Spectrin-based skeleton as an actor in cell signaling.

This review focuses on the recent advances in functions of spectrins in non-erythroid cells. We discuss new data concerning the commonly known role of the spectrin-based skeleton in control of membrane organization, stability and shape, and tethering protein mosaics to the cellular motors and to all major filament systems. Particular effort has been undertaken to highlight recent advances linking spectrin to cell signaling phenomena and its participation in signal transduction pathways in many cell types.

Determination of the relative avidity of the specific IgG antibodies in human toxocariasis.

Human toxocariasis is a zoonosis caused by infection with larvae of the ascarid nematode Toxocara canis and, less frequently, T. cati. Our study developed a method for distinguishing distant from recent human toxocariasis by assessing the avidity of the IgG antibodies. The avidity of specific antibodies increases with time after antigen challenge and assessment of the degree of avidity can be used to discriminate between recent and distant infections. The relative avidity was measured in 150 sera from children with visceral toxocariasis and in 46 sera from children with ocular toxocariasis. The probable time of infection was estimated on the basis of the medical history and clinical syndrome. Our study showed that 94.2% of positive sera collected from patients reporting infection > 6 months ago had high IgG avidity values, confirming distant toxocariasis, whereas 25.9% of positive sera taken < 6 months after infection showed low indices of IgG avidity. Our results suggest that measurement of the specific IgG avidity may assist in discriminating between recent and distant toxocariasis. The method can be used effectively to rule out (because of high avidity) a recently acquired infection. Low avidity is less reliable in discriminating between recent and distant infections.

Acid excreting mutants of yeast Saccharomyces cerevisiae.

Saccharomyces cerevisiae mutants acidifying glucose medium containing bromocresol purple were shown to excrete protons when placed in unbuffered water in the absence of any external carbon source. The mutants belong to 16 different complementation groups. Most of them do not grow on glycerol and the excreted protons are associated to particular sets of organic anions such as citrate, aconitate, succinate, fumarate or malate. These novel types of respiratory mutations seem to be located in genes operating in the Krebs or glyoxylate cycle.

Detection of Echinococcus multilocularis antigens in faeces by ELISA.

Faecal samples deriving from 391 animals belonging to nine species (polecats, badgers, martens, weasels, rats, dogs, cats, red foxes, raccoon-dogs) were examined by capture ELISA for the presence of the Echinococcus multilocularis coproantigen. The main claim of our studies is the reliable detection of E. multilocularis coproantigens, mainly in the faeces of foxes, dogs and cats. For the first time in coproantigen detection we used a "double-sandwich" ELISA. The main advantage of this method is the higher specificity and better differentiation of positive and negative faecal samples, in comparison with sandwich ELISA. The overall specificity of double-sandwich ELISA was 95.1% with only 16 of 327 E. multilocularis-free animals giving false-positive results. The E. multilocularis coproantigen was detected by double-sandwich ELISA in 37.5% of examined red foxes and in 8.0% of examined raccoon-dogs, compared with a prevalence of just 29.8% in red foxes and 8.0% in raccoon-dogs, as determined by parasitological techniques.

Detection of Trichinella spiralis antigens in urine of men and animals.

The practical inability to diagnose Trichinella spiralis antibodies in man before day 20 post infection (dpi) has stimulated interest in the development of immunodiagnostic test to detect circulating antigens. Our previous experience showed that soon after infection immune complexes as well as uncomplexed parasite antigens in sera of infected rats could be detected. To diagnose the presence of antigen in urine, double sandwich-capture ELISA was applied using a peroxidase-conjugated rabbit immunoglobulin to T. spiralis larval antigens. The plates were coated with metabolic (AES) or somatic (AS) larval antigens. Mice were infected with 500 T. spiralis larvae. The urine samples from experimentally infected mice taken from 1 to 41 dpi. and the urine samples from patients of the Clinical Hospital in Bialystok taken from 3 to 120 dpi were examined. Before testing, the urine samples were heated for 6 min. at 100 degrees C and centrifuged for 6 min. at 5000 g, supernatants were used in ELISA. The presence of T. spiralis antigens in mice urine samples was detected between 6-26 days post infection (dpi) using double sandwich-capture ELISA. All samples taken later were negative as samples taken from uninfected mice. 3 from 9 human urine samples taken 3-10 dpi were positive, the remaining samples taken 3-10 and 10-30 dpi showed values near to "cut-off". In both mice and human urine samples the higher level of antigens was detected in ELISA when somatic larval antigen was used. The T. spiralis antigens were present in urine of infected men and mice in the first phase of infection.

