Clint E. Carter

In vivo studies of cadmium-induced apoptosis in testicular tissue of the rat and its modulation by a chelating agent

In vivo CdCl2-induced apoptotic DNA fragmentation in the testes of the male Wistar rat has been demonstrated on agarose gel. Characteristic DNA migration patterns (laddering) provide evidence of apoptosis (programmed cell death) in testicular tissue of rats administered CdCl2 at a level of 0.03 mmol/kg 48 h previously. Evidence that administration of an appropriate cadmium chelating agent within the first 24 h can suppress some or all of the apoptotic changes in testicular DNA has also been obtained for the first time. A greater reduction in apoptosis is observed as the interval between the administration of the cadmium and that of the chelating agent is shortened. Administration of monoisoamyl meso-2,3-dimercaptosuccinate (Mi-ADMS) to male Wistar rats given CdCl2 is effective in the modulation of the typically apoptotic DNA fragmentation and associated histopathologic injury when the antagonist is given within approximately 1 h after the CdCl2 exposure. When the antagonist is given at later times there is a progressively more pronounced degradation of the DNA into oligonucleotides as seen in the typical electrophoretic DNA ladder pattern found with apoptosis. There is also a progressive increase in histopathological tissue changes as the antagonist is administered at progressively greater intervals after the cadmium.

An electrophoretic analysis of Schistosoma mansoni soluble egg antigen preparation.

Schistosoma mansoni soluble egg antigen (SEA) has been examined electrophoretically. Sodium dodecyl sulfate (SDS) electrophoresis of SEA reveals an extremely heterogeneous protein composition. At least 18-20 distinct bands stain with Coomassie blue and at least 6 bands stain with periodic acid Schiff (PAS). Four of the PAS-positive bands stain only faintly with Coomassie blue. The estimated molecular weight range for these proteins is between 16,000 and 200,000 daltons. An acid soluble fraction was isolated from SEA which contained 5 of the 6 glycoproteins. An immunoelectrophoretic analysis of SEA reveals at least 5 distinct precipitin arcs when developed with serum from mice infected with S. mansoni for 16 weeks.

CADMIUM-INDUCED APOPTOSIS IN THE UROGENITAL ORGANS OF THE MALE RAT AND ITS SUPPRESSION BY CHELATION

Cadmium-induced apoptosis is shown to occur, in vivo, in several organs of the male Wistar rat urogenital system, 48 h after cadmium administration i.p. at a dose of 0.03 mmol/kg. Characteristic DNA fragmentation (as measured by an enzyme-linked immunosorbent-assay, ELISA) and histopathologically observed changes characteristic of apoptosis are found in the kidney, prostate, seminal vesicles, testes, and epididymis. TUNEL assay also demonstrates the apoptosis. Such changes are absent from bladder and vas deferens tissue. Timely administration of an appropriate chelating agent capable of reaching intracellular cadmium binding sites can suppress the processes leading to apoptosis. Administration of monoisomyl meso-2,3-dimercaptosuccinate (Mi-ADMS, 0.5 mmol/kg i.p.) to cadmium-treated rats is effective in greatly reducing typical histopathologic signs of apoptosis and the associated chromatin DNA fragmentation as revealed by ELISA when the antagonist is administered 1 h after cadmium. Administration of the chelating agent at law times results in greater degradation of DNA into oligonucleotides and more prominent histopathological evidence of apoptotic changes in the affected organs of the rat urogenital system. There is also a progressive increase in apoptotic changes indicated by TUNEL assay, as the antagonist is administered at progressively greater intervals after cadmium.

Cloning of a Schistosoma japonicum gene encoding a major immunogen recognized by hyperinfected rabbits.

