Phillip D. Bowman

cDNA microarray reveals altered cytoskeletal gene expression in space-flown leukemic T lymphocytes (Jurkat)

Cytoskeletal disruption and growth arrest consistently occur in space‐flown human acute leukemic T cells (Jurkat). Although the microtubules appear to reorganize during spaceflight, cells remain nonproliferative. To test the hypothesis that spaceflight alters cytoskeletal gene expression and may thus affect cytoskeletal function, we flew Jurkat cells on Space Transportation System (STS) 95 and compared RNA message by cDNA microarray in space‐flown vs. ground controls at 24 h (4,324 genes) and 48 h (>20,000 genes). Messages for 11 cytoskeleton‐related genes, including calponin, dynactin, tropomodulin, keratin 8, two myosins, an ankyrin EST, an actinlike protein, the cytoskeletal linker (plectin), and a centriole‐associated protein (C‐NAP1), were up‐regulated in space‐flown compared with ground control cells; gelsolin precursor was down‐regulated. Up‐regulation of plectin and C‐NAP1 message in both space‐flown cells and vibrated controls is a novel finding and implies their role in vibration damage repair. This first report of cDNA microarray screening of gene expression in space‐flown leukemic T cells also identifies differential expression of genes that regulate growth, metabolism, signal transduction, adhesion, transcription, apoptosis, and tumor suppression. Based on differential expression of cytoskeletal genes, we conclude that centriole‐centriole, membrane‐cytoskeletal, and cytoskeletal filament associations are altered in the orbital phase of spaceflight.

Apoptosis and accidental cell death in cultured human keratinocytes after thermal injury.

The respective roles of apoptosis and accidental cell death after thermal injury were evaluated in normal human epidermal keratinocytes. By coupling the LIVE/DEAD fluorescence viability assay with the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method and ultrastructural morphology, these two processes could be distinguished. Cells were grown on glass coverslips with a microgrid pattern so that the results of several staining procedures performed sequentially could be visualized in the same cells after heating at temperatures of up to 72 degrees C for 1 second. After exposure to temperatures of 58 to 59 degrees C, cells died predominantly by apoptosis; viable cells became TUNEL positive, indicating degradation of DNA. After exposure to temperatures of 60 to 66 degrees C, both TUNEL-positive viable cells and TUNEL-positive nonviable cells were observed, indicating that apoptosis and accidental cell death were occurring simultaneously. Cells died almost immediately after exposure to temperatures above 72 degrees C, presumably from heat fixation. The fluorescent mitochondrial probe MitoTracker Orange indicated that cells undergoing apoptosis became TUNEL positive before loss of mitochondrial function. Nucleosomal fragmentation of DNA analyzed by enzyme-linked immunosorbent assay and gel electrophoresis occurred after exposure to temperatures of 58 to 59 degrees C. The characteristic morphological findings of cells undergoing apoptosis, by transmission electron microscopy, included cellular shrinkage, cytoplasmic budding, and relatively intact mitochondria. Depending on temperature and time of exposure, normal human epidermal keratinocytes may die by apoptosis, accidental cell death, or heat fixation.

Liver and Kidney Injury After Administration of Hemoglobin Cross-Linked with Bis(3,5-Dibromosalicyl) Fumarate

Human hemoglobin cross-linked between the alpha chains with bis (3,5-dibromosalicyl) fumarate (DBBF-Hb) was exchange transfused in swine and the histomorphologic changes were evaluated. Following exchange, animals were euthanized and tissues were taken for light and electron microscopy at 7.5 hours and days 1, 4, 7, and 15. Consistent hepatocellular and renal epithelial cell changes were seen. Hepatic injury, evident at 7.5 hours as cellular vacuolization, progressed to necrosis and acute inflammatory cell infiltration by days 1 and 4, was resolving by 7 days and was completely resolved by day 15. Cytochemical stains for iron and hemoglobin revealed positive material in Kupffer cells, endothelial cells, and necrotic hepatocytes. Rabbit anti-human hemoglobin antibody staining revealed immunoreactive material diffusely present at days 1 and 4 and limited to solitary hepatocytes by day 15. Kidney injury began as proximal tubular epithelial vacuolization and intraluminal casts progressing to tubular necrosis by 24 hours, with resolution by day 15. Iron and hemoglobin stains demonstrated these materials in the early lesions. Immunocytochemistries demonstrated human hemoglobin that remained as late as day 15. Electron microscopy revealed degeneration and regeneration of epithelial cells. The renal lesions were consistent with hemoglobinuria. The liver lesion was less well defined but was self limited.

