Comparison of DNA methylation patterns among mouse cell lines by restriction landmark genomic scanning.

Restriction landmark genomic scanning (RLGS) is a novel method which enables us to simultaneously visualize a large number of loci as two-dimensional gel spots. By this method, the status of DNA methylation can efficiently be determined by monitoring the appearance or disappearance of spots by using a methylation-sensitive restriction enzyme. In the present study, using RLGS with NotI, we examined, in comparison with a brain RLGS profile, the status of DNA methylation of more than 900 loci among three types of mouse cell lines: the embryonal carcinoma cell line P19, the stable mesenchymal cell line 10T1/2, and our established neuroepithelial (EM) cell lines. We found that the relative numbers of RLGS spots which appeared were less than 3.3% of those surveyed in all cell lines examined. However, 5 to 14% of spots disappeared, the numbers increasing with an increase in the length of the culture period, and many spots were commonly lost in 10T1/2 and in three EM cell lines. Thus, for these cell lines, many more spots disappeared than appeared. However, the numbers of spots disappearing and appearing were well balanced, and the ratio in P19 cells was almost equal to that in liver cells in vivo. These RLGS experimental observations suggested that permanent cell lines such as 10T1/2 are hypermethylated and that our newly established EM cell lines are also becoming heavily methylated at common loci. On the other hand, methylation and demethylation seem to be balanced in P19 cells in a manner similar to that in in vivo liver tissue.     

Transient expression of bile-duct-specific cytokeratin in fetal mouse hepatocytes

The differentiation of hepatocytes and biliary epithelial cells has been histochemically analyzed with anti-calf cytokeratin antiserum in the fetal mouse liver. Almost all young fetal hepatocytes transiently express bile-duct-specific cytokeratin; subsequently, the strong staining of the cytokeratin is confined to progenitor cells of intrahepatic biliary epithelial cells around portal veins. These results suggest that all fetal hepatocytes are bi-potent in terms of the differentiation of mature hepatocytes and intrahepatic bile-duct cells, and that the microenvironment around portal veins plays an important role in bile-duct differentiation. Large periportal hepatocytes continue to stain weakly for cytokeratin until 2 weeks after birth, although the number of positive hepatocytes decreases with development. The differentiation of bile ducts from periportal hepatocytes may continue for 2 weeks after birth.     

Efficient degradation of trichloroethylene by a hybrid aromatic ring dioxygenase.

Engineering of hybrid gene clusters between the toluene metabolic tod operon and the biphenyl metabolic bph operon greatly enhanced the rate of biodegradation of trichloroethylene. Escherichia coli cells carrying a hybrid gene cluster composed of todC1 (the gene encoding the large subunit of toluene terminal dioxygenase in Pseudomonas putida F1), bphA2 (the gene encoding the small subunit of biphenyl terminal dioxygenase in Pseudomonas pseudoalcaligenes KF707), bphA3 (the gene encoding ferredoxin in KF707), and bphA4 (the gene encoding ferredoxin reductase in KF707) degraded trichloroethylene much faster than E. coli cells carrying the original toluene dioxygenase genes (todC1C2BA) or the original biphenyl dioxygenase genes (bphA1A2A3A4).     

Induction of Manganese Superoxide Dismutase by Cytokines and Lipopolysaccharide in Cultured Mouse Astrocytes

Abstract: To determine whether cytokines or lipopolysaccharide (LPS) are involved in the induction of superoxide dismutase (SOD) in the nervous system, we examined the effects of these substances on the levels of SOD in cultured mouse astrocytes. Treatment of astrocytes with 10² to 10⁴ U/ml tumor necrosis factor-α for 3 days increased the levels of Mn SOD in a dose- and time-dependent manner to as much as six times the level under nontreated conditions. Treatment with 1.0 µg/ml LPS for 3 days elicited a fourfold increase in levels of Mn SOD, and the effect of LPS was also dose dependent. Furthermore, Mn SOD in astrocytes was induced by a 3-day exposure to interleukin-1α at concentrations of 10² or 10³ U/ml. However, these stimuli had no effect on levels of copper-zinc SOD (Cu/Zn SOD) in astrocytes. By contrast, interferon-γ did not change the levels of either Mn or Cu/Zn SOD in the cells. The results indicate that the selective induction of Mn SOD by cytokines and LPS, which has been observed in nonnervous tissues, may also occur in nervous tissues. The induction of Mn SOD may represent a mechanism for protection of cells from oxidative stress.     

Flash and smash: rapid freezing of muscle fibers activated by photolysis of caged ATP.

A new approach was used to study transient structural states of cross-bridges during activation of muscle fibers. Rabbit skinned muscle fibers were rapidly and synchronously activated from the rigor state by photolysis of caged ATP in the presence of Ca2+. At several different times during the switch from rigor to fully active tension development, the fibers were rapidly frozen on a liquid helium-cooled metal block, freeze-substituted, and examined in an electron microscope. The limits of structural preservation and resolution with this technique were analyzed. We demonstrate that the resolution of our images is sufficient to draw the following conclusions about cross-bridge structure. Rigor cross-bridges point away from the Z-line and most of them are wider near the thin filaments than near the backbone of the thick filaments. In contrast, cross-bridges in actively contracting fibers stretch between the thick and thin filaments at a variable angle, and are uniformly thin. Diffraction patterns computed from contracting muscle show layer lines both at 38 and 43 nm indicating that active cross-bridges contribute mass to both the actin- and myosin-based helical periodicities. The images obtained from fibers frozen 20 ms after release of ATP show a mixture of rigor and active type cross-bridge configurations. There is little evidence of cross-bridges with the rigor shape by 50 ms, and the difference in configurations between 50 and 300 ms after photolysis is surprisingly subtle.     

