Cell cycle specificity of tumor necrosis factor and its receptor

Phase specificity in the TNF cytotoxic effect and the number of TNF binding receptors was investigated using L-M cells incubated synchronously from the S phase. TNF cytotoxicity was observed to occur at various levels during the cell cycle, with peak effect in the G2-M phase. Analysis with 125I-labeled TNF to determine the number of receptors binding TNF in the various cell phases shewed a phase specificity with the maximum number occurring in the G2-M phase, similar to the peak in cytotoxicity. The results suggest the existence of a cell cycle specificity in the cytotoxicity of TNF which is apparently related to changes in the number of receptors capable of binding TNF.     

Induction of LAK cells by high density dialyzing culture device and its cytotoxicity

Lymphokine activated killer (LAK) cells are generated by culture of lymphocytes with interleukin 2 (IL-2) in short term culture (3 to 5 days) and are used for adoptive immunotherapy for advanced cancer patients. The culture condition hitherto reported are essentially based on the rotating culture system, in which the maximum cell density was at 2 X 10(6) cell/ml and the cell recovery was usually less than 100%. The inability to induce LAK cells efficiently in vitro made the culturing of cells for therapy rather difficult and costly work because the mean infusion dose of LAK cells of one patient requires more than 1 X 10(10)/ml. We have therefore attempted to culture lymphocytes in 10 times higher concentration comparing with conventional methods. By using a new dialyzing culture system under continuous regulation of the amount of infused IL-2, nutrition medium, and pO2 and pCO2, we could culture cells at 2 X 10(7)/ml for more than 21 days and the resulted LAK cells showed a 100 times increase of activity on a per cell basis. By limiting dilution procedure, these killer cells mostly express T cell markers such as CD3 and CD8 but dose not express CD16.     

Comparison of beta-D-galactosidase from Escherichia coli and horseradish peroxidase as labels of anti-human ferritin Fab' by sandwich enzyme immunoassay technique

beta-D-Galactosidase from Escherichia coli and horseradish peroxidase were compared as labels of anti-human ferritin Fab' by sandwich enzyme immunoassay technique using fluorogenic substrates for enzyme assay. The anti-human ferritin Fab'-peroxidase conjugates gave lower nonspecific bindings and higher specific bindings than the corresponding Fab'-beta-D-galactosidase conjugates. As a result, the former provided more sensitive dose response curves for human ferritin than the latter. However, the peroxidase conjugates were required in a larger quantity, since peroxidase assay was much less sensitive than beta-D-galactosidase assay.     

Rhombomere formation and hind-brain crest cell migration from prorhombomeric origins in mouse embryos

Prior to rhombomere development, structures called prorhombomeres appear in the mammalian hindbrain. This study clarifies the developmental relationship between prorhombomeres and their descendent rhombomeres and hindbrain crest cells in mouse embryos by focal dye injections at various levels of prorhombomere A (proRhA), proRhB, and proRhC, as well as at their boundaries. ProRhA gives rise to two rhombomeres, rhombomeres 1 and 2 (r1 and r2), as well as to crest cells that migrate into the first pharyngeal arch, including the trigeminal ganglion. ProRhB develops into r3 and r4 and produces crest cells populating the second arch and acousticofacial ganglion. The anterior portion of proRhC gives rise to r5 and r6 and to crest cells migrating into the third pharyngeal arch and the IXth ganglion; its posterior portion develops into r7 and releases crest cells into the fourth pharyngeal arch region as well as the Xth ganglion. These results suggest that the boundaries between prorhombomeres serve as lineage restrictions for both hind-brain neuroepithelial cells and for segmental origins of crest cell populations in mouse embryos. The Hox code of the mouse head can be schematized in a much simpler way based on this prorhombomeric organization of the hind-brain, suggesting that prorhombomeres primarily underlie mammalian hind-brain segmentation.     

Hyperactive TNF-α derivatives with combinational mutations in the amino and carboxyl terminal regions

Summary We prepared various TNF- derivatives by protein engineering techniques. Mutant 471, in which 7 N-terminal amino acids were deleted and Pro⁸Ser⁹Asp¹⁰ was replaced by ArgLysArg, had a 8-fold higher antitumor activity against mouse L929 cells than wild-type TNF-. The additional substitution of Ala¹⁵⁶ or Leu¹⁵⁷ by more hydrophobic amino acids enhanced the activity of mutant 471. These results suggested that the combinational mutations in the N- and C-terminal regions of TNF- are effective for the improvement of antitumor activity.     

Subcellular distribution of mitochondrial ribosomal RNA in the mouse oocyte and zygote

Mitochondrial ribosomal RNAs (mtrRNAs) have been reported to translocate extra-mitochondrially and localize to the germ cell determinant of oocytes and zygotes in some metazoa except mammals. To address whether the mtrRNAs also localize in the mammals, expression and distribution of mitochondrion-encoded RNAs in the mouse oocytes and zygotes was examined by whole-mount in situ hybridization (ISH). Both 12S and 16S rRNAs were predominantly distributed in the animal hemisphere of the mature oocyte. This distribution pattern was rearranged toward the second polar body in zygotes after fertilization. The amount of mtrRNAs decreased around first cleavage, remained low during second cleavage and increased after third cleavage. Staining intensity of the 12S rRNA was weaker than that of the 16S rRNA throughout the examined stages. Similar distribution dynamics of the 16S rRNA was observed in strontium-activated haploid parthenotes, suggesting the distribution rearrangement does not require a component from sperm. The distribution of 16S rRNAs did not coincide with that of mitochondrion-specific heat shock protein 70, suggesting that the mtrRNA is translocated from mitochondria. The ISH-scanning electron microscopy confirms the extra-mitochondrial mtrRNA in the mouse oocyte. Chloramphenicol (CP) treatment of late pronuclear stage zygotes perturbed first cleavage as judged by the greater than normal disparity in size of blastomeres of 2-cell conceptuses. Two-third of the CP-treated zygotes arrested at either 2-cell or 3-cell stage even after the CP was washed out. These findings indicate that the extra-mitochondrial mtrRNAs are localized in the mouse oocyte and implicated in correct cytoplasmic segregation into blastomeres through cleavages of the zygote.     

