Biomedical subjects

A Mondragon

Publications and source records attributed to A Mondragon.

11 recordsLinked to original sources

PubMed2000

Distinct mitochondrial and cytosolic enzymes mediate trypanothione-dependent peroxide metabolism in Trypanosoma cruzi.

The American trypanosome Trypanosoma cruzi is exposed to toxic oxygen metabolites that are generated by drug metabolism and immune responses in addition to those produced by endogenous processes. However, much remains to be resolved about the parasite oxidative defense system, including the mechanism(s) of peroxide reduction. Here we show that reduction of peroxides in T. cruzi is catalyzed by two distinct trypanothione-dependent enzymes. These were localized to the cytosol and mitochondrion. Both are members of the peroxiredoxin family of antioxidant proteins and are characterized by the presence of two conserved domains containing redox active cysteines. The role of these proteins in protecting T. cruzi from peroxide-mediated damage was demonstrated following overexpression of enzyme activity. The parasite-specific features of T. cruzi cytoplasmic peroxiredoxin and T. cruzi mitochondrial peroxiredoxin may be exploitable in terms of drug development.

Amino Acid Sequence↗

PubMed1999

Trypanosoma cruzi adenylyl cyclase is encoded by a complex multigene family.

The parasitic protozoan Trypanosoma cruzi undergoes several differentiation events during its life cycle. Some of these transitions are thought to involve activation of adenylyl cyclase via the binding of peptide ligands to the cell surface. Here we describe the characterisation of the adenylyl cyclase gene family of T. cruzi. Two complete genes and one pseudogene have been sequenced. The protein products appear to have a large extracellular domain, a single transmembrane helix and a cytosolic catalytic domain. The adenylyl cyclase genes are present on at least six chromosomes and are scattered rather than clustered. They form a large polymorphic family in which the extracellular domain is particularly variable. An Escherichia coli adenylyl cyclase mutant could be complemented by expression of the catalytic domain of the T. cruzi enzyme. The recombinant protein had adenylyl cyclase activity in vitro, which was enhanced by increasing concentrations of divalent cations (Mn2+ > Mg2+). This constitutively active recombinant protein will be a useful tool for dissecting the catalytic mechanism of adenylyl cyclase.

Adenylyl Cyclases↗

PubMed1999

Laparoscopic common bile duct exploration.

Since the introduction of laparoscopic cholecystectomy, the management of common bile duct (CBD) stones has undergone significant change. Preoperative endoscopic retrograde cholangiopancreatography (ERCP) with sphincterotomy is now routinely done in cases where the diagnosis of choledocholithiasis is suspected preoperatively, with clearance of the bile ducts before laparoscopic cholecystectomy. Intraoperative discovery of CBD stones by cholangiography represents a challenge to the surgeon, who must make a decision about when to perform laparoscopic CBD exploration, convert to open surgery, or send the patient for ERCP during the postoperative period. Because ERCP has a definite failure rate, laparoscopic CBD exploration can be a treatment option. Among 2500 laparoscopic cholecystectomies done by our group from January 1991 to June 1997, 50 patients (2%) underwent laparoscopic CBD exploration, 13 by the transcystic technique and 37 by choledocotomy, with a conversion rate of 8% and a hospital stay of 4.3 days. One patient died from complicated pancreatitis following ERCP and unsuccessful extraction of a CBD stone. We obtained our goal of a CBD free of stones in 92% of the cases. We conclude that laparoscopic CBD exploration is an effective method for treating choledocolithiasis that allows management of this pathology in one stage, although it requires advanced laparoscopic skills and adequate equipment.

Adult↗

PubMed1999

Optimization of conditions for growth of wild-type and genetically transformed Trypanosoma cruzi on agarose plates.

