Biogenesis of chromatin in animal cells. Stimulation of nuclear protein and DNA synthesis in hepatocytes after brief inhibition of translation by cycloheximide]

A drastic and brief inhibition of protein synthesis (up to 95% for 3--6 hrs) by cycloheximide in the liver of rats starved for 24 hrs results in a recovery and subsequent marked stimulation of non-histone proteins, histone chromosomal proteins and DNA. The stimulation of non-histone protein synthesis was observed after 1 hr (inhibition) 12--24 hrs (recovery and stimulation of protein synthesis) and 48--60 hrs (stimulation of DNA synthesis) following the administration of cycloheximide. Two periods of histone biosynthesis were observed. The first one (24--36 hrs) was not coupled and the second one (48--60 hrs) was coupled with DNA replication. During the recovery and stimulation of protein synthesis acetylation of the histone and non-histone proteins proceeds at an increased rate. Possible applicability of the model in question for investigations of chromatin biogenesis is discussed.     

DNA repair in mammalian nerve cells. II. DNA synthesis in neurons, induced by gamma-irradiation of isolated rat neocortex slices

DNA synthesis was studied in the neocortex neurons of 0-, 14- and 60-day old rats after gamma-irradiation in vitro of isolated slices of the neocortex on determining 3H-thymidine incorporation into DNA. Gamma-irradiation (20 Grays) increases levels of DNA synthesis for all the age groups of animals examined. The induced DNA synthesis in neurons of newborn rats is higher than that in 14- and 60-day old animals. Specificity of DNA synthesis in the process of nerve cell differentiation is discussed.     

The origin of DNA genomes and DNA replication proteins

In recent years, it has became clear that most proteins involved in cellular DNA precursor synthesis or DNA replication have been 'invented' more than once, indicating that the transition from RNA to DNA genomes was more complex than previously thought. Several authors have suggested that DNA viruses, which often encode their own version of these proteins, played an important role in this process. The nature of the genome of the last universal cellular ancestor (LUCA) -- that is, RNA or DNA, prokaryotic-like or eukaryotic-like -- remains in dispute. A hyperthermophilic LUCA would have suggested a circular, double-stranded DNA genome; however, recent data favor a mesophilic or moderately thermophilic LUCA.     

High mTORC1 signaling is maintained,while protein degradation pathways are perturbed in old murine skeletal muscles in the fasted state

This study investigated age-associated changes to protein synthesis and degradation pathways in the quadriceps muscles of male C57BL/6J mice at 5 ages, between 4 and 24 months (m). Sarcopenia was evident by 18 m and was accompanied by hyper-phosphorylation of S6K1, indicating increased mTORC1 signaling. Proteasomal and autophagosomal degradation pathways were also impacted by aging. In the 1% NP40 insoluble protein fraction, the abundance of MuRF1 increased at 24 m, while p62 increased at 15 m, and remained elevated at older ages. In addition, we investigated how protein synthesis and degradation pathways are modulated by fasting in young (4 m) and old (24 m) muscles, and showed that old mice respond to fasting less robustly compared with young. Overnight fasting for 16 h caused de-phosphorylation of AKT and molecules downstream of mTORC1 (S6K1, rpS6 and 4E-BP1) in young, but not old muscles. A longer time of fasting (24 h) was required to reduce phosphorylation of these molecules in old mice. Induction of MuRF1 and Fbxo32 mRNA was also more robust in young compared with old muscles following fasting for 16 h. In addition, a 16 h fast reduced ULK1 phosphorylation at the mTORC1 specific site Ser757 only in young muscles. The striking accumulation of insoluble p62 protein in muscles of all old male mice (fed or fasted), suggests age-related dysregulation of autophagy and protein aggregation. These data provide an insight into the mechanisms of metabolic responses that affect protein homeostasis in old skeletal muscles, with applications to design of clinical interventions that target sarcopenia.     

Estimation of the National Disease Burden of Influenza-Associated Severe Acute Respiratory Illness in Kenya and Guatemala: A Novel Methodology

Background

Knowing the national disease burden of severe influenza in low-income countries can inform policy decisions around influenza treatment and prevention. We present a novel methodology using locally generated data for estimating this burden.

