Structured triacylglycerol containing medium-chain fatty acids in sn-1(3) facilitates the absorption of dietary long-chain fatty acids in rats

A study was carried out to examine if the positional distribution of medium chain fatty acids (MCF) in triacylglycerol influences dietary fat absorption in rats. Two types of structure-specific fats, one predominantly composed of MCF in sn-1(3) and iinoleic acid in sn-2 [sn1(3)MCF-structured] and the others of MCF in sn-2 and linoleic acid in sn-1(3) [sn-2MCF-structured], were initially prepared, and the two structure-specific fats were interesterified and designated as sn-1(3)MCF-interesterified and sn-2MCF-interesterified. Synthetic fat was mixed with an equal amount of cocoa butter (103 g/kg of diet) and was supplemented to the AIN93G-based diet. Rats were fed on the diets for 4 wk. Long-chain saturated fatty acids were the predominant fatty acids excreted into the feces, and the positional distribution of MCF resulted in an altered fat absorption rate (%) of 81.8, 82.5, 84.2 and 86.3 for the rats fed on the diets containing sn-2MCF-structured, sn-1(3)MCF-interesterified, sn-2MCF-interesterified and sn-1(3)MCF-structured fats, respectively. The proportion of MCF in the serum, liver and adipose tissue triacylglycerols was not affected by the MCF distribution of the dietary fats. These results indicate that the distribution of MCF in dietary triacylglycerol is a determinant of intestinal fat absorption.     

Cryptochrome nucleocytoplasmic distribution and gene expression are regulated by light quality in the fern Adiantum capillus-veneris

Numerous cellular responses are reportedly regulated by blue light in gametophytes of lower plants; however, the molecular mechanisms of these responses are not known. Here, we report the isolation of two blue light photoreceptor genes, designated cryptochrome genes 4 and 5 (CRY4 and CRY5), from the fern Adiantum capillus-veneris. Because previously we identified three cryptochrome genes, this fern cryptochrome gene family of five members is the largest identified to date in plants. The deduced amino acid sequences of the five genes show remarkable similarities with previously identified cryptochromes as well as class I photolyases. Like the other plant cryptochromes, none of the cryptochromes of this fern possesses photolyase activity. RNA gel blot analysis and competitive polymerase chain reaction analysis indicate that the expression of the newly identified CRY4 and CRY5 genes is regulated by light and is under phytochrome control. The intracellular distribution of reporter beta-glucuronidase (GUS)-CRY fusion proteins indicates that GUS-CRY3 and GUS-CRY4 localize in fern gametophyte nuclei. The nuclear localization of GUS-CRY3 is regulated in a light-dependent manner. Together with our physiological knowledge, these results suggest that CRY3, CRY4, or both might be the photoreceptor that mediates inhibition of spore germination by blue light.     

Regulation of Kv4.3 currents by Ca2+/calmodulin-dependent protein kinase II

The voltage-dependent K+ channel 4.3 (Kv4.3) is one of the major molecular correlates encoding a class of rapidly inactivating K+ currents, including the transient outward current in the heart (Ito) and A currents (IA) in neuronal and smooth muscle preparations. Recent studies have shown that Ito in human atrial myocytes and IA in murine colonic myocytes are modulated by Ca2+/calmodulin-dependent protein kinase II (CaMKII); however, the molecular target of CaMKII in these studies has not been elucidated. We performed experiments to investigate whether CaMKII could regulate Kv4.3 currents directly. Inclusion of the autothiophosphorylated form of CaMKII in the patch pipette (10 nM) prolonged Kv4.3 currents such that the time required to reach 50% inactivation from peak more than doubled, with positive shifts in voltage dependence of both activation and inactivation. In contrast, the rate of recovery from inactivation was accelerated under these conditions. CaMKII-inhibitory peptide or KN-93 produced effects opposite to that above; thus the rate of inactivation was increased, and recovery from inactivation decreased. A number of mutagenesis experiments were conducted on the three candidate CaMKII consensus sequence sites on the channel. Mutations at S550A, located at the COOH-terminal region of the channel, resulted in currents that inactivated more rapidly but recovered from inactivation at a slower rate than that of wild-type controls. In addition, these currents were unaffected by dialysis with either autothiophosphorylated CaMKII or the specific inhibitory peptide of CaMKII, suggesting that CaMKII slows the inactivation and accelerates the rate of recovery from inactivation of Kv4.3 currents by a direct effect at S550A, located at the COOH-terminal region of the channel.     

