Internal transport and metabolism of cobalamin in Spirometra mansonoides (Cestoda) spargana

The anterior 2 mm of the sparganum of Spirometra mansonoides contained 35 to 48% of the total endogenous cobalamin (Cbl) with only 2 to 4% in the terminal 1 cm. [57Co]cobalamin taken up in the posterior region of spargana was transported directionally to the anterior region and concentrated there resulting in a uniform depletion of [57Co]Cbl along the remaining body length. The anterior 2 mm contained 24% of the sparganum's holo-MMCoAM activity with only 5% in the terminal 1 cm. When head and body preparations were chromatographed on Sephadex G-150 columns eluates from both regions exhibited single peaks of MMCoAM activity at an approximate molecular weight of 150,000 and two peaks of endogenous Cbl. The first Cbl peak cochromatographed with MMCoAM activity and the second Cbl peak eluted at the position of free Cbl. Spargana rapidly converted CN-[57Co]Cbl to hydroxocobalamin (OH-Cbl) and adenosylcobalamin (Ado-Cbl) in vitro. In the head region significant amounts of Ado-Cbl were present (25%) although OH-Cbl was the predominant form of Cbl (45%) after 264 hr in a Cbl-free medium. In the body Ado-Cbl was the predominant form of Cbl (44%) although significant amounts of OH-Cbl were present (27%) after 264 hr in a Cbl-free medium. No methylcobalamin (Me-Cbl) was detected, but an unidentified cobalamin-containing entity (Fraction 4) was present. Based on these results we propose that the sparganum takes up cobalamin, concentrates it in the anterior, "head" region, sequentially metabolizes it to the hydroxyl then the adenosyl forms, and that the adenosylated form is used in this region of high, anaerobic metabolism as an obligatory coenzyme for MMCoAM, in coordination with the differentiation of the sparganum to the adult cestode.     

Comparison of isozyme patterns between Spirometra erinacei and Spirometra mansonoides by isoelectric focusing.

No differences were observed in the isozyme patterns of 4 enzymes examined between fresh samples stored at -80 C and samples stored at room temperature for 10 days after lyophilization, which supports the validity of comparing lyophilized samples to fresh frozen tissue. Mature proglottids as well as plerocercoids of Spirometra erinacei from Japan and Australia were indistinguishable by comparison of isozyme patterns after isoelectric focusing. The isozyme patterns of acid phosphatase, glucosephosphate isomerase (GPI), and mannosephosphate isomerase from plerocercoids of Spirometra mansonoides were distinctly different from those of plerocercoids of S. erinacei. The adenylate kinase isozyme patterns of the mature proglottids of S. mansonoides were also distinctly different from those of the mature proglottids and the plerocercoids of S. erinacei. The GPI isozyme pattern of the mature proglottids of S. mansonoides was also distinguishable from the GPI patterns of those of S. erinacei. These electrophoretic data suggest that the S. erinacei from Japan and Australia are closely related, if not identical, but that S. mansonoides is genetically distinct from S. erinacei.     

Spirometra mansonoides: effects of plerocercoid infection on glycogen deposition in rats

The effects of infection with plerocercoids of Spirometra mansonoides on tissue glycogen deposition of rats was determined. Hypophysectomized rats infected for two days had higher liver glycogen concentrations than controls and this effect was greatest after one week. Elevated liver glycogen associated with plerocercoid infection was observed in fed animals both at the beginning and at the end of the light period as well as after an overnight fast. Glycogen phosphorylase (1,4 alpha D glucan: orthophosphate alpha glucosyltransferase EC 2.4.1.1.) was inhibited but glucose-6-phosphatase (EC 3.1.3.9) was unaffected in the livers of infected hypophysectomized rats. While this effect is similar to actions of both growth hormone and insulin, plerocercoid infection had no influence on glycogen of cardiac or skeletal muscle at any time. Plerocercoid infection had no effect on the glycogen concentration of any tissue of intact rats.     

