Senescent phenotype can be reversed by reduction of caveolin status

Hyporesponsiveness to growth factors is one of the fundamental characteristics of senescent cells. We previously reported that the up-regulation of caveolin attenuates the growth factor response and the subsequent downstream signal cascades in senescent human diploid fibroblasts. Therefore, in the present experiment, we investigated the modulation of caveolin status in senescent cells to determine the effect of caveolin on mitogenic signaling efficiency and cell cycling. We reduced the level of caveolin-1 in senescent human diploid fibroblasts using its antisense oligonucleotides and small interfering RNA, and this resulted in the restoration of normal growth factor responses such as the increased phosphorylation of Erk, the nuclear translocation of p-Erk, and the subsequent activation of p-Elk upon epidermal growth factor stimulation. Moreover, DNA synthesis and the re-entry of senescent cells into cell cycle were resumed upon epidermal growth factor stimulation concomitantly with decreases in p53 and p21. Taken together, we conclude that the loss of mitogenic signaling in senescent cells is strongly related to their elevated levels of caveolin-1 and that the functional recovery of senescent cells at least in the terms of growth factor responsiveness and cell cycle entry might be achieved simply by lowering the caveolin level.     

Rainfall, phycocyanin, and N:P ratios related to cyanobacterial blooms in a Korean large reservoir

Nutrient concentrations and other environmental factors were measured in the Daechung Reservoir for 25 weeks from spring until autumn in 1999. The high irradiance after heavy rainfall provided optimal meteorological conditions for bloom formation during summer, therefore, rain would also appear to forecast imminent bloom. The bloom formation was largely governed by cyanobacteria, in particular, Microcystis spp. and Anabaenaspp. Phycocyanin showed higher correlation with cyanobacteria (r = 0.744, P < 0.001) compared to chlorophyll-a(r = 0.599, P < 0.01). Therefore, phycocyanin was more accurate and useful than chlorophyll-a in quantitatively measuring cyanobacterial blooms. The atomic N:P ratio of the particulate form also showed a high correlation with cyanobacteria (r = 0.541, P < 0.01), increasing from 4.3 to 14.6 during bloom formation, while that of the dissolved form decreased from 25.5 to 8.7. These results indicated that the algae assimilated N significantly without comparable P uptake during the blooming season, which was in sharp contrast to the excessive storage of P during the spring.     

Identification of trait-improving quantitative trait loci alleles from a wild rice relative,Oryza rufipogon.

Wild species are valued as a unique source of genetic variation, but they have rarely been used for the genetic improvement of quantitative traits. To identify trait-improving quantitative trait loci (QTL) alleles from exotic species, an accession of Oryza rufipogon, a relative of cultivated rice, was chosen on the basis of a genetic diversity study. An interspecific BC2 testcross population (V20A/O. rufipogon//V20B///V20B////Ce64) consisting of 300 families was evaluated for 12 agronomically important quantitative traits. The O. rufipogon accession was phenotypically inferior for all 12 traits. However, transgressive segregants that outperformed the original elite hybrid variety, V20A/Ce64, were observed for all traits examined. A set of 122 RFLP and microsatellite markers was used to identify QTL. A total of 68 significant QTL were identified, and of these, 35 (51%) had beneficial alleles derived from the phenotypically inferior O. rufipogon parent. Nineteen (54%) of these beneficial QTL alleles were free of deleterious effects on other characters. O. rufipogon alleles at two QTL on chromosomes 1 and 2 were associated with an 18 and 17% increase in grain yield per plant, respectively, without delaying maturity or increasing plant height. This discovery suggests that the innovative use of molecular maps and markers can alter the way geneticists utilize wild and exotic germplasm.     

Gain in 1q is a common abnormality in phyllodes tumours of the breast.

We studied DNA copy number changes by CGH and allelic imbalance (AI) on 3p by LOH analysis on 22 phyllodes tumours (PT) of the breast in order to gain insight into the genetic basis of tumour progression in PT. Copy number changes were observed in 14 cases (63%). Gain in 1q with 1q21-23 as the minimal overlapping area was seen in 12 cases (55%). The gain was observed both in benign and malignant tumours. Our study did not reveal any DNA copy number changes or allelic loss on 3p. The results suggest that DNA copy number changes are not associated with the histological grade or clinical behaviour of PT and the chromosomal changes on 3p appear to be rare. Colour figure can be viewed on http://www.esacp.org/acp/2003/25-2/jee.htm     

Rapid increase in endothelial nitric oxide production by bradykinin is mediated by protein kinase A signaling pathway

