Green Fluorescent Protein (GFP) as a Marker for Cell Viability After UV Irradiation

Generation of Chinese Hamster Ovary (CHO) cell lines stably expressing green fluorescent protein (GFP) was achieved using a plasmid vector that encoded the red-shifted pCX-xGFP under the control of a strong hybrid promoter composed of a CMV enhancer and a -actin/-globin gene promoter. Cotransfection of the promoter-less pSV2-Neo helper plasmid transmitting neomycin resistance was followed by selection with the antibiotic G418. Constitutive GFP expression could be visualized in living and fixed cells using fluorescence spectroscopy, fluorescence microscopy, and flow cytometry. DNA repair-proficient (AA8) and deficient (UV5) CHO strains were used for survival tests after UVC irradiation. Cells carrying the GFP construct (AA8-pGFP, UV5-pGFP) show the same response to UV irradiation (colony forming ability) as their nontransformed parental cell lines (AA8, UV5). Using GFP as a marker for cell viability, cells were harvested after certain postirradiation growth periods and the numbers of GFP expressing cells and fluorescence intensities were determined by FACS analysis. Generally, GFP fluorescence in irradiated cells is not seen when cell membranes are damaged (leak-out of the soluble GFP). Irradiated cells without membrane damage express GFP continuously (leading to a dose-dependent increase in GFP contents).     

Raman spectroscopy as a tool for detecting mitochondrial fitness

Raman spectroscopy allows the molecular chemical analysis of whole living cells by comparing them to known Raman signatures of specific vibrational bonds. In this work we used Raman spectroscopy to differentiate between wild type yeast cells and mutants characterized by increased or reduced mitochondrial fragmentation. To associate mitochondrial fragmentation with biochemical markers, we performed Linear Discriminant Analysis (LDA) of whole cell Raman spectra (~50–100 cells/spectrum). We show that the long‐lived, less fragmented mutants fall into a significantly distant cluster from the wild type and short‐lived, more fragmented mutants. Clustering depends on respiratory growth and coincides with that of membrane phospholipids and some respiratory chain components. Spectral clustering is supported by enzymatic activity measurements of OXPHOS Complexes. In addition, we find that NAD(P)H autofluorescence also correlates with mitochondrial fragmentation, representing another likely aging biomarker, besides phospholipids and OXPHOS components. In summary, we demonstrate that Raman spectroscopy has the potential to become a powerful tool for differentiating healthy from unhealthy aged tissues, as well as for the prognostic evaluation of mitochondrial function and fitness. © 2016 The Authors Journal of Raman Spectroscopy Published by John Wiley & Sons Ltd     

BRCA1 involved in regulation of Bcl-2 expression and apoptosis susceptibility to ionizing radiation

BRCA1 has been proposed to be tightly linked to the resistance of tumor cells to ionizing radiation. The pathway leading to this phenomenon is not yet clear. In this work, we investigated the role of BRCA1 in the apoptosis regulation in response to carbon ion irradiation. We utilized three different cancer cell lines with various states for BRCA1 and p53 to identify the relationship between endogenous BRCA1 and the apoptosis-related genes, and determine whether p53 function would affect the role of BRCA1 in...     

Glutamate-induced apoptosis in neuronal cells is mediated via caspase-dependent and independent mechanisms involving calpain and caspase-3 proteases as well as apoptosis inducing factor (AIF) and this process is inhibited by equine estrogens

Background  

Glutamate, a major excitatory amino acid neurotransmitter, causes apoptotic neuronal cell death at high concentrations. Our previous studies have shown that depending on the neuronal cell type, glutamate-induced apoptotic cell death was associated with regulation of genes such as Bcl-2, Bax, and/or caspase-3 and mitochondrial cytochrome c. To further delineate the intracellular mechanisms, we have investigated the role of calpain, an important calcium-dependent protease thought to be involved in apoptosis along with mitochondrial apoptosis inducing factor (AIF) and caspase-3 in primary cortical cells and a mouse hippocampal cell line HT22.     

