A Host YB-1 Ribonucleoprotein Complex Is Hijacked by Hepatitis C Virus for the Control of NS3-Dependent Particle Production

Hepatitis C virus (HCV) orchestrates the different stages of its life cycle in time and space through the sequential participation of HCV proteins and cellular machineries; hence, these represent tractable molecular host targets for HCV elimination by combination therapies. We recently identified multifunctional Y-box-binding protein 1 (YB-1 or YBX1) as an interacting partner of NS3/4A protein and HCV genomic RNA that negatively regulates the equilibrium between viral translation/replication and particle production. To identify novel host factors that regulate the production of infectious particles, we elucidated the YB-1 interactome in human hepatoma cells by a quantitative mass spectrometry approach. We identified 71 YB-1-associated proteins that included previously reported HCV regulators DDX3, heterogeneous nuclear RNP A1, and ILF2. Of the potential YB-1 interactors, 26 proteins significantly modulated HCV replication in a gene-silencing screening. Following extensive interaction and functional validation, we identified three YB-1 partners, C1QBP, LARP-1, and IGF2BP2, that redistribute to the surface of core-containing lipid droplets in HCV JFH-1-expressing cells, similarly to YB-1 and DDX6. Importantly, knockdown of these proteins stimulated the release and/or egress of HCV particles without affecting virus assembly, suggesting a functional YB-1 protein complex that negatively regulates virus production. Furthermore, a JFH-1 strain with the NS3 Q221L mutation, which promotes virus production, was less sensitive to this negative regulation, suggesting that this HCV-specific YB-1 protein complex modulates an NS3-dependent step in virus production. Overall, our data support a model in which HCV hijacks host cell machinery containing numerous RNA-binding proteins to control the equilibrium between viral RNA replication and NS3-dependent late steps in particle production.     

An analytical model of traumatic diffuse brain injury

Diffuse axonal injury (DAI) with prolonged coma has been produced in the primate using an impulsive, rotational acceleration of the head without impact. This pathophysiological entity has been studied subsequently from a biomechanics perspective using physical models of the skull-brain structure. Subjected to identical loading conditions as the primate, these physical models permit one to measure the deformation within the surrogate brain tissue as a function of the forces applied to the head. An analytical model designed to approximate these experiments has been developed in order to facilitate an analysis of the parameters influencing brain deformation. These three models together are directed toward the development of injury tolerance criteria based upon the shear strain magnitude experienced by the deep white matter of the brain. The analytical model geometry consists of a rigid, right-circular cylindrical shell filled with a Kelvin-Voigt viscoelastic material. Allowing no slip on the boundary, the shell is subjected to a sudden, distributed, axisymmetric, rotational load. A Fourier series representation of the load allows unrestricted load-time histories. The exact solution for the relative angular displacement (V) and the infinitesimal shear strain (epsilon) at any radial location in the viscoelastic material with respect to the shell was determined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presence of dopamine-immunoreactive cell bodies in the catecholaminergic group A15 of the sheep brain

Summary Antisera were raised in rabbits against dopamine or noradrenaline conjugated to thyroglobulin with glutaraldehyde. These antisera, tested in enzyme linked immunosorbent assay and immunohistochemistry specifically recognized their homologous antigens.With the aid of anti-tyrosine hydroxylase, anti-aromatic aminoacid decarboxylase, anti-dopamine--hydroxylase, anti-dopamine, and anti-noradrenaline antisera, immunohistochemical reactions were performed on glutaraldehyde fixed sections of sheep diencephalon in order to determine the presence of dopamine in the catecholaminergic group A15. Perikarya of this nucleus were stained with anti-tyrosine hydroxylase, anti-aromatic aminoacid decarboxylase and anti-dopamine, but not with anti-dopamine--hydroxylase or anti-noradrenaline. Both of these latter antisera stained fibers within this area. So as recently found in the rat, we could conclude that dopamine is present in group A15 of the sheep.     