Bradykinin (BK) acutely increases endothelial nitric oxide (NO) production by activating endothelial NO synthase (eNOS), and this increase is in part correlated with enhanced phosphorylation/dephosphorylation of eNOS by several protein kinases and phosphatases. However, the signaling mechanisms producing this increase are still controversial. In an attempt to delineate the acute effect of BK on endothelial NO production, confluent bovine aortic endothelial cells were incubated with BK, and NO production was measured by NO-specific chemiluminescence. Significant increase in NO levels was detected as early as 1 min after BK treatment, with concomitant increase in the phosphorylation of Ser(1179) (bovine sequence) site of eNOS (eNOS-Ser(1179)). This acute effect of BK on both increases was blocked only by treatment of protein kinase A inhibitor H-89, but not by the inhibitors of calmodulin-dependent kinase II and protein kinase B, suggesting that the rapid increase in NO production by BK is mediated by the PKA-dependent phosphorylation of eNOS-Ser(1179).     

Identification of reference genes for normalization of gene expression in thoroughbred and Jeju native horse(Jeju pony) tissues

Quantitative analysis of horse gene expression profiles under diverse experimental conditions is limited by the lack of reliable reference genes for normalization of mRNA levels. Therefore, in this study, the expression of potential reference genes was compared between thoroughbred and Jeju native horse (Jeju pony). We compared the expression of nine genes by quantitative real-time RT-PCR in fourteen tissues between the two horse breeds and analyzed their stability using the geNorm and NormFinder programs. The data obtained in this study suggest that the UBB gene could serve as a reference gene in gene expression analysis of thoroughbred and Jeju native horses.     

Crystal structure of class I acetohydroxy acid isomeroreductase from Pseudomonas aeruginosa

Acetohydroxy acid isomeroreductase (AHIR) is a key enzyme in the biosynthesis of branched-chain amino acids. We have determined the first crystal structure of a class I AHIR from Pseudomonas aeruginosa at 2.0 A resolution. Its dodecameric architecture of 23 point group symmetry is assembled of six dimeric units and dimerization is essential for the formation of the active site. The dimeric unit of P.aeruginosa AHIR partially superimposes with a three-domain monomer of spinach AHIR, a class II enzyme. This demonstrates that the so-called plant-specific insert in the middle of spinach AHIR is structurally and functionally equivalent to the C-terminal alpha-helical domain of P.aeruginosa AHIR, and the C-terminal alpha-helical domain was duplicated during evolution from the shorter, class I AHIRs to the longer, class II AHIRs. The dimeric unit of P.aeruginosa AHIR possesses a deep figure-of-eight knot, essentially identical with that in the spinach AHIR monomer. Thus, our work lowers the likelihood of the previous proposal that "domain duplication followed by exchange of a secondary structure element can be a source of such a knot in the protein structure" being correct.     

Synaptic connections of cone bipolar cells that express the neurokinin 1 receptor in the rabbit retina

We have investigated and further characterized, in the rabbit retina, the synaptic connectivity of the ON-type cone bipolar cells that are immunoreactive for an antibody against the neurokinin-1 receptor (NK1R). NK1R-immunoreactive bipolar cell axons terminate in stratum 4 of the inner plexiform layer. The axons of NK1R-positive bipolar cells receive synaptic inputs from amacrine cells through conventional synapses and from putative AII amacrine cells via gap junctions. The major outputs from NK1R-positive bipolar cells make contacts with amacrine cell processes. The most frequent postsynaptic dyads comprise two amacrine cell processes. Double-labeling experiments with antibodies against NK1R and either calretinin or glycine have demonstrated that NK1R-immunoreactive bipolar cells form gap junctions with AII amacrine cells. Thus, NK1R-positive cone bipolar cells, together with calbindin-positive cone bipolar cells, may play an important role in transferring rod signals to the ON-type ganglion cells of the cone pathway in the rabbit retina.I.-B. Kim and M.R. Park contributed equally to this work.This work was supported by the Ministry of Science and Technology of Korea (grant no. M1-0108-00-0059; Neurobiology Support Grant).     

Chaperone-like activities of alpha-synuclein: alpha-synuclein assists enzyme activities of esterases

Alpha-synuclein, a major constituent of Lewy bodies (LBs), has been implicated to play a critical role in the pathogenesis of Parkinson's disease (PD), although the physiological function of alpha-synuclein has not yet been known. Here we have shown that alpha-synuclein, which has no well-defined secondary or tertiary structure, can protect the enzyme activity of microbial esterases against stress conditions such as heat, pH, and organic solvents. In particular, the flexibility of alpha-synuclein and its C-terminal region seems to be important for complex formation, but the structural integrity of the C-terminal region may not be required for stabilization of enzyme activity. In addition, atomic force microscopy (AFM) and in vivo enzyme assays showed highly specific interactions of esterases with alpha-synuclein. Our results indicate that alpha-synuclein not only protects the enzyme activity of microbial esterases in vitro, but also can stabilize the active conformation of microbial esterases in vivo.