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At least 811 records · Page 45Linked to original sources
PubMed2004

Molecular staging by RT-pCR analysis for PSA and PSMA in peripheral blood and bone marrow samples is an independent predictor of time to biochemical failure following radical prostatectomy for clinically localized prostate cancer.

Radical prostatectomy should ideally be curative for all patients with clinically localized prostate cancer (PrCa), yet a sizeable proportion of them eventually relapse. We examined in this setting the feasibility of pre-operative risk stratification for early post-operative relapse using reverse transcriptase polymerase chain reaction (RT-PCR) for prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) transcripts in preoperative bone marrow (BM) biopsies and peripheral blood (PBL) samples. Nested RT-PCR for PSA and PSMA transcripts were performed in RNA from BM biopsies and PBL samples prospectively obtained from 111 men newly diagnosed, by trans-rectal ultrasound (TRUS)-guided biopsy, with clinically localized PrCa and scheduled to undergo radical prostatectomy, according to their respective doctors' recommendation. Molecular analysis for each sample (PBL or BM) was considered positive only if both PSA and PSMA transcripts were detectable. Serial serum PSA level measurements served for biochemical follow-up and detection of biochemical failure (PSA >0.2 ng/ml). PBL and BM RT-PCR molecular staging delineated three groups of patients (a) PBL-BM- (72 patients, 65%), (b) PBL+BM+ (29 patients, 26%), and (c) PBL+BM- (10 patients, 9%). These three groups corresponded to low, high, and intermediate risk for early post-prostatectomy recurrence (median time to biochemical failure of >38, 8, and >28 months, respectively). Multivariate analysis confirmed that molecular staging status was independent predictor of disease-free survival, after adjusting for PSA levels and Gleason score. In clinically localized PrCa, combined PSA/PSMA RT-PCR in PBL and BM is an independent predictor of time to biochemical failure following radical prostatectomy.

Antigens, Surface↗
PubMed2006

Microarray analysis of MRI-defined tissue samples in glioblastoma reveals differences in regional expression of therapeutic targets.

Microarray technologies have come into prominence for the assessment of molecular diagnostic profiles in cancer tissue biopsies. To better understand the effect of sampling bias, we paired image-guided stereotactic biopsy and microarray technology to study regional intratumoral differences in tumor periphery and core regions of untreated glioblastoma. RNA was extracted from serial frozen sections using an integral histopathologic scoring approach. Gene expression analysis was performed using high-density oligonucleotide microarrays (22,283 probe sets). A consensus list of 643 genes (784 probe sets) with greater than 2-fold difference between intratumoral periphery and core samples was obtained using Microarray Suite 5.0, model-based expression indexes, and robust multiarray analysis algorithms. Results were validated using quantitative polymerase chain reaction and Western blotting analyses. Reproducible profiles emerged, in which multiple therapeutic targets significant to glioblastoma [matrix metalloproteinases, AKT1 (v-akt murine thymoma viral oncogene homolog 1), epidermal growth factor receptor, vascular endothelial growth factor] showed significant differences in regional expression that may affect treatment response. This study suggests important intratumoral regional differences in the molecular phenotype of glioblastoma.

Algorithms↗
PubMed1993

Rapid detection of cytomegalovirus structures in cerebrospinal fluid and brain biopsies by polymerase chain reaction.

The joint application of stereotactic brain biopsy and DNA amplification methods, like the polymerase chain reaction (PCR) are diagnostic purposes that allows in approximately 24 h to clarify the viral etiology of central nervous system infections. We demonstrate PCR results from the brain biopsy of an HIV-1 patient with two cerebral lesions. Beside the neuropathological diagnosis of a B-cell non-Hodgkin lymphoma the experimental results are indicative of active transcription of cytomegalovirus (CMV) in cerebral tissue. The markedly higher amount of CMV-specific RNA found in the biopsy specimens and CSF suggest the presence of an active infection with viral replication. CT-guided stereotactic brain biopsies followed by PCR fulfill the demands of a clinician, namely to get the results as fast as possible to initiate adequate treatment.

Adult↗
PubMed2005

Noncoding RNA of U87 host gene is associated with ribosomes and is relatively resistant to nonsense-mediated decay.

