KCl-induced depolarization facilitates neuronal differentiation of P19 embryonic carcinoma cells

Articlehttp://deepblue.lib.umich.edu/bitstream/2027.42/96992/1/UMURF-Issue06_2009-TMYang.pd

Cloning of CDP-Diacylglycerol Synthase from a Human Neuronal Cell Line

A critical step in the supply of substrate for the phosphoinositide signal transduction pathway is the formation of the liponucleotide intermediate, CDP-diacylglycerol, catalyzed by CDP-diacylglycerol synthase. Further insight into the regulation of phosphoinositide biosynthesis was sought by cloning of the gene for the vertebrate enzyme. Sequence of the corresponding gene from Drosophila was used to prepare a probe for screening of a human neuronal cell cDNA library. A cDNA was isolated with a predicted open reading frame of 1,332 bases, encoding a protein of 51 kDa. The amino acid sequence showed 50% identity (75% similarity) to that of Drosophila eye CDP-diacylglycerol synthase and substantial similarity to the Saccharomyces cerevisiae and Escherichia coli homologues. Northern blot analysis, with human cDNA riboprobes, suggested that the corresponding mRNA was expressed in all human tissues examined. Expression of the human cDNA in COS cells resulted in a more than fourfold increase in CDP-diacylglycerol synthase activity. Knowledge of the sequence of vertebrate CDP-diacylglycerol synthase should facilitate further investigations into its regulation and the possible existence of distinct isoforms.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65959/1/j.1471-4159.1996.67052200.x.pd

Enhancement of cancer chemotherapy in vitro by intense ultrawideband electric field pulses

Experiments have been performed to enhance the Jurkat cell-killing effects of the cancer chemotherapy agent bleomycin using electric field pulses of 50–200 kV/cm50–200kV∕cm peak electric field strength, ∼ 150 ns∼150ns duration, and nanosecond rise time. Dramatic increases in cell killing (factors of ∼ 1000∼1000) were observed with a low dose of bleomycin after treatment with trains of ten or more pulses at all electric field strengths tested, compared to pulse-only or drug-only treatments. Cell death occurred within 24 h24h for treated cells, with some evidence of membrane phosphatidylserine externalization at 6 h6h postexposure but no significant increase in caspase activity, indicating that the primary mode of cell death was not caspase-mediated apoptosis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/87477/2/094701_1.pd

Phosphatidylinositol 3‐kinase and Akt effectors mediate insulin‐like growth factor‐I neuroprotection in dorsal root ganglia neurons

Insulin‐like growth factor‐I (IGF‐I) protects neurons of the peripheral nervous system from apoptosis, but the underlying signaling pathways are not well understood. We studied IGF‐I mediated signaling in embryonic dorsal root ganglia (DRG) neurons. DRG neurons express IGF‐I receptors (IGF‐IR), and IGF‐I activates the phosphatidylinositol 3‐kinase (PI3K)/Akt pathway. High glucose exposure induces apoptosis, which is inhibited by IGF‐I through the PI3K/Akt pathway. IGF‐I stimulation of the PI3K/Akt pathway phosphorylates three known Akt effectors: the survival transcription factor cyclic AMP response element binding protein (CREB) and the pro‐apoptotic effector proteins glycogen synthase kinase‐3β (GSK‐3β) and forkhead (FKHR). IGF‐I regulates survival at the nuclear level through accumulation of phospho‐Akt in DRG neuronal nuclei, increased CREB‐mediated transcription, and nuclear exclusion of FKHR. High glucose increases expression of the pro‐apoptotic Bcl protein Bim (a transcriptional target of FKHR). However, IGF‐I does not regulate Bim or anti‐apoptotic Bcl‐xL protein expression levels, which suggests that IGF‐I neuroprotection is not through regulation of their expression. High glucose also induces loss of the initiator caspase‐9 and increases caspase‐3 cleavage, effects blocked by IGF‐I. These data suggest that IGF‐I prevents apoptosis in DRG neurons by regulating PI3K/Akt pathway effectors, including GSK‐3β, CREB, and FKHR, and by blocking caspase activation.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154325/1/fsb2fj041581fje.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/154325/2/fsb2fj041581fje-sup-0001.pd

