Expression and role of PAK6 after spinal cord injury in adult rat / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To observe p21-activated kinase 6 (PAK6) expression and its possible role after spinal cord injury (SCI) in adult rat.</p><p><b>METHODS</b>Sprague-Dawley rats were subjected to spinal cord injury. To explore the pathological and physiological significance of PAK6, the expression patterns and distribution of PAK6 were observed by Western blot, immunohistochemistry and immunofluorescence.</p><p><b>RESULTS</b>Western blot analysis showed PAK6 protein level was significantly up-regulated on day 2 and day 4, then reduced and had no up-regulation till day 14. Immunohistochemistry analysis showed that the expression of PAK6 was significantly increased on day 4 compared with the control group. Besides, double immunofluorescence staining showed PAK6 was primarily expressed in the neurons and astrocytes in the control group. While after injury, the expression of PAK6 was increased significantly in the astrocytes and neurons, and the astrocytes were largely proliferated. We also examined the expression of proliferating cell nuclear antigen (PCNA) and found its change was correlated with the expression of PAK6. Importantly, double immunofluorescence staining revealed that cell proliferation evaluated by PCNA appeared in many PAK6-expressing cells on day 4 after injury.</p><p><b>CONCLUSION</b>The up-regulation of PAK6 in the injured spinal cord may be associated with glial proliferation.</p>

Expression of β-1,4-galactosyltransferase I in a surgically-induced rat model of knee osteoarthritic synovitis / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>There are few reports of a biological role for glycosyltransferases in the infiltration of osteoarthritic synovitis. The aim of this research was to investigate the expression and cellular location of β-1,4-galactosyltransferase I (β-1,4-GalT-I) in a surgically-induced rat model of knee osteoarthritis (OA), and explore the role of β-1,4-GalT-I in the pathogenesis of OA.</p><p><b>METHODS</b>Male Sprague-Dawley rats were randomly divided into three groups: OA group, sham group and normal group. The model of OA was established in the right knees of rats by anterior cruciate ligament transaction (ACLT) with partial medial meniscectomy. Fibroblast-like synoviocytes (FLSs) obtained from normal rat synovial tissue were cultured. The expression of β-1,4-GalT-I mRNA in the synovial tissue, articular cartilage and FLSs treated with tumor necrosis factor-α (TNF-α) were assayed by real-time PCR. Western-blotting and immunohistochemisty were used to observe the expression of β-1,4-GalT-I at the protein level. Double immunofluorescent staining was used to define the location of the β-1,4-GalT-I with macrophage-like synoviocytes, FLSs, neutrophils, and TNF-α in the OA synovium. The alteration of TNF-α in FLSs which were treated with lipopolysaccharide (LPS) and β-1,4-GalT-I-Ab were detected by enzyme-linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>The mRNA and protein expression of β-1,4-GalT-I increased in synovial tissue of the OA group compared with the normal and sham groups at two and four weeks after the surgery, however, no significant difference appeared in the articular cartilage. Immunohistochemistry also indicated that the β-1,4-GalT-I expression in OA synovium at four weeks after surgery increased sharply compared with the control group. β-1,4-GalT-I co-localized with macrophage-like synoviocytes, FLSs, neutrophils and TNF-α in rat OA synovitis. Moreover, in vitro β-1,4-GalT-I mRNA in FLSs was affected in a dose- and time-dependent manner in response to TNF-α stimulation. ELISA revealed that the expression of TNF-α was attenuated in FLSs in vitro when treated with anti β-1,4-GalT-I antibody.</p><p><b>CONCLUSION</b>β-1,4-GalT-I may play an important role in the inflammation process of rat OA synovial tissue which would provide the foundation for further researching into the concrete mechanism of β-1,4-GalT-I in OA synovitis.</p>

Effect of atorvastatin on ACE2 expression in pressure overload induced cardiac hypertrophy in rats / 中南大学学报(医学版)

