Capacity of robot handling for Epstein-Barr virus transformation.

OBJECTIVES: Epstein-Barr virus (EBV) transformation has been described as a routine method to establish human B lymphoblastoid cell lines. Each established lymphoblastoid cell line represents one unique genetic information carrier and can produce unlimited quantities of DNA materials available for downstream applications and research. Undoubtedly, it is of great value to human clinical and experimental genetic studies. However, the current process of EBV transformation requires much manpower in the routine renewal of medium, which is time-consuming. This situation can become a serious problem especially when establishing a human B lymphoblastoid cell bank. A modified and cost-effective protocol for EBV transformation should be considered. MATERIALS AND METHODS: In the present study, process in EBV transformation was modified to fit the requirements of robot handling. RESULTS: 1 mL of whole blood was demonstrated to be sufficient to perform EBV transformation. Additionally, EBV transformation can performed in 96-deep-well plates that are directly and widely used with automatic work platforms. CONCLUSIONS: Based on these facts, a process of EBV transformation can be modified to fit the requirements of robot handling.

High-potentiality preliminary selection criteria and transformation time-dependent factors analysis for establishing Epstein-Barr virus transformed human lymphoblastoid cell lines.

Infection of freshly isolated and cryopreserved lymphocytes with Epstein-Barr virus (EBV) leads to the establishment of human B lymphoblastoid cell lines (LCLs). Techniques for optimal infection of the lymphocytes are vital for the establishment of a human biobank. The present study found that more than half (58-86%) of such established LCLs had transport times of less than 48 h, cell densities exceeding 10(6) cells/ml and cell viabilities greater than 90%. After EBV infection, 3306 freshly isolated lymphocytes required 30.0 +/- 0.1 days to become LCLs. Conversely, 1210 cryopreserved lymphocytes required 36.2 +/- 0.4 days. Cell density and viability of the culture affected transformation time in freshly isolated lymphocytes. On the other hand, blood transport time, cryopreservation time and initial cell viability were major factors in cryopreserved specimens. These results contribute to general information concerning the establishment of a human biobank for EBV infected cells.

In vivo measurement of tumor conductiveness with the magnetic bioimpedance method.

A noninvasive electromagnetic method has been developed that can effectively measure the in-vivo conductivity difference between rat tumor lines having a low and high metastatic potential. These tumor lines are used in the study of human prostate tumor.

NT-4/5 exacerbates free radical-induced neuronal necrosis in vitro and in vivo.

Neurotrophins render neurons highly vulnerable to certain injuries. We examined the possibility that NT-4/5 would enhance free radical neurotoxicity in vivo as well as in vitro. Striatal neurons exposed to 10 microM Fe(2+) or 1 mM l-buthionine-[S, R]-sulfoximine (BSO) underwent mild degeneration within 24 h. With concurrent addition of 10-100 ng/ml NT-4/5, neuronal death following exposure to Fe(2+) or BSO was significantly increased and suppressed by addition of 100 microM trolox, an antioxidant. In the adult brain, the intrastriatal injections of 20 nmol Fe(2+) revealed features of neuronal necrosis such as swelling cell body and mitochondria, fenestration of plasma membrane prior to nuclear membrane, and scattering condensation of nuclear chromatin. Cotreatment with 1.8 microg NT-4/5 augmented the striatal damage 24 h following the injections of Fe(2+). This study implies that free radicals produce necrotic degeneration in vivo as well as in vitro that becomes more sensitive in the presence of neurotrophins.

An automated, handheld biosensor for aflatoxin.

A new immunoaffinity fluorometric biosensor has been developed for detecting and quantifying aflatoxins, a family of potent fungi-produced carcinogens that are commonly found in a variety of agriculture products. They have also been cited as a biological agent under weapons development. The handheld, self-contained biosensor is fully automatic, highly sensitive, quick, quantitative, and requires no special storage. Approximately 100 measurements can be made before refurbishment is required, and concentrations from 0.1 parts per billion (ppb) to 50 ppb can be determined in <2 min with a 1 ml sample volume. The device operates on the principles of immunoaffinity for specificity and fluorescence for a quantitative assay. The analytic procedure is flexible so that other chemical and biological analytes could be detected with minor modifications to the current device. Advances in electro-optical components, electronics, and miniaturized fluidics were combined to produce this reliable, small, and versatile instrument.

Synergetic activation of p38 mitogen-activated protein kinase and caspase-3-like proteases for execution of calyculin A-induced apoptosis but not N-methyl-d-aspartate-induced necrosis in mouse cortical neurons.

