scholarly journals β-Tubulin mRNA as a Marker ofCryptosporidium parvum Oocyst Viability

1999 ◽  
Vol 65 (4) ◽  
pp. 1584-1588 ◽  
Author(s):  
Giovanni Widmer ◽  
Elizabeth A. Orbacz ◽  
Saul Tzipori
Keyword(s):  
Pcr Analysis ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Prolonged Incubation ◽  
Mrna Species ◽  
Viability Analysis ◽  
Viability Assay ◽  
Pcr Products ◽  
Tubulin Mrna ◽  
Β Tubulin

ABSTRACT Determining the viability of waterborne Cryptosporidium parvum oocysts remains a technical challenge. rRNA and mRNA were evaluated in a reverse transcription (RT)-PCR assay as potential markers of oocyst viability. The rationale for this approach is the rapid turnover and postmortem decay of cellular RNA. The β-tubulin mRNA and an anonymous mRNA transcript were chosen as potential markers because they are the only mRNA species in C. parvum known to possess introns. This feature facilitated the distinction between genuine RT-PCR products and PCR products originating from copurifying DNA. Prolonged incubation at room temperature of initially viable oocysts resulted in a gradual decrease in mRNA levels, which correlated with the loss of oocyst infectivity to neonatal mice. In contrast, oocysts stored at 4°C for over 39 weeks maintained their infectivity and displayed no decrease in the level of β-tubulin RT-PCR product. The postmortem decay of two mRNA species demonstrates that RT-PCR analysis can provide information on the viability of C. parvum oocysts. The methodological similarity between PCR detection and RT-PCR viability analysis could facilitate the development of a combined detection and viability assay.

scholarly journals Phenotypical and Functional Characteristics of Human Regulatory T Cells during Ex Vivo Maturation from CD4+ T Lymphocytes

2021 ◽  
Vol 11 (13) ◽  
pp. 5776
Author(s):  
Varvara G. Blinova ◽  
Natalia S. Novachly ◽  
Sofya N. Gippius ◽  
Abdullah Hilal ◽  
Yulia A. Gladilina ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Ex Vivo ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Inflammatory Reactions ◽  
Effector Cells ◽  
Cycle Regulation ◽  
Further Development

Regulatory T cells (Tregs) participate in the negative regulation of inflammatory reactions by suppressing effector cells. In a number of autoimmune disorders, the suppressive function and/or the number of Tregs is compromised. The lack of active functioning Tregs can be restored with adoptive transfer of expanded ex vivo autologous Tregs. In our study, we traced the differentiation and maturation of Tregs CD4+CD25+FoxP3+CD127low over 7 days of cultivation from initial CD4+ T cells under ex vivo conditions. The resulting ex vivo expanded cell population (eTregs) demonstrated the immune profile of Tregs with an increased capacity to suppress the proliferation of target effector cells. The expression of the FoxP3 gene was upregulated within the time of expansion and was associated with gradual demethylation in the promotor region of the T cell-specific demethylation region. Real-time RT-PCR analysis revealed changes in the expression profile of genes involved in cell cycle regulation. In addition to FOXP3, the cells displayed elevated mRNA levels of Ikaros zinc finger transcription factors and the main telomerase catalytic subunit hTERT. Alternative splicing of FoxP3, hTERT and IKZF family members was demonstrated to be involved in eTreg maturation. Our data indicate that expanded ex vivo eTregs develop a Treg-specific phenotype and functional suppressive activity. We suggest that eTregs are not just expanded but transformed cells with enhanced capacities of immune suppression. Our findings may influence further development of cell immunosuppressive therapy based on regulatory T cells.

Regulation of tubulin and actin mRNA production in rat brain: expression of a new beta-tubulin mRNA with development

1983 ◽  
Vol 3 (8) ◽  
pp. 1333-1342
Author(s):  
J F Bond ◽  
S R Farmer
Keyword(s):  
Rat Brain ◽  
Morphological Changes ◽  
In Vitro Translation ◽  
Brain Maturation ◽  
Cdna Clones ◽  
Mrna Levels ◽  
Beta Tubulin ◽  
Mrna Species ◽  
Tubulin Mrna ◽  
Actin Mrna

