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J A Giménez

Publications and source records attributed to J A Giménez.

15 recordsLinked to original sources
PubMed2005

Could the homologous sequence of anti-inflammatory pentapeptide (MLIF) produced by Entamoeba histolytica in the N protein of rabies virus affect the inflammatory process?

Amebiasis and rabies are public health problems, and they have in common a poor inflammatory effect in the target organs that they affect. In the GenBank, it was found that the anti-inflammatory peptide monocyte locomotion inhibitory factor (MLIF) produced by Entamoeba histolytica homologates 80%, with a fragment of the N protein of the rabies virus. We speculated if the N protein could contribute to the scant inflammatory reaction produced by rabies virus in central nervous system. The N protein was obtained and studied in vitro and in vivo. The N protein, as MLIF, inhibited the respiratory burst in human mononuclear phagocytes (43%, p<0.05), but in contrast to MLIF, it increased chemotaxis and it did not significantly inhibit delayed hypersensitivity skin reaction to 1-chloro-2-4-dinitrobenzene in guinea pigs. Therefore, the full peptide sequence has to be present or it has to be cleaved-free from the large recombinant N protein molecule (55 kDa) to become active.

Animals↗
PubMed2003

The monocyte locomotion inhibitory factor produced by Entamoeba histolytica inhibits induced nitric oxide production in human leukocytes.

The monocyte locomotion inhibitory factor, an anti-inflammatory pentapeptide produced by Entamoeba histolytica, inhibits the in vitro production of nitric oxide induced by cytokines (INF-gamma, TNF-alpha) or PMA in human leukocytes. This can be added to the other previously reported functional effects of this factor, such as the inhibition of monocyte locomotion and the synthesis of reactive oxygen intermediates in both monocytes and neutrophils. The decreased nitric oxide production may interfere with the killing of amebas by neutrophils in the early invasive stages of amebiasis, when oxidative mechanisms are used [reactive oxygen and nitrogen intermediates either individually or synergistically via peroxynitrite (ONOO(-))], and in the advanced stages, when both non-oxidative and oxidative (including nitric oxide) mechanisms are employed by macrophages. Diminished nitric oxide production by leukocytes may also contribute to the paucity of late inflammatory components in amebic abscess of the liver and other amebic lesions.

Animals↗
PubMed2001

A novel anti-inflammatory oligopeptide produced by Entamoeba histolytica.

The monocyte locomotion inhibitory factor (MLIF), a heat-stable oligopeptide found in the supernatant fluid of Entamoeba histolytica axenic cultures was isolated by ultra-filtration, gel-sieve chromatography and high powered liquid chromatography (HPLC), and its primary structure (Met-Gln-Cys-Asn-Ser) established by Edman sequencing and mass-spectrometry (MS). A synthetic peptide had the same selective anti-inflammatory features as the native material in comparable concentrations: in vitro inhibition of the locomotion in human peripheral blood monocytes, and of the respiratory burst in the same cells and in human neutrophil polymorphonuclear leucocytes; and in vivo depression of delayed hypersensitivity skin reactions to dinitrochlorobenzene in guinea pigs. This oligopeptide is apparently synthesized by the ameba as suggested by [(35)S]-Cys and Met incorporation, probably as part of a larger molecule, from which it is cleaved by proteolysis. The full sequence was not found in the 431 available E. histolytica protein sequences. The factor may contribute to the unexpected paucity of the late inflammatory reaction found in advanced invasive amebiasis and, perhaps in consequence, to the regeneration without scarring (restitutio ad integrum) of the affected organs that is observed following successful treatment of this disease

Adult↗
PubMed1995

The typing of botulinal neurotoxins.

The serological identification of Clostridium botulinum neurotoxin (BoNT) subtypes has shown to be elusive when current standard serologic tests are used. Based on (1) the in vivo response expected on quantitative BoNT-antitoxin systems and (2) the actual and the hypothetical antigenic makeup of BoNT subtypes, a comprehensive method for BoNTs typing is proposed.

Bacterial Typing Techniques↗
PubMed1992

Pepsin fragmentation of botulinum type E neurotoxin: isolation and characterization of 112, 48, 46, and 16 kD fragments.

