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PubMed2002

Molecular characterization of a senescence-associated gene encoding cysteine proteinase and its gene expression during leaf senescence in sweet potato.

The structure and expression of a senescence-associated gene (SPG31) encoding a cysteine proteinase precursor of sweet potato have been characterized. The coding region of the gene consists of two exons encoding an enzyme precursor of 341 amino acids with conserved catalytic amino acids of papain. Examination of the expression patterns of the SPG31 gene in sweet potato by Northern blot analyses reveals that the transcripts of SPG31 are specifically induced in the senescing leaves but not in other organs. The differential accumulation of the mature SPG31 protein in the senescing leaves was further identified by two-dimensional electrophoresis of leaf proteins and N-terminal sequencing. This result suggests the important role played by SPG31 in proteolysis and nitrogen remobilization during the leaf senescence process. Furthermore, treatment of mature green leaves with ethylene for 3 d resulted in a high-level induction of SPG31 transcripts. Ethylene-regulated expression of SPG31 is consistent with the presence of a number of putative ethylene-responsive elements in the 899-bp SPG31 promoter region.

Amino Acid Sequence↗
PubMed1994

Isolation of cDNA clones for genes that are expressed during leaf senescence in Brassica napus. Identification of a gene encoding a senescence-specific metallothionein-like protein.

cDNA clones representing genes that are expressed during leaf senescence in Brassica napus were identified by differential screening of a cDNA library made from RNA isolated from leaves at different stages of senescence. The expression of these genes at different stages of leaf development was examined by northern blot analysis, and several different patterns of expression were observed. One of the clones, LSC54, represented a gene that is expressed at high levels during leaf senescence. Analysis of this gene indicated strong expression in flowers as well as in senescing leaves. DNA sequence analysis of the LSC54 cDNA indicated a similarity between the deduced amino acid sequence and several metallothionein-like proteins previously identified in plants.

Amino Acid Sequence↗
PubMed1972

Role of Protein Synthesis in the Senescence of Leaves: II. The Influence of Amino Acids on Senescence.

When the first leaf of the oat (Avena sativa) seedling is detached and placed in the dark, yellowing and proteolysis take place rapidly. The earlier finding that d-serine promotes this process has led to a further study of the controlling roles of several amino acids. Since the action of serine was found to be more powerful in presence of kinetin than alone, the effects of other amino acids have been restudied in presence of kinetin. Cysteine emerges as a moderately strong promotor of senescence, with glycine and alanine having definite but weaker effects. The serine effect is antagonized by arginine, especially in presence of kinetin, and so is the cysteine effect. This is considered to indicate that these two amino acids act in the same way. The antagonism exerted by arginine is in turn antagonized by canavanine. The protease activities at two pH regions which increase in the oat leaf during senescence react to both p-chlorimercuri-phenylsulfonate and to phenylmethyl-sulfonyl fluoride, and thus may contain both SH and OH groups. The amounts of both these enzyme activities formed in the leaf during 3 days in the dark are increased over 50% by pretreatment with serine, and this increase is very largely prevented by arginine. The amounts of soluble proteins left in the leaf vary as expected in the opposite sense. It is deduced that control of the new formation of proteases plays an important part in senescence. A suggestion is made as to the mechanism of control of senescence in leaves.

Journal Article↗
PubMed1974

The Metabolism of Oat Leaves during Senescence: II. Senescence in Leaves Attached to the Plant.

The course of senescence in the first leaves of light-grown Avena seedlings when attached to the plant has been compared with that previously studied in detached leaves and leaf segments. Proteolysis in the leaf, whether attached or detached, is accompanied by markedly polar basipetal transport of amino acids. This polar transport can be superimposed on the known transport of amino acids towards a locally applied cytokinin. In the intact plant, it results in a strong movement into the roots. The reducing sugars, which are set free in senescence, do not participate appreciably in this polar transport phenomenon.If cytokinin is applied directly to the roots, then, if the plants are 7 days old, it becomes transported upward into the basal parts of the leaf and there delays the senescence process. However, if the plants are 10 days old, a "reverse Mothes effect" occurs, in which senescence of the leaf is actually promoted and part of the amino acids transported into the roots is synthesized into root protein.

Journal Article↗
PubMed1982

Senescence of Rice Leaves : VII. PROLINE ACCUMULATION IN SENESCING EXCISED LEAVES.

