Human immune suppression is inducible by trichosanthin via CD8 cell—mediated pathway

Trichosanthin(Tk),a polypeptide with 249 amino acid residues isolated and purified from a Chinese medicinal herb,showed the capability of inducing abortion and was able to inhibit tumor growth and HIV replication.Owing to sequence homology of the peptide with a ribosomeinactivating protein,the downward activity of Tk was suggested to be related to its cytotoxic property.We report here,however,that Tk could exert potent inhibitory effects on human lymphoproliferative responses in vitro to allogeneic,mitogenic and soluble antigens with 50% inhibition doses ranged between 0.05 and 0.5μg／ml.The lowresponsivenesss caused by Tk was not due to toxic cytolysis.Rather,evidences suggested that,in the dose range adopted,the Tk-induced inhibition was attributable,at least in part,to immune suppression,in view of (1) Tk was more effective in the early stage of alloreactivity;(2)Suppression also occurred if responder cells were pulsetreated with Tk rather than cocultured;(3)Irradiated Tk-pulsed cells were capable of inducing suppression in a Tk-free culture;(4)Suppression could also be transferred by the supernatants of Tk-pulsed cultured cells;(5)Tk-induced immune suppression was diminished by depletion of CD8^ cells from the culture,and,finally;(6)Adding CD8^ cells back to the culture could restore the suppres sion.Thus the possibility that Tk might function as a down-regulator by immunological mechanisms in human immune responses is discussed.     

ERK1/2 mediates lung adenocarcinoma cell proliferation and autophagy induced by apelin-13

The aim of this study was to investigate the role of apelin in the cell proliferation and autophagy of lung adenocarcin- oma. The over-expression of APJ in lung adenocarcinoma was detected by immunohistochemistry, while plasma apelin level in lung cancer patients was measured by enzyme-linked immunosorbent assay. Our findings revealed that apelin-13 significantly increased the phosphorylation of ERK1/2, the expression of cyclin D1, microtubule-associated protein 1 light chain 3A/B （LC3A/B）, and beclinl, and con- fwmed that apelin-13 promoted A549 cell proliferation and induced A549 cell autophagy via ERK1/2 signaling. More- over, there are pores on the surface of human lung adeno- carcinoma cell line A549 and apelin-13 causes cell surface smooth and glossy as observed under atomic force micros- copy. These results suggested that ERK1/2 signaling pathway mediates apelin-13-induced lung adenocarcinoma cell proliferation and autophagy. Under our experimental condition, autophagy associated with 3-methyladenine was not involved in cell proliferation.     

Regulation of membrane band 3 Tyr-phosphorylation by proteolysis of p72^syk and possible involvement in senescence process

Erythrocyte senescence is characterized by exposure of cell surface epitopes on cell membrane proteins leading to immune mediated removal of red blood cells. One mechanism for antigen formation is tyrosine phosphorylation （Tyr-P） of the transmembrane protein band 3 by Syk kinase. Our aim was to test the hypothesis that proteolytic activation of Syk kinase by conversion from 72 kDa （p72^syk） to the 36 kDa （p36^syk） isoform enhances its phosphorylating activity independently of the association of Syk kinase with the cytoskeleton. Tyr-P assay was conducted using quantification of 32p uptake into the cytoplasmic domain of band 3 after addition of p72^syk or p36syk. Effect of prephosphorylation of erythrocyte membrane band 3 protein by p36^syk on p72^syk-mediated phosphorylation and the effect of addition of a protease inhibitor （leupeptin） on p72s^yk-mediated phosphorylation were studied by autoradiographic visualization of 32p uptake. Tyr-P by Syk isoforms of membrane skeletal and soluble fractions of band 3 was visualized by immunoblotting. It was found that p36^Syk had a higher band 3 tyrosine phosphorylating activity compared with p72^syk. Pre-phosphorylation with p36^syk or p72^syk increased band 3 phosphorylating activity. Protease inhibition treatment reduced p72^syk but not p36^syk band 3 tyrosine phosphorylating activity significantly. Both soluble and membrane skeletal fractions of band 3 protein were equally tyrosine phosphorylated by each Syk isoform. In conclusion, we confirmed the hypothesis that proteolytic cleavage of p72^syk is an important regulatory step for band 3 Tyr-P and its independence of the association of band 3 with the cytoskeleton.     

