Effects of self-assembled type II collagen fibrils on the morphology and growth of pre-chondrogenic ATDC5 cells.

Objective: Although type II collagen could have marked potential for developing cartilage tissue engineering (CTE) scaffolds, its erratic supply and viscous nature have limited these studies, and there are no studies on the use of marine-derived type II collagen fibrils for CTE scaffold materials. In this study, we aimed to generate a fibril-based, thin-layered scaffold from marine-derived type II collagen and investigate its chondrogenic potential. Methods: Time-lapse observations revealed the cell adhesion process. The Cell Counting Kit-8 (CCK-8) assay, light microscopy, and scanning electron microscopy were performed to detect proliferation and filopodium morphology. Alcian blue staining was used to show the deposition of extracellular secretions, and qRT-PCR was performed to reveal the expression levels of chondrogenesis-related genes. Results: The cell adhesion speed was similar in both fibril-coated and control molecule-coated groups, but the cellular morphology, proliferation, and chondrogenesis activity differed. On fibrils, more elongated finer filopodia showed inter-cell communications, whereas the slower proliferation suggested an altered cell cycle. Extracellular secretions occurred before day 14 and continued until day 28 on fibrils, and on fibrils, the expression of the chondrogenesis-related genes Sox9 (p â€‹< â€‹0.001), Col10a1 (p â€‹< â€‹0.001), Acan (p â€‹< â€‹0.001), and Col2a1 (p â€‹= â€‹0.0049) was significantly upregulated on day 21. Conclusion: Marine-derived type II collagen was, for the first time, fabricated into a fibril state. It showed rapid cellular affinity and induced chondrogenesis with extracellular secretions. We presented a new model for studying chondrogenesis in vitro and a potential alternative material for cell-laden CTE research.

Properties of Grass Carp (Ctenopharyngodon idella) Collagen and Gel for Application in Biomaterials.

The biochemical properties of collagens and gels from grass carp (Ctenopharyngodon idella) were studied to explore the feasibility of their application in biomaterials. The yields of skin collagen (SC) and swim bladder collagen (SBC) extracted from grass carp were 10.41 ± 0.67% and 6.11 ± 0.12% on a wet basis, respectively. Both collagens were characterized as type I collagen. Denaturation temperatures of SC and SBC were 37.41 ± 0.02 °C and 39.82 ± 0.06 °C, respectively. SC and SBC had high fibril formation ability in vitro, and higher values of salinity (NaCl, 0-280 mM) and pH (6-8) in formation solution were found to result in faster self-assembly of SC and SBC fibrils as well as thicker fibrils. Further tests of SC gels with regular morphology revealed that their texture properties and water content were affected by pH and NaCl concentration. The hardness, springiness, and cohesiveness of SC gels increased and the chewiness and water content decreased as pH increased from 7 to 8 and NaCl concentration increased from 140 to 280 mM. These properties suggest that collagens from grass carp may be useful in biomaterial applications in the future.

Comparation of the structural characteristics and biological activities of chondroitin sulfates extracted from notochord and backbone of Chinese sturgeon (Acipenser sinensis).

To compare the structural properties and biological activities of chondroitin sulfate (CS) in two different tissues of Chinese sturgeon (Acipenser sinensis) and Russian sturgeon (Acipenser gueldenstaedti), we extracted their backbone cartilage CS (Cart-CS) and notochord CS (Noto-CS), and analyzed the CS structural properties using chromatographic and spectroscopic methods. The molecular weights of Chinese sturgeon Cart-CS and Noto-CS were 54.7 and 25.4 kDa, respectively, and the molecular weights of Russian sturgeon were 50.0 and 38.4 kDa, respectively. The disaccharide composition results showed that Cart-CS was mainly composed of CS-C, while Noto-CS was almost composed of pure CS-A. The antioxidant activity of sturgeon CS and its effect on collagen fibril formation were discussed. Sturgeon CS exhibited higher antioxidant activity than shark and bovine CSs. Sturgeon CS inhibited the self-assemble of type I collagen into fibrils. The inhibition effect of Cart-CS was higher than that of Noto-CS. The high value-added utilization of Cart-CS and Noto-CS will increase the value of sturgeon by-products. Furthermore, the disaccharide composition of CS in sturgeon depends on tissues of origin, but not on species. It means that the CS of Chinese sturgeon can be substituted by the CS of other commercial sturgeon. That will contribute to the protection of endangered species of Chinese sturgeon from illegal fishing and increase the value of commercial sturgeon by-products.