Proline content increased greatly in detached rice (Oryza sativa cv. Taichung Native 1) leaves during senescence. There was a slight but significant increase in proline level after one day of incubation, and, subsequently, proline accumulated relatively rapidly. By 4 days after excision, the level of proline had increased 30- to 50-fold, which is similar to the level seen in the water-stressed detached rice leaves. It is unlikely that the proline accumulation in detached leaves is to be derived solely from protein hydrolysis, since the addition of l-glutamic acid increased the proline level during senescence. The proline analog, 3,4-dehydroproline, did not affect the level of proline during senescence. It seems that accumulation of proline may, at least in part, result from an increased rate of synthesis, possibly due to a disruption of the normal feedback inhibition of proline synthesis. Potassium cyanide and 2,4-dinitrophenol strongly inhibited proline accumulation, indicating that some energy compound(s) may participate in proline accumulation during senescence of excised rice leaves.

Journal Article↗
PubMed1989

Dissociation of senescence-associated changes in differentiated gene expression and replicative senescence in cultured adrenocortical cells.

In bovine adrenocortical cells senescing in culture, the decline in expression of cyclic AMP-inducible 17 alpha-hydroxylase in mass cultures and clones of bovine adrenocortical cells results from a decrease in the number of cells in the population capable of expressing this gene. As the population of cells decreases in proliferative potential, the fraction of the population that expresses 17 alpha-hydroxylase after induction with cyclic AMP decreases, until the population comprises entirely nonexpressing cells. We used in situ hybridization to study the relationship between the loss of the ability to replicate and the loss of expression of this differentiated function gene. Colonies of cells from early passage cultures comprised complex mosaics of expressing and nonexpressing cells, indicating a high-frequency switching event. At an individual cell level, there was a complete independence of replicative senescence and loss of 17 alpha-hydroxylase expression. Groups of cells with a morphology characteristic of replicative senescence, occurring frequently in early-passage cultures, comprised both expressing and nonexpressing cells, as did cells that appeared to be in rapid growth. This was confirmed by prior incubation of cells with bromodeoxyuridine to label cycling cells; both cycling and noncycling populations comprised mixtures of expressing and nonexpressing cells. In contrast, replication was required for the loss of 17 alpha-hydroxylase expression, since cells rendered incapable of division by treatment with mitomycin C maintained 17 alpha-hydroxylase expression over long time periods. Thus, the process that results stochastically in replicative senescence and the process that results in loss of 17 alpha-hydroxylase expression are independent, but they have similar time-courses and both require replication for phenotypic switching to take place.

Adrenal Cortex↗
PubMed2002

Stress-induced premature senescence and replicative senescence are different phenotypes, proteomic evidence.

In this paper, we illustrate how a proteomic analysis can be useful to approach complex biological problems, in this case the concept of stress-induced premature senescence (SIPS). According to the stochastic theories of ageing, damage that accumulate with time in the cellular components are responsible for cellular ageing. As a corollary, some sort of premature senescence should appear if the damage level is artificially increased due to the presence of stressing agents at subcytotoxic level. It has been shown, in several different models, that at a long-term after subcytotoxic stresses of many different natures, human diploid fibroblasts (HDFs) display biomarkers of replicative senescence (RS), which led to the concept of SIPS as compared to telomere-dependent RS. We compared RS and SIPS of HDFs by proteome analysis. SIPS was induced by two very different stressors: tert-butyhydroperoxide or ethanol. First, only a part of the protein expression changes observed in RS were also observed in SIPS. Second, HDFs in SIPS show changes specific either to the long-term effects of t-BHP or ethanol or independent of the nature of the stress. These changes have been termed "molecular scars" of subcytotoxic stresses. This work is also an excellent opportunity to discuss on important methodological issue in proteomics: the absolute requirement to start from reliable and reproducible models, which was the case in this study. We also focus on the data handling and statistical analysis allowing to use two-dimensional gel electrophoresis patterns in a semi-quantitative analysis.

Aging, Premature↗
PubMed1997

Cellular and molecular biomarkers indicate precocious in vitro senescence in fibroblasts from SAMP6 mice. Evidence supporting a murine model of premature senescence and osteopenia.

