Effects of leaf age, nitrogen nutrition and photon flux density on the organization of the photosynthetic apparatus in leaves of a vine (Ipomoea tricolor Cav.) grown horizontally to avoid mutual shading of leaves

Effects of leaf age, nitrogen nutrition and photon flux density (PFD) on the organization of the photosynthetic apparatus in leaves were investigated in a vine, Ipomoea tricolor Cav., which was grown horizontally so as to avoid mutual shading of leaves. The plants were grown hydroponically at two nitrate levels under two growth light treatments. For one group of the plants, leaves were exposed to full sunlight. For another group, respective leaves were artificially shaded in a manner that simulated changes in the light gradient with the development of an erect herbaceous canopy: old leaves were placed under progressively shadier conditions with growth of the plants (canopy-type shading). In all the treatments, chlorophyll (Chl) content gradually decreased with leaf age. Photosystem I (PSI) per Chl was constant, independent of leaf age, nitrogen nutrition and/or PFD. Photosystem II (PSII) and cytochrome / per Chl, and Chl a/b ratio were independent of leaf age and/or nitrogen nutrition but decreased with the decrease in growth PFD. Ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39, RuBPCase) per Chl steeply decreased with decrease in PFD. When leaves grown at the same PFD were compared, RuBPCase/Chl was lower in the plants grown under lower nitrogen availability and also decreased with leaf age in the plants grown without shading. These decreases were attributed to the curvilinear relationship between RuBPCase and Chl in leaves grown at full sunlight, that was independent of nitrogen availability and leaf age. From these results, it is concluded that the composition of the photosynthetic apparatus is independent of leaf age but changes depending on the light environment and total amount of photosynthetic components of the leaf.Abbreviations Chl chlorophyll - cyt f cytochrome f - PFD photon flux density - RuBPCase ribulose-1,5-bisphosphate carboxylase The author thanks Drs. K. Sonoike, Y. Kashino, K. Okada, H. Hatanaka, Y. Suzuki and A. Aoyama for technical advise. The author also thanks Drs. I. Terashima, A. Makino (Tohoku University, Sendai, Japan), Dr. J.R. Evans (Research School of Biological Sciences, Australian National University, Canberra) and Prof. A. Watanabe for valuable suggestions.     

Estrogen receptor expression and vessel density in the vagina wall in postmenopausal women with prolapse

After menopause, critically estrogen low levels result in modifications in vaginal wall. This cross-sectional study aims to determine whether there is a change in the number of vessels in the lamina propria of the vagina after menopause in parallel to the ER-alpha expression on the vaginal wall. Twelve women who underwent a genital surgery for genital prolapse up to grade II were selected. They were divided into two groups: a premenopausal group (PG) consisting of six women who were 18–40 years old with FSH levels =12 mIU/ml and regular cycles, and a menopausal group (MG) consisting of six women at least one year after menopause who were <65 years old with FSH levels =40 mIU/ml. Slides were stained for ER-alpha immunohistochemistry, and an endothelial cell marker CD3 was used to label vessels which were identified by using a system for morphometry. The number of vessels was significantly higher in the PG than in the MG both on the anterior wall (PG: 1.055 ± 145.8 vessels/mm², MG: 346.6 ± 209.9 vessels/mm², p < 0.0001) and on the posterior wall (PG: 1064 ± 303.3 vessels/mm², MG: 348.6 ± 167.3 vessels/mm², p = 0.0005). The ER-alpha score was significantly higher in the PG than the score for the MG on both the anterior and posterior walls (PG: 6.0 ± 0.52, MG: 2.5 ± 0.89, p = 0.007; PG: 5.8 ± 0.79, MG: 2.7 ± 0.95, p = 0.03, respectively). There was a positive correlation between the ER-alpha score and the vessel concentration on the anterior (r = 0.6656, p = 0.018) and posterior (r = 0.6738, p = 0.016) vaginal walls. Age was strongly negatively correlated with vessel concentration on the vaginal walls (respectively r = -0.9033, p < 0.0001, r = -0.7440, p = 0.0055). Therefore, postmenopausal women with genital prolapse have a smaller number of vessels on the vaginal wall compared to normoestrogenic controls with the same pathological condition. Hypoestrogenism and advancing age are factors that are associated to these changes.     

