Recombinant human serotonin 5A receptors stably expressed in C6 glioma cells couple to multiple signal transduction pathways

Human serotonin 5A (5-HT5A) receptors were stably expressed in undifferentiated C6 glioma. In 5-HT5A receptors-expressing cells, accumulation of cAMP by forskolin was inhibited by 5-HT as reported previously. Pertussis toxin-sensitive inhibition of ADP-ribosyl cyclase was also observed, indicating a decrease of cyclic ADP ribose, a potential intracellular second messenger mediating ryanodine-sensitive Ca2+ mobilization. On the other hand, 5-HT-induced outward currents were observed using the patch-clamp technique in whole-cell configuration. The 5-HT-induced outward current was observed in 84% of the patched 5-HT5A receptor-expressing cells and was concentration-dependent. The 5-HT-induced current was inhibited when intracellular K+ was replaced with Cs+ but was not significantly inhibited by typical K+ channel blockers. The 5-HT-induced current was significantly attenuated by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) in the patch pipette. Depleting intracellular Ca2+ stores by application of caffeine or thapsigargin also blocked the 5-HT-induced current. Blocking G protein, the inositol triphosphate (IP3) receptor, or pretreatment with pertussis toxin, all inhibited the 5-HT-induced current. IP3 showed a transient increase after application of 5-HT in 5-HT5A receptor-expressing cells. It was concluded that in addition to the inhibition of cAMP accumulation and ADP-ribosyl cyclase activity, 5-HT5A receptors regulate intracellular Ca2+ mobilization which is probably a result of the IP3-sensitive Ca2+ store. These multiple signal transduction systems may induce complex changes in the serotonergic system in brain function.     

Hepatocyte growth factor exerts a proliferative effect on oval cells through the PI3K/AKT signaling pathway

Hepatocyte growth factor (HGF) is a potent mitogen for a variety of cells including hepatocytes. While rat oval cells are supposed to be one of hepatic stem cells, biological effects of HGF on oval cells and their relevant signal transduction pathways remain to be determined. We sought to investigate them on OC/CDE22 rat oval cells, which are established from the liver of rats fed a choline-deficient/DL-ethionine-supplemented diet. The oval cells were cultured on fibronectin-coated dishes and stimulated with recombinant HGF, transforming growth factor-alpha (TGF-alpha), and thrombopoietin (TPO) under the serum-free medium condition. HGF treatment enhanced [3H]thymidine incorporation into oval cells in a dose-dependent manner. On the contrary, treatment with TGF-alpha or TPO had no significant effects on [3H]thymidine incorporation into the oval cells. c-Met protein was phosphorylated at the tyrosine residues after the HGF treatment. AKT, extracellular signal-regulated kinase 1/2 (ERK1/2), and p70(s6k) were simultaneously activated after the HGF stimulation, peaking at 30min after the treatment. The activation of AKT, p70(s6k), and ERK1/2 induced by HGF was abolished by pre-treatment with LY294002, a phosphoinositide 3-OH kinase (PI3K) inhibitor, and U0126, a mitogen-activated protein kinase/ERK kinase (MEK) inhibitor, respectively. When the cells were pre-treated with LY294002 prior to the HGF stimulation, the proliferative action of HGF was completely abrogated, implying that the PI3K/AKT signaling pathway is responsible for the biological effect of HGF. These in vitro data indicate that HGF exerts a proliferative action on hepatic oval cells via activation of the PI3K/AKT signaling pathway.     

Tissue-specific expression of rice CYP72A21 induced by auxins and herbicides

Cytochrome monooxygenase P450s (CYPs) comprise one of the largest enzyme families in plants. Some P450s are involved in xenobiotic metabolism: they confer herbicide tolerance and are induced by chemical treatments. We isolated a novel P450 cDNA, CYP72A21 (accession number, AB237166), from rice (Oryza sativa L. cv. Nipponbare) seedlings treated with a mixture of 2,4-dichlorophenoxyacetic acid (2,4-D), chlorotoluron, phenobarbital, salicylic acid, and naphthalic anhydride (each 100 μM). We also isolated the gene’s promoter region. Endogenous CYP72A21 expression in rice seedlings treated with 2,4-D, herbicides esprocarb, or trifluralin was increased in the aerial part of seedlings. An expression plasmid, pI21pg, containing the GUS gene under the control of the CYP72A21 promoter was introduced into rice plants. GUS was expressed constitutively in roots, but this expression was suppressed by 2,4-D treatment. 2,4-D and other auxins induced GUS expression effectively in the stem and leaves. Histological observation revealed that GUS was expressed mainly in the base of the stem. Treatment with the herbicides acetochlor, esprocarb, and propyzamide induced GUS expression in the aerial parts of the seedlings. The CYP72A21 promoter was highly responsive to treatments with various chemicals, and thus might be useful for producing transgenic plants for biomonitoring of environmental chemicals.     

