Turnover of DNA in Ageing Tissues of Lycopersicon esculentum

Cut shoots of Lycopersicon esculentum were placed in [³H]thymidine(d[³H]Thd) for 24 h and then re-rooted. Immediately after theisotope pulse a variety of tissues in the lower internode regionwere found to have incorporated label into nuclear DNA. Duringthe prolonged chase period it was discovered that the amountof labelled DNA declined significantly in all tissues. Reasonsfor the apparent turnover or degradation of the labelled DNAare discussed for each tissue.     

Replication of DNA in larval salivary glands of Drosophila after in vivo synchronization

The replication of DNA in the giant chromosomes in different cells of Drosophila larval salivary glands is asynchronous. A method of in vivo synchronization of the nuclei has been successfully devised by a 5-fluorodeoxyuridine (FdU) block-release-thymidine chase technique, and the patterns of replication sequences have been examined by ³H-thymidine autoradiography. When the larvae of Drosophila melanogaster are fed on FdU for 48 h, and the block is released thereafter, most cells are found in mid-replication phase (termed 3C). When the larvae are subjected to a chase in normal Drosophila medium (or sucrose), a series of cells arrive at 3C phase about every 8 h. When they are chased in sucrose containing thymidine, the number of cells in 3C phase rises to 70%, and then drops rapidly to 1–2% of all labelled cells. The terminal phases (3D, 2D and 1D) reach a peak between 4–8 h. At 12–14 h of chase the 3D-1D peaks decline and a third peak consisting mostly of the initial phases (DD-1C) is found at 14–16 h. The replication of DNA in polytene chromosomes of Drosophila thus seems to proceed in a regular sequence of DD-3C-1D.Dedicated to Professor W. Beermann on the occasion of his 60th birthday     

Sodium tetraborate effects on mortality and reproduction of Anastrepha suspensa (Diptera: Tephritidae)

When flies were treated with 0- 0.5% sodium tetraborate by feeding for 24 h, mortality in treatments was not different from controls. Fecundity and fertility were reduced by 0.5% sodium tetraborate. When flies were fed for 48 h, mortality of both males and females increased in the 0.5% sodium tetraborate treatment; oviposition was eliminated for 20 d after treatment. When treatment was extended to 168 h, 0.1% sodium tetraborate caused increased mortality and decreased fecundity and fertility. Fed for 168 h, 0.2 and 0.5% sodium tetraborate killed almost all flies within the 7-d treatment. Oviposition of survivors in 0.1 and 0.2% sodium tetraborate treatments was arrested for 20 d after treatment.     

Food effect on the physiological condition of the mussel Perna viridis (Bivalvia: Mytilidae), using the RNA/DNA ratio

The green mussel, Perna viridis, became widespread in the northern coast of Sucre State since its arrival to Venezuela in 1993. RNA/DNA and Protein/DNA ratios were used to study the effect of starvation on its instantaneous growth. The mussels were collected in La Esmeralda and Chacopata, acclimatized in the laboratory for four weeks and maintained for another six weeks in two groups: one fed ad libitum and another without food (this later group was later fed for two additional weeks). Protein (colorimetric method), and nucleic acid concentrations (RNA and DNA, fluorometric method with ethidium bromide) were measured in adductor muscle, digestive gland and gills. The instantaneous growth was assessed using RNA/DNA and Protein/DNA rations. These indexes were always higher in the fed organisms. Animals from Chacopata were in better physiological condition that those from La Esmeralda during the abstinence time (six weeks). Muscle was the best tissue to determine instantaneous growth. The RNA/DNA ratio is a reliable index to determine the physiological condition and instantaneous growth of this species.     

