Human GAPDH Research Articles

In Vitro and in Vivo Enhanced Generation of Human A9 Dopamine Neurons from Neural Stem Cells by Bcl-XL

Human neural stem cells derived from the ventral mesencephalon (VM) are powerful research tools and candidates for cell therapies in Parkinson disease. Previous studies with VM dopaminergic neuron (DAn) precursors indicated poor growth potential and unstable phenotypical properties. Using the model cell line hVM1 (human ventral mesencephalic neural stem cell line 1; a new human fetal VM stem cell line), we have found that Bcl-X(L) enhances the generation of DAn from VM human neural stem cells. Mechanistically, Bcl-X(L) not only exerts the expected antiapoptotic effect but also induces proneural (NGN2 and NEUROD1) and dopamine-related transcription factors, resulting in a high yield of DAn with the correct phenotype of substantia nigra pars compacta (SNpc). The expression of key genes directly involved in VM/SNpc dopaminergic patterning, differentiation, and maturation (EN1, LMX1B, PITX3, NURR1, VMAT2, GIRK2, and dopamine transporter) is thus enhanced by Bcl-X(L). These effects on neurogenesis occur in parallel to a decrease in glia generation. These in vitro Bcl-X(L) effects are paralleled in vivo, after transplantation in hemiparkinsonian rats, where hVM1-Bcl-X(L) cells survive, integrate, and differentiate into DAn, alleviating behavioral motor asymmetry. Bcl-X(L) then allows for human fetal VM stem cells to stably generate mature SNpc DAn both in vitro and in vivo and is thus proposed as a helpful factor for the development of cell therapies for neurodegenerative conditions, Parkinson disease in particular.

Real-time PCR method for the quantification ofBurkholderia cepaciacomplex attached to lung epithelial cells and inhibition of that attachment

To develop a rapid method to quantify the attachment of the cystic fibrosis pathogen, Burkholderia multivorans, to lung epithelial cells (16HBE14o(-)) using real-time PCR with a view to monitoring potential inhibition of lung cell attachment. Mammalian and bacterial DNA were purified from bacteria attached to lung epithelial cells. The relative amount of bacteria attached was determined by amplification of the recA gene relative to the human GAPDH gene, in the presence of SYBR Green. The method was thoroughly validated and shown to correlate well with traditional plating techniques. Inhibition of bacterial attachment with simple sugars was then evaluated by real-time PCR. Of the sugars examined, pre-incubation of B. multivorans with lactose, mannose and xylitol all decreased bacterial adherence to 16HBE14o(-) cells, while glucose and galactose had no significant effect. Pre-incubation with lactose had the greatest effect, resulting in reduced adhesion to 35% of untreated controls. This method can be used to quickly and effectively screen novel agents with higher affinities for bacterial adhesins. This method will enable the rapid development of novel agents to inhibit colonization by this pathogen from the environment.

Analysis of Intracellular Molecules Using Nanoneedles Functionalized with FRET-based Molecular Probes

We have been developing a low invasive cell manipulation technology using a nanoneedle of diameter 200 nm and length 10 μm. The nanoneedle insertion is performed by using an atomic force microscope (AFM) to sense a force response when inserting the needle into a living cell body. In this study, nanoneedles functionalized with FRET-based molecular probe have been developed to detect the intracellular mRNA or protein of a living cell. A molecular beacon which targets on human GAPDH mRNA or a FRET-sensor protein which contains substrate motif of caspase-3 was immobilized on a nanoneedle and each functionalized nanoneedle was inserted into a healthy HeLa cell or an apoptotic HeLa cell, respectively. FRET disruptions on the nanoneedles caused by hybridizing with mRNA or by digesting of the sensor protein with caspase-3 could be detected with a confocal scanning fluorescent microscopy.

Quantitation of WT1 Transcripts by Copy Number Provides a Sensitive Indication of Minimal Residual Disease (MRD) and Response to Adoptive Cell Therapy in Patients Transplanted for Ph(+) CML.

