Characterization of reproductive hormones and related gene expression in the hypothalamus and pituitary gland in the egg-laying interval in White King pigeon.

The egg-laying interval (LI) directly reflects the laying performance of breeding pigeons, influenced by reproductive hormones. This study aimed to assess reproductive hormone levels in serum and the expression of related genes and their receptors in the hypothalamus and pituitary gland in 4 stages: first (LI1), third (LI3), fifth (LI5), and seventh (LI7) days. The results showed that serum gonadotropin-releasing hormone (GnRH) level decreased from LI1 to LI7 (P < 0.01) and peaked in LI1. The serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels stayed at high levels from LI1 to LI5. The FSH level decreased slightly from LI5 to LI7 (P > 0.05), but the LH level decreased rapidly (P < 0.01). The prolactin (PRL) levels significantly increased in LI5 (P < 0.01) compared with LI1 and then stayed at a high level. The GnRH1 expression in the hypothalamus had no significant change in LI (P > 0.05). However, the GnRHR first decreased from LI1 to LI3 (P < 0.05) and then increased. The FSH mRNA level in the pituitary gland decreased from LI1 to LI3 and slightly increased in LI5 (P > 0.05). The change pattern of FSHR was similar to that of FSH and peaked in LI5 (P < 0.05). The LH expression level was the highest in LI5 and significantly higher than that in LI3 and LI7 (P < 0.05). However, the LHR mRNA level decreased in LI (P < 0.05). The expression patterns of PRL and PRLR were similar; they were upregulated in LI and peaked in LI7 (P < 0.01). The expression pattern of GnRHR was similar to that of FSH, LH, and FSHR, suggesting the critical role of GnRHR in LI. Furthermore, the expression levels of these genes peaked in LI5, closely correlating with the maturation of the first largest follicle in pigeons. PRL-PRLR signaling inhibited GnRH activity to promote ovulation. This study provided a basis for further investigating the molecular mechanisms underlying the regulation of reproduction in pigeons.

Characterization of ovarian follicles, serum steroid hormone concentration, and steroidogenic gene expression profiles in the developing ovarian follicles in White King pigeons.

Paired pigeons only lay 2 eggs in a laying period, which is closely related to ovarian follicle development, but this process is not well understood. In this study, 60 pairs of 12-mo-old White King pigeons were selected and serum and follicles were collected at 4 stages of laying interval (LI), including the first (LI1), the third (LI3), the fifth (LI5), and the seventh day (LI7). Morphological results showed that paired pigeons normally had 2 preovulatory follicles and the second-largest follicle (F2) developed from LI3 and had been selected in LI5. Prehierarchical follicles were coupled and hierarchical, which was in accordance with its clutch size. The P4 concentration increased gradually from LI1 to LI5, reaching a maximum of 30.67 ng/mL in LI5 and decreasing to 27.83 ng/mL in LI7 (P < 0.05). The levels of T in LI1 and LI5 were higher than LI3 and LI7 (P < 0.05), although there was no significant difference in E2 in LI (P > 0.05), but it stayed at high levels. In the TCs of the largest follicle (F1), HSD3B1 mRNA and HSD17B1 mRNA levels peaked in LI7. The expression pattern of CYP17A1 and CYP19A1 was similar, increasing from LI3 to LI5 and then decreasing. In the TCs of F2, the expressions of HSD3B1 and CYP17A1 had no significant difference between LI5 and LI7 (P > 0.05), while the expression pattern of HSD17B1 and CYP19A1 was the opposite. In TCs of SF1, HSD3B1 mRNA level peaked in LI3 while CYP19A1 mRNA levels peaked in LI7. The expression of CYP17A1 had a minor change (P > 0.05) and the expression pattern of HSD17B1 was similar to F1. It was concluded that the morphological characteristics of follicles during the LI for the first time, including the number and diameter of small follicles (SFs) and hierarchical follicles in pigeon and the concentrations of steroid hormones and expressions of steroidogenic genes in TCs of different follicles could explain the growth and selection of 2 preovulatory follicles. This study facilitates further research into the regulation of ovulation and egg production in pigeons.

