Rhythmic electromyographic activities of trunk muscles characterize the sexual behavior in the Himé salmon (landlocked sockeye salmon,Oncorhynchus nerka)

Summary In order to describe precisely the fixed action patterns of salmon sexual behavior, we recorded the electromyographic (EMG) activities of trunk and jaw muscles from freely behaving male and female Himé salmon (landlocked sockeye salmon,Oncorhynchus nerka). A series of action patterns (quivering and spawning act in males, digging, covering, prespawning act and spawning act in females, and the swimming and turning movements in both sexes) were characterized by rhythmic activities of the trunk muscles. Each of these activity patterns is quantitatively distinct from the others in such parameters as frequency, bout duration, duty value, intersegmental phase delay, and spatial distribution of rhythmic activities. However, all of these rhythms share a qualitatively homologous pattern with the forward swimming movement: rhythmic activities alternate on both sides of the body (bilateral coupling) and are posteriorly propagated (intersegmental coupling). In addition, a 31 intersegmental phase coupling occurs in the most anterior trunk muscles during the spawning act in some males. Based on these observations, we discussed the biomechanics for these motor patterns (oviposition, ejaculation, body vibration, and mouth opening), and the neural mechanisms for the pattern generation. A possibility was pointed out that the locomotor pattern generator in the spinal cord may be modulated by descending supraspinal signals and recruited to generate such diverse forms of action patterns in sexual behavior.Abbreviations CPG central pattern generator - EMG electromyography - AC adductor mandibulae (cephalic portion) - AM adductor mandibulae (mandibular portion) - DO dilator operculi - GH geniohyoideus - LAP levator arcus palatitni - LPe musculus lateralis profundus (epaxial portion) - LPh musculus lateralis profundus (hypaxial portion) - LS musculus lateralis superficialis - PD protractor dorsalis - PI protractor ischii - RD retractor dorsalis - RI retractor ischii     

Brain-derived neurotrophic factor (BDNF) induces antagonistic action to Nogo signaling by the upregulation of lateral olfactory tract usher substance (LOTUS) expression

Neurons in the central nervous system (CNS) have limited capacity for axonal regeneration after trauma and neurological disorders due to an endogenous nonpermissive environment for axon regrowth in the CNS. Lateral olfactory tract usher substance (LOTUS) contributes to axonal tract formation in the developing brain and axonal regeneration in the adult brain as an endogenous Nogo receptor-1 (NgR1) antagonist. However, how LOTUS expression is regulated remains unclarified. This study examined molecular mechanism of regulation in LOTUS expression and found that brain-derived neurotrophic factor (BDNF) increased LOTUS expression in cultured hippocampal neurons. Exogenous application of BDNF increased LOTUS expression at both mRNA and protein levels in a dose-dependent manner. We also found that pharmacological inhibition with K252a and gene knockdown by siRNA of tropomyosin-related kinase B (TrkB), BDNF receptor suppressed BDNF-induced increase in LOTUS expression. Further pharmacological analysis of the TrkB signaling pathway revealed that BDNF increased LOTUS expression through mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) cascades, but not phospholipase C-γ (PLCγ) cascade. Additionally, treatment with c-AMP response element binding protein (CREB) inhibitor partially suppressed BDNF-induced LOTUS expression. Finally, neurite outgrowth assay in cultured hippocampal neurons revealed that BDNF treatment-induced antagonism for NgR1 by up-regulating LOTUS expression. These findings suggest that BDNF may acts as a positive regulator of LOTUS expression through the TrkB signaling, thereby inducing an antagonistic action for NgR1 function by up-regulating LOTUS expression. Also, BDNF may synergistically affect axon regrowth through the upregulation of LOTUS expression.

