Design of RGDS Peptide-Immobilized Self-Assembling ß-Strand Peptide from Barnacle Protein.

We designed three types of RGD-containing barnacle adhesive proteins using self-assembling peptides. In the present study, three types of RGD-containing peptides were synthesized by solid-phase peptide synthesis, and the secondary structures of these peptides were analyzed by CD and FT-IR spectroscopy. The mechanical properties of peptide hydrogels were characterized by a rheometer. We discuss the correlation between the peptide conformation, and cell attachment and cell spreading activity from the viewpoint of developing effective tissue engineering scaffolds. We created a peptide-coated cell culture substrate by coating peptides on a polystyrene plate. They significantly facilitated cell adhesion and spreading compared to a non-coated substrate. When the RGDS sequence was modified at N- or C-terminal of R-Y, it was found that the self-assembling ability was dependent on the strongly affects hydrogel formation and cell adhesion caused by its secondary structure.

Fermentation Characteristics, Tannin Contents and In vitro Ruminal Degradation of Green Tea and Black Tea By-products Ensiled at Different Temperatures.

Green and black tea by-products, obtained from ready-made tea industry, were ensiled at 10°C, 20°C, and 30°C. Green tea by-product silage (GTS) and black tea by-product silage (BTS) were opened at 5, 10, 45 days after ensiling. Fermentation characteristics and nutrient composition, including tannins, were monitored and the silages on day 45 were subjected to in vitro ruminal fermentation to assess anti-nutritive effects of tannins using polyethylene glycol (PEG) as a tannin-binding agent. Results showed that the GTS and BTS silages were stable and fermented slightly when ensiled at 10°C. The GTS stored at 20°C and 30°C showed rapid pH decline and high acetic acid concentration. The BTS was fermented gradually with moderate change of pH and acid concentration. Acetic acid was the main acid product of fermentation in both GTS and BTS. The contents of total extractable phenolics and total extractable tannins in both silages were unaffected by storage temperatures, but condensed tannins in GTS were less when stored at high temperature. The GTS showed no PEG response on in vitro gas production, and revealed only a small increase by PEG on NH3-N concentration. Storage temperature of GTS did not affect the extent of PEG response to both gas production and NH3-N concentration. On the other hand, addition of PEG on BTS markedly increased both the gas production and NH3-N concentration at any ensiled temperature. It can be concluded that tannins in both GTS and BTS suppressed rumen fermentation, and tannins in GTS did more weakly than that in BTS. Ensiling temperature for both tea by-products did not affect the tannin's activity in the rumen.

Assessment of Anti-nutritive Activity of Tannins in Tea By-products Based on In vitro Rumen Fermentation.

Nutritive values of green and black tea by-products and anti-nutritive activity of their tannins were evaluated in an in vitro rumen fermentation using various molecular weights of polyethylene glycols (PEG), polyvinyl pyrrolidone (PVP) and polyvinyl polypyrrolidone as tannin-binding agents. Significant improvement in gas production by addition of PEG4000, 6000 and 20000 and PVP was observed only from black tea by-product, but not from green tea by-product. All tannin binding agents increased NH3-N concentration from both green and black tea by-products in the fermentation medium, and the PEG6000 and 20000 showed relatively higher improvement in the NH3-N concentration. The PEG6000 and 20000 also improved in vitro organic matter digestibility and metabolizable energy contents of both tea by-products. It was concluded that high molecular PEG would be suitable to assess the suppressive activity of tannins in tea by-products by in vitro fermentation. Higher responses to gas production and NH3-N concentration from black tea by-product than green tea by-product due to PEG indicate that tannins in black tea by-product could suppress rumen fermentation more strongly than that in green tea by-product.

Repair potential of self-assembling peptide hydrogel in a mouse model of anterior cruciate ligament reconstruction.