Controlled digestion of approximately 150 kD single chain botulinum type E neurotoxin with pepsin at pH 6.0 produced 112, 48, 46, and 16 kD fragments. These were chromatographically purified; their locations in the approximately 1300 amino acid residue long neurotoxin were determined by identifying the amino terminal 10 residues of 112 and 48 kD fragments, 50 residues of 46 kD fragment, and 59 residues of 16 kD fragment. The 48 and 112 kD fragments contain the N-terminal segment of the neurotoxin (i.e., residue no. 1 to approximately 425 and 1 to approximately 990, respectively), the 46 kD fragment corresponds to approximately 407 residues of the C-terminal region, and the 16 kD fragment contains the approximately 140 residues from a segment nearer to the C-terminus. The 48 kD fragment is similar to the approximately 50 kD N-terminal light chain of the approximately 150 kD dichain neurotoxin, which is generated by tryptic cleavage of the approximately 150 kD single chain neurotoxin, and is separated from the approximately 100 kD C-terminal heavy chain by dithiothreitol (DTT) reduction of an intrachain disulfide bond in the presence of 2 M urea (Sathyamoorthy and DasGupta, J. Biol. Chem. 260, 10461, 1985). The pepsin-generated 48 kD fragment, unlike the light chain, was isolated without exposure to DTT and urea. The single chain 112 kD fragment following trypsin digestion yielded 48 and 60 kD fragments that were separable after DTT reduction of the intrachain disulfide which links them. The N-terminal residues of the smaller fragment were identical to that of the single chain 150 kD neurotoxin; the single chain 112 kD fragment is therefore the neurotoxin minus the approximately 50 kD C-terminal half of the heavy chain. The biological activities of the 48 and 112 kD fragments can be demonstrated in permeabilized PC12 cells (Lomneth et al., J. Neurochem. 57, 1413, 1991); they inhibit norepinephrine release.

Amino Acid Sequence↗
PubMed1992

Characterization of the neurotoxin isolated from a Clostridium baratii strain implicated in infant botulism.

Botulism is widely known to result from ingestion of food containing botulinum neurotoxin produced in situ by certain strains of Clostridium botulinum. Infant botulism caused by C. botulinum, unlike the food-borne intoxication, is the toxicoinfectious form of botulism (S. S. Arnon, p. 331-345, in G. E. Lewis, ed., Biomedical Aspects of Botulism, 1981). The strain of Clostridium baratii implicated in infant botulism produced a neurotoxin that was neutralized with antiserum for botulinum neurotoxin serotype F (J. D. Hall, L. M. McCroskey, B. J. Pincomb, and C. L. Hatheway, J. Clin. Microbiol. 21:654-655, 1985). We developed a procedure to culture the toxigenic C. baratii (strain 6341) in dialysis bags and a simple purification scheme (precipitation of 900-ml culture supernatant with ammonium sulfate and two anion-exchange chromatographic steps at pH 5.5 and 8.0) that yielded up to 150 micrograms of purified neurotoxin. It is an approximately 140-kDa single-chain protein and has the following sequence of amino acid residues at the N terminus: Pro-Val-Asn-Ile-Asn-Asn-Phe-Asn-Tyr-Asn-Asp-Pro-Ile-Asn-Asn-Thr-Thr-Ile- Leu. Comparison of this amino acid sequence with those of the botulinum neurotoxin serotypes A, B, and E showed 40 to 50% identical residues in comparable positions. The specific toxicity of the neurotoxin, approximately 2 x 10(6) 50% lethal doses for mice per mg of protein injected, was not enhanced significantly by mild trypsinization, although the protease cleaved the neurotoxin within a disulfide loop that generated at least two primary fragments, approximately 47 and approximately 86 kDa, that remained linked by an interchain disulfide. These two fragments resembled the light and heavy chains of the well-characterized neurotoxin serotypes A, B, C, D, E, and F produced by C. botulinum.

Amino Acid Sequence↗
PubMed1991

Comparative molecular topography of botulinum neurotoxins from Clostridium butyricum and Clostridium botulinum type E.

