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At least 1,675 records · Page 93Linked to original sources
PubMed1997

Thrombospondin-1 and -2 messenger RNA expression in normal, benign, and neoplastic human breast tissues: correlation with prognostic factors, tumor angiogenesis, and fibroblastic desmoplasia.

Thrombospondin-1 (TSP1) is a Mr 450,000 extracellular matrix glycoprotein that modulates tumor growth, angiogenesis, and metastasis. Of the five structurally different TSPs described to date, only TSP2 is similar to TSP1 in terms of its molecular architecture, and TSP2 also modulates angiogenesis. Angiogenesis plays a relevant role in the biological aggressiveness of breast cancer, and TSP1 is present in the tumor stroma (termed desmoplasia) of invasive human breast ductal carcinoma not otherwise specified (NOS). The present study was designed to identify and quantify TSP1 and TSP2 mRNAs in normal, benign, and neoplastic human breast tissues using the reverse transcriptase PCR technique. We found that TSP2, like TSP1, was expressed in human breast tissues, and that TSP1 and TSP2 mRNA expression in invasive breast carcinoma NOS was significantly increased compared to that observed in normal and benign tissues. The expression of TSP1 and TSP2 in invasive breast ductal carcinoma NOS did not significantly correlate with any of the prognostic factors studied (tumor size, lymph node status, morphology, and hormone receptor status). However, when our study population was divided according to the quantity of tumor stroma, TSP1 (and possibly TSP2) mRNA expression and microvessel counts in desmoplastic-rich stroma of breast carcinoma NOS were significantly increased compared to those observed in desmoplastic-poor stromata.

Adult↗
PubMed1998

Comparison of three RT-PCR methods.

Various approaches can now be taken for amplification of RNA transcripts using the polymerase chain reaction (PCR). Here, we compare three such methods: (i) uncoupled reverse transcription (RT)-PCR (using separate reactions for cDNA synthesis and PCR), (ii) continuous RT-PCR (in which RT and DNA amplification occur in an uninterrupted reaction) using either a single enzyme for both RT and DNA amplification or (iii) using two enzymes, one for each task. We have found that the continuous two-enzyme RT-PCR method is the most sensitive, followed by the uncoupled RT-PCR and then the continuous single-enzyme method. The continuous methods require less sample handling than the uncoupled method, and hence are less labor-intensive and less prone to contamination. The continuous single-enzyme method is the most expensive to perform in terms of reagents due to the quantity of DNA polymerase required; however, it does have advantages over the two enzyme methods in that the use of a thermostable enzyme for RT can alleviate certain problems by allowing RT to occur at higher temperatures than those tolerable by viral reverse transcriptases.

DNA-Directed DNA Polymerase↗
PubMed1991

Slowed transcription and rapid messenger RNA turnover contribute to a decline in synthesis of ovine trophoblast protein-1 during in vitro culture.

Ovine trophoblast protein-1 (oTP-1) is produced in massive amounts by conceptuses during the Day-12-20-period of early pregnancy. The rate of production of oTP-1 declines during culture of conceptuses, however, suggesting a role for critical intrauterine factors for continued production. The present study was conducted to define the mechanism responsible for the in vitro decline in oTP-1 synthesis. Over 24 h culture, synthesis of oTP-1 was initially rapid but then declined such that there was little increase in either the protein or its antiviral activity after 12 h. By contrast, the release of total protein into the medium continued at an approximately linear rate for the entire 24-h period of culture. By using in situ hybridization to tissue sections and dot-blot analysis of tissue extracts with labeled cDNA probes, it was shown that the quantity of oTP-1 mRNA fell 3- to 5-fold during culture, whereas the amount of actin mRNA remained relatively constant. To determine whether this selective fall in levels of oTP-1 mRNA resulted from decreased transcription rate and/or high turnover rate of existing mRNA, conceptuses were either provided continuously with [3H]uridine for 24 h or labeled for 9 h and then exposed to medium enriched in unlabeled uridine and cytidine for a further 15 h. Specific incorporation of 3H into oTP-1 mRNA was assessed by hybridization to excess oTP-1 cDNA immobilized on nitrocellulose membranes. In the continuous labeling study, 3H in total RNA increased at an approximately linear rate for at least 18 h, whereas the content of 3H in oTP-1 mRNA peaked at 9 h and declined about 10-fold by 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1978

Molecular and cellular aspects of facultative delayed implantation in the mouse.

