Histology of the articular cartilage of knee joints from CSS2^−/− (CSS2KO) mice and their wild-type (WT) littermates.

<p>Sections were stained with Safranin-O, which stains sulfated glycosaminoglycans, and fast green for counter staining. Each image shown is one representative section selected from 50 sections from an experimental group (10 sections from each knee joint and 5 knee joint in each experimental group). There were no obvious degradations of articular surface in knee joints shown and no morphologic differences among the groups at ages 1 and 6 months (1 M and 6 M).</p

Expression level of CSSs in cartilage of CSS2^−/− (CSS2KO) mice and their wild-type (WT) littermates.

<p>Realtime RT-PCR using cDNA synthesized from humerus of newborn mice were performed to investigate relative expression levels of CSS1, CSS2, ChSy3, Csgalnact1, and Csgalnact2. The amount of expression was normalized by GAPDH. All experiments were performed three times independently, and bars in the graphs were shown as a mean ± S.D. *; p<0.05.</p

Structure of CS Chains in cartilage of newborn mice.

<p><i>A</i>, CS extracted from proximal humeral cartilage of newborn mice was labeled with [³H]-sodium borohydride, applied to a Superose 6 column with effluent fractions of 0.5 ml each, and analyzed for radioactivity. The elution profiles of the samples obtained from CSS2^−/− (CSS2KO) (×) mice and wild-type (WT) (•) littermates are shown. Three independent experiments (n = 3) showed the same elution profile. <i>Numbered arrow 3.3</i>, <i>10</i>, and <i>30</i> indicate the eluted positions of chondroitin polysaccharides of known sizes (molecular size, 3,300, 10,000, and 30,000, respectively). <i>B</i>, Each total radioactivity of peaks of CSS2KO mice and WT littermates in Fig. 4A is shown. <i>C</i>, CS isolated from proximal humeral cartilage of newborn mice were digested with chondroitinase ABC and subjected to reverse-phase ion pair chromatography with postcolumn fluorescence labeling as described in “Experimental Procedures”. The histogram shows the amount and compositions of unsaturated disaccharide in the CS isolated from cartilage of CSS2KO (▪) mice and WT (□) littermates. All experiments were performed three times independently, and bars in the graphs were shown as a mean ± S.D. *, <i>p</i><0.01. <i>0S</i>, <i>4S</i>, and <i>6S</i>, represent ΔDi-0S, ΔDi-4S, and ΔDi-6S, respectively.</p

Primers for realtime PCR.

<p>Primers for realtime PCR.</p

Generation of CSS2^−/− mice.

<p>A, Genomic structure of CSS2 gene, the targeting vector, and the genome with homologous recombination and that whose fragments were excised by flippase and Cre systems. <i>A</i>, The genomic structure of CSS2 gene is depicted on the top line (a). CSS2 targeting vector (b) was constructed by flanking exon 1 with <i>loxP</i> sites, and flanking a Neo^R cassette with FRT sites. ES cell clones with homologous recombination of the vector segment (c) were obtained by positive selection, and used for generation of chimera mice, followed by germ line transmission. The FRT-flanked Neo cassette was subsequently deleted from the recombinant allele by crossing with CAG-Flp Tg mice (d). Then, a genomic fragment containing exon 1, flanked by loxP sites, was excised from the recombinant allele by crossing with CAG-Cre Tg mice (e). <i>B</i>, Genomic PCR. Genotyping was performed by PCR using tail DNA and primers shown in black arrows in Fig. 1A. The PCR product of wild-type allele and CSS2 mutant allele was 2.1 kb and 420 bp respectively. <i>C</i>, Immunoprecipitation of the CSS2, followed by western blot. The cell lysates obtained from WT MEFs were subjected to western blot analysis as described in “Experimental Procedures”. Western blot analysis shows bands of CSS2 (85 kDa) and the CSS2 variant (68 kDa).</p

Effects of CSS1 siRNA in CSS2^−/− (CSS2KO) and wild-type (WT) chondrocytes on CS synthesis.

<p>Primary chondrocytes obtained from CSS2KO and WT mice were downregulated of CSS1 using siRNA, as described in “Experimental Procedures”. All experiments were performed three times independently, and bars in the graphs were shown as a mean ± S.D. <i>A</i>, Relative expression level of CSS1 and CSS2 in WT and CSS2^−/− chondrocytes were shown. siRNA, cells transfected with CSS1 siRNA; control, cells transfected with control siRNA; mock, untransfected cells. <i>B</i>, The total radioactivity of the elution profiles in each sample. <i>C</i>, Chain length of CS in chondrocytes. Three independent experiments (n = 3) showed the same elution profile. <i>Numbered arrow 3.3</i>, <i>10</i>, and <i>20</i> indicate the eluted positions of chondroitin polysaccharides of known sizes (molecular size, 3,300, 10,000, and 20,000, respectively).</p

Skeletal analysis of CSS2^−/− (CSS2KO) mice and their wild-type (WT) littermates at newborn and age of 1 month.