     

Simvastatin enhances induction of inducible nitric oxide synthase in 3T3-L1 adipocytes

The present study was designed to determine whether hydroxymethylglutaryl-CoA reductase inhibitors (statins) modulate the NO production via iNOS in adipocytes stimulated by lipopolysaccharide (L) and tumour necrosis factor-α (T). Well-differentiated 3T3-L1 adipocytes significantly produced NO by LT-treatment. Pre-incubation with simvastatin, a lipophilic statin, pravastatin, a hydrophilic one, or Y27632, an inhibitor of Rho kinase, further enhanced the production of NO. The effect of simvastatin was offset by mevalonate and geranylgeranyl pyrophosphate (GGPP) but not by squalene. The mRNA level for iNOS parallelled the NO production. The NF-κB was activated by the LT-treatment and was further enhanced by simvastatin, pravastatin or Y27632 addition. Mevalonate and GGPP completely offset the effect of simvastatin. Statins and Y27632 also further increased the interleukin-6 secretion in the LT-treated 3T3-L1 adipocytes. These results suggest that statins, especially lipophilic type, enhance induction of iNOS by inhibiting the small GTP-binding protein signal in adipocytes.     

Coupling of GABAB receptor GABAB2 subunit to G proteins: evidence from Xenopus oocyte and baby hamster kidney cell expression system

Coupling of functional GABA_B receptors (GABA_BR) to G proteins was investigated with an expression system of baby hamster kidney (BHK) cells and Xenopus oocytes. Fluorescence resonance energy transfer (FRET) analysis of BHK cells coexpressing GABA_B1a receptor (GB_1aR) fused to Cerulean, a brighter variant of cyan fluorescent protein, and GABA_B2 receptor (GB₂R) fused to Venus, a brighter variant of yellow fluorescent protein, revealed that GB_1aR-Cerulean and GB₂R-Venus form a heterodimer. The GABA_BR agonists baclofen and 3-aminopropylphosphonic acid (3-APPA) elicited inward-rectifying K⁺ currents in a concentration-dependent manner in oocytes expressing GB_1aR and GB₂R, or GB_1aR-Cerulean and GB₂R-Venus, together with G protein-activated inward-rectifying K⁺ channels (GIRKs), but not in oocytes expressing GB_1aR alone or GB₂R alone together with GIRKs. Oocytes coexpressing GB_1aR + G_i2-fused GB₂R (GB₂R-G_i2) caused faster K⁺ currents in response to baclofen. Furthermore, oocytes coexpressing GB_1aR + GB₂R fused to G_qi5 (a chimeric G_q protein that activates PLC pathways) caused PLC-mediated Ca²⁺-activated Cl^– currents in response to baclofen. In contrast, these responses to baclofen were not observed in oocytes coexpressing GB_1aR-G_i2 or GB_1aR-G_qi5 together with GB₂R. BHK cells and Xenopus oocytes coexpressing GB_1aR-Cerulean + a triplet tandem of GB₂R-Venus-G_qi5 caused FRET and Ca²⁺-activated Cl^– currents, respectively, with a similar potency in BHK cells coexpressing GB_1aR-Cerulean + GB₂R-Venus and in oocytes coexpressing GB_1aR + GB₂R-G_qi5. Our results indicate that functional GABA_BR forms a heterodimer composed of GB₁R and GB₂R and that the signal transducing G proteins are directly coupled to GB₂R but not to GB₁R. fluorescence resonance energy transfer     

Characterization of newly established bovine intestinal epithelial cell line

Membranous epithelial cells (M cells) of the follicle-associated epithelium in Peyer’s patches have a high capacity for transcytosis of several viruses and microorganisms. Here, we report that we have successfully established a bovine intestinal epithelial cell line (BIE cells) and developed an in vitro M cell model. BIE cells have a cobblestone morphology and microvilli-like structures, and strongly express cell-to-cell junctional proteins and cytokeratin, which is a specific intermediate filament protein of epithelial cells. After co-culture with murine intestinal lymphocytes or treatment with supernatant from bovine PBMC cultured with IL-2, BIE cells acquired the ability of transcytosis. Therefore, BIE cells have typical characteristics of bovine intestinal epithelial cells and also have the ability to differentiate into an M cell like linage. In addition, our results indicate that contact between immune cells and epithelial cells may not be absolutely required for the differentiation of M cells. We think that BIE cells will be useful for studying the transport mechanisms of various pathogens and also the evaluation of drug delivery via M cells.     

