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PubMed2005

Roles for nutrients in epigenetic events.

The field of epigenetics is the study of modifications of DNA and DNA-binding proteins that alter the structure of chromatin without altering the nucleotide sequence of DNA; some of these modifications may be associated with heritable changes in gene function. Nutrients play essential roles in the following epigenetic events. First, folate participates in the generation of S-adenosylmethionine, which acts as a methyl donor in the methylation of cytosines in DNA; methylation of cytosines is associated with gene silencing. Second, covalent attachment of biotin to histones (DNA-binding proteins) plays a role in gene silencing and in the cellular response to DNA damage. Third, tryptophan and niacin are converted to nicotinamide adenine dinucleotide, which is a substrate for poly(ADP-ribosylation) of histones and other DNA-binding proteins; poly(ADP-ribosylation) of these proteins participates in DNA repair and apoptosis. Here we present a novel procedure to map nutrient-dependent epigenetic marks in the entire genomes of any given species: the combined use of chromatin immunoprecipitation assays and DNA microarrays. This procedure is also an excellent tool to map the enzymes that mediate modifications of DNA and DNA-binding proteins in chromatin. Given the tremendous opportunities offered by the combined use of chromatin immunoprecipitation assays and DNA microarrays, the nutrition community can expect seeing a surge of information related to roles for nutrients in epigenetic events.

Base Sequence↗
PubMed1994

Molecular characterization of the outer capsid spike protein (VP4) gene from human group C rotavirus.

A cDNA copy of the third genomic RNA segment of a noncultivatable human group C rotavirus (Bristol) was generated by single primer amplification. Human group C rotavirus genome segment 3 contains 2283 bp and encodes the VP4 gene with an open reading frame of 2232 nucleotides (744 amino acids) starting at nucleotide 21 and terminating at nucleotide 2251. PCR primers designed from the 5' and 3' terminal sequences of the C/Bristol VP4 gene were used to amplify the corresponding VP4 gene of a human group C rotavirus from Belém, Brazil. Nucleotide sequence comparisons of the Bristol and Belém VP4 genes revealed 45 differences of which only 6 were predicted to give amino acid changes. Alignment of the two human VP4 sequences with the prototype porcine group C/Cowden rotavirus VP4 showed only 71.2% nucleotide sequence identity. Protein sequence alignments showed that the human group C rotavirus VP4 sequences were 8 amino acids longer than the porcine VP4 sequence with an insertion of 6 amino acids, 252NSKLGD257 adjacent to the proposed proteolytic cleavage region (amino acids 231-250). The large overall number of differences between the human and porcine VP4 sequences strongly suggested that the porcine C/Cowden isolate may belong to a different group C rotavirus P "serotype." In contrast the very close similarity of the VP4 sequences of the UK and Brazilian group C rotaviruses support the hypothesis that these human isolates originate from a recent common ancestor.

Amino Acid Sequence↗
PubMedDate not supplied

Traceless, self-cleaving solid- and solution-phase parallel synthesis of 3,4,7-trisubstituted 3,4-dihydroquinoxalin-2-ones.

This article describes a new methodology for the parallel synthesis of 3,4-dihydroquinoxalin-2-ones containing three points of diversity. The synthesis begins with commercially available resin-bound alpha-amino acids as the source of the first diversity element and employs a combination of solid- and solution-phase chemistry to introduce the other two. The key step is an intramolecular cyclization and simultaneous traceless cleavage from the solid support to give a disubstituted 3,4-dihydroquinoxalin-2-one. The third substituent is introduced in solution by N-alkylation of the aniline nitrogen using a scavenger resin to dispose of excess reagent. All the reactions in the sequence take place at room temperature without the need to use strong acids or to maintain an inert atmosphere, thereby preserving the chiral integrity of the starting alpha-amino acid and facilitating the generation of libraries in a high-throughput parallel format.

Amino Acids↗
PubMed1998

Localization of autoepitopes on the PCM-1 autoantigen using scleroderma sera with autoantibodies against the centrosome.

