistance to cardiac ischemia/reperfusion injury. However, not much is known about the regulation of myocyte survival by Hsp27.

METHODSThe rat cardiac cell line H9c2, with a stable overexpression of Hsp27, was established, with empty vector transfected H9c2 cells as controls. Following the cells challenged by Hydrogen Peroxide (H2O2), lactate dehydrogenase (LDH) release, apoptosis, intracellular ROS, cell morphology, mitochondrial transmembrane potential and the activation of serine/threonine kinase Akt were determined.

RESULTSAlong with marked suppression of H2O2-induced injury by Hsp27 overexpression in H9c2 cells, ROS generation and the loss of mitochondrial membrane potential were also significantly depressed. Furthermore, augmented Akt activation was observed in Hsp27 overexpressed H9c2 cells following H2O2 exposure.

CONCLUSIONSHsp27 inhibits oxidative stress-induced H9c2 damage and inhibition of ROS generation and the augmentation of Akt activation may be involved in the protective signaling.

Animals ; Apoptosis ; Cell Line ; HSP27 Heat-Shock Proteins ; Heat-Shock Proteins ; physiology ; Humans ; Hydrogen Peroxide ; toxicity ; Myocytes, Cardiac ; pathology ; Neoplasm Proteins ; physiology ; Oxidative Stress ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats ; Reactive Oxygen Species ; metabolism

Cell Movement ; genetics ; physiology ; Humans ; Microfilament Proteins ; genetics ; metabolism ; Models, Biological ; Neoplasm Metastasis ; genetics ; physiopathology ; Protein Binding ; genetics ; physiology ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; Vesicular Transport Proteins ; genetics ; metabolism