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At least 811 records · Page 45Linked to original sources
PubMed2004

Impact of fixative on recovery of mRNA from paraffin-embedded tissue.

Due to the evolution of advanced tissue-analysis tools, such as proteomics and functional and structural genomics, the demands for handling and preserving samples are changing. For gene expression analysis, the presence of intact and extractable messenger RNA in the test material is mandatory. To find an optimal fixative for tissues aimed for such analyses, we evaluated the morphology-, protein antigen-, and RNA-maintaining abilities of 2 precipitating tissue fixatives, methanol-acetone and Carnoy's. Both fixatives preserved the morphology and protein epitopes of tissues and allowed extraction of total RNA that was of significantly higher quality than RNA extracted from formalin-fixed tissue. Carnoy's fixative performed better than methanol-acetone in maintaining the integrity of RNA, especially when the fixed, paraffin-embedded tissue blocks were stored at room temperature for more than 3 months. Total RNA extracted from epithelial cells microdissected from Carnoy's-fixed tissue samples contained intact template for up to a 977-base pair (bp) amplicon for beta-actin. Because of the emerging role of gene expression analyses in research, and in clinical work in the near future, an RNA-preserving fixative should replace formalin as the primary human tissue fixative. According to our data, Carnoy's fixative is an excellent candidate for a new primary fixing reagent for human tissue samples.

Acetic Acid↗
PubMed1999

Transcription of the adenine nucleotide translocase isoforms in various types of tissues in the rat.

Two different isoforms of the adenine nucleotide translocase (ANT1 and ANT2) have been identified in the rat. In order to obtain enhanced knowledge of the ANT isoform expression, we analyzed the transcription pattern of both isoforms and their mRNA levels in various tissues of the rat using the PCR technique. A predominant ANT1 mRNA percentage was recorded in the skeletal muscle, heart and brain, ranging from 81 to 58%. In contrast to these tissues, the percentages of ANT2 were dominant with a range from 59 to 75% in the kidney, lung, spleen and liver. The level of total ANT mRNA varied markedly in the various organs. Tissues with a dominant ANT1 percentage simultaneously showed a high level of total ANT transcription (24-41 attomol/ng total RNA). In comparison to the latter, tissues with a prevalent ANT2 transcription were shown to have an even lower ANT transcription level (2-5 attomol/ng total RNA). The predominance of the ANT1 expression appeared to be restricted to tissues with an inability to regenerate by means of mitotic division, whereas a prevalent ANT2 transcription is found in cell types able to proliferate. The level of total ANT transcription but not the individual ANT isoform expression depends to a great extent on the energy requirements of the tissue.

Animals↗
PubMed1975

Changes in insulin content of, and insulin and RNA synthesis in, isolated rat islets of Langerhans after hypophysectomy and short-term growth hormone replacement.

Three to four weeks after hypophysectomy, the insulin content and glucose-stimulated insulin or total RNA synthesis in the pancreatic islets were estimated. The ratio of the glucose-stimulated insulin release to the islet insulin content was lowered. After short-term GH replacement, insulin release and insulin content were slightly increased such that the ratio of release to content remained virtually unaltered. Glucose-stimulated insulin synthesis was also depressed but improved slightly after GH replacement. However, there was no significant change in the incorporation of [5-3H]uridine into islet total RNA after hypophysectomy or subsequent GH replacement.

Animals↗
PubMed1997

Use of internal controls to increase quantitative capabilities of the ribonuclease protection assay.

Through the use of two internal controls, we have developed an improved method of quantitating ribonuclease protection assay (RPA) results. A truncated sense RNA fragment and an antisense RNA fragment for the gene of interest were transcribed from PCR fragments containing T7 bacterial promoters. An 18S ribosomal RNA fragment was also used. When radiolabeled antisense and 18S probes, along with sense fragment and sample RNA, were hybridized, digested with RNase A/T1 and gel-electrophoresed, three distinct bands resulted. The antisense RNA fragment bound to the sense RNA fragment confirmed the integrity of the reaction. The antisense RNA fragment bound to endogenous mRNA measured the amount of specific gene expression in the sample. The 18S RNA fragment bound to endogenous mRNA determined the actual amount of sample added to the gel. Using the specific activities of the antisense and 18S transcripts, and scintillation counts of the protected fragments, we calculated the amounts of message and total RNA on the gel, determining picogram of message per microgram of total RNA. Final results were not based on assumed original amounts of RNA placed in the assay nor were they biased by lane-to-lane variations. Through the described adaptations, we have developed a well-controlled RPA that accurately and reproducibly quantifies gene expression.