Various aspects of RNA, DNA and protein synthesis, as well as cellular fine structure, were examined in mouse embryos during the developmental diapause associated with delayed implantation, and during the reactivation of the embryo either by hormonal administration or by culture in vitro. The findings from these studies demonstrate that a cessation of DNA synthesis and mitosis, and a marked decline in the level of protein synthesis, but not of RNA synthesis, accompany diapause. Reactivation of the blastocyst results in the resumption of DNA synthesis and cell division, as well as in quantitative and qualitative changes in protein synthesis. At the fine-structural level diapause is indicated by the accumulation of lipid-like vacuoles, microfilaments, a basal lamina coating trophectodermal cells and the disassembly of polysomes into ribosomes. Significantly, nucleolar morphology remains unaltered during diapause and cisternae of the rough-surfaced endoplasmic reticulum persist at least for the first five days of delay. Reactivation of diapausing blastocysts is associated with the reassembly of polysomes and the accumulation of large quantities of an amorphous material within the cisternae of the rough-surfaced endoplasmic reticulum. Studies of blastocyst growth and development in vitro suggest experimental approaches to the question of the regulation delayed implantation. Finally, the role of ionic balances and concentrations in the control of the onset, maintenance and termination of delayed implantation in the mouse is discussed.

Animals↗
PubMed2000

IL-11 expression is increased in severe asthma: association with epithelial cells and eosinophils.

BACKGROUND: IL-11 is a pleiotropic cytokine produced by a variety of stromal cells. Targeted overexpression of this cytokine in mice results in a remodeling of the airways and the development of airway hyperresponsiveness and airway obstruction. OBJECTIVES: Because these alterations mimic important pathologic and physiologic changes in the airways of some asthmatic patients, we investigated the expression of IL-11 messenger RNA (mRNA) within the airways of patients with mild to severe asthma and nonasthmatic control subjects. METHODS: Fiberoptic bronchoscopy to obtain bronchial biopsy specimens was performed on patients with mild (n = 13), moderate (n = 10), and severe (n = 9) asthma and on nonasthmatic control subjects (n = 9). RESULTS: These patients differed in their extent of airway fibrosis with types I and III collagens being noted in greater quantities in the biopsy specimens from the severe and moderate asthmatics than in those from controls (P <.05). IL-11 mRNA expression was observed in the epithelial and subepithelial layers of asthmatic and nonasthmatic control subjects. The number of cells within the epithelium and subepithelium expressing IL-11 mRNA was greater in those with moderate and severe asthma compared with mild asthma and nonasthmatic subjects (P <.001). There were also greater numbers of IL-11 mRNA-positive cells within the subepithelium in severe compared with moderate asthma (P <.001). Immunostaining for IL-11 within the airway tissues confirmed translation of the mRNA into IL-11-immunoreactive protein in airway epithelial cells. Colocalization of IL-11 mRNA and immunoreactivity with resident inflammatory cells demonstrated that this cytokine was also expressed by major basic protein-positive eosinophils. CONCLUSION: These results suggest that IL-11 is involved in the chronic remodeling seen in asthmatic airways and is associated with increasing severity of the disease.

Asthma↗
PubMed2006

Nuclear export of influenza A virus mRNAs requires ongoing RNA polymerase II activity.

