Increase In RNA Content Research Articles

Effects of dietary arginine supplementation on muscle structure, meat characteristics and lipid oxidation products in lambs and its potential mechanisms of action

This study aimed to assess the effect of dietary arginine supplementation on muscle structure and meat characteristics of lambs also considering lipid oxidation products and to contribute to reveal its mechanisms of action using tandem mass tagging (TMT) proteomics. Eighteen lambs were allocated to two dietary treatment groups: control diet or control diet with the addition of 1% L-arginine. The results revealed that dietary arginine supplementation increased muscle fibre diameter and cross-sectional area (P < 0.05), which was attributable to protein deposition, as evidenced by increased RNA content, RNA/DNA ratio, inhibition of apoptotic enzyme activity, and alterations in the IGF-1/Akt signaling pathway (P < 0.05). In addition, dietary arginine elevated pH24h, a* values, and IMF content, decreased shear force value and backfat thickness (P < 0.05), as well as decreased the formation of lipid oxidation products involved in meat flavor including hexanal, heptanal, octanal, nonanal and 1-octen-3-ol by increasing the antioxidant capacity of the muscle (P < 0.05). The proteomics results suggested that seven enrichment pathways may be potential mechanisms by which arginine affected the muscle structure and meat characteristics of lambs. In summary, arginine supplementation in lamb diets provides a safe and effective way to improve meat quality, and antioxidant capacity of muscle of lamb.

THE INFLUENCE OF ACUTE CEREBRAL BLOOD CIRCULATION DISORDERS IN THE POOL OF CAROTID ARTERIES ON THE RNA CONTENT IN THE CELLS OF THE LYMPHOID POPULATION OF THE RATS’ THYMUS OF DIFFERENT AGE GROUPS

The aim of the work – to find out the age-related features of the response of RNA cells ofthe lymphoid population of the thymus to an acute disturbance of cerebral blood flow inthe pool of carotid arteries in six- and nine-month-old rats.Materials and methods. Acute cerebrovascular accident was simulated in 6- and9-month-old white non-linear laboratory rats by bilateral carotid artery clamping for 20min. followed by reperfusion. The thymus was taken on the 12th day of the postischemicperiod, fixed in Buen’s solution, after standard histological processing it was embedded inparaffin, and serial sections with a thickness of 5 μm were made. After deparaffinization,rehydration, and staining with hallocyanine- chromium alum according to Einarson,histological sections were analyzed in a digital image analysis system VIDAS-386(Kontron Electronik, Germany) with a fluorescent microscope AXIOSKOP (Zeiss,Germany). Total RNA content was expressed in units of optical density (EOP per 1 mm2). The results. In the cells of the lymphoid population of all structural and functional zones ofthe thymus of control rats of both age groups, an increase in RNA content was establishedin the direction from immature to more mature classes (p&lt;0.05). This regularity persistsin the post-ischemic period. In the cells of the lymphoid population of the subcapsular anddeep cortical zones of the thymus, the RNA content significantly prevails in six-month-oldrats, in intralobular perivascular spaces – in nine-month-old rats, and in the medullaryzone there are no age differences. Postischemic changes in RNA content in six-month-oldrats in the subcapsular zone and intralobular perivascular spaces of the thymus consistin its decrease in immature cell classes and its increase in mature small thymocytes(р&lt;0.05); in the deep cortex – in growth in all classes of thymocytes (p&lt;0.05). In ninemonth-old animals, the differences were in the increase in RNA content in the subcapsularzone and in less mature middle thymocytes, in the deep cortex – in a decrease in RNAcontent in all classes, except small thymocytes, in intralobular perivascular spaces – ina decrease in RNA content in all classes of thymocytes (p&lt;0.05 for all cases). In themedullary zone of animals of both age groups, changes are unidirectional (decrease inRNA content, p&lt;0.05).Conclusion. Age differences in the nature of postischemic changes in the content of RNAin thymocytes are present in all structural and functional zones of the gland, with theexception of the medullary, in which the changes were unidirectional in both age groups.

