Detection of mitochondrial dysfunction by EPR technique in mouse model of dilated cardiomyopathy

Tgalphaq44 mice with targeted overexpression of activated Galphaq protein in cardiomyocytes mimic many of the phenotypic characteristics of dilated cardiomyopathy in humans. However, it is not known whether the phenotype of Tgalphaq44 mice would also involve dysfunction of cardiac mitochondria. The aim of the present work was to examine changes in EPR signals of semiquinones and iron in Fe-S clusters, as compared to classical biochemical indices of mitochondrial function in hearts from Tgalphaq44 mice in relation to the progression of heart failure. Tgalphaq44 mice at the age of 14 months displayed pulmonary congestion, increased heart/body ratio and impairment of cardiac function as measured in vivo by MRI. However, in hearts from Tgalphaq44 mice already at the age of 10 months EPR signals of semiquinones, as well as cyt c oxidase activity were decreased, suggesting alterations in mitochondrial electron flow. Furthermore, in 14-months old Tgalphaq44 mice loss of iron in Fe-S clusters, impaired citrate synthase activity, and altered mitochondrial ultrastructure were observed, supporting mitochondrial dysfunction in Tgalphaq44 mice. In conclusion, the assessment of semiquinones content and Fe(III) analysis by EPR represents a rational approach to detect dysfunction of cardiac mitochondria. Decreased contents of semiquinones detected by EPR and a parallel decrease in cyt c oxidase activity occurs before hemodynamic decompensation of heart failure in Tgalphaq44 mice suggesting that alterations in function of cardiac mitochondria contribute to the development of the overt heart failure in this model.     

In vivo spin trapping of nitric oxide from animal tumors.

Spin trapping/electron paramagnetic resonance (EPR) spectroscopy allows specific detection of nitric oxide (NO) generation, in vivo. However, in order to detect an EPR signal in living organism, usually a stimulation of immune system with LPS is used to achieve higher than physiological NO levels. Here, we report non-invasive spin trapping of NO in tumors of non-treated, living animals. EPR spectroscopy was performed at S-band to detect NO in Cloudman S91 melanoma tumors growing in the tail of living, syngeneic hosts-DBA/2 mice. Iron (II) N-(dithiocarboxy)sarcosine Fe2+(DTCS)(2) was used as the spin trap. The results were confirmed by X-band ex vivo study. A characteristic three-line spectrum of NO-Fe(DTCS)(2) (A(N)=13 G) was observed (n=4, out of total n=6) in non-treated tumors and in tumors of animals treated with l-arginine. Substrate availability did not limit the detection of NO by spin trapping. Half-life time of the NO-Fe(DTCS)(2) in tumor tissue was about 60 min. The feasibility of non-invasive spin trapping/EPR spectroscopic detection of NO generated in tumor tissue in living animals, without additional activation of the immune system, was demonstrated for the first time.     

The taste of polycose in hamsters

Hamsters show a preference for Polycose, a mixture of starch-derived glucose polymers, that is as strong as their preference for sucrose. However, in the hamster, taste aversions to Polycose may be less easily acquired than taste aversions to sucrose and the qualitative aspects of Polycose are unknown in this species. In order to examine the taste of Polycose in the hamster, we utilized a taste-aversion protocol with two conditioning trials. Animals were trained to avoid one of three different conditioning stimuli: 50 mM sucrose, 100 mM Polycose and a mixture of 50 mM sucrose with 100 mM Polycose. Control animals were conditioned with deionized water. After the second conditioning trial, generalization testing began for the three conditioning stimuli plus 3 mM citric acid, 300 mM KCI and 30 mM NaCl. The results showed that aversions to Polycose, sucrose or the Polycose/sucrose mixture cross- generalized, demonstrating that Polycose and sucrose share a common taste percept in the hamster. None of the aversions generalized to NaCl, citric acid or KCI. In addition, comparisons among the patterns of taste generalizations indicated that the tastes of Polycose and sucrose also had distinct qualitative components. Finally, although the taste of 100 mM Polycose was more salient than the taste of 50 mM sucrose, the taste of sucrose could still be detected in a mixture with Polycose.      