Methods and Findings

This method begins with calculating the hospitalized severe acute respiratory illness (SARI) incidence for children <5 years old and persons ≥5 years old from population-based surveillance in one province. This base rate of SARI is then adjusted for each province based on the prevalence of risk factors and healthcare-seeking behavior. The percentage of SARI with influenza virus detected is determined from provincial-level sentinel surveillance and applied to the adjusted provincial rates of hospitalized SARI. Healthcare-seeking data from healthcare utilization surveys is used to estimate non-hospitalized influenza-associated SARI. Rates of hospitalized and non-hospitalized influenza-associated SARI are applied to census data to calculate the national number of cases. The method was field-tested in Kenya, and validated in Guatemala, using data from August 2009–July 2011. In Kenya (2009 population 38.6 million persons), the annual number of hospitalized influenza-associated SARI cases ranged from 17,129–27,659 for children <5 years old (2.9–4.7 per 1,000 persons) and 6,882–7,836 for persons ≥5 years old (0.21–0.24 per 1,000 persons), depending on year and base rate used. In Guatemala (2011 population 14.7 million persons), the annual number of hospitalized cases of influenza-associated pneumonia ranged from 1,065–2,259 (0.5–1.0 per 1,000 persons) among children <5 years old and 779–2,252 cases (0.1–0.2 per 1,000 persons) for persons ≥5 years old, depending on year and base rate used. In both countries, the number of non-hospitalized influenza-associated cases was several-fold higher than the hospitalized cases.

Conclusions

Influenza virus was associated with a substantial amount of severe disease in Kenya and Guatemala. This method can be performed in most low and lower-middle income countries.     

Somatic mutagenesis at T-cell receptor locus in inhabitants of radiation polluted regions as a result of the Chernobyl disaster

In the period of 2001-2004, frequency of cells bearing mutations at T-cell receptor (TCR) locus was assessed in 553 inhabitants of radiation polluted regions of the Russian Federation and 154 unexposed control persons. The inhabitants were divided into three groups according to age at the moment of the Chernobyl disaster and 137Cs pollution density: 1) in utero, 37-555 kBq/m2; 2) 0-14 years old, 20-555 kBq/m2; 3) 18 and more years old, highest 137Cs density (185 more than 555 kBq/m2). The most intense changes of the TCR-mutant cell frequency were observed in the group of persons exposed to ionizing radiation in utero. The mean frequency of the mutant cells was higher in the first group than in age-matched control group by about 1.5-fold: 4.0 x 10(-4) vs 2.7 x 10(-4) accordingly (p < 0.0001). Elevation in the mean TCR-mutant cell frequency was less expressed in group of inhabitants aged 0-14 years at the moment of irradiation start: 1.3-fold increase in comparison to age-matched control (3.8 x 10(-4) vs 2.9 x 10(-4), p = 0.0002). It was not found significant differences in mutant cell frequencies between control group and adults consisting in the third group (18 and more years old at the moment of the Chernobyl accident). The changes of the TCR-mutant cell frequency in persons exposed in pre- and postnatal periods differ not only quantitatively, but qualitatively. In the fist case all persons react to irradiation by increasing number of the TCR-mutant cells in some degree. In the second case - only a part of population. Proportion of reacting persons depends on age at the start of irradiation and, perhaps, on dose absorbed. The TCR-mutant frequency was significantly higher in persons with benign tumors of different localizations and nodules in thyroid gland than in persons without this pathology.     

Evaluation of the reorganization in the high-order structure of DNA occurring during cell senescence

Electron microscopic pictures of loosened chromatin obtained from human fibroblasts aged in vitro were analyzed with an image processor. It was found that the density of the 30-nm chromatin fibers decreased progressively through the cell population lifespan. The spacing between the fibers increased abruptly during the last divisions (phase IV). The evolution of these 2 parameters, density and spacing, was identical, respectively, with that of the decline in the rate of initiating DNA synthesis after seeding the cells and with that of the maximal percent of cells synthesizing DNA during a 24-h period between seeding and resting phase. Cells from 2 human donors (30 and 60 years old) with Werner's syndrome were compared, using the same methodology, with those of 9 normal donors ranging from 29 to 98 years old. The spacing between chromatin fibers in the Werner's cells was significantly increased as compared to the age-matched normal controls while the density was within the normal range. The results are discussed with regard to other modifications in DNA structure previously reported in the same cell system.     