Characterization of epitope domains of autoantigens is important for deducing the cellular functions of autoantigens and may be important for understanding the autoimmune response. In the reported studies, epitope analysis of the centrosome autoantigen PCM-1 was performed. For these investigations, portion of the PCM-1 cDNA were subcloned into the pMAL expression plasmid, fusion proteins were induced, and aliquots of the extracts were probed by immunoblot analysis using two human autoimmune anticentrosome autoantisera. Immunoblotting identified three individual autoepitopes of 26-40 amino acid residues, amino acids 506-545, 1434-1465, and 1661-1686, within the PCM-1 protein. ELISA assays using non-denatured proteins did not identity any additional autoepitopes in the remainder of the PCM-1 molecule. To analyze the identified autoepitopes further, synthetic peptides were generated that covered each of the three autoepitopes and the synthetic peptides then were probed using the scleroderma sera. Peptides that covered the antigenic regions from amino acids 506-545 and 1434-1465 failed to react with the anticentrosome autoantisera suggesting that overall protein conformation may be important for the formation of those two autoepitopes. Peptides derived from the sequence of the third autoepitope were recognized by autoantibodies present in the anticentrosome autoantisera allowing the identification of the tripeptide KDC as the autoepitope in this region of the PCM-1 molecule. These studies lay the foundation for future investigations of the autoimmune response in scleroderma patients that are producing anticentrosome autoantibodies and should allow an investigation of the cellular role of the PCM-1 protein.

Autoantibodies↗
PubMed2025

The proteogenomic landscape of the human kidney and implications for cardio-kidney-metabolic health.

Nearly one-third of the global population is affected by cardio-kidney-metabolic (CKM) diseases; however, the molecular mechanisms underlying CKM diseases are poorly understood. Here we show that tissue proteomics provide critical insights not captured by tissue gene expression or blood proteomics information by performing whole-genome and RNA sequencing and proteomics analysis of human kidney samples (n = 337), and we generated a publicly available database. Via Bayesian co-localization and Mendelian randomization analyses of kidney protein quantitative trait loci and 36 CKM genome-wide association studies, we prioritized 89 proteins for CKM traits. We prioritized relationships that could underlie the interconnectedness of CKM traits and discovered multiple and targetable mechanisms for CKM diseases, including the potential role of kidney angiopoietin-like protein 3 (ANGPTL3) in serum lipid levels and kidney function as well as the role of charged multivesicular body protein 1A in kidney function and hypertension. Notably, we identify pathways with confluence of evidence from genetic loci, tissue gene expression and protein levels for CKM traits. In summary, our large-scale kidney proteomics study uncovers proteins and targetable mechanisms prioritized for CKM diseases.

Humans↗
PubMed1998

Different forms of the mRNA encoding the heat-shock transcription factor are expressed during the life cycle of the parasitic helminth Schistosoma mansoni.

Several cDNAs and a gene encoding the heat-shock transcription factor (HSF) of schistosome were cloned, and multiple forms of the mRNA were found at different developmental stages of the parasite. The encoded protein contained a DNA-binding domain with expected sequence identity (39-58%) to other HSF molecules, and two leucine zipper motifs (LZ123 and LZ4) involved in the oligomerization of HSF. Adult worms express three isoforms of HSF mRNA generated by alternative splicing inside the coding region that contains in-frame splice signals. Introns are not involved in the process since the deleted segments (36 bp or 45 bp) are not flanked by any intron in the gene. Structural variations generated by alternative splicing (insertion of 3 amino acids or 15 amino acids) are continual with LZ4 and added hydrophobic residues are in register with the hydrophobic heptad repeats of LZ4. Structural diversity at the C-terminus of LZ4 may affect the strength of LZ4 interaction with the oligomerization domain (LZ123) and thus modulate the DNA-binding activity of HSF. The conservation of this mechanism in mouse and schistosome may reflect evolutionary pressure to generate multiple HSF species exhibiting functional diversity and capable of responding to different stress signals and physiological signals. Adult worms express HSF mRNA of 2.5 kb, in agreement with the size of the cDNA, while cercariae (developmental stage preceding adult worm) show multiple bands in the range 2.5-3 kb. Available data indicate that the HSF mRNAs of cercariae are inactive. We propose that these mRNA species are generated by an alternative splicing that incorporates introns, which inactivate the mRNA by the insertion of termination codons and/or by shifting of the reading frame. Parasite HSF protein produced in bacteria showed DNA sequence recognition similar to that of HSF in parasite extracts, i.e. the recombinant HSF reacted better with a variant heat-shock element (HSE; one base change in the third NGAAN pentamer of the ideal HSE consensus sequence) than with the ideal HSE. The size of the HSF gene is 12 kb and it is composed of ten exons and nine introns. Excluding the introns, the gene and cDNA show 100% sequence identity. A plant HSF gene contains only a single intron, which matches with the position of intron I2 of schistosome. That the position of this intron is conserved in remote species is indicative of an important function during evolution of the HSF gene.

