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PubMed1996

Modulation of the levels of transforming growth factor beta messenger ribonucleic acids in human endometrial stromal cells.

We speculate that transforming growth factor beta (TGF beta) is involved in the predecidualization of human endometrial stromal cells (the progenitors of the decidual cells) during the late secretory phase of nonfertile, ovulatory cycles and in the completion of decidualization during fertile cycles after blastocyst implantation. In this study, the regulation of the levels of TGF beta mRNA levels in human endometrial stromal cells in culture was evaluated. TGF beta 1 and TGF beta 3 mRNAs were demonstrable by Northern analysis of total RNA from endometrial stromal cells maintained in serum-containing culture medium. TGF beta 2 mRNA was not detected by similar analyses of total RNA. The levels of TGF beta 3 mRNA in endometrial stromal cells increased in a time-dependent fashion after these cells were changed to serum-free medium. Platelet-derived growth factor acted, in a dose-dependent manner, to increase the levels of both TGF beta 1 and TGF beta 3 mRNAs in these cells. TGF beta 1 treatment caused an increase in the levels of TGF beta 1 mRNA and a decrease in the level of TGF beta 3 mRNA. The effects of platelet-derived growth factor and TGF beta 1 to increase the levels of TGF beta 1 mRNA were at least additive. Treatment of endometrial stromal cells with estradiol-17 beta caused an increase in the levels of TGF beta 1 and TGF beta 3 mRNAs. Treatment of stromal cells with medroxyprogesterone acetate (MPA, a synthetic progestin) also effected a small increase in the level of TGF beta 1 mRNA. The level of TGF beta 3 mRNA in endometrial stromal cells, however, was decreased by MPA treatment. The level of TGF beta 3 mRNA was greater in proliferative phase endometrial tissue than in secretory phase tissue. The levels of TGF beta 3 mRNA in decidua of the first trimester of pregnancy, in atrophic endometrium (from women treated with a GnRH agonist), and in endometrium from women ingesting oral progestin (MPA), were approximately one-fourth that in proliferative phase endometrium. These findings support the potential for modulation of TGF beta 1 and TGF beta 3 gene expression in endometrial stromal cells.

Cells, Cultured↗
PubMed2000

Effects of muscle type, castration, age, and compensatory growth rate on androgen receptor mRNA expression in bovine skeletal muscle.

The effect of testosterone on sexual dimorphism is evident by differential growth of forelimb and neck muscles in bulls and steers. Divergent hormone sensitivites may account for the differential growth rates of individual muscles. Therefore, the objective of this study was to compare androgen receptor (AR) expression in three different muscles of bulls and steers at various ages and growth rates. Thirty Montbéliard bulls and 30 steers were assigned to four slaughter age groups. Four or five animals of each sex were slaughtered at 4 and 8 mo of age. Animals in the remaining two slaughter groups (12 and 16 mo) were divided into groups of either restricted (R) or ad libitum (AL) access to feed. Five animals of each sex and diet were slaughtered at the end of the restricted intake period at 12 mo of age. To simulate compensatory growth, the remaining animals (R and AL) were allowed ad libitum access to feed until slaughter at 16 mo of age. Total RNA was extracted from samples of semitendinosus (ST), triceps brachii (TB), and splenius (SP) muscles. Androgen receptor mRNA was quantified in 200-ng total RNA preparations using an internally standardized reverse transcription (RT) PCR assay. Data were analyzed using 18S ribosomal RNA concentrations as a covariable. Steers had higher AR mRNA levels per RNA unit than bulls (P < .01). Androgen receptor mRNA levels differed between muscles (P < .05), with lowest expression in the SP. The pattern of AR expression differed (P < .05) for each muscle with increasing age. Between 4 and 12 mo of age, AR mRNA levels increased (P < .05) in SP but remained unchanged in the ST and TB. Feeding regimen had no effect on muscle AR expression, but steers exhibiting compensatory growth had higher AR mRNA levels than AL steers (P < .01) or bulls (P < .01). Our results show that AR expression is muscle-specific and may be modulated by circulating testicular hormones. These data suggest that the regulation of AR expression may be linked to allometric muscle growth patterns in cattle and compensatory gain in steers.

Aging↗
PubMed2006

WT1 gene expression lowered by IL-12 In vitro in peripheral blood mononuclear cells from patients with leukemia or myelodysplastic syndromes.