A library of randomly sheared Schistosoma japonicum genomic DNA fragments was constructed in the bacteriophage expression vector lambda gt11. A portion of the library was screened with sera collected from rabbits 8 weeks after they were infected with 1000 cercariae. Four clones whose recombinant gene products react with the rabbit sera were purified to homogeneity. Clone SjIR-12A was chosen for detailed study because of its very intense reaction with the rabbit sera. SjIR-12A was found to encode part of a 70 kDa protein (Sj70) that is present in both soluble egg antigen (SEA) and soluble worm antigen preparations (SWAP). Western blot analysis suggests that Sj70 is the only SWAP component that is strongly immunoreactive with the rabbit sera. Rabbit antibodies that react with the SjIR-12A fusion protein were immunoaffinity purified and used to localize immunoreactive product to the nervous tissue of male and female adult worms, the dorsal and lateral tegument of male adult worms, and in eggs to the miracidial tegument and the area between the eggshell and miracidium. Southern hybridization analysis suggests there are approximately four copies of the Sj70 gene per haploid genome.

Ultrastructural effects of the chelating agent 1,10-phenanthroline on Trypanosoma cruzi epimastigotes in vitro.

Abstract The in vitro effects of the metal chelator 1,10-phenanthroline (OPHEN) on the ultrastructure of Trypanosoma cruzi epimastigotes were investigated. Epimastigotes treated with OPHEN display swelling and electron-dense deposits in the kinetoplast, mitochondrion, and cisternae of the endoplasmic reticulum. These morphological alterations are dose-dependent and first appear at an OPHEN concentration of 5.0 μg/ml. Analytical electron microscope examination indicates that the metallic portion of the electron-dense deposits is predominantly calcium.

Immunocytochemical studies on Schistosoma mansoni. I. Soluble egg antigen in eggshell-enclosed miracidium.

The localization of soluble egg antigen (SEA) in the eggshell-enclosed miracidium of Schistosoma mansoni was performed at the light and electron microscope levels using the unlabeled antibody method. Reaction product was observed associated with the contents of the 3 major gland cells described previously. Additionally, smaller vesicles were observed that reacted positively for SEA. It was hypothesized that they may be responsible for the secretion of the hatching fluid. SEA components were also observed in the epidermis of the miracidium and in the area subjacent to the eggshell. Following oviposition, the egg of Schistosoma mansoni migrates through the tissue of the vertebrate host, its passage apparently facilitated by soluble secretions which pass to the exterior through micropores in the eggshell (see Warren, 1972, for a review). The biological function of these soluble secretions is probably twofold. Besides facilitating passage through the tissue as mentioned, they may also aid in the hatching process. Among the components of these secretions are soluble egg antigens (SEA) which are capable of inducing and eliciting the granulomatous response seen in several tissues of the vertebrate host, such as liver and intestine. It is the intent of this investigation to use immunocytochemical techniques at the electron microscope level to determine localization sites of the antigenic components of SEA in the egg and its enclosed miracidium. MATERIALS AND METHODS

In vitro effects of mannan and cytochalasin B on the uptake of horseradish peroxidase and [14C]sucrose by Trypanosoma cruzi epimastigotes

Ultrastructural and quantitative studies were conducted to determine the in vitro effects of mannan and cytochalasin B (CB) on the transport of horseradish peroxidase (HRP) and [14C]sucrose by epimastigotes of Trypanosoma cruzi (strain Y). Time-dependent changes in HRP uptake were observed in cells incubated with the actin inhibitor CB. After 60 min incubation in CB, HRP and sucrose uptakes were inhibited by 48 +/- 15.4% and 16.5 +/- 3.96%, respectively. Morphological changed included HRP reaction product on the cell surface and a reduction in the number of HRP-positive reservosomes when compared to controls. After 120 min incubation, no inhibition was measured for either molecule. However, electron microscopy revealed HRP reaction product on the surface of the cells and in the cytosol. Also, perturbation of the plasma membrane was evident, suggesting that CB compromised the integrity of the plasma membrane, allowing HRP and sucrose to diffuse into the cytosol, giving misleading quantitative results. Mannan displayed a concentration-dependent inhibitory effect on HRP uptake but had little effect on sucrose uptake. Electron microscopic analysis revealed no change in the number of reservosomes per cell but reduction in the amount of HRP in reservosomes concomitant with mannan concentration. These results suggest that T. cruzi epimastigotes transport HRP by receptor-mediated and fluid-phase pinocytosis.