NADPH oxidase NOX1 is involved in activation of protein kinase C and premature senescence in early stage diabetic kidney

Increased oxidative stress and activation of protein kinase C (PKC) under hyperglycemia have been implicated in the development of diabetic nephropathy. Because reactive oxygen species derived from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, NOX1 accelerate the translocation of PKC isoforms, NOX1 is postulated to play a causative role in the development of diabetic nephropathy. Hyperglycemia was induced in wild-type and Nox1-deficient mice (KO) by two doses of streptozotocin injection. At 3 weeks after the induction of hyperglycemia, glomeruli and cortical tubules were isolated from kidneys. The mRNA level of Nox1 was significantly upregulated in the renal cortex at 3 weeks of hyperglycemia. Urinary albumin and expression of inflammatory or fibrotic mediators were similarly elevated in diabetic wild-type and KO; however, increases in glomerular volume and mesangial matrix area were attenuated in diabetic KO. Nox1 deficiency significantly reduced the levels of renal thiobarbituric acid-reacting substances and 8-hydroxydeoxyguanosine, membranous translocation of PKCα/β, activity of PKC, and phosphorylation of p38 mitogen-activated protein kinase in the diabetic kidney. Furthermore, increased staining of senescence-associated β-galactosidase in glomeruli and cortical tubules of diabetic mice was significantly suppressed in KO. Whereas the levels of cyclin-dependent kinase inhibitors, p16^INK4A and p21^Cip1, were equivalent between the genotypes, increased levels of p27^Kip1 and γ-H2AX, a biomarker for DNA double-strand breaks, were significantly attenuated in isolated glomeruli and cortical tubules of diabetic KO. Taken together, NOX1 modulates the p38/p27^Kip1 signaling pathway by activating PKC and promotes premature senescence in early stage diabetic nephropathy.     

An activated-oxygen-mediated role for peroxisomes in the mechanism of senescence of Pisum sativum L. leaves

The possible involvement of peroxisomes and their activated-oxygen metabolism in the mechanism of leaf senescence was investigated in detached pea (Pisum sativum L.) leaves which were induced to senesce by incubation in complete darkness for up to 11 d. At days 0, 3, 8, and 11 of senescence, peroxisomes were purified from leaves and the activities of different peroxisomal and glyoxysomal enzymes were measured. Xanthine-oxidoreductase activity increased with senescence, especially the O ₂ ^{. -} -producing xanthine oxidase (EC 1.1.3.22). The activities of H₂O₂-generating Mn-superoxide dismutase (EC 1.15.1.1) and urate oxidase (EC 1.7.3.3) were also enhanced by senescence, whereas catalase (EC 1.11.1.6) activity was severely depressed. Hydrogen peroxide concentrations increased significantly in senescent leaf peroxisomes. During the progress of senescence, glycollate oxidase (EC 1.1.3.1) and hydroxypyruvate reductase (EC 1.1.1.81), two marker enzymes of photorespiratory metabolism, gradually decreased in activity and disappeared. At the same time, the activities of malate synthase (EC 4.1.3.2) and isocitrate lyase (EC 4.1.3.1), key enzymes of the glyoxylate cycle, which were undetectable in presenescent leaves, increased dramatically upon induction of senescence. Ultrastructural studies of intact leaves showed that the population of peroxisomes and mitochondria increased with senescence. Results indicate that peroxisomes could play a role, mediated by activated oxygen species, in the oxidative mechanism of leaf senescence, and further support the idea, proposed by other authors, that foliar senescence is associated with the transition of leaf peroxisomes into glyoxysomes.Abbreviation Mn-SOD (manganese-containing) superoxide dismutase The authors thank Dr. A.J. Sánchez-Raya (Unidad de Fisiología Vegetal, Estación Experimental del Zaidín, Granada, Spain) for his valuable help in measuring ethylene production, and Dr. G. Barja de Quiroga (Departamento de Biología Animal II, Universidad Complutense, Madrid, Spain) for carrying out the malondialdehyde determinations by HPLC. This work was supported by grant PB87-0404-01 from the DGICYT and the Junta de Andaluc'ia (Research Group # 3315), Spain.     

