Eclampsia in the United Kingdom.

Objectives: To measure the incidence of eclampsia, establish how often it is preceded by signs of pre-eclampsia, document the morbidity associated with eclampsia, and determine the maternal case fatality rates.

Design: A prospective, descriptive study of every case of eclampsia in the United Kingdom in 1992. Information was collected from reviews of hospital case notes and questionnaires to general practitioners.

Rational design of peptide-based inhibitors of trypanothione reductase as potential antitrypanosomal drugs.

The rational design of ligands for the substrate-binding site of a homology-modelled trypanothione reductase (TR) was performed. Peptides were designed to be selective for TR over human glutathione reductase (GR). The design process capitalized on the proposed differences between the activesites of TR and human GR, subsequently confirmed by the TR crystal structure.

Photoaffinity labelling of the active site of the rat glutathione transferases 3-3 and 1-1 and human glutathione transferase A1-1.

The glutathione transferases (GSTs) form a group of enzymes responsible for a wide range of molecular detoxications. The photoaffinity label S-(2-nitro-4-azidophenyl)glutathione was used to study the hydrophobic region of the active site of the rat liver GST 1-1 and 2-2 isoenzymes (class Alpha) as well as the rat class-Mu GST 3-3. Photoaffinity labelling was carried out using a version of S-(2-nitro-4-azidophenyl)glutathione tritiated in the arylazido ring.

Molecular design of DNA-directed ligands with specific interactions: solution NMR studies of the interaction of a m-hydroxy analogue of Hoechst 33258 with d(CGCGAATTCGCG)2.

We have used one-dimensional (1D) and two-dimensional (2D) proton nuclear magnetic resonance spectroscopy at 600 MHz for structural analysis of the complex formed between d(CGCGAATTCGCG)2 and 2-[2-(3-hydroxyphenyl)-6-benzimidazoyl]-6-(1-methyl-4-piperazinyl) benzimidazole (meta-Hoechst). This analogue differs from Hoechst 33258 only in the location of its meta rather than para phenolic hydroxyl group and was designed to introduce the possibility of intermolecular hydrogen bonding to DNA via the phenol. Complex formation was shown to be 1:1 at 25 degrees C in phosphate buffer in D2O by 1D NMR spectroscopic titration of a solution of d(CGCGAATTCGCG)2 with meta-Hoechst.

Ethnogeriatric assessment clinic in family medicine.

Background: The Department of Family Medicine at the University of California-Irvine needed a way to teach resident physicians care of ethnic elders and to provide specific training experiences in the use of geriatric assessment tools. The development of the ethnogeriatric assessment clinic presented the opportunity to do both.

Program Description: The clinic is part of a cross-cultural geriatrics curriculum spanning the 3-year residency program that includes didactic learning, clinical experience, and a research project.

Use of transferred nuclear-Overhauser-effect spectroscopy to measure the bound conformation of a disulphide-replaced analogue of glutathione disulphide as an inhibitor of yeast glutathione reductase.

The analogue of glutathione disulphide (GSSG) in which the disulphide bridge of GSSG is replaced by -CH2-S- was synthesised from L-cystathionine using t-butoxycarbonyl and t-butyl ester protection with triethylsilane-promoted deprotection. This analogue (GCSG) was found to be a linear, competitive inhibitor of yeast glutathione reductase (Ki value 981 microM at pH 7.0), a very poor substrate and not to act as an irreversible inhibitor of glutathione reductase.

Site-directed mutagenesis of glutamic acid 172 to glutamine completely inactivated human O6-alkylguanine-DNA-alkyltransferase.

DNA repair by O6-alkylguanine-DNA-alkyltransferase involves the stoichiometric transfer of the O6-alkyl group from the guanine lesion to the active-site cysteine residues of the protein. Site-directed mutagenesis of glutamic acid 172 of human O6-alkylguanine-DNA-alkyltransferase (EC 2.1.

A homology-derived structural model of the murine macrophage inflammatory protein, MIP-1 alpha.

Macrophage inflammatory protein 1 alpha (MIP-1 alpha), a monocyte cytokine, has roles postulated for it in neutrophil chemoattraction, the inflammatory response and the control of haemopoietic stem cell proliferation. The three-dimensional structure of MIP-1 alpha has been modelled structurally, based on its sequence similarity to interleukin-8 and related proteins. The predicted dimeric form of MIP-1 alpha contains two symmetry-related antiparallel alpha-helices lying at an angle across a beta-sheet.

