Studies on the fungitoxicity of captan

The cellular distribution of ³⁵S after incubating labelled captan with Neurospora crassa conidia has been determined. Nearly all the ³⁵S in the spores is bound to the water-soluble and protein fractions. Thin-layer chromatography of the hot-water extract of spores has shown that ³⁵S occurs largely in oxidized glutathione (GSSG) and in a product tentatively identified as a thiazolidine derivative of glutathione. It is suggested that this derivative, which only forms above pH 6·5, is produced by reaction between glutathione (GSH) and the thiocarbonyl chloride liberated on the decomposition of captan. Captan toxicity could not be completely reversed by pretreatment with thiols and disulphides capable of penetrating the cell membrane, confirming the previous hypothesis that fungitoxicity is due to irreversible changes following the oxidation of the protein thiols to disulphides.     

Fractionation of mouse T and B lymphocytes by preparative cell electrophoresis. Efficiency of the method

Mouse lymphocytes have been fractionated in preparative cell electrophoresis into two functionally viable populations, a high mobility cell (HMC) and a low mobility cell (LMC) population. The distribution of HMC in CBA spleen, blood, and lymph node corresponds to known proportions of θ-positive cells in these organs. The HMC carry the θ-isoantigen, respond to phytohemagglutinin in vitro, and induce a graft-versus-host reaction in newborn F₁ hybrid mice. Nearly all spleen LMC have complement receptors on their surface. About 70% of spleen LMC are sensitive to anti-MBLA serum and form “caps” when incubated with FITC-conjugated anti-Ig. Only LMC respond to E. coli lipopolysaccharide. Thus, T cells localize in the HMC population and B cells in the LMC population. There is no detectable contamination of T lymphocytes among the LMC, nor of B lymphocytes among the HMC.     

Polarization sensitivity in crayfish lamina monopolar neurons

1. Polarization sensitivity (PS) was examined in photoreceptors and lamina monopolar cells (LMCs) in two species of crayfish, Procambarus clarkii and Pacifasticus leniusculus. The measurements were made with intracellular recordings and broad field illumination.
2. PS is about 40% greater in Pacifasticus than in Procambarus (Table 1). In both species the LMC stationary PS profiles (estimated with flashes) are similar to those of receptors (Figs. 1 and 2). Both receptor and LMC sensitivity profiles are well described by cos² θ functions (Fig. 3). PS was observed in all receptors and 78% of LMCs.
3. When stimulated with a rotating polarizer, receptors and LMCs exhibit membrane potential modulation with phase predicted by the stationary PS profile (Fig. 5). In photoreceptors, the polarization-elicited percent modulation falls off steeply as intensity increases. The LMC modulation is stronger than that in receptors and relatively insensitive to the mean intensity (Figs. 6 to 8). For low intensities the LMC modulation is 100%. The LMC dynamic behavior is consistent with either an opponency mechanism or strong but polarization-insensitive lateral inhibition.
4. Receptors and LMCs exhibit steady-state differential sensitivity to stationary e-vector orientation (Fig. 9).
5. About 10% of the LMC neurons exhibit PS maxima separated by 90°. These results imply a nonlinear summation of signals from orthogonal receptor channels (Fig. 10).

     

The Structural Characterization of a Novel Water-Soluble Polysaccharide from Edible Mushroom Leucopaxillus giganteus and Its Antitumor Activity on H22 Tumor-Bearing Mice

In the present study, a novel cold water-soluble polysaccharide fraction (LGP) with the average molecular weight of 1.78×10⁶ Da was extracted and purified from Leucopaxillus giganteus and its primary structure as well as in vivo antitumor activity was evaluated. The monosaccharide composition of LGP was determined by ion chromatography to be galactose, xylose, glucose and fucose in a molar ratio of 2.568 : 1.209 : 1 : 0.853. Its backbone was composed of α-D-Glu, α-D-Xyl, α-D-Gal and α-L-Fuc. The results of in vivo antitumor experiment demonstrated that LGP could effectively protect immune organs, has excellent antitumor activity, and inhibit the proliferation of H22 solid tumors in a dose-dependent manner. By analyzing Annexin V-FITC/PI staining, cell cycle and mitochondrial membrane potential detection assay, we concluded that LGP induced apoptosis of H22 cells via S phase arrest and mitochondria-mediated apoptotic pathway. Our results could provide valuable information for the potential application of LGP as an anti-hepatoma agent.     

