Bactericidal and hemolytic activities of synthetic peptides derived from granulysin.

Granulysin is a human polypeptide produced by cytolytic cells active against a broad range of microbes. Three peptides covering the regions 25-50 (Gr-1 and Gr-2) and 39-62 (Gr-3) of granulysin were synthesized, and their in vitro activity against Mycobacterium tuberculosis was evaluated. The most active peptide was Gr-1C, containing a disulphide bridge, with Minimal Inhibitory Concentration value of 10.1 microM. In concentrations of up to 50 microM, Gr-1 and Gr2 didn't exceed 30% of hemolysis.

Characterization and purification of a new bacteriocin with a broad inhibitory spectrum produced by Lactobacillus plantarum lp 31 strain isolated from dry-fermented sausage.

AIMS: Characterization and purification of a new bacteriocin produced by Lactobacillus plantarum LP 31 strain, isolated from Argentinian dry-fermented sausage. METHODS AND RESULTS: Lactobacillus plantarum LP 31 strain produces an antimicrobial compound that inhibits the growth of food-borne pathogenic bacteria. It was inactivated by proteolytic enzymes, was stable to heat and catalase and exhibited maximum activity in the pH range from 5.0 to 6.0. Consequently, it was characterized as a bacteriocin. It was purified by RP (reverse-phase) solid-phase extraction, gel filtration chromatography and RP-HPLC. Plantaricin produced by Lact. plantarum LP 31 is a peptide with a molecular weight of 1558.85 Da as determined by Maldi-Tof mass spectrometry and contains 14 amino acid residues. It was shown to have a bactericidal effect against Pseudomonas sp., Staphylococcus aureus, Bacillus cereus and Listeria monocytogenes. CONCLUSIONS: The bacteriocin produced by Lact. plantarum LP 31 may be considered as a new plantaricin according to its low molecular weight and particular amino acid composition. SIGNIFICANCE AND IMPACT OF THE STUDY: In view of the interesting inhibitory spectrum of this bacteriocin and because of its good technological properties (resistance to heat and activity at acidic pH), this bacteriocin has potential applications as a biopreservative to prevent the growth of food-borne pathogens and food spoilage bacteria in certain food products.

[In-vitro activity of two hybrid synthetic peptides having antimicrobial activity against mycobacteria]. / Sensibilidad in vitro de micobacterias a dos péptidos sintéticos híbridos con actividad antimicrobiana.

The increase in both Mycobacterium tuberculosis human clinical isolates resistant to the essential drugs and cases of disseminated micobacteriosis due to Mycobacterium avium Complex, underlines the need to investigate new antimicobacterial agents. The antimicrobial peptides are a new group of active antibiotics with a particular mechanism of action. Some of them, like cecropin and melittin, isolated from insects, have demonstrated good in vitro activity against Gram-positive and Gram-negative bacteria. Synthetic hybrids of those peptides have been more active than individual peptides. In this study, the in vitro activity of two hybrid synthetic peptides from melittin and cecropin against M. tuberculosis, M. avium Complex, Mycobacterium fortuitum and Mycobacterium smegmatis has been evaluated. The minimal inhibitory concentration was determined by using the broth macrodilution technique. The minimal bactericide concentration in Lowenstein Jensen medium was then obtained. The peptides studied were active, in vitro, against M. smegmatis, but they did not show any activity against the other mycobacteria analyzed.

Antibodies and PMBC from EIAV infected carrier horses recognize gp45 and p26 synthetic peptides.

Equine infectious anemia virus (EIAV) is a lentivirus causing a persistent infection in horses characterized by recurrent febrile episodes and high levels of viremia associated with a novel antigenic strain of the virus. The virus contains two envelope glycoproteins, gp90 and gp45, and four internal proteins, p26, p15, p11 and p9. Considering that the most infected horses are able to restrict EIAV replication to very low levels and that gp45 and p26 contain highly conserved epitopes among lentiviruses, it would be necessary to identify those conserved epitopes stimulating cellular and humoral responses. The aims of this study were to determine if the synthetic peptides identified as gp45 (aa 523-547) and p26 (aa 318-346) representing two highly conserved and immunodominant regions of EIA virus are recognized by PBMC and antibodies to EIAV adult mixed-breed naturally infected carrier horses, and if these peptides are able to induce immune responses in mice. Antibodies from 100% of carrier horses, evaluated by ELISA, recognized both peptides; PBMC from 80% of carrier horses, evaluated by lymphoproliferation assay, recognized, at least, one peptide. Furthermore, immunization with 100 microg of each peptide elicited humoral and cellular responses in BALB/c mice, antibodies appeared at 48 or 63 days of immunization with gp45 or p26, respectively. Although the kinetics of gp45- and p26-specific antibody responses were similar, percentage of positivity was higher for gp45. The lymphoproliferation assay, evaluated by BrdU uptake, was higher in mice immunized with gp45 or p26 than in the control group (P<0.05). Based on our findings, we consider that both peptides could be included in an effective vaccine design to induce long-term immunological memory.