Insulin-like effects of fatty acid synthesis in liver of hamsters infected with plerocercoids of the tapeworm, Spirometra mansonoides

Elevated serum lipids are associated with infections of laboratory rodents with plerocercoids of Spirometra mansonoides. The effect of infection with these larval tapeworms on triglyceride degradation and hepatic de novo fatty acid synthesis was investigated in Syrian hamsters. Serum lipoprotein electrophoresis revealed a consistent elevation in very low density lipoproteins in the infected animals. Lipoprotein lipase activity was enhanced in the infected animals. After seven days of plerocercoid infection the activity of acetyl-CoA carboxylase (E.C. 6.4.1.2) was significantly elevated after 6, 12 and 18 hours of fasting. Fatty acid synthetase was significantly increased after 0, 6, 12, 18 and 24 hours of fasting. Therefore, a chronic insulin-like activity on lipid metabolism of hamsters is associated with plerocercoid infection.     

The growth hormone-like factor produced by the tapeworm Spirometra mansonoides specifically binds receptors on cultured human lymphocytes

Plerocercoid larvae of the tapeworm Spirometra mansonoides produce a factor with activities similar to those of growth hormone (GH). Highly selective receptors for GH have been described on cultured human lymphocytes (IM-9 cells) and these cells have been used as a model of binding essentially restricted to human GH (hGH). We compared the displacement of [125I]hGH by hGH and partially purified plerocercoid growth factor (PGF) in assays using rabbit hepatic membranes and IM-9 cells. PGF displaced [125I]hGH from both rabbit hepatic membranes and IM-9 cells in a dose-dependent manner (r greater than 0.98). These results show that PGF specifically binds to hGH receptors on human IM-9 cells and suggest the possibility that PGF will have somatotropic activity in humans.     

Survey of Spirometra erinaceieuropaei in frogs in Taiwan and its experimental infection in cats

Eighteen of 56 (32.1%) wild Rana limnocharis from central and south Taiwan were found to contain plerocercoids of Spirometra erinaceieuropaei. This is the first report of S. erinaceieuropaei infections in frogs in Taiwan, with the plerocercoids being recovered from the thigh and back muscles or under the skin. Other species of frogs examined, including nine wild R. latouchii, one wild Buergeria robustus and 110 cultured R. rugulosa were free of infection. The plerocercoids were orally inoculated into four cats; three of which were each given a single plerocercoid and one a dose of three plerocercoids. Daily faecal examination showed that two cats started shedding eggs of S. erinaceieuropaei on day 8 postinfection (PI) and the other two on day 10 PI. The highest eggs per gram and eggs per day for a single worm was found to be 428,000 and 14,416,000 respectively. Only the cat inoculated with three plerocercoids shed proglottids in its faeces during the 2 month observation period.     

Insulin-like effects in the rat of the purified growth factor from Spirometra mansonoides plerocercoids

The acute effects of injections of the human growth hormone-like factor purified from plerocercoids of the tapeworm Spirometra mansonoides on carbohydrate, lipid, and protein metabolisms were determined in intact rats. Male rats were injected ip with saline, insulin, or various doses of partially purified PGF. The rats injected with insulin had significantly reduced serum glucose concentrations but no dose of PGF caused a change in serum glucose levels. Insulin and PGF stimulated [14C]glucose and [14C]leucine oxidation to 14CO2 in adipose tissue and muscle and increased incorporation of both [14C]glucose carbons into lipids and [14C]leucine into protein in fat and muscle. The responses to PGF were dose-dependent and persisted after 3 hr of incubation in vitro. Injections of naloxone prior to injecting PGF to block the stress response did not prevent the stimulation of insulin-like responses by PGF. Therefore, PGF has intrinsic insulin-like activities in normal male rats.     