Preparation and properties of highly soluble chitosan–l-glutamic acid aerogel derivative

The objective of this work is to improve the solubility of chitosan at neutral or basic pH using the supercritical carbon dioxide (sc.CO₂). A novel water-soluble chitosan–l-glutamic acid (Cl-GA) aerogel derivative was synthesized by reaction of 85% deacetylated chitosan with l-glutamic acid (l-GA) in aq.AcOH subjected to solvent exchange prior to using sc.CO₂ as a nonsolvent for the polymer. The prepared aerogel derivative and molecular conformation of modified chitosan are characterized by using UV, FTIR, ¹H NMR, and CD techniques. Some physical properties and surface morphology were analyzed by X-ray diffraction, differential scanning calorimetry (DSC), thermogravimetry (TG), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and porosimetry analysis. Overall, the sc.CO₂ assisted chitosan aerogel derivative opens new perspectives in biomedical applications.     

Synthesis of novel β-carbolines with efficient DNA-binding capacity and potent cytotoxicity

A series of water-soluble β-carbolines, bearing a flexible amino side chain, was prepared and evaluated in vitro against a panel of human tumor cell lines. The N⁹-arylated alkyl substituted β-carbolines represented the most interesting cytotoxic activities, and compound 7b was found to be the most potent antitumor agent with IC₅₀ values lower than 10 μM against eight human tumor cell lines. The results confirmed that the N⁹-arylated alkyl substituents of β-carboline nucleus played an important role in the modulation of the cytotoxic potencies. In addition, these compounds were found to exhibit significant DNA-binding affinity.     

A Messenger RNA for Tobacco Mosaic Virus Coat Protein in Infected Tobacco Mesophyll Protoplasts

The low molecular weight tobacco mosaic virus (TMV)-specific RNA component (LMC) was demonstrated in tobacco mesophyll protoplasts by polyacrylamide gel electrophoresis of ¹⁴C-uridine-labelled RNA from infected protoplasts. Free and membrane-bound polysomes were isolated from infected protoplasts, and RNA extracted from them was analyzed. TMV-specific RNA species including full-length viral RNA, its replicative intermediate, and LMC were found in both free and membrane-bound polysomes, but were present in free polysomes in much larger amounts. In particular, as much as 37 % of total LMC in protoplasts was found in free polysomes. Fractionation of polysomes by sedimentation in sucrose gradients showed that LMC is associated with small-sized polysomes (mono- to tetrasomes). Polysomes of this size class produced viral coat protein in a cell-free protein synthesizing system from rabbit reticulocytes. On the other hand, full-length TMV-RNA was associated predominantly with larger polysomes which produced in the cell-free system TMV-specific high molecular weight polypeptides but no coat protein. These results indicated that LMC, a subgenomic RNA of TMV, in fact functions in vivo as messenger RNA for viral coat protein, as has been postulated on the basis of in vitro studies.     

Effects of heparin and benzyl alcohol on lymphocyte-mediated cytotoxicity in vitro

The effect of heparin on the in vitro lysis of EL4 tumor cells by immune BALB/C lymphocytes was investigated by using a ⁵¹Cr-release cytotoxicity. assay. Powdered heparin did not inhibit lymphocyte-mediated cytotoxicity (LMC) at concentrations up to 500 units/ml. Heparin containing 9 mg/ml of benzyl alcohol (BA), as preservative, significantly reduced the LMC. BA alone at 1 mg/ml inhibited LMC without any apparent toxic effect on the target cells or on the immune lymphocytes. The inhibitory effect of three different heparin preparations was found to be related to the BA concentration rather than the amount of heparin. However, low concentrations of heparin (0.5 or 1 unit/ml) significantly enhanced the LMC. Our findings are in contrast to previous reports suggesting a depressive effect of heparin on LMC.     

