Naturally occurring TAP-dependent specific T-cell tolerance for a variant of an immunodominant retroviral cytotoxic T-lymphocyte epitope

Upon immunization and restimulation with tumors induced by the endogenous AKR/Gross murine leukemia virus (MuLV), C57BL/6 mice generate vigorous H-2K(b)-restricted cytotoxic T-lymphocyte (CTL) responses to a determinant (KSPWFTTL) derived from the p15E transmembrane portion of the viral envelope glycoprotein. By contrast, the highly homologous determinant RSPWFTTL, expressed by tumor cells induced by Friend/Moloney/Rauscher (FMR) MuLV, is not immunogenic, even when presented to the immune system as vaccinia virus-encoded cytosolic or endoplasmic reticulum (ER)-targeted minigene products. Such minigene products are usually highly immunogenic since they bypass the need for cells to liberate the peptide or transport the peptide into the ER by the transporter associated with antigen processing (TAP). Using KSPWFTTL-specific CTLs that cross-react with RSPWFTTL, we previously demonstrated that presentation of RSPWFTTL from its natural viral gene product is TAP dependent. Here, we show first that C57BL/6 mice express mRNA encoding RSPWFTTL but not KSPWFTTL and second that the ER-targeted RSPWFTTL minigene product is highly immunogenic in C57BL/6 mice with a targeted deletion in TAP1. These findings provide the initial demonstration of TAP-dependent tolerance induction to a specific self peptide and demonstrate that this contributes to the differential recognition of RSPWFTTL and KSPWFTTL by C57BL/6 mice.     

An Olfactory Stimulus Modifies Nighttime Sleep in Young Men and Women

Aromatherapy is an anecdotal method for modifying sleep and mood. However, whether olfactory exposure to essential oils affects night‐time objective sleep remains untested. Previous studies also demonstrate superior olfactory abilities in women. Therefore, this study investigated the effects of an olfactory stimulus on subsequent sleep and assessed gender differences in such effects. Thirty‐one young healthy sleepers (16 men and 15 women, aged 18 to 30 yr, mean±SD, 20.5±2.4 yr) completed 3 consecutive overnight sessions in a sleep laboratory: one adaptation, one stimulus, and one control night (the latter 2 nights in counterbalanced order). Subjects received an intermittent presentation (first 2 min of each 10 min interval) of an olfactory (lavender oil) or a control (distilled water) stimulus between 23:10 and 23:40 h. Standard polysomnographic sleep and self‐rated sleepiness and mood data were collected. Lavender increased the percentage of deep or slow‐wave sleep (SWS) in men and women. All subjects reported higher vigor the morning after lavender exposure, corroborating the restorative SWS increase. Lavender also increased stage 2 (light) sleep, and decreased rapid‐eye movement (REM) sleep and the amount of time to reach wake after first falling asleep (wake after sleep onset latency) in women, with opposite effects in men. Thus, lavender serves as a mild sedative and has practical applications as a novel, nonphotic method for promoting deep sleep in young men and women and for producing gender‐dependent sleep effects.     

Use of moving aeration membrane bioreactor for the efficient production of tissue type plasminogen activator in serum free medium

A moving aeration-membrane (MAM) bioreactor was employed for the production of 2 μg/mL of tissue type Plasminogen Activator (tPA) in serum free medium from normal human fibroblast cells. This system could maintain high cell density for long periods of steady state conditions in perfusion cultivation. Under normal operating conditions, shear stress was as low as 0.65 dynes/cm² at the agitation speed of 80 rpm. Even though cell density gradually decreased with increasing agitation speed, tPA production increased linearly with increasing shear stress within a moderate range. This culture system allowed production of 2 μg tPA/mL while maintaining a high cell density of 1.0×10⁷ viable cells/mL.     

Induction of Apoptotic Cell Death on Human Cervix Cancer HeLa cells by Extract from <Emphasis Type="Italic">Loranthus yadoriki</Emphasis>

Loranthus yadoriki, one of the Korean mistletoe species, has been already known for anti-viral effects, but the molecular basis that it caused apoptosis in cancer cells was not definitely revealed yet. The aim of this study was to estimate the mechanisms of apoptotic cell death of the extract from Loranthus yadoriki (named as ELY) in human cervix HeLa cells. We identified that ELY prevented the proliferation of HeLa cells between 50 and 300 μg/mL which did not affect non-cancerous HaCaT cells. In addition, ELY induced a morphological change and nucleus disruption as well as an accumulation of sub-G1 phase in HeLa cells. The mechanism study, by using western blot analysis, showed that the phosphorylation of Fas-associated death domain (FADD), Bim and Bak was up-regulated by ELY treatment. Furthermore, the expression of cytochrome c and Apaf-1 was increased by ELY treatment. In immunofluorescence staining, the increased intensity of cleaved caspase-3 and cleaved PARP was also observed under ELY treatment. Sequentially, the caspase cascade was activated by ELY from caspase-8 to caspase-3 and from caspase-9 to caspase-3, in both extrinsic and intrinsic pathways. The results of this study demonstrate that ELY has anti-cancer effects on human cervix cancer HeLa cells via caspase cascade in apoptotic signaling pathways.     

