Changes In Isoforms Research Articles

Treatment of Higher Risk Myelodysplastic Syndrome Patients Unresponsive to Hypomethylating Agents with ON 01910.Na

AbstractAbstract 4010Patients with IPSS intermediate or high risk myelodysplastic syndrome (MDS) have few treatment options once their disease fails to respond to hypomethylating agent therapy. The styryl sulfone mitotic inhibitor ON 01910.Na, inhibits Polo-1 kinase, PI3-kinase and AKT pathways, and the drug has shown promising results in patients (pts) with advanced solid tumors (Jimeno et al, J Clin Onc 26:5504, 2008) and in Phase I/II studies of MDS pts, including those with trisomy 8 (Sloand at al, Proc ASH 2007, #822, Proc ASH 2008, #1651). In an ongoing Phase II clinical trial, we have treated 10 MDS pts unresponsive to at least 4 cycles (range 4–12 cycles) of hypomethylating agent therapy (5 post-azacytidine, 4 post-decitabine, and 1 pt treated with both agents) with ON 01910.Na. The pts had IPSS Intermediate-1 (n=3), Intermediate-2 (n=4) and High (n=3) risk MDS. The study cohort comprised pts with RAEB-1 (4 pts), RAEB-2 (3 pts) and RAEB-T (3 pts), with a median age of 80 years (range 65–86) and 2.3 year median (range 0.4–5.4) prior duration of MDS. Their cytogenetic profile included 5 pts with Good, 4 with Intermediate [t(8,10), t(14,18), +8, and (8+, 19+)], 1 with Poor risk cytogenetics (+8, 5q-, 1p-). At baseline, all pts were red blood cell transfusion-dependent. After the initial 2 pts were treated with 800mg/m2/day × 2day continuous IV infusion (CIVI)/week × 3weeks/month, the subsequent 8 patients received 1800mg/day × 3day CIVI q2weeks/month × 2 months, then monthly. Patients underwent bone marrow sampling and evaluation after every other cycle of treatment. To date, 7 pts have completed at least 2 cycles of therapy, with 2 pts having completed the full treatment course of 7 monthly cycles (median 4.7 for all pts). Responses according to IWG 2006 criteria were: Marrow CR (mCR) (2), Partial response (1), stable disease (SD) with hematologic improvement (HI) (2; 1 HI-E, N, 1 HI-P, N) = 5/10 overall responses (50%). Four pts had SD without HI, 1 pt progressed to AML. mCRs occurred in those with <10% marrow blasts. The 3 patients with trisomy 8 alone or with additional cytogenetic abnormalities achieved 1 mCR, 2 SD, associated with decrements in proportions of +8 karyotypes from 80% and 65% to 15% in the 2 pts with additional abnormalities. Grade 3/4 non-hematologic drug-related or possibly related toxicities occurred in 4 pts: 1 GI, 1 dysuria, 1 fatigue, 1 epistaxis; 2 pts had pulmonary infections unrelated to study drug. Two pts discontinued treatment early due to drug toxicity (1) or AML progression (1). No hematologic toxicities occurred. Nine of 10 pts remain alive (5.4 month median, range 2.5–13+) after study entry. To determine if a biologic response to ON 01910.Na treatment could be quantified, a novel nanoscale immunoassay (NIA) was used to measure changes in AKT kinase family activation in CD34+ cells isolated from the bone marrow specimens collected before and during treatment. In the subset of specimens analyzed to date, phosphorylation of AKT2 decreased by 36% in pts who had a response to therapy, suggesting that ON 01910.Na treatment altered oncogenic signaling in the MDS pts. Completion of NIA analysis will demonstrate whether changes in individual AKT isoforms (implicated in cell cycle arrest and apoptosis) occur uniquely in pts who have a clinical response to treatment. These preliminary data demonstrate encouraging efficacy and drug tolerance with ON 01910.Na in a substantial portion of higher risk MDS patients whose disease had been unresponsive to hypomethylating agent therapy. Accrual and continuing therapy for trial completion are ongoing. Disclosures:Off Label Use: The drug ON01910.Na is an investigational agent. Felsher:Cell Bioscience: On The Advisory Board. Wilhelm:Onconova Therapeutics Inc: Employment, Equity Ownership. Greenberg:Onconova Therapeutics Inc: Research Funding.

