Competitive Reverse Transcriptase-polymerase Chain Research Articles

ABC Assay: Method Development and Application to Quantify the Role of Three DWV Master Variants in Overwinter Colony Losses of European Honey Bees.

Deformed wing virus (DWV) is one of the most prevalent honey bee viral pathogens in the world. Typical of many RNA viruses, DWV is a quasi-species, which is comprised of a large number of different variants, currently consisting of three master variants: Type A, B, and C. Little is known about the impact of each variant or combinations of variants upon the biology of individual hosts. Therefore, we have developed a new set of master variant-specific DWV primers and a set of standards that allow for the quantification of each of the master variants. Competitive reverse transcriptase polymerase chain reaction (RT-PCR) experimental design confirms that each new DWV primer set is specific to the retrospective master variant. The sensitivity of the ABC assay is dependent on whether DNA or RNA is used as the template and whether other master variants are present in the sample. Comparison of the overall proportions of each master variant within a sample of known diversity, as confirmed by next-generation sequence (NGS) data, validates the efficiency of the ABC assay. The ABC assay was used on archived material from a Devon overwintering colony loss (OCL) 2006–2007 study; further implicating DWV type A and, for the first time, possibly C in the untimely collapse of honey bee colonies. Moreover, in this study DWV type B was not associated with OCL. The use of the ABC assay will allow researchers to quickly and cost effectively pre-screen for the presence of DWV master variants in honey bees.

Hormone-sensitive lipase – quantitation of enzyme and mRNA in small biopsies of human adipose tissue

Hormone-sensitive lipase (HSL) activity was first identified as an adrenaline-sensitive lipolytic activity in adipose tissue. Hormonal activation of HSL occurs via cAMP-dependent protein kinase, which phosphorylates HSL. HSL is the enzyme responsible for the release of free fatty acids (FFAs) from adipose tissue and, therefore, plays a pivotal role in providing the major source of energy for most tissues. Although its expression is highest in adipose tissue, HSL is also expressed in the adrenal glands, ovaries, testes and, to a lesser extent, in skeletal and cardiac muscle and macrophages. The aim of this article is to describe a modified method of determination of HSL activity and gene expression in 10 mg of frozen human adipose tissue samples obtained by biopsy. The enzymes were extracted from the frozen tissue and activity was tested against a fatty acid-labelled diacylglycerol emulsion. The released fatty acids were extracted using a liquid–liquid partition system. The mean±standard error of the mean (SEM) HSL activity in normal fasting individuals was 2.38±0.75 milliunits (mU) oleate/min/g at 37°C. HSL mRNA levels were assessed in 10 mg of adipose tissue using a competitive reverse transcriptase polymerase chain reaction (RT-PCR) assay and the mean ratio of HSL mRNA molecules to β2-microglobulin-mRNA molecules in normal individuals was 0.29±0.04 (mean±SEM). The methods described here are suitable for use in adipose tissue biopsies obtained from normal individuals and provide a means for the examination of the regulation of this enzyme. The importance of this is becoming increasingly apparent, as the inability to suppress lipolysis is associated with conditions of insulin resistance and an increase in propensity to atherosclerosis. This is associated with inappropriate release of FFAs in the postprandial period, resulting in reduced sensitivity of glucose metabolism and increased postprandial lipaemia.

Quantification of the Levels of CYP2D6 mRNA in Peripheral Blood Leukocytes by Reverse Transcriptase Polymerase Chain Reaction HPLC

We developed a sensitive high performance liquid chromatography (HPLC) method for determining CYP2D6 mRNA in peripheral blood leukocytes (PBL) by using competitive reverse transcriptase polymerase chain reaction (RT-PCR). The method is specific, reproducible, and sensitive enough to quantify the absolute amount of low and high abundant CYP2D6 mRNA. The native CYP2D6 transcript and the internal standard, a CYP2D6 deletion RNA, were amplified with similar efficiency in RT-PCR. The PCR products were separated as the corresponding peaks in optimized HPLC. The coefficients of variation for competitive RT-PCR and HPLC determination were 1.5-6.5% and 0.6-2.4%, respectively, showing high reproducibility and reliability. This approach could also be applicable to the quantification of mRNA expressing on various tissues, including PBL, of which the expression levels were so low that they were hard to determine by existing agarose gel electrophoresis methods.

