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PubMed1996

Very low level of major BCR-ABL expression in blood of some healthy individuals.

The nested reverse transcriptase polymerase chain reaction (RT-PCR) provides a powerful tool for detection of minimal residual disease in CML. The RT-PCR used in the present study for detection of the major bcr-abl fusion gene, the hallmark and presumably the cause of CML, was optimized by: (a) increasing the amount of total RNA involved in the reverse transcription reaction to correspond to total RNA extracted from 10(8) cells; (b) using a specific abl primer in this reverse transcriptase reaction, and (c) reamplifying 10% of the RT-PCR product in a nested amplification. This optimized RT-PCR permitted to detect up to 1 copy of RNA bcr-abl synthesized in vitro, mixed with yeast RNA in a quantity equivalent to 10(8) white blood cells (WBC). Using the highly sensitive RT-PCR, a systematic study of the possible expression of bcr-abl RNA in WBC of healthy adults, children and umbilical cord blood (UCB) revealed the presence of bcr-abl transcripts in blood cells of 22/73 adults, 1/22 children but not in 22 samples of UCB. The comparison of these three groups indicated a significant tendency for the anomaly to increase in frequency with age.

Adolescent↗

PubMedDate not supplied

Selection of a high activity ribozyme against cytostatic drug resistance-associated glypican-3 using an in vitro assay containing total tumor RNA.

We tested 11 hammerhead ribozymes for their ability to bind and cleave RNA transcripts of the cytostatic drug resistance-associated glypican-3-encoding gene (GPC3, MXR7, OCI-5). To select the optimum target sequence, the activity of each hammerhead ribozyme was tested in a short in vitro assay using truncated RNA substrates without time-consuming cloning procedures. Glypican-3-derived RNA was cleaved effectively by 3 of 11 hammerhead ribozymes. One of these, the hammerhead ribozyme Rz967, recognized the GUC sequence at nucleotides +965 to +967 of the open reading frame of the glypican-3-encoding mRNA. Rz967 cleaved in vitro-transcribed fragments derived from glypican-3 mRNA (nucleotides +803 to +1036) most efficiently. Cleavage efficiency was confirmed by a rapid in vitro assay using full-length total tumor cell RNA. We were able to demonstrate that this new in vitro assay is suitable for the selection of hammerhead ribozymes that have the capability to cleave glypican-3-encoding mRNA in human tumors.

Base Sequence↗

PubMedDate not supplied

Improved methods for extracting RNA from exfoliated human colonocytes in stool and RT-PCR analysis.

In order to diagnose colon cancer at an earlier, more localized stage, there is a need to develop diagnostic markers (genes) which can detect early patterns of gene expression in exfoliated colonocytes shed in the stool during routine screening for this disease. An RNA-based detection is more pertinent than either a DNA-based or a protein-based method as a screening procedure, but it has not been widely used as a cancer screen because of the difficulty of handling and stabilizing the RNA molecule. We describe a method that permits extraction of intact nondegraded total RNA from human colonocytes in stool and from normal and malignant colon tissues (which were employed for comparison with stool). Because it utilizes commercially available kits, this method is simpler than other published methods and does not require isolation of messenger (m)RNA, thereby reducing the chances of contaminating the preparations with degrading nucleases, and even a small amount of isolated total RNA can be adequately reverse transcribed, making high-quality copy (c) DNA. This is followed by PCR (either qualitative end point or semiquantitative real-time) using colon cancer-specific gene primers. By routinely and systematically being able to perform quantitative gene expression measurements on noninvasive samples, the goal of this pilot work is to lay the groundwork for conducting a large clinical study to identify groups of selected genes whose expression is consistently altered at an early stage in the neoplastic process. Such work will permit noninvasive monitoring of at-risk patients through the analysis of their stool samples. Correct diagnosis will allow for surgical and/or other interventions before the tumor is well established and, thus, should decrease mortality from this preventable disease.

Adenocarcinoma↗

PubMed1991

Injection of RNA from carp retina induces the formation of a membrane potassium channel in Xenopus oocytes.

