Early weaning induces anxiety and precocious myelination in the anterior part of the basolateral amygdala of male Balb/c mice.

Weaning is one of the most important events that occur during the early stages of life. For example, precocious weaning is known to increase anxiety-related behaviors in rodents. Here, we demonstrate that in addition to increasing anxiety, early-weaning manipulations alter the accumulation of galactosylceramide, a specific myelin constituent, and the axonal structure of myelinated fibers in the amygdala of male Balb/c mice. We found that early-weaned male mice entered the open arms of an elevated plus-maze less frequently than normally weaned mice at 3 and 5 weeks of age, which indicates persistently higher anxiety levels. However, early-weaned females exhibited fewer entries into the open arms only at 5 weeks of age. Lipid analysis of mice amygdalas showed the early accumulation of galactosylceramide in early-weaned male, but not female, mice at 5 weeks. The precocious accumulation of galactosylceramide was observed only in the amygdala; galactosylceramide accumulation was not observed in the prefrontal cortex or hippocampus of early-weaned male mice. Electron microscopy showed an increase in the number and a decrease in the diameter of myelinated axons in the anterior part of the basolateral amygdala in early-weaned male mice at 5 weeks. These results suggest that the higher anxiety levels observed in early-weaned male mice could be related to precocious myelin formation in the anterior part of the basolateral amygdala.

Reduced fidelity of DNA synthesis in cell extracts from chemically induced primary thymic lymphomas of mice.

To examine whether the fidelity of DNA synthesis is reduced in tumor cells, M13 mp2-based fidelity assays were carried out using 15 samples of whole-cell extracts from primary mouse thymic lymphomas induced by alkylating agents. We found that DNA synthesis activities of thymic lymphomas, detected as incorporation of [3H]TTP into acid-insoluble materials, were 2- to 10-fold higher compared to those of normal thymus. Furthermore, mutant frequencies in the forward mutation assay of DNA synthesis were increased 2- to 7-fold in cell extracts from thymic lymphomas compared to those from normal thymus. As the DNA polymerase beta (pol beta) activity was extremely high in the thymic lymphomas, we screened mutations in the pol beta gene to examine the possibility of involvement of mutated pol beta in reduction of the fidelity of DNA synthesis. Of 20 lymphomas, one case of point mutation (T to A) was found by reverse transcription-PCR single-strand conformation polymorphism analysis. These results suggest that the mutagenic DNA synthesis is involved in murine thymic lymphoma genesis, although mutation of the pol beta gene is not a major causal event.

Expression of UDP-glucose: poriferasterol glucosyltransferase in the process of differentiation of a true slime mold, Physarum polycephalum.

An expression of UDP-glucose:poriferasterol glucosyltransferase activity associated with differentiation of a true slime mold, Physarum polycephalum, from haploid myxoamoebae to diploid plasmodia was demonstrated. In the haploid cells, this enzyme activity was not detected, but after conjugation of the myxoamoebae, the enzyme activity was expressed and increased definitely. In the plasmodial stage, high enzyme activity was maintained constantly. The enzyme was partially purified (35-fold purification, and 28% yield), and molecular weight of 72,000, pH optimum of 7.0, and some characteristics were demonstrated.

Stimulation of sea urchin DNA polymerase by protein factors. II. Formation of active complex in DNA polymerase reaction.

A protein factor, SF I, which stimulated DNA polymerase activity severalfold was purified from nuclei of sea urchin embryos by phase separation, ammonium sulfate fractionation, DNA-cellulose, CM-cellulose and hydroxyapatitecolumn chromatography and gel filtration. The molecular weight of SF I was about 220 000, the S20,W value was about 8.5 and the isoelectric point was determined to be pH 5.1. In the presence of SF I,V of the DNA-polymerizing reaction was increased and Km values for the substrates of this reaction were not changed. Addition of polyamines increased the rate of stimulation. ATP which was required for stimulation could be substituted by other ribonucleoside triphosphates. SF I, nuclear DNA polymerase and ATP seemed to form an active complex, and in the complex, ATP was found to have been converted to AMP and inorganic pyrophosphate.

