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R Contreras

Publications and source records attributed to R Contreras.

At least 19 recordsLinked to original sources
PubMed2005

Comparison among four different ways to condense the Fukui function.

Four different ways to condense the Fukui function are compared. Three of them perform a numerical integration over different basins to define the condensed Fukui function, and the other one is the most traditional Fukui function using Mulliken population analysis. The basins are chosen to be the basins of the electron density (AIM), the basins of the electron localization function (ELF), and the basins of the Fukui function itself. The use of the last two basins is new and presented for the first time here. It is found that the last three methods yield results which are stable against a change in the basis set. The condensed Fukui function using the basins of the ELF is not able to give information on the reactivity of an acceptor molecule. In general, the condensed Fukui function using the basins of the density or the basins of the Fukui function describe the reactivity trends well. The latter is preferred, because it only contains information about the Fukui function itself and it gives the right information for donor as well as acceptor centers.

Journal Article↗
PubMed2004

Sigma-pi separation of the electron localization function and aromaticity.

The electron localization function (ELF) has been separated in its sigma and pi components. The topological analysis of the new ELFsigma and ELFpi functions has been used to quantify the concept of resonance. The highest bifurcation values of these functions describe in a correct way the aromaticity of classical ring molecules and some new aromatic compounds as B6CO6, Al4(2-), and N5-. In the case of Al4(2-), an important sigma delocalization contribution has been found, which is in agreement with previous interpretation.

Journal Article↗
PubMed2002

Novel tools for the study of class I alpha-mannosidases: a chromogenic substrate and a substrate-analog inhibitor.

The use of chromogenic substrates for evaluation of class I alpha-mannosidase is described. 2('),4(')-Dinitrophenyl-alpha-D-mannopyranoside allows rapid and sensitive assays of enzymatic activities, e.g., of heterologously expressed alpha-1,2-mannosidase from Trichoderma reesei. Interaction constants of several ligands with alpha-mannosidases from class I and II could also be determined. Furthermore, novel types of inhibitors derived from D-lyxose are presented. Methyl-alpha-D-lyxopyranosyl-(1(')-->2)-alpha-D-mannopyranoside is a potent inhibitor of the alpha-1,2-mannosidase from T. reesei (K(i)=600 microM) and since it probably spans subsites -1/+1, this disaccharide could be valuable in crystallographic studies of class I alpha-mannosidases.

Binding, Competitive↗
PubMed2001

Weak intramolecular proton-hydride and proton-fluoride interactions: experimental (NMR, X-ray) and DFT studies of the bis(NBH(3)) and bis(NBF(3)) adducts of 1,3-dimethyl-1,3-diazolidine.

Bis(NBH(3)), bis(NBF(3)), and NBF(3)/NBH(3) adducts 1-3 were prepared from 1,3-dimethyl-1,3-diazolidine and characterized by the (1)H, (13)C, (11)B, (19)F, 2D (1)H(-13)C HETCOR and NOESY NMR spectra. The structures and conformations of the adducts were established by the variable-temperature (1)H NMR spectra, the X-ray diffraction method (adduct 2A), and density functional calculations at different theoretical levels. The experimental and theoretical data have revealed that bis adducts 1-3 prefer trans orientations of the borane groups (1A, 2A, 3A) in solution, the solid state, and the gas phase. The studies have shown that the energetic preference of trans adducts with respect to cis compounds, decreasing as 2A (2.9 kcal/mol) > 3A (2.7 kcal/mol) > 1A (1.4 kcal/mol), is dictated by spatially repulsive interactions between the CH(3), BH(3), and BF(3) groups. The results of DFT calculations agree well with an experimental trans/cis isomeric ratio of 9:1 determined in solutions of adduct 1. The calculated geometry and energy, as well as the topological analysis of electronic densities, show that trans adducts 1-3 should exist in gas phase as twist conformations T-2 stabilized by the intramolecular C-H(delta+)...(-delta)H-B or C-H(delta+)...(-delta)F-B interactions. These interactions are characterized as closed-shell. The energy of one proton-hydride and proton-fluoride intramolecular contact, estimated as 1.9 (1A-T-2) and 0.7 (2A-T-2) kcal/mol, respectively, classifies the "elongated" intramolecular interactions CH(delta+)...(-delta)HB and CH(delta+)...(-delta)FB as weak ones. It has been established that, on going from gas phase to a condensed phase (solution and solid), the twist-conformations T-2 transform to conformations T-1, probably by intermolecular dipole-dipole interactions. The data presented in this work show that despite a weakness of the "elongated" proton-hydride and proton-fluoride interactions, they can play a significant role in the stabilization of conformational molecular states, especially when cooperativity is in action.

