[Show abstract][Hide abstract] ABSTRACT: Curcumin, a natural polyphenolic antioxidant compound, exerts well-known anti-inflammatory and immunomodulatory effects, the latter which can influence the activation of immune cells including T cells. Furthermore, curcumin can inhibit the expression of pro-inflammatory cytokines and chemokines, through suppression of the NF-κB signaling pathway. The beneficial effects of curcumin in diseases such as arthritis, allergy, asthma, atherosclerosis, diabetes and cancer may be due to its immunomodulatory properties. We studied the potential of curcumin to modulate CD4+ T cells-mediated autoimmune disease, by examining the effects of this compound on human CD4+ lymphocyte activation. Stimulation of human T cells with PHA or CD3/CD28 induced IL-2 mRNA expression and activated the endoplasmic reticulum (ER) stress response. The treatment of T cells with curcumin induced the unfolded protein response (UPR) signaling pathway, initiated by the phosphorylation of PERK and IRE1. Furthermore, curcumin increased the expression of the ER stress associated transcriptional factors XBP-1, cleaved p50ATF6α and C/EBP homologous protein (CHOP) in human CD4+ and Jurkat T cells. In PHA-activated T cells, curcumin further enhanced PHA-induced CHOP expression and reduced the expression of the anti-apoptotic protein Bcl-2. Finally, curcumin treatment induced apoptotic cell death in activated T cells via eliciting an excessive ER stress response, which was reversed by the ER-stress inhibitor 4-phenylbutyric acid or transfection with CHOP-specific siRNA. These results suggest that curcumin can impact both ER stress and mitochondria functional pathways, and thereby could be used as a promising therapy in the context of Th1-mediated autoimmune diseases.
International immunopharmacology 02/2013; · 2.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study investigates the mechanism through which increased 30K protein inhibits ecdysone-induced apoptosis in the Bm5 silkworm ovarian cell line. Treatment of Bm5 cells with 20-hydroxyecdysone (20E) after transfection with the pIZT/V5-His control vector triggered apoptosis, but 20E treatment did not trigger apoptosis in Bm5 cells transfected with the pIZT/30K/V5-His vector. To confirm its inhibitory effect on apoptosis, 30K protein was first purified from Escherichia coli transformed with a 30K expression vector and used to generate specific antibodies in mice. Anti-30K antiserum was used to confirm synthesis of the 30K protein in pIZT/30K/V5-His-transfected Bm5 cells and to detect 30K protein binding to the ecdysone receptor-B1 (EcR-B1). Anti-30K antiserum was used to immunoprecipitate protein complexes containing 30K from Bm5 cells transfected with pIZT/30K/V5-His vector and treated with 20E. We observed that 30K proteins bound primarily to the EcR-B1 and not to ultraspiracle (USP). Reciprocal immunoprecipitation of EcR-B1-containing complexes from Bm5 cells transfected with control pIZT/V5-His vector and treated with 20E showed that EcR-B1 bound to USP in the absence of 30K but did not bind to USP in pIZT/30K/V5-His-transfected Bm5 cells. These results demonstrate that 30K proteins block USP binding to EcR-B1 through formation of a 30K/EcR-B1 complex, resulting in inhibition of 20E-induced Bm5 cell apoptosis.
Archives of Insect Biochemistry and Physiology 08/2012; 81(3):136-47. · 1.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Using immunostaining methodology, we traced the axonal projection of FMRFamide (Phe-Met-Arg-Phe-NH2)-like immunoreactive (LI) medial neurosecretory cells (MNCs) and lateral neurosecretory cells (LNCs) from the brain into the ventral nerve cord (VNC) and retrocerebral complex in Bombyx mori (L.) (Lepidoptera: Bombycidae). Of the seven pairs of FMRFamide-LI MNCs, one pair extended its axons from the brain pars intercerebralis into the VNC ipsilateral connective where they appeared to terminate. The axons of the remaining MNCs ran through decussation in the brain median region and contralateral nervi corporis cardiaci (NCC) I out of the brain, and eventually innervated the contralateral corpus cardiacum (CC). Axons from the single pair of FMRFamide-LI LNCs projected into the ipsilateral NCC II fused with NCC I without decussation in the brain, and finally terminated in the CC. These results suggest that transport of the FMRFamide-like neuropeptide from may be related to the modulation of functions such as gut contraction in MNCs terminating in the VNC, and regulation of production and/or secretion of specific hormones such as juvenile hormone in MNCs and LNCs terminating in the CC.
