Dynamic properties of the haptenic site of lipid haptens in phosphatidylcholine membranes. Their relation to the phase transition of the host lattice.

The relation between the dynamic properties of the haptenic site of lipid haptens and the phase transition of the host lattice was investigated using head group spin-labeled phosphatidylethanolamines, that is, spin-label lipid haptens (Br?let, P., and H. M. McConnell, 1976, Proc. Natl. Acad. Sci. USA., 73:2977-2981; Br?let, P., and H. M. McConnell, 1977, Biochemistry, 16:1209-1217). The electron spin resonance (ESR) spectra of the lipid haptens in liposomal membranes showed three narrow resonance lines, whose widths and hyperfine splitting values suggested that the haptenic site, i.e., the spin-label moiety, should be exposed in the water phase. The line width of each peak depended on the host lipid species and on the incubation temperature. A temperature study using dipalmitoylphosphatidylcholine (DPPC) liposomes showed that the dynamic properties of the haptenic site were related to the main phase transition and the subphase transition of the host lattice but not to the prephase transition. The angular amplitudes of the tumbling motion of the haptenic site were estimated using oriented multibilayer systems. The angular amplitude of dipalmitoyl-phosphatidyl-N-[[N-(1-oxyl-2,2,6, 6-tetramethyl-4-piperidinyl)-carbamoyl]-methyl]-ethanolamine in DPPC membranes was 63 degrees at 2 degrees C, and it increased slightly with an increase in temperature regardless of the phase transition of the host lattice. The value for egg phosphatidylcholine (PC) at 25 degrees C was the same as for DPPC above its main phase transition temperature. Rotational correlation time analysis showed that the axial rotation of the haptenic site was preferable to the tumbling motion of the rotational axis, and the predominance depended on the phase transition, Lc----L beta' and P beta'----L alpha. Elongation of the spacer arm between the haptenic site and phosphate increased the angular amplitude of the tumbling motion but reduced the effect of the host lattice. Spin-label lipid haptens with unsaturated fatty acyl chains were distributed heterogeneously in DPPC membranes, whereas those with the same fatty acyl chain as the host lattice were distributed randomly. The ESR spectrum of a lipid hapten under its prephase transition temperature showed two components, broad and narrow. This suggests that at least two different domains, a hapten-rich domain and a hapten-poor one, may coexist in membranes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Enzymatic instability of NADH-cytochrome b5 reductase as a cause of hereditary methemoglobinemia type I (red cell type).

Nucleotide substitutions in the gene for NADH-cytochrome b5 reductase were identified in three independent probands of hereditary methemoglobinemia type I. Patients in Kagoshima and Okinawa in Japan were shown to possess the same base change, from guanine to adenine at codon 57, which results in amino acid substitution from Arg to Gln. This nucleotide change was the same as formerly found in a patient in Toyoake, Japan (Katsube, T., Sakamoto, N., Kobayashi, Y., Seki, R., Hirano, M., Tanishima, K., Tomoda, A., Takazakura, E., Yubisui, T., Takeshita, M., Sakaki, Y., and Fukumaki, Y. (1991) Am. J. Hum. Genet. 48, 799-808). A type I patient in Italy was shown to have a base change from guanine to adenine at codon 105 which causes substitution from Val to Met. To characterize the enzymes of type I patients, Arg-57----Gln and Val-105----Met mutant enzymes were overexpressed in Escherichia coli and purified to homogeneity. kcat/Km values (NADH) of these two enzymes were 25% in Arg-57----Gln and 14.5% in Val-105----Met compared with that of the wild type enzyme, while the value of type II (generalized, severe form of the disease) mutant enzyme was 3% of the normal value (Yubisui, T., Shirabe, K., Takeshita, M., Kobayashi, Y., Fukumaki, Y., Sakaki, Y., and Takano, T. (1991) J. Biol. Chem. 266, 66-70). The type I mutant enzymes were less heat-stable and more susceptible to proteinase treatment than the wild type. From these results we conclude that restriction of enzyme deficiency to red cells in hereditary methemoglobinemia type I may be generally derived from instability and increased proteolytic susceptibility of variant NADH-cytochrome b5 reductases due to a point mutation.     

Ontogeny of the response of the hypothalamo-pituitary-adrenal axis to maternal immobilization stress in rats.

In this study we investigated the response of the rat fetal hypothalamo-pituitary-adrenal (HPA) axis to an acute maternal stress in late gestation. On day 20 of gestation, pregnant rats were exposed to forced immobilization stress for up to 60 min. In mothers, a significant increase in plasma ACTH and corticosterone(B) was observed at 20 and 60 min. The ACTH content in the maternal pituitary decreased significantly at 60 min. Fetal blood pH was decreased by the maternal stress, showing a hypoxic condition of the fetus. Fetal plasma ACTH increased transiently at 20 min. Fetal plasma B increased at 20 and 60 min. ACTH in the fetal pituitary and the placenta did not show marked changes due to the maternal stress. Pregnant rats on day 18-21 of gestation were subjected to a 20 min maternal stress. In the basal condition without stress, fetal plasma ACTH and B showed parallel ontogenic patterns, having a peak value on day 19 of gestation. Fetal plasma ACTH as well as plasma B were increased significantly by the maternal stress at all points evaluated. These results indicate that fetal hypoxia is important in stress transmission to the fetal HPA axis in this type of maternal stress, and the fetal HPA axis responds to the stress as early as day 18 of gestation.     

