Conformational stability of antamanide and analogs Crystal structure of perhydrosymmetric antamanide,cyclic (-Val-Pro-Pro-Cha-Cha-Val-Pro-Pro-Cha-cha-)

The synthetic perhydrogenated symmetric analog of the cyclic decapeptide antamanide is biologically inactive, although the conformation of the molecule and the crystal packing are very similar to that of the active symmetric analog of antamanide. In fact, the same conformation for the molecule has now been found in six polymorphs of uncomplexed antamanide and its analogs. The differences between the active and inactive antamanide analogs are displayed dramatically in the conformations of their metal ion (Na⁺ or Li⁺) complexes, thus suggesting strongly that for physiological activity antamanide is not in the conformation assumed by the uncomplexed molecule, but rather in the conformation assumed by the complexed state of natural antamanide. The present structure crystallizes in space group P2₁2₁2₁ with a = 20.515(14) Å, b = 21.316(16) Å, c = 17.128(16) Å and four peptide molecules in the unit cell. There are three cocrystallized water molecules at full occupancy intrinsic to the peptide, and several more water molecules or other solvent molecules at partial occupancy. The formula of the peptide is C₆₆ H₁₀₆ N₁₀O₁₀· 4-H₂O·2X.     

Antibodies to Plasmodium falciparum gamete surface antigens in Papua New Guinea sera

Sera from individuals living in malaria endemic areas of Papua New Guinea were tested for their effect on infectivity of Plasmodium falciparum gametocytes grown in culture to Anopheles freeborni mosquitoes. Consistent reduction of infectivity to less than 5% of control was observed with nine out of the 41 sera from the endemic area tested and also with three out of seven sera tested from individuals rarely exposed to malaria infection. Gamete surface antigens recognized by the sera were investigated by immunoprecipitation from 125I surface-labelled gametes extracted in SDS and Triton X-100. The main antigens recognized were of the same mol. wt (230, 48 and 45 kD) as those known to be targets of transmission-blocking monoclonal antibodies. A significant negative correlation was observed between the total ct/min immunoprecipitated from surface-labelled gametes by the sera and the average number of oocysts per gut observed in membrane feeding experiments with these sera. Spearmann's rank correlation coefficient indicated that suppression of infectivity correlated strongly with the presence of antibodies against the 230 kD protein; there was no significant correlation between suppression and antibodies to the 48/45 kD proteins. The antibody response to the different gamete surface antigens varied greatly in sera from the endemic areas suggesting that individuals respond differently to each gamete antigen.     

Mature liver stages of cloned Plasmodium falciparum share epitopes with proteins from sporozoites and asexual blood stages

The liver merozoites of malaria parasites are of paramount importance, as they initiate the parasite invasion of red blood cells and start the cycle associated with the clinical features of malaria. Investigating liver merozoite antigen is difficult because of the lack of a rodent model of human malaria. In addition, only a low proportion of cells are obtained in vivo, the parasites from Cebus and Aotus monkeys are immature, and in-vitro experiments with liver cells are often confounded by contamination with the natural mosquito flora copurified with the sporozoites used for seeding the liver cultures. In our study, mature liver schizonts were shown to possess many of the antigenic determinants recognized by MoAbs and sera specific for defined sporozoite and blood-stage antigens. We employed an immunofluorescence procedure based on evaluating parasites in cryosections prepared from infected chimpanzee liver. Sufficient numbers of sectioned parasites were evaluated with each antibody to assure the reproducibility of the results, and the fixation procedure used was sufficiently non-destructive to parasite antigens so that clear differences between reactions of specific antibodies and negative controls were observed. Our evidence for sharing of epitopes by liver merozoites and sporozoites or by liver merozoites and asexual blood-stage parasites raises the possibility that immune responses elicited against sporozoites or asexual stage antigens being considered as vaccine candidates may also act against this important, little-studied stage of the parasite.     

