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PubMed1977

Effect of temperature on nuclear membranes and nucleo-cytoplasmic RNA-transport in Tetrahymena grown at different temperatures.

The effect of temperature on the nuclear envelope structure and the transport of total RNA and ribosomal subunits from nucleus to cytoplasm was examined in Tetrahymena cells propagated at two different temperatures. Freeze-etch electron microscopy of cells grown at 23 and 18 degrees C detects the emergence of smooth areas on the fracture faces of the nuclear membranes upon lowering the temperature below approximately 15 and approximately 12 degrees C, respectively. Coincident with these freeze-etch changes, a discontinuous decrease is observed in the nucleocytoplasmic RNA-transport; this is probably not due to a cease in RNA-synthesis. Below the thermotropic discontinuity observed in the transport of total RNA in 18 degrees-cells the nucleocytoplasmic transport of the small and large ribosomal subunits is equally retarded. Recent temperature studies on the endoplasmic reticulum membranes of Tetrahymena suggest that the freeze-etch changes in the nuclear membranes are induced by a thermotropic clustering of the membrane lipids. We conclude that this lipid clustering induces the permanent protein constituents in the nuclear envelope pore complexes to change from a relatively "open" into a relatively "closed" state thus causing the observed decrease in RNA-transport.

Biological Transport↗

PubMed1983

Age related changes in total DNA and RNA and incorporation of uridine and thymidine in rat brain.

1. Total brain DNA and total brain RNA and the incorporation of thymidine[14C] and uridine[3H] were measured in young and aged rats. 2. From 20 days to the time of sexual maturation, both DNA and RNA levels increase. Total RNA exceeds total DNA at all ages. Comparatively, the ratio of total DNA/RNA is higher in young than in aged animals. 3. The incorporation of thymidine[14C]/g of DNA and of uridine[3H]/g of RNA decreases with age. This decrease is rapid in young animals. After 350 days of age, the incorporation becomes very low. The significance of data is discussed.

Aging↗

PubMed1980

Biochemical Changes in Tuber-bearing Solanum Species in Relation to Frost Hardiness during Cold Acclimation.

Biochemical changes in potato leaves during cold acclimation have been examined and compared between a frost-tolerant S. acaule and a frost-susceptible S. tuberosum species. Changes were also examined in S. tuberosum, S. acaule, and S. commersonii species when they were hardened at different temperatures to varying hardiness levels.During three weeks of stepwise cold acclimation, S. acaule increased frost hardiness from -6.0 C (killing temperature) to -9.0 C, whereas frost hardiness of S. tuberosum remained unchanged at -3.0 C. Decreases in DNA content on a dry weight basis in both species suggest that matured leaf cells accumulated more dry matter during acclimation. The advantage of using DNA as a reference for comparing metabolite changes during cold acclimation is discussed.Under the stepwise acclimating conditions, both species showed the same trends for increasing total sugar and starch with an insignificant decrease in leaf water content. High levels of total RNA, rRNA, and total and soluble protein were observed in treated S. acaule plants as compared with controls, but not in S. tuberosum. Levels of total lipid and phospholipid also were high in treated S. acaule plants as compared with controls but decreased in S. tuberosum during acclimation.When S. tuberosum, S. acaule, and S. commersonii potatoes were cold-treated at constant day/night temperatures of 10, 5, and 2 C with 14-hour daylength, each species responds differently in terms of frost hardiness increase upon subjecting plants to a low temperature. For instance, after 20 days at 2 C, a net frost hardiness of 3 and 7 C was observed in S. acaule and S. commersonii, respectively, whereas the frost hardiness in S. tuberosum remained unchanged. Also, various levels of frost hardiness can be achieved in a species by subjecting plants to different low temperature treatments. Under a warm regime of 20/15 C day/night temperatures (14-hour light), both S. acaule and S. commersonii can survive at -4.5 C or colder, whereas S. tuberosum can survive only at -2.5 C.Biochemical changes in the leaf tissue of these species were investigated at 5-day intervals during low temperature treatments. Increases in total sugar and starch were found in all three species during hardening, although S. tuberosum failed to harden. Soluble protein contents were increased in both S. acaule and S. commersonii but decreased in S. tuberosum. RNA contents change in a pattern similar to the soluble protein. Net increases of the soluble proteins were positively and significantly correlated with net increases of frost hardiness in S. acaule and S. commersonii.