Changes in plasma müllerian-inhibiting substance and brain-derived neurotrophic factor after chemotherapy in premenopausal women

Eight premenopausal women with cancer had blood drawn for analysis of brain-derived neurotrophic factor (BDNF) and müllerian-inhibiting substance (MIS) before and 3 months after receiving chemotherapy. Unlike MIS, BDNF levels were not reduced after chemotherapy.

Mechanism of Regulation of Native Cardiac Muscle Thin Filaments by Rigor Cardiac Myosin-S1 and Calcium

We have studied the mechanism of activation of native cardiac thin filaments by calcium and rigor myosin. The acceleration of the rate of 2'-deoxy-3'-O-(N-methylanthraniloyl)ADP (mdADP) dissociation from cardiac myosin-S1-mdADP-P(i) and cardiac myosin-S1-mdADP by native cardiac muscle thin filaments was measured using double mixing stopped-flow fluorescence. Relative to inhibited thin filaments (no bound calcium or rigor S1), fully activated thin filaments (with both calcium and rigor-S1 bound) increase the rate of product dissociation from the physiologically important pre-power stroke myosin-mdADP-P(i) by a factor of ∼75. This can be compared with only an ∼6-fold increase in the rate of nucleotide diphosphate dissociation from nonphysiological myosin-mdADP by the fully activated thin filaments relative to the fully inhibited thin filaments. These results show that physiological levels of regulation are not only dependent on the state of the thin filament but also on the conformation of the myosin. Less than 2-fold regulation is due to a change in affinity of myosin-ADP-P(i) for thin filaments such as would be expected by a simple "steric blocking" of the myosin-binding site of the thin filament by tropomyosin. Although maximal activation requires both calcium and rigor myosin-S1 bound to the cardiac filament, association with a single ligand produces ∼70% maximal activation. This can be contrasted with skeletal thin filaments in which calcium alone only activated the rate of product dissociation ∼20% of maximum, and rigor myosin produces ∼30% maximal activation.

Proteomic alterations of Escherichia coli by paraquat.

Paraquat (PQ; a widely used herbicide) exerts its harmful effect to human, mammals and microorganisms upon intracellular conversion to superoxide radical. Cellular responses against toxic paraquat remain not fully understood, especially on the adaptive metabolic changes as a consequence of oxidative burden. In this study, alterations of metabolic processes of Escherichia coli (E. coli) by paraquat were systematically investigated by two-dimensional gel electrophoresis (2-DE) in conjunction with peptide mass fingerprinting (PMF). In host cells, the first line mechanism was scrutinized by a remarkable induction of endogenous superoxide dismutase (E. coli SOD). The second line involved in the metabolic adaptation and compensation for energy production by up- or down-regulation of the enzymes implicated in glycolysis and tricarboxylic acid cycle. Notably, down-regulation of aconitase enzyme and changes of enzyme isoform from the acidic (pI~5.29) to the higher basidic form (pI~5.59) were detected. Meanwhile, up-regulation of fumarase approximately 4-5 folds were observed. Importantly, overexpression of human manganese superoxide dismutase (human Mn-SOD) in E. coli cells could in turn down-regulate the expression of fumarase enzyme. This observation was not found when the cells expressing human catalase were tested. Other mechanisms such as changes of purine nucleoside phosphorylase and protein transporters (D-ribose-binding protein and oligopeptide binding protein) were also accounted. However, among all the differentially expressed proteins, the fumarase enzyme is evidenced to be a major target responsible for superoxide-generating paraquat, which may further be applied as a potential biomarker for paraquat toxicity in the future.

