Shinobu Watarai

A mutant form of the tax protein of bovine leukemia virus (BLV), with enhanced transactivation activity, increases expression and propagation of BLV in vitro but not in vivo.

ABSTRACT In a previous study, we identified an interesting mutant form of the Tax protein of bovine leukemia virus (BLV), designated D247G. This mutant protein strongly transactivated the long terminal repeat of BLV and was also able to transactivate the cellular proto-oncogene c-fos. This finding suggested that BLV that encode the mutant protein might propagate and induce lymphoma more efficiently than wild-type BLV. To characterize the effects of the strong transactivation activity of the mutant Tax protein, we constructed an infectious molecular clone of BLV that encoded D247G and examined the replication and propagation of the virus in vitro and in vivo. Cultured cells were transfected with the wild-type and mutant BLV, and then levels of viral proteins and particles and the propagation of viruses were compared. As expected, in vitro, mutant BLV produced more viral proteins and particles and was transmitted very effectively. We injected the wild-type and mutant BLV into sheep, which are easily infected with BLV, and monitored the proportion of BLV-positive cells in the blood and the expression of BLV RNA for 28 weeks. By contrast to the results of our analyses in vitro, we found no significant difference in the viral load or the expression of viral RNA between sheep inoculated with wild-type or mutant BLV. Our observations indicate that the mutant D247G Tax protein does not enhance the expansion of BLV and that there might be a dominant mechanism for regulation of the expression of BLV in vivo.

Antiglycolipid antibodies in normal and pathologic human sera and synovial fluids.

Antiglycolipid antibodies were measured in normal and pathologic sera and synovial fluids by means of a modified microplate method of complement‐mediated immune lysis of fluorescent dye‐trapped liposomes. All sera of normal subjects had antibodies against globopentaosylceramide (IV3 GalNAcGbOse4Cer), ganglioside GMI, gangliotriaosylceramide, gangliotetraosylceramide, and galactosylneolactotetraosylceramide antigens. Most sera of normal subjects had antibodies against lactotriaosylceramide, N‐glycolylneuraminosyl‐neolactotetraosylceramide (NeuGcnLcOse4Cer), GM3 ganglioside with N‐glycolylneuraminic acid (NeuGcGM3) and GDIa antigens. Differences of titers against IV3GalNAcGbOse4Cer, neolactotetraosylceramide, NeuGcGM3 and NeuGcnLcOse4Cer antigens were observed between sera of normal subjects and pathologic sera from cases of leukemias, lymphomas, several autoimmune diseases and liver diseases.

Occurrence of GD3 ganglioside in reactive astrocytes — an immunocytochemical study in the rat brain

Immunocytochemical study of ganglioside GD3 (II3 alpha(NeuAc alpha 2-8NeuAc)-LacCer) was performed in the cold lesions produced in the cerebral cortex of the rat brain, using mouse IgM anti-GD3 monoclonal antibody (DSG-1). Seven and 15 days after cold lesioning, GD3-like immunoreactivity was observed in reactive astrocytes. Thirty and 50 days after cold lesioning, GD3-like immunoreactivity was observed in the cells that formed glial scars. Normal astrocytes were not immunoreactive. Therefore, it is possible that GD3 may play an important role in the astrocytic functions required for the process of repair of edematous lesions in the central nervous system.

IMMUNOHISTOCHEMICAL DEMONSTRATION OF GANGLIOSIDE GD3 IN THE CENTRAL NERVOUS SYSTEM

Immunohistochemical staining for GD3 in adult rat brain was carried out using mouse IgM anti-GD3 monoclonal antibody (DSG-1). Neuronal cells in the cerebral cortex, striatum, hippocampus and various nuclei of the thalamus were immunoreactive. In the cerebellum, Purkinje cells, granule cells and also basket cells were immunoreactive. However, astrocytes and oligodendrocytes in the cerebrum and cerebellum were not immunoreactive. The GD3 immunoreactivity was located in the cytoplasm. These findings are of considerable interest, being the first reported demonstration of GD3 in the adult rat brain. The implications and possible significance of the presence of GD3 are discussed.

