Synthesis of Bis(silylene) Iron Chlorides and Their Catalytic Activity for Dinitrogen Silylation.

In this study, three tetracoordinated bis(silylene) iron(II) chlorides, namely, [SiCHRSi]FeCl2 (1) (R = H), (2) (R = CH3), and (3) (R = Ph), were synthesized through the reactions of the three different bis(silylene) ligands [LSiCHRSiL] (L = PhC(NtBu)2, L1 (R = H), L2 (R = CH3), L3 (R = Ph)) with FeCl2·(THF)1.5 in THF. The bis(silylene) Fe complexes 1-3 could be used as effective catalysts for dinitrogen silylation, with complex 3 demonstrating the highest turnover number (TON) of 746 equiv among the three complexes. The catalytic mechanism was explored, revealing the involvement of the pentacoordinated bis(dinitrogen) iron(0) complexes [SiCHRSi]Fe(N2)2(THF), (4)-(6), as the active catalysts in the dinitrogen silylation reaction. Additionally, the cyclic silylene compound 10 was obtained from the reaction of L1 with KC8. Single-crystal X-ray diffraction analyses confirmed the molecular structures of complexes 1-3 and 10 in the solid state.

Leptin accelerates BMSC transformation into vertebral epiphyseal plate chondrocytes by activating SENP1-mediated deSUMOylation of SIRT3.

Bone marrow mesenchymal stem cells (BMSCs) are capable of multidirectional differentiation, and engrafted BMSCs can be used to replace damaged chondrocytes for treatment of intervertebral disc disease. However, chondroblast differentiation of implanted BMSCs is inhibited by the anoxic environment of the articular cavity. Here, we found that leptin enhanced the transformation of BMSCs into chondrocytes under hypoxic conditions. BMSCs isolated from mice were cultured in medium supplemented with leptin under hypoxia. The expression of MFN1/2 and OPA1 were increased only in BMSCs cultured in an anoxic environment. In addition, in hypoxic environments cell energy metabolism relies on glycolysis regulated by leptin, rather than by mitochondrial oxidation. The expression of the de-SUMOylation protease SENP1 was elevated, leading to SIRT3-mediated activation of PGC-1α; these processes were regulated by CREB phosphorylation, and promoted mitochondrial fusion and cell differentiation. The chondrogenic activity of BMSCs isolated from SIRT3-knockout mice was lower than that of BMSCs isolated from wildtype mice. Implantation of SIRT3-knockout murine-derived BMSCs did not significantly improve the articular cartilage layer of the disc. In conclusion, the hypoxic microenvironment promoted BMSC differentiation into chondrocytes, whereas osteoblast differentiation was inhibited. SENP1 activated SIRT3 through the deSUMOylation of mitochondria and eliminated the antagonistic effect of SIRT3 acetylation on phosphorylation. When phosphorylation activity of CREB was increased, phosphorylated CREB is then transferred to the nucleus, affecting PGC-1α. This promotes mitochondrial fusion and differentiation of BMSCs. Leptin not only maintains chondrogenic differentiation homeostasis of BMSCs, but also provides energy for differentiation of BMSCs under hypoxic conditions through glycolysis.

The local concentration of Ca2+ correlates with BMP7 expression and osseointegration in patients with total hip arthroplasty.