[Long-term outcomes of Bryan artificial cervical disc replacement for degenerative cervical spondylosis].

Objective: To compare the long-term clinical effect and imaging results of Bryan artificial cervical disc replacement (ACDR) and anterior cervical discectomy and fusion (ACDF) and to explore whether ACDR can reduce the occurrence of postoperative adjacent segment degeneration (ASD) in the treatment of degenerative cervical spondylosis. Methods: It was a retrospective study. Total of 60 patients with degenerative cervical spondylosis, who had received operations by Bryan ACDR (n=27) and ACDF (n=33) in the Third Hospital of Hebei Medical University between January 2005 and December 2009 were enrolled in this study. The Japanese Orthopedic Association (JOA) scores, neck disability index (NDI), visual analogue scale (VAS), Odom's scale, cervical range of motion (ROM), surgical segment ROM, heterotopic ossification (HO) and ASD were used to evaluate the clinical and radiologic results. The indices abovementioned were compared between the two groups. Results: The VAS, NDI and JOA scores at each follow-up node in both ACDR and ACDF group were all significantly improved when compared with those before operation (all P<0.05), but there was no significant differences between the two groups (all P>0.05). Of all, 88.9%(24/27) of patients in ACDR group and 84.8%(28/33) of patients in ACDF group achieved excellent or good results, however, there was no significant difference in Odom's scale between the two groups (P>0.05). At the last follow-up, the cervical ROM in ACDR group was 43.2°±8.8°, which was slightly lower than that before operation (45.7°±10.4°), the difference was not statistically significant (t=0.954, P=0.345); the surgical segment ROM in ACDR group was 5.9°±3.6°, which was significantly reduced when compared with that before operation (8.8°±3.4°, t=3.043, P<0.01). However at the last follow-up, the cervical ROM in ACDF group was 36.4°±8.4°, which was significantly reduced when compared with that before operation (43.9°±11.1°), the difference was statistically significant (t=3.095, P<0.01). Although, there was no significant difference in the cervical ROM between the two groups before operation (t=0.643, P=0.523), and the difference was statistically significant at the last follow-up (t=3.054, P<0.01). At the last follow-up, the incidence of HO in ACDR group was 92.6%, and the high-grade HO was 37.0%. The incidence of ASD in ACDR group was 39.5%, which was much lower than that in ACDF group (61.1%, χ(2)=4.462, P=0.035). Conclusion: At minimum follow-up of 10 years, Bryan ACDR achieves a satisfactory clinical effect consistent with ACDF. In terms of advantages, ACDR could maintain the ROM of cervical and retain the ROM of the surgical segment, which reduces the occurrence of ASD by preserving motion.

[An analysis of the new "three tubes" method in the treatment of spontaneous esophageal rupture].

To investigate the clinical efficacy, feasibility and safety of new "three tubes" method in the treatment of spontaneous esophageal rupture. A total of 22 patients with spontaneous esophageal rupture were retrospectively analyzed. Through the new "three tubes" method of treatment, patients achieved leak cured with reduced hospital stay, less medical expenses and early resumption of oral diet. The new "three tubes" method for spontaneous esophageal rupture has the advantages of easy handling, minimal invasion, few complication and exact curative effect.

Overexpressed Sirt1 in MSCs Promotes Dentin Formation in Bmi1-Deficient Mice.