Journal Article↗

PubMed1988

Selective isolation of newly synthesized mammalian mRNA after in vivo labeling with 4-thiouridine or 6-thioguanosine.

Newly synthesized mRNA from hamster cells was labeled in vivo with the thionucleoside analogs 4-thiouridine (4-TU) and 6-thioguanosine (6-TG). The thio-substituted RNA was selectively recovered by Affi-Gel 501 phenylmercury affinity chromatography. Following a 1-h labeling period, enrichment for newly transcribed RNA after a single round of chromatography ranged between 10- and 15-fold when compared with total RNA. Exposure of CHO UrdA- cells, a uridine auxotrophic line, to 50 microM 4-TU allowed for optimal recovery of newly transcribed RNA. Increasing the concentration of 4-TU to 100 microM or labeling with 6-TG at concentrations of 3 microM or greater resulted in similar recoveries from uridine-prototrophic hamster cell lines. For shorter term labeling, exposure of prototrophic cells to 500 microM 4-TU or 100 microM 6-TG for 15 min allowed newly synthesized RNA to be selectively recovered. As a specific test case, enrichment for histone H3.2 mRNA was analyzed after hamster cells were labeled with 4-TU under conditions in which the gene was highly transcriptionally active. Northern blot analysis and the specific activity of thio-substituted RNA revealed a 15-fold enrichment when compared to total RNA. In vivo labeling of cellular RNA with 4-TU or 6-TG should provide a useful method for studying inducible gene expression and for isolating and cloning specific mRNAs from mammalian cells.

Affinity Labels↗

PubMed1976

Effect of the hepatocarcinogen ethionine on hepatic messenger RNA synthesis.

An experiment was conducted to determine if any changes resulted in the proportion of hepatic messenger RNA following treatment with ethionine, a hepatocarcinogen. The relative specific activity of the total RNA isolated from nontumor-like tissues was increased in Sprague-Dawley rats fed ethionine. However, the percentage of total RNA that was message was found to be decreased in the ethionine-treated rats.

Animals↗

PubMed1991

Expression of enzymatically active sucrase-isomaltase is a ubiquitous property of colon adenocarcinomas.

Adenocarcinoma of the colon is one of the most prevalent and lethal of all human malignancies. The early diagnosis and management of this disease could be improved if biological markers, whose expression was restricted to malignant colon cells, were identified. Sucrase-isomaltase is a glycoprotein hydrolase expressed throughout the small intestine and fetal colon but not in the normal adult colon. This study shows that the expression of enzymatically active sucrase-isomaltase is a ubiquitous property of primary and metastatic colon adenocarcinoma. Significant sucrase enzyme activity (i.e., greater than 5 mU/mg protein) was observed in 16 colon carcinomas but not in adjacent normal colon mucosa. Sucrase-isomaltase messenger RNA was identified in all tumors using reverse transcriptase polymerase chain reaction. Using a quantitative polymerase chain reaction analysis, this study shows that the amount of sucrase-isomaltase messenger RNA in tumors examined (3.4 x 10(-8) to 3.19 x 10(-7) micrograms/micrograms total RNA) was greater than in adjacent mucosa (0 to 3.4 x 10(-8) micrograms/micrograms total RNA). This induction of sucrase-isomaltase messenger RNA and enzyme activity was corroborated by immunostaining. Of 30 colon adenocarcinomas examined, 80% were positive for sucrase-isomaltase. In addition, all colon carcinoma metastases examined were positive for sucrase-isomaltase. The staining pattern was distinct and demarcated tumor cells from the surrounding histologically normal tissue. No sucrase-isomaltase staining was seen in normal mucosa from the same patients. With the exception of lung, no sucrase-isomaltase immunostaining was observed in a variety of examined noncolonic adenocarcinomas. Thus, the specificity and ubiquity of sucrase-isomaltase expression in adenocarcinomas of the colon can be exploited to improve the clinical management of this disease. In addition, studies on the structure of the sucrase-isomaltase gene and its regulatory elements should contribute toward understanding the alteration of gene expression by oncogenic transformation of the colonic mucosa.

