Plasmodium knowlesi: studies on invasion of rhesus erythrocytes by merozoites in the presence of protease inhibitors

The effect of protease inhibitors on invasion of rhesus erythrocytes by Plasmodium knowlesi merozoites was evaluated. Chymostatin, N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), and L-1-tosylamide-2-phenylethylchloromethyl ketone (TPCK) inhibited invasion. Leupeptin, antipain, pepstatin, and phenylmethylsulfonyl fluoride (PMSF) had no effect. TLCK and TPCK inhibited attachment of merozoites to host erythrocytes. Chymostatin had no adverse effect on attachment, and in its presence junction formation between the merozoite and host erythrocyte occurred. Both chymostatin and leupeptin inhibited normal rupture of schizont-infected erythrocytes. It is suggested that proteolytic activity may be important both in the rupture of schizont-infected erythrocytes and in the invasion of erythrocytes by malaria parasites.     

The role of protease in streptolysin S formation

Production of streptolysin S by streptococci was found to be inhibited by treatment with protease inhibitors, tosylphenylalanine chloromethyl ketone (TPCK), tosyllysine chloromethyl ketone (TLCK), or phenylmethylsulfonyl fluoride (PMSF), even in the presence of the inducer oligonucleotides. Other protease inhibitors, antipain, leupeptin, or pepstatin were found to have little or no effect. Trypsin reversed the effect of TPCK or TLCK. The reversal was dependent upon the amount of added trypsin and the incubation time at 37 degrees C, suggesting that a protease activity was involved in the hemolysin formation. The effect of trypsin was not observed if chloramphenicol was also added, suggesting that a precursor of streptolysin S was processed as it was synthesized and released into medium as the active hemolysin, by the concerted action of a protease and inducer oligonucleotides. Experiments with the subcellular fractions of streptococci indicated that the streptolysin precursor was localized in the insoluble fraction and the "processing" protease in the supernatant fraction.     

Plasmodium berghei: effect of protease inhibitors during gametogenesis and early zygote development

Plasmodium berghei: The effect of five protease inhibitors, TPCK, TLCK, PMSF, leupeptin, and 1,10-phenanthroline on in vitro gametogenesis and early zygote development of P. berghei was investigated. PMSF and leupeptin showed no effect. Cysteine/serine protease inhibitors TPCK/TLCK at concentrations of 75 and 100 microM were effective on inhibiting exflagellation center formation, and this effect was reversible with the addition of l-cysteine. Exflagellation center formation was most effectively blocked by 1,10-phenanthroline (1mM), and exflagellation center numbers were restored by the addition of Zn(2+). A reduction of ookinete production was observed when TPCK/TLCK (100 microM) was added at 2h after gametogenesis, but no effect was observed with 1,10-phenanthroline (1mM). Our results suggest that proteolysis is important in both gametocyte activation and sexual development of P. berghei.     

Down regulation of phorbol diester receptors by proteolytic degradation of protein kinase C in a cultured cell line of fetal rat skin keratinocytes

Down regulation of phorbol diester receptors was studied with respect to proteolysis of protein kinase C, which is activated by Ca2+, phospholipids, and diacylglycerols and which binds to phorbol diesters. We used FRSK cells, a cell line derived from fetal rat skin keratinocytes, because in these cells specific binding of phorbol 12,13-dibutyrate decreased rapidly (50% decrease in 30 min). This decrease (down regulation) was inhibited by some protease inhibitors, such as N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), N-p-tosyl-L-lysine chloromethyl ketone (TLCK), and leupeptin, but not by inhibitors of lysosomal hydrolases. On treatment with 12-O-tetradecanoylphorbol 13-acetate, protein kinase C was rapidly translocated from the cytosol to the membranes and then decreased. This decrease in protein kinase C was also inhibited by TPCK, TLCK, and leupeptin. The decrease in membrane activity of protein kinase C was associated with increase in cytosolic activity of a protein kinase that was smaller in molecular weight (Mr 40,000-60,000) than protein kinase C, did not depend on Ca2+/phosphatidylserine/diacylglycerol, and did not bind to phorbol 12,13-dibutyrate. These results indicate that down regulation of phorbol diester receptors is probably caused by nonlysosomal proteolysis of protein kinase C. The kinase formed by cleavage may be an active catalytic site of protein kinase C.     

