Anti-VSG antibodies induce an increase in Trypanosoma evansi intracellular Ca2+ concentration.

Trypanosoma evansi and Trypanosoma vivax have shown a very high immunological cross-reactivity. Anti-T. vivax antibodies were used to monitor changes in the T. evansi intracellular Ca2+ concentration ([Ca2+]i) by fluorometric ratio imaging from single parasites. A short-time exposure of T. evansi parasites to sera from T. vivax-infected bovines induced an increase in [Ca2+]i, which generated their complete lysis. The parasite [Ca2+]i boost was reduced but not eliminated in the absence of extracellular Ca2+ or following serum decomplementation. Decomplemented anti-T. evansi VSG antibodies also produced an increase in the parasite [Ca2+]i, in the presence of extracellular Ca2+. Furthermore, this Ca2+ signal was reduced following blockage with Ni2+ or in the absence of extracellular Ca2+, suggesting that this response was a combination of an influx of Ca2+ throughout membrane channels and a release of this ion from intracellular stores. The observed Ca2+ signal was specific since (i) it was completely eliminated following pre-incubation of the anti-VSG antibodies with the purified soluble VSG, and (ii) affinity-purified anti-VSG antibodies also generated an increase in [Ca2+]i by measurements on single cells or parasite populations. We also showed that an increase of the T. evansi [Ca2+]i by the calcium A-23187 ionophore led to VSG release from the parasite surface. In addition, in vivo immunofluorescence labelling revealed that anti-VSG antibodies induced the formation of raft patches of VSG on the parasite surface. This is the first study to identify a ligand that is coupled to calcium flux in salivarian trypanosomes.

Field evaluation of four novel enzyme immunoassays for Chagas' disease in Venezuela blood banks: comparison of assays using fixed-epimastigotes, fixed-trypomastigotes or trypomastigote excreted-secreted antigens from two Trypanosoma cruzi strains.

Many serological tests have been developed for the diagnosis of Chagas' disease, but few have been subjected to a rigorous field evaluation. We have recently described several novel enzyme immunoassays (EIAs) based on fixed-whole organisms or trypomastigote excretory-secretory antigens (TESA) from different Trypanosoma cruzi strains (Tulahuen or Brazil). This study evaluated the most promising of these novel assays (e.g. fixed-epimastigotes, fixed-trypomastigotes, TESA Brazil and TESA Tulahuen antigens) in a field study of Venezuelan blood bank specimens. The assays were tested in an operator-blinded fashion using 2038 blood bank samples obtained from low and high T.cruzi prevalence regions of Venezuela (n= 1050 and n= 988 from Bolivar and Portuguesa states, respectively). Based on National Laboratory for Chagas Immunodiagnosis (NLCI) 'gold standard' results, all novel EIAs were superior to the commercial kit currently used in Venezuela, achieving 100% sensitivity and >99% specificity at optimal cut-off values. The novel assays identified seven false-negative samples compared with the routine screening performed by the Venezuelan blood bank although two samples were also misclassified as positive. Minor differences in the performance of the four novel assays were observed at lower arbitrary cut-off values. This study confirms the potential utility of both the fixed-organism and the TESA-based assays in the diagnosis of T.cruzi infection.

Tight binding between a pool of the heterodimeric alpha/beta tubulin and a protein kinase CK2 in Trypanosoma cruzi epimastigotes.

Tubulin is the predominant phosphoprotein in Trypanosoma cruzi epimastigotes and is phosphorylated by a protein kinase CK2. Interestingly, the presence or absence of divalent cations affected the solubilization of a pool of the parasite tubulin and the CK2 responsible for its phosphorylation. This fraction of tubulin and its kinase co-eluted using phosphocellulose, DEAE-Sepharose and Sephacryl S-300 chromatographies. Anti-alpha tubulin antibodies co-immunoprecipitated both tubulin and the CK2 responsible for its phosphorylation, and anti-CK2 alpha-subunit antibodies immunoprecipitated radioactively labelled alpha and beta tubulin from phosphorylated epimastigote homogenates. Additionally, native polyacrylamide gel electrophoresis of the purified and radioactively labelled fraction containing tubulin and its kinase demonstrated the phosphorylation of a unique band that reacted with both anti-CK2 alpha-subunit and anti-tubulin antibodies. Together, these results establish a strong interaction between a pool of the heterodimeric alpha/beta tubulin and a CK2 in this parasite. Hydrodynamic measurements indicated that the T. cruzi tubulin-CK2 complex is globular with an estimated size of 145.4-147.5 kDa.

