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 Kv3.1b. We showed that caudal primary (CaP) motor neurons of zebrafish spinal cord transiently expressing fully glycosylated (WT) Kv3.1b have stereotypical morphology, while CaP neurons expressing partially glycosylated (N220Q) Kv3.1b showed severe maldevelopment with incomplete axonal branching and extension around the ventral musculature. Consequently, larvae expressing N220Q in CaP neurons had impaired swimming locomotor activity. We showed that replacement of complex N-glycans with oligomannose attached to Kv3.1b and at cell surface lessened Kv3.1b dispersal to outgrowths by altering the number, size, and density of Kv3.1b-containing particles in membranes of rat neuroblastoma cells. Opening and closing rates were slowed in Kv3 channels containing Kv3.1b with oligomannose, instead of complex N-glycans, which suggested a reduction in the intrinsic dynamics of the Kv3.1b α-subunit. Thus, N-glycosylation processing of Kv3.1b regulates neuronal development and excitability, thereby controlling motor activity.

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, MGAT2) protein substrates in neuroblastoma (NB) cells. MGAT2 was silenced in human BE(2)-C NB (HuNB) cells to generate a novel cell line, HuNB(-MGAT2), lacking complex type N-glycans, as in rat B35 NB cells. Changes in N-glycan types were confirmed by lectin binding assays in both cell lines, and the rescued cell line, HuNB(-/+MGAT2). Western blotting of cells heterologously expressing a voltage-gated K+ channel (Kv3.1b) showed that some hybrid N-glycans of Kv3.1b could be processed to complex type in HuNB(-/+MGAT2) cells. In comparing HuNB and HuNB(-MGAT2) cells, decreased complex N-glycans reduced anchorage-independent cell growth, cell proliferation, and cell invasiveness, while they enhanced cell-cell interactions. Cell proliferation, invasiveness and adhesion of the HuNB(-/+MGAT2) cells were more like the HuNB than HuNB(-MGAT2). Western blotting revealed lower protein levels of MMP-2, EGFR and Gab2 in glycosylation mutant cells relative to parental cells. Gelatin zymography demonstrated that decreased MMP-2 protein activity was related to lowered MMP-2 protein levels. Thus, our results support that decreased complex type N-glycans suppress cell proliferation and cell invasiveness in both NB cell lines via remodeling ECM.

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. Similar to nucleated cells, eryptotic cells lose membrane asymmetry and Ca2+ regulation, and undergo oxidative stress, shrinkage, and activation of a host of kinases. In this report, we sought to examine the hemolytic and eryptotic potential of TCS and dissect the underlying mechanistic scenarios involved there in. Hemolysis was spectrophotometrically evaluated by the degree of hemoglobin release into the medium. Flow cytometry was utilized to detect phosphatidylserine (PS) exposure by annexin-V binding, intracellular Ca2+ by Fluo-3/AM fluorescence, and oxidative stress by 2-,7-dichlorodihydrofluorescin diacetate (DCFH2-DA). Incubation of cells with 10-100 µM TCS for 1-4 h induced time- and dose-dependent hemolysis. Moreover, TCS significantly increased the percentage of eryptotic cells as evident by PS exposure (significantly enhanced annexin-V binding). Interestingly, TCS-induced eryptosis was preceded by elevated intracellular Ca2+ levels but was not associated with oxidative stress. Cotreatment of erythrocytes with 50 µM TCS and 50 µM SB203580 (p38 MAPK inhibitor), or 300 µM necrostatin-1 (receptor-interacting protein 1 (RIP1) inhibitor) significantly ameliorated TCS-induced PS externalization. We conclude that TCS is cytotoxic to erythrocytes by inducing hemolysis and stimulating premature death at least in part through Ca2+ mobilization, and p38 MAPK and RIP1 activation.

