Nuclear RNA Content Research Articles

Abstract 9528: Apical Resection in Newborn Pig Hearts Extends the Regenerative Window of Cardiomyocyte

Background: The neonatal porcine heart possesses proliferative cardiomyocyte capacity to recover heart function and structure after an acute myocardial injury during two days of birth. In this study, we have extended the myocardial regenerative window to postnatal day 28 (P28) by a double injury pig model (AR P1 MI P28 ), which performed apical resection at P1 (AR P1 ) followed by LAD ligation at P28 (MI P28 ). However, the molecular mechanism of regenerative window extension is unknown. Methods: The AR P1 MI P28 hearts showed no visible sign of myocardial damage or malfunction 4 weeks after LAD ligation. The nuclear RNA contents were isolated from hearts that received none, singe, and double injury hearts. They performed single nuclei RNA-seq to characterize the gene-expression profile of the AR P1 MI P28 in cardiomyocytes. Results: Gene-expression profiles revealed that regenerating AR P1 MI P28 hearts contained different subpopulations of cardiomyocytes early after 2 nd injury. Louvain clustering identified the six cardiomyocyte clusters (CM1-CM6). The CM6, CM3, and CM2 clusters have mainly comprised fetal (92.4%), CTL-P1 (95.7%), and CTL-P56 (96.2%) cardiomyocytes. In contrast, the CM1 cluster has comprised heterogeneous cardiomyocyte groups from all other animal groups, including less than 1% of fetal and CTL-P1 cardiomyocytes. The CM5 and CM4 clusters have mainly comprised AR P1 MI P28 -P35 (double injury hearts harvested at P35) cardiomyocytes, 97.8% and 68.6%, respectively. Sparse modeling analysis revealed that AR and MI injury, both alone and in combination, appeared to extend the regenerative capacity of cardiomyocytes and perturb cardiomyocyte maturation. Conclusion: We have demonstrated that the unique CM5 cluster appeared only after the 2 nd injury of AR P1 MI P28 -P35 hearts. This cell cluster was characterized by a highly expressed isoenzyme of the glycolytic enzyme pyruvate kinase M2 (Pkm2) and skeletal muscle filamentous gene Nebulin. Transient expression of the CM5 cluster induces by the AR P1 might contribute to the sustained remuscularization and myocardial function recovery following an acute MI in large mammals.

Single-nucleus and single-cell transcriptomes compared in matched cortical cell types.

Transcriptomic profiling of complex tissues by single-nucleus RNA-sequencing (snRNA-seq) affords some advantages over single-cell RNA-sequencing (scRNA-seq). snRNA-seq provides less biased cellular coverage, does not appear to suffer cell isolation-based transcriptional artifacts, and can be applied to archived frozen specimens. We used well-matched snRNA-seq and scRNA-seq datasets from mouse visual cortex to compare cell type detection. Although more transcripts are detected in individual whole cells (~11,000 genes) than nuclei (~7,000 genes), we demonstrate that closely related neuronal cell types can be similarly discriminated with both methods if intronic sequences are included in snRNA-seq analysis. We estimate that the nuclear proportion of total cellular mRNA varies from 20% to over 50% for large and small pyramidal neurons, respectively. Together, these results illustrate the high information content of nuclear RNA for characterization of cellular diversity in brain tissues.

Using measurements of muscle cell nuclear RNA with flow cytometry to improve assessment of larval condition of Walleye Pollock (Gadus chalcogrammus)

Nuclear RNA and DNA in muscle cell nuclei of laboratory-reared larvae of Walleye Pollock (Gadus chalcogrammus) were simultaneously measured through the use of flow cytometry for cell-cycle analysis during 2009–11. The addition of nuclear RNA as a covariate increased by 4% the classification accuracy of a discriminant analysis model that used cell-cycle, temperature, and standard length to measure larval condition, compared with a model without it. The greatest improvement, a 7% increase in accuracy, was observed for small larvae (<6.00 mm). Nuclear RNA content varied with rearing temperature, increasing as temperature decreased. There was a loss of DNA when larvae were frozen and thawed because the percentage of cells in the DNA synthesis cell-cycle phase decreased, but DNA content was stable during storage of frozen tissue.