Complexation and calcium-induced conformational changes in the cardiac troponin complex monitored by hydrogen/deuterium exchange and FT-ICR mass spectrometry

Cardiac muscle contraction is regulated by the heterotrimeric complex: troponin. We apply solution-phase hydrogen/deuterium exchange monitored by FT-ICR mass spectrometry to study the structural dynamics and the Ca-induced conformational changes of the cardiac isoform of troponin, by comparing H/D exchange rate constants for TnC alone, the binary TnC:TnI complex, and the ternary TnC:TnI:TnT complex for Ca-free and Ca-saturated states. The wide range of exchange rate constants indicates that the complexes possess both highly flexible and very rigid domains. Fast exchange rates were observed for the N-terminal extension of TnI (specific to the cardiac isoform), the DE linker in TnC alone, and the mobile domain of TnI. The slowest rates were for the IT coiled-coil that grants stability and stiffness to the complex. Ca2+ binding to site II of the N-lobe of TnC induces short-range allosteric effects, mainly protection for the C-lobe of TnC that transmits long-range conformational changes that reach the IT coiled-coil and even TnT1. The present results corroborate prior X-ray crystallography and NMR interpretations and also illuminate domains that were truncated or not resolved in those experiments.

YIA3 Proteomic analysis of the cardiac myofilament subproteome reveals dynamic alterations in phosphatase subunit distribution

RationaleMyofilament proteins are responsible for cardiac contraction. The myofilament subproteome, however, has not been comprehensively analysed thus far.MethodsCardiomyocytes were isolated from rodent hearts and stimulated with endothelin-1 and isoproterenol, potent...

Exon-Level Transcriptome Profiling in Murine Breast Cancer Reveals Splicing Changes Specific to Tumors with Different Metastatic Abilities

BackgroundBreast cancer is the second most frequent type of cancer affecting women. We are increasingly aware that changes in mRNA splicing are associated with various characteristics of cancer. The most deadly aspect of cancer is metastasis, the process by which cancer spreads from the primary tumor to distant organs. However, little is known specifically about the involvement of alternative splicing in the formation of macroscopic metastases. Our study investigates transcript isoform changes that characterize tumors of different abilities to form growing metastases.Methods and FindingsTo identify alternative splicing events (ASEs) that are associated with the fully metastatic phenotype in breast cancer, we used Affymetrix Exon Microarrays to profile mRNA isoform variations genome-wide in weakly metastatic (168FARN and 4T07) and highly metastatic (4T1) mammary carcinomas. Statistical analysis identified significant expression changes in 7606 out of 155,994 (4%) exons and in 1725 out of 189,460 (1%) intronic regions, which affect 2623 out of 16,654 (16%) genes. These changes correspond to putative alternative isoforms—several of which are novel—that are differentially expressed between tumors of varying metastatic phenotypes. Gene pathway analysis showed that 1224 of genes expressing alternative isoforms were involved in cell growth, cell interactions, cell proliferation, cell migration and cell death and have been previously linked to cancers and genetic disorders. We chose ten predicted splice variants for RT-PCR validation, eight of which were successfully confirmed (MED24, MFI2, SRRT, CD44, CLK1 and HNRNPH1). These include three novel intron retentions in CD44, a gene in which isoform variations have been previously associated with the metastasis of several cancers.ConclusionOur findings reveal that various genes are differently spliced and/or expressed in association with the metastatic phenotype of tumor cells. Identification of metastasis-specific isoforms may contribute to the development of improved breast cancer stage identification and targeted therapies.

The balance of TCF7L2 variants with differential activities in Wnt-signaling is regulated by lithium in a GSK3β-independent manner

TCF7L2 transcription factor is a downstream effector of the canonical Wnt/β-catenin signaling, which controls cell fate and homeostasis. However, the complexity of TCF7L2 expression with numerous mRNA isoforms coding for proteins with distinct N- and C-termini allows variability in TCF7L2 functions and regulations. Here, we show that although TCF7L2 mRNA isoforms distinguish fetal, immortalized and adult differentiated endothelial cells (EC), they cannot explain the lack of significant β-catenin/TCF7 activities in ECs. Lithium, a Wnt-signaling activator, increases TCF7L2 mRNA levels and induces an RNA isoform switch favoring the expression of TCF7L2-short forms lacking the C-termini domains. Although the latter occurs in different cell types, its extent depends on the overall increase of TCF7L2 transcription, which correlates with cell responsiveness to Wnt/β-catenin signaling. While GSK3β down-regulation increases TCF7L2 expression, there is no concomitant change in TCF7L2 mRNA isoforms, which demonstrate the dual effects of lithium on TCF7L2 expression via a GSK3β-dependent up-regulation and a GSK3β-independent modulation of RNA splicing. TCF7L2E-long forms display a repressor activity on TCF7L2-promoter reporters and lithium induces a decrease of the endogenous TCF7L2 forms bound to native TCF7L2-promoter chromatin at two novel distal TCF7-binding sites. Altogether our data reveal a lithium-induced RNA switch favoring the expression of TCF7L2-short forms, which results in a transcriptional de-repression of lithium target genes negatively regulated by TCF7L2-long forms, like TCF7L2, and thus to an amplification of Wnt-signaling in responsive cells.