OBJECTIVE@#To investigate the effect of atorvastatin on the expression of angiotensin converting enzyme 2 (ACE2) mRNA and its protein in hypertrophic myocardium in rats.@*METHODS@#Suprarenal abdominal aortic coarctation was performed to create the pressure overload induced left ventricular hypertrophy model in rats.@*RESULTS@#Rats were randomly divided into 5 groups: (1) normal control group (Group A); (2) normal control group treated with atorvastatin [(5 mg/(kg.dd), Group B]; (3) sham group (Group C); (4) atorvastatin given orally by gastric gavage for 4 weeks [5 mg/(kg.dd),Group D]; (5) vehicle group (Group E). Stained pathological section was observed under light microscope to measure cardiomyocyte diameter transversa and collagen volume fraction. ACE2 mRNA and its protein expression were detected by real-time RT-PCR and Western blot. Compared with Group A,B, and C, the left ventricular mass index, cardiomyocyte diameter transversa and collagen volume fraction in Group E increased statistically (P< 0.01), ACE2 mRNA and its protein expression also elevated remarkably (P< 0.01). Compared with Group E, the above mentioned indexes in Group D reduced significantly (P< 0.01).@*CONCLUSION@#ACE2 mRNA and its protein expression increase significantly in hypertrophic myocardium in rats; atorvastatin can attenuate cardiac hypertrophy due to pressure overload in rats effectively, and part of this anti-hypertrophy effect may be attributed to decrease ACE2 mRNA and protein expression.

Expression variation and significance of Skp2 and p27(kip1) during the proliferation of Jurkat cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the expression variation and significance of Skp2 and p27(kip1) during the proliferation of lymphoma cell line Jurkat cells.</p><p><b>METHODS</b>The binding of p27(kip1) and Skp2 in Jurkat cells were detected by immunoprecipitation. Jurkat cells were treated with serum starvation and release synchronization. The expression variation and subcellular localization of p27(kip1) and Skp2 were detected by subcellular fractionation, Western blot and double immunofluorescence labelling.</p><p><b>RESULTS</b>The results of immunoprecipitation suggested that p27(kip1) and Skp2 could bind each other in Jurkat cells. During the proliferation of Jurkat cells, the protein expression of p27(kip1) decreased and intranuclear p27(kip1) decreased significantly, while the Skp2 protein increased and cytoplasmic Skp2 increased significantly.</p><p><b>CONCLUSION</b>During the proliferation of Jurkat cells, the increased cytoplasmic synthesis of Skp2 may speed up p27(kip1) degradation via the ubiquitin-proteasome pathway, then intranuclear p27(kip1) decreases significantly, leading to an increased cell cycling activity.</p>

Growth inhibition of human leukemia cell line U937 by all-trans retinoic acid and its mechanism / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the effect of all-trans retinoic acid (ATRA) on U937 cell growth and its mechanism.</p><p><b>METHODS</b>Cell cycle was detected by flow cytometry (FCM), expressions of cell cycle associated protein and the p27 related protein were detected by Western blot. The binding of P27 and Skp2 was detected by immunoprecipitation.</p><p><b>RESULTS</b>FCM displayed that ATRA could inhibit the proliferation of U937 cells. At 72 h on 1 micromol/L ATRA treatment, 72% of the cells were arrested at G0/G1 phase. Western blot displayed that ATRA could decrease the expression of cyclin A, up-regulate the expression of p21 and p27, and down-regulate the expression of p27 related proteins Skp2. p27 could bind with Skp2 in U937 cells as detected by immunoprecipitation.</p><p><b>CONCLUSION</b>ATRA may arrest the proliferation of U937 cells through the reduction of Skp2 expression, and finally the induction of the accumulation of p27.</p>

Changes of Src-suppressed C kinase substrate expression in cytokine induced reactive C6 glioma cells / 神经科学通报·英文版

