Structure of the carboxypeptidase Y inhibitor IC in complex with the cognate proteinase reveals a novel mode of the proteinase-protein inhibitor interaction

Carboxypeptidase Y (CPY) inhibitor, IC, shows no homology to any other known proteinase inhibitors and rather belongs to the phosphatidylethanolamine-binding protein (PEBP) family. We report here on the crystal structure of the IC-CPY complex at 2.7 A resolution. The structure of IC in the complex with CPY consists of one major beta-type domain and a N-terminal helical segment. The structure of the complex contains two binding sites of IC toward CPY, the N-terminal inhibitory reactive site (the primary CPY-binding site) and the secondary CPY-binding site, which interact with the S1 substrate-binding site of CPY and the hydrophobic surface flanked by the active site of the enzyme, respectively. It was also revealed that IC had the ligand-binding site, which is conserved among PEBPs and the putative binding site of the polar head group of phospholipid. The complex structure and analyses of IC mutants for inhibitory activity and the binding to CPY demonstrate that the N-terminal inhibitory reactive site is essential both for inhibitory function and the complex formation with CPY and that the binding of IC to CPY constitutes a novel mode of the proteinase-protein inhibitor interaction. The unique binding mode of IC toward the cognate proteinase provides insights into the inhibitory mechanism of PEBPs toward serine proteinases and into the specific biological functions of IC belonging to the PEBP family as well.     

Type-3 ryanodine receptor involved in Ca2+-induced Ca2+ release and transmitter exocytosis at frog motor nerve terminals

Ca(2+)-induced Ca2+ release (CICR) occurs in frog motor nerve terminals after ryanodine receptors (RyRs) are primed for activation by conditioning large Ca2+ entry. We studied which type of RyR exists, whether CICR occurs without conditioning Ca2+ entry and how RyRs are primed. Immunohistochemistry revealed the existence of RyR3 in motor nerve terminals and axons and both RyR1 and RyR3 in muscle fibers. A blocker of RyR, 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8) slightly decreased rises in intracellular Ca2+ ([Ca2+]i) induced by a short tetanus (50 Hz, 1-2s), but not after treatment with ryanodine. Repetitive tetani (50 Hz for 15s every 20s) produced repetitive rises in [Ca2+]i, whose amplitude overall waxed and waned. TMB-8 blocked the waxing and waning components. Ryanodine suppressed a slow increase in end-plate potentials (EPPs) induced by stimuli (33.3 Hz, 15s) in a low Ca2+, high Mg2+ solution. KN-62, a blocker of Ca(2+)/calmoduline-activated protein kinase II (CaMKII), slightly reduced short tetanus-induced rises in [Ca2+]i, but markedly the slow waxing and waning rises produced by repetitive tetani in both normal and low Ca2+, high Mg2+ solutions. Likewise, KN-62, but not KN-04, an inactive analog, suppressed slow increases in EPP amplitude and miniature EPP frequency during long tetanus. Thus, CICR normally occurs weakly via RyR3 activation by single impulse-induced Ca2+ entry in frog motor nerve terminals and greatly after the priming of RyR via CaMKII activation by conditioning Ca2+ entry, thus, facilitating transmitter exocytosis and its plasticity.     

Treatment for psychological dependence on morphine: usefulness of inhibiting NMDA receptor and its associated protein kinase in the nucleus accumbens

A growing body of evidence indicates that the mesolimbic dopaminergic (DAergic) pathway projecting from the ventral tegmental area (VTA) to the nucleus accumbens (N.Acc.) play a critical role in the initiation of psychological dependence on morphine. As well as DAergic system, the involvement of non-DAergic neurotransmitter and neuromodulator systems in rewarding effects induced by morphine has been recently documented. We previously demonstrated that the morphine-induced rewarding effect was dramatically suppressed by co-treatment with NMDA receptor antagonists, such as dizocilpine (MK-801), ketamine and ifenprodil. Therefore, we propose here that inhibiting the N-methyl-D-aspartate (NMDA) receptor and its associated protein kinase in the N.Acc. is useful for the treatment for psychological dependence on morphine. The following review provides a summary of recent our findings regarding the role of NMDA receptor and its associated protein kinase in the development of psychological dependence on morphine.     