Abstract 4489▪The Wilm's tumor gene, WT-1, is markedly overexpressed in a high proportion of acute leukemias, advanced MDS and Ph(+) CML. Prior studies, employing semiquantitative PCR-based techniques, have suggested that elevated levels of WT-1 transcripts in bone marrow and PBMC are indicative of MRD, and predictive of relapse in AML and CML following chemotherapy. We have now developed a quantitative real time PCR-based assay for WT-1 transcripts and used it to evaluate the long term effects of donor lymphocyte infusions in a series of patients with Ph(+) CML treated for relapse or persistent MRD following allogeneic marrow transplants between 1993 and 2004 who are currently in molecular or cytogenetic remission.To quantitate WT-1 mRNA copy number in the marrow and PBMC, total RNA from patient samples received at different times that had been extracted with trizol reagent and stored at -20°C was transcribed into cDNA using MMLV reverse transcriptase. Real time PCR was performed with 1-2 μg of total RNA using the ABI 7300 instrument with a 25 base pair FAM labeled probe containing a sequence located at the boundary of exon 6 and 7 of the WT-1 gene in order to eliminate amplification of genomic DNA and insure measurement of all transcribed WT-1 isoforms. The forward and reverse primers were synthesized from sequences contained in exon 6 and 7 of the WT-1 gene respectively. Cloned plasmids containing defined copy numbers of WT-1 and human GAPDH were used to prepare standard curves using serial dilutions. Quantitation of WT-1 copy number expression was performed by calibration against these standard curves. The expression of WT-1 was normalized against the expression of the control GAPDH to adjust for variation in RNA and cDNA synthesis.Using this assay, we evaluated sequential samples from each patient for correlations between WT-1 copy number detected and the results of qualitative PCR assays for bcr/abl fusion gene transcripts previously performed on the same samples using nested primers as well as the results of clinical, hematologic and cytogenetic analyses of each patient obtained at each time of sampling. Blood samples from a group of 8 normal volunteers served as negative controls.The average WT-1 copy number in the blood of normal volunteers was 25 copies/ μg RNA (range 2-60). With one exception, blood and marrow samples from patients with CML in molecular remission (bcr/abl not detected) had WT-1 copy numbers < 50 copies/μg RNA. One sample of marrow from a patient who had been bcr/abl + until 6 months post transplant and had WT-1 copy numbers that had fallen from 12,932 one month post transplant to 1855 at that time, became bcr/abl negative at 11 months but still had a WT-1 copy number of 931/ μg RNA. In contrast, patients with molecular disease (+ bcr/abl) maintained WT-1 copy numbers ranging from 70-1000/ μg RNA. Samples from patients with cytogenetic relapse consistently had WT-1 copy numbers exceeding 1000/ μg RNA, with levels exceeding 10,000 in 4/6 such patients tested. In a subset of patients who have failed to achieve or sustain molecular remission following DLI, marrow and blood samples with detectable bcr/abl transcripts and WT-1 copy numbers ranging from 60-3000 have been repeatedly detected for periods of up to 6 years without additional treatment and without disease progression implying an ongoing mechanism of disease control. Therefore, quantitation of WT-1 copy number provides a useful technique for monitoring minimal residual disease and may be helpful in identification of patients at higher risk of cytogenetic relapse. Disclosures:No relevant conflicts of interest to declare.

Interleukin-25 Expressed by Brain Capillary Endothelial Cells Maintains Blood-Brain Barrier Function in a Protein Kinase Cϵ-dependent Manner

Interleukin (IL)-25, a member of the IL-17 family of cytokines, is expressed in the brains of normal mice. However, the cellular source of IL-25 and its function in the brain remain to be elucidated. Here, we show that IL-25 plays an important role in preventing infiltration of the inflammatory cells into the central nervous system. Brain capillary endothelial cells (BCECs) express IL-25. However, it is down-regulated by inflammatory cytokines, including tumor necrosis factor (TNF)-alpha, IL-17, interferon-gamma, IL-1beta, and IL-6 in vitro, and is also reduced in active multiple sclerosis (MS) lesions and in the inflamed spinal cord of experimental autoimmune encephalomyelitis, an animal model of MS. Furthermore, IL-25 restores the reduced expression of tight junction proteins, occludin, junction adhesion molecule, and claudin-5, induced by TNF-alpha in BCECs and consequently repairs TNF-alpha-induced blood-brain barrier (BBB) permeability. IL-25 induces protein kinase Cepsilon (PKCepsilon) phosphorylation, and up-regulation of claudin-5 is suppressed by PKCepsilon inhibitor peptide in the IL-25-stimulated BCECs. These results suggest that IL-25 is produced by BCECs and protects against inflammatory cytokine-induced excessive BBB collapse through a PKCepsilon-dependent pathway. These novel functions of IL-25 in maintaining BBB integrity may help us understand the pathophysiology of inflammatory brain diseases such as MS.

