Effect of n-3 fatty acids on serum lipid levels and hepatic fatty acid metabolism in BALB/c.KOR-Apoeshl mice deficient in apolipoprotein E expression

N-3 fatty acids exert a potent serum lipid-lowering effect in rodents mainly by affecting hepatic fatty acid oxidation and synthesis. However, it has been observed that fish oil and docosahexaenoic acid ethyl ester do not lower serum lipid levels in apolipoprotein E (apoE)-knockout (Apoetm1Unc) mice generated by gene targeting. To test the hypothesis that apoE expression is required for n-3 fatty acid-dependent regulation of serum lipid levels and hepatic fatty acid metabolism, we examined the effect of fish oil and n-3 fatty acid ethyl esters on the activity and gene expression of hepatic enzymes involved in fatty acid oxidation and synthesis using an alternative apoE-deficient mouse model with the BALB/c genetic background (BALB/c.KOR-Apoeshl). ApoE-deficient mice were fed diets containing 9.4% palm oil, fish oil, or 5.4% palm oil and 1% EPA plus 3% DHA ethyl esters for 15 days. In contrast to the reported data on apoE-knockout mice, fish oil and n-3 fatty acid ethyl esters greatly decreased serum triacylglycerol, cholesterol, and phospholipid levels in the Apoeshl mice. The decreases were greater with fish oil than with ethyl esters. The alterations by dietary n-3 fatty acids of serum lipid levels were accompanied by parallel changes in the activity and mRNA levels of enzymes involved in hepatic fatty acid oxidation and synthesis. The reason for the discrepancy between the results of the current study and previous studies is unknown. However, our study at least indicates that a lack of apoE expression does not necessarily accompany deficits in the n-3 fatty acid-dependent regulation of serum lipid levels and hepatic fatty acid metabolism.     

Imprinted polymer layer for recognizing double-stranded DNA

A method of preparing a thin polymer layer able to recognize double-stranded DNA (dsDNA) was developed by using 2-vinyl-4,6-diamino-1,3,5-triazine (VDAT) as a functional monomer for creating a DNA-imprinted polymer. The formation of hydrogen bonds between VDAT and A-T base pairs in dsDNA was confirmed by measuring the effects of VDAT on the melting point and the NMR and CD spectra of dsDNA. An imprinted polymer that can recognize dsDNA of the verotoxin gene was prepared by polymerizing VDAT, acrylamide, a crosslinking agent, and the template verotoxin dsDNA on a silanized glass surface. The specificity of this polymer layer for binding verotoxin dsDNA was investigated by using fluorescent-labelled dsDNAs. The fluorescence intensity of the polymer layer after binding verotoxin dsDNA was twice as high as after binding oligo(dG)-oligo(dC), indicating that verotoxin dsDNA was preferentially bound to the polymer imprinted with verotoxin dsDNA. The kinetics of verotoxin dsDNA binding to the imprinted polymer were analyzed by surface plasmon resonance measurements. The dissociation constant (KD) was low, of the order of 10(-9)M.     

Saturated glucose uptake capacity and impaired fatty acid oxidation in hypertensive hearts before development of heart failure

Abnormalities in energy metabolism may play an important role in the development of hypertensive heart failure. However, the transition from compensated hypertrophy to heart failure is not fully understood in terms of energy metabolism. In Dahl salt-sensitive (DS) and salt-resistant (DR) rats, myocardial fatty acid and glucose uptake values were determined using (131)I- or (125)I-labeled 9-methylpentadecanoic acid ((131)I- or (125)I-9MPA), and [(14)C]deoxyglucose ([(14)C]DG), fatty acid beta-oxidation was identified using thin-layer chromatography, and insulin-stimulated glucose-uptake was observed using a euglycemic hyperinsulinemic glucose clamp. Six-week-old rats were fed a diet that contained 8% NaCl, which resulted in development of compensated hypertrophy in DS rats at 12 wk of age and ultimately led to heart failure by 18 wk of age. Uptake of [(14)C]DG increased markedly with age in the DS rats, whereas (131)I-9MPA uptake was marginally but significantly increased only in animals aged 12 wk. The ratio of (125)I-9MPA beta-oxidation metabolites to total uptake in the DS rats was significantly lower (P < 0.05) at 12 (37%) and 18 (34%) wk compared with at 6 (45%) wk. Insulin increased [(14)C]DG uptake more than twofold in the DS rats at 6 wk, although this increase was markedly attenuated at 12 and 18 wk (11 and 8%, respectively). Our data suggest that in a hypertrophied heart before heart failure, fatty acid oxidation is impaired and the capacity to increase glucose uptake during insulin stimulation is markedly reduced. These changes in both glucose and fatty acid metabolism that occur in association with myocardial hypertrophy may have a pathogenic role in the subsequent development of heart failure.     