Journal Article↗
PubMed2001

Trichoderma reesei alpha-1,2-mannosidase: structural basis for the cleavage of four consecutive mannose residues.

The process of N-glycosylation of eukaryotic proteins involves a range of host enzymes that delete or add saccharide monomers. While endoplasmic reticulum (E.R.) mannosidases cleave only one mannose to produce the Man8B isomer, an alpha-1,2-mannosidase from Trichoderma reesei can sequentially cleave all four 1,2-linked mannose sugars from a Man(9)GlcNAc(2) oligosaccharide, a feature reminiscent of the activity of Golgi mannosidases. We now report the structure of the T. reesei enzyme at 2.37 A resolution. The enzyme folds as an (alpha alpha)(7) barrel. The substrate-binding site of the T. reesei mannosidase differs appreciably from the Saccharomyces cerevisiae enzyme. In the former, shorter loops at the surface allow substrate protein to come closer to the catalytic site. There is more internal space available, so that different oligosaccharide conformations are sterically allowed in the T. reesei alpha-1,2-mannosidase.

Amino Acid Sequence↗
PubMed2001

Use of HDEL-tagged Trichoderma reesei mannosyl oligosaccharide 1,2-alpha-D-mannosidase for N-glycan engineering in Pichia pastoris.

Therapeutic glycoprotein production in the widely used expression host Pichia pastoris is hampered by the differences in the protein-linked carbohydrate biosynthesis between this yeast and the target organisms such as man. A significant step towards the generation of human-compatible N-glycans in this organism is the conversion of the yeast-type high-mannose glycans to mammalian-type high-mannose and/or complex glycans. In this perspective, we have co-expressed an endoplasmic reticulum-targeted Trichoderma reesei 1,2-alpha-D-mannosidase with two glycoproteins: influenza virus haemagglutinin and Trypanosoma cruzi trans-sialidase. Analysis of the N-glycans of the two purified proteins showed a >85% decrease in the number of alpha-1,2-linked mannose residues. Moreover, the human-type high-mannose oligosaccharide Man(5)GlcNAc(2) was the major N-glycan of the glyco-engineered trans-sialidase, indicating that N-glycan engineering can be effectively accomplished in P. pastoris.

Animals↗
PubMed2001

Solvent effects on electrophilicity.

Continuum solvent effect on the electrophilicity index recently proposed by Parr and co-workers (Parr, R. G.; von Szentpaly, L.; Liu, S. J. Am. Chem. Soc. 1999, 121, 1922) is discussed in detail. Solvent effect is introduced using the self-consistent isodensity polarized continuum model (SCI-PCM). A linear relationship is found between the change in electrophilicity index and the solvation energy as represented in the frame of the reaction field theory. The effect of a polarizable environment on the global electrophilicity is examined for a series of 18 well-known electrophiles presenting a wide diversity in structure and bonding properties. It is found that solvation enhances the electrophilicity power of neutral electrophilic ligands but attenuates this power in charged and ionic electrophiles.

Journal Article↗
PubMed2001

An antisense-based functional genomics approach for identification of genes critical for growth of Candida albicans.

Converting the complete genome sequence of Candida albicans into meaningful biological information will require comprehensive screens for identifying functional classes of genes. Most systems described so far are not applicable to C. albicans because of its difficulty with mating, its diploid nature, and the lack of functional random insertional mutagenesis methods. We examined artificial gene suppression as a means to identify gene products critical for growth of this pathogen; these represent new antifungal drug targets. To achieve gene suppression we combined antisense RNA inhibition and promoter interference. After cloning antisense complementary DNA (cDNA) fragments under control of an inducible GAL1 promoter, we transferred the resulting libraries to C. albicans. Over 2,000 transformant colonies were screened for a promoter-induced diminished-growth phenotype. After recovery of the plasmids, sequence determination of their inserts revealed the messenger RNA (mRNA) they inhibited or the gene they disrupted. Eighty-six genes critical for growth were identified, 45 with unknown function. When used in high-throughput screening for antifungals, the crippled C. albicans strains generated in this study showed enhanced sensitivity to specific drugs.