Purification of a 64 kDa antigen from Trypanosoma evansi that exhibits cross-reactivity with Trypanosoma vivax.

Trypanosoma evansi and Trypanosoma vivax are the most extensively distributed trypanosomes responsible for diseases in livestock. Western blot and indirect immunofluorescence assays revealed a high immunological cross-reaction between these two parasites. An antigen with an apparent molecular mass of 64 kDa (p64), which exhibited cross-reactivity with T. vivax, was purified to homogeneity from a Venezuelan isolate of T. evansi. This antigen is glycosylated, contains a glycosyl-phosphatidylinositol anchor and appeared to be localized through the cell except in the nucleus, indicating that it could primarily be confined to the parasite surface. These results, together with its relative abundance and apparent molecular weight, suggest that p64 probably corresponds to the soluble form of a variable surface glycoprotein from T. evansi. Anti-p64 polyclonal antibodies, raised on mice, recognized a 53 kDa polypeptide band from a Venezuelan isolate of T. vivax on Western blots. Additionally, sera obtained from naturally infected animals also recognized p64, suggesting its potential use as a diagnostic reagent. Mild acid treatment only slightly decreased the immunorecognition of p64, suggesting its potential use as a diagnostic reagent. Mild acid treatment only slightly decreased the immunorecognition of p64, demonstrating that another relevant cross-reacting epitope, different than the inositol-1,2-cyclic phosphate of the cross-reacting determinant, must exist in p64. To date, p64 represents the first antigen isolated and partially characterized from T. evansi.

Chemical modification of transducin with iodoacetic acid: transducin-alpha carboxymethylated at Cys(347) allows transducin binding to Light-activated rhodopsin but prevents its release in the presence of GTP.

Modification of transducin (T) with iodoacetic acid (IAA) inhibited its light-dependent guanine nucleotide-binding activity. Approximately 1 mol of [(3)H]IAA was incorporated per mole of T. Cys(347), located on the alpha-subunit of T (T(alpha)), was identified as the major labeled residue in the [(3)H]IAA-modified holoenzyme. In contrast, Cys(135) and Cys(347) were modified with [(3)H]IAA in the isolated T(alpha). IAA-modified T was able to bind tightly to photoexcited rhodopsin (R*), but GTP did not promote the dissociation of the complex between alkylated T and R*. In addition, R* protected against the inhibition of T by IAA. A comparable inactivation of T and analogous interactions between T and R* were observed when 2-nitro 5-thiocyanobenzoic acid (NTCBA) was used as the modifying reagent (J. O. Ortiz and J. Bubis, 2001, Effects of differential sulfhydryl group-specific labeling on the rhodopsin and guanine nucleotide binding activities of transducin, Arch. Biochem. Biophys. 387, 233-242). However, while carboxymethylated T was capable of liberating GDP in the presence of R*, NTCBA-modified T was unable to release the guanine nucleotide diphosphate upon incubation with the photoactivated receptor. Thus, IAA-labeling stabilized a T:R* complex intermediate carrying the empty nucleotide pocket conformation of T. On the other hand, NTCBA-modified T seemed to be "locked" in the GDP-bound state of T, even in the presence of R*.

Effects of differential sulfhydryl group-specific labeling on the rhodopsin and guanine nucleotide binding activities of transducin.