BACKGROUND: A successful osseointegration of total hip arthroplasty (THA) relies on the interplay of implant surface and bone marrow microenvironment. This study was undertaken to investigate the impact of perioperative biochemical molecules (Ca2+, Mg2+, Zn2+, VD, PTH) on the bone marrow osteogenetic factors (BMP2, BMP7, Stro-1+ cells) in the metaphyseal region of the femoral head, and further on the bone mineral density (BMD) of Gruen R3. METHODS: Bone marrow aspirates were obtained from the discarded metaphysis region of the femoral head in 51 patients with THA. Flow cytometry was used to measure the Stro-1+ expressing cells. ELISA was used to measure the concentrations of bone morphologic proteins (BMP2 and BMP7) and the content of TRACP5b in serum. TRAP staining was used to detect the osteoclast activity in the hip joint. The perioperative concentrations of the biochemical molecules above were measured by radioimmunoassay. The BMD of Gruen zone R3 was examined at 6 months after THA, using dual-energy X-ray absorptiometry (DEXA). RESULTS: Our data demonstrated that the concentration of Ca2+ was positively correlated with BMP7 expression, and with the postoperative BMD of Gruen zone R3. However, the concentration of Mg2+ had little impact on the R3 BMD, although it was negatively correlated with the expression of BMP7. Osteoclast activity in hip joint tissue of patients with femoral neck fractures was increased. Compared with the patients before THA, the levels of TRACP5b in serum of patients after THA were decreased. The data also suggested that the other biochemical molecules, such as Zn2+, VD, and PTH, were not significantly correlated with any bone marrow osteogenetic factors (BMP2, BMP7, Stro-1+ cells). The postoperative R3 BMD of patients of different gender and age had no significant difference. CONCLUSIONS: These results indicate the local concentration of Ca2+ may be an indicator for the prognosis of THA patients.

Expression of tissue inhibitor of metalloproteinases-1 and B-cell lymphoma-2 in the synovial membrane in patients with knee osteoarthritis.

The purpose of this study was to determine the expression and impact of tissue inhibitor of metalloproteinases-1 (TIMP-1) and B-cell lymphoma-2 (Bcl-2) in knee osteoarthritis (KOA). We collected synovial fluids from the knee joint of 70 KOA patients and 30 controls. The expression levels of TIMP-1 and Bcl-2 were significantly higher in KOA patients than those in the control group (P<0.01). We also found positive correlation between the severity of KOA and the expression level of TIMP-1 (r=0.8027, P<0.05) and and Bcl-2 (r=0.5336, P<0.05). However, we found no correlation between the expression levels of TIMP-1 and Bcl-2 in the synovial membranes of KOA patients (P>0.05). Both TIMP-1 and Bcl-2 are expressed at high levels in the synovial membrane with KOA, and are closely related to the occurrence and development of KOA. Thus, detection of TIMP-1 and Bcl-2 in KOA patients can be helpful in diagnosing the state of KOA.

Small interfering RNA-mediated silencing of G-protein-coupled receptor 137 inhibits growth of osteosarcoma cells.

PURPOSE: Osteosarcoma is the most widespread primary carcinoma in bones. Osteosarcoma cells are highly metastatic and frequently develop resistance to chemotherapy making this disease harder to treat. This identifies an urgent need of novel therapeutic strategies for osteosarcoma. G-Protein-coupled receptor 137 (GPR137) is involved in several human cancers and may be a novel therapeutic target. METHODS: The expression of GPR137 was assessed in one osteoblast and three human osteosarcoma cell lines via the quantitative real-time polymerase chain reaction and western blot assays. Stable GPR137 knockdown cell lines were established using an RNA interference lentivirus system. Viability, colony formation, and flow cytometry assays were performed to measure the effects of GPR137 depletion on cell growth. The underlying molecular mechanism was determined using signaling array analysis and western blot assays. RESULTS: GPR137 expression was higher in the three human osteosarcoma cell lines, Saos-2, U2OS, and SW1353, than in osteoblast hFOB 1.19 cells. Lentivirus-mediated small interfering RNA targeting GPR137 successfully knocked down GPR137 mRNA and protein expression in both Saos-2 and U2OS cells. In the absence of GPR137, cell viability and colony formation ability were seriously impaired. The extent of apoptosis was also increased in both cell lines. Moreover, AMP-activated protein kinase α, proline-rich AKT substrate of 40 kDa, AKT, and extracellular signal-regulated kinase phosphorylation levels were down-regulated in GPR137 knockdown cells. CONCLUSIONS: The results of this study highlight the crucial role of GPR137 in promoting osteosarcoma cell growth in vitro. GPR137 could serve as a potential therapeutic target against osteosarcoma.

Asiaticoside protects cochlear hair cells from high glucose-induced oxidative stress via suppressing AGEs/RAGE/NF-κB pathway.