Incorporating spatial dependence into a multicellular tumor spheroid growth model

Recent models for organism and tumor growth yield simple scaling laws based on conservation of energy. Here, we extend such a model to include spatial dependence to model necrotic core formation. We adopt the allometric equation for tumor volume with a reaction-diffusion equation for nutrient concentration. In addition, we assume that the total metabolic energy and average cellular metabolic rate depend on nutrient concentration in a Michaelis-Menten-like manner. From experimental results, we relate the necrotic volume to nutrient consumption and estimate both the time and nutrient concentration at necrotic core formation. Based on experimental results, we demand that the necrotic core radius varies linearly with tumor radius after core formation and extend the equations for tumor volume and nutrient concentration to the postnecrotic core regime. In particular, we obtain excellent agreement with experimental data and the final steady-state viable rim thickness.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/87333/2/124701_1.pd

Behavioral Mechanism during Human Sperm Chemotaxis: Involvement of Hyperactivation

When mammalian spermatozoa become capacitated they acquire, among other activities, chemotactic responsiveness and the ability to exhibit occasional events of hyperactivated motility—a vigorous motility type with large amplitudes of head displacement. Although a number of roles have been proposed for this type of motility, its function is still obscure. Here we provide evidence suggesting that hyperactivation is part of the chemotactic response. By analyzing tracks of spermatozoa swimming in a spatial chemoattractant gradient we demonstrate that, in such a gradient, the level of hyperactivation events is significantly lower than in proper controls. This suggests that upon sensing an increase in the chemoattractant concentration capacitated cells repress their hyperactivation events and thus maintain their course of swimming toward the chemoattractant. Furthermore, in response to a temporal concentration jump achieved by photorelease of the chemoattractant progesterone from its caged form, the responsive cells exhibited a delayed turn, often accompanied by hyperactivation events or an even more intense response in the form of flagellar arrest. This study suggests that the function of hyperactivation is to cause a rather sharp turn during the chemotactic response of capacitated cells so as to assist them to reorient according to the chemoattractant gradient. On the basis of these results a model for the behavior of spermatozoa responding to a spatial chemoattractant gradient is proposed

Large-scale profiling of noncoding RNA function in yeast

Noncoding RNAs (ncRNAs) are emerging as key regulators of cellular function. We have exploited the recently developed barcoded ncRNA gene deletion strain collections in the yeast Saccharomyces cerevisiae to investigate the numerous ncRNAs in yeast with no known function. The ncRNA deletion collection contains deletions of tRNAs, snoRNAs, snRNAs, stable unannotated transcripts (SUTs), cryptic unstable transcripts (CUTs) and other annotated ncRNAs encompassing 532 different individual ncRNA deletions. We have profiled the fitness of the diploid heterozygous ncRNA deletion strain collection in six conditions using batch and continuous liquid culture, as well as the haploid ncRNA deletion strain collections arrayed individually onto solid rich media. These analyses revealed many novel environmental-specific haplo-insufficient and haplo-proficient phenotypes providing key information on the importance of each specific ncRNA in every condition. Co-fitness analysis using fitness data from the heterozygous ncRNA deletion strain collection identified two ncRNA groups required for growth during heat stress and nutrient deprivation. The extensive fitness data for each ncRNA deletion strain has been compiled into an easy to navigate database called Yeast ncRNA Analysis (YNCA). By expanding the original ncRNA deletion strain collection we identified four novel essential ncRNAs; SUT527, SUT075, SUT367 and SUT259/691. We defined the effects of each new essential ncRNA on adjacent gene expression in the heterozygote background identifying both repression and induction of nearby genes. Additionally, we discovered a function for SUT527 in the expression, 3’ end formation and localization of SEC4, an essential protein coding mRNA. Finally, using plasmid complementation we rescued the SUT075 lethal phenotype revealing that this ncRNA acts in trans. Overall, our findings provide important new insights into the function of ncRNAs