Previous studies demonstrated that interleukin-12 (IL-12) enhances the non-MHC-restricted cytotoxic activity of NK cells and facilitate specific allogeneic human cytotoxic T lymphocyte responses against fresh leukemia cells and cell lines. The Wilms' tumor gene, WT1 mRNA, has been used as a marker of minimal residual disease (MRD) for evaluating therapeutic efficacy of patients with leukemia or myelodysplastic syndrome (MDS). This study was aimed to investigate whether in vitro IL-12 can lower WT1 gene expression in peripheral blood monuclear cells (PBMNC) from patients with leukemia or MDS. PBMNC from these 30 patients and 5 healthy volunteers were cultured at 5 x 10(5) cells/ml alone with or without 100 units/ml of IL-12 for 3 days. WT1 mRNA was measured by competitive reverse transcription polymerase chain reaction (RT-PCR) since WT1 mRNA is considered as a marker of minimal residual disease (MRD) in leukemia and MDS. The results demonstrated that WT1 mRNA in PBMNC of 5 healthy volunteers was less than 10(3) copies/microg of total RNA. Following the 3-day IL-12 treatment, mean WT1 mRNA of PBMNC was reduced from 10(4.8) to 10(4.2) copies/microg of total RNA in 6 CML patients, from 10(5.4) to 10(4.8) copies/microg in 12 MDS patients and from 10(5.0) to 10(4.2) copies/microg in 5 AML patients in CR, but not reduced in 5 of 7 AML in non-CR. It is concluded that IL-12 significantly decrease the quantity of leukemia cells in PBMNC of most patients with MDS, CML and AML in CR. IL-12 may be of considerable benefit in the elimination of MRD in patients with hematological malignancies.

Adult↗
PubMed1998

Uterine androgen receptor mRNA expression in metestrous and anestrous bitches being healthy or suffering from Pyometra.

The importance of androgens for the female reproductive system has been investigated for decades and a number of androgen sensitive processes has now been identified in female reproductive organs. For carnivore species no data were available so far about uterine androgen sensitivity and its regulation. The present study therefore aimed to investigate whether androgen receptors (AR) are present in the dog uterus, whether they are regulated throughout the ovarian cycle and whether pyometra affects their expression rate. Uterine tissue samples were collected from 28 bitches of different ages and various breeds. The samples were grouped according to the stage of estrous cycle (metestrus ME or anestrus AE) and the pathological status of the uterus (i.e. suffering from pyometra or not). Androgen receptor mRNA (AR mRNA) was quantified from 500 ng of total RNA isolated from the tissue samples using an internally standardized reverse transcription polymerase chain reaction (RT-PCR) described previously. The amount of total RNA extractable per g tissue was elevated during pyometra. The successful amplification of the expected 172 bp fragment from canine uterine RNA together with the confirmation of the identity of this fragment by sequence analysis, demonstrates that AR is expressed in this particular tissue. Comparing the expression rates in uteri from bitches during ME or AE being healthy (H) or suffering from pyometra (P), the only significant (p < 0.01) difference was found between H and P uteri during ME with 3.5-fold lower expression rates in P. Although the same seems true for AE bitches, a significant difference could not be demonstrated due to the low number (n = 2) of diseased animals in the AE group. There was no evident effect of the stage of ovarian cycle on uterine AR mRNA levels.

Anestrus↗
PubMed1988

Posttranscriptional control of embryonic rat skeletal muscle protein synthesis. Control at the level of translation by endogenous RNA.

