Expression systems of human ß-defensins: vectors, purification and biological activities.

Human beta-defensins are 2-5 kDa, cationic, microbicidal peptides, which represent the first-line host defense against several Gram-negative and Gram-positive bacteria, fungi and viruses. They contain a conserved disulfide-bridge pattern of three pairs of intramolecular cystine bonds. The well-known public health problem related with the growing number of multiresistant bacteria has driven research to look for novel antibiotics, such beta-defensins and a feasible way to produce them. Heterologous expression of beta-defensins could be one way to generate large quantities of beta-defensins for clinical research; however, heterologous expression of beta-defensins has some biochemical problems, such toxicity toward the host cell, peptide degradation by proteolytic cell enzymes, size, folding constrains and low recombinant peptide yields. In this communication, several heterologous systems for producing human beta-defensins are reviewed.

N-acyl amino acid biosynthesis in marine bacterium, Deleya marina.

We reported previously that the marine bacterium, Deleya marina (ATCC 25374), produced N-acyl leucine and isoleucine, in which nonhydroxy fatty acid was linked to alpha-amino group of amino acid. Further analysis of bacterium lipids revealed the additional production of N-acyl ornithine. The N-acyl ornithine had a 3-hydroxy fatty acid linked by an amide bond to a-amino group of ornithine and a nonhydroxy fatty acid esterified to the hydroxy group of the 3-hydroxy fatty acid. N-acyl ornithine was located in the cell membrane and N-acyl leucine and isoleucine in cytoplasm. N-acyl ornithine is thought to be a functional analogue of phosphatidylethanolamine (PE) because of their similar structure. PE replacement into N-acyl ornithine in the cell membrane under phosphate-limited conditions was observed with other bacteria, so we anticipated the nonbiosynthesis of N-acyl ornithine under phosphate-sufficient conditions. We did not anticipate that N-acyl leucine and isoleucine in cytoplasm, whose structure is dissimilar to that of PE, would be replaced into PE in the cell membrane. Neither N-acyl leucine, N-acyl isoleucine, nor N-acyl ornithine was biosynthesized under phosphate-sufficient condition. Thus, we report here for the first time that N-acyl amino acids in cytoplasm were not biosynthesized under phosphate-sufficient conditions.

Solution structure of two insect-specific spider toxins and their pharmacological interaction with the insect voltage-gated Na+ channel.

Delta-paluIT1 and delta-paluIT2 are toxins purified from the venom of the spider Paracoelotes luctuosus. Similar in sequence to mu-agatoxins from Agelenopsis aperta, their pharmacological target is the voltage-gated insect sodium channel, of which they alter the inactivation properties in a way similar to alpha-scorpion toxins, but they bind on site 4 in a way similar to beta-scorpion toxins. We determined the solution structure of the two toxins by use of two-dimensional nuclear magnetic resonance (NMR) techniques followed by distance geometry and molecular dynamics. The structures of delta-paluIT1 and delta-paluIT2 belong to the inhibitory cystine knot structural family, i.e. a compact disulfide-bonded core from which four loops emerge. Delta-paluIT1 and delta-paluIT2 contain respectively two- and three-stranded anti-parallel beta-sheets as unique secondary structure. We compare the structure and the electrostatic anisotropy of those peptides to other sodium and calcium channel toxins, analyze the topological juxtaposition of key functional residues, and conclude that the recognition of insect voltage-gated sodium channels by these toxins involves the beta-sheet, in addition to loops I and IV. Besides the position of culprit residues on the molecular surface, difference in dipolar moment orientation is another determinant of receptor binding and biological activity differences. We also demonstrate by electrophysiological experiments on the cloned insect voltage-gated sodium channel, para, heterologuously co-expressed with the tipE subunit in Xenopus laevis oocytes, that delta-paluIT1 and delta-paluIT2 procure an increase of Na+ current. delta-PaluIT1-OH seems to have less effect when the same concentrations are used.

Novel peptides from assassin bugs (Hemiptera: Reduviidae): isolation, chemical and biological characterization.