Protease Inhibitors Cause Necrotic Cell Death in Chlamydomonas reinhardtii by Inducing the Generation of Reactive Oxygen Species

Protease inhibitors affecting the activity of the proteasome were reported to induce programmed cell death (apoptosis) in some mammalian cell lines. Proteasome activity can be suppressed by specific peptide derivatives and by N‐tosyl‐lysine‐chloromethyl‐ketone (TLCK) and N‐tosyl‐phenylalanine‐chloromethyl‐ketone (TPCK), which affect the trypsine‐ and chymotrypsine‐like activities of the proteasome, respectively. Particularly TLCK and TPCK caused necrotic cell death in the unicellular green alga Chlamydomonas reinhardtii. As a control, the effects of these protease inhibitors on the survival of human WISH cells were also studied. Bleaching of the Chlamydomonas cells after addition of TLCK or TPCK indicated that reactive oxygen species (ROS) were involved in this process. Indeed, increased levels of ROS were detected in Chlamydomonas cells treated with TLCK or TPCK. Furthermore, cell death induced by these protease inhibitors was accelerated by illumination and prevented or slowed down by scavengers of ROS.     

N-alpha-Tosyl-L-lysine chloromethyl ketone and N-alpha-tosyl-L-phenylalanine chloromethyl ketone inhibit protein kinase C

TLCK (N-alpha-tosyl-L-lysine chloromethyl ketone) inhibits protein kinase C whether or not the enzyme is under the regulation of Ca2+ and phospholipid. TLCK (IC50 = 1 mM) is a much more potent inhibitor of protein kinase C than TPCK (N-alpha-tosyl-L-phenylalanine chloromethyl ketone) (IC50 = 8 mM), suggesting that the lysyl moiety of TLCK may be specifically recognized by the active site of protein kinase C. These results extend the evidence that the active site of protein kinase C recognizes basic amino acids, and suggest that the active sites of protein kinase C and the cAMP-dependent protein kinase, which is also inhibited by TLCK and TPCK, are structurally related.     

Molecular interactions of cultured turkey kidney cells with specific antigens of Eimeria adenoeides sporozoites

Earlier studies suggested that specific communication between the parasite and the host cell may play a role in cellular invasion by sporozoites of species of avian Eimeria. In this study, quantification of cellular invasion and modified Western blot analysis were used to explore the possibility that parasite receptors for interaction with the host cell might be involved in the sporozoite-host cell communication. Invasion in cultured cells treated with a homogenate of Eimeria adenoeides sporozoites was approximately 50% lower than that in untreated cultures. When the sporozoite homogenate was solubilized in sodium dodecyl sulfate and electrophoretically separated, components of the cultured host cells bound consistently to sporozoite bands having Mr of 23 and 40 kDa. Biotinylation of intact sporozoites revealed at least 14 biotin-labeled bands, including bands at 23 and 40 kDa, that were considered to be surface molecules. If the sporozoites were incubated in trypsin after they were biotinylated, only two biotinylated bands at 18 and 23 kDa remained; the 40-kDa biotinylated band was not detected. Despite the removal of the majority of the surface molecules, the cell homogenate still bound to the trypsin-treated sporozoites; the intensity of the label was similar to that resulting from the binding of cell homogenate to untreated sporozoites. The data show specific interactions between 23- and 40-kDa sporozoite bands and host cell components, and provide evidence that the 23-kDa molecule may be located on the sporozoite surface and the 40-kDa molecule located intracellularly.     

