Concentration of serum lipids and aortic lesion size in female and male apo E-deficient mice fed docosahexaenoic acid

Apolipoprotein (apo) E-deficient mice were fed an atherogenic diet with either 1% ethyl ester docosahexaenoic acid (DHA) or safflower oil (SO) as a source of linoleic acid for 8 week. Both genders fed DHA had higher proportions of eicosapentaenoic acid and DHA, and lower proportions of linoleic and arachidonic acids in the liver and serum phospholipids than those fed SO. Males fed DHA had greater liver weight and tended to have higher concentrations of serum lipids and liver cholesterol than those fed SO, and there were opposite trends in females. Dietary fats and gender led to no significant effect on lesion sizes in aortic arch and thoracic plus abdominal aorta. These results indicate that the interactive action of sex-related factor(s) with dietary polyunsaturated fatty acids is involved in metabolic changes of serum lipids in apoE-deficient mice, and addition of DHA, compared with addition of SO, is not effective to abolish the atherosclerosis in this animal model.     

A new assay for the analysis of X-chromosome inactivation based on methylation-specific PCR

The pattern of X-chromosome inactivation in females is currently evaluated by assays of differential methylation in the genes between the active and the inactive X chromosomes, with methylation-sensitive enzymes. We report a new assay in the human androgen receptor (HUMARA) locus involving a methylation-specific polymerase chain reaction (M-PCR) technique, independent of the use of restriction enzymes. The assay involves the chemical modification of DNA with sodium bisulfite and subsequent PCR. By using the assay with specific primers for the methylated allele, we obtained an X-inactivation pattern based on the ratio of the maternal inactive X to the paternal inactive X. These patterns were consistent with those obtained by conventional PCR assay at the same locus in 48 female cases. We also obtained another X-inactivation pattern based on the ratio of the maternal active X to the paternal active X by using specific primers for the unmethylated allele. The latter pattern was complementary to the former pattern, and a combination of these patterns produced a reliable X-inactivation pattern. The assay revealed that 12 (11%) of the 105 normal females had non-random inactivation patterns (>80:20 or <20:80). Four patients with an X; autosome translocation showed extremely non-random patterns, and these results were consistent with those obtained by previous molecular/cytogenetic studies. We conclude that M-PCR provides an accurate assay for X-inactivation and that it can be performed on various DNA samples unsuitable for restriction digestion. Received: 3 September 1998 / Accepted: 10 October 1998     

Serum level of advanced glycation end-products (AGEs) is an independent determinant of plasminogen activator inhibitor-1 (PAI-1) in nondiabetic general population.

Glucose can react nonenzymatically with amino groups of proteins to form senescent macroprotein derivatives termed advanced glycation end-products (AGEs). Recently, AGEs have been shown to play an important role in atherosclerosis even in nondiabetic subjects. However, the molecular mechanism underlying this is not fully understood. We have now investigated whether serum AGE level was an independent determinant of plasminogen activator inhibitor-1 (PAI-1), a major physiological inhibitor of fibrinolysis, in nondiabetic general population. One-hundred and eighty-six nondiabetic Japanese subjects underwent a complete history and physical examination, determination of blood chemistries, PAI-1, and AGEs. Uni- and multivariate analyses were applied for the determinants of PAI-1 levels. The average PAI-1 levels were 29.7+/-23.8 ng/ml in males and 21.8+/-17.1 ng/ml in females, respectively. Univariate regression analysis showed that PAI-1 levels were associated with age (inversely, p=0.003), male (p=0.003), body mass index (BMI) (p<0.001), HDL-cholesterol (inversely, p<0.001), triglycerides (p<0.001), fasting plasma glucose (p<0.001), insulin (p<0.001), uric acids (p<0.001), AGEs (p=0.037), and alcohol intake (p<0.001). By the use of multiple regression analyses, BMI (p<0.001), male (p=0.003), fasting plasma glucose (p=0.005), age (inversely, p=0.017), and AGEs (p=0.034) remained significant. The present study is the first demonstration that serum AGE level was one of the independent determinants of PAI-1 in nondiabetic general population. The AGE-associated thrombogenic abnormality may be involved in atherogenesis in nondiabetic subjects.     

Oxysterol regulation of estrogen receptor alpha-mediated gene expression in a transcriptional activation assay system using HeLa cells

In order to test the estrogenic activity of sterol oxidation products from cholesterol and phytosterols, an estrogen-dependent gene expression assay was performed in estrogen receptor alpha-stably transformed HeLa cells. The ranking of the estrogenic potency of these compounds was different: 17beta-estradiol > genistein > beta-epoxycholesterol = daidzein = cholestanetriol = 22(R)-hydroxycholesterol = 20(S)-hydroxycholesterol = sitostanetriol > campestanetriol = beta-epoxysitosterol = 7beta-hydroxycholesterol. These compounds were not estrogenic in estrogen receptor-negative HeLa cells.     