Effect of bacterial endotoxin on secretion and synthesis of vasopressin during saline load in rats.

Secretion and synthesis of vasopressin was studied in adult male Wistar rats receiving lipopolysaccharide in a dose of 250 microg/100 g body weight and subjected to moderate osmotic stimulation (2% NaCl perorally for 6 days). Lipopolysaccharide stimulated secretion of vasopressin into the blood. It should be emphasized that the content of vasopressin mRNA in gigantocellular nuclei of the hypothalamus decreased, which probably reflected intensification of its translation. The observed changes and slight increase in transcription of the vasopressin gene (determined by the content of heterogeneous nuclear RNA) provide intensive secretion of this neurohormone into the blood.

Cytochrome P450 expression in human hepatocytes and hepatoma cell lines: molecular mechanisms that determine lower expression in cultured cells

1. Cultured hepatic cells have reduced cytochrome P450 (CYP) activities in comparison with human liver, but the mechanism(s) that underlies this circumstance is not clear. We investigated the causes of this low CYP activity by analysing the activity, protein, mRNA and heterologous nuclear RNA contents of the most important CYPs involved in drug metabolism (1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5) in cultured human hepatocytes, and in HepG2 and Mz-Hep-1 hepatoma cell lines. 2. After 24 h of culture, hepatocytes retained most of their CYP activities and protein contents, but the mRNA decreased 20-fold. However, the mRNA content of most CYPs in 24-h hepatocytes was still 400-fold higher than in hepatoma cells. When we examined the transcriptional activity of the CYP genes, this decreased during culture time in hepatocytes and it was poor in hepatoma cell lines. 3. We investigated the abundance of key hepatic transcription factors that govern CYP transcription (C/EBP- β : LAP and LIP, HNF-3 α, HNF-4 α, RXR- α) and observed that the expression of some factors was altered in the hepatoma cells. 4. In conclusion, the loss of biotransformation activity in cultured hepatic cells is caused by a decrease in CYP transcription, which correlates with an alteration in the expression of key transcription factors.

The transcriptional response to androgens of the rat VCSA1 gene is amplified by both binary and graded mechanisms.

In higher eukaryotes, gene expression can be highly modified in response to small variations of circulating hormonal inducers. To determine the mechanisms responsible for the 100- to 200-fold enhancement of expression of an androgen-regulated gene, VCSA1, in the acinar cells of rat submandibular glands during puberty, we performed a detailed analysis of VCSA1 expression at the single cell level. Using in situ detection of mature and primary VCSA1 transcripts, we show that VCSA1 expression is activated in only a small proportion of differentiated acinar cells in the presence of low levels of circulating androgens in prepubescent and in castrated males, as well as in females. During the time course of sexual maturation in males, we demonstrate an increase in the proportion of acinar cells expressing VCSA1 and an increase in VCSA1 heterogeneous nuclear RNA and mRNA content in the positive cell population. Finally, we show that changes in the methylation pattern of VCSA1 are correlated with VCSA1 transcriptional activation. These results demonstrate that androgens can, in physiological conditions, elicit both a binary and a graded response. They also provide evidence that the range of gene regulation may be expanded by a transcriptional repression in a majority of cells under basal conditions.

Differential induction of Cyp1a1, Cyp1b1, Ahd4, and Nmo1 in murine skin tumors and adjacent normal epidermis by ligands of the aryl hydrocarbon receptor.