Adenocarcinoma↗

PubMed1990

Histoautoradiographic study of rRNA and mRNA transcription activity related to secretory function in lizard epididymis.

The lizard epididymis is an androgen-dependent organ whose epithelial cells undergo marked changes in structure and secretory activity during the annual cycle. These changes are connected to fluctuations of testosterone levels. During the breeding season, the epididymis produces a major protein secretion, the L-proteins. In the present work we studied the fluctuations of RNA synthesis and accumulation during the annual cycle by means of histoautoradiographic methods. Total RNA synthesis was determined by uridine incorporation; accumulation of rRNAs and L-protein mRNAs were determined by in situ hybridization. Total RNA synthesis began during reorganization (Phase I), then increased gradually during differentiation and growth (Phase II). The synthesis peaked during maturation (Phase III), but stopped abruptly during hypersecretory activity (Phase IV). The rRNAs were very abundant from Phase II to Phase IV, which is related to the presence of many ribosomes as revealed by electron microscopy. The mRNAs of L-proteins were detected only during Phases III and IV in all epithelial cells. For every phase of the sexual cycle there exists a strong correspondence between the changes in transcriptional activity (rRNA and specific mRNA) of the epithelial cells and changes in the testosterone levels.

Animals↗

PubMedDate not supplied

[Dependence between the rate of RNA synthesis and the CTP-synthetase activity of different rat and chicken tissue].

The rate of total RNA synthesis determined by the method of Bukher and Sheffield in different tissues of rats and chickens decreases as follows: kidneys greater than liver greater than duodenum mucosa. The activity of CTP-synthetase (EC 6, 3, 4, 2) determined in vitro and in vivo by the method suggested correlates with the rate of total RNA synthesis in the kidneys and liver tissues of rats and chickens. It is shown that 34-35% of CTP synthetized de novo in the chicken tissues and 31-34% in the rat tissues are used for RNA synthesis. At the same time 75-90% of CTP incorporating into RNA is formed by salvage pathway.

Animals↗

PubMed2004

Isolation of high-quality RNA from gymnosperm and angiosperm trees.

An improved protocol was developed for efficient and reliable extraction of high-quality total RNA and mRNA from various tissues of spruce (Picea spp.) and poplar (Populus spp.) trees, as well as other plant species. This method was specifically optimized for tissues with high content of polysaccharides, oleoresin terpenoids, and phenolic secondary metabolites, which often co-precipitate with RNA and inhibit subsequent reverse transcription. The improved protocol yielded up to 600 micrograms of total RNA per gram of tissue suitable for standard expressed sequence tags (ESTs), full-length cDNA library construction, and for microarray applications.

Chemical Fractionation↗

PubMed1995

Estrogen receptor mRNA is expressed in vivo in rat calvarial periosteum.

Estrogen deficiency is well recognized as a cause of bone loss in rats and humans. Likewise, treatment with estrogen results in prevention of this loss. Initially, this effect was thought to be indirectly mediated but, more recently, estrogen receptors (ER) have been reported in osteosarcoma cells and primary cultures originating from surgical waste, suggesting a direct effect of this steroid hormone. Detection of ER in skeletal tissues, however, has remained elusive. The purpose of this investigation was to establish the efficacy of the highly sensitive reverse-transcription polymerase chain reaction (RT-PCR) technique to detect ER in a well defined skeletal tissue (calvarial periosteum) that is responsive to the hormone. Primers were made specific to rat ER sequences. Total RNA was extracted from rat uterus, liver, spleen, and the periosteum using an organic solvent method. cDNA was synthesized from 2 micrograms total RNA. cDNA corresponding to 40 ng total RNA/sample produced intense PCR products for ER. In descending order of intensity were uterus, liver, bone, and spleen. Importantly, a similar time-course for estrogen-induced down-regulation of steady-state mRNA levels for alkaline phosphatase and osteonectin was observed in calvarial periosteum and tissues known to express estrogen receptors. These data provide in vivo evidence of ER mRNA in bone and suggest that at least some of estrogen's action on bone is directly modulated.