Distribution of catecholaminergic and serotoninergic systems in forebrain and midbrain of the newt,Triturus alpestris (Urodela)

Summary Mapping of monoaminergic systems in the brain of the newt Triturus alpestris was achieved with antisera against (1) thyrosine hydroxylase (TH), (2) formaldehyde-conjugated dopamine (DA), and (3) formaldehyde-conjugated serotonin (5-HT). In the telencephalon, the striatum was densely innervated by a large number of 5-HT-, DA-and TH-immunoreactive (IR) fibers; IR fibers were more scattered in the amygdala, the medial and lateral forebrain bundles, and the anterior commissure. In the anterior and medial diencephalon, TH-IR perikarya contacting the cerebrospinal fluid (CSF-C perikarya) were located in the preoptic recess organ (PRO), the organum vasculosum laminae terminalis and the suprachiasmatic nucleus. Numerous TH-IR perikarya, not contacting the CSF, were present in the posterior preoptic nucleus and the ventral thalamus. At this level, DA-IR CSF-C neurons were only located in the PRO. In the posterior diencephalon, large populations of 5-HT-IR and DA-IR CSF-C perikarya were found in the paraventricular organ (PVO) and the nucleus infundibularis dorsalis (NID); the dorsal part of the NID additionally presented TH-IR CSF-C perikarya. Most regions of the diencephalon showed an intense monoaminergic innervation. In addition, numerous TH-IR, DA-IR and 5-HT-IR fibers, orginating from the anterior and posterior hypothalamic nuclei, extended ventrally and reached the median eminence and the pars intermedia of the pituitary gland. In the midbrain, TH-IR perikarya were located dorsally in the pretectal area. Ventrally, a large group of TH-IR cell bodies and some weakly stained DA-IR and 5-HT-IR neurons were observed in the posterior tuberculum. No dopaminergic system equivalent to the substantia nigra was revealed. The possible significance of the differences in the distribution of TH-IR and DA-IR neurons is discussed, with special reference to the CSF-C neurons.Abbreviations AM amygdala - CAnt commissura anterior - CH commissura hippocampi - CP commissura posterior - Ctm commissura tecti mesencephali - DH dorsal hypothalamus - DTh dorsal thalamus - FLM fasciculus longitudinalis medialis - Fsol fasciculus solitarius - H habenula - LFB lateral forebrain bundle - ME median eminence - MFB medial forebrain bundle - NID nucleus infundibularis dorsalis - nIP neuropil of nucleus interpeduncularis - NPOP nucleus preopticus posterior - NS nucleus septi - OVLT organum vasculosum laminae terminalis - PD pars distalis - Pdo dorsal pallium - PHi primordium hippocampi - PI pars intermedia - Pl lateral pallium - PN pars nervosa - PRO preoptic recess organ - Ptec pretectal area - PVO paraventricular organ - Ra nucleus raphe - Rm nucleus reticularis medius - SCO subcommisural organ - ST striatum; strm stria medullaris thalami - strt stria terminalis thalami - TM tegmentum mesencephali - TO tectum opticum - TP tuberculum posterius - trch tractus cortico-habenularis - trmp tractus mamillopeduncularis - VH ventral hypothalamus - Vm nucleus motorius nervi trigemini - VTh ventral thalamus - II optic nerve     

Comparative biological activities of ANF (Arg 101-Tyr 126) and the synthetic form of circulating ANF (Ser 99-Tyr 126)

The biological activities of ANF (Arg 101-Tyr 126) and of the circulating form, ANF (Ser 99-Tyr 126), were compared in the following assays: precontracted rabbit aortic strip and chick rectum, rat natriuresis, inhibition of aldosterone secretion and receptor affinity in bovine and rat adrenal zona glomerulosa cells, and receptor affinity in rabbit aorta and rat mesenteric artery cells. The results demonstrate that both peptides share the same biological activities. It is concluded that the addition of two amino acids to the N-terminal of ANF (Arg 101-Tyr 126) does not modify its biological characteristics, validating thus previous research employing this peptide.     

In vitro translation of human pheochromocytoma messenger RNAs: characterization of tyrosine-hydroxylase and dopamine-beta-hydroxylase

mRNAs extracted from human pheochromocytoma were translated in vitro in a lysate of a rabbit reticulocytes. Two enzymes of the biosynthetic pathway of the catecholamines, tyrosine-hydroxylase (TH) and dopamine-beta-hydroxylase (DBH), were characterized as translation products after immunoprecipitation by specific antisera and electrophoretic analysis. The precursor of TH is a polypeptide having a molecular mass of 62,000 identical to that found for the mature protein. The molecular mass of the precursor of DBH 73,000 while that of the mature form is 79,000. TH and DBH have been translated from mRNAs having sedimentation coefficients of 22S and 25S, respectively.     

Immunohistochemical demonstration of catecholaminergic cell bodies in the spinal cord of the rat

Summary In order to elucidate the anatomy of the spinal dopaminergic system, an immunohistochemical study using a tyrosine-hydroxylase (TH) antibody was undertaken in the rat. Intracisternal 6-hydroxydopamine (6-OHDA) injections were administered to destroy most of the noradrenergic fibres that descende to the spinal cord while preserving the dopaminergic fibres. The density of the remaining TH-like immunoreactive fibres was relatively low at all levels of the spinal cord; the highest density was observed in layers III, IV and X. In addition, we report the first evidence for the existence of TH-like immunoreactive cell bodies at definite levels (especially sacral) of the spinal cord.     

