Pg mRNA Research Articles

Abstract 5585: Extracellular vesicle poly(A)+ RNA analysis for cancer biomarker screening

Abstract Extracellular vesicles (EV) are a promising biomarker source and present in several biological fluids such as plasma (8.0 x 1011 particles/mL, 121 ± 2.3 nm in diameter) and urine (1.1 x 1011 particles/mL, 152 ± 11.2 nm in diameter). In order to streamline marker screening and validation in translational studies, we developed and optimized high throughput filter-based EV capture, poly(A)+ RNA isolation and gene expression assay protocols for RT-qPCR and RNA-seq. First, we confirmed that plasma and urinary EV mRNA profiles (12 to 15 genes by RT-qPCR) are stable up to 1 week at 4°C, and up to 5 repeats of freeze-thaw cycles, and urinary EV was stable up to 10 months at -80°C, indicating EV mRNA could be a useful biomarker source. Although EV mRNA is less abundant (550 pg mRNA from 1 mL plasma) compared to tissue mRNA or EV non-coding RNA, the optimized protocol allows quantification of about 9,000 genes in average by RNA-seq from 2 mL frozen plasma (N=3) with high reproducibility among the replicates (Spearman correlation: 0.9086 to 0.9326) and between the commercial EV kits (Spearman correlation: 0.9207 to 0.9683). About 77% of the 1000 most abundant genes in plasma EV are expressed ubiquitously throughout various organs but some are expressed specifically in blood (12%), spleen (4.4%), esophagus mucosa (1.2%), etc. according to the Genotype-Tissue Expression (GTEx) database. In urinary EV, other than ubiquitously expressed genes (88%), the genes specific to liver (1.5%), bladder (1%), brain (1%), etc. are present. These data suggest EV mRNA are released from various organs and circulating in biological fluids. The protocol was further applied to biomarker discovery for bladder cancer, and differential gene expression analysis of EV mRNA was conducted using bladder cancer (N=4), cancer remission (N=2), and healthy/disease control urine samples (N=4). Ingenuity pathway analysis indicated that cancer-related molecular and cellular functions are activated in bladder cancer urinary EV but not in remission, while immune-related functions are activated both in cancer and remission. These data indicate that EV mRNA is a promising biomarker source for cancer biomarker screening, and could be used in translational studies. Citation Format: Taku Murakami, Cindy Yamamoto, Mieko Ogura, Melanie Oakes, Hiroshi Harada. Extracellular vesicle poly(A)+ RNA analysis for cancer biomarker screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5585.

Overexpression of Calr Mutants Perturbs Developmental Hematopoiesis in Zebrafish Embryos

