Deleterious impact of elaidic fatty acid on ABCA1-mediated cholesterol efflux from mouse and human macrophages

Consumption of trans fatty acids (TFA) increase cardiovascular risk more than do saturated FA, but the mechanisms explaining their atherogenicity are still unclear. We investigated the impact of membrane incorporation of TFA on cholesterol efflux by exposing J774 mouse macrophages or human monocyte-derived macrophages (HMDM) to media enriched or not (standard medium) with industrially produced elaidic (trans-9 18:1) acid, naturally produced vaccenic (trans-11 18:1) acid (34 h, 70 μM) or palmitic acid. In J774 macrophages, elaidic and palmitic acid, but not vaccenic acid, reduced ABCA1-mediated efflux by ~23% without affecting aqueous diffusion, SR-BI or ABCG1-mediated pathways, and this effect was maintained in cholesterol-loaded cells. The impact of elaidic acid on the ABCA1 pathway was weaker in cholesterol-normal HMDM, but elaidic acid induced a strong reduction of ABCA1-mediated efflux in cholesterol-loaded cells (-36%). In J774 cells, the FA supplies had no impact on cellular free cholesterol or cholesteryl ester masses, the abundance of ABCA1 mRNA or the total and plasma membrane ABCA1 protein content. Conversely, TFA or palmitic acid incorporation induced strong modifications of the membrane FA composition with a decrease in the ratio of (cis-monounsaturated FA+polyunsaturated FA):(saturated FA+TFA), with elaidic and vaccenic acids representing each 20% and 13% of the total FA composition, respectively. Moreover, we demonstrated that cellular ATP was required for the effect of elaidic acid, suggesting that it contributes to atherogenesis by impairing ABCA1-mediated cholesterol efflux in macrophages, likely by decreasing the membrane fluidity, which could thereby reduce ATPase activity and the function of the transporter.     

Mechanical similarity across ontogeny of digging muscles in an Australian marsupial (Isoodon fusciventer)

Many mammals dig, either during foraging to access subsurface food resources, or in creating burrows for shelter. Digging requires large forces produced by muscles and transmitted to the soil via the skeletal system; thus fossorial mammals tend to have characteristic modifications of the musculoskeletal system that reflect their digging ability. Bandicoots (Marsupialia: Peramelidae) scratch-dig mainly to source food, searching for subterranean food items including invertebrates, seeds, and fungi. They have musculoskeletal features for digging, including shortened, robust forelimb bones, large muscles, and enlarged muscle attachment areas. Here, we compared changes in the ontogenetic development of muscles associated with digging in the Quenda (Isoodon fusciventer). We measured muscle mass (m _m), pennation angle, and fiber length (FL) to calculate physiological cross-sectional area (PCSA; a proxy of maximum isometric force) as well as estimate the maximum isometric force (Fmax) for 34 individuals ranging in body size from 124 to 2,390 g. Males grow larger than females in this bandicoot species, however, we found negligible sex differences in mass-specific m _m, PCSA or FL for our sample. Majority of the forelimb muscles PCSA showed a positive allometric relationship with total body mass, while m _m and FL in the majority of forelimb muscles showed isometry. Mechanical similarity was tested, and two thirds of forelimb muscles maximum isometric forces (Fmax) scaled with isometry; therefore the forelimb is primarily mechanical similar throughout ontogeny. PCSA showed a significant difference between scaling slopes between main movers in the power stroke, and main movers of the recovery stroke of scratch-digging. This suggests that some forelimb muscles grow with positive allometry, specially these associated with the power stroke of digging. Intraspecific variation in PCSA is rarely considered in the literature, and thus this is an important study quantifying changes in muscle architectural properties with growth in a mammalian model of scratch-digging.     

Thymic generation and regeneration: a new paradigm for establishing clinical tolerance of stem cell-based therapies

Tolerance to tissue-engineering products is a major obstacle hindering the clinical application of this rapidly advancing technology. Manipulation of central tolerance, by establishing thymus chimerism of both donor and host-derived haemopoietic cells (haemopoietic stem cell transplant--HSCT), should purge any T cells reactive to potential donor organ or tissue transplant. A functional thymus, however, is required to induce chimerism and repopulate the peripheral T cell pool, but age-related thymic atrophy and damage caused by ablative conditioning regimes significantly reduce thymic function and increase incident of infection-dependent morbidity and mortality. Thus rejuvenation of the thymus alongside HSCT may potentiate the use of this strategy in the clinic. In addition, the use of thymic epithelial progenitor cell technology may allow growth of ex vivo thymic tissue for use in clinical situations of immunodeficiency as well as in establishing tolerance to tissue/organ products derived from the same source.     

