Fine-tuning cell organelle dynamics during mitosis by small GTPases / 医学前沿

During mitosis, the allocation of genetic material concurs with organelle transformation and distribution. The coordination of genetic material inheritance with organelle dynamics directs accurate mitotic progression, cell fate determination, and organismal homeostasis. Small GTPases belonging to the Ras superfamily regulate various cell organelles during division. Being the key regulators of membrane dynamics, the dysregulation of small GTPases is widely associated with cell organelle disruption in neoplastic and non-neoplastic diseases, such as cancer and Alzheimer's disease. Recent discoveries shed light on the molecular properties of small GTPases as sophisticated modulators of a remarkably complex and perfect adaptors for rapid structure reformation. This review collects current knowledge on small GTPases in the regulation of cell organelles during mitosis and highlights the mediator role of small GTPase in transducing cell cycle signaling to organelle dynamics during mitosis.

Therapeutic regulation of autophagy in hepatic metabolism

Metabolic homeostasis requires dynamic catabolic and anabolic processes. Autophagy, an intracellular lysosomal degradative pathway, can rewire cellular metabolism linking catabolic to anabolic processes and thus sustain homeostasis. This is especially relevant in the liver, a key metabolic organ that governs body energy metabolism. Autophagy's role in hepatic energy regulation has just begun to emerge and autophagy seems to have a much broader impact than what has been appreciated in the field. Though classically known for selective or bulk degradation of cellular components or energy-dense macromolecules, emerging evidence indicates autophagy selectively regulates various signaling proteins to directly impact the expression levels of metabolic enzymes or their upstream regulators. Hence, we review three specific mechanisms by which autophagy can regulate metabolism: A) nutrient regeneration, B) quality control of organelles, and C) signaling protein regulation. The plasticity of the autophagic function is unraveling a new therapeutic approach. Thus, we will also discuss the potential translation of promising preclinical data on autophagy modulation into therapeutic strategies that can be used in the clinic to treat common metabolic disorders.

RNA transport proteins and their expression and function in tumors / 医学研究生学报

Malignant tumors, whose occurrence and development are related to a variety of RNA transporter proteins, seriously affect human health and quality of life. Under normal circumstances, RNA transport proteins help RNA shuttle between nucleus and cytoplasm and their precise localization, effectively coupling the life activities in the nucleus and cytoplasm. During the process of tumorigenesis and progression, the expression and localization of some RNA transporters are abnormal or dysfunctional, which can change the subcellular localization, expression level, transport efficiency of downstream key RNA molecules, and the decay rate of cytoplasmic mRNA, and affect the proliferation, invasion and metastasis of tumors. This paper mainly reviews RNA transport proteins and their expression changes and regulation in tumors.

RNA transport proteins and their expression and function in tumors / 医学研究生学报

Malignant tumors, whose occurrence and development are related to a variety of RNA transporter proteins, seriously affect human health and quality of life. Under normal circumstances, RNA transport proteins help RNA shuttle between nucleus and cytoplasm and their precise localization, effectively coupling the life activities in the nucleus and cytoplasm. During the process of tumorigenesis and progression, the expression and localization of some RNA transporters are abnormal or dysfunctional, which can change the subcellular localization, expression level, transport efficiency of downstream key RNA molecules, and the decay rate of cytoplasmic mRNA, and affect the proliferation, invasion and metastasis of tumors. This paper mainly reviews RNA transport proteins and their expression changes and regulation in tumors.

Role of transient receptor potential cation channel subfamily M member 2 in hepatic ischemia-reperfusion injury in the mouse and the underlying mechanisms / 中南大学学报(医学版)

