Pepsin Pretreatment Research Articles

Gelatin Gel from By-products of Sand Jellyfish (Rhopilema hispidum): Physicochemical and Biochemical Characterization

Salted sand-type jellyfish by-products are abundant in collagen, which may be processed into gelatin to decrease food waste. From the production standpoint, various factors affect gel qualities, including raw material used, pretreatment methods, and extraction times. So far, gelatin extracted from desalted sand-type jellyfish by-products (D-SJB) must be adequately characterized. Therefore, this research aimed to characterize gelatin from D-SJB using different pretreatment methods and extraction times. D-SJB was treated with 0.2 M hydrochloric acid (acid method) and extracted for 24 h at 60 °C (SA24), the optimal gelatin extraction condition with the highest gel qualities, while the jellyfish gelatin obtained after D-SJB was treated with pepsin and extracted for 48 h had the lowest gel qualities. The viscosity, gel strength, gelling temperature, and melting temperatures of SA24 were 20.80 cP, 352.22 g, 11.97 °C, and 22.70 °C, respectively. All jellyfish gelatin’s gelling and melting temperatures ranged from 6.13−11.97 °C and 15.85−22.70 °C, exhibiting a cold set gel and unstable gels at room temperature. The different pretreatment methods and extraction times during the jellyfish gelatin production resulted in the conversion of amides A, B, I, II, and III, especially the wavenumber of the amide I increased after pepsin pretreatment and increased with longer extraction time. Twenty-one collagen subtypes in bovine, fish, and jellyfish gelatin were analyzed using LC-MS/MS. The collagen alpha-2(I) chain, a key gelatin component, was identified in all gelatins. The research novelty showed the profound characterization results of gelatin gel produced from D-SJB. However, further experiments will be needed for pilot-scale production to be used in food and non-food applications.

Food-grade gelatin from camel skin: Extraction, characterisation and potential use for thin film packaging preparation

The present study aims on the extraction of gelatin from camel skin as a tool of agro-food by-products valorization. The extraction was performed through different methods, and the resulting gelatins served as biomaterial for edible films’ preparation. The results showed that Pepsin (P) pre-treatment increased the yield of gelatin extraction to reach 34 and 38%, at 70 and 90 °C, respectively, while it affected the molecular weight and amino acid composition, especially when the P-treatment was coupled with a high temperature (90 °C) in the extraction process. Nevertheless, gelatin extracted without pepsin at 70 °C (G70) showed the highest level of Gly, Pro and Hyp (530 residues/1000), reflecting its high polymerisation, compared to the other gelatins. Then, the extracted gelatins (G70, G90, G70-P and G90-P) were used for the preparation of thin films (∼60 μm), named F-G70, F-G90, F-G70-P and F-G90-P, respectively. Films possessed differences in the structural and techno-functional properties depending on the gelatin used for film formation. For instance, F-G70 was the strongest film and showed the best resistance against water solubility and vapor permeation. The highest mechanical and water resistance correlated with the thermal one, where F-G70 was characterized by the highest glass temperature (Tg = 74 °C) and this value tended to be decreased after increasing the gelatin extraction temperature and Pepsin pre-treatment. Overall, the present results proved that camel skin is a promising matrix of producing gelatin-based packaging intended for food applications.

Potential of Dry-Cured Ham Bones as a Sustainable Source to Obtain Antioxidant and DPP-IV Inhibitory Extracts.

The utilization of animal bones as a protein source could be used as a sustainable pathway for the production of bioactive compounds. In this study, bones were pretreated with pepsin enzyme (PEP) and then sequentially hydrolyzed with Alcalase (PA) and Alcalase, as well as Protana prime (PAPP). The degree of hydrolysis, antioxidant activity, and DPP-IV inhibitory activity were measured. All three hydrolysates showed antioxidant and DPP-IV inhibitory activity; however, the highest result in both bioactivities was obtained with the PAPP hydrolysate. The obtained free amino acid content was 54.62, 88.12, and 668.46 mg/100 mL of hydrolyzed in PEP, PA, and PAPP, respectively. Pepsin pretreatment did not significantly affect the degree of hydrolysis; however, it is suggested that it promoted the cleavage of certain bonds for subsequent protease action. Accordingly, a total of 550 peptides were identified in PEP hydrolysate, 1087 in PA hydrolysate, and 1124 in PAPP hydrolysate using an LC-MS/MS approach. Pepsin pretreatment could be an effective method in the utilization of bone sources for the production of antioxidant and hypoglycemic peptides.

