Removal of the projection domain of microtubule-associated protein 2 alters its interaction with tubulin

Microtubule-associated proteins (MAPs) can promote microtubule assemblyin vitro. One of these MAPs (MAP2) consists of a short promoter domain which binds to the microtubule and promotes assembly and a long projection domain which projects out from the microtubule and may interact wth other cytoskeletal elements. We have previously shown that MAP2 and another MAP, tau, differ in their interactions with tubulin in that tau, but not MAP2, promotes extensive aggregation of tubulin into spiral clusters in the presence of vinblastine and that microtubules formed with MAP2 are more resistant than those formed with tau to the antimitotic drug maytansine [Luduena, R. F.,et al. (1984),J. Biol. Chem. 259, 12890–12898; Fellous, A.,et al. (1985),Cancer Res. 45, 5004–5010]. Here we have used chymotryptic digestion to remove the projection domain of MAP2 and examined the interaction of the digested MAP2 (ctMAP2) with tubulin in the presence of vinblastine and maytansine. We have found that ctMAP2 behaves very much like tau, but not like undigested MAP2, in the presence of vinblastine, in that ctMAP2 causes tubulin to polymerize into large clusters of spirals. In contrast, microtubule assembly in the presence of ctMAP2 is much more resistant to maytansine inhibition than is assembly in the presence of tau or undigested MAP2. Our results suggest that the projection domain of MAP2 may play a role in the interaction of tubulin with MAP2 during microtubule assembly.Abbreviations MAPs microtubule-associated proteins - ctMAP2 MAP2 digested with-chymotrypsin - nMAP2 untreated MAP2 - PMSF phenylmethylsulfonyl fluoride - GMPCPP guanosine-5-(,-methylene)triphosphate     

Cu(II)–protamine interaction. II. The formation and structure of Cu(II) complexes of clupeine YII and of peptides mimicking clupeine N-terminals

The interaction of Cu(II) with the protamine clupeine YII (containing proline at the N-terminal) and with four peptides (H-Ala-Arg-OMe, H-Ala-Arg₂-OMe, H-Pro-Arg-OMe, and H-Arg₄-Tyr) has been studied by means of absorption, CD, and pH neasurements. The first two peptides mimic clupeine YI and Z N-terminals; the third, the clupeine YII N-terminal. At 1:1 molar ratio, clupeine YII yields two complexes: the first (I), at pH 6.6, through coordination via the N-terminal and the contiguous peptide nitrogen forming a five-membered chelate; the second (II), at pH 8.5, through the occupancy of the other two corners of the coordination square by amino nitrogens of the lateral chains. These complexes are strictly analogous and occur at the same pH as those formed with clupeine Z. Under the same conditions, all the peptides yield complex I in the first step, although the pH at which this complex is fully defined depends on the number of residues in the chain. It is 8.5 for dipeptides, decreases to 6.5 by the addition of a third residue to the chain, and remains constant when the number of residues is three or more. The amino nitrogens of lateral chains are unable to coordinate to the metal in a second step unless one additional peptide bond lies between the N-terminal residue and that containing the lateral chain bound to the metal. Thus, H-Ala-Arg-OMe and H-Pro-Arg-OMe form hydroxyl complexes in a second step (pH 11), by deprotonation of one of the water molecules coordinated to the metal; one of the lateral chains of H-Ala-Arg₂-OMe is able to coordinate in a second step (pH 8.5), but it is only with H-Arg₄-Tyr that a second complex (II) is obtained in which two amino nitrogens of lateral chains supersede the oxygens of water molecules in I, at pH 8.5.     