Sirt1 promotes odontoblastic gene expression in human dental pulp cells, whereas the inhibition of Sirt1 downregulates the expression of those genes. To investigate whether the overexpression of Sirt1 in mesenchymal stem cells (MSCs) driven by Prx1 promoter could rescue the dentin formation defects in Bmi1-deficient (Bmi1-/-) mice, we established the MSCs overexpressing Sirt1 in Bmi1 knockout mice (Sirt1TGBmi1-/-). First, we used Prx1-Cre/ROSAnTnG mice to demonstrate that Prx1 linage cells exist mainly in the pulp horns at 4 wk of age. Second, we found that 4-wk-old Sirt1TG mice had increased tooth volume as compared with wild-type (WT) littermates. The expression level of Sirt1 was significantly higher in dental papilla mesenchymal cells of Sirt1TG mice than WT mice. Furthermore, we demonstrated that the tooth mineralization, dental volume, dentin sialoprotein-immunopositive areas, odontoblastic gene expression, and percentage of proliferating BrdU-positive cells were significantly elevated in the Sirt1TG mice and dramatically reduced in the Bmi1-/- mice versus the WT littermates at 4 wk of age. However, the areas of predentin and the percentage of TUNEL-positive apoptotic cells were significantly reduced in the Sirt1TG mice but dramatically increased in the Bmi1-/- mice as compared with the WT littermates. All these parameters were rescued in the Sirt1TGBmi1-/- mice versus the Bmi1-/- mice. Finally, by using dental papilla mesenchymal cells, we found that the overexpression of Sirt1 rescued the reduced cell proliferation and differentiation and increased the cell apoptosis caused by Bmi1 deficiency, which was associated with increased p53 deacetylation. Therefore, this study indicates that Sirt1 is a potential therapeutic target for promoting dentin formation in an anabolic approach to the treatment of dental developmental defects.

Xuezhikang reduced arterial stiffness in patients with essential hypertension: a preliminary study.

This study aimed to test the effects of xuezhikang, a cholestin extract that contains statin-like components, on arterial stiffness in patients with essential hypertension. One hundred hypertensive patients from the Chinese PLA General Hospital were randomly allocated to receive xuezhikang (1200 mg/day, orally) or placebo (same capsules containing only pharmaceutical excipients). Physical examination outcomes, lipid profile, high sensitivity C-reactive protein (hs-CRP) levels, matrix metalloproteinases-9 (MMP-9) levels, and arterial outcomes, including stiffness parameter (ß), pressure-strain elasticity modulus (Ep), arterial compliance (AC), augmentation index (AI), and one-point pulse wave velocity (PWVß) were obtained at baseline and after 6 months of the intervention. Xuezhikang significantly reduced ß (8.4±3.1 vs 6.8±2.1, P=0.007), Ep (122.8±43.9 vs 100.7±33.2, P=0.009), PWVß (6.7±1.2 vs 6.1±1.0, P=0.013), low-density lipoprotein cholesterol (3.4±0.6 vs 2.9±0.5, P=0.001), hs-CRP [2.1 (0.4-10.0) vs 1.4 (0.3-4.1), P=0.020], and MMP-9 (17.2±2.4 vs 12.7±3.8, P <0.001) compared to baseline. The placebo had no effect on these parameters. The changes of PWVß in the xuezhikang group was significantly associated with the changes of hs-CRP and MMP-9 (r=0.144, P=0.043; r=0.278, P=0.030, respectively) but not with lipid profile changes. Our research showed xuezhikang can improve the parameters of arterial stiffness in hypertensive patients, and its effect was independent of lipid lowering.

Xuezhikang reduced arterial stiffness in patients with essential hypertension: a preliminary study

This study aimed to test the effects of xuezhikang, a cholestin extract that contains statin-like components, on arterial stiffness in patients with essential hypertension. One hundred hypertensive patients from the Chinese PLA General Hospital were randomly allocated to receive xuezhikang (1200 mg/day, orally) or placebo (same capsules containing only pharmaceutical excipients). Physical examination outcomes, lipid profile, high sensitivity C-reactive protein (hs-CRP) levels, matrix metalloproteinases-9 (MMP-9) levels, and arterial outcomes, including stiffness parameter (β), pressure-strain elasticity modulus (Ep), arterial compliance (AC), augmentation index (AI), and one-point pulse wave velocity (PWVβ) were obtained at baseline and after 6 months of the intervention. Xuezhikang significantly reduced β (8.4±3.1 vs 6.8±2.1, P=0.007), Ep (122.8±43.9 vs 100.7±33.2, P=0.009), PWVβ (6.7±1.2 vs 6.1±1.0, P=0.013), low-density lipoprotein cholesterol (3.4±0.6 vs 2.9±0.5, P=0.001), hs-CRP [2.1 (0.4-10.0) vs 1.4 (0.3-4.1), P=0.020], and MMP-9 (17.2±2.4 vs 12.7±3.8, P <0.001) compared to baseline. The placebo had no effect on these parameters. The changes of PWVβ in the xuezhikang group was significantly associated with the changes of hs-CRP and MMP-9 (r=0.144, P=0.043; r=0.278, P=0.030, respectively) but not with lipid profile changes. Our research showed xuezhikang can improve the parameters of arterial stiffness in hypertensive patients, and its effect was independent of lipid lowering.