Introduction: Calreticulin (CALR) is a 46-kDa highly conserved, multicompartmental and multifunctional protein. CALR acts as a Ca2+ binding chaperone protein and participates in ensuring proper protein and glycoprotein folding in the endoplasmic reticulum. CALR mutations have been identified in about 30% of JAK2 and MPL unmutated essential thrombocythemia and primary myelofibrosis. Although the expression of CALR mutants resulted in pathogenic thrombocytosis in adult mice, whether CALR mutants may disrupt normal hematopoiesis during early development remains unknown. Here we aimed to evaluate the effects of mutant CALR during embryonic hematopoietic development using the in vivo zebrafish model.Methods: Full-length CALR wild-type,and CALR-del52 and CALR-ins5 mutants cDNAs were subcloned in the pCS2+ vector and a bicistronic pSYC-102 T2A vector, respectively. Capped CALR mRNAs from the above vectors were micro-injected into 1-2 cells stage wild-type AB strain, Tg(cd41:GFP) and Tg(fli1:EGFP) zebrafish embryos, respectively. cd41+ thrombocytes were counted at 3 and 5 days post fertilization (dpf), respectively. Gene expression of hematopoietic lineage-specific and cytokine and cytokine receptor genes were evaluated by quantitative reverse-transcription and real-time polymerase chain reaction (Q-PCR) from 1 to 3 dpf. Morpholino (MO) was used to knock down cytokine receptor genes mpl, epor and csf3r.Results: The expression of CALR proteins from the injection of 100 pg mRNA was confirmed by CALR N-terminal and mutant specific antibodies, respectively. Expression of both CALR-del52 and CALR-ins5 mutant mRNA significantly increased the numbers of hematopoietic stem and progenitor cells in the caudal hematopoietic tissue when compared with CALR-wt mRNA at 3 dpf. No obvious changes in the angiogenesis were visualized in CALR-wt and mutant CALR expressing embryos at 3 dpf in the fli1:EGFP line when compared with uninjected control. Mutant CALR-del52 significantly increased the number of cd41+ thrombocytes at 5 dpf (mean 162.5±4.1 per embryo) when compared to CALR-wt (mean 117.1±3.1 per embryo, P<0.001), mutant CALR-ins5 (mean 128.3±6.1 per embryo, P<0.001) and uninjected control (mean 136.7±3.0 per embryo, P<0.001), respectively.At 5 dpf, the number of cd41+ thrombocytes significantly decreased upon mpl MO knockdown (mean 43.6±4.9 per embryo) when compared to the control MO group (mean 123.5±5.9 per embryo, P<0.001) and the mutant CALR-del52 group (P<0.001). Importantly, co-injection of CALR-del52 mutant mRNA (mean 73.7±5.1 per embryo) can only partially reverse the knockdown effect of mpl MO. In contrast, the numbers of cd41+ thrombocytes did not decrease significantly upon epor MO or csf3r MO knocked-down compared with the control MO group. When CALR-del52 mutant mRNA was co-injected with epor or csf3r MOs, the numbers of cd41+ thrombocytes were comparable to those of CALR-del52-injected embryos.In Q-PCR experiments, the expression of cmyb, runx1 and scl significantly increased in only CALR-ins5 mutant group at 3 dpf when compared to 2 dpf. The expression of αeHb and βeHb significantly decreased in both CALR-del52 and CALR-ins5 mutant groups at 2 dpf but the expression of gata1 remained unchange. The effects of CALR-del52 and CALR-ins5 mutant groups on the expression of cytokine and cytokine receptor genes included the upregulation of mpl at 1 dpf followed by progressive downregulation at 2 dpf and 3 dpf but relatively stable expression of tpo and epo during early development. In the group of genes related to thrombopoiesis, the expression of emilin1a and nbeal2 was significantly downregulated in CALR-del52 group at 3 dpf when compared to 2 dpf.Conclusions: In this study, we showed that the expression of mutant CALR causes thrombocytosis through an mpl-dependent mechanism and perturbs developmental hematopoiesis in zebrafish embryos. DisclosuresNo relevant conflicts of interest to declare.

Mild electrical stimulation with heat stimulation increase heat shock protein 70 in articular chondrocyte

The objective of this study is to investigate the effects of mild electrical stimulation (MES) and heat stress (HS) on heat shock protein 70 (HSP70), that protects chondrocytes and enhances cartilage matrix metabolism, in chondrocyte and articular cartilage. Rabbit articular chondrocytes were treated with MES and/or HS. The safeness was assessed by LDH assay and morphology. HSP70 protein, ubiquitinated proteins and HSP70 mRNA were examined by Western blotting and real-time PCR. Rat knee joints were treated with MES and/or HS. HSP70 protein, ubiquitinated proteins, HSP70 mRNA and proteoglycan core protein (PG) mRNA in articular cartilage were investigated. In vitro, HS increased HSP70 mRNA and HSP70 protein. MES augmented ubiquitinated protein and HSP70 protein, but not HSP70 mRNA. MES + HS raised HSP70 mRNA and ubiquitinated protein, and significantly increased HSP70 protein. In vivo, HS and MES + HS treatment augmented HSP70 mRNA. HS modestly augmented HSP70 protein. MES + HS significantly increased HSP70 protein and ubiquitinated proteins. PG mRNA was markedly raised by MES + HS. This study demonstrated that MES, in combination with HS, increases HSP70 protein in chondrocytes and articular cartilage, and promotes cartilage matrix metabolism in articular cartilage. MES in combination with HS can be a novel physical therapy for osteoarthritis by inducing HSP70 in articular cartilage.

Abstract 18703: Novel MicroRNA 874 is a Unique Angiogenic Modulator of Stem Cell Paracrine Factor Epidermacan

