Versatile Target Research Articles

Anti-primer quenching-based real-time PCR for simplex or multiplex DNA quantification and single-nucleotide polymorphism genotyping

Nucleic acid amplification and detection plays an increasingly important role in genetic analysis of clinical samples, medical diagnostics and drug discovery. We present a new quantitative PCR method that allows versatile and flexible nucleic acid target quantification. One of the PCR primers is modified by an oligonucleotide "tail" fluorescently labeled at the 5' end. An oligonucleotide complementary to this tail, carrying a 3'-quencher ("anti-primer"), is included in the PCR along with the two primers. Following primer extension, the reaction temperature is lowered such that the anti-primer hybridizes to and quenches the fluorescence of only the free primer and not the double-stranded PCR product, allowing real-time fluorescent quantification of the latter. This anti-primer-based quantitative real-time PCR (aQRT-PCR) allows simplex or multiplex quantification or single-nucleotide polymorphism genotyping in clinical samples of widely differing quality (e.g., fresh samples, formalin-fixed paraffin-embedded samples and plasma-circulating DNA) and provides a practical alternative to existing, more expensive approaches. The process of aQRT-PCR takes 1.5-2 h.

Protein-Peptide Interaction Studies Demonstrate the Versatility of Calmodulin Target Protein Binding

Calmodulin (CaM) is a prototypical Ca2+-sensor protein that can control many important biological functions by binding to hundreds of target proteins. To gain insight into the versatility of CaM-target recognition, we have analyzed the complex structures for many types of CaM-binding peptides and some target proteins. In particular, some recently reported novel complex structures reveal that the versatile target binding of CaM is accommodated by its flexible domain arrangement and the malleability of its interfaces.

Antiprimer Quenching-Based Real-Time PCR and Its Application to the Analysis of Clinical Cancer Samples

Nucleic acid amplification plays an increasingly important role in genetic analysis of clinical samples, medical diagnostics, and drug discovery. We present a novel quantitative PCR technology that combines the advantages of existing methods and allows versatile and flexible nucleic acid target quantification in clinical samples of widely different origin and quality. We modified one of the 2 PCR primers by use of an oligonucleotide "tail" fluorescently labeled at the 5' end. An oligonucleotide complementary to this tail, carrying a 3' quenching molecule (antiprimer), was included in the reaction along with 2 primers. After primer extension, the reaction temperature was lowered such that the antiprimer hybridizes and quenches the fluorescence of the free primer but not the fluorescence of the double-stranded PCR product. The latter provides real-time fluorescent product quantification. This antiprimer-based quantitative real-time PCR method (aQRT-PCR) was used to amplify and quantify minute amounts of input DNA for genes important to cancer. Simplex and multiplex aQRT-PCR demonstrated linear correlation (r(2) >0.995) down to a DNA input equivalent to 20 cells. Multiplex aQRT-PCR reliably identified the HER-2 gene in microdissected breast cancer samples; in formalin-fixed, paraffin-embedded specimens; and in plasma circulating DNA from cancer patients. Adaptation to multiplex single-nucleotide polymorphism detection via allele-specific aQRT-PCR allowed correct identification of apolipoprotein B polymorphisms in 51 of 51 human specimens. The simplicity, versatility, reliability, and low cost of aQRT-PCR make it suitable for genetic analysis of clinical specimens.

Estrogen receptors as targets for drug development for breast cancer, osteoporosis and cardiovascular diseases.

Estrogen receptors (ERs) are proteins that mediate the action of estradiol and a series of natural and synthetic chemicals that mimic the estradiol structure. Estrogenic action was initially attributed to a single type of ER, now known as ERalpha, but ERbeta was discovered in 1995. Tissue specific distribution and the intensity of expression of these proteins determine the first response of tissues to estrogenic compounds. Estrogens and ERs play a major role in the origin and progression of breast cancer, and antiestrogens that block ER function are useful for breast cancer prevention and treatment. Estrogen mimetics, however, do not fall into distinct categories of agonists and antagonists, since their action is regulated by tissue-specific expression of a number of auxiliary proteins called coactivators or corepressors. In addition, small molecules such as polyamines, fattyacids, and thioredoxin may modulate ER function. Estrogenic functions encompass multiple organ systems, including the reproductive, skeletal, cardiovascular, and nervous system. Estrogens are critical for bone remodeling and mineralization so that estrogen replacement therapy is proven to strengthen bone health in post-menopausal women. Ideally, selective blockade of ER function in breast epithelial cells should be accompanied by growth support on bone and cardiovascular systems. The details of estrogenic function in different organs are to be fully realized, in order to better utilize selective estrogen receptor modulators (SERMs) to fight not only breast cancer but also osteoporosis and cardiovascular diseases. Current research on SERMs points toward accomplishing this goal by exploiting ER as a versatile target against multiple diseases.