[Epidemiological study on the relationship between the siesta and blood pressure].

OBJECTIVE: Use epidemiological approaches to investigate the correlation between the siesta and blood pressure. METHOD: From March 1(st,) 2011 to June 30(th) 2013, a total of 950 people were collected from East Jiaozhou Qingdao region using variable sampling methods including stratified method, the entire group method, random and proportional methods. Medical professionals conducted a person-to-person survey, collecting the data and inputting it into computers, after which a database was established using STATA 12.0. We analyzed the correlation between the siesta time and blood pressure/hypertension by using rank correlation method (Spearman). Logistic regression method was used to analyze the relationship between high blood pressure and different time and habit of the siesta after adjusting age, sex and BMI. RESULTS: There was a negative correlation between the time of siesta and the systolic pressure with r=-0.18, P<0.001; there was no relationship between the time of siesta and the diastolic pressure with r=-0.07, P=0.02; also, there is a negative correlation between the time of siesta and the hypertension morbidity, with r=-0.22, P<0.001. In the Logistic regression analysis about the period of time to take a nap and the risk of hypertension, it was found that the relative risk factors for hypertension were more than 60-year-old, BMI >25 kg/m(2) and no siesta habits. CONCLUSIONS: The time of siesta is negatively correlated to the systolic pressure, rather than the diastolic pressure, and it can generally reduce the incidence of hypertension. The relative risk factors of hypertension are more than 60-year-old, BMI >25 kg/m(2) and no siesta habits in all four seasons. We recommend that take a nap a day, or it might be even better for systolic blood pressure to take longer siesta.

Effects of IL-10 and glucose on expression of OPG and RANKL in human periodontal ligament fibroblasts.

The effects of interleukin-10 (IL-10) and glucose on mRNA and protein expression of osteoprotegerin (OPG), and its ligand, receptor activator of nuclear factor-κB ligand (RANKL), were investigated in human periodontal ligament fibroblasts (HPDLFs). Primary HPDLFs were treated with different concentrations of IL-10 (0, 1, 10, 25, 50, and 100 ng/mL) or glucose (0, 5.5, 10, 20, 30, and 40 mmol/L). Changes in mRNA and protein expression were examined using the reverse-transcription polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. After IL-10 treatment, mRNA and protein levels of OPG were increased, while mRNA and protein levels of RANKL were decreased (P<0.05), both in a concentration-dependent manner. Glucose stimulation had the opposite concentration-dependent effect to that of IL-10 on OPG and RANKL expression. IL-10 upregulated OPG expression and downregulated RANKL expression, whereas high glucose upregulated RANKL and downregulated OPG in HDPLFs. Abnormal levels of IL-10 and glucose may contribute to the pathogenesis of periodontal disease.

Effects of IL-10 and glucose on expression of OPG and RANKL in human periodontal ligament fibroblasts

The effects of interleukin-10 (IL-10) and glucose on mRNA and protein expression of osteoprotegerin (OPG), and its ligand, receptor activator of nuclear factor-κB ligand (RANKL), were investigated in human periodontal ligament fibroblasts (HPDLFs). Primary HPDLFs were treated with different concentrations of IL-10 (0, 1, 10, 25, 50, and 100 ng/mL) or glucose (0, 5.5, 10, 20, 30, and 40 mmol/L). Changes in mRNA and protein expression were examined using the reverse-transcription polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. After IL-10 treatment, mRNA and protein levels of OPG were increased, while mRNA and protein levels of RANKL were decreased (P<0.05), both in a concentration-dependent manner. Glucose stimulation had the opposite concentration-dependent effect to that of IL-10 on OPG and RANKL expression. IL-10 upregulated OPG expression and downregulated RANKL expression, whereas high glucose upregulated RANKL and downregulated OPG in HDPLFs. Abnormal levels of IL-10 and glucose may contribute to the pathogenesis of periodontal disease.