Blue and Red Light Color Combinations Can Enhance Certain Aspects of Digestive and Anabolic Performance in Juvenile Steelhead Trout Oncorhynchus mykiss

The light spectrum varies with the altitude of the sun and shows different light colors in clear water. In this study, we aimed to investigate the response of juvenile steelhead trout Oncorhynchus mykiss (34.67 ± 2.69 g initial weight) under different light color conditions. The effects of different blue and red light combinations on plasma biochemical parameters, digestive enzyme activity, and RNA/DNA ratio were assessed in trout over 16 weeks. Six treatments were randomly assigned to 24 tanks with four replicates per treatment: a constant light intensity of 150 lx: 12 h white light then 12 h dark (12W); 12 h blue light then 12 h dark (12B); 12 h red light then 12 h dark (12R); 1.5 h blue light, 9 h red light, 1.5 h blue light, then 12 h dark (3B9R); 3 h blue light, 6 h red light, 3 h blue light, then 12 h dark (6B6R); and 12 h of both blue and red light then 12 h dark (T12BR). Fish exposed to the 3B9R light environment showed significantly increased plasma levels of total protein (TP), enhanced activities of midgut lipase, trypsin, and gastric lipase; and increased RNA content in the liver and muscle tissue to promote protein synthesis efficiency, thereby improving digestive and anabolic performance compared to fish in the other treatments. This indicates that steelhead trout have adapted well to such variable light conditions during long-term evolution. In contrast, trout exposed to the 6B6R light environment showed significant reductions in plasma glucose, TP, and triglyceride levels, decreased activity of gastrointestinal digestive enzymes, and reduced protein synthesis capacity in the muscle and liver, resulting in weakened digestive and anabolic performance. Furthermore, despite the high RNA content and RNA/DNA ratio in fish exposed to a 12R light environment, relatively high plasma cholesterol and triglycerides levels were observed, which might indicate oxidative stress. Therefore, this light is not considered suitable for long-term cultivation. In conclusion, the 3B9R treatment was the optimal light condition tested and can be used to improve the digestive and anabolic performance of steelhead trout.

Reticulated Platelets-Which Functions Have Been Established by In Vivo and In Vitro Data?

Reticulated platelets (RP) are the youngest platelet fraction released into the circulation. These immature platelets have increased RNA content, a larger cell volume, more dense granules, higher levels of surface activation markers and are thought to be more reactive compared to their mature counterparts. RP have been associated with cardiovascular disease, diabetes and increased mortality. Yet only a few animal studies investigating RP have been conducted so far and further investigations are warranted. Established methods to count RP are flow cytometry (staining with thiazole orange or SYTO13) or fully automated hematology analyzers (immature platelet fraction, IPF). IPF has been established as a diagnostic parameter in thrombocytopenia, cardiovascular disease and, in particular, the response to antiplatelet therapy. This review seeks to provide an overview of the key features of RP as well as preanalytical and analytical aspects that need to be considered when working with this platelet population.

Exosomes from IL-1β-Primed Mesenchymal Stem Cells Inhibited IL-1β- and TNF-α-Mediated Inflammatory Responses in Osteoarthritic SW982 Cells.

Exosomes from mesenchymal stem cells (MSCs) show anti-inflammatory effect on osteoarthritis (OA); however, their biological effect and mechanism are not yet clearly understood. This study investigated the anti-inflammatory effect and mechanism of MSC-derived exosomes (MSC-Exo) primed with IL-1β in osteoarthritic SW982 cells. SW982 cells were treated with interleukin (IL)-1β and tumor necrosis factor (TNF)-α to induce the OA phenotype. The effect of exosomes without priming (MSC-Exo) or with IL-1β priming (MSC-IL-Exo) was examined on the expression of pro- or anti-inflammatory factors, and the amount of IκBα was examined in SW982 cells. Exosomes were treated with RNase to remove RNA. The role of miR-147b was examined using a mimic and an inhibitor. MSC-IL-Exo showed stronger inhibitory effects on the expression of pro-inflammatory cytokines (IL-1β, IL-6, and monocyte chemoattractant protein-1) than MSC-Exo. The expression of anti-inflammatory factors (SOCS3 and SOCS6) was enhanced by MSCs-IL-Exo. Priming with IL-1β increased RNA content in MSC-IL-Exo, and pretreatment with RNase abolished anti-inflammatory effect in SW982 cells. miR-147b was found in much larger amounts in MSC-IL-Exo than in MSC-Exo. The miR-147b mimic significantly inhibited the expression of inflammatory cytokines, while the miR-147b inhibitor only partially blocked the anti-inflammatory effect of MSC-IL-Exo. MSC-IL-Exo and miR-147b mimic inhibited the reduction of IκBα, an nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) inhibitor, by IL-1β and TNF-α. This study showed that MSC exosomes with IL-1β priming exhibit significantly enhanced anti-inflammatory activity in osteoarthritic SW982 cells. The effect of IL-1β-primed MSC exosomes is mediated by miRNAs such as miR-147b and involves inhibition of the NF-κB pathway.

