Pttg1/securin is required for the branching morphogenesis of the mammary gland and suppresses mammary tumorigenesis.

Pituitary tumor transforming gene 1 (Pttg1) encodes the mammalian securin, which is an inhibitor of separase (a protease required for the separation of sister chromatids in mitosis and meiosis). PTTG1 is overexpressed in a number of human cancers and has been suggested to be an oncogene. However, we found that, in Pttg1-mutant females, the mammary epithelial cells showed increased proliferation and precocious branching morphogenesis. In accord with these phenotypic changes, progesterone receptor, cyclin D1, and Mmp2 were up-regulated whereas p21 (Cdkn1a) was down-regulated. These molecular changes provide explanation for the observed developmental defects, and suggest that Pttg1 is a tumor suppressor. Indeed, mice lacking Pttg1 developed spontaneous mammary tumors. Furthermore, in human breast tumors, PTTG1 protein levels were down-regulated and the reduction was significantly correlated with the tumor grade.

Proteolytic processing, deubiquitinase and interferon antagonist activities of Middle East respiratory syndrome coronavirus papain-like protease.

The emerging Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe pulmonary disease in humans and represents the second example of a highly pathogenic coronavirus (CoV) following severe acute respiratory syndrome coronavirus (SARS-CoV). Genomic studies revealed that two viral proteases, papain-like protease (PLpro) and 3C-like protease (3CLpro), process the polyproteins encoded by the MERS-CoV genomic RNA. We previously reported that SARS-CoV PLpro acts as both deubiquitinase (DUB) and IFN antagonist, but the function of the MERS-CoV PLpro was poorly understood. In this study, we characterized MERS-CoV PLpro, which is a protease and can recognize and process the cleavage sites (CS) of nsp1-2, nsp2-3 and nsp3-4. The LXGG consensus cleavage sites in the N terminus of pp1a/1ab, which is generally essential for CoV PLpro-mediated processing, were also characterized in MERS-CoV. MERS-CoV PLpro, like human SARS-CoV PLpro and NL63-CoV PLP2, is a viral deubiquitinating enzyme. It acts on both K48- and K63-linked ubiquitination and ISG15-linked ISGylation. We confirmed that MERS-CoV PLpro acts as an IFN antagonist through blocking the phosphorylation and nuclear translocation of IFN regulatory factor 3 (IRF3). These findings indicate that MERS-CoV PLpro acts as a viral DUB and suppresses production of IFN-ß by an interfering IRF3-mediated signalling pathway, in addition to recognizing and processing the CS at the N terminus of replicase polyprotein to release the non-structural proteins. The characterization of proteolytic processing, DUB and IFN antagonist activities of MERS-CoV PLpro would reveal the interactions between MERS-CoV and its host, and be applicable to develop strategies targeting PLpro for the effective control of MERS-CoV infection.

Liquiritin potentiate neurite outgrowth induced by nerve growth factor in PC12 cells.

Neurite outgrowth and neuronal differentiation play a crucial role in the development of the nervous system. Understanding of neurotrophins induced neurite outgrowth was important to develop therapeutic strategy for axon regeneration in neurodegenerative diseases as well as after various nerve injuries. It has been reported that extension of neurite and differentiation of sympathetic neuron-like phenotype was modulated by nerve growth factor (NGF) in PC12 cells. In this study, NGF mediated neurite outgrowth was investigated in PC12 cells after liquiritin exposure. Liquiritin is a kind of flavonoids that is extracted from Glycyrrhizae radix, which is frequently used to treat injury or swelling for its life-enhancing properties as well as detoxification in traditional Oriental medicine. The result showed that liquiritin significantly promotes the neurite outgrowth stimulated by NGF in PC12 cells in dose dependant manners whereas the liquiritin alone did not induce neurite outgrowth. Oligo microarray and RT-PCR analysis further clarified that the neurotrophic effect of liquiritin was related to the overexpression of neural related genes such as neurogenin 3, neurofibromatosis 1, notch gene homolog 2, neuromedin U receptor 2 and neurotrophin 5. Thus, liquiritin may be a good candidate for treatment of various neurodegenerative diseases such as Alzheimer's disease or Parkinson's disease.

Enhancing efficacy and mucosa-tropic distribution of an oral HIV-PsV DNA vaccine in animal models.