Reversible and nonoxidative gamma-resorcylic acid decarboxylase: characterization and gene cloning of a novel enzyme catalyzing carboxylation of resorcinol, 1,3-dihydroxybenzene, from Rhizobium radiobacter

We found a gamma-resorcylic acid (gamma-RA, 2,6-dihydroxybenzoic acid) decarboxylase, as a novel enzyme applicable to carboxylation of resorcinol (RE, 1,3-dihydroxybenzene) to form gamma-RA, in a bacterial strain Rhizobium radiobacter WU-0108 isolated through the screening of gamma-RA degrading microorganisms. The activities for carboxylation of RE and decarboxylation of gamma-RA were detected in the cell-free extracts of R. radiobacter WU-0108 grown aerobically with gamma-RA. The enzyme, gamma-RA decarboxylase, was purified to homogeneity on SDS-PAGE through the steps of one ion-exchange chromatography and two kinds of hydrophobic chromatography. The molecular weight of the enzyme was estimated to be 130 kDa by gel-filtration, and that of the subunit was determined to be 34 kDa by SDS-PAGE, suggesting that the enzyme is a homotetrameric structure. The enzyme catalyzed the decarboxylation of gamma-RA, but not alpha-RA or beta-RA. Without addition of any cofactors, the enzyme catalyzed the regio-selective carboxylation of RE to form gamma-RA, without formation of alpha-RA and beta-RA, and of catechol to 2,3-dihydroxybenzoic acid. In the presence of oxygen, this gamma-RA decarboxylase showed no decrease in both of the activities as for decarboxylation of gamma-RA and carboxylation of RE, different from other decarboxylases reported so far. The gene, rdc, encoding the gamma-RA decarboxylase was cloned into Escherichia coli, sequenced, and subjected to over-expression. The deduced amino acid sequence of the rdc gene consists of 327 amino acid residues corresponding to 34 kDa protein, and shows 42% and 30% identity to those of a 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus niger and a 5- carboxyvanillate decarboxylase from Sphingomonas paucimobilis SYK-6. A site-directed mutagenesis study revealed the two histidine residues at positions of 164 and 218 in Rdc to be essential for the catalytic activities of decarboxylation of gamma-RA and carboxylation of RE.     

Involvement of G protein betagamma-subunits in diverse signaling induced by G(i/o)-coupled receptors: study using the Xenopus oocyte expression system

We studied the functions of -subunits of G_i/o protein using the Xenopus oocyte expression system. Isoproterenol (ISO) elicited cAMP production and slowly activating Cl^– currents in oocytes expressing ₂-adrenoceptor and the protein kinase A-dependent Cl^– channel encoded by the cystic fibrosis transmembrane conductance regulator (CFTR) gene. 5-Hydroxytryptamine (5-HT), [D-Ala², D-Leu⁵]-enkephalin (DADLE), and baclofen enhanced ISO-induced cAMP levels and CFTR currents in oocytes expressing ₂-adrenoceptor-CFTR and 5-HT_1A receptor (5-HT_1AR), -opioid receptor, or GABA_B receptor, respectively. 5-HT also enhanced pituitary adenylate cyclase activating peptide (PACAP) 38-induced cAMP levels and CFTR currents in oocytes expressing PACAP receptor, CFTR and 5-HT_1AR. The 5-HT-induced enhancement of G_s-coupled receptor-mediated currents was abrogated by pretreatment with pertussis toxin (PTX) and coexpression of G transducin (G_t). The 5-HT-induced enhancement was further augmented by coexpression of the G-activated form of adenylate cyclase (AC) type II but not AC type III. Thus -subunits of G_i/o protein contribute to the enhancement of G_s-coupled receptor-mediated responses. 5-HT and DADLE did not elicit any currents in oocytes expressing 5-HT_1AR or -opioid receptor alone. They elicited Ca²⁺-activated Cl^– currents in oocytes coexpressing these receptors with the G-activated form of phospholipase C (PLC)-2 but not with PLC-1. These currents were inhibited by pretreatment with PTX and coexpression of G_t, suggesting that -subunits of G_i/o protein activate PLC-2 and then cause intracellular Ca²⁺ mobilization. Our results indicate that -subunits of G_i/o protein participate in diverse intracellular signals, enhancement of G_s-coupled receptor-mediated responses, and intracellular Ca²⁺ mobilization. G protein-coupled receptor; cystic fibrosis transmembrane conductance regulator gene; cross talk; electrophysiology     

