17beta-estradiol reduces tumor necrosis factor-alpha-mediated LDL accumulation in the artery wall. (1/1146)

Estrogens have direct effects on the vascular wall that may prevent the development of atherosclerosis. In particular, estrogens, such as 17beta-estradiol (estradiol), are known to have potent antioxidant activity. Tumor necrosis factor-alpha (TNF) is found in human atheroma and produces oxygen-derived free radicals. These oxygen-derived free radicals may modify low density lipoproteins (LDL) and increase LDL binding in the artery wall. We asked: 1) does TNF increase LDL accumulation in the artery wall and 2) can the TNF-mediated increase in LDL accumulation be prevented by the antioxidant activity of estradiol? Carotid arteries from ovariectomized 3-month-old rats were removed and perfused with fluorescently labeled LDL and arterial LDL flux was measured using quantitative fluorescence microscopy. In six arteries, addition of TNF (10 ng/ml) to the perfusate resulted in a 2.3-fold increase in the rate of LDL accumulation (1.50 +/- 0.37 ng/min per cm2 vs. 3.38 +/- 0.48 ng/min per cm2; P < 0.01). Estradiol (65 pg/ml) and alpha-tocopherol (6 mg/L) both attenuated TNF-mediated LDL accumulation (P < 0.05), indicating that TNF may exert its effects on LDL accumulation through cellular production of oxygen-derived free radicals. These results support an antioxidant role for estradiol in the protection against LDL accumulation in the artery wall and subsequent progression of atherosclerosis.  (+info)

Development of cephalic neural crest cells in embryos of Lampetra japonica, with special reference to the evolution of the jaw. (2/1146)

Neural crest cells contribute extensively to vertebrate head morphogenesis and their origin is an important question to address in understanding the evolution of the craniate head. The distribution pattern of cephalic crest cells was examined in embryos of one of the living agnathan vertebrates, Lampetra japonica. The initial appearance of putative crest cells was observed on the dorsal aspect of the neural rod at stage 20.5 and ventral expansion of these cells was first seen at the level of rostral somites. As in gnathostomes, cephalic crest cells migrate beneath the surface ectoderm and form three major cell populations, each being separated at the levels of rhombomeres (r) 3 and r5. The neural crest seems initially to be produced at all neuraxial levels except for the rostral-most area, and cephalic crest cells are secondarily excluded from levels r3 and r5. Such a pattern of crest cell distribution prefigures the morphology of the cranial nerve anlage. The second or middle crest cell population passes medial to the otocyst, implying that the otocyst does not serve as a barrier to separate the crest cell populations. The three cephalic crest cell populations fill the pharyngeal arch ventrally, covering the pharyngeal mesoderm laterally with the rostral-most population covering the premandibular region and mandibular arch. The third cell population is equivalent to the circumpharyngeal crest cells in the chick, and its influx into the pharyngeal region precedes the formation of postotic pharyngeal arches. Focal injection of DiI revealed the existence of an anteroposterior organization in the neural crest at the neurular stage, destined for each pharyngeal region. The crest cells derived from the posterior midbrain that express the LjOtxA gene, the Otx2 cognate, were shown to migrate into the mandibular arch, a pattern which is identical to gnathostome embryos. It was concluded that the head region of the lamprey embryo shares a common set of morphological characters with gnathostome embryos and that the morphological deviation of the mandibular arch between the gnathostomes and the lamprey is not based on the early embryonic patterning.  (+info)

Characterization of nodular neuronal heterotopia in children. (3/1146)

Neuronal heterotopia are seen in various pathologies and are associated with intractable epilepsy. We examined brain tissue from four children with subcortical or periventricular nodular heterotopia of different aetiologies: one with severe epilepsy following focal brain trauma at 17 weeks gestation, one with hemimegalencephaly and intractable epilepsy, one with focal cortical dysplasia and intractable epilepsy, and one dysmorphic term infant with associated hydrocephalus and polymicrogyria. The connectivity of nodules was investigated using histological and carbocyanine dye (DiI) tracing techniques. DiI crystal placement adjacent to heterotopic nodules revealed numerous DiI-labelled fibres within a 2-3 mm radius of the crystals. Although we observed labelled fibres closely surrounding nodules, the majority did not penetrate them. Placement of DiI crystals within nodules also identified a limited number of projections out of the nodules and in one case there was evidence for connectivity between adjacent nodules. The cellular and neurochemical composition of nodules was also examined using immunohistochemistry for calretinin and neuropeptide Y (NPY), which are normally expressed in GABAergic cortical interneurons. Within heterotopic nodules from all cases, numerous calretinin-positive neurons were identified, along with a few cell bodies and many processes positive for NPY. Calretinin-positive neurons within nodules were less morphologically complex than those in the cortex, which may reflect incomplete differentiation into an inhibitory neuronal phenotype. There were also abnormal clusters of calretinin-positive cells in the overlying cortical plate, indicating that the migratory defect which produces heterotopic nodules also affects development of the cortex itself. Thus, heterotopic nodules consisting of multiple neuronal cell types are associated with malformation in the overlying cortical plate, and have limited connectivity with other brain regions. This abnormal development of connectivity may affect neuronal maturation and consequently the balance of excitation and inhibition in neuronal circuits, leading to their epileptogenic potential.  (+info)

