Immunosuppression and ablastin

Ablastin formed by animals in response to infections by rodent trypanosomes possesses the characteristics of an antibody. Partial resistance to Trypanosoma lewisi is demonstrable in animals previously injected with live Trypanosoma musculi. Antisera from T. musculi infected mice do not inhibit reproduction by T. lewisi bloodstream forms in vitro as efficiently as homologous antisera collected at similar times during infections, indicating a degree of specificity. Ablastin activity in antisera is not altered by treatment with 2-mercaptoethanol or by heatng at 60 °C for 3 hr. Sephadex G-200 gel filtration of early and late antisera from T. lewisi infected rats and assays with bloodstream forms cultured at 37 °C detect ablastin activity in the second major fraction eluted from the columns. Ablastin appears to be an antibody of the immunoglobulin G species.Immunosuppressant procedures utilized in studies of the host responses to rodent trypanosomes are reviewed and include: chemical agents, irradiation, splenectomy, reticuloendothelia blockade and thymectomy, and treatment with antilymphocyte and antithymocyte sera. Evaluation of the results of the application of these procedures to rodent parasite systems indicates ablastin is an antibody and supports the concept that the inhibition of trypanosome reproduction is separate and distinct from the first trypanocidal event responsible for the decreasing parasitemias observed during the infections. Recent studies concur and suggest that the first crisis in the infections is mediated by the combined actions of a thymus-dependent ablastin and a thymus-independent trypanocidal antibody.     

Trypanosoma lewisi: Avidity and adsorbability of ablastin,the rat antibody inhibiting parasite reproduction

The relation of naturally acquired host IgG in the surface coat of bloodstream forms of Trypanosoma lewisi to ablastin was studied to determine whether, contrary to a long-held conclusion, the antibody is avid and adsorbable. It was found by immunofluorescence and agglutination tests with monospecific antisera to rat IgG that bloodstream forms collected from immunosuppressed hosts, in contrast to those from immunocompetent hosts, have little or no detectable surface IgO. Specificity of adsorption was also demonstrated in other immunofluorescence experiments in which bloodstream forms from immunosuppressed hosts adsorbed IgG from immune serum with ablastic activity only (previously adsorbed with trypanosomes from immunocompetent hosts to remove the trypanocidal antibodies), but did not adsorb IgG from normal rat serum. To determine whether this specific adsorption of IgG by the parasite could be correlated with a reduction in ablastic activity, immune sera were adsorbed with bloodstream forms from immunosuppressed hosts at packed cell/serum ratios of either 1.2 or 2.0, and the adsorbed sera were then tested for ablastic activity in vitro. With both cell/serum ratios, ablastic activity was reduced by 50%. In comparison, similar adsorptions of immune sera with trypanosomes from immunocompetent hosts resulted in reductions of ablastic activity of only about 9 and 27% with the low and high cell/serum ratios, respectively. It is concluded that the failure to effect significant adsorption of ablastin in earlier studies resulted from the use of ablastinsensitized trypanosomes from immunocompetent hosts.     

Serological and vaccination studies with bloodstream and culture forms of Trypanosoma musculi

Khazindar S. H. and Dusanic D. G. 1982. Serological and vaccination studies with blood-stream and culture forms for Trypanosoma musculi. International Journal for Parasitology12: 257–264. Trypanosoma musculi bloodstream forms (BSF) were collected from immunosuppressed infected mice and extracted with phosphate buffered saline (PBS). ML-15, O'Daly's and LMC media, each containing 5% fetal calf serum (FCS) and a dialysate medium were investigated to identify the medium providing the optimal growth of T. musculi culture forms (CF). Because of the ease of preparation, ML-15 containing 5% FCS was selected and the culture forms were harvested when the parasites attained concentrations of at least 1 × 10⁷ trypanosomes/ml. Cellular antigens present in PBS extracts of the BSF and CF parasites were analyzed with rabbit antisera by crossed immunoelectrophoresis and tandem crossed immuno-electrophoresis. Absorptions of rabbit antisera with CF, BSF, media and normal mouse blood extracts were performed on immunoabsorbent affinity columns prior to crossed immunoelectrophoresis to further study the unique and shared antigens of the parasites. A minimum of 13 antigens were shared by these trypanosomes. Four antigens appeared to be unique to BSF and a single antigen to CF. In immunization studies, two groups of C3H/Anf mice were immunized with the equivalent of 1 × 10⁸ frozen-thawed BSF or CF/injection. Two groups of five animals injected with PBS or uninoculated medium and one untreated group served as controls. Animals in each group received 6 injections administered at 3-day intervals. Three days following the last injection, all animals were challenged with 1 × 10⁴ BSF. Hemacytometer counts were performed every 4 days until no parasites were seen in wet blood preparations of the untreated group. None of the animals inoculated with BSF homogenate displayed parasitemias, while animals inoculated with CF homogenate were found to be infected. Parasitemias in mice immunized with CF were lower than those of the control mice.     

