Activation of the hypoxia-inducible factor-1 in overloaded temporomandibular joint, and induction of osteoclastogenesis

Vascular endothelial growth factor (Vegf) was previously shown to be expressed specifically in the condylar cartilage of temporomandibular joint-osteoarthritis (TMJ-OA) model rats. Here we demonstrate for the first time that hypoxia-inducible factor-1α (Hif-1α) is activated in mature chondrocytes of temporomandibular joint-osteoarthritis (TMJ-OA) model rat by mechanical overload, and that activated Hif-1 in chondrocytes can induce osteoclastogenesis via repression of osteoprotegerin (Opg) expression.In rat TMJs, degeneration of the condylar cartilage became prominent in proportion to the duration of overloading. Hif-1α expression was observed specifically in mature and hypertrophic chondrocytes, and Hif-1α-positivity, level of Vegf expression, and tartrate-resistant acid phosphatase (TRAP)-positive cell numbers all increased in the same manner. When ATDC5 cells induced differentiation by insulin were cultured under hypoxia, Hif-1α induction was observed in mature stage, but not in immature stage. Inductions of Hif-1-target genes showed a similar expression pattern. In addition, expression of Opg decreased in hypoxia, and Hif-1α played a role, in part, in its regulation.     

Estimating stand volume in broad-leaved forest using discrete-return LiDAR: plot-based approach

Quantitative assessment of forests is important at a variety of scales for forest planning and management. This study investigated the use of small-footprint discrete-return lidar for estimating stand volume in broad-leaved forest at plot level. Field measurements were conducted at 20 sample plots in the study area in western Japan, composed of temperate broad-leaved trees. Five height variables and two density variables were derived from the lidar data: 25th, 50th, 75th, and 100th percentiles, and mean of laser canopy heights as height variables (h ₂₅, h ₅₀, h ₇₅, h ₁₀₀, h _mean); and ground fraction and only-and-vegetation fraction (d _GF, d _OVF) as density variables, defined respectively as the proportion of laser returns that reached the ground, and the proportion of only echoes (i.e., single pulse returns for which the first and last pulses returned from the same point) within vegetation points. In addition, the normalized difference vegetation index (NDVI), which is often used as an estimator for leaf area index (LAI) and above-ground biomass, was derived from multispectral digital imagery as an alternative density variable (d _NDVI). Nonlinear least-square regression with cross-validation analysis was performed with single variables and combinations; a total of 23 models were studied. The best prediction was found when h ₇₅ and d _OVF were used as independent variables, resulting in adjusted R ² of 0.755 and root-mean-square error (RMSE) of 41.90 m³ ha⁻¹, corresponding to 16.4% of the mean stand volume, better than or comparable to the prediction models of previous studies.     

Incidence of symptomatic vertebral fractures in women of childbearing age newly treated with high-dose glucocorticoid

