Cytotoxic effect of 1-methyl-4-phenylpyridinium ion on human melanoma cell lines, HMV-II and SK-MEL-44, is dependent on the melanin contents and caused by inhibition of mitochondrial electron transport

MPP+, an oxidative metabolite of a neurotoxin, MPTP, was found to be cytotoxic to human melanoma cell lines, HMV-II and SK-MEL-44. After 3 days of culture in the presence of MPP+, a larger amount of MPP+ was accumulated in HMV-II cells than in SK-MEL-44 cells, which correlated well with the melanin contents; HMV-II cells contain larger amounts of melanin than SK-MEL-44 cells. After 6 days of culture in the presence of MPP+, the cytotoxicity of MPP+ on these cell types was evaluated by counting cell numbers with the dye exclusion test and double-layer soft agar clonogenic assay. It was found that exposure to MPP+ reduced the survival of HMV-II cells more significantly than that of SK-MEL-44 cells. In HMV-II cells, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was used to elucidate the mechanism of MPP+ lethality. The formazan formation was reduced markedly by the presence of MPP+ at concentrations much lower than those required for cell death. These results suggest that cytotoxicity of MPP+ may be ascribed to its accumulation due to high affinity for melanin, and to inhibition of the enzymes utilizing ubiquinone in the mitochondrial respiratory chain.     

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.     

Nutritional characteristics of a neoglycoprotein, casein modified covalently by glucose

Glucose was combined covalently with the epsilon-amino groups of lysyl residues of bovine casein in the presence of sodium cyanoborohydride as a reducing reagent by reductive alkylation, forming stable secondary amine linkages. Solubility characteristics and nutritional values of the neoglycoprotein were examined. The degree of modification (%) of the glucosylated casein was 82.5. Solubility of the modified casein was increased by the attachment of glucose. The modification did not disturb the digestion of casein by pepsin or trypsin. Rat feeding experiments using 10% protein diets demonstrated that the protein efficiency ratio (PER) of the modified casein was 0.35 +/- 0.33 compared with 2.99 +/- 0.29 for the unmodified casein. When the modified casein was supplemented with L-lysine to equal the level of total lysine of unmodified casein, the PER value was increased to 2.21 +/- 0.29. Nitrogen balance experiments showed that the modified casein was digested completely. On the other hand, biological value and net protein utilization of the modified protein were shown to be considerably lower than those of the unmodified casein.     

Induction of mutation in mouse FM3A cells by N4-aminocytidine-mediated replicational errors.

To explore the potential use of a nucleoside analog, N4-aminocytidine, in studies of cellular biology, the mechanism of mutation induced by this compound in mouse FM3A cells in culture was studied. On treatment of cells in suspension with N4-aminocytidine, the mutation to ouabain resistance was induced. The major DNA-replicating enzyme in mammalian cells, DNA polymerase alpha, was used to investigate whether the possible cellular metabolite of N4-aminocytidine, N4-aminodeoxycytidine 5'-triphosphate (dCamTP), can be incorporated into the DNA during replication. Using [3H]dCamTP in an in vitro DNA-synthesizing system, we were able to show that this nucleotide analog can be incorporated into newly formed DNA and that it can serve as a substitute for either dCTP or dTTP. dCamTP in the absence of dCTP maintained the activated calf thymus DNA-directed polymerization of deoxynucleoside triphosphates as efficiently as in its presence. Even in the presence of dCTP, dCamTP was incorporated into the polynucleotide. When dCamTP was used as a single substrate in the poly(dA)-oligo(dT)-directed polymerase reaction, it was incorporated into the polynucleotide fraction. The extent of incorporation was 4% of that of dTTP incorporation when dTTP was used as a single substrate. Even in the presence of dTTP, dCamTP incorporation was observed. A copolymer containing N4-aminocytosine residues was shown to incorporate guanine residues opposite the N4-aminocytosines. However, we were unable to observe adenine incorporation opposite N4-aminocytosine in templates. These cell-free experiments show that an AT-to-GC transition can take place in the presence of dCamTP during DNA synthesis, strongly suggesting that the mutation induced in the FM3A cells by N4-aminocytidine is due to replicational errors.     

