Distribution of bovine fetuin and albumin in plasma, allantoic and amniotic fluids during development

A radioimmunoassay for bovine fetuin was developed and its specificity and validity established. Albumin was measured by radial-immunodiffusion assay. Fetuin levels in fetal plasma increased from 10 to 15 mg/ml between 4 and 8 months of gestation; albumin levels remained higher than fetuin. Neonatal plasma fetuin levels rapidly declined during the first 14 days post partum, coincident with a marked reciprocal increase in albumin levels. In allantoic fluid fetuin and albumin concentrations reached a peak at 7 months but fetuin values were always higher. In amniotic fluid both proteins peaked at 8 months; albumin levels were similar to those in allantoic fluid but fetuin values remained consistently lower than those in allantoic fluid throughout gestation. Fetuin levels in maternal plasma declined from 0.7 to 0.4 mg/ml between 1 month and term. We conclude that (1) at term there is an abrupt change from fetuin synthesis to increased albumin synthesis by the neonatal liver; (2) fetuin appears to be preferentially accumulated in the allantois whereas albumin is equally concentrated in the allantois and amnion.     

Marked acceleration of exogenous fatty acid incorporation into cellular triglycerides by fetuin.

Fetuin belongs to a group of fetal glycoproteins whose specific function is not known. In this study we investigated the effect of bovine fetuin on exogenous fatty acid incorporation into lipid classes by fetal rabbit aortic smooth muscle cells (SMC) and human fetal skin fibroblasts. When compared with albumin, the addition of fetuin to the culture medium caused a dramatic increase in labeled fatty acid incorporation (nanomoles/mg of protein) by SMC into triglycerides (albumin (control) 2.8 +/- 0.3 + fetuin 178.3 +/- 13.7). This effect was noted at a wide range of fetuin concentrations (0.2-5%) at oleate:fetuin molar ratios of 3.3-0.13, respectively. Similar effects were noted using human fetal skin fibroblasts with both labeled oleic and arachidonic acids (0.1 mM) as substrates (arachidonic acid incorporation into triglycerides, albumin (control) 76.9 +/- 16.2 + fetuin 684.6 +/- 64.1). Stimulation of fatty acid incorporation into di- and monoglycerides was also noted. Although the amount of unbound fatty acid in the presence of fetuin was greater than with albumin, experiments done under conditions that create identical unbound oleate levels (by varying fatty acid concentration) still showed increased fatty acid incorporation into triglycerides by SMC when exposed to fetuin. This marked effect of fetuin on triglyceride accumulation in cells was confirmed by lipid analysis, strong positive staining with oil red O, and transmission of electron microscopy. Furthermore, the potential physiological role of fetuin in terms of fatty acid and transport was attested by (a) the presence of significant amounts of free fatty acids associated with fetuin; and (b) by the stimulatory effect of fetuin, even when added to culture media containing other fatty acid carriers. These results show that (a) fetuin is far more efficient than albumin in incorporating fatty acids into cells; and (b) this might represent a novel function for fetuin during development.     

Inhibition of sperm motility by bovine serum components.

Various sources and components of mammalian sera were evaluated for their ability to maintain or inhibit sperm motility. Human, rabbit, hamster, and porcine sera were equal in ability to maintain motility of human sperm. Four sources of fetal calf serum and one source of neonatal calf serum were unable to maintain motility of human sperm or sperm-fertilizing potential. In the presence of human serum, fetal calf serum actually inhibited human sperm motility. Fetuin, a component of fetal calf serum, contained the inhibitory activity. An inhibitory effect of fetuin on porcine and caprine sperm motility was also observed. The inhibitory activity resided in the second peak when fetuin was separated by isoelectric focusing. The sperm head membranes remained impermeable to dye, and mitochondrial membrane potential was maintained after motility had been reduced to almost zero by incubation with fetuin and fetuin fractions. Fetuin or the active portion of the molecule may be a useful component of a vaginal contraceptive and in research where inhibition of motility is desirable.     