Industrial application of fish cartilaginous tissues.

Cartilage is primarily composed of proteoglycans and collagen. Bioactive compounds derived from animal cartilage, such as chondroitin sulfate and type II collagen, have multiple bioactivities and are incorporated in popular health products. The aging population and increases in degenerative and chronic diseases will stimulate the rapid growth of market demand for cartilage products. Commercial production of bioactive compounds primarily involves the cartilages of mammals and poultry. However, these traditional sources are associated zoonosis concerns; thus, cartilage products from the by-products of fish processing has gained increasing attention because of their high level of safety and other activities. In this review, we summarize the current state of research into fish-derived cartilage products and their application, and discuss future trends and tasks to encourage further expansion and exploitation. At present, shark cartilage is the primary source of marine cartilage. However, the number of shark catches is decreasing worldwide, owing to overfishing. This review considers the potential alternative fish cartilage sources for industrialization. Three keys, the sustainable production of fish, new fish-processing model, and market demand, have been discussed for the future realization of efficient fish cartilage use. The industrialization of fish-derived cartilage products is beneficial for achieving sustainable development of local economies and society.

Effects of phosphate ion concentration on in-vitro fibrillogenesis of sturgeon type I collagen.

Nonmammalian collagens have attracted significant attention owing to their potential for use as a source of cell scaffolds for tissue engineering. Since the morphology of collagen fibrils controls cell proliferation and differentiation, its regulation is essential for fabricating scaffolds with desirable characteristics. In this study, we evaluated the effects of the phosphate ion (Pi) concentration on the characteristics of fibrils formed from swim bladder type I collagen (SBC) and skin type I collagen (SC) from the Bester sturgeon. An increase in the Pi concentration decreased the fibril formation rate, promoted the formation of thick fibrils, and increased the thermal stability of the fibrils for both SBC and SC. However, the SBC and SC fibrils exhibited different fibril formation rates, degrees of fibrillogenesis, morphologies, and denaturation temperatures for the same reaction conditions. Finally, by regulating the Pi concentration, various types of SBC and SC fibrils could be coated on cell culture wells, and fibroblasts could be cultured on them. The results showed that thin fibrils enhance fibroblast extension and proliferation, whereas thick fibrils restrain fibroblast extension but orient them in the same direction. The results of this study suggest that SBC fibrils, which exhibit diverse morphologies, are suitable for use as a novel scaffold material, whose characteristics can be tailored readily by varying the Pi concentration.

Self-assembled collagen fibrils from the swim bladder of Bester sturgeon enable alignment of MC3T3-E1 cells and enhance osteogenic differentiation.

Collagen is the most abundant protein in animals, and its polymer, collagen fibrils, regulate cellular proliferation, differentiation, and migration. Low antigenicity, biocompatibility, and biodegradability make collagen fibrils suitable functional scaffolds for tissue engineering. In a previous study, we found that the type I atelocollagen purified from the swim bladder of Bester sturgeon (swim bladder collagen, SBC) showed high fibril-forming ability, producing thicker fibrils faster than porcine collagen. In this study, we report a novel method to coat cell culture wells with highly aligned collagen fibrils using the SBC. Two types of fibrils with different thicknesses were prepared by changing the crosslinking treatment timing. The oriented, thick collagen fibrils induced pre-osteoblastic MC3T3-E1, pre-adipocytic 3T3-L1, pre-myocytic C2C12, and fibroblastic L929 cells to align in the same direction, whereas the oriented, fine fibrils made a cell network with their long pseudopods. Cellular proliferation was inhibited on both fibrils. Furthermore, both fibrils induced the early differentiation of MC3T3-E1 cells without differentiation stimuli. In contrast, the morphology of pre-chondrocytic ATDC5 cells on both fine and thick fibrils extended very short pseudopods and continued to maintain a spherical shape without stretching, suggesting a distinct effect by the fibrils. The newly developed fibril coatings are in the form of a thin film, thereby providing good visibility of the cell structure, including cell-cell and cell-ECM interactions, using a phase contrast microscope. The fibril coatings have high potential as a useful tool for tissue engineering research.

Properties of collagen extracted from Amur sturgeon Acipenser schrenckii and assessment of collagen fibrils in vitro.