OBJECTIVES@#To investigate the role of transient receptor potential cation channel subfamily M member 2 (TRPM2) in hepatic ischemia-reperfusion injury of mouse (HIRI) and the possible mechanisms.@*METHODS@#Sixty adult male C57BL/6 mice were randomly divided into 4 groups: a sham group (S group), a HIRI model group (M group), a TRPM2 adenovirus interference vector group (T group), and a TRPM2 adenovirus control vector group (C group) (=15 in each group). The liver tissues of mice before perfusion were obtained. The efficiency of adenovirus infection was detected by fluorescence microscopy, and the silencing efficiency of adenovirus against TRPM2 was detected by real-time PCR.The abdominal aorta blood and liver tissues were collected from mice at 2, 4 and 8 h after reperfusion. The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum of mice were detected. Hepatic pathological changes were examined by hematoxylin-eosin (HE) staining. The protein expression of TRPM2 and Rac family small GTPase 1 (RAC1) in liver tissues was detected by Western blotting. Changes of malondialdehyde (MDA), superoxide dismutase (SOD) and myeloperoxidase (MPO) activities in liver tissues were detected by enzyme-linked immunosorbent assay.@*RESULTS@#A strong signal of green fluorescence was observed in the liver tissues of mice in the T and C groups compared to the S or M group. Compared with the S, M or C group, the expression of TRPM2 mRNA in liver tissue in the T group was significantly down-regulated (all <0.05). The morphology of hepatocytes was normal in the S group under light microscope.Hepatic sinus dilatation, congestion, hepatocyte degeneration, central necrosis of lobule, and massive inflammatory granulocyte infiltration were observed in the M and C group, respectively. The degree of hepatocyte damage in the T group was significantly reduced compared with that in the M and C group, respectively. Compared with the S group, the serum ALT and AST activities in the M, T and C groups were significantly increased at 2, 4 and 8 h after reperfusion (all <0.05). Compared with the M or C group, the serum ALT and AST activities in the T group were significantly lower in serum of mice at 2, 4, and 8 h after reperfusion (all <0.05). Compared with the M or C group, the serum SOD activity in the T group was significantly increased at 2, 4, and 8 h after reperfusion (all <0.05), while the serum MDA and MPO activities were significantly decreased (all <0.05). The protein expression of TRPM2 and RAC1 in liver tissues in the T group were significantly lower than those in the M and C groups at 2, 4 and 8 h after reperfusion (all <0.05).@*CONCLUSIONS@#Pretreatment with TRPM2 adenovirus interference vector can effectively silence TRPM2 gene expression in liver tissues of mice and attenuate HIRI, which may be related to inhibiting oxidative stress and reducing the expression of RAC1 protein.

Effect of electroacupuncture on muscular atrophy and Slit/Robo signaling in sciatic nerve and lumbar spinal cord tissues in rats with sciatic nerve injury / 针刺研究

OBJECTIVE: To observe the effect of electroacupuncture (EA) of "Huantiao"(GB30) and" Zusanli"(ST36)on muscular atrophy and expression of Slit-Robo GTPase-activating protein(srGAP)1, 2 and 3 in the injured sciatic nerve and lumbar spinal cord tissues in sciatic nerve injury (SNI) rats, so as to reveal its mechanisms underlying improvement of peripheral nerve injury (PNI)．. METHODS: A total of 120 healthy male SD rats were randomly divided into control, sham-operation, model and EA groups (n＝30 rats in each) which were further divided into 7, 15 and 23 d subgroups (n＝10 rats in each subgroup). The SNI model was established by transecting the right sciatic nerve beneath the piriformis and immediately subsequent end-to-end suture. Rats of the sham operation group received an incision of the corresponding skin and suture. EA (5 Hz/20 Hz, 2－3 mA) was applied to the right GB30 and ST36 for 15 min, once daily, 6 days a week separately for 1，2 and 3 weeks. Rats in the sham-operation and model groups were grasped in the similar procedure as the EA group. The wet weight of gastrocnemius muscles (WWG) on both sides was measured to calculate the recovery rate (weight of the right WWG/weight of the left WWG×100%), and the expression levels of srGAP1, srGAP2 and srGAP3 proteins in the sciatic nerve and the spinal cord (L4-L6) tissues were detected by Western blot. RESULTS: After modeling and compared with the control and sham-operation groups, the recovery rate of WWG was significantly reduced, and the expression levels of srGAP1, srGAP2 and srGAP3 proteins of the sciatic nerve and lumbar spinal cord on day 7, 15 and 23 were considerably increased in the model group (P<0.01). Following the EA treatment, the reco-very rate of WWG was obviously increased and the expression levels of srGAP1, srGAP2 and srGAP3 proteins of both sciatic nerve and spinal cord on day 7, 15 and 23 were further significantly up-regulated in the EA group relevant to the model group (P<0.05，P<0.01). In addition, the expression levels of the 3 proteins in both sciatic nerve and lumbar spinal cord peaked on day 15 and attenuated on day 23. CONCLUSION: EA of GB30 and ST36 may relieve gastrocnemius atrophy in SNI rats, which is related to its function in up-regulating the Slit/Robo signaling in the sciatic nerve and lumbar spinal cord to promote the axonal targeting regeneration and repair of axonal plasma nutrition transportation.