Organopalladium compound 7b targets mitochondrial thiols and induces caspase-dependent apoptosis in human myeloid leukemia cells

The advances in the treatment of chronic myeloid leukemia (CML) during the last years were also accompanied by the development of evading strategies by tumor cells, resulting in chemotherapy resistance in some patients. Patented organopalladium compounds derived from the reaction of N,N-dimethyl-1-phenethylamine (dmpa) with [1,2-ethanebis(diphenylphosphine)] (dppe) exhibited a potent antitumor activity in vivo and in vitro in melanoma cells. We showed here that the cyclopalladated derivative [Pd₂(R₍₊₎)C², N-dmpa)₂(μ-dppe)Cl₂], named compound 7b, was highly effective to promote cell death in the K562 human leukemia cells and its mechanisms of action were investigated. It was shown that compound 7b was able to promote exclusively apoptotic cell death in K562 cells associated to cytochrome c release and caspase 3 activation. This cytotoxic effect was not observed in normal peripheral mononuclear blood cells. The compound 7b-induced intrinsic apoptotic pathway was triggered by the protein thiol oxidation that resulted in the dissipation of the mitochondrial transmembrane potential. The preventive effect of the dithiothreitol on the compound 7b-induced cell death and all downstream events associated to apoptosis confirmed that death signal was elicited by the thiol oxidation. These findings contribute to the elucidation of the palladacycle 7b-induced cell death mechanism and present this compound as a promising drug in the CML antitumor chemotherapy.     

Evidence for chitin synthesis in an insect cell line

Cells from the continuous MRRL-CH line derived from embryos of the tobacco hornworm synthesized chitin. Digestion of the washed pellet from [¹⁴C]-N-acetylglucosamine-labeled cells by chitinase yielded a water-soluble labeled compound. The lyophilized residue from the supernatant of the chitin digestion was analyzed by gas-liquid chromatography as its trimethylsilyl derivative. The major component cochromatographed with derivitized chitobiose. The presence of chitobiose was confirmed by gas chro-matography-mass spectrometry. The synthesis of chitin by this cell line is inhibited by diflubenzuron.     

Inability of EDTA to prevent damage mediated by cytolytic T-lymphocytes.

To analyze the nature of the target cell determinants recognized and bound by killer lymphocytes during lymphocyte-mediated cytolysis (LMC), the specific binding of serologically active tumor cell membrane fractions to cytotoxic T lymphocytes has been investigated. Particulate membrane fractions and soluble antigen preparations (extracted by papain or 3 M KCl) from tumor target cells were tested for their ability to inhibit the destruction of intact ⁵¹Cr-labeled target cells by killer lymphocytes in vitro. The effect of papain-solubilized tumor cell antigen on the binding of killer lymphocytes to tumor cell monolayers was also evaluated. Direct assays to determine the extent of binding of unlabeled or radioiodinated soluble antigen (extracted by papain or deoxycholate) to cytotoxic lymphocytes were carried out. In marked contrast to their serological activity, all of these particulate and soluble preparations failed to inhibit LMC or bind to killer lymphocytes in an immunologically specific way. It is suggested that killer lymphocytes recognize and bind to an antigenic complex whose organization is dependent upon the integrity of the target cell membrane.     

Synthesis and characterization of quaternized β-chitin

Water-soluble 2′-O-hydroxypropyltrimethylammoniumchitin chloride (2′-O-HTACCt) was prepared directly from β-chitin and 3-chloro-2-hydroxypropyltrimethylammonium chloride (CTA) in basic medium. The effect of alkali concentration, reaction temperature, reaction time, and dosage of CTA on yield and degree of substitution (DS) of 2′-O-HTACCt were studied. These quaternized chitin derivatives were characterized by FTIR and ¹H NMR spectroscopy, conductometric titration, and elemental analysis methods. Research results indicate that β-chitin can react directly with CTA to produce a water-soluble 2′-O-HTACCt derivative with a high DS. The optimal preparation conditions were determined to be 35-40 wt % (aq NaOH), 40 °C (reaction temperature), 6 h (reaction time), and 4 (molar ratio of CTA to β-chitin unit).     