Three novel peptides were isolated from the venomous saliva of predatory reduviids. They were identified by mass spectrometry and HPLC analysis and consist of 34-36 amino acid residues. They are relatively homologous to the calcium channel blockers omega-conotoxins from marine cone snails and belong to the four-loop Cys scaffold structural class. Ptu1, the shortest peptide, was chemically synthesized (sPtu1) and co-eluted with its native form. Circular dichroism spectra of the sPtu1 showed a high content of beta-turns similar to that of omega-conotoxins GVIA and MVIIA. Electrophysiological experiments demonstrated that sPtu1 reversibly blocks the N-type calcium channels expressed in BHK cells.

Structure and pharmacology of spider venom neurotoxins.

Spider venoms are complex mixtures of neurotoxic peptides, proteins and low molecular mass organic molecules. Their neurotoxic activity is due to the interaction of the venom components with cellular receptors, in particular ion channels. Spider venoms have proven to be a rich source of highly specific peptide ligands for selected subtypes of potassium, sodium and calcium channels, and these toxins have been used to elucidate the structure and physiological roles of the channels in excitable and non-excitable cells. Spider peptides show great variability in their pharmacological activity and primary structure but relative homogeneity in their secondary structure. Following diverse molecular evolution mechanisms, and in particular selective hypermutation, short spider peptides appear to have functionally diversified while retaining a conserved molecular scaffold. This paper reviews the composition and pharmacology of spider venoms with emphasis on polypeptide toxin structure, mode of action and molecular evolution.

Rapid and efficient identification of cysteine-rich peptides by random screening of a venom gland cDNA library from the hexathelid spider Macrothele gigas.

We identified novel 10 multi-cysteine peptides, namely Magi 7-16, from the spider Macrothele gigas by simple random cDNA screening of the venom gland. Mass analysis of the crude venom detected the mass numbers of the cross-linked forms of all peptides, confirming their presence in the venom. Magi 11, a C-terminus amidated peptide, was chemically synthesized and was indistinguishable from the native peptide proving the feasibility of the method for peptide identification. Moreover, toxicological assays showed diverse lethal or paralytic activities of these peptide toxins on mice and/or insects.

[Occupational exposure to lead in production units in Maracaibo, Venezuela]. / Exposición ocupacional a plomo en unidades productivas en Maracaibo, Venezuela.

A medical occupational study was performed in 40 workers belonging to productive units in telecommunication works, 22 to car radiator mechanics and 11 to battery repairs. A practical medical and occupational study was applied to the group and also were determined their blood lead and air lead exposure levels. Seventy-three individuals, without risk of laboral exposure to lead, without familiar, pathological and occupational antecedents, and healthy at the time of the test, to whom the blood lead levels were determined served as control group. The mean values of plumbemia in exposure workers to inorganic lead exceed the level threshold of the COVENIN 2277-85 norm (30 micrograms/dl) (Telecommunication work, 40.10 micrograms/dl, radiators mechanics, 37.40 micrograms/dl and battery repairs, 45.77 micrograms/dl), values that were significantly higher (p < 0.0001) compared with the ones obtained in the non-exposed population. The factors that can influence the variability of the results were analyzed and it was established a correlation between the plumbemia of the radiator mechanics and battery repairmen and the length of occupational period and air lead levels (p < 0.0001). The inherent factors to the climatic, occupational and personal conditions of technicians in telecommunications, are presented as elements able to explain the lack of correlation between blood lead levels and length of occupational period and air lead. The clinical findings in exposed workers were unspecific. The workers do not practice or follow the basic sanitary regulations, personal protection and industrial security. This work will contribute to establish a basic description, to further and more complex observational prospective studies in order to determine the occurrence of alterations that are derived from occupational lead exposure.

[Spirometry in workers in a wheat-processing industry]. / Espirometría en trabajadores de una industria procesadora de trigo.

In order to determine both clinical and spirometric changes due to high environmental concentrations of wheat dust at a wheat processing plant mill, 48 exposed men and 48 age and antroprometrically-matched, non-exposed apparently healthy men were studied. In both groups a medical and occupational history were taken, and spirometric measurements were carried out, that included Forced Vital Capacity (FVC), Forced Expiratory Volume at the first second (FEV1), Peak Flow Rate (PFR), Forced Percentual Expiratory Volume (FEV%), Forced Percentual Vital Capacity (FVC%), Forced Expiratory Flow at 25% (FEV25%), at 50% (FEV50%) and at 75% (FEV75%) of their Forced Vital Capacity, which were analyzed through Corzo's predictive equations and the lung deterioration's criteria by USA's Thoracic Association. The environmental wheat dust was determined by gravimetry and its concentration was higher than the legally admitted (3/5, 60%). There was a decrease in the PFR, FEV%, FEV25% and FEV75%. (p < 0.05). In addition, 4 restrictive and 1 obstructive syndrome were detected in the exposed workers and none in the control group. The spirometric values diminished in a positive correlation with the time of exposure and smoking habits. There was no correlation between the clinical findings and the dust concentration but it did exist with the spirometric values. It is concluded that in this plant, the wheat dust exposed workers have a diminished spirometric values.