Molecular cloning and characterization of CALP/KChIP4, a novel EF-hand protein interacting with presenilin 2 and voltage-gated potassium channel subunit Kv4

Presenilin (PS) genes linked to early-onset familial Alzheimer's disease encode polytopic membrane proteins that are presumed to constitute the catalytic subunit of gamma-secretase, forming a high molecular weight complex with other proteins. During our attempts to identify binding partners of PS2, we cloned CALP (calsenilin-like protein)/KChIP4, a novel member of calsenilin/KChIP protein family that interacts with the C-terminal region of PS. Upon co-expression in cultured cells, CALP was directly bound to and co-localized with PS2 in endoplasmic reticulum. Overexpression of CALP did not affect the metabolism or stability of PS complex, and gamma-cleavage of betaAPP or Notch site 3 cleavage was not altered. However, co-expression of CALP and a voltage-gated potassium channel subunit Kv4.2 reconstituted the features of A-type K(+) currents and CALP directly bound Kv4.2, indicating that CALP functions as KChIPs that are known as components of native Kv4 channel complex. Taken together, CALP/KChIP4 is a novel EF-hand protein interacting with PS as well as with Kv4 that may modulate functions of a subset of membrane proteins in brain.     

Effects of acute vs. chronic phorbol ester exposure on transepithelial permeability and epithelial morphology.

In previous experiments we have shown that acute (30 minutes) exposure to phorbol esters or other protein kinase C activators causes increased transepithelial permeability, specifically by the increased paracellular permeability through tight junctions. However, the role of protein kinase C activators in carcinogenesis is predicted upon a chronic exposure of an effective dose at frequent intervals for a prolonged period of time. We therefore sought to determine the effect of chronic phorbol ester exposure on transepithelial permeability by exposing cells of the polar renal epithelial cell line, LLC-PK1, to phorbol esters for time periods as long as 16 weeks. The following changes ensued: (1) after the initial drop in transepithelial resistance due to phorbol ester exposure, i.e., an increase in transepithelial permeability (in the acute phase of exposure), an adaptive response occurs as transepithelial resistances in chronically exposed cultures recover to approximately 50% of control values, (2) the cell sheets in chronically exposed cultures lose their acute responsiveness of transepithelial permeability to phorbol ester exposure, (3) cell sheet architecture changes as cells occasionally multilayer and actual polyp-like cell masses appear at high frequency, and (4) cytosolic protein kinase C activity decreases to 50% of control level with acute exposure and then is further decreased to less than 1% of control level in chronically treated cells; membrane-associated PKC activity is not as sharply decreased. The possible role of transepithelial permeability in carcinogenesis and the value of chronically treated epithelial cell cultures as a model for two-stage carcinogenesis are discussed.     

The closest lineage of Archaeplastida is revealed by phylogenomics analyses that include Microheliella maris

By clarifying the phylogenetic positions of ‘orphan’ protists (unicellular micro-eukaryotes with no affinity to extant lineages), we may uncover the novel affiliation between two (or more) major lineages in eukaryotes. Microheliella maris was an orphan protist, which failed to be placed within the previously described lineages by pioneering phylogenetic analyses. In this study, we analysed a 319-gene alignment and demonstrated that M. maris represents a basal lineage of one of the major eukaryotic lineages, Cryptista. We here propose a new clade name ‘Pancryptista’ for Cryptista plus M. maris. The 319-gene analyses also indicated that M. maris is a key taxon to recover the monophyly of Archaeplastida and the sister relationship between Archaeplastida and Pancryptista, which is collectively called ‘CAM clade’ here. Significantly, Cryptophyceae tend to be attracted to Rhodophyta depending on the taxon sampling (ex., in the absence of M. maris and Rhodelphidia) and the particular phylogenetic ‘signal’ most likely hindered the stable recovery of the monophyly of Archaeplastida in previous studies.     

Identification of regulatory cis-acting elements for alternative splicing of presenilin 2 exon 5 under hypoxic stress conditions

An alternatively spliced form of the presenilin 2 (PS2) gene lacking exon 5 (PS2V) was found in human brains with sporadic Alzheimer's disease. PS2V was induced by hypoxic stress in human neuroblastoma SK-N-SH cells, indicating that hypoxic stress affects the splicing machineries for PS2 exon 5. Here, we identified the critical cis-acting element (sec 2) on the PS2 pre-mRNA responsible for the aberrant splicing of PS2 exon 5 under hypoxic stress conditions. The element was composed of 23 nucleotides in exon 5 and RNA structural analyses showed a stem-loop structure in this sequence. Treatment with an antisense oligonucleotide directed toward the cis-acting element caused an increase in exon 5 inclusion. These results indicate that the sec 2 identified in this study is a novel regulatory element for exon 5 splicing under stress conditions and that trans-acting factors could specifically bind to the element to skip exon 5 of PS2.