Green fluorescent protein (GFP) expression patterns in the olfactory epithelium of GFP transgenic cloned Jinhua pigs

Domestic pigs possess a well‐developed sense of smell. However, the morphology of the porcine olfactory epithelium (OE) is poorly understood. Recently, several strains of transgenic cloned pigs that are presumed to ubiquitously express green fluorescent protein (GFP) have been created. Thus, the purpose of this study was to elucidate the features of porcine OE using the tissues of GFP transgenic cloned pigs. Based on observations of Hematoxylin and Eosin staining and measurements of thickness, porcine OE tissue portions were classified into three categories (thick, standard, and thin). Cryosections revealed that the prominent GFP signals were expressed in olfactory sensory neurons (OSN), Bowman's glands, and olfactory nerve. A few GFP‐expressing sustentacular cells were seen; however, the intensity of GFP fluorescence was slight. In the thick portion, numerous GFP‐expressing polygonal OSN that did not possess dendrites were found. In the standard portions, GFP‐expressing cells had longitudinal dendrites. A few GFP‐expressing cells were found in the thin portion. In the thick and standard portions, most of the prominent GFP‐expressing cells were positive for olfactory marker protein. Moreover, double immunofluorescence staining with boiled GFP and Sox2 antibody revealed that GFP expression patterns in OSN are synchronized with Sox2 immunoreactive patterns.     

Olfactory marker protein (OMP) exhibits a beta-clam fold in solution: implications for target peptide interaction and olfactory signal transduction

Olfactory marker protein (OMP) is a ubiquitous, cytoplasmic protein found in mature olfactory receptor neurons of all vertebrates. Electrophysiological and behavioral studies demonstrate that it is a modulator of the olfactory signal transduction pathway. Here, we demonstrate that the solution structure of OMP, as determined by NMR studies, is a single globular domain protein comprised of eight beta-strands forming two beta-sheets oriented orthogonally to one another, thus exhibiting a "beta-clam" or "beta-sandwich" fold: beta-sheet 1 is comprised of beta3-beta8-beta1-beta2 and beta-sheet 2 contains beta6-beta5-beta4-beta7. Insertions include two, long alpha-helices located on opposite sides of the beta-clam and three flexible loops. The juxtaposition of beta-strands beta6-beta5-beta4-beta7-beta2-beta1-beta8-beta3 forms a continuously curved surface and encloses one side of the beta-clam. The "cleft" formed by the two beta-sheets is opposite to the closed end of the beta-clam. Using a peptide titration series, we have identified this cleft as the binding surface for a peptide derived from the Bex1 protein. The highly conserved Omega-loop structure adjacent to the Bex1 peptide-binding surface found in OMP may be the site of additional OMP-protein interactions related to its role in modulating olfactory signal transduction. Thus, the interaction between the OMP and Bex1 proteins could facilitate the interaction between OMP and other components of the olfactory signaling pathway.     

Comparative study of the olfactory epithelium of the three-spined stickleback (Gasterosteus aculeatus) and the nine-spined stickleback (Pungitius pungitius)

Summary The olfactory epithelium of the three-spined stickleback (Gasterosteus aculeatus) and the nine-spined stickleback (Pungitius pungitius) has been studied with a conventional histochemical and a novel immunological staining technique. In both species, the sensory epithelium is arranged in folds separated by non-sensory epithelial tissue. In the nine-spined stickleback, intrinsic folds consisting of non-sensory cells are found in the apical part of the sensory epithelium where they divide the surface of the sensory epithelium into small islets. These non-sensory cells are non-ciliated, flattened and piled on top of each other; they contain numerous electron-translucent vesicles. The intrinsic folds are absent from the sensory epithelium of the three-spined stickleback. In both species, axons of receptor cells form a layer of fibers in the sensory epithelium immediately above the basal cells. In the three-spined stickleback, thick branches of the olfactory nerve are frequently found in this layer. These branches are only occasionally observed in the sensory epithelium of the nine-spined stickleback. Thus, the three-spined stickleback and the nine-spined stickleback show considerable differences in the organization of the sensory regions of the olfactory epithelium.     