Interaction between transcobalamin II and immobilized Cibacron Blue F3GA

Human serum transcobalamin II (TC II), a vitamin B₁₂ (Cbl) transport protein, complexes with Cibacron Blue F3GA, a reactive blue dye which can bind to proteins that require nucleotides as cofactors. Apo-TC II and holo-TC II both bind, but intrinsic factor (IF) and R-type binders of Cbl do not. Other mammalian species TC II also complex with the dye. Greater than 87% of the applied TC II-CN-[⁵⁷Co]Cbl remains bound to the dye even at pH 4.0. At pH values below this, the CN-[⁵⁷Co]Cbl dissociates off TC II which remains bound to the dye. High salt concentrations will break the TC II-dye complex. Ionic forces were considered not to be involved since complexing also occurred at pH 9.0, 2.5 pH units above the isoelectric point of TC II. Failure to dissociate the TC II-dye complex with 50% glycerol makes hydrophobic interactions unlikely. In addition to the potential uses of TC II-Cibacron Blue F3GA complexes in a total scheme for protein purification, the possibility that TC II is a nucleotide-requiring protein should be explored.     

Evidence of the growth factor in mouse serum infected with Spirometra erinacei plerocercoids

To investigate the action of the growth factor secreted by Spirometra erinacei plerocercoids, various organ weights, body weight and head-body length were measured in Snell normal and dwarf mice after injection with the serum from mice and rats. Serum from mice infected with the plerocercoids caused significant increases in the weights of the liver and spleen, in the same manner as mice infected with the plerocercoids. However, serum from rats infected with plerocercoids did not cause significant changes in these parameters. The growth factor in the serum of mice infected with plerocercoids was stable at -20 degrees C for at least 6 months and easily passed through the peritoneum.     

Functional expression of intrinsic factor-cobalamin receptor by renal proximal tubular epithelial cells.

Previous studies from our laboratory (Seetharam, B., Levine, J. S., Ramasamy, M., and Alpers, D. H. (1988) J. Biol. Chem. 263, 4443-4449; Fyfe, J. C., Ramanujam, K. S., Ramaswamy, K., Patterson, D. F., and Seetharam, B. (1991) J. Biol. Chem. 266, 4489-4494) have identified and isolated a 230-kDa receptor from rat and canine kidney which binds with high affinity [57Co]cyanocobalamin (Cbl) complexed to gastric intrinsic factor (IF). Although these studies have identified a renal receptor which binds intrinsic factor-cobalamin (IFCR), it is not known whether the binding is specific for IF-Cbl and whether renal cells internalize [57Co]Cbl bound to IF and transport [57Co]Cbl across the cell. Using a variety of renal cells, our results show that IF-[57Co]Cbl binding activity is detected in proximal tubular-derived epithelial cells from opossum (OK) and porcine kidney (LLC-PK1) but not in distal tubular-derived cells from canine kidney cells (MDCK). Metabolic labeling studies with Tran 35S-label confirmed the presence of a 230-kDa IFCR in OK and LLC-PK1 cells. Cell surface labeling and binding studies demonstrated that IFCR is targeted to the apical membrane. This apical expression of IFCR in OK cells is inhibited by the microtubule-disruptive drugs, colchicine and nocodazole. Opossum kidney cells when grown on culture inserts are polarized and transport [57Co]Cbl only when bound to IF and not to other Cbl binders. Furthermore, the transport of [57Co]Cbl occurred unidirectionally from the apical to the basolateral surface. Treatment of cells with colchicine or nocodazole inhibited the surface binding of IF-[57Co]Cbl as well as the transcytosis of [57Co]Cbl by 70-75%. IFCR retained intracellualarly by incubation of cells with colchicine or nocodazole is degraded by leupeptin-sensitive proteases. Based on these results, we suggest that proximal tubular-derived epithelial cells transport [57Co]Cbl bound to IF in a saturable way via receptor-mediated endocytosis.     

Leupeptin and ammonium chloride inhibit intrinsic factor mediated transcytosis of [57Co]cobalamin across polarized renal epithelial cells.

The [125I] intrinsic factor (IF) mediated transcytosis of [57Co]Cyanocobalamin (Cbl) by polarized opossum kidney cells was inhibited (greater than 80%) by preincubation of the cells with lysosomotropic agents leupeptin or ammonium chloride. Inhibition of Cbl transcytosis resulted in the intracellular accumulation of both [125I]IF (48 kDa) and [57Co]Cbl. Intracellular degradation of [125I]IF occurred during normal cellular transcytosis of [57Co]Cbl and in one h following internalization the major intracellular degradation products of IF were two polypeptides of Mr 29 kDa and 19 kDa. The size of the major degradation product of IF in the basolateral media was 10 kDa. Based on these results, we suggest that IF is internalized by the renal epithelial cells and is degraded by leupeptin-sensitive acid proteases during Cbl transcytosis.     