Products of several phase I and II genes transcriptionally activated by ligands of the aryl hydrocarbon receptor (AHR) were quantitated in cutaneous samples isolated from non-tumor-bearing SENCAR or SSIN mice, and animals bearing skin tumors generated in initiation-promotion protocols. The constitutive 7-ethoxyresorufin O-deethylase (EROD) activities in papillomas and squamous cell carcinomas were less than or equal to 37% of the values measured in the adjacent normal cutaneoustissue. Dermal and epidermal EROD specific activities in microsomal samples prepared from both tumor-bearing and non-tumor-bearing mice were elevated 9- to 14- and 43- to 77-fold, respectively, above constitutive levels 16-20 h after a single topical application of 100 nmol of dibenz[a,c]anthracene (DB[a,c]A). EROD specific activities in tumors were maximally elevated two-fold after topical application of DB[a,c]A. Western blot, northern blot, and reverse transcription (RT)-polymerase chain reaction (PCR) analyses confirmed that the EROD measurements reflected cutaneous cytochrome P450 (CYP) 1A1 protein, mature mRNA, and heterogeneous nuclear RNA contents, respectively. Analyses of CYP1A1, CYP1B1, cytosolic aldehyde dehydrogenase class 3, and NAD(P)H:menadione oxidoreductase (NMO1) mRNA content by RT-PCR revealed significant increases in all four mRNAs in the normal tissue adjacent to papillomas after exposure to 4 nmol of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) but no increases in the tumors. NMO1 mRNA content in acetone-treated papillomas approached the levels detected in TCDD-treated normal skin. RT-PCR analyses also demonstrated elevated constitutive aryl hydrocarbon receptor nuclear translocator mRNA content (an approximately two-fold increase) in skin tumors. In contrast, AHR mRNA content in the tumors was about 20% of that measured in adjacent normal tissue. Collectively, these studies demonstrated that ligand-induced, AHR-mediated processes are absent in murine skin tumors that develop in initiation-promotion protocols.

Flow cytometric methods for RNA content analysis

Analysis of the RNA content of individual cells provides information on their translational capacity, which varies with cell differentiation and proliferation. In fact, because the rates of cell growth and proliferation are coupled, cellular or nuclear (nucleolar) RNA content indirectly serves as a marker of cell proliferation. The most common use of RNA measurement is in the discrimination of cycling from noncycling cells; RNA content is also a prognostic parameter in many malignancies. Two flow cytometric methods that simultaneously measure cellular RNA and DNA were developed. The first method is based on the use of the metachromatic dye acridine orange, which can differentially stain these nucleic acids. This method measures total cellular or nuclear RNA content and can be used in flow cytometers that have a single source of illumination. The second method uses a combination of fluorescent dyes, pyronin Y and Hoechst 33342. Only double-stranded RNA fluoresces when stained with pyronin Y. Simultaneous measurement of DNA and RNA utilizing Hoechst 33342 and pyronin Y requires the use of instruments having double excitation sources. Both methods are very sensitive to variations in dye concentration.

Flow Cytometry Measurement of Nuclear RNA Content in Uveal Melanoma

We retrospectively studied the nuclear RNA content in uveal melanomas with known, long-term follow-up. Thirty patients had spindle cell melanomas, 25 had mixed cell tumors, and 9 had epithelioid cell neoplasms. Epithelioid cell types had a higher nuclear RNA content compared to mixed or spindle cell types (p less than 0.05). Using the Cox proportional hazards model, the nuclear RNA content appeared to be an independent prognostic indicator in uveal melanomas, and an increased nuclear RNA content was associated with a worse prognosis (p = 0.001).

Cultured human keratinocytes: discrimination of different cell cycle compartments based upon measurement of nuclear RNA or total cellular RNA content

Correlated measurements of total cellular RNA and DNA of cultured human keratinocytes by flow cytometry, followed by multivariate analysis, discriminate three distinct subpopulations of cells differing in RNA content. The first subpopulation is comprised of small cells resembling basal cells of epidermis, with low RNA content and long (100-300 h) generation times. The second subpopulation consists of keratinocytes resembling cells in the spinous layer of epidermis, characterized by increased RNA content and shorter (35-40 h) generation times. The third subpopulation consists of the largest, keratinohyalin-containing cells which remain in G1 and undergo terminal differentiation. In contrast to total cellular RNA, correlated measurements of DNA and nuclear RNA reveal that: (1) entrance of all cultured cells from G1 into S phase occurs only after accumulation of the same, threshold amount of nuclear RNA; hence there is only a single population of S + G2 + M-phase cells; (2) there are two distinct subpopulations in G1, one with minimal nuclear RNA content and another with increased RNA. Stathmokinetic experiments indicate that the G1-phase cells with low nuclear RNA have distinctly longer residence times in G1 compared to cells with high nuclear RNA content. Thus, measurements of the total cellular RNA versus nuclear RNA content reveal kinetically distinct cell subpopulations. Whereas total cellular RNA content correlates more with differentiation, nuclear RNA content reflects primarily the kinetic properties of the cell.