A variety of short-lived mouse strains (SAMP strains) and control strains of less abbreviated life span (SAMR strains) have been proposed as murine models of accelerated senescence. Each SAMP strain, in addition to displaying "progeroid" traits of accelerated aging, exhibits a singular age-related pathology. The application of this animal model to the study of normal aging processes has been and remains controversial. Therefore, we have undertaken a study of dermal fibroblasts derived from the short-lived SAMP6 strain, which shows early-onset and progressive osteopenia. We have investigated cellular and molecular characteristics that are associated with in vitro aging of normal human fibroblasts, and which are exacerbated in fibroblasts from patients with Werner syndrome, a human model of premature senescence. We found that SAMP6 dermal fibroblasts, relative to SAMR1 and C57BL/6 controls, exhibit characteristics of premature or accelerated cellular senescence with regard to in vitro life span, initial growth rate, and patterns of gene expression.

Aging, Premature↗
PubMed2004

Modification of senescence in ryegrass transformed with IPT under the control of a monocot senescence-enhanced promoter.

We report here the genetic modification of ryegrass senescence. Embryogenic cell suspensions of Lolium multiflorum were transformed by microprojectile bombardment with plasmid constructs containing 1.98 kb of the 5' flanking sequence of SEE1 (a maize cysteine protease gene showing enhanced expression during senescence) fused either to the Agrobacterium tumefaciens cytokinin biosynthesis gene IPT (designated PSEE1::IPT) or to the beta-glucuronidase reporter gene UIDA (PSEE1::UIDA). Plants were regenerated under selection for the HPH hygromycin resistance gene in the vector. PSEE1::UIDA transformants confirmed that the SEE1 flanking sequence functioned as a senescence-enhanced promoter in ryegrass. The IPT transgene was detected in 28 regenerants (PSEE1::IPT) from five independent transformation events. PSEE1::IPT leaves displayed a stay-green phenotype. Some PSEE1::IPT lines developed spontaneous lesions.

Aging↗
PubMed1989

Cellular senescence involves stochastic processes causing loss of expression of differentiated function genes: transfection with SV40 as a means for dissociating effects of senescence on growth and on differentiated function gene expression.

In the accompanying work we demonstrated that the decline in expression of steroid 17 alpha-hydroxylase in mass cultures and clones of adrenocortical cells is the result of a stochastic switching process which yields mixtures of expressing and nonexpressing cells. There is an apparent positive correlation between the replicative potential of adrenocortical cell cultures and the number of cells in the culture that can express 17 alpha-hydroxylase. We investigated this by extending the cells' replicative potential by transfecting them with cloned SV40 virus. Cells from a senescent subclone, with very limited remaining replicative potential, were transfected. The cell population showed a progressive increase in growth rate and gave rise to a line of cells that expressed T antigen and which was apparently immortalized. Induction of mRNA for 17 alpha-hydroxylase by cyclic AMP was absent in this line of cells, as it was in the senescent cells prior to transfection. The cells remained responsive to gene induction by cyclic AMP as evidenced by increases in mRNA and activity for cholesterol side-chain cleavage. The absence of 17 alpha-hydroxylase expression in this line was not the result of interference by SV40 T antigen. When early passage cells were transfected with pSV3neo, which contains the early region of SV40 and neo, and were selected with G418, SV40 T antigen-expressing lines were derived which showed high levels of expression of 17 alpha-hydroxylase after induction with cyclic AMP. These cells maintained high levels of expression of 17 alpha-hydroxylase through four successive recloning events, over a period of replication much longer than that achievable by nontransfected cells. Thus, transfection by SV40 can be used to dissociate effects of senescence on growth and differentiated gene expression. T antigen expression selectively affects growth, but preserves the state of expression of a differentiated function gene as it was prior to transfection.

Adrenal Cortex↗
PubMed1994

Age-related changes in levels of the beta-subunit of nerve growth factor in selected regions of the brain: comparison between senescence-accelerated (SAM-P8) and senescence-resistant (SAM-R1) mice.

