Investigation of the Effect of Nutritional Supplementation with Whey Protein and Vitamin D on Muscle Mass and Muscle Quality in Subacute Post-Stroke Rehabilitation Patients: A Randomized, Single-Blinded, Placebo-Controlled Trial.

In post-stroke hemiparesis patients, the skeletal muscle mass decrease rapidly with the histological degradation. We investigated the effect of nutritional supplementation with whey protein and vitamin D on the muscle mass and muscle quality, in post-stroke convalescent rehabilitation patients in a randomized, single-blinded, placebo-controlled trial. Fifty patients were randomly assigned to two groups; HP group received supplemental jelly (100 kcal; whey protein 10 g; vitamin D 20 µg) twice a day throughout up to 16-week period, the control group received placebo jelly. Cross-sectional area (CSA) of thigh muscle, skeletal muscle index (SMI), muscle strength, activity of daily living (ADL), and some nutritional indicators in blood were measured. Although no significant difference was observed in CSA and SMI between the groups, fat infiltration into the thighs muscle was singnificantly lower in the HP group. There were no significant difference in muscle strength and ADL between the groups. Blood urea nitrogen and serum 25(OH)D at endpoint were significantly higher in the HP group but physiological normal ranges. Supplementation with whey protein and vitamin D in post-stroke patients led to suppression of fat infiltration into the muscle. Long-term follow-up studies are needed to verify whether this nutritional intervention provides substantial benefits for the prognosis of stroke survivors.

Damage sensitivity of dental zirconias to simulated occlusal contact.

OBJECTIVE: Mechanical damages can occur from dental restoration processing and fitting, or while it is in-service. This study evaluates the damage sensitivity of translucent zirconia (5Y-PSZ) relative to conventional 3Y-PSZ following mouth-motion simulations at various loads. METHODS: 5Y-PSZ and 3Y-PSZ discs were adhesively bonded to a dentin-like substrate and divided into groups according to the load (50 N or 200 N) and number of cycles (up to 106) used in the chewing simulation. Specimens were mounted with 30° inclination in an electrodynamic mouth-motion simulator, and subjected to contact-slide-liftoff cyclic loading in water. Surface and sub-surface damages were analyzed using a sectioning technique. After the simulation, specimens were removed from the substrate and loaded with the damaged surface in tension for biaxial strength testing to assess their damage tolerance. RESULTS: The strength of both ceramics underwent significant degradation after mouth-motion simulations. For 5Y-PSZ, the strength degradation was greater (∼60%) and occurred at a lower number of cycles than 3Y-PSZ. Herringbone cracks emerged on 3Y-PSZ and 5Y-PSZ surfaces under a 200-N load after 50 and 10 cycles, respectively. Meanwhile at a 50-N load, cracks formed at ∼1000 cycles in both ceramics. Further increasing the number of cycles only had moderate effects on the strength of both ceramics, despite an increase in surface and sub-surface damage. More significantly, a 50-N occlusal load can debase the zirconia strengths as much as a 200-N load. SIGNIFICANCE: Surface flaws produced during the chewing simulation are capable of significant strength degradation in zirconia, even after a small number of low-load cycles.

Epicutaneous immunogenicity of partially hydrolyzed whey protein evaluated using tape-stripped mouse model.

BACKGROUND: Hydrolyzed cow's milk protein formulas are widely used for infants with a history or risk of cow's milk allergy. Based on the current theory that food allergen sensitization occurs via the skin, we investigated the epicutaneous immunogenicity of partially hydrolyzed whey proteins, which are ingredients in infant formulas. METHODS: BALB/c mice were exposed epicutaneously to whey protein concentrate (WPC) or partial whey protein hydrolysates (PWH1 or PWH2) on tape-stripped skin. Sensitization was assessed by evaluating serum ß-lactoglobulin (ß-LG)-specific antibodies, basophil activation, and cytokine production from ß-LG-stimulated lymphoid cells. The anaphylaxis reaction was evaluated by measuring the rectal temperature and plasma level of mouse mast cell protease-1 after oral ß-LG challenge. Immune cell accumulation in the skin was also analyzed. RESULTS: Substantive sensitization and ß-LG-induced anaphylaxis reaction were observed in WPC-exposed mice, whereas no significant changes were observed in PWH1- or PWH2-exposed mice. The basophil and eosinophil counts increased in WPC-exposed murine skin, not but in PWH1- or PWH2-exposed mice. CONCLUSION: The epicutaneous immunogenicity of PWH1 and PWH2 is markedly decreased, which may reduce the risk of allergen sensitization. Further studies are required to investigate the clinical value of these partial hydrolysates for high-risk infants.