Characterization of a new gene WX2 in Toxoplasma gondii

Using hybridization techniques, we prepared the monoclonal antibody （Mab） 7C3-C3 against Toxoplasma gondii. The protection tests showed that the protein （Mab7C3-C3） inhibited the invasion and proliferation of T. gondii RH strain in HeLa cells. The passive transfer test indicated that the antibody significantly prolonged the survival time of the challenged mice. It was also shown that the antibody could be used for the detection of the circulating antigen of T. gondii. After immunoscreening the T. gondii tachyzoite cDNA library with Mab7C3-C3, a new gene wx2 of T. gondii was obtained. Immunofluorescence analysis showed that the WX2 protein was located on the membrane of the parasite. Nucleotide sequence comparison showed 28% identity to the calcium channel α-IE unit and shared with the surface antigen related sequence in some conservative residues. However, no match was found in protein databases. Therefore, it was an unknown gene in T. gondii encoding a functional protein on the membrane of T. gondii. Because it has been shown to have a partial protective effect against T. gondii infection and is released as a circulating antigen, it could be a candidate molecule for vaccine or a novel target for new drugs.     

Preliminary computational modeling of nitric oxide synthase 3 interactions with caveolin-1： influence of exon 7 Glu298Asp polymorphism

The significance of endothelial nitric oxide synthase 3 （NOS3） activity has been recognized for many years, however it was only recently that the complicated regulation of this constitutively expressed enzyme in endothelial cells was identified. A critical component of the NOS3 regulatory cyde in endothelial cells is its intracellnlar localization to caveolae. The caveolar coordination of NOS3, more specifically its interaction with caveolin-1 （Cav-1）, plays a major role in normal endothelial NOS3 activity and vascular bioavailability of nitric oxide. We have recently shown that the presence of NOS3 exon 7 Glu298Asp polymorphism caused diminished shear-dependent NOS activation, was less extensively associated with caveolae, and had a decreased degree of interaction with Cav-1. Here, we carried out preliminary investigations to identify possible mechanisms of the genotype-dependent endothelial cell responses we observed in our previous investigations. Through this approach we tested the hypothesis that computer simulations could provide insights regarding the contribution of this single nucleotide polymorphism to regulation of the NOS3 isoform. We observed that in the Glu/Asp and Asp/Asp mutant genotypes, the amount of NOS3 associated with Cav-1 was significantly lower. Additionally, we have shown, using a theoretical computational model, that mutation of an amino acid at position 298 might affect the protein-protein interactions and localization of the NOS3 protein. These alterations might also affect the protein function and explain the enhanced disease risk associated with the presence of Glu298Asp polymorphism in the NOS3 protein.     

mad—overexpression down regulates the malignant growth and p53 mediated apoptosis in human hepatocellular carcinoma BEL—7404 cells

Mad protein has been shown as an antagonist of cMyc protein in some cell lines.The effect of Mad protein to the malignant phenotype of human hepatoma BEL-7404 cell line was investigated experimentally.An eukarryotic vector pCDNA Ⅲ containing full ORF fragment of mad cDNA was transfected into targeted cells.Under G418 selection,stable Mad-overexpressed cells were cloned.Studies on the effect of Mad over-expression in cell proliferation and cell cycle revealed that cell morphology of the Mad-overexpressed BEL-7404-M1 cells was significantly different from the parent and control vector transfected cells.DNA synthesis,cell proliferation and anchorage-independent growth in soft-agar of the madtransfected cells were partially inhibited in comparison to control cells.Flos cytometry analysis indicated that mad over-expression might block more transfectant cells at G0/G1 phase,resulting in the retardation of cell proliferation.RT-PCR detected a marked inhibition of the expression of cdc25A,an important regulator gene of G0/G1 to S phase in cell cycle.It was also found that Mad protein overexpression could greatly suppress p53-mediated apoptosis in BEL-74040M1 cells in the absence of serume.Thus,Mad proteins may function as a negative regulator antagonizing c-Myc activity in the control of cell growth and apoptosis in human hepatocellular carcinoma BEL-7404 cells.     

Lamprey buccal gland secretory protein-2 (BGSP-2) inhibits human T lymphocyte proliferation

<正> Lamprey is a representative of the agnathans, the most ancient class of vertebrates. Parasitic lampreys secrete anticoagulantfrom their buccal glands and prevent blood coagulation of host fishes. We identified a buccal gland secretory protein-2(BGSP-2) from a buccal gland cDNA library of Lampetra japonica. The full-length BGSP-2 gene was cloned and the recombinantBGSP-2 protein was generated. The role of BGSP-2 on lymphocyte proliferation was studied by examining its effects onhuman T lymphocytes. We found that lamprey BGSP-2 was able to effectively block the proliferation of T cells in vitro by inducingG_1/S cell cycle arrest. Furthermore, it inhibited the proliferation of human T lymphocytes stimulated by phytohemagglutinin(PHA) at a minimum concentration of 0.1μg/ml. Our data suggest that lamprey BGSP-2 is able to block the mitosis of humanT lymphocytes at the G1/S point, and has the potential of anti-proliferative effect on PHA-activated T lymphocytes     