Animals↗

PubMed2003

Expression, menstrual cycle-dependent activation, and bimodal mitogenic effect of transforming growth factor-beta1 in human myometrium and leiomyoma.

OBJECTIVE: Transforming growth factor-beta1 is the prototype of a bimodal regulator of cell growth, which can either inhibit or stimulate the proliferation of smooth muscle cells. Part of transforming growth factor-beta1-mediated stimulation of growth is associated with the increased production of platelet-derived growth factor. The conversion of latent-to-active transforming growth factor-beta provides a pivotal mechanism for the regulation of the biologic activity of transforming growth factor-beta. We investigated the differential expression and production of the active form of transforming growth factor-beta1 in the myometrium and leiomyoma throughout the menstrual cycle. We also studied the mitogenic effects of transforming growth factor-beta1 and platelet derived growth factor on myometrial and leiomyoma cells in culture. STUDY DESIGN: Myometrium and leiomyoma tissue pairs were obtained from 28 women who underwent hysterectomy. Total RNA from each tissue was extracted, and Northern blot analysis was performed for the detection of TGF-beta1 messenger RNA. Active and total transforming growth factor-beta1 protein was quantified with enzyme-linked immunosorbent assay. Cell proliferation of cultured human myometrial and leiomyoma cells that are treated with TGF-beta1 (0.01-1 ng/mL), anti-transforming growth factor-beta antibody (0.01-10 ng/mL), or platelet-derived growth factor (10 ng/mL) was assessed by the [(3)H]thymidine incorporation method. RESULTS: Overall, the transforming growth factor-beta1 messenger RNA level in myometrial samples was 1.2-fold higher than in the leiomyoma samples (P <.05). Active transforming growth factor-beta1 protein levels in follicular and luteal phase myometrial and leiomyoma samples were significantly greater than the levels in samples from women with atrophic endometrium (P < 0.05). Transforming growth factor-beta1, at low concentrations (0.01 ng/mL), induced an increase in cell proliferation (2- to 3-fold; P <.05). When cells were treated with anti-transforming growth factor-beta antibody, there was a larger magnitude of increase observed (7- to 20-fold; P <.05). Platelet-derived growth factor (10 ng/mL) consistently increased the rate of cell proliferation both in myometrium and leiomyoma cells (5- to 6-fold; P <.05). CONCLUSION: Levels of active transforming growth factor-beta1 that were produced in follicular and luteal phases indicate a stimulatory role for ovarian hormones. The finding that transforming growth factor-beta1, only at low concentrations, stimulates cell proliferation mainly in leiomyoma cells is in agreement with the bimodal and dose-dependent effects of transforming growth factor-beta1 that is observed in smooth muscle cells of other tissues. The persistent and high rate of cell proliferation with platelet-derived growth factor suggests that the growth stimulatory effect of transforming growth factor-beta1 may be mediated through its up-regulatory effect on platelet-derived growth factor.

Antibodies↗

PubMed2006

[The influence of downregulation of ezrin expression by RNA interference on the growth and metastasis of hepatocellular carcinoma: experiment in vitro].

