Entrapment of Ovalbumin into Liposomes—Factors Affecting Entrapment Efficiency,Liposome Size,and Zeta Potential

Various amounts of Ovalbumin (OVA) were encapsulated into positively and negatively charged multilamellar liposomes, with the aim to investigate the entrapment efficiency in different buffers and to study their effects on the liposome size and zeta potential. Results showed that the entrapment efficiency of OVA in anionic liposomes was the same in 10 mM Phosphate Buffer (PB) as in Phosphate-Buffered Saline (PBS; PB?+?0.15 M NaCl). Also, liposome size was approximately 1200 nm for all anionic liposomes incorporating OVA. The entrapment efficiency of OVA in cationic liposomes was highly dependent on ionic strength. The size of cationic liposomes was approximately 1200 nm in PBS, regardless of protein content, but increased with the amount of the incorporated protein in PB. Aggregation of cationic liposomes in PB was observed when the mass of the protein was 2.5 mg or greater. The zeta potential of anionic liposomes was negative and of cationic liposomes positive in the whole range of protein mass tested. These results show how different compositions of lipid and aqueous phases can be used to vary the entrapment efficiency, liposome size, and zeta potential—the factors that are of great importance for the use of liposomes as drug carriers.     

Effects of caffeic acid phenethyl ester (CAPE) on angiogenesis,apoptosis and oxidatıve stress ın various cancer cell lines

ABSTRACT

Cancer is a common cause of death worldwide. Approximately 80% of cancer patients use complementary or alternative medicines for treatment. Caffeic acid phenethyl ester (CAPE), the main active component of propolis, exhibits cytotoxic, antiproliferative and anti-cancer effects. Despite its anticancer effects CAPE exhibits no known harmful effects toward normal cells. We investigated the effects of CAPE on angiogenesis, apoptosis and oxidative stress using MDA MB-231, N2a and COLO 320 cell lines and CAPE treatments at 24 and 48 h. A two dimensional cell culture system was used and the findings were evaluated by an indirect immunohistochemical method and H-scores were calculated. CAPE was effective for all three cancer cell lines. After 24 and 48 h, we found a significant decrease in live cells and increased stress in the cells based on e-NOS and i-NOS levels.     

High relative air humidity and continuous light reduce stomata functionality by affecting the ABA regulation in rose leaves

Plants developed under high (90%) relative air humidity (RH) have previously been shown to have large, malfunctioning stomata, which results in high water loss during desiccation and reduced dark induced closure. Stomatal movement is to a large extent regulated by abscisic acid (ABA). It has therefore been proposed that low ABA levels contribute to the development of malfunctioning stomata. In this study, we investigated the regulation of ABA content in rose leaves, through hormone analysis and β‐glucosidase quantification. Compared with high RH, rose plants developed in moderate RH (60%) and 20 h photoperiod contained higher levels of ABA and β‐glucosidase activity. Also, the amount of ABA increased during darkness simultaneously as the ABA‐glucose ester (GE) levels decreased. In contrast, plants developed under high RH with 20 h photoperiod showed no increase in ABA levels during darkness, and had low β‐glucosidase activity converting ABA‐GE to ABA. Continuous lighting (24 h) resulted in low levels of β‐glucosidase activity irrespective of RH, indicating that a dark period is essential to activate β‐glucosidase. Our results provide new insight into the regulation of ABA under different humidities and photoperiods, and clearly show that β‐glucosidase is a key enzyme regulating the ABA pool in rose plants.     

Chemical modification of chitosan. Part 15: synthesis of novel chitosan derivatives by substitution of hydrophilic amine using N-carboxyethylchitosan ethyl ester as an intermediate

The Michael type reaction of chitosan with ethyl acrylate has been investigated. Although this reaction was quite slow in the case of chitosan, the reiteration of the reaction was an effective means for increasing the degree of substitution (DS) of ethyl ester. The N-carboxyethylchitosan ethyl ester as an intermediate was successfully substituted with various hydrophilic amines, although the simultaneous hydrolysis of the ester to carboxylic acid also occurred. Water-soluble chitosan derivatives were obtained by substitution with hydroxyalkylamines and diamines.     

