Biomedical subjects

R W King

Publications and source records attributed to R W King.

At least 19 recordsLinked to original sources

PubMed2001

New technologies for chemical genetics.

Chemical genetics, in which small molecules are used in lieu of mutations to study biological processes, requires large and diverse chemical libraries to specifically perturb different biological pathways. Here we describe a suite of technologies that enable chemical libraries prepared by split-pool solid phase synthesis to be screened in a diverse range of chemical genetic assays. Compounds are synthesized on 500 micron high-capacity polystyrene beads, and arrayed into individual wells of 384-well plates using a hand-held bead arrayer. Compounds are cleaved from synthesis beads using a chemically-resistant ceramic dispensing system, producing individual stock solutions of single compounds. Nanoliter volumes of these solutions are then transferred into assay plates using an array of stainless steel pins mounted on a robotic arm. We have designed reusable 1536- and 6144-well assay plates made of silicone rubber that can be cast in the laboratory and filled by hand. This integrated technology platform enables hundreds of biological assays to be performed from the product of a single synthesis bead, enabling the results of different chemical genetic experiments to be directly compared.

Cell Extracts↗

PubMed2001

A one-bead, one-stock solution approach to chemical genetics: part 2.

BACKGROUND: Chemical genetics provides a systematic means to study biology using small molecules to effect spatial and temporal control over protein function. As complementary approaches, phenotypic and proteomic screens of structurally diverse and complex small molecules may yield not only interesting individual probes of biological function, but also global information about small molecule collections and the interactions of their members with biological systems. RESULTS: We report a general high-throughput method for converting high-capacity beads into arrayed stock solutions amenable to both phenotypic and proteomic assays. Polystyrene beads from diversity-oriented syntheses were arrayed individually into wells. Bound compounds were cleaved, eluted, and resuspended to generate 'mother plates' of stock solutions. The second phase of development of our technology platform includes optimized cleavage and elution conditions, a novel bead arraying method, and robotic distribution of stock solutions of small molecules into 'daughter plates' for direct use in chemical genetic assays. This library formatting strategy enables what we refer to as annotation screening, in which every member of a library is annotated with biological assay data. This phase was validated by arraying and screening 708 members of an encoded 4320-member library of structurally diverse and complex dihydropyrancarboxamides. CONCLUSIONS: Our 'one-bead, multiple-stock solution' library formatting strategy is a central element of a technology platform aimed at advancing chemical genetics. Annotation screening provides a means for biology to inform chemistry, complementary to the way that chemistry can inform biology in conventional ('investigator-initiated') small molecule screens.

Bromodeoxyuridine↗

PubMed2001

A florigenic effect of sucrose in Fuchsia hybrida is blocked by gibberellin-induced assimilate competition.

The use of gas chromatography-mass spectrometry-selected ion monitoring along with a (13)C internal standard has allowed sensitive measurements of the sucrose (Suc) content of individual shoot apices of Fuchsia hybrida. With intact plants, as the photosynthetic irradiance increased, so did shoot apex Suc content, reaching saturation at about 500 micromol m(-2) s(-1). These same plants flowered at the higher irradiances, remaining vegetative in 10-h short days at an irradiance of 230 micromol m(-2) s(-1). The strong correlation (r = 0.93) in these studies between flowering and shoot apex Suc content indicates a role for Suc as a stimulus to flowering in this species. However, Suc is not the long-day (LD) "florigen" of F. hybrida because 2 to 4 LD given as a 14-h low-irradiance photoperiod extension (10-15 micromol m(-2) s(-1)) induced flowering but without increase in shoot apex Suc content. Flowering induced by either pathway, the LD- or the Suc-mediated one, was inhibited by applying gibberellin (GA) to the shoot tip. Such inhibition of flowering by GA, at least for the LD pathway, was associated with a reduced apex Suc content, enhanced elongation of subapical stem tissue, and a reduced import into the shoot apex of leaf-sourced assimilate. Thus, our findings show how GA inhibits flowering of F. hybrida and confirm the importance of nutrient diversion in regulating flowering.

Flowers↗

PubMed2001

Evolution of floral meristem identity genes. Analysis of Lolium temulentum genes related to APETALA1 and LEAFY of Arabidopsis.

Flowering (inflorescence formation) of the grass Lolium temulentum is strictly regulated, occurring rapidly on exposure to a single long day (LD). During floral induction, L. temulentum differs significantly from dicot species such as Arabidopsis in the expression, at the shoot apex, of two APETALA1 (AP1)-like genes, LtMADS1 and LtMADS2, and of L. temulentum LEAFY (LtLFY). As shown by in situ hybridization, LtMADS1 and LtMADS2 are expressed in the vegetative shoot apical meristem, but expression increases strongly within 30 h of LD floral induction. Later in floral development, LtMADS1 and LtMADS2 are expressed within spikelet and floret meristems and in the glume and lemma primordia. It is interesting that LtLFY is detected quite late (about 12 d after LD induction) within the spikelet meristems, glumes, and lemma primordia. These patterns contrast with Arabidopsis, where LFY and AP1 are consecutively activated early during flower formation. LtMADS2, when expressed in transgenic Arabidopsis plants under the control of the AP1 promoter, could partially complement the organ number defect of the severe ap1-15 mutant allele, confirming a close relationship between LtMADS2 and AP1.