Antibody recognition of synthetic peptides mimicking immunodominant regions of HIV-1 p24 and p17 proteins / Reconocimiento por anticuerpos de péptidos sintéticos que imitan regiones inmunodominantes de las proteínas p24 y p17 de VIH-1

The gag gene of HIV-1 encodes a single open reading frame of 55 kDa that contains three subdomains: the matrix domain (p17), the capsid domain (p24) and the nucleocapsid domain (p15). The p24 and p17 proteins have a predominant a-helical structure and perform important functions throughout thevirallife-cycle. The determination of gag-specific antibodies is important because declining titers of these antibodies herald clinical deterioration.In this work we present the results obtained on immunoreactiviy of synthetic peptides that mimic immunogenic a-helical regions of p24 and p17. The influence on the immunoreactivity of structural modifications in native sequences, including the addition of non immunogenic side chains: AAAC- and -CAAA on both side of minimal epitopes was evaluated in indirect and competitive enzymeimmunoassays. The conformational characteristcs to the peptides were analysed by circular dichroism and these results were correlated with that obtained in the immunoassays. It was shown that the reactivity of peptides mimicking short a-helical regions of p24 and p17 is improved by adding short non immunogenic chains on both N- and C- terminus. These modifications enhanced the immobilization of the peptides onto the solid support and allowed more accesibility to the minimal epitopes byspecific antibodies, in solution.

El gen gag del VIH-1 codifica una región de 55kDA que contiene tres subdominios: matriz (p17), cápside (p24) y nucleocápside (p15). Las proteínas p24 y p17 tienen una estructura predominante helicoidal y cumplen un rol importante en el ciclo de vida del virus. En este trabajo presentamos los resultados de inmunorreactividad de péptidos sintéticos que imitan regiones helicoidales de p24 y p17. Utilizando enzimoinmunoensayos se evaluó la influencia de modificaciones en las secuencias nativas sobre la capacidad de reconocimiento de anticuerpos específicos en solución y en fase sólida, incluyendo el agregado de cadenas no inmunogénicas en ambos extremos de los epitopes mínimos. La conformación de los péptidos se determinó por dicroísmo circular y los resultados se correlacionaron con los de inmunorreactividad. Se observó que la capacidad de reconocimiento de anticuerpos por péptidos pequeños que imitan estructuras helicoidales de p24 y p17 mejoró con el agregado de cadenas no inmunogénicas en ambos extremos de los epitopes. Estas modificaciones mejoran la inmovilización sobre las superficies sólidas y permiten una mayor accesibilidad de los anticuerpos a los epitopes mínimos en solución.

Antibody recognition of synthetic peptides mimicking immunodominant regions of HIV-1 p24 and p17 proteins.

The gag gene of HIV-1 encodes a single open reading frame of 55 kDa that contains three subdomains: the matrix domain (p17), the capsid domain (p24) and the nucleocapsid domain (p15). The p24 and p17 proteins have a predominant alpha-helical structure and perform important functions throughout the viral life-cycle. The determination of gag-specific antibodies is important because declining titers of these antibodies herald clinical deterioration. In this work we present the results obtained on immunoreactiviy of synthetic peptides that mimic immunogenic alpha-helical regions of p24 and p17. The influence on the immunoreactivity of structural modifications in native sequences, including the addition of non immunogenic side chains: AAAC- and -CAAA on both side of minimal epitopes was evaluated in indirect and competitive enzyme immunoassays. The conformational characteristcs to the peptides were analysed by circular dichroism and these results were correlated with that obtained in the immunoassays. It was shown that the reactivity of peptides mimicking short alpha-helical regions of p24 and p17 is improved by adding short non immunogenic chains on both N- and C-terminus. These modifications enhanced the immobilization of the peptides onto the solid support and allowed more accessibility to the minimal epitopes by specific antibodies, in solution.