Bmi1 regulates auditory hair cell survival by maintaining redox balance.

Reactive oxygen species (ROS) accumulation are involved in noise- and ototoxic drug-induced hair cell loss, which is the major cause of hearing loss. Bmi1 is a member of the Polycomb protein family and has been reported to regulate mitochondrial function and ROS level in thymocytes and neurons. In this study, we reported the expression of Bmi1 in mouse cochlea and investigated the role of Bmi1 in hair cell survival. Bmi1 expressed in hair cells and supporting cells in mouse cochlea. Bmi1(-/-) mice displayed severe hearing loss and patched outer hair cell loss from postnatal day 22. Ototoxic drug-induced hair cells loss dramatically increased in Bmi1(-/-) mice compared with that in wild-type controls both in vivo and in vitro, indicating Bmi1(-/-) hair cells were significantly more sensitive to ototoxic drug-induced damage. Cleaved caspase-3 and TUNEL staining demonstrated that apoptosis was involved in the increased hair cell loss of Bmi1(-/-) mice. Aminophenyl fluorescein and MitoSOX Red staining showed the level of free radicals and mitochondrial ROS increased in Bmi1(-/-) hair cells due to the aggravated disequilibrium of antioxidant-prooxidant balance. Furthermore, the antioxidant N-acetylcysteine rescued Bmi1(-/-) hair cells from neomycin injury both in vitro and in vivo, suggesting that ROS accumulation was mainly responsible for the increased aminoglycosides sensitivity in Bmi1(-/-) hair cells. Our findings demonstrate that Bmi1 has an important role in hair cell survival by controlling redox balance and ROS level, thus suggesting that Bmi1 may work as a new therapeutic target for the prevention of hair cell death.

Deficiency of osteoblastic Arl6ip5 impaired osteoblast differentiation and enhanced osteoclastogenesis via disturbance of ER calcium homeostasis and induction of ER stress-mediated apoptosis.

ADP-ribosylation-like factor 6 interacting protein 5 (Arl6ip5), which belongs to the prenylated rab-acceptor-family, has an important role in exocytic protein trafficking, glutathione metabolism and involves in cancer progression. However, its expression pattern and functional role in bone are unknown. Here we demonstrate that Arl6ip5 knock-out mice (Arl6ip5 (Δ2/Δ2)) show marked decrease of bone mineral density, trabecular bone volume and trabecular thickness. Histomorphometric studies reveal that bone formation parameters are decreased but bone resorption parameters and mRNA level of osteoclast-specific markers are increased in Arl6ip5(Δ2/Δ2) mice. In osteoblast, we demonstrate that Arl6ip5 abundantly expresses in osteoblastic cells and is regulated by bone metabolism-related hormones and growth factors. In vitro analysis reveals that osteoblast proliferation and differentiation are impaired in Arl6ip5 knocked-down and deficient primary osteoblast. Arl6ip5 is also found to function as an ER calcium regulator and control calmodulin signaling for osteoblast proliferation. Moreover, Arl6ip5 insufficiency in osteoblast induces ER stress and enhances ER stress-mediated apoptosis. CCAAT/enhancer-binding protein homologous protein (Chop) is involved in the regulation of apoptosis and differentiation in Arl6ip5 knocked-down osteoblasts. For osteoclastogenesis, Arl6ip5 insufficiency in osteoclast precursors has no effect on osteoclast formation. However, knocked-down osteoblastic Arl6ip5 induces receptor activator of nuclear factor-κB ligand (RANKL) expression and enhances osteoclastogenesis. In addition, ER stress and Chop are involved in the RANKL expression in Arl6ip5 knocked-down osteoblasts. In conclusion, we demonstrate that Arl6ip5 is a novel regulator of bone formation in osteoblasts.

Serum levels of high-sensitivity troponin T: a novel marker for left ventricular remodeling and performance in hypertensive subjects.

