Activity of BK(Ca) channel is modulated by membrane cholesterol content and association with Na+/K+-ATPase in human melanoma IGR39 cells

Interaction of large conductance Ca(2+)- and voltage-activated K(+) (BK(Ca)) channels with Na(+)/K(+)-ATPase, caveolin-1, and cholesterol was studied in human melanoma IGR39 cells. Functional BK(Ca) channels were enriched in caveolin-rich and detergent-resistant membranes, i.e. rafts, and blocking of the channels by a specific BK(Ca) blocker paxilline reduced proliferation of the cells. Disruption of rafts by selective depletion of cholesterol released BK(Ca) channels from these domains with a consequent increase in their activity. Consistently, cholesterol enrichment of the cells increased the proportion of BK(Ca) channels in rafts and decreased their activity. Immunocytochemical analysis showed that BK(Ca) channels co-localize with Na(+)/K(+)-ATPase in a cholesterol-dependent manner, thus suggesting their co-presence in rafts. Supporting this, ouabain, a specific blocker of Na(+)/K(+)-ATPase, inhibited BK(Ca) whole-cell current markedly in control cells but not in cholesterol-depleted ones. This inhibition required the presence of external Na(+). Collectively, these data indicate that the presence of Na(+)/K(+)-ATPase in rafts is essential for efficient functioning of BK(Ca) channels, presumably because the pump maintains a low intracellular Na(+) proximal to the BK(Ca) channel. In conclusion, cholesterol could play an important role in cellular ion homeostasis and thus modulate many cellular functions and cell proliferation.     

Persisting chronic gastritis and elevated Helicobacter pylori antibodies after successful eradication therapy

AIM: The persistence of chronic inflammation in gastric mucosa and elevated Helicobacter pylori antibodies after successful eradication therapy are common findings in clinical practice. We studied their possible association with each other and disappearance in long-term follow up, as well as their possible connection with gastric atrophy. PATIENTS AND METHODS: The study population consisted of 108 dyspeptic patients with successful eradication therapy median 6.4 years earlier. The patients underwent gastroscopy, and biopsies from antrum and corpus were evaluated by an experienced pathologist. Serum samples collected from 77 patients were studied for H. pylori antibodies, parietal cell antibodies, as well as for pepsinogen I, pepsinogen II, and gastrin-17 levels. RESULTS: The prevalence of chronic gastric inflammation and elevated H. pylori antibodies after successful eradication therapy decreased by time, but still after 5 years, 17 of 51 (33%) subjects had elevated H. pylori antibodies and 14 of 68 (21%) had a mild inactive chronic inflammation in gastric mucosa. In patients with and without chronic inflammation in gastric mucosa, elevated H. pylori antibodies were detected in three of 10 (30%) and 14 of 41 (34%), elevated parietal cell antibodies in one of 10 (10%) and six of 41 (15%), low pepsinogen I in one of 10 (10%) and none of 41, and elevated gastrin-17 in three of 10 (30%) and six of 41 (15%), respectively. CONCLUSION: More than 5 years after successful H. pylori eradication therapy, mild persistent chronic inflammation may occur in gastric mucosa in up to one-fifth and elevated H. pylori antibodies even in one-third of patients, although these two are independent phenomena.     

Functional characterization of enzymes catalyzing ceramide phosphoethanolamine biosynthesis in mice

Besides bulk amounts of SM, mammalian cells produce small quantities of the SM analog ceramide phosphoethanolamine (CPE). Little is known about the biological role of CPE or enzymes responsible for CPE production. Heterologous expression studies revealed that SM synthase (SMS)2 is a bifunctional enzyme producing both SM and CPE, whereas SMS-related protein (SMSr) serves as monofunctional CPE synthase. Acute disruption of SMSr catalytic activity in cultured cells causes a rise in endoplasmic reticulum (ER) ceramides, fragmentation of ER exit sites, and induction of mitochondrial apoptosis. To address the relevance of CPE biosynthesis in vivo, we analyzed the tissue-specific distribution of CPE in mice and generated mouse lines lacking SMSr and SMS2 catalytic activity. We found that CPE levels were >300-fold lower than SM in all tissues examined. Unexpectedly, combined inactivation of SMSr and SMS2 significantly reduced, but did not eliminate, tissue-specific CPE pools and had no obvious impact on mouse development or fertility. While SMSr is widely expressed and serves as the principal CPE synthase in the brain, blocking its catalytic activity did not affect ceramide levels or secretory pathway integrity in the brain or any other tissue. Our data provide a first inventory of CPE species and CPE-biosynthetic enzymes in mammals.     