Animals↗
PubMed2001

Regional variation in myosin isoforms and phosphorylation at the resting tone in urinary bladder smooth muscle.

Urinary bladder filling and emptying requires coordinated control of bladder body and urethral smooth muscles. Bladder dome, midbladder, base, and urethra showed significant differences in the percentage of 20-kDa myosin light chain (LC20) phosphorylation (35.45 +/- 4.6, 24.7 +/- 2.2, 13.6+/- 2.1, and 12.8 +/- 2.7%, respectively) in resting muscle. Agonist-mediated force was associated with a rise in LC20 phosphorylation, but the extent of phosphorylation at all levels of force was less for urethral than for bladder body smooth muscle. RT-PCR and quantitative competitive RT-PCR analyses of total RNA from bladder body and urethral smooth muscles revealed only a slight difference in myosin heavy chain mRNA copy number per total RNA, whereas mRNA copy numbers for NH2-terminal isoforms SM-B (inserted) and SM-A (noninserted) in these muscles showed a significant difference (2.28 x 10(8) vs. 1.68 x 10(8) for SM-B and 0.12 x 10(8) vs. 0.42 x 10(8) for SM-A, respectively), which was also evident at the protein level. The ratio of COOH-terminal isoforms SM2:SM1 in the urethra was moderately but significantly lower than that in other regions of the bladder body. A high degree of LC20 phosphorylation and SM-B in the bladder body may help to facilitate fast cross-bridge cycling and force generation required for rapid emptying, whereas a lower level of LC20 phosphorylation and the presence of a higher amount of SM-A in urethral smooth muscle may help to maintain the high basal tone of urethra, required for urinary continence.

Animals↗
PubMed2004

Hyperexpression of low-density lipoprotein receptors and hydroxy-methylglutaryl-coenzyme A-reductase in human pinguecula and primary pterygium.

PURPOSE: There is now increasing evidence that pterygium and pinguecula are tumorlike tissues and that cell growth and DNA replication are closely linked to cholesterol metabolism. In this study, the expression of two main genes correlated to cholesterol metabolism--namely, the low-density lipoprotein receptor (LDL-R) gene and the hydroxy-methylglutaryl-coenzyme A-reductase (HMG-CoA-R) gene--were investigated in primary pterygium, pinguecula, and normal conjunctiva. METHODS: Pterygium, pinguecula, and normal conjunctiva samples were obtained from 30 eyes (50% men) at the time of surgery. Total RNA extracted from the specimens was subjected to semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Equal amounts of total RNA were reverse transcribed into cDNA. The cDNA was subsequently amplified by the PCR in the presence of specific primers for low-density lipoprotein receptor (LDL-R) and for hydroxy-methylglutaryl coenzyme A reductase (HMG-CoA-R). RESULTS: Semiquantitative RT-PCR analysis revealed that the mRNA levels of LDL-R and HMG-CoA-R were increased in pterygia, compared with levels in both pingueculae and normal conjunctivae. Differences were statistically significant (P <0.05), including pingueculae versus normal conjunctivae. CONCLUSIONS: This study indicates that pterygium and pinguecula have an altered metabolism of cholesterol-namely increased LDL-R and HMG-CoA-R mRNAs-as is characteristic of tumorlike tissues, and that the high expression of LDL receptors renders them amenable to be treated by photodynamic therapy with intravenously injected verteporfin.

Cholesterol↗
PubMed1999

[Expression of soluble 55 kD receptor for human tumor necrosis factor using baculovirus vector].

Total RNA was isolated from HeLa cells and about 530 bp cDNA encoding shTNFR55 was amplified with RT-PCR from the total RNA. The cDNA was cloned into the downstream of polyhedrin gene promoter in transfer vector pAcGP67B, and a recombinant transfer plasmid pAcTNFR was constructed. Insect cell sf9 was co-transferred with recombinant plasmid pAcTNFR and baculovirus AcNPV DNA. Recombinant virus AcNPV-TNFR containing shTNFR55 gene were formed by homologous recombination in insect cells. They were purified by plaque assay and were confirmed by DNA dot blot. The biological activity of expressed product was determined with culturing TNF-susceptible L929 cell line, and the result showed that the expressed product could neutralize the cytotoxicity of TNF on L929 cell line. Ligand blot analysis revealed the expressed protein three discrete bands between 20-25 kD.

Animals↗
PubMed1978

Analysis of RNA initiated in isolated mouse myeloma nuclei using purine nucleoside 5'[gamma-S]triphosphates as affinity probes.