Total RNA was purified from freshly isolated retinas of adult carp and injected into oocytes of Xenopus laevis (stages 5-6). Two to six days after injection, depolarizing voltage-clamp steps evoked a slowly activated outward current as large as 3 microA. This current inactivated slowly with a single time constant (tau = 3.1 +/- 0.24 S.E.M., for Vm = +30 mV). The current was inhibited by tetraethylammonium (3.8 mM for half-maximal inhibition). In the presence of Co2+ (1 mM) or barium methanesulfonate (40 mM), the current-voltage relationship shifted to slightly more depolarized values (5-10 mV); the maximal value of the current that was sensitive to Co2+ or Ba2+ treatments was only a small fraction (about 10%) of the TEA-sensitive current, and its current-voltage relationship was similar to that for uninjected oocytes. The reversal potential of the membrane current was studied with [K+]0 of 1-77 mM. For [K+]0 greater than 20 mM, the reversal potential changed with a slope of 63 mV (+/- 2 mV S.E.M.) per 10-fold change in [K+]0. The conductance was induced half-maximally at 17 mV (+/- 0.9 mV S.E.M.). The depolarization required for an e-fold increase in conductance was 13 mV (+/- 0.6 mV S.E.M.). From these results, we conclude that the injection of total RNA from carp retinas induces the formation of a membrane K+ channel in Xenopus oocytes. The channel formed has many of the properties reported for the maintained outward current of goldfish horizontal and bipolar cells.

Animals↗

PubMed2003

Validation of cDNA microarray gene expression data obtained from linearly amplified RNA.

BACKGROUND: DNA microarray technology has permitted the analysis of global gene expression profiles for several diseases, including cancer. However, standard hybridisation and detection protocols require micrograms of mRNA for microarray analysis, limiting broader application of this technology to small excisional biopsies, needle biopsies, and/or microdissected tissue samples. Therefore, linear amplification protocols to increase the amount of RNA have been developed. The correlation between the results of microarray experiments derived from non-amplified RNA and amplified samples needs to be evaluated in detail. METHODS: Total RNA was amplified and replicate hybridisation experiments were performed with linearly amplified (aRNA) and non-amplified mRNA from tonsillar B cells and the SUDHL-6 cell line using cDNA microarrays containing approximately 4500 genes. The results of microarray differential expression using either source of RNA (mRNA or aRNA) were also compared with those found using real time quantitative reverse transcription polymerase chain reaction (QRT-PCR). RESULTS: Microarray experiments using aRNA generated reproducible data displaying only small differences to data obtained from non-amplified mRNA. The quality of the starting total RNA template and the concentration of the promoter primer used to synthesise cDNA were crucial components of the linear amplification reaction. Approximately 80% of selected upregulated and downregulated genes identified by microarray analysis using linearly amplified RNA were confirmed by QRT-PCR using non-amplified mRNA as the starting template. CONCLUSIONS: Linear RNA amplification methods can be used to generate high fidelity microarray expression data of comparable quality to data generated by microarray methods that use non-amplified mRNA samples.

Gene Amplification↗

PubMed1996

Detection of a unique gamma-glutamyl transpeptidase messenger RNA species closely related to the development of hepatocellular carcinoma in humans: a new candidate for early diagnosis of hepatocellular carcinoma.

Many studies concerning gamma-glutamyl transpeptidase (GGTP) in hepatocellular carcinoma (HCC) have suggested that changes in hepatic GGTP expression may be closely related to the development of HCC. However, its mechanisms are not well known, and genomic analysis of the specific GGTP to HCC is also lacking. Recently, the human GGTP complementary DNA (cDNA) sequences from fetal liver, placenta, and HepG2 cells have been published. In the present study, we sought to clarify the distribution of the GGTP messenger RNA (mRNA) molecular species in human liver and determine whether alterations in GGTP mRNA expression occur upon the development of HCC. The specific primer sets for reverse-transcription polymerase chain reaction (PCR) corresponding to the 5'-noncoding human GGTP mRNA of fetal liver (type A), HepG2 cells (type B), and placenta (type C) were prepared. Oligonucleotide probes specific for each type of mRNA were also synthesized. Liver tissues were obtained from patients with or without HCC, and total RNA was extracted. Total RNA was also extracted from various organs obtained from one male patient upon autopsy. Types of GGTP mRNAs were analyzed using type-specific primer sets and oligonucleotide probes. The types of GGTP mRNA varied in different organs. In normal liver and diseased liver without HCC, the main type of GGTP mRNA was type A. The expression was monogenic in most cases but was polygenic in some cases. In the polygenic cases, type C was common, but type B was found occasionally. On the other hand, type B was predominant in cancerous tissues with HCC. In noncancerous tissues of livers with HCC, the main types were types A and B. The prevalence of type B was significantly higher in both cancerous and noncancerous tissues of livers with HCC than in livers without HCC. The prevalence of type A in cancerous tissue, but not in noncancerous tissue, was significantly lower than in livers without HCC. These results strongly suggested that the GGTP mRNA expression in human liver may shift from type A to type B during the development of HCC. The high prevalence of type B in noncancerous tissues suggested that the shift of the GGTP mRNA may occur from the preneoplastic stage of hepatocytes.