A culture and biochemical assay system for Trypanosoma lewisi

Trypanosoma lewisi was cultivated as forms which appeared to be physiologically similar to those found in vivo. The medium consisted of 1.0 g peptone, 1.0 g glucose, 10 ml rat serum, 10,000 units penicillin G, 10,000 μg streptomycin and 90 ml Hank's Balanced Salt Solution. It was supplemented with 8.0 × 10⁸ rat erythrocytes per milliliter. In the complete medium trypanosomes multiplied for 48–72 hr. Cultured forms were lethal to newborn rats and infective to adults.Adsorbed early immune serum inhibited the growth of the trypanosomes in vitro and the percentage of reproductives declined from 66 to 45%. The cultured trypanosomes were also susceptible to both trypanocidal antibodies.     

Theileria parva: cell culture analysis of clones of macroschizont-infected bovine lymphoblastoid cells

Three clones (H7, D7, and C5) were established from single cells of a bovine lymphoblastoid cell line (IR.TPM.1) infected with macroschizonts of the protozoan parasite Theileria parva. The cloning efficiency using feeder layers was 0.3–0.4. The mean parasite size (the number of parasite nuclei per cell) was different in each clone and was correlated to the growth rate. The fast growing clone, C5 (population doubling time 24 hr), contained smaller (mean parasite nuclear number, 12) parasites than a slow growing clone, D7 (population doubling time, 73 hr; mean number of parasite nuclei per cell, 35.3). The third clone, H7, had an intermediate growth rate (population doubling time, 49 hr) and parasite size (mean nuclei number, 18.1). There was variation in the incidence of microschizonts among the clones but microschizont-free clones were not isolated. When the clones were subjected to 4.3 × 10^?7M aminopterin, 20–25% of the cell population of clones H7 and C5 and the uncloned parent line lost their parasites in 4 days, while it took 7 days to reach a similar result (31% parasite-free cells) in clone D7. We were unable to isolate parasite-free clones from cells treated with aminopterin. Hydroxyurea (4 × 10^?4M) inhibited the growth of clone C5, but the macroschizonts continued to proliferate, and the incidence of cells with microschizonts increased. The size profile analysis showed that most of the aminopterin-treated cells were 9.0 μm, the hydroxyurea-treated cells 14.7 μm, and the untreated cells 10.8 μm in diameter.     

Trypanosoma congolense: isolation and purification.

Yields of Trypanosoma congolense grown in rats may be increased by placing the rats in a 37 °C environment for 1 hr prior to sacrifice. A further increase in the number of parasites recovered per rat may be achieved by replacement of blood removed by a lactated Ringer's solution with 5% glucose as the rat is being bled from the abdominal aorta. The Ringer's solution serves to maintain intravascular volume during the bleeding procedure and thereby prevents premature cardiac arrest. Erythrocytes in infected blood may be then lysed by raising and rapidly lowering the osmolarity of the blood. This permits separation of the trypanosomes from 95% of the erythrocytes by differential centrifugation. The remaining blood cell contamination may then be removed on a small DEAE-cellulose column. The purified trypanosomes are motile, infective, and intact as judged by electron microscopy. More than 10¹⁰ purified T. congolense can be obtained from three adult rats by these methods.     