Antifungal Agents↗
PubMed2001

Characterization of sialyltransferase mutants using surface plasmon resonance.

Sialyltransferases are enzymes responsible for the important sialylation of glycoconjugates. Since crystal structures are not available, other tools are needed to study enzymatic mechanisms. As a model, we used human alpha2,6-sialyltransferase. A putative acceptor-binding domain containing the small and the very small sialyl motifs was randomly mutated. This resulted in enzymes with altered enzymatic activity. Affinity chromatography demonstrated that their binding to donor substrate was maintained. To illustrate the role of the mutated domain in acceptor binding, a method based on surface plasmon resonance was set up. Only at low salt and high acceptor concentration was association of wild-type ST6GalI with asialofetuin demonstrated. As expected, this interaction was affected by cytidine 5'-monophospho-N-acetylneuraminic acid, the donor substrate, which proves the specificity of the interaction. Different types of mutants were found. For some, the drop in activity could be explained by loss in affinity for the acceptor. For others, the catalytic center, but not the acceptor-binding site, was affected. Neither acceptor binding nor catalytic activity were limited to the sialyl motifs. To our knowledge, this is the first example in which surface plasmon resonance is successfully used to demonstrate the binding of a glycosyltransferase to its natural acceptor.

Amino Acid Sequence↗
PubMed2001

Ultrasensitive profiling and sequencing of N-linked oligosaccharides using standard DNA-sequencing equipment.

The analysis of protein-linked glycans is of increasing importance, both in basic glycobiological research and during the production process of glycoprotein pharmaceuticals. In many cases, the amount of glycoprotein available for typing the glycans is very low. This, combined with the high branching complexity typical for this class of compounds, makes glycan typing a challenging task. We present here methodology allowing the medium-throughput analysis of N-glycans derived from low picomole amounts of glycoproteins using the standard DNA-sequencing equipment available in any life sciences laboratory. The high sensitivity of the overall analytical process (from glycoprotein to results) is obtained using state-of-the-art deglycosylation procedures combined with a highly efficient and reproducible novel postderivatization cleanup step involving Sephadex G10 packed 96-well filterplates. All sample preparation steps (enzymatic deglycosylation with PNGase F, desalting, derivatization with 8-amino-1,3,6-pyrenetrisulfonic acid, and postderivatization cleanup) are performed using 96-well-based plates. This integrated sample preparation scheme is also compatible with capillary electrophoresis and MALDI-TOF-MS platforms already in use in some glycobiology labs and anticipates the higher throughput that will be offered by the capillary-array-based DNA sequencers currently penetrating the market. The described technology should bring high-performance glycosylation analysis within reach of each life sciences lab and thus help expedite the pace of discovery in the field of glycobiology.

Carbohydrate Conformation↗
PubMed2000

MET15 as a visual selection marker for Candida albicans.

To develop better molecular genetic tools for the diploid yeast Candida albicans, the suitability of the MET15 gene as a visual selection marker was studied. Both MET15 alleles of C. albicans CAI-4 were isolated by functional complementation of a Saccharomyces cerevisiae strain lacking the MET15 gene. Growth of this complemented strain on Pb(2+)-containing medium was associated with a colour shift of brown into white colonies. The MET15 alleles of C. albicans were located on chromosome 4 by pulsed-field gel electrophoresis and Southern blotting. A met15-deficient strain of C. albicans CAI-4 was generated using the ura blaster technique. This strain showed a brown colony colour on Pb(2+)-containing medium, which corresponded with the colony colour of a S. cerevisiae strain lacking the MET15 gene. Unexpectedly, the met15-deficient strain of C. albicans still grew on methionine-depleted medium. However, this growth was severely delayed. In addition, complementation of this strain with an integrative or replicative plasmid containing either of the MET15 alleles resulted in the formation of white transformants on Pb(2+)-containing medium. These transformants grew very well on methionine-depleted medium. Colony sectoring was obtained with the replicative plasmid and not with the integrative one. This study demonstrates that the MET15 gene of C. albicans is suitable as a visual marker and therefore can be used to identify transformants and study plasmid stability. GenBank Accession Nos for MET15 nucleotide sequences are AF188273, AF188274 and AF188275.