Day-night changes in production of carbohydrate and protein by natural phytoplankton population from Lake Biwa, Japan

The ¹³C and gas chromatography-mass spectrometry (¹³C-GC-MS)method was applied to determine the day-night changes in thecomposition of photosynthetic products of the natural phytoplanktonpopulation from Lake Biwa, Japan. Glucose is the most abundantmonosaccharide in acid-hydrolyzable carbohydrate. The contributionof glucose was high in incubatesd samples in daytime and decreasedduring the night. Other monosaccharides (rhamnose, fucose, ribose,arabinose, xylose, mannose and galactose) and amino acids tendedto be produced throughout both day- and night-time. These resultssuggested that the carbon flows from glucose, which might constitutereserve glucan, to other monosaccharides and amino acids duringnight-time. The disproportionate production of glucose (reservedglucan) during daytime was thus partly cancelled out at night.     

Effects of denaturation with HCl on the immunological staining of bromodeoxyuridine incorporated into DNA

Immunochemical procedures for detection of BrdUrd incorporated into DNA require a denaturation step of DNA. Denaturation with HCl is widely used for flow cytometric analysis of the cell cycle and for histological preparations. This brief communication describes an attempt to standardize a denaturation procedure with HCl. Various denaturation conditions at 20 degrees C were examined for human promyelocytic leukemia cells (HL-60 cells) fixed in ethanol. After denaturation of DNA, the cells were stained by an indirect immunofluorescence method using a commercially available monoclonal anti-BrdUrd antibody or by propidium iodide. The relative fluorescence intensities of stained BrdUrd and double-stranded DNA were altered reciprocally by changing HCl concentration and/or denaturation time. Treatment with 4N HCl for 10-20 min at 20 degrees C allowed denaturation of more than 80% of DNA and the maximum BrdUrd-linked immunofluorescence. Under this condition, the coefficient of variation of the DNA histograms remained relatively small.     

Novel hemoglobin components and their amino acid sequences from the crab-eating macaque (Macaca fascicularis)

Summary We found two types of hemoglobin, T and R, from the crab-eating macaque and compared those to A and Q previously reported. The 22 animals studied showed six different phenotypes, A, R, QA, QT, QAT, and QAR. Analysis of the complete amino acid sequences for the chains of hemoglobins Q, A, T, and R revealed that amino acids at four positions, 8, 55, 71, and 78 from the N-terminal, are variable. In the ^A chain, Thr, Val, Gly, and Gln occupy these positions, and in the ^Q chain the analogous amino acids are Thr, Val, Asp, and Gln, respectively. In the newly found ^T chain they are Thr, Val, Gly, and His; and in the ^R chain, they are Ser, Ile, Gly, and His, respectively. Two amino acids (8 Thr and 79 Gln) in ^A of the crab-eating macaque were found to be different from those in the chain of the Japanese macaque.     

Isolation and Characterization of a Photosystem II Core Complex Depleted in the 43 kDa-Chlorophyll-Binding Subunit

The photosystem II core complex purified from digitonin extractsof spinach chloroplasts was resolved into two chlorophyll-proteincomplexes by digitonin polyacrylamide gel electrophoresis aftertreatment with 1 M potassium thiocyanate. One of the chlorophyll-proteincomplexes resolved consisted of 47, 32, 30 and 9 kDa polypeptidesand the other was complementally composed of only the 43 kDapolypeptide. The former complex was highly active in the photoreductionof 2, 6-dichlorophenol indophenol by 1,5-diphenylcarbazide andretained all of the components responsible for the electrontransport from the secondary electron donor (Z) to the primaryelectron acceptor (Q_A). EPR signal II_fast and II_slow were alsopreserved in this complex although their hyperfine structureswere largely modified. The complex was estimated to contain1.8 molecules of plastoquinone A as well as 1.5, 3.7 and 3.9molecules of cytochrome b559, pheophytin and ß-carotene,respectively, per Q_A. These results indicate that potassiumthiocyanate specifically removes the 43 kDa polypeptide fromthe PS II core complex leaving the electron transport systemin an almost intact state. (Received June 17, 1987; Accepted October 23, 1987)     

Salt tolerance of the reed plant Phragmites communis

Reed plants ( Phragmites communis Trinius) were grown at NaCl concentrations up to 500 m M and their growth, mineral contents and leaf blade osmotic potential were determined. Addition of NaCl up to 300 m M did not affect growth significantly. Sucrose, Cl^-and Na⁺ concentrations in the shoots increased with the salinity of the medium and the shoot water content decreased. K⁺ always contributed most to the leaf osmotic potential. Even in the presence of 250 m M NaCl in the rooting medium, the leaf blade contained only 50 mM Na⁺, suggesting that the plants have an efficient mechanism for Na⁺ exclusion. ²²Na⁺ uptake experiments suggested that the retranslo-cation of absorbed Na⁺ from shoots to the rooting medium lowered the uptake of Na⁺.