Background: The treatment and prevention of glucocorticoid (GC)-induced osteoporosis have been controversial in premenopausal women during their childbearing years.Objective: This study assessed the incidence and risk factors for symptomatic vertebral fracture in women of childbearing age newly treated with high-dose GC.Methods: An observational cohort study was conducted at the rheumatic center of Shimoshizu National Hospital in Chiba, Japan, from 1986 to 2006. The prevalence of symptomatic vertebral fractures, as determined by x-rays, was assessed in premenopausal (aged <50 years) women with collagen vascular disease newly treated with high-dose GC (≥20 mg/d prednisolone equivalent) compared with their counterparts who did not receive GC. Differences in the incidences of vertebral fractures were compared between groups by the Kaplan-Meier method and evaluated by the log-rank test. Hazard ratios (HRs) with 95% CIs were estimated using the Cox proportional hazards regression model.Results: A total of 373 women were assessed: 292 patients in the high-dose GC treatment group (mean [SD] initial age, 32.4 [8.2] years; initial dose, 43.8 [14.9] mg/d; follow-up time, 124.2 [75.4] months) and 81 patients in the non-GC control group (initial age, 39.3 [7.8] years; follow-up time, 106.5 [79.7] months). Symptomatic vertebral fractures occurred more frequently in the high-dose GC group (11.3%) than in the non-GC group (1.2%). Using the Cox model, the adjusted HR for the high-dose GC group was 13.96 (95% CI, 1.87–104.22) relative to the non-GC group. In the high-dose GC group, Kaplan-Meier analyses revealed that the incidence of fractures in women in their forties was significantly higher in comparison with those in their twenties (P < 0.001) and thirties (P < 0.05), and that the incidence of fractures in those who consumed alcohol (>80 g/wk of pure alcohol) was significantly higher than in those who did not (P < 0.05). The Cox model also revealed that the risk was independently higher with every 10-year increment of initial age (HR = 2.27; 95% CI, 1.46–3.53), with every GC dose increase (HR = 2.28; 95% CI, 1.58–3.31), and with each 1-gram decrease of cumulative GC dose (HR = 0.95; 95% CI, 0.93–0.98).Conclusions: In this study, high-dose GC use was associated with a significantly high prevalence of symptomatic vertebral fractures in premenopausal women with collagen vascular disease during their childbearing years. However, the fracture risk was relatively low in women of childbearing age, especially those in their twenties and thirties during the early years of treatment.     

Combined genotoxic effects of radiation and a tobacco-specific nitrosamine in the lung of gpt delta transgenic mice

It is important to evaluate the health effects of low-dose-rate or low-dose radiation in combination with chemicals as humans are exposed to a variety of chemical agents. Here, we examined combined genotoxic effects of low-dose-rate radiation and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), the most carcinogenic tobacco-specific nitrosamine, in the lung of gpt delta transgenic mice. In this mouse model, base substitutions and deletions can be separately analyzed by gpt and Spi- selections, respectively. Female gpt delta mice were either treated with gamma-irradiation alone at a dose rate of 0.5, 1.0 or 1.5 mGy/h for 22 h/day for 31 days or combined with NNK treatments at a dose of 2 mg/mouse/day, i.p. for four consecutive days in the middle course of irradiation. In the gpt selection, the NNK treatments enhanced the mutation frequencies (MFs) significantly, but no obvious combined effects of gamma-irradiation were observable at any given radiation dose. In contrast, NNK treatments appeared to suppress the Spi- large deletions. In the Spi- selection, the MFs of deletions more than 1 kb in size increased in a dose-dependent manner. When NNK treatments were combined, the dose-response curve became bell-shaped where the MF at the highest radiation dose decreased substantially. These results suggest that NNK treatments may elicit an adaptive response that eliminates cells bearing radiation-induced double-strand breaks in DNA. Possible mechanisms underlying the combined genotoxicity of radiation and NNK are discussed, and the importance of evaluation of combined genotoxicity of more than one agent is emphasized.     

Altered B:9-23 insulin, when administered intranasally with cholera toxin adjuvant, suppresses the expression of insulin autoantibodies and prevents diabetes

Insulin peptide B:9-23 is a major autoantigen in type 1 diabetes that contains two distinct CD4 epitopes (B:9-16 and B:13-23). One of the two epitopes, B:13-23, overlaps with a CTL epitope (B:15-23). In this study, we report that the elimination of the CTL epitope from the B:9-23 peptide by amino acid substitution (with alanine) at positions B:16 and 19 (A16,19 altered peptide ligand) or truncation of the C-terminal amino acids from the peptide (B:9-21), neither of which stimulated the proliferation of insulin B:15-23 reactive CD8 T cells, provided significant intranasally induced suppression of diabetes when coadministered with a potent mucosal adjuvant cholera toxin (CT). Intranasal treatment with A16,19 resulted in the elimination of spontaneous insulin autoantibodies, significant inhibition of insulitis and remission from hyperglycemia, and prevented the progression to diabetes. Intranasal administration of native B:9-23/CT or B:11-23/CT resulted in a significant enhancement of insulin autoantibody expression and severity of insulitis and failed to prevent diabetes. Our present study indicates that elimination of the CTL epitope from the B:9-23 peptide was critically important for mucosally induced diabetes prevention. The A16,19 altered peptide ligand, but not other native insulin peptides, suppresses insulin autoantibodies associated with protection from and remission of diabetes.     

