Partial Enzymatic Degradation Research Articles

Enzymatic pre-treatment defines the water-binding and rheological properties of dynamic ultra-high-pressure homogenised pea hull suspensions

Pulse hulls are composed of cell wall polysaccharides and are a concentrated source of dietary fibre. However, the dense microstructure hinders their industrial application, resulting in poor techno-functional properties. To improve their functionality, the cell wall (cellulose, hemicellulose, and pectin) is disrupted by combining enzymatic pre-treatments and microfluidization. This work aims to study the impact of a fixed reduction of the insoluble mass by selective enzymatic pre-treatment of pulse hulls on the composition, microstructure, water binding, and rheological properties of microfluidized suspensions. To that end, enzymatic pre-treatments using cellulase, hemicellulase, pectinase, and their binary and ternary mixtures were conducted to reduce insoluble mass content. The combined treatment improved the water-binding properties, leading to the formation of viscoelastic gels. Microfluidized suspensions enzymatically pre-treated with a binary mixture of cellulase and hemicellulase presented the highest water binding capacity, viscosity, and viscoelasticity. The partial enzymatic degradation of the cellulose-hemicellulose network weakened the interactions between both polysaccharides. It facilitates the disruption of the particles and the release of high molecular weight soluble dietary fibre (HMWSDF), which are gelling substances. On the contrary, an enzymatic pre-treatment using only cellulase increased the content of low molecular weight soluble compounds, resulting in low viscous and viscoelastic suspensions. Thus, the concentration of remaining microfibrillated cellulose and HMWSDF are key parameters controlling the rheological behaviour of the suspensions. This study enhances comprehension regarding pea hulls' molecular and macromolecular modifications, potentially enabling the production of tailor-made pulse hull suspensions.

Update on Perineuronal Net Staining With Wisteria floribunda Agglutinin (WFA).

As chemically specialized forms of the extracellular matrix in the central nervous system, polyanionic perineuronal nets (PNs) contain diverse constituents, including chondroitin sulfate proteoglycans (CSPGs), hyaluronic acid, and tenascins. They are detectable by various histological approaches such as colloidal iron binding and immunohistochemical staining to reveal, for instance, the CSPGs aggrecan, neurocan, phosphacan, and versican. Moreover, biotin, peroxidase, or fluorescein conjugates of the lectins Vicia villosa agglutinin and soybean agglutinin enable the visualization of PNs. At present, the N-acetylgalactosamine-binding Wisteria floribunda agglutinin (WFA) is the most widely applied marker for PNs. Therefore, this article is largely focused on methodological aspects of WFA staining. Notably, fluorescent WFA labeling allows, after its conversion into electron-dense adducts, electron microscopic analyses. Furthermore, the usefulness of WFA conjugates for the oftentimes neglected in vivo and in vitro labeling of PNs is emphasized. Subsequently, we discuss impaired WFA-staining sites after long-lasting experiments in vitro, especially in autoptic brain samples with long postmortem delay and partial enzymatic degradation, while immunolabeling of aggrecan and CSPG link proteins under such conditions has proven more robust. In some hippocampal regions from perfusion-fixed mice, more PNs are aggrecan immunoreactive than WFA positive, whereas the retrosplenial cortex displays many WFA-binding PNs devoid of visible aggrecan immunoreactivity. Additional multiple fluorescence labeling exemplarily revealed in ischemic tissue diminished staining of WFA-binding sites and aquaporin 4 and concomitantly upregulated immunolabeling of neurofilament, light chains, and collagen IV. Finally, we briefly discuss possible future staining approaches based on nanobodies to facilitate novel technologies revealing details of net morphology.

Трипсинолиз полиуретановых частиц на основе циклодекстрина с инкапсулированным моксифлоксацином

Abstract-Moxifloxacin encapsulation in polymer cyclodextrin-based particles with an average hydrodynamic diameter of 150--200 nm has led to the formation of potential delivery systems with a degree of moxifloxacin inclusion of more than 80%. Cross-linking of the moxifloxacin-cyclodextrin complexes caused a pronounced slowdown in the release of the drug molecules in acidic media to less than 10% per day. In the presence of trypsin, the drug release was accelerated by 15--20% within 90 min. It was shown by Fourier-transform infrared spectroscopy that this acceleration was due to the partial enzymatic degradation of the urethane bonds of the polymer matrix near the surface of the particles. The results obtained are important for the development of highly effective oral dosage forms of prolonged action. Key words: fluoroquinolones, cyclodextrins, FTIR spectroscopy, trypsin

