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1.  Cardiac Transplantation in Friedreich Ataxia 
Journal of child neurology  2012;27(9):1193-1196.
In this paper, we describe a 14-year-old boy with a confirmed diagnosis of Friedreich ataxia who underwent cardiac transplantation for left ventricular failure secondary to dilated cardiomyopathy with restrictive physiology. His neurological status prior to transplantation reflected early signs of neurologic disease, with evidence of dysarthria, weakness, mild gait impairment, and limb ataxia. We review the ethical issues considered during the process leading to the decision to offer cardiac transplantation.
PMCID: PMC3671892  PMID: 22752490
cardiac transplantation; cardiomyopathy; Friedreich ataxia
2.  Regulation of an IMP Dehydrogenase Gene and Its Overexpression in Drug-sensitive Transcription Elongation Mutants of Yeast* 
The Journal of Biological Chemistry  2001;276(35):32905-32916.
IMP dehydrogenase is a rate-limiting enzyme involved in the synthesis of GTP. In mammalian cells it is regulated with respect to growth rate and is the target of numerous therapeutic agents. Mutations in the RNA polymerase II elongation machinery render yeast sensitive to inhibitors of IMP dehydrogenase and defective in inducing transcription of one of the IMP dehydrogenase-encoding genes, IMD2. Here we show that loss of IMD2, but not IMD1, IMD3, or IMD4, conferred upon yeast the same drug sensitivity found in elongation mutants. We tested whether the drug sensitivity of elongation mutants is due to their inability to induce IMD2 by providing them with exogenous copies of the gene. In some elongation mutants, overexpression reversed drug sensitivity and a transcriptional defect. Overexpression in mutants with a more severe phenotype partially suppressed drug sensitivity but was inconsequential in reversing a defect in transcription. These findings suggest that the drug sensitivity of elongation mutants is largely but not solely attributable to defects in the ability to induce IMD2, because transcription is compromised even when IMD2 mRNA levels are adequate. We describe two DNA sequence elements in the promoter of the gene that regulate it. We also found that IMD2 mRNA abundance is coupled to cell growth rate. These findings show that yeast possess a conserved system that gauges nucleotide pools and cell growth rate and responds through a uniquely regulated member of the IMD gene family.
PMCID: PMC3371605  PMID: 11441018
3.  An Elective Course to Engage Student Pharmacists in Elementary School Science Education 
Objective. To develop and assess the impact of an elective course (HealthWISE) on student pharmacists’ skills in communication and health promotion and elementary school students’ knowledge of and attitudes toward science.
Design. Three colleges and schools of pharmacy collaborated to develop a 1-credit elective course that used online and classroom teaching and learning techniques to prepare student pharmacists to teach science in elementary school classrooms. Student pharmacists delivered 6 science lessons to elementary students over the course of 2 months.
Assessment. In weekly journal reflections and a final paper, student pharmacists reported improved communication and health promotion skills. Elementary teachers reported they were satisfied with student pharmacists’ performance in the classroom. On pretest and posttest evaluations, elementary students demonstrated increased science knowledge and enhanced enthusiasm for science following the lessons taught by student pharmacists.
Conclusions. The HealthWISE elective course provided positive benefit for student pharmacists, elementary school teachers, and elementary students.
PMCID: PMC3279034  PMID: 22345722
service-learning; communication skills; health promotion; STEM education
4.  Gene Dosage–limiting Role of Aire in Thymic Expression, Clonal Deletion, and Organ-specific Autoimmunity 
The Journal of Experimental Medicine  2004;200(8):1015-1026.
Inactivation of the autoimmune regulator (Aire) gene causes a rare recessive disorder, autoimmune polyendocrine syndrome 1 (APS1), but it is not known if Aire-dependent tolerance mechanisms are susceptible to the quantitative genetic changes thought to underlie more common autoimmune diseases. In mice with a targeted mutation, complete loss of Aire abolished expression of an insulin promoter transgene in thymic epithelium, but had no effect in pancreatic islets or the testes. Loss of one copy of Aire diminished thymic expression of the endogenous insulin gene and the transgene, resulting in a 300% increase in islet-reactive CD4 T cells escaping thymic deletion in T cell receptor transgenic mice, and dramatically increased progression to diabetes. Thymic deletion induced by antigen under control of the thyroglobulin promoter was abolished in Aire homozygotes and less efficient in heterozygotes, providing an explanation for thyroid autoimmunity in APS1. In contrast, Aire deficiency had no effect on thymic deletion to antigen controlled by a systemic H-2K promoter. The sensitivity of Aire-dependent thymic deletion to small reductions in function makes this pathway a prime candidate for more subtle autoimmune quantitative trait loci, and suggests that methods to increase Aire activity would be a potent strategy to lower the incidence of organ-specific autoimmunity.