Proteolytic Cleavage of HIV-1 GFP-Vpr Fusions at Novel Sites Within Virions and Living Cells: Concerns for Intracellular Trafficking Studies

Fluorescent labelling of the highly conserved HIV-1 accessory protein Vpr (Viral Protein R) with GFP or variants thereof has proved a valuable approach to track Vpr and/or HIV-1 subcellular localisation in vivo. Our analysis in transfected mammalian cells expressing GFP-Vpr fusion protein, as well as within virus derived there from, documents site-specific proteolytic cleavage of the GFP-Vpr fusion protein. Western analysis revealed that transfected mammalian cells harbour a C-terminally truncated variant of Vpr in addition to full-length GFP-Vpr. Further, virions derived from these GFP-Vpr expressing cells show protein in which the GFP-tag has been additionally cleaved from the Vpr protein. Endogenous HIV protease (PR) activity was shown to be responsible for the latter, as addition of Saquinavir™, a potent PR inhibitor abolished the cleavage. Since many previous studies have relied on imaging the GFP fluorescence of GFP-Vpr, it would appear that the results may not reflect intact GFP-Vpr.     

辐射诱导促进AdCMV-p53 转染前列腺癌细胞

用腺病毒重组体（AdCMV p53/GFP）转染经0.5, 1.0和2.0 Gy γ射线辐射处理的前列腺癌细胞[PC 3（ nullp53）], 用克隆形成法检测细胞增殖能力, 用流式细胞分析法测定腺病毒重组体转染率和外源性p53蛋白表达。 结果提示, 辐射诱导使腺病毒重组体转染PC 3细胞提高7%—39%。 辐射联合 AdCMV p53 转染组p53表达水平提高18.5%—35.4%。 与单纯 AdCMV p53 转染组和单纯辐射组相比, 辐射联合 AdCMV-p53 转染组细胞存活率分别降低25%—64%和22%—65%。 To determine whether low dose pre irradiation could enhance adenovirus mediated p53 transfer and expression in human prostate adenocarcinoma, the PC 3 cells were pre exposed to γ rays, and then infected with replication deficient adenovirus recombinant vectors, containing human wild type p53 (AdCMV p53) or green fluorescent protein gene (AdCMV GFP) respectively (γ ray irradiation + AdCMV p53 /GFP infection). The exogenous gene transfer and expression were detected by flow cytometric analysis. The GFP transfer frequencies in γ irradiation + AdCMV GFP infection groups were 7%—39% more than those in AdCMV GFP infection groups. The p53 levels in the γ irradiation + AdCMV p53 infection groups were 18.5%—35.4% more than those in AdCMV p53 infection groups (p<0.05),suggesting that low dose (less than or equal to 1.0 Gy) irradiation could significantly promote exogenous p53 transfer and expression in the PC 3 cells. The survival fractions for the γ irradiation + AdCMV p53 infection groups were 25%—64%, 22%—65% less than those for AdCMV p53 infection, or γ irradiation groups, respectively (p<0.05).     

Gene transfer into retinal ganglion cells by in vivo electroporation: a new approach

We developed a new in vivo electroporation method to deliver genes into retinal ganglion cells (RGCs). Efficiency and degree of tissue damage were evaluated using green fluorescent protein (GFP) gene and TUNEL. Soon after the intravitreous injection of the GFP gene, electroporation (five electric pulses of 99 ms duration each and 12V/cm delivered twice 5 min apart) was carried out on the adult rat eyeball with the aid of tweezer-type disc electrodes attached to corneal (cathode) and scleral (anode) surfaces. GFP expression, exhibiting a maximum on day 7, was detectable for up to 21 days. DiI retrograde labeling of RGCs showed that 41.5% of the total ganglion cells in the electroinjected area were GFP-positive. Therefore, this new method may be a useful tool for the delivery of genes into RGCs.     

