Continuous-flow solid-phase synthesis of potential antigenic peptides of hepatitis B virus

The synthesis of three hepatitis B surface antigens derived from S and pre-S proteins (adw S(140-147), [Tyr148] adw S(139-148), and adw pre-S(120-145)) has been accomplished by the continuous flow Fmoc-polyamide solid phase method. The use of different scavengers and trimethylsilyl bromide (TMSBr) in trifluoroacetic acid as deprotecting procedures is discussed.     

Interaction of morphine and naloxone with mixed monolayers of lecithin and gangliosides

Monomolecular films of lecithin, gangliosides or lecithin/gangliosides mixtures were studied on a Langmuir through in order to examine the interactions between these lipids and opioid agonists or antagonists. Lecithin alone did not interact in a monolayer structure with opioids. However, gangliosides and lecithin/gangliosides mixtures were expanded by both morphine and naloxone. The expansion of ganglioside-containing monolayers was greater with morphine than with the antagonist, naloxone.     

Monoclonal antibodies to neurospora adenylate cyclase

A monoclonal antibody against $\text{Neurospora}$ soluble adenylate cyclase was obtained. The antibody inhibits cyclase activities from several lower eucaryotic organisms but not activities associated to testicular cytosol or turkey erythrocyte membranes.     

NMDA Receptor antagonists prevent acute ammonia toxicity in mice

We proposed that acute ammonia toxicity is mediated by activation of NMDA receptors. To confirm this hypothesis we have tested whether different NMDA receptor antagonists, acting on different sites of NMDA receptors, prevent death of mice induced by injection of 14 mmol/Kg of ammonium acetate, a dose that induces death of 95% of mice. MK-801, phencyclidine and ketamine, which block the ion channel of NMDA receptors, prevent death of at least 75% of mice. CPP, AP-5, CGS 19755, and CGP 40116, competitive antagonists acting on the binding site for NMDA, also prevent death of at least 75% of mice. Butanol, ethanol and methanol which block NMDA receptors, also prevent death of mice. There is an excellent correlation between the EC₅₀ for preventing ammonia-induced death and the IC₅₀ for inhibiting NMDA-induced currents. Acute ammonia toxicity is not prevented by antagonists of kainate/AMPA receptors, of muscarinic or nicotinic acetylcholine receptors or of GABA receptors. Inhibitors of nitric oxide synthase afford partial protection against ammonia toxicity while inhibitors of calcineurin, of glutamine synthetase or antioxidants did not prevent ammonia-induced death of mice. These results strongly support the idea that acute ammonia toxicity is mediated by activation of NMDA receptors.     

DNA/DNA hybridization studies of carnivorous marsupials. III. Relationships among species ofDidelphis (Didelphidae)

We report three sets of DNA hybridization experiments conducted to determine relationships among species ofDidelphis (D. albiventris, D. marsupialis, D. virginiana). The 1989 and 1991 sets had fewer replicates per cell than the 1990 series (3.4 and 5.4 vs 9), but in 1991 we distinguished two populations ofD. marsupialis and utilized several individuals for each heterologous comparison. BothPhilander opossum andLutreolina crassicaudata were used as outgroups in 1989, but onlyLutreolina was included in subsequent sets. For each set, we calculated all four standard indices of thermal stability (T _mode,T _m,T ₅₀ H, and NPH) and constructed trees by least-squares (FITCH) and neighbor-joining methods, both before and after correction for asymmetric reciprocal cell values. Subsets of the 1989 data lacking eitherPhilander orLutreolina were analyzed similarly. To explore measurement imprecision, the corrected and uncorrected matrices for each of the four indices were bootstrapped 100 times for the 1989 set and subsets and 1000 times for the 1990 and 1991 sets. Again, for the 1990 and 1991 data, an additional 100 bootstrapped distances were fitted to user trees representing the three possible pairings ofDidelphis spp. to determine the significance of the FITCH branch lengths. The successive experimental sets generated increasingly consistent evidence for pairingD. marsupialis withD. albiventris. The 1989 experiments involved just 85 comparisons, and only T _mode's pairedD. marsupialis withD. albiventris (at bootstrap percentages of 70% or above), but did so whetherPhilander orLutreolina or both were included as the outgroup(s). FITCH and neighbor-joining trees had identical topologies for T _mode's but sometimes differed for the other measures. In contrast, all but one of the corresponding FITCH and neighborjoining trees matched for the 1990 and 1991 data, and three of the four distance measures (T _mode, T _m, and T ₅₀ H) unitedD. marsupialis withD. albiventris at bootstrap percentages averaging 81%; NPH gave a different result for 1990, associatingD. marsupialis withD. virginiana. Further, all but 2 of the 16 matrices for 1990 and 1991 gave mean bootstrapped branch lengths for a consensus pairing that were positive and at least one standard deviation from zero, despite the very short internodes recovered. These results illustrate that the potential of DNA hybridization for resolving very close relationships depends on both the index and the experimental design employed. We conclude that of the three species,D. albiventris andD. marsupialis shared a more recent common ancestor and estimate thatDidelphis spp. have diverged at about 0.39% in nucleotide sequence per myr.Deceased.     