IgE-antibody-dependent immunotherapy of solid tumors: cytotoxic and phagocytic mechanisms of eradication of ovarian cancer cells

Abs have a paramount place in the treatment of certain, mainly lymphoid, malignancies, although tumors of nonhemopoietic origin have proved more refractory ones. We have previously shown that the efficacy of immunotherapy of solid tumors, in particular ovarian carcinoma, may be improved by the use of IgE Abs in place of the conventional IgG. An IgE Ab (MOv18 IgE) against an ovarian-tumor-specific Ag (folate binding protein), in combination with human PBMC, introduced into ovarian cancer xenograft-bearing mice, greatly exceeded the analogous IgG1 in promoting survival. In this study, we analyzed the mechanisms by which MOv18 IgE may exert its antitumor activities. Monocytes were essential IgE receptor-expressing effector cells that mediated the enhanced survival of tumor-bearing mice by MOv18 IgE and human PBMC. Monocytes mediated MOv18 IgE-dependent ovarian tumor cell killing in vitro by two distinct pathways, cytotoxicity and phagocytosis, acting respectively through the IgE receptors FcepsilonRI and CD23. We also show that human eosinophils were potent effector cells in MOv18 IgE Ab-dependent ovarian tumor cell cytotoxicity in vitro. These results demonstrate that IgE Abs can engage cell surface IgE receptors and activate effector cells against ovarian tumor cells. Our findings offer a framework for an improved immunotherapeutic strategy for combating solid tumors.     

Structural analogues of schweinfurthin F: Probing the steric,electronic, and hydrophobic properties of the D-ring substructure

The natural tetracyclic schweinfurthins are potent and selective inhibitors of cell growth in the National Cancer Institute’s 60-cell line screen. An interest in determination of their cellular or molecular target has inspired our efforts to prepare both the natural products and analogues. In this paper, chemical synthesis of analogues modified in different olefinic positions, and preliminary results from studies of their biological activity, are reported.     

Early-onset and robust cerebral microvascular accumulation of amyloid beta-protein in transgenic mice expressing low levels of a vasculotropic Dutch/Iowa mutant form of amyloid beta-protein precursor

Cerebrovascular deposition of amyloid beta-protein (Abeta) is a common pathological feature of Alzheimer's disease and related disorders. In particular, the Dutch E22Q and Iowa D23N mutations in Abeta cause familial cerebrovascular amyloidosis with abundant diffuse amyloid plaque deposits. Both of these charge-altering mutations enhance the fibrillogenic and pathogenic properties of Abeta in vitro. Here, we describe the generation of several transgenic mouse lines (Tg-SwDI) expressing human neuronal Abeta precursor protein (AbetaPP) harboring the Swedish K670N/M671L and vasculotropic Dutch/Iowa E693Q/D694N mutations under the control of the mouse Thy1.2 promoter. Tg-SwDI mice expressed transgenic human AbetaPP only in the brain, but at levels below those of endogenous mouse AbetaPP. Despite the paucity of human AbetaPP expression, quantitative enzyme-linked immunosorbent assay measurements revealed that Tg-SwDI mice developed early-onset and robust accumulation of Abeta in the brain with high association with isolated cerebral microvessels. Tg-SwDI mice exhibited striking perivascular/vascular Abeta deposits that markedly increased with age. The vascular Abeta accumulations were fibrillar, exhibiting strong thioflavin S staining, and occasionally presented signs of microhemorrhage. In addition, numerous largely diffuse, plaque-like structures were observed starting at 3 months of age. In vivo transport studies demonstrated that Dutch/Iowa mutant Abeta was more readily retained in the brain compared with wild-type Abeta. These results with Tg-SwDI mice demonstrate that overexpression of human AbetaPP is not required for early-onset and robust accumulation of both vascular and parenchymal Abeta in mouse brain.     

