Effects of swim stress on mRNA and protein levels of steroid 5alpha-reductase isozymes in prefrontal cortex of adult male rats

The metabolite of progesterone, allopregnanolone, is among the most potent known ligands of the gamma-aminobutyric acid receptor complex (GABA(A)-R) in the central nervous system. This neuroactive steroid is markedly increased in an animal model of acute stress. Allopregnanolone is synthesized from progesterone by steroidogenic enzymes 5alpha-reductase (5alpha-R) and 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD), with the former being the rate-limiting enzyme in this reaction sequence. In this paper, a quantitative RT-PCR method coupled to laser-induced fluorescence capillary electrophoresis (LIF-CE) and Western blot were used to measure both mRNA and protein levels of 5alpha-R type 1 (5alpha-R1) and 5alpha-R type 2 (5alpha-R2) isozymes in prefrontal cortex of male rats after acute swim stress situations. Our results demonstrate that both 5alpha-R isozymes are significantly higher in prefrontal cortex of male rats after acute swim stress in comparison with control rats. These data may open up a new research line that could improve our understanding of the role of 5alpha-R isozymes in processes that accompany stress situations.     

Effects of sulpiride on mRNA levels of steroid 5alpha-reductase isozymes in prostate of adult rats

Prolactin (PRL) is implicated in prostate growth and in the development and regulation of benign prostatic hypertrophy (BPH) and prostate cancer (PCa). PRL may exert its effects on prostate in synergism with androgens. The most active androgen in the prostate is the 5alpha-dihydrotestosterone (DHT) obtained from testosterone by the 5alpha-reductase (5alpha-R) enzyme, which is expressed in the prostate as two isozymes, 5alpha-R1 and 5alpha-R2. In this study, sulpiride, a prolactin-secretion inductor, was administered to male rats. mRNA levels of 5alpha-R1 and 5alpha-R2 were measured in prostate of controls and sulpiride-treated rats, using one-step quantitative RT-PCR coupled with laser-induced fluorescence capillary electrophoresis (LIF-CE). Results demonstrated that sulpiride-induced hyperprolactinemia is associated with an increase in mRNA levels of both 5alpha-R1 and 5alpha-R2 in prostate of adult rats. Although a direct effect of sulpiride on prostate gland cannot be ruled out, hyperprolactinemia may be a factor to be considered in aging males, in whom prostatic diseases such as BPH and PCa are more frequent.     

Effects of testosterone on brain mRNA levels of steroid 5alpha-reductase isozymes in early postnatal life of rat

The enzyme 5alpha-reductase (5alpha-R) (EC 1.3.99.5) exists as two isoforms, 5alpha-R type 1 (5alpha-R1) and 5alpha-R type 2 (5alpha-R2), and both are present in the brain. 5alpha-R1 has been proposed as a constitutive enzyme that essentially plays a catabolic and neuron protective role whereas 5alpha-R2 has been associated with sexually dimorphic functions of the male. In this work, we studied the effects of testosterone (T), the masculinizing hormone of the central nervous system (CNS), on mRNA levels of both 5alpha-R isoforms in the prefrontal cortex of male and female rats during the postnatal sexual differentiation of the CNS in the rat, using one-step quantitative RT-PCR coupled with laser-induced fluorescence capillary electrophoresis (LIF-CE). We found an increase in 5alpha-R2 mRNA levels in both male and female rats after T treatment, while 5alpha-R1 mRNA levels were decreased in the same experimental conditions. Our results clearly indicated that T regulates the expression of both 5alpha-R1 and 5alpha-R2 genes in an opposite manner and independently of the sex. This could point to a crucial role of T in the sexual dimorphism for both 5alpha-R isozymes in the neonatal brain. These results open up a new research line that could improve understanding of the role of 5alpha-R isozymes in the physiology of the CNS.     

