Cryptosporidium parvum Sporozoite Pellicle Antigen Recognized by a Neutralizing Monoclonal Antibody Is a β-Mannosylated Glycolipid

ABSTRACT The protozoan parasite Cryptosporidium parvum is an important cause of diarrhea in humans, calves, and other mammals worldwide. No approved vaccines or parasite-specific drugs are currently available for the control of cryptosporidiosis. To effectively immunize against C. parvum, identification and characterization of protective antigens are required. We previously identified CPS-500, a conserved, neutralization-sensitive antigen ofC. parvum sporozoites and merozoites defined by monoclonal antibody 18.44. In the present study, the biochemical characteristics and subcellular location of CPS-500 were determined. CPS-500 was chloroform extractable and eluted with acetone and methanol in silicic acid chromatography, consistent with being a polar glycolipid. Following chloroform extraction and silicic acid chromatography, CPS-500 was isolated by high-pressure liquid chromatography for glycosyl analysis, which indicated the presence of mannose and inositol. To identify which component of CPS-500 comprised the neutralization-sensitive epitope recognized by 18.44, the ability of the monoclonal antibody to bind CPS-500 treated with proteases, or with α- or β-glycosidases, was determined. Monoclonal antibody 18.44 did not bind antigen treated with β-d-mannosidase but did bind antigen treated with α-d-mannosidase, other α- or β-glycosidases, or a panel of proteases. These data indicated that the target epitope was dependent on terminal β-d-mannopyranosyl residues. By immunoelectron microscopy, 18.44 binding was localized to the pellicle and an intracytoplasmic tubulovesicular network in sporozoites. Monoclonal antibody 18.44 also bound to antigen deposited and released onto substrate over the course travelled by gliding sporozoites and merozoites. Surface localization, adhesion and release during locomotion, and neutralization sensitivity suggest that CPS-500 may be involved in motility and invasion processes of the infective zoite stages.

Changes in Cuticular Compounds of Developing Pecan Leaves

Leaf surface compounds of pecan [Carya illinoensis (Wangenh.) C. Koch] were analyzed with regard to developmental stage and to susceptibility to infection by Cladosporium caryigenum (Ell. et Lang. Gottwald). Immature and mature leaves of two resistant (`Elliott' and `Sumner') and two susceptible (`Wichita' and `Schley') cultivars were extracted with methylene chloride. Extracts were separated by silicic acid chromatography into polar and nonpolar fractions. Constituents of each fraction were subsequently separated by gas chromatography and were identified by gas chromatography-mass spectroscopy. Leaf surface constituents characterized included long-chain aliphatic hydrocarbons, aliphatic wax esters, triterpenoid constituents, aliphatic alcohols, fatty acids, and diacyl glycerides. The predominant surface compounds on immature leaves were lipids such as fatty acids, fatty alcohols, and glycerides. On mature leaves, lipids declined and aliphatic hydrocarbons and triterpenoids became predominant leaf surface constituents. The changes were observed for all cultivars, regardless of genotypic response to C. caryigenum. Thus, we conclude that cuticular chemicals change dramatically during leaf maturation but do not correlate with resistance to scab disease common to certain pecan cultivars.

Phospholipids as modulators of hepatic recognition of chylomicron remnants. Observations with emulsified lipoprotein lipids

The lipids extracted from chylomicrons, chylomicron remnants generated in vivo and hepatic-lipase-treated chylomicrons were emulsified by sonication. These emulsified particles retained the capacity of the native lipoproteins to be differentiated by the liver in vivo, i.e. only the particles derived from remnant and hepatic-lipase-treated chylomicron lipids were efficiently taken up by the liver. To investigate the role of phospholipids in this differentiation process, the phospholipids of all three lipoprotein preparations were separated from the remaining lipids by silicic acid chromatography. The phospholipid-free lipid fraction of chylomicrons was then emulsified with the phospholipids derived from each of the three lipoprotein preparations. Only the particles emulsified with phospholipids derived from remnants and hepatic-lipase-treated chylomicrons were efficiently taken up by the liver in vivo. These results support the proposition that phospholipids modulate the hepatic differentiation between chylomicrons and remnants in vivo.