A β-catenin/IQGAP1 regulatory feedback loop and its effects on the proliferation of colon cancer cells / 生物医学工程学杂志

The aim of this article is to study the regulatory feedback loop between β-catenin and IQ motif containing GTPase activating protein 1 (IQGAP1), as well as the effect of this regulation loop in colon cancer cell proliferation. Western blot was used to detect the expression of IQGAP1 and β-catenin after changing their expression respectively by transfection in SW1116 cells. CCK-8 cell proliferation assay was used to detect the effect of IQGAP1 involved in the proliferation of SW1116 cells promoted by β-catenin. The results of Western blot indicated that β-catenin could positively regulate IQGAP1, while IQGAP1 silencing could up-regulate β-catenin, forming a negative feedback loop. The results of CCK-8 showed that IQGAP1 silencing inhibited β-catenin-mediated proliferation in SW1116 cells. In conclusion, our research reveals a negative regulatory feedback loop between β-catenin and IQGAP1 which has a remarkable effect on the proliferation ability of colon cancer cells.

Effect of left atrial enlargement on expression of the angiotensinⅡ, signal transducers and activators of transcription 3 and Rac GTPase activating protein 1 signaling pathways in patients with persistent atrial fibrillation and rheumatic heart disease / 中国胸心血管外科临床杂志

@#Objective To evaluate the effect of left atrial enlargement on atrial myocardial fibrosis degree and levels of the angiotensinⅡ (AngⅡ)/Rac GTPase activating protein 1 (Rac1)/signal transducersand activators of transcription 3 (STAT3) signaling pathways expressing in patients with persistent atrial fibrillation and rheumatic heart disease (RHD). Methods From March to December 2011, 30 patients with RHD who underwent prosthetic valve replacement in our hospital were enrolled, including 16 males and 14 females, aged 42-70 (56.9±6.8) years. Twenty RHD patients with persistent atrial fibrillation as a research group and ten RHD patients with sinus rhythm as a control group (group A) underwent transthoracic echocardiography and right atrial appendage (RAA) tissue samples were obtained from these patients during mitral/aortic valve replacement operation. The research group according to left atrial diameter (LAD) was divided into two groups, ten patients in each group: a group B with LAD of 50–65 mm and a group C with LAD of LAD>65 mm. For each sample, histological examination was performed by hematoxylin-eosin and Masson’s trichrome staining. Light-microscopic pictures of atrial tissues samples were stained and tissue fibrosis degree in each group was analyzed. AngⅡ concentration was measured by enzyme linked immunosorbent assay. Rac1 and STAT3 were measured by western blotting. Results LAD was significantly greater in AF patients with RHD than in the control group. Hematoxylin-eosin staining demonstrated highly organized arrangement of atrial muscles in the control group and significant derangement in both group B and group C with reduced cell density and increased cell size. Moreover, Masson’s trichrome staining showed that atrial myocytes were surrounded by large trunks of collagen fibers in both group B and group C, but not in the group A. There was a positive correlation between atrial tissue fibrosis and LAD. AngⅡ content was positively correlated with LAD. Similarly, Rac1 and STAT3 protein levels were found considerably higher in the group C and group B than in the group A with excellent correlation to LAD. Conclusion In patients with RHD complicated with persistent atrial fibrillation, the degree of atrial fibrosis and the expression level of AngⅡ/Rac1/STAT3 signaling pathways significantly increase with the left atrialenlargement.

Research progress of relevant signals and mechanisms in clearance of apopto-sis cells / 中国免疫学杂志

Our bodies produce a lot of apoptotic cells every day,and the timely removal of these apoptotic cells is essential to maintain the immune balance of the body. In the removal of apoptotic cells,macrophages play a major role,and their removal process is divided into three stages:recruitment,identification and phagocytosis. In the recruitment stage,apoptotic cells secrete′find me′signals, and phagocytes respond and are recruited to apoptotic cells. At the identification stage, the ′eat-me′ signal of apoptotic cells was identified with the phagocytic receptor on the surface of phagocytes. Phagocytic phase, the ′eat-me′ signal transmits signals to macrophages,such as activating the small GTPase Rac1,which leads to actin polymerization and cytoskeleton rearrangement to promote the phagocytosis of apoptotic cells. If the removal mechanism of apoptotic cells is obstructive,the apoptotic cells that have not been cleared in time will enter the secondary necrotic state and release the self-antigens. These self-antigens may stimulate the body′s immune system to produce autoantibodies,leading to autoimmune diseases such as SLE. The research progress of the macrophage on the removal mechanism of apoptosis cells is reviewed in this paper.