Precise quantitation of 5alpha-reductase type 1 mRNA by RT-PCR in rat liver and its positive regulation by testosterone and dihydrotestosterone

The liver is a multifunctional organ responsible for steroid hormones catabolism. Thus, the enzymes responsible for steroid catabolism are located in the liver, including the steroid 5alpha-Reductase (5alpha-R) (EC 1.3.99.5) which catalyzes the conversion of compounds with Delta(4,5) double bonds such as testosterone (T) into their respective reduced derivatives such as dihydrotestosterone (DHT), which are more hydrosoluble, therefore facilitating their excretion. We present precise measurements of mRNA levels of steroid 5alpha-Reductase type 1 isozyme (5alpha-R1) in the liver of male rats with different androgen status, using a quantitative RT-PCR coupled to laser-induced fluorescence capillary electrophoresis (LIF-CE). By means of this technique, we demonstrate a high level of expression of the gene that encodes 5alpha-R1 isozyme in male rat liver, and both T and DHT exert a positive control on the genetic expression of liver 5alpha-R1 isozyme. Since DHT does not contain a Delta(4,5) double bond, our results raise the possibility that hepatic 5alpha-R type 1 not only participates in the catabolism of steroids with Delta(4,5) double bonds, but also in other physiological functions, perhaps in the masculinization of the external genitalia in males with 5alpha-R type 2 gene deficiency.     

Quantification of androgen receptor mRNA in tissues by competitive co-amplification of a template in reverse transcription—Polymerase chain reaction

We describe a polymerase chain reaction (PCR)-based method for the quantification of androgen receptor (AR) mRNA in tissues. The amount of PCR products depends on the exponential amplification of the initial cDNA copy number; therefore minor differences in the efficiency of amplification may dramatically influence the final product yield. To overcome these tube-to-tube differences in reaction efficiency, an internal control AR cRNA was reverse transcribed along with the target mRNA using the same primers. This standard was obtained by deleting a 38 bp fragment from an amplified bovine AR sequence, which was then subcloned and transcribed into cRNA. Known dilutions of the competitor cRNA were spiked into a series of RT-PCR reaction tubes containing equal amounts of the target mRNA. Following RT-PCR, the co-amplified specimens obtained were separated by gel electrophoresis and quantified by densitometric analysis of ethidium bromide stain. We applied this method to quantify the AR-mRNA in skeletal muscle of castrated as well as from intact male cattle. The applicability of the quantification system for AR-mRNA described herein was demonstrated for other species, e.g. man.     

Quantitative detection of Sphingomonaschlorophenolica in soil via competitive polymerase chain reaction

The 16S ribosomal RNA gene sequence of the pentachlorophenol degrader Sphingomonas chlorophenolica strain RA2 was used to generate specific polymerase chain reaction (PCR) primers for the detection of this strain in soil, whereas a region internal to the two primers was used to provide an S. chlorophenolica strain RA2-specific oligonucleotide probe. The PCR detection system resulted in a 727 bp product detectable via gel electrophoresis and hybridization. It was specific for strain RA2 and its close relative, S. chlorophenolica ATCC 39723, as evidenced by PCR amplifications of a range of bacterial genomic DNAs. Tests of total microbial community DNA obtained from five uninoculated and two RA2-inoculated soils confirmed this specificity for introduced S. chlorophenolica RA2. Strain RA2 could be detected in soil down to a level of 10³ cfu g⁻¹ soil. Two strategies were followed to generate internal standard DNA for competitive PCR. First, a 479 bp MIMICS fragment was obtained based on a previously constructed gene cassette; however, this standard did not reliably quantify RA2 targets. Low stringency PCR performed with a range of bacterial genomic DNAs resulted in the generation of an amplicon with a Paenibacillus azotofixans strain that was slightly smaller than the RA2-derived product. Both products were easily separable via conventional gel electrophoresis. The use of this competitor in a threefold dilution scheme applied to the target DNA allowed for the quantitative detection of RA2-specific target DNA molecules from pure culture and from soil. The fate of strain RA2 in pentachlorophenol-contaminated soil was described using this competitive PCR approach, and the organism was shown to persist at two inoculum levels over prolonged periods of time.     