Vanadium inhibits placental glutathione S-transferase (GST-P) positive foci in 1,2-dimethyl hydrazine induced rat colon carcinogenesis

Vanadium (V) has recently been found to possess potent anti-neoplastic activity in rat colon carcinogenesis. In the present study attempts have been made to investigate the expression of the number and area of aberrant crypt foci positive for placental glutathione S-transferase (GST-P) during 1,2-dimethyl hydrazine (DMH)-induced rat colon carcinogenesis. Male Sprague Dawley rats were randomly divided into four groups. Rats in group A were designed as normal controls. Group B animals received DMH once a week (20 mg/kg body wt.) intraperitoneally for 16 weeks. Group C rats received the same treatment of DMH as in group B, along with 0.5-ppm vanadium as ammonium monovanadate ad libitum in drinking water throughout the experiment. Vanadium alone was given to Group D rats without any DMH injection. The expression of the number and the area of aberrant crypt foci (ACF) positive for GST-P was maximum in DMH-treated group. Vanadium-treated rats significantly reduced (P < 0.001) the expression of GST-P positive ACF cells (by 71.13%) for the entire period of the study. Moreover the histopathological examination also showed that vanadium action could minimize the aberrant crypt foci (P < 0.001). Furthermore, vanadium supplementation also elevated SOD activities in both liver and colon (P < 0.01, P < 0.02 and P < 0.01, P < 0.02 respectively) when compared to their carcinogen counterparts. Our results confirm that vanadium is particularly effective in limiting the action of the carcinogen, thereby establishing its anticarcinogenicity in chemically induced rat colon carcinogenesis.     

A novel population of neuronal cells expressing the olfactory marker protein (OMP) in the anterior/dorsal region of the nasal cavity

The olfactory marker protein (OMP) is expressed in mature chemosensory neurons in the nasal neuroepithelium. Here, we report the identification of a novel population of OMP-expressing neurons located bilaterally in the anterior/dorsal region of each nasal cavity at the septum. These cells are clearly separated from the regio olfactoria, harboring the olfactory sensory neurons. During mouse development, the arrangement of the anterior OMP-cells undergoes considerable change. They appear at about stage E13 and are localized in the nasal epithelium during early stages; by epithelial budding, ganglion-shaped clusters are formed in the mesenchyme during the perinatal phase, and a filiform layer directly underneath the nasal epithelium is established in adults. The anterior OMP-cells extend long axonal processes which form bundles and project towards the brain. The data suggest that the newly discovered group of OMP-cells in the anterior region of the nasal cavity may serve a distinct sensory function.     

