Rapid cell corpse clearance by stabilin-2, a membrane phosphatidylserine receptor.

Rapid phagocytic clearance of apoptotic cells is crucial for the prevention of both inflammation and autoimmune responses. Phosphatidylserine (PS) at the external surface of the plasma membrane has been proposed to function as a general 'eat me' signal for apoptotic cells. Although several soluble bridging molecules have been suggested for the recognition of PS, the PS-specific membrane receptor that binds directly to the exposed PS and provides a tickling signal has yet to be definitively identified. In this study, we provide evidence that stabilin-2 is a novel PS receptor, which performs a key function in the rapid clearance of cell corpses. It recognizes PS on aged red blood cells and apoptotic cells, and mediates their engulfment. The downregulation of stabilin-2 expression in macrophages significantly inhibits phagocytosis, and anti-stabilin-2 monoclonal antibody provokes the release of the anti-inflammatory cytokine, transforming growth factor-beta. Furthermore, the results of time-lapse video analyses indicate that stabilin-2 performs a crucial function in the rapid clearance of aged and apoptotic cells. These data indicate that stabilin-2 is the first of the membrane PS receptors to provide tethering and tickling signals, and may also be involved in the resolution of inflammation and the prevention of autoimmunity.

Technical note: Improved extraction method with hexane for gas chromatographic analysis of conjugated linoleic acids.

Extraction properties of different solvents (chloroform/methanol, hexane/isopropanol, and hexane) were studied for the gas chromatographic analysis of conjugated linoleic acids (CLA) from probiotic bacteria grown in de Man, Rogosa, and Sharpe medium. As compared with chloroform/methanol and hexane/isopropanol, hexane showed comparable extraction efficiency for CLA from unspent de Man, Rogosa, and Sharpe medium, but showed minimal extraction of oleic acid originated from the emulsifier in broth. The extraction efficiency of CLA by hexane was influenced by the broth pH, showing the optimal pH of 7.0. Repeated extraction with hexane increased the yield. Extraction with hexane showed excellent recovery of spiked CLA from the spent broth with up to 97.2% (standard deviation of 1.74%). This represents the highest recovery of CLA from culture broth ever reported. The sample size was also successfully reduced to 0.5 mL to analyze CLA from the broth without impairment of analytical data. This smaller sample size in the 1.5-mL microcentrifuge tube using a small bench-top centrifuge reduced analytical time significantly.

Chemopreventive allylthiopyridazine derivatives induce apoptosis in SK-Hep-1 hepatocarcinoma cells through a caspase-3-dependent mechanism.

Dietary organosulphur compounds including diallylsulphide, a component of garlic oil, were shown to inhibit the proliferation of tumour cells. Since hepatocellular carcinoma is one of the most lethal malignancies and there is no effective preventive measure to date, we wished to pursue the chemopreventive potential of the synthetic allylthiopyridazine derivatives (K compounds) on hepatocarcinoma cells. Here, we report that the K compounds efficiently inhibited SK-Hep-1 cell proliferation through induction of apoptosis. Increased chain length at the 3-position of allylthiopyridazine ring improved the potency of growth inhibition. K compounds downregulated Bcl-2, while Bax remained unchanged, reducing the ratio of Bcl-2 to Bax. We also provide evidence that the K compound-induced apoptosis involves cytochrome c release and caspase-3 activation. These results suggest that the allythiopyridazine derivatives, especially 3-propoxy-6-allylthiopyridazine, induce apoptosis in SK-Hep-1 cells through a caspase-3-dependent mechanism, which may contribute to the chemopreventive function for hepatocellular carcinoma.

Effects of temperature and agitation rate on the formation of conjugated linoleic acids in soybean oil during hydrogenation process.

The effects of hydrogen temperature and agitation rate on the formation of total conjugated linoleic acids (CLA) and CLA isomers were studied during hydrogenation with a selective Ni catalyst. The CLA isomers were identified by using a 100-m cyano-capillary column gas chromatograph and a silver ion-impregnated HPLC. Reaction temperature and agitation rate greatly affected the quantities of total CLA and individual CLA isomers, and the time to reach the maximum quantity of CLA in the partially hydrogenated soybean oil. As the hydrogenation temperature increased, the maximum quantity of CLA in soybean oil increased, but the time to reach the maximum CLA content decreased. By increasing the hydrogenation temperature from 170 to 210 degrees C, the quantity of CLA obtained was about 2.6 times higher. As the agitation rate decreased, the CLA formation in soybean oil increased, and the time to reach the maximum CLA content also increased. The maximum CLA contents in soybean oil obtained during hydrogenation at 210 degrees C with agitation rates of 300, 500, and 700 rpm were 162.82, 108.62, and 66.15 mg total CLA/g oil, respectively. The present data showed that it is possible to produce high-CLA-content soybean oil without major modification of fatty acid composition by short-time (10 min) selective hydrogenation under high temperature and low agitation rate conditions.

