Lethal effects of freezing Echinococcus multilocularis eggs at ultralow temperatures.

Methods for killing Echinococcus multilocularis eggs within stool or intestinal samples, without damaging the diagnostic value of the sample, would significantly reduce the risk of animal health providers acquiring alveolar hydatid disease. The first objective of this study was to determine whether E. multilocularis eggs located in fox intestines can survive storage at -70 C for at least 4 days. Results showed that none of 72,000 E. multilocularis eggs remained infectious to defined strains of mice under these conditions, yet, similar eggs recovered from nonfrozen carcasses stored at 4 C for the same time period were viable. The structural identities of adult worms and eggs were not significantly altered by the freezing and thawing processes. These results indicate that ultracold temperatures can be used to kill or inactivate E. multilocularis eggs, making them safe to handle when diagnosing this parasite in definitive hosts. The second objective of this study was to determine whether E. multilocularis eggs could survive freezing to -70 C if commonly used cryopreservation protocols were used. The use of the cryoprotectant solution, 5% dimethyl sulfoxide-35% saline-60% lamb serum, with a -1 C/min freezing rate was unable to prevent the eggs from being killed by freezing to -70 C. Rapid cooling by plunge freezing into liquid nitrogen was also lethal to E. multilocularis eggs. Only a few of the many potential cryopreservation protocols were tested in this study, so it is not yet possible to completely rule out the possibility of preserving these eggs at ultralow temperatures, but it does indicate that temperatures below -70 C are lethal to eggs even under favorable storage conditions.

Inhibition of selenite cataract by S-diethylsuccinyl glutathione isopropyl ester.

PURPOSE: Previous studies have demonstrated that glutathione derivatives can partially prevent loss of hepatic glutathione levels, in vivo, during periods of oxidative stress. Since cataracts in animal model systems and in humans are thought to be the direct result of oxidative insult, the following study tested the possibility that treatment with these glutathione analogues may affect the progression of lens opacification. METHODS: Glutathione esters were tested for their ability to inhibit the selenite-induced cataract in rats. RESULTS: The S-alkyl glutathione ester Et(2)Sc-GS-iPr, but not a similar glutathione derivative S-succinyl glutathione (Sc-GS), at 0.5 mmoles/kg body weight, had anti-cataract activity in the selenite-induced cataract of rats. Analysis of lenses from treated and untreated animals demonstrated that Et(2)Sc-GS-iPr partially prevented the loss of reduced glutathione levels. CONCLUSIONS: The results demonstrate for the first time that an S-alkylated glutathione ester has anti-cataractogenic potential in the selenite-treated rat, and suggest possible mechanisms involving glutathione in the prevention of this lens opacification.

Confocal microscopy of cataracts from animal model systems: relevance to human nuclear cataract.

A recent study demonstrated that cytosolic lipid membrane structures, independent of the plasma membrane, preferentially occurred in human cataractous lenses. Animal model systems of cataractogenesis (selenite treated rats: galactose fed rats; buthionine-sulfoxime treated mice; Emory mice) were screened for possible relevant structures using the lipid membrane probe DiI and confocal microscopy. Well delineated plasma membranes of lens fiber cells with independent cytosolic staining structures were only observed in the selenite model system. These cytosolic structures were not observed in aged matched control lenses or within the transparent cortical regions of selenite treated animals with intense nuclear opacification. These results suggested that the morphological changes in DiI staining structures seen in the nucleus of the human cataractous lens were best approximated by those seen in the selenite model system.

Molecular karyotype analysis of Cryptosporidium parvum: evidence for eight chromosomes and a low-molecular-size molecule.

We report improved separation of chromosome-sized DNA molecules of the coccidian parasite Cryptosporidium parvum with contour-clamped homogeneous electric fields (CHEF). We used scanning densitometry to determine that the most likely number of chromosomes is eight. Molecular probes consisting of cloned genes were used to distinguish each of five bands visible on CHEF gels. We have also identified a low-molecular-size DNA molecule possibly related to the 35-kb circular DNAs found in other Apicomplexa.

Sequence of the parasitic protozoan, Cryptosporidium parvum, putative protein disulfide isomerase-encoding DNA.

A composite 1876-bp DNA encoding a putative protein disulfide isomerase (PDI) has been constructed from clones isolated from Cryptosporidium parvum (C. parvum) genomic and cDNA libraries and the nucleotide sequence determined. As predicted from the open reading frame (ORF), the protein product has a predicted molecular size of 54 kDa and a high degree of homology to PDIs from other species.

The putative acetyl-CoA synthetase gene of Cryptosporidium parvum and a new conserved protein motif in acetyl-CoA synthetases.

We determined the nucleotide (nt) sequence of the putative gene encoding acetyl-coenzyme A synthetase (ACS) from the parasitic protozoan Cryptosporidium parvum. The gene is single copy, located on a chromosome of approximately 1.08 mb, and has no introns. The gene is characterized by low codon usage bias and encodes a 694-amino acid (aa) protein with a predicted molecular size of 78 kDa, similar to other ACSs from different prokaryotic and eukaryotic species. Comparison of multiple protein alignments of ACSs revealed a new conserved sequence motif PKT(R/V/L)SGK(I/V/T)(T/M/V/K)R(R/N) near the C-terminus, which may be a signature for ACSs. This motif shares significant homology with sequences from other members of the AMP-binding family, has secondary structure similar to the purine-binding motif of ATP- and GTP-ases, and may play a role in the enzymatic activity of proteins from the AMP-binding family.

Cloning and analysis of a Cryptosporidium parvum gene encoding a protein with homology to cytoplasmic form Hsp70.

An intronless gene encoding a protein of 674 amino acid residues with a molecular mass of 73,403 Da showing homology to the cytoplasmic form of the 70 kDa heat shock proteins has been cloned and sequenced from the intestinal pathogen Cryptosporidium parvum. Monospecific polyclonal antibodies obtained to recombinant protein recognized a single band with an approximate molecular mass of 70 kDa on a Western blot of C. parvum proteins, as well as the 70 kDa heat shock protein from bovine brain. Southern blot analysis suggested the gene was single copy in the C. parvum genome. Eleven perfect repeats of the sequence GGMP were found in the predicted protein near the carboxyl terminus.