Identification of megalin/gp330 as a receptor for lipoprotein(a) in vitro. (4/1146)

Lipoprotein(a) [Lp(a)] is an atherogenic lipoprotein of unknown physiological function. The mechanism of Lp(a) atherogenicity as well as its catabolic pathways are only incompletely understood at present. In this report, we show that the low density lipoprotein receptor (LDLR) gene family member megalin/glycoprotein (gp) 330 is capable of binding and mediating the cellular uptake and degradation of Lp(a) in vitro. A mouse embryonic yolk sac cell line with native expression of megalin/gp330 but genetically deficient in LDLR-related protein (LRP) and a control cell line carrying a double knockout for both LRP and megalin/gp330 were compared with regard to their ability to bind, internalize, and degrade dioctadecyltetramethylindocarbocyanine perchlorate (DiI)-fluorescence-labeled Lp(a) as well as equimolar amounts of 125I-labeled Lp(a) and LDL. Uptake and degradation of radiolabeled Lp(a) by the megalin/gp330-expressing cells were, on average, 2-fold higher than that of control cells. This difference could be completely abolished by addition of the receptor-associated protein, an inhibitor of ligand binding to megalin/gp330. Mutual suppression of the uptake of 125I-Lp(a) and of 125I-LDL by both unlabeled Lp(a) and LDL suggested that Lp(a) uptake is mediated at least partially by apolipoprotein B100. Binding and uptake of DiI-Lp(a) resulted in strong signals on megalin/gp330-expressing cells versus background only on control cells. In addition, we show that purified megalin/gp330, immobilized on a sensor chip, directly binds Lp(a) in a Ca2+-dependent manner with an affinity similar to that for LDL. We conclude that megalin/gp330 binds Lp(a) in vitro and is capable of mediating its cellular uptake and degradation.  (+info)

Endocytic sorting of lipid analogues differing solely in the chemistry of their hydrophobic tails. (5/1146)

To understand the mechanisms for endocytic sorting of lipids, we investigated the trafficking of three lipid-mimetic dialkylindocarbocyanine (DiI) derivatives, DiIC16(3) (1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), DiIC12(3) (1,1'- didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), and FAST DiI (1,1'-dilinoleyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate), in CHO cells by quantitative fluorescence microscopy. All three DiIs have the same head group, but differ in their alkyl tail length or unsaturation; these differences are expected to affect their distribution in membrane domains of varying fluidity or curvature. All three DiIs initially enter sorting endosomes containing endocytosed transferrin. DiIC16(3), with two long 16-carbon saturated tails is then delivered to late endosomes, whereas FAST DiI, with two cis double bonds in each tail, and DiIC12(3), with saturated but shorter (12-carbon) tails, are mainly found in the endocytic recycling compartment. We also find that DiOC16(3) (3,3'- dihexadecyloxacarbocyanine perchlorate) and FAST DiO (3, 3'-dilinoleyloxacarbocyanine perchlorate) behave similarly to their DiI counterparts. Furthermore, whereas a phosphatidylcholine analogue with a BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorophore attached at the end of a 5-carbon acyl chain is delivered efficiently to the endocytic recycling compartment, a significant fraction of another derivative with BODIPY attached to a 12-carbon acyl chain entered late endosomes. Our results thus suggest that endocytic organelles can sort membrane components efficiently based on their preference for association with domains of varying characteristics.  (+info)

Reactive oxygen metabolites increase mitochondrial calcium in endothelial cells: implication of the Ca2+/Na+ exchanger. (6/1146)