Peripheral purinergic receptor modulation influences the trigeminal ganglia nitroxidergic system in an experimental murine model of inflammatory orofacial pain

ATP plays an important role as an endogenous pain mediator generating and/or modulating pain signaling from the periphery to the central nervous system. The aim of this study was to analyze the role of peripheral purinergic receptors in modulation of the nitroxidergic system at a trigeminal ganglia level by monitoring changes in nitric oxide synthase isoforms. We also evaluated Fos-positive neurons in brainstem (spinal trigeminal nucleus) and pain-related behavior. We found that local administration of the P2 purinergic receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) decreased face-rubbing activity, nitric oxide synthase isoform expression in trigeminal ganglia, and Fos expression in spinal trigeminal nucleus after subcutaneous injection of formalin. These results suggest a role for peripheral P2 purinergic receptors in orofacial pain transmission through modulation of the nitroxidergic system. .

Skeletal muscle fibre diversity and the underlying mechanisms

The review first briefly summarizes how myosin isoforms have been identified as the major determinant of the functional variability among skeletal muscle fibres. The latter feature is a major characteristic of muscle fibres and a major basis of skeletal muscle heterogeneity and plasticity in vivo. Then, evidence is reported, which indicates that the properties of muscle fibres can vary with no change in the myosin isoform they express. Moreover, the physiological and pathological conditions (ageing, disuse, exercise training, muscular dystrophy) in which such myosin isoform independent change in functional properties occurs and the possible underlying mechanisms are considered. Finally, the known molecular bases of the functional differences among slow and fast isoforms are briefly dealt with.

The role of tropomyosin isoforms and phosphorylation in force generation in thin-filament reconstituted bovine cardiac muscle fibres

The thin filament extraction and reconstitution protocol was used to investigate the functional roles of tropomyosin (Tm) isoforms and phosphorylation in bovine myocardium. The thin filament was extracted by gelsolin, reconstituted with G-actin, and further reconstituted with cardiac troponin together with one of three Tm varieties: phosphorylated alphaTm (alphaTm.P), dephosphorylated alphaTm (alphaTm.deP), and dephosphorylated betaTm (betaTm.deP). The effects of Ca, phosphate, MgATP and MgADP concentrations were examined in the reconstituted fibres at pH 7.0 and 25 degrees C. Our data show that Ca(2+) sensitivity (pCa(50): half saturation point) was increased by 0.19 +/- 0.07 units when betaTm.deP was used instead of alphaTm.deP (P < 0.05), and by 0.27 +/- 0.06 units when phosphorylated alphaTm was used (P < 0.005). The cooperativity (Hill factor) decreased (but insignificantly) from 3.2 +/- 0.3 (5) to 2.8 +/- 0.2 (7) with phosphorylation. The cooperativity decreased significantly from 3.2 +/- 0.3 (5) to 2.1 +/- 0.2 (9) with isoform change from alphaTm.deP to betaTm.deP. There was no significant difference in isometric tension or stiffness between alphaTm.P, alphaTm.deP, and betaTm.deP muscle fibres at saturating [Ca(2+)] or after rigor induction. Based on the six-state cross-bridge model, sinusoidal analysis indicated that the equilibrium constants of elementary steps differed up to 1.7x between alphaTm.deP and betaTm.deP, and up to 2.0x between alphaTm.deP and alphaTm.P. The rate constants differed up to 1.5x between alphaTm.deP and betaTm.deP, and up to 2.4x between alphaTm.deP and alphaTm.P. We conclude that tension and stiffness per cross-bridge are not significantly different among the three muscle models.