Purpose: Establishing zonal tendon-to-bone attachment could accelerate the anterior cruciate ligament reconstruction (ACLR) rehabilitation schedule and facilitate an earlier return to sports. KI24RGDS is a self-assembling peptide hydrogel scaffold (SAPS) with the RGDS amino acid sequence. This study aimed to elucidate the therapeutic potential of KI24RGDS in facilitating zonal tendon-to-bone attachment after ACLR. Methods: Sixty-four C57BL/6 mice were divided into the ACLR + SAPS and ACLR groups. ACLR was performed using the tail tendon. To assess the maturation of tendon-to-bone attachment, we quantified the area of mineralized fibrocartilage (MFC) in the tendon graft with demeclocycline. Immunofluorescence staining of α-smooth muscle actin (α-SMA) was performed to evaluate progenitor cell proliferation. The strength of tendon-to-bone attachment was evaluated using a pull-out test. Results: The MFC and maximum failure load in the ACLR + SAPS group were remarkably higher than in the ACLR group on Day 14. However, no significant difference was observed between the two groups on Day 28. The number of α-SMA-positive cells in the tendon graft was highest on Day 7 after ACLR in both the groups and was significantly higher in the ACLR + SAPS group than in the ACLR group. Conclusion: This study highlighted the latent healing potential of KI24RGDS in facilitating early-stage zonal attachment of tendon grafts and bone tunnels post-ACLR. These findings may expedite rehabilitation protocols and shorten the timeline for returning to sports. Level of Evidence: Not applicable.

Improving hemocompatibility of decellularized vascular tissue by structural modification of collagen fiber.

Decellularized vascular tissue has high potential as a tissue-engineered vascular graft because of its similarity to native vessels in terms of mechanical strength. However, exposed collagen on the tissue induces blood coagulation, and low hemocompatibility is a major obstacle to its vascular application. Here we report that freeze-drying and ethanol treatment effectively modify collagen fiber structure and drastically reduce blood coagulation on the graft surface without exogenous chemical modification. Decellularized carotid artery of ostrich was treated with freeze-drying and ethanol solution at concentrations ranging between 5 and 99.5 %. Collagen fiber distance in the graft was narrowed by freeze-drying, and the non-helical region increased by ethanol treatment. Although in vitro blood coagulation pattern was similar on the grafts, platelet adhesion on the grafts was largely suppressed by freeze-drying and ethanol treatments. Ex vivo blood circulation tests also indicated that the adsorption of platelets and Von Willebrand Factor was largely reduced to approximately 80 % by ethanol treatment. These results indicate that structural modification of collagen fibers in decellularized tissue reduces blood coagulation on the surface by inhibiting platelet adhesion.

Study on the potential of RGD- and PHSRN-modified alginates as artificial extracellular matrices for engineering bone.

Alginate is a polysaccharide that can be crosslinked by divalent cations, such as calcium ions, to form a gel. Chemical modification is typically used to improve its cell adhesive properties for tissue engineering applications. In this study, alginates were modified with peptides containing RGD (arginine-glycine-aspartic acid) or PHSRN (proline-histidine-serine-arginine-asparagine) sequences from fibronectin to study possible additive and synergistic effects on adherent cells. Alginates modified with each peptide were mixed at different ratios to form gels containing various concentrations and spacing between the RGD and PHSRN sequences. When normal human osteoblasts (NHOsts) were cultured on or in the gels, the ratio of RGD to PHSRN was found to influence cell behaviors, especially differentiation. NHOsts cultured on gels composed of RGD- and PHSRN-modified alginates showed enhanced differentiation when the gels contained >33 % RGD-alginate, suggesting the relative distribution of the peptides and the presentation to cells are important parameters in this regulation. NHOsts cultured in gels containing both RGD- and PHSRN-alginates also demonstrated a similar enhancement tendency of calcium deposition that was dependent on the peptide ratio in the gel. However, calcium deposition was greater when cells were cultured in the gels, as compared to on the gels. These results suggest that modifying this biomaterial to more closely mimic the chemistry of natural cell adhesive proteins, (e.g., fibronectin) may be useful in developing scaffolds for bone tissue engineering and provide three-dimensional cell culture systems which more closely mimic the environment of the human body.

In vivo MR imaging for tumor-associated initial neovascularization by supramolecular contrast agents.

Microvascular imaging is required to understand tumor angiogenesis development; however, an appropriate whole-body imaging method has not yet been established. Here, we successfully developed a supramolecular magnetic resonance (MR) contrast agent for long-term whole-tissue observation in a single individual. Fluorescein- and Gd-chelate-conjugated polyethylene glycols (PEGs) were synthesized, and their structures were optimized. Spectroscopic and pharmacokinetic analyses suggested that the fluorescein-conjugated linear and 8-arm PEGs with a molecular weight of approximately 10 kDa were suitable to form a supramolecular structure to visualize the microvessel structure and blood circulation. Microvascular formation was evaluated in a glioma cell transplantation model, and neovascularization around the glioma tissue at 5 days was observed, with the contrast agent leaking out into the cancer tissue. In contrast, after 12 days, microvessel structures were formed inside the glioma tissue, but the agents did not leak out. These imaging data for the first time proved that the microvessels formed inside cancer tissues at the early stage are very leaky, but that they form continuous microvessels after 12 days.

