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PubMed2005

Allosteric aptamers: targeted reversibly attenuated probes.

Aptamers are unique nucleic acids with regulatory potentials that differ markedly from those of proteins. A significant feature of aptamers not possessed by proteins is their ability to participate in at least two different types of three-dimensional structure: a single-stranded folded structure that makes multiple contacts with the aptamer target and a double-helical structure with a complementary nucleic acid sequence. We have made use of this structural flexibility to develop an aptamer-based biosensor (a targeted reversibly attenuated probe, TRAP) in which hybridization of a cis-complementary regulatory nucleic acid (attenuator) controls the ability of the aptamer to bind to its target molecule. The central portion of the TRAP, between the aptamer and the attenuator, is complementary to a target nucleic acid, such as an mRNA, which is referred to as a regulatory nucleic acid (regNA) because it regulates the activity of the aptamer in the TRAP by hybridization with the central (intervening) sequence. The studies reported here of the ATP-DNA TRAP suggest that, as well as inhibiting the aptamer, the attenuator also acts as a structural guide, much like a chaperone, to promote proper folding of the TRAP such that it can be fully activated by the regDNA. We also show that activation of the aptamer in the TRAP by the complementary nucleic acid at physiological temperatures is sensitive to single-base mismatches. Aptamers that can be regulated by a specific nucleic sequence such as in an mRNA have potential for many in vivo applications including regulating a particular enzyme or signal transduction pathway or imaging gene expression in vivo.

Adenosine Triphosphate↗

PubMed2004

The initiation of Hox gene expression in Xenopus laevis is controlled by Brachyury and BMP-4.

Hox genes encode a family of transcription factors that specify positional identities along the anterior-posterior (AP) axis during the development of vertebrate embryos. The earliest Hox expression in vertebrates is during gastrulation, at a position distant from the organiser or its equivalent. However, the mechanism that initiates this early expression is still not clear. Guided by the expression pattern, we identified upstream regulators in Xenopus laevis. The mesodermal transcription factor brachyury (Xbra) controls the early Hox expression domain in the animal-vegetal direction and the secreted growth factor BMP-4 limits it in the organiser/non-organiser direction. The overlap of these two signals, indicated by a Cartesian coordinate system, defines the initial Hox expression domain. We postulate that this system is a general mechanism for the activation of all Hox genes expressed during gastrulation.

Animals↗

PubMed2004

A field guide to the Mps1 family of protein kinases.

Cell cycle events must be faithfully executed and properly integrated to ensure genetic stability. The Mps1 family of protein kinases has recently emerged as a critical regulator of genetic stability, because they regulate several processes central to mitotic fidelity. The spindle checkpoint monitors alignment of mitotic chromosomes, and centrosomes control cell cycle entry, mitotic spindle assembly, and cytokinesis. Several studies have shown that vertebrate orthologues of budding yeast Mps1p regulate the spindle checkpoint. More recently it has been demonstrated that human Mps1 is also required for centrosome duplication, normal mitotic progression, and cytokinesis.

Animals↗

PubMed2002

Intrinsic differences between the superficial and deep layers of the Xenopus ectoderm control primary neuronal differentiation.

In Xenopus, primary neurons differentiate early, in the deep layer of the neuroectoderm. In contrast, the neural precursors of the superficial layer continue to proliferate. We report that superficial layer precursors differ from deep layer precursors in that they are refractory to the neuronal-promoting activity of bHLH genes, dominant-negative X-Delta-1, FGF-8, or signals from the organizer. In this system, neuronal differentiation is guided by an early established, intrinsic, cell-autonomous difference in the competence of the precursor cells to differentiate. This difference may be controlled in part by ESR6e, a bHLH gene of the Enhancer-of-split family, which is expressed in the superficial layer of the late blastula and when expressed ectopically suppresses primary neurogenesis in the deep layer.

Animals↗

PubMed2006

mRNA expression analysis of human kallikrein 11 (KLK11) may be useful in the discrimination of benign prostatic hyperplasia from prostate cancer after needle prostate biopsy.