Cryopreservation of in vitro-grown shoot-tips of <Emphasis Type="Italic">Rabdosia rubescens</Emphasis> by encapsulation-dehydration and evaluation of their genetic stability

Shoot-tips of Rabdosia rubescens, excised from in vitro-grown proliferating shoots that were cold-hardened at 5°C for 3 weeks, were encapsulated in alginate beads. Subsequently, these were precultured in a mixture of 0.4 M sucrose and 2 M glycerol for 1 h and then desiccated with silica-gel to about 21% water content prior to freezing in liquid nitrogen. After thawing, about 85% of cryopreserved shoot-tips grew into true-to-type shoots and with enhanced rooting capacity. Eight single-bud sibling lines were used to assess genetic stability of these encapsulated shoot-tips. When the relative DNA content was measured by flow cytometry (FCM), no changes were observed between controls and cryopreserved shoots. Using a sequence-related amplified polymorphism (SRAP) assay, it was observed that seven out of eight cryopreserved lines showed identical banding patterns; while the eighth line displayed an absent band, amounting to a low variance rate of 0.01%. These findings suggested that it was necessary to monitor the genetic stability of recovered cryopreserved R. rubescens shoots.     

Translocation and metabolism of glycine betaine in nodulated alfalfa plants subjected to salt stress

The fate of radioactive glycine betaine was investigated in 31-day-old alfalfa ( Medicago sativa L. cv Europe) plants nodulated by Rhizobium meliloti 102 F 34. Radioactive [methyl-¹⁴C]- or [1,2-¹⁴C]glycine betaine was fed for 6 h to plants subjected or not to stress by 0.2 M NaCl. A 36% decrease in glycine betaine uptake was observed in salinized plants. No loss of radioactivity in the gas phase or the growth medium was ever observed from either stressed or unstressed plants, even after a 4-day chase period. Glycine betaine catabolism was negligible in shoots of both control and salinized plants, but it was important in roots and even more significant in nodules of unstressed plants. In unstressed nodules, 52% of the labelled betaine was metabolized after 4 days, and the half-life of glycine betaine was estimated at ca 4 days. On the contrary, catabolism was dramatically reduced in stressed roots and, particularly, nodules in which the half-life of glycine betaine increased to at least 16 days. Analysis of the redistribution of radioactivity among plant organs during the chase period shows that glycine betaine was translocated from the roots to the nodules of salinized plants, so that during this period salinization resulted in a 91% increase in nodule radioactivity, whereas a 34% decrease was observed in control plants. Altogether, reduced catabolism and increased translocation of glycine betaine to stressed nodules favored its accumulation in these organs. The high level of glycine betaine might contribute to maintain a better water status in the nodule and, thus, protect the nitrogen fixation activity against the deleterious effects of elevated osmolarity in the nutrient solution.     

The influence of energy restriction and developmental state on DNA 5-methyldeoxycytidine in rat mammary and liver tissues

Female rats were fed either ad libitum or 30% energy-restricted diets from 5 weeks through 25 weeks of age. Genomic DNA was extracted from mammary tissue and liver at 7, 9, 14 (mid-pregnancy), 16.5 (mid-lactation), and 25 weeks of age. The 5-methyldeoxycytidine content of DNA was determined by high pressure liquid chromatography. As animals aged from 7–25 weeks, 5-methyldeoxycytidine increased in mammary tissue, whereas in liver it decreased. This suggests that 5-methyldeoxycytidine exhibits tissue specific patterns. No changes in 5-methyldeoxycytidine content due to 30% energy restriction were observed in either mammary tissue or liver.     

Evidence for highly stable nuclear poly(A) in cultured mammalian cells.

The kinetics of turnover of nuclear poly(A) were determined under conditions which facilitated the detection of relatively stable classes of the molecule. Growing 3T6 or HeLa cells were labeled with [3H]adenosine for several hours. The turnover of nuclear poly(A) was then followed over long time intervals using a variety of chase conditions. When a cordycepin chase was employed, a class of nuclear poly(A) with a half life of 2.5 h was observed. When the chase was effected by allowing the intracellular ATP pool specific activity to decay as a result of normal metabolic processes, a more stable class of nuclear poly(A) was detected (half life = 8--12 h). These results indicate that a significant portion of poly(A)-hnRNA has a long half-life.     