Vascular tissue reconstruction by monocyte subpopulations on small-diameter acellular grafts via integrin activation.

Although the clinical application of cell-free tissue-engineered vascular grafts (TEVGs) has been proposed, vascular tissue regeneration mechanisms have not been fully clarified. Here, we report that monocyte subpopulations reconstruct vascular-like tissues through integrin signaling. An Arg-Glu-Asp-Val peptide-modified acellular long-bypass graft was used as the TEVG, and tissue regeneration in the graft was evaluated using a cardiopulmonary pump system and porcine transplantation model. In 1 day, the luminal surface of the graft was covered with cells that expressed CD163, CD14, and CD16, which represented the monocyte subpopulation, and they exhibited proliferative and migratory abilities. RNA sequencing showed that captured cells had an immune-related phenotype similar to that of monocytes and strongly expressed cell adhesion-related genes. In vitro angiogenesis assay showed that tube formation of the captured cells occurred via integrin signal activation. After medium- and long-term graft transplantation, the captured cells infiltrated the tunica media layer and constructed vascular with a CD31/CD105-positive layer and an αSMA-positive structure after 3 months. This finding, including multiple early-time observations provides clear evidence that blood-circulating monocytes are directly involved in vascular remodeling.

Accelerated tissue regeneration in decellularized vascular grafts with a patterned pore structure.

Decellularized tissue is expected to be utilized as a regenerative scaffold. However, the migration of host cells into the central region of the decellularized tissues is minimal because the tissues are mainly formed with dense collagen and elastin fibers. This results in insufficient tissue regeneration. Herein, it is demonstrated that host cell migration can be accelerated by using decellularized tissue with a patterned pore structure. Patterned pores with inner diameters of 24.5 ± 0.4 µm were fabricated at 100, 250, and 500 µm intervals in the decellularized vascular grafts via laser ablation. The grafts were transplanted into rat subcutaneous tissue for 1, 2, and 4 weeks. All the microporous grafts underwent faster recellularization with macrophages and fibroblast cells than the non-porous control tissue. In the case of non-porous tissue, the cells infiltrated approximately 50% of the area four weeks after transplantation. However, almost the entire area was occupied by the cells after two weeks when the micropores were aligned at a distance of less than 250 µm. These results suggest that host cell infiltration depends on the micropore interval, and a distance shorter than 250 µm can accelerate cell migration into decellularized tissues.

Nutrient digestibility, fecal output of fractionated proteins, and ruminal fermentation parameters of goats fed a diet supplemented with spent green tea and black tea leaf silage.

Spent green and black tea leaf silage (GTS and BTS, respectively) was offered as a protein supplement to goats to examine in vivo digestibility, nitrogen balance, urinary excretion of purine derivatives, and ruminal fermentation. Four castrated goats were fed a basal diet supplemented with alfalfa hay cube (AHC), GTS, or BTS in a 4 × 4 Latin square design. Digestibilities of various nutrients except for nitrogen (N) fraction were unaffected by the type of supplement. Digestibility of acid detergent insoluble N (ADIN) in BTS treatment was a negative value and significantly lower than those in other treatments. Urinary N output and retained N were not significantly affected by the diets. The fecal output of neutral detergent insoluble nitrogen (NDIN) and ADIN in the BTS treatment was significantly higher than those in other treatments. Urinary excretion of purine derivatives was not affected by the treatments. Ruminal NH3 -N concentration in AHC and GTS treatments were not significantly different, but that in the BTS treatment was significantly lower than others. These results indicated that GTS is substitutable for AHC as a protein supplement, whereas BTS was able to bind proteins tightly in the digestive tract, which lowered ruminal N degradability and increase fecal N output.

Life cycle of the multiarmed sea star Coscinasterias acutispina (Stimpson, 1862) in laboratory culture: sexual and asexual reproductive pathways.

