The Carboxyl-Terminus of Human Immunodeficiency Virus Type 2 Circulating Recombinant form 01_AB Capsid Protein Affects Sensitivity to Human TRIM5α

Human immunodeficiency virus (HIV) type 2 shows limited geographical distribution compared with HIV type 1. Although 8 genetic groups of HIV type 2 (HIV-2) have been described, recombinant viruses between these groups are rarely observed. Recently, three HIV-2 patients in Japan were described with rapidly progressive, acquired immunodeficiency. These patients were infected with an A/B inter-group recombinant designated CRF01_AB. Here, we characterize the capsid protein (CA) encoded by the viruses from these patients. HIV-2 CRF01_AB CA showed unique amino acid sequence almost equally distinct from group A and group B viruses. Notably, HIV-2 CRF01_AB CA showed potent resistance to human TRIM5α. In addition to the previously identified amino acid position 119 in the N-terminal domain of CA, we found that HIV-2 CRF01_AB-specific amino acid substitutions in the C-terminal domain also were necessary for resistance to human TRIM5α. These results indicate that retroviruses can evade TRIM5α by substitution at residues within the C-terminal domain of CA.     

Inhibitory effect of chicken gonadotropin-releasing hormone II on food intake in the goldfish, Carassius auratus

Gonadotropin-releasing hormone (GnRH) is an evolutionarily conserved neuropeptide with 10 amino acid residues, which possesses some structural variants. A molecular form known as chicken GnRH II ([His⁵ Trp⁷ Tyr⁸] GnRH, cGnRH II) is widely distributed in vertebrates, and has recently been implicated in the regulation of sexual behavior and food intake in an insectivore, the musk shrew. However, the influence of cGnRH II on feeding behavior has not yet been studied in model animals such as rodents and teleost fish. In this study, therefore, we investigated the role of cGnRH II in the regulation of feeding behavior in the goldfish, and examined its involvement in food intake after intracerebroventricular (ICV) administration. ICV-injected cGnRH II at graded doses, from 0.1 to 10 pmol/g body weight (BW), induced a decrease of food consumption in a dose-dependent manner during 60 min after treatment. Cumulative food intake was significantly decreased by ICV injection of cGnRH II at doses of 1 and 10 pmol/g BW during the 60-min post-treatment observation period. ICV injection of salmon GnRH ([Trp⁷ Leu⁸] GnRH, sGnRH) at doses of 0.1-10 pmol/g BW did not affect food intake. The anorexigenic action of cGnRH II was completely blocked by treatment with the GnRH type I receptor antagonist, Antide. However, the anorexigenic action of cGnRH II was not inhibited by treatment with the corticotropin-releasing hormone (CRH) 1/2 receptor antagonist, α-helical CRH₍₉₋₄₁₎, and the melanocortin 4 receptor antagonist, HS024. These results suggest that, in the goldfish, cGnRH II, but not sGnRH, acts as an anorexigenic factor, as is the case in the musk shrew, and that the anorexigenic action of cGnRH II is independent of CRH- and melanocortin-signaling pathways.     

Alpha-melanocyte-stimulating hormone mediates melanin-concentrating hormone-induced anorexigenic action in goldfish

In goldfish, intracerebroventricular (ICV) administration of melanin-concentrating hormone (MCH) inhibits feeding behavior, and fasting decreases hypothalamic MCH-like immunoreactivity. However, while MCH acts as an anorexigenic factor in goldfish, in rodents MCH has an orexigenic effect. Therefore, we examined the involvement of two anorexigenic neuropeptides, alpha-melanocyte-stimulating hormone (alpha-MSH) and corticotropin-releasing hormone (CRH), in the anorexigenic action of MCH in goldfish, using an alpha-MSH receptor antagonist, HS024, and a CRH receptor antagonist, alpha-helical CRH((9-41)). ICV injection of HS024, but not alpha-helical CRH((9-41)), suppressed MCH-induced anorexigenic action for a 60-min observation period. We then examined, using a real-time PCR method, whether MCH affects the levels of mRNAs encoding various orexigenic neuropeptides, including neuropeptide Y (NPY), orexin, ghrelin and Agouti-related peptide (AgRP), in the goldfish diencephalon. ICV administration of MCH at a dose sufficient to inhibit food consumption decreased the expression of mRNAs for NPY and ghrelin, but not for orexin and AgRP. These results indicate that the anorexigenic action of MCH in the goldfish brain is mediated by the alpha-MSH signaling pathway and is accompanied by inhibition of NPY and ghrelin synthesis.     

