Molecular characterization, expression analysis and function identification of Pf_TNF-α and its two receptors Pf_TNFR1 and Pf_TNFR2 in yellow catfish (Pelteobagrus fulvidraco).

Inflammation is a common manifestation of body immunity and mediates a cascade of cytokines. Tumor necrosis factor-α (TNF-α), as a multi-effect cytokine, plays an important role in the inflammatory response by interacting with its receptor (TNFR). In this study, Pf_TNF-α, Pf_TNFR1 and Pf_TNFR2 genes were cloned from yellow catfish (Pelteobagrus fulvidraco), and bioinformatics analyses showed that the three genes were conserved and possessed similar sequence characteristics as those of other vertebrates. The qPCR results showed that Pf_TNF-α, Pf_TNFR1 and Pf_TNFR2 mRNAs were constitutively expressed in 14 tissues and the lymphocytes of four tissues from healthy adults. The mRNA expression levels of Pf_TNF-α and Pf_TNFR1 genes were significantly up-regulated in the spleen, liver, trunk kidney, head kidney and gill after Edwardsiella ictaluri infection, while the mRNA expression of Pf_TNFR2 was significantly up-regulated in the spleen, and down-regulated in the liver and gill. In the isolated peripheral blood leukocytes (PBLs) of yellow catfish, the expression of Pf_TNF-α mRNA was notably up-regulated and the two Pf_TNFR transcripts were distinctly down-regulated after stimulation with lipopolysaccharides (LPS), peptidoglycan (PGN), polyinosinic-polycytidylic acid (Poly I:C) and phytohaemagglutinin (PHA). After stimulated by recombinant (r) Pf_sTNF protein, the mRNA expressions of various inflammatory factors genes were up-regulated in the PBLs. Meanwhile, rPf_sTNF promoted the phagocytic activity of leukocytes, whereas the activity mediated by rPf_sTNF could be inhibited by rPf_TNFR1CRD2/3 and rPf_TNFR2CRD2/3. The up-regulation of TNF-α and IL-1ß mRNAs expression triggered by rPf_sTNF could be inhibited by MAPK inhibitor (VX-702) and NF-κB inhibitor (PDTC). rPf_sTNF induced the expression of FADD mRNA in PBLs and increased the apoptotic rate of PBLs, and inhibiting the NF-κB and MAPK signal pathways could enhance the apoptosis of PBLs. The results indicate that Pf_TNF-α, Pf_TNFR1 and Pf_TNFR2 play important roles in the immune response of yellow catfish to bacterial invasion.

Forkhead transcription factor O1 (FoxO1) in torafugu pufferfish Takifugu rubripes: Molecular cloning, in vitro DNA binding, and target gene screening in fish metagenome.

The fish insulin/insulin-like growth factor (IGF) pathway has weak control over carbohydrate metabolism. To understand the molecular basis for the metabolic diversity, we characterized the forkhead box transcription factor O1A (FoxO1A), a downstream target of the insulin/IGF pathway, in torafugu Takifugu rubripes. The cloned torafugu FoxO1A cDNA contained all conserved features critical for its transcriptional activity and a unique unspliced intron encoding a poly-glutamine stretch. Torafugu FoxO1A showed the IGF-dependent nuclear exclusion and in vitro binding to the well-conserved FoxO1 binding site, DAF-16 binding element (DBE), but failed to bind to the insulin-responsive element by which mammalian FoxO1 mediates insulin effects. The subsequent in silico genomic screening provided a list of 587 potential torafugu FoxO1A target genes containing the DBE. Some carbohydrate metabolic genes regulated by FoxO1 in mammals were not included in the list. We further identified about 250 potential fish FoxO1 target genes by integrating results of the DBE screening against fish metagenome that contained 262 species. Neuronal processes appeared to be the common major function of fish FoxO1, although further annotation of the potential target genes is required. These results provide a part of the molecular basis underlying the weak association between the insulin/IGF pathway and carbohydrate metabolism in fish.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO)-mediated de novo synthesis of glycolate-based polyhydroxyalkanoate in Escherichia coli.

Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (RuBisCO) generates 2-phosphoglycolate (2PG) as one of the metabolites from the Calvin-Benson-Bassham (CBB) cycle. In this study, we focused on the fact that glycolate (GL) derived from 2PG can be incorporated into the bacterial polyhydroxyalkanoate (PHA) as the monomeric constituent by using the evolved PHA synthase (PhaC1PsSTQK). In this study, the function of the RuBisCO-mediated pathway for GL-based PHA synthesis was evaluated using Escherichia coli JW2946 with the deletion of glycolate oxidase gene (ΔglcD) as the model system. The genes encoding RuBisCO, phosphoribulokinase and 2PG phosphatase (PGPase) from several photosynthetic bacteria were introduced into E. coli, and the cells were grown on xylose as a sole carbon source. The functional expression of RuBisCO and relevant enzymes was confirmed based on the increases in the intracellular concentrations of RuBP and GL. Next, PHA biosynthetic genes encoding PhaC1PsSTQK, propionyl-CoA transferase and 3-hydroxybutyryl(3HB)-CoA-supplying enzymes were introduced. The cells accumulated poly(GL-co-3HB)s with GL fractions of 7.8-15.1 mol%. Among the tested RuBisCOs, Rhodosprium rubrum and Synechococcus elongatus PCC7942 enzymes were effective for P(GL-co-3HB) production as well as higher GL fraction. The heterologous expression of PGPase from Synechocystis sp. PCC6803 and R. rubrum increased GL fraction in the polymer. These results demonstrated that the RuBisCO-mediated pathway is potentially used to produce GL-based PHA in not only E. coli but also in photosynthetic organisms.

Molecular Characterization of a Dirofilaria immitis Cysteine Protease Inhibitor (Cystatin) and Its Possible Role in Filarial Immune Evasion.

Infection with canine heartworm (Dirofilaria immitis), spread via mosquito vectors, causes coughing, asthma, pneumonia, and bronchitis in humans and other animals. The disease is especially severe and often fatal in dogs and represents a serious threat to public health worldwide. Cysteine protease inhibitors (CPIs), also known as cystatins, are major immunomodulators of the host immune response during nematode infections. Herein, we cloned and expressed the cystatin Di-CPI from D. immitis. Sequence analysis revealed two specific cystatin-like domains, a Q-x-V-x-G motif, and a SND motif. Phylogenetic analysis indicates that Di-CPI is a member of the second subgroup of nematode type II cystatins. Probing of D. immitis total proteins with anti-rDi-CPI polyclonal antibody revealed a weak signal, and immunofluorescence-based histochemical analysis showed that native Di-CPI is mainly localized in the cuticle of male and female worms and the gut of male worms. Treatment of canine peripheral blood mononuclear cells (PMBCs) with recombinant Di-CPI induced a Th2-type immune response characterized by high expression of the anti-inflammatory factor interleukin-10. Proliferation assays showed that Di-CPI inhibits the proliferation of canine PMBCs by 15%. Together, the results indicate that Di-CPI might be related to cellular hyporesponsiveness in dirofilariasis and may help D. immitis to evade the host immune system.

Molecular cloning, characterization of dax1 gene and its response to progesterone in Misgurnus anguillicaudatus.

Progesterone (P4) are aquatic contaminants that can impair fish reproduction even in low concentrations. The aim of this study was to investigate the effects of P4 on the sex differentiation, by quantitative determination of transcriptional changes of a candidate target gene (dax1, has a function in the sex determination and gonadal differentiation of several vertebrate species) in Misgurnus anguillicaudatus. We first cloned and characterized the full-length cDNAs for the dax1 in M. anguillicaudatus (designated as Ma-dax1). Sequence analysis reveals that Ma-dax1 shares high homology with dax1 in other species. Quantitative real-time PCR (qRT-PCR) and in situ hybridization showed that Ma-dax1 gene was highly conserved during vertebrate evolution and involved in a wide range of developmental processes including embryogenesis, central nervous system development and gonad development. For the P4 administration assay, groups of mature fish were exposed for 1, 7, 14, 21 and 28 days to nominal concentrations of 10, 100, and 1000 ng/L P4 in a flow-through system. Quantification of Ma-dax1 transcripts revealed the expression of Ma-dax1 mRNA is altered after P4 treatment in mature gonads. Those showed that P4 could influence the sexual development and sex differentiation in M. anguillicaudatus by disturbing sex differentiation-associated gene expression, and dax1 can be used as a sensitive molecular biomarker for early warning to monitor the environmental progestins chemicals in fresh water environment.