The large monopolar cells L1 and L2 are responsible for ERG transients inDrosophila

Summary The mutantVam (Vacuolar medulla ^KS74) has age specific degeneration of the large monopolar cells L1 and L2 (LMC) in the lamina and reduced ERG transients. The size of the on- and off-transients in the ERG is negatively correlated with degeneration of the LMCs (Figs. 1 and 2) as assessed by examination of ultrastructure. The retinula cells function normally as indicated by the induction of a prolonged depolarizing after potential inw Vam flies (Fig. 7) and genetic mosaic analysis (Figs. 5, 6). This indicates that the LMCs are probably the major contributers to ERG transients inDrosophila. Independent evidence in support of this hypothesis comes from the triple mutantrol mnb sol. This mutant has an abnormal lamina with some cartridges with no LMC and others with a single LMC (Fig. 8). The ERG ofrol mnb sol shows reduced on- and off-transients (Fig. 9) which is as expected if the LMCs are principally responsible for ERG transients.Abbreviations ERG electroretinogram - PDA prolonged depolarizing afterpotential - LMC large monopolar cell - EM electron microscope - LED light emitting diode     

Synthesis,characterization and antitumor activity of a series of N-substituted iminodiacetato(diammine) platinum(II) complexes

A series of water-soluble N-substituted iminodiacetato (diammine)platinum(II) complexes [Pt(NRIDA)(NH₃)₂] have been synthesized and characterized by measurement of physical properties (conductivity and pH) and by various spectroscopic techniques (infrared, ¹H and ¹³C{¹H} nuclear magnetic resonance). The iminodiacetate ligand is coordinated to platinum through an O,N linkage. The results obtained suggest that these complexes are relatively stable for more than 24 h in aqueous solution. Preliminary in vitro and in vivo screening test for antitumor activity of these complexes against L1210 murine leukemia were performed. Many of complexes had acceptable in vitro cytotoxicity, but none displayed a significant level of in vivo antitumor efficacy.     

The unsaturated acyclic nucleoside analogues bearing a sterically constrained (Z)-4′-benzamido-2′-butenyl moiety: Synthesis,X-ray crystal structure study,cytostatic and antiviral activity evaluations

A series of the novel acyclic unsaturated pyrimidine (1–12) and adenine (13) nucleoside analogues bearing conformationally restricted (Z)-2′-butenyl moiety were synthesized and evaluated for their antiviral and cytostatic activity potency against malignant tumor cell lines and normal human fibroblast (WI38). The N-1 and/or N-3 acyclic side chain substitution in pyrimidine ring in N-3 substituted 5-trifluoromethyluracil derivative (11), N-1, N-3 disubstituted 5-fluorouracil derivative (12) and adenine derivative (13) was deduced from their ¹H and ¹³C NMR spectra and confirmed by single crystal X-ray structure analysis. The X-ray crystal structure analysis 11–13 revealed also supramolecular self-assemblies, in which infinite chains or dimers built two- and three-dimensional networks. The results of the in vitro cytostatic activity evaluations of 1–13 indicate that the majority of the compounds tested exhibited a non-specific and moderate antiproliferative effect at the highest concentration (100 μM). Of all evaluated compounds on the cell lines tested only the N-1 4″-fluoro-substituted-benzamide uracil derivative (7) showed rather marked and selective inhibitory activity against the growth of MCF-7 cells at a concentration of 2.7 μM and no cytotoxic effect on normal fibroblasts WI38. This compound can be therefore considered as a potential antitumor lead compound for further synthetic structure modification.     