Production of human monoclonal antibodies to i blood group by EBV-induced transformation: possible presence of a new glycolipid in cord red cell membranes and human hematopoietic cell lines

To study the differentiation-associated glycolipid two anti-i mAb producers, GL-1 and GL-2, were established from the combination of EBV-induced transformation of normal PBL and immune lysis of fluorescent dye-trapped liposome-containing bovine i-active glycolipid. The mAb GL-1 reacted with both sialosylparagloboside and pentahexosyl ceramide and the bovine i-active glycolipid whereas mAb GL-2 reacted only with the bovine i-active glycolipid in LILA. Both mAbs cold-agglutinate human cord red cells but not adult red cells. However, unexpectedly, the majority of the reactivity of these mAbs in human cord red cells on TLC was not identical to the i-active glycolipid. The GL-1 antigenic substance is considered to be a glycolipid distinct from the i-active glycolipid because the immunoreactivity was canceled with endoglycoceramidase which cleaves a linkage between the oligosaccharide and ceramide. Based on complement cytolysis with the mAb, 15 hematopoietic cell lines and normal peripheral lymphocytes were screened for susceptibility to the mAbs. A Burkitt lymphoma cell line, Ramos, was most sensitive among those tested, and BJA-B, Daudi, Namalwa in the B cell lines, TALL-1, Jurkatt in the T-cell lines and HL-60 in the non-lymphoid cell lines were sensitive whereas normal lymphocytes or other 8 cell lines were not. An immunoreactive spot with the same Rf with cord red cells was also detected in sensitive cell lines. The possible presence of a new glycolipid antigen determined from the mAb and related to the differentiation of hematopoietic cells was speculated.

A MONOCLONAL ANTIBODY THAT RECOGNIZES GANGLIOSIDE GD1B IN THE RAT CENTRAL NERVOUS SYSTEM

A monoclonal antibody (MAb), generated by immunizing BALB/c mice with homogenized bovine retinal tissue, was specific to ganglioside GD1b incorporated into liposome membranes. The antibody (MAb-5G6), classified as IgM, immunostained intensely the perikaryon and processes of motoneurons in the cranial motor nuclei and spinal cord. Spinal and trigeminal ganglion cells were also immunopositive to the MAb. Some fiber tract systems, such as the spinal and mesencephalic trigeminal tracts, the solitary tract and the posterior funiculus, were also immunoreactive to the MAb. These findings suggest that MAb-5G6 labeled specifically neurons with axons extending outside of the central nervous system as a peripheral nerve. The immunoreactive substances were visualized under electron microscopy just beneath the postsynaptic membrane and just inside the plasmalemma of the thick dendrites. No axon terminal was immunolabeled by the MAb. In the rat embryos, immunoreactivity to MAb-5G6 was found in the dorsal and ventral root fibers on the 15th embryonic day (E15). However, cell bodies of the spinal ganglion cells and motoneurons were immunostained by MAb-5G6 at a later stage (E20). The ventral commissure fibers in the floor plate of the spinal cord were transiently immunolabeled during E13-15.

Suppressive Effect of Liposomes Containing DNA Coding for Diphtheria Toxin A-Chain on Cells Transformed with Bovine Leukemia Virus

A recombinant plasmid which contained a gene for diphtheria toxin A‐chain (DT‐A) under the control of the long terminal repeat (LTR) of bovine leukemia virus (BLV) (BLV‐LTR) was constructed to test a novel application of liposomes as antiviral agents. The promoter activity of BLV‐LTR was estimated by the chloramphenicol acetyltransferase (CAT) assay using a plasmid which contains the coding sequence of CAT under the control of BLV‐LTR (pBLVCAT). When BLV‐infected cells were transfected with pBLVCAT, CAT activity was detected. BLV‐uninfected cell lines, however, showed no detectable CAT activity. The plasmid DNA entrapped in liposomes was added to BLV‐infected cells in culture. Syncytium formation induced by BLV‐infected cells was effectively suppressed by the liposomes containing the gene for DT‐A under the control of BLV‐LTR. Conversely, liposomes containing the gene for DT‐A without a promoter showed no such effect. DT‐A gene‐containing liposomes with BLV‐LTR did not affect formation of syncytium induced by bovine immunodeficiency virus. These observations indicate that BLV‐infected cells were readily targeted on the level of gene expression. This strategy could be applied to the treatment of BLV‐induced B‐cell proliferation of cattle, and further to other viral/neoplastic diseases where specific gene expression is exerted.