The Canadian Entomologist 06/2012; 144(03). · 0.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study demonstrates that a 30K protein was gradually synthesized in primary-cultured motoneurons from the accessory planta retractor (APR) of the 6th abdominal ganglion (APR6) in silkworm ventral ganglia through stimulation of hemolymph. An increase in 30K protein synthesis resulted in an inhibition of programmed cell death (PCD) of APR6 motoneurons. The 30K protein was gradually synthesized from the 30Kc6 gene of identified APR6s in day-6 4th instars to day-9 5th instar larvae, but synthesis of the 30K protein ceased in isolated APR6s of day-1 pupa, which normally begin to undergo PCD. When pupal APR6s were treated with larval hemolymph, however, the 30K protein was synthesized suggesting the existence of an anti-PCD factor in the larval hemolymph. An increase of 30K protein within the APR6s was confirmed by antiserum made against the recombinant 30K protein that originated from the APR 30Kc6 gene. Larval APR6, in which PCD was induced with 20-hydroxyecdysone (20E) added to the primary culture, exhibited some PCD characteristics of shrinkage of cell bodies, axonal fragmentation and loss of mitochondrial function. These results provide new insights on the survival or PCD of insect motoneurons through stimulation of hemolymph.
Journal of insect physiology 03/2012; 58(5):756-62. · 2.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: IgE‐mediated allergic reactions caused by mosquito bites are a common problem all over the world. This study was undertaken to determine IgE levels in subjects, to elucidate human IgE and mouse IgG1 binding patterns and to investigate the cross‐reactivity of salivary gland antigens with three mosquitoes. Mosquito larvae of Aedes togoi, Culex tritaeniorhynchus and Culex pipiens pallens were collected and maintained in the laboratory. Salivary gland extracts (SGE) and whole‐body extracts (WBE) were prepared from female mosquitoes of each species. The 9 sera out of 12 with positive skin reactions to SGE of A. togoi by skin prick test showed significantly higher anti‐mosquito SGE IgE levels than in those without skin reactions. Protein band patterns of the SGE and WBE of the three species were different from one another. There were specific mouse IgG1 reactions to the bands of 30.5, 33, 37 and 57.5 kD in the SGE of A. togoi. The ELISA inhibition studies disclosed almost no crossreactivities between A. togoi, C. tritaeniorhynchus and C. pipiens pallens. Immunoblot analysis disclosed that allergenic proteins in the SGE of mosquitoes and their patterns were remarkably similar between human and mouse sera to the SGE of A. togoi. Species shared allergens may not exist among the three mosquito species prevalent in Korea.
[Show abstract][Hide abstract] ABSTRACT: We assessed the validity of monitoring changes in mitochondrial membrane potential (ΔΨ) with a fluorescent probe, JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl benzimidazolo-carbocyanine iodide), for the quantitative evaluation of neonatal hypoxic-ischemic brain injury. Seven-day-old rat pups were subjected to 2h of 8% oxygen following unilateral carotid artery ligation. Brain tissue was obtained for JC-1 staining at 24h after hypoxia ischemia (HI), and the results were compared with those of other simultaneous measurements such as flow cytometry with fluoresceinated annexin V/propidium iodide (PI), terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) staining, triphenyl tetrazolium chloride (TTC) infarct area and western blot for cytosolic cytochrome c. Flow cytograms of JC-1 showed two distinct sub-populations with different ΔΨ, red with high ΔΨ and green with low ΔΨ, at 24h after HI. This shift of JC-1 fluorescence from red to green indicated a collapse of ΔΨ. The increased percentage of low ΔΨ with JC-1 showed a significant positive correlation with a simultaneous increase in annexin V(+)/PI(+) necrotic cells, TUNEL-positive cells, TTC infarct area and western blot of cytosolic cytochrome c, and negative correlation with annexin V(-)/PI(-) live cells. In summary, low ΔΨ measured with JC-1 was significantly correlated with results from other methods used to assess the extent of brain damage after HI. Therefore, fluorocytometric analysis of ΔΨ with JC-1 might be a sensitive and reliable technique in the quantitative evaluation of neonatal brain injury.