Kallikrein 11 (KLK11, TLSP, hippostatin) is a member of the human kallikrein gene family, which includes PSA, KLK2 and 12 other members, all localized on chromosome 19q13.4. The aim of this study was to investigate whether KLK11 expression could be used to discriminate prostate cancer (CaP) from benign prostatic hyperplasia (BPH) in needle prostate biopsies. We analyzed the expression of the prostate-type variant of the KLK11 gene in 64 CaP and BPH tissues obtained by transrectal ultrasound-guided needle biopsy. Reverse transcription (RT), PCR and image analysis methodologies were developed and used. Of the 42 BPH tissues examined, only 10 (23.8%) were positive for prostate-type KLK11 expression, while of the 22 CaP patients, 12 (54.5%) were KLK11-positive (p=0.025). Logistic regression and receiver operating characteristic curve analyses demonstrated that KLK11 expression has a significant discriminatory value (crude odds ratio=3.84, p=0.016; area under the curve, 0.65, 95% CI 0.51-0.80) between CaP and BPH in needle prostate biopsies.

Age Distribution↗

PubMed2005

Molecular prognostic factors in diffuse large B-cell lymphoma.

The treatment of patients with diffuse large B-cell lymphoma (DLBCL) has been guided traditionally by clinical parameters such as the Ann Arbor Staging Classification for Hodgkin's disease. Although the International Prognostic Index (IPI) represents the most widely accepted prognostic model, there is still a marked variability in outcome within identical IPI subgroups, reflecting the heterogeneity of this malignancy. Use of DNA microarray, real-time reverse transcription polymerase chain reaction, and tissue array immunohistochemistry methodologies makes the development of new classifications possible based on molecular profiling. The molecular classification of DLBCL may lead to the grouping of specific disease entities sharing similar biologic features, clinical behavior, and outcome. Once tested and validated, this new generation of prognostic models should become an integral part of the daily practice, providing valuable additional information to the currently existing clinically based predictive models. To accomplish these goals and to be in a position in which existing or new prognostic models can be easily tested and validated, there is a strong need to collect frozen and paraffin-embedded material that can be used for RNA extraction and construction of tissue arrays, respectively. Such materials should be gathered as an integral part of any planned study.

Biomarkers, Tumor↗

PubMed2007

Characterization and functional analysis of a novel double-guide C/D box snoRNA in the fission yeast.

Ribose methylation of eukaryotic rRNA is directed by box C/D small nucleolar RNAs (snoRNAs), which pinpoint the nucleotide to be methylated in specific position within the rRNA sequence. Here, we report the identification of a novel double-guide C/D box snoRNA termed snR88 that directs methylation of two previously undetermined sites in 25S rRNA from the fission yeast. Knockout of the predicted TATA box of the snR88 gene resulted in the complete blocking of its expression, showing that snR88 is an independently transcribed gene and dispensable for yeast viability. The depletion of snR88 abolished 25S rRNA methylation at U2304 and U2497 simultaneously. Interestingly, an unusual pause of reverse transcription at U2495 was observed, which implies an unknown structure of 25S rRNA related to ribose methylation at U2497 in the fission yeast.

Base Sequence↗

PubMed1999

Nucleic acid based techniques for the detection of rare cancer cells in clinical samples.

