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PubMed2025

Concurrent stimulation of diflufenican biodegradation and changes in the active microbiome in gravel revealed by Total RNA.

The use of slowly degraded pesticides poses a particular problem when these are applied to urban areas such as gravel paths. The urban gravel provides an environment very different from agricultural soils; i.e., it is both lower in carbon and microbial activity. We, therefore, endeavored to stimulate the degradation of the pesticide diflufenican added to urban gravel microcosms amended with dry alfalfa to increase microbial activity. In the present study, alfalfa addition significantly increased the formation of diflufenican's primary metabolite, 2-[3-(trifluoromethyl)phenoxy]nicotinic acid (AE-B), indicating stimulated biotransformation. The concurrent changes of the active microbial communities within the gravel were explored using shotgun metatranscriptomic sequencing of ribosomal RNA and messenger RNA. Although bacterial taxa remained dominant (87.0%-98.5% relative abundance), the alfalfa treatment led to a 4-5-fold increase in eukaryotic groups, including fungi and microbial grazers. Several microbial taxa potentially involved in the degradation of complex carbon compounds and aromatic pollutants-including Bacteroidetes, Verrucomicrobia, Sordariomycetes, Mortierellales, Tremellales, Sphingopyxis, and Phenylobacterium-increased in relative abundance following alfalfa amendment. Functional gene profiling revealed elevated expression of genes related to microbial activity and biomass production. Genes with potential roles in the breakdown of complex carbon structures (e.g., xylanases/chitin deacetylases) and in the transformation of aromatic compounds (e.g., ring-cleaving dioxygenases) were revealed. We conclude that complex carbon amendments can enhance the microbial activity, promoting the biotransformation of diflufenican in urban gravel environments. These findings provide new insights into the interactions between microbial community dynamics, gene expression profiles, and pesticide biotransformation in non-agricultural matrices.IMPORTANCEPesticides used on urban areas, e.g., gravel paths, are likely to have different effects and fates than when these are used on agricultural soils. Hence, studies into the degradation of pesticides applied to urban matrices are needed. We have previously shown that metabolites of the persistent pesticide diflufenican are even more persistent in urban soils, and it has also previously been shown that these metabolites leach from gravel surfaces. The reasons behind this are that the urban gravel provides an environment very different from agricultural soils; i.e., it is both lower in carbon and microbial activity. In the present study, we, therefore, endeavored to stimulate the degradation of the pesticide diflufenican added to urban gravel microcosms amended with dry alfalfa to increase microbial activity, concurrently studying the changes in the active microbiome by Total RNA-metatranscriptomics.

Biodegradation, Environmental↗
PubMed1983

Translation in vitro and regulation of mouse liver S-adenosylmethionine synthetase messenger RNA.

Total RNA was isolated from adult mouse liver tissues. The alpha- and beta-form isozymes of S-adenosylmethionine synthetase existing in liver were synthesized in a reticulocyte lysate cell-free system under the direction of total RNA and were immunoprecipitated with antibody to the beta-form. The newly synthesized and the in vivo labeled S-adenosylmethionine synthetase subunits were compared by SDS-polyacrylamide gel electrophoresis. Both the alpha- and beta-forms consist of the same size Mr 48 000 subunit. The level of the beta-form mRNA activity in mouse liver was shown to increase following intraperitoneal transplantation of Ehrlich ascites tumor cells and the changes in the mRNA activity parallel those in the cellular level of S-adenosylmethionine synthetase beta.

Animals↗
PubMed1992

Selective amplification of cDNA sequence from total RNA by cassette-ligation mediated polymerase chain reaction (PCR): application to sequencing 6.5 kb genome segment of hantavirus strain B-1.