OBJECTIVE: To investigate the effect of membrane-cytoskeleton linker ezrin on the growth and metastasis of human hepatocellular carcinoma cell (HCC) lines. METHODS: Human HCC cells of the lines SF/SMMC7721, MHCC97-H, MHCC-1, and HepG2 were cultured. Four pairs of small interfering RNA (siRNA) targeting erzin were designed and transfected into the HCC cells. 48 h after transfection the cell total RNA was extracted and 72 h later the total cell protein was extracted. RT-PCR and Western blotting were used to detect the transfection rates so as to screen the most effective siRNA to be transfected into the HCC cells. HCC cells were collected every day for 7 days to extract the total RNA and protein. Real-time PCR and Western blotting were used to detect the downregulation rate of erzin at different times. MTT method was used to detect the proliferation of the cells. Flow cytometry was used to detect the cell cycle and apoptosis. Scanning electron microscopy was used to observe the cell pseudopods. Transwell test was used to detect the invasion ability of the transfected HCC cells. RESULTS: Real-time PCR and western-blotting revealed that ezrin siRNA notably down-regulated ezrin expression at both mRNA and protein levels. Down-regulation of ezrin expression distinctly decreased the proliferation rates of these 4 kinds of HCC line. After RNAi treatment the cell proportion in G(2)-M phase decreased from 28.07% to 21.53% in the SF/SMMC7721 cells, from 24.94% to 13.92% in the MHCC97-H cells, from 19.30% to 13.2% in the MHCC-1cells, and from 7.73% to 4.24% in the HepG2 cells. After RNAi treatment, the number of pseudopods decreased from 20.8 +/- 3.0 to 13.2 +/- 2.4 in the SF/SMMC7721: cells (P < 0.05), from 18.4 +/- 2.7 to 14.0 +/- 2.9 in the MHCC97-H cells (P < 0.01), from 22.6 +/- 3.5 to 13.3 +/- 1.9 in the MHCC-1: cells (P < 0.01), and from 31.0 +/- 2.9 to 17.8 +/- 2.3 in the HepG2 cells (P < 0.01); and the motility and invasiveness decreased from 49.9 +/- 7.7 to 31.9 +/- 5.2 in the SF/SMMC7721 cells (P < 0.05), from 58.5 +/- 4.2 to 33.0 +/- 3.3 in the MHCC97-H cells (P < 0.01), from 57.6 +/- 6.1 to 28.3 +/- 3.4 in the MHCC-1 cells (P < 0.01), and from 37.3 +/- 3.0 to 25.3 +/- 2.3 in the HepG2 cells (P < 0.01). The pseudopods of the HCC cells remarkably shortened and decreased in number (for the SF/SMMC7721 cells: t = 4.95, P < 0.05, for the MHCC97-H cells: t = 5.88, P < 0.01, for the MHCC-1 cells: t = 5.56, P < 0.01, and for the HepG2 cells: t = 5.71, P < 0.01) after siRNA interference. CONCLUSION: Ezrin is necessary for HCC proliferation and invasion. It is probably an important factor to inhibit tumor reoccurrence and metastasis.

Blotting, Western↗

PubMed1991

Xenopus liver ferritin H subunit: cDNA sequence and mRNA production in the liver following estrogen treatment.

In vitro translation of liver mRNA from estrogen-treated Xenopus frogs yields two abundant polypeptides in the range of 20 kDa. DNA clones for one of these translation products were isolated and shown to be complementary to mRNA for the heavy subunit of ferritin. The predicted Xenopus amino acid sequence shares about 86% identity with the ferritin heavy chain from bullfrogs and about 70% identity with the comparable mammalian and avian proteins. Clone identity was confirmed by hybridization selection followed by in vitro translation into translation products of 19.5-20 kDa. The nearly full-length cDNA clone, termed XlferH1, comprises 868 nucleotides plus 22 adenosines of the poly(A) tail, including 134 nucleotides of the 5'-untranslated region, a 528-base coding region for 176 amino acids, and a 206-nucleotide 3'-untranslated region. The clone lacks 22 nucleotides from the 5' end of the mRNA. The level of ferritin mRNA in the liver of estrogen-treated frogs was determined over time. The amount of this mRNA relative to total RNA decreased about 3-fold 14 days after estradiol-17 beta was administered. However, the hormone also elevated total RNA in the liver about 24-fold. Hence, the total ferritin mRNA content of the liver increased to about 8 times its initial amount. This pattern of gene expression was very similar to that for serum retinol binding protein. The estrogen induction of these two mRNAs appeared to parallel the overall stimulation of hepatic RNA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed2004

[DNA microarray reveals changes in gene expression of endothelial cells under shear stress].