Trypanosoma acomys (Wenyon, 1909): Continuous Culturing with a Mouse Fibroblast Cell-Line (A9)

The continuous culturing of Trypanosoma acomys in the presence of a murine areolar-adipose cell line (A9) was possible for the 1st time. The trypanosomes were cultured at 37° C with A9 in DMEM supplemented with 20% heat inactivated fetal bovine serum, using an initial inoculum from primary cultures of lung or blood clots from infected spiny mice. The cultures were maintained for 115 days and underwent 15 passages before termination and cryopreservation. Using this culture system T. acomys subcultures were initiated from 3 different initial inocula (3 × 10⁴, 1.5 × 10⁵ and 7.4 × 10⁵ parasites/ml) and growth curves revealed that the lowest inoculum gave the best growth pattern. This inoculum yielded a population doubling time of less than 12 h for 4 days, a high peak density of 7 × 10⁶ parasites/ml and the most gradual decline compared to the other 2 inocula. Rosetting epimastigotes and nests of amastigotes were observed in close association with the feeder layer cells. Epimastigotes were the most predominant form in culture supernatants but other morphological forms observed included trypomastigotes and sphaeromastigotes.     

Flow Cytometric Analysis of Immunoglobulin and Complement Component C3 on the Surface of Trypanosoma lewisi1

ABSTRACT. We have reinvestigated whether surface immunoglobulin (sIg) on Trypanosoma lewisi is antibody directed toward parasite antigen by using flow cytometry to analyze parasites stained with fluoresceinated F(ab′)₂ fragments of antibodies to rat IgG and IgM. We have confirmed that IgG antibody to the parasites is present both in the serum of rats and on the surface of parasites between the fourth and twentieth days of infection, that the amount of sIg per cell increases as the infection progresses, and that considerably more IgG is present on parasites harvested from intact rats than on those from rats that had been immunosuppressed by whole body γ-irradiation. In addition sIgM was detected on trypanosomes from intact, but not on parasites from irradiated rats. We have also made two observations suggesting that not all sIg is specific antibody made in response to T. lewisi. First, a low but significant amount of sIgG was detected on parasites throughout infection in irradiated rats; no sIgM was detected on these parasites. Second, when parasites harvested from immunosuppressed rats were incubated in normal rat serum, the amount of both sIgG and sIgM detected by flow immunofluorescence increased. Parasites harvested from intact animals bound IgM but not IgG from normal rat serum. These results suggest either that natural antibody to the trypanosomes is present in the serum of uninfected rats or that some rat immunoglobulins bind to structures on the trypanosome surface in ways that do not depend on usual antigen-antibody interactions. Finally, flow immunofluorescence was also used to detect complement component C3 on the surface of both intact and trypsinized bloodstream forms harvested from intact or immunosuppressed rats. The amount of sC3 per cell did not increase until late in the infection and consequently did not correlate with the increase of sIgG. Therefore, T. lewisi avoids destruction by the immune system although immune effector molecules, IgG, IgM, and C3, are on its surface.     

Trypanosoma lewisi: alterations in membrane function in the rat.

DEAE-cellulose-purified Trypanosoma lewisi from 4-day (dividing trypanosomes) and 7-day (non-dividing trypanosomes) infections in rats were compared for initial uptake of glucose, leucine, and potassium. Glucose entered the parasitic cells by mediated (saturable) processes, whereas leucine and K⁺ entered by mediated processes and diffusion. Glucose entry was significantly elevated in 4-day cells (V_max 4.00 ± 1.02 nmoles/ 1 × 10⁸ cells/min) with respect to 7-day cells (V_max 1.83 ± 0.62 nmoles 1 × 10⁸ cells/min). Likewise, the affinity of the glucose carrier was significantly greater in 4-day cells (K_m = 0.30 ± 0.02 mM) than in 7-day cells (K_m = 0.59 ± 0.11 mM). When leucine and K⁺ transport were compared in 4- and 7-day populations, significant elevations in the rate of entry (V_max) of both substrates were observed for 4-day cells; K_m values for leucine and K⁺ were not altered by the stage of infection. For leucine, the V_max and K_m for 4-day cells were 2.40 ± 0.50 nmoles/1 × 10⁸ cells/30 sec and 78 ± 7 μM, respectively; corresponding values in 7-day cells were 1.06 ± 0.02 nmoles/1 × 10⁸ cells/30 sec and 66 ± 11 μM. For K⁺, the V_max and K_m for 4-day cells were 15.97 ± 0.38 nmoles/1 × 10⁸ cells/min and 1.2 mM, respectively; corresponding values in 7-day cells were 4.76 ± 1.82 nmoles/1 × 10⁸ cells/min and 1.05 mM. The observed increase in the rate of K⁺ entry into 4-day cells was attributable to enhanced influx; no significant difference in the rate of K⁺ efflux was noted when 4- and 7-day cells were compared ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of K⁺ leak for 4- and 7-day cells were 68.1 ± 9.3 and 67.9 ± 15.2 min, respectively). Potassium influx was ouabain insensitive. Membrane function in 7-day cells was not uniformly inhibited. No significant difference in the activity of the membrane-bound enzyme, 5′-nucleotidase, was observed when 4- and 7-day cells were compared.     