Renal brush border membrane bound intrinsic factor

A highly active receptor for intrinsic factor (IF)-cobalamin (Cbl) complex has been detected and reported in mammalian kidney earlier (Seetharam, B., et al. (1988) J. Biol. Chem. 263, 4443-4449). The physiological role of this receptor in normal Cbl homeostasis is not known. In addition to binding of exogenously added IF-[57Co]Cbl, the renal apical membranes contain endogenous IF or IF-Cbl. Washing with pH 5/EDTA buffer enhanced the binding of exogenously added IF-[57Co]Cbl to renal apical but not basolateral membranes. The pH 5/EDTA extract from renal apical membranes bound [57Co]Cbl. The complex also bound to rat ileal brush border membrane and promoted ileal transport of [57Co]Cbl. On immunoblots using monospecific antiserum to IF a 62 kDa protein was identified in renal and intestinal apical membranes, serum and in tissue extracts of unperfused rat liver, kidney and heart. The 62 kDa band was eliminated from the renal apical membranes following pH 5/EDTA wash. Rat urine demonstrated unsaturated [57Co]Cbl binding (0.2 to 0.4 pmol/day) of which only 30-40% was immunoprecipitated with anti IF and could be identified on immunoblots. The identification of IF in rat renal apical membranes (160-200 ng/mg protein) and secretion of only traces of IF in urine suggest that the renal IF-Cbl receptor may play a role in sequestering IF/IF-Cbl and prevent urinary loss of Cbl.     

Some biochemical effects of the growth hormone analogue produced by plerocercoids of the tapeworm Spirometra mansonoides on carbohydrate metabolism of adipose tissue from normal, diabetic, and hypophysectomized rats

Plerocercoids of Spirometra mansonoides produce a functional analogue of mammalian growth hormone (GH). Plerocercoid growth factor (PGF) mimics the growth-promoting actions of GH, but has not been shown to duplicate all of the actions reported for GH. The purpose of this study was to determine the effects of plerocercoid infection (chronic PGF treatment) on glucose metabolism of adipose tissue and to compare the effects to those elicited by insulin and GH in intact, diabetic, and hypophysectomized male rats. Groups of rats were constantly exposed to PGF (via plerocercoid infection) or injected twice daily with bovine GH, insulin, or saline for 10 days. Basal oxidation rates of [U-14C]glucose to 14CO2 in adipose tissue segments were measured in vitro immediately after tissue removal. Other aliquots of adipose tissue were preincubated in hormone-free medium for 3 hr prior to testing the ability of the tissue to respond to insulin or human GH (hGH) added in vitro. Adipose tissue from PGF-treated intact and hypophysectomized rats had significantly elevated basal glucose oxidation rates, and the tissue was sensitive to further stimulation by insulin or hGH. The results obtained with intact and hypophysectomized rats were essentially the same, indicating that the effects of PGF were not due to suppression of endogenous GH. The basal glucose oxidation rate in adipose tissue from diabetic rats was stimulated (P less than 0.01) by PGF, but the tissue was not sensitive to insulin added in vitro. Furthermore, PGF had no effect on body growth or blood glucose concentrations of diabetic rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

The growth factor from plerocercoid larvae of the tapeworm, Spirometra mansonoides, stimulates growth but is not diabetogenic

