Schistosoma japonicum: immunoinhibitory studies on hemoglobin digestion using heterologous antiserum to bovine cathepsin D.

Antibody against purified bovine cathepsin D was raised in rabbits, and the polyclonal antiserum was tested to determine its ability to inhibit the hemoglobinolytic activity of the crude enzyme preparation (CEP) from adult Schistosoma japonicum and its effect upon in vitro cultured Schistosoma mansoni schistosomules. The 100,000 g supernatant fraction (CEP) from lyophilized adult worms was preincubated with antiserum and subsequently incubated with hemoglobin. Hemoglobinolytic activity was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoretic procedures. Five hours of incubation failed to diminish hemoglobin concentration in experimentals, whereas controls treated with preimmune serum displayed hemoglobin degradation. Pepstatin inhibited hemoglobin degradation. Western blot analysis of the CEP revealed a broad band of activity at approximately 45 kDa. Schistosomules incubated in vitro either in the antiserum or pepstatin and subsequently exposed to host erythrocytes showed a marked inhibition of digestive activities. Although structural changes were not evident in the gastrodermis, some perturbation of the tegument was observed. Schistosomules fed host erythrocytes and postincubated in the antiserum displayed increased tegumental perturbation and extensive alteration of the gastrodermis, including dilation of cisternae and membrane disruption. Schistosomules exposed to preimmune serum were normal in all respects.     

Comparative pepstatin inhibition studies on individual human pepsins and pepsinogens 1,3 and 5(gastricsin) and pig pepsin A

Human gastric juice contains 3 major proteolytic components (pepsins1,3 and 5 or gastricsin). Pepsin 1 is increased in peptic ulcer and it's properties are relatively poorly understood. Studies with pepstatin the highly specific aspartic-protease inhibitor have therefore been carried out on individual active and proenzymes to assess any enzymic similarities. Human pepsin 1 was inhibited with high affinity similar to pepsin 3, whereas pepsin 5(gastricsin) was at least 40 times less sensitive. Inhibition of human pepsinogens 1,3 and 5 and pig pepsinogen A showed similar trends to the active enzymes. Studies using Sephadex gel filtration showed that pepstatin does not bind to pepsinogens and inhibition arises from pepstatin binding the pepsins released upon activation. Pepstatin inhibition was shown to be relatively independent of pH between 1.5 and 3.8 although at higher pH inhibition was less effective. The evidence suggests that pepsin 1 is similar to pepsin 3 and pepstatin inhibits by a one to one molecular binding to the active site. The explanation for the reduced affinity of pepstatin to pepsin 5(gastricsin) needs further study by co-crystallisation X-ray analysis.     

Comparative Pepstatin Inhibition Studies on Individual Human Pepsins and Pepsinogens 1,3 and 5(gastricsin) and Pig Pepsin A

Human gastric juice contains 3 major proteolytic components (pepsins1,3 and 5 or gastricsin). Pepsin 1 is increased in peptic ulcer and it's properties are relatively poorly understood. Studies with pepstatin the highly specific aspartic-protease inhibitor have therefore been carried out on individual active and proenzymes to assess any enzymic similarities. Human pepsin 1 was inhibited with high affinity similar to pepsin 3, whereas pepsin 5(gastricsin) was at least 40 times less sensitive. Inhibition of human pepsinogens 1,3 and 5 and pig pepsinogen A showed similar trends to the active enzymes. Studies using Sephadex gel filtration showed that pepstatin does not bind to pepsinogens and inhibition arises from pepstatin binding the pepsins released upon activation. Pepstatin inhibition was shown to be relatively independent of pH between 1.5 and 3.8 although at higher pH inhibition was less effective. The evidence suggests that pepsin 1 is similar to pepsin 3 and pepstatin inhibits by a one to one molecular binding to the active site. The explanation for the reduced affinity of pepstatin to pepsin 5(gastricsin) needs further study by co-crystallisation X-ray analysis.     