Suppression of Cancer Cell Growth by Promoting Cyclin D1 Degradation

The cyclin D1 proto-oncoprotein is a crucial regulator in cell-cycle progression, and aberrant overexpression of cyclin D1 is linked to tumorigenesis of many different cancer types. By screening ubiquitinated cyclin D1 as a substrate with a deubiquitinase library, we have identified USP2 as a specific deubiquitinase for cyclin D1. USP2 directly interacts with cyclin D1 and promotes its stabilization by antagonizing ubiquitin-dependent degradation. Conversely, USP2 knockdown destabilizes cyclin D1 and induces growth arrest in the human cancer lines where cell growth is dependent on cyclin D1 expression. Of note, cyclin D1 is not universally required for cell-cycle progression. Inactivation of USP2 has either very mild effects on cell growth in normal human fibroblasts or no effect in the cancer cells that do not express cyclin D1. These findings suggest that targeting USP2 is an effective approach to induce growth suppression in the cancer cells addicted to cyclin D1 expression.

Hemin Counteracts the Repression of Bcl-2 and Nrf2 Genes and the Cell Killing Induced by Imatinib in Human Bcr-Abl(+) CML Cells

Imatinib is a targeted selective inhibitor of chimaeric Bcr-Abl tyrosine kinase developed for effective therapy of chronic myelogenous leukemia (CML) and acute lymphocytic leukemia (ALL) patients. Unfortunately, evidence now exists to indicate that a portion of such patients treated with imatinib acquire resistance and subsequently relapse. To understand the heterogeneous basis of imatinib resistance, we have investigated the possible mechanism(s) via which hemin, a key regulator of hematopoiesis that is converted to heme intracellularly, renders CML cells less susceptible to imatinib. Hemin at 30-90 aM protected a substantial proportion (>40%) of human Bcr-Abl(+) CML cells (K-562 and KU-812) from imatinib-induced cell killing by increasing the imatinib IC50 value, reducing DNA damage, and promoting erythroid differentiation. RT-PCR assessment of RNA transcripts encoded by human GAPDH, Ggamma-globin, Bcr-Abl, HO-2, Hpr-6, CEBPa, Bcl-2a, Bcl-2b, and Nrf2 genes revealed that hemin selectively counteracted the repression of antiapoptotic Bcl-2a, Bcl-2b, and Nrf2 genes in imatinib-treated cells. These genes are markedly repressed by imatinib alone in human K-562 CML cells. Hemin, however, had no detectable effect on the expression of the Bcr-Abl gene. Moreover, inhibition of de novo heme biosynthesis by succinyl-acetone enhanced the killing effect of imatinib. These data clearly indicate that: (a) cellular heme resulted from de novo biosynthesis and hemin uptake alters the developmental stage of human Bcr-Abl(+) CML cells and their susceptibility to imatinib; (b) cellular heme counteracts the ability of imatinib to repress Bcl-2 and Nrf2 gene expression; and (c) inhibitors of de novo biosynthesis can be developed and combined with imatinib to enhance its antileukemic activity.

Overexpression of malignancy‐associated laminins and laminin receptors by angiotropic human melanoma cells in a chick chorioallantoic membrane model

As distinct from intravascular/lymphatic dissemination, extravascular migratory metastasis (EVMM) has been described as a potential additional mechanism of melanoma spread in which tumor cells migrate along the external surfaces of vessels. Angiotropic melanoma cells are linked to the endothelium by a matrix containing laminin. In addition, it has been shown that C16 laminin-derived peptide increases extravascular migration of human green fluorescent protein (GFP) melanoma cells along vessels in a chicken chorioallantoic membrane model (CAM). In this study, we have tested the hypothesis that expression levels of some genes related to lamimin and metastasis are differentially expressed in vascularized angiotropic melanoma areas vs. avascular melanoma areas from the same tumor. C8161 human melanoma cells in a shell-less chick CAM assay were used to study EVMM associated with the presence of vascularized angiotropic melanoma areas. For both high-quality histomorphology and RNA preservation in paraffin-embedded tissue, we used a methanol-based fixative coupled with microwave-assisted rapid tissue processing as previously described. Using laser capture microdissection, angiotropic melanoma areas as well as avascular areas were microdissected. Using quantitative real time polymerase chain reaction (QRT-PCR), six genes have been studied: LAMC2 (laminin gamma2 chain), LAMA4 (laminin alpha4 chain), ITGB1 (integrin beta1), ITGB3 (integrin beta3), RSPA (ribosomal protein), and MMP2 (matrix metallopeptidase 2). QRT-PCR data were normalized to human GAPDH housekeeping gene and values were compared against Human Total RNA. Final results were expressed as percentage of expression. All tumors demonstrated a similar pattern, i.e. EVMM of angiotropic melanoma cells. The microdissected histopathological sections presented both angiotropic areas and avascular areas. All genes were overexpressed in angiotropic melanoma areas vs. avascular melanoma areas, especially LAMC2, LAMA4 and ITGB3 (respectively, 165.18, 208.86, and 483.69%). This study shows that several genes related to laminin are overexpressed in angiotropic melanoma areas vs. avascular melanoma areas. Since extravascular migration of melanoma cells along vessels has been demonstrated in the CAM model, taken together these results suggests that some laminins and laminin receptors may play a role in extravascular migratory metastasis. This model may represent a promising strategy to analyze differential gene expression in EVMM.