The multiarmed sea star Coscinasterias acutispina generally has 7-10 arms and 2-5 madreporites. It is known to be able to reproduce by asexual fission, and we have previously observed that this species also has the ability to reproduce sexually; however, there has been no report until now of spawning in this species. We succeeded in establishing a long-term culture of juveniles produced by artificial fertilization. Twelve months after the completion of metamorphosis, three individuals had six arms of the same length and a madreporite. At this time, fission occurred in two of these individuals, while the remaining individual underwent fission four months later. Each sea star divided into two halves, provided with three arms each. Thereafter, four or five new arms and two or four madreporites were formed anew in each of the six daughter sea-stars, so that by 30 days after the first fission the number of arms and madreporites in each was similar to that in adults. A second fission occurred in four of these six individuals, four or five months after the first fission, and in three of them the plane of division was the same as that of the first fission. The original three individuals eventually proliferated to 12 by undergoing fission. All individuals had fully developed gonads by 1-3 months after the second fission. Some of them eventually spawned under laboratory culture, and the resulting larvae metamorphosed into juveniles. Our observations demonstrate that individuals of C. acutispina possess the potential for both sexual and asexual reproduction.

Calreticulin inhibits prion protein PrP-(23-98) aggregation in vitro.

Because prion protein PrP-(23-98) was recently found to polymerize into amyloid-like and proteinase K-resistant spherical aggregates in the presence of NADPH plus copper ions, we tested to determine whether calreticulin (CRT) inhibits PrP-(23-98) aggregation in vitro. The results indicated that CRT suppressed PrP-(23-98) aggregation, and that CRT-mediated solubilization occurred in the aggregates.

Feasibility of a self-assembling peptide hydrogel scaffold for meniscal defect: An in vivo study in a rabbit model.

The inner avascular zone of the meniscus has limited healing capacity as the area is poorly vascularized. Although peptide hydrogels have been reported to regenerate bone and cartilage, their effect on meniscus regeneration remains unknown. We tested whether the self-assembling peptide hydrogel scaffold KI24RGDS stays in the meniscal lesion and facilitates meniscal repair and regeneration in an induced rabbit meniscal defect model. Full-thickness (2.0 mm diameter) cylindrical defects were introduced into the inner avascular zones of the anterior portions of the medial menisci of rabbit knees (n = 40). Right knee defects were left empty (control group) while the left knee defects were transplanted with peptide hydrogel (KI24RGDS group). Macroscopic meniscus scores were significantly higher in the KI24RGDS group than in the control group at 2, 4, and 8 weeks after surgery. Histological examinations including quantitative and qualitative scores indicated that compared with the control group, the reparative tissue in the meniscus was significantly enhanced in the KI24RGDS group at 2, 4, 8, and 12 weeks after surgery. Immunohistochemical staining showed that the reparative tissue induced by KI24RGDS at 12 weeks postimplantation was positive for Type I and II collagen. KI24RGDS is highly biocompatible and biodegradable, with strong stiffness, and a three dimensional structure mimicking native extracellular matrix and RGDS sequences that enhance cell adhesion and proliferation. This in vivo study demonstrated that KI24RGDS remained in the meniscal lesion and facilitated the repair and regeneration in a rabbit meniscal defect model.

Combination of Copper Ions and Nucleotide Generates Aggregates from Prion Protein Fragments in the N-Terminal Domain.

BACKGROUND: It has been previously found that PrP23-98, which contains four highly conserved octarepeats (residues 60-91) and one partial repeat (residues 92-96), polymerizes into amyloid-like and proteinase K-resistant spherical aggregates in the presence of NADPH plus copper ions. OBJECTIVE: We aimed to determine the requirements for the formation of these aggregates. METHODS: In this study, we performed an aggregation experiment using N-acetylated and Camidated PrP fragments of the N-terminal domain, Octa1, Octa2, Octa3, Octa4, PrP84-114, and PrP76-114, in the presence of NADPH with copper ions, and focused on the effect of the number of copper-binding sites on aggregation. RESULTS: Among these PrP fragments, Octa4, containing four copper-binding sites, was particularly effective in forming aggregates. We also tested the effect of other pyridine nucleotides and adenine nucleotides on the aggregation of Octa4. ATP was equally effective, but NADH, NADP, ADP, and AMP had no effect. CONCLUSION: The phosphate group on the adenine-linked ribose moiety of adenine nucleotides and pyridine nucleotides is presumed to be essential for the observed effect on aggregation. Efficient aggregation requires the presence of the four octarepeats. These insights may be helpful in the eventual development of therapeutic agents against prion-related disorders.