SASH1-IQGAP1-E-cadherin signal cascade may regulate breast cancer metastasis / 肿瘤

Objective: To explore the molecular mechanism of SAM-and SH3-domain containing 1 (SASH1) which serves as a novel tumor suppressor gene to regulate breast cancer metastasis. Methods: The expressions of SASH1, IQ motif-containing GTPase activating protein 1 (IQGAP1) and E-cadherin in breast cancer tissues were analyzed by immunohistochemistry (IHC). The correlation among SASH1, IQGAP1 and E-cadherin, as well as the association of SASH1 and IQGAP1 expressions with the clinical parameters of breast cancer patients were analyzed, respectively. The recombinant plasmids HAIQGAP1-pcDNA3.0 and pEGFP-C3-SASH1 were cloned and transfected into human embryonic kidney HEK-293T cells. The interaction of SASH1 and IQGAP1 was analyzed by immunoprecipitation-Western blotting. Results: In breast cancer tissues, there was a correlation between the expressions of SASH1 and IQGAP1 (P 0.05). The recombinant plasmids HAIQGAP1-pcDNA3.0 and pEGFP-C3-SASH1 were constructed successfully. After these recombinant plasmids were transfected into HEK-293T cells, the interaction between SASH1 and IQGAP1 was found. Conclusion: SASH1 interacts with IQGAP1, and which is closely related to the expression of E-cadherin. Therefore, it is suggested that SASH1 may form a new signaling cascade with IQGAP1 and E-cadherin to regulate breast cancer metastasis.

Role of immune-related GTPase M I in cortical neurons autophagy of mice with sepsis-induced brain injury / 中南大学学报(医学版)

Objective:To investigate the role of immune-related GTPase M1 (IRGM1) in cortical neurons autophagy in mice with sepsis induced brain injury (SIBI).Methods:Sixty wild-type C57BL/6 mice and sixty IRGM1 gene knockout C57BL/6 mice were randomly divided into 4 groups:a sham-operated wild-type (SWT) group,a cecal ligation and puncture (CLP) model wild-type (MWT) group,a sham-operated knockout (SKO) group,and a CLP model knockout (MKO) group.Models of mice with sepsis were established by CLP.Six hours of after CLP,the neurobehavioral scores for mice were recorded.The mice were diagnosed with SIBI and enrolled for the studies in next step if the neurobehavioral score was less than 6 in the MWT and MKO groups.The sham operation group only opened the abdominal cavity without CLP.Pathological changes in mouse cerebral cortex were observed by HE staining.Electron microscope was used to observe the ultrastructure of autophagy in cortical neurons.The expression of IRGM1 and INF-γ mRNA in the cerebral cortex of mice were detected by Real time quantitative PCR.The protein expression of microtubule-associated protein 1 light chain 3 (LC3)-Ⅱ,LC3-Ⅰ,sequestosome-1 (SQSTM1) and IRGM1 were measured by Western blot.Immunofluorescence staining was used to examine the expression of IRGM 1 in mouse cortical neurons.Results:In the MWT group,the cortical neurons showed dilated endoplasmic reticulum,swelling mitochondria,and increased number of autophagosomes after 6 or 24 h of CLP in contrast to the SWT group.At 6 h after CLP,the expression of LC3-Ⅱ in the cerebral cortex began to up-regulate,and the up-regulation was maintained till 96 h after CLP;on the contrary,SQSTM1 began to decline after 6 h of CLP.Compared with SWT group,IRGM1 was strongly up-regulated in the cerebral cortex of mice at both mRNA and protein levels in the MWT group after 12 h of CLP,and the mRNA expression of IFN-γ was also increased significantly (P＜0.05).At 24 h after CLP,the IRGM1 expression of cortical neurons in the MWT group was significantly higher than that in the SWT group.The baseline of autophagy activity was quite low in the cerebral cortex cells in the SWT and the SKO groups.There was almost no detected expression of LC3-Ⅱ;conversely,the expression of SQSTM1 was very high after 12 h of CLP.However,the expression of LC3-Ⅱ was significantly up-regulated and the expression of SQSTM1 was down-regulated in the MWT group (P＜0.05).On the other hand,there was almost no detected LC3-Ⅱ expression in cerebral cortex in the MKO group,and the expression of SQSTMI was up-regulated.At 6 h after CLP,the incidence of SIBI was 90％ (27/30) in the MWT group,and 96.67％ (29/30) in the MKO group.At 12 h of CLP,the neurobehavioral scores in the MKO group was significantly lower than that in the MWT group (4.97±0.71 vs 5.43±0.86;t=2.284,P=0.026).HE staining showed that mice in the MKO group suffered severe cerebral cortex injury,and the number of nerve cells was significantly reduced compared with that in the MWT group.Conclusion:The IRGM1 exerts a protective effect on the brain of the mice with SIBI,and its mechanism might be related to the regulation of autophagy in mouse cortical neurons.