The objective of this study was to assess the nature of the collagens from the Amur sturgeon to determine its possibility as a potential collagen source for biomedical applications. From a sturgeon (1.22 kg), 6.0 g (dry wt) of skin collagen (SC), 4.1 g of swim bladder collagen (SBC), and 0.4 g of notochord collagen (NC) were obtained. SC and SBC were characterized as type I, and NC as type II collagen. Denaturation temperatures of SC, SBC, and NC were calculated as 28.5, 30.5, and 33.5 °C, respectively. Gene expression of the type I procollagen α2 chain of Amur sturgeon (ascol1a2) was specifically higher than ascol1a1 expression in the swim bladder, suggesting a unique composition of α chains in this organ. SC and SBC had better abilities of fibril formation with unique higher-order structures compared with porcine type I collagen. The maximum transition temperature (Tm) of reassembled fibrils formed in a buffer solution containing NaCl at 0 and 140 mM was 34.4 °C and 38.9 °C in SC, and 40.1 °C and 40.7 °C in SBC, respectively. These characteristic features suggested that sturgeon collagens could be used in the biomedical industries in future applications.

Antioxidant activity of proteins extracted from red alga dulse harvested in Japan.

In this study, we investigated antioxidant activity of proteins from the red alga dulse (Palmaria sp.) harvested in Hokkaido, Japan. The dulse proteins that contain phycoerythrin (PE) as the main component showed a high radical scavenging activity. To clarify the key constituent of antioxidant activity in dulse proteins, we prepared recombinant dulse PE ß-subunit (rPEß) (apoprotein) and chromophores from the dulse proteins. As a result, the rPEß showed lower radical scavenging activity than that of dulse proteins. On the other hand, the dulse chromophores composed mainly of phycoerythrobilin (PEB) indicated extremely higher radical scavenging activity (90.4% ± 0.1%) than that of dulse proteins (17.9% ± 0.1%) on ABTS assay. In addition, on cell viability assay using human neuroblastoma SH-SY5Y cells, the dulse chromophores showed extracellular and intracellular cytoprotective effects against H2 O2 -induced cell damage. From these data, we concluded that the dulse proteins have antioxidant ability and the activity principally derives from the chromophores. PRACTICAL APPLICATION: Dulse is an abundant and underused resource, which contains a lot of proteins, especially phycoerythrin. We here demonstrated that the practically prepared dulse proteins possessed antioxidant activity and clarified that chromophores from the dulse proteins were the key components. Therefore, the dulse proteins have a potential for functional material.

Improvement of Muscle Quality of Grass Carp (Ctenopharyngodon idellus) With a Bio-Floating Bed in Culture Ponds.

Muscle quality and the physiological condition of fish are affected greatly by the culture environment. In aquaculture ponds, a bio-floating bed planted with vegetation is often used to improve water quality. This study investigated the growth and muscle quality of grass carp (Ctenopharyngodon idellus) cultured in ponds equipped with bio-floating beds. Fish were cultured in two replicated pond groups from May to November. Fish in the first group were cultured in experimental ponds equipped with a bio-floating bed planted with Ipomoea aquatica, whereas fish in the other group were reared in control ponds without a bio-floating bed. Compared with control ponds, the experimental ponds had better water quality with significantly lower concentrations of nitrite and ammonia. Grass carp in the experimental group had greater muscle weight gain, a significantly higher content of crude protein, and a significantly lower crude fat level than fish in the control group. The levels of pH, water-holding capacity, and antioxidant capacity of muscle decreased significantly in the control group compared to the experimental group. Texture profile analysis revealed higher values of hardness, springiness, gumminess, and chewiness and lower values of cohesiveness and resilience of white muscle in the experimental group compared to the control group. The filets of fish in the experimental group also received higher grades in the sensory evaluation of springiness, overall acceptability, aroma, and palatability. These results indicate that growth performance and muscle quality of grass carp were improved by the presence of bio-floating beds in the culture ponds.

Anti-obesity effects of chondroitin sulfate oligosaccharides from the skate Raja pulchra.