<p><b>OBJECTIVE</b>To investigate effect of tumor necrosis factor-alpha (TNF-alpha) on the Src-suppressed C kinase substrate (SSeCKS) in C6 glioma cells.</p><p><b>METHODS</b>Cultured C6 glioma cells were randomly divided into two groups. In time-dependent group, cells were cultured with TNF-alpha (2 ng/mL) for 0 h, 1 h, 3 h, 6 h, 12 or 24 h, respectively; in dose-dependent group, cells were cultured with TNF-alpha (0 ng/mL, 0.02 ng/mL, 0.2 ng/mL, or 2 ng/mL) for 6 h. The expression of SSeCKS was detected by Realtime PCR and Western blot analysis, and immunocytochemistry was used to investigate SSeCKS's subcellular localization.</p><p><b>RESULTS</b>TNF-alpha induced rapid phosphorylations of protein kinase C (PKC) substrates in C6 glioma cells, and upregulated SSeCKS expression in a time and concentration dependent manner. Immunocytochemistry suggested that SSeCKS was localized in the cyroplasm and the leading end of podosomal extensions in control groups, while TNF-alpha induced translocation of SSeCKS perinuclear. This effect could be partly reversed by PKC inhibitor Ro-31-8220.</p><p><b>CONCLUSION</b>TNF-alpha activates PKC and upregulates SSeCKS expression in C6 glioma cells. These effects are associated with PKC activity, suggesting that SSeCKS plays a role in response to glia activation in PKC mediated pathway.</p>

Promoting lumbar spinal fusion by adenovirus-mediated bone morphogenetic protein-4 gene therapy / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To determine whether an adenoviral construct containing bone morphogenetic protein-4 (BMP-4) gene can be used for lumbar spinal fusion.</p><p><b>METHODS</b>Twelve New Zealand white rabbits were randomly divided into two groups, 8 in the experimental group and 4 in the control group. Recombinant, replication-defective type 5 adenovirus with the cytomegalovirus (CMV) promoter and BMP-4 gene (Ad-BMP-4) was used. Another adenovirus constructed with the CMV promoter and beta-galactosidase gene (Ad-beta-gal) was used as control. Using collagen sponge as a carrier, Ad-BMP-4 (2.9 multiply 10(8) pfu/ml ) was directly implanted on the surface of L(5)-L(6) lamina in the experimental group, while Ad-beta-gal was implanted simultaneously in the control group. X-ray was obtained at 3, 6, and 12 weeks postoperatively to observe new bone formation. When new bone formation was identified, CT scans and three-dimensional reconstruction were obtained. After that, the animals were killed and underwent histological inspection.</p><p><b>RESULTS</b>In 12 weeks after operation, new bone formation and fusion were observed on CT scans in the experimental group, without the evidence of ectopic calcification in the canal. Negative results were found in the control group. Histological analysis demonstrated endochondral bone formation at the operative site and fusion at early stage was testified.</p><p><b>CONCLUSIONS</b>In vivo gene therapy using Ad-BMP-4 for lumbar posterolateral spinal fusion is practicable and effective.</p>

Expression and relationship of p27(kip1) and its related molecules Jab1 and CRM1 during proliferation of lymphoma cells U937 / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the expression and relationship of p27(kip1) and its related molecules Jab1 and CRM1 during proliferation of lymphoma cells U937.</p><p><b>METHODS</b>U937 cells were treated with serum starvation and release, and the effects of these treatments on the cell growth was tested with cell number counting. The expression and localization of p27(kip1), Jab1 and CRM1 in U937 cells were detected by Western blot, double immunolabelling and laser scanning confocal microscopy.</p><p><b>RESULTS</b>The growth of U937 cells was blocked by serum starvation. The total protein of p27(kip1) was increased while Ser10-phosphorylated p27(kip1) -related molecules Jab1 and CRM1 were decreased. Meanwhile, the location of p27(kip1) was changed from cytoplasm into nuclei. After serum release, the location of p27(kip1) expression reappeared in the cytoplasm again.</p><p><b>CONCLUSION</b>During the proliferation process of lymphoma U937 cells, Jab1 and CRM1 may influence the location and expression of p27kip1, and may participate in regulation of growth of NHL cells.</p>