Alternative Splicing↗
PubMed1986

Specialized nucleoprotein structures at the origin of replication of bacteriophage lambda: localized unwinding of duplex DNA by a six-protein reaction.

The O protein of bacteriophage lambda localizes the initiation of DNA replication to a unique site on the lambda genome, ori lambda. By means of electron microscopy, we infer that the binding of O to ori lambda initiates a series of protein addition and transfer reactions that culminate in localized unwinding of the origin DNA, generating a prepriming structure for the initiation of DNA replication. We can define three stages of this prepriming reaction, the first two of which we have characterized previously. First, dimeric O protein binds to multiple DNA binding sites and self-associates to form a nucleoprotein structure, the O-some. Second, lambda P and host DnaB proteins interact with the O-some to generate a larger complex that includes additional DNA from an A + T-rich region adjacent to the O binding sites. Third, the addition of the DnaJ, DnaK, and Ssb proteins and ATP results in an origin-specific unwinding reaction, probably catalyzed by the helicase activity of DnaB. The unwinding reaction is unidirectional, proceeding "rightward" from the origin. The minimal DNA sequence competent for unwinding consists of two O binding sites and the adjacent A + T-rich region to the right of the binding sites. We conclude that the lambda O protein localizes and initiates a six-protein sequential reaction responsible for but preceding the precise initiation of DNA replication. Specialized nucleoprotein structures similar to the O-some may be a general feature of DNA transactions requiring extraordinary precision in localization and control.

Adenosine Triphosphate↗
PubMed2006

Molecular epidemiology of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae in a neonatal intensive care unit.

OBJECTIVES: To investigate the molecular epidemiology of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae in the neonatal intensive care unit of a university hospital in Italy. METHODS: Antibiotic susceptibility was evaluated by disc diffusion and Etest. ESBLs were identified by isoelectric focusing, PCR and DNA sequencing analysis. Genotyping was performed by PFGE analysis. Conjugation was performed by broth mating. RESULTS: Molecular typing of K. pneumoniae isolates identified three distinct PFGE patterns. Isolates of PFGE profile A were isolated during an epidemic in 1996, while isolates of PFGE profiles B and C were sequentially isolated from September 2002 to December 2004, when 233 colonizations and 19 infections by K. pneumoniae occurred. All K. pneumoniae strains of different PFGE types were identified as ESBL producers. DNA sequencing of amplified beta-lactamase genes identified a novel bla(TEM) ESBL (bla(TEM-136)) along with bla(SHV-1) in chromosomal and plasmid DNA from K. pneumoniae of PFGE type A, respectively, and bla(TEM-1) and bla(SHV-12) in plasmid DNA from K. pneumoniae of PFGE types B and C. Conjugation experiments demonstrated that resistance to third-generation cephalosporins, along with an approximately 80 kb plasmid containing bla(SHV-12) and bla(TEM-1), was transferred from K. pneumoniae epidemic strains of PFGE types B and C to a susceptible Escherichia coli host at a frequency of 4 x 10(-6) and 1 x 10(-6) cfu/recipient cell, respectively. CONCLUSIONS: The selection of ESBL-producing clones and the transfer of the bla(SHV-12) ESBL gene between different clones were responsible for the spread of K. pneumoniae in the neonatal intensive care unit.