Non-coding RNAs are involved in many cellular processes. In particular, most of C/D box small nucleolar RNAs (snoRNAs) function as guide RNAs in site-specific 2'-O-methylation of rRNAs. While most snoRNA genes reside in introns of protein-coding genes, here we demonstrated an unusual snoRNA gene occupying an intron of a previously unknown non-protein-coding gene U87HG. We characterized this host gene in human, mouse, rat, and dog. It is a member of 5'TOP gene family, which includes many translation apparatus genes. U87HG RNA carried multiple stop-codons and was associated with ribosomes, suggesting that it may be a target for nonsense-mediated mRNA decay (NMD), a process that eliminates transcripts bearing nonsense mutations. Surprisingly, we found that U87HG RNA was hardly susceptible to NMD. Possible mechanisms (translation reinitiation, ribosomal leaky scanning, and low efficiency of translation) of this phenomenon are discussed. Unlike transcripts of four other known non-protein-coding host genes, U87HG RNA shows a relatively high degree of conservation suggesting a selective pressure and a possible functional activity of U87HG apart from producing U87 snoRNA.

Animals↗
PubMed2005

MicroRNA-dependent trans-acting siRNA production.

Less than a year has elapsed between the discovery of trans-acting small interfering RNAs (tasiRNAs) in plants and the elucidation of the major steps of the corresponding pathway. During tasiRNA biogenesis, polyadenylated RNAs transcribed from non-protein-coding TAS genes are cleaved by a microRNA (miRNA)-programmed RNA-induced silencing complex. In contrast to classical miRNA targets, RDR6 and SGS3 convert one of the TAS RNA cleavage products into double-stranded RNA, which is subsequently processed, in a phase determined by the initial miRNA cleavage site, by DICER-LIKE 4 to generate a 21-nucleotide tasiRNA population. tasiRNAs guide endogenous mRNA cleavage through the action of AGO1 or, perhaps in some cases, AGO7. Some of the tasiRNA targets probably regulate the juvenile-to-adult phase transition, but the roles of other tasiRNA targets remain to be determined.

Arabidopsis↗
PubMed2006

Molecular dissection of arginyltransferases guided by similarity to bacterial peptidoglycan synthases.

Post-translational protein arginylation is essential for cardiovascular development and angiogenesis in mice and is mediated by arginyl-transfer RNA-protein transferases Ate1-a functionally conserved but poorly understood class of enzymes. Here, we used sequence analysis to detect the evolutionary relationship between the Ate1 family and bacterial FemABX family of aminoacyl-tRNA-peptide transferases, and to predict the functionally important residues in arginyltransferases, which were then used to construct a panel of mutants for further molecular dissection of mouse Ate1. Point mutations of the residues in the predicted regions of functional importance resulted in changes in enzymatic activity, including complete inactivation of mouse Ate1; other mutations altered its substrate specificity. Our results provide the first insights into the mechanisms of Ate1-mediated arginyl transfer reaction and substrate recognition, and define a new protein superfamily called Dupli-GNAT to reflect its origin by the duplication of the GNAT acetyltransferase domain.

Amino Acid Sequence↗
PubMed1982

Molecular mechanism of promoter selection in gene transcription. II. Kinetic evidence for promoter search by a one-dimensional diffusion of RNA polymerase molecule along the DNA template.

The rapid mixing-photocrosslinking technique, in conjunction with an immunoprecipitation assay developed to measure the change in the distribution of Escherichia coli RNA polymerase molecules bound to T7 DNA, has been applied to investigate the molecular mechanism of promoter search by RNA polymerase. The binding of RNA polymerase to the DNA template can be divided into at least two steps. The initial binding is rapid and occurs at nonspecific sites randomly distributed throughout the DNA molecule. This is followed by a relatively slow promoter search in which RNA polymerase is transferred from nonspecific sites to promoter sites through a series of intramolecular processes. The rate of polymerase loss from a segment of DNA which does not contain promoter sites is a function of the distance from this segment to both the promoter sites and the ends of the DNA molecule. The kinetic data are consistent with a molecular mechanism in which RNA polymerase undergoes a bidirectional linear diffusion along the DNA template to search for the promoter site. This interpretation is supported by the computer simulation which correctly predicts the relative rates of polymerase loss from various DNA segments. The mechanism derived from these studies is in accordance with the notion that the whole DNA molecule serves as an effective sink for trapping and guiding polymerase molecules during promoter search.