Changes in the nitric oxide level in the rat liver as a response to brain injury

Liver disturbances stimulate inflammatory reaction in the brain but little is known if injury to the brain can significantly influence liver metabolism. This problem is crucial in modern transplantology, as the condition of the donor brain seems to strongly affect the quality (viability) of the graft, which is often obtained from brain-dead donors, usually after traumatic brain injury. Because nitric oxide is one of the significant molecules in brain and liver biology, we examined if brain injury can affect NO level in the liver. Liver samples of Wistar rats were collected and studied with EPR NO-metry to detect NO level changes at different time points after brain injury. Shortly after the trauma, NO level in the liver was similar to the control. However, later there was a significant increase in the NO content in the livers starting from the 2nd day after brain injury and lasting up to the 7th day. It seems that the response to a mechanical brain injury is of the systemic, rather than local character. Therefore brain metabolism disturbances can influence liver metabolism at least by stimulating the organ to produce NO.     

Palatability and efficacy to possums and rats of pest control baits containing bird repellents

Repellents used to reduce by-kill of birds during pest control must not compromise acceptance by target species. Two repellents combined, anthraquinone (AQ; 0.4 g kg^?1) and d-pulegone (DP; 1.0) did not reduce the palatability of blue-coloured carrot baits to laboratory rats (Rattus norvegicus); nor did DP (2.0). Green-coloured carrot baits coated with AQ, DP or AQ + DP were taken from bait stations by wild possums (Trichosurus vulpecula) and rats. Toxic (1080) bait coated with AQ (0.4) and peanut oil (0.1) had reduced palatability but was accepted by laboratory rats. However, laboratory rats did not consume enough baits coated with AQ and bacon, peanut butter, cinnamon or DP to be killed. Anthraquinone (0.4 or 0.8) plus cinnamon and DP (0.5) did not affect palatability or lethality to captive ship rats (R. rattus) or possums. Anthraquinone and DP as surface coatings on baits are therefore acceptable to possums and possibly rats, at concentrations that deter some bird species.     

Isolation of zoosporogenous actinomycetes from desert soils

We have undertaken a study to estimate the species diversity of zoosporogenous actinomycetes that can be isolated from an arid environment. The study site encompassed an area of approximately 22 000 square kilometers of the Mojave Desert along the California-Nevada border. A series of 29 soil samples was collected along two intersecting transects of approximately 190 and 240 km which traversed a number of distinct ecosystems. A₀ horizon soils were collected from the rhizosphere of the predominant vegetation at each sampling site and screened for the target genera using selective isolation techniques: chemoattraction (xylose and -collidine) and baiting with hair. Following incubation of primary isolation plates for 28 days at 28°C, all colonies that exhibited filamentous growth, presence of sporangia and/or motile spores upon direct microscopic observation (450 and 1000×) were further characterized by fatty acid analysis (FAME). Most of the isolates fell into three broad clusters that roughly correlated with presumptive genus assignments. Individual isolates could be assigned to 226 FAME biotypes based on chromatographic similarity (85%). The dominant species (514/826 isolates) belong to a previously undescribed taxon that morphologically resemblesGeodermatophilus but possesses unique FAME profiles that include at least three novel lipids. The remainder of the isolates were species ofActinoplanes, indeterminate species or vagrant isolates ofStreptomyces.     

