Primary culture of lobster (Homarus americanus) olfactory sensory neurons

Lobster olfactory sensory neurons have contributed to a number of advances in our understanding of olfactory physiology. To facilitate further study of their function, we have developed conditions allowing primary culture of the olfactory sensory neurons in a defined medium. The most common cells in the culture were round cell bodies with diameters of 10-15 micro m that often extended fine processes, features resembling olfactory sensory neurons. We discovered that acetylcholinesterase acted as a growth factor for these cells, improving their survival in culture. We also confirmed previous evidence from spiny lobsters that poly-D-lysine was a superior substrate for olfactory cells of this size and morphology. We then identified olfactory sensory neurons in the culture in two ways. Almost half the cells tested responded to application of a complex odorant with an inward current. An even more rigorous test was made possible by the development of an antiserum to OET-07, an ionotropic glutamate receptor homolog specifically expressed by Homarus americanus olfactory sensory neurons. It labeled a majority of the round cells in the culture, unequivocally identifying them as olfactory sensory neurons.     

Molecular Remodeling of Tip Links Underlies Mechanosensory Regeneration in Auditory Hair Cells

Sound detection by inner ear hair cells requires tip links that interconnect mechanosensory stereocilia and convey force to yet unidentified transduction channels. Current models postulate a static composition of the tip link, with protocadherin 15 (PCDH15) at the lower and cadherin 23 (CDH23) at the upper end of the link. In terminally differentiated mammalian auditory hair cells, tip links are subjected to sound-induced forces throughout an organism''s life. Although hair cells can regenerate disrupted tip links and restore hearing, the molecular details of this process are unknown. We developed a novel implementation of backscatter electron scanning microscopy to visualize simultaneously immuno-gold particles and stereocilia links, both of only a few nanometers in diameter. We show that functional, mechanotransduction-mediating tip links have at least two molecular compositions, containing either PCDH15/CDH23 or PCDH15/PCDH15. During regeneration, shorter tip links containing nearly equal amounts of PCDH15 at both ends appear first. Whole-cell patch-clamp recordings demonstrate that these transient PCDH15/PCDH15 links mediate mechanotransduction currents of normal amplitude but abnormal Ca²⁺-dependent decay (adaptation). The mature PCDH15/CDH23 tip link composition is re-established later, concomitant with complete recovery of adaptation. Thus, our findings provide a molecular mechanism for regeneration and maintenance of mechanosensory function in postmitotic auditory hair cells and could help identify elusive components of the mechanotransduction machinery.     

Reversal of the Mitochondrial Phenotype and Slow Development of Oxidative Biomarkers of Aging in Long-lived Mclk1+/? Mice

