Relationship between Urinary N-Desmethyl-Acetamiprid and Typical Symptoms including Neurological Findings: A Prevalence Case-Control Study

Neonicotinoid insecticides are nicotinic acetylcholine receptor agonists used worldwide. Their environmental health effects including neurotoxicity are of concern. We previously determined a metabolite of acetamiprid, N-desmethyl-acetamiprid in the urine of a patient, who exhibited some typical symptoms including neurological findings. We sought to investigate the association between urinary N-desmethyl-acetamiprid and the symptoms by a prevalence case-control study. Spot urine samples were collected from 35 symptomatic patients of unknown origin and 50 non-symptomatic volunteers (non-symptomatic group, NSG, 4–87 year-old). Patients with recent memory loss, finger tremor, and more than five of six symptoms (headache, general fatigue, palpitation/chest pain, abdominal pain, muscle pain/weakness/spasm, and cough) were in the typical symptomatic group (TSG, n = 19, 5–69 year-old); the rest were in the atypical symptomatic group (ASG, n = 16, 5–78 year-old). N-desmethyl-acetamiprid and six neonicotinoids in the urine were quantified by liquid chromatography-tandem mass spectrometry. The detection of N-desmethyl-acetamiprid was the most frequent and highest in TSG (47.4%, 6.0 ppb (frequency, maximum)), followed by in ASG (12.5%, 4.4 ppb) and in NSG (6.0%, 2.2 ppb), however acetamiprid was not detected. Thiamethoxam was detected in TSG (31.6%, 1.4 ppb), in ASG (6.3%, 1.9 ppb), but not in NSG. Nitenpyram was detected in TSG (10.5%, 1.2 ppb), in ASG (6.3%, not quantified) and in NSG (2.0%, not quantified). Clothianidin was only detected in ASG (6.3%, not quantified), and in NSG (2.0%, 1.6 ppb). Thiacloprid was detected in ASG (6.3%, 0.1 ppb). The cases in TSG with detection of N-desmethyl-acetamiprid and thiamethoxam were aged 5 to 62 years and 13 to 62 years, respectively. Detection of N-desmethyl-acetamiprid was associated with increased prevalence of the symptoms (odds ratio: 14, 95% confidence interval: 3.5–57). Urinary N-desmethyl-acetamiprid can be used as a biomarker for environmental exposure to acetamiprid. Further multi-centered clinical research in larger patients groups with more metabolites analysis is needed.     

Inhibitory effect of OPC-15161, a component of fungus Thielavia minor,on proliferation and extracellular matrix production of rat cultured hepatic stellate cells

A component of fungus Thielavia minor, OPC-15161, has been shown to inhibit the proliferation and extracellular matrix production of extracellular matrix-producing mesangial cells in the kidney in vivo. In this study, we examined the effects of OPC-15161 on the proliferation and extracellular matrix production of rat cultured hepatic stellate cells (HSCs). To determine the effect of OPC-15161 on proliferation of HSCs, the cell number and the uptake of [³H]thymidine were investigated in the presence and absence of interleukin-1β (IL-1β). IL-1β significantly increased the uptake of [³H]thymidine in the HSCs, and the addition of OPC-15161 inhibited the uptake in a dose-dependent manner. The cell number of HSCs was also increased by IL-1β, which was inhibited by OPC-15161. Production of extracellular matrix by OPC-15161 was studied by the production of [³H]-hydroxyproline in the presence and absence of transforming growth factor-β1 (TGF-β1). TGF-β1 significantly increased the production of [³H]-hydroxyproline in the cells, whereas the addition of OPC-15161 inhibited this effect dose dependently. We also investigated the effects of OPC-15161 on Ca²⁺ mobilization and measured D-myo-inositol 1,4,5-triphosphate (IP₃) in the HSCs. IL-1β induced the increase of intracellular Ca²⁺ and IP₃ concentrations in the HSCs, which were decreased by OPC-15161. Based on these results, we conclude that OPC-15161 inhibited the proliferation and production of hydroxyproline in cultured rat HSCs, and thus, it may have a role in prevention of liver fibrosis in vivo. J. Cell. Physiol. 174:398–406, 1998. © 1998 Wiley-Liss, Inc.     