Crystal Structure of Carbapenam Synthetase (CarA)

Carbapenam synthetase (CarA) is an ATP/Mg2+-dependent enzyme that catalyzes formation of the beta-lactam ring in (5R)-carbapenem-3-carboxylic acid biosynthesis. CarA is homologous to beta-lactam synthetase (beta-LS), which is involved in clavulanic acid biosynthesis. The catalytic cycles of CarA and beta-LS mediate substrate adenylation followed by beta-lactamization via a tetrahedral intermediate or transition state. Another member of this family of ATP/Mg2+-dependent enzymes, asparagine synthetase (AS-B), catalyzes intermolecular, rather than intramolecular, amide bond formation in asparagine biosynthesis. The crystal structures of apo-CarA and CarA complexed with the substrate (2S,5S)-5-carboxymethylproline (CMPr), ATP analog alpha,beta-methyleneadenosine 5'-triphosphate (AMP-CPP), and a single Mg2+ ion have been determined. CarA forms a tetramer. Each monomer resembles beta-LS and AS-B in overall fold, but key differences are observed. The N-terminal domain lacks the glutaminase active site found in AS-B, and an extended loop region not observed in beta-LS or AS-B is present. Comparison of the C-terminal synthetase active site to that in beta-LS reveals that the ATP binding site is highly conserved. By contrast, variations in the substrate binding pocket reflect the different substrates of the two enzymes. The Mg2+ coordination is also different. Several key residues in the active site are conserved between CarA and beta-LS, supporting proposed roles in beta-lactam formation. These data provide further insight into the structures of this class of enzymes and suggest that CarA might be a versatile target for protein engineering experiments aimed at developing improved production methods and new carbapenem antibiotics.

Solid targetry at the TESLA Accelerator Installation

According to the concept of the TESLA Accelerator Installation, the channel for production of radioisotopes has to routinely produce 201Tl, 111In, 67Ga, 123I and 18F, and a number of other radionuclides for experimental purposes. The production of 123I and 18F will be performed in dedicated, commercial target stations, while a versatile solid target irradiation system is designed for the routine and experimental production of all other radioisotopes. The solid target station is designed to accept targets for both the 7° and 90° irradiation geometry. The targets used for the routine production will be prepared by electroplating on a silver substrate. They can be irradiated with a 1.5 kW beam using the 7° geometry. The cooling of these targets is enhanced by fins on the back of the silver substrate designed so that the highest temperature on the surface of the target does not exceed 110°C. The irradiation procedures will conform to the GMP requirements for the production of radiopharmaceuticals. The irradiated targets will be transported directly into the appropriate hot cell for radiochemical processing. All cells will be equipped with a target dissolution unit for etching the irradiated, electroplated film. After decontamination and sufficient cooling down, these targets will be reused several times.

Regulation of both gene expression and protein stability provides genetically assisted target evaluation (GATE) for microbial target validation.

The attempt to develop novel antibiotics, active against organisms resistant to current therapies, has led researchers to seek and explore new drug targets. The rapid sequencing and analysis of entire microbial genomes has identified large numbers of genes that may be sufficiently different from their human counterparts to be exploited as targets for antimicrobial treatment. As a first step, the importance of the various putative targets for microbial growth and survival must be assessed. Emerging validation technologies are becoming increasingly sophisticated and, in certain cases, allow prioritisation of the best targets. In this paper, genetically assisted target evaluation (GATE) is introduced as a versatile target validation technology. GATE concomitantly manipulates both synthesis and stability of the targeted protein using copper ions as an effector. This technology allows rapid quantitation of the lethal consequences of inactivation of targeted gene products in Saccharomyces cerevisiae. Additional tools can then be applied to extend these results into pathogenic organisms, such as Candida albicans.

Production of medical radioisotopes in the ORNL high flux isotope reactor (HFIR) for cancer treatment and arterial restenosis therapy after PICA

The High Flux Isotope Reactor (HFIR) at the Oak Ridge National Laboratory (ORNL) represents an important resource for the production of a wide variety of medical radioisotopes. First beginning operation in 1965, the high thermal neutron flux (2.5×1015 neutrons/cm2/sec at 85 MW) and versatile target irradiation and handling facilities provide the opportunity for production of a wide variety of neutron-rich medical radioisotopes of current interest for therapy. In addition to serving as a key production site for californium-252 and other transuranic elements, important examples of therapeutic radioisotopes which are currently routinely produced in the HFIR for distribution include dysprosium-166 (parent of holmium-166), rhenium-186, tin-117m and tungsten-188 (parent of rhenium-188). The nine hydraulic tube (HT) positions in the central high flux region permit the insertion and removal of targets at any time during the operating cycle (22–24 days) and have traditionally represented a major site for production of medical radioisotopes. To increase the irradiation capabilities of the HFIR, special target holders have recently been designed and fabricated which will be installed in the six Peripheral Target Positions (PTP), which are also located in the high flux region. These positions are only accessible during reactor refueling and will be used for long-term irradiations, such as required for the production of tin-117m and tungsten-188. Each of the PTP tubes will be capable of housing a maximum of eight HT targets, thus increasing the total maximum number of HT targets from the current nine, to a total of 57. In this paper the therapeutic use of reactor-produced radioisotopes for bone pain palliation and vascular brachytherapy and the therapeutic medical radioisotope production capabilities of the ORNL HFIR are briefly discussed.