Synthesis of a series of caffeic acid phenethyl amide (CAPA) fluorinated derivatives: Comparison of cytoprotective effects to caffeic acid phenethyl ester (CAPE)

A series of catechol ring-fluorinated derivatives of caffeic acid phenethyl amide (CAPA) were synthesized and screened for cytoprotective activity against H2O2 induced oxidative stress in human umbilical vein endothelial cells (HUVEC). CAPA and three fluorinated analogs were found to be significantly cytoprotective when compared to control, with no significant difference in cytoprotection between caffeic acid phenethyl ester (CAPE) and CAPA.

Toxicity of Aluminum Silicates Used in Hemostatic Dressings Toward Human Umbilical Veins Endothelial Cells, HeLa Cells, and RAW267.4 Mouse Macrophages

BACKGROUND Aluminum silicates have been used to control bleeding after severe traumatic injury. QuikClot (QC) was the first such product, and WoundStat (WS) is the most recent. We recently observed that WS caused vascular thrombosis when applied to stop bleeding. This study investigated the cellular toxicity of WS in different cell types that may be exposed to this mineral and compared the results with other minerals such as bentonite, kaolin, and QuikClot ACS+ (QC+). METHODS Human umbilical vein endothelial cells (HUVEC), HeLa cells, and RAW267.4 mouse macrophage-like cells (RAW) were incubated directly with different concentrations of each mineral for 24 hours. Cell viability was determined metabolically using the AlamarBlue fluorescent technique. In another experiment, minerals were exposed to HUVEC via Transwell inserts with a polycarbonate filter (0.4-μm pore size) to prevent direct contact between cells and minerals for determining whether direct exposure or leaching compounds from minerals cause cytotoxicity. RESULTS Incubation of HUVEC and RAW cells with 1 to 100 μg/mL of the minerals for 24 hours resulted in differential toxicities. The cytotoxicity of WS was equal to that of bentonite and higher than kaolin and QC+. Neither cell type survived for 24 hours in the presence of 100 μg/mL WS or bentonite. These minerals, however, had little effect on the viability of HeLa cells. In the second HUVEC experiment, a 10 times higher concentration of these compounds placed in Transwell inserts yielded no decrease in cell viability. This result indicates that leaching toxicants or binding of nutrients by the ion-exchange properties of minerals did not cause the toxicity. CONCLUSIONS Although aluminum silicates seem relatively innocuous to epithelial cells, all produced some toxicity toward endothelial cells and macrophages. WS and bentonite were significantly more toxic than kaolin and zeolite present in QC+, respectively, at equivalent doses. The cytotoxic effect seemed to be caused by the direct contact of the minerals with the cells present in wounds. These data suggest that the future clearance of mineral-based hemostatic agents should require more extensive cytotoxicity testing than the current Food and Drug Administration requirements.

Myopathic Alterations in Extraocular Muscle of Rats Subchronically Fed Pyridostigmine Bromide

To determine if alterations in extraocular muscle morphology occur after subchronic oral administration of pyridostigmine bromide, rats were continuously fed 90 mg/kg in meal and examined at 1,2, 4, 7, and 15 days. Within the first day, blood acetylcholinesterase activity was reduced by 87% and remained inhibited by 74-91% during the study. Light microscopy demonstrated that by day 1 approximately 3% of the extraocular myofibers were shrunken and invaded by inflammatory cells. The most severe degenerative changes consisting of vacuoles and inflammatory cell infiltration occurred at day 1 with progressively less severe changes at days 2 and 4. At days 7 and 15,1.3-4.5% of the myofibers still exhibited damage. Ultrastructurally, all presynaptic areas were normal but the postsynaptic areas of affected myofibers at days 1,2, and 4 showed myofilament and Z-band dissolution, mitochondrial inclusions, subneural fold and T-tubule/sarcoplasmic reticulum vacuolization and subneural fold depth reduction. By days 7 and 15, these changes were diminished in some cases and in others alterations appeared similar to day 1. We conclude that subchronic feeding of pyridostigmine bromide induces myopathic rather than neurogenic changes in rat extraocular muscle and that the myopathy is different in these muscles than in the diaphragm from the same rats.