DNA, Viral↗
PubMed2004

Rapid and sensitive p53 alteration analysis in biopsies from lung cancer patients using a functional assay and a universal oligonucleotide array: a prospective study.

PURPOSE: Molecular profiling of alterations associated with lung cancer holds the promise to define clinical parameters such as response to treatment or survival. Because <5% of small cell lung cancers and <30% of non-small cell lung cancers are surgically resectable, molecular analysis will perforce rely on routinely available clinical samples such as biopsies. Identifying tumor mutations in such samples will require a sensitive and robust technology to overcome signal from excess amounts of normal DNA. EXPERIMENTAL DESIGN: p53 mutation status was assessed from the DNA and RNA of biopsies collected prospectively from 83 patients with lung cancer. Biopsies were obtained either by conventional bronchoscopy or computed tomography-guided percutaneous biopsy. Matched surgical specimens were available for 22 patients. Three assays were used: direct sequencing; a functional assay in yeast; and a newly developed PCR/ligase detection reaction/Universal DNA array assay. RESULTS: Using the functional assay, p53 mutation was found in 62% of biopsies and 64% of surgical specimens with a concordance of 80%. The sensitivity of the functional assay was determined to be 5%. Direct sequencing confirmed mutations in 92% of surgical specimens but in only 78% of biopsies. The DNA array confirmed 100% of mutations in both biopsies and surgical specimens. Using this newly developed DNA array, we demonstrate the feasibility of directly identifying p53 mutations in clinical samples containing <5% of tumor cells. CONCLUSIONS: The versatility and sensitivity of this new array assay should allow additional development of mutation profiling arrays that could be applied to biological samples with a low tumor cell content such as bronchial aspirates, bronchoalveolar lavage fluid, or serum.

Aged↗
PubMed2004

Targeting Alzheimer's disease genes with RNA interference: an efficient strategy for silencing mutant alleles.

Tau and amyloid precursor protein (APP) are key proteins in the pathogenesis of sporadic and inherited Alzheimer's disease. Thus, developing ways to inhibit production of these proteins is of great research and therapeutic interest. The selective silencing of mutant alleles, moreover, represents an attractive strategy for treating inherited dementias and other dominantly inherited disorders. Here, using tau and APP as model targets, we describe an efficient method for producing small interfering RNA (siRNA) against essentially any targeted region of a gene. We then use this approach to develop siRNAs that display optimal allele-specific silencing against a well-characterized tau mutation (V337M) and the most widely studied APP mutation (APPsw). The allele-specific RNA duplexes identified by this method then served as templates for constructing short hairpin RNA (shRNA) plasmids that successfully silenced mutant tau or APP alleles. These plasmids should prove useful in experimental and therapeutic studies of Alzheimer's disease. Our results suggest guiding principles for the production of allele-specific siRNA, and the general method described here should facilitate the production of gene-specific siRNAs.

Alleles↗
PubMed1996

Regulation of corticotropin-releasing factor type 1 (CRF1) receptor messenger ribonucleic acid in the paraventricular nucleus of rat hypothalamus by exogenous CRF.