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. Although red blood cells (RBCs) lack intracellular organelles, they nevertheless undergo programmed cell death termed eryptosis. Compromised RBC health contributes to the development of anemia; a condition affecting 25% of the global population. This study investigated the interaction between DEET and human RBCs, and explored accompanying biochemical and molecular alterations. RBCs at 5% hematocrit were incubated in presence and absence of 1-5 mM (0.02%-0.1%) of DEET for 6 h at 37 °C. Hemolysis was spectrophotometrically determined by hemoglobin release, while major eryptotic events were analyzed by flow cytometer. Phosphatidylserine (PS) exposure was detected with Annexin-V-FITC, cell volume by forward scatter (FSC) of light, intracellular calcium with Fluo-3/AM, and reactive oxygen species with 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA). DEET caused slight hemolysis at 4 and 5 mM, and significantly increased Annexin-V-FITC and Fluo3 fluorescence, with reduced FSC at 5 mM. Removal of extracellular Ca2+ abolished DEET-induced Fluo3 fluorescence but had no effect on Annexin-V binding. Importantly, blockade of eryptotic signaling mediators p38 MAPK, caspases, protein kinase C, casein kinase 1, or necroptotic kinases receptor-interacting protein 1 and mixed lineage kinase domain-like protein, with small molecule inhibitors, did not ameliorate DEET-mediated PS externalization. In conclusion, DEET elicits suicidal erythrocyte death; an event characterized by loss of membrane asymmetry, cell shrinkage, and elevations in intracellular Ca2+ mainly through dysregulated Ca2+ influx.

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

Modifications in surface glycans attached to proteins via N-acetylglucosamine-ß1-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). By comparing the aberrant cell properties of the NB_1(-Mgat2) cell line to the parental cell line (NB_1), we investigated the impact of eliminating complex type N-glycans on NB cell behavior. Further, the N-glycosylation pathway in the NB_1(-Mgat2) cell line was rescued by transiently transfecting cells with Mgat2, thus creating the NB_1(-/+Mgat2) cell line. Changes in the N-glycosylation pathway were verified by enhanced binding of E-PHA and L-PHA to proteins in the rescued cell line relative to those of the NB_1(-Mgat2) cell line. Also, western blotting of total membranes from the rescued cell line ectopically expressing a voltage-gated K+ channel (Kv3.1b) revealed that N-glycans of Kv3.1b were processed to complex type. By employment of various cell lines, we demonstrated that reduction of the complex type N-glycans diminished anchorage-independent cell growth, and enhanced cell-cell interactions. Two independent cell invasion assays showed that cell invasiveness was markedly lessened by lowering the levels of complex type N-glycans while cell mobility was only slightly modified. Neurites of NB cells were shortened by the absence of complex type N-glycans. Cell proliferation was reduced in NB cells with lowered levels of complex type N-glycans which resulted from hindered progression through G1+Go phases of the cell cycle. Overall, our results illustrate that reducing the ratio of complex to hybrid types of N-glycans diminishes aberrant NB cell behavior and thereby has a suppressive effect in cell proliferation, and cell dissociation and invasion phases of NB.

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. Western and lectin blotting, flow cytometry, TIRF and DIC microscopy, and patch clamp studies were conducted. Lectin binding revealed the predominant type of N-glycans expressed in NB_1 (-Mgat2) is hybrid while those of NB and NB_1 are complex. Kv3.1 b-expressing cells with complex N-glycans localized more glycosylated Kv3.1b to the neurites than cells with hybrid N-glycans. Further the absence of N-glycan attachment to Kv3.1b was critical for sub-plasma distribution of Kv3.1b to neurites in primary adult mammalian neurons, along with NB cells. Replacement of complex type N-glycans with hybrid type hindered the opening and closing rates of outward ionic currents of Kv3.1 b-expressing NB cells. The lacks of N-glycan attachment hindered the rates even more but were not significantly different between the NB cell lines. Taken together, our evidence supports N-glycosylation impacts the sub-plasma membrane localization and activity of Kv3.1 b-containing channels. We propose that N-glycosylation processing of Kv3.1 b-containing channels contributes to neuronal excitability, and abnormal modifications in N-glycosylation processing of Kv3.1b could contribute to neurological diseases.