Isolation and Expression Analysis of Three Types of α-Carbonic Anhydrases from the Antarctic Alga Chlamydomonas sp. ICE-L under Different Light Stress Treatments.

Carbonic anhydrases (CAs) are a class of zinc-containing metalloenzymes that can reversibly catalyse the hydration reaction of carbon dioxide. Antarctic algae are the most critical component of the Antarctic ecosystem; algae can enter the carbon cycle food chain by fixing carbon dioxide from the air. In this study, the complete open reading frames (ORFs) of CA1 (GenBank ID KY826431), CA2 (GenBank ID KY826432), and CA3 (GenBank ID KY826433), encoding CAs in the Antarctic ice microalga Chlamydomonas. sp. ICE-L, were successfully cloned using reverse transcription-polymerase chain reaction (RT-PCR). In addition, the expression patterns of CAs under blue light, under UV light, and in the dark were determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The CA1, CA2, and CA3 ORFs encode proteins of 376, 430, and 419 amino acids, respectively. Phylogenetic analysis revealed that all amino acid sequences showed high homology with those of C. sp. ICE-L. There are six types of algal CAs; we hypothesised that the CAs studied here are most likely α-CAs. Expression analysis showed that the transcription level of the CAs was influenced by both UV light and blue light. These findings provide additional insight into the molecular mechanisms of CAs and will accelerate the development of CAs for applications in agriculture and environmental governance.

Light-enhanced expression of Carbonic Anhydrase 4-like supports shell formation in the fluted giant clam Tridacna squamosa.

Giant clams represent symbiotic associations between a host clam and its extracellular zooxanthellae. They are able to grow in nutrient-deficient tropical marine environments and conduct light-enhanced shell formation (calcification) with the aid of photosynthates donated by the symbiotic zooxanthellae. In light, there is a high demand for inorganic carbon (Ci) to support photosynthesis in the symbionts and light-enhanced calcification in the host. In this study, we cloned and characterized a host Carbonic Anhydrase 4 homolog (CA4-like) from the whitish inner mantle of the giant clam Tridacna squamosa. The full cDNA coding sequence of CA4-like consisted of 1002 bp, encoding for 334 amino acids of 38.5 kDa. The host CA4-like was phenogramically distinct from algal CAs. The transcript level of CA4-like in the inner mantle was ~3-fold higher than those in the colorful outer mantle and the ctenidium. In the inner mantle, CA4-like was immunolocalized in the apical membrane of the seawater-facing epithelial cells, but absent from the shell-facing epithelium. Hence, CA4-like was positioned to catalyze the conversion of HCO3- to CO2 in the ambient seawater which would facilitate CO2 uptake. The absorbed CO2 could be converted back to HCO3- by the cytoplasmic CA2-like. As the protein abundance of CA4-like increased in the inner mantle after 6 or 12 h of light exposure, there could be an augmentation of the total CA4-like activity to increase Ci uptake in light. It is plausible that the absorbed Ci was allocated preferentially for shell formation due to the close proximity of the seawater-facing epithelium to the shell-facing epithelium in the inner mantle that contains only few zooxanthellae.

Development of a high efficient biocatalyst by oriented covalent immobilization of a novel recombinant 2'-N-deoxyribosyltransferase from Lactobacillus animalis.