A strategy combined the oral delivery route and bovine papillomavirus (BPV) pseudovirus (PsV)-based human immunodeficiency virus (HIV) DNA vaccine, which has been proven to enhance the mucosal immunization compared with the systemic immunization and in general does not induce effective mucosal immune responses. In this study, the immune responses against the BPV expressing HIV gp41 epitopes (ELDKWA, NWFDIT) after oral administration in Cynomolgus monkeys (Macaca fascicularis) were assessed, and the biodistribution of plasmid DNA encapsulated in the papillomavirus-like particles (VLPs) were evaluated in murine models. Results showed that oral immunization with the HIV-PsV DNA vaccine in monkey generated p24 and gp41 epitopes-specific serum IgG. Importantly, these induced antibodies had been shown to neutralize HIV-1 primary strain. In addition, the advantage of VLPs as vehicles delivering genes had been first revealed in biodistribution results. Therefore, orally administered HIV-PsV DNA vaccine was well-tolerated, enhanced the mucosa targeting property of the plasmid DNA, and reduced the nontargeting distribution, which indicate that it would reduce stress associated with systemic vaccination.

[Neuroprotection and neurotrophism effects of liquiritin on primary cultured hippocampal cells].

OBJECTIVE: To observe the neuroprotective and neurotrophic effects ofliquiritin (LQ) on rats primary cultured hippocampal neuronal damage induced by Abeta25-35. METHOD: The rats hippocampal neuronal damage model by Abeta25-35 was established. The protective effects of LQ to the cells were observed through the MTU assay. The apoptosis of neurons was detected by flow cytometer and the concentration of intracellular calcium by fluorescence probe dying. LQ' s neurotrophic effects were observed through measuring the neurite outgrowth induced by LQ in primary cultured hippocampal neurons and the differentiation of LQ on hippocampal stem cells to cholinergic neurons was assayed by flow cytometry. RESULT: Treatment of the cells with 10 micromol x L(-1) Abeta25-35 could induce a significant decrease of cell viability, enhance the level of intracellular [Ca2+] and increase the percentage of apoptosis to 28%. However, pretreatment with LQ (0.1, 1, and 10 micromol x L(-1)) for 6 hours exhibited cytoprotective effects, inhibited the cells' s death induced by Abeta25-35, prevented the accumulation of [Ca2+], and decreased the apoptosis neurons significantly to 10%, 15% and 9%, which meaned that LQ could antagonize Abeta25-35 induced apoptosis. LQ together with NGF had a dramatic prolonged effect on the neurite of the primary cultured hippocampal neurons, which was blocked by a specific MAPK kinase inhibitor to some extent. In addition, LQ could induce the differentiation of hippocampal stem cells to cholinergic neurons in vitro. CONCLUSION: These results demonstrate that LQ has the neuroprotective capacity to cell damage iduced by Abeta25-35 in primary cultured hippocampal neurons, and also has the neurotrophic effects.

The thrombolytic effect of miniplasmin in a canine model of femoral artery thrombosis.

BACKGROUND AND PURPOSE: Miniplasmin was a des-kringle variant of plasminogen with potential pharmacological application. We investigated the thrombolytic effect of miniplasmin in a canine model of femoral artery thrombosis. METHODS: In anesthetized dogs, a stable occlusive thrombus was formed by mechanical and electrolytic injury of the vessel wall, that the animals were later injected with miniplasmin (0.75 mg/kg, 1.5 mg/kg and 3.0 mg/kg, i.a.) and rt-PA (0.5 mg/kg, i.a.) intra-arterially. Hemodynamic parameters and hemorrhage status were monitored for 2 h. Thrombin time, activated partial thromboplastin time, prothrombin time and fibrinogen concentration were tested at 2 h after administration. Fibrin degradation product and D-dimer concentration were tested by ELISA. RESULTS: The incidence of reperfusion in the miniplasmin (3.0 and 1.5 mg/kg) groups was 100%, and time to reperfusion was (3.3+/-1.0) and (7.0+/-2.3) min, which was shorter than rt-PA. After reperfusion, none of the vessels in the miniplasmin (1.5 and 3.0 mg/kg) groups reoccluded, whereas 20% of vessels reoccluded in the rt-PA group. Rudimental thrombus mass in the miniplasmin (1.5 and 3.0 mg/kg) groups were smaller than rt-PA. The operative wounds in all miniplasmin groups had no hemorrhage within 2 h. There were no significant differences in thrombin time, activated partial thromboplastin time and prothrombin time. Fibrinogen concentration in the miniplasmin (3.0 mg/kg) group reduced significantly as compared with baseline and thrombosis values, whereas these values in the miniplasmin (1.5 and 0.75 mg/kg) groups were unchanged. Fibrin degradation product and D-dimer concentration increased significantly after thrombolysis. CONCLUSIONS: The results suggest that miniplasmin may be useful for the treatment of thrombosis and without complication of hemorrhage. This is in contrast to rt-PA, which intrinsically has a higher risk of occurring the hemorrhage risk.

Madecassoside reduces ischemia-reperfusion injury on regional ischemia induced heart infarction in rat.