Site-specific mutagenesis using a gapped duplex vector: a study of translesion synthesis past 8-oxodeoxyguanosine in E. coli

We have constructed a gapped plasmid vector in which a single defined lesion is introduced, site-specifically, within a single-strand region. Efficiency of translesional synthesis is determined by the number of colonies recovered following transformation of E. coli. The nucleotide sequence of progeny plasmids in the gapped region of the vector reflects incorporation of bases opposite and near the lesion. The analysis detects non-mutagenic as well as mutagenic events. This system was used to establish the mutagenic potential of 2'-deoxy-7,8-dihydro-8-oxoguanosine (8-oxodG), a lesion produced by the action of active oxygen species on DNA. The presence of 8-oxodG did not affect the number of transformants recovered. Most transformants (greater than 99%) contained G:C pairs at the site of the lesion; however, a limited number of targeted G----T transversions were observed in the presence and absence of SOS induction. Base substitutions neighboring the lesion, reported for an in vitro system, were not observed. We conclude that the 8-oxodG lesion in DNA is weakly mutagenic in E. coli.     

ESR measurement of radical clearance in lung of whole mouse.

Clearance of the nitroxide radicals, hydroxy-TEMPO and carboxy-PROXYL, in whole-mouse lung was directly measured by in vivo ESR. After injecting a nitroxide radical, distribution of the nitroxide radical all over the lung was confirmed by ESR imaging. The ESR signal of hydroxy-TEMPO was reduced in the lung and the clearance obeyed first-order kinetics, whereas the signal of carboxy-PROXYL remained constant. Comparison of the clearance rates of live and dead mice indicated the presence of 2 different clearance systems in the lung: loss of its paramagnetism in the lung, and transfer from alveolar to the blood circulation system.     

Proliferating cells in histiocytic necrotizing lymphadenitis.

The phenotypes of proliferating cells in histiocytic necrotizing lymphadenitis (HNL) were examined. The affected areas consisted mainly of CD 8-positive (suppressor/cytotoxic T-cells) and CD 4-positive (helper/inducer T-cells) in association with some CD 15-positive cells (monocytes). A marker of proliferating cells (Ki-67) and monoclonal antibodies for determining the phenotypes of cells (CD 4, CD 8, CD 15) in the affected areas were applied using a double-staining method. Ki-67-positive proliferating cells were mainly CD 8-positive. A few CD 4-positive cells and rare CD 15-positive cells were also Ki-67-positive. The percentage of CD 8-positive cells increased gradually over time and the ratio of CD 8-positive to proliferating cells did not decrease throughout the observation period of 6 weeks. These results suggest that the proliferation of CD 8-positive T-cells together with the accumulation of CD 4- and CD 15-positive cells is the main phenomenon occurring in HNL.     

Embryonic, larval and juvenile development of the roughskin sculpin,Trachidermus fasciatus (Scorpaeniformes: Cottidae)

Embryonic, larval and juvenile development of the catadromous roughskin sculpin,Trachidermus fasciatus, were described using eggs spawned in an aquarium. The eggs, measuring 1.98–2.21 mm in diameter, were light reddish-yellow and had many oil globules, 0.05–0.18 mm in diameter. Hatching occurred 30 days after spawning at 2.3–11.3°C. The newly-hatched larvae, measuring 6.9–7.3 mm BL, had a single oil globule, 9–10+25–26=34–36 myomeres and 6 or 7 large stellate melanophores dorsally along the gut. The yolk was almost resorbed, number of pectoral-fin rays attained 16–17, and two parietal, one nuchal and four preopercular spines were formed, 5 days after hatching, at 8.2–8.4 mm BL. The oil globule disappeared, and one supracleithral spine was formed, 11 days after hatching, at 8.9–9.5 mm BL. Notochord flexion began 15 days after hatching, at 9.7–10.3 mm BL. A posttemporal spine was formed 20 days after hatching, at 10.7–10.9 mm BL. The first dorsal fin spines (VII–VIII), second dorsal fin and anal fin rays (18–19, 16–18, respectively) appeared 23 days after hatching, at 12.0–13.7 mm BL. The pelvic fin spine and rays (I, 4) were formed and black bands on the head and sides of the body began to develop 27 days after hatching, at 13.8–15.8 mm BL. Newly-hatched larvae swam just below the surface in the aquaria. Preflexion larvae (8.9–9.5 mm BL), in which the oil globule had disappeared, swam in the middle layer, while juveniles (13.8–15.8 mm BL) began swimming on the bottom of the aquaria. Swimming behavior observed in the aquaria suggested that the fish started to change to a demersal existence at the juvenile stage.     

Illegitimate recombination leading to allelic loss and unbalanced translocation in p53-mutated human lymphoblastoid cells.

Allelic loss and translocation are critical mutational events in human tumorigenesis. Allelic loss, which is usually identified as loss of heterozygosity (LOH), is frequently observed at tumor suppressor loci in various kinds of human tumors. It is generally thought to result from deletion or mitotic recombination between homologous chromosomes. In this report, we demonstrate that illegitimate (nonhomologous) recombination strongly contributes to the generation of allelic loss in p53-mutated cells. Spontaneous and X-ray-induced LOH mutations at the heterozygous thymidine kinase (tk) gene, which is located on the long arm of chromosome 17, from normal (TK6) and p53-mutated (WTK-1) human lymphoblastoid cells were cytogenetically analyzed by chromosome 17 painting. We observed unbalanced translocations in 53% of LOH mutants spontaneously arising from WTK-1 cells but none spontaneously arising from TK6 cells. We postulate that illegitimate recombination was occurring between nonhomologous chromosomes after DNA replication, leading to allelic loss and unbalanced translocations in p53-mutated WTK-1 cells. X-ray irradiation, which induces DNA double-strand breaks (DSBs), enhanced the generation of unbalanced translocation more efficiently in WTK-1 than in TK6 cells. This observation implicates the wild-type p53 protein in the regulation of homologous recombination and recombinational DNA repair of DSBs and suggests a possible mechanism by which loss of p53 function may cause genomic instability.