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At least 901 records · Page 50Linked to original sources
PubMed2001

Early changes in lapine menisci during osteoarthritis development: Part II: molecular alterations.

OBJECTIVE: Osteoarthritis (OA) is the most common form of arthritis and patients with meniscal and ligament injuries of the knee are at high risk to develop the disease. The purpose of this study was to evaluate changes occurring in both medial and lateral menisci from the knees of anterior cruciate ligament (ACL) transected rabbits at 3 and 8 weeks post-surgery. This study describes both molecular and cellular alterations in menisci during the early stages of OA development. DESIGN: Rabbit meniscal tissues were processed for molecular analysis: DNA and RNA concentrations were assessed, as well as semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for a subset of relevant molecules was performed. In situ DNA fragmentation was evaluated using the TUNEL assay. RESULTS: Total RNA yields from the medial meniscus were significantly elevated at both 3 and 8 weeks post-ACL transection, while in the lateral meniscus total RNA levels were unchanged following ACL transection. DNA concentrations were significantly decreased in the medial menisci only at 8 weeks post-ACL transection. Following ACL transection, analysis of in situ DNA fragmentation using the TUNEL assay demonstrated an increase in the number of apoptotic cells in the medial meniscus only, in particular at 3 weeks post-ACL transection, a finding which correlates with declines in DNA content. Analysis of specific mRNA levels by RT-PCR demonstrated complex changes in both menisci following ACL transection. At 3 and 8 weeks post-ACL transection, in both medial and lateral menisci, mRNA levels for type I collagen and TIMP-1 were significantly increased, while mRNA levels for decorin, TNF-alpha and IGF-2 were significantly depressed. In the medial meniscus, significant increases in mRNA levels for type II collagen, biglycan as well as iNOS and PAI-1 were detected at both time periods, while mRNA levels for aggrecan, type III collagen and COX-2 were significantly elevated at 3 weeks post-ACL transection and mRNA levels for MMP-1 were significantly elevated at 8 weeks post-ACL transection. In contrast, mRNA levels for COL2 and aggrecan were unchanged in the lateral meniscus following ACL transection. In the lateral meniscus, at 3 weeks post-ACL transection, type III collagen mRNA levels were dramatically increased while fibromodulin mRNA levels were significantly depressed. In the lateral meniscus, significant increases in mRNA levels for biglycan were detected at 8 weeks post-ACL transection. CONCLUSION: These results show that after ACL transection complex molecular changes, as well as apoptosis, occur early, particularly in the medial meniscus.

Animals↗
PubMed1990

GFAP mRNA levels following stab wounds in rat brain.

We previously reported that glial fibrillary acidic protein (GFAP) levels increased significantly at 3 days after stab wounds, relative to sham-operated controls, reaching a maximum of 200% of control value at 5-7 days. They then fell to near-normal values by 21 days. To determine whether these protein changes correlated with changes in GFAP mRNA we performed Northern blot analyses. Total RNA, isolated from lesioned, sham-operated and intact rat forebrains, was hybridized with 32P-labeled mouse GFAP cDNA and quantified by densitometry. The maximum increase in total RNA content in lesioned animals was only 20% over controls at 12 h. GFAP mRNA levels increased to 2-fold control values at 6 h and reached 5-fold at 12 h. Thereafter they remained at 3.5- to 6-fold until 5 days and then declined to 1.5-fold by 21 days. The rapid increase of GFAP message at 12 h preceded a significant increase in GFAP by 2 days and the decrease of message after 5 days was more precipitate than the slow decrease in GFAP content. Sham-operated animals showed no significant changes in GFAP mRNA, compared to intact controls, during the period 3 h to 14 days postoperation. GFAP mRNA and GFAP in the stab-wound model reached levels similar to those found in the experimental autoimmune encephalomyelitis (EAE) model, but returned to normal much more rapidly.

Animals↗
PubMed1976

The effect of heat shock on RNA synthesis in Drosophila tissues.