Asiaticoside (AC) has been known to have anti-oxidative activity, however, the effect of AC on the progression of high glucose-induced hearing loss has not been studied. This study aims to analyze the effect of AC on cochlear hair cells under the treatment of high glucose in vitro and the hearing function in vivo. The results of MTT showed that high glucose decreased the activity of HEI-OC1 cells, but AC increased the activity of HEI-OC1 cells compared with high glucose group. The results of flow cytometry showed that AC decreased the degree of apoptosis induced by high levels of glucose. The results of DCFH-DA staining showed that AC inhibited the ROS production induced by high glucose levels. The results of JC-1 staining showed that AC inhibited the mitochondrial depolarization induced by high glucose levels. Furthermore, AC decreased the threshold, and protected inner and outer hair cells from damage in rats with hearing loss induced by diabetes mellitus. Moreover, AC decreased the activity of MDA, but, increased the activity of SOD, CAT and GSH-Px in vivo. AC also decreased the expression of AGEs, RAGE and NF-κB p65. Collectively, these results suggest that AC protects cochlear hair cells from high glucose-induced injury by increasing anti-oxidative activity and suppressing the AGEs/RAGE/NF-κB pathway.

Survivin activates NF­κB p65 via the IKKß promoter in esophageal squamous cell carcinoma.

Survivin and transcription factor p65 (NF­κB p65) participate in the progression of esophageal squamous cell carcinoma (ESCC). However, the mechanism of NF­κB p65 activation in ESCC remains to be elucidated. The aim of the present study was to investigate the role of survivin in the activation of NF­κB p65 in ESCC. The expression levels of survivin, NF­κB p65, inhibitor of nuclear factor κB kinase subunit α (IKKα) and inhibitor of nuclear factor κB kinase subunit ß (IKKß) were detected in ESCC tissue samples. Eca109 and KYSE150 cells were cultured and survivin activity was modulated via transfection with an overexpression plasmid, a small hairpin RNA plasmid and a specific inhibitor. Quantitative reverse transcription-polymerase chain reaction and western blotting assays were conducted to assess the effects of survivin on the expression levels of IKKα, IKKß and NF­κB p65. Cell cycle and apoptosis assays were conducted to detect surviving-dependent cellular behavior changes. In addition, the luciferase reporter gene assay and chromatin immunoprecipitation assay were conducted to determine the genomic sites responsible for surviving-induced activation of NF­κB p65. The present study demonstrated that the expression of survivin is positively correlated with IKKα and IKKß in ESCC tissues. Survivin affected the mRNA and protein expression levels of IKKα, IKKß, and NF­κB p65 in Eca109 and KYSE150 cells. Furthermore, survivin increased the transcriptional activity of the IKKß promoter and bound to the IKKß promoter region in the Eca109 cells. Downregulation of survivin arrested the cell cycle at the G2/M phase and induced apoptosis. Results of the present study suggest that survivin activates NF­κB p65 in Eca109 cells via binding to the IKKß promoter region and upregulating IKKß promoter transcriptional activity. Survivin overexpression activates NF­κB p65, which is important in the acquisition and maintenance of the oncogenic characteristics of ESCC.

[Children's OSAHS caused by the huge fibrolipoma in pharynx nasalis: a case report].

We reported a case of children's OSAHS caused by the huge fibrolipoma in pharynx nasalis. The patient was a 10-years-old child who went to the hospital with the chief complaint of "Snoring and mouth breathing during sleep for 10 years". Imaging tests found one huge tumor in pharynx nasalis before the operation. The tumor was resected totally. The postoperative pathological diagnosis was fibrolipoma. No recurrence was noted during the follow-up visit one year postoperatively. The clinical features, diagnosis, treatment, pathology and prognosis were reviewed herein.

A 3D finite element model to investigate prosthetic interface stresses of different posterior tibial slope.

