Epitope-Based Chicken-Derived Novel Anti-PAD2 Monoclonal Antibodies Inhibit Citrullination.

The aberrant upregulation of protein arginine deiminase 2- (PAD2-) catalyzed citrullination is reported in various autoimmune diseases (rheumatoid arthritis and multiple sclerosis) and several cancers. Currently, there are no anti-PAD2 monoclonal antibodies (mAbs) that can inhibit the citrullination reaction. Here, an epitope 341YLNRGDRWIQDEIEFGY357 was examined as an antigenic site of PAD2. Chickens were immunized with this epitope, and the generated mAbs were screened for its reactivity against the full-length PAD2. Enzyme-linked immunosorbent assay revealed that six mAbs, which were screened from the phage display library, crossreacted with mouse PAD2. Kinetic analysis revealed that mAbs are bound to PAD2 in the nanomolar range, which indicated a strong binding. Results of the in vitro citrullination inhibition assay revealed that the half-maximal effective concentration values of mAbs for the inhibition of histone or benzoyl-L-arginine ethyl ester citrullination were in the range of 6-75 nM which supports strong inhibition capabilities. Alanine scanning of epitope revealed that the peptide fragment 344RGDRWIQDEIEF355 was responsible for generating strong antibody responses that inhibit the PAD2-catalyzed citrullination reaction. These antibodies can aid in understanding the extracellular PAD2 function and treating diseases associated with aberrant citrullination.

Naturally Occurring Hair Growth Peptide: Water-Soluble Chicken Egg Yolk Peptides Stimulate Hair Growth Through Induction of Vascular Endothelial Growth Factor Production.

Alopecia is divided into two categories: androgenic alopecia and nonandrogenic alopecia. An androgen-dependent abnormality of biological functions causes alopecia in males, but the role of androgens is not yet elucidated in female pattern hair loss (FPHL). Modulation of androgenic activity is not effective in certain kinds of androgenic alopecia in females, as well as in cases of nonandrogenic alopecia in males and females. The hair growth drug, minoxidil, stimulates vascular endothelial growth factor (VEGF) production as well as vascularization and hair growth in females. Yet, because minoxidil has side effects with long-term use, a safe alternative hair growth agent is needed. Whereas hair develops after birth in mammalian species, hair mostly grows in a precocial bird, in the chicken, between hatching days 14 and 15. Therefore, we hypothesized that the chicken egg contains a key hair growth factor. In this study, we demonstrated that water-soluble peptides derived from the egg yolk stimulate VEGF production and human hair follicle dermal papilla cell growth. We also found that these peptides enhance murine hair growth and improve hair growth in FPHL. Finally, we characterized that water-soluble egg yolk peptides induce VEGF expression through insulin growth factor-1 receptor activation-induced hypoxia-inducible factor-1α transcription pathway. We have given the name "hair growth peptide (HGP)" to this water-soluble egg yolk peptide.

The beneficial effect of folate-enriched egg on the folate and homocysteine levels in rats fed a folate- and choline-deficient diet.

We investigated the effects of folate-enriched egg yolk powder on folate and homocysteine levels in plasma and liver of rats fed the folate- and choline-deficient diet to determine bioavailability in vivo. Three-wk-old Wistar rats were fed (1) the pteroylglutamate (PteGlu), (2) the choline, (3) the PteGlu and choline, (4) the folate-enriched egg yolk powder diet for 4 wk after having been fed the folate- and choline-deficient diet. The hepatic folate level in the folate-enriched egg yolk powder group was significantly higher than that in the folate- and choline-deficient or the control groups. The homocysteine concentration in plasma and liver of the folate-enriched egg yolk powder group was significantly lower than that of the folate- and choline-deficient or the PteGlu groups. The S-adenosyl-methionine (SAM)/S-adenosyl-homocysteine (SAH) ratio in the folate-enriched egg yolk powder group was significantly higher than that in the folate- and choline-deficient group. These effects were similar in the PteGlu and choline, but not the PteGlu or the choline groups. These data suggest that the intake of folate-enriched eggs, as well as of both folate and choline, induced the beneficial effects on folate and homocysteine metabolism. Thus, folate-enriched eggs could be used as beneficial source of folate with a high bioavailability.

Epigallocatechin-3-gallate has an anti-platelet effect in a cyclic AMP-dependent manner.

AIM: In this study, we investigated the effect of (-)-epigallocatechin-3-gallate (EGCG) on cyclic nucleotide production and vasodilator-stimulated phosphoprotein (VASP) phosphorylation in collagen (10 µg/mL)-stimulated platelet aggregation. METHODS: Washed platelets (10(8)/mL) from Sprague-Dawley rats (6-7 weeks old, male) were preincubated for 3 min at 37°C in the presence of 2 mM exogenous CaCl(2) with or without EGCG or other materials, stimulated with collagen (10 µg/mL) for 5 min, and then used for the determination of intracellular cytosolic Ca(2+) ([Ca(2+)](i)), thromboxane A(2) (TXA(2)), adenosine 3',5'-cyclic monophosphate (cAMP), guanosine 3',5'-cyclic monophosphate (cGMP), and VASP phosphorylation. RESULTS: EGCG dose-dependently inhibited collagen-induced platelet aggregation by inhibiting both [Ca(2+)](i) mobilization and TXA(2) production. Of two aggregation-inhibiting molecules, cAMP and cGMP, EGCG significantly increased intracellular levels of cAMP, but not cGMP. EGCG-elevated cAMP level was decreased by SQ22536, an adenylate cyclase inhibitor, but not by etazolate, a cAMPspecific phosphodiesterase inhibitor. In addition, EGCG elevated the phosphorylation of VASP-Ser(157), a cAMP-dependent protein kinase (A-kinase) substrate, but not the phosphorylation of VASP-Ser(239), a cGMP-dependent protein kinase substrate, in intact platelets and collagen-induced platelets, and VASP-Ser(157) phosphorylation by EGCG was inhibited by both an adenylate cyclase inhibitor SQ22536 and an A-kinase inhibitor Rp-8-Br-cAMPS. We have demonstrated that EGCG increases cAMP via adenylate cyclase activation and subsequently phosphorylates VASP-Ser(157) through A-kinase activation to inhibit [Ca(2+)](i) mobilization and TXA(2) production on collagen-induced platelet aggregation. CONCLUSIONS: These results strongly indicate that EGCG is a beneficial compound elevating cAMP level in collagen-platelet interaction, which may result in the prevention of platelet aggregation-mediated thrombotic diseases.