A case of α-fetoprotein–producing pulmonary carcinoma with restricted expression of hepatocyte nuclear factor-4 α in hepatoid foci: a case report with studies of previous cases

We report a case of *-fetoprotein (AFP)-producing pulmonary carcinoma with studies for messenger RNA (mRNA) expression of hepatocyte nuclear factor (HNF)-4*, which is a transcription factor that is highly expressed in the process of liver development. The patient was a 64-year-old man with a pulmonary tumor in his left lower lobe. Serum AFP was 673 ng/mL. The microscopic analysis of the surgical specimen revealed a large cell neuroendocrine carcinoma with occasional hepatoid foci. The competitive reverse transcriptase polymerase chain reaction analysis revealed that HNF-4* mRNA was expressed on the order of 10(2)- to 10(3)-fold more abundantly than control pulmonary carcinomas and normal lung tissues. In addition, AFP and HNF-4* expression was restricted in hepatoid foci. Two previously reported cases of AFP-producing pulmonary carcinoma were also positive, whereas all 18 control pulmonary carcinomas were negative for HNF-4*. These findings suggest that aberrant expression of HNF-4* is implicated in the emergence or maintenance of hepatoid foci in AFP-producing pulmonary carcinomas.

Effects of chronic desipramine treatment on α 2-adrenoceptors and μ-opioid receptors in the guinea pig cortex and hippocampus

The existence of a close relation between presynaptic inhibitory α 2-adrenoceptor and μ-opioid receptor pathways is well established. Such interplay may occur during chronic conditions that give rise to neuroadaptive changes involving both receptor systems. The aim of this study was to examine the effect of chronic treatment with the tricyclic antidepressant drug, desipramine, on α 2-adrenoceptors and μ-opioid receptors in the guinea pig brain. Guinea pigs were treated with 10 mg/kg desipramine, injected i.p. for 21 days, every 24 h. The levels of expression of α 2-adrenoceptors and μ-opioid receptors, the G protein receptor regulatory kinase, GRK2/3 and signal transduction inhibitory G proteins in synaptosomes of the guinea pig hippocampus and cortex were evaluated by immunoblotting. Quantitative analysis of α 2-adrenoceptor and μ-opioid receptor mRNA levels has been carried out by competitive reverse transcriptase polymerase chain reaction. The expression levels of α 2-adrenoceptors and μ-opioid receptors and the respective mRNAs were found unchanged in the cortex, after chronic desipramine treatment. In these experimental conditions α 2-adrenoceptor and μ-opioid receptor levels decreased, while the relevant transcripts increased, in the hippocampus. GRK2/3 levels remained unchanged and increased, respectively, in the cortex and the hippocampus, after chronic exposure to desipramine. In the same experimental conditions, Gα i1, Gα i2, Gα o and Gα z levels remained unchanged, while Gα i3 levels decreased, in the cortex; whereas, Gα i1, Gα i2 and Gα i3 levels significantly increased, and Gα o and Gα z levels remained unchanged, in the hippocampus. On the whole, the present data suggest that α 2-adrenoceptor and μ-opioid receptor expression and transcription are similarly influenced by chronic treatment with desipramine, in the guinea pig cortex and hippocampus. Furthermore, alterations in the levels of regulatory GRK2/3 and of inhibitory signal transduction G proteins, relevant to activation of both receptor pathways, have been documented. The distinct pattern of adaptations of the different protein studied in response to chronic desipramine treatment in both regions is discussed.