PURPOSE: Posterior tibial slope that is created during proximal tibial resection in total knee arthroplasty has emerged as an important factor in the mechanics of the knee joint and the surgical outcome. But the ideal degree of posterior tibial slope for recovery of the knee joint function and preventions of complications remains controversial and should vary in different racial groups. The objective of this paper is to investigate the effects of posterior tibial slope on contact stresses in the tibial polyethylene component of total knee prostheses. METHODS: Three-dimensional finite element analysis was used to calculate contact stresses in tibial polyethylene component of total knee prostheses subjected to a compressive load. The 3D finite element model of total knee prosthesis was constructed from the images produced by 3D scanning technology. Stresses in tibial polyethylene component were calculated with four different posterior tibial slopes (0°, 3°, 6° and 9°). RESULTS: The 3D finite element model of total knee prosthesis we presented was well validated. We found that the stress distribution in the polythene as evaluated by the distributions of the von Mises stress, the maximum principle stress, the minimum principle stress and the Cpress were more uniform with 3° and 6° posterior tibial slopes than with 0° and 9° posterior tibial slopes. Moreover, the peaks of the above stresses and trends of changes with increasing degree of knee flexion were more ideal with 3° and 6° posterior slopes. CONCLUSIONS: The results suggested that the tibial component inclination might be favourable to 7°-10° so far as the stress distribution is concerned. The range of the tibial component inclination also can decrease the wear of polyethylene. Chinese posterior tibial slope is bigger than in the West, and the current domestic use of prostheses is imported from the West, so their demands to tilt back bone cutting can lead to shorten the service life of prostheses; this experiment result is of important clinical significance, guiding orthopaedic surgeon after the best angle to cut bone.

Stro-1-positive BMSCs predict postoperative periprosthetic bone mineral density outcomes in uncemented total hip arthroplasty patients.

BACKGROUND: Bone marrow cell profiles are variable after total hip arthroplasty (THA), including variable levels of Stro-1+ and bone morphogenetic protein receptor (BMPRs)+ cells. We investigated the impact of bone marrow cell profiles on changes in periprosthetic bone mineral density (BMD) in uncemented THA patients. MATERIAL AND METHODS: Bone marrow aspirates were collected from the metaphyseal region of discarded femoral heads from 24 consecutive THA patients (12 men and 12 women; mean age 66.7 ± 11.0 years; range 52-87 years) treated from March 2009 to March 2011 at a single facility. Perioperative proportions of Stro-1+ and BMPR+ cells in femoral heads were assessed by flow cytometry. Follow-up examined the proximal femur Gruen zones R1 and R7 at 1 week and at 3, 6, and 12 months after THA, using dual-energy X-ray absorptiometry. Associations between BMD loss and age, gender, BMPRs+, and Stro-1+ were analyzed. RESULTS: At 3 months, R1 and R7 BMD decreased by 4.4% and 6.4%, respectively (P<0.05). At 12 months, the overall BMD decreases in R1 and R7 were 10.2% and 1%, respectively (P<0.05). Higher Stro-1+ cells proportion predicted R7 BMD increases at all time points (P<0.05) and R1 BMD increases at 6 and 12 months (P<0.05). BMPR1a+ proportion was associated with BMD increases at 6 months in the R1 region. BMPR2+ was not significantly associated with BMD (P>0.05). CONCLUSIONS: Elevated Stro-1+ bone marrow cell profile may be a useful prognostic indicator for uncemented THA patients.

Melatonin treatment increases the transcription of cell proliferation-related genes prior to inducing cell death in C6 glioma cells in vitro.

