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Biomedical subjects

R Perez-Montfort

Publications and source records attributed to R Perez-Montfort.

16 recordsLinked to original sources
PubMed1999

The efficacy of pentamidine combined with allopurinol and immunotherapy for the treatment of patients with diffuse cutaneous leishmaniasis.

A new treatment regimen was tested on patients with incurable diffuse cutaneous leshmaniasis (DCL) infected with Leishmania mexicana mexicana in Mexico. Two patients with advanced stages of the disease were treated with polychemotherapy (pentamidine and allopurinol) combined with recombinant human interferon-gamma (rIFN-gamma). For determination of the best medication, parasites isolated from patient lesions were exposed to available drugs both as promastigotes and as intracellular amastigotes. A synergistic effect was observed in vitro for the combination of pentamidine and allopurinol. Both patients were treated and recovered rapidly, but one of them developed insulin-dependent type I diabetes because of pentamidine toxicity. The complication was controlled and both patients were discharged with an apparent parasitologic cure, but after 3 months the two patients began to relapse. Our results suggest that allopurinol-pentamidine polychemotherapy, involving reduced dosage of pentamidine, combined with rIFN-gamma is an alternative for DCL patients infected with L. m. mexicana.

Adult↗
PubMed1998

Reactivation of triosephosphate isomerase from three trypanosomatids and human: effect of suramin.

The reactivation of the homodimeric triosephosphate isomerases (TIMs) from Trypanosoma brucei, T. cruzi, Leishmania mexicana and humans was determined after their denaturation with guanidine hydrochloride. In the range of 2-32 microg of T. brucei TIM per ml and 0.2-5 microg of the other enzymes per ml, the rate and extent of TIM reactivation depended on protein concentration, indicating that at these protein concentrations, the rate-limiting step of reactivation is monomer association and not monomer folding. The rate of monomer association was more than one order of magnitude lower in the T. brucei enzyme than in the other three enzymes. Suramin is a drug of choice in the treatment of sleeping sickness, but its mechanism of action is not known. At micromolar concentrations, Suramin inhibited the reactivation of the four enzymes, but the extent of inhibition by Suramin decreased with increasing protein concentration as consequence of a diminution of the life time of the folded monomer. Since the life time of the monomer of T. brucei TIM is longer than that of the other enzymes, Suramin is a more effective inhibitor of the reactivation of TIM from T. brucei, particularly at monomer concentrations above 1 microg of protein per ml (monomer concentration approx. 37 nM). Compounds that are structurally related to Suramin also inhibit TIM reactivation; their effect was about five times more pronounced in the enzyme from T. brucei than in human TIM.

Animals↗
PubMed1998

Expression and characterization of recombinant pyruvate phosphate dikinase from Entamoeba histolytica.

The parasite Entamoeba histolytica is an organism whose main energetic source comes from glycolysis. It has the singularity that several of its glycolytic enzymes use pyrophosphate as an alternative phosphate donor. Thus, pyruvate phosphate dikinase (PPDK), an inorganic pyrophosphate (PPi)-dependent enzyme, substitutes pyruvate kinase present in humans. We previously cloned and sequenced the gene that codifies for PPDK in E. histolytica. We now report its expression in a bacterial system and its purification to 98% homogeneity. We determined its K(m) for phosphoenolpyruvate, AMP and PPi (21, < 5 and 100 microM, respectively). Unlike PPDK from maize and bacteria and pyruvate kinase from other cells, EhPPDk is dependent on divalent cations but does not require monovalent cations for activity. The enzyme has an optimum pH of 6.0, it is labile to low temperatures and has a tetrameric structure. Since EhPPDK is a PPi-dependent enzyme, we also tested the effect of some pyrophosphate analogs as inhibitors of activity. Studies on the function and structure of this enzyme may be important for therapeutic research in several parasitic diseases, since it has no counterpart in humans.

Animals↗
PubMed1998

Differences in the intersubunit contacts in triosephosphate isomerase from two closely related pathogenic trypanosomes.