Changes in the expression of steroid metabolism-related genes in rat adrenal glands during selective REM sleep deprivation

Selective REM sleep deprivation was carried out under the conditions designed to minimize the adverse influence of environmental conditions and restricted movement, and the influence of REM sleep deprivation on adrenocortical steroid metabolism was investigated by measuring the steady-state levels of mRNAs encoding steroid metabolism-related genes, steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme cytochrome P450 (P450scc) and steroid 5alpha-reductase (5alpha-R), in rat adrenal glands. Selective REM sleep deprivation caused a significant decrease in StAR mRNA and an increase in 5alpha-R mRNA levels without any notable change in P450scc mRNA levels in the adrenal gland. In contrast, non-selective sleep disturbance, resulting in the partial reductions of non-REM and REM sleep, tended to increase both StAR and P450scc mRNA levels without any statistical significance. These results indicate that REM sleep deprivation by itself may affect the expression of steroid metabolism-related genes in the adrenal gland, suggesting a possible relation between REM sleep and adrenocortical steroid metabolism.     

Kinetic analysis of LY320236: competitive inhibitor of type I and non-competitive inhibitor of type II human steroid 5alpha-reductase

Type I and type II steroid 5alpha-reductases (5alpha-R) catalyze the conversion of testosterone (T) to dihydrotestosterone (DHT). LY320236 is a benzoquinolinone (BQ) that inhibits 5alpha-R activity in human scalp skin (Ki(typeI)=28.7+/-1.87 nM) and prostatic homogenates (Ki(typeII)=10.6+/-4.5 nM). Lineweaver-Burk, Dixon, and non-linear analysis methods were used to evaluate the kinetics of 5alpha-R inhibition by LY320236. Non-linear modeling of experimental data evaluated V(max) in the presence or absence of LY320236. Experimental data modeled to the following equation 1v=+ fixing the In0c value equal to 1.0 or 0 are consistent with non-competitive or competitive inhibition, respectively. LY320236 is a competitive inhibitor of type I 5alpha-R (In0c=0, Ki=3.39+/-0.38, RMSE = 1.300) and a non-competitive inhibitor of type II 5alpha-R (In0c=1, Ki=29. 7+/-3.4, RMSE = 0.0592). These data are in agreement with linear transformation of the data using Lineweaver-Burk and Dixon analyses. These enzyme kinetic data support the contention that the BQ LY320236 is a potent dual inhibitor with differing modes of activity against the two known human 5alpha-reductase isozymes. LY320236 represents a class of non-steroidal 5alpha-R inhibitors with potential therapeutic utility in treating a variety of androgen dependent disorders.     

Quantification of mRNA Levels by Fluorescently Labelled Reversde Transcription Competitive PCR

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Detection and quantitative analysis of zoospores of Pythium porphyrae, causative organism of red rot disease in Porphyra, by competitive PCR

The detection and quantitative analysis of Pythium porphyraezoospores was performed by PCR using PP-1 and PP-2 primers specific tothe internal transcribed spacer region of P. porphyrae. To estimatethe amount of fungal zoospores of P. porphyrae, an internal standardplasmid (pPPISC) containing a modified DNA fragment was constructed. Both ends of this fragment were complementary to the PCR primers. Amplification using primers PP-1 and PP-2 produced DNA fragments ofapproximately 700 and 400 bp from the target DNA of P. porphyraezoospores and from the pPPISC, respectively. To perform quantitativePCR, known quantities of pPPISC were added to reaction mixturescontaining the experimental DNAs extracted from zoospores. After aco-amplification reaction, the two different sized PCR products wereseparated by agarose gel electrophoresis and visualized by ethidium bromidestaining. The number of zoospores was estimated by comparing thefluorescence intensities of the PCR products using a charge-coupled deviceimage analyzer. The results show that competitive PCR using P.porphyrae specific primers and competitor pPPISC are useful tools for thequantitative analysis of P. porphyrae zoospores in seawater from Porphyra cultivation farms.     

