Dietary interaction of high fat and marginal copper deficiency on cardiac contractile function

Objective: High‐fat and marginally copper‐deficient diets impair heart function, leading to cardiac hypertrophy, increased lipid droplet volume, and compromised contractile function, resembling lipotoxic cardiac dysfunction. However, the combined effect of the two on cardiac function is unknown. This study was designed to examine the interaction between high‐fat and marginally copper‐deficient diets on cardiomyocyte contractile function. Research Methods and Procedures: Weanling male rats were fed diets incorporating a low‐ or high‐fat diet (10% or 45% of kcal from fat, respectively) with adequate (6 mg/kg) or marginally deficient (1.5 mg/kg) copper content for 12 weeks. Contractile function was determined with an IonOptix system including peak shortening (PS), time‐to‐PS, time‐to‐90% relengthening, maximal velocity of shortening/relengthening, and intracellular Ca²⁺ ([Ca²⁺]_I) rise and decay. Results: Neither dietary treatment affected blood pressure or glucose levels, although the high‐fat diet elicited obesity and glucose intolerance. Both diets depressed PS, maximal velocity of shortening/relengthening, and intracellular Ca²⁺ ([Ca²⁺]_I) rise and prolonged time‐to‐90% relengthening and Ca²⁺ decay without an additive effect between the two. Ca²⁺ sensitivity, apoptosis, lipid peroxidation, nitrosative damage, tissue ceramide, and triglyceride levels were unaffected by either diet or in combination. Phospholamban (PLB) but not sarco(endo)plasmic reticulum Ca²⁺‐ATPase was increased by both diets. Endothelial NO synthase was depressed with concurrent treatments. The electron transport chain was unaffected, although mitochondrial aconitase activity was inhibited by the high‐fat diet. Discussion: These data suggest that high‐fat and marginally copper deficient diets impaired cardiomyocyte contractile function and [Ca²⁺]_i homeostasis, possibly through a similar mechanism, without obvious lipotoxicity, nitrosative damage, and apoptosis.     

Changes in copper and zinc status and response to dietary copper deficiency in metallothionein-overexpressing transgenic mouse heart

Previous studies have shown that cardiac-specific overexpression of metallothionein (MT) inhibits progression of dietary copper restriction-induced cardiac hypertrophy. Because copper and zinc are critically involved in myocardial response to dietary copper restriction, the present study was undertaken to understand the effect of MT on the status of copper and zinc in the heart and the subsequent response to dietary copper restriction. Dams of cardiac-specific MT-transgenic (MT-TG) mouse pups and wild-type (WT) littermates were fed copper-adequate (CuA) or copper-deficient (CuD) diet starting on the fourth day post delivery, and the weanling mice were continued on the same diet until they were sacrificed. Zinc and copper concentrations were significantly elevated in MT-TG mouse heart, but the extent of zinc elevation was much more than that of copper. Dietary copper restriction significantly decreased copper concentrations to the same extent in both MT-TG and WT mouse hearts, and decreased zinc concentrations along with a decrease in MT concentrations in the MT-TG mouse heart. Copper deficiency-induced heart hypertrophy was significantly inhibited, but copper deficiency-induced suppression of serum ceruloplasmin or hepatic Cu,Zn-SOD activities was not inhibited in the MT-TG mice. These results suggest that elevation in zinc but not in copper in the heart may be involved in the MT inhibition of copper deficiency-induced cardiac hypertrophy.     

Reconstruction of cell population dynamics using CFSE

Background  

Quantifying cell division and death is central to many studies in the biological sciences. The fluorescent dye CFSE allows the tracking of cell division in vitro and in vivo and provides a rich source of information with which to test models of cell kinetics. Cell division and death have a stochastic component at the single-cell level, and the probabilities of these occurring in any given time interval may also undergo systematic variation at a population level. This gives rise to heterogeneity in proliferating cell populations. Branching processes provide a natural means of describing this behaviour.     