We examined the possibility that p38 mitogen-activated protein kinase and caspase-3 would be activated for execution of apoptosis and excitotoxicity, the two major types of neuronal death underlying hypoxicischemic and neurodegenerative diseases. Mouse cortical cell cultures underwent widespread neuronal apoptosis 24 h following exposure to 10-30 nM calyculin A, a selective inhibitor of Ser/Thr phosphatase I and IIA. Activity of p38 was increased 2-4 h following exposure to 30 nM calyculin A. Addition of 3-10 microM PD169316, a selective p38 inhibitor, partially attenuated calyculin A neurotoxicity. Activity of caspase-3-like proteases was increased in cortical cell cultures exposed to 30 nM calyculin A for 8-16 h as shown by cleavage of DEVD-p-nitroanilide and phosphorylated tau. Proteolysis of tau was completely blocked by addition of 100 microM N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk), a broad-spectrum inhibitor of caspases, but incompletely by 10 microM PD169316. Calyculin A neurotoxicity was partially sensitive to 100 microM z-VAD-fmk. Cotreatment with 10 microM PD169316 and 100 microM z-VAD-fmk showed additive neuroprotection against calyculin A. Neither PD169316 nor z-VAD-fmk showed a beneficial effect against excitotoxic neuronal necrosis induced by exposure to 20 microM NMDA. Thus, caspase-3-like proteases and p38 likely contribute to calyculin A-induced neuronal apoptosis but not NMDA-induced neuronal necrosis.

Nuclear factor kappa B-mediated kainate neurotoxicity in the rat and hamster hippocampus.

Administration of the excitotoxin kainate produces seizure activity and selective neuronal death in various brain areas. We examined the degeneration pattern of hippocampal neurons following systemic injections of kainate in the hamster and the rat. As reported, treatment with kainate resulted in severe neuronal loss in the hilus and CA3 in the rat. While the hilar neurons were also highly vulnerable to kainate in the hamster, neurons in the CA1 area, but not CA3, were highly sensitive to kainate. In both animals, immunoreactivity to anti-p50 nuclear factor kappa B antibody was increased in nuclei of the hilar neurons within 4 h following administration of kainate. Kainate treatment also increased the nuclear factor kappa B immunoreactivity in hamster CA1 neurons and rat CA3 neurons 24 h later. Neurons showing intense nuclear factor kappa B signal were stained with acid fuchsin. Kainate also increased DNA binding activity of p50 and p65 nuclear factor kappa B in the nuclear extract of the hippocampal formation as analysed by electrophoretic mobility shift assay in the hamster, suggesting that activation of nuclear factor kappa B may contribute to kainate-induced hippocampal degeneration. Administration of 100 nmol dizocilpine maleate 3 h prior to kainate attenuated kainate-induced activation of nuclear factor kappa B and neuronal death in CA1 in the hamster. The present study provides evidence that the differential vulnerability of neurons in the rat and the hamster hippocampus to kainate is partly mediated by mechanisms involving N-methyl-D-aspartate-dependent activation of nuclear factor kappa B.

Phosphatidylinositol 3-kinase-mediated regulation of neuronal apoptosis and necrosis by insulin and IGF-I.

We examined effects of two insulin-like growth factors, insulin and insulin-like growth factor-I (IGF-I), against apoptosis, excitotoxicity, and free radical neurotoxicity in cortical cell cultures. Like IGF-I, insulin attenuated serum deprivation-induced neuronal apoptosis in a dose-dependent manner at 10-100 ng/mL. The anti-apoptosis effect of insulin against serum deprivation disappeared by addition of a broad protein kinase inhibitor, staurosporine, but not by calphostin C, a selective protein kinase C inhibitor. Addition of PD98059, a mitogen-activated protein kinase kinase (MAPKK) inhibitor, blocked insulin-induced activation of extracellular signal-regulated protein kinases (ERK1/2) without altering the neuroprotective effect of insulin. Cortical neurons underwent activation of phosphatidylinositol (PI) 3-kinase as early as 1 min after exposure to insulin. Inclusion of wortmannin or LY294002, selective inhibitors of PI 3-K, reversed the insulin effect against apoptosis. In contrast to the anti-apoptosis effect, neither insulin nor IGF-I protected excitotoxic neuronal necrosis following continuous exposure to 15 microM N-methyl-D-aspartate or 40 microM kainate for 24 h. Surprisingly, concurrent inclusion of 50 ng/mL insulin or IGF-I aggravated free radical-induced neuronal necrosis over 24 h following continuous exposure to 10 microM Fe2+ or 100 microM buthionine sulfoximine. Wortmannin or LY294002 also reversed this potentiation effect of insulin. These results suggest that insulin-like growth factors act as anti-apoptosis factor and pro-oxidant depending upon the activation of PI 3-kinase.