Characterization of a large gene family in Schistosoma japonicum that encodes an immunogenic miracidial antigen.

Three of eleven clones isolated from a genomic expression library of Schistosoma japonicum DNA using chronically infected human sera also react with chronically infected mouse sera. Characterization of these three clones showed that they contain different members of the same gene family. One clone contains two members of the gene family approximately 2 kb apart and in opposite orientation to each other. DNA sequence homologies between pairs of genes range from 98% to 99.5%. Southern hybridization results indicate there are approximately 40 copies of these genes per haploid genome. Sera from mice immunized with purified fusion protein detected immunoreactive products in the central ganglion and ciliated epidermal cells of miracidia.

PROPERTIES OF GLYCOGEN SYNTHASE AND PHOSPHORYLASE FROM BIOMPHALARIA GLABRATA (MOLLUSCA)

Glycogen synthase and phosphorylase were characterized from the cephalopedal region of the snail, Biomphalaria glabrata. Glycogen synthase exhibited increases and decreases in its activity ratio (-G6P/+G6P) under conditions that are known to cause conversion of the two forms of the enzyme from mammalian systems, implying that the snails synthase also possesses interconvertible forms. Each form had a distinct pH optimum, with the G6P-independent form (synthase alpha) exhibiting maximum activity at pH 7.4, whereas the G6P-dependent form (synthase beta) had optimal activity at pH 8.3. Both synthase alpha and beta displayed classical Michaelis-Menten kinetics for the substrates UDP-glucose and glycogen, and the beta form displayed sigmoidal kinetics for its modulator, G6P. Only UDP-glucose could function as a glucosyl donor in the synthase-catalyzed reaction. ADP, GDP, UDP, and ATP were all competitive inhibitors of synthase alpha, although at varying degrees of efficiency. Glycogen phosphorylase also demonstrated interconversion of its two forms (alpha and beta), as evidenced by changes in its activity ratio (-AMP/+AMP). AMP elicited hyperbolic kinetics from this enzyme. Concentrations of KF above 20 mM were found to inhibit glycogen synthase alpha while stimulating phosphorylase beta, thus causing erroneous activity ratios for both enzymes.

Schistosoma japonicum: Excretory-secretory products of the eggs during miracidial development

The release pattern of excretory-secretory (E-S) products of Schistosoma japonicum eggs was investigated using eggs cultured in a chemically defined medium (MEMSE-J) for 16 days. The amount of protein released in culture supernatants was greater in 0- to 4-day and 12- to 16-day cultures than in 4- to 12-day cultures. The protein composition of E-S products and soluble extracts of newly laid eggs (N-SEA) and in vitro matured eggs (M-SEA) was analyzed by SDS-PAGE. Silver staining patterns of N-SEA and M-SEA were found to be similar except for the band at approximately 66 kDa, which appeared in highest concentrations in N-SEA. Western blot analysis with human infected sera showed antibody recognition of a 140- to 160-kDa antigen present in E-S products from mature eggs, while E-S products from immature eggs were unreactive. When either [35S]methionine or [3H]glucosamine was added to the culture medium, newly synthesized proteins or glycoproteins of the SEA and E-S products were labeled. Incorporation of both isotopes into SEA appears to correlate with developmental activity of the eggs. In contrast, release of E-S proteins and glycoproteins is more apparent as the miracidium matures. These results suggested that the source of E-S products from immature eggs is likely to be the collapsing vitelline cells and that of E-S products from mature eggs to be mainly miracidial secretions.