Role of C/EBPβ in two luminal progenitor populations in the mouse mammary gland

The mammary gland is a branched epithelial organ comprised of myoepithelial, ductal and alveolar cells that are derived from resident stem and progenitor cells. The progression from mammary gland stem cell(s) to the differentiated mammary gland cell types is poorly understood. Here, I describe the identification and characterization of two luminal progenitor cell populations in the mouse mammary gland, and investigate the role of the transcription factor C/EBPβ in their development. In Chapter 2, I describe the isolation of two luminal progenitor cell populations (Sca1+ and Sca1- luminal cells) and show that they are differentially primed in their gene expression towards ductal and alveolar cell fates, respectively. Furthermore, I show that in vivo genetic priming affects the in vitro differentiation potential of Sca1+ and Sca1- luminal cells. In Chapter 3, I show that C/EBPβ is required for the appropriate specification of ductal and alveolar lineages, and in its absence, alveolar lineage priming is lost, and ductal lineage priming is up-regulated in both Sca1+ and Sca1- cells. Preliminary data also shows that in addition to severe proliferation defects, the changes in in vivo lineage priming in Cebpb-/- mice also affect the in vitro differentiation potential of Cebpb-/- Sca1+ and Sca1- luminal progenitors. Lastly, in Chapter 4, I describe the genome-wide binding characteristics of C/EBPβ in Sca1+, Sca1- and P16.5 alveolar cells. These experiments reveal that genome-wide C/EBPβ occupancy is correlated with alveolar cells fate, and that C/EBPβ target genes perform distinct cellular functions in alveolar cells (Sca1- cells and P16.5). Furthermore, I show that Elf5 is directly regulated by C/EBPβ, and posit that direct regulation of Elf5 by C/EBPβ may be one mechanism through which C/EBPβ exerts its alveolar cell fate programming

FOG-1 and GATA-1 act sequentially to specify definitive megakaryocytic and erythroid progenitors

During haematopoiesis, megakaryocytes and erythrocytes derive from a common precursor called preMegE. This study reports a role for the transcription factor FOG-1 in specification of preMegEs, while GATA-1 is subsequently required for erythroid-lineage commitment

Homozygous N396T mutation in Gaucher disease:Portuguese sisters with markedly different phenotypes

Samantha Kimball1,2, Francis Choy4, Agnes Zay5, Dominick Amato31Department of Nutritional Sciences, University of Toronto, Canada; 2Department of Laboratory Medicine and Pathology, 3Department of Medicine, Division of Hematology, Mt Sinai Hospital, Toronto, Canada; 4Department of Biology, University of Victoria, Victoria, Canada; 5MRC Center for Regenerative Medicine, University of Edinburgh, Edinburgh, ScotlandAbstract: Gaucher disease (GD) is characterized by reduced activity of glucocerebrosidase leading to complications in the reticuloendothelial system. N396T, a rarer mutation of the glucocerebrosidase gene, has been encountered in Portuguese populations and has generally been associated with milder phenotypes. This report presents brief histories of two Portuguese sisters, both with homozygous N396T mutations. These patients are phenotypically very different despite the fact that in both patients residual enzyme activity is very low. The case of patient 1 is complicated by comorbid diabetes mellitus and human immunodeficiency virus (HIV) infection. Enzyme replacement therapy (ERT) improved this patient's clinical picture sufficiently to enable antiretroviral treatment to proceed for the HIV. This report demonstrates the poor correlation of clinical GD with genotype as well as with residual enzyme activity. It further illustrates how treatment of the underlying GD with ERT improved symptoms allowing for antiretroviral therapy thereby improving both the GD and HIV.Keywords: Gaucher disease, N396T mutation, glucocerebrosidase, HI

Glycine cleavage enzyme complex:molecular cloning and expression of the H-protein cDNA from cultured human skin fibroblasts

The human H-protein is one of four essential components (H-, L-, P-, and T-proteins) of the mammalian glycine cleavage enzyme complex and its function is involved in the pathogenesis and diagnosis of glycine encephalopathy. A transcript corresponding to the glycine cleavage H-protein functional gene was isolated from cultured human skin fibroblasts along with a transcript for a putative processed pseudogene on chromosome 2q33.3. Sequence analysis of the fibroblast H-protein functional gene transcript showed complete identity to that reported from human liver. The H-protein cDNA was subsequently cloned with a hexahistidine affinity tag in the Pichia pastoris plasmid vector pPICZαA and recombined into the yeast genome downstream of the alcohol oxidase promoter for methanol-induced expression. The recombinant H-protein was secreted into the culture medium and purified to homogeneity using a one-step nickel-nitrilotriacetic acid resin column. Approximately 4 mg of homogeneous H-protein was obtained from 1 L of culture medium. Since the attachment of a lipoic acid prosthetic group is required for H-protein function, we have expressed and purified E. coli lipoate protein ligase and succeeded in lipoylating H-protein, converting the apo-H-protein to the functional holo-H-protein. A lipoamide dehydrogenase assay was performed to confirm that the apo-H-protein was inactive, whereas the holo-H-protein was approximately 2.3-fold more active than free lipoic acid as a hydrogen donor in driving the reaction. The availability of copious amounts of human recombinant H-protein by using Pichia pastoris expression and affinity purification will facilitate the elucidation of the structure and function of the H-protein and its relationship to the P-, T-, and L-proteins in the glycine cleavage enzyme complex. In view of the fact that there is no detectable glycine cleavage enzyme activity in human skin fibroblasts, we speculate that a plausible function of the H-protein is to interact with the L-protein, which is also part of the l-ketoglutarate dehydrogenase complex present in fibroblasts.</p