We aimed to investigate the anti-obesity effects of chondroitin sulfate (CS) oligosaccharides obtained from cartilage of the skate Raja pulchra and to compare them with those of CSs of other molecular weights (MWts) (skate CS polysaccharides) and origins (shark CS, bovine CS). CSs suppressed pancreatic lipase activity as well as proliferation and lipid accumulation in mature adipocytes. Higher MWt CS had a greater lipase inhibitory activity than lower MWt CS. CSs of different origin show differing potencies for lipase inhibition and effects on adipocytes. Also, dietary intake of skate CS oligosaccharides could ameliorate obesity in high-fat diet mice model: it prevented gaining in body weight, liver weight and adipose tissue weight, maintained lower food consumption, inhibited intestinal absorption of triglyceride, and adjusted the serum endotoxin level. In conclusion, skate CS oligosaccharides have an anti-obesity activity, and the MWt and origin of the CSs may affect this activity.

The effect of alkaline pretreatment on the biochemical characteristics and fibril-forming abilities of types I and II collagen extracted from bester sturgeon by-products.

Non-mammalian collagens have attracted increasing attention for industrial and biomedical use. We have therefore evaluated extraction conditions and the biochemical properties of collagens from aquacultured sturgeon. Pepsin-soluble type I and type II collagen were respectively extracted from the skin and notochord of bester sturgeon by-products, with yields of 63.9 ±â€¯0.19% and 35.5 ±â€¯0.68%. Collagen extraction efficiency was improved by an alkaline pretreatment of the skin and notochord (fewer extraction cycles were required), but the final yields decreased to 56.2 ±â€¯0.84% for type I and 31.8 ±â€¯1.13% for type II. Alkaline pretreatment did not affect the thermal stability or triple-helical structure of both types of collagen. Types I and II collagen formed re-assembled fibril structures in vitro, under different conditions. Alkaline pretreatment slowed down the formation of type I collagen fibrils and specifically inhibited the formation of thick fibril-bundle structures. In contrast, alkaline pretreatment did not change type II collagen fibril formation. In conclusion, alkaline pretreatment of sturgeon skin and notochord is an effective method to accelerate collagen extraction process of types I and II collagen without changing their biochemical properties. However, it decreases the yield of both collagens and specifically changes the fibril-forming ability of type I collagen.

Anisotropic tough double network hydrogel from fish collagen and its spontaneous in vivo bonding to bone.

Soft supporting tissues in the human body, such as cartilages and ligaments, are tough materials and firmly fixed to bones. These soft tissues, once injured, cannot regenerate spontaneously in vivo. Developing tough and biocompatible hydrogels as artificial soft supporting tissues would substantially improve outcomes after soft tissue injury. Collagen is the main rigid component in soft connective tissues which is organized in various hierarchical arrays. We have successfully developed a novel class of collagen fibril-based tough hydrogels based on the double network (DN) concept using swim bladder collagen (SBC) extracted from Bester sturgeon fish. The DN hydrogels, SBC/PDMAAm, are composed of physically/chemically crosslinked anisotropic SBC fibril as the first network and neutral, biocompatible poly(N,N'-dimethylacrylamide) (PDMAAm) as the second network. The anisotropic structure of SBC fibril network, which is well retained in the DN hydrogels, is formed by free injection method, taking advantage of the excellent fibrillogenesis capacity of SBC. The denaturation temperature of collagen is improved in the DN hydrogels. These DN gels possess anisotropic swelling behavior, exhibit excellent mechanical properties comparable to natural cartilage. The 4 weeks implantation of the gels in the osteochondral defect of rabbit knee also shows excellent biomechanical performance in vivo. Furthermore, the hydroxyapatite (HAp) coated DN gels, HAp/SBC/PDMAAm gels, strongly bond to bone after 4 weeks. This new class of collagen-based hybrid DN gels, as soft and elastic ceramics, having good biomechanical performance and strong bonding ability with bone would expand the choice for designing next-generation orthopedic implants such as artificial cartilage, bone defect repair material in the load-bearing region of the body.

Double-Network Hydrogels Strongly Bondable to Bones by Spontaneous Osteogenesis Penetration.

On implanting hydroxyapatite-mineralized tough hydrogel into osteochondral defects of rabbits, osteogenesis spontaneously penetrates into the gel matrix owing to the semi-permeablility of the hydrogel. The gradient layer (around 40 µm thick) contributes quite strong bonding of the gel to bone. This is the first success in realizing the robust osteointegration of tough hydrogels, and the method is simple and feasible for practical use.

Characterization of type I and II procollagen α1 chain in Amur sturgeon (Acipenser schrenckii) and comparison of their gene expression.