Introduction: We have previously shown that treatment with epidermacan a novel stem cell derived paracrine factor promoted angiogenesis in vitro accompanied with an increase in angiogenic miRNA expression. Here, we identified novel microRNAs regulated by epidermacan and validated their importance in modulating the epidermacan angiogenic effects in vitro and in vivo . Methods and Results: Increased levels of pro-angiogenic miRNAs such as miR-10b, miR-21 and miR-424 were discovered in MicroRNA profiling of HUVEC cells treated with epidermacan and then confirmed by qRT-PCR. We also identified miR-874, a relatively unknown miRNA but for its role as a tumor suppressor. Investigation of the downstream microRNA/gene networks revealed that epidermacan regulated known angiogenic genes such as VEGFC, TBX1, HIF-1a and NRP1 . Importantly, treatment with epidermacan decreased the levels of HoxD10, a known target of miR-10b in thrombin induced angiogenesis. Inhibition of miR-10b or miR-874 via anti-miR transfection repressed the effects of epidermacan in HUVEC tube formation on matrigel. Interestingly inhibition of miR-10b also reversed the angiogenic effects of VEGF; however inhibition of mir-874 only suppressed the effects of epidermacan, suggesting miR-874 as an epidermacan specific target. The fli:GFP transgenic zebrafish model was used to study the role of epidermacan in angiogenesis in vivo . Morpholino against zebrafish epidermacan gene was injected into the yolk of fli:GFP transgenic zebrafish embryos at 1-4 cell stage, and larvae were collected at 4 days post fertilization to monitor vessel development. The trunk vascular network was disorganized in larvae injected with the morpholino of epidermacan compared to uninjected control. The vascular defects were rescued by co-injection of 100 pg mRNA of human NDNF (epidermacan homolog), suggesting that vessel malformation is specifically due to knockdown of epidermacan. Conclusion: Our data confirm the role of epidermacan as an angiogenic factor and identify the novel miR-874 as specific modulator of epidermacan’s angiogenic effect. Further analysis to elucidate the microRNA/gene networks downstream of epidermacan will provide new insights on the regulatory networks of angiogenesis.

Exogenous Ghrelin Administration Can Ameliorate Indomethacin-Induced Small Intestinal Damage in Mice

Purpose: The arrival of video capsule endoscopy and balloon enteroscopy revealed that non-steroidal anti-inflammatory drugs (NSAIDs) can cause more frequent and more serious complications than formerly believed in the small intestine. However, safe and effective treatment is not available for NSAIDs-induced small intestinal damage. As a possible candidate, we focused on ghrelin, an orexigenic peptide secreted mainly from stomach. Although previous reports revealed that this peptide might show gastroprotection, the effect of ghrelin on NSAIDs-induced small intestinal damage was still vague. In order to examine its therapeutic potential for NSAIDs-induced small intestinal damage, we analyzed the influence of exogenous ghrelin administration on small intestinal protection using experimental mouse model. Methods: Indomethacin or vehicle was subcutaneously administrated to C57BL/6J mice, and ghrelin or vehicle was intraperitoneally administrated before 30 minutes and 2 hours afterwards (vehicle/vehicle group, vehicle/ghrelin group, indomethacin/vehicle group, and indomethacin/ghrelin group). Small intestinal injuries were assessed by macroscopical and microscopical alterations, myeloperoxidase (MPO) activity, prostaglandin E2 (PG E2) and expression of mRNA for cox-1, cox-2, iNOS, TNF-alpha, IL-1beta, and IL-6. Results: Vehicle/vehicle group and vehicle/ghrelin group did not show small intestinal alterations. Indomethacin/ghrelin group significantly modified macroscopical and microscopical scores, and also attenuated NSAIDs-induced changes in MPO activity, PG E2 level and mRNA expression compared those in indomethacin/vehicle group. Conclusion: Ghrelin shows a strong protective efficacy against indomethacin-induced small intestinal damage in mice. Also considering the previous reports on gastroprotective role, ghrelin can prevent NSAIDs-induced mucosal damage through the whole digestive tract, which will lead to a new treatment approach toward the increase of ghrelin expression.

The vestibuloocular reflex of tadpoles (Xenopus laevis) after knock-down of the isthmus related transcription factor XTcf-4

Development of the amphibian vestibular organ is regulated by molecular and neuronal mechanisms and by environmental input. The molecular component includes inductive signals derived from neural tissue of the hindbrain and from the surrounding mesoderm. The integrity of hindbrain patterning, on the other hand, depends on instructive signals from the isthmus organizer of the midbrain, including the transcription factor XTcf-4. If the development of the vestibular system depends on the integrity of the isthmus as the organizing centre, suppression of isthmus maintenance should modify vestibular morphology and function. We tested this hypothesis by downregulation of the transcription factor XTcf-4. 10 pmol l(-1) XTcf-4-specific antisense morpholino oligonucleotide was injected in one blastomere of two-cell-stage embryos of Xenopus laevis. For reconstitution experiments, 500 pg mRNA of the repressing XTcf-4A isoform or the activating XTcf-4C isoform were co-injected. Overexpression experiments were included using the same isoforms. Otoconia formation and vestibular controlled behaviour such as the roll-induced vestibuloocular reflex (rVOR) and swimming were recorded two weeks later. In 50% of tadpoles, downregulation of XTcf-4 induced (1) a depression of otoconia formation accompanied by a reduction of the rVOR, (2) abnormal tail development and (3) loop swimming behaviour. (4) All effects were rescued by co-injection of XTcf-4C but not, or only partially, by XTcf-4A. (5) Overexpression of XTcf-4A caused similar morphological and rVOR modifications as XTcf-4 depletion, while overexpression of XTcf-4C had no effect. Because XTcf-4C has been described as an essential factor for isthmus development, we postulate that the isthmus is strongly involved in vestibular development.

Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming

Correlating gene expression with cell behavior is ideally done at the single-cell level. However, this is not easily achieved because the small amount of labile mRNA present in a single cell (1-5% of 1-50 pg total RNA, or 0.01-2.5 pg mRNA, per cell) mostly degrades before it can be reverse transcribed into a stable cDNA copy. For example, using standard laboratory reagents and hardware, only a small number of genes can be qualitatively assessed per cell. One way to increase the efficiency of standard laboratory reverse transcriptase (RT) reactions (i.e. standard reagents in microliter volumes) comprising single-cell amounts of mRNA would be to more rapidly mix the reagents so the mRNA can be converted to cDNA before it degrades. However this is not trivial because at microliter scales liquid flow is laminar, i.e. currently available methods of mixing (i.e. shaking, vortexing and trituration) fail to produce sufficient chaotic motion to effectively mix reagents. To solve this problem, micro-scale mixing techniques have to be used. A number of microfluidic-based mixing technologies have been developed which successfully increase RT reaction yields. However, microfluidics technologies require specialized hardware that is relatively expensive and not yet widely available. A cheaper, more convenient solution is desirable. The main objective of this study is to demonstrate how application of a novel "micromixing" technique to standard laboratory RT reactions comprising single-cell quantities of mRNA significantly increases their cDNA yields. We find cDNA yields increase by approximately 10-100-fold, which enables: greater numbers of genes to be analyzed per cell; more quantitative analysis of gene expression; and better detection of low-abundance genes in single cells. The micromixing is based on acoustic microstreaming, a phenomenon where sound waves propagating around a small obstacle create a mean flow near the obstacle. We have developed an acoustic microstreaming-based device ("micromixer") with a key simplification; acoustic microstreaming can be achieved at audio frequencies by ensuring the system has a liquid-air interface with a small radius of curvature. The meniscus of a microliter volume of solution in a tube provides an appropriately small radius of curvature. The use of audio frequencies means that the hardware can be inexpensive and versatile, and nucleic acids and other biochemical reagents are not damaged like they can be with standard laboratory sonicators.

Development of a Sensitive and Specific Quantitative Reverse Transcription-Polymerase Chain Reaction Assay for Blood Thyroglobulin Messenger Ribonucleic Acid in the Follow-Up of Patients with Differentiated Thyroid Carcinoma

Serum thyroglobulin is a sensitive tumor marker in the follow-up of patients with differentiated thyroid carcinoma (DTC), but the presence of endogenous anti-thyroglobulin antibodies (TgAb) can interfere on its measurement. To prevent interference by TgAb, several investigators have tried to quantify blood mRNA Tg by real-time RT-PCR, but the results have been variable, not reporting a correlation between mRNA Tg and the presence of metastases. The aim of the study was to evaluate the development of a sensitive and specific quantitative RT-PCR assay for blood mRNA Tg in the follow-up of patients with DTC. An assay employing primers located in a region not affected by alternative splicing or single nucleotide polymorphisms was developed to study 104 DTC patients (13 of 104 with positive TgAb). The assay is specific for thyroid tissue because we found mRNA Tg expression in normal thyroid tissue, but we did not find any mRNA Tg expression in any extrathyroidal tissues. Quantitative mRNA Tg levels were significantly different between patients "free of disease" (82 of 104) and those with metastases (22 of 104) (2.61 +/- 0.26 vs. 27.58 +/- 1.62 pg mRNA Tg/microg RNA) (P < 0.0001). A cutoff point of 5.51 was able to discriminate between the two groups. In addition, the measurement of mRNA Tg was not affected by the presence of TgAb. This new mRNA Tg quantification is a reliable method that allowed us to differentiate patients free of disease from those with metastases, and it could represent an appropriate molecular marker for the follow-up of patients with DTC, especially those with positive TgAb.

