Effects of orexins/hypocretins on neuronal activity in the paraventricular nucleus of the thalamus in rats in vitro

Orexin-A (ORX-A) and orexin-B (ORX-B), also called hypocretin-1 and hypocretin-2, respectively, act upon orexin 1 (OX1R) and orexin 2 (OX2R) receptors, and are involved in the regulation of sleep-wakefulness and energy homeostasis. Orexin neurons in the lateral hypothalamic perifornical region project heavily to the paraventricular nucleus of the thalamus (PVT), which is deeply involved in the control of motivated behaviors. In the present study, electrophysiological and cytosolic Ca2+ concentration ([Ca2+]i) imaging studies on the effects of ORX-A and ORX-B on neurons in the PVT were carried out in rat brain slice preparations. ORX-A and/or ORX-B were applied extracellularly in the perfusate. Extracellular recordings showed that about 80% of the PVT neurons were excited dose-dependently by both ORX-A and ORX-B at concentrations of 10(-8) to 10(-6)M, and the increase in firing rate was about three times larger for ORX-B than for ORX-A at 10(-7)M. When both ORX-A and ORX-B were applied simultaneously at 10(-7)M, the increase in firing rate was almost equal to that of ORX-B at 10(-7)M, suggesting that the PVT neurons do not show a high affinity to ORX-A which is expected if they have OX1R receptors. The excitatory effect of ORX-B was seen in low Ca2+ and high Mg2+ ACSF as well as in normal ACSF, and the increase in firing rate was greater in low Ca2+ and high Mg2+ ACSF than in normal ACSF. [Ca2+]i imaging studies demonstrated that [Ca2+]i was increased in about 50% of the PVT neurons by both 10(-7)M ORX-A and ORX-B with a stronger effect for ORX-B, and the increase in [Ca2+]i induced by ORX-B was abolished in Ca2+-free ACSF, suggesting that ORX-B does not release Ca2+ from intracellular Ca2+ stores. Subsequent whole cell patch clamp recordings revealed that an after hyperpolarization seen following each action potential in normal ACSF disappeared in Ca2+-free ACSF, and the mean magnitude of the depolarization induced by ORX-B was same in normal, Ca2+-free and TTX-containing Ca2+-free ACSFs. Furthermore, ORX-B-induced depolarization was reversed to hyperpolarization when membrane potential was lowered to about -97 mV, and an increase of extracellular K+ concentration from 4.25 to 13.25 mM abolished the ORX-B-induced depolarization, indicating that the ORX-B-induced depolarization is associated with an increase in the membrane resistance resulting from a closure of K+ channels. These results suggest that orexins depolarize and excite post-synaptically PVT neurons via OX2R receptors, and that orexin-activated PVT neurons play a role in the integration of sleep-wakefulness and energy homeostasis, and in the control of motivated behaviors.     

The neck domain of myosin II primarily regulates the actomyosin kinetics, not the stepsize

In order to study the role of the neck domain of myosin in muscle contraction, we measured the steps of individual myosin II molecules engineered to have no neck domain (light chain-binding domain) by optical trapping nanometry. The actin filament and myosin cofilaments interacted on a glass surface to minimize the angle between them, and to minimize the interaction between myosin and the glass surface. The results showed that the average myosin stepsize did not change much when the neck domain was removed, but the sliding velocity decreased approximately fivefold. Furthermore, the duration of steps for neckless myosin was several times longer at saturated ATP concentration, indicating that the slower velocity was due to a slower dissociation rate of myosin heads from actin. From these data, we conclude that the neck domain of myosin-II primarily regulates the actomyosin kinetics, not the mechanics.     