Supramolecular Antifouling Additives for Robust and Efficient Functionalization of Elastomeric Materials: Molecular Design Matters

AbstractThe ultimate functionality of elastomeric materials can be largely influenced by the molecular design of antifouling additives that interact through directed hydrogen bonding bisurea motifs. Herein, three additives, composed of matching bisurea groups and antifouling oligo(ethylene glycol) (OEG) functionalities, are judiciously designed. The first additive is composed of one bisurea and one OEG, the second additive of one bisurea and two OEGs, and the third additive of two bisurea and one OEG. On solution‐cast films, non‐cell adhesive properties are dependent on the amount of incorporated OEG irrespective of the bisurea design; however, on 3D electrospun scaffolds only the additive that consists of two bisurea moieties connected via an OEG functionality ensures proper non‐cell adhesive properties. Interestingly, robust non‐cell adhesive properties are maintained, both with repeated cell seeding and after partial enzymatic degradation of the scaffold. These results highlight the importance of additive design in supramolecular functionalization and show that translation from simple 2D solution‐cast films to 3D electrospun scaffolds is not trivial with respect to additive presentation and functionality.

Possible Mechanisms by Which Enzymatic Degradation of Human Serum Albumin Can Lead to Bioactive Peptides and Biomarkers.

Partial enzymatic degradation of human serum albumin in vivo can lead to the generation of peptides with novel functions or to peptides that might serve as biomarkers for disease. In pathological conditions, biomarkers are possibly produced from the protein in the lysosomes and set free by cell death, or cell death could release acid endoproteases which produce biomarkers by degrading extracellular albumin. Alternatively, lysosomes or secretory granules can be stimulated to release enzymes which produce bioactive peptides from albumin. In physiological conditions, it is proposed that bioactive peptides can be made by enzymatic attack on the protein bound to the endosomal neonatal Fc receptor. The peptides formed could leave the cell, together with native albumin, by exocytosis. Thus, the receptor could have a new function in addition to saving albumin from degradation in the lysosomes. Large amounts of albumin are degraded every day, and this fact can compensate for the short in vivo half-lives of the bioactive peptides. One or more of the procedures outlined above could also apply to other plasma proteins or to structural proteins.

A simple, rapid and cost-effective process for production of latex clearing protein to produce oligopolyisoprene molecules

Aiming at finding feasible alternatives for rubber waste disposal, the partial enzymatic degradation of poly(cis-1,4-isoprene)-containing materials represents a potential solution. The use of rubber-degrading enzymes and the biotransformation of rubber into new materials is limited by the high costs associated with the production and purification of the enzyme and the complexity of the process. This study presents a simple and low-cost procedure to obtain purified latex clearing protein (Lcp), an enzyme capable of cleaving the double bonds of poly(cis-1,4-isoprene) in presence of oxygen to produce different size of oligomers with terminal aldehyde and ketone groups, respectively. The gene coding for Lcp1VH2 from Gordonia polyisoprenivorans strain VH2 was overexpressed in Escherichia coli C41 (DE3), and by using an auto-induction medium high protein yields were obtained. The cultivation process was described and compared with an IPTG-inducible medium previously used. Purification of the enzyme was performed using salting out precipitation with ammonium sulfate. Different salt concentrations and pH were tested in order to find the optimal for purification, obtaining a concentration of 60mg Lcp per l. The enzymatic activity of the purified enzyme was measured by an oxygen consumption assay in the presence of polyisoprene latex. Volumetric activities of 0.16Uml−1 were obtained at optimal conditions of temperature and pH. The results showed an active and partial purified fraction of Lcp1VH2, able to cleave the backbone of poly(cis-1,4-isoprene) and to produce degradation products that were identified with staining methodologies (Schiff reagent for aldehyde groups and 2,4-DNPH for carbonyl groups) and characterized using nuclear magnetic resonance (NMR). Thirteen different storage conditions were tested for the purified enzyme analyzing the enzymatic activity after 1 and 3 months. Lcp1VH2, as an ammonium sulfate precipitate, was stable, easy to handle and sufficiently active for further analysis. The described methodology offers the possibility to upscale the process and to produce large amounts of this protein.

