Effects of γ-IFN and NGF on subpopulations in a human neuroblastoma cell line: Flow cytometric and morphological analysis

Summary Neuroblastomas are neural crest-derived tumors that contain neuronal, melanocyte, and Schwann cell precursors. We examined the effects of treatment with γ-interferon (γ-IFN) and nerve growth factor (NGF), alone, and in combination, on these progenitor subpopulations in the human neuroblastoma cell line, SH-SY5Y. Using fluorescence-activated flow cytometry (FACS), changes in expression of three differentiation-specific or-associated marker proteins, the 200 kD neurofilament protein, the myelin basic protein, and the S-100 protein, were analyzed. Growth rates and morphological changes associated with each treatment over the 2-wk incubation period were noted. The greatest effects were observed with combined IFN +NGF treatment. These were significant increases in expression of all three proteins distinctive morphological signs of differentiation, and extensive inhibition of proliferation compared to control cultures. Treatment with NGF alone resulted in increased neurofilament protein expression and in the length and number of neurite extensions, but there was no effect on the growth rate. IFN induced striking morphological changes, significant inhibition of growth, and changes in protein expression that correlated with neuronal to non-neuronal subpopulation shifts due to the death of differentiated cells. When treatment was discontinued after 15 d, the morphological changes induced by NGF were reversed within 2–3 d, while those induced by IFN±NGF were present up to 4 wk post-treatment. Small, neuroblastic colonies were observed throughout the treatment period and within 4–6 wk after the cessation of treatment this cell-type fully reconstituted the cultures suggesting the presence of a stem cell. Our results indicate that treatment with γ-IFN±NGF can regulate growth and induce, either stem cells or progenitor neuronal, Schwann and melanocyte subpopulations in the SH-SY5Y cell line to irreversibly differentiate.     

Basis for selectivity of cationic antimicrobial peptides for bacterial versus mammalian membranes

Novel cationic antimicrobial peptides typified by structures such as KKKKKKAAXAAWAAXAA-NH2, where X = Phe/Trp, and several of their analogues display high activity against a variety of bacteria but exhibit no hemolytic activity even at high dose levels in mammalian erythrocytes. To elucidate their mechanism of action and source of selectivity for bacterial membranes, phospholipid mixtures mimicking the compositions of natural bacterial membranes (containing anionic lipids) and mammalian membranes (containing zwitterionic lipids + cholesterol) were challenged with the peptides. We found that peptides readily inserted into bacterial lipid mixtures, although no insertion was detected in model "mammalian" membranes. The depth of peptide insertion into model bacterial membranes was estimated by Trp fluorescence quenching using doxyl groups variably positioned along the phospholipid acyl chains. Peptide antimicrobial activity generally increased with increasing depth of peptide insertion. The overall results, in conjunction with molecular modeling, support an initial electrostatic interaction step in which bacterial membranes attract and bind peptide dimers onto the bacterial surface, followed by the "sinking" of the hydrophobic core segment to a peptide sequence-dependent depth of approximately 2.5-8 A into the membrane, largely parallel to the membrane surface. Antimicrobial activity was likely enhanced by the fact that the peptide sequences contain AXXXA sequence motifs, which promote their dimerization, and possibly higher oligomerization, as assessed by SDS-polyacrylamide gel analysis and fluorescence resonance energy transfer experiments. The high selectivity of these peptides for nonmammalian membranes, combined with their activity toward a wide spectrum of Gram-negative and Gram-positive bacteria and yeast, while retaining water solubility, represent significant advantages of this class of peptides.     

Directional surface plasmon-coupled emission: application for an immunoassay in whole blood