Vascular endothelial growth factor-A isoform and (co)receptor expression are differentially regulated by 17β-oestradiol in the ovariectomised mouse uterus

The angiogenic effects of 17beta-oestradiol (E(2)) in the mouse endometrium are mediated by vascular endothelial growth factor-A (VEGFA). We analysed the temporal and spatial changes in VEGFA isoform and (co)receptor expression in ovariectomised mouse uteri following E(2) treatment. VEGFA isoform and receptor mRNA were quantified in whole uterine tissue collected 2, 6, 12 and 24 h after E(2) or vehicle treatment. Laser capture microdissection was used to investigate mRNA expression in epithelial, stromal and myometrial tissues separately. Endothelial cell proliferation, VEGFA and VEGF receptor-2 (VEGFR2) protein were visualised using immunohistochemistry. Endometrial endothelial cell proliferation was only observed 24 h after E(2) treatment. In whole uterine tissue, total Vegfa, Vegfa(164) and Vegfa(120) mRNA expression increased 2 h post E(2) treatment, and then decreased by 24 h. Vegfa(188) expression was lower in E(2)-treated animals at all time points relative to control animals. Vegfr2 and neuropilin-1 (Nrp1) mRNA expression did not change following E(2) treatment; Nrp2 expression decreased by 24 h. When uterine compartments were considered separately at 24 h post E(2) or vehicle, stromal Vegfa(120), Vegfa(188) and Vegfr2 mRNA expression and myometrial Vegfa(120) and Vegfa(188) mRNA expression were reduced in E(2)-treated mice relative to controls, whereas epithelial Vegfa(188) mRNA expression increased. The highest VEGFA immunoexpression was observed in luminal epithelium; expression increased at 24 h relative to other time points. No changes were noted in VEGFR2 immunoexpression among treatment groups. We have provided the first evidence that VEGFA isoform and receptor mRNA expression are differentially regulated by E(2) in different uterine cell compartments.

THYROID STATUS MODIFIES CARDIAC NITRIC OXIDE SYNTHASES DURING HEMORRHAGIC SHOCK: PP.22.400

The regulation of cardiac function is a complex process in which nitric oxide (NO) is a mediator and the thyroid status has a role. Objectives: To study the involvement of thyroid status and NO synthases (NOS) on hemodynamic changes induced by acute hemorragic shock. Animals euthyroid (E), hyperthyroid (H) and hypothyroid (h), are divided into two groups, control (C), and hemorrhaged rats (H) (withdrawal of 20 % of blood volume). Right atrium (A) and left ventricule (V) were removed at 120 min of hemorrhage. Histochemical NOS activity (NADPH-diaphorase technique), NOS, caveolin 1 and 3 (cav 1 and 3) protein levels (western blot) were evaluated. The results are expresed as X ± ES, n = 9/ group. Analysis of variance (ANOVA) followed by the ad hoc Bonferroni test was used for multiple comparisons. The 5% probability level was used as a criterion for biological significance. Results: Blood loss did not change neither eNOS and nNOS protein levels in A and V nor cav-3 protein levels in V. Conclusions: Acute blood loss increased NOS activity in A and V of euthyroid rats that correlated with changes in i-NOS protein levels. Hypovolemic state induced by acute hemorrhage induced changes in the cav-1 protein levels in A of hyperthyroid rats and in V of hyper and hypothyroid animals. The production of NO and its involvement in the cardiac adaptation to hemorrhage depend on the thyroid status. The coordinated expressional changes in NOS isoforms and their allosteric regulators, such as caveolin, may be viewed as a compensatory mechanism to maintain the production of bioactive NO.

