NMR protocol for determination of oxidation susceptibility of serum lipids and application of the protocol to a chocolate study

A protocol for determination of oxidation susceptibility of serum lipids based on proton nuclear magnetic resonance ((1)H NMR) spectroscopy is presented and compared to the commonly used spectrophotometric method. Even though there are methodological differences between these two methods, the NMR-based oxidation susceptibility correlates well (r(2)?=?0.73) with the lag time determined spectrophotometrically. In addition to the oxidizability of serum lipids, the NMR method provides also information about the lipid profile. The NMR oxidation assay was applied to the chocolate study including fasting serum samples (n?=?45) from subjects who had consumed white (WC), dark (DC) or high-polyphenol chocolate (HPC) daily for 3?weeks. The oxidation susceptibility of serum lipids decreased in the HPC group, and there was a significant difference between the WC and HPC groups (P?=?0.031). According to the random forest analysis, the consumption of the HPC chocolate induced changes to the amounts of HDL, phosphatidylcholine, sphingomyelin, and nervonic, docosahexaenoic and myristic acids. Furthermore, arachidonic, docosahexaenoic, docosapentaenoic and palmitic acids, gamma-glutamyl transferase, hemoglobin, HDL, phosphatidylcholine and choline containing phospholipids explained about 60% of the oxidation susceptibility values. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11306-011-0323-2) contains supplementary material, which is available to authorized users.     

Cys-27 variant of human δ-opioid receptor modulates maturation and cell surface delivery of Phe-27 variant via heteromerization

The important role of G protein-coupled receptor homo/heteromerization in receptor folding, maturation, trafficking, and cell surface expression has become increasingly evident. Here we investigated whether the human δ-opioid receptor (hδOR) Cys-27 variant that shows inherent compromised maturation has an effect on the behavior of the more common Phe-27 variant in the early secretory pathway. We demonstrate that hδOR-Cys-27 acts in a dominant negative manner and impairs cell surface delivery of the co-expressed hδOR-Phe-27 and impairs conversion of precursors to the mature form. This was demonstrated by metabolic labeling, Western blotting, flow cytometry, and confocal microscopy in HEK293 and human SH-SY5Y neuroblastoma cells using differentially epitope-tagged variants. The hδOR-Phe-27 precursors that were redirected to the endoplasmic reticulum-associated degradation were, however, rescued by a pharmacological chaperone, the opioid antagonist naltrexone. Co-immunoprecipitation of metabolically labeled variants revealed that both endoplasmic reticulum-localized precursors and mature receptors exist as homo/heteromers. The existence of homo/heteromers was confirmed in living cells by bioluminescence resonance energy transfer measurements, showing that the variants have a similar propensity to form homo/heteromers. By forming both homomers and heteromers, the hδOR-Cys-27 variant may thus regulate the levels of receptors at the cell surface, possibly leading to altered responsiveness to opioid ligands in individuals carrying the Cys-27 variant.     

Kinematic and kinetic changes in obese gait in bariatric surgery-induced weight loss

This study examines the effects of a radical bariatric surgery-induced weight loss on the gait of obese subjects. We performed a three-dimensional motion analysis of lower limbs, and collected force platform data in the gait laboratory to calculate knee and hip joint moments. Subjects (n=13) performed walking trials in the laboratory before and 8.8 months (SD 4.2) after the surgical procedure at two gait speeds (1.2m/s and 1.5m/s). The average weight loss was 26.7kg (SD 9.2kg), corresponding to 21.5% (SD 6.8%) of the initial weight. We observed a decrease in step width at both gait speeds, but no changes in relative double support or swing time or stride length. A significant decrease was noted in the absolute values of peak knee abductor, peak knee flexor and peak hip extensor moments. However, the moment values normalized by the body weight and height remained unchanged in most cases. Thus, we conclude that weight loss reduces hip and knee joint moments in proportion to the amount of weight lost.     

Adenosine kinase and 5'-nucleotidase activity after prolonged wakefulness in the cortex and the basal forebrain of rat

The effect of prolonged wakefulness on adenosine kinase (AK), ecto-5'-nucleotidase and endo-5'-nucleotidase activity was assessed in the present study. Rats were sleep deprived for 3 or 6h, and one group was allowed to sleep 2h of recovery sleep after the 6h deprivation. The cortex and the basal forebrain were dissected, and frozen rapidly on dry ice. The enzyme activity of adenosine kinase was measured by monitoring the conversion of [2-3H]-adenosine into [3H]-adenosine monophosphate (AMP) and the ecto-5'-nucleotidase and endo-5'-nucleotidase activities by monitoring the conversion of [2-3H]-AMP into [3H]-adenosine. The enzyme activities did not change during deprivation or recovery sleep in either cortex or basal forebrain when compared to unhandled controls. Significant diurnal variation in enzyme activities was noted in both brain areas. In the basal forebrain adenosine kinase and both nucleotidases showed their lowest activity in the middle of the rest phase, 6h after lights on, suggesting a low level of adenosine metabolism, both production and degradation at this time point. In the cortex adenosine kinase had a diurnal activity pattern similar to the basal forebrain and the ecto-5'-nucleotidase activity was low already early in the rest phase, 3h after lights on, and remained low until the end part of the rest phase, 8h after lights on. Endo-5'-nucleotidase lacked diurnal variation. These activity patterns may be associated with the lower level of energy metabolism during sleep compared to wakefulness.     