Immunomodulative effect of muramyldipeptide in mice with larval toxocarosis.

The phagocytic ability of polymorphonuclear leukocytes, the metabolic activity of peritoneal macrophages, the proliferative response of T- and B-cells, and the production of specific anti-Toxocara antibodies were studied in paratenic hosts with experimental larval toxocarosis after treatment with the immunomodulator muramyldipeptide for 119 days. Peroral infection of mice with 2,500 Toxocara canis eggs partially reduced the numbers of cells and inhibited the activity of all cellular immune-response parameters studied. During most of the experiment the greatest suppression was recorded for the peritoneal-macrophage metabolic activity. Parenteral administration of muramyldipeptide to mice at two doses prior to and after infection restored and significantly stimulated the parasite-inhibited phagocytic ability of blood polymorphonuclear leukocytes, metabolic activity of macrophages, and T-lymphocyte proliferative response. It also elevated the production of specific circulating anti-Toxocara antibodies. The immunomodulator significantly reduced the count of migrating T. canis larvae in the host organism (30.6%) and decreased by half the percentage of larvae in the brain.

Inhibition of lymphoproliferative response and its restoration with a glucan immunomodulator in mice with experimental larval toxocarosis.

A high infective dose of Taxocara canis eggs (2,500 eggs per mouse) induced a partial immunosuppression in mice, manifested by inhibition of the proliferative response of splenic T and B cells to polyclonal activators. A glucan immunomodulator given to infected animals at the beginning of the experiment showed a marked stimulative and restorative effect on the parasite-suppressed lymphoproliferative response. The ability of T. canis to migrate in the host was reduced in glucan-treated animals by 27%.

Detection of Taenia saginata antigens in faeces by ELISA.

Stool samples were examined by capture ELISA for the presence of T. saginata antigens. The specimens were from 303 patients infected with T. saginata, 43 samples from individuals suffering from bacterial, fungal, protozoan and nematode infections and 36 ones deriving from uninfected persons. The samples were diluted 1:10 (w:v) in PBS + 0.35% Tween 20, centrifuged twice at 5000 g for 20 min at room temperature and resulting supernatant at 12,000 g for 30 min at 0 degree C. The capturing and detecting polyclonal antibodies were prepared in rabbits immunizing them with T. saginata surface antigens. The stool samples freshly obtained, stored at 4 degrees C not longer than 72 h, or kept at at -20 to -30 degrees C even for 24 months showed reliable results in ELISA. The samples kept for a long time at 4 degrees C, dried and covered with moult were negative in the test. All samples taken from patients infected with other than T. saginata parasites or uninfeced were negative.

Factors conditioning detection of Taenia saginata antigens in faeces.

The conditions which optimize detection of T. saginata antigens in faeces were subject of examination. The most appropriate appeared diluent of PBS + 0.3% Tween 20. The highest effectiveness was achieved using capturing and detecting antibodies to surface antigens in comparison with somatic and metabolic ones. Two step centrifugation and final dilution of samples 1:10 (w:v) were recommended. The stability of freezed faecal supernatants after the first centrifugation was limited to 2 months. The sensitivity of capture ELISA established in our examinations allow to signalize 1 ng/ml antigen in high absorbance values. The antigens recognized by the test had MW bigger than 100,000.

Thymidylate synthases from Hymenolepis diminuta and regenerating rat liver: purification, properties, and inhibition by substrate and cofactor analogues.