Wheat Flour Proteases and Their Action on Gluten Proteins in Dilute Acetic Acid

The presence in wheat flour of several kinds of proteases was shown on the basis of pH-activity profile, substrate specificity, and response to inhibitors. Among them, ¹⁴C-hemoglobin and cbz-Phe-Ala hydrolases (¹⁴C-Hbase and CPAase) showed optimal activity around pH 4. ¹⁴C-Hbase was inhibited almost completely by pepstatin, and CPAase was partially inhibited by DFP. About 85% of ¹⁴C-Hbase activity and 40% of CPAase activity in the original flour were retained in gluten fraction. The decrease in viscosity of the gluten solution in dilute acetic acid was effectively prevented by pepstatin. An SDS-PAGE pattern showed that hydrolysis of gluten proteins was prevented effectively by pepstatin, although not completely, and that the simultaneous addition of pepstatin and DFP prevented completely self-digestion of the gluten proteins. Therefore, pepstatin and DFP sensitive proteases were shown to be responsible for the self-digestion of gluten proteins. The mode of action of these enzymes was relatively specific for the second highest molecular weight subunit of glutenin and for some other proteins.     

Developmentally regulated localization of endosymbiotic dinoflagellates in different tissue layers of coral larvae

In adult cnidarians, symbiotic dinoflagellate Symbiodinium are usually located in the gastrodermis. However, the onset of this endosymbiotic association and its regulation during larval development are unclear. This study examined the distribution of the Symbiodinium population in tissue layers of planula larvae released from the stony coral Euphyllia glabrescens. Symbiodinium were redistributed from the epidermis to the gastrodermis, at a rate that was fastest during early planulation and then decreased prior to metamorphosis. This process indicates that the endosymbiotic activity of coral tissues is developmentally regulated. During the early larval stage, both the epidermis and gastrodermis contained Symbiodinium; then, as the larvae developed toward metamorphosis, the numbers in the epidermis gradually diminished until they were only found in the gastrodermis. The mechanism of redistribution remains unknown, but may be due to a direct translocation and/or change in the proliferation of symbionts in different tissue layers.     

Receptor-mediated introduction of pepstatin-asialofetuin conjugate into lysosomes of rat hepatocytes

Pepstatin was linked through a carboxyl group to asialofetuin (PS-ASF). An analysis by separation of hepatocytes from nonparenchymal cells showed that PS-ASF was taken up by hepatocytes, following intravenous injection into rats. After the injection of PS-ASF, pepstatin concentration in the liver reached a maximum at 2 h and then decreased. In an analysis by differential centrifugation of the liver homogenate from rats injected with PS-ASF, pepstatin showed a lysosomal type subcellular distribution pattern. Isolation studies of tritosomes clearly demonstrated the exclusive accumulation of pepstatin within the lysosomes of livers from rats given PS-ASF (at 2 h after administration). Pepstatin contained in tritosomes was in a free form, as determined by column chromatography of Sephadex G-15. The activity of cathepsin D in the livers was markedly inhibited in rats given PS-ASF. However, the treatment of rats with PS-ASF had no effect on the hepatic lysosomal degradation of endocytosed FITC-labeled asialofetuin (FITC-ASF). Introduction of PS-ASF into the hepatocytes was followed by the immediate and time-dependent excretion of free pepstatin into the bile. Quantification of pepstatin excreted into the bile revealed that the biliary excretion route can account for the disappearance of pepstatin from the liver.     

Schistosoma mansoni: an in vivo study of drug-induced autophagy in the gastrodermis

The effects of Astiban, Lucanthone, Hycanthone and Niridazole on autophagic activities in the gastrodermis of Schistosoma mansoni were determined in vivo, using different dosage levels and dosage times. With Astiban, high levels of autophagy were observed in the gastrodermis 2 hours after an injection of the drug into the mouse, and this response had declined by 20 hours, marking a recovery by the parasite from the drug. Hycanthone and Lucanthone produced an autophagic response several days after the onset of treatment, and no recovery was observed in the morphology of the gastrodermis after the drug was discontinued. The effects of Niridazole on the gastrodermis were to produce the most dramatic ultrastructural changes after high doses and over several days of treatment. With all the drugs examined, gastrodermal autophagy was characterized by the formation of vacuoles containing cell components, lipid droplets and sometimes hydrolytic enzyme reaction product. The autophagic vacuoles appeared to be formed by the sequestration of cytoplasmic material by the basal membrane infoldings, and the transfer of enzymes into the vacuole from within the limiting membrane. The residues from intracellular digestion appeared to be emptied into the caecal lumen.     

pH-dependent reversible inhibition of violaxanthin de-epoxidase by pepstatin related to protonation-induced structural change of the enzyme

The redox enzyme violaxanthin de-epoxidase (VDE) was found to be sensitive to pepstatin, a specific inhibitor of aspartic protease. The inhibition was similar to that of aspartic protease in that it was reversible and accompanied by the protonation of the enzyme. Of the two peaks of VDE appearing on anion exchange chromatography, VDE-I predominated at pH 7.2. On lowering the pH of the chromatography, VDE-I decreased and VDE-II increased. Furthermore, re-chromatography of either peak yielded both peaks. These results suggest that VDE-I and VDE-II are interconvertible depending on pH, and thus, they represent the de-protonated and protonated forms of the enzyme, respectively. Presumably the protonation-induced structural change of the enzyme is responsible for the interaction with pepstatin, and also with substrate.     