Initiation of Plasmodium sporozoite motility by albumin is associated with induction of intracellular signalling

Malaria infection is initiated when a mosquito injects Plasmodium sporozoites into a mammalian host. Sporozoites exhibit gliding motility both in vitro and in vivo. This motility is associated with the secretion of at least two proteins, circumsporozoite protein (CSP) and thrombospondin-related anonymous protein (TRAP). Both derive from micronemes, which are organelles that empty out of the apical end of the sporozoite. Sporozoite motility can be initiated in vitro by albumin added to the medium. To investigate how albumin functions in this process, we studied second messenger signalling within the sporozoite. Using pharmacological activators and inhibitors, we have concluded that gliding motility is initiated when albumin interacts with the surface of the sporozoite and that this leads to a signal transduction cascade within the sporozoite, including the elevation of intracellular cAMP, the modulation of sporozoite motility by Ca²⁺ and the release of microneme proteins.     

Studies on the Motility of Plasmodium Sporozoites*

Albumin was found to have a striking stimulatory effect on motility of Plasmodium sporozoites, while serum globulins had an inhibitory effect. Albumin also preserved viability of sporozoites in vitro at 4 C for several days. P. berghei, P. cynomolgi, and P. falciparum sporozoites each had a distinct and characteristic type of motility. P. berghei sporozoites from oocysts had a different type of motility from that of salivary gland sporozoites, each type presumably associated with different invasive capacities at different times during the life cycle of the parasite. This change in sporozoite motility during development was also associated with other physiologic developmental changes in the sporozoite. The degree of motility of a given pool of sporozoites was to some degree associated with other parameters of metabolic activity of these sporozoites, i.e. infectivity, immunogenicity, and secretory activity. Secretions of the rhoptry-microneme complex may play a role in sporozoite motility.     

Effects of antiphagocytic agents on penetration of Eimeria magna sporozoites into cultured cells.

Madin-Darby Bovine Kidney cells were treated with sodium flouride, iodoacetate, and 2-deosyglucose, reagents that block glycolysis, and thus reduce phagocytosis. Sporozoites readily entered cells whose ATP stores were largely depleted. They also entered cells treated with colchicine, colcemid, and vinblastine. These latter agents did not inhibit sporozite motility after 6 hr incubation. Cytochalasin B prevented penetration of cells by inhibiting the motility of sporozoites. This effect was reversible. Warm sporozoites entered cold cells 4 times more radily than cold sporozoites into warm cells. The above findings suggest that phagocytosis is not the mechanism for entry of E. magna sporozoites into cultured cells, but that sporozoite motility is of primary importance.     

Effect of Fetal Bovine Serum Glycoproteins on the In Vitro Proliferation of the Oyster Parasite Perkinsus marinus: Development of a Fully Defined Medium

ABSTRACT. The oyster parasite Perkinsus marinus replicates in our medium consisting of Dulbecco modified Eagle's medium: Ham's F12 nutrient mixture (1:1) supplemented with 1–5% fetal bovine serum, with a doubling time of 24 hours during the exponential phase of the culture. Fetal bovine serum concentrations above 5% dramatically reduced parasite proliferation in a dose-dependent manner. We tested the individual effects of the three major protein components of fetal bovine serum (fetuin, transferrin and albumin) on the replication of the parasite in a serum-free medium. At the concentrations tested, fetuin enhanced parasite growth, whereas albumin had a modest positive effect and transferrin was inhibitory. Proteolytic digestion of fetuin, strongly diminished its growth-enhancing properties, indicating that the overall glycoprotein architecture may be required for activity. On the contrary, desialylation of fetuin slightly enhanced its growth-promoting activity. The addition of fetuin at 1.7 mg/ml to the serum-free DME: Ham's F12 medium yielded growth rates that are comparable to those obtained with our standard culture methodology. This has resulted in a fully defined culture medium that will allow for a rigorous characterization of excretory/secretory products involved in modulating or blocking the host's humoral and cellular defense mechanisms.     

