Insertion/deletion polymorphism in clusterin gene influences serum lipid levels and carotid intima-media thickness in hypertensive Japanese females

Clusterin has been implicated in lipid metabolism and atherogenesis, however, the influence of genetic variation has not been examined in Japanese. In this study, we identified 11 single nucleotide polymorphisms (SNPs) of clusterin gene by direct sequencing. Among them, one promoter SNP (-4453T>G), one missense SNP (4183G>A), and 2 common SNPs (5608T>C and 6316delT) were genotyped in 525 asymptomatic hypertensives not treated with lipid lowering agents. -4453T>G, 4183G>A, and 5608T>C showed no correlation with the clinical characteristics, however, in the 6316delT, an insertion (I)/deletion (D) polymorphism, D/D subjects had significantly higher levels of total cholesterol and low-density lipoprotein (LDL)-cholesterol than I/I subjects in females but not in males. Female subjects with the D allele (D/D+I/D) had greater intima-media thickness of the carotid artery than I/I subjects. In a multiple logistic regression analysis, the D allele of 6316delT was detected as an independent predictor for the plaque prevalence. In conclusion, the clusterin gene polymorphism may contribute to the serum lipid levels and the progression of carotid atherosclerosis in hypertensive Japanese females.     

Analysis of the molecular interaction of the farnesyl moiety of transducin through the use of a photoreactive farnesyl analogue

Farnesylation of the gamma-subunit of the retinal G-protein, transducin (Talpha/Tbetagamma), is indispensable for light-initiated signaling in photoreceptor cells. However, the farnesyl-mediated molecular interactions important for signaling are not well understood. To explore this issue, we created a functional Tbetagamma analogue in which the farnesyl group was replaced with a (3-azidophenoxy)geranyl (POG) group, a novel farnesyl analogue with a distal photoreactive azido group. In the presence of lipid membranes and/or Talpha-GDP, UV irradiation of POG-modified Tbetagamma (POG-Tbetagamma) invariably yielded a cross-linked product Tgamma-Tbeta, reflecting a constitutive interaction of the Tgamma C-terminal lipid with Tbeta. In addition to the Tgamma-Tbeta adduct, a Tgamma-Talpha cross-link was detected in the aqueous fraction. Reconstitution of POG-Tbetagamma with Talpha and light-activated rhodopsin (Rh) in photoreceptor membranes resulted in cross-linking of Tgamma with a glycerophospholipid, indicating molecular interaction of the farnesyl group with cellular membranes. The Tgamma-phospholipid cross-link was observed only in the presence of both Talpha-GDP and Rh, and was abolished by the addition of GTPgammaS or by replacing Rh with opsin. These findings suggest a transient farnesyl-membrane interaction occurs only in a signaling state formed in a transducin-Rh ternary complex. On the other hand, UV irradiation of POG-Tbetagamma in a soluble complex with phosducin, a negative regulator of G-protein, yielded a Tgamma-phosducin adduct in addition to the Tgamma-Tbeta cross-link. These results illustrate that, rather than being a static membrane anchor, the farnesyl moiety plays an active role in the dynamics of protein-protein and protein-membrane interactions at defined steps in the signal transduction process.     

Retinosomes: new insights into intracellular managing of hydrophobic substances in lipid bodies

Lipid bodies form autonomous intracellular structures in many model cells and in some cells of specific tissue origin. They contain hydrophobic substances, a set of structural proteins such as perilipin or adipose differentiation-related protein, enzymes implicated in lipid metabolism, and proteins that participate in signaling and membrane trafficking. Retinosomes, particles reminiscent of lipid bodies, have been identified in retinal pigment epithelium as distinct structures compartmentalizing a metabolic intermediate involved in regeneration of the visual chromophore. These observations suggest that lipid bodies, including retinosomes, carry out specific functions that go beyond those of mere lipid storage organelles.     

Noninvasive two-photon imaging reveals retinyl ester storage structures in the eye

Visual sensation in vertebrates is triggered when light strikes retinal photoreceptor cells causing photoisomerization of the rhodopsin chromophore 11-cis-retinal to all-trans-retinal. The regeneration of preillumination conditions of the photoreceptor cells requires formation of 11-cis-retinal in the adjacent retinal pigment epithelium (RPE). Using the intrinsic fluorescence of all-trans-retinyl esters, noninvasive two-photon microscopy revealed previously uncharacterized structures (6.9 +/- 1.1 microm in length and 0.8 +/- 0.2 microm in diameter) distinct from other cellular organelles, termed the retinyl ester storage particles (RESTs), or retinosomes. These structures form autonomous all-trans-retinyl ester-rich intracellular compartments distinct from other organelles and colocalize with adipose differentiation-related protein. As demonstrated by in vivo experiments using wild-type mice, the RESTs participate in 11-cis-retinal formation. RESTs accumulate in Rpe65-/- mice incapable of carrying out the enzymatic isomerization, and correspondingly, are absent in the eyes of Lrat-/- mice deficient in retinyl ester synthesis. These results indicate that RESTs located close to the RPE plasma membrane are essential components in 11-cis-retinal production.     