[Lung function in workers in a chicken slaughterhouse in the city of Maracaibo, Venezuela]. / Estudio de la función pulmonar en trabajadores de una industria beneficiadora de pollos en la ciudad de Maracaibo, Venezuela.

In order to evaluate the respiratory health status in workers exposed to antigenic substances (chicken feathers, serum and dropping), typical of usual practice in the avian slaughter-house, pulmonary function was studied on 49 exposed workers, and in a sample of 49 people with similar anthropometric characteristics, non exposed to these substances, by means of occupational-medical history, spirometric tests, hematologic and biochemical tests, and postero-anterior chest x-rays. The values for the spirometric parameters varied with sex, age, weight, size, smoking habits, length of employment and exposure time, and there were no significant differences between exposed and control groups as a whole; showing significant differences with decreasing values for CVF, VEF1, PFE, FEF-25% and FEF-50% in the intermediate zone workers, and in subjects with short exposure time (< 1 year). Prevalence of clinical findings in the exposed population was significantly higher than the non exposed group (p < 0.001). Laboratory tests showed reduction of monocytes cells in the exposed group (p < 0.05) in addition, in the exposed women there was an increase of the eosinophiles, total proteins and globulines (p < 0.05). The frequency of radiographic findings was significantly higher in the exposed group (p < 0.006), and they were no specific. The lack of association between clinical findings, laboratory and radiographic findings, with the spirometric results, could be explained by the short period of exposure, individual and collectives hygienic conditions and size of the sample.

Improved protease stability of the antimicrobial peptide Pin2 substituted with D-amino acids.

Cationic antimicrobial peptides (AMPs) have attracted a great interest as novel class of antibiotics that might help in the treatment of infectious diseases caused by pathogenic bacteria. However, some AMPs with high antimicrobial activities are also highly hemolytic and subject to proteolytic degradation from human and bacterial proteases that limit their pharmaceutical uses. In this work a D-diastereomer of Pandinin 2, D-Pin2, was constructed to observe if it maintained antimicrobial activity in the same range as the parental one, but with the purpose of reducing its hemolytic activity to human erythrocytes and improving its ability to resist proteolytic cleavage. Although, the hydrophobic and secondary structure characteristics of L- and D-Pin2 were to some extent similar, an important reduction in D-Pin2 hemolytic activity (30-40 %) was achieved compared to that of L-Pin2 over human erythrocytes. Furthermore, D-Pin2 had an antimicrobial activity with a MIC value of 12.5 µM towards Staphylococcus aureus, Escherichia coli, Streptococcus agalactiae and two strains of Pseudomonas aeruginosa in agar diffusion assays, but it was half less potent than that of L-Pin2. Nevertheless, the antimicrobial activity of D-Pin2 was equally effective as that of L-Pin2 in microdilution assays. Yet, when D- and L-Pin2 were incubated with trypsin, elastase and whole human serum, only D-Pin2 kept its antimicrobial activity towards all bacteria, but in diluted human serum, L- and D-Pin2 maintained similar peptide stability. Finally, when L- and D-Pin2 were incubated with proteases from P. aeruginosa DFU3 culture, a clinical isolated strain, D-Pin2 kept its antibiotic activity while L-Pin2 was not effective.

Scorpion and spider venom peptides: gene cloning and peptide expression.

This communication reviews most of the important findings related to venom components isolated from scorpions and spiders, mainly by means of gene cloning and expression. Rather than revising results obtained by classical biochemical studies that report structure and function of venom components, here the emphasis is placed on cloning and identification of genes present in the venomous glands of these arachnids. Aspects related to cDNA library construction, specific or random ESTs cloning, transcriptome analysis, high-throughput screening, heterologous expression and folding are briefly discussed, showing some numbers of species and components already identified, but also shortly mentioning limitations and perspectives of research for the future in this field.

