Effect of cytokines on Japanese encephalitis virus production by human monocytes

Japanese encephalitis (JE) virus was shown to grow in in vitro cultures of human monocytes. Interferon (IFN)-alpha and IFN-gamma inhibited JE virus production by the infected monocytes in the absence of anti-JE virus antibody, but interleukin (IL)-1 alpha, IL-2, IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte-CSF (G-CSF), and tumor necrosis factor (TNF)-alpha did not show a significant inhibition. Antibody against JE virus increased the JE virus production by the infected monocytes probably by enhanced uptake of virus-antibody complexes via Fc receptors. IFN-gamma and GM-CSF increased JE virus production by monocytes in the presence of anti-JE virus antibody, whereas IFN-alpha inhibited JE virus production even in the presence of the antibody. The other 5 cytokines (IL-1 alpha, IL-2, IL-3, G-CSF, and TNF-alpha) did not show a significant effect on JE virus production by monocytes in the presence or absence of the antibody.     

Alterations of human erythrocyte membrane fluidity by oxygen-derived free radicals and calcium

Two possible reasons for the structural alterations of cell membranes caused by free radicals are lipid peroxidation and an increase in the intracellular calcium ion concentration. To characterize the alterations in membrane molecular dynamics caused by oxygen-derived free radicals and calcium, human erythrocytes were spin-labeled with 5-doxyl stearic acid, and alterations in membrane fluidity were quantified by electron spin resonance oxidase (0.07 U/mL) decreased membrane fluidity, and the addition of superoxide dismutase and catalase inhibited the effect on membrane fluidity of the hypoxanthine-xanthine oxidase system. Hydrogen peroxide (0.1 and 1 nM) also decreased membrane fluidity and caused alterations to erythrocyte morphology. In addition, a decrease in membrane fluidity was observed in erythrocytes incubated with 2.8 mM CaCl₂. On the other hand, incubation of erythrocytes with calcium-free solution decreased the changes in membrane fluidity caused by hydrogen peroxide.

These results suggest that changes in membrane fluidity are directly due to lipid peroxidation and are indirectly the result of increased intracellular calcium concentration. We support the hypothesis that alterations of the biophysical properties of membranes caused by free radicals play an important role in cell injury, and that the accumulation of calcium amplifies the damge to membranes weakened by free radicals.     

Zinc and copper accumulation and isometallothionein induction in mouse ascites sarcoma S180A cells.

To investigate Zn and Cu accumulation and isometallothionein (iso-MT) induction in ascites-sarcoma S180A cells, 5 micrograms of Zn2+ or Cu2+/g body weight was administered to tumour-bearing mice intraperitoneally. In the tumour cells the Zn or Cu concentration increased more than in the host liver, which is the target organ for those metals; the maximum Zn or Cu level was about 2-3 times that in the host liver. The amounts of Zn-MT or Cu-MT accumulated in the tumour cells and host liver were proportional to such dose accumulation levels in the each cytosol; the maximum level of Zn-MT or Cu-MT was 4 or 2 times higher than in the host liver. MT accumulated in the tumour cells showed two subfractions (MT-1 and MT-2); the ratio of Zn (or Cu) bound to MT-1 to that bound to MT-2 in the host liver and tumour cells was 1.0 (or 1.0) and 0.7 (or 0.25) respectively, suggesting that the induction level of MT-2 in the tumour cells is more than that of MT-1. The h.p.l.c. profiles (using an anion-exchange column) of the isolated MT-1 and MT-2 subfractions from Zn-treated normal-mouse liver showed a single peak (MT-1-1) and two peaks (MT-2-1 and MT-2-2) respectively; mouse MTs were separated into three isoforms. In the ascites cells, the MT fraction obtained by a gel filtration was also separated into three isoforms; however, the amount of MT-2-1 isoform was 3 times that in the Zn-treated normal-mouse liver.     

Modification of regression of virally xenogenized tumor cells by cyclophosphamide and busulfan

Summary Rat fibrosarcoma cells infected with Friend leukemia virus (FV-KMT-17) grow for a short time and then regress spontaneously in syngeneic hosts. This regression mechanism was examined by analyzing the immunomodulating action of the antitumor drugs busulfan (BU) and cyclophosphamide (CY). In preliminary experiments, the optimum dosages of BU and CY for the enhancement of DTH responses to SRBC were 10 mg/kg and 40 mg/kg respectively. Treatment of rats with BU (10 mg/kg) on day 5 induced the regression of KMT-17 cells, while in contrast, the same drug delayed the spontaneous regression of FV-KMT-17 cells. Pretreatment with CY (40 mg/kg) on day 5 did not affect the growth of KMT-17 or FV-KMT-17 cells. After the same treatment schedule, BU inhibited humoral antibody formation against SRBC and against virus-associated antigen (VAA), NK cell activity, and ADCC effector cell activity. On the other hand, CY did not affect the activities of NK cells or ADCC effector cells, although it significantly augmented the DTH responses to SRBC and the production of antibody to VAA but had no effect on production of antibodies to SRBC. These results suggest that NK cells and ADCC may play an important role in the initial stage of the spontaneous regression of FV-KMT-17 cells.Supported in part by a Grant-in-Aid for Cancer Research from the Japanese Ministry of Education Abbreviations used: BU, busulfan; CY, cyclophosphamide; PFC assay, plaque forming cell assay; VAA, virus-associated antigen; NK cell, natural killer cell; ADCC, antibody dependent cellular cytotoxicity; MuLV, murine leukemia virus; DTH, delayed type hypersensitivity; SRBC, sheep red blood cells; C.I., cytotoxic index; CRBC, chicken red blood cells; IL-1, interleukin 1; IL-2, interleukin 2; IFN, interferon     

