A case of hyperlipidemia with homozygous apolipoprotein E5 (Glu3-->Lys)

In this study, we present clinical feature of a novel case with homozygous apolipoprotein (apo) E5.The patient was a 53-year-old Japanese woman. She was from a small island off the coast of Kagoshima Prefecture, Japan. Her parents were first degree cousins. No corneal opacification, xanthomatosis, lymphadenopathy, or hepatosplenomegaly was observed. There have been no signs of clinically overt atherosclerosis to date. Her serum total cholesterol, triglycerides (TG) and high-density lipoprotein (HDL)-cholesterol levels were 11.6, 6.1 and 1.2 mmol/l, respectively, and apo A-I, A-II, B, C-II, C-III and E levels were 121, 34.8, 269, 10.4, 25.7 and 10.3 mg/dl, respectively. Serum lipoprotein profile analyzed by agarose gel electrophoresis and differential staining revealed markedly increased cholesterol and TG in both beta and prebeta-migrated lipoproteins, whereas alpha-migrated lipoprotein showed decreased cholesterol. Her apo E isoform analyzed by isoelectric focusing (IEF) was found to be homozygous apo E5.Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) analysis of her apo E and lipoprotein lipase (LPL) genes revealed that she had a homozygous apo E (Glu3-->Lys) and heterozygous LPL variant Ser447 to Ter. Her son and daughter, both of whom had hyperlipidemia, were found to have apo E3/5 phenotype. Direct sequencing analysis of her apo E gene confirmed a homozygous one nucleotide change: G to A at nucleotide position of 2836 in the exon 3, resulting in Glu3-->Lys mutation.This is the first report of lipids and lipoprotein profiles in patients with homozygous apo E5 (Glu3-->Lys).     

Acerogenin A, a natural compound isolated from Acer nikoense Maxim, stimulates osteoblast differentiation through bone morphogenetic protein action

We investigated the effects of acerogenin A, a natural compound isolated from Acer nikoense Maxim, on osteoblast differentiation by using osteoblastic cells. Acerogenin A stimulated the cell proliferation of MC3T3-E1 osteoblastic cells and RD-C6 osteoblastic cells (Runx2-deficient cell line). It also increased alkaline phosphatase activity in MC3T3-E1 and RD-C6 cells and calvarial osteoblastic cells isolated from the calvariae of newborn mice. Acerogenin A also increased the expression of mRNAs related to osteoblast differentiation, including Osteocalcin, Osterix and Runx2 in MC3T3-E1 cells and primary osteoblasts: it also stimulated Osteocalcin and Osterix mRNA expression in RD-C6 cells. The acerogenin A treatment for 3 days increased Bmp-2, Bmp-4, and Bmp-7 mRNA expression levels in MC3T3-E1 cells. Adding noggin, a BMP specific-antagonist, inhibited the acerogenin A-induced increase in the Osteocalcin, Osterix and Runx2 mRNA expression levels. These results indicated that acerogenin A stimulates osteoblast differentiation through BMP action, which is mediated by Runx2-dependent and Runx2-independent pathways.     

Hyperactive TNF-α derivatives with combinational mutations in the amino and carboxyl terminal regions

Summary We prepared various TNF- derivatives by protein engineering techniques. Mutant 471, in which 7 N-terminal amino acids were deleted and Pro⁸Ser⁹Asp¹⁰ was replaced by ArgLysArg, had a 8-fold higher antitumor activity against mouse L929 cells than wild-type TNF-. The additional substitution of Ala¹⁵⁶ or Leu¹⁵⁷ by more hydrophobic amino acids enhanced the activity of mutant 471. These results suggested that the combinational mutations in the N- and C-terminal regions of TNF- are effective for the improvement of antitumor activity.     

Effect of the dietary alpha-linolenate/linoleate balance on leukotriene production and histamine release in rats

Rats were fed semi-purified diets supplemented either with safflower seed oil rich in linoleate (18:2n-6) or with perilla seed oil rich in alpha-linolenate (18:3n-3) through two generations. In the major phospholipids of polymorphonuclear leukocytes (PMNs), the proportions of n-6 polyunsaturated fatty acids (18:2, 20:4, 22:4 and 22:5) were higher but those of n-3 acids (20:5, 22:5 and 22:6) were lower in the safflower group than in the perilla group. When stimulated with a calcium ionophore, the PMNs from the safflower group produced 27% more leukotriene (LT)B4 than those from the perilla group. The formation of LTB5 which has biological activities less than 1/10 those of LTB4, was negligible in the safflower group but was 40 ng/10(7) PMN cells in the perilla group. The amount of the total LTB formed in the perilla group tended to be more than in the safflower group. The formation of SRS-A (slow-reacting substances of anaphylaxis) by PMNs was determined by measuring the spasmogenic activities of LTs on guinea pig ileum. SRS-A activity was 59% higher in the safflower group than in the perilla group. In contrast, histamine release from rat peritoneal mast cells was not significantly different between the two groups. Thus, the increasing the alpha-linolenate/linoleate ratio of diets results in the decreased formation of LTs derived from 20:4n-6 in PMNs. This may be beneficial in lowering the severity of allergic and inflammatory responses caused by LTs, and thereby shifting the pathological symptoms to normal self-defense mechanism.     