Production of botulinum-like neurotoxin by a non-Clostridium botulinum organism has profound implications in the epidemiology of the disease botulism. Molecular topography of the approximately 150 kDa neurotoxic protein produced by Clostridium butyricum (strain 5839) and its activation kinetics were examined and compared with a serologically related botulinum neurotoxin produced by C. botulinum type E to further characterize the butyricum neurotoxin. Botulinum neurotoxin was fully activated within 30 min of incubation with trypsin, whereas butyricum neurotoxin achieved maximum activation within 5 min of incubation. Molecular topography of the two neurotoxins was analyzed in terms of secondary structures and the surface accessibilities of the polypeptide domains containing aromatic amino acids. The secondary structure parameters of the butyricum neurotoxin (alpha-helix 22%, beta-sheet 41% and random coil 37%), as estimated from the far ultraviolet circular dichroic spectra, appeared similar to that of botulinum neurotoxin. (Singh, B.R. and DasGupta, B.R., (1989) Mol. Cell. Biochem. 86, 87). Second derivative ultraviolet spectral analysis revealed 37 and 41 Tyr residues exposed on the surface of butyricum and botulinum neurotoxins, respectively, suggesting a differential surface accessibility of polypeptide segments containing Tyr residues. Fluorescent Trp residues in both the botulinum type E and butyricum neurotoxins were in a relatively hydrophobic environment as indicated by the blue-shifted emission maxima (334 nm). About half of the fluorescent Trp residues of both proteins were accessible to acrylamide, a neutral fluorescence quencher, and appeared to be in a similar molecular environment. The ionic surface probe, I-, quenched the Trp fluorescence of botulinum significantly, but not that of butyricum neurotoxin. Thus, a considerable number of fluorescent Trp residues were apparently located on the surface of the botulinum, but not on that of the butyricum neurotoxin. Botulinum and butyricum neurotoxins, indistinguishable by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, migrated differently in the absence of sodium dodecyl sulfate suggesting difference(s) in their surface charge distribution. These results provide the first report of the secondary and tertiary structure parameters of the neurotoxin produced by a non-botulinum species and comparison of the molecular topography of the neurotoxin with the antigenically related botulinum neurotoxin type E.

Animals↗
PubMed1990

Botulinum neurotoxin type E fragmented with endoproteinase Lys-C reveals the site trypsin nicks and homology with tetanus neurotoxin.

Botulinum neurotoxin type E, a 150 kDa single chain protein, cleaved with endoproteinase Lys-C yielded 113, 73, and 50 kDa fragments. The N-terminal sequence of the 113 kDa fragment, Gly-Ile-Arg-Lys-Ser-Ile-Cys-Ile, overlaps the N-terminal sequence, Lys-Ser-Ile-Cys-Ile, of the 103 kDa heavy chain produced by nicking the neurotoxin with trypsin. The -Arg-Lys- bond is therefore the site on the single chain type E NT where trypsin nicks generating the 50 kDa light and 103 kDa heavy chains of the dichain NT. The sequence of the first 50 N-terminal residues of the 73 kDa fragment were determined. This fragment is a segment of the heavy chain; 50% of the 50 residues are present in identical positions in a similar segment of the heavy chain of tetanus neurotoxin.

Amino Acid Sequence↗
PubMed1988

Comparison of toxins of Clostridium butyricum and Clostridium botulinum type E.

The toxin of Clostridium butyricum strains isolated from two infants with botulism is neutralized by antitoxin for type E botulinum toxin. This toxin and that of a C. botulinum type E strain were purified by the same protocol. Both toxins were Mr 145,000 proteins which, when activated with trypsin, were composed of an H subunit of Mr 105,000 and an L subunit of Mr 50,000. The activated specific toxicity of purified butyricum toxin based on an intravenous assay was 2 X 10(8) mouse 50% lethal doses (LD50s)/mg of protein, but that based on an intraperitoneal assay was 7 X 10(7) LD50s/mg, compared with 6 X 10(7) LD50s/mg for type E toxin as determined by both methods. Immunodiffusion tests with antitoxin raised with type E toxin indicated that the two toxins were serologically very similar except for a spur formed by type E toxin. The close similarities of the two toxins suggest that toxigenic C. butyricum could arise when a wild-type strain, which is normally nontoxigenic, acquires the toxin gene of a C. botulinum type E strain.