CSF orexin-A/hypocretin-1 concentrations in patients with intracerebral hemorrhage (ICH)

Orexins/hypocretins are neuropeptides that have various physiological effects, including the regulation of both the feeding behavior neuroendocrine functions and sleep-wakefulness cycle. Recent studies have suggested that the orexin system may also be involved in neuronal damage in the clinical setting and animal experiments. The aim of this study was to examine the role of the hypothalamic orexin-A/hypocretin-1 system in patients with intracerebral hemorrhage (ICH). The CSF orexin-A/hypocretin-1 levels were measured in 11 ICH patients. CSF orexin-A/hypocretin-1 levels were low in ICH patients during the 13 days following the ICH event. The mean CSF orexin-A/hypocretin-1 levels were 61.1+/-22.3 (S.D.) pg/ml (range 27.5-106.9 pg/ml).The decreasing in the CSF orexin-A/hypocretin-1 levels was not related to the severity of ICH. The CSF orexin-A/hypocretin-1 levels were lower in the thalamic hemorrhage patients than those in other patients (48.5+/-23.3 pg/ml vs. 65.2+/-21.2 pg/ml; p=0.03.) These data indicate that orexin-A/hypocretin-1 may therefore play an important role in the various physiological responses including sleep, feeding, and the overall metabolism in ICH patients.     

Neuropeptide W in the rat pancreas: potentiation of glucose-induced insulin release and Ca2+ influx through L-type Ca2+ channels in beta-cells and localization in islets

Neuropeptide W (NPW) is a regulatory peptide that acts via two subtypes of G protein-coupled receptors, GPR7 and GPR8. Evidence has been provided that NPW is involved in the central regulation of energy homeostasis and feeding behavior. In this study, we examined the effects of NPW on insulin release and localization of NPW in the rat pancreas. NPW (10-100 nM) significantly increased insulin release in the presence of 8.3 mM, but not 2.8 mM, glucose in the isolated rat islets. By fura-2 microfluorometry, NPW (1-100 nM) concentration-dependently increased cytosolic Ca(2+) concentration ([Ca(2+)](i)) at 8.3 mM glucose in rat single beta-cells. The NPW-induced [Ca(2+)](i) increase was abolished under external Ca(2+)-free conditions and by an L-type Ca(2+) channel blocker nifedipine (10 microM). RT-PCR analysis revealed that mRNA for NPW was expressed in the rat pancreas and hypothalamus. Double immunohistochemical analysis showed that NPW-immunoreactivity was found in islets and co-localized with insulin-containing beta-cells, but not glucagon-containing alpha-cells and somatostatin-containing delta-cells. These results suggest that NPW could serve as a local modulator of glucose-induced insulin release in rat islets. NPW directly activates beta-cells to enhance Ca(2+) influx through voltage-dependent L-type Ca(2+) channels and potentiates glucose-induced insulin release.     

Neuronal interaction between melanin-concentrating hormone- and alpha-melanocyte-stimulating hormone-containing neurons in the goldfish hypothalamus

Intracerebroventricular (ICV) administration of melanin-concentrating hormone (MCH) inhibits food intake in goldfish, unlike in rodents, suggesting that its anorexigenic action is mediated by alpha-melanocyte-stimulating hormone (alpha-MSH) but not corticotropin-releasing hormone. This led us to investigate whether MCH-containing neurons in the goldfish brain have direct inputs to alpha-MSH-containing neurons, using a confocal laser scanning microscope, and to examine whether the anorexigenic action of MCH is also mediated by other anorexigenic neuropeptides, such as cholecystokinin (CCK) and pituitary adenylate cyclase-activating polypeptide (PACAP), using their receptor antagonists. MCH- and alpha-MSH-like immunoreactivities were distributed throughout the brain, especially in the diencephalon. MCH-containing nerve fibers or endings lay in close apposition to alpha-MSH-containing neurons in the hypothalamus in the posterior part of the nucleus lateralis tuberis (NLTp). The inhibitory effect of ICV-injected MCH on food intake was not affected by treatment with a CCK A/CCK B receptor antagonist, proglumide, or a PACAP receptor (PAC(1) receptor) antagonist, PACAP((6-38)). ICV administration of MCH at a dose sufficient to inhibit food consumption also did not influence expression of the mRNAs encoding CCK and PACAP. These results strongly suggest that MCH-containing neurons provide direct input to alpha-MSH-containing neurons in the NLTp of goldfish, and that MCH plays a crucial role in the regulation of feeding behavior as an anorexigenic neuropeptide via the alpha-MSH (melanocortin 4 receptor)-signaling pathway.     