It has been well established that high-sensitivity cardiac troponin T (hs-TnT) is a specific and highly sensitive marker in acute coronary syndromes. On the other hand, studies on serum concentrations of hs-TnT in patients with hypertension in the absence of significant coronary stenosis are limited. Therefore, we hypothesized that hs-TnT levels are related to left ventricular (LV) remodeling and performance in hypertension. We included 537 hemodynamically stable hypertensive subjects, 247 males aged 60.7 ± 11.1 years, and 100 normotensive subjects of similar age and gender. Clinical examination, clinical assessment and laboratory assays were performed for all hypertensive and normotensive subjects. The detectable rate (>0.003 ng/mL) and elevated rate (>0.013 ng/mL) of hs-TnT were higher in hypertensive subjects than those in normotensive subjects. hs-TnT level gradually increased in hypertensive subjects with LV normal geometry, concentric remodeling, concentric hypertrophy and eccentric hypertrophy. hs-TnT was independently related to age, gender, hypertension, fasting blood glucose, renal function, and LV hypertrophy, and diastolic function on multiple analysis during the whole participation. An increase in hs-TnT levels could be a reliable biomarker of cardiac remodeling and function in hypertension, as an indicator of subclinical ongoing cardiomyocyte injury.

Adipocytes derived from human bone marrow mesenchymal stem cells exert inhibitory effects on osteoblastogenesis.

The infiltration of adipocytes in osteoporotic patients' bone marrow suggests an important regulatory function of bone marrow fat on the development of aged bone. Therefore, we have examined the effects of adipocytes derived from bone mesenchymal stem cell (MSC) on osteoblast differentiation using two different co-culture modes (direct mode and indirect mode). Alkaline phosphatase (ALP)-positive areas and mineralized areas of MSC-derived osteoblasts decrease similarly in the two co-culture modes as the amount of MSC-derived adipocytes increases, suggesting that the crosstalk between adipocytes and osteoblasts may be mainly through secretory factors in the medium. To further understand the molecular mechanisms, both mRNA and protein expressions in osteoblasts in the lower layer of the indirect mode were analyzed, leading to identification of 12 differential genes/proteins. Among them, S100A6 and calreticulin are possibly related to bone formation. S100A6 was down-regulated and calreticulin was up-regulated as MSC-derived adipocytes increased. Similarly, differential expression of these proteins was also observed in bone tissue slides from young (1-month-old) and old (6-month-old) mice. The expression level of ß-catenin in osteoblasts of bone tissues was lower in 6-month-old mice compared to 1-month-old mice. Total TGF-ß analyzed with antibody-based protein microarray and active TGF-ß analyzed with ELISA in the co-cultured cell medium increased consistently as the amount of adipocytes increased. Taken together, our results suggest that MSC-derived adipocytes may regulate osteoblast differentiation in the aged bone through TGF-ß-mediated canonical Wnt signaling.

Immunomodulatory activities of a new pentapeptide (Bursopentin) from the chicken bursa of Fabricius.

The bursa of Fabricius (BF) is a central immune organ in birds, and some peptides from chicken BF have demonstrated important immune functions. Here, a new 626.27 Da pentapeptide, Bursopentin (BP5, Cys-Lys-Arg-Val-Tyr) was isolated and purified by reverse-phase high-performance liquid chromatography. In this study, we examined the effects of BP5 on antigen-specific immune response in BALB/c mice sensitized with inactivated avian influenza virus (AIV) [A/Duck/Jiangsu/NJ08/05 (AIV H9N2 subtype)]. The results suggested that BP5 enhanced anti-hemagglutinin antibody (IgG, the isotypes IgG1 and IgG2a) production, induced both of Th1- (IL-2 and IFN-γ) and Th2-type (IL-4 and -10) cytokines, increased proliferations of splenic lymphocyte subsets CD4+ T cells (CD3+CD4+), CD8+ T cells (CD3+CD8+) and B cells, and enhanced cytotoxic T-lymphocyte activity of the activated splenocytes against NIH3T3 cells. The effects of BP5 on the proliferation of isolated T- and/or B-cell populations of BALB/c mice were assessed, and the data suggested that BP5 promoted spleen lymphocyte proliferation by activating B cells directly and T cells indirectly. Further analysis revealed that B-lymphocyte proliferation induced by BP5 is mediated by reactive oxygen species generated from thiol auto-oxidation of BP5. Furthermore, our data indicated that protein kinase C, mitogen-activated protein kinase, and nuclear factor kappa B are involved in the signal transductions during the BP5-induced B lymphocyte proliferation. This study indicates that BP5 could be a potential immunomodulator for future immuno-pharmacological use.