Six Weeks of Low-Load Blood Flow Restricted and High-Load Resistance Exercise Training Produce Similar Increases in Cumulative Myofibrillar Protein Synthesis and Ribosomal Biogenesis in Healthy Males.

Purpose: High-load resistance exercise contributes to maintenance of muscle mass, muscle protein quality, and contractile function by stimulation of muscle protein synthesis (MPS), hypertrophy, and strength gains. However, high loading may not be feasible in several clinical populations. Low-load blood flow restricted resistance exercise (BFRRE) may provide an alternative approach. However, the long-term protein synthetic response to BFRRE is unknown and the myocellular adaptations to prolonged BFRRE are not well described.Methods: To investigate this, 34 healthy young subjects were randomized to 6 weeks of low-load BFRRE, HLRE, or non-exercise control (CON). Deuterium oxide (D2O) was orally administered throughout the intervention period. Muscle biopsies from m. vastus lateralis were collected before and after the 6-week intervention period to assess long-term myofibrillar MPS and RNA synthesis as well as muscle fiber-type-specific cross-sectional area (CSA), satellite cell content, and myonuclei content. Muscle biopsies were also collected in the immediate hours following single-bout exercise to assess signaling for muscle protein degradation. Isometric and dynamic quadriceps muscle strength was evaluated before and after the intervention.Results: Myofibrillar MPS was higher in BFRRE (1.34%/day, p < 0.01) and HLRE (1.12%/day, p < 0.05) compared to CON (0.96%/day) with no significant differences between exercise groups. Muscle RNA synthesis was higher in BFRRE (0.65%/day, p < 0.001) and HLRE (0.55%/day, p < 0.01) compared to CON (0.38%/day) and both training groups increased RNA content, indicating ribosomal biogenesis in response to exercise. BFRRE and HLRE both activated muscle degradation signaling. Muscle strength increased 6–10% in BFRRE (p < 0.05) and 13–23% in HLRE (p < 0.01). Dynamic muscle strength increased to a greater extent in HLRE (p < 0.05). No changes in type I and type II muscle fiber-type-specific CSA, satellite cell content, or myonuclei content were observed.Conclusions: These results demonstrate that BFRRE increases long-term muscle protein turnover, ribosomal biogenesis, and muscle strength to a similar degree as HLRE. These findings emphasize the potential application of low-load BFRRE to stimulate muscle protein turnover and increase muscle function in clinical populations where high loading is untenable.

Antihypertensive and cardioprotective effects of new compound 1-(β-phenylethyl)-4-amino-1,2,4-triazolium bromide (Hypertril)

Despite the success which was achieved in the treatment of arterial hypertension, the problem remains actual. At the departments of pharmaceutical chemistry and pharmacology of the Zaporozhye State Medical Institute (Ukraine), our research team isolated the compound 1-(β-phenylethyl)-4-amino-1,2,4-triazolium bromide (Hypertril) as derivative of 4-amino-1,2,4-triazole. The objectives of this investigation were the study of cardioprotective and antihypertensive activities of this new compound Hypertril and we used the Spontaneously hypertensive rats (SHR) as an experimental model. We discovered that Hypertril has a reliable dose-dependent antihypertensive effect in the dose range 5–20 mg/kg after 30-day administration and this antihypertensive effect of Hypetril competes or significantly exceeds Metoprolol (20 mg/kg). Our studies obtained evidence of a dose-dependent improvement of myocardial energy metabolism. Hypertril reduces the manifestations of secondary mitochondrial dysfunction due to arterial hypertension. Hypertril can prevent oxidative modification of the protein; also Hypertril reduces the insufficiency of mitochondrial pores. As a result, Hypertril increases the content of ATP in the myocardium of SHR, normalizes the activity of mitochondrial enzymes, decreases lactate production and increases pyruvate. Hypertril enhances the cardioprotective effects of NO and increases the resistance of the cardiomyocytes to ischemia. The use of Hypertril leads to a dose-dependent increase of the density of cardiomyocyte nuclei, significant increase RNA content in nuclei and the cytoplasm of cardiomyocytes, and an increase of the nuclear-cytoplasmic index. These changes indicate a decrease of myocardial hypertrophy.