Growth of Trypanosoma cruzi as colonies on solid medium has not been widely used as an experimental procedure. We therefore sought to establish a reliable and routine plating method. The optimal results were achieved with a matrix of 0.65% low melting point agarose onto which epimasigotes from the mid-to-late logarithmic phase of growth were spread. Colonies could be isolated after incubation for 21 days in a humidified 5% CO2 environment at 28 degrees C. Plating efficiencies in the range of 40% were obtained by this method and clones could be recovered into liquid medium or onto blood-agar slopes with a high success rate. The procedure has also been adapted for the isolation of genetically transformed clones after electroporation of epimastigotes with either plasmid or cosmid vectors. This was best achieved by inclusion of the electroporated cell inoculum in a 0.6% agarose overlay containing G418 as the selective drug, on top of a 0.8% agar base. Transformation efficiencies were as high as 10(-5) cells per microgram of DNA. A reliable plating method for T. cruzi will have many applications and is a significant step towards the use of 'shotgun transformation' to generate libraries of T. cruzi recombinants.

Animals↗

PubMed1998

beta-D-glucosyl-hydroxymethyluracil is a conserved DNA modification in kinetoplastid protozoans and is abundant in their telomeres.

The unusual DNA base beta-D-glucosyl-hydroxymethyluracil, called "J, " replaces approximately 0.5-1% of Thy in DNA of African trypanosomes but has not been found in other organisms thus far. In Trypanosoma brucei, J is located predominantly in repetitive DNA, and its presence correlates with the silencing of telomeric genes. Using antibodies specific for J, we have developed sensitive assays to screen for J in a range of organisms and have found that J is not limited to trypanosomes that undergo antigenic variation but is conserved among Kinetoplastida. In all kinetoplastids tested, including the human pathogens Leishmania donovani and Trypanosoma cruzi, J was found to be abundantly present in the (GGGTTA)n telomere repeats. Outside Kinetoplastida, J was found only in Diplonema, a small phagotrophic marine flagellate, in which we also identified 5-MeCyt. Fractionation of Diplonema DNA showed that the two modifications are present in a common genome compartment, which suggests that they may have a similar function. Dinoflagellates appear to contain small amounts of modified bases that may be analogs of J. The evolutionary conservation of J in kinetoplastid protozoans suggests that it has a general function, repression of transcription or recombination, or a combination of both. T. brucei may have recruited J for the control of genes involved in antigenic variation.

Animals↗

PubMed1997

Overexpression and purification of human calcineurin alpha from Escherichia coli and assessment of catalytic functions of residues surrounding the binuclear metal center.

Calcineurin is an important signal-transducing enzyme in many cell types including T lymphocytes and is a common target for the immunosuppressants cyclosporin A and FK506. The crystal structures of both calcineurin [Griffith et al. (1995) Cell 82, 507-522; Kissinger et al. (1995) Nature 378, 641-644] and a related enzyme, protein phosphatase-1 [Goldberg et al. (1995) Nature 376, 745-753], revealed that this class of serine/threonine phosphatases contain in their putative active sites a binuclear metal center formed by an Asn, two Asp, and three His residues. In addition, one His and two Arg residues lie in close vicinity of the binuclear metal centers. The importance of the binuclear metal center and its surrounding residues in catalysis by calcineurin has not been investigated experimentally. Herein, we report an efficient bacterial expression and purification system for human calcineurin alpha. Using this system, a systematic alanine-scan mutagenesis on the residues surrounding the putative active site was performed. It was found that an intact binuclear metal center is essential for the catalytic activity of the enzyme. In addition, His151, Arg122, and Arg254 also exhibited either a loss or a dramatic decrease in catalytic activity upon mutation into alanines. Interestingly, the Arg254Ala mutant retained a small but significant amount of catalytic activity toward the small substrate p-nitrophenyl phosphate, but is completely inactive toward a phosphopeptide substrate, suggesting that this arginine may be involved in the binding of phosphoprotein substrates as well as in catalysis. As all the residues in the putative active site are conserved between different eukaryotic serine/threonine phosphatases, these results should apply to all members of this family of protein phosphatases.

Arginine↗

PubMed1996

Mucin-like glycoprotein genes are closely linked to members of the trans-sialidase super-family at multiple sites in the Trypanosoma cruzi genome.