Bacterial Toxins↗
PubMed1987

Characterization of proteins, insoluble at low temperature, produced by Clostridium botulinum type C and C. subterminale. Their antigenic relationship with a similar protein synthesized by C. botulinum type G.

The production of a protein insoluble at low temperature ("cryoprotein"), by cultures of Clostridium botulinum type G has been shown to be a metabolic characteristic also shared by C. botulinum type C and by C. subterminale. These new cryoproteins have been purified and some of their chemical and immunological properties studied. It was found that both proteins were chemically very similar among themselves and to the cryoprotein isolated from C. botulinum type G. All these proteins are formed by a single polypeptide chain of approximately Mr = 180,000, with closely related amino acid compositions, isoelectric points and do not contain either free cysteine or disulfide bridges. Homologous and heterologous radioimmunoassays established the existence of an antigenic similitude among the cryoproteins from C. botulinum type G and C. subterminale thus becoming the first purified antigens which relate both bacterial species. If the production of cryoproteins can be shown to be a generalized phenomenon within the genus Clostridium these substances would provide an important tool to examine immunological and genetical relatedness between strains in this bacterial group.

Amino Acids↗
PubMed1987

Simplified purification method for Clostridium botulinum type E toxin.

Clostridium botulinum type E toxin was purified in three chromatography steps. Toxin extracted from cells was concentrated by precipitation and dissolving in a small volume of citrate buffer. When the extract was chromatographed on DEAE-Sephadex without RNase or protamine treatment, the first protein peak had most of the toxin but little nucleic acid. When the toxic pool was applied to a carboxymethyl Sepharose column, toxin was recovered in the first protein peak in its bimolecular complex form. The final chromatography step at 4 degrees C on a DEAE-Sephacel column at a slightly alkaline pH purified the toxin (Mr, 145,000) by separating the nontoxic protein from the complex. At least 1.5 mg of pure toxin was obtained from each liter of culture, and the toxicity was 6 X 10(7) 50% lethal doses per mg of protein. These values are significantly higher than those previously reported.

Botulinum Toxins↗
PubMed1986

Molecular characterization of a protein, insoluble at low temperature, produced by Clostridium botulinum type G.

A preliminary study of a low-toxicity protein, called cryoprotein, produced by Clostridium botulinum type G, led to a better characterization of this substance and to discriminate its relationship with type G botulinum toxin. This sparingly soluble protein has been characterized as an aggregated form of a soluble precursor with an Mr of 170,000. This phenomenon is temperature-dependent. The monomeric protein is usually contaminated with a lower Mr form (150,000) quite probably originated by a limited proteolytic process. The amino acid composition of this protein is relatively analogous to that of the botulinum toxins A and B, the only notable exception being the absence of cysteine. The N-terminal amino acid is alanine and the C-terminal sequence is Val-Ala-Leu-OH. The low toxicity which is usually demonstrable in samples of this protein disappears after a reductive treatment, strongly suggesting that it is not an intrinsic property. Taking into account that some of its physiochemical properties are similar to those of the known botulinal toxins, it is quite probable that this substance accompanies G toxin preparations currently obtained by routine methods, increasing its non-toxic antigenic mass. This fact could be critical to the sensitivity and specificity of G toxin immunological detection methods.

Amino Acids↗
PubMed1983

[Identification of strain B 657 of Clostridium botulinum].

Strain 657 has been described as a toxin variant of Clostridium botulinum type B. Toxin neutralization tests performed with types B and A botulinum antitoxins of known potency, avidity and specificity at 20, 200, 2.000 and 20.000 DL50/mouse level of testing have shown that 657 toxin is a mixture of B (90 to 99% of the complex) and A antigenic fractions. The possibility of a cross contamination between A and B serotypes has been, in principle, ruled out through the serologic screening of 33 toxin samples coming one each from 33 well isolated colonies taken from two colony variants of this strain. Strain 657 produces a new complex toxin and is proposed as the candidate for the prototype of a new C. botulinum serotype: subtype Ba.

Animals↗