Generation of constitutively active calcineurin by calpain contributes to delayed neuronal death following mouse brain ischemia

Calpain, a Ca(2+)-dependent cysteine protease, in vitro converts calcineurin (CaN) to constitutively active forms of 45 kDa and 48 kDa by cleaving the autoinhibitory domain of the 60 kDa subunit. In a mouse middle cerebral artery occlusion (MCAO) model, calpain converted the CaN A subunit to the constitutively active form with 48 kDa in vivo. We also confirmed increased Ca(2+)/CaM-independent CaN activity in brain extracts. The generation of constitutively active and Ca(2+)/CaM-independent activity of CaN peaked 2 h after reperfusion in brain extracts. Increased constitutively active CaN activity was associated with dephosphorylation of dopamine-regulated phosphoprotein-32 in the brain. Generation of constitutively active CaN was accompanied by translocation of nuclear factor of activated T-cells (NFAT) into nuclei of hippocampal CA1 pyramidal neurons. In addition, a novel calmodulin antagonist, DY-9760e, blocked the generation of constitutively active CaN by calpain, thereby inhibiting NFAT nuclear translocation. Together with previous studies indicating that NFAT plays a critical role in apoptosis, we propose that calpain-induced CaN activation in part mediates delayed neuronal death in brain ischemia.     

Rho-kinase phosphorylates PAR-3 and disrupts PAR complex formation

A polarity complex of PAR-3, PAR-6, and atypical protein kinase C (aPKC) functions in various cell polarization events. PAR-3 directly interacts with Tiam1/Taim2 (STEF), Rac1-specific guanine nucleotide exchange factors, and forms a complex with aPKC-PAR-6-Cdc42*GTP, leading to Rac1 activation. RhoA antagonizes Rac1 in certain types of cells. However, the relationship between RhoA and the PAR complex remains elusive. We found here that Rho-kinase/ROCK/ROK, the effector of RhoA, phosphorylated PAR-3 at Thr833 and thereby disrupted its interaction with aPKC and PAR-6, but not with Tiam2. Phosphorylated PAR-3 was observed in the leading edge, and in central and rear portions of migrating cells having front-rear polarity. Knockdown of PAR-3 by small interfering RNA (siRNA) impaired cell migration, front-rear polarization, and PAR-3-mediated Rac1 activation, which were recovered with siRNA-resistant PAR-3, but not with the phospho-mimic PAR-3 mutant. We propose that RhoA/Rho-kinase inhibits PAR complex formation through PAR-3 phosphorylation, resulting in Rac1 inactivation.     

Morphological interaction between galanin-like peptide- and dopamine-containing neurons in the rat arcuate nucleus

Galanin-like peptide (GALP) is a 60-amino acid neuropeptide that plays an important role in the neuronal regulation of feeding, energy balance and reproduction. GALP is produced in the hypothalamic arcuate nucleus, an area containing, amongst other neuron types, two populations of neurons in which we were interested: a population of GALP-containing neurons which regulate energy balance and reproduction, and a second population consisting of tuberoinfundibular dopaminergic neurons which suppress prolactin secretion from the adenohypophysis. To characterize morphologically the relationship between GALP and dopamine-containing neurons in the arcuate nucleus, a double immunofluorescence study was performed on cryosections from rat brain. Immunohistochemical double labeling studies revealed that GALP-immunoreactive nerve fibers made direct contact on tyrosine hydroxylase (TH)-containing neuronal cell bodies in the arcuate nucleus. These results suggest that GALP-containing neurons innervate tuberoinfundibular dopaminergic neurons.