Inhibitory effects of oligopeptides from hen egg white on both human platelet aggregation and blood coagulation.

Egg white proteins have many biological functions and substantial nutritional benefits when used as a food source; however, they also contain allergens such as ovalbumin, ovomucoid, and ovotransferrin. We prepared oligopeptides without allergens from hen egg whites via the use of several proteases, and assessed their effects on platelet aggregation and blood coagulation, known to both of which are known to be major risk factors in thrombogenesis. Egg white oligopeptides (EWOP) inhibited collagen-induced human platelet aggregation in a dose-dependent manner. Additionally, we attempted to determine whether cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), aggregation-inhibiting intracellular molecules, regulate EWOP-inhibited platelet aggregation. EWOP caused an increase in cAMP levels, but did not affect cGMP levels, which suggests that the anti-platelet activity of EWOP operates in a cAMP-dependent manner, rather than via a cGMP-dependent process, in collagen-induced platelet aggregation. In addition, EWOP induced a significantly prolonged prothrombin time (PT) as compared with the controls. These data show that EWOP inhibits the conversion of fibrinogen to fibrin in a plasmatic atmosphere on an extrinsic pathway. Accordingly, these findings suggest that EWOP may be an excellent candidate as a crucial inhibitor of platelet activation, and its anti-platelet effects appear to involve the inhibition of both platelet aggregation and blood coagulation within the cardiovascular system.

Protective effect of lipoic acid against methylglyoxal-induced oxidative stress in LLC-PK(1) cells.

Methylglyoxal (MG), a reactive dicarbonyl compound, is a metabolic byproduct of glycolysis often found at high levels in blood from diabetic patients. The effect of lipoic acid on MG-induced oxidative stress was investigated using LLC-PK(1) renal tubular epithelial cells, which are susceptible to oxidative stress. MG (500 microM) treatment induced LLC-PK(1) cell death to nearly 50% compared with non-treated control cells, but lipoic acid significantly inhibited the MG-induced cytotoxicity in a concentration-dependent manner. In addition, lipoic acid treatment dose-dependently reduced the intracellular reactive oxygen species level increased by 500 microM MG. The nitric oxide level was also increased by 500 microM MG treatment, but it was significantly inhibited by lipoic acid. Furthermore, lipoic acid treatment at 50 microM inhibited the nuclear translocation of nuclear factor-kappa B induced by MG treatment in LLC-PK(1) cells. These findings indicate that lipoic acid has potential as a therapeutic agent against the development of diabetic complications related to MG-induced oxidative stress in diabetes.

Egg yolk soluble protein stimulates the proliferation and differentiation of osteoblastic MC3T3-E1 cells.

We determined the effects of yolk water-soluble protein (YSP) on bone formation in pre-osteoblastic MC3T3-E1 cells. YSP (50-5,000 microg/ml) increased cell proliferation and collagen content. Alkaline phosphatase (ALP) activity was also increased by YSP treatment. After enhancement of ALP activity, significant augmentation of calcification was observed. These results suggest that YSP is a promising agent for the prevention and treatment of bone loss.

Therapeutic potential of (-)-epigallocatechin 3-O-gallate on renal damage in diabetic nephropathy model rats.

Previous investigations have demonstrated that green tea polyphenols and partially hydrolyzed guar gum as dietary fiber have antioxidative and hypolipidemic activity, respectively, supporting their reduction of risk factors in the course of diabetic nephropathy via a hypoglycemic effect and ameliorating the decline of renal function through their combined administration to rats with subtotal nephrectomy plus streptozotocin (STZ) injection. As a further study, we examined whether (-)-epigallocatechin 3-O-gallate (EGCg), the main polyphenolic compound, could ameliorate the development of diabetic nephropathy. Rats with subtotal nephrectomy plus STZ injection were orally administrated EGCg at doses of 25, 50, and 100 mg/kg body weight/day. After a 50-day administration period, EGCg-treated groups showed suppressed hyperglycemia, proteinuria, and lipid peroxidation, although there were only weak effects on the levels of serum creatinine and glycosylated protein. Furthermore, EGCg reduced renal advanced glycation end-product accumulation and its related protein expression in the kidney cortex as well as associated pathological conditions. These results suggest that EGCg ameliorates glucose toxicity and renal injury, thus alleviating renal damage caused by abnormal glucose metabolism-associated oxidative stress involved in renal lesions of diabetic nephropathy.

Effects of green tea polyphenol on cognitive and acetylcholinesterase activities.

The effect of tea polyphenol (TP) on cognitive and anti-cholinesterase activity was examined in scopolamine-treated mice. Chronic administration of TP significantly reversed scopolamine-induced retention deficits in both step-through passive avoidance and spontaneous alternation behavior tasks. Furthermore, TP exhibited a dramatic inhibitory effect on acetylcholinesterase activity. This finding suggests that TP might be useful in the treatment of Alzheimer's disease.