Population genetic structure of the char species of the Northern Kuril Islands and the rank of the Dolly Varden Char in the system of the genus Salvelinus (Salmonidae: Teleostei)

Analysis of the taxonomic position of most species and forms of the char (genus Salvelinus, Salmonidae: Teleostei) was made based on RAPD-PCR. The material was represented by samples from 29 populations from the Kuril Islands, coast of the Sea of Okhotsk, Kamchatka, Chukotka, Taymyr, Transbaikalia, the Kola Peninsula, Svalbard, Finland, and North America. It was shown that the genus Salvelinus splits into three well-justified clusters: (1) all the forms assigned to the Salvelinus alpinus--S. malma complex; (2) two samples of the White-Spotted Char from the Southern Kuril Islands and from Kamchatka; (3) two North American species, S. fontinalis and S. namaycush (samples of the North American species S. confluentis were absent from the collection). Analysis of the absolute values of genetic disctances of the S. alpinus--S. malma forms relative to S. leucomaenis, S. fontinalis, and S. namaycush revealed distances approaching the species rank between the following isolates: Frolikh Char, Mountain Char, Black Lake Char, Goggle-Eyed Char, and Neyva Char. Samples of Dolly Varden currently considered as "S. malma", do not constitute a separate cluster, falling within the group of the Arctic char S. alpinus. This conclusion is supported by the analysis of the results of three series of experiments by R. Phillips on ITS1 ribosome genes (Pleute et al., 1992; Phillips et al., 1995; Phillips et al., 1999). This indicates the infraspecific rank of malma within S. alpinus. Isolated populations of "Salvethymus svetovidovi" from the lake Elgygytgyn (Chukotka Peninsula) and of the char from the lake Chyornoye (Onekotan Island), recently described as S. gritzenkoi (Vasil'eva, Stygar, 2000), fell withing the S. alpinus--S. malma complex, the Onekotan char grouped together with another isolate from the same island. Comparison of genetic distances between the samples showed that the differences between the two isolated of Onekotan and migratory forms of the Kuril Islands are approximately equal, yet the homogeneity of the Chyornoye sample is higher than that of the other samples. The revealed 330-nucleotide diagnostic sequence of the Onekotan lake isolate showed identity of part of the fragment with a section of expressed DNA from the library of EST clones derived from the gills of Salmo salar, this possibly indicates the adaptive character of the evolution.     

Age-related changes in the functioning of the mitochondrial potassium-transporting system

Changes in the rate of respiration and functioning of the ATP-dependent potassium channel in liver and heart mitochondria of one-, three-, eight-, and 24-month-old Wistar male rats have been investigated. It was shown that the activity of the channel in the mitochondria of both tissues in 24-month-old animals decreases more than three times, and the content of potassium, 1.5–2 times compared with young one-month-old rats. The changes occur against the background of age-related decrease of energy supply in mitochondria, the respiratory complex-I undergoing the greatest changes upon aging. The decrease of channel activity may be the result of changes in channel sensitivity to modulators and a decrease in the expression of mitochondrial K+-transporting channel-protein with a molecular mass of 5.5 kDa upon aging found in this work. As a result, the functioning of not only the mitoK_ATP but also the whole mitochondrial potassium cycle is impaired.     

Isolation and purification of human blood plasma proteins able to form potassium channels in artificial bilayer lipid membrane

A protein fraction able to induce K⁺-selective transport across a bilayer lipid membrane was isolated from human blood plasma with the use of the detergent- and proteolytic enzyme-free method developed in our laboratory. After addition of the studied sample to the artificial membrane in the presence of 100 mM KCl, a discrete current change was observed. No channel activity was recorded in the presence of calcium and sodium ions. Channel-forming activity of the fraction was observed only in the presence of K⁺. Using a threefold gradient of KCl in the presence of studied proteins, a potassium-selective potential balanced by voltage of ?29 mV was registered. This value is very close to the theoretical Nernst potential in this case. This means that the examined ion channel is cation-selective. According to the data obtained with MS-MALDI-TOF/TOF and NCBI database, three protein components were identified in the isolated sample.     

Reversed-phase chromatography with multiple fraction concatenation strategy for proteome profiling of human MCF10A cells

In this study, we evaluated a concatenated low pH (pH 3) and high pH (pH 10) reversed-phase liquid chromatography strategy as a first dimension for two-dimensional liquid chromatography tandem mass spectrometry ("shotgun") proteomic analysis of trypsin-digested human MCF10A cell sample. Compared with the more traditional strong cation exchange method, the use of concatenated high pH reversed-phase liquid chromatography as a first-dimension fractionation strategy resulted in 1.8- and 1.6-fold increases in the number of peptide and protein identifications (with two or more unique peptides), respectively. In addition to broader identifications, advantages of the concatenated high pH fractionation approach include improved protein sequence coverage, simplified sample processing, and reduced sample losses. The results demonstrate that the concatenated high pH reversed-phased strategy is an attractive alternative to strong cation exchange for two-dimensional shotgun proteomic analysis.     