A novel L-glutamate transporter inhibitor reveals endogenous D-aspartate homeostasis in rat pheochromocytoma MPT1 cells

We previously reported for the first time that D-aspartate (D-Asp) is biosynthesized by cultured mammalian cells such as pheochromocytoma (PC)12 cells and its subclone MPT1 (FEBS Lett. 434 (1998) 231, Arch. Biochem. Biophys. 404 (2002) 92). We speculated that D-Asp levels in the intra- and extracellular spaces of the cultured cells are maintained in a dynamic state of homeostasis. To test this here, we utilized a novel and potent L-Glu transporter inhibitor, TFB-TBOA. This inhibitor proved to be a genuine nontransportable blocker of the transporter even during long periods of culture. Use of this inhibitor with MPT1 cells confirmed that D-Asp levels are in a dynamic steady state where it is constantly released into the extracellular space by a yet undefined mechanism as well as being constantly and intensively taken up by the cells via the L-Glu transporter. We estimated the rate with which D-Asp is constitutively released from MPT1 cells is approx. 3.8 pmol/h/1x10(5) cells.     

Potential ability of hot water adzuki (Vigna angularis) extracts to inhibit the adhesion, invasion, and metastasis of murine B16 melanoma cells

The 40% ethanol eluent of the fraction of hot-water extract from adzuki beans (EtEx.40) adsorbed onto DIAION HP-20 resin has many biological activities, for example, antioxidant, antitumorigenesis, and intestinal alpha-glucosidase suppressing activities. This study examined the inhibitory effect of EtEx.40 on experimental lung metastasis and the invasion of B16-BL6 melanoma cells. EtEx.40 was found significantly to reduce the number of tumor colonies. It also inhibited the adhesion and migration of B16-BL6 melanoma cells into extracellular matrix components and their invasion into reconstituted basement membrane (matrigel) without affecting cell proliferation in vitro. These in vivo data suggest that EtEx.40 possesses a strong antimetastatic ability, which might be a lead compound in functional food development.     

Mode of action on deacetylation of acetylated methyl glycoside by cellulose acetate esterase from Neisseria sicca SB

The regioselective deacetylation of purified cellulose acetate esterase from Neisseria sicca SB was investigated on methyl 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside and 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranoside. The substrates were used as model compounds of cellulose acetate in order to estimate the mechanism for deacetylation of cellulose acetate by the enzyme. The enzyme rapidly deacetylated at position C-3 of methyl 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside to accumulate 2,4,6-triacetate as the main initial reaction product in about 70% yield. Deacetylation was followed at position C-2, and generated 4,6-diacetate in 50% yield. The enzyme deacetylated the product at positions C-4 and C-6 at slower rates, and generated 4- and 6-monoacetates at a later reaction stage. Finally, it gave a completely deacetylated product. For 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranoside, CA esterase deacetylated at positions C-3 and C-6 to give 2,4,6- and 2,3,4-triacetate. Deacetylation proceeded sequentially at positions C-3 and C-6 to accumulate 2,4-diacetate in 55% yield. The enzyme exhibited regioselectivity for the deacetylation of the acetylglycoside.     

Hypoxic stress enhances osteoclast differentiation via increasing IGF2 production by non-osteoclastic cells

Development of bone depends on a continuous supply of bone-degrading osteoclasts. Although several factors such as cytokines and integrins have been shown to be important for osteoclast recruitment, their mechanism of action is poorly understood. In this study, we demonstrated the enhancement of osteoclast formation by hypoxia and investigated the molecular mechanisms involved. Primary mouse bone marrow cells were cultured in normoxic and hypoxic conditions, and RNA was prepared from each group of cells. Total RNAs were applied to a DNA microarray analysis and then RT-PCR was performed to confirm the microarray data. The most interesting finding of our microarray analysis was upregulation of insulin-like growth factor 2 (IGF2) and stromal cell-derived factor 1 (SDF1) under hypoxic conditions. RT-PCR analysis revealed that IGF2 expression was markedly upregulated in the non-osteoclastic cells. The addition of exogenous IGF2 increased the number of osteoclastic TRAP-positive multinuclear cells formed under normoxic conditions, whereas the addition of exogenous SDF1 did not change osteoclast formation. These results suggest that the upregulation of IGF2 derived from non-osteoclastic cells might be a crucial factor for osteoclast differentiation.     