Bcl6 acts as an amplifier for the generation and proliferative capacity of central memory CD8+ T cells

Central memory CD8(+) T cells (T(CM)) are considered to be more efficient than effector ones (T(EM)) for mediating protective immunity. The molecular mechanism involved in the generation of these cells remains elusive. Because Bcl6 plays a role in the generation and maintenance of memory CD8(+) T cells, we further examined this role in the process in relation to T(CM) and T(EM) subsets. In this study, we show that T(CM) and T(EM) were functionally identified in CD62L(+) and CD62L(-) memory (CD44(+)Ly6C(+)) CD8(+) T cell subsets, respectively. Although T(CM) produced similar amounts of IFN-gamma and IL-2 to T(EM) after anti-CD3 stimulation, the cell proliferation capacity after stimulation and tissue distribution profiles of T(CM) differed from those of T(EM). Numbers of T(CM) were greatly reduced and elevated in spleens of Bcl6-deficient and lck-Bcl6 transgenic mice, respectively, and those of T(EM) were constant in nonlymphoid organs of these same mice. The majority of Ag-specific memory CD8(+) T cells in spleens of these mice 10 wk after immunization were T(CM), and the number correlated with Bcl6 expression in T cells. The proliferation of Ag-specific memory CD8(+) T cells upon secondary stimulation was dramatically up-regulated in lck-Bcl6 transgenic mice, and the adoptive transfer experiments with Ag-specific naive CD8(+) T cells demonstrated that some of the up-regulation was due to the intrinsic effect of Bcl6 in the T cells. Thus, Bcl6 is apparently a crucial factor for the generation and secondary expansion of T(CM).     

Expression of cytochrome c oxidase subunit 1 gene in the brain at an early stage in the termination of pupal diapause in the sweet potato hornworm, Agrius convolvuli

Suppression-subtractive hybridization was used to isolate cDNAs specifically expressed in the brain at the termination of pupal diapause in Agriusconvolvuli. One of the isolated clones shows similarity to the cytochrome c oxidase subunit 1 (COX1) gene. The full-length cDNA was obtained from brain mRNA by rapid amplification of cDNA ends (RACE). The insert is 1.65 kb in length and has an open reading frame of 1.46 kb which encodes a putative protein of 486 amino acid residues. RT-PCR reveals that the mRNA increases dramatically at an early stage of diapause termination. Activity of cytochrome c oxidase in the brain also increases at the same time. The up-regulation of this gene suggests that expression of the COX1 gene and ATP synthesis are initiated in the brain in association with diapause termination.     

Effects on rotavirus cell binding and infection of monomeric and polymeric peptides containing alpha2beta1 and alphaxbeta2 integrin ligand sequences

Integrin-using rotaviruses bind MA104 cell surface alpha2beta1 integrin via the Asp-Gly-Glu (DGE) sequence in virus spike protein VP4 and interact with alphaxbeta2 integrin during cell entry through outer capsid protein VP7. Infection is inhibited by the alpha2beta1 ligand Asp-Gly-Glu-Ala (DGEA) and the alphaxbeta2 ligand Gly-Pro-Arg-Pro (GPRP), and virus-alpha2beta1 binding is increased by alpha2beta1 activation. In this study, we analyzed the effects of monomers and polymers containing DGEA-, GPRP-, and DGEA-related peptides on rotavirus binding and infection in intestinal (Caco-2) and kidney (MA104) cells and virus binding to recombinant alpha2beta1. Blockade of rotavirus-cell binding and infection by peptides and anti-alpha2 antibody showed that Caco-2 cell entry is dependent on virus binding to alpha2beta1 and interaction with alphaxbeta2. At up to 0.5 mM, monomeric DGEA and DGAA inhibited binding to alpha2beta1 and infection. At higher concentrations, DGEA and DGAA showed a reduced ability to inhibit virus-cell binding and infection that depended on virus binding to alpha2beta1 but occurred without alteration in cell surface expression of alpha2, beta2, or alphavbeta3 integrin. This loss of DGEA activity was abolished by genistein treatment and so was dependent on tyrosine kinase signaling. It is proposed that this signaling activated existing cell surface alpha2beta1 to increase virus-cell attachment and entry. Polymeric peptides containing DGEA and GPRP or GPRP only were inhibitory to SA11 infection at approximately 10-fold lower concentrations than peptide monomers. As polymerization can improve peptide inhibition of virus-receptor interactions, this approach could be useful in the development of inhibitors of receptor recognition by other viruses.     

The novel gene fad104, containing a fibronectin type III domain, has a significant role in adipogenesis

A novel gene named fad104 (factor for adipocyte differentiation-104), whose expression level quickly increased in the early stage of adipogenesis, was isolated and characterized. The deduced amino acid sequence of fad104 revealed the possible presence of a fibronectin type III domain and transmembrane domain. The expression of fad104 was detected in adipocyte differentiable 3T3-L1 cells but not observed in the non-adipogenic cell line NIH-3T3. Moreover, the ability of 3T3-L1 cells to differentiate declined with the knockdown of fad104 by RNA interference, strongly indicating that fad104 functions as a positive regulator of adipogenesis.     

Abnormal angiogenesis in Foxo1 (Fkhr)-deficient mice

Members of the Foxo family, Foxo1 (Fkhr), Foxo3 (Fkhrl1), and Foxo4 (Afx), are mammalian homologs of daf-16, which influences life span and energy metabolism in Caenorhabditis elegans. Mammalian FOXO proteins also play important roles in cell cycle arrest, apoptosis, stress resistance, and energy metabolism. In this study, we generated Foxo1-deficient mice to investigate the physiological role of FOXO1. The Foxo1-deficient mice died around embryonic day 11 because of defects in the branchial arches and remarkably impaired vascular development of embryos and yolk sacs. In vitro differentiation of embryonic stem cells demonstrated that endothelial cells derived from wild-type and Foxo1-deficient embryonic stem cells were able to produce comparable numbers of colonies supported by a layer of OP9 stromal cells. Although the morphology of the endothelial cell colonies was identical in both genotypes in the absence of exogenous vascular endothelial growth factor (VEGF), Foxo1-deficient endothelial cells showed a markedly different morphological response compared with wild-type endothelial cells in the presence of exogenous VEGF. These results suggest that Foxo1 is essential to the ability of endothelial cells to respond properly to a high dose of VEGF, thereby playing a critical role in normal vascular development.