Platelet Derived Growth Factor-A mRNA Levels in Diabetic and Nondiabetic Subjects at Risk of Coronary Heart Disease

Coronary heart disease (CHD) is the number one cause of death in developed countries. We investigated the quantity of platelet derived growth factor-A mRNA (PDGF-A mRNA) among four different groups of diabetic and nondiabetic Saudi subjects (n = 40) at risk of CHD. Total RNA was derived from peripheral blood mononuclear cells (PBMN) and PDGF-A mRNA reverse transcribed into cDNA in the presence of internal standard (cRNA) and amplified by competitive reverse transcriptase polymerase chain reaction (cRT-PCR). The gel was silver stained after polyacrylamide gel electrophoresis (PAGE) and PDGF-A mRNA quantitated by scanning densitometry. We demonstrate a significantly high expression of PDGF-A mRNA in hyperlipidaemic and diabetic subjects (41 ± 4 A— 104 copies/I¼g of RNA) compared to normal healthy individuals (29.3 ± 5 A— 104 copies/I¼g of RNA). These findings suggest an important role of PDGF-A mRNA in progression of CHD among diabetics.

The effects of debanding on the lung expression of ET-1, eNOS, and cGMP in rats with left ventricular pressure overload.

Pulmonary hypertension (PH) usually develops secondary to left ventricular (LV) dysfunction; therefore, it is also called retrograde PH. To investigate our hypothesis that PH is at least partially reversible, as in some congenital heart diseases, in a rat model we investigated whether release of constriction could attenuate pulmonary vascular remodeling and change the expression of endothelin (ET)-1 and endothelial nitric oxide synthase (eNOS). We used rats with LV dysfunction produced by an ascending aortic banding. In this study, there were four groups enrolled: 4-weeks banded (AOB(1-28); n = 7), 7-weeks banded (AOB(1-49); n = 7), debanded groups (AOB(1-28)/DeB(29-49); n = 7), and rats receiving a sham operation (n = 7). Subsequently, there was significant attenuation of medial hypertrophy in pulmonary arterioles and reversal of PH in the AOB(1-28)/DeB(29-49) group (sham, 19 +/- 1.3 mm Hg; AOB(1-28), 31 +/- 2.7 mm Hg; AOB(1-49), 32 +/- 2.7 mm Hg; and AOB(1-28)/DeB(29-49), 20 +/- 1.3 mm Hg). PreproET-1 mRNA and eNOS mRNA were measured by competitive reverse transcriptase (RT) polymerase chain reaction (PCR), and eNOS was measured by Western blotting. Compared with the banded groups, debanding significantly decreased pulmonary preproET-1 mRNA, pulmonary ET-1 (sham, 210 +/- 12 pg/g protein; AOB(1-28), 242 +/- 12 pg/g protein; AOB(1-49), 370 +/- 49 pg/g protein; and AOB(1-28)/DeB(29-49), 206 +/- 1.9 pg/g protein), and plasma ET-1 levels (sham, 10.1 +/- 1.5 pg/ml; AOB(1-28), 13.4 +/- 2.0 pg/ml; AOB(1-49), 15.4 +/- 2.0 pg/ml; and AOB(1-28)/DeB(29-49), 10.3 +/- 0.9 pg/ml protein). Debanding could not, however, alter pulmonary eNOS, eNOS mRNA, or cGMP. These findings suggest that pulmonary vascular remodeling, increased pulmonary arterial pressure, and upregulation of ET-1 gene expression are all reversible. We infer that it is the upregulated gene expression of ET-1, not eNOS, that is closely related to the development of the PH secondary to 4 weeks of aortic banding.