DNA synthesis by the isolated nuclear matrix from synchronized plasmodia of Physarum polycephalum.

Nuclear matrices were isolated from plasmodia of a true slime mold, Physarum polycephalum, and the DNA synthetic activity in vitro was examined. These matrices isolated in S-phase catalyzed DNA synthesis requiring Mg2+, deoxyribonucleoside 5'-triphosphates and ATP, without exogenous templates. The activity changed during S-phase with the rate of in vivo DNA replication. Product analysis by gel electrophoresis revealed that the matrices produced Okazaki fragments. These results suggest that DNA synthesis partially reflects in vivo DNA replication. DNA synthesis was sensitive to aphidicolin, heparin and N-ethylmaleimide, indicating involvement of the alpha-like DNA polymerase of Physarum. Exogenous addition of activated DNA stimulated DNA synthesis 4-10-fold and suggested that only some of the existing enzymes are involved in endogenous DNA synthesis. Matrices isolated in G2-phase were also associated with a similar DNA synthetic activity, but they did not produce Okazaki fragments in vitro. It is, therefore, concluded that nuclear matrices are associated with alpha-like DNA polymerase throughout the cell cycle, and that some of the enzymes participate in in vivo DNA replication in S-phase; thus, DNA replication is possibly controlled by this process. The relationship between DNA synthetic activities by the isolated nuclei and matrices was also discussed.

Changes in phospholipid composition and phospholipase D activity during the differentiation of Physarum polycephalum.

Changes in phospholipid composition and phospholipase D activity were observed during a differentiation from haploid myxoamoebae to diploid plasmodia of a true slime mold, Physarum polycephalum. In the amoeboid stage, the main components of phospholipid fraction were phosphatidylethanolamine (PE, 43.3%), phosphatidylcholine (PC, 28.8%) and phosphatidylinositol (PI, 8.0%), but in the plasmodial stage, PC was dominant (40.7%) and other main components were PE (31.5%) and phosphatidic acid (PA, 11.0%). The specific activity of phospholipase D in the plasmodia was 5.7-times higher than that in the myxoamoebae when measured in the presence of Ca2+ at the alkaline pH. In the amoeboid stage, phospholipase A activity (A1 or A2) was detected at the alkaline pH with Ca2+. Phospholipase D activity in the plasmodia was characterized: pH optimum was 6.0; Ca2+ was required for the reaction and Ba2+ could substitute partly for Ca2+; PE was the best substrate for the hydrolytic activity and PC and PI were not appreciably hydrolyzed; and all detergents tested inhibited the enzyme activity.

Side-chain structure is critical for the transport of sterols from lysosomes to cytoplasm.

Macrophages take up and metabolize negatively charged liposomes containing free cholesterol efficiently, resulting in a massive accumulation of cholesteryl esters and triacylglycerols in their cytoplasm (Nishikawa, K., Arai, H. and Inoue, K. (1990) J. Biol. Chem. 265, 5226-5231). This system was used to assess the effects of structural variation of sterol on the intracellular transport and the metabolism of endocytosed sterols by the cells. Liposomes containing phytosterols with an extra one (campesterol) or two (beta-sitosterol, stigmasterol, fucosterol) carbons at the C-24 position of the cholesterol side-chain were endocytosed as efficiently as those containing cholesterol without exhibiting any apparent toxicity on the cells. Esterification of endocyotosed phytosterols was, however, extremely low; campesterol esterification was only 20% that of cholesterol and either beta-sitosterol or stigmasterol was not esterified appreciably. A morphological study showed that the endocytosed phytosterols were accumulated in the phagolysosomes of the cells. Blocking of esterification of endocytosed cholesterol by an acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor did not lead to cholesterol accumulation in the phagolysosomes. These data suggest that accumulation of endocytosed phytosterols in phagolysosomes is not a consequence of the inability of the cell to esterify sterols in the endoplasmic reticulum. In the light of these observations, we conclude that cultured macrophages can discriminate between sterols that differ only by a methyl or ethyl group at the C-24 position at their lysosomal compartment.