Schistosoma japonicum: Immunocytochemistry of adults using heterologous antiserum to bovine cathepsin D

Results from previous biochemical and cytochemical studies show that a pepstatin-sensitive hemoglobinase is present in the gastrodermis of adult Schistosoma japonicum. To determine whether there is structural similarity to mammalian cathepsin D in addition to inhibition similarity, an immunocytochemical study was initiated using heterologous antiserum to bovine cathepsin D. With light microscopy, immunostaining was observed in the cecal lumen, the gastrodermis, and on the dorsal tegument and tubercles of male worms. With transmission electron microscopy, immunostaining was observed in gastrodermal autophagosomes and tegumental invaginations of the dorsal tegument of males. Immunostaining was also observed within the tubercles, but the reaction product did not appear to be associated with any definite structure or organelle. Heavy endogenous peroxidase activity in the cecal lumen due to ingested hemoglobin obscured with immunostaining. Assuming that all immunostaining is due to molecules that function in a manner similar to that of cathepsin D, it is suggested that such molecules may regulate some aspect of the host-parasite relationship and/or the parasite's metabolism. Alternatively, the immunostained molecules may be structural proteins with epitopes similar to those of mammalian cathepsin D. Reactions associated with the cecum, however, on the basis of previous studies, are believed to derive from molecules that are proteolytic enzymes.     

Mechanisms of cholinesterase inhibition by inorganic mercury

The poorly known mechanism of inhibition of cholinesterases by inorganic mercury (HgCl2) has been studied with a view to using these enzymes as biomarkers or as biological components of biosensors to survey polluted areas. The inhibition of a variety of cholinesterases by HgCl2 was investigated by kinetic studies, X-ray crystallography, and dynamic light scattering. Our results show that when a free sensitive sulfhydryl group is present in the enzyme, as in Torpedo californica acetylcholinesterase, inhibition is irreversible and follows pseudo-first-order kinetics that are completed within 1 h in the micromolar range. When the free sulfhydryl group is not sensitive to mercury (Drosophila melanogaster acetylcholinesterase and human butyrylcholinesterase) or is otherwise absent (Electrophorus electricus acetylcholinesterase), then inhibition occurs in the millimolar range. Inhibition follows a slow binding model, with successive binding of two mercury ions to the enzyme surface. Binding of mercury ions has several consequences: reversible inhibition, enzyme denaturation, and protein aggregation, protecting the enzyme from denaturation. Mercury-induced inactivation of cholinesterases is thus a rather complex process. Our results indicate that among the various cholinesterases that we have studied, only Torpedo californica acetylcholinesterase is suitable for mercury detection using biosensors, and that a careful study of cholinesterase inhibition in a species is a prerequisite before using it as a biomarker to survey mercury in the environment.     

Cytochemical and biochemical observations on the digestive tracts of digenetic trematodes. 8. Acid phosphatase activity in Schistosoma mansoni and Schistosomatium douthitti

At the light microscope level, nonspecific acid phosphatase (AcPase) (EC 3.1.3.2) and N-acetyl glucosaminidase (NAGase) (EC 3.2.1.29) activities are in the esophageal gland cells of Schistosoma mansoni and Schistosomatium douthitti and in the gastrodermis of S. mansoni. The gastrodermis of S. douthitti is negative for these two enzymes. At the electron microscope level, AcPase activity in the esophageal gland cells of both species is observed in cytoplasmic vesicles. In S. mansoni, AcPase activity is also observed associated with the infoldings of the basal plasma membranes of the esophagus and the gastrodermis. It is hypothesized that this enzyme(s) is involved with membrane transport. AcPase activity is also associated with “droplets” and vesicles in the gastrodermis of S. mansoni. It is believed that the digestion of foodstuffs in both species occurs extracellularly.     