罗非鱼微囊藻毒素去毒相关基因克隆与活体表达研究

可溶性谷胱甘肽S-转移酶(Soluble glutathione S-transferase,sGST)催化微囊藻毒素(Microcystins,MCs)与还原型谷胱甘肽(GSH)的加合去毒代谢过程,谷胱甘肽过氧化物酶(Glutathione peroxidase,GPX)为sGST的去毒反应提供GSH,解偶联蛋白2(Uncoupling protein 2,UCP2)则可抑制微囊藻毒素诱发活性氧导致的肝细胞凋亡。本研究从罗非鱼肝脏通过简并引物克隆sGST、GPX与UCP2基因cDNA核心序列,并应用5’RACE和3’RACE技术分别扩增罗非鱼肝脏sGST基因cDNA序列5’末端和3’末端序列而获得其cDNA全序列。罗非鱼肝脏sGST基因cDNA全序列长861 bp,其中5’非翻译区(5-’UTR)为25 bp,3’非翻译区(3-’UTR)为167 bp,开放阅读框(ORF)为669 bp,编码222个氨基酸,包含脊椎动物完整sGST的2个功能域:N-末端功能域(GSH结合位点)和C-末端功能域(底物结合位点)。罗非鱼sGST与真鲷、条石鲷(Oplegnathus fasciatus)、斑马鱼同源性较高,达到64.3%—78.5%,而与人、大鼠、小鼠、牛、猪、鸡差异较大,氨基酸同源性为48.2%—55.9%。罗非鱼肝脏GPX、UCP2基因cDNA核心序列长280 bp、776 bp,分别编码92、258个氨基酸。罗非鱼GPX与条石鲷、虹鳟、斑马鱼、人、大鼠、小鼠、牛、猪GPX同源性均较高,达到69.6%—85.9%。罗非鱼UCP2与真鲷、斑马鱼、鲤鱼、欧洲白鲑(Leuciscus cephalus)、草鱼、人、大鼠、小鼠UCP2同源性更高,达到71.8%—93.8%。通过对罗非鱼(5—8 g)活体腹腔注射亚致死量MC-LR(50μg/kg bwt),发现微囊藻毒素对罗非鱼肝脏sGST基因表达有显著的诱导作用(p<0.05),注射微囊藻毒素24h后sGST基因mRNA表达水平上调80%。注射微囊藻毒素24h后,虽然罗非鱼肝脏GPX与UCP2基因mRNA表达水平亦出现明显的升高趋势,但两者均未出现显著性的变化(p>0.05)。本研究从基因表达调控的角度证实,罗非鱼肝脏sGST在微囊藻毒素去毒过程中可能发挥关键作用,同时也说明罗非鱼肝脏GPX、UCP2基因可能在微囊藻去毒过程中发挥协同作用。     

Mating behavior induces changes of expression of Fos protein,plasma testosterone and androgen receptors in the accessory olfactory bulb (AOB) of the male mandarin vole Microtus mandarinus

In order to investigate the neuroendocrine mechanism of the mating behavior in the adult male mandarin voles Microtus mandarinus,the radioimmunoassay(RIA)and immunohistochemistry methods were used to investigate the differences in plasma testosterone(T)concentrations and distribution of T immunoreactive neurons(T-IRs),androgen receptor immunoreactive neurons(AR-IRs)and Fos protein immunoreactive neurons(Fos-IRs)in the accessory olfactory bulb(AOB)and the main olfactory bulb(MOB)following exposure to clean h...     

Primary structure and function of a cytotoxic outer-membrane protein (ComP) of Plesiomonas shigelloides

We previously isolated and characterized a 40-kDa cytotoxic outer-membrane protein (ComP) produced by Plesiomonas shigelloides strain P-1 (P-1). Sequence analysis of the comP gene revealed a coding region of 1068 bp, with a predicted mature protein composed of 335 amino acids and a molecular mass of 38.597 kDa. Three-dimensional structural modeling of ComP suggests that it has a beta-barrel structure with 16 transmembrane strands, eight short periplasmic turns and eight external loops. blast search results and protein modeling suggest that ComP may be a novel porin protein of P. shigelloides. In order to understand the role of ComP during P. shigelloides infection, we constructed a deletion mutant strain (P. shigelloides DeltacomP; P-1201), and compared the pathogenicity of P-1201 vs. the wild-type strain P-1 in Caco-2 cells. Unlike P-1, the deletion strain P-1201 was not cytotoxic to Caco-2 cells and did not lead to apoptosis. These data indicate that ComP may be the predominant virulence factor that triggers cell death in the host cells following infection.     

The metabolic bioactivation of caffeic acid phenethyl ester (CAPE) mediated by tyrosinase selectively inhibits glutathione S-transferase

