Effect of cell cholesterol content on apolipoprotein B secretion and LDL receptor activity in the human hepatoma cell line, HepG2

Human hepatoma HepG2 cells were used to study the effects of cholesterol loading and depletion on apolipoprotein B (apoB) secretion and low-density lipoprotein (LDL) receptor activity. Exposure of HepG2 cells to cholesterol and oleic acid, which elevated intracellular cholesterol levels, stimulated apoB secretion and reduced receptor-mediated uptake of LDL, whereas recombinant complexes of apolipoprotein A-I with dimyristoylphosphatidylcholine, which depleted the cellular cholesterol pool, inhibited apoB secretion and up-regulated LDL receptors. Significant negative correlation (r = -0.92, P less than 0.001) between the levels of apoB secretion and LDL uptake was found. These data suggest that the cholesterol content of the cells may induce concomitant changes in apoB secretion and LDL receptor activity.     

A real time learning algorithm for recurrent analog neural networks

A new learning algorithm is described for a general class of recurrent analog neural networks which ultimately settle down to a steady state. Recently, Pineda (Pineda 1987; Almeida 1987; Ikeda et al. 1988) has introduced a learning rule for the recurrent net in which the connection weights are adjusted so that the distance between the stable outputs of the current system and the desired outputs will be maximally decreased. In this method, many cycles are needed in order to get a target system. In each cycle, the recurrent net is run until it reaches a stable state. After that, the weight change is calculated by using a linearized recurrent net which receives the current error of the system as a bias input. In the new algorithm the weights are changed so that the total error of neuron outputs over the entire trajectory is minimized. The weights are adjusted in real time when the network is running. In this method, the trajectory to the target system can be controlled, whereas Pineda's algorithm only controls the position of the fixed point. The relation to the back propagation method (Hinton et al. 1986) is also discussed.     

A quantitative comparison of foliage development among allopatric ferns,Dryopteris crassirhizoma,D. coreano-montana andD. filix-mas

A quantitative comparison was conducted on the foliage development during sporophyte development of three allopatric ferns in cool temperate and subalpine regions of Hokkaido and Tirol, European Alps. The foliage development ofDryopteris crassirhizoma, D. coreano-montana andD. filix-mas was quantitatively described by the leaf development (NV, number of veins); NV correlates the leaf-shape complexity from a circle (DI, L/2(3.14×S)^1/2). Nearly similar patterns were detected on frequency distribution of fertile leaves, fertility increase and number of leaves in threeDryopteris ferns which exhibit funnel-shaped foliage arrangements in mature sporophyte. No difference was found in number of leaves, maximum NV, fertility rate and leaf-shape parameters among three ferns. A positive difference was found only on changes in order of pinnae with maximum number of costa branches (NVP) and the DI of outline of pinnae betweenD. crassirhizoma andD. filix-mas. These allopatricDryopteris ferns seem to have a similar foliage structure, in spite of some sympatricDryopteris ferns capable of producing putative hybrids (D. austriaca andD. amurensis; D. tokyoensis andD. monticola) having different foliage structures in Hokkaido. Contribution No. 3346 from the Institute of Low Temperature Science, Hokkaido University.     

Heterocyclic amine-DNA adducts analyzed by 32P-postlabeling method

DNA adducts formed by 12 heterocyclic amines were analyzed by 32P-postlabeling method. Several DNA adducts were detected in rat liver by administration of each heterocyclic amine. Total adduct levels ranged from 0.5 for 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) to more than 250 for 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) per 10(7) nucleotides 24 hr after intragastric administration of these compounds. The N-hydroxy derivative of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) was reactive toward DNA in vitro to form adducts. Addition of acetic anhydride to N-OH-MeIQx greatly enhanced its reactivity to DNA. 32P-Postlabeling analysis revealed that the MeIQx-DNA adducts formed in vivo and in vitro were identical. Thus, MeIQx would be metabolized in vivo to N-hydroxy form and further esterified to produce more reactive species, such as N-acetoxy form, which modify DNA to form adducts.     

Processing, secretion, and biological properties of a novel growth factor of the fibroblast growth factor family with oncogenic potential.

We recently reported that the protein encoded in a novel human oncogene isolated from Kaposi sarcoma DNA was a growth factor with significant homology to basic and acidic fibroblast growth factors (FGFs). To study the properties of this growth factor (referred to as K-FGF) and the mechanism by which the K-fgf oncogene transforms cells, we have studied the production and processing of K-FGF in COS-1 cells transfected with a plasmid encoding the K-fgf cDNA. The results show that, unlike basic and acidic FGFs, the K-FGF protein is cleaved after a signal peptide, glycosylated, and efficiently secreted as a mature protein of 176 or 175 amino acids. Inhibition of glycosylation impaired secretion, and the stability of the secreted K-FGF was greatly enhanced by the presence of heparin in the cultured medium. We have used the conditioned medium from transfected COS-1 cells to test K-FGF biological activity. Similar to basic FGF, the K-FGF protein was mitogenic for fibroblasts and endothelial cells and induced the growth of NIH 3T3 mouse cells in serum-free medium. Accordingly, K-fgf-transformed NIH 3T3 cells grew in serum-free medium, consistent with an autocrine mechanism of growth. We have also expressed the protein encoded in the K-fgf protooncogene in COS-1 cells, and it was indistinguishable in its molecular weight, glycosylation, secretion, and biological activity from K-FGF. Taken together, these results suggest that the mechanism of activation of this oncogene is due to overexpression rather than to mutations in the coding sequences.     

