Multiple modes of ligand recognition: Crystal structures of cyclin-dependent protein kinase 2 in complex with ATP and two inhibitors,olomoucine and isopentenyladenine

Cyclin-dependent kinases (CDKs) are conserved regulators of the eukaryotic cell cycle with different isoforms controlling specific phases of the cell cycle. Mitogenic or growth inhibitory signals are mediated, respectively, by activation or inhibition of CDKs which phosphorylate proteins associated with the cell cycle. The central role of CDKs in cell cycle regulation makes them a potential new target for inhibitory molecules with anti-proliferative and/or anti-neoplastic effects. We describe the crystal structures of the complexes of CDK2 with a weakly specific CDK inhibitor, N6-(δ2-isopentenyl)adenine, and a strongly specific inhibitor, olomoucine. Both inhibitors are adenine derivatives and bind in the adenine binding pocket of CDK2, but in an unexpected and different orientation from the adenine of the authentic ligand ATP. The N6-benzyl substituent in olomoucine binds outside the conserved binding pocket and is most likely responsible for its specificity. The structural information from the CDK2-olomoucine complex will be useful in directing the search for the next generation inhibitors with improved properties. © 1995 Wiley-Liss, Inc.     

Establishing a minor illness nurse in a busy general practice.

OBJECTIVE--To study the feasibility of a practice nurse caring for patients with minor illnesses. DESIGN--Nurse given training in dealing with patients with minor illnesses. Patients requesting a same day appointment were offered a nurse consultation. SETTING--Group practice in Stockton on Tees. MAIN OUTCOME MEASURES--Number of consultations which required a doctor contact, treatment, and rate of reconsultation. RESULTS--Of 696 consultations in six months, 602 (86%) required no doctor contact. 549 (79%) patients did not reconsult about the episode of illness, and 343 (50%) patients were given advice on self care only. CONCLUSION--Trained nurses could diagnose and treat a large proportion of patients currently consulting general practitioners about minor illness provided that the nurse has immediate access to a doctor.     

Oxidation of Indole-3-acetic Acid and Oxindole-3-acetic Acid to 2,3-Dihydro-7-hydroxy-2-oxo-1H Indole-3-acetic Acid-7′-O-β-d-Glucopyranoside in Zea mays Seedlings

Radiolabeled oxindole-3-acetic acid was metabolized by roots, shoots, and caryopses of dark grown Zea mays seedlings to 2,3-dihydro-7-hydroxy-2-oxo-1H indole-3-acetic acid-7′-O-β-d-glycopyranoside with the simpler name of 7-hydroxyoxindole-3-acetic acid-glucoside. This compound was also formed from labeled indole-3-acetic acid supplied to intact seedlings and root segments. The glucoside of 7-hydroxyoxindole-3-acetic acid was also isolated as an endogenous compound in the caryopses and shoots of 4-day-old seedlings. It accumulates to a level of 4.8 nanomoles per plant in the kernel, more than 10 times the amount of oxindole-3-acetic acid. In the shoot it is present at levels comparable to that of oxindole-3-acetic acid and indole-3-acetic acid (62 picomoles per shoot). We conclude that 7-hydroxyoxindole-3-acetic acid-glucoside is a natural metabolite of indole-3-acetic acid in Z. mays seedlings. From the data presented in this paper and in previous work, we propose the following route as the principal catabolic pathway for indole-3-acetic acid in Zea seedlings: Indole-3-acetic acid → Oxindole-3-acetic acid → 7-Hydroxyoxindole-3-acetic acid → 7-Hydroxyoxindole-3-acetic acid-glucoside.     

Identifying sites of simultaneous DNA replication in eukaryotes by N-methyl-N''-nitro-N-nitrosoguanidine multiple mutagenesis.