Capsaicin-induced apoptosis in SK-Hep-1 hepatocarcinoma cells involves Bcl-2 downregulation and caspase-3 activation.

Hepatocellular carcinoma is one of the most lethal malignancies and there is no effective preventive measure in this highly malignant disease to date. In the present study, we investigated the chemopreventive potential of capsaicin (8-methyl-N- vanillyl-6-nonenamide), the principal pungent ingredient found in hot red pepper, in SK-Hep-1 hepatocellular carcinoma cells. Treatment of capsaicin inhibited growth of SK-Hep-1 cells in a concentration-dependent manner while 4-methoxy capsaicin (Met-capsaicin) was less potent. This inhibitory effect of capsaicin on SK-Hep-1 cell growth was mainly due to the induction of apoptosis as evidenced by DNA fragmentation and nuclear condensation. Furthermore, capsaicin prominently reduced the ratio of anti-apoptotic Bcl-2 to pro-apoptotic Bax and consequently increased caspase-3 activity. These results demonstrate that capsaicin efficiently induced apoptosis in SK-Hep-1 cells through a caspase-3-dependent mechanism, which may contribute to its chemopreventive function.

Riboflavin-sensitized photochemical changes in beta-lactoglobulin in an aqueous buffer solution as affected by ascorbic acid.

The effects of ascorbic acid on the riboflavin-sensitized photochemical changes in beta-lactoglobulin in an aqueous buffer solution as determined by high performance gel permeation liquid chromatography (HPGPLC), insoluble protein content, and individual amino acid content during fluorescent light illumination were studied. The riboflavin-sensitized photochemical degradation of beta-lactoglobulin was effectively inhibited by ascorbic acid, and its inhibitory effectiveness was concentration dependent. The 0.1% ascorbic acid treatment showed 74.4% inhibition of beta-lactoglobulin degradation as determined by a HPGPLC during 6 h light illumination. Insolubility of beta-lactoglobulin in a buffer solution during light illumination was also effectively decreased by ascorbic acid treatment. The riboflavin-sensitized photochemical reduction of cysteine, histidine, lysine, methionine, and tryptophan in beta-lactoglobulin was high during 6 h fluorescent light illumination. The 0.1% ascorbic acid treatment exhibited 20.8% inhibition of total amino acid degradation in beta-lactoglobulin during 6 h light illumination, showing strong inhibitory activity against the degradation of arginine, aspartic acid, cystein, glycine, histidine, phenylalanine, proline, serine, and tryptophan.

Antiphotooxidative activity of protoberberines derived from Coptis japonica makino in the chlorophyll-sensitized photooxidation of oil.

Antiphotooxidative components were isolated from the methanolic extract of Coptis japonica Makino by liquid-liquid partitioning fractionation, subsequent column chromatography on Sephadex LH-20 and silica gel, and preparative silica gel TLC. The isolated compounds were identified as coptisine, jatrorrizhine, berberine, and magnoflorine by a combination of spectroscopic studies using UV-visible, IR, mass-spectrometry, and NMR. Coptisine, jatrorrizhine, and berberine isolated from Coptis japonica Makino showed strong antiphotooxidative activity in the chlorophyll-sensitized photooxidation of linoleic acid. However, these compounds did not show either inhibitory activity against lipid peroxidation in rat liver microsomes nor DPPH radical scavenging activity, indicating that their antiphotooxidative activity was not due to the radical chain reaction breaking ability but due to singlet oxygen quenching activity. Commercially available authentic protoberberines (berberine chloride and palmatine chloride) also showed strong antioxidative activity in the chlorophyll-sensitized photooxidation of linoleic acid. The antiphotooxidative activities of the berberine chloride and palmatine chloride were significantly higher than that of ascorbyl palmitate in the chlorophyll-sensitized photooxidation of linoleic acid. These results clearly showed for the first time the antiphotooxidative properties of protoberberines in chlorophyll-sensitized photooxidation of oil.