Protective mucosal immunity in aging is associated with functional CD4+ T cells in nasopharyngeal-associated lymphoreticular tissue

Our previous studies showed that mucosal immunity was impaired in 1-year-old mice that had been orally immunized with OVA and native cholera toxin (nCT) as mucosal adjuvant. In this study, we queried whether similar immune dysregulation was also present in mucosal compartments of mice immunized by the nasal route. Both 1-year-old and young adult mice were immunized weekly with three nasal doses of OVA and nCT or with a nontoxic chimeric enterotoxin (mutant cholera toxin-A E112K/B subunit of native labile toxin) from Brevibacillus choshinensis. Elevated levels of OVA-specific IgG Abs in plasma and secretory IgA Abs in mucosal secretions (nasal washes, saliva, and fecal extracts) were noted in both young adult and 1-year-old mice given nCT or chimeric enterotoxin as mucosal adjuvants. Significant levels of OVA-specific CD4(+) T cell proliferative and OVA-induced Th1- and Th2-type cytokine responses were noted in cervical lymph nodes and spleen of 1-year-old mice. In this regard, CD4(+), CD45RB(+) T cells were detected in greater numbers in the nasopharyngeal-associated lymphoreticular tissues of 1-year-old mice than of young adult mice, but the same did not hold true for Peyer's patches or spleen. One-year-old mice given nasal tetanus toxoid plus the chimeric toxin as adjuvant were protected from lethal challenge with tetanus toxin. This result reinforced our findings that age-associated immune alterations occur first in gut-associated lymphoreticular tissues, and thus nasal delivery of vaccines for nasopharyngeal-associated lymphoreticular tissue-based mucosal immunity offers an attractive possibility to protect the elderly.     

Recycle Use of Sphingomonas sp. CDH-7 Cells for Continuous Degradation of Carbazole in the Presence of MgCl2

Carbazole (CA) is a heterocyclic nitrogen compound contained in the crude petroleum oil and recalcitrant to removal through the refinery processes. For development of the efficient CA-degradation bioprocess, conditions for the recycle use of Sphingomonas sp. CDH-7 resting cells were examined. When the resting cells (O.D.₆₆₀ 3.3) were shaken in 50 mM K₂HPO₄-KH₂PO₄ buffer (pH 7.0) containing CA 1000 mg/L, CA 880 mg/L was degraded within 3 h, but thereafter the activity decreased markedly. However, the activity was found to be restored to the initial level after the shaking treatment for 3 h in CA-free medium solution or in the buffer containing 20 mM MgCl₂. Although the CA-degradation activity of CDH-7 resting cells was lost after 3 h of shaking in the buffer containing 100 mM EDTA, it was restored through the shaking treatment for 3 h in the buffer containing 20 mM MgCl₂. When CA was periodically added eight times at a concentration of 100 mg/L (0.599 mM) to the reaction mixture containing the resting cells, CA 778 mg/L (4.66 mM) was continuously degraded within 35 h by the recycle use of resting cells, with the restoration treatment after each CA-degradation reaction by the resting cells. Received: 28 June 2001 / Accepted: 30 July 2001