Expression of the ubiquitin-proteasome pathway and muscle loss in experimental cancer cachexia

Muscle protein degradation is thought to play a major role in muscle atrophy in cancer cachexia. To investigate the importance of the ubiquitin-proteasome pathway, which has been suggested to be the main degradative pathway mediating progressive protein loss in cachexia, the expression of mRNA for proteasome subunits C2 and C5 as well as the ubiquitin-conjugating enzyme, E214k, has been determined in gastrocnemius and pectoral muscles of mice bearing the MAC16 adenocarcinoma, using competitive quantitative reverse transcriptase polymerase chain reaction. Protein levels of proteasome subunits and E214k were determined by immunoblotting, to ensure changes in mRNA were reflected in changes in protein expression. Muscle weights correlated linearly with weight loss during the course of the study. There was a good correlation between expression of C2 and E214k mRNA and protein levels in gastrocnemius muscle with increases of 6–8-fold for C2 and two-fold for E214k between 12 and 20% weight loss, followed by a decrease in expression at weight losses of 25–27%, although loss of muscle protein continued. In contrast, expression of C5 mRNA only increased two-fold and was elevated similarly at all weight losses between 7.5 and 27%. Both proteasome functional activity, and proteasome-specific tyrosine release as a measure of total protein degradation was also maximal at 18–20% weight loss and decreased at higher weight loss. Proteasome expression in pectoral muscle followed a different pattern with increases in C2 and C5 and E214k mRNA only being seen at weight losses above 17%, although muscle loss increased progressively with increasing weight loss. These results suggest that activation of the ubiquitin-proteasome pathway plays a major role in protein loss in gastrocnemius muscle, up to 20% weight loss, but that other factors such as depression in protein synthesis may play a more important role at higher weight loss.

Upregulation of the BRCA1 gene in human germ cells and in preimplantation embryos

The quantification of BRCA1 messenger RNA molecules by a quantitative competitive one-step reverse transcriptase polymerase chain reaction method indicates that BRCA1 is upregulated both in human male and female germ cells and in preimplantation embryos. Because BRCA1 is involved in several pathways that participate in preserving intact chromosome and genome integrity, these data suggest that BRCA1 dysfunction might alter human embryogenesis or fertility.

Parathyroid hormone-related peptide expression in rat collagen-induced arthritis

The aim of this study was to describe expression of parathyroid hormone-related peptide (PTHrP) in collagen-induced arthritis (CIA), a well-established animal model for rheumatoid arthritis. CIA was induced in female dark agouti rats. Inguinal (ILNs) and popliteal (PLNs) lymph nodes and distal interphalangeal joints (DIP) were retrieved at different time points. Tissues were processed for detection of PTHrP and cell marker proteins by immunohistochemistry. Lymph node RNA was extracted, and PTHrP mRNA quantified using competitive reverse transcriptase polymerase chain reaction. Hyperplasia of ILNs was observed 2 days after injection, coinciding with the peak in PTHrP expression in ILNs (1240 +/- 373 gene copies/ng RNA vs normal 339 +/- 120, P < 0.05). Hyperplasia of PLNs was first seen at 1 day after onset of arthritis, coinciding with the peak in PTHrP expression in PLNs (2267 +/- 697 vs normal 781 +/- 136, P < 0.01). PTHrP expression in PLNs remained increased 5 days after onset (1361 +/- 302 vs normal 781 +/- 136, P < 0.05). In both PLNs and ILNs PTHrP protein was localized to high endothelial venules, lymphocytes and monocytes/macrophages. In DIP joint synovium PTHrP staining was first detected on day 10 after onset, and was most abundant at day 20 after onset, at sites of bone resorption and deposition, where it was localized to neutrophils, cells of monocyte lineage and osteoblasts. Changes in ILN and PLN PTHrP mRNA expression suggest that elevated levels of the cytokine are associated with aggravation of the inflammatory immune response. Changes in PTHrP in DIP joints indicate its involvement in late rather than early pathogenic events in CIA joints.