Combined apoptotic effects of peptide and miRNA in a peptide/miRNA nanocomplex

The present study investigated combined biological effects of peptide and miRNA in a peptide/miRNA nanocomplex. We utilized TatBim peptide as a cell-penetrating peptide-based RNA carrier with apoptotic activity. miRNA with apoptotic activity (miR-34a) was used for complex formation to investigate the additional effects of the combination with TatBim peptide. TatBim peptide and the miRNA formed nanocomplexes (approximately 250nm in diameter), and these complexes were efficiently internalized by cells. Despite its efficient cell internalization, apoptotic activity of the nanocomplex decreased with increasing RNA content. However, photosensitizer-attachment to TatBim and photoirradiation significantly improved the apoptotic activity of the nanocomplex by facilitating dispersion of the peptide and RNA in the cytoplasm. Combined apoptotic activity of both TatBim peptide and miR-34a in the nanocomplex was demonstrated by substituting TatBim with Lipofectamine and by substituting miR-34a with scrambled siRNA.

Total RNA content in sheep oocytes and developing embryos produced in vitro, a comparative study between spectrophotometric and fluorometric assay

Isolation of total RNA from limited number of oocytes and embryos is a big challenge. DNA free RNA and assessment of RNA integrity are crucial to the success of gene expression studies because poor quality RNA give misleading results. The objective of the present study was to establish a suitable protocol to isolate good quality total RNA from a minimal number of sheep oocytes and embryos that enables the downstream applications, as well as to estimate RNA content in oocytes and developmental stages of embryos. Five protocols were approached to isolate total RNA from oocytes and embryos. Four methods were by standard Trizol protocols and its modification whereas fifth method was by commercial kit (RNeasy mini kit, Quiagen). Total RNA isolated by modified Trizol protocol with coprecipitants (acrylamide and glycogen) showed significantly (P < 0.05) more spectrophotometric reading of RNA concentration than by modified Trizol protocol without coprecipitant followed by commercial kit and conventional Trizol protocol. RNA quality, purity, concentration, RNA per oocyte and expression of GAPDH (house keeping gene) were compared to find the best RNA isolated by different protocols. Spectrophotometric and fluorometric assay were compared to quantify the total RNA concentration in sheep oocytes and different stages of developing embryos. RNA yield by spectrophotometer analysis showed 5–100 times more reading than fluorometer. Significant (P < 0.05) reduction in RNA content was observed in matured oocytes than that of immature oocytes. There was significant (P < 0.05) increase in RNA content after fertilization upto 2–4 cells stage followed by significant (P < 0.05) decrease at 8–16 cells and increased at morula. RNA concentration at blastocyst was significantly low than at morula. From the protocols approached modified Trizol protocol with coprecipitant was most efficient and suitable method over other protocols approached to isolate RNA from few sheep oocytes and embryos for gene expression study.

Molecular breeding of Saccharomyces cerevisiae with high RNA content by harnessing essential ribosomal RNA transcription regulator

As yeast is commonly used for RNA production, it is industrially important to breed strains with high RNA contents. The upstream activating factor (UAF) plays an important role in transcription of ribosomal RNA (rRNA), a major constituent of intracellular RNA species. Here, we targeted the essential rRNA transcription regulator Rrn5 of Saccharomyces cerevisiae, a component of the UAF complex, and disrupted the genomic RRN5 gene using a helper plasmid carrying an RRN5 gene. Then we isolated nine suppressor mutants (Sup mutants) of RRN5 gene disruption, causing deficiency in rRNA transcription. The Sup mutants had RNA contents of approximately 40% of the wild type level and expansion of rDNA repeats to ca. 400–700 copies. Reintroduction of a functional RRN5 gene into Sup mutants caused a reduction in the number of rDNA repeats to close to the wild type level but did not change RNA content. However, we found that reintroduction of RRN5 into the Sup16 mutant (in which the FOB1 gene encoding the rDNA replication fork barrier site binding protein was disrupted) resulted in a significant increase (17%) in RNA content compared with wild type, although the rDNA repeat copy number was almost identical to the wild type strain. In this case, upregulated transcription of non-transcribed spacers (NTS) occurred, especially in the NTS2 region; this was likely mediated by RNA polymerase II and accounted for the increased RNA content. Thus, we propose a novel breeding strategy for developing high RNA content yeast by harnessing the essential rRNA transcription regulator.

Local trauma in human patellar tendon leads to widespread changes in the tendon gene expression.