Total RNA from Drosophila imaginal discs, labeled under conditions of heat shock, is analyzed by hybridization in situ to salivary gland polytene chromosomes. Grain densities over the hybridizing bands are compared, showing that the response to heat shock is similar for several disc types and fly stocks. Alteration of the culture medium used for labeling during heat shock results in the specific induction (in discs, salvary glands, or fat body) of one of the heat-shock loci to a level far beyond that normally seen. We discuss the implications of this specific induction regarding the mechanism of response. We also discuss the observed difference between transcription labeling of salivary gland chromosomes and labeling by in situ hybridization.

Culture Media↗
PubMed1991

Ontogenesis of transthyretin gene expression in chicken choroid plexus and liver.

1. Chicken liver transthyretin cDNA hybridizes strongly with choroid plexus transthyretin mRNA from chickens, pigeons, quails and ducks. 2. In the chicken at hatching the choroid plexus has reached 70%, total brain 30%, and liver 5.8% of their organ masses in adults. 3. The proportion of transthyretin mRNA in total RNA is 0.45-times the adult value in the choroid plexus of the chicken at hatching. 4. In the liver at hatching, the proportion of transthyretin mRNA in total RNA is 1.1-times the value in adult chickens. 5. The pattern of maturation of transthyretin gene expression in chicken liver is comparable to that in precocial, but differs from that in altricial mammals.

Animals↗
PubMed1999

Burn injury with infection alters prostaglandin E2 synthesis and metabolism.

OBJECTIVE: The aim of this study was to examine the relationship between prostaglandin synthesis and prostaglandin degradation in a model of burn injury with infection. METHODS: Male B2D6F1 mice were assigned to control, burn (16% dorsal scald burn), or burn with infection (burn with topical application of 1,000 colony forming units of Pseudomonas aeruginosa) groups. Lung tissue was harvested at 1, 2, and 3 days after burn injury and subsequently processed for total RNA and protein. Northern and Western blot analyses were used to examine differences in cyclooxygenase 2 (COX-2) and prostaglandin 15-OH dehydrogenase (PGDH) protein and mRNA expression. Total RNA was probed with the riboprobe for murine PGDH and COX-2 and the 100,000g protein fraction was immunoblotted by using an rabbit anti-murine PGDH and anti-murine COX-2 antibody. RESULTS: COX-2 expression was elevated in the burn with infection animals on day 1 and day 2 after burn injury. At these time points in the burn + infection group, PGDH was significantly depressed. Burn injury increased COX-2 expression on day 1, but by day 2, COX-2 expression had decreased to control values. A corresponding increase in PGDH expression was observed on day 2 in the burned mice. The mRNA expression of COX-2 was followed by a similar increase in COX-2 protein expression at all time points in the injured animals. This was not the case with PGDH expression. On day 1, PGDH mRNA expression was depressed in the burn with infection mice with no change in PGDH protein expression. This finding indicates that PGDH is subject to regulation at both the transcriptional and posttranscriptional levels. CONCLUSION: Burn wound infection depressed both PGDH mRNA and protein expression and increased COX-2 mRNA and protein expression. Therefore, increases in circulating prostaglandin E2 levels during septic injury are derived from alterations in synthesis and degradation of prostaglandin E2.

Animals↗
PubMed1993

Isolation and in vitro translation of messenger RNA from the American cockroach.

Total RNA was extracted from the whole body of American cockroach (Periplaneta americana) using chaotropic salt guanidine isothiocyanate in the presence of 2-mercaptoethanol (2-ME). Polyadenylated mRNA was isolated by oligothymidylic acid-cellulose chromatography and mRNA was translated using a rabbit reticulocyte lysate system. The translation, as judged by the incorporation of 35S-methionine, was obtained with poly(A)+ RNA, where an approximately 9.5-fold increase in label incorporation over control was achieved. Analysis of translation products by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) in combination with autoradiography showed that many proteins with apparent molecular weights ranging from 12 to 200 kD were synthesized, and no labelled proteins were found with negative RNA control and poly(A)- RNA. Immunoprecipitation studies performed using polyclonal and monoclonal antibodies revealed that synthesized proteins of MW 90, 78, 72, 49, 45, and 26 kD corresponded with previously identified principal and major allergens of American cockroach from our laboratory. In addition, the allergenicity of the translation mixtures was also confirmed by fluoroallergosorbent test (FAST) inhibition studies with IgE antibodies of human reaginic serum pool.