Differential expression of gonadotropin-releasing hormone (GnRH) in pancreas during rat pregnancy

Many studies have shown that there is a relationship between gonadotropin-releasing hormone (GnRH) and glucose metabolism, but little is known about the effects of GnRH on the pancreas. Our experiment investigated the effect of GnRH on pancreatic islet cell in Sprague-Dawley (SD) rats fed with high-cholesterol diet before and during pregnancy. We found that although high-cholesterol diet led to no significant difference of GnRH mRNA levels in pancreas in nonpregnant rats, it led to a marked increase of those in pregnant rats. Furthermore, in rats fed with standard laboratory chow, no significant differences were apparent in GnRH mRNA levels before and during gestation; however, when fed with high-cholesterol diet, the GnRH mRNA levels increased significantly in pregnant rats. As results indicated both diets could lead to increase of PG mRNA in pancreas of pregnant rats. It is also demonstrated that the GnRH mRNA levels are positively associated with PG mRNA levels. Moreover, our data showed a significant increase in fasting insulin level in the Gestation group compared with Control. Such changes were contrary to the changes of GnRH level in the pancreas. This may imply that GnRH influences hormones secretion in the pancreas by autocrine and paracrine effects on islet cells.

Effects of heat stimulation via microwave applicator on cartilage matrix gene and HSP70 expression in the rabbit knee joint

The objective of this study was to investigate the effects of heat stimulation on the expression of extracellular matrix genes and heat-shock protein 70 (HSP70) in rabbit articular cartilage in vivo. Heat stimulation was applied to the knee joints of Japanese white rabbits for 20 min using a microwave (MW) applicator (2.45-GHz, 0-80 W). After 8-72 h, the articular cartilage was removed from the knee joints and proteins and total RNA were extracted. As controls, knee joints without heat stimulation were analyzed. The expression of HSP70 was confirmed by real-time PCR and Western blotting. The expression of proteoglycan core protein (PG) and type II collagen (Col II) was quantified using real-time PCR to assess cartilage matrix metabolism. Compared to controls, HSP70 expression was higher with more than 40 W of heat stimulation. The expression of PG and Col II mRNA was higher, with more than 20 W of heat stimulation and peaked with 40 W. When quercetin was used to inhibit the induction of HSP70 expression, PG mRNA expression did not increase. External MW application stimulated HSP70 expression in the articular cartilage in vivo. The expression of extracellular matrix genes was increased by appropriate heat stimulation.

Murine cytomegalovirus retinitis during MAIDS: Susceptibility correlates with elevated intraocular levels of interleukin-4 mRNA

Purpose. To determine if murine cytomegalovirus (MCMV)- infected eyes of mice with murine acquired immunodeficiency syndrome (MAIDS) that are destined to develop MCMV retinitis display elevated intraocular levels of interleukin-4 (IL-4) mRNA when compared with uninfected eyes of mice with MAIDS and unmanipulated, uninfected, eyes of normal healthy mice. Methods. Groups of C57BL/6 mice with MAIDS and normal C57BL/6 mice were infected uniocularly with MCMV by subretinal MCMV injection. IL-4 levels in individual spleens collected five days later from groups of MAIDS mice and normal mice were assessed by quantitative ELISA. MCMV-infected eyes and uninfected contralateral eyes from another group of mice with MAIDS were also collected at five days postinfection and individually subjected to competitive RT-PCR assay and real-time RT-PCR assay for detection and quantification of IL-4 mRNA. Unmanipulated eyes from normal C57BL/6 mice served as controls. Results. IL-4 mRNA was detected at a level of 9.7 ± 3.4 pg mRNA per 1000 ng total RNA in 100% of MCMV-infected eyes of mice with MAIDS by competitive RT-PCR assay, but could not be detected in any of the uninfected eyes of MAIDS mice. In comparison, the more sensitive technique of real-time RT-PCR assay detected copies of IL-4 cDNA in both MCMV-infected eyes and uninfected eyes of MAIDS mice. MCMV-infected eyes showed a 16-fold increase in the number of IL-4 cDNA copies when compared with uninfected eyes. Neither technique detected IL-4 mRNA in unmanipulated eyes of normal mice. As expected, spleen cells from mice with MAIDS expressed significantly greater levels of IL-4 when compared with spleen cells from normal mice. Conclusions. MCMV-infected mice with MAIDS exhibited an expected preferential activation of Th2 cells as determined by increased levels of IL-4 in spleen cells when compared with spleen cells of normal mice. MCMV-infected eyes destined to develop retinitis during MAIDS also showed increased levels of detectable IL-4 mRNA when compared with uninfected eyes of mice with MAIDS. It is therefore possible that IL-4 synthesis by Th2 CD4+ T cells during retrovirus-induced immunosuppression serves to inhibit the perforin cytotoxic pathway that subsequently allows susceptibility to MCMV retinitis during MAIDS.

Polygalacturonase: a candidate gene for the soft flesh and deciduous fruit mutation in Capsicum.