A factor produced by plerocercoids of the tapeworm Spirometra mansonoides is similar to human growth hormone (hGH) in that it stimulates body growth, binds to hGH receptors, cross-reacts with anti-hGH antibodies, and has lactogenic and insulin-like activities. The purpose of this study was to determine whether plerocercoid growth factor (PGF) is similar to hGH in expressing diabetogenic activity in the genetically obese (ob/ob) mouse. To determine an effective dose for use in the obese mice, the ability of daily injections of PGF to stimulate growth of phenotypically normal mice of the same strain was assessed in a 10-day weight gain assay. Injections of PGF stimulated a dose-dependent weight gain (r = 0.83) and 25 ng eq/day of PGF stimulated a response not significantly different from that produced by 100 micrograms of bovine growth hormone/day. Diabetogenicity was assessed using fasting blood glucose and glucose tolerance tests in obese mice that had been injected for 3 days with saline, hGH, or PGF. Human growth hormone caused a significant increase (P less than 0.005) in fasting blood glucose and glucose tolerance of the obese mice was impaired (P less than 0.01). All of the doses of PGF used to test diabetogenicity in the obese mice were at least twice that required to stimulate a maximal growth response in normal mice, yet none of the doses of PGF increased fasting blood glucose or decreased glucose tolerance. These results show that PGF was a potent growth stimulant but was not diabetogenic.     

Perturbation of neuronal cobalamin transport by lysosomal enzyme inhibition

Cbl (cobalamin) utilization as an enzyme cofactor is dependent on its efficient transit through lysosomes to the cytosol and mitochondria. We have previously proposed that pathophysiological perturbations in lysosomal function may inhibit intracellular Cbl transport with consequences for down-stream metabolic pathways. In the current study, we used both HT1080 fibroblasts and SH-SY5Y neurons to assess the impact that protease inhibitors, chloroquine and leupeptin (N-acetyl-L-leucyl-L-leucyl-L-argininal), have on the distribution of [⁵⁷Co]Cbl in lysosomes, mitochondria and cytosol. Under standard cell culture conditions the distribution of [⁵⁷Co]Cbl in both neurons and fibroblasts was ~5% in lysosomes, 14% in mitochondria and 81% in cytosol. Treatment of cells with either 25 μM chloroquine or 40 μM leupeptin for 48 h significantly increased the lysosomal [⁵⁷Co]Cbl levels, by 4-fold in fibroblasts and 10-fold in neurons, and this was associated with reduced cytosolic and mitochondrial [⁵⁷Co]Cbl concentrations. Based on Western blotting of LAMP2 in fractions recovered from an OptiPrep density gradient, lysosomal Cbl trapping was associated with an expansion of the lysosomal compartment and an increase in a subpopulation of lysosomes with increased size and density. Moreover, the decreased mitochondrial Cbl that was associated with lysosomal Cbl trapping was correlated with decreased incorporation of [¹⁴C] propionate into cellular proteins/macromolecules, indicating an inhibition of Cbl-dependent Mm-CoA (methylmalonyl-coenzyme A) mutase activity. These results add support to the idea that lysosomal dysfunction may significantly impact upon Cbl transport and utilization.     

Cobalamin metabolism in cultured human chorionic villus cells

Cobalamin (Cbl, vitamin B₁₂) metabolism was analyzed in cultures of human chorionic villus (CV) cells obtained at 9–10 weeks of gestation. CV cells were shown to synthesize transcobalamin II (TCII) and to possess a high affinity receptor for that molecule. The cells bound and internalized radioactive cyanocobalamin (CN[⁵⁷Co]Cbl) complexed to TCII. This internalized CN[⁵⁷Co]Cbl was found to be converted to both methylCbl and adenosylCbl, the two intracellular coenzyme forms of Cbl, and bound to the two known intracellular Cbl requiring enzymes, methionine synthase (MS) and methylmalonyl-CoA mutase. Both enzyme systems were found to be functional in the intact cell by demonstrating the incorporation of the radioactive label from both [¹⁴C]CH₃-tetrahydrofolate and [¹⁴C]propionate into acid insoluble products. MS activity was also detected in lysed cell material. CV cells were shown not to be auxotrophic for methionine since they were able to utilize homocysteine in place of methionine for cell division. Since CV cells are capable of performing many of the complex events associated with Cbl metabolism, it may be possible to use these cells to diagnose genetic defects of Cbl metabolism. © 1993 Wiley-Liss, Inc.     