Quantitative PCR analysis of laryngeal muscle fiber types

Voice and swallowing dysfunction as a result of recurrent laryngeal nerve paralysis can be improved with vocal fold injections or laryngeal framework surgery. However, denervation atrophy can cause late-term clinical failure. A major determinant of skeletal muscle physiology is myosin heavy chain (MyHC) expression, and previous protein analyses have shown changes in laryngeal muscle fiber MyHC isoform with denervation. RNA analyses in this setting have not been performed, and understanding RNA levels will allow interventions better designed to reverse processes such as denervation in the future. Total RNA was extracted from bilateral rat thyroarytenoid (TA), posterior cricoarytenoid (PCA), and cricothyroid (CT) muscles in rats. Primers were designed using published MyHC isoform sequences. SYBR Green real-time reverse transcription-polymerase chain reaction (SYBR-RT-PCR) was used for quantification. The electropherogram showed a clear separation of total RNA to 28S and 18S subunits. Melting curves illustrated single peaks for all type MyHC primers. All MyHC isoforms were identified in all muscles with various degrees of expression. Quantitative PCR is a sensitive method to detect MyHC isoforms in laryngeal muscle. Isoform expression using mRNA analysis was similar to previous analyses but showed some important differences. This technique can be used to quantitatively assess response to interventions targeted to maintain muscle bulk after denervation. Learning outcomes: (1) Readers will be able to describe the relationship between myosin heavy chain expression and muscle contractile properties. (2) Readers will be able to separate myosin heavy chain isoforms into slow and fast twitch phenotypes. (3) Readers will be able to describe differential muscle isoform expression between different laryngeal muscles. (4) Readers will be able to compare this study to other modalities of determining muscle fiber type.

AMPA receptor downregulation induced by ischaemia/reperfusion is attenuated by age and blocked by meloxicam

Stroke prevalence increases with age, while alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) and inflammation have been related to ischaemia-induced damage. This study shows how age and treatment with an anti-inflammatory agent (meloxicam) modify the levels of AMPAR subunits GluR1 and GluR2, as well as the mRNA levels of the GluR2-editing enzyme, ADAR2, in a global brain ischaemia/reperfusion (I/R) model. Two days after global ischaemia CA1, CA3, dentate gyrus and cerebral cortex were obtained from sham-operated and I/R-injured 3- and 18-month-old Sprague-Dawley rats. Real time polymerase chain reaction, Western blotting and immunohistochemical assays were performed. Meloxicam treatment was assayed on young animals. Data showed that age attenuates the downregulation induced by I/R in the AMPAR subunits GluR1 and GluR2 and modifies the GluR1/GluR2 mRNA level ratio in a structure-dependent way. The study of the ADAR2 mRNA levels showed more downregulation in older animals than young ones. Meloxicam treatment prevented the transcriptional arrest induced by I/R. Our data suggest that changes in the AMPAR isoforms could be associated with ageing in the different structures studied. Although GluR2 editing seems to be involved in age-dependent vulnerability to ischaemia supporting the 'GluR2 hypothesis', this alone does not explain the differential vulnerability in the different brain regions. Finally, inflammation could play a role in protection from I/R-induced injury.

1683 EFFECT OF DETRUSOR OVERACTIVITY INDUCED BY BLADDER OUTLET OBSTRUCTION ON INTERSTITIAL CELLS IN THE URINARY BLADDER OF RATS