Base Sequence↗

PubMed2005

Fluorescent methods to study DNA, RNA, proteins and cytoplasmic membrane polarization in the pentachlorophenol-mineralizing bacterium Sphingomonas sp. UG30 during nutrient starvation in water.

The effect of sodium pentachlorophenolate (NaPCP) exposure on the nutrient-starved pentachlorophenol (PCP)-mineralizing bacterium Sphingomonas sp. UG30 was assessed using fluorescent methods to measure DNA, RNA, total cellular protein, and cytoplasmic membrane proteins. UG30 cells were inoculated into sterilized Speed River (Guelph, ON, Canada) water samples in the presence of 50, 100, and 250 ppm NaPCP. No marked changes were observed in the total cellular DNA, RNA or protein levels over 90 d, indicating the macromolecular composition of UG30 was not affected by both nutrient limitation and NaPCP. Total cell counts as determined by DAPI staining also did not change over 90 d. Over the same period, viable counts decreased with increasing concentrations of NaPCP. At 250 ppm NaPCP, viable cell counts decreased over 6 orders of magnitude after 1 hr exposure. Cell numbers partially recovered once NaPCP was degraded. The UG30 cytoplasmic membrane polarization ratio also decreased after NaPCP was depleted. The decreased polarization value at the end of the study period suggested the UG30 membrane was more fluid and that this increase in fluidity was due to nutrient starvation effects rather than exposure to NaPCP. These results indicated that UG30 is a robust organism that is able to degrade NaPCP even under adverse conditions and fluorescent methods are useful for determining macromolecular concentrations and cytoplasmic membrane polarization values.

Bacterial Proteins↗

PubMed2004

Reactive oxygen species induce RNA damage in human atherosclerosis.

BACKGROUND: Reactive oxygen species (ROS)-induced DNA damage has recently been identified in both human and experimental atherosclerosis. This study was undertaken to investigate whether RNA damage occurs in human atherosclerotic plaques and whether this could be related to oxidative stress. MATERIALS AND METHODS: The integrity of total RNA isolated from carotid endarterectomy specimens (n = 20) and nonatherosclerotic mammary arteries (n = 20) was analyzed using an Agilent 2100 Bioanalyser (Agilent Technologies, Palo Alto, CA). Oxidative modifications of RNA were detected by immunohistochemistry. RESULTS: Eleven out of 20 atherosclerotic plaques showed a significant reduction of the 18S/28S rRNA peaks and a shift in the RNA electropherogram to shorter fragment sizes. In contrast, all mammary arteries showed good-quality RNA with clear 18S and 28S rRNA peaks. Strong nuclear and cytoplasmic immunoreactivity for oxidative damage marker 7,8-dihydro-8-oxo-2'-guanosine (8-oxoG) could be detected in the entire plaque in smooth muscle cells (SMCs), macrophages and endothelial cells, but not in SMCs of adjacent normal media or in mammary arteries. Cytoplasmic 8-oxoG staining in the plaque clearly diminished when tissue sections were pretreated with RNase A, suggesting oxidative base damage of RNA. In vitro treatment of total RNA with ROS-releasing compounds induced RNA degradation. CONCLUSION: Both loss of RNA integrity and 8-oxoG oxidative modifications were found in human atherosclerotic plaques. Because RNA damage may affect in vitro transcript quantification, RT-PCR results must be interpreted cautiously if independent experimental validation (e.g. evaluation of RNA integrity) is lacking.

