Transport of Human Lactoferrin into Mouse Brain: Administration Routes and Distribution.

We studied different ways of transport of human lactoferrin to the brain of C57Bl/6 mice after its administration via different routes, analyzed its distribution in the brain, and determined the phenotype of lactoferrin-containing cells. Colocalization of lactoferrin and markers of various cell types was estimated by fluorescent immunohistochemical analysis. Lactoferrin was detected in mouse brain sections after its intranasal, sublingual, and intraperitoneal administration, but not after conjunctival administration. After intranasal administration, lactoferrin rapidly penetrated into the brain and accumulated in the cytoplasm of vascular endothelial cells in the neocortex, striatum, hippocampus, and thalamus. After application of protein solution onto fixed floating sections, highly specific binding of lactoferrin was found in the nuclei of neurons, astrocytes, and microglia cells, but not in the nuclei of endothelial cells of mouse brain.

Receptor/ligand interactions between Cryptosporidium parvum and the surface of the host cell.

The ability of membrane antigens on sporozoites of the intestinal pathogen, Cryptosporidium parvum, to bind host cell surface antigens was investigated. A novel membrane-associated protein of approximately 47 kDa, designated CP47, was found to possess significant binding affinity for the surface of both human and animal ileal cells. This protein was purified by a combination of anion-exchange chromatography on FPLC and immunoaffinity chromatography. Purified CP47 demonstrated competitive binding with parasite-associated membrane antigens to membranes of HCT-8 and ileal cells in a dose-dependent manner. Furthermore, the binding activity of CP47 was found to be Mn2+-sensitive, and was completely inhibited in the presence of 10 mM MnCl2. These results were consistent with earlier findings demonstrating the inhibitory effect of Mn2+ ions on Cryptosporidium infection both in vitro and in vivo (Nesterenko et al., Biol. Trace Elem. Res. 56 (1997) 243-253). Immunoelectron microscopy using gold-conjugated antibodies revealed CP47 to be localized at the apical end of the sporozoites. A single protein with an electrophoretic mobility of 57 kDa was purified from host cell membranes using CP47-Affigel. Similarly, affinity purification of this protein was abrogated in the presence of Mn2+. These data suggest that a novel parasite protein, CP47, may play an important role in sporozoite/host cell attachment.

Effects of manganese salts on the AIDS-related pathogen, Cryptosporidium parvum in vitro and in vivo.

The authors examined the effects of manganese salts on the interaction of the AIDS-related pathogen, Cryptosporidium parvum, with human ileoadenocarcinoma (HCT-8) cells in vitro. Manganese (Mn) inhibited binding of C. parvum sporozoite membrane antigens to intact, fixed HCT-8 cells in a dose-dependent fashion, whereas Ca++, Mg++, and Zn++ salts had no effect. Manganese was also found to affect sporozoite penetration of live HCT-8 cells, which resulted in a dose-dependent inhibition of parasite development. However, the levels of Mn++ needed in the live cell assays was approx 10-fold greater than in the fixed-cell assays. This inhibition of parasite development was not reversible when Ca++ or Mg++ were used as competitors. Oral supplementation of suckling mice infected with C. parvum with MnSO4 resulted in significant reductions and, in some cases, elimination of intestinally derived oocysts.

Virus-like, double-stranded RNAs in the parasitic protozoan Cryptosporidium parvum.

We have discovered and analysed two novel, linear extrachromosomal double-stranded RNAs (dsRNAs) within oocysts of major north Amercian isolates of Cryptosporidium parvum, a parasitic protozoan that infects the gastrointestinal tract of a variety of mammals, including humans. These dsRNAs were found to reside within the cytoplasm of sporozoites, and were not detected in other species of the genus. cDNAs representing both dsRNA genomes were cloned and sequenced, 1786 and 1374 nt, and each encoded one large open reading frame (ORF). The deduced protein sequence of the larger dsRNA (L-dsRNA) had homology with viral RNA-dependent RNA polymerases (RDRP), with more similarity to polymerases from fungi than those from other protozoa. The deduced protein sequence from the smaller dsRNA (S-dsRNA) had limited similarity with mitogen-activated c-June NH2 terminal protein kinases (JNK) from mammalian cells. Attempts to visually identify or purify virus-like particles associated with the dsRNAs were unsuccessful. Sensitivity of the dsRNAs to RNase A also suggests that the dsRNAs may be unencapsidated. A RDRP activity was identified in crude extracts from C. parvum sporozoites and products of RNA polymerase activity derived in vitro were similar to the dsRNAs purified directly from the parasites.

Efficacy of 101 antimicrobials and other agents on the development of Cryptosporidium parvum in vitro.

An in-situ ELISA was used as a primary screen to test the effects of 101 antimicrobials and other agents on the development of Cryptosporidium parvum in vitro. Over 40 of the compounds displayed some form of anticryptosporidial activity, and dose-response curves were generated for 40 of these. The in-situ ELISA makes a highly effective primary, pharmaceutical screen for C parvum, to be used prior to more detailed microscopical, toxicological or in-vivo assays.

Development of a microtitre ELISA to quantify development of Cryptosporidium parvum in vitro.