The purine ribonucleoside triphosphate analogues adenosine 5'[gamma-S]triphosphate and guanosine 5'[gamma-T]triphosphate were used as affinity probes for studying RNA chain initiation in isolated nuclei from the mouse myeloma 66.2 cell line. Transcripts initiated with either nucleotide analogue were isolated by affinity chromatography on a mercury-agarose affinity column. The binding was specific and dependent upon the inclusion of the sulfur nucleotide analogues in the in vitro synthetic reaction. Several lines of evidence indicate that the affinity-labeled RNA is initiated in vitro. First, the sulfur nucleotide is recovered in high yield as a single nucleoside 5'[gamma-S]triphosphate 2',3'-monophosphate product following alkaline hydrolysis of RNA bound to the affinity column. Second, authentic ribosomal 5S RNA is known to initiate with GTP; in vitro 5S RNA is bound to mercury-agarose only if [gamma-S]GTP is used as the affinity label in the synthesis, and not if [gamma-S]ATP is used. Under the conditions studied, nuclei incorporated 1.2--2.4 pmole of UMP per 10(6) nuclei per min, and the rate of synthesis was unaffected by substitution of the nucleotide analogues for the normal nucleotides. The percentage of the total RNA synthesized that was incorporated into sequences initiated in vitro was 7.8 +/- 1.5% with [gamma-S]ATP and 9.6 +/- 6.4% with [gamma-S]GTP. The size of the total RNA synthesized, determined by sedimentation on sucrose density gradients containing dimethylsulfoxide, ranged from less than 5S to 45S, and the size of the affinity-labeled sequences ranged from less than 5S to 28S. Approximately half of the incorporation into RNA initiated in vitro was sensitive to a concentration of alpha-amanitin which selectively inhibits polymerase II activity. Most of the remaining incorporation into initiated sequences can be abolished by concentrations of alpha-amanitin that are inhibitory for polymerase III activity. Over 70% of the total incorporation into ribosomal 5S RNA transcripts was into sequences initiated in vitro. This initiation was catalyzed by polymerase III and was specific for GTP as the initiating nucleotide. The RNAase T1 fingerprint of the newly initiated 5S RNA indicates that this gene is accurately initiated and faithfully elongated in vitro. The use of these affinity label probes provides much greater sensitivity for studying the initiation of RNA in vitro.

Amanitins↗
PubMed1991

Histochemical study of RNA content of neurones in the dorsal lateral geniculate nucleus during postnatal development.

Nuclear and cytoplasmic dLGN neurons were investigated by cytophotometric measurements of RNA. This study has been carried out in rats from birth to adulthood. In order to quantify the RNA content a cytophotometer was used. Extinction mean values were obtained which indicated RNA concentrations per surface unit. The nuclear and cytoplasmic surface were calculated simultaneously and from the product of the mean extinction and the surface the RNA total content was calculated. Our results have suggested that the changes are age-related. From day 1 to day 21 the neuronal size and RNA content increase; this may somehow be involved with the differentiation process. Around post-natal day 21 neuronal maturation may begin, reaching its optimal phase around day 42, on which the RNA concentration per surface unit, surface neuronal content and RNA total content are stable.

Analysis of Variance↗
PubMed1992

Regulation of hepatic angiotensinogen synthesis and secretion by steroid hormones.

The regulation of angiotensinogen gene expression by steroid hormones in the rat liver has been examined. In the intact animal, dexamethasone (7 mg/kg ip) and estradiol (7 mg/kg sc) caused an increase in plasma angiotensinogen, which became first apparent after 5 or 9 h, respectively, and resulted in plasma concentrations 4.6- and 1.9-fold higher than in controls at 24 h. These changes were preceded by comparable increases in hepatic angiotensinogen messenger RNA (mRNA). In contrast, dihydrotestosterone (10 mg/kg sc) failed to alter plasma angiotensinogen, although hepatic angiotensinogen mRNA and total RNA were slightly elevated. In isolated hepatocytes exposed to either dexamethasone or estradiol (10 microM each) angiotensinogen mRNA started to increase within less than 1 or 3 h, respectively, followed, with a further time lag of about 2 h, by an increase in secretion rate of angiotensinogen. Dihydrotestosterone (10 and 100 microM) induced a rapid increase in total hepatocyte RNA (1.3-fold) and angiotensinogen mRNA (2-fold) with a peak at 2 h. Surprisingly, angiotensinogen secretion remained either unaltered (10 microM dihydrotestosterone) or even decreased (100 microM dihydrotestosterone). In a hepatoma cell line (FT02B) and a subclone (Fe 33) stably transfected with the human estrogen receptor, dexamethasone and estradiol induced an increase in angiotensinogen mRNA and secretion with the same characteristics as in hepatocytes. In conclusion, in this study a direct effect of estradiol on angiotensinogen mRNA and secretion in hepatocytes could be established, which differs from that of dexamethasone by a delayed onset of action. The observation, both in vivo and in vitro, that dihydrotestosterone induced an increase in total RNA and angiotensinogen mRNA, which is not accompanied by an increased angiotensinogen secretion, cannot be explained at present. This study also demonstrates the usefulness of a hepatoma cell line stably transfected with the estrogen receptor gene for the investigation of estrogen-dependent effects in vitro.

