好アルカリ性Bacillus A-007株のK^+ : 促進ATPaseについて

1.好アルカリ性Bacillus A-007株の生育にとってK^+は必須であった.2.K^+-濃度を制限した培地(1.5mMK^+)で生育させた細胞の膜画分に, K^+により促進されるATPase活性が認められた.3.K^+促進ATPaseは, 動力学的特性及びウワバイン, NaN_3, PCMBに対する感受件において, 同菌株のH^+-ATPaseと明らかに異なっていた.1. K^+ was essential for the growth of an alkalophilic Bacillus A-007. 2. Membrane fraction, which was prepared from the cells grown in K^+ -limited medium (1.5mM K^+), showed K^+ -stimulated ATPase activity. 3. The K^+ -stimulated ATPase was clearly different from H^+ -ATPase on kinetical profile and ouabain-, NaN_3- and PCMB-sensvtivity

TNF Accelerates Death of Mandibular Condyle Chondrocytes in Rats with Biomechanical Stimulation-Induced Temporomandibular Joint Disease

<div><p>Objective</p><p>To determine if temporomandibular joint chondrocyte apoptosis is induced in rats with dental biomechanical stimulation and what a role TNF takes.</p><p>Methods</p><p>Thirty-two rats were divided into 4 groups (n = 8/group) and exposed to incisor mal-occlusion induced by unilateral anterior crossbite biomechanical stimulation. Two groups were sampled at 2 or 4 weeks. The other two groups were treated with local injections of a TNF inhibitor or PBS into the temporomandibular joints area at 2 weeks and then sampled at 4 weeks. Twenty-four rats either served as unilateral anterior crossbite mock operation controls (n = 8/group) with sampling at 2 or 4 weeks or received a local injection of the TNF inhibitor at 2 weeks with sampling at 4 weeks. Chondrocytes were isolated from the temporomandibular joints of 6 additional rats and treated with TNF <i>in vitro</i>. Joint samples were assessed using Hematoxylin&eosin, Safranin O, TUNEL and immunohistochemistry staining, real-time PCR, fluorogenic activity assays and Western blot analyses. The isolated chondrocytes were also analyzed by flow cytometry.</p><p>Results</p><p>Unilateral anterior crossbite stimulation led to temporomandibular joint cartilage degradation, associated with an increase in TUNEL-positive chondrocytes number, caspase-9 expression levels, and the release of cytochrome c from mitochondria at 2 weeks without changes in TNF and caspase-8 levels until after 4 weeks. TNF stimulated apoptosis of the isolated chondrocytes and up-regulated caspase-8 expression, but did not change caspase-9 expression levels. Local injection of TNF inhibitor down-regulated caspase-8 expression and reduced TUNEL-positive cell number, but did not reverse cartilage thickness reduction, caspase-9 up-regulation or cytochrome c release.</p><p>Conclusions</p><p>Unilateral anterior crossbite stimulation induces mitochondrion-mediated apoptosis of articular chondrocytes. TNF accelerated the unilateral anterior crossbite induced chondrocytes apoptosis via death-receptor pathway. However, anti-TNF therapy does not prevent cartilage loss in this model of temporomandibular joint.</p></div

Condylar cartilage Safranin O staining.

<p><b>(a).</b> Safranin O staining between groups: 2-week Control (2w Control), 2-week UAC (2w UAC), 4-week Control (4w Control), 4-week UAC (4w UAC), UAC+Inhibitor (UAC+Inhib), Control+Inhibitor (Control+Inhib) and UAC+PBS (UAC+PBS). <b>(b).</b> Comparison of Safranin O staining positive areas in the central and posterior thirds. The data are expressed as the means and 95% CIs. n = 3.</p

Cell identification and detection of apoptosis in cultured chondrocytes.