Absence of Nceh1 augments 25-hydroxycholesterol-induced ER stress and apoptosis in macrophages

An excess of cholesterol and/or oxysterols induces apoptosis in macrophages, contributing to the development of advanced atherosclerotic lesions. In foam cells, these sterols are stored in esterified forms, which are hydrolyzed by two enzymes: neutral cholesterol ester hydrolase 1 (Nceh1) and hormone-sensitive lipase (Lipe). A deficiency in either enzyme leads to accelerated growth of atherosclerotic lesions in mice. However, it is poorly understood how the esterification and hydrolysis of sterols are linked to apoptosis. Remarkably, Nceh1-deficient thioglycollate-elicited peritoneal macrophages (TGEMs), but not Lipe-deficient TGEMs, were more susceptible to apoptosis induced by oxysterols, particularly 25-hydroxycholesterol (25-HC), and incubation with 25-HC caused massive accumulation of 25-HC ester in the endoplasmic reticulum (ER) due to its defective hydrolysis, thereby activating ER stress signaling such as induction of CCAAT/enhancer-binding protein-homologous protein (CHOP). These changes were nearly reversed by inhibition of ACAT1. In conclusion, deficiency of Nceh1 augments 25-HC-induced ER stress and subsequent apoptosis in TGEMs. In addition to reducing the cholesteryl ester content of foam cells, Nceh1 may protect against the pro-apoptotic effect of oxysterols and modulate the development of atherosclerosis.     

Synchronous intra-Golgi transport induces the release of Ca from the Golgi apparatus

The mechanisms of secretory transport through the Golgi apparatus remain an issue of debate. The precise functional importance of calcium ions (Ca²⁺) for intra-Golgi transport has also been poorly studied. Here, using different approaches to measure free Ca²⁺ concentrations in the cell cytosol ([Ca²⁺]_cyt) and inside the lumen of the Golgi apparatus ([Ca²⁺]_GA), we have revealed transient increases in [Ca²⁺]_cyt during the late phase of intra-Golgi transport that are concomitant with a decline in the maximal [Ca²⁺]_GA restoration ability. Thus, this redistribution of Ca²⁺ from the Golgi apparatus into the cytosol during the movement of cargo through the Golgi apparatus appears to have a role in intra-Golgi transport, and mainly in the late Ca²⁺-dependent phase of SNARE-regulated fusion of Golgi compartments.     

The AAT1 locus is critical for the biosynthesis of esters contributing to ‘ripe apple’ flavour in ‘Royal Gala’ and ‘Granny Smith’ apples

The ‘fruity’ attributes of ripe apples (Malus × domestica) arise from our perception of a combination of volatile ester compounds. Phenotypic variability in ester production was investigated using a segregating population from a ‘Royal Gala’ (RG; high ester production) × ‘Granny Smith’ (GS; low ester production) cross, as well as in transgenic RG plants in which expression of the alcohol acyl transferase 1 (AAT1) gene was reduced. In the RG × GS population, 46 quantitative trait loci (QTLs) for the production of esters and alcohols were identified on 15 linkage groups (LGs). The major QTL for 35 individual compounds was positioned on LG2 and co‐located with AAT1. Multiple AAT1 gene variants were identified in RG and GS, but only two (AAT1‐RGa and AAT1‐GSa) were functional. AAT1‐RGa and AAT1‐GSa were both highly expressed in the cortex and skin of ripe fruit, but AAT1 protein was observed mainly in the skin. Transgenic RG specifically reduced in AAT1 expression showed reduced levels of most key esters in ripe fruit. Differences in the ripe fruit aroma could be perceived by sensory analysis. The transgenic lines also showed altered ratios of biosynthetic precursor alcohols and aldehydes, and expression of a number of ester biosynthetic genes increased, presumably in response to the increased substrate pool. These results indicate that the AAT1 locus is critical for the biosynthesis of esters contributing to a ‘ripe apple’ flavour.     