Although there is a consensus that mitochondrial function is somehow linked to the aging process, the exact role played by mitochondria in this process remains unresolved. The discovery that reduced activity of the mitochondrial enzyme CLK-1/MCLK1 (also known as COQ7) extends lifespan in both Caenorhabditis elegans and mice has provided a genetic model to test mitochondrial theories of aging. We have recently shown that the mitochondria of young, long-lived, Mclk1^+/− mice are dysfunctional, exhibiting reduced energy metabolism and a substantial increase in oxidative stress. Here we demonstrate that this altered mitochondrial condition in young animals paradoxically results in an almost complete protection from the age-de pend ent loss of mitochondrial function as well as in a significant attenuation of the rate of development of oxidative biomarkers of aging. Moreover, we show that reduction in MCLK1 levels can also gradually prevent the deterioration of mitochondrial function and associated increase of global oxidative stress that is normally observed in Sod2^+/− mutants. We hypothesize that the mitochondrial dysfunction observed in young Mclk1^+/− mutants induces a physiological state that ultimately allows for their slow rate of aging. Thus, our study provides for a unique vertebrate model in which an initial alteration in a specific mitochondrial function is linked to long term beneficial effects on biomarkers of aging and, furthermore, provides for new evidence which indicates that mitochondrial oxidative stress is not causal to aging.Because it is well known that the aging process is characterized by declines in basal metabolic rate and in the general performance of energy-dependent processes, many aging studies have focused on mitochondria because of their central role in producing chemical energy (ATP) by oxidative phosphorylation (1). Among the various theories of aging that have been proposed, the mitochondrial oxidative stress theory of aging is the most widely acknowledged and studied (2–4). It is based on the observation that mitochondrial energy metabolism produces reactive oxygen species (ROS),² that mitochondrial components are damaged by ROS, that mitochondrial function is progressively lost during aging, and that the progressive accumulation of global oxidative damage is strongly correlated with the aged phenotype. However, the crucial question of whether these facts mean that mitochondrial dysfunction and the related ROS production cause aging remains unproven (5–7). Furthermore, recent observations made in various species, including mammals, have begun to directly challenge this hypothesis, notably by relating oxidative stress to long (8) or increased (9) lifespans, by demonstrating that overexpression of the main antioxidant enzymes does not extend lifespan (10) as well as by showing that mitochondrial dysfunction could protect against age-related diseases (11).A direct and powerful approach to attempt to clarify this major question and to test the theory is to characterize the mitochondrial function of long-lived mutants (12). CLK-1/MCLK1 is an evolutionary conserved protein (13) and has been found to be located in the mitochondria of yeast (14), worms (15), and mice (16). The inactivation of the Caenorhabditis elegans gene clk-1 substantially increases lifespan (17). Moreover, the elimination of one functional allele of its murine orthologue also resulted in an extended longevity for Mclk1^+/− mice in three distinct genetic backgrounds (18). These findings have provided for an evolutionarily conserved pathways of animal aging that is affected by the function of a mitochondrial protein (19, 20). In mitochondria CLK1/MCLK1 acts as an hydroxylase and is implicated in the biosynthesis of ubiquinone (coenzyme Q or UQ), a lipid-like molecule primarily known as an electron carrier in the mitochondrial respiratory chain and as a membrane antioxidant but which is also associated with an increasing number of different aspects of cellular metabolism (20, 21). Taken together, these observations indicate that the long-lived Mclk1^+/− mouse is a model of choice for the understanding of the links between mitochondrial energy metabolism, oxidative stress, and the aging process in mammals.Previous analysis of Mclk1^+/− mice, which show the expected reduction of MCLK1 protein levels (22), have revealed that their tissues as well as their mitochondria contain normal levels of UQ at 3 months of age (23). Yet the same study also revealed a host of phenotypes induced by Mclk1 heterozygosity (see below). Thus, it appears that MCLK1 has an additional function that is unrelated to UQ biosynthesis but responsible for the phenotypes observed in young Mclk1^+/− mutants. This is consistent with several results from nematodes which also strongly suggest that CLK-1 has other functions (24, 25).In depth characterization of the phenotype of young Mclk1^+/− mutants has revealed that the reduction of MCLK1 levels in these animals profoundly alters their mitochondrial function despite the fact that UQ production is unaffected (23). In fact, we have shown that Mclk1 heterozygosity induces a severe impairment of mitochondrial energy metabolism as revealed by a reduction in the rates of mitochondrial electron transport and oxygen consumption as well as in ATP synthesis and ATP levels in both the mitochondria and the whole cell. ATP levels in several organs were surprisingly strongly affected with, for example, a 50% reduction of overall cellular ATP levels in the livers of Mclk1^+/− mutants (23). Moreover, we have found that the Mclk1^+/− mice sustain high mitochondrial oxidative stress by a variety of measurements, including aconitase activity, protein carbonylation, and ROS production (23). Additionally, we have shown that this early mitochondrial dysfunction is associated with a reduction in some aspects of cytosolic oxidative damage and global oxidative stress that can be measured via recognized plasma biomarkers such as 8-isoprostanes and 8-hydroxy-2-deoxyguanosine (8-OHdG). Considering that the accumulation of global oxidative damage is known to be tightly linked to the aging process (26), this latter result suggests that the anti-aging effect triggered by low MCLK1 levels might already act at a young age.To further investigate the clk-1/Mclk1-dependent mechanism of aging as well as to try to elucidate the still unclear relation between mitochondrial dysfunction, oxidative stress, and aging, we have now carefully analyzed the evolution of the phenotype of Mclk1^+/− mutants over time. We have also studied the effects of reduced MCLK1 levels on the phenotype of mice heterozygous for the mitochondrial superoxide dismutase (Sod2), which represent a well known model of mitochondrial oxidative stress (27). In addition of confirming the long lifespan phenotype of the Mclk1^+/− mutants in a mixed background (129S6 x BALB/c), we also report here a study of mutants and controls on a completely isogenic background where we find that the condition of Mclk1^+/− mutants unexpectedly results in protection against the age-dependent loss of mitochondrial function. Moreover, we found that the mutants are characterized by a significant attenuation of the age-associated increase in global oxidative stress normally observed in mammals. We also show that the Mclk1^+/− condition can gradually reverse the deterioration of mitochondrial function and the associated increase of global oxidative stress that is normally observed in Sod2^+/− mutants. Thus, this study provides for a unique vertebrate model in which reduced levels of a specific mitochondrial protein causes early mitochondrial dysfunction but has long term beneficial effects that slow down the rate of aging, as established with appropriate biomarkers, and can ultimately prolong lifespan in mice. Furthermore, in line with recent studies that have raised doubts about the validity of the mitochondrial oxidative stress theory of aging (4, 8, 10), our results, which relate to a recognized long-lived mice model, represent a novel and crucial indication that mitochondrial oxidative stress might not by itself be causal to aging.     