Naphthalenesulfonamide derivatives ML9 and W7 inhibit catecholamine secretion in intact and permeabilized chromaffin cells

The role of protein phosphorylation in catecholamine secretion from bovine adrenomedullary chromaffin cells was studied using different protein kinase inhibitors. Naphthalenesulfonamide derivatives as ML9 and ML7, more specific for the myosin light chain kinase, and the calmodulin antagonist W7 inhibited catecholamine secretion 20 and 40% respectively in digitonin-permeabilized chromaffin cells. ML9 also decreased calcium evoked protein phosphorylation of different proteins including tyrosine hydroxylase in permeabilized cells. These naphthalenesulfonamide derivatives showed also an effect in intact cells, ML9 and W7 produced 50% inhibition in catecholamine secretion and⁴⁵Ca²⁺ uptake, however H8 had no effect. The partial [³H]nitrendipine binding displacement of these drugs to adrenomedullary membranes suggests that these sulfonamide derivatives could interact directly with L-type calcium channels in intact cells. The results obtained in permeabilized cells suggest a possible role of protein phosphorylation in the regulation of catecholamine secretion in chromaffin cells.The abbreviations used are ML9 1-(5-Chloronaphthalene-1-sulfonyl)1H-hexahydro-1,4-diazepine hydrochloride - ML7 1-(5-Iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4 diazepine hydrochloride - H7 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride - H8 N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride - W7 N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride - PKI protein kinase A inhibitor - HEPES N-(2-hydroxyethylpiperazine-N-(2 ethanesulfonic acid) - PIPES piperazine-N, N-bis (2-ethanesulfonic acid) - EGTA [ethylene-bis (oxyethylenenitrilo)] tetraacetic acid - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - DMEM Dulbecco's Modified Eagle's medium - MLC myosin light chain - MLCK myosin light chain kinase - TH tyrosine hydroxylase     

Anti-Hepatitis A Virus Antibody Response Elicited in Mice by Different Forms of a Synthetic VP1 Peptide

Peptide VP1 (11-25) of the capsid of hepatitis A virus was synthesized by the Fmoc-polyamide solid phase method, and administered to mice in different forms: (1) free, (2) encapsulated in multilamellar liposomes, (3) coupled to keyhole limpet hemocyanin (KHL), and (4) incorporated into a tetrameric branched lysine core. The highest anti-VP1 peptide responses were generated by synthetic peptides entrapped into liposomes and coupled to KLH. No anti-HAV response was generated with the free peptide, while all the other forms induced both anti-HAV and HAV-neutralizing antibodies. Maximum neutralization indices were observed in ascites from mice treated with liposome-entrapped and KLH peptides.     

New Ctenomys karyotypes (Rodentia,Octodontidae) from north-eastern Argentina and from Paraguay confirm the extreme chromosomal multiformity of the genus

Bone-marrow karyotypes of 68 specimens of the subterranean octodontid rodent genus Ctenomys from 16 different populations of north east Argentina and one from Paraguay have been studied. A surprising variety of chromosome numbers was found, ranging from 2n=42 to 2n=70. Some of the karyomorphs are clearly assigned to named species by topotypy: C. conoveris 2n=50, FN=56; C. argentinus, 2n=44, FN=54; C. perrensi, 2n=50, FN=84; C. dorbignyi, 2n=70, FN=84; C. roigi, 2n=48, FN=80; C. yolandae, 2n=50, FN=78. Four populations of Corrientes Province similar in morphology to C. perrensi were found to be polymorphic and polytypic; they maintain the same FN=84, but diploid numbers increase from 2n=54 to 2n=58 from SW to the NE, thus suggesting Robertsonian rearrangements. In the middle of this cline, a stable karyomorph of 2n=62, FN=84 was found in two different populations, suggesting to belong to an undescribed species. Another karyomorph of 2n=42, FN=76 found in Curuzú Laurel, Corrientes, may also prove to represent another undescribed species. One karyomorph of 2n=52, FN=74, and another of 2n=56, FN=78 from Paraná and Ubajaý (Entre Ríos Province, Argentina) respectively are close to C. rionegrensis. The relationships among these karyomorphs is considered in light of data on sperm morphology. The hypothesis is advanced that karyotypic rearrangements among the FN=84 group may be the result of Robertsonian repatterning from a 2n=70 original widespread form. Fixation of chromosomal variants is correlated with patchy distribution and small size of unstable demes, and may or may not have resulted in reproductive isolation.     

Serotypes and Clonal Diversity of Streptococcus pneumoniae Causing Invasive Disease in the Era of PCV13 in Catalonia,Spain

The aim of this study was to study the serotypes and clonal diversity of pneumococci causing invasive pneumococcal disease in Catalonia, Spain, in the era of 13-valent pneumococcal conjugate vaccine (PCV13). In our region, this vaccine is only available in the private market and it is estimated a PCV13 vaccine coverage around 55% in children. A total of 1551 pneumococcal invasive isolates received between 2010 and 2013 in the Molecular Microbiology Department at Hospital Sant Joan de Déu, Barcelona, were included. Fifty-two serotypes and 249 clonal types—defined by MLST—were identified. The most common serotypes were serotype 1 (n = 182; 11.7%), 3 (n = 145; 9.3%), 19A (n = 137; 8.8%) and 7F (n = 122; 7.9%). Serotype 14 was the third most frequent serotype in children < 2 years (15 of 159 isolates). PCV7 serotypes maintained their proportion along the period of study, 16.6% in 2010 to 13.4% in 2013, whereas there was a significant proportional decrease in PCV13 serotypes, 65.3% in 2010 to 48.9% in 2013 (p<0.01). This decrease was mainly attributable to serotypes 19A and 7F. Serotype 12F achieved the third position in 2013 (n = 22, 6.4%). The most frequent clonal types found were ST306 (n = 154, 9.9%), ST191 (n = 111, 7.2%), ST989 (n = 85, 5.5%) and ST180 (n = 80, 5.2%). Despite their decrease, PCV13 serotypes continue to be a major cause of disease in Spain. These results emphasize the need for complete PCV13 vaccination.