The role of transducin sulfhydryl groups was examined by chemical modification with four different reagents: 4-acetamido-4'-maleimidyl-stilbene-2, 2' disulfonic acid (AMDA); 4-vinyl pyridine (VP); 2-nitro-5-thiocyano benzoic acid (NTCBA); and 2, 5-dimethoxystilbene-4'-maleimide (DM). All these compounds rapidly inhibited the [3H]GMPpNp-binding activity of transducin stimulated by photoexcited rhodopsin (R*). Sedimentation experiments showed that the labeling of transducin with AMDA or VP hindered its binding to R* while NTCBA-modified transducin was capable of interacting with the photoreceptor protein. In contrast, DM-labeled transducin precipitated even in the absence of R*. Photoactivated rhodopsin was capable of protecting against the observed AMDA and NTCBA inhibition in transducin function, but not against the inactivation caused by VP or DM. These results suggest the existence of different functional cysteines on transducin that are located in the proximity of the interaction site with the photoreceptor protein, near the guanine nucleotide binding site, or in regions involved in the structural changes taking place upon protein activation. With the use of these reagents, transducin appears to be "frozen" in various conformational stages of its cycle, providing conditions for studying two of the initial steps of the visual process: the light-dependent binding of transducin to rhodopsin and the transducin guanine nucleotide exchange reaction.

Effect of detergents and lipids on transducin photoactivation by rhodopsin.

Rhodopsin samples, isolated using four different extraction procedures, were used to investigate the photodependent activation of the GTPase activity of transducin. A complete inhibition of transducin light-dependent GTP hydrolytic activity was observed when rhodopsin purified in the presence of 1% digitonin, following rod outer segment (ROS) solubilization with 1% 3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate (CHAPS), was used for its activation [0 pmol of inorganic phosphate (Pi) released/min/pmol of rhodopsin]. Rhodopsin, isolated in the presence of 1% digitonin following ROS solubilization with 1% digitonin, was capable of stimulating slightly transducin GTPase activity, with an initial rate of 1 pmol of GTP hydrolyzed/min/pmol of rhodopsin. However, rhodopsin purified in the presence of 0.2% n-dodecyl-beta-D-maltoside (DM), following ROS solubilization with either 1% CHAPS or 1% DM, stimulated the enzymatic activity of transducin in a light-dependent manner, with an initial rate of 5 pmol of Pi released/min/pmol of rhodopsin. Addition of 0.075% egg phosphatidylcholine (PC) to the four different solubilized rhodopsin samples significantly enhanced light-stimulated GTP hydrolysis by transducin, with initial rates increasing from 0 to 1, 1 to 2, and 5 to 30 pmol of Pi released/min/pmol of rhodopsin, respectively. Furthermore, DM-solubilized rhodopsin induced the hydrolysis of the maximum amount of GTP by transducin at 0.0075% PC, while digitonin-solubilized rhodopsin only stimulated the GTPase activity of transducin to a similar value, when the amount of the photoreceptor protein was increased 4-fold and 0.15% PC was added to the assay mixture. These results suggest that the effective photoactivation of transducin by rhodopsin requires phospholipids, which seem to be differentially eliminated with the detergent extraction procedure utilized during ROS membranes solubilization and photopigment isolation.

Percutaneous laser nucleolysis of the intervertebral lumbar disc. An experimental study.

Percutaneous laser discectomy has developed as a new modality in the treatment of herniated intervertebral discs. An experimental study of percutaneous nucleolysis using a carbon dioxide laser beam was performed in 10 dogs, each dog having 2 intervertebral disc spaces treated, for a total of 20 discs. The intradiscal pressure decreased in all instances with the drop ranging from 10% to 55% at the L2-L3 disc and 40% to 69% at the L4-L5 disc. The macroscopic and microscopic studies showed vaporization of the nucleus pulposus, and in 8 spaces, thermal damage to the end plate was observed. This study suggests that carbon dioxide laser nucleolysis is easy and effective, but 300 J of laser energy can damage the end plates in some intervertebral discs in dogs.

Improved purification of transducin subunits from bovine retinal rod outer segments.

Transducin serves as a mediator between the receptor protein, rhodopsin, and the effector protein, cGMP phosphodiesterase, in the visual process. Transducin is a protein composed of three polypeptides: T alpha, T beta, and T gamma, and acts as two functional units, the alpha-subunit and the beta gamma-complex. In the present study, I describe an efficient and fast method of purifying T alpha and T beta gamma using chromatography on a blue agarose column connected in tandem with an omega-amino octylagarose column. The recombination of T alpha and T beta gamma reconstitutes the functional heterotrimeric holoprotein, as demonstrated by the recovery of three native properties of transducin: 1) its capacity to exchange guanine nucleotide, 2) its GTP hydrolytic activity, and 3) the ADP-ribosylation of T alpha catalysed by pertussis toxin.