Immunosuppressive properties of cloned bone marrow mesenchymal stem cells

Mesenchymal stem cells(MSCs),derived from adult tissues,are multipotent progenitor cells,which hold greatpromise for regenerative medicine.Recent studies have shown that MSCs are immunosuppressive in vivo and in vitro inboth animals and humans.However,the mechanisms that govern these immune modulatory functions of MSCs remainlargely elusive.Some studies with bulk populations of MSCs indicated that soluble factors such as PGE2 and TGFβ areimportant,while others support a role for cell-cell contact.In this study,we intended to clarify these issues by examin-ing immunosuppressive effects of cloned MSCs.We derived MSC clones from mouse bone marrow and showed thatthe majority of these clones were able to differentiate into adipocytes and osteoblast-like cells.Importantly,cells fromthese clones exhibited strong inhibitory effects on TCR activation-induced T cell proliferation in vitro,and injection ofa small number of these cells promoted the survival of allogeneic skin grafts in mice.Conditioned medium from MSCcultures showed some inhibitory effect on anti-CD3 induced lymphocyte proliferation independent of PGE2 and TGFβ.In comparison,direct co-culture of MSCs with stimulated lymphocytes resulted in much stronger immunosuppressiveeffect.Interestingly,the suppression was bi-directional,as MSC proliferation was also reduced in the presence of lym-phocytes.Taking together,our findings with cloned MSCs demonstrate that these cells exert their immunosuppressiveeffects through both soluble factor(s)and cell-cell contact,and that lymphocytes and MSCs are mutually inhibitory ontheir respective proliferation.     

Dynamics of CD4^＋CD25^＋ T Cells in Spleens and Mesenteric Lymph Nodes of Mice Infected with Schistosomajaponicum

CD4~ CD25~ T cells play a major role in modulating immune response,but few reports havebeen published about schistosomiasis.Here,we investigated the changes in CD4~ CD25~ T cell populations inspleens and mesenteric lymph nodes of mice infected with Schistosoma japonicum.The proportions ofCD4~ CD25~ T cells in total CD4~ T cells were analyzed by flow cytometry.CD25 and Foxp3 expression wasmeasured by real-time quantitative polymerase chain reaction.The suppressive activities of CD4~ CD25~ Tcells were detected by in vitro proliferation of splenocytes.Evidence showed that the percentage of CD4~ CD25~ T cells was the same as controls 3 weeks post-infection.At the acute stage of infection,the percentagedecreased significantly.However,at the chronic stage of infection,it rebounded to normal levels or evenhigher.The expression of the CD25 and Foxp3 showed gradual increase along with the infection progress.Invitro experiment also showed the strong suppressive effect of CD4~ CD25~ T cells,isolated during the chronicstage,on proliferation of the CD25~-splenocytes.This is the first time that the dynamics of CD4~ CD25~ T cellpopulations was demonstrated in mice infected with schistosomiasis.In conclusion,our data indicated thatCD4~ CD25~ cells might be involved in the immune modulation during S.japonicum infection,which en-hances current knowledge of the mechanisms of the immuno-downregulation and re-infection inschistosomiasis.     

Growth induction of hepatic stimulator substance in hepatocytes through its regulation on EGF receptors

The cytosolic liver-specific growth factor-hepatic stimulator substance (HSS) has been shown to be able to amplify the rat hepatkocyte proliferation responded to BGF.In order to get more insight into the mechanism,the regulatory effect of HSS on EGF-receptor (EGF-R) and the receptor phosphorylation at molecular level was studied.HSS partially purified from weanling rat liver was given to cultured hepatocytes and its influence on EGF-R specific binding and internalization as well as mRNA expression were investigated.The results showed that preincubation of hepatocytes with HSS could lead to an increase in [^125I]-EGF binding to its receptors and inhibit EGF-induced receptor down-regulation.Furthermore,the overexpression of EGF-R mRNA stimulated by HSS was seen during 2-12 h after the incubation.Additionally,it was demonstrated with human hepatoma SMMC-7721 cells in Western blot that the EGF-R expression and the receptor autophosphorylation were increased with dose/timedependency after HSS treatment.These results strongly suggest that the mechanism of HSS action on hepatocyte growth might be related to its modulation on EGF-R and receptor-mediated signaling transfuction.     