Allelic characterization of the leaf-variegated mutation <Emphasis Type="Italic">var2</Emphasis> identifies the conserved amino acid residues of FtsH that are important for ATP hydrolysis and proteolysis

Arabidopsis var1 and var2 mutants exhibit leaf variegation. VAR1 and VAR2 encode similar FtsH metalloproteases (FtsH5 and FtsH2, respectively). We have previously found many variegated mutants to be allelic to var2. Each mutant was shown to express a different degree of variegation, and the formation of white sectors was enhanced in severely variegated alleles when these alleles were grown at low temperature. VAR1/FtsH5 and VAR2/FtsH2 levels were mutually affected even in the weak alleles, confirming our previous observation that the two proteins form a hetero complex. In this study, the sites of the mutations in these var2 alleles were determined. We isolated eight point mutations. Five alleles resulted in an amino acid substitution. Three of the five amino acid substitutions occurred in Walker A and B motifs of the ATP-binding site, and one occurred in the central pore motif. These mutations were considered to profoundly suppress the ATPase and protease activities. In contrast, one mutation was found in a region that contained no obvious signature motifs, but a neighboring sequence, Gly–Ala–Asp, was highly conserved among the members of the AAA protein family. Site-directed mutagenesis of the corresponding residue in E. coli FtsH indeed showed that this residue is necessary for proper ATP hydrolysis and proteolysis. Based on these results, we propose that the conserved Gly–Ala–Asp motif plays an important role in FtsH activity. Thus, characterization of the var2 alleles could help to identify the physiologically important domain of FtsH.     

A highly bioactive lignophenol derivative from bamboo lignin exhibits a potent activity to suppress apoptosis induced by oxidative stress in human neuroblastoma SH-SY5Y cells

Approaches to protection against neurodegenerative diseases, in which oxidative stress and inflammation are implicated, should be based on the current concept on the etiology of these diseases. Recently, a new therapeutic strategy has been proposed to protect neurons from cell death by attenuating the apoptotic signal transduction. Lignin, a durable aromatic network polymer second to cellulose in abundance, was able to be converted into highly active lignophenol derivatives with antioxidant activity by using our newly developed phase-separation technique. These lignophenol derivatives were found to show the potent neuroprotective activity against oxidative stress. Among the compounds examined, a lignocresol derivative from bamboo (lig-8) exhibited the most potent neuroprotective activity against hydrogen peroxide (H(2)O(2))-induced apoptosis in human neuroblastoma cell line SH-SY5Y by preventing the caspase-3 activation via either caspase-8 or caspase-9. Furthermore, it was found that lig-8 exerted the antiapoptotic effect by inhibiting dissipation of the mitochondrial membrane permeability transition induced by H(2)O(2) or by the peripheral benzodiazepin receptor ligand PK11195. Lig-8 was also shown to be potent in the antioxidant activity in the cells exposed to H(2)O(2), as assessed by flow cytometry using 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate and in vitro reactive oxygen species-scavenging potency. These data suggest that lig-8 is a promising neuroprotector, which affects the signaling pathway of neuronal cell death and that it would be of benefit to delay the progress of neurodegenerative diseases.     

Effects of prolonged delivery of brain-derived neurotrophic factor on the fate of neural stem cells transplanted into the developing rat retina

One of the major roles of brain-derived neurotrophic factor (BDNF) is to promote the differentiation and support the survival of neurons in the central nervous system. The objective of the present study was to evaluate the effect of BDNF on the fate of adult rat hippocampus-derived neural stem cells (AHPCs) transplanted into the developing rat retina. Immunohistochemical analysis showed a significant increase in the ratio of grafted AHPCs stained for MAP2ab (P<0.05) and a marked decrease in the ratio of nestin-positive grafted cells in the slow-releasing BDNF group compared with the control group. The respective changes in the ratios of MAP5 and GFAP-positive grafted cells were comparable for the two groups. The results reported here suggest a potentially beneficial role for extended delivery of BDNF in the differentiation of grafted neural stem cells, which may lead to a novel modification of stem cell transplantation.     