Vascularized periosteal grafts: an experimental study using two different forms of tibial periosteum in rabbits

Three groups of rabbits were studied to investigate the osteogenic capacity of vascularized periosteum. It was found that such tissue, especially in the form of musculoperiosteal flaps, produced a significant amount of bone. It is concluded that vascularized periosteal flaps have an excellent osteogenic capacity even though they are not subjected to weight or stress.     

The role of transfectedHLA-DQ genes in the mixed lymphocyte reaction-like condition

DR gene products are commonly thought to be involved in the induction of the mixed lymphocyte reaction (MLR). However, very little is known about the role of HLA DQ antigens in the MLR. To address this question, we introduced DQ and chain genes into mouse L cells, a human T -cell line, and a human premonocytoid cell line using a liposome-mediated transfer technique. The DQ and DQ genomic clones were isolated from a DR2 DQw1 and a DR3 DQw2 phage library, respectively. ThepSV2-Neo gene was introduced as a selection marker with both DQ and DQ . The resultant transfected cells were able to bind several HLA class II monoclonal antibodies. In addition, these cells were found to be efficient in stimulating peripheral blood lymphocyte proliferation under MLR-like conditions, implying a role for HLA-DQ molecules in HLA-D typing differences.     

Self-incompatibility (S) alleles of the rosaceae encode members of a distinct class of the T2/S ribonuclease superfamily

Stylar riboncleases (RNases) are associated with gametophytic self-incompatibility in two plant families, the Solanaceae and the Rosaceae. The self-incompatibility-associated RNases (S-RNases) of both the Solanaceae and the Rosaceae were recently reported to belong to the T2 RNase gene family, based on the presence of two well-conserved sequence motifs. Here, the cloning and characterization of S-RNase genes from two species of Rosaceae, apple (Malus × domestica) and Japanese pear (Pyrus serotina) is described and these sequences are compared with those of other T2-type RNases. The S-RNases of apple specifically accumulated in styles following maturation of the flower bud. Two cDNA clones for S-RNases from apple, and PCR clones encoding a further two apple S-RNases as well as two Japanese pear S-RNases were isolated and sequenced. The deduced amino acid sequences of the rosaceous S-RNases contained two conserved regions characteristic of the T2/S-type RNases. The sequences showed a high degree of diversity, with similarities ranging from 60.4% to 69.2%. Interestingly, some interspecific sequence similarities were higher than those within a species, possibly indicating that diversification of S-RNase alleles predated speciation in the Rosaceae. A phylogenetic tree of members of the T2/S-RNase superfamily in plants was obtained. The rosaceous S-RNases formed a new lineage in the tree that was distinct from those of the solanaceous S-RNases and the S-like RNases. The findings suggested that self-incompatibility mechanisms in Rosaceae and Solanaceae are similar but arose independently in the course of evolution.     

Lysophospholipids and ATP mutually suppress maturation and release of IL-1 beta in mouse microglial cells using a Rho-dependent pathway

The P2X7 receptor (P2X7R), an ATP-gated ion channel, plays essential roles in the release and maturation of IL-1beta in microglial cells in the brain. Previously, we found that lysophosphatidylcholine (LPC) potentiated P2X7R-mediated intracellular signals in microglial cells. In this study, we determined whether the lysophospholipids, i.e., LPC and sphingosylphosphorylcholine (SPC), modulate the ATP-induced release and processing of IL-1beta mediated by P2X7R in mouse MG6 microglial cells. LPC or SPC alone induced the release of precursor (pro-IL-1beta) and mature IL-1beta (mIL-1beta) from LPS-primed MG6 cells, possibly due to lytic functions. However, these lysophospholipids inhibited ATP-induced caspase-1 activation that is usually followed by the release of mIL-1beta. Conversely, ATP inhibited the release of pro-IL-1beta and mIL-1beta induced by LPC/SPC. This suggests that lysophospholipids and ATP mutually suppressed each function to release IL-1beta. P2X7R activation resulted in microtubule reorganization in the MG6 cells that was blocked in the presence of LPC and SPC. LPC/SPC reduced the amount of activated RhoA after stimulation with ATP, implying that these lysophospholipids block ATP-induced microtubule reorganization by interfering with RhoA activation. In addition, the microtubule inhibitor colchicine inhibited ATP-induced release of mIL-1beta similar to that of LPC and SPC. This suggests that the impairment of the microtubule reassembly may be associated with the inhibitory effects of LPC/SPC on ATP-induced mIL-1beta release. Mutual suppression by ATP and LPC/SPC on the maturation of IL-1beta was observed in LPS-primed primary microglia. Collectively, these data suggest opposing functions by lysophospholipids, either proinflammatory or anti-inflammatory, in regard to the maturation and release of IL-1beta from microglial cells.     