Summary N-methyl-N-nitro-N-nitrosoguanidine (NG) induces certain classes of multiple mutations in yeast at high frequency. By selecting for mutation at one locus (his4 or leu1) one frequently obtains double mutants where another mutation to temperature sensitivity has also been induced. This multiple mutagenesis exhibits a considerable specificity: for mutation at one particular locus there is a high chance that another mutation will be found in the same cell at one of a restricted number of other loci. For any given locus (e.g. his4) there is a spectrum of sites at which temperature-sensitivity mutations are coinduced. This spectrum differs for different loci, such that the spectrum of sites co-mutating with leul differs completely from that for sites co-mutating with his4. This NG-induced co-mutation is interpreted in terms of NG acting to enhance mutagenesis at sites of simultaneous DNA replication within the cell. The results so obtained indicate a very strict control over the order and timing of gene replication in Saccharomyces cerevisiae, and it is suggested that it is now possible to use NG double mutagenesis to try and locate origins of replication in yeast.     

Dominant and recessive informational suppressors of a missense mutation in Coprinus

Summary Twenty-one suppressor gene mutations which suppress the met-5.1 missense mutation of Coprinus were separated into six groups (A-F) on the basis of dominance or recessiveness, linkage to the met-5 locus, comlementation in heterozygous cells and growth behaviour. The actual number of suppressor loci could not be determined because crosses between suppressed mutants were inviable. The allele specificity of group A, C, D and F suppressors was confirmed by appropriate crosses. Group B and E suppressors were not tested because of close linkage to the met-5 locus. No evidence for functional suppression of met-5 mutations was obtained thus it is likely that all the suppressors cause translational corelation of met-5.1. Suppressors in four groups (C-F) have properties expected of tRNA structural gene mutations: the group C mutation is dominant, the other mutations are recessive but do not complement in heterozygous cells. The relative efficiencies of the tRNA species involved was assessed by comparing the degree to which the different sup ⁺ mutations depressed the growth rate on methionine supplemented medium. The dominant mutation depressed growth to the greatest extent and is, therefore, the most efficient suppressor. The least efficient suppressors did not depress growth at all. When growth was compared on minimal medium it was found that the more efficient the suppressor the less well it restored growth. The mutations in groups A and B depressed growth more than the tRNA mutations but affect some other component in translation because they are recessive and complement normally. It is suggested that they may act to alter tRNA modifying enzymes.     

ULTRASTRUCTURE OF THE RED TIDE DINOFLAGELLATE GYMNODINIUM BREVE. I. GENERAL DESCRIPTION1,2,3

Gymnodimium breve Davis, an unarmored marine dinoflagellate has a cell covering (theca) composed of four membranes. The inner two membranes represent a vesicular layer and in tangential section, the theca appears composed of polygonal areas. Unusual threat ridges are located in the cingular region between the epi- and hypocone. This osmotically sensitive species is extremely vesiculate with dispersed areas of cytoplasm containing typical eukaryotic organelles as well as other organelles found only in dinoflagellates. The non-vesiculated cytoplasm is continuous in serial sections. The chloroplasts can contain either quasi-radial or parallel lamellae typically consisting of three thylakoids each. The pyrenoid is multiple-stalked and lacks a starch cap. The dinophycean pusule is simple and similar to those found in several unarmored marine species. The nucleus is typically dinophycean but the chromosomes appear to lack nonfibrillar material.     

Iron enhances the bactericidal action of streptonigrin

The lethal action of streptonigrin on strains of $\text{Escherichia}\text{coli}$ is greatly enhanced by citrate (10^?2 M). Desferrioxamine (2×10^?4 M), when added with streptonigrin and citrate, eliminates the citrate enhancement. These observations point to a role for iron in the bactericidal mechanism of streptonigrin. Extracellular citrate is known to promote the acquisition of iron by $\text{E.}\text{coli}$ by delivering it as a ferric citrate complex to a specific transport apparatus on the cell envelope. Therefore, it may promote action of streptonigrin by increasing the intracellular concentration of available iron. Desferrioxamine, which forms a much stronger complex with ferric ion than does citrate, would be expected to suppress the ferric citrate effect, and this was observed.