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Biomedical subjects

A C Anderson

Publications and source records attributed to A C Anderson.

At least 19 recordsLinked to original sources
PubMed2006

Towards species-specific antifolates.

Dihydrofolate reductase (DHFR) plays an essential role in cellular biochemistry and has been a well-recognized drug target for over fifty years. Antifolate inhibitors of DHFR, including clinically used therapeutics such as methotrexate, trimethoprim, and pyrimethamine have been successful as anticancer, antibacterial, antifungal and antiparasitic agents. As resistant strains of these microorganisms evolve and as new disease threats arise, the need for new antifolates that are potent and specific for infectious organisms becomes more pressing. Several new antifolates have been reported over the past decade; many of these are potent against a particular species of DHFR, but achieving the goal of potency and selectivity has proven to be more difficult. This review will describe recent advances in attaining species selectivity in developing new antifolates. Specifically, advances in developing inhibitors against Pneumocystis jirovecii and Plasmodium falciparum, the causative agents in pneumocystis pneumonia and malaria, respectively, will be presented.

Amino Acid Sequence↗
PubMed2001

Notch signaling in lymphocyte development.

Signaling through Notch has been implicated in many cell-fate decisions during lymphocyte development. Recent studies have provided new clues--and raised new controversies--regarding the exact role that Notch signaling plays in the commitment of cells to the T-cell lineage. Progress has also been made in deducing the transcriptional program induced by Notch and the mechanism of oncogenic transformation by Notch in lymphocytes.

Animals↗
PubMed2001

Approaches to solving the rigid receptor problem by identifying a minimal set of flexible residues during ligand docking.

BACKGROUND: Using fixed receptor sites derived from high-resolution crystal structures in structure-based drug design does not properly account for ligand-induced enzyme conformational change and imparts a bias into the discovery and design of novel ligands. We sought to facilitate the design of improved drug leads by defining residues most likely to change conformation, and then defining a minimal manifold of possible conformations of a target site for drug design based on a small number of identified flexible residues. RESULTS: The crystal structure of thymidylate synthase from an important pathogenic target Pneumocystis carinii (PcTS) bound to its substrate and the inhibitor, BW1843U89, is reported here and reveals a new conformation with respect to the structure of PcTS bound to substrate and the more conventional antifolate inhibitor, CB3717. We developed an algorithm for determining which residues provide 'soft spots' in the protein, regions where conformational adaptation suggests possible modifications for a drug lead that may yield higher affinity. Remodeling the active site of thymidylate synthase with new conformations for only three residues that were identified with this algorithm yields scores for ligands that are compatible with experimental kinetic data. CONCLUSIONS: Based on the examination of many protein/ligand complexes, we develop an algorithm (SOFTSPOTS) for identifying regions of a protein target that are more likely to accommodate plastically to regions of a drug molecule. Using these indicators we develop a second algorithm (PLASTIC) that provides a minimal manifold of possible conformations of a protein target for drug design, reducing the bias in structure-based drug design imparted by structures of enzymes co-crystallized with inhibitors.

Algorithms↗
PubMed2001

The origin and regulation of autopathogenic T cells.

A clear understanding of the events surrounding the selection of autoreactive T cells in the thymus and their regulation in the periphery has eluded immunologists for years. However, recent work examining the expression of tissue-specific antigens in the thymus and the biochemistry of disease associated MHC alleles has provided important clues into the generation of the autoreactive T cell repertoire in the thymus. In addition, recent studies focusing on the role of immunoregulatory cytokines and cross-reactive peptide ligands has provided information regarding both the regulation and activation of autoreactive cells in the periphery. An improved understanding of the selection and regulation of autoreactive T cells will undoubtedly aid in the development of strategies for treating autoimmune disease.

Animals↗
PubMed2001

Assessing the professional development needs of public health professionals.

This article describes the major activities associated with designing and implementing a comprehensive, professional development needs assessment of public health professionals in four states of the South Central region of the United States. The instrumentation, research design, and summary results of the needs assessment described in this article may facilitate similar efforts by interested researchers and program developers to assess the public health professional workforce training needs. Results of needs assessments can be useful in designing and evaluating professional development curricula and activities to strengthen public health services in the United States.