The aligned amino acid sequences of TIM from Trypanosoma cruzi (TcTIM) and Trypanosoma brucei (TbTIM) have a positional identity of 68%. The two enzymes have markedly similar catalytic properties. Agents that interact with their interface Cys inhibit TcTIM and TbTIM; and those TIMs that lack this Cys (such as human TIM) are largely or completely insensitive to these agents. The susceptibility of TcTIM to the agents is approximately 100 times higher than that of TbTIM. To ascertain the cause of this large difference, the crystal structure of TcTIM was solved at 1.83 A resolution. The two enzymes are very similar homodimers. In TcTIM and TbTIM their respective Cys, 15 or 14, forms part of the dimer interface. In both, the contacts of the Cys with residues of the other subunit are almost identical. Nevertheless, there are noteworthy differences between the two; the existence of glutamine 18 in TbTIM instead of glutamic acid in TcTIM at the beginning of helix 1 decreases the contacts between this portion of the protein and helix 3 of the other subunit. In addition, TcTIM has proline at position 24 in the first helix of the TIM barrel; this is absent in the other TIM. Pro24 disrupts the regular helix arrangement, making the pitch of this helix 1.2 A longer than in TbTIM. When Pro24 of TcTIM was substituted for Glu, the sensitivity of TcTIM to sulfhydryl reagents increased about fivefold, possibly as a consequence of an increase in the space between the first portion of helix 1 and helix 3 of the other subunit. Therefore, it may be concluded that the geometry of the latter region is central in the accessibility to agents that perturb the interface Cys. In human TIM this region is more compact.

Amino Acid Sequence↗
PubMed1998

Urokinase-type plasminogen activator and plasminogen activator inhibitors (PAI-1 and PAI-2) in extracts of invasive cervical carcinoma and precursor lesions.

In a previous study we reported a direct correlation between the degree of total proteolytic activity and the natural history of cervical carcinoma. The present work examined whether an increase in the urokinase-type plasminogen activator (uPA) and the plasminogen activator inhibitors (PAIs) is correlated with the natural history of cervical carcinoma. We measured uPA and PAI-1 activities and uPA, PAI-1 and PAI-2 antigen concentrations in cervical extracts from normal, squamous intraepithelial lesions (SIL) or invasive carcinoma patients. The uPA activity in invasive carcinoma extracts was 8.46 times that of normal extracts and 4.9 times that of SIL extracts. The PAI-1 activity in invasive carcinoma extracts was 1.3 times that of normal extracts and 1.24 times that of SIL extracts. uPA, PAI-1 and PAI-2 amounts were 25.7-, 12.1- and 7.9-fold higher, respectively, in invasive carcinoma than in SIL, and 39.1-, 21.38- and 27.3-fold higher, respectively, than in normal extracts. uPA and PAI-1 activities were 2.02- and 1.42-fold higher in extracts from patients with stages II-IV than those from stage I extracts, respectively. uPA, PAI-1 and PAI-2 amounts were 3.06-, 4.2- and 1.4-fold higher in extracts from patients with stages II-IV than those from stage I extracts, respectively. The increase in uPA activity and the antigen levels of uPA and PAIs (PAI-1 and PAI-2) in stages II-IV of invasive carcinoma of the cervix suggests that these components play an important role in invasion and metastasis in advanced stages of this tumour.

Adult↗
PubMed1995

Proteolytic activity in extracts of invasive cervical carcinoma and precursor lesions.

In this preliminary report, we showed that proteolytic activity of extracts from 85 cervical samples of patients with normal cervix, low and high-grade squamous intra-epithelial lesions and invasive carcinoma, increased according to the natural history of the cervical cancer when measured with three different substrates. Inhibitor assays for four different catalytic classes of endopeptidases indicated that the predominant catalytic class in extracts of all groups was that of metalloproteinases. Substrate gel electrophoresis revealed that invasive carcinoma extracts had two bands with proteolytic activity (with M(r) of 72 and 52 kDa) which were not present in normal tissue or biopsies with precursor lesions. Immunological and molecular characterization of these bands may provide information relevant to cervical cancer biology and clinical applications.

Adult↗
PubMed1990

Causes of the decrease in fluorescence due to proteolysis of alpha-casein.