A chymotrypsin-like proteinase from the midgut of Tenebrio molitor larvae

A chymotrypsin-like proteinase was isolated from the posterior midgut of larvae of the yellow mealworm, Tenebrio molitor, by ion-exchange and gel filtration chromatography. The enzyme, TmC1, was purified to homogeneity as determined by SDS-PAGE and postelectrophoretic activity detection. TmC1 had a molecular mass of 23.0 kDa, pI of 8.4, a pH optimum of 9.5, and the optimal temperature for activity was 51 degrees C. The proteinase displayed high stability at temperatures below 43 degrees C and in the pH range 6.5-11.2, which is inclusive of the pH of the posterior and middle midgut. The enzyme hydrolyzed long chymotrypsin peptide substrates SucAAPFpNA, SucAAPLpNA and GlpAALpNA and did not hydrolyze short chymotrypsin substrates. Kinetic parameters of the enzymatic reaction demonstrated that the best substrate was SucAAPFpNA, with k(cat app) 36.5 s(-1) and K(m) 1.59 mM. However, the enzyme had a lower K(m) for SucAAPLpNA, 0.5 mM. Phenylmethylsulfonyl fluoride (PMSF) was an effective inhibitor of TmC1, and the proteinase was not inhibited by either tosyl-l-phenylalanine chloromethyl ketone (TPCK) or N(alpha)-tosyl-l-lysine chloromethyl ketone (TLCK). However, the activity of TmC1 was reduced with sulfhydryl reagents. Several plant and insect proteinaceous proteinase inhibitors were active against the purified enzyme, the most effective being Kunitz soybean trypsin inhibitor (STI). The N-terminal sequence of the enzyme was IISGSAASKGQFPWQ, which was up to 67% similar to other insect chymotrypsin-like proteinases and 47% similar to mammalian chymotrypsin A. The amino acid composition of TmC1 differed significantly from previously isolated T. molitor enzymes.     

Differential effects of protease inhibitors on 1,25-dihydroxyvitamin D3 receptors

We describe herein two different effects of protease inhibitors and substrates on receptors for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) obtained from the intestinal mucosa of vitamin D-deficient chicks: inhibition of binding of 1,25(OH)2D3 to its receptor and stabilization of the receptor. Both L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK), a chymotrypsin inhibitor, and N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), a trypsin inhibitor, block [3H]1,25(OH)2D3 binding to the receptor. Fifty per cent inhibition of binding occurs at 20 microM TPCK, and 100% inhibition at 100-200 microM; TLCK is about 25-fold less effective. At higher concentrations (10-100 mM), the chymotrypsin substrates N alpha-p-tosyl-L-arginine methyl ester and tryptophan methyl ester and the cathepsin B inhibitor leupeptin also inhibit [3H] 1,25(OH)2D3 binding to its receptor. Different inhibitors and substrates interact with the receptor differently: TPCK (20 microM) and N alpha-p-tosyl-L-arginine methyl ester (10 mM) are reversible, noncompetitive inhibitors, L-tryptophan methyl ester (20 mM) is a reversible competitive inhibitor, and phenylmethylsulfonyl fluoride (300 microM) shows no effect on [3H]1,25(OH)2D3 binding to its receptor. The most stable form of unoccupied 1,25(OH)2D3 receptors from chick intestinal mucosa was that obtained from a low salt chromatin preparation (t 1/2 = 6.0 h). The presence of KCl drastically decreased receptor stability (t 1/2 = 1.8 h); and the addition of 2.5 mM CaCl2 further reduced their stability. Phenylmethylsulfonyl fluoride and Trasylol inhibited the KCl-induced receptor instability, but did not prevent the additional instability in the presence of CaCl2. In summary, TPCK and TLCK exert direct effects on the 1,25(OH)2D3 receptor molecule, independent of their protease inhibitor function. These compounds may prove useful as covalent affinity labels for the receptor. On the other hand, phenylmethylsulfonyl fluoride and Trasylol stabilize 1,25(OH)2D3 receptors, probably via inhibition of KCl-activated nuclear protease(s). This receptor stabilization will be advantageous in receptor assays and/or purification procedures.     