Isolation and ldentification of Aza-Arenes of Tobacco Smoke

The nitrogen analogues of polynuclear aromatic hydrocarbons (aza-arenes) were isolated and identified in a basic fraction of cigarette smoke condensate. Silicic acid chromatography removed the predominant nicotine alkaloids, while gel chromatography on Bio-Beads S-X12 in benzene effectively separated the aza-arenes from interfering aliphatic compounds. In addition, the gel columns separated the aza-arenes by ring number and degree of alkylation on the basis of an adsorption-type mechanism. These gel characteristics facilitated the identifications of a large number of isomeric aza-arenes. Compounds identified included 2-vinylpyridine, 3-vinylpyridine and 2-phenylpyridine as well as quinoline, isoquinoline, 4-azafluorene, benzoquinolines, benzoisoquinolines, 1-azafluoranthene, 7-azafluoranthene, 4-aza-pyrene, 7- azaindole, pyrroloquinoline and their mono-, di- and trimethyl derivatives. All eight possible isomers of benzoquinoline and benzoisoquinoline were found, four of which are being reported for the first time. Evidence was also found for the probable presence of 5,6-benzo-7-azaindole.

Dynamics of leukotriene C production by macrophages.

A method for the radiochemical assay of LTC production by mouse peritoneal macrophages in vitro is presented. The method involves labeling macrophages in culture with [5,6,8,9,11,12,14,15-3H]20:4 followed by stimulation of arachidonic acid (20:4) release under the experimental conditions desired. Radiolabeled leukotriene C (LTC) is recovered from the culture medium by extraction and silicic acid chromatography in 40% yield with full retention of biological activity. Because this LTC is radiochemically pure, the quantity of LTC release may be estimated from the amount of radioactivity in the sample. Use of the radioassay to study parameters affecting LTC synthesis by macrophages indicated that the time course of LTC synthesis and its relationship to the dose of a phagocytic stimulus (zymosan) were very similar to those of prostaglandin (PG) release. LTC release was also similar to that of PG in that lower levels of both metabolites were produced by Corynebacterium parvum-elicited macrophages than by resident cells. Finally, LTC release was stimulated in response to a challenge with antigen-antibody complexes, but lower maximal levels were attained than those with zymosan. The data presented here are consistent with the hypothesis that challenge of macrophages with a phagocytic stimulus leads to the release of 20:4 by an inducible phospholipase. Cyclooxygenase and lipoxygenase then compete for the released 20:4, leading to the production of PG, hydroxyeicosatetraenoic acids, and LTC.

3-hydroxydecanedioic acid and related homologues: urinary metabolites in ketoacidosis.

Urine from patients with ketoacidosis was found to contain a number of aliphatic 3-hydroxy dicarboxylic acids. The acids were purified by silicic acid chromatography and their structures determined by gas chromatography-mass spectrometry of different derivatives. The major compound was 3-hydroxydecanedioic acid. Minor compounds were 3-hydroxyoctanedioic acid, 3-hydroxyoctenedioic acid, 3-hydroxydecenedioic acid, 3-hydroxydodecanedioic acid, 3-hydroxydodecenedioic acid, 3-hydroxytetradecenedioic acid, and 3-hydroxytetradecadienedioic acid. The excretion of 3-hydroxydecanedioic acid correlated positively with the excretion of hexanedioic acid, another metabolite constantly found in ketoacidosis (Pettersen et al., Clin. Chim. Acta 38: 17-24, 1972). We suggest that the 3-hydroxy dicarboxylic acids are formed from fatty acids by a combination of omega-oxidation and incomplete beta-oxidation.

3-hydroxydecanedioic acid and related homologues: urinary metabolites in ketoacidosis.

Urine from patients with ketoacidosis was found to contain a number of aliphatic 3-hydroxy dicarboxylic acids. The acids were purified by silicic acid chromatography and their structures determined by gas chromatography-mass spectrometry of different derivatives. The major compound was 3-hydroxydecanedioic acid. Minor compounds were 3-hydroxyoctanedioic acid, 3-hydroxyoctenedioic acid, 3-hydroxydecenedioic acid, 3-hydroxydodecanedioic acid, 3-hydroxydodecenedioic acid, 3-hydroxytetradecenedioic acid, and 3-hydroxytetradecadienedioic acid. The excretion of 3-hydroxydecanedioic acid correlated positively with the excretion of hexanedioic acid, another metabolite constantly found in ketoacidosis (Pettersen et al., Clin. Chim. Acta 38: 17-24, 1972). We suggest that the 3-hydroxy dicarboxylic acids are formed from fatty acids by a combination of omega-oxidation and incomplete beta-oxidation.