Mammalian protein methylesterase. Physical and enzymic properties.

Protein methylesterase (PME) amino acid composition and substrate specificity towards methylated normal and deamidated protein substrates were investigated. The enzyme contained 23% acidic and 5% basic residues. These values are consistent with a pI of 4.45. The product formed from methylated protein by PME was confirmed as methanol by h.p.l.c. The kcat. and Km values for several methylated protein substrates ranged from 20 x 10(-6) to 560 x 10(-6) s-1 and from 0.5 to 64 microM respectively. However, the kcat./Km ratios ranged within one order of magnitude from 11 to 52 M-1.s-1. Results with the irreversible cysteine-proteinase inhibitor E-64 suggested that these low values were in part due to the fact that only one out of 25 molecules in the PME preparations was enzymically active. When PME was incubated with methylated normal and deamidated calmodulin, the enzyme hydrolysed the latter substrate at a higher rate. The Km and kcat. for methylated normal calmodulin were 0.9 microM and 31 x 10(-6) s-1, whereas for methylated deamidated calmodulin values of 1.6 microM and 188 x 10(-6) s-1 were obtained. The kcat./Km ratios for methylated normal and deamidated calmodulin were 34 and 118 M-1.s-1 respectively. By contrast, results with methylated adrenocorticotropic hormone (ACTH) substrates indicated that the main difference between native and deamidated substrates resides in the Km rather than the kcat. The Km for methylated deamidated ACTH was 5-fold lower than that for methylated native ACTH. The kcat./Km ratios for methylated normal and deamidated ACTH were 43 and 185 M-1.s-1 respectively. These results indicate that PME recognizes native and deamidated methylated substrates as two different entities. This suggests that the methyl groups on native calmodulin and ACTH substrates may not be on the same amino acid residues as those on deamidated calmodulin and ACTH substrates.     

Research perspectives and the anomalous status of modern ecology

Ecology has often been characterized as an immature scientific discipline. This paper explores some of the sources of this alleged immaturity. I argue that the perception of immaturity results primarily from the fact that historically ecologists have based their work upon two very different approaches to research.     

Determination of the enantiomeric composition of (R*,S*)-1-ethylpropyl 2-methyl-3-hydroxypentanoate, the male-produced aggregation pheromone of Sitophilus granarius

The enantiomeric composition of sitophilate, the granary weevil [Sitophilus granarius (L.)] male-produced aggregation pheromone [(R^*,S^*)-1-ethylpropyl 2-methyl-3-hydroxypentanoate)], was determined by three methods: (1) bioassaying the synthetic (2S,3R) and (2R,3S) enantiomers of the active (R^*,S^*) diastereomer; (2) ¹H NMR spectroscopy of Mosher ester derivatives of the natural pheromone and synthetic (2S,3R)-and (2R,3S)-sitophilate; and (3) capillary GLC comparisons of the retention times of derivatized natural pheromone and the two synthetic enantiomers. The combined methods confirmed the (2S,3R) enantiomer as the active form of sitophilate. Male granary weevils were shown to produce >96% (2S,3R)-sitophilate. No significant attraction of S. granarius by the (2R,3S) enantiomer was observed. Rice and maize weevils [S. oryzae (L.) and S. zeamais Motschulsky] were not attracted by (2S,3R)-sitophilate. S. granarius L. est un déprédateur important des grains stockés. Le (R^*,S^*)-1-éthylpropyl 2-méthyl-3-hydroxypentanoate a été identifié en 1987 comme le principal composé du sitophilate, la phéromone mâle d'agrégation de S. granarius. La composition énantiométrique du sitophilate a été déterminée par 3 méthodes:

Marfan syndrome: exclusion of genetic linkage to five genes coding for connective tissue components in the long arm of chromosome 2

Summary Marfan syndrome represents a heterogeneous connective tissue disease, the symptoms arising in several tissues and organs. The defective gene(s) behind this autosomal dominant condition has not been found despite considerable research. The main targets of the research have been the genes coding for connective tissue components. Several of the candidate genes suspected to be defective in Marfan syndrome are located on the long arm of chromosome 2. These genes include a cluster of two genes coding for fibrillar collagens COL3A1 and COL5A2, and a third member of the collagen gene family: COL6A3. Furthermore, genes for elastin (ELN) and fibronectin (FN) are also located in this area of chromosome 2. We studied this chromosomal area using restriction fragment length polymorphism (RFLP) linkage analysis in five Finnish Marfan families with affected members in three generations. In two point linkage analyses, Lod scores of –3.192 ( = 0.1) to COL3A1, –1.683 ( = 0) to COL6A3 and –2.664 ( = 0.01) to FN were obtained, whereas the linkage analysis between elastin and the disease was non-informative (Lod score 0.444, = 0). With the multipoint linkage analysis that permits simultaneous examination of several loci and more efficient use of family data, we obtained an exclusion of all these loci as the site of the mutation leading to Marfan syndrome in these families.     