Arteriosclerosis↗

PubMed1997

Effects of the dihydropyridine calcium channel blocker amlodipine on ventricular and atrial protein synthesis in an aortic constriction model of hypertension and, following chronic treatment, in the left ventricle of SHR rats.

The dihydropyridine calcium channel blocking agent amlodipine is an effective anti-hypertensive agent and its use (in doses of 5 or 10 mg/day/kg body weight) was investigated in male Wistar rats with hypertension induced by aortic constriction. Controls were sham-operated and pair-fed. At the end of the study, rates of protein synthesis were measured with radiolabelled phenylalanine to calculate fractional rates of protein synthesis (ks), absolute rates of protein synthesis (Vs) and synthesis rates relative to RNA (kRNA). After 30 days of aortic constriction, weights of the left ventricle and left atrium were significantly increased by hypertension. The weights of the right ventricle and right atrium were relatively unaffected. Hypertension was accompanied by significant increases in the protein and RNA contents of the left ventricle and left atrium. The contractile and non-contractile protein contents were also increased in the left ventricles of hypertensive rats as were total proteins and total RNA. In the myofibrillary fraction, ks decreased. The right ventricle and right atrium were generally unaffected except for a decline in mixed protein ks. Many of these changes in hypertension were ameliorated by treatment with amlodipine, particularly at the higher dose (i.e. 10 mg/kg body weight/day) implicating an effect on protein metabolism. In the left ventricle these included amelioration of the increases in mixed and contractile proteins, total RNA contents, mixed Vs and Vs for sarcoplasmic and stromal proteins. The ameliorating effects of amlodipine were moderate in the left atrium. Furthermore, amlodipine also retarded the hypertension-induced reduction in right ventricule rates of protein synthesis. Although the preceding study emphasises the preventative aspects of amlodipine's efficacy, an additional study was carried out in SHR rats to ascertain the applicability for regression per se. Amlodipine (10 mg/kg/body weight) therapy for 30 weeks caused regression of LV mass, total protein, RNA and DNA contents. We conclude that amlodipine, is an efficient agent in ameliorating the hypertension-induced changes in protein metabolism in an aortic constriction model.

Amlodipine↗

PubMed1995

Transcriptional up-regulation of the mouse gene for the muscle-specific subunit of enolase during terminal differentiation of myogenic cells.

The glycolytic enzyme enolase (EC 4.2.1.11) exists as dimers formed from three structurally related subunits alpha, beta, and gamma, encoded by separate genes. The gene encoding the beta-subunit is expressed only in striated muscles. We have previously shown that the beta-enolase gene belongs to a small subset of muscle-specific genes showing transcriptional activity in cultured myoblasts, prior to withdrawal from the cell cycle. An increase in the level of beta-enolase mRNA occurs during terminal differentiation of myoblasts. To investigate the mechanisms underlying this increase, we have simultaneously estimated, under steady state conditions, the rate of synthesis and the stability of beta-enolase mRNA in proliferating C2.7 myoblasts as well as in differentiating myotubes. The method used is based on the isolation of newly synthesized RNA from the total RNA pool, following pulse-labeling of intact cells in the presence of 4-thiouridine. The results described here demonstrate a coordinate increase in newly synthesized and total beta-enolase mRNA, while the mRNA half-life, about 4 hr, remains unchanged in the course of terminal differentiation. The expression of the gene for insulin-like growth factor-II (IGF-II), a major positive regulator of myogenesis, was analyzed using the same approach. It is concluded that the up-regulation of beta-enolase as well as IGF-II gene expression in differentiating muscle cells reflects an increased rate of entry of newly synthesized mRNAs into the general pool of transcripts without changes in their respective half-lives.

Animals↗

PubMedDate not supplied

Development stage-specific expression of fibroin in the silk worm Bombyx mori is regulated translationally.