Catalytic properties of enzymes from Erwinia carotovora involved in transamination of phenylpyruvate

K _m for L-phenylalanine, L-glutamic acid, L-aspartic acid, and the corresponding keto acids were calculated, as well as V _max was measured for the following pairs of substrates: L-phenylalanine-2-ketoglutarate, L-phenylalanine-oxaloacetate, L-glutamic acid-phenylpyruvate, and L-aspartic acid-phenylpyruvate for aminotransferases PAT1, PAT2, and PAT3 from Erwinia carotovora catalyzing transamination of phenylpyruvate. The ping-pong bi-bi mechanism was shown for the studied aminotransferases. The substrate inhibition (K _s) of PAT3 with 2-ketoglutarate and oxaloacetate was 10.23 ± 3.20 and 3.73 ± 1.99 mM, respectively. It was shown that L-β-(N-benzylamino)alanine was a competitive inhibitor with respect to L-phenylalanine for PAT1 (K _i = 0.32 ± 0.07 mM, K _m = 0.45 ± 0.1 mM, V _max = 11. 6 ± 0.4 U/mg) at 25 mM concentration of 2-ketoglutarate in the reaction medium. L-β-(N-methylamino)alanine is a noncompetitive inhibitor with respect to L-phenylalanine for PAT3 (K _I = 138.4 ± 95.4 mM, K _m = 13.7 ±3.9 mM, V _max = 18.6 ± 4.1 U/mg) at 2 mM concentration of 2-ketoglutarate in the reaction medium. L-stereo isomers of nonprotein analogues of aromatic amino acids were studied as substrates for PAT1, PAT2, and PAT3. L-β-(2-Br-phenyl)alanine, L-β-(4-Br-phenyl)alanine, L-β-(2-F-phenyl)alanine, and L-(2-F)tryptophan were good substrates for all three aminotransferases; L-α-methyl-β-(2-Br-phenyl)alanine and L-O-benzyltyrosine were substrates only for PAT3; L-β-(4-F-phenyl)alanine was a substrate for PAT1 and PAT3. Thus, these analogues of aromatic amino acids can be stereoselectively synthesized using the studied aminotransferases in the presence of the corresponding keto acids.     

Effect of oil and oil products on morphofunctional parameters of marine macrophytes

In the present paper, the authors analyzed and generalized data on the oil pollution effect on marine macrophytes at various structural levels.     

EEG reactions during creative activity

Evoked activity in response to light was recorded in students performing a verbal creative task. The changes in the amplitude of the N200 negative component of evoked potentials were analyzed. The amplitude of the N200 component was significantly increased in the frontal and anterior frontal areas of the left hemisphere, which indicated increased activity in the cortical structures involved in divergent thinking. The amplitude of the N200 component was increased in the temporo-parieto-occipital area of the right hemisphere, which indicated that the posterior associative region of the right hemisphere was also involved in the creative activity. The data obtained suggest that the frontal and temporo-parieto-occipital areas of the cerebral cortex actively participate in the performance of a creative test with distinct elements of complexity.     