The highly stable alcohol dehydrogenase of Thermomicrobium roseum: purification and molecular characterization

An alcohol dehydrogenase (ADH) was purified to electrophoretic homogeneity from an extremely thermophilic bacterium, Thermomicrobium roseum. The native enzyme was found to be a homo-dimer of 43-kDa subunits. The pI of the enzyme was determined to be 6.2, while its optimum pH is 10.0. The enzyme oxidized mainly primary aliphatic alcohols and exhibited high substrate specificity towards ethanol, n-propanol and crotyl alcohol. The highest reaction rate was observed when ethanol was used as substrate and the K(m) value of the enzyme for ethanol was 24.2 mM. Pyrazole notably inhibited the enzymatic activity. The enzyme had the optimal temperature of 70 degrees C and was highly stable against high temperature.     

EGCG, a green tea polyphenol, improves endothelial function and insulin sensitivity, reduces blood pressure, and protects against myocardial I/R injury in SHR

Epigallocatechin gallate (EGCG), a bioactive polyphenol in green tea, may augment metabolic and vascular actions of insulin. Therefore, we investigated effects of EGCG treatment to simultaneously improve cardiovascular and metabolic function in spontaneously hypertensive rats (SHR; model of metabolic syndrome with hypertension, insulin resistance, and overweight). In acute studies, EGCG (1-100 microM) elicited dose-dependent vasodilation in mesenteric vascular beds (MVB) isolated from SHR ex vivo that was inhibitable by N(omega)-nitro-L-arginine methyl ester (L-NAME; nitric oxide synthase antagonist) or wortmannin [phosphatidylinositol (PI) 3-kinase inhibitor]. In chronic studies, 9-wk-old SHR were treated by gavage for 3 wk with EGCG (200 mg.kg(-1).day(-1)), enalapril (30 mg.kg(-1).day(-1)), or vehicle. A separate group of SHR receiving L-NAME (80 mg/l in drinking water) was treated for 3 wk with either EGCG or vehicle. Vasodilator actions of insulin were significantly improved in MVB from EGCG- or enalapril-treated SHR (when compared with vehicle-treated SHR). Both EGCG and enalapril therapy significantly lowered systolic blood pressure (SBP) in SHR. EGCG therapy of SHR significantly reduced infarct size and improved cardiac function in Langendorff-perfused hearts exposed to ischemia-reperfusion (I/R) injury. In SHR given L-NAME, beneficial effects of EGCG on SBP and I/R were not observed. Both enalapril and EGCG treatment of SHR improved insulin sensitivity and raised plasma adiponectin levels. We conclude that acute actions of EGCG to stimulate production of nitric oxide from endothelium using PI 3-kinase-dependent pathways may explain, in part, beneficial effects of EGCG therapy to simultaneously improve metabolic and cardiovascular pathophysiology in SHR. These findings may be relevant to understanding potential benefits of green tea consumption in patients with the metabolic syndrome.     

A role of retinoic acid in the regulation of the morphology and the levels of intermediate filament proteins and mRNAs in PC12 cells.

An adrenal tumor-derived cell line (PC12W) cultured in the presence of nerve growth factor exhibited a spindle-shaped cell morphology resembling neuronal cells. The shape of these cells can be specifically changed in vitamin A-depleted medium supplemented with retinoic acid. Retinoic acid promoted an epithelial-like cell morphology except for occasional neuronal processes. These morphological results were correlated with differential expression of intermediate filaments at the mRNA and protein levels in these cells. Retinoic acid suppressed the synthesis of peripherin, an intermediate filament protein predominantly found in peripheral nerve cells, but a high level of simple keratins, normally found in simple epithelial cells, was present in retinoic acid-treated PC12 cells. The neurofilaments typically expressed in neurons remained virtually unaffected under the same conditions. In contrast, nerve growth factor induced the production of neurofilaments, but suppressed the synthesis of simple keratins. Since intermediate filament expression is known to be tissue-specific, these changes in expression together with the cell morphology changes are consistent with PC12 cells undergoing an epithelial-like differentiation in the presence of retinoic acid and a neuronal-like differentiation in the presence of nerve growth factor. These results suggest that retinoic acid and nerve growth factor are both effective regulators of PC12 cell differentiation but stimulate opposing pathways.