Antibody recognition of synthetic peptides mimicking immunodominant regions of HIV-1 p24 and p17 proteins.

The gag gene of HIV-1 encodes a single open reading frame of 55 kDa that contains three subdomains: the matrix domain (p17), the capsid domain (p24) and the nucleocapsid domain (p15). The p24 and p17 proteins have a predominant alpha-helical structure and perform important functions throughout the viral life-cycle. The determination of gag-specific antibodies is important because declining titers of these antibodies herald clinical deterioration. In this work we present the results obtained on immunoreactiviy of synthetic peptides that mimic immunogenic alpha-helical regions of p24 and p17. The influence on the immunoreactivity of structural modifications in native sequences, including the addition of non immunogenic side chains: AAAC- and -CAAA on both side of minimal epitopes was evaluated in indirect and competitive enzyme immunoassays. The conformational characteristcs to the peptides were analysed by circular dichroism and these results were correlated with that obtained in the immunoassays. It was shown that the reactivity of peptides mimicking short alpha-helical regions of p24 and p17 is improved by adding short non immunogenic chains on both N- and C-terminus. These modifications enhanced the immobilization of the peptides onto the solid support and allowed more accessibility to the minimal epitopes by specific antibodies, in solution.

[Evaluation of synthetic peptides for the detection of antibodies against human immunodeficiency virus]. / Evaluación de péptidos sintéticos para la detección de anticuerpos contra el virus de inmunodeficiencia humana.

The serologic diagnosis of human immunodeficiency virus (HIV) infection is currently done by detecting the presence of antibodies against the different antigenic viral proteins through immunoassays and later confirmation by Western blot. Several types of antigens can be used in immunoassays, but recombinant proteins and synthetic peptides are the most frequently used. In this paper, peptides mimicking antigenic regions from p24 (region 196-224), gp41 (region 600-614) and gp120 (region 303-338, Loop V3) proteins of HIV-1 have been used as antigens in an enzyme-linked immunosorbent assay and their reactivity was screened against a panel of positive and negative sera. Six antigenic mixtures containing different amounts of each peptide were prepared, and the one consisting of 1 microgram of gp41-15, 0.5 microgram of p24-1 and 0.5 microgram of gp120-1 per well has shown the best performance to differentiate positive and negative serum samples, with sensitivity and specificity values of 99.18% and 100%, respectively. Considering the potential utilization of this system for screening of HIV infection, it would be relevant to evaluate the additional incorporation of sequences derived from Argentine local circulating viral variants to improve the diagnostic sensitivity of the assay, allowing the development of an ELISA based on specific viral sequences.

Evaluación de péptidos sintéticos para la detección de anticuerpos contra el virus de inmunodeficiencia humana. / [Evaluation of synthetic peptides for the detection of antibodies against human immunodeficiency virus]

The serologic diagnosis of human immunodeficiency virus (HIV) infection is currently done by detecting the presence of antibodies against the different antigenic viral proteins through immunoassays and later confirmation by Western blot. Several types of antigens can be used in immunoassays, but recombinant proteins and synthetic peptides are the most frequently used. In this paper, peptides mimicking antigenic regions from p24 (region 196-224), gp41 (region 600-614) and gp120 (region 303-338, Loop V3) proteins of HIV-1 have been used as antigens in an enzyme-linked immunosorbent assay and their reactivity was screened against a panel of positive and negative sera. Six antigenic mixtures containing different amounts of each peptide were prepared, and the one consisting of 1 microgram of gp41-15, 0.5 microgram of p24-1 and 0.5 microgram of gp120-1 per well has shown the best performance to differentiate positive and negative serum samples, with sensitivity and specificity values of 99.18

and 100

, respectively. Considering the potential utilization of this system for screening of HIV infection, it would be relevant to evaluate the additional incorporation of sequences derived from Argentine local circulating viral variants to improve the diagnostic sensitivity of the assay, allowing the development of an ELISA based on specific viral sequences.