Impact of antigen retrieval protocols on the immunohistochemical detection of epigenetic DNA modifications

This study compares three different pretreatment protocols for the immunohistochemical detection of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in nuclear DNA. The human biological samples analyzed included formalin-fixed and paraffin-embedded (FFPE) normal squamous epithelium, ethanol-fixed cultured cells, and metaphase chromosomes. The antigen retrieval methods included low pH Citrate and high pH Tris–ethylenediaminetetraacetic acid (EDTA) protocols, as well as a method using Pepsin pretreatment combined with HCl for DNA denaturation. A gradual increase in the detection levels of 5-mC and 5-hmC was observed when going from Citrate via Tris/EDTA to Pepsin/HCl retrieval. While the Citrate retrieval protocol was the least efficient for the detection of 5-mC and 5-hmC, it did preserve nuclear morphology and enabled visualization of differences in intra- and internuclear distribution patterns in tissue and cell culture samples by single- and double-fluorescence detection. Quantification of (hydroxy)methylation levels in FFPE material demonstrated a significant heterogeneity and differences in 5-mC and 5-hmC levels within and between nuclei in the different compartments of normal squamous epithelium. It was concluded that immunohistochemical detection of 5-mC and 5-hmC enables the correlation of these DNA modifications with histomorphological features in heterogeneous tissues, but this is influenced by different pretreatment protocols that must be carefully chosen to allow an appropriate interpretation of these epigenetic switches.

Pepsin pretreatment corrects underestimation of 25-hydroxyvitamin D measurement by an automated immunoassay in subjects with high vitamin D binding protein levels

Article Pepsin pretreatment corrects underestimation of 25-hydroxyvitamin D measurement by an automated immunoassay in subjects with high vitamin D binding protein levels was published on January 1, 2022 in the journal Clinical Chemistry and Laboratory Medicine (CCLM) (volume 60, issue 1).

Removing Cross-Linked Telopeptides Enhances the Production of Low-Molecular-Weight Collagen Peptides from Spent Hens.

The low-molecular-weight (LMW) peptides derived from collagen have shown a potential for various nutritional and pharmaceutical applications. However, production of LMW peptides from vertebrate collagen remains a challenge. Herein, we report a new method to produce LMW collagen peptides using pepsin pretreatment that removed cross-linked telopeptides in collagen molecules. After the pretreatment, the proportion of LMW collagen peptides (<1.4 kDa) that were obtained from pepsin-soluble collagen increased to 32.59% compared to heat-soluble collagen peptides (16.10%). Fourier transform infrared spectroscopy results indicated that telopeptide cleavage retained the triple-helical conformation of collagen. Liquid chromatography-tandem mass spectrometry analysis suggested that Gly-X-Y (X is often proline, while Y is either hydroxyproline or hydroxylysine) repeats were not the main factors that hindered the enzymatic hydrolysis of collagen molecules. However, cross-link quantification demonstrated that trivalent cross-links that included pyridinolines and pyrroles were the primary obstacles to producing small peptides from collagen of spent hens. This study demonstrated for the first time that removing cross-linked telopeptides could enhance the production of LMW peptides from spent hen collagen, which is also of interest to manufacturers who produce LMW collagen peptides from other vertebrate animals, such as bovids and porcids.