Comparative studies of thymidylate synthases, isolated from the tapeworm, Hymenolepis diminuta, and regenerating liver of its host, rat, aimed at a possibility of specific inhibition of the helminthic enzyme, are presented. While similar in structure (dimers with monomer molecular masses of 33.7 kDa and 34.9 kDa, respectively) and parameters describing interactions with substrates and products, the tapeworm and rat enzymes differed in the dependences of reaction velocity on temperature (Arrhenius plots biphasic and linear, respectively). The tapeworm, compared with the host, enzyme was less sensitive to the competitive slow-binding inhibition by 5-fluoro-dUMP and its 2-thio congener, but equally sensitive to inhibition by 4-thio-5-fluoro-dUMP, N4-hydroxy-dCMP and N4-hydroxy-5-fluoro-dCMP, the latter being more potent inhibitor of the parasite enzyme than 5-fluoro-dUMP. alpha-Anomer of 5-fluoro-dUMP behaved as a very weak competitive slow-binding inhibitor of both enzymes. Both enzymes differed markedly in sensitivity to inhibition by 10-propargyl-5,8-dideazafolate and its di- and triglutamates (pddPteGlu1-3), with pddPteGlu1 being stronger inhibitor of the mammalian enzyme, but pddPteGlu3 showing opposite specificity. Sulfonamidobenzoylglutamate analogue of pddPteGlu (pddPteSO2Glu) and 2-desamino-2-methyl derivative of this analogue (CH3pddPteSO2Glu) were weaker inhibitors of both enzymes than the parent compound. Substitution of the glutamyl residue in CH3pddPteSO2Glu with either norvaline or alanine increased inhibition potency, whereas similar substitutions with glycine, valine or phenylglycine were without a distinct effect with the host enzyme but weakened inhibition of the tapeworm enzyme.

[Comparison of thymidylate synthase properties isolated from the rat tapeworm, hymenolepis diminuta, with properties of enzyme isolated from the host in regenerating rat liver]. / Porównanie wlasciwosci syntazy tymidylanowej oczyszczonej z tasiemca szczurzego, Hymenolepis diminuta, z wlasciwosciami enzymu zywiciela wyizolowanego z regenerujacej watroby szczura.

Thymidylate synthases (TS) from the tapeworm, Hymenolepis diminuta, and regenerating rat liver have been purified by means of affinity chromatography on immobilized 10-formyl-5,8-dideazafolate and concentrated on immobilized p-aminophenyl-5-fluoro-2'-deoxyuridine monophosphate. Molecular weights of native TS from the tapeworm and regenerating rat liver were 62 kD and 81.5 kD, respectively, and molecular weights of the monomers were 34.4 kD and 34.9 kD, respectively, pointing to dimeric structures of both enzymes. The dependence of TS activity on temperature (Arrhenius plot) was biphasic for the parasite enzyme, with lower activation energy above 32 degrees C, and monophasic for the host enzyme. 2'-deoxyuridine-5'-monophosphate (dUMP) analogues, 5-fluoro-2'-deoxyuridine-5'-monophosphate (5-FdUMP), 2-tio-5-FdUMP,N4-hydroxy-2'-deoxycytidine-5'-monophosphate (N4-hydroxy-dCMP) and N4-hydroxy-5-FdCMP, were competitive with respect to dUMP, slow-binding inhibitors of TS from both sources, with K1 values in 10(-6)-10(-9) M range. 5-FdUMP was distinctly stronger inhibitor of the host than the tapeworm TS, whereas N4-hydroksy-5-FdCMP inhibited stronger the parasite enzyme. Interaction of 5,10-methylenetetrahydrofolate (CH2H4PteGlu) analogue, 10-propargyl-5,8-dideazafolate (pddPteGlu), and its di- and triglutamates with both enzymes were studied. Inhibition of the parasite and host enzymes by pddPteGlu was of mixed-type with respect to CH2H4PteGlu, with K1 values in 10(-8) M range. Introduction of additional glutamate residues changed inhibition type to noncompetitive with respect to Ch2H4PteGlu and lowered K1 values (pddPteGlu3 < pddPteGlu2 < pddPteGlu1).(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and immunologic reactivity of Hymenolepis diminuta surface antigens.

