Preparation of biologically active Ascaris suum mitochondrial tRNAMet with a TV-replacement loop by ligation of chemically synthesized RNA fragments.

Ascaris suum mitochondrial tRNA Met lacking the entire T stem was prepared by enzymatic ligation of two chemically synthesized RNA fragments. The synthetic tRNA could be charged with methionine by A.suum mitochondrial extract, although the charging activity was considerably low compared with that of the native tRNA, probably due to lack of modification. Enzymatic probing of the synthetic tRNA showed a very similar digestion pattern to that of the native tRNA Met, which has already been concluded to take an L-shape-like structure [Watanabe et al. (1994) J. Biol. Chem., 269, 22902-22906]. These results suggest that the synthetic tRNA possesses almost the same conformation as the native one, irrespective of the presence or absence of modified residues. The method of preparing the bizarre tRNA used here will provide a useful tool for elucidating the tertiary structure of such tRNAs, because they can be obtained without too much difficulty in the amounts necessary for physicochemical studies such as NMR spectroscopy.     

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.     

Oral administration of antigen does not influence the proliferation and IFN-γ production of responsive CD8+ T cells but enables to establish T cell clones with different lymphokine production profile.

Feeding of a whole casein diet, which abolished the α_s1-casein-specific proliferation and IFN-γ productivity of CD⁴⁺ T cells, did not affect the proliferative response of CD8⁺ T cells with regard to the antigen dose response, cell dose response, kinetics of the proliferation and epitope specificity, as well as IFN-γ production. To assess the characteristics of the CD8⁺ T cells, we established α_s1-casein-specific CD8⁺ T cell clones from both casein-fed and control mice. The established clones produced different amount of IFN-γ and IL-10, and one clone derived from the casein-fed mice produced a remarkable amount of IL-10. The clones from casein-fed mice produced considerable amounts of TGF-β, while those from control mice produced only small amounts. The possible role of CD8⁺ T cells in oral tolerance is discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Partial tetrasomy 9(9pter to 9q2101) due to an extra iso-dicentric chromosome.

A 3-year-old boy with partial No. 9 tetrasomy is described. The patient showed markedly retarded physical and mental development as well as multiple congenital anomalies. Routine chromosome analysis revealed an extra C-group chromosome. It had a pronounced secondary constriction at the proximal part of its long arm. Based on studies by a variety of banding techniques, the extra chromosome was identified to be an iso-dicentric No. 9 chromosome with inactivation of one of the two centromeres, the karyotype being 47,XY, + DIC (9)(Q2101). The value of BrdUrd treatment was emphasized in the detection of a very small piece of euchromatin within a long stretch of constitutive heterochromatin.     

Stimulation of prolyl hydroxylase activity by chelating agents.

The activity of purified prolyl hydroxylase was enhanced several fold by addition of some chelating agents to the assay medium. Chelating agents could be classified into three groups. The chelating agents of Group I such as α, α′-dipyridyl were inactive until they reached equimolar concentration with ferrous ion in the assay mixture. The Group II agents, EDTA, diethylenetriaminepentaacetic acid, etc., stimulated the enzymatic activity 1.5- to 3-fold at equimolar concentration with ferrous ion. But the agents of both groups precipitously inhibited the enzymatic activity at concentrations greater than ferrous ion. On the other hand, Group III chelating agents, such as nitrilotriacetic acid, enhanced the enzymatic activity 5- to 10-fold at concentrations greater than ferrous ion. Nucleoside triphosphates, which also stimulate the enzymatic activity several fold and whose optimal concentrations are 1–3 × 10^?m, may be analogous to nitrilotriacetic acid of Group III.     

Developmental changes in soluble proteins during the early development of Dictyostelium discoideum

Changes in the pattern of soluble proteins that accumulatedat the growth phase, interphase and late-aggregation phase ofthe cellular slime mold Dictyostelium discoideum were studiedby two-dimensional polyacrylamide gel electrophoresis. Amongthe 300 proteins detected during the early development, themost soluble do not change during the growth and aggregationphases, but about 90 proteins show changes in their relativeintensities on staining. During the transition from growth tothe interphase, the predominant changes were the disappearanceof 16 spots, the decrease in 30 spots, the appearance of 13new spots, and the increase in 14 spots. In contrast, from theinterphase to the late-aggregation phase, there were remarkableprogressive increases in 13 spots, an overall increase in 6spots, a decrease in 16 spots, the appearance of 8 new spotsand the disappearance of 4 spots. (Received July 13, 1979; )     

Different structural requirements of endotoxic glycolipid for tumor regression and endotoxic activity

Endotoxic glycolipid extracted from the heptose-less mutant of $\text{Salmonella typhimurium}$ was treated with alkali and acid reagents. The glycolipid freed of all O-ester linked fatty acids by hydroxylamine had lost tumor regression activity and toxicity, whereas a partial removal of O-ester linked fatty acids by mild alkali did not impair with these activities. The glycolipid retained both activities after removal of 2-keto-3-deoxyotonate by sodium acetate (pH 4.5) but was rendered nontoxic while retaining antitumor activity when hydrolyzed by 0.1N HCl whereby 2-keto-3-deoxyoctonate and glycosidic phosphate was split off the glycolipid molecule. Nontoxic and tumor regressive fractions were separated by means of preparative thin layer chromatography of glycolipid hydrolyzed by mild acid. Thus, it was concluded that glycosidic bound phosphate and at least a portion of fatty acids of the lipid A moiety were essential for toxicity, but that this phosphate is not essential for tumor regression activity.     

A case of renin producing leiomyosarcoma originating in the lung.

A 54-year-old woman was referred to our hospital for the treatment of a tumor of the right chest wall. Clinical examination revealed hypertension, hypokalemia, metabolic alkalosis, hyperaldosteronism and hyperreninemia. Computed tomography and an abdominal echogram indicated a tumor in the right phrenic area and two tumors in the retroperitoneum near the pancreas head. After the surgical resection of these tumors, the primary reninism was diminished. The pathological diagnosis of these tumors was leiomyosarcoma. Plasma active and inactive (trypsin-activated) renin activities (PRA) were 85.7 and 38.9 ng angiotensin I/ml/h, respectively. These PRA did not respond to either postural stimulation or suppression by the volume expansion. Active and inactive renin activities in a right phrenic area tumor were 208 and 32 ng angiotensin I/mg protein /h, respectively. Those of an abdominal tumor were 196 and 30 ng angiotensin I/mg protein/h, respectively. Renin mRNA identical in molecular size to that of the human kidney was identified by northern blot analysis. This is the first case report of renin producing leiomyosarcoma derived from the lung, which is characterized by relatively lower plasma prorenin concentrations.