The glutamyl binding site of trypanothione reductase from Crithidia fasciculata: enzyme kinetic properties of gamma-glutamyl-modified substrate analogues.

Trypanothione reductase, central to the redox defense systems of parasitic trypanosomes and leishmanias, is sufficiently different in its substrate-specificity from mammalian glutathione reductase to represent an attractive target for chemotherapeutic intervention. Previous studies of the physiological substrates trypanothione (N1,N8-bis(glutathionyl)spermidine) and N1-glutathionylspermidine disulphide established that the spermidine moiety of these substrates can be replaced by the 3-dimethyl-propylamide group (N1-glutathionyl-N3-dimethyl-propylamide). With this modification, the specificity for the gamma-glutamyl moiety of the substrate was examined.

A molecular model for cinnamyl alcohol dehydrogenase, a plant aromatic alcohol dehydrogenase involved in lignification.

The plant aromatic alcohol dehydrogenase, cinnamyl alcohol dehydrogenase (CAD2 from Eucalyptus) was found by sequence analysis of its cloned gene to be homologous to a range of dehydrogenases including alcohol dehydrogenases, L-threonine-3-dehydrogenase, D-xylose reductase and sorbitol dehydrogenase. A homology model of CAD2 was built using the X-ray crystallographic coordinates of horse-liver alcohol dehydrogenase to provide the template, with additional modelling input from other analogous regions of structure from similar enzymes where necessary. The structural model thus produced rationalised the Zn-binding properties of CAD2, indicated the possession of a Rossmann fold (GXGXXG motif), and explained the class A stereospecificity (pro-R hydrogen removal from substrate alcohol) and aromatic substrate specificity of the enzyme.

Invasive sinusitis with intracranial extension caused by Curvularia lunata.

Curvularia lunata has previously been linked to localized sinus infection in immunocompetent hosts. We treated a case of pansinusitis with extensive bone destruction and intracranial extension caused by C lunata. Curvularia lunata was identified when typical fungal macroconidia appeared on culture.

A direct link between LY83583, a selective repressor of cyclic GMP formation, and glutathione metabolism.

LY83583 (6-anilino-5,8-quinolinedione), considered to be a relatively specific repressor of cyclic GMP formation, is shown in the present study to inhibit (K(i) = 3 microM) glutathione reductase from bovine intestinal mucosa. As glutathione disulphide has been reported to inhibit guanylate cyclase irreversibly [Braughler, Biochem Pharmacol 32: 811-818, 1983], the inhibition of glutathione reductase should affect the activity of guanylate cyclase and may thus have physiological implications in the action of endothelium-derived relaxation factor and the design of muscle relaxants. LY83583 is reduced by NADPH and glutathione reductase in aerobic media and this may offer a route to the metabolic activation of LY83583.

Synthesis of substrate analogues for trypanothione reductase.

The synthesis and chemical characterisation of a range of substrate analogues for trypanothione reductase are described, with the spermidine portion of trypanothione replaced by the 3-dimethylaminopropylamide moiety. Using 1-hydroxybenzotriazole/N-hydroxysuccinimide coupling, products were obtained which had a range of replacements of the gamma-glutamyl groups of the enzyme substrate. The materials were characterised by FPLC, 1H/13C NMR spectroscopy and FAB mass spectroscopy.

Sequence-dependent reactivity of linear DNA to chemical cleavage by Cu(II):thiol combinations including cysteine or glutathione.

The chemical cleavage of linear DNA by Cu(II):thiol combinations was investigated using end-labelled double-stranded restriction fragments. Single-strand cleavage of the target DNA occurred with apparent sequence preference, but there appeared to be no apparent consensus sequence for such preferentially cleaved sites based on studies of three different restriction fragments. For any given restriction fragment, the observed Cu(II):thiol DNA-cleavage sequence preference was found to be independent of the nature of the thiol used.

Direct observation of defect structure in protein crystals by atomic force and transmission electron microscopy.