Pharmacologically active spider peptide toxins.

Advances in mass spectrometry and peptide biochemistry coupled to modern methods in electrophysiology have permitted the isolation and identification of numerous novel peptide toxins from animal venoms in recent years. These advances have also opened up the field of spider venom research, previously unexplored due to methodological limitations. Many peptide toxins from spider venoms share structural features, amino acid composition and consensus sequences that allow them to interact with related classes of cellular receptors. They have become increasingly useful agents for the study of voltage-sensitive and ligand-gated ion channels and the discrimination of their cellular subtypes. Spider peptide toxins have also been recognized as useful agents for their antimicrobial properties and the study of pore formation in cell membranes. Spider peptide toxins with nanomolar affinities for their receptors are thus promising pharmacological tools for understanding the physiological role of ion channels and as leads for the development of novel therapeutic agents and strategies for ion channel-related diseases. Their high insecticidal potency can also make them useful probes for the discovery of novel insecticide targets in the insect nervous system or for the development of genetically engineered microbial pesticides.

Bile salt hydrolase activity of three strains of Lactobacillus acidophilus.

Three strains of Lactobacillus acidophilus, two from human intestinal origin (016 and L1) and one from porcine intestinal origin (ATCC 43121), were tested for their bile salt deconjugation activity. The L. acidophilus ATCC 43121 had more deconjugating activity of both sodium glycocholate and sodium taurocholate at pH 6.5 than did either L. acidophilus 016 or L1. The activity of intracellular bile salt hydrolase found in strain ATCC 43121 was 14-fold higher than that in either of the other two strains. The optimum pH for deconjugation of sodium glycocholate was between 4 and 5.5 for all three strains. For deconjugation of sodium taurocholate, the optimum pH was between 3.5 and 4.5 for strains L1 and ATCC 43121 and was between pH 5 and 6 for strain O16. The molecular mass of the enzyme in all three strains of L. acidophilus was estimated to be 126 kDa by Sephadex G-200 gel filtration. All three strains exhibited more bile salt hydrolase activity towards sodium glycocholate than towards sodium taurocholate.

Measurement of bile salt hydrolase activity from Lactobacillus acidophilus based on disappearance of conjugated bile salts.

Bile salt hydrolase activity of Lactobacillus acidophilus was measured based on the disappearance of sodium glycocholate and sodium taurocholate from the reaction mixture using HPLC. The amount of sodium glycocholate and sodium taurocholate that disappeared was proportional to the amount of sodium cholate that appeared in the mixture as detected by HPLC. Sodium glycocholate did not precipitate at the enzyme reaction conditions (37 degrees C and pH 5.4) for determining bile salt hydrolase activity. The bile salt hydrolase assay was insensitive to low oxidation-reduction potential when measuring bile salt hydrolase from L. acidophilus, an intestinal microorganism. However, EDTA and freezing temperatures were necessary to maintain stability of the partially purified enzyme during storage.

Synthesis and paralytic activities of squaryl amino acid-containing polyamine toxins.

Eight analogs 4A-7A and 4B-7B of philanthotoxin (PhTX) from wasp venom and nephilatoxin-8 (NPTX-8) from spider venom whose tyrosine or asparagine linker is replaced by squaryl (sq) amino acid or 4-amino squaryl (4-asq) amino acid have been synthesized in an efficient manner via coupling of N-acyl squaryl amino acid intermediate 19 or 26 with the corresponding polyamine part. Preliminary bioassay using crickets revealed that the analogs substituted by glutamate-type squaryl amino acid-containing NPTX 7A and 7B showed more potent paralytic activities than that of NPTX-8.

Solution structure of Ptu1, a toxin from the assassin bug Peirates turpis that blocks the voltage-sensitive calcium channel N-type.