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. Herein, clustered regularly interspaced short palindromic repeats/caspase9 (CRISPR/Cas9) technology was employed to engineer a glycosylation mutant Chinese Hamster Ovary (CHO) cell line, K16, which expresses predominantly hybrid type N-glycans. This newly engineered cell line enabled us to compare N-glycan effects on cellular properties of hybrid type N-glycans, to the well-established Pro(-)5 and Lec1 cell lines, which express complex and oligomannose types of N-glycans, respectively. Lectin binding studies revealed the predominant N-glycan expressed in K16 is hybrid type. Cell dissociation and migration assays demonstrated the greatest strength of cell-cell adhesion and fastest migratory rates for oligomannose N-glycans, and these properties decreased as oligomannose type were converted to hybrid type, and further decreased upon conversion to complex type. Next, we examined the roles of three general types of N-glycans on ectopic expression of E-cadherin, a cell-cell adhesion protein. Microscopy revealed more functional E-cadherin at the cell-cell border when N-glycans were oligomannose and these levels decreased as the oligomannose N-glycans were processed to hybrid and then to complex. Thus, we provide evidence that all three general types of N-glycans impact plasma membrane architecture and cellular properties.

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. Heme-containing soluble guanylyl cyclase (sGC) synthesizes cyclic guanosine monophosphate (cGMP) and maintains vascular homeostasis predominantly through cGMP-dependent protein kinase (PKG) signaling. Considering that reactive oxygen species (ROS) can interfere with appropriate sGC signaling by oxidizing the cyclase heme moiety and so are associated with several CVD pathologies, the current study was designed to test the hypothesis that heme-independent sGC activation by BAY 60-2770 (BAY60) maintains cGMP levels despite heme oxidation and inhibits ASM cell migration through phosphorylation of the PKG target and actin-binding vasodilator-stimulated phosphoprotein (VASP). First, using the heme oxidant ODQ, cGMP content was potentiated in the presence of BAY60. Using a rat model of arterial growth, BAY60 significantly reduced neointima formation and luminal narrowing compared to vehicle (VEH)-treated controls. In rat ASM cells BAY60 significantly attenuated cell migration, reduced G:F actin, and increased PKG activity and VASP Ser239 phosphorylation (pVASP·S239) compared to VEH controls. Site-directed mutagenesis was then used to generate overexpressing full-length wild type VASP (FL-VASP/WT), VASP Ser239 phosphorylation-mimetic (FL-VASP/239D) and VASP Ser239 phosphorylation-resistant (FL-VASP/239A) ASM cell mutants. Surprisingly, FL-VASP/239D negated the inhibitory effects of FL-VASP/WT and FL-VASP/239A cells on migration. Furthermore, when FL-VASP mutants were treated with BAY60, only the FL-VASP/239D group showed reduced migration compared to its VEH controls. Intriguingly, FL-VASP/239D abrogated the stimulatory effects of FL-VASP/WT and FL-VASP/239A cells on PKG activity. In turn, pharmacologic blockade of PKG in the presence of BAY60 reversed the inhibitory effect of BAY60 on naïve ASM cell migration. Taken together, we demonstrate for the first time that BAY60 inhibits ASM cell migration through cGMP/PKG/VASP signaling yet through mechanisms independent of pVASP·S239 and that FL-VASP overexpression regulates PKG activity in rat ASM cells. These findings implicate BAY60 as a potential pharmacotherapeutic agent against aberrant ASM growth disorders such as CAD and also establish a unique mechanism through which VASP controls PKG activity.