Gaucher disease among Chinese patients:review on genotype/phenotype correlation from 29 patients and identification of novel and rare alleles

Gaucher disease, the most prevalent lysosomal storage disease, results from an inherited deficiency in the enzyme glucocerebrosidase. Three clinical forms of Gaucher disease have been described: Type 1 non-neuronopathic, Type 2 acute neuronopathic, and Type 3 subacute neuronopathic. Although Gaucher disease is panethnic, its presentation reveals some ethnic-specific characteristics. The Type 1 form is most common among Caucasian patients. In contrast, the majority of Chinese Gaucher disease patients have early age of onset, severe hematological and skeletal complications, and often neurological involvement, resulting in early childhood death. In this report, we review 29 cases of Gaucher disease from 23 unrelated patients and 6 patients from 3 non-consanguineous families. Among these patients, 13 were diagnosed as Type 1, 10 as Type 2, and 6 as Type 3. A novel mutation, del 205-209ACCTT, was identified in the heterozygous form with mutation R353W (c.1174C&gt;T) by DNA sequence analysis in 2 Type 1 patients who are sibs. Mutation R353W was also found in the heterozygous form in 3 other Type 1 patients, with mutation L444P in 2 sibs and a second unknown Gaucher allele in the third patient. The Gaucher genotypes of the remaining Type 1 patients were F37V/L444P, G46E/L444P, R48W/R120W, N188S/L444P, Y205C/L444P, N370S/L444P, and L444P/unknown. It was noted that mutation N370S in the patient was linked to the pv1.1(-)(1) haplotype present in Jewish patients. Among the Type 2 patients, L444P was present in the heterozygous form with mutation F213I, L385P, or the complex allele (RecNci) in 5 patients. The second most common mutation, F213I, was found in the heterozygous form in 6 patients with mutations N382K, L383R, or L444P. The other mutations found in the Type 2 patients were P122L, V375L, Y363C, M416V, and 383-400del. The genotypes of the 6 Type 3 patients identified were D409H/D409H, D409H/G202R, G46E/N188S, N188S/unknown, and L444P/L444P. While D409H has been reported as being associated with cardiovascular/ocular involvements in Gaucher disease, there have been no such complications in these patients. As noted, the majority of the Gaucher mutations we identified in the Chinese patients were either rare or absent in other populations. With the exception of N370S and R353W found only in the Type 1 form, the majority of these mutations are severe ones that result in poor prognosis and often Types 2 and 3 Gaucher disease.</p

Empagliflozin in Patients with Chronic Kidney Disease

Background The effects of empagliflozin in patients with chronic kidney disease who are at risk for disease progression are not well understood. The EMPA-KIDNEY trial was designed to assess the effects of treatment with empagliflozin in a broad range of such patients. Methods We enrolled patients with chronic kidney disease who had an estimated glomerular filtration rate (eGFR) of at least 20 but less than 45 ml per minute per 1.73 m(2) of body-surface area, or who had an eGFR of at least 45 but less than 90 ml per minute per 1.73 m(2) with a urinary albumin-to-creatinine ratio (with albumin measured in milligrams and creatinine measured in grams) of at least 200. Patients were randomly assigned to receive empagliflozin (10 mg once daily) or matching placebo. The primary outcome was a composite of progression of kidney disease (defined as end-stage kidney disease, a sustained decrease in eGFR to &lt; 10 ml per minute per 1.73 m(2), a sustained decrease in eGFR of &amp; GE;40% from baseline, or death from renal causes) or death from cardiovascular causes. Results A total of 6609 patients underwent randomization. During a median of 2.0 years of follow-up, progression of kidney disease or death from cardiovascular causes occurred in 432 of 3304 patients (13.1%) in the empagliflozin group and in 558 of 3305 patients (16.9%) in the placebo group (hazard ratio, 0.72; 95% confidence interval [CI], 0.64 to 0.82; P &lt; 0.001). Results were consistent among patients with or without diabetes and across subgroups defined according to eGFR ranges. The rate of hospitalization from any cause was lower in the empagliflozin group than in the placebo group (hazard ratio, 0.86; 95% CI, 0.78 to 0.95; P=0.003), but there were no significant between-group differences with respect to the composite outcome of hospitalization for heart failure or death from cardiovascular causes (which occurred in 4.0% in the empagliflozin group and 4.6% in the placebo group) or death from any cause (in 4.5% and 5.1%, respectively). The rates of serious adverse events were similar in the two groups. Conclusions Among a wide range of patients with chronic kidney disease who were at risk for disease progression, empagliflozin therapy led to a lower risk of progression of kidney disease or death from cardiovascular causes than placebo