Role of Rho GTPases in the immune regulation of infection and inflamma-tion / 中国血吸虫病防治杂志

The Rho subfamily of GTPase belongs to the Ras superfamily of small GTP binding protein,it is a nucleotide de-pendent protein,which plays a"molecular switch"function in the signal transduction process and control of numerous signaling pathways. Rho protein has many biological effects on cytoskeleton or target proteins as a signal converter in signal transduction , such as the regulation of membrane transport function,cell migration,cell adhesion,and cell proliferation. It also plays a very important role in the infection and immune inflammation of the body. Rho protein is widely distributed in related immune cells , such as T cells,B cells,NK cells and so on. When the body is infected by microorganism,the immune inflammatory reaction will be regulated through a series of signal transduction mechanism,and Rho GTPases signal transduction mechanism is one of the important signal pathways. In this paper,we conclude that Rho GTPases how to regulate the body's immune response through its signal pathway,and ultimately affect the body's immune response.

Species Specific Antiviral Activity of Porcine Interferon-α8 (IFNα8)

Interferons (IFNs) have been known as antiviral genes and they are classified by type 1, type 2, and type 3 IFN. The type 1 IFN consists of IFNα, IFNβ, IFNτ, and IFNω whereas the type 2 IFN consists of only IFNγ, which is a key cytokine driving T helper cell type 1 immunity. IFNλ belongs to the type 3 IFN, which is also known as IL-28 and IL-29 possessing antiviral activities. Type 1 IFN is produced by viral infection whereas type 2 IFN is induced by mitogenic or antigenic T-cell stimuli. The IFNτ of bovine was first discovered in an ungulate ruminant recognition hormone. IFNτ belongs to the type 1 IFN with the common feature of type 1 IFN such as antiviral activity. IFNs have been mostly studied for basic research and clinical usages therefore there was no effort to investigate IFNs in industrial animals. Here we cloned porcine IFNα8 from peripheral blood mononuclear cells of Korean domestic pig (Sus scrofa domestica). The newly cloned IFNα8 amino acid sequence from Korean domestic pig shares 98.4% identity with the known porcine IFNα8 in databank. The recombinant porcine IFNα8 showed potent antiviral activity and protected bovine Madin-Darby bovine kidney epithelial (MDBK) cells from the cytopathic effect of vesicular stomatitis virus, but it failed to protect human Wistar Institute Susan Hayflick (WISH) cells and canine Madin-Darby canine kidney epithelial-like (MDCK) cells. The present study demonstrates species specific antiviral activity of porcine IFNα8.

Relationship between methylation status of Rap1 GTPase activating protein and colon cancer / 肿瘤研究与临床

Objective To explore the methylation status of Rap1 GTPase activating protein (Rap1GAP) promoter in colon cancer, and to provide the oretical basis and research direction for the early diagnosis, targeted therapy, anti-multidrug resistance of colon cancer and so on. Methods The paraffin embedded specimens of 33 patients with colonic adenocarcinoma diagnosed by pathology were analyzed from Department of Pathology of Xinzhou City People′s Hospital from January 2010 to September 2014, including 19 males and 14 females, and aged 41-72 years old. The paraffin embedded specimens of 16 patients with colonic adenoma were enrolled, including 9 males and 7 females, and aged 34-58 years old. 13 normal tissues from the tumor distal margin (from the tumor > 15 cm) were selected. Quantitative methylation specific PCR (q-MSP) was applied to detect methylation level of Rap1GAP gene promoter. The methylation level differences of Rap1GAP gene promoter region among 3 groups or between different clinicopathologic factor subgroups were compared. Results The methylation rates [median (interquartile range)] of Rap1GAP promoter were 65.43 % (50.35 %), 21.37 % (8.39 %) and 17.43 % (15.71 %) in colonic adenocarcinoma group, colonic adenoma group and adjacent normal tissue group, respectively. The methylation rate of colonic adenocarcinoma group was significantly higher than that of colon adenoma group or that of adjacent normal tissue group (P60yearsold:36.26%(62.62%)and26.23%(76.42 %);well-differentiated vs. moderately/poorly-differentiated: 21.98 % (40.32 %) vs. 42.74 % (74.20 %); TNM Ⅰ-Ⅱ vsⅢ-Ⅳ: 25.31 % (48.27 %) vs. 36.26 % (75.55 %); all P> 0.05]. Conclusion The methylation status of RAP1GAP promoter maybe associate with genesis and development of colon cancer, which might be used as a target for early diagnose of colon cancer.