Amino Acid Sequence↗

PubMed2001

Inhibition of the bovine viral diarrhoea virus NS3 serine protease by a boron-modified peptidyl mimetic of its natural substrate.

Bovine viral diarrhoea virus (BVDV) is closely related to hepatitis C virus (HCV), and has been used as a surrogate virus in drug development for HCV infection. Similar to HCV, BVDV-encoded NS3 serine proteinase is responsible for multiple cleavages in the viral polyprotein, generating mature NS4A, NS4B, NS5A and NS5B proteins. NS3-dependent cleavage sites of BVDV contain a strictly conserved leucine at P1, and either serine or alanine at P1'. The full length BVDV NS3/4A serine protease has been cloned and expressed in bacterial cells. The enzyme has been purified from the soluble portion of Escherichia coli via a two-step purification procedure employing chromatography on heparin resin and gel filtration. The protease activity was characterized using in vitro translated BVDV NS4A/B and NS5A/B polyprotein substrates. A boronic acid analogue of the BVDV NS4A/NS4B cleavage site was synthesized and shown to be an efficient inhibitor of the NS3 serine protease in vitro. The compound, designated DPC-AB9144-00, inhibited approximately 75% of the NS3/4 activity at 10 microM with the NS4A/B substrate. However, no antiviral activity was detected with DPC-AB9144-00 in BVDV-infected Madin-Darby bovine kidney cells at concentrations as great as 90 pM, suggesting permeability or that other cellular-derived limitations were present.

Animals↗

PubMed2001

Long-day induction of flowering in Lolium temulentum involves sequential increases in specific gibberellins at the shoot apex.

One challenge for plant biology has been to identify floral stimuli at the shoot apex. Using sensitive and specific gas chromatography-mass spectrometry techniques, we have followed changes in gibberellins (GAs) at the shoot apex during long day (LD)-regulated induction of flowering in the grass Lolium temulentum. Two separate roles of GAs in flowering are indicated. First, within 8 h of an inductive LD, i.e. at the time of floral evocation, the GA(5) content of the shoot apex doubled to about 120 ng g(-1) dry weight. The concentration of applied GA(5) required for floral induction of excised apices (R.W. King, C. Blundell, L.T. Evans [1993] Aust J Plant Physiol 20: 337-348) was similar to that in the shoot apex. Leaf-applied [(2)H(4)] GA(5) was transported intact from the leaf to the shoot apex, flowering being proportional to the amount of GA(5) imported. Thus, GA(5) could be part of the LD stimulus for floral evocation of L. temulentum or, alternatively, its increase at the shoot apex could follow import of a primary floral stimulus. Later, during inflorescence differentiation and especially after exposure to additional LD, a second GA action was apparent. The content of GA(1) and GA(4) in the apex increased greatly, whereas GA(5) decreased by up to 75%. GA(4) applied during inflorescence differentiation strongly promoted flowering and stem elongation, whereas it was ineffective for earlier floral evocation although it caused stem growth at all times of application. Thus, we conclude that GA(1) and GA(4) are secondary, late-acting LD stimuli for inflorescence differentiation in L. temulentum.

Deuterium↗

PubMed2001

GAMYB-like genes, flowering, and gibberellin signaling in Arabidopsis.

We have identified three Arabidopsis genes with GAMYB-like activity, AtMYB33, AtMYB65, and AtMYB101, which can substitute for barley (Hordeum vulgare) GAMYB in transactivating the barley alpha-amylase promoter. We have investigated the relationships between gibberellins (GAs), these GAMYB-like genes, and petiole elongation and flowering of Arabidopsis. Within 1 to 2 d of transferring plants from short- to long-day photoperiods, growth rate and erectness of petioles increased, and there were morphological changes at the shoot apex associated with the transition to flowering. These responses were accompanied by accumulation of GAs in the petioles (GA(1) by 11-fold and GA(4) by 3-fold), and an increase in expression of AtMYB33 at the shoot apex. Inhibition of GA biosynthesis using paclobutrazol blocked the petiole elongation induced by long days. Causality was suggested by the finding that, with GA treatment, plants flowered in short days, AtMYB33 expression increased at the shoot apex, and the petioles elongated and grew erect. That AtMYB33 may mediate a GA signaling role in flowering was supported by its ability to bind to a specific 8-bp sequence in the promoter of the floral meristem-identity gene, LEAFY, this same sequence being important in the GA response of the LEAFY promoter. One or more of these AtMYB genes may also play a role in the root tip during germination and, later, in stem tissue. These findings extend our earlier studies of GA signaling in the Gramineae to include a dicot species, Arabidopsis, and indicate that GAMYB-like genes may mediate GA signaling in growth and flowering responses.