Choline acetyltransferase activity of spinal cord cell cultures increased by co-culture with muscle and by muscle-conditioned medium

Activity of the enzyme choline acetyltransferase (CAT), which mediates the synthesis of the neurotransmitter, acetylcholine, was increased up to 20- fold in spinal cord (SC) cells grown in culture with muscle cells for 2 wk. This increase was directly related to the duration of co-culture as well as to the cell density of both the SC and muscle involved and was not affected by the presence of the acetylcholine receptor blocking agent, α-bungarotoxin. Glutamic acid decarboxylase (GAD) activity was often markedly decreased in SC-muscle cultures while the activities of acetylcholinesterase and several other enzymes were little changed. Increased CAT activity was also observed when SC cultures were maintained in medium which had been conditioned by muscle cells or by undifferentiated cells from embryonic muscle. Muscle-conditioned medium (CM) did not affect the activities of SC cell GAD or acetylcholinesterase. Dilution or concentration of the CM directly affected its ability to increase SC CAT activity , as did the duration and timing of exposure of the SC cells to the CM. The medium could be conditioned by muscle cells in the presence or absence of serum, and remained effective after dialysis or heating to 58 degrees C. Membrane filtration data were consistent with the conclusion that the active material(s) in CM had a molecular weight in excess of 50,000 daltons. We conclude that large molecular weight material that is released by muscle cells is capable of producing a specific increase in CAT activity of SC cells.     

Splenic eumelanin differs from hair eumelanin in C57BL/6 mice

The presence of melanin in spleens of black C57BL/6 mice has been known for long. Although its origin and biological functions are still obscure, the relation of splenic melanin to the hair follicle and skin pigmentation was suggested. Here, we demonstrated using for the first time electron paramagnetic resonance spectroscopy that black-spotted C57BL/6 spleens contain eumelanin. Its presence here is a "yes or no" phenomenon, as even in the groups which revealed the highest percentage of spots single organs completely devoid of the pigment were found. Percentage of the spotted spleens decreased, however, with the progress of telogen after spontaneously-induced hair growth. The paramagnetic properties of the spleen eumelanin differed from the hair shaft or anagen VI skin melanin. The splenic melanin revealed narrower signal, and its microwave power saturability betrayed more heterogenous population of paramagnetic centres than in the skin or hair shaft pigment. Interestingly, the pigment of dry hair shafts and of the wet tissue of depilated anagen VI skin revealed almost identical properties. The properties of splenic melanin better resembled the synthetic dopa melanin (water suspension, and to a lesser degree - powder sample) than the skin/hair melanin. All these findings may indicate a limited degradation of splenic melanin as compared to the skin/hair pigment. The splenic eumelanin may at least in part originate from the skin melanin phagocyted in catagen by the Langerhans cells or macrophages and transported to the organ.     

Critical temperatures for xylogenesis in conifers of cold climates

Aim To identify temperatures at which cell division and differentiation are active in order to verify the existence of a common critical temperature determining growth in conifers of cold climates. Location Ten European and Canadian sites at different latitudes and altitudes. Methods The periods of cambial activity and cell differentiation were assessed on a weekly time‐scale on histological sections of cambium and wood tissue collected over 2 to 5 years per site from 1998 to 2005 from the stems of seven conifer species. All data were compared with daily air temperatures recorded from weather stations located close to the sites. Logistic regressions were used to calculate the probability of xylogenesis and of cambium being active at a given temperature. Results Xylogenesis lasted from May to October, with a growing period varying from 3 to 5 months depending on location and elevation. Despite the wide geographical range of the monitored sites, temperatures for onset and ending of xylogenesis converged towards narrow ranges with average values around 4–5, 8–9 and 13–14 °C for daily minimum, mean and maximum temperature, respectively. On the contrary, cell division in the cambium stopped in July?August, when temperatures were still high. Main conclusions Wood formation in conifers occurred when specific critical temperatures were reached. Although the timing and duration of xylogenesis varied among species, sites and years, the estimated temperatures were stable for all trees studied. These results provide biologically based evidence that temperature is a critical factor limiting production and differentiation of xylem cells in cold climates. Although daily temperatures below 4?5 °C are still favourable for photosynthesis, thermal conditions below these values could inhibit the allocation of assimilated carbon to structural investment, i.e. xylem growth.