Integrated Approach to Identify Heparan Sulfate Ligand Requirements of Robo1.

An integrated methodology is described to establish ligand requirements for heparan sulfate (HS) binding proteins based on a workflow in which HS octasaccharides are produced by partial enzymatic degradation of natural HS followed by size exclusion purification, affinity enrichment using an immobilized HS-binding protein of interest, putative structure determination of isolated compounds by a hydrophilic interaction chromatography-high-resolution mass spectrometry platform, and chemical synthesis of well-defined HS oligosaccharides for structure-activity relationship studies. The methodology was used to establish the ligand requirements of human Roundabout receptor 1 (Robo1), which is involved in a number of developmental processes. Mass spectrometric analysis of the starting octasaccharide mixture and the Robo1-bound fraction indicated that Robo1 has a preference for a specific set of structures. Further analysis was performed by sequential permethylation, desulfation, and pertrideuteroacetylation followed by online separation and structural analysis by MS/MS. Sequences of tetrasaccharides could be deduced from the data, and by combining the compositional and sequence data, a putative octasaccharide ligand could be proposed (GlA-GlcNS6S-IdoA-GlcNS-IdoA2S-GlcNS6S-IdoA-GlcNAc6S). A modular synthetic approach was employed to prepare the target compound, and binding studies by surface plasmon resonance (SPR) confirmed it to be a high affinity ligand for Robo1. Further studies with a number of tetrasaccharides confirmed that sulfate esters at C-6 are critical for binding, whereas such functionalities at C-2 substantially reduce binding. High affinity ligands were able to reverse a reduction in endothelial cell migration induced by Slit2-Robo1 signaling.

Molecular Adhesion between Cartilage Extracellular Matrix Macromolecules

In this study, we investigated the molecular adhesion between the major constituents of cartilage extracellular matrix, namely, the highly negatively charged proteoglycan aggrecan and the type II/IX/XI fibrillar collagen network, in simulated physiological conditions. Colloidal force spectroscopy was applied to measure the maximum adhesion force and total adhesion energy between aggrecan end-attached spherical tips (end radius R ≈ 2.5 μm) and trypsin-treated cartilage disks with undamaged collagen networks. Studies were carried out in various aqueous solutions to reveal the physical factors that govern aggrecan–collagen adhesion. Increasing both ionic strength and [Ca2+] significantly increased adhesion, highlighting the importance of electrostatic repulsion and Ca2+-mediated ion bridging effects. In addition, we probed how partial enzymatic degradation of the collagen network, which simulates osteoarthritic conditions, affects the aggrecan–collagen interactions. Interestingly, we found a significant increase in aggrecan–collagen adhesion even when there were no detectable changes at the macro- or microscales. It is hypothesized that the aggrecan–collagen adhesion, together with aggrecan–aggrecan self-adhesion, works synergistically to determine the local molecular deformability and energy dissipation of the cartilage matrix, in turn, affecting its macroscopic tissue properties.

Transition of spherulite morphology in a crystalline/crystalline binary blend of biodegradable microbial polyesters

Crystalline/crystalline binary blend films of microbial polyesters composed of poly[(R)-3-hydroxybutyrate-co-(R)-3hydroxyhexanoate] (P(3HB-co-3HH)) and poly[(R)-3-hydroxybutyrate] (P(3HB)) that exhibit a morphological change are prepared by solvent casting. Differential scanning calorimetry measurements indicate that P(3HB-co-3HH) and P(3HB) are miscible for all blend ratios because a single glass transition temperature is observed. Polarization optical microscopy is used to investigate the transition of spherulite morphology and measure the radial growth rate of spherulites in the blend films. P(3HB-co-3HH) and P(3HB) contain positive spherulites, whereas in the binary blends, spherulite morphology changes from positive to negative. This change is related to the different growth rates of P(3HB-co-3HH) and P(3HB) lamellar crystals. Partial enzymatic degradation of the film surfaces reveals that the lamellar crystals of negative spherulites are oriented both perpendicular and parallel to the radial direction of spherulites. A new growth mechanism for spherulites in crystalline/crystalline blends is constructed from the results obtained for the blend films.