Solid tumors evolve from cells which have lost control functions safeguarding their genomic integrity by mutation within specific genes. Consequently, proliferating cells within a tumor differ slightly from generation to generation. This permits the continuous production of new and subsequent selection of the best adapted cells, which retain this capacity of self-evolution. The threat of neoplastic diseases is due to the clinical experience, that surgical resection of all cells with this potential for autonomous evolution is often not achievable, since they were spread to distant non-resectable sites in the organism before diagnosis and surgical removal of the primary tumor and might later grow out as metastatic lesion. Lack of diagnostic techniques to detect small preneoplastic lesions as well as single spread cancer cells often causes delayed diagnosis when curative therapy is no longer achievable. Recent advances in characterizing the molecular basis of genomic instability, identifying specific gatekeeper mutations and their functional consequences in neoplastic cells now permit the development of new highly sensitive tests to identify preneoplastic lesions as well as spread single cancer cells. These techniques carry a tremendous potential for simple cost effective cancer early detection and screening assays as well as for diagnostic applications to identify spread cancer cells. Thus, they most likely will soon guide indication for surgical and adjuvant therapy protocols for patients with preneoplastic or neoplastic lesions. However, due to the complexity of the assays and the great variety of technical aspects involved almost all diagnostic applications are not yet standardized. This poses significant problems for quality control as well as inter-laboratory comparability of the results and underlines the urgent demand for well controlled collaborative efforts to evaluate indications and diagnostic standards for these assays. Here, the theoretical background, basic principles and some diagnostic applications of these new tests are reviewed.

Animals↗

PubMed1992

Splice site selection by intron aI3 of the COX1 gene from Saccharomyces cerevisiae.

Interactions of the 5' and 3' splice sites with intron internal sequences of the yeast mitochondrial group I intron aI3 were studied using mutation analysis. The results can be fully explained by the splice guide model in which the splice sites are defined by the Internal Guide Sequence. No evidence was found for an alternative interaction between intron nucleotides preceding the 3' splice site and nucleotides in the vicinity of the core region as was found for the Tetrahymena intron. Our results also suggest that binding of the 5' and 3' splice site nucleotides to the IGS can not take place simultaneously. The intron must therefore undergo conformational changes as the reaction proceeds from the first step of self splicing, GTP attack at the 5' splice site, to exon ligation, the second step.

Base Sequence↗

PubMed1998

A case of well-differentiated minute hepatocellular carcinoma with extrahepatic metastasis.

A rare case of well-differentiated minute hepatocellular carcinoma (HCC) metastasizing to distant sites in a 77-year-old man with hepatitis C virus (HCV)-related cirrhosis is presented. Ultrasonography (US) disclosed a 9 mm hypoechoic lesion in segment seven (S7) of the liver, although computed tomography (CT), magnetic resonance imaging (MRI) and angiography did not reveal any space-occupying lesion. Ultrasound-guided biopsy showed the histological features of well-differentiated HCC. A plain film of the abdomen and CT revealed osteolytic changes in the sacrum and the lumbar vertebra. Ultrasound-guided biopsy of the sacrum revealed well-to-moderately differentiated HCC metastasizing from the liver. Percutaneous ethanol injection therapy (PEIT) effected complete response and completely eliminated the abnormal findings on US. Three months after PEIT, metastasis to the thoracic vertebra was revealed by CT, despite negative alpha-fetoprotein-mRNA in serum. This is the first report describing a well-differentiated HCC with metastatic potential. Further studies may provide insights into metastasis of well-differentiated HCC.

Aged↗

PubMed2003

Nucleoside and nucleotide analogue reverse transcriptase inhibitors: a clinical review of antiretroviral resistance.

Although advances in highly active antiretroviral therapy (HAART) have made long-term suppression of HIV an achievable goal of therapy, a substantial proportion of first-line regimens will eventually fail. Successful longterm treatment requires consideration of downstream treatment options at the time of initiating or changing regimens. An understanding of the patterns and interactions of resistance mutations, and the appropriate use of genotypic and phenotypic testing is an important component of successful drug sequencing. Resistance to multiple nucleoside reverse transcriptase inhibitors (NRTIs) may result from several genotypically distinct pathways, including the Q151M (151 complex), the 69 insertion complex, two distinct thymidine analogue mutational pathways and the K65R mutation. Knowledge of the clinical implications of these and other resistance pathways, as well as the antagonism or synergy between mutations, helps guide individualized treatment choices from initial therapy in the treatment-naive patient to salvage therapy in the highly treatment-experienced individual. The development of effective sequencing strategies will depend upon the continued understanding of drug resistance mutation patterns and their associations with specific HAART combinations. This review summarizes research advances that further the understanding of nucleoside and nucleotide analogue resistance mutations, and their interplay.