Journal of neuroscience methods 11/2010; 193(2):232-8. · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nattokinase was produced by batch and fed-batch culture of Bacillus subtilis in flask and fermentor. Effect of supplementing complex media (peptone, yeast extract, or tryptone) was investigated on the production of nattokinase. In flask culture, the highest cell growth and nattokinase activity were obtained with 50 g/L of peptone supplementation. In this condition, nattokinase activity was 630 unit/ml at 12 h. In batch culture of B. subtilis in fermentor, the highest nattokinase activity of 3400 unit/ml was obtained at 10h with 50 g/L of peptone supplementation. From the batch kinetics data, it was shown that nattokinase production was growth-associated and culture should be harvested before stationary phase for maximum nattokinase production. In fed-batch culture of B. subtilis using pH-stat feeding strategy, cell growth (optical density monitored at 600 nm) increased to ca. 100 at 22 h, which was 2.5 times higher than that in batch culture. The highest nattokinase activity was 7100 unit/ml at 19 h, which was also 2.1 times higher than that in batch culture.
New Biotechnology 09/2010; 27(4):341-6. · 1.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Polyclonal antisera to Manduca sexta allatotropin and allatostatin were utilized to localize allatotropin- and allatostatin-immunoreactivities in the central nervous system of larvae, pupae and adults from the silk moth Bombyx mori. In larva the first allatotropin-immunoreactivity appeared in the brain and terminal abdominal ganglion of first instar larva. In the third, fourth and fifth instar larvae, there was allatotropinimmunoreactivity in the suboesophageal ganglion, three thoracic ganglia, and eight abdominal ganglia with immunoreactivity in some axons of N 1 and N 2. Allatostatin-immunoreactivity, which could be not demonstrated in the first and second instar larvae, appeared first in the brain and suboesophageal ganglion of the third instar larva. Allatostatin-immunoreactive cells increased to seven pairs in brain of the fifth instar larva, in which immunreactivity also appeared in eight abdominal ganglia. Allatotropin- and allatostatinimmunoreactive cell bodies in the brain projected their axons into corpora allata without terminations in the corpora cardiaca. During pupal and adult stages, brains had no allatotropin-immunoreactivity in the brains, but most ventral ganglia contained allatotropin-immunoreactive cells. There was allatostatin-immunoreactivity in the brains of the 5- and 7-day-old pupae and adult and suboesophageal ganglion of the 7-day-old pupa.
[Show abstract][Hide abstract] ABSTRACT: To investigate the effects of intravesical electrical stimulation (IVES) on bladder function and synaptic neurotransmission in the lumbosacral spinal cord in the spinalized rat, as the clinical benefits of IVES in patients with increased residual urine or reduced bladder capacity have been reported but studies on the mechanism of IVES have mainly focused on bladder A delta afferents in central nervous system-intact rats.
In all, 30 female Sprague-Dawley rats were divided equally into three groups: normal control rats, sham-stimulated spinalized rats and IVES-treated spinalized rats. IVES was started 5 weeks after spinal cord injury (SCI) and was performed 20 min a day for 5 consecutive days. At 7 days after IVES, conscious filling cystometry was performed. Sections from the L6 and S1 spinal cord segments were examined for n-methyl-d-aspartic acid receptor 1 (NMDAR1) subunit and gamma-aminobutyric acid (GABA) immunoactivity.