Schistosoma mansoni: Decarboxylation of 5-hydroxytryptophan, l-dopa,and l-histidine in adult and larval schistosomes

Homogenates of adult and larval Schistosoma mansoni, decarboxylate 5-hydroxytryptophan (5-HTP) and l-dopa in vitro, but not l-histidine. The enzyme responsible, similar to mammalian aromatic-l-amino acid carboxy-lyase (EC 4.1.1.28), has a K_m of 5.1 × 10^?5M, a V_max of 1.1 nmole/30 min/mg protein, and high activity at pH 7.9. No evidence for a specific l-histidine decarboxylase (EC 4.1.1.22) was found in either stage of the schistosome. In vivo, schistosomules form serotonin from 5-HTP, detectable by thin-layer chromatography, within 3–4 hr after addition of ¹⁴C-labeled 5-HTP to the medium. Addition of ¹⁴C-labeled tryptophan under similar conditions results in no detectable formation of 5-HTP.     

Size and specific activity of the UTP pool and overall rates of RNA synthesis in early mouse embryos

Mouse embryos from the one-cell to the blastocyst stage were cultured for 2 hr in the presence of 5 μM [³H]uridine or 10 μM [³H]adenosine, and the size and specific activity of the UTP and ATP pools were determined by an Escherichia coli RNA polymerase assay using synthetic poly(dA-dT) as template. The total UTP pool increased in size and specific activity with development from 0.05 pmole (0.06% labeled) in the one-cell stage to 0.54 pmole (27% labeled) in the blastocyst stage. The total ATP pool remained relatively constant in size at about 1 pmole/embryo, but increased in specific activity from 2.6 to 52% from one-cell to blastocyst. The turnover of the [³H]UTP pool was also examined under pulse-chase conditions in eight-cell and morula-stage embryos. The UTP pool decayed with approximately first-order kinetics up to 20 hr of chase, but the rate of decay was slower in eight-cell embryos (t_0.5 = 5.5 hr) than in morulae (t_0.5 = 2.8 hr). The observed specific activities of the UTP pools were used to calculate the overall rates of uridine incorporation into acid-precipitable material during early development. The rate of uridine incorporation per embryo increased from 3.6 × 10^?3 pmole/2 hr in the two-cell embryo to 1.8 × 10^?1 pmole/2 hr in the blastocyst. The rate of RNA synthesis per cell over a 2-hr period was estimated at 2.5 pg in the two- to four-cell embryo, 5 pg in the eight-cell, and 10 pg in the morula-early blastocyst.     

