Lateral diffusion coefficients of separate lipid species in a ternary raft-forming bilayer: a Pfg-NMR multinuclear study

By isotopical labeling lipid lateral diffusion coefficients for each of the membrane constituents, including cholesterol, have been measured by 1H, 2H, and 19F pulsed field gradient NMR spectroscopy in macroscopically oriented lipid bilayers. This provides a means of obtaining detailed dynamic and compositional information in raft-forming lipid bilayers without introducing foreign molecules into the systems. The raft systems studied contained dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DPPC)/cholesterol at the molar ratios of 42.5:42.5:15 and 35:35:30 in excess water. At temperatures below 30 degrees C the raft system forms large (>1 microm) domains of a liquid ordered (l(o)) phase, in which the lipid lateral diffusion was approximately 5 times slower than for the lipids in the surrounding liquid disordered (l(d)) phase. Within each domain all lipid species showed the same diffusion coefficient, despite the very different structures of cholesterol and phospholipids. DPPC partitions exclusively into the l(o) domains, whereas cholesterol and dioleoylphosphatidylcholine were distributed in both l(o) and l(d) phases. The cholesterol concentration was found to be 10-20 mol % in the l(d) domain and 30-40 mol % in the l(o) domain. Comparison of these results with data from sphingomyelin-containing systems suggests that DPPC interacts more weakly with cholesterol than does sphingomyelin.     

The effect of peptide/lipid hydrophobic mismatch on the phase behavior of model membranes mimicking the lipid composition in Escherichia coli membranes

The effect of hydrophobic peptides on the lipid phase behavior of an aqueous dispersion of dioleoylphosphatidylethanolamine and dioleoylphosphatidylglycerol (7:3 molar ratio) was studied by (31)P NMR spectroscopy. The peptides (WALPn peptides, where n is the total number of amino acid residues) are designed as models for transmembrane parts of integral membrane proteins and consist of a hydrophobic sequence of alternating leucines and alanines, of variable length, that is flanked on both ends by tryptophans. The pure lipid dispersion was shown to undergo a lamellar-to-isotropic phase transition at approximately 60 degrees C. Small-angle x-ray scattering showed that at a lower water content a cubic phase belonging to the space group Pn3m is formed, suggesting also that the isotropic phase in the lipid dispersion represents a cubic liquid crystalline phase. It was found that the WALP peptides very efficiently promote formation of nonlamellar phases in this lipid system. At a peptide-to-lipid (P/L) molar ratio of 1:1000, the shortest peptide used, WALP16, lowered the lamellar-to-isotropic phase transition by approximately 15 degrees C. This effect was less for longer peptides. For all of the WALP peptides used, an increase in peptide concentration led to a further lowering of the phase transition temperature. At the highest P/L ratio (1:25) studied, WALP16 induced a reversed hexagonal liquid crystalline (H(II)) phase, while the longer peptides still promoted the formation of an isotropic phase. Peptides with a hydrophobic length larger than the bilayer thickness were found to be unable to inhibit formation of the isotropic phase. The results are discussed in terms of mismatch between the hydrophobic length of the peptide and the hydrophobic thickness of the lipid bilayer and its consequences for lipid-protein interactions in membranes.     

Cloning and characterization of SK2 channel from chicken short hair cells

In the inner ear of birds, as in mammals, reptiles and amphibians, acetylcholine released from efferent neurons inhibits hair cells via activation of an apamin-sensitive, calcium-dependent potassium current. The particular potassium channel involved in avian hair cell inhibition is unknown. In this study, we cloned a small-conductance, calcium-sensitive potassium channel (gSK2) from a chicken cochlear library. Using RT-PCR, we demonstrated the presence of gSK2 mRNA in cochlear hair cells. Electrophysiological studies on transfected HEK293 cells showed that gSK2 channels have a conductance of approximately 16 pS and a half-maximal calcium activation concentration of 0.74±0.17 M. The expressed channels were blocked by apamin (IC₅₀=73.3±5.0 pM) and d-tubocurarine (IC₅₀=7.6±1.0 M), but were insensitive to charybdotoxin. These characteristics are consistent with those reported for acetylcholine-induced potassium currents of isolated chicken hair cells, suggesting that gSK2 is involved in efferent inhibition of chicken inner ear. These findings imply that the molecular mechanisms of inhibition are conserved in hair cells of all vertebrates.     

Signaling pathway of nitric oxide-induced acrosome reaction in human spermatozoa

Nitric oxide (NO) has been recently shown to modulate in vitro motility, viability, the acrosome reaction (AR), and metabolism of spermatozoa in various mammalian species, but the mechanism or mechanisms through which it influences sperm functions has not been clarified. In human capacitated spermatozoa, both the intracellular cGMP level and the percentage of AR-positive cells were significantly increased after 4 h of incubation with the NO donor, sodium nitroprusside (SNP). SNP-induced AR was significantly reduced in the presence of the soluble guanylate cyclase (sGC) inhibitors, LY83583 and ODQ; this block was bypassed by adding 8-bromo-cGMP, a cell-permeating cGMP analogue, to the incubation medium. Finally, Rp-8-Br-cGMPS and Rp-8-pCPT-cGMPS, two inhibitors of the cGMP-dependent protein kinases (PKGs), inhibited the SNP-induced AR. Furthermore, SNP-induced AR did not occur in Ca2+ -free medium or in the presence of the protein kinase C (PKC) inhibitor, calphostin C. This study suggests that the AR-inducing effect of exogenous NO on capacitated human spermatozoa is accomplished via stimulation of an NO-sensitive sGC, cGMP synthesis, and PKG activation. In this effect the activation of PKC is also involved, and the presence of extracellular Ca2+ is required.     