Increased expression of proteasome subunits in skeletal muscle of cancer patients with weight loss

Atrophy of skeletal muscle is common in patients with cancer and results in increased morbidity and mortality. In order to design effective therapy the mechanism by which this occurs needs to be elucidated. Most studies suggest that the ubiquitin-proteasome proteolytic pathway is most important in intracellular proteolysis, although there have been no reports on the activity of this pathway in patients with different extents of weight loss. In this report the expression of the ubiquitin-proteasome pathway in rectus abdominis muscle has been determined in cancer patients with weight loss of 0–34% using a competitive reverse transcriptase polymerase chain reaction to measure expression of mRNA for proteasome subunits C2 and C5, while protein expression has been determined by western blotting. Overall, both C2 and C5 gene expression was increased by about three-fold in skeletal muscle of cachectic cancer patients (average weight loss 14.5 ± 2.5%), compared with that in patients without weight loss, with or without cancer. The level of gene expression was dependent on the amount of weight loss, increasing maximally for both proteasome subunits in patients with weight loss of 12–19%. Further increases in weight loss reduced expression of mRNA for both proteasome subunits, although it was still elevated in comparison with patients with no weight loss. There was no evidence for an increase in expression at weight losses less than 10%. There was a good correlation between expression of proteasome 20Sα subunits, detected by western blotting, and C2 and C5 mRNA, showing that increased gene expression resulted in increased protein synthesis. Expression of the ubiquitin conjugating enzyme, E2 14k, with weight loss followed a similar pattern to that of proteasome subunits. These results suggest variations in the expression of key components of the ubiquitin-proteasome pathway with weight loss of cancer patients, and suggest that another mechanism of protein degradation must be operative for patients with weight loss less than 10%.

β‐Defensin Expression in Immunocompetent and Immunodeficient Germ‐Free andCandida albicans–Monoassociated Mice

Defensins are important components of innate immunity and play a key role in the fight against infectious diseases; however, little is known about their role in resistance to fungal infection. We examined gene expression of mouse beta -defensins (mBDs) 1-4 in orogastric tissues from germ-free (gf) and Candida albicans-monoassociated immunocompetent (C57BL/6) and immunodeficient (Tg epsilon 26 or gp91(phox-/-)/NOS2(-/-)) mice, using competitive reverse-transcriptase polymerase chain reaction. The basal levels of beta -defensin gene expression in orogastric tissues from gf mice varied significantly between immunodeficient and immunocompetent mice and by the particular tissue analyzed. During gastric and lethal oroesophageal candidiasis, expression of mBD1, -3, and -4 was induced at the infection sites (stomach and tongue) and was concomitant with an induction of tumor necrosis factor- alpha expression in Tg epsilon 26 mice, compared with that in tissues from gf mice. Induction of mBD4 expression in response to gastric candidiasis, however, was dependent on the immune competency of the host. A C. albicans mutant that lacked key genes important for filamentation and virulence also significantly induced expression of mBD1, -3, and -4 in Tg epsilon 26 mice. These data not only demonstrate quantitative and qualitative differences in beta -defensin expression in gf and gnotobiotic mice, they also suggest a role for these peptides in resistance to gastric and lethal oroesophageal candidiasis.

Intragraft iNOS induction during human liver allograft rejection depresses cytochrome p450 activity

Abstract Allograft function may become impaired during rejection after human liver transplantation. Cytokines induce nitric oxide (NO) production in hepatocytes, Kupffer cells and infiltrating mononuclear cells. NO inhibits cytoplasmatic cytochrome p450 (CYP) enzyme activity in vitro. It is not known whether this mechanism plays a role in vivo. In order to characterize the role of locally produced cytokines in the pathogenesis of liver dysfunction, we analysed human liver transplant biopsy material for the expression of proinflammatory cytokines as well as for NO synthase and we compared these results to the microsomal liver function in vivo [aminopyrine breath test (ABT)] and in vitro (enzymatic analysis of CYP). Microsomal liver function decreased in vivo during rejection while ABT levels decreased by 40% and increased again by 59% after the acute rejection episode. Similarly, CYP 1A2 and 2E1 activity dropped 42% and 24% in rejecting samples, respectively. Competitive reverse transcriptase polymerase chain reaction (RT-PCR) showed a fivefold upregulation of interferon gamma (IFN-γ) gene expression. Inducible, but not constitutive NO-synthase gene expression was upregulated fivefold in samples from rejecting patients suggesting a local induction of NO in response to immune events. Our data show a marked impairment of CYP enzyme activity during allograft rejection which is presumably secondary to an increased intragraft production of proinflammatory cytokines and NO.

