Synthesis and application of a new phosphorylating agent: S-(N-monomethoxytritylaminoethyl)-O-(o-chlorophenyl)phosphorothi oate

A new phosphorylating agent, S-(N-monomethoxytritylaminoethyl)-O-(O-chlorophenyl) phosphorothioate, was prepared and reacted with a 5'-hydroxyl group of an oligonucleotide using 1-mesitylene-sulfonyl-3-nitrotriazole (MSNT) as a condensing agent. After base labile protecting groups were removed, the partially deprotected oligonucleotide was separated on a reversed phase column and converted to the oligonucleotide with an aminoethyl or a phosphoryl group at the 5'-end by treatment with 80% acetic acid or iodine-water, respectively. The syntheses of ppT, pppT, A5'pp5'T and A5'ppp5'T were also performed by treatment of 5'-O-(N-monomethoxytritylaminoethylthiophosphoryl) thymidine with tri-n-octylammonium salt of phosphoric acid, pyrophosphoric acid, pA and ppA, respectively.     

The augmentation of tumor-specific immunity using haptenic muramyl dipeptide (MDP) derivatives

Summary A new haptenic compound, a muramyl dipeptide (MDP) derivative (designated as L4-MDP-ONB) cross-reactive with Bacillus Calmette Guerin (BCG) was synthesized. The cross-reactivity of L4-MDP hapten to BCG was demonstrated from the following evidence; (a) lymph node cells from BCG-primed C3H/HeN mice exhibited appreciable L4-MDP-specific proliferative responses to the in vitro stimulation of L4-MDP-modified syngeneic cells (L4-MDP-self); (b) inoculation of L4-MDP-self into footpads of BCG-primed C3H/HeN mice elicited ample delayed type-hypersensitivity (DTH) responses in vivo as measured by footpad swelling; and (c) BCG-primed mice contained L4-MDP-reactive helper T cell activity which functions to augment the generation of effector T cell responses to cell surface antigens. This crossreactivity between L4-MDP hapten and BCG as measured by the helper T cell activity was applied to enhanced induction of tumor-specific immunity. When BCG-primed C3H/HeN mice were immunized with L4-MDP-modified syngeneic X5563 tumor cells, these mice could generate augmented tumor-specific in vivo protective (tumor neutralizing) immunity as well as in vitro cytotoxic T cell responses. These results indicate the effectiveness of L4-MDP hapten in augmenting tumor-specific immunity. The present approach is discussed in the context of potential advantages of this new hapten for its future application to clinical tumor systems.     

Solution Radioactivated by Hadron Radiation Can Increase Sister Chromatid Exchanges

When energetic particles irradiate matter, it becomes activated by nuclear reactions. Radioactivation induced cellular effects are not clearly understood, but it could be a part of bystander effects. This investigation is aimed at understanding the biological effects from radioactivation in solution induced by hadron radiation. Water or phosphate buffered saline was activated by being exposed to hadron radiation including protons, carbon- and iron-ions. 1 mL of radioactivated solution was transferred to flasks with Chinese hamster ovary (CHO) cells cultured in 5 mL of complete media. The induction of sister chromatid exchanges (SCE) was used to observe any increase in DNA damage responses. The energy spectrum and the half-lives of the radioactivation were analyzed by NaI scintillation detector in order to identify generated radionuclides. In the radioactivated solution, 511 keV gamma-rays were observed, and their half-lives were approximately 2 min, 10 min, and 20 min. They respectively correspond to the beta+ decay of ¹⁵O, ¹³N, and ¹¹C. The SCE frequencies in CHO cells increased depending on the amount of radioactivation in the solution. These were suppressed with a 2-hour delayed solution transfer or pretreatment with dimethyl sulfoxide (DMSO). Our results suggest that the SCE induction by radioactivated solution was mediated by free radicals produced by the annihilated gamma-rays. Since the SCE induction and DMSO modulation are also reported in radiation-induced bystander effects, our results imply that radioactivation of the solution may have some contribution to the bystander effects from hadron radiation. Further investigations are required to assess if radioactivation effects would attribute an additional level of cancer risk of the hadron radiation therapy itself.     

Global and temporal regulation of gene expression in Xenopus kidney cells in response to presumed microgravity generated by 3D clinostats.