Pine microsatellite markers allow roots and ectomycorrhizas to be linked to individual trees

Linking roots and ectomycorrhizas (EcM) to individual host trees in the field is required to test whether individual trees support different ectomycorrhizal communities. Here we describe a method that identifies the source of EcM roots by PCR of polymorphic pine nuclear microsatellite loci using fluorescently labelled primers and high-throughput fragment analysis. ITS-PCR can also be performed on the same EcM DNA extract for fungal identification. The method was tested on five neighbouring Scots pine (Pinus sylvestris var scotica) trees in native woodland. Successful host tree identification from DNA extracts of EcM root tips was achieved for 93% of all root fragments recovered from soil cores. It was estimated that each individual mature pine sampled was colonised by between 15 and 19 EcM fungi. The most abundant fungal species were found on all five trees, and within the constraints of the sampling scheme, no differences between trees in EcM fungal community structure or composition were detected.     

Impaired deformability of copper-deficient neutrophils

We have previously shown that dietary copper deficiency augments neutrophil accumulation in the lung microvasculature. The current study was designed to determine whether a diet deficient in copper promotes neutrophil chemoattraction within the lung vasculature or if it alters the mechanical properties of the neutrophil, thus restricting passage through the microvessels. Sprague-Dawley rats were fed purified diets that were either copper adequate (6.3 microg Cu/g diet) or copper deficient (0.3 microg Cu/g diet) for 4 weeks. To assess neutrophil chemoattraction, bronchoalveolar lavage fluid was assayed for the neutrophil chemokine macrophage inflammatory protein-2 (MIP-2) by enzyme-linked immunosorbent assay. Neutrophil deformability was determined by measuring the pressure required to pass isolated neutrophils through a 5-microm polycarbonate filter. The MIP-2 concentration was not significantly different between the dietary groups (Cu adequate, 435.4 +/- 11.9 pg/ml; Cu deficient, 425.6 +/- 14.8 pg/ml). However, compared with controls, more pressure was needed to push Cu-deficient neutrophils through the filter (Cu adequate, 0.150 +/- 0.032 mm Hg/sec; Cu deficient, 0.284 +/- 0.037 mm Hg/sec). Staining of the filamentous actin (F-actin) with FITC-Phalloidin showed greater F-actin polymerization and shape change in the Cu-deficient group. These results suggest that dietary copper deficiency reduces the deformability of neutrophils by promoting F-actin polymerization. Because most neutrophils must deform during passage from arterioles to venules in the lungs, we propose that copper-deficient neutrophils accumulate in the lung because they are less deformable.     

Winter hardening and glutathione status in the bilberry (Vaccinium myrtillus) in response to trace gases (CO2, O3) and nitrogen fertilization

Bilberry plants (Vaccinium myrtillus L.) at a field site in northern Finland (65°N) were subjected to nitrogen fertilization [6.5 mmol m^?2 NH₄NO₃× Ca(OH)₂] at the beginning of 3 growing seasons in late May and to trace gas fumigation (CO₂ and O₃) for 5 months (May–September) in 1993–1995 in order to investigate frost resistance and glutathione concentrations during the winter hardening period, and to assess the correlation between these variables. Harvesting was performed twice in the autumn of both 1994 and 1995, and the two-year data for each harvest were pooled. The frost resistance of the bilberry stems increased by about 10°C during the hardening period between the two harvests. Nitrogen fertilization increased the frost resistance towards late autumn. The fumigation treatments had no marked effect on it. The combination of elevated CO₂ and nitrogen fertilization induced a decrease in frost resistance. Increases in total glutathione concentrations and the proportion of reduced glutathione (GSH) in the stems were evident during hardening. Nitrogen fertilization positively affected the total glutathione concentration and the proportion of GSH at the beginning of the hardening period but the effect disappeared during the hardening process. Trace gas fumigation as such had no marked effect on glutathione concentration. Increases in glutathione concentrations during hardening did not correlate with frost resistance, possibly due to different timing of the appearence of the response to fertilization treatment, i.e., glutathione responded in the beginning of hardening while frost resistance at the end. The lack of correlation with frost resistance, and especially the different responses to nitrogen fertilization, may reflect the indirect role of glutathione in the development of winter hardening, as a transport and storage form of reduced nitrogen and sulphur. In conclusion, winter hardening and glutathione status in the bilberry seems to be sensitive to nitrogen fertilization, and not affected by elevated CO₂ and O₃.     