Crucial role of the small GTPase ARF6 in hepatic cord formation during liver development

The mammalian small GTPase ADP-ribosylation factor 6 (ARF6) plays important roles in a wide variety of cellular events, including endocytosis, actin cytoskeletal reorganization, and phosphoinositide metabolism. However, physiological functions for ARF6 have not previously been examined. Here, we described the consequence of ARF6 ablation in mice, which manifests most obviously in the context of liver development. Livers from ARF6-/- embryos are smaller and exhibit hypocellularity, due to the onset of midgestational liver cell apoptosis. Preceding the apoptosis, however, defective hepatic cord formation is observed; the liver cells migrate abnormally upon exiting the primordial hepatic epithelial sheet and clump rather than becoming dispersed. Consistent with this observation, the ability of hepatocyte growth factor/scatter factor (HGF) to induce hepatic cord-like structures from ARF6-/- fetal hepatocytes cultured in vitro in collagen gel matrix is impaired. Finally, we show that endogenous ARF6 in wild-type fetal hepatocytes is activated in response to HGF stimulation. These results provide evidence that ARF6 is an essential component in the signaling pathway coupling HGF signaling to hepatic cord formation.     

Separation of DNA binding domain from hormone and core histone binding domains by trypsin digestion of rat liver nuclear thyroid hormone receptor

Rat liver nuclear thyroid hormone receptor was subjected to limited trypsin digestion, and the tryptic fragment of the 3,5,3'-triiodo-L-thyronine (T3)-receptor complex was characterized. Rat liver nuclear thyroid hormone receptor is an asymmetrical protein with Stokes radius of 34 A, sedimentation coefficient of 3.4 S, and molecular weight of 49,000. A globular T3-receptor complex with Stokes radius of 22 A, sedimentation coefficient of 2.8 S, and molecular weight of 26,000 was obtained by tryptic digestion. This fragment had no DNA binding activity, whereas undigested receptor showed significant DNA binding activity. Addition of undigested receptor to the tryptic fragment did not restore DNA binding activity of digested receptor, nor did mixing inhibit DNA binding activity of undigested receptor complex. Undigested receptor bound to core histones, and this activity was stronger than with other proteins tested (H1 histone, cytochrome c, and ovalbumin). The tryptic fragment of receptor maintained core histone binding activity comparable to that of undigested receptor. The tryptic fragment had affinity for T3 comparable to undigested receptor as assessed by Scatchard analysis and the same rate for dissociation of [125I]T3 from receptor. The tryptic fragment of the T3-receptor complex was more stable than undigested receptor at 43 degrees C. Digestion of receptor unoccupied by T3 caused a significantly larger loss of T3 binding capacity than did digestion of T3-occupied receptor, suggesting a protective effect of T3 on a second trypsin-sensitive site on the receptor, which, when cut, destroys T3 binding activity.     

Structural insight into receptor-selectivity for lurasidone

Lurasidone is a novel antipsychotic agent with high affinity for dopamine D₂, 5-hydroxyltryptamine 5-HT_2A, and 5-HT₇ receptors. Lurasidone has negligible affinity for histamine H₁ and muscarinic M₁ receptors, which are thought to contribute to side effects such as weight gain, sedation, and worsening of cognitive deficits. Our interests focus on why lurasidone has such high selectivity for only a part of these aminergic G-protein coupled receptors (GPCRs) and the different binding profile from ziprasidone, which has the same benzisothiazolylpiperazine moiety as lurasidone. In order to address these issues, we constructed structural models of lurasidone–GPCR complexes by homology modeling of receptors, exhaustive docking of ligand, and molecular dynamics simulation-based refinement of complexes. This computational study gave reliable structural models for D₂, 5-HT_2A, and 5-HT₇, which had overall structural complementarities with a salt bridge anchor at the center of the lurasidone molecule, but not for H₁ and M₁ owing to steric hindrance between the norbornane-2,3-dicarboximide and/or cyclohexane part of lurasidone and both receptors. By comparison with the structural models of olanzapine–GPCRs and ziprasidone–GPCRs constructed using the same computational protocols, it was suggested that the bulkiness of the norbornane-2,3-dicarboximide part and the rigidity and the bulkiness of the cyclohexyl linker gave lurasidone high selectivity for the desired aminergic GPCRs. Finally, this structural insight was validated by a binding experiment of the novel benzisothiazolylpiperazine derivatives. This knowledge on the structural mechanism behind the receptor selectivity should help to design new antipsychotic agents with preferable binding profiles, and the established computational protocols realize virtual screening and structure-based drug design for other central nervous system drugs with desired selectivity for multiple targets.     

LRRK2 phosphorylates tubulin-associated tau but not the free molecule: LRRK2-mediated regulation of the tau-tubulin association and neurite outgrowth

Leucine-rich repeat kinase 2 (LRRK2), a large protein kinase containing multi-functional domains, has been identified as the causal molecule for autosomal-dominant Parkinson's disease (PD). In the present study, we demonstrated for the first time that (i) LRRK2 interacts with tau in a tubulin-dependent manner; (ii) LRRK2 directly phosphorylates tubulin-associated tau, but not free tau; (iii) LRRK2 phosphorylates tau at Thr181 as one of the target sites; and (iv) The PD-associated LRRK2 mutations, G2019S and I2020T, elevated the degree of tau-phosphorylation. These results provide direct proof that tau is a physiological substrate for LRRK2. Furthermore, we revealed that LRRK2-mediated phosphorylation of tau reduces its tubulin-binding ability. Our results suggest that LRRK2 plays an important role as a physiological regulator for phosphorylation-mediated dissociation of tau from microtubules, which is an integral aspect of microtubule dynamics essential for neurite outgrowth and axonal transport.