Hepatocyte Nuclear Factor 1α Plays a Critical Role in PCSK9 Gene Transcription and Regulation by the Natural Hypocholesterolemic Compound Berberine

PCSK9 is a natural inhibitor of LDL receptor (LDLR) that binds the extracellular domain of LDLR and triggers its intracellular degradation. PCSK9 and LDLR are coordinately regulated at the transcriptional level by sterols through their promoter-imbedded sterol response elements (SRE) and co-induced by statins. Identification of regulatory networks modulating PCSK9 transcription is important for developing selective repressors of PCSK9 to improve statin efficacy by prolonging the up-regulation of LDLR. Interestingly, the plant-derived hypocholesterolemic compound berberine (BBR) up-regulates LDLR expression while down-regulating PCSK9. In our investigations to define mechanisms underlying the transcriptional suppression of PCSK9 by BBR in HepG2 cells, we have identified a highly conserved hepatocyte nuclear factor 1 (HNF1) binding site residing 28 bp upstream from SRE as a critical sequence motif for PCSK9 transcription and its regulation by BBR. Mutation of the HNF1 site reduced PCSK9 promoter activity >90%. A battery of functional assays identified HNF1α as the predominant trans-activator for PCSK9 gene working through this sequence motif. We further provide evidence suggesting that HNF1 site works cooperatively with SRE as HNF1 mutation significantly attenuated the activity of nuclear SREBP2 to transactivate PCSK9 promoter. Finally, we show that a coordinate modest reduction of HNF1α and nuclear SREBP2 by BBR led to a strong suppression of PCSK9 transcription through these two critical regulatory sequences. This is the first described example of SREBP pairing with HNF1 to control an important regulatory pathway in cholesterol homeostasis. This work also provides a mechanism for how BBR suppresses PCSK9 transcription.

Toward Regenerating a Human ThumbIn Situ

Regenerative technology promises to alleviate the problem of limited donor supply for bone or organ transplants. Most expensive and time consuming is cell expansion in laboratories. We propose a method of magnetically enriched osteoprogenitor stem cells, dispersed in self-assembling hydrogels and applied onto new ultra-high resolution, jet-based, three-dimensional printing of living human bone in a single-step for in situ bone regeneration. Human bone marrow-derived mesenchymal stem cells (hBMSCs) were enriched with CD 117+ cells, dispersed in different collagen I and RAD 16I hydrogel mixes, and applied onto three-dimensional printed btricalcium phosphate=poly(lactic-co-glycolic acid) scaffolds, printed from ultra-high-resolution volumetric CT images of a human thumb. Constructs were directly implanted subcutaneously into nude mice for 6 weeks. In vivo radiographic volumetric CT scanning and histological evaluations were performed at 1, 2, 4, and 6 weeks, and expression of bone-specific genes and biomechanical compression testing at 6 weeks endpoint. Time-dependant accumulation of bone-like extracellular matrix was most evident in CD 117+ hBMSCs using collagen I=RAD 16I hydrogel mix. This was shown histologically by Toluidine blue, von Kossa, and alkaline phosphatase staining, paralleled by increased radiological densities within implants approximating that of human bone, and confirmed by high expression of bone-specific osteonectin and biomechanical stiffness at 6 weeks. Human origin of newly formed tissue was established by expression of human GAPDH using RT-PCR. Statistical analysis confirmed high correlations between biomechanical stiffness, radiological densities, and bone markers. Bone tissue can be successfully regenerated in vivo using a single-step procedure with constructs composed of RAD 16I=collagen I hydrogel, CD 117+-enriched hBMSCs, and porous b-tricalcium phosphate=poly(lactic-co-glycolic acid) scaffolds.