The soft flesh and deciduous fruit of pepper (Capsicum spp.) originated from the wild C. frutescens BG 2816 accession is a complete dominant trait controlled by the S gene. We constructed an F2 population from a cross of BG 2816 (SS) and the bell-type C. annuum cultivar Maor (ss) and determined that S cosegregated with the tomato fruit-specific endo-polygalacturonase (PG) gene. The soft flesh and deciduous fruit phenotypes were observed together in all F2 individuals, indicating a pleiotropic effect of PG on the two traits. We mapped S to pepper chromosome 10 in the region corresponding to that in which PG was previously mapped in tomato. Northern, RT-PCR and western analyses and enzyme activity assays, collectively, indicated that PG is not detected in green, breaker or red fruits of Maor, nor in green fruits of BG 2816. Accumulation of PG mRNA and protein was detected in the fruits of BG 2816, and it increased during ripening from breaker to red stages. The sequence analysis of partial PG cDNA isolated from BG 2816 revealed high homology (87% identity) with the tomato PG. The resemblance of the soft flesh and deciduous fruit phenotypes to PG-associated phenotypes in other fruit crops, the complete linkage between S and PG, and the greater expression of PG in the fruits of BG 2816 than in those of Maor, all strongly indicate that PG is a candidate gene for S.

Expression of proteoglycan mRNA in human temporomandibular joint disks in temporomandibualr disorders.

The purpose of this study was to investigate the expression of PG mRNA in human temporomandibular joint (TMJ ) disks from patients with temporomandibular disorders (TMD). Total RNA was isolated from the TMJ disks. All patients had undergone diskectomy under general anethesia. RT-PCR was performed using primers for the proteoglycans, aggrecan and decorin. On electrophoresis of a lysate of human TMJ disks, decorin was recognized in each sample. In the calcified part of the disk which we recognized visually, aggrecan was also recognized. These results suggest that TMJ disks contain decorin, and are fibrous tissue. If the disk was subjected to excessive compression in addition to tension, cells similar to chondrocytes and expression of aggrecan was induced. Calcification would be initiated in the part of the disk experiencing the increased compression.

Doxycycline-induced expression of sense and inverted-repeat constructs modulates phosphogluconate mutase (Pgm) gene expression in adult Drosophila melanogaster.

A tetracycline-regulated (conditional) system for RNA interference (RNAi) would have many practical applications. Such a strategy was developed using RNAi of the gene for phosphogluconate mutase (Pgm). Pgm is a candidate lifespan regulator: PgmS allele frequency is increased by selection for increased lifespan, whereas PgmM and PgmF allele frequencies are decreased. The Pgm alleles were cloned and sequenced and were found to differ by amino-acid substitutions consistent with the relative electrophoretic mobilities of the proteins. The 'tet-on' doxycycline-regulated promoter system was used to overexpress PgmS in a wild-type (PgmM) background. Enzyme activity increases of two- to five-fold were observed in five independent transgenic lines. Tet-on was also used to drive expression of an inverted-repeat fragment of Pgm coding region. The inverted-repeat transcript was expected to form a dsRNA hairpin, induce RNAi, and thereby reduce endogenous Pgm gene expression at the RNA level. Endogenous Pgm RNA levels in adult flies were found to be reduced or eliminated by doxycycline treatment in five independent inverted-repeat transgenic lines. Our results show that doxycycline-regulated expression of inverted-repeat constructs can cause a conditional reduction in specific gene expression. The effect of sense and inverted-repeat construct expression on lifespan was assayed in multiple transgenic lines. Under the conditions tested, altered Pgm gene expression had no detectable effect on adult Drosophila lifespan. A system for conditional RNAi in Drosophila adults shows promise for assay of gene functions during aging. Our results indicate that Pgm does not have a simple strong effect on longevity.

Effect of glucocorticoid on expression of rat MUC5AC mRNA in rat gastric mucosa in vivo and in vitro.

The gastric mucus is an important factor in the gastric mucosal protection from acid, pepsin and various reagents (alcohol, aspirin, etc.). MUC5AC is the mucin secreted from surface mucous cells, and belongs to the gel-forming mucin. We examined the regulation of rat MUC5AC (rMUC5AC) mRNA by glucocorticoid in vivo and in vitro, comparing it with that of pepsinogen (Pg) mRNA. By adrenal gland resection, rMUC5AC and Pg mRNA levels and Pg content in rats significantly decreased to 70%, 46% and 42% of those in the sham operated controls, respectively. With the treatment of hydrocortisone (1, 5 and 50 mg/kg), Pg mRNA level and Pg content in adrenalectomized rats was restored. On the other hand, the rMUC5AC mRNA level exceeded the control with 1 or 5 mg/kg injections of hydrocortisone, but drastically decreased to 18% of sham operation levels with it (50 mg/kg). Similar results were obtained in normal rats with the treatment of hydrocortisone (50 mg/kg). Mucus and DNA content of cultured rat gastric epithelial cells were not affected by hydrocortisone, but rMUC5AC mRNA level was significantly decreased in a dose-dependent manner. From the in vivo and in vitro results, at least a physiological concentration of glucocorticoid was necessary in the expression of rMUC5AC mRNA. However, high dose of hydrocortisone directly suppressed the expression of rMUC5AC mRNA. These results suggested that hydrocortisone might directly cause the suppression and indirectly the enhancement of the mucin biosynthesis.