A number of studies have suggested that melatonin possesses anticancer properties. However, conflicting data exists with regard to the role of melatonin in the treatment of cancer. In the present study, the effects of melatonin on the transcriptional regulation of three genes associated with cell proliferation (Nestin, Bmi-1 and Sox2), and on C6 glioma cell survival and viability, were investigated in vitro to evaluate the use of melatonin in cancer therapy. Melatonin was shown to increase the mRNA levels of Nestin, Bmi-1 and Sox2 in a similar pattern, with the highest mRNA levels noted at a concentration of 3 mM. At higher concentrations of melatonin (5 mM), the mRNA levels of Nestin, Bmi-1 and Sox2 were reduced from their peak levels, and were correlated with changes observed in immunofluorescence morphology studies, cell viability and survival assays. Immunofluorescence studies of Nestin-stained cells demonstrated that treatment with a higher concentration of melatonin (3 and 5 mM) led to the Nestin filaments condensing and rearranging around the cell nuclei, and an alteration in the cell morphology. C6 cell viability was also significantly decreased at 3 mM melatonin, and cell death was observed at 5 and 10 mM melatonin. These results suggested that Nestin, Bmi-1 and Sox2 were strongly correlated with the survival of C6 cells following treatment with melatonin, and that high therapeutic concentrations of melatonin (>5 mM) were required to induce cell death. These findings suggested that the implementation of melatonin in the treatment of glioma and other types of cancer may be inhibited by conflicting cell growth signals in cells. Therefore, adjunct therapy is required to improve the efficacy of melatonin in the treatment of cancer.

Optimizing the transduction efficiency of capsid-modified AAV6 serotype vectors in primary human hematopoietic stem cells in vitro and in a xenograft mouse model in vivo.

BACKGROUND AIMS: Although recombinant adeno-associated virus serotype 2 (AAV2) vectors have gained attention because of their safety and efficacy in numerous phase I/II clinical trials, their transduction efficiency in hematopoietic stem cells (HSCs) has been reported to be low. Only a few additional AAV serotype vectors have been evaluated, and comparative analyses of their transduction efficiency in HSCs from different species have not been performed. METHODS: We evaluated the transduction efficiency of all available AAV serotype vectors (AAV1 through AAV10) in primary mouse, cynomolgus monkey and human HSCs. The transduction efficiency of the optimized AAV vectors was also evaluated in human HSCs in a murine xenograft model in vivo. RESULTS: We observed that although there are only six amino acid differences between AAV1 and AAV6, AAV1, but not AAV6, transduced mouse HSCs well, whereas AAV6, but not AAV1, transduced human HSCs well. None of the 10 serotypes transduced cynomolgus monkey HSCs in vitro. We also evaluated the transduction efficiency of AAV6 vectors containing mutations in surface-exposed tyrosine residues. We observed that tyrosine (Y) to phenylalanine (F) point mutations in residues 445, 705 and 731 led to a significant increase in transgene expression in human HSCs in vitro and in a mouse xenograft model in vivo. CONCLUSIONS: These studies suggest that the tyrosine-mutant AAV6 serotype vectors are the most promising vectors for transducing human HSCs and that it is possible to increase further the transduction efficiency of these vectors for their potential use in HSC-based gene therapy in humans.

Conditional deletion of Jak2 reveals an essential role in hematopoiesis throughout mouse ontogeny: implications for Jak2 inhibition in humans.

Germline deletion of Jak2 in mice results in embryonic lethality at E12.5 due to impaired hematopoiesis. However, the role that Jak2 might play in late gestation and postnatal life is unknown. To understand this, we utilized a conditional knockout approach that allowed for the deletion of Jak2 at various stages of prenatal and postnatal life. Specifically, Jak2 was deleted beginning at either mid/late gestation (E12.5), at postnatal day 4 (PN4), or at ∼2 months of age. Deletion of Jak2 beginning at E12.5 resulted in embryonic death characterized by a lack of hematopoiesis. Deletion beginning at PN4 was also lethal due to a lack of erythropoiesis. Deletion of Jak2 in young adults was characterized by blood cytopenias, abnormal erythrocyte morphology, decreased marrow hematopoietic potential, and splenic atrophy. However, death was observed in only 20% of the mutants. Further analysis of these mice suggested that the increased survivability was due to an incomplete deletion of Jak2 and subsequent re-population of Jak2 expressing cells, as conditional deletion in mice having one floxed Jak2 allele and one null allele resulted in a more severe phenotype and subsequent death of all animals. We found that the deletion of Jak2 in the young adults had a differential effect on hematopoietic lineages; specifically, conditional Jak2 deletion in young adults severely impaired erythropoiesis and thrombopoiesis, modestly affected granulopoiesis and monocytopoiesis, and had no effect on lymphopoiesis. Interestingly, while the hematopoietic organs of these mutant animals were severely affected by the deletion of Jak2, we found that the hearts, kidneys, lungs, and brains of these same mice were histologically normal. From this, we conclude that Jak2 plays an essential and non-redundant role in hematopoiesis during both prenatal and postnatal life and this has direct implications regarding the inhibition of Jak2 in humans.