Quantitative Determination of Fluorinated Caffeic Acid Phenethyl Ester Derivative From Rat Blood Plasma by Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry

The quantitative determination of caffeic acid phenethyl ester (CAPE) and its fluorinated derivative (FCAPE) from rat plasma using ultra-performance liquid chromatography with electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) is reported. CAPE and FCAPE were extracted using ethyl acetate in the presence of methyl caffeate (MC) as internal standard. Separation was achieved using a C(18) column (2.1 mm x 50 mm, 1.7 microm) and gradient elution with water and acetonitrile containing 0.2% and 0.1% formic acid, respectively. A non-linear response over a broad concentration range (1-1000 ng/ml, r(2)>0.995 using a quadratic regression model and 1/concentration weighting) was obtained. The inter-day and intra-day variability for CAPE and FCAPE were found to be less than 14.2% and 9.5%, respectively. Data are presented to illustrate the practicality of the method for the pharmacokinetic evaluation of CAPE and FCAPE after intravenous administration to rats.

Reuse of cDNA Microarrays Hybridized With cRNA by Stripping With RNase H

DNA microarrays are powerful tools for global analysis of gene transcript expression. However, their high cost and the need for replication have limited their use. Here, we report a new stripping technique applicable to microarrays hybridized with cRNA with RNase H that is reproducible, leaving the DNA oligonucleotide probes intact and available for adding two additional uses. A Pearson correlation was used to assess the agreement between the first-round hybridization and the second- and third-round hybridizations. Significant correlations (R2, 0.9893 and 0.975; P < 0.001) were observed among virgin arrays and stripped arrays hybridized with the same sample. Additionally, statistical class comparison analysis globally indicated that there were essentially no differences detected following three hybridizations. Dye-swapped microarrays produced similar results. However, arrays stripped with RNase H exhibited decreased efficiency of hybridization signal with increasing use. In the present study, the oligonucleotide microarrays can be used three times.

MORPHOLOGIC ALTERATIONS IN RAT RETINA AFTER HYPERVOLEMIC INFUSION OF CROSS-LINKED HEMOGLOBIN*

Hypervolemic infusion in rats of bis (3,5-dibromosalicyl) fumarate cross-linked hemoglobin (DBBF-Hb) to 40-60% of blood volume produced histologic lesions in retina which were not observed in rats similarly infused with human serum albumin or lactated Ringers solution. Rats treated with 40% DBBF-Hb, exhibited intermittent zones of dense retinal pigmented epithelium while 60% DBBF-Hb animals exhibited severe inner retinal edema and retinal pigmented epithelium vacuolization, large focal zones of photoreceptor outer segment disruption and in one animal, subretinal hemorrhage. Light microscopic immunocytochemical evaluation of retinas with antibodies directed to human hemoglobin and albumin, showed the presence of both hemoglobin and albumin in this tissue. Transmission electron microscopy of the lesions demonstrated vacuolated retinal pigmented epithelial cells and large areas of focal photoreceptor outer segment disruption. We conclude that hypervolemic infusion disrupts the blood retinal barrier and that although both DBBF Hb and albumin cross, only hemoglobin produced damage in the retina.

Cytoprotection of Human Endothelial Cells from Oxidant Stress with CDDO Derivatives: Network Analysis of Genes Responsible for Cytoprotection

Aim: To identify drugs that may reduce the impact of oxidant stress on cell viability. Methods: Human umbilical vein endothelial cells were treated with 200 nmol/l CDDO-Im (imidazole) and CDDO-Me (methyl) after exposure to menadione and compared to vehicle-treated cells. Cell viability and cytotoxicity were assessed, and gene expression profiling was performed. Results: CDDO-Im was significantly more cytoprotective and less cytotoxic (p < 0.001) than CDDO-Me. Although both provided cytoprotection by induction of gene transcription, CDDO-Im induced more genes. In addition to a higher induction of the key cytoprotective gene heme oxygenase-1 (38.9-fold increase for CDDO-Im and 26.5-fold increase for CDDO-Me), CDDO-Im also induced greater expression of heat shock proteins (5.5-fold increase compared to 2.8-fold for CDDO-Me). Conclusions: Both compounds showed good induction of heme oxygenase, which largely accounted for their cytoprotective effect. Differences were detected in cytotoxicity at higher doses, indicating that CDDO-Me was more cytotoxic than CDDO-Im. Significant differences were detected in the ability of CDDO-Im and CDDO-Me to affect differential gene transcription. CDDO-Im induced more genes than did CDDO-Me. The source of the differences in gene expression patterns between CDDO-Im and CDDO-Me was not determined but may be important in long-term use of this class of drugs.