Evaluación de péptidos sintéticos para la detección de anticuerpos contra el virus de inmunodeficiencia humana / [Evaluation of synthetic peptides for the detection of antibodies against human immunodeficiency virus]

The serologic diagnosis of human immunodeficiency virus (HIV) infection is currently done by detecting the presence of antibodies against the different antigenic viral proteins through immunoassays and later confirmation by Western blot. Several types of antigens can be used in immunoassays, but recombinant proteins and synthetic peptides are the most frequently used. In this paper, peptides mimicking antigenic regions from p24 (region 196-224), gp41 (region 600-614) and gp120 (region 303-338, Loop V3) proteins of HIV-1 have been used as antigens in an enzyme-linked immunosorbent assay and their reactivity was screened against a panel of positive and negative sera. Six antigenic mixtures containing different amounts of each peptide were prepared, and the one consisting of 1 microgram of gp41-15, 0.5 microgram of p24-1 and 0.5 microgram of gp120-1 per well has shown the best performance to differentiate positive and negative serum samples, with sensitivity and specificity values of 99.18

, respectively. Considering the potential utilization of this system for screening of HIV infection, it would be relevant to evaluate the additional incorporation of sequences derived from Argentine local circulating viral variants to improve the diagnostic sensitivity of the assay, allowing the development of an ELISA based on specific viral sequences.

Design and validation of an ELISA for equine infectious anemia (EIA) diagnosis using synthetic peptides.

Three peptides derived from the equine infectious anemia virus (EIAV) surface proteins were synthesized to design and validate an ELISA for EIA diagnosis. Peptides identified as gp90-I and gp90-II correspond to the N- and C-terminal part of the surface glycoprotein gp90. Peptide gp45-1 overlaps the immunodominant epitope CIERTHVFC of the transmembrane glycoprotein gp45, and includes a hydrophilic chain close to the N-terminal end of this nonapeptide loop. Serum samples from 140 naturally infected horses with EIAV and a panel of 167 non-immune equine sera obtained from non-infected animals were used. Differences in reactivity between positive and negative serum samples were clearly distinguished. Samples considered weak positive to the agar gel immunodiffusion (AGID) test were "true" positive in the ELISA. These results are consistent with the improved sensitivity of the ELISA in comparison with the AGID test. The cyclic peptide that mimics the immunodominant sequence of gp45 showed excellent reactivity, thus suggesting that its functional activity depends significantly on its conformation, since very low reactivity was observed in the linear form of the peptide. The detectability indices of positive and negative sera reached 98% when gp90-II and gp45-I synthetic peptides were used in the same assay, illustrating the high specificity and sensitivity of the assay. Our study represents a first approach for the design of a diagnostic kit, which would allow the rapid analysis of a large numbers of serum samples from horses, and could be applied in endemic areas with different prevalence of infection.

Differential distribution of gonadotropin-releasing hormone variants in the brain of Hydrochaeris hydrochaeris (Mammalia, Rodentia).

1. In a previous paper we reported evidence for the presence of mGnRH- and sGnRH-like peptides in the preoptic-hypothalamic region of the capybara Hydrochaeris hydrochaeris (Montaner et al., 1998). In that study, the presence of a cGnRH-II like molecule in olfactory bulb extracts was suggested. 2. The capybara, the largest living rodent in the world, belongs to the order Hystricomorpha, which is considered to be one of the oldest groups of rodents. Some authors consider that this group is the ancestor of all remaining rodents. 3. In this study we have characterized GnRH molecular variants found in extracts from the olfactory bulbs and the mesencephalic region of capybara. These regions represent the two GnRH neuronal systems: the terminal nerve-septopreoptic and the midbrain systems. 4. An indirect method combining reverse-phase high-performance liquid chromatography (RP-HPLC) and radioimmunoassay (RIA) was used to characterize GnRH variants. The analysis of both extracts with two different RIA systems revealed three immunoreactive GnRH peaks, coeluting with mGnRH, cIIGnRH, and sGnRH synthetic standards. These results were additionally supported by serial dilution studies with specific antisera. 5. To our knowledge this the first report on the presence of three GnRH variants in the brain of an eutherian mammal. These results suggest that, similarly to other vertebrates, the expression of multiple GnRH variants may also be a common pattern in mammals.