Some factors which influence intrinsic factor content and its mRNA level in the rat stomach.

Factors which regulate Intrinsic Factor (IF) content and IF mRNA level were examined with an abc-ELISA and Northern blot analysis in growing rats, and compared with pepsinogen (Pg) and in terms of the increased need for vitamin B12 (V.B12). Increases in IF content and IF mRNA level gradually occurred from day 13 after birth, whereas those of Pg and Pg mRNA started from day 16. The effects of a few related hormones on the expression of IF mRNA were examined. The injection of hydrocortisone induced IF and Pg mRNA expression in 5-d-old postnatal rats. Furthermore, adrenalectomy-induced decreases in IF content and IF mRNA level in adult male rats were recovered with hydrocortisone administration. IF content and IF mRNA level were measured in the artificially and physiologically created needs for V.B12, the first being regeneration of the liver, the V.B12 storing tissue, following partial hepatectomy, and the second pregnancy or lactation. During regeneration of the liver, increases in IF content and IF mRNA level were marked, followed by reduction toward the original level after accomplishment of regeneration. Increases in IF content and IF mRNA level were also seen in lactating rats, but no increases were obtained in pregnant rats. These results suggest that the IF content and IF mRNA level are regulated not only by corticosteroids but also by the increased need for V.B12.

Regulation of tomato fruit polygalacturonase mRNA accumulation by ethylene: A Re-examination

Polygalacturonase (PG) is the major enzyme responsible for pectin disassembly in ripening fruit. Despite extensive research on the factors regulating PG gene expression in fruit, there is conflicting evidence regarding the role of ethylene in mediating its expression. Transgenic tomato (Lycopersicon esculentum) fruits in which endogenous ethylene production was suppressed by the expression of an antisense 1-aminocyclopropane-1-carboxylic acid (ACC) synthase gene were used to re-examine the role of ethylene in regulating the accumulation of PG mRNA, enzyme activity, and protein during fruit ripening. Treatment of transgenic antisense ACC synthase mature green fruit with ethylene at concentrations as low as 0.1 to 1 L/L for 24 h induced PG mRNA accumulation, and this accumulation was higher at concentrations of ethylene up to 100 L/L. Neither PG enzyme activity nor PG protein accumulated during this 24-h period of ethylene treatment, indicating that translation lags at least 24 h behind the accumulation of PG mRNA, even at high ethylene concentrations. When examined at concentrations of 10 L/L, PG mRNA accumulated within 6 h of ethylene treatment, indicating that the PG gene responds rapidly to ethylene. Treatment of transgenic tomato fruit with a low level of ethylene (0.1 L/L) for up to 6 d induced levels of PG mRNA, enzyme activity, and protein after 6 d, which were comparable to levels observed in ripening wild-type fruit. A similar level of internal ethylene (0.15 L/L) was measured in transgenic antisense ACC synthase fruit that were held for 28 d after harvest. In these fruit PG mRNA, enzyme activity, and protein were detected. Collectively, these results suggest that PG mRNA accumulation is ethylene regulated, and that the low threshold levels of ethylene required to promote PG mRNA accumulation may be exceeded, even in transgenic antisense ACC synthase tomato fruit.

A new quantitative solution hybridisation-RNase protection assay for APP and APLP2 mRNA