BRCA1 mutations in a population-based study of breast cancer in Stockholm County

The mutation frequency of BRCA1 and BRCA2 in women with breast cancer varies according to family history, age at diagnosis and ethnicity. The contribution of BRCA1 and BRCA2 mutations in breast cancer populations, unselected for age and family history, has been examined in several studies reporting mutation frequencies between 1% and 12% by screening methods, population sizes, and to what extent the gene/s were screened differed in the studies. We wanted to clarify the proportion of breast cancer attributable to mutations in BRCA1 in an unselected breast cancer population from the Stockholm region. All incident breast cancer patients treated surgically in a 19-month period were eligible for the study and 70% (489/696) participated. Exon 11 of BRCA1 was screened for mutations using the protein truncation test, and the mutation frequency was estimated from that. In previous studies on high-risk families from Stockholm, more than 70% of the mutations were detected in exon 11. Two mutations were found, both in patients with a family history or their own medical history of ovarian cancer, giving a mutation frequency in exon 11 of 0.4% and an estimated BRCA1 mutation frequency of <1%. Mutations in BRCA1 in unselected breast cancer cases in our region are rare and likely to be found only in high-risk families. Our BRCA1 prevalence is the lowest of all studies on unselected breast cancer patients, probably reflecting the comparatively low rates detected also in high-risk breast cancer families from the region.     

Phase equilibria of the ternary system 1-palmitoyl-sn-glycero-3-phosphocholine/oleic acid/water studied by NMR

Part of a phase diagram for the system 1-palmitoyl-sn-glycero-3-phosphocholine (PamGroPCho)/oleic acid/water has been constructed from mainly 31P-NMR data and a previous determination of the phase equilibria of the binary PamGroPCHo/water system. It was found that the appearance of the phase diagram is very similar to those found for several simple soap/fatty acid/water or soap/long-chain alcohol/water systems. The most striking features observed are: (1) the lamellar phase can swell towards very high water contents (2) vesicles are formed after sonication and (3) the cubic liquid crystalline phase disappears upon addition of very small amounts of oleic acid. The self-association of the amphiphiles and the shape of the aggregates are discussed in terms of existing first-order approximative theories.     

Do selenium hyperaccumulators affect selenium speciation in neighboring plants and soil? An X-Ray Microprobe Analysis

Neighbors of Se hyperaccumulators Stanleya pinnata and Astragalus bisulcatus were found earlier to have elevated Se levels. Here we investigate whether Se hyperaccumulators affect Se localization and speciation in surrounding soil and neighboring plants. X-ray fluorescence mapping and X-ray absorption near-edge structure spectroscopy were used to analyze Se localization and speciation in leaves of Artemisia ludoviciana, Symphyotrichum ericoides and Chenopodium album growing next to Se hyperaccumulators or non-accumulators at a seleniferous site. Regardless of neighbors, A. ludoviciana, S. ericoides and C. album accumulated predominantly (73–92%) reduced selenocompounds with XANES spectra similar to the C-Se-C compounds selenomethionine and methyl-selenocysteine. Preliminary data indicate that the largest Se fraction (65–75%), both in soil next to hyperaccumulator S. pinnata and next to nonaccumulator species was reduced Se with spectra similar to C-Se-C standards. These same C-Se-C forms are found in hyperaccumulators. Thus, hyperaccumulator litter may be a source of organic soil Se, but soil microorganisms may also contribute. These findings are relevant for phytoremediation and biofortification since organic Se is more readily accumulated by plants, and more effective for dietary Se supplementation.     

Variants of the PPARD Gene and Their Clinicopathological Significance in Colorectal Cancer

Background

Peroxisome proliferator-activated receptor delta (PPARD) is nuclear hormone receptor involved in colorectal cancer (CRC) differentiation and progression. The purpose of this study was to determine prevalence and spectrum of variants in the PPARD gene in CRC, and their contribution to clinicopathological endpoints.

Methods and Findings

Direct sequencing of the PPARD gene was performed in 303 primary tumors, in blood samples from 50 patients with ≥3 affected first-degree relatives, 50 patients with 2 affected first-degree relatives, 50 sporadic patients, 360 healthy controls, and in 6 colon cancer cell lines. Mutation analysis revealed 22 different transversions, 7 of them were novel. Three of all variants were somatic (c.548A>G, p.Y183C, c.425-9C>T, and c.628-16G>A). Two missense mutations (p.Y183C and p.R258Q) were pathogenic using in silico predictive program. Five recurrent variants were detected in/adjacent to the exons 4 (c.1-87T>C, c.1-67G>A, c.130+3G>A, and c.1-101-8C>T) and exon 7 (c.489T>C). Variant c.489C/C detected in tumors was correlated to worse differentiation (P = 0.0397).

Conclusions

We found 7 novel variants among 22 inherited or acquired PPARD variants. Somatic and/or missense variants detected in CRC patients are rare but indicate the clinical importance of the PPARD gene.