Cytometric analysis of growth-regulator-dependent transcription and cell-cycle progression in Petunia protoplast cultures

Acridine orange simultaneously stains DNA and RNA. Using flow cytometry, synthesis of these nucleic acids can be related throughout a culture time-course. This technique has been used with nuclei isolated from Petunia hybrida protoplasts during 48 h of culture. Nuclear RNA content has been evaluated with respect to DNA levels, namely the cell-cycle phase.Nuclear RNA synthesis was not dependent upon exogeneous hormones during the first 18 h of culture, but either auxin (2,4-dichlorophenoxyacetic acid, 2,4-D) or cytokinin (N(6)-benzyladenine) were necessary for entry into the S phase. Cytokinin alone could stimulate maximal RNA synthesis within each cell-cycle phase up to 24 h. In complete medium, DNA synthesis only began from a phase "G1B" having substantial RNA, although a subnormal amount of RNA (in protoplasts cultivated only with 2,4-D) did not prevent protoplast entry into the S phase. However, both hormones were necessary for highest RNA levels and G2 frequencies after 48 h. As in mammalian cells, the mean RNA level in plant 4C nuclei is double that of 2C nuclei. G2 nuclei are larger than G1 nuclei, but upon activation G1 nuclei in fact diminsh in size.This study aimed to identify restriction points in the cell cycle as affected by growth regulators and the specific synthesis of nucleic acids. For example, the RNA levels induced by N(6)-benzyladenine, although similar to those in complete medium, were not sufficient to induce mitosis. Conversely, 2,4-D action was probably limited by low nucleotide synthesis in the absence of cytokinin.

Spliceosome assembly involves the binding and release of U4 small nuclear ribonucleoprotein.

Splicing complexes that form a rabbit beta-globin precursor mRNA (pre-mRNA) have been analyzed for their small nuclear RNA (snRNA) content by both affinity chromatography and specific probe hybridization of replicas of native electrophoretic gels. A pathway of spliceosome assembly was deduced that has at least three stages. (i) U2 small nuclear ribonucleoprotein (snRNP) alone binds to sequences of mRNA upstream of the 3' splice site. (ii) U4, U5, and U6 snRNPs bind, apparently simultaneously. (iii) U4 snRNP is released to generate a spliceosome that contains U2, U5, and U6 snRNPs together with the RNA intermediates in splicing. U1 snRNP was not detected in association with any of these complexes. A parallel analysis of the spliceosome found with an adenovirus precursor mRNA substrate yielded an identical snRNP composition with one additional, unidentified RNA species, called X. This latter RNA species was not detected in the spliceosome bound to the beta-globin substrate.

Does the interchromatin compartment contain actin?

Transient blockage of transcription in chick liver by alpha-amanitin results in a temporary clearing of nucleoplasmic regions in affected nuclei and concurrent exposure in situ of a discrete interchromatin framework. This is accompanied by sharp decreases in total nuclear RNA (~28%) and nonhistone protein content (~18%). Electrophoretic analysis of matrix fraction proteins from such highly denuded nuclei shows a marked relative enrichment in actin and RNP-derived polypeptides. These observations suggest that actin is a real constituent of the interchromatin region and supports various reports that this protein may have a basic structural role in nuclei.

Cell and nuclear area, RNA and protein content, and chromosome counts in meristematic cells of secondary roots and callus of Cocos nucifera

The relationships between cell area, nuclear area, and cellular RNA and protein content were determined for mitotic cells in callus lines D-80, D-81, H-81, and H-83, for which ploidy levels were determined, and compared with the corresponding relationships for cells in the secondary root meristem of dwarf, hybrid, and tall varieties of Cocos nucifera L. Mitotic cells in hypotetraploid line D-80 and diploid H-83 showed increases in cell size and cellular RNA and protein content compared with mitotic cells from dwarf and hybrid root tips. However, there was a disproportionate increase in cell size in lines D-80 and H-83. Probit analysis revealed that cell size and protein and RNA content per cell were less tightly coupled in these callus lines. Apart from line D-80, nuclear area for prophase cells in the other three diploid callus lines was very similar to that in cells of the variety from which each was established. Overall, our results suggest that nuclear and cellular parameters are free to change independently of each other when cells are transferred to culture media. Furthermore, the failure to increase RNA and protein contents by amounts equivalent to changes in cell size may have detrimental effects on the cell cycle in callus cells.