In Trypanosoma cruzi a cell surface enzyme with trans-sialidase (TS) activity has been implicated as an important factor in establishing infection. The enzyme is encoded by genes belonging to a large super-family which on the basis of sequence has been subdivided into 4 groups. TS mediates the transfer of sialic acid residues from host glycoconjugates to acceptor molecules on the parasite surface. To study the organisation of the TS genes we isolated several distinct cosmids from a library constructed with DNA from the T. cruzi X10.6 clone. In these cosmids, the TS genes (group I) were present either as single copies or as a direct tandem repeat. A common feature of the cosmids was the presence of a related group III gene located 10-12kb downstream of the TS gene(s) and arranged in the same orientation. In several of the cosmids we also identified a mucin-like glycoprotein gene located between the group I and group III genes. The mucin-like genes are part of a large polymorphic family and contour clamped homogeneous electric field electrophoresis (CHEFE) analysis showed that they were linked to members of the TS super-family at multiple sites in the X10.6 genome. Screening of a second cosmid library made with DNA from the CL-Brener clone confirmed this multiple linkage suggesting that it is a common feature of the species. This genetic organisation may have important functional significance since the mucin-like glycoproteins are the major cell surface acceptors of sialic acid.

Amino Acid Sequence↗

PubMed1988

Recognition of DNA sequences by the repressor of bacteriophage 434.

The structure of a complex between the DNA-binding domain of phage 434 repressor and a 14 base-pair synthetic DNA operator reveals the molecular interactions important for sequence-specific recognition. A set of contacts with DNA backbone, notably involving hydrogen bonds between peptide-NH groups and DNA phosphates, position the repressor and fix the DNA configuration. Direct interactions between amino acid side chains and DNA bases involve nonpolar van der Waals contacts as well as hydrogen bonds. The structures of the repressor domain and of the 434 cro protein are extremely similar. There appear to be no major conformational changes in the proteins when they bind to DNA.

Bacteriophages↗

PubMed1986

Oligonucleotide binding to the coat protein disk of tobacco mosaic virus. Possible steps in the mechanism of assembly.

Binding of the oligoribonucleotides AAG, AAGAAG and AAGAAGUUG to the disk aggregate of tobacco mosaic virus coat protein has been studied in solution under conditions favourable for virus assembly. The two longer oligomers bind strongly with Kd around 1 microM, approach complete saturation of binding sites and cause the formation of long, nicked helical rods resembling the virus. It is suggested that the binding of these oligomers, with sequences chosen from the assembly origin of the viral RNA, simulates the tobacco mosaic virus assembly process. No binding could be detected for AAG, indicating that chain length is a crucial determinant in the interaction. The binding of AAGAAG to coat protein crystals is very much weaker than that observed in solution, and the crystals crack at high oligomer concentrations. The corresponding oligodeoxyribonucleotide, d(AAGAAG), shows no binding to the protein in solution; the interaction is extremely specific for RNA.

Base Sequence↗

PubMed1979

Direct duplication 2p14 to 2p23.

A malformed male newborn was first diagnosed as having Smith-Lemli-Opitz syndrome. Extensive cytogenetic studies, including Q, G, C, R and T banding and BudR treatment, were applied, finally leading the authors to conclude that the patient had a partial 2p trisomy caused by direct duplication 2p14 to 2p23. This was a de novo chromosome abnormality, as both parents had normal karyotypes.

Abnormalities, Multiple↗

PubMedDate not supplied

Correlation between segmental mobility and the location of antigenic determinants in proteins.

Most continuous antigenic determinants of tobacco mosaic virus protein (TMVP), myoglobin and lysozyme correspond to those surface regions in the protein structure, as determined by X-ray crystallography, which possess a run of high-temperature factors along the polypeptide backbone, that is, a high segmental mobility. The mobility of an antigenic determinant may make it easier to adjust to a pre-existing antibody site not fashioned to fit the exact geometry of a protein. The correlation found between temperature factors and antigenicity is better than that between hydrophilicity and antigenicity.

Antigens, Viral↗