The contents of fibroin H RNA as a function of development have been quantitated in the posterior silk glands of Bombyx mori larvae on different days of 4th and 5th instars. The fibroin RNA levels increased during the feeding stages of larvae and the RNA got completely degraded during the interim moult. The patterns of accumulation of fibroin RNA were similar in both the instars. Although there was considerable increase in the fibroin RNA content during the 5th larval instar, the relative abundance of fibroin RNA in the total RNA was fairly constant during the 4th and 5th instars. The increased content of fibroin RNA in 5th instar was the consequence of an overall increase in transcription accompanying the development progress, rather than specific increase only in fibroin transcription. The contents of fibroin protein in the 4th and 5th instars of development have also been quantitated making use of a sensitive radioimmune assay with a purified, antifibroin antibody. There were substantial differences between 4th and 5th instars in the absolute fibroin contents as well as the relative proportion of fibroin in the total proteins. These results implied that although the fibroin gene was transcribed at the same efficiency during the 4th and 5th instars, the translational efficiency was much lower during the 4th instar. The extent of polyadenylation of fibroin RNA was similar in both instars. However, there was a two-fold increase in the polysome association of fibroin RNA in the 5th instar. Over and above this, there was substantial increase during the 5th instar in the contents of those tRNAs. (e.g. Gly, Ala and Ser) which are abundantly represented in fibroin and therefore directly related to the expression of fibroin. The increased polysome association of fibroin mRNA and the adequate supply of cognate tRNAs in the 5th instar, together contributes to the translational regulation of fibroin in a developmental stage-specific manner. Based on these observations, we propose that translational regulation plays a major role in the development stage-specific synthesis of fibroin in Bombyx mori.

Animals↗

PubMed1989

The cyclo-oxygenase inhibitors indomethacin and ibuprofen inhibit the insulin-induced stimulation of ribosomal RNA synthesis in L6 myoblasts.

Insulin stimulated total RNA accretion and the incorporation of [3H]uridine into RNA in L6 skeletal-muscle myoblasts. Incorporation of uridine into the rRNA was measured after either separation of 18 S and 28 S rRNA species by agarose-gel electrophoresis or separation of dissociated 40 S and 60 S ribosomal subunits on sucrose density gradients. Both methods showed a stimulation by insulin of uridine incorporation into the RNA of the two subunits. Two non-steroidal anti-inflammatory drugs, indomethacin and ibuprofen, which inhibit the metabolism of arachidonic acid by the cyclo-oxygenase pathway, inhibited the insulin-induced accretion of total cellular RNA and the incorporation of uridine into the RNA of both ribosomal subunits. The effect of insulin was observed both by using a tracer dose of [3H]uridine (5 microM) and in the presence of a high concentration (1 mM) of uridine to minimize possible changes in intracellular precursor pools. Neither insulin nor indomethacin was found to affect the incorporation of uridine into the total intracellular nucleotide pool, or the conversion of uridine into UTP. The ability of inhibitors of arachidonic acid metabolism to prevent insulin-induced increases in RNA metabolism suggests that a prostaglandin or other eicosanoid is involved in the signal mechanism whereby insulin stimulates RNA synthesis.

Animals↗

PubMed1996

Suppression of IFN-gamma production from Listeria monocytogenes-specific T cells by endogenously produced nitric oxide.

The induction of nitric oxide (NO) by IFN-gamma has been well documented in a variety of experimental settings, but so far there has been no report on whether the endogenously produced NO can suppress IFN-gamma production. In the present study, CD4+ T cells from Listeria monocytogenes-immune mice produced IFN-gamma upon stimulation with specific antigen and NO was generated in culture. When NG-monomethyl-L-arginine (NMMA) was added to the culture at a dose sufficient for the complete blockade of NO production, there was a significant level of enhancement of IFN-gamma production, which was also dose dependently correlated with addition of NMMA. RT-PCR revealed that IFN-gamma mRNA per given amount of total RNA remained the same irrespective of NO blockade by NMMA; however, total RNA recovery was significantly higher in the culture with NMMA. The endogenously produced NO suppressed T-cell proliferation which can be restored by the addition of NMMA. Sodium nitroprusside, a spontaneous NO generator, inhibited T-cell proliferation dose dependently and suppressed IFN-gamma production. Taken together, it may be concluded that NO down-regulates IFN-gamma production mainly by inhibiting T-cell proliferation.