In vitro model for the activation of CD4 and CD8 T cell receptors

Previously, most models that sought to explain the misregulation of immune cell function assumed molecular similarities between the disease-causing pathogens and the host's proteins. In recent time several different models have been proposed and in this study, these concepts are compared to a new hypothesis proposing another explanation for this immune dysregulation: the possibility that the mislocalization of proteins may be responsible for autoimmune activity. Based on this hypothesis, proteins are recognized as self or non-self depending on where they appear in sufficiently high concentrations. To examine this new idea, the intracellular human proteins β-actin, GAPDH, and hemoglobin as well as the extracellular human proteins insulin and albumin, were added to human whole blood samples. After an incubation period, the activation of whole-blood T lymphocytes in the samples was measured. The observed activation pattern of the T lymphocytes fit well with the proposed hypothesis. Therefore, these data suggest that protein mislocalization and/or errors within protein trafficking might be important in the development of autoimmune diseases.

GTP Cyclohydrolase I Expression, Protein, and Activity Determine Intracellular Tetrahydrobiopterin Levels, Independent of GTP Cyclohydrolase Feedback Regulatory Protein Expression

GTP cyclohydrolase I (GTPCH) is a key enzyme in the synthesis of tetrahydrobiopterin (BH4), a required cofactor for nitricoxide synthases and aromatic amino acid hydroxylases. Alterations of GTPCH activity and BH4 availability play an important role in human disease. GTPCH expression is regulated by inflammatory stimuli, in association with reduced expression of GTP cyclohydrolase feedback regulatory protein (GFRP). However, the relative importance of GTPCH expression versus GTPCH activity and the role of GFRP in relation to BH4 bioavailability remain uncertain. We investigated these relationships in a cell line with tet-regulated GTPCH expression and in the hph-1 mouse model of GTPCH deficiency. Doxycycline exposure resulted in a dose-dependent decrease in GTPCH protein and activity, with a strong correlation between GTPCH expression and BH4 levels (r(2) = 0.85, p < 0.0001). These changes in GTPCH and BH4 had no effect on GFRP expression or protein levels. GFRP overexpression and knockdown in tet-GCH cells did not alter GTPCH activity or BH4 levels, and GTPCH-specific knockdown in sEnd.1 endothelial cells had no effect on GFRP protein. In mouse liver we observed a graded reduction of GTPCH expression, protein, and activity, from wild type, heterozygote, to homozygote littermates, with a striking linear correlation between GTPCH expression and BH4 levels (r(2) = 0.82, p < 0.0001). Neither GFRP expression nor protein differed between wild type, heterozygote, nor homozygote mice, despite the substantial differences in BH4. We suggest that GTPCH expression is the primary regulator of BH4 levels, and changes in GTPCH or BH4 are not necessarily accompanied by changes in GFRP expression.

Angiopoietin-1 Induces Krüppel-like Factor 2 Expression through a Phosphoinositide 3-Kinase/AKT-dependent Activation of Myocyte Enhancer Factor 2

Angiopoietin-1 (Ang1) regulates both vascular quiescence and angiogenesis through the receptor tyrosine kinase Tie2. We and another group have recently shown that Ang1 and Tie2 form distinct signaling complexes at cell-cell and cell-matrix contacts and further demonstrated that the former selectively induces expression of Krüppel-like factor 2 (KLF2), a transcription factor involved in vascular quiescence. Here, we investigated the mechanism of how Ang1/Tie2 signal induces KLF2 expression to clarify the role of KLF2 in Ang1/Tie2 signal-mediated vascular quiescence. Ang1 stimulated KLF2 promoter-driven reporter gene expression in endothelial cells, whereas it failed when a myocyte enhancer factor 2 (MEF2)-binding site of KLF2 promoter was mutated. Depletion of MEF2 by siRNAs abolished Ang1-induced KLF2 expression, indicating the requirement of MEF2 in KLF2 induction by Ang1. Constitutive active phosphoinositide 3-kinase (PI3K) and AKT increased the MEF2-dependent reporter gene expression by enhancing its transcriptional activity and stimulated the KLF2 promoter activity cooperatively with MEF2. Consistently, inhibition of either PI3K or AKT and depletion of AKT abrogated Ang1-induced KLF2 expression. In addition, we confirmed the dispensability of extracellular signal-regulated kinase 5 (ERK5) for Ang1-induced KLF2 expression. Furthermore, depletion of KLF2 resulted in the loss of the inhibitory effect of Ang1 on vascular endothelial growth factor (VEGF)-mediated expression of vascular cell adhesion molecule-1 in endothelial cells and VEGF-mediated monocyte adhesion to endothelial cells. Collectively, these findings indicate that Ang1/Tie2 signal stimulates transcriptional activity of MEF2 through a PI3K/AKT pathway to induce KLF2 expression, which may counteract VEGF-mediated inflammatory responses.