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. To extrapolate this mechanism to other N-glycosylated Kv channels, we evaluated the impact of N-glycosylation occupancy of Kv3.1a and Kv1.1 channels. Western blots revealed that wild type Kv3.1a and Kv1.1 α-subunits had complex and oligomannose N-glycans, respectively, and that abolishment of the N-glycosylation site(s) generated Kv proteins without N-glycans. Total internal reflection fluorescence microscopy images revealed that N-glycans of Kv3.1a contributed to its placement in the cell membrane while N-glycans had no effect on the distribution of Kv1.1. Based on particle analysis of EGFP-Kv proteins in the adhered membrane, glycosylated forms of Kv3.1a, Kv1.1, and Kv3.1b had differences in the number, size or density of Kv protein clusters in the cell membrane of neurites and cell body of B35 cells. Differences were also observed between the unglycosylated forms of the Kv proteins. Cell dissociation assays revealed that cell-cell adhesion was increased by the presence of complex N-glycans of Kv3.1a, like Kv3.1b, whereas cell adhesion was similar in the oligomannose and unglycosylated Kv1.1 subunit containing B35 cells. Our findings provide direct evidence that N-glycans of Kv3.1 splice variants contribute to the placement of these glycoproteins in the plasma membrane of neuronal-derived cells while those of Kv1.1 were absent. Further when the cell membrane distribution of the Kv channel was modified by N-glycans then the cell-cell adhesion properties were altered. Our study demonstrates that N-glycosylation of Kv3.1a, like Kv3.1b, provides a mechanism for the distribution of these proteins to the cell body and outgrowths and thereby can generate different voltage-dependent conductances in these membranes.

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. We conclude that N-glycans at the cell surface are tightly linked to the placement of E-cadherin at the cell-cell border and thereby control E-cadherin mediated cell-cell adhesion.

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, Kv3.1 and E-cadherin, at the adherent membrane which in turn alter cellular properties. Distinct N-glycan structures were generated by heterologous expression of these glycoproteins in parental and glycosylation mutant Chinese hamster ovary cell lines. Unlike the N-linked glycans, the O-linked glycans of the mutant cell lines are similar to those of the parental cell line. Western and lectin blots of total membranes and GFP immunopurified samples, combined with glycosidase digestion reactions, were employed to verify the glycoproteins had predominantly complex, oligomannose, and bisecting type N-glycans from Pro(-)5, Lec1, and Lec10B cell lines, respectively. Based on total internal reflection fluorescence and differential interference contrast microscopy techniques, and cellular assays of live parental and glycosylation mutant CHO cells, we propose that glycoproteins with complex, oligomannose or bisecting type N-glycans relay information for localization of glycoproteins to various regions of the plasma membrane in both a glycan-specific and protein-specific manner, and furthermore cell-cell interactions are required for deciphering much of this information. These distinct spatial arrangements also impact cell adhesion and migration. Our findings provide direct evidence that N-glycan structures of glycoproteins contribute significantly to the information content of cells.

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. Our study demonstrates clearly the significant benefits of quantitative 3D morphological study in illuminating cellular functions and development of novel morphology based cell assay methods.

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. A set of biotinylated DNA target sequence heteroduplexes were also utilized for binding hDDB2 and hMSH2, prepared by heating a biotinylated oligonucleotide strand with an equimolar amount of the complementary strand to form a DNA duplex for hMSH2; a UV-irradiated duplex was employed for hDDB2 instead of an irradiated single-strand DNA to enhance binding. SDS was used to regenerate heteroduplex-modified chips that were used in a BIAcore 2000 SPR instrument at 25°C. Results showed that hMSH2 does not bind preferentially to the heteroduplex-complementary pair. In contrast, hDDB2 was found to bind preferentially to the UV-irradiated version of the heteroduplex-complementary pair. It is concluded that the choice of antibodies with appropriate epitopes is crucial to the success of these SPR binding studies because of enhanced specificity.

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. DNA from cells treated with 2 and 8 microM B[alpha]P exhibited increases of 3- and 4-fold in APE1 (p<0.001), 11- and 19-fold in Endo III (p<0.001), and 8- and 15-fold in hOGG1 (p<0.001) OCDLs, respectively, compared to the 0 microM B[alpha]P-treated (control) group. Mammary cells treated with 8 microM B[alpha]P produced 0.12 aberrations per cell (p<0.05) and there was a strong positive correlation (r=0.91) between the levels of OCDLs and those of chromosomal aberrations. Finally, TAC was decreased by 25% (p<0.02), whereas ROS production increased by 2-fold (p<0.02) in cells treated with 8 microM B[alpha]P compared to the control group. In conclusion, oxidatively induced clustered DNA damage mediated through differential expression of APE1, reduced TAC, and increased ROS may play a significant role in the chemically induced transformation of normal primary mammary cells.