Intragraft iNOS induction during human liver allograft rejection depresses cytochrome p450 activity.

Allograft function may become impaired during rejection after human liver transplantation. Cytokines induce nitric oxide (NO) production in hepatocytes, Kupffer cells and infiltrating mononuclear cells. NO inhibits cytoplasmatic cytochrome p450 (CYP) enzyme activity in vitro. It is not known whether this mechanism plays a role in vivo. In order to characterize the role of locally produced cytokines in the pathogenesis of liver dysfunction, we analysed human liver transplant biopsy material for the expression of proinflammatory cytokines as well as for NO synthase and we compared these results to the microsomal liver function in vivo [aminopyrine breath test (ABT)] and in vitro (enzymatic analysis of CYP). Microsomal liver function decreased in vivo during rejection while ABT levels decreased by 40% and increased again by 59% after the acute rejection episode. Similarly, CYP 1A2 and 2E1 activity dropped 42% and 24% in rejecting samples, respectively. Competitive reverse transcriptase polymerase chain reaction (RT-PCR) showed a fivefold upregulation of interferon gamma (IFN-gamma) gene expression. Inducible, but not constitutive NO-synthase gene expression was upregulated fivefold in samples from rejecting patients suggesting a local induction of NO in response to immune events. Our data show a marked impairment of CYP enzyme activity during allograft rejection which is presumably secondary to an increased intragraft production of proinflammatory cytokines and NO.

Development of Quantitative Competitive—Reverse Transcriptase—Polymerase Chain Reaction for Detection and Quantitation of Avian Leukosis Virus Subgroup J

Infection with avian leukosis virus subgroup J (ALV-J) causes severe economic losses in the broiler industry by increasing mortality, producing tumors, and decreasing weight gain in chickens. The quantitation of ALV-J is difficult because of its failure to produce a cytopathic effect in cell culture systems and the nonspecificity of antigen-capture enzyme-linked immunosorbent assay (ELISA) tests. This study was performed to develop a quantitative competitive-reverse transcriptase-polymerase chain reaction (QC-RT-PCR) method based on coamplification of ALV-J genomic RNA and a known amount of a synthesized RNA competitor. The 369 bp RNA competitor was constructed by restriction enzyme treatment of an ALV-J specific 545 bp PCR product, ligation, transformation into Escherichia coli, and in vitro transcription. The competitor contained the same amplification primer annealing sites and sequence as the original viral RNA, except that it had a 176 bp internal deletion. Coamplified RT-PCR products were visualized by electrophoresis and ethidium bromide staining, and fluorescences were quantified using computer-assisted image analysis. The sensitivity of this new QC-RT-PCR method was 25 fg of viral RNA, and 10-fold dilutions were differentiable. This method allowed absolute and relative quantification of ALV-J RNA copy numbers and was simpler than previously published methods for ALV-J quantification.