A Monoclonal Antibody Recognizing Nonderivative 13-Hydroxy Gibberellins and Their Glucosides

The production and characterization of a monoclonal antibody (MAb AB10) against GA3-glucoside as well as GA3 is described. MAb AB10 was derived from an immunogen in which human serum albumin (HSA) was linked to GA3 at carbon-3. This antibody showed high affinity for GA3-glucoside as well as for 13-hydroxy gibberellins (GA1, GA3, GAs, etc). The affinity of MAb AB10 for 13-hydroxy GAs was significantly reduced by methylation of the 7-oic acid but not by glycosylation of 3-hydroxyl group. Based on this antibody, both of competitive enzyme-linked immunosorbent assays (ELISAs) for GA3-glucoside and for GA3 were developed. These two ELISAs displayed linear detection ranges from 0.2 pmol to 20 pmol. Using these assays, the fluctuation of GA3-1ike and GA3-glucoside-like sub-stances in the leaves of Rurnex japonicus was investigated. The results indicated that the glycosylation of free GAs was connected with leaf senescence and that the function of 6-ben- zyl amino purine in retarding the leaf senescence was probably related to delaying the process of glycosylation of free GAs.     

Replicative senescence, telomere shortening and cell proliferation rate in Gaddi goat's skin fibroblast cell line

We assessed aging in continuous donor skin fibroblast cell line GGM5 up to the 25th passage by in vitro replicative senescence, telomere dynamics and chromosomal abnormalities. Cell proliferation rate increased from 0.84+/-0.26 (primary cells) to 1.20+/-0.17 (13-15 passage group) per day and reduced to 0.65+/-0.14 in 22-25 passages. Cell proliferation rate was reduced by 45.7% after 87.62 CPDs. Cell viability reduced from 100% to 97.4% up to the 25th passages. Frequency of beta gal(+) cells increased in successive passages and days in culture. The correlation coefficient between frequency of beta gal(+) cells and growth rate was -0.50 to -0.61. Loss of mean TRF length was 13.8 nucleotides (passage 15) to 95.4 nucleotides per cell division in later passages. All cells showed Robertsonian translocation in 22-25 passaged cells. The SCNT pre-implantation embryos production was highest (22.5%) in donor cells used from 10-15 passages as compared to early (相似文献   

Vascular endothelial cells senescence is associated with NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation via reactive oxygen species (ROS)/thioredoxin-interacting protein (TXNIP) pathway

Endothelial dysfunction caused by endothelial cells senescence and chronic inflammation is tightly linked to the development of cardiovascular diseases. NLRP3 (NOD-like receptor family pyrin domain-containing3) inflammasome plays a central role in inflammatory response that is associated with diverse inflammatory diseases. This study explores the effects and possible mechanisms of NLRP3 inflammasome in endothelial cells senescence. Results show an increment of pro-inflammatory cytokine interleukin (IL) −1β secretion and caspase-1 activation during the senescence of endothelial cells induced by bleomycin. Moreover, secreted IL-1β promoted endothelial cells senescence through up-regulation of p53/p21 protein expression. NLRP3 inflammasome was found to mediate IL-1β secretion through the production of ROS (reactive oxygen species) during the senescence of endothelial cells. Furthermore, the association of TXNIP (thioredoxin-interacting protein) with NLRP3 induced by ROS promoted NLRP3 inflammasome activation in senescent endothelial cells. In addition, the expressions of NLRP3 inflammasome related genes, ASC (apoptosis associated speck-like protein containing a CARD), TXNIP, cleaved caspase-1 and IL-1β, were also increased in vitro and in vivo studies. These findings indicate that endothelial senescence could be mediated through ROS and NLRP3 inflammasome signaling pathways, suggesting a potential target for the prevention of endothelial senescence-related cardiovascular diseases.     