Exogenous cell-permeable C6 ceramide sensitizes multiple cancer cell lines to Doxorubicin-induced apoptosis by promoting AMPK activation and mTORC1 inhibition.

New chemotherapy-enhancing strategies are needed for better cancer therapy. Previous studies suggest that exogenous cell-permeable C6 ceramide may be a useful adjunct to the anti-tumor effects of chemotherapeutic agents (such as Taxol) against multiple cancers. Here we demonstrate that exogenous cell-permeable C6 ceramide largely sensitizes multiple progressive cancer cell lines to Doxorubicin-induced cell death and apoptosis. We found for the first time that Doxorubicin induces AMP-activated protein kinase (AMPK) activation in a reactive oxygen species-dependent manner. Activation of AMPK contributes to Doxorubicin-induced cancer cell death and apoptosis. Inhibition of AMPK by small interfering RNA knockdown or a pharmacological inhibitor reduces Doxorubicin-induced cancer cell apoptosis, whereas AMPK activator AICAR enhances it. Importantly, we found that C6 ceramide largely enhances Doxorubicin-induced activation of AMPK, which leads to mTOR complex 1 inhibition and chemo-sensitization. Our data suggest that the combination of C6 ceramide with traditional chemotherapy drugs such as Doxorubicin may have the potential to be used as a new therapeutic intervention against multiple cancers.

Therapeutic potential of non-adherent BM-derived mesenchymal stem cells in tissue regeneration.

We demonstrated that non-adherent BM cells (NA-BMCs) can be expanded in suspension and give rise to multiple mesenchymal phenotypes including fibroblastic, osteoblastic, chondrocytic and adipocytic as well as glial cell lineages in vitro using the 'pour-off' BMC culture method. Mesenchymal stem cells (MSCs) derived from NA-BMCs (NA-MSCs) from wild-type mice were transplanted into VDR gene knockout (VDR(-/-)) mice that had received a lethal dose of radiation. Results revealed that NA-MSC can be used to rescue lethally irradiated mice and become incorporated into a diverse range of tissues. After lethal dose irradiation, all untransplanted mice died within 2 weeks, whereas those transplanted with NA-MSCs were viable for at least 3 months. Transplantation rescued these mice by reconstructing a hematopoietic system and repairing other damaged tissues. WBC, RBC and platelet counts recovered to normal after 1 month, and VDR gene expression was found in various tissues of viable VDR(-/-) recipients. Adult BM harbors pluripotent NA-MSCs, which can migrate in vivo into multiple body organs. In an appropriate microenvironment, they can adhere, proliferate and differentiate into specialized cells of target tissues and thus function in damaged tissue regeneration and repair.

A comparison of a blocking ELISA and a haemagglutination inhibition assay for the detection of antibodies to Avibacterium (Haemophilus) paragallinarum in sera from artificially infected chickens.

The ability of blocking ELISAs and haemagglutination-inhibition (HI) tests to detect antibodies in sera from chickens challenged with either Avibacterium (Haemophilus) paragallinarum isolate Hp8 (serovar A) or H668 (serovar C) was compared. Serum samples were examined weekly over the 9 weeks following infection. The results showed that the positive rate of serovar A specific antibody in the B-ELISA remained at 100% from the second week to the ninth week. In chickens given the serovar C challenge, the highest positive rate of serovar C specific antibody in the B-ELISA appeared at the seventh week (60% positive) and was then followed by a rapid decrease. The B-ELISA gave significantly more positives at weeks 2, 3, 7, 8 and 9 post-infection for serovar A and at week 7 post-infection for serovar C. In qualitative terms, for both serovar A and serovar C infections, the HI tests gave a lower percentage of positive sera at all time points except at 9 weeks post-infection with serovar C. The highest positive rate for serovar A HI antibodies was 70% of sera at the fourth and fifth weeks post-infection. The highest rate of serovar C HI antibodies was 20% at the fifth and sixth weeks post-infection. The results have provided further evidence of the suitability of the serovar A and C B-ELISAs for the diagnosis of infectious coryza.