Improved production of L-lysine by disruption of stationary phase-specific rmf gene in Escherichia coli

Growth and rate, at which fermentation products are formed in cells, generally decreases during the stationary phase as a result of changes in gene expression. We focused on the rmf gene, which encodes the ribosome modulation factor protein, as a target for strain modification in order to improve the rate of L-lysine production in Escherichia coli. Increased expression of the rmf gene during the stationary phase was confirmed under various cultivation conditions using DNA macroarray analysis. Mutants with disrupted rmf were then generated from an L-lysine-producing E. coli strain. The rates of L-lysine accumulation and production were significantly increased in disruptants that were cultivated with excess phosphate. By contrast, a higher biomass was generated in disruptants that were grown under limited phosphate conditions. These results demonstrate that disruption of the rmf gene significantly affects L-lysine production and growth in E. coli.     

Activators of AMP-activated protein kinase enhance GLUT4 translocation and its glucose transport activity in 3T3-L1 adipocytes

To determine whether the increase in glucose uptake following AMP-activated protein kinase (AMPK) activation in adipocytes is mediated by accelerated GLUT4 translocation into plasma membrane, we constructed a chimera between GLUT4 and enhanced green fluorescent protein (GLUT4-eGFP) and transferred its cDNA into the nucleus of 3T3-L1 adipocytes. Then, the dynamics of GLUT4-eGFP translocation were visualized in living cells by means of laser scanning confocal microscopy. It was revealed that the stimulation with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) and 2,4-dinitrophenol (DNP), known activators of AMPK, promptly accelerates its translocation within 4 min, as was found in the case of insulin stimulation. The insulin-induced GLUT4 translocation was markedly inhibited after addition of wortmannin (P < 0.01). However, the GLUT4 translocation through AMPK activators AICAR and DNP was not affected by wortmannin. Insulin- and AMPK-activated translocation of GLUT4 was not inhibited by SB-203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK). Glucose uptake was significantly increased after addition of AMPK activators AICAR and DNP (P < 0.05). AMPK- and insulin-stimulated glucose uptake were similarly suppressed by wortmannin (P < 0.05-0.01). In addition, SB-203580 also significantly prevented the enhancement of glucose uptake induced by AMPK and insulin (P < 0.05). These results suggest that AMPK-activated GLUT4 translocation in 3T3-L1 adipocytes is mediated through the insulin-signaling pathway distal to the site of activated phosphatidylinositol 3-kinase or through a signaling system distinct from that activated by insulin. On the other hand, the increase of glucose uptake dependent on AMPK activators AICAR and DNP would be additionally due to enhancement of the intrinsic activity in translocated GLUT4 protein, possibly through a p38 MAPK-dependent mechanism.     

Effect of peroxisome proliferator-activated receptor-gamma ligands on the expression of retinoic acid-inducible gene-I in endothelial cells stimulated with lipopolysaccharide