Production of 5' Nucleotide by Using Halophilic Nuclease H Preferentially Adsorbed on Flocculated Cells of the Halophile Micrococcus varians subsp. halophilus

A bioreactor with a column of flocculated cells of the moderate halophile Micrococcus varians subsp. halophilus which adsorbed the halophilic nuclease H was designed to be used in the production of 5' nucleotides from RNA. A remarkable characteristic of the flocculated cells was that they preferentially adsorbed much exogenous nuclease, excluding adsorbed 5' nucleotidase. Furthermore, desalting treatment of the flocculated cells in the presence of 2% MgSO(4) . 7H(2)O gave rise to selective inactivation of 5' nucleotidase without the loss of nuclease H activity, and 5'-guanylic acid was produced with the bioreactor.     

Levels of IAA, Cytokinins, ABA and Ethylene in Rice Plants as Affected by a Gibberellin Biosynthesis Inhibitor, Uniconazole-P.

Effects of uniconazole-P, a triazole-type growth retardant,on endogenous levels of IAA, cytokinins, ABA and ethylene inrice seedlings were investigated. Endogenous levels of IAA andABA were similar between control and uniconazole-P-treated riceshoots. Evolution of ethylene was promoted slightly, being 1.8times greater under 0.3 ppm uniconazole-P treatment than thatof control. The most obvious effect was the increase of trans-Zand trans-RZ in shoots. Shoots treated with uniconazole-P (10mg/m² nursery box) contained 3.4 times and 3 times more trans-Zand trans-RZ than control, respectively. No significant differencesof cytokinin levels were recognized in roots except for cis-RZ.The increase of ethylene and active forms of cytokinins, andthe decrease of gibberellin in the shoots may be the basis forphysiological phenomena caused by uniconazole-P, namely thepromotion of flowering in woody plants and the enhancement offemaleness in cucumber. (Received September 9, 1987; Accepted October 20, 1987)     

Existence of a Common Glycoprotein Homologous to S-Glycoproteins in Two Self-incompatible Homozygotes of Brassica campestris

S-Glycoproteins (S-locus-specific glycoproteins) in Brassicaspecies are present only in stigmas and thought to play an importantrole in self-incompatibility system. The stigma extract containsalso several other glycoproteins reacting with the antiserumto S-glycoproteins, among which some glycoproteins from S₈S₈-and S₉S₉-homozygotes have the same pI value. Both of the glycoproteinswhich were tentatively termed NS₈- and NS₈S₉-glycoproteins,respectively, were isolated and analyzed. Those were revealedto be identical. Its amino acid sequence was homologous withthe S-glycoproteins in Brassica species. The NS-glycoproteinswere expressed at the same time and only in stigma as S-glycoproteins. (Received July 19, 1988; Accepted September 7, 1988)     

DNA Methylation Occurred around Lowly Expressed Genes of Plastid DNA during Tomato Fruit Development

We have analyzed DNA methylation of plastid DNA from fully ripened red fruits, green mature fruits, and green leaves of tomato (Lycopersicon esculentum var. Firstmore). Essentially identical restriction profiles were obtained between chromoplast and chloroplast DNAs by EcoRI digestion. BstNI/EcoRII and HpaII/MspI are pairs of isoschizomers that can discriminate between methylated and unmethylated DNAs. These endonucleases produced different restriction patterns of plastid DNAs from tomato fruits compared to tomato leaves. Moreover, we have found from Southern blots that methylation was not detected in DNA fragments containing certain genes that are actively expressed in chromoplasts, whereas DNA fragments bearing genes that are barely transcribed in chromoplasts are methylated.     

Regulation of microtubule protein levels during cellular morphogenesis in nerve growth factor-treated PC12 cells

Nerve growth factor induces neurite process formation in pheochromacytoma (PC12) cells and causes the parallel increase in levels of the microtubule-associated proteins, tau and MAP1, as well as increases in tubulin levels. Mechanisms to insure balanced accumulation of microtubule proteins and make their levels highly responsive to nerve growth factor were investigated. The effects on tau, MAP1, and tubulin are due to changes in protein synthesis rates, which for tau and tubulin we could show are due in part to changes in the mRNA levels. Whereas tubulin shows feedback regulation to modulate synthesis up or down, tau protein synthesis is not affected in a straightforward way by microtubule polymerization and depolymerization. The degradation of tau, MAP1, and both tubulin polypeptides, however, are stimulated by microtubule depolymerization caused by colchicine, or nerve growth factor removal. Combined feedback on synthesis and stability make tubulin levels highly responsive to assembly states. In addition, the linkage of tau and MAP1 turnover with the state of microtubule polymerization amplifies any change in their rate of synthesis, since tau and MAP1 promote microtubule polymerization. This linkage lends itself to rapid changes in the state of the system in response to nerve growth factor.