Compromised N-Glycosylation Processing of Kv3.1b Correlates with Perturbed Motor Neuron Structure and Locomotor Activity.

Neurological difficulties commonly accompany individuals suffering from congenital disorders of glycosylation, resulting from defects in the N-glycosylation pathway. Vacant N-glycosylation sites (N220 and N229) of Kv3, voltage-gated K+ channels of high-firing neurons, deeply perturb channel activity in neuroblastoma (NB) cells. Here we examined neuron development, localization, and activity of Kv3 channels in wildtype AB zebrafish and CRISPR/Cas9 engineered NB cells, due to perturbations in N-glycosylation processing of Kv3.

Knockdown of N-Acetylglucosaminyltransferase-II Reduces Matrix Metalloproteinase 2 Activity and Suppresses Tumorigenicity in Neuroblastoma Cell Line.

Neuroblastoma (NB) development and progression are accompanied by changes in N-glycans attached to proteins. Here, we investigated the role of N-acetylglucosaminyltransferase-II (GnTII, ) protein substrates in neuroblastoma (NB) cells. was silenced in human BE(2)-C NB (HuNB) cells to generate a novel cell line, HuNB(-), lacking complex type N-glycans, as in rat B35 NB cells.

Disruption of erythrocyte membrane asymmetry by triclosan is preceded by calcium dysregulation and p38 MAPK and RIP1 stimulation.

Triclosan (TCS) is a broad-spectrum antimicrobial used in personal care products, household items, and medical devices. Owing to its apoptotic potential against tumor cells, TCS has been proposed for the treatment of malignancy. A major complication of chemotherapy is anemia, which may result from direct erythrocyte hemolysis or premature cell death known as eryptosis.

Stimulation of eryptosis by broad-spectrum insect repellent N,N-Diethyl-3-methylbenzamide (DEET).

N,N-Diethyl-3-methylbenzamide (DEET) is the most widely used insect repellent in the world. Adverse effects following DEET exposure are well documented. Moreover, DEET has been shown to possess cytotoxic and apoptotic properties in nucleated cells.

Lack of complex type N-glycans lessens aberrant neuronal properties.

Modifications in surface glycans attached to proteins via N-acetylglucosamine-β1-N-asparagine linkage have been linked to tumor development and progression. These modifications include complex N-glycans with high levels of branching, fucose and sialic acid residues. Previously, we silenced Mgat2 in neuroblastoma (NB) cells, which halted the conversion of hybrid type N-glycans to complex type, to generate a novel cell line, NB_1(-Mgat2).

Membrane Distribution and Activity of a Neuronal Voltage-Gated K+ Channel is Modified by Replacement of Complex Type N-Glycans with Hybrid Type.

Abnormal modifications in N-glycosylation processing are commonly associated with neurological disorders, although the impact of specific N-glycans on neuronal excitability is unknown. By replacement of complex types of N-glycans with hybrid types in neuroblastoma cells, we provide the first study that addresses how distinct N-glycan types impact neuronal excitability. Using CRISPR/Cas9 technology, NB_1, a clonal cell line derived from rat neuroblastoma cells (NB), was modified to create an N-glycosylation mutant cell line, NB_1 (-Mgat2), which expresses predominantly hybrid type N-glycans.

Predominant Expression of Hybrid N-Glycans Has Distinct Cellular Roles Relative to Complex and Oligomannose N-Glycans.

Glycosylation modulates growth, maintenance, and stress signaling processes. Consequently, altered N-glycosylation is associated with reduced fitness and disease. Therefore, expanding our understanding of N-glycans in altering biological processes is of utmost interest.

Soluble guanylyl cyclase-activated cyclic GMP-dependent protein kinase inhibits arterial smooth muscle cell migration independent of VASP-serine 239 phosphorylation.

Coronary artery disease (CAD) accounts for over half of all cardiovascular disease-related deaths. Uncontrolled arterial smooth muscle (ASM) cell migration is a major component of CAD pathogenesis and efforts aimed at attenuating its progression are clinically essential. Cyclic nucleotide signaling has long been studied for its growth-mitigating properties in the setting of CAD and other vascular disorders.