Silencing of Cav1.2 gene in Rat Neonatal Cardiomyocytes by Lentiviral delivered shRNA

Background: Two types of L-type Ca Channels are expressed in the heart: Cav1.2 (α1C) and Cav1.3 (α1D). In contrast to α1C, α1D Ca channel is highly expressed in the sinoatrial node and atria, and is involved in the impulse generation and propagation through the AV node. Deletion of the α1C gene results in embryonic lethality before E14.5 and there are no pharmacological or biophysical means to separate α1D from α1C Ca currents. The aim of this study was to establish a model to characterize α1D Ca current using the novel technology of RNA interference.Methods and Results: Rat neonatal cardiomyocytes (RNC) were transfected with α1C specific siRNA using lipofectamine which resulted in 50% silencing at the mRNA/protein level. Limited by the low transfection effieciency (50%) in the RNC, we cotransfected α1C siRNA with cy3 labeled Human GAPDH siRNA and sorted out the fluorescent cells using FACS to separate tranfected and non-transfected cells. The level of silencing of α1C in enriched transfected cells reached only 65% efficiency. To achieve higher transfection efficiency, we generated and infected RNC with a lentivirus construct carrying the α1C siRNA sequence under a U6 promoter. Using this model, we achieved 100% transfection efficiency, and more than 90% silencing of the α1C gene confirmed by real-time PCR, Western blot, and immunoflourecence. These biochemical results were confirmed electrophysiologically by measurements of total L-type Ca current which was reduced by 80% in transfected cells.Conclusion: Lentiviral shRNA is an efficient model for post-transcriptional gene silencing of ion channels in primary cardiomyocytes. This novel approach provides a valuable mean for assessing the differential roles of α1C and α1D Ca channels in native cardiomyocytes and could be used to examine their roles in physiological and pathological settings.

Neprilysin gene expression requires binding of the amyloid precursor protein intracellular domain to its promoter: implications for Alzheimer disease

Amyloid beta-peptide (Abeta) accumulation leads to neurodegeneration and Alzheimer disease; however, amyloid metabolism is a dynamic process and enzymic mechanisms exist for Abeta removal. Considerable controversy surrounds whether the intracellular domain of the amyloid precursor protein (AICD) regulates expression of the Abeta-degrading metalloprotease, neprilysin (NEP). By comparing two neuroblastoma cell lines differing substantially in NEP expression, we show by chromatin immunoprecipitation (ChIP) that AICD is bound directly to the NEP promoter in high NEP-expresser (NB7) cells but not in low-expresser (SH-SY5Y) cells. The methylation status of the NEP promoter does not regulate expression in these cells, whereas the histone deacetylase inhibitors trichostatin A and valproate partly restore NEP expression and activity in SH-SY5Y cells. ChIP analysis also reveals AICD binding to the NEP promoter in rat primary neurons but not in HUVEC cells. Chromatin remodelling of crucial Alzheimer disease-related genes by valproate could provide a new therapeutic strategy.

Role of Homing Regulation in Coculturing Human Cord blood–derived Mesenchymal Stem cells with CD34-Positive Cells from Umbilical Cord Blood