Selective inhibition of DNA polymerase-alpha family with chemically synthesized derivatives of PHYLPA, a unique Physarum lysophosphatidic acid.

PHYLPA, a unique Physarum lysophosphatidic acid (LPA), showed selective inhibition of a family of DNA polymerase alpha, including DNA polymerases alpha, delta and epsilon; but no inhibition of DNA polymerase beta or gamma was observed. To reveal the molecular mechanism of inhibition of DNA polymerases by PHYLPA, four stereoisomers and some other derivatives were synthesized and their effects on DNA polymerases were studied. Among eight derivatives synthesized, PHYLPA-1 (the natural PHYLPA; sodium 1-O-[(9'S,10'R)-9',10'-methanohexadecanoyl]-sn-glycerol 2,3-cyclic phosphate) and PHYLPA-2 (sodium 3-O-[9'S,10'R)-9',10'-methanohexadecanoyl]-sn-glycerol 1,2-cyclic phosphate) were strong and specific inhibitors of a family of DNA polymerase alpha. But their stereoisomers PHYLPA-3 (sodium 1-O-[9'R,10'S)-9',10'-methanohexadecanoyl]-sn-glycerol 2,3-cyclic phosphate) and PHYLPA-4 (sodium 3-O-[9'R,10'S)-9',10'-methanohexadecanoyl-sn-glycerol 1,2 cyclic phosphate) were weak inhibitors, showing the critical importance of stereochemistry of a cyclopropane-containing fatty acid for the inhibitory activity. Some derivatives having no cyclopropane-containing fatty acids--palmitoyl-, oleoyl-, and palmitoleoyl-PHYLPA--showed inhibition to some extent; but 1-palmytoyl and 1-oleoyl lysophosphatidic acid, which has no cyclic phosphate, did not show an apparent inhibitor activity on DNA polymerases. Hence, the extent of the inhibition apparently depends on the stereochemistry of both the fatty acid moiety and the cyclic phosphate.

Expression of cholesteryl glucoside by heat shock in human fibroblasts.

We investigated the heat-induced alteration of glycolipids in human cultured cells, TIG-3 fibroblasts, to show the expression of steryl glucoside by heat shock. A glycolipid band was detected on a thin-layer chromatography plate in lipid extracts from TIG-3 cells exposed to high temperature (42 degrees C) for 15 and 30 minutes, while it was hardly detectable without heat shock. Both cholesterol and glucose were almost exclusively detected by gas liquid chromatography as degradation products of the lipid. The structure of the lipid molecule was elucidated by electrospray mass spectrometry to be a cholesteryl glucoside. This is the first report to show the occurrence of a steryl glucoside in mammalian cells, and this substance is considered to have a significant role in heat shock responses in mammalian cells.

A cytoplasmic inhibitor of DNA polymerase from the plasmodia of Physarum polycephalum.

A factor which inhibited DNA polymerase [EC 2.7.7.7] activity was isolated from the cytoplasm of plasmodia of true slime mold, Physarum polycephalum. This factor was purified by DEAE-Sephadex and CM-cellulose column chromatographies, heat treatment and gel filtration. This inhibitor was heat-stable, insensitive to trypsin [EC 3.4.21.4] and was not digested by RNase [EC 3.1.4.22] or DNase [EC 3.1.4.5]. The molecular weight was 16,000 as determined by gel filtration, and the isoelectric point was determined to be pH 10.1. In the presence of the inhibitor, Km for DNA in the DNA polymerizing reaction was markedly increased. The inhibitory effect was eliminated by addition of excess DNA, but the addition of excess enzyme or deoxyribonucleoside triphosphates had no effect on the inhibition.

