Adaptation and Therapeutic Exploitation of the Plasma Membrane of African Trypanosomes.

African trypanosomes are highly divergent from their metazoan hosts, and as part of adaptation to a parasitic life style have developed a unique endomembrane system. The key virulence mechanism of many pathogens is successful immune evasion, to enable survival within a host, a feature that requires both genetic events and membrane transport mechanisms in African trypanosomes. Intracellular trafficking not only plays a role in immune evasion, but also in homeostasis of intracellular and extracellular compartments and interactions with the environment.

Enhanced snoMEN Vectors Facilitate Establishment of GFP-HIF-1α Protein Replacement Human Cell Lines.

The snoMEN (snoRNA Modulator of gene ExpressioN) vector technology was developed from a human box C/D snoRNA, HBII-180C, which contains an internal sequence that can be manipulated to make it complementary to RNA targets, allowing knock-down of targeted genes. Here we have screened additional human nucleolar snoRNAs and assessed their application for gene specific knock-downs to improve the efficiency of snoMEN vectors. We identify and characterise a new snoMEN vector, termed 47snoMEN, that is derived from box C/D snoRNA U47, demonstrating its use for knock-down of both endogenous cellular proteins and G/YFP-fusion proteins.

Targeted Knock-Down of miR21 Primary Transcripts Using snoMEN Vectors Induces Apoptosis in Human Cancer Cell Lines.

We have previously reported an antisense technology, 'snoMEN vectors', for targeted knock-down of protein coding mRNAs using human snoRNAs manipulated to contain short regions of sequence complementarity with the mRNA target. Here we characterise the use of snoMEN vectors to target the knock-down of micro RNA primary transcripts. We document the specific knock-down of miR21 in HeLa cells using plasmid vectors expressing miR21-targeted snoMEN RNAs and show this induces apoptosis.

Umbilical cord blood as an alternative source of reduced-intensity hematopoietic stem cell transplantation for chronic Epstein-Barr virus-associated T or natural killer cell lymphoproliferative diseases.

Chronic Epstein-Barr virus-associated T/natural killer cell lymphoproliferative diseases represented by chronic active Epstein-Barr virus infection are lethal but are curable with several courses of chemotherapy and allogeneic hematopoietic stem cell transplantation (HSCT). Recently, we reported that reduced-intensity conditioning (RIC) provided better outcomes than myeloablative conditioning because RIC was less toxic. However, it was unclear whether cord blood transplantation (CBT) works in the context of RIC.

FMN2 is a novel regulator of the cyclin-dependent kinase inhibitor p21.

We have identified the human FMN2 gene as a novel target regulated by induction of p14ARF and by multiple other stress responses, including DNA damage and hypoxia, which have in common activation of cell cycle arrest. We showed that increased expression of the FMN2 gene following p14ARF induction is caused, at the transcriptional level, by relief of repression by RelA and E2F1, which, under non-induced conditions, bind the FMN2 promoter. Increased FMN2 protein levels promote cell cycle arrest by inhibiting the degradation of p21, and our data show that control of p21 stability is a key part of the mechanism that regulates p21 induction.

Analysis of human protein replacement stable cell lines established using snoMEN-PR vector.

The study of the function of many human proteins is often hampered by technical limitations, such as cytotoxicity and phenotypes that result from overexpression of the protein of interest together with the endogenous version. Here we present the snoMEN (snoRNA Modulator of gene ExpressioN) vector technology for generating stable cell lines where expression of the endogenous protein can be reduced and replaced by an exogenous protein, such as a fluorescent protein (FP)-tagged version. SnoMEN are snoRNAs engineered to contain complementary sequences that can promote knock-down of targeted RNAs.

Feasibility of reduced-intensity allogeneic stem cell transplantation with imatinib in children with philadelphia chromosome-positive acute lymphoblastic leukemia.

Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph + ALL) in children is one of the highest-risk ALL groups. Improved outcome in combination with imatinib has been reported. However, intensive chemotherapy or myeloablative conditioning followed by hematopoietic stem cell transplantation (HSCT) can be associated with significant adverse late effects.

Identification and functional characterization of FMN2, a regulator of the cyclin-dependent kinase inhibitor p21.

The ARF tumor suppressor is a central component of the cellular defense against oncogene activation in mammals. p14ARF activates p53 by binding and inhibiting HDM2, resulting, inter alia, in increased transcription and expression of the cyclin-dependent kinase inhibitor p21 and consequent cell-cycle arrest. We analyzed the effect of p14ARF induction on nucleolar protein dynamics using SILAC mass spectrometry and have identified the human Formin-2 (FMN2) protein as a component of the p14ARF tumor suppressor pathway.

Human box C/D snoRNA processing conservation across multiple cell types.