Complex N-Glycans Influence the Spatial Arrangement of Voltage Gated Potassium Channels in Membranes of Neuronal-Derived Cells.

The intrinsic electrical properties of a neuron depend on expression of voltage gated potassium (Kv) channel isoforms, as well as their distribution and density in the plasma membrane. Recently, we showed that N-glycosylation site occupancy of Kv3.1b modulated its placement in the cell body and neurites of a neuronal-derived cell line, B35 neuroblastoma cells.

Cell surface N-glycans influence the level of functional E-cadherin at the cell-cell border.

E-cadherin is crucial for adhesion of cells to each other and thereby development and maintenance of tissue. While it is has been established that N-glycans inside the cell impact the level of E-cadherin at the cell surface of epithelial-derived cells, it is unclear whether N-glycans outside the cell control the clustering of E-cadherin at the cell-cell border. Here, we demonstrate reduction of N-glycans at the cell surface weakened the recruitment and retention of E-cadherin at the cell-cell border, and consequently reduced the strength of cell-cell interactions.

Glycan structures contain information for the spatial arrangement of glycoproteins in the plasma membrane.

Glycoconjugates at the cell surface are crucial for cells to communicate with each other and the extracellular microenvironment. While it is generally accepted that glycans are vectorial biopolymers, their information content is unclear. This report provides evidence that distinct N-glycan structures influence the spatial arrangement of two integral membrane glycoproteins, Kv3.

Comparative study of 3D morphology and functions on genetically engineered mouse melanoma cells.

Quantification of 3D morphology and measurement of cellular functions were performed on the mouse melanoma cell lines of B16F10 to investigate the intriguing problem of structure-function relations in the genetically engineered cells with GPR4 overexpression. Results of 3D analysis of cells in suspension and phase contrast imaging of adherent cells yield consistent evidence that stimulation of the proton-sensing GPR4 receptor in these cells may modify significantly their morphology with diminishing ability to produce membrane protrusions and to migrate. Examination of the 3D parameters of mitochondria provide further insights on the measured variation of the maximal capacity of oxygen consumption rate among the genetically modified cells, indicating that the proton-sensing receptor may regulate cancer cell metabolism with increased mitochondrial surface area.

Surface plasmon resonance (SPR) spectrometry as a tool to analyze nucleic acid-protein interactions in crude cellular extracts.

This study presents proof-of-principle application showing that label-free affinity enrichment surface plasmon resonance (SPR) biosensor binding is able to semiquantitatively detect molecular DNA-protein interactions in crude cellular extracts in a real-time ligand fishing analysis study. Crude cell extracts obtained from a confluent HT-28 human adenocarcinoma cell line, synchronized to the G(0)/G(1) phase of the cell cycle, were extracted in a chaotropic medium and cryopreserved in liquid nitrogen. Various immunoprecipitation antibodies were used against defective human excision and mismatch repair genes, hDDB2 and hMSH2, respectively, which theoretically allow for protein binding to DNA ligands in their native conformation.

Role of benzo[alpha]pyrene in generation of clustered DNA damage in human breast tissue.

Complex DNA damage may manifest in double-strand breaks (DSBs) and non-DSB, bistranded, oxidatively induced clustered DNA lesions (OCDLs). Although the carcinogen benzo[alpha]pyrene (B[alpha]P) has been shown to induce chromosomal aberrations and transformation of mammary cells, it is not known whether this compound engenders clustered DNA damage. Normal primary breast tissue-derived cells were treated with B[alpha]P, and the levels of DNA lesions, chromosomal aberrations, total antioxidant capacity (TAC), and reactive oxygen species (ROS) were determined.

Differences in mRNA and microRNA microarray expression profiles in human colon adenocarcinoma HT-29 cells treated with either Intensity-modulated Radiation Therapy (IMRT), or Conventional Radiation Therapy (RT).

We carried out this in vitro molecular study to investigate the effect of two clinical X-irradiation modalities (a two-dimensional external beam radiotherapy referred to in this article as conventional RT, and a three dimensional conformal intensity-modulated radiation therapy (IMRT) on a colon adenocarcinoma HT-29 cell line. Cells were synchronized by serum deprivation 48 h before irradiation so that >90% of them were in the G(0)/G(1) phase of the cell cycle. Cells were allowed to recover 3 h after irradiation before total RNA extraction.