High-efficiency transduction of primary human hematopoietic stem cells and erythroid lineage-restricted expression by optimized AAV6 serotype vectors in vitro and in a murine xenograft model in vivo.

We have observed that of the 10 AAV serotypes, AAV6 is the most efficient in transducing primary human hematopoietic stem cells (HSCs), and that the transduction efficiency can be further increased by specifically mutating single surface-exposed tyrosine (Y) residues on AAV6 capsids. In the present studies, we combined the two mutations to generate a tyrosine double-mutant (Y705+731F) AAV6 vector, with which >70% of CD34(+) cells could be transduced. With the long-term objective of developing recombinant AAV vectors for the potential gene therapy of human hemoglobinopathies, we generated the wild-type (WT) and tyrosine-mutant AAV6 vectors containing the following erythroid cell-specific promoters: ß-globin promoter (ßp) with the upstream hyper-sensitive site 2 (HS2) enhancer from the ß-globin locus control region (HS2-ßbp), and the human parvovirus B19 promoter at map unit 6 (B19p6). Transgene expression from the B19p6 was significantly higher than that from the HS2-ßp, and increased up to 30-fold and up to 20-fold, respectively, following erythropoietin (Epo)-induced differentiation of CD34(+) cells in vitro. Transgene expression from the B19p6 or the HS2-ßp was also evaluated in an immuno-deficient xenograft mouse model in vivo. Whereas low levels of expression were detected from the B19p6 in the WT AAV6 capsid, and that from the HS2-ßp in the Y705+731F AAV6 capsid, transgene expression from the B19p6 promoter in the Y705+731F AAV6 capsid was significantly higher than that from the HS2-ßp, and was detectable up to 12 weeks post-transplantation in primary recipients, and up to 6 additional weeks in secondary transplanted animals. These data demonstrate the feasibility of the use of the novel Y705+731F AAV6-B19p6 vectors for high-efficiency transduction of HSCs as well as expression of the b-globin gene in erythroid progenitor cells for the potential gene therapy of human hemoglobinopathies such as ß-thalassemia and sickle cell disease.

Molecular mechanisms of platelet and stem cell rebound after 5-fluorouracil treatment.

Sublethal irradiation and 5-fluorouracil (5-FU) treatment are two commonly used myelosuppressive methods used in the study of hematopoiesis. These methods have been considered interchangeable by some researchers because the morphological changes in the bone marrow to these treatments are similar. Here, we sought to compare the responses of hematopoietic cells, stem and progenitor cells and the bone marrow microenvironment to these treatments. Although bone marrow cellularity decreased after both treatments, the underlying mechanism of the bone marrow cell regression and recovery were very different between the two models. We found: 1. Myeloid cells and lymphoid cells had different sensitivity to the different treatments. 2. Following an initial decrease in stem cell number, 5-FU treated mice had profound thrombopoietin (Tpo) dependent stem cell rebound above baseline levels. 3. Platelet rebound in 5-FU treated animals was not the result of stem cell rebound. 4. Stem cell and platelet rebound did not occur in sub-lethally irradiated mice. 5. Platelet rebound resulted from an indirect effect of 5-FU on the microenvironment cells, but not a direct effect on the stem cells. 6. Microarray studies demonstrated that up-regulation of the angiopoietin-1/Tie2 signaling pathway coincided with platelet rebound. 7. Suppression of genes involved in chromosomal organization coincided with stem cell and platelet rebound.

Association of maximum weight with hyperuricemia risk: a retrospective study of 21,414 Chinese people.