Arabidopsis↗

PubMed2000

Phenylpropenamide derivatives as inhibitors of hepatitis B virus replication.

A non-nucleoside class of compounds that inhibits the replication of hepatitis B virus (HBV) in cell culture has been discovered. A series of substituted analogues of phenylpropenamide 6 has been prepared and evaluated in the HepAD38 cellular assay. Structure-activity relationships of this series are discussed.

Antiviral Agents↗

PubMed2000

Dissecting cellular processes using small molecules: identification of colchicine-like, taxol-like and other small molecules that perturb mitosis.

BACKGROUND: Understanding the molecular mechanisms of complex cellular processes requires unbiased means to identify and to alter conditionally gene products that function in a pathway of interest. Although random mutagenesis and screening (forward genetics) provide a useful means to this end, the complexity of the genome, long generation time and redundancy of gene function have limited their use with mammalian systems. We sought to develop an analogous process using small molecules to modulate conditionally the function of proteins. We hoped to identify simultaneously small molecules that may serve as leads for the development of therapeutically useful agents. RESULTS: We report the results of a high-throughput, phenotype-based screen for identifying cell-permeable small molecules that affect mitosis of mammalian cells. The predominant class of compounds that emerged directly alters the stability of microtubules in the mitotic spindle. Although many of these compounds show the colchicine-like property of destabilizing microtubules, one member shows the taxol-like property of stabilizing microtubules. Another class of compounds alters chromosome segregation by novel mechanisms that do not involve direct interactions with microtubules. CONCLUSIONS: The identification of structurally diverse small molecules that affect the mammalian mitotic machinery from a large library of synthetic compounds illustrates the use of chemical genetics in dissecting an essential cellular pathway. This screen identified five compounds that affect mitosis without directly targeting microtubules. Understanding the mechanism of action of these compounds, along with future screening efforts, promises to help elucidate the molecular mechanisms involved in chromosome segregation during mitosis.

Animals↗

PubMed2000

Calculated electric field induced in bundles of long cells in the human body when it is exposed to low-frequency electric fields.

A theoretical analysis is made of the electric field induced in the interior of long cylindrical cells arranged in bundles of parallel cells embedded in the saline fluid in the human body exposed to extremely low-frequency (ELF) and very low-frequency (VLF) electric fields parallel to the length of the cells. Such bundles characterize muscle. The analysis parallels in many respects the earlier study of isolated long cells (King and Wu, Phys. Rev. E 58, 2363-2369, 1998) but has important added complications. It leads to a similar result: The field inside the membrane of each long cell is the same as the field just outside the membrane in the saline fluid; i.e., the membrane provides no shielding. While one might expect the presence of the neighboring cells to provide a shielding effect for each cell, the results of this study show quantitatively that this is not the case. In fact, the electric field in the interior of each of a bundle of long cells is seen to increase very slightly (of the order of 0.4%) when the cells in the bundle are moved close enough to be practically in contact.

Cell Membrane↗

PubMed1999

Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen.

Small molecules that perturb specific protein functions are valuable tools for dissecting complex processes in mammalian cells. A combination of two phenotype-based screens, one based on a specific posttranslational modification, the other visualizing microtubules and chromatin, was used to identify compounds that affect mitosis. One compound, here named monastrol, arrested mammalian cells in mitosis with monopolar spindles. In vitro, monastrol specifically inhibited the motility of the mitotic kinesin Eg5, a motor protein required for spindle bipolarity. All previously known small molecules that specifically affect the mitotic machinery target tubulin. Monastrol will therefore be a particularly useful tool for studying mitotic mechanisms.

Actins↗

PubMed1999

Chemistry or biology: which comes first after the genome is sequenced?

In drug discovery, chemical screening is performed after a protein's function has been determined. By screening for ligands that alter the function of a cell or an organism, new proteins that participate in poorly understood biological processes can be identified. Genomic and expression-cloning technologies can rapidly identify the protein targets of these ligands, enhancing the power of chemical screening as a tool for the initial stages of biological discovery.

Cloning, Molecular↗

PubMed1999

Long-day up-regulation of a GAMYB gene during Lolium temulentum inflorescence formation.