Genetic transformation and gene silencing mediated by multiple copies of a transgene in eastern white pine.

An efficient transgenic eastern white pine (Pinus strobus L.) plant regeneration system has been established using Agrobacterium tumefaciens strain GV3850-mediated transformation and the green fluorescent protein (gfp) gene as a reporter in this investigation. Stable integration of transgenes in the plant genome of pine was confirmed by polymerase chain reaction (PCR), Southern blot, and northern blot analyses.

Efficient delivery of small interfering RNA to plant cells by a nanosecond pulsed laser-induced stress wave for posttranscriptional gene silencing.

Small interfering RNA (siRNA) induced posttranscriptional gene silencing (PTGS) has been an efficient method for genetic and molecular analysis of certain developmental and physiological processes and represented a potential strategy for both controlling virus replication and developing therapeutic products. However, there are limitations for the methods currently used to deliver siRNA into cells. We report here, to our knowledge, the first efficient delivery of siRNA to plant cells by a nanosecond pulsed laser-induced stress wave (LISW) for posttranscriptional gene silencing.

Raf/MEK/ERK signalling triggers reactivation of Kaposi's sarcoma-associated herpesvirus latency.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman's disease. KSHV infection of cells produces both latent and lytic cycles of infection. In vivo, the virus is found predominantly in the latent state.

Differential gene silencing induced by short interfering RNA in cultured pine cells associates with the cell cycle phase.

The double-stranded short interfering RNA (siRNA) molecules can silence targeted genes through sequence-specific cleavage of the cognate RNA transcript. The rapid adoption of technologies based on this siRNA interference mechanism has been a widely used method to analyze gene function in plants, invertebrates, and mammalian systems. In order to understand the dynamics of siRNA-mediated gene inactivation during cell division, we have investigated the relationship between the cell cycle phase and the post-transcriptional gene silencing mediated by siRNA in gfp transgenic Virginia pine (Pinus virginiana Mill.

Estrogen increases retrograde labeling of motoneurons: evidence of a nongenomic mechanism.

Estrogen has a variety of neurotrophic effects mediated via different signaling cascades, including ERK and phosphatidylinositol 3-kinase (PI3K) pathways. In this study, we investigated effects of estrogen and inhibitors for ERK and PI3K applied directly onto the cut sciatic nerve on retrograde labeling of lumbar motoneurons. A mix of retrograde tracer (Fluorogold) and 17beta-estradiol, in combination with an antagonist for estrogen receptors ICI 182,780, an inhibitor of ERK1/2 pathway (U0126), an inhibitor of PI3K (LY-294002), or a protein synthesis inhibitor (cycloheximide), was applied to the proximal stump of the transected sciatic nerve for 24 h.

Protein kinase Cepsilon interacts with Bax and promotes survival of human prostate cancer cells.

Prostatic glandular epithelial cells express protein kinase Cepsilon (PKCepsilon ), an oncoprotein that coordinately disrupts the reactivation of the tumor suppressor Rb, derepressess transcriptional elongation of the c-myc oncogene, and propagates survival signals in LNCaP cells. Since the activation of such a program may contribute to the progression of human prostate cancer, a proteomic analysis was performed to gain a more global perspective on the signaling network that PKCepsilon might be capable of engaging in prostate cancer cells. Using CWR22 xenografts, we identified at least 18 different structural, signaling, and stress-related proteins that associated with PKCepsilon, including an interaction with the proapoptotic protein Bax that was novel to recurrent CWR22 tumors.

Induction of apoptosis by bexarotene in cutaneous T-cell lymphoma cells: relevance to mechanism of therapeutic action.

Purpose: Bexarotene is the first synthetic rexinoid approved for the treatment of all stages of cutaneous T-cell lymphoma (CTCL) however the mechanism of bexarotene action is unknown. We examined the effects of bexarotene on induction of apoptosis and expression of its cognate receptors in well-established CTCL cell lines (MJ, Hut78, and HH).

Experimental Design: CTCL cells were treated with 0.