Surface binding properties of aged and fresh (recently excreted) Toxoplasma gondii oocysts

The surfaces of aged (10 years) and fresh (recently excreted) oocysts of Toxoplasma gondii were investigated using monoclonal antibody (mAb) and lectin-binding assays. Fresh oocysts bound a wall-specific mAb labelled with fluorescein isothiocyanate while aged oocysts did not. In contrast, the walls of aged oocysts bound a lectin (wheat germ agglutinin, WGA), but not the walls of fresh oocysts. Exposure of oocysts to detergent solutions or trypsin did not affect the binding properties of the walls of the oocysts. However, exposure of fresh oocysts to acidified pepsin enabled labelling of the walls with WGA, presumably due to the relevant moieties on the oocyst walls becoming exposed. WGA binding, but not mAb binding, was partially abrogated with periodate exposure. These findings reveal a significant difference in the binding properties of oocyst walls from “aged” and “fresh” oocysts. The results are of relevance when considering technologies for isolating or detecting T. gondii oocysts in environmental samples based on oocyst surface properties, as used for other protozoan parasites. Our results suggest the possibility of developing a WGA-based separation procedure for isolating Toxoplasma oocysts from environmental matrices, in which pepsin pre-treatment would be included to ensure that both fresh and aged oocysts were isolated.

Process integration for recovery of recombinant collagen type I α1 from corn seed.

Because of safety concerns and product consistency issues with the use of animal-derived collagen, several recombinant protein expression hosts have been considered for recombinant collagen corn seed. Full length, triple-helical, recombinant collagen (rCIα1) is expressed as a fusion with a foldon domain, which must later be removed. Here we have examined integration of purification and foldon removal by comparing advantages of removal before or after purification, using salt precipitation as the main purification step. Because expression levels in available maize lines are low, Pichia-produced recombinant collagens, both with and without foldon, were added to corn seed germ at the extraction step. Salt precipitation of an acidic corn seed extract yielded 100% of the collagen without foldon at >70% purity without the pepsin pretreatment. With pepsin pretreatment, yield was 94.0% with purity of 76.5%. Analysis of the protein molecular weight distribution of the pre- and post-treatment extracts showed that the corn proteins are largely resistant to pepsin proteolysis, explaining why little benefit was obtained by pepsin treatment. In the absence of pepsin treatment, the recovery of rCIα1 with foldon was still above 90% but the purity was only 44%. This still represented at about 13-fold purification with a 2.7-fold volume reduction which would reduce the pepsin requirement for post-recovery foldon cleavage.

Improvement of the ACE‐inhibitory and DPPH radical scavenging activities of soya protein hydrolysates through pepsin pretreatment

SummaryThis study aimed to investigate the effect of pepsin pretreatment on the ACE‐inhibitory and DPPH radical scavenging activities of soya protein hydrolysates prepared with alcalase and protamex. The protein recovery, TCA‐soluble peptide content, surface hydrophobicity, particle size and zeta potential were evaluated. Results showed that the hydrolysates exhibited varying ACE‐inhibitory (i.e. the highest value 72.6% by alcalase and 84.3% by protamex) and DPPH radical scavenging activities (i.e. the highest value 51.9% by alcalase and 51.7% by protamex). Pepsin pretreatment could make soya proteins more susceptible for hydrolysis, and the results showed that the ACE‐inhibitory and DPPH radical scavenging activities of the resultant hydrolysates were improved. A highly significant positive correlation between ACE‐inhibitory and DPPH radical scavenging activities was observed in the alcalase hydrolysates, while no significant correlation among other treatments. The physical properties of hydrolysates, that is surface hydrophobicity, particle size and zeta potential, could influence their ACE‐inhibitory and DPPH radical scavenging activities.