Improved purification of transducin subunits from bovine retinal rod outer segments

Transducin serves as a mediator between the receptor protein, rhodopsin, and the effector protein, cGMP phosphodiesterase, in the visual process. Transducin is a protein composed of three polypeptides: T alpha, T beta, and T gamma, and acts as two functional units, the alpha-subunit and the beta gamma-complex. In the present study, I describe an efficient and fast method of purifying T alpha and T beta gamma using chromatography on a blue agarose column connected in tandem with an omega-amino octylagarose column. The recombination of T alpha and T beta gamma reconstitutes the functional heterotrimeric holoprotein, as demonstrated by the recovery of three native properties of transducin: 1) its capacity to exchange guanine nucleotide, 2) its GTP hydrolytic activity, and 3) the ADP-ribosylation of T alpha catalysed by pertussis toxin

Identification of guanine nucleotide binding proteins from Trypanosoma cruzi.

Guanine nucleotide binding proteins (GTP-binding proteins) function as transducers of signals in different cellular processes. We have identified several GTP-binding proteins in Trypanosoma cruzi by Western blot analyses. Six polypeptide bands, p20, p25, p28, p31, p37 and p38, were specifically detected in epimastigote crude extracts, using polyclonal antibodies directed against transducin (T) or the alpha-subunit of transducin (T alpha). Four of these bands, p28, p31, p37 and p38, were found in both the soluble and the particulate epimastigote fractions. On the other hand, two of the polypeptides, p20 and p25, were observed only in the particulate fraction, and were not solubilized using 0.2% Triton X-100 and 0.2% Nonidet P-40. A rat monoclonal antibody directed against the ras oncogene, immunorecognized a band with molecular mass of 20,000 daltons, in epimastigote homogenates. In view of their identical apparent molecular weight and solubilization properties, p20, recognized by anti-T or anti-T alpha antibodies, and the 20 KDa band, recognized by anti-ras antibodies, seem to correspond to the same polypeptide. [3H] GDP and [3H] GMP-PNP binding experiments revealed the presence of guanine nucleotide binding proteins in total epimastigote crude extracts, as well as, in the soluble, detergent soluble, and particulate fractions. A primary screening of a T. cruzi cDNA library with anti-T alpha antibodies, followed by secondary and tertiary screenings with anti-ras antibodies yielded six positive clones. One of these clones (Tc-ras1) contains a 600 bp insert which we believe encodes for the ras protein from T. cruzi. On a Northern blot, this cDNA hybridizes to a unique mRNA band of 2.0 Kilobases in epimastigotes.

cAMP receptor protein from Trypanosoma cruzi: purification and cloning of a short sequence of the corresponding cDNA.

cAMP is involved in the differentiation of Trypanosoma cruzi, the causative agent of Chagas' disease. cAMP levels are elevated in the infective, non-dividing metacyclic trypomastigote stage, with respect to the non-infective, proliferative, epimastigote stage. In both stages three is a cAMP receptor protein (CARPT), with unique properties that differentiate it from the regulatory subunits of the cAMP-dependent protein kinase (RI and RII). The CARPT from T. cruzi epimastigotes was purified using ion-exchange chromatography, affinity chromatography and gel filtration. After the final step of purification, two protein bands were obtained, p89 and p70, corresponding to the intact CARPT and its proteolytic product. These two CARPT polypeptides were utilized to prepare polyclonal antibodies in rabbits. Previous results from our laboratory showed that CARPT cross-reacts with polyclonal antibodies prepared against the regulatory subunit (RII) of the cAMP-dependent protein kinase (PKA). As expected from these results, the anti-CARPT antibody recognized purified RII protein in an ELISA assay. The anti-CARPT antibodies were used for immunoblot analyses of epimastigote lysates. The two bands corresponding to the CARPT (p89 and p70), as well as a p40 band, were recognized. Immunoscreening of a T. cruzi lambda ZAP cDNA library with these anti-CARPT polyclonal antibodies yielded one positive clone (pBSCARPT) which contained a 540 bp insert. Northern analyses using the pBSCARPT clone as a probe, showed a 5.2 kb mRNA band in epimastigotes, which were grown in culture from 2 to 10 days in LIT medium. Sequence analyses of the 540 bp insert have failed to show homology to other gene sequences in the database.(ABSTRACT TRUNCATED AT 250 WORDS)

Post-traumatic reactive and resorptive lesions of the medial end of the clavicle.