Biological effects of Spirometra erinacei plerocercoids in several species of rodents

Observations were made of the biological effects on infection with plerocercoids of Spirometra erinacei on normal female Snell mice, male chinese hamsters, golden hamsters, normal and hypox rats. Plerocercoid infection caused the strongest growth-promoting effect on normal Snell mice. In mice, this effect appears to be independent of strain. Chinese hamsters infected with these larvae showed similar growth. The infected normal rats and golden hamsters, however, showed a weight increase in the skeletal muscle only, while the hypox rats exhibited no effect at all. The elevation in the concentration of serum triglyceride was observed in all the animals investigated except for rats. Golden hamsters, in particular, exhibited a marked increase in the concentration of serum free fatty acids and total cholesterol. There was close correlation between the concentrations of serum triglyceride and free fatty acids, and the regression coefficient of the resulting linear regression equation for the experimentals was higher than that for the controls. This suggests that serum triglyceride results from an increased concentration of serum free fatty acids derived from stimulated lipolysis. The total cholesterol concentration in the serum decreased in chinese hamsters infected with larvae. The serum glucose concentration increased in normal Snell mice but decreased in chinese and golden hamsters. No difference in glycerol and free fatty acid concentration was observed in infected animals except for golden hamsters.     

Chemical structures and immunolocalization of glycosphingolipids isolated from Diphyllobothrium hottai adult worms and plerocercoids.

Glycosphingolipids (GSLs) were purified from adults and plerocercoids of the tapeworm Diphyllobothrium hottai, and their chemical structures were determined. Total lipid fractions prepared from chloroform/methanol extracts of whole tissues were fractionated successively on ion-exchange chromatography, silicic acid column chromatography, and preparative TLC. The purified GSLs were characterized by methylation analysis, TLC-immunostaining, liquid secondary ion MS, MALDI-TOF MS, and 1H-NMR. Ten GSLs were isolated from adult worms and four from plerocercoids, comprising mono-, di-, tri-, tetra-, and pentasaccharides. The GSL Gal beta 1-4(Fuc alpha 1-3)Glc beta 1-3Gal beta 1-Cer was found in adult worms but not in plerocercoids, whereas Ga lbeta 1-4 (Fuc alpha 1-3)Glc beta 1-3(Gal beta 1-6)Gal beta 1-Cer was found in both adult worms and plerocercoids. We previously found a similar series of GSLs in plerocercoids of the cestode Spirometra erinaceieuropaei, and termed them 'spirometosides'[Kawakami, Y. et al. (1996) Eur J. Biochem. 239, 905-911]. The core structure of spirometosides, Gal beta 1-4Glc beta 1-3 Gal beta 1-Cer, may have taxonomic significance, being characteristic of pseudophyllidean tapeworms. In the present study, GSL compositions were significantly different between adults and plerocercoids, and growth-dependent changes in composition were documented. We found a novel dihexosylceramide, Glc beta 1-3Gal beta 1-Cer, which is a possible precursor for spirometosides. Immunohistochemical examination showed that spirometoside GSLs are highly enriched in the inner surface of bothria, the major point of contact between the adult worm and the host's intestine. Our findings indicate that spirometosides are involved in host-parasite interaction.     

Hymenolepis diminuta: ultrastructural abnormalities in worms from C57 mice

Ultrastructural studies, including stereological analyses of micrographs, have been made of five-worm primary infections of Hymenolepis diminuta from C57 mice to determine whether the immune destrobilation/rejection process was accompanied by significant changes in the fine structure of the scolex tegument. Destrobilation/rejection of worms occurred from Days 9-12 after infection. For the first 5 days after infection, the scolex tegument showed no detectable differences in ultrastructure compared with that of "control" worms from either Wistar rats or immunosuppressed C57 mice. By Day 6, large lipid deposits were observed in the tegument and associated musculature of worms from untreated C57 mice. Further, worms recovered from Days 6-8 after infection also showed increased activity of the Golgi apparatus, GER, and mitochondria of the tegument, resulting in increased numbers of discoidal secretory bodies. Concomitant with destrobilation/rejection from Day 9 was a drop in the number of secretory bodies, an increase in autophagic activity throughout the tegument, and a blistering of the tegument surface plasma membrane. The possible functional significance of the results is discussed in relation to host immunity.