口腔へん平上皮癌における核ＤＮＡ‐ＲＮＡ量の解析とその臨床的意義に関する研究

In this study, the author has analyzed cell population kinetics in oral squamous cell carcinoma from the distribution patterns of nuclear DNA and RNA contents in the lesion and pursued the relationship of the result with the clinicopathological findings and the effects of radiation and chemotherapeutic agents. The number of subject cases was 48 and the determination of nuclear DNA and RNA contents was made using DNA-RNA cytofluorometry with acridine orange fluorescence stain. The auther found the following:1) DNA-RNA scattergrams (cytograms) made for the initial medical examination were divided into 4 types: type I had many cells with low RNA contents (that is, Go phase); type II had many cells going into cell cycle and had some cell populations belonging to different cell cycles; type i increased RNA content along with an increase in DNA content. The presence of a single cell population was also estimated; type Ilf, cell groups with abnormally high RNA contents in the 2C DNA range were seen in addition to the presence of the distribution seen and described in type III.2) The clinical effect of external irradiation or the administration of Peplomycin was observed mainly in type III followed by type N. The cases with poor effect were seen often in type I. The cases in type II showed variance in the clinical effect.3) In the cytograms made during the treatment, type I showed a relative abundance of cells at the G0 phase even after the treatment. Type III and IV showed an early decrease in the cells at the G0 phase and an increase in the cells with high contents of both DNA and RNA, that is, those at the G2M phase or abortive mitotic cells. On the other hand, the cases with high clinical effects among type II underwent changes similar to those of type III and IV, and the cases with low effects tended to show no change in the cytogram even after the treatment or accumulation of the cells at the G0 phase.Thus, it was found that analysis of nuclear DNA and RNA contents in oral squamous cell carcinoma could clarify presence of the cells at the G0 phase and some cell populations belonging to other cell cycles. In addition, analysis made it possible to determine cell population kinetics and was useful as an objective index for clinical sensitivity and evaluation.

Alzheimer's disease: a correlative study.

In a study of 17 patients with histologically proven Alzheimer's disease the relationship between psychological, pathological and chemical measures of disorder was examined. Severity of dementia, determined by mental test performance, correlated highly with pathological change in large cortical neurons (cell loss and reduction in nuclear and nucleolar volume and cytoplasmic RNA content), to a lesser extent with cortical senile plaque and neurofibrillary tangle frequency and reduction in acetylcholine (ACh) synthesis, and not with reduction in choline acetyltransferase (CAT) activity. A strongly significant relationship was demonstrated between cell loss and reductions in nuclear and nucleolar volume and cytoplasmic RNA content. Reduction in CAT activity and senile plaque frequency were significantly correlated, thereby linking changes in the sub-cortical projection system of the nucleus basalis with the cortical pathology. The pattern of correlations suggests that the dementia of Alzheimer's disease is largely a reflection of the state of large cortical neurons, and it is argued that abnormalities in the latter may not be directly related to primary loss of cholinergic neurons in the subcortex.

Procedures for two-dimensional electrophoretic analysis of nuclear proteins

A series of mehtods designed for electrophoresis of nuclear proteins is described. They deal with the low solubility of many nuclear proteins and with the presence of large amounts of nucleic acids. These were eliminated by enzymatic digestion, centrifugation, partition or precipitation. A combination of RNAse digestion and centrifugation is the method of choice when proteolysis is low. In the opposite case, precipitationor partition methods are preferred, at the expense of precipitation of some proteins. When the nuclear RNA content is low, centrifugation in an Airfuge is the simplest and the most efficient mehtod, superseding the widely used S1 nuclease method.