Animals↗

PubMed1995

Cadmium chloride (CdCl2)-induced metallothionein (MT) expression in neonatal rat primary astrocyte cultures.

Metallothionein (MT) protein and mRNA levels were studied following exposure of rat neonatal primary astrocyte cultures to cadmium chloride (CdCl2). MT mRNA was probed on Northern blots with a 32P labeled synthetic cDNA probe specific for rat MT mRNA. The probe hybridizes to a single mRNA with a size appropriate for MT, approximately 550 bases. Expression of MT-I mRNA in astrocyte monolayers exposed to 2 x 10(-6) M CdCl2 for 6 h was increased approximately 5-fold (9.7 fg/micrograms total RNA) over MT-I mRNA levels in controls (2 fg/micrograms total RNA). MT-I mRNA could also be detected in untreated cells, suggesting constitutive MT expression in these cells. Western-blot analysis revealed a marked increase in MT protein levels upon exposure to CdCl2 (1 x 10(-6) M; 96 h). Consistent with the constitutive expression of MTs both at the mRNA level and protein level, we have also demonstrated a time-dependent increase in MT-immunoreactivity in astrocytes exposed to CdCl2. The present study suggests that astrocytes constitutively express MTs, and that MT-induction by CdCl2 may be an example of a generalized increase in MTs in response to heavy metal exposure, thus protecting astrocytes, and perhaps also indirectly, juxtaposed neurons from the neurotoxic effects of heavy metals.

Animals↗

PubMed2006

IGF-I is distinctly located in the bony fish pituitary as revealed for Oreochromis niloticus, the Nile tilapia, using real-time RT-PCR, in situ hybridisation and immunohistochemistry.

In bony fish, IGF-I released from the liver under the control of pituitary GH is the main endocrine regulator of growth, maintenance and development, and the amount of circulating IGF-I regulates synthesis and release of GH. In mammals and amphibia, evidence indicates that anterior pituitary endocrine cells also contain IGF-I. However, only preliminary and conflicting data exist on IGF-I gene expression in bony fish pituitary. Thus, we investigated the presence of IGF-I in the tilapia (Oreochromis niloticus) pituitary by quantitative real-time RT-PCR, in situ hybridisation and immunohistochemistry. The absolute amount of IGF-I mRNA in the whole pituitary (7.4+/-3.3 x 10(-3)pg/microg total RNA) was 1000-times lower than in liver (7.5+/-3.1 pg/microg total RNA). IGF-I peptide occurred in both neuro- and adenohypophysis but IGF-I gene expression was mainly restricted to the adenohypophysis. In the neurohypophysis, only few cells, probably pituicytes, contained IGF-I mRNA whereas IGF-I peptide was found also in numerous axons in the pars nervosa. In the adenohypophysis, both IGF-I mRNA and peptide were present in the majority of ACTH cells in all individuals investigated. In alpha-MSH cells, only IGF-I mRNA but no IGF-I peptide was detected likely suggesting an immediate release of IGF-I after synthesis. IGF-I mRNA and peptide were further observed in GH cells but their presence showed pronounced inter-individual differences likely due to the physiological, e.g., nutritional, status of the individual. IGF-I released from the GH cells may serve as auto/paracrine mediator of a negative feedback mechanism in addition to liver-derived endocrine IGF-I. Generally, the constitutive synthesis of IGF-I in ACTH cells and the varying content in GH and alpha-MSH cells suggest particular roles for IGF-I. Local IGF-I may regulate synthesis and release of pituitary hormones in an autocrine and/or paracrine manner as well as prevent apoptosis and stimulate proliferation of endocrine cells.

Adrenocorticotropic Hormone↗

PubMed2004

Selenoprotein mRNA is expressed in blood at levels comparable to major tissues in rats.