Cytogenetic Analysis of Lagria villosa (Coleoptera, Tenebrionidae): Emphasis on the Mechanism of Association of the Xyp Sex Chromosomes

The Xy_p sex determination mechanism is the system most frequent and ancestral to Coleoptera. Moreover, the presence of argyrophilous material associated with the sex bivalent is described as being responsible for the maintenance and association of these chromosomes. There are no karyotype data available regarding the genus Lagria and no consensus in the literature regarding the argyrophilous material present in the lumen of sex bivalent. Therefore, the aim of this work was to investigate the mechanism of sex chromosome bivalent association in Lagria villosa by analyzing the argyrophilous nature of the material present in the Xy_p lumen. It was also intended to characterize L. villosa cytogenetically. The analysis of meiotic cells showed 2n = 18 = 16+Xy_p for males and 2n = 18 = 16+XX in females and the meiotic formula was 2n = 8^II+Xy_p. The C-banding showed blocks of pericentromeric heterochromatin in all chromosomes except in the y_p chromosome. In these regions, the use of fluorochromes revealed the presence of heterochromatin containing GC rich DNA sequences. The study of synaptonemal complex showed a gradual increase in the electron-density of the axial elements of the sex chromosomes and their association with strongly electron-dense material. The pepsin pretreatment revealed that the material impregnated by silver is protein.

Short Communication: Use of a Prepaid Cellulase Solution for Screening Forage Grass Germplasm for Digestibility

A reliable rapid and inexpensive laboratory procedure is needed to screen native African forage grass species for forage quality. This study determined the potential of using a prepared cellulase solution to assess in vitro dry matter solubility of 22 native Kenyan forage grasses and 18 native warm season grasses from Missouri, USA. A prepared cellulase solution was used for screening the grass species for digestibility using two procedures. One procedure involved digesting grass samples in prepared cellulose solution without any pre-treatment (CSD), and the other procedure used an acid pepsin pretreatment prior to digestion in the prepared cellulose solution (APCS). The CSD procedures in comparison to APCS generally underestimated in vitro dry matter solubility by a rand of up to 23%. The variations were highly species dependent. Although the APCS procedure more time consuming, it made it possible to identify a group of species resolvable into high (42.6-61.4%), medium (36-40%) and low (29-34%) digestibility. With intensified efforts it is possible for one person to obtain in vitro dry matter solubility estimates on up to 200 samples in 30 days with minimal expense. Results of this investigation points to the end need for further research on the potential of the two digestibility procedures for rationalizing numbers in germplasm collection for initial screening.Keywords: Cellulase solution, forage grass germplasm, screening

Alcohol based tissue fixation as an alternative for formaldehyde: influence on immunohistochemistry

BackgroundFormaldehyde is commonly used in histopathology to fix tissue. Not only are the carcinogenic properties of this solution a hazard to the people in the workplace, it is also a...

Brain-Specific Deletion of Extracellular Signal-Regulated Kinase 2 Mitogen-Activated Protein Kinase Leads to Aberrant Cortical Collagen Deposition

The mitogen-activated protein kinases extracellular signal-regulated kinase (ERK)1 and 2 are essential intracellular mediators of numerous transmembrane signals. To investigate neural-specific functions of ERK2 in the brain, we used a Cre/lox strategy using Nestin:Cre to drive recombination in neural precursor cells. Nestin:Cre;ERK2(fl/fl) conditional knockout (cKO) mice have architecturally normal brains and no gross behavioral deficits. However, all cKO mice developed early-onset (postnatal day 35 to 40) frontal cortical astrogliosis, without evidence of neuronal degeneration. Frontoparietal cortical gray matter, but not underlying white matter, was found to contain abundant pericapillary and parenchymal reticulin fibrils, which were shown by immunohistochemistry to contain fibrillar collagens, including type I collagen. ERK1 general KO mice showed neither fibrils nor astrogliosis, indicating a specific role for ERK2 in the regulation of brain collagen. Collagen fibrils were also observed to a lesser extent in GFAP:Cre;ERK2(fl/fl) mice but not in CamKII-Cre;ERK2(fl/fl) mice (pyramidal neuron specific), consistent with a possible astroglial origin. Primary astroglial cultures from cKO mice expressed elevated fibrillar collagen levels, providing further evidence that the phenotype may be cell autonomous for astroglia. Unlike most other tissues, brain and spinal cord parenchyma do not normally contain fibrillar collagens, except in disease states. Determining mechanisms of ERK2-mediated collagen regulation may enable targeted suppression of glial scar formation in diverse neurological disorders.