Three soldiers presented with a painful swelling of the sternoclavicular area. Computed tomography (CT) studies showed sclerosis as well as lysis of the medial end of the clavicle with adjacent soft-tissue swelling. Bone biopsy revealed new bone formation and inflammatory reaction; cultures were negative, no tumor cells were found. The patients recovered after antibiotic treatment. Although the CT findings cannot completely rule out a malignant lesion, these changes in the medial end of the clavicle in young people may suggest trauma as the etiology.

Connective tissue nevi collagens. Study with picrosirius red and polarizing microscopy.

Biopsy specimens of five connective tissue nevi were examined under crossed polars after staining with Picrosirius red. One biopsy specimen was from a solitary nevus, another from a Shagreen patch. The other three specimens were of erupted nevi. In all cases, thick (as well as thin) collagen fibers appeared green to yellow. In contrast, thick fibers of normal human dermis appeared orange to red. The findings indicate that the collagen of collagenous connective tissue nevi is less well packed than normal collagen. Examination of the polarization colors of Picrosirius red-stained sections is a useful procedure for diagnosing collagenous connective tissue nevi.

Clustering of colorectal neoplasia: characteristics of coexisting adenomas in patients with severely dysplastic polyps or invasive (malignant) polyps as compared to patients with benign adenomas or carcinomas.

This study compared the size, histology and morphology of coexisting neoplastic polyps found in colonoscopy, and evaluated the clustering of these polyps in patients with either colorectal carcinoma, cancerous polyps or benign adenomas. Patients were divided by their most malignant form of neoplasia: Group A included 58 patients with early invasive cancerous polyps, and Group B included 73 patients with in situ carcinoma within an adenoma. Group C consisted of 335 patients with benign adenomas, and Group D had 289 patients with colorectal carcinoma. There were no significant differences between the four groups regarding sex, age and ethnicity. The cancerous polyps were significantly larger than the benign polyps. There were significantly (P less than 0.01) more patients with multiple (greater than or equal to 5) colonic lesions in Groups A or B than in Groups C or D. Coexisting polyps were much closer to the index growth, and demonstrated more severe dysplastic changes in the case of cancerous polyps than those associated with benign polyps or cancer. Based on our data we speculate that cancerous polyps are not merely a middle link in adenoma-carcinoma sequence, but rather mark a subset of patients who are especially prone to develop neoplastic changes in their colonic mucosa. These patients should be included in a more strict colonoscopic surveillance programme.

Sites of interaction in the complex between beta- and gamma-subunits of transducin.

Transducin, the guanine nucleotide-binding regulatory protein in rod outer segments, is a heterotrimer consisting of alpha-, beta-, and gamma-subunits. Activation of the photoreceptor, rhodopsin, by light, results in activation of transducin which cleaves to form transducin alpha. GTP and a complex of beta gamma-subunits. We have investigated the point(s) of contact between the subunits of transducin by analyzing for the formation of intersubunit disulfide bond(s) in the presence of copper phenanthroline. The formation of a new species with an apparent molecular mass of 43 kDa was observed which had resulted from the formation of a disulfide bond between the beta- and gamma-subunits. The amino acid residues participating in the disulfide bond were identified as Cys-25 in the beta-subunit and Cys-36 and/or Cys-37 in the gamma-subunit. Thus, these cysteine residues and, probably, some of the adjacent amino acid residues form a point of contact between the beta- and gamma-subunits of transducin in the stable complex.

Invasive colorectal adenomas: surgery versus colonoscopic polypectomy.

Of 6,426 colonoscopies performed in 1978-1987, 66 invasive colorectal adenomatous polyps were removed in 58 examinations. The study group included 36 (62%) men and 22 (38%) women with an age range of 42-96 years. Forty-three patients had invasive pedunculated polyps and 15 had invasive sessile polyps. Following the colonoscopic polypectomy, secondary surgical resection was done in 19 patients with pedunculated polyps and in 13 patients with sessile polyps. The operative specimens showed that the colonoscopic polypectomy removed the entire cancerous focus in all patients with pedunculated polyps, including those with stalk invasion. In contrast, most cases with sessile polyps turned out on operation to be Dukes' B or C carcinoma. Follow-up (mean 4.4 years) was available for 53 (93%) patients: none of 24 unoperated patients with pedunculated polyps suffered from local recurrence. We conclude that colonoscopic polypectomy is sufficient for invasive pedunculated polyps, provided that histology shows that the resection margins are free of tumoral cells. Surgery is recommended for all invasive sessile polyps.