The effect of pepstatin A, an inhibitor of the pro-opiomelanocortin (POMC)-converting enzyme, on POMC processing in mouse intermediate pituitary

In our previous studies, we have purified a unique, paired basic residue-specific, prohormone-converting enzyme from pituitary intermediate lobe secretory vesicles. This enzyme, an aspartyl protease, was shown to cleave the intermediate lobe prohormone, pro-opiomelanocortin (POMC), to adrenocorticotropin, beta-endorphin and a 16 kDa NH2-terminal glycopeptide, in vitro [(1985) J. Biol. Chem. 260, 7194-7205]. To provide some evidence that this enzyme plays a role in prohormone conversion in the intact cell, the ability of pepstatin A, an aspartyl protease inhibitor, to block POMC processing in the mouse intermediate pituitary was investigated. By the use of a radioactive pulse-chase paradigm, [3H]POMC processing was found to be inhibited by 36.4% in pepstatin A-treated intermediate lobes. This result is consistent with the inactivation of pro-opiomelanocortin-converting enzyme by pepstatin A in the intact pituitary and further supports a role of this enzyme in POMC processing in vivo.     

Mercury toxicity to terrestrial biota

The heavy metal mercury is a non-essential hazardous element which concentrates up the food chain. It is necessary to assess the ecological risk of mercury to establish proper regulatory guideline levels. Most of the toxicological assessment of mercury has been focused on aquatic organisms, however in terrestrial bodies the information is limited. Hence this review critically discusses the toxicity of inorganic mercury to key terrestrial biota from recent literature and evaluate whether these information are adequate to establish safe regulatory limits or precautionary values which is invaluable for risk assessment of mercury in soil. Till date soil microorganisms, plants and invertebrates have been utilized for assessing mercury toxicity; among them, microorganisms have been observed to be the most sensitive indicators to mercury stress. Large inconsistency among the measured toxic concentrations indicates that measuring mercury toxicity in soil may be influenced by soil characteristics and ageing period of contamination. This review warrants more studies to obtain widely acceptable safe limit of soil mercury.     

Isolation of tissue layers in hermatypic corals by N-acetylcysteine: morphological and proteomic examinations

Corals are diploblastic in body pattern and include two tissue layers, the epidermis and gastrodermis, interconnected by an acellular matrix mesoglea. During development, cells in these tissue layers differentiate morphologically and functionally. In most hermatypic corals, the gastrodermis further develops an ability to associate with microalgae dinoflagellates. This endosymbiosis occurs inside specific host gastrodermal cells, and its mechanism still remains unclear notwithstanding decades of research. The delay in progress is partly due to the difficulty in separating the gastrodermis and its symbionts from the epidermis for detailed cellular and biochemical investigations. The present study reports a simple method to separate these two tissue layers in hermatypic corals using the reducing agent, N-acetylcysteine (NAC). Efficient tissue and proteomic isolations are demonstrated by microscopy and two-dimensional SDS polyacrylamide gel electrophoresis (2D SDS-PAGE). The NAC treatment was able to separate tissue layers without inducing protein degradation. Furthermore, the sensitivity of protein detection greatly increases in the isolated tissue layers. The application of the present technique provides future research on endosymbiosis and coral development with a tool for higher accuracy and sensitivity.     

The relationship of mouse spermatozoal to mouse testicular cathepsins

Mouse testicular and sperm-associated cathepsin activities were compared for optimal conditions of assay, molecular sieving properties, inhibition data, and electrophoretic mobility. An extract of testes had several isoenzymes of cathepsin among which cathepsin B1 may be a major form while a sperm-associated component may be a minor form. The sperm-associated component was highly sensitive to pepstatin and may be cathepsin D.     

Mercury in bald eagle nestlings from South Carolina,USA

Bald eagles (Haliaeetus leucocephalus) may be at risk from contaminants in their diet and young birds may be particularly sensitive to contaminant exposure. To evaluate potential risks from dietary mercury exposure to eagle nestlings in South Carolina (USA), we surveyed mercury concentrations in 34 nestlings over two breeding seasons (1998 and 1999). Samples were also obtained from several post-fledging eagles in the region. Nestling feather mercury ranged from 0.61-6.67 micrograms Hg/g dry weight, nestling down mercury from 0.50-5.05 micrograms Hg/g dry weight, and nestling blood mercury from 0.02-0.25 microgram Hg/g wet weight. We did not detect significant differences in tissue mercury between nestlings from coastal and inland regions in contrast to some other studies of piscivorous birds. Mercury concentrations were much higher in the post fledging birds we sampled. Our data show that nestling eagles in South Carolina are accumulating mercury, and that concentrations in older birds may exceed regulatory guidelines.     