Antigenized antibodies expressing Vbeta8.2 TCR peptides immunize against rat experimental allergic encephalomyelitis

BACKGROUND: Immunity against the T cell receptor (TCR) is considered to play a central role in the regulation of experimental allergic encephalomyelitis (EAE), a model system of autoimmune disease characterized by a restricted usage of TCR genes. Methods of specific vaccination against the TCR of pathogenetic T cells have included attenuated T cells and synthetic peptides from the sequence of the TCR. These approaches have led to the concept that anti-idiotypic immunity against antigenic sites of the TCR, which are a key regulatory element in this disease. METHODS: The present study in the Lewis rat used a conventional idiotypic immunization based on antigenized antibodies expressing selected peptide sequences of the Vbeta8.2 TCR (93ASSDSSNTE101 and 39DMGHGLRLIHYSYDVNSTEKG59). RESULTS: The study demonstrates that vaccination with antigenized antibodies markedly attenuates, and in some instances, prevents clinical EAE induced with the encephalitogenic peptide 68GSLPQKSQRSQDENPVVHF88 in complete Freunds' adjuvant (CFA). Antigenized antibodies induced an anti-idiotypic response against the Vbeta8.2 TCR, which was detected by ELISA and flowcytometry. No evidence was obtained of a T cell response against the corresponding Vbeta8.2 TCR peptides. CONCLUSIONS: The results indicate that antigenized antibodies expressing conformationally-constrained TCR peptides are a simple means to induce humoral anti-idiotypic immunity against the TCR and to vaccinate against EAE. The study also suggests the possibility to target idiotypic determinants of TCR borne on pathogenetic T cells to vaccinate against disease.     

Radiation damage to DNA: the importance of track structure

A wide variety of biological effects are induced by ionizing radiation, from cell death to mutations and carcinogenesis. The biological effectiveness is found to vary not only with the absorbed dose but also with the type of radiation and its energy, i.e., with the nature of radiation tracks. An overview is presented of some of the biological experiments using different qualities of radiation, which when compared with Monte Carlo track structure studies, have highlighted the importance of the localized spatial properties of stochastic energy deposition on the nanometer scale at or near DNA. The track structure leads to clustering of damage which may include DNA breaks, base damage etc., the complexity of the cluster and therefore its biological repairability varying with radiation type.

The ability of individual tracks to produce clustered damage, and the subsequent biological response are important in the assessment of the risk associated with low-level human exposure. Recent experiments have also shown that biological response to radiation is not always restricted to the ‘hit’ cell but can sometimes be induced in ‘un-hit’ cells near by.     

Contribution of constitutively proliferating precursor cell subtypes to dentate neurogenesis after cortical infarcts

Background  

It is well known that focal ischemia increases neurogenesis in the adult dentate gyrus of the hippocampal formation but the cellular mechanisms underlying this proliferative response are only poorly understood. We here investigated whether precursor cells which constitutively proliferate before the ischemic infarct contribute to post-ischemic neurogenesis. To this purpose, transgenic mice expressing green fluorescent protein (GFP) under the control of the nestin promoter received repetitive injections of the proliferation marker bromodeoxyuridine (BrdU) prior to induction of cortical infarcts. We then immunocytochemically analyzed the fate of these BrdU-positive precursor cell subtypes from day 4 to day 28 after the lesion.     

Recent developments in monitoring calcium and protein interactions in cells using fluorescence lifetime microscopy

Time-resolved fluorescence lifetime microscopy (TRFLM) allows the combination of the sensitivity of fluorescence lifetime to environmental parameters to be monitored in a spatial manner in single living cells, as well as providing more accurate, sensitive, and specific diagnosis of certain clinical diseases and chemical analyses. Here we discuss two applications of TRFLM: (1) the use of nonratiometric probes such as Calcium Crimson, for measuring Ca²⁺; and (2) quantification of protein interaction in living cells using green and blue fluorescent protein (GFP and BFP, respectively) expressing constructs in combination with fluorescence resonance energy transfer microscopy (FRET). With respect to measuring Ca²⁺ in biological samples, we demonstrate thatintensity-based measurements of Ca²⁺ with single-wavelength Ca²⁺ probes such as Calcium Crimson may falsely report the actual Ca²⁺ concentration. This is due to effects of hydrophobicity of the local environment on the emission of Calcium Crimson as well as interaction of Calcium Crimson with proteins, both of which are overcome by the use of TRFLM. The recent availability of BFP (P4-3) and GFP (S65T) (which can serve as donor and acceptor, respectively) DNA sequences which can be attached to the carboxy-or amino-terminal DNA sequence of specific proteins allows the dual expression and interaction of proteins conjugated to BFP and GFP to be monitored in individual cells using FRET. Both of these applications of TRFLM are expected to enhance substantially the information available regarding both the normal and the abnormal physiology of cells and tissues.     