Low cellular activity and slow tissue turnover in human tendon may prolong resolution of tendinopathy. This may be stimulated by moderate localized traumas such as needle penetrations, but whether this results in a widespread cellular response in tendons is unknown. In an initial hypothesis-generating study, a trauma-induced tendon cell activity (increased total RNA and collagen I mRNA) was observed after repeated patellar tendon biopsies in young men. In a subsequent controlled study, 25 young men were treated with two 0.8-mm-diameter needle penetrations [n = 13, needle-group (NG)] or one 2.1-mm-diameter needle biopsy [n = 12, biopsy-group (BG)] in one patellar tendon. Four weeks later biopsies were taken from treated (5 mm lateral from trauma site) and contralateral tendons for analyses of RNA content (ribogreen assay), DNA content (PCR based), and gene expression for relevant target genes (Real-time RT-PCR) (NG, n = 11 and BG, n = 8). Intervention increased RNA content, and mRNA expression of collagen I and III and TGF-β1 (P < 0.05), with biopsy treatment having greatest effect (tendency for RNA and collagen I). Results for DNA content were inconclusive, and no changes were detected in expression of insulin-like growth factor-I, connective tissue growth factor, scleraxis, decorin, fibromodulin, tenascin-C, tenomodulin, VEGFa, CD68, IL-6, MMP12, and MMP13. In conclusion, a moderate trauma to a healthy human tendon (e.g., biopsy sampling) results in a widespread upregulation of tendon cell activity and their matrix protein expression. The findings have implications for design of studies on human tendon and may provide perspectives in future treatment strategies in tendinopathy.

Accurate Assessment of Cell Death by Imaging Flow Cytometry.

The number of investigators using cell death analysis applications has greatly expanded since the introduction of flow cytometry. The Annexin V/propidium iodide (PI) method is among the most commonly used procedures and allows users to determine if cells are viable, apoptotic, or necrotic, based on changes in membrane lipid composition, integrity, and permeability. Unfortunately, PI can intercalate into RNA, in addition to DNA, which contributes to a large number of events showing PI staining within the cytoplasmic compartment. We show that this occurs across a broad range of animal primary cells and commonly used cell lines, and is most prevalent in large cells (nuclear:cytoplasmic ratio <0.5). Any cellular system where RNA levels change throughout an experiment will be particularly affected, such as those that utilize virally infected cells. As two examples, we highlight our recent work on cells infected with vesicular stomatitis virus (VSV), an RNA virus, and herpes simplex virus-1 (HSV-1), a DNA virus. Similarly, these issues are relevant to experimental systems where cells have increased RNA content such as during genotoxic stress, following exposure to cell cycle arrest drugs such as thymidine or hydroxyurea, or where developmental progression promotes discrete changes in cellular RNA synthesis. This chapter outlines a modified Annexin V/PI method that addresses cytoplasmic RNA staining issues to allow for accurate assessment of cell death. This protocol takes advantage of an additional cellular permeability caused by fixation to promote RNase A entry into the cell. Based on our observations, cell morphological parameters are well maintained and less than 5 % of total cellular events exhibit cytoplasmic PI staining under this protocol.

Abstract 78: Extracellular RNA Induces Venous Thrombosis Through TLR3

Venous thromboembolism (VTE) afflicts 117 people per 100,000 each year and is an important cause of morbidity and mortality. Vascular and circulating cells may sense ‘pathologic ligands’ accumulating in the circulation via toll-like receptor (TLR) and translate signals into a pro-thrombotic state. TLR3 recognizes self-molecules generated during tissue damage and inflammation such as extracellular RNA (eRNA). We hypothesize that eRNA and TLR3 are involved in the development of venous thrombosis after vessel injury. Injection of RNase1 decreased the size of thrombi after FeCl3-induced inferior vena cava injury (IVC) compared to mice treated with vehicle. Using a specific fluorescent probe for RNA, we found that FeCl3 induced RNA release and thus increased RNA content in the thrombus. WT or TLR3 deficient (-/-) mice received an injection of vehicle, eRNA or poly(I:C) prior to thrombosis induction. In WT mice, thrombus size was increased by eRNA or poly(I:C). Interestingly, no change in thrombus size was observed in TLR3-/- mice regardless of the treatment. Poly(I:C) treatment was associated with higher amount of neutrophils within the thrombus of WT mice compared with vehicle. In TLR3-/- mice, poly(I:C) failed to increase the recruitment of neutrophils. Staining for citrulinated H3, a marker of neutrophil extracellular traps (NETs) formation, was increased by poly(I:C) in WT mice. Interestingly, in TLR3-/- mice, no increase of NET formation was found after poly(I:C) injection as compared to vehicle control. Poly(I:C) also increased the recruitment of monocytes within thrombi in WT but not in TLR3-/- mice. Preliminary results suggest that injection of eRNA may also promote the recruitment of monocytes, neutrophils and the formation of NETs in WT but not in TLR3-/- mice. Human neutrophils were then treated with conditioned media from HUVEC treated with poly(I:C) in presence of control siRNA or TLR3 siRNA. Media from HUVEC transfected with control siRNA and treated with poly(I:C) induced the migration of neutrophils, whereas media from TLR3 siRNA and poly(I:C) treated cells did not. These results suggest that eRNA and TLR3 may participate to thrombus formation by inducing a pro-inflammatory response leading to the recruitment of monocytes and neutrophils.

