Searching for genes involved in arteriosclerosis: proteomic analysis of cultured human umbilical vein endothelial cells undergoing replicative senescence

It is known that replicative senescence of endothelium in vivo contributes at least partially to age-related vascular disorders such as arteriosclerosis. However, the genes involved in this process remain to be identified. In this study, we employed a proteomics-based approach to identify candidate genes using in vitro cultured human umbilical vein endothelial cells (HUVECs) as an experimental model for replicative senescence. By comparing protein spots from young and senescent HUVECs using two-dimensional electrophoresis, we identified three up-regulated proteins and five down-regulated proteins in senescent HUVECs as compared to young HUVECs, whose alteration was not observed during replicative senescence of primary human fibroblasts. Consistent results were obtained in Western blotting analysis using specific antibodies raised against some of these proteins, whereas there were no significant changes in the mRNA levels of these genes during senescence of HUVECs. Among them, cathepsin B, a protease participating in both intracellular proteolysis and extracellular matrix remodeling was observed to be dramatically up-regulated in senescent HUVECs and whose activity is known to be up-regulated in atherosclerotic lesions with senescence-associated phenotypes in vivo. Additional proteins, including cytoskeletal proteins and proteins involved in the processes of synthesis, turnover and modification of protein, were identified, whose function in endothelium was previously unsuspected. These proteins identified by a proteomics-based approach using cultured HUVECs may be involved not only in replicative senescence but also in functional alterations in vascular endothelial cells with senescence-associated phenotypes and may serve as molecular markers for these processes.     

Three nicotianamine synthase genes isolated from maize are differentially regulated by iron nutritional status

Nicotianamine synthase (NAS) is an enzyme that is critical for the biosynthesis of the mugineic acid family of phytosiderophores in graminaceous plants, and for the homeostasis of metal ions in nongraminaceous plants. We isolated one genomic NAS clone, ZmNAS3, and two cDNA NAS clones, ZmNAS1 and ZmNAS2, from maize (Zea mays cv Alice). In agreement with the increased secretion of phytosiderophores with Fe deficiency, ZmNAS1 and ZmNAS2 were positively expressed only in Fe-deficient roots. In contrast, ZmNAS3 was expressed under Fe-sufficient conditions, and was negatively regulated by Fe deficiency. This is the first report describing down-regulation of NAS gene expression in response to Fe deficiency in plants, shedding light on the role of nicotianamine in graminaceous plants, other than as a precursor in phytosiderophore production. ZmNAS1-green fluorescent protein (sGFP) and ZmNAS2-sGFP were localized at spots in the cytoplasm of onion (Allium cepa) epidermal cells, whereas ZmNAS3-sGFP was distributed throughout the cytoplasm of these cells. ZmNAS1 and ZmNAS3 showed NAS activity in vitro, whereas ZmNAS2 showed none. Due to its duplicated structure, ZmNAS2 was much larger (65.8 kD) than ZmNAS1, ZmNAS3, and previously characterized NAS proteins (30-38 kD) from other plant species. We reveal that maize has two types of NAS proteins based on their expression pattern and subcellular localization.     

Variations in plasma melatonin levels of the rainbow trout (Oncorhynchus mykiss) under various light and temperature conditions

Daily variations in plasma melatonin levels in the rainbow trout Oncorhynchus mykiss were studied under various light and temperature conditions. Plasma melatonin levels were higher at mid-dark than those at mid-light under light-dark (LD) cycles. An acute exposure to darkness (2 hr) during the light phase significantly elevated the plasma melatonin to the level that is comparable with those at mid-dark, while an acute exposure to a light pulse (2 hr) during the dark phase significantly suppressed melatonin to the level that is comparable with those at mid-light. Plasma melatonin kept constantly high and low levels under constant darkness and constant light, respectively. No circadian rhythm was seen under both conditions. When the fish were subjected to simulative seasonal conditions (simulative (S)-spring: under LD 13.1:10.9 at 13 degrees C; S-summer: under LD 14.3:9.7 at 16.5 degrees C; S-autumn: under LD 11.3:12.7 at 13 degrees C; S-winter: under LD 10.1:13.9 at 9 degrees C), melatonin levels during the dark phase were significantly higher than those during the light phase irrespective of simulative seasons. The peak melatonin level in each simulative season significantly correlated with temperature but not with the length of the dark phase employed. In addition, the peak melatonin level in S-autumn was significantly higher than those in S-spring although water temperature was the same under these conditions. These results indicate that the melatonin rhythm in the trout plasma is not regulated by an endogenous circadian clock but by combination of photoperiod and water temperature.     