Preparation and Characterization of Polyhydroxyalkanoates Macroporous Scaffold Through Enzyme-Mediated Modifications

Polyhydroxyalkanoates (PHAs) are hydrophobic biodegradable thermoplastics that have received considerable attention in biomedical applications due to their biocompatibility, mechanical properties, and biodegradability. In this study, the degradation rate was regulated by optimizing the interaction of parameters that influence the enzymatic degradation of P(3HB) film using response surface methodology (RSM). The RSM model was experimentally validated yielding a maximum 21 % weight loss, which represents onefold increment in percentage weight loss in comparison with the conventional method. By using the optimized condition, the enzymatic degradation by an extracellular PHA depolymerase from Acidovorax sp. DP5 was studied at 37 °C and pH 9.0 on different types of PHA films with various monomer compositions. Surface modification of scaffold was employed using enzymatic technique to create highly porous scaffold with a large surface to volume ratio, which makes them attractive as potential tissue scaffold in biomedical field. Scanning electron microscopy revealed that the surface of salt-leached films was more porous compared with the solvent-cast films, and hence, increased the degradation rate of salt-leached films. Apparently, enzymatic degradation behaviors of PHA films were determined by several factors such as monomer composition, crystallinity, molecular weight, porosity, and roughness of the surface. The hydrophilicity and water uptake of degraded salt-leached film of P(3HB-co-70%4HB) were enhanced by incorporating chitosan or alginate. Salt-leached technique followed by partial enzymatic degradation would enhance the cell attachment and suitable for biomedical as a scaffold.

The non-traditional growth regulator pectimorf is an elicitor of defense responses and protects Arabidopsis against Botrytis cinerea

Pectimorf is a non-traditional growth regulator produced in Cuba by partial enzymatic degradation of the cell wall of citrus fruit rinds. It is mainly composed of oligogalacturonides (OGs) and is currently used for in vitro micropropagation of different crops. Since OGs are known to elicit plant defense responses and are able to protect plants against fungal infection, we have investigated whether Pectimorf also has elicitor properties in Arabidopsis thaliana. The activity of Pectimorf was similar to that observed with similar doses of OGs in different bioassays (induction of gene expression, elicitation of oxidative burst, activation of MAP kinases). Furthermore, Pectimorf pre-treatments increased resistance of Arabidopsis leaves against infection by Botrytis cinerea. Pectimorf, as previously observed with OGs, also showed antagonistic effects with exogenous auxin. Taken together, these data indicate that Pectimorf is a potent elicitor of plant innate immunity and may be used as an inexpensive natural product to protect crops against diseases.

AN ENZYMATIC METHOD TO OBTAIN A NEW SCAFFOLD FOR ENGINEERING CARTILAGE

ABSTRACTThe purpose of this study was to achieve a descellularized scaffold from cartilage tissue, which can be used as xenograft for cartilage tissue regeneration.This work presents the results obtained using one method to wash porcine trachea in order to remove cellular material from the extracellular matrix and to avoid the immune reaction using enzymatic detergent and partial enzymatic degradation with Deoxyribonuclease I (DNase-I), Ethylenediaminetetraacetic Acid (EDTA) and Trypsin. This treatment was qualitatively evaluated by Scanning Electron Microscopy (SEM), and H&E Stain (Histology), and quantitatively evaluated by DNA quantification. The thermal characterization of the descellularized scaffold was carried out using Termogravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC). The type of collagen obtained from the scaffold was determined through SDS-PAGE electrophoresis. When using Enzymatic Treatment (ET) to wash trachea tissue, it is possible to obtain an acellular xenograft; this procedure has the potential to avoid rejection reactions of the xenograft.

Evaluation of Cell Adhesion and Proliferation on a Novel Tissue Engineering Scaffold Containing Chitosan and Hydroxyapatite

Porous chitosan-hxdroxyapatite hybrids were developed by partial enzymatic degradation [14] of the chitin/chitosan surface using chitosanase and lysozyme. This article evaluates the influence of chitosan and hydroxyapatite components, substrate roughness, stability, as well as surface porosity produced by enzymatic hydrolysis to cell adhesion and proliferation. L929 mouse fibroblastic lung cells were cultured on enzymatic degraded porous chitosan-hydroxyapatite hybrids. The presence of hydroxyapatite and porosity produced by partial lysozyme hydrolysis enhance cell proliferation. Besides, cell adhesion and proliferation are primarily dependent on substrate roughness and stability.

