Docosahexaenoic acid supplementation-increased oxidative damage in bone marrow DNA in aged rats and its relation to antioxidant vitamins

We compared the influence of docosahexaenoic acid (DHA) supplementation on oxidative DNA damage in bone marrow between young and aged rats. As a marker of oxidative DNA damage, 8-hydroxydeoxy-guanosine (8-OHdG) in DNA was analyzed. Young (5-week-old) and aged (100-week-old) female Wistar rats were given DHA (300mg/kg body weight/day) or vehicle (control) orally for 12 weeks. The 8-OHdG in the bone marrow in the aged DHA group was significantly higher than that in the other groups. Vitamin E concentrations, however, did not differ among the groups regardless of the DHA supplementation. Vitamin C (ascorbic acid) concentrations in the aged control group were approximately 1/2 those in the young control group. The concentrations of vitamin C tended to be higher in the young DHA group and lower in the aged DHA group when compared to their respective control groups. Changes in the concentrations of vitamin C and vitamin E in plasma were similar to those in the bone marrow. The activity of hepatic l-gulono- γ -lactone oxidase, an enzyme responsible for vitamin C synthesis, corresponded well to the concentrations of vitamin C in the bone marrow and the plasma. These results suggest that in aged rats, but not young rats, excess supplementation of DHA induces oxidative DNA damage in bone marrow and that the decrease in vitamin C synthesis in aged rats is involved in the mechanisms of DNA damage.     

Confirmation of the anomeric structure of galacturonic acid in the galacturonosyl-ceramide of Sphingomonas yanoikuyae

The anomeric structure of glycosphingolipids significantly influences their activity to stimulate natural killer T cells. In this study the chemical structure of the galacturonosyl-ceramide in Sphingomonas yanoikuyae, designated GSL-1'sy, was re-examined to prove the anomeric structure of the Dgalacturonic acid (GalA) in the lipid, which was reported as beta-configuration by Naka et al., but was suggested as alpha-configuration in our preliminary study. GSL-1'sy was purified from the bacterial cells with the same procedure as Naka et al. The 1H-NMR analysis of GSL-1'sy revealed that the coupling constant of the anomeric proton of GalA was 3.0 Hz, indicating that GalA in GSL-1'sy is alpha-anomer, the configuration active for the stimulation of natural killer T cells.     

Personalized medicine and proteomics: lessons from non-small cell lung cancer

Personalized medicine allows the selection of treatments best suited to an individual patient and disease phenotype. To implement personalized medicine, effective tests predictive of response to treatment or susceptibility to adverse events are needed, and to develop a personalized medicine test, both high quality samples and reliable data are required. We review key features of state-of-the-art proteomic profiling and introduce further analytic developments to build a proteomic toolkit for use in personalized medicine approaches. The combination of novel analytical approaches in proteomic data generation, alignment and comparison permit translation of identified biomarkers into practical assays. We further propose an expanded statistical analysis to understand the sources of variability between individuals in terms of both protein expression and clinical variables and utilize this understanding in a predictive test.     

High Cell Density Upregulates Calcium Oscillation by Increasing Calcium Store Content via Basal Mitogen-Activated Protein Kinase Activity

Calcium releases of non-excitable cells are generally a combination of oscillatory and non-oscillatory patterns, and factors affecting the calcium dynamics are still to be determined. Here we report the influence of cell density on calcium increase patterns of clonal cell lines. The majority of HeLa cells seeded at 1.5 x 10⁴/cm² showed calcium oscillations in response to histamine and ATP, whereas cells seeded at 0.5 x 10⁴/cm² largely showed transient and sustained calcium increases. Cell density also affected the response of HEK293 cells to ATP in a similar manner. High cell density increased the basal activity of the mitogen-activated protein (MAP) kinase and calcium store content, and both calcium oscillation and calcium store content were down-regulated by a MAP kinase inhibitor, U0126. Thus, MAP kinase-mediated regulation of calcium store likely underlie the effect of cell density on calcium oscillation. Calcium increase patterns of HeLa cells were conserved at any histamine concentrations tested, whereas the overexpression of histamine H1 receptor, which robustly increased histamine-induced inositol phospholipid hydrolysis, converted calcium oscillations to sustained calcium increases only at high histamine concentrations. Thus, the consequence of modulating inositol phospholipid metabolism was distinct from that of changing cell density, suggesting the effect of cell density is not attributed to inositol phospholipid metabolism. Collectively, our results propose that calcium increase patterns of non-excitable cells reflect calcium store, which is regulated by the basal MAP kinase activity under the influence of cell density.     