Identification of L3 leukemia and Burkitt's lymphoma cells by flow cytometric quantitation of nuclear and cellular RNA and DNA content

Separate quantitations of nucleic acids of isolated nuclei and cells of L3 leukemia/Burkitt's lymphoma cell populations of peripheral blood (PB), bone marrow (BM) and lymph node (LN) cell suspensions of 17 patients were performed by acridine orange (AO) flow cytometry (FCM). The cell populations were analysed with respect to cell cycle characteristics and DNA/RNA distribution histograms of cells in various compartments of the cell cycle using mean value, coefficient of variation (CV), third moment about the mean as a measure of skewness (MOM3). The correlations between numbers of L3 blasts detected by microscopy and aneuploid cells quantified by FCM in 10 patients with aneuploid disease were r = 0.75 and r = 0.85 for BM and PB cell populations, respectively. Content of both nuclear and cellular RNA was 3–4 times higher in L3 cells than in normal donor lymphocytes. Percentages of cells in SG 2M phase of L3 populations were significantly different from control populations in BM ( p < 0.01) and PB ( p < 0.0001). All samples with L3 blasts had abnormal CV of the DNA, and abnormal CV and MOM3 of the RNA peaks of G 0/1 cell histograms. All samples with no blasts by morphology had normal DNA and RNA patterns. AO FCM correctly classified all specimens as involved or not involved with disease in accordance with the cytomorphological diagnoses. Combining AO FCM with prior cell separation increased the sensitivity of the method to detect abnormal cells to 0.02%, or 0.5 L3 cells per μl of blood.

Cell cycle compartments of adult mouse hepatocytes identified by flow cytometric analysis of total cellular and nuclear RNA content: Effect of aging on G1 substates

A model of the hepatocyte cell cycle was computer-constructed using two-parameter cytochemical data obtained by flow cytometric measurements of the RNA and DNA content of individual liver cells (or isolated liver cell nuclei). In young mice (under 10 weeks post-partum), the diploid (2C DNA) G1 phase was resolved into 3 distinct subpopulations of liver cells distinguishable on the basis of RNA content. These substates were conventionally designated G1Q (quiescent non-cycling cells; lowest RNA content), G1A (cells of the indeterminate phase; do not directly enter the DNA synthetic compartment; intermediate RNA content) and G1B (immediate pre-DNA synthetic cells; highest G1 RNA content). An early G1-associated event during postnatal aging of the mouse liver (pre-10 weeks post-partum) involved a shift from a predominant G1A/B substate to a predominant G1Q substate. Later stages in aging were characterized by a gradual decline in the percentage of G1A/B cells in the 2C G1 compartment. Greater than 80% of the total 2C G1 liver cell population of animals approaching 2 years of age consisted of GIQ hepatocytes. This increase in the quiescent cell population (G1Q) and decrease in the cycling G1 substates (G1A/B) within the mouse liver as a function of age may be involved in the aging-associated diminution of the hepatic compensatory regenerative response in mammals.

Changes in DNA and in nuclear and nucleolar dry mass and area in senescing parenchyma cells of corn cob and stalk tissues

Corn ( Zea mays L.) cob and stalk parenchyma tissues were used for a study of senescence using quantitative interference microscopy and quantitative Feulgen cytophotometry. In both parenchyma tissues, nuclear dry mass (NDM), nucleolar dry mass (NoDM), nuclear area (NA), and nucleolar area (NoA) increased during cell elongation and then decreased prior to cell death. These changes were more distinct in corn cob parenchyma tissue than in stalk parenchyma tissue. DNA content (determined for cob parenchyma, center section, only) decreased over the entire study period, slowly at first, them more rapidly. Because NoDM and NoA trends in cob parenchyma tissue were similar to those of NDM and NA but with earlier decreases, we conclude that nucleolar degeneration is an early event in corn parenchyma cell senescence. Loss of DNA also occurs earlier than loss of NDM in cob parenchyma tissue. It is further suggested that the increases in NDM as DNA content decreased represents nuclear protein and RNA content increases prior to senescence. However, no indication of nucleoli degeneration was found in stalk parenchyma cells even though NDM and NA decreased prior to cell death.