The infectivity of Plasmodium yoelii in different strains of mice

We evaluated the effect of using Medium 199 alone and Medium 199 supplemented with 5% normal mouse serum, 5% fetal calf serum, 5% bovine serum albumin or 5% Albumax on Plasmodium yoelii sporozoite yield from infected mosquitoes and infectivity in BALB/c mice. The sporozoites yield, as well as their infectivity, was statistically lower (P = 0.0031) when unsupplemented Medium 199 was used to separate sporozoites from infected mosquitoes. Although Medium 199 supplemented with Albumax led to lower sporozoite yield (P < 0.0009), infectivity of the sporozoites was similar to those obtained with the other medium supplements. Because normal mouse serum supports good sporozoite infections and is also the supplement that can be used repeatedly in mice during multiple sporozoite injections without inducing anaphylaxis, we selected it to evaluate the infectivity of P. yoelii sporozoites in different strains of mice. After injecting mice with serial dilutions of sporozoites and detecting patent infections, we determined that the infective dose 50 (ID50) for BALB/c, C57Bl/6, A/J, and B10BR mice ranged between 4.9 and 10.6 sporozoites. The ID50 obtained for CD-1 mice (147 sporozoites) was significantly higher.     

Malaria sporozoites leave behind trails of circumsporozoite protein during gliding motility

As Plasmodium sporozoites undergo gliding motility in vitro, they leave behind trails of circumsporozoite (CS) protein that correspond to their patterns of movement. This light microscopic observation was made using Plasmodium berghei sporozoites, a monoclonal antibody (MAb H4) directed against the immunodominant repetitive epitope of the CS protein of P. berghei, and an immunogold-silver staining (IGSS) technique. Sporozoites pretreated with agents that inhibit sporozoite motility and invasiveness did not produce trails. Sporozoites that glided on microscope slides coated with MAb H4 left behind considerably longer CS protein trails than those on uncoated slides, and the staining of these trails was more intense. The fact that the CS protein is an exoantigen continuously released as trails by motile sporozoites, together with our previous finding that anti-CS protein antibodies inhibit sporozoite motility, strongly suggests that the CS protein plays a role in gliding motility. The sensitive IGSS technique used in this study may be a useful tool in the study of the translocation of surface proteins during gliding of other apicomplexans, other protists, and bacteria.     

Factors affecting motility and morphology of Cryptosporidium sporozoites in vitro

In vitro motility and morphology of Cryptosporidium sporozoites were examined in the presence of various solutions. Crude preparations of the bile salt, taurocholic acid, maintained both motility and morphology in a dose-dependent manner. These effects appeared to be due to the taurocholic acid itself, and not simply due to pH variations, osmotic factors, or contaminants. Lysis of sporozoites was also observed and was found to be dependent on pH, with acidic conditions (pH less than 6.2) triggering the lysis.     

In Vitro Excystation of Caryospora simplex (Apicomplexa: Eimeriorina)1

In vitro excystation of sporozoites of the heteroxenous coccidian Caryospora simplex Léger, 1904 (Apicomplexa: Eimeriorina) is described. Sporocysts freed mechanically from oocysts released a maximum of 51% of their sporozoites within 45 min at 25°C and a maximum of 74% within 20 min at 37°C when incubated in a 0.25% (w/v) trypsin–0.75% (w/v) sodium taurocholate (bile salt) excystation solution. At emergence from sporocysts, sporozoites were weakly motile then became highly active after about 2 min in excystation solution. Sporozoites within sporocysts exposed to bile salt only became highly motile within 25 min at 25°C and within 15 min at 37°C but did not excyst. When exposed only to trypsin at the above temperatures, the Stieda body dissolved; the substieda body remained intact, and the sporozoites exhibited only limited motility within sporocysts; only a few excysted. Intact, sporulated oocysts incubated at 25° or 37°C in 0.02 M cysteine-HC1 and a 50% CO₂ atmosphere for 18 h had no morphologic changes in the oocyst wall. Further incubation of these intact oocysts in excystation solution for 30 min at 37°C caused neither motility of sporozoites within sporocysts nor excystation. Grinding oocysts for 30 sec in a motor-driven, teflon-coated tissue grinder caused motility of some sporozoites within sporocysts but did not result in excystation.     

Factors Affecting Motility and Morphology of Cryptosporidium Sporozoites In Vitro1

In vitro motility and morphology of Cryptosporidium sporozoites were examined in the presence of various solutions. Crude preparations of the bile salt, taurocholic acid, maintained both motility and morphology in a dose-dependent manner. These effects appeared to be due to the taurocholic acid itself, and not simply due to pH variations, osmotic factors, or contaminants. Lysis of sporozoites was also observed and was found to be dependent on pH, with acidic conditions (pH < 6.2) triggering the lysis.     