Anti-Bacterial Agents↗
PubMed2003

Genetic markers and their application in poultry breeding.

The current chicken genetic map contains at least 1,965 loci within 50 linkage groups, and it covers about 4,000 cM. About 235 of these loci have homology with known human or mammalian genes. The remaining loci are anonymous molecular DNA markers, including microsatellites, amplified fragment length polymorphism (AFLP), randomly amplified polymorphic DNA (RAPD), CR1 elements, and others. A third generation genetic map for human uses single nucleotide polymorphisms (SNP), which have allowed the mapping of complex traits by linkage disequilibrium. One advantage of SNP is that they are usually linked to the gene of interest, and association of the SNP with traits of economic importance can be analyzed using candidate gene approaches. With the tremendous advancements in characterizing chicken expressed sequence tags (EST), the identification of genetic polymorphisms such as SNP in chicken genes has become a reality. Our laboratory has undertaken an in silico analysis of the chicken EST at the University of Delaware by using a Phred/Phrap/Polyphred/Consed pipeline to identify candidate chicken SNP. Initial scanning of 23,427 chicken EST identified a total of 1,209 candidate SNP, with at least 182 non-synonymous SNP that result in an amino acid change observed. Validation of these candidate chicken SNP is ongoing. Placement of the SNP on the chicken genetic map will enhance marker density, thus allowing for mapping of complex traits through linkage analysis and linkage disequilibrium. Application of SNP to identify disease resistance genes in chickens is of special interest to our laboratory, especially in regards to Marek's disease and coccidiosis.

Animal Husbandry↗
PubMed1992

The respiratory burst oxidase.

Sbarra and Karnovsky were the first to present evidence suggesting the presence in phagocytes of a special enzyme designed to generate reactive oxidants for purposes of host defense. In the years since their report appeared, a great deal has been learned about this enzyme, now known as the respiratory burst oxidase. It has been found to be a plasma membrane-bound heme- and flavin-containing enzyme, dormant in resting cells, that catalyzes the one-electron reduction of oxygen to O2- at the expense of NADPH: O2 + NADPH----O2- + NADP+ + H+ Its behavior in whole cells and its response to various activating stimuli have been described in detail, although important insights continue to emerge, as for example a very interesting new series of observations on differences in oxidase activation patterns between suspended and adherent cells. The enzyme has been shown by biochemical and genetic studies to consist of at least six components. In the resting cell, three of these components are in the cytosol and three in the plasma membrane, but when the cell passes from its resting to its activated state the cytosolic components are all transferred to the plasma membrane, presumably assembling the oxidase. Of the components initially bound to the membrane, two constitute cytochrome b558, a heme protein characteristic of the respiratory burst oxidase, and the third may represent an oxidase flavoprotein. With regard to the cytosolic components, one is a phosphoprotein and another is the NADPH-binding component, possibly a second oxidase flavoprotein. The nature of the third (p67phox) is a puzzle. Four of the six oxidase components have now been cloned and sequenced. These findings only scratch the surface, however, and many questions remain. How many oxidase components, for example, remain to be discovered, and how do they fit together to form the active enzyme? How is the route of activation of the oxidase integrated into the general signal transduction systems of the cell? How did the oxidase come to be? Could there be a widespread system that generates small amounts of O2- as an intercellular signaling molecule, as recent work is beginning to suggest, and did the ever-destructive respiratory burst oxidase arise from that innocuous system as the creation of some evolutionary Frankenstein--an oxidase from hell? Finally, will it be possible to develop drugs that specifically block the respiratory burst oxidase, and will such drugs prove to be clinically useful as anti-inflammatory agents?(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1993

Multiple domains of the N-formyl peptide receptor are required for high-affinity ligand binding. Construction and analysis of chimeric N-formyl peptide receptors.