Fluorometric microassay of trypsin and enteropeptidase in children — comparison with a titrimetic assay

Abstract Two trypsin assay methods for the estimation of this enzyme in duodenal fluid from children have been compared. Assay results for a fluorometric method based on the use of N-carbobenzoxy-diglycyl-L-arginyl-2-naphthylamide hydrochloride (GANA) as the trypsin substrate were found to correlate well (r=0.91, P<0.001) with those obtained with a much less sensitive titrimetric assay which used benzoylarginine ethylester hydrochloride (BAEE) as substrate. The higher sensitivity of the fluorometric assay has allowed accurate determination of trypsin activity in 10 μl aliquots of duodenal fluid. This low volume requirement makes the assay suitable for studies on infants of all ages and conserves duodenal fluid for use in other investigations often warranted during the assessment of childhood malabsorption.
The fluorometric assay has also been used to monitor the separation of enteropeptidase from trypsin(ogen) by chromatography on Sephacryl S-200 in samples of duodenal fluid from two children. Different proteolytic pathway deficiencies were confirmed in these children.     

Fourth polymorph of [Phe 4 Val 6] antamanide (pentahydrate)

The cyclic decapeptide antamanide and the synthetic, biologically active analog [Phe⁴ Val⁶] antamanide (cyclic[ValProProPhePhe]₂) crystallize in various crystal forms as a function of the solvent. The present crystalline polymorph obtained from acetone/water (also from ethanol/water and DMSO/water) crystallizes in space group P2₁2₁2₁ with a = 20.194 (30) Å, b = 21.118 (31) Å, c = 16.132 (25) Å and four molecules of peptide in the unit cell. There are five cocrystallized water molecules per peptide molecule, of which four water molecules are intrinsic to the peptide molecule. Although the molecular packing is entirely different in each of the polymorphs, the conformation of the peptide molecule, including the intrinsic water molecules, is very similar in all the polymorphs.     

Water structure in [Phe4 Val6] antamanide • 12H2O crystallized from dioxane

Crystals of [Phe⁴ Val⁶] antamanide (cyclic[ValProProPhePhe]₂) grown from dioxane/H₂O, with space group P2₁2₁2 and cell parameters a= 15.099(4), b = 22.008(5) and c = 11.024(3) Å, are almost identical to crystals grown from H₂O/acetone, the structure of which was determined a number of years ago. Perpeptide molecule there are the equivalent of 12 water molecules occupying 16 sites in both crystals; however, in the new investigation a number of water molecules present at one-half occupancy have been found in different positions than in the earlier analysis. The interpretation of the hydrogen bonding between peptide/water and between water/water is much more satisfactory. Pentagonal water assemblies are present in the solvent channel. There is a distinct indication of the occurrence of a bifurcated bond between two water molecules, as well as the presence of three-center hydrogen bonds joining three water molecules. This may be the first experimental example of a bifurcated bond between two water molecules.     

Parallel and antiparallel aggregation of α-helices

An apolar helical decapeptide with different end groups, Boc- or Ac-, crystallizes in a completely parallel fashion for the Boc-analog and in an antiparallel fashion for the Ac-analog. In both crystals, the packing motif consists of rows of parallel molecules. In the Boc-crystals, adjacent rows assemble with the helix axes pointed in the same direction. In the Ac-crystals, adjacent rows assemble with the helix axes pointed in opposite directions. The conformations of the molecules in both crystals are quite similar, predominantly α-helical, except for the tryptophanyl side chain where χ¹? 60° in the Boc- analog and ? 180° in the Ac-analog. As a result, there is one lateral hydrogen bond between helices, N(lε)…O(7), in the Ac-analog. The structures do not provide a ready rationalization of packing preference in terms of side-chain interactions and do not support a major role for helix dipole interactions in determining helix orientation in crystals. The crystal parameters are as follow. Boc-analog: C₃H₇,N₁₁O₁₃ C₃H₇OH, space group P1 with a = 10.250(3) Å, b = 12.451(4) Å, c = 15.077(6) Å, x= 96.55(3)°, β= 92.31(3)°, y= 106.37(3)°, Z = 1, R = 5.5% for 5581 data (|F| > 3.0a(F)), resolution 0.89 Å. Ac-analog: C₅₇,H₉₁,N₁₁,O₁₂, space group P2₁, with a = 9.965(1) Å, b = 19.707(3) Å, c = 16.648(3) Å, β= 94.08(1), Z = 2, R = 7.2% for 2530 data (|F| > 3.0σ(F)), resolution 1.00 Å.     