Dynamic release of riboflavin from a starch-based semi IPN via partial enzymatic degradation: part II

The dynamic release of riboflavin from the starch – cross-linked polyacrylic acid semi IPN has been studied at the physiological temperature 37 °C. The gel demonstrated nearly 39% drug in the amylase-free phosphate buffer of pH 7.4 while in the presence of 1.30 IU per ml enzyme activity in the release media, the gel underwent enzymatic degradation to release nearly 75% drug. The gel exhibited complex H-bonded structure in the lower pH thus demonstrating almost minimum release (i.e. 20%) in the release medium of pH 2.0. The amount of drug released was found to increase with the starch content in the gels. Similarly, with the increase in the initial water content, the quantity of drug released was also observed to increase. The degree of cross-linking was found to affect the release behavior. However, the gels demonstrated nearly `zero-order release' for maximum as well as minimum concentration of cross-linker in the gels. A linear dependency between square of diameter of the gel and t 1/2 i.e. time required for 50% release was also observed. Finally, the gastrointestinal transit time and agitation speed also affected the release behavior.

Enzymatically degradable and pH‐sensitive hydrogels for colon‐targeted oral drug delivery. I. Synthesis and characterization

AbstractCylindrical hydrogels, composed of starch and poly(acrylic acid), were synthesized, and their swelling behavior was studied as a function of the pH of the medium. The gels underwent a sharp transition from Fickian swelling behavior (swelling exponent n = 0.30) to non‐Fickian swelling behavior (n = 0.96) as the pH of the swelling medium changed from 2.0 to 7.4. The hydrogels also underwent partial enzymatic degradation in an amylase‐containing medium of pH 7.4 at 37°C. The effects of the enzyme concentration in the swelling media, the amount of starch present in the gel, the initial water content, the degree of crosslinking, and the diameter of cylindrical hydrogels on the degradation behavior were studied. The degradation of the gels followed Michaelis–Menten kinetics, and the value of the Menten constant was 41.62 × 10−2. The gels exhibited minimum swelling in an acidic pH medium through the formation of a complex hydrogen‐bonded structure and underwent enzymatic degradation in a medium of pH 7.4 (i.e., simulating intestinal fluid) along with chain‐relaxation‐controlled swelling. Therefore, the gels have potential for colon‐targeted drug delivery. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 92: 3630–3643, 2004

Structural Characterization and Enzymatic Degradation of α‐, β‐, and γ‐Crystalline Forms for Poly(β‐propiolactone)

AbstractA structural comparison of three different crystalline forms of poly(β‐propiolactone) (PPL) was carried out by wide‐angle X‐ray diffraction, Fourier‐transform infrared spectroscopy, and differential scanning calorimetry. The α‐form in a hot‐drawn and annealed film represents a 21 helix conformation. The β‐form in a cold‐drawn and annealed film represents a planar zigzag conformation. The γ‐form in an oriented sedimented mat of solution‐grown chain‐folded lamellar crystals also implies a planar zigzag conformation. The solution‐cast film depicts similar outlines with the γ‐form in lamellar crystals in all the experimental measurements, suggesting that the molecular chain in the solution‐cast film has a planar zigzag conformation. While elongation at break decreased, tensile strength and Young's modulus increased with an increase in the crystallinity, independent of the crystalline forms. The influence of the enzymatic degradation of these crystal structures has been investigated by using an extracellular PHB depolymerase purified from Ralstonia pickettii T1. The rate of degradation was in the order of β‐form > α‐form > solution‐cast (γ‐form) film, and the different surface morphologies after partial enzymatic degradation were observed in scanning electron micrographs. It is suggested that the crystal structure is one of the important factors for determining the rate of degradation together with crystallinity.Enzymatic degradation profiles of poly(β‐propiolactone) films.magnified imageEnzymatic degradation profiles of poly(β‐propiolactone) films.

Degradation of apple cell wall material by commercial enzyme preparations.

