Use of A-549 cells in a clinical virology laboratory.

A-549 cells were compared with other cell lines for virus recovery, except from specimens submitted specifically for detection of cytomegalovirus. Of 589 specimens submitted specifically for detection of herpes simplex virus (HSV), 163 (28%) were positive for HSV--159 (97.5%) in A-549 cells and 156 (96%) in primary rabbit kidney cells. HSV cytopathic effect was identified an average of 0.6 day earlier in A-549 cells. Virus was recovered from 194 (11%) of 1,790 specimens submitted for general virus isolation. Of 40 HSV isolates, 85% were positive in A-549 cells, 72.5% were positive in MRC-5/WI-38 cells, and 42.5% were positive in HEp-2 cells. With adenovirus, 96% of 45 isolates were detected in A-549 cells, 62% were detected in HEp-2 cells, 38% were detected in MRC-5/WI-38 cells, and 31% were detected in PMK cells. Of the 76 enterovirus-positive specimens, 71% were positive in PMK cells, 62% were positive in A-549 cells, and 62% were positive in MRC-5/WI-38 cells. None of 12 respiratory syncytial virus, 14 rhinovirus, or 7 influenza A virus isolates were detected in A-549 cells. Of the cell lines examined, A-549 cells performed optimally for recovery of HSV and adenovirus, they allowed good growth of many of the enterovirus isolates, but they did not allow recovery of any of the respiratory syncytial virus, rhinovirus, or influenza A virus isolates.     

Eotaxin and nitric oxide production as markers of inflammation in allergic cynomolgus monkeys

BACKGROUND: Cynomolgus monkeys have a natural hypersensitivity to Ascaris suum antigen. Inhalation of antigen produces immediate and delayed allergic reactions and an influx of inflammatory cells into the lungs. This study investigated the production of nitric oxide (NO) and the chemokine eotaxin during this allergic response. The effect of bronchoscopy alone on lung inflammatory cells was also investigated along with the time course of the eosinophil influx into the lung. METHODS: Allergic cynomolgus monkeys were challenged with antigen. Bronchoalveolar lavage (BAL) was performed before and after challenge, and end-tidal NO was measured before and 24 h after challenge. Eotaxin was measured in the BAL fluid 6, 24 and 72 h after challenge. One group of animals was treated with dexamethasone before challenge to block the influx of cells into the lung. RESULTS: BLA alone induced an influx of neutrophils, but not eosinophils, into the lung 24 h later. A single antigen challenge produced a marked increase in BAL eosinophils that was apparent at 6 h but increased at 72 h after challenge. The increase at 6 h was largely blocked by dexamethasone. Three antigen challenges produced elevated BAL eosinophil levels that persisted for at least 8 weeks. Eotaxin levels rose dramatically 6 h after challenge and remained the same after 24 h. By 72 h, the eotaxin levels had returned to baseline. The increase in eotaxin at 6 h was nonsignificantly reduced by dexamethasone. Exhaled NO levels doubled 24 h after challenge and were not affected by dexamethasone. CONCLUSIONS: Eotaxin and NO production were increased after airway challenge in allergic monkeys. The rise in NO was not blocked by dexamethasone. The effects of bronchoscopy on the BAL can be avoided by using alternate lungs on consecutive occasions. Eosinophils persist in the BAL for many weeks after antigen challenge.     

Use of egg yolk-derived immunoglobulin as an alternative to antibiotic treatment for control of Helicobacter pylori infection

The present study evaluated the potential use of immunoglobulin prepared from the egg yolk of hens immunized with Helicobacter pylori (immunoglobulin Y [IgY]-Hp) in the treatment of H. pylori infections. The purity of our purified IgY-Hp was 91.3%, with a yield of 9.4 mg of IgY per ml of egg yolk. The titer for IgY-Hp was 16 times higher than that for IgY in egg yolk from nonimmunized hens, and IgY-Hp significantly inhibited the growth and urease activity of H. pylori in vitro. Bacterial adhesion on AGS cells was definitely reduced by preincubation of both H. pylori (10(8) CFU/ml) and 10 mg of IgY-Hp/ml. In Mongolian gerbil models, IgY-Hp decreased H. pylori-induced gastric mucosal injury as determined by the degree of lymphocyte and neutrophil infiltration. Therefore, in this experimental model, H. pylori-associated gastritis could be successfully treated by orally administered IgY-Hp. The immunological activity of IgY-Hp stayed active at 60 degrees C for 10 min, suggesting that pasteurization can be applied to sterilize the product. Fortification of food products with this immunoglobulin would significantly decrease the H. pylori infection. In conclusion, the IgY-Hp obtained from hens immunized by H. pylori could provide a novel alternative approach to treatment of H. pylori infection.     