The 2'-N-deoxyribosyltransferases [NDT; EC 2.4.2.6] are a group of enzymes widely used as biocatalysts for nucleoside biosynthesis. In this work, the molecular cloning, expression and purification of a novel NDT from Lactobacillus animalis (LaNDT) have been reported. On the other hand, biocatalyst stability has been significantly enhanced by multipoint covalent immobilization using a hetero-functional support activated with nickel-chelates and glyoxyl groups. The immobilized enzyme could be reused for more than 300 h and stored during almost 3 months without activity loss. Besides, the obtained derivative (Ni2+-Gx-LaNDT) was able to biosynthesize 88 mg floxuridine/g biocatalyst after 1 h of reaction. In this work, a green bioprocess by employing an environmentally friendly methodology was developed, which allowed the obtaining of a compound with proven anti-tumor activity. Therefore, the obtained enzymatic biocatalyst meets the requirements of high activity, stability, and short reaction times needed for low-cost production in a future preparative application.

An NADPH-dependent Lactobacillus composti short-chain dehydrogenase/reductase: characterization and application to (R)-1-phenylethanol synthesis.

A new anti-Prelog short-chain dehydrogenase/reductase (SDR) encoding gene lcsdr was cloned from Lactobacillus composti DSM 18527, and heterologously expressed in Escherichia coli. LcSDR is nicotinamide adenine dinucleotide phosphate (NADPH)-dependent and has a molecular weight of approximately 30 kDa. The optimal pH and temperature were 6.5 and 30 °C, respectively. The maximal reaction rate Vmax was 133.9 U mg-1; the Michaelis-Menten constant K m of LcSDR were 0.345 mM for acetophenone (1a), and 0.085 mM for NADPH. Through introducing an EsGDH-catalyzed NADPH regeneration system, a biocatalytic process for (R)-1-phenylethanol ((R)-1b) was developed with outstanding time-space yield. Under the optimized conditions, 50 g l-1 1a was converted to (R)-1b in 2 h with a yield of 93.8%, enantiomeric excess of product (e.e.p) above 99% and space-time yield of 562.8 g l-1 d-1.

Characterization and Functional Analysis of 4-Coumarate:CoA Ligase Genes in Mul-berry.

A small, multigene family encodes 4-coumarate:CoA ligases (4CLs) that catalyze the ligation of CoA to hydroxycinnamic acids, a branch point directing metabolites to flavonoid or monolignol pathways. In this study, we characterized four 4CL genes from M. notabilis Genome Database, and cloned four Ma4CL genes from M. atropurpurea cv. Jialing No.40. A tissue-specific expression analysis indicated that Ma4CL3 was expressed at higher levels than the other genes, and that Ma4CL3 was strongly expressed in root bark, stem bark, and old leaves. Additionally, the expression pattern of Ma4CL3 was similar to the trend of the total flavonoid content throughout fruit development. A phylogenetic analysis suggested that Mn4CL1, Mn4CL2, and Mn4CL4 belong to class I 4CLs, and Mn4CL3 belongs to class II 4CLs. Ma4CL genes responded differently to a series of stresses. Ma4CL3 expression was higher than that of the other Ma4CL genes following wounding, salicylic acid, and ultraviolet treatments. An in vitro enzyme assay indicated that 4-coumarate acid was the best substrate among cinnamic acid, 4-coumarate acid, and caffeate acid, but no catalytic activity to sinapate acid and ferulate acid. The results of subcellular localization experiments showed that Ma4CL3 localized to the cytomembrane, where it activated transcription. We used different vectors and strategies to fuse Ma4CL3 with stilbene synthase (STS) to construct four Ma4CL-MaSTS co-expression systems to generate resveratrol. The results indicated that only a transcriptional fusion vector, pET-Ma4CL3-T-MaSTS, which utilized a T7 promoter and lac operator for the expression of MaSTS, could synthesize resveratrol.

Cytochrome P450 20A1 in zebrafish: Cloning, regulation and potential involvement in hyperactivity disorders.