Adult↗
PubMed2001

Androgen suppression of GnRH-stimulated rat LHbeta gene transcription occurs through Sp1 sites in the distal GnRH-responsive promoter region.

Steroids may regulate LH subunit gene transcription by modulating hypothalamic GnRH pulse patterns or by acting at the pituitary gonadotrope to alter promoter activity. We tested direct pituitary effects of the androgen dihydrotestosterone (DHT) to modulate the rat LHbeta promoter in transfected LbetaT2 clonal gonadotrope cells and in pituitaries of transgenic mice expressing LHbeta-luciferase. The LHbeta promoter (-617 to +44 bp)-luciferase construct was stimulated in LbetaT2 cells 7- to 10-fold by GnRH. Androgen treatment had little effect on basal promoter activity but suppressed GnRH stimulation by approximately 75%. GnRH stimulation of LHbeta was also suppressed by DHT in isolated pituitary cells from male or female mice with functional nuclear ARs, but not in male littermates with mutant AR. GnRH stimulation of the LHbeta promoter requires interactions between a complex distal response element containing two specificity protein-1 (Sp1) binding sites and a CArG box, and a proximal element with two bipartite binding sites for steroidogenic factor-1 and early growth response protein-1 (Egr-1). DHT effectively suppressed promoter constructs with an intact distal response element. The distal response element does not bind AR, but AR reduces Sp1 binding to this region. Glutathione-S-transferase pull-down studies demonstrated direct interactions of AR with Sp1, which requires the DNA-binding domain of AR, and weaker interactions with Egr-1. We conclude that androgen suppression of the rat LHbeta promoter occurs primarily through direct interaction of AR with Sp1, with some possible role through binding to Egr-1. These interactions result in interference with GnRH-stimulated gene transcription by reducing cooperation between the distal and proximal GnRH response elements.

Animals↗
PubMed2000

Autoantigen-responsive T cell clones demonstrate unfocused TCR cross-reactivity toward multiple related ligands: implications for autoimmunity.

It has been suggested that the cross-reaction of a single T cell receptor with multiple different peptide ligands is a mechanism for maintaining a diverse yet compact immune repertoire. In the context of autoimmune disease it is important to understand how this property is balanced against the maintenance of self-tolerance. Specifically, whether the cross-reactivity inherent in the immune system is focused or unfocused will have important consequences for the development of autoimmune disease. If cross-reactivity is "focused," then in an immune response to a foreign antigen all T cell receptors that recognize the foreign antigen will cross-react with a specific autoantigenic peptide. However, if cross-reactivity is "unfocused," an immune response to a foreign antigen will result in the activation of a small number of self-reactive cells within a larger pool of cells specific for the foreign antigen. We have tested whether cross-reactivity is focused or unfocused by generating a panel of T cell clones that respond to two closely related ligands. W144 is an autoantigenic peptide of myelin proteolipid protein, PLP 139-151 (HSLGKWLGHPDKF), and Q144 is an altered peptide of PLP 139-151 bearing a glutamine for tryptophan substitution at position 144. The Q144-responsive clones have a broad degree of cross-reactivity with other position 144 substituted peptides. We find that despite their characteristic responses to Q144 and W144, the patterns of responses of these clones to other structurally related ligands are random, demonstrating that cross-reactivity is unfocused in the absence of selection. Maintaining a diverse range of cross-reactive interactions may limit nonspecific responses to autoantigens.

Amino Acid Sequence↗
PubMed2000

The crystal structure of thymidylate synthase from Pneumocystis carinii reveals a fungal insert important for drug design.

Thymidylate synthase from Pneumocystis carinii (PcTS) is an especially important drug target, since P. carinii is a fungus that causes opportunistic pneumonia infections in immune-compromised patients and is among the leading causes of death of AIDS patients. Thymidylate synthase (TS) is the sole enzyme responsible for the de novo production of deoxythymidine monophosphate and hence is crucial for DNA replication in every organism. Inhibitors selective for P. carinii TS over human TS would be greatly beneficial in combating this disease. The crystal structure of TS from P. carinii bound to its substrate, dUMP, and a cofactor mimic, CB3717, was determined to 2.6 A resolution. A comparison with other species of TS shows that the volume of the closed PcTS active-site is 20 % larger than that of five other TS closed active-sites. A two-residue proline insert that is strictly conserved among all fungal species of TS, and a novel C-terminal closing interaction involving a P. carinii-specific tyrosine residue are primarily responsible for this increase in volume. The structure suggests several options for designing an inhibitor specific to PcTS and avoiding interactions with human TS. Taking advantage of the residue substitutions of P. carinii TS over human TS enables the design of a selective inhibitor. Additionally, the larger volume of the active-site of PcTS is an important advantage for designing de novo inhibitors that will exclude the human TS active-site through steric hindrance.