Influenza A virus transcribes its segmented negative sense RNA genome in the nuclei of infected cells in a process long known to require host RNA polymerase II (RNAP-II). RNA polymerase II synthesizes pre-mRNAs whose 5'-cap structures are scavenged by the viral RNA-dependent RNA polymerase during synthesis of viral mRNAs. Drugs that inhibit RNAP-II therefore block viral replication, but not necessarily solely by denying the viral polymerase a source of cap-donor molecules. We show here that 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB), a compound that prevents processive transcription by RNAP-II, inhibits expression of the viral HA, M1 and NS1 genes at the post-transcriptional level. Abundant quantities of functionally and structurally intact viral mRNAs are made in the presence of DRB but with the exception of NP and NS2 mRNAs, are not efficiently translated. Taking M1 and NP mRNAs as representatives of DRB-sensitive and insensitive mRNAs, respectively, we found that the block to translation operates at the level of nuclear export. Furthermore, removal of DRB reversed this block unless a variety of chemically and mechanistically distinct RNAP-II inhibitors were added instead. We conclude that influenza A virus replication requires RNAP-II activity not just to provide capped mRNA substrates but also to facilitate nuclear export of selected viral mRNAs.

Active Transport, Cell Nucleus↗
PubMed1999

Proviral load and expression of avian leukosis viruses of subgroup C in long-term persistently infected heterologous hosts (ducks).

Proviral DNA load and expression of avian leukosis viruses of subgroup C (ALV-C) in ducks infected in mid embryogenesis were studied using quantitative PCR, RT-PCR, in situ hybridization employing ALV-specific riboprobe, and immunohistochemistry. A group of long-term surviving, non-reviremic ducks was selected for the study and compared to control reviremic animals in order to obtain information about persisting retroviruses in different duck tissues. A widespread distribution of proviruses in the tested tissues was found, but the proviral load was significantly lower in non-reviremic in comparison to reviremic animals. The only exception were brain and blood cells, in which no significant difference in the quantity of integrated proviruses was found between both categories of ducks, thus indicating an exceptional position of the brain and blood cells among all tested tissues. Contrary to reviremic, the proviruses were not transcribed in non-reviremic ducks, with the exception of brain and thymus. In the majority of non-reviremic ducks viral RNA was revealed in the brain, but no infectious virus could be recovered from this tissue. The opposite situation was observed in the thymus, where infectious virus was recovered but viral RNA remained below the detection limit of the assay. As revealed by in situ analysis, infected cells were either disseminated or focally distributed in tissues. From the long-term follow up of ALV-C in intraembryonally infected ducks we conclude that this model is suitable for the study of retrovirus persistence accompained both by the presence and absence of reviremias. The possible consequences of transmission and long-term persistence of retroviruses in the heterologous host for retroviral evolution are discussed.

Animals↗
PubMed1997

Detection of coxsackievirus B3 in intestinal tissue of orally-infected mice by a standardized RT-PCR assay.

BACKGROUND: Previous studies have reported the role of enteroviruses in chronic diseases, using in-house RT-PCR protocols. A well-standardized PCR assay (Amplicor enterovirus, Produits Roche) designed for the diagnosis of enterovirus meningitis in cerebrospinal fluids (CSF) was recently described. OBJECTIVES: To evaluate this commercially-available PCR assay for the detection of enterovirus in intestinal biopsies. STUDY DESIGN: In order to obtain large quantities of infected material, eight mice were inoculated orally with 2 x 10(5) 50% tissue culture infective doses (TCID50) of coxsackievirus B3 (CBV3); two mice were sacrificed every day from day 1 to day 4 post-infection. Stool specimens and small bowel fragments were taken from infected animals and controls. Four protocols of RNA extraction from intestinal tissue were compared. Extracted RNA was then tested by the Amplicor assay and by a seminested in-house PCR. RESULTS: The best results were obtained with a commercial reagent using a combination of guanidium thiocyanate and phenol (TRI Reagent, Sigma). This procedure allowed the detection of enteroviral RNA in intestinal samples of 7/8 and 8/8 infected mice by Amplicor assay and seminested PCR, respectively, whereas only five samples were tested positive by conventional cell culture. When tested on serial dilutions of CBV3 mixed with intestinal tissue, a sensitivity of 0.2 TCID50/mg was achieved with both PCR assays. CONCLUSIONS: The data demonstrate that the Amplicor enterovirus assay, which is designed to avoid false-positive amplifications, can be used, with a slight modification of the RNA extraction step, for the detection of enterovirus in specimens different from CSF such as intestinal tissue.