Prostaglandin E2 modulates F-actin stress fiber in FSS-stimulated MC3T3-E1 cells in a PKA-dependent manner

The effect of prostaglandin E2（PGE2） on bone mass has been well-established in vivo. Previous studies have showed that PGE2 increases differentiation, proliferation, and regu- lates cell morphology through F-actin stress fiber in statically cultured osteoblasts. However, the effect of PGE2 on osteo- blasts in the presence of fluid shear stress （FSS）, which could better uncover the anabolic effect of PGEz in vivo, has yet to be examined. Here, we hypothesized that PGE2 modulates F-actin stress fiber in FSS-stimulated MC3T3-E1 osteoblastic cells through protein kinase A （PKA） pathway. Furthermore, this PGE2-induced F-actin remodeling was associated with the recovery of cellular mechanosensitivity. Our data showed that treatment with 10 nM dmPGE2 for 15 rain significantly suppressed the F-actin stress fiber intensity in FSS-stimulated cells in a PKA-dependent manner. In addition, dmPGE2 treatment enhanced the cells＇ calcium peak magnitude and the percentage of responding cells in the second FSS stimulation, though these effects were abolished and attenuated by co-treatment with phalloidin. Our results demonstrated that 10 nM dmPGE2 was able to accelerate the ＇reset＇ process of F-actin stress fiber to its pre-stimulated level partially through PKA pathway, and thus promoted the recovery of cellular mechanosensitivity. Our finding provided a novel cellular mechanism by which PGE2 increased bone forma- tion as shown in vivo, suggesting that PGE2 could be a potential target for treatments of bone formation-related diseases.     

Extracellular calmodulin: A polypeptide signal in plants?

Traditionally, calmodulin (CaM) was thought to be a multi-functional receptor for intra-cellular Ca2+ signals. But in the last ten years, it was found that CaM also exists and acts extracel-lularly in animal and plant cells to regulate many important physiological functions. Laboratory studies by the authors showed that extracellular CaM in plant cells can stimulate the proliferation of suspension cultured cell and protoplast; regulate pollen germination and pollen tube elongation, and stimulate the light-independent gene expression of Rubisco small subunit (rbcS). Furthermore, we defined the trans-membrane and intracellular signal transduction pathways for extracellular CaM by using a pollen system. The components in this pathway include heterotrimeric G-protein, phospholipase C, IP3, calcium signal and protein phosphorylation etc. Based on our findings, we suggest that extracellular CaM is a polypeptide signal in plants. This idea strongly argues against the traditional concept that there is no interce     

Phosphorylation of Microtubule-associated Protein SB401 from Solanum berthaultii Regulates Its Effect on Microtubules

We reported previously that the protein SB401 from Solanum berthaultii binds to and bundles both microtubules and F-actin. In the current study, we investigated the regulation of SB401 activity by its phosphorylation. Our experimental results showed that the phosphorylation of SB401 by casein kinase II （CKII） downregulates the activities of SB401, namely the bundling of microtubules and enhancement of the polymerization of tubulin. However, phosphorylation of SB401 had no observable effect on its bundling of F-actin. Further investigation using extract of potato pollen indicated that a CKIl-like kinase may exist in potato pollen. Antibodies against CKII alpha recognized specifically a major band from the pollen extract and the pollen extract was able to phosphorylate the SB401 protein in vitro. The CKIl-like kinase showed a similar ability to downregulate the bundling of microtubules. Our experiments demonstrated that phosphorylation plays an important role in the regulation of SB401 activity. We propose that this phosphorylation may regulate the effects of SB401 on microtubules and the actin cytoskeleton.     

Advanced glycation end products of bovine serum albumin affect the cell growth of human umbilical vein endothelial cells via modulation of MEG3/miR-93/p21 pathway

Advanced glycation end products of BSA (AGE-BSA) contribute to the pathogenesis of diabetic vascular diseases.However,the roles and underlying mechanisms of AGE-BSA in diabetic vascular diseases remain largely unclear.Long non-coding RNAs (lncRNAs) are widely identified and known as gene regulators.However,the roles of lncRNAs in diabetic vascular disease are still vague.In this study,we sought to investigate the contributions of lncRNAs in human umbilical vein endothelial cells (HUVECs) treated with AGE-BSA.We first demonstrated that AGE-BSA reduced the cell viability and inhibited the cell proliferation of HUVECs.Then,we found that lncRNA MEG3 was up-regulated in HUVECs treated with AGE-BSA.Furthermore,inhibition of MEG3 restored the AGE-BSA-induced repression of cell viability and proliferation.In addition,our results revealed that MEG3 played its role via modulation of miR-93 expression in HUVECs treated with AGE-BSA.Furthermore,we illustrated that miR-93 played its role via regulation of p21 in HUVECs treated with AGE-BSA.Ultimately,our study displayed that AGE-BSA exerted its function via modulation of MEG3/miR-93/p21 pathway in HUVECs.Thus,for the first time,we identified the MEG3/miR-93/p21 axis in HUVECs treated with AGE-BSA,which might be a novel regulatory network in diabetic vascular cells,and possess the potential therapeutic value for diabetes mellitus.