Characterization of a de novo conversion in human complement C4 gene producing a C4B5-like protein

Complement C4 is a highly polymorphic protein essential for the activation of the classical complement pathway. Most of the allelic variation of C4 resides in the C4d region. Four polymorphic amino acid residues specify the isotype and an additional four specify the Rodgers and Chido determinants of the protein. Rare C4 allotypes have been postulated to originate from recombination between highly homologous C4 genes through gene conversions. Here we describe the development of a de novo C4 hybrid protein with allotypic and antigenic diversity resulting from nonhomologous intra or interchromosomal recombination of the maternal chromosomes. A conversion was observed between maternal C4A3a and C4B1b genes producing a functional hybrid gene in one of the children. The codons determining the isotype, Asp(1054), Leu(1101), Ser(1102), Ile(1105) and His(1106), were characteristic of C4B gene, whereas the polymorphic sites in exon and intron 28 were indicative of C4A3a sequence. The protein produced by this hybrid gene was electrophoretically similar to C4B5 allotype. It also possesses reversed antigenicity being Rodgers 1, 2, 3 and Chido-1, -2, -3, 4, -5, and -6. Our case describes the development of a rare bimodular C4B-C4B haplotype containing a functional de novo C4 hybrid gene arisen through gene conversion from C4A to C4B. Overall the data supports the hypothesis of gene conversions as an ongoing process increasing allelic diversity in the C4 locus.     

Effects of aluminium and lead on ATPase activity of knockout +/- mouse cerebral synaptosomes in vitro

In this in vitro study, changes in the activity of the neural membrane integral protein, ATPase, were recorded after the exposure of isolated synaptosomes to different concentrations of aluminium and lead. Both total ATPase activity and Mg(2+)-ATPase activity were studied. A specific mouse strain, heterozygous for a glial cell line-derived neurotrophic factor (GDNF), and the corresponding wildtype mouse cerebral tissue, were used for the synaptosome isolations. The ATPase activities of the +/- mouse synaptosomes were compared with those of wild-type synaptosomes. The decrease in total ATPase activity was similar in both types of synaptosomes, but after exposure to aluminium, the decrease of Mg(2+)-ATPase activity in the GDNF+/- synaptosomes was smaller than that in the wild-type synaptosomes. After exposure to lead, the protective effect of GDNF was not so clear. The synaptosomal effects of lead were already found at concentrations lower than those where cell toxicity appeared in SH-SY5Y cell cultures. Thus, synaptosomal ATPase activity was considered to be a sensitive marker for the detection of lead-induced neurotoxicity.     

Hereditary prostate cancer in Finland: fine-mapping validates 3p26 as a major predisposition locus

In a recent genome-wide linkage (GWL) analysis of Finnish families at high risk for prostate cancer, we found two novel putative susceptibility loci at 3p25-p26 and 11q14. Here, we report the fine-mapping of these two critical regions at high resolution with 39 microsatellite markers in 16 families, including multiplex families that were not used in the GWL scan. The maximum multipoint HLOD was 3.39 at 3p26 and 1.42 at 11q14. The highest LOD scores were seen around markers D3S1270 and D3S4559 (=0.89), covering approximately two megabases. The two known genes in this region CHL1 (cell adhesion molecule with homology to L1CAM) and CNTN6 (contactin 6) were screened for exonic mutations in the families showing the strongest linkage, but no disease-segregating sequence variants were observed. The recombination map pointed to a region proximal to the area of best linkage, suggesting that more genes may need to be investigated as candidates. These results provide strong evidence for the existence of a prostate cancer susceptibility gene at 3p26 in Finnish prostate cancer families. This locus has not been strongly linked with hereditary prostate cancer in other populations. However, the mildly positive 3p LOD scores in a recent GWL analysis of patients from the United States suggest that the locus may also be important in other populations.     

S-Nitrosothiols modulate G protein-coupled receptor signaling in a reversible and highly receptor-specific manner

Background  

Recent studies indicate that the G protein-coupled receptor (GPCR) signaling machinery can serve as a direct target of reactive oxygen species, including nitric oxide (NO) and S-nitrosothiols (RSNOs). To gain a broader view into the way that receptor-dependent G protein activation – an early step in signal transduction – might be affected by RSNOs, we have studied several receptors coupling to the G_i family of G proteins in their native cellular environment using the powerful functional approach of [³⁵S]GTPγS autoradiography with brain cryostat sections in combination with classical G protein activation assays.     

The spectrum of mitochondrial DNA mutations in families with Leber hereditary optic neuroretinopathy

The mitochondrial complex I genes were sequenced in seven Leber hereditary optic neuroretinopathy (LHON) families without the ND4/11778 and ND1/3460 mutations. Four replacement mutations restricted only to LHON families were found, one in the ND1 gene at nt 4025, and three in the ND5 gene at nt 12811, 13637, and 13967. The mutations did not change evolutionarily conserved amino acids suggesting that they are not primary LHON mutations in these families. They may be considered as secondary LHON mutations serving as exacerbating factors in an appropriate genetic background. A complex III mutation, cyt b/15257, has been suggested to be one of the primary mutations causing LHON. Its presence was determined for 23 Finnish LHON families, and it was detected in two families harboring the ND4/11778 mutation. Similarly, complex IV mutation COI/7444 was screened in Finnish LHON families, and it was found in one family carrying the ND1/3460 mutation.