Reparative DNA synthesis in the lymphocytes of persons coming in contact with chromium compounds in industry

The intensity of repair DNA synthesis in the lymphocytes of persons who were in contact with chromium compounds was studied depending on the contact duration. It is discovered that the intensity of repair DNA synthesis is reduced with the increase of the contact duration with chromium compounds. It is suggested that the mutagenic effect of chromium compounds is connected with the influence on the repair DNA synthesis in human cells.     

Surface analysis and depth profiles of calcium in hepatoma cells during pyruvate-induced DNA synthesis

Induction of DNA synthesis is associated with increased uptake of calcium in cultured cells. Calcium distribution within the plasma membrane and adjacent cytoplasmic layers of hepatoma cells was investigated with X-ray photoelectron spectroscopy and oxygen plasma etching. Cells in minimal growth medium initiate active DNA synthesis 16 h after addition of sodium pyruvate. Cells stimulated with pyruvate and pyruvate-free controls were analysed by X-ray photoelectron spectroscopy--oxygen plasma etching at 0--40 A (layer I), 0--450 A (layer II) and 0--4000 A (layer III from the outer cell surface. Calcium concentrations were elevated in induced cells compared with controls: +20% in layer I, +60% in layer II, and +300% in layer III. As the plasma membrane is 90--120 A thick, these results indicate that pyruvate-induced DNA synthesis is preceded by an increase in calcium, most marked in the cytoplasm subjacent to the plasma membrane, moderate at its inner surface, and minimal at its outer surface.     

流行性感冒裂解疫苗临床免疫效果观察

兰州生物制品研究所研制的流行性感冒病毒裂解疫苗于2003年9月~12月在广西进行Ⅰ~Ⅲ临床试验,对疫苗的安全性和免疫原性进行考核评价。试验中随机选取852人(6月龄~67岁)接种试验疫苗,227人接种对照疫苗。6~36月龄的婴幼儿接种2针,每次0.25m l,间隔28天;成人接种0.5 m l。所有接种对象均未见红肿和硬结等局部反应;发生低热反应(37.1℃~37.5℃)率为3.5%,均于48小时内恢复正常。疫苗接种后易感人群的H I抗体总阳转率为100%,非易感人群的H I抗体几何平均效价增长7.1~16.8倍,抗体4倍增长率为73.1%~91.7%。证实该疫苗具有良好的安全性和免疫原性。     

Replicon size and rate of DNA replication in the macronucleus of Tetrahymena pyriformis]

The size of replication units (or replicons) measured in Tetrahymena pyriformis GL macronuclear DNA reaches 20--30 microns, according to the two independent methods: DNA fiber autoradiography, and alkaline isokinetic sucrose gradient centrifugation. The synthesis of new DNA fragments--replicons and their subsequent assembly are separated by time intervals (30 min). The rate of DNA synthesis for one fork averaged 0.6--0.7 microns/min. These data were obtained for cells of cultures being both in the expotential phase of growth, and those synchronized by starvation-refeeding. The generation time of T. pyriformis cells, calculated by the increase of the part of labeled nuclei, is almost 2 hours; the synthesis lasts 1 hour. Total amount of replication units in polyploid (polygenomic) Tetrahymena macronucleus is about 3000. Their initiation during S-period is presumably asynchronous.     

The tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate enhances the proliferative response of Balb/c-3T3 cells to hormonal growth factors.

Stimulation of Balb/c-3T3 cell growth by TPA requires factors found in serum. We examined the interaction between TPA and serum growth factors in the stimulation of cell growth. The number of cells synthesizing DNA (incorporating 3H-thymidine) within 24 to 30 hours after the addition of TPA and the growth factors to density-inhibited Balb/c-3T3 cultures in serum-free medium was determined by autoradiography. With no additions or with TPA (30--300 ng/ml) alone, only 3--7% of cells synthesized DNA. However, TPA synergistically promoted DNA synthesis in combination with each of the defined serum growth fractions, platelet derived growth factor and platelet poor plasma. TPA also synergistically promoted DNA synthesis in combination with purified growth factors including fibroblast growth factor, insulin (10(-6)--10(-5)M), and epidermal growth factor. In all conditions, TPA enhancement of DNA synthesis also resulted in an increase in cell number. Because TPA synergistically enhanced the activity of each growth factor tested, it did not act identically to any of the growth factors.     