We have examined the structure of S-layers isolated from Sulfolobus acidocaldarius using atomic force microscopy (AFM) and transmission electron microscopy (TEM). From the AFM images, we were able to directly observe individual dimers of the crystal, defects in the crystal structure, and twin boundaries. We have identified two types of boundaries, one defined by a mirror plane and the other by a glide plane.

Rationally designed selective inhibitors of trypanothione reductase. Phenothiazines and related tricyclics as lead structures.

Trypanothione reductase, an essential component of the anti-oxidant defences of parasitic trypanosomes and Leishmania, differs markedly from the equivalent host enzyme, glutathione reductase, in the binding site for the disulphide substrate. Molecular modelling of this region suggested that certain tricyclic compounds might bind selectively to trypanothione reductase without inhibiting host glutathione reductase. This was confirmed by testing 30 phenothiazine and tricyclic antidepressants, of which clomipramine was found to be the most potent, with a K(i) of 6 microM, competitive with respect to trypanothione.

C-terminally truncated human O6-alkylguanine-DNA alkyltransferase retains activity.

A cDNA encoding the human O6-alkylguanine-DNA alkyltransferase (ATase; EC 2.1.1.

Alteration of enzyme specificity and catalysis.

In the past year, site-directed mutagenesis and other forms of protein engineering have been used to reverse the substrate specificity of several pairs of enzymes, including disulphide oxidoreductases, proteases, sugar-processing enzymes, and nucleases, as well as the specificity of hormones and their receptors. Mutations have been found that affect rate-determining steps, allowing normally transient intermediates to accumulate. Other mutations endow enzymes with totally new chemical reactions, and even novel biological functions.

Transfer of biologically derived nanometer-scale patterns to smooth substrates.

Atomic force microscopy has been used to measure the surface profile of a periodic array of 10-nanometer (nm)-diameter holes fabricated by fast-atom beam milling of a smooth graphite surface in which a 3.5-nm-thick titanium oxide screen was used as a mask. The nanostructured titanium oxide mask was itself derived from a protein crystal template.

Management of severe pre-eclampsia and eclampsia by UK consultants.

Objective: To determine the current management of severe pre-eclampsia and eclampsia in the United Kingdom.

Design: One-page postal survey to all (1007) UK consultant obstetricians with questions about use of antihypertensive and anticonvulsant drugs in severe pre-eclampsia and eclampsia, other management strategies, definition of factors determining severity, protocol development and regional review.

Results: 688 replies (69.

Synthesis and cytotoxicity of shikimate analogues. Structure:activity studies based on 1-crotonyloxymethyl-3R,4R,5R-trihydroxycyclohex-2-enone.

Syntheses are described for and structure:activity studies undertaken of the anti-tumour activity of (2-crotonyloxymethyl-(4R,5R,6R)-4,5,6-trihydroxycyclohex-2-+ ++enone) (1) and its analogues 1-crotonyloxymethyl-(3R,4S,5R)-3,4,5-trihydroxycyclohex-1-en e (3), 1-crotonyloxymethyl-(3R,4S,5S)-3,4,5-trihydroxycyclohexene (4) and 2-crotonyloxymethyl-2-cyclohexenone (5), which differ from 1 in the presence/absence of the cyclic keto group and/or the stereochemistry at one of the -OH bearing carbon atoms. None of the above compounds, including 1, directly inhibited glyoxalase I, isolated for the first time to homogeneity from rat Yoshida sarcomas and for which a purification protocol was developed. The apparent inhibition of glyoxalase I by 1 and 5 (but not detected for 4 or 3) could be explained by reaction of 1 and 5 with the glutathione present in the assay buffer and the consequent depletion of substrate.

Isolation and partial characterization of murine O6-alkylguanine-DNA-alkyltransferase: comparative sequence and structural properties.

A cDNA encoding murine O6-alkylguanine-DNA-alkyltransferase (ATase) has been sequenced after isolation from total liver RNA by the polymerase chain reaction using oligonucleotide primers derived from the rat ATase cDNA sequence. Functionally active murine ATase protein has been expressed in Escherichia coli at high levels (about 2% of total protein) and purified to apparent homogeneity (molecular mass 26 kDa). In liquid hybridization experiments, anti-human ATase polyclonal antibodies inhibited human but not rat or mouse ATase, whereas anti-rat polyclonal antibodies inhibited rat and mouse but not human ATase.