Chemical composition of the plasma membrane from epimastigote forms of Trypanosoma cruzi

The chemical composition of two plasma membrane fractions from epimastigote forms of Trypanosoma cruzi is reported. Fraction M, a preparation obtained by conventional methods of cell fractionation is composed of 31% proteins, 34% lipids, 16% carbohydrates and 3% of the lipopeptidophosphoglycan. Phospholipids and sterols account for 7.5 and 9%, respectively, of the total mass. Phosphatidylethanolamine is the major phospholipid in fraction M, representing 45% of the total membrane phospholipids. The other fraction, fraction V (vesicles), was obtained by treatment of the cell with a vesiculating agent. This fraction contains 42% lipids, 20% carbohydrates, 13% proteins and 21% of the lipopeptidophosphoglycan. Phospholipids and sterols make up 17 and 8%, respectively, of the total mass of this fraction. Phosphatidylcholine and phosphatidylethanolamine are the main phospholipids found in fraction V. Phosphonolipids and sialic acid have not been detected in either membrane fraction. Sodium dodecyl sulphate polyacrylamide gel electrophoretic analysis show that the glycoproteins ABC and the lipopeptidophosphoglycan are 50- and 10-times more concentrated, respectively, in fractions V and M than in the whole cell homogenate. The high molar sterol/phospholipid ratio found in fraction M suggests that this fraction is less fluid than fraction V, perhaps reflecting a migration of certain membrane components in the presence of the vesiculating agent. Hence, fraction M is, probably, more representative of the epimastigote plasma membrane as a whole than fraction V.     

The epoxide hydrolase catalyzed hydrolysis of trans-3-bromo-1,2-epoxycyclohexane. A direct proof for a general base catalyzed mechanism of the enzymatic hydration

The hydrolysis of (±)-$\text{trans}$-3-bromo-1,2-epoxycyclohexane in the presence of rabbit liver microsomes was investigated, and found to yield, beside $\text{c}$-3-bromocyclohexane-$\text{r}$-1,$\text{t}$-2-diol, 2,3-epoxycyclohexanol. It was demonstrated that the latter compound was the only product of the enzymatic reaction, whereas the diol resulted from a non enzymatic hydration in the reaction medium. These data provide the first direct proof for a general base catalysis in the enzymatic epoxide hydration, previously hypothesized on the basis of several lines of indirect evidence, and disprove alternative mechanisms involving protonation of the oxirane oxygen.     

Methane- and n-butaneboronates: nw derivatives for gas-chromatographic analysis of 3-methoxy-4-hydroxyphenylethylenglycol.

Several methods were described for visualization of proteolytic activity on electrophoregrams obtained with agar, agarose, starch, or acrylamide gels as supporting media. In most of these reports casein or hemoglobin were used as nonspecific substrates (1–3). Recently, colorimetric assays for trypsin, using α-benzoyl-d,l-arginine-p-nitroanilide (4), and for subtilisin, using Z-glycyl-glycyl-l-leucine-p-nitroanilide (5) were introduced for the localization of these proteases after acetate celulose and acrylamide gel electrophoresis. No convenient and simple methods were in practice for detection of leucineaminopeptidase (3), although this enzyme was assayed in solution with specific chromogenic substrates such as l-leucine-p-nitroanilide or l-leucine-β-naphtylamide (6,7).The present report describes the use of p-nitroanilide substrate-l-leucine-p-nitroanilide for detection of leucineaminopeptidase activity after acrylamide gel electrophoresis. The method allows a rapid and sensitive localization of leucineaminopeptidases.     

Fingerprinting of Mixed Bacterial Strains and BIOLOG Gram-Negative (GN) Substrate Communities by Enterobacterial Repetitive Intergenic Consensus Sequence-PCR (ERIC-PCR)

PCR-based genomic fingerprinting by use of enterobacterial repetitive intergenic consensus primers (ERIC-PCR) was evaluated for its use in fingerprinting DNA of mixed Gram-negative bacterial strains and BIOLOG Gram-negative (GN) microplate substrate communities. ERIC-PCR fingerprints of six different pure bacterial strains and a combined mixture of the strains were compared with fingerprints obtained by two more established methods: amplified ribosomal DNA restriction analysis (ARDRA) and random amplified polymorphic DNA analysis (RAPD-PCR). The ERIC-PCR fingerprint of the mixed strains was highly reproducible and was more species-specific and representative of the individual strain fingerprints than the ARDRA and RAPD-PCR fingerprints, respectively. ERIC-PCR fingerprinting of model and rhizosphere BIOLOG GN substrate communities also provided clearly distinguishable fingerprints. Results of this study suggest that ERIC-PCR represents a rapid and highly discriminating method for fingerprinting DNA of mixed Gram-negative bacterial strains and BIOLOG GN substrate communities. Received: 11 September 1998 / Accepted: 29 October 1998     

Selective antibacterial activity of the cationic peptide PaDBS1R6 against Gram-negative bacteria

Infections caused by Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa foremost among them, constitute a major worldwide health problem. Bioinformatics methodologies are being used to rationally design new antimicrobial peptides, a potential alternative for treating these infections. One of the algorithms used to develop antimicrobial peptides is the Joker, which was used to design the peptide PaDBS1R6. This study evaluates the antibacterial activities of PaDBS1R6 in vitro and in vivo, characterizes the peptide interaction to target membranes, and investigates the PaDBS1R6 structure in contact with mimetic vesicles. Moreover, we demonstrate that PaDBS1R6 exhibits selective antimicrobial activity against Gram-negative bacteria. In the presence of negatively charged and zwitterionic lipids the structural arrangement of PaDBS1R6 transits from random coil to α-helix, as characterized by circular dichroism. The tertiary structure of PaDBS1R6 was determined by NMR in zwitterionic dodecylphosphocholine (DPC) micelles. In conclusion, PaDBS1R6 is a candidate for the treatment of nosocomial infections caused by Gram-negative bacteria, as template for producing other antimicrobial agents.     

Synthesis,semipreparative HPLC separation,biological evaluation,and 3D-QSAR of hydrazothiazole derivatives as human monoamine oxidase B inhibitors

The present study reports on synthesis in high yields (70–99%), HPLC enantioseparation, inhibitory activity against human monoamino oxidases, and molecular modeling including 3D-QSAR studies, of a large series of (4-aryl-thiazol-2-yl)hydrazones (1–45). Most of the synthesized compounds proved to be potent and selective inhibitors of hMAO-B isoform in the micromolar or nanomolar range, thus demonstrating that hydrazothiazole could be considered a good pharmacophore to design new hMAO-B inhibitors. Due to the presence in some derivatives of a chiral center, we also performed a semipreparative chromatographic enantioseparation of these compounds obtained by a stereoconservative pattern. The separated enantiomers were submitted to in vitro biological evaluation to point out the stereorecognition of the active site of the enzyme towards these structures. Finally, a 3D-QSAR study was carried out using Comparative Molecular Field Analysis (CoMFA), aiming to deduce rational guidelines for the further structural modification of these lead compounds.     

Plant cyclotides: an unusual class of defense compounds

Plant cyclotides are unusual peptides with low molecular masses and a three-dimensional structure characterized by the presence of a cyclic fold. Synthetic peptides can adopt this circular conformation, but it is not a common feature for most members of other peptide groups. Cyclotides present a wide range of functions, such as the ability to induce stronger contractions during childbirth and anti-tumor activity. Additionally, some cyclotides present anti-viral, insecticidal or proteinase inhibitory activity. In this paper, we describe the structural and functional characteristics of plant cyclotides, their most conserved features and the development of these peptides for human health and biotechnological applications.