Antiretroviral Therapy, Highly Active↗

PubMed2005

Nutrient-gene interaction: metabolic genotype-phenotype relationship.

The U.S. Department of Health and Human Services (DHHS)/USDA Dietary Guidelines for Americans is a science and population evidence-based guide on diet and physical activity, providing advice and recommendations to promote a healthier lifestyle and reduce the risk of chronic diseases, including cancer. These recommendations are supported by the comprehensive evidence-based review on diet and cancer prevention conducted by the American Institute for Cancer Research, National Cancer Institute, World Health Organization/International Agency for Research on Cancer, and others. However, influencing dietary effects are the individual genetic predispositions that are the basis for considerable interindividual variations in cancer risk within the population and in nutrient homeostasis, which is maintained by genomic-nutrient and metabolic-phenotype interactions. Although genetics is an important component, it accounts for only a portion of this variation. An individual's overall phenotype, including health status, is achieved and maintained by the sum of metabolic activities functioning under differing circumstances within the life cycle and the complex interactions among genotype, metabolic phenotype, and the environment. In this postgenomic era, high-throughput groups of technologies in genomics, proteomics, and metabolomics measure and analyze DNA sequences, RNA transcripts, proteins, and nutrient-metabolic fluxes in a single experiment. These advances have transformed biomarker studies on nutrient-gene interactions from a reductionist concept into a holistic practice in which many regulated genes involved in metabolism, along with its metabolic phenotypes, can be measured through functional genomics and metabolic profiling. The overall integration of data and information from the building blocks of metabolism-based nutrient-gene interaction can lead to future individualized dietary recommendations to diminish cancer risk.

Diet↗

PubMed2003

Germ cell transplantation in an azoospermic Klinefelter bull.

Germ cell transplantation is a technique that transfers donor testicular cells into recipient testes. A population of germ cells can colonize the recipient testis, initiate spermatogenesis, and produce sperm capable of fertilization. In the present study, a nonmosaic Klinefelter bull was used as a germ cell recipient. The donor cell suspension was introduced into the rete testis using ultrasound-guided puncture. A pulsatile administration of GnRH was performed to stimulate spermatogenesis. The molecular approach to detect donor cells was done by a quantitative polymerase chain reaction with allele discrimination based on a genetic mutation between donor and recipient. Therefore, a known genetic mutation, associated with coat-color phenotype, was used to calculate the ratio of donor to recipient cells in the biopsy specimens and ejaculates for 10 mo. After slaughtering, meiotic preparations were performed. The injected germ cells did not undergo spermatogenesis. Six months after germ cell transplantation, the donor cells were rejected, which indicates that the donor cells could not incorporate in the testis. The hormone stimulation showed that the testosterone-producing Leydig cells were functionally intact. Despite subfertility therapy, neither the recipient nor the donor cells underwent spermatogenesis. Therefore, nonmosaic Klinefelter bulls are not suitable as germ cell recipients. Future germ cell recipients in cattle could be mosaic Klinefelters, interspecies hybrids, bulls with Sertoli cell-only syndrome, or bulls with disrupted germ cell migration caused by RNA interference.

Animals↗

PubMed2005

Design of a genome-wide siRNA library using an artificial neural network.

The largest gene knock-down experiments performed to date have used multiple short interfering/short hairpin (si/sh)RNAs per gene. To overcome this burden for design of a genome-wide siRNA library, we used the Stuttgart Neural Net Simulator to train algorithms on a data set of 2,182 randomly selected siRNAs targeted to 34 mRNA species, assayed through a high-throughput fluorescent reporter gene system. The algorithm, (BIOPREDsi), reliably predicted activity of 249 siRNAs of an independent test set (Pearson coefficient r = 0.66) and siRNAs targeting endogenous genes at mRNA and protein levels. Neural networks trained on a complementary 21-nucleotide (nt) guide sequence were superior to those trained on a 19-nt sequence. BIOPREDsi was used in the design of a genome-wide siRNA collection with two potent siRNAs per gene. When this collection of 50,000 siRNAs was used to identify genes involved in the cellular response to hypoxia, two of the most potent hits were the key hypoxia transcription factors HIF1A and ARNT.