Arg-Glu-Asp-Val Peptide Immobilized on an Acellular Graft Surface Inhibits Platelet Adhesion and Fibrin Clot Deposition in a Peptide Density-Dependent Manner.

Acellular blood vessels possess high potential to be used as tissue-engineered vascular scaffolds. Previously, a high patency was achieved for an Arg-Glu-Asp-Val (REDV) peptide-immobilized small-diameter acellular graft in a minipig model. Results revealed the potential of the peptide to capture a circulating cell and also to suppress fibrin clot deposition. Here, the effect of REDV peptide density on the blood response under ex vivo blood perfusion conditions was investigated. When endothelial cells or platelets were seeded under static conditions, the number of adherent endothelial cells increased with the increase in peptide density. Platelets scarcely adhered on the surface where the peptide density was above 18.9 × 10-4 molecules per nm3. Fibrin clot deposition and circulating cell capture were evaluated in a minipig extracorporeal circulatory system. The fibrin clot did not form on the peptide-immobilized surface, in the range of peptide modification density that was evaluated, whereas the unmodified surface was covered with microthrombi. REDV-specific blood circulating cells were captured on the peptide-immobilized surface with a density above 18.9 × 10-4 molecules per nm3. These results illustrated, under ex vivo blood perfusion conditions, that the REDV-immobilized acellular surface was able to capture cells and also suppress platelet adhesion and fibrin clot deposition in a peptide density-dependent manner.

Artificial switching of the metabolic processing pathway of an etiologic factor, ß2-microglobulin, by a "navigator" molecule.

Metabolic pathways in the body are highly specific. Dysfunction of a metabolic pathway triggers the accumulation of its target substance. For example, kidney failure results in increased ß2-microglobulin blood levels, causing dialysis-related amyloidosis. Previously, we proposed a novel therapeutic concept, that is a removal of an etiologic factor of metabolic disease by artificial switching of its metabolic processing pathway, and tested this concept using in cultured cells. However, the feasibility of artificial metabolic switching in vivo remained unknown. Here, we show that a newly developed "navigator" molecule changes the metabolic processing pathway of ß2-microglobulin from the kidney to the liver in mouse. The artificial metabolic switching is achieved by the capture of the etiologic factor by the navigator, which then steers the etiologic factor to hepatic lysosomes via low-density lipoprotein receptors. These findings demonstrate that navigator-based artificial metabolic switching can be a therapeutic strategy for various diseases caused by metabolic disorders.

Thermal behavior of J-aggregates in a Langmuir-Blodgett film of pure merocyanine dye investigated by UV-visible and IR absorption spectroscopy.

We have characterized the structure of J-aggregate in a Langmuir-Blodgett film of pure merocyanine dye (MS18) fabricated under an aqueous subphase containing a cadmium ion (Cd2+) and have investigated its thermal behavior by UV-visible and IR absorption spectroscopy in the range from 25 to 250 degrees C with a continuous scan. The results of both UV-visible and IR absorption spectra indicate that temperature-dependent changes in the MS18 aggregation state in the pure MS18 system are closely and mildly linked with the MS18 intramolecular charge transfer and the behavior of the packing, orientation, conformation, and thermal mobility of MS18 hydrocarbon chain, respectively. The J-aggregate in the pure MS18 system dissociates from 25 to 150 degrees C, and the dissociation temperature at 150 degrees C is higher by 50 degrees C than that in the previous MS18- arachidic acid (C20) binary system. The lower dissociation temperature in the binary system originates from the fact that temperature-dependent structural disorder of cadmium arachidate (CdC20), being phase-separated from MS18, has an influence on the dissociation of J-aggregate. From 160 to 180 degrees C, thermally induced blue-shifted bands, caused by the oligomeric MS18 aggregation, appear at around 520 nm in the pure MS18 system by contraries, regardless of the lack of driving force by the melting phenomenon of CdC20. The temperature at which the 520 nm bands occur is in good agreement with the melting point (160 degrees C) of hydrocarbon chain in MS18 with Cd2+, whereas its chromophore part is clearly observed to melt near 205 degrees C by UV-visible spectra. Therefore, it is suggested that the driving force that induces the 520 nm band in the pure MS18 system arises from the partial melting of hydrocarbon chain in MS18 with Cd2+.