Cytochrome P450 (CYP) enzymes for which there is no functional information are considered "orphan" CYPs. Previous studies showed that CYP20A1, an orphan, is expressed in human hippocampus and substantia nigra, and in zebrafish (Danio rerio) CYP20A1 maternal transcript occurs in eggs, suggesting involvement in brain and in early development. Moreover, hyperactivity is reported in humans with chromosome 2 microdeletions including CYP20A1. We examined CYP20A1 in zebrafish, including impacts of chemical exposure on expression. Zebrafish CYP20A1 cDNA was cloned, sequenced, and aligned with cloned human CYP20A1 and predicted vertebrate orthologs. CYP20A1s share a highly conserved N-terminal region and unusual sequences in the I-helix and the heme-binding CYP signature motifs. CYP20A1 mRNA expression was observed in adult zebrafish organs including the liver, heart, gonads, spleen and brain, as well as the eye and optic nerve. Putative binding sites in proximal promoter regions of CYP20A1s, and response of zebrafish CYP20A1 to selected nuclear and xenobiotic receptor agonists, point to up-regulation by agents involved in steroid hormone response, cholesterol and lipid metabolism. There also was a dose-dependent reduction of CYP20A1 expression in embryos exposed to environmentally relevant levels of methylmercury. Morpholino knockdown of CYP20A1 in developing zebrafish resulted in behavioral effects, including hyperactivity and a slowing of the optomotor response in larvae. The results suggest that altered expression of CYP20A1 might be part of a mechanism linking methylmercury exposure to neurobehavioral deficits. The expanded information on CYP20A1 brings us closer to "deorphanization", that is, identifying CYP20A1 functions and its roles in health and disease.

Restriction-ligation-free (RLF) cloning: a high-throughput cloning method by in vivo homologous recombination of PCR products.

In this study, we optimized a restriction-ligation-free (RLF) method to save time and cost of constructing multiple plasmids with the same gene insert, and examined the efficacy of RLF on high-throughput multi-plasmid cloning. This method utilizes the precise DNA repair and recombination systems within Escherichia coli, which allows to bypass the in vitro restriction and ligation enzyme reactions commonly included in routine cloning procedures. A homologous arm is linked to the 5'-end of the forward primer used to amplify both the target gene and vector. A different homologous arm is linked to the 5'-end of the reverse primer. Therefore, genes can be cloned into the vectors by homologous recombination after co-transformation of the amplified target gene and the linearized vector, which bear the same homologous arm on either end. More than twenty-four different plasmids were generated by this method, which uses two simple polymerase chain reaction steps. This method is highly efficient in cloning any gene of interest into any vector at any site without sequence constraints, as no restriction and ligation reactions are required.

Escherichia coli expression, purification, crystallization, and structure determination of bacterial cohesin-dockerin complexes.

Cellulosomes are highly efficient nanomachines that play a fundamental role during the anaerobic deconstruction of complex plant cell wall carbohydrates. The assembly of these complex nanomachines results from the very tight binding of repetitive cohesin modules, located in a noncatalytic molecular scaffold, and dockerin domains located at the C-terminus of the enzyme components of the cellulosome. The number of enzymes found in a cellulosome varies but may reach more than 100 catalytic subunits if cellulosomes are further organized in polycellulosomes, through a second type of cohesin-dockerin interaction. Structural studies have revealed how the cohesin-dockerin interaction mediates cellulosome assembly and cell-surface attachment, while retaining the flexibility required to potentiate catalytic synergy within the complex. Methods that might be applied for the production, purification, and structure determination of cohesin-dockerin complexes are described here.

A novel approach for secretion of heterologous proteins with correct n-terminal processing by using α-factor pre sequence in Pichia pastoris.

Numerous proteins have been secreted in P. pastoris by fusing the target gene with α-factor pre-pro sequence at Kex2 endopeptidase cleavage site. However, in some instances the product cannot be correctly processed due to aberrant cleavage by Kex2 endopeptidase such as aprotinin. In this study, an aprotinin gene was cloned into pPIC9K at the signal peptidase cleavage site through a single NheI restriction site designed at the 3'end of the α-factor signal sequence preregion, and transformed into GS115 host cell. By G418 resistance and ELISA assay, a high-yield recombinant was selected. After fed-batch cultivation in a 7-L bioreactor, the product was efficiently secreted into culture medium and accumulated up to ~4.7 mg L⁻¹. MALDI-TOF/MS and N-terminal analyses confirmed its authenticity. Thus, a novel cloning strategy for secretion of aprotinin with correct N-terminal processing in P. pastoris has been developed which can be potentially applied to other proteins.

