HDLs induce raft domain vanishing in heterogeneous giant vesicles

Cholesterol efflux from the plasma membrane to HDLs is essential for cell cholesterol homeostasis. Recently, cholesterol-enriched ordered membrane domains, i.e. lipid rafts have been proposed to play an important role in this process. Here we introduce a new method to investigate the role of HDL interactions with the raft lipid phase and to directly visualize the effects of HDL-induced cholesterol efflux on rafts in model membranes. Addition of HDLs to giant lipid vesicles containing raft-type domains promoted decrease in size and disappearance of such domains as visualized by fluorescence microscopy. This was interpreted as resulting from cholesterol efflux from the vesicles to the HDLs. The raft vanishing rate was directly related to the HDL concentration. Evidence for a direct interaction of HDLs with the membrane was obtained by observing mutual adhesion of vesicles. It is suggested that the present method can be used to study the selective role of the bilayer lipid phase (raft and non-raft) in cholesterol efflux and membrane-HDL interaction and their underlying mechanisms. Such mechanisms may contribute to cholesterol efflux in vivo.     

A comparison of chemical systems for luminometric determination of antioxidant capacity towards individual reactive oxygen species.

The aim of the study was to investigate the reactive oxygen species (ROS) production in the hypoxanthine-xanthinoxidase (HX-XO), hydrogen peroxide-ferrous sulphate (H2O2-FeSO4) and hydrogen peroxide (H2O2) systems by using various concentrations of ROS scavengers, such as superoxide dismutase (SOD), dimethylthiourea (DMTU) or catalase (CAT). Luminol (0.8 mmol/L) was dissolved in a borate buffer, pH 9.0, and was used as a luminophor in the chemiluminescence (CL) measurements. In the HX-XO system SOD, CAT and DMTU deepened the CL signal, whereas in the H2O2-FeSO4 system, only CAT and DMTU deepened the CL signal, and in the H2O2 system SOD and CAT increased and DMTU deepened the CL signal. Electron spin resonance (ESR) measurements were performed only in the H2O2-FeSO4 system. 5,5-dimethyl-pyrroline-N-oxide (DMPO) was used as a spin trap. According to typical ESR spectra, .OH was produced in this chemical system. It can be concluded that the chemical systems do not produce single reactive oxygen species but a mixture of them.     

Spindle pole centrosomes of sea urchin embryos are partially composed of material recruited from maternal stores

The spindle poles of fertilized sea urchin eggs have commonly been modeled as being derived from the centrosomes of the fertilizing spermatozoon. Boveri's theory of fertilization, proposed at the turn of the century, states that the maternal centrosome is suppressed or inactivated during oogenesis and that the sperm centrosome is functionally dominant. In support of this proposal, more recent studies have shown that the sperm imports a determinant that is involved in centrosomal replication. Examination of sea urchin zygotes immunofluorescently labeled with a new anti-centrosomal antibody by quantitative confocal laser-scanning microscopy shows, however, that spindle pole centrosomes are not exclusively paternal structures, but additionally contain material derived from maternal pools. Furthermore, this maternal centrosomal material is divided among daughter blastomeres during cleavage. It therefore appears that although the sperm centrosome plays a dominant role in organizing the spindle poles, much of the centrosomal material within the spindle poles of the zygote is actually recruited from preexisting egg cytoplasmic stores. These data indicate that centrosomes of sea urchin embryos are biparentally derived, composite organelles.     

Hirschsprung's disease prevalence in Europe: A register based study

Background: Hirschsprung's disease is a congenital gut motility disorder, characterised by the absence of the enteric ganglion cells along the distal gut. The aim of this study was to describe the epidemiology of Hirschsprung's disease, including additional congenital anomalies, total prevalence, trends, and association with maternal age. Methods: Cases of Hirschsprung's disease delivered during 1980 to 2009 notified to 31 European Surveillance of Congenital Anomaly registers formed the population‐based case‐series. Prevalence rates and 95% confidence intervals were calculated as the number of cases per 10,000 births. Multilevel Poisson regression was performed to investigate trends in prevalence, geographical variation and the association with maternal age. Results: There were 1,322 cases of Hirschsprung's disease among 12,146,210 births. The total prevalence was 1.09 (95% confidence interval, 1.03–1.15) per 10,000 births and there was a small but significant increase in prevalence over time (relative risk = 1.01; 95% credible interval, 1.00–1.02; p = 0.004). There was evidence of geographical heterogeneity in prevalence (p < 0.001). Excluding 146 (11.0%) cases with chromosomal anomalies or genetic syndromes, there were 1,176 cases (prevalence = 0.97; 95% confidence interval, 0.91–1.03 per 10,000 births), of which 137 (11.6%) had major structural anomalies. There was no evidence of a significant increased risk of Hirschsprung's disease in cases born to women aged ≥35 years compared with those aged 25 to 29 (relative risk = 1.09; 95% credible interval, 0.91–1.31; p = 0.355). Conclusion: This large population‐based study found evidence of a small increasing trend in Hirschsprung's disease and differences in prevalence by geographic location. There was also no evidence of an association with maternal age. Birth Defects Research (Part A), 100:695–702, 2014. © 2014 Wiley Periodicals, Inc.     

