OBJECTIVETo investigate the expression of RhoGDIalpha in human testes and spermatozoa, and compare the expression of RhoGDIalpha in the ejaculated spermatozoa from normozoospermic me and infertile patients men receiving in vitro fertilization (IVF).

METHODSThe localization of RhoGDIalpha in the human testis was determined by immunohistochemistry, and that in the pre-capacitated, capacitated and acrosome-reacted sperm by immunofluorescence. Western blot was used to detect the expression of RhoGDIalpha in the semen samples obtained from normozoospermic males (n = 10), IVF patients with high fertilization rates (> or = 60%, n = 12) and those with low fertilization rates (< 60%, n = 13).

RESULTSImmunohistochemistry showed that the RhoGDIalpha protein was located in all spermatogenic cells and highly expressed in the elongated spermatids. Immunofluorescence exhibited a high expression of RhoGDIalpha in the acrosome and flagellum of human sperm, which decreased in the acrosome after capacitation and disappeared after acrosome reaction. Western blot revealed an obviously decreased expression of RhoGDIalpha in the spermatozoa of the IVF patients with low fertilization rates (0.66 +/- 0.18), with statistically significant difference from those with high fertilization rates (0.97 +/- 0.17) and the normozoospermic men (1.13 +/- 0.21).

CONCLUSIONThe RhoGDIalpha protein is located in the acrosome and flagellum of human sperm, and might be involved in sperm movement, capacitation and acrosome reaction. The significantly reduced expression of RhoGDIalpha in the sperm of low-fertilization patients suggests that it may be a new diagnostic biomarker for male infertility, and has a potential application value in sperm selection for IVF.

Fertilization in Vitro ; Guanine Nucleotide Dissociation Inhibitors ; metabolism ; Humans ; Infertility, Male ; Male ; Sperm Motility ; Spermatozoa ; metabolism ; Testis ; metabolism ; rho Guanine Nucleotide Dissociation Inhibitor alpha ; rho-Specific Guanine Nucleotide Dissociation Inhibitors

OBJECTIVETo investigate the expression of Rho-GDI in the decidual tissues of patients preeclampsia and explore its clinical implication.

METHODSReal-time PCR, Western blotting and immunohistochemistry were used to detect the mRNA and protein expressions of Rho-GDI in the decidual tissues from 30 normal women with full-term pregnancy, 30 patients with early-onset severe preeclampsia and 30 with late-onset severe preeclampsia.

RESULTSRho-GDI expression was found mainly on the cell membrane and in the cytoplasm and nuclei of the decidual cells, occasionally occurring in the stroma. Both the mRNA and protein expressions of Rho-GDI in the decidual tissues were significantly higher in the normal pregnancy group than in the two severe preeclampsia groups (P<0.05), and the patients with late-onset severe preeclampsia had the lowest expressions of Rho-GDI.

CONCLUSIONThe lowered expression of Rho-GDI in the deciduas might be involved in the pathogenesis and progression of preeclampsia.

Adult ; Decidua ; metabolism ; Female ; Guanine Nucleotide Dissociation Inhibitors ; genetics ; metabolism ; Humans ; Pre-Eclampsia ; metabolism ; Pregnancy ; RNA, Messenger ; genetics ; metabolism ; Real-Time Polymerase Chain Reaction ; methods ; Young Adult ; rho-Specific Guanine Nucleotide Dissociation Inhibitors

Annexin A3 ; genetics ; metabolism ; Cells, Cultured ; Guanine Nucleotide Dissociation Inhibitors ; genetics ; metabolism ; Hepatocytes ; drug effects ; metabolism ; Humans ; Lactoylglutathione Lyase ; genetics ; metabolism ; RNA, Messenger ; genetics ; Trichloroethylene ; toxicity ; rho-Specific Guanine Nucleotide Dissociation Inhibitors

BACKGROUNDBeta(2)-adrenoceptor (beta(2)AR) desensitization is a common problem in clinical practice. beta(2)AR desensitization proceeds by at least such three mechanisms as heterologous desensitization, homologous desensitization and a kind of agonist-induced rapid phosphorylation by a variety of serine/threonine kinases. It is not clear whether there are other mechanisms. This study aimed to investigate potential mechanisms of beta(2)AR desensitization.