Senescence-accelerated mice (SAM-P8) are characterized as mice in which aging is accelerated and memory disturbances occur. In several regions in the brain of SAM-P8 mice at 2, 4 and 8 months of age, we examined the concentrations of the beta-subunit of nerve growth factor (beta-NGF) and nine kinds of proteins such as S100 beta and alpha B-crystallin, and compared them with those in senescence-resistant mice (SAM-R1, as controls) at corresponding ages. Levels of beta-NGF in the hippocampus of SAM-R1 and SAM-P8 mice were reduced at 8 months of age. However, the decrease was more conspicuous in SAM-R1 than in SAM-P8, resulting in a significant difference between them (P < 0.01). The concentrations of beta-NGF in the cerebral cortex and cerebellum decreased to some extent with age in the control mice while it remained unchanged in the mutant mice. By contrast, the olfactory bulbs from SAM-R1 and SAM-P8 retained almost constant levels of beta-NGF during the first 8 months. However, its level was already higher in SAM-P8 at 2 months than in SAM-R1. Among nine proteins measured here, the acceleration of age-related increase was apparent in the levels of S100 beta and Mn-SOD in the cerebral cortex from SAM-P8. By contrast, the cerebral cortex and cerebellum from SAM-P8 showed tendencies to contain significantly high levels of alpha B-crystallin. These results suggest, at least, the presence of fibrous gliosis at quite an early age as well as the acceleration of senescence, in selected regions of the brain of SAM-P8.

Aging↗
PubMed1988

Senescence in Podospora anserina: a possible role for nucleic acid interacting proteins suggested by the sequence analysis of a mitochondrial DNA region specifically amplified in senescent cultures.

In Podospora anserina, the phenomenon of senescence was previously shown to be correlated with the presence of a senescence-specific DNA (sen-DNA) resulting from the amplification of some regions (alpha, beta, gamma, epsilon) of the mitochondrial chromosome. The beta region gives rise to sen-DNAs with variable sizes and junctions which share a 1,100-bp common sequence. Here we report the complete nucleotide sequence of one 4-kb beta sen-DNA. Included in the sequence are a large part of the first intron open reading frame (ORF) of the gene ND4L and three short unidentified ORFs more precisely located in the common beta region. The primary structure of the polypeptide possibly encoded by one of them is very similar to the glycine-rich domains present in various single-stranded DNA-binding proteins. The comparison of the information content of this beta sen-DNA with that of other previously sequenced sen-DNAs suggests that the role in the senescence process attributed to the sen-DNAs could be related to the overproduction of a variety of proteins which interact with nucleic acids.

Amino Acid Sequence↗
PubMed1997

Leaf senescence in Brassica napus: cloning of senescence related genes by subtractive hybridisation.

A subtractive hybridisation technique was developed to clone cDNAs representing genes that showed enhanced expression during leaf senescence in Brassica napus. A number of different genes were identified that, when analysed by northern hybridisation, showed different patterns of expression during leaf development but were all expressed at increased levels during senescence. Sequence analysis of these cDNAs showed that several types of genes were found including two different proteases, glutamine synthetase, ATP sulphurylase, catalase, metallothionein, ferritin and an antifungal protein. The possible roles of these gene products in the senescence process are discussed.

Amino Acid Sequence↗
PubMed1979

Relation between senescence and stomatal opening: Senescence in darkness.

THE SENESCENCE (PROTEOLYSIS AND LOSS OF CHLOROPHYLL) OF ISOLATED LEAVES OF OAT SEEDLINGS IN THE DARK IS INHIBITED OR DELAYED BY COMPOUNDS OF SIX DIFFERENT TYPES: phenazine methosulfate, fusicoccin, alpha,alpha'-dipyridyl, cycloheximide, spermidine, and two cytokinins. In every case but the last, these compounds in optimum concentration caused the stomata to open and remain partly or completely open throughout the 72- or 96-hr experimental period. The cytokinins caused only a partial opening, which is ascribed to their exerting two different effects. Taken together with the previous report that five different treatments that accelerated or promoted senescence in the light caused stomatal closure or occlusion, these data establish a general parallel between stomatal aperture and senescence, with strong indication that the stomatal aperture is the causal factor. A possible explanation of the relationship is proposed.

Journal Article↗
PubMed1985

Chronic food restriction modulates the advance of senescence in the senescence accelerated mouse (SAM).

The effects of chronic food restriction on grading scores of senescence, deposition of senile amyloid (ASSAM), mean life span and 10th decile were investigated by using animal models for accelerated senescence (SAM-P/1) and for normal aging (SAM-R/1). The experimental groups consisted of control (ad libitum fed), 80% (fed 80% of control intake), and 60% (fed 60% of control intake) groups. The grading score of SAM-P/1 mice was significantly improved in the 60% group, but not in the 80% group, compared to the control group. The grading score of SAM-R/1 mice, however, was significantly less than that in the control group in both the 60 and 80% groups. In SAM-P/1 mice liver, skin and testis, the severity of senile amyloid deposition was significantly less with 40% food restriction (60% group) than in the control group. A restriction of 20% (80% group) had no influence on amyloid deposition. A definite tendency to prolong mean life span (24.3%) and 10th decile (65.9%, mean life span of the last 10th of survivors of a group) was observed in the 60% group of SAM-P/1 mice, but the changes were not statistically significant. In the 80% group of SAM-P/1 mice and also in either restriction group of SAM-R/1 mice, however, such a tendency was not evident. These results indicate that 40% food restriction modulates the advance of senescence in these mice.

