Search

Acute promyelocytic leukemia (APL) is characterized by the t(15;17) chromosomal translocation, which results in fusion of the retinoic acid receptor [alpha] (RARA) gene to another gene, most commonly promyelocytic leukemia (PML). The resulting fusion protein, PML-RARA, initiates APL, which is a subtype (M3) of acute myeloid leukemia (AML). In this report, we identify a gene expression signature that is specific to M3 samples; it was not found in other AML subtypes and did not simply represent the normal gene expression pattern of primary promyelocytes. To validate this signature for a large number of genes, we tested a recently developed high throughput digital technology (NanoString nCounter). Nearly all of the genes tested demonstrated highly significant concordance with our microarray data (P < 0.05). The validated gene signature reliably identified M3 samples in 2 other AML datasets, and the validated genes were substantially enriched in our mouse model of APL, but not in a cell line that inducibly expressed PML-RARA. These results demonstrate that nCounter is a highly reproducible, customizable system for mRNA quantification using limited amounts of clinical material, which provides a valuable tool for biomarker measurement in low-abundance patient samples., Introduction Here we describe what is, to our knowledge, the first use of a high-throughput digital system to assay the expression of a large number of genes in primary clinical [...]

Objectives: CYP2D6 levels are higher in many brain regions of human smokers in comparison with nonsmokers. We have shown that CYP2D is expressed in rat brain regions and that enzyme [...]

Administration of oxidized oils to rats or pigs causes a reduction of their cholesterol concentrations in liver and plasma. The reason for this effect is unknown. We tested the hypothesis that oxidized oils lower cholesterol concentrations by inhibiting the proteolytic activation of sterol regulatory element-binding protein (SRFBP)-2 in the liver and transcription of its target genes involved in cholesterol synthesis and uptake through an upregulation of gene expression of insulin-induced genes (Insig). For 6 d, 18 rats were orally administered either sunflower oil (control group) or an oxidized oil prepared by heating sunflower oil. Rats administered the oxidized oil had higher messenger RNA (mRNA) concentrations of acyl-CoA oxidase and cytochrome P450 4A1 in the liver than control rats (P < 0.05), indicative of activation of PPAR[alpha]. Furthermore, rats administered the oxidized oil had higher mRNA concentrations of Insig-1 and Insig-2a, a lower concentration of the mature SREBP-2 in the nucleus, lower mRNA concentrations of the SREBP-2 target genes 3-hydroxy-3-methyiglutaryl CoA reductase and LDL receptor in their livers, and a lower concentration of cholesterol in liver, plasma, VLDL, and HDL than control rats (P < 0.05). In conclusion, this study shows that reduced cholesterol concentrations in liver and plasma of rats administered an oxidized oil were due to an inhibition of the activation of SREBP-2 by an upregulation of Insig, which in turn inhibited transcription of proteins involved in hepatic cholesterol synthesis and uptake.

The objectives of the current study were to evaluate the correlation between reproductive status and steady-state levels of Myxovirus resistance gene (MX2) mRNA in peripheral blood leukocytes (PBL) of dairy heifers and the reliability of using change in MX2 messenger RNA (mRNA) for identification of nonpregnant heifers 18 to 19 d after AI. Holstein heifers (n = 266), 13 [+ or -] 1 mo of age, were assigned randomly to be inseminated (BRED; n = 214) or not (NONBRED; n = 52). Estrous cycles of all heifers were synchronized with an intravaginal insert containing progesterone for 7 d. At insert removal, heifers received an injection of PG[F.sub.2[alpha]]. Heifers in the BRED group were inseminated on detection of estrus or at a fixed time, 72 h after insert removal concomitant with a GnRH treatment. Heifers in the NONBRED group received an injection of GnRH 48 h after insert removal. Blood samples collected on d 0 (d of AI or estrus) and 18 were used to determine steady-state levels of MX2 mRNA. Samples collected on d 0, 7, 14, and 21 were analyzed for progesterone concentration. Pregnancy was determined retrospectively by progesterone concentration on d 21 and was diagnosed at 30 [+ or -] and 60 [+ or -]3 d after AI. The fold change in levels of MX2 mRNA from d 0 to 18 was greater for heifers classified and diagnosed as pregnant on d 21 (P < 0.05) and 30 [+ or -]1 (P < 0.05) and 60 [+ or -] 3 (P < 0.05) d after AI compared with nonpregnant (bred but not pregnant) and NONBRED heifers. Heifers that experienced pregnancy loss from 21 to 30 [+ or -] 1 (P = 0.11) or 21 to 60 [+ or -] 3 (P = 0.08) d of gestation tended to have smaller fold increases in MX2 mRNA expression than those that maintained pregnancy. The sensitivity (range 57.1 to 65.6%) and negative predictive values (range 47.9 to 57.1%) of determining reproductive status on d 18 according to the change in the level of MX2 mRNA expression in PBL were low, and the correlation between diagnosis of pregnancy by fold change in MX2 mRNA expression and other methods was small (r = 0.20 to 0.36). The current study indicates that increased expression of MX2 mRNA in PBL is related to pregnancy approximately 21, 30, and 60 d after AI in dairy heifers and that losses that occurred later in pregnancy were associated with lower fold increases in MX2 mRNA. However, using the change in MX2 mRNA expression was not a reliable method for diagnosis of pregnancy at 18 d after AI because of the low sensitivity and negative predictive value. Key words: gene expression, heifer, leukocyte, pregnancy