Binding of the chemotactic tripeptide fMet-Leu-Phe (fMLP) to its receptor on phagocytes activates these cells through a G protein-coupled pathway. To delineate the structural requirement of the N-formyl peptide receptor (FPR) for ligand binding and signaling, we constructed chimeric receptors between FPR and a recently identified granulocyte receptor, FPR2 (Ye, R. D., Cavanagh, S. L., Quehenberger, O., Prossnitz, E. R., and Cochrane, C. G. (1992) Biochem. Biophys. Res. Commun. 184, 582-589). FPR2 shares 69% sequence homology with the FPR; yet it binds fMLP with a low affinity (Kd = 430 nM), as compared with the high affinity (Kd = 1 nM) displayed by the FPR. This property of the FPR2 was utilized for mapping the FPR ligand binding domains. Seven chimeric FPR/FPR2 receptors were generated by sequential replacement of the FPR segments with the corresponding regions from FPR2. Three reciprocal FPR2/FPR chimeric receptors were also constructed by selective substitution of the FPR segments into FPR2. These chimeric receptors were stably expressed in transfected fibroblasts and analyzed for their ligand binding and transmembrane signaling properties. Replacement of the FPR domains, including the first and the third extracellular loops, resulted in 275- and 85-fold decrease in ligand binding affinity, respectively. Introduction of both domains into the FPR2 significantly increased ligand binding affinity (Kd = 18 nM), whereas substitution of the domains containing the first or third extracellular loop alone improved ligand binding to a lesser degree (Kd = 90 and 372 nM, respectively). In contrast, substitution of either the amino or the carboxyl-terminal regions with those of the FPR2 had little effect on ligand binding affinity. An analysis of the sequences of the two receptors revealed several key residues in the first and the third extracellular loops of the FPR and their adjacent transmembrane domains that may be essential for binding of fMLP. We propose that multiple domains of the FPR are required for high-affinity ligand binding, with a major determinant located in the first extracellular loop and its adjacent transmembrane domains.

Amino Acid Sequence↗
PubMed2004

Construction and validation of the Rhodobacter sphaeroides 2.4.1 DNA microarray: transcriptome flexibility at diverse growth modes.

A high-density oligonucleotide DNA microarray, a genechip, representing the 4.6-Mb genome of the facultative phototrophic proteobacterium, Rhodobacter sphaeroides 2.4.1, was custom-designed and manufactured by Affymetrix, Santa Clara, Calif. The genechip contains probe sets for 4,292 open reading frames (ORFs), 47 rRNA and tRNA genes, and 394 intergenic regions. The probe set sequences were derived from the genome annotation generated by Oak Ridge National Laboratory after extensive revision, which was based primarily upon codon usage characteristic of this GC-rich bacterium. As a result of the revision, numerous missing ORFs were uncovered, nonexistent ORFs were deleted, and misidentified start codons were corrected. To evaluate R. sphaeroides transcriptome flexibility, expression profiles for three diverse growth modes--aerobic respiration, anaerobic respiration in the dark, and anaerobic photosynthesis--were generated. Expression levels of one-fifth to one-third of the R. sphaeroides ORFs were significantly different in cells under any two growth modes. Pathways involved in energy generation and redox balance maintenance under three growth modes were reconstructed. Expression patterns of genes involved in these pathways mirrored known functional changes, suggesting that massive changes in gene expression are the major means used by R. sphaeroides in adaptation to diverse conditions. Differential expression was observed for genes encoding putative new participants in these pathways (additional photosystem genes, duplicate NADH dehydrogenase, ATP synthases), whose functionality has yet to be investigated. The DNA microarray data correlated well with data derived from quantitative reverse transcription-PCR, as well as with data from the literature, thus validating the R. sphaeroides genechip as a powerful and reliable tool for studying unprecedented metabolic versatility of this bacterium.

Adaptation, Physiological↗
PubMed1995

Rearranging the domains of pepsinogen.