Mechanically induced intracellular calcium waves in osteoblasts demonstrate calcium fingerprints in bone cell mechanotransduction

An early response to mechanical stimulation of bone cells in vitro is an increase in intracellular calcium concentration ([Ca ²⁺]_i). This study analyzed the [Ca ²⁺]_i wave area, magnitude, duration, rise time, fall time, and time to onset in individual osteoblasts for two identical bouts of mechanical stimulation separated by a 30-min rest period. The area under the [Ca ²⁺]_i wave increased in the second loading bout compared to the first. This suggests that rest periods may potentiate mechanically induced intracellular calcium signals. Furthermore, many of the [Ca ²⁺]_i wave parameters were strongly, positively correlated between the two bouts of mechanical stimulation. For example, in individual primary osteoblasts, if a cell had a large [Ca ²⁺]_i wave area in the first bout it was likely to have a large [Ca ²⁺]_i wave area in the second bout (r ² = 0.933). These findings support the idea that individual bone cells have “calcium fingerprints” (i.e., a unique [Ca ²⁺]_i wave profile that is reproducible for repeated exposure to a given stimulus).     

Development of transgenic rice seed accumulating a major Japanese cedar pollen allergen (Cry j 1) structurally disrupted for oral immunotherapy

Rice seed-based edible vaccines expressing T-cell epitope peptides derived from Japanese cedar major pollen allergens have been used to successfully suppress allergen-specific Th2-mediated immunoglobulin E (IgE) responses in mouse experiments. In order to further expand the application of seed-based allergen-specific immunotherapy for controlling Japanese cedar pollinosis, we generated transgenic rice plants that specifically express recombinant Cry j 1 allergens in seeds. Cry j 1 allergens give low specific IgE-binding activity but contain all of the T-cell epitopes. The allergens were expressed directly or as a protein fusion with the major rice storage protein glutelin. Fusion proteins expressed under the control of the strong rice endosperm-specific GluB-1 promoter accumulated in rice endosperm tissue up to 15% of total seed protein. The fusion proteins aggregated with cysteine-rich prolamin and were deposited in endoplasmic reticulum-derived protein body I. The production of transgenic rice expressing structurally disrupted Cry j 1 peptides with low IgE binding activity but spanning the entire Cry j1 region can be used as a universal, safe and effective tolerogen for rice seed-based oral immunotherapy for cedar pollen allergy in humans and other mammals.     

Clock gene-dependent glutamate dynamics in the bean bug brain regulate photoperiodic reproduction

Animals adequately modulate their physiological status and behavior according to the season. Many animals sense photoperiod for seasonal adaptation, and the circadian clock is suggested to play an essential role in photoperiodic time measurement. However, circadian clock-driven neural signals in the brain that convey photoperiodic information remain unclear. Here, we focused on brain extracellular dynamics of a classical neurotransmitter glutamate, which is widely used for brain neurotransmission, and analyzed its involvement in photoperiodic responses using the bean bug Riptortus pedestris that shows clear photoperiodism in reproduction. Extracellular glutamate levels in the whole brain were significantly higher under short-day conditions, which cause a reproductive diapause, than those under long-day conditions. The photoperiodic change in glutamate levels was clearly abolished by knockdown of the clock gene period. We also demonstrated that genetic modulation of glutamate dynamics by knockdown of glutamate-metabolizing enzyme genes, glutamate oxaloacetate transaminase (got) and glutamine synthetase (gs), attenuated photoperiodic responses in reproduction. Further, we investigated glutamate-mediated photoperiodic modulations at a cellular level, focusing on the pars intercerebralis (PI) neurons that photoperiodically change their neural activity and promote oviposition. Electrophysiological analyses showed that L-Glutamate acts as an inhibitory signal to PI neurons via glutamate-gated chloride channel (GluCl). Additionally, combination of electrophysiology and genetics revealed that knockdown of got, gs, and glucl disrupted cellular photoperiodic responses of the PI neurons, in addition to reproductive phenotypes. Our results reveal that the extracellular glutamate dynamics are photoperiodically regulated depending on the clock gene and play an essential role in the photoperiodic control of reproduction via inhibitory pathways.     

Screening of Peptides that Inhibit Bacterial Binding to Fibronectin using Combinatorial Peptide Libraries

Infective endocarditis is often caused by passage of oral endogenous bacteria into the blood stream. Such bacteremia occurs after tooth extraction, and occasionally even after brushing of the teeth. Abnormal or damaged heart valves, including artificial valves, show high risk of infection. Antibiotics are widely used to prevent this infection, however, frequent use of these have resulted in the generation of resistant mutants, which generate serious social problems. Thus, the development of more effective and safer drugs for the prevention of such infections is very desirable. The adhesion of bacteria to fibronectin, one of the major extracellular matrix (ECM) protein exposed on the wound endocardia, is considered critical for the infection. We have previously found a novel mode of interaction between endocarditis-causing bacteria and human fibronectin. The present study focuses on the discovery of candidate compounds that inhibit the association between microorganisms and fibronectin. Positional scanning libraries (PSL) with N-terminal biotinylated 6-mer peptides have been constructed and screened for binding to a monoclonal antibody for fibronectin that inhibits the bacterial fibronectin-binding. The consensus sequences derived from these experiments are expected to be structural mimetics of the local structure of fibronectin involved in the bacterial adhesion. Since individual synthetic 6-mer peptides did not show the desired action, discontinuous epitopes can be envisaged and therefore a 9-mer-PSL was constructed to reveal conformational epitopes. In the second library, several 9-mer peptides based on the screening were synthesized and gave improved results.Australian Peptide Conference Issue