Animals↗
PubMed2026

Genome-targeted enrichment and sequencing of human-infecting Cryptosporidium spp.

Cryptosporidium spp. are parasites that cause severe illness in vulnerable human populations. Obtaining pure and sufficient Cryptosporidium DNA from clinical and environmental samples is a challenging task. Oocysts shed in available fecal samples can be limited in quantity, require purification (biased towards dominant strains), and yield limited DNA (&#x2009;<&#x2009;40 fg/oocyst). Here, we use updated genomic sequences from a broad diversity of Cryptosporidium species that have been found to infect humans (C. cuniculus, C. hominis, C. meleagridis, C. parvum, C. tyzzeri, and C. viatorum) to develop and validate a set of 100,000 RNA baits (CryptoCap_100k) with the aim of enriching Cryptosporidium DNA from varied samples. Compared to unenriched libraries, CryptoCap_100k increases the percentage of reads mapping to target genome sequences, increases the depth and breadth of genome coverage, and facilitates analyses of genetic variants in many samples, while decreasing overall costs.

Cryptosporidium↗
PubMed1980

Protein synthesis in cell-free extracts of Coxiella burnetti.

Some aspects of protein biosynthesis were investigated in extracts of the obligate intracellular bacterium Coxiella burnetti. Sucrose gradient analysis revealed small quantities of 30S and 50S ribosomal subunits, few 70S ribosomes and no polysomes. Functional endogenous mRNA was not detected. In translation of exogenously added poly(U), extracts required Mg2+ (17 mM) and NH4+ (60 mM) for optimal polyphenylalanine synthesis; the optimum MG2+ requirement differed from that of Escherichia coli. The translation of coliphage Q beta RNA by C. burnetti extracts required Mg2+ (13 mM), NH4+ (60 mM) and an energy source for polypeptide synthesis, and was sensitive to chloramphenicol but not to cycloheximide. Under optimal conditions, the translation of Q beta RNA proceeded at a rate and to an extent equal to that obtained in a conventional E. coli system. Electrophoretic analysis of translation products made during incubation of C. burnetti extracts with polycistronic Q beta RNA revealed a major product with a molecular weight of about 14 000; this product co-electrophoresed with the coat protein extracted from Q beta phage propagated in E. coli. The results suggested that the extracellular form of the rickettsia-like organism, C. burnetti, possessed the full array of components necessary for the initiation, elongation and termination of polypeptides.

Ammonia↗
PubMed1983

Circadian rhythmicity of polyamine urinary excretion.

Polyamines are small, highly charged, organic cations of possible regulatory importance in RNA-dependent protein synthesis, the production of which reflects cellular growth and division. The cytokinetics of normal cell populations is circadian rhythmic. This is reflected by a circadian rhythmicity undescribed previously in urinary monoacetylputrescine and the ratio of N1-acetylspermidine to N8-acetylspermidine in healthy individuals. Patients harboring advanced cancers sometimes excrete abnormal quantities of certain acetylated polyamines, and their urine samples may exhibit an abnormally high ratio of N1-acetylspermidine to N8-acetylspermidine. Changes in polyamine production and excretion associated with cancer may be best perceived by rhythmometric analysis of carefully timed samplings.

Circadian Rhythm↗
PubMed1994

[A rare etiology for HBs-Ag negative acute hepatitis B--coinfection by hepatitis B and delta].