Most eukaryotic aspartic protease zymogens are synthesized as a single polypeptide chain that contains two distinct homologous lobes and a pro peptide, which is removed upon activation. In pepsinogen, the pro peptide precedes the N-terminal lobe (designated pep) and the C-terminal lobe (designated sin). Based on the three-dimensional structure of pepsinogen, we have designed a pepsinogen polypeptide with the internal rearrangement of domains from pro-pep-sin (native pepsinogen) to sin-pro-pep. The domain-rearranged zymogen also contains a 10-residue linker designed to connect sin and pro domains. Recombinant sin-pro-pep was synthesized in Escherichia coli, refolded from 8 M urea, and purified. Upon acidification, sin-pro-pep autoactivates to a two-chain enzyme. However, the emergence of activity is much slower than the conversion of the single-chain zymogen to a two-chain intermediate. In the activation of native pepsinogen and sin-pro-pep, the pro region is cleaved at two sites between residues 16P and 17P and 44P and 1 successively, and complete activation of sin-pro-pep requires an additional cleavage at a third site between residues 1P and 2P. In pepsinogen activation, the cleavage of the first site is rate limiting because the second site is cleaved more rapidly to generate activity. In the activation of sin-pro-pep, however, the second site is cleaved slower than the first, and cleavage of the third site is the rate limiting step.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Cloning, sequencing and expression of two isoforms of the murine oct-1 transcription factor.

Oct-1 is a ubiquitously expressed regulatory gene of the POU domain family. The Oct-1 protein binds to the octamer motif present in the control regions of a variety of genes such as the immunoglobulins, histone H2B and snRNAs. To learn about Oct-1 and its possible role in B-cell maturation, we have used oct-2 cDNA to screen a murine pre-B cell, cDNA library. Two cDNA clones were identical in their POU-homeo box DNA binding domain, but differed in their 3'-region. Whereas one clone (oct-1a) was very similar to its human oct-1 homologue, the other (oct-1b), contained an additional 72 bp sequence (designated E1) at the serine threonine rich coding region (position 1485 of the human oct-1), and a deletion of another 72 bp sequence (designated E2) downstream (position 1920). These changes preserve the protein reading frame. DNA blot analysis indicates that murine oct-1 is a single copy gene and that the two oct-1 isoforms oct-1 is expressed as a large approximately 10 kb transcript in all the cell are generated by alternative RNA splicing. RNA blots showed that oct-1 is expressed as a large approximately 10 kb transcript in all the cell lines tested. PCR analysis of the E1 and E2 72 bp regions, indicated the presence of a third isoform containing both E1 and E2 (Oct-1c). Oct-1a and Oct-1b were present in all cell types examined, but the level of expression was lower in liver and spleen as compared to testis, thymus and kidney. The ratio of Oct-1b to Oct-1a ranged between 0.2 to 0.5, for all tissues examined except for testis which expressed higher amounts of oct-1b and/or oct-1c. Our findings thus show that the pattern of expression of the oct-1 gene is more complex than hitherto thought.

Amino Acid Sequence↗
PubMed1987

Perspectives on antigenicity and idiotypy.

The recent crystal determination of a lysozyme-antilysozyme complex provides a three-dimensional prototype of the manner in which contacts in idiotype-anti-idiotype interactions may be realized. Such interactions can be approximated by two complementary "flat" surfaces. Each IDR (autoantigenic locus) location might provide a particular recognition feature between two interacting partners. The combinatorial manner in which IDR domains are recognized by anti-idiotypic antibodies describe the repertoire of private and public (crossreactive) idiotopes of an antibody. Several interesting features emerge from consideration of the Ab contact residues in the crystal structure. First, framework residues are implicated in contacting the antigen: Thr 30 (FR1) of the heavy chain and Tyr 49 (FR2) of the kappa light chain. Both of these residues lie within predicted IDRs. Framework regions have recently been suggested to be involved in several anti-idiotypic systems, although such regions have, in the past, been disregarded based solely upon sequence analysis. The surface variability analysis, which identifies the repertoire of complementary interacting surfaces, depicts the immunoglobulin as having more variability than generally thought. This variability may also extend to T cell receptors since T cell chains express an extensive surface variable repertoire similar to that of the immunoglobulin light chains (Kieber-Emmons and Köhler, unpublished). Second, the D region plays a critical role in the generation of the antilysozyme combining sites. Similarly, the D segment makes up the largest component of an IDR. Third, while the CDR3 of the heavy chain contributes most to the antibody-lysozyme complex it is not the most surface-exposed (see Novotny, this issue). Nevertheless, surface variability analysis indicates that this region is generally immunodominant which is also observed experimentally. Together, these results indicate that perhaps certain IDR regions are intrinsically more antigenic. Idiotypic structures must be accessible for antibody recognition and binding. From a structural viewpoint, a single antibody molecule has a continuum or several different combining sites. Subsequently, a single residue can be contained in several overlapping idiotypic determinants. Surface variability analysis suggests that the hypervariable regions of Igs provide a diverse idiotope repertoire that can be utilized for binding. Monoclonal antibodies have been shown to have multiple specificities and this capacity for multiple binding is also intrinsic to the definitions that have emerged for anti-idiotypic antibodies.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1991