BacMam virus transduced cardiomyoblasts can be used for myocardial transplantation using AP-PEG-A microcapsules: molecular cloning, preparation, and in vitro analysis.

The potential of genetically modified cardiomyoblasts in treating damaged myocardium is well known. However, efficient delivery of these cells is of major concern during treatment. The limiting factors are the massive cell death that occurs soon after their intramyocardial transplantation into the beating heart. To address these problems, we generated recombinant baculoviruses (BacMam viruses) which efficiently transduced cardiomyoblast cells under optimized conditions. These genetically modified cells were then protected in a new polymeric microcapsule using poly-ethylene-glycol (PEG), alginate, and poly-L-lysine (PLL) polymers for efficient delivery. Results showed that microcapsules maintain cell viability and support cell proliferation for at least 30 days. The capsules exhibit strong immunoprotective potential and have high mechanical and osmotic stability with more than 70% intact capsules. The encased transduced cells showed a rapid transgene expression inside the capsule for at least 15 days. However, preclinical studies are needed to further explore its long-term functional benefits.

Analogues of Y27632 increase gap junction communication and suppress the formation of transformed NIH3T3 colonies.

BACKGROUND: Constitutive activation of RhoA-dependent RhoA kinase (ROCK) signalling is known to promote cellular transformation and the ROCK inhibitor Y-27632 has the ability to suppress focus formation of RhoA transformed NIH3T3 cells. METHODS: Sixty-four novel structural analogues of Y27632 were synthesised and tested for their ability to persistently inhibit the transformation of NIH3T3 cells by Rho guanidine exchange factor 16 (ARHGEF16) or Ras. In vitro kinase inhibitor profiling, co-culture of transformed cells with non-transformed cells and a novel Lucifer yellow/PKH67 dye transfer method were used to investigate their mode of action. RESULTS: Four Y27632 analogues inhibited transformed focus formation that persisted when the compound was withdrawn. No toxicity was observed against either transformed or non-transformed cells and the effect was dependent on co-culture of these two cell types. In vitro kinase inhibitor profiling indicated that these compounds had reduced activity against ROCK compared with Y27632, targeting instead Aurora A (AURKA), p38 (MAPK14) and Hgk (MAP4K4). Dye transfer analysis showed they increased gap junction intercellular communication (GJIC) between transformed and non-transformed cells. CONCLUSIONS: These data are the first to suggest that transient blockade of specific kinases can induce a persistent inhibition of non-contact inhibited transformed colony formation and can also remove pre-formed colonies. These effects could potentially be mediated by the observed increase in GJIC between transformed and non-transformed cells. Selection of kinase inhibitors with this property may thus provide a novel strategy for cancer chemoprevention.

Culture of Escherichia coli in SOC medium improves the cloning efficiency of toxic protein genes.

In this paper, we report a useful protocol for cloning toxic protein genes. Use of the SOC medium, which is a glucose-containing rich medium, significantly improved the transformation efficiency of a recombinant plasmid containing a toxic plant subtilase SaSBT1 cDNA. Both glucose and rich nutrients present in the SOC medium prevented the unintended activation of the lac promoter carried on the cloning vector, and led to significantly improved transformation efficiency of recombinant plasmids containing toxic protein genes and an increased rate of transformant growth.

Differential effects of linear and cyclic polyamines on NMDA receptor activities.

