Analysis of neutral glycosphingolipids from Trypanosoma brucei

Neutral glycosphingolipids (GSLs) were isolated from Trypanosoma brucei and analyzed by thin-layer chromatography (TLC), TLC/secondary ion mass spectrometry (TLC/SIMS), and liposome immune lysis assay (LILA). Three species of neutral GSLs, designated as N-1, -2, and -3 were separated on TLC. N-1 GSL migrated very close to glucosylceramide (GlcCer) and N-2 GSL showed the same mobility as lactosylceramide (LacCer). On the other hand, the mobility of N-3 GSL on the TLC plate was slower than globotetraosylceramide (Gb4). In order to characterize the molecular species of neutral GSLs from T. brucei, N-1, -2 and -3 GSLs were analyzed by TLC/SIMS. The TLC/SIMS analysis of N-1 of the parasites revealed a series of (M–H)⁻ ions from m/z 698 to 825 representing the molecular mass range of ceramide monohexoside (CMH) (GlcCer or galactosylceramide). On the other hand, the TLC/SIMS spectra of N-2 GSL revealed a series of (M–H)⁻ ions from m/z 944–987 indicating the molecular mass range of LacCer. In the TLC/SIMS analysis of N-3 GSL, however, the characteristic molecular ions that can elucidate the structure of N-3 GSL were not obtained. In order to confirm the results obtained from TLC/SIMS, N-1, -2, and -3, GSLs were tested by LILA with specific antibodies against GlcCer, LacCer, and Gb4, respectively. N-1 GSL had reactivity to anti-GlcCer antibody and N-2 GSL reacted with the antibody against LacCer. However, N-3 GSL was not recognized by anti-Gb4 antibody. Using anti-GlcCer and anti-LacCer antibodies, furthermore, we studied the expression of GlcCer and LacCer in T. brucei parasites. Both GlcCer and LacCer were detected on the cell surface of T. brucei.     

A Case of Spontaneous Malignant Hibernoma in a Crl:CD(SD)IGS Rat

A firm, tan, well-circumscribed mass that measured 25 × 30 × 35 mm was observed in the thoracic cavity of a 53-week-old male Crl:CD(SD) IGS rat. Histologically, the mass was encapsulated by fibrous tissue and contained fibrovascular septae. Tumor cells were compactly arranged, and most were oval to polygonal in shape with multivacuolated cytoplasm and a centrally located nucleus. In some parts of the tumor, marked cellular atypia and frequent mitoses were evident. Vacuoles in cytoplasm were positive for oil red O. The tumor cells were characterized ultrastructurally by abundant, round to oval mitochondria with transverse closely-packed cristae. Tumor cells were immunohistochemically positive for uncoupling protein 1 (UCP-1). Several thrombi and hemorrhagic or necrotic foci were also observed within the tumor mass. Vascular invasion of the tumor capsule was observed; however, invasion of surrounding tissues or metastases were not observed. Based on the pathology findings, this case was diagnosed as a malignant hibernoma.     

Differential glutathione S-transferase isozyme activities in rice and early watergrass seedlings

Glutathione S -transferase (GST) isozymes were investigated in one-leaf-stage rice ( Oryza sativa L. cv. Nipponbare) and early watergrass ( Echinochloa oryzicola Vasing) shoots after being induced by treatment with a combination of pretilachlor [2-chloro-2',6'-diethyl-N-(2-propoxyethyl)acetanilide] and fenclorim (4,6-dichloro-2-phenylpyrimidine) using DEAE-Sephacel anion exchange chromatography. Non-treated plants contained GST isozymes that had activity to the following substrates: three isozymes for l-chloro-2,4-dinitrobenzene (CDNB), six isozymes for pretilachlor and three isozymes for fenclorim in rice shoots; and four isozymes for CDNB, three or four isozymes for pretilachlor and two or three isozymes for fenclorim in early watergrass shoots. Glutathione S -transferase isozyme activities of non-treated plants were higher in rice than in early watergrass, especially in the case of GST activity with fenclorim as a substrate. Pretreatment of rice roots with a combination of pretilachlor and fenclorim increased the activity of the constitutively expressed isozymes that exhibited activity with CDNB, pretilachlor and fenclorim. This pretreatment also caused the appearance of one new GST(fenclorim) isozyme. Pretreatment of early watergrass roots with a combination of pretilachlor and fenclorim produced almost no increase in activity of some constitutively expressed isozymes that exhibited activity to CDNB and fenclorim, although it partly increased the peaks to corresponding to pretilachlor. The induction of GST was higher in rice than that in early watergrass. These results indicated that the isozyme pattern and substrate specificity of GST isozymes in rice were different from those in early watergrass. Furthermore, the selectivity of pretilachlor between rice and early watergrass may be related to different constitutively expressed GST(pretilachlor) isozyme activities and the induction of GST(pretilachlor) isozyme activities in the combination treatment.     