cDNA microarray technology is used as a powerful tool for rapid, comprehensive, and quantitative analysis of gene profiles of cultured human umbilical vein endothelial cells(HUVECs) in the normal static group and the shear stressed (4.20 dyne/cm2, 2 h) group. The total RNA from normal static cultured HUVECs was labeled by Cy3-dCTP, and total RNA of HUVECs from the paired shear stressed experiment was labeled by Cy5-dCTP. The expression ratios reported are the average from the two separate experiments. After bioinformatics analysis, we identified a total of 108 genes (approximately 0.026%) revealing differential expression. Of these 53 genes expressions were up-regulated, the most enhanced ones being human homolog of yeast IPP isomerase, human low density lipoprotein receptor gene, Squalene epoxidase gene, 7-dehydrocholesterol reductase, and 55 were down-regulated, the most decreased ones being heat shock 70 kD protein 1, TCB gene encoding cytosolic thyroid hormone-binding protein in HUVECs exposed to low shear stress. These results indicate that the cDNA microarray technique is effective in screening the differentially expressed genes in endothelial cells induced by various experimental conditions and the data may serve as stimuli to further researches.

Cell Division↗

PubMed2004

Clinical evaluation of a new molecular method for detection of micrometastases in head and neck squamous cell carcinoma.

OBJECTIVE: To better detect occult cervical metastases. DESIGN: RNA from 153 cervical lymph nodes was analyzed for the presence of squamous cell carcinoma using quantitative cytokeratin (CK) 14 real-time reverse transcription polymerase chain reaction (RT-PCR). Detection of CK RNA in pathologically negative nodes was further analyzed by semi-step sectioning and CK immunohistochemistry. Subjects Thirteen consecutive patients with head and neck squamons cell carcinoma (HNSCC) presenting to the Department of Otolaryngology/Head and Neck Surgery of the University of North Carolina at Chapel Hill for neck dissection. RESULTS: Cytokeratin detection was deemed nonspecific if expressed at fewer than 50 molecules of CK 14 RNA per nanogram total RNA. Of 35 HNSCCs, 33 expressed CK 14 RNA, and 15 lymph nodes with routine pathologically positive metastasis were also positive for CK 14 RNA. Four lymph nodes that were pathologically negative nodes were positive for CK 14 RT-PCR, with 2 containing metastases detected by semi-step sectioning. CONCLUSIONS: Cytokeratin 14 RT-PCR is very sensitive for detecting micrometastasis in lymph nodes that are negative by routine pathological examination, with a relatively high false-positive rate. Quantitative CK 14 RT-PCR could be used to identify nodes negative for tumor by standard pathological analysis that should be examined by step sectioning and CK immunohistochemistry. Identification of micrometastases in patients with HNSCC will allow for appropriate and aggressive treatment of patients with metastatic disease.

Biomarkers, Tumor↗

PubMed2026

Dual Aberrant Splicing Caused by an Apparently Missense CHD7 Variant, c.5273A>G (p.Asp1758Gly), in CHARGE Syndrome.

CHARGE syndrome is a rare congenital disorder primarily attributed to heterozygous pathogenic variants of the CHD7 gene. Most pathogenic CHD7 variants are loss-of-function (LoF) variants, whereas the interpretation of missense variants remains challenging in the absence of functional evidence for their pathogenicity. We report a female infant presenting with clinical features characteristic of CHARGE syndrome. Targeted sequencing identified a heterozygous CHD7 variant (NM_017780.4:c.5273A>G), initially annotated as a missense substitution p.Asp1758Gly. This variant has been previously reported and registered with conflicting pathogenicity classifications; however, its transcript-level consequences remain unclear. Long-PCR-based RNA sequencing of total RNA from peripheral blood mononuclear cells revealed two aberrant splicing patterns associated with the variant: a predominant transcript carrying a 28-bp deletion due to cryptic donor splice-site activation, and a minor transcript with partial intron 24 retention. Both transcripts were predicted to result in premature termination codons. These findings demonstrate that c.5273A>G functions as a LoF variant through dual aberrant splicing rather than a simple missense substitution. This case underscores the importance of RNA-level splicing analysis for the accurate interpretation and classification of CHD7 missense variants.