Conjugated linoleic acid isomers in partially hydrogenated soybean oil obtained during nonselective and selective hydrogenation processes.

Partially hydrogenated soybean oil samples were collected during selective and nonselective hydrogenation processes. The formation of conjugated linoleic acids (CLAs) during hydrogenation was greatly dependent on the types and duration of hydrogenation processes. During hydrogenation processes, CLA contents increased initially. After reaching maximum CLA content, the content decreased during hydrogenation. Selective hydrogenation was much more favorable for the formation of conjugated linoleic acids. With nonselective hydrogenation process, the total CLA content was a maximum (9.06 mg total CLA/g oil) at 35 min. However, with the selective hydrogenation process, the total CLA content was a maximum (98.27 mg total CLA/g oil) at 210 min. The CLA contents in some of the tested selectively hydrogenated soybean oils were among the highest ever reported in foods.

Effects of roasting on pyrazine contents and oxidative stability of red pepper seed oil prior to its extraction.

Red pepper seeds were roasted with constant stirring for 6, 9, 10, and 12 min at 210 degrees C, and oils were extracted from the roasted red pepper seeds using an expeller. The iodine values and fatty acid compositions of red pepper seed oils did not change with roasting time. The fatty acid composition of the oil obtained from the red pepper seeds roasted for 6 min was 0.24% myristic acid, 13. 42% palmitic acid, 0.33% palmitoleic acid, 2.07% stearic acid, 10. 18% oleic acid, 73.89% linoleic acid, and 0.37% linolenic acid, showing a fatty acid composition similar to that of high-linoleate safflower oil. Thirteen alkylpyrazines were identified in the roasted red pepper seed oils: 2-methylpyrazine, 2,5-dimethylpyrazine, 2,6-dimethylpyrazine, 2-ethylpyrazine, 2-ethyl-6-methylpyrazine, 2-ethyl-5-methylpyrazine, trimethylpyrazine, 2,6-diethylpyrazine, 2-ethyl-3,5-dimethylpyrazine, tetramethylpyrazine, 2, 3-diethyl-5-methylpyrazine, 2-isobutyl-3-methylpyrazine, and 3, 5-diethyl 2-methylpyrazine. The pyrazine content increased markedly as the roasting time increased, showing 2.63, 5.01, 8.48, and 13.10 mg of total pyrazine/100 g of oils from the red pepper seeds roasted for 6, 8, 10, and 12 min, respectively, at 210 degrees C. 2, 5-Dimethylpyrazine in the roasted red pepper seed oil seemed to be the component most responsible for the pleasant nutty aroma of the oils. The oxidative stabilities of oils increased greatly as the roasting time increased.

Decreased c-fos responses to dopamine D(1) receptor agonist stimulation in mice deficient for D(3) receptors.

The acute administration of dopamine D(1) receptor agonists induces the expression of the immediate early gene c-fos. In wild type mice, this induction is completely abolished by pretreatment with the D(1)-selective antagonist SCH23390, and pretreatment with the D(2)-like receptor antagonist eticlopride reduces the levels of c-fos expressed in response to D(1) receptor stimulation. Mice deficient for the dopamine D(3) receptor express levels of D(1) agonist-stimulated c-fos immunoreactivity that are lower than c-fos levels of their wild type littermates. Moreover, the acute blockade of D(2) receptors in D(3) mutant mice further reduces c-fos expression levels. These data indicate that the basal activity of both D(2) and D(3) receptors contributes to D(1) agonist-stimulated c-fos responses. The findings therefore indicate that not only D(2) but also D(3) receptors play a role in dopamine-regulated gene expression.

Proteolytic processing is required for viral superantigen activity.

The mouse mammary tumor virus-7 superantigen (vSAG7) is proteolytically processed in B cells at as many as three positions. Proteolytic processing appears to be important for superantigen activity because a processed form of vSAG7 was predominant among those forms that were found to bind to major histocompatibility complex class II molecules. To determine the functional significance of proteolytic processing, a mutation was introduced in vSAG7 at one of the sites where proteolytic cleavage is thought to take place in B cells. Elimination of the putative processing site at position 171 abrogated detectable vSAG7 surface expression in B cells, indicating that proteolytic processing is required for vSAG7 function. Coexpression in insect cells of vSAG7 and furin, a proprotein-processing enzyme, also demonstrated that furin could process vSAG7 at position 171.