Allergens↗
PubMed1979

Transcription of the bovine parvovirus genome in isolated nuclei.

Transcription of the genome of the nondefective parvovirus BPV was examined in nuclei isolated from synchronized bovine fetal spleen cells. The relative levels of total RNA polymerase and RNA polymerase I, II, and III activities in nuclei isolated from BPV-infected and mock-infected cells were found to be similar throughout the course of infection. Hybridization of RNA synthesized in isolated nuceli indicated that BPV-specific RNA synthesis began during the period of 8 to 12 h postinfection and proceeded linearly until at least 20 h postinfection. By 20 h postinfection, 5% of the total RNA synthesized in nuclei from infected cells was virus specific. BPV-specific RNA synthesis was inhibited by 95% in the presence of 0.1 microgram of alpha-amanitin per ml, suggesting that the viral genome is transcribed by cellular RNA polymerase II.

Amanitins↗
PubMed1988

[Template activity of poly(A+) and poly(A-) messenger RNA of pathogenic arenaviruses].

The total RNA from cells infected with Machupo and Lassa viruses as well as poly(A+) and poly(A-) fractions of the RNA were translated in the cell-free protein synthesizing system from rabbit reticulocytes. The translated products were treated with specific antibodies and analyzed in polyacrylamide gel electrophoresis. Only poly(A-) fraction of RNA coded for the synthesis of NP protein in vitro. The mRNAs for NP protein of Machupo and Lassa viruses are supposed to contain no poly(A) sequences at 3'end, or if they really do, the size of the sequences is not adequate for binding with oligo(dT)-cellulose.

Animals↗
PubMed1995

Use of differential display to identify differentially expressed mRNAs induced by rat carotid artery balloon angioplasty.

BACKGROUND: We have used differential display technology in an animal model of arterial restenosis to study the molecular mechanisms of neointima formation. EXPERIMENTAL DESIGN: Balloon angioplasty of the rat common carotid artery was performed, and tissues were examined from naive animals (control) and from animals that had undergone angioplasty 6 hours, 3 days, and 14 days earlier. Total RNA was isolated, and differential display was performed to identify mRNA transcripts whose expression is modulated in a temporal fashion as a consequence of balloon angioplasty. RESULTS: Using total RNA isolated from vessels excised from naive rats and those from rats that underwent angioplasty up to 14 days earlier, we describe two differentially regulated transcripts by differential display and verify the expression pattern of these same transcripts by Northern analysis. DNA sequence analysis has identified one of these angioplasty-induced transcripts as a novel sequence and the other as the tryptophan hydroxylase and protein kinase C regulatory protein 14-3-3 gamma. CONCLUSIONS: In this study, we describe the in vivo application of the differential display technique in a rat carotid artery angioplasty model to identify mRNA transcripts whose expression is modulated selectively in vessels undergoing neointima formation. Use of this technique in animal models of disease should facilitate our understanding of gene expression patterns in pathogenesis and may serve as a reliable technique to "fingerprint" disease processes.

14-3-3 Proteins↗
PubMed2002

InsP(3)-mediated calcium release induced by heterologous expression of total chicory Leaf RNA.

A calcium dependent-chloride current (I(ni)) was recorded in Xenopus oocytes injected with total RNA from chicory leaf tissues, following depolarization from -35 to +60 mV. However, the signal transduction mechanism mediating I(ni) is unknown. The development of this current was mimicked by intracellular injection of the second messenger InsP(3) in control (non-injected) oocytes. Moreover, InsP(3) injection after I(ni) rundown did not reinitiate the current. The same phenomenon was observed following a second injection into control oocytes. Measurement of InsP(3) production in injected oocytes showed a net increase in the InsP(3) level on depolarization. Moreover, extracellular application of caffeine (5 mM) significantly reduced the number of oocytes displaying I(ni). Also, extracellular application of U-73122, a potent PLC inhibitor, clearly reduced the occurrence of I(ni). These data provide the first evidence that the calcium homeostasis mechanism induced by heterologous expression of total RNA from chicory leaves involves the InsP(3) signaling pathway.