Alternative splicing of pre-mRNA encoding the Musca domestica latrophilin-like protein(LLP): primary structures of four spliced forms of mRNA and their protein products

An internal DNA fragment (approximately 2000 bp) homologous to the conserved regions of genes encoding latrophilin-like proteins (LLPs) was obtained by the PCR technique using degenerate primers to these gene regions. The gene-specific primers were synthesized based on the results of sequencing of the isolated fragment, and all overlapping cDNA fragments of the llp gene encoding the Musca domestica LLP were obtained by the rapid amplification of cDNA 5'- and 3'-ends (5'- and 3'-RACE). Four alternatively spliced mRNAs were found while sequencing the obtained cDNA fragments. Two long mRNAs (approximately 6000 nt) differ in the structures of both the sites encoding signal peptides and 5'-terminal untranslated regions. They encode large proteins (approximately 1800 aa), whose domain organization is similar to that of mammalian latrophilins. Each deduced protein contains a domain with seven transmembrane regions followed by an extended cytoplasmic C-terminal domain. Two other mRNA forms are derived from these long mRNAs; they encode proteins severly truncated at their C-termini (approximately 900 aa). They are composed of only three transmembrane regions and a short unique cytoplasmic C-terminal domain (23 aa). The limitations and drawbacks of the existing 3'-RACE techniques found during study of the long alternatively spliced cDNAs are analyzed, and ways for overcoming these difficulties are proposed.     

Detection of thyrotropin-releasing hormone (TRH) mRNA by the reverse transcription-polymerase chain reaction in the human normal and tumoral anterior pituitary.

To investigate the presence of TRH mRNA in the human anterior pituitary tissue, total RNA from human normal and tumoral anterior pituitary, hypothalamus (positive control) and muscle tissues (negative control) was reverse transcribed (RT) to the first strand of cDNA. RT products were then amplified by polymerase chain reaction (PCR) using a set of three exon-specific primers (two external 5' and 3' primers and one internal 3' primer) for a target sequence of the TRH gene including an intronic sequence of about 650 base pairs (bp). Southern analysis of the RT-PCR products specifically hybridizing with a 45-mer TRH probe showed two bands of the predicted sizes (399 and 351 bp) far more intense in hypothalamus than in normal and tumoral anterior pituitary tissue. The 399 and 351 bp RT-PCR products contained the BglII enzyme restriction site included in the TRH cDNA sequences spanned by the primers and the two respective digested fragments which were, as predicted, 337 and 289 bp long, hybridized with the TRH probe. Based on these results, we can conclude that the RT-PCR products generated from RNA tissue were the target TRH sequences in the human normal and tumoral anterior pituitary tissue as well as in the hypothalamus. Our data imply TRH gene expression in the human anterior pituitary.     

脂蛋白脂酶基因的克隆、序列测定及定点突变

 以人的脂肪组织总RNA为模板 ,参考已报道的脂蛋白脂酶 (lipoproteinlipase ,LPL)cDNA设计引物 ,利用RT PCR方法扩增得到了LPLcDNA ,并经序列测定证实其序列是正确的 .在冠心病患者LPL基因第 5外显子的 830位碱基处发现了G→A的转换 ,该变异导致LPL基因第 192位的密码子CGA被CAA取代 ,使LPL第 192位精氨酸改变为谷氨酰胺 .在变异碱基附近设计合成两条引物 ,其中一条包含所要改变的碱基 ,利用基于PCR的定点突变技术和体外重组的方法获得了G830A变异的LPLcDNA     