The onset of muscle cell differentiation is associated with increased transcription of muscle-specific mRNA. Studies from this laboratory using 19-d embryonic rat skeletal muscle, suggest that additional, posttranscriptional controls regulate maturation of muscle tissue via a quantitative effect upon translation, and that the regulatory component may reside within the poly A- RNA pool (Nathanson, M.A., E.W. Bush, and C. Vanderburg. 1986. J. Biol. Chem. 261:1477-1486). To further characterize muscle cell translational control, embryonic and adult total RNA were separated into oligo(dT)cellulose-bound (poly A+) and -unbound (poly A-) pools. Unbound material was subjected to agarose gel electrophoresis to resolve constituents of varying molecular size and mechanically cut into five fractions. Material of each fraction was electroeluted and recovered by precipitation. Equivalent loads of total RNA from 19-20-d embryonic rat skeletal muscle exhibited a 40% translational inhibition in comparison to its adult counterpart. Inhibition was not due to decreased message abundance because embryonic, as well as adult muscle, contained equivalent proportions of poly A+ mRNA. An inhibition assay, based upon the translatability of adult RNA and its inhibition by embryonic poly A- RNA, confirmed that inhibition was associated with a 160-2,000-nt poly A- fraction. Studies on the chemical composition of this fraction confirmed its RNA composition, the absence of ribonucleoprotein, and that its activity was absent from similarly fractionated adult RNA. Rescue of inhibition could be accomplished by addition of extra lysate or mRNA; however, smaller proportions of lysate were required, suggesting a strong interaction of inhibitor and components of the translational apparatus. Additional studies demonstrated that the inhibitor acted at the level of initiation, in a dose-dependent fashion. The present studies confirm the existence of translational control in skeletal muscle and suggest that it operates at the embryonic to adult transition. A model of muscle cell differentiation, based upon transcriptional control at the myoblast level, followed by translational regulation at the level of the postmitotic myoblast and/or myotube, is proposed.

Animals↗
PubMed1976

The conformation of 16S RNA in the 30S ribosomal subunit from Escherichia coli.

The digestion of E. coli 16S RNA with a single-strand-specific nuclease produced two fractions separable by gel filtration. One fraction was small oligonucleotides, the other, comprising 67.5% of the total RNA, was highly structured double helical fragments of mol. wt. 7,600. There are thus about 44 helical loops of average size corresponding to 12 base pairs in each 16S RNA. 10% of the RNA could be digested from native 30S subunits. Nuclease attack was primarily in the intraloop single-stranded region but two major sites of attack were located in the interloop single-stranded regions. Nuclease digestion of unfolded subunits produced three classes of fragments, two of which, comprising 80% of the total RNA, were identical to fragments from 16S RNA. The third, consisting of 20% RNA, together with an equal weight of peotein, was a resistant core (sedimentation coefficient 7S).

Circular Dichroism↗
PubMed1983

Influence of thyroid hormone on the in vitro translational activity of specific mRNAs in the rat heart.

The influence of thyroid hormone on the translational activity of specific cardiac mRNA was determined by in vitro translation of RNA isolated from the heart of normal, hypothyroid, and 3,3',5-triiodo-L-thyronine-injected hypothyroid rats. Proteins synthesized in vitro in the presence of [35S]methionine were separated by two-dimensional gel electrophoresis and quantitated by a novel scanning procedure using digital matrix photometry. A total of 421 translational products were detected by fluorography and changes in the predominance of 12 of these were influenced by the thyroid state of the animals. The relative predominance of 8 species was increased in euthyroid animals, whereas 4 translational products were increased in hypothyroid animals. The majority of these thyroid hormone-related alterations occurred in spot pairs of similar molecular weights, but slightly different isoelectric points. In contrast, the relative predominance of mRNAs coding for the major contractile proteins, light chain 1, light chain 2, tropomyosin, actin, and myosin heavy chain was not altered by the thyroid status of the animals. The relative levels of these abundant mRNA species remained unaltered in spite of a thyroid hormone-related increase in total RNA levels. In vivo effects of thyroid hormone on cardiac RNA levels are complex. In addition to a general increase in total RNA and mRNA levels, increases or attenuations in the predominance of a small number of specific mRNA species are observed when euthyroid and hypothyroid animals are compared.

Animals↗
PubMed1993

Trabecular cells of the eye express messenger RNA for transforming growth factor-beta 1 and secrete this cytokine.