The expression of glucose-6-phosphatase (G6Pase) mRNA is repressed by insulin and stimulated by cAMP and dexamethasone, with the insulin effect dominant. Both lipids and glucose increase the expression of G6Pase mRNA under conditions in which insulin is either absent or at basal levels. The aim of the present study was to investigate dietary nutrient regulation of G6Pase mRNA and protein under postprandial conditions. Male rats (n = 6-8/group) were deprived of food for 48 h and then either remained food deprived (FD) or were refed diets containing 68% cornstarch and 12% corn oil (ST; % energy), 68% sucrose and 12% corn oil (SU) or 35% cornstarch and 45% corn oil (HF) for 3 h. Rats were anesthetized, blood was drawn from the portal vein, and the liver was removed and immediately processed for subsequent analyses. Energy intake over the 3-h refeeding period did not differ among groups (209 [+ or -] 25 kJ). Portal vein glucose and insulin were 5.0 [+ or -] 0.2 mmol/L and 90 [+ or -] 18 pmol/L, respectively, in FD rats and were not significantly different among the refed groups (14.5 [+ or -] 1.2 mmol/L and 1302 [+ or -] 154 pmol/L, respectively). Compared with the FD rats, G6Pase mRNA was ~50% lower in ST and HF groups, whereas it was ~1.6 fold higher in SU-refed rats (P < 0.05). G6Pase activity in whole liver homogenates was lower in ST and HF rats than in FD and SU rats. Insulin receptor substrate (IRS) phosphorylation, IRS-association with phosphatidylinositol 3 (PI3)-kinase and activation of protein kinase B (PKB) were not significantly different among the refed groups. However, glycogen synthase kinase-3[alpha] phosphorylation was lower and cAMP response element binding protein (CREB) phosphorylation was higher in SU rats than in ST and HF refed groups. Thus, the postprandial environment after ingestion of sucrose appears to overcome the dominant effects of insulin on G6Pase mRNA, perhaps via cellular changes that reduce phosphorylation of, and therefore activate, glycogen synthase kinase-3[alpha]. KEY WORDS: * liver * glucose uptake * fructose * rats

cDNA array technology has proven to be a powerful way to monitor global changes in gene expression patterns. Here, we present an approach that extends the current utility of cDNA arrays to allow the evaluation of the relative roles of transcription and mRNA turnover in governing gene expression on a global basis, compared with current individual gene-by-gene analyses. This method, which involves comparison of large-scale hybridization patterns generated with steady-state mRNA versus newly transcribed (nuclear run-on) RNA, was used to demonstrate the importance of mRNA turnover in regulating gene expression following several conditions of stress. transcription | stress response | gene expression

Analysis of p53-regulated (tumor suppressor gene-regulated) gene expression patterns has been carried out using oligonucleotide arrays. The oligonucleotide microarrays were used to measure mRNA levels from a large number of genes regulated by the p53 transcription factor. It was found that the nature to the p53 response in different mRNA species depends on the cell type, levels of p53 protein in a cell, and the type of inducing agent or event.

We examined the ontogeny of mRNA levels of IGF-I and -II, IGF type 1 (IGFI-R) and type II receptors (IGFII-R), IGF binding protein-1 and -3 (IGFBP-1 and -3), GH receptor (GHR), and tissue concentrations of IGF and IGFBP in the pancreas of pigs. Tissues were collected from fetuses at 90 and 110 d of gestation and from pigs at 1, 21, 90 and 180 d of age. Northern blots were performed using total RNA hybridized with 32p-labeled cDNA probes (human IGF-I and human IGFI-R) and cRNA probes (rat IGFII, human IGFII-R, human IGFBP-1, pig IGFBP-3, and pig GHR). There were two accelerated growth stages of the pancreas: the first one at 90 d of fetal life, which is characterized by cell hyperplasia (high ratio of DNA to body weight), and the second one at postnatal 90 d, which is attributed to cell hypertrophy (high ratios of pancreatic weight, RNA, and protein to DNA). The level of IGF-II mRNA and its tissue concentration were predominant during fetal life and low thereafter. The IGF-I mRNA level was high during fetal and early postnatal life and decreased thereafter. Messenger RNA levels of IGFI-R, IGFBP-3, and GHR and concentrations of IGFBP-1 and -2 were abundant during fetal and early postnatal life. In conclusion, IGF may be involved in various physiological periods of pancreatic development in pigs. Key Words: Pigs, Pancreas, Age, Messenger RNA, Insulin-Like growth factor