A trisialosyl ganglioside GT3 of hog kidney. Structure and biosynthesis in vitro.

A trisialosyl ganglioside was isolated from hog kidney cortex and purified with a yield of 6.8 nmol/g tissue or 13 mol % of total gangliosides. The homogeneous ganglioside was subjected to graded neuraminidase treatment, mild acid hydrolysis, periodate oxidation-borohydride reduction, permethylation analysis and chromium trioxide oxidation. The results suggested that the structure of this ganglioside is NeuAc alpha 2-8NeuAc alpha 2-8NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1Cer (GT3). On the addition of exogenous disialosyl ganglioside GD3 and radioactive CMP-NeuAc, a GT3 synthesis activity was demonstrated in a crude membrane fraction of hog kidney. The activity was greatest in the presence of detergents and had a pH optimum of 6.4. Apparent Km values for CMP-NeuAc and GD3 were about 0. 6 and 0.1 mM, respectively. The reaction produce was assigned as GT3 by chromatographic separation, carbohydrate analysis and periodate oxidation followed by borohydride reduction.

A novel 66-kDa stress protein, p66, associated with the process of cyst formation of Physarum polycephalum is a Physarum homologue of a yeast actin-interacting protein, AIP1.

When exposed to various stresses including heat shock, myxoamoebae, growing haploid cells of Physarum polycephalum, show marked morphological changes and consequently become disk-shaped microcysts. We have found that p66 is induced exclusively in the course of microcyst formation and has an actin-binding activity. In this study, we purified p66 to homogeneity and isolated a p66 cDNA. The deduced protein sequence contained 601 amino acids and showed 31% identity to a yeast actin-interacting protein, AIP1. Northern blot analysis revealed that the amount of p66 mRNA was significantly increased by heat shock in myxoamoebae but not in plasmodia. Thus, p66 seems to be a developmentally-expressed stress protein which regulates the rearrangement of actin organization during microcyst formation in P. polycephalum.

Existence of a bioactive lipid, cyclic phosphatidic acid, bound to human serum albumin.

A novel bioactive lipid, cyclic phosphatidic acid (cPA), was identified in lipids bound to human serum albumin. A cPA fraction was extracted and purified from human serum albumin by use of a combination of preparative TLC and HPLC. Electrospray ionization mass spectrometry of the purified fraction showed molecular ions corresponding to cPA, which was composed of some different fatty acid species. The most abundant component was identified as palmitoyl-cPA by tandem mass spectrometry using collision-induced dissociation. These data have established that cPA is a naturally occurring lipid bound to human serum albumin.

Unique characteristics of radiation-induced apoptosis in the postnatally developing small intestine and colon of mice.

Abstract We examined the response of the developing mouse intestine to X radiation using neonates (1 day postpartum), infants (2 weeks postpartum) and adults (7 weeks postpartum). Irradiated adult small intestinal crypts displayed two waves of apoptosis. The first wave peaked at 3 h and was followed by a broad wave with a peak persisting from 24 to 48 h. p53 was expressed during the first wave but not the second wave. For the infant small intestine, the intensity of the first wave was approximately half that of the adult wave, and for the colon the intensity was even smaller. In neonates, apoptosis was delayed, peaking at 6 h for small intestinal crypts and at 24 h for colonic crypts. Although no apoptosis occurred at 3 h postirradiation in neonates, p53 was present in both the small intestine and colon, owing at least in part to the inability of p53 to increase the level of Noxa, a p53-dependent pro-apoptosis protein, suggesting a discontinuity in the p53-Noxa-caspase pathway in neonates. By contrast, the induction of p21, a pro-survival protein, was greater in neonatal cells than in adult cells. Thus it appears that the developing and adult intestine mount distinct apoptotic responses to radiation.