Characterization of a serine protease activity in Sarcocystis neurona merozoites

Sarcocystis neurona merozoites were examined for their ability to invade and divide in bovine turbinate (BT) cell cultures after treatment with cysteine (iodoacetamide), aspartic (pepstatin A), metallo-(1,10-phenanthroline and ethylene glycol-bis(aminoethylether)-tetraacetic acid [EGTA]), or serine (4-[2-aminoethyl]-benzenesulfonyl fluoride hydrochloride [AEBSF], phenylmethane sulphonyl fluoride [PMSF], and tosyl lysyl chloramethyl ketone [TLCK]) protease inhibitors. Significant (P < 0.01) inhibition of serine protease activity by PMSF and TLCK led to a reduction of 86 and 78% in merozoites produced in BT cell cultures, respectively, whereas AEBSF (1 mM) led to a 68% reduction in merozoites produced in BT cell cultures and a reduction of 84 and 92% at higher AEBSF concentrations (2 and 3 mM, respectively). Pepstatin A and iodoacetamide failed to cause any inhibition in merozoite production, whereas 1,10-phenanthroline and EGTA caused slight, but not significant, inhibition at 6 and 17%, respectively. In zymograms, 2 bands of protease activity between 65- and 70-kDa molecular weight were seen. The protease activity was inhibited by AEBSF but not by E-64 (cysteine protease inhibitor), EGTA, iodoacetamide, or pepstatin A. In native zymograms, the protease activity was highest between a pH range of 8 and 10. These data suggest that merozoites of S. neurona have serine protease activity with a relative molecular weight range between 65 and 70 kDa and optimal pH range between 8 and 10, which is essential for host cell entry at least in vitro. The protease activity described here could be a potential target for chemotherapy development.     

Giardia lamblia: characterization of proteinase activity in trophozoites

The proteinase activity of Giardia lamblia trophozoites, Portland 1 strain, was characterized with respect to substrate specificities and inhibitor sensitivities. Proteinase activity with urea-denatured hemoglobin (UDH), alpha-N-benzoyl-DL-arginine-2-naphthylamide (BANA), and alpha-N-benzoyl-argininamide (BAA) as substrates exhibited pH optima of 5.8, 3.8, and 5.0, respectively. For BANA, the apparent Km was 0.20 mM and the Vmax was 2.56 microM. For BAA, the apparent Km was 4.0 mM and the Vmax was 8.69 microM. Dithiothreitol (DTT, 5 mM) enhanced proteinase activity threefold for UDH, fourfold for BAA, and fivefold for BANA. Iodoacetamide, L-tosylamide-2-phenylethyl chloromethyl ketone (TPCK), and N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), each at 1 mM, inhibited proteinase activity by greater than 90% with BANA and BAA. Iodoacetamide inhibited proteinase activity by 35% with UDH; TPCK and TLCK inhibited activity greater than 70% with UDH. Activity on BAA was inhibited by 91% with Zn2+ and activity on UDH was inhibited by 30% with Cu2+. Virtually complete inhibition of proteinase activity on BANA and BAA was obtained with leupeptin and chymostatin at 1 microgram/ml. Pepstatin A, chelators, and other heavy metals had no apparent effect on proteinase activity. Two polypeptide bands (ca. 105 and 40 kDa) indicative of proteinase activity were visualized by sodium dodecyl sulfate-gelatin polyacrylamide gel electrophoresis. The 105 kDa band was visible over the pH range of 4 to 7, but with greater intensity from pH 5 to 7. The 40 kDa band, while present at pH 5, was most intense at pH 6 and 7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell: sporozoite interactions and invasion by apicomplexan parasites of the genus Eimeria