The linear polyamine spermine enhances N-methyl-d-aspartate (NMDA) receptors activity at depolarized membrane potential and shows a voltage-dependent block. Spermine potentiates NMDA receptor currents in the presence of saturating concentrations of glutamate and glycine, but cyclic polyamines such as CP2323 do not. CP2323 inhibited the currents most potently amongst 10 kinds of cyclic polyamines tested. The inhibition was prominent at heteromeric NR1/NR2A and NR1/NR2B receptors but not at NR1/NR2C and NR1/NR2D receptors expressed in Xenopus oocytes. Inhibition by CP2323 was voltage-dependent, because the degree of inhibition was in the order -100mV>-70mV>-20mV. It was 10-100 times more prominent than inhibition by spermine. The inhibitory potency of both CP2323 and spermine was attenuated by the mutations around the vestibule of the channel pore at NR1 W563, N650, T807, and NR2B Y646. Inhibition by CP2323 was hardly affected by the mutations of NR1 N616 and E621, whereas inhibition by spermine was reduced by these mutations. The results suggest that CP2323 interacts with the vestibule region of the NMDA receptor and does not enter deep into the channel. Mutations of NR2B W607 greatly reduced the inhibition by CP2323 and spermine, suggesting that the mutation of this residue may cause the change of the channel structure. Neuroprotective effects of cyclic polyamines against cell damage caused by NMDA were compared with those of spermine in cultured rat hippocampal neurons. Addition of CP2323, but not spermine, into the medium attenuated the neurotoxicity induced by NMDA. These results indicate that CP2323 functions as a channel blocker of the NMDA receptor.

Molecular and functional characterization of an I(h)-channel from lobster olfactory receptor neurons.

We isolated a cDNA named PAIH encoding a member of the I(h)-channel family expressed in olfactory receptor neurons (ORNs) of the spiny lobster Panulirus argus. Functional expression of recombinant PAIH in HEK293 cells generated a slowly activating, noninactivating inward current under whole-cell voltage-clamp to hyperpolarizing voltage steps, the amplitude and activation rate of which increase with increasing hyperpolarization. The channel is weakly selective for K+. Intracellular cAMP or cGMP shifts activation of the current to less negative potentials in a concentration-dependent manner. Finally, the channel is blocked by the I(h)-channel blocker ZD7288. An I(h)-channel sharing the properties of the recombinant channel occurs in cultured lobster ORNs. PAIH immunoreactivity localizes the protein to the transduction compartment of the ORNs in situ, and selectively applying the blocker to the transduction compartment reduces spontaneous activity in the ORN. Collectively, these results implicate for the first time a functional role for an I(h)-channel in olfactory signal transduction.

Cross-talk between beta(1)-adrenoceptors and ET(A) receptors in modulation of the slow component of delayed rectifier K(+) currents.

Delayed rectifier K(+) currents (I(K)) play a critical role in determining cardiac action potential duration (APD). Modulation of I(K) affects cardiac excitability critically. There are three components of cardiac delayed rectifier, and the slowly activating component (I(Ks)) is influenced strongly by a variety of stimuli. Plasma levels of noradrenaline and endothelin are elevated in heart failure, and arrhythmias are promoted by such humoral abnormalities through modulation of ion channels. It has been reported that protein kinase A (PKA) and protein kinase C (PKC) modulate I(Ks) from human minK in a complex manner. In the present study, we coexpressed human minK with the human beta(1)-adrenoceptor (hbeta(1)AR) and the endothelin receptor subtype A (hET(A)R) in Xenopus oocytes and investigated the effects of receptor activation on the currents (I(Ks)) flowing through the oocytes. ET-1 modulated I(Ks) biphasically: a transient increase followed by a decrease. The PKC inhibitor chelerythrine completely inhibited the effects of ET-1. Intracellular EGTA abolished the transient increase by ET-1 and partially inhibited the subsequent decrease in the currents. When I(Ks) was increased by 10(-6) M isoproterenol (ISO), ET-1 did not increase but rather decreased the current to an even greater extent than under control conditions. In addition, the effects of ISO on I(Ks) were suppressed by ET(A)R stimulation. These data indicate that I(Ks) can be regulated by cross-talk between the ET(A)R and beta(1)AR systems in addition to direct regulation by each receptor system.