Role of simian virus 40 Vp1 cysteines in virion infectivity.

We have developed a new nonoverlapping infectious viral genome (NO-SV40) in order to facilitate structure-based analysis of the simian virus 40 (SV40) life cycle. We first tested the role of cysteine residues in the formation of infectious virions by individually mutating the seven cysteines in the major capsid protein, Vp1. All seven cysteine mutants-C9A, C49A, C87A, C104A, C207S, C254A, and C267L-retained viability. In the crystal structure of SV40, disulfide bridges are formed between certain Cys104 residues on neighboring pentamers. However, our results show that none of these disulfide bonds are required for virion infectivity in culture. We also introduced five different mutations into Cys254, the most strictly conserved cysteine across the polyomavirus family. We found that C254L, C254S, C254G, C254Q, and C254R mutants all showed greatly reduced (around 100,000-fold) plaque-forming ability. These mutants had no apparent defect in viral DNA replication. Mutant Vp1's, as well as wild-type Vp2/3, were mostly localized in the nucleus. Further analysis of the C254L mutant revealed that the mutant Vp1 was able to form pentamers in vitro. DNase I-resistant virion-like particles were present in NO-SV40-C254L-transfected cell lysate, but at about 1/18 the amount in wild-type-transfected lysate. An examination of the three-dimensional structure reveals that Cys254 is buried near the surface of Vp1, so that it cannot form disulfide bonds, and is not involved in intrapentamer interactions, consistent with the normal pentamer formation by the C254L mutant. It is, however, located at a critical junction between three pentamers, on a conserved loop (G2H) that packs against the dual interpentamer Ca(2+)-binding sites and the invading C-terminal helix of an adjacent pentamer. The substitution by the larger side chains is predicted to cause a localized shift in the G2H loop, which may disrupt Ca(2+) ion coordination and the packing of the invading helix, consistent with the defect in virion assembly. Our experimental system thus allows dissection of structure-function relationships during the distinct steps of the SV40 life cycle.

[Differential regulation of the release of norepinephrine and neuropeptide Y]. / Régulation différentielle de la libération de noradrénaline et de neuropeptide Y.

The release of catecholamines and their co-neurotransmitter neuropeptide Y was investigated in conscious dogs with neurogenic arterial hypertension elicited by sinoaortic denervation. One month after denervation, an elevation of catecholamine levels (measured by HPLC) without elevation of neuropeptide Y levels in plasma (evaluated by RIA) has been found. This dissociation could be explained by a transient release of neuropeptide Y during the first weeks after surgery; a depletion of neuronal neuropeptide Y due to the permanent sympathetic stimulation; or an insufficient increase in sympathetic tone. To test these three hypotheses, we investigated the time courses of catecholamine and neuropeptide Y levels in arterial plasma during the first five weeks after sinoaortic denervation; and the responses to yohimbine (an alpha 2 antagonist which enhances transmitter release). Resting neuropeptide Y levels in plasma remained normal during the first five weeks after sinoaortic denervation. In normal dogs, a high dose of yohimbine (0.5 mg/kg i.v.) elevated both catecholamine (6-fold) and neuropeptide Y levels (1.5-fold), whereas a lower dose (0.05 mg/kg i.v.) induced a two fold elevation of catecholamine levels without changing neuropeptide Y concentrations. In sinoaortically denervated dogs, yohimbine elicited elevation of both catecholamines and neuropeptide Y whatever the dose used. Thus, neurogenic arterial hypertension in dogs seams to involve catecholamines but not neuropeptide Y. Moreover, the present work suggests that a high level of sympathetic stimulation is required for a co-release of catecholamines and neuropeptide Y.

Is neuropeptide Y co-released with catecholamines in experimental arterial hypertension following sinoaortic denervation?

The release of catecholamines and their coneurotransmitter neuropeptide Y (NPY) was investigated in conscious dogs with neurogenic arterial hypertension elicited by sinoaortic denervation. One month after denervation, an elevation of catecholamine levels (measured by HPLC) without elevation of NPY-like immunoreactivity (NPY-LI) levels in plasma (evaluated by RIA) has been found. This dissociation could be explained by 1) a transient release of NPY during the first weeks after surgery, 2) a depletion of neuronal NPY due to the permanent sympathetic stimulation, or 3) an insufficient increase in sympathetic tone. To test these hypotheses, we investigated the time courses of catecholamine and NPY-LI levels in arterial plasma during the first five weeks after sinoaortic denervation and responses to yohimbine (an alpha 2 antagonist which enhances transmitter release). Resting NPY-LI levels in plasma remained normal during the first five weeks after sinoaortic denervation. In normal dogs, a high dose of yohimbine (0.5 mg/kg i.v.) elevated both catecholamine (6-fold) and NPY-LI levels (1.5-fold), whereas a lower dose (0.05 mg/kg i.v.) induced a two fold elevation of catecholamine levels without changing NPY-LI concentrations. In sinoaortically denervated dogs, yohimbine elicited elevation of both catecholamines and NPY-LI whatever the dose used. Thus, neurogenic arterial hypertension in dogs seems to involve catecholamines but not NPY. Moreover, the present work suggests that a high level of sympathetic stimulation is required for a co-release of catecholamines and NPY.

Absence of arterial hypertension despite chronic plasma noradrenaline elevation: evidence for down-regulation of alpha-adrenoceptors. A case report.