BACKGROUND: Obesity has been demonstrated to be associated with increased serum uric acid (SUA); however, little is known regarding the relationship between maximum weight, or maximum weight fluctuation, and uric acid concentration. Through retrospective means, we determined the association of maximum weight with SUA risk. METHODS: Data of 21,414 participants (8,630 males and 12,784 females) from the 2007-8 China National Diabetes and Metabolic Disorders Study were analyzed for parameters including lifestyle habits, biochemical blood analysis and self-reported maximum weight. RESULTS: Elevated SUA subjects shared a cluster of demographic features. After adjustment for age, gender, education, smoking, drinking, physical activity, WHR, height, eGFR(evaluate glomerular filtration rate), and diuretic usage, multivariate logistic regression models demonstrated maximum weight was associated with increased risk of elevated SUA level (P<0.001). Duration of maximum weight was related with decreased risk of elevated SUA level (P<0.001). There was a significant correlation between time of weight loss and risk of increased SUA level reduction (P<0.001). Furthermore, our data indicated that the degree of weight loss from maximum weight was another important factor for the risk of increased SUA level reduction (P<0.001). Finally, ROC curve analysis revealed area under the curve was 0.661 (95% CI, 0.647-0.674), statistically significant for maximum weight association with hyperuricemia (P<0.001). CONCLUSIONS: Maximum weight is a strong risk factor for increased uric acid level in the Chinese population, which might serve as a novel clinical indicator suggesting hyperuricemia. Controlling maximum weight, keeping weight to the appropriate range, and maintaining the stable weight may be conducive for decreasing risk of hyperuricemia.

Impacts of age and gender on bone marrow profiles of BMP7, BMPRs and Stro-1⁺ cells in patients with total hip replacement.

PURPOSE: A successful osseointegration relies on the interplay of implant surface and surrounding bone marrow cells. This study was undertaken to investigate the impact of age and gender on the bone marrow composition. METHODS: Bone marrow aspirates were obtained from the discarded metaphysis region of the femoral head in 24 patients with total hip replacement. Flow cytometry was used to measure the expression of Stro-1(+) cells and BMP receptors (BMPRs)-expressing cells. ELISA was used to measure bone marrow aspirate bone morphology protein7 (BMP7) concentration. RESULTS: Our data demonstrates that there are diverse bone marrow profiles (Stro-1(+) cell and BMPRs(+) cells). There are no differences of Stro-1(+) cells, BMPRs(+) cells, and BMP7 concentration between male and female patients. Though there are slight increases in the number of Stro-1(+) cells and BMPRs(+) cells in younger patients (<70 years old) than those of old patients (≥ 70 years old), the difference is not statistically significant. However, we found a close association between the Stro-1(+) cells, BMPR1a cells and BMP7 concentration. In addition, a correlation exists between the number of Stro-1(+) cells and BMIs of these patients. CONCLUSION: Our data suggests that the age and gender of THR patients have little impact on their bone marrow osteogenic potential. The significance of the number of the Stro-1(+) with BMPRs expression on the implant fixation and osseointegration warrants further investigation.

Intrinsic and extrinsic effects of mafG deficiency on hematopoietic recovery following bone marrow transplant.

OBJECTIVE: MafG is the small subunit of the transcription factor NF-E2 that controls terminal megakaryocyte maturation and platelet release. Studies were conducted to evaluate the intrinsic and extrinsic effects of mafG deficiency on bone marrow engraftment kinetics. MATERIALS AND METHODS: We used mafG knockout mice either as donors or recipients in bone marrow transplantations with wild-type mice and compared the engraftment kinetics to transplantations using wild-type donors and recipients. We measured peripheral cell counts, the presence of circulating donor-derived cells by flow cytometry, changes in the cellularity of the bone marrow and splenic weight on day 5, 7, 14, and 1 month post-transplantation. RESULTS: Compared to wild-type recipients, mafG recipients had delayed platelet and leukocyte recovery and lower spleen weight at early time points after transplantation. Intrinsic effects: When mafG-deficient bone marrow served as donor source, we observed more rapid recovery of bone marrow cellularity and increased splenic hematopoiesis. The finding of increased short-term hematopoietic stem cells and progenitors in the mafG-deficient bone marrow could explain the accelerated hematopoietic recovery after transplantation. Furthermore, the expression of Bach 2, which can form a heterodimer with mafG protein, was found to be greatly reduced, while Notch 1 expression was increased in mafG-deficient mice. Extrinsic effects: When mafG-deficient mice were transplant recipients, there were delays in recovery of normal levels of marrow and splenic hematopoiesis as well as circulating leukocytes and platelets. CONCLUSIONS: Our study demonstrates that mafG expression has intrinsic and extrinsic effects on hematopoietic engraftment following bone marrow transplantation.