Crystal structure of chartreusin derivative A132

The crystal structure of chartreusin derivative A132 (benzilidene chartreusin) has been determined by single-crystal X-ray diffraction. The space group is C2 with unit cell dimensions, a=18.482(4), b=8.749(3), c=43.906(2) A, beta=94.87(2) degrees, and the structure was refined to R-factors of 0.2365 (6585 all unique reflections) and 0.087 (2914 reflections with F(o)>4 sigma(F(o))) by a full-matrix least-squares method. There are two molecules in an asymmetric unit. Both molecules have similar structures, which are favorable to bind with DNA in the minor groove. A modeling study of the A132-DNA complex based on the X-ray structures suggests that the sugar moiety of A132 may play an important role in recognizing the sequence of DNA base pairs.     

Interaction of the heart-specific LIM domain protein, FHL2, with DNA-binding nuclear protein, hNP220

Using a yeast two-hybrid library screen, we have identified that the heart specific FHL2 protein, four-and-a-half LIM protein 2, interacted with human DNA-binding nuclear protein, hNP220. Domain studies by the yeast two-hybrid interaction assay revealed that the second LIM domain together with the third and the fourth LIM domains of FHL2 were responsible to the binding with hNP220. Using green fluorescent protein (GFP)-FHL2 and blue fluorescent protein (BFP)-hNP220 fusion proteins co-expressed in the same cell, we demonstrated a direct interaction between FHL2 and hNP220 in individual nucleus by two-fusion Fluorescence Resonance Energy Transfer (FRET) assay. Besides, Western blot analysis using affinity-purified anti-FHL2 antipeptide antibodies confirmed a 32-kDa protein of FHL2 in heart only. Virtually no expression of FHL2 protein was detected in brain, liver, lung, kidney, testis, skeletal muscle, and spleen. Moreover, the expression of FHL2 protein was also detectable in the human diseased heart tissues. Our results imply that FHL2 protein can shuttle between cytoplasm and nucleus and may act as a molecular adapter to form a multicomplex with hNP220 in the nucleus, thus we speculate that FHL2 may be particularly important for heart muscle differentiation and the maintenance of the heart phenotype.     

Mitochondrial permeability transition mediates apoptosis induced by N-methyl(R)salsolinol,an endogenous neurotoxin,and is inhibited by Bcl-2 and rasagiline,N-propargyl-1(R)-aminoindan

The role of mitochondrial permeability transition (PT) in apoptosis induced by an endogenous neurotoxin, N-methyl(R)salsolinol [NM(R)Sal], was studied by use of dopaminergic neuroblastoma SH-SY5Y cells. NM(R)Sal reduced mitochondrial membrane potential, DeltaPsim, in the early phase of apoptosis, which was not suppressed by a pan-caspase inhibitor, but was antagonized by Bcl-2 and cyclosporin A, suggesting the involvement of the PT in NM(R)Sal-induced loss of DeltaPsim. NM(R)Sal-induced apoptosis was completely inhibited not only by Bcl-2 and a pan-caspase inhibitor, but also by cyclosporin A, suggesting the essential role of the PT in NM(R)Sal-induced apoptosis. In mitochondria isolated from rat liver, NM(R)Sal induced swelling and reduced DeltaPsim, which was inhibited by cyclosporin A and Bcl-2 overexpression. These results indicate that NM(R)Sal induced the PT by direct action on the mitochondria. Rasagiline, N-propargyl-1(R)-aminoindan, which is a now under a clinical trial for Parkinson's disease, suppressed the DeltaPsim reduction, release of cytochrome c, and apoptosis induced by NM(R)Sal in SH-SY5Y cells. Rasagiline also inhibited the NM(R)Sal-induced loss of DeltaPsim and swelling in the isolated mitochondria, proving that rasagiline directly targets the mitochondria also. Altogether, mitochondrial PT plays a key role both in NM(R)Sal-induced cell death and the neuroprotective effect of rasagiline.