Glutathione S-transferase (GST) and multidrug resistance-associated proteins (MRPs) play major roles in drug resistance in melanoma. In this study, we investigated caffeic acid phenethyl ester (CAPE) as a selective GST inhibitor in the presence of tyrosinase, which is abundant in melanoma cells. Tyrosinase bioactivates CAPE to an o-quinone, which reacts with glutathione to form CAPE-SG conjugate. Our findings indicate that 90% CAPE was metabolized by tyrosinase after a 60-min incubation. LC–MS/MS analyses identified a CAPE-SG conjugate as a major metabolite. In the presence of tyrosinase, CAPE (10–25 μM) showed 70–84% GST inhibition; whereas in the absence of tyrosinase, CAPE did not inhibit GST. CAPE-SG conjugate and CAPE-quinone (25 μM) demonstrated ?85% GST inhibition via reversible and irreversible mechanisms, respectively. Comparing with CDNB and GSH, the non-substrate CAPE acted as a weak, reversible GST inhibitor at concentrations >50 μM. Furthermore, MK-571, a selective MRP inhibitor, and probenecid, a non-selective MRP inhibitor, decrease the IC₅₀ of CAPE (15 μM) by 13% and 21%, apoptotic cell death by 3% and 13%, and mitochondrial membrane potential in human SK-MEL-28 melanoma cells by 10% and 56%, respectively. Moreover, computational docking analyses suggest that CAPE binds to the GST catalytic active site. Caffeic acid, a hydrolyzed product of CAPE, showed a similar GST inhibition in the presence of tyrosinase. Although, as controls, 4-hydroxyanisole and l-tyrosine were metabolized by tyrosinase to form quinones and glutathione conjugates, they exhibited no GST inhibition in the absence and presence of tyrosinase. In conclusion, both CAPE and caffeic acid selectively inhibited GST in the presence of tyrosinase. Our results suggest that intracellularly formed quinones and glutathione conjugates of caffeic acid and CAPE may play major roles in the selective inhibition of GST in SK-MEL-28 melanoma cells. Moreover, the inhibition of MRP enhances CAPE-induced toxicity in the SK-MEL-28 melanoma cells.     

Replication protein A (AtRPA1a) is required for class I crossover formation but is dispensable for meiotic DNA break repair

Replication protein A (RPA) is involved in many aspects of DNA metabolism including meiotic recombination. Many species possess a single RPA1 gene but Arabidopsis possesses five RPA1 paralogues. This feature has enabled us to gain further insight into the meiotic role of RPA1. Proteomic analysis implicated one of the AtRPA1 family (AtRPA1a) in meiosis. Immunofluorescence studies confirmed that AtRPA1a is associated with meiotic chromosomes from leptotene through to early pachytene. Analysis of an Atrpa1a mutant revealed that AtRPA1a is not essential at early stages in the recombination pathway. DNA double‐strand breaks are repaired in Atrpa1a, but the mutant is defective in the formation of crossovers, exhibiting a 60% reduction in chiasma frequency. Consistent with this, localization of recombination proteins AtRAD51 and AtMSH4 appears normal, whereas the numbers of AtMLH1 and AtMLH3 foci at pachytene are significantly reduced. This suggests that the defect in Atrpa1a is manifested at the stage of second‐end capture. Analysis of Atrpa1a/Atmsh4 and Atrpa1a/Atmlh3 double mutants indicates that loss of AtRPA1a predominantly affects the formation of class I, interference‐dependent crossovers.     

Induction of expression and co-localization of heat shock polypeptides with the polyalanine expansion mutant of poly(A)-binding protein N1 after chemical stress

Formation of nuclear inclusions consisting of aggregates of a polyalanine expansion mutant of nuclear poly(A)-binding protein (PABPN1) is the hallmark of oculopharyngeal muscular dystrophy (OPMD). OPMD is a late onset autosomal dominant disease. Patients with this disorder exhibit progressive swallowing difficulty and drooping of their eye lids, which starts around the age of 50. Previously we have shown that treatment of cells expressing the mutant PABPN1 with a number of chemicals such as ibuprofen, indomethacin, ZnSO₄, and 8-hydroxy-quinoline induces HSP70 expression and reduces PABPN1 aggregation. In these studies we have shown that expression of additional HSPs including HSP27, HSP40, and HSP105 were induced in mutant PABPN1 expressing cells following exposure to the chemicals mentioned above. Furthermore, all three additional HSPs were translocated to the nucleus and probably helped to properly fold the mutant PABPN1 by co-localizing with this protein.     