Analysis of Variance↗
PubMed1984

Frequency of N-benzyladenine incorporation into major RNA fractions of tobacco cell suspensions grown at stimulatory or cytostatic cytokinin concentration.

The frequency of incorporation of the cytokinin N(6)-[p-(3)H]benzyladenine into major RNA species of tobacco (Nicotiana tabacum cv W 38) cells steadily increased as a function of its concentration in the culture medium, up to a 10 micromolar cytostatic overdose. During a 55-hour incubation of cells with 0.4 micromolar benzyladenine (BA), which is the optimal concentration for cell division, the incorporation frequency increased to one BA per 1.5 to 2.0 x 10(4) conventional bases in total RNA. Frequencies of BA incorporation into 18S and 25S rRNA and into RNA precursors were very similar, 2- to 3-fold higher than the frequency of BA incorporation into the 4S + 5S RNA fraction. In cells incubated with 10 micromolar BA, the rate of RNA synthesis between 24 and 55 hours was lower than at optimal growth conditions; 18S and 25S rRNA synthesis was depressed more than the synthesis of 4S + 5S RNA. At 55 hours, BA was incorporated into total RNA at the steady state frequency of one per 1,300 conventional bases. All major RNA species were BA-labeled to approximately the same level, except that the labeling of the RNA precursors was 2-fold higher than the labeling of mature RNA species. These results may reflect an alteration in the processing of the RNA precursors at supra-optimal cytokinin concentration.

Journal Article↗
PubMed2004

Real-time RT-PCR for gene expression profiling in blood of heart failure patients-a pilot study: gene expression in blood of heart failure patients.

Cardiovascular diseases are associated with multiple changes in gene expression. In general, cardiac tissue is not accessible to expression analysis. This study was designed to investigate expression of cardiac significant genes in white blood cells of heart failure patients and to identify differentially expressed genes. The quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) method was used for quantification of messenger RNA (mRNA) transcripts in blood samples of 20 patients (NYHA III-IV) with severe heart failure and of 20 healthy controls (NYHA I). Total RNA was extracted from leukocytes, reverse transcribed into cDNA, amplified and quantitated by SYBR Green detection. Relative mRNA expression was calculated using phosphoglycerate kinase-1 ( PGK-1) gene expression as an endogenous reference. Identified were 14 genes relevant to cardiomyocyte excitability or contractility. Most of them had not been previously reported to be expressed in blood cells. Data was based on 0.5 micro g total RNA applied to RT-PCR and on leukocyte number. In both, an increased transcription level of the Na/Ca exchanger ( NCX) was found in blood of heart failure patients as compared to controls (p < 0.02), in line with an upregulated NCX expression known from myocardial tissue of heart failure patients. This pilot study demonstrates that NCX transcription increased in potential relation to heart failure disease.

Adult↗
PubMed2002

Linear amplification of catalyzed reporter deposition technology on nylon membrane microarray.

The application of microarray analysis to gene expression from limited tissue samples has not been very successful because of the poor signal qualityfrom the genes expressed at low levels. Here we discussed the use of catalyzed reporter deposition (CARD) technology to amplify signals from limited RNA samples on nylon membrane cDNA microarray. When the input RNA level was greater than 10 microg, the genes expressed at high levels did not amplify in proportion to those expressed at low levels. Compared to conventional colorimetric detection, the CARD method requires less than 10% of the total RNA used for amplification of signal displayed onto a nylon membrane cDNA microarray. Total RNA (5-10 microg), as one can extract from a limited amount of specimen, was determined to produce a linear correlation between the colorimetric detection and CARD methods. Beyond this range, it can cause a nonlinear amplification of highly expressed and low-abundance genes. These results suggest that when amplification is needed for any applications using the CARD method, including DNA microarray experiments, precaution has to be taken in the amount of RNA used to avoid skew amplification and thus misleading conclusions.