Phosphatidohydrolase and base-exchange activity of commercial phospholipase D

Base-exchange activity was contrasted to the usual phosphatidohydrolase activity of commercial phospholipase D preparation from cabbage. The former activity was assayed by measuring the incorporation of labeled ethanolamine and choline into phospholipids. The latter activity was assayed by measuring the formation of phosphatidic acid with radioactive phosphatidylcholine microdispersion as substrate. The pH optimum for the base-exchange activity was about 9.0, whereas the phosphatidohydrolase activity had a pH optimum around 5.6. The incorporation of ethanolamine and choline into phospholipid was dependent upon the amount of acceptor asolectin microdispersion present. The optimum concentration of Ca²⁺ in the base-exchange reaction was about 4 mm, whereas the optimum concentration for the phosphatidohydrolase activity was greater than 28 mm. The incorporation of ethanolamine into phospholipid was decreased 50% by heating the enzyme preparation at 50°C for about 10 min, whereas the choline incorporation decreased approximately 20% and the phosphatidohydrolase activity decreased by about 10% under these conditions.Hemicholinium-3 was found to be a noncompetitive inhibitor for the incorporation of both ethanolamine and choline into phospholipid with respective K_i, values of 1.25 × 10^?3 and 2.50 × 10^?3m. The K_m values for ethanolamine and choline in the base-exchange reaction were 1.25 × 10^?3 and 2.50 × 10^?3m, respectively. The apparent K_m for phosphatidylcholine for the phosphatidohydrolase activity was about 1.5 × 10^?3m, and there was no inhibition by hemicholinium-3.     

Transmembrane potential and amino acid transport in rat hepatocytes in primary monolaver culture

Transmembrane potential (E_m) and alpha-aminoisobutyric acid (AIB) transport were measured in primary monolayer cultures of rat hepatocytes obtained from unoperated control rats and from rats 12 hr following partial hepatectomy. Measurements were performed 20–24 hr after plating the cells. The capacity of both kinds of cells to concentrate AIB depended upon extracellular sodium; however, the steady-state accumulation in regenerating cells was twice that of control cells. Transmembrane potentials, recorded with glass microelectrodes, were –13 ± 0.6 mV and –27 ± 1.6 mV in control and regenerating cells, respectively. Ouabain (1 mM) depolarlized regenerating cells to –18 ± 1.0 mV, but it had no effect on control cells. The initial rates of 1 mM AIB transport into control and regenerating cells were 1.2 ± 0.1 and 3.1 ± 0.1 nanomoles/mg protein × 4 min, respectively. Ouabain (1 mM) reduced the initial rate of AIB transport into regenerating cells to 2.7 ± 0.1 nanomoles/mg protein × 4 min, but it had no effect on AIB transport into control cells. Glucagon (10^?7 M) added to control cells 12 hr before measurements hyperpolarized E_m to –31 ± 1.3 mV and increased AIB transport rate to 3.1 nanomoles/mg protein × 4 min. The results suggest a relationship between increases in E_m and increases in AIB transport in rat hepatocytes. An electrogenic Na-K pump may be involved in both of these events.     

Chlorpromazine increases sodium permeability across the isolated toad skin

The effects of Chlorpromazine (Cpz) on the potential difference (PD), short-circuit current (SCC), and total conductance (G_T) on Pleurodema thaul skin and on the skin response to dopamine were analysed. Cpz applied to the serosal surface in concentrations ranging from 1.25 × 10⁻⁵ to 1.25 × 10⁻⁴ M significantly increased the PD, the SCC and the GT. The effect of Cpz was abolished by BaCl₂ but not by alpha or beta adrenergic receptor antagonists. Cpz decreased the skin response to noradrenaline and to angiotensin 11. Dopamine (5 × 10⁻⁷ M to 5 × 10⁻⁶M) also induced a significant increase in the PD, SCC and G_T. This response was antagonized by propranolol but not by dibenamine. Additive effects of dopamine and Cpz were also found. The amiloride test showed that Cpz decreased E_Na., the driving force of sodium and increased g_na, which represents active sodium conductance. These results are consistent with the hypothesis that Cpz increases transport across the isolated toad skin by increasing mucosal and serosal permeability. The results also suggest that Cpz decreases membrane cabnodulin availability.     