H3-THYMIDINE DERIVATIVE POOLS IN RELATION TO MACRONUCLEAR DNA SYNTHESIS IN TETRAHYMENA PYRIFORMIS

The formation of a soluble H³-thymidine derivative pool has been examined in Tetrahymena pyriformis as a function of macronuclear DNA synthesis during the cell life cycle. An autoradiographic technique which allows the detection of water-soluble materials within a cell has shown that these cells do not take up and retain exogenous H³-thymidine during G1 or G2. Uptake of H³-thymidine is restricted to the S period of the cell cycle. Additional autoradiographic experiments show, however, that a soluble pool of H³-thymidine derivatives persists from the end of one DNA synthesis period to the beginning of the next synthesis period in the subsequent cell cycle. Since this persisting pool cannot be labeled with H³-thymidine, the pool does not turn over during non-S periods.     

Synthesis of a cyclic hexapeptide with sequence corresponding to murine tumor necrosis factor-(127-132) as a novel potential antitumor agent

A cyclic hexapeptide cyclo(Lys-Gly-Asp-Gln-Leu-Ser-) 10 was synthesized stepwise in solution by acylation of peptide ester trifluoroacetates directly with preactivated Boc-amino acids using the DCC/HOBt method; the final cyclization reaction was performed using the pentafluorophenyl ester method in solution (1-4). This peptide is a cyclic derivative of murine tumor necrosis factor-(127-132) and is designed as a potential antitumor agent. The cyclic peptide 10 displayed weak cytotoxic activity on three of the four human tumor cell lines tested.     

A convenient preparation of 6-oligo(lactic acid)cyclomaltoheptaose as kinetically degradable derivative for controlled release of amoxicillin

6-Oligo(lactic acid)cyclomaltoheptaose (6-OLA-βCD) with an average substitution of about 7.0 lactic acid units was prepared as a new water-soluble cyclomaltoheptaose (βCD) derivative (solubility of about 70.7-fold that of βCD), based on the ring-opening polymerization of 3,6-dimethyl-1,4-dioxane-2,5-dione (lactide). The product was characterized by ¹H NMR, ¹³C NMR, IR, and MS spectroscopy. The complexation of amoxicillin with 6-OLA-βCD was found to be much stronger than that with βCD at first, and then 6-OLA-βCD was shown to decompose moderately into βCD and lactic acid. 6-OLA-βCD might be greatly valuable in a controlled release system for Amoxicillin (AMX).     

Pyrrolidinium-type fullerene derivative-induced apoptosis by the generation of reactive oxygen species in HL-60 cells

The biological activities of C₆₀-bis(N,N-dimethylpyrrolidinium iodide), a water-soluble cationic fullerene derivative, on human promyeloleukaemia (HL-60) cells were investigated. The pyrrolidinium fullerene derivative showed cytotoxicity in HL-60 cells. The characteristics of apoptosis, such as DNA fragmentation and condensation of chromatin in HL-60 cells, were observed by exposure to the pyrrolidinium fullerene derivative. Caspase-3 and -8 were activated and cytochrome c was also released from mitochondria. The generation of reactive oxygen species (ROS) by the pyrrolidinium fullerene derivative was observed by DCFH-DA, a fluorescence probe for the detection of ROS. Pre-treatment with α-tocopherol suppressed cell death and intracellular oxidative stress caused by the pyrrolidinium fullerene derivative. The apoptotic cell death induced by the pyrrolidinium fullerene derivative was suggested to be mediated by ROS generated by the pyrrolidinium fullerene derivative.     

Synthesis and Biological Properties of New Phosmidosine Analogs Having an N-Acylsulfamate Linkage

A new phosmidosine analog 10, in which the proline and 8-oxoadenosine moieties were linked by an N-acyl sulfamate linkage, was successfully synthesized by the sulfamoylation of an 8-oxoadenosine derivative 5 followed by coupling with an L-proline derivative 8. An L-alanine-substituted derivative 13 and its derivative 14 without the alanyl residue were also synthesized. The morphological reversion activity of these synthetic compounds in v-src ^ts NRK cells and their antitumor activity in L1210 and KB cells were studied. As the result, neither L-proline- nor L-alanine-substituted phosmidosine analogs 10 and 13 showed any antitumor activity. Contrary to these results, the derivative 14 lacking the amino acid residue showed potent antitumor activities against cancer cells.