We documented changes in morphology and gene expression of the renal epithelial cell line A6 derived from Xenopus leavis adult kidney induced by long-term culturing with three dimensional clinostats. An oligo microarray analysis on A6 cells showed that mRNA levels of 52 out of 8091 genes were significantly altered in response to clinorotation. Upregulation or downregulation of gene expression became evident on day 8 and day 10 while there was no significant change on day 5. However, on day 15, expression of 18 out of 52 genes resumed to the levels similar to its original levels while remaining 33 genes maintained altered levels of expression. Quantitative analyses of gene expression by real-time PCR confirmed that changes in mRNA levels of selected genes were found only under clinorotation but not under hypergravity (7 G) and ground control (1 G). Morphological changes including loss of dome-like structures, disassembly of E-cadherin adherence junctions and disassembly of cortical actin were also observed over 10 days of culturing with clinorotation. The results revealed genes which expression was altered specifically in A6 cells cultured under clinorotation.     

Effect of Osmotic Stress on Turgor Pressure in Mung Bean Root Cells

Turgor pressure in cells of the elongating region of intactmung bean roots was directly measured by using the pressure-probetechnique. After the external osmotic pressure had been increasedfrom 0 MPa to 0.5 MPa, turgor pressure rapidly decreased byabout 0.5 MPa from 0.65 MPa to 0.14 MPa and root elongationstopped. Subsequent turgor regulation was clearly confirmed,which followed the osmotic adjustment to maintain a constantdifference in the osmotic pressure between root-cell sap andthe external medium ( II). It took at least 6 h for turgor pressureto recover to an adjusted constant level of about 0.5 MPa dueto turgor regulation, but rootelongation resumed within onlyan hour after the osmotic treatment. Therefore, the resumptionof root elongation under osmotic stress could not have beendirectly connected with turgor regulation. Furthermore, sincethe amounts of decrease in turgor pressure just after applicationsof various degrees of osmotic stress could be interpreted inrelation to those in II, hydraulic conductivity between theinside and the outside of root cells must be large enough toattain water potential equilibrium rapidly in response to osmoticstress. We conclude that turgor pressure in the cells of theelongating region of mung bean roots is determined mainly by II because of water potential equilibrium. (Received January 27, 1987; Accepted May 21, 1987)     

Studies on the structure and stabilizing factor of the CUUCGG hairpin RNA using chemically synthesized oligonucleotides.

A tridecaribonucleotide, r-UGAGCUUCGGCUC, and two analogues r(UGAGC)d(UUCG)r(GCUC) and r-UGAGCUUCIGCUC, which form a hairpin structure with a four-base-paired stem and a UUCG loop, were synthesized by the solid-phase phosphoramidite method. Properties of these three oligomers and d-TGAGCTTCGGCTC, the DNA analogue, were studied by UV, CD and NMR spectroscopy. The melting temperature (Tm) data suggest that the 2'-hydroxy1 groups and the 2-amino group of guanosine in the loop (9G) stabilize the CUUCGG hairpin which is known to have an unusually high Tm. NMR studies show that this 9G takes a syn conformation and the phosphodiester backbone has a turn at 9G-10G which is a junction of the stem and loop.     

The augmentation of tumor-specific immunity using haptenic muramyl dipeptide (MDP) derivatives

Summary A previous paper has demonstrated that enhanced tumor-specific immunity could be induced by priming mice with Bacillus Calmette Guerin (BCG) and subsequently immunizing them with syngeneic tumor cells modified with BCG-cross-reactive muramyl dipeptide (MDP) hapten [15]. The present study establishes a tumorspecific immunotherapy protocol for a murine chronic leukemia based on the above T-T cell collaboration between antitumor effector T cells and anti-MDP hapten helper T cells induced by BCG priming. BALB/c mice which had been primed to BCG were injected intravenously (i.v.) with viable, syngeneic BCL1 leukemia cells. One week later, these mice were immunized intraperitoneally (i.p.) with unmodified or MDP hapten-modified, 10,000 R X-irradiated BCL1 cells, followed by 4 booster immunizations at 5-day intervals. The administration of unmodified BCL1 tumor cells into BCG-primed mice failed to prevent them from tumor death due to the persistent growth of preinjected BCL1 cells. In contrast, the immunization of BCG-primed, BCL1 leukemia-cell-bearing mice with MDP-modified BCL1 cells resulted in a high growth inhibition of leukemia cells and protection of these mice from death by leukemia. It was also revealed that potent tumorspecific, T-cell-mediated immunity was generated in mice which survived in this immunotherapy model. Thus, these results indicate that administration of MDP hapten-modified, syngeneic leukemia cells into leukemia-bearing mice which have been primed with BCG results in potent tumor-specific, T-cell-mediated immunity attributable to preventing the growth of disseminated leukemic cells.This work was supported by a Grant-in-Aid for the Special Project Cancer-Bioscience from the Ministry of Education, Science, and Culture, Japan Abbreviations used: TATA, tumor-associated transplantation antigens; MDP, muramyl dipeptide; MTP, muramyl tripeptide; BCG, Bacillus Calmette Guerin     