An unusual case of a 25-year-old male Italian is reported. The patient endured an acute hepatitis without detectable HBs-antigen by coinfection with hepatitis-B and Delta. Coincidently, a cured hepatitis-C was present. Firstly hepatitis-B-virus DNA could be demonstrated in a small quantity by serodiagnosis (6 pg/ml, hybridization technique). Subsequently, the identification of B-virus DNA was only possible in liver tissue (PCR-technique), but no longer by serodiagnosis. The probable enduring inhibition of hepatitis-B-virus replication by Delta virus resulted in a self limitation of the disease within 2 months (HDV-RNA negative, HBs-Ag and HBe-Ag negative; Anti-HBs negative, Anti-HBe and Anti-HBc positive). In spite of negativation of replication markers for hepatitis-B a subsequent reactivation of the infection was possible by viral material which persisted in liver tissue.

Acute Disease↗
PubMed1991

Pretranslational regulation of albumin synthesis in tumor-bearing mice. The role of anorexia and undernutrition.

Hepatic albumin synthesis, serum albumin turnover, and hepatic albumin messenger RNA (mRNA) content were evaluated in mice bearing a transplantable low differentiated tumor (MCG 101). Results obtained on tumor-bearing mice were compared with results obtained from non-tumor-bearing animals that were either freely fed, food restricted so that their body composition was similar to tumor-bearing animals (pair-weighed), fed a protein-free diet for 5 days, or fasted for 48 hours. Tumor-bearing animals became hypoalbuminemic (33 +/- 5 vs. 44 +/- 3 g/L in freely fed mice), which could be explained by both depressed albumin synthesis (1.95% +/- 0.20% vs. 2.67% +/- 0.27%/h in freely fed mice) and increased albumin degradation. Pair-weighed and protein-calorie malnourished controls had reductions in albumin synthesis (1.81% +/- 0.18% and 1.67% +/- 0.17%/h, respectively) similar to tumor-bearing animals, and the starved controls had the lowest synthetic rates (1.07% +/- 0.10%/h). Albumin degradation was increased only in tumor-bearing animals. Hepatic albumin mRNA in undernourished animals was less (tumor bearing, 32% +/- 5%; pair weighed, 47% +/- 4%; 48 hours fasted, 18% +/- 2%; and protein-calorie malnourished, 26% +/- 3%) than 50% of the mRNA content in the livers of freely fed control mice. Messenger RNA-directed synthesis of albumin in vitro was also depressed to a variable degree in tumor-bearing and malnourished non-tumor-bearing controls. The hypoalbuminemia in tumor-bearing animals could not be prevented by daily injections of a prostaglandin synthesis inhibitor (indomethacin, 1 microgram/g body wt), but the hepatic acute phase protein serum amyloid P decreased from 157 +/- 12 to 103 +/- 9 micrograms/mL in indomethacin-treated tumor-bearing mice (P less than 0.01). It is concluded that increased albumin degradation seen in tumor-bearing animals cannot be explained by associated malnutrition, whereas tumor-associated malnutrition can explain to a large extent the depressed albumin synthesis. Decreased albumin synthesis in tumor-bearing animals correlated in part with a decreased quantity of liver albumin mRNA. The results of the current study are consistent with either a reduced transcription of the albumin gene or a change in albumin mRNA processing and stability communicated by anorexia and malnutrition.

Albumins↗
PubMed1996

Regulation of gene expression in plant mitochondria.

Many genes is plant mitochondria have been analyzed in the past 15 years and regulatory processes controlling gene expression can now be investigated. In vitro systems capable of initiating transcription faithfully at promoter sites have been developed for both monocot and dicot plants and will allow the identification of the interacting nucleic acid elements and proteins which specify and guide transcriptional activities. Mitochondrial activity, although required in all plant tissues, is capable of adapting to specific requirements by regulated gene expression. Investigation of the factors governing the quality and quantity of distinct RNAs will define the extent of interorganelle regulatory interference in mitochondrial gene expression.