Retinoic acid-inducible gene-I (RIG-I) is a member of the DExH box protein family and designated as a putative RNA helicase. RIG-I is implicated in host defense and inflammatory reactions by regulating the expression of various genes. RIG-I is expressed in endothelial cells and upregulated with lipopolysaccharide (LPS). Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a nuclear hormone receptor and regulates gene expressions in response to its specific ligands. In the present study, we examined the effect of PPAR-gamma ligands on the LPS-induced RIG-I expression in cultured human umbilical vein endothelial cells (HUVEC). 15-Deoxy-Delta(12,14)-prostaglandin J2 (15d-PGJ2), a metabolite of PGD2, is a natural ligand for PPAR-gamma and known to modulate inflammatory reactions by regulating the expression of various genes in PPAR-gamma-dependent and -independent manners. LPS-induced RIG-I expression in HUVEC was inhibited by pretreatment of the cells with 15d-PGJ2 in time-and concentration-dependent manners. However, ciglitazone and bisphenol A diglycide ether, authentic and specific ligands for PPAR-gamma, did not affect the RIG-I expression. These results suggest that 15d-PGJ2 inhibits LPS-induced RIG-I expression through a mechanism independent on PPAR-gamma. 15d-PGJ2 may regulate inflammatory reactions, at least in part, by inhibiting the expression of RIG-I.     

Desferrioxamine, an iron chelator, upregulates cyclooxygenase-2 expression and prostaglandin production in a human macrophage cell line

Prostaglandins (PGs) play regulatory roles in a variety of physiological and pathological processes, including the immune response, cytoprotection and inflammation. Desferrioxamine (DFX), an iron chelator, is known to reduce free radical-mediated cell injury and to upregulate certain inflammatory mediators. We investigated the effects of DFX on the production of PGs and the expression of cyclooxygenase-2 (COX-2), the rate-limiting enzyme in the synthesis of PGs, using a human macrophage cell line, U937. Our results showed that COX-2 expression and PGE(2) production are upregulated by DFX treatment and that this upregulation is dependent on both COX-2 promoter activity and alteration of mRNA stability. COX-2 promoter activity may be, at least in part, mediated by activation of the extracellular signal-regulated kinase pathway. These findings suggest that iron metabolism may regulate inflammatory processes by modulating PGs as well as other inflammatory mediators.     

Increased production of beta-amyloid and vulnerability to endoplasmic reticulum stress by an aberrant spliced form of presenilin 2

An alternative spliced form of the presinilin 2 (PS2) gene (PS2V) lacking exon 5 has previously been reported to be expressed in human brains in sporadic Alzheimer's disease (AD). PS2V encodes the amino-terminal portion of PS2, which contains residues Met1-Leu119 and 5 additional amino acid residues (SSMAG) at its carboxyl terminus. Here we report that PS2V protein impaired the signaling pathway of the unfolded protein response, similarly to familial AD-linked PS1 mutants and caused significant increases in the production of both amyloid beta40 and beta42. Interestingly, PS2V-encoding protein was expressed in neuropathologically affected neurons of the hippocampal CA1 region and temporal cortex in AD patients. These findings suggest that the aberrant splicing of the PS2 gene may be implicated in the neuropathology of sporadic AD.     

Lymphatic transport of dietary cholesterol oxidation products,cholesterol and triacylglycerols in rats

Rats were fed on a diet containing 0.5% cholesterol oxidation products (oxysterols) or 0.5% cholesterol for 30 min, and their lymph was collected for 7 h. The amount of each of the individual oxysterols absorbed in the lymph depended on the ingested amounts, but the recovery was the highest for 5alpha,6alpha-epoxycholesterol (10.5%), this being followed by 7-ketocholesterol (5.8%), cholestanetriol (5.2%), 7beta-hydroxycholesterol (4.8%), 7alpha-hydroxycholesterol (3.4%), 5beta,6beta-epoxycholesterol (2.2%), and 25-hydroxycholesterol (1.8%). A diet enriched with oxysterol, but not cholesterol, resulted in increased transport of triacylglycerols in the lymph. These results suggest that the absorption rate of oxysterols depends on the type, and indicate that the effect of dietary oxysterols on the lymphatic transport of triacylglycerols differs from that of dietary cholesterol. It therefore remains to be determined which oxysterol was responsible for the triacyglycerol transport.