Amino Acid Sequence↗
PubMed2000

High frequency of autoreactive myelin proteolipid protein-specific T cells in the periphery of naive mice: mechanisms of selection of the self-reactive repertoire.

The autoreactive T cells that escape central tolerance and form the peripheral self-reactive repertoire determine both susceptibility to autoimmune disease and the epitope dominance of a specific autoantigen. SJL (H-2(s)) mice are highly susceptible to the induction of experimental autoimmune encephalomyelitis (EAE) with myelin proteolipid protein (PLP). The two major encephalitogenic epitopes of PLP (PLP 139-151 and PLP 178-191) bind to IA(s) with similar affinity; however, the immune response to the PLP 139-151 epitope is always dominant. The immunodominance of the PLP 139-151 epitope in SJL mice appears to be due to the presence of expanded numbers of T cells (frequency of 1/20,000 CD4(+) cells) reactive to PLP 139-151 in the peripheral repertoire of naive mice. Neither the PLP autoantigen nor infectious environmental agents appear to be responsible for this expanded repertoire, as endogenous PLP 139-151 reactivity is found in both PLP-deficient and germ-free mice. The high frequency of PLP 139-151-reactive T cells in SJL mice is partly due to lack of thymic deletion to PLP 139-151, as the DM20 isoform of PLP (which lacks residues 116-150) is more abundantly expressed in the thymus than full-length PLP. Reexpression of PLP 139-151 in the embryonic thymus results in a significant reduction of PLP 139-151-reactive precursors in naive mice. Thus, escape from central tolerance, combined with peripheral expansion by cross-reactive antigen(s), appears to be responsible for the high frequency of PLP 139-151-reactive T cells.

Animals↗
PubMed2000

Methadone in the treatment of chronic nonmalignant pain: a 2-year follow-up.

OBJECTIVE: To examine the longitudinal use of methadone in a pain clinic. DESIGN: Follow-up study of 40 patients initially treated with methadone and re-evaluated 2 years later, comparing those maintained on methadone with those who were switched to other opioids. SETTING: Pain clinic at a university hospital. RESULTS: The 14 patients (35%) who stayed on methadone for the duration of the study, had higher employment rates (P <.05) and higher functional ratings (P <.02) than those on other opioids. Side effects were the most common reason (33.4%) for discontinuation of methadone. Dose escalation occurred in 11 of 14 patients (78.6%). CONCLUSIONS: Chronic pain patients may be safely and effectively treated with methadone. Those not responding or tolerating methadone may be benefited by treatment with other opioids.

Journal Article↗
PubMed2000

Biological derivation of a range of cephalometric norms for children of African American descent (after Steiner).

The purpose of this study was to report a normal range of anteroposterior apical base differences and concomitant interincisor inclinations and locations derived from a sample of American children (12 to 16 years) of African descent with normal occlusion (statistically defined). Standard cephalometric radiographs of 40 boys and 40 girls were traced and the Steiner Analysis performed. In addition to the ANB angle, 6 supplemental anteroposterior apical base separation estimators were measured, mean values established, and correlation (r) associations performed. The range of anteroposterior apical base difference was -0.5 degrees to 9.5 degrees estimated by the ANB angle or a linear distance of +/-6.5 mm using the Wits Appraisal. The angular and linear distance of the upper incisor to NA line (compensations) ranged from a low of 12 degrees and 3 mm to a high of 39 degrees and 14 mm. The angular and linear distance of the lower incisor to the NB line ranged from a low of 17 degrees and 3 mm to a high of 47.5 degrees and 17.5 mm. Biologically, the results suggested a wide range of equally acceptable sagittal apical base relationships and associated compensations in upper and lower incisor inclinations; normal occlusion was viewed as the reference point. Considering the usage of such descriptive terms as "ideal," "acceptable compromises," and "individualized treatment goals," to describe the angulation and inclination of the incisor teeth, the need for a distinction between biologically derived reference norms and esthetic preference reference norms is apparent when analyzing normal occlusion.