Novel antifungal compounds produced by Sterile Dark, an unidentified wheat rhizosphere fungus

Two novel antifungal compounds were isolated from a culture broth of Sterile Dark, an unidentified fungus isolated from the rhizosphere of wheat grown in a continuous cropping field. These compounds were elucidated to be phthalide-based compounds by spectroscopic analyses.     

Osmotic stress in barley regulates expression of a different set of genes than salt stress does

Under high salt conditions, plant growth is severely inhibited due to both osmotic and ionic stresses. In an effort to dissect genes and pathways that respond to changes in osmotic potential under salt stress, the expression patterns were compared of 460 non-redundant salt-responsive genes in barley during the initial phase under osmotic versus salt stress using cDNA microarrays with northern blot and real-time RT-PCR analyses. Out of 52 genes that were differentially expressed under osmotic stress, 11, such as the up-regulated genes for pyrroline-5-carboxylate synthetase, betaine aldehyde dehydrogenase 2, plasma membrane protein 3, and the down-regulated genes for water channel 2, heat shock protein 70, and phospholipase C, were regulated in a virtually identical manner under salt stress. These genes were involved in a wide range of metabolic and signalling pathways suggesting that, during the initial phase under salt stress, several of the cellular responses are mediated by changes in osmotic potential.     

In vivo VL-targeted activation-induced apoptotic supraclonal deletion by a microbial B cell toxin

To interfere with host immune responses, some microbial pathogens produce proteins with the properties of superantigens, which can interact via conserved V region framework subdomains of the Ag receptors of lymphocytes rather than the complementarity-determining region involved in the binding of conventional Ags. In recent studies, we have elucidated how a model B cell superantigen affects the host immune system by targeting a conserved V(H) site on the Ag receptors of B lymphocytes. To determine whether these findings represent a general paradigm, we investigated the in vivo immunobiologic properties of protein L of Peptostreptococcus magnus (PpL), a microbial Ig-binding protein specific for a V region site on Ig L chains. Our studies confirmed that PpL binding is restricted to a subset of murine Vkappa-expressing B cells, and found that B cells with stronger PpL-binding activity are associated with certain B cell subsets: splenic marginal zone (CD21(high) CD23(low)), splenic CD1(+), peritoneal B-1a (IgD(low) CD5(+)), and CD21(high) CD24(high) B cells in peripheral lymph nodes, mesenteric lymph nodes, and Peyer's patches. Infusion of PpL triggered a sequence of events in B cell receptor (BCR)-targeted B cells, with rapid down-regulation of BCR, the induction of an activation phenotype, and limited rounds of proliferation. Apoptosis followed through a process heralded by the dissipation of mitochondrial membrane potential, the induction of the caspase pathway, DNA fragmentation, and the deposition of B cell apoptotic bodies. These studies define a common pathway by which microbial toxins that target V region-associated BCR sites induce programmed cell death.     

Molecular mechanism of changes in the morphine-induced pharmacological actions under chronic pain-like state: suppression of dopaminergic transmission in the brain

In the present study, we demonstrated whether a neuropathic pain-like state induced by sciatic nerve ligation in rodents could cause a long-lasting change in intracellular signaling in both supraspinal and spinal cord related to the suppression of morphine's effect. Mice with sciatic nerve ligation exhibited a significant suppression of the morphine-induced antinociception. Under this condition, phosphorylated-conventional protein kinase C-like immunoreactivity (p-cPKC-IR) and phosphorylated-micro-opioid receptor (p-MOR)-IR were clearly increased on the ipsilateral side in the dorsal horn of the spinal cord of nerve-ligated mice. It is of interest to note that astroglial hypertrophy as well as its proliferation was also noted in this area of sciatic nerve-ligated mice. Like nerve injury, the increase in cPKC activities and astroglial hypertrophy/proliferation in this region was observed by repeated morphine treatment. These findings suggest that the phosphorylation of both cPKC and MOR in the dorsal horn of the spinal cord by sciatic nerve ligation may play a substantial role in the suppression of morphine-induced antinociception under a neuropathic pain-like state. Sciatic nerve injury also caused a significant inhibition of MOR-mediated G-protein activation onto GABAergic neurons and a dramatic reduction in ERK activities onto dopaminergic neurons in the ventral tegmental area (VTA) regulating the rewarding effect of opioids. Furthermore, we found that the inhibition of ERK cascade in the VTA by treatment with specific inhibitors suppressed the morphine-induced rewarding effect in normal mice. These findings provide evidence that the direct reduction in MOR function and the persistent decrease in ERK activity of dopaminergic neurons in the VTA may contribute to the suppression of the morphine-induced rewarding effect under a neuropathic pain-like state. Conclusively, our recent findings provide novel evidences for the mechanism underlying the less sensitivity to opioids under a neuropathic pain-like state.     