Relationship of butterfly diversity with nectar plant species richness in and around the Aokigahara primary woodland of Mount Fuji,central Japan

We examined the relationships between the diversities of vegetation, adult nectar plants, and butterflies in and around the Aokigahara primary woodland on the northwestern footslopes of Mount Fuji, central Japan. The results showed that the nectar resource utilization by adult butterflies was significantly biased to herbaceous plants, especially to perennials, compared to woody species, although most of the study area was in and near a primary woodland. There were greater nectar plant species in sites with greater plant species richness. Among the butterfly community indices analyzed, the strongest correlation was detected between butterfly species richness and nectar plant species richness at each site. Another close correlation was detected between the species richness of nectar plants and herbaceous plants at each site. These results suggest that herbaceous plant species richness in a habitat plays a central role in its nectar plant species richness, and the nectar plant richness is a highly important factor supporting its adult butterfly species richness. Consequently, we propose that the maintenance and management of herbaceous plant species richness in a butterfly habitat, which lead to those of its nectar plant species richness, are very important for conservation of butterfly diversity even in and around woodland landscapes of temperate regions.     

CUL1 regulates TOC1 protein stability in the Arabidopsis circadian clock

The circadian clock is the endogenous timer that coordinates physiological processes with daily and seasonal environmental changes. In Arabidopsis thaliana , establishment of the circadian period relies on targeted degradation of TIMING OF CAB EXPRESSION 1 (TOC1) by the 26S proteasome. ZEITLUPE (ZTL) is the F-box protein that associates with the SCF (Skp/Cullin/F-box) E3 ubiquitin ligase that is responsible for marking TOC1 for turnover. CULLIN1 (CUL1) is a core component of SCF complexes and is involved in multiple signaling pathways. To assess the contribution of CUL1-containing SCF complexes to signaling within the plant oscillator, circadian rhythms were examined in the recessive, temperature-sensitive CUL1 allele axr6-3 . The activity of CUL1 in this mutant declines progressively with increasing ambient temperature, resulting in more severe defects in CUL1-dependent activities at elevated temperature. Examination of circadian rhythms in axr6-3 revealed circadian phenotypes comparable to those observed in ztl null mutants; namely, lengthened circadian period, altered expression of core oscillator genes, and limited degradation of TOC1. In addition, treatment of seedlings with exogenous auxin did not alter TOC1 stability. These results demonstrate that CUL1 is required for TOC1 degradation and further suggest that this protein is the functional cullin for the SCF^ZTL complex.     

Functional reconstitution of G-protein-coupled receptor-mediated adenylyl cyclase activation by a baculoviral co-display system

Recently, evidence has accumulated in support of the heterologous expression of functional membrane proteins and their complexes on extracellular baculovirus particles (budded virus, BV). In this study, we attempted to apply this BV display system to detect G-protein-coupled receptor (GPCR) signaling. We infected Sf9 cells with a combination of four recombinant baculoviruses individually encoding the dopamine D1 receptor (DR-D1), G-protein -subunit (G_s), G-protein β₁γ₂ subunit dimer (Gβ₁γ₂), and adenylyl cyclase type VI (ACVI). The recovered BV fraction produced cAMP in response to the stimulation with dopamine. Co-expression of all three G-protein subunits in addition to receptor and ACVI led to a maximal response. BV co-expressing DR-D1, G_s, Gβ₁γ₂, and ACVI also responded to dopamine agonists and an antagonist. Furthermore, BV expressing two other G_s-coupled receptors together with G_s, Gβ₁γ₂, and ACVI also produced cAMP in response to their specific ligands. These results indicate the functional coupling of receptor, G_s and ACVI is reconstituted on BV. Since BV is essentially free of endogenous GPCRs, this BV co-display system should prove highly useful for the development of functional assay systems for GPCRs.