Ingestion of Casein Hydrolysate Induces Oral Tolerance and Suppresses Subsequent Epicutaneous Sensitization and Development of Anaphylaxis Reaction to Casein in Mice.

BACKGROUND: Casein is the most dominant causal allergen in cow's milk allergy (CMA). Casein hydrolysates are frequently applied in infant formulas for children with a risk or history of CMA. However, there is limited information on the oral tolerance-inducing ability of casein hydrolysates. OBJECTIVES: The aim of this study was to investigate whether the ingestion of casein hydrolysate induces tolerance to casein, ultimately preventing subsequent epicutaneous sensitization and development of an anaphylaxis reaction. METHODS: BALB/c mice were orally administered casein or a casein hydrolysate (CNH) via the drinking water and were then epicutaneously sensitized by repeated exposure of casein on tape-stripped skin. Sensitization was assessed by basophil activation tests, the serum levels of casein-specific antibodies, and cytokine production from casein-stimulated spleen and mesenteric lymph node (MLN) cells. Occurrence of an anaphylaxis reaction was evaluated by measuring rectal temperature and the plasma level of mouse mast cell protease-1 (mMCP-1) after oral casein challenges. The T cell population in the spleen and MLN was assessed by flow cytometry. Intestinal mast cells and basophils were analyzed histologically. RESULTS: Sensitization and anaphylaxis reaction to casein were significantly suppressed in casein- or CNH-fed mice compared to controls. Prior ingestion of casein or CNH had no effect on the population of regulatory T cells and activated T cells in lymphoid tissues. Intestinal basophils increased by the epicutaneous sensitization of casein, which was suppressed in casein- or CNH-fed mice. Although the increase in the plasma level of mMCP-1 after oral challenge was suppressed in casein- or CNH-fed mice, there was no change in the number of intestinal mast cells. CONCLUSION: Prior ingestion of casein or CNH induced oral tolerance and suppressed subsequent epicutaneous sensitization and development of systemic anaphylaxis to casein.

Ingestion of partially hydrolyzed whey protein suppresses epicutaneous sensitization to ß-lactoglobulin in mice.

BACKGROUND: Epicutaneous sensitization to food allergens can occur through defective skin barriers. However, the relationship between oral tolerance and epicutaneous sensitization remains to be elucidated. We aimed to determine whether prior oral exposure to whey proteins or their hydrolysates prevents epicutaneous sensitization and subsequent food-allergic reaction to the whey protein, ß-lactoglobulin (ß-LG), and investigated the underlying mechanisms. METHODS: BALB/c mice were given whey protein concentrate (WPC), two kinds of partial whey protein hydrolysate (PWH1 or PWH2), or extensive whey protein hydrolysate (EWH) in drinking water for 21 days. The mice were then epicutaneously sensitized with ß-LG on tape-stripped skin. Sensitization was assessed by basophil activation tests and by measuring the level of serum ß-LG-specific antibodies and cytokines secreted from ß-LG-restimulated spleen and mesenteric lymph node (MLN) cells. Development of an allergic reaction was assessed by monitoring body temperature and by measuring mast cell protease-1 level in plasma after the ß-LG oral challenge. Activated T-cell population among ß-LG-restimulated MLN cells was also analyzed. RESULTS: In mice fed with WPC, PWH1, or PWH2, sensitization and the development of an allergic reaction were totally reduced. The acceleration of cytokine release from the spleen and MLN cells or T-cell activation was not evident after ß-LG restimulation. In EWH-fed mice, a suppressive effect, though milder than that in WPC-, PWH1-, or PWH2-fed mice, was observed during the development of the allergic reaction. CONCLUSIONS: Prior oral exposure to partially hydrolyzed whey protein prevents epicutaneous sensitization and subsequent allergic response to ß-LG in mice.