Cbln1 accumulates and colocalizes with Cbln3 and GluRδ2 at parallel fiber–Purkinje cell synapses in the mouse cerebellum

Cbln1 (a.k.a. precerebellin) is secreted from cerebellar granule cells as homohexamer or in heteromeric complexes with Cbln3. Cbln1 plays crucial roles in regulating morphological integrity of parallel fiber (PF)-Purkinje cell (PC) synapses and synaptic plasticity. Cbln1-knockout mice display severe cerebellar phenotypes that are essentially indistinguishable from those in glutamate receptor GluRdelta2-null mice, and include severe reduction in the number of PF-PC synapses and loss of long-term depression of synaptic transmission. To understand better the relationship between Cbln1, Cbln3 and GluRdelta2, we performed light and electron microscopic immunohistochemical analyses using highly specific antibodies and antigen-exposing methods, i.e. pepsin pretreatment for light microscopy and postembedding immunogold for electron microscopy. In conventional immunohistochemistry, Cbln1 was preferentially associated with non-terminal portions of PF axons in the molecular layer but rarely overlapped with Cbln3. In contrast, antigen-exposing methods not only greatly intensified Cbln1 immunoreactivity in the molecular layer, but also revealed its high accumulation in the synaptic cleft of PF-PC synapses. No such synaptic accumulation was evident at other PC synapses. Furthermore, Cbln1 now came to overlap almost completely with Cbln3 and GluRdelta2 at PF-PC synapses. Therefore, the convergence of all three molecules provides the anatomical basis for a common signaling pathway regulating circuit development and synaptic plasticity in the cerebellum.

Pepsin pretreatment allows collagen IV immunostaining of blood vessels in adult mouse brain

While the brain vasculature can be imaged with many methods, immunohistochemistry has distinct advantages due to its simplicity and applicability to archival tissue. However, immunohistochemical staining of the murine brain vasculature in aldehyde fixed tissue has proven elusive and inconsistent using current protocols. Here we investigated whether antigen retrieval methods could improve vascular staining in the adult mouse brain. We found that pepsin digestion prior to immunostaining unmasked widespread collagen IV staining of the cerebrovasculature in the adult mouse brain. Pepsin treatment also unmasked widespread vascular staining with laminin, but only marginally improved isolectin B4 staining and did not enhance vascular staining with fibronectin, perlecan or CD146. Collagen IV immunoperoxidase staining was easily combined with cresyl violet counterstaining making it suitable for stereological analyses of both vascular and neuronal parameters in the same tissue section. This method should be widely applicable for labeling the brain vasculature of the mouse in aldehyde fixed tissue from both normal and pathological states.

Lumican expression is associated with the formation of mesenchyme-like elements in salivary pleomorphic adenomas.

Pleomorphic adenomas are the most common salivary gland tumour. Although this tumour is considered to be of epithelial origin, it contains 'mesenchyme'-like elements histologically. Lumican is a keratan sulphate proteoglycan that belongs to the small leucine-rich repeat (LRR) proteoglycans and has been reported to be associated with cartilage formation. These findings suggest that lumican expression may be related to the chondroid component in pleomorphic adenomas. To investigate this hypothesis, the present study investigated the expression and localization of lumican in 20 normal human salivary glands and 35 pleomorphic adenomas. Firstly, immunohistochemistry for lumican was performed with pepsin pretreatment. In normal salivary glands, lumican was deposited in the periductal regions. In pleomorphic adenomas, it was predominantly deposited in the hyaline (100%) and fibrous areas (89.4%). In 16 tumours (66.7%), lumican was also deposited in the chondroid areas. Without pepsin pretreatment, lumican was identified in myoepithelial cells in myxoid areas, lacuna cells in chondroid areas, and in the cytoplasm of inner ductal cells. In situ hybridization revealed lumican mRNA expression mainly in the inner cells, the neoplastic myoepithelial cells, and the lacuna cells. These results suggest that lumican is associated with the formation of 'mesenchyme'-like structures in pleomorphic adenomas. In conclusion, normal salivary glands express lumican, which appears to be related to stromal maintenance, and pleomorphic adenomas express lumican mRNA and protein, which may play important roles in the formation of 'mesenchyme'-like areas in this type of tumour.