Adolescent↗
PubMed2000

Tuning T cell activation threshold and effector function with cross-reactive peptide ligands.

We have generated a panel of cross-reactive T cells by immunizing SJL mice (I-A(s)) with Q144 peptide, an analog of an autoantigenic peptide (W144) of myelin proteolipid protein (PLP) 139-151 (HSLGKWLGHPDKF) in which W was replaced by Q at position 144. Following immunization with Q144, T cells were expanded in vitro with W144, which is a cross-reactive, suboptimal ligand, for Q144-specific T cells. The T cell clones responded to both ligands and grew normally on the peptide W144, but were hyperstimulated when activated by Q144 in vitro. This hyperstimulation results in a heteroclitic proliferative response with secretion of additional cytokines not induced by W144. Thus expansion of T cells by a suboptimal cross-reactive ligand effectively lowers the activation threshold so that the immunizing antigen becomes a hyperstimulating ligand for the clones. Surprisingly, when the T cell clones are grown on the hyperstimulating ligand Q144, some adapt by increasing their activation threshold. This desensitization results in a loss of response to a number of cross-reactive ligands and the appearance of a more specific T cell response. Long-term culture with the hyperstimulating ligand is sometimes associated with down-regulation of CD4 expression. These results provide an explanation for the common finding of T cell heteroclicity, and suggest that although the specificity and hierarchy of the response of T cells to peptides is determined by the TCR, activation threshold and effector functions are modified by exposure to cross-reactive ligands. This observation has implications for the development and regulation of autoimmune disease.

Animals↗
PubMed2000

Divergent and composite gonadotropin-releasing hormone-responsive elements in the rat luteinizing hormone subunit genes.

GnRH pulses regulate gonadotropin subunit gene transcription in a frequency-dependent, subunit-specific manner. The alpha-subunit gene is stimulated by constant GnRH and by rapid to intermediate pulse frequencies, while stimulation of LHbeta subunit gene transcription requires intermediate frequency pulses. We have defined the GnRH-responsive elements of the rat LH subunit gene promoters by deletion/mutation analysis and transfection studies in rat pituitary cells and two clonal gonadotrope cell lines. The alpha-subunit gene GnRH-responsive region lies between -411 and -375 bp. The region contains two Ets-domain protein binding sites, and mutating either site obliterates the response. DNA protein binding studies demonstrate the two sites are not equivalent, and that Ets-1 does not mediate this response. Studies of the LHbeta promoter reveal a major GnRH-responsive region between -456 and -342 bp. Within this region, two Sp1 binding sites contribute to the GnRH response, and the 3'Sp1 site is also critical for basal expression. The 5'Sp1 site partially overlaps a CArG box, and mutating the CArG element specifically eliminates the response to pulsatile GnRH. DNA containing this mutation cannot form intermediate mobility complexes with nuclear proteins, but retains Sp1 binding. Mutation of the 3'Sp1 site and either the 5'Sp1 or CArG element partially restores GnRH stimulation, suggesting a downstream element contributes to the full GnRH response. These studies demonstrate that unique composite elements and transcription factors are responsible for GnRH stimulation of the LH subunit genes and may contribute to their differential responses to GnRH pulses.

Animals↗
PubMed1999

The structural mechanism for half-the-sites reactivity in an enzyme, thymidylate synthase, involves a relay of changes between subunits.

Thymidylate synthase (TS), a half-the-sites reactive enzyme, catalyzes the final step in the de novo biosynthesis of deoxythymidine monophosphate, dTMP, required for DNA replication. The cocrystal structure of TS from Pneumocystis carinii (PcTS), a new drug target for an important pathogen, with its substrate, deoxyuridine monophosphate (dUMP), and a cofactor mimic, CB3717, was determined. The structure, solved at 2.6 A resolution, shows an asymmetric dimer with two molecules of the substrate dUMP bound yet only one molecule of cofactor analogue bound. The structural evidence reveals that upon binding cofactor analogue and forming a covalent bond from the nucleophilic cysteine to the substrate, dUMP, at one active site, PcTS undergoes a conformational change that renders the opposite monomer incapable of forming a covalent bond or binding a molecule of cofactor analogue. The communication pathway between the two active sites is evident, allowing a structural definition of the basis of half-the-sites reactivity for thymidylate synthase and providing an example of such a mechanism for other half-the-sites reactive enzymes.