Auxin signalling: protein stability as a versatile control target.

Two components of an auxin signalling pathway in Arabidopsis have been found to be homologous to budding yeast enzymes that are known to be involved in regulating the stability of key cell-cycle regulatory proteins, such as the cyclin-dependent kinase inhibitor Sic1p.

Producing ??d and ??t in vacuum

After the feasibility of vacuum isolated μ−d production was demonstrated at TRIUMF in 1989, development was begun on a target system that would take advantage of the process to aid in the understanding of the muon catalyzed fusion cycle. Minimal neutron backgrounds, the ability to use silicon detectors, and compatibility with tritium were considered important for a very versatile target system. The advantages which the target gives in isolating μCF process will be outlined.

Multichain interleukin-2 receptor: a target for immunotherapy in lymphoma.

Activation of resting T cells induces synthesis of interleukin-2 (IL-2) and expression of its specific high-affinity receptor. We proposed a multichain model for the high-affinity IL-2 receptor in which both a 55-kilodalton IL-2-binding peptide identified by the anti-Tac monoclonal antibody and a 70/75-kilodalton IL-2-binding peptide are associated in a receptor complex. The IL-2 receptor is proving to be an extraordinarily versatile therapeutic target, since it is expressed by the abnormal T cells in patients with certain lymphoid malignancies or autoimmune disorders and in individuals rejecting allografts, whereas it is not expressed by normal resting cells. Monoclonal antibodies and toxin-lymphokine conjugates directed toward IL-2 receptors represent novel therapeutic agents for these clinical conditions.

A versatile target chamber in an ion implanter for metallurgical research

In an ion implanter for metallurgical research the target chamber must be easily adaptable for use with various samples and must also permit implantation under controlled conditions. The complexity of the samples movements and the length of the implantation process requires the automatic control of the chamber. This paper describes a target chamber designed to match these requirements. The sample holder is driven by three step-motors operated by a microprocessor control system. Depending on the sample and the desired implantation geometry, interchangeable supports may be used. The rotations are transmitted to them by stepping motors and these can be used independently or combined in order to produce the required movements. The control system is made up of an independent stepping motor driver interface and a microcomputer assembled with standard PCBs for industrial control. The microprocessor is a Z-80 model. The system software is based on FORTH, a high level structured language that permits flexibility and simplicity in the programming and use of the system.

A versatile target manipulator for use in ultra-high vacuum

A target manipulator for use in ultra-high vacuum is described. With this manipulator a target can be translated 20 mm in three mutually perpendicular directions and rotated around three mutually perpendicular rotation axes. The directions of the rotation axes are independent of each other. A special feature of this design is that the third rotation (the tilt) does not change the direction of the azimuthal rotation axis. The azimuthal and polar rotation have a range of 360°. The target temperature and beam currents can be measured at the back of the target. The target can be heated by electron bombardment on the front or by an electrical heater at the back. The whole manipulator can be mounted on a 4″ flange of the target chamber. To perform the rotation the manipulator head requires a volume with diameter of 50 mm at the centre of the target chamber. Targets with a maximum diameter of 13 cm can be inserted. Only materials suited for ultra-high vacuum are used in the construction.

Production en routine par cyclotron de fluor-18 et potassium-43 à usage médical au moyen d'une cible gazeuse télécommandée

In order to routinely produce high sterile activities of 18F and 43K for medical use, an electrically remote-controlled and versatile gaseous target has been designed for the preparation of both nuclides. This static Ne or Ar gas target system gives the following production rates: 20 mC of 18F/μA·h at 10.6 MeV and more than 60 μC of 43K/μA·h at 21.0 MeV. In a production run of 200 mC ( 18F) and 3 mC ( 43K) the maximum handling dose never reaches more than 5 mR (finger dose) which means considerable reduction of radiation dose and absence of health hazard of such a procedure. This target system can also be used in a dynamic mode now under investigation for the labelling of organic compounds in organic liquid solvent or gaseous phase.

A low cost multi-purpose target chamber

The characteristics and specifications of a 10 in. (25.4 cm) diameter highly versatile target chamber are presented. Designed to be an inexpensive multi-purpose chamber, it facilities γ−γ, γ-particle, γ−γ-particle correlations, angular distributions of both particles and γ-rays, perturbed angular correlations, and many other experiments.

Etude d'une source d'ions lourds sans electrode d'extraction

Resume An experimental study of the properties of an rf ion source which does not require an extraction electrode is described. The beam is extracted by means of a converging-diverging nozzle. The absence of any electrode drastically reduces the maintenance time required by the more common types of ion source. Beams of CO 2 + , O 2 + , O + , N 2 + and N + have been obtained with an intensity up to 150 μA for the N + ions when the optimum operating conditions of our present limited equipment are achieved. Suggestions for improving the intensity and the efficiency of the source are presented. The present system can also be used as a versatile gas target.