Surface Charge Density Estimation of Vigna mungo Protoplasts Using a Fluorescent Dye, 9-Aminoacridine

We showed that the surface charge density of protoplasts canbe estimated by the 9-aminoacridine method. The estimated surfacecharge density of the protoplasts isolated from elongating regionsof Vigna mungo root was – 39 ? 8 mC/m². The negative surfacecharge density increased when protoplasts were treated withglutaraldehyde or when EDTA was added to the protoplast suspensionmedium. These results support the validity of our estimationof the surface charge density of protoplasts by the 9-aminoacridinemethod. The concentration of amino groups at the surface ofthe protoplasts was estimated to be 34 mC/m². (Received June 19, 1987; Accepted April 11, 1988)     

Application of 39K NMR Spectroscopy to Potassium Transport in Mung Bean Root Tips

The intracellular K⁺ concentration and its change in mung bean[Vigna mungo (L.) Hepper] root tips were investigated non-invasivelywith ³⁹K nuclear magnetic resonance spectroscopy using a membraneimpermeable shift reagent, dysprosium (III) tripolyphosphate[Dy(PPP_i)^7–₂]. The K⁺ resonance was shifted to highermagnetic field in proportion to the concentration of the shiftreagent. In addition to a reference capillary peak for measuringthe K⁺ concentration, two well-resolved peaks (intra- and extracellularK⁺ resonances) were observed in the 39K NMR spectra of mungbean root tips. The intracellular K⁺ concentration was determinedto be 41 mM, which was similar to the value obtained by flamephotometry. When 20 mM KCl was added to the external medium,the intensity of the intracellular K⁺ resonance gradually increasedand the net K⁺ uptake rate was calculated to be 4.1 micromolesper gram fresh weight per hour. After removal of KCl from theperfusion medium, the intracellular K⁺ concentration considerablydecreased. With ³¹P NMR method, 2.5 mM Dy(PPP_j)^7–1₂ and20 mM KCl had little effect on the ATP level in the cells. Wehave indicated that the ³⁹K NMR method can be used to determinethe K⁺ levels and net fluxes of the K⁺ transport in perfusedroot tips successively. (Received April 6, 1988; Accepted September 29, 1988)     

Down-regulation of macrophage Ia mRNA expression by interferon (IFN)-alpha and IFN-beta mediated by de novo synthesized protein

We investigated the regulation of class I and class II major histocompatibility complex (MHC) antigen expression of murine peritoneal macrophages (M phi) by interferons (IFNs) at the mRNA level. Enhancement of class I antigen expression by IFNs (IFN-alpha, beta, and gamma), induction of class II antigen expression by IFN-gamma, and inhibition of class II antigen expression by IFN-alpha or IFN-beta all corresponded to steady-state levels of these MHC-specific mRNAs. Cycloheximide (CHX), a protein synthesis inhibitor, was used to elucidate whether IFN regulation of MHC mRNA expression depends on the newly synthesized proteins. CHX concentration was carefully chosen so that M phi viability was not decreased, total protein synthesis was considerably but not completely inhibited, and suppression of surface class II expression was virtually perfect. Under these conditions CHX did not affect the levels of either class I or class II mRNA, but it prevented IFN-beta from interfering with class II mRNA induction by IFN-gamma. These results indicate that the augmentation of induction and/or accumulation of MHC mRNA by IFNs is not dependent on the de novo synthesis of protein, but the down-regulation of class II mRNA level by IFN-beta is mediated by some newly synthesized protein(s).