Ethylene formation from 1-aminocyclopropane-1-carboxylic acid by microsomal membranes from senescing carnation flowers

Isolated membranes from the petals of senescing carnation flowers (Dianthus caryophyllus L. cv. White-Sim) catalyze the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) to ethylene. A microsomal membrane fraction obtained by centrifugation at 131,000 g for 1 h proved to be more active than the membrane pellet isolated by centrifugation at 10,000 g for 20 min. The ethylene-producing activity of the microsomal membranes is oxygen-dependent, heat-denaturable, sensitive to n-propyl gallate, and saturable with ACC. Corresponding cytosol fractions from the petals are incapable of converting ACC to ethylene. Moreover, the addition of soluble fraction back to the membrane fraction strongly inhibits the ACC to ethylene conversion activity of the membranes. The efficiency with which isolated membranes convert ACC to ethylene is lower than that exhibited by intact flowers based on the relative yield of membranes per flower. This may be due to the presence of the endogenous soluble inhibitor of the reaction, for residual soluble fraction inevitably remains trapped in membrane vesicles isolated from a homogenate.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AOA aminoxyacetic acid - AVG aminoethoxyvinylglycine - EPPS N-2-hydroxyethylpiperazine propane sulfonic acid     

Telomerase activity is sufficient to bypass replicative senescence in human limbal and conjunctival but not corneal keratinocytes

The human ocular surface is covered by the conjunctival, corneal and limbal stratified epithelia. While conjunctival stem cells are distributed in bulbar and forniceal conjunctiva, corneal stem cells are segregated in the basal layer of the limbus, which is the transitional zone between the cornea and the bulbar conjunctiva. Keratinocyte stem and transient amplifying (TA) cells when isolated in culture give rise to holoclones and paraclones, respectively. Keratinocyte replicative senescence ensues when all holoclones have generated paraclones which express high levels of p16(INK4a). In the present study, we show that enforced telomerase activity induces the bypass of replicative senescence in limbal and conjunctival keratinocytes, without the inactivation of the p16(INK4a)/Rb pathway or the abrogation of p53 expression. hTERT-transduced limbal and conjunctival keratinocytes are capable to respond to both growth inhibitory and differentiation stimuli, since they undergo growth arrest in response to phorbol esters, and activate p53 upon DNA damage. Following a sustained PKC stimulation, occasional clones of p16(INK4a)-negative cells emerge and resume ability to proliferate. Telomerase activity, however, is unable to induce the bypass of senescence in corneal TA keratinocytes cultured under the same conditions. These data support the notion that telomere-dependent replicative senescence is a general property of all human somatic cells, including keratinocytes, and suggest that telomerase activity is sufficient to extend the lifespan only of keratinocytes endowed with high proliferative potentials (which include stem cells), but not of TA keratinocytes.     

Loss of p21 expression in senescent cells after DNA damage accompanied with increase of miR-93 expression and reduced p53 interaction with p21 gene promoter

To answer what is a critical event for higher incidence of tumor development in old than young individuals, primary culture of human diploid fibroblasts were employed and DNA damage was induced by doxorubicin or X-ray irradiation. Response to the damage was different between young and old cells; loss of p21^sdi1 expression in spite of p53^S15 activation in old cells along with [³H]thymidine and BrdU incorporation, but not in young cells. The phenomenon was confirmed by other tissue fibroblasts obtained from different donor ages. Induction of miR-93 expression and reduced p53 binding to p21 gene promoter account for loss of p21^sdi1 expression in senescent cells after DNA damage, suggesting a mechanism of in vivo carcinogenesis in aged tissue without repair arrest.