Fluorescence decrease in casein solutions induced by proteolytic enzymes is mainly due to cleavage of alpha-casein, and in particular to alpha S1-casein, which is quantitatively the main component of commercial casein. Treatment of alpha-casein with o-iodosobenzoic acid, diminished its intrinsic fluorescence considerably and abolished the decrease in fluorescence induced by proteolytic cleavage. The carboxyterminal Trp at position 199 in alpha S1-casein contributes approximately 30% to the overall effect, while the Trp at position 164 contributes about 70%. Treatment of alpha-casein with cyanogen bromide lowered the initial fluorescence of the preparation, but, in the resulting fragment, trypsin still diminished some of the residual fluorescence. The velocity of decrease in fluorescence correlates with the distance from the Trp in position 164 at which the peptide bond is broken. This effect seems to be rather unique for the caseins, but particularly for alpha S1-casein; this is due to the existence of a Trp that is in the vicinity of hydrophobic amino acids and which upon hydrolysis, becomes exposed to a more hydrophilic environment.

Amino Acid Sequence↗
PubMed1989

Measurement of casein digestion by a fluorometric method.

A qualitative and quantitative method to assay proteolytic degradation of casein with a spectrofluorometer was developed. Proteolysis produced by different pure or mixed proteinases in a pH range 2 to 7.4 quenches the fluorescence emitted at a wavelength of 350 nm by casein excited at 300 nm in less than 5 min. This method is very sensitive, fast, and requires minimal sample preparation. Proteinases that do not generate peptides appropriate for fluorescence quenching cannot be detected with this assay and proteinases with intrinsic fluorescence may require special adjustments of the spectrofluorometer. This method monitors the disappearance of intact substrate proteins continuously, omitting the separation step necessary in other methods to measure product peptides.

Animals↗
PubMed1989

Proteinases of Entamoeba histolytica associated with different subcellular fractions.

Crude lysates of Entamoeba histolytica (strain HM 1:IMSS) analyzed by substrate gel electrophoresis in 12.5% acrylamide separating gels with reducing agents showed six hydrolysis zones with apparent molecular weights of 73,000 (high), 45,000, 36,000 (intermediate), 30,000, 26,000 and 23,000 (low molecular weight proteinases). Amebic lysates fractionated using the procedure of Aley et al. or the procedure of Rosenberg and Gitler and analyzed by the same method show all enzymes in the fractions with the soluble components and only the intermediate and low molecular weight proteinases in the fraction containing internal vesicles or membranes and plasma membrane. Some of these proteinases seem to be integral membrane proteins since they resist treatment with high salt, high urea buffer. All fractions are capable of digesting azocasein. Fractionation of amebic lysates by hydrophobic chromatography using phenyl-Sepharose or phase separation of amebic extracts with Triton X-114 show that proteinases with high, intermediate and low molecular weight behave as hydrophilic proteins while only proteinases of intermediate and low molecular weight behave as hydrophobic proteins. These results suggest that some proteinases are segregated in different compartments of the cell.

Animals↗
PubMed1987

Catalytic classes of proteinases of Entamoeba histolytica.

Endopeptidase inhibitors were used to determine the catalytic classes of proteinases present in extracts of Entamoeba histolytica (strain HM 1:IMSS) axenically grown in vitro. Cysteine proteinases account for most of the proteolytic activity; one or more proteinases with different catalytic mechanisms are also present but could not be unambiguously assigned to a particular catalytic class. Proteinases in amebic lysates were resolved by polyacrylamide gel electrophoresis with sodium dodecyl sulfate. The detergent was exchanged with Triton X-100 and the proteolytic activity in the gels was demonstrated by overlaying it on another gel containing the substrate. Four lysis zones were observed corresponding to molecular weights of 66,000, 56,000, 40,000 and 27,000. The first cannot be classified yet, but the last three showed properties consistent with those of cysteine proteinases. Finally, a novel technique is described which uses purified human alpha-2-macroglobulin to trap, purify and characterize proteases from amebic lysates. The results obtained with this technique confirm those of the overlay technique, since both methods reveal four distinct proteinases in the two different amebic preparations examined.

Animals↗
PubMed1985

Noncovalently and covalently bound lipid on the receptor for immunoglobulin E.