Long-day exposure of the grass Lolium temulentum may regulate flowering via changes in gibberellin (GA) levels. Therefore, we have examined both GA levels and expression of a MYB transcription factor that is specific to the GA signal transduction pathway in monocots. This MYB gene from L. temulentum shows over 90% nucleotide identity with the barley and rice GAMYB genes, and, like them, gibberellic acid (GA3) up-regulates its expression in the seed. Furthermore, cDNAs of both the barley and L. temulentum GAMYB show the same simple patterns of hybridization with digests of L. temulentum genomic DNA. Compared with vegetative shoot apices of L. temulentum, the in situ mRNA expression of LtGAMYB does not change during the earliest steps of "floral" initiation at the apex. However, by 100 h (the double-ridge stage of flowering) its expression increased substantially and was highest in the terminal and lateral spikelet sites. Thereafter, expression declined overall but then increased within stamen primordia. Prior to increased LtGAMYB expression, long-day exposure sufficient to induce flowering led to increased (5- to 20-fold) levels of GA1 and GA4 in the leaf. Thus, increases first in GA level in the leaf followed by increased expression of LtGAMYB in the apex suggest important signaling and/or response roles in flowering.

Amino Acid Sequence↗

PubMed1999

The electric field induced in the human body when exposed to electromagnetic fields at 1-30 MHz on shipboard.

The electric field induced in the body of a man standing on the metal deck of a ship near a vertical antenna is determined analytically. Typical antennas for radio communication in the 1-30 MHz band are described and their near fields are calculated. The current induced in the man is determined by solving the relevant integral equation. Explicit formulas are obtained for the total axial current and the associated current densities and electric fields.

Electric Conductivity↗

PubMed1999

Nerves in a human body exposed to low-frequency electromagnetic fields.

Following a summarizing introduction in which the background research is reviewed and referenced, a detailed description is given of the properties of the elongated cell that constitutes a nerve axon. The functioning of the cell membrane is reviewed with reference to the transmission of a frequency-modulated signal. The need for successive regeneration by means of action potentials is described. Propagation within both myelinated and unmyelinated membranes is discussed. Currents and electric fields induced in the organs of the human body by external electric and magnetic fields are introduced and their determination reviewed. The interaction between these currents and electric fields and those involved in the propagation of a signal along a nerve axon is analyzed. It is shown that incident 60-Hz electric fields near high-voltage transmission lines do not induce large enough currents and fields in a nerve axon in the leg to disrupt a propagating signal. Scaling with respect to frequency and size is discussed. Surface sensations due to exposure to electric fields in the 5-15-kV/m range are analyzed. It is concluded that exposure to the electromagnetic field of a 60-Hz high-voltage transmission line or a 10-30-kHz high-power transmitting antenna should have no observable effect on the normal functioning of nerves.

Action Potentials↗

PubMed1999

A standardized letter of recommendation for residency application.

Emergency medicine (EM) program directors have expressed a desire for more evaluative data to be included in application materials. This is consistent with frustrations expressed by program directors of multiple specialties, but mostly by those in specialties with more competitive matches. Some of the concerns about traditional narrative letters of recommendation included lack of uniform information, lack of relative value given for interval grading, and a perception of ambiguity with regard to terminology. The Council of Emergency Medicine Residency Directors established a task force in 1995 that created a standardized letter of recommendation form. This form, to be completed by EM faculty, requests that objective, comparative, and narrative information be reported regarding the residency applicant.

Correspondence as Topic↗

PubMed1998

Identification of a cullin homology region in a subunit of the anaphase-promoting complex.

The anaphase-promoting complex is composed of eight protein subunits, including BimE (APC1), CDC27 (APC3), CDC16 (APC6), and CDC23 (APC8). The remaining four human APC subunits, APC2, APC4, APC5, and APC7, as well as human CDC23, were cloned. APC7 contains multiple copies of the tetratrico peptide repeat, similar to CDC16, CDC23, and CDC27. Whereas APC4 and APC5 share no similarity to proteins of known function, APC2 contains a region that is similar to a sequence in cullins, a family of proteins implicated in the ubiquitination of G1 phase cyclins and cyclin-dependent kinase inhibitors. The APC2 gene is essential in Saccharomyces cerevisiae, and apc2 mutants arrest at metaphase and are defective in the degradation of Pds1p. APC2 and cullins may be distantly related members of a ubiquitin ligase family that targets cell cycle regulators for degradation.

Amino Acid Sequence↗

PubMed1998

Fields and currents in the organs of the human body when exposed to power lines and VLF transmitters.

A study is made of the electric fields and currents induced in the organs of the human body when exposed to high-voltage 50-60-Hz transmission lines and 10-30-kHz high-power transmitters. Relevant analyses previously carried out are summarized and supplemented with detailed investigations that complete the picture. Incomplete, misleading, and incorrect statements and methods in the related literature are pointed out, completed, and corrected. The major contribution is to provide quantitatively accurate, relatively simple analytic formulas that relate the incident electric field to the induced field in the organs of the body. The formulation and solution of the underlying integral equation are carried out in the Appendix.

Electric Impedance↗