The expression of alpha-tubulin, beta-tubulin, and actin mRNA during rat brain development has been examined by using specific cDNA clones and in vitro translation techniques. During brain maturation (0 to 80 days postnatal), these mRNA species undergo a significant decrease in abundance. The kinetics of this decrease varies between the cerebrum and the cerebellum. These mRNAs are most abundant in both tissues during week 1 postnatal, each representing 10 to 15% of total mRNA activity. Both alpha- and beta-tubulin mRNA content decreases by 90 to 95% in the cerebrum after day 11 postnatal, and 70 to 80% decreases in the cerebellum after day 16. Actin sequences also decrease but to a lesser extent in both tissues (i.e., 50%). These decreases coincide with the major developmental morphological changes (i.e., neurite extension) occurring during this postnatal period. These studies have also identified the appearance of a new 2.5-kilobase beta-tubulin mRNA species, which is more predominant in the cerebellar cytoplasm. The appearance of this form occurs at a time when the major 1.8-kilobase beta-tubulin mRNA levels are declining. The possibility that the tubulin multigene family is phenotypically expressed and then this expression responds to the morphological state of the nerve cells is discussed.

RT-PCR analysis of Na+/H+ exchanger mRNAs in rat medullary thick ascending limb

1995 ◽  
Vol 268 (6) ◽  
pp. F1224-F1228 ◽  
Author(s):  
P. Borensztein ◽  
M. Froissart ◽  
K. Laghmani ◽  
M. Bichara ◽  
M. Paillard
Keyword(s):  
Rat Kidney ◽  
Pcr Amplification ◽  
Restriction Enzymes ◽  
Pcr Analysis ◽  
Thick Ascending Limb ◽  
Rt Pcr ◽  
Medullary Thick Ascending Limb ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Nhe Isoforms

The thick ascending limb (TAL) of rat kidney absorbs bicarbonate secondary to proton secretion, but displays both basolateral and luminal Na+/H+ exchange (NHE) activity. Several NHE genes, including NHE-1, NHE-2, NHE-3, and NHE-4, are expressed in the kidney. To identify the NHE isoforms expressed in the rat medullary TAL (MTAL), we used the reverse transcription-polymerase chain reaction (RT-PCR) to detect the mRNAs for NHE in microdissected MTAL. RT-PCR amplification from total RNA was performed between two specific primers for each NHE isoform. In rat kidney homogenate, the four NHE isoform mRNAs were detected, and the identity of the PCR products was demonstrated by the sizes of the fragments, digestion with restriction enzymes, and Southern blot analysis. In microdissected rat MTAL, NHE-3 was strongly expressed and NHE-1 mRNA was also detected, whereas NHE-2 and NHE-4 mRNAs were not detected. Therefore, NHE-3 could be the apical Na+/H+ exchanger, and NHE-1 could be the basolateral isoform in the MTAL.

scholarly journals Expression and homologous regulation of GH secretagogue receptor mRNA in rat adrenal gland

2002 ◽  
pp. 677-688 ◽  
Author(s):  
ML Barreiro ◽  
L Pinilla ◽  
E Aguilar ◽  
M Tena-Sempere
Keyword(s):  
Biological Effects ◽  
Biologically Active ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
R Gene ◽  
Rt Pcr ◽  
Short Term ◽  
Opposite Pattern ◽  
Corticosterone Secretion

OBJECTIVE: GH secretagogues (GHSs) elicit a variety of biological effects in several endocrine and non-endocrine target tIssues, including activation of the hypothalamic-pituitary-adrenal axis. The latter is mainly carried out through a central hypothalamic action; yet the possibility of additional effects directly at the adrenal level cannot be ruled out. The aims of this study were to evaluate the expression and homologous regulation of the GHS-receptor (GHS-R) gene in rat adrenal and to assess the effects of synthetic (GH releasing peptide-6 - GHRP-6) and natural (ghrelin) ligands of GHS-R upon basal and ACTH-stimulated corticosterone secretion in vitro. DESIGN AND METHODS: Analysis of adrenal expression of target mRNAs (GHS-R, GHS-R1a, ghrelin, and several steroidogenic factors) was conducted by means of primer-specific, semi-quantitative RT-PCR. Evaluation of corticosterone secretion by incubated adrenal tIssue was carried out by specific RIA. RESULTS: RT-PCR analysis demonstrated expression of the GHS-R gene, but not of the gene encoding the cognate ligand ghrelin, in rat adrenal. Moreover, expression of the mRNA coding for the type 1a GHS-R (GHS-R1a), i.e. the biologically active receptor form, was demonstrated. The adrenal expression of the GHS-R message appeared under the regulation of homologous signals in vitro, as short-term incubation of adrenal samples in serum-free medium induced a significant increase in GHS-R mRNA levels that was inhibited by exposure to different doses of GHRP-6 (10(-9)-10(-5) mol/l) or ghrelin (10(-7) mol/l). Notably, an opposite pattern of homologous regulation of GHS-R gene expression was observed at the pituitary. Finally, short-term stimulation with increasing concentrations of GHRP-6 (10(-9)-10(-5) mol/l) or ghrelin (10(-7) mol/l) failed to alter basal and ACTH-stimulated corticosterone secretion in vitro, neither did it modify ACTH-stimulated mRNA expression levels of several upstream elements in the steroidogenic route: the steroidogenic acute regulatory (StAR) protein, and the enzymes P450 cholesterol side-chain cleavage (P450scc) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD). CONCLUSIONS: Our study provides novel evidence for the expression and homologous regulation of the GHS-R gene in rat adrenal. However, our results cast doubts on the possibility of direct adrenal actions of ligands of the GHS-R in the regulation of corticosterone secretion in the rat.