Enhancement by transition metals of unscheduled DNA synthesis induced by isoniazid and related hydrazines in cultured normal and xeroderma pigmentosum human cells

In combination with transition metals (Mn(II), Cu(II), and Fe(III)), isoniazid and related hydrazine compounds induced unscheduled DNA synthesis (DNA repair) in cultured human fibroblasts. Manganese at 10(-5) and 10(-4) M strongly enhanced DNA repair induced by isoniazid, iproniazid, nialamide and hydrazine. Peak levels of DNA repair occurred at 5 x 10(-4)--10(-3) M of the 4 hydrazine compounds. Copper caused less enhancement of DNA repair while iron had no detectable effect. Without added metal, unscheduled DNA synthesis was not observed in cells treated with any of the 4 freshly-prepared hydrazine compounds. However, following preincubation in medium for 6--12 h, isoniazid alone at high concentrations (10(-2) M--10(-1) M) induced DNA repair. With isoniazid/manganese mixtures, preincubation did not further enhance DNA repair except at low concentrations of isoniazid (2--5 x 10(-4) M). Catalase reduced the DNA damage caused by preincubated isoniazid and by the isoniazid/metal mixtures. Exposure of repair-deficient xeroderma pigmentosum cells to isoniazid plus manganese resulted in a DNA-repair profile similar to that of normal cells. The results are consistent with hydrogen peroxide being a critical intermediate for the production of free radicals which cause the observed DNA damage.     

DNA synthesis, methylation and degradation during conjugation in Tetrahymena thermophila.

We have investigated the timing of DNA synthesis, methylation and degradation during macronuclear development in the ciliate, Tetrahymena thermophila. DNA synthesis was first detected in the anlagen early in macronuclear development, but the majority of DNA synthesis occurred later, after pair separation. Anlagen DNA was first detectably methylated at GATC sites 3-5 hours after its synthesis. Once initiated, de novo methylation was rapid and complete, occurring between 13.5 and 15 hours of conjugation. The level of methylation of GATC sites was constant throughout the remainder of conjugation, and was similar to that in mock-conjugated cells. Degradation of DNA in the old macronucleus and DNA synthesis in the anlagen began at about the same time. Upon pair separation, less than 20% of old macronuclear DNA remained. A small percentage of nucleotides prelabeled prior to conjugation were recycled in the developing anlagen.     

A pharmaceutical preparation of Salvia miltiorrhiza protects cardiac myocytes from tumor necrosis factor-induced apoptosis and reduces angiotensin II-stimulated collagen synthesis in fibroblasts

Salvia miltiorrhiza is a medicinal herb commonly used in traditional Chinese medicine for the prevention and treatment of cardiovascular disease. This study investigated the effects of Cardiotonic Pill (CP), a pharmaceutical preparation of Salvia miltiorrhiza, on cardiac myocytes and fibroblasts with respect to the viability, proliferation, and collagen synthesis in these cells under various conditions. A cardiac myocyte line, H9c2, and primarily cultured fibroblasts from rat hearts were incubated with CP over a broad concentration range (50–800 μg/ml) under normal cultures, conditions of ischemia (serum-free culture), and stimulation by angiotensin II (AII, 100 nM), hydrogen peroxide (H₂O₂, 50–200 μM), or tumor necrosis factor α (TNFα, 40 ng/ml) for 24–48 h. Cell growth, apoptosis, DNA and collagen synthesis, and expression of relevant genes were assessed via cell number study, morphological examination, Annexin-V staining, flow-cytometry, [³H]-thymidine or [³H]-proline incorporation assay, and Western blotting analysis. It was found that (1) at therapeutic (50 μg/ml) and double therapeutic (100 μg/ml) concentrations, CP did not significantly affect normal DNA synthesis and cell growth in these cardiac cells, while at higher (over 4-fold therapeutic) concentrations (200–800 μg/ml), CP decreased DNA synthesis and cell growth and increased cell death; (2) CP treatment (50 μg/ml) significantly inhibited TNFα-induced apoptosis in myocytes, with 12.3±1.46% cells being apoptosis in CP treatment group and 37.0±7.34% in the control (p<0.01), and simultaneously, expression of activated (phosphorylated) Akt protein was increased by about 2 folds in the CP-treated cells; and (3) in cultured fibroblasts, CP significantly reduced AII-induced collagen synthesis in a concentration-dependent manner (by ～50% and ～90% reduction of AII-induced collagen synthesis at 50 and 100 μg/ml, respectively). Thus, Salvia miltiorrhiza preparation CP is physiologically active on cardiac cells. The actions by CP to reduce apoptotic damage in myocytes and collagen synthesis in fibroblasts may help to preserve the heart function and reduce heart failure risk. The actions by CP to inhibit DNA synthesis and cell growth, which occurred at over therapeutic doses, may weaken the ability of heart repair. Further studies are needed to identify the chemical compounds in this herbal product that are responsible for these observed physiological effects.     