Ultrastructural observations on gastrodermal regeneration in the planarian Dugesia japonica (Turbellaria)

The earliest detectable change during regeneration of the gastrodermis in Dugesia japonica was an aggregation of regenerative cells underneath the gastrodermis remaining at the wound margin. The gastrodermal cells in experimental regenerates retained some of their original characters and presented no indication of cell dedifferentiation. The regenerative cells came into contact with the basal surface of gastrodermal cells, forming stratified cell layers. Differentiation of these cells into gastrodermal cells was initiated by the development of synthetic organelles within their cytoplasm. These differentiating cells gave rise to two different types of gastrodermal cells, namely phagocytic cells and sphere cells. In later stages, there was an apparent movement of differentiated gastrodermal cells towards the parenchyma.     

New renin inhibitors homologous with pepstatin.

Four homologues of pepstatin, the potent but poorly soluble inhibitor of aspartic proteinases, were synthesized by coupling to the C-terminus of the natural pentapeptide the following amino acid residues: L-arginine methyl ester, L-aspartic acid, L-glutamic acid and the dipeptide L-aspartyl-L-arginine. The peptide-coupling reagent we used, benzotriazolyloxytris(dimethylamino)phosphonium hexafluorophosphate, allowed us to obtain readily pure pepstatin homologues with high yields (60-83%). Pepstatylarginine methyl ester and pepstatylglutamic acid were about one order of magnitude more water-soluble than pepstatin. The four homologues and pepstatin were tested in vitro as inhibitors for highly purified pig and human renins acting on the N-acetyltetradecapeptide substrate. The 50% inhibitory concentrations (IC50) of the homologues were ranged from 0.01 to 1 microM against porcine renin at pH 6.0 (pepstatin IC50 approximately 0.32 microM) and from 5.8 to 41 microM against human renin at pH 6.5 (pepstatin IC 50 approximately 17 microM). By three different graphical methods we showed that pepstatin and the four homologues behaved as competitive inhibitors for porcine renin. The most potent inhibitors were pepstatylaspartic acid and pepstatylglutamic acid, with inhibitory constants respectively 2- and 10-fold smaller than that of pepstatin. By coupling glutamic acid to pepstatin, the ratio solubility/Ki was increased by two orders of magnitude.     

Interaction of human cathepsin D with the inhibitor pepstatin.

1. Because of the proposed role of cathepsin D in a variety of biological and pathological processes, the characteristics of inhibition by the potentially useful agent, pepstatin, were determined. 2. The beta and gamma forms of human cathepsin D, separated by isoelectric focusing, have identical specific extinction coefficients and specific activity in the degradation of haemoglobin. 3. Cathepsin D showed tight binding of 1 mol of pepstatin per 43000 g of protein, indicating that titration with the inhibitor represents a useful method for determination of absolute concentrations of the enzyme. 4. The titration curves were used to determine apparent dissociation constants (KD) for the binding of pepstatin and pepstatin methyl ester at pH3.5; values of approx. 5 X 10(-10)M were obtained. 5. Pepstatinyl-[3H]glycine was synthesized and shown to have a KD similar to that of pepstatin. Gel-chromatographic experiments showed that the binding of pepstatin and its derivatives is strongly pH-dependent. 6. The effect of pH on the KD for pepstatinyl-glycine was determined by equilibrium dialysis. As the pH was raised from 5.0 to 6.4, KD rose from 5 X 10(-10)M to 2 X 10(-6)M. 7. The catalytic activity of cathepsin D declines essentially to zero on going from pH5.0 to pH7.0, and we suggest that the binding site for substrate and pepstatin is abolished by a conformational change in the enzyme molecule. 8. The data indicate that, in biological experiments near neutral pH, large molar excesses of pepstatin over cathepsin D will be required for efficient inhibition.     

Biological activity of aspartic proteinase inhibitors related to pepstatin

We have synthesized eight tripeptide analogs of pepstatin in which both the side-chain and stereochemistry of the novel amino acid statine have been altered. They have been compared to pepstatin for inhibition of pepsin and cathepsin D activity, inhibition of autolysis at pH 4, and inhibition of protein degradation in cultured cells. Effective inhibition of aspartic proteinase activity appears to require the novel amino acid to have a bulky hydrophobic side-chain and the S-configuration at both chiral centers. However, the Cbz-Val-Val-(3S4S)-statine peptide was more effective than pepstatin in cultured cells, and inhibition was also achieved, and in some cases enhanced relative to pepstatin, by its stereoisomers and by tripeptides containing valyl and alanyl analogs of statine.