Research progress of Rho/ROCK signal pathway / 药学学报

Rho GTPases belong to Ras superfamily, which is reported to involve in cell migration, phagocytosis, contraction and adhesion. ROCK (also known as Rho-associated kinase) is considered to be one of the most important downstream targets of Rho that is widely investigated. Rho/ROCK signal pathway induces cytoskeletal reorganization, cell migration and stress fiber formation, affects endothelial permeability, tissue constriction and growth, involves in diabetic nephropathy, eye disease, cancer, heart disease, nerve injury disease, hypertension, radiation injury and leukemia. As a novel drug research target, Rho/ROCK signal pathway has received more and more attention. This review provides the basic characteristics and physiological effects of Rho/ROCK signal pathway, the relationships between Rho/ROCK signal pathway and diseases, and the therapeutic methods based on the Rho/ROCK signal pathway.

Correlation between δ-catenin and Cdc42 expression in non-small cell lung cancer / 中国癌症杂志

Background and purpose:δ-catenin is a member of the p120 catenin subfamily, which can directly bind to E-cadherin on the cell membrane, forming E-cadherin/catenin complex. δ-catenin can also affect the cytoskeleton assembly by regulating the activity of Cdc42 (Small GTPase). Therefore, this study detected the expression of δ-catenin and Cdc42 in non-small cell lung cancer (NSCLC) and investigated the relationship between them.Methods:The expressions of δ-catenin and Cdc42 in 122 cases of NSCLC were detected by immunohistochem-istry. This study also used Western blot and real-time lfuorescent quantitative polymerase chain reaction (RTFQ-PCR) to detect the protein and mRNA expressions of δ-catenin and Cdc42 in lung cancer tissues. After up-regulating or down-regulating δ-catenin in lung cancer cell line, the activity of Cdc42 and invasion ability of lung cancer cells were detect-ed by G-LISA and Transwell.Results:The mRNA and protein expression of δ-catenin and Cdc42 in lung cancer tissues was signiifcantly higher than that in normal lung tissues. In 122 NSCLC cases, the δ-catenin positive expression rate was 65.57% (80/122), and the Cdc42 overexpression rate was 68.03% (83/122). There was a good correlation betweenδ-catenin positive expression and Cdc42 overexpression (P<0.001). The co-expression of δ-catenin and Cdc42 was related to the high clinical stage, poor differentiation, adenocarcinoma and lymph node metastasis of lung cancer (P<0.05), and was signiifcantly associated with poor prognosis in patients with lung cancer. In the lung cancer cell line, the expression and the activity of Cdc42 were changed by regulating the δ-catenin expression, which affected invasion ability of the lung cancer cells.Conclusion:The δ-catenin expression was significantly correlated with the Cdc42 expression. The co-expression of δ-catenin and Cdc42 in lung cancer was correlated with the poor prognosis of lung cancer.

Effect of immunity-related GTPase Irgm1 gene knockout on CD4+T cell subsets in experimental autoimmune encephalomyelitis / 中国免疫学杂志

Objective: To investigate whether Irgm 1 impact CD4+T cell subsets in the experimental autoimmune encephalomyelitis.Methods: The Irgm1 heterozygous mice were backcrossed with C 57BL/6 Wt.mice for 10 generations to produce C57BL/6 Irgm1+/-mouse.C57BL/6 Irgm1+/-mice were intercrossed to obtain three genotypes:Irgm1-/-, Irgm1+/-and Irgm1+/+.To establish model of EAE ,C57BL/6 Wt.mice and Irgm1 knock out mice were immunized with myelin oligodendrocyte glycolprotein 33-55 ( MOG33-55 ) and the clinical symptoms were observed.The proliferation of lymphocytes to MOG antigen was detected with Methyl Thiazolyl Tetrazolium ( MTT).The infiltration of inflammatory cells in the spinal cords was observed through HE staining.The CD4+T cell subsets from lymph nodes ,spleens and CNS were detected by flow cytometry.Results:EAE model was induced successfully.The proliferation of T cells in lymph nodes in Irgm 1-/-mice was lower than Wt.mice.Quantitative analysis of flow cytometry indicates that , compared with Wt.mice,the level of Th1 subset was higher,Th17 was lower relatively in lymph nodes and CNS of Irgm1 knock out mice.Conclusion:Irgm1 knockout mice can be partially protected EAE spinal cord function and clinical symptoms .Irgm1 may play a key role at early stage of EAE ,which may use as an important molecular target for treatment of EAE.