Gene cloning and function analysis of replication factor C from Thermococcus kodakaraensis KOD1

Replication factor C (RFC) catalyzes the assembly of circular proliferating cell nuclear antigen (PCNA) clamps around primed DNA, enabling processive synthesis by DNA polymerase. The RFC-like genes, arranged in tandem in the Thermococcus kodakaraensis KOD1 genome, were cloned individually and co-expressed in Escherichia coli cells. T. kodakaraensis KOD1 RFC homologue (Tk-RFC) consists of the small subunit (Tk-RFCS: MW=37.2 kDa) and the large subunit (Tk-RFCL: MW=57.2 kDa). The DNA elongation rate of the family B DNA polymerase from T. kodakaraensis KOD1 (KOD DNA polymerase), which has the highest elongation rate in all thermostable DNA polymerases, was increased about 1.7 times, when T. kodakaraensis KOD1 PCNA (Tk-PCNA) and the Tk-RFC at the equal molar ratio of KOD DNA polymerase were reacted with primed DNA.     

Monitoring the cellular activity of a cultured single cell by scanning electrochemical microscopy (SECM). A comparison with fluorescence viability monitoring

The respiratory activities of cultured HeLa cells were monitored at a single cell level using scanning electrochemical microscopy (SECM) that produces images of the localized distribution of oxygen around the cell. The change in the cellular activity was traced after exposures to KCN, ethyl alcohol and the antibiotic drug, Antimycin A. The results were compared with those from the conventional fluorescence monitoring using Calcein-AM that is sensitive to deformation of the cell membrane. The SECM-based measurement follows the decrease in the cellular activity upon exposure to KCN and Antimycin A more rapidly than the fluorescence-based measurements, demonstrating that SECM is suitable for studying the cellular influence of respiration inhibitors.     

Imaging of immobilized enzyme spots by scanning chemiluminescence microscopy with electrophoretic injection

Scanning chemiluminescence microscopy (SCLM) with electrophoretic injection was developed and applied to visualize enzyme reactions localized in an enzyme microspot. The SCLM uses a tapered glass capillary as a probe for injecting a small amount of luminol onto the substrate to generate localized chemiluminescence. The electrophoretic injection by application of a constant current between the inside and outside of the capillary enabled the continuous and controllable injection of a minute quantity of luminol in the range of 0.1 pmol/s. The image of enzyme activity in a monolayer spot of horseradish peroxidase was obtained by using the electrophoretic injection-based SCLM system.     

Induction of oxidatively modified proteins in skeletal muscle by electrical stimulation and its suppression by dietary supplementation of (-)-epigallocatechin gallate

The oxidative stress produced by electrical stimulation-induced muscle contraction was examined in the skeletal muscle proteins of rats that had been fed on the dietary flavonoid, (-)-epigallocatechin gallate (EGCg). Electrical stimulation of the rat leg muscle every second day for a two-week period resulted in an increased (p < 0.05) muscle weight and accumulation of oxidatively induced modified proteins. Similar stimulation conducted every day for only one week had no effect on the muscle weight or protein oxidation, although the rate of protein degradation increased. Rats fed on a 20% casein diet supplemented with 0.1% EGCg for 2 weeks responded to the electrical stimulation of muscle contraction by reducing the increased muscle protein carbonyl content when compared to their counterparts fed on a control diet. There was no change in activity of antioxidative enzymes in muscle tissue of the EGCg-fed rats receiving electrical stimulation. The results of this study show that the antioxidative property of EGCg was effective for suppressing oxidative modification of the skeletal muscle protein induced by electrical stimulation. This finding demonstrates that EGCg has a beneficial effect in vivo on the free radical-mediated oxidative damage to muscle proteins.