Algorithms↗

PubMed2002

Role of Epstein-Barr virus DNA load monitoring in prevention and early detection of post-transplant lymphoproliferative disease.

Posttransplant lymphoproliferative disease (PTLD) is a severe and life-threatening complication after stem cell or solid-organ transplantation, virtually always associated with presence of Epstein-Barr virus (EBV) in the proliferating cells. PTLD is probably caused by the iatrogenically impaired T-cell response allowing outgrowth of EBV-positive B-cells. Quantitative EBV DNA load monitoring is a minimally invasive technique increasingly recognized as a valuable tool in posttransplant patient management. In this review, we focus on the clinical utility of EBV DNA load monitoring in the peripheral blood of transplant recipients using PCR and we discuss the currently most-widely used techniques and their value and limitations in predicting and diagnosing PTLD. Options for EBV DNA load-guided pre-emptive therapy and application of monitoring EBV DNA load dynamics in the prediction of clinical response after therapy are described. Origins of elevated EBV DNA loads in immunosuppressed patients and recent insights in the EBV life cycle in the immuncompromised host are discussed. Finally, a standardization of methodology, clinical specimen type, and cut-off values is proposed. This is essential for comparisons between different institutes and more adequate patient management.

DNA, Viral↗

PubMed2000

Olfactory based spatial learning in neonatal mice and its dependence on CaMKII.

Spatial learning and memory involves the ability to encode geometric relationships between perceived cues and depends critically on the hippocampus. Visually guided spatial learning has been demonstrated in adult animals. As infant animals rely heavily on olfaction, olfactory based spatial learning was assessed in infant mice. When 12-day-old pups were displaced from their nest, they learned within a few training trials to use the spatial pattern of odor cues to move back to the nest. However, mouse pups that over-expressed Ca2+/calmodulin-dependent protein kinase (CaMKII) in hippocampal neurons were impaired in olfactory based spatial learning.

Age Factors↗

PubMed2002

Immunopurified small nucleolar ribonucleoprotein particles pseudouridylate rRNA independently of their association with phosphorylated Nopp140.

The isomerization of up to 100 uridines to pseudouridines (Psis) in eukaryotic rRNA is guided by a similar number of box H/ACA small nucleolar RNAs (snoRNAs), each forming a unique small nucleolar ribonucleoprotein particle (snoRNP) with the same four core proteins, NAP57 (also known as dyskerin or Cbf5p), GAR1, NHP2, and NOP10. Additionally, the nucleolar and Cajal body protein Nopp140 (Srp40p) associates with the snoRNPs. To understand the role of these factors in pseudouridylation, we established an in vitro assay system. Short site-specifically (32)P-labeled rRNA substrates were incubated with subcellular fractions, and the conversion of uridine to Psi was monitored by thin-layer chromatography after digestion to single nucleotides. Immunopurified box H/ACA core particles were sufficient for the reaction. SnoRNPs associated quantitatively and reversibly with Nopp140. However, pseudouridylation activity was independent of Nopp140, consistent with a chaperoning role for this highly phosphorylated protein. Although up to 14 bp between the snoRNA and rRNA were required for the in vitro reaction, rRNA pseudouridylation and release occurred in the absence of ATP and magnesium. These data suggest that substrate release takes place without RNA helicase activity but may be aided by the snoRNP core proteins.

Animals↗

PubMed2002

Differential display technology: a general guide.

The 10 years since the invention of differential display technology (DD) has produced a massive amount of literature detailing problems and improvements to the technique, successful gene expression studies and studies done using genes found through the use of DD. In this review we summarise the results of 10 years of research that has focussed on improving DD and discuss how some of the problems associated with DD can be resolved or minimised. In addition to discussing DD, we address issues related to other differential gene expression analysis techniques and try to illustrate how these techniques can be used to complement one's use of DD. This review also serves as an introduction to the taxa-specific DD review articles that are found in this issue.

Adenosine↗