Differential properties between TRK-820 and U-50,488H on the discriminative stimulus effects in rats

It has been demonstrated that the newly synthesized kappa-opioid receptor agonist TRK-820, which has a unique structure that is different from those of other prototypical kappa-opioid receptor agonists such as U-50,488H, exert some behavioral effects that differ from those induced by U-50,488H. Therefore, the present study was designed to examine the possible difference between the discriminative stimulus effects of TRK-820 and U-50,488H in rats. Substitution tests with several kappa-opioid receptor agonists were initiated in rats trained to discriminate between TRK-820 (40 microg/kg) or U-50,488H (3.0 mg/kg) and saline. In the cross-substitution tests, U-50,488H substituted for the discriminative stimulus effects of TRK-820, whereas TRK-820 did not substitute completely for those of U-50,488H, indicating that the discriminative stimulus effects of TRK-820 and U-50,488H were somewhat different. In the substitution tests, E-2078, but not R-84760, substituted for the discriminative stimulus effects of both TRK-820 and U-50,488H. KT-90, CI-977 and ICI-199441 each substituted for the discriminative stimulus effects of U-50,488H, but not to those of TRK-820. These results imply that these kappa-opioid receptor agonists possess U-50,488H-like discriminative stimulus effects. Furthermore, that U-50,488H and the other kappa-opioid receptor agonists substituted for the discriminative stimulus effects of U-50,488H, produced aversive effects in rats. These findings suggest the possibility that unlike those of TRK-820, the cue of the discriminative stimulus effects of U-50,488H may be, at least in part, associated with its aversive effects.     

3-(2-Aminocarbonylphenyl)propanoic acid analogs as potent and selective EP3 receptor antagonists. Part 1: Discovery and exploration of the carboxyamide side chain

A series of 3-(2-aminocarbonyl-4-phenoxymethylphenyl)propanoic acid analogs were synthesized and evaluated for their EP3 antagonist activity in the presence of additive serum albumin. Several compounds were biologically evaluated for their in vivo efficacy with respect to the PGE₂-induced uterine contraction in pregnant rats as well as their pharmacokinetics. The discovery process of these potent and selective EP3 antagonists and their structure activity relationship are also presented.     

Improvement of protein production in lactic acid bacteria using 5′-untranslated leader sequence of slpA from Lactobacillus acidophilus

The 5′-untranslated leader sequence (UTLS) of the slpA gene from Lactobacillus acidophilus contributes to mRNA stabilization by producing a 5′ stem and loop structure, and a high-level expression system for the lactic acid bacteria was developed using the UTLS in this study. A plasmid, which expresses α-amylase under the control of the ldh promoter, was constructed by integrating the core promoter sequence with the UTLS. The role of the UTLS in increasing the copies of the α-amylase mRNA was proved by measuring α-amylase activity in the culture supernatant and the relative expression of α-amylase mRNA was determined by the quantitative real-time PCR analysis. Moreover, several expression systems were constructed by combining the core promoter sequence with the UTLS or with the partially deleted UTLS and the expression level was evaluated. The use of the UTLS led to the success in improving α-amylase expression in the two strains of Lactobacillus casei and Lactococcus lactis. The current study showed that the improvement in protein production using the UTLS could be applied to the expression system in the lactic acid bacteria.