Expression of Rap1 GTPase-activating protein 1, matrix metalloproteinase 2 and matrix metalloproteinase 9 in colorectal carcinoma and their significance / 肿瘤研究与临床

Objective To investigate the expression of Rap1 GTPase-activating protein 1 (Rap1GAP),matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9),and their relation with clinical patterns in colorectal carcinoma.Methods Immunohistochemistry was used to detect the expression of Rap1GAP,MMP-2 and MMP-9 in colorectal carcinoma,villous adenoma,tubular adenoma and normal colorectal tissue,and their relationship with clinicopathological parameters was analyzed.Results The positive rate of Rap1GAP expression was 30.4 ％ (14/46),77.8 ％ (14/18),69.6 ％ (16/23) and 95.2 ％ (20/21) in colorectal carcinoma,villous adenoma,tubular adenoma,and normal colorectal tissue,respectively (x2 =30.659,P=0.000).The figures were 71.7 ％ (33/46),55.6 ％ (10/18),52.2 ％ (12/16) and 9.5 ％ (2/21) for the positive rate of MMP-2 expression (x2 =22.459,P =0.000),as well as for 76.1％ (35/46),61.1％ (11/18),56.5 ％ (13/23) and 14.3 ％ (3/21) for the positive rate of MMP-9 expression,respectively (x2 =22.643,P =0.000).In patients with colorectal carcinoma,the expression of Rap1GAP was correlated with tumor differentiation (x2 =5.275,P =0.022),but not sex,age,or lymphatic metastasis (all P ＞ 0.05).The expression of MMP-2 and MMP-9 were correlated with lymphatic metastasis (x2 =6.661,P =0.010;x2 =8.475,P =0.040),but not sex,age or tumor differentiation(all P ＞ 0.05).There was a negative correlation between expression of Rap1GAP and MMP-2,MMP-9 in colorectal carcinoma,respectively (r =-0.424,P =0.003;r =-0.294,P =0.048),but no correlation between the expression of MMP-2 and MMP-9 (r =0.101,P =0.505).Conclusions Rap1GAP,MMP-2 and MMP-9 play important roles in the malignant biological behavior of colorectal carcinoma,and the expression of Rap1GAP is negatively correlated with MMP-2 and MMP-9.The interactions among the three affect the occurrence and development of colorectal carcinoma.

O papel de RhoA e Rac1 GTPases nas respostas celulares após danos no DNA induzidos por radiação ionizante gama / The role of RhoA and Rac1 GTPases in cellular responses after DNA damage induced by ionizing gamma radiation

O mecanismo pelo qual uma célula responde a algum dano no seu material genético é extremamente importante. Isto ocorre pela rápida ativação da maquinaria de reparo de danos no DNA, a qual é composta por uma rede intrincada de sinalização proteica, culminando no reparo do DNA; porém se o dano for irreparável ocorre ativação de mecanismos de morte celular. RhoA,e Rac1 pertencem a família das pequenas proteínas sinalizadoras Rho GTPases, as quais atuam como interruptores moleculares ciclando entre estado ativo (ligada a GTP) e inativo (ligada a GDP). Os componentes desta família estão relacionados ao controle dos mais diversos processos celulares como, por exemplo, remodelamento do citoesqueleto, migração, adesão, endocitose, progressão do ciclo celular e oncogênese. No entanto, apesar das proteínas Rho GTPases estarem envolvidas em um amplo espectro de atividades biológicas, há poucas informações sobre seu papel na manutenção da integridade genômica quando células são submetidas a algum agente genotóxico. Para investigar o envolvimento das GTPases RhoA e Rac1 nas respostas de células submetidas a radiação gama, foram gerados, a partir de células de carcinoma de cervix humano - HeLa, sublinhagens clonais mutantes de RhoA e Rac1 expressando exogenamente RhoA constitutivamente ativa (HeLa-RhoA V14), RhoA dominante negativa (HeLa-RhoA N19), Rac1 constitutivamente ativa (HeLa-Rac1 V12) e Rac1 dominante negativa (HeLa-Rac N17). Após estas linhagens celulares serem expostas a diferentes doses de radiação gama, observamos que ambas GTPases, RhoA e Rac1, são ativadas em resposta aos efeitos da radiação. Além disso, a modulação da atividade destas enzimas, através das mutações, levou a uma alteração das respostas celulares frente aos danos no DNA, como uma redução da capacidade de reparar quebras simples e duplas nas fitas do DNA. Por outro lado, a deficiência de RhoA ou Rac1 GTPase levou a uma redução da ativação de Chk1 e Chk2 ou da fosforilação da histona H2AX, respectivamente, prejudicando os mecanismos de detecção de danos no DNA e levando as células a permanecerem mais tempo nos pontos de checagem G1/S e/ou G2/M do ciclo celular. Esses fatores contribuíram de modo expressivo para a redução da proliferação e sobrevivência celular levando as células à morte. Por fim, ensaios celulares de reparo de danos de um DNA exógeno através de mecanismos de Recombinação Homóloga (HR) e Recombinação Não-Homóloga de extremidades (NHEJ), demonstraram que a inibição da atividade de RhoA reduz significativamente a eficiência de ambas vias de reparo. Desta maneira, este trabalho demonstra e reforça a existência de mais um viés de atuação das pequenas GTPases RhoA e Rac1, agora em células HeLa, nas respostas celulares aos danos induzidos por exposição a radiação gama, modulando a sobrevivência, proliferação e indiretamente modulando resposta ao reparo do DNA através da via de Recombinação Homóloga e Não-Homóloga