COX-1 is coupled with mPGES-1 and ABCC4 in human cervix cancer cells

Cyclooxygenases are key enzymes in the arachidonic acid metabolism. Their unstable intermediate, prostaglandin H₂, is further metabolized to bioactive lipids by various downstream enzymes. In this study, utilizing short hairpin RNAs, we prepared a cell line of human cervix carcinoma with stable down-regulated cyclooxygenase-1 (COX-1) to assess the impact of COX-1 reduction on the downstream enzymes. We found a significant microsomal prostaglandin E synthase-1 (mPGES-1) suppression. In addition, mRNA expression of multidrug resistance protein 4 (MRP4, ABCC4), supposed to take part in antiviral and anticancer drug transport from cells, was up-regulated after COX-1 down-regulation. Our findings indicate that mPGES-1, believed to be coexpressed preferentially with cyclooxygenase-2, may be coupled to COX-1. ABCC4 up-regulation further supports the assumption of its involvement in prostanoid transport.     

Lipid domain separation,bilayer thickening and pearling induced by the cell penetrating peptide penetratin

Protein membrane transduction domains are able to translocate through cell membranes. This capacity resulted in new concepts on cell communication and in the design of vectors for internalization of active molecules into cells. Penetratin crosses the plasma membrane by a receptor and metabolic energy-independent mechanism which is at present unknown. A better knowledge of its interaction with phospholipids will help to understand the molecular mechanisms of cell penetration. Here, we investigated the role of lipid composition on penetratin induced membrane perturbations by X-ray diffraction, microscopy and ³¹P-NMR. Penetratin showed the ability to induce phospholipid domain separation, membrane bilayer thickening, formation of vesicles, membrane undulations and tubular pearling. These data demonstrate its capacity to increase membrane curvature and suggest that dynamic phospholipid–penetratin complexes can be organized in different structural arrangements. These properties and their implications in peptide membrane translocation capacity are discussed.     

Disruption of nucleocytoplasmic trafficking of cyclin D1 and topoisomerase II by sanguinarine

Background  

The quaternary isoquinoline alkaloid sanguinarine is receiving increasing attention as a potential chemotherapeutic agent in the treatment of cancer. Previous studies have shown that this DNA-binding phytochemical can arrest a number of different types of transformed cells in G0/G1, and upregulate the CKIs p21 and p27 while downregulating multiple cyclins and CDKs. To more closely examine the responses of some of these cell cycle regulatory molecules to sanguinarine, we used immunocytochemical methods to visualize cyclin D1 and topoisomerase II behavior in MCF-7 breast cancer cells.     

Carbon : nitrogen : phosphorus ratios influence biofilm formation by Enterobacter cloacae and Citrobacter freundii

AIMS: To test the effects of C : N : P ratio modification of a well-known nutrient medium formulation, the Endo formulation on biofilm formation by Enterobacter cloacae Ecl and Citrobacter freundii Cf1 in both single-species and binary species biofilms. METHODS AND RESULTS: The C : N : P atom : atom ratio of a well-known nutrient medium formulation, the Endo formulation, that has been applied in fermentative biohydrogen studies, was modified to include two different C concentrations, one containing 17.65 g l(-1) and the other 8.84 g l(-1) sucrose, each containing four different C : N : P ratios, two at higher C : N : P ratios (334 : 84 : 16.8 and 334 : 84 : 3) and two at lower C : N : P ratios (334 : 28 : 5.6 and 334 : 28 : 1). Attached cells were enumerated after dislodging the biofilms that had formed on granular activated carbon (GAC). The modified medium containing 17.65 g l(-1) sucrose and having a C : N : P ratio of 334 : 28 : 5.6 resulted in significantly (P < 0.05) higher counts of attached cells for both single-species biofilms at 7.73 log(10) CFU g(-1) GAC and 9.3 log(10)CFU g(-1) GAC for Ent. cloacae Ecl and Cit. freundii Cf1, respectively, and binary species biofilms at 8.2 log(10) CFU g(-1) GAC and 6.34 log(10) CFU g(-1) GAC for Ent. cloacae Ecl and Cit. freundii Cf1, respectively. Scanning electron micrographs showed qualitative evidence that the 334 : 28 : 5.6 ratio encouraged more complex and extensive biofilm growth for both single-species and binary species biofilms. CONCLUSIONS: The differences in the attachment numbers between the different ratios were found not to be a result of the individual actions of the bacterial isolates involved but rather because of the effects of the various C : N : P ratios. The 334 : 28 : 5.6 ratio showed significantly (P < 0.05) higher counts of attached cells for both single-species and binary species biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates that C : N : P ratios should be a key consideration with regard to maximizing biofilm formation in shake flask and fluidized bed bioreactor studies as well as understanding fundamental factors affecting biofilm growth in natural environments.