Are the polarization colors of picrosirius red-stained collagen determined only by the diameter of the fibers?

Polarization colors of various purified collagens were studied in fibers of similar thickness. Three different soluble collagens of type I, insoluble collagen type I, lathyritic collagen type I, two p-N-collagens type I, pepsin extract collagen type II, two soluble collagens type III, p-N-collagen type III, and soluble collagen type V were submitted to a routine histopathologic procedure of fixation, preparation of 5-microns-thick sections, staining with Picrosirius red and examination under crossed polars. Polarization colors were determined for thin fibers (0.8 micron or less) an thick fibers, (1.6-2.4 microns). Most thin fibers of collagens and p-N-collagens showed green to yellowish-green polarization colors with no marked differences between the various samples. Thick fibers of all p-N-collagens, lathyritic and normal 0.15 M NaCl-soluble collagens showed green to greenish-yellow polarization colors, while in all other collagens, polarization colors of longer wavelengths (from yellowish-orange to red) were observed. These data suggested that fiber thickness was not the only factor involved in determining the polarization colors of Picrosirius red-stained collagens. Tightly packed and presumably, better aligned collagen molecules showed polarization colors of longer wavelengths. Thus, packing of collagen molecules and not only fiber thickness plays a role in the pattern of polarization colors of Picrosirius red-stained collagens.

Aging of wound healing in an experimental model in mice.

Healing of incised wounds in the backs of mice was studied to establish criteria that would allow the timing of stages during cutaneous wound healing. Starting from day 4 after the incisions were made, three mice were killed every 3 days until day 40. Sections from wounds were stained with hematoxylin-eosin, a reticulin procedure, and picrosirius red. Unstained sections were used for blue autofluorescence. The significance of the findings from the ultraviolet study was limited, but the picrosirius red-polarized light procedure was found to be useful for estimating the age of the repair process as polarization colors of greater wavelengths progressively increased with the age of the lesion.

Deletion of cAMP-binding site B in the regulatory subunit of cAMP-dependent protein kinase alters the photoaffinity labeling of site A.

Photoaffinity labeling with 8-azidoadenosine 3':5'-monophosphate is a highly selective method for probing the cAMP-binding sites of the regulatory subunits of cAMP-dependent protein kinase and for identifying specific residues that are in close proximity to the cAMP-binding sites. The cAMP-binding site of a mutant RI-subunit has been characterized here and contrasted to the native RI-subunit. This mutant RI-subunit was generated by oligonucleotide-directed muta-genesis and lacks the entire second cAMP-binding domain which includes both of the residues, Trp260 and Tyr371, that are photolabeled in the native RI-subunit. The mutant RI-subunit, nevertheless, is photoaffinity-labeled with high efficiency, and the residue covalently modified was identified as Tyr244. The position of Tyr244 based on a computer graphic model of cAMP-binding site A is proposed and correlated with the presumed locations of Tyr371 and Trp260 in the native R-subunit. Photoaffinity labeling also can be used to detect functional cAMP-binding sites following electrophoretic transfer of the denatured protein to nitrocellulose. Labeling of the immobilized protein on nitrocellulose required a functional cAMP-binding site A that can be photoaffinity-labeled in solution based on the following criteria. 1) The type I R-subunit is photolabeled, whereas the type II R-subunit is not. A primary feature which distinguishes these two R-subunits is that the RI-subunit is photolabeled at both sites A and B, whereas covalent modification of the RII-subunit occurs only at site B. 2) The truncated mutant of the RI-subunit which lacks the entire second cAMP-binding domain can be photolabeled on nitrocellulose. 3) A mutant RI-subunit which can no longer be photolabeled in site B is still photolabeled on nitrocellulose. 4) A mutation which abolished cAMP binding to site A also abolished photoaffinity labeling after transfer to nitrocellulose.