To characterize type I and II collagen in the Amur sturgeon at the molecular level, mRNAs encoding the proα chain of both types of collagen were cloned and sequenced. Full sequences of both were obtained, and the molecular phylogeny based on the deduced amino acid sequence indicated that the correct sequences of the target genes were obtained. Analyses of primary structure of the proα chains revealed that type I and II collagen share the basic structure of the proα chain of fibril collagen, but have different characteristics, especially in residues related to thermal stability. In the triple helical domain, Gly-Pro-Pro sequence stabilizing the tripeptide unit was more frequent in type II than in type I, and Gly-Gly, which likely decline in thermal stability, was more frequent in type I than in type II. These results suggested that the denaturation temperature of type II would be remarkably higher than type I. The spatial pattern of gene expression was analyzed by quantitative real-time PCR, which showed that relatively ubiquitous type I gene and strongly skewed distribution of type II gene, which highly expressed only in vertebra, snout cartilage, and notochord. This pattern was similar to the distribution pattern of each collagen protein detected by previous biochemical analyses using Amur and Bester sturgeons. The present study is the first report of the cloning of the full-length cDNAs for both of type I and type II collagen in the Amur sturgeon, and is the first comparative analysis of type I and II collagens in a sturgeon species at the molecular level. The results provide basic and general information on collagens in sturgeons.

Swim bladder collagen forms hydrogel with macroscopic superstructure by diffusion induced fast gelation.

Marine collagen has been attracting attention as a medical material in recent times due to the low risk of pathogen infection compared to animal collagen. Type I collagen extracted from the swim bladder of Bester sturgeon fish has excellent characteristics such as high denaturation temperature, high solubility, low viscosity and an extremely fast rate to form large bundle of fibers under certain conditions. These specific characteristics of swim bladder collagen (SBC) permit us to create stable, disk shaped hydrogels with concentric orientation of collagen fibers by the controlled diffusion of neutral buffer through collagen solution at room temperature. However, traditionally used animal collagens, e.g. calf skin collagen (CSC) and porcine skin collagen (PSC), could not form any stable and oriented structure by this method. The mechanism of the superstructure formation of SBC by a diffusion induced gelation process has been explored. The fast fibrillogenesis rate of SBC causes a quick squeezing out of the solvent from the gel phase to the sol phase during gelation, which builds an internal stress at the gel-sol interface. The tensile stress induces the collagen molecules of the gel phase to align along the gel-sol interface direction to give this concentric ring-shaped orientation pattern. On the other hand, the slow fibrillogenesis rate of animal collagens due to the high viscosity of the solution does not favor the ordered structure formation. The denaturation temperature of SBC increases significantly from 31 °C to 43 °C after gelation, whereas that of CSC and PSC were found to increase a little. Rheology experiment shows that the SBC gel has storage modulus larger than 15 kPa. The SBC hydrogels with thermal and mechanical stability have potential as bio-materials for tissue engineering applications.

Biochemical characterisation and assessment of fibril-forming ability of collagens extracted from Bester sturgeon Huso huso × Acipenser ruthenus.

Collagens purified from Bester sturgeon organs were characterised biochemically, and their fibril-forming abilities and fibril morphologies formed in vitro clarified. Yields of collagens were 2.1%, 11.9%, 0.4%, 18.1%, 0.4%, 0.8% and 0.03% (collagen dry weight/tissue wet weight) from scales, skin, muscle, swim bladder, digestive tract, notochord and snout cartilage, respectively. Using SDS-PAGE and amino acid composition analyses, collagens from scales, skin, muscle, the swim bladder and digestive tract were characterised as type I, and collagens from the notochord and snout cartilage as type II. Denaturation temperatures of the collagens, measured using circular dichroism, were 29.6, 26.8, 29.0, 32.9, 31.6 and 36.3 °C in scales, skin, muscle, swim bladder, digestive tract, and notochord, respectively. For fibril formation, swim bladder and skin collagen showed a more rapid rate of increase in turbidity, a shorter time to attain the maximum turbidity, and formed thicker fibrils compared with porcine tendon type I collagen.

Dorsal hump morphology in pink salmon (Oncorhynchus gorbuscha).