An immunocytochemical study of pulpal responses to cavity preparation by laser ablation in rat molars by using antibodies to heat shock protein (Hsp) 25 and class II MHC antigen

Initial responses of odontoblasts and immunocompetent cells to cavity preparation by laser ablation were investigated in rat molars. In untreated control teeth, intense heat shock protein (Hsp) 25 immunoreactivity was found in the cell bodies of odontoblasts, whereas cells immunopositive for the class II major histocompatibility complex (MHC) antigen were predominantly located beneath the odontoblast layer in the dental pulp. Cavity preparation caused the destruction of the odontoblast layer and the shift of most class-II-MHC-positive cells from the pulp-dentin border toward the pulp core at the affected site. Twelve hours after cavity preparation, numerous class-II-MHC-positive cells appeared along the pulp-dentin border and extended their processes deep into the exposed dentinal tubules, but subsequently disappeared from the pulp-dentin border together with Hsp-25-immunopositive cells by 24 h after the operation. By 3–5 days postoperation, distinct abscess formation consisting of polymorphonuclear leukocytes was found in the dental pulp. The penetration of masses of oral bacteria was recognizable in the dentinal tubules beneath the prepared cavity. These findings indicate that cavity preparation by laser ablation induces remarkable inflammation by continuous bacterial infections via dentinal tubules in this experimental model, thereby delaying pulpal regeneration.This work was supported by Grant-in-Aid for Scientific Research to promote 2001-Multidisciplinary Research Projects in 2001–2005, and KAKENHI (C) (nos. 12671765 and 14571727 to H.O.) from MEXT     

The crystal structure of death receptor 6 (DR6): a potential receptor of the amyloid precursor protein (APP)

Death receptors belong to the tumor necrosis factor receptor (TNFR) super family and are intimately involved in the signal transduction during apoptosis, stress response and cellular survival. Here we present the crystal structure of recombinantly expressed death receptor six (DR6), one family member that was recently shown to bind to the amyloid precursor protein (APP) and hence to be probably involved in the development of Alzheimer's disease. The extracellular cysteine rich region of DR6, the typical ligand binding region of all TNFRs, was refined to 2.2 Å resolution and shows that its four constituting cysteine rich domains (CRDs) are arranged in a rod-like overall structure, which presents DR6-specific surface patches responsible for the exclusive recognition of its ligand(s). Based on the structural data, the general ligand binding modes of TNFRs and molecular modeling experiments we were able to elucidate structural features of the potential DR6-APP signaling complex.     

Crystal structure of the conserved protein TTHA0727 from Thermus thermophilus HB8 at 1.9 A resolution: A CMD family member distinct from carboxymuconolactone decarboxylase (CMD) and AhpD

TTHA0727 is a conserved hypothetical protein from Thermus thermophilus HB8, with a molecular mass of 12.6 kDa. TTHA0727 belongs to the carboxymuconolactone decarboxylase (CMD) family (Pfam 02627). A sequence comparison with its homologs suggested that TTHA0727 is a distinct protein from alkylhydroperoxidase AhpD and gamma-carboxymuconolactone decarboxylase in the CMD family. Here we report the 1.9 A crystal structure of TTHA0727 (PDB ID: 2CWQ) determined by the multiwavelength anomalous dispersion method. The TTHA0727 monomer structure consists of seven alpha-helices (alpha1-alpha7) and one short 3(10)-helix. The crystal structure and the analytical ultracentrifugation revealed that TTHA0727 forms a hexameric ring structure in solution. The electrostatic potential distribution on the solvent-accessible surface of the TTHA0727 hexamer showed that positively charged regions exist on the side of the ring structure, suggesting that TTHA0727 interacts with some negatively charged molecules. A structural homology search revealed that the structure of three alpha-helices (alpha4-alpha6) is remarkably conserved, suggesting that it is the common structural motif for the CMD family proteins. In addition, the nine residues of the N-terminal tag bound to the cleft region between alpha1 and alpha3 in chains A and B of TTHA0727, implying that this region is the putative binding/active site for some small molecules.