A computer management system for patient simulations.

A series of interactive videodisc patient simulations is being used to teach clinical problem-solving skills, including diagnosis and management, to dental students. This series is called Oral Disease Simulations for Diagnosis and Management (ODSDM). A computer management system has been developed in response to the following needs. First, the sequence in which students perform simulations is critical. Second, maintaining records of completed simulations and student performance on each simulation is a time-consuming task for faculty. Third, the simulations require ongoing evaluation to ensure high quality instruction. The primary objective of the management system is to ensure that each student masters diagnosis. Mastery must be obtained at a specific level before advancing to the next level. The management system does this by individualizing the sequence of the simulations to adapt to the needs of each student. The management system generates reports which provide information about students or the simulations. Student reports contain demographic and performance information. System reports include information about individual patient simulations and act as a quality control mechanism for the simulations.

Computer Simulation↗
PubMedDate not supplied

"In silico" design of new uranyl extractants based on phosphoryl-containing podands: QSPR studies, generation and screening of virtual combinatorial library, and experimental tests.

This paper is devoted to computer-aided design of new extractants of the uranyl cation involving three main steps: (i) a QSPR study, (ii) generation and screening of a virtual combinatorial library, and (iii) synthesis of several predicted compounds and their experimental extraction studies. First, we performed a QSPR modeling of the distribution coefficient (logD) of uranyl extracted by phosphoryl-containing podands from water to 1,2-dichloroethane. Two different approaches were used: one based on classical structural and physicochemical descriptors (implemented in the CODESSA PRO program) and another one based on fragment descriptors (implemented in the TRAIL program). Three statistically significant models obtained with TRAIL involve as descriptors either sequences of atoms and bonds or atoms with their close environment (augmented atoms). The best models of CODESSA PRO include its own molecular descriptors as well as fragment descriptors obtained with TRAIL. At the second step, a virtual combinatorial library of 2024 podands has been generated with the CombiLib program, followed by the assessment of logD values using developed QSPR models. At the third step, eight of these hypothetical compounds were synthesized and tested experimentally. Comparison with experiment shows that developed QSPR models successfully predict logD values for 7 of 8 compounds from that "blind test" set.

Journal Article↗
PubMed1985

Renaturation of skeletal muscle tropomyosin: implications for in vivo assembly.

The observation that the alpha beta heterodimer is the predominant species of tropomyosin in rabbit skeletal muscles has led to the suggestion that this species assembles preferentially. To understand the molecular basis of this assembly process, we have studied renaturation under conditions that favor heterodimer formation. When skeletal muscle tropomyosin composed of equal amounts of alpha and beta subunits is renatured either by cooling or by dialysis a distribution that favors homodimers is generated. In contrast, rapid renaturation by dilution from urea favors the heterodimer. Further analysis of this latter renaturation procedure with cysteine-cleavage fragments of tropomyosin using circular dichroic measurements shows that as few as 30 residues in the NH2-terminal third of each tropomyosin subunit are involved in the initial interaction that results in heterodimer formation. Based on the density of sequence substitutions between the alpha and beta subunits, that region probably includes residues 36-64.

Animals↗