Should we screen for globin gene mutations in blood samples with mean corpuscular volume (MCV) greater than 80 fL in areas with a high prevalence of thalassaemia?

AIMS: To investigate whether it is worthwhile, in areas where thalassaemia is common, to screen for globin gene mutations in subjects with a mean corpuscular volume (MCV) above 80 fL, especially in partners of known thalassaemia carriers. METHODS: Blood samples from 95 subjects with MCV between 80 and 85 fL were screened for the presence of alpha globin gene mutations and the haemoglobin (Hb) E mutation. RESULTS: Thirty four subjects harboured globin gene mutations. Of these, 31 had deletions of one alpha globin gene, one had Hb Constant Spring, and three had Hb E mutations. CONCLUSION: Based on the above figures and known prevalence rates of thalassaemia carriers, it would seem worthwhile to screen for globin gene mutations in partners of known thalassaemia carriers, regardless of MCV, to identify pregnancies at risk of Hb H disease or Hb E/beta thalassaemia.     

Comparison of haemoglobin H inclusion bodies with embryonic zeta globin in screening for alpha thalassaemia.

AIMS--To compare the haemoglobin (Hb) H inclusion test with immunocytochemical detection of embryonic zeta chains in screening for alpha thalassaemia. METHODS--Blood samples from 115 patients with relevant clinical history and hypochromic microcytic indexes were screened using the HbH inclusion test and the Variant Hemoglobin Testing System (BioRad, Hercules, CA, USA). RESULTS--The HbH inclusion test was positive in 61 of 115 cases, three of whom had HbH disease confirmed by electrophoresis. The remaining 58 had alpha thalassaemia 1. All three HbH cases and 56 of 58 cases of alpha thalassaemia 1 expressed embryonic zeta chains, giving a specificity of 96.7%. Fifty four of 115 cases had a negative HbH inclusion test, of whom 50 had beta thalassaemia trait and three had iron deficiency. No diagnosis was reached for the remaining patient. CONCLUSION--The immunocytochemical test is as sensitive as the HbH inclusion test in screening for alpha thalassaemia. The presence of zeta chains is highly specific for alpha thalassaemia 1 incorporating the (--/SEA) deletion. The specificity and simplicity of the immunocytochemical test make it the test of choice in screening for alpha thalassaemia.     

Role of hydrophobic interaction in the enantioselective solvolyses of amino acid p-nitrophenyl esters by optically active imidazole-containing polymers

Solvolytic reactions of N-benzyloxycarbonyl-D -phenylalanine p-nitrophenyl ester and N-benzyloxycarbonyl-L -phenylalanine p-nitrophenyl ester by optically active imidazole-containing polymers were performed at different temperatures and in aq. ethanol of different water contents. The catalyst polymers employed were homopolymers of N-methacryloyl-L -histidine ( 1a ) and N-methacryloyl-L -histidine methyl ester ( 1b ), as well as copolymers of 1a with dodecyl methacrylate (DMA) and 1b with DMA. In 30 vol. -% aqueous ethanol at pH 7,02 the homopolymers did not show any enantioselective catalysis. However, the copolymers did exhibit enantioselective catalysis, viz., k_cat(L )/k_cat(D ) = 1,25 for poly ( 1b -co-DMA) containing 5,7 mol-% of DMA. As the reaction temperature was lowered, the reaction rate increased and the enantioselectivity was enhanced (k_cat(L )/k_cat(D ) = 1,67 for poly ( 1b -co-DMA) at 10°C). When the ethanol content was decreased, enhanced reaction rates and enantioselectivity (k_cat(L )/k_cat(D ) = 1,65 for poly ( 1 b -co-DMA) in 20 vol.-% aqueous ethanol) were observed. From these results it is concluded that hydrophobic interaction plays an important role in the enantioselective catalysis of optically active imidazole-containing polymers.     