Endogenously released DOPA is a causal factor for glutamate release and resultant delayed neuronal cell death by transient ischemia in rat striata

Glutamate is implicated in neuronal cell death. Exogenously applied DOPA by itself releases neuronal glutamate and causes neuronal cell death in in vitro striatal systems. Herein, we attempt to clarify whether endogenous DOPA is released by 10 min transient ischemia due to four-vessel occlusion during rat striatal microdialysis and, further, whether DOPA, when released, functions to cause glutamate release and resultant delayed neuronal cell death. Ischemia increased extracellular DOPA, dopamine, and glutamate, and elicited neuronal cell death 96 h after ischemic insult. Inhibition of striatal L-aromatic amino acid decarboxylase 10 min before ischemia increased markedly basal DOPA, tripled glutamate release with a tendency of decrease in dopamine release by ischemia, and exaggerated neuronal cell death. Intrastriatal perfusion of 10-30 nM DOPA cyclohexyl ester, a competitive DOPA antagonist, 10 min before ischemia, concentration-dependently decreased glutamate release without modification of dopamine release by ischemia. At 100 nM, the antagonist elicited a slight ceiling effect on decreases in glutamate release by ischemia and protected neurons from cell death. Glutamate was released concentration-dependently by intrastriatal perfusion of 0.3-1 mM DOPA and stereoselectively by 0.6 mM DOPA. The antagonist elicited no hypothermia during and after ischemia. Endogenously released DOPA is an upstream causal factor for glutamate release and resultant delayed neuronal cell death by brain ischemia in rat striata. DOPA antagonist has a neuroprotective action.     

Genetic architecture,biochemical underpinnings and ecological impact of floral UV patterning

Floral attraction traits can significantly affect pollinator visitation patterns, but adaptive evolution of these traits may be constrained by correlations with other traits. In some cases, molecular pathways contributing to floral attraction are well characterized, offering the opportunity to explore loci potentially underlying variation among individuals. Here, we quantify the range of variation in floral UV patterning (i.e. UV ‘bulls‐eye nectar guides) among crop and wild accessions of Brassica rapa. We then use experimental crosses to examine the genetic architecture, candidate loci and biochemical underpinnings of this patterning as well as phenotypic manipulations to test the ecological impact. We find qualitative variation in UV patterning between wild (commonly lacking UV patterns) and crop (commonly exhibiting UV patterns) accessions. Similar to the majority of crops, recombinant inbred lines (RILs) derived from an oilseed crop × WI fast‐plant^® cross exhibit UV patterns, the size of which varies extensively among genotypes. In RILs, we further observe strong statistical‐genetic and QTL correlations within petal morphological traits and within measurements of petal UV patterning; however, correlations between morphology and UV patterning are weak or nonsignificant, suggesting that UV patterning is regulated and may evolve independently of overall petal size. HPLC analyses reveal a high concentration of sinapoyl glucose in UV‐absorbing petal regions, which, in concert with physical locations of UV‐trait QTLs, suggest a regulatory and structural gene as candidates underlying observed quantitative variation. Finally, insects prefer flowers with UV bulls‐eye patterns over those that lack patterns, validating the importance of UV patterning in pollen‐limited populations of B. rapa.     

Effects of disialoganglioside GD3 on the mitochondrial membrane potential

GD3 is an intracellular mediator of apoptotic signaling. Although GD3 is known to directly act on mitochondria, the dynamic responses of individual mitochondria to GD3 remain to be elucidated. In the current study, the membrane potential of single mitochondria is observed in the presence of GD3 or its analogues. Here, we report that (1) GD3 specifically induces gradual depolarizations of the inner membrane by a mechanism that differs from the permeability transition, and (2) the GD3-induced depolarizations are suppressed by cyclosporin A. These results suggest that GD3 depolarizes mitochondria by a mechanism distinct from but relevant to the permeability transition.