Evaluation of the components of the chylomicron remnant removal mechanism by use of the isolated perfused mouse liver.

The isolated perfused mouse liver was utilized to evaluate the relative contribution of various molecules believed to participate in the removal of chylomicron remnants by the liver. Sixty percent of asialofetuin was removed from the perfusate per pass; bovine serum albumin was not removed. Normal mouse livers removed chylomicron remnants more efficiently (40-50%/pass) than nascent chylomicrons (10-20%/pass). The fractional removal rate of remnants decreased as their concentration in the perfusate increased demonstrating saturability. Remnant removal by livers of low density lipoprotein receptor-deficient (LDLRD) mice paralleled that of normal mice at low remnant concentrations (0.05, 0.2 microg protein/ml); as concentration increased (4-16 microg protein/ml), removal by LDLRD livers was reduced. About 50% of the capacity to remove remnants was due to the LDL receptor. The role of the LDLR-related protein (LRP) was estimated using the receptor-associated protein (RAP). Four microg/ml of RAP inhibited only LRP; it reduced the removal of remnants by 30-40% in normal livers. When RAP was included in the perfusate of LDLRD livers, remnant removal persisted but was diminished, particularly late in the perfusion; the capacity was approximately 30% of controls. The present study has established that there is more than one mechanism operating for the removal of chylomicron remnants by the liver, provides estimates of the concentration of each to the removal of remnants, and indicates a method for further studies.It is concluded that in normal livers, the LDL receptor has the greatest capacity for removing chylomicron remnants. The LRP contributes to the process as well and a third component, perhaps "sequestration," accounts for up to 30% of the capacity for the initial removal of chylomicron remnants.     

Altered levels of primary antioxidant enzymes in progeria skin fibroblasts

Free radicals are involved in the aging process. In this study, the profile of primary antioxidant enzymes that scavenge reactive oxygen species (ROS) was examined for the first time in human skin fibroblasts from progeria, a premature aging disease. Altered levels of antioxidant enzymes were found in progeria cells. Basal levels of MnSOD were decreased in progeria cells as well as a blunted induction in response to chronic stress. This change may contribute to the accelerated aging process in progeria cells. In contrast, the levels of CuZnSOD showed no progeria-related change. Two H2O2 removing enzymes demonstrated a significant reduction in progeria cells: only 50% of normal CAT activity and 30% of normal GPX activity can be detected in progeria cells. This diminished H2O2 removing capacity in progeria cells may lead to an imbalance of intracellular ROS and therefore may play an important role in the development of progeria.     

pH-dependent conformational changes of ferricytochrome c induced by electrode surface microstructure

pH-dependent processes of bovine heart ferricytochrome c have been investigated by electronic absorption and circular dichroism (CD) spectra at functionalized single-wall carbon nanotubes (SWNTs) modified glass carbon electrode (SWNTs/GCE) using a long optical path thin layer cell. These methods enabled the pH-dependent conformational changes arising from the heme structure change to be monitored. The spectra obtained at functionalized SWNTs/GCE reflect electrode surface microstructure-dependent changes for pH-induced protein conformation, pK_a of alkaline transition and structural microenvironment of the ferricytochrome c heme. pH-dependent conformational distribution curves of ferricytochrome c obtained by analysis of in situ CD spectra using singular value decomposition least square (SVDLS) method show that the functionalized SWNTs can retain native conformational stability of ferricytochrome c during alkaline transition.     

Distinct Wnt members regulate the hierarchical morphogenesis of skin regions (spinal tract) and individual feathers

Skin morphogenesis occurs in successive stages. First, the skin forms distinct regions (macropatterning). Then skin appendages with particular shapes and sizes form within each region (micropatterning). Ectopic DKK expression inhibited dermis formation in feather tracts and individual buds, implying the importance of Wnts, and prompted the assessment of individual Wnt functions at different morphogenetic levels using the feather model. Wnt 1, 3a, 5a and 11 initially were expressed moderately throughout the feather tract then were up-regulated in restricted regions following two modes: Wnt 1 and 3a became restricted to the placodal epithelium, then to the elongated distal bud epidermis; Wnt 5a and 11 intensified in the inter-tract region and interprimordia epidermis or dermis, respectively, then appeared in the elongated distal bud dermis. Their role in feather tract formation was determined using RCAS mediated misexpression in ovo at E2/E3. Their function in periodic feather patterning was examined by misexpression in vitro using reconstituted E7 skin explant cultures. Wnt 1 reduced spinal tract size, but enhanced feather primordia size. Wnt 3a increased dermal thickness, expanded the spinal tract size, reduced interbud domain spacing, and produced non-tapering "giant buds". Wnt 11 and dominant negative Wnt 1 enhanced interbud spacing, and generated thinner buds. In cultured dermal fibroblasts, Wnt 1 and 3a stimulated cell proliferation and activated the canonical beta-catenin pathway. Wnt 11 inhibited proliferation but stimulated migration. Wnt 5a and 11 triggered the JNK pathway. Thus distinctive Wnts have positive and negative roles in forming the dermis, tracts, interbud spacing and the growth and shaping of individual buds.     