Image reconstruction of anisotropic conductivity tensor distribution in MREIT: computer simulation study

We describe a novel method of reconstructing images of an anisotropic conductivity tensor distribution inside an electrically conducting subject in magnetic resonance electrical impedance tomography (MREIT). MREIT is a recent medical imaging technique combining electrical impedance tomography (EIT) and magnetic resonance imaging (MRI) to produce conductivity images with improved spatial resolution and accuracy. In MREIT, we inject electrical current into the subject through surface electrodes and measure the z-component Bz of the induced magnetic flux density using an MRI scanner. Here, we assume that z is the direction of the main magnetic field of the MRI scanner. Considering the fact that most biological tissues are known to have anisotropic conductivity values, the primary goal of MREIT should be the imaging of an anisotropic conductivity tensor distribution. However, up to now, all MREIT techniques have assumed an isotropic conductivity distribution in the image reconstruction problem to simplify the underlying mathematical theory. In this paper, we firstly formulate a new image reconstruction method of an anisotropic conductivity tensor distribution. We use the relationship between multiple injection currents and the corresponding induced Bz data. Simulation results show that the algorithm can successfully reconstruct images of anisotropic conductivity tensor distributions. While the results show the feasibility of the method, they also suggest a more careful design of data collection methods and data processing techniques compared with isotropic conductivity imaging.     

N-methylated beta-carbolines protect PC12 cells from cytotoxic effect of MPP+ by attenuation of mitochondrial membrane permeability change

Opening of the mitochondrial permeability transition pore has been recognized to be involved in cell death. The present study investigated the effect of beta-carbolines (harmaline and harmalol) on the MPP(+)-induced change in the mitochondrial membrane permeability and cell death in differentiated PC12 cells. beta-Carbolines and antioxidants (superoxide dismutase, catalase, ascorbate or rutin) prevented the loss of cell viability in PC12 cells treated with 250 microM MPP(+), while the effects of N-acetylcysteine and dithiothreitol were not observed. beta-Carbolines reduced the condensation and fragmentation of nuclei caused by MPP(+) in PC12 cells. beta-Carbolines alone did not exhibit a significant cytotoxic effect on PC12 cells. beta-Carbolines (50 microM) inhibited the decrease in mitochondrial transmembrane potential, cytochrome c release, activation of caspase-3, formation of reactive oxygen species (ROS) and depletion of GSH caused by MPP(+) in PC12 cells. beta-Carbolines reduced the hydrogen peroxide- or SIN-1-induced cell death in PC12 cells. The results suggest that beta-carbolines may attenuate the MPP(+)-induced viability loss in PC12 cells by inhibition of change in the mitochondrial membrane permeability and by antioxidant effect.     

Protein synthesis is required for the enhancement of long-term potentiation and long-term memory by spaced training

Spaced training is generally more effective than massed training for learning and memory, but the molecular mechanisms underlying this trial spacing effect remain poorly characterized. One potential molecular basis for the trial spacing effect is the differential modulation, by distinct temporal patterns of neuronal activity, of protein synthesis-dependent processes that contribute to the expression of specific forms of synaptic plasticity in the mammalian brain. Long-term potentiation (LTP) is a type of synaptic modification that may be important for certain forms of memory storage in the mammalian brain. To explore the role of protein synthesis in the trial spacing effect, we assessed the protein synthesis dependence of hippocampal LTP induced by 100-Hz tetraburst stimulation delivered to mouse hippocampal slices in either a temporally massed (20-s interburst interval) or spaced (5-min interburst interval) fashion. To extend our studies to the behavioral level, we trained mice in fear conditioning using either a massed or spaced training protocol and examined the sensitivity of long-term memory to protein synthesis inhibition. Larger LTP was induced by spaced stimulation in hippocampal slices. This improvement of synaptic potentiation following temporally spaced synaptic stimulation in slices was attenuated by bath application of an inhibitor of protein synthesis. Further, the maintenance of LTP induced by spaced synaptic stimulation was more sensitive to disruption by anisomycin than the maintenance of LTP elicited following massed stimulation. Temporally spaced behavioral training improved long-term memory for contextual but not for cued fear conditioning, and this enhancement of memory for contextual fear was also protein synthesis dependent. Our data reveal that altering the temporal spacing of synaptic stimulation and behavioral training improved hippocampal LTP and enhanced contextual long-term memory. From a broad perspective, these results suggest that the recruitment of protein synthesis-dependent processes important for long-term memory and for long-lasting forms of LTP can be modulated by the temporal profiles of behavioral training and synaptic stimulation.