The effect of plant identity and the level of plant decay on molecular gut content analysis in a herbivorous soil insect

Plant roots represent an important food source for soil‐dwelling animals, but tracking herbivore food choices below‐ground is difficult. Here, we present an optimized PCR assay for the detection of plant DNA in the guts of invertebrates, using general plant primers targeting the trnT‐F chloroplast DNA region. Based on this assay, we assessed the influence of plant identity on the detectability of ingested plant DNA in Agriotes click beetle larvae. Six different plant species were fed to the insects, comprising a grass, a legume and four nonlegume forbs. Moreover, we examined whether it is possible to amplify DNA of decaying plants and if DNA of decayed plant food is detectable in the guts of the larvae. DNA of the ingested roots could be detected in the guts of the larvae for up to 72‐h post‐feeding, the maximum digestion time tested. When fed with living plants, DNA detection rates differed significantly between the plant species. This may be ascribed to differences in the amount of plant tissue consumed, root palatability, root morphology and/or secondary plant components. These findings indicate that plant identity can affect post‐feeding DNA detection success, which needs to be considered for the interpretation of molecularly derived feeding rates on plants. Amplification of plant DNA from decaying plants was possible as long as any tissue could be retrieved from the soil. The consumption of decaying plant tissue could also be verified by our assay, but the insects seemed to prefer fresh roots over decaying plant material.     

Body fat-lowering effect of conjugated linoleic acid is not due to increased lipolysis

The ability of conjugated linoleic acid (CLA) to reduce adiposity may be due to changes in energy expenditure and/or direct effects on adipocyte lipid metabolism. The aim of the present work was to analyse if CLA supplementation modifies lipolytic activity in adipose tissue from hamsters fed on high-fat diet. Hamsters were divided into two groups and fed on diets supplemented with either 0.5% linoleic acid (control) or 0.5% trans-10,cis-12 CLA. After 6 weeks, animals were fasted overnight and adipose tissues were dissected and weighed. Adipocytes were isolated by collagenase digestion and incubated in Krebs-Ringer bicarbonate buffer with or without several agents acting at different levels of the lipolytic cascade. Adipocyte diameters were measured by microscopy. Adipose tissue DNA content was assessed by spectrophotometry. Animals fed on CLA diet showed significantly reduced adipose tissue mass. No differences between both groups was found for basal lipolysis, lipolytic effects of isoproterenol, forskolin, dibutyryl-cAMP and isobutylmethylxanthine, and pD2 for isoproterenol. A similar total DNA amount was found in adipose tissue of both groups, showing that CLA diet had no effect on total cell number per fat pad. Although DNA content per gram tissue, an indirect reverse index of cell size, was significantly increased in CLA fed hamsters, microscopy did not reveal differences in medium mature adipocyte diameter, nor in cell size distribution between both groups. These results suggest that adipose tissue size reduction induced by trans-10,cis-12 CLA intake is not due to changes in lipolysis. Reduced preadipocyte differentiation into mature adipocytes may account for this fat-lowering effect.     

The effect of protein depletion and repletion on muscle-protein turnover in the chick.

Rates of growth and protein turnover in the breast muscle of young chicks were measured in order to assess the roles of protein synthesis and degradation in the regulation of muscle mass. Rates of protein synthesis were measured in vivo by injecting a massive dose of L-[1-14C]valine, and rates of protein degradation were estimated as the difference between the synthesis rate and the growth rate of muscle protein. In chicks fed on a control diet for up to 7 weeks of age, the fractional rate of synthesis decreased from 1 to 2 weeks of age and then changed insignificantly from 2 to 7 weeks of age, whereas DNA activity was constant for 1 to 7 weeks. When 4-week-old chicks were fed on a protein-free diet for 17 days, the total amount of breast-muscle protein synthesized and degraded per day and the amount of protein synthesized per unit of DNA decreased. Protein was lost owing to a greater decrease in the rate of protein synthesis, as a result of the loss of RNA and a lowered RNA activity. When depleted chicks were re-fed the control diet, rapid growth was achieved by a doubling of the fractional synthesis rate by 2 days. Initially, this was a result of increased RNA activity; by 5 days, the RNA/DNA ratio also increased. There was no evidence of a decrease in the fractional degradation rate during re-feeding. These results indicate that dietary-protein depletion and repletion cause changes in breast-muscle protein mass primarily through changes in the rate of protein synthesis.     