Androgenic regulation of epidermal growth factor in the mouse ventral prostate

Castration of adult male mice caused a marked reduction in the amount of immunoreactive epidermal growth factor (EGF) in the ventral prostate, and the treatment of such castrated mice with testosterone increased the EGF level significantly. Gel filtration of prostate extract showed that the immunoreactive EGF in the prostate had the same molecular weight (6,045) as the submandibular gland EGF. Moreover, its isoelectric point (pH 4.5) was almost similar to that (pH 4.55) of the submandibular gland peptide. These results suggest that under the control of androgens, mouse ventral prostate synthesizes EGF structurally and functionally identical to the submandibular gland EGF.     

Comparative Study of Glial Marker Proteins in the Hypoplastic Cerebellum of Jaundiced Gunn Rats

The behavior of marker proteins of glial cells [alpha-enolase, beta-S100 protein, and glial fibrillary acidic protein (GFAP)] was investigated quantitatively by using enzyme immunoassay systems during the development of cerebellar hypoplasia in jaundiced Gunn rats. A neuronal marker protein, gamma-enolase, was also measured as a reference. At postnatal day 8 corresponding to the early stage of cerebellar damage, the amount of beta-S100 on a protein basis was significantly higher in jaundiced homozygotes (jj) than in control nonjaundiced heterozygotes (j+), whereas no differences in alpha- and gamma-enolases and GFAP were observed between the two groups of rats. At days 15 and 30, which correspond, respectively, to the advanced and late stages of cerebellar damage, the three glial proteins, especially GFAP, were higher and the neuronal protein was lower in the jj rat cerebellum than in the control. These results are consistent with the reported histological observations that neuronal cells are vulnerable and damaged by bilirubin, whereas glial cells seem to be less sensitive. On the other hand, the amounts of beta-S100 and alpha-enolase per cerebellum were significantly lower in jj rats at days 15 and 30, as in the case of gamma-enolase, whereas that of GFAP remained at the same level as the control at day 15 and showed a slight but significant decrease at day 30. The possibility is suggested that beta-S100 and GFAP may be available as biochemical indicators of glial cells, especially in the early and advanced stages of cerebellar damage, respectively, but that alpha-enolase is less available.     

Characterization of the nontransformed and transformed androgen receptor and heat shock protein 90 with high-performance hydrophobic-interaction chromatography

The hydrophobicity of the nontransformed and transformed androgen receptor from rat submandibular gland and heat shock protein 90 (hsp90) from rat submandibular gland and liver was characterized by using high-performance hydrophobic-interaction chromatography on TSK gel Ether-5PW. In the absence of molybdate, cytosol [3H]R1881-androgen receptor complexes were mainly eluted in the 1.3 M region (Peak 1) with a small peak in the 0.8 M region (Peak 2) of a descending salt gradient (2 to 0 M) of ammonium sulfate. In the presence of molybdate, Peak 2 was predominant. When labeled-cytosol was applied after being heated at 25 degrees C for 30 min, a third peak (Peak 3) at around 0.64 M ammonium sulfate was newly observed. Peaks 2 and 3 were observed, while Peak 1 completely disappeared with the labeled-cytosol precipitated at 40% saturated ammonium sulfate. The Stokes radius of Peak 1 was 7 nm, and of Peak 2 was 8 nm. Both peaks were retained poorly by DNA-cellulose but bound rather well to DEAE-cellulose. These results suggest that these two peaks represent the nontransformed receptor, indicating that there are isoforms of the nontransformed androgen receptor which are distinguished by their hydrophobic properties and Stokes radii. Peak 3 had a Stokes radius of 5 nm and preferentially bound to DNA-cellulose, suggesting that this peak corresponds to the transformed receptor. These results indicated that the transformation of the androgen receptor accompanies the enrichment of the hydrophobicity of the receptor molecule. Hsp90 purified from rat livers and hsp90 in the cytosol both from livers and submandibular glands were eluted from Ether-5PW at 0.8 M ammonium sulfate, at almost the same position as Peak 2. This finding suggests that the enrichment of hydrophobicity on transformation is due to dissociation of hsp90 from the nontransformed androgen receptor.     

Possible involvement of calcium ions in the hatching of Schistosoma mansoni eggs in water

The possibility of involvement of calcium ions in the hatching of Schistosoma mansoni eggs in water is described. The hatching of S. mansoni eggs under low osmotic pressure was partially inhibited by EGTA (5 mM), lanthanum chloride (1-5 mM), and ruthenium red (0.1-1 mM). The reagents used in these experiments were not toxic to the eggs however, because miracidia hatched normally when the reagents were removed.