Malaria Sporozoites Leave Behind Trails of Circumsporozoite Protein During Gliding Motility1

As Plasmodium sporozoites undergo gliding motility in vitro, they leave behind trails of circumsporozoite (CS) protein that correspond to their patterns of movement. This light microscopic observation was made using Plasmodium berghei sporozoites, a monoclonal antibody (MAb H4) directed against the immunodominant repetitive epitope of the CS protein of P. berghei, and an immunogold-silver staining (IGSS) technique. Sporozoites pretreated with agents that inhibit sporozoite motility and invasiveaess did not produce trails. Sporozoites that glided on microscope slides coated with MAb H4 left behind considerably longer CS prolem trails than those on uncoated slides, and the staining of these trails was more intense. The fact that the CS protein is an exoantigen continuously released as trails by motile sporozoites, together with our previous finding that anti-CS protein antibodies inhibit sporozoite motility, strongly suggests that the CS protein plays a role in gliding motility. The sensitive IGSS technique used in this study may be a useful tool in the study of the translocation of surface proteins during gliding of other apicomplexans, other protists, and bacteria.     

Factors influencing ascorbate free radical formation

Several constituents of serum and related substances were examined in order to find factors influencing ascorbate radical formation. Human ceruloplasmin and albumin catalyzed singly ascorbate oxidation and caused a remarkable increase in ESR intensity of the ascorbate radical. Although, however, the combination of these two factors showed synergic effects on catalysis of ascorbate oxidation, the radical intensity significantly decreased. Fibrinogen and fetuin showed inhibitory effects on catalysis of ascorbate oxidation, whereas transferrin, citric acid, or other related substances exhibited no effect. A new factor which inhibited ascorbate oxidation was found in a serum fraction. These results indicate there is a counterbalanced equilibrium in the redox process of ascorbate in serum and the intensity of ascorbate radical is influenced by the summation of the complicated effects of many factors.     

The Alveolin IMC1h Is Required for Normal Ookinete and Sporozoite Motility Behaviour and Host Colonisation in Plasmodium berghei

Alveolins, or inner membrane complex (IMC) proteins, are components of the subpellicular network that forms a structural part of the pellicle of malaria parasites. In Plasmodium berghei, deletions of three alveolins, IMC1a, b, and h, each resulted in reduced mechanical strength and gliding velocity of ookinetes or sporozoites. Using time lapse imaging, we show here that deletion of IMC1h (PBANKA_143660) also has an impact on the directionality and motility behaviour of both ookinetes and sporozoites. Despite their marked motility defects, sporozoites lacking IMC1h were able to invade mosquito salivary glands, allowing us to investigate the role of IMC1h in colonisation of the mammalian host. We show that IMC1h is essential for sporozoites to progress through the dermis in vivo but does not play a significant role in hepatoma cell transmigration and invasion in vitro. Colocalisation of IMC1h with the residual IMC in liver stages was detected up to 30 hours after infection and parasites lacking IMC1h showed developmental defects in vitro and a delayed onset of blood stage infection in vivo. Together, these results suggest that IMC1h is involved in maintaining the cellular architecture which supports normal motility behaviour, access of the sporozoites to the blood stream, and further colonisation of the mammalian host.     

Immunolocalization of an enterotoxic glycoprotein exoantigen on the secretory organelles of Cryptosporidium parvum sporozoites

In this study, the fine ultrastructures of the secretory organelles of C. parvum sporozoites were demonstrated using transmission electron microscopy (TEM). Meanwhile, a previously identified enterotoxic 18-20 kDa copro-antigen (18-20 kDa CCA), associated with cryptosporidiosis in both human and calves, was isolated and immunolocalized on C. parvum sporozoites. Using immunoelectron microscopy and anti-18-20 kDa monospecific antibody demonstrated marked existence of the 18-20 kDa CCA on the apical organelles and at the trilaminar pellicles. An anterior extrusion-of this protein was demonstrated around the excysted and released sporozoites. However, non excysted sporozoites did not show this protein. Affinity blotting, with biotinylated jacalin, demonstrated the O-linked oligosaccharide moiety of this protein. The potential role of this protein in the host cell invasion and/or gliding motility remains unelucidated. However, its enterotoxicity, location and secretory nature suggest that it may be a target for neutralization or invasion inhibition of Cryptosporidium.     

