Solution structure of the superactive monomeric des-[Phe(B25)] human insulin mutant: elucidation of the structural basis for the monomerization of des-[Phe(B25)] insulin and the dimerization of native insulin

The three-dimensional solution structure of des-[Phe(B25)] human insulin has been determined by nuclear magnetic resonance spectroscopy and restrained molecular dynamics calculations. Thirty-five structures were calculated by distance geometry from 581 nuclear Overhauser enhancement-derived distance constraints, ten phi torsional angle restraints, the restraints from 16 helical hydrogen bonds, and three disulfide bridges. The distance geometry structures were optimized using simulated annealing and restrained energy minimization. The average root-mean-square (r.m.s.) deviation for the best 20 refined structures is 1.07 angstroms for the backbone and 1.92 angstroms for all atoms if the less well-defined N and C-terminal residues are excluded. The helical regions are more well defined, with r.m.s. deviations of 0.64 angstroms for the backbone and 1.51 angstroms for all atoms. It is found that the des-[Phe(B25)] insulin is a monomer under the applied conditions (4.6 to 4.7 mM, pH 3.0, 310 K), that the overall secondary and tertiary structures of the monomers in the 2Zn crystal hexamer of native insulin are preserved, and that the conformation-averaged NMR solution structure is close to the structure of molecule 1 in the hexamer. The structure reveals that the lost ability of des-[Phe(B25)] insulin to self-associate is caused by a conformational change of the C-terminal region of the B-chain, which results in an intra-molecular hydrophobic interaction between Pro(B28) and the hydrophobic region Leu(B11)-Leu(B15) of the B-chain alpha-helix. This interaction interferes with the inter-molecular hydrophobic interactions responsible for the dimerization of native insulin, depriving the mutant of the ability to dimerize. Further, the structure displays a series of features that may explain the high potency of the mutant on the basis of the current model for the insulin-receptor interaction. These features are: a change in conformation of the C-terminal region of the B-chain, the absence of strong hydrogen bonds between this region and the rest of the molecule, and a relatively easy accessibility to the Val(A3) residue.     

The prognostic value of NCAM, p53 and cyclin D1 in resected non-small cell lung cancer

Expression of the neural cell adhesion molecule, NCAM, in frozen sections has been associated with decreased postoperative survival in non-small cell lung carcinoma. Of the various isoforms of NCAM described, the highly sialylated isoform plays a role in the migration of embryonal cells from the neural crest and is expressed by highly malignant tumours such as small cell lung carcinomas. We investigated the clinical significance of expression of this NCAM isoform as a prognostic factor in a series of 96 non-small cell lung carcinomas resected with curative intent. We also evaluated the effect of microwave pre-treatment of formalin-fixed, paraffin-embedded sections on the NCAM immunostaining and related the outcome to the postoperative clinical course of disease. In addition, in an attempt to extend our search for possible molecular markers of unfavourable prognosis in lung cancer, we evaluated increased immunostaining for p53 and cyclin D1 in the same series. We did not find a significant relation between expression of NCAM or its highly sialylated isoform and the length of postoperative survival. The numbers of positive cases (9 and 14, respectively) were relatively low. Increased p53 and cyclin D1 immunostaining (50 and 55 of the 96 tumours) failed to show a significant relation with postoperative survival. In our material, tumour stage was the only significant prognostic factor.     

Poly(A) metabolism in Xenopus laevis embryos: substrate-specific and default poly(A) nuclease activities are mediated by two distinct complexes

The metabolism of the poly(A) tail is a process important for the translational regulation of maternal mRNAs in Xenopus laevis oocytes and early embryos. Two poly(A) nuclease (PAN) activities have been described in Xenopus embryo or activated egg extracts (Legagneux et al (1995) RNA 1, 1001-1008). These activities (default PAN and EgPAN) are distinguishable by their deadenylation kinetics and their substrate specificities. In this report, we show that these activities display different sensitivities to biochemical treatments. Urea and, to a lesser extent, spermidine, inhibit EgPAN at concentrations which have no effect on default PAN. Heparin activates default PAN but inhibits EgPAN. When extracts are fractionated by ultracentrifugation, the default activity is recovered in one unique fraction, whereas two fractions must be combined to reconstitute the EgPAN activity. Moreover, these two deadenylation activities are separable by size exclusion chromatography under native conditions. We conclude that these two deadenylation activities are mediated by two protein complexes.     