We present a new approach for performing fluorescence immunoassay in whole blood using fluorescently labeled anti-rabbit immunoglobulin G (IgG) on a silver surface. This approach, which is based on surface plasmon-coupled emission (SPCE), provides increased sensitivity and substantial background reduction due to exclusive selection of the signal from the fluorophores located near a bioaffinity surface. This article describes the effect of an optically dense sample matrix, namely human whole blood and serum, on the intensity of the SPCE. An antigen (rabbit IgG) was adsorbed to a slide covered with a thin silver metal layer, and the SPCE signal from the fluorophore-labeled anti-rabbit antibody, binding to the immobilized antigen, was detected. The effect of the sample matrix (buffer, human serum, or human whole blood) on the end-point immunoassay SPCE signal was studied. It was demonstrated that the kinetics of binding could be monitored directly in whole blood or serum. The results showed that human serum and human whole blood attenuate the SPCE end-point signal and the immunoassay kinetic signal only approximately two- and threefold, respectively, as compared with buffer, resulting in signals that are easily detectable even in whole blood. The high optical absorption of the hemoglobin can be tolerated because only fluorophores within a couple of hundred nanometers from the metallic film contribute to SPCE. Excited fluorophores outside the 200-nm layer do not contribute to SPCE, and their free space emission is not transmitted through the opaque metallic film into the glass substrate. We believe that SPCE has the potential of becoming a powerful approach for performing immunoassays based on surface-bound analytes or antibodies for many biomarkers directly in dense samples such as whole blood with no need for washing steps.     

Multi-wavelength immunoassays using surface plasmon-coupled emission

We describe a new method for multi-wavelength immunoassays using surface plasmon-coupled emission (SPCE). This phenomenon is coupling of excited fluorophores with a nearby thin metal film, in our case silver, resulting in strongly directional emission into the underlying glass substrate. The angle at which the radiation propagate through the prism depends on the surface plasmon angle for the relevant wavelength. These angles depend on emission wavelength, allowing measurement of multiple analytes using multiple emission wavelengths. We demonstrated this possibility using antibodies labeled with either Rhodamine Red-X or AlexaFluor 647. These antibodies were directed against an antigen protein bound to the silver surface. The emission from each labeled antibody occurred at a different angle on the glass prism, allowing independent measurement of surface binding of each antibody. This method of SPCE immunoassays can be readily extended to 4 or more wavelengths.     

Metal-enhanced fluorescence immunoassays using total internal reflection and silver island-coated surfaces

We present a generic immunoassay platform that uses enhanced total internal reflection fluorescence in the proximity of silver island films (SIFs), a surface coating consisting of metal (silver) particles. This platform is used with a model immunoassay where a protein antigen, rabbit immunoglobulin G, was immobilized on the SIF-coated glass surface. The signal from a fluorescent dye-labeled anti-rabbit antibody binding to the surface antigen was detected; different color dyes have been tested. Close placement of the fluorophore to surface-bound silver nanostructures results in dramatic signal enhancement (up to 40-fold) on the SIFs as compared with the glass slides. Use of the total internal reflection mode of excitation has significant advantages (over classic front-face excitation) for practical assay development. The limited evanescent wave excitation volume makes it possible to minimize the background signal and use the immunoassay with no need for any washing steps.     

Regulation of telomere length and suppression of genomic instability in human somatic cells by Ku86

Ku86 plays a key role in nonhomologous end joining in organisms as evolutionarily disparate as bacteria and humans. In eukaryotic cells, Ku86 has also been implicated in the regulation of telomere length although the effect of Ku86 mutations varies considerably between species. Indeed, telomeres either shorten significantly, shorten slightly, remain unchanged, or lengthen significantly in budding yeast, fission yeast, chicken cells, or plants, respectively, that are null for Ku86 expression. Thus, it has been unclear which model system is most relevant for humans. We demonstrate here that the functional inactivation of even a single allele of Ku86 in human somatic cells results in profound telomere loss, which is accompanied by an increase in chromosomal fusions, translocations, and genomic instability. Together, these experiments demonstrate that Ku86, separate from its role in nonhomologous end joining, performs the additional function in human somatic cells of suppressing genomic instability through the regulation of telomere length.     

Sindbis virus with a tricomponent genome

We established a system for propagation of Sindbis virus (SIN)-based replicons in tissue culture in the form of a tricomponent genome virus. Three RNA fragments containing complementing genetic information required for virus replication are packaged into separate viral particles, and each cell produces at least 1,000 packaged replicons and the number of packaged helpers sufficient to perform the next passage. This system can be used to generate large stocks of packaged replicons. The formation of infectious recombinant SIN virus was not detected in any experiments. These features make multicomponent genome SIN an attractive system for a variety of research and biotechnology applications.     