An in situ enzyme-linked immunosorbent assay (ELISA) was developed to evaluate growth of Cryptosporidium parvum in vitro. Ninety-six-well tissue culture microtitre plates were each seeded with 4.0 x 10(4) human ileocecal adenocarcinoma (HCT-8) cells, then infected with CsCl-purified oocysts 24 h later. The growth medium consisted of RPMI 1640 supplemented with 10% fetal bovine serum, 15 mM HEPES (N-2-hydroxyethylpiperazine N'-2-ethanesulfonic acid), 50 mM glucose, 1 microgram ml-1 folic acid, 4 micrograms ml-1 4-aminobenzoic acid, 2 micrograms ml-1 pantothenic acid and 35 micrograms ml-1 ascorbic acid. Incubation conditions were at 37 degrees C in a 5% CO2/95% humidified air incubator. Oocysts were allowed to excyst in situ so that sporozoites could infect cells directly. Monolayers were then washed, new medium added, and infected cells re-incubated. Levels of infection were assessed 48 h later using a rat anti-C. parvum polyvalent antiserum directed against purified parasite membranes, followed by a goat anti-rat IgG conjugated to horseradish peroxidase and 3,3',5,5'-tetramethyl-benzidine as substrate. Using various parasite inoculating doses and incubation times, optimal results were obtained using a 90-min exposure of host cells to 2.5-3.0 x 10(4) oocysts/well. Evaluation of various concentrations of four anti-microbials (monensin, lasalocid, paromomycin and sulfadimethoxine) in the system resulted in the acquisition of precise dose-response curves for each compound.

A metallo-dependent cysteine proteinase of Cryptosporidium parvum associated with the surface of sporozoites.

A proteinase of 24 kD was found associated with sporozoites of Cryptosporidium parvum. Optimal hydrolysis of azocasein, casein, bovine serum albumin, and gelatin occurred at a pH of 6.5-7.0. Activity against azocasein was inhibited by ethylenediaminotetraacetic acid (EDTA), iodoacetic acid (IAA), trans-epoxysuccinyl-L-leucylamido(4-guanido) butane (E-64), and phosphoramidon, suggesting that the enzyme was a metallo-dependent cysteine proteinase. Both serine and aspartate protease inhibitors failed to inhibit enzyme activity. The enzyme was partially purified by preparative isoelectric focusing of parasite membrane proteins. Polyclonal antiserum to parasite membrane proteins was generated in rats. The enzyme-containing fraction was subjected to SDS-PAGE and probed with antiserum, and the antibodies against the protease were eluted directly from nitrocellulose blots. An indirect immunofluorescence assay using these monospecific antibodies revealed that the protease occurred on the surface of sporozoites, but was not associated with oocyst walls, rhoptries, or micronemes.

A simple and reliable method of producing in vitro infections of Cryptosporidium parvum (Apicomplexa).

A variety of techniques have been used to infect cell monolayers in culture with the protozoan, Cryptosporidium parvum. However, most of these methods rely on the use of trypsin and/or bile salts to excyst sporozoites in vitro, followed by washing sporozoites free of excystation solution prior to their addition to subconfluent monolayers. This method not only increases the amount of time required to establish infections in vitro, but also results in prolonged exposure of free sporozoites to environmental conditions. Here we report a simple, fast, and efficient method of obtaining consistent infections of C. parvum in cell monolayers. This technique relies on the ability of the parasite to excyst at 37 degrees C but not at room temperature following pretreatment with sodium hypochlorite. By adding surface-sterilized oocysts directly to monolayers, sporozoites have access to host cells immediately upon excystation.

A simple modification of Blum's silver stain method allows for 30 minute detection of proteins in polyacrylamide gels.

A simple and rapid protocol for silver staining of proteins following electrophoresis in polyacrylamide gels (PAGE) is described. We have reduced the number of steps in the procedure of Blum et al. (Electrophoresis (1987) 8, 93-99), and shortened fixation and washing times so that efficient detection of proteins can be achieved within 30 min. In common with more time-consuming silver-staining methods, the present protocol is capable of detecting nanogram quantities of proteins on a colorless background and is suitable for rapid screening of large numbers of samples.

Factors affecting the binding between fusicoccin and plasma membranes from maize roots.

Plasma membranes have been purified from roots of maize (Zea mays L.) using a two-phase aqueous polymer system, dextran-polyethylene glycol. The plant material was homogenized in the presence of a mixture of natural protease inhibitors from potato (Solanum tuberosum L.); these inhibitors have been shown to be more effective than phenylmethylsulfonyl fluoride in suppressing the endogenous proteases in maize roots. Inhibition of proteolysis in the homogenization medium markedly increased (about tenfold) the number of lowaffinity binding sites for fusicoccin (FC). In addition, storage of plasma membranes at -20° C decreased both the number of the low-affinity sites and their dissociation constant (KD); this effect was in all probability caused by lipid peroxidation. The presence of EDTA throughout isolation and storage of the plasma membranes stabilized the parameters of FC binding to the membranes. The kinetics of binding of [(3)H]dihydroFC and the competition between [(3)H]dihydroFC and FCs A, C, J, and H were determined for the low-affinity sites. It was found that (i) the rate constant of association between FC and the low-affinity binding sites is about two orders of magnitude lower than that for the high-affinity sites; (ii) different FCs can be arranged in the order of decreasing avidity for the low-affinity FCbinding site: FC A>FC C>FC J>FC H.