Liver glutathione peroxidase-1 (GPX1) mRNA is highly regulated by Se status relative to other parameters, but is of limited use for determining Se requirements in humans. To examine the efficacy of using blood for Se status assessment using molecular biology markers, we used a ribonuclease protection assay (RPA) to study mRNA levels in whole blood relative to 16 other rat tissues. Significant amounts of total RNA (>50 microg) were obtained from 1 mL of whole blood. Total RNA from 28-d postweaning Se-adequate (0.2 microg Se/g diet) male rats was analyzed for GPX1, GPX4, GPX3, thioredoxin reductase-1 (TRR1), and selenoprotein-P (SelP). RPA detected significant mRNA expression for at least 1 selenoprotein in all tissues except pancreas. GPX1 mRNA expression using this mix of RPA probes yielded the highest signal for GPX1 relative to the other selenoprotein signals in all tissues except testis; GPX1 expression was 4th highest in blood and similar to the major organs (liver, 1st; heart, 5th; kidney, 6th). Kidney was highest for GPX3, and testes was highest for GPX4, TRR1, and SelP. This study is the first to report the gene expression pattern for a number of selenoproteins and across a comprehensive set of tissues. The mRNA levels for all selenoproteins in blood were comparable to levels in the major organs, and decreases in blood and liver GPX1 mRNA levels in Se deficiency were similar, supporting potential use of whole blood for assessing Se status using molecular biology markers.

Animals↗

PubMed2000

Sympathetic activity in early renal posttransplantation hypertension in rats.

The contribution of elevated sympathetic activity to the development of renal posttransplantation hypertension was investigated. F1 hybrids (F1H) from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) were transplanted with either an SHR or an F1H kidney and bilaterally nephrectomized. Three weeks after transplantation, sympathetic activity was assessed by measuring adrenal tyrosine hydroxylase (TH) mRNA content and recording splanchnic nerve activity (SNA) in conscious animals. To investigate the dependence of arterial pressure on sympathetic activity, animals were treated with the alpha(2)-adrenoceptor agonist guanabenz intracerebroventricularly. Mean arterial pressure (MAP) was 143 +/- 4 mmHg in recipients of an SHR kidney (n = 15) versus 110 +/- 3 mmHg in recipients of an F1H kidney (n = 10; P < 0.001). Adrenal TH mRNA content was 1.93 +/- 0.15 fmol/microg total RNA in recipients of an SHR kidney versus 1.96 +/- 0.17 fmol/microg total RNA in recipients of an F1H kidney (not significant). SNA did not differ significantly between recipients of an SHR kidney (n = 8) and recipients of an F1H kidney (n = 7) in terms of frequency and amplitude of synchronized nerve discharges. In response to cumulative intracerebroventricular administration of 10 and 20 microg guanabenz, SNA fell to 51 +/- 5% of control in recipients of an SHR kidney versus 44 +/- 6% of control in recipients of an F1H kidney (not significant) accompanied by a slight fall in MAP in either group. The results suggest that elevated sympathetic activity is not a major contributor to the development of renal posttransplantation hypertension.

Animals↗

PubMed1990

[Reconstitution of rabbit brain GABA binding protein (receptor) on Xenopus oocytes].

Gurdon's Xenopus laevis oocyte translation system has been extensively employed to investigate the molecular biology of gamma-aminobutyric acid (GABA) receptors in the brains of embryonic chick, rat, etc. As GABA and its receptor may play a role in the pathogenesis of hepatic encephalopathy and a rabbit model of fulminant hepatitis has been established, we reconstituted the rabbit model of fulminant hepatitis has been established, we reconstituted the rabbit brain GABA binding protein (receptor) on Xenopus laevis oocytes to offer a base for further study of the pathological effects of GABA receptor at molecular level. In this report, total RNA was extracted from rabbit brain by guanidine hydrochloride method. The total RNA was then subjected to oligo-(dT)-cellulose affinity chromatography to isolate mRNA. All the RNA samples achieved a high purity with an average OD260/OD280 ratio of about 2.10. The clear bands of 18 S and 28S rRNA in electrophoresis implied that the RNA was not degraded and was therefore available for expression. After two days of incubation of the oocytes injected with mRNA, radioreceptor assay indicated that saturable and specifically displaceable GABA binding sites were implanted onto the oocyte membrane. This result led us to the conclusion that rabbit brain GABA binding protein (receptor) can be reconstituted by injecting exogenous mRNA inclusive of those encoding the receptor into Xenopus oocytes. However, the functional activity of the reconstituted receptor as a GABA gated chloride ion channel needs to be further characterized by patch clamping.

Animals↗