Dietary supplementation with shiikuwasha extract attenuates dexamethasone-induced skeletal muscle atrophy in aged rats.

BACKGROUND: Skeletal muscle atrophy is caused by a variety of diseases and conditions. In particular, skeletal muscle atrophy in the elderly contributes to a loss of independence with advanced age and increases the risk of falling. However, the effect of food consumed on a daily basis on skeletal muscle atrophy has been the focus of little research. In this study, the effects of dietary supplementation with shiikuwasha extract or grape extract on dexamethasone-induced skeletal muscle atrophy were evaluated in aged rats. METHODS: Aged male rats (15-month-old) were fed a diet supplemented with either 1 % shiikuwasha extract or 1 % grape extract for 19 days. During the last 5 days of the feeding period, rats were injected with dexamethasone to induce muscle atrophy. RESULTS: Body weight and hind-limb muscle weight were significantly decreased by dexamethasone treatment. The supplementation of shiikuwasha extract showed no effect on body weight loss, but markedly attenuated tibialis anterior muscle weight loss induced by dexamethasone. On the other hand, grape extract did not affect muscle weight loss. Furthermore, shiikuwasha extract significantly reduced dexamethasone-induced expression of atrogin-1 and MuRF1 mRNA, but did not reduce LC3B-II protein levels. CONCLUSION: These results suggest that shiikuwasha extract may partially inhibit the activation of the ubiquitin-proteasome system and may consequently attenuate skeletal muscle atrophy induced by dexamethasone in aged rats.

Multiple-turnover cleavage of double-stranded DNA by sandwiched zinc-finger nuclease.

To refine zinc-finger nuclease (ZFN) technology, we constructed a sandwiched ZFN, in which a DNA cleavage enzyme was sandwiched with two artificial zinc-finger proteins (AZPs). Because the sandwiched ZFN is designed to cleave the DNA between the two AZP-binding sites, the sandwiched ZFN is expected to bind preferentially to a DNA substrate rather than to cleavage products and thereby cleave it with multiple turnovers. To prove the concept, we sandwiched a staphylococcal nuclease (SNase), which cleaves DNA as a monomer, between two 3-finger AZPs. The AZP-sandwiched SNase cleaved large amounts of dsDNA site-specifically. Such multiple-turnover cleavage was not observed with control nucleases that possess a single AZP.

Enhanced cleavage of double-stranded DNA by artificial zinc-finger nuclease sandwiched between two zinc-finger proteins.

To enhance DNA cleavage by zinc-finger nucleases (ZFNs), we sandwiched a DNA cleavage enzyme with two artificial zinc-finger proteins (AZPs). Because the DNA between the two AZP-binding sites is cleaved, the AZP-sandwiched nuclease is expected to bind preferentially to a DNA substrate rather than to cleavage products and thereby cleave it with multiple turnovers. To demonstrate the concept, we sandwiched a staphylococcal nuclease (SNase), which cleaves DNA as a monomer, between two three-finger AZPs. The AZP-sandwiched SNase cleaved large amounts of dsDNA site-specifically. Such multiple-turnover cleavage was not observed with nucleases that possess a single AZP. Thus, AZP-sandwiched nucleases will further refine ZFN technology.

Application of artificial zinc-finger proteins to inhibition of DNA replication of human papillomavirus.