Amino Acid Sequence↗
PubMed2000

Analysis of intracardiac electrograms showing monomorphic ventricular tachycardia in patients with implantable cardioverter-defibrillators.

Ventricular tachycardia (VT) initiation and its relation to various clinical factors was studied by reviewing intracardiac electrograms from patients with implantable cardioverter-defibrillators. Events were divided into (1) sudden onset without preceding ventricular premature complexes (VPCs), (2) extrasystolic onset with VPCs, or (3) paced, depending on the type and morphology of the last 5 beats before initiation of VT. Prematurity index, sinus rate, cycle length, and presence of short-long-short sequence for each episode was noted. A total of 268 episodes of VT among 52 patients were analyzed. Extrasystolic initiation was the most frequent pattern (177; 66%) followed by sudden onset (75; 28%) and paced (16; 6%). Among extrasystolic onset, 99 episodes (56%) were due to multiple VPCs and 149 episodes (84%) had different VPC morphology than the subsequent VT. Among pacing-induced VT, 13 of 16 episodes were due to inappropriate pacing due to undersensing of prior R waves. Sudden-onset episodes were slower (mean cycle length 383+/-97 ms) than extrasystolic (mean cycle length 336+/-88 ms, p = 0.002) and paced (mean cycle length 313+/-85 ms, p = 0.01) onset. Patients in the sudden-onset group had better left ventricular ejection fraction (33+/-15%) than the extrasystolic (29+/-11%, p<0.001) and paced (28+/-14%, p<0.01) groups. Extrasystolic onset with multiple, late coupled VPCs was the most common pattern of VT initiation and was associated with lower ejection fraction. Sudden-onset initiation was more common with better preserved systolic function.

Aged↗
PubMed2000

Molecular cloning and enzymatic characterization of a Trichoderma reesei 1,2-alpha-D-mannosidase.

A cDNA encoding 1,2-alpha-D-mannosidase mds 1 from Trichoderma reesei was cloned. The largest open reading frame occupied 1571 bp. The predicted sequence contains 523 amino acid residues for a calculated molecular mass of 56,266 Da and shows high similarity to the amino acid sequences of 1,2-alpha-D-mannosidases from Aspergillus saitoi and Penicillium citrinum (51.6 and 51.0% identity, respectively). T. reesei mannosidase was produced as a recombinant enzyme in the yeast Pichia pastoris. Replacement of the N-terminal part with the prepro-signal peptide of the Saccharomyces cerevisiae alpha-mating factor resulted in high amounts of secreted enzyme. A three-step purification protocol was designed and the enzymatic properties were analyzed. The enzyme was characterized as a class-I mannosidase.

Amino Acid Sequence↗
PubMed2000

Cloning of Trypanosoma cruzi trans-sialidase and expression in Pichia pastoris.

Trypanosoma cruzi, the agent causing Chagas' disease, expresses an enzyme that transfers sialic acids among glycoproteins and glycolipids both from the host cell surface and its own surface. This enzyme, called trans-sialidase, is different from higher eukaryotic sialyltransferases in that it does not accept cytidine 5'-monophospho-N-acetylneuraminic acid as a donor substrate. Also, the common glycosyltransferase structure is not present. To study this enzyme, an active member was cloned and expressed in higher eukaryotic cells. Expression of recombinant enzyme was achieved in the methylotrophic yeast Pichia pastoris. The N-terminal fusion of a secretion signal and the C-terminal addition of an epitope tag resulted not only in high expression levels, but also enabled easy detection and purification. Using P. pastoris, we obtained about 5 mg of enzymatically active trans-sialidase per liter of induced culture medium.