The mechanism by which a cell responds to DNA damage is extremely important. This occurs by a quick activation of the DNA damage repair machinery, which consists of an intricate protein signaling network culminating in DNA repair. But if the damages are irreparable occurs there is activation of cell death mechanisms. RhoA and Rac1 belong to family of small Rho GTPases, signaling proteins that act as molecular switches cycling between the active state (GTP-bound) and inactive state (GDP-bound). Members of this family are implicated in the control of diverse cellular process such as cytoskeletal remodeling, migration, adhesion, endocytosis, cell cycle progression, and oncogenesis. However, despite Rho proteins are involved in a broad spectrum of biological activities, there is just a few information about their roles in the maintenance of genomic integrity, that is, when the cells are subjected to some kinf of genotoxic agent. To investigate the involvement of the GTPases RhoA and Rac1 in cellular responses to gamma radiation, we generated from human cervix carcinoma cells - HeLa, clonal sublines of RhoA and Rac1 mutants, exogenous and stably expressing the constitutively active RhoA (HeLa-RhoA V14), the dominant negative RhoA (HeLa-RhoA N19), the constitutively active Rac1 (HeLa-Rac1 V12) and the dominant negative Rac1 (HeLa-Rac1 N17). After all these cell lines have been exposed to different doses of gamma radiation, we found that both GTPases, RhoA and Rac1, are activated in response to the radiation effects. Furthermore, the modulation of two enzymes activity, by using the mutant clones, led to a change in cellular responses to the DNA damage, as the reduction in the capacity of repairing DNA single and double strand breaksr. On the other hand, the deficiency of RhoA or Rac1 GTPase led to a reduction of Chk1 and Chk2 activation, or on the phosphorylation of histone H2AX, respectively, hindering the mechanisms of DNA damage detection and arresting cells in the G1/S and/or G2/M checkpoints of cell cycle. These factors significantly contributed to the reduction of cell proliferation and survival, leading cells to death. Finally, cellular assays of DNA damage repair of exogenous DNA by Homologous Recombination (HR) and Non-Homologous End Joining (NHEJ), demonstrated that RhoA inhibition significantly reduced the repair efficiency of both pathways. Thus, this work demonstrates and reinforces the existence of other biological functions of small GTPases RhoA and Rac1 in HeLa cells, by regulating cellular responses to DNA damage induced by exposure to gamma radiation, modulating the survival, proliferation and indirectly modulating the response to DNA damage repair pathway through the Homologous Recombination and Non-Homologous Recombination

Modulation of phagolysosome maturation by bacterial tlyA gene product.

The pathogenic traits of TlyA proteins of Mycobacterium tuberculosis are not known. Expressions of TlyA in bacteria that do not express endogenous TlyA adhere better to RAW264.7 macrophages and get phagocytosed efficiently. The internalized bacteria avoid acidification to the extent of >65% in the case of both TlyA-expressing E. coli and M. smegmatis. Consistent with this observation, we have observed decreased co-localizaton of Lysosomal Membrane Associated Protein-1 (~35%), Early Endosomal Antigen-1 (~34%), Rab5 (~30%) and Rab7 (~35%) and enhanced colocalizaton of Rab14 (~80%) on both TlyA-expressing bacteria as well as on TlyA-coated latex beads. These results suggest that the mycobacterial TlyA, in general, can modulate phagolysosome maturation pathway immediately after entry into macrophages, while other important molecules may aid the bacterium for long-term, intracellular survival at later point of time.