The site specificity that avian Eimeria sporozoites and, to a more limited degree, other apicomplexan parasites exhibit for invasion in vivo suggests that specific interactions between the sporozoites and the target host cells may mediate the invasion process. Although sporozoite motility and structural and secreted antigens appear to provide the mechanisms for propelling the sporozoite into the host cell,there is a growing body of evidence that the host cell provides characteristics by which the sporozoites recognise and interact with the host cell as a prelude to invasion. Molecules on the surface of cells in the intestinal epithelium, that act as receptor or recognition sites for sporozoite invasion, may be included among these characteristics. The existence of receptor molecules for invasion by apicomplexan parasites was suggested by in vitro studies in which parasite invasion was inhibited in cultured cells that were treated with a variety of substances designed to selectively alter the host cell membrane. These substance included cationic compounds or molecules, enzymes that cleave specific linkages, protease inhibitors, monoclonal antibodies, etc. More specific evidence for the presence of receptors was provided by the binding of parasite antigens to specific host cell surface molecules.Analyses of host cells have implicated 22, 31, and 37 kDa antigens, surface membrane glycoconjugates,conserved epitopes of host cells and sporozoites, etc., but no treatment that perturbs these putative receptors has completely inhibited invasion of the cells by parasites. Regardless of the mechanism,sporozoites of the avian Eimeria also invade the same specific sites in foreign host birds that they invade in the natural host. Thus, site specificity for invasion may be a response to characteristics of the intestine that are shared by a number of hosts rather than to a unique trait of the natural host. Protective immunity elicited against avian Eimeria species is not manifested in a total blockade of parasite invasion. In fact, the effect of immunity on invasion differs according to the eliciting species and depends upon the area of the intestine that is invaded. Immunity produced against caecal species of avian Eimeria, for example Eimeria tenella and Eimeria adenoeides, inhibits subsequent invasion by homologous or heterologous challenge species, regardless of the area of the intestine that the challenge species invade. Conversely, in birds immunised with upper intestinal species, Eimeria acervulina and Eimeria meleagrimitis, invasion by challenge species is not decreased and often is significantly increased.     

Expression of flotillin-1 on Eimeria tenella sporozoites and its role in host cell invasion

Lipid rafts are detergent-resistant, liquid-ordered microdomains in plasma membranes that are enriched in cholesterol and sphingolipids and involved in intracellular signal transduction, membrane trafficking, and molecular sorting. In this study, we investigated the possibility that lipid rafts on Eimeria tenella sporozoites may act as platforms for host cell invasion. Flotillin-1, a resident protein of lipid rafts, was identified on E. tenella sporozoites and was prominently expressed at the apex of the cells, a region mediating host cell invasion. Pretreatment of sporozoites with antibody against flotillin-1 blocked parasite invasion. Furthermore, the anticoccidial drug, monensin, disrupted the localization of flotillin-1 within raft structures resulting in loss of invasion. We conclude that Eimeria sporozoites utilize lipid rafts containing flotillin-1 for internalization into host cells.     

The Characterization of Cell Death Induced by 1-(3-C-Ethynyl-β-D-ribo-Pentofuranosyl)Cytosine (ECyd) in FM3A Cells

Abstract

The characterization of cell death induced by 1-(3-C-ethynyl-β-D-ribo-pentofuranosyl)cytosine(ECyd), a potent inhibitor of RNA synthesis, was performed using mouse mammary tumor FM3A cells in vitro. Accompanied with the cell death induced by ECyd (3.0 μM)-treatment, about 100–200 kbp-sized and internucleosomal DNA fragmentation were observed by orthogonal-field-alternation gel electrophoresis (OFAGE) and conventional gel electrophoresis, respectively. Protease inhibitors, carbobenzoxy-L-aspart-1-yl[(2,6-dichlorobenzoyl)oxy]methane (Z-Asp-CH₂-DCB), Nα-p-tosyl-L-lysine chloromethyl ketone (TLCK) and N-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK), effectively blocked the cell death, suggesting that the proteases inhibited by Z-Asp-CH₂-DCB, TLCK or TPCK were involved in the process of the cell death.     