Subcellular localization of the cyanogenic glucoside of sorghum by autoradiography

The use of microautoradiography at the electron microscopic level indicates that the vacuole is the site of accumulation of the cyanogenic glucoside of Sorghum bicolor. When a specific biosynthetic precursor of dhurrin, p-hydroxy[3,5-(3)H]phenylacetaldoxime, was used, 90% of the tritium label was recovered in the vacuoles of tissue prepared for microautoradiography. l-[3,5-(3)H]Tyrosine and d-[1-(3)H(N)]glucose, nonspecific precursors of dhurrin, of differing solubilities and biosynthetic capacity, were also fed to the shoots. The data obtained from these controls indicated that the high recovery of label in the vacuole of aldoxime-fed shoots was not indicative simply of the size of the vacuole, nor was it a result of movement of labeled compounds during preparation of the tissue for electron microscopy. The problem of movement of these labeled compounds during dehydration of tissue was dramatically reduced by chemical dehydration in 2,2-dimethoxypropane in less than 30 minutes rather than with routine dehydration in acetone or alcohol series for 24 hours.     

Loss of regulator of G protein signaling 5 exacerbates obesity, hepatic steatosis, inflammation and insulin resistance

Background

The effect of regulator of G protein signaling 5 (RGS5) on cardiac hypertrophy, atherosclerosis and angiogenesis has been well demonstrated, but the role in the development of obesity and insulin resistance remains completely unknown. We determined the effect of RGS5 deficiency on obesity, hepatic steatosis, inflammation and insulin resistance in mice fed either a normal-chow diet (NC) or a high-fat diet (HF).

Methodology/Principal Findings

Male, 8-week-old RGS5 knockout (KO) and littermate control mice were fed an NC or an HF for 24 weeks and were phenotyped accordingly. RGS5 KO mice exhibited increased obesity, fat mass and ectopic lipid deposition in the liver compared with littermate control mice, regardless of diet. When fed an HF, RGS5 KO mice had a markedly exacerbated metabolic dysfunction and inflammatory state in the blood serum. Meanwhile, macrophage recruitment and inflammation were increased and these increases were associated with the significant activation of JNK, IκBα and NF-κBp65 in the adipose tissue, liver and skeletal muscle of RGS5 KO mice fed an HF relative to control mice. These exacerbated metabolic dysfunction and inflammation are accompanied with decreased systemic insulin sensitivity in the adipose tissue, liver and skeletal muscle of RGS5 KO mice, reflected by weakened Akt/GSK3β phosphorylation.

Conclusions/Significance

Our data suggest that loss of RGS5 exacerbates HF-induced obesity, hepatic steatosis, inflammation and insulin resistance.     

In vitro adventitious shoot formation from embryonic and cotyledonary tissues of Pinus brutia Ten.

Adventitious buds were induced when isolated whole embryos, and excised cotyledons from treated seeds of Calabrian pine (Pinus brutia Ten.) were cultured on a cytokinin-supplemented medium. The adventitious buds formed directly from the cotyledons. The highest number of bud primordia were formed, with both embryos and excised cotyledons, after 6 weeks on a BAP — supplemented Schenk and Hildebrandt medium under a 16 h photoperiod. The buds, when separated and maintained individually on a full-strength medium without growth regulators, developed into well-formed shoots within 4 weeks. The average number of harvested shoots obtained (>1 cm in height) per seed over 24 weeks was 55; however, a maximum number of 152 shoots was obtained from one individual over the same period. The shoot forming capacity of the meristematic tissue was not lost after seven harvests.     

DNA synthesis in HeLa cell nuclei isolated in a non-aqueous medium.