γ脉宽对电子器件瞬时辐照效应的影响

 采用了2种γ脉冲辐射源,在脉冲宽度分别约为20,50,150 ns,剂量率为10⁶～10⁹ Gy(Si)·s^-1下,对5种不同类型的电子器件进行了辐照试验并对其辐照响应进行了分析,比较了不同脉冲宽度条件下辐照响应的差异。实验结果表明：脉冲宽度是影响瞬时辐照效应的重要因素,γ脉冲宽度越宽,辐照响应越强,分离器件比集成电路受脉宽的影响更明显。     

Scanning Near-Field Optical Microscopy and Microspectroscopy of Green Fluorescent Protein in Intact Escherichia coli Bacteria

Scanning near-field optical microscopy (SNOM) yields high-resolution topographic and optical information and constitutes an important new technique for visualizing biological systems. By coupling a spectrograph to a near-field microscope, we have been able to perform microspectroscopic measurements with a spatial resolution greatly exceeding that of the conventional optical microscope. Here we present SNOM images of Escherichia coli bacteria expressing a mutant green fluorescent protein (GFP), an important reporter molecule in cell, developmental, and molecular biology. Near-field emission spectra confirm that the fluorescence detected by SNOM arises from bacterially expressed GFP molecules.     

Study of the response of silicon detectors for alpha particles

Numerous radon measurements are carried out using silicon detectors directly in the environment. This new kind of alpha radiation measurement has been developed because the reduced cost makes it possible to replace the usual plastic track detectors. At our laboratory, an alpha particle detector has been designed from a commercial silicon photodiode. This type of detector can determine the device response perfectly in any kind of environment. Different spectrum analyses have been conducted in the laboratory and field to define the exact origin of counted alpha particles. We studied the response for different radon and thoron concentration levels and observed the energy of the detected alpha particles. We carried out some of these experiments with gas flux, and some without, to show the effects of interactions with surfaces to obtain thermodynamic equilibrium in the detection chamber. Finally, the silicon diodes that we tested measure the alpha particles of the decay products (polonium) from the radon and the thoron, but very weakly from the gases themselves. Thus, it is possible to make mistakes when measuring the radon if the count of alpha particles is performed without spectrum analysis. One reason for this is that the decay progenies of the radon are solid radio-elements with thermodynamic proprieties different from gases.     

High resolution laser-based detection of ammonia

In the present paper we compare the response of two types of photoacoustic cells (resonant and nonresonant) to determine the amount of ammonia volatilized from biological liquid samples at constant temperature, pressure and pH. The home made detector was a photoacoustic spectroscopy apparatus developed by Molecular Spectroscopy Laboratory staff at ENEA Frascati Research Centre in Italy. The sensor makes use of photo-acoustic cells equipped with commercially available high sensitivity miniaturized microphones. The radiation source was a line-tunable stabilized 10 W CW CO₂ laser. Ammonia measurements were performed by tuning the laser source on the 9R30 laser line (9.2197 µm radiation wavelength). Ammonium chloride standard solutions were prepared by us in laboratory, to serve as reproducible ideal samples. The photoacoustic response of the two type of photoacoustic cells was determined and compared. The feasibility study was reported.     

Non‐thermal plasma‐induced apoptosis in yeast Saccharomyces cerevisiae

The effect of non‐thermal plasma generated by the direct current (DC) corona discharge in the mode of transition spark is studied on a yeast Saccharomyces cerevisiae. The exposure to plasma increases production of reactive oxygen species (ROS) in cells, possibly causing the induction of apoptosis. To clarify the mechanism of apoptosis, its induction is tested not only on a wild strain of S. cerevisiae, but also on mutant strains: A deletion mutant Δyca1 without yeast metacaspase proves that in S. cerevisiae the apoptosis occurs partly by the caspase‐independent pathway. A petite strains with mutation in the mitochondria do not show pronounced ROS formation, but in spite of this, apoptosis is detected. Hence, mitochondrial ROS probably do not play an important role in induction of apoptosis.