The outer part of Hymenolepis diminuta tegument was extracted with 3 M KCl. The antigen was adsorbed from the extract on an affinity column containing H. diminuta-infected rat immunoglobulin immobilized on Sepharose 4B and could be eluted with 2.5 M urea at pH 2.8. Analysis of polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of both the crude extract and material eluted from the column showed that the latter was markedly purified. Double diffusion and immunoelectrophoresis revealed 11 and 4 antigenic components in the crude extract and purified preparation, respectively.

Binding of dexamethasone and its effect on histamine release from rat mast cells.

Purified rat peritoneal mast cells were incubated for 20 h with or without dexamethasone (4 x 10(-6) M) and then passively sensitized with serum from Trichinella spiralis-infected rats. The release of histamine using various secretagogues (concanavalin A, crude antigen of T. spiralis and polymyxin B) was determined. Dexamethasone treatment markedly inhibited IgE-dependent release of histamine (from 33.9 +/- 5.0% to 12.4 +/- 5.1% and from 39.8 +/- 7.9% to 14.2 +/- 6.5% of total cellular histamine content, respectively) whereas histamine release stimulated by the nonimmunological stimulus, polymyxin B was unaffected by this steroid. This suggests that the effects of dexamethasone cannot be exclusively explained by inhibition of phospholipases. Specific binding of 3H-dexamethasone to purified mast cells displayed sigmoidal dependence on concentration which may be the result of either negative cooperativity or the presence of a different class of binding sites. Two saturation plateaux at 20-30 x 10(-9) M and 70-90 x 10(-9) M were observed. The equilibrium dissociation constant for the higher affinity binding sites was Kd1 = 1.9 x 10(-8) M and represented 25,290 sites/cell, whereas the apparent Kd2 for lower affinity sites amounted to 5.5 x 10(-8) M and represented about 120,000 sites/cell.

Thymidylate synthase activity in the development of Hymenolepis diminuta.

Extracts of the tapeworm, Hymenolepis diminuta, catalyse N5,10-methylene-tetrahydrofolate-dependent release of tritium from [5-3H]dUMP, indicating the presence of thymidylate synthase. The enzyme activity was found in immature, mature and gravid proglottids, as well as in immature and mature oncospheres. The reaction showed pH optimum at 7.5. Its Michaelis constants were approximately 2 and 15 microM for dUMP and (+/-), L-N5,10-methylenetetrahydrofolate, respectively. Incubation of the tapeworm extracts with 5-F-[3H]dUMP and N5,10-methylenetetrahydrofolate resulted in formation of a labelled complex, separable under conditions of SDS polyacrylamide electrophoresis (mol. wt. of approx. 34,000), corresponding to thymidylate synthase subunit. Results of gel filtration of the above complex, under nondenaturing conditions, pointed to a dimeric structure of the enzyme.

Influence of 0.02 M EDTA and 3 M KCl on surface of Hymenolepis diminuta and composition of isolated proteins.

The glycocalyx of Hymenolepis diminuta (Cestoda, Cyclophyllidea) was isolated using 0.02 M EDTA or 3 M KCl. It was shown in the electron micrographs that 0.02 M EDTA did not damage the tapeworm plasma membrane, eliminating glycocylax only, in contrast to 3 M KCl which disrupted tegument up to the basal membrane. The protein analysis of extracts and the supernatant of homogenate of the whole tapeworm strobila by polyacrylamide gel electrophoresis (PAGE) and dodecyl sulphate-polyacrylamide gel electrophoresis (SDS) electrophoresis revealed that the substance extracted with 3 M KCl was more abundant in protein fractions than the two remaining ones. The substance extracted with 0.02 EDTA, collecting the tapeworm glycocalyx possessed the smallest amount of protein fractions, however, some of them were more abundant.

Host serum proteins in Taenia saginata metacestode fluid.

Taenia saginata cyst fluid was examined for host proteins; IgG1 and IgG2 as well as haemolytic complement activity were detected. Polyacrylamide gel electrophoresis revealed differences in proteinograms among the samples taken from 1-, 4-, and 10-month old cysts. Fluid from older cysts had fewer protein components and showed a weaker antigenic reaction with sera of bovines infected with T. saginata than that of younger cysts. The roles of antibody and complement in initiating degeneration of the parasite are discussed.