Cell Line↗
PubMed1987

Accumulation kinetics of cotton late embryogenesis-abundant mRNAs and storage protein mRNAs: coordinate regulation during embryogenesis and the role of abscisic acid.

The accumulation of total RNA transcripts of 18 late embryo-abundant (Lea) gene families, each encoding two closely related Lea mRNAs, was measured in cotyledon total RNA during embryogenesis and germination of Gossypium hirsutum L. by RNA dot hybridization. Transcript abundance of the three storage protein families was also followed. The Lea mRNAs belong to only two related groups of commonly regulated mRNAs. The transcript level of each of the 6 members of Class I has two transient maxima during early maturation and a maximum level at 3 days prior to desiccation. The transcript level of each of the 12 members of Class II increases abruptly in late maturation with a maximum concentration at 3 days before desiccation (Class IIA) or at desiccation (Class IIB). Several patterns of early accumulation also exist within Class II, some overlapping with those of storage protein or Lea Class I mRNAs. The concentrations of Lea mRNAs increase at least 10- to 1700-fold during embryogenesis and decline 15- to 220-fold during the first day of germination. Earlier studies indicated that most Lea mRNAs, but no storage protein mRNAs, are induced in excised embryos exposed to abscisic acid (ABA). Free (+)ABA was measured during embryogenesis using a monoclonal antibody-based ELISA. The ABA concentration shows maxima correlating with the maxima in early Class I Lea mRNA abundance. However, ABA declines during the accumulation of Class I and Class II Lea mRNAs in late embryogenesis. Consequently, ABA could be an endogenous regulator of Class I Lea mRNAs during early maturation but is not the primary regulator of Lea mRNAs during late maturation. In contrast, cotyledon water potential exhibits a decrease that correlates with the late induction of Lea mRNAs. These findings are consistent with the hypothesis that low water potential is the endogenous regulator of some ABA-inducible mRNAs. The cessation of vascular flow to the embryo may also be involved in the disappearance of storage protein mRNAs and the late induction of Lea mRNAs.

Abscisic Acid↗
PubMed2005

Differential expression of the duplicated cellular retinoic acid-binding protein 2 genes (crabp2a and crabp2b) during zebrafish embryonic development.

The cellular retinoic acid-binding protein 2 (CRABP2) is believed to be involved in regulating access of retinoic acid to nuclear retinoic acid receptors. We have determined the cDNA sequence and the genomic organization of the duplicated crabp2 gene (crabp2b) in zebrafish. The crabp2b cDNA was 522bp in length and encodes a polypeptide consisting of 146 amino acids. Radiation hybrid mapping assigned the crabp2b gene to zebrafish linkage group 19. The comparison of the mapped human CRABP2 gene, zebrafish crabp2a and zebrafish crabp2b genes revealed that human chromosome 1 has a syntenic relationship to zebrafish linkage groups 16 and 19. Reverse transcription-polymerase chain reaction (RT-PCR) detected crabp2b mRNA in total RNA extracted from whole adult zebrafish, but not in any of the adult zebrafish tissues examined. The crabp2a mRNA was detected in total RNA extracted from whole adult zebrafish, adult zebrafish muscle, testes, and skin and to a lesser extent in heart, ovary and brain. No crabp2a mRNA-specific product was detected in kidney, liver or intestine of the adult zebrafish. Whole mount in situ hybridization detected crabp2b and crabp2a mRNA in a number of structures known to require retinoic acid signaling during embryonic development. The crabp2b mRNA was detected in the central nervous system, branchial arches, pectoral fins, retina (dorsal to the lens), epidermis and otic vesicle of the developing zebrafish. The crabp2a transcripts were detected by whole mount in situ hybridization in the central nervous system, epidermis, proliferative zone of the retina, intestinal bulb, oesophagus, pectoral fins and branchial arches during zebrafish embryonic development.

Amino Acid Sequence↗
PubMed2002

Assessment of specific mRNA levels in cartilage regions in a lapine model of osteoarthritis.