Separation of growth-promoting activity for human muscle cells from fetuin.

Whether the growth-promoting activity of Pedersen fetuin is due to fetuin itself or to a contaminant(s) has been a long-standing puzzle. The possibility that the growth-promoting activity of Pedersen fetuin for human muscle satellite cells (HMSC) could be caused by some other component of fetal bovine serum (FBS) that remained in the fetuin as a contaminant has been investigated. One liter of FBS was first precipitated with 50% saturated ammonium sulfate, which leaves the serum albumin in solution, and then with 25% polyethylene glycol, which leaves the fetuin in solution, to generate a fraction 50 PEG 2x that was enriched 11-fold in growth-promoting activity for HMSC, with 68% recovery of total activity. Further purification with FPLC anion exchange chromatography achieved 99-fold enrichment of the activity with 30% overall recovery. The activity is heat labile and pH sensitive, suggesting that it is of protein nature, and the size of the activity is above 70 kDa. SDS-PAGE of the most active fractions shows that they are virtually free of fetuin. Thus, although the active fractions are not homogeneous, these studies demonstrate that the growth-promoting activity for HMSC can be fully separated from fetuin.     

Effects of intestinal contents from normal and immunized mice on sporozoites of Eimeria falciformis

The interaction of Eimeria falciformis sporozoites with the intestinal epithelium and with the intestinal contents from the cecum and colon of normal and specifically immunized mice was studied by light (LM) and scanning electron (SEM) microscopy. Fecal (FM) and enterocyte-associated (EAM) mucus were removed from the cecum and colon of normal mice and mice that had been immunized 1, 6, 12, or 20 days earlier with a series of oral inoculations of E. falciformis oocysts. Sporozoite-specific IgA, but neither IgM nor IgG, was detected by the immunofluorescent antibody test in FM and EAM from immunized mice. No sporozoite-specific immunoglobulin was detected in normal mice. When examined by LM, sporozoites exposed to all FM and EAM preparations exhibited greater motility and excystation from sporocysts. At 4 h after incubation in FM or EAM from normal or immune mice, about 10% of the sporozoites appeared damaged, being non-motile and non-refractile. Immune FM and EAM caused agglutination of sporozoites and sporocysts and oocyst walls of E. falciformis, but did not agglutinate those of E. ferrisi. Scanning electron microscopy of in vitro interactions between E. falciformis sporozoites and intestinal contents revealed that sporozoites exposed to immune EAM were coated with particulate material whereas those exposed to normal EAM were relatively clean. Sporozoites exposed to immune FM were usually embedded within the mucus whereas those exposed to normal FM were situated on top of the mucus. No significant differences occurred between the length/width (L/W) ratios of sporozoites incubated in normal and EAM or in PBS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteins in the amniotic and allantoic fluids of fetal pigs

Seven proteins were identified in the amniotic and allantoic fluids of fetal pigs (Sus scrofa domesticus) using crossed immunoelectrophoresis: albumin, fetuin, transferrin, alpha-fetoprotein (AFP), alpha 1-acid glycoprotein, alpha 1-antitrypsin and alpha 2-macroglobulin. The total protein concentrations were determined by the method of Bradford and individual protein concentrations by radial immunodiffusion or rocket immunoelectrophoresis. Transferrin and fetuin were the major proteins in amniotic fluid during the second trimester of gestation and together with AFP and albumin accounted for the majority of the total protein in amniotic, but not allantoic fluid.     

Plasmodium berghei: energy metabolism of sporozoites.

The energy metabolism of Plasmodium berghei sporozoites was studied by using their motility as an indicator of energy production and consumption. Sporozoites suspended in medium without sugars or amino acids ceased to move. Motility was restored by the addition of any of several sugars or amino acids to the medium. Inhibition of sporozoite motility, under otherwise favorable conditions, was induced by fluoride, malonate, cyanide, amytal, rotenone, antimycin A, arsenate, 2,4-DNP, and diphenylamine. The results suggest that these sporozoites utilize glycolysis, the Krebs' cycle, and conventional electron transport through the cytochrome chain.