Electrochemical evaluation of cellular physiological status under stress in Escherichia coli with the rpoS-lacZ reporter gene

We developed an electrochemical detection method for evaluating cellular physiological status based on the stringent response as a means to monitor cell viability. A reporter plasmid was constructed by inserting the beta-galactosidase gene (lacZ) under the control of the rpoS promoter, and then used to transform E. coli cells. Electrochemical responses from the products catalyzed by beta-galactosidase expressed by these E. coli cells were detected using the chronoamperometric technique in a nondestructive manner. Comparisons of response currents between the relA-positive strain and relA-negative strain revealed that increases in these currents were caused by the stringent response due to the stressful alcoholic environment, and thus as a model of stressful cultivating conditions. The current was proportional to the beta-galactosidase activity assayed by a conventional method that required the destruction of cells. The cellular physiological status, which depends on the stringent response as a viability marker, therefore, could then be evaluated online with a current using the rpoS-lacZ reporter gene in the relA-positive strain without pretreatment.     

Tollip and Tom1 form a complex and recruit ubiquitin-conjugated proteins onto early endosomes

Tom1 (target of Myb1) is a protein of unknown function. Tom1 and its relative Tom1L1 have an N-terminal VHS (Vps27p/Hrs/Stam) domain followed by a GAT (GGA and Tom1) domain, both of which are also found in the GGA (Golgi-localizing, gamma-adaptin ear domain homology, ADP-ribosylation factor-binding protein) family of proteins. Although the VHS and GAT domains of GGA proteins bind to transmembrane cargo proteins and the small GTPase ADP-ribosylation factor, respectively, the VHS and GAT domains of Tom1 are unable to interact with these proteins. In this study, we show that the GAT domains of Tom1 and Tom1L1 interact with ubiquitin and Tollip (Toll-interacting protein). Ubiquitin bound the GAT domains of Tom1, Tom1L1, and GGA proteins, whereas Tollip interacted specifically with Tom1 and Tom1L1. Ubiquitin and Tollip bound to an overlapping region of the Tom1-GAT domain in a mutually exclusive manner. Tom1 was predominantly cytosolic when expressed in cells. On the other hand, Tollip was localized on early endosomes and recruited Tom1 and ubiquitinated proteins. These observations suggest that Tollip and Tom1 form a complex and regulate endosomal trafficking of ubiquitinated proteins.     

Antinociceptive effect of 7-hydroxymitragynine in mice: Discovery of an orally active opioid analgesic from the Thai medicinal herb Mitragyna speciosa

Mitragynine is an indole alkaloid isolated from the Thai medicinal plant Mitragyna speciosa. We previously reported the morphine-like action of mitragynine and its related compounds in the in vitro assays. In the present study, we investigated the opioid effects of 7-hydroxymitragynine, which is isolated as its novel constituent, on contraction of isolated ileum, binding of the specific ligands to opioid receptors and nociceptive stimuli in mice. In guinea-pig ileum, 7-hydroxymitragynine inhibited electrically induced contraction through the opioid receptors. Receptor-binding assays revealed that 7-hydroxymitragynine has a higher affinity for micro-opioid receptors relative to the other opioid receptors. Administration of 7-hydroxymitragynine (2.5-10 mg/kg, s.c.) induced dose-dependent antinociceptive effects in tail-flick and hot-plate tests in mice. Its effect was more potent than that of morphine in both tests. When orally administered, 7-hydroxymitragynine (5-10 mg/kg) showed potent antinociceptive activities in tail-flick and hot-plate tests. In contrast, only weak antinociception was observed in the case of oral administration of morphine at a dose of 20 mg/kg. It was found that 7-hydroxymitragynine is a novel opioid agonist that is structurally different from the other opioid agonists, and has potent analgesic activity when orally administered.