Self-Rated Health in the Last 12 Years of Life Compared to Matched Surviving Controls: The Health and Retirement Study

Self-rated health (SRH) is a valid measure of health status and associated with mortality. Based on individual-level biannual repeat data on SRH we sought to characterize the natural history of poor SRH during the 12 years prior to death in men and women in different age groups. We conducted a retrospective analysis of the Health and Retirement Study participants who died between 1998 and 2010 and had at least two SRH measurements in the 12 years prior to death. We used a nested case-control design to compare SRH trajectories of deceased men and women aged 30–64, 65–79 and 80 years versus surviving participants. The cases comprised 3,350 deceased participants who were matched to surviving controls (n = 8,127). SRH was dichotomized into good vs. poor health. Men and women dying at age 65–79 and ≥80 years had 1.5 to 3 times higher prevalence of poor SRH already 11–12 years prior to death compared to surviving controls. The risk estimates remained statistically significant even after adjusting for life-style related risk factors and diagnosed diseases. Prevalence of poor SRH before death was lowest among those aged ≥80 years and highest in 30–64 year-olds. In conclusion, men and women who subsequently die perceive their health worse already 11–12 years prior to death compared to their surviving controls.     

Quantifying neighbourhood socioeconomic effects in clustering of behaviour-related risk factors: a multilevel analysis

Background

The extent to which neighbourhood characteristics explain accumulation of health behaviours is poorly understood. We examined whether neighbourhood disadvantage was associated with co-occurrence of behaviour-related risk factors, and how much of the neighbourhood differences in the co-occurrence can be explained by individual and neighbourhood level covariates.

Methods

The study population consisted of 60 694 Finnish Public Sector Study participants in 2004 and 2008. Neighbourhood disadvantage was determined using small-area level information on household income, education attainment, and unemployment rate, and linked with individual data using Global Positioning System-coordinates. Associations between neighbourhood disadvantage and co-occurrence of three behaviour-related risk factors (smoking, heavy alcohol use, and physical inactivity), and the extent to which individual and neighbourhood level covariates explain neighbourhood differences in co-occurrence of risk factors were determined with multilevel cumulative logistic regression.

Results

After adjusting for age, sex, marital status, and population density we found a dose-response relationship between neighbourhood disadvantage and co-occurrence of risk factors within each level of individual socioeconomic status. The cumulative odds ratios for the sum of health risks comparing the most to the least disadvantaged neighbourhoods ranged between 1.13 (95% confidence interval (CI): 1.03–1.24) and 1.75 (95% CI, 1.54–1.98). Individual socioeconomic characteristics explained 35%, and neighbourhood disadvantage and population density 17% of the neighbourhood differences in the co-occurrence of risk factors.

Conclusions

Co-occurrence of poor health behaviours associated with neighbourhood disadvantage over and above individual''s own socioeconomic status. Neighbourhood differences cannot be captured using individual socioeconomic factors alone, but neighbourhood level characteristics should also be considered.     

Tracking Sphingosine Metabolism and Transport in Sphingolipidoses: NPC1 Deficiency as a Test Case

The late endosomal/lysosomal compartment (LE/LY) plays a key role in sphingolipid breakdown, with the last degradative step catalyzed by acid ceramidase. The released sphingosine can be converted to ceramide in the ER and transported by ceramide transfer protein (CERT) to the Golgi for conversion to sphingomyelin. The mechanism by which sphingosine exits LE/LY is unknown but Niemann-Pick C1 protein (NPC1) has been suggested to be involved. Here, we used sphingomyelin, ceramide and sphingosine labeled with [(3) H] in carbon-3 of the sphingosine backbone and targeted them to LE/LY in low-density lipoprotein (LDL) particles. These probes traced LE/LY sphingolipid degradation and recycling as suggested by (1) accumulation of [(3) H]-sphingomyelin-derived [(3) H]-ceramide and depletion of [(3) H]-sphingosine upon acid ceramidase depletion, and (2) accumulation of [(3) H]-sphingosine-derived [(3) H]-ceramide and attenuation of [(3) H]-sphingomyelin synthesis upon CERT depletion. NPC1 silencing did not result in the accumulation of [(3) H]-sphingosine derived from [(3) H]-sphingomyelin/LDL or [(3) H]-ceramide/LDL. Additional evidence against NPC1 playing a significant role in LE/LY sphingosine export was obtained in experiments using the [(3) H]-sphingolipids or a fluorescent sphingosine derivative in NPC1 knock-out cells. Instead, NPC1-deficient cells displayed an increased affinity for sphingosine independently of protein-mediated lipid transport. This likely contributes to the increased sphingosine content of NPC1 cells.     