You have accessJournal of UrologyUrodynamics/Incontinence/Female Urology: Non-Neurogenic Voiding Dysfunction II1 Apr 20101683 EFFECT OF DETRUSOR OVERACTIVITY INDUCED BY BLADDER OUTLET OBSTRUCTION ON INTERSTITIAL CELLS IN THE URINARY BLADDER OF RATS Byung Seok Oh, Kim Sun-Ouck, Jun Back Park, Chang Min Im, Kyung Jin Oh, Seung Il Jung, Taek Won Kang, Kwon Dongdeuk, Park Kwangsung, and Soo Bang Ryu Byung Seok OhByung Seok Oh More articles by this author , Kim Sun-OuckKim Sun-Ouck More articles by this author , Jun Back ParkJun Back Park More articles by this author , Chang Min ImChang Min Im More articles by this author , Kyung Jin OhKyung Jin Oh More articles by this author , Seung Il JungSeung Il Jung More articles by this author , Taek Won KangTaek Won Kang More articles by this author , Kwon DongdeukKwon Dongdeuk More articles by this author , Park KwangsungPark Kwangsung More articles by this author , and Soo Bang RyuSoo Bang Ryu More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.1508AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Recent studies have shown that interstitial cells (ICs) are widely distributed in the genitourinary tract, and their involvement in spontaneous electrical activity and muscle contraction has been suggested. However, nitric oxide (NO) is thought to play a role in bladder overactivity related to partial bladder outlet obstruction (BOO). The purpose of this study was to investigate the effect of BOO on ICs and nitric oxide synthase (NOS) isoforms in the urinary bladder of rats. METHODS Female Sprague-Dawley rats (230-240 g, n=60) were divided into 2 groups: a control group (group Con, n=30) and a group that underwent partial BOO (group BOO, n=30). After 4 weeks, a urodynamic study was conducted to measure the contraction interval and contraction pressure. The expression and cellular localization of ICs, endothelial NOS, and neuronal NOS in the urinary bladder of rats were determined by Western blot and immunohistochemical analysis, respectively. RESULTS Four weeks after surgery, voiding frequency increased and voiding volume decreased in group BOO. Filling cystometry showed a reduction in the interval between voiding contractions and an increase in voiding pressure in the bladders of group BOO. The estimated contraction interval (min, mean ?SE) was significantly lower in group BOO (2.9?.35) than in group Con (6.1?.05) (p<0.05). The thickness of and the population of kit-positive ICs were significantly greater in the suburothelial and detrusor smooth muscle layers of bladders in group BOO than in group Con. ICs had close contact with each other and with neighboring neuronal NOS–expressing nerves. CONCLUSIONS The results indicate that the population of ICs is greater in rats with than in those without partial BOO and suggest that functional changes in ICs and NOS isoforms may contribute to the pathophysiology of bladder overactivity induced by BOO. Gwangju, Republic of Korea© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e650 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Byung Seok Oh More articles by this author Kim Sun-Ouck More articles by this author Jun Back Park More articles by this author Chang Min Im More articles by this author Kyung Jin Oh More articles by this author Seung Il Jung More articles by this author Taek Won Kang More articles by this author Kwon Dongdeuk More articles by this author Park Kwangsung More articles by this author Soo Bang Ryu More articles by this author Expand All Advertisement Advertisement Loading ...

Metallothionein-1 Isoforms and Vimentin Are Direct PU.1 Downstream Target Genes in Leukemia Cells

PU.1 is a key transcription factor for hematopoiesis and plays important roles in various hematological malignancies. To clarify the molecular function of PU.1, we initially tried to identify bona fide target genes regulated by PU.1. Dual microarrays were employed for this study to compare PU.1-knockdown K562 cells (K562PU.1KD) stably expressing PU.1 short inhibitory RNAs versus control cells and PU.1-overexpressing K562 cells (K562PU.1OE) versus control cells. In these analyses, we found that several genes, including metallothionein (MT)-1 isoforms (MT-1G and MT-1A) and vimentin (VIM), were markedly induced while Jun dimerization protein (JDP) 2 was suppressed in K562PU.1KD cells. Furthermore, the mRNA expressions of the MT-1 and VIM genes were inversely correlated and the mRNA expression of JDP2 was positively correlated with PU.1 mRNA expression in 43 primary acute myeloid leukemia specimens (MT-1G: R = -0.50, p < 0.001; MT-1A: R = -0.58, p < 0.0005; VIM: R = -0.39, p < 0.01; and JDP2: R = 0.30, p < 0.05). Next, we analyzed the regulation of the MT-1 and VIM genes. We observed increased associations of acetylated histones H3 and H4 with the promoters of these genes in K562PU.1KD cells. Sequence analyses of the regions approximately 1 kb upstream from the transcription start sites of these genes revealed numerous CpG sites, which are potential targets for DNA methylation. Chromatin immunoprecipitation assays revealed that methyl CpG-binding protein 2 (MeCP2) and PU.1 bound to the CpG-rich regions in the MT-1 and VIM promoters. Bisulfite sequencing analyses of the PU.1-bound regions of these promoters revealed that the proportions of methylated CpG sites were tightly related to the PU.1 expression levels.