Recently, we have demonstrated that plant DNA virus replication could be inhibited in Arabidopsis thaliana by using an artificial zinc-finger protein (AZP) and created AZP-based transgenic A. thaliana resistant to DNA virus infection. Here we apply the AZP technology to inhibition of replication of a mammalian DNA virus, human papillomavirus (HPV) type 18. Two AZPs, designated AZP(HPV)-1 and AZP(HPV)-2, were designed to block binding of the HPV-18 E2 replication protein to the replication origin. Both the designed AZPs had much higher affinities towards the replication origin than did the E2 protein, and efficiently blocked E2 binding in vitro. In transient replication assays, both AZPs inhibited the viral DNA replication: AZP(HPV)-2, especially, reduced the replication level to approximately 10%. Thus, it was demonstrated that the AZP technology could be applied not only to plant DNA viruses, but also to mammalian DNA viruses.

Homodimeric cross-over structure of the human granulocyte colony-stimulating factor (GCSF) receptor signaling complex.

A crystal structure of the signaling complex between human granulocyte colony-stimulating factor (GCSF) and a ligand binding region of GCSF receptor (GCSF-R), has been determined to 2.8 A resolution. The GCSF:GCSF-R complex formed a 2:2 stoichiometry by means of a cross-over interaction between the Ig-like domains of GCSF-R and GCSF. The conformation of the complex is quite different from that between human GCSF and the cytokine receptor homologous domain of mouse GCSF-R, but similar to that of the IL-6/gp130 signaling complex. The Ig-like domain cross-over structure necessary for GCSF-R activation is consistent with previously reported thermodynamic and mutational analyses.

Crystallization of a 2:2 complex of granulocyte-colony stimulating factor (GCSF) with the ligand-binding region of the GCSF receptor.

The granulocyte-colony stimulating factor (GCSF) receptor receives signals for regulating the maturation, proliferation and differentiation of the precursor cells of neutrophilic granulocytes. The signalling complex composed of two GCSFs (GCSF, 19 kDa) and two GCSF receptors (GCSFR, 34 kDa) consisting of an Ig-like domain and a cytokine-receptor homologous (CRH) domain was crystallized. A crystal of the complex was grown in 1.0 M sodium formate and 0.1 M sodium acetate pH 4.6 and belongs to space group P4(1)2(1)2 (or its enantiomorph P4(3)2(1)2), with unit-cell parameters a = b = 110.1, c = 331.8 A. Unfortunately, this crystal form did not diffract beyond 5 A resolution. Since the heterogeneity of GCSF receptor appeared to prevent the growth of good-quality crystals, the GCSF receptor was fractionated by anion-exchange chromatography. Crystals of the GCSF-fractionated GCSF receptor complex were grown as a new crystal form in 0.2 M ammonium phosphate. This new crystal form diffracted to beyond 3.0 A resolution and belonged to space group P3(1)21 (or its enantiomorph P3(2)21), with unit-cell parameters a = b = 134.8, c = 105.7 A.

Thermodynamic analysis of the activation mechanism of the GCSF receptor induced by ligand binding.

The granulocyte colony-stimulating factor receptor (GCSFR), containing the Ig-like domain (Ig) and cytokine receptor homologous region (CRH), was prepared as a preformed dimer (Ig-CRH-Fc)(2) after fusion to the mouse Fc region via an eight-residue linker (approximately 55 A). Monomer Ig-CRH was also prepared after the Fc region was removed from (Ig-CRH-Fc)(2). GCSF binding to Ig-CRH and (Ig-CRH-Fc)(2) was investigated using light scattering and isothermal titration calorimetry. The average molecular mass determined by light scattering showed that both Ig-CRH and (Ig-CRH-Fc)(2) formed a 2:2 dimer with GCSF. Moreover, isothermal titration calorimetry showed that the thermodynamic parameters upon binding of GCSF to Ig-CRH and (Ig-CRH-Fc)(2) were comparable, suggesting a similar binding stoichiometry and interface [including similar buried surface area (5700-6000 A(2))] despite the presence of the eight-residue linker. The buried surface area is much larger than that calculated from our previous report of the crystal structure of the GCSF-CRH complex [Aritomi, M., et al. (1999) Nature 401, 713-717], suggesting a substantial contribution of the Ig domain to GCSF binding. The data also indicate that the distance (55 A) between two CRH domains in the 2:2 complex is much shorter than in our previous model (approximately 90 A) predicted from the same crystal structure of the GCSF-CRH complex.