Structural characterisation of a neutral antitumour β-d-glucan extracted with hot sodium hydroxide from cultured fruit bodies of Grifola frondosa

The structural characterisation of an antitumour β-d-glucan (grifolan-7N), obtained from the hot sodium hydroxide extract of Grifola frandosa, is described. Grifolan-7N, purified by digestion with alpha-amylase, precipitation with ethanol, and chromatography on Con A-Sepharose, gave a single and symmetrical peak on gel filtration with Sepharose CL-4B (0.2m NaOH/8m urea) and had a molecular weight of ～1,200,000. The results of methylation analysis, ¹³C-n.m.r. spectroscopy, Smith degradation, and enzymic digestion indicated grifolan-7N to be a (1→3)-linked β-d-glucan having a single β-d-glucopyranosyl group attached to position 6 of almost every third backbone unit. Grifolan-7N showed potent activity against the solid Sarcoma 180 in mice.     

Pituitary estrogen receptor α and dopamine subtype 2 receptor gene expression in transgenic mice with overproduction of heterologous growth hormones

 Pituitary somatotrophs are suppressed in mice transgenic for human (h) or bovine (b) growth hormone (GH) genes fused with metallothionein (MT) or phosphoenolpyruvate carboxykinase (PEPCK) promoters. Previous morphologic studies revealed that lactotrophs are inhibited in hGH transgenic lines probably due to prolactin-like effects of hGH whereas in female bGH transgenics, the lactotrophs are stimulated. In the present study, estrogen receptor (ERα) mRNA was studied by autoradiographic in situ hybridization (ISH), ERα protein by immunocytochemistry, and dopamine subtype 2 receptor (D2R) mRNA by ISH. In MT/ and PEPCK/hGH transgenic mice, silver grains signaling ERα mRNA were significantly decreased compared to controls; the reduction was stronger in males (8.6 and 37%) than in females (4.6 and 11%). The decrease in the number of ERα-immunoreactive nuclei followed the same pattern (13.3 and 6% in males vs 3.2 and 5.2% in females). In MT/hGH mice the D2R mRNA signal was significantly increased in males (6 and 15.4%) and females (16%). In MT/bGH transgenics, ERα mRNA and ERα-immunoreactive nuclei were significantly increased (25 and 6%) only in males; D2R mRNA was more decreased in females (23%) than in males (15%). In conclusion, the opposite changes in ERα and D2R gene expressions are correlated with lactotroph inhibition in hGH transgenic mice and their stimulation in bGH transgenic mice. The changes in ERα expression were stronger in males, whereas those of D2R were more pronounced in females. Accepted: 16 November 1998     

Activation of calcium-sensing receptor accelerates apoptosis in hyperplastic parathyroid cells

Calcimimetic compounds inhibit not only parathyroid hormone (PTH) synthesis and secretion, but also parathyroid cell proliferation. The aim of this investigation is to examine the effect of the calcimimetic compound NPS R-568 (R-568) on parathyroid cell death in uremic rats. Hyperplastic parathyroid glands were obtained from uremic rats (subtotal nephrectomy and high-phosphorus diet), and incubated in the media only or the media which contained high concentration of R-568 (10(-4)M), or 10% cyclodextrin, for 6h. R-568 treatment significantly suppressed medium PTH concentration compared with that of the other two groups. R-568 treatment not only increased the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay-positive cells, but also induced the morphologic changes of cell death determined by light or electron microscopy. These results suggest that CaR activation by R-568 accelerates parathyroid cell death, probably through an apoptotic mechanism in uremic rats in vitro.     

Mechanisms of spectral tuning in the RH2 pigments of Tokay gecko and American chameleon

At present, molecular bases of spectral tuning in rhodopsin-like (RH2) pigments are not well understood. Here, we have constructed the RH2 pigments of nocturnal Tokay gecko (Gekko gekko) and diurnal American chameleon (Anolis carolinensis) as well as chimeras between them. The RH2 pigments of the gecko and chameleon reconstituted with 11-cis-retinal had the wavelengths of maximal absorption (lambda(max)'s) of 467 and 496 nm, respectively. Chimeric pigment analyses indicated that 76-86%, 14-24%, and 10% of the spectral difference between them could be explained by amino acid differences in transmembrane (TM) helices I-IV, V-VII, and amino acid interactions between the two segments, respectively. Evolutionary and mutagenesis analyses revealed that the lambda(max)'s of the gecko and chameleon pigments diverged from each other not only by S49A (serine to alanine replacement at residue 49), S49F (serine to phenylalanine), L52M (leucine to methionine), D83N (aspartic acid to asparagine), M86T (methionine to threonine), and T97A (threonine to alanine) but also by other amino acid replacements that cause minor lambda(max)-shifts individually.     