Amyloid precursor protein (APP) and amyloid precursor-like protein 2 (APLP2) are members of a multigene family of proteins implicated in the pathogenesis of Alzheimer's disease. We describe the development of an RNA-RNA solution hybridisation-RNase protection assay to quantify APP mRNA, APP mRNA splice forms containing the Kunitz-type protease inhibitor (KPI) insert, and APLP2 mRNA in total nucleic acid extracts from a range of tissue types. Solution hybridisation-RNase protection assay enables absolute quantification of target mRNA, by conversion of the hybridisation signal to pg mRNA using a standard curve. The assay is sensitive, capable of detecting 1 pg target mRNA, and reproducible, with an inter-assay variability of less than 10% and an intra-assay variability of 3–4%. We quantified APP and APLP2 mRNA in cell lines and post-mortem human brain tissue samples. To test whether we could detect physiological differences in APP mRNA levels, a fibroblast cell line with a paternal chromosome 21 deletion of the region including the APP gene was analysed and found to express half as much APP mRNA as control fibroblasts. In addition, a reversible, approx. 30% increase in APP mRNA levels was detected in human lymphoblastoid cell lines following heat shock, a physical stimulus previously shown to increase APP expression. Regional differences in the expression of APP and APLP2 were seen in human post-mortem cerebral cortex and cerebellum. Levels of APP and APLP2 mRNA were highest in the temporal cortex, slightly lower in frontal and occipital cortices, and lowest in the cerebellum. The highest proportion of KPI-containing APP was seen in the frontal and temporal cortices. The ratio of APP:APLP2 mRNA was 1:0.3 in the cortical tissue and 1:0.8 in the cerebellum. In conclusion, quantitative solution hybridisation-RNase protection assay of total APP, APP KPI and APLP2 mRNA provides a new tool to improve the resolution of studies of potentially subtle alterations in the expression of these genes in both cell culture model systems and Alzheimer's disease post-mortem human brain tissue.

Differential regulation of polygalacturonase and pectin methylesterase gene expression during and after heat stress in ripening tomato (Lycopersicon esculentum Mill.) fruits

The effects of extended heat stress on polygalacturonase (PG; EC 3.2.1.15) and pectin methylesterase (PME; EC 3.1.1.11) gene expression at mRNA, protein and activity levels in ripening tomato fruits were investigated. Steady state levels of PG mRNA declined at temperatures of 27 degrees C and above, and a marked reduction in PG protein and activity was observed at temperatures of 32 degrees C and above. Exogenous ethylene treatment did not reverse heat stress-induced inhibition of PG gene expression. Transfer of heat-stressed fruits to 20 degrees C partly restored PG mRNA accumulation, but the rate of PG mRNA accumulation declined exponentially with duration of heat stress. Heat stress-induced inhibition of PME mRNA accumulation was recoverable even after 14 days of heat stress. In fruits held at 34 degrees C, both PG and PME protein and activity continued to accumulate for about 4 days, but thereafter PG protein and activity declined while little change was observed in PME protein and activity. In spite of increases in mRNA levels of both PG and PME during the recovery of heat-stressed fruit at 20 degrees C, levels of PG protein and activity declined in fruits heat-stressed for four or more days while PME protein and activity levels remained unchanged. Collectively, these data suggest that PG gene expression is being gradually and irreversibly shut off during heat stress, while PME gene expression is much less sensitive to heat stress.

Evidence for differences in cultured left ventricular fibroblast populations isolated from spontaneously hypertensive and Wistar???Kyoto rats

To compare fibroblast populations derived from spontaneously hypertensive rats (SHRLJ) and normotensive Wistar-Kyoto rats (WKYLJ) for angiotensin II receptor binding, gene expression of the AT1 receptor and angiotensinogen, hormone responsiveness and phenotypic changes. Fibroblasts were isolated by either collagenase B or collagenase P and grown to confluency in the presence of 10% fetal bovine serum. Angiotensin II receptor binding was assessed under both serum and serum-free conditions. Hormonal treatment of cells was conducted in a serum-free background. The concentrations of AT1 receptor and angiotensinogen messenger RNA (mRNA) were determined by liquid hybridization. Phenotypic changes in fibroblast populations were analysed by visualization of lipid-containing vacuoles (oil red O stain) or of alpha-smooth muscle actin-containing fibres (immunostain). SHRLJ collagenase-B cells grew more slowly and had nearly twofold fewer angiotensin II receptors than WKYLJ cells as measured by both radioligand binding and AT1 mRNA content (SHRLJ 1.34 +/- 0.05 versus WKYLJ 5.94 +/- 0.41 pg mRNA per microgram total RNA) but contained significantly more angiotensinogen mRNA (SHRLJ 147 +/- 12 versus WKYLJ 98 +/- 8 fg mRNA per microgram total RNA). Collagenase-P cells from the two strains exhibited similar binding and growth properties. Collagenase-B fibroblasts also exhibited greater responses to exogenous steroids, including a greater shift towards an adipocyte phenotype, than collagenase-P cells. Exogenous angiotensin II promoted transformation towards a myofibroblast cell type, especially in collagenase-P SHRLJ cells. Our results indicate that subsets of fibroblasts that differ in growth rate, angiotensin II receptor binding, AT1 and angiotensinogen mRNA levels, structure and steroid responsiveness may be isolated from the left ventricle. The potential importance of these altered phenotypes to cardiac remodelling and hypertrophy warrants further examination.