Binding Sites↗
PubMed1999

Crystal structure of a brominated RNA helix with four mismatched base pairs: An investigation into RNA conformational variability.

The X-ray crystal structure of a brominated RNA helix with four mismatched base pairs and sequence r(UG(Br)C(Br)CAGUUCGCUGGC)(2) was determined to 2.1 A using the methods of multiwavelength anomalous diffraction (MAD) applied to the bromine K-absorption edge. There are three molecules in the asymmetric unit with unique crystal-packing environments, revealing true conformational variability at high resolution for this sequence. The structure shows that the sequence itself does not define a consistent pattern of solvent molecules, with the exception of the mismatched base pairs, implying that specific RNA-protein interactions would occur only with the nucleotides. There are a number of significant tertiary interactions, some of which are a result of the brominated base pairs and others that are directly mediated by the RNA 2' hydroxyl groups. The mismatched base pairs exhibit a solvent network as well as a stacking pattern with their nearest neighbors that validate previous thermodynamic analysis.

Base Pair Mismatch↗
PubMed1999

Effect of metoclopramide dose on preventing emesis after oral administration of N-acetylcysteine for acetaminophen overdose.

OBJECTIVE: To determine the effect of the metoclopramide dose on the prevention of vomiting of N-acetylcysteine in acetaminophen overdose. METHODS: Patients with acetaminophen ingestions receiving metoclopramide prior to emergency department administration of N-acetylcysteine were included. Emergency Department and poison center records were reviewed for administration of metoclopramide pre-N-acetylcysteine and incidence of subsequent vomiting. The treatment group was defined as patients receiving high-dose metoclopramide (20-50 mg intravenously) prior to the loading dose of N-acetylcysteine. Controls were patients receiving standard-dose (< 20 mg intravenously) metoclopramide prior to loading dose of N-acetylcysteine. Outcome was vomiting within 60 minutes of N-acetylcysteine administration. RESULTS: Twelve of 19 patients (63%) receiving standard-dose metoclopramide vomited N-acetylcysteine. Only 5 of 23 patients (22%) receiving high-dose metoclopramide vomited N-acetylcysteine (crude odds ratio: 6.2; 95% CI [1.3-30.3]). After controlling for confounding in the logistic regression model, the effect of high-dose metoclopramide in preventing vomiting of N-acetylcysteine remained significant (adjusted odds ratio: 17.0; 95% CI [2.6-110.0]). CONCLUSION: This study supports the efficacy of high-dose metoclopramide to prevent emesis after the oral loading dose of N-acetylcysteine.

Acetaminophen↗
PubMed1999

Osmol gaps in the pediatric population.

OBJECTIVE: To define the osmol gap (OG) range in pediatric ED (PED) patients. METHODS: This was a blinded, observational patient series involving an urban PED with an annual census of 35,000. All patients presenting to the Hasbro Children's Hospital Emergency Department who required electrolyte determination during their evaluations were enrolled into the study. Exclusionary criteria included the presence of urinary ketones, alcohol ingestion within the preceding 24 hours, or illnesses that are known to change serum osmolarity. Electrolytes, BUN, glucose, and freezing point depression osmolality were measured on a single serum specimen. Additional laboratory information included ethanol and anion gap. The OG was determined using each of three equations previously described in the literature (see Results). The best coefficients for sodium, BUN, and glucose were determined by multiple linear regression. RESULTS: 192 children (90 girls, 102 boys) with a median age of 6.6 years (mean: 7.3 years; range: 7 days to 17.9 years) made up the study population. The mean measured osmolality (+/-SD) for the entire sample was 284.2+/-6.9 mOsm/dL with a range of 265-311 mOsm/dL. Mean osmol gaps with standard deviations varied with the equation used for calculation. CONCLUSION: Regardless of the equation used, the range of "normal" osmol gaps in the pediatric population is approximately 22 mOsm.

Adolescent↗