A method, referred to as cassette-ligation mediated polymerase chain reaction (PCR), has been developed to permit selective and specific amplification of cDNA sequence from total cellular RNA. This technique comprises (i) digestion of cDNA with multiple restriction enzymes, (ii) ligation of cleavage products to double-stranded DNA cassettes possessing a corresponding restriction site and (iii) amplification of cassette-ligated restriction fragments containing a short, known sequence (but not all the other ligation products) by PCR using the specific and cassette primers; the specific primer is designed to prime synthesis from the known sequence of the cDNA whereas the cassette primer anneals to one strand of the cassette. Sequencing from the cassette primer provides information to design a new primer for the next walking step. The amplified cDNA fragments are often larger than the maximum DNA fragments (500-600 bp) that can be sequenced without the need of synthesizing internal sequencing primer. Each of such large cDNA fragments is dissected into smaller DNA fragments by repeating cassette-ligation mediated PCR exploiting different restriction sites and different sets of cassette primers. This dissection process reduces the number of specific primers to a minimum, thereby increasing the speed of sequencing and minimizing the overall cost. We have successfully applied this cDNA walking and sequencing by the cassette-ligation mediated PCR to the sequencing of an entire 6.5 kb genome segment of hantavirus strain B-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1977

[Transforming action of the total RNA preparations isolated from Rous virus-induced sarcomas].

The authors compared the effect of RNA substances isolated from chick sarcomas producing Rous virus and hamster sarcomas not producing the virus. In both species of animals the tumors were induced by the same Rous virus strain (Carr-Zilber). The treatment of embryonal cells cultures both of the chick and hamster with RNA preparations resulted in the morphological transformation. The action of RNA-s would reduce considerably or eliminated totally the effect, while DNA-s failed to influence the activity of the preparations under study. Embryonal cells culture in hamsters proved to be more sensitive to RNA action than chick cells.

Animals↗
PubMed1984

[Characterization of total RNA, messenger poly(A)+ RNA and homologous complementary DNA in experimental cardiac hypertrophy].

Two biological modifications are observed during adaptation of cardiac tissue to work overload: an increase in total protein synthesis and a redistribution of myosin isoenzymes. These modifications suggest that changes in DNA transcription are involved in the early response of cardiac tissue to overload. Results are reported in this paper that show a parallel increase in total and polyadenylated RNA content and concentration in cardiac overloaded tissue in the rat. The characterization of cardiac poly (A)+ mRNA by mRNA X cDNA hybridization and the identification of specific mRNAs with recombinant plasmid cDNAs are given in this report as preliminary results.

Animals↗
PubMed2000

Electrolytes, water, RNA, total creatine and calculated resting membrane potential in muscle tissue from pregnant women.

BACKGROUND: The nutritional situation of the fetus and pregnant woman is important for human health, but knowledge of how nutrition affects maternal metabolism and physiology during pregnancy is limited. Such knowledge is important, for example in body composition studies, when information about lean tissue composition is needed. Muscle, a main part of lean tissue, changes its composition in response to age and sex, but the effect of pregnancy on this composition is unknown. METHODS: Muscle samples from 11 pregnant and 16 nonpregnant women were analyzed for water, electrolytes, total creatine, alkali-soluble protein (ASP), DNA and RNA. Plasma was analyzed for electrolytes. The amount of extracellular and intracellular water as well as the resting membrane potential (RMP) in muscle were calculated. RESULTS: Pregnant women had lower plasma concentrations of potassium and sodium but higher muscle concentrations of sodium and water (total and extracellular) than nonpregnant women. RMP was more negative in pregnant than in nonpregnant women. Total creatine in muscle (per kilogram ASP) was increased during pregnancy. The muscle content of RNA (per kilogram DNA) was lower in gestational week 18 than in nonpregnant controls. CONCLUSION: Pregnancy influences muscle composition in several ways that are relevant for an increased understanding of interactions between nutrition and reproduction.

Adult↗
PubMed1998

Bacterial transcript imaging by hybridization of total RNA to oligonucleotide arrays.