Results show that the use of a real-time reverse transcription-polymerase chain reaction assay system for correlating aflatoxin production by Aspergillus flavus is based on the assay system's ability to quantitatively estimate the aflatoxin biosynthetic gene nor-1 expression. Data indicate that after 4 days of incubation nor-1 mRNA can be detected, which increases rapidly and maximum aflatoxin B(sub)1 is reaced by 9 days of incubation.

Evaluation of ovarian POMC mRNA through quantitative RT-PCR analysis in Rana esculenta. Am J Physiol Cell Physiol 280: C1038-C1044, 2001.--The evaluation of changes in the expression of specific genes requires accurate measurement of the corresponding mRNA concentration, especially when the gene is expressed at a very low level. We previously showed that the proopiomelanocortin (POMC) gene is expressed in the ovary of the frog Rana esculenta, and, to evaluate its mRNA content in frog ovary, we have now developed a sensitive quantitative RT-PCR method. This study provides evidence for the validation of this method and for the effects of captivity and hypophysectomy on POMC gene expression in the ovary of this anuran. Our data indicate that ovarian POMC gene is involved in short-term captivity stress response and seems not influenced by pituitary. These results are discussed taking into account the knowledge of the role played by opioids in stress response; moreover, a local control of POMC gene expression is also suggested. frog; ovary; proopiomelanocortin gene expression; captivity; hypophysectomy

A 970 bp cDNA [Na.sup.+]/glucose cotransporter (SGLT1) was isolated and sequenced from chicken jejunum by reverse transcriptase polymerase chain reaction (RT-PCR) using primers based on conserved regions. Using the 970 bp PCR product as a specific probe, Northern Blot hybridization indicated a transcript of ca. 4 kb. The isolated chicken intestinal SGLT1 cDNA was used to quantitate mRNA expression. Glucose uptake activity and kinetics were determined in brush border membrane vesicles (BBMV) from jejunum tissue of chickens which were either fed, food-deprived or refed following food deprivation. Net glucose uptake to BBMV was higher (P [is less than] 0.02) in the control and refed chicks (149 [+ or -] 11.9, 139.6 [+ or -] 7.43 pmol [multiplied by] mg [protein.sup.-1] [multiplied by] [s.sup.-1]) than in food-deprived chicks (107 [+ or -] 4.23 pmol [multiplied by] mg [protein.sup.-1] [multiplied by] [s.sup.-1]). The [k.sub.m] (150 [micro]mol/L) and Vmax (1111.1 pmol [multiplied by] mg [protein.sup.-1] [multiplied by] [s.sup.-1]) were higher in the food-deprived chicks compared to control and refed birds (25, 24 [micro]mol/L and 227, 142 pmol [multiplied by] mg [protein.sup.-1] [multiplied by] [s.sup.-1], respectively). Expression of SGLT1 mRNA was significantly enhanced in the food-deprived and refed birds. In food-deprived chicks the lower affinity and higher activity of the SGLT1 transporter for glucose were accompanied by higher expression of mRNA which might indicate that the transporter was upregulated by low substrate concentration. Quantification of expression of intestinal mRNA of SGLT1 provides important information concerning control of nutrient uptake. J. Nutr. 130: 2174-2179, 2000. KEY WORDS: * chickens * small intestine * [Na.sup.+] * Glucose cotransporter (SGLT1) * gene sequence.

A specific, sensitive and reliable index for assessment of human zinc status has not been developed, and continues to present a considerable challenge for nutritionists in the trace element field. We have focused on metallothionein (MT) expression as a potential index. A protocol involving 16 men and a 10-d supplementation period plus a 4-d postsupplementation period was used to examine the relative response of MT expression in erythrocytes, monocytes, peripheral blood mononuclear cells (PBMC) and cells from a dried blood spot (DBS). Zinc was supplemented at the current adult male recommended dietary allowance (RDA) of 15 mg. Erythrocyte MT protein, as measured by ELISA, increased gradually to about twofold over the placebo group during zinc supplementation and remained elevated for 4 d postsupplementation. Competitive reverse transcriptase-polymerase chain reaction showed that MT mRNA levels in both monocytes and PBMC increased (up to 4.7- and 2.7-fold, respectively) after 2 d of supplementation, with greater expression in monocytes compared with PBMC. Total RNA extracted from dried blood spots, representing cells from 50 [micro]L of blood, showed a comparable change in MT mRNA upon zinc supplementation. In each leukocyte population isolated, when zinc supplementation was withdrawn, MT mRNA levels decreased. Collectively, these experiments show that, in men, MT gene expression increases during supplementation at the RDA, and that the DBS sampling method will be of value in measuring MT expression in a variety of clinical and survey situations. J. Nutr. 130: 2180-2187, 2000. KEY WORDS: * zinc * metallothionein * monocytes * lymphocytes * gene expression * humans