Mechanism of Sulfonylurea Herbicide Resistance in the Broadleaf Weed, Kochia scoparia

Selection of kochia (Kochia scoparia) biotypes resistant to the sulfonylurea herbicide chlorsulfuron has occurred through the continued use of this herbicide in monoculture cereal-growing areas in the United States. The apparent sulfonylurea resistance observed in kochia was confirmed in greenhouse tests. Fresh and dry weight accumulation in the resistant kochia was 2- to >350-fold higher in the presence of four sulfonylurea herbicides as compared to the susceptible biotype. Acetolactate synthase (ALS) activity isolated from sulfonylurea-resistant kochia was less sensitive to inhibition by three classes of ALS-inhibiting herbicides, sulfonylureas, imidazolinones, and sulfonanilides. The decrease in ALS sensitivity to inhibition (as measured by the ratio of resistant I₅₀ to susceptible I₅₀) was 5- to 28-fold, 2- to 6-fold, and 20-fold for sulfonylurea herbicides, imidazolinone herbicides, and a sulfonanilide herbicide, respectively. No differences were observed in the ALS-specific activities or the rates of [¹⁴C]chlorsulfuron uptake, translocation, and metabolism between susceptible and resistant kochia biotypes. The K_m values for pyruvate using ALS from susceptible and resistant kochia were 2.13 and 1.74 mm, respectively. Based on these results, the mechanism of sulfonylurea resistance in this kochia biotype is due solely to a less sulfonylurea-sensitive ALS enzyme.     

Mitochondrial respiration in heart, liver, and kidney of copper-deficient rats

Morphological observations in some tissues indicate that dietary copper deficiency results in structural damage to mitochondria. The purpose of this study was to determine whether mitochondrial function is impaired as well. Male, weanling Sprague-Dawley rats were fed diets deficient or sufficient in copper for 4 weeks. Mitochondria were isolated from heart, liver, kidney cortex, and kidney medulla. P/O ratio, state 3 and state 4 respiration rates (oxygen consumed in the presence and absence of ADP, respectively), and acceptor control index (ratio of state 3:state 4) were determined using succinate or pyruvate/malate as substrate. State 3 respiration rate in mitochondria from copper-deficient hearts and livers was lower than in mitochondria from copper-sufficient hearts. Copper deficiency reduced the state 4 respiration rate only in cardiac mitochondria. Neither respiration rate was affected by copper deficiency in mitochondria from kidney medulla or cortex. P/O ratio was not significantly affected by copper deficiency in any tissue examined. Acceptor control index was reduced only in liver mitochondria. The observed decreases in respiration rates are consistent with decreased cytochrome c oxidase activity, shown by others to occur in mitochondria isolated from hearts and livers of copper-deficient rats.     

Evaluation of commercial soy sauce <Emphasis Type="Italic">koji</Emphasis> strains of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> for γ-aminobutyric acid (GABA) production

In this study, four selected commercial strains of Aspergillus oryzae were collected from soy sauce koji. These A. oryzae strains designated as NSK, NSZ, NSJ and NST shared similar morphological characteristics with the reference strain (A. oryzae FRR 1675) which confirmed them as A. oryzae species. They were further evaluated for their ability to produce γ-aminobutyric acid (GABA) by cultivating the spore suspension in a broth medium containing 0.4 % (w/v) of glutamic acid as a substrate for GABA production. The results showed that these strains were capable of producing GABA; however, the concentrations differed significantly (P < 0.05) among themselves. Based on the A. oryzae strains, highest GABA concentration was obtained from NSK (194 mg/L) followed by NSZ (63 mg/L), NSJ (51.53 mg/L) and NST (31.66 mg/L). Therefore, A. oryzae NSK was characterized and the sequence was found to be similar to A. oryzae and A. flavus with 99 % similarity. The evolutionary distance (K _nuc) between sequences of identical fungal species was calculated and a phylogenetic tree prepared from the K _nuc data showed that the isolate belonged to the A. oryzae species. This finding may allow the development of GABA-rich ingredients using A. oryzae NSK as a starter culture for soy sauce production.