A 44 year old-healthy female presented chronic and stable high levels of plasma noradrenaline (NA) without any major change in adrenaline. The diagnosis of phaeochromocytoma was discarded. These increased levels of NA offered an unique opportunity to investigate under in vivo conditions a putative regulation of alpha-adrenoceptors by this endogenous catecholamine. Infusion rates of exogenous NA up to 0.74 micrograms/kg per min were unable to induce any change in blood pressure (or heart rate) in the subject, In contrast, in normotensive controls, an increase in blood pressure (+ 15 mm Hg) was observed with 0.39 micrograms/kg per min. The magnitude of yohimbine-induced increase in plasma NA was similar in the subject and in the controls. Platelet alpha 2-adrenoceptors evaluated by specific [3H]-yohimbine binding showed a significantly lower level in the subject when compared to controls. The results show that a sustained increase in plasma NA is able to induce down-regulation of alpha-adrenoceptors. This down-regulation can explain the lack of arterial hypertension despite the increased sympathetic tone.

Effect of clomipramine treatment on dog platelet alpha 2-adrenergic receptivity.

The effects of clomipramine treatment (5 mg/kg s.c. a day during 21 days) on dog platelet alpha 2-adrenoreceptivity were investigated. The radioligand binding studies on platelet membrane preparations showed that the antidepressant treatment promotes a decrease in the number of [3H]-yohimbine binding sites. The measurement of the inhibition of PGE1-induced cyclic AMP accumulation in the whole platelet indicated that the biological event immediately associated with alpha 2-adrenergic stimulation was not modified. The aggregatory response to ADP or ADP plus adrenaline was totally abolished in clomipramine-treated platelets. The diastolic pressor responses to noradrenaline or adrenaline injections (after propranolol and prazosin administration) in chloralose anaesthetized dogs were not affected by clomipramine treatment. This work shows that variations in the binding capacities of alpha 2-adrenergic sites are not always linked to modifications of related physiological events.

Adrenergic neurohumoral influences on FFA release from bone marrow adipose tissue.

It is well known that both the sympathetic nervous system and several hormones control adipose tissue lipolysis. However, the relative importance of these different lipolytic agents may vary according to the animal species and according to the adipose territories in a same species. The action of noradrenaline administration on tibial intraosseous lipolysis was compared with the effects of splanchnic nerve and lumbar sympathetic chain stimulation in chloralose-anaesthetized dogs. The osseous nutrient artery was infused through the homolateral femoral artery in order to check constant blood flow. The levels of free fatty acids (FFA) were compared in femoral artery blood and in the osseous nutrient vein. Noradrenaline was injected in the infusion line. Lumbar sympathetic chain and splanchnic nerve were stimulated at frequencies of 3-10 Hz and 5-10 Hz respectively. Noradrenaline (at 411 p mol/min i.e. a dose three times higher than that required for adrenaline) induced FFA mobilization from tibial adipose bone marrow. Splanchnic nerve stimulation elicited an increase in the FFA levels in the nutrient vein whereas lumbar sympathetic chain stimulation had no remained without any effect. It is concluded that circulating adrenaline seems more important than noradrenaline released by sympathetic nerve endings in the control of activity of tibial bone marrow adipocytes.

Interplay of alpha-2 and beta adrenoceptors in the control of free fatty acid release from bone marrow adipose tissue.

The effect of various adrenergic drugs on the free fatty acid (FFA) output from bone marrow was studied in dog tibia after constant flow autoperfusion of the nutrient artery by femoral arterial blood and cannulation of the nutrient vein. Clonidine, an alpha-2 adrenoceptor agonist, inhibited the isoproterenol-induced FFA outflow from bone marrow adipose tissue. This effect was suppressed by the alpha-2 antagonists (phentolamine and yohimbine). Moreover, clonidine inhibited the FFA outflow initiated by theophylline associated with adenosine deaminase. When the alpha-2 sites were blocked by phentolamine or yohimbine, epinephrine exerted an enhanced effect on FFA outflow in comparison with its effect when infused alone. These in vivo experiments clearly demonstrate that alpha-2 adrenoceptor stimulation is able to inhibit beta-stimulated or theophylline plus adenosine deaminase-promoted lipid mobilization from bone marrow fat stores. The simultaneous stimulation of alpha-2 and beta adrenoceptors initiated by epinephrine could be responsible for the weaker lipid-mobilizing effect of the endogenous catecholamine. It is concluded that adrenoceptors are operative in bone marrow adipose tissue and control FFA outflow in physiological conditions. Moreover, the existence of an interplay between alpha-2 and beta adrenergic effects in in vivo conditions is demonstrated.

Effects of catecholamines on free fatty acid release from bone marrow adipose tissue.

We have studied the effect of epinephrine and isoproterenol on free fatty acid (FFA) mobilization from bone marrow adipose tissue in dog tibia after constant-flow autoperfusion of the nutrient artery by ipsilateral femoral arterial blood. The perfusions of epinephrine (0.025 microgram/min) or isoproterenol (0.005 microgram/min) significantly increased the FFA level in the nutrient vein of the tibia. Moreover, our data demonstrate that in vitro the bone marrow adipose tissue was less responsive to catecholamines than omental adipose tissue. It can be concluded that bone marrow adipose tissue is able to release FFA after administration of catecholamines but to a lesser extent than in other adipose tissue (omental adipose tissue). These results support the hypothesis that the bone marrow adipose tissue is involved in local nutrition rather than in the total energy supply of the animal.