Complement activation on surfaces carrying amino groups.

The complement system is strongly activated by surfaces carrying nucleophilic groups, such as hydroxyl (OH) groups, and triggered by deposition of complement protein fragment, C3b. Surfaces carrying amino groups, the other representative nucleophilic group, are expected to be potential activators of the complement system through the alternative pathway. Few studies thus far have examined the potential of artificial materials carrying amino groups in activating the complement system. In this study, we employed a self-assembled monolayer (SAM) of 11-amino-1-undecanethiol (NH2-SAM) and a polyethyleneimine (PEI)-coated surface as model surfaces to study interactions between amino groups and serum complement pathway. SAMs of 11-mercaptoundecanol (OH-SAM) and 1-dodecanethiol (CH3-SAM) were used as control surfaces, respectively. Although much protein was adsorbed from serum solutions on the two types of amino surfaces, amounts of C3b deposition were much less than those observed on OH-SAM. Amounts of C3a released on the amino surfaces were same levels as that of CH3-SAM, but significantly smaller than that on OH-SAM. These facts suggest that the nucleophilic amino groups on NH2-SAM and PEI-coated surfaces do not directly activate the alternative pathway, but the protein adsorbed layers formed on amino surfaces activate it, but to an extent much smaller than that on OH-SAM. In addition, we found no deposition of C1q molecules on the amino surfaces, suggesting that these surfaces fail to activate the classical pathway. However, more careful studies are needed to conclude it, because it is known that C1q is only transiently detected at typical classical activation interfaces.

Thermal behavior of H-aggregate in a mixed Langmuir-Blodgett film of merocyanine dye, arachidic acid, and n-octadecane ternary system investigated by UV-visible and IR absorption spectroscopy.

We have investigated the thermal behavior of H-aggregate in a mixed Langmuir-Blodgett (LB) film of the merocyanine dye (MS18)-arachidic acid (C20)- n-octadecane (AL18) ternary system by means of UV-visible and IR absorption spectroscopy in the range from 25 to 250 degrees C with a continuous scan. The results of both UV-visible and IR spectra indicate that the temperature-dependent variation in MS 18 aggregation state is linked not only with the degree of intramolecular charge transfer and the behavior of packing, orientation, conformation, and thermal mobility of the MS18 hydrocarbon chain but also with the presence and absence of AL18. The H-aggregate dissociates from 25 up to 50 degrees C, which is caused by the AL18 evaporation from the mixed LB film and the increment of thermal mobility of the MS18 hydrocarbon chain. From 110 to 160 degrees C, blue-shifted bands, attributed to the oligomeric MS18 aggregation, appear near 515 nm in the MS18-C 20-AL18 ternary system as well. The temperature at which the 515 nm band occurs is identical for both present ternary system and previously investigated MS18-deuterated arachidic acid (C20- d) binary system, and it is in good agreement with the melting point (110 degrees C) of cadmium arachidate (CdC20). Therefore, it is indicated that the driving force which induces the 515 nm band comes from the melting phenomenon of CdC20 molecules which are phase-separated from MS 18 molecules in as-deposited LB films.

Nutritive evaluation of spent green and black tea leaf silages by in vitro gas production characteristics, ruminal degradability and post-ruminal digestibility assessed with inhibitory activity of their tannins.

Spent tea leaf contains high levels of crude protein, suggesting that it may be used as an alternative source for ruminant feeding. We assessed the nutritive characteristics of spent green tea leaf silage (GTS) and black tea leaf silages (BTS) in comparison with soybean meal (SBM) and alfalfa hay cube (AHC) using in vitro assay. The effects of tannin on the nutritive characteristics were also evaluated by adding polyethylene glycol (PEG) as a tannin-binding agent. The amount of gas production was greater for SBM, followed by AHC, GTS, and BTS. A significant improvement in gas production upon addition of PEG was observed only for BTS. Ruminal protein degradability and post-ruminal digestibility was higher for SBM, followed by AHC, GTS, and BTS. The presence of PEG significantly increased ruminal degradability and post-ruminal protein digestibility for GTS and BTS, but not for AHC. The increment of protein digestibility by PEG was much greater for BTS than for GTS, indicating that GTS tannins suppress protein digestibility slightly, whereas BTS tannins do so strongly. According to these results, GTS but not BTS has a potential as an alternative to AHC as a ruminant feedstuff.