In IVES-treated spinalized rats, the number and maximal pressure of nonvoiding detrusor contractions were significantly less than in sham-stimulated spinalized rats. The mean maximal voiding pressure was also lower in IVES-treated than in sham-stimulated spinalized rats. IVES significantly reduced the interval between voiding contractions compared with the untreated spinalized rats. There was an overall increase in NMDAR1 immunoactivity after SCI, which was significantly lower in IVES-treated spinalized rats. Immunoactivity of GABA after SCI was significantly lower than in the control group and was significantly higher in IVES-treated spinalized rats.
Our results suggest that IVES might affect voiding contractions in addition to inhibiting C-fibre activity and that IVES seems to have a more complex effect on the bladder control pathway. For synaptic neurotransmission in the spinal cord, IVES could possibly shift the balance between excitation and inhibition towards inhibition.
BJU International 12/2008; 103(8):1136-41. · 3.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: c-fos expression in spinal neurons that are activated by lower urinary tract stimulation are not organ specific. In this experiment, we demonstrated changes of c-fos expression in bladder-specific preganglionic neurons (PGNs) and interneurons using pseudorabies virus (PRV).
Forty Sprague-Dawley rats were used. We identified the neuronal pathway associated with the bladder by injecting PRV into the detrusor. An immunohistochemical method was used to stain Fos-protein encoded by the c-fos gene. Immunofluorescent staining for PRV was performed to evaluate changes in bladder-specific spinal neurons.
Immunofluorescent staining with choline acetyltransferase (ChAT) revealed that the sacral parasympathetic nucleus (SPN) regions contained 9.8 PGNs/section. In rats with chronic spinal cord injury by intravesical saline instillation, 82.4+/-10.3% of PGNs in SPN exhibited Fos-immunoreactive (IR). Two and a half days after PRV infection, PRV-IR PGNs were observed at 5.4 PGNs/section, and 2.7+/-1.6% of them exhibited Fos-IR. Unlike ChAT-IR PGNs, PRV-IR PGNs are bladder-specific neurons and PRV-IR and Fos-IR cells found in the back of PRV-IR PGNs are bladder-specific interneurons. Three days after PRV infection, we observed many PRV-IR and Fos-IR cells in the dorsal commissure. These neurons are interneurons distributed in the bladder.
We confirmed that in chronic spinal cord injury, the patterns of c-fos expression in bladder-specific spinal neurons were similar to those in voiding-reflex related spinal neurons, which had already been demonstrated earlier. We believe that our methodology can be applied to study interactions between voiding and other organs as well, such as the urethra and prostate.
Yonsei Medical Journal 07/2008; 49(3):479-85. · 1.31 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Molecularly imprinted polymers (MIPs) are artificial materials containing recognition sites with high affinity and selectivity. This study aims to immobilize MIPs on the surface of carbon nanotube (CNT) in an effort to develop biosensor system based on CNT transistor using MIPs as a probe material. As a linking molecule of MIPs to CNT, acrylated Tween 20 was synthesized by reacting Tween 20 with acryloyl chloride, and then immobilized on CNT by hydrophobic interactions. 1H NMR and FT-IR spectra confirmed the synthesis of acrylated Tween 20. MIPs were formed for theophylline as a model template on the surface of CNT with methacrylic acid (functional monomer) and ethylene glycol dimethacrylate (crosslinking agent) using a photografting polymerization technique. The adsorbed layer of 2,2-dimethoxy-2-phenylacetophenone initiated a radical polymerization near the surface by UV-light irradiation. AFM images displayed the formation of MIPs on CNT with a size less than 10 nm. The theophylline-imprinted polymer on CNT showed higher binding capacity for theophylline than non-imprinted polymer (NIP) on CNT and selectivity for theophylline over caffeine (similar structure molecules).
Colloids and Surfaces A: Physicochemical and Engineering Aspects. 01/2008;
[Show abstract][Hide abstract] ABSTRACT: This study was conducted to investigate effects of brain-derived neurotrophic factor (BDNF) on the neurite growth of deutocerebral neurons in vitro, and production of BDNF-like neuropeptide from brain of the silk moth, Bombyx mori. In primary culture of antennal lobe (AL) neurons with BDNF, it promoted a significant neurite extension of putative AL projection neurons and an outgrowth of branches from principal neurites of putative AL interneurons. Results from immunolabeling of brain and retrocerebral complex showed that BDNF -like neuropeptide labeled in brain was synthesized by median and lateral neurosecretory cells, then transported to corpora allata for storage.
