Specialized structures on the border between rhizocephalan parasites and their host's nervous system reveal potential sites for host-parasite interactions.

Rhizocephalan barnacles are a unique group of endoparasitic crustaceans. In their extreme adaptation to endoparasitism, rhizocephalan adults have lost almost all features of their free-living relatives but acquired an outstanding degree of control over the body of their hosts (mostly decapods). The subtle influence exercised by rhizocephalans on the physiology, morphology and behaviour of their hosts is a vivid example of the most intimate host-parasite interactions but their mechanisms are very poorly known. In this study we examined the morphology and the adaptive ultrastructure of the organs invading the nervous system of the host in two rhizocephalan species from the families Peltogastridae, (Peltogaster paguri) and Peltogasterellidae (Peltogasterella gracilis). We found two essentially different types of structures involved in interactions of these two rhizocephalans with the nervous system of their hosts: modified rhizocephalan rootlets lying inside the ganglia and the neural fibres of the host enlacing the trophic rootlets of the parasites. We suggest that both these structures may be highly specialized tools allowing the parasite to interact with the host on the humoral level via neuromediators, hormones, attractants and trophic factors.

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.

[Use of antihypoxant/antioxidant treatment of asthenic syndrome in elderly patients].

The article is devoted to the study of asthenic disorders in the elderly people and their correction which is the actual problem in modern medicine. It is shown that asthenia in the elderly people appears as a painful condition, accompanied by fatigue and exhaustion with the extreme instability of mood, the weakening of self-control, impatience, restlessness, sleep disturbances, lack of capacity for long-term mental and physical tension, intolerance to loud noises, bright lights, odors. The clinical effi cacy of the drug «Cytofl avin¼ therapy in asthenic syndrome in 43 elderly patients, aged 68 to 86 years with established diagnosis of asthenia was evaluated. According to the results of clinical and psychopathological analysis indicated a reliable contribution Cytofl avin therapy in combination with Fluvoxamine in the correction of asthenic symptoms in elderly patients.

Pharmacokinetics of disappearance of cocaine from hair after discontinuation of drug use.

UNLABELLED: Methods that employ detection of drugs of abuse in hair are important for monitoring compliance with drug abstinence. Understanding the mechanisms and timeline of drug disappearance from hair is critical for clinical and forensic application of hair testing. We aimed to evaluate the kinetics of disappearance of cocaine and its metabolite, benzoylecgonine (BE), from hair after discontinuation of drug use. METHODS: The Motherisk laboratory at the Hospital for Sick Children in Toronto routinely receives hair samples for toxicology analysis. Cocaine and BE hair results were obtained from the Motherisk Database for calculation of half-life of these compounds in hair. Subjects were included in the study if they had gradually decreasing concentrations of cocaine and/or BE in sequential hair samples, with higher levels in the 1-3 cm distal segments (i.e. earlier in time) and low or non-measurable levels in the segment closest to the scalp (i.e. closer to the date of sampling). Elimination half-life of cocaine and BE in hair was calculated using standard kinetics calculations. The study was anonymous, and received ethics approval by the Ethics Review Board of our institution. RESULTS: 137 subjects met the inclusion criteria for the study. The median half-life of cocaine in hair was 1.5 months (95% CI 1.2-1.8) in females and 1.5 months (95% CI 1.1-1.8) in males. The median half-life of BE was 1.5 months (95% CI 1.1-2) in females and 1.5 months (95% CI 0.8-1.8) in males. Half lives of cocaine or BE were not statistically different between males and females (Mann-Whitney U-test; P=0.93 for cocaine, P=0.99 for BE). Half lives of cocaine and BE were strongly correlated (Spearman rank rho=0.73; P<0.001). CONCLUSION: Cocaine and BE could be detected in hair of former drug users for several months after abstinence. The calculated half-life of over 1 month for cocaine implies that, assuming first order elimination, approximately 3-4 months have to pass for hair testing to become negative in the segment proximal to the scalp. This finding should be incorporated in interpreting compliance with abstinence of former drug users, and suggests that caution has to be exerted when evaluating potential breaches of abstinence.

Computing with almost periodic functions.

This paper develops a method for discrete computational Fourier analysis of functions defined on quasicrystals and other almost periodic sets. A key point is to build the analysis around the emerging theory of quasicrystals and diffraction in the setting on local hulls and dynamical systems. Numerically computed approximations arising in this way are built out of the Fourier module of the quasicrystal in question and approximate their target functions uniformly on the entire infinite space. The methods are entirely group theoretical, being based on finite groups and their duals, and they are practical and computable. Examples of functions based on the standard Fibonacci quasicrystal serve to illustrate the method (which is applicable to all quasicrystals modeled on the cut-and-project formalism).

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.