Aging↗
PubMed1993

Dietary soybean protein compared with casein retards senescence in the senescence accelerated mouse.

The effects of replacing dietary casein with soybean protein on mean life span, mean life span of the last one-tenth of a group, grading scores of senescence and deposition of senile amyloid were investigated in senescence accelerated mice (SAM-P/1) compared with a control strain (SAM-R/1). SAM-R/1 mice fed the soybean protein-containing diet had mean life spans of 618 +/- 42 d (males) and 578 +/- 62 d (females), 58% (males) and 44% (females) longer than those of corresponding casein fed mice (P < 0.01). Similarly, in SAM-P/1 mean life-spans were 265 +/- 16 d (males) and 307 +/- 23 d (females) in the soybean diet group, 27% (males) and 30% (females) longer than in the casein diet groups (P < 0.01). The mean life span of the last one-tenth of each group fed soybean protein was significantly longer than the corresponding group fed casein. In SAM-R/1 mice, pathological studies revealed that severe secondary amyloid deposition (amyloid A protein) in the kidneys, spleen, stomach and liver was significantly suppressed, in males only, by replacing casein with soybean protein (P < 0.01). The occurrence of contracted kidneys caused by the infiltration of amyloid A protein was suppressed in SAM-R/1 mice fed the soybean protein-containing diet (P < 0.05). The deposition of senile amyloid in SAM-P/1 mice with aging was retarded by replacing casein with soybean protein (P < 0.01). These results indicate that dietary protein source is important in modulating the advance of senescence in SAM mice.

Aging↗
PubMed1998

Analysis of the incubation periods, induction of obesity and histopathological changes in senescence-prone and senescence-resistant mice infected with various scrapie strains.

The similarity in histopathological changes seen in scrapie-infected mice and in an uninfected senescence-accelerated mouse strain led to a study in which the mouse strain that is prone to senescence (SAMP8), a strain that is resistant to senescence (SAMR1) and a progenitor strain (AKR) of these two strains were infected with three different scrapie strains, ME7, 139A and 22L. For each scrapie strain, the incubation period was shortest in AKR mice and longest in SAMR1 mice. The induction of obesity was a function of scrapie strain and not mouse strain; ME7 caused obesity in all mouse strains, whereas the average weights of mice injected with 139A and 22L did not differ significantly from mice injected with homogenates of normal mouse brain. The pattern of vacuolation seen in the brain of each mouse strain was primarily dependent on the scrapie strain injected. There were, in general, similarities to the patterns induced in other inbred strains; e.g. ME7 caused extensive forebrain vacuolation, 22L caused prominent vacuolation in the cerebellum, and the 139A strain induced characteristic white matter vacuolation. Vacuolation was also seen in the medulla and midbrain of SAMP8 mice injected with normal mouse brain, which is consistent with the occurrence of accelerated ageing changes in the brain of this strain. Further analysis of the differences among these mouse strains should provide information relating to the observed differences in scrapie incubation periods.

Aging↗
PubMed1975

The Metabolism of Oat Leaves during Senescence: IV. The Effects of alphaalpha'-Dipyridyl and other Metal Chelators on Senescence.

The senescence of the first leaves of light-grown Avena seedlings when detached and placed in the dark is inhibited by alpha, alpha'-dipyridyl and alpha, alpha', alpha''-tripyridyl at concentrations between 10(-5) and 10(-4) M. Five other chelating agents exert similar inhibiting effects at concentrations 3 to 30 times higher. The senescence of etiolated leaves, as shown by loss of carotenoid and protein, is similarly inhibited. Ethylene-diaminetetraacetate has a similar effect in the dark, though only at 10 mM and above, but in the light it causes bleaching of chlorophyll. It is deduced that an iron-containing system plays an essential part in the initiation of the senescence process.

Journal Article↗