Nonselective voltage-gated ionic channels in type II taste cells

In cells of different types outward voltage-gated (VG) ion currents are generally carried by potassium ions. However, in mouse type II taste cells these currents persist when K⁺-selective ion channels are inhibited. In this study, we examined the ion channels that provide a pathway for atypical VG outward currents in type II taste cells. These channels are found to be weakly selective and permeabile to large molecules such as NMDG, gluconate, and ATP. According to non-stationary fluctuation analysis, single channel conductance is about 200 pS. The data obtained suggest that the nonselective ion channels are similar to hemichannels formed by connexins, the gap-junction proteins, in the plasma membrane of vertebrate cells.     

Skeletal Cell Differentiation Is Enhanced by Atmospheric Dielectric Barrier Discharge Plasma Treatment

Enhancing chondrogenic and osteogenic differentiation is of paramount importance in providing effective regenerative therapies and improving the rate of fracture healing. This study investigated the potential of non-thermal atmospheric dielectric barrier discharge plasma (NT-plasma) to enhance chondrocyte and osteoblast proliferation and differentiation. Although the exact mechanism by which NT-plasma interacts with cells is undefined, it is known that during treatment the atmosphere is ionized generating extracellular reactive oxygen and nitrogen species (ROS and RNS) and an electric field. Appropriate NT-plasma conditions were determined using lactate-dehydrogenase release, flow cytometric live/dead assay, flow cytometric cell cycle analysis, and Western blots to evaluate DNA damage and mitochondrial integrity. We observed that specific NT-plasma conditions were required to prevent cell death, and that loss of pre-osteoblastic cell viability was dependent on intracellular ROS and RNS production. To further investigate the involvement of intracellular ROS, fluorescent intracellular dyes Mitosox (superoxide) and dihydrorhodamine (peroxide) were used to assess onset and duration after NT-plasma treatment. Both intracellular superoxide and peroxide were found to increase immediately post NT-plasma treatment. These increases were sustained for one hour but returned to control levels by 24 hr. Using the same treatment conditions, osteogenic differentiation by NT-plasma was assessed and compared to peroxide or osteogenic media containing β-glycerolphosphate. Although both NT-plasma and peroxide induced differentiation-specific gene expression, neither was as effective as the osteogenic media. However, treatment of cells with NT-plasma after 24 hr in osteogenic or chondrogenic media significantly enhanced differentiation as compared to differentiation media alone. The results of this study show that NT-plasma can selectively initiate and amplify ROS signaling to enhance differentiation, and suggest this technology could be used to enhance bone fusion and improve healing after skeletal injury.     

Inhibition of Mitogen-Activated Kinase Signaling Sensitizes HeLa Cells to Fas Receptor-Mediated Apoptosis

The Fas receptor (FasR) is an important physiological mediator of apoptosis in various tissues and cells. However, there are also many FasR-expressing cell types that are normally resistant to apoptotic signaling through this receptor. The mitogen-activated protein kinase (MAPK) signaling cascade has, apart from being a growth-stimulating factor, lately received attention as an inhibitory factor in apoptosis. In this study, we examined whether MAPK signaling could be involved in protecting FasR-insensitive cells. To this end, we used different approaches to inhibit MAPK signaling in HeLa cells, including treatment with the MAPK kinase inhibitor PD 98059, serum withdrawal, and expression of dominant-interfering MAPK kinase mutant protein. All of these treatments were effective in sensitizing the cells to FasR-induced apoptosis, demonstrating that MAPK indeed is involved in the control of FasR responses. The MAPK-mediated control seemed to occur at or upstream of caspase 8, the initiator caspase in apoptotic FasR responses. Transfection with the constitutively active MAPK kinase abrogated FasR-induced apoptosis also in the presence of cycloheximide, indicating that the MAPK-generated suppression of FasR-mediated apoptotic signaling is protein synthesis independent. In cells insensitive to FasR-induced apoptosis, stimulation of the FasR with an agonistic antibody resulted in significant MAPK activation, which was inhibited by PD 98059. When different cell types were compared, the FasR-mediated MAPK activation seemed proportional to the degree of FasR insensitivity. These results suggest that the FasR insensitivity is likely to be a consequence of FasR-induced MAPK activation, which in turn interferes with caspase activation.     

Dielectric parameters of ascitic and pleural fluids in the microwave range in different nosologies

The dielectric parameters of ascitic and pleural fluids formed in the human body in oncological and nononcological diseases of different nosology have been estimated in the range between 400 MHz and 1.2 GHZ. The dependence of refractive and absorption indices of ascitic and pleural liquids on the signal frequency and mass concentration of dissolved substances was found. Common regularities and distinctions in the behavior of their dielectric properties were revealed.