scholarly journals Asma ve Narlardan İzole Edilen Grapevine leafroll-associated virus-1 İzolatlarının Kısmi Sekanslarının Karşılaştırmalı Genomik Analizleri

2017 ◽  
Vol 5 (10) ◽  
pp. 1136
Author(s):  
Eminur Elçi ◽  
Mona Gazel ◽  
Kadriye Çağlayan
Keyword(s):  
Movement Protein ◽  
Economic Importance ◽  
Pcr Analysis ◽  
Yield Losses ◽  
Grapevine Virus ◽  
Rt Pcr ◽  
Virus Diseases ◽  
New Host ◽  
Pcr Products ◽  
Leafroll Disease

Grapevine leafroll disease is one of the worldwide diseases with economic importance among grapevine virus diseases since many years. Grapevine leafroll-associated virus-1 (GLRaV-1) is the first identified virus related to leafroll viruses which are belonged to Closterovirus and Ampelovirus. Leafroll symptoms are typical for this virus and it causes yield losses on grapevines. Pomegranates are also economically important trees and up to now; only a few viral agents were identified in this plant species and in the last years, it was reported that pomegranate could be as a new host of GLRaV-1. Aim of this study was to compare GLRaV-1 isolates from grapevine and pomegranates. For this purpose, dsRNA and total RNA isolations were done and RT-PCR analysis were conducted by using primers of movement protein (p24) and heatshock70 protein (HSP70h) genes of GLRaV-1 and PCR products were cloned and sequenced in the collected samples from Hatay and Niğde in 2014. -PCR analysis was done by using degenerated primer heatshock 70 homolog protein of Closterovirus. Blast and phylogenetic analysis were performed with the obtained partial nucleotid sequences. At the end of this study, it was ensured that GLRaV-1 isolates isolated from the pomegranate, which is thought to be a new host, were analysed comparatively with the grapevine isolates and high similarities were detected between isolates.

scholarly journals The der(11)-encoded MLL/AF-4 fusion transcript is consistently detected in t(4;11)(q21;q23)-containing acute lymphoblastic leukemia

Blood ◽  
1994 ◽  
Vol 83 (2) ◽  
pp. 330-335 ◽  
Author(s):  
JR Downing ◽  
DR Head ◽  
SC Raimondi ◽  
AJ Carroll ◽  
AM Curcio-Brint ◽  
...  
Keyword(s):  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cell ◽  
Fusion Transcript ◽  
Pcr Analysis ◽  
Chromosome 11 ◽  
Rt Pcr ◽  
Pcr Products ◽  
Contrast Analysis ◽  
Leukemic Clone

The t(4;11)(q21;q23) is the most common translocation involving band 11q23 and is found predominantly in acute lymphoblastic leukemias (ALLs) of infants. Recent studies have shown that this translocation involves the MLL gene on chromosome 11 and the AF-4 gene on chromosome 4. Using oligonucleotide primers derived from these genes, we established reverse transcription-polymerase chain reaction (RT-PCR) assays for the detection of the fusion transcripts from both the der(11) and der(4) chromosomes. Using these assays we analyzed 23 pediatric cases of t(4;11) containing ALL. RT-PCR analysis for the der(11)-derived MLL/AF-4 fusion transcript resulted in its detection in every case at a sensitivity of greater than 1 leukemic cell in 10(5) cells. Sequence analysis of MLL/AF-4 PCR products demonstrated fusion mRNAs resulting from breaks in MLL introns 6, 7, or 8, with alternative splicing to one of three exons in the AF-4 gene. In contrast, analysis for the der(4)-derived transcript resulted in the detection of this chimeric mRNA in only 84% of the cases analyzed. These data suggest that the critical chimeric gene product involved in the establishment of the leukemic clone is derived from the der(11) chromosome. Moreover, these data demonstrate the utility of the RT-PCR assay for the der(11)- encoded message both for diagnosing t(4;11)-containing leukemia and for monitoring patients for minimal residual disease.