Association of a regulatory gene, slyA with a mouse virulence of Salmonella serovar Choleraesuis

The influence of slyA gene, originally found in Salmonella serovar Typhimurium as a regulatory gene for the expression of virulence genes, on a mouse virulence of S. serovar Choleraesuis was investigated by using an slyA-defective mutant. The defective mutant was constructed by the insertion of a kanamycin-resistance gene (aph) into the cloned slyA gene, and the homologous recombination with the intact slyA gene on the chromosome. The mutant strain showed the LD50 value for BALB/c mouse approximately 10(5) higher than that of the parent strain. The increase of the LD50 value was the same order as that shown by the mutation of the slyA gene of S. serovar Typhimurium, although LD50 of the wild-type strain of S. serovar Choleraesuis was 40-fold higher than that of S. serovar Typhimurium. The time course of infection observed in the mice organs also proved the clear difference of the virulence between the parent and the mutant strains. These results suggested that the slyA gene product functions as a virulence-associated regulator also in S. serovar Choleraesuis.     

A miRNA involved in phosphate-starvation response in Arabidopsis

Although microRNAs (miRNAs) have been documented to regulate development in plants and animals , the function of miRNAs in physiology is unclear. miR399 has multiple target sites in the 5' untranslated region (UTR) of a gene encoding a putative ubiquitin-conjugating enzyme (UBC) in Arabidopsis thaliana. We report here that miR399 was highly induced, whereas the target UBC mRNA was reduced by low-phosphate (Pi) stress. In transgenic plants with constitutive expression of miR399, UBC mRNA accumulation was suppressed even under high Pi. The expression of transgene UBC mRNA with 5' UTR miR399 target sites, but not the one without 5' UTR, was reduced under low-Pi condition. Furthermore, transgenic Arabidopsis plants with constitutive expression of miR399 accumulated more Pi than the wild-type, and transgenic plants expressing the UBC mRNA without 5' UTR (miRNA-deregulated) showed less inhibition of primary root growth and less induction of a Pi transporter gene by low-Pi stress than those of wild-type plants. We conclude that miR399 downregulates UBC mRNA accumulation by targeting the 5' UTR, and this regulation is important for plant responses to Pi starvation. The results suggest that miRNAs have functional roles for plants to cope with fluctuations in mineral-nutrient availability in the soil.     

Heterogeneity in smooth muscle cell population accumulating in the neointimas and the media of poststenotic dilatation of the rabbit carotid artery.

Rabbit smooth muscles contain at least three types of myosin heavy chain (MHC) isoforms; SM1, SM2 and SMemb (NMHC-B), the expression of which is developmentally regulated. We have recently reported that smooth muscles with the embryonic phenotype accumulate in the neointimas produced by endothelial denudation or high-cholesterol feeding. In this study, we examined MHC isoform expression in the neointimas and the media of poststenotic dilatation of the rabbit carotid artery, and determined the phenotype of the smooth muscle cell in the dilated segment. We report here that neointimal cells in the dilated segment are smooth muscle cells with the embryonic phenotype as previously reported in our ballooning-injury study. The medial smooth muscles, however, are composed of heterogeneous population of smooth muscles which differ in stage of differentiation as determined by the MHC isoform expression. These results indicate that MHC isoforms are useful molecular markers to identify abnormally proliferating smooth muscles in diseased arteries and to understand the process of atherogenesis occurring following vascular injury.     

Saturated glucose uptake capacity and impaired fatty acid oxidation in hypertensive hearts before development of heart failure