Expression and relationship of p27(kip1) and its nuclear export factor Jab1 in lymphoma cell Jurkat / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the expression and relationship of p27(kip1) and its nuclear export factor Jab1 during proliferation process of lymphoma cell.</p><p><b>METHODS</b>Jurkat and Raji cells were treated with serum starvation and then serum release. The protein and mRNA expression of p27(kip1), Jab1 in the cells were detected by Western blot and RT-PCR respectively. LMB were used for stimulating Jurkat cells during their proliferation process, and then the expression changes of p27(kip1) and Jab1 were detected. An eukaryotic expression plasmid(pcDNA3. 1-myc) containing Jab1 was constructed. Jurkat cell were transfected in vitro with or without pcDNA3. 1-myc-Jab1. Double immunolabelling was used to identify the localization of p27(kip1). Immunoprecipitation was used to detect the combination of p27(kip1) and Jab1.</p><p><b>RESULTS</b>The growth of Jurkat and Raji cells were blocked by serum starvation. The total protein amount of p27(kip1) increased while that of Jab1 decreased. The reverse changes were happened after serum release, but the mRNA expression of p27(kip1) has no significant change. LMB could inhibit the cell proliferation caused by serum release. The expression of p27(kip1) was up-regulated and Jab1 down-regulated when Jurkat cells were treated with LMB. After pcDNA3. 1-myc-Jab1 infected Jurkat cells for 48 h, the distribution of p27(kip1) was translocated from nucleus into cytoplasma. p27(kip1) and Jab1 could form compound in Jurkat and Raji cells detected by Immunoprecipitation.</p><p><b>CONCLUSION</b>Jab1 may influence the location and expression of p27(kip1) through integrating with p27(kip1), and then participates in regulating the growth of NHL cell through interfering with the function of p27(kip1).</p>

Expression and correlation of Skp2 and p27(kip1) in 92 cases of non-Hodgkin's lymphoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the expression and correlation of Skp2 and p27kipl in non-Hodgkin's lymphoma.</p><p><b>METHODS</b>The expression of Skp2, p27(kip1) and Ki-67 (the proliferation index)were detected in sections of 92 cases of non-Hodgkin's lymphoma (NHL) and 14 cases of reactive lymph nodes by immunohistochemistry and histopathology. The expression of Skp2 and p27(kip1) in 4 NHL cell lines were detected by Western blot.</p><p><b>RESULTS</b>The expression of Skp2 in NHL cases were significantly higher than that in reactive lymph nodes (except the germinal centers), positively correlated with proliferation activity, and an increasing tumor aggressiveness was associated with the increased expression of Skp2. The expression of p27(kip1) protein in NHL cases were significantly lower than that in reactive lymph nodes (except the germinal centers), negatively correlated with proliferation activity, and an increasing tumor aggressiveness was associated with decreased expression of p27(kip1). The statistical analysis indicated that there was no obvious correlation between Skp2 and p27(kip1) expression in NHL tissues.</p><p><b>CONCLUSION</b>The higher expression of Skp2 and lower expression of p27(kip1) in NHL tissues may play a role in the tumorigenesis and development of NHL.</p>

Expression, localization and interrelationship of P27kip1 and cyclin D3 in non-Hodgkin's lymphoma / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the expression, localization and interrelationship of P27(kip1) and cyclin D3 in non-Hodgkin's lymphoma (NHL).</p><p><b>METHODS</b>The expressions of P27(kip1), cyclin D3 and index Ki-67 was detected in 100 NHL and 20 reactive lymph nodes by immunohistochemical technique. The expression and localization of P27(kip1) and cyclin D3 in 3 NHL cell lines were detected by Western blot, double immunolabelling and laser scanning confocal microscopy, respectively.</p><p><b>RESULTS</b>In general the expression of P27(kip1) in NHL was lower than in control group, and was negatively related to the tumor aggressiveness and proliferating activity; the expression of cyclin D3 in NHL was higher than in control group, and was positively related to the tumor aggressiveness and proliferating activity. There was a negative correlation between P27(kip1) and cyclin D3. Nevertheless, anomalous high P27(kip1) expression was found in a few NHL tissues with high expression of cyclin D3 and Ki-67. Overexpression and colocalization of P27(kip1) and cyclin D3 was found in Raji cell line.</p><p><b>CONCLUSIONS</b>Under expression of P27(kip1) and overexpression of cyclin D3 may play a role in the occurrence and development of NHL. Anomalous high P27(kip1) expression and its interaction with cyclin D3 may be another mechanism for tumor genesis of NHL.</p>