Animals↗
PubMed1995

Microdensitometric measures of cytoplasmic RNA and total protein in pyramidal neurons of the insular cortex and midfrontal gyrus in patients with Alzheimer's disease.

Scanning and integrating microdensitometry of azure B- and Coomassie brilliant blue G-stained tissue sections was used to measure the levels of RNA and protein, respectively, in pyramidal neurons of the insular cortex (INS) and midfrontal gyrus (MFG) in patients with Alzheimer's disease (AD) and age-matched, nondemented control subjects. AD was associated with a decreased neuronal RNA (by 7.4 per cent) and protein (by 28.7 per cent) content in INS. Although the neuronal RNA content was maintained at the control amounts in MFG, the average protein level was lower (14.7 per cent) in AD patients. These results demonstrate a disease-related impairment in metabolic function in two brain regions connected via discrete corticocortical pathways. Such findings support the hypothesis that a primary site of pathology occurs in AD, and specific neural deficits occur secondarily in certain connected brain regions.

Aged↗
PubMed2005

Intestinal microsomal triglyceride transfer protein in type 2 diabetic and non-diabetic subjects: the relationship to triglyceride-rich postprandial lipoprotein composition.

BACKGROUND: Microsomal triglyceride transfer protein (MTP) is responsible for the assembly of the triglyceride-rich lipoproteins (TRLs) and is increased in diabetic animal models. Human intestinal MTP expression has not been previously reported. This study examined the relationship between intestinal MTP gene expression and postprandial TRL composition in diabetic and non-diabetic subjects. Since the MTP promoter region has a sterol response element the effect of statins on intestinal MTP mRNA was analysed. METHODS: Twenty-seven diabetic and 24 non-diabetic subjects were examined. Duodenal biopsies were taken during gastroscopy and MTP mRNA was measured by RNase protection assay. Postprandial lipoprotein composition was determined. RESULTS: Diabetic subjects had significantly higher MTP mRNA than non-diabetic subjects. Statin therapy was associated with lower MTP mRNA in both groups. In the untreated diabetic patients compared to the untreated non-diabetic patients MTP mRNA was 25.0 +/- 25.1 amol/microg versus 13.1 +/- 5.6 amol/microg total RNA (p < 0.05). In the statin-treated diabetic group compared to statin-treated non-diabetic group MTP mRNA was 17.7 +/- 8.6 amol/microg versus 5.8 +/- 4.1 amol/microg total RNA (p < 0.05). In the whole group there was a positive correlation between the MTP mRNA and postprandial chylomicron cholesterol/B48 (r = 0.36, p < 0.01). CONCLUSIONS: This is the first study to demonstrate increased MTP expression in diabetic subjects. MTP mRNA expression was lower in statin-treated patients confirming the suggestion that the insulin and sterol response elements of the MTP gene are important regulators of MTP transcription in diabetes. Our results show that MTP plays a central role in regulating the cholesterol content of the chylomicron particle.

Biopsy↗
PubMed1992

Changes in Cytosolic Glutamine Synthetase Polypeptide and its mRNA in a Leaf Blade of Rice Plants during Natural Senescence.