Immunization of mice with irradiated Trypanosoma cruzi grown in cell culture: Relation of numbers of parasites,immunizing injections,and route of immunization to resistance

Mice were given 5 or 8 weekly injectins of either 2·0 × 10⁶ or 20·0 × 10⁶ irradiated T. cruzi from cell culture (ratio of trypomastigotes to amastigotes, 1 : 1) via the intraperitoneal route or via the subcutaneous route and challenged via the subcutaneous route one week after the last injection with 5·0 × 10⁴T. cruzi in mouse blood. The irradiated parasites used were not capable of producing infections in either Vero cell cultures or C₃H mice. Mice receiving irradiated parasites were significantly protected against the challenge infection as evidenced by significantly lower mean parasitemia, lessened signs of acute disease, and reduced mortality than that observed in untreated controls. Mice receiving 5 weekly immunizing injections of irradiated parasites were more resistant to challenge than those receiving 3 in previous work. Mice receiving 8 weekly immunizing injections were not significantly more protected against challenge than those receiving 5. Mice given 5 weekly injections of 20·0 × 10⁶ irradiated parasites were significantly more resistant to challenge than those receiving 2·0 × 10⁶ irradiated parasites on the same schedule. Mice given 5 weekly intraperitoneal injections of 20·0 × 10⁶ irradiated parasites were significantly more resistant to challenge than those receiving an equivalent number of immunizing injections via the subcutaneous route.     

Heterotrophic microorganisms and viruses in the water of the Gorky reservoir during a period of anomalously high water temperature

During the anomalously hot summer of 2010, the water temperature in the Gorky reservoir reached 27–33°C. Pronounced cyanobacterial blooms occurred in the limnetic part of the reservoir. The average values for bacterioplankton abundance (11.58 ± 1.25 × 10⁶ cell/mL), biomass (886 ± 96 mg/m³), and production [169 ± 32 mg C/(m³ day)] were twice as high as in the year with temperatures comparable to long-term average values. These parameters were higher in the limnetic part than in the river one. The abundance (4.86 ± 0.75 × 10³ cell/mL) and biomass (138 ± 9 mg/m³) of heterotrophic nanoflagellates were 2.3 and 1.7 times higher, respectively, than in years with regular temperature regimes. The average number of plank-tonic viral particles (N _v) in 2010 was 48.89 ± 9.54 × 10⁶ particles/mL, while virus-induced bacterial mortality (VMB) accounted for 26.9 ± 4.6% of the bacterial production. The N _v and VMB values in the limnetic part of the reservoir were, respectively, 1.5 and 1.8 times higher than in the river one.     

Plasmodium lophurae: Quantitative in vitro incorporation of 14C-1-acetate into lipids

Blood from ducks parasitized with Plasmodium lophurae and normal duck blood were incubated with sodium ¹⁴C-1-acetate. After release of the parasites from infected red blood cells (RBC) and concurrent treatment of normal blood, lipids were extracted from cellular material and plasma and lipid classes separated by thin-layer chromatography. Specific activity (dpm/mg lipid) of lipid classes was measured quantitatively by liquid scintillation radioassay and gravimetric analysis. The data indicated that the parasite within the RBC incorporated ¹⁴C-labeled lipid precursors.Experiments employing sodium ¹⁴C-1-acetate in two concentrations, 50 μCi ¹⁴C in 0.91 μmole sodium acetate/50 ml blood and 500 μCi ¹⁴C in 9.1 μmole sodium acetate/50 ml blood (1.82 × 10^?5M and 1.82 × 10^?4M), showed higher ¹⁴C incorporation into parasitized blood than normal blood preparations at the higher substrate concentration at 5 hr of incubation. At $\text{1.82 × 10}{}^{\mathrm{?5}}\text{M}{}^{14}$C-1-acetate, the highest specific activity in P. lophurae was associated with lipid alcohols. Monoglycerides and diglycerides were significantly labeled. At the higher acetate concentration (1.82 × 10^?4M), monoglyceride and diglyceride lipid classes had the highest specific activity in preparations of partially purified P. lophurae.Lipids of plasma from parasitized blood incubated for 5 hr with both concentrations of labeled acetate exhibited the highest specific activity in the free fatty acid class and sterols.At 24 hr of incubation, the lipids of partially purified P. lophurae had increased specific activity in free fatty acids, diglycerides, monoglycerides, phospholipids, and triglycerides.In plasma from parasitized blood incubated 24 hr with ¹⁴C-1-acetate, the highest specific activity and greatest percent of ¹⁴C incorporation was found in free fatty acids.     