[Show abstract][Hide abstract] ABSTRACT: Programmed cell death was characterized in the silkworm thoracic ganglia TG1, TG2 and TG3 during postembryonic periods by TUN EL assay. Apoptotic cells were detected in the three TGs of all larval stages except for day‐1, 2 1stinstar larvae, in which no apoptotic cells were found. From day‐7 5th larva, the numbers of apoptotic cells were dramatically increased and peaked on day‐1 pupa and day‐2 pupa and then abruptly decreased. Apoptotic cells finally disappeared in day‐1 adult. In‐vivo injection of 20‐hydroxyecdysone (20E) into day‐8 5th larva resulted in a striking decrease of apoptotic cells. Actinomycin D (Act D) or cycloheximide (CHX), injected into hemolymph of day‐8 5th larva, resulted in a decrease of apoptotic cells in the three TGs. Injection of caspase‐8 and ‐3 inhibitors also blocked cellular apoptosis. These results will provide valuable information for understanding of cellular changes in the three TGs during metamorphosis of the insect species.
[Show abstract][Hide abstract] ABSTRACT: Several pharmacological and physiological studies have suggested that GABA(A) receptors (GABA(A) Rs) may exist in the rat major pelvic ganglion (MPG), a large coalescent pelvic ganglion that contains both sympathetic and parasympathetic components which innervates pelvic organs. However, the presence of GABA(A) R in the MPG has never been demonstrated directly by morphological studies. In the present study, we used immunohistochemistry to demonstrate the existence of GABA(A) R beta2/3 subunits for the first time in the rat MPG. We also analyzed the neurochemical properties of MPG neurons expressing GABA(A) R beta2/3 subunits. GABA(A) R beta2/3-immunoreactive (-IR) neurons occupied 27.4+/-7.0% of the whole neuronal population, and many of these (77.6%) were co-localized with tyrosine hydroxylase (TH). Likewise, most (86.5%) of TH-IR neurons were GABA(A) R beta2/3-positive. GABA(A) R beta2/3 subunits were also expressed in a few VIP- or NOS-IR neurons, the cholinergic or non-adrenergic, non-cholinergic (NANC) neurons. These results suggest that GABA(A) Rs are involved in the modulation of most sympathetic, noradrenergic neurons and also a subset of VIP and NOS neurons of the rat MPG.
[Show abstract][Hide abstract] ABSTRACT: Responses to energy restriction tend to vary within the population because of genetic differences. In this study, we have genotyped 6 uncoupling protein 3 (UCP-3) polymorphisms (-55C/T, Int2-143G/C, Tyr99Tyr, Int3-47G/A, Int4-498C/T, and Tyr210Tyr) among 214 overweight Korean female subjects recruited from an obesity clinic. Three major haplotypes, identified with frequencies in excess of 0.04, were constructed from 6 single nucleotide polymorphisms. Association studies were then undertaken, involving the measurement of anthropometric characteristics and body composition both before and after 1 month of a energy-restriction regimen. At baseline, haplotype 1 (ht1) [CGTACC] was associated with elevated anthropometric characteristics, including body weight, waist-hip ratio, and body mass index, as well as body components, including body fat mass and body fat-free mass. After the completion of the 1-month weight control program, which involved a very low-energy (2900 kJ/d) diet, we analyzed the outcomes according to the UCP-3 genetic polymorphisms. Among the 3 principal haplotypes, ht1 [CGTACC] was significantly associated with an increased reduction in body weight, in the codominant (P=.022), dominant (P=.016), and recessive (P=.041) models. Body mass index reduction was associated with the ht1 haplotype in a similar fashion. Among the body components, changes in body fat mass were significantly associated with ht1 [CGTACC] (P=.028), but changes in body fat-free mass were not significantly associated with the UCP-3 polymorphism.