Background and Objectives Mesenchymal stem cells plays an important role in the hematopoietic stem cell engraftment condition with SDF-1 (CXCL12)-CXCR4 signaling and in their homing in various tissues. In this study, we evaluated that the regulation of homing efficiency for mesenchymal stem cells to support ex vivo expansion of hematopoietic stem cells from umbilical cord blood.Methods We investigated the expression of CXCR4 and Stromal-Derived Factor-1 (SDF-1) in cocultured mesenchymal stem cell with umbilical cord blood-derived CD34-positive cell, which stimulated with granulocyte macrophage-colony stimulating factor (GM-CSF) and stem cell factor (SCF) cytokine.Results In this study, we evaluated that coculturing of SDF-1+ mesenchymal stem cells with stimulated CD34+ cells significantly increased the expression of CD34, CD45, and CD19 for myeloid surface marker and intracellular CXCR4 within a few hours as compared with culturing of CD34-positive cells alone or with SDF-1− mesenchymal stem cells or untreated mesenchymal stem cells by Flow cytometre. In the result of stimulation for 48 hours with various cytokines in CD34-positive cells, CXCR4 gene and ERK-1,2 protein up-regulated, and increased in vitro migration capacity of cocultured SDF-1+ mesenchymal stem cell with CD34+ cells as examined by quantitative RT-PCR of human GAPDH. To enhance homing effect by mesenchymal stem cell, we maintained expanded mesenchymal stem cells for up to 5–10 passages with monitoring of the expression of various tissue surface antigens, such as skeletal muscle, neural, liver, and endothelial cells. SDF-1+ mesenchymal stem cells induced the homing of cellular products of stimulated cord blood-derived CD34-positive cells for 10 days. Moreover, the tranfected SDF-1+ cells with a green fluorescent protein gene using lentivirus maintained their capacities of protein release and homing in culture system. SDF-1− mesenchymal stem cells reduced CXCR4 expression in cocultured CD34-positive cells.Conclusions: These results demonstrate that the role of the SDF-1/CXCR4 axis is an important rold in the regulation of homing and engraftment of mesenchymal and hematopoietic stem cells. SDF-1+ mesenchymal stem cells have clinical potential to regulate homing and short-term engraftment for hematopoietic stem cell transplantation.

Group IVA Phospholipase A2 Is Necessary for the Biogenesis of Lipid Droplets

Lipid droplets (LD) are organelles present in all cell types, consisting of a hydrophobic core of triacylglycerols and cholesteryl esters, surrounded by a monolayer of phospholipids and cholesterol. This work shows that LD biogenesis induced by serum, by long-chain fatty acids, or the combination of both in CHO-K1 cells was prevented by phospholipase A(2) inhibitors with a pharmacological profile consistent with the implication of group IVA cytosolic phospholipase A(2) (cPLA(2)alpha). Knocking down cPLA(2)alpha expression with short interfering RNA was similar to pharmacological inhibition in terms of enzyme activity and LD biogenesis. A Chinese hamster ovary cell clone stably expressing an enhanced green fluorescent protein-cPLA(2)alpha fusion protein (EGFP-cPLA(2)) displayed higher LD occurrence under basal conditions and upon LD induction. Induction of LD took place with concurrent phosphorylation of cPLA(2)alpha at Ser(505). Transfection of a S505A mutant cPLA(2)alpha showed that phosphorylation at Ser(505) is key for enzyme activity and LD formation. cPLA(2)alpha contribution to LD biogenesis was not because of the generation of arachidonic acid, nor was it related to neutral lipid synthesis. cPLA(2)alpha inhibition in cells induced to form LD resulted in the appearance of tubulo-vesicular profiles of the smooth endoplasmic reticulum, compatible with a role of cPLA(2)alpha in the formation of nascent LD from the endoplasmic reticulum.

An Oncogenic Role for the Phosphorylated h-Subunit of Human Translation Initiation Factor eIF3

Dysregulation of protein synthesis has been implicated in oncogenesis through a mechanism whereby "weak" mRNAs encoding proteins involved in cell proliferation are strongly translated when the protein synthesis apparatus is activated. Previous work has determined that many cancer cells contain high levels of eIF3h, a protein subunit of translation initiation factor eIF3, and overexpression of eIF3h malignantly transforms immortal NIH-3T3 cells. This is a general feature of eIF3h, as high levels also affect translation, proliferation, and a number of malignant phenotypes of CHO-K1 and HeLa cells and, most significantly, of a primary prostate cell line. Furthermore, overexpressed eIF3h inhibits Myc-dependent induction of apoptosis of primary prostate cells. eIF3h appears to function through translation, as the initial appearance of overexpressed eIF3h in rapidly induced NIH-3T3 cells correlates tightly with the stimulation of protein synthesis and the generation of malignant phenotypes. This oncogenic potential of eIF3h is enhanced by phosphorylation at Ser(183). Finally, reduction of eIF3h levels in breast and prostate cancer cell lines by short interfering RNA methods reduces their rates of proliferation and anchorage-independent growth in soft agar. The results provide compelling evidence that high eIF3h levels directly stimulate protein synthesis, resulting in the establishment and maintenance of the malignant state in cells.