Abnormalities in energy metabolism may play an important role in the development of hypertensive heart failure. However, the transition from compensated hypertrophy to heart failure is not fully understood in terms of energy metabolism. In Dahl salt-sensitive (DS) and salt-resistant (DR) rats, myocardial fatty acid and glucose uptake values were determined using (131)I- or (125)I-labeled 9-methylpentadecanoic acid ((131)I- or (125)I-9MPA), and [(14)C]deoxyglucose ([(14)C]DG), fatty acid beta-oxidation was identified using thin-layer chromatography, and insulin-stimulated glucose-uptake was observed using a euglycemic hyperinsulinemic glucose clamp. Six-week-old rats were fed a diet that contained 8% NaCl, which resulted in development of compensated hypertrophy in DS rats at 12 wk of age and ultimately led to heart failure by 18 wk of age. Uptake of [(14)C]DG increased markedly with age in the DS rats, whereas (131)I-9MPA uptake was marginally but significantly increased only in animals aged 12 wk. The ratio of (125)I-9MPA beta-oxidation metabolites to total uptake in the DS rats was significantly lower (P < 0.05) at 12 (37%) and 18 (34%) wk compared with at 6 (45%) wk. Insulin increased [(14)C]DG uptake more than twofold in the DS rats at 6 wk, although this increase was markedly attenuated at 12 and 18 wk (11 and 8%, respectively). Our data suggest that in a hypertrophied heart before heart failure, fatty acid oxidation is impaired and the capacity to increase glucose uptake during insulin stimulation is markedly reduced. These changes in both glucose and fatty acid metabolism that occur in association with myocardial hypertrophy may have a pathogenic role in the subsequent development of heart failure.     

Construction of hybrid photosynthetic units using peripheral and core antennae from two different species of photosynthetic bacteria: detection of the energy transfer from bacteriochlorophyll a in LH2 to bacteriochlorophyll b in LH1

Typical purple bacterial photosynthetic units consist of supra-molecular arrays of peripheral (LH2) and core (LH1-RC) antenna complexes. Recent atomic force microscopy pictures of photosynthetic units in intact membranes have revealed that the architecture of these units is variable (Scheuring et al. (2005) Biochim Bhiophys Acta 1712:109–127). In this study, we describe methods for the construction of heterologous photosynthetic units in lipid-bilayers from mixtures of purified LH2 (from Rhodopseudomonas acidophila) and LH1-RC (from Rhodopseudomonas viridis) core complexes. The architecture of these reconstituted photosynthetic units can be varied by controlling ratio of added LH2 to core complexes. The arrangement of the complexes was visualized by electron-microscopy in combination with Fourier analysis. The regular trigonal array of the core complexes seen in the native photosynthetic membrane could be regenerated in the reconstituted membranes by temperature cycling. In the presence of added LH2 complexes, this trigonal symmetry was replaced with orthorhombic symmetry. The small lattice lengths for the latter suggest that the constituent unit of the orthorhombic lattice is the LH2. Fluorescence and fluorescence-excitation spectroscopy was applied to the set of the reconstituted membranes prepared with various proportions of LH2 to core complexes. Remarkably, even though the LH2 complexes contain bacteriochlorophyll a, and the core complexes contain bacteriochlorophyll b, it was possible to demonstrate energy transfer from LH2 to the core complexes. These experiments provide a first step along the path toward investigating how changing the architecture of purple bacterial photosynthetic units affects the overall efficiency of light-harvesting.     

In vitro reconstitution of plant Atg8 and Atg12 conjugation systems essential for autophagy

Genetic and biochemical analyses using yeast Saccharomyces cerevisiae showed that two ubiquitin-like conjugation systems, the Atg8 and Atg12 systems, exist and play essential roles in autophagy, the bulk degradation system conserved in yeast and mammals. These conjugation systems are also conserved in Arabidopsis thaliana; however, further detailed study of plant ATG (autophagy-related) conjugation systems in relation to those in yeast and mammals is needed. Here, we describe the in vitro reconstitution of Arabidopsis thaliana ATG8 and ATG12 (AtATG8 and AtATG12) conjugation systems using purified recombinant proteins. AtATG12b was conjugated to AtATG5 in a manner dependent on AtATG7, AtATG10, and ATP, whereas AtATG8a was conjugated to phosphatidylethanolamine (PE) in a manner dependent on AtATG7, AtATG3, and ATP. Other AtATG8 homologs (AtATG8b-8i) were similarly conjugated to PE. The AtATG8 conjugates were deconjugated by AtATG4a and AtATG4b. These results support the hypothesis that the ATG conjugation systems in Arabidopsis are very similar to those in yeast and mammals. Intriguingly, in vitro analyses showed that AtATG12-AtATG5 conjugates accelerated the formation of AtATG8-PE, whereas AtATG3 inhibited the formation of AtATG12-AtATG5 conjugates. The in vitro conjugation systems reported here will afford a tool with which to investigate the cross-talk mechanism between two conjugation systems.