The effect of free and polyethylene glycol-liposome-entrapped albendazole on larval mobility and number in Toxocara canis infected mice

As part of our exploratory drug research on the larva migrans that causes roundworm in dogs and cats, this study was carried out to clarify the effect of free and liposome-entrapped (LE) albendazole in Toxocara canis infected mice. In infected mice, evaluation of mobility and number of larva were examined in detail in the brain, skeletal muscle and liver. Larva mobility was evaluated by using the relative mobility (RM) value. Albendazole was LE as one of the drug delivery systems (DDSs). Polyethylene glycol (PEG) was added to the liposome in order to avoid evoking a response by the reticuloendothelial system (RES). By using the albendazole PEG-LE delivery system, it was possible to target the larvae in the mouse brain and liver resulting in a decrease in the number of larvae. In the skeletal muscle of the infected mice, the intraperitoneal dosages of PEG-LE albendazole did not cause a complete decrease in the number of larvae, even though free albendazole did cause the number to decrease. Therefore, it is necessary to take into consideration the migrating stage of the larvae before the initiation of any drug administration.     

Induction of immune responses against glycosphingolipid antigens: comparison of antibody responses in mice immunized with antigen associated with liposomes prepared from various phospholipids

The immune responses of mice against glycosphingolipid (GSL) antigens and the effect of the phospholipid composition of liposomes on the immunogenicity in mice of liposome-associated GSL antigens were examined. The immunization with GSL antigen alone was unable to induce any detectable anti-GSL antibody responses. On the other hand, the immune responses against GSL antigens were detected after immunization with liposomes composed of dipalmitoylphosphatidylcholine (DPPC) (0.5 micromol), cholesterol (Chol) (0.5 micromol), Salmonella minnesota R595 lipopolysaccharides (LPS) (10 microg) and GSL (0.05 micromol) (DPPC-liposome). However, the administration with liposome composed of dimyristoylphosphatidylcholine (DMPC) (0.5 micromol), Chol (0.5 micromol), S. minnesota R595 LPS (10 microg) and GSL (0.05 micromol) and with liposomes composed of distearylphosphatidylcholine (DSPC) (0.5 micromol), Chol (0.5 micromol), and S. minnesota R595 LPS (10 microg) and GSL (0.05 micromol) was ineffective for the induction of the immune responses against GSL antigens. These results suggest that DPPC-liposome would serve effectively as a delivery vehicle for inducing immune responses against GSL antigen.     

Macroscopic study of the functional significance of the forearm muscles in the giant panda

The extensor and flexor group muscles and their related muscles were functional-morphologically observed in the dead body of the giant panda to clarify the action of the forearm and the palm in the manipulation of the species. The Musculus flexor carpi ulnaris had two developed heads, however, we can conclude that the contraction of this muscle slightly changes the angle of the accessory carpal bone to the ulna. The data pointed out that the accessory carpal bone acts as a supporting post, when the giant panda seizes the object. The M. abductor digiti I longus possessed the well-developed origin in both ulna and radius. These findings suggest that this muscle may function as a supinator of the forearm. We also suggest that the well-developed M. pronator quadratus and M. pronator teres, and the proximal part of the M. abductor digiti I longus and the M. supinator may efficiently contribute to the pronator-spinator action of the forearm, when the giant panda brings the food to its mouth using the manipulation system equipped in the palm region.     

