Enhancement of Nitrogen Dioxide-Induced Lipid Peroxidation and Dna Strand Breaking by Cysteine and Glutathione

Nitrogen dioxide less than 100 ppm in air induced lipid peroxidation of liposome composed of l-palmitoyl-2-arachidonylphosphatidylcholine as assessed by thiobarbituric acid reactivity. The nitrogen dioxide-induced lipid peroxidation was enhanced by cysteine, glutathione and bovine serum albumin. While the activity of nitrogen dioxide in air to induce single strand breaks of supercoiled plasmid DNA was low, the breaking was remarkably enhanced by cysteine, glutathione and bovine serum albumin. ESR spin trapping using 5,5-dimethyl-1-pyrroline N-oxide showed that certain strong oxidant(s) were generated by interaction of nitrogen dioxide and cysteine. The spin trapping using 3,5-dibromo-4-nitrosobenzene-sulfonate suggested that sulfur-containing radicals were generated by interaction of nitrogen dioxide and cysteine or glutathione. Hence, certain sulfur-containing radicals generated by the interaction which could effectively induce lipid peroxidation and DNA strand breaks.     

Ontogenesis of gastrin cells in the pyloric antrum and duodenum of the mouse

Summary Ontogenesis of gastrin cells was studied in the pyloroduodenal mucosa of the mouse using anti-human G17 serum, R-1301, and anti-human G34(1–15) serum, R-2703. R-1301-immunostained cells first appeared in the pyloric mucosa of 14-day-old fetuses. Cells stained with both R-1301 and R-2703 appeared immediately after birth, and gradually increased in number to the adult level. Most R-1301-reactive cells were also reactive to R-2703, whereas some cells that reacted with R-1301 exhibited very weak or no reaction with R-2703. The discrepancy between these two immunoreactivities is discussed.In the duodenum, a considerable number of R-1301-reactive cells were present from the perinatal stage and through out adult development. A few R-2703-reactive cells were seen in the duodenum of young mice but not of the adult.     

Forward mutation assay of V79 cells to 6-thioguanine resistance in a soft agar technique that eliminates effects of metabolic co-operation

A technique involving culture in soft agar was used for the assay of forward mutation of V79 cells to 6-thioguanine (6TG) resistance. The main reason for the use of soft agar was to prevent reduction in recovery of mutants depending on the cell density plated for mutation selection, which is the chief problem in the liquid method, and which results mainly from metabolic co-operation due to cell-to-cell contact.V79 cells grew well in fortified soft agar medium (DMEM + 20% FBS) showing cloning efficiencies (>80%) as high as in liquid culture. Therefore, V79/HGPRT mutagenesis could be assayed quantitatively in soft agar culture.The frequency of 6TG-resistant colonies in agar selective medium increased linearly with increase in concentration of EMS. Toxicity and mutagenic responses were greater in soft agar than in liquid culture.In cultures of untreated and EMS-treated cells, more than 95% of the 6TG-resistant colonies isolated were aminopterin-sensitive.Use of soft agar for selection prevented the reduction in the number of mutants with increase in the size of incula on plating up to 1?2 × 10⁶ cells per 9-cm dish: in liquid culture, even with a lower plating number (2 × 10⁵ cells per 9-cm dish), a notable reduction in numbers of mutants was observed. This character was re-examined in a reconstruction experiment. The results show that, when up to 2 × 10⁶ cells were plated per 9-cm dish, 6TG-resistant cells were almost completely recovered from the soft agar medium, whereas only 10% were recovered from liquid culture.     

Sexual differentiation,gonad development,and spawning seasonality of the Hawaiian butterflyfish,Chaetodon multicinctus

Synopsis The reproductive biology of the coral reef butterflyfish,Chaetodon multicinctus, was investigated by histological examination of gonads sampled over an 18 month period from a shallow inshore population on Oahu, Hawaii. Most gonads developed directly from previously undifferentiated tissue. Ovarian development (the structural formation of lamellae and primary oocytes) was observed in fish ≥44 mm and testicular development (the formation of spermatogenic crypts) in fish ≥62 mm standard length (SL). In addition, testis formation was identified within the ovarian lamellae of several differentiated but immature fish. It is hypothesized that prematurational sex change may facilitate monogamy within the highly competitive social structure of this site attached species. Oocyte development in mature females was marked by distinct phases of primary growth, the formation of yolk vesicles, and vitellogenesis. Spawning activity was histologically identified by the maturation and hydration of fully yolked oocytes, and presence of postovulatory follicles. Recently spawned females from field collections and experimental gonadotropin-treatments exhibited postovulatory follicles that were estimated to persist at least 24 h after ovulation. Atresia of yolked oocytes was classified into four stages of cell degeneration and resorption. Monthly analyses of oocyte development and atresia within the sample population show thatC. multicinctus has a protracted annual spawning season with a major peak during the early spring and evidence of spawning activity among some individuals in the fall. Histological analyses of spawning activity provide more accurate and unambiguous information than do traditional gonadosomatic assays in this and probably other coral reef fishes.     