NMDA receptor subunits GluRepsilon1, GluRepsilon3 and GluRzeta1 are enriched at the mossy fibre-granule cell synapse in the adult mouse cerebellum.

Cerebellar N-methyl-D-aspartate (NMDA) receptors are concentrated in the granular layer and are involved in motor coordination and the induction of long-term potentiation at mossy fibre-granule cell synapses. In the present study, we used immunohistochemistry to examine the distribution of NMDA receptor subunits in the adult mouse cerebellum. We found that appropriate pepsin pretreatment of sections greatly enhanced the sensitivity and specificity of immunohistochemical detection. As a result, intense immunolabelling for GluRepsilon1 (NR2A), GluRepsilon3 (NR2C), and GluRzeta1 (NR1) all appeared in synaptic glomeruli of the granular layer. Double immunofluorescence showed that these subunits were colocalized in individual synaptic glomeruli. Within the glomerulus, NMDA receptor subunits were located between centrally-located huge mossy fibre terminals and peripherally-located tiny Golgi axon terminals. By immunoelectron microscopy, all three subunits were detected at the postsynaptic junction in granule cell dendrites, forming synapses with mossy fibre terminals. Consistent with the known functional localization, GluRepsilon1, GluRepsilon3, and GluRzeta1 are, thus, anatomically concentrated at the mossy fibre-granule cell synapse. By contrast, immunohistochemical signals were very low in Purkinje cell somata and dendrites in the molecular layer. The lack of GluRzeta1 immunolabelling in Purkinje cells was unexpected because the cells express GluRzeta1 mRNA at high levels and high levels of GluRzeta1 protein in the molecular layer were revealed by immunoblot. As Purkinje cells are exceptionally lacking GluRepsilon expression, the discrepant result may provide in vivo evidence suggesting the importance of accompanying GluRepsilon subunits in synaptic localization of GluRzeta1.

Improved immunohistochemical detection of postsynaptically located PSD‐95/SAP90 protein family by protease section pretreatment: A study in the adult mouse brain

Postsynaptic density (PSD)–95, SAP102, and Chapsyn-110 are members of the PSD-95/SAP90 protein family, which interact with the C-terminus of N-methyl-D-aspartate (NMDA) receptor and shaker-type potassium channel subunits. Here we report that appropriate section pretreatment with pepsin has led to qualitative and quantitative changes in light microscopic immunohistochemical detection of the protein family. First, pepsin pretreatment lowered the concentration of affinity-purified primary antibodies, while it greatly increased the intensity of immunoreactions. Second, the resulting overall distributions of PSD-95, SAP102, and Chapsyn-110 in the adult mouse brain were consistent with their mRNA distributions. Third, instead of the reported patterns of somatodendritic labeling, tiny punctate staining in the neuropil became overwhelming. Fourth, many PSD-95-immunopositive puncta were apposed closely to synaptophysin-positive nerve terminals and overlapped with NMDA receptor subunits. By postembedding immunogold, the PSD-95 antibody was shown to label exclusively the postsynaptic density at asymmetrical synapses. Based on these results, we conclude that antibody access and binding to the postsynaptically located PSD-95/SAP90 protein family are hindered when conventional immunohistochemistry is adopted, and that pepsin pretreatment effectively unmasks the postsynaptic epitopes. On the other hand, PSD-95 in axon terminals of cerebellar basket cells, where high levels of potassium channels are present, was detectable irrespective of pepsin pretreatment, suggesting that PSD-95 antibody is readily accessible to the presynaptic epitopes. Consequently, the present immunohistochemical results have provided light microscopic evidence supporting the prevailing notion that the PSD-95/SAP90 protein family interacts with NMDA receptor subunits and potassium channel subunits. J. Comp. Neurol. 426:572–586, 2000. © 2000 Wiley-Liss, Inc.