Development of Peyer's patch and cecal tonsil in gut-associated lymphoid tissues in the chicken embryo

It is well known that chicken B cells develop in the bursa of Fabricius (BF), which is categorized as gut-associated lymphoid tissue (GALT). Chicken GALT also includes Peyer's patch (PP) and cecal tonsil (CT). The relationship between these tissues in GALT during B cell development is currently unknown. In this study, we conducted comparative examination of PP, CT and BF development during embryogenesis using immunohistochemical staining. On day 13 of embryogenesis (E13), accumulation of MHC class II(+) cells was observed in the intestine. Thereafter, Bu-1(+) cells and IgM(+) cells appeared, and their number continuously increased at the same sites where MHC class II(+) cells were present. Similar results were obtained in the CT. The locations of embryonic PP were limited to two sites; near the Meckel's diverticulum and the ileocecal junction. Anlage of bursal follicles first appeared at E13 and developed thereafter. Immigration of Bu-1(+) cells to bursal follicles began at E13, and the number of Bu-1(+) cell subsequently increased. When the follicle of BF was eliminated from the embryo by treatment with testosterone, development of PP and CT were observed. We concluded therefore that the development of PP and CT start during late embryogenesis at the same time as the follicle of BF, and that appearance of surface IgM(+) cells in PP and CT is independent form the development of the follicle of BF.     

Redescription of Pterygodermatites (Mesopectines) nycticebi (Mönnig, 1920) (Nematoda: Rictulariidae), a parasite of slow loris Nycticebus coucang (Mammalia: Primates)

Pterygodermaties (Mesopectines) nycticebi (M?nnig, 1920) (Nematoda: Spirurida: Rictulariidae) is redescribed based on immature and mature adults collected from the stomach and small intestine at autopsy of a slow loris, Nycticebus coucang (Boddaert, 1785) (Mammalia: Primates), in a zoological garden in Japan. It is first demonstrated that male possesses a minute telamon and a left lateral pore in the preanal part of body. The cause of death of the slow loris is strongly surmised to be related to the nematode infection, which was apparently acquired under captivity in the zoological garden.     

Seroepidemiologic studies on Babesia caballi and Babesia equi infections in Japan

Antibodies to Babesia caballi and Babesia equi were examined on a total of 2,019 horse serum samples that had been collected in 1971-1973 by the National Institute of Animal Health by enzyme-linked immunosorbent assay (ELISA) using recombinant proteins and by Western-blot analysis. Based on the criterion for positivity by ELISA, 5.4% (109/2,019) and 2.2% (44/2,019) had antibodies against B. caballi and B. equi, respectively. The ELISA-positive sera were further examined by Western blot; 30/109 for B. caballi and 2/ 44 for B. equi were positive for native B. caballi or B. equi, but none of them was seropositive for both infections. Based on the results of this study, further investigations should be required to survey horses that have arrived in Japan relatively recently and tick vectors of equine Babesia using ELISA with some recombinant protein, a parasite detection method in an in vitro culture of equine Babesia, and PCR testing.     

Cryopreservation of the semen collected by electroejaculation from the Hokkaido brown bear (Ursus arctos yesoensis)

Four adult Hokkaido brown bears were used as semen donors, and semen characteristics were examined before freezing and after thawing. A total of 10 electroejaculates were diluted with Tris-egg yolk extender and cooled to 4 degrees C over 90 min. Spermatozoa were equilibrated with 4.7% glycerol for 80 min. Semen packed in 0.25 ml plastic straws were frozen with liquid nitrogen vapor. Percentages (mean +/- SD) of motile and live sperm were 96+/-2 and 86.5+/-7.2% before freezing, and 43+/-5 and 67.4+/-3.9% after thawing, respectively. Although the number of progressively motile sperm after thawing varied among samples (1.8+/-1.2 x 10(8) cells/ejaculate), frozen semen in the present study might serve for artificial insemination.