Madecassoside (MA), one of the principle terpenoids in Centella asiatica, has shown protect effect on isolated rat hearts and isolated cardiomyocytes against reperfusion injury in our previous studies. The aim of this study is to investigate if MA also protected against myocardial ischemia-reperfusion injury in vivo. The ischemia infarction model was established in rats. Left ventricular function was monitored during the ischemia-reperfusion period by a multi-channel recorder. After the ischemia-reperfusion process the infarcted areas were assessed. The levels of lactate dehydrogenase (LDH), creatinephosphokinase (CK), malondialdehyde (MDA), super-oxide dismutase (SOD) and C-reactive protein (CRP) in serum were determined. Cardiomyocytic apoptosis was measured by terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) staining. Pre-treatment with MA (50, 10 mg/kg) attenuated myocardial damage characteristic of decreasing infarct size, decreasing LDH and CK release. Activities of SOD were increased and MDA level increased obviously in control group whereas pretreatment with MA blunted the decrease of SOD activity, markedly reduced the level of MDA and the activity of CRP, and relieved myocardial cell apoptosis. These results suggest that MA has the protective effect on myocardial ischemia-reperfusion injury. This protection ability possibly due to its anti-lipid peroxidation, anti-inflammation and anti-apoptosis function and the enhancement of SOD activity.

GLP-1 C-terminal structures affect its blood glucose lowering-function.

Glucagon-like peptide-1 (GLP-1), which is an endogenous insulinotropic peptide that can stimulate islet cells to secret insulin, is a promising new drug candidate for the treatment of type 2 diabetes. However, due to the very short half-life of this peptide, the clinical value of GLP-1 is restricted. A GLP-1 peptide analog that had been altered by deletion of five amino acids from the C-terminus (sGLP-1) was selected and investigated in vivo for the therapeutic effect on GK rats with type II DM (T2DM). The results revealed that sGLP-1 exhibited decreased blood glucose-lowering ability compared to GLP-1 in the first week, as measured after once-daily administration. However, after drug administration for 2 weeks, the blood glucose-lowering effect of sGLP-1 became superior to that of GLP-1. sGLP-1 reduced apoptosis of the old islets, enhanced insulin production, and promoted new islets replication. sGLP-1 is a shorter but more efficient GLP-1 analog for type 2 diabetes management. Because sGLP-1 prolonged the proliferation and recovery of islet cells, the ability to maintain blood glucose (BG) within a normal range was still present 2 weeks after drug withdrawal. These results confirmed the importance of the C-terminus of GLP-1 molecule, and further demonstrated that GLP-1 (7-37) can be truncated till the 32nd amino acid to have a better long-term BG lowing function. This result may imply for the presence of glucagon family clearance receptors in vivo and demonstrates that the C-terminus participates in GLP-1 clearance.

[Protective effect of madecassoside against reperfusion injury after regional ischemia in rabbit heart in vivo].

This study is to investigate if madecassoside can protect against myocardial reperfusion injury in rabbit heart in vivo. The ischemia reperfusion model was established. Left ventricular function and ECG were monitored at the ischemia and reperfusion period. The infarct areas were expressed as percentage. The levels of LDH, CK, MDA and SOD were measured and C-reactive protein (CRP) in serum was measured by ELISA kit. Cardiomyocyte apoptosis were measured by TUNEL staining. A monoclonal rabbit anti-goat Bcl-2 proteins as primary antibody was used for Bcl-2 immunohistochemical staining. Treatment with madecassoside (3.2, 1.6 and 0.8 mg x kg(-1)) i.v. during ischemia reperfusion injury attenuated myocardial damage, that is, characteristic of decreasing infarct size, decreasing LDH and CK release. Activities of SOD were diminished and MDA level increased obviously in control group whereas pretreatment with madecassoside significantly blunted the decrease of SOD activity, markedly reduced the levels of MDA, CRP and cardiomyocyte apoptosis, and upregulated the expression of Bcl-2. Madecassoside has the protective effect against myocardial ischemia reperfusion injury, and effects of anti-lipid peroxidation, enhancement of SOD activity, anti-inflammation and anti-apoptosis.

[Effect of recombinant microplasmin on acute cerebral infarction in rats].

The effect of recombinant microplasmin (micro-plasmin) on acute cerebral infarction was evaluated in rats, and compared with recombinant tissue plasminogen activator (rt-PA). After the model of middle cerebral artery occlusion (MCAO) was established by autologous blood clots, different doses of micro-plasmin (2.5, 5, and 10 mg x kg(-1)) were administered into the thrombus intra-arterial. Twelve hours after administration of micro-plasmin, the neurological deficit score of rats was recorded and the infarct volumes were determined. Bleeding time (BT), fibrin degradation product (FDP) concentration in serum and thrombin time (TT), prothrombin time (PT) and fibrinogen (FIB) concentration in plasma were tested after administration. Intra-arterial administration of micro-plasmin could reduce significantly neurological deficit score and infarct volumes in MCAO rats. FDP concentration increased significantly as compared with model group. There were no significant differences in TT, PT and BT. FIB concentration reduced markedly as compared with model group, but had no significant difference as compared with sham group. The results suggest that micro-plasmin is effective in treatment of rat acute cerebral infarction, and has no significant influence on fibrinolytic system and blood clotting system, indicating that micro-plasmin may be useful for treatment of acute cerebral infarction, and not lead to hemorrhage. Micro-plasmin seems to be distinguished from clinical used rt-PA by its no hemorrhage effect.