In endothelial cells, a bolus of hydrogen peroxide (H2O2) or oxygen metabolites generated by hypoxanthine-xanthine oxidase (HX-XO) increased the mitochondrial calcium concentration [Ca2+]m. Both agents caused a biphasic increase in [Ca2+]m which was preceded by a rise in cytosolic free calcium concentration [Ca2+]c (18 and 6 seconds for H2O2 and HX-XO, respectively). The peak and plateau elevations of [Ca2+] were consistently higher in the mitochondrial matrix than in the cytosol. In Ca2+-free/EGTA medium, the plateau phase of elevated [Ca2+] evoked by H2O2 due to capacitative Ca2+ influx was abolished in the cytosol, but was maintained in the mitochondria. In contrast to H2O2 and HX-XO, ATP which binds the P2Y purinoceptors induced an increase in [Ca2+]m that was similar to that of [Ca2+]c. When cells were first stimulated with inositol 1,4, 5-trisphosphate-generating agonists or the Ca2+-ATPase inhibitor cyclopiazonic acid (CPA), subsequent addition of H2O2 did not affect [Ca2+]c, but still caused an elevation of [Ca2+]m. Moreover, the specific inhibitor of the mitochondrial Ca2+/Na+ exchanger, 7-chloro-3,5-dihydro-5-phenyl-1H-4.1-benzothiazepine-2-on (CGP37157), did not potentiate the effects of H2O2 and HX-XO on [Ca2+]m, while causing a marked increase in the peak [Ca2+]m and a significant attenuation of the rate of [Ca2+]m efflux upon addition of histamine or CPA. In permeabilized cells, H2O2 mimicked the effects of CGP37157 causing an increase in the basal level of matrix free Ca2+ and decreased efflux. Dissipation of the electrochemical proton gradient by carbonylcyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), and blocade of the Ca2+ uptake by ruthenium red prevented [Ca2+]m increases evoked by H2O2. These results demonstrate that the H2O2-induced elevation in [Ca2+]m results from a transfer of Ca2+ secondary to increased [Ca2+]c, and an inhibition of the Ca2+/Na+ electroneutral exchanger of the mitochondria.  (+info)

Optical mapping of neural network activity in chick spinal cord at an intermediate stage of embryonic development. (7/1146)

We have applied multiple-site optical recording of transmembrane potential changes to recording of neuronal pathway/network activity from embryonic chick spinal cord slice preparations. Spinal cord preparations were dissected from 8-day-old chick embryos at Hamburger-Hamilton stage 33, and transverse slice preparations were prepared with the 13th cervical spinal nerve or with the 2nd or 5th lumbosacral spinal nerve intact. The slice preparations were stained with a voltage-sensitive merocyanine-rhodanine dye (NK2761). Transmembrane voltage-related optical (dye-absorbance) changes evoked by spinal nerve stimulation with positive square-current pulses using a suction electrode were recorded simultaneously from many loci in the preparation, using a 128- or 1,020-element photodiode array. Optical responses were detected from dorsal and ventral regions corresponding to the posterior (dorsal) and anterior (ventral) gray horns. The optical signals were composed of two components, fast spike-like and slow signals. In the dorsal region, the fast spike-like signal was identified as the presynaptic action potential in the sensory nerve and the slow signal as the postsynaptic potential. In the ventral region, the fast spike-like signal reflects the antidromic action potential in motoneurons, and the slow signal is related to the postsynaptic potential evoked in the motoneuron. In preparations in which the ventral root was cut microsurgically, the antidromic action potential-related optical signals were eliminated. The areas of the maximal amplitude of the evoked signals in the dorsal and ventral regions were located near the dorsal root entry zone and the ventral root outlet zone, respectively. Quasiconcentric contour-line maps were obtained in the dorsal and ventral regions, suggesting the functional arrangement of the dorsal and ventral synaptic connections. Synaptic fatigue induced by repetitive stimuli in the ventral synapses was more rapid than in the dorsal synapses. The distribution patterns of the signals were essentially similar among C13, LS2, and LS5 preparations, suggesting that there is no difference in the spatiotemporal pattern of the neural responses along the rostrocaudal axis of the spinal cord at this developmental stage. In the ventral root-cut preparations, comparing the delay times between the ventral slow optical signals, we have been able to demonstrate that neural network-related synaptic connections are generated functionally in the embryonic spinal cord at Hamburger-Hamilton stage 33.  (+info)

Correct targeting of dihydropyridine receptors and triadin in dyspedic mouse skeletal muscle in vivo. (8/1146)

Excitation-contraction coupling in skeletal muscle involves junctions (triads and dyads) between sarcoplasmic reticulum (SR) and transverse (T) -tubules. Two proteins of the junctional SR, ryanodine receptors (RyRs) and triadin and one protein of T tubules, dihydropyridine receptors (DHPRs) are located at these junctions. We studied the targeting of DHPRs and triadin to T-tubules and SR in skeletal muscles of dyspedic mouse embryos lacking RyR1. In normal differentiating muscle fibers DHPRs, triadin and RyRs are located in intensely immunolabeled foci that are randomly distributed across the fiber. Correlation with electron microscopy and with previous studies indicates that the foci represent the location of triads and dyads. In dyspedic fibers DHPRs and triadin antibodies stain internal foci of the two proteins; RyR antibodies are completely negative. The appearance and location of the foci in dyspedic fibers is similar to that of normal muscle, but their fluorescent intensity is weaker. The SR Ca-ATPase has more diffuse distribution than triadin in both normal and dyspedic fibers. These observations indicate that an interaction with RyRs is not necessary for the appropriate targeting of DHPRs or triadin to junctional domains of T tubules and SR respectively.  (+info)

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