Rubralactone, rubralides A, B and C, and rubramin produced by Penicillium rubrum

Rubralactone (1), rubralides A, B and C (2-4), rubramin (5), and 2-formyl-3,5-dihydroxy-4-methylbenzoic acid (6), were isolated from Penicillium rubrum, and their structures established by spectroscopic methods including 2D NMR. The effects on plant growth of 1-6 were examined using the lettuce seedling bioassay. Compound 1 promoted root growth. Compounds 2, 3 and 5 inhibited the growth of lettuce seedlings, but 4 and 6 did not have any inhibitory effect on their growth.     

BEN1, a gene encoding a dihydroflavonol 4-reductase (DFR)-like protein, regulates the levels of brassinosteroids in Arabidopsis thaliana

The ben1-1D (bri1-5 enhanced 1-1dominant) mutant was identified via an activation-tagging screen for bri1-5 extragenic modifiers. bri1-5 is a weak mutant allele of the brassinosteroid receptor gene, BRI1. Overexpression of BEN1 greatly enhances the defective phenotypes of bri1-5 plants. Removal of BEN1 by gene disruption in a Col-0 wild-type background, on the other hand, promotes the elongation of organs. Because BEN1 encodes a novel protein homologous to dihydroflavonol 4-reductase (DFR) and anthocyanidin reductase (BAN), BEN1 is probably involved in a brassinosteroid metabolic pathway. Analyses of brassinosteroid profiles demonstrated that BEN1 is indeed responsible for regulating the levels of several brassinosteroids, including typhasterol, castasterone and brassinolide. In vivo feeding and in vitro biochemical assays suggest that BEN1 is probably involved in a new mechanism to regulate brassinosteroid levels. These results provide additional insight into the regulatory mechanisms of bioactive brassinosteroids.     

Controlling the surface density of DNA on gold by electrically induced desorption

We report on a method to control the packing density of sulfur-bound oligonucleotide layers on metal electrodes by electrical means. In a first step, a dense nucleic acid layer is deposited by self-assembly from solution; in a second step, defined fractions of DNA molecules are released from the surface by applying a series of negative voltage cycles. Systematic investigations of the influence of the applied electrode potentials and oligonucleotide length allow us to identify a sharp desorption onset at -0.65 V versus Ag/AgCl, which is independent of the DNA length. Moreover, our results clearly show the pronounced influence of competitive adsorbents in solution on the desorption behavior, which can prevent the re-adsorption of released DNA molecules, thereby enhancing the desorption efficiency. The method is fully bio-compatible and can be employed to improve the functionality of DNA layers. This is demonstrated in hybridization experiments revealing almost perfect yields for electrically "diluted" DNA layers. The proposed control method is extremely beneficial to the field of DNA-based sensors.     

Possible Involvement of Interleukin-1 in Cyclooxygenase-2Induction After Spinal Cord Injury in Rats

Abstract : A standardized compression injury of rat spinal cord brought about a time-dependent biphasic production of thromboxane A₂ (detected as thromboxane B₂) and prostaglandin I₂ (detected as 6-ketoprostaglandin F_1α. Thromboxane B₂ was predominant during the first 1 h, whereas the 6-ketoprostaglandin F_1α level exceeded that of thromboxane B₂ at 8 h postinjury. As examined by inhibitor experiments and northern blotting, cyclooxygenase-1 was responsible for the first phase, and cyclooxygenase-2 was involved in the second phase. On compression injury the levels of interleukin-1α and -1β detected as mRNA and protein increased and peaked at 2-4 h. Injection of exogenous interleukin-1 α into the spinal cord resulted in an increase of cyclooxygenase-2 mRNA content and a predominant production of 6-ketoprostaglandin F_1α resembling the second phase of eicosanoid production. Concomitantly, extravascular migration of polymorphonuclear leukocytes was enhanced after the interleukin-1α injection. These cells together with vascular endothelial cells and glial cells were stained positively with an anti-cyclooxygenase-2 antibody. The results suggest that the immediate eicosanoid synthesis after spinal cord injury was due to the constitutive cyclooxygenase-1 and the delayed synthesis of eicosanoids was attributable to the induction of cyclooxygenase-2 mediated by interleukin-1 α.     

Phenolic compounds and flavonoids as plant growth regulators from fruit and leaf of Vitex rotundifolia

Five phenolic compounds, 4-hydroxybenzoic acid methyl ester (1), vanillic acid methyl ester (2), 4-hydroxy benzaldehyde (3), 4-hydroxybenzoic acid (4) and ferulic acid (5), and four flavonoids, 5,5'-dihydroxy-4',6,7-trimethoxyflavanone (6), luteolin (7), vitexicarpin (8) and artemetin (9), were isolated from fruits and leaves of Vitex rotundifolia L. The biological activities of these nine compounds have been examined using a bioassay with lettuce seedlings.