PURPOSE: To determine whether trabecular tissue in vivo and cultured trabecular cells have the messenger RNA transcript for transforming growth factor-beta 1 (TGF-beta 1), and to examine whether these cells synthesize and secrete TGF-beta 1 in vitro. METHODS: Total RNA was isolated from the trabecular meshwork, iris, and ciliary body freshly excised from porcine eyes as well as from cultured trabecular cells, and the reverse transcriptase-polymerase chain reaction and Southern hybridization were used for detection of TGF-beta 1 messenger RNA. The amount of TGF-beta 1 secreted by trabecular cells in culture was determined by radioimmunoassay. RESULTS: Excised whole trabecular tissue, iris, and ciliary body, as well as cultured trabecular cells expressed messenger RNA transcripts for TGF-beta 1. On the ethidium bromide-stained agarose gel, two PCR-amplified products (161 and 400 base pairs) were found in the total RNA isolated from cultured trabecular cells. The oligonucleotide probe specific for TGF-beta 1 detected only one band with the expected length of 161 base pairs. The secretion of TGF-beta 1 into conditioned medium was at the level of 16.7-20 pg/ml per 2 million trabecular cells during a 24-hr period. CONCLUSIONS: These investigations show that the trabecular meshwork, iris, and ciliary body in vivo express the messenger RNA transcript for TGF-beta 1, and that trabecular cells in vitro synthesize and secrete this cytokine. The TGF-beta 1 present in normal aqueous humor may be derived locally, at least in part, from the cells of the trabecular meshwork, iris, and ciliary body. Abnormal synthesis, secretion, activation, and clearance of TGF-beta 1 may contribute to the pathogenesis of many ocular disorders, including primary open-angle glaucoma.

Animals↗
PubMed1999

Expression of the thrombopoietin gene in human fetal and neonatal tissues.

Thrombopoietin (TPO) regulates megakaryopoiesis and platelet production. In the adult, TPO is mainly produced by the liver and the kidneys. This study focuses on fetal and neonatal TPO mRNA expression. In 26 human fetuses and preterm neonates, samples from liver, kidney, spleen, lung, and bone marrow were extracted for total RNA. We measured platelet counts, TPO serum concentrations by enzyme-linked immunosorbent assay, and TPO mRNA contents by reverse transcription/competitive polymerase chain reaction. TPO mRNA concentrations per microgram total RNA were similar in liver, spleen, and bone marrow, slightly lower in kidney, and significantly lower in lung. When related to gram tissue, TPO mRNA levels were highest in the liver. Considering the total amount of TPO mRNA produced in liver, kidney, and spleen, the liver accounted for 95.3%. No correlations between TPO mRNA expression and serum TPO concentration, blood platelet count, or gestational age were observed. In conclusion, the liver is the primary site of TPO gene expression in human fetuses and neonates. The spleen may contribute to TPO production during fetal life. Like in the adult, TPO mRNA is expressed in fetal bone marrow.

Adult↗
PubMed2004

Optimized procedures for microarray analysis of histological specimens processed by laser capture microdissection.

Analysis of cell-specific gene expression patterns using microarrays can reveal genes that are differentially expressed in diseased and normal tissue, as well as identify genes associated with specialized cellular functions. However, the cellular heterogeneity of the tissues precludes the resolution of expression profiles of specific cell types. While laser capture microdissection (LCM) can be used to obtain purified cell populations, the limited quantity of RNA isolated makes it necessary to perform an RNA amplification step prior to microarray analysis. The linearity and reproducibility of two RNA amplification protocols--the Baugh protocol (Baugh et al., 2001, Nucleic Acids Res 29:E29) and an in-house protocol have been assessed by conducting microarray analyses. Cy3-labeled total RNA from the colorectal cell line Colo-205 was compared to Cy5-labeled Colo-205 amplified RNA (aRNA) generated with each of the two protocols, using a human 10K cDNA array. The correlation of the gene intensities between amplified and total RNA measured in the two channels of each microarray was 0.72 and 0.61 for the Baugh protocol and the in-house protocol, respectively. The two protocols were further evaluated using aRNA obtained from normal colonic crypt cross-sections isolated via LCM. In both cases a microarray profile representative of colonic mucosa was obtained; statistically, the Baugh protocol was superior. Furthermore, a substantial overlap between highly expressed genes in the Colo-205 cells and colonic crypts underscores the reliability of the microarray analysis of LCM-derived material. Taken together, these results demonstrate that LCM-derived tissue from histological specimens can generate abundant amounts of high-quality aRNA for subsequent microarray analysis.