Invasion of different cell types by sporozoites of Eimeria species and effects of monoclonal antibody 1209-C2 on invasion of cells by sporozoites of several apicomplexan parasites

Sporozoites of avian Eimeria species differed markedly in their ability to invade cells in vitro. Invasion by E. tenella and E. adenoeides was significantly greater in baby hamster kidney (BHK) and chicken cecal cell (CC) cultures than in primary chicken (PCK) or turkey kidney (PTK) cell cultures. Moreover, invasion of BHK cell cultures by E. adenoeides was significantly greater than that of other Eimeria species, and invasion by E. acervulina sporozoites was significantly lower. Monoclonal antibody 1209-C2 (MAb 1209-C2) reacted by immunofluorescent labeling (IFA) with refractile bodies of sporozoites of 5 species of Eimeria and Caryospora bigenetica, but not with sporozoites of Toxoplasma gondii, Hammondia hammondi, or Cryptosporidium parvum, which have no refractile bodies. The MAb also cross-reacted with formalin-fixed BHK, CC, turkey cecal (TC) cells, and PTK. Pretreatment of BHK cells with MAb 1209-C2 significantly reduced invasion of the cells by sporozoites of E. tenella, E. acervulina, E. meleagrimitis, and C. bigenetica, but did not alter invasion by T. gondii, C. parvum, or H. hammondia. Apparently, reactivity of MAB 1209-C2 with the sporozoites was required for inhibition of invasion despite the fact that the inhibition resulted from pre-treatment of the host cell. Conversely, although MAb 1209-C2 also reacted moderately with PTK and TC cells, pre-treatment of these cell cultures with the MAb did not inhibit invasion by either MAB 1209-C2-reactive or -nonreactive parasites. Collectively, the data indicated that refractile body antigens of sporozoites of Eimeria and Caryospora, which are recognized by MAb 1209-C2, may function in cellular invasion, but also suggest that cellular invasion is probably not mediated by interactions between the conserved epitopes in sporozoites and cultured host cells that are recognized by the MAb.     

Assay for enzyme inhibition: detection of natural inhibitors of trypsin and chymotrypsin

A technique for quickly detecting nanogram quantities of low- and high-molecular-weight inhibitors of some serine proteases is described. The inhibitor solutions are spotted onto agar films which contain either L-1-p-tosylamino-2-phenylethyl chloromethyl ketone (TPCK)-trypsin or tosyl lysine chloromethyl ketone (TLCK)-chymotrypsin. Enzyme inhibition is visualized as colorless zones on a pink background after the films were stained with the chromogenic substrate N-acetyl-DL-phenylalanine-beta-naphthyl ester. The method is used for rapidly testing both high-performance liquid chromatography fractions and thin-layer chromatograms to identify the inhibitors of trypsin and chymotrypsin in complex microbial extracts. The assay is quantitative so that it is possible to compare the specificity of the inhibitory fractions for trypsin and chymotrypsin. Results with standard inhibitors demonstrate the high sensitivity of the method, e.g., inhibition is detected with 1 ng of soybean trypsin inhibitor and 0.3 ng of antipain or chymostatin.     

Chloromethyl ketones are insulin-like stimulators of lipid synthesis in locust fat body

Nα-p-tosyl-L-lysine chloromethyl ketone (TLCK) stimulates lipid synthesis in locust fat body in vitro, and is able to reverse the inhibitory effects of AKH-I on lipid synthesis. Effective stimulatory concentrations of TLCK were in the range of 0.2–1.0 mM. Similar stimulatory effects were also achieved with phenylalanine chloromethyl ketone (PheCK) and leucine chloromethyl ketone (LeuCK), but not with tosyl-phenylalanine chloromethyl ketone (TPCK), dansyl-glu-gly-arg-CK, chloroacetone, chloroacetic acid, chloroacetamide, chloroacetaldehyde, chloroacetyl-L-leucine or acetylated or fluorescamine-labelled TLCK, PheCK, and LeuCK. The level of stimulation caused by TLCK was dependent on incubation time, so that after a 5-h preincubation of fat body tissue with TLCK the stimulated rate was severalfold higher than the control. TLCK also increased the rate of uptake of trehalose and uridine, but not glucose, deoxyglucose or glycine. Increasing concentrations of bovine serum albumin (BSA) in the incubation medium caused a reduction in the rate of TLCK-stimulated acetate uptake, such that levels of uptake were no higher with 1% BSA than in the controls. A range of more specific protease and kinase inhibitors was tested, but none caused stimulation; thus the mode of action of TLCK on the stimulation of acetate uptake has yet to be identified. Elucidation of the mode of action of TLCK may facilitate the development of novel compounds for insect pest control. Arch. Insect Biochem. Physiol. 39:9–17, 1998. © 1998 Wiley-Liss, Inc.     