The Relationship between Systemic Resistance Induced by Pruning and Accumulation of Antifungal Substances in Barley Seedlings

Infection by a compatible race of Erysiphe graminis f. sp. hordei on barley secondary leaves was significantly suppressed upon pruning of the primary leaves when E. graminis hordei was inoculated 3–12 h after the pruning, but it, was rather enhanced during 15–21 h. The accumulation of antifungal substances was detected in hot ethanol extracts of barley seedlings from 15–27 h after pruning the primary leaves. Taking the time of the infection process of a challenger (E. graminis, hordei) into consideration, timing of systemic resistance induced upon pruning coincided with the accumulation of antifungal substances.     

Observations and measurements of sea urchin eggs with a centrifuge microscope

Eggs of the sea urchins, Arbacia punctulata and Lytechinus variegatus were observed with a centrifuge microscope. The protoplasmic viscosity calculated from the displacement velocity of the nucleus in a centrifugal field is about 30 poises. The surface forces of the unfertilized egg, which were determined from the relationship between the deformations of the egg in centrifugal fields and the magnitudes of the centrifugal forces, are 0.046 dyne/cm in Arbacia and 0.087 dyne/cm in Lytechinus (mean values). Both of these values increase as the deformation of the egg increases. The cleavage plane of the first cleavage of the egg in the centrifugal field (35–140 × g) is parallel to the direction of the centrifugal force. The flattening of the fertilized egg in a constant centrifugal field changes during development, probably owing to the change in the surface force. The flattening attains a minimum shortly before the onset of cleavage and another minimum during the cleavage, corresponding to a peak of the surface before the cleavage and another peak during the cleavage.     

Vitamin D2 interacts with Human PrPc (90–231) and breaks PrPc oligomerization in vitro

PrP^sc, the pathogenic isoform of PrP^c, can convert PrP^c into PrP^sc through direct interactions. PrP^c oligomerization is a required processing step before PrP^sc formation, and soluble oligomers appear to be the toxic species in amyloid-related disorders. In the current study, direct interactions between vitamin D₂ and human recombinant PrP^c (90–231) were observed by Biacore assay, and 3F4 antibody, specific for amino acid fragment 109–112 of PrP^c, inhibited this interaction. An ELISA study using3F4 antibody showed that PrP^c (101–130), corresponding sequence to human PrP, was affected by vitamin D₂, supporting the results of Biacore studies and suggesting that the PrP^c sequence around the 3F4 epitope was responsible for the interaction with vitamin D₂. Furthermore, the effects of vitamin D₂ on disruption of PrP^c (90–231) oligomerization were elucidated by dot blot analysis and differential protease k susceptibilities. While many chemical compounds have been proposed as potential therapeutic agents for the treatment of scrapie, most of these are toxic. However, given the safety and blood brain barrier permeability of vitamin D₂, we propose that vitamin D₂ may be a suitable agent to target PrP^c in the brain and therefore is a potential therapeutic candidate for prion disease.     

Invariant Asp-1122 and Asp-1124 are essential residues for polymerization catalysis of family D DNA polymerase from Pyrococcus horikoshii

Family D DNA polymerase has recently been found in the Euryarchaeota subdomain of Archaea. Its genes are adjacent to several other genes related to DNA replication, repair, and recombination in the genome, suggesting that this enzyme may be the major DNA replicase in Euryarchaeota. Although it possesses strong polymerization and proofreading activities, the motifs common to other DNA polymerase families are absent in its sequences. Here we report the mapping of the catalytic residues in a family D DNA polymerase from Pyrococcus horikoshii. Site-directed alanine mutants for 28 conserved aspartic acid or glutamic acid residues were screened for polymerization and 3'-5' exonuclease activities. We identified the invariant aspartates Asp-1122 and Asp-1124 within the most conserved motif as the catalytic residues involved in DNA polymerization. Alanine mutation at either site caused a loss of polymerization activity, whereas the conserved mutants, D1122E, D1124N, and D1124E, had slightly reduced polymerization activity. We also found that the 3'-5' exonuclease activity remains in D1122A and D1124A, indicating that the catalytic residues of DNA polymerization are different from those of the 3'-5' exonuclease activity. Furthermore we determined the molecular mass of the recombinant enzyme by gel filtration and proposed a heterotetrameric structure for this enzyme.     

Effective inhibition by beta-carotene of cellular DNA breaking induced by peroxynitrous acid

Peroxynitrous acid synthesized by reaction of hydrogen peroxide and nitrite and generated from 3-morpholinosydononimine (SIN-1) induced cellular DNA breaking of human promyelocytic leukemia HL-60 cells in phosphate buffer (pH 7.5) as assessed by alkaline single cell gel electrophoresis (comet) assay and quantification of comet types. Ascorbate and Trolox inhibited cellular DNA breaking induced by peroxynitrous acid, but the concentrations of these antioxidants required for effective inhibition was about 50-fold higher than that of peroxynitrous acid. βCarotene protected DNA breaking by peroxynitrous acid in 20% tetrahydrofuran-phosphate buffer (pH 7.5) much more effectively than ascorbate and Trolox. The concentrations of β-carotene required for effective inhibition was lower than the concentration of peroxynitrous acid.