Cell Line, Tumor↗
PubMed2005

Four-color multiplex reverse transcription polymerase chain reaction--overcoming its limitations.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) conducted in real time is a powerful tool for measuring messenger RNA (mRNA) levels in biological samples. Multiplex PCR is defined as the simultaneous amplification of two or more DNA (cDNA) targets in a single reaction vessel and may be carried out only using uniquely labeled probes for each target. Up to four genes can be detected in a multiplex 5' nuclease assay when using the appropriate instrument and the right combination of fluorophores. One of the more important advantages of multiplexing is a reduced sample requirement, which is especially important when sample material is scarce. Additional benefits are saving time on reaction setup and lower cost compared to singleplex reactions. Although multiplexing has several advantages over singleplex qRT-PCR, limited work has been done to show its feasibility. Few publications on four-color multiplex qRT-PCR have been reported, and to our knowledge no work has been done to explore the assay's limitations. In this paper, we report the first in-depth analysis of a four-gene multiplex qRT-PCR. To achieve a better understanding of the potential limitations of the qRT-PCR assay, we used in vitro transcribed RNA derived from four human genes. To emulate gene expression experiments, we developed a model system in which the in vitro transcripts were spiked with plant total RNA. This model allowed us to develop an artificial system closely resembling differential gene expression levels varying up to a million fold. We identified a single "universal" reaction condition that enabled optimal amplification in real time of up to four genes over a wide range of template concentrations. This study shows that multiplexing is a feasible approach applicable to most qRT-PCR assays performed with total RNA, independent of the expression levels of the genes under scrutiny.

Color↗
PubMed1976

Studies on secondary structure of single-stranded RNA from bacteriophage MS2 by electron microscopy.

A method allowing the demonstration and study by electron microscopy of secondary structure of viral RNA has been developed. Single-stranded RNA from the bacteriophage MS2 has been analyzed in the electron microscope in the presence of various concentrations of MgCl2. Depending on the salt concentration, the molecules display one to three large open loops which range in size from 10 to 20% of the total RNA length, and smaller closed loops which are approximately 3-5% of the total RNA length. Within one spreading, the conformation of the molecules is variable. However, the average complexity of the molecules increases with increasing salt, and individual loops which are infrequent at low salt increase in frequency with increasing salt. By analyzing the manner in which the individual loop appeared, it was possible to show that all molecules could be described by one basic pattern of secondary structure formation.

Coliphages↗
PubMed2001

A quantitative analysis of purinoceptor expression in the bladders of patients with symptomatic outlet obstruction.

OBJECTIVE: To compare the expression of the seven known P2X receptors in human bladder from male patients with detrusor instability caused by symptomatic bladder outlet obstruction with that from control bladders, using a quantitative reverse transcription-polymerase chain reaction (RT-PCR) method. PATIENTS AND METHODS: Real-time quantitative RT-PCR provides a system for detecting and analysing RNA. Bladder biopsies were obtained from nine patients undergoing prostate surgery and control biopsies were obtained from eight age-matched men undergoing routine bladder endoscopy studies, and who were asymptomatic. Total RNA was extracted from each sample and 10 ng of this used for individual PCR reactions. The expression levels of the seven P2X genes in the total RNA were then determined. RESULTS: In the control bladder, P2X1 was by far the predominant purinergic receptor at the RNA level, the remainder consistently present in the order P2X1 >> P2X4 > P2X2 > P2X7 > P2X5 >> P2X3 = P2X6 = 0. Calponin, a smooth muscle-specific protein, was used as a marker for smooth muscle content. In bladder from symptomatic patients, the P2X1/calponin ratio was greater than that in controls (P = 0.016). There appeared to be no difference in P2X2, but P2X4, P2X5, and P2X7 were all greater in the symptomatic bladder than in the controls, although these differences were not significant. CONCLUSION: P2X1 is the predominant purinoceptor subtype in the human male bladder, consistent with pharmacological evidence. The amount of P2X1 receptor per smooth muscle cell is greater in the obstructed than in control bladder, suggesting an increase in purinergic function in the unstable bladder arising from bladder outlet obstruction.

Aged↗
PubMed1998

Lactoferrin and interleukin-6 interaction in amniotic infection.