Species difference in increased vascular permeability by synthetic leukotriene C4 and D4

The activity of synthetic LTC₄ was tested in guinea-pig ileum and was 200 times more potent than histamine in contraction of the ileum (3 × 10^?11 M- 3 × 10^?9 M). The activities of LTC₄ and LTD₄ in increased vascular permeability in guinea pigs, rats and rabbits were compared with those histamine, bradykinin and prostaglandin (PG) E₂. LTC₄ was approximately equipotent to bradykinin on a molar basis in guinea pigs and rats and 5–100 times more potent than histamin. LTD₄ was about 10 times more potent than LTC₄ in guinea pigs and as equipotent to LTC₄ in rats. On the contrary, in rabbits, neither LTC₄ (upto 30 nmole/site) nor LTD₄ (1 nmole/site) induced the dye exduation. These results show that species difference is present in activity of LTC₄ and LTD₄ in vascular permeability. Furthermore, in guinea pigs, the vascular permeability increased by LTC₄ was not affected after pretreatment with pyrilamine (2.5 mg/kg, i.v.), and LTC₄ and LTD₄ did not potenciate the activity of bradykinin in vascular permeability.     

Trypanosoma brucei: An evaluation of salicylhydroxamic acid as a trypanocidal drug

The chemotherapeutic potential of salicylhydroxamic acid (SHAM) was studied in adult rats infected with a strain of Trypanosoma brucei that kills the rats in about 100 hr. The median lethal dose, administered intraperitoneally in a carboxymethyl-cellulose suspension, is approximately 820 mg/kg body weight for male and 850 mg/kg for female rats. The apparent cause of death is severe depression of the central nervous system.Half-maximal inhibition of O₂ uptake by trypanosomes in vitro requires 15 μM SHAM, whereas 100 μM inhibits over 90%. This inhibitory effect on trypanosome respiration was used as a biological assay for the effective SHAM concentration in rat plasma. After administration of a sublethal SHAM dose to rats, the effective plasma SHAM concentration rose rapidly to about 500 μM and then fell to about 10 μM at 4 hr. Nevertheless, this dose did not significantly affect the survival time of rats infected with T. brucei. Even if, by repeated SHAM administration, the plasma SHAM concentration was kept at around 100 μM for more than 4 hr, no therapeutic effect was observed.These results show that O₂ uptake is not essential for the survival of trypanosomes in rats and they support the idea that bloodstream trypanosomes have an alternative pathway for glycolysis, allowing energy production in the absence of respiration.The possibility that SHAM or other inhibitors of trypanosome respiration could stilll be trypanocidal if used in conjunction with another inhibitor of glycolysis is discussed.     

Nematodirus battus: Permeability changes,calcium binding,and phosphorylation of the eggshell during hatching

Eggshells of Nematodirus battus leaked trehalose 4 hr after being stimulated to hatch, and became permeable to trypan blue at their poles; 80% of eggs were stained blue 24 hr later. Exogenous application of ruthenium red significantly inhibited chill- and sodium fluoride-stimulated hatching, 50% hatch inhibition occurring in 44.67 ± 2.2 and 8.5 ± 1.5 μM, respectively. Lanthanum chloride, however, was not as inhibitory as ruthenium red on fluoride-stimulated hatching, 50% occurring at 31.60 ± 1.25 μM. A Scatchard plot of the competitive binding of ruthenium red to eggshells demonstrated a high-affinity binding site for calcium, K_Ca′ = 1.92 μM and a second, low-affinity site, K_Ca′ = 1169.60 μM. Ruthenium red binding was significantly reduced by several enzymes, e.g., EGTA-buffered trypsin reduced binding by 73%. Radioiodinated concanavalin A also bound competitively to the eggshells in the presence of α-d-glucosyl-α-d-glucopyranoside and α-methyl-d-mannopyranoside. Eggshells incorporated phosphorus-32 from ATP after chilling or on exposure to sodium fluoride; gel filtration of solubilized homogenates of these samples showed that two proteins were radiolabelled with molecular weights of 38 × 10³ and 8 × 10³ Da, respectively. This phosphorylation was inhibited by N-ethylmaleimide, which also prevented hatching.