Effect of enalapril (MK-421), an orally active angiotensin I converting enzyme inhibitor, on blood pressure, active and inactive plasma renin, urinary prostaglandin E2, and kallikrein excretion in conscious rats

The angiotensin I converting enzyme (ACE) inhibitor enalapril (MK-421), at a dose of 1 mg/kg or more by gavage twice daily, effectively inhibited the pressor response to angiotensin I for more than 12 h and less than 24 h. Plasma renin activity (PRA) did not change after 2 or 4 days of treatment at 1 mg/kg twice daily despite effective ACE inhibition, whereas it rose significantly at 10 mg/kg twice daily. Blood pressure fell significantly and heart rate increased in rats treated with 10 mg/kg of enalapril twice daily, a response which was abolished by concomitant angiotensin II infusion. However, infusion of angiotensin II did not prevent the rise in plasma renin. Enalapril treatment did not change urinary immunoreactive prostaglandin E2 (PGE2) excretion and indomethacin did not modify plasma renin activity of enalapril-treated rats. Propranolol significantly reduced the rise in plasma renin in rats receiving enalapril. None of these findings could be explained by changes in the ratio of active and inactive renin. Water diuresis, without natriuresis and with a decrease in potassium urinary excretion, occurred with the higher dose of enalapril. Enalapril did not potentiate the elevation of PRA in two-kidney one-clip Goldblatt hypertensive rats. In conclusion, enalapril produced renin secretion, which was in part beta-adrenergically mediated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ammonia exchange and photorespiration in chlamydomonas

Two hours after the addition of l-methionine-dl-sulfoximine to the cell suspension, glutamine synthetase activity was inhibited by more than 90% in air-grown Chlamydomonas reinhardii. Cells continued to take up NH₃ from the medium provided that the concentration of dissolved CO₂ was high (equilibrated with 4% CO₂ in air). This NH₃ uptake, about 30% of the control, is discussed in terms of glutamate dehydrogenase activity. Without CO₂, or with a low CO₂ level, a NH₃ excretion was observed, the rate of which depended on the actual concentration of the dissolved CO₂. Experiments with ¹⁵NH₃ demonstrated that no NH₃ uptake was masked by this excretion and inversely that no excretion occurred during the uptake in the conditions where it took place. Furthermore, the NH₃ excretion observed in the absence of CO₂ increased when O₂ concentration rose to 15% and was inhibited when 10 millimolar isonicotinic acid hydrazide was supplied to the algal suspension. Thus, NH₃ excretion in the presence of l-methionine-dl-sulfoximine seems to be related to a photorespiratory process inasmuch as it presents the same properties with regard to the O₂ and the isonicotinic acid hydrazide effects. These results favor the hypothesis that NH₃ produced in the medium originates from the glycine to serine reaction. On the other hand, partial inhibition (50%) of photosynthesis by l-methionine-dl-sulfoximine was attributed to uncoupling between electron transfer and photophosphorylation due to NH₃ accumulation into the cell.     

Critical review on quantitative autoradiography of D1 and D2 dopaminergic receptors in the striatum of the mammalian brain: differential localization and plastic changes after pharmacological manipulation and dopaminergic input disruption

Major technical progress in the development of computer-based image analysis systems has made possible the entry of autoradiographic and immunohistochemical techniques into a new era where quantification via densitometry and morphometry has become easily accessible. In this context, quantitative biochemical data can be adapted to anatomical and histological resolution. This adaptation is most efficient in the neuroscience fields because of the huge importance of cellular communication via neuronal networks in the nervous system. Therefore, any experimental approach to the brain which considers the brain as a 'black box' appears now as very crude. In fact, subtle heterogeneity in the distribution of biochemical markers can now be demonstrated, as illustrated here by the use of quantitative autoradiography of D1 and D2 dopaminergic receptors in the striatum of the mammalian brain. Also, local adaptive changes resulting from chronic blockade of the dopaminergic input can be detected after repeated treatments with dopaminergic antagonists selective for D1 or D2 receptors or with surgical lesioning of the dopaminergic nigrostriatal pathway. The resulting plastic changes are unevenly distributed throughout the striatal target organ and vary according to the mode of suppressing the dopaminergic flow: direct destruction of the dopaminergic pathway or selective pharmacological manipulation without physical elimination of the dopaminergic cells themselves. All these results are discussed and reviewed in light of the most recent reports in this field.