Biomedical subjects
Publications and source records attributed to H J Martin.
[reaction: see text] Here we describe the proline-catalyzed Michael addition of unmodified ketones to nitro olefins. This novel reaction provides gamma-nitro ketones in modest enantioselectivity yet excellent yields.
A highly convergent and stereocontrolled synthesis of epothilone B (1) has been developed. The epoxide moiety in 1 was generated by regioselective mesylation and base treatment of the 12,13-diol 30 which was formed by a chelate Cram controlled Grignard addition of 14 and methyl ketone 13. Both fragments were synthesized from the chiral carbon pool precursors (S)-citronellol and (S)-lactic acid, respectively. A highly diastereoselective aldol addition of epoxy-aldehyde 7 and the known Southern hemisphere ketone 8 delivered the full carbon skeleton, containing all the stereogenic centers of 1. Functional group manipulation, macrolactonization and removal of two protecting groups then yielded 1. The spatial closeness of the C4-beta-methyl and C6-methyl group in the crystal structure of 1 inspired us to connect them through a methylene bridge to give a cyclohexanone derivative. Thus, the Northern hemisphere aldehyde 7 was added to the enolate of the cyclohexanone 47. Further manipulations and macrolactonization delivered the conformationally restrained epothilone derivative 42.
11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD 1) is a microsomal enzyme responsible for the reversible interconversion of active 11beta-hydroxyglucocorticoids into inactive 11-ketosteroids and by this mechanism regulates access of glucocorticoids to the glucocorticoid receptor. The enzyme has also been proven to participate in xenobiotic carbonyl compound detoxification. 11beta-HSD 1 is anchored within the membranes of the endoplasmic reticulum (ER) by its N-terminus, whereby its active site protrudes into the lumen of the ER. In the primary structure of 11beta-HSD 1 three Asn-X-Ser glycosylation motifs have been identified. However, the importance of N-linked glycosylation of 11beta-HSD 1 for catalytic activity has been controversely discussed. To clarify if glycosylation is essential for enzyme activity, we performed deglycosylation experiments of native 11beta-HSD 1 from human liver as well as site-directed mutagenesis to remove potential glycosylation sites upon overexpression in Pichia pastoris. The altered proteins were examined regarding their catalytic activity towards their physiological glucocorticoid substrates. The molecular size of the various 11beta-HSD 1 forms was analyzed by immunoblotting with a polyclonal antibody raised against 11beta-HSD 1 protein from human liver. By stepwise enzymatic deglycosylation of native 11beta-HSD 1 we could demonstrate that all potential glycosylation sites carry N-linked oligosaccharide residues under physiological conditions. Interestingly, complete deglycosylation did not affect enzyme activity, neither in the reductive (cortisone) nor in the oxidative (cortisol) direction. Upon overexpression in the yeast P. pastoris, 11beta-HSD 1 did not undergo glycosylation, but, in spite of this, yielded a fully active enzyme. Our results conclusively demonstrate that 11beta-HSD 1 does not need to be glycosylated to perform its physiological role as glucocorticoid oxidoreductase.
Detoxification of aldehydes and ketones generally proceeds via reduction to their corresponding alcohols, which are then conjugated and eliminated. We focused our interest on 11beta-hydroxysteroid-dehydrogenase type 1 (11beta-HSD 1), a pluripotent enzyme which physiologically performs the interconversion of active and inactive glucocorticoid hormones, and which also participates in xenobiotic carbonyl compound detoxification. 11beta-HSD 1 belongs to the protein superfamily of the short-chain dehydrogenases/reductases (SDR), and has been structurally and functionally characterized. 11beta-HSD 1 is a glycosylated membrane protein which is very difficult to purify in an active state. In addition, expression levels in humans differ in a wide range. In order to facilitate biochemical and molecular studies on the significance of human 11beta-HSD 1 in detoxification processes, we have successfully performed the overexpression of recombinant human 11beta-HSD 1 in the yeast Pichia pastoris and in Escherichia coli. Recombinant 11beta-HSD 1 from E. coli was purified to homogeneity and used to generate a polyclonal antibody. The enzyme had no enzymatic activity, possibly due to the lack of glycosylation and/or incorrect folding in E. coli. In contrast, 11beta-HSD 1 overexpressed in P. pastoris was enzymatically active towards its physiological glucocorticoid substrates as well as towards xenobiotic carbonyl compounds. In western blot experiments the antibody crossreacted with both recombinant 11beta-HSD 1 forms and with the native enzyme from mouse and human liver. In conclusion, recombinant 11beta-HSD 1 from P. pastoris serves as a valuable tool for future studies on carbonyl compound detoxification.