Expression of angiopoietin 1 and 2 in ectopic endometrium on the chicken chorioallantoic membrane

Objective To investigate the role of angiopoietin 1 and 2 (ANGPT1/ANGPT2) in angiogenesis of the ectopic endometrium as a crucial step in the development of an endometriotic lesion, we analyzed their expression patterns in an experimental model of endometriosis. Design Experimental prospective study. Setting University hospital. Patient(s) Endometrium samples obtained from healthy, ovulating women undergoing hysterectomy for benign gynecologic conditions. Intervention(s) Endometrial fragments were transplanted to the chicken chorioallantoic membrane (CAM) and cultivated for 0, 24, 48, and 72 hours. Main outcome measure(s) Expression of ANGPT1 and ANGPT2 mRNA was quantified by competitive reverse transcriptase polymerase chain reaction (RT-PCR) and normalized to expression of the housekeeping gene human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA. The expression of ANGPT1 and ANGPT2 protein was analyzed by immunohistochemical staining. Result(s) All grafts expressed ANGPT1 and ANGPT2 mRNA. The mRNA concentration of both factors decreased after cultivation, but the ANGPT2/ANGPT1 ratio increased considerably during the first 24 hours of cultivation. The immunohistochemical investigation for ANGPT1 and ANGPT2 revealed presence of both proteins at all the times but no obvious correlation with the duration of cultivation. Conclusion(s) The ratio of ANGPT2/ANGPT1 mRNA in endometrial grafts increased after 24 hours of cultivation on the chicken chorioallantoic membrane and shows a shift toward a more dominant role of ANGPT2. This agrees with the current model of angiopoietin action in angiogenesis and might indicate angiogenic activity in the endometrial graft. The angiopoietins are likely to play an important role in the pathogenesis of endometriosis.

Immobilization stress elevates tryptophan hydroxylase mRNA and protein in the rat raphe nuclei

Background Stress triggers adaptive and maladaptive changes in the central nervous system, including activation of the hypothalamic–pituitary–adrenal axis, and can trigger mood disorders and posttraumatic stress disorder. We examined the effect of immobilization stress (IMO) on gene expression of tryptophan hydroxylase (TPH), the rate-limiting enzyme in serotonin biosynthesis, and the role of cortisol in that response. Methods Regular and adrenalectomized Sprague-Dawley rats were exposed to various repetitions of IMO. Tryptophan hydroxylase messenger ribonucleic acid (mRNA) was determined by competitive reverse transcriptase polymerase chain reaction, and TPH protein was examined by immunoblot and immunocytochemistry. Results Elevation of TPH mRNA by IMO was tissue-specific and dose-dependent. A single IMO elicited a threefold rise in TPH mRNA in median raphe nucleus (MRN), but repeated (3×) IMOs were needed for similar response in dorsal raphe nucleus (DRN). Repeated daily IMO, up to 7 days, triggered a robust induction (6–10-fold) in TPH mRNA, accompanied by corresponding rise in TPH protein levels in raphe nuclei but not in the pineal gland. The rise in TPH immunoreactivity was widespread throughout the DRN and MRN. Bilateral adrenalectomy did not prevent the IMO-triggered increase in TPH immunoreactive protein in the raphe nuclei. Conclusions This study reveals adrenal glucocorticoid–independent induction of TPH gene expression in raphe nuclei in response to immobilization stress.

Proteomic Changes during Disturbance of Cholesterol Metabolism by Azacoprostane Treatment in Caenorhabditis elegans