Plasma cholesterol concentration is reduced by feeding some dietary fibers and mushroom fruit body, but the mechanism is not fully understood. We examined the effects of mushroom (Agaricus bisporus) fiber and sugar beet fiber on serum cholesterol and hepatic LDL receptor mRNA in rats. Rats were fed a cholesterol-free diet with 50 g/kg cellulose powder (CP), 50 g/kg mushroom (Agaricus bisporus) fiber (MSF) or 50 g/kg sugar beet fiber (BF) for 4 wk. There were no significant differences in the body weight, food intake and cecum weight among the groups. The relative liver weight in the CP group was significantly greater than that in the MSF and BF groups. The cecal pH in the CP and MSF groups was significantly higher than that in the BF group. Cecal acetic acid, butyric acid and total short-chain fatty acid (SCFA) concentrations in the BF group were significantly higher than those in the other groups. The serum total cholesterol, VLDL + intermediate density lipoprotein (IDL) + LDL cholesterol concentrations in the CP group were significantly greater than those in the MSF and BF groups. The HDL cholesterol concentration in the MSF group was significantly lower than that in the CP group. The hepatic LDL receptor mRNA level in the MSF and BF groups was significantly higher than that in the CP group. The results of this study demonstrate that mushroom fiber and sugar beet fiber lowered the serum total cholesterol level by enhancement of the hepatic LDL receptor mRNA. J. Nutr. 130: 2151-2156, 2000. KEY WORDS: * rats * mushroom (Agaricus bisporus) fiber * sugar beet fiber * cholesterol * LDL receptor mRNA

Severely vitamin B-12 (B-12)-deficient rats were produced by feeding a B-12-deficient diet. The status of B-12 deficiency was confirmed by an increase in urinary methylmalonate excretion and decreases in liver B-12 concentrations and cobalamin-dependent methionine synthase activity. Rat liver methionine synthase existed almost exclusively as the holoenzyme. In B-12-deficient rats, the level of methionine synthase protein was lower, although the mRNA level was not significantly different from that of control rats. When methylcobalamin, the coenzyme for methionine synthase, was administered to the B-12-deficient rats, growth, liver B-12 concentrations and urinary excretion of methylmalonate were reversed although not always to control (B-12-sufficient) levels in a short period. During this recovery process, methionine synthase activity and its protein level increased, whereas the mRNA level was unaffected. We reported previously that rat apomethionine synthase is very unstable and is stabilized by forming a complex with methylcobalamin. Thus, the extremely low activity of methionine synthase in B-12-deficient rats may be related to effects on 'coenzyme stabilization' (stabilization of the enzyme by cobalamin binding) rather than to changes in 'coenzyme induction.' J. Nutr. 130: 1894-1900, 2000. KEY WORDS: * vitamin B-12. * cobalamin * methionine synthase * vitamin B-12 deficiency * rats

The multiplex messenger assay (MMA) is a new method for measuring the gene activity of different cell lines. Based on the polymerase chain reaction technique, MMA utilizes immobilized cDNA as targets for labeled RNA, and determines the total amount of messenger RNA (mRNA) based on the signal emitted on the X-ray film. Some also called it the 'reverse Northern blot' because the DNA is probed with RNA, which is the reverse of the normal procedure for performing Northen blots. Variations of this method have also been tested using samples which have low amounts of mRNA.

Methods and reagents is a unique monthly column that highlights current discussions in the newsgroup bionet.molbio.methds-reagnts, available on the Internet. This month's column discusses quantitative measurements of messenger RNA levels by using reverse transcription polymerase chain reaction (RT-PCR). For details on how to partake in the newsgroup, see the accompanying box., The use of reverse transcription polymerase chain reaction (RT-PCR) for quantitative measurements of messenger ribonucleic acid suffers from a poor reputation because of its misuse. Since it requires the approximation of the intensity of the amplified deoxyribonucleic acid fragment via densitometry, the northern blotting and RNase protection assay methods are recommended to confirm results obtained from RT-PCR.