The development of emotion regulation ability between the ages of 13 and 18 months in Japanese infants during the strange situation as a function of mothers' styles of emotion expression

The purposes of this study were two-fold. First, it compared Japanese infants' (N=129) abilities to regulate emotions at 13 months and 18 months of age, using the Strange Situation procedure. Second, it examined the relationship between the development of emotion regulation and the mother's emotion expression style as assessed by the Emotion Expression Style Questionnaire (EESQ). The total number of subjects who successfully completed all 8 episodes of the Strange Situation procedure increased significantly during the aging between 13 and 18 months of age, indicating that as a group these infants increased their ability to cope with stressful situations. However, infants who had mothers with negative emotion expression styles did not show greater capacity for emotion regulation at 18 months. These findings suggest that the development of emotion regulation is mediated by the mother's emotion expression style.     

cDNA structure, expression and nucleic acid-binding properties of three RNA-binding proteins in tobacco: occurrence of tissue-specific alternative splicing.

Three cDNAs encoding RNA-binding proteins were isolated from a tobacco (Nicotiana sylvestris) cDNA library. The predicted proteins (RGP-1) are homologous to each other and consist of a consensus-sequence type RNA-binding domain of 80 amino acids in the N-terminal half and a glycine-rich domain of 61-78 amino acids in the C-terminal half. Nucleic acid-binding assay using the in vitro synthesized RGP-1 protein confirmed that it is an RNA-binding protein. Based on its strong affinity for poly(G) and poly(U), the RGP-1 proteins are suggested to bind specifically to G and/or U rich sequences. All three genes are expressed in leaves, roots, flowers and cultured cells, however, the substantial amount of pre-mRNAs are accumulated especially in roots. Sequence analysis and ribonuclease protection assay indicated that significant amounts of alternatively spliced mRNAs, which are produced by differential selection of 5' splice sites, are also present in various tissues. Tissue-specific alternative splicing was found in two of the three genes. The alternatively spliced mRNAs are also detected in polysomal fractions and are suggested to produce truncated polypeptides. A possible role of this alternative splicing is discussed.     

Isolation of cDNA for a Plasma Membrane H+-ATPase from Guard Cells of Vicia faba L.

cDNA encoding the plasma membrane H⁺-ATPase of guard cells ofVicia faba L. was isolated. The clone encoded a 105-kDa polypeptide(956 amino acids) that was 79–85% identical in terms ofamino acid sequence to other plant H⁺-ATPases. High levels ofmRNA explain the high H⁺-ATPase activity of these plasma membranes. (Received December 24, 1994; Accepted April 12, 1995)     

Seasonal body weight, lipid content, and impact of starvation and water stress on adult survivorship and longevity ofNezara viridula andEuschistus heros

Fresh and dry body weights (FW, DW) were greater for adult southern green stink bug,Nezara viridula (L.) than for the brown stink bug,Euschistus heros F. throughout the year in southern Brazil. FemalesN. viridula significantly increased FW and DW in late summer-early autumn, and during mid-spring; femaleE. heros did not show the same rates of increase in FW and DW. FemaleN. viridula were heavier than males, particularly during summer; however, female and maleE. heros were generally similar in weight.E. heros contained significantly greater amounts of lipid thanN. viridula, during mid-autumn to early-spring (April–September). Survivorship (%) and total longevity ofE. heros adults provided water only was greater (34.6–24.6 days, for females and males) than that forN. viridula (14.8–13.0 days); without water and food, longevity was drastically reduced (<7 days) for both species.     

Protective Effects of Pyridoxal Phosphate Against Glucose Deprivation-Induced Damage in Cultured Hippocampal Neurons

Abstract: When hippocampal cultures were deprived of glucose, massive release of lactate dehydrogenase (LDH), an indicator of neuronal death, occurred via NMDA receptor activation. Addition of pyridoxal phosphate (PLP; 1 and 10 µ M ) inhibited this LDH release in a concentration-dependent manner. Prior exposure to PLP evoked more potent inhibitory effects on LDH release compared with those treated at the onset of glucose deprivation. Furthermore, PLP inhibited the reduction of intracellular content of pyruvate induced by glucose deprivation, which was accompanied by the reversal of intracellular ATP depletion. A noteworthy elevation of extracellular glutamate in response to glucose deprivation was completely reversed by addition of PLP. Aminooxyacetic acid, a potent inhibitor of PLP-dependent enzymes, antagonized the effects of PLP on LDH release, pyruvate production, and ATP formation. These results suggest that PLP protects neurons from glucose deprivation-induced damage by enhancing the formation of energy-yielding products and relieving extracellular load of glutamate. The observed phenomena further indicate that PLP might be used prophylactically against neuronal death induced by metabolic disorders.