Mature male Pacific salmon (Genus Oncorhynchus) develop a dorsal hump, as a secondary male sexual characteristic, during the spawning period. Previous gross anatomical studies have indicated that the dorsal humps of salmon are mainly composed of cartilaginous tissue (Davidson [1935] J Morphol 57:169-183.) However, the histological and biochemical characteristics of such humps are poorly understood. In this study, the detailed microstructures and components of the dorsal humps of pink salmon were analyzed using histochemical techniques and electrophoresis. In mature males, free interneural spines and neural spines were located in a line near to the median septum of the dorsal hump. No cartilaginous tissue was detected within the dorsal hump. Fibrous and mucous connective tissues were mainly found in three regions of the dorsal hump: i) the median septum, ii) the distal region, and iii) the crescent-shaped region. Both the median septum and distal region consisted of connective tissue with a high water content, which contained elastic fibers and hyaluronic acid. It was also demonstrated that the lipid content of the dorsal hump connective tissue was markedly decreased in the mature males compared with the immature and maturing males. Although, the crescent-shaped region of the hump consisted of connective tissue, it did not contain elastic fibers, hyaluronic acid, or lipids. In an ultrastructural examination, it was found that all of the connective tissues in the dorsal hump were composed of collagen fibers. Gel electrophoresis of collagen extracts from these tissues found that the collagen in the dorsal hump is composed of Type I collagen, as is the case in salmon skin. These results indicate that in male pink salmon the dorsal hump is formed as a result of an increase in the amount of connective tissue, rather than cartilage, and the growth of free interneural spines and neural spines.

Spatiotemporal expression of the dermatopontin gene in zebrafish Danio rerio.

There has been significant interest in the expression and function of dermatopontin (DPT) in mammals owing to recent evidence pointing to its critical role in collagen fibrillogenesis. Despite this interest, limited information is available about the site/s of DPT mRNA expression or changes in expression in vivo. We used reverse-transcription polymerase chain reaction and in situ hybridization to evaluate the spatial and temporal pattern of DPT mRNA expression in zebrafish, Danio rerio, a widely used vertebrate model. We observed that DPT transcripts were expressed in zebrafish embryos at all developmental stages in a range of tissues, including the brain and optic neuron cells. Based on our results, we hypothesize that DPT may also play a role in neural functions in vivo.

Expression patterns of runx2, sparc, and bgp during scale regeneration in the goldfish Carassius auratus.

Teleost fish scale is a dermal skeleton equipped with a strong regenerative ability. Owing to this regenerative ability, teleost fish scale can be used as a model for the regeneration of the dermal skeleton. However, there is insufficient fundamental knowledge of the regeneration, and this limits the usage of fish scale. In this study, as a first step toward understanding the molecular mechanism of the cellular differentiation during scale regeneration, we cloned the cDNAs for osteoblast-related proteins (Runx2, Sparc, and Bgp) in goldfish, and analyzed their expressions during scale regeneration. The expression profiles of these genes during scale regeneration were similar to those during mammalian osteoblastic differentiation. Specifically, runx2 expression was increased at the earliest time point, followed by sparc expression and then bgp expression. In the earlier stages, these genes were expressed in cells that formed cellular condensations and the flat cells surrounding them in the scale pocket. As the regeneration proceeded, the expressions became restricted to the episquamal, hyposquamal, and marginal scleroblasts and the cells around the marginal area of the regenerating scale. These results strongly suggest that (1) the differentiation mechanism of scleroblasts is similar to that of mammalian osteoblasts and odontoblasts, (2) scleroblast differentiation occurs around the cellular condensations at the early regeneration stage and is restricted to the marginal area of the scale at the later stage, and (3) the differentiation mechanisms are similar between the episquamal scleroblasts that produce the external layer and the hyposquamal scleroblasts that produce the basal plate.

Immunohistochemical analysis of the proteolytic enzyme subtilase in the digestive organs of the sea urchin Strongylocentrotus intermedius.

Subtilase, a major protease in the short-spined sea urchin (Strongylocentrotus intermedius), was isolated and used as antigen for the subsequent production of a specific polyclonal antibody. Immunoreactive cells were observed by immunohistochemical analysis in granules in the anterior and posterior stomach and the anterior intestine. These granules, which were most numerous in the anterior stomach, also stained intensely with methylene blue-Azure II. However, granules in cells of the esophagus, posterior intestine, and rectum were not stained by this antibody. We conclude that subtilase mainly localizes in the stomach and anterior intestine of the sea urchin.