Karyotypes of some Cypriniform fishes from the water bodies of Armenia

Karyotypes of six species of Cypriniformes from the water bodies of Armenia—blackbrow Acanthalburnus microlepis, white bream Blicca bjoerkna transcaucasica, chub Leuciscus cephalus orientalis, stone moroco Pseudorasbora parva, mursa Barbus mursa, and Angora stone loach Barbatula angorae were studied. The karyotype of A. microlepis is represented by 50 chromosomes (2n = 50), 10M + 28SM + 12STA, NF = 88; of B. bjoerkna transcaucasica, by 2n = 50, 12M + 24SM + 14STA, NF = 86; of L. cephalus orientalis, by 2n = 50, 12M + 18SM + 20STA, NF = 80; of P. parva, by 2n = 50, 8M + 16SM + 26STA, NF = 74; of B. mursa, by 2n = 100, 6M + 36SM + 58STA, NF = 142; and of B. angorae, by 2n = 50, 8M + 24SM + 18STA, NF = 86. The intraspecific and interspecific chromosome polymorphism of species of the genera Blicca, Leuciscus and Pseudorasbora is described.     

The location of the maximum amplitude focus of cortical evoked potential component N200 in subjects solving a maze problem

The dependence of the efficiency of maze problem solution on the “commitment” of the frontal, parietal, temporal, and occipital cortices to the activity was studied. For this purpose, topographic neurophysiological characteristics of component N200 of cortical evoked potentials were determined and used to locate the maximum amplitude focus (MAF). Right-and left-handed subjects of both sexes (50 persons aged 18–23 years) were divided into five groups: (I) women with a low nonverbal IQ, (II) women with a high nonverbal IQ, (III) men with a low nonverbal IQ, (IV) men with a high nonverbal IQ, and (V) left-handed men with a high nonverbal IQ. In subjects from group III, a successful solution of a maze problem was associated with a high activity only in the occipital cortex. In groups II and V, in addition to the strong commitment of the occipital cortex, about the same activity was observed in the frontal cortex. In groups I and IV, and MAF appeared irregularly if at all, which may have accounted for the low efficiency of maze problem solution in these subjects.     

Dietary Exposure Assessment of Potentially Toxic Trace Elements in Fruits and Vegetables Sold in Town of Kapan,Armenia

Fruits and vegetables grown under the impact of Armenia’s mining industry are widely sold in markets of adjacent towns. As the share of fruits and vegetables in Armenians’ diet is significant, the present study aims to assess the dietary exposure of potentially toxic trace elements through the intake of fruits and vegetables sold in Kapan town, located in the biggest mining region of Armenia. The concentrations of Cu, Mo, Ni, Cr, Pb, Zn, Hg, As, and Cd in 15 types of fruits and vegetables were determined. Non-carcinogenic and carcinogenic risks were assessed. Although the estimated daily intakes of trace elements for each studied food item did not exceed health-based guidelines values, in case of the combined consumption of fruits and vegetables estimated cumulative daily intakes exceeded reference doses for Cu and Mo. Moreover, carcinogenic risk for the majority of fruits and vegetables exceeded the EPA recommended risk level of 10⁻⁶, indicating adverse health effect to local population. The outcomes of this study can serve as a basis for further research that will consider many other exposure pathways (i.e., inhalation or dermal pathways) in order to ensure the safety of the residents living under the impact of mining industry.

     

Dinitrosyl iron complexes with cysteine suppress the development of experimental endometriosis in rats

Administration of dinitrosyl iron complexes (DNIC) with cysteine suppressed the development of experimental (surgically induced) endometriosis in rats: the mean size of endometrioma was 1.85 times smaller if 0.5 mL of a 5 mM aqueous solution of DNIC had been injected daily for 10 days. It is supposed that NO molecules and nitrosonium ions (NO⁺), released from DNIC rapidly decomposed in the organism, prove cytotoxic for endometrioid tissue.