[Utility of gas chromatography for the identification of mycobacterial species]. / Utilidad de la cromatografia gaseosa para la identificación de micobacterias.

The human immunodeficiency virus (HIV) epidemic has altered the epidemiological profile of tuberculosis in both industrialized and developing countries. Serious diseases caused by mycobacteria other than Mycobacterium tuberculosis, mostly belonging to the M. avium-intracellulare complex (MAC), have become very common in association with severe immunosuppression. The increase in mycobacterial disease complexity has stimulated the development of more rapid and efficient methods for diagnosis. In the present study, we investigated and assessed the suitability of a gas-liquid chromatography technique for diagnosis of clinically important mycobacteria in Argentina. An identification scheme was developed from the results obtained in a previous study where we characterized the cellular fatty acids and the mycolic acid cleavage products from most frequent species in Argentina. Of 183 isolates tested, 69% were correctly identified to species level and 5% were incorrectly classified. If we only take into account the isolates that could be identified, 93% were correctly identified. Although all of the isolates of M. tuberculosis were correctly identified, four isolates of MAC incorrectly matched by M. tuberculosis. Gas chromatography provides a rapid technique of highly predictive value for mycobacteria identification; it could be used in reference laboratories as a rapid presumptive identification until the biochemical tests are completed.

Caracterización de ácidos grasos y productos de degradación de ácidos micólicos en especies micobacterianas de mayor incidencia en Argentina / Characterization of fatty acids and mycolic acid cleavage products in mycobacteria of major incidence in Argentina

El virus de la inmunodeficiencia humana (HIV) causa un profundo impacto sobre el problema de la tuberculosis tanto en los países industrializados como en los en vías de desarrollo. Enfermedades graves causadas por micobacterias no tuberculosas, la mayoría correspondiente al complejo Mycobacterium avium-intracellulare (MAC), se han vuelto muy comunes en asociación con la inmunosupresión severa. El aumento de la complejidad de las enfermedades micobacterianas ha estimulado el desarrollo de métodos de diagnóstico más rápidos y eficientes. En el presente estudio se caracterizaron los ácidos grasos y los productos de degradación de los ácidos micólicos celulares de las especies micobacterianas más frecuentes en la Argentina empleando cromatografía gaseosa (CG), para luego poder desarrollar una técnica rápida de identificación de especies. Los ácidos grasos y los ácidos micólicos de las células micobacterianas saponificadas fueron analizados como ésteres metílicos por CG capilar. Los principales ácidos grasos detectados en todas las especies estudiadas, con excepción de M. smegmatis, fueron los ácidos octadecenoico (18:1) y hexadecanoico (16:0). Los perfiles cromatográficos presentaron diferencias cuantitativas y no cualitativas entre las distintas especies. El ácido tuberculoesteárico se detectó en todas las micobacteias analizadas. Se observaron diferencias significativas (p<0,01) en las medias de las cantidades relativas de algunos ácidos grasos entre aislamientos clínicos de M. tuberculosis, M. bovis y MAC. Se detectaron trazas de 2-elcosanol en cepas de M. tuberculosis H37Rv. Aunque se estudió un número limitado de cepas y de especies, los resultados preliminares indican que este método podría ser usado para caracterizar cultivos micobacterianos (AU)

Caracterización de ácidos grasos y productos de degradación de ácidos micólicos en especies micobacterianas de mayor incidencia en Argentina / Characterization of fatty acids and mycolic acid cleavage products in mycobacteria of major incidence in Argentina