CHD7↗

PubMed2005

Classification of human tumors using gene expression profiles obtained after microarray analysis of fine-needle aspiration biopsy samples.

BACKGROUND: Gene expression profiling using gene-discovery, high-density microarray technologies is a powerful tool. One potential application is the development of tumor classifiers that predict the site of origin. For this technology to be relevant, however, it must be applicable to tumor biopsy samples, which most often are fine-needle aspiration biopsy (FNAB) samples. METHODS: Surgically resected tumors were sampled by FNAB using different gauge needles. A portion of the excised tumor was also collected. RNA samples were extracted using standard techniques and the quality and quantity of the RNA samples were measured for each sample. Thirteen representative FNAB samples and two representative tissue samples were submitted for microarray analysis and then subjected to a tumor classifier. RESULTS: Fourteen of 18 samples analyzed for quantity and quality of RNA yielded an adequate amount of RNA (> 1 microg total RNA). Tumor type contributed to the RNA yield because one of the four inadequate samples was retrieved from a patient with lobular carcinoma of the breast and the other three samples were retrieved from patients with retroperitoneal sarcomas. Of the 13 samples submitted for microarray analysis, 9 were classified correctly as to tumor type using a tissue-based tumor classifier. CONCLUSIONS: The authors demonstrated that FNABs reproducibly obtained an adequate amount of RNA for microarray analysis when a standardized collection procedure was used. Furthermore, the samples generated interpretable gene expression profiles that could be matched accurately with a tumor classifier established on tissue specimens. The current study showed that FNAB produced adequate material for microarray analysis when utilizing a standardized collection procedure.

Biopsy, Fine-Needle↗

PubMed1997

Localization and cellular sources of activins in normal and fibrotic rat liver.

UNLABELLED: Activins are dimeric proteins, members of the transforming growth factor beta (TGF-beta) gene superfamily, consisting of the beta-subunits of inhibin (betaA and betaB). Recently, it was shown that activin A (betaA:betaA) inhibits DNA replication and induces apoptosis in rat parenchymal cells in vitro and in vivo. Cryostat sections of normal livers and livers of rats treated with intraperitoneal injections of CCl4 were stained for the different inhibin subunits and desmin, a marker for stellate cells (SC). Staining for inhibin-alpha was invariably negative, both in normal and fibrotic rat liver. In normal liver, inhibin-betaA subunit immunoreactivity was localized in parenchymal cells (PC). Staining for inhibin-betaB was weaker but similarly distributed. In fibrotic livers, connective tissue septa were strongly immunoreactive for inhibin-betaA. Desmin-positive stellate cells (SC) accumulated in areas strongly immunoreactive for inhibin-betaA and several cells were positive for both desmin and inhibin-betaA. Staining for inhibin-betaB was weaker but similarly distributed. As above data pointed to a possible role for PC and SC, we examined by Northern blot analysis, the expression of inhibin-alpha, -betaA, and -betaB messenger RNA (mRNA) in total RNA extracted from freshly isolated SC and PC of normal and CCl4-treated liver and in cultured SC. Inhibin-betaA mRNA was predominantly expressed in PC of normal liver. Expression was lost in PC of CCl4-treated liver. Inhibin-betaB mRNA expression was induced in SC of CCl4-treated liver. Inhibin-betaA mRNA, and to a lesser extent, inhibin-betaB mRNA expression was rapidly induced in cultured SC. The presence of activin A in conditioned media of cultured SC was shown by Western blotting. Apoptotic cells, identified by terminal deoxy-transferase mediated X-dUTP nick end labeling (TUNEL)-staining, were found predominantly in and near the fibrotic septa. IN CONCLUSION: 1) while activin A was constitutively expressed in PC of normal liver, its expression was lost in PC of fibrotic liver; 2) expression of activins was induced in activated SC; and 3) apoptotic cells were found predominantly near the septa, in support of the hypothesis that activin A, derived from activated SC in the septa, contributes to the induction of cell death in neighboring PC.

Activins↗