Increase in rRNA content in a Saccharomyces cerevisiae suppressor strain from rrn10 disruptant by rDNA cluster duplication

Breeding of yeast strains with higher RNA content is important because yeast RNA is a significant source of 5'-ribonucleotides, which have considerable use in both the food and pharmaceutical industries. Ribosomal RNA (rRNA) is an important source of yeast RNA as it accounts for about 80 % of total RNA content. We previously reported a dominant suppressor mutant of an rrn10 disruptant named SupE, which displays the ability not only to restore diminished RNA content caused by rrn10 disruption but also to increase the transcription level of ribosomal protein (RP) genes on an ∆rrn10 background in Saccharomyces cerevisiae. Here, to construct an S. cerevisiae strain with higher RNA content, we investigated the effect of increasing the copy number of the rDNA gene on a ∆rrn10 SUPE background. We successfully constructed a SupE strain with two copies of the rDNA cluster (ca. 300 rDNA genes) by using chromosome-splitting technology. The RNA content of this strain was 61 % higher than that of the SupE strain with a single copy of the rDNA cluster (ca. 150 rDNA genes), and 40 % higher than that of the wild-type strain with two copies of the rDNA cluster. A further increase in RNA content of 47 % was achieved by multicopy expression of the RPL40A gene in the SupE strain with two copies of the rDNA cluster. These observations suggest that we have constructed an S. cerevisiae strain with two copies of the rDNA cluster, which has achieved a considerably higher RNA content. Furthermore, the strategy taken in this study provides an effective approach to constructing S. cerevisiae strains with high potential for yeast food biotechnology.

Increased transcription of RPL40A and RPL40B is important for the improvement of RNA production in Saccharomyces cerevisiae

Yeast (Saccharomyces cerevisiae) RNA is an important source of 5'-ribonucleotides that is used in both the food and pharmaceutical industries. Efficient transcription of rDNA is very important to construct yeast strains with high RNA content. The gene RRN10, which encodes, a component of the upstream activation factor, is essential to promote high-level transcription of rDNA. In our previous study, we isolated SupE strain as a dominant suppressor, which showed the ability to restore the severe growth defects and reduced RNA content caused by disruption of the RRN10 gene. SupE strain has multiple mutations which we designated collectively as SUPE. Further analysis on SUPE mutation indicated that RPL40A was responsible for suppression of defect caused by rrn10 disruption. However, there were no base changes in this gene as compared with the parental Δrrn10 strain, thus suggesting that an additional copy of RPL40A suppress the defects caused by Δrrn10 disruption, and that, in SupE strain, these defects are suppressed by increased transcription of RPL40A whose copy is doubled. When multiple copies of RPL40A were combined with SUPE mutation on an RRN10⁺ background, the resultant SupE strain had significantly higher RNA content than wild-type strain. In addition, increased transcription of RPL40B also showed significant effect to restore the growth defect and reduced RNA content caused by Δrrn10 disruption. We propose a model to explain how SUPE mutation increases the transcription of ribosomal protein genes such as RPL40A and RPL40B in SupE strain, resulting in an increase in RNA content.