El virus de la inmunodeficiencia humana (HIV) causa un profundo impacto sobre el problema de la tuberculosis tanto en los países industrializados como en los en vías de desarrollo. Enfermedades graves causadas por micobacterias no tuberculosas, la mayoría correspondiente al complejo Mycobacterium avium-intracellulare (MAC), se han vuelto muy comunes en asociación con la inmunosupresión severa. El aumento de la complejidad de las enfermedades micobacterianas ha estimulado el desarrollo de métodos de diagnóstico más rápidos y eficientes. En el presente estudio se caracterizaron los ácidos grasos y los productos de degradación de los ácidos micólicos celulares de las especies micobacterianas más frecuentes en la Argentina empleando cromatografía gaseosa (CG), para luego poder desarrollar una técnica rápida de identificación de especies. Los ácidos grasos y los ácidos micólicos de las células micobacterianas saponificadas fueron analizados como ésteres metílicos por CG capilar. Los principales ácidos grasos detectados en todas las especies estudiadas, con excepción de M. smegmatis, fueron los ácidos octadecenoico (18:1) y hexadecanoico (16:0). Los perfiles cromatográficos presentaron diferencias cuantitativas y no cualitativas entre las distintas especies. El ácido tuberculoesteárico se detectó en todas las micobacteias analizadas. Se observaron diferencias significativas (p<0,01) en las medias de las cantidades relativas de algunos ácidos grasos entre aislamientos clínicos de M. tuberculosis, M. bovis y MAC. Se detectaron trazas de 2-elcosanol en cepas de M. tuberculosis H37Rv. Aunque se estudió un número limitado de cepas y de especies, los resultados preliminares indican que este método podría ser usado para caracterizar cultivos micobacterianos

[Characterization of fatty acids and mycolic acid degradation products in mycobacterial species of major incidence in Argentina]. / Caracterización de ácidos grasos y productos de degradación de ácidos micólicos en especies micobacterianas de mayor incidenica en Argentina.

The human immunodeficiency virus (HIV) epidemic has altered the epidemiological profile of tuberculosis in both industrialized and developing countries. Serious diseases caused by mycobacteria other that M. tuberculosis, mostly belonging to the M. avium-intracellulare complex (MAC), have become very common in association with severe immunosuppression. The increase in mycobacterial disease complexity has stimulated the development of more rapid and efficient methods of diagnosis. In the present study we characterized the cellular fatty acids and the mycolic acid cleavage product from most frequent mycobacteria species in Argentina using gas chromatography in order to develop a rapid technique for their identification. Fatty acids and mycolic acids extracted from saponified mycobacterial cells were examined as methyl esters by capillary has chromatography. The major constituent fatty acids in all species, with the exception of M. smegmatis, were octadecenoic (18:1) and hexadecanoic (16:1) acids. The fatty acids and mycolic acid cleavage product profiles from the studied species were quantitatively but not qualitatively different. Tuberculostearic acid was found in all species. Significantly different amounts of some fatty acids (p < 0.01) were observed among clinical isolates of M. tuberculosis, M. bovis and MAC. Traces of 2-eicosanol were detected in the M. tuberculosis H37Rv strain. Although a limited number of strains and species were tested, preliminary results indicate that this method could be used to characterize mycobacterial cultures.

Epitope contiguous chains and antibody recognition in HIV-1 synthetic peptide antigens.

Five peptides derived from human immuno deficiency virus (HIV-1) gp41 transmembrane protein have been synthesized: M9 (610-618), M12 (598-609), M15 (600-614), M21 (584-604) and M23 (587-609). These sequences partially overlap in the region vicinal to the immunodominant epitope CSGKLIC, between two cysteine residues 603-609 and three of them (M12, M15 and M23) include this complete heptapeptide. M23, the longer peptide, includes an hydrophilic chain in addition to the heptapeptide loop. The purpose of this work was to determine the influence of contiguous chains to the heptapeptide loop on antibody recognition in fluid and solid phases, and dissociation constants (KD) of each sequence with human anti-HIV-1 antibodies. Two peptides, M13 and M23, overlapped on this loop, were found to be more reactive. Antigen-antibody dissociation constants were determined for both peptides by competition enzyme-linked immunosorbent assay, using each peptide alternatively as the solid phase-immobilized antigen. In addition to the influence of solid-phase antigen on calculated dissociation constants (a phenomenon described by Seligman, 1994), the inhibitory effect of M15 in liquid phase on antibody binding to solid phase M23 was higher than exerted by M23 in solution over antibody binding to M15 on solid phase. On the basis of peptide sequence and predicted antigenicity, this behavior appeared to be contradictory. It is assured that the possible origin of this phenomenon is due to unfavorable conformation of the longer peptide. Even though synthetic peptides mimic mainly sequential epitopes, conformational preferences in fluid or solid phase play an important role in epitope functionality. In particular, addition of residues to known immunodominant sequences may not always amplify antibody recognition if conformation provokes steric hindrance in the native epitope.