The Functional Interrelationship between Gap Junctions and Fenestrae in Endothelial Cells of the Liver Organoid

Functional intact liver organoid can be reconstructed in a radial-flow bioreactor when human hepatocellular carcinoma (FLC-5), mouse immortalized sinusoidal endothelial M1 (SEC) and A7 (HSC) hepatic stellate cell lines are cocultured. The structural and functional characteristics of the reconstructed organoid closely resemble the in vivo liver situation. Previous liver organoid studies indicated that cell-to-cell communications might be an important factor for the functional and structural integrity of the reconstructed organoid, including the expression of fenestrae. Therefore, we examined the possible relationship between functional intact gap junctional intercellular communication (GJIC) and fenestrae dynamics in M1-SEC cells. The fine morphology of liver organoid was studied in the presence of (1) irsogladine maleate (IM), (2) oleamide and (3) oleamide followed by IM treatment. Fine ultrastructural changes were studied by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) and compared with control liver organoid data. TEM revealed that oleamide affected the integrity of cell-to-cell contacts predominantly in FLC-5 hepatocytes. SEM observation showed the presence of fenestrae on M1-SEC cells; however, oleamide inhibited fenestrae expression on the surface of endothelial cells. Interestingly, fenestrae reappeared when IM was added after initial oleamide exposure. GJIC mediates the number of fenestrae in endothelial cells of the liver organoid.     

Protein kinase C-delta phosphorylates Ebp1 and prevents its proteolytic degradation, enhancing cell survival

ErbB3-binding protein (Ebp1) promotes cell survival by preventing apoptotic DNA fragmentation through a complex with active nuclear Akt. Ebp1 phosphorylation by protein kinase C (PKC)-delta mediates its binding to nuclear Akt. In this study, we show that Ebp1 itself acts as a substrate of active caspase 3 during the programmed cell death. PKC-delta phosphorylation on Ebp1 protects it from apoptotic degradation initiated in cell-free apoptotic solution. Moreover, Ebp1 is evidently cleaved in PKC-delta-deficient cells but not in wild-type cells. Ebp1 translated from first ATG is resistant to apoptotic cleavage; by contrast, Ebp1 from second and third ATG demonstrates robust degradation, and PKC phosphorylation on S360 suppresses its cleavage by active caspase 3. Ebp1 can be digested at both D53 and D196 sites, but cleavage at D196 appears to be a prerequisite for its further degradation at D53 site. Compared with wild-type Ebp1, D196A mutant markedly protects cells from apoptosis. Thus, PKC-delta antagonizes apoptosis through phosphorylating Ebp1 and protects it from apoptotic degradation.     

Facile determination of DNA-binding nuclear factor-kappaB by chemiluminescence detection

A simple, rapid, and sensitive method for the assay of a sequence-specific DNA-binding protein, nuclear factor-kappaB (NF-kappaB), has been developed by using a DNA-detectable chemiluminogenic reagent and a centrifugal filter that distinguishes different molecular sizes. After the formation of a complex between NF-kappaB and DNA, the unbound DNA is separated from the complex by the centrifugal filter. The amount of the bound NF-kappaB is estimated by chemiluminescence detection of the bound DNA. This detection is performed within 2 min at room temperature by the use of a chemiluminogenic reagent, 3',4',5'-trimethoxyphenylglyoxal, which selectively recognizes guanine moiety in oligonucleotides or DNAs. This method does not require any labeled probes or antibodies and can determine a concentration as low as 5 nM of DNA-binding NF-kappaB. The sensitivity is nearly the same as that of other methods such as gel shift assay using fluorescence-labeled probes and enzyme-linked immunosorbent assay. Therefore, the current method provides a convenient tool for surveying various DNA-binding proteins.