Secretory leukoprotease inhibitor and pulmonary surfactant serve as principal defenses against influenza A virus infection in the airway and chemical agents up-regulating their levels may have therapeutic potential

Influenza A virus (IAV) is one of the most common infectious pathogens in humans. Entry of this virus into cells is primarily determined by host cellular trypsin-type processing proteases, which proteolytically activate viral membrane fusion glycoprotein precursors. Human IAV and murine parainfluenza virus type 1 Sendai virus are exclusively pneumotropic, and the infectious organ tropism of these viruses is determined by the susceptibility of the viral envelope glycoprotein to cleavage by proteases in the airway. Proteases in the upper respiratory tract are suppressed by secretory leukoprotease inhibitor, and those in the lower respiratory tract are suppressed by pulmonary surfactant, which by adsorption inhibits the interaction between the proteases and viral membrane proteins. Although the protease activities are predominant over the activities of inhibitory compounds under normal airway conditions, intranasal administration of inhibitors was able to significantly suppress multi-cycles of viral replication in the airway. In addition, we identified chemical agents that could act as defensive factors by up-regulating the levels of the natural inhibitors and immunoglobulin A (IgA) in airway fluids. One of these compounds, ambroxol, is a mucolytic and anti-oxidant agent that stimulates the release of secretory leukoprotease inhibitor and pulmonary surfactant in the early phase, and IgA in the late phase of infection at an optimal dose, i.e. a dose sufficient to inhibit virus proliferation and increase the survival rate of animals after treatment with a lethal dose of IAV. Another agent, clarithromycin, is a macrolide antibiotic that increases IgA levels through augmentation of interleukin-12 levels and mucosal immunization in the airway. In addition to the sialidase inhibitors, which prevent the release of IAV from infected cells, inhibitors of the processing proteases and chemical agents that augment mucosal immunity and/or levels of the relevant defensive compounds may also ultimately prove to be useful as new anti-influenza agents.     

Apoptosis-inducing neurotoxicity of dopamine and its metabolites via reactive quinone generation in neuroblastoma cells

Neurotoxic properties of L-dopa and dopamine (DA)-related compounds were assessed in human neuroblastoma SH-SY5Y cells with reference to their structural relationship. L-Dopa and its metabolites containing two free hydroxyl residues on their benzene ring showed toxicity in the cell, which was prevented by superoxide dismutase (SOD) and reduced glutathione (GSH), but not by catalase. Furthermore, a synthetic derivative of DA, 3-hydroxy-4-methoxyphenethylamine (HMPE) containing methoxy residue at position 4 in the benzene ring, exerted partial cytotoxicity, which was not prevented by SOD, GSH or catalase. However, the metabolites containing methoxy residue at position 3 failed to show a toxic effect in the SH-SY5Y cells. Moreover, DA induced apoptotic cell death, which was observed by nuclear and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining and measurement of caspase-3 activity; this compound up-regulated apoptotic factor p53 while down-regulating anti-apoptotic factor Bcl-2. In the cell-free in vitro electron spin resonance (ESR) spectrometry, DA possessing two hydroxyl groups showed generation of DA-semiquinone radicals, which were markedly prevented by addition of SOD or GSH but not by catalase. On the other hand, methylation of one of the hydroxyl residues on the benzene ring of DA converted DA to an unoxidizable compound (3-MT or HMPE), and caused it to lose the property to produce semiquinone radicals. It has been previously reported that SOD acting as a superoxide:semiquinone oxidoreductase prevents quinone formation, and that reduced GSH through forming a complex with DA-quinone prevents quinone binding to the thiol group of the intact protein. Therefore, the present results suggest that DA and its metabolites containing two hydroxyl residues exert cytotoxicity mainly due to generation of highly reactive quinones.     

Direct measurement of renal sympathetic nervous activity in high-fat diet-related hypertensive rats

The elevation of renal sympathetic nervous activity (SNA) is a possible cause of blood pressure (BP) elevation. Although a high-fat diet (FAT) often induces BP elevation in animals, the effect of FAT on renal SNA in animals is not consistent between studies. Thus, we compared the basal levels of efferent renal SNA and BP in FAT- or high-carbohydrate diet (CHO)-fed rats. Twenty-four male Sprague-Dawley rats were fed FAT (P/F/C=20/45/35% cal) or CHO (20/5/75) from 5 weeks of age. After 20-21 weeks of feeding, a 24-h urine sample was collected to measure sodium excretion. The next day, blood (0.2 ml) was withdrawn from a femoral artery, and basal efferent renal nerve discharges and mean arterial pressure (MAP) were recorded under anesthesia. Immediately after the experiment, abdominal (epididymal, perirenal and mesenteric) adipose tissues were dissected. Total abdominal fat weight was significantly greater in the FAT group than in the CHO group. The plasma level of leptin was significantly higher in the FAT group, but blood glucose and plasma insulin levels did not differ between the two groups. MAP and renal SNA were significantly higher in the FAT group. In addition, the ratio of urinary sodium excretion to dietary sodium intake was significantly lower in the FAT group than in the CHO group. The data suggest that the increased renal SNA may contribute to BP elevation in FAT-fed rats. The present study firstly demonstrated that renal SNA was elevated with FAT-related BP elevation.