Circular dichroism evidence of the left-handed conformation adopted by cyclopropylpyrroloindole antitumor antibiotics in solution

The purpose of this work was to determine the conformation adopted in solution by adozelesin, carzelesin, and their derivatives obtained under basic or acidic conditions. Circular dichroism in the 270–330 nm wavelengths region was studied. In solution adozelesin can adopt two different conformations: a left-handed one which is thermodynamically favoured at low temperature and is mainly present at room temperature, and a right-handed one which is observed either at high temperature or at room temperature in the presence of DNA. This was ascertained by the presence of circular dichroism signals of the couplet type. The active form of carzelesin, i.e., U-76074, also exhibited a left-handed conformation in solution. Carzelesin and the derivatives obtained under acidic conditions that lack the cyclopropyl ring, and cyclopropylpyrroloindole, obtained under basic conditions, cannot adopt such a conformation. Chirality 8:585–589, 1996. © 1997 Wiley-Liss, Inc.     

Screening for complex carbohydrates inhibiting hemagglutinations by CFA/I- and CFA/II-expressing enterotoxigenic Escherichia coli strains

Abstract Numerous structural families of naturally occurring glycopeptides and oligosaccharides have been evaluated as potential inhibitors of hemagglutinations mediated by CFA/I- and CFA/II-positive enterotoxigenic Escherichia coli strains. Among the preparations tested were glycopeptides with short O-linked (mucin-type) chains, various mixtures containing N-linked glycans (either oligomannoside-, hybrid- or complex-type), three fractions of human milk oligosaccharides, and glycopeptides derived from either pooled new-born meconiums or pooled human red blood cell membranes. In almost all cases, the same inhibitory preparations were active toward all E. coli strains. This emphasizes the close analogy between the carbohydrate specificities of the colonization factors concerned. Such inhibitors always contained lactosamine units in their oligosaccharide backbones, but this structural requirement alone was not sufficient for activity. The glycopeptide mixture derived from human erythrocyte membranes (known to contain blood group-related carbohydrate antigens carried by a lactosaminoglycan backbone) behaved as a potent hemagglutination inhibitor, especially towards CFA/II-expressing strains. This last result clearly indicates the structural family in which complex carbohydrates should be selected to establish precisely the specificity of these CFA/II adhesins.     

Can laboratory-based tennis profiles predict field tests of tennis performance?

The present study examined the impact of physical characteristics of adolescent competitive tennis players (13-18 years) on field tests of tennis performance. Results (n = 33) showed that boys were taller (p = 0.001), possessed greater wingspan (p = 0.030), had greater maximum oxygen consumption (p = 0.001), and performed better on isokinetic strength measurements. Multiple regression analyses controlling for age and sex showed that height (p = 0.025), maximum minute ventilation (p = 0.005), and isokinetic strength measurements significantly and positively affected ball velocity. Knee extension average power was the only variable to positively and significantly affect ball placement (p = 0.040); however, several isokinetic strength measurements negatively affected ball placement. For the nondominant side, down-the-line strokes showed reduced accuracy (p = 0.001) and ball velocity (p = 0.001) compared with cross-court strokes. Given our results, resistance training may be beneficial for the performance of on-court tennis play.     

Epidemiology of Schistosomiasis and Usefulness of Indirect Diagnostic Tests in School-Age Children in Cubal,Central Angola

Introduction

Schistosomiasis remains a public health major problem and little is known in many areas, mainly in Sub-Saharan Africa

Objectives

To assess the burden and risk factors of schistosomiasis and intestinal parasitic helminthes in the children of Cubal, Angola, and to compare different diagnostic approaches for urinary schistosomiasis under field conditions.

Methods

A cross-sectional study was conducted. Urine and faeces samples of school children were microscopically studied. A random sample of children was obtained from an alphabetically arranged list of children, taking one of two children. Urine dipstick, colorimetric test and macrohaematuria were considered as indirect diagnostic methods and compared to direct urine examination. Possible risk factors for the infection were sex, age, distance to the river and previous treatment with praziquantel; the assessment was performed using Chi-square test.

Results

A total of 785 (61.18%) children showed S. haematobium eggs in urine; children living within 500 meters from the river had a higher odds for infection: Odds ratio 1.97 (1.45–2.7 CI 95%); urine dipstick showed sensitivity of 96% and specificity of 61.3%, with a positive predictive value; colorimetric test showed sensitivity of 52.5%, specificity of 74.6% and a positive predictive value of 77%. Proteinuria was present in 653 (51.1%) children, being more frequent in children with S. haematobium in urine (75.2%); 32 of 191 stool samples (16%) showed the presence of other intestinal parasites and 8 (4%) for S. haematobium.