Single-nucleotide polymorphism in populations of sockeye salmon Oncorhynchus nerka from Kamchatka Peninsula

The genetic variability of 45 single-nucleotide polymorphism loci was examined in the four largest wild populations of sockeye salmon Oncorhynchus nerka from drainages of the Asian coast of the Pacific Ocean (Eastern and Western Kamchatka). It was demonstrated that sockeye salmon from the Palana River were considerably different from all other populations examined. The most probable explanation of the observed differences is the suggestion on possible demographic events in the history of this population associated with the decrease in its effective number. To study the origin, colonization patterns, and evolution of Asian sockeye salmon, as well as to resolve some of the applied tasks, like population assignment and genetic identification, a differential approach to SNP-marker selection was suggested. Adaptively important loci that evolve under the pressure of balancing (stabilizing) selection were identified, owing to this fact the number of loci that provide the baseline classification error rates in the population assignment tests was reduced to 30. It was demonstrated that SNPs located in the MHC2 and GPH genes were affected by diversifying selection. Procedures for selecting single-nucleotide polymorphisms for phylogenetic studies of Asian sockeye salmon were suggested. Using principal-component analysis, 17 loci that adequately reproduce genetic differentiation within and among the regions of the origin of Kamchatka sockeye salmon were selected.     

Functioning of the mitochondrial ATP-dependent potassium channel in rats varying in their resistance to hypoxia. Involvement of the channel in the process of animal’s adaptation to hypoxia

The mechanism of tissue protection from ischemic damage by activation of the mitochondrial ATP-dependent K⁺ channel (mitoK_ATP) remains unexplored. In this work, we have measured, using various approaches, the ATP-dependent mitochondrial K⁺ transport in rats that differed in their resistance to hypoxia. The transport was found to be faster in the hypoxia-resistant rats as compared to that in the hypoxia-sensitive animals. Adaptation of animals to the intermittent normobaric hypoxia increased the rate of transport. At the same time, the intramitochondrial concentration of K⁺ in the hypoxia-sensitive rats was higher than that in the resistant and adapted animals. This indicates that adaptation to hypoxia stimulates not only the influx of potassium into mitochondria, but also K⁺/H⁺ exchange. When mitoK_ATP was blocked, the rate of the mitochondrial H₂O₂ production was found to be significantly higher in the hypoxia-resistant rats than that in the hypoxia-sensitive animals. The natural flavonoid-containing adaptogen Extralife, which has an evident antihypoxic effect, increased the rate of the mitochondrial ATP-dependent K⁺ transport in vitro and increased the in vivo tolerance of hypoxia-sensitive rats to acute hypoxia 5-fold. The involvement of the mitochondrial K⁺ transport in the mechanism of cell adaptation to hypoxia is discussed.     

Proteomic Analysis of Saliva in HIV-Positive Heroin Addicts Reveals Proteins Correlated with Cognition

The prevalence of HIV-associated neurocognitive disorders (HAND) remains high despite effective antiretroviral therapies. Multiple etiologies have been proposed over the last several years to account for this phenomenon, including the neurotoxic effects of antiretrovirals and co-morbid substance abuse; however, no underlying molecular mechanism has been identified. Emerging evidence in several fields has linked the gut to brain diseases, but the effect of the gut on the brain during HIV infection has not been explored. Saliva is the most accessible gut biofluid, and is therefore of great scientific interest for diagnostic and prognostic purposes. This study presents a longitudinal, liquid chromatography-mass spectrometry-based quantitative proteomics study investigating saliva samples taken from 8 HIV-positive (HIV⁺), 11 −negative (HIV⁻) heroin addicts. In addition, saliva samples were investigated from 11 HIV⁻, non-heroin addicted healthy controls. In the HIV⁺ group, 58 proteins were identified that show significant correlations with cognitive scores, implicating disruption of protein quality control pathways by HIV. Notably, only one protein from the HIV⁻ heroin addict cohort showed a significant correlation with cognitive scores, and no proteins correlated with cognitive scores in the healthy control group. In addition, the majority of correlated proteins have been shown to be associated with exosomes, allowing us to propose that the salivary glands and/or oral epithelium may modulate brain function during HIV infection through the release of discrete packets of proteins in the form of exosomes.     

Applying a targeted label-free approach using LC-MS AMT tags to evaluate changes in protein phosphorylation following phosphatase inhibition

To identify phosphoproteins regulated by the phosphoprotein phosphatase (PPP) family of S/T phosphatases, we performed a large-scale characterization of changes in protein phosphorylation on extracts from HeLa cells treated with or without calyculin A, a potent PPP enzyme inhibitor. A label-free comparative phosphoproteomics approach using immobilized metal ion affinity chromatography and targeted tandem mass spectrometry was employed to discover and identify signatures based upon distinctive changes in abundance. Overall, 232 proteins were identified as either direct or indirect targets for PPP enzyme regulation. Most of the present identifications represent novel PPP enzyme targets at the level of both phosphorylation site and protein. These include phosphorylation sites within signaling proteins such as p120 Catenin, A Kinase Anchoring Protein 8, JunB, and Type II Phosphatidyl Inositol 4 Kinase. These data can be used to define underlying signaling pathways and events regulated by the PPP family of S/T phosphatases.