Squid glycoproteins with structural similarities to Thy-1 and Ly-6 antigens

In an attempt to identify invertebrate homologs of Thy-1 antigen, the optic and central nervous tissue of squid was solubilized in deoxycholate and fractionated by lentil lectin affinity chromatography and gel filtration to yield small abundant glycoproteins. Material with biochemical similarities to Thy-1 was found and shown to consist of two glycoproteins that were ultimately purified using monoclonal antibody affinity columns. Both glycoproteins were sequenced to yield sequences of 84 residues for Sgp-1 and 92 residues for Sgp-2. The sequences were analyzed for similarities to Thy-1 and other Ig-related sequences, and Sgp-1 showed some similarities that were > 3 standard deviation units away from mean random scores when tested with the ALIGN program. However, the sequence patterns were not typical of Ig-related domains and the relationship of Sgp-1 to the Ig superfamily remains problematical. Sgp-2 showed no relationship to the Ig superfamily, but similarities to Ly-6 antigen sequences were noted that are in accord with an evolutionary relationship. The similarities included ten Cys residues in each sequence of which eight were matched in the best alignment given by the ALIGN program. Chemical evidence was obtained for glycophospholipid tails at the COOH-termini of Sgp-1 and Sgp-2 as is the case for Thy-1 and Ly-6 antigens.Abbreviations used in this paper GPL glycophospholipid - mAb monoclonal antibody - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Subcellular Distribution of UDP-Galactose:Ceramide Galactosyltransferase in Rat Brain Oligodendroglia

Oligodendrocytes isolated from 18-19-day-old rat brain were homogenized in 0.32 M sucrose. The homogenate was centrifuged at 100,000 g for 50 min in a gradient containing 0.8, 1.05, and 1.3 M sucrose. Three discrete bands were obtained at the interfaces 0.32-0.8 (F1), 0.8-1.05 (F2), and 1.05-1.3 M (F3). The distribution of UDP-galactose:ceramide galactosyltransferase (CgalT) activity in each fraction was measured using liposomes containing normal fatty acid-containing ceramides (NFA-CgalT activity) or 2-hydroxy fatty acid-containing ceramides (HFA-CgalT activity). Although detection of both CgalT activities was possible in all fractions, HFA-CgalT activity was enriched in F1 and F2 fractions, which also showed an enrichment of Golgi and endoplasmic reticulum markers, respectively. It is interesting that NFA-CgalT activity was significantly enriched in the F2 fraction. These results suggest that hydroxylated and nonhydroxylated galactocerebrosides may be synthesized at different intracellular locations.     

How can the functional reponse best be determined?

Summary We evaluated three methods for the analysis of functional response data by asking whether a given method could discriminate among functional responses and whether it could accurately identify regions of positive density-dependent predation. We evaluated comparative curve fitting with foraging models, linear least-squares analysis using the angular transformation, and logit analysis. Using data from nature and simulations, we found that the analyses of predation rates with the angular transformation and logit analysis were best at consistently determining the true functional response, i.e. the model used to generate simulated data. These methods also produced the most accurate estimates of the true regions of density dependence. Of these two methods, functional response data best fulfill the assumptions of logit analysis. Angularly transformed predation rates only approximate the assumptions of linear leastsquares analysis for predation rates between 0.1 and 0.9. Lack-of-fit statistics can reveal inadequate fit of a model to a data set where simple regression statistics might erroneously suggest a good match.     

Death and brain death: a new formulation for Canadian medicine. Canadian Congress Committee on Brain Death.

Between June 1984 and December 1986, 35 patients with acute myocardial infarction received streptokinase intravenously within 3 hours after the beginning of chest pain and underwent percutaneous transluminal coronary angioplasty (PTCA) either immediately (in 2 cases) or 1 to 19 (mean 4.4) days later (in 33). The rate of successful PTCA was 89%. Reocclusion occurred in one patient. The mean percentage of stenosis decreased from 86% to 11%. The mean trans-stenotic gradient was reduced from 41 to 11 mm Hg. The results suggest that in patients whose condition is stable, PTCA performed a few days after thrombolysis is a valuable alternative to more aggressive treatment with immediate PTCA.     

Transthyretin Pro55, a variant associated with early-onset,aggressive, diffuse amyloidosis with cardiac and neurologic involvement

Summary Mutations in the protein transthyretin cause amyloidosis involving the heart, peripheral nerves, and other organs. A family from West Virginia developed an unusually aggressive form of widespread transthyretin amyloidosis. Single-strand conformation polymorphism analysis revealed a variant in the transthyretin gene, which was found on sequencing to be a TC transversion at position 2 of codon 55, corresponding to a Leu Pro substitution. The variant sequence was confirmed by restriction analysis and polymerase chain reaction (PCR)-primer introduced restriction analysis.     

A semiempirical study of the prototropic tautomerism of hypoxanthine

The total and relative energies, bond order matrices and localized MOs for the eight possible tautomers of hypoxanthine (HYP) have been calculated, with full geometry optimization, using both AM1 and MNDO methods. The AM1 relative energies show that HYP(9,1), HYP(7,1) and HYP (9,10) are the predominant species at room temperature, the two former being in larger concentration that the latter. The calculated IR spectra for these species agree well with the reported spectrum in an isolated matrix, which has been interpreted in terms of the presence of these three tautomeric forms. The MNDO method does not predict the right order, and the more stable tautomer would be HYP(9,10). The calculated structure for the HYP(9,1) species shows that the molecule is essentially planar. The bond distances compare well with those of hypoxanthine hydrochloride and guanine and also correlate well with the calculated bond orders. The proton affinities for the three more stable tautomers have also been calculated. For HYP(9,1) the prefered site of protonation is N7, whereas for HYP(7,1) the protonation occurs rather at N9. These results agree well with¹⁵N and¹³C NMR studies in DMSO.