The present study sought to examine the effects of intracerebroventricular (icv) administration of corticotropin-releasing factor (CRF) on the expression of CRF1 receptor messenger RNA (mRNA) within the hypothalamus as determined by quantitative in situ hybridization histochemistry. Adult male Sprague-Dawley rats were stereotaxically implanted with guide cannulae directed towards the right lateral ventricle. After 8-10 days of recovery, either 10 microliters CRF (5 micrograms) or vehicle solution was injected into the lateral ventricle over a 2-min period. The rats were then deeply anesthetized at 15, 60, and 180 min after icv injection, transcardially perfused, and their brains cut into 30-micron coronal sections. Brain sections were then processed using standard radioactive in situ hybridization histochemistry revealing the expression of the CRF1 receptor mRNA. Low to moderate basal levels of CRF1 receptor transcript were observed in several regions of the forebrain. However, the hybridization signal for the mRNA encoding the CRF1 receptor was barely detectable in the paraventricular nucleus of the hypothalamus (PVN) of vehicle-injected rats. In contrast, 180 min after icv administration of CRF, a significant increase in CRF1 receptor transcript was measured specifically in the PVN, despite having virtually any hybridization signal before 180 min. This increase in the level of receptor transcription by CRF was restricted to the type 1 receptor subtype because the hybridization signal for the CRF2 alpha receptor mRNA was unaffected in the brain regions in which it was located. Moreover, we confirmed previous findings of a CRF-induced neuronal activation of parvocellular neurosecretory cells of the PVN, as assessed by c-fos mRNA expression. This neuronal activation induced by exogenous CRF was also associated with a rapid and strong induction of CRF heteronuclear RNA selectively in the rat PVN, a phenomenon abolished by a pretreatment with a CRF receptor antagonist. These results provide evidence that elevated levels of central CRF may trigger CRF1 receptor transcription selectively in the PVN. This positive feedback of CRF on its own receptor may represent a functional adaptation of the hypothalamic-pituitary-adrenal axis in response to stress.

Analysis of Variance↗
PubMed2006

On the art of identifying effective and specific siRNAs.

Small interfering RNAs (siRNAs) have been widely exploited for sequence-specific gene knockdown, predominantly to investigate gene function in cultured vertebrate cells, and also hold promise as therapeutic agents. Because not all siRNAs that are cognate to a given target mRNA are equally effective, computational tools have been developed based on experimental data to increase the likelihood of selecting effective siRNAs. Furthermore, because target-complementary siRNAs can also target other mRNAs containing sequence segments that are partially complementary to the siRNA, most computational tools include ways to reduce potential off-target effects in the siRNA selection process. Though these methods facilitate selection of functional siRNAs, they do not yet alleviate the need for experimental validation. This perspective provides a practical guide based on current wisdom for selecting siRNAs.

Animals↗
PubMed2026

Multi-sampling allows intra-tumoral heterogeneity querying and vulnerability profiling in glioblastoma.

BACKGROUND: Glioblastoma (GBM) remains a devastating cancer with limited treatment options, largely due to its heterogeneity. While supramaximal resection has recently provided survival benefits, therapeutic profiling of different tumor compartments, particularly its infiltrative edge remains largely unexplored. METHODS: Here, we leveraged magnetic resonance imaging (MRI)-guided multi-sampling, collecting 2 cores and 2 margins per case, to query GBM heterogeneity. Whole-exome and RNA-seq with drug testing in two patient-derived 3D models were used to reveal similarities and differences in genomic and transcriptomic makeups, cellular compositions, and drug responses across cores and margins. Bioinformatics interrogations further identified response biomarkers. RESULTS: Mutation analysis showed that oncogenes exhibited a higher degree of spatial heterogeneity than tumor suppressor genes, regardless of MRI status. While the mesenchymal transcriptional subtype with extracellular matrix remodeling, stress response, and immune programs were preferentially enriched in enhancing cores, proneural tumors with neurological processes favored non-enhancing margins. Using a 15-drug GBM-targeted panel, ERK (ulixertinib) and PI3K pathway (paxalisib, CC-115) inhibitors showed preferential efficacy in enhancing cores and non-enhancing margins, respectively. The anti-apoptosis, pan-Bcl2 agent navitoclax and the epigenetic drug trotabresib represented the most effective, tumor-wide monotherapies. Importantly, drug combinations generally outperformed single agents across all regions. CONCLUSIONS: This work demonstrates the regional heterogeneity of therapeutic vulnerabilities in GBM ex vivo, showing various drugs with tumor-wide or MRI-enhancement informed activity. These findings offer preclinical bases of numerous monotherapies and drug combinations for future clinical trial design.

Humans↗
PubMed2006

Specific association of Piwi with rasiRNAs derived from retrotransposon and heterochromatic regions in the Drosophila genome.