小麦矮腥黑穗菌差异片段筛选与分子检测体系的建立

采取随机扩增DNA多态性(Random amplified polymorphic DNA,RAPD)引物介导的半特异PCR技术(RAPD primer mediated hemi-specific PCR,RM-PCR),在从不同地域征集的18个小麦矮腥黑穗菌(Tilletia controversa Kühn,TCK)菌株和29个小麦网腥黑穗病菌(Tilletia caries(DC)Tul,TCT)菌株的总基因组DNA中筛选鉴定出TCK独有的大小为1322bp差异基因组片段。根据该片段序列设计筛选出2对特异性引物CQUTCK2/CQUTCK3和CQUTCK4/CQUTCK5,均可以从18个TCK菌株的菌丝体和冬孢子DNA中稳定地扩增出747bp和200bp的单一靶带DNA,而在29个TCT菌株的菌丝体或冬孢子DNA均无任何扩增产物。以腥黑穗菌属通用引物对CQUK6/CQUK7为内置对照,可以确定被检样品是否含PCR抑制物质进而判断检测体系是否正确,同时有效地排除样品检测结果的假阳性和假阴性。采用建立的TCK特异PCR检测技术体系,实现简单而快速地鉴定小麦矮腥黑穗菌冬孢子或罹病小麦组织中侵染菌丝体的目的。     

PCR-based quantitation of Cryptosporidium parvum in municipal water samples.

A PCR method for the quantitation of Cryptosporidium parvum oocysts in municipal drinking water samples was investigated. Quantitative PCR uses an internal standard (IS) template with unknown target numbers to compare to standards of known concentrations in a standard curve. The IS template was amplified using the same primers used to amplify a portion of a 358 bp gene fragment that encodes a repetitive oocyst wall protein in C. parvum. Municipal water samples spiked with known numbers of C. parvum oocysts were tested by quantitative PCR using the IS and the Digene SHARP Signal System Assay for PCR product detection. The absorbance readings for target DNA and IS templates versus the number of molecules of the target DNA were plotted to generate standard curves for estimating oocyst numbers. The method allowed the quantitation of oocysts from log 3 to log 5 spiked into municipal water samples.     

胰岛素样生长因子1(IGF-1)mRNA同源模板竞争性PCR定量方法的建立

胰岛素样生长因子 1(IGF 1)是一种多功能的细胞增殖调控因子 ,其表达水平受多种因素的影响 ,为了研究IGF 1基因在转录水平上的调控机制 ,建立了定量测定IGF 1mRNA的竞争性PCR方法 .同时 ,也建立了一种简便的制备同源性竞争模板的方法 .以构建好的重组pUC IGF 1质粒为基础 ,利用IGF 1mRNA序列上唯一存在 ,但是在pUC18质粒上多拷贝的MspⅠ酶切位点 ,以该限制性内切酶处理重组pUC IGF 1质粒 .在T4DNA连接酶作用下对酶切产物进行随机连接 ,以连接产物作为模板 ,用可扩增IGF 1cDNA的引物进行PCR ,由此得到因含有随机插入序列而与原IGF 1cDNA产生明显长度差别的重组IGF 1.以不同浓度的该DNA片段作为同源竞争模板与大鼠肝组织cDNA在同一反应体系中进行PCR ,对PCR产物进行分析 ,计算出样本中IGF 1cDNA的初始浓度 .成功地建立了IGF 1mRNA的竞争性PCR定量检测方法 ,为研究IGF 1基因的表达调控奠定了基础 ,同时也为对已克隆的基因进行mRNA定量测定提供了一种简便和灵敏的手段     

Quantitation of polymerase chain reaction products by capillary electrophoresis using laser fluorescence

In samples where the amount of DNA is limited, the polymerase chain reaction (PCR) can amplify specific regions of the DNA. A quantitative analysis of the PCR product would be desirable to ensure sufficient DNA is available for analysis. In this study, we examine the use of capillary electrophoresis (CE) with laser fluorescence detection for quantitation of PCR products. A coated open tubular capillary was used with a non-gel sieving buffer and a fluorescent intercalating dye to obtain results within 20 minutes. Using an internal standard, peak migration time was below 0.1% relative standard deviation (R.S.D.) with a peak area precision of 3% R.S.D. In comparison to quantitation by hybridization, (i.e., slot blot) and spectrophotometric analysis, capillary electrophoresis shows distinct advantages due to its ability to separate unincorporated primers and PCR byproducts from the targeted PCR product. The results demonstrate that CE can be used to monitor the quality and quantity of the PCR product.