Gene Expression Regulation, Developmental↗
PubMed2004

Comparing field and microcosm experiments: a case study on methano- and methylo-trophic bacteria in paddy soil.

Methane-oxidising bacteria (MOB) play an important role in the reduction of methane emissions from rice agriculture. In rice fields, they are subjected to many environmental and field management parameters, which may have a significant impact on their community composition. To study this in greater detail, the community structure of methano- and methylo-trophic bacteria was investigated in a rice field in northern Italy during the summer 1999 and compared to a microcosm study described previously. We used PCR-based denaturing gradient gel electrophoresis applying 16S rDNA (9alpha and 10gamma) and mxaF (methanol-dehydrogenase) primer sets. In parallel, population size and activity of MOB were determined. This study provides the first comprehensive investigation of different compartments (bulk soil, rhizosphere, rhizoplane, and homogenate) throughout an entire rice-growing season in the field. Lower cell numbers of MOB were detected in the field compared to the microcosms, possibly due to lower CH4 concentrations in the soil pore water. In both studies, growth of MOB occurred predominantly at the root surface (rhizoplane) and in the root (homogenate), whereas cell numbers in bulk soil showed only minor changes throughout the season. Molecular analysis detected only few changes in alpha-proteobacterial methylotrophs during the season, whereas a higher variability was detected in gamma-proteobacteria. Nevertheless, the sequences of electrophoretic bands showed that the diversity in the field study and in the microcosms was comparable. Activity patterns of MOB and the population structure of methylotrophic bacteria agreed well between both studies, even though the detected quantities differed. Extrapolations of microcosm data to the field scale are thus possible, but should be used carefully when concerning quantitative changes.

Agriculture↗
PubMed2003

Expression of granzyme B mRNA is altered in human immunodeficiency virus infected patients.

CD8-positive cytotoxic T lymphocytes and natural killer cells are the major cytotoxic components of the antiviral immune response. The major pathway used by these cells in response to viral-infected cells involves granzymes, cytotoxic granule serine proteases involved in the pathway leading to target cell DNA fragmentation and apoptosis. The levels of granzyme B mRNA in peripheral blood cells of human immunodeficiency virus (HIV) type-1 infected patients in comparison to noninfected individuals were assessed by quantitative competitive reverse transcriptase polymerase chain reaction. Expression of granzyme B mRNA is altered in HIV-1 infected patients. Significantly fewer HIV patients had detectable granzyme B mRNA levels than controls. The one HIV-infected patient with detectable granzyme B mRNA displayed a much higher level of this mRNA than all healthy controls. Cell-mediated cytotoxicity during HIV-1 infection may be impaired due to a deficient quantity of active cytotoxic granules or to their abnormal regulation.

CD8-Positive T-Lymphocytes↗
PubMed1991

Unknown sequence amplification: application to in vitro genome walking in Chlamydia trachomatis L2.

A recently described technique, 'Chemical Genetics' unknown sequence amplification method, which requires only one specific oligonucleotide, has broadened the applicability of the polymerase chain reaction to DNA of unknown sequence. We have adapted this technique to the study of the genome of Chlamydia trachomatis, an obligate intracellular bacterium, and describe modifications that significantly improve the utility of this approach. These techniques allow for rapid genomic analysis entirely in vitro, using DNA of limited quantity or purity.

Bacterial Outer Membrane Proteins↗
PubMed2003

Comparison of transfection conditions for a lentivirus vector produced in large volumes.

A number of different transfection reagents have been used for lentiviral vector production. We directly compared transfection buffers, DNA purification methods, chemical facilitators, and DNA concentrations to optimize production. The use of N,N-bis (2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), sodium butyrate, and one fourth the total amount of DNA used in standard transient transfection protocols were the best conditions for virus production. These reagents were combined into a single protocol and scaled-up to produce liter quantities of virus in a multitray tissue culture vessel.

Alkanesulfonic Acids↗