Low cholesterol efflux capacity and abnormal lipoprotein particles in youth with type 1 diabetes: a case control study

Background

Patients with type 1 diabetes (T1DM) have increased mortality from cardiovascular disease (CVD). Risk factors for CVD include an elevation of LDL (LDLp) and small HDL (sHDLp) particles, and a decrease in reverse cholesterol transport i.e. HDL-cholesterol efflux capacity (CEC). Our objective was to compare lipoprotein particles and CEC between T1DM and healthy controls (HC) and to explore the associations between NMR lipid particles and cholesterol efflux.

Methods

78 patients with T1DM and 59 HC underwent fasting lipoprotein profile testing by NMR and measurements of CEC by cell-based method. The associations between NMR lipid particles with CEC were analyzed using multivariable linear regression models.

Results

Youth with T1DM had higher total LDLp 724 [(563–985) vs 622 (476–794) nmol/L (P?=?0.011)] (Maahs et al. in Circulation 130(17):1532–58, 2014; Shah et al. in Pediatr Diabetes 16(5):367–74, 2015), sHDLp [11.20 (5.7–15.3) vs 7.0 (3.2–13.1) μmol/L (P?=?0.021)], and lower medium HDLp [11.20 (8.5–14.5) vs 12.3 (9–19.4), (P?=?0.049)] and lower CEC (0.98?±?0.11% vs 1.05?±?0.15%, P?=?0.003) compared to HC. Moreover, CEC correlated with sHDLp (β?=???0.28, P?=?0.045) and large HDLp (β?=?0.46, P?<?0.001) independent of age, sex, ethnicity, BMIz, HbA1c, hsCRP and total HDLp in the diabetic cohort.

Conclusions

Youth with T1DM demonstrated a more atherogenic profile including higher sHDL and LDLp and lower CEC. Future efforts should focus on considering adding lipoprotein particles and CEC in CVD risk stratification of youth with T1DM.Trial registration Clinical Trials Registration Number NCT02275091

     

Cryopreservation of the Norway spruce tissue culture line able to produce extracellular lignin

Somatic hybridization has been used in potato to overcome the sexual barriers between the cultivated (Solanum tuberosum L.) and wild species. To date hundreds of inter/intra-specific somatic hybrids have been produced via protoplast fusions using 23 Solanum species and characterized for multiple traits such as agronomic, disease/pest resistance, salinity, frost and others. With increasing success in recovery of fusion products, somatic hybrids have been exploited in potato genetics, breeding and genomics studies. Here, we report on progress in somatic hybridization research in potato during the past 40 years.     

Lactobacillus casei Exerts Anti-Proliferative Effects Accompanied by Apoptotic Cell Death and Up-Regulation of TRAIL in Colon Carcinoma Cells

Probiotic microorganisms such as lactic acid bacteria (LAB) exert a number of strain-specific health-promoting activities attributed to their immunomodulatory, anti-inflammatory and anti-carcinogenic properties. Despite recent attention, our understanding of the biological processes involved in the beneficial effects of LAB strains is still limited. To this end, the present study investigated the growth-inhibitory effects of Lactobacillus casei ATCC 393 against experimental colon cancer. Administration of live Lactobacillus casei (as well as bacterial components thereof) on murine (CT26) and human (HT29) colon carcinoma cell lines raised a significant concentration- and time-dependent anti-proliferative effect, determined by cell viability assays. Specifically, a dramatic decrease in viability of colon cancer cells co-incubated with 10⁹ CFU/mL L. casei for 24 hours was detected (78% for HT29 and 52% for CT26 cells). In addition, live L. casei induced apoptotic cell death in both cell lines as revealed by annexin V and propidium iodide staining. The significance of the in vitro anti-proliferative effects was further confirmed in an experimental tumor model. Oral daily administration of 10⁹ CFU live L. casei for 13 days significantly inhibited in vivo growth of colon carcinoma cells, resulting in approximately 80% reduction in tumor volume of treated mice. Tumor growth inhibition was accompanied by L. casei-driven up-regulation of the TNF-related apoptosis-inducing ligand TRAIL and down-regulation of Survivin. Taken together, these findings provide evidence for beneficial tumor-inhibitory, anti-proliferative and pro-apoptotic effects driven by this probiotic LAB strain.