METHODSTwenty-four BALB/c (6-8 weeks old) mice were divided into three groups, which is, group A, phosphate buffered saline (PBS)-treated; group B, ovalbumin (OVA)-induced; and group C, salbutamol-treated. Inflammatory cell counts, cytokine concentrations of bronchoalveolar lavage fluid (BALF), pathological sections, total serum IgE, airway responsiveness, membrane receptor numbers and total amount of beta(2)AR were observed. Asthmatic mouse model and beta(2)AR desensitization asthmatic mouse model were established. Groups B and C were selected for two-dimensional gel electrophoresis (2DE) analysis so as to find key protein spots related to beta(2)AR desensitization.

RESULTSAsthmatic mouse model and beta(2)AR desensitization asthmatic mouse model were verified by inflammatory cell count, cytokine concentration of BALF, serum IgE level, airway hyperreactivity measurement, radioligand receptor binding assay, Western blot analysis, and pathologic examination. Then the two groups (groups B and C) were subjected to 2DE. Two key protein spots associated with beta(2)AR desensitization, Rho GDP-dissociation inhibitor 2 (RhoGDI(2)) and peroxiredoxin 5, were found by comparative proteomics (2DE and mass spectrum analysis).

CONCLUSIONOxidative stress and small G protein regulators may play an important role in the process of beta(2)AR desensitization.

Albuterol ; therapeutic use ; Animals ; Asthma ; drug therapy ; metabolism ; Disease Models, Animal ; Electrophoresis, Gel, Two-Dimensional ; Female ; Guanine Nucleotide Dissociation Inhibitors ; analysis ; Lung ; chemistry ; pathology ; Mice ; Mice, Inbred BALB C ; Oxidative Stress ; Peroxiredoxins ; analysis ; Proteomics ; Receptors, Adrenergic, beta-2 ; physiology ; rho-Specific Guanine Nucleotide Dissociation Inhibitors

PURPOSE: The mechanism including changes of proteome within cavernosal tissue after cavernous nerve injury were not evaluated. We performed proteomics and functional analysis to identify proteins of penile corpus cavernosum whose expression was or was not altered by cavernous nerve resection (CNR). MATERIALS AND METHODS: Using 8-week-old male WKY rats, sham and CNR operation under microscope were performed. After 8 weeks, penile tissues of sham and CNR group were harvested. We used 2-DE and MALDI-TOF/TOF (AB 4700) to identify of differently expressed penile proteins. 2-DE gels were stained with silver nitrate and the gels were analyzed with PDQuest. RESULTS: We isolated more than 950 proteins on silver-stained gels of whole protein extracts from normal rat penile corpus cavernous. Of these proteins, 48 prominent proteins were identified using MALDI-TOF/TOF. Protein characterization revealed that the most prominent penile corpus cavernous proteins were those with antioxidant, chaperone, or cytoskeletal structure. Moreover, 11 proteins having levels elevated by CNR were annexin proteins, endoplasmic reticulum protein 29, glutathione s-transferase w-1, and others. In addition, Rho-GDP dissociation inhibitor (RhoGDI), a regulator of Rho proteins, was also increased in CNR rats compared with sham-operated control rats. CONCLUSIONS: The apoptotic signals observed in penile tissues was greatly increased in CNR rats than in sham-operated rats. These results suggest that RhoGDI is one of the proteins regulated by CNR in penile smooth muscle strips, and has a crucial role in the early stage of penile apoptosis.
Animals ; Apoptosis ; Caves ; Dissociative Disorders ; Endoplasmic Reticulum ; Erectile Dysfunction ; Gels ; Glutathione Transferase ; Humans ; Male ; Muscle, Smooth ; Proteins ; Proteome ; Proteomics ; Rats ; Rats, Inbred WKY ; rho-Specific Guanine Nucleotide Dissociation Inhibitors ; Salicylamides ; Silver Nitrate

