Enhanced dopaminergic differentiation of human neural stem cells by synergistic effect of Bcl-xL and reduced oxygen tension

Neural stem cells constitute a promising source of cells for transplantation in Parkinson's disease, but a protocol for controlled dopaminergic differentiation is not yet available. Here we investigated the effect of the anti-apoptotic protein Bcl-x_L and oxygen tension on dopaminergic differentiation and survival of a human ventral mesencephalic stem cell line (hVM1). hVM1 cells and a Bcl-x_L over-expressing subline (hVMbcl-x_L) were differentiated by sequential treatment with fibroblast growth factor-8, forskolin, sonic hedgehog, and glial cell line-derived neurotrophic factor. After 10 days at 20% oxygen, hVMbcl-x_L cultures contained proportionally more tyrosine hydroxylase(TH)-positive cells than hVM1 control cultures. This difference was significantly potentiated from 11 ± 0.8% to 17.2 ± 0.2% of total cells when the oxygen tension was lowered to 3%. Immunocytochemistry and Q-PCR-analysis revealed expression of several dopaminergic markers besides of TH just as dopamine was detected in the culture medium by HPLC analysis. Although Bcl-x_L-over-expression reduced cell death in the cultures, it did not alter the relative content of GABAergic, neurons, while the content of astroglial cells was reduced in hVMbcl-x_L cell cultures compared with control. We conclude that Bcl-x_L and lowered oxygen tension act in concert to enhance dopaminergic differentiation and survival of human neural stem cells.     

K2P18.1 translates T cell receptor signals into thymic regulatory T cell development

It remains largely unclear how thymocytes translate relative differences in T cell receptor (TCR) signal strength into distinct developmental programs that driv...     

A deliberate approach to screening for initial crystallization conditions of biological macromolecules

A method to rationally predict crystallization conditions for a previously uncrystallized macromolecule has not yet been developed. One way around this problem is to determine initial crystallization conditions by casting a wide net, surveying a large number of chemical and physical conditions to locate crystallization leads. A facility that executes the rapid survey of crystallization lead conditions is described in detail. Results and guidelines for the initial screening of crystallization conditions, applicable to both manual and robotic setups, are discussed.     

Hemopoietic cell expression of the chemokine decoy receptor D6 is dynamic and regulated by GATA1

D6 scavenges inflammatory chemokines and is essential for the regulation of inflammatory and immune responses. Mechanisms explaining the cellular basis for D6 function have been based on D6 expression by lymphatic endothelial cells. In this study, we demonstrate that functional D6 is also expressed by murine and human hemopoietic cells and that this expression can be regulated by pro- and anti-inflammatory agents. D6 expression was highest in B cells and dendritic cells (DCs). In myeloid cells, LPS down-regulated expression, while TGF-beta up-regulated expression. Activation of T cells with anti-CD3 and soluble CD28 up-regulated mRNA expression 20-fold, while maturation of human macrophage and megakaryocyte precursors also up-regulated D6 expression. Competition assays demonstrated that chemokine uptake was D6 dependent in human leukocytes, whereas mouse D6-null cells failed to uptake and clear inflammatory chemokines. Furthermore, we present evidence indicating that D6 expression is GATA1 dependent, thus explaining D6 expression in myeloid progenitor cells, mast cells, megakaryocytes, and DCs. We propose a model for D6 function in which leukocytes, within inflamed sites, activate D6 expression and thus trigger resolution of inflammatory responses. Our data on D6 expression by circulating DCs and B cells also suggest alternative roles for D6, perhaps in the coordination of innate and adaptive immune responses. These data therefore alter our models of in vivo D6 function and suggest possible discrete, and novel, roles for D6 on lymphatic endothelial cells and leukocytes.     

Validation of candidate genes putatively associated with resistance to SCMV and MDMV in maize (Zea mays L.) by expression profiling

Background  

The potyviruses sugarcane mosaic virus (SCMV) and maize dwarf mosaic virus (MDMV) are major pathogens of maize worldwide. Two loci, Scmv1 and Scmv2, have ealier been shown to confer complete resistance to SCMV. Custom-made microarrays containing previously identified SCMV resistance candidate genes and resistance gene analogs were utilised to investigate and validate gene expression and expression patterns of isogenic lines under pathogen infection in order to obtain information about the molecular mechanisms involved in maize-potyvirus interactions.     

Human immunodeficiency virus-specific CD8+ T-cell activity is detectable from birth in the majority of in utero-infected infants

Human immunodeficiency virus (HIV)-infected infants in sub-Saharan Africa typically progress to AIDS or death by 2 years of life in the absence of antiretroviral therapy. This rapid progression to HIV disease has been related to immaturity of the adaptive immune response in infants. We screened 740 infants born to HIV-infected mothers and tracked development and specificity of HIV-specific CD8⁺ T-cell responses in 63 HIV-infected infants identified using gamma interferon enzyme-linked immunospot assays and intracellular cytokine staining. Forty-four in utero-infected and 19 intrapartum-infected infants were compared to 45 chronically infected children >2 years of age. Seventy percent (14 of 20) in utero-infected infants tested within the first week of life demonstrated HIV-specific CD8⁺ T-cell responses. Gag, Pol, and Nef were the principally targeted regions in chronic pediatric infection. However, Env dominated the overall response in one-third (12/36) of the acutely infected infants, compared to only 2/45 (4%) of chronically infected children (P = 0.00083). Gag-specific CD4⁺ T-cell responses were minimal to undetectable in the first 6 months of pediatric infection. These data indicate that failure to control HIV replication in in utero-infected infants is not due to an inability to induce responses but instead suggest secondary failure of adaptive immunity in containing this infection. Moreover, the detection of virus-specific CD8⁺ T-cell responses in the first days of life in most in utero-infected infants is encouraging for HIV vaccine interventions in infants.     

EST-based gene discovery in pig: virtual expression patterns and comparative mapping to human

A molecular understanding of porcine reproduction is of biological interest and economic importance. Our Midwest Consortium has produced cDNA libraries containing the majority of genes expressed in major female reproductive tissues, and we have deposited into public databases 21,499 expressed sequence tag (EST) gene sequences from the 3 end of clones from these libraries. These sequences represent 10,574 different genes, based on sequence comparison among these data, and comparison with existing porcine ESTs and genes indicate as many as 4652 of these EST clusters are novel. In silico analysis identified sequences that are expressed in specific pig tissues or organs and confirmed the broad expression in pig for many genes ubiquitously expressed in human tissues. Furthermore, we have developed computer software to identify sequence similarity of these pig genes with their human counterparts, and to extract the mapping information of these human homologues from genome databases. We demonstrate the utility of this software for comparative mapping by localizing 61 genes on the porcine physical map for Chromosomes (Chrs) 5, 10, and 14. The following Accession numbers were assigned to our deposited sequences: BF701840 – BF704551, BF708383, BF708386 – BF713604, BG322266 – BG322271, BI398567 – BI405235, BQ597354 – BQ605166.