In Drosophila, Piwi (P-element-induced wimpy testis), which encodes a protein of the Argonaute family, is essential for germ stem cell self-renewal. Piwi has recently been shown to be a nuclear protein involved in gene silencing of retrotransposons and controlling their mobilization in the male germline. However, little is known about the molecular mechanisms of Piwi-dependent gene silencing. Here we show that endogenous Piwi immunopurified from ovary specifically associates with small RNAs of 25-29 nucleotides in length. Piwi-associated small RNAs were identified by cloning and sequencing as repeat-associated small interfering RNAs (rasiRNAs) derived from repetitive regions, such as retrotransposon and heterochromatic regions, in the Drosophila genome. Northern blot analyses revealed that in vivo Piwi does not associate with microRNAs (miRNAs) and that guide siRNA was not loaded onto Piwi when siRNA duplex was added to ovary lysate. In vitro, recombinant Piwi exhibits target RNA cleavage activity. These data together imply that Piwi functions in nuclear RNA silencing as Slicer by associating specifically with rasiRNAs originating from repetitive targets.

Animals↗
PubMed1980

An examination of the relationship between experimentally altered rates of epidermal proliferations and rates of epidermal metabolism assayed in vitro.

Continuously regenerating stratified squamous epithelia form an interesting model for examining mechanisms controlling the balance between rates of cell formation and cell maturation and death. Previous investigations of epidermal metabolism have been mainly based on single enzyme assays which may not form a reliable guide to changing rates of flux through metabolic pathways. Methods for in vitro assays of rates of glycolysis, protein synthesis and RNA synthesis of epidermal sheets free from dermal contamination were developed and used to examine rates of epidermal metabolism after experimental alteration of rates of epidermal proliferation. Starvation resulted in a 45-53% reduction in the in vivo epidermal labeling index and a 49-56% reduction in glycolysis and incorporation of amino acids assayed in vitro. Induction of epidermal hyperplasia with hexadecane resulted in a 4-fold increase in labeling index, a 6-fold increase in vitro glycolysis and a 3 to 4-fold increase in in vitro assays of incorporation of amino acids and uridine. Hyperplastic epidermis also showed an increased rate of incorporation of histidine (a marker for keratokyalin synthesis) relative to leucine (a marker for basal cell protein synthesis) indicating a change in maturation. The results suggest mechanisms linking rates of cell proliferation and death and indicate the possible value of such assays investigating these mechanisms.

Animals↗
PubMed2003

A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development.

Plant microRNAs (miRNAs) show a high degree of sequence complementarity to, and are believed to guide the cleavage of, their target messenger RNAs. Here, I show that miRNA172, which can base-pair with the messenger RNA of a floral homeotic gene, APETALA2, regulates APETALA2 expression primarily through translational inhibition. Elevated miRNA172 accumulation results in floral organ identity defects similar to those in loss-of-function apetala2 mutants. Elevated levels of mutant APETALA2 RNA with disrupted miRNA172 base pairing, but not wild-type APETALA2 RNA, result in elevated levels of APETALA2 protein and severe floral patterning defects. Therefore, miRNA172 likely acts in cell-fate specification as a translational repressor of APETALA2 in Arabidopsis flower development.

Antisense Elements (Genetics)↗
PubMed1990

Direct measurement of oligonucleotide substrate binding to wild-type and mutant ribozymes from Tetrahymena.

Like protein enzymes, RNA enzymes (ribozymes) provide specific binding sites for their substrates. We now show that equilibrium dissociation constants for complexes between the Tetrahymena ribozyme and its RNA substrates and products can be directly measured by electrophoresis in polyacrylamide gels containing divalent cations. Binding is 10(3)- to 10(4)-fold tighter (4-5 kcal/mol at 42 degrees C) than expected from base-pairing interactions alone, implying that tertiary interactions also contribute to energetic stabilization. Binding decreases with single base changes in the substrate, substitution of deoxyribose sugars, and lower Mg2+ concentration. Ca2+, which enables the ribozyme to fold but is unable to mediate efficient RNA cleavage, promotes weaker substrate binding than Mg2+. This indicates that Mg2+ has special roles in both substrate binding and catalysis. Mutagenesis of a region near the internal guide sequence disrupts substrate binding, whereas binding is not significantly affected by a mutation of the guanosine-binding site. This approach should be generally useful for analysis of ribozyme variants independent of their catalytic activities.

Animals↗