Animals↗
PubMed2000

Molecular identification of Dunaliella sp. utilizing the 18S rDNA gene.

The utilization of micro-algae for the production of food and fine chemicals is growing in importance. However, confusion of names and species makes comparison of results by different authors very difficult. In this work, five species of Dunaliella were characterized using their 18S ribosomal RNA genes. Conserved oligonucleotides complementary to 5' and 3' termini of the 18S rDNAs were designed and utilized to amplify theme, restriction fragment length polymorphism analysis of the polymerase chain reaction products was developed. Species-specific primers were also designed and utilized to corroborate the identification.

DNA Primers↗
PubMed2000

Supercritical fluid extraction and gas chromatography or electroanalysis of metal chelates from different sample matrices

The supercritical fluid extraction of Pb(DDC)2 and MoO2(acac)2 complexes is performed. The previously formed complexes are used in order to simplify the extraction process. In the extraction cell, 9.0 mg of Pb(DDC)2 or 30.0 mg of MoO2(acac)2 is added. With these two complexes, a study of static and dynamic extraction as a function of pressure (1000-2500 psi), temperature (40-160 degrees C), and presence of modifier (methanol) is performed. Under the best conditions, 5.6 mg of Pb(DDC)2 (2.3 mg of Pb2+) is recovered. The parameters are 2500 psi of pressure, 160 degrees C of temperature, 0.5 mL methanol (placed in a 10-mL extraction cell), 60.0 min of static extraction, and 2.0 min of dynamic extraction. It is necessary to add 3.0 mL of methanol to enhance efficiency on the MoO2(acac)2 complex recovery. Quantitative extractions of MoO2(acac)2 (9.0 mg of MoVI) are obtained when the experiments are carried out under 1000-2500 psi of pressure, 140 degrees C, and times no longer than 10.0 min. Then, the study is carried out forming the in situ complexes. For this purpose, metallic ion and ligand are added. Under these conditions, the Pb2+ recovery decreases from 2.3 to 1.9 mg, and the MoVI recovery decreases from 9.0 to 1.0 mg. When 1.9 mg of Pb2+ and 1.0 mg of MoVI or less is placed in the extraction cell, the recoveries are always 100%. The Pb2+ extracts are directly accomplished using gas chromatography-flame ionization detection (GC-FID), and the MoVI extracts are analyzed using GC-FID and catalytic adsorption voltammetry. The quantitation of pure extracts is carried out by constructing calibration curves with complex solutions and sample solutions using the standard addition method. This method is applied by determination of Pb2+ in sodium alginate extracted from algae and blood, urine, and human milk from patients with diagnosed plumbunemy. MoVI is determined in irrigation water and pasture of animal intake.

Journal Article↗
PubMed2000

The influence of intrapartum antibiotics on the clinical spectrum of early-onset group B streptococcal infection in term infants.

OBJECTIVE: The use of intrapartum antibiotics to prevent early-onset group B streptococcal (EOGBS) infection has left pediatricians in a quandary about the appropriate evaluation and treatment of infants at risk for this infection. The aim of this study was to determine whether intrapartum antibiotic prophylaxis changed the constellation and timing of onset of clinical signs of group B streptococcal (GBS) infection in term infants. METHODOLOGY: We conducted a retrospective chart review of infants who had EOGBS infection and were born in Southern California Kaiser Permanente Hospitals from 1988 through 1996. Objective criteria were used to ascertain maternal risk of infection, intrapartum antibiotic prophylaxis, and onset of clinical signs of infection. RESULTS: Three hundred nineteen infants with EOGBS sepsis, bacteremia, or clinically suspected infection were identified from a population of 277 912 live births. Of the 172 term infants with culture-positive infection who had clinical signs of infection, 95% exhibited them in the first 24 hours of life. All of the infants exposed to intrapartum antibiotics became ill within the first 24 hours of life. CONCLUSIONS: Exposure to antibiotics during labor did not change the clinical spectrum of disease or the onset of clinical signs of infection within 24 hours of birth for term infants with EOGBS infection. A 48-hour stay is not required to monitor asymptomatic term infants exposed to intrapartum antibiotics for onset of GBS infection.

Antibiotic Prophylaxis↗