Protein kinase C mRNA and protein expressions in hypobaric hypoxia‐induced cardiac hypertrophy in rats

Protein kinase C (PKC), cloned as a serine/threonine kinase, plays key roles in diverse intracellular signalling processes and in cardiovascular remodelling during pressure overload or volume overload. We looked for correlations between changes in PKC isoforms (levels and/or subcellular distributions) and cardiac remodelling during experimental hypobaric hypoxic environment (HHE)-induced pulmonary hypertension. To study the PKC system in the heart during HHE, 148 male Wistar rats were housed for up to 21 days in a chamber at the equivalent of 5500 m altitude level (10% O(2)). At 14 or more days of exposure to HHE, pulmonary arterial pressure (PAP) was significantly increased. In the right ventricle (RV): (1) the expression of PKC-alpha protein in the cytosolic and membrane fractions was increased at 3-14 days and at 5-7 days of exposure respectively; (ii) the cytosolic expression of PKC-delta protein was increased at 1-5, 14 and 21 days of exposure; (3) the membrane expressions of the proteins were decreased at 14-21 (PKC-betaII), 14-21 (PKC-gamma), and 0.5-5 and 21 (PKC-epsilon) days of exposure; (4) the expression of the active form of PKC-alpha protein on the plasma membrane was increased at 3 days of exposure (based on semiquantitative analysis of the immunohistochemistry). In the left ventricle, the expressions of the PKC mRNAs, and of their cytosolic and membrane proteins, were almost unchanged. The above changes in PKC-alpha, which were strongly evident in the RV, occurred alongside the increase in PAP. PKC-alpha may help to modulate the right ventricular hypertrophy caused by pulmonary hypertension in HHE.

SplicerAV: a tool for mining microarray expression data for changes in RNA processing

BackgroundOver the past two decades more than fifty thousand unique clinical and biological samples have been assayed using the Affymetrix HG-U133 and HG-U95 GeneChip microarray platforms. This substantial repository has been used extensively to characterize changes in gene expression between biological samples, but has not been previously mined en masse for changes in mRNA processing. We explored the possibility of using HG-U133 microarray data to identify changes in alternative mRNA processing in several available archival datasets.ResultsData from these and other gene expression microarrays can now be mined for changes in transcript isoform abundance using a program described here, SplicerAV. Using in vivo and in vitro breast cancer microarray datasets, SplicerAV was able to perform both gene and isoform specific expression profiling within the same microarray dataset. Our reanalysis of Affymetrix U133 plus 2.0 data generated by in vitro over-expression of HRAS, E2F3, beta-catenin (CTNNB1), SRC, and MYC identified several hundred oncogene-induced mRNA isoform changes, one of which recognized a previously unknown mechanism of EGFR family activation. Using clinical data, SplicerAV predicted 241 isoform changes between low and high grade breast tumors; with changes enriched among genes coding for guanyl-nucleotide exchange factors, metalloprotease inhibitors, and mRNA processing factors. Isoform changes in 15 genes were associated with aggressive cancer across the three breast cancer datasets.ConclusionsUsing SplicerAV, we identified several hundred previously uncharacterized isoform changes induced by in vitro oncogene over-expression and revealed a previously unknown mechanism of EGFR activation in human mammary epithelial cells. We analyzed Affymetrix GeneChip data from over 400 human breast tumors in three independent studies, making this the largest clinical dataset analyzed for en masse changes in alternative mRNA processing. The capacity to detect RNA isoform changes in archival microarray data using SplicerAV allowed us to carry out the first analysis of isoform specific mRNA changes directly associated with cancer survival.

Age-related changes in hepatic expression and activity of cytochrome P450 in male rats

Age-related changes in hepatic expression and activity of cytochrome P450 (CYP) were investigated in male rats aged 3 (weanling), 12 (young), 26 (adult), and 104 (old) weeks. Levels of microsomal protein, total CYP, and cytochrome b(5) increased fully after puberty. CYP1A1 was detected only in 3-week-old rats, and CYP1A2, CYP2B1, and CYP2E1 were maximally expressed at 3 weeks but decreased at 12 and 26 weeks. CYP2C11 and CYP3A2 increased markedly after puberty and decreased with aging. Ethoxyresorufin-O-deethylase, methoxyresorufin-O-demethylase, pentoxyresorufin-O-depenthylase, and p-nitrophenol hydroxylase activities were at their highest in 3-week-old rats, and midazolam hydroxylase activity was at a maximum in 12-week-old rats but decreased with aging. The present results show that increasing age caused significant alterations in hepatic expression/activity of CYP isoforms in an isoform-specific manner. These results suggest that age-related changes in hepatic CYP isoforms may be an important factor for deciding the efficacy and safety of xenobiotics.