<p><i>A</i>, skeletal structure of CSS2KO and WT newborn mice stained with alcian blue and alizarin red. Whole body (a, b), upper extremity (c, d), and lower extremity (e, f) of WT littermates and CSS2KO mice, respectively, were shown. Scale bar: 500 µm. <i>B</i>, Proximal humerus of WT and CSS2KO newborn mice stained with hematoxylin-eosin is shown. The area size, the number and orientation of chondrocytes are similar between WT littermates (g) and CSS2KO (h) mice. The lengths of proliferative (P), prehypertrophic (PH), and hypertrophic (H) zones of WT (□) littermates and CSS2KO (▪) mice are shown in the histogram. Bar graph shown in the graphs indicate a mean ± S.D. of 10 samples’ measurements (n = 10). Large scale bar: 50 µm, small scale bar: 5 µm. <i>C</i>, skeletal structure of CSS2KO and WT 1-month-old mice stained with alcian blue and alizarin red. Whole body (i, j), upper extremity (k, l), and lower extremity (m, n) of WT littermates and CSS2KO mice, respectively, were shown. Scale bar: 5 mm. <i>D</i>, The histogram shows comparison of the length of humerus, ulna, femur, and tibia of WT (□) littermates and CSS2KO (▪) mice. Bar graph shown in the graphs indicate a mean ± S.D. of 10 samples’ measurements (n = 10). *; p<0.005.</p

Versican A-subdomain is required for its adequate function in dermal development

<p>Versican, a large chondroitin sulfate (CS) proteoglycan, serves as a structural macromolecule of the extracellular matrix (ECM) and regulates cell behavior. We determined the function of versican in dermal development using Vcan^Δ3/Δ3 mutant mice expressing versican with deleted A-subdomain of the N-terminal G1 domain. The mutant versican showed a decreased hyaluronan (HA)-binding ability and failed to accumulate in the ECM. In the early developmental stage, Vcan^Δ3/Δ3 dermis showed a decrease in versican expression as compared with WT. As development proceeded, versican expression further decreased to a barely detectable level, and Vcan^Δ3/Δ3 mice died at the neonatal period (P0). At P0, Vcan^Δ3/Δ3 dermis exhibited an impaired ECM structure and decreased cell density. While the level of collagen deposition was similar in both genotypes, collagen biosynthesis significantly decreased in Vcan^Δ3/Δ3 fibroblasts as compared with that in wild type (WT). Transforming growth factor β (TGFβ) signaling mediated through the Smad2/3-dependent pathway was down-regulated in Vcan^Δ3/Δ3 fibroblasts and a reduced TGFβ storage in the ECM was observed. Microarray analysis revealed a decrease in the expression levels of transcription factors, early growth response (Egr) 2 and 4, which act downstream of TGFβ signaling. Thus, our results suggest that A-subdomain is necessary for adequate versican expression in dermis and that versican is involved in the formation of the ECM and regulation of TGFβ signaling.</p

Data_Sheet_1_Ketogenic effects of medium chain triglycerides containing formula and its correlation to breath acetone in healthy volunteers: a randomized, double-blinded, placebo-controlled, single dose-response study.docx

The efficacy of low-carbohydrate, high-fat diets, such as ketogenic diets, for cancer patients is of research interest. We previously demonstrated the efficacy of the ketogenic diet in a case study in which medium-chain triglycerides (MCTs) or MCT-containing formula (ketogenic formula) was used as a supplement to increase blood ketone bodies. However, little is known about the amounts needed to induce ketogenic effects and about the usefulness of monitoring of breath acetone. To investigate the pharmacokinetics of MCTs and their metabolites, blood ketone bodies and breath acetone, 24 healthy subjects received one of four single oral doses of the ketogenic formula (equivalent to 0, 10, 20, and 30 g of MCTs) under fasting conditions. Total blood ketone bodies, β-hydroxybutyrate, octanoic acid, and decanoic acid were increased in a dose-dependent manner. The ketogenic effect was considered to depend on octanoic and decanoic acids, because a positive correlation was observed between them. A strong positive correlation was also observed between total serum ketone bodies and breath acetone at each time points. Therefore, monitoring breath acetone levels seems a less invasive method to predict blood concentrations of ketone bodies during ketogenic diet therapy.Clinical trial registration:https://rctportal.niph.go.jp/en/detail?trial_id=UMIN000032634, UMIN-CTR UMIN000032634.</p