In vitro development of exoerythrocytic forms of Plasmodium gallinaceum sporozoites in avian macrophages

Exoerythrocytic forms of Plasmodium gallinaceum were cultured in vitro using salivary gland sporozoites extracted from experimentally infected Aedes fluviatilis mosquitoes. The host cells were macrophage precursors from chicken bone marrow. At various times after introduction of sporozoites, the cultures were stained by Giemsa or by immunofluorescence assay (IFA) using anti-sporozoite-specific monoclonal antibodies (MAb). The time to complete parasite development in vitro was 50-70 h. By 70 h, ruptured segmenters and free merozoites were visible within the cells. Inoculation of normal chickens with infected cultures induced parasitemia after a pre-patent period of 10-11 days. In vitro young exoerythrocytic forms, late schizonts that include the matured segmenters, and free merozoites shared common antigens with the sporozoites as revealed by IFA using anti-sporozoite-specific MAbs. Our data indicate that macrophages support development of P. gallinaceum sporozoites and that the circumsporozoite proteins are present until the end of the primary exoerythrocytic schizogony.     

Antiproteinases protect cultured lung endothelial cells from endotoxin injury

To determine whether the effects of endotoxin on cultured lung endothelium involve proteolytic mechanisms, we incubated bovine pulmonary arterial endothelial cells with endotoxin in medium 199 + 10% fetal bovine serum (FBS) in the presence and absence of several proteinase inhibitors. Three chloromethyl ketone (CK) derivatives [N-tosyl-L-lysine (CK)-(TLCK), N-tosyl-L-phenylalanine CK(TPCK), methoxysuccinyl-Ala-Ala-Pro-Val CK(SPCK)] and a single synthetic proteinase substrate [N-alpha-p-tosyl-L-arginine methyl ester hydrochloride (TAME)] attenuated endotoxin-induced cytotoxicity (lactate dehydrogenase release) and prostacyclin production in a dose-related fashion. The most effective inhibitors of endotoxin-induced cytotoxicity were TLCK and TPCK. TLCK and TAME most effectively attenuated endotoxin-stimulated prostacyclin production. Two chemically unrelated substances, soybean trypsin inhibitor and alpha 1 proteinase inhibitor also attenuated the endotoxin response. In the absence of FBS or in the presence of 10% heat-inactivated FBS, antiproteases attenuated endotoxin-induced prostacyclin production but had less effect on cytotoxicity than with 10% FBS. We also measured the capacity of the CK inhibitors to scavenge superoxide radicals generated in a cell-free xanthine/xanthine oxidase system by measuring inhibition of cytochrome c reduction. Percent scavenging of superoxide by these inhibitors was as follows: TLCK, 62.7 +/- 5.8 (SE); TPCK, 83.9 +/- 7.7; TAME, 24.5 +/- 6.4; SPCK, 0. We conclude that certain proteinase inhibitors attenuate endotoxin-induced endothelial cytotoxicity and prostacyclin production and that direct scavenging of superoxide radicals fails to explain the protective effects of proteinase inhibition. We speculate that the effects of endotoxin on lung endothelium may involve proteolytic mechanisms even in the absence of neutrophils.