Ptu1 is a toxin from the assassin bug Peirates turpis which has been demonstrated to bind reversibly the N-type calcium channels and to have lower affinity than the omega-conotoxin MVIIA. We have determined the solution structure of Ptu1 by use of conventional two-dimensional NMR techniques followed by distance-geometry and molecular dynamics. The calculated structure of Ptu1 belongs to the inhibitory cystin knot structural family (ICK) that consists of a compact disulfide-bonded core from which four loops emerge. Analysis of the 25 converged solutions indicates that the molecular structure of Ptu1 contains a 2-stranded antiparallel beta-sheet (residues 24-27 and 31-34) as the only secondary structure. The loop 2 that has been described to be critical for the binding of the toxin on the channel is similar in Ptu1 and MVIIA. In this loop, the critical residue, Tyr13, in MVIIA is retrieved in Ptu1 as Phe13, but the presence of an acidic residue (Asp16) in Ptu1 could disturb the binding of Ptu1 on the channel and could explain the lower affinity of Ptu1 toward the N-type calcium channel compared to the one of MVIIA. Analysis of the electrostatic charge's repartition gives some insights about the importance of the basic residues, which could interact with acidic residues of the channel and then provide a stabilization of the toxin on the channel.

IsCT, a novel cytotoxic linear peptide from scorpion Opisthacanthus madagascariensis.

A novel cytotoxic linear peptide, IsCT, was characterized from scorpion Opisthacanthus madagascariensis. It is a linear peptide with a molecular weight of 1501.9 Da composed of 13 amino acid residues without cysteines. MS/MS analysis showed that its C-terminal is amidated. The identity of IsCT is re-confirmed by comparing the chemical synthesized peptide with the natural one. IsCT demonstrated antimicrobial activity against both gram-positive and gram-negative bacteria and hemolytic activity to sheep red blood cells. Also, it can release histamine from rat peritoneal mast cells. The CD absorption suggested that IsCT had an alpha-helix configuration in aqueous TFE. IsCT is one of the shortest natural cytotoxic peptides described, and it will be a suitable model for studying peptide-lipid interactions.

Isolation, synthesis and pharmacological characterization of delta-palutoxins IT, novel insecticidal toxins from the spider Paracoelotes luctuosus (Amaurobiidae).

Four novel insecticidal toxins were isolated from the venom of the spider Paracoelotes luctuosus (Araneae: Amaurobiidae) and named delta-palutoxins IT1 to IT4. The four toxins are homologous 36-37 amino acid peptides reticulated by four disulfide bridges and three have amidated C-terminal residues. The delta-palutoxins are highly homologous with the previously described mu-agatoxins and curtatoxins (77-97%). The four peptides demonstrated significant toxicity against larvae of the crop pest Spodoptera litura (Lepidoptera: Noctuidae) in a microinjection bioassay, with LD50 values in the 9-50 microg per g of insect range. This level of toxicity is equivalent to that of several of the most active scorpion toxins used in the development of recombinant baculoviruses, and the delta-palutoxins appear to be insect specific. Electrophysiological experiments demonstrated that delta-palutoxin IT1, the most active toxin acts by affecting insect sodium channel inactivation, resulting in the appearance of a late-maintained sodium current, in a similar fashion to insecticidal scorpion alpha and alpha-like toxins and is thus likely to bind to channel receptor site 3. However, delta-palutoxin IT1 was distinguished by its lack of effect on peak sodium conductance, on the early phase of sodium current inactivation and the absence of a shift in the activation voltage of the sodium channels. delta-Palutoxins are thus proposed as new insecticidal toxins related to the alpha and alpha-like scorpion toxins. They will be useful both in the development of recombinant baculoviruses in agrochemical applications and also as molecular probes for the investigation of molecular mechanisms of insect selectivity and structure and function of sodium channels.

A comparison of matrix-assisted laser desorption/ionization time-of-flight and liquid chromatography electrospray ionization mass spectrometry methods for the analysis of crude tarantula venoms in the Pterinochilus group.

The search for novel pharmacological tools in spider venoms involves the need for precise and reproducible species identification methods. As an addition to morphological analysis, we have developed venom fingerprinting by reversed-phase chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) as an efficient and precise venom identification tool. In order to compare the possible use of liquid chromatography electrospray ionization mass spectrometry (LC/ESI-MS) as an additional venom characterization tool, we have applied both methodologies to the study of several tarantula venom samples in the Pterinochilus murinus group. These species possess highly active venoms yet their taxonomy remains difficult. We demonstrate that both methodologies can be successfully applied to tarantula venom characterization. MALDI-TOFMS and ESI-MS gave similar overall profiles and allowed fine discrimination of samples. At least one venom sample was proven to belong to a completely different venom group. Coupling of ESI-MS with HPLC separation afforded a new dimension in venom analysis, with clear discrimination between components of similar Mr and gave a finer picture of venom composition, number of molecular species and molecular weight distribution.