Conclusions

Prevalence of urinary schistosomiasis in our study area is much higher than the national average, considering it as a high-risk community. Proximity to a source of water was a risk factor for the infection. Indirect tests, as urine dipstick and colorimetric test, were useful tools for diagnosis, due to ease of use and low cost. Proteinuria was a common finding, probably showing an early structural damage due to schistosomiasis in this group of children.     

Point Mutations in FimH Adhesin of Crohn's Disease-Associated Adherent-Invasive Escherichia coli Enhance Intestinal Inflammatory Response

Adherent-invasive Escherichia coli (AIEC) are abnormally predominant on Crohn''s disease (CD) ileal mucosa. AIEC reference strain LF82 adheres to ileal enterocytes via the common type 1 pili adhesin FimH and recognizes CEACAM6 receptors abnormally expressed on CD ileal epithelial cells. The fimH genes of 45 AIEC and 47 non-AIEC strains were sequenced. The phylogenetic tree based on fimH DNA sequences indicated that AIEC strains predominantly express FimH with amino acid mutations of a recent evolutionary origin - a typical signature of pathoadaptive changes of bacterial pathogens. Point mutations in FimH, some of a unique AIEC-associated nature, confer AIEC bacteria a significantly higher ability to adhere to CEACAM-expressing T84 intestinal epithelial cells. Moreover, in the LF82 strain, the replacement of fimH _LF82 (expressing FimH with an AIEC-associated mutation) with fimH _K12 (expressing FimH of commensal E. coli K12) decreased the ability of bacteria to persist and to induce severe colitis and gut inflammation in infected CEABAC10 transgenic mice expressing human CEACAM receptors. Our results highlight a mechanism of AIEC virulence evolution that involves selection of amino acid mutations in the common bacterial traits, such as FimH protein, and leads to the development of chronic inflammatory bowel disease (IBD) in a genetically susceptible host. The analysis of fimH SNPs may be a useful method to predict the potential virulence of E. coli isolated from IBD patients for diagnostic or epidemiological studies and to identify new strategies for therapeutic intervention to block the interaction between AIEC and gut mucosa in the early stages of IBD.     

Electron spin resonance detection of extracellular superoxide anion released by cultured endothelial cells

Objective and Methods Endothelium produces oxygen-derived free radicals which play a major role in vessel wall physiology and pathology. Whereas NO^· production from endothelium has been extensively characterized, little is known about endothelium-derived O^-·₂. In the present study, we determined the O^-·₂ production of bovine aortic endothelial cells (BAEC) using the spin trap 5,5-dimethyl-1 pyrroline-N-oxide (DMPO) and electron spin resonance (ESR) spectroscopy.

Results An ESR adduct DMPO-OH detected in the supernatant of BAEC after stimulation with the calcium ionophore A23187 originated from the trapping of extracellular O^-·₂, because coincubation with superoxide dismutase (30 U/ml) completely suppressed the ESR signal, whereas catalase (2000 U/ml) had no effect. A23187 stimulated extracellular O^-·₂ production in a time- and dose-dependent manner. The coenzymes NADH and NADPH both increased the ESR signal, whereas a flavin antagonist, diphenylene iodonium, abolished the ESR signal. Phorbol myristate acetate potentiated, whereas bisindolylmaleimide I inhibited the A23187-stimulated O^-·₂ production, suggesting the involvement of protein kinase C. These signals were not altered L-NAME, a NO-synthase inhibitor, suggesting that the endogenous production of NO^· did not alter O^-·₂ production. Finally, the amount of O^-·₂ generated by A23187-stimulated post-confluent BAEC was one order of magnitude higher than that evoked by rat aortic smooth muscle cells stimulated under the same conditions.