OBJECTIVE: To evaluate and compare protein expression profiles of synovial fibroblasts using proteome analysis in swine knee injuries with joint instability, during early post-traumatic arthritis (PTA) development. METHOD: Experimental PTA was induced by transection of the anterior cruciate ligament (ACL) in swine left knee joints. After sacrifice at 8 weeks, cartilage and synovium obtained from both knee joints were prepared for histopathologic examination. Cultured synovial fibroblasts were processed for 2-dimensional electrophoresis and mass spectrometric analysis. Histopathologic examination showed overt arthritic changes that supported the development of early PTA. RESULTS: Proteome analyses led to the identification of more than 1,500 protein spots and of 11 differently expressed protein spots. Of those, six proteins were down-regulated (cytoskeletal beta actin, cofilin-1, destrin, Rho GDP dissociation inhibitor alpha, and unnamed protein product), and five proteins were up-regulated (alpha-B crystallin, smooth muscle protein 22-alpha, and cytoskeletal beta actin) in ACL-transected synovial fibroblasts. That is, proteins related to cellular organization and signal transduction are down-regulated, and those related to cell rescue, defence, and stress are up-regulated. CONCLUSION: These results may suggest that joint instability contributes to the development of PTA and is one of the major etiologic factors of PTA. In addition, this suggests that the proteome analysis of synovial fibroblasts is a useful approach in examining a joint after an injury and can be used to understand the pathogenesis of PTA.
Actins ; Anterior Cruciate Ligament ; Arthritis* ; Cartilage ; Crystallins ; Destrin ; Electrophoresis ; Fibroblasts* ; Guanine Nucleotide Dissociation Inhibitors ; Joint Instability ; Joints ; Knee Injuries ; Knee Joint ; Muscle, Smooth ; Proteome ; Signal Transduction ; Swine ; Synovial Membrane

The release of neurotransmitter is regulated in the processes of membrane docking and membrane fusion between synaptic vesicles and presynaptic plasma membranes. Synaptic vesicles contain a diverse set of proteins that participate in these processes. Small GTP-binding proteins exist in the synaptic vesicles and are suggested to play roles for the regulation of neurotransmitter release. We have examined a possible role of GTP-binding proteins in the regulation of protein phosphorylation in the synaptic vesicles. GTPgammaS stimulated the phosphorylation of 46 kappa Da protein (p46) with pI value of 5.0-5.2, but GDPbetaS did not. The p46 was identified as protein interacting with C-kinase 1 (PICK-1) by MALDI-TOF mass spectroscopy analysis, and anti-PICK-1 antibody recognized the p46 spot on 2-dimensional gel electrophoresis. Rab guanine nucleotide dissociation inhibitor (RabGDI), which dissociates Rab proteins from SVs, did not affect phosphorylation of p46. Ca2+/ calmodulin (CaM), which causes the small GTP- binding proteins like Rab3A and RalA to dissociate from the membranes and stimulates CaM- dependnet protein kinase(s) and phosphatase, strongly stimulate the phosphorylation of p46 in the presence of cyclosporin A and cyclophylin. However, RhoGDI, which dissociates Rho proteins from membranes, reduced the phosphorylation of p46 to the extent of about 50%. These results support that p46 was PICK-1, and its phosphorylation was stimulated by GTP and Ca2+/CaM directly or indirectly through GTP-binding protein(s) and Ca2+/CaM effector protein(s). The phosphorylation of p46 (PICK-1) by GTP and Ca2+/CaM may be important for the regulation of transporters and neurosecretion.
Animals ; Calcium/*metabolism ; Calmodulin/*metabolism ; Carrier Proteins/*chemistry/*metabolism ; Guanine Nucleotide Dissociation Inhibitors/metabolism ; Guanosine Triphosphate/metabolism/*pharmacology ; Molecular Weight ; Monomeric GTP-Binding Proteins/metabolism ; Phosphorylation/drug effects ; Rats ; Recombinant Fusion Proteins/*chemistry/*metabolism ; Synaptic Membranes/chemistry/drug effects/*metabolism ; Synaptic Vesicles/chemistry/drug effects/*metabolism