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 molecular and structural changes occurring in four articular cartilage (AC) regions from the knees of anterior cruciate ligament (ACL)-transected rabbits at 3 and 8 weeks post-surgery. Rabbit AC from the lateral and medial femoral condyles (LFC and MFC) as well as from the medial and lateral tibial plateau (MTP and LTP) were processed for histology and for semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for a subset of relevant molecules (collagen II, aggrecan. biglycan, decorin, fibromodulin, MMP-1, -3, -13, and TIMP-1). While the most severe histological changes were observed in the MTP starting as early as 3 weeks post-ACL transection based on Mankin scores, histological examination demonstrated a progression of osteoarthritic changes in the MFC from 3 to 8 weeks post-surgery. In contrast, very few changes were observed within both the LFC and LTP, and these changes did not worsen with increasing time after surgery. The water content increased significantly in the MFC at 8 weeks post-ACL transection and at both 3 and 8 weeks post-ACL transection in the MTP. Significant decreases in DNA content were observed for the MFC, LTP and MTP at 8 weeks post-ACL transection. Total RNA yields from the MFC and MTP were significantly elevated at 8 weeks post-ACL transection, while in the lateral compartment total RNA was unchanged following ACL transection. Analysis of mRNA levels for a subset of matrix molecules, proteinases and proteinase inhibitors, by RT-PCR demonstrated significant region-specific changes at the mRNA level following ACL transection. These results show that following ACL transection, complex molecular, as well as structural changes occur early in cartilage and that the observed changes are both region-specific and time-dependent.

Animals↗
PubMed2003

Influence of zinc deficiency on the mRNA expression of zinc transporters in adult rats.

The accumulation of zinc in the cell is a sum of influx and efflux processes via transporter proteins, like the four Zn transporters (ZnT1-4), the divalent cation transporter 1 (DCT1) and of storage processes mainly bound to metallothionein (MT). To study the effect of Zn deficiency on mRNA expression levels, adult rats were used as an animal model. Food intake was restricted to 8 g/day containing 2 microg Zn/g fortified with pure phytate in Zn deficiency rats and 58 microg Zn/g in controls (n = 7). At day 1, 2, 4, 7, 11, 16, 22, and 29 of Zn deficiency, 3 animals were sacrificed, respectively (n = 24). Zn deficiency was evident from reduced plasma Zn, plasma alkaline phosphatase activity and severe mobilization of Zn from tissue stores (mainly skeleton), while food intake and body weight remained unaffected. Tissues representing Zn absorption (jejunum, colon), Zn storage and utilization (muscle, liver), and Zn excretion (kidney) were retrieved. Total RNA contents increased in colon (p = 0.003) and trend to decrease in liver (p = 0.086). Zn deficiency was without effect on tissue total RNA concentrations in muscle tissue and kidney. Real-time reverse transcription (RT) polymerase chain reaction (PCR) assays were developed and a relative quantification on the basis of GAPDH was applied. Assays allowed a relative and accurate quantification of mRNA molecules with a sufficiently high sensitivity and repeatability. All known Zn transporter subtypes were found in the tissues. ZnT3 was newly elucidated and sequenced in rat tissues. Expression patterns and reactions to Zn deficiency were specific for the tissue analysed. Expression results imply that some transporters are expressed constitutively, whereas others are highly regulated in tissues responsible for Zn homeostasis. The most distinct changes of expression levels were shown in colon which can therefore be postulated as a highly Zn sensitive tissue. MT was down-regulated in all tissues, massively in liver (p < 0.001) and in colon (p = 0.002) and in tendency also in the jejunum and kidney. In parallel with intracellular Zn status it is a potent candidate gene for Zn deficiency. ZnT1 and ZnT2 showed a significant up-regulation of mRNA expression in colon (p = 0.032 and p = 0.026) and for ZnT2 a trend of down regulation in jejunum (p = 0.098). This study provides the first comparative view of regulation of gene expression and fully quantitative expression analysis of all known Zn transporters in a non growing adult rat model on a constant platform and therefore allows a direct comparison.

Animals↗
PubMed1992

Analysis of muscarinic receptor subtypes in the mouse cochlea by means of the polymerase chain reaction.

Total RNA was extracted with guanidine thiocyanate from the cochleas of 16-day-old CBAJ mice. The mRNA was purified from the total RNA using oligo-dT cellulose, and the mRNA was treated with DNase to degrade genomic DNA. After reverse transcription, resulting cDNA was amplified by polymerase chain reaction (PCR), using primers specific for the nucleotide sequences m1-m5, representing subtypes of muscarinic acetylcholine receptors. PCR products corresponding to subtypes m1, m3, and m5, but not to m2 and m4, were amplified. These results suggest that muscarinic acetylcholine receptors of these odd-numbered subtypes are expressed in the mammalian cochlea.

Animals↗