Expression patterns of ClC-3 mRNA and protein in aortic smooth muscle, kidney and brain in diabetic rats.

ClC-3, a member of the ClC family of voltage-gated chloride channels, regulates cell proliferation of cultured rat aortic vascular smooth muscle cells, pathogenesis of allergic rhinitis and tumor cell migration. However, its role in diabetic animals is still unknown. To address this issue, we investigated the expression patterns of ClC-3 in diabetic rats. Five-week-old Sprague-Dawley rats were divided into two groups, 50 non-diabetic control rats (non-DM) and 50 diabetic model rats (DM). ClC-3 mRNA and protein expression in aortic smooth muscle, kidney and brain tissues were examined by fluorimeter-based quantitive RT-PCR assay and Western blot analysis, respectively. ClC-3 mRNA and protein were endogenously expressed in aortic smooth muscle, kidney (cortex and medulla) and brain tissues of both control and streptozotocin-induced diabetic rats. ClC-3 mRNA and protein expression levels were significantly higher in aortic smooth muscle and brain tissues of diabetic rats, but significantly decreased in kidney medulla tissue, relative to non-DM controls. There were no significant differences in ClC-3 mRNA and protein expression in kidney cortex between non-diabetic control and diabetic rats. Furthermore, the altered ClC-3 expression patterns in diabetic rat aortic smooth muscle, brain, and kidney medulla tissues all correlated with the changes in blood glucose levels (p < 0.05). In conclusion, our data show for the first time that diabetes alters both the gene and protein expression of ClC-3 channels. These changes may contribute to the impaired vascular, brain and kidney functions observed in diabetes.

Upregulation of PTEN by peroxynitrite contributes to cytokine-induced apoptosis in pancreatic beta-cells.

Phosphatase and tensin homolog (PTEN), a tumor suppressor gene, by negatively regulating the PI3K-Akt signaling pathway, participates in multiple biological processes such as cell proliferation, apoptosis, differentiation, and migration. Recent studies show that selective deletion of PTEN in pancreatic beta-cells leads to resistance to streptozotocin (STZ)-induced diabetes, but the mechanism is unclear. One major mechanism underlying STZ toxicity is cytokine-mediated beta-cell destruction in which oxidative stress plays a key role. The present study investigated the role of PTEN in cytokine-induced beta-cell apoptosis, and further explored whether oxidative stress, particularly peroxynitrite formation, could regulate PTEN-Akt pathway. Incubation of betaTC-6 cells with cytokine mixture (IL-1beta, TNF-alpha, and IFN-gamma) or exogenous peroxynitrite significantly increased apoptotic cell percentage, elevated PTEN and p-PTEN levels, and inhibited Akt activation. Transfection with PTEN-specific siRNA protected betaTC-6 cells from cytokine or peroxynitrite-mediated cell apoptosis and partially reversed Akt inhibition. Furthermore, nitrotyrosine formation, an indicator of peroxynitrite production, was significantly elevated after cytokine treatment. Preventing peroxynitrite formation by administrating NAC/L: -NMMA, or scavenging peroxynitrite directly by UA, attenuated cytokine-induced PTEN upregulation, Akt inhibition, and beta-cell apoptosis. These findings suggest that peroxynitrite-mediated PTEN upregulation plays an important role in cytokine-induced pancreatic beta-cell apoptosis.