We have used high-density oligonucleotide probe arrays (chips) for bacterial transcript imaging. We designed a chip containing probes representing 106 Hemophilus influenzae genes and 100 Streptococcus pneumoniae genes. The apparent lack of polyadenylated transcripts excludes enrichment of mRNA by affinity purification and we thus used total, chemically biotinylated RNA as hybridization probe. We show that hybridization of Streptococcus RNA to a chip allows simultaneous quantification of the transcript levels. The sensitivity was found to be in the range of one to five transcripts per cell. The quantitative chip results were in good agreement with conventional Northern blot analysis of selected genes. This technology allows simultaneous and quantitative measurement of the transcriptional activity of entire bacterial genomes on a single oligonucleotide probe array.

Biotinylation↗
PubMed2006

[Expression of total RNA and amplification of myocardial troponin gene during monitoring viral cardiomyocyte injury.].

BACKGROUND: To investigate the value of circulating cTnI-mRNA detection for monitoring myocardial injury development and prognosis. METHODS: Viral myocardial injury models in BALB/c mice were created by intraperitoneal inoculation with Coxsackievirus B3 (CVB3,1x108 TCID50) for inducing myocardial injury. The total RNAs were extracted and cTnI-mRNA in mice cardiac tissues and circulating blood were amplified by RT-PCR during mice myocardial injury. RESULTS: In virus infected mice, the mRNA abundance for cTnI was up-regulated in heart and circulating blood and associated with salient myocardial histopathologic features, including myocardial swelling, inflammatory cell infiltration, pyknosis, karyorrhexis, karyolysis, denaturalization, necrosis, and calcification. The cTnI-mRNA form infected mice heart and circulating cardiac myocytes were analyzed by RT-PCR, the amplified gene fragments were found in all heart tissues. The incidence of cTnI-mRNA was 0, 0, 0, 16.7%, 40.0%, 71.4%, 83.3% and 87.5% in the controls, the 3rd, 6th, 9th, 12th, 15th, 18th,and 21st day in circulating bloods from the infected mice, respectively. CONCLUSION: The present data suggest that cTnI-mRNA expression is up-regulated and released into blood on viral myocardial injury, and detection of circulating cTnI-mRNA is a sensitive genetic marker for monitoring myocardial injury development and prognosis.

Animals↗
PubMed2005

Comparison of a pump-around, a diffusion-driven, and a shear-driven system for the hybridization of mouse lung and testis total RNA on microarrays.

In the present study, we demonstrate the benefits of a shear-driven rotating microchamber system for the enhancement of microarray hybridizations, by comparing the system with two commonly used hybridization techniques: purely diffusion-driven hybridization under coverslip and hybridization using a fully automated hybridization station, in which the sample is pumped in an oscillating manner. Starting from the same amount of DNA for the three different methods, a series of hybridization experiments using mouse lung and testis DNA is presented to demonstrate these benefits. The gain observed using the rotating microchamber is large: both in terms of analysis speed (up to tenfold increase) and in final spot intensity (up to sixfold increase). The gain is due to the combined effect of the hybridization chamber miniaturization (leading to a sample concentration increase if comparing iso-mass conditions) and the transport enhancement originating from the rotational shear-driven flow induced by the rotation of the chamber bottom wall.

Animals↗
PubMed2000

Effective isolation of high-quality total RNA from human adult articular cartilage.

The isolation of large quantities of good-quality RNA from human articular cartilage has been a long-standing problem for researchers working with human articular cartilage. In this paper we report a protocol which we have developed based on the Qiagen RNeasy procedure to produce high yields of purified, DNA-free RNA from normal and osteosteoarthritic human articular cartilage. The average yield of RNA was 8.39 microg/g (n = 59) for normal and 6.69 microg/g (n = 58) for osteoarthritic cartilage (average ratio OD 260/280 = 1.8-1.9). Quantitative PCR, cDNA array technology, and Northern blot analysis were used to verify the quality of the RNA.

Adult↗