Influence of Natural Thermal Gradients on Whole Animal Rates of Protein Synthesis in Marine Gammarid Amphipods

Although temperature is known to have an important effect on protein synthesis rates and growth in aquatic ectotherms held in the laboratory, little is known about the effects of thermal gradients on natural populations in the field. To address this issue we determined whole-animal fractional rates of protein synthesis (ks) in four dominant species of gammarid amphipods with different distributions along the coasts of Western Europe from arctic to temperate latitudes. Up to three populations of each species were collected in the summer and ks measured within 48 h. Summer ks values were relatively high in the temperate species, Gammarus locusta, from Portugal (48°N) and Wales (53°N) and were maintained across latitudes by the conservation of translational efficiency. In sharp contrast, summer ks remained remarkably low in the boreal/temperate species G. duebeni from Wales, Scotland (58°N) and Tromsø (70°N), probably as a temporary energy saving strategy to ensure survival in rapidly fluctuating environments of the high intertidal. Values for ks increased in acclimated G. duebeni from Scotland and Tromsø showing a lack of compensation with latitude. In the subarctic/boreal species, G. oceanicus, summer ks remained unchanged in Scotland and Tromsø but fell significantly in Svalbard (79°N) at 5°C, despite a slight increase in RNA content. At 79°N, mean ks was 4.5 times higher in the circumpolar species G. setosus than in G. oceanicus due to a doubling in RNA content. The relationship between whole-animal protein synthesis rates and natural thermal gradients is complex, varies between species and appears to be associated with local temperatures and their variability, as well as changes in other environmental factors.

Changes in the Nuclei of Infected Cells at Early Stages of Infection with EMCV

By the methods of quantitative cytophotometry, we have identified the changes in the nucleus and of some intranuclear compartments in the early stages of infection with encephalomyocarditis virus (EMCV). They can be characterized as early 1 - 2 hours post infection (hpi) and temporary increase (duration about 1 hour) in the content of the acidic proteins of the nucleolus, changing their decline to the control values. Then (after 1 - 2 hours) follows an increase in RNA content of nucleoli to 4 hours post infection (the process takes about 2 hours). The increase in RNA content in nucleoli is in approximately the same time (slightly behind) with the activation of PML bodies (2 - 4 hpi). Then, the RNA content in nucleoli decreased to the control values, while simultaneously decreasing activity of PML bodies (ranging from 5 - 6 hpi). The early stages of infection EMCV are also characterized by the tendency to increase in the size of the nuclei of infected cells, and preserve at a later time. Then there is an increase in RNA content in the nucleus, roughly coinciding with the increased content of RNA in the nucleoli.

Physiological responses of dietary tryptophan fedLabeo rohitato temperature and salinity stress

Two experiments were conducted to elucidate the possible effects of dietary L-tryptophan (TRP) in Labeo rohita based on growth performance and physio-biochemical responses. In the experiment I, a 60-day feeding trial was carried out to elucidate the effects of dietary TRP enrichment on growth performance and physio-biochemical responses. In the experiment II, the TRP pre-fed L.rohita, from experiment I, was exposed to temperature and salinity stress to evaluate stress-mitigating efficacy of TRP. In L.rohita, dietary supplementation of TRP showed significant effect on weight gain percentage and feed conversion ratio but not on blood glucose. A significant increase in RNA content and RNA/DNA ratio upon TRP supplementation was observed and was positively correlated with growth performance. The results of experiment II indicated that weight gain percentage, serum T3 and T4 levels were significantly reduced in groups that were exposed to temperature and salinity stress and fed diets without TRP supplementation. However, dietary supplementation of TRP significantly augmented weight gain percentage in stress-exposed groups. Tryptophan supplementation helped in bringing back T3 and T4 levels comparable with control. A significant increase in superoxide dismutase, catalase, Adenosine triphosphatase, blood glucose and serum cortisol was observed in temperature- and salinity-exposed groups fed without TRP-supplemented diets. However, TRP supplementation was found to be effective in restoring the above parameters. The results of these experiments suggest that dietary TRP supplementation augments growth, lowers energy demand and helps in mitigating thermal and salinity stress in L.rohita.

Some histochemical features of anther wall of Leucojum aestivum (Amaryllidaceae) during pollen development

In this study, polysaccharide and RNA contents of anthers were investigated on different phases of sporogenesis by using light microscopy techniques from histological and cytological point of view in Leucojum aestivum. Paraffin and semi-thin sections of anthers were stained with toluidine blue and PAS. Anthers were tetrasporangiate. The wall of the anther consists of an epidermis, endothecium, middle layer and glandular tapetum. During one nucleated microspore and mature pollen phase microspores and tapetum cells began to degenerate and they were become very rich of RNA in L. aestivum. And also RNA content was increased in endothecium and middle layer cells except the epidermis cells of anther wall. An increase in RNA content indicates cell activation. Polysaccharides were not seen in young anther wall but they were seen in older ones. They were generally condensed in the cell walls and especially in the cell walls of vascular bundles of connective tissue. This could be thought that insoluble polysaccharides were used in metabolic events in early developmental stages. Appearance of polysaccharides in late phases was indicated that polysaccharides were used in the formation of cuticule and differentiation of endothelium cell walls.