Bioimpedance: novel use of a minimally invasive technique for cancer localization in the intact prostate.

BACKGROUND: Prostate cancer is presently diagnosed by transrectal ultrasound (TRUS)-guided sextant needle biopsy. While echo texture of the tissue can prompt localization of tumor, it is presently imprecise. From 50-75% of men biopsied, based on an abnormal digital rectal examination (DRE) or elevated prostate-specific antigen (PSA) level, have negative biopsy results. Improvements in tumor localization during TRUS-guided prostate biopsy are greatly needed. Bioimpedance is an electrical property of biologic tissue. Electric current is limited in living tissue by highly insulating cell membranes; however, different tissue architecture such as cancer may impede current differently and allow detection of differences between normal and abnormal or malignant prostate tissue. Our goal was to assess the utility of bioimpedance measurements in differentiating tumor from normal prostatic tissue in an ex vivo model. METHODS: Bioimpedance was measured in six ex vivo prostates, which were removed for clinically localized prostate cancer. Two bioimpedance needles, 1 mm apart, were inserted 3 mm into the posterior surface of the prostate an average of 16 times per gland. Frequencies ranging from 100 kHz-4 MHz were used to obtain 594 bioimpedance measurements from the six glands. These measurements were then correlated with histology to determine the presence or absence of prostate cancer. RESULTS: Prostate cancer was found to have a higher impedance, of 932+/-170 ohms, compared to areas of no cancer within the same prostate, 751+/-151 ohms, P < 0.0001, at 2 MHz. This phenomenon was observed across all frequencies tested. CONCLUSIONS: This study demonstrates for the first time application of bioimpedance to distinguish areas of prostate cancer from areas of normal prostate. This technology may improve identification and localization of cancer within the prostate. Moreover, bioimpedance can potentially guide needle placement during prostate biopsy and thus improve sampling of tumors. Currently, our ex vivo model is limited by variables such as temperature and lack of blood flow. Further studies in an in vivo model will be needed to assess their effect.

Necrosis and apoptosis after retinal ischemia: involvement of NMDA-mediated excitotoxicity and p53.

PURPOSE: Accumulated evidence has shown that apoptosis and necrosis contribute to neuronal death after ischemia. The present study was performed to study the temporal and spatial patterns of neuronal necrosis and apoptosis after ischemia in retina and to outline mechanisms underlying necrosis and apoptosis. METHODS: Retinal ischemia was induced by increasing intraocular pressure to a range of 160 mm Hg to 180 mm Hg for 90 minutes in adult rats. The patterns of neuronal cell death were determined using light and electron microscopy and were visualized by TdT-dUTP nick-end labeling (TUNEL). The mRNA expression profile of p53 was examined using reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization histochemistry. Immunohistochemistry was performed using anti-p53, anti-microtubule associated protein-2, and anti-glial fibrillary acidic protein antibodies. RESULTS: Within 4 hours after ischemia, neurons in the inner nuclear cell layer (INL) and ganglion cell layer (GCL) underwent marked necrosis, made apparent by swelling of the cell body and mitochondria, early fenestration of the plasma membrane, and irregularly scattered condensation of nuclear chromatin. After 3 days, the INL and GCL neurons showed further degeneration through apoptosis marked by cell body shrinkage, aggregation, and condensation of nuclear chromatin. Apoptotic neurons were also observed sparsely in the outer nuclear cell layer. Intravitreal injections of MK-801 prevented early neuronal degeneration after ischemia. Of note, mRNA and protein levels of p53, the tumor suppressor gene known to induce apoptosis, were increased in the retinal areas undergoing apoptosis 1 to 3 days after ischemic injury. CONCLUSIONS: Ischemia produces the N-methyl-D-aspartate-mediated necrosis and slowly evolving apoptosis of neurons in the retina. The latter may depend on the expression of the p53 proapoptosis gene.

A neuron-specific gene transfer by a recombinant defective Sindbis virus.

We examined the possibility that Sindbis virus, an alpha virus with a single-stranded RNA genome, would be applied for neuronal gene transfer. The recombinant defective Sindbis viruses were constructed by replacing the structural genes of Sindbis virus with genes encoding beta-galactosidase (rdSind-lacZ) or enhanced green fluorescent protein (rdSind-EGFP). In neuron-glia cocultures prepared from the neocortex, hippocampus, and striatum, EGFP or beta-galactosidase was expressed selectively in neurons 24 h after infection with rdSind-EGFP or rdSind-lacZ. Most cortical neurons were infected with rdSind-lacZ at a multiplicity of infection (M.O.I.) of 5 while glial cells were little infected. In addition, transient neuron-specific expression of beta-galactosidase was observed near injection sites over the next 3 d following administration of rdSind-lacZ in adult rat. In the cortical neurons infected with rdSind-EGFP, treatment with NMDA induced neuritic blebs and cell body swelling in a Na+-dependent manner. Therefore, recombinant defective Sindbis viruses can be used as an efficient and selective vector for gene transfer into neurons and applied to investigate biological role of target genes delivered into neurons in vitro and in vivo.