Detection and clinical significance of multidrug resistance-1 mRNA in bone marrow cells in children with acute lymphoblastic leukemia by real-time fluorescence quantitative RT-PCR

Objective Multidrug resistance(MDR) is one of the most important reasons for treatment failure and recurrence of acute leukemia. Its manifestations are different in children with acute lymphoblastic leukemia(ALL) which may be due to different detection methods. This study was to detect the expression of MDR1 mRNA in bone marrow cells of children with ALL by real-time fluorescence-quantitative reverse transcription polymerase-chain reaction(FQ-RT-PCR), and combine minimal residual desease(MRD) detection by flow cytometry(FCM) and to study their relationship with treatment response and prognosis of ALL. Methods The MDR1 mRNA levels in bone marrow cells from 67 children with ALL[28 had newly diagnosed disease, 27 had achieved complete remission(CR), 12 recurrent] and 22 children without leukemia were detected by FQ-RT-PCR. MRD was detected by FCM. The patients were observed for 9∼101 months, with a median of 64 months. Results Standard curves of human MDR1 and GAPDH genes were constructed successfully. MDR1 mRNA was detected in all children with a positive rate of 100%. The mRNA level of MDR1 was similar among the newly diagnosed ALL group, CR group, and control group( P > 0.05), but significantly higher in the recurrence group than that in newly diagnosed disease group and control group(0.50 ±0.55 vs. 0.09 ±0.26 and 0.12 ±0.23, P < 0.05). 54 ALL patients were followed up, and it was found that MDR1 mRNA level was significantly higher in ALL patients within 3 years duration than that of ALL patients with 3∼6 years and over 6 years duration(0.63 ±0.56 vs. 0.11 ±0.12 and 0.04 ±0.06, P < 0.01). For the 28 children with newly diagnosed disease, the MDR1 mRNA level was similar between WBC > 50×10 9 group and WBC<50×10 9 group( P > 0.05). In the 33 CR patients, the MDR1 mRNA level was significantly higher in MRD>10 3 group than that in MRD<10 3 group(0.39 ±0.47 vs. 0.03 ±0.03, P < 0.05). Conclusion The sensitivity and specificity of FQ-RT-PCR in detecting MDR1 mRNA in bone marrow cells of children with ALL patients are high. MDR1 mRNA is expressed in children with and without leukemia. MDR1 mRNA is highly expressed in the CR ALL patients with high MRD, recurrence and short duration(within 3 years). Monitoring MRD and the MDR1 mRNA level might be helpful for individual treatment.

Specific Binding of GAPDH in the Presence or Absence of Selenium to SPS Monitored by Affinity Chromatography

The methods of synthesis and insertion of selenium into selenocysteine (Sec)_containing enzymes has been recognized in prokaryotes, eukaryotes and archaebacteria. Yet, the transport and intracellular trafficking of selenium is incompletely characterized. Therefore, considerable attention has reallocated to the identification and characterization of selenium adducts or delivery proteins required in specific metabolic pathways. It has been documented the Escherichia coli glyceraldehyde_3_phosphate dehydrogenase (GAPDH; EC ) actively bound selenium possibly as a perselenide derivative following in vivo labeling with 75Se[selenite] [Lacourciere, G.M., Levine, R.L. and Stadtman, T.C. (2002) PNAS USA 99, 9150_9153]. Subsequently, Yuki and coworkers [Ogasawara, Y., Lacourciere, G.M., Ishii, K. and Stadtman, T.C. (2005) PNAS USA 102, 1012_1016] provided in vitro evidence that each subunit of human erythrocyte GAPDH possessed a reactive cysteine residue, which can act as the binding site for selenium supplied as selenodiglutathione from selenite and glutathione. These findings suggest GAPDH participated as a component of a selenium delivery system for the biosynthesis of selenophosphate, a reactive selenium compound, catalyzed by the E. coli selenophosphate synthetase (SPS). To date, there are no reports relating to the binding capability of SPS to native and 75Se_labeled GAPDH. Consequently, the work presented herein reports the application of affinity chromatography as a screening tool to emphasize the specificity of the Se enzymes. This research was fully supported by the National Heart, Lung, and Blood Institute, National Institutes of Health.