Functional Response to Cardiac Resynchronization Therapy is Associated with Improved Clinical Outcome and Absence of Appropriate Shocks

Clinical Outcome in Cardiac Resynchronization Therapy (CRT) Functional Responders . Introduction : We evaluated clinical outcome and incidence of (in)appropriate shocks in consecutive chronic heart failure (CHF) patients treated with CRT with a defibrillator (CRT‐D) according to functional response status. Furthermore, we investigated which factors predict such functional response. Methods and Results : In a large teaching hospital, 179 consecutive CHF patients received CRT‐D in 2005–2010. Patients were considered functional responders if left ventricular ejection fraction (LVEF) increased to ≥35% postimplantation. Analysis was performed on 142 patients, who had CRT‐D as primary prevention, complete data and a baseline LVEF <35%. Endpoints consisted of all‐cause mortality, heart failure (HF) hospitalizations, appropriate shocks and inappropriate shocks. Median follow‐up was 3.0 years (interquartile range [IQR] 1.6–4.4) and median baseline LVEF was 20% (IQR 18–25%). The functional response‐group consisted of 42 patients. In this group no patients died, none were hospitalized for HF, none received appropriate shocks and 3 patients (7.1%) received ≥1 inappropriate shocks. In comparison, the functional nonresponse group consisted of 100 patients, of whom 22 (22%) died (P = 0.003), 17 (17%) were hospitalized for HF (P = 0.007), 17 (17%) had ≥1 appropriate shocks (P = 0.003) and 8 (8.1%) received ≥1 inappropriate shocks (P = 0.78). Multivariable analysis showed that left bundle branch block (LBBB), QRS duration ≥150 milliseconds and no need for diuretics at baseline are independent predictors of functional response. Conclusion : Functional responders to CRT have a good prognosis and rarely need ICD therapy. LBBB, QRS duration ≥150 milliseconds and lack of chronic diuretic use predict functional response. (J Cardiovasc Electrophysiol, Vol. 24, pp. 316‐322, March 2013)     

Crystal structures of a nonapeptide helix containing α, α-di-n-butylglycine (Dbg), Boc-G1y-Dbg-Ala-Val-Ala-Leu-Aib-Val-Leu-OMe

Two crystal structures of a nonapeptide (anhydrous and hydrated) containing the amino acid residue α,α-di-n-butylglycyl, reveal a mixed 3₁₀-α-helical conformation. Residues 1-7 adopt φ, ψ values in the helical region, with Val(8) being appreciably distorted. The Dbg residue has φ, ψ values of -40, -37° and -46, -407° in the two crystals with the two butyl side chains mostly extended in each. Peptide molecules in the crystals pack into helical columns. The crystal parameters are: C₅₀ H₉₁ N₉ O₁₂, space group P2₁, with a= 9.789(1)Å;, b= 20.240(2) Å. c= 15.998(3) Å. β= 103.97(1): Z= 2, R=10.3% for 1945 data observed < 3σ(F) and C₅₀H₉₁N₉O₁₂· 3H₂O, space group P2₁ with a= 9.747(3)Å, b= 21.002(8) Å, c= 15.885(6) Å, β= 102.22(3). Z= 2. R=13.6% for 2535 data observed < 3σ(F) The observation of a helical conformation at Dbg suggests that the higher homologs in the α,α-dialkylated glycine series also have a tendency to stabilize peptide helices. © Munksgaard 1996.