Kinetic analysis of the coding properties of O6-methylguanine in DNA: the crucial role of the conformation of the phosphodiester bond

Production by N-nitroso compounds of O6-alkylguanine (O6-alkylG) in DNA directs the misincorporation of thymine during DNA replication, leading to G:C to A:T transition mutations, despite the fact that DNA containing O6-alkylG:T base pairs is less stable than that containing O6-alkylG:C pairs. We have examined the kinetics of incorporation by Klenow fragment (KF) of Escherichia coli DNA polymerase I of thymine (T) and of cytosine (C) opposite O6-MeG in the template DNA strand. Both T and C were incorporated opposite O6-MeG much slower than nucleotides forming regular A:T or G:C base pairs. Using various concentrations of dTTP, dCTP, or their phosphorothioate (Sp)-dNTP alpha S analogues, or a mixture of dTTP and dCTP, the progress of incorporation of a single nucleotide in a single catalytic cycle of a preformed KF-DNA complex was measured (pre-steady-state kinetics). The results were consistent with the kinetic scheme (Kuchta, R. D., Benkovic, P., & Benkovic, S. J. (1988) Biochemistry 27, 6716-6725): (1) binding of dNTP to polymerase-DNA; (2) conformational change in polymerase; (3) formation of phosphodiester between the dNTP and the 3'-OH of the primer; (4) conformational change of polymerase; (5) release of pyrophosphate. The results were analyzed mathematically to identify the steps at which the rate constants differ significantly between the incorporation of T and C. The only significant difference was the 5-fold difference in the rates of formation of the phosphodiester bond (for dTTP, kforward = 3.9 s-1 and kback = 1.9 s-1; for dCTP, kforward = 0.7 s-1 and kback = 0.9 s-1). These pre-steady-state progress curves were biphasic with a rapid initial burst followed by an apparently steady-state rise. Deconvolution of these curves gave direct evidence for the importance of the conformational change after polymerization by showing that the curves represented the sum of the rapid accumulation of the product of step 3 followed by the slow conversion of that to the product of step 5 (because of the rapidity of the release of pyrophosphate there was no significant accumulation of the product of step 4). The equilibrium constants for each step suggest that the greatest change in the Gibbs free energy occurs at the conformational change after polymerization and that while the formation of the phosphodiester bond to T is slightly exothermic, that to C is slightly endothermic.(ABSTRACT TRUNCATED AT 400 WORDS)     

3-Alkanoyl-5-hydroxymethyl tetronic acid homologues and resistomycin: new inhibitors of HIV-1 protease. I. Fermentation, isolation and biological activity

In the course of a screening program for HIV-1 protease inhibiting activity, six new homologues of 3-alkanoyl-5-hydroxymethyl tetronic acids (1 approximately 6) and the known natural product resistomycin (7) were isolated from cultures of the Actinomycete strain DSM 7357. The substituted tetronic acids belong to a recently described structural class of secondary metabolites. The HIV-1 activity of resistomycin (7) has not been reported before.     

Magnetic resonance imaging of disseminated sclerosis

Multiple sclerosis is a chronic demyelinating disease. Paraclinical examinations may contribute to the diagnosis of multiple sclerosis. Magnetic resonance imaging (MRI) has a very high sensitivity concerning multiple sclerosis, and has made it possible to visualize multiple sclerosis plaques in vivo, to follow each plaque over the course of time and in this way to obtain information about the pathogenesis. MRI has shown that the size of plaques may vary considerably, and that plaques are dynamic structures with the ability to change in size over few weeks. By using MRI and the contrast agent Gadolinium-DTPA, it is possible to distinguish a newly developed plaque from an older one. Therefore, MRI has become an important examination in therapeutic trials. Just now, MRI with Gadolinium-DTPA is being used to evaluate the efficacy of plasmapheresis and immunoglobulin treatment in a joint study between Rigshospitalet and Hvidovre Hospital.     

Changing clinical practice. Prospective study of the impact of continuing medical education and quality assurance programs on use of prophylaxis for venous thromboembolism

OBJECTIVE: To determine the effect of continuing medical education (CME) with and without a quality assurance component (CME+QA) on physician practices in the prevention of venous thromboembolism. METHODS: A communitywide study was performed in 15 short-stay hospitals in central Massachusetts. The study population included 3158 patients in acute-care hospitals with multiple risk factors for venous thromboembolism. Study hospitals were randomly assigned to one of two educational strategies or to a control group that received no intervention. RESULTS: The proportion of patients at high risk for venous thromboembolism who received effective methods of prophylaxis increased significantly from 29% in 1986 to 52% in 1989 (P < .001). This increase was seen in all study groups: control hospitals, 40% to 51% (P < .001); CME hospitals, 21% to 49% (P < .0001); and CME+QA hospitals, 27% to 55% (P < .0001). The increase in prophylaxis use from 1986 to 1989 was significantly greater among patients cared for in hospitals whose physicians participated in a formal CME program (an increase of 28%) than in control hospitals (an increase of 11%) (P < .001). There was no significant difference in the use of prophylaxis in hospitals whose physicians received CME+QA interventions compared with hospitals whose physicians received CME interventions alone (identical increases of 28%). CONCLUSION: A formal CME program significantly increased the frequency with which physicians prescribed prophylaxis for venous thromboembolism. We believe the key factor in our CME interventions that motivated clinicians to change their practices was the provision of hospital-specific data demonstrating a compelling need for improvement. Despite the substantial investment by hospitals in QA, traditional QA intervention appeared to provide no additional benefit. Even after extensive CME/QA interventions, prophylaxis for venous thromboembolism remained underutilized, suggesting the need to develop new approaches to changing clinical practice.