Improved immunohistochemical detection of postsynaptically located PSD-95/SAP90 protein family by protease section pretreatment: A study in the adult mouse brain

Postsynaptic density (PSD)-95, SAP102, and Chapsyn-110 are members of the PSD-95/SAP90 protein family, which interact with the C-terminus of N-methyl-D-aspartate (NMDA) receptor and shaker-type potassium channel subunits. Here we report that appropriate section pretreatment with pepsin has led to qualitative and quantitative changes in light microscopic immunohistochemical detection of the protein family. First, pepsin pretreatment lowered the concentration of affinity-purified primary antibodies, while it greatly increased the intensity of immunoreactions. Second, the resulting overall distributions of PSD-95, SAP102, and Chapsyn-110 in the adult mouse brain were consistent with their mRNA distributions. Third, instead of the reported patterns of somatodendritic labeling, tiny punctate staining in the neuropil became overwhelming. Fourth, many PSD-95-immunopositive puncta were apposed closely to synaptophysin-positive nerve terminals and overlapped with NMDA receptor subunits. By postembedding immunogold, the PSD-95 antibody was shown to label exclusively the postsynaptic density at asymmetrical synapses. Based on these results, we conclude that antibody access and binding to the postsynaptically located PSD-95/SAP90 protein family are hindered when conventional immunohistochemistry is adopted, and that pepsin pretreatment effectively unmasks the postsynaptic epitopes. On the other hand, PSD-95 in axon terminals of cerebellar basket cells, where high levels of potassium channels are present, was detectable irrespective of pepsin pretreatment, suggesting that PSD-95 antibody is readily accessible to the presynaptic epitopes. Consequently, the present immunohistochemical results have provided light microscopic evidence supporting the prevailing notion that the PSD-95/SAP90 protein family interacts with NMDA receptor subunits and potassium channel subunits.

Effects of acid pepsin pretreatment, bile acids and reductants on the excystation of Clonorchis sinensis (Trematoda: Opisthorchiidae) metacercariae in vitro

The effects of acid pepsin pretreatment, bile acids and reductants on the excystation of Clonorchis sinensis metacercariae were studied. The velocities of metacercarial excystation, digestion of the metacercarial outer layer and tryptic BAEE hydrolysis were measured in various media. Pretreatment with acid or acid pepsin accelerated the excystation, but was not required under adequate tryptic activity. Taurocholate and deoxycholate evoked no excystation by themselves. However, in the trypsin medium with taurocholate and deoxycholate, they accelerated both the velocity of metacercarial excystation and of digestion of the outer layer, although the accelerative effect did not increase above 4 −2% deoxycholate. Taurocholate below 1% and deoxycholate below 4 −2% did not affect the rate of tryptic hydrolysis, while this was depressed by deoxycholate over 4 −2%. Addition of deoxycholate over 4 −2% also depressed the final percentage of excystation, as well as worm motility. By themselves, cysteine and 2-mercaptoethanol (MCE) swelled and deformed the cyst wall and evoked metacercarial excystation without trypsin, however, ascorbate and Na dithionite evoked no excystation. Addition of ascorbate to the trypsin medium did not change the velocity of excystation, but this was completely inhibited by the addition of dithionite and was accelerated by the addition of cysteine or MCE. Although excystation was accelerated with an increase of cysteine concentration, this acceleration was depressed at over 0.01 M cysteine. The rate of tryptic hydrolysis was depressed at over 0.002 M cysteine. The present study suggests that the excystation facilitative effects of pretreatment of acid pepsin, bile acids and reductants (cysteine and MCE) in trypsin medium are due to denaturation of protein, detergent action and cleavage of SS-linkage, respectively, on the digestion step of the outer layer of the metacercariae and cessation of the facilitative effect at a high concentration of deoxycholate is related to the rupturing step of the inner layer by larval movement.