Ca2+-mediated activation of c-Jun N-terminal kinase and nuclear factor kappa B by NMDA in cortical cell cultures.

We examined the possibility that c-Jun N-terminal kinase (JNK) and nuclear factor kappaB (NF-kappaB) might be involved in intracellular signaling cascades that mediate NMDA-initiated neuronal events. Exposure of cortical neurons to 100 microM NMDA induced activation of JNK within 1 min. Activity of JNK was further increased over the next 5 min and then declined by 30 min. Similarly, ionomycin, a selective Ca2+ ionophore, induced activation of JNK. The NMDA-induced activation of JNK was abrogated in the absence of extracellular Ca2+, suggesting that Ca2+ entry is necessary and sufficient for the JNK activation. Immunohistochemistry with anti-NF-kappaB antibody demonstrated nuclear translocation of NF-kappaB within 5 min following NMDA treatment. NMDA treatment also enhanced the DNA binding activity of nuclear NF-kappaB in a Ca2+-dependent manner. Treatment with 3 mM aspirin blocked the NMDA-induced activation of JNK and NF-kappaB. Neuronal death following a brief exposure to 100 microM NMDA was Ca2+ dependent and attenuated by addition of aspirin or sodium salicylate. The present study suggests that Ca2+ influx is required for NMDA-induced activation of JNK and NF-kappaB as well as NMDA neurotoxicity. This study also implies that aspirin may exert its neuroprotective action against NMDA through blocking the NMDA-induced activation of NF-kappaB and JNK.

The cDNA cloning and ontogeny of mouse alpha-synuclein.

Alpha-synuclein has been implicated in the pathogenesis of Parkinson's disease. To investigate the role of alpha-synuclein in the brain, the cDNA clone encoding the mouse cognate of the human alpha-synuclein was isolated from a mouse brain cDNA library. The open reading frame coded for 140 amino acids that share 95% identity with human alpha-synuclein. Northern blot analysis showed that alpha-synuclein mRNA was primarily expressed in brain and spleen of adult mouse. In situ hybridization histochemistry revealed the highest expression of alpha-synuclein mRNA in the hippocampal formation and neocortex of the adult mouse. alpha-Synuclein mRNA expression in the brain was first observed in the hippocampus and neocortex on postnatal day 1. Levels of alpha-synuclein mRNA in these forebrain areas were nearly maximal at postnatal day 7 and remained relatively high until the adult stage. alpha-Synuclein mRNA was expressed in the liver transiently during embryogenesis.

Development of a computerized EEG imaging system with a personal computer.

The authors developed a computerized electroencephalography imaging system with an IBM PC AT. The EEG signals amplified with a 16 channel EEG machine were digitized at 51.2 Hz (512 samples per epoch). The shifted DC potential and 60Hz artificats were removed by a high pass filter and 60Hz notch filter. A window function consisting of a 10% cosine taper was obtained by weighting the points at either end of the epoch by a cosine bell. A fast Fourier transform was applied to every epoch and the power spectrum estimates were computed in 0.39 Hz steps. The activity estimates for the delta, theta, alpha and beta bands were computed by summimg adjacent values. The outline of the top-down maps was formed from a series of sagittal cuts, then 32 electrodes were placed on the map. A file was created which contained a table of weighting parameters for calculating the interpolated values for every point within the outline. Each weight was in inverse linear proportion to the distance of the pixel to the nearest four electrodes. The map was finally generated with computation of the spectral EEG in each pixel according to the weighting parameter. The functioning of this system was tested with a functional generator and a human subject.

Practical limits on the biomagnetic inverse process determined from in vitro measurements in spherical conducting volumes.

A technique of locating current dipoles in spherical conducting volumes by determining the location of the magnetic field maximum and inverting the magnetic field equations was developed and the expected localisation errors were predicted. AC current dipoles were placed in spheres of uniform conductivity. Each dipole's magnetic field was measured and its location was calculated by determining the angle between the magnetic field null and maximum and using an iterative inverse solution to the magnetic field equations. Absolute agreement between predicted magnetic field strengths and actual magnetic field measurements was within 5%. A study of the effect of signal to noise ratio and number of data points in the analysis indicates that dipole localisation of approximately 1 mm is achievable for a signal to noise ratio greater than 10 decibels (S/N greater than 10 db).