[Establishment of a reporter gene-based cell screening model for discovering new agonists of estrogen receptor beta subtype].

AIM: To establish a sensitive and efficient reporter gene-based screening model for finding agonists of estrogen receptor beta subtype. METHODS: A recombinant vector pTAL-ERE-SEAP was constructed by inserting a synthetic sequence composed of five estrogen responsive elements in front of promoter of pTAL-SEAP vector. pTAL-ERE-SEAP was then transfected into human embryonic kidney (HEK293) cells. G418 (200 microg x mL(-1)) was added to select positive clones that can be induced by E2 to express reporter gene SEAP. The speciality was tested by several ligands of relative nuclear receptors of the same family. The stability of the model, the time-effect relationship, the dose-response relationship, and the immunocytochemistry staining of ERbeta expression after transfection were observed. 2 622 compounds were screened by using this model. RESULTS: Stably transfected clones were obtained. The expression levels of reporter gene SEAP of positive clones was induced by E2 in a dose-response and time-effect relationship manners. The Z' factor value was 0.7. The expression levels of dexamethasone and other ligands were low. The result of immunocytochemistry staining showed the expression of ERbeta. E2 had no proliferating effects on stably transfected clones. CONCLUSION: Stably transfected positive clones transfected with recombinant vector pTAL-ERE-SEAP were obtained. The positive clones may be used to screen for agonists of estrogen receptor beta subtype by measurement of luminescent value of expressed SEAP in wells of microlitre plate.

Radioimmunotherapy of carcinoma of colon with [131I]-labeled recombinant chimeric monoclonal antibodies to carcinoembryonic antigen.

AIM: To study the distribution of [(131)I]-labeled anti-CEA MoAbs and its therapeutic effect on the human colonic cancer model in nude mice. METHODS: A nude mice model of human colonic cancer was established. [(131)I]-labeled anti-CEA MoAbs were injected intravenously into mice. The distribution of the MoAbs was then determined and the effect of RIT on human colonic cancer was observed. RESULTS: The [(131)I]-labeled anti-CEA MoAbs had a specific distribution after injection. Tumor/non-tumor ratios for [(131)I]-labeled anti-CEA MoAbs were 10-20 times higher than [(131)I]-labeled IgG 96 h after injection. Thirty days after injection, significant inhibition of the volume and weight of tumor was observed in the treated mice compared with the control. The tumor growth inhibition rate of 3.1 mCi/kg CEA MoAbs group (LS180, LS174T, SW1116) was 47.8%-64.0%. This was 69.6%-78.6% in the 6.25 mCi/kg CEA MoAbs group, and 81.8%-86.2% in the 12.5 mCi/kg [(131)I]-labeled anti-CEA MoAbs group. The plasma CEA level was also lower in treated mice. CONCLUSION: The results indicate that [(131)I]-labeled anti-CEA MoAbs can be effective in RIT on colonic cancers.

Profiling of differentially expressed chemotactic-related genes in MCP-1 treated macrophage cell line using human cDNA arrays.

AIM: To study the global gene expression of chemotactic genes in macrophage line U937 treated with human monocyte chemoattractant protein-1 (MCP-1) through the use of ExpreeChipHO2 cDNA array. METHODS: Total RNA was extracted from MCP-1 treated macrophage line U937 and normal U937 cells, reversely transcribed to cDNA, and then screened in parallel with HO2 human cDNA array chip. The scanned result was additionally validated using RT-PCR. RESULTS: The result of cDNA array showed that one chemotactic-related gene was up-regulated more than two-fold (RANTES) and seven chemotactic-related genes were down-regulated more than two-fold (CCR1, CCR5, ccl16, GRObeta, GROgamma, IL-8 and granulocyte chemotactic protein 2) in MCP-1 treated U937 cells at mRNA level. RT-PCR analysis of four of these differentially expressed genes gave results consistent with cDNA array findings. CONCLUSION: MCP-1 could influence some chemokine and receptor expressions in macrophages in vitro. MCP-1 mainly down-regulates the expression of chemotactic genes influencing neutrophilic granulocyte expression (GRObeta, GROgamma, IL-8 and granulocyte chemotactic protein 2), and the mRNA level of CCR5, which plays a critical role in many disorders and illnesses.