BACKGROUND & AIMS: Vitamins A and E are the most light-sensitive vitamins. Vitamin A is degraded by photolysis, while vitamin E degrades by photo-oxidation. The composition of the parenteral nutrition mixture and the container could therefore influence degradation during daylight administration. The aim of this study was therefore to determine the influence of fat emulsion and the type of bag on the photo-degradation of vitamins A and E in Parenteral Nutrition (PN) mixtures during simulated infusion in daylight. METHODS: Representative adult PN mixtures, with and without fat emulsion, were prepared. Samples for analysis were taken from infusates and each bag during simulated infusion. Degradation of vitamins A and E was determined by stability-indicating HPLC analysis. RESULTS: Results indicated that vitamin A loss proceeded rapidly during infusion, resulting in up to 80% loss in 6 hours, even with light protection of the bag. The presence of fat emulsion did not provide significant light protection. Vitamin E degradation was substantial if mixtures were prepared in EVA bags but was largely prevented if PN mixtures were compounded and stored in multi-layered bags. CONCLUSIONS: It is recommended that all PN bags should be light-protected during infusion in daylight. The use of multi-layered bags will prevent vitamin E losses during infusion.
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The analysis of initial glucose uptake in Saccharomyces cerevisiae at 25 degrees, 20 degrees, 15 degrees and 10 degrees C by computer-assisted nonlinear regression analysis predicts two transport systems. The first demonstrates Michaelis-Menten kinetics and the second shows first order behaviour. The activation energies of these two systems were calculated by the Arrhenius equation at four different growth phases, namely early exponential (EE), middle exponential (ME2), late exponential (LE) and early stationary (ES) with 2% glucose in the batch medium. The activation energies calculated from the V(m) values in EE, ME, LE and ES growth phases were 15.8 +/- 1.7, 13.5 +/- 1.0, 15.1 +/- 0.8 and 13.5 +/- 0.7 kcal/mol. These values are in agreement with activation energies calculated for the first mechanism, facilitated diffusion, which is the mechanism deduced from countertransport experiments. The activation energies derived for the second transport system from the first order rate constants in cells grown to EE, ME2, LE and ES were 8.0 +/- 2.1, 8.1 +/- 1.3, 9.6 +/- 3.0 and 7.5 +/- 2.6 kcal/mol. These values are still significantly higher than for free diffusion of glucose in water and lower as predicted for passage of glucose through the lipid phase. Therefore, we assume in addition to carrier-mediated facilitated diffusion the entrance of glucose into the cell through a pore.
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The stability of cimetidine (Tagamet) was investigated in total parenteral nutrition (TPN) mixtures containing different amino acid sources. TPN mixtures were stored at 5 degrees C in ethylvinyl acetate bags for 28 days and analysed by stability-indicating high-pressure liquid chromatography. Results indicated that cimetidine was physically compatible and chemically stable (less than 5% degradation) for at least 28 days in TPN mixtures containing either Freamine III, Vamin 14 or Aminoplex 12 as the amino acid source.
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It has been asserted that physicians hold the key to success in hospital efforts to increase admissions and contain costs. While there is a great need to forge a partnership with the medical staff in achieving mutual goals, little is known about what physicians want from hospitals. A survey was completed by 177 physicians in two Cleveland-area hospitals that assessed their preferences on issues concerning hospital governance and control, hospital services, and hospital employment of physicians. Results showed that physicians want greater involvement in hospital decision making and desire services that facilitate their practice of medicine. Responses varied significantly according to physician age, specialty, HMO participation, and multiple-staff membership. Greater effort on the part of hospital administrators to assess and understand medical staff needs is suggested.
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A case of astereognosis and amyotrophy of the right hand is reported, which was caused by a large extradural neurinoma of the C2 nerve root. The quick and complete recovery which followed surgery suggested a vascular mechanism as the cause of the neurological deficits.
In a neonate who had seizures in the second day of life, the EEG showed - during waking and sleep alike - periodic bursts of asymmetrical high voltage on a low amplitude background tracing. From the third week head jerks occurred, followed by repetitive spasms and then adversive tonic seizures. A severe psychomotor retardation was noted. A CT examination demonstrated a right hemispheric hypertrophy. The pathological examination revealed astrocytic gliosis of the cortex associated with a decrease in the oxidative enzyme activity of neurons, and characterized electron microscopically, by the presence of spheroid bodies in the neuropile. This case was different from those published by Aicardi and Goutières (encéphalopathie myoclonique néo-natale) because there was no erratic myoclonus. It is more similar to the 'early infantile epileptic encephalopathy with suppression bursts' described by Ohtahara. This electro-clinical condition should be characterized as the earliest type of secondary generalized epilepsy.
Chesney and Tasto's (1975a) Menstrual Symptom Questionnaire (MSQ) was developed on the basis of Dalton's theory of spasmodic and congestive dysmenorrhea, which states that the two types are caused by opposite hormonal imbalances and therefore should not be found in the same woman. Evidence is offered which suggests that the theory is incorrect. A multiple-group factor analysis failed to adequately support the two-component model. Subsequently, a factor analysis of 275 MSQs revealed seven factors rather than the two predicted by the theory. In addition, the incidence of both high premenstrual and menstrual symptoms in the same women was demonstrated. Finally, symptoms reported by women using oral contraceptives differed little from those reported by women not using oral contraceptives. It is concluded that scoring for the MSQ should be modified to include two subscales for premenstrual and menstrual symptoms, and that the labels "spasmodic" and "congestive" be discontinued until some evidence is found for their support.
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