Difference in growth factor requirements of rat 3Y1 cells among growth in mass culture,clonal growth in low density culture,and stimulation to enter S phase in resting culture

Summary A semiserum-free medium was developed for monolayer culture of rat 3Y1 fibroblastic cells. The main components of the developed medium added to Dulbecco's modified Eagle's medium (DMEM) were insulin transferrin epidermal growth factor, poly-d-lysine, bovine albumin, oleic acid, and bovine α-globulin. In this medium, 3Y1 cells grew in mass culture at much the same rate as in DMEM supplemented with 10% fetat bovine serum (FBS), and colonies, albeit of smaller sizes, diddform. Virally transformed derivatives of 3Y1 (simian virus 40-3Y1, polyoma virus-3Y1 and adenovirus type 12-3Y1) also formed colonies in the semiserum-free medium. When trypsinized 3Y1 cells were seeded with the medium lacking α-globulin, neither growth in the mass culture nor clonal growth in the low density culture (clonal growth) occurred. In this case, cell spreading was inhibited by albumin, and this inhibition was overcome by adding α-globulin or treating dishes with serum. When albumin was excluded from the semiserum-free medium, clonal growth did not occur, whereas growth in mass culture and stimulation of DNA synthesis in the resting mass culture (stimulation of DNA synthesis) were not so drastically affected. When oleic acid was removed, growth in mass culture was inhibited considerably, but no considerable effect was seen on clonal growth or on stimulation of DNA synthesis. In the absence of insulin, stimulation of DNA synthesis was inhibited more markedly than when other components were removed, but such was not the case with growth in mass culture and clonal growth.     

Chemical characteristics of pond waters in the Labyrinth of southern Victoria Land,Antarctica

A large number of fresh water and saline ponds are located in the Labyrinth (77° 33 S, 160° 50 E) of the upper Wright Valley in the Dry Valleys region of southern Victoria Land, Antarctica. It is situated near the terminus of the Wright Upper Glacier between 800–1000 m above sea level. From a limnological point of view, the most interesting problems concerning these saline ponds are the origin of their salts and their evolutional history.Chloride ion contents vary remarkably among the ponds ranging from 0.0049 to 52.4 g kg^–1. Surprisingly, more than a half of the ponds are saline with the highest chloride ion content being 2.7 times greater than that of seawater. The D and ¹⁸O values of the pond waters indicate a snow and/or glacial meltwaters origin, and that the ponds underwent subsequent alteration due to evaporation or freezing. The composition of chemical components reveal no evidence of trapped seawater. Thus the salt concentrations in the Labyrinth pond waters must be explained principally by the accumulation of atmospheric salts and subsequent repeated cycles of evaporation and freezing of the pond waters over considerable time periods.     

Insertion sequence-based cassette PCR: cultivation-independent isolation of γ-hexachlorocyclohexane-degrading genes from soil DNA

gamma-Hexachlorocyclohexane (gamma-HCH) is a highly chlorinated pesticide that has caused serious environmental problems. Based on the frequently observed association of insertion sequence IS6100 with lin genes for gamma-HCH degradation in several gamma-HCH-degrading bacterial strains isolated to date, DNA fragments flanked by two copies of IS6100 were amplified by nested polymerase chain reaction (PCR) technique using a DNA sample extracted from soil contaminated with HCH. Four distinct DNA fragments with sizes of 6.6, 2.6, 1.6, and 1.3 kb were obtained, three of which carried lin genes: the 6.6-kb fragment carried linD and linE as well as linR; the 2.6-kb fragment showed a truncated form of linF; and the 1.6-kb fragment carried linB. Our approach, named as insertion sequence (IS)-based cassette PCR, was successful in the isolation of the lin genes from HCH-contaminated soil without cultivation of host cells and is applicable for the culture-independent isolation of other functional genes bordered by other IS elements.     

Phytochromes and cryptochromes regulate the differential growth of Arabidopsis hypocotyls in both a PGP19-dependent and a PGP19-independent manner

Photoreceptors, phytochromes and cryptochromes regulate hypocotyl growth under specific conditions, by suppressing negative gravitropism, modulating phototropism and inhibiting elongation. Although these effects seem to be partially caused via the regulation of the phytohormone auxin, the molecular mechanisms underlying this process are still poorly understood. In our present study, we demonstrate that the flabby mutation enhances both phytochrome- and cryptochrome-inducible hypocotyl bending in Arabidopsis. The FLABBY gene encodes the ABC-type auxin transporter, PGP19, and its expression is suppressed by the activation of phytochromes and cryptochromes. Our current results therefore indicate that the phytochromes and cryptochromes have at least two effects upon the tropic responses of the hypocotyls in Arabidopsis: the enhancement of hypocotyl bending through the suppression of PGP19, and a PGP19-independent mechanism that induces hypocotyl bending. By the using an auxin polar transport assay and DR5:GUS expression analysis, we further find that the phytochromes inhibit basipetal auxin transport, and induce the asymmetric distribution of auxin in the hypocotyls. These data suggest that the control of auxin transport by phytochromes and cryptochromes is a critical regulatory component of hypocotyl growth in response to light.     