Lactoferrin (Lf) has been found in most biological fluids including amniotic fluid and cervical mucoids in pregnant women, and released from neutrophils in response to the inflammation. As Lf possesses antimicrobial properties, it is widely considered to be an important component of the host defence against microbial infections. It is known that premature labor is caused by amniotic infection with the increase of prostaglandin production. High concentration of the inflammatory cytokines: interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) in the amniotic fluid has been known. However, changes of Lf in amniotic fluid with infection has not been reported. In the present study, Lf concentrations in amniotic fluid were measured under the intra-uterine infections state and the biological significance of Lf was investigated. The effects of Lf on the IL-6 and IL-6mRNA production in cultured amnion cells were also investigated. The concentrations of Lf and IL-6 in amniotic fluid with CAM were 8.76 +/- 0.65 micrograms/ml and 6.92 +/- 4.88 ng/ml (n = 28) respectively and both were significantly higher (p < 0.01) than those without CAM [0.86 +/- 0.81 microgram/ml and 0.34 +/- 0.25 ng/ml (n = 31)]. Significant positive correlation (r = 0.91, p < 0.01) between Lf and IL-6 levels in amniotic fluid was found. IL-6 production induced by lipopolysaccharide (LPS) (100 ng/ml) in cultured amnion cells was significantly inhibited (p < 0.05) under the physiological concentration of Lf in amnion. Total RNA was extracted from the amniotic cells by guianizine solution. RT-PCR procedure and product analysis were performed from one microgram aliquote of total RNA. beta-actin was used as an international standard and c-DNA samples were followed by 30 cycles of PCR. RT-PCR product of IL-6 mRNA was detected by Southern hybridization. Expression of IL-6 mRNA was inhibited by the addition of Lf. From the results, the possibility that Lf might suppress amniotic IL-6 production under the condition of amniotic infection is suggested. It is also suggested that Lf might act as self defence mechanism from intra-uterine infection.

Amnion↗
PubMed1995

Quantitative analysis of messenger ribonucleic acid encoding natriuretic peptide receptors in aldosterone-producing adenoma.

There exist conflicting data regarding the inhibitory effect of atrial natriuretic peptide on aldosterone production from aldosterone-producing adenoma (APA). Natriuretic peptides mediate their actions through natriuretic peptide receptors (NPRs). Whether or not NPRs are present in the tumors remains controversial. To elucidate this paradox, gene expression of NPRs was examined by Northern blot analysis and competitive polymerase chain reaction in tumorous and non-tumorous portions of APA, and in normal adrenal gland from patients with renal cell carcinoma. The results of Northern blot analysis showed the presence of messenger ribonucleic acid (mRNA) of three NPRs in all adrenal tissues, including APA. The proportional expression of NPR gene transcripts in APA was type A (0.6%), type B (18.7%), and type C (80.7%). The levels, but not the proportions, of type C and possibly type B NPR mRNAs were lower in tumorous and non-tumorous portions of APA compared to those in normal adrenal gland (type C 190.2 +/- 24.5 [means +/- SEM, normal adrenal gland] > 168.1 +/- 20.8 [non-tumorous portion] > 112.2 +/- 15.5 [tumorous portion] pg/10 micrograms total RNA, F = 3.82, P < 0.05; type B 45.2 +/- 8.5 [normal adrenal gland] > 30.0 +/- 5.2 [non-tumorous portion] > 25.1 +/- 4.1 [tumorous portion] pg/10 micrograms total RNA, F = 3.03, P = 0.065). The mRNA levels of type C, rather than type A or type B, NPR were correlated with the percentage of zona fasciculata-like cells in APA (r = 0.90, P < 0.05). In conclusion we have demonstrated the presence of mRNA encoding the three NPRs in APA.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenoma↗
PubMed1995

A mutualistic fungal symbiont of perennial ryegrass contains two different pyr4 genes, both expressing orotidine-5'-monophosphate decarboxylase.