<p><b>(a).</b> Chondrocytes identified by toluidine blue staining. <b>(b).</b> Aggrecan staining (Primary antibody: abcam, Ab36861. Secondary antibody: Zhongshan Co. Ltd, China, Cy3-conjugated goat anti-rabbit IgG). <b>(c).</b> Type II collagen dyeing (Primary antibody: Santa Cruz, sc7763. Secondary antibody: Zhongshan Co. Ltd, China, Cy3-conjugated donkey anti-goat IgG). <b>(d).</b> Type I collagen dyeing (Primary antibody: Abcam, ab90395. Secondary antibody: Zhongshan Co. Ltd, China, FITC-conjugated goat anti-mouse IgG). <b>(e).</b> Chondrocyte apoptosis detected by flow cytometry after stimulation with TNF at dosages of 10 ng/ml, 50 ng/ml or 100 ng/ml for 24 h.</p

Gene primers.

<p>Gene primers.</p

Sagittal central section of the TMJ in the control group stained with hematoxylin & eosin.

<p><b>(a).</b> Six squares denoting the areas on slides where immunohistochemical and TUNEL staining were performed. D, articular disc; C, mandibular condyle. (<b>b).</b> Sagittal central section of the TMJ condylar cartilage showing the four cellular layers.</p

The mRNA and protein levels of apoptotic pathway components <i>in vivo</i>.

<p>A comparison of the apoptotic activity in the mandibular condylar cartilage between the 2-week Control (2w Control), 2-week UAC (2w UAC), 4-week Control (4w Control), 4-week UAC (4w UAC), UAC+Inhibitor (UAC+Inhib), Control+Inhibitor (Control+Inhib) and UAC+PBS (UAC+PBS) groups. The mRNA expression levels of caspase-8 <b>(a)</b> and caspase-9 (<b>b</b>). The activities of caspase-8 <b>(c)</b> and caspase-9 <b>(d)</b> units. Western blot for the release of cytochrome c from the mitochondria into the cytoplasm <b>(e)</b> and its quantification <b>(f)</b>. The data are expressed as the means and 95% CIs. n = 3.</p

The mRNA and protein levels of apoptosis <i>in vitro</i>.

<p>A comparison of the mRNA expression levels of caspase-8 <b>(a)</b> and caspase-9 <b>(b)</b> in the cultured chondrocytes between groups with or without TNF (10 ng/ml, 50 ng/ml and 100 ng/ml) stimulation for 24 h. Comparison of the activities of caspase-8 <b>(c)</b> and caspase-9 <b>(d)</b> units between the groups with or without TNF (10 ng/ml, 50 ng/ml and 100 ng/ml) stimulation for 24 h. Western blot measuring the release of cytochrome c from the mitochondria to the cytoplasm <b>(e)</b> and comparisons between the groups with or without TNF (10 ng/ml, 50 ng/ml and 100 ng/ml) stimulation for 24 h <b>(f)</b>. The columns represent the mean values with 95% CIs. n = 3.</p

The mRNA and protein levels <i>in vitro</i> after treatment with TNF inhibitor.

<p>A comparison of the mRNA expression levels of caspase-8 <b>(a)</b> and caspase-9 <b>(b)</b> in the cultured chondrocytes stimulated for 24 h between the groups: control (Con), TNF 100 ng/ml (TNF) and TNF 100 ng/ml plus TNF inhibitor 10ug/ml (Inhib). Comparison of the activities of caspase-8 <b>(c)</b> and caspase-9 <b>(d)</b> units between the groups with stimulation for 24 h. Western blot measuring the release of cytochrome c from the mitochondria to the cytoplasm <b>(e)</b> and comparisons between the groups with stimulation for 24 h <b>(f)</b>. The columns represent the mean values with 95% CIs. n = 3.</p

Condylar cartilage histomorphology staining.

<p><b>(a).</b> A comparison of the TMJ condylar cartilage histomorphology between groups: 2-week Control (2w Control), 2-week UAC (2w UAC), 4-week Control (4w Control), 4-week UAC (4w UAC), UAC+Inhibitor (UAC+Inhib), Control+Inhibitor (Control+Inhib) and UAC+PBS (UAC+PBS). Areas in 2w UAC and 4w UAC where cells have died are indicated by points. <b>(b).</b> Comparison of cartilage thickness in the central and posterior thirds. The data are expressed as the means and 95% CIs. n = 3.</p