Changes in the levels of cytosolic glutamine synthetase (GS1) and chloroplastic glutamine synthetase (GS2) polypeptides and of their corresponding mRNAs have been investigated in segments of the 13th leaf of hydroponically grown rice (Oryza sativa L.) plants during natural senescence. The leaf blade on the main stem at early (0 day), middle (15 days), and late (25 days) stages of senescence was harvested and cut into 18 or 19 segments, 2 centimeters in length from the base to the tip. The amount of GS1 polypeptide, detected with specific antibody for the GS1, was greatest near the middle of the leaf blade (segments 11-13). There was little difference in the GS1 content between corresponding leaf segments obtained at the early and middle stages of senescence. At the late senescence stage, all segments had lost some GS1 polypeptide, but more than 50% of GS1 detected at both the early and middle stages was still detectable in segments. The relative content of mRNA for GS1 in the total RNA in all segments was very low during early senescence but increased in all leaf segments during later senescence. At the late stage of senescence, GS1 mRNA in the total RNA increased about 4.2- to 4.6-fold in segments 12 to 16 in the day-25 samples compared with those in the early stage. The content of the GS2 polypeptide, as well as ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) protein, was highest in segment 17 in the 0-day samples. During senescence, this peak became lower and broader, and finally disappeared, i.e. approximately 80% of GS2 polypeptide and Rubisco protein in segment 17 were lost by day 25. In contrast with GS2 polypeptide, the relative level of GS2 mRNA increased 1.8- to 2.9-fold in individual segments at the middle stage of senescence. Even at the late stage, the transcript signals remained slightly higher than those at the early stage in all segments. Thus, GS1 and GS2 polypeptides and corresponding mRNAs responded in a different manner within an attached rice leaf during natural senescence. The contents of GS1 and GS2 polypeptides were not simply determined by the abundance of their corresponding mRNAs in the rice leaf blades during natural senescence.

Journal Article↗
PubMed1991

Ontogeny of corticosteroid-binding globulin biosynthesis in the rat.

In plasma, glucocorticoids are transported by corticosteroid-binding globulin (CBG), which is synthesized primarily in the liver. Plasma levels of maternal and fetal CBG fluctuate during gestation, and this may be due to changes in the biosynthesis and/or clearance of the protein. We have, therefore, studied the ontogeny of CBG biosynthesis in the rat by using a solution hybridization assay to measure hepatic CBG mRNA levels. The results indicate that the concentration of CBG mRNA is exceptionally high in 15-day-old fetal livers (55.1 pg CBG mRNA/micrograms total RNA), but declines to very low levels at birth (day 21). During the same period, CBG mRNA levels in maternal livers remained relatively constant (18.9-23.1 pg CBG mRNA/micrograms total RNA). Hepatic CBG mRNA levels were barely detectable 1 week after birth, and a sex difference was apparent by 2 weeks of age, with higher levels in female livers. Although adult CBG mRNA levels were attained by 3 weeks of age, serum CBG concentrations did not reach adult values for an additional 3 weeks. To determine whether age-related differences in the clearance of CBG are responsible for this, CBG from infant or adult animals was radiolabeled and administered iv to infant and adult rats. When this was done, the half-life of CBG in infants (approximately 6.9 h) was consistently less than that in adults (approximately 14.5 h) regardless of the source of the labeled CBG, and we conclude that variations in CBG biosynthesis and clearance may influence glucocorticoid action during fetal and postnatal development.

Aging↗
PubMed1992

Growth hormone-releasing hormone-like messenger ribonucleic acid and immunoreactive peptide are present in human testis and placenta.

Although the sequence of human GH-releasing hormone (GHRH) has been determined, all of the information concerning gene expression has been based on pathological sources of ectopic GHRH, since the only established physiological source of GHRH in humans is the hypothalamus. We recently reported the presence of extrahypothalamic GHRH-like mRNA and immunoreactive material in rat testis and placenta. To determine if human testis and placenta also contain immunoreactive GHRH-like peptides, tissue extracts were analyzed using enzyme-linked immunosorbent assay methodology. Both tissues had detectable quantities of immunoreactive peptide recognized by a monoclonal antibody to synthetic human GHRH-(1-44) (testis, 2.4 ng/g tissue, 0.68 ng/mg protein; placenta, 2.6 ng/g tissue, 0.36 ng/mg protein). The origin of these peptides was confirmed by extracting total RNA from human testis and placenta, with analysis on Northern blots probed with riboprobes for rat hypothalamic GHRH cDNA and human pancreatic tumor GHRH cDNA. Human testis and placenta total RNA both contain an approximately 790-nucleotide RNA species similar in size to that reported in ectopic GHRH-producing human tumors. In addition, two larger hybridization signals were seen at 3000 and 4900 nucleotides. These data suggest that testis and placenta are extrahypothalamic sites of expression of the human GHRH gene. Normal expression of the GHRH gene in extrahypothalamic sites may include transcription of larger mRNA species than those observed in ectopic pathological sources of GHRH expression.