We describe experiments which indicate that lipids interact with the receptor for immunoglobulin E (IgE) in several ways. Evidence for loosely bound lipid comes from observations on the special conditions that are required in order to oxidatively iodinate the alpha chain of the receptor in IgE-receptor complexes. Evidence for tightly, but still noncovalently, bound lipids comes from studies on the lipids required to inhibit dissociation of the subunits of the receptor in micellar detergent. Finally, biosynthetic studies indicate that the subunits of the receptor contain ester-linked fatty acids. The latter modification appears to take place on receptors that have already been inserted into the plasma membrane.

Animals↗
PubMed1983

A previously unrecognized subunit of the receptor for immunoglobulin E.

Our laboratory previously found that under conditions that stabilized the interaction between the alpha and beta subunits of the receptor for immunoglobulin E, two new components were recovered having apparent molecular weights of 45 000 and 20 000, respectively. In this paper, we characterize the 20-kDa material. We demonstrate that it consists of a disulfide-linked dimer of 10-kDa polypeptides and that these have all the characteristics expected for subunits of the receptor. We propose that they be termed gamma chains and that the receptor consists of four chains: one alpha, one beta, and two gamma chains. The gamma chains share many of the labeling properties of the beta chain and, like the latter, are likely to be embedded in the plasma membrane and exposed on the internal but not the external surface of the bilayer.

Animals↗
PubMed1983

Covalent cross-linking of subunits of the receptor for immunoglobulin E induced by immunoprecipitation.

The receptor on rat basophilic leukemia and related normal cells that binds monomeric immunoglobulin E (IgE) with high affinity contains four polypeptide chains: alpha (to which the IgE binds), beta, and a disulfide-linked dimer of gamma chains. In this study, we have analyzed a further component variably seen when the purified receptors are analyzed on polyacrylamide gels. This component has an apparent Mr of approximately 43 000 and, after treatment with reducing agents, yields one beta and two gamma chains. This complex is generated by immunoprecipitation of preparations totally lacking in it. This novel in vitro phenomenon has provided additional information about the structure of the receptor. Its possible relationship to in vivo aggregation that triggers degranulation of the cells is of interest.

Animals↗
PubMed1983

Changes in the receptor for immunoglobulin E coincident with receptor-mediated stimulation of basophilic leukemia cells.

Aggregation of the receptor for immunoglobulin E on mast cells and related tumor cells initiates exocytosis. We examined tumor cells that had incorporated [3H]leucine and 32P to see if stimulating them produced modifications in the receptors themselves. No changes were observed in the yield of receptors or in the relative proportion and the molecular weights of their alpha, beta, and gamma subunits. In addition, no new "receptor-associated" components were observed. However, after the cells were stimulated, the gamma chains of the receptors showed an average 35% decrease in their associated 32P. Changes in the beta subunits were more variable but on the average showed a similar-sized increase in 32P. Using a novel protocol that permitted examination of aggregated and unaggregated receptors from the same cell, we found that changes in the unaggregated receptors were quantitatively indistinguishable from those exhibited by the aggregated receptors. These findings raise the possibility that the changes are related to one of the inactivation reactions thought to accompany the activation sequence.

Animals↗
PubMed1982

Proteolysis of soluble IgE-receptor complexes: localization of sites on IgE which interact with the Fc receptor.

Mouse and rat IgE and the respective soluble IgE-receptor complexes purified from rat basophilic leukemia cells were digested with trypsin. The end product in each case was F(ab')2-like. It contained the Ce2 regions, had intact antigen combining sites but had lost all ability to bind to the cell receptor for IgE. With mouse IgE the two principal sites of cleavage are likely to be the interdomain regions between Ce4:Ce3 and Ce3:C32 respectively. Cleavage at these sites occurs sequentially with the rate constant for the cleavage at the second site being approximately four-fold greater than that for the initial cleavage. When IgE is bound to the receptor the rates of cleavage are inhibited approximately three-fold. With rat IgE, the principal initial cleavage occurs within the intrachain disulfide loop in the Ce3 domain. Even when this disulfide bond in the digested protein is reduced, the product retains a substantial binding activity. A second cleavage occurs at a similar rate as the first and at a site analogous to that seen with mouse IgE, i.e. between the Ce3 and Ce2 domains. Notably, when bound to the receptor, the rate of cleavage at the first site is inhibited approximately three-fold but at the second site more than or equal to 40-fold. These results strongly implicate thd Ce3 domain as the principal site of interaction between rodent IgE and its receptor.

Amino Acid Sequence↗