Small nucleolar RNAs (snoRNAs) function mainly as guides for the post-transcriptional modification of ribosomal RNAs (rRNAs). In recent years, several studies have identified a wealth of small fragments (<35 nt) derived from snoRNAs (termed sdRNAs) that stably accumulate in the cell, some of which may regulate splicing or translation. A comparison of human small RNA deep sequencing data sets reveals that box C/D sdRNA accumulation patterns are conserved across multiple cell types although the ratio of the abundance of different sdRNAs from a given snoRNA varies.

Tea catechins modulate the glucose transport system in 3T3-L1 adipocytes.

In this study, we investigated the effects of tea catechins on the translocation of glucose transporter (GLUT) 4 in 3T3-L1 adipocytes. We found that the ethyl acetate fraction of green tea extract, containing abundant catechins, most decreased insulin-induced glucose uptake activity in 3T3-L1 cells. When the cells were treated with 50 μM catechins in the absence or presence of insulin for 30 min, nongallate-type catechins increased glucose uptake activity without insulin, whereas gallate-type catechins decreased insulin-induced glucose uptake activity.

Quantitative proteomic analysis of chromatin reveals that Ctf18 acts in the DNA replication checkpoint.

Yeast cells lacking Ctf18, the major subunit of an alternative Replication Factor C complex, have multiple problems with genome stability. To understand the in vivo function of the Ctf18 complex, we analyzed chromatin composition in a ctf18Δ mutant using the quantitative proteomic technique of stable isotope labeling by amino acids in cell culture. Three hundred and seven of the 491 reported chromosomal proteins were quantitated.

Severe persistent bone marrow failure following therapy with 2-chlorodeoxyadenosine for relapsing juvenile xanthogranuloma of the brain.

2-Chlorodeoxyadenosine (2-CdA) has been successfully used in children to treat refractory Langerhans cell histiocytosis and juvenile xanthogranuloma (JXG) as salvage therapy. Although 2-CdA is generally well-tolerated, with temporary myelosuppression as the primary dose-limiting toxicity, prolonged myelosuppressive, and immunosuppressive effects have been reported. We describe an adolescent patient with refractory multiple central nervous system JXG, with the lesion size markedly reduced after treatment with 2-CdA.

Identification of human miRNA precursors that resemble box C/D snoRNAs.

There are two main classes of small nucleolar RNAs (snoRNAs): the box C/D snoRNAs and the box H/ACA snoRNAs that function as guide RNAs to direct sequence-specific modification of rRNA precursors and other nucleolar RNA targets. A previous computational and biochemical analysis revealed a possible evolutionary relationship between miRNA precursors and some box H/ACA snoRNAs. Here, we investigate a similar evolutionary relationship between a subset of miRNA precursors and box C/D snoRNAs.

Analysis of human small nucleolar RNAs (snoRNA) and the development of snoRNA modulator of gene expression vectors.

Human small nucleolar RNAs (snoRNAs) that copurify with nucleoli isolated from HeLa cells have been characterized. Novel fibrillarin-associated snoRNAs were detected that allowed the creation of a new vector system for the targeted knockdown of one or more genes in mammalian cells. The snoMEN (snoRNA modulator of gene expressioN) vector technology is based on snoRNA HBII-180C, which contains an internal sequence that can be manipulated to make it complementary to RNA targets.

Cell fate decisions are specified by the dynamic ERK interactome.

Extracellular signal-regulated kinase (ERK) controls fundamental cellular functions, including cell fate decisions. In PC12, cells shifting ERK activation from transient to sustained induces neuronal differentiation. As ERK associates with both regulators and effectors, we hypothesized that the mechanisms underlying the switch could be revealed by assessing the dynamic changes in ERK-interacting proteins that specifically occur under differentiation conditions.

Factitious purpura in a 10-year-old girl.

We describe a 10-year-old girl who presented with bizarre purpura. Both congenital and autoimmune hemorrhagic disorders were excluded based on her past medical history and physical and laboratory findings. Child abuse was also ruled out as purpura continued to develop after child-family separation.

Mammalian Sox15 gene: promoter analysis and implications for placental evolution.

Sox15 belongs to the Sox (Sry-type HMG box) protein family, which is involved in placental development and muscle regeneration. Previously, we showed that the Sox15 gene is highly expressed in the trophoblast giant cells of the mouse placenta. To elucidate the molecular mechanisms of the tissue-dependent transcription of the gene, we isolated approximately 2.

Sox15 enhances trophoblast giant cell differentiation induced by Hand1 in mouse placenta.

Some members of the Sry-type HMG box (Sox) protein family play important roles in embryogenesis as transcription factors. Here, we report that Sox15 transcripts were much more abundant in mouse placenta than in the fetus, the yolk sac, or several adult tissues. In situ hybridization analysis of the mouse E8.

Transcriptional regulation of the rainbow trout CYP19a gene by FTZ-F1 homologue.

In rainbow trout, there are at least two CYP19 genes (CYP19a and CYP19b). They encode distinct P450arom isozymes that are differentially expressed in the ovary and brain. To understand the transcriptional regulation of the rainbow trout CYP19a (rtCYP19a) gene in the ovary, we isolated its 5'-flanking region.