A fragment of the Claviceps purpurea pyr4 gene, encoding orotidine-5'-monophosphate decarboxylase (OMP decarboxylase), was used to screen a genomic library from an isolate of a fungus, Acremonium sp. (designated Lp1), which grows as an endophyte in perennial ryegrass (Lolium perenne). Three positive clones, lambda MC11, lambda MC12 and lambda MC14, were isolated. Two of these clones, lambda MC12 and lambda MC14, were overlapping clones from the same locus, while lambda MC11 was from a different locus. Fragments of these clones which hybridised with C. purpurea pyr4 were sequenced and found to have similarity with pyr4 from other Pyrenomycete fungi. The pyr4 gene from lambda MC12 and lambda MC14 was designated pyr4-1 and that from lambda MC11 was designated pyr4-2. The predicted ORFs of the two genes were highly conserved, with 97.5% identity at the nucleotide level, the 5' non-coding sequences were the least conserved with 88.5% identity and the 3' non-coding sequences had 93.0% identity. RT-PCR analysis of total RNA from Lp1 demonstrated that transcripts from the two genes were present at similar levels, and hybridisation of pyr4-1 to Northern blots of total RNA from Lp1 showed that full-length transcripts were being produced. Genomic fragments containing pyr4 were transformed into a strain of Aspergillus nidulans which has a mutation in pyrG (encoding OMP decarboxylase). Both pyr4-1 and pyr4-2 complemented the pyrG mutation in A. nidulans, indicating that both encode functional OMP decarboxylases. It has been proposed [Schardl et al., Genetics 136 (1994) 1307-1317] that the two pyr4 in Lp1 arose by interspecific hybridisation, most likely between the ryegrass choke pathogen, Epichloë typhina, and another endophyte from perennial ryegrass, Acremonium lolii. Analysis by PCR amplification and direct sequencing of the variable 5' non-coding regions of pyr4, from possible ancestors to Lp1 supports this hypothesis. Comparisons of these sequences to the 5' non-coding sequences from pyr4-1 and pyr4-2 demonstrated that E. typhina and A. lolii were the most likely ancestors of the two pyr4 found in Lp1.

Acremonium↗
PubMed2000

The decompensated detrusor III: impact of bladder outlet obstruction on sarcoplasmic endoplasmic reticulum protein and gene expression.

PURPOSE: Regulation of calcium ion homeostasis has a significant role in smooth muscle contractility. The sarcoplasmic endoplasmic reticulum, calcium, magnesium, adenosine triphosphatase (SERCA) is a regulatory ion pump that may have a role in the functional outcome after outlet obstruction. We investigate what correlation if any existed between SERCA protein and gene expression, and the contractile properties in the same bladder. MATERIALS AND METHODS: Standardized partial bladder outlet obstructions were created in adult New Zealand white rabbits, which were divided into control, sham operated and obstructed groups. Muscle strip studies subcategorized the obstructed group into compensated (force greater than 50% of control) and decompensated (force less than 50% of control). Microsomal membrane and total RNA fractions were prepared from the same bladder tissue. Membrane proteins were used for Western blot analysis using a SERCA specific monoclonal antibody, and total RNA was assessed with Northern blot analysis. RESULTS: The relative intensities of signals for the Western and Northern blots demonstrated a strong correlation between protein and gene expression. Furthermore there was a strong association between the loss of SERCA messenger RNA and protein expression and loss of bladder function. CONCLUSIONS: Bladder contractility after outlet obstruction is influenced in part by smooth muscle cell ability to maintain calcium homeostasis via SERCA. The loss of SERCA protein expression is mediated by down-regulation in gene expression in the same bladder. These data suggest that smooth muscle ion pump gene expression is in part mechanically (pressure work) regulated.

Animals↗
PubMed1996

Expression of aminopeptidase A, an angiotensinase, in glomerular mesangial cells.

Glomerular mesangial cells are known to express angiotensin II type 1 receptors and contract in response to circulating and/or locally produced angiotensin II. In addition, stimulation of mesangial cell matrix protein synthesis by elevated levels of angiotensin II is known to contribute to the development of glomerulosclerosis. Previously, we reported that mesangial cells were positively immunostained with antiserum directed against aminopeptidase A, the principal angiotensinase in the metabolism of angiotensin II. Here we demonstrate directly that aminopeptidase A is expressed in mesangial cells cultured from rat kidney. First, cultured mesangial cells had measurable aminopeptidase A enzymatic activity. Second, immunoblots for aminopeptidase A were positive for isolated glomeruli and mesangial cells, although two bands were seen for mesangial cells (approximately 138 and 144 kD), and only the larger band was seen for isolated glomeruli and kidney. Third, Northern blot hybridizations of total RNA from mesangial cells or kidney were positive and labeled similarly sized bands. Fourth, reverse transcription-polymerase chain reaction amplification of mesangial cell total RNA yielded a partial cDNA of the expected size that was confirmed by sequencing to be identical to rat kidney aminopeptidase A. These results indicate that aminopeptidase A is expressed within mesangial cells. These results further suggest that metabolism of angiotensin II by aminopeptidase A could play a protective role in minimizing the adverse effects of angiotensin II stimulation of mesangial cells.

Aminopeptidases↗