G1 cell cycle progression and the expression of G1 cyclins are regulated by PI3K/AKT/mTOR/p70S6K1 signaling in human ovarian cancer cells

Ovarian cancer is one of the most common cancers among women. Recent studies demonstrated that the gene encoding the p110alpha catalytic subunit of phosphatidylinositol 3-kinase (PI3K) is frequently amplified in ovarian cancer cells. PI3K is involved in multiple cellular functions, including proliferation, differentiation, antiapoptosis, tumorigenesis, and angiogenesis. In this study, we demonstrate that the inhibition of PI3K activity by LY-294002 inhibited ovarian cancer cell proliferation and induced G(1) cell cycle arrest. This effect was accompanied by the decreased expression of G(1)-associated proteins, including cyclin D1, cyclin-dependent kinase (CDK) 4, CDC25A, and retinoblastoma phosphorylation at Ser(780), Ser(795), and Ser(807/811). Expression of CDK6 and beta-actin was not affected by LY-294002. Expression of the cyclin kinase inhibitor p16(INK4a) was induced by the PI3K inhibitor, whereas steady-state levels of p21(CIP1/WAF1) were decreased in the same experiment. The inhibition of PI3K activity also inhibited the phosphorylation of AKT and p70S6K1, but not extracellular regulated kinase 1/2. The G(1) cell cycle arrest induced by LY-294002 was restored by the expression of active forms of AKT and p70S6K1 in the cells. Our study shows that PI3K transmits a mitogenic signal through AKT and mammalian target of rapamycin (mTOR) to p70S6K1. The mTOR inhibitor rapamycin had similar inhibitory effects on G(1) cell cycle progression and on the expression of cyclin D1, CDK4, CDC25A, and retinoblastoma phosphorylation. These results indicate that PI3K mediates G(1) progression and cyclin expression through activation of an AKT/mTOR/p70S6K1 signaling pathway in the ovarian cancer cells.     

Blood pressure regulates the activity and function of the Na-K-2Cl cotransporter in vascular smooth muscle

The Na-K-2Cl cotransporter (NKCC1) is one of several transporters that have been linked to hypertension, and its inhibition reduces vascular smooth muscle tone and blood pressure. NKCC1 in the rat aorta is stimulated by vasoconstrictors and inhibited by nitrovasodilators, and this is linked to the contractile state of the smooth muscle. To determine whether blood pressure also regulates NKCC1, we examined the acute effect of hypertension on NKCC1 in rats after aortic coarctation. In the hypertensive aorta (28-mmHg rise in mean blood pressure), an increase in NKCC1 activity (measured as bumetanide-sensitive (86)Rb efflux) was apparent by 16 h and reached a plateau of 62% greater than control at 48 h. In contrast, there was a slight decrease in NKCC1 activity in the hypotensive aorta (21% decrease in mean blood pressure). Measurement of NKCC1 mRNA by real-time PCR revealed a fivefold increase in the hypertensive aorta compared with the hypotensive aorta or sham aorta. The inhibition by bumetanide of isometric force response to phenylephrine was significantly greater in the hypertensive aorta than in the control aorta or hypotensive aorta. We conclude that NKCC1 in rat aortic smooth muscle is regulated by blood pressure, most likely through changes in transporter abundance. This upregulation of NKCC1 is associated with a greater contribution to force generation in the hypertensive aorta. This is the first demonstration that NKCC1 in vascular smooth muscle is regulated by blood pressure and indicates that this transporter is important in the acute response of vascular smooth muscle to hypertension.     

Diarrhea-associated HIV-1 APIs potentiate muscarinic activation of Cl- secretion by T84 cells via prolongation of cytosolic Ca2+ signaling

Aspartyl protease inhibitors (APIs) effectively extend the length and quality of life in human immunodeficiency virus (HIV)-infected patients, but dose-limiting side effects such as lipodystrophy, insulin resistance, and diarrhea have limited their clinical utility. Here, we show that the API nelfinavir induces a secretory form of diarrhea in HIV-infected patients. In vitro studies demonstrate that nelfinavir potentiates muscarinic stimulation of Cl^- secretion by T84 human intestinal cell monolayers through amplification and prolongation of an apical membrane Ca²⁺-dependent Cl^- conductance. This stimulated ion secretion is associated with increased magnitude and duration of muscarinically induced intracellular Ca²⁺ transients via activation of a long-lived, store-operated Ca²⁺ entry pathway. The enhanced intracellular Ca²⁺ signal is associated with uncoupling of the Cl^- conductance from downregulatory intracellular mediators generated normally by muscarinic activation. These data show that APIs modulate Ca²⁺ signaling in secretory epithelial cells and identify a novel target for treatment of clinically important API side effects. nelfinavir; clotrimazole; barium     

Relationship between contents of adrenomedullin and distributions of neutral endopeptidase in blood and tissues of rats in septic shock

Adrenomedullin (ADM), a multifunction peptide with important roles in regulating cardiovascular homeostasis, has the vasodilatory properties and is of particular interest in the pathophysiology of sepsis. ADM levels in plasma and tissues are regulated by the proteolysis of neutral endopeptidase (NEP), the major enzyme of ADM degradation. We observed the NEP activity in the plasma, the activity and distribution of NEP and its mRNA expression in the tissues of rats in septic shock to study the possible role of NEP in elevating tissue ADM concentration during sepsis. ADM level increases progressively during sepsis except in the jejunum. Rats in early phase of shock (ES) showed diverse changes in tissue NEP activity. Plasma NEP activity, tissue NEP activity and its protein and mRNA expression in the left ventricle, aorta, jejunum and lung in the late phase of shock (LS) rats were lower than those in ES and the control, but no statistical change of NEP activity in the kidney was observed. The level of ADM was inversely correlated with NEP activity in the plasma, ventricle and aorta and positively correlated with NEP activity in the jejunum. Thus, in sepsis, the local concentration and action of ADM in tissues may be differentially regulated by NEP.