Occurrence of 2-hydroxy acids in microalgae

2-Hydroxy acids were believed to be absent in algae until this study, in which the analysis of microalgae belonging to Chlorophyta (Chlamydomonas reinhardtii and Chlorella pyrenoidosa), Rhodophyta (Cyanidium caldarium M-8 and Cyanidium caldarium RK-1) and Cyanophyta (Anbaena variabilis, Anacystis nidulans, Oscillatoria species and Phormidium foveolarum) is reported. 2-Hydroxy adds with carbon chain lengths of C₁₆-C₂₆, were found in all the algal samples studied, ranging in concentrations from 4.0 to 320μg/g dry alga. The dominant constituents are 2-hydroxyhexadecanoic, 2-hydroxynonadecanoic, 2-hydroxyhexacosanoic and a branched 2-hydroxy-C₁₉ acid. The distribution patterns of the acids differed significantly among the algal samples. Hence 2-hydroxy acids may be useful for the classification of algal species as well as being an important source of 2-hydroxy acids in the natural environment.     

Isopoly-L-ornithine Derivatives of Thymine and Thymidine†

Abstract

L-Ornithine derivatives of thymine, and thymidine gave oligomers by solid phase elongation reactions. These oligomers 2 and 3, however, hardly interact with the complementary polynucleotide. Conformational studies of the oligomers with CD and NMR revealed that stable intramolecular hydrogen bonding was formed between thymine base and ornithine unit.

     

Microarray Analysis of Perinatal-Estrogen-Induced Changes in Gene Expression Related to Brain Sexual Differentiation in Mice

Sexual dimorphism of the behaviors or physiological functions in mammals is mainly due to the sex difference of the brain. A number of studies have suggested that the brain is masculinized or defeminized by estradiol converted from testicular androgens in perinatal period in rodents. However, the mechanisms of estrogen action resulting in masculinization/defeminization of the brain have not been clarified yet. The large-scale analysis with microarray in the present study is an attempt to obtain the candidate gene(s) mediating the perinatal estrogen effect causing the brain sexual differentiation. Female mice were injected with estradiol benzoate (EB) or vehicle on the day of birth, and the hypothalamus was collected at either 1, 3, 6, 12, or 24 h after the EB injection. More than one hundred genes down-regulated by the EB treatment in a biphasic manner peaked at 3 h and 12-24 h after the EB treatment, while forty to seventy genes were constantly up-regulated after it. Twelve genes, including Ptgds, Hcrt, Tmed2, Klc1, and Nedd4, whose mRNA expressions were down-regulated by the neonatal EB treatment, were chosen for further examination by semiquantitative RT-PCR in the hypothalamus of perinatal intact male and female mice. We selected the genes based on the known profiles of their potential roles in brain development. mRNA expression levels of Ptgds, Hcrt, Tmed2, and Nedd4 were significantly lower in male mice than females at the day of birth, suggesting that the genes are down-regulated by estrogen converted from testicular androgen in perinatal male mice. Some genes, such as Ptgds encoding prostaglandin D2 production enzyme and Hcrt encording orexin, have been reported to have a role in neuroprotection. Thus, Ptgds and Hcrt could be possible candidate genes, which may mediate the effect of perinatal estrogen responsible for brain sexual differentiation.     

Distribution of Intravenously Administered Acetylcholinesterase Inhibitor and Acetylcholinesterase Activity in the Adrenal Gland: 11C-Donepezil PET Study in the Normal Rat

Purpose

Acetylcholinesterase (AChE) inhibitors have been used for patients with Alzheimer''s disease. However, its pharmacokinetics in non-target organs other than the brain has not been clarified yet. The purpose of this study was to evaluate the relationship between the whole-body distribution of intravenously administered ¹¹C-Donepezil (DNP) and the AChE activity in the normal rat, with special focus on the adrenal glands.

Methods

The distribution of ¹¹C-DNP was investigated by PET/CT in 6 normal male Wistar rats (8 weeks old, body weight  = 220±8.9 g). A 30-min dynamic scan was started simultaneously with an intravenous bolus injection of ¹¹C-DNP (45.0±10.7 MBq). The whole-body distribution of the ¹¹C-DNP PET was evaluated based on the Vt (total distribution volume) by Logan-plot analysis. A fluorometric assay was performed to quantify the AChE activity in homogenized tissue solutions of the major organs.

Results

The PET analysis using Vt showed that the adrenal glands had the 2nd highest level of ¹¹C-DNP in the body (following the liver) (13.33±1.08 and 19.43±1.29 ml/cm³, respectively), indicating that the distribution of ¹¹C-DNP was the highest in the adrenal glands, except for that in the excretory organs. The AChE activity was the third highest in the adrenal glands (following the small intestine and the stomach) (24.9±1.6, 83.1±3.0, and 38.5±8.1 mU/mg, respectively), indicating high activity of AChE in the adrenal glands.

Conclusions

We demonstrated the whole-body distribution of ¹¹C-DNP by PET and the AChE activity in the major organs by fluorometric assay in the normal rat. High accumulation of ¹¹C-DNP was observed in the adrenal glands, which suggested the risk of enhanced cholinergic synaptic transmission by the use of AChE inhibitors.     

Structure and photodimerizations of 1-alkylthymines in single crystals.

1-Alkylthymine gave four kinds of crystals depending on the solvents used. Photodimerizations of 1-alkylthymine in single crystals were found to depend on the crystal structure. In inactive crystals, the terminal methyl group of alkyl chains approached to the double bond of thymine, and inhibited rotation of thymine bases during photodimerization.