PMCID: PMC2211852  PMID: 15492124
diabetes mellitus type I; autoimmune diseases; clonal deletion; immune tolerance; thymus
5.  Failure to Censor Forbidden Clones of CD4 T Cells in Autoimmune Diabetes 
The Journal of Experimental Medicine  2002;196(9):1175-1188.
Type 1 diabetes and other organ-specific autoimmune diseases often cluster together in human families and in congenic strains of NOD (nonobese diabetic) mice, but the inherited immunoregulatory defects responsible for these diseases are unknown. Here we track the fate of high avidity CD4 T cells recognizing a self-antigen expressed in pancreatic islet β cells using a transgenic mouse model. T cells of identical specificity, recognizing a dominant peptide from the same islet antigen and major histocompatibility complex (MHC)-presenting molecule, were followed on autoimmune susceptible and resistant genetic backgrounds. We show that non-MHC genes from the NOD strain cause a failure to delete these high avidity autoreactive T cells during their development in the thymus, with subsequent spontaneous breakdown of CD4 cell tolerance to the islet antigen, formation of intra-islet germinal centers, and high titre immunoglobulin G1 autoantibody production. In mixed bone marrow chimeric animals, defective thymic deletion was intrinsic to T cells carrying diabetes susceptibility genes. These results demonstrate a primary failure to censor forbidden clones of self-reactive T cells in inherited susceptibility to organ-specific autoimmune disease, and highlight the importance of thymic mechanisms of tolerance in organ-specific tolerance.
PMCID: PMC2194101  PMID: 12417628
autoimmune disease; diabetes mellitus type I; clonal deletion; T lymphocytes; genetic predisposition to disease
6.  Methionine Regeneration and Aspartate Aminotransferase in Parasitic Protozoa 
Journal of Bacteriology  2001;183(15):4421-4434.
Aspartate aminotransferases have been cloned and expressed from Crithidia fasciculata, Trypanosoma brucei brucei, Giardia intestinalis, and Plasmodium falciparum and have been found to play a role in the final step of methionine regeneration from methylthioadenosine. All five enzymes contain sequence motifs consistent with membership in the Ia subfamily of aminotransferases; the crithidial and giardial enzymes and one trypanosomal enzyme were identified as cytoplasmic aspartate aminotransferases, and the second trypanosomal enzyme was identified as a mitochondrial aspartate aminotransferase. The plasmodial enzyme contained unique sequence substitutions and appears to be highly divergent from the existing members of the Ia subfamily. In addition, the P. falciparum enzyme is the first aminotransferase found to lack the invariant residue G197 (P. K. Mehta, T. I. Hale, and P. Christen, Eur. J. Biochem. 214:549–561, 1993), a feature shared by sequences discovered in P. vivax and P. berghei. All five enzymes were able to catalyze aspartate-ketoglutarate, tyrosine-ketoglutarate, and amino acid-ketomethiobutyrate aminotransfer reactions. In the latter, glutamate, phenylalanine, tyrosine, tryptophan, and histidine were all found to be effective amino donors. The crithidial and trypanosomal cytosolic aminotransferases were also able to catalyze alanine-ketoglutarate and glutamine-ketoglutarate aminotransfer reactions and, in common with the giardial aminotransferase, were able to catalyze the leucine-ketomethiobutyrate aminotransfer reaction. In all cases, the kinetic constants were broadly similar, with the exception of that of the plasmodial enzyme, which catalyzed the transamination of ketomethiobutyrate significantly more slowly than aspartate-ketoglutarate aminotransfer. This result obtained with the recombinant P. falciparum aminotransferase parallels the results seen for total ketomethiobutyrate transamination in malarial homogenates; activity in the latter was much lower than that in homogenates from other organisms. Total ketomethiobutyrate transamination in Trichomonas vaginalis and G. intestinalis homogenates was extensive and involved lysine-ketomethiobutyrate enzyme activity in addition to the aspartate aminotransferase activity. The methionine production in these two species could be inhibited by the amino-oxy compounds canaline and carboxymethoxylamine. Canaline was also found to be an uncompetitive inhibitor of the plasmodial aspartate aminotransferase, with a Ki of 27 μM.