Long-term prevention of diabetes and marked suppression of insulin autoantibodies and insulitis in mice lacking native insulin B9-23 sequence.

We analyzed double native insulin gene knockout NOD mice with a mutated (B16:alanine) proinsulin transgene at multiple ages for the development of insulin autoantibodies, insulitis, and diabetes. In contrast to mice with at least one copy of a native insulin gene that expressed insulin antibodies, only 2 out of 21 (10%) double native insulin gene knockout mice with a mutated insulin transgene developed insulin autoantibodies. Of 21 double insulin knockout mice sacrificed between 10 to 48 weeks of age, only 5 showed minimal insulitis versus 100% of wild-type NOD and more than 90% of insulin 1 knockout mice. Consistent with robust suppression of insulin autoantibodies and insulitis, no double insulin knockout mice developed diabetes. In that the B9-23 peptide with B16A is an altered peptide ligand inducing Th2 responses, we analyzed transfer of splenocytes into NOD.SCID mice. There was no evidence for regulatory T cells able to inhibit transfer of diabetes by diabetogenic NOD splenocytes. Insulin peptide B9-23 is likely a crucial target for initiation of islet autoimmunity and further mutation of the sequence will be tested to attempt to eliminate all anti-islet autoimmunity.

A new model of insulin-deficient diabetes: male NOD mice with a single copy of Ins1 and no Ins2.

AIMS/HYPOTHESIS: We describe a novel model of insulin-deficient diabetes with a single copy of the gene encoding insulin 1 (Ins1) and no gene encoding insulin 2 (Ins2). MATERIALS AND METHODS: We constructed five lines of mice: mice with two copies of Ins1 (NOD( Ins1+/+,Ins2-/-)), mice with a single copy of Ins1 (NOD( Ins1+/-,Ins2-/-)), mice with two copies of Ins2 (NOD( Ins1-/-,Ins2+/+)), mice with a single copy of Ins2 (NOD( Ins1-/-,Ins2+/-)) and NOD( Ins1+/-,Ins2-/-) mice with a transgene encoding B16:Ala proinsulin. RESULTS: By 10 weeks of age, all male NOD( Ins1+/-,Ins2-/-) mice were diabetic, whereas all female NOD( Ins1+/-,Ins2-/-) were not diabetic (p < 0.0001). In contrast, neither male nor female NOD( Ins1-/-,Ins2+/-) with a single copy of Ins2 (rather than single copy of Ins1) developed early diabetes and no mice with two copies of either gene developed early diabetes. Islets of the diabetic male NOD( Ins1+/-,Ins2-/-) at this early age had no lymphocyte infiltration. Instead there was heterogeneous (between islet cells) weak staining for insulin. Although only male NOD( Ins1+/-,Ins2-/-) mice developed diabetes, both male and female NOD( Ins1+/-,Ins2-/-) mice had markedly decreased insulin content. In NOD( Ins1+/+,Ins2-/-), there was also a significant decrease in insulin content, whereas NOD( Ins1-/-,Ins2+/+) mice, and even NOD( Ins1-/-,Ins2+/-) mice, were normal. Male NOD( Ins1+/-,Ins2-/-) mice were completely rescued from diabetes by introduction of a transgene encoding proinsulin. On i.p. insulin tolerance testing, male mice had insulin resistance compared with female mice. CONCLUSIONS/INTERPRETATION: These results suggest that Ins1 is a 'defective gene' relative to Ins2, and that the mouse lines created provide a novel model of sex-dimorphic insulin-deficient diabetes.