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to investigate the secretion of brain-derived neurotrophic factor (BDNF)-like neuropeptide in the silkworm, Bombyx mori , by using immunocytochemical techniques on the brain and retrocerebral complex of fifth instar larvae. In the brain, four pairs of median neurosecretory cell (MNC) bodies and six pairs of lateral neurosecretory cell (LNC) bodies had distinct immunoreactivities to this peptide, suggesting that this peptide is produced from two types of brain neuron. These reactivities were much stronger in the MNC than in the LNC. Labeled MNC projected their axons into the contralateral corpora allata, to which axons of labeled MNC were eventually innervated, through decussation in the median region, contralateral nerve corporis cardiaci I and nerve corpora allata I. Labeled LNC extended their axons into the ipsilateral corpora allata to be innervated through the ipsilateral nerve corporis cardiaci II and nerve corpora allata I. These results suggest that BDNF is secreted as a neurohormone from MNC and LNC of the brain into the corpora allata.
[Show abstract][Hide abstract] ABSTRACT: A cuticle protein gene, AyCP12, from the Japanese oak silkmoth, Antheraea yamamai, was isolated and characterized. The gene spans 1107 bp and consists of one intron and two exons coding for a 112 amino acid polypeptide with a predicted molecular mass of 12,163 Da and a pI of 4.4. The AyCP12 protein contained a type-specific consensus sequence identifiable in other insect cuticle proteins and the deduced amino acid sequence of the AyCP12 cDNA is most homologous to another silkmoth, A. pernyi, cuticle protein ApCP13 (82% protein sequence identity). Northern blot analysis revealed that AyCP12 showed the epidermis-specific expression.
[Show abstract][Hide abstract] ABSTRACT: The ventriculus terminalis (VT) is a dilated cavity within the conus medullaris of the spinal cord. Although the VT was discovered in the mid-nineteenth century, little is known about its characteristics during development in human fetuses. Ependymal cells lining the cavities within the CNS retain high differentiation potential, and are believed to be responsible for the postnatal neurogenesis. To evaluate the differentiation capacity of the ependymal cells lining the VT during development, we examined glial fibrillary acidic protein (GFAP) and proliferating cell nuclear antigen (PCNA) expression in the spinal cord of 18-24-week-old human fetuses. GFAP is a marker for the degree of ependymal cell differentiation in the human fetus, and PCNA is a well-known marker for cell division. Morphological characteristics of the VT were also examined. At the lower portion of the conus medullaris, the central canal abruptly expands dorsally to become the VT. Then the VT widens bilaterally while its anteroposterior diameter reduces gradually in a caudal direction. Finally, the VT becomes a narrow, transverse slit at the level of the lowermost conus medullaris. Compared with those lining the central canal, more numerous ependymal cells lining the VT showed more intensive GFAP and PCNA expression throughout all gestational ages examined. This suggests that, in the developing human spinal cord, ependymal cells lining the VT retain their differentiation potential, including a higher proliferative capacity, until a later stage of development than those lining the central canal.
[Show abstract][Hide abstract] ABSTRACT: This study was conducted to investigate effects of brain-derived neurotrophic factor on the neurite growth and the survival rate of antennal lobe neurons in vitro, and secretion of brain-derived neurotrophic factor-like neuropeptide from brain into hemolymph in the silk moth, Bombyx mori. In primary culture of antennal lobe neurons with brain-derived neurotrophic factor, it promoted both a neurite extension of putative antennal lobe projection neurons and an outgrowth of branches from principal neurites of putative antennal interneurons with significance (p<0.05). Brain-derived neurotrophic factor also increased significantly a survival rate of antennal lobe neurons (p<0.05). Results from immunolabeling of brain and retrocerebral complex, and ELISA assay of hemolymph showed that brain-derived neurotrophic factor-like neuropeptide was synthesized by both median and lateral neurosecretory cells of brain, then transported to corpora allata for storage, and finally secreted into hemolymph for action. These results will provide valuable information for differentiation of invertebrate brain neurons with brain-derived neurotrophic factor.