The action of commercial enzyme preparations on the release of cell wall constituents from alcohol-insoluble substance prepared from apples without skins and cores as well as their influence on the water binding of remaining residues is described as a model for the enzymatic cell wall destruction during production of liquid fruit products. Besides 'normal' enzyme concentrations adapted from the usual industrial dosage, 'tenfold' enzyme concentrations were applied. Dependent on enzyme spectrum and activities, concentrations of dietary fibre, e.g., pectin, increased in the soluble fractions using conditions of enzymatic 'mash treatment'. A further release of these cell wall constituents occurred when cellulase containing enzyme preparations were used under conditions of 'pomace treatment', especially with the 'tenfold' enzyme dosage. The partial enzymatic degradation of the cell wall material is connected with a decrease in water binding of the remaining residues during both simulated mash treatment of pomace treatment. Alcohol-insoluble substance from apples is a suitable model for the determination of complex enzymatic actions of enzyme preparations containing pectolytic, hemicellulolytic, and/or cellulolytic activities under standardised conditions.

Inorganic-organic polymer hybrid scaffold for tissue engineering - II: Partial enzymatic degradation of hydroxyapatite-chitosan hybrid

This article describes the preparation of porous chitosan, a hydroxyapatite hybrid, by partial enzymatic degradation. Two enzymes, chitosanase and lysozyme, were selected to hydrolyze a chitosan-reinforced matrix and create pores within the chitosan-hydroxyapatite composite. The degree of enzymatic hydrolysis of the chitosan-hydroxyapatite composite was determined by measuring the % weight loss of the chitosan matrix and the hydroxyapatite component. Hydroxyapatite loss from the chitosan matrix increased with the degree of enzymatic hydrolysis of the chitosanreinforced matrix. After hydrolysis, the composite was further characterized by FTIR. Quantitative analysis revealed a decrease in the characteristic pyranose ring peak (1072 cm-1), compared with PO2- 4 (1110 cm-1), showing that the chitosan matrices were enzymatically hydrolyzed. The surface 4 of the porous chitosan-hydroxyapatite composite, prepared by controlling enzymatic hydrolysis, was also observed by SEM.

Osteoarthritis‐related changes in human articular cartilage

We report the use of immunologic methods for detecting specific alterations in human osteoarthritic cartilage. Monoclonal antibodies with specificities for proteoglycans and link proteins have been used to define immunogenic sites in these molecules. We have identified 1 site in the protein core, 1 site in the link proteins, and another site that is common to both proteoglycan and link proteins, which are not modified in osteoarthritic cartilage. In addition, an antigenic determinant in link proteins that is altered in osteoarthritic cartilage has been identified. These results suggest that the structure of the ternary complex (hyaluronic acid binding region-link proteins-hyaluronic acid) could be altered in osteoarthritic cartilage. These modifications may be due to a genetic defect or to partial enzymatic degradation of these sites.

Diversity in immunoreactivity of tumor-derived cytokeratin monoclonal antibodies.

The cytokeratins 8, 18, and 19, expressed in many normal and malignant epithelial cells, were purified from human gastrointestinal tumors and used as immunogen for hybridoma generation. The reactivity pattern of five of the generated ten monoclonal antibodies (MAb) was characterized biochemically and immunohistochemically. All of the generated MAb were reactive with the central rod portion of the cytokeratins, as determined after partial enzymatic degradation, and displayed characteristic reactivity patterns. MAb TS 4 exhibits pan-epithelial immunohistochemical reactivity staining of all epithelial structures, including all layers of epidermis and non-keratinizing squamous epithelium and myoepithelial cells. The determinant involved is present on several different cytokeratins, i.e., nos. 1, 5, 7, 8, and 15, as determined by immunoblotting experiments from different tissues and cell lines. MAb TS 1, TS 3, and TS 7 reveal pluri-epithelial reactivity pattern immunohistochemically, similar to TS 4, but they are unreactive with whole epidermis and with superficial cell layers of non-keratinizing squamous cells. MAb TS 1 was found to be highly specific and reactive only with cytokeratin 8. Furthermore, the TS 1 MAb alone can precipitate the antigen, indicating reactivity with repetitive epitopes on cytokeratin 8. MAb TS 3 and 7 bind to cytokeratins 7 and 8. Finally, MAb TS 8 was found to be immunohistologically the most restricted, in general lacking reactivity to hepatocytes, pancreatic and salivary gland acinar cells, proximal renal tubules, and luminal cells of the epididymis. TS 8 was mainly reactive with cytokeratin 19 and showed weak binding to cytokeratin 8 and 14.