ISOLATION AND CHARACTERIZATION OF GANGLIOSIDES FROM TRYPANOSOMA BRUCEI

Gangliosides were isolated from Trypanosoma brucei and analyzed by thin-layer chromatography (TLC) and TLC immunostaining test. Four species of gangliosides, designated as G-1, G-2, G-3, and G-4, were separated by TLC. G-1 ganglioside had the same TLC migration rate as GM3. In contrast, G-2, G-3, and G-4 gangliosides migrated a little slower than GM1, GD1a, and GD1b, respectively. To characterize the molecular species of gangliosides from T. brucei, G-1, G-2, G-3, and G-4 gangliosides were purified and analyzed by TLC immunostaining test with monoclonal antibodies against gangliosides. G-1 ganglioside showed the reactivity to the monoclonal antibody against ganglioside GM3. G-2 was recognized by the anti-GM1 monoclonal antibody. G-3 showed reaction with the monoclonal antibody to GD1a. G-4 had the reactivity to anti-GD1b monoclonal antibody. Using 4 kinds of monoclonal antibodies, we also studied the expression of GM3, GM1, GD1a, and GD1b in T. brucei parasites. GM3, GM1, GD1a, and GD1b were detected on the cell surface of T. brucei. These results suggest that G-1, G-2, G-3, and G-4 gangliosides are GM3 (NeuAcα2-3Galβ1-4Glcβ1-1Cer), GM1 (Galβ1-3GalNAcβ1-4[NeuAcα2-3]Galβ1-4Glcβ1-1Cer), GD1a (NeuAcα2-3Galβ1-3GalNAcβ1-4[NeuAcα2-3]Galβ1-4Glcβ1-1Cer), and GD1b (Galβ1-3GalNAcβ1-4[NeuAcα2-8NeuAcα2- 3]Galβ1-4Glcβ1-1Cer), respectively, and also that they are expressed on the cell surface of T. brucei.

Developmental profile of ganglioside GD3 in the central nervous system: an immunocytochemical study in the rat

Light and electron microscopy observations were made of developing rat brain at gestation days (E) E13, E16, E19, and postnatal days (P) P1, P3, P5, P7 after immunocytochemical staining for ganglioside GD3 (II3 alpha(NeuAc alpha 2-8NeuAc)-LacCer, GD3) using mouse IgM anti-GD3 monoclonal antibody (DSG-1). Immunoreactivity was observed in neuroblasts (E13, E16) and immature neuronal cells (P1, P3, P5, P7), and also in glioblasts (E19). Electron microscopy revealed that at E13 peroxidase reaction product for GD3 (RP) was present on the plasma membrane and in the cytoplasm of neuroblasts, with accentuation in the former. At E16, RP was observed predominantly in the cytoplasm of neuroblasts. At E19, EP was seen mainly in the cytoplasm of glioblasts. At P1, P3, P5, and P7, immature neuronal cells in the cerebrum were immunoreactive. In the cerebellum, immature Purkinje cells and immature neuronal cells in the external and internal granular layers were also immunoreactive for GD3, the reaction product being located in the cytoplasm. The present findings suggest that changes in the localization of GD3 in neuroblasts were correlated with the alteration in their biological functions.

Natural transmission of bovine leukemia virus among cattle.

Misao ONUMA,* Shinobu WATARAI, Shigeru ICHIJO, Katsuya ISHIHARA, Tsuyoshi OHTANI, Mitsuo SONODA, Takeshi MIKAMI, Hisao IZAWA, and Tatsuo KONISHI *Department of Epizootiology , Faculty of Veterinary Medicine, Hokkaido University, Sapporo, Hokkaido 060, Department of Veterinary Internal Medicine, College of Dairy Agriculture, Ebestu, Hokkaido 069-01, Department of Veterinary Internal Medicine, Obihiro University ofAgriculture and Veterinary Medicine Obihiro, Hokkaido 080, Department of Veterinary Internal Medicine, Faculty of Agriculture, Gifu University, Kakamihara, Gifu 504, and Livestock Bureau, Prefecture ofGifu, Gifu 500