The Cellular Classification and Separation of Nucleated Blood Cells by Supravital Cell Staining with Acridine Orange

Abstract 4907Immunohistochemical staining and flow cytometry are performed on the tissue to distinguish specific cells. The detection of diseases is made by the flow cytometry using monoclonal antibodies on peripheral blood (PB) or bone marrow cells. The diagnosis of leukemia is made with PB and marrow cells using antibodies to cluster of differentiation (CD) antigens specific to leukemic cells (e.g.,CD13, CD33). Therefore, conventional approaches to identify cellular phenotypes are being replaced by immunophenotyping using flow cytometry. However antibodies satisfying for needs are not always available. In this case, the generation of such antibody to each specific antigen also causes problems of producing the respective recombinant antigen for immunization. When attempting to construct new animal models using other than mice and humans, this issue becomes one of the serious limiting factors in developing research. In addition, the opportunity is increasing to classify unexperienced types of cells such as cells derived from iPS cells and others. We therefore consider that it is important to develop methods to classify and separate specific cells of interest without antibodies. As supravital cell staining with acridine orange (AO) is introduced in 1960's (Jacson JF, Blood, 1961; Lewis M. et al. Blood, 1962), this metachromatic fluorescent dye rapidly stains DNA and RNA independently. Morphological abnormalities of human erythrocytes such as red cell fragment and large platelets are detectable (Nagai Y et al., Int. J Lab Hematol., 2008). AO emits green fluorescence when it binds to the double-stranded DNA and also red fluorescence when it binds to the single-stranded RNA. Consequently flow cytometry of cells stained with AO is suitable for analyzing blood cells in four parameter, which are scattered light intensity (FSC and SSC) and fluorescence intensity (DNA content and RNA content). This analysis method has the possibility of developing the classifying and separating method for abnormalities.Xenopus laevis(X. laevis) has various nucleated blood cells, which are erythrocytes, leukocytes, and thrombocytes, and their progenitors that are not classified yet. As the first step to identify cells with increased RNA content, we chose X. laevis blood cells as a new model of AO staining for flow cytometry by FACS Aria II cell sorter based on the content of DNA and RNA. Since collected cells were stained by May-Griinwald-Giemsa on cytocentrifuge preparations. The population in lower content of DNA and RNA is composed of erythrocytes (95.7±1.3% of whole PB cells). The population in higher concentration of DNA and RNA was pure leukocytes fraction (0.7±0.6% of whole PB cells) expressing the mRNA of X. laevis myeloperoxidase. This population was then fractionated with the higher content of RNA containing eosinophils and basophils (48.7±29.9%), and lower content of RNA contained neutrophils (50.9±29.9%). The proportion of the peripheral thrombocytes in lower forward light scatter (FSC) and side light scatter (SSC) ranges was 0.8±0.8% of whole PB cells, and the expression of c-Mpl, CD41 and Fli-1 mRNA was detected in the sorted cells by RT-PCR. Furthermore, sorted cells were confirmed by immunohistochemical staining by anti X. laevis thrombocyte monoclonal antibody. To reveal the characteristics of the abnormal PB cells, we compared the normal PB with the PB of phenylhydrazine (PHZ) induced anemic X. laevis. At 8 days after treated with PHZ, immature hematopoietic cells were appeared in circulating blood. We isolated these cells with high content of DNA and RNA, and the population in lower FSC and higher SSC with crossover analysis of light emission by AO, suggesting that this method could identify abnormalities in whole PB. The character of these cells was morphologically small and high nuclear cytoplasmic ratio. We also applied this method to blood cells of Rana catesbeiana, American bull frog. Despite the differences in size of erythrocytes, thrombocytes and leukocytes, Rana catesbeiana PB was similarly sorted to X. laevis PB by cellular DNA/RNA content. These results suggest that this method can sort nucleated cells in various species by crossover analysis of light emission by AO. Our study showed that this method has the advantage to characterize of human leukemia cells, tumor cells, iPS-derived cells and nucleated blood cells. [Display omitted] Disclosures:No relevant conflicts of interest to declare.