scholarly journals The der(11)-encoded MLL/AF-4 fusion transcript is consistently detected in t(4;11)(q21;q23)-containing acute lymphoblastic leukemia

Blood ◽  
1994 ◽  
Vol 83 (2) ◽  
pp. 330-335 ◽  
Author(s):  
JR Downing ◽  
DR Head ◽  
SC Raimondi ◽  
AJ Carroll ◽  
AM Curcio-Brint ◽  
...  
Keyword(s):  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cell ◽  
Fusion Transcript ◽  
Pcr Analysis ◽  
Chromosome 11 ◽  
Rt Pcr ◽  
Pcr Products ◽  
Contrast Analysis ◽  
Leukemic Clone

Abstract The t(4;11)(q21;q23) is the most common translocation involving band 11q23 and is found predominantly in acute lymphoblastic leukemias (ALLs) of infants. Recent studies have shown that this translocation involves the MLL gene on chromosome 11 and the AF-4 gene on chromosome 4. Using oligonucleotide primers derived from these genes, we established reverse transcription-polymerase chain reaction (RT-PCR) assays for the detection of the fusion transcripts from both the der(11) and der(4) chromosomes. Using these assays we analyzed 23 pediatric cases of t(4;11) containing ALL. RT-PCR analysis for the der(11)-derived MLL/AF-4 fusion transcript resulted in its detection in every case at a sensitivity of greater than 1 leukemic cell in 10(5) cells. Sequence analysis of MLL/AF-4 PCR products demonstrated fusion mRNAs resulting from breaks in MLL introns 6, 7, or 8, with alternative splicing to one of three exons in the AF-4 gene. In contrast, analysis for the der(4)-derived transcript resulted in the detection of this chimeric mRNA in only 84% of the cases analyzed. These data suggest that the critical chimeric gene product involved in the establishment of the leukemic clone is derived from the der(11) chromosome. Moreover, these data demonstrate the utility of the RT-PCR assay for the der(11)- encoded message both for diagnosing t(4;11)-containing leukemia and for monitoring patients for minimal residual disease.

scholarly journals The fluorene catabolic linear plasmid in Terrabacter sp. strain DBF63 carries the β-ketoadipate pathway genes, pcaRHGBDCFIJ, also found in proteobacteria

Microbiology ◽  
2005 ◽  
Vol 151 (11) ◽  
pp. 3713-3722 ◽  
Author(s):  
Hiroshi Habe ◽  
Jin-Sung Chung ◽  
Ayako Ishida ◽  
Kano Kasuga ◽  
Kazuki Ide ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Tricarboxylic Acid Cycle ◽  
Bacterial Species ◽  
Linear Plasmid ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Gene Encoding ◽  
Positive Trait ◽  
Tricarboxylic Acid Cycle Intermediates

Terrabacter sp. strain DBF63 is capable of degrading fluorene (FN) to tricarboxylic acid cycle intermediates via phthalate and protocatechuate. Genes were identified for the protocatechuate branch of the β-ketoadipate pathway (pcaR, pcaHGBDCFIJ) by sequence analysis of a 70 kb DNA region of the FN-catabolic linear plasmid pDBF1. RT-PCR analysis of RNA from DBF63 cells grown with FN, dibenzofuran, and protocatechuate indicated that the pcaHGBDCFIJ operon was expressed during both FN and protocatechuate degradation in strain DBF63. The gene encoding β-ketoadipate enol-lactone hydrolase (pcaD) was not fused to the next gene, which encodes γ-carboxymuconolactone decarboxylase (pcaC), in strain DBF63, even though the presence of the pcaL gene (the fusion of pcaD and pcaC) within a pca gene cluster has been thought to be a Gram-positive trait. Quantitative RT-PCR analysis revealed that pcaD mRNA levels increased sharply in response to protocatechuate, and a biotransformation experiment with cis,cis-muconate using Escherichia coli carrying both catBC and pcaD indicated that PcaD exhibited β-ketoadipate enol-lactone hydrolase activity. The location of the pca gene cluster on the linear plasmid, and the insertion sequences around the pca gene cluster suggest that the ecologically important β-ketoadipate pathway genes, usually located chromosomally, may be spread widely among bacterial species via horizontal transfer or transposition events.

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