Although nematodes like Caenorhabditis elegans are incapable of de novo cholesterol biosynthesis, they can utilize nonfunctional sterols by converting them into cholesterol and other sterols for cellular function. The results reported previously and presented here suggest that blocking of sterol conversion to cholesterol in C. elegans by 25-azacoprostane-HCl (azacoprostane) treatment causes a serious defect in germ cell development, growth, cuticle development, and motility behavior. To establish a biochemical basis for these physiological abnormalities, we performed proteomic analysis of mixed stage worms that had been treated with the drug. Our results from a differential display proteomic analysis revealed significant decreases in the levels of proteins involved in collagen and cytoskeleton organization such as protein disulfide isomerase (6.7-fold), beta-tubulin (5.41-fold), and NEX-1 protein (>30-fold). Also reduced were enzymes involved in energy production such as phosphoglycerate kinase (4.8-fold) and phosphoenolpyruvate carboxykinase (8.5-fold), a target for antifilarial drugs such as azacoprostane. In particular, reductions in the expression of lipoprotein families such as vitellogenin-2 (7.7-fold) and vitellogenin-6 (5.4-fold) were prominent in the drug-treated worms, indicating that sterol metabolism disturbance caused by azacoprostane treatment is tightly coupled with suppression of the lipid transfer-related proteins at the protein level. However, competitive quantitative reverse transcriptase polymerase chain reaction showed that the transcriptional levels of vit-2, vit-6, and their receptors (e.g. rme-2 and lrp-1) in drug-treated worms were 3- to 5-fold higher than those in the untreated group, suggesting a presence of a sterol regulatory element-binding protein (SREBP)-like pathway in these genes. In fact, multiple predicted sterol regulatory elements or related regulatory sequences responding to sterols were found to be located at the 5'-flanking regions in vit-2 and lrp-1 genes, and their transcriptional activities fluctuated highly in response to changes in sterol concentration. Thus, many physiological abnormalities caused by azacoprostane-mediated sterol metabolism disturbance appear to be exerted at least in part through SREBP pathway in C. elegans.

Intestinal defensin gene expression in human populations

Defensins are thought to play a major role in the defence of small intestinal crypts against colonisation by potential pathogens. In humans two α-defensins, HD5 and HD6 and two β-defensins, hBD1 and hBD2, probably contribute to the antimicrobial barrier, but there are no data to indicate how the expression of these defensin genes might vary in individuals and in populations. To begin to address this question we developed a competitive reverse transcriptase polymerase chain reaction (RT-PCR) assay to quantify HD5 and HD6 mRNA and used it to measure transcripts in small intestinal biopsy tissue from adults living in London, UK, or in Lusaka, Zambia. We also measured α- and β-defensin mRNA in biopsies collected in London from different regions of the small intestine. Jejunal biopsies ( n=169) from 83 adults in Lusaka contained approximately one order of magnitude less HD5 and HD6 mRNA than biopsies ( n=33) obtained from 27 adults in London. HD5 and HD6 transcript levels were high throughout duodenum, jejunum and ileum. hBD1 and hBD2 mRNA were detected in some, but not all, biopsies from normal small intestine. These data suggest that α-defensin expression is down-regulated in tropical populations, and that there are distinct pathways regulating transcription of α- and β-defensins.

GABA A α 1 and α 2 receptor subunit expression in rostral ventrolateral medulla in nonpregnant and pregnant rats

Pregnancy results in attenuated baroreflex mediated sympathoexcitatory responses which may be due to potentiation of γ-aminobutyric acid (GABA) inhibition in the rostral ventrolateral medulla (RVLM). The major metabolite of progesterone, 3α-hydroxy-dihydroprogesterone (3α-OH-DHP), which is elevated in pregnancy, is a potent neurosteroid positive modulator of GABA A receptors, and sensitivity of GABA A receptors to 3α-OH-DHP is dependent on the receptor subunit composition. The purpose of this study was to evaluate the GABA A α 1 and α 2 receptor subunit mRNA and protein expression in the RVLM of nonpregnant and late term pregnant rats. Micropunches of RVLM were collected from nonpregnant and late term pregnant rats and the expression levels of GABA A α 1 and α 2 receptor subunits were analyzed using quantitative competitive reverse transcriptase polymerase chain reaction (RT-PCR) and immunoblot techniques. The competitive RT-PCR analysis allows comparison of expression levels between different mRNA, and the mRNA expression level of GABA A α 1 was several hundred fold greater than GABA A α 2 in both groups. However, this relative distribution of GABA A α 1 and α 2 receptor subunits protein or mRNA expression was not altered in late term pregnant compared to nonpregnant rats. These data demonstrate, that within the RVLM of both nonpregnant and late term pregnant rats, the relative expression levels of GABA A α 1,2 receptor subunits favor GABA A receptors susceptible to positive modulation by progesterone metabolites.