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. Two types of arrays, namely Affymetrix Human HG U133A 2.0 oligonucleotide microarrays and Ambion mirVana bioarrays, were employed to study mRNA and microRNA expressions, respectively. Three flasks were used per irradiation dose, and an additional three unirradiated flasks served as control. Microarray data were validated by reverse transcriptase quantitative polymerase chain reaction, and proteins of some expressed genes were determined by Western blots. Results showed the existence of differences in expression profiles between the two irradiation modalities. IMRT appeared to influence expression of some DNA repair genes, whereas in conventional RT, some DNA repair and cell cycle-related genes that initially seemed to be preferentially expressed dwindled to normal levels. Earlier in vitro experiments using cell survival to study sublethal damage repair support our conclusions. Bioinformatic investigation revealed a correlation of gene expression with derepression effects of microRNA molecules. We have presented opinions as to how microRNAs might influence gene expression during radiation-induced stress and have suggested future avenues for research.

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. Transgene expression was analysed in pine T-DNA transformants carrying different numbers of copies of T-DNA insertions. Post-transcriptional gene silencing (PTGS) was mostly obtained in transgenic lines with more than three copies of T-DNA, but not in transgenic lines with one copy of T-DNA. In situ hybridization chromosome analysis of transgenic lines demonstrated that silenced transgenic lines had two or more T-DNA insertions in the same chromosome. These results suggest that two or more T-DNA insertions in the same chromosome facilitate efficient gene silencing in transgenic pine cells expressing green fluorescent protein. There were no differences in shoot differentiation and development between transgenic lines with multiple T-DNA copies and transgenic lines with one or two T-DNA copies.

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. In vitro, a lytic infection can be induced in KSHV-infected cells by treating with phorbol ester (TPA). However, the exact signalling events that lead to the reactivation of KSHV lytic infection are still elusive. Here, a role is demonstrated for B-Raf/MEK/ERK signalling in TPA-induced reactivation of KSHV latent infection. Inhibiting MEK/ERK signalling by using MEK-specific inhibitors decreased expression of the TPA-induced KSHV lytic-cycle gene ORF8. Transfection of BCBL-1 cells with B-Raf small interfering RNA inhibited TPA-induced KSHV lytic infection significantly. Additionally, overexpression of MEK1 induced a lytic cycle of KSHV infection in BCBL-1 cells. The significance of these findings in understanding the biology of KSHV-associated pathogenesis is discussed.

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.) cells. Among the different phases of the cell cycle, transgenic cells at the M phase gave 2-3 times lower gfp silencing than those at the G1, S, and G2 phases. The similar results of the siRNA-mediated gfp silencing were obtained in three transgenic cell lines. Differential gfp silencing induced by siRNA has been confirmed by northern blot, laser scanning microscopy, and siRNA analysis. These data suggested that siRNA-mediated gene inactivation is associated with the cell cycle phase in Virginia pine.

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. Using LISW, we are able to silence gene expression in cell cultures of three different plant species rice (Oryza sativa L.), cotton (Gossypium hirsutum L.), and slash pine (Pinus elliottii Engelm.). Gene silencing induced by siRNA has been confirmed by northern blot, laser scanning microscopy, and siRNA analysis. These data suggested that LISW-mediated siRNA delivery can be a reliable and effective method for inducing PTGS in cultured cells.

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. Coapplication of Fluorogold with 17beta-estradiol produced a significant increase in the number of retrograde-labeled lumbar motoneurons, compared with Fluorogold alone. Estrogen potentiation of retrograde labeling was inhibited by application of ICI 182,780, U0126, LY-294002, and cycloheximide. Immunohistochemical analysis of the sciatic nerve, 24 h following crush injury, revealed accumulation of phospho-ERK in regenerating nerve fibers. The data suggest a role for estrogen, ERK, PI3K, and protein synthesis in the uptake and retrograde transport of Fluorogold. We propose that estrogen action in peripheral nerve fibers is mediated via the ERK and PI3K signaling pathways and is reliant on local protein synthesis.