Conserved domains and evolution of secreted phospholipases A(2)

Secreted phospholipases A(2) (sPLA(2) s) are lipolytic enzymes present in organisms ranging from prokaryotes to eukaryotes but their origin and emergence are poorly understood. We identified and compared the conserved domains of 333 sPLA(2) s and proposed a model for their evolution. The conserved domains were grouped into seven categories according to the in silico annotated conserved domain collections of 'cd00618: PLA(2) _like' and 'pfam00068: Phospholip_A2_1'. PLA(2) s containing the conserved domain cd04706 (plant-specific PLA(2) ) are present in bacteria and plants. Metazoan PLA(2) s of the group (G) I/II/V/X PLA(2) collection exclusively contain the conserved domain cd00125. GIII PLA(2) s of both vertebrates and invertebrates contain the conserved domain cd04704 (bee venom-like PLA(2) ), and mammalian GIII PLA(2) s also contain the conserved domain cd04705 (similar to human GIII PLA(2) ). The sPLA(2) s of bacteria, fungi and marine invertebrates contain the conserved domain pfam09056 (prokaryotic PLA(2) ) that is the only conserved domain identified in fungal sPLA(2) s. Pfam06951 (GXII PLA(2) ) is present in bacteria and is widely distributed in eukaryotes. All conserved domains were present across mammalian sPLA(2) s, with the exception of cd04706 and pfam09056. Notably, no sPLA(2) s were found in Archaea. Phylogenetic analysis of sPLA(2) conserved domains reveals that two main clades, the cd- and the pfam-collection, exist, and that they have evolved via gene-duplication and gene-deletion events. These observations are consistent with the hypothesis that sPLA(2) s in eukaryotes shared common origins with two types of bacterial sPLA(2) s, and their persistence during evolution may be related to their role in phospholipid metabolism, which is fundamental for survival.     

Comparison of rat epidermal keratinocyte organotypic culture (ROC) with intact human skin: lipid composition and thermal phase behavior of the stratum corneum.

The present report is a part of our continuing efforts to explore the utility of the rat epidermal keratinocyte organotypic culture (ROC) as an alternative model to human skin in transdermal drug delivery and skin irritation studies of new chemical entities and formulations. The aim of the present study was to compare the stratum corneum lipid content of ROC with the corresponding material from human skin. The lipid composition was determined by thin-layer chromatography (TLC) and mass-spectrometry, and the thermal phase transitions of stratum corneum were studied by differential scanning calorimetry (DSC). All major lipid classes of the stratum corneum were present in ROC in a similar ratio as found in human stratum corneum. Compared to human skin, the level of non-hydroxyacid-sphingosine ceramide (NS) was increased in ROC, while alpha-hydroxyacid-phytosphingosine ceramide (AP) and non-hydroxyacid-phytosphingosine ceramides (NP) were absent. Also some alterations in fatty acid profiles of ROC ceramides were noted, e.g., esterified omega-hydroxyacid-sphingosine contained increased levels of oleic acid instead of linoleic acid. The fraction of lipids covalently bound to corneocyte proteins was distinctly lower in ROC compared to human skin, in agreement with the results from DSC. ROC underwent a lipid lamellar order to disorder transition (T2) at a slightly lower temperature (68 degrees C) than human skin (74 degrees C). These differences in stratum corneum lipid composition and the thermal phase transitions may explain the minor differences previously observed in drug permeation between ROC and human skin.     

Genetic differentiation among European whitefish ecotypes based on microsatellite data

The amount of genetic differentiation at DNA microsatellite loci in European whitefish (Coregonus lavaretus) was assessed among ecotypes, populations and run-timing types. The magnitude of genetic changes potentially caused by hatchery broodstock rearing were also compared with those observed in corresponding natural populations. A total of 35 populations were studied, including 33 Coregonus lavaretus populations and two samples of Coregonus peled. Five of the six whitefish ecotypes in Finland were represented within C. lavaretus populations. Genetic diversity among C. lavaretus populations proved to be high compared to two C. peled populations. The genetic D(A) distance between these two species was as high as 0.86. The genetic differentiation among ecotypes was generally low and thus gives support for the hypothesis of one native European whitefish species in Scandinavia. Among the ecotypes the northern, large sparsely-rakered, bottom-dwelling whitefish was most unique. Thus, observed genetic differences in quantitative traits have either developed independently of phylogenetic lineages, or have mixed and later changed according to environments and selection pressures. Overall genetic distances between the anadromous whitefish populations along the Finnish coast, especially in the Bothnian Bay area, were small. Populations of this area have been heavily influenced by human activities, and they also have the highest probability of mixing by natural means. In two cases, the Rivers Iijoki and Tornionjoki, statistically significant genetic differences could be observed between summer- and autumn-run spawning-time types. Wild populations had slightly higher allelic diversity than hatchery-reared populations of corresponding rivers. Although some reduction in genetic diversity during hatchery rearing is possible, it is an important aid in maintaining endangered populations.