PMCID: PMC95336  PMID: 11443076
7.  Tyrosine Aminotransferase Catalyzes the Final Step of Methionine Recycling in Klebsiella pneumoniae 
Journal of Bacteriology  1999;181(6):1739-1747.
An aminotransferase which catalyzes the final step in methionine recycling from methylthioadenosine, the conversion of α-ketomethiobutyrate to methionine, has been purified from Klebsiella pneumoniae and characterized. The enzyme was found to be a homodimer of 45-kDa subunits, and it catalyzed methionine formation primarily using aromatic amino acids and glutamate as the amino donors. Histidine, leucine, asparagine, and arginine were also functional amino donors but to a lesser extent. The N-terminal amino acid sequence of the enzyme was determined and found to be almost identical to the N-terminal sequence of both the Escherichia coli and Salmonella typhimurium tyrosine aminotransferases (tyrB gene products). The structural gene for the tyrosine aminotransferase was cloned from K. pneumoniae and expressed in E. coli. The deduced amino acid sequence displayed 83, 80, 38, and 34% identity to the tyrosine aminotransferases from E. coli, S. typhimurium, Paracoccus denitrificans, and Rhizobium meliloti, respectively, but it showed less than 13% identity to any characterized eukaryotic tyrosine aminotransferase. Structural motifs around key invariant residues placed the K. pneumoniae enzyme within the Ia subfamily of aminotransferases. Kinetic analysis of the aminotransferase showed that reactions of an aromatic amino acid with α-ketomethiobutyrate and of glutamate with α-ketomethiobutyrate proceed as favorably as the well-known reactions of tyrosine with α-ketoglutarate and tyrosine with oxaloacetate normally associated with tyrosine aminotransferases. The aminotransferase was inhibited by the aminooxy compounds canaline and carboxymethoxylamine but not by substrate analogues, such as nitrotyrosine or nitrophenylalanine.
PMCID: PMC93571  PMID: 10074065
8.  Massive Scalp Haemorrhage after Fetal Blood Sampling due to Haemorrhagic Disease 
British Medical Journal  1972;4(5836):321-322.
Massive subaponeurotic haematoma occurred in a baby after suture of bleeding scalp blood sampling stabs made before delivery. Eighteen hours after delivery blood samples showed marked prolongation of the prothrombin time. The condition was successfully treated with vitamin K1 and blood transfusion.
PMCID: PMC1786565  PMID: 4637510
9.  A Novel Selection Regime for Differentiation Defects Demonstrates an Essential Role for the Stumpy Form in the Life Cycle of the African Trypanosome 
Molecular Biology of the Cell  2000;11(5):1905-1917.
A novel selection scheme has been developed to isolate bloodstream forms of Trypanosoma brucei, which are defective in their ability to differentiate to the procyclic stage. Detailed characterization of one selected cell line (defective in differentiation clone 1 [DiD-1]) has demonstrated that these cells are indistinguishable from the wild-type population in terms of their morphology, cell cycle progression, and biochemical characteristics but are defective in their ability to initiate differentiation to the procyclic form. Although a small proportion of DiD-1 cells remain able to transform, deletion of the genes for glycophosphatidyl inositol-phospholipase C demonstrated that this enzyme was not responsible for this inefficient differentiation. However, the attenuated growth of the Δ-glycophosphatidyl inositol-phospholipase C DiD-1 cells in mice permitted the expression of stumpy characteristics in this previously monomorphic cell line, and concomitantly their ability to differentiate efficiently was restored. Our results indicate that monomorphic cells retain expression of a characteristic of the stumpy form essential for differentiation, and that this is reduced in the defective cells. This approach provides a new route to dissection of the cytological and molecular basis of life cycle progression in the African trypanosome.
PMCID: PMC14892  PMID: 10793160

Results 1-9 (9)