Effects of formic acid,molasses and inoculant additives on corn silage composition,organic matter digestibility and microbial protein synthesis in sheep

This study examined the production of com silages with low or high lactic acid concentrations, provided by the addition of formic acid (0.5%), molasses (5%) or microbial inoculant (homofermentative lactic acid bacteria, 10 g/tonne). After the fermentation period, sheep were fed the silages to determine true and apparent digestibility of the organic matter and microbial protein synthesis. The experiment were carried out with four KıvırcıkxMorkaraman sheep, 1.5 years old, fixed with cannula in their rumen and duodenum.Lactic acid concentrations were significantly higher in silages treated with enzyme or molasses compared to other specific treatments. Acetic acid concentration was highest in silage treated with formic acid, and lowest in silage treated with molasses (P < 0.05). The by-pass of crude protein was highest in silage treated with formic acid.     

Retrospective molecular detection of transhyretin Met 111 mutation in a Danish kindred with familial amyloid cardiomyopathy,using DNA from formalin-fixed and paraffin-embedded tissues

Severe familial amyloid cardiomyopathy (FAC) in a Danish kindred is associated with a specific mutation (Met for Leu 111) in the transthyretin (TTR) gene. The mutation causes the loss of a DdeI restriction site in the gene, allowing molecular diagnostic studies. We studied formalin-fixed, paraffin-embedded tissues, up to 39 years old, from 29 family members of this kindred. DNA was partially purified from deparaffinized tissue sections and a DNA sequence of the TTR gene flanking the mutation site was amplified by the polymerase chain reaction (PCR), followed by restriction enzyme analysis. Amplified DNA was obtained from tissues representing 23 of the 29 persons. Ten out of the 23 family members were found to carry the TTR Met 111 mutation, whereas 13 were not affected. The results were consistent with known clinical data and with corresponding serum TTR examinations. This retrospective study shows that archival tissues can be used to confirm the diagnosis and disease pattern in members of families affected by hereditary diseases.     

Increased synthesis of abundant poly(A)-containing RNA in a DNA defective mutant of Saccharomyces cerevisiae containing the "killer character".

A Saccharomyces cerevisiae strain which contains both the "killer character" and a ts mutation in the initiation of nuclear DNA synthesis (cdc4) was studied. Incubation of this strain at the restrictive temperature caused a 3--4 fold increase in the relative rate of synthesis of abundant RNA which contains poly(A) and a 2--3-fold increase in the relative rate of synthesis of killer dsRNA. Thus, the amount of killer dsRNA found in these cells seems to be correlated to the amount of abundant poly(A)-RNA.     

Mitochondrial DNA sequences are present inside nuclear DNA in rat tissues and increase with age

Mitochondrial DNA (mtDNA) mutations increase with age. However, the number of cells with predominantly mutated mtDNA is small in old animals. Here a new hypothesis is proposed: mtDNA fragments may insert into nuclear DNA contributing to aging and related diseases by alterations in the nucleus. Real-time PCR quantification shows that sequences of cytochrome oxidase III and 16S rRNA from mtDNA are present in highly purified nuclei from liver and brain in young and old rats. The sequences of these insertions revealed that they contain single nucleotide polymorphisms identical to those present in mtDNA of the same animal. Interestingly, the amount of mitochondrial sequences in nuclear DNA increases with age in both tissues. In situ hybridization of mtDNA to nuclear DNA confirms the presence of mtDNA sequences inside nuclear DNA in rat hepatocytes. Bone marrow metaphase cells from both young and old rats show mtDNA at centromeric regions in 20 out of the 2n = 40 chromosomes. Consequently, mitochondria can be a major trigger of aging but the final target could also be the nucleus.