Nuclei were isolated from synchronized HeLa cells in the S-phase by a modification of the non-aqueous method described by Kirsch et al. (Science (1970) 168, 1592-1595). The method involved lyophilization of the cells, homogenization in non-aqueous glycerol and centrifugation in a gradient of 0-35% (w/w) 3-chloro-1,2-propanediol in glycerol. Such nucleic incorporated deoxyribonucleotides into DNA when incubated in an aqueous buffer containing Mg2+, ATP, dATP, dGTP, dCTP and dTTP. The product was sensitive to DNAase and banded with bulk DNA in isopycnic centrifugation. Sedimentation of the product in alkaline sucrose gradients after labelling of the nuclei for 2 min revealed labelled material in the 5 S peak and in the 18 S area. The material in the 5 S peak moved into the 12 S area after a 13 min chase.     

The effect of monensin on thyroglobulin secretion

The effect of monensin on the secretion of thyroglobulin was studied in open follicles isolated from pig thyroid tissue; in this system, thyroglobulin is secreted into the incubation medium. When monensin was present during a 4-h chase incubation after pulse-labelling with 3H-leucine, the secretion of labelled thyroglobulin was reduced by about 85%; in electron-microscopic autoradiographs of rat thyroid lobes labelled and chase-incubated under similar conditions the relative number of grains over follicle lumina was strongly reduced when monensin was present during the chase. These observations are in agreement with the consensus that monensin arrests transport of secretory proteins in the Golgi complex. In other experiments, pulse-labelled follicles were chase-incubated for 1.5 h whereby labelled thyroglobulin was transported from the RER to exocytic vesicles. Monensin present during a subsequent chase of 0.5 h caused only a moderate decrease of labelled thyroglobulin secretion. TSH present during the second chase-stimulated secretion in both control and monensin-exposed follicles. TSH also caused a drastic reduction of exocytic vesicles in rat thyroid lobes, and the number of vesicles remaining in the cells was the same in controls and lobes exposed to the ionophore. The observations are interpreted to show that monensin does not inhibit the basal or TSH-stimulated transport of thyroglobulin from the site of monensin-induced arrest in the Golgi complex to the apical cell surface or the exocytosis of thyroglobulin.     

The role of sink demand in carbon partitioning and photosynthetic reinvigoration following shoot decapitation

Photosynthesis, growth, and carbon partitioning of vigorous coppice shoots were compared with the slower growing intact shoots of Populus maximowiczii × nigra L. MN9 to determine the relationship between carbon partitioning and photosynthetic rate. Relative height growth rate of coppice shoots was 2.2 times that of intact shoots with net photosynthetic rate 1.9 times that of intact shoots. Coppice leaves exported a larger proportion of newly-fixed assimilate (11% compared with 6%) after a 4-h chase. The greater export from coppice leaves was correlated with a greater proportion of [¹⁴C]-labelled photosynthate deposited as starch in stems 4 cm below the point of label application. Coppice leaf assimilate levels were reduced to 15% that of leaves on intact plants, but coppice leaves had twice the concentration of labelled sucrose. Carbohydrates constituted 55% of the water-soluble [¹⁴C]-labelled photosynthate in leaves of coppice shoots compared with 40% in intact shoots. The results suggest that carbon allocation and partitioning in coppice shoots were altered towards production and export of new assimilate, and support the hypothesis that photosynthetic rate is responsive to sink demand for assimilates.     

Decreased hepatic retinyl palmitate hydrolase activity in protein-deficient rats

28-day-old weanling rats were fed a diet containing 3% casein as the only source of protein for eight weeks to induce protein deficiency. When compared to control animals (fed a diet containing 25% casein), these rats had significantly lowered body (5.2-fold reduction) and liver (2.5-fold reduction) weights. The circulatory level of retinol (nmol per ml plasma) as well as retinol (nmol per g tissue) in the liver of these protein-deficient animals were also reduced significantly, although their liver concentration of retinyl palmitate (nmol per g tissue) was comparable to that of the control group. Assay of liver tissue for retinyl palmitate hydrolase activity revealed a 4-fold reduction (compared to that of control animals) of specific enzyme activity (nmol retinol formed per g protein per h). These findings suggest that severe protein deficiency results in a decreased hydrolysis of retinyl esters in the liver, which may be in part responsible for the reduced level of metabolically 'active' retinoids available for normal physiological functions.