Animals↗
PubMed2005

RNA constitution and estrogen-responsive gene expression in the ovariectomized rat uterus.

Because reproductive tracts in ovariectomized rodents, which are commonly used in endocrinological studies, exhibit drastic changes in response to exogenous estrogens, quantitative evaluation of gene expression requires extra caution. We found that the whole mRNA content of total RNA in the uterus of the ovariectomized rat was dose-dependently reduced by treatment with 17alpha-ethynylestradiol (EE) for 3 consecutive days. Because of the decline in the ratio of mRNA/total RNA, real-time reverse transcriptase-polymerase chain reaction analysis seemingly showed that the relative ratios of the levels of stable RNAs, rRNA18S, rRNA5S, and tRNA-Gly, to whole mRNA were increased by EE. These results indicate that applying a fixed concentration of total RNA and stable RNA as an internal control to uterine mRNA quantification should be reconsidered. On the other hand, the beta-actin gene showed the most stable expression among the housekeeping genes examined. Using beta-actin mRNA as an internal control, we observed that tissue inhibitor of metalloproteinase-3 (TIMP3) mRNA was dose-dependently reduced 24 h after treatment with EE or bisphenol A for 3 days. Continued investigation of TIMP3 with an appropriate internal control may be helpful in elucidating the mechanisms involved in uterine activation.

Animals↗
PubMed1979

Construction and properties of a cell-free system for bacteriophage T4 late RNA synthesis.

A cell-free system for synthesizing bacteriophage T4 late RNA is described. The system, which is based on the "cellophane disc" technique introduced by Schaller and co-workers (Schaller, H., Otto, B., Nüsslein, V., Huf, J., Hermann, R., and Bonnhoeffer, F. (1972) J. Mol. Biol. 63, 183-200), provides favorable conditions for T4 DNA and RNA synthesis in vitro. Total RNA synthesis can be sustained for more than 1 h at 25 degrees C and initiation of early and late RNA chains occurs in vitro. The capacity to yield cell-free systems which make T4 late RNA in vitro is acquired by virus-infected cells as they make late RNA in vivo. The in vitro synthesized RNA is highly asymmetric. The conditions which optimize the in vitro system with respect to several parameters (total extent of T4 transcription, rate of transcription, asymmetry, fraction of T4 late RNA, and sensitivity to inhibition by rifampicin and streptolydigin) are described.

Coliphages↗
PubMed1997

Specific polyadenylation and purification of total messenger RNA from Escherichia coli.

Obtaining pure mRNA preparations from prokaryotes has been difficult, if not impossible, for want of a poly(A) tail on these messages. We have used poly(A) polymerase from yeast to effect specific polyadenylation of Escherichia coli polysomal mRNA in the presence of magnesium and manganese. The polyadenylated total mRNA, which could be subsequently purified by binding to and elution from oligo(dT) beads, had a size range of 0.4-4.0 kb. We have used hybridization to a specific plasmid-encoded gene to further confirm that the polyadenylated species represented mRNA. Withdrawal of Mg2+ from the polyadenylation reaction resulted in addition of poly(A) to 16S rRNA despite the presence of Mn2+, indicating the vital role of Mg2+ in maintaining the native structure of polysomes. Complete dissociation of polysomes into ribosomal subunits resulted in quantitative polyadenylation of both 16S and 23S rRNA species. Chromosomal lacZ gene-derived messages were quantitatively recovered in the oligo(dT)-bound fraction, as demonstrated by RT-PCR analysis. Potential advantages that accrue from the availability of pure total mRNA from prokaryotes is discussed.

Blotting, Northern↗