OsRhoGDI2 was isolated as a putative partner of Rho protein family member OsRacD from rice panicles by yeast two-hybrid, but its function remains unknown. In order to identify the function of OsRhoGDI2, OsRhoGDI2 knockout mutants were created by CRISPR/Cas9 technology. The results showed that two different homozygous mutants were obtained in T0 generation, and eight kinds homozygous mutants were identified in T1 generation. Sequence analysis revealed that the base substitution or base deletion occurred near the editing targets of the gene in knockout rice, and it could be expected that the truncated OsRhoGDI2 proteins lacking the RhoGDI conserved domain would be generated. Phenotype analysis showed that the OsRhoGDI2 knockout rice plants were significantly lower than the control plants. Statistical analysis confirmed that the significant decrease of plant height was due to the shortening of the second and third internodes, suggesting that OsRhoGDI2 gene may be related with rice height control.
CRISPR-Cas Systems ; Genes, Plant ; genetics ; Oryza ; genetics ; growth & development ; Plants, Genetically Modified ; rho Guanine Nucleotide Dissociation Inhibitor beta ; genetics

PURPOSE: Recent research has identified many genes and proteins that play specific roles in the process of systemic metastasis in various types of cancer. Rho GDP dissociation inhibitor 2 (RhoGDI2) has been shown to inhibit metastasis in human bladder cancer, but its role in breast cancer is controversial. MATERIALS AND METHODS: We examined the regulation and clinical significance of RhoGDI2 in Korean breast cancer patients by using proteomic approaches. RESULTS: By using a proteomic approach, we observed an increased expression of RhoGDI2 in human breast cancer tissues when compared to that of the normal breast tissues, and we validated its up-regulation in an independent cohort of 8 breast cancer patients. The clinical implication of a RhoGDI2 expression was investigated in 57 breast cancer patients by performing immunohistochemistry. RhoGDI2 did not show a significant association with the tumor size, lymph node metastasis, the histologic grade or the hormone receptor status. However, the patients with RhoGDI2-expressing tumors had significantly shorter disease-free survival (p=0.043; hazard ratio, 3.87) and distant metastasis-free survival (p=0.039; hazard ratio, 5.15). CONCLUSION: Our results demonstrated a potential role of RhoGDI2 as a poor prognostic marker as well as a potential therapeutic target. The pro-metastatic nature of RhoGDI2 shown in our study may indicate its organ-specific role in cancer metastasis.
Breast ; Breast Neoplasms ; Cohort Studies ; Disease-Free Survival ; Humans ; Immunohistochemistry ; Lymph Nodes ; Neoplasm Metastasis ; Prognosis ; Proteins ; Proteomics ; rho Guanine Nucleotide Dissociation Inhibitor beta ; Up-Regulation ; Urinary Bladder Neoplasms

OBJECTIVETo identify novel multi-drug resistance-related genes, and to explore the mechanisms of multi-drug resistance.

METHODSMulti-drug resistant cell line Lovo/5-FU was established by incubation with increasing dose of 5-FU. The sensitivity to 5-FU and cis-diaminodichloroplatinum (CDDP) was measured by MTT assay. Two dimensional electrophoresis plus mass spectrum(2-DE/MS) was used to identify the differentially expressed protein between Lovo and Lovo/5-FU. The identified protein was then verified by Western blot analysis.

RESULTSThe IC50 concentrations of Lovo/5-FU to 5-FU and CDDP were increased by 31 and 3 times, compared with Lovo (both P<0.01). 2DE-MS showed that CAP-G and RhoGDI2 were up-regulated, whereas 6-PGL, DCI, Prdx-6 and Maspin were down-regulated in Lovo/5-FU. Western blot analysis confirmed that the expression levels of RhoGDI2 and CAP-G in Lovo/5-FU were increased by 6.14 and 2.98 fold respectively (both P<0.01), whereas Maspin was decreased to 5.2% of Lovo(P<0.01).

CONCLUSIONSMulti-gene and multi-pathway are involved in the development of multi-drug resistance of colorectal cancer cells. CAP-G, RhoGDI2 and Maspin are potential multi-drug resistant genes.

Cell Line, Tumor ; Colonic Neoplasms ; genetics ; Drug Resistance, Multiple ; genetics ; Drug Resistance, Neoplasm ; genetics ; Humans ; Microfilament Proteins ; genetics ; Nuclear Proteins ; genetics ; Serpins ; genetics ; rho Guanine Nucleotide Dissociation Inhibitor beta ; genetics